Tuned IL3-Zetakine Coupled to a CD33 Costimulatory Receptor As a Dual CAR for Safer and Selective Targeting of Acute Myeloid Leukemia

Clinical application of chimeric antigen receptor (CAR) immune-effector cells in Acute Myeloid Leukemia (AML) needs a strategy for safer and durable disease remission, especially in elderly patients. To date, the translation of CAR-T cell therapy in AML is limited by the absence of an ideal tumor-specific antigen. Indeed, the overlapping expression of the target antigen between healthy tissues and tumor cells represents one of the main challenges to develop safe immunotherapies devoid of on-target/off-tumor toxicities.

CD123 and CD33 are the two most widely overexpressed biomarkers of both AML blasts and leukemic stem cells, but their shared expression with endothelial and hematopoietic stem progenitor cells (HSPCs) increases the risk of undesired vascular and hematologic toxicities. To counteract this issue, we established a balanced dual CAR strategy aimed at reducing off-target toxicities while retaining full functionality against AML.

Cytokine-induced killer (CIK) effector cells were genetically modified to express both a first generation anti-CD123 IL-3 zetakine (IL3z.CAR), thus carrying membrane tethered-IL3 ligand as extracellular domain and providing the activation signal 1 (CD3ζ), and a CD33 chimeric costimulatory receptor (CCR) providing the costimulatory signal 2 (4-1BB and CD28 double costimulation). Moreover, this design has been optimized to elicit a productive CIK cell activation only after the simultaneous engagement of both receptors by CD123+/CD33+ leukemic cells by (1) lowering the IL-3 zetakine affinity by point-mutations screened by molecular dynamics in the IL-3 cytokine (DC mut) to minimize recognition and toxicity against single low-CD123 expressing endothelium and HSPCs without compromising the reactivity towards the CD123+/CD33+ leukemic cells and (2) selecting optimal CAR spacers to avoid receptor dimerization and therefore unwanted activation following single CD33 engagement.

The CD33 trans-activation contribution in the Dual CAR full activation was verified by measuring the differential avidity force during the immune synapse formation between the Dual CAR and the wild type (23% of T-cells bound) and CRISPR-Cas9 knockout CD33- (13%) and CD123-CD33- (5,7%) edited AML cells. In vitro efficacy has been confirmed in short- and long- term cytotoxicity and cytokine production assays upon challenge with different CD123/CD33 positive AML cell lines and primary blasts. The lower affinity of the IL3z.CAR has allowed to reduce the toxicity profile towards the CD123 positive human endothelial cell line (TIME). Furthermore, methylcellulose-based clonogenic assay performed on CD34+ HSPCs plated after 24 hours exposure to NT and CAR-CIK cells, showed that the optimized Dual-CAR (DC-mut) does not significantly affect the myeloid colony-forming potential. Significant anti-leukemic activity has also been observed in vivo, where Dual-CAR CIK cell treatment resulted in better tumor control and improved survival of KG-1 engrafted NSG mice (median survival time increased from 39 in the untreated group to 76,5 and 83 days in the DC wt-and DC-mut CIK groups, respectively, p < 0.01, log-rank test).

In conclusion, low affinity Dual CAR with trans-acting costimulation provides improved efficacy and high specificity for CD123 and CD33 recognition on leukemic cells as well as tolerable toxicity on endothelium and HSPCs, thus highlighting how this Dual CAR approach unleashes the potential of targeting CD123 and CD33, reducing the risk of life-threatening toxicities.

Martelli:Pfizer: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisor; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisor; Jazz: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisor; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisor; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisor; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisor; AbbVie: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisor. Falini:Rasna Therapeutics, Inc: Honoraria. Biondi:CoImmune: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; CoImmune: Honoraria, Research Funding.