From the enediyne class of antitumor antibiotics, uncialamycin is among the rarest and most potent, yet one of the structurally simpler, making it attractive for chemical synthesis and potential applications in biology and medicine. In this article we describe a streamlined and practical enantioselective total synthesis of uncialamycin that is amenable to the synthesis of novel analogues and renders the natural product readily available for biological and drug development studies. Starting from hydroxy- or methoxyisatin, the synthesis features a Noyori enantioselective reduction, a Yamaguchi acetylide-pyridinium coupling, a stereoselective acetylide-aldehyde cyclization, and a newly developed annulation reaction that allows efficient coupling of a cyanophthalide and a p-methoxy semiquinone aminal to forge the anthraquinone moiety of the molecule. Overall, the developed streamlined synthesis proceeds in 22 linear steps (14 chromatographic separations) and 11% overall yield. The developed synthetic strategies and technologies were applied to the synthesis of a series of designed uncialamycin analogues equipped with suitable functional groups for conjugation to antibodies and other delivery systems. Biological evaluation of a select number of these analogues led to the identification of compounds with low picomolar potencies against certain cancer cell lines. These compounds and others like them may serve as powerful payloads for the development of antibody drug conjugates (ADCs) intended for personalized targeted cancer therapy.

To enhance the therapeutic index of T-cell engagers (TCEs), we engineered masked, precision-activated TCEs (XPAT proteins), targeting a tumor antigen (human epidermal growth factor receptor 2 (HER2) or epidermal growth factor receptor (EGFR)) and CD3. Unstructured XTEN polypeptide masks flank the N and C termini of the TCE and are designed to be released by proteases in the tumor microenvironment. In vitro, unmasked HER2-XPAT (uTCE) demonstrates potent cytotoxicity, with XTEN polypeptide masking providing up to 4-log-fold protection. In vivo, HER2-XPAT protein induces protease-dependent antitumor activity and is proteolytically stable in healthy tissues. In non-human primates, HER2-XPAT protein demonstrates a strong safety margin (>400-fold increase in tolerated maximum concentration versus uTCE). HER2-XPAT protein cleavage is low and similar in plasma samples from healthy and diseased humans and non-human primates, supporting translatability of stability to patients. EGFR-XPAT protein confirmed the utility of XPAT technology for tumor targets more widely expressed in healthy tissues.

Background: TCEs have proven to be effective in creating durable remissions in hematologic malignancies but have been challenging in solid tumors due to on-target, off-tumor toxicity. To circumvent the toxicity, many have tried step-up, fractionated dosing or complex formats, but these have largely been unsuccessful due to toxicity and/or enhanced immunogenicity. To address this challenge, Amunix has developed a conditionally active TCE, the XPAT or XTENylated Protease-Activated T Cell Engager, that exploits the protease activity present in tumors vs. healthy tissue, enabling expansion of the therapeutic index (TI). The core of the XPAT consists of 2 tandem scFVs targeting CD3 and a tumor antigen. Two unstructured and modular polypeptide masks (XTEN) are attached to the core that sterically reduce target engagement and extend T1/2. Protease cleavage sites at the base of these XTEN polypeptide masks are then proteolytically released in the tumor microenvironment to unleash a highly potent TCE with a short T1/2, further improving the TI. Methods: HER2-XPAT and EGFR-XPATs were each purified from E.coli expressed from a single plasmid. The activity of both the prodrugs (XPATs) and their protease-activated counterparts (PATs) were characterized for cytotoxicity in vitro and in huPBMC-transduced tumor-bearing mice. Pilot toxicity studies were conducted in NHPs (cyno). Results: Both protease-activated (PATs) forms of EGFR-XPAT and HER2-XPAT demonstrated potent in vitro T cell directed cytotoxicity against tumor cells (EC50s 1-2pM), while the masked XPAT provided 3,000-14,000-fold protection against target cell killing. Intact EGFR-XPAT demonstrated dose-dependent complete tumor responses (CRs) in HT-29 CRC xenografts, with HER2-XPAT demonstrating similar efficacy and CRs in both BT-474 and SK-OV-3 tumors. In cynos, 1 mg/kg of EGFR-XPAT was the MTD and 1.5 mg/kg exceeded the MTD, while the activated PAT was lethal at 0.132mg/kg/day by continuous infusion (with single dose 1 hour bolus of 0.066ug/kg). This indicated a near 2 log protection or more in predicted Cmax of XPAT vs PAT. A HER2-XPAT has been dose escalated up to 21 mg/kg in cynos and was well-tolerated, with the MTD still unreached. At doses starting from 2.5mg/kg of a HER2-XPAT, lymphocyte margination and laboratory abnormalities were observed, indicating evidence of biologic activity well below the tolerated 21 mg/kg dose. An estimated T1/2 of ~4 days was observed. Conclusions: EGFR-XPAT and HER2-XPAT are novel T cell engagers with protease-cleavable XTEN masks with preclinical evidence of potent anti-tumor activity and a wide margin of protection in cynos. With the prior XTEN clinical data of low immunogenicity, the XPAT TCEs provide a promising solution to overcome On-target, Off-tumor toxicity. Additional PD, safety and efficacy data will be presented. Citation Format: Fiore Cattaruzza, Ayesha Nazeer, Zachary Lange, Mikhail Hammond, Caitlin Koski, Angela Henkensiefken, Mika K. Derynck, Bryan Irving, Volker Schellenberger. HER2-XPAT and EGFR-XPAT: Pro-drug T-cell engagers (TCEs) engineered to address on-target, off-tumor toxicity with potent efficacy in vitro and in vivo and large safety margins in NHP [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3376.