Fig. 7. Cotinine-drug conjugate can eliminate allogeneic CAR-T cells after tumor eradication to reduce GVHD side effect.

a Experimental scheme for alleviation of GVHD by Cot-Saporin in an allogeneic Cot CAR-T cell therapy model in haploidentical HSCT recipients. Lethally irradiated CB6F1 mice were injected with T cell-depleted B6 BM cells. After 4 days, A20-Luc cells were injected to mimic tumor recurrence after HSCT. Four days later, Cot CAR-T cells derived from Thy1.1-congenic B6 mice and C1C02-Cot proteins were administered as in Fig. 4a for tumor eradication. b, c Survival (b, n = 10) and clinical GVHD score (c, n = 5) were monitored periodically. Survival data were pooled from two replicate experiments. d–f The same experiment as in (b, c) was performed except that when GVHD began to develop after CAR-T cell injection, Cot-saporin (0.5 μg/head) was administered intraperitoneally three times at 3-day intervals (days 31, 34, and 37 after CAR-T cell injection). Cot CAR-T cells were traced by flow cytometry from 19 days after CAR-T cell transfer. Numbers are percentages (black) or absolute numbers (brown) of Cot CAR-T cells (boxes, Thy1.1⁺CD19^- cells) in a fixed volume (7 μl) of peripheral blood (d). Relative kinetics of Cot CAR-T cell expansion in peripheral blood (e). Absolute numbers of Cot CAR-T cells at each time point in (d) were normalized to the numbers on day 19 (n = 5). *: p = 0.0053 on day 42 and p = 0.0015 on day 49. Clinical GVHD score (n = 5) was monitored periodically (f). *: p = 0.0355 on day 36, p = 0.0108 on day 40, p = 0.0059 on day 43, p = 0.0033 on day 47, p = 0.0269 on day 50, and p = 0.0311 on day 57. Data in (c, e, f) are presented as mean ± SEM. Statistical significance was determined by log-rank (Mantel-Cox) test (b) or by unpaired two-tailed t test (e, f). Results are representative of at least two independent experiments (b–f). p: p-value. Source data are provided in the Source Data file.