Targeted delivery of cytotoxic drugs to tumor tissues is an effective strategy to minimize drug exposure of normal tissues and thus improve the toxicity and efficacy profiles of these agents. A tumor targeting system consists of a tumor recognition moiety linked to a cytotoxic payload. Antibody-drug conjugates represent the most advanced form of this approach. These systems are dependent on the chemical conjugation of drug molecules to the targeting moieties through various linker chemistries. The need for chemical modification and coupling steps adds significant cost and complexity to the manufacturing process. Additionally, there remain concerns that the linkers may have inappropriate stability profiles, the drugs may not be released in their active states or in quantities needed to achieve efficacy, and the conjugation process will perturb mAb binding characteristics. We thus seek a universal solution that would circumvent the need for complex chemical conjugation processes and that would be directly applicable to a wide variety of targeting modalities. We envision a protein-based domain that would bind small molecule drugs non-covalently. The binding and stability profile can be directly customized to the environment where the drug is targeted for release. Importantly, these drug-binding domains can be genetically fused to targeting domains. Microproteins, which are very small proteins with high disulfide bond densities, possess distinctive properties which make them particularly suited for this purpose. Their unique structure allows the accommodation of large degrees of both sequence and structural diversity. Their small sizes, stability and non-immunogenicity are also attractive therapeutic attributes. As an initial proof of concept, which can be immediately extended to a therapeutic product concept, we propose to develop microprotein domains that can specifically bind the commonly used cancer drug, doxorubicin, and release it in acidic or reducing environments - conditions which prevail after intracellular uptake and not within the systemic circulation. We have designed and constructed 10 phage display libraries based on different microprotein scaffold families. The total diversity in these libraries exceeds 1011 unique sequences. We plan to test the feasibility of our approach through the systematic set of specific aims below. 1) Pan phage display libraries for microprotein-displaying phages which bind to immobilized doxorubicin. 2) Confirm ability of enriched microproteins to specifically bind to immobilized doxorubicin. Our goal is to identify at least 5 different lead variants. 3) Characterize binding properties and serum stability of the selected microproteins. Completion of these Phase I milestones will enable us to obtain important proof of concept and validation of our strategy for developing microproteins as drug-binding domains for targeted delivery of cancer therapeutics. Our ultimate goal would be to advance optimized microprotein drug-binding domains, which are fused to clinically important targeting moieties with specificity for tumor antigens such as CD22, CD30, or CD74, into clinical studies. ? ? ?
