GENERATING VIRUS OR OTHER ANTIGEN-SPECIFIC T CELLS FROM A NAÏVE T CELL POPULATION

Abstract

Safe, rapid and efficient methods for producing virus-specific or other antigen-specific T-cells from cord blood and other samples containing naive immune cells.

Claims

1. A process for producing a virus- or other antigen-specific T cell comprising: (a) dividing mononuclear cells from a cord blood sample or other sample of nave immune cells into two portions; (b) contacting a first portion of said sample with PHA or another mitogen and, optionally with IL-2, to produce ATCs (activated T cells) and treating the ATCs with radiation or another agent to inhibit their outgrowth; (c) separating non-adherent T-cells and T-cell precursor cells from adherent dendritic cells and dendritic precursor cells); (d) cryopreserving or otherwise reserving the non-adherent cells; (e) contacting the adherent cells in the second portion with IL-4 and GM-CSF or other cytokine(s) and/or other agent(s) that generate and mature dendritic cells and with at least one peptide antigen to produce antigen-presenting dendritic cells that present the at least one peptide antigen, and treating said antigen-presenting dendritic cells with radiation or another agent sufficient to inhibit their outgrowth; (f) contacting the cryopreserved or otherwise reserved non-adherent cells from (d) with the dendritic antigen-presenting cells produced in (e) in the presence of IL-7 and IL-15 to produce antigen-specific T-cells that recognize the at least one peptide antigen; (g) contacting the antigen-specific T-cells produced by (f) with the ATCs of (b) in the presence of the at least one peptide antigen in the presence if K562 cells or other accessory cells and in the presence of IL-15; optionally, repeating (g) one or more times; (h) recovering the antigen-specific T-cells that recognize the at least one peptide antigen; and (i) optionally, administering said antigen-specific T-cells to a subject in need thereof or banking or storing said antigen-specific T-cells.

2. The process of claim 1, further comprising separating mononuclear cells from cord blood or another sample containing nave T-cells prior to (a).

3. The process of claim 1, wherein the mononuclear cells are obtained from cord blood.

4. The process of claim 1, wherein the mononuclear cells are obtained from stem cells nave to the at least one peptide antigen.

5. The process of claim 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 nave to the at least one peptide antigen.

6. The process of claim 1, wherein (b) comprises contacting a first portion of said sample with PHA and with and IL-2 to produce ATCs (activated T cells).

7. The process of claim 1 that comprises contacting about 1 to 20 million mononuclear cord blood cells with PHA and IL-2 in (b).

8. The process of claim 1, wherein (b) comprises producing T-blasts, B-blasts, lymphoblastoid cells, or CD3-CD28 blasts.

9. The process of claim 1, wherein T-cells and T-cell precursor cells are separated from dendritic cells and dendritic precursor cells by contacting the second portion with a solid medium for a time and under conditions sufficient for cells in the second portion to adhere to the solid medium and then removing non-adherent T-cells and T-cell precursor cells from the solid medium and recovering the dendritic cells and dendritic precursor cells attached to the solid medium.

10. The process of claim 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 process of claim 1, wherein in (e) the dendritic cells and dendritic precursor cells are contacted with a dendritic cell-maturing cytokine or agent selected from the group consisting of LPS, TNF-alpha, IL-1 beta, IL-6, PGE-1 and PGE-2; along with IL-4 and GM-CSF.

12. The process of claim 1, wherein in or prior to (f) the dendritic cells and dendritic precursor cells are treated to expand CD45RA positive cells.

13. The process of claim 1, wherein in or prior to (f) the dendritic cells and dendritic precursor cells are treated to deplete CD45RO positive cells.

14. The process of claim 1, wherein said at least one peptide antigen comprises a series of overlapping peptides.

15. The process of claim 1, wherein said at least one peptide antigen comprises a tumor-associated or tumor-specific antigen.

16. (canceled)

17. The process of claim 1, wherein said at least one peptide antigen comprises a virus antigen.

18.-28. (canceled)

29. The process of claim 1, wherein (g) further comprises contacting said peptide antigen-specific T-cells with K562 cells, modified HLA-negative, K562cs cells that express CD80, CD83, CD86, and/or 4-1BBL cells.

30. (canceled)

31. The process of claim 1, further comprising repeating (g) with the peptide antigen-specific T-cells recovered in (h) in the presence of IL-2.

32. An in vitro composition comprising antigen-specific T-cells produced by the process of claim 1.

33. (canceled)

34. A method of treatment comprising administering antigen-specific T-cells produced by the process of claim 1 to a subject in need thereof.

35.-51. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0080] The figures describe particular, non-limiting embodiments of the invention.

[0081] FIG. 1. Dendritic cell, PHA blast initiation, and cryopreservation of non-adherent cells.

[0082] FIG. 2. Dendritic cell maturation and pulsing with peptide antigens.

[0083] FIG. 3. 1.sup.st T-cell stimulation with dendritic cells.

[0084] FIG. 4. 2.sup.nd and subsequent T-cell stimulations.

[0085] FIG. 5. A general description of one embodiment of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0086] Accessory cell is a cell, such as a K562 cell, that provides costimulation for recognition of peptide antigens by T-cells or that otherwise assists a T-cell recognize, become primed or expand in the presence of a peptide antigen.

[0087] An activated T-cell or ATC according to the invention is obtained by exposing mononuclear cells in cord blood or another sample containing nave immune cells to a mitogen, such as Phytohemagglutinin (PHA) and Interleukin (IL)-2.

[0088] An antigen includes molecules, such as polypeptides, peptides, or glyco- or lipo-peptides that are recognized by the immune system, such as by the cellular or humoral arms of the human immune system. the term antigen includes antigenic determinants, such as peptides with lengths of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more amino acid residues that bind to MHC molecules, form parts of MHC Class I or II complexes, or that are recognized when complexed with such molecules.

[0089] An antigen presenting cell (APC) refers to a class of cells capable of presenting one or more antigens in the form of peptide-MHC complex recognizable by specific effector cells of the immune system, and thereby inducing an effective cellular immune response against the antigen or antigens being presented. Examples of professional APCs are dendritic cells and macrophages, though any cell expressing MHC Class I or II molecules can potentially present a peptide antigen.

[0090] A control is a reference sample or subject used for purposes of comparison with a test sample or test subject. Positive controls measure an expected response and negative controls provide reference points for samples where no response is expected.

[0091] Cord blood has its normal meaning in the art and refers to blood that remains in the placenta and umbilical cord after birth and contains hematopoietic stem cells. Cord blood may be fresh, cryopreserved or obtained from a cord blood bank.

[0092] The term cytokine has its normal meaning in the art. Examples of cytokines used in the invention include IL-2, IL-7 and IL-15.

[0093] The term dendritic cell or Dc describes a diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues, see Steinman, Ann. Rev. Immunol. 9:271-296 (1991). One embodiment of the invention involves dendritic cells and dendritic cell precursors derived from cord blood.

[0094] The term effector cell describes a cell that can bind to or otherwise recognize an antigen and mediate an immune response. Virus- or other antigen-specific T-cells are effector cells.

[0095] The term isolated means separated from components in which a material is ordinarily associated with, for example, an isolated cord blood mononuclear cell can be separated from red blood cells, plasma, and other components of cord blood.

[0096] A naive T-cell or other immune effector cell is one that has not been exposed to or primed by an antigen or to an antigen-presenting cell presenting a peptide antigen capable of activating that cell.

[0097] A peptide library or overlapping peptide library within the meaning of the application is a complex mixture of peptides which in the aggregate covers the partial or complete sequence of a protein antigen, especially those of opportunistic viruses. Successive peptides within the mixture overlap each other, for example, a peptide library may be constituted of peptides 15 amino acids in length which overlapping adjacent peptides in the library by 11 amino acid residues and which span the entire length of a protein antigen. Peptide libraries are commercially available and may be custom-made for particular antigens. Methods for contacting, pulsing or loading antigen-presenting cells are well known and incorporated by reference to Ngo, et al. (2014). Peptide libraries may be obtained from JPT and are incorporated by reference to the website at https://www.jpt.com/products/peptrack-peptide-libraries/ (last accessed Mar. 21, 2016).

[0098] The term precursor cell refers to a cell which can differentiate or otherwise be transformed into a particular kind of cell. For example, a T-cell precursor cell can differentiate into a T-cell and a dendritic precursor cell can differentiate into a dendritic cell.

[0099] A subject is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to humans, simians, equines, bovines, porcines, canines, felines, murines, other farm animals, sport animals, or pets. Subjects include those in need of virus- or other antigen-specific T-cells, such as those with lymphocytopenia, those who have undergone immune system ablation, those undergoing transplantation and/or immunosuppressive regiments, those having nave or developing immune systems, such as neonates, or those undergoing cord blood or stem cell transplantation.

[0100] In one nonlimiting embodiment of the invention, cord blood is used to produce the virus- or other antigen-specific T-cells as described by FIGS. 1, 2, 3 and 4 and as explained in more detail below.

[0101] Step 1. As shown in FIG. 1, cord blood unit is processed to isolate the mononuclear cells (MNC). From the MNC three subsets were isolated and expanded: 1) the immature dendritic cells (DCs), which are isolated by plastic adherence, 2) the T cell-containing fraction, the non-adherent cells, which are cryopreserved for later use, and 3) PHA blasts, which are non-specifically activated T cells that are used later as antigen presenting cells. These are generated from 5 million MNC. Once adherent, the adherent cells (DCs) are fed with IL-4 and GM-CSF. This method is novel in that the PHA blasts are generated from the starting product (which is typically cryopreserved).

[0102] Step 2. As shown in FIG. 2, about 5 days after initiation, the dendritic cells are matured by adding a cytokine cocktail containing IL-4, GM-CSF, IL-1beta, TNF-alpha, PGE-2, IL-6, and LPS. LPS is novel in this application. From the peripheral blood setting the use of adherence for DCs is also different (they use CD14-selection to enrich for DC precursors).

[0103] In step 3, as shown in FIG. 3, at initiation, the matured dendritic cells are pulsed with overlapping peptides, irradiated so that they do not expand, and they are then combined with the non-adherent cells (which are thawed in the presence if IL-7 and IL-15. IL-12 is no longer used.

[0104] In step 4 as shown in FIG. 4, which is about 14-16 days from initiation of the culture (7-9 days from the first T cell stimulation), PHA blasts (derived from the same cord blood are pulsed with the same overlapping peptides, irradiated, and then combined with K562 cells; the combination of these two act as the antigen-presenting cells for the previously-expanded T cells. The use of the peptide-pulsed PHA-blasts and K562 differs from previous cord blood generation protocols. In this embodiment, advantageously the T cells do not need to be frozen after one expansion. Prior methods required one to wait for the LCL to be ready before continuing. Since no waiting for the LCL is required, the antigen-specific T-cells can be manufactured in about 30 days instead of 60. Another difference with prior methods is that PHA blasts are used instead of CD3/CD28 blasts and because T cells responding to the PHA are nave T cells, unlike in prior protocols which used peripheral blood where the majority of T-cells were memory cells.

EXAMPLE

Production and Expansion of Virus- or Other Antigen-Specific T Cells From Cord Blood

[0105] Non-adherent mononuclear cells (e.g., nave T cells) isolated from cord blood were simulated by contact with irradiated peptide-pulsed antigen presenting cells prepared from non-adherent cells (e.g., monocytes, dendritic cells, etc.) in cord blood and then by irradiated peptide-pulsed antigen presenting cells non-specifically expanded from cord blood. This method was produced virus- or other antigen specific T-cells from cord blood cells.

[0106] Specifically, mononuclear cells were isolated from cord blood by centrifugation at 800g for 20 minutes with little acceleration and brake and at room temperate on a Ficoll gradient. Approximately 10 million of the isolated mononuclear cells were reserved to produce non-specifically expanded T cells (antigen-presenting cells) also known as Activated T Cells or ATCs. In this case, Phytohemagglutinin (PHA) was used to stimulate the ATCs.

[0107] 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 was washed with PBS to remove non-adherent cells which were then cryopreserved and saved for later use.

[0108] The cells that adhered to the cell culture plates after washing were mixed with cytokines to generate dendritic cells (DC). This was done by contacting the cells with 1000 U/mL Interleukin (IL)-4, and 800 U/mL Granulocyte-Macrophage/Colony Stimulating Factor (GM-CSF) and then with 30 ng/mL Lipopolysaccharide (LPS), 10 ng/mL Tumor Necrosis Factor Alpha (TNF-), 10 ng/mL IL-1, 100 ng/mL IL-6, and 1 ug/mL Prostaglandin (PGE)-2 or PGE-1, along with 1000 U/mL IL-4 and 800 U/mL GM-CSF.

[0109] Once the dendritic cells matured for 7 days from initiation and they were pulsed with a pool of overlapping peptides containing about 200 ng of each peptide per million cells obtained from an overlapping peptide library. In this case we used the overlapping peptides from JPT 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 that span the entire protein (antigen) and overlap neighboring peptides by 11 amino acids. This allows for the expansion of both CD4+ and CD8+ T cells, regardless of the MHC class-restriction. Following the pulsing of the mature dendritic cells with the pool of overlapping peptides the cells were irradiated at 25 Gy to prevent their outgrowth.

[0110] At this time, the cryopreserved non-adherent cells previously washed off the cell culture plates were thawed and plated with the peptide-pulsed dendritic cells at an approximate ratio of 1 DC to 10 non-adherent cells in the presence of the cytokines 10 ng/mL IL-7 and 5 ng/mL IL-15. This represented an initial antigen-stimulation of the cryopreserved non-adherent mononuclear cells (e.g., nave T cells). Cells were grown in a nave T cell-specific medium containing 45% Advanced RPMI, 45% Click's (EHAA) medium, 10% human AB serum, and 200 mM Glutamax.

[0111] The cryopreserved non-adherent cells were cultured for 8-10 days in the presence of the irradiated (25 Gy for DC, 75 Gy for ATCs and K562) peptide-pulsed non-adherent cells (e.g., naive T cells) and then harvested, the number of T-cells determined, and resuspended in a T cell medium.

[0112] The T-cells in the resuspension were contacted with irradiated ATCs, which have been pulsed with the same pool of overlapping peptides that were present on the irradiated mature dendritic cells derived from the adherent mononuclear cells of cord blood, at a ratio of 1 T-Cells to 1 irradiated ATC to 5 K562 cells in the presence of cytokine IL-15 (5 ng/mL) followed by twice-weekly feeds with the IL-2 cytokine (50-100 U/mL). After this secondary stimulation, T-cells which recognized antigenic determinants in the pool of overlapping peptides were recovered. This was achieved by assessing T cell activation via IFN-gamma ELISPOT assay and assessing the cytolytic ability of the T cells in a chromium release cytotoxicity assay.

[0113] 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.

[0114] The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the following claims.