EBV status as a predictor of response to immunotherapy

Abstract

Provided herein are methods related to detection of Epstein-Barr virus (EBV) status as a predictor of human leukocyte antigen (HLA)-I loss-of-heterozygosity (LOH) and / or responses to treatment comprising an immunotherapy.

Description

EBV STATUS AS A PREDICTOR OF RESPONSE TO IMMUNOTHERAPYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 528,227, filed July 21, 2023, which is hereby incorporated by reference in its entirety.FIELD

[0002] Provided herein are methods related to detection of Epstein-Barr virus (EBV) status as a predictor of human leukocyte antigen (HLA)-I loss-of-heterozygosity (LOH) and / or responses to treatment comprising an immunotherapy.BACKGROUND

[0003] Immunotherapies have revolutionized current treatments for advanced cancer patients. Some (e.g., cell-based therapies) provide or stimulate an immune response to the cancer, while others (e.g., immune checkpoint inhibitors or ICIs) are thought to reinvigorate the patient’s own T-cell mediated immune response (Reck, M., et al. N Engl J Med 375, 1823-1833 (2016); Hellmann, M.D., et al. N Engl J Med M , 2093-2104 (2018); Nghiem, P.T., et al. NEnglJMed 374, 2542-2552 (2016); Robert, C„ et al. N Engl J MedMZ, 2521-2532 (2015); Le, D.T., et al. N Engl J Med 372, 2509-2520 (2015)). However, responses to immunotherapies such as ICI treatment have been found to be variable among different patients.

[0004] Patients with oncoviral infections are predicted to be good candidates for immunotherapy since viral antigen presentation is believed to be a mechanism akin to tumor neoantigen presentation. Patients who are tumor mutational burden (TMB) low are often not considered good candidates for immune checkpoint inhibitor (ICI) therapy. However, virus-positive patients, who are often TMB low, may be a subset of TMB low patients who could be good ICI responders. Previous research indicated that patients who were virus-positive tended to have higher rates of HLA-I LOH than patients who were virus-negative, suggesting that viral antigen presentation is highly immunogenic and that HLA-I LOH was a mechanism of immune evasion in these patients (Montesion, M. et al. (2021) Cancer Discov. 11(2):282-292).

[0005] Epstein-Barr virus (EBV) is a ubiquitous pathogen that, despite resulting largely in asymptomatic infections, can be linked to the development of certain cancers such as lymphoma, nasopharyngeal carcinoma, and gastric carcinoma. Although some studies suggest that patients with oncoviral infections exhibit superior responses to immunotherapy, information is lacking regarding whether response differs based on viral subtype.

[0006] Thus, there is a need for predicting which patients exhibit HLA-I LOH and / or would likely show better survival in response to treatment with immunotherapeutic approaches, such as treatment with an ICI.

[0007] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.SUMMARY

[0008] In one aspect, provided herein are methods of treating or delaying progression of cancer in an individual, comprising: acquiring knowledge of at least Epstein-Barr virus (EBV) subtype 2-positive (EBV-2+) status in a sample from the individual; and administering to the individual an effective amount of an immunotherapy responsive to said knowledge.

[0009] In another aspect, provided herein are methods of treating or delaying progression of cancer in an individual, comprising: detecting EBV-2+ status in a sample from the individual; and administering to the individual an effective amount of an immunotherapy.

[0010] In another aspect, provided herein are methods of predicting survival of an individual having cancer, comprising acquiring knowledge of EBV-2+ status in a sample from the individual, wherein responsive to said knowledge, the individual is predicted to have a higher likelihood of survival when treated with an immunotherapy as compared to an individual with EBV-negative (EBV-) status or EBV subtype 1-positive (EBV-1+) and EBV-2 negative (EBV-2- ) status.

[0011] In another aspect, provided herein are methods of predicting response to immunotherapy of an individual having cancer, comprising acquiring knowledge of EBV-2+ status in a sample from the individual, wherein responsive to said knowledge, the individual is predicted to have a better response to treatment when treated with an immunotherapy as compared to response to treatment of an individual with EBV-negative (EBV-) status or EBV subtype 1 -positive (EBV- 1+) and EBV-2 negative (EBV-2-) status.

[0012] In another aspect, provided herein are methods of identifying one or more treatment options for an individual having cancer, comprising detecting EBV-2+ status in a sample from the individual; and generating a report comprising one or more treatment options identified for the individual based at least in part on the detection of EBV-2+ status in the sample, wherein the one or more treatment options comprise an immunotherapy.

[0013] In another aspect, provided herein are methods of predicting human leukocyte antigen class I (HLA-I) loss-of-heterozygosity (LOH) of a cancer of an individual, comprising acquiring knowledge of EBV-2+ status in a sample from the individual, wherein responsive to said knowledge, the cancer of the individual is predicted to have a higher likelihood of exhibiting HLA-I LOH as compared to a cancer of an individual with EBV-negative (EBV-) status or EBV subtype 1-positive (EBV-1+) and EBV-2 negative (EBV-2-) status.

[0014] In some embodiments according to any of the embodiments described herein, the methods further comprise acquiring knowledge of or detecting EBV-1+ status in a sample from the individual. In some embodiments, the method comprises acquiring knowledge of or detecting EBV subtype 1 and EBV subtype 2-positive (EBV- 1 / 2+) status in a sample from the individual. In some embodiments, the cancer exhibits HLA-I LOH. In some embodiments, the methods further comprise acquiring knowledge of or detecting HLA-I LOH in a sample obtained from the cancer of the individual. In some embodiments, the methods further comprise acquiring knowledge of or detecting tumor mutational burden (TMB) in a sample obtained from the cancer of the individual. In some embodiments, the cancer is a TMB low cancer. In some embodiments, the cancer has a TMB of less than 10 mutations per megabase (muts / Mb). In some embodiments, the cancer is gastric or nasopharyngeal cancer. In some embodiments, the cancer is advanced or metastatic.

[0015] In some embodiments according to any of the embodiments described herein, the immunotherapy is administered as a monotherapy. In some embodiments, the immunotherapy is administered in combination with one or more additional anti-cancer therapies or treatments. In some embodiments, the immunotherapy comprises one or more of a checkpoint inhibitor, cancer vaccine, cell-based therapy, T cell receptor (TCR)-based therapy, adjuvant immunotherapy, cytokine immunotherapy, and oncolytic virus therapy. In some embodiments, the immunotherapy comprises a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor targets PD-L1, PD-1, CTLA-4, CEACAM, LAIR1, CD160, 2B4, CD80, CD86, CD276,VTCN1, HVEM, KIR, A2AR, MHC class I, MHC class II, GALS, adenosine, TGFR, 0X40, CD137, CD40, IDO, CSF1R, TIM-3, BTLA, VISTA, LAG-3, TIGIT, IDO, MICA / B, or arginase. In some embodiments, the checkpoint inhibitor is an agent that inhibits PD-1. In some embodiments, the agent that inhibits PD-1 is a small molecule, a nucleic acid, a polypeptide, carbohydrate, a lipid, a metal, or a toxin. In some embodiments, the agent that inhibits PD-1 is a PD-1 binding antagonist. In some embodiments, the PD-1 binding antagonist is an antibody, antibody-drug conjugate, antibody fragment, or immunoadhesin. In some embodiments, the PD- 1 binding antagonist is pembrolizumab, nivolumab, cemiplimab, spartalizumab, genolimzumab, SHR1210, JS001, BGB-108, BGB-A317, IBI308, GLS-010, BMS-936558, BCD-100, REGN2810, MGA-012, BI 754091, STI-Al l 10, INCSHR-1210, PF-06801591, TSR-042, AM0001, JNJ-63723283, or ENUM 244C8. In some embodiments, the checkpoint inhibitor is an agent that inhibits PD-L1 and / or PD-L2. In some embodiments, the agent that inhibits PD-L1 and / or PD-L2 is a small molecule, a nucleic acid, a polypeptide, carbohydrate, a lipid, a metal, or a toxin. In some embodiments, the agent that inhibits PD-L1 is a PD-L1 binding antagonist. In some embodiments, the PD-L1 binding antagonist is an antibody, antibody-drug conjugate, antibody fragment, or immunoadhesin. In some embodiments, the PD-L1 binding antagonist is atezolizumab, avelumab, durvalumab, KN035, CS1001, MDX-1105, LY3300054, STLA1014, FAZ053, or CX-072. In some embodiments, the checkpoint inhibitor is an agent that inhibits CTLA4. In some embodiments, the agent that inhibits CTLA4 is a small molecule, a nucleic acid, a polypeptide, carbohydrate, a lipid, a metal, or a toxin. In some embodiments, the agent that inhibits CTLA4 is an antibody, antibody-drug conjugate, antibody fragment, or immunoadhesin. In some embodiments, the agent that inhibits CTLA4 is ipilimumab, APL-509, AGEN1884, or CS 1002.

[0016] In some embodiments according to any of the embodiments described herein, the sample comprises a tissue biopsy sample or a liquid biopsy sample. In some embodiments, the sample is a tissue biopsy and comprises a tumor biopsy, tumor specimen, or circulating tumor cells. In some embodiments, the sample is a liquid biopsy sample and comprises blood, serum, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some embodiments, the sample comprises cells and / or nucleic acids from the cancer. In some embodiments, the sample comprises mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, cell-free RNA from the cancer, or any combination thereof. In some embodiments, the sample is a liquid biopsy sample and comprisescirculating tumor cells (CTCs). In some embodiments, the sample is a liquid biopsy sample and comprises cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof. In some embodiments, the methods further comprise obtaining the sample from the individual. In some embodiments, EBV-2+ status is detected in the sample by sequencing nucleic acids encoding EBV-2 nuclear antigens from the sample. In some embodiments, EBV- 2+ status is detected in the sample by sequencing nucleic acids encoding an EBV-2 Epstein-Barr nuclear antigen 3 (EBNA-3) gene from the sample. In some embodiments, the sequencing comprises a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique; and optionally wherein the massively parallel sequencing (MPS) technique comprises next-generation sequencing (NGS). In some embodiments, the sample is an enriched sample. In some embodiments, the sequencing comprises: (a) providing a plurality of nucleic acid molecules obtained from the sample, wherein the plurality of nucleic acid molecules comprises a mixture of tumor nucleic acid molecules and non-tumor nucleic acid molecules; (b) optionally, ligating one or more adapters onto one or more nucleic acid molecules from the plurality of nucleic acid molecules; (c) amplifying nucleic acid molecules from the plurality of nucleic acid molecules; (d) optionally, capturing nucleic acid molecules from the amplified nucleic acid molecules, wherein the captured nucleic acid molecules are captured from the amplified nucleic acid molecules by hybridization to one or more bait molecules; and (e) sequencing, by a sequencer, the captured nucleic acid molecules to obtain a plurality of sequence reads corresponding to EBV-2 nucleic acids in the sample. In some embodiments, the adapters comprise one or more of amplification primer sequences, flow cell adapter hybridization sequences, unique molecular identifier sequences, substrate adapter sequences, or sample index sequences. In some embodiments, amplifying nucleic acid molecules comprises performing a polymerase chain reaction (PCR) technique, a non-PCR amplification technique, or an isothermal amplification technique. In some embodiments, the one or more bait molecules comprise one or more nucleic acid molecules, each comprising a region that is complementary to a region of a captured nucleic acid molecule. In some embodiments, the one or more bait molecules each comprise a capture moiety. In some embodiments, the capture moiety is biotin.

[0017] In some embodiments according to any of the embodiments described herein, the individual is a human.

[0018] In another aspect, provided herein is an immunotherapy (e.g., an effective amount of an immunotherapy) for treating or delaying progression of cancer in an individual according to any of the methods of the present disclosure, wherein EBV-2+ status has been detected in a sample from the individual.

[0019] In another aspect, provided herein is the use of an immunotherapy (e.g., an effective amount of an immunotherapy) in the manufacture of a medicament for treating or delaying progression of cancer in an individual, wherein EBV-2+ status has been detected in a sample from the individual.

[0020] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art. These and other embodiments of the invention are further described by the detailed description that follows.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Various aspects of the disclosed methods, devices, and systems are set forth with particularity in the appended claims. A better understanding of the features and advantages of the disclosed methods, devices, and systems will be obtained by reference to the following detailed description of illustrative embodiments and the accompanying drawings, of which:

[0022] FIGS. 1A & IB show overall survival in TMB low (<10 muts / Mb) advanced gastric cancer patients treated with ICI monotherapy (any line) in EBV+ vs. EBV- patients. FIG. 1A shows survival probability, and FIG. IB shows hazard ratio.

[0023] FIGS. 2A & 2B show overall survival in TMB low (<10 muts / Mb) advanced gastric cancer patients treated with ICI monotherapy (any line) in EBV-1+ patients, EBV-1+ / EBV-2+ (EBV-1 / 2+) patients, or EBV- patients. FIG. 2A shows survival probability, and FIG. 2B shows hazard ratio.DETAILED DESCRIPTION

[0024] The present disclosure relates generally to detection of Epstein-Barr virus (EBV) status as a predictor of human leukocyte antigen (HLA)-I loss-of-heterozygosity (LOH) and / or responses to treatment comprising an immunotherapy.

[0025] As noted above, it is still difficult to predict whether some patients, particularly those with oncoviral infections, would be good candidates for immunotherapy. While viral antigen presentation is believed to be a mechanism akin to tumor neoantigen presentation, virus-positive patients are also often TMB low, which can be a negative predictor for response to immunotherapy. In addition, whether different viral subtypes may have different correlations with response to treatment has not been well explored. In previous studies (see, e.g., Montesion, M. et al. (2021) Cancer Discov. 11(2):282-292), HLA-I LOH was found to be prevalent in tumor types with known oncoviral associations, including HPV+ head and neck squamous cell carcinoma (HNSCC), HPV+ cervical cancer, EBV+ gastric cancer, EBV+ nasopharyngeal cancer, and HBV+ hepatocellular cancer, but not in other tumor types such as EBV+ nasopharyngeal cancer.

[0026] The present disclosure is based at least in part on the discovery demonstrated herein that the rate of HLA-I LOH was significantly higher in EBV1 / 2+ patients as compared to EBV- negative (EBV-) patients in nasopharyngeal and gastric carcinoma, whereas the rate of HLA-I LOH in EBV1+ patients within either tumor type was not. In addition, when examining overall survival (OS) in patients treated with ICI monotherapy, patients who were EBV1 / 2+ had significantly longer OS as compared to patients who were EBV-, whereas patients who were EBV1+ had no improvement in OS as compared to patients who were EBV-. As such, EBV infection status (particularly EBV2+ status) can be used as a predictor of HLA-I LOH and higher survival in response to immunotherapy treatment.I. General Techniques

[0027] The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press;Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRE Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993).II. Definitions

[0028] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and / or” unless otherwise stated.

[0029] The terms “about” and “approximately” as used herein refer to the usual error range for the respective value readily known to the skilled person in this technical field. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values. Reference to “about” or “approximately” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0030] It is understood that aspects and embodiments of the invention described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0031] The terms “cancer” and “tumor” are used interchangeably herein. These terms refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certaincharacteristic morphological features. Cancer cells are often in the form of a tumor, but such cells can exist alone within an animal, or can be a non-tumorigenic cancer cell, such as a leukemia cell. These terms include a solid tumor, a soft tissue tumor, or a metastatic lesion. As used herein, the term “cancer” includes premalignant, as well as malignant cancers.

[0032] “Polynucleotide,” “nucleic acid,” or “nucleic acid molecule” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. Thus, for instance, polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be singlestranded or, more typically, double-stranded or include single- and double-stranded regions. In addition, the term “polynucleotide” as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. The term “polynucleotide” specifically includes cDNAs.

[0033] A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by nonnucleotide components. A polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, “caps,” substitution of one or more of the naturally-occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, and the like) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, and the like), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, and the like), those with intercalators (e.g., acridine, psoralen, and the like), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, and the like), thosecontaining alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-0-methyl-, 2'-0-allyl-, 2'-fluoro-, or 2'- azido-ribose, carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S ("thioate"), P(S)S ("dithioate"), "(0)NR2 ("amidate"), P(0)R, P(0)OR', CO or CH2 ("formacetal"), in which each R or R' is independently H or substituted or unsubstituted alkyl (1 -20 C) optionally containing an ether (- 0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. A polynucleotide can contain one or more different types of modifications as described herein and / or multiple modifications of the same type. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0034] “Oligonucleotide,” as used herein, generally refers to short, single stranded, polynucleotides that are, but not necessarily, less than about 250 nucleotides in length. Oligonucleotides may be synthetic. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.

[0035] The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0036] An “isolated” antibody is one which has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its naturalenvironment are materials which would interfere with research, diagnostic, and / or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, an antibody is purified (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of, for example, a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using, for example, Coomassie blue or silver stain. An isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0037] “Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.

[0038] The “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa (“K”) and lambda (“X”), based on the amino acid sequences of their constant domains.

[0039] The term “constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain contains the CHI, CH2, and CH3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.

[0040] The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain maybe referred to as “VH.” The variable domain of the light chain may be referred to as “VL.” These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[0041] The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs) both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991 )). The constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibodydependent cellular toxicity.

[0042] The term “hypervariable region,” “HVR,” or “HV,” as used herein, refers to the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). In native antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248: 1 -25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1 993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0043] A number of HVR delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md. (1 991 )). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901 -917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The“contact” HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below.Loop Kabat AbM Chothia ContactLI L24-L34 L24-L34 L26-L32 L30-L36L2 L50-L56 L50-L56 L50-L52 L46-L55L3 L89-L97 L89-L97 L91-L96 L89-L96Hl H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering)Hl H31-H35 H26-H35 H26-H32 H30-H35 (Chothia numbering)H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

[0044] HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (Hl), 50-65 or 49-65 (H2) and 93-102, 94- 102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[0045] “Framework” or “FR” residues are those variable domain residues other than the HVR residues as herein defined.

[0046] The term “variable domain residue numbering as in Kabat” or “amino acid position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.

[0047] The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1 -107 of the light chain and residues 1 -1 13 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991 )). The “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The “EU index as in Kabat” refers to the residue numbering of the human IgGl EU antibody.

[0048] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain an Fc region.

[0049] “Antibody fragments” comprise a portion of an intact antibody comprising the antigenbinding region thereof. In some embodiments, the antibody fragment described herein is an antigen-binding fragment. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0050] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that a selected target-binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target-binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target-binding sequence is also a monoclonal antibody of this invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a singledeterminant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

[0051] The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature 256:495-97 (1975); Hongo et al., Hybridoma 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981 )), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g., Clackson et al., Nature, 352: 624-628 (1991 ); Marks et al., J. Mol. Biol. 222: 581 -597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-31 0 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101 (34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1 -2): 1 1 9-132 (2004)), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741 ; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1 993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg et al., Intern. Rev. Immunol. 13: 65- 93 (1995)).

[0052] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non- human antigen-binding residues.

[0053] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human framework regions (FRs). Incertain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.

[0054] A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0055] A “blocking” antibody or an “antagonist” antibody is one which inhibits or reduces biological activity of the antigen it binds. For example, blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0056] As used herein, the term “binds”, “specifically binds to” or is “specific for” refers to measurable and reproducible interactions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antibody that binds to or specifically binds to a target (which can be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and / or with greater duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among the protein from different species. In another embodiment, specific binding can include, but does not require exclusive binding.

[0057] The terms “homology” or “identity,” as used herein, refer to sequence similarity between two polynucleotide sequences or between two polypeptide sequences. The phrases “percent identity or homology” and “% identity or homology” refer to the percentage of sequence similarity found in a comparison of two or more polynucleotide sequences or two or more polypeptide sequences. Identity or similarity can be determined by comparing a position in each sequence that can be aligned for purposes of comparison. When a position in the compared sequences is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position.

[0058] The term “detection” includes any means of detecting, including direct and indirect detection. The term “biomarker” as used herein (e.g., a “biomarker” such as an EBV nucleic acid molecule or EBV infection status described herein) refers to an indicator, e.g., predictive, diagnostic, and / or prognostic, which can be detected in a sample. The biomarker may serve as an indicator of a particular subtype of a disease or disorder (e.g., cancer) characterized by certain, molecular, pathological, histological, and / or clinical features (e.g., responsiveness to therapy).

[0059] “Amplification,” as used herein generally refers to the process of producing multiple copies of a desired sequence. “Multiple copies” mean at least two copies. A “copy” does not necessarily mean perfect sequence complementarity or identity to the template sequence. For example, copies can include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced through a primer comprising a sequence that is hybridizable, but not complementary, to the template), and / or sequence errors that occur during amplification.

[0060] The technique of “polymerase chain reaction” or “PCR” as used herein generally refers to a procedure wherein minute amounts of a specific piece of nucleic acid, RNA and / or DNA, are amplified as described, for example, in U.S. Pat. No. 4,683,195. Generally, sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage, or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51 :263 (1987) and Erlich, ed., PCR Technology (Stockton Press, NY, 1989). As used herein, PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, comprising the use of a known nucleic acid (DNA or RNA) as a primer and utilizes a nucleic acid polymerase to amplify or generate a specific piece of nucleic acid or to amplify or generate a specific piece of nucleic acid which is complementary to a particular nucleic acid.

[0061] The term “diagnosis” is used herein to refer to the identification or classification of a molecular or pathological state, disease or condition (e.g., cancer). For example, “diagnosis” may refer to identification of a particular type of cancer. “Diagnosis” may also refer to theclassification of a particular subtype of cancer, for instance, by histopathological criteria, or by molecular features (e.g., a subtype characterized by expression of one or a combination of biomarkers (e.g., particular genes or proteins encoded by said genes)).

[0062] The term “aiding diagnosis” is used herein to refer to methods that assist in making a clinical determination regarding the presence, or nature, of a particular type of symptom or condition of a disease or disorder (e.g., cancer). For example, a method of aiding diagnosis of a disease or condition (e.g., cancer) can comprise measuring certain somatic mutations in a biological sample from an individual.

[0063] The term “sample,” as used herein, refers to a composition that is obtained or derived from a subject and / or individual of interest that contains a cellular and / or other molecular entity that is to be characterized and / or identified, for example, based on physical, biochemical, chemical, and / or physiological characteristics. For example, the phrase “disease sample” and variations thereof refers to any sample obtained from a subject of interest that would be expected or is known to contain the cellular and / or molecular entity that is to be characterized. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, plasma, serum, blood-derived cells, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, tumor tissue, cellular extracts, and combinations thereof. In some instances, the sample is a whole blood sample, a plasma sample, a serum sample, or a combination thereof. In some embodiments, the sample is from a tumor e.g., a “tumor sample”), such as from a biopsy. In some embodiments, the sample is a formalin-fixed paraffin-embedded (FFPE) sample.

[0064] A “tumor cell” as used herein, refers to any tumor cell present in a tumor or a sample thereof. Tumor cells may be distinguished from other cells that may be present in a tumor sample, for example, stromal cells and tumor-infiltrating immune cells, using methods known in the art and / or described herein.

[0065] A “reference sample,” “reference cell,” “reference tissue,” “control sample,” “control cell,” or “control tissue,” as used herein, refer to a sample, cell, tissue, standard, or level that is used for comparison purposes.

[0066] By “correlate” or “correlating” is meant comparing, in any way, the performance and / or results of a first analysis or protocol with the performance and / or results of a second analysis or protocol. For example, one may use the results of a first analysis or protocol in carrying out a second protocol and / or one may use the results of a first analysis or protocol to determine whether a second analysis or protocol should be performed. With respect to the embodiment of polypeptide analysis or protocol, one may use the results of the polypeptide expression analysis or protocol to determine whether a specific therapeutic regimen should be performed. With respect to the embodiment of polynucleotide analysis or protocol, one may use the results of the polynucleotide expression analysis or protocol to determine whether a specific therapeutic regimen should be performed.

[0067] “Individual response” or “response” can be assessed using any endpoint indicating a benefit to the individual, including, without limitation, (1) inhibition, to some extent, of disease progression (e.g., cancer progression), including slowing down or complete arrest; (2) a reduction in tumor size; (3) inhibition (i.e., reduction, slowing down, or complete stopping) of cancer cell infiltration into adjacent peripheral organs and / or tissues; (4) inhibition (i.e. reduction, slowing down, or complete stopping) of metastasis; (5) relief, to some extent, of one or more symptoms associated with the disease or disorder (e.g., cancer); (6) increase or extension in the length of survival, including overall survival and progression free survival; and / or (7) decreased mortality at a given point of time following treatment.

[0068] An “effective response” of a patient or a patient's “responsiveness” to treatment with a medicament and similar wording refers to the clinical or therapeutic benefit imparted to a patient at risk for, or suffering from, a disease or disorder, such as cancer. In one embodiment, such benefit includes any one or more of: extending survival (including overall survival and / or progression-free survival); resulting in an objective response (including a complete response or a partial response); or improving signs or symptoms of cancer.

[0069] An “effective amount” refers to an amount of a therapeutic agent to treat or prevent a disease or disorder in a mammal. In the case of cancers, the therapeutically effective amount of the therapeutic agent may reduce the number of cancer cells; reduce the primary tumor size; inhibit (i.e., slow to some extent and in some embodiments stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and in some embodiments stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more ofthe symptoms associated with the disorder. To the extent the drug may prevent growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. For cancer therapy, efficacy in vivo can, for example, be measured by assessing the duration of survival, time to disease progression (TTP), response rates (e.g., CR and PR), duration of response, and / or quality of life.

[0070] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0071] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0072] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention (e.g., administration of an anti-cancer agent or anticancer therapy) in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0073] As used herein, the terms “individual,” “patient,” or “subject” are used interchangeably and refer to any single animal, e.g., a mammal (including such non-human animals as, for example, dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and non-human primates) for which treatment is desired. In particular embodiments, the patient herein is a human.

[0074] As used herein, “administering” is meant a method of giving a dosage of an agent or a pharmaceutical composition (e.g., a pharmaceutical composition including the agent) to a subject (e.g., a patient). Administering can be by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single ormultiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0075] The term “concurrently” is used herein to refer to administration of two or more therapeutic agents, where at least part of the administration overlaps in time. Accordingly, concurrent administration includes a dosing regimen when the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s).

[0076] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings concerning the use of such therapeutic products.

[0077] An “article of manufacture” is any manufacture (e.g., a package or container) or kit comprising at least one reagent, e.g., a medicament for treatment of a disease or disorder (e.g., cancer), or a reagent for specifically detecting a biomarker (e.g., an EBV nucleic acid molecule or EBV infection status described herein) described herein. In certain embodiments, the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0078] The phrase “based on”, “responsive to”, and the like, when used herein mean that the information about one or more biomarkers (e.g., an EBV nucleic acid molecule or EBV infection status described herein) is used to inform a treatment decision, information provided on a package insert, or marketing / promotional guidance, etc.

[0079] The terms “allele frequency” and “allele fraction” are used interchangeably herein and refer to the fraction of sequence reads corresponding to a particular allele relative to the total number of sequence reads for a genomic locus. The terms “variant allele frequency” and “variant allele fraction” are used interchangeably herein and refer to the fraction of sequence reads corresponding to a particular variant allele relative to the total number of sequence reads for a genomic locus.III. Methods, Systems, and Devices

[0080] In some aspects, provided herein are methods for treating or delaying progression of cancer in an individual. In some embodiments, the methods comprise acquiring knowledge of at least Epstein-Barr virus (EBV) subtype 2-positive (EBV-2+) status in a sample from theindividual; and administering to the individual an effective amount of an immunotherapy, e.g., responsive to said knowledge. In some embodiments, the methods comprise detecting EBV-2+ status in a sample from the individual; and administering to the individual an effective amount of an immunotherapy, e.g., based at least in part on the detection of EBV-2+ status in the sample.

[0081] In other aspects, provided herein are methods of predicting survival of an individual having cancer, comprising acquiring knowledge of EBV-2+ status in a sample from the individual, wherein responsive to said knowledge, the individual is predicted to have a higher likelihood of survival when treated with an immunotherapy as compared to an individual with EBV-negative (EBV-) status or EBV subtype 1-positive (EBV-1+) and EBV-2 negative (EBV-2- ) status. In some embodiments, the survival is overall survival (OS). In some embodiments, the survival is progression free survival (PFS).

[0082] In other aspects, provided herein are methods of predicting response to immunotherapy of an individual having cancer, comprising acquiring knowledge of EBV-2+ status in a sample from the individual, wherein responsive to said knowledge, the individual is predicted to have a better response to treatment when treated with an immunotherapy as compared to response to treatment of an individual with EBV-negative (EBV-) status or EBV subtype 1 -positive (EBV- 1+) and EBV-2 negative (EBV-2-) status. In some embodiments, the response is objective response (including a complete response or a partial response), i.e., the individual is predicted to have an increased likelihood of a complete or partial response to treatment when treated with an immunotherapy as compared to likelihood of a complete or partial response to treatment of an individual with EBV-negative (EBV-) status or EBV subtype 1-positive (EBV-1+) and EBV-2 negative (EBV-2-) status. In some embodiments, the response refers to duration of response, e.g., the individual is predicted to have a longer response to treatment when treated with an immunotherapy as compared to duration of response to treatment of an individual with EBV- negative (EBV-) status or EBV subtype 1-positive (EBV-1+) and EBV-2 negative (EBV-2-) status.

[0083] In other aspects, provided herein are methods of identifying one or more treatment options for an individual having cancer, comprising: detecting EBV-2+ status in a sample from the individual; and generating a report comprising one or more treatment options identified for the individual based at least in part on the detection of EBV-2+ status in the sample, wherein the one or more treatment options comprise an immunotherapy.

[0084] In other aspects, provided herein are methods of predicting human leukocyte antigen class I (HLA-I) loss-of-heterozygosity (LOH) of a cancer of an individual, comprising acquiring knowledge of EBV-2+ status in a sample from the individual, wherein responsive to said knowledge, the cancer of the individual is predicted to have a higher likelihood of exhibiting HLA-I LOH as compared to a cancer of an individual with EBV-negative (EBV-) status or EBV subtype 1-positive (EBV-1+) and EBV-2 negative (EBV-2-) status.

[0085] In some embodiments according to any of the embodiments described herein, the individual is a human.EBV status

[0086] Certain aspects of the present disclosure relate to EBV status, e.g., EBV subtype 2- positive (EBV-2+), EBV-2 negative (EBV-2-), EBV subtype 1-positive (EBV-1+), or EBV subtype 1 -negative (EBV-1-) status. In some embodiments, the methods of the present disclosure comprise acquiring knowledge of and / or detecting EBV status in a sample, e.g., an EBV-2+ status, an EBV-1 / 2+ status, an EBV-1+ status, or an EBV- status (e.g., an EBV-1- and / or EBV-2- status).

[0087] In some embodiments, detecting EBV status (e.g., an EBV-2+ status) as described herein comprises detecting an EBV nucleic acid molecule of the disclosure (e.g., an EBV-2 nucleic acid molecule) in a sample. In some embodiments, detecting EBV-2+ status in a sample comprises detecting an EBV-2 nucleic acid molecule of the disclosure in the sample. In some embodiments, detecting EBV-1+ status in a sample comprises detecting an EBV-1 nucleic acid molecule of the disclosure in the sample. In some embodiments, detecting EBV- 1 / 2+ status in a sample comprises detecting an EBV-2 nucleic acid molecule of the disclosure in the sample, since as disclosed herein more than 20,000 EBV+ samples have been subtyped, and no sample was found to have EBV-2+ status without also displaying EBV-1+ status. In other embodiments, detecting EBV-1 / 2+ status in a sample comprises detecting an EBV-1 nucleic acid molecule of the disclosure and detecting an EBV-2 nucleic acid molecule of the disclosure in a sample.

[0088] In some embodiments, the methods comprise detecting EBV DNA (e.g., EBV-1 or EBV- 2 DNA), or a lack thereof, in a sample. In some embodiments, an EBV nucleic acid molecule of the disclosure (e.g., EBV DNA) may be detected by sequencing part or all of an EBV gene, e.g.,an EBV-1 or EBV-2 gene. For example, in some embodiments, EBV-1+ or EBV-2+ status can be detected by sequencing one or more nucleic acids encoding an EBV-1 or EBV-2 nuclear antigen, e.g., in a sample. In some embodiments, EBV-2+ status is detected (e.g., in a sample of the present disclosure) by sequencing one or more nucleic acids encoding an EBV-2 Epstein- Barr nuclear antigen 3 (EBNA-3) gene, e.g., from the sample. In some embodiments, the methods comprise aligning one or more sequencing reads to an EBV genome or sequence thereof, e.g., an EBV-1 or EBV-2 genome or sequence thereof. Methods for EBV subtyping are known in the art and / or exemplified infra. See, e.g., Zanella, L. et al. (2019) Sci Reports 9:9829. Suitable detection assays are described infra and / or otherwise known in the art.

[0089] In some embodiments, the methods further comprise contacting a sample, nucleic acid, or nucleic acid library with a plurality of target capture reagents in order to select and capture a plurality of specific target sequences (e.g, EBV gene sequences or fragments thereof) for analysis. In some instances, a target capture reagent (i.e., a molecule which can bind to and thereby allow capture of a target molecule) is used to select the subject intervals to be analyzed, e.g, EBV genes (such as EBV-1 and / or EBV-2 genes). For example, a target capture reagent can be a bait molecule, e.g., a nucleic acid molecule (e.g., a DNA molecule or RNA molecule) which can hybridize to (z.e., is complementary to) a target molecule, and thereby allows capture of the target nucleic acid. In some instances, the target capture reagent, e.g., a bait molecule (or bait sequence), is a capture oligonucleotide (or capture probe). In some instances, the target nucleic acid is a genomic DNA molecule, an RNA molecule, a cDNA molecule derived from an RNA molecule, a microsatellite DNA sequence, and the like. In some instances, the target capture reagent is suitable for solution-phase hybridization to the target. In some instances, the target capture reagent is suitable for solid-phase hybridization to the target. In some instances, the target capture reagent is suitable for both solution-phase and solid-phase hybridization to the target. The design and construction of target capture reagents is described in more detail in, e.g., International Patent Application Publication No. WO 2020 / 236941.

[0090] In some embodiments, a bait provided herein includes a label, a tag or detection reagent. In some embodiments, the label, tag or detection reagent is a radiolabel, a fluorescent label, an enzymatic label, a sequence tag, biotin, or another ligand. In some embodiments, a bait provided herein includes a detection reagent such as a fluorescent marker. In some embodiments, a bait provided herein includes (e.g., is conjugated to) an affinity tag or reagent, e.g., that allowscapture and isolation of a hybrid formed by a bait and a nucleic acid molecule hybridized to the bait. In some embodiments, the affinity tag or reagent is an antibody, an antibody fragment, biotin, or any other suitable affinity tag or reagent known in the art. In some embodiments, a bait is suitable for solution phase hybridization.

[0091] Baits can be produced and used according to methods known in the art, e.g., as described in WO2012092426A1 and / or or in Frampton et al (2013) Nat Biotechnol, 31 : 1023-1031. For example, biotinylated baits (e.g., RNA baits) can be produced by obtaining a pool of synthetic long oligonucleotides, originally synthesized on a microarray, and amplifying the oligonucleotides to produce the bait sequences. In some embodiments, the baits are produced by adding an RNA polymerase promoter sequence at one end of the bait sequences, and synthesizing RNA sequences using RNA polymerase. In one embodiment, libraries of synthetic oligodeoxynucleotides can be obtained from commercial suppliers, such as Agilent Technologies, Inc., and amplified using known nucleic acid amplification methods.

[0092] In some embodiments, a bait provided herein is between about 100 nucleotides and about 300 nucleotides in length. In some embodiments, a bait provided herein is between about 130 nucleotides and about 230 nucleotides in length. In some embodiments, a bait provided herein is between about 150 nucleotides and about 200 nucleotides in length. In some embodiments, a bait provided herein comprises a target-specific bait sequence and universal tails on each end. In some embodiments, the target-specific sequence is between about 40 nucleotides and about 300 nucleotides in length. In some embodiments, the target-specific sequence is between about 100 nucleotides and about 200 nucleotides in length. In some embodiments, the target-specific sequence is between about 120 nucleotides and about 170 nucleotides in length. In some embodiments, the target-specific sequence is about 150 nucleotides or about 170 nucleotides in length. In some embodiments, a bait provided herein comprises an oligonucleotide comprising about 200 nucleotides, of which about 150 nucleotides or about 170 nucleotides are targetspecific, and the other 50 nucleotides or 30 nucleotides (e.g., 25 or 15 nucleotides on each end of the bait) are universal arbitrary tails, e.g., suitable for PCR amplification.

[0093] The baits described herein can be used for selection of exons and short target sequences. In some embodiments, a bait of the disclosure distinguishes a target nucleic acid molecule, e.g., a genomic or transcribed nucleic acid molecule, e.g., a cDNA or RNA from a reference nucleotide sequence.

[0094] In some embodiments, detecting EBV- status (e.g., an EBV-1- and / or EBV-2- status) as described herein comprises running an EBV detection assay of the present disclosure without detecting an EBV nucleic acid molecule of the disclosure in a sample. In some embodiments, detecting EBV- status (e.g., an EBV-1- and / or EBV-2- status) as described herein comprises detecting the lack of an EBV nucleic acid molecule of the disclosure in a sample.

[0095] In some embodiments of any of the methods provided herein, the cancer is gastric cancer, nasopharyngeal cancer, or lymphoma. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is gastric carcinoma. In some embodiments, the cancer is nasopharyngeal cancer. In some embodiments, the cancer is nasopharyngeal carcinoma. In some embodiments, the cancer is lymphoma. In some embodiments, the cancer is EBV-2+. In some embodiments, the cancer is EBV-1 / 2+. In some embodiments, the cancer is advanced or metastatic.

[0096] In some embodiments, an EBV nucleic acid molecule of the disclosure is detected using any suitable method known in the art, such as a nucleic acid hybridization assay, an amplification-based assay (e.g., polymerase chain reaction, PCR), a PCR-RFLP assay, real-time PCR, sequencing (e.g., Sanger sequencing or next-generation sequencing), a screening analysis (e.g., using karyotype methods), fluorescence in situ hybridization (FISH), break away FISH, spectral karyotyping, multiplex-FISH, comparative genomic hybridization, in situ hybridization, single specific primer-polymerase chain reaction (SSP-PCR), high performance liquid chromatography (HPLC), or mass-spectrometric genotyping. Methods of analyzing samples, e.g., to detect a nucleic acid molecule, are described in U.S. Patent No. 9,340,830 and in WO2012092426A1. In some embodiments, an EBV nucleic acid molecule of the disclosure is detected by sequencing. In some embodiments, the sequencing comprises a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique. In some embodiments, the massively parallel sequencing (MPS) technique comprises next-generation sequencing (NGS).

[0097] In some embodiments, an EBV nucleic acid molecule of the disclosure is detected using an amplification-based method. As is known in the art, in such amplification-based methods, a sample of nucleic acids, such as a sample obtained from an individual, a tumor or a tissue or liquid biopsy, is used as a template in an amplification reaction (e.g., Polymerase Chain Reaction(PCR)) using one or more oligonucleotides or primers, e.g., such as one or more oligonucleotides or primers provided herein. The presence of an EBV nucleic acid molecule of the disclosure in the sample can be determined based on the presence or absence of an amplification product. Quantitative amplification methods are also known in the art and may be used according to the methods provided herein. Methods of measurement of DNA copy number at microsatellite loci using quantitative PCR analysis are known in the art. The known nucleotide sequence for genes is sufficient to enable one of skill in the art to routinely select primers to amplify any portion of the gene. Fluorogenic quantitative PCR can also be used. In fluorogenic quantitative PCR, quantitation is based on the amount of fluorescence signals, e.g., TaqMan and Sybr green.

[0098] Other amplification methods suitable for use according to the methods provided herein include, e.g., ligase chain reaction (LCR), transcription amplification, self-sustained sequence replication, dot PCR, and linker adapter PCR.

[0099] In some embodiments, an EBV nucleic acid molecule of the disclosure is detected using a sequencing method. Any method of sequencing known in the art can be used to detect an EBV nucleic acid molecule provided herein. Exemplary sequencing methods that may be used to detect a fusion nucleic acid molecule provided herein include those based on techniques developed by Maxam and Gilbert or Sanger. Automated sequencing procedures may also be used, e.g., including sequencing by mass spectrometry.

[0100] In some embodiments, an EBV nucleic acid molecule of the disclosure is detected using hybrid capture-based sequencing (hybrid capture-based NGS), e.g., using adaptor ligation-based libraries. See, e.g., Frampton, G.M. et al. (2013) Nat. Biotech. 31 :1023-1031. In some embodiments, an EBV nucleic acid molecule of the disclosure is detected using next-generation sequencing (NGS). Next-generation sequencing includes any sequencing method that determines the nucleotide sequence of either individual nucleic acid molecules or clonally expanded proxies for individual nucleic acid molecules in a highly parallel fashion e.g., greater than 105molecules may be sequenced simultaneously). Next generation sequencing methods suitable for use according to the methods provided herein are known in the art and include, without limitation, massively parallel short-read sequencing, template-based sequencing, pyrosequencing, real-time sequencing comprising imaging the continuous incorporation of dyelabeling nucleotides during DNA synthesis, nanopore sequencing, sequencing by hybridization, nano-transistor array based sequencing, polony sequencing, scanning tunneling microscopy(STM)-based sequencing, or nanowire-molecule sensor based sequencing. See, e.g., Metzker, M. (2010) Nature Biotechnology Reviews 11 :31-46. Exemplary NGS methods and platforms that may be used to detect an EBV nucleic acid molecule provided herein include, without limitation, the HeliScope Gene Sequencing system from Helicos BioSciences (Cambridge, MA., USA), the PacBio RS system from Pacific Biosciences (Menlo Park, CA, USA), massively parallel shortread sequencing such as the Solexa sequencer and other methods and platforms from Illumina Inc. (San Diego, CA, USA), 454 sequencing from 454 LifeSciences (Branford, CT, USA), Ion Torrent sequencing from ThermoFisher (Waltham, MA, USA), or the SOLiD sequencer from Applied Biosystems (Foster City, CA, USA). Additional exemplary methods and platforms that may be used to detect an EBV nucleic acid molecule provided herein include, without limitation, the Genome Sequencer (GS) FLX System from Roche (Basel, CHE), the G.007 polonator system, the Solexa Genome Analyzer, HiSeq 2500, HiSeq3000, HiSeq 4000, and NovaSeq 6000 platforms from Illumina Inc. (San Diego, CA, USA).

[0101] In some embodiments of any of the methods provided herein, the methods may comprise one or more of the steps of: (i) obtaining a sample from an individual (e.g., an individual suspected of having or determined to have cancer), (ii) extracting nucleic acid molecules (e.g., tumor or cancer nucleic acid molecules, or a mixture of tumor or cancer nucleic acid molecules and non-tumor or non-cancer nucleic acid molecules) from the sample, (iii) ligating one or more adapters to the nucleic acid molecules extracted from the sample (e.g., one or more amplification primers, flow cell adaptor sequences, substrate adapter sequences, sample index sequences, or unique molecular identifier (UMI) sequences), (iv) amplifying the nucleic acid molecules (e.g., using a polymerase chain reaction (PCR) amplification technique, a non-PCR amplification technique, or an isothermal amplification technique), (v) capturing nucleic acid molecules from the amplified nucleic acid molecules (e.g., by hybridization to one or more bait molecules, where the bait molecules each comprise one or more nucleic acid molecules (e.g., capture nucleic acid molecules) that each comprise a region that is complementary to a region of a captured nucleic acid molecule), (vi) sequencing the nucleic acid molecules extracted from the sample (or library proxies derived therefrom) using, e.g., a next-generation (massively parallel) sequencing technique, a whole genome sequencing (WGS) technique, a whole exome sequencing technique, a targeted sequencing technique, a direct sequencing technique, or a Sanger sequencing technique) using, e.g., a next-generation (massively parallel) sequencer, and (vii) generating,displaying, transmitting, and / or delivering a report (e.g., an electronic, web-based, or paper report) to the individual (or patient), a caregiver, a healthcare provider, a physician, an oncologist, an electronic medical record system, a hospital, a clinic, a third-party payer, an insurance company, or a government office. In some instances, the report comprises output from the methods described herein. In some instances, all or a portion of the report may be displayed in a graphical user interface of an online or web-based healthcare portal. In some instances, the report is transmitted via a computer network or peer-to-peer connection.

[0102] In some embodiments of any of the methods provided herein, the methods may comprise one or more of the steps of: (a) providing a plurality of nucleic acid molecules obtained from a sample from an individual (e.g., an individual suspected of having or determined to have cancer), wherein the plurality of nucleic acid molecules comprises nucleic acid molecules corresponding to an EBV nucleic acid molecule of the disclosure; (b) ligating one or more adapters onto one or more nucleic acid molecules from the plurality of nucleic acid molecules; (c) amplifying the one or more ligated nucleic acid molecules from the plurality of nucleic acid molecules; (d) capturing amplified nucleic acid molecules from the amplified nucleic acid molecules; (e) sequencing, by a sequencer, the captured nucleic acid molecules to obtain a plurality of sequence reads that represent the captured nucleic acid molecules, wherein one or more of the plurality of sequence reads correspond to the EBV nucleic acid molecule; (f) analyzing the plurality of sequence reads; and (g) based on the analysis, detecting the presence or absence of the EBV nucleic acid molecule in the sample. In some embodiments, the method further comprises subtyping one or more detected EBV nucleic acid molecules from the sample, e.g., as described herein, to determine if they are EBV-1 or EBV-2 nucleic acid molecules. In some embodiments, the methods further comprise receiving, at one or more processors, sequence read data for the plurality of sequence reads. In some embodiments, the analyzing the plurality of sequence reads comprises identifying, using the one or more processors, the presence or absence of sequence reads corresponding to the EBV nucleic acid molecule. In some embodiments, the amplified nucleic acid molecules are captured by hybridization with one or more bait molecules. In some embodiments, the analyzing the plurality of sequence reads comprises aligning one or more sequence reads to an EBV-1 or EBV-2 nucleic acid sequence.

[0103] In some embodiments of any of the methods provided herein, the methods may comprise one or more of the steps of: (a) providing a sample from an individual (e.g., an individualsuspected of having or determined to have cancer), wherein the sample comprises a plurality of nucleic acid molecules; (b) preparing a nucleic acid sequencing library from the plurality of nucleic acid molecules in the sample; (c) amplifying said library; (d) selectively enriching for one or more nucleic acid molecules comprising nucleotide sequences corresponding to an EBV nucleic acid molecule of the disclosure in said library to produce an enriched sample; (e) sequencing the enriched sample, thereby producing a plurality of sequence reads; (f) analyzing the plurality of sequence reads for the presence of the EBV nucleic acid molecule; (g) detecting, based on the analyzing step, the presence or absence of the EBV nucleic acid molecule in the sample from the individual. In some embodiments, the method further comprises subtyping one or more detected EBV nucleic acid molecules from the sample, e.g., as described herein, to determine if they are EBV-1 or EBV-2 nucleic acid molecules.

[0104] In some embodiments of any of the methods provided herein, the plurality of nucleic acid molecules comprises cancer nucleic acid molecules. In some embodiments of any of the methods provided herein, the plurality of nucleic acid molecules comprises a mixture of cancer nucleic acid molecules and non-cancer nucleic acid molecules. In some embodiments, the cancer nucleic acid molecules are derived from a tumor portion of a heterogeneous tissue biopsy sample, and the non-cancer nucleic acid molecules are derived from a normal portion of the heterogeneous tissue biopsy sample. In some embodiments, the sample comprises a liquid biopsy sample, and the cancer nucleic acid molecules are derived from a circulating tumor DNA (ctDNA) fraction of the liquid biopsy sample; and the non-cancer nucleic acid molecules are derived from a non-tumor fraction of the liquid biopsy sample or a cell-free DNA (cfDNA) fraction of the liquid biopsy sample.

[0105] In some embodiments of any of the methods, the one or more adapters comprise amplification primers, flow cell adaptor sequences, substrate adapter sequences, sample index sequences, or unique molecular identifier (UMI) sequences. In some embodiments, the one or more adapters comprise one or more sample index sequences. As is known in the art, sample indexes allow the sequencing of multiple samples on the same instrument flow cell or chip (i.e., multiplexing). Sample indexes are typically between about 8 and about 10 bases in length, and comprise a nucleotide sequence specific to a sample that is used to assign sequence reads to the correct sample during data analysis. In some embodiments, the one or more adapters comprise one or more unique molecule identifiers (UMIs). As is known in the art, UMIs comprise shortnucleotide sequences that include a unique barcode that is incorporated into each molecule in a given sample library. UMIs are useful for identifying PCR duplicates created during library amplification steps, and / or for reducing the rate of false-positive variant calls and increasing variant detection, since variant alleles present in the original sample (true variants) can be distinguished from errors introduced during library preparation, target enrichment, or sequencing.

[0106] In some embodiments of any of the methods provided herein, the methods comprise selectively enriching for one or more nucleic acids in a sample comprising nucleotide sequences corresponding to an EBV nucleic acid molecule of the disclosure. In some embodiments, selectively enriching comprises: (a) combining one or more bait molecules with a sequencing library, thereby hybridizing the one or more bait molecules to one or more nucleic acid molecules comprising nucleotide sequences corresponding to an EBV nucleic acid molecule and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids to produce an enriched sample. In other embodiments, the selectively enriching comprises: (a) combining one or more bait molecules with a sample, thereby hybridizing the one or more bait molecules to one or more nucleic acids in the sample comprising nucleotide sequences corresponding to the EBV nucleic acid molecule and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids to produce an enriched sample. In other embodiments, the selectively enriching comprises amplifying one or more nucleic acids comprising nucleotide sequences corresponding to an EBV nucleic acid molecule using a polymerase chain reaction (PCR) to produce an enriched sample. In other embodiments, nucleic acid molecules comprising nucleotide sequences corresponding to an EBV nucleic acid molecule are captured from amplified nucleic acid molecules by hybridization to one or more bait molecules. In some embodiments, the methods further comprise sequencing the enriched sample or the captured nucleic acid molecules. In some embodiments, the sequencing comprises use of a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique. In some embodiments, the sequencing comprises a massively parallel sequencing technique, and the massively parallel sequencing technique comprises next generation sequencing (NGS). In some embodiments, the sequencing is performed using a sequencer, optionally a next generation sequencer.

[0107] In some embodiments of any of the methods provided herein, the methods further comprise analyzing sequence data (e.g., obtained from sequencing as described above), for the presence or absence of one or more EBV nucleic acid molecules, e.g., EBV-1 or EBV-2 nucleic acid molecules. In some embodiments, the method further comprises subtyping one or more detected EBV nucleic acid molecules from the sample, e.g., as described herein, to determine if they are EBV-1 or EBV-2 nucleic acid molecules. In some embodiments, the analyzing the plurality of sequence reads comprises aligning one or more sequence reads to an EBV-1 or EBV- 2 nucleic acid sequence.

[0108] The disclosed methods may be used with any of a variety of samples, e.g., as described in further detail below. For example, in some instances, the sample may comprise a tissue biopsy sample, a liquid biopsy sample, or a normal control. In some instances, the sample may be a liquid biopsy sample and may comprise blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some instances, the sample may be a liquid biopsy sample and may comprise circulating tumor cells (CTCs). In some instances, the sample may be a liquid biopsy sample and may comprise cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof.

[0109] In some instances, nucleic acid molecules extracted from a sample may comprise a mixture of tumor or cancer nucleic acid molecules and non-tumor or non-cancer nucleic acid molecules. In some instances, the tumor nucleic acid molecules may be derived from a tumor portion of a heterogeneous tissue biopsy sample, and the non-tumor nucleic acid molecules may be derived from a normal portion of the heterogeneous tissue biopsy sample. In some instances, the sample may comprise a liquid biopsy sample, and the tumor or cancer nucleic acid molecules may be derived from a circulating tumor DNA (ctDNA) fraction of the liquid biopsy sample while the non-tumor or non-cancer nucleic acid molecules may be derived from a non-tumor or non-cancer, cell-free DNA (cfDNA) fraction of the liquid biopsy sample. In some embodiments of any of the methods provided herein, the method further comprises determining the circulating tumor DNA (ctDNA) fraction of a liquid biopsy sample.Loss of Heterozygosity of Human Leukocyte Antigen Class I Genes

[0110] Certain immunotherapies may depend at least in part on T cell recognition of tumor cells and / or tumor-specific epitopes. For example, immune checkpoint inhibitors (ICIs) function byreinvigorating the immune system and enabling T cell-mediated tumor elimination. This elimination is dependent upon T cell recognition, achieved through presentation of tumorspecific antigens by human leukocyte antigen class I (HLA-I) gene-encoded major histocompatibility complex class I (MHC-I) proteins. The HLA-I genes in humans include the genes HLA-A, HLA-B and HLA-C. Disruption of MHC-I presentation, through mechanisms such as loss of heterozygosity (LOH) of one or more of the HLA-I genes, is one potential mechanism of immune evasion and could affect responses to immune checkpoint inhibitors.[OHl] In some embodiments, a cancer of the present disclosure exhibits HLA-I LOH. Previous research indicated that patients who were virus-positive tended to have higher rates of HLA-I LOH than patients who were virus-negative, suggesting that viral antigen presentation is highly immunogenic and that HLA-I LOH was a mechanism of immune evasion in these patients (Montesion, M. et al. (2021) Cancer Discov. 11 (2) :282-292). The prevalence of HLA-I LOH in tumor types with known oncoviral associations has been examined, including HPV+ head and neck squamous cell carcinoma (HNSCC), HPV+ cervical cancer, EBV+ gastric cancer, EBV+ nasopharyngeal cancer, and HBV+ hepatocellular cancer. While some tumor types such as HPV+ HNSCC, HPV+ cervical, and EBV+ gastric cancer showed a significant association between viral infection and prevalence of HLA-I LOH, other cancer types such as EBV+ nasopharyngeal cancer did not. The present disclosure demonstrates that the rate of HLA-I LOH was significantly higher in EBV1 / 2+ patients as compared to EBV-negative (EBV-) patients in multiple tumor types (e.g., nasopharyngeal and gastric carcinoma).

[0112] In some embodiments, the methods of the present disclosure comprise acquiring knowledge of or detecting HLA-I LOH in a sample. LOH of one or more HLA-I genes, e.g., one or more of HLA-A, HLA-B or HLA-C, may be assessed using any suitable method known in the art, including, without limitation, sequencing (e.g., whole exome sequencing, whole genome sequencing, gene-targeted sequencing, methylation sequencing, or next-generation sequencing) and hybrid-capture-based sequencing methods.

[0113] In some embodiments, LOH of one or more HLA-I genes, e.g., one or more of HLA-A, HLA-B ox HLA-C, is assessed using a hybrid-capture-based sequencing method. Hybrid capture processes are described in U.S. Pat. No. 9,340,830; Frampton, G.M. et al. (2013) Nat. Biotech. 31 : 1023-1031; and Montesion, M., et al., Cancer Discovery (2021) 11(2):282-92. For example, detection of LOH can include preparing a population of DNA fragments from a sample from anindividual, some of which correspond to an HLA-I gene. If the subject or a cancer thereof is heterozygous at an HLA-I gene, the population of DNA fragments will contain different alleles in roughly equal amounts; if the subject or cancer thereof has undergone LOH, one of the alleles will be absent or significantly decreased in the population of on-target DNA fragments. In some embodiments, detection of HLA-I LOH comprises hybridizing of one or more nucleic acid(s) to one or more bait molecules corresponding to an HLA-I gene. Exemplary and non-limiting methods for detection of HLA-I LOH can be found, e.g., in International Pub. Nos. WO2022 / 272309 and WO2021 / 174052.Tumor mutational burden (TMB)

[0114] In some embodiments, the methods of the present disclosure comprise acquiring knowledge of or detecting tumor mutational burden (TMB) in a sample. In some embodiments, tumor mutational burden is assessed in sample from an individual, such as sample described herein. In some embodiments, the sample from the individual comprises fluid, cells, or tissue. In some embodiments, the sample from the individual comprises a tumor biopsy or a circulating tumor cell. In some embodiments, the sample from the individual comprises nucleic acids. In some embodiments, the sample from the individual comprises mRNA, DNA, circulating tumor DNA, cell-free DNA, or cell-free RNA.

[0115] In some embodiments, tumor mutational burden is measured using any suitable method known in the art. For example, tumor mutational burden may be measured using whole-exome sequencing (WES), next-generation sequencing, whole genome sequencing, gene-targeted sequencing, or sequencing of a panel of genes, e.g., panels including cancer-related genes. See, e.g., Melendez et al., Transl Lung Cancer Res (2018) 7(6):661-667. In some embodiments, tumor mutational burden is measured using gene-targeted sequencing, e.g., using a nucleic acid hybridization-capture method, e.g., coupled with sequencing. See, e.g., Fancello et al., J Immunother Cancer (2019) 7: 183. In some embodiments, tumor mutational burden is measured according to the methods provided in WO2017151524A1. In some embodiments, tumor mutational burden is measured according to the methods described in Montesion, M., et al., Cancer Discovery (2021) 11(2):282-92.

[0116] In some embodiments, tumor mutational burden is measured according to the methods described in Chalmers et al., Genome Med (2017) 19;9(1):34. In some embodiments, tumormutational burden is assessed as the number of somatic, coding, base substitution, and indel mutations per megabase of genome examined. In some embodiments, all base substitutions and indels in the coding regions of targeted genes, including synonymous alterations, are counted. In some embodiments, assessment of tumor mutational burden further comprises filtering / counting of the base substitutions and indels, comprising one or more, or all, of the following steps: (a) synonymous mutations are counted in order to reduce sampling noise; (b) non-coding alterations are not counted; (c) alterations listed as known somatic alterations in COSMIC (see, e.g., cancer.sanger.ac.uk / cosmic) and truncations in tumor suppressor genes are not counted; (d) alterations predicted to be germline by a somatic-germline zygosity algorithm (Sun et al., Cancer Res. 2014;74(19S): 1893) are not counted; (e) alterations that are recurrently predicted to be germline are not counted; (f) known germline alterations in dbSNP (see, e.g., www.ncbi.nlm.nih.gov / snp / ) are not counted; and (g) germline alterations occurring with two or more counts in the ExAC database (Lek et al., Nature. 2016;536:285-91.) are not counted. In some embodiments, to calculate the tumor mutational burden per megabase, the total number of mutations counted (e.g., as described above) is divided by the size of the coding region of the targeted territory. In some embodiments, the nonparametric Mann-Whitney U test is used to test for significance in difference of means between two populations.

[0117] In some embodiments, tumor mutational burden is assessed based on the number of nondriver somatic coding mutations / megabase (mut / Mb) of genome sequenced.

[0118] In some embodiments, tumor mutational burden is measured in the sample by whole exome sequencing. In some embodiments, tumor mutational burden is measured in the sample using next-generation sequencing. In some embodiments, tumor mutational burden is measured in the sample using whole genome sequencing. In some embodiments, tumor mutational burden is measured in the sample by gene-targeted sequencing. In some embodiments, tumor mutational burden is measured on between about 0.7 Mb and about 1.3 Mb of sequenced DNA.

[0119] In some embodiments, a cancer of the disclosure has a low tumor mutational burden (e.g., the cancer is a TMB low cancer), wherein the cancer has a tumor mutational burden of at less than about 10 mutations per megabase (mut / Mb).

[0120] In some embodiments, measuring tumor mutational burden comprises assessing mutations in a sample derived from a cancer in an individual. In some embodiments, measuring tumor mutational burden comprises assessing mutations in a sample derived from a cancer in anindividual and in a matched normal sample, e.g., a sample from the individual derived from a tissue or other source that is free of the cancer.

[0121] In some embodiments, tumor mutational burden is obtained from a plurality of sequence reads, e.g., a plurality of sequence reads obtained by sequencing nucleic acids corresponding to at least a portion of a genome (such as from an enriched or unenriched sample), e.g., according to any sequencing method known in the art or described herein. In some embodiments, tumor mutational burden is determined based on the number of non-driver somatic coding mutations per megabase of genome sequenced.Reporting

[0122] In some embodiments, the methods provided herein comprise generating a report, and / or providing a report to party.

[0123] In some embodiments of any of the methods provided herein, the methods comprise generating a report comprising one or more treatment options identified for the individual based at least in part on detection of the EBV-2+ or EBV-1 / 2+ status in the sample, wherein the one or more treatment options comprise an immunotherapy.

[0124] In some embodiments of any of the methods provided herein, responsive to acquisition of knowledge of EBV-2+ or EBV-1 / 2+ status in a sample from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer): (i) the individual is classified as a candidate to receive a treatment comprising an immunotherapy; and / or (ii) the individual is identified as likely to respond to a treatment that comprises an immunotherapy. In some embodiments, responsive to acquisition of knowledge of EBV-2+ or EBV-1 / 2+ status in a sample from the individual, the individual is predicted to have longer survival when treated with a treatment comprising an immunotherapy, as compared to survival of an individual whose cancer does not comprise or exhibit EBV-2+ or EBV-1 / 2+ status (e.g., EBV- status or EBV-1+ / EBV-2- status).

[0125] In some embodiments, responsive to acquisition of knowledge or detection of EBV-2+ or EBV- 1 / 2+ status in a sample from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer), the methods comprise administering to the individual an effective amount of a treatment that comprises an immunotherapy.

[0126] In some embodiments, the report can include information on the sample, cancer, or individual other than EBV status. For example, in some embodiments, the report can contain information on HLA-I LOH of the sample or cancer and / or information on TMB of the sample or cancer, e.g., as described herein.

[0127] In some embodiments, the report includes information on the role of EBV status, such as EBV-2+ or EBV- 1 / 2+ status in disease, such as in cancer. Such information can include one or more of: information on prognosis of a cancer, such as a cancer provided herein, e.g., comprising EBV-2+ or EBV- 1 / 2+ status; information on resistance of a cancer, such as a cancer provided herein (e.g., comprising EBV-2+ or EBV- 1 / 2+ status) to one or more treatments; information on potential or suggested therapeutic options (e.g., such as an immunotherapy provided herein); or information on therapeutic options that should be avoided. In some embodiments, the report includes information on the likely effectiveness, acceptability, and / or advisability of applying a therapeutic option (e.g., such as an immunotherapy provided herein) to an individual having a cancer, such as a cancer provided herein (e.g., comprising EBV-2+ or EBV-1 / 2+ status) and identified in the report. In some embodiments, the report includes information or a recommendation on the administration of a treatment (e.g., an immunotherapy provided herein). In some embodiments, the information or recommendation includes the dosage of the treatment and / or a treatment regimen (e.g., in combination with other treatments, such as a second therapeutic agent). In some embodiments, the report comprises information or a recommendation for at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more treatments.

[0128] Also provided herein are methods of generating a report according to the present disclosure. In some embodiments, a report according to the present disclosure is generated by a method comprising one or more of the following steps: obtaining one or more samples, such as sample(s) described herein, from an individual, e.g., an individual having a cancer, such as a cancer provided herein; detecting EBV-2+ or EBV- 1 / 2+ status in the sample(s), or acquiring knowledge of the presence of EBV-2+ or EBV-1 / 2+ status in the sample(s); and generating a report. In some embodiments, a report generated according to the methods provided herein comprises one or more of: information about the presence or absence of EBV-2+ or EBV-1 / 2+ status in the sample(s); an identifier for the individual from which the sample(s) was obtained; information on the role of the EBV-2+ or EBV- 1 / 2+ status in disease (e.g., such as in cancer);information on prognosis, resistance, or potential or suggested therapeutic options (such as an immunotherapy provided herein, or a treatment selected or identified according to the methods provided herein); information on the likely effectiveness, acceptability, or the advisability of applying a therapeutic option (such as an immunotherapy provided herein, or a treatment selected or identified according to the methods provided herein) to the individual; a recommendation or information on the administration of a treatment (such as an immunotherapy provided herein, or a treatment selected or identified according to the methods provided herein); or a recommendation or information on the dosage or treatment regimen of a treatment (such as an immunotherapy provided herein, or a treatment selected or identified according to the methods provided herein), e.g., in combination with other treatments (e.g., a second therapeutic agent). In some embodiments, the report generated is a personalized cancer report.

[0129] A report according to the present disclosure may be in an electronic, web-based, or paper form. The report may be provided to an individual or a patient (e.g., an individual or a patient having, suspected of having, or being tested for a cancer, such as a cancer provided herein, e.g., comprising EBV-2+ or EBV-1 / 2+ status), or to an individual or entity other than the individual or patient, such as one or more of a caregiver, a physician, an oncologist, a hospital, a clinic, a third party payor, an insurance company, or a government entity. In some embodiments, the report is provided or delivered to the individual or entity within any of about 1 day or more, about 7 days or more, about 14 days or more, about 21 days or more, about 30 days or more, about 45 days or more, or about 60 days or more from obtaining a sample from the individual. In some embodiments, the report is provided or delivered to an individual or entity within any of about 1 day or more, about 7 days or more, about 14 days or more, about 21 days or more, about 30 days or more, about 45 days or more, or about 60 days or more from detecting EBV-2+ or EBV-1 / 2+ status in one or more samples obtained from the individual. In some embodiments, the report is provided or delivered to an individual or entity within any of about 1 day or more, about 7 days or more, about 14 days or more, about 21 days or more, about 30 days or more, about 45 days or more, or about 60 days or more from acquiring knowledge of the presence of EBV-2+ or EBV-1 / 2+ status in one or more samples obtained from the individual. In some instances, all or a portion of the report may be displayed in a graphical user interface of an online or web-based healthcare portal.Immunotherapy

[0130] Certain aspects of the present disclosure relate to immunotherapies. Examples of immunotherapies include, without limitation, a checkpoint inhibitor (ICI), cancer vaccine, cellbased therapy, T cell receptor (TCR)-based therapy, adjuvant immunotherapy, cytokine immunotherapy, or oncolytic virus therapy. In some embodiments, the cancer immunotherapy comprises a small molecule, nucleic acid, polypeptide, carbohydrate, toxin, cell-based agent, or cell-binding agent. Examples of cancer immunotherapies are described in greater detail herein but are not intended to be limiting. In some embodiments, the cancer immunotherapy activates one or more aspects of the immune system to attack a cell (e.g., a tumor cell) that expresses a neoantigen, e.g., a viral neoantigen corresponding to an EBV nucleic acid molecule or polypeptide of the disclosure. The cancer immunotherapies of the present disclosure are contemplated for use as monotherapies, or in combination approaches comprising two or more in any combination or number, subject to medical judgement. Any of the cancer immunotherapies (optionally as monotherapies or in combination with another cancer immunotherapy or other therapeutic agent described herein) may find use in any of the methods described herein.

[0131] In some embodiments, the cancer immunotherapy comprises a cancer vaccine. A range of cancer vaccines have been tested that employ different approaches to promoting an immune response against a cancer (see, e.g., Emens L A, Expert Opin Emerg Drugs 13(2): 295-308 (2008) and US20190367613). Approaches have been designed to enhance the response of B cells, T cells, or professional antigen-presenting cells against tumors. Exemplary types of cancer vaccines include, but are not limited to, DNA-based vaccines, RNA-based vaccines, virus transduced vaccines, peptide-based vaccines, dendritic cell vaccines, oncolytic viruses, whole tumor cell vaccines, tumor antigen vaccines, etc. In some embodiments, the cancer vaccine can be prophylactic or therapeutic. In some embodiments, the cancer vaccine is formulated as a peptide-based vaccine, a nucleic acid-based vaccine, an antibody based vaccine, or a cell based vaccine. For example, a vaccine composition can include naked cDNA in cationic lipid formulations; lipopeptides (e.g., Vitiello, A. et al, J. Clin. Invest. 95:341, 1995), naked cDNA or peptides, encapsulated e.g., in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge, et ah, Molec. Immunol. 28:287-294, 1991 : Alonso et al, Vaccine 12:299- 306, 1994; Jones et al, Vaccine 13:675-681, 1995); peptide composition contained in immune stimulating complexes (ISCOMS) (e.g., Takahashi et al, Nature 344:873-875, 1990; Hu et al, Clin. Exp.Immunol. 113:235-243, 1998); or multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J.P., J. Immunol. Methods 196: 17-32, 1996). In some embodiments, a cancer vaccine is formulated as a peptide-based vaccine, or nucleic acid based vaccine in which the nucleic acid encodes the polypeptides. In some embodiments, a cancer vaccine is formulated as an antibody -based vaccine. In some embodiments, a cancer vaccine is formulated as a cell based vaccine. In some embodiments, the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine. In some embodiments, the cancer vaccine is a multivalent long peptide, a multiple peptide, a peptide mixture, a hybrid peptide, or a peptide pulsed dendritic cell vaccine (see, e.g., Yamada et al, Cancer Sci, 104: 14-21, 2013). In some embodiments, such cancer vaccines augment the anti-cancer response.

[0132] In some embodiments, the cancer vaccine comprises a polynucleotide that encodes a neoantigen, e.g., a viral neoantigen corresponding to an EBV nucleic acid molecule or polypeptide of the disclosure. In some embodiments, the cancer vaccine comprises DNA that encodes a neoantigen, e.g., a viral neoantigen corresponding to an EBV nucleic acid molecule or polypeptide of the disclosure. In some embodiments, the cancer vaccine comprises RNA that encodes a neoantigen, e.g., a viral neoantigen corresponding to an EBV nucleic acid molecule or polypeptide of the disclosure. In some embodiments, the cancer vaccine comprises a polynucleotide that encodes a neoantigen, e.g., a viral neoantigen corresponding to an EBV nucleic acid molecule or polypeptide of the disclosure. In some embodiments, the cancer vaccine further comprises one or more additional antigens, neoantigens, or other sequences that promote antigen presentation and / or an immune response. In some embodiments, the polynucleotide is complexed with one or more additional agents, such as a liposome or lipoplex. In some embodiments, the polynucleotide(s) are taken up and translated by antigen presenting cells (APCs), which then present the neoantigen(s) via MHC class I on the APC cell surface.

[0133] In some embodiments, the cancer vaccine is selected from sipuleucel-T (e.g., Provenge®, Dendreon / Valeant Pharmaceuticals), which has been approved for treatment of asymptomatic, or minimally symptomatic metastatic castrate-resistant (hormone-refractory) prostate cancer; and talimogene laherparepvec (e.g., Imlygic®, BioVex / Amgen, previously known as T-VEC), a genetically modified oncolytic viral therapy approved for treatment of unresectable cutaneous, subcutaneous and nodal lesions in melanoma. In some embodiments, the cancer vaccine isselected from an oncolytic viral therapy such as pexastimogene devacirepvec (PexaVec / JX-594, SillaJen / formerly Jennerex Biotherapeutics), a thymidine kinase- (TK-) deficient vaccinia virus engineered to express GM-CSF, for hepatocellular carcinoma (NCT02562755) and melanoma (NCT00429312); pelareorep (e.g., Reolysin®, Oncolytics Biotech), a variant of respiratory enteric orphan virus (reovirus) which does not replicate in cells that are not RAS-activated, in numerous cancers, including colorectal cancer (NCTO 1622543), prostate cancer (NCT01619813), head and neck squamous cell cancer (NCTO 1166542), pancreatic adenocarcinoma (NCT00998322), and non-small cell lung cancer (NSCLC) (NCT 00861627); enadenotucirev (NG-348, PsiOxus, formerly known as ColoAdl), an adenovirus engineered to express a full length CD80 and an antibody fragment specific for the T-cell receptor CD3 protein, in ovarian cancer (NCT02028117), metastatic or advanced epithelial tumors such as in colorectal cancer, bladder cancer, head and neck squamous cell carcinoma and salivary gland cancer (NCT02636036); ONCOS-102 (Targovax / formerly Oncos), an adenovirus engineered to express GM-CSF, in melanoma (NCT03003676), and peritoneal disease, colorectal cancer or ovarian cancer (NCT02963831); GL-ONC1 (GLV-lh68 / GLV-lhl53, Genelux GmbH), vaccinia viruses engineered to express beta-galactosidase (beta-gal) / beta-glucoronidase or beta-gal / human sodium iodide symporter (hNIS), respectively, were studied in peritoneal carcinomatosis (NCT01443260), fallopian tube cancer, ovarian cancer (NCT 02759588); or CG0070 (Cold Genesys), an adenovirus engineered to express GM-CSF in bladder cancer (NCT02365818); anti-gplOO; STINGVAX; GV AX; DCVaxL; and DNX-2401. In some embodiments, the cancer vaccine is selected from JX-929 (SillaJen / formerly Jennerex Biotherapeutics), a TK- and vaccinia growth factor-deficient vaccinia virus engineered to express cytosine deaminase, which is able to convert the prodrug 5 -fluorocytosine to the cytotoxic drug 5 -fluorouracil; TGO1 and TG02 (Targovax / formerly Oncos), peptide-based immunotherapy agents targeted for difficult-to- treat RAS mutations; and TILT-123 (TILT Biotherapeutics), an engineered adenovirus designated: Ad5 / 3-E2F-delta24-hTNFa-IRES-hIL20; and VSV-GP (ViraTherapeutics) a vesicular stomatitis virus (VSV) engineered to express the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), which can be further engineered to express antigens designed to raise an antigen-specific CD8+ T cell response. In some embodiments, the cancer vaccine comprises a vector-based tumor antigen vaccine. Vector-based tumor antigen vaccines can be used as a way to provide a steady supply of antigens to stimulate an anti-tumor immuneresponse. In some embodiments, vectors encoding for tumor antigens are injected into an individual (possibly with pro-inflammatory or other attractants such as GM-CSF), taken up by cells in vivo to make the specific antigens, which then provoke the desired immune response. In some embodiments, vectors may be used to deliver more than one tumor antigen at a time, to increase the immune response. In addition, recombinant virus, bacteria or yeast vectors can trigger their own immune responses, which may also enhance the overall immune response.

[0134] In some embodiments, the cancer vaccine comprises a DNA-based vaccine. In some embodiments, DNA-based vaccines can be employed to stimulate an anti-tumor response. The ability of directly injected DNA that encodes an antigenic protein, to elicit a protective immune response has been demonstrated in numerous experimental systems. Vaccination through directly injecting DNA that encodes an antigenic protein, to elicit a protective immune response often produces both cell-mediated and humoral responses. Moreover, reproducible immune responses to DNA encoding various antigens have been reported in mice that last essentially for the lifetime of the animal (see, e.g., Yankauckas et al. (1993) DNA Cell Biol., 12: 771-776). In some embodiments, plasmid (or other vector) DNA that includes a sequence encoding a protein operably linked to regulatory elements required for gene expression is administered to individuals (e.g. human patients, non-human mammals, etc.). In some embodiments, the cells of the individual take up the administered DNA and the coding sequence is expressed. In some embodiments, the antigen so produced becomes a target against which an immune response is directed.

[0135] In some embodiments, the cancer vaccine comprises an RNA-based vaccine. In some embodiments, RNA-based vaccines can be employed to stimulate an anti-tumor response. In some embodiments, RNA-based vaccines comprise a self-replicating RNA molecule. In some embodiments, the self-replicating RNA molecule may be an alphavirus-derived RNA replicon. Self-replicating RNA (or "SAM") molecules are well known in the art and can be produced by using replication elements derived from, e.g., alphaviruses, and substituting the structural viral proteins with a nucleotide sequence encoding a protein of interest. A self-replicating RNA molecule is typically a +-strand molecule which can be directly translated after delivery to a cell, and this translation provides a RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomictranscripts, may be translated themselves to provide in situ expression of an encoded polypeptide, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the antigen.

[0136] In some embodiments, the cancer immunotherapy comprises a cell-based therapy. In some embodiments, the cancer immunotherapy comprises a T cell-based therapy. In some embodiments, the cancer immunotherapy comprises an adoptive therapy, e.g., an adoptive T cellbased therapy. In some embodiments, the T cells are autologous or allogeneic to the recipient. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are CD4+ T cells. Adoptive immunotherapy refers to a therapeutic approach for treating cancer or infectious diseases in which immune cells are administered to a host with the aim that the cells mediate either directly or indirectly specific immunity to (i.e., mount an immune response directed against) cancer cells. In some embodiments, the immune response results in inhibition of tumor and / or metastatic cell growth and / or proliferation, and in related embodiments, results in neoplastic cell death and / or resorption. The immune cells can be derived from a different organism / host (exogenous immune cells) or can be cells obtained from the subject organism (autologous immune cells). In some embodiments, the immune cells (e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or gamma-delta T cells), NK cells, invariant NK cells, or NKT cells) can be genetically engineered to express antigen receptors such as engineered TCRs and / or chimeric antigen receptors (CARs). For example, the host cells (e.g., autologous or allogeneic T-cells) are modified to express a T cell receptor (TCR) having antigenic specificity for a cancer antigen. In some embodiments, NK cells are engineered to express a TCR. The NK cells may be further engineered to express a CAR. Multiple CARs and / or TCRs, such as to different antigens, may be added to a single cell type, such as T cells or NK cells. In some embodiments, the cells comprise one or more nucleic acids / expression constructs / vectors introduced via genetic engineering that encode one or more antigen receptors, and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and / or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature (e.g. chimeric). In some embodiments, a population of immune cells can be obtained from a subjectin need of therapy or suffering from a disease associated with reduced immune cell activity. Thus, the cells will be autologous to the subject in need of therapy. In some embodiments, a population of immune cells can be obtained from a donor, such as a histocompatibility-matched donor. In some embodiments, the immune cell population can be harvested from the peripheral blood, cord blood, bone marrow, spleen, or any other organ / tissue in which immune cells reside in said subject or donor. In some embodiments, the immune cells can be isolated from a pool of subjects and / or donors, such as from pooled cord blood. In some embodiments, when the population of immune cells is obtained from a donor distinct from the subject, the donor may be allogeneic, provided the cells obtained are subject-compatible, in that they can be introduced into the subject. In some embodiments, allogeneic donor cells may or may not be human-leukocyteantigen (HLA)-compatible. In some embodiments, to be rendered subject-compatible, allogeneic cells can be treated to reduce immunogenicity.

[0137] In some embodiments, the cell-based therapy comprises a T cell-based therapy, such as autologous cells, e.g., tumor-infiltrating lymphocytes (TILs); T cells activated ex-vivo using autologous DCs, lymphocytes, artificial antigen-presenting cells (APCs) or beads coated with T cell ligands and activating antibodies, or cells isolated by virtue of capturing target cell membrane; allogeneic cells naturally expressing anti-host tumor T cell receptor (TCR); and non- tumor-specific autologous or allogeneic cells genetically reprogrammed or "redirected" to express tumor-reactive TCR or chimeric TCR molecules displaying antibody-like tumor recognition capacity known as "T- bodies". Several approaches for the isolation, derivation, engineering or modification, activation, and expansion of functional anti-tumor effector cells have been described in the last two decades and may be used according to any of the methods provided herein. In some embodiments, the T cells are derived from the blood, bone marrow, lymph, umbilical cord, or lymphoid organs. In some embodiments, the cells are human cells. In some embodiments, the cells are primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and / or persistence capacities, antigen- specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. In some embodiments, thecells may be allogeneic and / or autologous. In some embodiments, such as for off-the-shelf technologies, the cells are pluripotent and / or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs).

[0138] In some embodiments, the T cell-based therapy comprises a chimeric antigen receptor (CAR)-T cell-based therapy. This approach involves engineering a CAR that specifically binds to an antigen of interest and comprises one or more intracellular signaling domains for T cell activation. The CAR is then expressed on the surface of engineered T cells (CAR-T) and administered to a patient, leading to a T-cell-specific immune response against cancer cells expressing the antigen. In some embodiments, the CAR specifically binds a neoantigen, such as a viral neoantigen corresponding to an EBV nucleic acid molecule or polypeptide of the disclosure.

[0139] In some embodiments, the T cell-based therapy comprises T cells expressing a recombinant T cell receptor (TCR). This approach involves identifying a TCR that specifically binds to an antigen of interest, which is then used to replace the endogenous or native TCR on the surface of engineered T cells that are administered to a patient, leading to a T-cell-specific immune response against cancer cells expressing the antigen. In some embodiments, the recombinant TCR specifically binds a neoantigen corresponding to an EBV nucleic acid molecule or polypeptide of the disclosure.

[0140] In some embodiments, the T cell-based therapy comprises tumor-infiltrating lymphocytes (TILs). For example, TILs can be isolated from a tumor or cancer of the present disclosure, then isolated and expanded in vitro. Some or all of these TILs may specifically recognize an antigen expressed by the tumor or cancer of the present disclosure. In some embodiments, the TILs are exposed to one or more neoantigens, e.g., a neoantigen corresponding to an EBV nucleic acid molecule or polypeptide of the disclosure, in vitro after isolation. TILs are then administered to the patient (optionally in combination with one or more cytokines or other immune-stimulating substances).

[0141] In some embodiments, the cell-based therapy comprises a natural killer (NK) cell-based therapy. Natural killer (NK) cells are a subpopulation of lymphocytes that have spontaneous cytotoxicity against a variety of tumor cells, virus-infected cells, and some normal cells in the bone marrow and thymus. NK cells are critical effectors of the early innate immune response toward transformed and virus-infected cells. NK cells can be detected by specific surfacemarkers, such as CD16, CD56, and CD8 in humans. NK cells do not express T-cell antigen receptors, the pan T marker CD3, or surface immunoglobulin B cell receptors. In some embodiments, NK cells are derived from human peripheral blood mononuclear cells (PBMC), unstimulated leukapheresis products (PBSC), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood by methods well known in the art.

[0142] In some embodiments, the cell-based therapy comprises a dendritic cell (DC)-based therapy, e.g., a dendritic cell vaccine. In some embodiments, the DC vaccine comprises antigen- presenting cells that are able to induce specific T cell immunity, which are harvested from the patient or from a donor. In some embodiments, the DC vaccine can then be exposed in vitro to a peptide antigen, for which T cells are to be generated in the patient. In some embodiments, dendritic cells loaded with the antigen are then injected back into the patient. In some embodiments, immunization may be repeated multiple times if desired. Methods for harvesting, expanding, and administering dendritic cells are known in the art; see, e.g., W02019178081. Dendritic cell vaccines (such as Sipuleucel-T, also known as APC8015 and PROVENGE®) are vaccines that involve administration of dendritic cells that act as APCs to present one or more cancer-specific antigens to the patient’s immune system. In some embodiments, the dendritic cells are autologous or allogeneic to the recipient.

[0143] In some embodiments, the cancer immunotherapy comprises a TCR-based therapy. In some embodiments, the cancer immunotherapy comprises administration of one or more TCRs or TCR-based therapeutics that specifically bind an antigen expressed by a cancer of the present disclosure, e.g., a neoantigen corresponding to an EBV nucleic acid molecule or polypeptide of the disclosure. In some embodiments, the TCR-based therapeutic may further include a moiety that binds an immune cell (e.g., a T cell), such as an antibody or antibody fragment that specifically binds a T cell surface protein or receptor (e.g., an anti-CD3 antibody or antibody fragment).

[0144] In some embodiments, the immunotherapy comprises adjuvant immunotherapy. Adjuvant immunotherapy comprises the use of one or more agents that activate components of the innate immune system, e.g., HILTONOL® (imiquimod), which targets the TLR7 pathway.

[0145] In some embodiments, the immunotherapy comprises cytokine immunotherapy. Cytokine immunotherapy comprises the use of one or more cytokines that activate componentsof the immune system. Examples include, but are not limited to, aldesleukin (e.g., PROLEUKIN®; interleukin-2), interferon alfa-2a (e.g., ROFERON®-A), interferon alfa-2b (e.g., INTRON®-A), and peginterferon alfa-2b (e.g., PEGINTRON®).

[0146] In some embodiments, the immunotherapy comprises oncolytic virus therapy. Oncolytic virus therapy uses genetically modified viruses to replicate in and kill cancer cells, leading to the release of antigens that stimulate an immune response. In some embodiments, replication- competent oncolytic viruses expressing a tumor antigen comprise any naturally occurring (e.g., from a “field source”) or modified replication-competent oncolytic virus. In some embodiments, the oncolytic virus, in addition to expressing a tumor antigen, may be modified to increase selectivity of the virus for cancer cells. In some embodiments, replication-competent oncolytic viruses include, but are not limited to, oncolytic viruses that are a member in the family of myoviridae, siphoviridae, podpviridae, teciviridae, corticoviridae, plasmaviridae, lipothrixviridae, fuselloviridae, poxyiridae, iridoviridae, phycodnaviridae, baculoviridae, herpesviridae, adnoviridae, papovaviridae, polydnaviridae, inoviridae, microviridae, geminiviridae, circoviridae, parvoviridae, hcpadnaviridae, retroviridae, cyctoviridae, reoviridae, birnaviridae, paramyxoviridae, rhabdoviridae, filoviridae, orthomyxoviridae, bunyaviridae, arenaviridae, Leviviridae, picornaviridae, sequiviridae, comoviridae, potyviridae, caliciviridae, astroviridae, nodaviridae, tetraviridae, tombusviridae, coronaviridae, glaviviridae, togaviridae, and barnaviridae. In some embodiments, replication-competent oncolytic viruses include adenovirus, retrovirus, reovirus, rhabdovirus, Newcastle Disease virus (NDV), polyoma virus, vaccinia virus (VacV), herpes simplex virus, picornavirus, coxsackie virus and parvovirus. In some embodiments, a replicative oncolytic vaccinia virus expressing a tumor antigen may be engineered to lack one or more functional genes in order to increase the cancer selectivity of the virus. In some embodiments, an oncolytic vaccinia virus is engineered to lack thymidine kinase (TK) activity. In some embodiments, the oncolytic vaccinia virus may be engineered to lack vaccinia virus growth factor (VGF). In some embodiments, an oncolytic vaccinia virus may be engineered to lack both VGF and TK activity. In some embodiments, an oncolytic vaccinia virus may be engineered to lack one or more genes involved in evading host interferon (IFN) response such as E3L, K3L, B18R, or B8R. In some embodiments, a replicative oncolytic vaccinia virus is a Western Reserve, Copenhagen, Lister or Wyeth strain and lacks a functional TK gene. In some embodiments, the oncolytic vaccinia virus is a Western Reserve, Copenhagen, Lister or Wyethstrain lacking a functional B18R and / or B8R gene. In some embodiments, a replicative oncolytic vaccinia virus expressing a tumor antigen may be locally or systemically administered to a subject, e.g. via intratumoral, intraperitoneal, intravenous, intra-arterial, intramuscular, intradermal, intracranial, subcutaneous, or intranasal administration.

[0147] In some embodiments, an immunotherapy of the disclosure comprises an immune checkpoint inhibitor. In some embodiments, the methods provided herein comprise administering to an individual an effective amount of an immune checkpoint inhibitor. As is known in the art, a checkpoint inhibitor targets at least one immune checkpoint protein to alter the regulation of an immune response. Immune checkpoint proteins include, e.g., CTLA4, PD-L1, PD-1, PD-L2, VISTA, B7-H2, B7-H3, B7-H4, B7-H6, 2B4, ICOS, HVEM, CEACAM, LAIR1, CD80, CD86, CD276, VTCN1, MHC class I, MHC class II, GALS, adenosine, TGFR, CSF1R, MICA / B, arginase, CD 160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, LAG-3, BTLA, IDO, 0X40, and A2aR. In some embodiments, molecules involved in regulating immune checkpoints include, but are not limited to: PD-1 (CD279), PD-L1 (B7-H1, CD274), PD-L2 (B7- CD, CD273), CTLA-4 (CD 152), HVEM, BTLA (CD272), a killer-cell immunoglobulin-like receptor (KIR), LAG-3 (CD223), TIM-3 (HAVCR2), CEACAM, CEACAM-1, CEACAM-3, CEACAM-5, GAL9, VISTA (PD-1H), TIGIT, LAIR1, CD160, 2B4, TGFRbeta, A2AR, GITR (CD357), CD80 (B7-1), CD86 (B7-2), CD276 (B7-H3), VTCNI (B7-H4), MHC class I, MHC class II, GALS, adenosine, TGFR, B7-H1, 0X40 (CD134), CD94 (KLRD1), CD137 (4-1BB), CD137L (4-1BBL), CD40, IDO, CSF1R, CD40L, CD47, CD70 (CD27L), CD226, HHLA2, ICOS (CD278), ICOSL (CD275), LIGHT (TNFSF14, CD258), NKG2a, NKG2d, OX40L (CD134L), PVR (NECL5, CD155), SIRPa, MICA / B, and / or arginase. In some embodiments, an immune checkpoint inhibitor (i.e., a checkpoint inhibitor) decreases the activity of a checkpoint protein that negatively regulates immune cell function, e.g., in order to enhance T cell activation and / or an anti-cancer immune response. In other embodiments, a checkpoint inhibitor increases the activity of a checkpoint protein that positively regulates immune cell function, e.g., in order to enhance T cell activation and / or an anti-cancer immune response. In some embodiments, the checkpoint inhibitor is an antibody. Examples of checkpoint inhibitors include, without limitation, a PD-1 axis binding antagonist, a PD-L1 axis binding antagonist (e.g., an anti-PD-Ll antibody, e.g., atezolizumab (MPDL3280A)), an antagonist directed against a co-inhibitorymolecule (e.g., a CTLA4 antagonist (e.g., an anti-CTLA4 antibody), a TIM-3 antagonist (e.g., an anti-TIM-3 antibody), or a LAG-3 antagonist (e.g., an anti-LAG-3 antibody)), or any combination thereof. In some embodiments, the immune checkpoint inhibitors comprise drugs such as small molecules, recombinant forms of ligand or receptors, or antibodies, such as human antibodies (see, e.g., International Patent Publication W02015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012). In some embodiments, known inhibitors of immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized or human forms of antibodies may be used.

[0148] In some embodiments, the checkpoint inhibitor is a PD-L1 axis binding antagonist. PD-1 (programmed death 1) is also referred to in the art as "programmed cell death 1," "PDCD1," "CD279," and "SLEB2." An exemplary human PD-1 is shown in UniProtKB / Swiss-Prot Accession No. Q15116. PD-L1 (programmed death ligand 1) is also referred to in the art as "programmed cell death 1 ligand 1,” "PDCD1 LG1," "CD274," "B7-H," and "PDL1." An exemplary human PD-L1 is shown in UniProtKB / Swiss-Prot Accession No.Q9NZQ7.1. PD-L2 (programmed death ligand 2) is also referred to in the art as "programmed cell death 1 ligand 2," "PDCD1 LG2," "CD273," "B7-DC," "Btdc," and "PDL2." An exemplary human PD-L2 is shown in UniProtKB / Swiss-Prot Accession No. Q9BQ51. In some instances, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1 and PD-L2.

[0149] In some instances, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific embodiment, the PD-1 ligand binding partners are PD-L1 and / or PD-L2. In another instance, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding ligands. In a specific embodiment, PD-L1 binding partners are PD-1 and / or B7-1. In another instance, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its ligand binding partners. In a specific embodiment, the PD-L2 binding ligand partner is PD-1. The antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the PD-1 binding antagonist is a small molecule, a nucleic acid, a polypeptide (e.g., antibody), a carbohydrate, a lipid, a metal, or a toxin.

[0150] In some instances, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), for example, as described below. In some instances, the anti-PD-1 antibody is one or more of MDX-1 106 (nivolumab), MK-3475(pembrolizumab, e.g., Keytruda®), MEDI-0680 (AMP-514), PDR001, REGN2810, MGA-012, JNJ-63723283, BI 754091, or BGB-108. In other instances, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). In some instances, the PD-1 binding antagonist is AMP -224. Other examples of anti-PD-1 antibodies include, but are not limited to, MEDI-0680 (AMP-514; AstraZeneca), PDR001 (CAS Registry No. 1859072-53-9; Novartis), REGN2810 (e.g., LIBTAYO® or cemiplimab-rwlc; Regeneron), BGB-108 (BeiGene), BGB-A317 (BeiGene), BI 754091, JS-001 (Shanghai Junshi), STI-Al l 10 (Sorrento), INCSHR-1210 (Incyte), PF-06801591 (Pfizer), TSR-042 (also known as ANB011; Tesaro / AnaptysBio), AM0001 (ARMO Biosciences), ENUM 244C8 (Enumeral Biomedical Holdings), or ENUM 388D4 (Enumeral Biomedical Holdings). In some embodiments, the PD-1 axis binding antagonist comprises tislelizumab (BGB-A317), BGB-108, STI-Al l 10, AM0001, BI 754091, sintilimab (IBI308), cetrelimab (JNJ-63723283), toripalimab (JS-001), camrelizumab (SHR-1210, INCSHR-1210, HR-301210), MEDI-0680 (AMP-514), MGA-012 (INCMGA 0012), nivolumab (BMS-936558, MDX1106, ONO-4538), spartalizumab (PDR001), pembrolizumab (MK-3475, SCH 900475, e.g., Keytruda®), PF-06801591, cemiplimab (REGN-2810, REGEN2810), dostarlimab (TSR-042, ANB011), FITC-YT-16 (PD-1 binding peptide), APL-501 or CBT-501 or genolimzumab (GB-226), AB-122, AK105, AMG 404, BCD-100, F520, HLX10, HX008, JTX-4014, LZM009, Sym021, PSB205, AMP-224 (fusion protein targeting PD-1), CX-188 (PD-1 probody), AGEN-2034, GLS-010, budigalimab (ABBV-181), AK-103, BAT- 1306, CS-1003, AM-0001, TILT- 123, BH-2922, BH-2941, BH- 2950, ENUM-244C8, ENUM-388D4, HAB-21, H EISCOI 11-003, IKT-202, MCLA-134, MT- 17000, PEGMP-7, PRS-332, RXL762, STI-1110, VXM-10, XmAb-23104, AK-112, HLX-20, SSI-361, AT-16201, SNA-01, AB122, PD1-PIK, PF-06936308, RG-7769, CAB PD-1 Abs, AK- 123, MEDI-3387, MEDI-5771, 4H1128Z-E27, REMD-288, SG-001, BY-24.3, CB-201, IBL319, ONCR-177, Max-1, CS-4100, JBI-426, CCC-0701, or CCX- 4503, or derivatives thereof.

[0151] In some embodiments, the PD-L1 binding antagonist is a small molecule that inhibits PD- 1. In some embodiments, the PD-L1 binding antagonist is a small molecule that inhibits PD-L1. In some embodiments, the PD-L1 binding antagonist is a small molecule that inhibits PD-L1 and VISTA or PD-L1 and TIM3. In some embodiments, the PD-L1 binding antagonist is CA-170 (also known as AUPM-170). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-Ll antibody can bind to a human PD-L1, for example a human PD-L1 as shown in UniProtKB / Swiss-Prot Accession No.Q9NZQ7.1, or a variant thereof. In some embodiments, the PD-L1 binding antagonist is a small molecule, a nucleic acid, a polypeptide (e.g., antibody), a carbohydrate, a lipid, a metal, or a toxin.

[0152] In some instances, the PD-L1 binding antagonist is an anti-PD-Ll antibody, for example, as described below. In some instances, the anti-PD-Ll antibody is capable of inhibiting the binding between PD-L1 and PD-1, and / or between PD-L1 and B7-1. In some instances, the anti- PD-Ll antibody is a monoclonal antibody. In some instances, the anti-PD-Ll antibody is an antibody fragment selected from a Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment. In some instances, the anti-PD-Ll antibody is a humanized antibody. In some instances, the anti-PD-Ll antibody is a human antibody. In some instances, the anti-PD-Ll antibody is selected from YW243.55.S70, MPDL3280A (atezolizumab), MDX-1 105, MEDI4736 (durvalumab), or MSB0010718C (avelumab). In some embodiments, the PD-L1 axis binding antagonist comprises atezolizumab, avelumab, durvalumab (imfinzi), BGB-A333, SHR-1316 (HTI-1088), CK-301, BMS-936559, envafolimab (KN035, ASC22), CS1001, MDX-1105 (BMS-936559), LY3300054, STLA1014, FAZ053, CX-072, INCB086550, GNS-1480, CA-170, CK-301, M- 7824, HTI-1088 (HTI-131, SHR-1316), MSB-2311, AK- 106, AVA-004, BBI-801, CA-327, CBA-0710, CBT-502, FPT-155, IKT-201, IKT-703, 10-103, JS-003, KD-033, KY-1003, MCLA-145, MT-5050, SNA-02, BCD-135, APL-502 (CBT-402 or TQB2450), IMC-001, KD- 045, INBRX-105, KN-046, IMC-2102, IMC-2101, KD-005, IMM-2502, 89Zr-CX-072, 89Zr- DFO-6E11, KY-1055, MEDL1109, MT-5594, SL-279252, DSP-106, Gensci-047, REMD-290, N-809, PRS-344, FS-222, GEN-1046, BH-29xx, or FS-118, or a derivative thereof.

[0153] In some embodiments, the checkpoint inhibitor is an antagonist of CTLA4. In some embodiments, the checkpoint inhibitor is a small molecule antagonist of CTLA4. In some embodiments, the checkpoint inhibitor is an anti-CTLA4 antibody. CTLA4 is part of the CD28- B7 immunoglobulin superfamily of immune checkpoint molecules that acts to negatively regulate T cell activation, particularly CD28-dependent T cell responses. CTLA4 competes for binding to common ligands with CD28, such as CD80 (B7-1) and CD86 (B7-2), and binds to these ligands with higher affinity than CD28. Blocking CTLA4 activity (e.g., using an anti- CTLA4 antibody) is thought to enhance CD28-mediated costimulation (leading to increased T cell activation / priming), affect T cell development, and / or deplete Tregs (such as intratumoralTregs). In some embodiments, the CTLA4 antagonist is a small molecule, a nucleic acid, a polypeptide (e.g., antibody), a carbohydrate, a lipid, a metal, or a toxin. In some embodiments, the CTLA-4 inhibitor comprises ipilimumab (IB 1310, BMS-734016, MDX010, MDX-CTLA4, MEDI4736), tremelimumab (CP-675, CP-675,206), APL-509, AGEN1884, CS1002, AGEN1181, Abatacept (Orencia, BMS-188667, RG2077), BCD-145, ONC-392, ADU-1604, REGN4659, ADG116, KN044, KN046, or a derivative thereof.

[0154] In some embodiments, the anti-PD-1 antibody or antibody fragment is MDX-1106 (nivolumab), MK-3475 (pembrolizumab, e.g., Keytruda®), MEDI-0680 (AMP-514), PDR001, REGN2810, MGA-012, JNJ-63723283, BI 754091, BGB-108, BGB-A317, JS-001, STI-Al l 10, INCSHR-1210, PF-06801591, TSR-042, AM0001, ENUM 244C8, or ENUM 388D4. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 immunoadhesin. In some embodiments, the anti-PD-1 immunoadhesin is AMP-224. In some embodiments, the anti-PD-Ll antibody or antibody fragment is YW243.55.S70, MPDL3280A (atezolizumab), MDX-1105, MEDI4736 (durvalumab), MSB0010718C (avelumab), LY3300054, STI-A1014, KN035, FAZ053, or CX-072.

[0155] In some embodiments, the immune checkpoint inhibitor comprises a LAG-3 inhibitor (e.g., an antibody, an antibody conjugate, or an antigen-binding fragment thereof). In some embodiments, the LAG-3 inhibitor comprises a small molecule, a nucleic acid, a polypeptide (e.g., an antibody), a carbohydrate, a lipid, a metal, or a toxin. In some embodiments, the LAG-3 inhibitor comprises a small molecule. In some embodiments, the LAG-3 inhibitor comprises a LAG-3 binding agent. In some embodiments, the LAG-3 inhibitor comprises an antibody, an antibody conjugate, or an antigen-binding fragment thereof. In some embodiments, the LAG-3 inhibitor comprises eftilagimod alpha (IMP321, IMP-321, EDDP-202, EOC-202), relatlimab (BMS-986016), GSK2831781 (IMP-731), LAG525 (IMP701), TSR-033, EVIP321 (soluble LAG-3 protein), BI 754111, IMP761, REGN3767, MK-4280, MGD-013, XmAb22841, INCAGN-2385, ENUM-006, AVA-017, AM-0003, iOnctura anti-LAG-3 antibody, Arcus Biosciences LAG-3 antibody, Sym022, a derivative thereof, or an antibody that competes with any of the preceding.

[0156] In some embodiments, an immunotherapy of the disclosure comprises an immunoregulatory molecule or a cytokine. An immunoregulatory profile is required to trigger an efficient immune response and balance the immunity in a subject. Examples of suitableimmunoregulatory cytokines include, but are not limited to, interferons (e.g., IFNa, IFNP and IFNy), interleukins (e.g, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 and IL- 20), tumor necrosis factors (e.g, TNFa and TNFP), erythropoietin (EPO), FLT-3 ligand, glplO, TCA-3, MCP-1, MIF, MIP-la, MIP-ip, Rantes, macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), or granulocyte-macrophage colony stimulating factor (GM-CSF), as well as functional fragments thereof. In some embodiments, any immunomodulatory chemokine that binds to a chemokine receptor, i.e, a CXC, CC, C, or CX3C chemokine receptor, can be used in the context of the present disclosure. Examples of chemokines include, but are not limited to, MIP-3a (Lax), MIP-3P, Hcc-1, MPIF-1, MPIF-2, MCP-2, MCP-3, MCP-4, MCP-5, Eotaxin, Tare, Elc, 1309, IL-8, GCP-2 Groa, Gro-P, Nap-2, Ena-78, Ip-10, MIG, LTac, SDF-1, or BCA-1 (Bic), as well as functional fragments thereof. In some embodiments, the immunoregulatory molecule is included with any of the treatments provided herein.

[0157] In some embodiments, the immune checkpoint inhibitor is monovalent and / or monospecific. In some embodiments, the immune checkpoint inhibitor is multivalent and / or multispecific.

[0158] In some embodiments, the methods further comprise administering to an individual an effective amount of a second anti-cancer therapy or agent, e.g, in combination with an immunotherapy of the present disclosure. In some embodiments, the second anti-cancer therapy or agent comprise a chemotherapy, anti-angiogenic agent, anti-DNA repair therapy, radiosensitizer, anti-inflammatory agent, anti-hormonal agent, antimetabolite, or platinum-based chemotherapeutic agent. Examples of chemotherapeutic agents include 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, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlomaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites, such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues, such as denopterin, 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 floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane; folic acid replenishers such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an 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; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2”- trichlorotri ethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; 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-1 1); topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, famesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0159] Some non-limiting examples of chemotherapeutic drugs which can be combined with immunotherapies of the present disclosure are carboplatin (Paraplatin), cisplatin (Platinol, Platinol-AQ), cyclophosphamide (Cytoxan, Neosar), docetaxel (Taxotere), doxorubicin (Adriamycin), erlotinib (Tarceva), etoposide (VePesid), fluorouracil (5-FU), gemcitabine (Gemzar), imatinib mesylate (Gleevec), irinotecan (Camptosar), methotrexate (Fol ex, Mexate, Am ethopterin), paclitaxel (Taxol, Abraxane), sorafinib (Nexavar), sunitinib (Sutent), topotecan (Hycamtin), vincristine (Oncovin, Vincasar PFS), and vinblastine (Velban).

[0160] In some embodiments, an immunotherapy of the disclosure is administered as a monotherapy. In some embodiments, the immunotherapy is administered in combination with one or more additional anti-cancer therapies or treatments, e.g., as described herein. In some embodiments, the one or more additional anti-cancer therapies or treatments include one or more anti-cancer therapies described herein. In some embodiments, the methods of the present disclosure comprise administration of any combination of any of the anti-cancer therapies provided herein. In some embodiments, the additional anti-cancer therapy comprises one or more of surgery, radiotherapy, chemotherapy, anti-angiogenic therapy, anti-DNA repair therapy, and anti-inflammatory therapy. In some embodiments, the additional anti-cancer therapy comprises an anti-neoplastic agent, a chemotherapeutic agent, a growth inhibitory agent, an anti-angiogenic agent, a radiation therapy, a cytotoxic agent, or combinations thereof. In some embodiments, an anti-cancer therapy may be administered in conjunction with a chemotherapy or chemotherapeutic agent. In some embodiments, the chemotherapy or chemotherapeutic agent is a platinum-based agent (including, without limitation cisplatin, carboplatin, oxaliplatin, and staraplatin). In some embodiments, an anti-cancer therapy may be administered in conjunction with a radiation therapy.Samples

[0161] In some embodiments, an EBV nucleic acid molecule of the disclosure is detected in a cancer or tumor cell, e.g., using tumor tissue, such as from a tumor biopsy or other tumor specimen; in a circulating cancer or tumor cell, e.g., using a liquid biopsy, such as from blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva; or in circulating tumor DNA (ctDNA), e.g., using a liquid biopsy, such as from blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva.

[0162] The disclosed method may be used with any of a variety of samples (also referred to herein as specimens) comprising nucleic acids (e.g., DNA) that are collected from a subject (e.g., a patient). Examples of a sample include, but are not limited to, a liquid biopsy sample, a blood sample (e.g., a peripheral whole blood sample), a blood plasma sample, a blood serum sample, a lymph sample, a saliva sample, a sputum sample, a urine sample, a gynecological fluid sample, a circulating tumor cell (CTC) sample, a cerebral spinal fluid (CSF) sample, a pericardial fluid sample, a pleural fluid sample, an ascites (peritoneal fluid) sample, a feces (or stool) sample, or other body fluid, secretion, and / or excretion sample (or sample derived therefrom), as well as a tissue sample. In some embodiments, the plurality of nucleic acid molecules is obtained from a liquid biopsy sample or a tissue sample.

[0163] In some embodiments, the sample may be collected by needle biopsy, fine needle aspiration, collection cup or tube, oral swab, nasal swab, vaginal swab or a cytology smear, etc.

[0164] In some embodiments, the sample is a liquid biopsy sample, and may comprise, e.g., whole blood, blood plasma, blood serum, buffy coat, urine, stool, sputum, saliva, or cerebrospinal fluid. In some embodiments, the sample may be a liquid biopsy sample and may comprise circulating tumor cells (CTCs). In some embodiments, the sample may be a liquid biopsy sample and may comprise cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof. In some embodiments, the sample is a tissue sample, and may fresh frozen tissue, FFPE tissue, PBMC, tumor biopsy, organ biopsy, bone marrow aspirate, cell lines, hair follicles, or any combination thereof.

[0165] In some embodiments, the nucleic acids extracted from the sample may comprise deoxyribonucleic acid (DNA) molecules. Examples of DNA that may be suitable for analysis by the disclosed methods include, but are not limited to, genomic DNA or fragments thereof, mitochondrial DNA or fragments thereof, cell-free DNA (cfDNA), and circulating tumor DNA(ctDNA). Cell-free DNA (cfDNA) is comprised of fragments of DNA that are released from normal and / or cancerous cells during apoptosis and necrosis and circulate in the blood stream and / or accumulate in other bodily fluids. Circulating tumor DNA (ctDNA) is comprised of fragments of DNA that are released from cancerous cells and tumors that circulate in the blood stream and / or accumulate in other bodily fluids.

[0166] In some embodiments, a sample is a primary sample obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by a method chosen from biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, or collection of body fluid (e.g., blood, lymph, or feces). In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. Such a processed sample may comprise, for example, nucleic acids (e.g., for use in any of the methods for detection of EBV nucleic acid molecules provided herein) extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification methods, reverse transcription of mRNA, or isolation and / or purification of certain components such as nucleic acids and / or proteins.

[0167] In some embodiments, the sample comprises nucleic acids, e.g., genomic DNA, cDNA, or mRNA. In some embodiments, the sample comprises cell-free DNA (cfDNA). In some embodiments, the sample comprises cell-free RNA (cfRNA). In some embodiments, the sample comprises circulating tumor DNA (ctDNA). In certain embodiments, the nucleic acids are purified or isolated (e.g., removed from their natural state). In some embodiments, the sample comprises tumor or cancer nucleic acids, such as nucleic acids from a tumor or cancer sample, e.g., genomic DNA, RNA, or cDNA derived from RNA, or from a liquid biopsy, e.g., ctDNA from blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In certain embodiments, a tumor or cancer nucleic acid sample, or a ctDNA sample, is purified or isolated (e.g., it is removed from its natural state).

[0168] In some embodiments, the sample is obtained from an individual having a cancer, such as a cancer described herein. In some embodiments, the sample comprises an EBV nucleic acid molecule of the disclosure. In some embodiments, the sample is obtained from an individual known or suspected of having a cancer.

[0169] In some embodiments, nucleic acids may be extracted from liquid biopsy samples, including but not limited to blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva or other bodily fluid samples using any of a variety of techniques known to those of skill in the art (see, e.g., Example 1 of International Patent Application Publication No. WO 2012 / 092426; Tan, et al. (2009), “DNA, RNA, and Protein Extraction: The Past and The Present”, J. Biomed. Biotech. 2009:574398; the technical literature for the Maxwell® 16 LEV Blood DNA Kit (Promega Corporation, Madison, WI); and the Maxwell 16 Buccal Swab LEV DNA Purification Kit Technical Manual (Promega Literature #TM333, January 1, 2011, Promega Corporation, Madison, WI)).

[0170] A typical DNA extraction procedure, for example, comprises (i) collection of the fluid sample from which DNA is to be extracted, (ii) treatment of the fluid sample with a concentrated salt solution to precipitate proteins, lipids, and RNA, followed by centrifugation to separate out the precipitated proteins, lipids, and RNA, and (iii) purification of DNA from the supernatant to remove detergents, proteins, salts, or other reagents used during previous steps. The DNA sample optionally may be further treated with an RNase for digestion of RNA in the sample.

[0171] Examples of suitable techniques for DNA purification include, but are not limited to, (i) precipitation in ice-cold ethanol or isopropanol, followed by centrifugation (precipitation of DNA may be enhanced by increasing ionic strength, e.g., by addition of sodium acetate), (ii) phenol-chloroform extraction, followed by centrifugation to separate the aqueous phase containing the nucleic acid from the organic phase containing denatured protein, and (iii) solid phase chromatography where the nucleic acids adsorb to the solid phase (e.g., silica or other) depending on the pH and salt concentration of the buffer.

[0172] In some instances, cellular and histone proteins bound to the DNA may be removed either by adding a protease or by having precipitated the proteins with sodium or ammonium acetate, or through extraction with a phenol-chloroform mixture prior to a DNA precipitation step.

[0173] In some instances, DNA may be extracted using any of a variety of suitable commercial DNA extraction and purification kits. Examples include, but are not limited to, the QIAamp (for isolation of genomic DNA from human samples) and DNAeasy (for isolation of genomic DNA from animal or plant samples) kits from Qiagen (Germantown, MD) or the Maxwell® and ReliaPrep™ series of kits from Promega (Madison, WI). After isolation, the nucleic acids aretypically dissolved in a slightly alkaline buffer, e.g., Tris-EDTA (TE) buffer, or in ultra-pure water.

[0174] In some instances, the nucleic acids isolated from the sample may be used to construct a library (e.g., a nucleic acid library as described herein). In some instances, the nucleic acids are fragmented using any of the methods described above, optionally subjected to repair of chain end damage, and optionally ligated to synthetic adapters, primers, and / or barcodes (e.g., amplification primers, sequencing adapters, flow cell adapters, substrate adapters, sample barcodes or indexes, and / or unique molecular identifier sequences), size-selected (e.g., by preparative gel electrophoresis), and / or amplified (e.g., using PCR, a non-PCR amplification technique, or an isothermal amplification technique). In some instances, the fragmented and adapter-ligated group of nucleic acids is used without explicit size selection or amplification prior to hybridization-based selection of target sequences. In some instances, the nucleic acid is amplified by any of a variety of specific or non-specific nucleic acid amplification methods known to those of skill in the art. In some instances, the nucleic acids are amplified, e.g., by a whole-genome amplification method such as random-primed strand-displacement amplification. Examples of nucleic acid library preparation techniques for next-generation sequencing are described in, e.g., van Dijk, et al. (2014), Exp. Cell Research 322: 12 - 20, and Illumina’s genomic DNA sample preparation kit.

[0175] In some instances, the resulting nucleic acid library may contain all or substantially all of the complexity of the genome. The term “substantially all” in this context refers to the possibility that there can in practice be some unwanted loss of genome complexity during the initial steps of the procedure. The methods described herein also are useful in cases where the nucleic acid library comprises a portion of the genome, e.g., where the complexity of the genome is reduced by design. In some instances, any selected portion of the genome can be used with a method described herein. For example, in certain embodiments, the entire exome or a subset thereof is isolated. In some instances, the library may include at least 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of the genomic DNA. In some instances, the library may consist of cDNA copies of genomic DNA that includes copies of at least 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of the genomic DNA. In certain instances, the amount of nucleic acid used to generate the nucleic acid library may be less than 5 micrograms, less than 1microgram, less than 500 ng, less than 200 ng, less than 100 ng, less than 50 ng, less than 10 ng, less than 5 ng, or less than 1 ng.

[0176] In some instances, a library (e.g., a nucleic acid library) includes a collection of nucleic acid molecules. As described herein, the nucleic acid molecules of the library can include a target nucleic acid molecule (e.g., a tumor nucleic acid molecule, a reference nucleic acid molecule and / or a control nucleic acid molecule; also referred to herein as a first, second and / or third nucleic acid molecule, respectively). The nucleic acid molecules of the library can be from a single subject or individual. In some instances, a library can comprise nucleic acid molecules derived from more than one subject (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or more subjects). For example, two or more libraries from different subjects can be combined to form a library having nucleic acid molecules from more than one subject (where the nucleic acid molecules derived from each subject are optionally ligated to a unique sample barcode corresponding to a specific subject). In some instances, the subject is a human having, or at risk of having, a cancer or tumor.Reporting

[0177] In some embodiments, the methods provided herein comprise generating a report, and / or providing a report to party.

[0178] In some embodiments, a report according to the present disclosure comprises information about one or more of: an EBV status of the disclosure, an EBV nucleic acid molecule, a cancer of the disclosure, e.g., comprising an EBV nucleic acid molecule of the disclosure; or a treatment, a therapy, or one or more treatment options for an individual having a cancer, such as a cancer of the disclosure (e.g., comprising an EBV nucleic acid molecule described herein). In some embodiments, the cancer is EBV-2+. In some embodiments, the cancer is EBV-1 / 2+.

[0179] In some embodiments, a report according to the present disclosure comprises information about an EBV status of a sample obtained from an individual or the presence or absence of an EBV nucleic acid molecule in a sample obtained from an individual, such as an individual having a cancer, e.g., a cancer provided herein. In one embodiment, a report according to the present disclosure indicates an EBV+ status (e.g, EBV-2+ status), or that an EBV nucleic acid molecule of the disclosure (e.g, an EBV-2 nucleic acid molecule of the disclosure) is present in a sample obtained from the individual. In one embodiment, a report according to the presentdisclosure indicates an EBV- status (e.g., an EBV-2- status) or that an EBV nucleic acid molecule of the disclosure (e.g., an EBV-2 nucleic acid molecule of the disclosure) is not present in a sample obtained from the individual. In one embodiment, a report according to the present disclosure indicates an EBV+ status (e.g., EBV-2+ status), or that an EBV nucleic acid molecule of the disclosure (e.g., an EBV-2 nucleic acid molecule of the disclosure) has been detected in a sample obtained from the individual. In one embodiment, a report according to the present disclosure indicates an EBV- status (e.g., an EBV-2- status) or that an EBV nucleic acid molecule of the disclosure (e.g., an EBV-2 nucleic acid molecule of the disclosure) has not been detected in a sample obtained from the individual. In some embodiments, the report comprises an identifier for the individual from which the sample was obtained.

[0180] In some embodiments, the report includes information on the role of an EBV fusion nucleic acid molecule of the disclosure in disease, such as in cancer. Such information can include one or more of: information on prognosis of a cancer, such as a cancer provided herein; information on an EBV status of the disclosure, information on resistance of a cancer, such as a cancer provided herein, to one or more treatments; information on potential or suggested therapeutic options (e.g., such as an immunotherapy provided herein, or a treatment selected or identified according to the methods provided herein); or information on therapeutic options that should be avoided. In some embodiments, the report includes information on the likely effectiveness, acceptability, and / or advisability of applying a therapeutic option (e.g., such as an immunotherapy provided herein, or a treatment selected or identified according to the methods provided herein) to an individual having a cancer, such as a cancer provided herein. In some embodiments, the report includes information or a recommendation on the administration of a treatment (e.g., an anti-cancer therapy provided herein, or a treatment selected or identified according to the methods provided herein). In some embodiments, the information or recommendation includes the dosage of the treatment and / or a treatment regimen (e.g., in combination with other treatments, such as a second therapeutic agent). In some embodiments, the report comprises information or a recommendation for at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more treatments.

[0181] Also provided herein are methods of generating a report according to the present disclosure. In some embodiments, a report according to the present disclosure is generated by amethod comprising one or more of the following steps: obtaining a sample, such as a sample described herein, from an individual, e.g., an individual having a cancer, such as a cancer provided herein; detecting an EBV status or EBV nucleic acid molecule of the disclosure or detectable lack thereof in the sample, or acquiring knowledge of an EBV status or the presence of the EBV nucleic acid molecule of the disclosure or detectable lack thereof in the sample; and generating a report. In some embodiments, the report generated is a personalized cancer report.

[0182] A report according to the present disclosure may be in an electronic, web-based, or paper form. The report may be provided to an individual or a patient (e.g., an individual or a patient having, suspected of having, or being tested for a cancer, such as a cancer provided herein), or to an individual or entity other than the individual or patient, such as one or more of a caregiver, a physician, an oncologist, a hospital, a clinic, a third party payor, an insurance company, or a government entity. In some embodiments, the report is provided or delivered to the individual or entity within any of about 1 day or more, about 7 days or more, about 14 days or more, about 21 days or more, about 30 days or more, about 45 days or more, or about 60 days or more from obtaining a sample from the individual. In some embodiments, the report is provided or delivered to an individual or entity within any of about 1 day or more, about 7 days or more, about 14 days or more, about 21 days or more, about 30 days or more, about 45 days or more, or about 60 days or more from detecting EBV status or an EBV nucleic acid molecule or detectable lack thereof in a sample obtained from the individual. In some embodiments, the report is provided or delivered to an individual or entity within any of about 1 day or more, about 7 days or more, about 14 days or more, about 21 days or more, about 30 days or more, about 45 days or more, or about 60 days or more from acquiring knowledge of the EBV status or an EBV nucleic acid molecule or detectable lack thereof in a sample obtained from the individual.IV. Exemplary Embodiments

[0183] The following exemplary embodiments are representative of some aspects of the invention.

[0184] Embodiment 1. A method of treating or delaying progression of cancer in an individual, comprising: acquiring knowledge of at least Epstein-Barr virus (EBV) subtype 2-positive (EBV-2+) status in a sample from the individual; andadministering to the individual an effective amount of an immunotherapy responsive to said knowledge.

[0185] Embodiment 2. A method of treating or delaying progression of cancer in an individual, comprising: detecting EBV-2+ status in a sample from the individual; and administering to the individual an effective amount of an immunotherapy.

[0186] Embodiment 3. A method of predicting survival of an individual having cancer, comprising acquiring knowledge of EBV-2+ status in a sample from the individual, wherein responsive to said knowledge, the individual is predicted to have a higher likelihood of survival when treated with an immunotherapy as compared to an individual with EBV-negative (EBV-) status or EBV subtype 1-positive (EBV-1+) and EBV-2 negative (EBV-2-) status.

[0187] Embodiment 4. A method of predicting response to immunotherapy of an individual having cancer, comprising acquiring knowledge of EBV-2+ status in a sample from the individual, wherein responsive to said knowledge, the individual is predicted to have a better response to treatment when treated with an immunotherapy as compared to response to treatment of an individual with EBV-negative (EBV-) status or EBV subtype 1-positive (EBV-1+) and EBV-2 negative (EBV-2-) status.

[0188] Embodiment 5. A method of identifying one or more treatment options for an individual having cancer, comprising detecting EBV-2+ status in a sample from the individual; and generating a report comprising one or more treatment options identified for the individual based at least in part on the detection of EBV-2+ status in the sample, wherein the one or more treatment options comprise an immunotherapy.

[0189] Embodiment 6. A method of predicting human leukocyte antigen class I (HLA-I) loss-of-heterozygosity (LOH) of a cancer of an individual, comprising acquiring knowledge of EBV-2+ status in a sample from the individual, wherein responsive to said knowledge, the cancer of the individual is predicted to have a higher likelihood of exhibiting HLA-I LOH as compared to a cancer of an individual with EBV-negative (EBV-) status or EBV subtype 1- positive (EBV-1+) and EBV-2 negative (EBV-2 -) status.

[0190] Embodiment 7. The method of any one of embodiments 1-6, further comprising acquiring knowledge of or detecting EBV-1+ status in a sample from the individual.

[0191] Embodiment 8. The method of embodiment 7, wherein the method comprises acquiring knowledge of or detecting EBV subtype 1 and EBV subtype 2-positive (EBV-1 / 2+) status in a sample from the individual.

[0192] Embodiment 9. The method of any one of embodiments 1-5, 7, and 8, wherein the cancer exhibits HLA-I LOH.

[0193] Embodiment 10. The method of any one of embodiments 1-5 and 7-9, further comprising acquiring knowledge of or detecting HLA-I LOH in a sample obtained from the cancer of the individual.

[0194] Embodiment 11. The method of any one of embodiments 1-10, further comprising acquiring knowledge of or detecting tumor mutational burden (TMB) in a sample obtained from the cancer of the individual.

[0195] Embodiment 12. The method of any one of embodiments 1-11, wherein the cancer is a TMB low cancer.

[0196] Embodiment 13. The method of embodiment 12, wherein the cancer has a TMB of less than 10 mutations per megabase (muts / Mb).

[0197] Embodiment 14. The method of any one of embodiments 1-13, wherein the cancer is gastric or nasopharyngeal cancer.

[0198] Embodiment 15. The method of any one of embodiments 1-14, wherein the cancer is advanced or metastatic.

[0199] Embodiment 16. The method of any one of embodiments 1-5 and 7-15, wherein the immunotherapy is administered as a monotherapy.

[0200] Embodiment 17. The method of any one of embodiments 1-5 and 7-16, wherein the immunotherapy comprises one or more of a checkpoint inhibitor, cancer vaccine, cell-based therapy, T cell receptor (TCR)-based therapy, adjuvant immunotherapy, cytokine immunotherapy, and oncolytic virus therapy.

[0201] Embodiment 18. The method of embodiment 17, wherein the immunotherapy comprises a checkpoint inhibitor.

[0202] Embodiment 19. The method of embodiment 18, wherein the checkpoint inhibitor targets PD-L1, PD-1, CTLA-4, CEACAM, LAIR1, CD160, 2B4, CD80, CD86, CD276, VTCN1, HVEM, KIR, A2AR, MHC class I, MHC class II, GALS, adenosine, TGFR, 0X40, CD137, CD40, IDO, CSF1R, TIM-3, BTLA, VISTA, LAG-3, TIGIT, IDO, MICA / B, or arginase.

[0203] Embodiment 20. The method of embodiment 19, wherein the checkpoint inhibitor is an agent that inhibits PD-1.

[0204] Embodiment 21. The method of embodiment 20, wherein the agent that inhibits PD- 1 is a small molecule, a nucleic acid, a polypeptide, carbohydrate, a lipid, a metal, or a toxin.

[0205] Embodiment 22. The method of embodiment 21, wherein the agent that inhibits PD- 1 is a PD-1 binding antagonist.

[0206] Embodiment 23. The method of embodiment 22, wherein the PD-1 binding antagonist is an antibody, antibody-drug conjugate, antibody fragment, or immunoadhesin.

[0207] Embodiment 24. The method of embodiment 22, wherein the PD-1 binding antagonist is pembrolizumab, nivolumab, cemiplimab, spartalizumab, genolimzumab, SHR1210, JS001, BGB-108, BGB-A317, IBI308, GLS-010, BMS-936558, BCD-100, REGN2810, MGA- 012, BI 754091, STI-Al l 10, INCSHR-1210, PF-06801591, TSR-042, AM0001, JNJ-63723283, or ENUM 244C8.

[0208] Embodiment 25. The method of embodiment 19, wherein the checkpoint inhibitor is an agent that inhibits PD-L1 and / or PD-L2.

[0209] Embodiment 26. The method of embodiment 25, wherein the agent that inhibits PD- L1 and / or PD-L2 is a small molecule, a nucleic acid, a polypeptide, carbohydrate, a lipid, a metal, or a toxin.

[0210] Embodiment 27. The method of embodiment 26, wherein the agent that inhibits PD- L1 is a PD-L1 binding antagonist.

[0211] Embodiment 28. The method of embodiment 27, wherein the PD-L1 binding antagonist is an antibody, antibody-drug conjugate, antibody fragment, or immunoadhesin.

[0212] Embodiment 29. The method of embodiment 27, wherein the PD-L1 binding antagonist is atezolizumab, avelumab, durvalumab, KN035, CS1001, MDX-1105, LY3300054, STI-A1014, FAZ053, or CX-072.

[0213] Embodiment 30. The method of embodiment 19, wherein the checkpoint inhibitor is an agent that inhibits CTLA4.

[0214] Embodiment 31. The method of embodiment 30, wherein the agent that inhibits CTLA4 is a small molecule, a nucleic acid, a polypeptide, carbohydrate, a lipid, a metal, or a toxin.

[0215] Embodiment 32. The method of embodiment 31, wherein the agent that inhibits CTLA4 is an antibody, antibody-drug conjugate, antibody fragment, or immunoadhesin.

[0216] Embodiment 33. The method of embodiment 31, wherein the agent that inhibits CTLA4 is ipilimumab, APL-509, AGEN1884, or CS1002.

[0217] Embodiment 34. The method of any one of embodiments 1-33, wherein the sample comprises a tissue biopsy sample or a liquid biopsy sample.

[0218] Embodiment 35. The method of embodiment 34, wherein the sample is a tissue biopsy and comprises a tumor biopsy, tumor specimen, or circulating tumor cells.

[0219] Embodiment 36. The method of embodiment 34, wherein the sample is a liquid biopsy sample and comprises blood, serum, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva.

[0220] Embodiment 37. The method of any one of embodiments 1-36, wherein the sample comprises cells and / or nucleic acids from the cancer.

[0221] Embodiment 38. The method of embodiment 37, wherein the sample comprises mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, cell-free RNA from the cancer, or any combination thereof.

[0222] Embodiment 39. The method of embodiment 37, wherein the sample is a liquid biopsy sample and comprises circulating tumor cells (CTCs).

[0223] Embodiment 40. The method of embodiment 37, wherein the sample is a liquid biopsy sample and comprises cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof.

[0224] Embodiment 41. The method of any one of embodiments 1-40, further comprising obtaining the sample from the individual.

[0225] Embodiment 42. The method of any one of embodiments 1-41, wherein EBV-2+ status is detected in the sample by sequencing nucleic acids encoding EBV-2 nuclear antigens from the sample.

[0226] Embodiment 43. The method of embodiment 42, wherein EBV-2+ status is detected in the sample by sequencing nucleic acids encoding an EBV-2 Epstein-Barr nuclear antigen 3 (EBNA-3) gene from the sample.

[0227] Embodiment 44. The method of embodiment 42 or embodiment 43, wherein the sequencing comprises a massively parallel sequencing (MPS) technique, whole genomesequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique; and optionally wherein the massively parallel sequencing (MPS) technique comprises next-generation sequencing (NGS).

[0228] Embodiment 45. The method of any one of embodiments 1-44, wherein the sample is an enriched sample.

[0229] Embodiment 46. The method of any one of embodiments 42-45, wherein the sequencing comprises: (a) providing a plurality of nucleic acid molecules obtained from the sample, wherein the plurality of nucleic acid molecules comprises a mixture of tumor nucleic acid molecules and non-tumor nucleic acid molecules; (b) optionally, ligating one or more adapters onto one or more nucleic acid molecules from the plurality of nucleic acid molecules; (c) amplifying nucleic acid molecules from the plurality of nucleic acid molecules; (d) optionally, capturing nucleic acid molecules from the amplified nucleic acid molecules, wherein the captured nucleic acid molecules are captured from the amplified nucleic acid molecules by hybridization to one or more bait molecules; and (e) sequencing, by a sequencer, the captured nucleic acid molecules to obtain a plurality of sequence reads corresponding to EBV-2 nucleic acids in the sample.

[0230] Embodiment 47. The method of embodiment 46, wherein the adapters comprise one or more of amplification primer sequences, flow cell adapter hybridization sequences, unique molecular identifier sequences, substrate adapter sequences, or sample index sequences.

[0231] Embodiment 48. The method of embodiment 46 or embodiment 47, wherein amplifying nucleic acid molecules comprises performing a polymerase chain reaction (PCR) technique, a non-PCR amplification technique, or an isothermal amplification technique.

[0232] Embodiment 49. The method of any one of embodiments 46-48, wherein the one or more bait molecules comprise one or more nucleic acid molecules, each comprising a region that is complementary to a region of a captured nucleic acid molecule.

[0233] Embodiment 50. The method of any one of embodiments 46-49, wherein the one or more bait molecules each comprise a capture moiety.

[0234] Embodiment 51. The method of embodiment 50, wherein the capture moiety is biotin.

[0235] Embodiment 52. The method of any one of embodiments 1-51, wherein the individual is a human.EXAMPLESExample 1: HLA-ILOH prevalence in EBV-positive tumors

[0236] Patients with oncoviral infections are predicted to be good candidates for immunotherapy since viral antigen presentation is believed to be a mechanism akin to tumor neoantigen presentation. Patients who are tumor mutational burden (TMB) low are often not considered good candidates for immune checkpoint inhibitor (ICI) therapy. However, virus-positive patients, who are often TMB low, may be a subset of TMB low patients who could be good ICI responders. Previous research indicated that patients who were virus-positive tended to have higher rates of HLA-I LOH than patients who were virus-negative, suggesting that viral antigen presentation is highly immunogenic and that HLA-I LOH was a mechanism of immune evasion in these patients (Montesion, M. et al. (2021) Cancer Discov. 11(2):282-292).

[0237] Epstein-Barr virus (EBV) is a ubiquitous pathogen that, despite resulting largely in asymptomatic infections, can be linked to the development of certain cancers such as lymphoma, nasopharyngeal carcinoma, and gastric carcinoma. Although some studies suggest that patients with oncoviral infections exhibit superior responses to immunotherapy, information is lacking regarding whether response differs based on viral subtype.

[0238] Montesion, M. et al. at Figure 3C showed the prevalence of HLA-I LOH in tumor types with known oncoviral associations, including HPV+ head and neck squamous cell carcinoma (HNSCC), HPV+ cervical cancer, EBV+ gastric cancer, EBV+ nasopharyngeal cancer, and HBV+ hepatocellular cancer. While some tumor types such as HPV+ HNSCC, HPV+ cervical, and EB V+ gastric cancer showed a significant association between viral infection and prevalence of HLA-I LOH, other cancer types such as EBV+ nasopharyngeal cancer did not.

[0239] Example 1 investigates whether EBV infection status is correlated with HLA-I LOH in various cancers. Specifically, real world clinico-genomics data were used to assess whether EBV subtypes differ in rate of co-occurring mechanisms of immune evasion as well as overall survival (OS) after treatment with an immune checkpoint inhibitor (ICI).Materials and methods

[0240] Patients underwent comprehensive genomic profiling as part of routine clinical care. EBV detection was performed by competitively mapping sequencing reads acquired by adaptor- ligated hybridization capture to the NCBI nucleotide database. A positive EBV status (EBV+)was assigned to samples with de novo assembled contigs >80 nucleotides in length that had a >97% identity to the BLAST sequence. Subtyping was performed by referencing the accession number of the mapped sequence to previously published phylogenetic EBV classifications (see, e.g., Zanella, L. et al. (2019) Sci Reports 9:9829). Genomic ancestry was predicted using a single nucleotide polymorphism approach and a sample was considered positive for HLA-I LOH if at least one of the HLA class I (HLA-I) genes was predicted to be under loss of heterozygosity (LOH). Patient outcomes were assessed using a nationwide (US-based) gastric clinico-genomic database containing de-identified data that originated from -280 US cancer clinics (-800 sites of care).Results

[0241] The hypothesis that EBV infection status was not significantly associated with HLA-I LOH in previous studies because of differing patterns due to EBV subtype was investigated. There are two major subtypes of EBV (subtype 1 and subtype 2). EBV type 1 is known as the dominant subtype, whereas EBV type 2 is more common in certain regions like central Africa and is more often reported as dual infection with EBV type 1.

[0242] Of the 21,034 EBV+ samples that were subtyped, 3,049 (14.5%) were co-positive for both EBV-1 and EBV-2 (EBV1 / 2+) while all other samples were positive for EBV-1 alone (EBV1+). No sample was identified as having only an EBV-2 infection. No significant difference was detected in the median age of patients that were EBV1+ (62 years) vs EBV1 / 2+ (63 years). However, EBV1+ patients were more likely to be female (52.8%) than EBV1 / 2+ patients (41.7%) (P<0.001). When assessing EBV subtype prevalence across the five genomic superpopulations, patients who were predominantly East Asian were found to have significantly lower rates of EBV1 / 2+ (11.8%), and patients who were predominantly South Asian had significantly higher rates of EBV1 / 2+ (16.9%) as compared to the other genomic ancestries (African (14.1%), Admixed American (14.7%), and European (14.8%)) (P=0.04).

[0243] To investigate if EBV subtypes elicit different immune selective pressures, rates of HLA- I LOH were compared in nasopharyngeal and gastric carcinoma. In both tumor types, the rate of HLA-I LOH was significantly higher in EBV1 / 2+ patients as compared to EBV-negative (EBV-) patients (24.2% vs 5.9% in nasopharyngeal (P=0.02, HR=5.1) and 29.2% vs 17.8% in gastric(P=0.05, HR=1.9). In contrast, this trend was not observed in the EBV1+ patients within either tumor type (HLA-I LOH rates: 0% in nasopharyngeal and 21.6% in gastric).

[0244] To assess any clinical implications of these findings, a preliminary analysis was performed in the Clinico-Genomic Database (CGDB). OS was assessed in a subset of patients with gastric carcinoma from the start of advanced line ICI monotherapy. Patients who were EBV1 / 2+ (n=12) had significantly longer OS as compared to patients who were EBV- (n=127), tripling the median OS (mOS) from 3.2 months in the EBV- cohort to 9.5 months in the EBV1 / 2+ cohort (P=0.04, HR=0.47). By comparison, patients who were EBV1+ (n=12) had no improvement in OS as compared to patients who were EBV- (mOS=3.3 months, P=0.8, HR=0.9). Without wishing to be bound to theory, it is thought that these results may indicate that EBV1 / 2+ patients elicit a stronger immune response than EBV1+ patients, and that EBV1 / 2+ status can be used as a positive biomarker of ICI response in advanced gastric cancer.

[0245] The number of nasopharyngeal cancer samples was low, and overall survival was evaluated from the start of ICI monotherapy. As shown in FIGS. 1A & IB, no difference in overall survival was observed when comparing EBV+ vs EBV- gastric patients when all EBV subtypes were grouped together. However, a statistically significant difference in overall survival was observed when patients were separated as being EBV-1+ vs. EBV-1 / 2+ (FIGS. 2A & 2B). Patients who were EBV-1 and EBV-2 co-positive had a significantly longer overall survival than patients who were positive for EBV-1 alone or negative for EBV entirely.

[0246] It should be understood from the foregoing that, while particular implementations of the disclosed methods have been illustrated and described, various modifications can be made thereto and are contemplated herein. It is also not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the preferable embodiments herein are not meant to be construed in a limiting sense. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. Various modifications in form and detail of the embodiments of the invention will be apparent to a person skilled in the art. It is therefore contemplated that the invention shall also cover any such modifications, variations and equivalents.

Claims

CLAIMSWhat is claimed is:

1. A method of treating or delaying progression of cancer in an individual, comprising: detecting EBV-2+ status in a sample from the individual; and administering to the individual an effective amount of an immunotherapy.

2. A method of treating or delaying progression of cancer in an individual, comprising: acquiring knowledge of at least Epstein-Barr virus (EBV) subtype 2-positive (EBV-2+) status in a sample from the individual; and administering to the individual an effective amount of an immunotherapy responsive to said knowledge.

3. A method of identifying one or more treatment options for an individual having cancer, comprising detecting EBV-2+ status in a sample from the individual; and generating a report comprising one or more treatment options identified for the individual based at least in part on the detection of EBV-2+ status in the sample, wherein the one or more treatment options comprise an immunotherapy.

4. The method of claim 1, further comprising acquiring knowledge of or detecting EBV-1+ status in a sample from the individual.

5. The method of claim 4, wherein the method comprises detecting EBV subtype 1 and EBV subtype 2-positive (EBV-1 / 2+) status in a sample from the individual.

6. The method of claim 1, wherein the cancer exhibits HLA-I LOH.

7. The method of claim 1, further comprising acquiring knowledge of or detecting HLA-I LOH in a sample obtained from the cancer of the individual.

8. The method of claim 1, further comprising acquiring knowledge of or detecting tumor mutational burden (TMB) in a sample obtained from the cancer of the individual.

9. The method of claim 1, wherein the cancer is a TMB low cancer, optionally wherein the cancer has a TMB of less than 10 mutations per megabase (muts / Mb).

10. The method of claim 1, wherein the cancer is gastric or nasopharyngeal cancer, optionally wherein the cancer is advanced or metastatic.

11. The method of claim 1, wherein the immunotherapy is administered as a monotherapy.

12. The method of claim 1, wherein the immunotherapy comprises one or more of a checkpoint inhibitor, cancer vaccine, cell-based therapy, T cell receptor (TCR)-based therapy, adjuvant immunotherapy, cytokine immunotherapy, and oncolytic virus therapy.

13. The method of claim 12, wherein the immunotherapy comprises a checkpoint inhibitor that targets PD-L1, PD-1, CTLA-4, CEACAM, LAIR1, CD160, 2B4, CD80, CD86, CD276, VTCN1, HVEM, KIR, A2AR, MHC class I, MHC class II, GALS, adenosine, TGFR, 0X40, CD137, CD40, IDO, CSF1R, TIM-3, BTLA, VISTA, LAG-3, TIGIT, IDO, MICA / B, or arginase; optionally wherein the checkpoint inhibitor is an agent that inhibits PD-1, an agent that inhibits PD-L1 and / or PD-L2, or an agent that inhibits CTLA4.

14. The method of claim 1, wherein the sample comprises a tissue biopsy sample or a liquid biopsy sample; and / or wherein the sample comprises cells, nucleic acids, or cells and nucleic acids from the cancer.

15. The method of claim 14, wherein the sample is a tissue biopsy and comprises a tumor biopsy, tumor specimen, or circulating tumor cells; or wherein the sample is a liquid biopsy sample and comprises blood, serum, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva.

16. The method of claim 14, wherein the sample:(a) comprises mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, cell-free RNA from the cancer, or any combination thereof;(b) is a liquid biopsy sample and comprises circulating tumor cells (CTCs); or(c) is a liquid biopsy sample and comprises cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof.

17. The method of claim 1, wherein EBV-2+ status is detected in the sample by sequencing nucleic acids encoding EBV-2 nuclear antigens from the sample, optionally wherein EBV-2+ status is detected in the sample by sequencing nucleic acids encoding an EBV-2 Epstein-Barr nuclear antigen 3 (EBNA-3) gene from the sample.

18. The method of claim 1, wherein the sample is an enriched sample.

19. The method of claim 17, wherein the sequencing comprises: (a) providing a plurality of nucleic acid molecules obtained from the sample, wherein the plurality of nucleic acid molecules comprises a mixture of tumor nucleic acid molecules and non-tumor nucleic acid molecules; (b) optionally, ligating one or more adapters onto one or more nucleic acid molecules from the plurality of nucleic acid molecules; (c) amplifying nucleic acid molecules from the plurality of nucleic acid molecules; (d) optionally, capturing nucleic acid molecules from the amplified nucleic acid molecules, wherein the captured nucleic acid molecules are captured from the amplified nucleic acid molecules by hybridization to one or more bait molecules; and (e) sequencing, by a sequencer, the captured nucleic acid molecules to obtain a plurality of sequence reads corresponding to EBV-2 nucleic acids in the sample.

20. The method of claim 1, wherein the individual is a human.