Precision-engineered t cells targeting cancer and viral antigens and methods of using them

Abstract

Methods of obtaining a population of cells enriched in CD4+ and / or CD8+ T-cells specific for a pre-determined antigen and populations of cells enriched in CD4+ and / or CD8+ T-cells specific for a pre-determined antigen useful in immunotherapies.

Description

Docket: 93597 / 7618 - 92499-A-PCTPRECISION-ENGINEERED T CELLS TARGETING CANCER AND VIRAL ANTIGENS AND METHODS OF USING THEM CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 779,704, filed March 28, 2025, the entirety of which is hereby incorporated by reference.BACKGROUND OF THE INVENTION

[0002] Throughout this application, various publications are referenced, including referenced in parenthesis. The disclosures of all publications mentioned in this application in their entireties are hereby incorporated by reference into this application in order to provide additional description of the art to which this invention pertains and of the features in the art which can be employed with this invention.

[0003] Typically, tumor infiltrating lymphocyte (TIL) or virus specific T cell (VST) products are created from the mixed T cell populations containing only rare antigen-specific precursors. Conventional polyclonal T cells usually exhibit high patient-to-patient variability and limited potency, as they are dominated by non-specific “passenger” cells, whereas only a relatively minor fraction of the immune effectors displays the desired antigenic specificity. Irrelevant cells not only diminish / dilute the overall potency of the product, but they might be actively detrimental by acting as competing “cytokine sinks”, promoting senescence and contributing to off-target toxi cities, such as GVHD in the allogeneic or “off-the-shelf’ setting, thus necessitating low cell doses as safety precautions. Consequently, partial or failed clinical responses are often seen, whereas complete responses remain rare. Elimination of competing irrelevant T cells is needed to increase potency and uniformity of the cell therapy product, but clinical-grade cell sorting remains costly and complex. Thus, efficient, simplified, and cost-effective strategies are needed.BRIEF SUMMARY OF THE INVENTION

[0004] TNFa is an effector cytokine rapidly and ubiquitously produced by antigen-activated T cells. Unlike other cytokines, TNFa is exported from the cytoplasm but remains anchored to the cell membrane (Fig. 1). The final release step requires an active cleavage by a matrix metalloproteinase known as ADAMI 7 or TNFa-converting enzyme (TACE). Without this proteolytic step TNFa remains in a transmembrane form (TM-TNFa). Pharmacological inhibition14914-3364-4440v.lof TACE induces retention of TM-TNFa on the surface of the activated cells, thus antigen-specific T cells can be identified using common anti-TNFa antibodies. Many pharmacological TACE inhibitors (e.g. TAPI-1) exist and some have been tested as candidate anti-inflammatory agents in patients with lupus and rheumatoid arthritis. The biological impact of TACE inhibition and TM-TNFa on differentiation, phenotypes and functionality of the ex vivo expanded immune effector T cells has not been studied and the net effects of this manipulation are unclear. However, TM-TNFa retains biological activity, binding the TNF Receptor 2 (TNFR2) as well to the TNFR1, whereas soluble TNFa preferentially binds to TNFR1. Furthermore, TM-TNFa might also act as an active immune receptor in a retrograde manner (Fig.2), likely with pro-survival and anti-apoptotic signaling effects. The capture strategy described herein can actively reprogram the T cells for improved functionality. TACE / AD AMI 7 and a closely related ADAM 10 have a highly pleotropic effects, targeting multiple cell membrane bound and soluble molecules, such as TNFR2, IL-6R, IL2R, interferon (IFN)y, NOTCH and dozens of other proteins modulating the immune microenvironment.

[0005] Here we disclose a novel simplified method for highly efficient capture of the antigenspecific T cells with enhanced potency for use in manufacturing of multi-epitope specific antiviral and anti-cancer (TIL and TIL-like) T cell products using TM-TNFa as the isolation marker.

[0006] Clinical-grade cell sorting is needed to improve the potency of TILs and similar multispecific T cells, but it has not been broadly adapted due to many limitations including low efficiency, technical complexities, excessive cost, time and labor intensity. Several methods for identification and isolation of antigen-specific T cells are currently in use. Multimer labeling identifies T cells with specificity for a single epitope recognized in the context of its restriction element, but is not applicable when broad polyclonal responses are desired, especially in the context of MHC class II. Activation induced markers (AIM) such as CD137 (4-1BB) can be used for isolation of reactive T cells although prolonged / toxic (app. 12-24hrs) antigenic stimulation period is needed . Interferon y (IFN-y) capture is commercially available, but is a complex method, only applicable to rare (<5%) populations to avoid false positive labeling and relatively inefficient, as only a fraction of relevant T cells might secrete IFN-y, whereas TNF-a is usually produced by majority of specific human T cells.24914-3364-4440v.l

[0007] The strategy described herein has not been previously exploited in the setting of manufacturing of the effector T cells for therapeutic use, and offers practical and biological advantages over the standard methods:(a) Labeling is highly efficient, as TNFa is the most ubiquitous effector cytokine produced by human immune effectors, secreted by most antigen-specific T cells upon stimulation.(b) The procedure is simple, with only a few steps and a short (e.g., 4hr) stimulation time.(c) Equally applicable to T cell populations containing low and high (>5%) frequency of specific T cell precursors of interest without the risk for false positive labeling. (d) Broadly applicable as a stand-alone system for engineering of specific T cells directly from blood or ex vivo cultures for downstream processing, TCR identification, generation of GVHD-free “off-the-shelf’ VST T cells and as a rapid immunomonitoring or potency assay etc.

[0008] TACE inhibition and TM-TNFa capture process may actively reprogram the immune effectors for superior activity (Fig. 2). Specifically, TM-TNFa can behave as a costimulatory molecule promoting enhanced functionality, survival, and self-renewal, and likely resulting in improved in vivo potency of isolated T cells unlike traditional expansion and selection methods that foster senescence of the immune effectors.

[0009] In some embodiments, the techniques described herein relate to a method of obtaining a population of cells enriched in CD4+ and / or CD8+ T-cells specific for a pre-determined antigen, the method including contacting a preparation of cells including T-cells obtained from a tumor environment in a subject, a viral infection site in a subject, or from peripheral blood of a subject having a tumor or a viral infection, with an agent that captures transmembrane TNF alpha (TM-TNFalpha), (a) in the presence of, or wherein said cells have been previously contacted with, a TACE inhibitor or ADAMS 17 inhibitor or (b) in the presence of an agent that blocks the TACE cleavage site on a TM-TNFalpha, so as to thereby isolate CD4+ and / or CD8+ T-cells specific for the pre-determined antigen, so as to obtain the population.

[0010] In some embodiments, the techniques described herein relate to a method of selectively depleting a T-cell population for stem cell transplant immune reconstitution of allo-reactive T-34914-3364-4440v.lcells in a subject, the method including contacting the T-cell population for stem cell transplant immune reconstitution with an agent that captures transmembrane TNF alpha, wherein said cells have been previously contacted with, or in the presence of, a TACE inhibitor or ADAMS 17 inhibitor or presence of an agent that blocks the TACE cleavage site on a TM- TNF alpha, so as to thereby remove allo-reactive T-cells from the population.

[0011] In some embodiments, the techniques described herein relate to a population of cells enriched in CD4+ and / or CD8+ T-cells specific for a pre-determined antigen versus a natural population of CD4+ and / or CD8+ T-cells specific for the pre-determined antigen, wherein the population exhibits enhanced IL-2 production relative to a predetermined control level of IL-2 production associated with a naturally-occurring CD4+ and / or CD8+ T-cell population.

[0012] In some embodiments, the techniques described herein relate to a method of treating a tumor in a subject including administering to the subject a population of cells enriched in CD4+ and / or CD8+ T-cells specific for an antigen of the tumor.

[0013] In some embodiments, the techniques described herein relate to a method of treating an infection in a subject including administering to the subject a population of cells enriched in CD4+ and / or CD8+ T-cells specific for an antigen of a pathogen associated with the infection.

[0014] In some embodiments, the techniques described herein relate to the method of any of Clams 1-19, 21-23, or 27-40, wherein the subject is a human.

[0015] In some embodiments, the techniques described herein relate to a method of obtaining a population of cells enriched in CD4+ and / or CD8+ T-cells specific for tumor antigen(s), the method including contacting a preparation of cells including T-cells wherein said cells have been stimulated, are stimulated, or re-stimulated with a whole tumor cell, a tumor lysate thereof, or an antigen presenting cell, optionally a dendritic cell, that has been pulsed or loaded with a tumor lysate, with an agent that captures transmembrane TNFalpha (TM-TNFalpha), (a) in the presence of, or wherein said cells have been previously contacted with, a TACE inhibitor or ADAMS 17 inhibitor or (b) in the presence of an agent that blocks the TACE cleavage site on a TM-TNFalpha, so as to thereby isolate CD4+ and / or CD8+ T-cells specific for an antigen of the tumor or tumor lysate so as to obtain the population.44914-3364-4440v.l

[0016] In some embodiments, the techniques described herein relate to a method of obtaining a population of cells enriched in CD4+ and / or CD8+ T-cells specific for senescence-associated antigen(s), the method including contacting a preparation of cells including T-cells wherein said cells have been stimulated, are stimulated, or re-stimulated with, a whole senescent cell, a senescent cell lysate, or an antigen presenting cell, optionally a dendritic cell, that has been pulsed or loaded with apoptotic body(ies) or senescent cell lysate, with an agent that captures transmembrane TNF alpha (TM-TNF alpha), (a) in the presence of, or wherein said cells have been previously contacted with, a TACE inhibitor or ADAMS 17 inhibitor or (b) in the presence of an agent that blocks the TACE cleavage site on a TM-TNF alpha, so as to thereby isolate CD4+ and / or CD8+ T-cells specific for senescence-associated antigen(s) so as to obtain the population.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Further objects, features and advantages of the present disclosure will become apparent from the following detailed description taken in conjunction with the accompanying Figures showing illustrative embodiments of the present disclosure, in which:

[0018] Fig. 1 depicts a schematic representation of TNFa secretion via active cleavage mediated by TACE. TACE / ADAM17 inhibitor (TAPI-1) induces retention of TNFa on the cell surface in a transmembrane form (TM-TNF a).

[0019] Fig. 2 depicts hypothetical pro-survival effect of TM-TNFa retrograde signaling; adapted from Zhang, et. al.

[0020] Fig. 3 depicts kinetics of TM-TNFa labeling of Cytomegalovirus (CMV)-specific T cells in ex vivo expanded VST product specific for the immunodominant CMV antigens. CMV-VST cells from two representative donors are shown.

[0021] Fig. 4 depicts generation of highly enriched CMV-specific VST cells from in vitro expanded standard CMV-VST cells. CMV-VST cells were generated in G-Rex bioreactors from healthy donor PBMCs by stimulation with CMV pp65 and IE1 pepmixes. T cells were maintained in media containing IL-7, IL-15 and IL-2 (added at 72hrs) for 14 days. CMV-VSTs were stimulated pp65 and IE1 antigens as described in Fig 3 for 4 hrs in presence of TAPL1 and anti-TNFa antibody, purified by flow cytometric sorting and placed in media with cytokines for 1454914-3364-4440v.ldays. Final T cells were tested for reactivity (see Fig 3). Intracellular TNFa and IL-2 secretion are shown for sorted and unsorted populations. Representative experiment.

[0022] Figs. 5A-5C depicts TM-TNFa-based generation of VST cells targeting Epstein-Barr Virus (EBV) oncoproteins for adoptive immunotherapy of EBV-mediated hematological malignancies (e.g. EBV-PTLD; EBV-lymphoma) Cells were expanded for 14 days in presence of indicated EBV peptide libraries. Cells were then challenged with the same antigens and sorted using TM-TNF capture method or left unsorted. A. Comparison of EBV-specific T cell frequency between sorted and unsorted EBV-VST Thl cultures at 14 days post-sort. B. Representative dot plots showing comparison of sorted and unsorted EBV specific T cells. C. Comparison of %IL2 among antigen-reactive (TNFaHI) EBV-VST cells.

[0023] Figs. 6A-6F depicts identification and generation of virus-specific T cells directly from peripheral blood: A. Dot plots showing antigen specific upregulation of indicated activation markers (AIM) upon stimulation with the immunodominant antigens of indicated viral pathogens as compared to TM-TNFa labeling. PBMCs were stimulated with indicated antigens overnight for CD 134, CD 137, CD 154 and for 4 hours for TNFa in presence of TAPI1. Analyses was performed upon gating on viable CD3+ T cells. B. Comparison of frequency of T cells upregulating the indicated AIM various activation markers upon antigen stimulation. C-F. CMV-pp65 reactive cells were sorted from donor PBMCs using TM-TNFa capture method and expanded for 14 days. Cells were counted and analyzed for cytokine production by intracellular flow cytometric staining and enrichment.

[0024] Figs. 7A-7C depicts TM-TNFa-based generation of T cells targeting PRAME cancer testis antigen for adoptive immunotherapy of cancer and hematological malignancies A Comparison of PRAME- specific T cell frequency between baseline and post-sort PRAME specific T cell population. B. Representative dot plots showing comparison of sorted and unsorted EBV specific T cells. Cells were expanded for 14 days in presence of indicated EBV peptide libraries. Cells were then challenged with the same antigens and sorted using TM-TNF capture method or left unsorted. C. (Left panel) Percent enrichment after further expansion of 14days post sorting. C. (Right panel) Comparison of %IL-2 secreting T cells among TNFaHIT cells as a measure of polyfunctionality (n=7).64914-3364-4440v.l

[0025] Fig. 8 depicts TM-TNFa-based labeling identifies higher frequency of tumor-reactive T cells as compared to the standard CD 154 AIM approach. CD4+ T cells stimulated for two rounds with antigen presenting cells pulsed with peptide libraries of cancer testis antigens (PRAME, MAGE-A3, WT-1, NYESO1) and expanded for 24 days in vitro. Cells were then re-challenged with antigens in presence of TAPI-1 for 4 hours and expression of surface TNFa and CD154 was analyzed. A. Comparison of CD154+ and TNF+ CD4+ T cells in three donors. B. Dot plot showing antigen-specific upregulation of CD154 and TNFa.

[0026] Fig. 9 TM-TNFa-based manufacture of high-potency T cells with enhance polyfunctionality for adoptive immunotherapy of Merkel Cell Carcinoma (MCC): CD4+ T cells were stimulated for two rounds with antigen presenting cells (DCs) pulsed with peptide libraries representing MCPy V LTt viral oncoprotein and expanded in vitro in presence of proinflammatory cytokines. T-cells were then re-challenged with LTt antigen in presence of TAPI-1 TACE inhibitor for 4 hours and labeled with anti-TNFa PE monoclonal antibody. TM-TNFaHIand TM-TNFaLOT cells were sorted or left unsorted as control. Sorted or Unsorted cells were then expanded for 14 days before testing for antigen-specific reactivity by intracellular secretion assay to measure TNFa, IFN-y, IL-2 and GZMB production upon LTt stimulation.

[0027] Figs. 10A-10B depicts kinetics of TM-TNFa labeling of CMV-specific T cells in a VST product (A) and purification of T cells from MCC TAA-specific T cell cultures generated ex vivo (B).DETAILED DESCRIPTION OF THE INVENTIONDefinitions and general

[0028] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.74914-3364-4440v.l

[0029] In the discussion unless otherwise stated, adjectives such as “substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. In some embodiments, about means within a standard deviation using measurements generally acceptable in the art. In some embodiments, about means a range extending to + / - 10% of the specified value. In some embodiments, about includes the specified value. Unless otherwise indicated, the word “or” in the specification and claims is considered to be the inclusive “or” rather than the exclusive or, and indicates at least one of and any combination of items it conjoins.

[0030] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a,” “an” and “at least one” are used interchangeably in this application.

[0031] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0032] In the description and claims of the present application, each of the verbs, “comprise,” “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb. Other terms as used herein are meant to be defined by their well-known meanings in the art.

[0033] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections. All combinations of the various elements disclosed herein are within the scope of the invention.84914-3364-4440v.lOverview of methods and cells

[0034] In some embodiments, the present invention includes methods of obtaining a population of cells enriched in CD4+T cells, or CD8+ T cells, or both, specific for a pre-determined antigen.

[0035] In some embodiments, the present invention includes methods of selectively depleting a T cell population for stem cell transplant immune reconstitution of allo-reactive T cells in a subject

[0036] In some embodiments, the present invention includes a population of cells enriched in CD4+ and / or CD8+ T-cells specific for a pre-determined antigen versus a natural population of CD4+ and / or CD8+ T-cells specific for the pre-determined antigen.

[0037] In some embodiments, the present invention includes methods of treating a tumor in a subject comprising administering to the subject a population of cells enriched in CD4+ and / or CD8+ T-cells specific for an antigen of the tumor.

[0038] In some embodiments, the present invention includes methods of treating an infection in a subject comprising administering to the subject a population of cells enriched in CD4+ and / or CD8+ T-cells specific for an antigen of a pathogen associated with the infection.T-cells

[0039] In some embodiments, the present invention is directed to a population of cells enriched in a T-cells specific for a pre-determined antigen, or methods of obtaining the population.

[0040] In some embodiments, the T-cells produce TNFa. In some embodiments, the T-cells include one or both of CD4+T cells, or CD8+ T cells. In some embodiments, the T-cells are CD4+T cells, or CD8+ T cells, or both. In some embodiments, the T-cells are specific for a predetermined antigen. In some embodiments, the T-cells are TIL, or TIL like, or are VST.

[0041] In some embodiments, a natural population of cells contains 5% or less CD4+ and / or CD8+ T-cells specific for the pre-determined antigen. In some embodiments, the natural population of cells contains 5% or more CD4+ and / or CD8+ T-cells specific for the pre-determined antigen. In some embodiments, the T-cells are contained in a preparation of cells which comprises T-cells. In some embodiments, the preparation is obtained from a tumor environment in a subject,94914-3364-4440v.la viral infection site in a subject, or from peripheral blood of a subject having a tumor or a viral infection.Antigens

[0042] In some embodiments, the present invention is directed to a population of cells enriched in a T-cells specific for a pre-determined antigen. In some embodiments, the antigen is a human cancer antigen, viral antigen, bacteria antigen, parasite antigen, or a fungal antigen. In some embodiments, the antigens may also be associated with its label, ex. a fungal antigen may be associated with a fungus. In some embodiments, the antigens may be amino acid sequence identical to the antigen.

[0043] In some embodiments of the viral antigen, the virus is a virus that infects humans. In some embodiments the antigen is an antigen of a bacteria or a parasite or a fungus. In some embodiments the bacteria or a parasite or a fungus is one that infects humans.Human tumor antigens

[0044] Human tumor antigens are well known in the art, for example see www.zhang-lab.org / caatlas / caantigen (1007 members as of March 27, 2025 - The cancer antigen atlas (caAtlas)). See Yi, X., Liao, Y., Wen, B„ Li, K., Dou, Y., Savage, S. R., & Zhang, B. (2021). caAtlas: an immunopeptidome atlas of human cancer. Iscience, 24. https: / / doi.Org / 10.1016 / j.isci.2021.103107, hereby incorporated by reference. See also the cancer testis CTantigen list at www.zhang-lab.org / caatlas / ctantigen and post-translationally-modified PTM antigen list at www.zhang-lab.org / caatlas / ptmantigen. In some embodiments, the tumor antigen includes one or more of the following: PRAME, MAGE-A3, WT-1, NYESO1, a KRAS mutation-derived neoantigen, a BRAF mutation-derived neoantigen, MCPyV Large T antigen, or truncated MCPyV Large T antigen.

[0045] In some embodiments, the tumor is a hematological malignancy or a solid tumor, including melanoma, acute myeloid leukemia, myelodysplastic syndrome, or Merkel Cell Carcinoma.Viral antigens

[0046] In some embodiments, viral antigens are viruses or compounds associated with a virus or a viral infection. Human viruses are known in the art and include the listing at104914-3364-4440v.lviralzone.expasy.org / 678. Human viruses include Adeno-Associated Virus (AAV), Adenovirus, African Swine Fever Virus (ASFV), Chikungunya Virus, Coronavirus, Coxsackie Virus, Crimean-Congo Hemorrhagic Fever Virus, Cytomegalovirus (CMV), Dengue Virus, Eastern Equine Encephalitis Virus (EEEV), Ebola Virus, Echovirus, Enterovirus, Epstein-Barr Virus (EBV), Hepatitis A Virus (HAV), Hepatitis B Virus (HBV), Human Cytomegalovirus (HCMV), Hepatitis C Virus (HCV), Hepatitis D Virus (HDV), Hepatitis E Virus (HEV), Human Immunodeficiency Virus (HIV), Human Papillomavirus (HPV), Herpes Simplex Virus (HSV), Human T-Lymphotropic Virus (HTLV), Influenza A Virus, Influenza B Virus, Japanese Encephalitis Virus, Leukemia Virus, Marburg Virus, Measles Virus, Metapneumovirus, Molluscum Contagiosum Virus, Mumps Virus, Nipah Virus, Norovirus, Orf Virus, Parainfluenza Virus, Parvovirus, Poliovirus, Rabies Virus, Respiratory Syncytial Virus (RSV), Rhinovirus, Rift Valley Fever Virus, Rotavirus, Rubella Virus, Simian Immunodeficiency Virus (SIV), Tick-Borne Encephalitis Virus (TBEV), Tobacco Etch Virus, Varicella Zoster Virus (VZV), Variola Virus, Venezuelan Equine Encephalitis Virus (VEEV), West Nile Virus, Yellow Fever Virus, or Zika Virus.

[0047] In some embodiments, the viral antigen includes one or more of the following: CMV pp65, CMV IE1, EBV EBNA1, EBV LMP1, EBV LMP2, MCPyV Large T antigen, MCPyV truncated Large T antigen, BKV Large T antigen, BKV VP1, SARS-CoV-2 Spike, SARS-CoV-2 Nucleocapsid, or SARS-CoV-2 Membrane protein.Bacterial antigens

[0048] In some embodiments, bacterial antigens are bacteria or compounds associated with a bacteria or bacterial infection. In some embodiments, bacterial antigens include Burkholderia Pseudomallei antigens, Legionella Pneumophila antigens, Neisseria Gonorrhoeae antigens, Salmonella antigens, Staphylococcus antigens, Ureaplasma Urealyticum antigens, Vibrio Cholerae antigens, Aeromonas antigens, Arthrobacter Globiformis antigens, Bacillus antigens, Bordetella Pertussis antigens, Borrelia antigens, Brucella Abortus antigens, Campylobacter Jejuni antigens, Candida Albicans antigens, Chlamydia Trachomatis antigens, Chlamydophila Pneumoniae antigens, Clostridium Tetani antigens, Corynebacterium Diphtheriae antigens, E. coli antigens, Haemophilus Influenza antigens, Helicobacter Pylori antigens, Leptospira Biflexa antigens, Listeria Monocytogenes antigens, Mycobacterium Tuberculosis antigens, Mycoplasma Pneumoniae antigens, Salmonella Typhimurium antigens, Streptococcus Pneumoniae antigens,114914-3364-4440v.lStreptomyces Avidinii antigens, Treponema Pallidum antigens, or Yersinia Enterocolitica antigens.Fungal antigens

[0049] In some embodiments, fungal antigens are fungus or compounds associated with a fungus or a fungal infection. Fungal antigens include, for example, aspergillus and saccharomyces antigens.Parasitic antigens

[0050] In some embodiments, parasitic antigens are parasites or compounds associated with a parasite or a parasitic infection. Parasitic antigens include: Leishmania antigens, Plasmodium Falciparum antigens, Plasmodium Vivax antigens, Plasmodium antigens, Toxoplasma Gondii antigens, Acanthamoeba antigens, Caenorhabditis Elegans antigens, Echinococcus Granulosus antigens, Malaria antigens, Schistosoma Japonicum antigens, Trichomonas Vaginalis antigens, and, Trypanosoma cruzi (T. cruzi) antigens.Agents that capture transmembrane TNFalpha and / or block the TACE cleavage site

[0051] In some embodiments, the present invention employs an agent that captures transmembrane TNFalpha. In some embodiments, the present invention employs an agent blocks the TACE cleavage site on a TM-TNFalpha. In some embodiments, the same agent captures transmembrane TNFalpha and blocks the TACE cleavage site on a TM-TNFalpha. In some embodiments, the agent comprises an anti-TNF alpha antibody or anti-TNF alpha aptamer.

[0052] TNFalpha antibodies, including monoclonal antibodies, that bind TMTNFalpha are well-known in the art, including the medical literature. Laboratory-ready TNFalpha antibodies can be bought from lab suppliers. Medically-used TNFalpha antibodies are also available. For example, anti-TNFalpha antibodies are available from Bioss Inc., Leinco Technologies, Inc., MyBioSource.com, BosterBio, Santa Cruz Biotechnology, Inc., Biorbyt, BioLegend, GeneTex, Bio-Rad, R&D Systems, Aladdin Scientific, Bioassay Technology Laboratory, Miltenyi Biotec, RayBiotech, LSBio, United States Biological, Antibodies.com, Huabio, Novus Biologicals, Ampersand Biosciences, BD Biosciences, G Biosciences, Elabscience Bionovation Inc., AAT Bioquest, Inc., Fine Biotech Co., Ltd, Ichorbio, AntibodySystem, Biomatik, Cell Sciences, Cedarlane, NSJ Bioreagents, Sino Biological, Inc., Abeam, Abbexa Ltd., Creative Biolabls.124914-3364-4440v.l

[0053] TNFalpha aptamers are known in the art and have been described in the scientific and patent literature. Laboratory-grade TNFalpha aptamers can be synthesized or ordered from commercial oligonucleotide manufacturers and biotechnology suppliers. For example, custom DNA or RNA aptamers targeting TNFalpha can be obtained from companies such as Integrated DNA Technologies, Eurofins Genomics, TriLink BioTechnologies, Base Pair Biotechnologies, Aptamer Group, SomaLogic, and other nucleic acid synthesis providers.TACE or ADAMS17 inhibitors

[0054] In some embodiments, the present invention employs TACE or ADAMS 17 inhibitors.

[0055] TACE inhibitors or ADAMS17 inhibitors are known in the art. For example, TAPI-1 (e.g. from MedChemExpress). See also the list of TACE inhibitors at / www.scbt.com / browse / tace-inhibitors?srsltid=AfmBOopCN4UPZ_vtnPIJqHBdRNU_TKcNB8OQy51J4oVzIznJLiLawAcH as sold by Santa Cruz Biotechnology, including TAPI-2, Marimastat, TAPL0, Batimastat, Prinomastat, RO-4929097 and GW 280264X. TAPL1 is also known as N-(R)-[2-(Hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-L-naphthylalanyl-L-alanine, 2-aminoethyl Amide.

[0056] In some embodiments, not only does TACE inhibitors result in T-cell TM-TNF retention, but using TACE inhibitors also has a retrograde signaling effect that reduces senescence, increase self-renewal, and / or increase IL2 secretion compared to natural CD4+ and / or CD8+ T-cells.Enriched population of cells

[0057] In some embodiments, the present invention provides a population of cells enriched in CD4+ and / or CD8+ T-cells specific for a pre-determined antigen.

[0058] In some embodiments, the population has higher purity of CD4+ and / or CD8+ T-cells specific for a pre-determined antigen than a natural population obtained from a subject of CD4+ and / or CD8+ T-cells specific for the pre-determined antigen. In some embodiments, the population enriched in CD4+ and / or CD8+ T-cells specific for the pre-determined antigen contains 50%, 60%, 70%, 80% or 90% or more CD4+ and / or CD8+ T-cells specific for the pre-determined antigen. In some embodiments, the population enriched in CD4+ and / or CD8+ T-cells specific for the predetermined antigen contains 80% or more CD4+ and / or CD8+ T-cells specific for the pre-134914-3364-4440v.ldetermined antigen. In some embodiments, the population enriched in CD4+ and / or CD8+ T-cells specific for the pre-determined antigen contains 90% or more CD4+ and / or CD8+ T-cells specific for the pre-determined antigen.

[0059] In some embodiments, T-cells of the enriched population are further expanded, optionally in the presence of a type 17-like cytokine.

[0060] In some embodiments, T-cells of the enriched population exhibit a proliferative capacity of over 1000% expansion after 14-28 days in culture.

[0061] In some embodiments, T-cells of the enriched population exhibit enhanced IL-2 secretion relative to otherwise identical cells not contacted with a TACE inhibitor.

[0062] In some embodiments, T-cells of the enriched population include TILs or VSTs orboth.Methods of obtaining enriched population of cells

[0063] In some embodiments, the present invention provides methods for obtaining a population of cells enriched in CD4+ and / or CD8+ T-cells specific for a pre-determined antigen.

[0064] In some embodiments, the methods include contacting a preparation of cells comprising T-cells obtained from a tumor environment in a subject, a viral infection site in a subject, or from peripheral blood of a subject having a tumor or a viral infection, with an agent that captures transmembrane TNFalpha, (a) in the presence of, or wherein said cells have been previously contacted with, a TACE inhibitor or ADAMS 17 inhibitor or (b) in the presence of an agent that blocks the TACE cleavage site on a TM-TNF alpha , so as to thereby isolate CD4+ and / or CD8+ T-cells specific for the pre-determined antigen., so as to obtain the population. In some embodiments, the preparation comprises TILs. In some embodiments, the preparation comprises VSTs.

[0065] In some embodiments, the methods include comprising washing or otherwise removing cells of the preparation not bound to the agent that captures transmembrane TNFalpha so as to further enrich the population obtained.

[0066] In some embodiments, the T-cells have been stimulated, are stimulated, or restimulated with an antigen obtained from a tumor environment in a subject, viral infection site in a subject, or from peripheral blood, or having an amino acid sequence identical thereto. In some embodiments, the T-cells have been stimulated, are stimulated, or re-stimulated with an antigen 144914-3364-4440v.lobtained from a tumor environment in a subject, viral infection site in a subject, or from peripheral blood, or having an amino acid sequence identical thereto, during when the cells are contacted with, or in the presence of, a TACE inhibitor or ADAMS 17 inhibitor or presence of an agent that blocks a TACE cleavage site on a TM-TNF alpha.Additional methods

[0067] In some embodiments, the present invention provides methods of selectively depleting a T-cell population for stem cell transplant immune reconstitution of allo-reactive T-cells in a subject, the method including contacting the T-cell population for stem cell transplant immune reconstitution with an agent that captures transmembrane TNFalpha, wherein said cells have been previously contacted with, or in the presence of, a TACE inhibitor (or ADAMS 17 inhibitor) or presence of an agent that blocks the TACE cleavage site on a TM- TNFalpha, so as to thereby remove allo-reactive T-cells from the population.

[0068] In some embodiments, the T-cell population has been stimulated, is stimulated, or is re-stimulated with an antigen that is an allo-antigen, optionally, a donor antigen. In some embodiments, the antigen is a donor antigen. In some embodiments, the donor is a donor of stem cells.

[0069] In some embodiments, the subject is immunocompromised.

[0070] In some embodiments, the present invention provides methods of treating a tumor in a subject comprising administering to the subject a population of cells enriched in CD4+ and / or CD8+ T-cells specific for an antigen of the tumor.

[0071] In some embodiments, the population of cells is enriched in CD4+ and / or CD8+ T-cells specific for antigen of the tumor versus a natural population containing CD4+ and / or CD8+ T-cells specific for the antigen of the tumor, optionally, wherein the natural population containing CD4+ and / or CD8+ T-cells specific for the antigen of the tumor is obtained from the subject.

[0072] In some embodiments, the preparation of cells comprising T-cells obtained from a tumor environment in a subject comprises T-cells obtained from the tumor environment of the subject. In some embodiments, the preparation of cells comprising T-cells obtained from a tumor environment in a subject comprises T-cells obtained from a tumor environment of a tumor of the same type in another subject. In some embodiments, the T-cells are TILs.154914-3364-4440v.l

[0073] In some embodiments, the present invention provides methods of method of treating an infection in a subject comprising administering to the subject a population of cells enriched in CD4+ and / or CD8+ T-cells specific for an antigen of a pathogen associated with the infection. In some embodiments, the T-cells are VSTs.

[0074] In some embodiments, the pathogen is a virus. In some embodiments, the pathogen is a bacterium.

[0075] In some embodiments, the subject is immunocompromised.

[0076] In some embodiments, the preparation of cells comprising T-cells obtained from a viral infection site in a subject comprises T-cells obtained from the viral infection site in the subject or from peripheral blood of the subject

[0077] In some embodiments, the preparation of cells comprising T-cells obtained from a viral infection site in a subject comprises T-cells obtained from a viral infection site in another subject having the same type of viral infection or from peripheral blood of said subject.

[0078] In some embodiments, the pathogen is a virus which infects humans.

[0079] In some embodiments, the subject is a human.Additional embodiments

[0080] In some embodiments, the techniques described herein relate to a method of obtaining a population of cells enriched in CD4+ and / or CD8+ T-cells specific for a pre-determined antigen, the method including contacting a preparation of cells including T-cells obtained from a tumor environment in a subject, a viral infection site in a subject, or from peripheral blood of a subject having a tumor or a viral infection, with an agent that captures transmembrane TNF alpha (TM-TNFalpha), (a) in the presence of, or wherein said cells have been previously contacted with, a TACE inhibitor or ADAMS 17 inhibitor or (b) in the presence of an agent that blocks the TACE cleavage site on a TM-TNFalpha, so as to thereby isolate CD4+ and / or CD8+ T-cells specific for the pre-determined antigen, so as to obtain the population.

[0081] In some embodiments, the techniques described herein relate to a method, further including washing or otherwise removing cells of the preparation not bound to the agent that captures TM-TNFalpha so as to further enrich the population obtained.164914-3364-4440v.l

[0082] In some embodiments, the techniques described herein relate to a method, wherein the T-cells have been stimulated, are stimulated, or re-stimulated with an antigen obtained from a tumor environment in a subject, viral infection site in a subject, or from peripheral blood, or having an amino acid sequence identical thereto.

[0083] In some embodiments, the techniques described herein relate to a method, wherein the T-cells have been stimulated, are stimulated, or re-stimulated with an antigen obtained from a tumor environment in a subject, viral infection site in a subject, or from peripheral blood, or having an amino acid sequence identical thereto, during when the cells are contacted with, or in the presence of, a TACE inhibitor or ADAMS 17 inhibitor or presence of an agent that blocks a TACE cleavage site on a TM-TNF alpha.

[0084] In some embodiments, the techniques described herein relate to a method, wherein the population has higher purity of CD4+ and / or CD8+ T-cells specific for a pre-determined antigen than a natural population obtained from a subject of CD4+ and / or CD8+ T-cells specific for the pre-determined antigen.

[0085] In some embodiments, the techniques described herein relate to a method, wherein the agent that captures transmembrane TNF alpha includes an anti -TNF alpha antibody or anti-TNF alpha aptamer.

[0086] In some embodiments, the techniques described herein relate to a method, wherein the agent that blocks the TACE cleavage site on a TM-TNF alpha is used and which includes an anti-TNF alpha antibody or anti-TNF alpha aptamer.

[0087] In some embodiments, the techniques described herein relate to a method, wherein the ADAMS 17 inhibitor is used.

[0088] In some embodiments, the techniques described herein relate to a method, wherein the same agent captures transmembrane TNF alpha and blocks the TACE cleavage site on a TM-TNF alpha, and is an anti-TNF alpha antibody or anti-TNF alpha aptamer.

[0089] In some embodiments, the techniques described herein relate to a method, wherein an anti-TNF alpha antibody is used.174914-3364-4440v.l

[0090] In some embodiments, the techniques described herein relate to a method, wherein the anti-TNF alpha antibody-captured T-cell is purified by magnetic bead positive selection or flowbased sorting.

[0091] In some embodiments, the techniques described herein relate to a method, wherein a preparation of cells includes T-cells obtained from a tumor fragment obtained from a tumor environment in a subject.

[0092] In some embodiments, the techniques described herein relate to a method, wherein T-cells of the enriched population are further expanded, optionally in the presence of a type 17-like cytokine.

[0093] In some embodiments, the techniques described herein relate to a method, wherein T-cells of the enriched population exhibit a proliferative capacity of over 1000% expansion after 14-28 days in culture.

[0094] In some embodiments, the techniques described herein relate to a method, wherein T-cells of the enriched population exhibit enhanced IL-2 secretion relative to otherwise identical cells not contacted with a TACE inhibitor.

[0095] In some embodiments, the techniques described herein relate to a method, wherein T-cells of the enriched population in CD4+ and / or CD8+ T-cells specific for the pre-determined antigen are enriched to 85% or more CD4+ and / or CD8+ T-cells of the population being specific for the pre-determined antigen.

[0096] In some embodiments, the techniques described herein relate to a method, wherein the pre-determined antigen is a human cancer antigen.

[0097] In some embodiments, the techniques described herein relate to a method, wherein the pre-determined antigen is a viral antigen, wherein the virus is a virus that infects humans.

[0098] In some embodiments, the techniques described herein relate to a method, wherein the pre-determined antigen is an antigen of a bacteria or a parasite or a fungus which infects humans.

[0099] In some embodiments, the techniques described herein relate to an enriched population 1-19.184914-3364-4440v.l

[0100] In some embodiments, the techniques described herein relate to a method of selectively depleting a T-cell population for stem cell transplant immune reconstitution of allo-reactive T-cells in a subject, the method including contacting the T-cell population for stem cell transplant immune reconstitution with an agent that captures transmembrane TNF alpha, wherein said cells have been previously contacted with, or in the presence of, a TACE inhibitor or ADAMS 17 inhibitor or presence of an agent that blocks the TACE cleavage site on a TM-TNE alpha, so as to thereby remove allo-reactive T-cells from the population.

[0101] In some embodiments, the techniques described herein relate to a method, wherein the T-cell population has been stimulated, is stimulated, or is re-stimulated with an antigen that is an allo-antigen, optionally, a donor antigen.

[0102] In some embodiments, the techniques described herein relate to a method, wherein the subject is immunocompromised.

[0103] In some embodiments, the techniques described herein relate to a T-cell population, depleted 21-23.

[0104] In some embodiments, the techniques described herein relate to a population of cells enriched in CD4+ and / or CD8+ T-cells specific for a pre-determined antigen versus a natural population of CD4+ and / or CD8+ T-cells specific for the pre-determined antigen, wherein the population exhibits enhanced IL-2 production relative to a predetermined control level of IL-2 production associated with a naturally-occurring CD4+ and / or CD8+ T-cell population.

[0105] In some embodiments, the techniques described herein relate to a population 25 of CD4+ and / or CD8+ T-cells specific for the pre-determined antigen containing 50%, 60%, 70%, 80% or 90% or more CD4+ and / or CD8+ T-cells specific for the pre-determined antigen.

[0106] In some embodiments, the techniques described herein relate to a method of treating a tumor in a subject including administering to the subject a population of cells enriched in CD4+ and / or CD8+ T-cells specific for an antigen of the tumor.

[0107] In some embodiments, the techniques described herein relate to a method, wherein the population of cells is enriched in CD4+ and / or CD8+ T-cells specific for antigen of the tumor versus a natural population containing CD4+ and / or CD8+ T-cells specific for the antigen of the194914-3364-4440v.ltumor, optionally, wherein the natural population containing CD4+ and / or CD8+ T-cells specific for the antigen of the tumor is obtained from the subject.

[0108] In some embodiments, the techniques described herein relate to a method, wherein the population 1-19.

[0109] In some embodiments, the techniques described herein relate to a method, wherein the preparation of cells including T-cells obtained from a tumor environment in a subject includes T-cells obtained from the tumor environment of the subject.

[0110] In some embodiments, the techniques described herein relate to a method, wherein the preparation of cells including T-cells obtained from a tumor environment in a subject includes T-cells obtained from a tumor environment of a tumor of the same type in another subject.

[0111] In some embodiments, the techniques described herein relate to a method of treating an infection in a subject including administering to the subject a population of cells enriched in CD4+ and / or CD8+ T-cells specific for an antigen of a pathogen associated with the infection.

[0112] In some embodiments, the techniques described herein relate to a method, wherein the pathogen is a virus.

[0113] In some embodiments, the techniques described herein relate to a method, wherein the pathogen is a bacterium.

[0114] In some embodiments, the techniques described herein relate to a method, wherein the population of cells is enriched in CD4+ and / or CD8+ T-cells specific for antigen of the pathogen versus a natural population containing CD4+ and / or CD8+ T-cells specific for the antigen of the pathogen, optionally, wherein the natural population containing CD4+ and / or CD8+ T-cells specific for the antigen of the pathogen is obtained from the subject.

[0115] In some embodiments, the techniques described herein relate to a method, wherein the subject is immunocompromised.

[0116] In some embodiments, the techniques described herein relate to a method, wherein the population 1-19.204914-3364-4440v.l

[0117] In some embodiments, the techniques described herein relate to a method, wherein the preparation of cells including T-cells obtained from a viral infection site in a subject includes T-cells obtained from the viral infection site in the subject or from peripheral blood of the subject.

[0118] In some embodiments, the techniques described herein relate to a method, wherein the preparation of cells including T-cells obtained from a viral infection site in a subject includes T-cells obtained from a viral infection site in another subject having the same type of viral infection or from peripheral blood of said subject.

[0119] In some embodiments, the techniques described herein relate to a method, wherein the pathogen is a virus which infects humans.

[0120] In some embodiments, the techniques described herein relate to the method of any of Clams 1-19, 21-23, or 27-40, wherein the subject is a human.

[0121] In some embodiments, the techniques described herein relate to a method of obtaining a population of cells enriched in CD4+ and / or CD8+ T-cells specific for tumor antigen(s), the method including contacting a preparation of cells including T-cells wherein said cells have been stimulated, are stimulated, or re-stimulated with a whole tumor cell, a tumor lysate thereof, or an antigen presenting cell, optionally a dendritic cell, that has been pulsed or loaded with a tumor lysate, with an agent that captures transmembrane TNFalpha (TM-TNF alpha), (a) in the presence of, or wherein said cells have been previously contacted with, a TACE inhibitor or ADAMS17 inhibitor or (b) in the presence of an agent that blocks the TACE cleavage site on a TM-TNF alpha, so as to thereby isolate CD4+ and / or CD8+ T-cells specific for an antigen of the tumor or tumor lysate so as to obtain the population.

[0122] In some embodiments, the techniques described herein relate to a method, wherein the stimulated T-cells population includes tumor-infiltrating lymphocytes.

[0123] In some embodiments, the techniques described herein relate to a method, wherein the tumor cell or lysate has been obtained from a subject.

[0124] In some embodiments, the techniques described herein relate to a method of obtaining a population of cells enriched in CD4+ and / or CD8+ T-cells specific for senescence-associated antigen(s), the method including contacting a preparation of cells including T-cells wherein said cells have been stimulated, are stimulated, or re-stimulated with, a whole senescent cell, a214914-3364-4440v.lsenescent cell lysate, or an antigen presenting cell, optionally a dendritic cell, that has been pulsed or loaded with apoptotic body(ies) or senescent cell lysate, with an agent that captures transmembrane TNF alpha (TM-TNF alpha), (a) in the presence of, or wherein said cells have been previously contacted with, a TACE inhibitor or ADAMS 17 inhibitor or (b) in the presence of an agent that blocks the TACE cleavage site on a TM-TNF alpha, so as to thereby isolate CD4+ and / or CD8+ T-cells specific for senescence-associated antigen(s) so as to obtain the population.

[0125] In some embodiments, the techniques described herein relate to a method, wherein the T-cells have been obtained from peripheral blood or are ex vivo expanded T-cells.

[0126] A population of cells enriched in CD4+ and / or CD8+ T-cells specific for a predetermined antigen versus a natural population of CD4+ and / or CD8+ T-cells specific for the predetermined antigen, wherein the population exhibits enhanced IL-2 production relative to a predetermined control level of IL-2 production associated with a naturally-occurring CD4+ and / or CD8+ T-cell population.

[0127] In some embodiments, a natural population of CD4+ and / or CD8+ T-cells specific for the pre-determined antigen contains 5% or less CD4+ and / or CD8+ T-cells specific for the predetermined antigen. In some embodiments, the population enriched in CD4+ and / or CD8+ T-cells specific for the pre-determined antigen contains 50%, 60%, 70%, 80% or 90% or more CD4+ and / or CD8+ T-cells specific for the pre-determined antigen. In some embodiments, the population enriched in CD4+ and / or CD8+ T-cells specific for the pre-determined antigen contains 80% or more CD4+ and / or CD8+ T-cells specific for the pre-determined antigen. In some embodiments, the population enriched in CD4+ and / or CD8+ T-cells specific for the pre-determined antigen contains 90% or more CD4+ and / or CD8+ T-cells specific for the pre-determined antigen.

[0128] In some embodiments, the antigen is a human cancer antigen. In some embodiments, the antigen is a viral antigen. In some embodiments of the viral antigen, the virus is a virus that infects humans.

[0129] A method of obtaining a population of cells containing a high purity of CD4+ and / or CD8+ T-cells specific for a pre-determined antigen, wherein the population has higher purity than a natural population of CD4+ and / or CD8+ T-cells specific for the pre-determined antigen, the method comprising contacting a preparation of cells comprising T-cells obtained from a tumor environment in a subject, viral infection site in a subject, or from peripheral blood of a subject 224914-3364-4440v.lhaving a tumor or a viral infection, with an agent that captures transmembrane TNF alpha, in the presence of, or wherein said cells have been previously contacted with, a TACE inhibitor (or ADAMS 17 inhibitor) or presence of an agent that blocks the TACE cleavage site on a TM-TNFalpha, so as to thereby isolate CD4+ and / or CD8+ T-cells specific for the pre-determined antigen. In some embodiments, the T-cells are stimulated or re-stimulated with an antigen obtained from a tumor environment in a subject, viral infection site in a subject, or from peripheral blood, or having an amino acid sequence identical thereto. In some embodiments, the T-cells are stimulated or re-stimulated with an antigen obtained from a tumor environment in a subject, viral infection site in a subject, or from peripheral blood, or having an amino acid sequence identical thereto, during the time that the cells are contacted with, or in the presence of, a TACE inhibitor (or ADAMS 17 inhibitor) or presence of an agent that blocks the TACE cleavage site on a TM-TNF alpha,

[0130] In some embodiments, the agent that captures transmembrane TNF alpha includes an anti-TNF alpha antibody or anti-TNFalpha aptamer. In some embodiments, the agent that blocks the TACE cleavage site on a TM-TNFalpha comprises an anti-TNFalpha antibody or anti-TNFalpha aptamer. In some embodiments the same agent captures transmembrane TNF alpha and blocks the TACE cleavage site on a TM-TNFalpha, and is an anti-TNFalpha antibody or anti-TNFalpha aptamer. In some embodiments, the anti-TNFalpha antibody-captured T-cell is purified by magnetic bead positive selection or flow-based sorting.

[0131] In some embodiments, the T-cells are obtained from a tumor fragment obtained from a tumor environment in a subject. In embodiment, the T-cells are expanded in the presence of a type 17-like cytokine.

[0132] In some embodiments, the TM-TNFa-captured T cells exhibit a proliferative capacity of over 1000% expansion after 14-28 days in culture, which is not seen in natural populations. In some embodiments, wherein the cells are contacted with a TACE inhibitor, the TM-TNFa-captured T cells exhibit enhanced IL -2 secretion relative to otherwise identical cells not contacted with a TACE inhibitor.

[0133] In some embodiments, the T-cells are expanded in vitro after being obtained by the method disclosed herein.234914-3364-4440v.l

[0134] In some embodiments, a population of cells containing a high purity of CD4+ and / or CD8+ T-cells specific for a pre-determined antigen obtained by the method disclosed herein is provided.

[0135] A method of selectively depleting a T cell population for stem cell transplant immune reconstitution of allo-reactive T cells, the method comprising contacting the T cell population for stem cell transplant immune reconstitution with an agent that captures transmembrane TNFalpha, wherein said cells have been previously contacted with, or in the presence of, a TACE inhibitor (or ADAMS 17 inhibitor) or presence of an agent that blocks the TACE cleavage site on a TM-TNF alpha, so as to thereby remove allo-reactive T cells from the population. In some embodiments, a T cell population for stem cell transplant immune reconstitution depleted of allo-reactive T cells is provided.EXAMPLES

[0136] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0137] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0138] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only.244914-3364-4440v.lTM-TNFa-based generation of highly potent virus specific T cells for adoptive immunotherapy of refractory viral infections.Example 1: The use of TM-TNFa for identification of antigen-specific T cells in ex vivo expanded VST cell products

[0139] TM-TNFa labeling was tested using clinical-grade VST cells specific for CMV, generated from the peripheral blood mononuclear cells (PBMCs) upon stimulation with 15-aminoacid (AA)- long overlapping peptide libraries (pepmixes) representing pp65 and IE1 antigens in cGMP-compliant G-Rex containers. Final CMV-VSTs were re-stimulated in presence of a selective ADAM17 / TACE inhibitor TAPI-1 (Sigma) and stained for T cell markers (CD3, CD4, CD8) and surface TNFa at hourly intervals to establish kinetics of TM-TNFa expression (Fig. 3). TM-TNFa was detected at 2hrs after stimulation, reaching peak at 4hrs. Resting (nonstimulated) CMV-VST cells were used as a control, confirming that only antigen-reactive T cells were detected using this approach.Example 2: TM-TNFa capture as a strategy for generation of highly purified virus specific T cells

[0140] Next, it was tested if it is possible to apply TM-TNFa capture strategy to create highly-enriched CMV specific T cells from the standard ex vivo expanded VST cultures targeting the immunodominat antigens (IE-1 and pp65) of CMV. Therefore, the CMV-VST cells generated using standard methodologies (as shown for Fig. 3) were stimulated for 4 hours with pp65 and IE-1 pepmixes in presence of TAPI-1 and anti-TNFa PE antibody. TNFaHI T cells were purified using standard flow sorter, with gating set based on unstimulated control CMV-VST cells used as negative control. TM-TNFa-captured CMV-VST cells were subsequently placed in media with cytokines (IL-2 and IL-7) and expanded for 14 days. A robust expansion (30-50-fold range) of captured T cells was observed in all tested cultures. Final CMV-VSTs were analyzed for reactivity, demonstrating marked enhancement in frequency of CMV-reactive T cells upon challenge with cognate pp65 and IE1 antigens. Importantly, a marked increase in frequency of IL-2 producing T cells among the CMV-reactive (TNFa-producing) fraction was seen (Fig 4), as compared to the baseline activity, suggesting that TM-TNFa capture might promote polyfunctionality, whereas multiple rounds of in vitro stimulation / expansion typically lead to loss of IL-2 production by the immune effectors indicating terminally differentiated / senescent state of expanded immune254914-3364-4440v.leffectors. Thus, TM-TNF -capture strategy not only allows for generation of T cell effectors with superior purity and lower frequency of irrelevant passenger cells, but it also may elicit a paradoxical biological effect of enhanced polyfunctionality consistent with functional preservation of less differentiated, non-terminal state of the immune effectors.Example 3: TM-TNFa-enriched EBV-specific T cells for adoptive immunotherapy of EBV-PTLD and other EBV-associated hematological malignancies

[0141] EBV-VST cells were generated from PBMCs of healthy donors using standard methodologies analogous to the inventor’s current cGMP process used for Phase 1 clinical trial. Briefly, PBMCs expended in vitro upon stimulation with 15 amino acid (AA) peptide libraries spanning the full length of immunodominant EBV proteins associated with EBV latency I and latency II, including EBNA1, LMP1 and LMP2 pulsed into irradiated autologous PBMCs used as stimulators. Cultures were maintained in G-rex 10 bioreactors with media containing IL-7 and IL-2 (starting from day +3). Fresh media was added every 2-3 days. Final cultures were harvested on day +14 and used for the TM-TNFa capture experiments.

[0142] In brief, EBV-VST cells were exposed to the same (cognate) EBV libraries for 4 hours in presence of TAPI-1 and anti-TNFa PE antibody and isolated using standard flow cytometric sorting, whereas unsorted but restimulated / labeled EBV-VSTs were used as control. Following the flow sorting EBV-VST cells were placed again in complete culture media supplemented with IL-2 and IL-7 and expanded for 14 days. Resulting TM-TNFa-captured EBV-VST cells displayed a significantly higher total reactivity against EBV antigens (Fig.4A and B). Importantly, final TM-TNFa-captured cells displayed markedly increased frequency of antigen-specific (TNFa111) CD3+T cells producing IL -2, indicating enhanced poly functionality of the EBV-VST cells generated using TM-TNFa capture strategy (Fig 4B and C). This observation is paradoxical, as typically the loss of IL-2 secretion (i.e. polyfunctionality) is expected following multiple rounds stimulation / expansion ex vivo expansion, consistent with terminal differentiation and acquisition of functional senescence. This loss of polyfunctionality is associated with decreasing capacity to self-renew, proliferate and persist in vivo and with reduction in anti-viral or anti-tumor therapeutic activity. Therefore, the TM-TNFa-based isolation strategy actively reprograms the immune effectors for polyfunctionality and enhances overall functionality of the therapeutic T cell products.264914-3364-4440v.lExample 4: Generation of VST cells for adoptive immunotherapy using direct TM-TNFa capture of memory T cell precursors from peripheral blood

[0143] Several methods for direct isolation of antigen specific T cells from the peripheral blood are currently used, including some that are explored in the clinical settings. These methods predominantly utilize activation induced markers (AIMs). Therefore the following were compared the frequency of T cells displaying common AIM markers 4-1BB (CD137), 0X40 (CD134), CD40L (CD154) vs. the frequency of T cells upregulating TM-TNFa upon stimulation with a panel of pepmixes from the following viral pathogens: BK Polyomavirus (BKV) LT and VP1, CMV pp65 and IE1 and human Coronavirus 2 (hCoV2) Spike (S), nucleocapsid (NP) and membrane (M). As shown in Fig 5 A and B, in every case the highest frequency of antigen specific T cells was seen when TM-TNFa labeling was used, establishing the notion that the proposed strategy might have the highest efficacy when isolation of the rare memory precursors is attempted from PBMCs of donors, as compared with AIM approach.

[0144] Next, the feasibility of generating VST cells from antigen-specific T cells captured directly from PBMCs using the TM-TNFa was tested. To that end, healthy donor PBMCs were stimulated with CMV pp65 peptide library in presence of TAPL1 and anti-TNFa PE. Baseline flow cytometric analysis revealed app. 1.13% pp65-reactive cells (Fig. 5C), predominantly within the CD4+ T cell compartment. TM-TNFa labeled T cells were then sorted by flow cytometry and placed in culture for 14 days in complete media supplemented with IL-7 and IL-2. Approximately 60-fold expansion of T cells was seen (Fig. 5D), with app. 85% of VST cells specifically recognizing cognate CMV-pp65 antigen (Fig. 5E and F) and displaying excellent viability (not shown). Thus, it is possible to capture and robustly expand antigen specific T cells for adoptive immunotherapy application. Moreover, the final products generated from TM-TNFa captured precursors display remarkable specificity with minimal content of irrelevant passenger cells. TM-TNFa capture for generation of high-potency tumor-specific T cells targeting common tumor antigens for immunotherapy of cancer and hematological malignancies Example 5: TM-TNFa-enriched multi-epitope specific T cells targeting common tumor antigens

[0145] CD4+ T cells were isolated from PBMCs of healthy donors and stimulated in vitro with autologous monocyte-derived dendritic cells (DCs) pulsed with 15AA-long peptide library spanning the full length of PRAME, a cancer testis antigen commonly overexpressed in solid274914-3364-4440v.ltumors (e g. melanoma) and hematological malignancies (e.g. AML / MDS). Cultures were maintained in media supplemented with cytokines for app. 14 days. Resulting T cell population was restimulated with cognate PRAME library in presence of TAPI- 1 and anti-TNFa PE antibody for 4 hours and sorted by FACS. As shown in Fig. 6A, a marked enrichment of antigen-specific T cells was reached post-sort Isolated T cells were subsequently placed in culture for 14 days, as described previously and maintained in cytokine-supplemented media. Final cultures displayed marked antigen-specific reactivity and polyfunctionality with increased fraction of reactive T cells retaining capacity to secrete IL-2 (Fig. 6B and C), indicating that despite multiple rounds of stimulation, TM-TNF -capture either preserved or in many cases enhanced polyfunctionality of the immune effectors and suggesting less terminal differentiation and more robust capacity to persist and function in vivo upon adoptive transfer in patients with cancer.

[0146] Furthermore, when the labeling efficiency tumor-specific T cells using AIM CD154 with TM-TNFa labeling (Fig. 8) were compared it was observed a significant positive correlation, as the majority of reactive T cells co-expressed CD 154 and TM-TNFa upon antigenic challenge (CD154HITM-TNFaHI), but there was also a significant subset of antigen-reactive T cells only producing TM-TNFa (single-positive) but remaining CD154LOW, as illustrated in Fig. 8, right panel. Therefore, TM-TNFa-based capture of tumor-specific T cells is significantly more efficient that a commonly-used surface marker AIM-based strategy, as it identifies and captures the highest numbers of reactive T cells within a given T cell population. This observation is consistent with data shown in Fig. 3, where efficacy of TM-TNFa labeling of virus-specific T cells in PBMCs was superior to the conventional AIM-based approaches.Example 6: TM-TMFa-enriched multi-epitope specific Thl / 17 cytotoxic cells targeting Merkel Cell Carcinoma

[0147] CD4+ T cells from healthy donors were stimulated twice as described above 14 days apart with autologous DCs pulsed with truncated Large T (LT-t) antigen peptide library derived from Merkel Cell Polyomavirus (MCPy V), a viral oncoprotein driver of Merkel Cell Carcinoma (MCC). Cultures were maintained in media containing IL-2 and a proprietary cocktail of inflammatory cytokines developed in our laboratory (patent pending). TM-TNFaHI T cells were purified by flow cytometric sorting using TM-TNFa-capture strategy as described above and further expanded for 14 days before testing for reactivity against the cognate LT-t. Unsorted, but284914-3364-4440v.lrestimulated cultures and sorted TM-TNFaLOW were used as controls. In both tested donors (Fig.7A-C) a marked enrichment of LTt-reactive cells was seen in TM-TNFa -captured T cells cultures (86.1% and 94.89% respectively), whereas activity of TM-TNFaLOW T cells and unsorted but restimulated T cells was significantly lower in both cases. Furthermore, a marked upregulation of polyfunctionality was seen in both donors, with increases in subset of T cells secreting IL-2 and also T cells capable of producing granzyme B, a marker of cytotoxic capability (Fig. 7B). Thus, TM-TNFa capture not only generates highly specific T cells with reduced number of irrelevant passenger T cells, but it reprograms the effector cells by promoting or preserving the capacity to produce IL-2 and overall polyfunctionality (evidenced by enhanced co-secretion of other cytokines). This characteristic is linked to robust in vivo self-renewal, proliferative capacity and long-term persistence of T cells, correlating closely with superior therapeutic potency and improved outcomes in adoptive transfer immunotherapy of cancer.Additional examplesExample 7: Potent high-purity tumor antigen specific T cells for adoptive immunotherapy of cancer (PBMC derived)

[0148] TNFa capture upon TACE inhibition or TM-TNFa-specific antibody or aptamer blocking cleavage, site on TM-TNFa allows for efficient enrichment and massive expansion of highly pure tumor associated antigen-specific CD4+ and / or CD8+ T cells from the mixed population with low frequency of these T cells.

[0149] TAA-specific T cells generated from PBMCs of patients with hematological or solid malignancies are stimulated with respective TAAs in presence of TACE inhibitor, labeling with anti-TNFa monoclonal antibody and / or TNFa-specific aptamer followed by sorting using either magnetic bead positive selection or flow-based sorting.

[0150] Enrichment will be carried out by either TM-TNFa-biding antibody or an aptamer that binds to TACE cleavage site, accomplishing blockade and labeling in a single step TM-TNFa-captured T cells allow for dramatic enhancement in activity of the TAA-specific cell population (app. 90% purity vs. <5% for control population), while retaining or enhancing polyfunctionality, including markedly increased ability to produce IL-2, a key correlate to in vivo engraftment, persistence and anti -cancer activity in vivo.294914-3364-4440v.l

[0151] This strategy can be applied to common / shared TAAs (e.g. viral oncoproteins, cancer testis antigens and others) and neoantigens either patient-specific or recurrent / shared (e.g. KRAS or BRAF mutations and others)

[0152] This strategy can be used to massively expand pure populations of multi-epitope specific T cells as well as further identification of T cells for TCR cloning and further gene engineering.

[0153] TAA-specific T cells initially expanded in presence of inflammatory cytokines (type 17-like) particularly benefit from TM-TNFa-based enrichment.Example 8: Tumor Infiltrating Lymphocytes (TILs) with enhanced potency

[0154] TNFa capture upon TACE inhibition or TM-TNFa-specific antibody or aptamer blocking TACE cleavage allows for efficient enrichment and massive expansion of autologous TILs derived directly from tumor fragments resected from patients with cancer. These TM-TNFa-captured TILs display superior purity, polyfunctionality, proliferative capacity and anti-tumor activity.

[0155] Highly-potent enriched TILs were generated from tumor fragments cultured in vitro, followed by antigen restimulation with either autologous tumor, tumor-derived organoids, autologous dendritic cells expressing TAA-encoding genes or pulsed with TAA antigenic peptides or tumor lysate or apoptotic bodies or TAAs in presence of TACE inhibitor, labeling with anti-TNFa monoclonal antibody and / or TNFa-specific aptamer and sorted using either magnetic bead positive selection or flow-based sorting.

[0156] TIL enrichment can be accomplished by either TACE inhibitor or TM-TNFa-biding antibody or aptamer specific for TACE cleavage site, accomplishing blockade and labeling in a single step.

[0157] TM-TNFa-captured TILs demonstrate robust proliferation, polyfunctionality and are highly specific towards the autologous tumor target.

[0158] TILs initially expanded in presence of inflammatory cytokines (type 17-like) particularly benefit from TM-TNFa-based enrichment.304914-3364-4440v.lExample 9: High-potency antigen-specific VST cells for allogeneic and off-the-shelf application

[0159] Currently used allogeneic VST cells are typically a variable-frequencies of antigenspecific T cells with a large population of passenger, non-specific cells of unknown specificity. It was discovered that TM-TNF-a sorting strategy generate large numbers of almost pure (>95%) VST cells targeting difficult viral antigens.

[0160] This population is almost completely devoid of “passenger” T cells of unknown specificity - thus less likely to cause GVHD and / or off target toxicity - thus might be used in at higher doses in allogeneic setting, where typically only limited cell doses are used.

[0161] TM-TNFa-captured T cells display robust proliferative capacity (>1000% expansion) after 14-28 days in culture

[0162] Final TM-TNFa captured VST product displays improved polyfunctionality and viability.

[0163] Furthermore, TACE inhibition during sorting improves IL-2 secretion in population and generates a cell therapy product with desirable “early” functional characteristics despite multiple rounds of expansion in vitro that typically results in exhaustion.Example 10: Rapidly isolated antigen-specific T cells captured directly from peripheral blood for adoptive immunotherapy or downstream processing

[0164] Labeled T cells specific to common viral pathogens or viral oncoproteins and common or private tumor antigens were successfully isolated from either autologous or allogeneic sources and the feasibility of expanding them under GMP -grade conditions was tested. VST cells were generated directly from the peripheral blood of the donor.

[0165] It was found that even small number (as low as 0.25%) of TM-TNF -lab eled T cells (CD4+ and / or CD8+) remain highly viable following the TNFa capture and sorting.

[0166] TM-TNFa-captured cells undergo a dramatic proliferation when cultured in vitro (>2000-5000%).

[0167] Thus the procedure allows for generation of very high numbers of highly specific T cells.314914-3364-4440v.l

[0168] At the end of the expansion these cells retained features that correlating with long-term antitumor clinical efficacy, such as polyfunctionality and ability to produce IL-2, thus they were protected from terminal differentiation and senescence that frequently affects in vitro expanded effector cells used for immunotherapy.

[0169] Therefore, TACE inhibitor and TM-TNFa-based not only allows for identification and enrichment of antigen-specific T cells, but also modulates / improves the quality of the resulting population, preventing premature exhaustion.

[0170] TM-TNFa-captured T cells can be used for identification / cloning of relevant TCRs for immunotherapy or rapid immunomonitoring or potency testing.Example 11: Selective Depletion of the allo-reactive T cell population for stem cell transplant immune reconstitution:

[0171] Membrane-bound TNFa following TACE inhibition can be used to selectively eliminate allo-reactive T cell populations, thus providing a novel strategy for generating the immune population with reduced ability to cause GVHD.

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Claims

CLAIMSWhat is claimed is:

1. A method of obtaining a population of cells enriched in CD4+ and / or CD8+ T-cells specific for a pre-determined antigen, the method comprisingcontacting a preparation of cells comprising T-cells obtained from a tumor environment in a subject, a viral infection site in a subject, or from peripheral blood of a subject having a tumor or a viral infection, with an agent that captures transmembrane TNF alpha (TM-TNF alpha), (a) in the presence of, or wherein said cells have been previously contacted with, a TACE inhibitor or ADAMS 17 inhibitor or (b) in the presence of an agent that blocks the TACE cleavage site on a TM-TNF alpha, so as to thereby isolate CD4+ and / or CD8+ T-cells specific for the predetermined antigen, so as to obtain the population.

2. The method of Claim 1, further comprising washing or otherwise removing cells of the preparation not bound to the agent that captures TM-TNF alpha so as to further enrich the population obtained.

3. The method of Claim 1 or 2, wherein the T-cells have been stimulated, are stimulated, or re-stimulated with an antigen obtained from a tumor environment in a subject, viral infection site in a subject, or from peripheral blood, or having an amino acid sequence identical thereto.

4. The method of Claim 3, wherein the T-cells have been stimulated, are stimulated, or restimulated with an antigen obtained from a tumor environment in a subject, viral infection site in a subject, or from peripheral blood, or having an amino acid sequence identical thereto, during when the cells are contacted with, or in the presence of, a TACE inhibitor or ADAMS 17 inhibitor or presence of an agent that blocks a TACE cleavage site on a TM-TNF alpha.

5. The method of any of Claims 1-4, wherein the population has higher purity of CD4+ and / or CD8+ T-cells specific for a pre-determined antigen than a natural population obtained from a subject of CD4+ and / or CD8+ T-cells specific for the pre-determined antigen.454914-3364-4440v.l6. The method of any of Claims 1 -4, wherein the agent that captures transmembrane TNF alpha comprises an anti-TNF alpha antibody or anti-TNF alpha aptamer.

7. The method of any of Claims 1-4, wherein the agent that blocks the TACE cleavage site on a TM-TNF alpha is used and which comprises an anti-TNF alpha antibody or anti-TNF alpha aptamer.

8. The method of any of Claims 1-4, wherein the ADAMS17 inhibitor is used.

9. The method of any of Claims 1-7, wherein the same agent captures transmembrane TNF alpha and blocks the TACE cleavage site on a TM-TNF alpha, and is an anti-TNF alpha antibody or anti-TNF alpha aptamer.

10. The method of any of Claims 1-7 or 9, wherein an anti-TNF alpha antibody is used.

11. The method of Claim 10, wherein the anti-TNF alpha antibody-captured T-cell is purified by magnetic bead positive selection or flow-based sorting.

12. The method of any of Claims 1-11, wherein a preparation of cells comprises T-cells obtained from a tumor fragment obtained from a tumor environment in a subject.

13. The method of any of Claims 1-12, wherein T-cells of the enriched population are further expanded, optionally in the presence of a type 17-like cytokine.

14. The method of any of Claims 1-13, wherein T-cells of the enriched population exhibit a proliferative capacity of over 1000% expansion after 14-28 days in culture.

15. The method of any of Claims 1-14, wherein T-cells of the enriched population exhibit enhanced IL-2 secretion relative to otherwise identical cells not contacted with a TACE inhibitor.464914-3364-4440v.l16. The method of any of Claims 1-15, wherein T-cells of the enriched population in CD4+ and / or CD8+ T-cells specific for the pre-determined antigen are enriched to 85% or more CD4+ and / or CD8+ T-cells of the population being specific for the pre-determined antigen.

17. The method of any of Claims 1-16, wherein the pre-determined antigen is a human cancer antigen.

18. The method of any of Claims 1-16, wherein the pre-determined antigen is a viral antigen, wherein the virus is a virus that infects humans.

19. The method of any of Claims 1-16, wherein the pre-determined antigen is an antigen of a bacteria or a parasite or a fungus which infects humans.

20. An enriched population of cells containing a high purity of CD4+ and / or CD8+ T-cells specific for a pre-determined antigen obtained by the method of any of Claims 1-19.

21. A method of selectively depleting a T-cell population for stem cell transplant immune reconstitution of allo-reactive T-cells in a subject, the method comprisingcontacting the T-cell population for stem cell transplant immune reconstitution with an agent that captures transmembrane TNFalpha, wherein said cells have been previously contacted with, or in the presence of, a TACE inhibitor or ADAMS 17 inhibitor or presence of an agent that blocks the TACE cleavage site on a TM- TNFalpha, so as to thereby remove allo-reactive T-cells from the population.

22. The method of Claim 21, wherein the T-cell population has been stimulated, is stimulated, or is re-stimulated with an antigen that is an allo-antigen, optionally, a donor antigen.

23. The method of Claim 21 or 22, wherein the subject is immunocompromised.

24. A T-cell population, depleted of allo-reactive T-cells, for stem cell transplant immune reconstitution obtained by the method of any of Claims 21-23.474914-3364-4440v.l25. A population of cells enriched in CD4+ and / or CD8+ T-cells specific for a predetermined antigen versus a natural population of CD4+ and / or CD8+ T-cells specific for the pre-determined antigen, wherein the population exhibits enhanced IL-2 production relative to a predetermined control level of IL-2 production associated with a naturally-occurring CD4+ and / or CD8+ T-cell population.

26. The population of cells of Claim 25 of CD4+ and / or CD8+ T-cells specific for the predetermined antigen containing 50%, 60%, 70%, 80% or 90% or more CD4+ and / or CD8+ T-cells specific for the pre-determined antigen.

27. A method of treating a tumor in a subject comprising administering to the subject a population of cells enriched in CD4+ and / or CD8+ T-cells specific for an antigen of the tumor.

28. The method of Claim 27, wherein the population of cells is enriched in CD4+ and / or CD8+ T-cells specific for antigen of the tumor versus a natural population containing CD4+ and / or CD8+ T-cells specific for the antigen of the tumor, optionally, wherein the natural population containing CD4+ and / or CD8+ T-cells specific for the antigen of the tumor is obtained from the subject.

29. The method of Claim 27 or 28, wherein the population of cells enriched in CD4+ and / or CD8+ T-cells specific for antigen of the tumor has been obtained by the method of any of Claims 1-19.

30. The method of Claim 29, wherein the preparation of cells comprising T-cells obtained from a tumor environment in a subject comprises T-cells obtained from the tumor environment of the subject.

31. The method of Claim 29, wherein the preparation of cells comprising T-cells obtained from a tumor environment in a subject comprises T-cells obtained from a tumor environment of a tumor of the same type in another subject.484914-3364-4440v.l32. A method of treating an infection in a subject comprising administering to the subject a population of cells enriched in CD4+ and / or CD8+ T-cells specific for an antigen of a pathogen associated with the infection.

33. The method of Claim 32, wherein the pathogen is a virus.

34. The method of Claim 32, wherein the pathogen is a bacterium.

35. The method of Claim 32, 33, or 34, wherein the population of cells is enriched in CD4+ and / or CD8+ T-cells specific for antigen of the pathogen versus a natural population containing CD4+ and / or CD8+ T-cells specific for the antigen of the pathogen, optionally, wherein the natural population containing CD4+ and / or CD8+ T-cells specific for the antigen of the pathogen is obtained from the subject.

36. The method of any of Claims 32-35, wherein the subject is immunocompromised.

37. The method of any of Claims 32-36, wherein the population of cells enriched in CD4+ and / or CD8+ T-cells specific for the antigen of the pathogen has been obtained by the method of any of Claims 1-19.

38. The method of Claim 37, wherein the preparation of cells comprising T-cells obtained from a viral infection site in a subject comprises T-cells obtained from the viral infection site in the subject or from peripheral blood of the subject.

39. The method of Claim 38, wherein the preparation of cells comprising T-cells obtained from a viral infection site in a subject comprises T-cells obtained from a viral infection site in another subject having the same type of viral infection or from peripheral blood of said subject.

40. The method of any of Claims 32-39, wherein the pathogen is a virus which infects humans.494914-3364-4440v.l41. The method of any of Clams 1-19, 21-23, or 27-40, wherein the subject is a human.

42. A method of obtaining a population of cells enriched in CD4+ and / or CD8+ T-cells specific for tumor antigen(s), the method comprisingcontacting a preparation of cells comprising T-cells wherein said cells have been stimulated, are stimulated, or re-stimulated with a whole tumor cell, a tumor lysate thereof, or an antigen presenting cell, optionally a dendritic cell, that has been pulsed or loaded with a tumor lysate, with an agent that captures transmembrane TNFalpha (TM-TNF alpha), (a) in the presence of, or wherein said cells have been previously contacted with, a TACE inhibitor or ADAMS 17 inhibitor or (b) in the presence of an agent that blocks the TACE cleavage site on a TM-TNF alpha, so as to thereby isolate CD4+ and / or CD8+ T-cells specific for an antigen of the tumor or tumor lysate so as to obtain the population.

43. The method of Claim 42, wherein the stimulated T-cells population comprises tumorinfiltrating lymphocytes.

44. The method of Claim 42 or 43, wherein the tumor cell or lysate has been obtained from a subject.

45. A method of obtaining a population of cells enriched in CD4+ and / or CD8+ T-cells specific for senescence-associated antigen(s), the method comprisingcontacting a preparation of cells comprising T-cells wherein said cells have been stimulated, are stimulated, or re-stimulated with, a whole senescent cell, a senescent cell lysate, or an antigen presenting cell, optionally a dendritic cell, that has been pulsed or loaded with apoptotic body(ies) or senescent cell lysate, with an agent that captures transmembrane TNFalpha (TM-TNF alpha), (a) in the presence of, or wherein said cells have been previously contacted with, a TACE inhibitor or ADAMS 17 inhibitor or (b) in the presence of an agent that blocks the TACE cleavage site on a TM-TNF alpha, so as to thereby isolate CD4+ and / or CD8+ T-cells specific for senescence-associated antigen(s) so as to obtain the population.504914-3364-4440v.l46. The method of any of Claims 42-45, wherein the T-cells have been obtained from peripheral blood or are ex vivo expanded T-cells.514914-3364-4440v.l

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