RESUMEN
Rare individuals can naturally clear chronic hepatitis B virus (HBV) infection and acquire protection from reinfection as conferred by vaccination. To examine the protective humoral response against HBV, we cloned and characterized human antibodies specific to the viral surface glycoproteins (HBsAg) from memory B cells of HBV vaccinees and controllers. We found that human HBV antibodies are encoded by a diverse set of immunoglobulin genes and recognize various conformational HBsAg epitopes. Strikingly, HBsAg-specific memory B cells from natural controllers mainly produced neutralizing antibodies able to cross-react with several viral genotypes. Furthermore, monotherapy with the potent broadly neutralizing antibody Bc1.187 suppressed viremia in vivo in HBV mouse models and led to post-therapy control of the infection in a fraction of animals. Thus, human neutralizing HBsAg antibodies appear to play a key role in the spontaneous control of HBV and represent promising immunotherapeutic tools for achieving HBV functional cure in chronically infected humans.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Virus de la Hepatitis B/inmunología , Animales , Linfocitos B/inmunología , Reacciones Cruzadas/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Citometría de Flujo , Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Humanos , Memoria Inmunológica/inmunología , Ratones , Pruebas de NeutralizaciónRESUMEN
This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformedNicotiana tabacum Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy's 13 antibody heavy and light chain mutant combinations were expressed transiently inN. tabacumand demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.-Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Nicotiana/metabolismo , Hojas de la Planta/metabolismo , Proteínas Recombinantes/metabolismo , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Sitios de Unión/genética , Western Blotting , Ratones , Mutagénesis Sitio-Dirigida , Mutación , Péptido Hidrolasas/metabolismo , Hojas de la Planta/genética , Plantas Modificadas Genéticamente , Proteínas Recombinantes/química , Análisis de Secuencia de Proteína , Nicotiana/genéticaRESUMEN
The unique specificity of the B cell receptor is generated by an ordered sequence of gene rearrangement events. Once IGH genes have rearranged, rearrangement at the IGK locus is initiated followed by the IGL locus if functional IGK rearrangement is not achieved. Receptor specificity can subsequently be altered by secondary light chain editing based on the features of the heavy and light chain combination. The final profile of expressed genes is not random and biases in this profile are associated with several autoimmune diseases. However, how and when biases are created is not known. To increase our understanding of the processes of selection and editing of IGK rearrangements, we compared four groups of rearrangements of IGK acquired by next generation sequencing. First, expressed rearrangements of IGK from cDNA of IGK expressing B cells. Second, productive rearrangements of IGK from DNA of the same kappa expressing B cells. Third, non-productive rearrangements of IGK from DNA of IGK and IGL expressing B cells, and fourth productively rearranged IGK from DNA of IGL expressing B cells. The latter group would have been rejected during B cell development in favour of rearrangement at the IGL locus and are therefore selected against. We saw evidence that rearranged IGK segments can be selected at a checkpoint where the decision to rearrange the IGL locus is made. In addition, our data suggest that mechanisms regulating the expression or not of IGK rearrangements may also contribute to repertoire development and also that this latter component of the selection process is defective in SLE.
Asunto(s)
Linfocitos B/inmunología , Reordenamiento Génico de Cadena Ligera de Linfocito B/inmunología , Sitios Genéticos/inmunología , Región Variable de Inmunoglobulina/inmunología , Cadenas kappa de Inmunoglobulina/inmunología , Lupus Eritematoso Sistémico/inmunología , Adulto , Linfocitos B/patología , ADN Complementario/genética , ADN Complementario/inmunología , Femenino , Regulación de la Expresión Génica/inmunología , Reordenamiento Génico de Cadena Ligera de Linfocito B/genética , Humanos , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/patología , MasculinoRESUMEN
Plants are promising hosts for the production of monoclonal antibodies (mAbs). However, proteolytic degradation of antibodies produced both in stable transgenic plants and using transient expression systems is still a major issue for efficient high-yield recombinant protein accumulation. In this work, we have performed a detailed study of the degradation profiles of two human IgG1 mAbs produced in plants: an anti-HIV mAb 2G12 and a tumour-targeting mAb H10. Even though they use different light chains (κ and λ, respectively), the fragmentation pattern of both antibodies was similar. The majority of Ig fragments result from proteolytic degradation, but there are only a limited number of plant proteolytic cleavage events in the immunoglobulin light and heavy chains. All of the cleavage sites identified were in the proximity of interdomain regions and occurred at each interdomain site, with the exception of the VL /CL interface in mAb H10 λ light chain. Cleavage site sequences were analysed, and residue patterns characteristic of proteolytic enzymes substrates were identified. The results of this work help to define common degradation events in plant-produced mAbs and raise the possibility of predicting antibody degradation patterns 'a priori' and designing novel stabilization strategies by site-specific mutagenesis.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Inmunoglobulina G/metabolismo , Nicotiana/genética , Proteolisis , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Glicosilación , Immunoblotting , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Análisis de Secuencia de ProteínaRESUMEN
Molecular Pharming represents an unprecedented opportunity to manufacture affordable modern medicines and make these available at a global scale. The area of greatest potential is in the prevention of infectious diseases, particular in underdeveloped countries where access to medicines and vaccines has historically been limited. This is why, at St. George's, we focus on diseases such as HIV, TB and rabies, and aim to develop production strategies that are simple and potentially easy to transfer to developing countries.
Asunto(s)
Agricultura Molecular/métodos , Vacunas/biosíntesis , Vacunas contra el SIDA/biosíntesis , Adyuvantes Inmunológicos/biosíntesis , Animales , Anticuerpos Monoclonales/biosíntesis , Complejo Antígeno-Anticuerpo/inmunología , Ensayos Clínicos como Asunto/métodos , Países en Desarrollo , Aprobación de Drogas , Industria Farmacéutica , Humanos , Hidroponía , Propiedad Intelectual , Ratones , Desarrollo de la Planta , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Vacunas Antirrábicas/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Transferencia de Tecnología , Vacunas contra la Tuberculosis/biosíntesisRESUMEN
BACKGROUND: Interest in using plants for production of recombinant proteins such as monoclonal antibodies is growing, but proteolytic degradation, leading to a loss of functionality and complications in downstream purification, is still a serious problem. RESULTS: In this study, we investigated the dynamics of the assembly and breakdown of a human IgG(1)κ antibody expressed in plants. Initial studies in a human IgG transgenic plant line suggested that IgG fragments were present prior to extraction. Indeed, when the proteolytic activity of non-transgenic Nicotiana tabacum leaf extracts was tested against a human IgG1 substrate, little activity was detectable in extraction buffers with pH > 5. Significant degradation was only observed when the plant extract was buffered below pH 5, but this proteolysis could be abrogated by addition of protease inhibitors. Pulse-chase analysis of IgG MAb transgenic plants also demonstrated that IgG assembly intermediates are present intracellularly and are not secreted, and indicates that the majority of proteolytic degradation occurs following secretion into the apoplastic space. CONCLUSIONS: The results provide evidence that proteolytic fragments derived from antibodies of the IgG subtype expressed in tobacco plants do not accumulate within the cell, and are instead likely to occur in the apoplastic space. Furthermore, any proteolytic activity due to the release of proteases from subcellular compartments during tissue disruption and extraction is not a major consideration under most commonly used extraction conditions.