Potent and Effective Targeting of AML with ‘Off-the-Shelf’ Engineered CD45-Targeting CAR T-Cells
Authors: Shaw Matthew , Liao Aiyin , Khan Naeem , Freeman Sylvie D , Whittaker Thomas E. , Thrasher Adrian J. , Pule Martin , Chester Kerry , Yeung Jenny , Amrolia Persis 
Published: December 2024

Introduction

To date the outcomes of CAR T-cell therapy trials in AML have been disappointing. This likely reflects both poor fitness of autologous T-cells due to prior chemotherapy and leukemic escape due to the heterogeneous expression of existing target antigens on leukemic stem cells (LSCs). To overcome these challenges, we have developed CAR T-cells from healthy 3rd party donors directed against CD45 which is uniformly expressed on AML LSCs and expressed solely in the hemopoietic lineage. To prevent fratricide and GVHD, CAR T-cells were gene-edited to knockout CD45 and the T cell receptor (TCR). Our hypothesis is that 3rd party double knockout (DKO) CD45CAR T-cells could potentially be used as an “off-the-shelf”, universal immunotherapy to induce deep remission prior to Stem Cell Transplant (SCT) for high risk hematological malignancies.

Methods

CyTOF analysis was used to compare CD45, CD33, CD123 and CLL-1 expression on leukemic blasts and LSCs from a cohort of poor risk adult AML diagnostic blood/BM samples. CRISPR/Cas9 editing of PTPRC and TRAC was utilized to knockout expression of CD45 and TCR in healthy donor T cells. DKO T cells were transduced with lentiviral vectors encoding CD45-targeting, second generation CARs derived from different antibody clones. An inducible suicide gene system was incorporated to enable deletion of DKO CD45CAR T-cells. In vitro51Cr-release and flow-based cytotoxicity, 3H-thymidine uptake and ELISAs were performed to select the DKO CD45CAR with optimum functional activity prior to assessment for in vivo efficacy in xenogeneic models of AML.

Results

CyTOF demonstrated that CD45 was uniformly expressed on LSCs in almost all AML patients whereas existing CAR T-cell targets (CD33, CD123, CLL-1) were only expressed uniformly in a minority. DKO CD45CAR T-cells were generated with efficient knockout of CD45 and TCR with evidence of fratricide of residual CD45+ cells leading to self-enrichment of the DKO CD45CAR T-cells. DKO CD45CAR T-cells expressing a YTH24.5-CD28zeta CAR showed optimal function, efficiently killed CD45+ AML cell lines in vitro and demonstrated antigen-specific proliferation and cytokine secretion. DKO CD45CAR T-cells completely prevented engraftment of human HSCs in NSG mice. To prevent targeting of donor HSCs by residual DKO CD45CAR T-cells during SCT, we incorporated an iCaspase9 suicide gene and showed that DKO CD45CAR T-cells were efficiently deleted by treatment with Rimiducid in vitro. DKO CD45CAR T-cells prevented development of AML in an NSG-MOLM14 xenogeneic mouse model leading to improved survival compared to mice treated with DKO CD19CAR T-cells. Further, we demonstrated that DKO CD45CAR T-cells were able to induce complete regression of AML in an established MOLM14 tumor model. Finally, we compared the functionality of our CD45CAR against the BC8-4-1BBzeta CD45CAR developed by Wellhausen et al (Sci. Transl. Med. 2023). DKO T cells transduced with our CD45CAR showed markedly enhanced cytotoxicity against CD45+ cells (particularly against targets expressing CD45 at low antigen density) and enhanced proliferation/IL-2 secretion in response to CD45+ targets compared to the UPenn CD45CAR T-cells. Further, in contrast to our DKO CD45CAR T-cells, the BC8-based DKO CAR T-cells did not induce regression of AML in our xenogeneic MOLM14 model demonstrating the superiority of our CAR in the context of CD45 knockout.

Conclusions

Our data confirm that CD45 is an excellent candidate for CAR T-cell therapy in AML. We have demonstrated that DKO CD45CAR T-cells show potent anti-leukemic activity against AML both in vitro and in vivo and are functional despite the loss of endogenous CD45 expression. DKO CD45CAR T-cells depleted human HSCs in vivo, suggesting they would render patients aplastic and would need to be deleted prior to consolidative SCT, and we have shown that this can be achieved using a Rimiducid inducible Caspase 9 suicide gene. This approach avoids the need to epitope edit donor HSCs representing a major safety advantage. Moreover, generating DKO CD45CAR T-cells from an allogeneic healthy donor will enhance CAR T-cell fitness and allows a bank of “off-the-shelf” CAR T-cells to be used for multiple patients, reducing complexity and cost. Based on these data we are now planning to test this approach in a clinical study where DKO CD45CAR T-cells will be used as adjunctive immunotherapy prior to SCT in adult patients with ELN high risk AML.