Optimization algorithms for functional deimmunization of therapeutic proteins

<p>Abstract</p> <p>Background</p> <p>To develop protein therapeutics from exogenous sources, it is necessary to mitigate the risks of eliciting an anti-biotherapeutic immune response. A key aspect of the response is the recognition and surface display by antigen-presenting cells of epitopes, short peptide fragments derived from the foreign protein. Thus, developing minimal-epitope variants represents a powerful approach to deimmunizing protein therapeutics. Critically, mutations selected to reduce immunogenicity must not interfere with the protein's therapeutic activity.</p> <p>Results</p> <p>This paper develops methods to improve the likelihood of simultaneously reducing the anti-biotherapeutic immune response while maintaining therapeutic activity. A dynamic programming approach identifies optimal and near-optimal sets of conservative point mutations to minimize the occurrence of predicted T-cell epitopes in a target protein. In contrast with existing methods, those described here integrate analysis of immunogenicity and stability/activity, are broadly applicable to any protein class, guarantee global optimality, and provide sufficient flexibility for users to limit the total number of mutations and target MHC alleles of interest. The input is simply the primary amino acid sequence of the therapeutic candidate, although crystal structures and protein family sequence alignments may also be input when available. The output is a scored list of sets of point mutations predicted to reduce the protein's immunogenicity while maintaining structure and function. We demonstrate the effectiveness of our approach in a number of case study applications, showing that, in general, our best variants are predicted to be better than those produced by previous deimmunization efforts in terms of either immunogenicity or stability, or both factors.</p> <p>Conclusions</p> <p>By developing global optimization algorithms leveraging well-established immunogenicity and stability prediction techniques, we provide the protein engineer with a mechanism for exploring the favorable sequence space near a targeted protein therapeutic. Our mechanism not only helps identify designs more likely to be effective, but also provides insights into the interrelated implications of design choices.</p

Optimization Algorithms for Site-directed Protein Recombination Experiment Planning

Site-directed protein recombination produces improved and novel protein variants by recombining sequence fragments from parent proteins. The resulting hybrids accumulate multiple mutations that have been evolutionarily accepted together. Subsequent screening or selection identifies hybrids with desirable characteristics. In order to increase the hit rate of good variants, this thesis develops experiment planning algorithms to optimize protein recombination experiments. First, to improve the frequency of generating novel hybrids, a metric is developed to assess the diversity among hybrids and parent proteins. Dynamic programming algorithms are then created to optimize the selection of breakpoint locations according to this metric. Second, the trade-off between diversity and stability in recombination experiment planning is studied, recognizing that diversity requires changes from parent proteins, which may also disrupt important residue interactions necessary for protein stability. Accordingly, methods based on dynamic programming are developed to provide combined optimization of diversity and stability, finding optimal breakpoints such that no other experiment plan has better performance in both aspects simultaneously. Third, in order to support protein recombination with heterogeneous structures and focus on functionally important regions, a general framework for protein fragment swapping is developed. Differentiating source and target parents, and swappable regions within them, fragment swapping enables asymmetric, selective site-directed recombination. Two applications of protein fragment swapping are studied. In order to generate hybrids inheriting functionalities from both source and target proteins by fragment swapping, a method based on integer programming selects optimal swapping fragments to maximize the predicted stability and activity of hybrids in the resulting library. In another application, human source protein fragments are swapped into therapeutic exogenous target protein to minimize the occurrence of peptides that trigger immune response. A dynamic programming method is developed to optimize fragment selection for both humanity and functionality, resulting in therapeutically active variants with decreased immunogenicity

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

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs

T-cell dependent immunogenicity of protein therapeutics: Preclinical assessment and mitigation

Protein therapeutics hold a prominent and rapidly expanding place among medicinal products. Purified blood products, recombinant cytokines, growth factors, enzyme replacement factors, monoclonal antibodies, fusion proteins, and chimeric fusion proteins are all examples of therapeutic proteins that have been developed in the past few decades and approved for use in the treatment of human disease. Despite early belief that the fully human nature of these proteins would represent a significant advantage, adverse effects associated with immune responses to some biologic therapies have become a topic of some concern. As a result, drug developers are devising strategies to assess immune responses to protein therapeutics during both the preclinical and the clinical phases of development. While there are many factors that contribute to protein immunogenicity, T cell- (thymus-) dependent (Td) responses appear to play a critical role in the development of antibody responses to biologic therapeutics. A range of methodologies to predict and measure Td immune responses to protein drugs has been developed. This review will focus on the Td contribution to immunogenicity, summarizing current approaches for the prediction and measurement of T cell-dependent immune responses to protein biologics, discussing the advantages and limitations of these technologies, and suggesting a practical approach for assessing and mitigating Td immunogenicity

PeptX: Using Genetic Algorithms to optimize peptides for MHC binding

<p>Abstract</p> <p>Background</p> <p>The binding between the major histocompatibility complex and the presented peptide is an indispensable prerequisite for the adaptive immune response. There is a plethora of different <it>in silico </it>techniques for the prediction of the peptide binding affinity to major histocompatibility complexes. Most studies screen a set of peptides for promising candidates to predict possible T cell epitopes. In this study we ask the question vice versa: Which peptides do have highest binding affinities to a given major histocompatibility complex according to certain <it>in silico </it>scoring functions?</p> <p>Results</p> <p>Since a full screening of all possible peptides is not feasible in reasonable runtime, we introduce a heuristic approach. We developed a framework for Genetic Algorithms to optimize peptides for the binding to major histocompatibility complexes. In an extensive benchmark we tested various operator combinations. We found that (1) selection operators have a strong influence on the convergence of the population while recombination operators have minor influence and (2) that five different binding prediction methods lead to five different sets of "optimal" peptides for the same major histocompatibility complex. The consensus peptides were experimentally verified as high affinity binders.</p> <p>Conclusion</p> <p>We provide a generalized framework to calculate sets of high affinity binders based on different previously published scoring functions in reasonable runtime. Furthermore we give insight into the different behaviours of operators and scoring functions of the Genetic Algorithm.</p

Immunglobulin-basierte Strategien zur Verlängerung der Halbwertszeit rekombinanter Proteine

With numerous small recombinant proteins being developed for treatment of various diseases, half-life extension strategies are becoming increasingly vital for the creation of long lasting and efficient biotherapeutics. Because of their small size, these proteins are rapidly cleared from plasma circulation and lose a lot of their potential as therapeutic. Using immunoglobulin-binding domains (IgBD), in particular domain C3 from Streptococcus protein G (SpGC3), can substantially increase the half-life by forming transient complexes with endogenous IgG molecules. This complex formation needs to occur at neutral pH to prevent rapid renal clearance, but also under acidic conditions, facilitating the salvage from lysosomal degradation via the neonatal Fc receptor (FcRn). In order to enable FcRn recycling, the major binding site of SpGC3 to the IgG-Fc part was eliminated, since it is overlapping with the FcRn binding site, generating the CH1 specific domain SpGC3Fab. Affinity maturation towards the Fab fragment resulted in domains SpGC3FabRR and SpGC3FabRR,E15V showing significantly increased terminal half-lives as scDb-IgBD fusion proteins. Since the IgBDs originate from bacterial proteins, they might provoke immunogenic reactions and the production of anti-drug antibodies. For deimmunization, possible T-cell epitopes were identified by sequence and structure-based analysis and eliminated by combination of single amino acid substitutions. This resulted in deimmunized IgBDs that were able to strongly increase the terminal half-life as scDb fusion proteins. The application of SpGC3FabRR to recombinant human erythropoietin (huEPO) resulted in a roughly 5-fold increased terminal half-life compared to the unmodified huEPO that also translated into increased hemoglobin concentration after one single intravenous injection. Furthermore, a comparative study of various Fc fusion proteins revealed that a multitude of factors is jointly responsible for the long half-life of full-length IgG molecules. Although possessing an identical huIgG1 Fc part and affinity to the moFcRn, neither an scFv-Fc nor an scDb-Fc or a single-chain version of the IgG molecule (scFv-scCLCH1-Fc) could reach the pharmacokinetic properties of the IgG molecule, not fully explainable by factors like size, isoelectric point and stability. All in all, the utilization of immunoglobulins and FcRn-mediated salvage via IgBDs and Fc fusion proteins is a powerful tool for the generation of recombinant fusion proteins with favorable pharmacokinetic properties.Angesichts der Entwicklung einer Vielzahl kleiner rekombinanter Proteine, die fu&#776;r die Behandlung verschiedenster Krankheiten eingesetzt werden, nimmt das Interesse an Strategien zur Halbwertszeitverlängerung zu, um lang zirkulierende und effiziente Biotherapeutika zu entwerfen. Aufgrund ihrer geringen Größe werden diese Proteine schnell aus dem Blutkreislauf eliminiert und verlieren einen Großteil ihrer therapeutischen Wirksamkeit. Die Verwendung von Immunglobulin-bindenden Domänen (IgBD), insbesondere Domäne C3 von Protein G aus Streptococcus (SpGC3), fu&#776;hrt zu einer deutlichen Erhöhung der Halbwertszeit rekombinanter Proteine, indem sie transiente Komplexe mit körpereigenen IgG Moleku&#776;len bildet. Diese Komplexbildung muss sowohl bei neutralem als auch saurem pH im Endosom auftreten und kann eine schnelle Ausscheidung in der Niere sowie den lysosomalen Abbau durch Recycling u&#776;ber den neonatalen Fc Rezeptor (FcRn) verhindern. Da die Bindestelle von SpGC3 und FcRn am IgG-Fc Teil u&#776;berlappen, wurde diese bei der IgBD beseitigt und die CH1 spezifische Domäne SpGC3Fab generiert, um besseres FcRn Recycling zu ermöglichen. Die Domänen SpGC3FabRR und SpGC3FabRR,E15V, die durch CH1-gerichtete Affinitätsreifungen entstanden, zeigten signifikant erhöhte terminale Halbwertszeiten als scDb-IgBD Fusionsproteine. Da die IgBDs aus bakteriellen Proteinen hervorgingen, ist das Einsetzen einer Immunantwort und die damit verbundene Produktion von Antikörpern gegen das Medikament möglich. Fu&#776;r eine Deimmunisierung wurden daher mögliche T-Zell Epitope mittels sequenz- und strukturbasierter Untersuchung identifiziert und durch eine Kombination einzelner Aminosäureaustausche beseitigt. Die so entstandenen deimmunisierten IgBDs konnten die terminale Halbwertszeit als scDb Fusionsproteine deutlich steigern. Die Fusion von SpGC3FabRR mit rekombinantem humanem Erythropoietin (huEPO) erhöhte dessen Halbwertszeit ungefähr 5-fach, verglichen mit nicht modifiziertem huEPO. Diese verbesserte Pharmakokinetik spiegelte sich auch in einer erhöhten Hämoglobin Konzentration nach einmaliger intravenöser Gabe wieder. Außerdem zeigte eine vergleichende Studie verschiedener Fc Fusionsproteine, dass eine Vielzahl von Faktoren fu&#776;r die lange Halbwertszeit von ganzen IgG Moleku&#776;len mitverantwortlich ist. Obwohl alle Fusionsproteine einen identischen huIgG1 Fc Teil mit gleicher Affinität zum FcRn besaßen, konnten weder ein scFv-Fc, noch ein scDb-Fc oder eine einzelkettige Version des IgG Moleku&#776;ls (scFv-scCLCH1-Fc) die pharmakokinetischen Eigenschaften des IgG Moleku&#776;ls erreichen. Faktoren wie Größe, isoelektischer Punkt und Stabilität sind nicht in der Lage dies hinreichend zu erklären. Zusammenfassend kann gesagt werden, dass die Verwendung von Immunglobulinen und FcRn-abhängigem Recycling durch IgBDs und Fc Fusionsproteine rekombinante Fusionsproteine mit vorteilhaften pharmakokinetischen Eigenschaften schaffen kann

Synthesis Cost-Optimal Targeted Mutant Protein Libraries

Protein variant libraries produced by site-directed mutagenesis are a useful tool utilized by protein engineers to explore variants with potentially improved properties, such as activity and stability. These libraries are commonly built by selecting residue positions and alternative beneficial mutations for each position. All possible combinations are then constructed and screened, by incorporating degenerate codons at mutation sites. These degenerate codons often encode additional unwanted amino acids or even STOP codons. Our study aims to take advantage of annealing based recombination of oligonucleotides during synthesis and utilize multiple degenerate codons per mutation site to produce targeted protein libraries devoid of unwanted variants. Toward this goal we created an algorithm to calculate the minimum number of degenerate codons necessary to specify any given amino acid set, and a dynamic programming method that uses this algorithm to optimally partition a DNA target sequence with degeneracies into overlapping oligonucleotides, such that the total cost of synthesis of the target mutant protein library is minimized. Computational experiments show that, for a modest increase in DNA synthesis costs, beneficial variant yields in produced mutant libraries are increased by orders of magnitude, an effect particularly pronounced in large combinatorial libraries

Circulating Biomarkers for Cancer Immunoprofiling

abstract: Biomarkers find a wide variety of applications in oncology from risk assessment to diagnosis and predicting and monitoring recurrence and response to therapy. Developing clinically useful biomarkers for cancer is faced with several challenges, including cancer heterogeneity and factors related to assay development and biomarker performance. Circulating biomarkers offer a rapid, cost-effective, and minimally-invasive window to disease and are ideal for population-based screening. Circulating immune biomarkers are stable, measurable, and can betray the underlying antigen when present below detection levels or even no longer present. This dissertation aims to investigate potential circulating immune biomarkers with applications in cancer detection and novel therapies. Over 600,000 cancers each year are attributed to the human papillomavirus (HPV), including cervical, anogenital and oropharyngeal cancers. A key challenge in understanding HPV immunobiology and developing immune biomarkers is the diversity of HPV types and the need for multiplexed display of HPV antigens. In Project 1, nucleic acid programmable protein arrays displaying the proteomes of 12 HPV types were developed and used for serum immunoprofiling of women with cervical lesions or invasive cervical cancer. These arrays provide a valuable high-throughput tool for measuring the breadth, specificity, heterogeneity, and cross-reactivity of the serologic response to HPV. Project 2 investigates potential biomarkers of immunity to the bacterial CRISPR/Cas9 system that is currently in clinical trials for cancer. Pre-existing B cell and T cell immune responses to Cas9 were detected in humans and Cas9 was modified to eliminate immunodominant epitopes while preserving its function and specificity. This dissertation broadens our understanding of the immunobiology of cervical cancer and provides insights into the immune profiles that could serve as biomarkers of various applications in cancer.Dissertation/ThesisDoctoral Dissertation Molecular and Cellular Biology 201