Poster Presentation NSW State Cancer Conference 2023

Taming the Immune Response: CRISPR Based Knockout of HLA Class I and II to Prevent Allorejection of Off-the-Shelf Chimeric Antigen Receptor T-cells (#214)

Melanie Mach 1 2 , David Bishop 1 2 3 4 5 , Kavitha Gowrishankar 2 6 , Kenneth Micklethwaite 1 2 3 4
  1. The Westmead Institute for Medical Research, Westmead, NSW, Australia
  2. Faculty of Medicine and Health, The University of Sydney, Sydney
  3. Department of Haematology, Westmead Hospital, Sydney
  4. Blood Transplant and Cell Therapies Program, Westmead Hospital , Sydney
  5. Department of Laboratory Haematology, ICPMR-Westmead, NSW Health Pathology, Sydney
  6. Children's Cancer Research Unit, Kids Research, Sydney

Background: T-cells can be genetically modified to express artificial Chimeric Antigen Receptors (CARs) enabling them to target and lyse cancer cells. CAR T-cells targeting the B-cell antigen, CD19, have induced complete remissions in >90% of patients with relapsed/refractory Acute Lymphoblastic Leukemia and >50% in relapsed/refractory Diffuse Large B-cell Lymphoma. Currently, CAR T-cells are produced with random gene integrating techniques to introduce the CAR transgene. Additionally, they are manufactured individually for each patient from autologous T-cells which is time-consuming and expensive. To mitigate these problems, off-the-shelf CAR T-cells can be produced from a healthy donor. However, their efficacy may be limited by recipient T-cell mediated rejection of mismatched human leukocyte antigens (HLA), while they may also cause toxicity in the form of graft-versus-host disease (GVHD) via their native T-cell receptor (TCR).

Aims: To prevent GVHD, we generated off-the-shelf CAR T-cells by CRISPR/Cas9 knockout of the TCR complex. Additionally, we investigated knockout of various HLA Class I and II-associated gene targets, to prevent CAR T-cell rejection.

Methods: T-cells from healthy donors were transfected by electroporation with Cas9 enzyme, single guide RNA and CAR19 transgene, expanded for 3 weeks, and stimulated with interleukin-15 and irradiated autologous peripheral blood mononuclear cells.

Results: CRISPR-edited CAR T-cells exhibited comparable expansion, memory phenotypes and expression of immunoinhibitory molecules to our Phase I clinical trial CAR T-cells produced with the non-specific Super PiggyBac transposon system. In screening potential HLA-associated targets for HLA downregulation, we discovered a single novel target gene that efficiently downregulates HLA Class I and II expression on CAR T-cells.

Translational significance: Our data demonstrates that CRISPR/Cas9 directed against novel targets can be used to efficiently generate CAR T-cells lacking TCR and HLA. This developmental research will lay the crucial groundwork for the development of future off-the-shelf T-cell therapy products to target various malignancies and infections.