US 12,221,627 B2
Methods of enriching cell populations for cancer-specific T cells using in vitro stimulation of memory T cells
Gal Cafri, Kibbutz Nir David (IL); and Steven A. Rosenberg, Potomac, 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. 16/768,930
Filed by The United States of America, as represented by the Secretary, Department of Health and Human Services, Bethesda, MD (US)
PCT Filed Dec. 3, 2018, PCT No. PCT/US2018/063563
§ 371(c)(1), (2) Date Jun. 2, 2020,
PCT Pub. No. WO2019/112932, PCT Pub. Date Jun. 13, 2019.
Claims priority of provisional application 62/594,262, filed on Dec. 4, 2017.
Prior Publication US 2021/0052642 A1, Feb. 25, 2021
Int. Cl. C12N 5/0784 (2010.01); A61K 39/00 (2006.01); C07K 14/725 (2006.01); C12N 5/0783 (2010.01)
CPC C12N 5/0639 (2013.01) [A61K 39/4611 (2023.05); A61K 39/4632 (2023.05); A61K 39/46447 (2023.05); C07K 14/7051 (2013.01); C12N 5/0636 (2013.01); A61K 2239/50 (2023.05); C12N 2506/115 (2013.01)] 11 Claims
 
1. An in vitro method of obtaining a cell population enriched for T cells having antigenic specificity for a cancer-specific mutation, the method comprising:
(a) providing monocytes from an epithelial cancer patient;
(b) differentiating the monocytes into dendritic cells (DCs);
(c) inducing the DCs to present one or more mutated amino acid sequences, each mutated amino acid sequence being encoded by a gene comprising a cancer-specific mutation;
(d) providing a bulk population of peripheral blood mononuclear cells (PBMCs) from the epithelial cancer patient;
(e) specifically selecting cells with a T cell phenotype from the bulk population, wherein the T cell phenotype is a central memory T cell phenotype comprising all of CCR7+, CD62L+, CD45RO+, and CD45RA−;
(f) separating the cells with the T cell phenotype selected in (e) from cells which lack the T cell phenotype;
(g) stimulating the separated cells with the T cell phenotype of (f) with the dendritic cells of (c) in vitro;
(h) re-stimulating the cells with the T cell phenotype of (g) with the dendritic cells of (c) in vitro, wherein the re-stimulating of (h) occurs 11 to 16 days after the stimulating of (g);
(i) specifically selecting the re-stimulated cells of (h) which express one or more markers of T cell stimulation;
(j) separating the selected cells of (i) which express the one or more markers of T cell stimulation from the cells which do not express the one or more markers of T cell stimulation;
(k) screening the cells of (j) which express the one or more markers of T cell stimulation for recognition of the one or more mutated amino acid sequences; and
(l) selecting the cells of (k) which have antigenic specificity for the one or more mutated amino acid sequences to provide a cell population enriched for T cells having antigenic specificity for the cancer-specific mutation of (c),
wherein the one or more mutated amino acid sequences comprise(s) one or more neoantigen(s).