title: Development of a recombinant antibody to target peptides and proteins to sialoadhesin-expressing macrophages.
abstract: BACKGROUND
  Sialoadhesin (Sn)-expressing monocytes/macrophages have been associated with several diseases like inflammatory and autoimmune disorders as well as viral infections, and they also appear to play a role in the initiation of an adaptive immune response. This makes Sn-expressing cells not only attractive targets for cell-directed therapies, but also an appealing target for vaccination. Furthermore, since Sn was shown to be an endocytic receptor, the conjugation of effector molecules to an Sn-specific ligand should allow intracellular delivery of these conjugates. Previously, we developed functional Sn-specific immunoconjugates that were generated via chemical coupling. Although successful, the system requires significant optimization for each immunoconjugate to be made. To generate a more flexible and controlled system, we developed a recombinant antibody vector allowing the creation of genetic antibody fusion constructs. This paper reports on the characterization of the recombinant antibody and the evaluation of its use for Sn-directed targeting.
  
  
  RESULTS
  The variable domains of the porcine Sn-specific monoclonal antibody 41D3 were sequenced and cloned in frame with a mouse IgG1 backbone. Transfection of HEK293T cells with the resulting plasmid led to the secretion of fully assembled IgG into the culture medium. This recombinant antibody rec41D3 was shown to specifically bind to porcine Sn with a comparable affinity as the native monoclonal antibody. In addition, rec41D3 also induced Sn endocytosis in primary macrophages and resided for prolonged times in early/late endosomes. To allow the generation of antibody fusion constructs, a multiple cloning site was introduced at the C-terminus of the heavy chain. Two fusion constructs were generated, one containing a V5 peptide tag and one containing an eGFP molecule. Both constructs were shown to be efficiently produced in HEK293T cells and easily purified using standard protein G chromatography. In addition, both V5 and eGFP were shown to be co-internalized together with rec41D3 into Sn-expressing primary macrophages.
  
  
  CONCLUSIONS
  A recombinant antibody allowing targeted delivery of peptides and proteins to Sn-expressing macrophages was developed. Production and purification of antibody fusion constructs was possible without major optimization and with batch to batch consistency, confirming the development of a versatile antibody vector to evaluate Sn-directed targeting strategies in a porcine animal model.
authors.affiliation: Laboratory of Virology, Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, Merelbeke, 9820, Belgium. Karen.Ooms@UGent.be
authors.forename: Karen
authors.initials: K
authors.lastname: Ooms
authors.forename: Hanne
authors.initials: H
authors.lastname: Van Gorp
authors.forename: Tim
authors.initials: T
authors.lastname: Van Gaever
authors.forename: Hans J
authors.initials: HJ
authors.lastname: Nauwynck
authors.forename: Peter L
authors.initials: PL
authors.lastname: Delputte
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000276:immunology; D057809:HEK293 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008264:Macrophages / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D063267:Sialic Acid Binding Ig-like Lectin 1 / Q000276:immunology*; D013552:Swine; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D063267:Sialic Acid Binding Ig-like Lectin 1; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins