LAUSR

Purpose: A key challenge in cancer drug development is optimizing the therapeutic index (TI), a measure of a treatment’s antitumor efficacy and its toxicity, such that the agent is effective in targeting the tumor with an acceptable safety profile. Despite the growing list of emerging treatment modalities, successful translation of a drug candidate into an effective therapeutic agent may be hampered by inability to dose the therapy within the TI. We have developed a novel platform for protein and/or antibody-based therapies by combining protein engineering with biopolymer physics to enhance antitumor mechanisms at lower treatment doses. Our long-term goal is to improve the TI of multiple therapeutic classes. Methods: A drug-engineering platform was developed based on multivalent protein (MVP) conjugates, wherein multiple copies (i.e., valency) of a therapeutic protein, such as monoclonal single-domain antibodies or interleukins, were covalently bound to soluble biopolymers, including heparin and modified celluloses (15 kDa-1.5 MDa). MVP valencies ranging from 2 to 200 protein copies (±10%) per polymer were generated, and the reproducibility of this process was verified by UV analysis and chromatography. The binding affinity of the MVPs to their targets was determined using biolayer interferometry and cell bioassays, and the hydrodynamic radius of the MVPs were measured using dynamic light scattering. Using longitudinal in vitro and in vivo fluorescence measurements of drug signal, the intratumoral (IT) retention, cellular internalization, and systemic distribution of each MVP was compared to that of equimolar unconjugated controls of the corresponding protein. Results: At high valency, the target binding affinity of MVPs was substantially greater than that of the unconjugated protein controls. MVPs with improved cell-receptor engagement could modulate the internalization/processing of antibody-drug conjugates. Multivalent conjugation also increased the hydrodynamic radius of the MVPs to >10-times larger than corresponding unconjugated proteins. This resulted in a high concentration of MVP therapeutics in solid tumors, as demonstrated by an extension of their IT half-lives by >5 times versus unconjugated controls in mouse models of multiple tumor types. Conclusions: An MVP platform can be applied to a wide range of therapeutic mechanisms. Our data demonstrate that MVP therapeutics can be localized within the tumor and potentially enable more potent tumor cell-killing responses compared with equimolar unconjugated protein controls. Thus, the MVP platform represents a compelling new tool for designing protein-based therapies. Further development of our MVP pipeline is continuing, with the aim of identifying a candidate for IND-enabling studies. Citation Format: Amy A. Twite, Adam Barnebey, Livia W. Brier, Brian Barnett, Wesley M. Jackson. A novel platform for protein engineering and modification to expand the therapeutic index of anti-tumor therapeutics [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 1525.