Activating mutations of epidermal growth factor receptor (EGFR) are associated with the high sensitivity of non-small cell lung cancer (NSCLC) patients to EGFR tyrosine kinase inhibitors (TKIs) like erlotinib. However, acquired resistance limits the clinical efficacy of EGFR-TKIs, which is the most commonly caused by T790M mutation in EGFR. Second generation EGFR-TKIs such as afatinib are able to inhibit T790M mutation but the clinical efficacy in T790M positive patients is limited due to severe side effects associated with wild type (WT) EGFR inhibition. ASP8273 is a mutant-selective irreversible EGFR inhibitor currently in clinical trials (ClinicalTrials.gov Identifier: NCT02113813, NCT02192697). We have previously reported that ASP8273 covalently binds to mutant EGFR via C797 and inhibits kinase activity of mutant EGFR, which results in anti-tumor activity in xenograft models. To further explore the selectivity and the activity of ASP8273 on mutant EGFR, we evaluated effects of ASP8273 and other EGFR-TKIs on EGFR signal pathway, cell growth and anti-tumor activity. Phosphorylation of EGFR, ERK and Akt was determined by Western blot after treatment with EGFR-TKIs at 10, 100 and 1000nM. Apoptosis induction was analyzed by detecting caspase activity after 24h treatment with EGFR-TKIs. Anti-tumor activity of ASP8273 was evaluated in mice xenografted with HCC827 (deletion in exon 19 [del ex19]), NCI-H1975 (T790M/L858R), A431 (WT), and a patient derived LU1868 (T790M/L858R). ASP8273 selectively inhibited phosphorylation of EGFR and its down-stream signal pathway, ERK and Akt from 10nM in HCC827 and NCI-H1975 while inhibitory effects were only detected at 1000nM in A431.In NCI-H1650 (del ex19), ASP8273 inhibited cell growth with an IC50 value of 70nM while other EGFR-TKIs were only partially effective. ERK and Akt phosphorylation were diminished after ASP8273 treatment at 1000nM, however, other EGFR-TKIs only partially reduced the phosphorylation levels of these proteins. ASP8273 potently enhanced the caspase activity in NCI-H1650 after 24h treatment, which is concordant with signal and cell growth inhibitory effect. In HCC827 and NCI-H1975 xenograft models, ASP8273 induced tumor regression at 10, 30 and 100mg/kg without affecting body weight. ASP8273 also produced tumor growth inhibition from 10mg/kg in the NSCLC patient derived tumor xenograft, LU1868 which express T790M/L858R. On the other hand, ASP8273 did not produce significant tumor growth inhibition at 10 and 30mg/kg in A431 xenograft model. ASP8273 selectively inhibited mutant EGFR compared to WT EGFR in preclinical models, showing activity in mutant EGFR cell line which is resistant to other EGFR-TKIs including AZD9291 and CO-1686. These results indicate the potential of ASP8273 to induce tumor shrinkage in patients with mutant EGFR positive tumors including those that do not respond to other EGFR-TKIs despite EGFR mutation. Citation Format: Satoshi Konagai, Hideki Sakagami, Hiroko Yamamoto, Hiroaki Tanaka, Takahiro Matsuya, Shinya Mimasu, Yusuke Tomimoto, Masamichi Mori, Hiroyuki Koshio, Masaaki Hirano, Sadao Kuromitsu, Masahiro Takeuchi. ASP8273 selectively inhibits mutant EGFR signal pathway and induces tumor shrinkage in EGFR mutated tumor models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2586. doi:10.1158/1538-7445.AM2015-2586
The implementation of efficient technologies for the production of recombinant mammalian membrane receptors is an outstanding challenge in understanding receptor-ligand actions and the development of therapeutic antibodies. In order to improve the solubility of recombinant extracellular domains of human membrane receptors expressed in Escherichia coli, proteins were synthesized by an E. coli in vitro translation system supplemented with bacterial molecular chaperones, such as GroEL-GroES (GroEL/ES), Trigger factor (TF), a DnaK-DnaJ-GrpE chaperone system (DnaKJE), and/or a heat shock protein Hsp100, ClpB. The following three proteins that are prone to aggregation were examined: the extracellular domain (ECD) or the second immunoglobulin-like domain (IgII) of the human neurotrophin receptor TrkC (TrkC-ECD and TrkC-IgII), and the C-type lectin carbohydrate recognition domain of the human asialoglycoprotein receptor (ASGPR HI CRD). The cooperative chaperone system including GroEL/ES, DnaKJE and ClpB had a marked effect on the solubility of TrkC-ECD and TrkC-IgII, and the GroEL/ES-DnaKJE-TF chaperone system was more effective for TrkC-IgII. The GroEL/ES-DnaKJE-TF chaperone network increased the yield of soluble ASGPR HI CRD. The present findings demonstrate that E. coli molecular chaperones are useful in improving the yield of soluble recombinant extracellular domains of human membrane receptors in an E. coli expression system.
VHH, the antigen-binding fragment of a heavy chain-only antibody, is a useful component of antibody-based therapeutics. Thermal stability, represented by the melting temperature (Tm), is one of the key factors affecting the developability of antibody-based therapeutics. In this study, we examined whether the in silico free energy score dStability can be used to design mutants with improved Tm compared to the anti-lysozyme VHH, D3-L11. After verifying that exhaustive mutagenesis was inefficient for improving Tm, we performed a two-round rational approach that combined dStability calculations with a small number of experiments. This method improved the Tm by more than 5 °C in several single mutants including A79I. It reduced the affinity for the antigen by less than 1.6-fold. We speculate that stabilization of A79I required exquisite compatibility among neighboring residues to fill in the internal cavity in the protein. Given that we identified only one mutation that could simultaneously improve Tm and almost maintain affinity, we concluded that achieving both is extremely difficult, even with single mutations that are not located in the paratope. Therefore, we recommend using a variety of approaches when trying to achieve such a feat. Our method will be a useful complementary approach to other existing methods.
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