Choice of costimulatory domains and of cytokines determines CAR T-cell activity in neuroblastoma

Abstract

Chimeric antigen receptor (CAR) T-cell therapy has been shown to be dramatically effective in the treatment of B-cell malignancies. However, there are still substantial obstacles to overcome, before similar responses can be achieved in patients with solid tumors. We evaluated both in vitro and in a preclinical murine model the efficacy of different 2nd and 3rd generation CAR constructs targeting GD2, a disial-ganglioside expressed on the surface of neuroblastoma (NB) tumor cells. In order to address potential safety concerns regarding clinical application, an inducible safety switch, namely inducible Caspase-9 (iC9), was also included in the vector constructs. Our data indicate that a 3rd generation CAR incorporating CD28.4-1BB costimulatory domains is associated with improved anti-tumor efficacy as compared with a CAR incorporating the combination of CD28.OX40 domains. We demonstrate that the choice of 4-1BB signaling results into significant amelioration of several CAR T-cell characteristics, including: 1) T-cell exhaustion, 2) basal T-cell activation, 3) in vivo tumor control and 4) T-cell persistence. The fine-tuning of T-cell culture conditions obtained using IL7 and IL15 was found to be synergic with the CAR.GD2 design in increasing the anti-tumor activity of CAR T cells. We also demonstrate that activation of the suicide gene iC9, included in our construct without significantly impairing neither CAR expression nor anti-tumor activity, leads to a prompt induction of apoptosis of GD2.CAR T cells. Altogether, these findings are instrumental in optimizing the function of CAR T-cell products to be employed in the treatment of children with NB.

Selecting costimulatory domains for chimeric antigen receptors: functional and clinical considerations

“…However, preclinical models and exploratory comparisons between single-arm clinical studies suggest CAR T cells incorporating each of these domains may differ qualitatively. 11,24,25,29,50,56,68 In animal studies, CD28-costimulated CAR T cells exhibit improved early expansion and cytotoxic activity compared to their 41BB-costimulated counterparts in vivo, while 41BB-costimulated CAR T cells exhibit better long-term persistence. 27 Consistent with this, pharmacokinetic analyses of phase 2 CAR T cell clinical trials in r/r B-NHL suggest that CD28-containing second-generation anti-CD19 CAR T cells peak earlier but persist less well than 41BB-incorporating CAR T cells.…”

Treating osteosarcoma with CAR T cells

“…However, to compare the outcome using two different antibodies, recognizing different epitopes, might not have a clear scientific value. One would need to put FRP5 scFv on a third‐generation construct or trastuzumab on the second generation to be able to conclude on the main cause of this discrepancy, as recently depicted with GD2 CAR . One potential strategy for improving cancer specificity and clinical response is to target not only one, but two tumour‐associated antigens on a single cancer cell by a bispecific CAR T‐cell molecule .…”

Switching on the green light for chimeric antigen receptor T‐cell therapy

“…Combination therapy using a-PD-1 demonstrated superior antitumor efficacy in an in vivo model compared to conventional CAR T cells that correlated with enhanced effector function of the CAR T cells such as granzyme B and IFNc upon PD-1 blockade. 16,17 In the context of costimulation using exogenous antibodies, a recent preclinical study tested the combination of Her2-specific CAR T cells with a-4-1BB therapy against Her2-expressing solid tumors. 12 The clinical translation of CAR T-cell and a-PD-1 mAb is now underway with multiple clinical trials currently recruiting patients.…”

“…16 Both 4-1BB-and/or OX40-containing CAR T cells have been tested in various preclinical studies; however, comparisons between the two domains remain inconclusive in terms of overall antitumor effect observed given variability in the models used from different groups. 16,17 In the context of costimulation using exogenous antibodies, a recent preclinical study tested the combination of Her2-specific CAR T cells with a-4-1BB therapy against Her2-expressing solid tumors. The combination treatment resulted in significantly enhanced tumor regression compared to CAR T-cell therapy alone or control T cells in combination with a-4-1BB mAb.…”