Abstract 3523: A high-throughput screening strategy for the identification of novel lymphoproliferative elements

Abstract

Abstract Introduction - Chimeric Antigen Receptor (CAR) T-cell therapy is effective against certain leukemias and lymphomas and shows promise for other incurable malignancies. Considerable challenges remain however to expand this platform technology beyond transplant-oriented hospital care. Centralized manufacturing of genetically modified T cells, lymphodepleting chemotherapy and patient management of current CAR-T therapies are associated with significant costs and treatment complexity. As a first step to reduce this treatment complexity, the present study describes a high throughput combinatorial domain library screening method to identify synthetic lymphoproliferative elements capable of driving in vivo expansion and survival of CAR-T cells in a lymphoreplete host without the homeostatic proliferation signals generated by lymphodepleting chemotherapy. Methods - High-diversity semi-rationally-designed combinatorial libraries of putative lymphoproliferative protein subdomains were DNA barcoded and assembled into a lentiviral vector co-expressing a ROR2-targeted CAR. Human PBMC were transduced with the library and cultured in vitro for several days. Purified cells were injected into mice bearing xenograft tumors modified to express the ROR2 antigen and compared to unmodified xenograft controls. The expansion rate of integrated cells was monitored weekly by quantitative PCR and, after 21 days of exposure, genomic DNA was isolated from blood, spleen and xenograft tumor tissues. Enriched barcodes were amplified using PCR and amplicons were subjected to HiSeq Next-Generation Sequencing. Barcode decoding was achieved using PacBio long read sequencing analysis to align full-length construct sequences with barcode quantitation. Results - Using this approach, putative CAR-T cell driver candidates and common key protein subdomains were identified that support selective in vivo expansion and survival of human lymphocytes in a tumor-bearing mouse model. Conclusion - Taken together, these results demonstrate that a high throughput combinatorial screening strategy with quantitative bioinformatics is a viable method for identifying protein domain combinations capable of selectively driving human CAR-T cells in vivo. These small synthetic combinatorial protein domains may facilitate lymphodepleting chemotherapy-free regimens and lower CAR-T cell doses in the future. Citation Format: Laurence Jadin, Hiba Shaban, Anirban Kundu, Gregory Schreiber, Scooter Willis, Farzad Haerizadeh, James Onuffer, Gregory Frost. A high-throughput screening strategy for the identification of novel lymphoproliferative elements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3523.