RESUMO
Novel COVID-19 therapeutics are urgently needed. We generated a phage-displayed human antibody VH domain library from which we identified a high-affinity VH binder ab8. Bivalent VH, VH-Fc ab8, bound with high avidity to membrane-associated S glycoprotein and to mutants found in patients. It potently neutralized mouse-adapted SARS-CoV-2 in wild-type mice at a dose as low as 2 mg/kg and exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection, possibly enhanced by its relatively small size. Electron microscopy combined with scanning mutagenesis identified ab8 interactions with all three S protomers and showed how ab8 neutralized the virus by directly interfering with ACE2 binding. VH-Fc ab8 did not aggregate and did not bind to 5,300 human membrane-associated proteins. The potent neutralization activity of VH-Fc ab8 combined with good developability properties and cross-reactivity to SARS-CoV-2 mutants provide a strong rationale for its evaluation as a COVID-19 therapeutic.
Assuntos
Infecções por Coronavirus/tratamento farmacológico , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Região Variável de Imunoglobulina/administração & dosagem , Biblioteca de Peptídeos , Pneumonia Viral/tratamento farmacológico , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/ultraestrutura , Afinidade de Anticorpos , COVID-19 , Cricetinae , Feminino , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/ultraestrutura , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Pandemias , Peptidil Dipeptidase A/metabolismo , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Tratamento Farmacológico da COVID-19RESUMO
Mycobacterium tuberculosis (Mtb) 16.3 kDa heat shock protein 16.3 (HSP16.3) is a latency-associated antigen that can be targeted for latent tuberculosis (TB) diagnostic and therapeutic development. We have previously developed human VH domain antibodies (dAbs; clone E3 and F1) specific against HSP16.3. In this work, we applied computational methods to optimise and design the antibodies in order to improve the binding affinity with HSP16.3. The VH domain antibodies were first docked to the dimer form of HSP16.3 and further sampled using molecular dynamics simulation. The calculated binding free energy of the HSP16.3-dAb complexes showed non-polar interactions were responsible for the antigen-antibody association. Per-residue free energy decomposition and computational alanine scanning have identified one hotspot residue for E3 (Y391) and 4 hotspot residues for F1 (M394, Y396, R397 and M398). These hotspot residues were then mutated and evaluated by binding free energy calculations. Phage ELISA assay was carried out on the potential mutants (E3Y391W, F1M394E, F1R397N and F1M398Y). The experimental assay showed improved binding affinities of E3Y391W and F1M394E against HSP16.3 compared with the wild type E3 and F1. This case study has thus showed in silico methods are able to assist in optimisation or improvement of antibody-antigen binding.
Assuntos
Anticorpos/química , Proteínas de Bactérias/química , Chaperoninas/química , Simulação por Computador , Modelos Moleculares , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Chaperoninas/genética , Chaperoninas/imunologia , Bases de Dados de Proteínas , Humanos , Mutação Puntual , Ligação Proteica , Conformação Proteica , Domínios Proteicos , TermodinâmicaRESUMO
Interest in single-domain antibodies (sdAbs) stems from their unique structural/pronounced, hence therapeutically desirable, features. From the outset-as therapeutic modalities-human antibody heavy chain variable domains (VHs) attracted a particular attention compared with 'naturally-occurring' camelid and shark heavy-chain-only antibody variable domains (VHHs and VNARs, respectively) due to their perceived lack of immunogenicity. However, they have not quite lived up to their initial promise as the VH hits, primarily mined from synthetic VH phage display libraries, have too often been plagued with aggregation tendencies, low solubility and low affinity. Largely unexplored, synthetic camelized human VH display libraries appeared to have remediated the aggregation problem, but the low affinity of the VH hits still persisted, requiring undertaking additional, laborious affinity maturation steps to render VHs therapeutically feasible. A wholesome resolution has recently emerged with the development of non-canonical transgenic rodent antibody discovery platforms that appear to facilely and profusely generate high affinity, high solubility and aggregation-resistant human VHs.
Assuntos
Cadeias Pesadas de Imunoglobulinas , Anticorpos de Domínio Único , Anticorpos/genética , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Anticorpos de Domínio Único/genéticaRESUMO
Incorporating virtual human (VH) technology into simulation programs for perioperative education and training can improve interdisciplinary teamwork and communication. The development of VHs allowed interdisciplinary teams at the University of Florida Health Shands Hospital to overcome obstacles during training, such as communication gaps. A multidisciplinary team that included perioperative leaders and quality specialists developed three scenarios for interdisciplinary training to provide participants with the opportunity to practice their communication skills and improve patient safety. Using validated tools to promote standardized communication, participants had to address patient safety concerns assertively during the simulations. After each simulation session, quality specialists conducted a debriefing, collected data via surveys, and identified areas for improvement. As a result of the VH simulation sessions, perioperative staff members reported increased confidence and competence when providing patient care. Using VHs to enhance education is an innovative strategy that is useful for both novice and experienced perioperative nurses.
Assuntos
Equipe de Assistência ao Paciente , Treinamento por Simulação , Competência Clínica , Comunicação , Humanos , Segurança do Paciente , TecnologiaRESUMO
Single-domain antibodies represent an emerging class of antibody fragments with promising therapeutic and diagnostic potential. As a result, multiple strategies have been developed in order to improve their biophysical and/or biological properties. In particular, the fusion of single-domain antibodies to the Fc part of an IgG molecule has become a common protein engineering approach toward this aim. Here, we describe a detailed protocol for a streamlined laboratory-scale production of VH single-domain antibodies as Fc fusions in mammalian cells. Firstly, DNA sequence encoding VH domain of interest fused to an IgG Fc is synthesized as a double-stranded gene fragment. Secondly, the DNA fragment is directly assembled into a restriction enzyme-digested vector in an assembly reaction. Finally, vector carrying the VH-Fc-fusion construct is introduced into suspension-adapted mammalian cells for transient expression of the Fc chimeric fusion. One-week post-transfection, the expressed Fc-fusion protein is purified using protein A/G affinity chromatography. Using this protocol, we were able to clone, express, and purify milligrams of isolated anti-HER2 VH domain as a mouse IgG2c Fc fusion in less than 2 weeks. This protocol can be readily modified to express proteins of interest other than VH domains as Fc fusions.
Assuntos
Fragmentos Fc das Imunoglobulinas/genética , Proteínas Recombinantes de Fusão/genética , Anticorpos de Domínio Único/genética , Animais , Biotinilação , Linhagem Celular , Cromatografia de Afinidade/métodos , Clonagem Molecular/métodos , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Camundongos , Plasmídeos/genética , Receptor ErbB-2/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/isolamento & purificação , Transfecção/métodosRESUMO
Human autonomous VH/VL single-domain antibodies (sdAbs) are attractive therapeutic molecules, but often suffer from suboptimal stability, solubility and affinity for cognate antigens. Most commonly, human sdAbs have been isolated from in vitro display libraries constructed via synthetic randomization of rearranged VH/VL domains. Here, we describe the design and characterization of three novel human VH/VL sdAb libraries through a process of: (i) exhaustive biophysical characterization of 20 potential VH/VL sdAb library scaffolds, including assessment of expression yield, aggregation resistance, thermostability and tolerance to complementarity-determining region (CDR) substitutions; (ii) in vitro randomization of the CDRs of three VH/VL sdAb scaffolds, with tailored amino acid representation designed to promote solubility and expressibility; and (iii) systematic benchmarking of the three VH/VL libraries by panning against five model antigens. We isolated ≥1 antigen-specific human sdAb against four of five targets (13 VHs and 7 VLs in total); these were predominantly monomeric, had antigen-binding affinities ranging from 5 nM to 12 µM (average: 2-3 µM), but had highly variable expression yields (range: 0.1-19 mg/L). Despite our efforts to identify the most stable VH/VL scaffolds, selection of antigen-specific binders from these libraries was unpredictable (overall success rate for all library-target screens: ~53%) with a high attrition rate of sdAbs exhibiting false positive binding by ELISA. By analyzing VH/VL sdAb library sequence composition following selection for monomeric antibody expression (binding to protein A/L followed by amplification in bacterial cells), we found that some VH/VL sdAbs had marked growth advantages over others, and that the amino acid composition of the CDRs of this set of sdAbs was dramatically restricted (bias toward Asp and His and away from aromatic and hydrophobic residues). Thus, CDR sequence clearly dramatically impacts the stability of human autonomous VH/VL immunoglobulin domain folds, and sequence-stability tradeoffs must be taken into account during the design of such libraries.
RESUMO
Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, nonaggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain antibody libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant- and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell-penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell-specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb-DNA or -protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic.
Assuntos
Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Anticorpos de Cadeia Única/química , Proteínas Virais/antagonistas & inibidores , Viroses/metabolismo , Animais , Citosol/metabolismo , Epitopos/química , Epitopos/genética , Epitopos/metabolismo , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Viroses/genéticaRESUMO
Several technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and variable light (VL) domains in combinatorial display libraries or isolation of cognate pairs of VH and VL domains from human B cells or from transgenic mice carrying human immunoglobulin loci followed by single-cell sorting, single-cell RT-PCR, and bulk cloning of isolated natural VH-VL pairs. Heavy-chain-only antibodies (HCAbs) that naturally occur in camelids require only heavy immunoglobulin chain cloning. Here, we present an automatable novel, high-throughput technology for rapid direct cloning and production of fully human HCAbs from sorted population of transgenic mouse plasma cells carrying a human HCAb locus. Utility of the technique is demonstrated by isolation of diverse sets of sequence unique, soluble, high-affinity influenza A strain X-31 hemagglutinin-specific HCAbs.