CRISPR-mediated generation of a tumor-associated antigen-deficient Raji platform to investigate antigen loss in CAR-T cell therapy

Abstract

Tumor-associated antigen (TAA) loss remains a significant mechanism of resistance to chimeric antigen receptor (CAR) T cell therapy, leading to relapse in patients with B-cell malignancies and representing a major clinical challenge. Recent clinical data suggest that CD19 antigen loss triggers relapse in more than 40% of patients undergoing CD19 CAR-T cell therapy. To rigorously validate antigen loss, robust in vitro models that mimic the dynamic process of antigen escape are essential. However, the current absence of these models hampers our ability to fully evaluate and optimize treatment strategies. To model this clinically relevant phenomenon, we generated single (sKO), double (dKO), and triple (tKO) knockout Raji lymphoma cell lines targeting CD19, CD20, and CD22 using CRISPR/Cas9 genome editing. Initially, we established a dual-reporter cell line expressing the fluorescent marker mCherry and the bioluminescent marker Luciferase, enabling a uniform luminescence background across all the knockout cell lines before performing the CRISPR/Cas9 editing. The loss of individual or combinatorial TAAs was validated at the genomic, transcript, and protein levels. Functional co-culture assays with antigen-specific CAR-T cells showed that antigen-deficient Raji cells resisted CAR-T cell-mediated killing, closely mimicking clinical relapse. The triple knockout (tKO) model, in particular, provided a superior system compared to commonly used K562 models, as it retains the same lymphoma background while eliminating the crucial antigenic targets, thus better simulating resistance to CAR-T cell therapy. These antigen-loss models serve as valuable tools for studying mechanisms of CAR-T cell resistance and evaluating next-generation, multi-targeting CAR-T cell therapies.

Keywords: B-cell malignancies; CAR-T cell therapy; CRISPR/Cas9; antigen escape; antigen loss.

FIGURE 1

Raji-Luc-WT stably express functional luciferase-mCherry and retain parental features. (A) Schematic representation of the experimental workflow for generating Raji-Luciferase line from Raji WT. (B) Flow cytometry analysis confirming stable expression of mCherry in the Raji-Luc-WT. (C) qRT-PCR analysis showing robust luciferase gene expression in Raji-Luc-WT cells while no detectable amplification was observed in untransduced Raji-WT cells (Ct > 35). GAPDH was used as a housekeeping control. Data shown is the representative of three independent experiments. (D) Luciferase activity assay demonstrating dose-dependent luminescence in Raji-Luc-WT cells across increasing cell numbers. Parental Raji-WT cells showed negligible luminescence under the same conditions, confirming functional transgene expression. (E) Proliferation assay comparing Raji-Luc-WT and parental Raji-WT cells over 96 h. Both cell lines exhibited similar growth kinetics. Data plotted represents mean ± SD. Statistical analysis was performed using multiple unpaired t-tests with Holm–Šidák correction for multiple comparisons. ****p < 0.0001; ns = not significant.

FIGURE 2

Genomic deletion of target antigens in Raji-Luc lines does not alter proliferation or viability. (A) Schematic representation of CRISPR-Cas9–mediated strategy to generate single, double, and triple knockout (KO) Raji-Luc cell lines. (B–D) Amplicon sequencing data showing genomic deletions at the CD19 (B), CD20 (C), and CD22 (D) loci in the respective KO lines, confirming targeted gene disruption. (E) Proliferation assay comparing all the Raji-Luc KO lines and control Raji-Luc-WT over 96 h. Data plotted as fold change normalized to Raji WT. (F) Viability analysis after 2 h incubation with AlamarBlue reveals no significant differences between Raji WT, Raji-Luc WT, and KO lines. Statistical analysis was performed using ordinary one-way ANOVA followed by the Dunnett’s multiple comparisons test. *, p < 0.05; ns = not significant.

FIGURE 3

Raji knockout lines generated via CRISPR-Cas9 exhibit complete loss of CD19, CD20, and CD22 transcript and protein expression. (A–C) Transcript level analysis via qRT-PCR revealed significant reduction in the mRNA levels of (A) CD19, (B) CD20 and (C) CD22. K562 was used as CD-marker negative control. Gene expression was normalized to Raji WT and GAPDH was used as an internal control. Data plotted as fold change (mean ± SD, n = 3). (D) Immunofluorescence staining for CD19 (yellow), CD20 (green), CD22 (red), and Hoechst (blue) in all Raji cell lines and K562. Loss of surface antigen staining is evident in all corresponding knockout lines. Images are representative of three independent experiments. Scale bar = 50 μm. (E–G) Flow cytometry analysis of surface CD19 (E), CD20 (F), and CD22 (G) expression in nonpermeabilized Raji-derived knockout cells and K562 control. Data represent mean ± SD of fluorescence intensity (MFI) from three independent experiments, confirming complete loss of surface expression of the respective target antigen in each knockout line. Statistical analysis was performed using ordinary one-way ANOVA followed by the Dunnett’s multiple comparisons test. ****, p < 0.0001; ***, p < 0.001; ns = not significant.

FIGURE 4

Raji KO lines model antigen loss and resist CAR-T cell–mediated cytotoxicity. (A) Schematic representation of CAR constructs targeting CD19, CD20, and CD22. (B) Flow cytometric analysis of isolated T cells confirmed the expression of T cell–specific markers and negligible expression of non–T cell markers. (C) Expression of T-cell activation markers on isolated T cells post 48 h of activation with CD3/CD28 beads. (D) Flow cytometry of GFP expression in T cells 48 h post-transduction. Histogram shown from one of three independent transductions using T cells from three different donors. (E–G) Viability of Raji KO lines assessed by bioluminescence following 16 h co-culture with CAR T cells expressing CAR19 (E), CAR20 (F) or CAR22 (G) at indicated effector-to-target (E:T) ratios. Percent live cells was calculated based on luminescence signal relative to target-only control and normalized to untransduced controls. CAR-T cells selectively eliminated WT Raji cells expressing the corresponding target antigen, while respective single, double, and triple KO lines remained resistant. K562 cells served as antigen negative control. Data are presented as mean ± SD for three independent biological replicates. Statistical analysis was performed using multiple unpaired t-tests (one per row) with Holm–Šidák correction for multiple comparisons (C) or by comparing area under the curve for each cell line group in an ordinary one-way ANOVA with Dunnett’s multiple comparisons test (E–G). ***, p < 0.001; ****, p < 0.0001; ns = not significant.