Nx-2127 and Nx-5948, Two Clinical Stage Cereblon-Recruiting BTK Degraders, Facilitate T Cell Functionality in Chronic Lymphocytic Leukemia

Introduction: Inhibitors targeting Bruton’s tyrosine kinase (BTKi) have revolutionized treatment of chronic lymphocytic leukemia (CLL), but resistance develops due to BTK mutation. Targeted protein degradation has emerged as a strategy to overcome this resistance. NX-2127 and NX-5948 are degraders designed to induce BTK degradation by recruiting the cereblon (CRBN) E3 ubiquitin ligase complex. Of those, only NX-2127 has CRBN immunomodulatory activity. While BTKi’s are known to positively modulate T cell function in CLL, the effects of BTK degradation on T cell functionality are unknown. Furthermore, it is unclear to what extent the addition of CRBN neosubstrate activity may contribute to T cell modulation and ultimately degrader function. Here, we begin to address these questions by comparing NX-2127 and NX-5948 to ibrutinib, a BTK inhibitor, and lenalidomide, a CRBN immunomodulatory compound.

Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from CLL patients. T cells were purified using Dynabeads and stimulated with αCD3/CD28 and then analyzed by flow cytometry. Naïve T cells were studied under Th1/Th2/Treg-polarizing conditions. For immunological synapse assays, T cells and CLL cells were treated with drugs, combined at a 1:1 ratio, and imaged using confocal microscopy. Cytotoxicity of BTK degrader-treated T cells against OCI-LY19 cells was assessed by flow cytometry (E:T ratio 1:10). PBMCs from CLL patients treated with NX-2127 on a phase 1a/1b clinical trial (NCT04830137) were collected at baseline, C1D8, and C2D1 and analyzed by flow cytometry.

Results: Treatment with NX-2127 and NX-5948 resulted in BTK degradation in CLL cells. NX-2127, but not NX-5948, induced dose-dependent degradation of Aiolos and Ikaros in CLL and T cells, confirming its CRBN immunomodulatory activity. Neither degrader induced apoptosis or reduced proliferation of CD3⁺ T cells. T cell activation markers (CD69, CD25, HLA-DR, PD-1) on CD4⁺ and CD8⁺ T cells were unaffected by treatment with either NX-2127 or NX-5948. NX-2127 upregulated CD38 expression in CD4⁺ and CD8⁺ T cells, which was not seen with NX-5948 or ibrutinib/lenalidomide controls. In vitro polarization assays showed that NX-2127, but not NX-5948, significantly upregulated IFN-γ and IL-2 under Th1 conditions (to a degree that exceeded the effect of either lenalidomide or ibrutinib), without altering Th2 differentiation markers (IL-4, GATA3). NX-2127 and lenalidomide reduced Treg differentiation, distinct from NX-5948 or ibrutinib.

NX-2127, but not NX-5948 or ibrutinib, enhanced immunological synapse formation to levels comparable to those of lenalidomide, accompanied by upregulation of ICAM-1. Treatment with NX-2127 and lenalidomide, but not NX-5948 or ibrutinib, enhanced T cell-mediated killing of OCI-LY19 cells in cytotoxicity assays, which was accompanied by increased levels of granzyme B secretion by CD8⁺ T cells.

Analysis of clinical trial material demonstrated that T cells from NX-2127-treated CLL patients showed no changes in Th1/Th2 cytokine secretion or exhaustion markers (CTLA-4, PD-1) but did indicate decreased IL-4 and IL-17 expression. Bulk RNA sequencing of PBMCs was consistent with preclinical data and identified differential gene signatures in NX-2127 responders compared to non-responders, with decreased Th2 and Th17 pathway genes at baseline. Further, T cell modulation signatures were evident post dose. Single-cell RNA-seq data is currently being analyzed.

Conclusion: CRBN-recruiting BTK degraders NX-2127 and NX-5948 induce BTK degradation in primary CLL cells without compromising T cell activation and survival in vitro. CRBN-immunomodulatory activity was required to upregulate CD38 (an IFN response gene), promote Th1 phenotype, downregulate Tregs, and facilitate synapse formation and cytotoxicity in vitro, suggesting NX-2127's potential to reverse CLL-associated immunosuppression. RNA sequencing highlighted distinct gene expression in NX-2127-treated patients. Overall, our findings provide a strong rationale for continued investigation of BTK degraders in CLL and lymphoid malignancies.