Bovine ultralong CDR-H3 derived knob paratopes elicit potent TNF-α neutralization and enable the generation of novel adalimumab-based antibody architectures with augmented features.

In this work we have generated cattle-derived chimeric ultralong CDR-H3 antibodies targeting tumor necrosis factor α (TNF-α) via immunization and yeast surface display. We identified one particular ultralong CDR-H3 paratope that potently neutralized TNF-α. Interestingly, grafting of the knob architecture onto a peripheral loop of the CH3 domain of the Fc part of an IgG1 resulted in the generation of a TNF-α neutralizing Fc (Fcknob) that did not show any potency loss compared with the parental chimeric IgG format. Eventually, grafting this knob onto the CH3 region of adalimumab enabled the engineering of a novel TNF-α targeting antibody architecture displaying augmented TNF-α inhibition.

Cattle-derived knob paratopes grafted onto peripheral loops of the IgG1 Fc region enable the generation of a novel symmetric bispecific antibody format.

In this work we present a novel symmetric bispecific antibody format based on engraftments of cattle-derived knob paratopes onto peripheral loops of the IgG1 Fc region. For this, knob architectures obtained from bovine ultralong CDR-H3 antibodies were inserted into the AB loop or EF loop of the CH3 domain, enabling the introduction of an artificial binding specificity into an IgG molecule. We demonstrate that inserted knob domains largely retain their binding affinities, resulting into bispecific antibody derivatives versatile for effector cell redirection. Essentially, generated bispecifics demonstrated adequate biophysical properties and were not compromised in their Fc mediated functionalities such as FcRn or FcγRIIIa binding.

Antibody display technologies: selecting the cream of the crop.

Antibody display technologies enable the successful isolation of antigen-specific antibodies with therapeutic potential. The key feature that facilitates the selection of an antibody with prescribed properties is the coupling of the protein variant to its genetic information and is referred to as genotype phenotype coupling. There are several different platform technologies based on prokaryotic organisms as well as strategies employing higher eukaryotes. Among those, phage display is the most established system with more than a dozen of therapeutic antibodies approved for therapy that have been discovered or engineered using this approach. In recent years several other technologies gained a certain level of maturity, most strikingly mammalian display. In this review, we delineate the most important selection systems with respect to antibody generation with an emphasis on recent developments.

Miniaturized Forced Degradation of Therapeutic Proteins and ADCs by Agitation-Induced Aggregation Using Orbital Shaking of Microplates.

Microplate-based formulation screening is a powerful approach to identify stabilizing excipients for therapeutic proteins while reducing material requirements. However, this approach is sometimes not representative of studies conducted in relevant container closures. The present study aimed to identify critical parameters for a microplate-based orbital shaking method to screen biotherapeutic formulations by agitation-induced aggregation. For this purpose, an in-depth methodological study was conducted using different shakers, microplates, and plate seals. Aggregation was monitored by size exclusion chromatography, turbidity, and backgrounded membrane imaging. Both shaker quality and liquid-seal contact had substantial impacts on aggregation during shaking and resulted in non-uniform sample treatment when parameters were not suitably selected. The well volume to fill volume ratio (Vwell/Vfill) was identified as an useful parameter for achieving comparable aggregation levels between different microplate formats. An optimized method (2400 rpm [ac 95 m/s2], Vfill 60-100 µL [Vwell/Vfill 6-3.6], 24 h, RT, heat-sealed) allowed for uniform sample treatment independent of surface tension and good agreement with vial shaking results. This study provides valuable guidance for miniaturization of shaking stress studies in biopharmaceutical drug development, facilitating method transfer and comparability between laboratories.

Rapid cGMP manufacturing of COVID-19 monoclonal antibody using stable CHO cell pools.

Therapeutic proteins, including monoclonal antibodies, are typically manufactured using clonally derived, stable host cell lines, since consistent and predictable cell culture performance is highly desirable. However, selecting and preparing banks of stable clones takes considerable time, which inevitably extends overall development timelines for new therapeutics by delaying the start of subsequent activities, such as the scale-up of manufacturing processes. In the context of the coronavirus disease 2019 (COVID-19) pandemic, with its intense pressure for accelerated development strategies, we used a novel transposon-based Leap-In Transposase® system to rapidly generate high-titer stable pools and then used them directly for large scale-manufacturing of an anti-severe acute respiratory syndrome coronavirus 2 monoclonal antibody under cGMP. We performed the safety testing of our non-clonal cell bank, then used it to produce material at a 200L-scale for preclinical safety studies and formulation development work, and thereafter at 2000L scale for supply of material for a Phase 1 clinical trial. Testing demonstrated the comparability of critical product qualities between the two scales and, more importantly, that our final clinical trial product met all pre-set product quality specifications. The above expediated approach provided clinical trial material within 4.5 months, in comparison to 12-14 months for production of clinical trial material via the conventional approach.

Grabbing the Bull by Both Horns: Bovine Ultralong CDR-H3 Paratopes Enable Engineering of 'Almost Natural' Common Light Chain Bispecific Antibodies Suitable For Effector Cell Redirection.

A subset of antibodies found in cattle comprises ultralong CDR-H3 regions of up to 70 amino acids. Interestingly, this type of immunoglobulin usually pairs with the single germline VL gene, V30 that is typically very conserved in sequence. In this work, we have engineered ultralong CDR-H3 common light chain bispecific antibodies targeting Epidermal Growth Factor Receptor (EGFR) on tumor cells as well as Natural Cytotoxicity Receptor NKp30 on Natural Killer (NK) cells. Antigen-specific common light chain antibodies were isolated by yeast surface display by means of pairing CDR-H3 diversities following immunization with a single V30 light chain. After selection, EGFR-targeting paratopes as well as NKp30-specific binders were combined into common light chain bispecific antibodies by exploiting the strand-exchange engineered domain (SEED) technology for heavy chain heterodimerization. Biochemical characterization of resulting bispecifics revealed highly specific binding to the respective antigens as well as simultaneous binding to both targets. Most importantly, engineered cattle-derived bispecific common light chain molecules elicited potent NK cell redirection and consequently tumor cell lysis of EGFR-overexpressing cells as well as robust release of proinflammatory cytokine interferon-γ. Taken together, this data is giving clear evidence that bovine bispecific ultralong CDR-H3 common light chain antibodies are versatile for biotechnological applications.

Affinity Maturation of B7-H6 Translates into Enhanced NK Cell-Mediated Tumor Cell Lysis and Improved Proinflammatory Cytokine Release of Bispecific Immunoligands via NKp30 Engagement.

Activating NK cell receptors represent promising target structures to elicit potent antitumor immune responses. In this study, novel immunoligands were generated that bridge the activating NK cell receptor NKp30 on NK cells with epidermal growth factor receptor (EGFR) on tumor cells in a bispecific IgG-like format based on affinity-optimized versions of B7-H6 and the Fab arm derived from cetuximab. To enhance NKp30 binding, the solitary N-terminal IgV domain of B7-H6 (ΔB7-H6) was affinity matured by an evolutionary library approach combined with yeast surface display. Biochemical and functional characterization of 36 of these novel ΔB7-H6-derived NK cell engagers revealed an up to 45-fold-enhanced affinity for NKp30 and significantly improved NK cell-mediated, EGFR-dependent killing of tumor cells compared with the NK cell engager based on the wild-type ΔB7-H6 domain. In this regard, potencies (EC50 killing) of the best immunoligands were substantially improved by up to 87-fold. Moreover, release of IFN-γ and TNF-α was significantly increased. Importantly, equipment of the ΔB7-H6-based NK cell engagers with a human IgG1 Fc part competent in Fc receptor binding resulted in an almost 10-fold superior killing of EGFR-overexpressing tumor cells compared with molecules either triggering FcγRIIIa or NKp30. Additionally, INF-γ and TNF-α release was increased compared with molecules solely triggering FcγRIIIa, including the clinically approved Ab cetuximab. Thus, incorporating affinity-matured ligands for NK cell-activating receptors might represent an effective strategy for the generation of potent novel therapeutic agents with unique effector functions in cancer immunotherapy.

Biophysical and biochemical characterization of a VHH-based IgG-like bi- and trispecific antibody platform.

Here, we report the characterization of a VHH-derived IgG-like bi- and trispecific antibody platform that essentially relies on the replacement of the VH and VL regions of a conventional antibody by two independently functioning VHH domains. Consequently, a VHH is engrafted onto constant region CH1 while the other VHH-based paratope is engrafted on the constant region of the light chain, Cκ or Cλ, resulting in a tetravalent bispecific IgG-like molecule. Combined with a heavy chain heterodimerization technique, this platform allows facile engineering of bi- and trispecific antibodies with flexible valencies. We demonstrate the general applicability of this generic platform approach and elaborate on the limitations of specific formats.

A One-Step Process for the Construction of Phage Display scFv and VHH Libraries.

In this work we present a one-step cloning approach for the establishment of antibody phage display libraries relying on type IIs restriction enzymes. We show that single chain variable fragment (scFv) libraries with adequate qualities can readily be cloned in a 'scar-less' manner and that the isolation of antigen-specific antibodies from immunized chickens is feasible within three selection rounds. Moreover, we demonstrate the general applicability of this method by rapidly constructing and panning VHH single domain antibody phage display libraries from immunized Llama repertoires.

A novel one-step approach for the construction of yeast surface display Fab antibody libraries.

BACKGROUND: Yeast surface display (YSD) has proven to be a versatile platform technology for antibody discovery. However, the construction of antibody Fab libraries typically is a tedious three-step process that involves the generation of heavy chain as well as light chain display plasmids in different haploid yeast strains followed by yeast mating. RESULTS: Within this study, we aimed at implementing a focused Golden Gate Cloning approach for the generation of YSD libraries. For this, antibodies heavy and light chains were encoded on one single plasmid. Fab display on yeast cells was either mediated by a two-directional promoter system (2dir) or by ribosomal skipping (bicis). The general applicability of this methodology was proven by the functional display of a therapeutic antibody. Subsequently, we constructed large antibody libraries with heavy chain diversities derived from CEACAM5 immunized animals in combination with a common light chain. Target-specific antibodies from both display systems were readily obtained after three rounds of fluorescence activated cell sorting. Isolated variants exhibited high affinities in the nanomolar and subnanomolar range as well as appropriate biophysical properties. CONCLUSION: We demonstrated that Golden Gate Cloning appears to be a valid tool for the generation of large yeast surface display antibody Fab libraries. This procedure simplifies the hit discovery process of antibodies from immune repertoires.

Isolation of pH-Sensitive Antibody Fragments by Fluorescence-Activated Cell Sorting and Yeast Surface Display.

Fluorescence-activated cell sorting (FACS) in combination with yeast surface display (YSD) has proven to be a valuable tool for the engineering of antibodies. It enables the fast and robust identification and isolation of candidates with prescribed characteristics from combinatorial libraries. A novel application for FACS and YSD that has recently evolved addresses the engineering of antibodies toward pH-switchable antigen binding, aiming at reduced binding at acidic pH, compared to neutral pH. Therefore, we give guidance for the incorporation of such pH switches into antibody variable domains using combinatorial histidine scanning libraries. The protocol describes a flow cytometric sorting technique for the enrichment of antigen-specific molecules. Moreover, we provide information on how to screen the obtained antibody pools from initial sorting to isolate and characterize pH-sensitive variants.

Camelid and shark single domain antibodies: structural features and therapeutic potential.

In addition to canonical antibodies composed of heavy and light chains, the adaptive immune systems of camelids and cartilaginous fish comprise heavy-chain only isotypes (HcAb) devoid of light chains, where antigen-binding is mediated exclusively by one variable domain. Due to their inherent favorable attributes, such as high affinity and specificity for their cognate antigen, extraordinary stability, small size and, most importantly, the possibility to complement classical antibodies in terms of 'drugable' target-space, HcAb-derived entities evolved as promising candidates for biomedical applications of which many have already proven to be successful in early stage clinical trials.

An Apoptosis-Inducing Peptidic Heptad That Efficiently Clusters Death Receptor†5.

Multivalent ligands of death receptors hold particular promise as tumor cell-specific therapeutic agents because they induce an apoptotic cascade in cancerous cells. Herein, we present a modular approach to generate death receptor 5 (DR5) binding constructs comprising multiple copies of DR5 targeting peptide (DR5TP) covalently bound to biomolecular scaffolds of peptidic nature. This strategy allows for efficient oligomerization of synthetic DR5TP-derived peptides in different spatial orientations using a set of enzyme-promoted conjugations or recombinant production. Heptameric constructs based on a short (60-75 residues) scaffold of a C-terminal oligomerization domain of human C4b binding protein showed remarkable proapoptotic activity (EC50=3 nm) when DR5TP was ligated to its carboxy terminus. Our data support the notion that inter-ligand distance, relative spatial orientation and copy number of receptor-binding modules are key prerequisites for receptor activation and cell killing.

Coupled reactions on bioparticles: Stereoselective reduction with cofactor regeneration on PhaC inclusion bodies.

Chiral alcohols are important building blocks for specialty chemicals and pharmaceuticals. The production of chiral alcohols from ketones can be carried out stereo selectively with alcohol dehydrogenases (ADHs). To establish a process for cost-effective enzyme immobilization on solid phase for application in ketone reduction, we used an established enzyme pair consisting of ADH from Rhodococcus erythropolis and formate dehydrogenase (FDH) from Candida boidinii for NADH cofactor regeneration and co-immobilized them on modified poly-p-hydroxybutyrate synthase (PhaC)-inclusion bodies that were recombinantly produced in Escherichia coli cells. After separate production of genetically engineered and recombinantly produced enzymes and particles, cell lysates were combined and enzymes endowed with a Kcoil were captured on the surface of the Ecoil presenting particles due to coiled-coil interaction. Enzyme-loaded particles could be easily purified by centrifugation. Total conversion of 4'-chloroacetophenone to (S)-4-chloro-α-methylbenzyl alcohol could be accomplished using enzyme-loaded particles, catalytic amounts of NAD(+) and formate as substrates for FDH. Chiral GC-MS analysis revealed that immobilized ADH retained enantioselectivity with 99 % enantiomeric excess. In conclusion, this strategy may become a cost-effective alternative to coupled reactions using purified enzymes.

Single-domain antibodies for biomedical applications.

Single-domain antibodies are the smallest antigen-binding units of antibodies, consisting either only of one variable domain or one engineered constant domain that solely facilitates target binding. This class of antibody derivatives comprises naturally occurring variable domains derived from camelids and sharks as well as engineered human variable or constant antibody domains of the heavy or light chain. Because of their high affinity and specificity as well as stability, small size and benefit of multiple re-formatting opportunities, those molecules emerged as promising candidates for biomedical applications and some of these entities have already proven to be successful in clinical development.

Cystine-knot peptides targeting cancer-relevant human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4).

Cystine-knot peptides sharing a common fold but displaying a notably large diversity within the primary structure of flanking loops have shown great potential as scaffolds for the development of therapeutic and diagnostic agents. In this study, we demonstrated that the cystine-knot peptide MCoTI-II, a trypsin inhibitor from Momordica cochinchinensis, can be engineered to bind to cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), an inhibitory receptor expressed by T lymphocytes, that has emerged as a target for the treatment of metastatic melanoma. Directed evolution was used to convert a cystine-knot trypsin inhibitor into a CTLA-4 binder by screening a library of variants using yeast surface display. A set of cystine-knot peptides possessing dissociation constants in the micromolar range was obtained; the most potent variant was synthesized chemically. Successive conjugation with neutravidin, fusion to antibody Fc domain or the oligomerization domain of C4b binding protein resulted in oligovalent variants that possessed enhanced (up to 400-fold) dissociation constants in the nanomolar range. Our data indicate that display of multiple knottin peptides on an oligomeric scaffold protein is a valid strategy to improve their functional affinity with ramifications for applications in diagnostics and therapy.