A tandem CD19/ROR1 CAR-NK to overcome Wnt5a-mediated apoptotic resistance in chronic lymphocytic leukemia Free

Introduction While autologous CD19-directed CAR-T cell therapy has been highly efficacious in certain B cell malignancies, its efficacy in chronic lymphocytic leukemia (CLL) has been limited by toxicity and T cell dysfunction characteristic of CLL pathophysiology. CAR natural killer (CAR-NK) therapy holds promise as a possible alternative due to a favorable toxicity profile and off-the-shelf allogeneic sourcing potential. A potential challenge for CAR-NK therapy in CLL is the emergence of compensatory pro-survival signaling in tumor cells which may inhibit tumor cell apoptosis. Receptor tyrosine kinase-like orphan receptor (ROR1) is ubiquitously expressed on CLL cells, and signaling via its ligand Wnt5a has been associated with both ibrutinib and venetoclax resistance; however, its connection to cell therapy resistance remains underexplored. Here, we studied Wnt5a-induced ROR1 signaling as an underlying CAR-NK resistance mechanism in CLL and leveraged our findings to develop a novel dual CAR-NK construct designed to overcome apoptotic resistance in CLL.

Methods ROR1 expression on peripheral blood-derived primary CLL cells was measured via flow cytometry. CLL cells were incubated with 200ng/mL of recombinant Wnt5a and were assessed for their apoptotic priming via BH3 profiling, a functional assay that measures the proximity of tumor cells to the apoptotic threshold and cellular dependence on specific anti-apoptotic proteins. Resistance to NK-mediated killing was evaluated by treating CLL cells with 1ug/mL Wnt5a and exposing them to either CD19-CAR-NK or untransduced primary NK cells, with Annexin V and propidium iodide (PI) staining used to assess CLL cell apoptosis. The ROR1 inhibiting monoclonal antibody (mAb) zilovertamab was used to investigate attenuation of Wnt5a/ROR1-associated survival. Tandem, 2^nd generation anti-CD19/ROR1 (dual) CAR constructs were designed by joining an anti-CD19 scFv with an anti-ROR1 scFv via a (G4S)4 linker, followed by a 4-1BB costimulatory domain and a CD3Z signaling domain. CAR constructs were transduced into Jurkat and peripheral blood-derived primary NK cells via an optimized baboon lentivirus protocol. Effector cell activation and cytotoxicity against primary CLL cells, a CD19+ CLL-derived cell line MEC-1, and the ROR1+ NCCIT cell line were evaluated via flow cytometry-based readouts of NK activation marker expression and Annexin/PI staining.

Results ROR1 expression was confirmed as present in primary CLL cells in all samples (n=33, ΔMFI [ROR1-Isotype] range: 346–2199). Stimulation of the cells with Wnt5a led to reduced apoptotic priming by 11–18% at 4h and 14–42% at 24h (BIM 0.1 µM), with greater reductions observed in high-ROR1 samples. Correspondingly, high-ROR1+ CLL samples incubated with Wnt5a exhibited up to 20% reduction in cell death compared to untreated conditions when co-cultured with either CD19 CAR-NK or untransduced primary NK cells. In venetoclax-treated CLL cells, zilovertamab reversed Wnt5a-induced resistance to NK-mediated killing, restoring cell death levels from 7.6% to 13.9% and restored apoptotic priming by increasing BIM-induced cytochrome C release from 5% to 18%.

Dual CAR-Jurkat cells showed increased expression of CD69 in the presence of either CD19+ MEC1 or ROR1+ NCCIT cell lines, while single-directed CARs exhibited no increase in activation against nonspecific targets. Dual CAR-NK cells similarly demonstrated higher CD69 expression in primary CLL patient cells (n=6) compared to single-targeting and untransduced NK cells at 16h. Cytotoxicity assays also showed that the dual CAR-NK kills at least as well as CD19-CAR-NK, and outperformed ROR1-CAR-NK and untransduced NK cells across high- and low-ROR1 patients.

Conclusion We demonstrated that Wnt5a-induced ROR1 signaling is a key pathway that reduces CLL cell priming for apoptosis and enhances CLL cell survival against NK cells, a process reversible ex vivo by zilovertamab. We subsequently developed a novel tandem CD19/ROR1 CAR-NK product with enhanced activation against CLL cells compared to single-directed CARs. This dual CAR-NK also has the potential to subvert single antigen escape. In vivo studies are underway using NSG mice engrafted with HG3 CLL cells engineered to express both ROR1 and constitutive Wnt5a to assess whether a novel dual CAR-NK product expressing the D10-derived anti-ROR1 scFv (derived from zilovertamab) more effectively reduces tumor burden compared to non-ROR1-inhibiting CAR constructs.