Katharina Stadlbauer

Conformational plasticity allows class I viral fusion proteins, including the SARS‐CoV‐2 spike, to undergo major structural rearrangements that support receptor binding and membrane fusion, making them key vaccine and antiviral targets. Using high‐speed atomic force microscopy (AFM) and single‐molecule force spectroscopy, we analyzed the ancestral and nine spike variants and found an evolutionary trend from increased flexibility in early strains to greater rigidification beginning with Delta, fluctuating plasticity in Omicron sublineages, and pronounced compaction in JN.1. Delta favored multivalent ACE2 engagement through its three receptor‐binding domains, whereas later Omicron variants shifted toward predominantly single‐site interactions. This change was compensated by stronger RBD–ACE2 bond stability. Overall, spike evolution appears to balance openness and compaction to regulate receptor accessibility, mobility, and immune escape while preserving or enhancing entry efficiency. These single‐molecule measurements define functional constraints on viral adaptation and may inform prediction of future variants and design of improved countermeasures.

Immunoglobulin (Ig) A has attracted interest as a proposed therapeutic agent due to its ability to engage cell groups differently compared to an IgG scaffold and elicit tumor eradication. Further, its multimeric forms enable increased flexibility in the design of available paratopes. The latter is particularly advantageous for bi- and multispecific antibody formats, which are unparalleled in their enhanced selectivity and unique biological functions. We engineered bispecific heterodimeric IgA-based antibodies using the strand-exchanged engineered domain (SEED) technology, which relies on intertwined segments of IgA and IgG in the C_H3 domain, and applied mutagenesis to introduce two additional binding sites to enable the interaction of IgA-Fc with the myeloid cell-activating receptor CD89 (FcαR). These antibodies exhibited good biophysical properties and thermostability similar to the parental SEED molecule. Binding capacity to both antigens recognized by variable domains, epidermal growth factor receptor (EGFR) and receptor tyrosine kinase like orphan receptor 1 (ROR1), was not impaired, and in contrast to the original SEED-IgA, trispecific mutants could bind to CD89-expressing cells, mediate tumor cell-effector cell clustering, and induce neutrophil-mediated specific lysis of tumor cells. Trispecific design was applicable to both SEED-IgA1 and -IgA2 scaffolds. Interestingly, HEK-expressed mutants featured a CH2-linked N-glycan pattern more similar to wild-type IgA, with reduced core fucosylation in comparison with IgA-SEED. Collectively, the presented format combines the mobilization of CD89-positive effector cells with the flexibility of incorporating antigen specificities of choice into the variable domains, and thus is a promising basis for biochemically stable multispecific IgA with high therapeutic potential.

High numbers of membrane immunoglobulin E (IgE)-positive cells are characteristic of allergic conditions, atopic dermatitis, or IgE myeloma. Antibodies targeting the extracellular membrane-proximal domain of the membranous IgE-B-cell receptor (BCR) fragment can be used for specific depletion of IgE-BCR-positive cells. In this study, we derivatized such an antibody with a toxin and developed an antibody–drug conjugate (ADC) that showed strong cytotoxicity for an IgE-positive target cell line. Site-specific conjugation with maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl-monomethyl-auristatin E via a newly introduced single cysteine residue was used to prepare a compound with a drug–antibody ratio of 2 and favorable biophysical properties. The antibody was rapidly taken up by the target cells, showing almost complete internalization after 4 h of treatment. Its cytotoxic effect was potentiated upon cross-linking mediated by an anti-human IgG F(ab’)₂ fragment. Because of its fast internalization and strict target specificity, this antibody–drug conjugate presents a valuable starting point for the further development of an anti-IgE cell-depleting agent, operating by the combined action of receptor cross-linking and toxin-mediated cytotoxicity.

Inhibition of complement activation via the overexpression of complement-regulatory proteins (CRPs), most notably CD46, CD55 and CD59, is an efficient mechanism of disguise of cancer cells from a host immune system. This phenomenon extends to counteract the potency of therapeutic antibodies that could lyse target cells by eliciting complement cascade. The manifold functions and ubiquitous expression of CRPs preclude their systemic specific inhibition. We selected CD59-specific Fc fragments with a novel antigen binding site (Fcabs) from yeast display libraries using recombinant antigens expressed in bacterial or mammalian cells. To produce a bispecific antibody, we endowed rituximab, a clinically applied anti-CD20 antibody, used for therapy of various lymphoid malignancies, with an anti-CD59 Fcab. This bispecific antibody was able to induce more potent complement-dependent cytotoxicity for CD20 and CD59 expressing Raji cell line measured with lactate dehydrogenase-release assay, but had no effect on the cells with lower levels of the primary CD20 antigen or CD20-negative cells. Such molecules are promising candidates for future therapeutic development as they elicit a higher specific cytotoxicity at a lower concentration and hence cause a lower exhaustion of complement components.

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 increased incidence of allergies and asthma has sparked interest in IgE, the central player in the allergic response. Interaction with its high-affinity receptor FcεRI leads to sensitization and allergen presentation, extracellular membrane-proximal domain in membrane IgE can act as an antigen receptor on B cells, and the interaction with low-affinity IgE receptor CD23 additionally influences its homeostatic range. Therapeutic anti-IgE antibodies act by the inhibition of IgE functions by interfering with its receptor binding or by the obliteration of IgE-B cells, causing a reduction of serum IgE levels. Fusion proteins of antibody fragments that can act as bispecific T-cell engagers have proven very potent in eliciting cytotoxic T-lymphocyte-mediated killing. We have tested five anti-IgE Fc antibodies, recognizing different epitopes on the membrane-expressed IgE, for the ability to elicit specific T-cell activation when expressed as single-chain Fv fragments fused with anti-CD3ε single-chain antibody. All candidates could specifically stain the cell line, expressing the membrane-bound IgE-Fc and bind to CD3-positive Jurkat cells, and the specific activation of engineered CD3-overexpressing Jurkat cells and non-stimulated CD8-positive cells was demonstrated for 8D6- and ligelizumab-based bispecific antibodies. Thus, such anti-IgE antibodies have the potential to be developed into agents that reduce the serum IgE concentration by lowering the numbers of IgE-secreting cells.

Since two decades, yeast display methodology is a popular tool for discovery, stability improvement, and affinity maturation of diverse protein scaffolds, intended for antigen recognition. Yeast display is particularly well suited for the selection of heterodimeric proteins, such as antibodies and T-cell receptors (TCRs), as it allows rapid library creation via gap-repair-driven homologous recombination and subsequent construction of a combinatorial library after mating of yeast of opposite mating types. Certain properties of the TCR scaffold, such as its stability, inferior to most antibody fragments, require modifications of traditional antigen selection strategies. Their selection can be monitored and guided upon staining with the soluble versions of their original antigen, peptide-major histocompatibility complex (MHC), or clonotypic antibodies, whose binding is critically dependent on the TCR structural integrity. Overall, this chapter underlines the importance of the versatile yeast display technique for the diversification of the TCR scaffold for antigen recognition and optimization of its stability.

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.

Since the introduction of the yeast display platform, this method has increasingly gained popularity for the discovery and affinity maturation of antibodies and other protein scaffolds intended for antigen recognition. Yeast display is particularly well suited for the selection of antigen-binding Fc fragments (Fcabs) as it allows rapid combinatorial library construction via gap repair-driven homologous recombination and an efficient display of a glycosylated Fc able to interact with Fcγ receptors. Apart from expression-related normalization, isolation of properly folded Fcabs can be guided efficiently by simultaneous staining with ligands such as protein A, FcγRI, or the conformation-sensitive anti-FigC_H2 antibody, whose binding is critically dependent on the integrity of the Fc structure. The particular properties of the Fcab scaffold, such as its homodimeric state which can promote binding to multiple antigen molecules, require modifications of traditional affinity maturation strategies. Preferred to equilibrium selections are kinetically driven antigen selections, designed to specifically influence the binding off-rate, which in many cases augments the desired biological effect. A simple design of a yeast-displayed heterodimeric Fc fragment is described and can be used as a general guideline for affinity selection of Fcabs with an asymmetric binding site. Overall, this chapter underlines the importance of the versatile yeast display technique for the optimization of the novel Fcab scaffold for antigen recognition.