Adoptive t cell therapy for cmv infection and cmv-associated disease

Abstract

Provided herein are immunogenic polypeptides, compositions, and methods related to the development of CMV-specific prophylactic and / or therapeutic immunotherapies based on cytotoxic T cell (CTL)-recognized T cell epitopes (e.g., CMV epitopes), and can be used to prevent and / or treat CMV infection, reactivation, and / or disease (e.g., CMV-associated end-organ disease), especially in solid organ transplant recipients.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 673,260, filed May 18, 2018, the entire contents of which are incorporated herein by reference. Background Technology

[0003] Herpesviruses represent a large and nearly ubiquitous family of eukaryotic viruses associated with a wide variety of animal and human diseases. The Herpesviridae family shares several common structures, such as a double-stranded, linear DNA genome, and a virion containing an icosahedral capsid, which itself encloses the viral cutaneous layer and a lipid bilayer (viral membrane). Additionally, herpesviruses contain characteristic and highly conserved glycoproteins carried on the lipid bilayer membrane of the herpesvirus virion. At least some of these glycoproteins play a role in the initial viral adhesion to the cell surface and subsequent penetration into the cell.

[0004] Members of the herpesvirus family represent important human pathogens, including human cytomegalovirus (CMV). CMV is ubiquitous across all geographic locations and socioeconomic groups, infecting 60% to 90% of individuals. In healthy individuals, CMV establishes a latent state after initial infection, periodically reactivating and shed from mucosal surfaces, and may present with clinical symptoms resembling mononucleosis, similar to those caused by Epstein-Barr virus (EBV), but is usually asymptomatic. CMV utilizes a variety of immunomodulatory strategies to evade the host's immune response. Examples of such strategies include inhibiting interferon (IFN) and IFN-stimulated genes, degrading HLA to prevent antigen presentation to cytotoxic T cells, and regulating activating and repressive ligands to inhibit the function of natural killer (NK) cells.

[0005] However, in some cases, CMV can lead to significant morbidity and mortality. For example, the clinical management of CMV infection in solid organ transplant (SOT) recipients remains a major challenge. Since the advent of ganciclovir-based antiviral therapy, the incidence of early CMV-related complications in SOT recipients has significantly decreased. Therefore, suppressing viral reactivation through prophylactic or preemptive administration of ganciclovir has become crucial in the prevention of CMV-related disease. However, late CMV reactivation can be more difficult to manage, especially in patients who cannot reconstitute antiviral T-cell immunity. Furthermore, the reactivation or onset of ganciclovir-resistant CMV presents significant challenges to clinical management, with significant morbidity and mortality due to drug-related toxicity, immunomodulatory effects, and allogeneic graft loss.

[0006] For ganciclovir-resistant CMV, there are no safe and effective alternative treatment options. Other antiviral management strategies using foscarnet or cidofovir are associated with nephrotoxicity and require intravenous administration and hospitalization. Genes conferring ganciclovir resistance are also associated with resistance to foscarnet and cidofovir. Reducing immunosuppression can be used to improve viral control, but increases the risk of transplant rejection.

[0007] Therefore, there is a great need for new and improved methods and compositions for treating CMV infection, reactivation, and related complications and diseases in SOT recipients and other patients with CMV-related diseases. Summary of the Invention

[0008] This article provides immunogenic peptides, compositions, and methods relating to the development of CMV-specific prophylactic and / or therapeutic immunotherapies based on T-cell epitopes (e.g., CMV epitopes) recognized by cytotoxic T cells (CTLs), and which can be used to prevent and / or treat CMV infection, reactivation, and / or disease (e.g., CMV-related end-organ disease), particularly in solid organ transplant recipients. In some embodiments, CMV infection, reactivation, and / or disease is persistent. In some embodiments, CMV infection, reactivation, and / or disease is resistant to antiviral therapy.

[0009] This document also provides a library of immunogenic peptides comprising HLA class I and II restricted cytomegalovirus (CMV) peptide epitopes capable of inducing peptide-specific T cell proliferation. In some embodiments, the library of immunogenic peptides comprises at least one, or a combination thereof, of the epitope amino acid sequences shown in SEQ ID NO: 25 to 29. In some embodiments, the peptide library comprises at least one peptide epitope derived from each of the CMV antigens pp50, pp65, IE-1, gB, and gH. Preferably, such an immunogenic peptide library also comprises at least one of the CMV peptide epitope amino acid sequences listed in Table 1. More preferably, the immunogenic peptide library of the present invention comprises each CMV peptide epitope amino acid sequence listed in Table 1. In some embodiments, each epitope of the immunogenic peptide library disclosed herein is selected from any of the following HLA-specific restriction epitopes: HLA-A*01:01, -A*02:01, -A*23:01, -A*24:02, -B*07:02, -B*08:01, -B*18:01, -B*35:01, -B*35:08, -B*40:01, -B*40:02, -B*41.01, -B*44:02, -C*06:02, -C*07:02, -DRB1*01:01, -DRB1*03:01, -DRB1*04:01, -DRB1*07, or -DRB1*11:01.

[0010] In some aspects, this document provides a method for preparing a formulation comprising multifunctional CMV-specific cytotoxic T cells (CTLs), comprising the steps of: a) isolating a sample containing CTLs; b) exposing said sample to a library of immunogenic peptides according to any one of claims 1-6; and c) collecting the CTLs. In some embodiments, the library of immunogenic peptides consists substantially of the amino acid sequence of each CMV peptide epitope listed in Table 1. In some embodiments, the sample containing CTLs comprises peripheral blood mononuclear cells (PBMCs) from a healthy donor. In some such embodiments, the donor is immune-naïve. In some embodiments, the donor is undergoing immunosuppressive therapy. Preferably, the donor is a solid organ transplant recipient. In some preferred embodiments, the donor is receiving antiviral therapy.

[0011] In some embodiments, the exposed sample from step b) is incubated for at least 14 days. Cytokines can be used in the method of the present invention and may include, but are not limited to, IL-1, IL-2, IL-4, IL-6, IL-7, IL-12, IL-15, and / or IL-21. For example, the exposed sample from step b) may be incubated with IL-21 on day 0. In some such embodiments, the exposed sample from step b) is incubated with IL-2 on day 2. Preferably, the sample is incubated with IL-2 every three days.

[0012] In certain aspects of the invention, methods for treating or preventing CMV infection in subjects are provided herein, the methods comprising administering to the subject a CTL or a composition thereof produced by the methods disclosed herein. In some embodiments, the subject has CMV reactivation or a CMV-related condition (e.g., CMV-related end-organ disease), or is at risk of such condition. In some preferred embodiments, the subject has received a solid organ transplant. Methods for reducing or eliminating the need for antiviral therapy in subjects who have received solid organ transplants are also provided herein, such methods comprising administering to the subject a CTL produced by the methods disclosed herein. Attached Figure Description

[0013] Figure 1Phenotypic and functional characteristics of CMV-specific T cells expanded for adoptive immunotherapy are shown. (A) Phenotypic characteristics of T cells expanded from the CMV peptide library were assessed using standard TBNK (T cell, B cell, NK cell) analysis, measuring the surface expression of CD3 (T cells), CD8 (CD8+ T cells), CD4 (CD4+ T cells), CD16 and CD56 (NK cells), and CD19 (B cells). (B) PBMCs (ex vivo; prior to peptide exposure) or expanded T cells (day 14) were assessed for intracellular IFN-γ production following restimulation with the CMV peptide library or a single HLA-matched peptide. Data represent the proportion of CD8+ T cells producing IFN-γ. (C) Comparison of CMV-specific T cell responses from kidney or heart / lung transplant recipients. (D) Comparison of CMV-specific T cell responses from CMV seronegative recipients (R-) or CMV seronegative recipients (R+). (E) After recall with the CMV peptide library, intracellular production of cytokines (IFN-γ, TNF, IL-2) and degranulation (CD107a) of CMV peptide library-stimulated T cells was assessed. Data represent the proportion of total antigen-specific T cells producing effector functions (i.e., pluripotency) for each combination.

[0014] Figure 2 Immunological and virological effects following adoptive cell therapy are shown. (A) CMV-specific T cells producing IFN-γ were assessed in PBMC samples from patients before and after T-cell therapy following stimulation with a CMV peptide library. Data represent coverage plots of IFN-γ-producing CD8+ T cell counts and CMV load (in copies / mL) from four patients who responded to therapy. Shaded areas indicate the time period prior to adoptive T-cell therapy, and arrows indicate T-cell infusion. (B) Utilitarianism, i.e., cytokine production (IFN-γ, TNF, IL-2) and degranulation (CD107a), was assessed in PBMC samples following stimulation with a CMV peptide library. Heatmaps represent the proportion of total antigen-specific T cells producing each effector functional combination.

[0015] Figure 3 Multicolor analysis of T cell phenotypes was shown. Figure 3 Representative t-distribution random neighbor embedding (tSNE) analysis of the upper group showed the expression of T cell phenotypic markers and CMV-specific T cells (VTE) in patient P1553PAH08 before and after treatment, indicating increased CD57 expression. Figure 3The data in the lower group represent a graph showing the percentage of CD8+ T cells expressing CD57 and the percentage of cells producing CMV-specific IFN-γ after T-cell therapy in three SOT recipients (P1553PAH08, 1553PCH02, and 1553PCH04) who responded to adoptive T-cell therapy and one SOT recipient (P1553RAH01) who did not show any clinical response. Detailed Implementation

[0016] Overview

[0017] Reconstructing CMV immunity through the application of CMV-specific T cells offers an attractive option for enhancing CMV control. As disclosed herein, the use of multiple epitopes derived from multiple CMV antigens can induce a broad spectrum of virus-specific immune responses to provide more effective protection against virus-associated pathogenesis. Most preferably, the present invention relates to the stimulation and expansion of pluripotent T cells, i.e., T cells capable of inducing multiple immune effector functions, which provide a more effective immune response against pathogens compared to cells that, for example, produce only a single immune effector (e.g., a single biomarker, such as a cytokine or CD107a). During chronic infection, less pluripotent, monofunctional, or even “exhausted” T cells may dominate the immune response, thereby negatively impacting protection against virus-associated complications.

[0018] However, in the case of SOT recipients, autologous immune cells from highly immunosuppressed individuals are required to generate effective T-cell therapy. Although the use of autologous CMV-specific T-cell therapy in SOT patients has shown some promising results, previous case studies have also raised concerns about potential safety (Brestrich et al. (2009) Am J Transplant 9(7):1679-84). As a result, the development of this approach has been limited due to the recognized difficulty in generating T cells from highly immunosuppressed subjects (e.g., SOT recipients) and the potential risks associated with transplant rejection after T-cell administration.

[0019] definition

[0020] For convenience, certain terms used in the specification, embodiments and appended claims are collected herein.

[0021] The articles “a” and “an” are used in this text to refer to one or more (i.e., at least one) of the grammatical objects of the article. For example, “an element” refers to one element or more elements.

[0022] As used herein, the term "administration" refers to the provision of a pharmaceutical agent or composition to a subject, including but not limited to administration by a medical professional and self-administration. Such agents may contain, for example, peptides described herein, antigen-presenting cells provided herein, and / or CTLs provided herein.

[0023] As used herein, the terms “subject” or “recipient” refer to a human or non-human animal selected for treatment or therapy.

[0024] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of an individual's disease within the context of clinicopathology. Ideal treatment outcomes include slowing the rate of progression, improving or alleviating the pathological condition, and mitigating or improving the prognosis of a specific disease, disorder, or symptom. For example, an individual can be successfully "treated" if one or more symptoms associated with a specific disease, disorder, or symptom are relieved or eliminated.

[0025] As used in this article, a "preventive" treatment agent for a disease refers to a compound that, when administered to a statistical sample prior to the onset of a disease or condition, reduces the occurrence of the disease or condition in the treated sample compared to an untreated control sample, or delays the onset of one or more symptoms of the disease or condition or reduces its severity compared to an untreated control sample.

[0026] As used herein, the phrase “pharmaceutically acceptable” means that reagents, compounds, materials, compositions and / or dosage forms are suitable for use in contact with human and animal tissues, within reasonable medical judgment, without excessive toxicity, irritation, allergic reactions or other problems or complications, and are equivalent to a reasonable benefit / risk ratio.

[0027] As used herein, the phrase “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, or transporter, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, which relates to carrying or transporting a reagent from one organ or part of the body to another organ or part of the body. The carrier must be “acceptable” in the sense that it is compatible with other components of the formulation and is harmless to the patient. 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) astragalus gum powder; (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) Ethylene glycol, such as propylene glycol; (12) Polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (13) Esters, such as ethyl oleate and ethyl laurate; (14) Agar; (15) Buffers, such as magnesium hydroxide and aluminum hydroxide; (16) Alginate; (17) Atherless water; (18) Isotonic saline; (19) Ringer's solution; (10) Ethanol; (21) pH buffer solution; (22) Polyesters, polycarbonates and / or polyanhydrides; (23) Other non-toxic and compatible substances used in pharmaceutical preparations.

[0028] The terms “binding” or “interaction” refer to association, which can be a stable association between two molecules, such as the stable association between a TCR and a peptide / MHC, which is caused by, for example, electrostatic, hydrophobic, ionic and / or hydrogen bonding interactions under physiological conditions.

[0029] As used herein, "specific binding" refers to the ability of a TCR to bind peptides presented on MHCs (e.g., class I or class II MHCs). Typically, a TCR binds at least about 10 -4 M or smaller K D The affinity specifically binds to its peptide / MHC, and its affinity for binding nonspecific, unrelated peptide / MHC complexes (e.g., peptide / MHC complexes containing BSA peptides or casein peptides) is at least 10-fold, at least 100-fold, or at least 1000-fold lower (in K0.05) compared to its affinity for binding to nonspecific, unrelated peptide / MHC complexes (e.g., peptide / MHC complexes containing BSA peptides or casein peptides). D (Indicates) binding to a predetermined antigen / binding partner.

[0030] The terms “biological sample,” “tissue sample,” or simply “sample” refer to a collection of cells obtained from the tissues of a subject. Tissue samples can be derived from solid tissues, such as fresh, frozen, and / or preserved organs, tissue samples, biopsies, or aspirates; blood or any blood component, serum, or blood; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage of the subject’s pregnancy or development.

[0031] As used herein, the term "cytokine" refers to any secreted polypeptide that affects cellular function; it is a molecule that regulates cell-cell interactions in immune, inflammatory, or hematopoietic responses. Cytokines include, but are not limited to, monokines and lymphokines, regardless of the type of cell that produces them. For example, monokines are generally referred to as factors produced and secreted by mononuclear cells, such as macrophages and / or monocytes. However, many other cells also produce monokines, such as natural killer cells, fibroblasts, basophils, neutrophils, endothelial cells, brain astrocytes, bone marrow stromal cells, epidermal keratinocytes, and B lymphocytes. Lymphokines are generally referred to as factors produced by lymphocytes. Examples of cytokines include, but are not limited to, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNFα), and tumor necrosis factor-β (TNFβ).

[0032] The term "epitope" refers to a protein determinant that specifically binds to an antibody or TCR. Epitopes typically consist of a group of chemically active surface components of a molecule, such as amino acids or sugar side chains. Some epitopes can be defined by specific amino acid sequences that antibodies can bind to.

[0033] The terms “polynucleotide” and “nucleic acid” are used interchangeably. They refer to polymeric forms of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides or their 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 segments, one or more loci as defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogues. If present, nucleotide structural modifications can be conferred before or after polymer assembly. For example, polynucleotides can be further modified by conjugation with labeled components. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.

[0034] The term "vector" refers to a means by which nucleic acids can proliferate and / or transfer between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophages, proviruses, phage particles, transposons, and artificial chromosomes, which may or may not replicate autonomously, and may or may not integrate into the host cell's chromosome.

[0035] peptides

[0036] This document provides peptides comprising herpesvirus epitopes recognized by cytotoxic T lymphocytes (CTLs), which may be used for the prevention and / or treatment of CMV infection, reactivation and / or diseases and / or cancers caused by CMV infection (e.g., end-organ disease in solid organ transplant recipients). In some embodiments, the CMV epitopes are those listed in Table 1.

[0037] Table 1: Exemplary CMV Tabletops

[0038]

[0039] *For patient P1553PAH01, the HLA-B*31:01 restriction epitope KARAKKDELR (KAR) encoded by IE-1 was supplemented into the CMV peptide library.

[0040] In some aspects, this document provides a library of immunogenic peptides comprising HLA class I and II restricted cytomegalovirus (CMV) peptide epitopes capable of inducing peptide-specific T cell proliferation. In some embodiments, the library of immunogenic peptides comprises at least one, or a combination thereof, of the epitope amino acid sequences shown in SEQ ID NO: 25 to 29. In some such embodiments, the peptide library comprises at least one peptide epitope derived from each of the CMV antigens pp50, pp65, IE-1, gB, and gH. Preferably, the library of immunogenic peptides further comprises at least one, or a combination thereof, of the CMV peptide epitope amino acid sequences listed in Table 1. Most preferably, such a peptide library comprises the amino acid sequence of each CMV peptide epitope listed in Table 1.

[0041] "HLA restriction (i.e., MHC restriction)" means that a given T cell recognizes and responds to a peptide only when the peptide binds to a specific HLA molecule. In some embodiments, each epitope of the immunogenic peptide library disclosed herein is selected from any of the following HLA-specific restriction epitopes: HLA-A*01:01, -A*02:01, -A*23:01, -A*24:02, -B*07:02, -B*08:01, -B*18:01, -B*35:01, -B*35:08, -B*40:01, -B*40:02, -B*41.01, -B*44:02, -C*06:02, -C*07:02, -DRB1*01:01, -DRB1*03:01, -DRB1*04:01, -DRB1*07, or -DRB1*11:01.

[0042] Most preferably, the immunogenic peptides and their libraries can induce the proliferation of peptide-specific cytotoxic T cells (CTLs).

[0043] In some embodiments, the peptides provided herein are full-length CMV polypeptides. In some embodiments, the peptides provided herein comprise fewer than 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, or 10 consecutive amino acids of a CMV viral polypeptide. In some embodiments, the peptides provided herein comprise two or more CMV epitopes listed in Table 1. For example, in some embodiments, the peptides provided herein comprise two or more CMV epitopes listed in Table 1 linked by a polypeptide linker. In some embodiments, the peptides provided herein comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or all of the epitopes listed in Table 1.

[0044] In some embodiments, the peptide sequence comprises a CMV viral polypeptide sequence other than 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" refers to an amino acid modification that does not significantly affect or alter the interaction between the peptide containing the amino acid sequence presented on the T cell receptor (TCR) and the major histocompatibility complex (MHC). Such conserved modifications include amino acid substitution, addition (e.g., adding an amino acid to the N- or C-terminus of the peptide), and deletion (e.g., deleting an amino acid from the N- or C-terminus of the 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 peptides described herein can be substituted with other amino acid residues from the same side chain family, and the retention of TCR binding by the altered peptides 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.

[0045] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences, and dissimilar sequences may be ignored for comparison purposes). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. The molecules are identical at that position when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. The percentage of identity between two sequences is a function of the number of common positions shared by the sequences, taking into account the number of gaps that need to be introduced to achieve optimal alignment and the length of each gap.

[0046] This document also provides chimeric or fusion proteins. As used herein, a “chimeric protein” or “fusion protein” comprises one or more peptides provided herein (e.g., peptides comprising the epitopes listed in Table 1) linked to different peptides that are not naturally linked. For example, different peptides may be fused directly to the N-terminus or C-terminus of the peptide via peptide bonds or indirectly via chemical linkers. In some embodiments, the peptides provided herein are linked to polypeptides comprising other CMV epitopes. In some embodiments, the peptides provided herein are linked to peptides comprising epitopes from other viruses and / or infectious diseases. In some embodiments, the peptides provided herein are linked to peptides encoding cancer-related epitopes.

[0047] The chimeric or fusion peptides presented herein can be generated using standard recombinant DNA techniques. For example, DNA fragments encoding different peptide sequences can be joined together within a frame using conventional techniques, such as ligation using blunt or staggered ends, restriction enzyme digestion to provide appropriate ends, filling in sticky ends as needed, alkaline phosphatase treatment to avoid unwanted ligation, and enzymatic ligation. Similarly, fusion genes can be synthesized using conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that generate complementary overhangs between two consecutive gene fragments, which can then be annealed and re-amplified to produce chimeric gene sequences (see, for example, Current Protocols in Molecular Biology, edited by Ausubel et al., John Wiley & Sons: 1992). Furthermore, many expression vectors encoding fusion motifs are commercially available.

[0048] In some aspects, this document provides cells that present the peptides described herein (e.g., peptides containing epitopes listed in Table 1). In some embodiments, the cells are mammalian cells. The cells may be 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 the peptides described herein can be generated using standard techniques known in the art. For example, cells can be pulsed to promote peptide uptake. In some embodiments, cells are transfected with nucleic acids encoding the peptides provided herein.

[0049] In some respects, this document provides methods for generating antigen-presenting cells (APCs) that include using peptide pulsed cells as described herein. Exemplary methods for generating antigen-presenting cells can be found in WO2013088114, which is incorporated herein in its entirety.

[0050] The peptides described herein can be isolated from cellular or tissue sources using standard protein purification techniques with appropriate purification protocols, generated using recombinant DNA technology, and / or chemically synthesized 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. The expression of heteropeptides in recombinant hosts, the chemical synthesis of peptides, and in vitro translation methods are well known in the field and have been documented in Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd edition, Cold Spring 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 Further descriptions can be found in Kent, SBH (1988) Annu. Rev. Biochem. 57:957; and Ofowe, RE (1980) Semisynthetic Proteins, Wiley Publishing, which are cited herein as references.

[0051] cell

[0052] In some aspects, this document provides antigen-presenting cells (APCs) that express MHCs on their surface, said MHCs presenting one or more peptides containing CMV epitopes as described herein (e.g., APCs presenting one or more CMV epitopes listed in Table 1). In some embodiments, the MHC is a class I MHC. In some embodiments, the MHC is a class II MHC. In some embodiments, class I MHCs have an α-chain polypeptide that is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-g, HLA-K, or HLA-L. In some embodiments, class II MHCs have an α-chain polypeptide that is HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA, or HLA-DRA. In some embodiments, class II MHCs have a β-chain polypeptide that is HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB, or HLA-DRB.

[0053] 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 this process can be prepared by extracting PBMCs from a patient sample and adhering them to a plastic. Typically, a monocyte colony adheres while all other cells can be washed away. The adhered colony 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 transduced with one or more peptides provided herein.

[0054] 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 incorporated herein by reference.

[0055] In some aspects, this document provides a method for generating an APC that presents one or more CMV epitopes as described herein, the method comprising contacting the APC with a peptide containing a CMV epitope, or a library of CMV epitope peptides as described herein, and / or a nucleic acid encoding one or more CMV epitope peptides as described herein. In some embodiments, the APC is irradiated.

[0056] In some aspects, this document provides T cells (e.g., CD4 T cells and / or CD8 T cells) that express TCRs (e.g., αβTCR or γδTCR) that recognize peptides described herein presented on MHC (e.g., HLA-restricted) (peptides containing CMV epitopes listed in Table 1). In some embodiments, the T cells are CD8+ T cells (CTLs) that express TCRs that recognize peptides described herein presented on class I MHC (e.g., HLA-A, -B, and -C). In some embodiments, the T cells are CD4+ T cells (helper T cells) that recognize peptides described herein presented on class II MHC (e.g., HLA-DP, -DM, -DOA, -DOB, -DQ, and -DR). In some embodiments, such T cells are prepared by any of the methods disclosed herein.

[0057] In some embodiments, the T cells provided herein can be engineered to express chimeric antigen receptors (CARs). Various CARs have been described in the scientific literature. Typically, a CAR includes an extracellular antigen-binding domain (e.g., scFv derived from antibody variable heavy and light chains), a spacer domain, a transmembrane domain, and an intracellular signaling domain. Therefore, in some embodiments, CMV-specific T cells (e.g., CTLs stimulated by a provided CMV peptide epitope pool) express a CAR that targets diseased cells, such as cancer cells (e.g., tumor cells), and associated extracellular molecules (e.g., tumor antigens, such as HER2).

[0058] In some aspects, this document provides methods for generating, activating, and / or inducing the proliferation of T cells (e.g., CTLs) that recognize one or more CMV epitopes described herein. In some embodiments, a sample containing CTLs (e.g., a PBMC sample), exposes it to a library of immunogenic peptides disclosed herein, and collects the stimulated CTLs. Preferably, the library of immunogenic peptides consists substantially of the amino acid sequence of each CMV peptide epitope listed in Table 1. In some embodiments, the exposed sample is incubated for at least 14 days. In some such embodiments, the exposed sample is incubated with IL-21 on day 0. Preferably, the exposed sample is incubated with IL-2 on day 2. In a more preferred embodiment, the incubation of the exposed sample comprises adding IL-2 every three days.

[0059] In some implementations, the PBMC sample is derived from a healthy donor. In some implementations, the PBMC is derived from a donor with no immune response. In some such implementations, the donor is undergoing immunosuppressive therapy. In some implementations, the donor is a solid organ transplant recipient. In other implementations, the donor is receiving antiviral therapy.

[0060] In some embodiments, a sample containing CTLs (e.g., a PBMC sample) is incubated with an APC (e.g., an APC presenting a peptide containing a CMV epitope described herein on a class I MHC complex). The APC may be autologous for the subject from whom T cells are obtained. In some embodiments, a sample containing T cells is incubated with an APC provided herein two or more times. In some embodiments, T cells are incubated with an APC in the presence of at least one cytokine (e.g., IL-2, IL-4, IL-7, IL-15, and / or IL-21). Exemplary methods for inducing T cell proliferation using APCs are provided, for example, in U.S. Patent Publication No. 2015 / 0017723, which is incorporated herein by reference.

[0061] In some aspects, this document provides compositions comprising T cells (e.g., CMV peptide-specific CTLs provided herein) and / or APCs provided herein (e.g., therapeutic compositions). In some embodiments, such compositions are used to treat and / or prevent CMV infection, reactivation, and / or disease in a subject by administering an effective amount of the composition to the subject. The T cells and / or APCs may be autologous or non-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. In some embodiments, the subject may be a solid organ transplant recipient.

[0062] Pharmaceutical Composition

[0063] In some respects, this article provides compositions (e.g., pharmaceutical compositions) containing CTLs formulated with pharmaceutically acceptable carriers, and methods of administering such pharmaceutical compositions.

[0064] In some embodiments, the composition may further comprise an adjuvant. As used herein, the term "adjuvant" broadly refers to an immunological or pharmacological agent that alters or enhances the immune response to the composition, either in vitro or in vivo. For example, adjuvants may increase the presence of antigens over time, aid in the uptake of antigens on antigen-presenting cells, activate macrophages and lymphocytes, and support the production of cytokines. By altering the immune response, adjuvants can allow for smaller doses of the immune-interacting agent or formulation to increase dose efficacy or safety. For example, adjuvants can prevent T-cell exhaustion, thereby increasing the efficacy or safety of a particular immune-interacting agent or formulation. 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 DNA, GPI-0100, lipid A and its modified forms (e.g., monophosphorylated lipid A, lipopolysaccharide, Lipovant, Montanide, N-acetyl-murayl-L-alanyl-D-isoglutamine, Pam3CSK4, quil A, and trehalose dimethicone).

[0065] Methods for preparing these formulations or compositions include the steps of combining the reagents described herein with a carrier and, optionally, one or more auxiliary components. Typically, formulations are prepared by uniformly and tightly combining the reagents described herein with a liquid carrier or a subdivided solid carrier, or both, and then, if desired, shaping the product.

[0066] The pharmaceutical compositions of the present invention suitable for parenteral administration comprise one or more of the reagents described herein, and one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injections or dispersions prior to use, wherein 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.

[0067] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., 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, in the case of dispersions, appropriate flowability can be maintained by using a coating material (e.g., lecithin) and by using a surfactant.

[0068] Regardless of the chosen route of administration, the reagents of the present invention (which may be used in a suitable hydrated form) and / or the pharmaceutical compositions of the present invention can be formulated into pharmaceutically acceptable dosage forms using conventional methods known to those skilled in the art.

[0069] Treatment

[0070] In some embodiments, this document provides methods for treating or preventing CMV infection, reactivation, and / or disease (e.g., end-organ disease in solid organ transplant recipients) in subjects, said methods comprising administering to the subject peptide-specific T cells (or pharmaceutical compositions comprising said T cells) prepared according to the methods provided herein.

[0071] In some embodiments, this document provides methods for treating or preventing CMV infection in a subject. In some embodiments, this document provides methods for treating or preventing CMV reactivation or CMV-related conditions in a subject. In a preferred embodiment, the method includes administering a CTL prepared according to the methods provided herein to the subject. For example, but not limited to, exposing an isolated PBMC sample to a library of immunogenic peptides according to the methods provided herein. In some such embodiments, the library of immunogenic peptides induces stimulation and proliferation of CMV peptide-specific T cells. In some embodiments, the CTL administered to the subject is autologous. In some embodiments, the infection is a recurrent CMV infection. In some embodiments, the subject being treated is immune-naïve. For example, in some embodiments, the subject has a T-cell deficiency. In some embodiments, the subject has leukemia, lymphoma, or multiple myeloma. In some embodiments, the subject is infected with HIV and / or has AIDS. In some embodiments, the subject has undergone tissue, organ, and / or bone marrow transplantation. In some such embodiments, the subject is a solid organ transplant recipient. In some embodiments, an immunosuppressive drug is administered to the subject. In some embodiments, the subject has undergone and / or is undergoing chemotherapy. In some implementation schemes, the subject has already undergone and / or is undergoing radiation therapy.

[0072] In some embodiments, an antiviral drug is also administered to the subject. In some such embodiments, the antiviral drug is used to treat CMV infection (e.g., the antiviral drug inhibits CMV replication). For example, in some embodiments, ganciclovir, valganciclovir, foscarnet, cidofovir, acyclovir, fomivir, malivavir, BAY38-4766, or GW275175X is administered to the subject. In some embodiments, the CMV infection is drug-resistant. For example, in some embodiments, the CMV infection is ganciclovir-resistant.

[0073] Biomarkers of CMV peptide-specific T cell expression can be assessed using any suitable method, such as flow cytometry. In some embodiments, CMV peptide-specific T cells are stimulated with a CMV-specific peptide and then sorted by flow cytometry. Preferably, CMV peptide-specific T cells are stimulated and / or surface stained according to the protocols illustrated in Examples 1, 4, 5, or any combination thereof. In some embodiments, CMV peptide-specific T cells are incubated with one or more antibodies specific to CD107a and then sorted by flow cytometry. In some embodiments, CMV peptide-specific T cells are incubated with one or more antibodies that bind intracellular cytokines, such as antibodies specific to IFNγ, IL-2, and / or TNF. In some embodiments, CMV peptide-specific T cells are incubated with antibodies targeting intracellular cytokines and then sorted by flow cytometry.

[0074] In some respects, this article provides a method for selecting subjects for adoptive immunotherapy, the method being performed by: obtaining PMBC samples from the subject, isolating autologous T cells, determining the CMV reactivity of the autologous T cells, and selecting the subject for adoptive immunotherapy if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the autologous T cells are CMV-reactive.

[0075] In some respects, this article provides a method for selecting subjects for adoptive immunotherapy, the method being performed by: obtaining a sample containing T cells (e.g., CTLs) from the subject, isolating autologous T cells, and determining CD107a expression in the autologous T cells, selecting the subject for adoptive immunotherapy if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the autologous T cells express CD107a.

[0076] In some respects, this article provides a method for selecting subjects for adoptive immunotherapy, the method being performed by: obtaining a sample containing T cells (e.g., CTLs) from the subject, isolating autologous T cells, determining IFNγ expression in the autologous T cells, and selecting the subject for adoptive immunotherapy if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the autologous T cells express IFNγ.

[0077] In some respects, this article provides a method for selecting subjects for adoptive immunotherapy, the method being performed by: obtaining a sample containing T cells (e.g., CTLs) from the subject, isolating autologous T cells, determining TNF expression in the autologous T cells, and selecting the subject for adoptive immunotherapy if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the autologous T cells express TNF.

[0078] In some respects, this article provides a method for selecting subjects for adoptive immunotherapy, the method being performed by: obtaining a sample containing T cells (e.g., CTLs) from the subject, isolating autologous T cells, determining IL-2 expression in the autologous T cells, and selecting the subject for adoptive immunotherapy if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the autologous T cells express IL-2.

[0079] In some embodiments, the method further includes obtaining a sample containing T cells from a subject (e.g., obtaining a PBMC sample from a subject). In some embodiments, autologous T cells (e.g., CD4+ T cells or CD8+ T cells) are isolated from the sample. In some embodiments, the sample consists primarily or entirely of autologous T cells.

[0080] This document provides a method for treating or preventing CMV infection in a subject, the method comprising administering to the subject T cells stimulated by an immunogenic peptide library expressing a T cell receptor (e.g., autologous CMV peptide-specific CTLs), said T cell receptor specifically binding to one or more CMV peptides presented on class I and / or class II MHCs (e.g., any one of the peptides or combinations thereof listed in Table 1). In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the T cells (e.g., CTLs) in the sample express CD107a. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the T cells (e.g., CTLs) in the sample express IFNγ. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the T cells (e.g., CTLs) in the sample express TNF. In some implementations, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the T cells (e.g., CTLs) in the sample express IL-2.

[0081] In some implementations, the sample contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48% of the sample. 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of T cells (e.g., CTLs) express CD107a and IFNγ.

[0082] In some implementations, the sample contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 4 8%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of T cells (e.g., CTLs) express CD107a and TNF.

[0083] In some implementations, the sample contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48% of the sample. 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of T cells (e.g., CTLs) express CD107a and IL-2.

[0084] In some embodiments, the sample contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of T cells (e.g., CTLs) express IFNγ and TNF.

[0085] In some implementations, the sample contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 4 8%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of T cells (e.g., CTLs) express IFNγ and IL-2.

[0086] In some embodiments, the sample contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of T cells (e.g., CTLs) express TNF and IL-2.

[0087] In some implementations, the sample contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, and 48% of the sample. 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of T cells (e.g., CTLs) express IFNγ, TNF, and IL-2.

[0088] In some embodiments, the sample contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of T cells (e.g., CTLs) express CD107a, TNF, and IL-2.

[0089] In some embodiments, the sample contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of T cells (e.g., CTLs) express CD17a, IFNγ, and IL-2.

[0090] In some embodiments, the sample contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of T cells (e.g., CTLs) express CD107a, IFNγ, and TNF.

[0091] In some implementations, the sample contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%... 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of T cells (e.g., CTLs) express CD107a, IFNγ, TNF, and IL-2.

[0092] In some embodiments of the methods disclosed herein, T cells (e.g., CTLs) exhibit responsiveness to multiple peptide epitopes derived from various CMV antigens. 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of T cells (e.g., CTLs) are responsive to more than one CMV epitope. In some embodiments, T cells (e.g., CTLs) are responsive to any one of the CMV peptide epitope amino acid sequences or combinations thereof listed in Table 1. In some implementations, T cells (e.g., CTLs) are responsive to any one or a combination of pp50, pp65, IE-1, gB, gH.

[0093] The expression of T cell biomarkers and / or CMV responsiveness can be measured and / or analyzed before or after T cell (e.g., CTL) expansion using any of the methods disclosed herein, such as by exposure to a library of immunogenic CMV peptide epitopes.

[0094] In some embodiments, CMV responsiveness and biomarker expression are quantified prior to stimulation of T cells (e.g., CTLs). Alternatively or additionally, CMV responsiveness and biomarker expression may be quantified after stimulation of T cells (e.g., CTLs). In some embodiments, CMV responsiveness is measured by quantifying the percentage of T cells expressing CD107a in a sample. In some embodiments, CMV responsiveness is measured by quantifying the percentage of T cells expressing IFNγ in a sample. In some embodiments, CMV responsiveness is measured by quantifying the percentage of T cells expressing TNF in a sample. In some embodiments, CMV responsiveness is measured by quantifying the percentage of T cells expressing IL-2 in a sample. In some embodiments, CMV responsiveness is measured as the percentage of T cells expressing multiple biomarkers (e.g., two or more, preferably all four, of CD107a, IFNγ, TNF, and IL-2). In some embodiments, CMV responsiveness is calculated by quantifying the percentage of autologous T cells expressing CD107a, IFNγ, TNF, and IL-2 in a sample. T cells can be isolated from a sample (e.g., a PBMC sample or a sample containing T cells) before or after CMV reactivity percentage quantification. Therefore, in some embodiments, CMV reactivity is the percentage of T cells in a sample primarily containing T cells that possess one or more desired characteristics.

[0095] In some embodiments, CMV reactivity is measured by quantifying the percentage of CD8+ lymphocytes expressing CD107a in a sample. In some embodiments, CMV reactivity is measured by quantifying the percentage of CD8+ lymphocytes expressing IFNγ in a sample. In some embodiments, CMV reactivity is measured by quantifying the percentage of CD8+ lymphocytes expressing TNF in a sample. In some embodiments, CMV reactivity is measured by quantifying the percentage of CD8+ lymphocytes expressing IL-2 in a sample. In some embodiments, CMV reactivity is measured as the percentage of CD8+ lymphocytes expressing multiple biomarkers (e.g., two or more, preferably all four, of CD107a, IFNγ, TNF, and IL-2). CD8+ lymphocytes can be isolated from a sample (e.g., a PBMC sample or a sample of CD8+ lymphocytes) before or after CMV reactivity percentage quantification. Therefore, in some embodiments, CMV reactivity is the percentage of CD8+ lymphocytes in a sample that primarily contains CD8+ lymphocytes and possesses one or more desired characteristics.

[0096] In some embodiments, CMV reactivity is measured by quantifying the percentage of CD3+ lymphocytes expressing CD107a in a sample. In some embodiments, CMV reactivity is measured by quantifying the percentage of CD3+ lymphocytes expressing IFNγ in a sample. In some embodiments, CMV reactivity is measured by quantifying the percentage of CD3+ lymphocytes expressing TNF in a sample. In some embodiments, CMV reactivity is measured by quantifying the percentage of CD3+ lymphocytes expressing IL-2 in a sample. In some embodiments, CMV reactivity is measured as the percentage of CD3+ lymphocytes expressing multiple biomarkers (e.g., two or more, preferably all four, of CD107a, IFNγ, TNF, and IL-2). CD3+ lymphocytes can be isolated from a sample (e.g., a PBMC sample or a sample of CD3+ lymphocytes) before or after CMV reactivity percentage quantification. Therefore, in some embodiments, CMV reactivity is the percentage of CD3+ lymphocytes in a sample that primarily contains CD3+ lymphocytes and possesses one or more desired characteristics.

[0097] In some embodiments, the method further includes analyzing the expression of CD107a, IFNγ, TNF, or IL-2 in CMV peptide-specific T cells (e.g., CTLs), if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%... If 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of CMV peptide-specific T cells (e.g., CTLs) express CD107a, IFNγ, TNF, or IL-2, then CMV peptide-specific autologous T cells (e.g., CTLs) are administered to the subject.

[0098] In some implementations, the method further includes analyzing the expression of multiple biomarkers of CMV peptide-specific T cells (e.g., CTLs), and administering CMV peptide-specific T cells to the subject if the CMV peptide-specific T cells express at least two biomarkers. In some embodiments, the method further includes analyzing the expression of CD107a and TNF in CMV peptide-specific T cells (e.g., CTLs), if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, ... If 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of CMV peptide-specific T cells (e.g., CTLs) express CD107a and TNF, then peptide-specific autologous T cells (e.g., CTLs) are administered to the subject.

[0099] In some embodiments, the method further includes analyzing the expression of CD107a and IFNγ in CMV peptide-specific T cells (e.g., CTLs), if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 4 If 7%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of CMV peptide-specific T cells (e.g., CTLs) express CD107a and IFNγ, then CMV peptide-specific T cells (e.g., CTLs) are administered to the subject.

[0100] In some embodiments, the method further includes analyzing the expression of CD107a and IL-2 in proliferating peptide-specific autologous T cells (e.g., CTLs), if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, ... If 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of CMV peptide-specific T cells (e.g., CTLs) express CD107a and IL-2, then CMV peptide-specific T cells (e.g., CTLs) are administered to the subject.

[0101] In some embodiments, the method further includes analyzing the expression of TNF and IL-2 in CMV peptide-specific T cells (e.g., CTLs), if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%... If 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of CMV peptide-specific T cells (e.g., CTLs) express TNF and IL-2, then CMV peptide-specific autologous T cells (e.g., CTLs) are administered to the subject.

[0102] In some embodiments, the method further includes analyzing the expression of IFNγ and IL-2 in CMV peptide-specific T cells (e.g., CTLs), if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%... If 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of CMV peptide-specific autologous T cells (e.g., CTLs) express IFNγ and IL-2, then CMV peptide-specific T cells (e.g., CTLs) are administered to the subject.

[0103] In some embodiments, the method further includes analyzing the expression of IFNγ and TNF in proliferating CMV peptide-specific T cells (e.g., CTLs), if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, or 46%. If 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of CMV peptide-specific T cells (e.g., CTLs) express IFNγ and TNF, then CMV peptide-specific T cells (e.g., CTLs) are administered to the subject.

[0104] In some embodiments, the method further includes analyzing the expression of CD107a, IFNγ, and TNF in CMV peptide-specific T cells (e.g., CTLs), if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 4 If 7%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of CMV peptide-specific T cells (e.g., CTLs) express CD107a, IFNγ, and TNF, then CMV peptide-specific T cells (e.g., CTLs) are administered to the subject.

[0105] In some embodiments, the method further includes analyzing the expression of CD107a, IFNγ, and IL-2 in CMV peptide-specific T cells (e.g., CTLs), if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 4 If 7%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of CMV peptide-specific T cells (e.g., CTLs) express CD107a, IFNγ, and IL-2, then CMV peptide-specific T cells (e.g., CTLs) are administered to the subject.

[0106] In some embodiments, the method further includes analyzing the expression of CD107a, IL-2, and TNF in CMV peptide-specific T cells (e.g., CTLs), if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, or 46%. If 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of CMV peptide-specific T cells (e.g., CTLs) express CD107a, IL-2, and TNF, then peptide-specific T cells (e.g., CTLs) are administered to the subject.

[0107] In some embodiments, the method further includes analyzing the expression of IFNγ, IL-2, and TNF in CMV peptide-specific T cells (e.g., CTLs), if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 4 If 7%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of CMV peptide-specific T cells (e.g., CTLs) express IFNγ, IL-2, and TNF, then CMV peptide-specific T cells (e.g., CTLs) are administered to the subject.

[0108] In some implementations, if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53% If 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of CMV peptide-specific autologous T cells (e.g., CTLs) express CD107a, IFNγ, TNF, and IL-2, then the autologous T cells (e.g., CTLs) are administered to the subject.

[0109] CMV peptide-specific autologous T cells (e.g., CTLs) can have at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, or 44% of the cells. 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of CMV reactivity.

[0110] In some embodiments, the method further includes analyzing the CMV responsiveness of CMV peptide-specific T cells (e.g., CTLs), and, if the responsiveness is for more than one epitope, and at least a threshold percentage of CMV peptide-specific T cells (e.g., CTLs) (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%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 3...), is considered. If 9%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% are CMV-responsive, then CMV peptide-specific T cells (e.g., CTLs) are administered to the subject.

[0111] In some implementation schemes, approximately 1 × 10⁻⁶ doses are administered to the subjects per dose. 5 To approximately 1×10 810 T cells. In some implementations, approximately 1 × 10⁶ T cells are administered to the subject per dose. 6 To approximately 1×10 7 10 T cells. In some implementations, 5 × 10 T cells are administered to the subject. 6 1×10 7 1.5×10 7 Or 2×10 7 10 T cells (e.g., CTLs). Multiple doses may be administered to the subject. In some embodiments, an initial dose of T cells (e.g., autologous CTLs) is administered, and one or more additional doses of T cells (e.g., autologous CTLs) are administered, for example, at increased doses during treatment. In some embodiments, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more doses are administered. Other doses, the same as or different from the initial dose, may be administered to the subject. For example, a lower dose may be administered, followed by a higher dose. Dosage may be administered once daily, twice weekly, once weekly, once every two weeks, once monthly, once every two months, once every three months, or once every six months. In some embodiments, the subject does not experience any side effects due to the administration of T cells (e.g., autologous CTLs).

[0112] In some aspects, the method further includes assessing the efficacy of adoptive immunotherapy by measuring the CMV viral load in subjects with CMV infection, reactivation, or related disease. In some embodiments, the subject has already received a solid organ transplant. As a non-limiting example, CMV viral load can be measured by obtaining a first sample (e.g., a blood sample) from the subject, assessing the viral load in the first sample using methods known in the art (preferably before CTL administration), obtaining a second sample from the subject after a period of time (preferably after CTL administration), assessing the viral load in the second sample, and if the viral load in the second sample is less than that in the first sample, then the CMV infection, reactivation, or related disease has improved and / or has not progressed. Other samples may be obtained and compared with previous samples. This document also provides a method for reducing viral load in subjects with CMV infection, reactivation, or related disease by administering immunogenic peptide library-stimulated T cells (e.g., CMV peptide-specific autologous CTLs disclosed herein) to the subject. Changes in viral load (e.g., reduction) can be measured using methods known in the art, such as nucleic acid-based assays (e.g., nucleic acid tests (NAT) and nucleic acid amplification tests (NAAT)) or non-nucleic acid tests (e.g., quantitative enzyme immunoassay). After T-cell administration, viral load can be reduced by approximately 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0113] In some embodiments, the method includes improving or stabilizing symptoms or conditions in a subject suffering from CMV infection, reactivation, or related disease by administering immunogenic peptide-stimulated T cells (e.g., CTLs, such as the CMV peptide-specific autologous CTLs described herein) to the subject. This document also provides methods for reducing or resolving DNAemia; and / or reducing, stabilizing, or stopping CMV-related end-organ disease in subjects infected with CMV, the methods including administering immunogenic peptide-stimulated T cells (e.g., CTLs, such as the CMV peptide-specific autologous CTLs described herein) to the subject. In some embodiments, this document also provides methods for using antiviral therapy to reduce or stop CMV infection, the methods including administering immunogenic peptide-stimulated T cells (e.g., CTLs, such as the CMV peptide-specific autologous CTLs described herein) to the subject. In a preferred embodiment, the subject has received a solid organ transplant. In a more preferred embodiment, the subject suffers from ganciclovir-resistant CMV infection, reactivation, or related disease.

[0114] In some embodiments, the subject has cancer. In some embodiments, the methods described herein can be used to treat any cancerous or precancerous tumor. In some embodiments, the cancer expresses one or more CMV epitopes provided herein (e.g., CMV epitopes listed in Table 1). In some embodiments, the cancer includes solid tumors. Cancers that can be treated by the methods and compositions provided herein include, but are not limited to, cancer cells originating 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, this cancer can specifically be the following histological types, although it is not limited to these: malignant tumor; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatric carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial adenomatous polyposis; solid carcinoma; malignant carcinoid; bronchoalveolar adenocarcinoma; papillary adenocarcinoma; chromogenic carcinoma; eosinophilic carcinoma;Chromophobe carcinoma, eosinophilic carcinoma, adenocarcinoma, basophilic granulosa carcinoma, clear cell adenocarcinoma, granulosa cell carcinoma, follicular adenocarcinoma, papillary and follicular adenocarcinoma, non-capsular sclerosing carcinoma, adrenocortical carcinoma, endometrioid carcinoma, skin adnexal carcinoma, apocrine gland carcinoma, sebaceous gland carcinoma, ceruminous gland carcinoma, mucoepidermoid carcinoma, cystadenocarcinoma, papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, mucinous cystadenocarcinoma, mucinous gland carcinoma, 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 of squamous metaplasia, malignant thymoma, malignant ovarian stromal tumor, malignant theca cell tumor, malignant granulosa cell tumor, and malignant blastoma, Sertoli cell carcinoma, malignant Leydig cell carcinoma. Dig cell tumor, malignant lipocytoma, malignant paraganglionic tumor, malignant breast paraganglioma, pheochromocytoma, angiosarcoma, malignant melanoma, amelanoma, superficial spreading melanoma, malignant melanoma in giant pigmented nevus, epithelioid cell melanoma, malignant blue nevus, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, myosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, stromal sarcoma, malignant mixed tumor, Müllerian mixed tumor, nephroblastoma, hepatoblastoma, carcinosarcoma, malignant mesothelioma, malignant Blenner tumor, malignant phyllodes tumor, synovial sarcoma, malignant mesothelioma, dysgerminoma, embryonal carcinoma, malignant teratoma, malignant ovarian thyroid tumor Choriocarcinoma, Malignant Mesonephric Tumor, Angiosarcoma, Malignant Hemangioendothelioma, Kaposi's Sarcoma, Malignant Pericytoma, Lymphangiosarcoma, Osteosarcoma, Cortical Osteosarcoma, Chondrosarcoma, Malignant Chondroblastoma, Mesenchymal Chondrosarcoma, Giant Cell Tumor of Bone, Ewing's Sarcoma, Malignant Odontogenic Tumor, Ameloblastic Odonosarcoma, Malignant Ameloblastoma, Ameloblastic Fibrosarcoma, Malignant Pineal Tumor, Chordoma, Malignant Glioma, Ependymoma, Astrocytoma, Protoplasmic Astrocytoma, Fibroblastoma, Astroblastoma, Glioblastoma, Glioblastoma Multiforme (GBM), Oligodendroglioma, Oligodendroblastoma, Primitive Neuroectodermal, Cerebellar Sarcoma, Ganglioblastoma, Neuroblastoma, Visual Tissue Retinoblastoma, olfactory neurogenic tumors, malignant meningiomas, neurofibrosarcomas, malignant neuromas, malignant granular cell tumors, malignant lymphomas, Hodgkin's disease, Hodgkin's lymphoma, paragangliomas, small lymphocytic malignant lymphomas, diffuse large cell malignant lymphomas, follicular malignant lymphomas, mycosis fungoides, other specified non-Hodgkin's lymphomas, malignant histiocytic proliferative diseases, multiple myeloma, mast cell sarcoma, immunoproliferative small bowel diseases, leukemia, lymphocytic leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma cell leukemia, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryocytic leukemia, myeloid sarcoma, and piloblastic leukemia.

[0115] In some implementations, an anticancer compound is also administered to the subject. Exemplary anticancer compounds include, but are not limited to, aleizumab. alivitic acid Anastrozole bevacizumab Besarodine Bortezomib Bosutinib Bentuximab Cabozantinib (Cometriq) TM ), Kafilzomi (Kyprolis) TM Cetuximab Crizotinib Dasatinib Denileukindiftitox Erlotinib Hydrochloride Everolimus Isimmetan Flucelestane Gefitinib Etemozolomide Tishumab Imatinib Mesylate Yervoy TM Lapatinib dimethylbenzenesulfonate Letrozole Nilotinib Ophamumab Panitumumab Pazopanib Hydrochloride Pertuzumab (Perjeta) TM ), Prattria Regofini Rituximab Romidesin Sorafenib Tosylate Sunitinib malate Tamoxifen, Temoromosifen Torremifen Tocetomab and 131I-Tocetomab Trastuzumab Retinoic acid Van der Thani Verafinil Vorinostat and Abercet

[0116] In some implementations, a chemotherapeutic agent is also administered to the subject. Examples of such chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethiylenethiophosphoramide, and trimethylolamine. trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analogue topotecan); bromostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin;Nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, and uracil. Mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammal I and calicheamicin omegal I); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin;And neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, arrnycin, azaserine, bleomycins, cactinomycin C, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-leucine. 5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, and mitomycins, such as mitomycin C. C) Mycophenolic acid, nogalamycin, oligomycins, peplomycin, potfiromycin, puromycin, quercetin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, antimetabolites such as methotrexate and 5-fluorouracil (5-FU);Folic acid analogs, such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and fluxuridine; and androgens, such as calusterone and dromostanolone. Propionate, epitiostanol, mepitiostane, testolactone; anti-adrenergics, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate); epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A) A) and guanidine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactal; piperobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, such as paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine (mercaptopurine); methotrexate; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda;Ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; and any pharmaceutically acceptable salts, acids, or derivatives thereof.

[0117] In some implementations, immunotherapeutic agents are also administered to the subject. Immunotherapy refers to treatments that utilize a subject's immune system to treat and prevent disease; examples include cancer vaccines, cytokines, target-specific antibodies, T-cell therapy, and dendritic cell therapy.

[0118] In some implementations, immunomodulatory proteins are also administered to the subject. Examples of immunomodulatory proteins include, but are not limited to, B lymphocyte chemokine (“BLC-C), C2 motif chemokine 11 (“C1-C),” eosinophil chemokine 2 (“eosinophil chemokine),” granulocyte colony-stimulating factor (“granulocyte colony-stimulating factor”), granulocyte-macrophage colony-stimulating factor (“granulocyte-macrophage 1,” IL-309), intercellular adhesion molecule 1 (“intercellular adhesion molecule,” interferon-γ (“interferon-γ molecule,” interleukin-1 alpha (“IL-1α,” interleukin-1 beta (“IL-1β”), interleukin-1 receptor antagonist (“IL-1 antagonist β”), interleukin-2 (“IL-2 antagonist,” interleukin-4 (“IL-4 antagonist,” interleukin-2), and interleukin-4 (“IL-4 antagonist,” interleukin-2, interleukin-3, interleukin-4, interleukin-4, interleukin-4, interleukin-3 ... Interleukin-4 (“interleukin-5”), Interleukin-6 (“interleukin-6”), Interleukin-6 soluble receptor (“soluble receptor β”), Interleukin-7 (“interleukin-7”), Interleukin-8 (“interleukin-8”), Interleukin-10 (“interleukin-10 β”), Interleukin-11 (“interleukin-11 β”), Interleukin-12 subunit beta (“interleukin-12 β” colony spike or “interleukin-13 β”), Interleukin-15 (“interleukin-15 β”), Interleukin-16 (“interleukin-16 β”), Interleukin-17 (“interleukin-17 β”), Chemokinetic (CC motif) ligand 2 (“MCP-1”), Macrophage colony-stimulating factor (“Macrophage-17 β”), Colony spikes), gamma interferon-induced mononuclear factor (“interferon-induced”), chemokine (CC motif) ligand 2 (“somatic C factor α”), chemokine (CC motif) ligand 4 (“somatic C factor β”), macrophase inflammatory protein-1-delta (“1-delδ”), platelet-derived growth factor subunit B (“platelet-derived growth”), chemokine (CC motif) ligand 5, regulation of activation, normal T cell expression and secretion (“cell expression and secretion”), TIMP metallopeptidase inhibitor 1 (“belonging to peptidase inhibitor”), TIMP metallopeptidase inhibitor 2 (“belonging to peptidase inhibitor”), tumor necrosis factor, lymphotoxin-alpha (“alpha”) Tumor necrosis factor, lymphotoxin-beta (“beta factor”), type 1 soluble TNF receptor (“sigmoid factor α”), sTNFRIA, brain-derived neurotrophic factor (“brain-derived neurotrophic factor”), basic fibroblast growth factor (“basic fibroblast”), bone morphogenetic protein 4 (“bone morphogenetic protein”), bone morphogenetic protein 5 (“bone morphogenetic protein”), bone morphogenetic protein 7 (“bone morphogenetic protein”), nerve growth factor (“nerve growth factor protein”), epidermal growth factor (“epidermal growth factor”), epidermal growth factor receptor (“epidermal growth factor”), endocrine gland-derived vascular endothelial growth factor (“endocrine gland-derived vascular endothelial growth factor”), fibroblast growth factor 4 (“fibroblast growth factor”)Keratinocyte growth factor (“Keratinocyte growth factor”), growth differentiation factor 15 (“G5 growth differentiation factor”), glial cell-derived neurotrophic factor (“GLA-derived neurotrophic factor”), growth hormone, heparin-bound EGF-like growth factor (“GGF-binding”), hepatocyte growth factor (“Hepatocyte growth factor”), insulin-like growth factor binding protein 1 (“Insulin-like growth factor”), insulin-like growth factor binding protein 2 (“Insulin-like growth factor”), insulin-like growth factor binding protein 3 (“Insulin-like growth factor”), insulin-like growth factor binding protein 4 (“Insulin-like growth factor”), insulin-like growth factor binding protein 6 (“Insulin-like growth factor”), insulin-like growth factor 1 (“Insulin-like growth factor”), insulin, macrophage colony-stimulating factor (“Insulin-macrophage”), nerve growth factor receptor (“GGF receptor”). Neurotrophin-3 (“3-neurotrophin”), Neurotrophin-4 (“4-neurotrophin”), Osteoclastogenesis Inhibitor (“Osteoclastogenesis Inhibitor Stimulatory Factor”), Platelet-Derived Growth Factor Receptor (“Platelet-Derived Growth Factor”), Phosphatidylinositol-Glycan Biosynthesis (“Glycan Biosynthesis”), Skp, Cullin, F-box-containing Complex (“Complex X”), Stem Cell Factor Receptor (“Stem Cell Factor Receptor”), Transforming Growth Factor Alpha (“alpha”), Transforming Growth Factor Beta-1 (“eta-1”), Transforming Growth Factor Beta-3 (“eta-3”), Vascular Endothelial Growth Factor (“VEGF”), Vascular Endothelial Growth Factor Receptor 2 (“VEGF”), Vascular Endothelial Growth Factor Receptor 3 (“VEGF”), VEGF-D 6Ckine, tyrosine protein kinase receptor UFO (“FO acid protein”), cytokinin (“cytokinin protein”), mucosa-associated epithelial chemokine (“mucosa-associated epithelial chemokine”), chemokine (CC motif) ligand 27 (“7C factor”), chemokine (CXC motif) ligand 16 (“6X-C epithelial chemokine”), CXC motif chemokine 5 (“motif chemokine epithelial chemokine”), chemokine (CC motif) ligand 26 (“6C factor epithelial chemokine”), granulocyte chemoattractant protein 2 (“granulocyte chemoattractant protein”), GRO, chemokine (CC motif) ligand 14 (“4C factor chemokine”). ), chemokine (CC motif) ligand 16 (“6C factor protein”), interleukin-9 (“9-interleukin protein”), interleukin-17F (“17F protein”), interleukin-18 binding protein (“synthesis protein F protein chemokine”), interleukin-28A (“28A protein”), interleukin-29 (“9-interleukin A protein”), interleukin-31 (“1-interleukin A protein”), CXC motif chemokine 10 (“0 chemokine protein”), chemokine receptor CXCR3 (“XCR3 receptor”), leukemia inhibitory factor (“leukemia suppressor”), chemokine (C motif) ligand (“body,Chemokines (e.g., chemokines), monocyte chemoattractant protein 2 (“monocyte CP-2”), monocyte chemoattractant protein 3 (“monocyte chemotaxis”), monocyte chemoattractant protein 4 (“monocyte chemotaxis”), macrophage-derived chemokines (“macrophages”), macrophage migration inhibitory factor (“macrophages”), chemokine (CC motif) ligand 20 (“OC factor migration inhibitor”), CC motif chemokine 19 (“9 chemokine migration inhibitor”), chemokine (CC motif) ligand 23 (“3C factor migration inhibitor”), macrophages... Phagocytocyte stimulatory protein α chain (“Phrenic spur”), nucleosome assembly protein 1-like 4 (“Nucleosome assembly protein”), secreted phosphoprotein 1 (“Osteopontin”), lung and activation-regulated cytokines (“Lung and Activation-Regulated Cytokines”), platelet factor 4 (“Platelet Factor”), stromal cell-derived factor-1α (“1α Cell-Derived”), chemokine (CC motif) ligand 17 (“7C Factor Derived”), thymic expressed chemokines (“Thymic Expressed”), thymic stromal lymphopoietin (“Thymic Stromal Lymphopoietin”), CD4+ 166 antigen (“Proto-166 Bar”), differentiation cluster 80 (“O-Cluster 66”), tumor necrosis factor receptor superfamily member 17 (“7-Tumor Necrosis Factor”), differentiation cluster 14 (“4-Cluster Death Factor”), differentiation cluster 30 (“O-Cluster Death Factor”), differentiation cluster 40 (“O-Cluster Death Ligand”), carcinoembryonic antigen-associated cell adhesion molecule 1 (bile glycoprotein) (“glycoprotein-associated cell adhesion molecule”), death receptor 6 (“death receptor”), deoxythymidine kinase (“oxythymidine kinase”), type 1 membrane glycoprotein (“membrane glycoprotein-associated cell”), receptor tyrosine protein kinase erbB-3 (“rbB-3 protein”), endothelial cell-leukocyte adhesion molecule 1 (“cytoselectin”), apoptosis antigen 1 (“apoptosis antigen”), Fms-like tyrosine kinase 3 (“tyrosine kinase protein”), tumor necrosis factor receptor superfamily member 1 (“tumor necrosis factor”), tumor necrosis factor receptor superfamily member 14 (“tumor necrosis factor”), intercellular adhesion molecule 3 (“intercellular adhesion molecule”), IL-1R4, IL-1RI, IL-10Rbeta, IL-17R, IL-2Rgamma, IL-21R, lysosomal membrane protein 2 (“lysosomal membrane protein m”), neutrophil gelatinase-associated lipid transporter (“neutrophil gelatinase-associated lipid”), CD62L (“D6 selectin”), lymphatic endothelium (“lymphatic endothelial gelatin”), MHC Class I polypeptide-related sequence A (“Polypeptide-related sequence”), MHC Class I polypeptide-related sequence B (“Polypeptide-related sequence”), NRG1-betal, β-platelet-derived growth factor receptor (“Platelet-derived growth factor receptor”), platelet endothelial cell adhesion molecule (“Platelet endothelial cell adhesion molecule”), RAGE, hepatitis A virus cell receptor 1 (“Hepatitis A virus”), tumor necrosis factor receptor superfamily member IOC (“OC necrosis factor receptor superfamily”).Trappin protein transglutaminase-binding domain (“transglutaminase-binding domain”), urokinase receptor (“urokinase receptor”), vascular cell adhesion protein 1 (“vascular cell adhesion”), XEDAR, activin A, Agouti-related protein (“antigen 1”), ribonuclease 5 (“antigen 1”), angiopoietin 1, angioinhibin, cathepsin S, CD40, recessive family protein IB (“B-family protein group”), DN, Dickkopf-related protein 1 (“antigen 1”), E-cadherin, epithelial cell adhesion molecule (“antigen 1”), Fas ligand (FasL or CD95L), Fcg RIIB / C, Fuuistatin, galactagogue-7, intercellular adhesion molecule 2 (“Intercellular adhesion molecule”), IL-13R1, IL-13R2, IL-17B, IL-2Ra, IL-2Rb, IL-23, LAP, neuronal cell adhesion molecule (“Neuronal cell adhesion molecule”), plasminogen activator inhibitor-1 (“1-lysozyme activator”), platelet-derived growth factor receptor (“P-DG”), resistin, stromal cell-derived factor 1 (“Resistin, stromal cell”), sgpl 30. Secretory coil-associated protein 2 (“Secretory coil”), sialic acid-binding immunoglobulin type lectin (“White type lectin immunoglobulin”), ST2, transforming growth factor-transformation (“Transforming growth factor”), Tie-2, thrombopoietin (“Thrombopoietin”), tumor necrosis factor receptor superfamily member 10D (“0D Necrosis factor receptor super”), trigger receptor 1 expressed on myeloid cells (“On myeloid cell surface”), vascular endothelial growth factor C (“VEN”), VEGFR1, adiponectin, lipase (Adips) (in) (“Adip”), Alpha-fetoprotein (“AFP”), Angiopoietin-like 4 (“Angiopoietin-like n”), Beta-2-microglobulin (“Globulin-”), Basal cell adhesion molecule (“Basal cell adhesion”), Carbohydrate antigen 125 (“Compound Anti-25”), Cancer antigen 15-3 (“Compound Anti-5-3”), Carcinoembryonic antigen (“Carcinoembryonic antigen”), cAMP receptor protein (“Somatic proteinogen”), Human epidermal growth factor receptor 2 (“Human epidermal growth factor”), Follicle-stimulating hormone (“Follicle-stimulating hormone”), Chemokines Matrix metalloproteinase-1 (CXC motif) ligand 1 (“XC”), human chorionic gonadotropin (“βHCG”), insulin-like growth factor 1 receptor (“XC”), IL-1 sRII, IL-3, IL-18Rb, IL-21, leptin, matrix metalloproteinase-1 (“1”), matrix metalloproteinase-2 (“2”), matrix metalloproteinase-3 (“3”), matrix metalloproteinase-8 (“8”), matrix metalloproteinase-9 (“9”).Matrix metalloproteinase-10 (“MMP-10”), matrix metalloproteinase-13 (“MMP-13”), neural cell adhesion molecule (“NCAM-1”), nestin (“Nidogen-1”), neuron-specific enolase (“NSE”), oncogene M (“OSM”), procalcitonin, prolactin, prostate-specific antigen (“prolactin”), sialic acid-binding Ig-like lectin 9 (“Gland-specific lectin”), ADAM 17 endopeptidase (“Peptidase M1”), thyroglobulin, inhibitor of metalloproteinases 4 (“Thyroglobulin, gold”), TSH2B4, protein 9 containing depolymerization and metalloproteinase domains (“Contains depolymerization and gold”), angiopoietin 2, tumor necrosis factor ligand superfamily member 13 / acid-rich leucine nucleoprotein 32 family member B (“Family member leucine”), bone morphogenetic protein 2 (“Bone morphogenetic protein”), bone morphogenetic protein 9 (“Bone morphogenetic protein”), complement component 5a (“A-component”), cathepsin L, CD200, CD97, chemerin, tumor necrosis factor receptor superfamily member 6B (“B-tumor necrosis factor”), fatty acid-binding protein 2 (“Fatty acid-binding protein”), fibroblast activation protein, α (“Fibroblast”), fibroblast growth factor 19 (“9-fibroblast growth factor”), galactagogue-3, hepatocyte growth factor receptor (“Hepatocyte growth factor”), IFN-alpha / beta R2, Insulin-like growth factor 2 (“Insulin-like growth”), Insulin-like growth factor 2 receptor (“Insulin-like growth factor”), Interleukin-1 receptor 6 (“Interleukin-like growth factor”), Interleukin-24 (“Interleukin-4”), Interleukin-33 (“Interleukin-3”), Kallikrein 14, Asparagine endopeptidase (“Asparagine”), Oxidized low-density lipoprotein receptor 1 (“Oxidized low-density”), Mannose-binding lectin (“Mannose knot”), Enkephalin (“Enkephalin”), Notch homolog 1, Translocation-associated (Drosophila) (“Translocation-associated lectin”), Nephroblastoma overexpression (“Nephroblastoma”), Bone activator, Programmed cell death protein 1 (“Programmed cell death”), N-acetylmurayl-L-alanine amidase (“L-alanine amidase”), Serpin A4, secreted coil-associated protein 3 (“secreted coil-associated protein”), thrombomodulin, Toll-like receptor 2 (“receptor 1 receptor protein”), tumor necrosis factor receptor superfamily member 10A (“0A necrosis factor receptor superfamily”), transferrin (“transferrin”), WIF-1ACE-2, albumin, AMICA, angiopoietin 4, B cell activating factor (“cell activating factor”), carbohydrate antigen 19-9 (“9-9 complex antigen”), CD163, clusteringin, CRT AM, chemokine (CXC motif) ligand 14 (“4X-CM antigen”), cystatin C, Decorin (“ecor”)Dickkopf-related protein 3 (“Kopf”), Delta-like protein 1 (“Kopf”), Fetoprotein A, Heparin-binding growth factor 1 (“Heparin-binding growth factor”), Folate receptor α (“Folic acid receptor α”), Furin protease, GPCR-related sorting protein 1 (“Kopf”), GPCR-related sorting protein 2 (“Kopf”), Granulocyte colony-stimulating factor receptor (“GCL-2”), Serine protease heparin (“Protease heparin spike”), Interleukin-17B receptor (“Interleukin-17B heparin-stimulating factor”), Interleukin-27 (“Interleukin-27”), Lymphocyte activation gene 3 (“Lymphocyte activation”), Apolipoprotein AV (“-V protein activation”), Pepsinogen I. Retinol-binding protein 4 (“Retinol-binding”), SOST, heparan sulfate proteoglycan (“Heparan sulfate proteoglycan”), tumor necrosis factor receptor superfamily member 13B (“3B Necrosis Factor”), tissue factor pathway inhibitor (“Tissue Factor Pathway”), TSP-1, tumor necrosis factor receptor superfamily member 10B (“0B Necrosis Factor Receptor Superfamily”), TRANCE, troponin I, urokinase plasminogen activator (“Urokinase Plasminogen Activator”), cadherin 5, type 2 or VE-cadherin (vascular endothelium) also known as CD144 (“D14 cadherin”), WNT1-induced signal transduction pathway protein 1 (“WNT1-induced signal transduction pathway”), and nuclear factor kappa nucleus receptor activator (“Receptor Activator”).

[0119] In some implementations, an immune checkpoint inhibitor is also administered to the subject. Immune checkpoint inhibition, broadly speaking, refers to the inhibition of checkpoints that impair or downregulate the immune response, particularly those associated with cancer cells. 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 or antigen-binding fragments thereof that bind to and inhibit immune checkpoint proteins. Examples of immune checkpoint inhibitors include, but are not limited to, nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, STI-A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736, MSB-0020718C, AUR-012, and STI-A1010.

[0120] In some embodiments, the compositions provided herein (e.g., the vaccine compositions provided herein) are administered prophylactically to prevent cancer and / or CMV infection. In some embodiments, the vaccine is administered to inhibit tumor cell proliferation. The vaccine may be administered before or after testing a patient for cancer cells or CMV-infected cells. Inhibition of tumor cell proliferation is understood to mean preventing, stopping, slowing the growth of tumor cells, or killing tumor cells. In some embodiments, a pro-inflammatory response is induced after administration of a vaccine comprising the peptides, nucleic acids, antibodies, or APCs described herein. Pro-inflammatory immune responses include the production of pro-inflammatory cytokines and / or chemokines, such as the production of interferon-γ (IFN-γ) and / or interleukin-2 (IL-2). Pro-inflammatory cytokines and chemokines are well known in the art.

[0121] Combination therapy includes the sequential, simultaneous, and separate administration of active compounds in a manner such that the therapeutic effect of the first agent is not completely lost upon administration of subsequent treatments. In some embodiments, the second agent may be formulated together with the first agent or as a separate pharmaceutical composition.

[0122] The actual dosage level of the active ingredient in the pharmaceutical composition provided herein can be altered to obtain an amount of active ingredient that effectively achieves the desired therapeutic response for a particular patient, composition, and method of administration, without being toxic to the patient.

[0123] The chosen dose level will depend on a variety of factors, including the activity of the specific agent used, the route of administration, the time of administration, the excretion or metabolic rate of the specific compound used, the duration of treatment, other drugs, compounds and / or substances used in combination with the specific compound used, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical field.

[0124] In some embodiments, the methods provided herein further include treating the identified subject with the treatment methods provided herein (e.g., by administering the pharmaceutical composition provided herein to the subject).

[0125] Example

[0126] Example 1: Patient Characteristics

[0127] To assess the safety of autologous T-cell therapy in solid organ transplant (SOT) recipients with CMV-related complications, patients were selected and considered eligible once they met one of the following four criteria:

[0128] (A) CMV reactivation or disease (as defined by histology) following successful initial therapy, such as ganciclovir-resistant CMV reactivation;

[0129] (B) Persistent CMV disease, i.e., no response to 2 weeks of salvage phosphonoformic acid or other second-line antiviral agents, for example, relapse of CMV due to incompatibility with second-line drug therapy;

[0130] (C) CMV replication persists despite appropriate antiviral therapy (more than 6 weeks as confirmed by PCR); or

[0131] (D) Any CMV reactivation or disease that is contraindicated for antiviral therapy due to intolerance or end-organ limitations (e.g., renal insufficiency, bone marrow insufficiency), such as end-organ CMV disease or intolerance to antiviral drug therapy.

[0132] Antiviral therapy was administered according to institutional guidelines. Patients received up to six doses of 1-2 x 10 every two weeks. 7 cells / m 2 In vitro expanded T cells. Safety, clinical symptoms, viral load, and immune reconstitution were monitored in each participant for 28 weeks after the completion of adoptive T-cell therapy. Viral load was monitored using an in-house quantitative assay, as previously described (Hill et al. 2016 Am J Transplant 2010; 10(1):173-9).

[0133] result

[0134] Table 2 provides the clinical characteristics of the participants in this study. A total of 21 SOT recipients were included in the study (13 kidney, 8 lung, and 1 heart). Two lung transplant patients included in the follow-up analysis had previously received treatment under the Therapeutic Goods Administration's special access protocol (Holmes-Liew et al., Clinical & Translational Immunology 2015; 4(3):e35; Pierucci et al., J Heart Lung Transplant 2016; 35(5):685-7). Of the 21 patients analyzed, 13 SOT recipients were designated for intervention, receiving up to six doses of adoptive T-cell therapy. One patient discontinued therapy after one dose without immune monitoring. Of the remaining eight patients, seven did not receive adoptive T-cell therapy due to improvement in their clinical condition, and no therapy was prepared for any patient.

[0135] Table 2: Clinical data of SOT recipients participating in the study

[0136]

[0137]

[0138]

[0139] NA was not obtained;

[0140] A: CMV reactivation or disease (as defined histologically) following successful initial treatment.

[0141] B: Persistent CMV disease, i.e., no response to salvage phosphonoformic acid or other second-line antiviral agents for 2 weeks.

[0142] C: Despite appropriate antiviral therapy, CMV replication persists (as confirmed by PCR for more than 6 weeks).

[0143] D: Any CMV reactivation or disease that contraindicates antiviral therapy due to intolerance or end-organ limitations (e.g., renal insufficiency, bone marrow insufficiency).

[0144] AZA: Azathioprine;

[0145] CSA: Cyclosporin

[0146] EVR: Everolimus;

[0147] LEF: Leflunomide;

[0148] MePRD: Methylprednisolone

[0149] MMF: Mycophenolate;

[0150] PRD: Prednisolone;

[0151] TAC: Tacrolimus

[0152] CDV: Cidofovir;

[0153] FOS: Foscarnet;

[0154] GCV: Gancyclovir;

[0155] VGCV: Valgancyclovir

[0156] Example 2: Preparation for T-cell therapy

[0157] To generate CMV-specific T-cell therapy, peripheral blood mononuclear cells (PBMCs) obtained from each patient were stimulated individually with a clinical-grade CMV peptide library in the presence of IL-21 (day 0, 40 ng / mL). This library included predefined HLA class I and II restricted peptide epitopes for pp65, pp50, IE-1, gH, and gB (Table 1). The stimulated samples were then cultured in Grex-10 flasks (WilsonWolf Corporation, Saint Paul, MN) at 2–5 × 10⁻⁵ ng / mL. 6 cells / cm 2 The cells were cultured at the initial cell density. These cultures were supplemented with IL-2 (120 IU / mL) on day 2 and every three days thereafter. On day 14, expanded T cells were harvested and frozen in 1 mL aliquots of Alpexex 4 (CSLBehring, Broadmeadows, Australia) containing 10% dimethyl sulfoxide (WAK-Chemie Medical GmbH, Steinbach, Germany). T cells were tested for microbial contamination prior to infusion and characterized for phenotype and function using Multitest 6-color TBNK reagent (BD Biosciences, San Jose, CA) and intracellular cytokine staining (detailed below). For adoptive transfer, T cells were infused into 19 mL of clinical-grade saline and infused intravenously over a period of 5–10 minutes.

[0158] result

[0159] CMV-specific T cells were successfully expanded from 20 of the 21 patients, and their antigen specificity was assessed by intracellular IFN-γ analysis (Table 3). The cells expanded from the CMV peptide library were mainly CD3+CD8+ T cells. Figure 1 A), with a median specificity of 51.2% (A). Figure 1 B). Kidney and lung / heart transplant recipients ( Figure 1 C) or individuals who were CMV serologically positive or CMV serologically negative before transplantation ( Figure 1 There was no significant difference in the frequency of CD8+ T cells producing IFN-γ between D) and D. After in vitro expansion, the pluripotency of CMV-specific T cells was significantly improved, with an increased proportion of cells capable of producing IFN-γ, TNF, and CD107a. Figure 1 E). T cells generated from most patients showed responsiveness to multiple peptide epitopes encoded by multiple CMV antigens (Table 3).

[0160] Table 3: CMV-specific reactivity of T cells expanded in vitro from SOT recipients

[0161]

[0162]

[0163] NA was not obtained;

[0164] #CMV reaction assay measures the proportion of CD8+ T cells producing IFN-γ.

[0165] * Add KAR peptide to the CMV peptide library for stimulation.

[0166] **An HLA-specific peptide library was generated to produce T cells for these patients.**

[0167] Example 3: Clinical outcomes after adoptive immunotherapy

[0168] No treatment-related grade 3, 4, or 5 adverse events were observed in patients receiving adoptive CMV-specific T-cell therapy (Table 4). All adverse events considered at least attributable to T-cell infusion were grade 1 and 2, including fatigue and discomfort. Importantly, no adverse events related to changes in graft status were detected. Clinical follow-up of patients receiving the designated T-cell therapy intervention showed that 11 out of 13 patients exhibited objective symptom improvement. These improvements included reduction or resolution of CMV reactivation and / or disease, as well as improved response to antiviral drug therapy. Among the 11 patients who demonstrated a clinical response, the median peak viral load prior to adoptive T-cell therapy was 3.2 × 10⁻⁶. 4 CMV copies / mL blood (range 1.4 × 10⁻⁶) 3 –3.44×10 5 (Copy). Following adoptive immunotherapy, the median viral load decreased to 1.2 × 10⁻⁶. 3 CMV copies / mL blood (range 0-7.9×10⁻⁶) 3 (Copy; Table 4). Furthermore, many of these patients showed resolution of CMV disease symptoms (Table 4). More importantly, antiviral drug therapy was completely discontinued (5 / 11) or significantly reduced (6 / 11; Table 5) after completion of adoptive T-cell therapy.

[0169] result

[0170] In one cohort of patients (recruited due to evidence of drug resistance / intolerance, persistent viral reactivation, or related disease), there was no evidence of serious adverse events or any negative impact on the graft following T-cell administration (see Table 4).

[0171] Table 4: Safety assessment after T-cell therapy

[0172]

[0173]

[0174] *Events that may or are probable to be related to T-cell therapy. No adverse events were considered to be clearly related to T-cell therapy.

[0175] Table 5: Clinical responses after adoptive T-cell therapy

[0176]

[0177]

[0178] CDV: Sidofovir; FOS: Foscarnet; GCV: Ganciclovir; IVIG: Intravenous CMV Immunoglobulin; LEF: Leflunomide; VGCV: Valganciclovir

[0179] Example 4: Virological and immunological monitoring after T-cell therapy

[0180] To assess the impact of adoptive T-cell therapy on CMV-specific T-cell immune reconstitution, longitudinal intracellular cytokine analysis was performed after immunotherapy, and virological surveillance was conducted in each patient. Briefly, to characterize T-cell therapy and PBMCs isolated from follow-up blood samples, cells were stimulated with CMV peptide epitopes, and the expression of IFN-γ, TNF, and IL-2, as well as CD107 mobilization, was assessed using intracellular cytokine assays, as previously described (Smith C et al., Oncoimmunology 2017; 6(2):e1273311). Cells were acquired using a BD LSR Fortessa equipped with FACSDiva software (BD Biosciences). Following collection, Boolean analysis was performed using FlowJo software (FlowJo LLC, Ashland, OR).

[0181] result

[0182] Figure 2 Representative data from four SOT patients who demonstrated an objective response to adoptive immunotherapy are presented. Shaded boxes represent pretreatment during the analysis, and arrows represent each infusion of autologous, in vitro expanded CMV-specific T cells. This analysis reveals evidence of post-treatment immune reconstitution associated with viremia control. This is best illustrated in patient 1553PAH08, whose proportion of IFN-γ-producing CMV-specific T cells increased from 0.03% before the first infusion to 9.3% at the end of follow-up, while viral load decreased and antiviral therapy was discontinued. Figure 2A). Similar improvements in peripheral blood T-cell immunity after the infusion of T cells were also evident in other patients, including those with 1553PAH09, 1553PCH02, and 1553PCH04. Figure 2 A). Despite continued immunosuppressive therapy prior to adoptive T-cell therapy, immune reconstitution was observed in these patients (Table 2). Along with immune reconstitution, an improved functional quality of CMV-specific T-cell responses was also observed, characterized by an increased proportion of T cells co-expressing IFN-γ, TNF, and CD107. Figure 2 B). Conversely, patient 1553RAH01, who did not clinically respond to therapy, showed no evidence of immune reconstitution after treatment (data not shown). Patient 1553PCH03 was unable to undergo follow-up immunological analysis and died early after therapy initiation due to CMV infection-related complications. Although patients 1553PAH06 and 1553PCH05 showed clinical improvement, the frequency of peripheral blood CMV-specific T cells did not change after adoptive T-cell therapy (data not shown).

[0183] Example 5: Multicolor analysis of T cell phenotypes

[0184] To characterize the phenotype of adoptive T-cell therapy and reconstituted CMV-specific T cells, T cells obtained from each patient were incubated with allophycocyanin-labeled MHC class I multimers that are specific for HLA-A2-restricted epitopes NLV (pp65), HLA-A1-restricted epitopes VTE (pp65), HLA-B7-restricted epitopes TPR and RPH (pp65), or HLA-B8-restricted epitopes ELR and ELK (IE-1). To assess surface phenotype, cells were then further incubated at 4°C for 30 min with the following antibodies: anti-CD45RA FITC, anti-CD8 PerCP-Cy5.5, anti-CCR7 AF700, anti-CD95 BV421, anti-CD28 BV480, anti-CD57-Biotin, followed by SA-BV605, anti-CD27 PE, anti-CD19 PE-Cy5, anti-CD4 PE-Cy7, and live / dead NIRs; (cells were acquired using a BD LSR Fortessa (BD Biosciences) with FACSDiva software). Post-acquisition analyses were performed using FlowJo software (TreeStar) and t-distributed random neighbor embedding (tSNE) analysis to define changes in immunophenotype following therapy.

[0185] result

[0186] Figure 3The representative tSNE analysis of the upper group showed the expression of T cell phenotypic markers and CMV-specific T cells (VTE) in patients P1553PAH08 before and after treatment, and demonstrated increased CD57 expression. Figure 3 The data in the lower group represent the overlap of the percentage of CD8+ T cells expressing CD57 and the percentage of cells producing CMV-specific IFN-γ after T-cell therapy in three SOT recipients (P1553PAH08, 1553PCH02, and 1553PCH04) who responded to adoptive T-cell therapy and one SOT recipient (P1553RAH01) who did not show any clinical response.

[0187] Conclusion Summary

[0188] In contrast to the CMV-specific T cells generated in healthy CMV seropositive individuals, the administration of autologous CMV-specific immunotherapy to hematopoietic stem cell transplant (HSCT) recipients (Fuji et al. Current opinion in infectious diseases 2017; 30(4):372-6; Tzannou et al. J Clin Oncol 2017; 35(31):3547-57) depends on the ability to generate CMV-specific T cells from immunosuppressed individuals. However, as disclosed in this paper, CMV-specific T cells were successfully generated from 20 out of 21 patients. Despite a strict immunosuppressive protocol to prevent graft rejection, most patients were able to elicit a CMV-specific T cell response and, in some cases, had a high precursor frequency in their PBMCs prior to T cell expansion. According to a recent report (Snyder LD, Chan C, Kwon D et al., Polyfunctional T-Cell Signatures to Predict Protection from Cytomegalovirus after Lung Transplantation. Am J Respir Crit Care Med 2016; 193(1):78-85), functional defects were observed in CMV-specific T cells in the peripheral blood of SOT recipients; characterized by reduced ability to express TNF and IFN-γ. Importantly, this phenotype could be reversed after in vitro stimulation, in which most of the expanded CMV-specific T cells co-expressed CD107a, TNF, and IFN-γ.

[0189] Virological and immunological surveillance has provided evidence demonstrating the potential benefit of immune reconstitution following adoptive immunotherapy for viral control in SOT patients. Clear evidence in multiple patients indicates that immune reconstitution is consistent with a reduction or resolution of viral reactivation. This is particularly important for SOT recipients who have developed drug resistance, are experiencing CMV-related end-organ disease, or have a history of such disease. Furthermore, the adoptive T-cell therapy disclosed in this article can be safely used concurrently with immunosuppressive therapy to prevent CMV-related complications in patients who cannot tolerate standard antiviral therapy.

Claims

1. A library of immunogenic peptides comprising HLA class I and II restricted cytomegalovirus (CMV) peptide epitopes capable of inducing peptide-specific T cell proliferation, wherein the peptide library comprises each of the amino acid sequences of the epitopes listed in SEQ ID No: 1 to 31.

2. The library of immunogenic peptides according to claim 1, wherein the peptide library comprises at least one peptide epitope derived from each of the CMV antigens pp50, pp65, IE-1, gB, and gH.

3. The library of immunogenic peptides according to claim 1 or 2, wherein each epitope is restricted by HLA specificity selected from any of the following: HLA-A*01:01, -A*02:01, -A*23:01, -A*24:02, -B*07:02, -B*08:01, -B*18:01, -B*35:01, -B*35:08, -B*40:01, -B*40:02, -B*41.01, -B*44:02, -C*06:02, -C*07:02, -DRB1*01:01, -DRB1*03:01, -DRB1*04:01, -DRB1*07, or -DRB1*11:

01.

4. The library of immunogenic peptides according to any one of claims 1-3, wherein the immunogenic peptides are capable of inducing the proliferation of peptide-specific cytotoxic T cells (CTLs).

5. A method for producing a formulation that co-recognizes each epitope shown in SEQ ID NO: 1-31, comprising: a) Separate samples containing CTL; b) Exposing the sample to a library of immunogenic peptides according to any one of claims 1-4; and c) Collect CTLs.

6. The method of claim 5, wherein the sample containing CTLs comprises peripheral blood mononuclear cells (PBMCs) from a healthy donor.

7. The method of claim 5, wherein the sample containing CTL comprises PBMCs from a non-immunely responsive donor.

8. The method of claim 7, wherein the donor is undergoing immunosuppressive therapy.

9. The method according to any one of claims 7 or 8, wherein the donor is a solid organ transplant recipient.

10. The method according to any one of claims 7 or 8, wherein the donor is a donor receiving antiviral therapy.

11. The method according to any one of claims 5 to 8, wherein the exposed sample of step b) is incubated for at least 14 days.

12. The method of claim 11, further comprising incubating the sample exposed in step b) with IL-21 on day 0.

13. The method of claim 11, further comprising incubating the exposed sample from step b) with IL-2 on day 2.

14. The method of claim 13, further comprising adding IL-2 every three days.

15. CTL prepared by any one of claims 5 to 13.

16. Use of the CTL of claim 15 in the preparation of a medicament for treating or preventing CMV infection in a subject.

17. The use according to claim 16, wherein exposure to the library of immunogenic peptides induces stimulation and proliferation of CMV peptide-specific T cells.

18. The use according to claim 16, wherein the CTL administered to the subject is autologous.

19. The use according to claim 16, wherein the infection is a recurrent CMV infection.

20. The use according to any one of claims 16 or 19, wherein the CMV infection is drug-resistant.

21. The use according to claim 20, wherein the CMV infection is ganciclovir resistant.

22. The use according to any one of claims 16-19, wherein the subject is a recipient of a solid organ transplant.

23. The use according to any one of claims 16-19, wherein at least 5% of the CTLs express CD107a.

24. The use according to any one of claims 16-19, wherein at least 10% of the CTLs express CD107a.

25. The use according to any one of claims 16-19, wherein at least 20% of the CTLs express CD107a.

26. The use according to any one of claims 16-19, wherein at least 60% of the CTLs express CD107a.

27. The use according to any one of claims 16-19, wherein at least 90% of the CTLs express CD107a.

28. The use according to any one of claims 16-19, wherein at least 5% of the CTLs express IFNγ.

29. The use of claim 28, wherein at least 10% of the CTLs express IFNγ.

30. The use according to claim 29, wherein at least 20% of the CTLs express IFNγ.

31. The use according to claim 30, wherein at least 60% of the CTLs express IFNγ.

32. The use according to claim 31, wherein at least 90% of the CTLs express IFNγ.

33. The use according to any one of claims 16-19, wherein at least 5% of the CTLs express TNF.

34. The use according to claim 33, wherein at least 10% of the CTLs express TNF.

35. The use according to claim 34, wherein at least 20% of the CTLs express TNF.

36. The use according to claim 35, wherein at least 60% of the CTLs express TNF.

37. The use according to claim 36, wherein at least 90% of the CTLs express TNF.

38. The use according to any one of claims 16-19, wherein at least 1% of the CTLs express IL-2.

39. The use according to claim 38, wherein at least 5% of the CTLs express IL-2.

40. The use according to claim 39, wherein at least 10% of the CTLs express IL-2.

41. The use according to claim 40, wherein at least 20% of the CTLs express IL-2.

42. The use according to any one of claims 16-19, wherein at least 20% of the CTLs express CD107a, IFNγ and TNF.

43. The use according to claim 42, wherein at least 43% of the CTLs express CD107a, IFNγ and TNF.

44. The use according to claim 43, wherein at least 55% of the CTLs express CD107a, IFNγ and TNF.

45. The use according to claim 44, wherein at least 90% of the CTLs express CD107a, IFNγ, and TNFα.

46. ​​The use according to any one of claims 15-19, wherein the CTL exhibits responsiveness to multiple peptide epitopes derived from multiple CMV antigens.

47. The use according to claim 46, wherein the CTL has at least 11% CMV reactivity to more than one CMV epitope.

48. The use according to claim 47, wherein the CTL has at least 43% CMV reactivity to more than one CMV epitope.

49. The use according to claim 48, wherein the CTL has at least 48% CMV reactivity to more than one CMV epitope.

50. The use according to claim 49, wherein the CTL has at least 66% CMV reactivity to more than one CMV epitope.

51. The use according to claim 50, wherein the CTL has at least 77% CMV reactivity to more than one CMV epitope.

52. The use according to claim 51, wherein the CTL has at least 79% CMV reactivity to more than one CMV epitope.

53. The use according to claim 46, wherein the CTL is reactive to any CMV peptide epitope amino acid sequence or combination thereof listed in Table 1.

54. The use according to any one of claims 40-41 and 46, wherein the CTL is reactive to any one or a combination of pp50, pp65, IE-1, gB, gH.

55. Use of the CTL of claim 15 in the preparation of a medicament for treating or preventing CMV reactivation or CMV-related conditions in a subject.

56. The use according to claim 55, wherein the CTL is self.

57. The use according to any one of claims 55 or 56, wherein the use further comprises analyzing the expression of multiple biomarkers of a CMV peptide-specific CTL, and administering the CTL to the subject if the CTL expresses at least two biomarkers.

58. The use according to claim 57, wherein the expression of CD107a, TNF, and IFNγ of the CMV peptide-specific CTL is analyzed, and if at least 10% of the CTLs express CD107a, TNF, and IFNγ, the peptide-specific CTL is administered to the subject.

59. The use according to claim 58, wherein the CTL is applied if at least 20% of the CMV peptide-specific CTLs in the sample express CD107a, TNF, and IFNγ.

60. The use of claim 58, wherein the CTL is administered if at least 60% of the proliferating peptide-specific CTLs in the sample express CD107a, TNF, and IFNγ.

61. The use according to claim 58, wherein the CTL is administered if at least 90% of the proliferating peptide-specific CTLs in the sample express CD107a, TNF, and IFNγ.

62. The use according to any one of claims 55 or 56, wherein the use further comprises analyzing the CMV reactivity of the CMV peptide-specific CTL, and administering the peptide-specific CTL to the subject if the reactivity is for more than one peptide epitope and is above a predetermined threshold.

63. The use according to claim 62, wherein the threshold is 11%.

64. The use according to claim 62, wherein the threshold is 43%.

65. The use according to claim 62, wherein the threshold is 48%.

66. The use according to claim 62, wherein the threshold is 66%.

67. The use according to claim 62, wherein the threshold is 77%.

68. The use according to claim 62, wherein the threshold is 79%.

69. The use according to any one of claims 55 or 56, wherein in step (a) the sample is incubated with one or more cytokines.

70. The use according to any one of claims 55 or 56, wherein the sample comprises PBMC.

71. The use according to claim 70, wherein the subject is without an immune response.

72. The use according to claim 70, wherein the subject is undergoing immunosuppressive therapy.

73. The use according to claim 70, wherein the subject is a solid organ transplant recipient.

74. The use according to claim 70, wherein the subject is receiving antiviral therapy.

75. The use according to any one of claims 16 to 19, said use comprising administering to a subject about 1 × 10⁻⁶ of a dose. 7 CTL.

76. The use according to any one of claims 16 to 19, said use comprising administering to a subject about 1.5 × 10⁻⁶ of a dose. 7 CTL.

77. The use according to any one of claims 16 to 19, said use comprising administering to a subject about 2 × 10⁻⁶ of a dose. 7 CTL.

78. The use according to claim 75, wherein the dose is administered once every two weeks.

79. The use according to any one of claims 16 to 19, wherein the subject does not experience significant adverse effects due to CTL administration.

80. The use according to any one of claims 16 to 19, wherein the use further comprises evaluating the efficacy of adoptive T-cell therapy by measuring the CMV viral load in the subject.

81. The use according to claim 80, wherein the CMV viral load is reduced by approximately 82% after CTL administration.

82. The use according to claim 80, wherein the CMV viral load is reduced by approximately 95% after CTL administration.

83. The use according to claim 80, wherein the CMV viral load is reduced by approximately 100% after CTL administration.

84. The use according to any one of claims 16 to 19, wherein the clinical symptoms of the subject are improved.

85. The use according to claim 84, wherein the subject experienced a reduction or resolution of DNA emia.

86. The use according to claim 84, wherein the subject experienced a reduction or cessation of CMV-related end-organ disease.

87. The use according to claim 84, wherein the subject experienced cessation or reduction of antiviral therapy.

88. Use of the CTL of claim 15 in the preparation of a medicament for reducing CMV viral load in subjects who have received solid organ transplants.

89. Use of the CTL of claim 15 in the preparation of a medicament for the treatment or prevention of CMV-related end-organ disease in subjects who have received solid organ transplants.

90. Use of the CTL of claim 15 in the preparation of a medicament for reducing or eliminating the need for anti-CMV therapy in subjects who have received solid organ transplants.

91. Use of the CTL of claim 15 in the preparation of a medicament for treating drug-resistant CMV infection, reactivation, or related diseases.

92. The use according to any one of claims 88 to 91, wherein the subject suffers from ganciclovir-resistant CMV infection, reactivation, or related disease.

93. The use according to any one of claims 88 to 91, wherein the CTL is an autologous CTL.

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