Positive charge in the complementarity-determining regions of synthetic nanobody prevents aggregation.

In the past, specificity and affinity were the priority for synthetic antibody library. However, therapeutic antibodies need good stability for medical use. Through carefully adjust the chemical diversity in CDRs, one hopes to design a synthetic antibody library with good developability. Here we thoroughly analyzed 296 nanobody sequences and structures, constructed a fully-functional synthetic nanobody library, evaluated the relationship between aggregation and isoelectric point, and found that high-pI nanobodies were more resistant to aggregation than low-pIs. As we used the same framework for constructing the library, CDRs charge played a crucial role in mediating nanobody aggregation. We also analyzed the theoretical pI of 296 nanobodies from PDB, about 75% had basic pI, only 25% were acidic. Those results provided useful guidelines for designing next-generation synthetic nanobody libraries and for identifying potent and safe nanobody therapeutics.

An adapted consensus protein design strategy for identifying globally optimal biotherapeutics.

Biotherapeutic optimization, whether to improve general properties or to engineer specific attributes, is a time-consuming process with uncertain outcomes. Conversely, Consensus Protein Design has been shown to be a viable approach to enhance protein stability while retaining function. In adapting this method for a more limited number of protein sequences, we studied 21 consensus single-point variants from eight publicly available CD3 binding sequences with high similarity but diverse biophysical and pharmacological properties. All single-point consensus variants retained CD3 binding and performed similarly in cell-based functional assays. Using Ridge regression analysis, we identified the variants and sequence positions with overall beneficial effects on developability attributes of the CD3 binders. A second round of sequence generation that combined these substitutions into a single molecule yielded a unique CD3 binder with globally optimized developability attributes. In this first application to therapeutic antibodies, adapted Consensus Protein Design was found to be highly beneficial within lead optimization, conserving resources and minimizing iterations. Future implementations of this general strategy may help accelerate drug discovery and improve success rates in bringing novel biotherapeutics to market.

Thermostability enhancement of l-glutamate oxidase from Streptomyces sp. NT1 by full consensus protein design.

Thermostable l-glutamate oxidases (LGOXs) are desirable for use in l-glutamate (L-Glu) assay kits, enzymatic synthesis of α-ketoglutarate and for biosensor development. However, protein engineering efforts to improve thermostability often lead to a decrease in enzymatic activity. In this report, we aimed to enhance the thermostability (melting temperature, Tm) of a mesophilic LGOX from Streptomyces sp. NT1 (LGOXNT1) without a reduction in activity by a sequence-based protein design approach, termed full consensus (Fc) protein design. Among the 690 amino acids of LGOXNT1, 104 amino acids were substituted by the Fc protein design. The mutant gene was artificially synthesized and expressed in Escherichia coli BL21(DE3) cells. The Tm of the purified, recombinant LGOX mutant (FcLGOX) was determined to be ∼72 °C, which is an increase on the Tm of 65 °C for LGOXNT1 and the highest among known LGOXs. Importantly, purified FcLGOX showed no loss of specific activity or substrate specificity after a 30-min incubation at 70 °C. Our findings provide a new approach to improve the thermostability of enzymes.