Development of ZE66-0205, a Novel MALT1 Degrader for Treatment of B-Cell Malignancies

Introduction: Inhibiting B-cell receptor (BCR) signaling with Bruton’s tyrosine kinase (BTK) inhibitors in diffuse large B cell lymphoma (DLBCL) has benefited a subset of patients. The activated B-cell (ABC) subtype of DLBCL is critically dependent upon chronically active BCR signaling that promotes the assembly of the CBM adapter complex (CARD11, BCL10, and MALT1). The CBM complex enables MALT1-dependent recruitment of additional signaling proteins and unleashes MALT1 protease activity, engaging the downstream NF-ΚΒ pathway. While MALT1 promotes proper lymphocyte activation and development, it is also a significant contributor to the survival of aggressive lymphomas and other cancers. MALT1 is a promising target for chronic lymphocytic leukemia (CLL) in patients developing resistance to BTK inhibitors. Within the CBM complex, MALT1 provides both molecular scaffolding and protease activity. The scaffolding function of MALT1 provides early signaling events leading to NF-κB activation, while the protease activity of MALT1 serves to amplify and prolong this effect, and targeting both functions is optimal for an effective inhibitor. However, many existing MALT1 allosteric inhibitors only inhibit a single function through protease inhibition. Therefore, we describe here ZE66-0205, a novel, oral bioavailable MALT1 degrader with potent in vitro and in vivo activity.

Results: Using CADD approaches and available crystal structures of the MALT1 protease we developed a highly active and selective MALT1 degrader, confirmed by proteomics studies in hPBMC cells. In vitro experiments with ZE66-0205 demonstrate inhibition of paracaspase activity in TMD8 cells (IC50 38 nM); inhibition of MALT1 dependent IL2 release in Jurkat cells (EC50 9 nM); and degradation of MALT1 protein in hPBMC cells (DC50 3 nM). In vivo PK experiments with ZE66-0205 demonstrated acceptable bioavailability, 20-25% in mice and 20-25% in dogs. To evaluate the MALT1 degrader ZE66-0205, the ABC-DLBCL cell line OCI-Ly3 (CARD11, MyD88 mutant) was treated with DMSO or ZE66-0205 (50, 500, 5000 nM) for 48 hrs. Immunoblots showed complete MALT1 degradation at all doses. Additionally, a dose dependent increase in full length RelB and CYLD was observed. Similar results were achieved in the TMD8 cell line (CD79b, MyD88 mutant). Interestingly, TMD8 cell lines CRISPR-edited to exclusively express BTK mutations seen with non-covalent (pirtobrutinib) T474I or covalent (acalabrutinib or zanubrutinib) C481S BTK inhibitors responded similarly to ZE66-0205. Furthermore, treatment of OCI-Ly3 at 500 nM ZE66-0205 led to >50% reduction of viable cells. To examine the effectiveness of ZE66-0205 in primary CLL B-cells, cells were treated with 1 or 10 μM for 48 hrs. and immunoblot showed complete degradation of MALT1 in comparison to DMSO treated control. Treatment with 10 μM of ZE66-0205 resulted in an 87% reduction (+19.7%) in viability versus DMSO control (n=3). To assess the efficacy of ZE66-0205 in degrading MALT1 in vivo, we utilized both wildtype and human celebron mutated mice (hu-CRBN, PubMed:30064974). The hu-CRBN mice are necessary to elicit any immunomodulatory function of immunomodulatory drug (IMiD) based degrader compounds. Mice were dosed with vehicle,10 mg/kg or 50 mg/kg ZE66-0205. A subset of mice (n=4 per group) received the drug orally for 4 days after which the mice were euthanized for pharmacodynamic analysis. Immunoblots in spleen lysates showed dose dependent degradation of MALT1 in the hu-CRBN mice. To assess survival efficacy of ZE66-0205 we used the OCI-Ly3 cell line disseminated xenograft model. The allosteric MALT1 inhibitor MLT-985 was used as a positive control. NCG mice were engrafted with OCI-Ly3 cells via the tail vein and mice received daily oral gavage of vehicle (n=11), 10 mg/kg ZE66-0205 (n=13) or 30 mg/kg MLT-985 (n=13) starting day 13 post-engraftment. Median survival was 37, 48, and 41 days, respectively. Analysis via Cox's proportional-hazards model shows superiority for the 10 mg/kg ZE66-0205 in comparison to vehicle group (p= 0.033).

Conclusion: We describe a novel, orally bioavailable potent MALT1 degrader compound with good PK properties and in vivo efficacy. ZE66-0205 leads to on target degradation of CBM target proteins and represents a promising new treatment strategy for DLBCL and other B-cell malignancies. Future clinical development of ZE66-0205 is warranted and ongoing.