Extended Data Fig. 9: Toxicity of αFITC-FMC63-mCAR-T cells and amph-FITC vaccine therapy in a hCD19+ Eμ-Myc B-ALL/Lymphoma mouse model.
(a) Experimental setup and whole animal imaging of disease progression. B6 albino mice were lymphodepleted (L.D.) and injected with 0.5x106 hCD19+ Eμ-Myc cells. On day 4, mice were adoptively transferred with 2×106 αFITC-FMC63-mCAR-T cells or FMC63-mCAR-T (mCAR-T), then vaccinated 1 day later with 10 nM amph-FITC (amph-FITC vax) or 10 μg amph-F12-A1 (amph-mimotope Vax) (n = 8 animals/group). Vaccinations were repeated 7 and 14 days later. mFMC63-MCAR-T, and FMC63-mCAR-T + Amph-mimotope Vax are the same as presented in Fig. 6. (b) Quantification of total photon counts over time in different treatment groups (n = 8 animals/group). (c) Eμ-Myc-injected mice were monitored for overall survival (****, p < 0.0001). (d) Toxicity score of each treatment group. Mice were monitored daily for clinical signs of toxicity before CAR-T injection and until mice reached the endpoint of the study. The toxicity of therapy was monitored using a toxicity score as described before44. A toxicity score of 0 corresponded to active, well-groomed animals, with well-kept hair coat and invisible spine. A score of 1 was assigned to mice with signs of hypomotility, tousled hair coat and a partially visible spine. A score of 2 was given in case of somnolence, rough, dull or soiled hair coat, hunched back with visible spine (n = 8 animals/group). (e) Enumeration of circulating CAR-T cells or control CD45.1+ T cells by flow cytometry on day 11. Control mice, mFMC63-MCAR-T, and FMC63-mCAR-T + Amph-mimotope Vax are the same as presented in Fig. 6c. (n = 8 animals/group; n = 5 for αFITC-FMC63-mCAR-T + Amph-FITC group). (FMC63-mCAR-T vs FMC63-mCAR-T + Amph-mimotope Vax, ****, p < 0.0001; Control T vs FMC63-mCAR-T + Amph-mimotope Vax, ****, p < 0.0001; αFITC-FMC63-mCAR-T vs αFITC-FMC63-mCAR-T + Amph-FITC Vax, *, p = 0.0316; FMC63-mCAR-T + Amph-mimotope Vax vs αFITC-FMC63-mCAR-T + Amph-FITC Vax, *, p = 0.0480; Control T vs αFITC-FMC63-mCAR-T + Amph-FITC Vax, **, p = 0.0088). (f) Serum cytokine analysis. Blood was collected on day 11 or day 7 for amph-FITC-treated mice (light yellow points), and a Cytometric Bead Array (CBA) was performed to determine the concentration of IL-6, IL-10, TNF-α, IFN-γ, and IL-2 in the serum (n = 8 animals/group; n = 7 for αFITC-FMC63-mCAR-T + Amph-FITC group) (****, p < 0.0001; **, p = 0.0057). (g-h) Cytokine responses of CAR-T cells to amph-vax labeled K562 cells. FMC63-mCAR-T and αFITC-FMC63-mCAR-T were co-cultured with K562 labeled with 500 nM Amph-F12-A2 or 500 nM Amph-FITC at a 10:1 E:T ratio for 6 hours followed by a measurement of IFN-γ and TNF-α concentration in supernatant by ELISA (n = 3). (g, ****, p < 0.0001; FMC63-mCAR-T + Amph-F12-A2 vs αFITC-FMC63-mCAR-T + Amph-F12-A2, **, p = 0.0017; αFITC-FMC63-mCAR-T + unlabeled vs αFITC-FMC63-mCAR-T + Amph-F12-A2, **, p = 0.0038. h, ****, p < 0.0001; αFITC-FMC63-mCAR-T + unlabeled vs αFITC-FMC63-mCAR-T + Amph-F12-A2, **, p = 0.0010). (i) Experimental setup and color coding used for j-l. B6 albino mice were lymphodepleted (L.D.) and injected with 0.5×106 hCD19+ Eμ-Myc cells. On day 4, mice were adoptively transferred with 2×106 αFITC-FMC63-mCAR-T cells +/- anti-IL-6 (αIL-6), then vaccinated 1 day later with 10 nM amph-FITC (amph-FITC vax) +/- dexamethasone (dexa). Vaccinations and αIL-6/dexamethasone treatment were repeated 7 and 14 days later (n = 5 animals/group). (j) Whole animal imaging of disease progression over time. (k) Total flux at day 20 (control T cells vs mCAR-T, *, p = 0.0118; mCAR-T + αIL-6/dexa vs mCAR-T + αIL-6/dexa + Vax, *, p = 0.0164; control T vs mCAR-T + αIL-6/dexa + Vax, ***, p = 0.0009). (l) Toxicity score of each treatment group. (m) Experimental setup and tumor size measurements. B6 albino mice were subcutaneously injected with 0.5×106 hCD19+ B16-F10 cells. On day 5, mice were lymphodepleted (L.D.) followed by adoptive transfer of 2×106 αFITC-FMC63-mCAR-T cells on day 6. On day 7, mice were vaccinated with 10 nM amph-FITC (amph-FITC vax). Vaccinations were repeated 7 and 14 days later (n = 5 animals/group). (n) Serum cytokine analysis. Blood was collected 6 days after vaccination, day 11 for Eμ-Myc-bearing mice or day 13 for B16F10-earing mice, and the CBA was performed to determine the concentration of IL-6, TNF-α, and IFN-γ in the serum (n = 5 animals/group) (****, p < 0.0001, ***, p = 0.0002). Error bars show mean±95% CI for B-F, J-N or mean±s.e.m. for G, H. *, p < 0.05, **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, non-significant by Long-rank (Mantel-Cox) test for C, one-way ANOVA with Tukey’s post-test for E-H, K, N; by two-way ANOVA with Tukey’s post-test for M.
