IL-10 gene transfer to peripheral mononuclear cells
View Patent ↗The present invention to methods for producing mononuclear cells overexpressing IL-10. The method comprises the ex vivo introduction of an expression construct comprising a nucleotide sequence encoding a polypeptide having IL-10 activity into peripheral blood mononuclear cells of a subject. The thus obtained peripheral blood mononuclear cells have an altered phenotype as a result of the expression of an introduced IL-10 transgene. In particular the invention relates to CD4 + T cells that functionally behave as regulatory T cells as a result of the expression of an IL-10 transgene. The IL-10 transgenic mononuclear cells may be used to treat a variety of inflammatory diseases, particularly T helper 1-mediated inflammatory diseases.
1. A method for producing an anti-inflammatory, antigen-non-specific population of lymphocytes overexpressing interleukin-10 (IL-10) polypeptide that are useful for treating a Th1-mediated inflammatory disease in an antigen-independent manner when administered to a subject, the method comprising:
(a) modifying at least a portion of a peripheral blood mononuclear cell population from a healthy donor, among which cells lymphocytes are not selected or enriched on the basis of antigen specificity, by introducing into said cells an expression construct that comprises a nucleotide sequence encoding an IL-10 polypeptide; and,
(b) recovering, from said modified mononuclear cells, lymphocytes or a subset thereof that overexpress the IL-10 polypeptide,
thereby producing said anti-inflammatory, antigen non-specific lymphocyte population.
2. A method according to claim 1 , wherein prior to the introducing of step (a), the cells are induced to, or allowed to, proliferate.
3. A method according to claim 2 , wherein the cells are induced to proliferate by exposure to a proliferating agent.
4. A method according to claim 3 , wherein the proliferating agent is one or more of
(a) an anti-CD3 antibody;
(b) an anti-CD28 antibody; or
(c) phytohemagglutinin.
5. A method according to claim 1 , wherein prior to or subsequent to step (a), the mononuclear cells are fractionated to yield an enriched fraction or subset of lymphocytes.
6. A method for producing a pharmaceutical composition comprising lymphocytes overexpressing IL-10, which method comprises
(a) producing the lymphocytes overexpressing IL-10 in accordance with claim 1 , and
(b) combining said lymphocytes with an acceptable pharmaceutical carrier.
7. The method according to claim 1 wherein the lymphocyte subset comprises an enriched population of B lymphocytes or T lymphocytes.
8. The method according to claim 7 , wherein the lymphocyte subset comprises an enriched population of T lymphocytes.
9. The method according to claim 8 wherein the T lymphocytes are CD4+ T lymphocytes.
10. A composition comprising lymphocytes from a healthy donor that are:
(i) not selected to be specific for a predetermined antigen, and
(ii) which cells are modified to comprise and overexpress an IL-10 transgene.
11. The composition according to claim 10 wherein the lymphocytes are T lymphocytes.
12. A T lymphocyte composition according to claim 11 , wherein the T cells functionally mimic regulatory T cells in that they inhibit:
(a) proliferation of autologous responder cells, and/or
(b) production of pro-inflammatory cytokine IL-12 by dendritic cells.
13. A pharmaceutical composition comprising the composition according to claim 11 , and a pharmaceutically acceptable carrier.
14. A method of treating a Th1-mediated inflammatory disease or condition associated with undesired activation and/or expansion of T cells in a subject in need thereof, comprising administering an effective amount of a pharmaceutical composition according to claim 13 to said subject.
15. A composition according to claim 11 wherein the T lymphocytes are CD4+ T lymphocytes.
16. A pharmaceutical composition comprising the composition according to claim 15 , and a pharmaceutically acceptable carrier.
17. A method of treating a Th1-mediated inflammatory disease or condition associated with undesired activation and/or expansion of T cells in a subject in need thereof, comprising administering an effective amount of a pharmaceutical composition according to claim 16 to said subject.
18. A pharmaceutical composition comprising the composition according to claim 10 , and a pharmaceutically acceptable carrier.
19. A method of treating a Th1-mediated inflammatory disease or condition associated with undesired activation and/or expansion of T cells in a subject in need thereof, comprising administering an effective amount of a pharmaceutical composition according to claim 18 to said subject.
20. The method according to claim 19 , wherein the Th1-mediated inflammatory disease or condition is Crohn's disease, reactive arthritis, insulin-dependent diabetes, colitis, pancreatitis, an inflammatory lung disease, an inflammatory eye disease, multiple sclerosis, Hashimoto's thyroiditis, Graves' disease, chronic articular rheumatism, contact dermatitis, psoriasis, graft rejection, graft-versus-host disease, or sarcoidosis.
21. The method according to claim 20 wherein the Th1-mediated inflammatory disease Crohn's disease or colitis.