Mapping the Pareto Optimal Design Space for a Functionally Deimmunized Biotherapeutic Candidate

The immunogenicity of biotherapeutics can bottleneck development pipelines and poses a barrier to widespread clinical application. As a result, there is a growing need for improved deimmunization technologies. We have recently described algorithms that simultaneously optimize proteins for both reduced T cell epitope content and high-level function. In silico analysis of this dual objective design space reveals that there is no single global optimum with respect to protein deimmunization. Instead, mutagenic epitope deletion yields a spectrum of designs that exhibit tradeoffs between immunogenic potential and molecular function. The leading edge of this design space is the Pareto frontier, i.e. the undominated variants for which no other single design exhibits better performance in both criteria. Here, the Pareto frontier of a therapeutic enzyme has been designed, constructed, and evaluated experimentally. Various measures of protein performance were found to map a functional sequence space that correlated well with computational predictions. These results represent the first systematic and rigorous assessment of the functional penalty that must be paid for pursuing progressively more deimmunized biotherapeutic candidates. Given this capacity to rapidly assess and design for tradeoffs between protein immunogenicity and functionality, these algorithms may prove useful in augmenting, accelerating, and de-risking experimental deimmunization efforts.Author Summary: Protein therapeutics have created a revolution in disease therapy, providing improved outcomes for prevalent illnesses and conditions while at the same time yielding treatments for diseases that were previously intractable. However, this powerful class of drugs is subject to their own unique challenges and risk factors. In particular, the biological origins of therapeutic proteins predispose them towards eliciting a detrimental immune response from the patient's own body. Therefore, fully capitalizing on the medicinal reservoir of natural and engineered proteins will require efficient, effective, and broadly applicable deimmunization technologies. We have developed deimmunization algorithms that simultaneously optimize therapeutic candidates for both low immunogenicity and high-level activity and stability. Here, we combine computational modeling and experimental analysis to show that the process of protein deimmunization manifests inherent tradeoffs between immunogenic potential and biomolecular function. Our experimental results demonstrate that dual objective optimization allows us to assess and design for these tradeoffs, thereby enabling facile construction of deimmunized variants that span a broad range of immunogenicity and functionality performance parameters. Thus, we can rapidly map the design space for deimmunized drug candidates, and we can use this information to guide selection of engineered proteins that are most likely to meet performance benchmarks for a given clinical application.