Development of next-generation biologics programmed with multifunctional synthetic chemistries

PROJECT SUMMARY Protein biologics constitute an important class of therapeutics owing to their high specificity, bioactivity and potential for tunability. Cytokine (CK) proteins applied as biotherapeutics show great promise for several disease indications, including cancer, multiple sclerosis, and hepatitis C infection. However, major barriers preventing the advancement and widespread clinical use of CKs to address critical public health needs include: (I) tendencies to cause systemic immune activation as a result of their on-target, off-tumor effects (ii) rapid proteolytic degradation and clearance by the kidneys as small proteins (<20 KDa) with short half-lives in circulation and (iii) frequent and high dosing leading to undesirable “peak-and-valley” pharmacokinetic (PK) profile which result in (iv) reduced therapeutic efficacy, dose-limiting toxicities and reduce patient compliance. New strategies are urgently needed for simultaneously targeting tumor antigens to build specificity and localization while also tuning half-life. A multi-functionalized CK with tunable properties could lead to the next generation of clinical biologics. Existing approaches to extend half-life such as the attachment of polyethylene glycol moieties (PEG), tend to elicit undesired immunogenicity and new tools are required solve this defining challenge. Pearl Bio’s proprietary synthetic biology platform technology is built on three core technologies: Genomically Recoded Organisms (GROs), engineered orthogonal translation systems (OTS), and tethered ribosomes for production of novel proteins incorporating synthetic amino acids (sAAs) including novel sAAs previously inaccessible for precise functionalization. In proposed studies, we will leverage this platform to demonstrate the tunability of CK half-life as well as tumor-targeting through precise and multi-site incorporation and functionalization of sAAs with fatty acid groups. Specifically, we will (1) Demonstrate multisite incorporation of sAAs into IL-15, (2a) Perform efficient lipidation and purification of synthetic IL-15 biologics with fatty acid for tuning half-life, (2b) Enable tumor-targeting functionalization using engineered antibody/nanobody (multiple instances if desired) and (3) Measure immunogenicity, bioactivity and albumin/tumor-antigen binding of these IL-15 variants in vitro. In summary, during Phase 1 we will develop multi-site fatty acid functionalization of cytokines and perform in vitro testing for bioactivity and immunogenicity. In Phase 2 studies, albumin and tumor targeting capabilities would be combined to create a multi-functionalized CK with tunable half-life and tumor specificity. Phase 2 in vivo studies will be used to test half-life tuning, tumor homing and efficacy of the multi- functionalized CKs for further therapeutic development. We envision that this project could establish the basis for entirely novel and programmable protein biologics with utility across a broad range of disease indications.