Combining deep mutational scanning to heatmap of HLA class II binding of immunogenic sequences to preserve functionality and mitigate predicted immunogenicity.

Removal of CD4 T cell epitopes from therapeutic antibody sequences is expected to mitigate their potential immunogenicity, but its application is complicated by the location of their T cell epitopes, which mainly overlap with complementarity-determining regions. We therefore evaluated the flexibility of antibody sequences to reduce the predicted affinity of corresponding peptides for HLA II molecules and to maintain antibody binding to its target in order to guide antibody engineering for mitigation of predicted immunogenicity. Permissive substitutions to reduce affinity of peptides for HLA II molecules were identified by establishing a heatmap of HLA class II binding using T-cell epitope prediction tools, while permissive substitutions preserving binding to the target were identified by means of deep mutational scanning and yeast surface display. Combinatorial libraries were then designed to identify active clones. Applied to adalimumab, an anti-TNFα human antibody, this approach identified 200 mutants with a lower HLA binding score than adalimumab. Three mutants were produced as full-length antibodies and showed a higher affinity for TNFα and neutralization ability than adalimumab. This study also sheds light on the permissiveness of antibody sequences with regard to functionality and predicted T cell epitope content.

Combining deep mutational scanning to heatmap of HLA class II binding of immunogenic sequences to preserve functionality and mitigate predicted immunogenicity

Removal of CD4 T cell epitopes from therapeutic antibody sequences is expected to mitigate their potential immunogenicity, but its application is complicated by the location of their T cell epitopes, which mainly overlap with complementarity-determining regions. We therefore evaluated the flexibility of antibody sequences to reduce the predicted affinity of corresponding peptides for HLA II molecules and to maintain antibody binding to its target in order to guide antibody engineering for mitigation of predicted immunogenicity. Permissive substitutions to reduce affinity of peptides for HLA II molecules were identified by establishing a heatmap of HLA class II binding using T-cell epitope prediction tools, while permissive substitutions preserving binding to the target were identified by means of deep mutational scanning and yeast surface display. Combinatorial libraries were then designed to identify active clones. Applied to adalimumab, an anti-TNFα human antibody, this approach identified 200 mutants with a lower HLA binding score than adalimumab. Three mutants were produced as full-length antibodies and showed a higher affinity for TNFα and neutralization ability than adalimumab. This study also sheds light on the permissiveness of antibody sequences with regard to functionality and predicted T cell epitope content.

Deep mutational engineering of broadly-neutralizing nanobodies accommodating SARS-CoV-1 and 2 antigenic drift.

Here, we report the molecular engineering of nanobodies that bind with picomolar affinity to both SARS-CoV-1 and SARS-CoV-2 receptor-binding domains (RBD) and are highly neutralizing. We applied deep mutational engineering to VHH72, a nanobody initially specific for SARS-CoV-1 RBD with little cross-reactivity to SARS-CoV-2 antigen. We first identified all the individual VHH substitutions that increase binding to SARS-CoV-2 RBD and then screened highly focused combinatorial libraries to isolate engineered nanobodies with improved properties. The corresponding VHH-Fc molecules show high affinities for SARS-CoV-2 antigens from various emerging variants and SARS-CoV-1, block the interaction between ACE2 and RBD, and neutralize the virus with high efficiency. Its rare specificity across sarbecovirus relies on its peculiar epitope outside the immunodominant regions. The engineered nanobodies share a common motif of three amino acids, which contribute to the broad specificity of recognition. Our results show that deep mutational engineering is a very powerful method, especially to rapidly adapt existing antibodies to new variants of pathogens.

An antibody targeting type III secretion system induces broad protection against Salmonella and Shigella infections.

Salmonella and Shigella bacteria are food- and waterborne pathogens that are responsible for enteric infections in humans and are still the major cause of morbidity and mortality in the emerging countries. The existence of multiple Salmonella and Shigella serotypes as well as the emergence of strains resistant to antibiotics requires the development of broadly protective therapies. Recently, the needle tip proteins of the type III secretion system of these bacteria were successfully utilized (SipD for Salmonella and IpaD for Shigella) as vaccine immunogens to provide good prophylactic cross-protection in murine models of infections. From these experiments, we have isolated a cross-protective monoclonal antibody directed against a conserved region of both proteins. Its conformational epitope determined by Deep Mutational Scanning is conserved among needle tip proteins of all pathogenic Shigella species and Salmonella serovars, and are well recognized by this antibody. Our study provides the first in vivo experimental evidence of the importance of this common region in the mechanism of virulence of Salmonella and Shigella and opens the way to the development of cross-protective therapeutic agents.

Functional dissection of the retrograde Shiga toxin trafficking inhibitor Retro-2.

The retrograde transport inhibitor Retro-2 has a protective effect on cells and in mice against Shiga-like toxins and ricin. Retro-2 causes toxin accumulation in early endosomes and relocalization of the Golgi SNARE protein syntaxin-5 to the endoplasmic reticulum. The molecular mechanisms by which this is achieved remain unknown. Here, we show that Retro-2 targets the endoplasmic reticulum exit site component Sec16A, affecting anterograde transport of syntaxin-5 from the endoplasmic reticulum to the Golgi. The formation of canonical SNARE complexes involving syntaxin-5 is not affected in Retro-2-treated cells. By contrast, the interaction of syntaxin-5 with a newly discovered binding partner, the retrograde trafficking chaperone GPP130, is abolished, and we show that GPP130 must indeed bind to syntaxin-5 to drive Shiga toxin transport from the endosomes to the Golgi. We therefore identify Sec16A as a druggable target and provide evidence for a non-SNARE function for syntaxin-5 in interaction with GPP130.

Fab is the most efficient format to express functional antibodies by yeast surface display.

Multiple formats are available for engineering of monoclonal antibodies (mAbs) by yeast surface display, but they do not all lead to efficient expression of functional molecules. We therefore expressed four anti-tumor necrosis factor and two anti-IpaD mAbs as single-chain variable fragment (scFv), antigen-binding fragment (Fab) or single-chain Fabs and compared their expression levels and antigen-binding efficiency. Although the scFv and scFab formats are widely used in the literature, 2 of 6 antibodies were either not or weakly expressed. In contrast, all 6 antibodies expressed as Fab revealed strong binding and high affinity, comparable to that of the soluble form. We also demonstrated that the variations in expression did not affect Fab functionality and were due to variations in light chain display and not to misfolded dimers. Our results suggest that Fab is the most versatile format for the engineering of mAbs.