Poster Presentation Asia-Pacific Vaccine and Immunotherapy Congress 2023

Using CRISPR activation applications to identify novel tumour resistance mechanisms of aggressive lymphomas in CAR-T cell therapy. (#172)

Christina Koenig 1 2 , Sarah Diepstraten 1 3 , Paul Beavis 4 5 , Gemma Kelly 1 3 , Marco Herold 1 3 6 7
  1. Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia
  2. DFG (German Research Foundation) funded postdoctoral research fellow, Germany
  3. Department of Medical Biology, University of Melbourne, Melbourne, VIC, Australia
  4. Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
  5. Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, VIC, Australia
  6. Olivia Newton-John Cancer Research Institute, Austin Health, Heidelberg, VIC, Australia
  7. School of Cancer Medicine, La Trobe University, Heidelberg, VIC, Australia

A particularly aggressive subtype of Diffuse Large B Cell Lymphoma (DLBCL) called Double Hit Lymphoma (DHL), expresses high levels of the pro-survival protein BCL-2 in addition to high cMYC. The standard of care for patients presenting with DLBCL that have relapsed or are refractory to standard therapies undergo CD19+CAR-T cell therapy. However, many patients respond poorly or not at all, and the mechanisms of resistance are not well understood.

To address this, we are using mouse DHL cells generated by unique CRISPR activator (dCas9A) technology (see Deng Y. et al., Nature Comm. 2022), together with CD19+CAR-T cell mediated killing assays to identify novel resistance mechanisms of DHL cells to CAR-T cell therapy. Therefore, we transduced dCas9A+ DHL cells with genome wide lentiviral CRISPRa sgRNA libraries able to induce robust expression of genes when introduced into dCas9A expressing cells. Successfully transduced dCas9A+ DHL cells were co-cultured with CD19+CAR-T cells for a period of 16h and surviving DHL cells resistant against CAR-T cell killing mechanisms isolated. Since CAR-T cell mediated killing is mainly induced in a cytokine dependent manner post 4 hours of co-culture with their target cells, we cultured sgRNA-library transduced dCas9A+ DHL cells with CD19+CAR-T cells in sequential shorter killing cycles. This allows the identification of novel resistance mechanisms evolved specifically towards contact-dependent CAR-T killing mechanisms. Surviving DHL cells were recovered, genomic DNA isolated, and resistance-promoting sgRNAs identified by next generation sequencing. Top candidate hits are currently validated in CD19+CAR-T killing assays in vitro. Finally, in vitrovalidated resistance genes will be tested in an in vivo setting by transplanting dCas9A+ DHL cells expressing these genes into immunocompromised RAG1 knockout animals. Upon lymphoma development, CD19+CAR-T cells will be transplanted and their efficiency of clearing the tumour evaluated.      

We expect that the findings upon completion of this project will enhance our understanding of tumour resistance mechanisms and provide us with novel targets to be used in future CAR-T therapies against DHL.