SBIR-STTR Award

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

Treatment of metastatic tumors is a major health challenge. Recently, antibody-based therapies have been developed that are more specific and have fewer side effects compared with conventional chemotherapy. However, the potency of most antibody therapeutics is limited by their inadequate ability to kill tumor cells. Consequently, there is an urgent, unmet need to develop therapeutics that combine the specificity of antibodies for tumor tissues with a potent cytotoxic function. The development of tumor-targeted toxins has yielded promising results and led to one approved product, Ontak (Denileukin). Most molecules however are immunogenic and aggregation prone, have limited stability and require complex manufacturing routes. To achieve clinical and commercial success it is critical for candidates to meet following criteria: 1) high potency; 2) low systemic toxicity; 3) low immunogenicity; 4) high protein stability, lack of aggregation; 5) robust manufacturing. This proposal aims to develop tumor-targeted toxins by combining three elements that confer significant advantages over current approaches: microproteins for tumor binding/internalization; RNAse for cell killing; rPEG to optimize PK properties. In the successful Phase I of this project, we developed tumor-specific microproteins with the following properties: 1) efficient production in E. coli; 2) efficient phage display that enables rapid specificity optimization; 3) effective internalization of toxic payloads; 4) excellent serum stability. In a separate phase I SBIR project, we developed rPEGs, hydrophilic protein sequences that mimic the properties of chemical polyethylene glycol (PEG) but can be directly fused to other proteins. rPEGs optimize the pharmacokinetics of a product, reduce product immunogenicity, and greatly reduce protein aggregation. Our Phase II goal is to optimize the specificity of our lead microproteins to achieve a >1000x ration of tumor/normal affinity. Subsequently, we will fuse these optimized microproteins to RNAse as toxic payload and rPEG to optimize PK, PD and protein manufacturing. The resulting fusion proteins will be thoroughly evaluated for in vitro and in vivo performance. In addition, we will develop an effective manufacturing process that can be transferred with minor modifications to a GMP manufacturer. We aim to generate two lead molecules that will be ready to enter preclinical followed by clinical development. In addition we will generate microprotein-rPEG fusions with defined conjugation sites that will be uniquely suitable for the chemical conjugation of toxic payloads.

Public Health Relevance:
The development of tumor-targeted toxins have yielded promising results and led to one approved product, Denileukin. However, existing molecules have significant limitations especially immunogenicity and complex manufacturing requirements. This project will use tumor-specific microproteins to address these limitations and develop targeted toxins with the following characteristics: 1) high potency; 2) low systemic toxicity; 3) low immunogenicity to allow repeat dosing; 4) good protein stability; 5) lack of aggregation; 6) robust manufacturing process.

Public Health Relevance:
- 1 - Project Narrative The development of tumor-targeted toxins have yielded promising results and led to one approved product, Denileukin. However, existing molecules have significant limitations especially immunogenicity and complex manufacturing requirements. This project will use tumor-specific microproteins to address these limitations and develop targeted toxins with the following characteristics: 1) high potency; 2) low systemic toxicity; 3) low immunogenicity to allow repeat dosing; 4) good protein stability; 5) lack of aggregation; 6) robust manufacturing process.

Thesaurus Terms:
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