Immunotherapy for polyomaviruses

Abstract

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

Description

[0001] Related applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 62 / 878,105, filed on July 24, 2019 (which is incorporated in its entirety by reference).

[0003] background

[0004] Polyomaviruses are ubiquitous viruses that infect many different mammalian species. Currently, more than 12 different human polyomavirus species have been identified, including BK polyomavirus (BKV / human polyomavirus 1), John Cunningham polyomavirus (JCV / human polyomavirus 2), and Merkel cell polyomavirus (MCV / human polyomavirus 5)).

[0005] Most such polyomaviruses are typically asymptomatic in humans. However, disease-associated human polyomaviruses are often acquired in childhood and / or in hosts with compromised immune systems. For example, initial JCV infection can occur via the tonsils or gastrointestinal tract and remain latent in the gastrointestinal tract and possibly in lymphoid organs, neuronal tissue, and the kidneys. The virus continues to multiply and shed viral particles in the gastrointestinal tract and possibly in lymphoid organs, neuronal tissue, and the kidneys. Subsequently, in the absence of immunity, immunosuppression, or immunodeficiency, both JCV and BKV can be reactivated and develop into serious organ diseases.

[0006] Of particular note is JC virus, which can cross the blood-brain barrier to enter the central nervous system (CNS). In the CNS, JC virus is neurotropic, thereby infecting glial cells (e.g., oligodendrocytes and astrocytes) and meningeal cells. Once reactivated in the brain (e.g., in subjects with compromised immune systems), JCV infection is associated with white matter demyelination and several pathological syndromes, such as JCV granule cell layer neuron disease (JCV GCN), JCV encephalopathy (JCV CPN / JCVE), JCV meningitis (JCVM), and especially progressive multifocal leukoencephalopathy (PML) (a demyelinating disease of the central nervous system with a high mortality rate). Polyomavirus (PML) has been observed almost exclusively in patients with severe immunodeficiency (such as those with acquired immunodeficiency syndrome (AIDS)) and in patients receiving immunosuppressive therapy (e.g., steroids, cell inhibitors and antiproliferators, therapeutic antibodies, calcineurin inhibitors, anti-rejection drugs, etc.) (such as organ transplant recipients, patients with Hodgkin's lymphoma, multiple sclerosis, psoriasis, and other autoimmune diseases). Currently, there are no effective drugs to suppress or cure the viral infection; treatment primarily relies on reversing or alleviating the patient's immunodeficiency to slow or halt disease progression. Unfortunately, such strategies require pausing or discontinuing therapy in immunosuppressed patients, creating a predicament that makes these patients susceptible to one of their two conditions. Therefore, new therapies are needed to treat and prevent polyomavirus infection and / or polyomavirus-related diseases.

[0007] Overview

[0008] This document provides compositions and methods relating to polyomavirus epitopes (e.g., epitopes listed in Tables 1, 2, 3, 4, 5, and / or 6), which are recognized by T lymphocytes (e.g., cytotoxic T lymphocytes (CTLs) and / or helper T lymphocytes) and are useful in the prevention and / or treatment of polyomavirus infection (e.g., JCV infection) and / or cancer (e.g., polyomavirus-associated cancers such as JCV-associated cancers). In some embodiments, the compositions and methods relate to JCV epitopes (e.g., epitopes listed in Tables 1, 2, and 3). In some embodiments, the compositions and methods relate to heterozygous epitopes (e.g., epitopes listed in Table 4) that are incorporated into viral strains and / or pervade sequence variations found in relevant viral strains.

[0009] In some aspects, this document provides peptides (e.g., isolated polypeptides and / or recombinant polypeptides) comprising one or more epitopes from one or more JCV antigens (e.g., epitopes from LTA virus antigens, STA virus antigens, or VP1 virus antigens (as listed in Tables 1, 2, and 3)) and / or one or more heterozygous epitopes (e.g., epitopes listed in Table 4). In some embodiments, the polypeptide comprises multiple such epitopes. In some embodiments, the polypeptide further comprises an intercalated amino acid sequence between at least two of the multiple epitopes. In some embodiments, the peptide is capable of evoking an immune response upon administration to a subject (e.g., a mammalian subject, such as a human subject)).

[0010] In some implementations, epitopes are selected to provide broad coverage of the human population. In some implementations, epitopes are HLA Class I restricted to HLA-A1, HLA-A2, HLA-A3, HLA-A11, HLA-A23, HLA-A24, HLA-A26, HLA-A29, HLA-A30, HLA-B7, HLA-B8, HLA-B27, HLA-B35, HLA-B38, HLA-B40, HLA-B41, HLA-B44, HLA-B51, HLA-B56, HLA-B57, or HLA-B58. In some implementations, epitopes are HLA Class II restricted to HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR. In some implementations, epitopes are HLA Class II restricted to HLA-DRB or HLA-DQB. In some embodiments, the peptide comprises, or consists substantially of, the epitope amino acid sequences shown in SEQ ID NO:1 to SEQ ID NO:21. In some embodiments, pharmaceutical compositions comprising the peptides provided herein are provided.

[0011] In some aspects, this document provides nucleic acids (e.g., isolated nucleic acids) encoding peptides disclosed herein. In some embodiments, this document provides expression constructs comprising such nucleic acids. In some embodiments, this document provides host cells comprising such expression constructs. In some aspects, this document provides methods for producing isolated peptides, the methods comprising expressing the isolated peptides in host cells provided herein and at least partially purifying the isolated peptides. In some embodiments, this document provides pharmaceutical compositions comprising nucleic acids provided herein.

[0012] In some aspects, this document provides T lymphocytes (e.g., isolated T lymphocytes, CD4+ T lymphocytes, CD8+ T lymphocytes) comprising T cell receptors (TCRs) that specifically bind to epitopes described herein presented on HLA (e.g., class I HLA, class II HLA). In some embodiments, this document provides a method for expanding BK virus-specific T lymphocytes for adoptive immunotherapy, the method comprising: (i) contacting one or more cells isolated from a subject, one or more of which comprise T lymphocytes, wherein an antigen-presenting cell presents an epitope provided herein; and (ii) culturing one or more cells under conditions that cause BK virus-specific T lymphocytes to expand from said one or more cells. In a particular embodiment, the operation of culturing one or more cells is performed in the presence of IL-2 and / or IL-21. In some implementations, the concentrations of 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, 20 ng / ml, 21 ng / ml, 22 ng / ml, and 23 ng / ml are present. Cells are cultured at 24 ng / ml, 25 ng / ml, 26 ng / ml, 27 ng / ml, 28 ng / ml, 29 ng / ml, or 30 ng / ml of IL-2 and / or IL-21. In some embodiments, cells are cultured at 30 ng / ml, 35 ng / ml, 40 ng / ml, 45 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml of IL-2 and / or IL-21. In some embodiments, cells are cultured at 10-50 ng / ml, 20-40 ng / ml, 25-35 ng / ml, or about 30 ng / ml of IL-2 and / or IL-21. In some embodiments, cells are cultured at 30 ng / ml of IL-2 and / or IL-21. In some implementations, amplification in the presence of IL-2 and / or IL-21 results in an increase in the ratio of the absolute number of polyomavirus-specific CD8 T cells to the absolute number of polyomavirus-specific CD4 T cells in the amplified T lymphocyte population compared to amplification in the absence of IL-2 and / or IL-21.

[0013] In some embodiments, this document provides methods for treating or preventing polyomavirus infection (e.g., JCV infection) and / or treating polyomavirus-associated cancers (e.g., JCV-associated cancers) and / or inducing a T-lymphocyte immune response in a subject, the methods comprising administering to the subject a peptide, nucleic acid, T cell, or pharmaceutical composition provided herein. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject has an impaired immune system.

[0014] In some aspects, this document provides a method for detecting JC virus infection in a subject, comprising detecting the presence of JCV-specific T lymphocytes by contacting T lymphocytes isolated from the subject with isolated peptides provided herein. In some embodiments, the method further includes treating the subject for JC virus infection according to the methods described herein. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject has an compromised immune system.

[0015] In some aspects, this document provides methods for treating cancer in subjects (e.g., polyomavirus-associated cancers such as JCV-associated cancers). In some embodiments, the method includes administering to the subject a pharmaceutical composition comprising cytotoxic T cells (CTLs) including T cell receptors (TCRs) that recognize one or more epitopes listed in Tables 1, 2, 3, and / or 4 (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more). In some embodiments, the subject expresses human leukocyte antigens (HLA) that restrict one or more epitopes. In some embodiments, the CTLs are autologous to the subject. In some embodiments, the CTL is not autologous to the subject. In some embodiments, the CTL is obtained from a CTL library or CTL database. In some embodiments, the method includes administering a vaccine composition to the subject, the vaccine composition comprising one or more epitopes listed in Tables 1, 2, 3 and / or 4 (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more). In some embodiments, the method includes administering to a subject a pharmaceutical composition comprising antigen-presenting cells (APCs) that present one or more epitopes listed in Tables 1, 2, 3, and / or 4 (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more). In some embodiments, the subject expresses a human leukocyte antigen (HLA) that restricts one or more epitopes.

[0016] In some aspects, this document provides methods for treating polyomavirus infection (e.g., JCV infection) in subjects. In some embodiments, the subject has an compromised immune system. In some embodiments, the method includes administering to the subject a pharmaceutical composition comprising a CTL comprising a TCR that recognizes one or more epitopes (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) listed in Tables 1, 2, 3, and 4. In some embodiments, the subject expresses an HLA that restricts one or more epitopes. In some embodiments, the CTL is autologous to the subject. In some embodiments, the CTL is not autologous to the subject. In some embodiments, CTLs are obtained from a CTL library or CTL database. In some embodiments, the method includes administering a vaccine composition to a subject, the vaccine composition comprising one or more epitopes listed in Tables 1, 2, 3 and / or 4 (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more). In some embodiments, the method includes administering to a subject a pharmaceutical composition comprising antigen-presenting cells (APCs) that present one or more epitopes listed in Tables 1, 2, 3, and / or 4 (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more). In some embodiments, the subject expresses a human leukocyte antigen (HLA) that restricts one or more epitopes.

[0017] In some respects, this document provides a population of CTLs that include T-cell receptors (TCRs) that recognize one or more of the epitopes listed in Tables 1, 2, 3 and / or 4 (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more).

[0018] In some aspects, this document provides populations of APCs that present one or more epitopes listed in Tables 1, 2, 3, and / or 4 (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more). In some embodiments, APCs include B cells, antigen-presenting T cells, dendritic cells, and / or artificial antigen-presenting cells (e.g., aK562 cells). In some aspects, antigen-presenting cells (e.g., aK562 cells) express CD80, CD83, 41BB-L, and / or CD86. In some implementations, this document provides methods for treating or preventing cancer (e.g., polyomavirus-associated cancers such as JCV-associated cancers) and / or polyomavirus (e.g., JCV) infection in subjects, methods comprising administering the APC described herein to the subject.

[0019] In some respects, this document provides polypeptides comprising one or more epitopes listed in Tables 1, 2, 3 and / or 4 (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more). In some aspects, this document provides nucleic acid molecules (e.g., DNA or RNA molecules) encoding polypeptides, the polypeptides comprising one or more epitopes listed in Tables 1, 2, 3, and / or 4 (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more). In some embodiments, the nucleic acid molecule is a vector (e.g., an adenovirus vector). In some embodiments, this document provides vaccine compositions comprising the polypeptides and / or nucleic acid molecules described herein.

[0020] In some embodiments, this document provides methods for generating, activating, and / or inducing the proliferation of polyomavirus-specific CTLs (e.g., JCV-specific CTLs), including methods comprising contacting the CTLs with one or more epitopes (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more) of APCs presenting in Tables 1, 2, 3, and / or 4. In some embodiments, the CTLs are contacted with APCs in vitro. In some embodiments, APCs include B cells, antigen-presenting T cells, dendritic cells, and / or artificial antigen-presenting cells (e.g., aK562 cells). In some aspects, antigen-presenting cells (e.g., aK562 cells) express CD80, CD83, 41BB-L, and / or CD86. In some embodiments, CTLs contact APCs in the presence of one or more cytokines.

[0021] In some implementations, this document provides a method for generating an APC that presents the tabletops provided herein, the method comprising making the APC with one or more tabletops including those listed in Tables 1, 2, 3, and / or 4 (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, ...). Nucleic acid contacts of 29, 30 or more peptides and / or peptides encoding one or more epitopes listed in Tables 1, 2, 3 and / or 4 (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more). In some embodiments, APC expression restricts HLA to one or more epitopes.

[0022] In some embodiments, one or more epitopes include epitopes shared by two or more polyomaviruses. In some embodiments, the shared epitopes include sequence homology regions between at least two polyomaviruses, and the sequence homology regions are at least 3, 4, 5, 6, or 7 amino acids spanning the entire length of the epitope sequence. In some embodiments, the two polyomaviruses are BKV and JCV. In some embodiments, at least three amino acids are LLL.

[0023] In other respects, this document provides methods for identifying subjects suitable for the treatments provided herein (e.g., administration of the CTL, APC, or vaccine composition provided herein), methods comprising isolating a sample (e.g., a blood sample or tumor sample) from the subject and detecting the presence of an epitope provided herein or a nucleic acid encoding an epitope provided herein. In some embodiments, a subject is identified as suitable for the treatments provided herein if the subject expresses an HLA that restricts one or more of the epitopes described herein. In some embodiments, the subject identified as suitable for the treatments provided herein is treated with the treatment method.

[0024] Brief description of the attached figures

[0025] Figure 1 T cell responses to JCV antigens are shown. In short, PBMCs from 17 healthy subjects were stimulated with JCV overlapping peptide libraries (OPPs), and T cells expanded for up to 14 days in the presence of IL-2. Cell expression for IFN-γ was measured on day 14 after stimulation with their respective peptide libraries using flow cytometry. The figures show compiled data from all 17 donors after stimulation with their respective BKVOPPs; (A) shows BKV-specific CD8. + T cell response, and (B) shows CD4 + T cell response.

[0026] Figure 2 Representative data demonstrating the identification of T-cell determinants using a two-dimensional peptide matrix are depicted. JCV-specific T cells expanded in vitro using OPP were further characterized by the identification of specific T-cell determinants. Individual overlapping peptides were used to prepare sub-libraries (24 libraries; LTA1–LTA24), and T-cell responses for each library were measured by intracellular cytokine staining (ICS) IFN-γ assay, as shown in the bar graph of Figure A. The responses of the sub-libraries were superimposed on a two-dimensional matrix, thus revealing the individual peptides commonly found in the libraries. The FAC plot in Figure B demonstrates the T-cell responses of each individual peptide (P29, P30, and P32) used in the IFN-γ ICS assay, thus confirming the peptides responsible for inducing JCV-specific T-cell responses.

[0027] Figure 3Representative data from HLA class II restriction analysis of epitopes localized from the JCV-LT antigen are presented. Specifically, HLA class II restriction is shown for the VDLHAFLSQAVFSNR (LT29) peptide, FLSQAVFSNRTVASF (LT30) peptide, and TVASFAVYTTKEKAQ (LT32) peptide as HLA DRB1*10:01. In short, a set of lymphoblasts (LCLs) with a single allele matching the donor's HLA type were selected and loaded with their respective peptides for 1 hour. The loaded LCLs were then used as stimulating cells in an IFN-γ ICS assay. Upon presentation by MHC, the peptides elicited an IFN-γ response in JCV-specific T cells.

[0028] Figure 4 T cell cross-reactivity between BKV and JCV epitopes was demonstrated. ICS FACS plots show IFN-γ expression in T cells amplified with either the BKV epitope (SSGTQQWRGLARYFK) or the JCV epitope (RSGSQQWRGLSRYFK). Stimulation of these antigen-exposed T cells with both BKV and JCV peptides demonstrated that T cells amplified with one of these epitopes recognize both the BKV and JCV peptide sequences.

[0029] Figure 5 The illustration shows JCV-specific T cell expansion from healthy subjects. CD4 cells expressing IFN-γ were assessed. + The frequency of T cells, and the response of each individual subject is shown in the graph.

[0030] Figure 6 This demonstrates the pluripotency of JCV-specific T cells expanded using a pooled peptide library. A representative FAC dot plot illustrates CD4+ after restimulation with a JCV peptide library. + Expression of individual effector molecules in T cells (a). Images (b) and (c) illustrate the versatility of JCV-specific T cells expressing multiple cytokines.

[0031] Figure 7 The transcription factor and effector characteristics of JCV-specific T cells grown in vitro are shown.

[0032] Detailed description

[0033] Overview

[0034] This document provides compositions and methods relating to polyomavirus epitopes (e.g., epitopes listed in Tables 1, 2, 3, and / or 4), which are transmitted via T lymphocytes (e.g., cytotoxic (CD8) cells). + T lymphocytes (CTLs) and / or helper (CD4+) cells +T lymphocytes are identified and are useful in the prevention and / or treatment of polyomavirus infection (e.g., JCV infection) and / or cancer (e.g., polyomavirus-associated cancers such as JCV-associated cancers). In some embodiments, the compositions and methods provided herein relate to JCV epitopes (e.g., epitopes listed in Tables 1, 2, and 3). In some embodiments, the compositions and methods relate to heterozygous epitopes, which include variations found within or throughout BKV and JCV epitopes (e.g., epitopes listed in Table 4).

[0035] definition

[0036] For convenience, certain terms used in the specification, embodiments and appended claims are collected herein.

[0037] In this text, the articles “a” and “an” refer to one or more (i.e., at least one) grammatical object of the article. For example, “an element” means one element or more elements.

[0038] As used herein, the term "administration" means providing a medicine or pharmaceutical composition to a subject and includes (but is not limited to) administration by a healthcare professional and self-administration. Such medicines may contain, for example, peptides described herein, antigen-presenting cells provided herein, and / or CTLs provided herein.

[0039] The term "amino acid" is intended to include all molecules (whether natural or synthetic) that contain both amino and acid functionalities and can be contained in polymers of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; their analogs, derivatives, and homologs; amino acid analogs with variant side chains; and all stereoisomers of any of the foregoing.

[0040] The terms “binding” or “interaction” refer to the association (which can be stable) between two molecules (e.g., between a TCR and a peptide / HLA) due to electrostatic interactions, hydrophobic interactions, ionic interactions, and / or hydrogen bonding interactions under physiological conditions. When an epitope is present on the appropriate HLA, the TCR “recognizes” the T cell epitope it can bind to.

[0041] The terms “biological sample,” “tissue sample,” or simply “sample” each refer to a collection of cells obtained from the tissues of a subject. Tissue samples can originate from solid tissues such as fresh, frozen, and / or preserved organs, tissue samples, biopsies, or extracts; blood or any blood component, serum, blood; bodily fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid, urine, saliva, feces, tears; or cells from any stage of the subject’s pregnancy or development.

[0042] As used herein, the term "cancer" includes (but is not limited to) solid tumors and blood-borne tumors. The term cancer encompasses diseases of the skin, tissues, organs, bones, cartilage, blood, and blood vessels. The term "cancer" also includes primary cancers and metastatic cancers.

[0043] As used herein, the term "homologous" refers to sequence similarity (e.g., nucleic acid or amino acid sequences) between two regions of the same sequence strand or between two regions of different sequence strands. The term "homologous" can also be used to refer to sequence similarity between two regions of the same sequence strand or between two regions of different sequence strands. For example, regions are homologous at a position when the amino acid residues in two regions are occupied by the same amino acid residues. A first region and a second region are homologous if at least one nucleotide residue in each region is occupied by the same residue. Homologousness between two regions is expressed based on the proportion of nucleotide or amino acid residue positions occupied by the same nucleotide or amino acid residues in the two regions. For example, a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology. Preferably, the first region comprises a first 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 95% of the nucleotide residue positions in each of the portions are occupied by the same nucleotide residues. More preferably, all nucleotide residue positions in each of the portions are occupied by the same nucleotide residues.

[0044] The term "separated" refers to material that has been removed from its natural state or otherwise subjected to human manipulation. Separated material may be substantially or essentially free of the components that normally accompany it in its natural state, or it may be manipulated to be in an artificial state together with the components that normally accompany it in its natural state.

[0045] The term “peptide” refers to two or more amino acids linked together by peptide bonds or modified peptide bonds. The terms “peptide,” “polypeptide,” and “protein” as used herein are used interchangeably. In some embodiments, peptides are prepared from recombinant DNA or recombinant RNA, and the peptides are of synthetic origin, or combinations thereof, which (1) are not related to peptides commonly found in nature, (2) are isolated from the cells in which they are normally present, (3) are isolated from the same cellular source without containing other peptides, (4) are expressed by cells from a different species, or (5) are not found in nature.

[0046] The term "epitaph" refers to a peptide determinant that can specifically bind to an antibody or TCR. Epitopes are typically composed of chemically active surface groups of a molecule (such as amino acids or sugar side chains). Some epitopes may be defined by specific amino acid sequences that antibodies can bind to.

[0047] As used herein, the phrase “pharmaceutically acceptable” means, to a reasonable extent of medical judgment, that a pharmaceutical preparation, compound, material, composition, and / or dosage form is suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0048] As used herein, the phrase “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle (such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material) that involves carrying or transporting a pharmaceutical preparation from one organ or part of the body to another organ or part of the body. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the preparation and is harmless to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars (such as lactose, glucose, and sucrose); (2) starches (such as corn starch and potato starch); (3) cellulose and its derivatives (such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate); (4) powdered astragalus gum; (5) malt; (6) gelatin; (7) talc; (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); (10) (11) Diols (such as propylene glycol); (12) Polyols (such as glycerol, sorbitol, mannitol and polyethylene glycol); (13) Esters (such as ethyl oleate and ethyl dodecanoate); (14) Agar; (15) Buffers (such as magnesium hydroxide and aluminum hydroxide); (16) Alginate; (17) Atherless water; (18) Isotonic saline; (19) Ringer's solution; (20) Ethanol; (21) pH buffer solutions; (22) Polyesters, polycarbonates and / or polyanhydrides; and (23) Other non-toxic and compatible substances used in pharmaceutical preparations.

[0049] The terms “polynucleotide” and “nucleic acid” are used interchangeably. They refer to polymeric forms of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, or analogues. Polynucleotides can have any three-dimensional structure and can perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci (locus) determined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribonucleases, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogues. Modifications to the nucleotide structure can be conferred before or after polymer assembly, if present. Polynucleotides can be further modified, such as by coupling with a labeling component. In all nucleic acid sequences provided herein, U nucleotides and T nucleotides are interchangeable.

[0050] As used herein, a "preventive" treatment agent for a condition refers to a compound that, when administered to a statistical sample prior to the onset of a disorder or condition, reduces the occurrence of the disorder or condition in a treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of a disorder or condition or reduces the severity of one or more symptoms of a disorder or condition relative to an untreated control sample.

[0051] As used herein, "specific binding" 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, antibodies or peptides are defined in terms of approximately 10... -7 M or smaller K D The affinity of the antigen to bind specifically to its predetermined antigen or predetermined binding partner, and with an affinity at least 10-fold, at least 100-fold, or at least 1000-fold less than its affinity for binding to nonspecific and unrelated antigen / binding partners (e.g., BSA, casein) (as shown by K). D (Indicated) binds to the predetermined antigen / binding partner.

[0052] As used in this article, the term “subject” means a human or non-human animal selected for treatment or therapy.

[0053] As used herein, the phrases “therapeutic effective amount” and “effective amount” mean the amount of a drug that, when administered to a subject, elicits a sufficient therapeutic response in the subject to provide a beneficial outcome in the subject with a reasonable benefit / risk ratio applicable to any medical treatment.

[0054] "Treating" a disease in a subject or "treating" a subject with a disease refers to subjecting the subject to drug treatment (e.g., administration of a drug) such that at least one symptom of the disease is relieved or prevented from worsening.

[0055] The term "vector" refers to means by which nucleic acids can be replicated and / or transferred between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophages, proviruses, phage particles, transposons, and artificial chromosomes, which may or may not be able to autonomously replicate or integrate into the host cell's chromosome.

[0056] Epitope

[0057] In some embodiments, methods and compositions are provided herein that relate to polyomavirus epitopes (such as JCV epitopes) that are recognized by immune effector cells (e.g., cytotoxic T cells / CTLs) when presented on HLA. In some embodiments, the epitopes described herein are useful in the prevention and / or treatment of polyomavirus infections (e.g., JCV virus infection) and / or cancers (e.g., JCV-associated cancers expressing epitopes provided herein), and / or for the generation of pharmaceutical agents and compositions thereof (e.g., sensitized immune effector cells and / or APCs) useful in the prevention and / or treatment of polyomavirus infections (e.g., JCV virus infection) and / or cancers (e.g., polyomavirus-associated cancers expressing epitopes provided herein). In some embodiments, the epitopes are JCV epitopes listed in Tables 1, 2, and / or 3. In some embodiments, the epitopes are heterozygous epitopes comprising amino acids from both BKV epitopes and homologous JCV epitopes, and / or amino acid variants found in different BKV strains or JCV strains. Exemplary heterozygous epitopes are listed in Table 4. In some embodiments, the compositions and methods described herein also involve epitopes derived from additional viruses such as EBV, CMV, or ADV. In some embodiments, the epitopes are HLA class I restricted T-cell epitopes. In other embodiments, the epitopes are HLA class II restricted T-cell epitopes.

[0058] Table 1: Exemplary JCV HLA-restricted T-cell epitopes

[0059]

[0060] Table 2: List of JCV epitope peptide sequences and homologous BKV epitope peptide sequences

[0061] JCV epitope peptide sequence SEQ ID NO.: Homologous BKV epitope peptide sequence SEQ ID NO.: IDQFMVVFEDVKGTG 1 IDQYMVVFEDVKGTG 22 VDLHAFLSQAVFSNR 2 SDLHQFLSQAVFSNR 23 FLSQAVFSNRTVASF 3 FLSQAVFSNRTLACF 24 TVASFAVYTTKEKAQ 4 TLACFAVYTTKEKAQ 25 ERLNFELGVGIDQFM 5 ERLTFELGVAIDQYM 26 TCGNILMWEAVTLKT 6 TCGNLLMWEAVTVKT 27 RYWLFKGPIDSGKTT 7 RYWLFKGPIDSGKTT 28 MTREEMLVERFNFLL 8 MTREEMLTERFNHIL 29 EQYMAGVAWIHCLLP 9 EQYMAGVAWLHCLLP 30 RKAYLKKCKELHPDK 10 RKAYLRKCKEFHPDK 31 GGHNILFFLTPHRHR 11 AGHNIIFFLTPHRHR 32 RSGSQQWRGLSRYFK 12 SSGTQQWRGLARYFK 33 DPDMMRYVDKYGQLQ 13 DPDMIRYIDRQGQLQ 34 MDKVLNREESMELMD 14 MDKVLNREESMELMD 35 SITEVECFLTPEMGD 15 AITEVECFLNPEMGD 36 SKNQKSICQQAVDTV 16 SKNQKSICQQAVDTV 37 SICQQAVDTVAAKQR 17 SICQQAVDTVLAKKR 38 RNRKFLRSSPLVWID 18 LNRKFLRKEPLVWID 39 LRSSPLVWIDCYCFD 19 LRKEPLVWIDCYCID 40 KMKRMNFLYKKMEQG 20 KMKRMNTLYKKMEQD 41 NFLYKKMEQGVKVAH 21 NTLYKKMEQDVKVAH 42

[0062] Table 3: Exemplary epitope sequences from JCVs homologous to BKV epitope sequences

[0063]

[0064]

[0065] *The amino acid residues from variants derived from BKV epitopes are bolded and underlined.

[0066] **Unrestricted

[0067] Table 4: Exemplary epitope sequences from JCV / BKV heterozygous epitope sequences

[0068]

[0069]

[0070] **Unrestricted

[0071] In some embodiments, this document provides peptides (e.g., polypeptides) comprising one or more epitopes from Tables 1, 2, 3, and / or 4. In some embodiments, the peptides disclosed herein are full-length viral proteins (e.g., full-length BKV proteins and / or full-length JCV proteins). In some embodiments, the peptides are not full-length viral proteins (e.g., not full-length BKV proteins and / or full-length JCV proteins). In some embodiments, the peptides disclosed herein comprise BKV and JCV epitopes with sequence homology (e.g., epitopes listed in Tables 2, 3, and 4). In some embodiments, the peptides disclosed herein comprise fewer than 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, or 10 adjacent amino acids of a viral protein. In some embodiments, the peptides disclosed herein comprise two or more epitopes listed in Tables 1, 2, 3, and / or 4. For example, in some embodiments, the peptides disclosed herein include two or more epitopes listed in Tables 1, 2, 3 and / or 4 linked by a polypeptide linker. In some embodiments, the peptides provided herein include 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, or 27 epitopes (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 of the epitopes listed in Tables 1, 2, 3, and / or 4). In a preferred embodiment, the peptides disclosed herein include the JCV epitopes shown in Table 1 (i.e., any one or any combination of the JCV epitopes shown in SEQ ID No: 1-21). For example, the peptide may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or all 21 epitopes encoded by the amino acid sequences shown in SEQ ID No: 1-21.

[0072] In some aspects, this document provides polypeptides (e.g., isolated polypeptides and / or recombinant polypeptides) comprising multiple epitopes from BKV antigens or JCV antigens (e.g., epitopes from large T antigen (LTA), small T antigen (STA), or major capsid protein VP1 viral antigens (such as those listed in Tables 1, 2, 3, or 4, preferably those shown in Table 1)). More preferably, the polypeptides disclosed herein comprise any one or a combination of the JCV epitopes shown in SEQ ID Nos: 1-21. In some such embodiments, the polypeptide further comprises an intercalated amino acid sequence between at least two of the multiple epitopes. In some embodiments, the intercalated amino acid or amino acid sequence is a proteasome-released amino acid or amino acid sequence. Non-limiting examples of a proteasome-released amino acid or amino acid sequence are or include AD, K, or R. In some embodiments, the intercalated amino acid or amino acid sequence is a TAP recognition motif. Typically, a TAP recognition motif may conform to the following formula: (R / N:I / Q:W / Y) n , where n is any integer ≥1. Non-limiting examples of TAP recognition motifs include RIW, RQW, NIW, and NQY. In some embodiments, the epitopes provided herein are linked or conjugated via a proteasome-released amino acid sequence and optionally a TAP recognition motif at the carboxyl terminus of each epitope. In some such embodiments, the polypeptide comprises, or is substantially composed of, each of the epitopes encoded by the amino acid sequences shown in SEQ ID No:1-21.

[0073] In some embodiments, the peptides provided herein further include epitopes from at least one additional virus (e.g., Epstein-Barr virus (EBV), cytomegalovirus (CMV), and / or adenovirus (ADV)). In some embodiments, the peptide includes epitopes from two or more viruses. In some embodiments, the peptide includes epitopes from three or more viruses. In some embodiments, the peptide includes epitopes from four or more viruses. In some embodiments, the peptide includes epitopes from five or more viruses. For example, in some embodiments, the peptide includes sequences from at least two, three, four, or five of JCV, BKV, EBV, CMV, and / or ADV.

[0074] In some embodiments, this document provides multiepitope peptides comprising two or more of the epitopes described herein (i.e., single-stranded peptides comprising amino acid residues of multiple T-cell epitopes that are not linked in nature). In some embodiments, the T-cell epitopes in the peptide are linked via amino acid linkers. In some embodiments, the T-cell epitopes in the peptide are directly linked without the need for amino acid insertion. Examples of multiepitope peptides, methods for generating multiepitope peptides, and vectors encoding multiepitope peptides can be found in Dasari et al., Molecular Therapy-Methods & Clinical Development (2016) 3, 16058 (which is hereby incorporated in its entirety by reference).

[0075] In some respects, this article provides a library of immunogenic peptides, which includes HLA class I and II restricted polyomavirus peptide epitopes capable of inducing peptide-specific T cell proliferation (e.g., epitopes listed in Tables 1, 2, 3, 4, 5 and / or 6). In some embodiments, the library of immunogenic peptides includes 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, or 27 epitopes (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 of the epitopes listed in Tables 1, 2, 3, and / or 4), or combinations thereof. In a preferred embodiment, the peptide library includes at least one JCV epitope shown in Table 1 (i.e., any one or any combination of the JCV epitopes shown in SEQ ID No: 1-21). For example, a library of immunogenic peptides may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or all 21 epitopes encoded by the amino acid sequences shown in SEQ ID No: 1-21. Most preferably, such a peptide library includes each of the amino acid sequences of the JCV peptide epitopes shown in SEQ ID No: 1-21. Immunogenic peptides and libraries thereof are capable of inducing peptide-specific T cells (e.g., peptide-specific cytotoxic T cells and / or CD4+). + Proliferation of T cells.

[0076] In some embodiments, the compositions and methods provided herein include or relate to naturally occurring variants of the epitopes listed in Tables 1, 2 and / or 3. For example, in some embodiments, this document provides multi-epitope peptides that include two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9 or 10) naturally occurring variants of the epitopes listed in Tables 1, 2 and / or 3.

[0077] In some embodiments, the epitopes provided herein have sequences disclosed herein, except for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) conserved sequence modifications. As used herein, the term "conserved sequence modification" is intended to refer to an amino acid modification that does not significantly affect or alter the interaction between the TCR and a peptide containing an HLA-presented amino acid sequence. Such conserved modifications include amino acid substitutions, additions (e.g., adding an amino acid to the N-terminus or C-terminus of a peptide), and deletions (e.g., deleting an amino acid from the N-terminus or C-terminus of a peptide). A conserved amino acid substitution is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues of the peptide described herein can be substituted with other amino acid residues from the same side chain family, and the retention of TCR binding (e.g., antigenicity) of the altered peptide can be tested using methods known in the art. Modifications can be introduced into antibodies using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0078] In some embodiments, the peptides (e.g., polypeptides) described herein are immunogenic and capable of evoking an immune response upon administration to a subject (e.g., a mammalian subject, such as a human subject). In other embodiments, the peptides (e.g., polypeptides) described herein are capable of evoking an immune response following endogenous processing and / or presentation of the peptide by immune cells (e.g., the subject's immune cells and / or donor-derived immune cells, such as those comprising allogeneic PBMCs) or exogenous processing and / or presentation of the peptide.

[0079] In some aspects, this document provides cells that present one or more of the peptides described herein (e.g., peptides including at least one epitope listed in Tables 1, 2, 3, and / or 4). In some embodiments, the cells are mammalian cells. In some embodiments, the cells are antigen-presenting cells (APCs) (e.g., antigen-presenting T cells, dendritic cells, B cells, macrophages, or artificial antigen-presenting cells (such as aK562 cells)). Cells that present peptides described herein can be generated using standard techniques known in the art. For example, cells may be pulsed to promote peptide uptake. In some embodiments, cells are transfected with nucleic acids encoding the peptides provided herein. In some aspects, this document provides methods for generating antigen-presenting cells (APCs) including pulsed application of the peptides described herein to the cells. Exemplary examples of generating antigen-presenting cells can be found in WO2013088114 (which is hereby incorporated herein in its entirety).

[0080] The peptides provided herein can be isolated from cell or tissue sources using standard protein purification techniques with appropriate purification protocols, can be generated using recombinant DNA technology, and / or can be synthesized chemically using standard peptide synthesis techniques. The peptides described herein can be generated in prokaryotic or eukaryotic host cells by expressing nucleotides encoding one or more peptides of the present invention. Alternatively, such peptides can be synthesized chemically. Methods for expressing heteropeptides in recombinant hosts, methods for the chemical synthesis of peptides, and methods for in vitro translation are well known in the art and have been further documented in Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., ColdSpring Harbor, NY; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.; Merrifield, J. (1969) J. Am. Chem. Soc. 91:501; Chaiken IM (1981) CRC Crit. Rev. Biochem. 11:255; Kaiser et al. (1989) Science 243:187; Merrifield, B. (1986) Science 232:342; Kent, SBH (1988) Annu. Rev. Biochem. 57:957; and Ofow, RE (1980) Semisynthetic Proteins, Wiley Publishing (which is incorporated herein by reference).

[0081] Nucleic acid molecules

[0082] This document provides nucleic acid molecules encoding the epitopes and peptides described herein. Nucleic acids may be present, for example, in whole cells, in cell lysates, or in partially purified or substantially pure forms. The nucleic acid molecules described herein can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, oligonucleotides corresponding to one or more of the epitopes listed in Tables 1, 2, 3, or 4 can be prepared using standard synthetic techniques (i.e., using an automated DNA synthesizer).

[0083] In some embodiments, this document provides vectors containing the nucleic acid molecules described herein (e.g., viral vectors, such as adenovirus-based expression vectors). Viral vectors may contain additional DNA segments that can be linked to a viral genome. Some vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., bacterial vectors with bacterial origins of replication, free-living mammalian vectors). Other vectors (e.g., non-free-living mammalian vectors) can be integrated into the host cell genome upon introduction into the host cell and thus replicated along with the host genome. Furthermore, some vectors are capable of directing gene expression. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”). In some embodiments, this document provides nucleic acids operatively linked to one or more regulatory sequences (e.g., promoters) of an expression vector. In some embodiments, cells transcribe the nucleic acids provided herein and thereby express antibodies, antigen-binding fragments of antibodies, or peptides described herein. The nucleic acid molecules may be integrated into the cell's genome, or they may be extrachromosomal.

[0084] In some embodiments, the nucleic acid vectors or recombinant adenoviruses provided herein encode one or more epitopes listed in Tables 1, 2, 3, and / or 4. For example, a nucleic acid vector or recombinant adenovirus may consist of one or more epitopes from the same table (e.g., one or more epitopes from Table 1, one or more epitopes from Table 2, one or more epitopes from Table 3, or one or more epitopes from Table 4). Alternatively, a nucleic acid vector or recombinant adenovirus may consist of one or more epitopes from the same table (e.g., Table 1) and one or more epitopes from different tables (e.g., Table 2). In some implementations, in addition to the epitopes listed in Tables 1, 2, 3 and / or 4, the nucleic acid vectors or recombinant adenoviruses provided herein encode no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids.

[0085] In some embodiments, the nucleic acid vector comprises a nucleic acid sequence that has undergone codon optimization. In such embodiments, the coding sequence is constructed by changing the codons in each nucleic acid used to assemble the coding sequence. Typically, a method for identifying and optimizing the nucleotide sequence used for codons to produce a peptide includes at least the following steps (a) through (e). In step (a), an oligomer is provided that encodes a portion of a polypeptide containing a degenerate form of the codons for the encoded amino acids in the portion, wherein the oligomer is extended to provide flanking coding sequences with overlapping sequences. In step (b), the oligomer is treated to achieve the assembly of the coding sequence of the peptide. The reassembled peptide is contained in an expression system operatively linked to a control sequence to achieve its expression. In step (c), the expression system is transfected into a culture of compatible host cells. In step (d), the peptide production level of colonies obtained from the transformed host cells is tested. In step (e), at least one colony with the highest or satisfactory peptide production is obtained from the expression system. The sequence of the expression system portion encoding the protein is determined. Further description of codon optimization is provided in U.S. Patent Publication No. US2010 / 035768 (which is incorporated herein by reference in its entirety).

[0086] Antigen-presenting cells

[0087] In some aspects, this document provides an APC that presents (e.g., on HLA) one or more T-cell epitopes provided herein (e.g., one or more T-cell epitopes listed in Tables 1, 2, 3, and / or 4). In some embodiments, the HLA is class I HLA. In some embodiments, the HLA is class II HLA. In some embodiments, class I HLA has an α-chain polypeptide, which is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-g, HLA-K, or HLA-L. In some embodiments, class II HLA has an α-chain polypeptide, which is HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA, or HLA-DRA. In some embodiments, class II HLA has a β-chain polypeptide, which is HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB, or HLA-DRB. In some implementations, APCs present at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 T-cell epitopes (e.g., Table 1). (At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 of the T-cell epitopes listed in Tables 2, 3, and / or 4).

[0088] In some implementations, APCs are B cells, antigen-presenting T cells, dendritic cells, or artificial antigen-presenting cells (e.g., aK562 cells). Dendritic cells for the process can be prepared by removing PBMCs from a patient sample and adhering them to a plastic. Typically, a population of monocytes adheres, and all other cells can be washed away. The adhered population is then differentiated with IL-4 and GM-CSF to generate monocyte-derived dendritic cells. These cells can be matured by adding IL-1β, IL-6, PGE-1, and TNF-α (which upregulate important co-stimulatory molecules on the surface of dendritic cells) and then contacted with the recombinant adenovirus described herein.

[0089] In some embodiments, the APC is an artificial antigen-presenting cell (such as aK562 cells). In some embodiments, the artificial antigen-presenting cell is engineered to express CD80, CD83, 41BB-L, and / or CD86. Exemplary artificial antigen-presenting cells (including aK562 cells) are described in U.S. Patent Publication No. 2003 / 0147869 (which is hereby incorporated by reference).

[0090] In some aspects, this document provides a method for generating APCs that present two or more of the T-cell epitopes described herein, the method comprising contacting the APCs with a nucleic acid vector and / or recombinant adenovirus encoding the T-cell epitopes described herein and / or with multiple epitopes generated by the nucleic acid vector and / or recombinant adenovirus described herein. In some embodiments, the APCs are irradiated.

[0091] T cells

[0092] In some aspects, this document provides T cells and populations of T cells (e.g., CD4 T cells and / or CD8 T cells) that express TCRs (e.g., αβTCR or γδTCR) that recognize HLA-presented peptides described herein (e.g., epitopes listed in Tables 1, 2, 3, and / or 4). In some embodiments, the T cells are CD8 T cells (CTLs) that express TCRs recognizing class I HLA-presented peptides described herein. In some embodiments, the T cells are CD4 T cells (helper T cells) that recognize class II HLA-presented peptides described herein. Most preferably, this disclosure relates to the stimulation and expansion of pluripotent T cells (i.e., those T cells capable of inducing multiple immune effector functions and providing a more effective immune response against epitopes (e.g., epitopes listed in Tables 1, 2, 3, and / or 4) compared to cells that produce only a single immune effector (e.g., a single biomarker, such as a cytokine or CD107a). During chronic infection or disease states (e.g., cancer), less pluripotent, monofunctional, or even “exhausted” T cells can dominate the immune response, negatively impacting the treatment or prevention of virus-related complications. The functional capacity and activity of such T cells can be further assessed by determining the expression patterns of transcription factors (such as T-bet and Eomes) and / or cytotoxic effector molecules (such as perforin and granzyme B) (e.g., expression profiles as determined by ICS). In some embodiments, the expression of each of T-bet, Eomes, perforin, and granzyme B is determined for the T cells disclosed herein. Such expression levels can be determined and evaluated as relative measures (e.g., ratios). In a preferred embodiment, the expression profiles of T-bet / Eomes and / or granzyme B / perforin are determined. In a preferred embodiment, the T cells disclosed herein (e.g., JCV-specific T cells) exhibit high expression of T-bet and low expression of Eomes (i.e., T-bet...). 高 / Eomes 低 Similarly, the T cells disclosed in this paper can exhibit high expression of granzyme B and low expression of perforin (i.e., granzyme B is less expressed than perforin). 高 / perforin 低 In some such implementations, T-bet is demonstrated. 高 / Eomes 低 and / or granzyme 高 / perforin 低T cells with specific expression profiles (e.g., JCV-specific T cells) are identified as having functional capacity and activity. Such T cells can be selected for expansion and / or use in adoptive T-cell immunotherapy. Most preferably, the T cells disclosed herein (e.g., JCV-specific T cells) are multifunctional (i.e., producing two or more cytokines described herein) and exhibit T-bet... 高 / Eomes 低 and / or granzyme 高 / perforin 低 Expression spectrum.

[0093] In some aspects, this document provides methods for generating T cells (e.g., CTLs), activating T cells (e.g., CTLs), and / or inducing the proliferation of T cells (e.g., CTLs) that recognize one or more epitopes described herein. In some embodiments, a sample comprising CTLs (i.e., a PBMC sample) is incubated in a culture medium with an APC (e.g., an APC presenting a peptide comprising a BKV and / or JCV epitope described herein on a class I HLA complex). In some embodiments, the sample containing T cells is incubated with the APC provided herein two or more times. In some embodiments, the T cells are incubated with the APC in the presence of at least one cytokine. In some embodiments, the cytokine is IL-4, IL-7, and / or IL-15. For example, U.S. Patent Publication No. 2015 / 0017723 (which is hereby incorporated by reference) provides an exemplary method for inducing T cell proliferation using an APC.

[0094] In some aspects, this document provides a population of CTLs that collectively includes T cell receptors that recognize one or more T cell epitopes (e.g., one or more T cell epitopes listed in Tables 1, 2, 3, and / or 4). In some embodiments, CTLs recognize two or more T cell epitopes from Tables 1, 2, 3, and / or 4. In some embodiments, the population of CTLs collectively includes T cell receptors that recognize any combination of T cell epitopes from JCV, BKV, EBV, CMV, ADV, and / or from other viruses. In some implementations, the CTL population collectively includes T cell receptors, which recognize at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 3 T cell receptors. Eight T cell epitopes (e.g., at least 1, 2, 3, 4, 5, 6 or 7 T cell epitopes from Table 1 and / or 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 or 30 of the epitopes listed in Tables 1, 2, 3 and / or 4).

[0095] In some aspects, this document provides methods for preventing or treating polyomavirus infection (e.g., JCV infection) or cancer (e.g., polyomavirus-associated cancers, such as JVC-associated cancers) in subjects, the methods comprising administering to the subject a composition (e.g., a therapeutic composition) comprising a nucleic acid vector as described herein, a peptide derived from the nucleic acid vector as described herein, a CTL and / or APC provided herein (e.g., comprising the nucleic acid vector as described herein), and a pharmaceutically acceptable carrier. In some embodiments, the CTL and / or APC are not autologous to the subject (i.e., the CTL and / or APC are allogeneic to the subject). In some embodiments, the T cells and / or APCs are autologous to the subject. In some embodiments, the T cells and / or APCs are stored in a cell bank prior to administration to the subject.

[0096] Pharmaceutical Composition

[0097] In some aspects, this document provides compositions (e.g., pharmaceutical compositions, such as vaccine compositions) and methods of using such pharmaceutical compositions to treat cancer (e.g., polyomavirus-associated cancers, such as JVC-associated cancers) or polyomavirus infections (e.g., JCV infection, CMV infection, EBV infection, or ADV infection), the compositions comprising peptides (e.g., including epitopes from Table 1), nucleic acids, nucleic acid vectors, recombinant adenoviruses, antibodies, CTLs, or APCs as described herein, formulated with a pharmaceutically acceptable carrier. In some embodiments, the compositions comprise a combination of multiple (e.g., two or more) pharmaceutical agents provided herein.

[0098] In some embodiments, the pharmaceutical composition further includes an adjuvant. As used herein, the term "adjuvant" broadly refers to an agent that affects the immune or physiological response in a patient or subject. For example, an adjuvant can increase the presence of an antigen over time or, for a region of interest (like a tumor), aid in the uptake of antigens by antigen-presenting cells, activate macrophages and lymphocytes, and support the production of cytokines. By altering the immune response, adjuvants can allow smaller doses of immune interactants to increase the efficacy or safety of a particular dose of the immune interactant. For example, an adjuvant can prevent T cell depletion and thus increase the efficacy or safety of a particular immune interactant. Examples of adjuvants include (but are not limited to) immunomodulatory proteins, adjuvant 65, α-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, β-glucan peptide, CpG oligodeoxynucleotides, non-CpG oligodeoxynucleotides, GPI-0100, lipid A and its modified forms (e.g., monophosphorylated lipid A), lipopolysaccharides, Lipovant, Montanide, N-acetyl-muramycin-L-alanyl-D-isoglutamine, Pam3CSK4, quil A, TLR9 agonists, ODN1a, cationic antimicrobial peptides (CAMP) (such as KLK), IC31, and trehalose dimethicone.

[0099] Methods for preparing these formulations or compositions involve associating the agents described herein with a carrier and optionally one or more adjuvants. Typically, formulations are prepared by uniformly and closely associating the agents described herein with a liquid carrier or a finely pulverized solid carrier, or both, and then shaping the product (if necessary).

[0100] Pharmaceutical compositions of the present invention suitable for parenteral administration comprise combinations of one or more agents described herein with one or more pharmaceutically acceptable sterile isotonic or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders (which may be reconstituted into sterile injectable solutions or dispersions prior to use), which may contain sugars, alcohols, antioxidants, buffers, antibacterial agents, solutes that make the formulation isotonic with the blood of the intended recipient, or suspending agents or thickeners. Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). For example, appropriate flowability can be maintained by using coating materials (such as lecithin), by maintaining the particle size required for the dispersion, and by using surfactants.

[0101] Regardless of the chosen route of administration, the pharmaceutical agents of the present invention (which may be used in a suitable hydrated form) and / or the pharmaceutical compositions of the present invention are formulated into pharmaceutically acceptable dosage forms using conventional methods known to those skilled in the art.

[0102] Treatment

[0103] JCV sequence and / or protein expression can be observed in several malignancies and is frequently reported in patients with and without PML who have compromised immune systems (e.g., immunodeficient, immunocompromised, and / or immunosuppressed). Therefore, in some respects, this article provides methods for treating and / or preventing cancer (e.g., polyomavirus-associated cancers, such as JVC-associated cancers) or polyomavirus infection (e.g., JCV infection). In some embodiments, the method includes administering a pharmaceutical composition to a subject, the pharmaceutical composition comprising CTLs, APCs, peptides, and / or nucleic acid molecules described herein.

[0104] In some implementations, the treated subject has a compromised immune system. For example, in some implementations, the subject has a T-cell deficiency. In some implementations, the subject has leukemia, lymphoma (e.g., Hodgkin's lymphoma), or multiple myeloma. In some implementations, the subject has multiple sclerosis, psoriasis, and / or other autoimmune diseases. In some implementations, the subject is infected with HIV and / or has AIDS. In some implementations, the subject has undergone tissue, organ, and / or bone marrow transplantation. In some implementations, the subject is receiving immunosuppressive therapy (e.g., steroids, cell inhibitors and antiproliferators, therapeutic antibodies, calcineurin inhibitors, anti-rejection drugs, etc., or combinations thereof). In some implementations, the subject has undergone and / or is undergoing chemotherapy. In some implementations, the subject has undergone and / or is undergoing radiation therapy.

[0105] In a preferred embodiment, the subject is suffering from a JCV infection in the central nervous system (e.g., reactivation of a JCV infection or inoculation with a newly reactivated virus). In some such embodiments, the JCV infection is associated with oligodendrocyte destruction and / or white matter demyelination. In a further embodiment, the subject is suffering from JCV granule cell layer neuron disease (JCV GCN), JCV encephalopathy (JCV CPN / JCVE), JCV meningitis (JCVM), and / or progressive multifocal leukoencephalopathy (PML) (preferably PML). In some such embodiments, the pathogen (e.g., JCV) is detectable in the subject's cerebrospinal fluid.

[0106] In some embodiments, the subject has cancer. In some embodiments, the methods described herein can be used to treat any cancerous tumor or precancerous tumor. In some embodiments, the cancer expresses one or more of the polyomaviral epitopes provided herein (e.g., BKV epitopes / JCV epitopes listed in Tables 1, 2, 3, and / or 4). In some embodiments, the cancer is JCV-related cancer. In some embodiments, the cancer comprises a solid tumor. Preferably, the cancer is a gastrointestinal malignancy (e.g., colon cancer, gastric cancer, gastrointestinal tumors, etc.). Most preferably, the cancer is a CNS malignancy (e.g., glioma and all its subtypes (e.g., ependymoma, astrocytoma, brainstem glioma, oligodendroglioma, optic nerve glioma, mixed glioma, etc.), medulloblastoma, primitive neuroectodermal tumor, and neuroblastoma). For illustrative purposes and not for limitation, cancers that can be treated by the methods and compositions provided herein include cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, cancer can specifically be, but is not limited to, the following histological types: malignant neoplasms; carcinoma; undifferentiated carcinoma; giant spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatal carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; hepatocellular carcinoma with cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous polyp; adenocarcinoma; familial adenomatous polyposis; solid carcinoma; malignant carcinoid tumor; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic adenocarcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerotic carcinoma. Sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine gland adenocarcinoma; sebaceous gland carcinoma; ceruminous gland adenocarcinoma; mucoepidermoid carcinoma; cystadenoma; papillary cystadenoma; papillary serous cystadenoma; mucinous cystadenoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant theca cell tumor; malignant granulosa cell tumor; and malignant neuroblastoma (malignant Roblastoma; Setoli cell carcinoma; Malignant Ledich cell tumor; Malignant lipocyte tumor; Malignant paraganglioma; Malignant extramammary paraganglioma; Pheochromocytoma; Balloon sarcoma; Malignant melanoma; Amelanoma; Superficial expansile melanoma; Malignant melanoma within a giant nevus; Epithelioid cell melanoma; Malignant blue nevus; Sarcoma; Fibrosarcoma; Malignant dermatofibroma;Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic Rhabdomyosarcoma; Acinar Rhabdomyosarcoma; Stromal Sarcoma; Malignant Mixed Tumor; Müllerian Mixed Tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Malignant Mesenchymal Tumor; Malignant Ovarian Fibroepithelial Tumor; Malignant Phyloid Tumor; Synovial Sarcoma; Malignant Mesothelioma; Dysgerminoma; Embryonic Carcinoma; Malignant Teratoma; Malignant Goiter-like Ovarian Tumor; Choriocarcinoma; Malignant Mesonephric Tumor; Angiosarcoma; Malignant Hemangioendothelioma; Kaposi's Sarcoma; Malignant Hemangiopericytoma; Lymphangosarcoma; Osteosarcoma; Paracortical Osteosarcoma; Chondrosarcoma Malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pineal tumor; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astrocytoma; glioblastoma; glioblastoma multiforme; pineal blastoma; gliosarcoma Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal tumor; Cerebellar sarcoma; Ganglioblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Malignant meningioma; Neurofibrosarcoma; Malignant schwannoma; Malignant granular cell tumor; Malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; Granuloma-like tumor; Small lymphocytic malignant lymphoma; Diffuse large cell malignant lymphoma; Follicular malignant lymphoma; Mycosis fungoides; other specified non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphocytic leukemia; plasmacytic leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0107] In some implementations, subjects are also administered antiviral drugs that inhibit polyomavirus replication. For example, in some implementations, subjects are administered ganciclovir, valganciclovir, foscarnet, cidofovir, acyclovir, formivirsen, maribavir, BAY 38-4766, or GW275175X.

[0108] In some implementations, the subject is also administered an immune checkpoint inhibitor. Immune checkpoint inhibition broadly refers to the inhibition of checkpoints that cancer cells can produce to prevent or downregulate the immune response. Examples of immune checkpoint proteins include (but are not limited to) CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, TIM-3, or VISTA. Immune checkpoint inhibitors can be antibodies that bind to and inhibit immune checkpoint proteins, or antigen-binding fragments of said antibodies. Examples of immune checkpoint inhibitors include (but are not limited to) atezolizumab, avelumab, camrelizumab, cemiplimab, cetrelimab, durvalumab (MEDI-4736), genolimzumab, ipilimumab, nivolumab, pembrolizumab, and sintilimab. (i.e., imab), spartalizumab, tislelizumab, toripalimab, AMP-224, AMP-514, AK-104, ASP-8374, AUR-012, BCD-135, BGB-A333, BMS-936559, CBT-502, MCLA-145, KN-046, MGD-019, MK-4830, MSB-0020718C, RG-7446, SL-279252, STI-A1010, STI-A1110, TSR-042, XmAb20717, and XmAb23104.)

[0109] In some embodiments, the compositions provided herein are administered prophylactically to prevent cancer and / or polyomavirus infection (e.g., JCV infection). In some embodiments, the compositions may be administered before or after detection of cancer cells or polyomavirus-infected cells in a subject. Thus, in some such embodiments, the compositions provided herein are administered before or after administration of immunosuppressive therapy (e.g., steroids, cell inhibitors and antiproliferators, therapeutic antibodies, calcineurin inhibitors, anti-rejection drugs, etc., or combinations thereof). In some such embodiments, the compositions are administered before or after chemotherapy. Similarly, in some embodiments, the compositions are administered before or after radiotherapy. In some embodiments, a pro-inflammatory response is induced after administration of a composition comprising the peptides, nucleic acids, CTLs, and / or APCs described herein. The pro-inflammatory immune response includes the production of pro-inflammatory cytokines and / or chemokines (e.g., interferon-γ (IFN-γ) and / or interleukin-2 (IL-2)).

[0110] Combination therapy comprises the sequential, simultaneous, and individual, and / or co-administration of active compounds in such a manner that the therapeutic effect of the first agent has not yet completely worn off when subsequent treatments are administered. In some embodiments, the second agent may be formulated together with the first agent or in a separate pharmaceutical composition.

[0111] In some aspects, this document provides methods for identifying subjects suitable for the therapies provided herein (e.g., methods for treating polyomavirus infections (such as JCV infection) and / or cancer in subjects, methods including administering the pharmaceutical compositions provided herein to the subject). In some embodiments, the methods include isolating samples (e.g., blood samples, tissue samples, tumor samples) from the subject and detecting the presence of epitopes listed in Table 1, Table 2, or Table 3 in the samples. In some embodiments, epitopes are detected using ELISA assays, Western blot assays, FACS assays, fluorescence microscopy assays, Edman degradation assays, and / or mass spectrometry assays (e.g., protein sequencing). In some such embodiments, the presence of JCV epitopes is detected, for example, by detecting nucleic acids encoding JCV epitopes. In some embodiments, nucleic acids encoding JCV epitopes are detected using nucleic acid probes, nucleic acid amplification assays, and / or sequencing assays. Notably, the JC virus genome consists of two conserved coding regions separated by a highly variable non-coding control region (NCCR), which contains both the replication-required sequence (ORI, the origin of viral replication) and the transcription-required sequence (several promoters and cis-regulatory elements). The highly conserved region containing the ORI is followed by segments a, b, c, d, e, and f. JC viruses found in the CNS of PML patients are frequently found to have rearranged NCCRs (e.g., absence of segments b and d, and duplication of the ace sequence). Differences in the NCCR sequence can contribute to viral adaptation in the CNS and thus to the development of PML. Therefore, this paper provides a method for identifying subjects suitable for the therapy presented herein, comprising isolating samples from the subjects (e.g., blood samples, urine samples, tissue samples, cerebrospinal fluid samples, tumor samples) and detecting the presence of PML-related JCV sequence rearrangements (e.g., using nucleic acid amplification techniques such as nested PCR). Such sequences and detection methods are known in the art (e.g., in L'Honner et al., PLoS ONE, 13(6), 2018 (which is incorporated in its entirety by reference)).

[0112] In some embodiments, the method includes HLA typing of the subject. In some embodiments, the subject is identified as suitable for treatment using the method provided herein if the subject expresses an HLA that restricts the epitope provided herein. In some embodiments, the method provided herein further includes treating the identified subject with the treatment method provided herein (e.g., by administering a pharmaceutical composition provided herein to the subject). In some embodiments, the subject is administered a composition described herein comprising a CTL, wherein the CTL comprises a TCR that recognizes an epitope provided herein, said epitope being HLA-restricted by the HLA expressed by the subject. In some embodiments, the subject is administered a composition comprising a peptide that comprises an epitope provided herein, said epitope being HLA-restricted by the HLA expressed by the subject. In some embodiments, the subject is administered a composition comprising an APC that presents a peptide, said peptide comprising an epitope provided herein, said epitope being HLA-restricted by the HLA expressed by the subject. In some embodiments, the subject is administered a composition comprising a nucleic acid encoding a peptide, said peptide comprising an epitope provided herein, said epitope being HLA-restricted by the HLA expressed by the subject. Example

[0113] Example 1: CD8 targeting LTA JCV antigen, VP1 JCV antigen and STA JCV antigen + T cell response and CD4 + T Cellular response

[0114] PBMCs from 17 healthy volunteers were incubated with the JVC overlapping peptide library (OPP) for 14 days in the presence of IL-2. The peptide matrices for each of the large T antigen (LTA), small T antigen (STA), and viral protein 1 (VP1), and the composition of the peptide library for each matrix, are arranged as follows.

[0115] Large T antigen matrix

[0116] LTA library LTA 15 LTA 16 LTA 17 LTA 18 LTA 19 LTA 20 LTA 21 LTA 22 LTA 23 LTA 24 LTA 1 P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 LTA 2 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 LTA 3 P21 P22 P23 P24 P25 P26 P27 P28 P29 P30 LTA 4 P31 P32 P33 P34 P35 P36 P37 P38 P39 P40 LTA 5 P41 P42 P43 P44 P45 P46 P47 P48 P49 P50 LTA 6 P51 P52 P53 P54 P55 P56 P57 P58 P59 P60 LTA 7 P61 P62 P63 P64 P65 P66 P67 P68 P69 P70 LTA 8 P71 P72 P73 P74 P75 P76 P77 P78 P79 P80 LTA 9 P81 P82 P83 P84 P85 P86 P87 P88 P89 P90 LTA 10 P91 P92 P93 P94 P95 P96 P97 P98 P99 P100 LTA 11 P101 P102 P103 P104 P105 P106 P107 P108 P109 P110 LTA 12 P111 P112 P113 P114 P115 P116 P117 P118 P119 P120 LTA 13 P121 P122 P123 P124 P125 P126 P127 P128 P129 P130 LTA 14 P131 P132 P133 P134 P135 P136 P137

[0117] small T antigen

[0118] VP1 library VP 10 VP 11 VP 12 VP 13 VP 14 VP 15 VP 16 VP 17 VP 1 V1 V2 V3 V4 V5 V6 V7 V8 VP 2 V9 V10 V11 V12 V13 V14 V15 V16 VP 3 V17 V18 V19 V20 V21 V22 V23 V24 VP 4 V25 V26 V27 V28 V29 V30 V31 V32 VP 5 V33 V34 V35 V36 V37 V38 V39 V40 VP 6 V41 V42 V43 V44 V45 V46 V47 V48 VP 7 V49 V50 V51 V52 V53 V54 V55 V56 VP 8 V57 V58 V59 V60 V61 V62 V63 V64 VP 9 V65 V66 V67 V68 V69 V70 V71

[0119] Viral protein 1

[0120] STA Library STA 7 STA 8 STA 9 STA 10 STA 11 STA 12 STA 1 S1 S2 S3 S4 S5 S6 STA 2 S7 S8 S9 S10 S11 S12 STA 3 S13 S14 S15 S16 S17 S18 STA 4 S19 S20 S21 S22 S23 S24 STA 5 S25 S26 S27 S28 S29 S30 STA 6 S31 S32 S33

[0121] On day 14, the JVC specificity of these T cell cultures was assessed using an intracellular cytokine (ICS) assay. Notably, culturing T cells in vitro with JVC peptides for 14 days resulted in the expansion of virus-specific T cells. These preliminary analyses clearly demonstrate that the T cell response targets LTA, VP1, and STA (see [link to analysis]). Figure 1 ).

[0122] Example 2: JCV epitope HLA restriction

[0123] To precisely localize HLA class I and HLA class II restricted T cell responses, individual overlapping peptides (15 amino acids long with 10 overlapping amino acids) derived from LTA, STA, and VP1 proteins were used for T cell epitope localization. Two-dimensional peptide matrices were used to distribute all individual peptides into small overlapping peptide libraries. For example, in the case of large T antigens, the matrix was arranged such that each peptide in the library (LTA1 to LTA24) appeared once on the ordinate. T cell responses for each library were measured by intracellular cytokine staining (ICS) IFN-γ assay (see [link to documentation]). Figure 2 A), and the data is overlaid on a two-dimensional matrix as follows.

[0124] Peptide library LTA1-LTA24 T cell response matrix

[0125] LTA15 LTA16 LTA17 LTA18 LTA19 LTA20 LTA21 LTA22 LTA23 LTA24 LTA1 P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 LTA2 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 LTA3 P21 P22 P23 P24 P25 P26 P27 P28 P29 P30 LTA4 P31 P32 P33 P34 P35 P36 P37 P38 P39 P40 LTA5 P41 P42 P43 P44 P45 P46 P47 P48 P49 P50 LTA6 P51 P52 P53 P54 P55 P56 P57 P58 P59 P60 LTA7 P61 P62 P63 P64 P65 P66 P67 P68 P69 P70 LTA8 P71 P72 P73 P74 P75 P76 P77 P78 P79 P80 LTA9 P81 P82 P83 P84 P85 P86 P87 P88 P89 P90 LTA10 P91 P92 P93 P94 P95 P96 P97 P98 P99 P100 LTA11 P101 P102 P103 P104 P105 P106 P107 P108 P109 P110 LTA12 P111 P112 P113 P114 P115 P116 P117 P118 P119 P120 LTA13 P121 P122 P123 P124 P125 P126 P127 P128 P129 P130 LTA14 P131 P132 P133 P134 P135 P136

[0126] Therefore, individual peptides commonly found in the library that elicit T-cell responses were identified (i.e., peptide 32 (P32) at the intersection of row LTA4 and column LTA16, and peptides P29 and P30 at the intersection of row LTA3 and columns LTA23 and LTA24). Fluorescence activated cell sorting (FACS) confirmed that peptides P29, P30, and P32 alone elicited JCV-specific T-cell responses (see [link to Facility]). Figure 2 B).

[0127] Further evaluation of these individual peptides using T-cell expansion and ICS analysis was conducted to identify potential JCV antigens. The resulting peptides provide a high percentage of HLA allele coverage for JCV (see [link to study]). Figure 3 (and Table 5).

[0128] Table 5: HLA restriction mapping and allele coverage

[0129]

[0130] Example 3: JCV-specific T cell expansion and characterization

[0131] JCV-specific T cells were expanded in vitro following stimulation with consorted JCV epitopes. Specifically, PBMCs from healthy volunteers were stimulated with synthetic JCV peptides (Table 1) for 1 hour and then cultured for 12–14 days in the presence of different combinations of cytokines (containing IL-2 (10 ng / ml), IL-7 (10 ng / ml), IL-12 (10 ng / ml), and / or IL-15 (10 ng / ml)). The JCV specificity of the expanded T cells was assessed using standard intracellular cytokine assays (Table 6).

[0132] Table 6: CD4 response of JCV peptide + T cell response

[0133]

[0134] Example 4: T cell cross-reactivity between JCV and BKV epitopes

[0135] Peptide-specific T cells were expanded in vitro after stimulation with JCV or BKV epitopes and then restimulated with the corresponding homologous peptide epitopes (see Table 2) to observe the cross-reactivity between JCV and BKV epitopes. Specifically, PBMCs from healthy volunteers were cultured with either the synthetic JCV peptide epitope RSGSQQWRGLSRYFK or the synthetic BKV peptide epitope SSGTQQWRGLARYFK. After initial expansion, each sample was restimulated (recovered) with either the JCV epitope RSGSQQWRGLSRYFK or the BKV epitope SSGTQQWRGLARYFK (with samples recovered with the same epitope serving as internal controls). The responsiveness of the expanded T cells was assessed using standard intracellular cytokine assays (see Table 2). Figure 4 T cells amplified using either homologous epitope recognize both the BKV peptide sequence and the JCV peptide sequence.

[0136] Example 5: Analysis of the functional and phenotypic characteristics of JCV-specific T cells in healthy individuals and transplant recipients

[0137] In recent years, T-box transcription factor (T-bet) and amesoderm protein (Eomes) have been shown to play a role in identifying CD8 during infection. +T cells play a crucial role in T cell fate. High levels of T-bet are associated with cytotoxic T cell differentiation in antigen-specific cells and the upregulation of perforin and granzyme B. High levels of amesericulture protein are associated with long-term memory formation. Their cooperative expression has been shown to be critical for infection control in various studies. In mouse studies, the absence of any of these transcription factors has also been shown to result in uncontrolled infection. Therefore, studying the expression of these transcription factors is crucial, as it can help understand the phenotypic characteristics of T cells and T cell differentiation in both acute and chronic viral infections. The expression patterns of T-bet and amesericulture protein in JCV-specific T cells are not yet understood, and analysis of these transcription factors on such T cells could enable a deeper understanding of JCV-specific T cell differentiation. Detailed studies of the functional characteristics of T cells could also lead to the development of effective immunotherapies for JCV-related diseases. A preliminary set of experimental studies investigated the transcription factors on T cells that regulate their differentiation. The expression of T-bet, amesericulture protein, perforin, and granzyme B was measured on JCV-specific T cells and CMV-specific T cells using ICS. Preliminary analysis revealed moderate to low levels of T-bet expression in JCV-specific T cells, compared to high levels in CMV-specific T cells. Very low levels of demesoblastic proteins were observed in JCV-specific T cells compared to CMV-specific T cells. Similarly, low levels of perforin and granzyme B were also observed in JCV-specific T cells. This indicates that JCV-specific T cells have lower effector function compared to CMV-specific T cells. Therefore, investigating the effector function of JCV-specific CTLs is a key area of ​​research for developing effective adoptive T-cell immunotherapies.

[0138] Example 6: Feasibility of T cell expansion using the proposed peptide library:

[0139] PBMCs from 15 healthy donors were randomly selected, regardless of their HLA type, and JCV-specific T cells were expanded upon stimulation with a peptide library including the peptides disclosed herein (i.e., peptides comprising the amino acid sequences shown in SEQ ID NO. 1-21). T cells were allowed to expand for 17 days, and JCV responses were assessed using intracellular cytokine staining assays. T cells from 13 of the 15 donors exhibited JCV-specific T cell responses, demonstrated by the production of IFN-γ upon restimulation with the peptide library (see [link to relevant documentation]). Figure 5 ).

[0140] Example 7: Functional characterization of JCV-specific T cell products:

[0141] To determine the pluripotency of JCV-specific T cells expanded using a peptide library, T cells were analyzed by intracellular staining targeting the expression of IL-2, TNF, IFN-γ, and CD107 (see [link to article]). Figure 6 a). JCV-specific T cells (along with other cytokines) showed high TNF expression.

[0142] Boolean analysis of expression patterns for different combinations of cytokines revealed that JCV-specific T cell products are multifunctional and produce two or more cytokines (see [link to article]). Figure 6 (b and c). To further characterize the expression of JCV-specific T cell products, transcription factors (T-bet and Eomes) and effector molecules (perforin and granzyme B) were observed. A large proportion of cells possessed T-bet. 高 / Eomes 低 and granzyme 高 / perforin 低 These characteristics reveal that JCV-specific T cells possess functional activity and can exhibit cytotoxic effects against JCV-infected cells. Such T cells can be expanded in vitro and used to treat JCV-related diseases (see [link to relevant documentation]). Figure 7 ).

Claims

1. A library of immunogenic peptides comprising HLA class I and / or II restricted JCV peptide epitopes capable of inducing the proliferation of peptide-specific T cells, wherein the peptide library comprises at least three of the epitope amino acid sequences shown in SEQ ID NO: 1-21, and wherein the at least three of the epitope amino acid sequences comprise the epitope amino acid sequence shown in SEQ ID NO:

12.

2. An in vitro method for expanding JC virus-specific T cells for adoptive immunotherapy, the method comprising: (i) Contacting one or more cells isolated from a subject with a library of immunogenic peptides, wherein the library of immunogenic peptides comprises at least three of the epitope amino acid sequences shown in SEQ ID NO: 1-21, and wherein the at least three epitope amino acid sequences comprise the epitope amino acid sequence shown in SEQ ID NO: 12; and (ii) The one or more cells are cultured under conditions that allow JC virus-specific T cells to expand from the one or more cells.

3. Cytotoxic T cells (CTLs) prepared by the method of claim 2.

4. Use of a medicament comprising a CTL and a T-cell receptor in the preparation of a medicament for treating or preventing polyomavirus infection in a subject, wherein the T-cell receptor recognizes at least three of the epitope amino acid sequences shown in SEQ ID NO: 1-21, wherein the at least three of the epitope amino acid sequences include the epitope amino acid sequence shown in SEQ ID NO: 12, wherein the polyomavirus is JCV or BKV.

5. An in vitro method for inducing the proliferation of polyomavirus-specific CTLs, the method comprising contacting the CTLs with antigen-presenting cells (APCs) presenting three or more polyomavirus peptides, the polyomavirus peptides comprising at least three of the epitope amino acid sequences shown in SEQ ID NO: 1-21, wherein the at least three of the epitope amino acid sequences comprise the epitope amino acid sequence shown in SEQ ID NO: 12.

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