54P - Superior antitumor activities of fourth-generation CAR-T cells containing three costimulatory domains targeting GD2-positive tumors

Background

Disialoganglioside (GD2) is a glycolipid antigen highly expressed in several solid tumors. A chimeric antigen receptor (CAR)-T cell targeting GD2 has been developed and recently showed promising results in clinical trials for neuroblastoma. Previous generations of aGD2-CAR-T cells belong to second—and third-generation CARs, which harbor one and two costimulatory domains within their CAR structures. Incorporation of co-stimulatory domains may help improve CAR T-cell functions. We aim to validate whether incorporating three costimulatory domains within fourth-generation CAR (CAR4) can improve antitumor efficiency against GD2-positive tumors in vitro.

Methods

A fourth-generation CAR containing humanized scFv-GD2, hu3F8, and three costimulatory domains composed of CD28, 4-1BB, and CD27 linked to a CD3 zeta was produced in primary human T cells by using a lentiviral vector system. Two versions of third-generation CAR were also maced (CAR3.1 and CAR3.2 containing co-stimulatory parts of CD28/BB or CD28/CD27, respectively), linked to a CD3z signaling domain. Antitumor activity, CAR-T cell proliferation, and cytokine production were compared between CAR3 and CAR4-T cells against human neuroblastoma and retinoblastoma cell lines.

Results

In vitro, co-culture assays showed that aGD2-CAR4-T exerted superior anti-tumor efficiency than aGD2-CAR3-T cells for killing neuroblastoma. In contrast, these aGD2-CAR-T did not destroy a normal retinal epithelial cell that lacks GD2 expression. However, upon long-term co-culture for 72 h, anti-GD2-CAR4-T showed more excellent cytolytic activities than CAR3-T cells. Interestingly, cytokine production levels, IFN-γ and TNF-α, secreted from anti-GD2-CAR4-T cells were slightly lower than those from CAR3-T cells. We observed that CAR3.2 containing CD27 (CD28/CD27z) had the highest proliferative capacity upon antigen engagement.

Conclusions

Our fourth-generation CAR T-cells targeting GD2 showed superior cytolytic activities than third-generation CAR T-cells against GD2-positive tumor cells in vitro. Anti-GD2-CAR4-T cells will be a promising therapeutic strategy for GD2-positive cancers.

Legal entity responsible for the study

The authors.

Funding

The Office of National Higher Education Science Research and Innovation Policy Council (NXPO) by Program Management Unit-Competitiveness (PMU-C) (grant no. C10F630063).

Disclosure

All authors have declared no conflicts of interest.