US 12,241,081 B2
B cell receptor modification in B cells
James Even Voss, San Diego, CA (US); Raiees Andrabi, San Diego, CA (US); Dennis R. Burton, La Jolla, CA (US); Deli Huang, La Jolla, CA (US); and Alicia Gonzalez-Martin, Madrid (ES)
Assigned to The Scripps Research Institute, La Jolla, CA (US)
Appl. No. 16/642,810
Filed by The Scripps Research Institute, La Jolla, CA (US)
PCT Filed Aug. 3, 2018, PCT No. PCT/US2018/045255
§ 371(c)(1), (2) Date Feb. 27, 2020,
PCT Pub. No. WO2019/028417, PCT Pub. Date Feb. 7, 2019.
Claims priority of provisional application 62/540,702, filed on Aug. 3, 2017.
Prior Publication US 2023/0060376 A1, Mar. 2, 2023
Int. Cl. C12N 15/90 (2006.01); C07K 16/10 (2006.01); C12N 9/22 (2006.01); C12N 15/11 (2006.01); C12N 15/63 (2006.01)
CPC C12N 15/907 (2013.01) [C07K 16/1045 (2013.01); C12N 9/22 (2013.01); C12N 15/11 (2013.01); C12N 15/63 (2013.01); C07K 2317/76 (2013.01); C12N 2310/20 (2017.05); C12N 2800/80 (2013.01)] 16 Claims
 
1. A method of producing modified antibody producing cells, said method comprising:
(a) introducing only one double-stranded cut on a 3′ side of a genomic segment within one or more antibody producing cells, wherein the genomic segment comprises sequences encoding a recipient immunoglobulin variable region, wherein said cut at the 3′ side is located at a 3′ side of the sequences encoding the recipient immunoglobulin variable region, downstream to a immunoglobulin Joining (J) gene and upstream of a constant region gene; and
(b) inserting a donor nucleic acid into the cut of the genomic segment, said donor nucleic acid comprises a V gene promoter, a replacement variable region, and a J gene splice site followed by a sequence homologous to a J gene intron sequence to guide incorporation of the donor nucleic acid to said cut, wherein said donor nucleic acid comprises a J gene splice donor sequence located 3′ to sequences encoding said replacement variable region and is post-transcriptionally spliced to a downstream endogenous constant region gene, thereby generating a population of cells comprising one or more modified antibody producing cells, wherein each modified antibody producing cell comprises the replacement variable region derived from a segment of the donor nucleic acid.