US 12,215,349 B2
Methods of preparing an isolated or purified population of thymic emigrant cells and methods of treatment using same
Raul E. Vizcardo, Derwood, MD (US); Nicholas D. Klemen, Carmel, IN (US); and Nicholas P. Restifo, Chevy Chase, MD (US)
Assigned to The United States of America, as represented by the Secretary, Department of Health and Human Services, Bethesda, MD (US)
Appl. No. 16/468,890
Filed by The United States of America, as represented by the Secretary, Department of Health and Human Services, Bethesda, MD (US)
PCT Filed Dec. 13, 2017, PCT No. PCT/US2017/065986
§ 371(c)(1), (2) Date Jun. 12, 2019,
PCT Pub. No. WO2018/111981, PCT Pub. Date Jun. 21, 2018.
Claims priority of provisional application 62/433,591, filed on Dec. 13, 2016.
Prior Publication US 2020/0080057 A1, Mar. 12, 2020
Int. Cl. C12N 5/0783 (2010.01); A61K 39/00 (2006.01); A61P 35/00 (2006.01)
CPC C12N 5/0637 (2013.01) [A61K 39/461 (2023.05); A61K 39/4611 (2023.05); A61K 39/4632 (2023.05); A61K 39/464499 (2023.05); A61P 35/00 (2018.01); C12N 5/0636 (2013.01); C12N 5/0638 (2013.01); C12N 2502/1185 (2013.01); C12N 2506/45 (2013.01); C12N 2513/00 (2013.01)] 10 Claims
 
1. A method of preparing an isolated or purified population of thymic emigrant cells in vitro, comprising:
modifying T cells that have antigenic specificity for a cancer antigen into pluripotent cells, multipotent cells, or T-lineage cells;
culturing the pluripotent cells, multipotent cells, or T-lineage cells in the presence of a Notch receptor agonist to produce CD45+ cells;
culturing the CD45+ cells in the presence of thymic tissue in a hanging drop of medium, wherein culturing the CD45+ cells in the presence of thymic tissue comprises seeding the thymic tissue with the CD45+ cells and migrating the cells into the thymic tissue;
egressing the cells from the thymic tissue in the hanging drop of medium, wherein the cells egressing from the thymic tissue are thymic emigrant cells and wherein the cells begin to egress from the thymic tissue 2 to 5 days after seeding the thymic tissue with the CD45+ cells; and
isolating the thymic emigrant cells from the thymic tissue without disrupting the thymic tissue, wherein the thymic emigrant cells are CD8α+CD8β+CD4− or CD8α31 CD8β−CD4+, wherein the thymic emigrant cells have the capacity to differentiate into a T cell with a T cell receptor having antigenic specificity for the cancer antigen, and wherein the thymic emigrant cells do not express Ptcra, Rag1, Rag2 and Rorc.