Neutralizing Antibody Fragment Research Articles

Production of neutralizing antibody fragment variants in the cytoplasm of E. coli for rapid screening: SARS-CoV-2 a case study

Global health challenges such as the coronavirus pandemic warrant the urgent need for a system that allows efficient production of diagnostic and therapeutic interventions. Antibody treatments against SARS-CoV-2 were developed with an unprecedented pace and this enormous progress was achieved mainly through recombinant protein production technologies combined with expeditious screening approaches. A heterologous protein production system that allows efficient soluble production of therapeutic antibody candidates against rapidly evolving variants of deadly pathogens is an important step in preparedness towards future pandemic challenges. Here, we report cost and time-effective soluble production of SARS-CoV-2 receptor binding domain (RBD) variants as well as an array of neutralizing antibody fragments (Fabs) based on Casirivimab and Imdevimab using the CyDisCo system in the cytoplasm of E. coli. We also report variants of the two Fabs with higher binding affinity against SARS-CoV-2 RBD and suggest this cytoplasmic production of disulfide containing antigens and antibodies can be broadly applied towards addressing future global public health threats.

An effective strategy for the humanization of antibody fragments under an accelerated timeline

The use of monoclonal antibodies (mAbs) in therapy is gradually advancing and discussions entail its safety, rentability and effectiveness. To this date, around a hundred mAbs have been approved by the FDA for the treatment of various diseases. Aiming for their large-scale production, recombinant DNA technology is mainly employed, and antibodies can be expressed in various eukaryotic and prokaryotic systems. Moreover, considering their heterologous origin and potential immunogenicity, various strategies have been developed for mAb humanization, considering that around 50 % of commercial mAbs are humanized. Hence, we introduce LimAb7, a mouse mAb capable of binding and neutralizing brown spider's Loxosceles intermedia dermonecrotic toxins in vivo/in vitro. This antibody has been produced in mouse and humanized scFv and diabody formats, however results indicated losses in antigen-binding affinity, stability, and neutralizing ability. Intending to develop evolved, stable, and neutralizing antibody fragments, we report for the first time the design of humanized antibody V-domains produced as Fab fragments, against spider venom toxins. Improvements in constructs were observed regarding their physicochemical stability, target binding and binding pattern maintenance. As their neutralizing features remain to be characterized, we believe this data sheds new light on antibody humanization by producing a parental molecule in different recombinant formats.

501Y.V2 spike protein resists the neutralizing antibody in atomistic simulations

SARS-CoV-2 outbreaks worldwide caused COVID-19 pandemic, which is related to several million deaths. In particular, SARS-CoV-2 Spike (S) protein is a major biological target for COVID-19 vaccine design. Unfortunately, recent reports indicated that Spike (S) protein mutations can lead to antibody resistance. However, understanding the process is limited, especially at the atomic scale. The structural change of S protein and neutralizing antibody fragment (FAb) complexes was thus probed using molecular dynamics (MD) simulations. In particular, the backbone RMSD of the 501Y.V2 complex was significantly larger than that of the wild-type one implying a large structural change of the mutation system. Moreover, the mean of Rg, CCS, and SASA are almost the same when compared two complexes, but the distributions of these values are absolutely different. Furthermore, the free energy landscape of the complexes was significantly changed when the 501Y.V2 variant was induced. The binding pose between S protein and FAb was thus altered. The FAb-binding affinity to S protein was thus reduced due to revealing over steered-MD (SMD) simulations. The observation is in good agreement with the respective experiment that the 501Y.V2 SARS-CoV-2 variant can escape from neutralizing antibody (NAb).

Engineering and Functional Evaluation of Neutralizing Antibody Fragments Against Congenital Toxoplasmosis.

Maternal-fetal transmission of Toxoplasma gondii tachyzoites acquired during pregnancy has potentially dramatic consequences for the fetus. Current reference-standard treatments are not specific to the parasite and can induce severe side effects. In order to provide treatments with a higher specificity against toxoplasmosis, we developed antibody fragments-single-chain fragment variable (scFv) and scFv fused with mouse immunoglobulin G2a crystallizable fragment (scFv-Fc)-directed against the major surface protein SAG1. After validating their capacity to inhibit T. gondii proliferation in vitro, the antibody fragments' biological activity was assessed in vivo using a congenital toxoplasmosis mouse model. Dams were treated by systemic administration of antibody fragments and with prevention of maternal-fetal transmission being used as the parameter of efficacy. We observed that both antibody fragments prevented T. gondii dissemination and protected neonates, with the scFv-Fc format having better efficacy. These data provide a proof of concept for the use of antibody fragments as effective and specific treatment against congenital toxoplasmosis and provide promising leads.

Cryo-electron microscopy structures of the N501Y SARS-CoV-2 spike protein in complex with ACE2 and 2 potent neutralizing antibodies.

The recently reported “UK variant” (B.1.1.7) of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-Å resolution cryo-electron microscopy (cryo-EM) structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. This additional interaction provides a structural explanation for the increased ACE2 affinity of the N501Y mutant, and likely contributes to its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to 2 representative potent neutralizing antibody fragments.

Injectable hydrogels functionalized with antibody fragments as intra-articular cytokine sinks neutralizing pro-catabolic cytokines

Purpose: Intra-articular (IA) injection with biologics like cytokine neutralizing antibodies to treat osteoarthritis (OA) have been explored, but so far success is limited. One of the key obstacle for the efficacy of intra-articular therapies is the short retention time of these biologics in the joint space due to rapid clearing by the synovial membrane. To overcome these limitations, we developed a new concept that potentially can increase the therapeutic value of pro-inflammatory cytokine neutralizing antibody fragments in the management of OA. The concept is based on the use of injectable hydrogels functionalized with the variable domain of single chain heavy chain only antibodies (VHHs) for cytokine capture. VHHs can be easily isolated using phage-display technology, recombinantly engineered and produced in yeast. These hydrogels can either be pre-crosslinked in microgels before injection or injected as polymer-VHH conjugates that cross link in a hydrogel in situ. Here, hyaluronic acid was used as model polymer after substitution with tyramine and maleimide groups. The tyramine moiety is used in an enzymatic cross-linking reaction to yield stable macromolecular hydrogel networks. The maleimide group is used for thiol-maleimide chemistry coupling a recombinantly engineered VHH with a free Cysteine in its C-terminal tail to the polymer backbone. We hypothesized that this strategy to functionalize hydrogels with the VHH can retain biological activity of neutralizing VHH. To proof the concept of these so-called cytokine sinks we have selected an anti-TNFα VHH that effectively neutralizes TNFα, which plays an essential role in cartilage degradation in inflammatory arthritis. Methods: To achieve directed conjugation of the VHH to the polymer, we have introduced an unpaired cysteine by using recombinant DNA technology in the C-terminus of the anti-TNFα VHH. Afterwards, the modified VHH was produced in yeast and purified. The conjugation of the VHH to the hyaluronic acid was performed by incubating the VHH with tyramine and maleimide functionalized hyaluronic acid. Characterization of binding affinity for both the non-conjugated VHH and conjugated VHH were measured by ELISA. hydrogels functionalized with the VHH was prepared by mixing the hyaluronic acid-VHH conjugates with enzyme peroxidase and H2O2. Biological activity of the conjugated VHH and hydrogels functionalized with the VHH was measured using an NFκb responsive luciferase reporter cell line after stimulation with TNFα. An equimolar concentration of non-conjugated VHH was used as a control. Results: We show successful conjugation of the VHH to the polymer backbone. The VHH functionalized polymer could be used for making stable hydrogels after tyramine mediated cross linking. This modification did not affect the biological activity of the VHH. Using an NFκb responsive luciferase reporter cell line we demonstrated that hydrogels functionalized with the VHH efficiently inactivated TNFα and that this inhibition was comparable to inhibition by an equimolar concentration of non-conjugated VHH. Conclusions: We successfully developed a biocompatible and efficient way to couple VHH to hyaluronic acid. These conjugates could be used for in situ generation of cytokine sinks capable of capturing different pro-catabolic cytokines in treatment of OA. Our Results demonstrate that cytokine sinks have great potential for neutralizing inflammatory pro-catabolic cytokines after intra-articular injection by increasing the retention time of neutralizing antibody fragments in the joint cavity.

HCV Glycoprotein Structure and Implications for B-Cell Vaccine Development.

Despite the approval of highly efficient direct-acting antivirals in the last decade Hepatitis C virus (HCV) remains a global health burden and the development of a vaccine would constitute an important step towards the control of HCV. The high genetic variability of the viral glycoproteins E1 and E2, which carry the main neutralizing determinants, together with their intrinsic structural flexibility, the high level of glycosylation that shields conserved neutralization epitopes and immune evasion using decoy epitopes renders the design of an efficient vaccine challenging. Recent structural and functional analyses have highlighted the role of the CD81 receptor binding site on E2, which overlaps with those neutralization epitopes within E2 that have been structurally characterized to date. This CD81 binding site consists of three distinct segments including “epitope I”, “epitope II” and the “CD81 binding loop”. In this review we summarize the structural features of the HCV glycoproteins that have been derived from X-ray structures of neutralizing and non-neutralizing antibody fragments complexed with either recombinant E2 or epitope-derived linear peptides. We focus on the current understanding how neutralizing antibodies interact with their cognate antigen, the structural features of the respective neutralization epitopes targeted by nAbs and discuss the implications for informed vaccine design.

Development of Neutralizing and Non-neutralizing Antibodies Targeting Known and Novel Epitopes of TcdB of Clostridioides difficile.

Clostridioides difficile is the causative bacterium in 15–20% of all antibiotic associated diarrheas. The symptoms associated with C. difficile infection (CDI) are primarily induced by the two large exotoxins TcdA and TcdB. Both toxins enter target cells by receptor-mediated endocytosis. Although different toxin receptors have been identified, it is no valid therapeutic option to prevent receptor endocytosis. Therapeutics, such as neutralizing antibodies, directly targeting both toxins are in development. Interestingly, only the anti-TcdB antibody bezlotoxumab but not the anti-TcdA antibody actoxumab prevented recurrence of CDI in clinical trials. In this work, 31 human antibody fragments against TcdB were selected by antibody phage display from the human naive antibody gene libraries HAL9/10. These antibody fragments were further characterized by in vitro neutralization assays. The epitopes of the neutralizing and non-neutralizing antibody fragments were analyzed by domain mapping, TcdB fragment phage display, and peptide arrays, to identify neutralizing and non-neutralizing epitopes. A new neutralizing epitope within the glucosyltransferase domain of TcdB was identified, providing new insights into the relevance of different toxin regions in respect of neutralization and toxicity.

The European AntibotABE Framework Program and Its Update: Development of Innovative Botulinum Antibodies.

The goal of the AntiBotABE Program was the development of recombinant antibodies that neutralize botulinum neurotoxins (BoNT) A, B and E. These serotypes are lethal and responsible for most human botulinum cases. To improve therapeutic efficacy, the heavy and light chains (HC and LC) of the three BoNT serotypes were targeted to achieve a synergistic effect (oligoclonal antibodies). For antibody isolation, macaques were immunized with the recombinant and non-toxic BoNT/A, B or E, HC or LC, followed by the generation of immune phage-display libraries. Antibodies were selected from these libraries against the holotoxin and further analyzed in in vitro and ex vivo assays. For each library, the best ex vivo neutralizing antibody fragments were germline-humanized and expressed as immunoglobulin G (IgGs). The IgGs were tested in vivo, in a standardized model of protection, and challenged with toxins obtained from collections of Clostridium strains. Protective antibody combinations against BoNT/A and BoNT/B were evidenced and for BoNT/E, the anti-LC antibody alone was found highly protective. The combination of these five antibodies as an oligoclonal antibody cocktail can be clinically and regulatorily developed while their high “humanness” predicts a high tolerance in humans.

Exploration of broadly neutralizing antibody fragments produced in bacteria for the control of HIV

ABSTRACTWhile broadly neutralizing antibodies (bnAbs) are a promising preventative and therapeutic tool for HIV infection, production is difficult and expensive. Production of antibody-like fragments in bacterial cytoplasm provides a cheaper alternative. This work explored the transplantation of the complementarity determining regions of the anti-HIV bnAbs PGT121 and 10E8 onto a single-chain variable fragment (scFv) scaffold, previously discovered through a novel screening platform. The scaffolded 10E8 scFv, but not the scaffolded PGT121 scFv, was soluble in bacterial cytoplasm, enabling efficient production in bacteria. Three additional multimeric constructs employing the scaffolded 10E8 scFv were also generated and soluble versions produced in bacteria. However, the constructs were found to have substantially lost anti-HIV binding function and had completely abrogated neutralizing activity. Overall, while this study provides a proof-of-concept for anti-HIV bnAb construct production in bacterial cytoplasm, future refinement of these technologies will be required to realize the goal of producing inexpensive and effective bnAb-like tools for the control of HIV.

Exceptionally long CDR3H of bovine scFv antigenized with BoHV-1 B-epitope generates specific immune response against the targeted epitope.

We discovered that some bovine antibodies are amongst the largest known to exist due to the presence of an exceptionally long CDR3H (≥49 amino acids) with multiple cysteines that provide a unique knob and stalk structure to the antigen binding site. The large CDR3H size, unlike mouse and human, provides a suitable platform for antigenization with large configurational B-epitopes. Here we report the identification of a B-epitope on the gC envelope protein of bovine herpes virus type-1 (BoHV-1) recognized by a bovine IgG1 antibody. The identified 156 amino acid long gC fragment (gC156) was expressed as a recombinant protein. Subsequently, a functional scFv fragment with a 61 amino-acid long CDR3H (scFv1H12) was expressed such that gC156 was grafted into the CDR3H, replacing the "knob" region (gC156scFv1H12 or Ag-scFv). Importantly, the Ag-scFv could be recognized by a neutralizing antibody fragment (scFv3-18L), which suggests that the engraftment of gC156 into the CDR3H of 1H12 maintained the native conformation of the BoHV-1 B-epitope. A 3D model of gC156 was generated using fold-recognition approaches and this was grafted onto the CDR3H stalk of the 1H12 Fab crystal structure to predict the 3D structure of the Ag-scFv. The grafted antigen in Ag-scFv is predicted to have a compact conformation with the ability to protrude into the solvent. Upon immunization of bovine calves, the antigenized scFv (gC156scFv1H12) induced a higher antibody response as compared to free recombinant gC156. These observations suggest that antigenization of bovine scFv with an exceptionally long CDR3H provides a novel approach to developing the next generation of vaccines against infectious agents that require induction of protective humoral immunity.

Broadening the neutralizing capacity of a family of antibody fragments against different toxins from Mexican scorpions

New approaches aimed at neutralizing the primary toxic components present in scorpion venoms, represent a promising alternative to the use of antivenoms of equine origin in humans. New potential therapeutics developed by these approaches correspond to neutralizing antibody fragments obtained by selection and maturation processes from libraries of human origin. The high sequence identity shared among scorpion toxins is associated with an important level of cross reactivity exhibited by these antibody fragments. We have exploited the cross reactivity showed by single chain variable antibody fragments (scFvs) of human origin to re-direct the neutralizing capacity toward various other scorpion toxins. As expected, during these evolving processes several variants derived from a parental scFv exhibited the capacity to simultaneously recognize and neutralize different toxins from Centruroides scorpion venoms. A sequence analyses of the cross reacting scFvs revealed that specific mutations are responsible for broadening their neutralizing capacity. In this work, we generated a set of new scFvs that resulted from the combinatorial insertion of these point mutations. These scFvs are potential candidates to be part of a novel recombinant antivenom of human origin that could confer protection against scorpion stings. A remarkable property of one of these new scFvs (ER-5) is its capacity to neutralize at least three different toxins and its complementary capacity to neutralize the whole venom from Centruroides suffusus in combination with a second scFv (LR), which binds to a different epitope shared by Centruroides scorpion toxins.

A cryo-electron microscopy study identifies the complete H16.V5 epitope and reveals global conformational changes initiated by binding of the neutralizing antibody fragment.

Human papillomavirus 16 (HPV16) is a worldwide health threat and an etiologic agent of cervical cancer. To understand the antigenic properties of HPV16, we pursued a structural study to elucidate HPV capsids and antibody interactions. The cryo-electron microscopy (cryo-EM) structures of a mature HPV16 particle and an altered capsid particle were solved individually and as complexes with fragment of antibody (Fab) from the neutralizing antibody H16.V5. Fitted crystal structures provided a pseudoatomic model of the virus-Fab complex, which identified a precise footprint of H16.V5, including previously unrecognized residues. The altered-capsid-Fab complex map showed that binding of the Fab induced significant conformational changes that were not seen in the altered-capsid structure alone. These changes included more ordered surface loops, consolidated so-called "invading-arm" structures, and tighter intercapsomeric connections at the capsid floor. The H16.V5 Fab preferentially bound hexavalent capsomers likely with a stabilizing effect that directly correlated with the number of bound Fabs. Additional cryo-EM reconstructions of the virus-Fab complex for different incubation times and structural analysis provide a model for a hyperstabilization of the capsomer by H16.V5 Fab and showed that the Fab distinguishes subtle differences between antigenic sites. Our analysis of the cryo-EM reconstructions of the HPV16 capsids and virus-Fab complexes has identified the entire HPV.V5 conformational epitope and demonstrated a detailed neutralization mechanism of this clinically important monoclonal antibody against HPV16. The Fab bound and ordered the apical loops of HPV16. This conformational change was transmitted to the lower region of the capsomer, resulting in enhanced intercapsomeric interactions evidenced by the more ordered capsid floor and "invading-arm" structures. This study advances the understanding of the neutralization mechanism used by H16.V5.

Mapping the Epitopes of a Neutralizing Antibody Fragment Directed against the Lethal Factor of Bacillus anthracis and Cross-Reacting with the Homologous Edema Factor

The lethal toxin (LT) of Bacillus anthracis, composed of the protective antigen (PA) and the lethal factor (LF), plays an essential role in anthrax pathogenesis. PA also interacts with the edema factor (EF, 20% identity with LF) to form the edema toxin (ET), which has a lesser role in anthrax pathogenesis. The first recombinant antibody fragment directed against LF was scFv 2LF; it neutralizes LT by blocking the interaction between PA and LF. Here, we report that scFv 2LF cross-reacts with EF and cross-neutralizes ET, and we present an in silico method taking advantage of this cross-reactivity to map the epitope of scFv 2LF on both LF and EF. This method identified five epitope candidates on LF, constituted of a total of 32 residues, which were tested experimentally by mutating the residues to alanine. This combined approach precisely identified the epitope of scFv 2LF on LF as five residues (H229, R230, Q234, L235 and Y236), of which three were missed by the consensus epitope candidate identified by pre-existing in silico methods. The homolog of this epitope on EF (H253, R254, E258, L259 and Y260) was experimentally confirmed to constitute the epitope of scFv 2LF on EF. Other inhibitors, including synthetic molecules, could be used to target these epitopes for therapeutic purposes. The in silico method presented here may be of more general interest.

Construction and characterization of neutralizing antibody fragments for efficient access to V3 epitope

Background The neutralizing antibody against HIV-1 is important for the vaccine development. However, primary isolates of HIV-1 have resistance to antibody neutralization. While a neutralization epitope in V3 is exposed by conformational change of gp120 following CD4-binding, anti-V3 IgG molecules are too large to access V3-epitope. The purpose of our study is to develop Fab and scFv fragments from the anti-V3 monoclonal neutralizing antibody 5G2 for efficient access to V3-epitope.

Generation and characterization of the human neutralizing antibody fragment Fab091 against rabies virus

To transform the human anti-rabies virus glycoprotein (anti-RABVG) single-chain variable fragment (scFv) into a Fab fragment and to analyze its immunological activity. The Fab gene was amplified using overlap PCR and inserted into the vector pComb3XSS. The recombinant vector was then transformed into E coli Top10F' for expression and purification. The purified Fab was characterized using SDS-PAGE, Western blotting, indirect ELISA, competitive ELISA, and the fluorescent antibody virus neutralization test (FAVN), respectively, and examined in a Kunming mouse challenge model in vivo. A recombinant vector was constructed. The Fab was expressed in soluble form in E coli Top10F'. Specific binding of the Fab to rabies virus was confirmed by indirect ELISA and immunoprecipitation (IP). The neutralizing antibody titer of Fab was 10.26 IU/mL. The mouse group treated with both vaccine and human rabies immunoglobulin (HRIG)/Fab091 (32 IU/kg) showed protection against rabies, compared with the control group (P<0.05, Logrank test). The antibody fragment Fab was shown to be a neutralizing antibody against RABVG. It can be used together with other monoclonal antibodies for post-exposure prophylaxis of rabies virus in future studies.

Isolation of a novel neutralizing antibody fragment against human vascular endothelial growth factor from a phage-displayed human antibody repertoire using an epitope disturbing strategy

Following the clinical success of Bevacizumab, a humanized monoclonal antibody that blocks the interaction between vascular endothelial growth factor (VEGF) and its receptors, the search for new neutralizing antibodies targeting this molecule has continued until now. We used a human VEGF variant containing three mutations in the region recognized by Bevacizumab to direct antibody selection towards recognition of other epitopes. A total of seven phage-displayed antibody fragments with diverse binding properties in terms of inter-species cross-reactivity and sensitivity to chemical modifications of the antigen were obtained from a human phage display library. All of them were able to recognize not only the selector mutated antigen, but also native VEGF. One of these phage-displayed antibody fragments, denominated 2H1, was shown to compete with the VEGF receptor 2 for VEGF binding. Purified soluble 2H1 inhibited in a dose dependent manner the ligand–receptor interaction and abolished VEGF-dependent proliferation of human umbilical vein endothelial cells. Our epitope disturbing strategy based on a triple mutant target antigen was successful to focus selection on epitopes different from a known one. Similar approaches could be used to direct phage isolation towards the desired specificity in other antigenic systems.

Topological Layers in the HIV-1 gp120 Inner Domain Regulate gp41 Interaction and CD4-Triggered Conformational Transitions

The entry of human immunodeficiency virus (HIV-1) into cells is initiated by binding of the gp120 exterior envelope glycoprotein to the receptor, CD4. How does CD4 binding trigger conformational changes in gp120 that allow the gp41 transmembrane envelope glycoprotein to mediate viral-cell membrane fusion? The transition from the unliganded to the CD4-bound state is regulated by two potentially flexible topological layers (layers 1 and 2) in the gp120 inner domain. Both layers apparently contribute to the noncovalent association of unliganded gp120 with gp41. After CD4 makes initial contact with the gp120 outer domain, layer 1-layer 2 interactions strengthen gp120-CD4 binding by reducing the off rate. Layer 1-layer 2 interactions also destabilize the activated state induced on HIV-1 by treatment with soluble CD4. Thus, despite lack of contact with CD4, the gp120 inner-domain layers govern CD4 triggering by participating in conformational transitions within gp120 and regulating the interaction with gp41.

Ebolavirus Glycoprotein Structure and Mechanism of Entry

Ebolavirus (EBOV) is a highly virulent pathogen capable of causing a severe hemorrhagic fever with 50-90% lethality. The EBOV glycoprotein (GP) is the only virally expressed protein on the virion surface and is critical for attachment to host cells and catalysis of membrane fusion. Hence, the EBOV GP is a critical component of vaccines as well as a target of neutralizing antibodies and inhibitors of attachment and fusion. The crystal structure of the Zaire ebolavirus GP in its trimeric, prefusion conformation (3 GP(1) plus 3 GP(2)) in complex with a neutralizing antibody fragment, derived from a human survivor of the 1995 Kikwit outbreak, was recently determined. This is the first near-complete structure of any filovirus glycoprotein. The overall molecular architecture of the Zaire ebolavirus GP and its role in viral entry and membrane fusion are discussed in this article.

Parvo Viruses Complexed with Neutralizing Antibody Fragments Studied by Cryo Electron Microscopy and Three-Dimensional Image Reconstruction

Parvo Viruses Complexed with Neutralizing Antibody Fragments Studied by Cryo Electron Microscopy and Three-Dimensional Image Reconstruction