Efficient Expression of Functionally Active Aflibercept with Designed N-glycans.

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 AFLIGnGn and AFLISia, 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 AFLIGnGn and AFLISia displayed similar binding potency to VEGF165, 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.

Easy assessment of the avidity of polyclonal allergen-specific serum antibodies.

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 (KD) 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 KD 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.

Development of a Cytotoxic Antibody-Drug Conjugate Targeting Membrane Immunoglobulin E-Positive Cells.

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.

Selection of High-Affinity Heterodimeric Antigen-Binding Fc Fragments from a Large Yeast Display Library.

Antigen-binding Fc (Fcab™) fragments, where a novel antigen binding site is introduced by the mutagenesis of the C-terminal loops of the CH3 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.

Bispecific mAb2 Antibodies Targeting CD59 Enhance the Complement-Dependent Cytotoxicity Mediated by Rituximab.

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.

Construction of Yeast Display Libraries for Selection of Antigen-Binding Variants of Large Extracellular Loop of CD81, a Major Surface Marker Protein of Extracellular Vesicles.

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.

Trispecific antibodies produced from mAb2 pairs by controlled Fab-arm exchange.

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 mAb2 format, which feature a novel antigen binding site in the CH3 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 mAb2 constructs.

Designed SARS-CoV-2 receptor binding domain variants form stable monomers.

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.

Bispecific T-Cell Engagers Targeting Membrane-Bound IgE.

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.

A Tetravalent Biparatopic Antibody Causes Strong HER2 Internalization and Inhibits Cellular Proliferation.

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.

Binding Characteristic of Various Antibody Formats Against Aflatoxins.

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.

An engineered CD81-based combinatorial library for selecting recombinant binders to cell surface proteins: Laminin binding CD81 enhances cellular uptake of extracellular vesicles.

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.

A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests.

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.

Bispecific antibodies with Fab-arms featuring exchanged antigen-binding constant domains.

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 mAb2 format where a set of mutated amino acid residues in the CH3 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 CH1/CL domain pair with a pair of antigen-binding CH3 domains in a model scaffold with trastuzumab variable domains and VEGF-binding CH3 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 CH3 domains offer an alternative solution in positioning and valency of antigen binding sites.

Beyond affinity: selection of antibody variants with optimal biophysical properties and reduced immunogenicity from mammalian display libraries.

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.

Stabilization of soluble high-affinity T-cell receptor with de novo disulfide bonds.

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 ß-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.

Constant domain-exchanged Fab enables specific light chain pairing in heterodimeric bispecific SEED-antibodies.

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 CH1 and CL domain pair in one of the Fab fragments is replaced with a CH3-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 CH3-exchanged constant domains is a promising tool for the generation of complete heterodimeric bispecific antibodies with correct light chain pairing.

Methods for Construction of Yeast Display Libraries of Four-Domain T-Cell Receptors.

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.

Yeast Surface Display and Cell Sorting of Antigen-Binding Fc Fragments.

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-FigCH2 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.