Second harmonic generation (SHG) is an emergent biophysical method that sensitively measures real-time conformational change of biomolecules in the presence of biological ligands and small molecules. This study describes the successful implementation of SHG as a primary screening platform to identify fragment ligands to oncogenic Kirsten rat sarcoma (KRas). KRas is the most frequently mutated driver of pancreatic, colon, and lung cancers; however, there are few well-characterized small molecule ligands due to a lack of deep binding pockets. Using SHG, we identified a fragment binder to KRasG12D and used 1H 15N transverse relaxation optimized spectroscopy (TROSY) heteronuclear single-quantum coherence (HSQC) NMR to characterize its binding site as a pocket adjacent to the switch 2 region. The unique sensitivity of SHG furthered our study by revealing distinct conformations induced by our hit fragment compared with 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI), a Ras ligand previously described to bind the same pocket. This study highlights SHG as a high-throughput screening platform that reveals structural insights in addition to ligand binding.

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Background: TCEs are effective in leukemias, but have been limited in solid tumors due to on-target, off-tumor toxicity. Attempts to circumvent cytokine release syndrome (CRS), including step-up dosing and complex designs, have had limited success due to toxicity and immunogenicity. AMX-818, HER2-XPAT, or XTENylated Protease-Activated T Cell Engager, is a prodrug TCE that exploits dysregulated protease activity present in tumors to expand the therapeutic index (TI). The core of the molecule (PAT) consists of 2 tandem scFvs targeting CD3 and HER2. Attached to the core are two unstructured polypeptides (XTEN) that function as universal masks, sterically reduce target engagement and extend T1/2. Protease cleavage sites at the base of the XTEN masks enable proteolytic activation of XPATs in the tumor microenvironment, releasing a potent TCE with short T1/2. Methods: Nonclinical studies were conducted with HER2-XPAT or AMX-818, unmasked HER2-PAT, HER2-NoClvSite (no protease linkers) or the fluorescent labeled counterparts in vitro and in vivo xenografts and NHP. Results: The unmasked HER2-PAT demonstrated potent in vitro T cell cytotoxicity (EC50 1-2pM), target-dependent T cell activation and cytokine production by hPBMCs (human mononuclear cells). HER2-XPAT demonstrated 14,000-fold protection against killing of HER2 tumor cells, and minimal cytotoxicity against cardiomyocytes up to 1uM. In vivo, HER2-XPAT or AMX-818 induced complete tumor regressions (CRs) in HER2+ BT-474 tumors (similar to effect with equimolar doses of HER2-PAT), whereas HER2-NoClvSite lacked efficacy, supporting the need for protease cleavage for activity. HER2-XPAT was also highly efficacious in the HER2 low-expressing HT-55 colorectal model. Labeled XPATs with the same protease cleavage sites in 9 different in vivo xenograft and PDX models demonstrated preferential unmasking into cleaved products in tumors in contrast to healthy organs, with average ~20% of drug in tumor cleaved to the fully active TCE form. In NHPs, AMX-818 caused early T-cell margination as low as 2mg/kg, but has been dose-escalated safely up to 42mg/kg (MTD) with no CRS. Doses of 0.5 to 42 mg/kg demonstrated dose proportional increases in HER2-XPAT exposure, with low to negligible levels of cleavage products present in systemic circulation. Continuous infusion of unmasked HER2-PAT induced lethal CRS and cytokine spikes at 0.3mg/kg/d but was tolerated at 0.2 mg/kg/d, providing AMX-818 with ~450-fold improvement in safety compared to HER2-PAT. Repeat-dose GLP toxicology studies have demonstrated that the highest dose evaluated (6 mg/kg) was the NOAEL, suggesting that HER2-XPAT has a wide TI based on predicted human PK and predicted efficacious exposures. Conclusions: AMX-818 is a potent, protease-activated prodrug TCE exhibiting preferential tumor unmasking, with minimal systemic unmasking and no CRS at high doses in NHP. AMX-818 represents a promising solution to widen the TI for TCE activity in HER2-high and HER2-low expressing tumors. Citation Format: Milton To, Pete Yeung, Michael Fox, Mikhail Hammond, Fiore Cattaruzza, Ayesha Nazeer, Caitlin Koski, Lucas Liu, Sina Khorsand, Deena Rennerfeldt, Kari Morrissey, Zachary Lange, Ming Dong, Sharon Lam, Mika K. Derynck, Bryan A. Irving, Volker Schellenberger. AMX-818, a novel prodrug HER2-XPAT T-cell engager (TCE) with potent T cell activation, proteolytic cleavage and efficacy in xenograft tumors, and wide safety margins in NHP (Non Human Primate) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P193.