Fig. 5: Amph-mimotopes require a threshold affinity to stimulate CAR-T proliferation in vivo.
a, Schematic of hybrid FMC63-mCAR design. Created in BioRender. Ma, L. (2025) https://BioRender.com/xd8q66t. b, Comparison with various amph-mimotopes in stimulating FMC63-mCAR-T cells. K562 cells were labelled with 100 nM of amph-mimotope variants for 30 min, washed and co-cultured with FMC63-mCAR-T cells at a 1:1 E:T ratio for 6 h followed by ELISA measurement of IFNγ secretion (amph-pepvIII versus amph-P1 P = 0.0016; amph-P1 versus amph-F12 P = 0.0137; amph-F12 versus amph-F12-A1 P = 0.0090; amph-F12-A1 versus NALM6 P = 0.3338). c, Amph-mimotope labelling of APCs in vivo. C57BL/6 mice (n = 3 mice per group) were vaccinated with 10 nmol amph-mimotope ± CDG adjuvant, and 24 h or 48 h later, mimotope uptake by macrophages and cDCs was detected by staining with FMC63 followed by flow cytometry analysis. Shown are representative histograms of FMC63 staining (**P = 0.0055; ***P = 0.0006; ****P < 0.0001). d, Amph-mimotope vaccine stimulation of CAR-T cells in vivo. Wild-type C57BL/6 mice (n = 3 mice per group) received adoptively transferred with 2 × 106 CTV-labelled FMC63-mCAR-T cells, and then vaccinated 1 day later with 10 nmol amph-peptides + 25 μg CDG adjuvant (Vax). Shown above is the timeline and below are representative histograms of FMC63-mCAR-T cell proliferation in LNs 48 h after vaccination. e, Longitudinal monitoring of CAR-T expansion in response to amph-mimotope vaccination. C57BL/6 mice (n = 5 mice per group) were lympho-depleted (LD), adoptively transferred with 106 FMC63 CAR-T cells and then vaccinated at indicated time points, and circulating FMC63-mCAR-T cells were quantified in the blood by flow cytometry over time. Error bars show mean ± s.d. with triplicate samples for B, mean ± 95% CI for c–e. ***P < 0.0001; **P < 0.01, by one-way ANOVA with Tukey’s post-test for b–d, and two-way ANOVA with Tukey’s post-test for e.
