Introduction:

Glioblastoma (GBM) is malignant and aggressive brain tumor with a median overall survival of 14.6 months, despite the development of new treatments. Chimeric Antigen Receptor (CAR) modified T cells are powerful new class of immune based therapies that have shown a high rate of long term durable response in CD19 expressing B-cell malignancies. However, this success has yet to be translated to solid tumors, in part due to a lack of surface antigens specifically expressed on tumor cells while not present on normal tissue and an immunosuppressive tumor microenvironment. Approximately 60% of GBMs contain a mutation, rearrangement, splicing alteration, and/or amplification of EGFR. Investigation of 411 GBM patient samples from University of Pennsylvania identified EGFR amplification in 38% of cases and EGFRvIII in 25% of cases and most common oncogenic missense mutations in extracellular domain (ECD) A289D/T/V, R108G/K, and G598V in 12-13% of cases. ECD missense mutations are associated with increased invasiveness aided by ligand independent activation of receptor and decreased patient survival compared to patients that are wild type EGFR at R108, A289 and G59V locus.

Methods:

We generated two structurally different CARs by fusing the scFv of mAb806 to 4-1BB and Killer immunoglobulin like receptor (KIR) co-stimulatory domains. mAb806 recognizes a conformationally exposed epitope of wild-type EGFR when it is overexpressed on tumor cells or in the presence of oncogenic mutations such as EGFRvIII. We tested in vitro cytolytic activity of 806-EGFR-CART cells against U87-MG (expressing low levels of EGFR) and U87-MG EGFR (U87-MG parental cell line transduced with EGFR wild type for amplified background) cell lines. These lines were engineered to ectopically express targeted EGFR-ECD missense mutations and EGFRvIII.

Results:

Both 4-1BB and KIR based EGFR806 CAR T cells specifically lysed tumor cells, proliferated, and secreted cytokines when co-cultured with target-expressing cell lines. To test exclusive specificity of EGFR806 CART to targeted mutations, K562 cells were transduced with EGFR-ECD mutations and co-cultured with 4-1BB and KIR CART cells. While the EGFR806 lysed K562 cells expressing wild type EGFR, EGFR-ECD Mutants, and EGFRvIII, it did not demonstrate any activity against K562-Parental cells. Unlike EGFR-specific cetuximab based CAR, EGFR-806CART cells did not kill EGFR wild type expressing fetal brain astrocytes and keratinocytes in vitro. We further evaluated the in vivo efficacy of CARs in NSG mice by injecting U87-MG, U87-MG EGFR and U87-MG EGFR expressing EGFRvIII subcutaneously and assessed tumor burden and mice survival. Though both 4-1BB and KIR based EGFR806 CART cells control tumor growth, KIR based CAR outperformed 4-1BB based CAR in terms of tumor burden and improved mice survival.

Conclusion:

Though our CART clinical trial for EGFRvIII positive recurrent GBM patients demonstrated CART therapy is safe and CART cells are able to successfully traffic to regions of active GBM, antigen loss and tumor heterogeneity resulted in limited therapeutic success. Broad specificity of EGFR806 CART cells to Amplified EGFR, EGFRvIII and EGFR-ECD mutants gives us the potential to clear various forms of EGFR. The enhanced anti-tumor efficacy by KIR based CAR in vivo setting provides us with additional therapeutic options for GBM.

Citation Format: Radhika Thokala, Zev Binder, Yibo Yin, Michael Milone, Donald M. Orourke. High affinity chimeric antigen receptors with cross reactivity to clinically relevant EGFR mutated proteins [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6596.