Chimeric antigen receptor preparation from hybridoma to T-cell expression

Abstract

The successful use of chimeric antigen receptor (CAR) for hematological cancer treatment has influenced the direction taken in translational research toward an increasing focus on personalized targeted immunotherapy. Thus, a growing number of labs worldwide are now interested in testing their old antibody collections in this format to broaden the spectrum of utility and improve safety and efficacy. We herein present a straightforward protocol for the identification of an antibody from a hybridoma and the design of the single chain fragment that will be placed on the extracellular part of the CAR construct. We further show how to test the expression and the activity of the construct in primary T cells. We illustrate our demonstration with two new CARs targeted against the B cell receptor, more precisely the light chains κ and λ, that represent potential alternatives to the CD19 CAR used in the treatment of B-cell malignancies. Statement of Significance Chimeric antigen receptor molecules are becoming increasingly popular, and an easy-to-follow method to isolate and express them is herein presented.

Combinatorial CAR design improves target restriction

“…The construct presented herein is a prototype and alternative versions will be designed in which the affinity of one of the scFv can be modified: it is tempting to speculate that a lower affinity CD19 CAR (32) would reduce the recognition of Igλ+/CD19+ targets. A similar observation was also noted with our high affinity anti-Igλ (IGL) CAR (33) when designed in a CD19-combinatorial construct (unpublished data). Another possible improvement would be the regulation of the expression: a lower or controlled presence of the construct could also affect the Igλ+/CD19+ target recognition as supported by our mRNA titration studies, where increase IGK part reduced CD19 CAR dominancy.…”

“…A DNA sequence encoding the anti-Igκ scFv was generated after sequencing the original hybridoma (FN162). Briefly, the sequences of the VL and VH regions were determined by 5’-RACE ( 33 ). The design consists in the linkage of the two chains with a (G 4 S) 4 linker.…”

Intracellular Delivery of mRNA in Adherent and Suspension Cells by Vapor Nanobubble Photoporation

“…Jurkat cells are, for instance, routinely used for initial in vitro screenings of novel CAR or engineered T cell receptor designs. A method that enables efficient and quick screening of different CAR constructs, without the need for designing a new dedicated viral vector for each construct, is therefore highly desirable [48][49][50][51][52]65]. As a consequence, we selected the Jurkat T cell line to deliver the proof-of-concept that photoporation holds promise for the production of engineered T cells by mRNA transfections.…”

“…We performed experiments on HeLa and Jurkat T cells as models for adherent and suspension cells. Jurkat T cells serve as a valid model for primary human T cells [47] and are routinely used for screening and optimization of CAR constructs [48][49][50][51][52]. We started by systematically optimizing several parameters related to the VNB photoporation procedure, including AuNP concentration, laser fluence and transfection buffer.…”

Efficient chimeric antigen receptor targeting of a central epitope of CD22

“…We identified the IS7 coding sequence and designed a scFv ( Figs. 1 C and S1 A ), which was subcloned into a second-generation CAR backbone composed of a CD8 hinge, a CD8 transmembrane domain, and a 4-1BB-CD3z signaling tail ( 29 ). J76 cells bearing an NFAT-GFP reporter construct, J76-NFAT-GFP, were transduced with IS7-CAR and labeled with a CD22-Fc chimera.…”