Lentivirus display: stable expression of human antibodies on the surface of human cells and virus particles.
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.
Taube
Ran
R
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America.
Zhu
Quan
Q
Xu
Chen
C
Diaz-Griffero
Felipe
F
Sui
Jianhua
J
Kamau
Erick
E
Dwyer
Markryan
M
Aird
Daniel
D
Marasco
Wayne A
WA
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*
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
J Virol. 2004 Oct;78(19):10628-35
15367630
J Biol Chem. 1994 Feb 18;269(7):4845-52
8106456
Protein Eng Des Sel. 2007 Feb;20(2):81-90
17242026
Nat Biotechnol. 2005 Sep;23(9):1105-16
16151404
Mol Med. 2001 Aug;7(8):569-79
11591893
J Virol Methods. 2004 Dec 15;122(2):131-9
15542136
Science. 1985 Jun 14;228(4705):1315-7
4001944
J Biol Chem. 2003 Jul 4;278(27):24243-6
12730193
Hum Gene Ther. 2001 Mar 1;12(4):323-32
11242525
Cell. 2003 Jul 25;114(2):161-70
12887918
Proc Natl Acad Sci U S A. 2004 Feb 24;101(8):2536-41
14983044
Proc Natl Acad Sci U S A. 2006 Aug 1;103(31):11479-84
16864770
Nat Biotechnol. 2005 Sep;23(9):1073-8
16151394
Nat Biotechnol. 2003 Feb;21(2):163-70
12536217
Expert Opin Biol Ther. 2003 Apr;3(2):305-18
12662144
Nucleic Acids Res. 2005 Feb 24;33(4):e35
15731328
J Biol Chem. 2003 Jan 17;278(3):1433-42
12401780
Nat Biotechnol. 2002 Nov;20(11):1129-34
12379869
Nat Biotechnol. 2003 May;21(5):569-72
12679787
Mol Cancer Ther. 2006 Aug;5(8):2096-105
16928831
Nat Med. 2003 Mar;9(3):269-77
12612576
Nat Rev Drug Discov. 2004 May;3(5):383-4
15152631
Biotechnol Prog. 2000 Jan-Feb;16(1):31-7
10662486
Nat Biotechnol. 2005 May;23(5):556-7
15877072
Mol Ther. 2000 Aug;2(2):170-6
10947945
Nat Biotechnol. 2005 Sep;23(9):1147-57
16151408
Nature. 2002 Jun 6;417(6889):584-6
12050630
J Immunol. 2007 Aug 15;179(4):2408-18
17675502
J Immunol Methods. 2000 Sep 21;243(1-2):211-27
10986416
Nat Biotechnol. 2005 Jun;23(6):731-5
15924134
Proc Natl Acad Sci U S A. 2006 Jun 20;103(25):9637-42
16763048
J Virol. 2005 May;79(10):5900-6
15857975
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):2706-11
14981267