Revolutionizing Synthetic Antibody Design: Harnessing Artificial Intelligence and Deep Sequencing Big Data for Unprecedented Advances.

Synthetic antibodies (Abs) represent a category of engineered proteins meticulously crafted to replicate the functions of their natural counterparts. Such Abs are generated in vitro, enabling advanced molecular alterations associated with antigen recognition, paratope site engineering, and biochemical refinements. In a parallel realm, deep sequencing has brought about a paradigm shift in molecular biology. It facilitates the prompt and cost-effective high-throughput sequencing of DNA and RNA molecules, enabling the comprehensive big data analysis of Ab transcriptomes, including specific regions of interest. Significantly, the integration of artificial intelligence (AI), based on machine- and deep- learning approaches, has fundamentally transformed our capacity to discern patterns hidden within deep sequencing big data, including distinctive Ab features and protein folding free energy landscapes. Ultimately, current AI advances can generate approximations of the most stable Ab structural configurations, enabling the prediction of de novo synthetic Abs. As a result, this manuscript comprehensively examines the latest and relevant literature concerning the intersection of deep sequencing big data and AI methodologies for the design and development of synthetic Abs. Together, these advancements have accelerated the exploration of antibody repertoires, contributing to the refinement of synthetic Ab engineering and optimizations, and facilitating advancements in the lead identification process.

Structure-guided design of a trivalent nanobody cluster targeting SARS-CoV-2 spike protein.

Nanobodies are natural anti-SARS-CoV-2 drug candidates. Engineering multivalent nanobodies is an effective way to improve the functional binding affinity of natural nanobodies by simultaneously targeting multiple sites on viral proteins. However, multivalent nanobodies have usually been engineered by trial and error, and rational designs are still lacking. Here, we describe a structure-guided design of a self-assembled trivalent nanobody cluster targeting the SARS-CoV-2 spike protein. Using the nanobody Nb6 as a monovalent binder, we first selected a human-derived trimerization scaffold evaluated by molecular dynamics simulations, then selected an optimal linker according to the minimum distance between Nb6 and the trimerization scaffold, and finally successfully engineered a trivalent nanobody cluster called Tribody. Compared with the low-affinity monovalent counterpart (Nb6), Tribody showed much higher target binding affinity (KD < 1 pM) and thus had a 900-fold increase in antiviral neutralization against SARS-CoV-2 pseudovirus. We determined the cryo-EM structure of the Tribody-spike complex and confirmed that all three Nb6 binders of Tribody collectively bind to the three receptor-binding domains (RBDs) of the spike and lock them in a 3-RBD-down conformation, fully consistent with our structure-guided design. This study demonstrates that synthetic nanobody clusters with human-derived self-assembled scaffolds are potential protein drugs against SARS-CoV-2 coronaviruses.

A bivalent form of a RBD-specific synthetic antibody effectively neutralizes SARS-CoV-2 variants.

Coronavirus Disease 2019 (COVID-19) pandemic is severely impacting the world, and tremendous efforts have been made to deal with it. Despite many advances in vaccines and therapeutics, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remains an intractable challenge. We present a bivalent Receptor Binding Domain (RBD)-specific synthetic antibody, specific for the RBD of wild-type (lineage A), developed from a non-antibody protein scaffold composed of LRR (Leucine-rich repeat) modules through phage display. We further reinforced the unique feature of the synthetic antibody by constructing a tandem dimeric form. The resulting bivalent form showed a broader neutralizing activity against the variants. The in vivo neutralizing efficacy of the bivalent synthetic antibody was confirmed using a human ACE2-expressing mouse model that significantly alleviated viral titer and lung infection. The present approach can be used to develop a synthetic antibody showing a broader neutralizing activity against a multitude of SARS-CoV-2 variants.

Structure-based design of nanobodies that inhibit seeding of Alzheimer's patient-extracted tau fibrils.

Despite much effort, antibody therapies for Alzheimer's disease (AD) have shown limited efficacy. Challenges to the rational design of effective antibodies include the difficulty of achieving specific affinity to critical targets, poor expression, and antibody aggregation caused by buried charges and unstructured loops. To overcome these challenges, we grafted previously determined sequences of fibril-capping amyloid inhibitors onto a camel heavy chain antibody scaffold. These sequences were designed to cap fibrils of tau, known to form the neurofibrillary tangles of AD, thereby preventing fibril elongation. The nanobodies grafted with capping inhibitors blocked tau aggregation in biosensor cells seeded with postmortem brain extracts from AD and progressive supranuclear palsy (PSP) patients. The tau capping nanobody inhibitors also blocked seeding by recombinant tau oligomers. Another challenge to the design of effective antibodies is their poor blood-brain barrier (BBB) penetration. In this study, we also designed a bispecific nanobody composed of a nanobody that targets a receptor on the BBB and a tau capping nanobody inhibitor, conjoined by a flexible linker. We provide evidence that the bispecific nanobody improved BBB penetration over the tau capping inhibitor alone after intravenous administration in mice. Our results suggest that the design of synthetic antibodies that target sequences that drive protein aggregation may be a promising approach to inhibit the prion-like seeding of tau and other proteins involved in AD and related proteinopathies.

A window into the human immune system: comprehensive characterization of the complexity of antibody complementary-determining regions in functional antibodies.

The human immune system uses antibodies to neutralize foreign antigens. They are composed of heavy and light chains, both with constant and variable regions. The variable region has six hypervariable loops, also known as complementary-determining regions (CDRs) that determine antibody diversity and antigen specificity. Knowledge of their significance, and certain residues present in these areas, is vital for antibody therapeutics development. This study includes an analysis of more than 11,000 human antibody sequences from the International Immunogenetics information system (IMGT). The analysis included parameters such as length distribution, overall amino acid diversity, amino acid frequency per CDR and residue position within antibody chains. Overall, our findings confirm existing knowledge, such as CDRH3's high length diversity and amino acid variability, increased aromatic residue usage, particularly tyrosine, charged and polar residues like aspartic acid, serine, and the flexible residue glycine. Specific residue positions within each CDR influence these occurrences, implying a unique amino acid type distribution pattern. We compared amino acid type usage in CDRs and non-CDR regions, both in globular and transmembrane proteins, which revealed distinguishing features, such as increased frequency of tyrosine, serine, aspartic acid, and arginine. These findings should prove useful for future optimization, improvement of affinity, synthetic antibody library design, or the creation of antibodies de-novo in silico.

Highly reliable GIGA-sized synthetic human therapeutic antibody library construction.

Background: Monoclonal antibodies (mAbs) and their derivatives are the fastest expanding category of pharmaceuticals. Efficient screening and generation of appropriate therapeutic human antibodies are important and urgent issues in the field of medicine. The successful in vitro biopanning method for antibody screening largely depends on the highly diverse, reliable and humanized CDR library. To rapidly obtain potent human antibodies, we designed and constructed a highly diverse synthetic human single-chain variable fragment (scFv) antibody library greater than a giga in size by phage display. Herein, the novel TIM-3-neutralizing antibodies with immunomodulatory functions derived from this library serve as an example to demonstrate the library's potential for biomedical applications. Methods: The library was designed with high stability scaffolds and six complementarity determining regions (CDRs) tailored to mimic human composition. The engineered antibody sequences were optimized for codon usage and subjected to synthesis. The six CDRs with variable length CDR-H3s were individually subjected to ß-lactamase selection and then recombined for library construction. Five therapeutic target antigens were used for human antibody generation via phage library biopanning. TIM-3 antibody activity was verified by immunoactivity assays. Results: We have designed and constructed a highly diverse synthetic human scFv library named DSyn-1 (DCB Synthetic-1) containing 2.5 × 1010 phage clones. Three selected TIM-3-recognizing antibodies DCBT3-4, DCBT3-19, and DCBT3-22 showed significant inhibition activity by TIM-3 reporter assays at nanomolar ranges and binding affinities in sub-nanomolar ranges. Furthermore, clone DCBT3-22 was exceptionally superior with good physicochemical property and a purity of more than 98% without aggregation. Conclusion: The promising results illustrate not only the potential of the DSyn-1 library for biomedical research applications, but also the therapeutic potential of the three novel fully human TIM-3-neutralizing antibodies.

Abiotic Synthetic Antibody Inhibitor with Broad-Spectrum Neutralization and Antiviral Efficacy against Escaping SARS-CoV-2 Variants.

The rapid emergence and spread of vaccine/antibody-escaping variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed serious challenges to our efforts in combating corona virus disease 2019 (COVID-19) pandemic. A potent and broad-spectrum neutralizing reagent against these escaping mutants is extremely important for the development of strategies for the prevention and treatment of SARS-CoV-2 infection. We herein report an abiotic synthetic antibody inhibitor as a potential anti-SARS-CoV-2 therapeutic agent. The inhibitor, Aphe-NP14, was selected from a synthetic hydrogel polymer nanoparticle library created by incorporating monomers with functionalities complementary to key residues of the SARS-CoV-2 spike glycoprotein receptor binding domain (RBD) involved in human angiotensin-converting enzyme 2 (ACE2) binding. It has high capacity, fast adsorption kinetics, strong affinity, and broad specificity in biologically relevant conditions to both the wild type and the current variants of concern, including Beta, Delta, and Omicron spike RBD. The Aphe-NP14 uptake of spike RBD results in strong blockage of spike RBD-ACE2 interaction and thus potent neutralization efficacy against these escaping spike protein variant pseudotyped viruses. It also inhibits live SARS-CoV-2 virus recognition, entry, replication, and infection in vitro and in vivo. The Aphe-NP14 intranasal administration is found to be safe due to its low in vitro and in vivo toxicity. These results establish a potential application of abiotic synthetic antibody inhibitors in the prevention and treatment of the infection of emerging or possibly future SARS-CoV-2 variants.

Computational Analysis of Antibody Paratopes for Antibody Sequences in Antibody Libraries.

To ensure the functionalities of the antibodies in phage-displayed synthetic antibody libraries, we use computational method to evaluate the designs of the antibody libraries. The computational methodologies developed in our lab for designing antibody library provide rich information on the function of the designed antibody sequences-adequate antibody designs for a specific antigen type should have predicted paratopes for the antigen type. This computational assessment of the designed antibody sequences helps eliminate non-functional designs before proceeding to construct the library designs in the wet lab. As such, only reasonable antibody designs are constructed for antibody discoveries.

Generation of synthetic antibody fragments with optimal complementarity determining region lengths for Notch-1 recognition.

Synthetic antibodies have been engineered against a wide variety of antigens with desirable biophysical, biochemical, and pharmacological properties. Here, we describe the generation and characterization of synthetic antigen-binding fragments (Fabs) against Notch-1. Three single-framework synthetic Fab libraries, named S, F, and modified-F, were screened against the recombinant human Notch-1 extracellular domain using phage display. These libraries were built on a modified trastuzumab framework, containing two or four diversified complementarity-determining regions (CDRs) and different CDR diversity designs. In total, 12 Notch-1 Fabs were generated with 10 different CDRH3 lengths. These Fabs possessed a high affinity for Notch-1 (sub-nM to mid-nM KDapp values) and exhibited different binding profiles (mono-, bi-or tri-specific) toward Notch/Jagged receptors. Importantly, we showed that screening focused diversity libraries, implementing next-generation sequencing approaches, and fine-tuning the CDR length diversity provided improved binding solutions for Notch-1 recognition. These findings have implications for antibody library design and antibody phage display.

Development, structure, and mechanism of synthetic antibodies that target claudin and Clostridium perfringens enterotoxin complexes.

Strains of Clostridium perfringens produce a two-domain enterotoxin (CpE) that afflicts humans and domesticated animals, causing prevalent gastrointestinal illnesses. CpE's C-terminal domain (cCpE) binds cell surface receptors, followed by a restructuring of its N-terminal domain to form a membrane-penetrating ß-barrel pore, which is toxic to epithelial cells of the gut. The claudin family of membrane proteins are known receptors for CpE and also control the architecture and function of cell-cell contacts (tight junctions) that create barriers to intercellular molecular transport. CpE binding and assembly disables claudin barrier function and induces cytotoxicity via ß-pore formation, disrupting gut homeostasis; however, a structural basis of this process and strategies to inhibit the claudin-CpE interactions that trigger it are both lacking. Here, we used a synthetic antigen-binding fragment (sFab) library to discover two sFabs that bind claudin-4 and cCpE complexes. We established these sFabs' mode of molecular recognition and binding properties and determined structures of each sFab bound to claudin-4-cCpE complexes using cryo-EM. The structures reveal that the sFabs bind a shared epitope, but conform distinctly, which explains their unique binding equilibria. Mutagenesis of antigen/sFab interfaces observed therein result in binding changes, validating the structures, and uncovering the sFab's targeting mechanism. From these insights, we generated a model for CpE's claudin-bound ß-pore that predicted sFabs would not prevent cytotoxicity, which we then verified in vivo. Taken together, this work demonstrates the development and mechanism of claudin/cCpE-binding sFabs that provide a framework and strategy for obstructing claudin/CpE assembly to treat CpE-linked gastrointestinal diseases.

Protein Engineering: Advances in Phage Display for Basic Science and Medical Research.

Functional Protein Engineering became the hallmark in biomolecule manipulation in the new millennium, building on and surpassing the underlying structural DNA manipulation and recombination techniques developed and employed in the last decades of 20th century. Because of their prominence in almost all biological processes, proteins represent extremely important targets for engineering enhanced or altered properties that can lead to improvements exploitable in healthcare, medicine, research, biotechnology, and industry. Synthetic protein structures and functions can now be designed on a computer and/or evolved using molecular display or directed evolution methods in the laboratory. This review will focus on the recent trends in protein engineering and the impact of this technology on recent progress in science, cancer- and immunotherapies, with the emphasis on the current achievements in basic protein research using synthetic antibody (sABs) produced by phage display pipeline in the Kossiakoff laboratory at the University of Chicago (KossLab). Finally, engineering of the highly specific binding modules, such as variants of Streptococcal protein G with ultra-high orthogonal affinity for natural and engineered antibody scaffolds, and their possible applications as a plug-and-play platform for research and immunotherapy will be described.

Synthetic antibodies block receptor binding and current-inhibiting effects of α-cobratoxin from Naja kaouthia.

Each year, thousands of people fall victim to envenomings caused by cobras. These incidents often result in death due to paralysis caused by α-neurotoxins from the three-finger toxin (3FTx) family, which are abundant in elapid venoms. Due to their small size, 3FTxs are among the snake toxins that are most poorly neutralized by current antivenoms, which are based on polyclonal antibodies of equine or ovine origin. While antivenoms have saved countless lives since their development in the late 18th century, an opportunity now exists to improve snakebite envenoming therapy via the application of new biotechnological methods, particularly by developing monoclonal antibodies against poorly neutralized α-neurotoxins. Here, we describe the use of phage-displayed synthetic antibody libraries and the development and characterization of six synthetic antibodies built on a human IgG framework and developed against α-cobratoxin - the most abundant long-chain α-neurotoxin from Naja kaouthia venom. The synthetic antibodies exhibited sub-nanomolar affinities to α-cobratoxin and neutralized the curare-mimetic effect of the toxin in vitro. These results demonstrate that phage display technology based on synthetic repertoires can be used to rapidly develop human antibodies with drug-grade potencies as inhibitors of venom toxins.

Synthetic Antibodies Detect Distinct Cellular States of Chromosome Passenger Complex Proteins.

High performance affinity reagents are essential tools to enable biologists to profile the cellular location and composition of macromolecular complexes undergoing dynamic reorganization. To support further development of such tools, we have assembled a high-throughput phage display pipeline to generate Fab-based affinity reagents that target different dynamic forms of a large macromolecular complex, using the Chromosomal Passenger Complex (CPC), as an example. The CPC is critical for the maintenance of chromosomal and cytoskeleton processes during cell division. The complex contains 4 protein components: Aurora B kinase, survivin, borealin and INCENP. The CPC acts as a node to dynamically organize other partnering subcomplexes to build multiple functional structures during mitotic progression. Using phage display mutagenesis, a cohort of synthetic antibodies (sABs) were generated against different domains of survivin, borealin and INCENP. Immunofluorescence established that a set of these sABs can discriminate between the form of the CPC complex in the midbody versus the spindle. Others localize to targets, which appear to be less organized, in the nucleus or cytoplasm. This differentiation suggests that different CPC epitopes have dynamic accessibility depending upon the mitotic state of the cell. An Immunoprecipitation/Mass Spectrometry analysis was performed using sABs that bound specifically to the CPC in either the midbody or MT spindle macromolecular assemblies. Thus, sABs can be exploited as high performance reagents to profile the accessibility of different components of the CPC within macromolecular assemblies during different stages of mitosis suggesting this high throughput approach will be applicable to other complex macromolecular systems.

Antibody heavy chain CDR3 length-dependent usage of human IGHJ4 and IGHJ6 germline genes.

Therapeutic antibody discovery using synthetic diversity has been proved productive, especially for target proteins not suitable for traditional animal immunization-based antibody discovery approaches. Recently, many lines of evidences suggest that the quality of synthetic diversity design limits the development success of synthetic antibody hits. The aim of our study is to understand the quality limitation and to properly address the challenges with a better design. Using VH3-23 as a model framework, we observed and quantitatively mapped CDR-H3 loop length-dependent usage of human IGHJ4 and IGHJ6 germline genes in the natural human immune repertoire. Skewed usage of DH2-JH6 and DH3-JH6 rearrangements was quantitatively determined in a CDR-H3 length-dependent manner in natural human antibodies with long CDR-H3 loops. Structural modeling suggests choices of JH help to stabilize antibody CDR-H3 loop and JH only partially contributes to the paratope. Our observations shed light on the design of next-generation synthetic diversity with improved probability of success.

Synthetic Antibody Mimics Based on Cancer-Targeting Immunostimulatory Peptides.

De novo cancer-targeting immunostimulatory peptides have been designed and developed as synthetic antibody mimics. A series of bifunctional peptides incorporating NKp30-binding and NK-cell-activating domains were synthesized as linear dimers and then extended into branching trimeric peptides by the incorporation of GRP78-targeting and tumor-cell-binding sequences. A selected trimeric peptide from this small set of peptides displayed binding capabilities on GRP78+ HepG2 and A549 target cells. Cell binding diminished in the presence of an anti-GRP78 peptide blocker, thus suggesting GRP78-binding dependence. Similarly, the selected trimeric peptide was also found to exhibit NK cell binding in an NKp30-dependent manner, which translated into NK cell activation as indicated by cytokine secretion. In co-culture, fluorescence microscopy revealed that the target GFP-expressing A549 cells were visibly associated with the effector NK cells when pre-activated with lead trimeric peptide. Accordingly, A549 cells were found to be compromised, as evidenced by the loss of GFP signal and notable detection of early-/late-stage apoptosis. Investigation of the immunological markers related to toxicity revealed detectable secretion of pro-inflammatory cytokines and chemokines, including IFN-γ, TNF-α, and IL-8. Furthermore, administration of peptide-activated NK cells into A549-tumor-bearing mice resulted in a consistent decrease in tumor growth when compared to the untreated control group. Taken together, the identification of a lead trimeric peptide capable of targeting and activating NK cells' immunotoxicity directly towards GRP78+ /B7H6- tumors provides a novel proof-of-concept for the development of cancer-targeting immunostimulatory peptide ligands that mimic antibody-targeting and -activating functions related to cancer immunotherapy applications.

Crystal Structure of ß-Arrestin 2 in Complex with CXCR7 Phosphopeptide.

ß-Arrestins (ßarrs) critically regulate G-protein-coupled receptor (GPCR) signaling and trafficking. ßarrs have two isoforms, ßarr1 and ßarr2. Receptor phosphorylation is a key determinant for the binding of ßarrs, and understanding the intricate details of receptor-ßarr interaction is the next frontier in GPCR structural biology. The high-resolution structure of active ßarr1 in complex with a phosphopeptide derived from GPCR has been revealed, but that of ßarr2 remains elusive. Here, we present a 2.3-Å crystal structure of ßarr2 in complex with a phosphopeptide (C7pp) derived from the carboxyl terminus of CXCR7. The structural analysis of C7pp-bound ßarr2 reveals key differences from the previously determined active conformation of ßarr1. One of the key differences is that C7pp-bound ßarr2 shows a relatively small inter-domain rotation. Antibody-fragment-based conformational sensor and hydrogen/deuterium exchange experiments further corroborated the structural features of ßarr2 and suggested that ßarr2 adopts a range of inter-domain rotations.

Genetically encoded intrabody sensors report the interaction and trafficking of ß-arrestin 1 upon activation of G-protein-coupled receptors.

Agonist stimulation of G-protein-coupled receptors (GPCRs) typically leads to phosphorylation of GPCRs and binding to multifunctional proteins called ß-arrestins (ßarrs). The GPCR-ßarr interaction critically contributes to GPCR desensitization, endocytosis, and downstream signaling, and GPCR-ßarr complex formation can be used as a generic readout of GPCR and ßarr activation. Although several methods are currently available to monitor GPCR-ßarr interactions, additional sensors to visualize them may expand the toolbox and complement existing methods. We have previously described antibody fragments (FABs) that recognize activated ßarr1 upon its interaction with the vasopressin V2 receptor C-terminal phosphopeptide (V2Rpp). Here, we demonstrate that these FABs efficiently report the formation of a GPCR-ßarr1 complex for a broad set of chimeric GPCRs harboring the V2R C terminus. We adapted these FABs to an intrabody format by converting them to single-chain variable fragments and used them to monitor the localization and trafficking of ßarr1 in live cells. We observed that upon agonist simulation of cells expressing chimeric GPCRs, these intrabodies first translocate to the cell surface, followed by trafficking into intracellular vesicles. The translocation pattern of intrabodies mirrored that of ßarr1, and the intrabodies co-localized with ßarr1 at the cell surface and in intracellular vesicles. Interestingly, we discovered that intrabody sensors can also report ßarr1 recruitment and trafficking for several unmodified GPCRs. Our characterization of intrabody sensors for ßarr1 recruitment and trafficking expands currently available approaches to visualize GPCR-ßarr1 binding, which may help decipher additional aspects of GPCR signaling and regulation.

High-Throughput Generation of In Silico Derived Synthetic Antibodies via One-step Enzymatic DNA Assembly of Fragments.

Phage-display technology offers robust methods for isolating antibody (Ab) molecules with specificity for different target antigens. Recent advancements couple Ab selections with in silico strategies, such as predictive computational models or next-generation sequencing metadata analysis of Ab selections. These advancements result in enhanced Ab clonal diversities with potential for enlarged epitope coverage of the target antigen. A current limitation however, is that de novo Ab sequences must undergo DNA gene synthesis, and subsequent expression as Ab proteins for downstream validations. Due to the high costs and time for commercially generating large sets of DNA genes, we report a high-throughput platform for the synthesis of in silico derived Ab clones. As a proof of concept we demonstrate the simultaneous synthesis of 96 unique Abs with varied lengths and complementary determining region compositions. Each of the 96 Ab clones undergoes a one-step enzymatic assembly of distinct DNA fragments that combine into a circularized Fab expression plasmid. This strategy allows for the rapid and efficient synthesis of 96 DNA constructs in a 3 day window, and exhibits high percentage fidelity-greater than 93%. Accordingly, the synthesis of Ab DNA constructs as Fab expression plasmids allow for rapid execution of downstream Ab protein validations, with potential for implementation into high-throughput Ab protein characterization pipelines. Altogether, the platform presented here proves rapid and also cost-effective, which is important for labs with limited resources, since it utilizes standard laboratory equipment and molecular reagents.

Evaluation of Molecularly Imprinted Polymers as Synthetic Virus Neutralizing Antibody Mimics.

Rapid development of antibody-based therapeutics are crucial to the agenda of innovative manufacturing of macromolecular therapies to combat emergent diseases. Although highly specific, antibody therapies are costly to produce. Molecularly imprinted polymers (MIPs) constitute a rapidly-evolving class of antigen-recognition materials that act as synthetic antibodies. We report here on the virus neutralizing capacity of hydrogel-based MIPs. We produced MIPs using porcine reproductive and respiratory syndrome virus (PRRSV-1), as a model mammalian virus. Assays were performed to evaluate the specificity of virus neutralization, the effect of incubation time and MIP concentration. Polyacrylamide and N-hydroxymethylacrylamide based MIPs produced a highly significant reduction in infectious viral titer recovered after treatment, reducing it to the limit of detection of the assay. MIP specificity was tested by comparing their neutralizing effects on PRRSV-1 to the effects on the unrelated bovine viral diarrhea virus-1; no significant cross-reactivity was observed. The MIPs demonstrated effective virus neutralization in just 2.5 min and their effect was concentration dependent. These data support the further evaluation of MIPs as synthetic antibodies as a novel approach to the treatment of viral infection.

Efficient Construction and Effective Screening of Synthetic Domain Antibody Libraries.

Phage display is a powerful technique for drug discovery in biomedical research in particular for antibody libraries. But, several technical challenges are associated with the selection process. For instance, during the panning step, the successful elution of the phages bound to the antigen is critical in order to avoid losing the most promising binders. Here, we present an efficient protocol to establish, screen and select synthetic libraries of domain antibodies using phage display. We do not only present suitable solutions to the above-mentioned challenges to improve elution by 50-fold, but we also present a step by step in-depth protocol with miniaturized volumes and optimized procedures to save material, costs and time for a successful phage display with domain antibodies. Hence, this protocol improves the selection process for an efficient handling process. The here presented library is based on the variable domain (vNAR) of the naturally occurring novel antibody receptor (IgNAR) from cartilage fishes. Diversity was introduced in the Complementarity-Determining Region 3 (CDR3) of the antigen-binding site with different composition and length.