Correction: Optimizing antibody affinity and stability by the automated design of the variable light-heavy chain interfaces.

[This corrects the article DOI: 10.1371/journal.pcbi.1007207.].

Design of a basigin-mimicking inhibitor targeting the malaria invasion protein RH5.

Many human pathogens use host cell-surface receptors to attach and invade cells. Often, the host-pathogen interaction affinity is low, presenting opportunities to block invasion using a soluble, high-affinity mimic of the host protein. The Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) provides an exciting candidate for mimicry: it is highly conserved and its moderate affinity binding to the human receptor basigin (KD ≥1 µM) is an essential step in erythrocyte invasion by this malaria parasite. We used deep mutational scanning of a soluble fragment of human basigin to systematically characterize point mutations that enhance basigin affinity for RH5 and then used Rosetta to design a variant within the sequence space of affinity-enhancing mutations. The resulting seven-mutation design exhibited 1900-fold higher affinity (KD approximately 1 nM) for RH5 with a very slow binding off rate (0.23 h-1 ) and reduced the effective Plasmodium growth-inhibitory concentration by at least 10-fold compared to human basigin. The design provides a favorable starting point for engineering on-rate improvements that are likely to be essential to reach therapeutically effective growth inhibition.

Optimizing antibody affinity and stability by the automated design of the variable light-heavy chain interfaces.

Antibodies developed for research and clinical applications may exhibit suboptimal stability, expressibility, or affinity. Existing optimization strategies focus on surface mutations, whereas natural affinity maturation also introduces mutations in the antibody core, simultaneously improving stability and affinity. To systematically map the mutational tolerance of an antibody variable fragment (Fv), we performed yeast display and applied deep mutational scanning to an anti-lysozyme antibody and found that many of the affinity-enhancing mutations clustered at the variable light-heavy chain interface, within the antibody core. Rosetta design combined enhancing mutations, yielding a variant with tenfold higher affinity and substantially improved stability. To make this approach broadly accessible, we developed AbLIFT, an automated web server that designs multipoint core mutations to improve contacts between specific Fv light and heavy chains (http://AbLIFT.weizmann.ac.il). We applied AbLIFT to two unrelated antibodies targeting the human antigens VEGF and QSOX1. Strikingly, the designs improved stability, affinity, and expression yields. The results provide proof-of-principle for bypassing laborious cycles of antibody engineering through automated computational affinity and stability design.

CRISPR-Cas9 engineering of the RAG2 locus via complete coding sequence replacement for therapeutic applications.

RAG2-SCID is a primary immunodeficiency caused by mutations in Recombination-activating gene 2 (RAG2), a gene intimately involved in the process of lymphocyte maturation and function. ex-vivo manipulation of a patient's own hematopoietic stem and progenitor cells (HSPCs) using CRISPR-Cas9/rAAV6 gene editing could provide a therapeutic alternative to the only current treatment, allogeneic hematopoietic stem cell transplantation (HSCT). Here we show an innovative RAG2 correction strategy that replaces the entire endogenous coding sequence (CDS) for the purpose of preserving the critical endogenous spatiotemporal gene regulation and locus architecture. Expression of the corrective transgene leads to successful development into CD3+TCRαß+ and CD3+TCRγδ+ T cells and promotes the establishment of highly diverse TRB and TRG repertoires in an in-vitro T-cell differentiation platform. Thus, our proof-of-concept study holds promise for safer gene therapy techniques of tightly regulated genes.

Multiplex HDR for disease and correction modeling of SCID by CRISPR genome editing in human HSPCs.

Severe combined immunodeficiency (SCID) is a group of disorders caused by mutations in genes involved in the process of lymphocyte maturation and function. CRISPR-Cas9 gene editing of the patient's own hematopoietic stem and progenitor cells (HSPCs) ex vivo could provide a therapeutic alternative to allogeneic hematopoietic stem cell transplantation, the current gold standard for treatment of SCID. To eliminate the need for scarce patient samples, we engineered genotypes in healthy donor (HD)-derived CD34+ HSPCs using CRISPR-Cas9/rAAV6 gene-editing, to model both SCID and the therapeutic outcomes of gene-editing therapies for SCID via multiplexed homology-directed repair (HDR). First, we developed a SCID disease model via biallelic knockout of genes critical to the development of lymphocytes; and second, we established a knockin/knockout strategy to develop a proof-of-concept single-allelic gene correction. Based on these results, we performed gene correction of RAG2-SCID patient-derived CD34+ HSPCs that successfully developed into CD3+ T cells with diverse TCR repertoires in an in vitro T cell differentiation platform. In summary, we present a strategy to determine the optimal configuration for CRISPR-Cas9 gene correction of SCID using HD-derived CD34+ HSPCs, and the feasibility of translating this gene correction approach in patient-derived CD34+ HSPCs.

Ultrahigh specificity in a network of computationally designed protein-interaction pairs.

Protein networks in all organisms comprise homologous interacting pairs. In these networks, some proteins are specific, interacting with one or a few binding partners, whereas others are multispecific and bind a range of targets. We describe an algorithm that starts from an interacting pair and designs dozens of new pairs with diverse backbone conformations at the binding site as well as new binding orientations and sequences. Applied to a high-affinity bacterial pair, the algorithm results in 18 new ones, with cognate affinities from pico- to micromolar. Three pairs exhibit 3-5 orders of magnitude switch in specificity relative to the wild type, whereas others are multispecific, collectively forming a protein-interaction network. Crystallographic analysis confirms design accuracy, including in new backbones and polar interactions. Preorganized polar interaction networks are responsible for high specificity, thus defining design principles that can be applied to program synthetic cellular interaction networks of desired affinity and specificity.