Generation of virus- or other antigen-specific T cells from naive T-cell populations

Abstract

To provide a cell-based therapy using virus and other antigen-specific T cells.SOLUTION: The present invention discloses a method for treating a human patient having a tumor expressing PRAME, WT1 and Survivin. The method includes administering, to the human subject in need thereof, an effective amount of an antigen-specific T cell population activated to a tumor-associated antigen consisting of PRAME, WT1 and Survivin. The antigen-specific T cell population is prepared from a naive immune cell of a non-autologous human donor naive to PRAME, WT1 and Survivin. The antigen-specific T cell population is a naive cell not prepared from cord blood.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Divisional Application No. 62 / 135,851, filed March 20, 2015, and U.S. Divisional Application No. 62 / 135,888, filed March 20, 2015, the entire disclosures of which are incorporated herein by reference. This application is related to PCT / US2014 / 62698, entitled "Expansion of CMV-Specific T Cells from CMV-Seronegative Donors," filed October 28, 2014, which claims priority to U.S. Divisional Application No. 61 / 896,296, filed October 28, 2013. The disclosures of all of the above documents are incorporated herein by reference.

[0002] Technical Field The present invention relates generally to the field of viral and other antigen-specific T cells, methods for their production from naive T cells, and cell-based therapies using viral and other antigen-specific T cells. [Background technology]

[0003] Existing T cell-based immunotherapies use virus- and tumor-specific T cells expanded from samples containing T cells and progenitor T cells. Virus-specific T cells have been shown to be effective against viral infections after stem cell transplantation, and T cell-based cell therapy using virus-specific T cell populations has been shown to provide protection from virus-infected cells with fewer side effects than many antiviral drug therapies. T cell-based therapy using expanded virus-specific populations has also demonstrated a graft-versus-leukemia effect, clearing circulating leukemia blasts. These immunotherapies have the advantage of providing lifelong protection through the generation of memory populations. Furthermore, these cells are easily expanded ex vivo because the donors from which they are derived are seropositive (implying the presence of memory) and virus-specific T cells rapidly expand in the presence of antigen. However, these methods suffer from the requirement for T cells obtained from donors whose immune systems already recognize viral or tumor antigens (e.g., donors seropositive for a particular virus) (see Ngo, et al., J. Immunother. 37(4): 192-203 (2014)).

[0004] For example, if naive T cells or T cell precursor populations in umbilical cord blood are not exposed to and stimulated by antigens or antigenic peptides, virus- and other antigen-specific T cells cannot proliferate. Such naive populations lack antigen-specific memory T cells that can rapidly proliferate when exposed to the antigens they recognize. For example, when a subject undergoes umbilical cord blood transplantation, the umbilical cord blood contains almost entirely naive T cells that do not provide protection against viruses, other pathogens, or tumors. Similar transplants, such as stem cell transplants from naive donors who are seronegative for certain viruses, pathogens, or tumor antigens, also lack rapidly proliferating memory T cells. As a result, the proliferation of virus-specific T cells for transplantation from umbilical cord blood or naive donors is limited and clinically unusable.

[0005] Difficulties associated with generating virus-specific T cells from these populations arise from (1) the need for stimulation of naive antigen-specific T cells and (2) the limited volume of umbilical cord blood. Umbilical cord blood units typically contain a total of 25 mL of blood. From this 25 mL, 20 mL is typically administered directly to the patient as a transplant to regenerate the immune system, while only 5 mL remains for potential T cell expansion. Furthermore, the naive T cells present in the product, which are also limited in quantity, have traditionally made this process undesirable for clinical situations, highlighting the need for the development of new processes to generate the types and numbers of virus- or other antigen-specific T cells required for successful immunotherapy.

[0006] Methods for stimulating and expanding virus- or other antigen-specific T cells from naive T cells have not yet been successful (see McGoldrick, et al., “Cytomegalovirus-specific T cells are primed early after cord blood transplant but fail to control virus in vivo”, Blood 121(14): 2796-2803 (Epub 2013)). This is consistent with the observation that the developing immune system of newborns has little immunological memory, increasing their vulnerability to infectious agents (see Basha, et al., “Immune responses in neonates”, Expert Rev. Clin. Immunol. 10(9):1171-1184 (2014)). Neonatal, congenital, and / or intrauterine pathogens include rubella, cytomegalovirus (CMV), parvovirus B19, varicella-zoster (VZV), enteroviruses, HIV, HTLV-1, hepatitis C, hepatitis B, Lassa fever, and Japanese encephalitis. Perinatal and neonatal infectious agents include herpes simplex virus (including human herpes simplex types 1 and 2), VZV, enteroviruses, HIV, hepatitis B, hepatitis C, and HTLV-1. Other pathogens include respiratory syncytial virus (RSV), metapneumovirus (hMPV), rhinovirus, parainfluenza (PIV), and human coronaviruses, norovirus, herpes simplex virus (HSV), Zika virus, and encephalitis virus.

[0007] An additional problem with many existing methods for expanding virus- and other antigen-specific T cells is that many current methods involve the use of infectious viruses, virus-infected cells, or virus-transformed cells, such as Epstein-Barr virus-transformed lymphoblastoid cell lines (Ngo, et al. (2014)). Methods for therapeutic use that involve the use of viruses to produce virus- and other antigen-specific T cells are undesirable because they are associated with clinical risks and significant regulatory obstacles.

[0008] One embodiment of the present invention advantageously enables the rapid and robust expansion of virus- and other antigen-specific T cells from naive populations, thereby providing virus- and other antigen-specific T cells that recognize therapeutically important antigens, such as opportunistic viral antigens and tumor antigens. This embodiment does not require the use of live viruses or virus-transformed cells, thereby making it more clinically acceptable. It also does not require the use of infectious or dangerous agents that are discouraged or prohibited by U.S. or international regulatory agencies. Furthermore, T cells expanded by this embodiment can be easily used in clinical practice or banked for convenient use as an off-the-shelf product. Summary of the Invention

[0009] In some embodiments, the present invention provides a powerful method for generating T cells that specifically recognize a particular antigen, such as an antigen derived from a virus, other pathogen, or tumor. The present invention also generates populations of T cells that recognize different or multiple epitopes of a pathogen, providing broad-spectrum cellular immunity. For example, to obtain a broad-spectrum cellular immune response, a naive cell population can be exposed to antigen-presenting cells that are stimulated with and present overlapping peptides corresponding to one or more antigens of a particular pathogen, such as cytomegalovirus. These peptides can stimulate different antigen-presenting cells (dendritic cells, monocytes, K562 cells, PHA blasts, B-blasts, lymphoblast cells, and CD3-28 blasts), and the method can utilize different stimulatory and proliferation cytokines (including, but not limited to, IL2, IL7, and IL15) and different selection methods (such as depletion of CD45RO). Virus or other antigen-specific T cells produced in this manner can be used to treat post-transplant viral infection, infection by a non-viral pathogen, or tumor recurrence in subjects who have received a transplant of naive cord blood, donor stem cells, or other cells. Moreover, the antigen-specific T cells can be advantageously banked or stored for later administration to a subject in need of treatment, e.g., a subject in need of T cells that recognize a particular virus or tumor.

[0010] In other embodiments, the present invention provides antigen-specific T cells, including populations of antigen-specific T cells that recognize multiple determinants of an antigen, which can be used to enhance or support the immune system of other subjects, including those who have not received umbilical cord blood or naive hematological cell transplants, if necessary. Examples of such subjects include those who have received organ transplants, those who have undergone immune system ablation, and those who are immunosuppressed or immunocompromised, such as those with opportunistic infections. The present invention produces T cells specific for multiple viral antigens from naive T cells in a clinically relevant manner that has never been achieved before from naive T cells. In some embodiments, the present invention itself is a method and use that can be readily applied to other opportunistic viruses, including, but not limited to, HHV6 and BK virus. It can be expanded to include virus-specific antigens from diseases associated with malignancies, such as, but not limited to, EBV and HIV. Other medical uses include promoting engraftment and providing therapy to immunocompromised patients prior to transplantation.

[0011] Without limitation, embodiments of the present invention can be combined with other therapies, such as cell products, lymphocyte depletion regimens, epigenetic modifiers, or other anti-bacterial or anti-tumor therapies.

[0012] In some embodiments, the present invention generates antigen-specific T cells using different overlapping peptide libraries that stimulate different antigen-presenting cells (dendritic cells, monocytes, K562 cells, PHA blasts, B-blasts, lymphoblasts, and CD3-CD28 blasts), different stimulatory and proliferation cytokines (including but not limited to IL2, IL7, IL15), and different selection methods (such as CD45RO depletion). These cells are used to treat viral or other microbial infections after transplantation.

[0013] In another embodiment, the invention involves third party deposit of antigen-specific T cells produced from naive T cells by a method that selects the most suitable donor.

[0014] Another advantageous feature of many embodiments of the invention is that they involve simple and repeatable steps that are compatible with good manufacturing practice. There is no need to perform multiple complex, potentially non-repeatable or non-standardizable steps. The methods of the invention are safe, simple, rapid, and reproducible, and can be used to produce T cells specific for viruses and other antigens for a variety of different patients.

[0015] The method of the present invention is broad in scope in that it can be targeted to different patients receiving different transplants, e.g., transplants of cord blood, stem cells, or other naive donor cells. For example, it is the only method for producing virus- and other antigen-specific T cells for cord blood transplant patients when the same cord blood unit is used in the transplant, and it can also be used to manufacture virus- and other antigen-specific T cells that protect patients from opportunistic infections.

[0016] Specific non-limiting embodiments of the present invention include: 1. A method for producing T cells specific for a virus or other antigen, the method comprising: (a) Mononuclear cells from a cord blood sample or other sample containing naive immune cells are divided into two portions; (b) contacting a first portion of said sample with PHA or other mitogen, and / or IL-2, to produce ATCs ("activated T cells"), and treating said ATCs with radiation or other agent that inhibits their growth; (c) T cells and T cell precursors (e.g., nonadherent cells, CD3 + cells) to dendritic cells and dendritic precursor cells (e.g., adherent cells, CD11C + or CD14 + cells); (d) cryopreserving or otherwise preserving the non-adherent cells; (e) contacting the adherent cells in the second portion with a cytokine or other agent that generates and matures dendritic cells and at least one virus or other peptide antigen to produce antigen-presenting dendritic cells that present at least one peptide antigen, and treating the antigen-presenting dendritic cells with radiation or other agent sufficient to inhibit their growth; (f) contacting the cryopreserved or otherwise preserved non-adherent cells obtained from (d) with the dendritic antigen-presenting cells produced in (e) in the presence of IL-7 and IL-15 to produce virus or other antigen-specific T cells that recognize at least one viral or peptide antigen; (g) contacting the virus or other antigen-specific T cells produced by (f) with the ATCs of (b) optionally in the presence of at least one peptide antigen in the presence of K562 cells or other accessory cells, and in the presence of IL-15; optionally repeating (g) one or more times; (h) recovering virus or other antigen-specific T cells that recognize at least one virus or other peptide antigen; and (i) Optionally, administering the antigen-specific T cells to a subject in need thereof, or banking or storing the antigen-specific T cells. 2. The method of embodiment 1, further comprising, prior to (a), isolating mononuclear cells from the cord blood or other sample containing naive T cells. 3. The method of embodiment 1 or 2, wherein said mononuclear cells are obtained from umbilical cord blood. 4. The method of embodiment 1, 2 or 3, wherein said mononuclear cells are obtained from stem cells naive to at least one viral or other peptide antigen. 5. The method of embodiment 1, 2, 3 or 4, wherein said mononuclear cells are obtained from a sample comprising stem cells, progenitor T cells, or T cells from a subject whose immune system is naive to at least one viral or other peptide antigen. 6. The method of embodiment 1, 2, 3, 4, or 5, wherein (b) comprises contacting a first portion of the sample with PHA and IL-2 to produce ATCs ("activated T cells"). These ATCs may be cryopreserved or otherwise banked for later use, or may be used immediately. Preferably, neither the ATCs nor the virus- or other antigen-specific T cells need to be cryopreserved; the ATCs are immediately mixed with the virus- or other antigen-specific T cells produced in (f). For example, the PHA blasts prepared in (b) can be used to provide a second stimulation to the virus- or other antigen-specific T cells produced in (f) 14 to 16 days after the start of the process. 7. The method of embodiment 1, 2, 3, 4, 5 or 6, comprising in (b) contacting about 1,000,000 to 20,000,000, preferably about 5,000,000 to 15,000,000, and most preferably about 8,000,000 to 12,000,000 monocytic cord blood cells with PHA and IL-2. 8. The method of embodiment 1, 2, 3, 4, 5, or 7, wherein (b) comprises production of T-blasts, B-blasts, lymphoblast cells, or CD3-CD28 blasts. 9. The method of any one of embodiments 1-8, wherein T cells and T cell precursors are separated from dendritic cells and dendritic precursor cells by contacting the second portion with a solid medium for a predetermined time under conditions sufficient for the cells in the second portion to adhere to the solid medium, and then removing the T cells and T cell precursors from the solid medium and recovering the dendritic cells and dendritic precursors that adhere to the solid medium. Alternatively, these two cell populations can be magnetically separated by the use of antibodies or other ligands that specifically recognize each population, or by other known cell sorting methods. The separated cell populations may be cryopreserved or otherwise deposited for later use or may be used immediately for the production of T cells or dendritic cells. These populations may be cryopreserved or otherwise deposited after subsequent processing steps described herein to produce mature dendritic cells loaded with viral or other peptide antigens, or T cells specific for viral or other antigens. 10. The method of any one of embodiments 1 to 9, wherein in (e), the dendritic cells and dendritic precursor cells are contacted with at least one dendritic cell-generating cytokine selected from the group consisting of IL-4 and GM-CSF.

[0017] 11. The method of any one of embodiments 1 to 10, wherein in (e), the dendritic cells and dendritic precursor cells are contacted with a dendritic cell maturation cytokine or agent selected from the group consisting of LPS, TNF-α, IL-1β, IL-6, PGE-1, and PGE-2, along with IL-4 and GM-CSF. 12. The method of any one of embodiments 1 to 11, wherein in (f) or before (f), the dendritic cells and dendritic precursor cells are treated to expand CD45RA-positive cells. 13. The method of any one of embodiments 1 to 12, wherein in (f) or before (f), the dendritic cells and dendritic precursor cells are treated to deplete CD45RO positive cells. 14. The method of any one of embodiments 1-14, wherein said at least one viral or other peptide antigen comprises a series of overlapping peptides. 15. The method of any one of embodiments 1-14, wherein said at least one viral or other antigen-specific peptide antigen comprises a tumor-associated or tumor-specific antigen. 16. The method of any one of embodiments 1-15, wherein said at least one viral or other peptide antigen is a determinant of a tumor-associated or tumor-specific antigen selected from the group consisting of PRAME, NYESO, MAGE A4, MAGE A3, MAGE A1, survivin, WT1, neuroelastase, proteinase 3, p53, CEA, claudin 6, histone H1, histone H2, histone H3, histone H4, MART1, gp100, PSA, SOX2, SSX2, Nanog, Oct4, Myc, and Ras. 17. The method of any one of embodiments 1-16, wherein the at least one peptide antigen comprises a viral determinant, including a peptide derived from or related to an MHC-I or MHC-II restricted virus, including opportunistic or emerging viral pathogens such as Zika virus and other disease-associated viruses. 18. The method of any one of embodiments 1-17, wherein said at least one peptide antigen comprises a filovirus determinant, for example, a GP, NP, VP40, VP35, VP30, or VP24 determinant from Ebola virus. 19. The method of any one of embodiments 1-18, wherein said at least one peptide antigen comprises a determinant of the measles virus, such as a determinant of antigens P, V, C, M, N, F, P, or L. 20. The method of any one of embodiments 1-19, wherein said at least one viral or other peptide antigen is a viral antigen from an opportunistic viral pathogen, a viral antigen from a neonatal congenital or intrauterine pathogen, such as rubella, cytomegalovirus (CMV), parvovirus B19, varicella-zoster (VZV), enterovirus, HIV, HTLV-1, hepatitis C, hepatitis B, Lassa fever, and Japanese encephalitis; or a set of overlapping peptides corresponding to a viral antigen from a perinatal or neonatal pathogen, such as human herpes simplex, VZV, enterovirus, HIV, hepatitis B, hepatitis C, HTLV-1, Zika virus, or encephalitis virus.

[0018] 21. The method of any one of embodiments 1-20, wherein said at least one viral peptide antigen is a series of overlapping peptides that correspond to or are constituents of overlapping fragments of all or part of a CMV antigen. 22. The method of any one of embodiments 1-21, wherein said at least one viral or other peptide antigen is a series of overlapping peptides corresponding to an Epstein-Barr virus (EBV) antigen or an adenovirus antigen. 23. The method of any one of embodiments 1-22, wherein said at least one viral or other peptide antigen comprises a peptide or set of peptides from multiple viral antigens of an opportunistic or emerging viral pathogen. 24. The method of any one of embodiments 1-23, wherein said at least one peptide antigen comprises a determinant of a bacterial antigen. 25. The method of any one of embodiments 1-24, wherein said at least one peptide antigen comprises a mycobacterial determinant, such as a determinant of ESAT6, HLPMt, PPE5, MVA85A, AG85, PSTS1, ACR, HSP65, GroES, EsxA, EsxB, MPB70 from Mycobacterium tuberculosis. 26. The method of any one of embodiments 1-25, wherein said at least one peptide antigen comprises a determinant of a fungal, parasitic, or other eukaryotic pathogen. 27. The method of any one of embodiments 1-26, wherein said at least one peptide antigen comprises a mammalian histocompatibility antigen or other mammalian antigen. 28. The method of any one of embodiments 1 to 27, wherein in (f), the non-adherent cells from (d) are contacted with dendritic antigen-presenting cells prepared in (e) at a ratio of (d):(e) in the range of 1:1 to 200:1, preferably in the range of 5:1 to 100:1, and most preferably in the range of 5:1 to 20:1. 29. The method of any one of embodiments 1 to 28, wherein (g) further comprises contacting the T cells specific for the viral antigen with HLA-negative modified K562 cells, K562cs cells expressing CD80, CD83, CD86, and / or 4-1BBL, or other accessory cells. 30. The method of any one of embodiments 1-29, wherein (g) comprises contacting the T cells produced in (f) with ATCs and K568 cells at a T cell to ATC ratio in the range of 10:1 to 1:1, preferably in the range of 5:1 to 2:1, and most preferably about 4:1.

[0019] 31. The method of any one of embodiments 1-30, further comprising repeating (g) in the presence of IL-2 for the T cells specific for the virus or other peptide antigen recovered in (h). 32. A composition comprising T cells specific for a virus or other antigen, obtained by the method of any one of embodiments 1 to 31. 33. A T cell bank specific for a virus or other antigen, comprising a plurality of frozen or otherwise preserved samples of viable T cells specific for a virus or other antigen obtained by the method of any one of embodiments 1 to 31. 34. A method of treatment comprising administering T cells specific for a virus or other antigen obtained by the method of any one of embodiments 1 to 31 to a subject in need thereof. 35. The method of embodiment 34, wherein said subject is partially histocompatible with T cells specific for said virus or other antigen. 36. The method of embodiment 34, wherein said subject is fully histocompatible with T cells specific for said virus or other antigen. 37. The method of any one of embodiments 34-36, wherein the subject's immune system has been reconstituted with the same umbilical cord blood cells or the same naive immune cells used to produce T cells specific for the virus or other antigen. 38. The method of any one of embodiments 34-37, wherein the subject is immunocompromised. 39. The method of embodiment 34, wherein the subject's immune system has been ablated or lymphocyte-depleted, for example, by radiation, chemotherapy, infection or immunosuppression. 40. The method of any one of embodiments 34-39, wherein the subject has undergone an allograft or other transplant.

[0020] 41. The method of any one of embodiments 34 to 40, wherein the subject's immune system is naive to the antigen recognized by the produced virus or other antigen-specific T cells. 42. The method of any one of embodiments 34-41, wherein the T cells specific for the virus or other antigen recognize a cytomegalovirus antigen or antigenic determinant, or the T cells specific for the virus or other antigen recognize an antigen or antigenic determinant of the Epstein-Barr virus. 43. The method of any one of embodiments 34-42, wherein T cells specific for said virus or other antigen recognize an adenoviral antigen or antigenic determinant. 44. The method of any one of embodiments 34-43, wherein said virus or other antigen-specific T cells recognize multiple antigens or antigenic determinants of one or more opportunistic viral pathogens. 45. The method of any one of embodiments 34-44, wherein the virus-specific T cells recognize at least one viral antigen of an opportunistic viral pathogen selected from the group consisting of CMV, adenovirus, BK virus, human herpesvirus-6 (HHV6) or other herpesvirus, influenza, respiratory syncytial virus, parainfluenza virus, and varicella-zoster virus. 46. The method of any one of embodiments 34-45, wherein the virus or other antigen-specific T cells recognize at least one antigen of an opportunistic viral pathogen that is hospital- or iatrogenically acquired, or transmitted to a subject in a hospital (e.g., hospital-acquired). 47. A composition comprising mononuclear cells isolated from umbilical cord blood or other sample containing naive immune cells, PHA or other mitogen, IL-2 and medium that maintains the viability of said cells, and optionally K562 cells or other non-autologous cells that costimulate T cells, wherein optionally said cells have been treated to prevent proliferation. 48. A composition comprising: (i) Dendritic cells and dendritic precursor cells (e.g., adherent cells, CD11C + or CD14 + T cells and T cell precursors (e.g., non-adherent cells, CD3 + cell), (ii) IL-7 and IL-15, and (iii) a medium that maintains the viability of said T cells and T cell progenitor cells. 49. The composition of any one of embodiments 47 or 48, wherein the mononuclear cells, T cells, or T cell precursors are contacted with dendritic cells that have been contacted or stimulated with at least one peptide antigen, wherein the composition comprises mononuclear cells, T cells, or T cell precursors that recognize the at least one peptide antigen. 50. T cells and T cell precursors (e.g., non-adherent cells, CD3 + Dendritic cells and dendritic precursor cells (e.g., adherent cells, CD11C + or CD14 + 1. A composition comprising a dendritic cell (a dendritic cell), at least one agent for generating and maturing dendritic cells, and a medium for maintaining viability of said cells, wherein optionally, said cells are contacted with one or more peptide antigens and, optionally, treated to prevent proliferation. 51. A cell bank or cell repository comprising one or more samples of the composition of any one of embodiments 47-50 in combination with storage or freezing medium, wherein said one or more samples are optionally linked, identified or indexed by information describing their source, including full or partial DNA sequence information, information describing histocompatibility including at least one major and / or minor histocompatibility antigen or marker, and / or information about the antigens it contains or recognizes. [Brief explanation of the drawings]

[0021] The drawings particularly show non-limiting embodiments of the invention. [Figure 1] Figure 1. Cryopreservation of dendritic cells, PHA blast-initiating, and nonadherent cells. [Figure 2] Figure 2. Dendritic cell maturation and stimulation with peptide antigens. [Figure 3] Figure 3. Primary T cell stimulation by dendritic cells. [Figure 4] Figure 4 Secondary subsequent T cell stimulation. [Figure 5] FIG. 5. General description of one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0022] "Accessory cells" are cells, such as K562 cells, that provide costimulation for or otherwise assist T cell recognition of peptide antigens, and are stimulated or proliferated in the presence of peptide antigens.

[0023] "Activated T cells" or "ATCs" in the present invention are obtained by exposing mononuclear cells in umbilical cord blood or other samples containing naive immune cells to mitogens such as phytohemagglutinin (PHA) and interleukin (IL)-2.

[0024] "Antigen" includes molecules such as polypeptides, peptides, or glyco- or lipopeptides that are recognized by, for example, the cellular or humoral arms of the human immune system. The term "antigen" includes antigenic determinants such as peptides of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more amino acid residues in length that bind to, form part of, or are recognized when complexed with an MHC molecule.

[0025] "Antigen-presenting cells (APCs)" refer to a class of cells that can present one or more antigens in the form of peptide-MHC complexes that are recognized by specific effector cells of the immune system, thereby inducing an effective cellular immune response to the antigen or antigens presented. Examples of professional APCs are dendritic cells and macrophages, but any cell that expresses MHC class I or II molecules can potentially present peptide antigens.

[0026] A "control" is a reference sample or control used to compare with a sample or control being tested. A positive control measures an expected response, while a negative control provides a reference point for samples that are not expected to respond.

[0027] "Umbilical cord blood" has its usual meaning in the art and refers to the blood remaining in the placenta and umbilical cord after birth, which contains hematopoietic stem cells. Umbilical cord blood may be fresh, frozen, or obtained from a cord blood bank.

[0028] The term "cytokine" has its normal meaning in the art. Examples of cytokines used in the present invention include IL-2, IL-7 and IL-15.

[0029] The term "dendritic cell" or "DC" refers to a diverse population of morphologically similar cell types found in many lymphoid and non-lymphoid tissues (see Steinman, Ann. Rev. Immunol. 9:271-296 (1991)). One embodiment of the present invention includes umbilical cord blood-derived dendritic cells and dendritic cell precursors.

[0030] The term "effector cell" refers to a cell that can bind to or recognize an antigen and mediate an immune response. T cells specific for a virus or other antigen are effector cells.

[0031] The term "isolated" means that the material is separated from components with which it is normally contained; for example, isolated cord blood mononuclear cells can be separated from red blood cells, plasma, and other components of cord blood.

[0032] A "naive" T cell or other immune effector cell is one that has not been exposed to or stimulated by an antigen-presenting cell that presents a peptide antigen capable of activating the cell.

[0033] A "peptide library" or "overlapping peptide library" in the sense of this application is a complex mixture that collectively covers a partial or complete sequence of a protein antigen, particularly that of an opportunistic virus. Consecutive peptides in the mixture overlap with each other; for example, a peptide library may be composed of 15 amino acid-long peptides, with adjacent peptides in the library overlapping by 11 amino acid residues and spanning the entire length of the protein antigen. Peptide libraries are commercially available or can be custom-produced for a specific antigen. Methods for contacting, stimulating, or loading antigen-presenting cells are well known and are incorporated herein by reference in Ngo, et al. (2014). Peptide libraries can be obtained from JPT, whose website https: / / www.jpt.com / products / peptrack-peptide-libraries / (last accessed March 21, 2016) is incorporated herein by reference.

[0034] The term "progenitor cell" refers to a cell that can differentiate or change into a specific type of cell. For example, a "T cell progenitor cell" can differentiate into a T cell, and a "dendritic progenitor cell" can differentiate into a dendritic cell.

[0035] A "subject" is a vertebrate, preferably a mammal, and more preferably a human. Mammals include, but are not limited to, humans, apes, horses, cows, pigs, dogs, cats, mice, other farm animals, sport animals, or pets. Subjects include those in need of T cells specific for a virus or other antigen, such as, for example, those suffering from lymphopenia, those undergoing immune system ablation, those undergoing transplantation and / or immunosuppressive regimens, those with naive or developing immune systems, e.g., newborns, or those undergoing cord blood or stem cell transplants.

[0036] In an embodiment of the invention, as depicted in Figures 1, 2, 3 and 4 and explained in detail below, umbilical cord blood is used to produce T cells specific for a virus or other antigen.

[0037] Step 1. As shown in Figure 1, a cord blood unit is processed to isolate mononuclear cells (MNCs). Three subsets are isolated and expanded from the MNCs: 1) immature dendritic cells (DCs), which are isolated by adherence to plastic; 2) a T cell-containing fraction, non-adherent cells, which are cryopreserved for later use; and 3) PHA blasts, which are nonspecifically activated T cells that are subsequently used as antigen-presenting cells. These are generated from approximately 5,000,000 MNCs. Once adherent, the adherent cells (DCs) are given IL-4 and GM-CSF. This method is novel in that PHA blasts are generated from starting material, typically cryopreserved.

[0038] Step 2. As shown in Figure 2, approximately 5 days after initiation, dendritic cells are matured by the addition of a cytokine cocktail containing IL-4, GM-CSF, IL-1β, TNF-α, PGE-2, IL-6, and LPS. LPS is novel in this application. Another difference is the use of adherence to DCs in a peripheral blood setting, which uses CD14 selection for enrichment of DC precursors.

[0039] In step 3, as shown in Figure 3, mature dendritic cells are initially stimulated with overlapping peptides, irradiated to prevent proliferation, and then combined with (thawed) non-adherent cells in the presence of IL-7 and IL-15. IL-12 is no longer used.

[0040] As shown in Figure 4, approximately 14-16 days after the initiation of culture (7-9 days after the first T cell stimulation), PHA blasts (derived from the same cord blood) are stimulated with the same overlapping peptide, irradiated, and combined with K562 cells; these two cells act as antigen-presenting cells for the pre-expanded T cells. The use of peptide-stimulated PHA blasts and K562 cells differs from previous cord blood generation procedures. This embodiment has the advantage of eliminating the need to freeze T cells after expansion. Previous methods required waiting for LCLs before continuing. Because there is no need to wait for LCLs, antigen-specific cells can be produced in approximately 30 days instead of 60 days. Other differences from previous methods include the use of PHA blasts instead of CD3 / CD28 blasts, and the T cells that respond to PHA are naive T cells, unlike previous procedures using peripheral blood, where many T cells are memory cells. [Example]

[0041] Production and expansion of virus- or other antigen-specific T cells from umbilical cord blood Non-adherent mononuclear cells (e.g., naive T cells) isolated from umbilical cord blood were stimulated by contact with irradiated, peptide-stimulated antigen-presenting cells prepared from non-adherent cells (e.g., monocytes, dendritic cells, etc.) in the cord blood, followed by stimulation with irradiated, peptide-stimulated antigen-presenting cells nonspecifically expanded from the cord blood. This method produced T cells specific for viruses or other antigens from the cord blood.

[0042] Specifically, mononuclear cells were isolated from umbilical cord blood by centrifugation at 800 × g for 20 minutes at room temperature with little acceleration or braking on a Ficoll density gradient. Approximately 10,000,000 isolated mononuclear cells were stored to produce nonspecifically proliferating T cells (antigen-presenting cells), also known as "activated T cells" or "ATCs." In this case, phytohemagglutinin (PHA) was used to stimulate the ATCs.

[0043] The remaining isolated mononuclear cells were plated onto tissue culture plates containing Cellgenix CellGro serum-free medium. After 1–2 hours, the tissue culture plates were washed with PBS to remove non-adherent cells and then frozen and stored for later use.

[0044] After washing, cells remaining attached to the cell culture plate were mixed with cytokines to generate dendritic cells (DCs) by contacting the cells with 1000 U / mL interleukin (IL)-4 and 800 U / mL granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by 1000 U / mL IL-4 and 800 U / mL GM-CSF plus 30 ng / mL lipopolysaccharide (LPS), 10 ng / mL tumor necrosis factor α (TNF-α), 10 ng / mL IL-1β, 100 ng / mL IL-6, and 1 μg / mL prostaglandin (PGE)-2 or PGE-1.

[0045] Dendritic cells were matured for 7 days from initiation and then stimulated with approximately 200 ng of overlapping peptide pools per cell, including each peptide from the overlapping peptide library. In this case, we used overlapping peptides derived from JTP, including IE-1 and pp65 from CMV, hexon and penton from adenovirus, and LMP2 and BZLF-1 from EBV. These overlapping peptide mixtures, or "pepmixes," consist of 15-amino acid peptides spanning the entire protein (antigen), with an 11-amino acid overlap between adjacent peptides. This allows for the proliferation of both CD4+ and CD8+ T cells, regardless of MHC class restriction. Following stimulation of mature dendritic cells with the overlapping peptide pools, the cells were irradiated at 25 Gy to prevent their proliferation.

[0046] At this time, cryopreserved nonadherent cells that had previously been washed off the cell culture plate were thawed and plated together with peptide-stimulated dendritic cells at a ratio of approximately 1:10 nonadherent cells to DCs in the presence of cytokines, 10 ng / mL IL-7 and 5 ng / mL IL-15. This corresponds to the initial antigen stimulation of cryopreserved nonadherent mononuclear cells (e.g., naive T cells). Cells were cultured in a naive T cell-specific medium containing 45% Advanced RPMI, 45% Click's (EHAA) medium, 10% human AB serum, and 200 mM Glutamax.

[0047] Cryopreserved nonadherent cells were cultured for 8–10 days in the presence of irradiated (25 Gy for DCs, 75 Gy for ATC and K562) peptide-stimulated nonadherent cells (e.g., naive T cells), then collected, T cell numbers determined, and resuspended in T cell medium.

[0048] The resuspended T cells were contacted with irradiated ATCs, which were stimulated with the same pool of overlapping peptides presented on irradiated mature dendritic cells derived from adherent cord blood mononuclear cells, at a ratio of 1:5, in the presence of the cytokine IL-15 (5 ng / mL), followed by IL-2 cytokine (50-100 U / mL) twice weekly. After this second stimulation, T cells recognizing antigenic determinants in the overlapping peptide pool were recovered. This was achieved by assessing T cell activation by IFN-γ in an ELISPOT assay and by assessing the cytolytic ability of T cells in a chromium release cytotoxicity assay.

[0049] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0050] With the present invention now fully disclosed, it will be apparent to those skilled in the art that many changes and modifications may be made thereto without departing from the spirit and scope of the following claims. The following claims as originally filed in this application are appended as embodiments. [1] A method for producing T cells specific for a virus or other antigen, the method comprising: (a) Mononuclear cells from a cord blood sample or other sample containing naive immune cells are divided into two portions; (b) contacting a first portion of said sample with PHA or other mitogen, and optionally IL-2, to produce ATCs ("activated T cells"), and treating said ATCs with radiation or other agent that inhibits their growth; (c) T cells and T cell precursors (e.g., nonadherent cells, CD3 + cells) to dendritic cells and dendritic precursor cells (e.g., non-adherent cells, CD11C + or CD14 + cells); (d) cryopreserving or otherwise preserving the non-adherent cells; (e) contacting the adherent cells in the second portion with IL-4 and GM-CSF or other cytokines and / or other agents that generate and mature dendritic cells, and at least one CMV peptide antigen or other peptide antigen, to produce antigen-presenting dendritic cells that present at least one peptide antigen, and treating the antigen-presenting dendritic cells with radiation or other agents sufficient to inhibit their growth; (f) contacting the cryopreserved or otherwise preserved non-adherent cells obtained from (d) with the dendritic antigen-presenting cells produced in (e) in the presence of IL-7 and IL-15 to produce virus or other antigen-specific T cells that recognize at least one peptide antigen; (g) contacting the virus or other antigen-specific T cells produced by (f) with the ATCs of (b) in the presence of at least one peptide antigen in the presence of K562 cells or other accessory cells, and in the presence of IL-15; optionally repeating (g) one or more times; (h) recovering virus or other antigen-specific T cells that recognize at least one virus or other type of peptide antigen; and (i) Optionally, administering the antigen-specific T cells to a subject in need thereof, or banking or storing the antigen-specific T cells. [2] The method according to [1], further comprising isolating mononuclear cells from umbilical cord blood or other sample containing naive T cells before (a). [3] The method according to [1], wherein the mononuclear cells are obtained from umbilical cord blood. [4] The method according to [1], wherein the mononuclear cells are obtained from stem cells that are naive to at least one virus or other peptide antigen. [5] The method according to [1], wherein the mononuclear cells are obtained from a sample containing stem cells, precursor T cells, or T cells from a subject whose immune system is naive to at least one virus or other peptide antigen. [6] The method of [1], wherein (b) comprises contacting a first portion of the sample with PHA and IL-2 to produce ATCs ("activated T cells"). [7] The method according to [1], wherein in (b), about 1,000,000 to 20,000,000, preferably about 5,000,000 to 15,000,000, most preferably about 8,000,000 to 12,000,000 monocytic cord blood cells are contacted with PHA and IL-2. [8] The method according to [1], wherein (b) comprises the production of T-blasts, B-blasts, lymphoblast cells or CD3-CD28 blasts. [9] The method of [1], wherein the T cells and T cell precursors are separated from the dendritic cells and dendritic precursors by contacting the second portion with a solid medium for a predetermined period of time under conditions sufficient for the cells in the second portion to adhere to the solid medium, and then removing the T cells and T cell precursors from the solid medium and recovering the dendritic cells and dendritic precursors that have adhered to the solid medium.

[10] The method according to [1], wherein in (e), the dendritic cells and dendritic precursor cells are contacted with at least one dendritic cell-generating cytokine selected from the group consisting of IL-4 and GM-CSF.

[11] The method according to [7], wherein in (e), the dendritic cells and dendritic precursor cells are contacted with a dendritic cell maturation cytokine or agent selected from the group consisting of LPS, TNF-α, IL-1β, IL-6, PGE-1, and PGE-2, together with IL-4 and GM-CSF.

[12] The method according to [1], wherein in (f) or before (f), the dendritic cells and dendritic precursor cells are treated to expand CD45RA-positive cells.

[13] The method according to [1], wherein in (f) or before (f), the dendritic cells and dendritic precursor cells are treated to deplete CD45RO-positive cells.

[14] The method of [1], wherein the at least one viral or other peptide antigen comprises a series of overlapping peptides.

[15] The method of [1], wherein the at least one viral or other peptide antigen comprises a tumor-associated or tumor-specific antigen.

[16] The method of [1], wherein the at least one viral or other peptide antigen is a determinant of a tumor-associated or tumor-specific antigen selected from the group consisting of PRAME, NYESO, MAGE A4, MAGE A3, MAGE A1, survivin, WT1, neuroelastase, proteinase 3, p53, CEA, claudin 6, histone H1, histone H2, histone H3, histone H4, MART1, gp100, PSA, SOX2, SSX2, Nanog, Oct4, Myc, and Ras.

[17] The method of [1], wherein the at least one viral or other peptide antigen comprises a viral determinant.

[18] The method of [1], wherein the at least one viral or other peptide antigen comprises a filovirus determinant, such as a GP, NP, VP40, VP35, VP30, or VP24 determinant from Ebola virus.

[19] The method of [1], wherein the at least one viral or other peptide antigen comprises a determinant of measles virus, such as a determinant of antigens P, V, C, M, N, F, P, or L.

[20] The method of [1], wherein the at least one viral or other peptide antigen is a viral antigen from an opportunistic viral pathogen, including CMV, a viral antigen from a neonatal congenital or intrauterine pathogen, such as rubella, cytomegalovirus (CMV), parvovirus B19, varicella-zoster (VZV), enterovirus, HIV, HTLV-1, hepatitis C, hepatitis B, Lassa fever, and Japanese encephalitis; or a set of overlapping peptides corresponding to a viral antigen from a perinatal or neonatal pathogen, such as human herpes simplex, VZV, enterovirus, HIV, hepatitis B, hepatitis C, or HTLV-1.

[21] The method of [1], wherein the at least one viral or other peptide antigen is a series of overlapping peptides corresponding to a CMV antigen.

[22] The method of [1], wherein the at least one viral or other peptide antigen is a series of overlapping peptides corresponding to an Epstein-Barr virus (EBV) antigen or an adenovirus antigen.

[23] The method of [1], wherein the at least one viral or other peptide antigen comprises a peptide or series of peptides from multiple viral antigens of an opportunistic or emerging viral pathogen.

[24] The method of [1], wherein the at least one viral or other peptide antigen comprises a determinant of a bacterial antigen.

[25] The method of [1], wherein the at least one viral or other peptide antigen comprises a mycobacterial determinant, such as a determinant of ESAT6, HLPMt, PPE5, MVA85A, AG85, PSTS1, ACR, HSP65, GroES, EsxA, EsxB, or MPB70 from Mycobacterium tuberculosis.

[26] The method of [1], wherein the at least one viral or other peptide antigen comprises a determinant of a fungal, parasitic, or other eukaryotic pathogen.

[27] The method of [1], wherein the at least one viral or other peptide antigen comprises a mammalian histocompatibility antigen or other mammalian antigen.

[28] The method according to [1], wherein in (f), the non-adherent cells from (d) are contacted with the dendritic antigen-presenting cells prepared in (e) at a ratio of (d):(e) in the range of 1:1 to 200:1, preferably in the range of 5:1 to 100:1, and most preferably in the range of 5:1 to 20:1.

[29] The method of [1], wherein (g) further comprises contacting T cells specific for the virus or other peptide antigen with HLA-negative modified K562 cells, K562cs cells expressing CD80, CD83, CD86, and / or 4-1BBL, or other accessory cells.

[30] The method according to [1], wherein (g) comprises contacting the T cells produced in (f) with ATCs and K568 cells at a T cell to ATC ratio in the range of 10:1 to 1:1, preferably in the range of 5:1 to 2:1, and most preferably about 4:1.

[31] The method according to [1], further comprising repeating (g) in the presence of IL-2 for the T cells specific to the virus or other peptide antigen recovered in (h).

[32] A composition comprising T cells specific for a virus or other antigen, obtained by the method described in [1].

[33] A virus or other antigen-specific T cell bank containing multiple samples of frozen or otherwise preserved viable virus or other antigen-specific T cells obtained by the method described in [1].

[34] A method of treatment comprising administering to a subject in need thereof T cells specific for a virus or other antigen obtained by the method described in [1].

[35] The method of

[34] , wherein the subject is partially histocompatible with T cells specific for the virus or other antigen.

[36] The method of

[34] , wherein the subject is fully histocompatible with T cells specific for the virus or other antigen.

[37] The method described in

[34] , wherein the subject's immune system is reconstituted with the same umbilical cord blood cells or the same naive immune cells used to produce the viral antigen-specific T cells.

[38] The method according to

[34] , wherein the subject is immunocompromised.

[39] The method of

[34] , wherein the subject's immune system has been ablated or lymphocyte-depleted.

[40] The method according to

[34] , wherein the subject has undergone an allograft or other transplant.

[41] The method of

[34] , wherein the subject's immune system is naive to the antigen recognized by the produced virus or other antigen-specific T cells.

[42] The method described in

[34] , wherein T cells specific to the virus or other antigen recognize a cytomegalovirus antigen or antigenic determinant, or T cells specific to the virus or other antigen recognize an Epstein-Barr virus antigen or its antigenic determinant.

[43] The method according to

[34] , wherein T cells specific to the virus or other antigen recognize an adenovirus antigen or antigenic determinant.

[44] The method of

[34] , wherein the virus or other antigen-specific T cells recognize multiple antigens or antigenic determinants of one or more opportunistic viral pathogens.

[45] The method according to

[34] , wherein the virus or other antigen-specific T cells recognize at least one viral antigen of an opportunistic viral pathogen selected from the group consisting of CMV, adenovirus, BK virus, human herpesvirus-6 (HHV6) or other herpesvirus, influenza, respiratory syncytial virus, parainfluenza virus, and varicella-zoster virus.

[46] The method of

[34] , wherein the virus or other antigen-specific T cells recognize at least one antigen of an opportunistic viral pathogen that is hospital- or iatrogenically acquired or transmitted to a subject in a hospital (e.g., hospital-acquired infection).

[47] A composition comprising mononuclear cells isolated from umbilical cord blood or other sample containing naive immune cells, PHA or other mitogen, IL-2 and medium that maintains the viability of the cells, and, optionally, K562 cells or other non-autologous cells that costimulate T cells, wherein, optionally, the cells have been treated to prevent proliferation.

[48] A composition comprising: (i) Dendritic cells and dendritic precursor cells (e.g., adherent cells, CD11C + or CD14 + T cells and T cell precursors (e.g., non-adherent cells, CD3 + cell), (ii) IL-7 and IL-15, and (iii) a medium that maintains the viability of said T cells and T cell progenitor cells.

[49] The composition described in

[47] or

[48] , wherein the monocytes, T cells, or T cell precursors are contacted with at least one peptide antigen or contacted with dendritic cells stimulated therewith, wherein the monocytes, T cells, or T cell precursors recognize the at least one peptide antigen.

[50] T cells and T cell precursors (e.g., nonadherent cells, CD3 + Dendritic cells and dendritic precursor cells (e.g., adherent cells, CD11C + or CD14 + 1. A composition comprising a dendritic cell (a dendritic cell), at least one agent for generating and maturing dendritic cells, and a medium for maintaining viability of said cells, wherein optionally, said cells are contacted with one or more peptide antigens and, optionally, treated to prevent proliferation.

[51] A cell bank or cell repository comprising one or more samples of the composition according to any one of

[47] to

[51] in combination with storage or freezing medium, wherein said one or more samples are optionally linked, identified or indexed by information describing their source, including full or partial DNA sequence information, information describing histocompatibility including major and / or minor histocompatibility antigens or markers, and / or information about the antigens they contain or recognize.

Claims

1. A method for producing a tumor-associated antigen-specific T cell population activated against tumor-associated antigens consisting of PRAME, WT1, and survivin, wherein the antigen-specific T cell population is prepared from naive immune cells of a non-autologous human donor subject that are naive to PRAME, WT1, and survivin, and the antigen-specific T cell population is naive cells not prepared from umbilical cord blood; (i) collecting mononuclear cells from a sample obtained from a human donor having an immune system naive to PRAME, WT1, and survivin, wherein the sample contains naive immune cells other than umbilical cord blood; (ii) dividing the mononuclear cells into a first portion and a second portion; (iii) contacting a first portion of the sample with PHA to produce activated T cells (ATCs), and treating the ATCs with radiation to inhibit their proliferation; (iv) separating the non-adherent T cells and T cell precursors in said second portion from the adherent dendritic cells and dendritic precursor cells; (v) storing the non-adherent T cells and T cell precursors obtained from (iv); (vi) contacting the adherent dendritic cells and dendritic precursor cells in the second portion with IL-4 and GM-CSF, and peptide antigens derived from PRAME, WT1, and survivin to produce antigen-presenting dendritic cells that present the peptide antigens, and treating the antigen-presenting dendritic cells with radiation sufficient to prevent their proliferation. (vii) contacting the stored non-adherent T cells and T cell precursors obtained from (v) with the antigen-presenting dendritic cells produced in (vi) in the presence of IL-7 and IL-15 to produce a tumor-associated antigen-specific T cell population that recognizes PRAME, WT1, and survivin; (viii) contacting the tumor-associated antigen-specific T cell population produced in (vii) with the ATC of (iii) in the presence of the peptide antigen and IL-15; (ix) recovering the tumor-associated antigen-specific T cell population. A method comprising:

2. A tumor-associated antigen-specific T cell population recovered in step (ix) of the method of claim 1 for use as a medicament in the treatment of a tumor expressing PRAME, WT1 and survivin in a human patient subject, wherein the medicament is administered in combination with another anti-tumor therapy.

3. A non-autologous tumor-associated antigen-specific T cell population recovered in step (ix) of the method of claim 1, which is specific for a tumor antigen consisting of PRAME, WT1 and survivin and for use as a medicament in the treatment of a tumor expressing PRAME, WT1 and survivin in a human patient subject, wherein the human patient subject shares at least one major histocompatibility antigen with the tumor-associated antigen-specific T cell population.

4. The tumor-associated antigen-specific T cell population of claim 3 , wherein the human patient subject is immunocompromised.

5. 5. The tumor-associated antigen-specific T cell population of claim 4, wherein the immune system of the human patient subject has been ablated or lymphocyte-depleted by radiation therapy or chemotherapy.

6. The tumor-associated antigen-specific T cell population of claim 3 , wherein the human patient subject has undergone an allogeneic transplant.

7. 10. The method of claim 1, wherein step (viii) is repeated one or more times.

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