US 12,258,397 B2
Production of engineered T-cells by sleeping beauty transposon coupled with methotrexate selection
Michael C. Jensen, Bainbridge Island, WA (US); Suzie Pun, Seattle, WA (US); and Nataly Kacherovsky, Seattle, WA (US)
Assigned to Seattle Children's Hospital, Seattle, WA (US); and University of Washington, Seattle, WA (US)
Filed by Seattle Children's Hospital, Seattle, WA (US); and University of Washington, Seattle, WA (US)
Filed on Nov. 12, 2020, as Appl. No. 17/096,138.
Application 17/096,138 is a division of application No. 15/302,449, abandoned, previously published as PCT/US2015/024868, filed on Apr. 8, 2015.
Claims priority of provisional application 62/090,845, filed on Dec. 11, 2014.
Claims priority of provisional application 62/089,730, filed on Dec. 9, 2014.
Claims priority of provisional application 62/088,363, filed on Dec. 5, 2014.
Claims priority of provisional application 62/058,973, filed on Oct. 2, 2014.
Claims priority of provisional application 61/986,479, filed on Apr. 30, 2014.
Claims priority of provisional application 61/977,751, filed on Apr. 10, 2014.
Prior Publication US 2021/0139583 A1, May 13, 2021
Int. Cl. C07K 16/28 (2006.01); A61K 35/12 (2015.01); A61K 35/28 (2015.01); A61K 38/17 (2006.01); A61K 39/00 (2006.01); A61K 39/395 (2006.01); C07K 14/705 (2006.01); C07K 14/71 (2006.01); C07K 14/715 (2006.01); C07K 14/725 (2006.01); C07K 16/32 (2006.01); C12N 5/0783 (2010.01); C12N 9/12 (2006.01); C12N 15/85 (2006.01)
CPC C07K 16/2803 (2013.01) [A61K 35/28 (2013.01); A61K 38/1774 (2013.01); A61K 38/179 (2013.01); A61K 38/1793 (2013.01); A61K 39/3955 (2013.01); A61K 39/4611 (2023.05); A61K 39/4631 (2023.05); A61K 39/464412 (2023.05); C07K 14/7051 (2013.01); C07K 14/70517 (2013.01); C07K 14/70521 (2013.01); C07K 14/70578 (2013.01); C07K 14/71 (2013.01); C07K 14/7151 (2013.01); C07K 16/2818 (2013.01); C07K 16/32 (2013.01); C12N 5/0636 (2013.01); C12N 5/0638 (2013.01); C12N 9/12 (2013.01); C12N 15/85 (2013.01); C12Y 207/10001 (2013.01); A61K 2035/124 (2013.01); A61K 2039/505 (2013.01); A61K 2039/572 (2013.01); C07K 2317/14 (2013.01); C07K 2317/524 (2013.01); C07K 2317/526 (2013.01); C07K 2317/53 (2013.01); C07K 2317/622 (2013.01); C07K 2317/73 (2013.01); C07K 2319/02 (2013.01); C07K 2319/03 (2013.01); C07K 2319/33 (2013.01); C12N 2510/00 (2013.01); C12N 2800/90 (2013.01)] 17 Claims
 
1. A method of generating engineered multiplexed T-cells for adoptive T-cell immunotherapy comprising:
(a) introducing into a population of T-cells a first minicircle and a second minicircle to obtain a first population of transfected T-cells, wherein each minicircle comprises a gene delivery polynucleotide, wherein the gene delivery polynucleotide comprises:
(i) a first inverted terminal repeat (ITR),
(ii) a promoter,
(iii) a nucleic acid encoding a polypeptide, wherein the polypeptide of the first minicircle is different from the polypeptide of the second minicircle,
(iv) a nucleic acid encoding a double mutant of dihydrofolate reductase (DHFR) comprising the nucleotide sequence as set forth in SEQ ID NO:2,
(v) a second ITR,
wherein (i) to (v) have a 5′ to 3′ order in the gene delivery polynucleotide;
(b) introducing a vector encoding a Sleeping Beauty transposase into the first population of transfected T-cells to obtain a second population of transfected T-cells;
(c) selecting for T-cells comprising the first minicircle and the second minicircle in the second population of transfected T-cells, wherein the selecting comprises a first round of selection comprising contacting the second population of transfected T-cells with methotrexate at a first concentration range, and a second round of selection comprising contacting the second population of transfected T-cells with methotrexate at a second concentration range, wherein the second concentration range is at least 1.5 fold higher than that of the first concentration range; and
(d) isolating the selected T-cells, wherein at least 75% of the selected T-cells express both (a) the polypeptide of the first minicircle, and (b) the polypeptide of the second minicircle.