11
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Design of a humanized anti vascular endothelial growth factor nanobody and evaluation of its in vitro function

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Objective(s):

          Nanobodies, the single domain antigen binding fragments of heavy chain-only antibodies occurring naturally in camelid sera, are the smallest intact antigen binding entities. Their minimal size assists in reaching otherwise largely inaccessible regions of antigens. However, their camelid origin raises a possible concern of immunogenicity when used for human therapy. Humanization is a promising approach to overcome the problem.

          Materials and Methods:

          Here, we designed a humanized version of previously developed nanobody (anti vascular endothelial growth factor nanobody), evaluated and compared its predicted 3D structure, affinity and biological activity with its original wild type nanobody.

          Results:

          Our in silico results revealed an identical 3D structure of the humanized nanobody as compare to original nanobody. In vitro studies also demonstrated that the humanization had no significant visible effect on the nanobody affinity or on its biological activity.

          Conclusion:

          The humanized nanobody could be developed and proposed as a promising lead to target pathologic angiogenesis.

          Related collections

          Most cited references26

          • Record: found
          • Abstract: found
          • Article: not found

          Structure validation by Calpha geometry: phi,psi and Cbeta deviation.

          Geometrical validation around the Calpha is described, with a new Cbeta measure and updated Ramachandran plot. Deviation of the observed Cbeta atom from ideal position provides a single measure encapsulating the major structure-validation information contained in bond angle distortions. Cbeta deviation is sensitive to incompatibilities between sidechain and backbone caused by misfit conformations or inappropriate refinement restraints. A new phi,psi plot using density-dependent smoothing for 81,234 non-Gly, non-Pro, and non-prePro residues with B < 30 from 500 high-resolution proteins shows sharp boundaries at critical edges and clear delineation between large empty areas and regions that are allowed but disfavored. One such region is the gamma-turn conformation near +75 degrees,-60 degrees, counted as forbidden by common structure-validation programs; however, it occurs in well-ordered parts of good structures, it is overrepresented near functional sites, and strain is partly compensated by the gamma-turn H-bond. Favored and allowed phi,psi regions are also defined for Pro, pre-Pro, and Gly (important because Gly phi,psi angles are more permissive but less accurately determined). Details of these accurate empirical distributions are poorly predicted by previous theoretical calculations, including a region left of alpha-helix, which rates as favorable in energy yet rarely occurs. A proposed factor explaining this discrepancy is that crowding of the two-peptide NHs permits donating only a single H-bond. New calculations by Hu et al. [Proteins 2002 (this issue)] for Ala and Gly dipeptides, using mixed quantum mechanics and molecular mechanics, fit our nonrepetitive data in excellent detail. To run our geometrical evaluations on a user-uploaded file, see MOLPROBITY (http://kinemage.biochem.duke.edu) or RAMPAGE (http://www-cryst.bioc.cam.ac.uk/rampage). Copyright 2003 Wiley-Liss, Inc.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay.

            Enzyme-linked immunoadsorbent assay (EIA) has widespread use for the measurement of antibody concentration. The affinity constant (Kaff) of the antibody has an effect upon the quantification by EIA. It is thus important to be able to measure Kaff by solid-phase EIA. Based upon the Law of Mass Action and using serial dilutions of both antigens (coating the plate) and antibody, Kaff has been measured by EIA. A microtiter plate was coated with antigen (Ag) and then incubated with monoclonal antibody (Ab). The plate was sequentially incubated with a second enzyme-antibody conjugate (EAC) and with the enzyme substrate. The amount of Ab adherent to Ag on the plate [Ag Ab] and [Ag2 Ab] was reflected by the enzyme product measured by OD. The use of serial dilutions of Ab resulted in a sigmoid curve of OD versus logarithm of total Ab added to the well. Comparison of the OD at the upper plateau (OD-100) for different antibodies was a reflection of the relative number of epitopes on the Ag that were identified by the different antibodies, provided excessive EAC was used. [Ab]t and [Ab']t were the measurable total antibody concentrations in the wells at OD-50 and OD-50' for plates coated with [Ag] and [Ag'], respectively. [Ag] and [Ag'] were not true antigen concentrations, but were a measure of antigen density on the plate. For [Ag'] = [Ag]/2, Kaff = 1/2(2[Ab']t-[Ab]t. Using five different anti-CEA antibodies and different proportions of CEA in the coating solution, Kaff was measured. Kaff determined by EIA correlated well with Kaff measured by soluble phase inhibition assay. This EIA method of estimation of Kaff is simple, rapid, and reliable.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Isolation of antigen specific llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae.

              Recently the existence of 'heavy chain' immunoglobulins in Camelidae has been described. However, as yet there is no data on the binding of this type of antibody to haptens. In addition, it was not a priori predictable whether the binding domains (VHH) of these antibodies could be produced and secreted by the lower eukaryotic micro-organism Saccharomyces cerevisiae. In the present study these questions are addressed. Heavy chain immunoglobulins directed against two hapten molecules, the azo-dyes RR6 and RR120 as well as the (proteinaceous) human pregnancy hormone, have been raised in Lama glama. We were able to select specific VHH fragments for all three antigens by direct screening of Escherichia coli or yeast libraries, even without prior enrichment via bio-panning. This is the first example of the isolation of llama anti-hapten VHH domains. Surprisingly, the affinities of the llama VHHs for the RR6 hapten obtained in this way are in the low nM range. Furthermore, some of the antigen specific VHHs were secreted by S. cerevisiae at levels over 100 mg l-1 in shake flask cultures. These two findings extend the possible application areas for the llama VHH fragments significantly.
                Bookmark

                Author and article information

                Journal
                Iran J Basic Med Sci
                Iran J Basic Med Sci
                Iranian Journal of Basic Medical Sciences
                Mashhad University of Medical Sciences (Iran )
                2008-3866
                2008-3874
                March 2018
                : 21
                : 3
                : 260-266
                Affiliations
                [1 ]Venom & Biotherapeutics Molecules Laboratory, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
                [2 ]Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, 1050 Brussels, Belgium
                [3 ]National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
                Author notes
                [* ] Corresponding author: Fatemeh Kazemi-Lomedasht, Venom & Biotherapeutics Molecules Laboratory, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran. Tel: +98-21-66480780; Email: fa_kazemi@ 123456pasteur.ac.ir
                Article
                IJBMS-21-260
                10.22038/ijbms.2018.24898.6183
                5817169
                766b6c9e-a91a-48b0-8065-3bb6f45183c4
                Copyright: © Iranian Journal of Basic Medical Sciences

                This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 12 July 2017
                : 28 September 2017
                Categories
                Original Article

                angiogenesis,antibody engineering,humanization,nanobody,vegf

                Comments

                Comment on this article