Page 78 - 2021_04-Haematologica-web

M. Guercio et al.
Dynamic evolution of the CD4/CD8 ratio in response to cytokine or tumor stimulation of CAR.CD30 T cells
We also evaluated the dynamic evolution of CD4+ and CD8+ T cells during prolonged in vitro culture in the pres- ence of either cytokine stimulation (days +5, +15 and +30 after transduction) or exposure to tumor cells. As shown by CFSE analysis, we observed that CD8+ T cells had a proliferative advantage compared to CD4+ T cells, whether stimulated with IL2 (Figure 5A) or IL7/IL15 (Figure 5B). This observation correlated with the evidence that, during prolonged in vitro culture, in the presence of cytokine stimulation, the CAR.CD30 T-cell population was significantly enriched for CD8+ cells (Figure 5C and D). Indeed, irrespectively of cytokine usage (IL2 or IL7/IL15) or choice of co-stimulatory domains (28.OX40 vs. 28.4-1BB), CD4+ T-cell percentage decreased over time up to day +30 of culture, compared with the percentage on day +5. Notably, the CD4+/CD8+ T-cell ratio remained stable over time when CAR.CD30 T cells were exposed to sequential re-challenge with Karpas-299 (Figure 5E and F), suggesting that repeated tumor stimulation induces equal expansion of CD4 and CD8 CAR+ T cells.
Evaluation of the efficacy of CAR.CD30 T-cells in a mouse model of Hodgkin lymphoma
We next assessed whether the choice of the co-stimula- tory combination or cytokines used during in vitro expan- sion might influence the in vivo activity of CAR.CD30 T cells against L428 cells (Figure 6A). Bioluminescence in HL-tumor-bearing mice, treated with NT T cells, rapidly increased up to 5 log in less than 50 days (Figure 6B and C) and mice either died or were sacrificed due to poor conditions. Macroscopic analysis of sacrificed mice showed large tumor masses located preferentially in the liver. HL-tumor-bearing mice treated with 28.4-1BB.ζ (IL2) survived on average significantly longer (79 ± 10 days) compared with mice treated with NT (IL2) (Figure 6D; 52 ± 9 days, P=0.008). The use of IL7/IL15 did not improve the anti-tumor effect of 28.4-1BB.ζ, in terms of both bioluminescence signal (Figure 6B and C) and overall survival (Figure 6D). The median survival of HL-tumor- bearing mice treated with 28.OX40.ζ (IL2) was signifi- cantly better than that of mice treated with either NT (IL2) (P=0.009) or 28.4-1BB.ζ (IL2) (P=0.008). Notably, the best outcome was observed in mice given 28.OX40.ζ T cells grown in the presence of IL7/IL15, as three out of five mice were still alive at the experimental end-point of day +165 (Figure 6D).
Although mice treated with NT T cells showed a signif- icant increase in human circulating CD45+CD3+ cells with a peak evaluated at day +56 (Figure 6E), we did not observe any tumor control. Only in mice given 28.OX40.ζ T cells did we observe a long-lasting persist- ence of circulating CAR T cells up to day +130 (Figure 6F). The percentage of circulating 28.OX40.ζ T cells (Figure 6F) remained stable during the first 100 days. On day +165, we found residual circulating T cells in only two out of four treated mice (0.06% ± 0.02%) although all four mice were cured at this time. In these two mice, cir- culating CAR.CD30 T cells were equally distributed between CD4+ and CD8+, as CM and EM (Online Supplementary Figure S8). These data further confirmed in vitro results about the superiority of IL7/IL15 over IL2 in expanding CAR T cells. Moreover, 28.4-1BB.ζ T cells showed only slight in vivo expansion and persistence
(Figure 6F). We also correlated the kinetics of tumor growth (bioluminescence) and CAR T-cell expansion, observing that mice receiving 28.OX40.ζ T cells showed a significant reduction of tumor burden concomitant with the CD3+ peak (Online Supplementary Figure S9A and B). By contrast, in mice treated with 28.41BB.ζ, the kinetics of circulating T cells (CD45+CD3+), as well as of CAR+ T cells, did not correlate with bioluminescence signaling (Online Supplementary Figure S9C and D).
Evaluation of persistence and establishment of long-term immunological memory in the re-challenged mouse model of non-Hodgkin lymphoma
We then evaluated in vivo efficacy, persistence and estab- lishment of long-term memory of CAR.CD30 T cells (IL7/IL15) in a Karpas-299 xenograft model with tumor re- challenge (Figure 7A). While tumor bioluminescence in the group treated with NT T cells progressively increased up to 5 log in less than 40 days (Figure 7B and C), in mice receiving 28.OX40.ζ T cells we observed significant tumor control (P=0.0075) as measured by reduction of the biolu- minescence signal. The median survival of mice treated with NT T cells was 45.5 days, while 30% of mice given 28.4-1BB.ζ and 60% of mice given 28.OX40.ζ experienced long-term tumor control (Figure 7D). In particular, the median survival of mice treated with 28.4-1BB.ζ was 58 days (P=0.05), and undetermined for mice given 28.OX40.ζ (P=0.0002) (Figure 7D).
After 140 days, cured mice were re-challenged i.v. with Karpas-299 cells, and followed for an additional 100 days. Bioluminescence analysis showed rapid progression of the tumor in control mice (CTR mice), as well as in 28.4- 1BB.ζ-treated mice. In contrast, in 28.OX40.ζ-treated mice, after an initial expansion of the tumor for the first 40 days, at day +100 (at day +240 overall), four of six mice were tumor-free, which translated into a statistically sig- nificant survival benefit (Figure 7D). To confirm the estab- lishment of long-term immunological memory, we ana- lyzed circulating CD45+CD3+ human cells over time in treated mice. We observed significantly greater expansion of circulating T cells in the first week after effector T-cell infusion in mice treated with 28.OX40.ζ CAR T cells (2.49% ± 1.03%) (Figure 7E) compared to mice given 28.4- 1BB.ζ CAR T cells (0.27% ± 0.11%; P<0.001) or NT T cells (0.35% ± 0.07%; P=0.005).
Interestingly, at day +132 (before the second infusion of tumor cells), the number of circulating CAR T cells was negligible in all cohorts. Forty days after tumor re-challenge (day +180), we observed a significant and impressive expansion of circulating human CD45+CD3+ cells in mice treated with 28.OX40.ζ T cells (Figure 7E) compared to that in mice receiving 28.4-1BB.ζ T cells. After re-expansion, CD3+ cells were significantly enriched in 28.OX40.ζ CAR+ T cells (Figure 7F, Online Supplementary Figure S10A-H). This enrichment was tumor-specific, since the complete eradica- tion of the second tumor infusion was associated with a simultaneous decline of circulating 28.OX40.ζ T cells to an undetectable percentage, as measured at day +221 (Figure 7E, Online Supplementary Figure S10A-H).
Finally, while human CAR.CD30 T cells were found to infiltrate tumor (Online Supplementary Figure S11A) inde- pendently of the co-stimulatory combination present in the CAR construct, the infiltration observed in 28.OX40.ζ (IL7/15) T-cell-treated mice was greater than that in all other conditions (Online Supplementary Figure S11B and C).
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haematologica | 2021; 106(4)