Panel A shows the CAIleR construct with EF1 alpha promoter, scFv from four antibody variants Ab 5, 8, 10, and 11, CD28 and CD3 zeta signaling domains, and mCherry reporter. Panel B shows the Bet v1 surface expression construct and lentiviral transduction of K562 wild type to K562 Bet v1 cells confirmed by a GFP histogram. Panel C shows the CAIleR cloning and transduction workflow into Jurkat NFAT luciferase cells followed by 24 hour coculture with K562 Bet v1 and luminescence readout. Panel D shows a bar graph of relative light units up to 25000, where CAIleR 5 and 8 reach approximately 20000 with K562 Bet v1 stimulation, significantly higher than other constructs. Panel E shows an experimental timeline from day 0 to day 8 covering isolation, transduction, and activation assay. Panel F shows flow cytometry plots confirming transduction efficiencies of 36.2, 43.4, 18.4, and 33.5 percent for CAIleR 5, 8, 10, and 11 respectively. Panel G shows transduction efficiency reaching approximately 40 percent across constructs, while MFI Bet v1 reaches approximately 4000 for CAIleR 5 and 8 compared to near 0 for CAlleR 10 and approximately 2000 for CAlleR 11. Panel H shows flow cytometry plots with CD25 positive CD71 positive percentages of 95.4, 91.8, 48.7, and 65.2 percent for CAIleR 5, 8, 10, and 11, respectively. Panel I shows activation reaching approximately 80 to 90 percent for CAIleR 5 and 8 at 1 to 1 ratio with K562 Bet v1. Panel J shows a positive correlation between MFI Bet v1 and CD25 positive CD71 positive expression with r of 0.7147 and p value of 0.0019.
Validation of four novel anti-Bet v1 CAlleRs. (A) Anti-Bet v1 CAlleR and anti-CD19 CAR lentiviral constructs. (B) Construct of lentivirus encoding surface expression of Bet v1 used to transduce K562 cells and phenotype check by GFP reporter expression. (C) Experimental design of a CAlleR screening platform using NFAT-Luciferase CD4+ Jurkat cells. (D) Luminescence signal measured in RLU of anti-Bet v1 CAlleR and control NFAT cell lines after 24 h coculture with K562 Bet v1 cells (ratio 1:2) (n = 3 from three independent experiments). (E) Experimental design of human anti-Bet v1 CAlleR T cell production. (F) Representative flow cytometry plots showing mCherry reporter expression and binding to Bet v1 of wild-type (WT) and transduced human T cells. (G) Cumulative percentages of transduction efficiency (mCherry+Bet v1+) of CAR CD19 and anti-Bet v1 CAlleRs and MFI of Bet v1 expression (n = 7–10 biologically independent experiments). (H) Representative flow cytometry plots of CD25 and CD71 expression on T cells after 48-h coculture with K562 WT or K562 Bet v1 (ratio 1:1). (I) Cumulative data showing percentages of CD25+CD71+ T cells after 48-h coculture (n = 4–5 biologically independent experiments). (J) Correlation between CAlleR Bet v1 binding and activation (CD25+CD71+ expression) is plotted with best-fit lines using simple linear regression and calculated using Pearson’s correlation coefficient. Data are represented as the mean ± SEM in D, G, and I. Exact P values were determined by two-way ANOVA with Dunnett’s test in D and I, and by one-way ANOVA with Tukey’s test in G. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. RLU, relative light units; MFI, mean fluorescence intensity.