Abstract 5871: Immunogenomic approaches to optimize immunotherapeutic targeting of neuroblastoma
Authors: Meijie Tian, Adam Tai-Chi Cheuk, Yong Song, Sivasish Sindiri, Nan Li, Christopher M. Dower, Mitchell Ho, Bradley St Croix, Javed Khan
Journal: Cancer Research (2020)

Abstract Background: Neuroblastoma (NB) is the most common extra-cranial solid cancer in children. Although multimodal therapies with differentiating agents and immunotherapy with anti-GD2 antibody and GM-CSF have shown promising results, it remains deadly in approximately 50% of patients with high-risk disease. Chimeric antigen receptor T-cell therapies (CAR-T) have been found to be effective in treating refractory and relapsed leukemia and lymphoma, and two of them have been recently approved by the FDA. However, current CARs frequently lose efficacy due to T cell exhaustion and CARs against solid tumor antigens often lack enough specificity due to a low incidence of somatic mutations resulting in a paucity of tumor neoantigens. There have not been effective CAR-T therapies against other solid cancers to date although many clinical trials are underway. We previously identified 2 cell surface cancer-associated antigens, GPC2 (Glypican-2) and CD276 (B7-H3), both highly expressed in NB tumor cells but expressed at low or undetectable levels in normal organs. Here we attempt to develop a high throughput way of identifying optimal CART cell binders that show activation and expansion in the presence of GPC2 and CD276 but lack of exhaustion. Methods: Binders targeting GPC2 or CD276 were cloned into CAR lentiviral constructs and then were separately transduced into T cells to develop CAR-T cells. Then we analyzed cytotoxicity of these CART cells individually. To identify the most effective GPC2 or CD276-specific targeting CAR-T cells, we utilized a combined proteomics and transcriptomics method for every single CAR-T cell to quantify RNA and protein at the same. All CAR-T cells were pooled and co-cultured with CD276/GPC2-expressing NB cancer cells (target cells) for 24 hr. Co-cultured CAR-T cells were examined for their activation, exhaustion, cytotoxicity state and distinguished different cell types by staining with CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) antibodies, and then molecularly barcoded using 10X Genomics platform for single cell RNA-sequencing (scRNA-seq). Results: We attempted to test the efficacy of CAR-T cells using 14 established and novel binders against GPC2 or CD276. According to cytotoxicity assay of these 14 CART cells, we found CT3 was the most effective GPC2 targeting CART cells but majority of CD276 CART cells showed high cytolytic activity in vitro. Conclusions and Future Directions: Using the CITE-seq and RNAseq analyses, we can identify which of the CARs are enriched and have an activated T cell signature, but lack exhaustion markers. Top candidate binders for each antigen are currently being developed into “AND” or “OR” CARs that are being tested in in-vitro and in-vivo models of NB. Thus, we have developed a high throughput method to identify high affinity functional binders against tumor cell surface antigens and provide a novel immunogenomics methods of CARs optimization for development of highly effective immunotherapies against NB and other cancers. Citation Format: Meijie Tian, Adam Tai-Chi Cheuk, Yong Song, Sivasish Sindiri, Nan Li, Christopher M. Dower, Mitchell Ho, Bradley St Croix, Javed Khan. Immunogenomic approaches to optimize immunotherapeutic targeting of neuroblastoma [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 5871.