RESUMEN
We have previously reported the isolation of a novel single-chain variable fragment (scFv) against vascular endothelial growth factor (VEGF), from a phage-displayed human antibody repertoire. This scFv, denominated 2H1, was shown to block the binding of VEGF to its receptor but exhibited a moderate binding affinity. Here, we describe the affinity maturation of the 2H1 scFv. Two phage-displayed libraries were constructed by diversification of the third complementarity-determining regions (CDRs) of the light (VL) and heavy (VH) chain variable domains of 2H1 using parsimonious mutagenesis. A competitive phage-selection strategy in the presence of the parental scFv as a competitor was used to eliminate low affinity binders. High affinity variants were retrieved from both libraries. An optimized VL variant was designed and constructed by combining recurrent replacements found among selected variants in a single molecule, resulting in an additional affinity increase. Further affinity improvements were achieved by combining this optimized VL with the best VH variants. The final variant obtained here, L3H6, showed an overall affinity improvement of 18-fold over the parental scFv and exhibited an enhanced potency to block the binding of VEGF to its receptor. Using phage display and extensive mutagenesis of VEGF, we determined the fine specificity of L3H6. This functional mapping revealed a novel neutralizing epitope on human VEGF defined by the residues Y25, T71, E72, N100, K101, E103 and R105. The conformational epitope recognized by L3H6 was recapitulated by grafting human VEGF residues into the mouse molecule, providing further confirmation of the nature of the identified epitope.
Asunto(s)
Mapeo Epitopo/métodos , Epítopos/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Anticuerpos de Cadena Única/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Animales , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Epítopos/genética , Epítopos/inmunología , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Biblioteca de Péptidos , Unión Proteica , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
A broad variety of foreign genes can be expressed in transgenic plants, which offer the opportunity for large-scale production of pharmaceutical proteins, such as therapeutic antibodies. Nimotuzumab is a humanized anti-epidermal growth factor receptor (EGFR) recombinant IgG1 antibody approved in different countries for the treatment of head and neck squamous cell carcinoma, paediatric and adult glioma, and nasopharyngeal and oesophageal cancers. Because the antitumour mechanism of nimotuzumab is mainly attributed to its ability to interrupt the signal transduction cascade triggered by EGF/EGFR interaction, we have hypothesized that an aglycosylated form of this antibody, produced by mutating the N(297) position in the IgG(1) Fc region gene, would have similar biochemical and biological properties as the mammalian-cell-produced glycosylated counterpart. In this paper, we report the production and characterization of an aglycosylated form of nimotuzumab in transgenic tobacco plants. The comparison of the plantibody and nimotuzumab in terms of recognition of human EGFR, effect on tyrosine phosphorylation and proliferation in cells in response to EGF, competition with radiolabelled EGF for EGFR, affinity measurements of Fab fragments, pharmacokinetic and biodistribution behaviours in rats and antitumour effects in nude mice bearing human A431 tumours showed that both antibody forms have very similar in vitro and in vivo properties. Our results support the idea that the production of aglycosylated forms of some therapeutic antibodies in transgenic plants is a feasible approach when facing scaling strategies for anticancer immunoglobulins.
Asunto(s)
Anticuerpos Monoclonales Humanizados/biosíntesis , Anticuerpos Monoclonales Humanizados/farmacología , Receptores ErbB/antagonistas & inhibidores , Inmunoglobulinas/biosíntesis , Nicotiana/genética , Nicotiana/metabolismo , Planticuerpos/farmacología , Animales , Antineoplásicos/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Desnudos , Agricultura Molecular/métodos , Fosforilación/efectos de los fármacos , Planticuerpos/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Ratas , Ratas Wistar , Proteínas Recombinantes , Tirosina/metabolismoRESUMEN
Because of the wide use and high demand in medicine, monoclonal antibodies are among the main recombinant pharmaceuticals at present, although present limitations of the productive platforms for monoclonal antibodies are driving the improvement of the large-scale technologies and the development of alternative expression systems. This has drawn the attention on plants as expression system for monoclonal antibodies and related derivatives, owning the capacity of plants to properly express and process eukaryotic proteins with biological activity resembling that of the natural proteins. In this chapter, the procedures from the isolation of the monoclonal antibody genes to the biochemical and biological characterization of the plant-expressed monoclonal antibody are described.
Asunto(s)
Nicotiana/genética , Planticuerpos/genética , Caulimovirus/genética , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos , Plantas Modificadas Genéticamente/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cancer is one of the most prevalent diseases worldwide, which explains why biological therapies for cancer are forecast to make up 35% of total recombinant pharmaceuticals by 2010. Because of the high demand for cancer drugs, the need to lower production costs and the constraints of present production technologies for recombinant pharmaceuticals (such as the difficulties involved in culturing bacteria, yeast and mammalian cells), attention has recently been focused on recombinant expression of pharmaceutical anti-cancer proteins in plants. This review aims to provide an update on the most recent publications about anti-cancer recombinant pharmaceuticals expressed in plants, as well as on the relevant technical issues, potential and prospects of this emerging production system.
Asunto(s)
Antineoplásicos/metabolismo , Antineoplásicos/uso terapéutico , Productos Biológicos/uso terapéutico , Extractos Vegetales/uso terapéutico , Plantas Modificadas Genéticamente/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapéutico , Productos Biológicos/metabolismo , Humanos , Extractos Vegetales/metabolismoRESUMEN
We report here a method for the identification of free or blocked N-terminal peptide of in-gel digested isolated proteins. The primary amino groups of the gel-entrapped protein are blocked with normal acetic or succinic anhydride, and the protein is digested with a high-specificity protease. The generated peptides are treated with an equimolar mixture of normal and deuterated acetic anhydride. Upon mass spectrometric analysis internal peptides display a complex isotopic ion distribution while the N-terminal peptide shows a normal isotopic ion distribution. The procedure was applied to the identification of the N-terminus of individual and protein mixtures isolated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE).
Asunto(s)
Aminoácidos/química , Electroforesis en Gel de Poliacrilamida/métodos , Mapeo Peptídico/métodos , Péptidos/química , Proteínas/química , Acilación , Espectrometría de Masas/métodos , Coloración y Etiquetado/métodosRESUMEN
In the present paper we report the development of a bivalent scFv (single-chain Fv) antibody fragment, starting from a mouse mAb (monoclonal antibody) specific for CEA (carcinoembryonic antigen) that has received approval for in vivo radioimmunodiagnosis in humans. The diabody is well expressed in Escherichia coli, is easily purified by a combination of immobilized metal ion affinity chromatography and gel filtration and exhibits high affinity and specificity for CEA, comparable with those of the original mAb. Biodistribution experiments in athymic nude mice transplanted with human CEA+ cancer cells showed that the 125I-labelled diabody preferentially localizes in the tumour tissue and that retention is still high 48 h after injection. The diabody can be advantageous for some in vivo tumour targeting applications, due to the faster clearance derived from its smaller molecular mass.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Antígeno Carcinoembrionario/inmunología , Fragmentos de Inmunoglobulinas/farmacología , Neoplasias/metabolismo , Proteínas Recombinantes/farmacocinética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Línea Celular Tumoral , Clonación Molecular , Escherichia coli/genética , Femenino , Humanos , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/inmunología , Radioisótopos de Yodo , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Trasplante de Neoplasias , Neoplasias/diagnóstico por imagen , Neoplasias/inmunología , Radioinmunodetección , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunologíaRESUMEN
When generating stably transformed transgenic plants, transient gene expression experiments are especially useful to rapidly confirm that the foreign molecule of interest is correctly assembled and retains its biological activity. TheraCIM(R) (CIMAB S.A., Havana) is a recombinant humanized antibody against the Epidermal Growth Factor receptor (EGF-R), now in clinical trials for cancer therapy in Cuba and other countries. An aglycosylated version (Asn 297 was mutated for Gln 297) of this antibody was transiently expressed in tobacco leaves after vacuum-mediated infiltration of recombinant Agrobacterium tumefaciens that carried a binary plasmid bearing the antibody heavy and light chain genes and plant regulation signals. Protein extracts from "agroinfiltrated" leaves were tested by ELISA and Western blot, showing that the fully assembled antibody was accumulated in plant tissues. The absence of plant specific glycans did not interfere in the assembling or in the activity of the plantibody, as demonstrated in this work. Indirect immunofluorescence demonstrated that the aglycosylated antibody expressed in plants recognizes the EGF-R expressed on the surface of A431 human tumor culture cells.