The receptor binding domain (RBD) of the SARS‐CoV‐2 spike (S)‐protein is a prime target of virus‐neutralizing antibodies present in convalescent sera of COVID‐19 patients and thus is considered a key antigen for immunosurveillance studies and vaccine development. Although recombinant expression of RBD has been achieved in several eukaryotic systems, mammalian cells have proven particularly useful. The authors aimed to optimize RBD produced in HEK293‐6E cells towards a stable homogeneous preparation and addressed its O‐glycosylation as well as the unpaired cysteine residue 538 in the widely used RBD (319‐541) sequence. The authors found that an intact O‐glycosylation site at T323 is highly relevant for the expression and maintenance of RBD as a monomer. Furthermore, it was shown that deletion or substitution of the unpaired cysteine residue C538 reduces the intrinsic propensity of RBD to form oligomeric aggregates, concomitant with an increased yield of the monomeric form of the protein. Bead‐based and enzyme‐linked immunosorbent assays utilizing these optimized RBD variants displayed excellent performance with respect to the specific detection of even low levels of SARS‐CoV‐2 antibodies in convalescent sera. Hence, these RBD variants could be instrumental for the further development of serological SARS‐CoV‐2 tests and inform the design of RBD‐based vaccine candidates.
Tetraspan proteins are significantly enriched in the membranes of exosomal vesicles (EVs) and their extracellular domains are attractive targets for engineering towards specific antigen recognition units. To enhance the tolerance of a tetraspanin fold to modification, we achieved significant thermal stabilization of the human CD81 large extracellular loop (hCD81 LEL) via de novo disulfide bonds. The best mutants were shown to exhibit a positive shift in the melting temperature (Tm) of up to 25 °C. The combination of two most potent disulfide bonds connecting different strands of the protein resulted in a mutant with a Tm of 109 °C, 43 °C over the Tm of the wild-type hCD81 LEL. A peptide sequence binding to the human transferrin receptor (hTfr) was engrafted into the D-segment of the hCD81 LEL, resulting in a mutant that still exhibited a compact fold. Grafting of the same peptide sequence between helices A and B resulted in a molecule with an aberrant profile in size exclusion chromatography (SEC), which could be improved by a de novo cysteine bond connecting both helices. Both peptide-grafted proteins showed an enhanced internalization into the cell line SK-BR3, which strongly overexpresses hTfr. In summary, the tetraspan LEL fold could be stabilized to enhance its amenability for engineering into a more versatile protein scaffold.
We have designed a complete antibody-like construct where the CH1 and Cκ domains are exchanged for a pair of the CH3 domains and efficient pairing of the heavy and light variable domain is achieved using “Knobs-into-Holes” strategy. This construct, composed of only naturally occurring immunoglobulin sequences without artificial linkers, expressed at a high level in mammalian cells, however exhibited low solubility. Rational mutagenesis aimed at the amino acid residues located at the interface of the variable domains and the exchanged CH3 domains was applied to improve the biophysical properties of the molecule. The domain-exchanged construct, including variable domains of the HER2/neu specific antibody trastuzumab, was able to bind to the surface of the strongly HER2/neu positive cell line SK-BR3 4-fold weaker than trastuzumab, but could nevertheless incite a more potent response in an antibody-dependent cell cytotoxicity (ADCC) reporter assay with FcγRIIIa-overexpressing T-cells. This could be explained with a stronger binding to the FcγRIIIa. Importantly, the novel construct could mediate a specific ADCC effect with natural killer cells similar to the parental antibody.
Fc fragment with antigen-binding (Fcab) is a novel construct which can be selected to recognize specifically a wide variety of target proteins. We describe the selection and affinity maturation of Fcab clones targeting VEGF, an important pro-angiogenesis factor. To investigate the extent of engineering permissible to Fcabs we applied targeted mutagenesis to all three C-terminal loop structures and the C-terminus of the CH3 domain to isolate high-affinity binders by directed evolution and yeast display. The matured clone, CT6, binds to VEGF with low nanomolar affinity and inhibits VEGF-stimulated proliferation of human umbilical vein endothelial cells in vitro. Molecular dynamics simulations were performed to address flexibility of the molecular structure of CT6 and to approximate a structural ensemble in aqueous solution. Significantly higher RMSF levels of CT6 in comparison to wild-type Fc were limited to the elongated CD-loop in the CH3 domain, while the overall structural integrity was retained. This allowed the Fcab to replace the Fc portion of a mAb, in which both the CH3 and Fab are capable of antigen engagement: a construct called mAb2 was assembled with CT6 and the Fab of bevacizumab. This bispecific molecule showed more potent antagonistic activity than bevacizumab in vitro. Further evaluation for the potential of the CT6 Fcab in targeted therapy is warranted due to the possibility of being combined with other therapeutically meaningful targets.
The overexpression of tyrosine kinase HER2 in numerous cancers, connected with fierce signaling and uncontrolled proliferation, makes it a suitable target for immunotherapy. The acquisition of resistance to currently used compounds and the multiplicity of signaling pathways involved prompted research into the discovery of novel binders as well as treatment options with multiple targeting and multispecific agents. Here we constructed an anti-HER2 tetravalent and biparatopic symmetrical IgG-like molecule by combining the Fab of pertuzumab with a HER2-specific Fcab (Fc fragment with antigen binding), which recognizes an epitope overlapping with trastuzumab. In the strongly HER2-positive cell line SK-BR-3, the molecule induced a rapid and efficient reduction in surface HER2 levels. A potent anti-proliferative effect, specific for the HER2-positive cell line, was observed in vitro, following the induction of apoptosis, and this could not be achieved with treatment with the mixture of pertuzumab and the parental Fcab. The inhibitory cytotoxic effect of our antibody as a single agent makes it a promising contribution to the armory of anti-cancer molecules directed against HER2-addicted cells.
The research of extracellular vesicles (EVs) has boomed in the last decade, with the promise of them functioning as target‐directed drug delivery vehicles, able to modulate proliferation, migration, differentiation, and other properties of the recipient cell that are vital for health of the host organism. To enhance the ability of their targeted delivery, we employed an intrinsically overrepresented protein, CD81, to serve for recognition of the desired target antigen. Yeast libraries displaying mutant variants of the large extracellular loop of CD81 have been selected for binders to human placental laminin as an example target. Their specific interaction with laminin was confirmed in a mammalian display system. Derived sequences were reformatted to full‐length CD81 and expressed in EVs produced by HeLa cells. These EVs were examined for the presence of the recombinant protein and were shown to exhibit an enhanced uptake into laminin‐secreting mammalian cell lines. For the best candidate, the specificity of antigen interaction was demonstrated with a competition experiment. To our knowledge, this is the first example of harnessing an EV membrane protein as mediator of de novo target antigen recognition via in vitro molecular evolution, opening horizons to a broad range of applications in various therapeutic settings.
Soluble T‐cell receptors (TCRs) have recently gained visibility as target‐recognition units of anticancer immunotherapeutic agents. Here, we improved the thermal stability of the well‐expressed high‐affinity A6 TCR by introducing pairs of cysteines in the invariable parts of the α‐ and β‐chain. A mutant with a novel intradomain disulfide bond in each chain also tested superior to the wild‐type in the accelerated stability assay. Binding of the mutant to the soluble cognate peptide (cp)–MHC and to the peptide‐loaded T2 cell line was equal to the wild‐type A6 TCR. The same stabilization motif worked efficiently in TCRs with different specificities, such as DMF5 and 1G4. Altogether, the biophysical properties of the soluble TCR molecule could be improved, without affecting its expression level and antigen‐binding specificity.
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