Engineering safe mutations to protect cells from antibody-based targeted therapy

Therapeutic depletion of diseased cells using antibody-based targeted therapies such as monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), T cell engagers (TCEs) or chimeric antigen receptor (CAR)-T cells is very effective for hematologic diseases. However, shared expression of antigens between diseased and healthy cells bears the risk for collateral killing of healthy cells leading to unwanted side-effects. For instance, myeloid tumor cells and hematopoietic stem and progenitor cells (HSPCs) express very similar surface proteins resulting in a risk for extensive myelotoxicity upon targeted depletion. If the healthy cell is essential (e.g. HSPCs) then such co-expression may constitute a major, possibly insurmountable, barrier. In order to enable therapeutic targeting of such proteins, it was proposed to transplant engineered hematopoietic stem cells in which the target was removed. However, since removing the protein abolishes its function, this approach is limited to redundant proteins. Here, we show feasibility for shielding the therapeutic cells from antibody-based targeted therapy while preserving the expression of the targeted receptors with two proteins. In chapter I we showed that CD123, the interleukin-3 (IL-3) receptor alpha-chain, could be engineered to protect cells from targeted depletion. We identified several CD123 point mutations which protected in vitro from antibody dependent cellular cytotoxicity, TCE and CAR T-cells killing. Preserved function of two shielding variants was shown in vitro using the IL-3 dependent cell line TF-1 and in vivo using engineered HSPCs. In chapter II, we showed that a second protein, CD45, the protein tyrosine phosphatase receptor type C, could be engineered into primary human T cells to shield them from ADC killing. To finish, we optimized genome engineering of CD45 mutations using base editors. We showed that point mutations could be introduced with strategies other than CRISPR/Cas9 and homology-directed repair engineering, demonstrating that multiple genome engineering approaches could be employed to shield cells.