Abstract 3638: AMX-168, a long-acting, tumor protease-sensitive bispecific precursor for the treatment of solid malignancies
Authors: Sim Bee-Cheng 
Published: July 2017

Abstract
Bispecific T cell engagers (BiTE®) recruit highly cytotoxic host CD3+ T cells for destruction of tumor antigen-specific malignant cells. Bearing a defined single-chain antibody fragment (scFv) binding to CD3 on T cells and a selected scFv binding to tumor-specific antigen, BiTE-based antibodies suffer from a narrow therapeutic window and short t1/2, requiring careful dose administration and continuous infusion for efficacy. To overcome these limitations, Amunix has developed the ProTIA (Protease Triggered Immune Activators) platform by incorporating its proprietary XTEN® protein polymer half-life extension technology into the design of such T cell-dependent bispecific cytotoxic payload (BCP). An engineered protease cleavage site positioned between the BCP moiety and adjoining XTEN creates the novel, long-acting prodrug ProTIA molecule. Insertion of the unique protease site enables selective cleavage of ProTIA by tumor-associated proteases, facilitating localized release of active BCPs in tumor tissues.
Amunix’ lead ProTIA molecule, AMX-168, is in development for treatment of epithelial cancer associated ascites. AMX-168 consists of an αEpCAM scFv coupled to an αCD3 scFv that is linked to an XTEN protein sequence of defined length containing the embedded protease cleavage site. AMX-168 is shown to release the active αEpCAM-αCD3 BCP by tumor-specific proteases cleaving intact XTEN. Importantly, protease-treated AMX-168 BCP exhibits (1), high specificity for EpCAM and CD3 targets; (2), achieves low picomolar caspase activity in a panel of EpCAM+ human cancer lines in the presence of human PBMC; (3), induces expression of CD25 and CD69 T cell activation markers; and (4) release of key hallmark cytokines within 24 h when incubated with a mixture of EpCAM+ tumor cells and PBMC.
The t1/2 of αEpCAM-αCD3 ProTIA in mice is 32 h vs 3.5 h for its BCP counterpart, indicating potential for weekly dosing in humans. The in vivo efficacy of AMX-168 was evaluated in human colorectal HCT-116 tumor cells + PBMC in NOD/SCID mice. AMX-168 and its BCP-only moiety demonstrated therapeutic efficacy vs vehicle, with %TGI of 97% and 82%, respectively. AMX-168 demonstrated considerable sustained tumor regression compared to the BCP-only moiety, supporting efficient conversion of AMX-168 to the active BCP at tumor site, and improved tumor exposure of the long-acting AMX-168 compared to the BCP-only entity. Safety assessment of αEpCAM-αCD3 ProTIA for uncontrolled cytokine release in the presence of PBMC but not EpCAM+ target cells, demonstrated ProTIA induces significantly less cytokine production compared to the BCP-only moiety.
AMX-168 is currently in preclinical development with an expected IND/CTA filing by the end of 2017. Successful proof-of-concept studies of AMX-168 support further development of a broad pipeline of ProTIA therapeutics addressing a spectrum of common and advanced malignancies.
Citation Format: Bee-Cheng Sim. AMX-168, a long-acting, tumor protease-sensitive bispecific precursor for the treatment of solid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3638. doi:10.1158/1538-7445.AM2017-3638