A nanobody recognizes a unique conserved epitope and potently neutralizes SARS-CoV-2 omicron variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) Omicron variant sub-lineages spread rapidly worldwide, mostly due to their immune-evasive properties. This has put a significant part of the population at risk for severe disease and underscores the need for effective anti-SARS-CoV-2 agents against emergent strains in vulnerable patients. Camelid nanobodies are attractive therapeutic candidates due to their high stability, ease of large-scale production, and potential for delivery via inhalation. Here, we characterize the receptor binding domain (RBD)-specific nanobody W25 and show superior neutralization activity toward Omicron sub-lineages in comparison to all other SARS-CoV2 variants. Structure analysis of W25 in complex with the SARS-CoV2 spike glycoprotein shows that W25 engages an RBD epitope not covered by any of the antibodies previously approved for emergency use. In vivo evaluation of W25 prophylactic and therapeutic treatments across multiple SARS-CoV-2 variant infection models, together with W25 biodistribution analysis in mice, demonstrates favorable pre-clinical properties. Together, these data endorse W25 for further clinical development.

Evaluation of Phage Display Biopanning Strategies for the Selection of Anti-Cell Surface Receptor Antibodies.

Monoclonal antibodies (mAbs) are one of the most successful and versatile protein-based pharmaceutical products used to treat multiple pathological conditions. The remarkable specificity of mAbs and their affinity for biological targets has led to the implementation of mAbs in the therapeutic regime of oncogenic, chronic inflammatory, cardiovascular, and infectious diseases. Thus, the discovery of novel mAbs with defined functional activities is of crucial importance to expand our ability to address current and future clinical challenges. In vitro, antigen-driven affinity selection employing phage display biopanning is a commonly used technique to isolate mAbs. The success of biopanning is dependent on the quality and the presentation format of the antigen, which is critical when isolating mAbs against membrane protein targets. Here, we provide a comprehensive investigation of two established panning strategies, surface-tethering of a recombinant extracellular domain and cell-based biopanning, to examine the impact of antigen presentation on selection outcomes with regards to the isolation of positive mAbs with functional potential against a proof-of-concept type I cell surface receptor. Based on the higher sequence diversity of the resulting antibody repertoire, presentation of a type I membrane protein in soluble form was more advantageous over presentation in cell-based format. Our results will contribute to inform and guide future antibody discovery campaigns against cell surface proteins.

Preclinical Evaluation of a Fluorescent Probe Targeting Receptor CDCP1 for Identification of Ovarian Cancer.

Optimal cytoreduction for ovarian cancer is often challenging because of aggressive tumor biology and advanced stage. It is a critical issue since the extent of residual disease after surgery is the key predictor of ovarian cancer patient survival. For a limited number of cancers, fluorescence-guided surgery has emerged as an effective aid for tumor delineation and effective cytoreduction. The intravenously administered fluorescent agent, most commonly indocyanine green (ICG), accumulates preferentially in tumors, which are visualized under a fluorescent light source to aid surgery. Insufficient tumor specificity has limited the broad application of these agents in surgical oncology including for ovarian cancer. In this study, we developed a novel tumor-selective fluorescent agent by chemically linking ICG to mouse monoclonal antibody 10D7 that specifically recognizes an ovarian cancer-enriched cell surface receptor, CUB-domain-containing protein 1 (CDCP1). 10D7ICG has high affinity for purified recombinant CDCP1 and CDCP1 that is located on the surface of ovarian cancer cells in vitro and in vivo. Our results show that intravenously administered 10D7ICG accumulates preferentially in ovarian cancer, permitting visualization of xenograft tumors in mice. The data suggest CDCP1 as a rational target for tumor-specific fluorescence-guided surgery for ovarian cancer.

Preclinical development of a molecular clamp-stabilised subunit vaccine for severe acute respiratory syndrome coronavirus 2.

OBJECTIVES: Efforts to develop and deploy effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue at pace. Here, we describe rational antigen design through to manufacturability and vaccine efficacy of a prefusion-stabilised spike (S) protein, Sclamp, in combination with the licensed adjuvant MF59 'MF59C.1' (Seqirus, Parkville, Australia). METHODS: A panel recombinant Sclamp proteins were produced in Chinese hamster ovary and screened in vitro to select a lead vaccine candidate. The structure of this antigen was determined by cryo-electron microscopy and assessed in mouse immunogenicity studies, hamster challenge studies and safety and toxicology studies in rat. RESULTS: In mice, the Sclamp vaccine elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S-specific CD4+ and cytotoxic CD8+ T cells in vivo. In the Syrian hamster challenge model (n = 70), vaccination results in reduced viral load within the lung, protection from pulmonary disease and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level. CONCLUSION: The SARS-CoV-2 Sclamp vaccine candidate is compatible with large-scale commercial manufacture, stable at 2-8°C. When formulated with MF59 adjuvant, it elicits neutralising antibodies and T-cell responses and provides protection in animal challenge models.

Recombinant Antibody Engineering Enables Reversible Binding for Continuous Protein Biosensing.

Engineering antibodies to improve target specificity, reduce detection limits, or introduce novel functionality is an important research area for biosensor development. While various affinity biosensors have been developed to generate an output signal upon varying analyte concentrations, reversible and continuous protein monitoring in complex biological samples remains challenging. Herein, we explore the concept of directed evolution to modulate dissociation kinetics of a high affinity anti-epidermal growth factor receptor (EGFR) single-chain variable antibody fragment (scFv) to enable continuous protein sensing in a label-free binding assay. A mutant scFv library was generated from the wild type (WT) fragment via targeted permutation of four residues in the antibody-antigen-binding interface. A single round of phage display biopanning complemented with high-throughput screening methods then permitted isolation of a specific binder with fast reaction kinetics. We were able to obtain ∼30 times faster dissociation rates when compared to the WT without appreciably affecting overall affinity and specificity by targeting a single paratope that is known to contribute to the binding interaction. Suitability of a resulting mutant fragment to sense varying antigen concentrations in continuous mode was demonstrated in a modified label-free binding assay, achieving low nanomolar detection limits (KD = 8.39 nM). We also confirmed these results using an independent detection mechanism developed previously by our group, incorporating a polarity-dependent fluorescent dye into the scFv and reading out EGFR binding based on fluorescence wavelength shifts. In future, this generic approach could be employed to generate improved or novel binders for proteins of interest, ready for deployment in a broad range of assay platforms.

Aggregates in blood filter chambers used from the plasma donations of anti-D donors: evaluation for monoclonal antibody discovery using phage display.

BACKGROUND: RhD-immunoglobulin (RhIg) prevents anti-D alloimmunisation in D-negative pregnant women when the fetus is D-positive, reducing the incidence of haemolytic disease of the fetus and newborn. Manufacturing RhIg is reliant on the limited supply of plasma donations with anti-D antibodies. Monoclonal antibody (mAb) development platforms such as phage display, require blood samples to be collected from anti-D donors, which may be a complicated process. The blood filter chamber (BFC) discarded after an anti-D donor's donation might provide a source of Ig-encoding RNA. This study aims to evaluate whether used BFCs are a suitable source of Ig-encoding RNA for phage display. MATERIAL AND METHODS: Haemonetics PCS2 BFCs were obtained from 10 anti-D donors for total RNA extraction, cDNA synthesis and amplification of VH and VL IgG sequences for assembly of single-chain variable fragments (scFvs). A scFv-phage display library was constructed and 3 rounds of biopanning were performed using D-positive and D-negative red blood cells (RBCs). Positive phage clones were isolated, Sanger sequenced and, where possible, reformatted into full-length human IgGs to define specificity. The BFC aggregates from 2 anti-D donors underwent a Wright-Giemsa stain and hematological cell count. RESULTS: Of 10 BFCs, a sufficient yield of total RNA for library construction was obtained from BFCs containing cellular aggregates (n=5). Aggregate analysis showed lymphocytes were the cellular source of Ig-encoding RNA. From the 5 samples with aggregates, scFvs were assembled from amplified IgG variable regions. The library constructed from 1 of these samples resulted in the isolation of clones binding to D-positive RBCs with IGHV3 gene usage. Of the 4 reformatted IgG, 3 were anti-D and 1 had undefined specificity. DISCUSSION: BFC aggregates are a new and convenient source of Ig-encoding RNA which can be used to construct Ig gene libraries for mAb isolation and discovery via antibody phage display.

Phage Display Derived Monoclonal Antibodies: From Bench to Bedside.

Monoclonal antibodies (mAbs) have become one of the most important classes of biopharmaceutical products, and they continue to dominate the universe of biopharmaceutical markets in terms of approval and sales. They are the most profitable single product class, where they represent six of the top ten selling drugs. At the beginning of the 1990s, an in vitro antibody selection technology known as antibody phage display was developed by John McCafferty and Sir. Gregory Winter that enabled the discovery of human antibodies for diverse applications, particularly antibody-based drugs. They created combinatorial antibody libraries on filamentous phage to be utilized for generating antigen specific antibodies in a matter of weeks. Since then, more than 70 phage-derived antibodies entered clinical studies and 14 of them have been approved. These antibodies are indicated for cancer, and non-cancer medical conditions, such as inflammatory, optical, infectious, or immunological diseases. This review will illustrate the utility of phage display as a powerful platform for therapeutic antibodies discovery and describe in detail all the approved mAbs derived from phage display.

Safety, tolerability, pharmacokinetics, and immunogenicity of a human monoclonal antibody targeting the G glycoprotein of henipaviruses in healthy adults: a first-in-human, randomised, controlled, phase 1 study.

BACKGROUND: The monoclonal antibody m102.4 is a potent, fully human antibody that neutralises Hendra and Nipah viruses in vitro and in vivo. We aimed to investigate the safety, tolerability, pharmacokinetics, and immunogenicity of m102.4 in healthy adults. METHODS: In this double-blind, placebo-controlled, single-centre, dose-escalation, phase 1 trial of m102.4, we randomly assigned healthy adults aged 18-50 years with a body-mass index of 18·0-35·0 kg/m2 to one of five cohorts. A sentinel pair for each cohort was randomly assigned to either m102.4 or placebo. The remaining participants in each cohort were randomly assigned (5:1) to receive m102.4 or placebo. Cohorts 1-4 received a single intravenous infusion of m102.4 at doses of 1 mg/kg (cohort 1), 3 mg/kg (cohort 2), 10 mg/kg (cohort 3), and 20 mg/kg (cohort 4), and were monitored for 113 days. Cohort 5 received two infusions of 20 mg/kg 72 h apart and were monitored for 123 days. The primary outcomes were safety and tolerability. Secondary outcomes were pharmacokinetics and immunogenicity. Analyses were completed according to protocol. The study was registered on the Australian New Zealand Clinical Trials Registry, ACTRN12615000395538. FINDINGS: Between March 27, 2015, and June 16, 2016, 40 (52%) of 77 healthy screened adults were enrolled in the study. Eight participants were assigned to each cohort (six received m102.4 and two received placebo). 86 treatment-emergent adverse events were reported, with similar rates between placebo and treatment groups. The most common treatment-related event was headache (12 [40%] of 30 participants in the combined m102.4 group, and three [30%] of ten participants in the pooled placebo group). No deaths or severe adverse events leading to study discontinuation occurred. Pharmacokinetics based on those receiving m102.4 (n=30) were linear, with a median half-life of 663·3 h (range 474·3-735·1) for cohort 1, 466·3 h (382·8-522·3) for cohort 2, 397·0 h (333·9-491·8) for cohort 3, and 466·7 h (351·0-889·6) for cohort 4. The elimination kinetics of those receiving repeated dosing (cohort 5) were similar to those of single-dose recipients (median elimination half-time 472·0 [385·6-592·0]). Anti-m102.4 antibodies were not detected at any time-point during the study. INTERPRETATION: Single and repeated dosing of m102.4 were well tolerated and safe, displayed linear pharmacokinetics, and showed no evidence of an immunogenic response. This study will inform future dosing regimens for m102.4 to achieve prolonged exposure for systemic efficacy to prevent and treat henipavirus infections. FUNDING: Queensland Department of Health, the National Health and Medical Research Council, and the National Hendra Virus Research Program.

Retooling phage display with electrohydrodynamic nanomixing and nanopore sequencing.

Phage display methodologies offer a versatile platform for the isolation of single-chain Fv (scFv) molecules which may be rebuilt into monoclonal antibodies. Herein, we report on a complete workflow termed PhageXpress, for rapid selection of single-chain Fv sequences by leveraging electrohydrodynamic-manipulation of a solution containing phage library particles to enhance target binding whilst minimizing non-specific interactions. Our PhageXpress technique is combined with Oxford Nanopore Technologies' MinION sequencer and custom bioinformatics to achieve high-throughput screening of phage libraries. We performed 4 rounds of biopanning against Dengue virus (DENV) non-structural protein 1 (NS1) using traditional methods (4 week turnaround), which resulted in the isolation of 19 unique scFv clones. We validated the feasibility and efficiency of the PhageXpress method utilizing the same phage library and antigen target. Notably, we successfully mapped 14 of the 19 anti-NS1 scFv sequences (∼74%) with our new method, despite using ∼30-fold less particles during screening and conducting only a single round of biopanning. We believe this approach supersedes traditional methods for the discovery of bio-recognition molecules such as antibodies by speeding up the process for the development of therapeutic and diagnostic biologics.

Canine CD117-Specific Antibodies with Diverse Binding Properties Isolated from a Phage Display Library Using Cell-Based Biopanning.

CD117 (c-Kit) is a tyrosine kinase receptor that is overexpressed in multiple dog tumors. There is 100% homology between the juxtamembrane domain of human and canine CD117, and many cancer-causing mutations occur in this region in both species. Thus, CD117 is an important target for cancer treatment in dogs and for comparative oncology studies. Currently, there is no monoclonal antibody (mAb) specifically designed to target the exposed region of canine CD117, although there exist some with species cross-reactivity. We panned a naïve phage display library to isolate antibodies against recombinant CD117 on whole cells. Several mAbs were isolated and were shown to bind recombinant canine CD117 at low- to sub-nanomolar affinity. Additionally, binding to native canine CD117 was confirmed by immunohistochemistry and by flow cytometry. Competitive binding assays also identified mAbs that competed with the CD117 receptor-specific ligand, the stem cell factor (SCF). These results show the ability of our cell-based biopanning strategy to isolate a panel of antibodies that have varied characteristics when used in different binding assays. These in vitro/ex vivo assessments suggest that some of the isolated mAbs might be promising candidates for targeting overexpressed CD117 in canine cancers for different useful applications.

Wavelength-Dependent Fluorescent Immunosensors via Incorporation of Polarity Indicators near the Binding Interface of Antibody Fragments.

Herein, we describe a fluorescent immunosensor designed by incorporating an unnatural amino acid fluorophore into the binding site of an EGFR-specific antibody fragment, resulting in quantifiable EGFR-dependent changes in peak fluorescence emission wavelength. To date, immunosensor design strategies have relied on binding-induced changes in fluorescence intensity that are prone to excitation source fluctuations and sample-dependent noise. In this study, we used a rational design approach to incorporate a polarity indicator (Anap) into specific positions of an anti-EGFR single chain antibody to generate an emission wavelength-dependent immunosensor. We found that when incorporated within the topological neighborhood of the antigen binding interface, the Anap emission wavelength is blue-shifted by EGFR-binding in a titratable manner, up to 20 nm, with nanomolar detection limits. This approach could be applicable to other antibody/antigen combinations for integration into a wide range of assay platforms (including homogeneous, solid-phase assay, or microfluidic assays) for one-step protein quantification.

Selection of Antibodies to Transiently Expressed Membrane Proteins Using Phage Display.

Cell membrane proteins serve as attractive targets for biopharmaceutical development in addition to gauging their fundamental process in a biological system. Approximately 38% of the entire genome codes for plasma membrane proteins; however the discovery and development of antibody binders to such targets are a technical challenge. Methods to raise binders against such targets by cloning and expressing soluble extracellular regions have been met with limited success due to the loss of critical epitopes, with the resulting antibodies failing to bind to their target in its native conformation. This chapter outlines a "cell based biopanning" method in order to isolate antibodies against membrane proteins in their native conformation using transiently expressed, GFP-tagged target proteins. This method overcomes the limitations of non-specific binding of phage to the cells, abundance of irrelevant antigens on the cell surface, while retaining the native structure of the antigen on the cell surface.

CD83 Antibody Inhibits Human B Cell Responses to Antigen as well as Dendritic Cell-Mediated CD4 T Cell Responses.

Anti-CD83 Ab capable of Ab-dependent cellular cytotoxicity can deplete activated CD83+ human dendritic cells, thereby inhibiting CD4 T cell-mediated acute graft-versus-host disease. As CD83 is also expressed on the surface of activated B lymphocytes, we hypothesized that anti-CD83 would also inhibit B cell responses to stimulation. We found that anti-CD83 inhibited total IgM and IgG production in vitro by allostimulated human PBMC. Also, Ag-specific Ab responses to immunization of SCID mice xenografted with human PBMC were inhibited by anti-CD83 treatment. This inhibition occurred without depletion of all human B cells because anti-CD83 lysed activated CD83+ B cells by Ab-dependent cellular cytotoxicity and spared resting (CD83-) B cells. In cultured human PBMC, anti-CD83 inhibited tetanus toxoid-stimulated B cell proliferation and concomitant dendritic cell-mediated CD4 T cell proliferation and expression of IFN-γ and IL-17A, with minimal losses of B cells (<20%). In contrast, the anti-CD20 mAb rituximab depleted >80% of B cells but had no effect on CD4 T cell proliferation and cytokine expression. By virtue of the ability of anti-CD83 to selectively deplete activated, but not resting, B cells and dendritic cells, with the latter reducing CD4 T cell responses, anti-CD83 may be clinically useful in autoimmunity and transplantation. Advantages might include inhibited expansion of autoantigen- or alloantigen-specific B cells and CD4 T cells, thus preventing further production of pathogenic Abs and inflammatory cytokines while preserving protective memory and regulatory cells.

Functional domain analysis of SOX18 transcription factor using a single-chain variable fragment-based approach.

Antibodies are routinely used to study the activity of transcription factors, using various in vitro and in vivo approaches such as electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, genome-wide method analysis coupled with next generation sequencing, or mass spectrometry. More recently, a new application for antibodies has emerged as crystallisation scaffolds for difficult to crystallise proteins, such as transcription factors. Only in a few rare cases, antibodies have been used to modulate the activity of transcription factors, and there is a real gap in our knowledge on how to efficiently design antibodies to interfere with transcription. The molecular function of transcription factors is underpinned by complex networks of protein-protein interaction and in theory, setting aside intra-cellular delivery challenges, developing antibody-based approaches to modulate transcription factor activity appears a viable option. Here, we demonstrate that antibodies or an antibody single-chain variable region fragments are powerful molecular tools to unravel complex protein-DNA and protein-protein binding mechanisms. In this study, we focus on the molecular mode of action of the transcription factor SOX18, a key modulator of endothelial cell fate during development, as well as an attractive target in certain pathophysiological conditions such as solid cancer metastasis. The engineered antibody we designed inhibits SOX18 transcriptional activity, by interfering specifically with an 8-amino-acid motif in the C-terminal region directly adjacent to α-Helix 3 of SOX18 HMG domain, thereby disrupting protein-protein interaction. This new approach establishes a framework to guide the study of transcription factors interactomes using antibodies as molecular handles.

Targeting mesothelin receptors with drug-loaded bacterial nanocells suppresses human mesothelioma tumour growth in mouse xenograft models.

Human malignant mesothelioma is a chemoresistant tumour that develops from mesothelial cells, commonly associated with asbestos exposure. Malignant mesothelioma incidence rates in European countries are still rising and Australia has one of the highest burdens of malignant mesothelioma on a population basis in the world. Therapy using systemic delivery of free cytotoxic agents is associated with many undesirable side effects due to non-selectivity, and is thus dose-limited which limits its therapeutic potential. Therefore, increasing the selectivity of anti-cancer agents has the potential to dramatically enhance drug efficacy and reduce toxicity. EnGeneIC Dream Vectors (EDV) are antibody-targeted nanocells which can be loaded with cytotoxic drugs and delivered to specific cancer cells via bispecific antibodies (BsAbs) which target the EDV and a cancer cell-specific receptor, simultaneously. BsAbs were designed to target doxorubicin-loaded EDVs to cancer cells via cell surface mesothelin (MSLN). Flow cytometry was used to investigate cell binding and induction of apoptosis, and confocal microscopy to visualize internalization. Mouse xenograft models were used to assess anti-tumour effects in vivo, followed by immunohistochemistry for ex vivo evaluation of proliferation and necrosis. BsAb-targeted, doxorubicin-loaded EDVs were able to bind to and internalize within mesothelioma cells in vitro via MSLN receptors and induce apoptosis. In mice xenografts, the BsAb-targeted, doxorubicin-loaded EDVs suppressed the tumour growth and also decreased cell proliferation. Thus, the use of MSLN-specific antibodies to deliver encapsulated doxorubicin can provide a novel and alternative modality for treatment of mesothelioma.

Isolation of serotype-specific antibodies against dengue virus non-structural protein 1 using phage display and application in a multiplexed serotyping assay.

The multidimensional nature of dengue virus (DENV) infections, which can be caused by four distinct serotypes of the virus, complicates the sensitivity of assays designed for the diagnosis of infection. Different viral markers can be optimally detected at different stages of infection. Of particular clinical importance is the early identification of infection, which is pivotal for disease management and the development of blood screening assays. Non-structural protein 1 (NS1) is an early surrogate marker of infection and its detection in serum coincides with detectable viraemia. The aim of this work was to isolate and characterise serotype-specific monoclonal antibodies that bind to NS1 for each of the four DENV serotypes. This was achieved using phage display and a subtractive biopanning strategy to direct the antibody selection towards serotype-specific epitopes. This antibody isolation strategy has advantages over immunisation techniques where it is difficult to avoid antibody responses to cross-reactive, immunodominant epitopes. Serotype specificity to recombinant antigen for each of the antibodies was confirmed by Enzyme Linked Immunosorbent Assay (ELISA) and Surface Plasmon Resonance. Confirmation of binding to native DENV NS1 was achieved using ELISA and immunofluorescence assay on DENV infected Vero cells. No cross-reactivity with Zika or Kunjin viruses was observed. A previously isolated pan-reactive antibody that binds to an immunodominant epitope was able to pair with each of the serotype-specific antibodies in a sandwich ELISA, indicating that the serotype specific antibodies bind to epitopes which are all spatially distinct from the immunodominant epitope. These antibodies were suitable for use in a multiplexed assay for simultaneous detection and serotyping of DENV NS1 in human serum. This work demonstrates that phage display coupled with novel biopanning strategies is a valuable in vitro methodology for isolation of binders that can discern amongst antigens with high homology for diagnostic applicability.

Computational Identification of Antibody Epitopes on the Dengue Virus NS1 Protein.

We have previously described a method to predict antigenic epitopes on proteins recognized by specific antibodies. Here we have applied this method to identify epitopes on the NS1 proteins of the four Dengue virus serotypes (DENV1-4) that are bound by a small panel of monoclonal antibodies 1H7.4, 1G5.3 and Gus2. Several epitope regions were predicted for these antibodies and these were found to reflect the experimentally observed reactivities. The known binding epitopes on DENV2 for the antibodies 1H7.4 and 1G5.3 were identified, revealing the reasons for the serotype specificity of 1H7.4 and 1G5.3, and the non-selectivity of Gus2. As DENV NS1 is critical for virus replication and a key vaccine candidate, epitope prediction will be valuable in designing appropriate vaccine control strategies. The ability to predict potential epitopes by computational methods significantly reduces the amount of experimental work required to screen peptide libraries for epitope mapping.

Strategies for Selecting Membrane Protein-Specific Antibodies using Phage Display with Cell-Based Panning.

Membrane proteins are attractive targets for monoclonal antibody (mAb) discovery and development. Although several approved mAbs against membrane proteins have been isolated from phage antibody libraries, the process is challenging, as it requires the presentation of a correctly folded protein to screen the antibody library. Cell-based panning could represent the optimal method for antibody discovery against membrane proteins, since it allows for presentation in their natural conformation along with the appropriate post-translational modifications. Nevertheless, screening antibodies against a desired antigen, within a selected cell line, may be difficult due to the abundance of irrelevant organic molecules, which can potentially obscure the antigen of interest. This review will provide a comprehensive overview of the different cell-based phage panning strategies, with an emphasis placed on the optimisation of four critical panning conditions: cell surface antigen presentation, non-specific binding events, incubation time, and temperature and recovery of phage binders.

Beyond Antibodies: Development of a Novel Protein Scaffold Based on Human Chaperonin 10.

Human Chaperonin 10 (hCpn10) was utilised as a novel scaffold for presenting peptides of therapeutic and diagnostic significance. Molecular dynamic simulations and protein sizing analyses identified a peptide linker (P1) optimal for the formation of the quarternary hCpn10 heptamer structure. hCpn10 scaffold displaying peptides targeting Factor VIIa (CE76-P1) and CD44 (CP7) were expressed in E. coli. Functional studies of CE76-P1 indicated nanomolar affinity for Factor VIIa (3 nM) similar to the E-76 peptide (6 nM), with undetectable binding to Factor X. CE76-P1 was a potent inhibitor of FX activity (via inhibition of Factor VIIa) and prolonged clot formation 4 times longer than achieved by E-76 peptide as determined by prothrombin time (PT) assays. This improvement in clotting function by CE76-P1, highlights the advantages of a heptamer-based scaffold for improving avidity by multiple peptide presentation. In another example of hCPn10 utility as a scaffold, CP7 bound to native CD44 overexpressed on cancer cells and bound rCD44 with high affinity (KD 9.6 nM). The ability to present various peptides through substitution of the hCpn10 mobile loop demonstrates its utility as a novel protein scaffold.