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

Lamdan, Humberto; Ayala, Marta; Rojas, Gertrudis; Muñoz, Yasmiana; Morera, Yanelys; Guirola, Osmany; Chinea, Glay; Gavilondo, Jorge V.

doi:10.1016/j.jbiotec.2010.12.007

Cited by 8 publications

(8 citation statements)

References 33 publications

(35 reference statements)

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“…We recently reported the isolation of a novel anti-VEGF single-chain variable fragment (scFv), 2H1, from a phage-displayed human antibody repertoire, using an epitope disturbing strategy. 8 This approach, based on disrupting the Bevacizumab epitope by site-directed mutagenesis of human VEGF, rendered 2H1 scFv that recognizes an epitope different from the one recognized by Bevacizumab. This scFv blocks the binding of VEGF to KDR but showed a moderate VEGF binding affinity (4.7 Â 10 À7 M).…”

Section: Introductionmentioning

confidence: 99%

Affinity maturation and fine functional mapping of an antibody fragment against a novel neutralizing epitope on human vascular endothelial growth factor

et al. 2013

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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.

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Section: Introductionmentioning

confidence: 99%

Affinity maturation and fine functional mapping of an antibody fragment against a novel neutralizing epitope on human vascular endothelial growth factor

et al. 2013

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“…To construct the plasmid pET28a-L36-TIE, a human TIE XVIII domain of was synthesized by GeneartAG (Life Technologies) and subcloned as Not I/ Bam HI into the vector pET28a-L36. To generate the E.coli expression plasmid pET28a-2H1-TIE, the DNA fragment coding for the anti-VEGF 2H1 scFv (Lamdan et al 2011 ) was synthesized by GeneArt AG and subcloned as Sfi I/ Not I into the vector pET28a-L36-TIE.…”

Section: Methodsmentioning

confidence: 99%

“…Here, we have studied the potential of E. coli for the production of scFv-based N-terminal trimerbodies using the anti-angiogenic scFv antibodies L36 and 2H1. The L36 scFv recognizes laminin-111, inhibits capillary morphogenesis of endothelial cells and prevents the establishment and growth of subcutaneous tumors in mice (Sanz et al 2002 ; Sánchez-Arevalo Lobo et al 2006 ), whereas the 2H1 scFv blocks the interaction between vascular endothelial growth factor (VEGF) and VEGF receptor-2 (KDR/Flk-1) and hampers endothelial cell proliferation in a dose-dependent manner (Lamdan et al 2011 ). Antibody genes were cloned into the pET28a expression vector under the control of a T7 promoter and the resulting plasmids were transformed into E. coli BL21(DE3) cells.…”

Section: Introductionmentioning

confidence: 99%

Bacterial secretion of soluble and functional trivalent scFv-based N-terminal trimerbodies

et al. 2015

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Recombinant antibodies are used with great success in many different diagnostic and therapeutic applications. A variety of protein expression systems are available, but nowadays almost all therapeutic antibodies are produced in mammalian cell lines due to their complex structure and glycosylation requirements. However, production of clinical-grade antibodies in mammalian cells is very expensive and time-consuming. On the other hand, Escherichia coli (E. coli) is known to be the simplest, fastest and most cost-effective recombinant expression system, which usually achieves higher protein yields than mammalian cells. Indeed, it is one of the most popular host in the industry for the expression of recombinant proteins. In this work, a trivalent single-chain fragment variable (scFv)-based N-terminal trimerbody, specific for native laminin-111, was expressed in human embryonic kidney 293 cells and in E. coli. Mammalian and bacterially produced anti-laminin trimerbody molecules display comparable functional and structural properties, although importantly the yield of trimerbody expressed in E. coli was considerably higher than in human cells. These results demonstrated that E. coli is a versatile and efficient expression system for multivalent trimerbody-based molecules that is suitable for their industrial production.

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“…Adsorption of β-casein and ovalbumin increased in the range of protein concentration of 0.1–0.6 mg mL −1 while for BSA and lysozyme no significant increase was observed even at concentration of 1 mg mL −1 showing potential of use of phosphate Zr 4+ -IMAM for enrichment of phosphorylated proteins [46]. The development of new stationary phases for specific and selective binding of proteins and good protein recovery lead to IMAC being extensively used in antibody purification [47–51]. Evaluation of performance of iminodiacetic acid (IDA) and Tris(2-aminoethyl)amine (TREN) as a chelating agents in purifications of IgG with immobilized nickel affinity polyethylene vinyl alcohol (PEVA) hollow fiber membrane chromatography showed that Ni(II)-TREN had lower binding capacity for IgG compared to NI(II)-IDA; 9.8 and 9.4 mg for Ni(II)-IDA-PEVA and 1.4 and 1.5 mg Ni(II)-TREN for protein elution and regeneration, respectively [52].…”

Section: Implementation Of Different Selectivities In Intact Protein mentioning

confidence: 99%

Different Stationary Phase Selectivities and Morphologies for Intact Protein Separations

2016

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The central dogma of biology proposed that one gene encodes for one protein. We now know that this does not reflect reality. The human body has approximately 20,000 protein-encoding genes; each of these genes can encode more than one protein. Proteins expressed from a single gene can vary in terms of their post-translational modifications, which often regulate their function within the body. Understanding the proteins within our bodies is a key step in understanding the cause, and perhaps the solution, to disease. This is one of the application areas of proteomics, which is defined as the study of all proteins expressed within an organism at a given point in time. The human proteome is incredibly complex. The complexity of biological samples requires a combination of technologies to achieve high resolution and high sensitivity analysis. Despite the significant advances in mass spectrometry, separation techniques are still essential in this field. Liquid chromatography is an indispensable tool by which low-abundant proteins in complex samples can be enriched and separated. However, advances in chromatography are not as readily adapted in proteomics compared to advances in mass spectrometry. Biologists in this field still favour reversed-phase chromatography with fully porous particles. The purpose of this review is to highlight alternative selectivities and stationary phase morphologies that show potential for application in top-down proteomics; the study of intact proteins.

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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

Cited by 8 publications

References 33 publications

Affinity maturation and fine functional mapping of an antibody fragment against a novel neutralizing epitope on human vascular endothelial growth factor

Affinity maturation and fine functional mapping of an antibody fragment against a novel neutralizing epitope on human vascular endothelial growth factor

Bacterial secretion of soluble and functional trivalent scFv-based N-terminal trimerbodies

Different Stationary Phase Selectivities and Morphologies for Intact Protein Separations

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