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Despite the immense clinical success of the antibody therapeutics that neutralize programmed death receptor ligand 1 (PD-L1) and thus resurrect T cell antitumor activity, the patient response rates remain low. In this issue of Cell Chemical Biology, Ludwig et al. 1 reveal novel topologies of multiparatopic antibodies that mediate potent PD-L1 downregulation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aflibercept is a therapeutic recombinant fusion protein comprising extracellular domains of human vascular endothelial growth factor receptors (VEGFRs) and IgG1-Fc. It is a highly glycosylated protein with five N-glycosylation sites that might impact it structurally and/or functionally. Aflibercept is produced in mammalian cells and exhibits large glycan heterogeneity, which hampers glycan-associated investigations. Here, we report the expression of aflibercept in a plant-based system with targeted N-glycosylation profiles. Nicotiana benthamiana -based glycoengineering resulted in the production of aflibercept variants carrying designed carbohydrates, namely, N-glycans with terminal GlcNAc and sialic acid residues, herein referred to as AFLI GnGn and AFLI Sia , respectively. Both variants were transiently expressed in unusually high amounts (2 g/kg fresh leaf material) in leaves and properly assembled to dimers. Mass spectrometric site-specific glycosylation analyses of purified aflibercept showed the presence of two to four glycoforms in a consistent manner. We also demonstrate incomplete occupancy of some glycosites. Both AFLI GnGn and AFLI Sia displayed similar binding potency to VEGF 165 , with a tendency of lower binding to variants with increased sialylation. Collectively, we show the expression of functionally active aflibercept in significant amounts with controlled glycosylation. The results provide the basis for further studies in order to generate optimized products in the best-case scenario.

Introduction: Allergen-specific IgE-blocking IgG antibodies contribute to successful allergen immunotherapy (AIT), however, not much is known about their affinity. Since affinity measurements of polyclonal antibodies in serum are technically challenging we evaluated the applicability of acidic disruption of antibody-allergen complexes by a modified ELISA protocol with monoclonal antibodies (mAbs) specific for the relevant major allergens Betv1 and Mald1. Then, AIT-induced blocking and non-blocking Mald1-specific antibodies in sera from individuals with or without reduced apple allergy were compared., Methods: After testing their pH stability coated recombinant allergens were incubated with (i) mAbs diluted in PBS or human serum and (ii) sera from individuals after sublingual administration of Mald1 or Betv1 for 16 weeks. Immune complexes were exposed to buffers in the pH range of 6.4-3.4 and residual antibodies were measured. Avidity indexes (AI), defined as the pH removing 50% of antibodies, were compared to the dissociation constants (K D ) of mAbs determined by surface plasmon resonance., Results: The selected pH range was applicable to disrupt allergen complexes with highly affine mAbs without compromising allergen integrity. AIs of mAbs accorded with K D values and were unaffected by epitope specificity or the presence of serum proteins. The inter-assay variability was <4% CV. Protective Mald1-specific IgG antibodies from individuals with reduced apple allergy showed a higher collective binding strength than that of the non-protective antibodies of individuals without reduced apple allergy., Conclusion: Acidic disruption of allergen-antibody complexes may be used to estimate the net-binding force of polyclonal serum antibodies and eases the investigation of affinity-related research questions in AIT., (© 2024 The Authors. Clinical & Experimental Allergy published by John Wiley & Sons Ltd.)

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.

Antigen-binding Fc (Fcab™) fragments, where a novel antigen binding site is introduced by the mutagenesis of the C-terminal loops of the C H 3 domain, function as parts of bispecific IgG-like symmetrical antibodies when they replace their wild-type Fc. Their homodimeric structure typically leads to bivalent antigen binding. In particular, biological situations monovalent engagement, however, would be preferred, either for avoiding agonistic effects leading to safety issues, or the attractive option of combining a single chain (i.e., one half) of an Fcab fragment reactive with different antigens in one antibody. We present the strategies for construction and selection of yeast libraries displaying heterodimeric Fcab fragments and discuss the effects of altered thermostability of the basic Fc scaffold and novel library designs that lead to isolation of highly affine antigen binding clones., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 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., (© 2022 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

Bispecific antibodies and antibody fragments are therapeutics of growing importance. They are clinically applied for effector cell engagement, enhanced targeting selectivity, addressing of multiple cellular pathways and active transfer of certain activities into difficult-to-reach compartments. These functionalities could profit from a third antigen specificity. In this work we have employed symmetrical bispecific parental antibodies of mAb 2 format, which feature a novel antigen binding site in the C H 3 domains, and engineered them with a minimal number of point mutations to guide the formation of a controlled Fab-arm exchanged trispecific antibody at a high yield after reduction and re-oxidation. Two model antibodies, one reactive with EGFR, Her2 and VEGF, and one with Fab-arms binding to Ang2 and VEGF and an Fc fragment binding to VEGF, were prepared and examined for heterodimeric status, stability, antigen binding properties and biological activity. Resulting molecules were of good biophysical characteristics and retained antigen reactivity and biological activity of the parental mAb 2 constructs., (© 2022 Veronica Natale et al., published by De Gruyter, Berlin/Boston.)

Over the last two decades, yeast display methodology has served as a popular tool for discovery, humanization, stability improvement, and affinity maturation of antibodies and antibody fragments, but also for development of diverse non-antibody protein scaffolds towards the ability of antigen recognition. Yeast display is particularly well suited for multiparametric analysis of properties of derivatized proteins, allowing the evolution of most diverse protein structures into antigen binding entities with favorable expression, stability, and folding properties. Here we present the methodological basics of a novel yeast display-based approach for the functionalization of the large extracellular loop of CD81 into a de novo antigen binding unit. CD81 is intrinsically overrepresented on the surface of extracellular vesicles (EVs), naturally occurring nanoparticle units that act as cell-to-cell messengers by delivering their intracellular cargo from the source cell into a recipient cell. This amazing feature makes them of highest biotechnological interest, yet methods for their targeted delivery are still in their infancy. As a novel approach for introducing EV surface modifications enabling specific target cell recognition and internalization, we have prepared yeast display libraries of CD81 large extracellular loop mutants, which are selected towards specific antigen binding and resulting mutants conveniently clicked into the full-length EV surface protein. Resulting EVs display wild-type-like characteristics regarding the expression level and distribution of recombinant proteins and are hence promising therapeutic tools., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

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 application of recombinant antibodies for the analysis of foods and food contaminants is now a major focus, given their capacity to be engineered to tailor their specificity, enhance their stability, and modify their structural formats to fit the desired analytical platform. In this study, human scFv antibody fragments generated against aflatoxin B1 (AFB1) were selected as the model antibody to explore the effect of antibody formats on their binding activity and to evaluate their potential use as immunoreagents for food contaminant analysis. Four human scFv antibody fragments against aflatoxin B1 (AFB1), previously isolated and engineered by chain shuffling, were converted into various formats, that is, scFv-AP fusions, scFv-Fc, and whole IgG molecules. The result indicated that the effects of the antibody format on the binding property varied, depending on the sequence of scFv. For all of the scFv clones, the scFv-AP fusion format showed the highest sensitivity by competitive ELISA, while the effects on the binding activity after conversion to scFv-Fc or IgG format varied, depending on the amino acid sequence of the antibodies. The sAFH-3e3 antibodies that showed the best performance by competitive ELISA were selected for further investigation. The sAFH-3e3 was converted to the scFv-GFP format and tested by fluorescence-linked immunosorbent assay (FLISA), which showed that its binding property was equivalent to those of scFv-Fc and IgG formats. The potential applications of the sAFH-3e3 in a rapid test kit format based on ELISA (scFv-AP) and in a lateral flow immunochromatography assay (LFIA) (IgG) were demonstrated. A comparison of methods for the extraction of AFB1 from matrices for use with these assay formats indicated that PBS and TBST are better than 70% methanol., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

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., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Background: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups., Methods: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests' broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification., Findings: Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus., Interpretation: We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms., Funding: WWTF, Project No. COV20-016; BOKU, LBI/LBG., Competing Interests: Declaration of Competing Interest Dr. Klausberger has nothing to disclose. Dr. Duerkop has nothing to disclose. Dr. Haslacher has nothing to disclose. Dr. Wozniak-Knopp has nothing to disclose. Dr. Cserjan-Puschmann has nothing to disclose. Dr. Perkmann has nothing to disclose. Dr. Lingg has nothing to disclose. Dr. Pereira Aguilar has nothing to disclose. Dr. Laurent has nothing to disclose. Dr. De Vos has nothing to disclose. Mag.rer.nat. Hofner has nothing to disclose. Dr. Holzer has nothing to disclose. Mrs. Stadler has nothing to disclose. Dipl.-Ing. Manhart has nothing to disclose. DI Vierlinger has nothing to disclose. Dr. Egger has nothing to disclose. Dipl. Ing. Milchram has nothing to disclose. Dr. Gludovacz has nothing to disclose. Dr. Marx has nothing to disclose. Dipl.-Ing. Köppl has nothing to disclose. Christopher Tauer, BSc has nothing to disclose. Jürgen Beck, MSc reports nothing to disclose. Daniel Maresch has nothing to disclose. Dr. Grünwald-Gruber has nothing to disclose. Mr. Strobl has nothing to disclose. Dr. Satzer has nothing to disclose. Dr. Stadlmayr has nothing to disclose. Ing. Vavra has nothing to disclose. Ms. Huber BSc has nothing to disclose. Dr. Wahrmann has nothing to disclose. Dr. Eskandary has nothing to disclose. Dr. Breyer has nothing to disclose. Dr. Sieghart has nothing to disclose. Dr. Quehenberger reports other from Roche Austria, personal fees from Takeda, outside the submitted work; . Dr. Leitner has nothing to disclose. Dr. Strassl has nothing to disclose. Dr. Egger has nothing to disclose. Dr. IRSARA has nothing to disclose. Dr. Griesmacher has nothing to disclose. Dr. Hoermann has nothing to disclose. Dr. Weiss has nothing to disclose. Dr. Bellmann-Weiler has nothing to disclose. Dr. Löffler-Ragg has nothing to disclose. Dr. Borth has nothing to disclose. Dr. Strasser has nothing to disclose. Dr. Jungbauer has nothing to disclose. Dr. Hahn has nothing to disclose. Dr. Mairhofer reports other from enGenes Biotech GmbH, outside the submitted work; In addition, Dr. Mairhofer has a patent PCT/EP2016/059597-Uncoupling growth and protein production issued. Dr. Hartmann has nothing to disclose. Dr. Binder reports grants from Vienna Business Agency, during the conduct of the study; and Employee of Technoclone Herstellung von Diagnostika und Arzneimitteln GmbH. Dr. Striedner reports other from enGenes GmbH, outside the submitted work; In addition, Dr. Striedner has a patent. US20180282737A1 issued to enGenes GmbH. Dr. Mach has nothing to disclose. Dr. Weinhaeusel has nothing to disclose. Dr. Dieplinger has nothing to disclose. Dr. Grebien has nothing to disclose. Dr. Gerner has nothing to disclose. Dr. Christoph Binder is board member of Technoclone GmbH. Dr. Grabherr has nothing to disclose., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Monoclonal antibodies can acquire the property of engagement of a second antigen via fusion methods or modification of their CDR loops, but also by modification of their constant domains, such as in the mAb 2 format where a set of mutated amino acid residues in the C H 3 domains enables a high-affinity specific interaction with the second antigen. We tested the possibility of introducing multiple binding sites for the second antigen by replacing the Fab C H 1/C L domain pair with a pair of antigen-binding C H 3 domains in a model scaffold with trastuzumab variable domains and VEGF-binding C H 3 domains. Such bispecific molecules were produced in a "Fab-like" format and in a full-length antibody format. Novel constructs were of expected molecular composition using mass spectrometry. They were expressed at a high level in standard laboratory conditions, purified as monomers with Protein A and gel filtration and were of high thermostability. Their high-affinity binding to both target antigens was retained. Finally, the Her2/VEGF binding domain-exchanged bispecific antibody was able to mediate a potentiated surface Her2-internalization effect on the Her2-overexpressing cell line SK-BR-3 due to improved level of cross-linking with the endogenously secreted cytokine. To conclude, bispecific antibodies with Fabs featuring exchanged antigen-binding C H 3 domains offer an alternative solution in positioning and valency of antigen binding sites., (© 2021 The Authors. Published by Elsevier B.V.)

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., (© 2019 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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.

The early phase of protein drug development has traditionally focused on target binding properties leading to a desired mode of therapeutic action. As more protein therapeutics pass through the development pipeline; however, it is clear that non-optimal biophysical properties can emerge, particularly as proteins are formulated at high concentrations, causing aggregation or polyreactivity. Such late-stage "developability" problems can lead to delay or failure in traversing the development process. Aggregation propensity is also correlated with increased immunogenicity, resulting in expensive, late-stage clinical failures. Using nucleases-directed integration, we have constructed large mammalian display libraries where each cell contains a single antibody gene/cell inserted at a single locus, thereby achieving transcriptional normalization. We show a strong correlation between poor biophysical properties and display level achieved in mammalian cells, which is not replicated by yeast display. Using two well-documented examples of antibodies with poor biophysical characteristics (MEDI-1912 and bococizumab), a library of variants was created based on surface hydrophobic and positive charge patches. Mammalian display was used to select for antibodies that retained target binding and permitted increased display level. The resultant variants exhibited reduced polyreactivity and reduced aggregation propensity. Furthermore, we show in the case of bococizumab that biophysically improved variants are less immunogenic than the parental molecule. Thus, mammalian display helps to address multiple developability issues during the earliest stages of lead discovery, thereby significantly de-risking the future development of protein drugs.

Background: Bispecific antibodies promise to broadly expand the clinical utility of monoclonal antibody technology. Several approaches for heterodimerization of heavy chains have been established to produce antibodies with two different Fab arms, but promiscuous pairing of heavy and light chains remains a challenge for their manufacturing., Methods: We have designed a solution in which the C H 1 and C L domain pair in one of the Fab fragments is replaced with a C H 3-domain pair and heterodimerized to facilitate correct modified Fab-chain pairing in bispecific heterodimeric antibodies based on a strand-exchange engineered domain (SEED) scaffold with specificity for epithelial growth factor receptor and either CD3 or CD16 (FcγRIII)., Results: Bispecific antibodies retained binding to their target antigens and redirected primary T cells or NK cells to induce potent killing of target cells. All antibodies were expressed at a high yield in Expi293F cells, were detected as single sharp symmetrical peaks in size exclusion chromatography and retained high thermostability. Mass spectrometric analysis revealed specific heavy-to-light chain pairing for the bispecific SEED antibodies as well as for one-armed SEED antibodies co-expressed with two different competing light chains., Conclusion: Incorporation of a constant domain-exchanged Fab fragment into a SEED antibody yields functional molecules with favorable biophysical properties., General Significance: Our results show that the novel engineered bispecific SEED antibody scaffold with an incorporated Fab fragment with C H 3-exchanged constant domains is a promising tool for the generation of complete heterodimeric bispecific antibodies with correct light chain pairing., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

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 H 2 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.

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 ( T m ) 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 T m of 109 °C, 43 °C over the T m 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.

Liposomes functionalized with monoclonal antibodies or their antigen-binding fragments have attracted much attention as specific drug delivery devices for treatment of various diseases including cancer. The conjugation of antibodies to liposomes is usually achieved by covalent coupling using cross-linkers in a reaction that might adversely affect the characteristics of the final product. Here we present an alternative strategy for liposome functionalization: we created a recombinant Fab antibody fragment genetically fused on its C-terminus to the hydrophobic peptide derived from pulmonary surfactant protein D, which became inserted into the liposomal bilayer during liposomal preparation and anchored the Fab onto the liposome surface. The Fab-conjugated liposomes specifically recognized antigen-positive cells and efficiently delivered their cargo, the Alexa Fluor 647 dye, into target cells in vitro and in vivo. In conclusion, our approach offers the potential for straightforward development of nanomedicines functionalized with an antibody of choice without the need of harmful cross-linkers., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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 Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Broadly neutralizing anti-HIV-1 monoclonal antibodies, such as PG9, and its derivative RSH hold great promise in AIDS therapy and prevention. An important feature related to the exceptional efficacy of PG9 and RSH is the presence of sulfated tyrosine residues in their antigen-binding regions. To maximize antibody functionalities, we have now produced glycan-optimized, fucose-free versions of PG9 and RSH in Nicotiana benthamiana. Both antibodies were efficiently sulfated in planta on coexpression of an engineered human tyrosylprotein sulfotransferase, resulting in antigen-binding and virus neutralization activities equivalent to PG9 synthesized by mammalian cells ((CHO)PG9). Based on the controlled production of both sulfated and nonsulfated variants in plants, we could unequivocally prove that tyrosine sulfation is critical for the potency of PG9 and RSH. Moreover, the fucose-free antibodies generated in N. benthamiana are capable of inducing antibody-dependent cellular cytotoxicity, an activity not observed for (CHO)PG9. Thus, tailoring of the antigen-binding site combined with glycan modulation and sulfoengineering yielded plant-produced anti-HIV-1 antibodies with effector functions superior to PG9 made in CHO cells.

Antigen-binding Fc fragments (Fcabs) are a new unique class of immunotherapeutics. They are small (50 kD) fully functional antibody alternatives that bind antigen and elicit effector functions such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity. Since Fcabs carry the natural FcRn binding site of antibodies, they have very favorable pharmacokinetics. We showed recently that Fcab H10-03-6 is a high-affinity binder of Her-2/neu (ErbB2/neu) mediating killing of Her-2/neu-overexpressing tumor cells in the presence of immune effector cells, strongly suggesting that the mechanism of killing is due to ADCC. The present study further confirms ADCC as the mechanism by which H10-03-6 mediates tumor cell killing, since H10-03-6 was shown to interact simultaneously with Her-2/neu and the Fc receptor CD16a. The epitope recognized by H10-03-6 overlaps with that of the clinically used monoclonal antibody trastuzumab. However, unlike trastuzumab, Fcab H10-03-6 did not inhibit proliferation of human tumor cells in vitro even under conditions favoring Her-2/neu crosslinking. Treatment of mice harboring human BT-474 cell xenograft tumors with Fcab H10-03-6 led to statistically significant retardation of tumor growth. For the first time, in vivo properties of an Fcab are presented, supporting the view that Fcabs could become highly efficacious immunotherapeutics for human use., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Antigen-binding Fc fragments (Fcab) are generated by engineering the C-terminal loop regions in the CH3 domain of human immunoglobulin G class 1-crystallizable fragment (IgG1-Fc). For an optimum library design with high percentage of well-folded clones for efficient binder selection, information about the correlation between primary structure and stability is needed. Here, we present a rapid method that allows determination of the overall stability of whole libraries of IgG1-Fc on the surface of yeast by flow cytometry. Libraries of IgG1-Fc mutants with distinct regions in AB-, CD- and EF-loops of the CH3 domains randomized or carrying therein insertions of five additional residues were constructed, incubated at increasing temperatures and probed for residual binding of generic Fc ligands. Calculated temperatures of half-maximal irreversible denaturation of the libraries gave a clear hierarchy of tolerance to randomization of distinct loop positions. Experimental data were evaluated by a computational approach and are discussed with respect to the structure of IgG1-Fc and variation in sequence and length of these loops in homologous Fc proteins. Generally, the described method allows for quick assessment of the effects of randomization of distinct regions on the foldability and stability of a yeast-displayed protein library.

We describe the stabilization of human IgG1 Fc by an engineered interdomain disulfide bond at the C-terminal end of the molecule. Covalently interconnecting the C-termini of the CH(3) domains led to an increase of the melting temperatures by 5.6 and 9.1 °C respectively as compared to CH(3) domains in the context of the wild-type Fc. Combined with a recently described additional intradomain disulfide bond, both novel disulfide bonds led to an increase of the Tm by about 18.1 °C to 100.7 °C. The interdomain disulfide bond had no impact on the thermal stability of the CH(2) domain. Far- and near-UV CD spectroscopy showed very similar overall CD profiles, indicating that secondary and tertiary structure of the Fc was not negatively affected. When introduced into an Fc fragment that had been engineered to bind to Her2/neu via a novel antigen binding site located at the C-terminus of the CH(3) domain, the novel inter- and intra-domain bonds also brought about a significant increase in thermostability. Using them in combination, the Tm of the CH(3) domain was raised by 18 °C and thus restored to the Tm of the wild-type CH(3) domain. Importantly, antigen binding of the modified Fc was not affected by the engineered disulfide bonds., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Antigen binding immunoglobulin Fc fragments (Fcab) are generated by engineering loop regions in the CH3 domain of human IgG1 Fc. Variants of an Fcab specific for Her-2 were designed to display either enhanced (S239D:A330L:I332E) or diminished (L234A:L235A) binding affinities to the Fc receptor CD16a based on mutations described previously. The two mutant Fcab proteins demonstrated the expected modulation of CD16a binding. Interaction with recombinant or cell surface expressed Her-2 was unaffected in both mutants compared to the parental Fcab. Binding affinities for CD16a correlated with the ADCC-potencies of the Fcab variants. Additional studies indicated that the L234A:L235A variant Fcab had equivalent structural features as the unmodified Fcab since their DSC profiles were similar and antigen binding after re-folding upon partial heat denaturation had not changed. Introduction of the S239D:A330L:I332E mutations resulted in a significant reduction of the CH2 domain melting temperature, a moderate decrease of the thermal transition of the CH3 domain and lower antigen binding after thermal stress compared to the parental Fcab. We conclude that the known correlation between CD16a binding affinity and ADCC potency is also valid in Fcab proteins and that antigen specific Fcab molecules can be further engineered for fine tuning of immuno effector functions., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We report the stabilization of the human IgG1 Fc fragment by engineered intradomain disulfide bonds. One of these bonds, which connects the N-terminus of the CH3 domain with the F-strand, led to an increase of the melting temperature of this domain by 10°C as compared to the CH3 domain in the context of the wild-type Fc region. Another engineered disulfide bond, which connects the BC loop of the CH3 domain with the D-strand, resulted in an increase of T(m) of 5°C. Combined in one molecule, both intradomain disulfide bonds led to an increase of the T(m) of about 15°C. All of these mutations had no impact on the thermal stability of the CH2 domain. Importantly, the binding of neonatal Fc receptor was also not influenced by the mutations. Overall, the stabilized CH3 domains described in this report provide an excellent basic scaffold for the engineering of Fc fragments for antigen-binding or other desired additional or improved properties. Additionally, we have introduced the intradomain disulfide bonds into an IgG Fc fragment engineered in C-terminal loops of the CH3 domain for binding to Her2/neu, and observed an increase of the T(m) of the CH3 domain for 7.5°C for CysP4, 15.5°C for CysP2 and 19°C for the CysP2 and CysP4 disulfide bonds combined in one molecule.

Recently, it has been demonstrated that loops of the crystallizable fragment of IgG1 (IgG1-Fc) can be engineered to form antigen-binding sites. In this work C-terminal structural loops in the CH3 domains of homodimeric IgG1-Fc have been functionalized to form integrin-binding sites in order to probe the effect of engineering on structural integrity and thermal stability of IgG1-Fc as well as on binding to the ligands Protein A, CD16 and FcRn, respectively. The peptide sequence GCRGDCL--a disulfide-bridged cyclic heptapeptide that confers binding to human αvβ3 integrin was introduced into AB, CD and/or EF loops and single and double mutants were heterologously expressed in Pichia pastoris. Integrin binding of engineered IgG-Fc was tested using both binding to coated αvβ3 integrin in ELISA or to αvβ3-expressing K562 cells in FACS analysis. Additionally, blocking of αvβ3-mediated cell adhesion to vitronectin was investigated. The data presented in this report demonstrate that bioactive integrin-binding peptide(s) can be grafted on the C-terminal loops of IgG-Fc without impairing binding to effector molecules. Observed differences between the investigated variants in structural stability and integrin binding are discussed with respect to the known structure of IgG-Fc and its structural loops., (Copyright © 2011 Elsevier B.V. All rights reserved.)