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338 Knockout of CBLB Greatly Enhances Anti-Tumor Activity of CAR T Cells

Program: Oral and Poster Abstracts
Type: Oral
Session: 625. Lymphoma: Pre-Clinical—Chemotherapy and Biologic Agents: Immunologic approaches
Hematology Disease Topics & Pathways:
Biological, Therapies, CAR-Ts, Technology and Procedures, gene editing
Sunday, December 2, 2018: 9:45 AM
Room 28D (San Diego Convention Center)

Kathryn Hooper, PhD1*, Kyle Havens, PhD1*, Anne-Rachel Krostag, BS1*, Michael S Magee, PhD2*, Unja Martin1*, Ankit Gupta, PhD2*, Yegor Smurnyy, PhD2*, Lisa Pechilis2*, Amanda Rode2*, Andrew Chavkin, MS2*, Shannon Grande, PhD2*, Richard A. Morgan, PhD2, Jordan Jarjour, PhD1* and Alexander Astrakhan, PhD1*

1bluebird bio, Seattle, WA
2bluebird bio, Cambridge, MA

Chimeric antigen receptor (CAR) T cell therapies continue to show excellent outcomes in hematological cancers. Achieving success in additional tumor indications, however, will likely require modulating inhibitory pathways that limit CAR T cell potency. We developed a megaTAL nuclease targeting the gene encoding Casitas B-lineage lymphoma proto-oncogene-b (CBLB), a ubiquitin ligase that serves as an intracellular checkpoint that negatively regulates T cell activation. The megaTAL nuclease platform has been previously shown to drive highly efficient genome editing in primary T cells. Electroporation of primary T cells with mRNA encoding the CBLB megaTAL resulted in >90% indels at the target locus and a concomitant reduction of Cbl-b protein levels. Specificity characterization studies revealed three detectable non-exonic off-target sites with near negligible indel frequencies. We next assessed the functional impact of CBLB disruption in CAR T cells engineered to target the epidermal growth factor receptor (EGFR). When co-cultured with EGFR+ target cells, CAR T cells with megaTAL-mediated CBLB gene knockout had a 2-fold increase in pro-inflammatory cytokine production compared with mock-treated CAR T cells. We developed an A549 tumor xenograft model to test the activity of CBLB megaTAL-treated CAR T cells in vivo. While mock-treated CAR T cells had a transient impact on tumor growth, we observed complete and durable tumor elimination in mice infused with the CBLB megaTAL-treated CAR T cells. Improved responses in the megaTAL treated animals were particularly pronounced at lower CAR T cell doses, suggesting that CBLB knockout enhances the potency of CAR T cells. In summary, the CBLB megaTAL is a highly efficient and specific gene editing nuclease that enhances CAR T cell anti-tumor responses in vitro and in vivo, and thus could potentially improve the efficacy of CAR T therapy.

Disclosures: Hooper: bluebird bio: Employment, Equity Ownership. Havens: bluebird bio: Employment, Equity Ownership. Krostag: bluebird bio: Employment, Equity Ownership. Magee: bluebird bio: Employment, Equity Ownership. Martin: bluebird bio: Employment, Equity Ownership. Gupta: bluebird bio: Employment, Equity Ownership. Smurnyy: bluebird bio: Employment, Equity Ownership. Pechilis: bluebird bio: Employment, Equity Ownership. Rode: bluebird bio: Employment, Equity Ownership. Chavkin: bluebird bio: Employment, Equity Ownership. Grande: bluebird bio: Employment, Equity Ownership. Morgan: bluebird bio: Employment, Equity Ownership. Jarjour: bluebird bio: Employment, Equity Ownership. Astrakhan: bluebird bio: Employment, Equity Ownership.

*signifies non-member of ASH