18
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      An Inside Job: Applications of Intracellular Single Domain Antibodies

      review-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

          Sera of camelid species contain a special kind of antibody that consists only of heavy chains. The variable antigen binding domain of these heavy chain antibodies can be expressed as a separate entity, called a single domain antibody that is characterized by its small size, high solubility and oftentimes exceptional stability. Because of this, most single domain antibodies fold correctly when expressed in the reducing environment of the cytoplasm, and thereby retain their antigen binding specificity. Single domain antibodies can thus be used to target a broad range of intracellular proteins. Such intracellular single domain antibodies are also known as intrabodies, and have proven to be highly useful tools for basic research by allowing visualization, disruption and even targeted degradation of intracellular proteins. Furthermore, intrabodies can be used to uncover prospective new therapeutic targets and have the potential to be applied in therapeutic settings in the future. In this review we provide a brief overview of recent advances in the field of intracellular single domain antibodies, focusing on their use as research tools and potential therapeutic applications. Special attention is given to the available methods that allow delivery of single domain antibodies into cells.

          Related collections

          Most cited references178

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

          Nanobodies: natural single-domain antibodies.

          Sera of camelids contain both conventional heterotetrameric antibodies and unique functional heavy (H)-chain antibodies (HCAbs). The H chain of these homodimeric antibodies consists of one antigen-binding domain, the VHH, and two constant domains. HCAbs fail to incorporate light (L) chains owing to the deletion of the first constant domain and a reshaped surface at the VHH side, which normally associates with L chains in conventional antibodies. The genetic elements composing HCAbs have been identified, but the in vivo generation of these antibodies from their dedicated genes into antigen-specific and affinity-matured bona fide antibodies remains largely underinvestigated. However, the facile identification of antigen-specific VHHs and their beneficial biochemical and economic properties (size, affinity, specificity, stability, production cost) supported by multiple crystal structures have encouraged antibody engineering of these single-domain antibodies for use as a research tool and in biotechnology and medicine.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Naturally occurring antibodies devoid of light chains.

            Random association of VL and VH repertoires contributes considerably to antibody diversity. The diversity and the affinity are then increased by hypermutation in B cells located in germinal centres. Except in the case of 'heavy chain' disease, naturally occurring heavy-chain antibodies have not been described, although antigen binding has been demonstrated for separated heavy chains or cloned VH domains. Here we investigate the presence of considerable amounts of IgG-like material of M(r) 100K in the serum of the camel (Camelus dromedarius). These molecules are composed of heavy-chain dimers and are devoid of light chains, but nevertheless have an extensive antigen-binding repertoire, a finding that calls into question the role of light chains in the camel. Camel heavy-chain IgGs lack CH1, which in one IgG class might be structurally replaced by an extended hinge. Heavy-chain IgGs are a feature of all camelids. These findings open new perspectives in the engineering of antibodies.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake.

              Cellular uptake of a family of cationic cell-penetrating peptides (examples include Tat peptides and penetratin) have been ascribed in the literature to a mechanism that does not involve endocytosis. In this work we reevaluate the mechanisms of cellular uptake of Tat 48-60 and (Arg)(9). We demonstrate here that cell fixation, even in mild conditions, leads to the artifactual uptake of these peptides. Moreover, we show that flow cytometry analysis cannot be used validly to evaluate cellular uptake unless a step of trypsin digestion of the cell membrane-adsorbed peptide is included in the protocol. Fluorescence microscopy on live unfixed cells shows characteristic endosomal distribution of peptides. Flow cytometry analysis indicates that the kinetics of uptake are similar to the kinetics of endocytosis. Peptide uptake is inhibited by incubation at low temperature and cellular ATP pool depletion. Similar data were obtained for Tat-conjugated peptide nucleic acids. These data are consistent with the involvement of endocytosis in the cellular internalization of cell-penetrating peptides and their conjugates to peptide nucleic acids.
                Bookmark

                Author and article information

                Journal
                Biomolecules
                Biomolecules
                biomolecules
                Biomolecules
                MDPI
                2218-273X
                12 December 2020
                December 2020
                : 10
                : 12
                : 1663
                Affiliations
                [1 ]VIB-UGent Center for Medical Biotechnology, VIB, B-9052 Ghent, Belgium; eline.soetens@ 123456vib-ugent.be (E.S.); marlies.ballegeer@ 123456vib-ugent.be (M.B.)
                [2 ]Department of Biochemistry and Microbiology, Ghent University, B-9000 Ghent, Belgium
                Author notes
                Author information
                https://orcid.org/0000-0002-4914-7455
                https://orcid.org/0000-0002-3861-6965
                Article
                biomolecules-10-01663
                10.3390/biom10121663
                7764588
                33322697
                f6e8c960-7e50-4693-943d-fefb5ea0452a
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 20 November 2020
                : 10 December 2020
                Categories
                Review

                single domain antibody,intrabody,delivery methods,therapy,research tool

                Comments

                Comment on this article