Identification and characterization of potent CD-19 scFv and CD-69 expression in CAR J Cells

Abstract

Background and Objective: CD19 is the primary target for generating chimeric antigen receptor (CAR) T cells against B-cell leukemia. This study aimed to test a screening protocol for evaluating the efficacy of the CAR constructs using patient-derived B-ALL cells and CAR–expressing Jurkat (CAR-J) cells.

Methodology: This study was conducted in the Molecular Research Department of Indus Hospital, Karachi, Pakistan, from January 2024 to August 2025. CAR-containing backbone plasmids (#135991, #135992, and #135993) obtained from Addgene were used to produce second-generation VSV-G pseudotyped lentiviruses. Jurkat cells were infected with these lentiviruses to create CAR-J cells. CAR-J cells were cocultured with patient-derived B-ALL CD-19-positive cells. Flow cytometry was used to evaluate CD69-APC MFI expression following co-culture (a marker of Jurkat cell activation), using APC-labelled antibodies. Increased CD69 expression in comparison with controls (uninfected Jurkat cells, acute myeloid leukemia) was taken as an indicator of the efficacy of CAR binding with CD19.

Results: Only two of the three anti-CD19 CAR-J constructs induced CD69 upregulation upon co-culture with patient-derived B-ALL cells (CAR-J 135992 and CAR-J 135993) in comparison with AML controls (p = 0.0044 and p = 0.0195, respectively). When compared with uninfected Jurkat cocultured with B-ALL, all three CAR-J constructs showed significant CD69 upregulation: 135991 (p = 0.0004), 135992 (p = 0.0063), and 135993 (p = 0.0187).

Conclusion: Two anti-CD19 CAR-J cell constructs (135992 and 135993) demonstrated a detectable increase in CD69 upregulation compared with AML controls, while one construct (135991) showed no activation. Once further validated, the method may have potential for screening of other CAR molecules.