G2 - Immune Targeting and Approaches with Genetically-Modified Cells and Cell Therapies (Including CAR-T, CAR-NK, TCR editing)
826: In Vivo Pooled Screening Platform for the Discovery of Optimized Chimeric Antigen Receptor (CAR) Design in T cells
Type: Poster Session
Poster Board Number: 826
Presentation Details
Session Title: Wednesday Posters: Immune Targeting and Approaches with Genetically-Modified Cells and Cell Therapies
While Chimeric Antigen Receptor expressing T cells (CAR-T cells) have shown high response rates against hematological cancers in the clinic, they have shown limited efficacy against solid tumors due to lack of persistence and susceptibility to various immunosuppressive factors in the tumor microenvironment (TME). To address the suppressive TME, recent efforts to systemically discover novel intracellular signaling domains (ICDs) at scale have demonstrated success in engineering CAR designs with enhanced solid tumor efficacy. However, in vitro screens are inherently limited in mimicking the TME and other physiologically relevant aspects of adoptive cell therapy (ACT). Here, we developed an in vivo pooled screen approach to identify potent CAR-T designs in a CHLA-20 neuroblastoma xenograft model. We utilized a DNA-barcoded 10,000-member CAR ICD library that we previously used to identify novel ROR1 CAR ICDs with enhanced anti-tumor function. This library was cloned into a GD2 CAR-T backbone, GD2 CAR library-expressing primary T cells were injected into tumor-bearing mice at >100x coverage of our library size, and 2 weeks post-ACT, spleens and tumors were collected. Sequencing genomic DNA quantified the abundance of barcodes linked to each CAR ICD combination. Based on previous literature, we hypothesized that any remaining CAR-T cells at the tumor site and spleen would represent T cells with long-term persistence elicited by antigen-specific CAR ICD signaling. Novel ICD combinations were preferentially enriched across multiple replicate mice. We selected the top 100 shared hits and validated their anti-tumor function in an arrayed screen, assessing cytotoxicity, cytokine secretion profiles, proliferation, and memory and exhaustion phenotypes in a tumor rechallenge setting. These functional datasets allowed us to dissect the effects of particular signaling components in T cells that endow long-term persistence and potentially resist immunosuppressive TME factors. In vivo pooled screen approach is readily applicable to identifying signaling modules driving favorable phenotypes of T cells against solid tumors. Further development of an in vivo screening platform in humanized mice could be a promising next step to discovering cell therapy products with strong translational potential.
Taeyoon Kyung1, Joshua Mace1, Matthew Forsberg2, Nina La Vonne Denne3,4, Siqi Zhao1, Khloe Gordon Wei1, Jai Raman1, Narendra Maheshri1, Christian Capitini2, Krishanu Saha3,4, Shawdee Eshghi1
1Ginkgo Bioworks, Boston, MA,2University of Wisconsin School of Medicine and Public Health, Madison, WI,3Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI,4Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI"
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