Reproducible Discovery of Cell-Binding Peptides "Lost" in Bulk Amplification via Emulsion Amplification in Phage Display Panning

Many pharmaceutically-relevant cell surface receptors are functional only in the context of intact cells. Phage display, while being a powerful method for the discovery of ligands for purified proteins often fails to identify a diverse set of ligands to receptors on a cell membrane mosaic. To understand this deficiency, we examined growth bias in naive phage display libraries and observed that it fundamentally changes selection outcomes: The presence of growth-biased (parasite) phage clones in a phage library is detrimental to selection and cell-based panning of such biased libraries is poised to yield ligands from within a small parasite population. Importantly, amplification of phage libraries in water-oil emulsions suppressed the amplification of parasites and steered the selection of biased phage libraries away from parasite population. Attenuation of the growth bias through the use of emulsion amplification reproducibly discovers the ligands for cell-surface receptors that cannot be identified in screen that use conventional "bulk" amplification.

Phage display as a tool for identifying HIV-1 broadly neutralizing antibodies

Combinatorial biology methods offer a good solution for targeting interactions of specific molecules by a high-throughput screening and are widely used for drug development, diagnostics, identification of novel monoclonal antibodies, search for linear peptide mimetics of discontinuous epitopes for the development of immunogens or vaccine components. Among all currently available techniques, phage display remains one of the most popular approaches. Despite being a fairly old method, phage display is still widely used for studying protein-protein, peptide-protein and DNA-protein interactions due to its relative simplicity and versatility. Phage display allows highly representative libraries of peptides, proteins or their fragments to be created. Each phage particle in a library displays peptides or proteins fused to its coat protein and simultaneously carries the DNA sequence encoding the displayed peptide/protein in its genome. The biopanning procedure allows isolation of specific clones for almost any target, and due to the physical link between the genotype and the phenotype of recombinant phage particles it is possible to determine the structure of selected molecules. Phage display technology continues to play an important role in HIV research. A major obstacle to the development of an effective HIV vaccine is an extensive genetic and antigenic variability of the virus. According to recent data, in order to provide protection against HIV infection, the so-called broadly neutralizing antibodies that are cross-reactive against multiple viral strains of HIV must be induced, which makes the identification of such antibodies a key area of HIV vaccinology. In this review, we discuss the use of phage display as a tool for identification of HIV-specific antibodies with broad neutralizing activity. We provide an outline of phage display technology, briefly describe the design of antibody phage libraries and the affinity selection procedure, and discuss the biology of HIV-1-specific broadly neutralizing antibodies. Finally, we summarize the studies aimed at identification of broadly neutralizing antibodies using various types of phage libraries.

Multivariate mining of an alpaca immune repertoire identifies potent cross-neutralising SARS-CoV-2 nanobodies

Conventional approaches to isolate and characterize nanobodies are laborious and cumbersome. Here we combine phage display, multivariate enrichment, and novel sequence analysis techniques to annotate an entire nanobody repertoire from an immunized alpaca. We combine this approach with a streamlined screening strategy to identify numerous anti-SARS-CoV-2 nanobodies, and use neutralization assays and Hydrogen/Deuterium exchange coupled to mass spectrometry (HDX-MS) epitope mapping to characterize their potency and specificity. Epitope mapping revealed that the binding site is a key determinant of neutralization potency, rather than affinity alone. The most potent nanobodies bind to the receptor binding motif of the RBD, directly preventing interaction with the host cell receptor ACE2, and we identify two exceptionally potent members of this category (with monomeric IC50s around 13 and 16 ng/ml). Other nanobodies bind to a more conserved epitope on the side of the RBD, and are able to potently neutralize the SARS-CoV-2 founder virus (42 ng/ml), the beta variant (B.1.351/501Y.V2) (35 ng/ml), and also cross-neutralize the more distantly related SARS-CoV-1 (0.46 μg/ml). The approach presented here is well suited for the screening of phage libraries to identify functional nanobodies for various biomedical and biochemical applications.

Semi-Automated Cell Panning for Efficient Isolation of FGFR3-Targeting Antibody

Phage display technology is a widely used practical tool for isolating binding molecules against the desired targets in phage libraries. In the case of targeting the membrane protein with its natural conformation, conventional bio-panning has limitations on the efficient screening of the functionally relevant antibodies. To enrich the single-chain variable fragment (scFv) pools for recognizing the natural conformation of the membrane targets, the conventional bio-panning and screening process was modified to include the semi-automated cell panning protocol. Using FGFR3-overexpressing patient-derived cancer cells, biotin-X-DHPE was introduced and coupled to Streptavidin-coated magnetic beads for use in the solution-phage bio-panning procedure. The resulting clones of scFv were compared to the diversity of the binding region, especially on CDR-H3. The clones enriched further by cell-based panning procedure possessed a similar binding site and the CDR-H3 loop structure. The resulting antibodies inhibited cell growth and induced target degradation. This process may be a useful tool for screening biologically related antibodies that recognize natural conformational structure on cell membrane protein. Furthermore, cell-based panning has the potential to further expand to a high-throughput screening (HTS) system and automation process.

Human single-chain antibodies neutralize SARS-CoV-2 variants by engaging an essential epitope of the spike: a new weapon against COVID-19

As of June 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global emergency and effective therapeutic interventions for the treatment and prevention of coronavirus disease 2019 (COVID-19) are urgently needed. SARS-CoV-2-neutralizing monoclonal antibodies (mAbs) represent a promising approach to COVID-19 therapy. However, the recently described accumulating mutations in the SARS-CoV-2 spike protein are challenging the efficacy of approved and investigational mAbs, whose widespread use is also hampered by their significant costs and possible side effects, including Antibody-Dependent Enhancement (ADE). Here we describe a cluster of SARS-CoV-2 neutralizing human single chain variable fragment antibodies, identified by phage display, sharing a common VH CDR3 sequence. Phage libraries were built by amplifying variable domains of immunoglobulin genes from cDNA derived from lymphocytes of COVID-19 convalescent subjects living in Bergamo, Italy. The scFv76-cluster antibodies (scFv76-cl Abs) exhibit high affinity for the spike receptor binding domain (RBD) of Wuhan strain and emerging variants, leading to inhibition of RBD/human ACE2 interaction. The antigenic epitope recognized by scFv76 was mapped in the receptor binding motif (RBM) of RBD at residues L455, F456, Y473, N487 and Y489. None of these residues has been to date listed among the RBD mutations of SARS-CoV-2 variants of concern (VOCs), suggesting an important role of such epitope in viral infectivity. Treatment with scFv76-cl Abs is effective against SARS-CoV-2, as determined by in vitro experiments of viral infection, replication, cytopathogenicity and spike-mediated syncytia formation. Moreover, their intranasal administration is shown to counteract infection in two independent animal models. Overall, the biochemical and biological characteristics of scFv76-cl Abs are compatible with their clinical use for COVID-19 therapy by intranasal or aerosol administration. To our knowledge, this is the first example of promising human anti-SARS-CoV-2 scFv antibodies as drug candidates for COVID-19 therapy.

Neisseria gonorrhoeae Multivalent Maxibody with a Broad Spectrum of Strain Specificity and Sensitivity for Gonorrhea Diagnosis

Gonorrhea is one of the most common, but still hidden and insidious, sexually transmitted diseases caused by Neisseria gonorrhoeae (gonococci). However, the diagnosis and treatment of gonorrhea are hampered by antigenic variability among gonococci, the lack of acquired immunity, and antimicrobial resistance. Further, strains resistant to cephalosporins, including ceftriaxone, the last line of defense, represent a growing threat, which prompted us to develop gonococci-specific diagnostic antibodies with broad-spectrum binding to gonococci strains to generate gonorrhea-detecting reagents. This study reports the identification of gonococci antibodies via bio-panning on gonococci cells using scFv-phage libraries. Reformatting the lead scFv-phage Clones 1 and 4 to a multivalent scFv1-Fc-scFv4 maxibody increased the sensitivity by up to 20-fold compared to the single scFv-Fc (maxibody) alone. Moreover, the multivalent maxibody showed broader cross-reactivity with clinical isolates and the ceftriaxone antibiotic-resistant World Health Organization (WHO) reference strain L. In contrast, the selected antibodies in the scFv-phage, maxibody, and multivalent maxibody did not bind to N. sicca, N. meningitides, and N. lactamica, suggesting the clinical and pharmaceutical diagnostic value of these selected antibodies for gonorrheal infections. The present study illustrates the advantages and potential application of multivalent maxibodies to develop rapid and sensitive diagnostic reagents for infectious diseases and cancer.

Tumor-Targeting Peptides Search Strategy for the Delivery of Therapeutic and Diagnostic Molecules to Tumor Cells

Background: The combination of the unique properties of cancer cells makes it possible to find specific ligands that interact directly with the tumor, and to conduct targeted tumor therapy. Phage display is one of the most common methods for searching for specific ligands. Bacteriophages display peptides, and the peptides themselves can be used as targeting molecules for the delivery of diagnostic and therapeutic agents. Phage display can be performed both in vitro and in vivo. Moreover, it is possible to carry out the phage display on cells pre-enriched for a certain tumor marker, for example, CD44 and CD133. Methods: For this work we used several methods, such as phage display, sequencing, cell sorting, immunocytochemistry, phage titration. Results: We performed phage display using different screening systems (in vitro and in vivo), different phage libraries (Ph.D-7, Ph.D-12, Ph.D-C7C) on CD44+/CD133+ and without enrichment U-87 MG cells. The binding efficiency of bacteriophages displayed tumor-targeting peptides on U-87 MG cells was compared in vitro. We also conducted a comparative analysis in vivo of the specificity of the accumulation of selected bacteriophages in the tumor and in the control organs (liver, brain, kidney and lungs). Conclusions: The screening in vivo of linear phage peptide libraries for glioblastoma was the most effective strategy for obtaining tumor-targeting peptides providing targeted delivery of diagnostic and therapeutic agents to glioblastoma.

Development of humanized tri-specific nanobodies with potent neutralization for SARS-CoV-2

SARS-CoV-2 is a newly emergent coronavirus, which has adversely impacted human health and has led to the COVID-19 pandemic. There is an unmet need to develop therapies against SARS-CoV-2 due to its severity and lack of treatment options. A promising approach to combat COVID-19 is through the neutralization of SARS-CoV-2 by therapeutic antibodies. Previously, we described a strategy to rapidly identify and generate llama nanobodies (VHH) from naïve and synthetic humanized VHH phage libraries that specifically bind the S1 SARS-CoV-2 spike protein, and block the interaction with the human ACE2 receptor. In this study we used computer-aided design to construct multi-specific VHH antibodies fused to human IgG1 Fc domains based on the epitope predictions for leading VHHs. The resulting tri-specific VHH-Fc antibodies show more potent S1 binding, S1/ACE2 blocking, and SARS-CoV-2 pseudovirus neutralization than the bi-specific VHH-Fcs or combination of individual monoclonal VHH-Fcs. Furthermore, protein stability analysis of the VHH-Fcs shows favorable developability features, which enable them to be quickly and successfully developed into therapeutics against COVID-19.