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Mapping the Pareto optimal design space for a functionally deimmunized biotherapeutic candidate.

Salvat RS, Parker AS, Choi Y, Bailey-Kellogg C, Griswold KE - PLoS Comput. Biol. (2015)

Bottom Line: As a result, there is a growing need for improved deimmunization technologies.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.

View Article: PubMed Central - PubMed

Affiliation: Thayer School of Engineering, Dartmouth, Hanover, New Hampshire, United States of America.

ABSTRACT
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.

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Global categorical immunoreactivity for full length protein designs.The binding strength of individual peptides for MHC II alleles DRB1*0101, 0401, 0701, and 1501 were binned as strong (IC50<1 µM, red), moderate (1 µM≤IC50<10 µM, orange), weak (10 µM≤IC50<100 µM, yellow), or non-binding (IC50 ≥100 µM, not shown). The counts for each enzyme's constituent peptides were summed and plotted by semi-quantitative category (y-axis). The horizontal hatched lines are visual guides for the wild type binding counts in each category.
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pcbi-1003988-g007: Global categorical immunoreactivity for full length protein designs.The binding strength of individual peptides for MHC II alleles DRB1*0101, 0401, 0701, and 1501 were binned as strong (IC50<1 µM, red), moderate (1 µM≤IC50<10 µM, orange), weak (10 µM≤IC50<100 µM, yellow), or non-binding (IC50 ≥100 µM, not shown). The counts for each enzyme's constituent peptides were summed and plotted by semi-quantitative category (y-axis). The horizontal hatched lines are visual guides for the wild type binding counts in each category.

Mentions: To enable comparison of whole protein immunoreactivity, MHC II binding data for individual peptides was integrated across the full length of each enzyme design. For each protein, a categorical immunoreactivity score was obtained by summing the number of strong, moderate, and weak MHC binders across the protein's component peptides (11 peptides • 4 MHC alleles  =  44 possible interactions). Consistent with the predicted Sseq epitope parameter (Table 1), the design series showed a general trend of decreasing experimental immunoreactivity moving from variant 1I to 8Z (Fig. 7). Of the 18 engineered designs, 11 had a net deletion of one or more high affinity interactions, 17 deleted one or more moderate affinity interactions, and 16 deleted one or more weak interactions. Importantly, none of the engineered enzymes suffered a net increase in total experimental epitopes. In only one case was a design found to have a net addition of epitopes in any single binding category. Namely, 4P possessed one additional weak binder, but at the same time it deleted four moderate and two strong binders, the latter two being most prone to drive a T cell mediated immune response [40]. Indeed, with respect to deleting moderate and strong binders, design 4P was bested only by 8Z. The latter was the single most aggressive design, and it was in fact found to have the lowest categorical immunoreactivity. Specifically, 8Z yielded a net deletion of three strong binders, four moderate binders, and one weak binder, thereby eliminating a full quarter of all experimentally identified MHC II binders. Considering only the higher affinity peptides (IC50<10 µM), 8Z benefitted from a 39% reduction in epitope content, similar to the 47% reduction predicted by the deimmunization algorithm. Thus, prediction of epitope disruption was borne out in the overall experimental analysis.


Mapping the Pareto optimal design space for a functionally deimmunized biotherapeutic candidate.

Salvat RS, Parker AS, Choi Y, Bailey-Kellogg C, Griswold KE - PLoS Comput. Biol. (2015)

Global categorical immunoreactivity for full length protein designs.The binding strength of individual peptides for MHC II alleles DRB1*0101, 0401, 0701, and 1501 were binned as strong (IC50<1 µM, red), moderate (1 µM≤IC50<10 µM, orange), weak (10 µM≤IC50<100 µM, yellow), or non-binding (IC50 ≥100 µM, not shown). The counts for each enzyme's constituent peptides were summed and plotted by semi-quantitative category (y-axis). The horizontal hatched lines are visual guides for the wild type binding counts in each category.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4288714&req=5

pcbi-1003988-g007: Global categorical immunoreactivity for full length protein designs.The binding strength of individual peptides for MHC II alleles DRB1*0101, 0401, 0701, and 1501 were binned as strong (IC50<1 µM, red), moderate (1 µM≤IC50<10 µM, orange), weak (10 µM≤IC50<100 µM, yellow), or non-binding (IC50 ≥100 µM, not shown). The counts for each enzyme's constituent peptides were summed and plotted by semi-quantitative category (y-axis). The horizontal hatched lines are visual guides for the wild type binding counts in each category.
Mentions: To enable comparison of whole protein immunoreactivity, MHC II binding data for individual peptides was integrated across the full length of each enzyme design. For each protein, a categorical immunoreactivity score was obtained by summing the number of strong, moderate, and weak MHC binders across the protein's component peptides (11 peptides • 4 MHC alleles  =  44 possible interactions). Consistent with the predicted Sseq epitope parameter (Table 1), the design series showed a general trend of decreasing experimental immunoreactivity moving from variant 1I to 8Z (Fig. 7). Of the 18 engineered designs, 11 had a net deletion of one or more high affinity interactions, 17 deleted one or more moderate affinity interactions, and 16 deleted one or more weak interactions. Importantly, none of the engineered enzymes suffered a net increase in total experimental epitopes. In only one case was a design found to have a net addition of epitopes in any single binding category. Namely, 4P possessed one additional weak binder, but at the same time it deleted four moderate and two strong binders, the latter two being most prone to drive a T cell mediated immune response [40]. Indeed, with respect to deleting moderate and strong binders, design 4P was bested only by 8Z. The latter was the single most aggressive design, and it was in fact found to have the lowest categorical immunoreactivity. Specifically, 8Z yielded a net deletion of three strong binders, four moderate binders, and one weak binder, thereby eliminating a full quarter of all experimentally identified MHC II binders. Considering only the higher affinity peptides (IC50<10 µM), 8Z benefitted from a 39% reduction in epitope content, similar to the 47% reduction predicted by the deimmunization algorithm. Thus, prediction of epitope disruption was borne out in the overall experimental analysis.

Bottom Line: As a result, there is a growing need for improved deimmunization technologies.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.

View Article: PubMed Central - PubMed

Affiliation: Thayer School of Engineering, Dartmouth, Hanover, New Hampshire, United States of America.

ABSTRACT
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.

Show MeSH