CAR T cells as micropharmacies against solid cancers: Combining effector T-cell mediated cell death with vascular targeting in a one-step engineering process.

To enhance the potency of chimeric antigen receptor (CAR) engineered T cells in solid cancers, we designed a novel cell-based combination strategy with an additional therapeutic mode of action. CAR T cells are used as micropharmacies to produce a targeted pro-coagulatory fusion protein, truncated tissue factor (tTF)-NGR, which exerts pro-coagulatory activity and hypoxia upon relocalization to the vascular endothelial cells that invade tumor tissues. Delivery by CAR T cells aimed to induce locoregional tumor vascular infarction for combined immune-mediated and hypoxic tumor cell death. Human T cells that were one-vector gene-modified to express a GD2-specific CAR along with CAR-inducible tTF-NGR exerted potent GD2-specific effector functions while secreting tTF-NGR that activates the extrinsic coagulation pathway in a strictly GD2-dependent manner. In murine models, the CAR T cells infiltrated GD2-positive tumor xenografts, secreted tTF-NGR into the tumor microenvironment and showed a trend towards superior therapeutic activity compared with control cells producing functionally inactive tTF-NGR. In vitro evidence supports a mechanism of hypoxia-mediated enhancement of T cell cytolytic activity. We conclude that combined CAR T cell targeting with an additional mechanism of antitumor action in a one-vector engineering strategy is a promising approach to be further developed for targeted treatment of solid cancers.

HLA-G and HLA-E Immune Checkpoints Are Widely Expressed in Ewing Sarcoma but Have Limited Functional Impact on the Effector Functions of Antigen-Specific CAR T Cells.

Immune-inhibitory barriers in the tumor microenvironment of solid cancers counteract effective T cell therapies. Based on our finding that Ewing sarcomas (EwS) respond to chimeric antigen receptor (CAR) gene-modified effector cells through upregulation of human leukocyte antigen G (HLA-G), we hypothesized that nonclassical HLA molecules, HLA-G and HLA-E, contribute to immune escape of EwS. Here, we demonstrate that HLA-G isotype G1 expression on EwS cells does not directly impair cytolysis by GD2-specific CAR T cells (CART), whereas HLA-G1 on myeloid bystander cells reduces CART degranulation responses against EwS cells. HLA-E was induced in EwS cells by IFN-γ stimulation in vitro and by GD2-specific CART treatment in vivo and was detected on tumor cells or infiltrating myeloid cells in a majority of human EwS biopsies. Interaction of HLA-E-positive EwS cells with GD2-specific CART induced upregulation of HLA-E receptor NKG2A. However, HLA-E expressed by EwS tumor cells or by myeloid bystander cells both failed to reduce antitumor effector functions of CART. We conclude that non-classical HLA molecules are expressed in EwS under inflammatory conditions, but have limited functional impact on antigen-specific T cells, arguing against a relevant therapeutic benefit from combining CART therapy with HLA-G or HLA-E checkpoint blockade in this cancer.