title: Lentivirus display: stable expression of human antibodies on the surface of human cells and virus particles.
abstract: BACKGROUND
  Isolation of human antibodies using current display technologies can be limited by constraints on protein expression, folding and post-translational modifications. Here we describe a discovery platform that utilizes self-inactivating (SIN) lentiviral vectors for the surface display of high-affinity single-chain variable region (scFv) antibody fragments on human cells and lentivirus particles.
  
  
  RESULTS
  Bivalent scFvFc human antibodies were fused in frame with different transmembrane (TM) anchoring moieties to allow efficient high-level expression on human cells and the optimal TM was identified. The addition of an eight amino acid HIV-1 gp41 envelope incorporation motif further increased scFvFc expression on human cells and incorporation into lentiviral particles. Both antibody-displaying human cells and virus particles bound antigen specifically. Sulfation of CDR tyrosine residues, a property recently shown to broaden antibody binding affinity and antigen recognition was also demonstrated. High level scFvFc expression and stable integration was achieved in human cells following transduction with IRES containing bicistronic SIN lentivectors encoding ZsGreen when scFvFc fusion proteins were expressed from the first cassette. Up to 10(6)-fold enrichment of antibody expressing cells was achieved with one round of antigen coupled magnetic bead pre-selection followed by FACS sorting. Finally, the scFvFc displaying human cells could be used directly in functional biological screens with remarkable sensitivity.
  
  
  CONCLUSIONS
  This antibody display platform will complement existing technologies by virtue of providing properties unique to lentiviruses and antibody expression in human cells, which, in turn, may aid the discovery of novel therapeutic human mAbs.
authors.affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America.
authors.forename: Ran
authors.initials: R
authors.lastname: Taube
authors.forename: Quan
authors.initials: Q
authors.lastname: Zhu
authors.forename: Chen
authors.initials: C
authors.lastname: Xu
authors.forename: Felipe
authors.initials: F
authors.lastname: Diaz-Griffero
authors.forename: Jianhua
authors.initials: J
authors.lastname: Sui
authors.forename: Erick
authors.initials: E
authors.lastname: Kamau
authors.forename: Markryan
authors.initials: M
authors.lastname: Dwyer
authors.forename: Daniel
authors.initials: D
authors.lastname: Aird
authors.forename: Wayne A
authors.initials: WA
authors.lastname: Marasco
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000821:virology*; D002469:Cell Separation; D005434:Flow Cytometry; D015967:Gene Expression Regulation, Viral; D005821:Genetic Techniques; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D016086:Lentivirus / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000737:chemistry; D019151:Peptide Library; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000737:chemistry; D014771:Virion / Q000378:metabolism*
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D008562:Membrane Glycoproteins; D019151:Peptide Library; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins
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