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130 Immunogenic potential of chimeric antigen receptor (CAR)-engineered T cells expressing inducible nuclease-deactivated SpCas9 (dCas9)
  1. Dharmeshkumar Patel1,
  2. Dharmeshkumar Patel1,
  3. Angshumala Goswami1,
  4. Vitaly Balan1,
  5. Zhifen Yang1,
  6. Lingyu Li1,
  7. Sowndharya Rajavel1,
  8. Alper Kearney1,
  9. Rona Harari-Steinfeld1,
  10. Maggie Bobbin1,
  11. Bing Wang1,
  12. Alessandra Cesano1,
  13. Stanley Qi2 and
  14. Francesco Marincola1
  1. 1Refuge Biotechnologies Inc., Menlo Park, CA, USA
  2. 2Stanford University, Stanford, CA, USA


Background The application of CRISPR-Cas9 for personalized medicine is potentially revolutionary for the treatment of several diseases including cancer. However, the bacterial origin of the Cas9 protein raises concerns about immunogenicity. Recent ELISA-based assays detected antibodies against Cas9 from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in 5–10% of sera from 343 normal healthy individuals.1,2 SpCas9-specific memory CD8 T cell responses were not demonstrated in those individuals. To date, there are no conclusive studies assessing whether CRISPR-Cas9-modified CAR-T could raise CD8 T cell-mediated immunogenicity in humans. Refuge CAR-T cell platform employs an inducible, non-gene editing, nuclease deactivated Cas9 (dCas9) to modulate gene expression in response to external stimuli such as antigen-dependent CAR signaling to suppress PD-1 expression.

Methods In the present study, we analyzed two putative HLA-A*02:01 and two HLA-B*07:02-associated SpCas9 T cell epitopes. The candidate epitopes were derived from a prediction algorithm that incorporates T cell receptor contact residue hydrophobicity and HLA binding affinity. We engaged in-vitro sensitization (IVS) assay to identify immunogenic potential of dCas9 peptides.

Results Autologous IVS assay of T cells in two healthy donor PBMCs identified CD8-T cell responses after two rounds of stimulation against only one HLA-A*02:01-associated Cas9 peptide (sequence NLIALSLGL) P1– while the other candidate epitopes did not elicit any response. Dextramer analysis demonstrated that 15% of CD8+ T cells were specific for P1 and ~11% of CD8+ cells produced INFG upon challenge with P1-loaded T2 cells.

Conclusions Our in-vitro sensitization assay was able to demonstrate that dCas9 epitope P1 is immunogenic and may elicit adaptive immune response against gene edited CAR-T cells. Endogenous processing and presentation of P1 and other putative epitopes by Refuge CAR-T cells are currently being analyzed.

Acknowledgements Refuge Biotechnologies Inc. Menlo Park, California, 94025

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  1. Simhadri VL, McGill J, McMahon S, Wang J, Jiang H, Sauna ZE. Prevalence of Pre-existing Antibodies to CRISPR-Associated Nuclease Cas9 in the USA Population. Mol Ther Methods Clin Dev 2018;10:105–112. Published 2018 Jun 15. doi:10.1016/j.omtm.2018.06.006

  2. Ferdosi SR, Ewaisha R, Moghadam F, et al. Multifunctional CRISPR-Cas9 with engineered immunosilenced human T cell epitopes. Nat Commun2019;10(1):1842. Published 2019 Apr 23. doi:10.1038/s41467-019-09693-x

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