As a key component of the B-cell receptor (BCR) signaling complex, Bruton’s tyrosine kinase (BTK) plays an essential role in B-cell malignancies via both kinase and non-kinase functions. This was revealed by early genetic studies and the emerging kinase-impaired mutations, which activate BCR signaling via intricate protein-protein interactions, conferring resistance to a range of covalent or non-covalent BTK inhibitors (BTKi). Therefore, a new targeting strategy is urgently needed for patients who acquired kinase-impaired mutations of BTK. Targeted protein degradation is a potential and powerful means to modulate the non-catalytic functions.

TGRX-3911 is a heterobifunctional molecule that promotes ubiquitination and proteasomal degradation of BTK. In a diffuse large B cell lymphoma (DLBCL) cell line TMD8 featuring chronic active BCR signaling, TGRX-3911 induced rapid (4h) and potent degradation of wildtype (WT) and knock-in mutant BTK proteins including the kinase-proficient C481S and T474I, the kinase-impaired L528W and V416L, and even the compound mutants C481S/T474I, C481S/L528W and T474I/L528W, with DC50 < 1 nM and near 100% Dmax. Functionally, in Ramos cells expressing WT or knock-in C481S, T474I, V416L or L528W mutant BTK, TGRX-3911 diminished anti-IgM induced phosphorylation of BTK (excluding V416L and L528W) and PLCγ2, and strongly blocked Ca2+ flux. TGRX-3911 also reduced surface CD86 expression in TMD8 cells, as well as in C481S, T474I, V416L and T474I/C481S mutant cells. These results confirm that degradation of BTK induced by TGRX-3911 abolishes the BCR signaling pathway. TGRX-3911 potently inhibited proliferation of TMD8 parental cells and those harboring knock-in BTK mutations that are resistant to currently approved ibrutinib, zanubrutinib and/or pirtobrutinib, including C481S, T474I, L528W, V416L, A428D, T474I/C481S, T474I/L528W and C481S/L528W (IC50 from 0.4 to 9.9 nM). Remarkably, TGRX-3911 potently inhibited the A428D mutant which is still resistant to current BTK degraders, NX-5948, BGB-16673 and ABBV-101. It also demonstrated higher potency against the T474I/L528W compound mutant. In vivo, oral dosing of TGRX-3911 led to tumor shrinkage in a mantle cell lymphoma (MCL) REC-1 xenograft model expressing BTK-C481S, and the effect was superior to pirtobrutinib. No sign of toxicity was observed.

In summary, TGRX-3911 has potent and robust degradation activity and effectively suppresses the BCR signaling pathway. It has potent anti-proliferation activity against kinase-proficient, -impaired and even compound mutants of BTK, and has in vivo anti-tumor activity. By eliminating both the catalytic and scaffold functions of BTK, TGRX-3911 has the potential to overcome clinical resistance which can hardly be achieved by conventional BTKi. The potential clinical application of TGRX-3911 for patients with B-cell malignancies is worth exploring.