No abstract

Biosensors for ligands are versatile tools for biological applications ranging from disease diagnosis to the detection of environmental chemicals. Thus, expanding the repertoire of biosensor toolkits provides novel insights into the unappreciated potentials of molecules. While the natural recognition domains have been employed in current biosensors, their use is limited to the methods such as fluorescence resonance energy transfer sensors or circular permutation. Here, we describe a first-of-its-kind approach that transforms a protein exhibiting ligand-mediated hinge-bending motion into a highly specific fluorescent biosensor. As a proof-of-concept, a glutamine sensor named Q-SHINE (Glutamine sensor via Split HINgE-motion binding protein) was designed by splitting a glutamine-binding protein into two separate stable domains that, in principle, only dimerize in the presence of glutamine. The application of Q-SHINE to determine glutamine concentrations in solution or mouse serum yielded comparable sensitivity and higher specificity to a conventional glutamine assay kit. Moreover, genetically encoded Q-SHINE could effectively monitor intracellular glutamine levels. Aside from highlighting the reliability of a versatile glutamine sensor, our study opens avenues for researchers who wish to explore the application of ligand-binding proteins as biosensors.

Background: EGFR mutation in non-small cell lung cancer (NSCLC) presents a viable therapeutic target for which tyrosine kinase inhibitor (TKI) therapy has shown impressive clinical benefit over the past 20 years. Unfortunately, most patients inevitably progress on earlier-generation EGFR TKIs due to various mechanisms after a period of therapy. The most commonly acquired on-target resistance mutations are T790M and C797S, often appearing after progression on 1st-, 2nd-generation, and 3rd-generation EGFR TKIs, respectively. To prevent the emergence of drug-resistant subclones, a next-generation EGFR TKI must have activity against both treatment-emergent and drug-naïve mutants. Further preclinical studies were conducted to evaluate the activity of BBT-207 in delaying tumor regrowth or prolonging survival in NSCLC tumor models driven by the EGFR T790M and C797S mutation. Method: We evaluated the inhibitory potency of BBT-207 against wild-type (WT) and mutated EGFR proteins. In vivo anti-tumor activity was evaluated in Ba/F3 EGFR ex19del/T790M (DT), L858R/T790M (LT), ex19del/C797S (DC), L858R/C797S (LC), ex19del/T790M/C797S (DTC) or L858R/T790M/C797S (LTC), H1975 EGFR LTC cell line-derived xenograft (CDX), and EGFR DTC-patient-derived xenograft (PDX) models. In vivo inhibition of brain metastases (BM) was evaluated in a luciferase-expressing PC-9 EGFR DTC BM model through direct intracranial implantation. Results: BBT-207 is broadly active against activating and acquired resistance EGFR mutants and displayed potent anti-proliferation activity against Ba/F3 EGFR DT, LT, DC, LC, DTC, LTC with 4, 4, 1, 16, 5 and 8nM, respectively while being highly selective against Ba/F3 EGFR WT (184 nM). BBT-207 also showed IC50 values <5 nM against Osimertinib-resistant EGFR L792H triple mutants, DTL and LTL in cell-free in vitro kinase assay. QD administration as a single agent resulted in significant tumor regression in the Ba/F3 EGFR DT, LT, DC, LC, DTC, LTC CDX models and EGFR DTC PDX model. Indeed, BBT-207 showed sustained anti-tumor efficacy in Ba/F3 EGFR DC CDX model. Furthermore, BBT-207 exerted dose-dependent intracranial anti-tumor activity and enhanced survival rate of mice in the PC-9/EGFR DTC_Luc BM model. PK studies revealed CNS penetration with good B/P ratios in animals. Conclusion: BBT-207 is a reversible, mutant-specific, broad-spectrum TKI, active to clinically observed mutations of EGFR and is expected to be compatible with monotherapy of QD schedule in humans. BBT-207 is well-positioned to augment the treatment of EGFR mutated NSCLC, either for acquired drug resistance or in earlier line, with potential to treat or prevent CNS metastasis. PK/TK evaluation justify exploration in humans. First-in-human study in patients harboring EGFRm and previously treated with EGFR TKI, is to begin enrollment in USA 1H 2023. Citation Format: Chulwon Kim, Youn Hee Jung, Naeun Jeon, Yong-Hee Lee, Sang-Yoon Lee, Jimmy Taiguang Jin. BBT-207 is a broad-spectrum, highly potent, 4th generation EGFR TKI with enhanced activity to both sensitizing and treatment-emergent EGFR mutations including T790M and C797S. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4018.