An Orally Active Pan-BCR::ABL1 Degrader Tgrx-3247 Potently Targets a Wide Spectrum of Orthosteric, Allosteric and Compound Mutations Resistant to TKI Therapies

BCR::ABL1 mutations that confer resistance to tyrosine kinase inhibitors (TKIs), regardless of ATP-competitive or allosteric inhibitors, remain challenging for the treatment of chronic myeloid leukemia (CML), especially for patients who developed the T315I, T315M or compound mutations that are highly resistant to currently approved therapies. Furthermore, many studies suggest that patients with fast and sustained deep molecular response (DMR) are more likely to achieve treatment-free remission (TRF). How to improve the clinical response rate and prevent the emergence of acquired resistance is a key issue in the discovery of next-generation therapeutics.

To address the unmet medical need, we designed TGRX-3247, a heterobifunctional molecule that recruits BCR::ABL1 and the E3 ligase CRBN, leading to ubiquitination and subsequent proteasomal degradation of the oncoprotein. At 48 hrs, TGRX-3247 efficiently induced BCR::ABL1 degradation in cell lines such as K562, KCL22s, KCL22s^T315I, KCL22s^T315M, Ba/F3-BCR::ABL1^T315I, and Ba/F3-BCR::ABL1^Y253H/T315I, with half-maximal degradation concentration (DC₅₀) ranging from 0.02 to 6.68 nM, and maximal level of degradation (D_max) from 87% to 98%. Proteasome inhibitors such as MG132 and bortezomib blocked the degradation. Importantly, in KCL22s cell lines with knock-in T315I or T315M mutation, the effects of transient exposure (4 hrs) of TGRX-3247 at a very low concentration (10 nM), including the degradation of BCR::ABL1 and the reduction of phosphorylation levels of ABL1, BCR and STAT5, sustained as long as 72 hrs after the degrader being washed out. In contrast, the prolonged effects were not achievable with the warhead only or a non-degrader control after washout.

In cell proliferation assays, TGRX-3247 potently inhibited CML cell lines such as K562, KCL22s and LAMA-84 with striking picomolar half-maximal inhibition concentrations (IC50 < 0.06 nM). We next analyzed the KCL22s cell lines with knock-in BCR::ABL1 mutations. TGRX-3247 potently inhibited the ATP competitive inhibitor-resistant mutants such as T315I andT315M (IC50 < 0.3 nM), the allosteric inhibitor-resistant A337V, P465S, V468F and I502L (IC50 < 8 nM), and even the compound mutants including E255V/T315I, F359V/T315I and Y253H/T315I (IC50 < 1 nM). The extraordinary anti-proliferation activity of TGRX-3247 was recapitulated in a plethora of Ba/F3 cell lines expressing BCR::ABL1 variants including the ATP site, allosteric site mutants, compound mutants, as well as the p190 variant. On the other hand, TGRX-3247 exhibited no cytotoxicity in the Ba/F3 parental cells or various other normal cell lines (IC50 >10 μM), suggesting its high selectivity.

In vivo, TGRX-3247 demonstrated dose-dependent anti-tumor effect. In mice with K562 xenograft, daily oral dosing at 0.3, 1 and 3 mpk achieved tumor growth inhibition (TGI) of 22.0%, 64.9% and 99.8%, respectively. In a BCR::ABL1^T315I xenograft model, oral dosing at 3, 10, 30 mpk daily or 10 mpk twice daily achieved TGI of 36.8%, 67.9%, 77.0% and 82.0%, respectively. In another xenograft model harboring the Y253H/T315I compound mutation, daily oral dosing at 3, 10 and 30 mpk achieved TGI of 24.8%, 54.9% and 61.5%, respectively. No significant body weight loss of the animals was observed during dosing.

In summary, TGRX-3247 has demonstrated robust and sustained targeted degradation activity, potent anti-proliferation activity and in vivo oral anti-tumor efficacy, against a wide spectrum of single- or compound-mutations of BCR::ABL1. With such potent and continuous depletion of the oncogenic signaling, coupled with a wide range of mutant coverage, TGRX-3247, as a targeted protein degrader, may potentially bring clinical benefits that are unseen with previous TKIs, including improved DMR, TFR rates, and even the possibility of functional cure. Its potential clinical application for CML patients is worth further investigation.