Retinoblastoma-independent antiproliferative activity of novel intracellular antibodies against the E7 oncoprotein in HPV 16-positive cells.
BACKGROUND
"High risk" human papillomavirus strains are the causative agents of the vast majority of carcinomas of the uterine cervix. In these tumors, the physical integration of the HPV genome is a frequent, though not invariable occurrence, but the constitutive expression of the E6 and E7 viral genes is always observed, suggesting key roles for the E6 and E7 oncoproteins in the process of malignant transformation. The "intracellular antibody" technology using recombinant antibodies in single-chain format offers the possibility of targeting a protein in its intracellular environment even at the level of definite domains thus representing a valuable strategy to "knock out" the function of specific proteins.


METHODS
In this study, we investigate the in vitro activity of two single-chain antibody fragments directed against the "high-risk" HPV 16 E7 oncoprotein, scFv 43M2 and scFv 51. These scFvs were expressed by retroviral system in different cell compartments of the HPV16-positive SiHa cells, and cell proliferation was analyzed by Colony Formation Assay and EZ4U assay. The binding of these scFvs to E7, and their possible interference with the interaction between E7 and its main target, the tumor suppressor pRb protein, were then investigated by immunoassays, PepSet™ technology and Surface Plasmon Resonance.


RESULTS
The expression of the two scFvs in the nucleus and the endoplasmic reticulum of SiHa cells resulted in the selective growth inhibition of these cells. Analysis of binding showed that both scFvs bind E7 via distinct but overlapping epitopes not corresponding to the pRb binding site. Nevertheless, the binding of scFv 43M2 to E7 was inhibited by pRb in a non-competitive manner.


CONCLUSIONS
Based on the overall results, the observed inhibition of HPV-positive SiHa cells proliferation could be ascribed to an interaction between scFv and E7, involving non-pRb targets. The study paves the way for the employment of specific scFvs in immunotherapeutic approaches against the HPV-associated lesions.
Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Rome, Italy. luisa.accardi@iss.it
Luisa
L
Accardi
Maria Gabriella
MG
Donà
Anna M
AM
Mileo
Marco G
MG
Paggi
Antonio
A
Federico
Paola
P
Torreri
Tamara C
TC
Petrucci
Rosita
R
Accardi
David
D
Pim
Massimo
M
Tommasino
Lawrence
L
Banks
Barbara
B
Chirullo
Colomba
C
Giorgi
D001667:Binding, Competitive; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018604:Epitope Mapping; D005260:Female; D005455:Fluorescent Antibody Technique; D057809:HEK293 Cells; D054884:Host-Pathogen Interactions; D052162:Human papillomavirus 16 / Q000276:immunology* / Q000502:physiology; D006801:Humans; D050725:Papillomavirus E7 Proteins / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014162:Transfection; D002583:Uterine Cervical Neoplasms / Q000276:immunology / Q000473:pathology / Q000821:virology
D050725:Papillomavirus E7 Proteins; D016160:Retinoblastoma Protein; D057127:Single-Chain Antibodies; C059731:oncogene protein E7, Human papillomavirus type 16
J Proteome Res. 2006 Aug;5(8):1829-42
16889404
Cancer Res. 2009 Jul 15;69(14):5656-63
19584294
PLoS One. 2009 Oct 13;4(10):e7254
19826491
Cancer Res. 2006 Oct 1;66(19):9393-400
17018593
Curr Med Chem. 2010;17(17):1730-55
20345346
Bioessays. 1999 Mar;21(3):221-30
10333731
J Virol. 2007 Dec;81(24):13533-43
17913829
J Med Virol. 1998 Mar;54(3):192-5
9515768
Expert Opin Biol Ther. 2007 May;7(5):677-89
17477805
Nat Rev Cancer. 2002 May;2(5):342-50
12044010
Mol Cell Biol. 2002 Oct;22(20):7041-52
12242284
Biochemistry. 2007 Sep 18;46(37):10405-12
17715947
Int J Cancer. 2005 Sep 10;116(4):564-70
15818614
J Cell Physiol. 2007 Jul;212(1):118-25
17348035
Methods Mol Biol. 2005;292:401-20
15507723
Antivir Ther. 2003 Aug;8(4):265-78
14518695
Oncogene. 2003 Sep 4;22(38):5938-45
12955072
J Mol Biol. 2006 Jan 20;355(3):360-78
16324714
Virology. 2009 Jan 5;383(1):60-8
18996550
Science. 1989 Feb 17;243(4893):934-7
2537532
J Gen Virol. 1997 Oct;78 ( Pt 10):2607-13
9349482
J Biol Chem. 1998 Aug 21;273(34):21769-76
9705314
Mol Cell Biol. 2007 Oct;27(20):7248-65
17709384
Carcinogenesis. 2003 Feb;24(2):159-69
12584163
Proc Natl Acad Sci U S A. 2005 Aug 9;102(32):11492-7
16061792
Oncogene. 2005 Nov 24;24(53):7830-8
16044149
Cancer Res. 1998 May 1;58(9):1893-900
9581830
Virology. 2009 Feb 20;384(2):335-44
19007963
J Virol. 2001 Aug;75(16):7583-91
11462030
J Biol Chem. 2006 Jan 6;281(1):578-86
16249186
Oncogene. 2000 Feb 10;19(6):821-6
10698500
Biochemistry. 2004 Mar 30;43(12):3310-7
15035602
BMC Cancer. 2007 Jan 31;7:25
17266749
J Virol. 2007 Sep;81(18):9737-47
17609271
Bioessays. 2010 Jul;32(7):589-98
20544739
Nat Rev Cancer. 2010 Aug;10(8):550-60
20592731
Mol Cell Biol. 2003 Dec;23(24):9094-103
14645521
Oncogene. 2001 Nov 26;20(54):7888-98
11753671