CAR-T Cells with High CAR Expression Intensity Have Improved In Vitro and In Vivo Anti-Lymphoma Effect without Functional Exhaustion

Background

While immunotherapy with anti-CD19 chimeric antigen receptor (CAR) T cells has shown significant efficacy in B-cell malignancies, CAR T cells directed against CD30 (CAR30) for the treatment of Hodgkin lymphoma (HL) showed modest antitumor effect, with more than 50% of patients being unresponsive. Several factors related to the infused product and persistence may be relevant to increase clinical efficacy, but further investigation is needed. In this way, CAR expression intensity may play an important role on CAR T cell function, but this has not been systematically explored.

Aim

We have evaluated the impact of CAR expression intensity on T cell function, cell exhaustion and antitumor efficacy against HL and B cell lymphoma.

Methods

T cells were generated as previously described (Alvarez-Fernández C et al. 2016) and transduced with third generation lentivirus encoding a 4-1BBz CAR (either CAR30 or CAR19). Two populations of CAR⁺ T cells were sorted according to mean fluorescence intensity (MFI) of CAR: CAR^HI (MFI> 5x10³) and CAR^LO (MFI <3x10³). Cytotoxicity assays were performed using Raji (CD19⁺) or L540 (CD30⁺) tumor cell lines. Multiparametric flow cytometry was used to analyze T-cell inhibition and activation markers. CAR^HI and CAR^LO in vivo antitumor effect was tested under stringent therapeutic conditions using 5x10⁶ T cells/mice (iv) in a HL NSG model.

Results

CAR30⁺ T cells were sorted into CAR^LO (MFI: 1064±124.7) and CAR^HI (MFI: 7068±1377) (p=0.01). T_SCM were highly represented in CAR^LO compared to CAR^HI (CD4⁺: 70.14±1.78% vs. 55.61±5.5%, CD8⁺: 83.78±3.8% vs 72.2±5.47%, respectively) (p<0.01). However, these differences disappear after 24h co-culture with tumor cells due to an increase of T_SCM in CAR^HI (CD4⁺: 72.52±7.54%, CD8⁺: 80.26±5.3%).

CAR^HI showed a significantly higher in vitro antitumor effect compared to CAR^LO (tumor death at 5:1 E:T ratio: 96.6±1.86% vs. 89.1±3.83%; 1.25:1 E:T ratio: 84.61±4.7% vs. 31.15±19.79%; CAR^HI vs. CAR^LO, respectively) (p<0.0001). No differences were observed in expression of activation markers (i.e.: CD25, CD69, and HLA-DR) among both populations. Generalizability of this finding was studied using a CAR19. Similarly, CAR19⁺ T cells were arranged into CAR^LO (MFI: 1610±187) and CAR^HI (MFI: 10810±1486) subgroups (p<0.01). T_SCM represented the most frequent subtype in both populations (CD4⁺: CAR^HI 70,22±9,87%, CAR^LO 69,22±9,33%; CD8⁺: CAR^HI 65,1±10,5%, CAR^LO 60,9±9,5%) and no differences in T cell subset composition between CAR^HI and CAR^LO were found. Again, CAR^HI exhibited superior antitumor effect compared to CAR^LO (tumor death at 5:1 E:T ratio:59.9±8.72% vs. 28.8±8.7%; 1.25:1 E:T ratio: 21.6±11.4% vs. 2.9±2.9%, CAR^HI vs. CAR^LO, respectively) (p<0.0001).

At 24h and 72h of antigen encounter, expression of inhibitory markers was determined in both CAR30⁺ populations. While CD4⁺ T cells showed significantly higher PD1 and TIM3 co-expression in CAR^HI compared to CAR^LO (p<0.05), CD8⁺ T cells showed similar co-expression (p=0.4 and p=0.8, at 24h and 72h, respectively).

A similar kinetics was observed in CAR19⁺ T cells, suggesting that it could be related to an inhibitory control of activation, but not cellular exhaustion. To confirm this, functional performance of CAR30^HI and CAR30^LO T cells was evaluated by continuous tumor exposure. CAR30^HI function persisted after sequential re-exposition (n=5) to tumor cells; in contrast, the CAR30^LO subpopulation showed progressive loss of cytotoxic activity (i.e., tumor death at ratio E:T 5:1 after 4 expositions: 0% vs. 91.96%, CAR30^LO and CAR30^HI respectively; representative of 2 independent studies with different donors).

To assess if these results were consistent in vivo, the antitumor effect of CAR30^HI and CAR30^LO were evaluated in a xenograft model of HL. Mice treated with CAR30^HI T cells showed reduced tumor growth compared to those treated with CAR30^LO T cells, which translated into an improved survival.

Conclusion

We have shown that high expression of a CAR (either CAR30 or CAR19) confers an enhanced in vitro antitumor effect against HL and B cell lymphoma. This effect is maintained after repetitive exposures to tumor cells and is not associated with T cell exhaustion or differentiation. Notably, this enhanced antitumor effect was also found in vivo. Our data shows that CAR expression intensity should be considered as an additional important factor to improve the efficacy of CAR T cells.