Method for preparation of cancer/testis antigen-specific t-cells
AssigneeCytovac A/S
InventorsAlexei Kirkine, Karine Dzhanzhugazyan
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Abstract
Provided is a method for preparation of a composition comprising activated human CD8+ and natural killer (NK) lymphocytes. The method entails use of mature dendritic cells as feeder cells added at an early stage in CD4+ mediated activation of the CD8+ cells. Also provided is a method for treatment of cancer using the cells obtained from the process.
Claims37
The method according to claim 14, comprising, prior to step i),
- 1) isolating mononuclear cells from a blood sample from a human donor and separating the PBMCs into a fraction enriched for monocytes and a fraction enriched for lymphocytes;
- 2) culturing a portion of the monocyte-enriched fraction under conditions that facilitate maturation of dendritic cells;
- 3) subsequently mixing a first portion of the mature dendritic cells obtained in step 2 with a first portion of the lymphocyte fraction obtained in step 1);
- 4) co-culturing the mixed cells obtained in step 3 to stimulate proliferation of CD4+ lymphocytes, thereby increasing the CD4+/CD8+ ratio compared to the lymphocytes obtained from step 1;
- and then
- 5) isolating proliferating lymphocytes from the co-cultured cells of step 4, and subsequently contacting them with an agent that induces expression of cancer/testis antigens followed by a period of culture that results in said expression of cancer/testis antigens;
- and wherein step i) and ii) comprises;
- 6) mixing the cancer/testis antigen expressing lymphocytes obtained in step 5 with a second portion of the fraction enriched for lymphocytes in step 1;
- 7) subsequently culturing the lymphocyte mixture from step 6 to stimulate proliferation of CD8+ and NK lymphocytes,
- wherein a second portion of mature dendritic cells obtained from step 2 are added to the proliferating lymphocytes in any of steps 5-7 and at the latest 6 days after step 6.
The method according to claim 14, comprising, prior to step 1) the steps of
- a) contacting a first composition of human cells comprising proliferating CD4+ lymphocytes with an agent that induces expression of cancer/testis antigens followed by a culturing period that results in said expression of cancer/testis antigens by cells in the first composition;
- and wherein steps i) and ii) comprise
- b) adding a second composition of human cells comprising unstimulated peripheral blood lymphocytes to the first composition of cells and culturing the combined compositions of cells to stimulate proliferation of CD8+ and NK lymphocytes;
- wherein a third composition of human cells comprising mature dendritic cells is added in step a or b and at the latest 6 days after initiation of step b, and wherein the first, second and third compositions of human cells are isogeneic.
A method for preparation of a composition comprising activated human CD8+ and natural killer (NK) lymphocytes, comprising the steps of
- i) mixing
- a first composition of human cells comprising cancer/testis antigen expressing CD4+ lymphocytes with
- a second composition of human cells comprising unstimulated peripheral blood lymphocytes and
- ii) culturing the combined compositions of cells to stimulate proliferation of CD8+ and NK lymphocytes;
- wherein a third composition of human cells comprising mature dendritic cells is added to the combined compositions at the latest 6 days after mixing the first and second composition and wherein the first, second and third compositions of cells are isogeneic.
25-28. (canceled)
Dependencies are inferred from the claim text.