Bispecific affibody molecule targeting HPV16 and HPV18E7 oncoproteins for enhanced molecular imaging of cervical cancer

Abstract

High-risk human papillomavirus (HPV16 and HPV18) are now widely recognized as responsible for cervical cancer, which remains to be the most common gynecologic malignancy in women worldwide. It is well known that viral oncoproteins E6/E7 play key roles in HPV-associated cervical carcinogenesis. Thus, in vivo detection of the two oncoproteins may provide important diagnostic information influencing patient management. More recently, affibody molecules have been demonstrated to be a promising candidate for development as molecular imaging probes. Based on the two monomeric affibody molecules (Z and Z) generated in our laboratory, here, we used a peptide linker (GlySer) to link Z and Z to develop a novel heterodimeric affibody Z-(GlySer)-Z Both biosensor and immunofluorescence assays have proved that the heterodimeric affibody molecule targeted simultaneously HPV16 and HPV18E7 proteins by binding to the viral oncoproteins. In vivo tumor-imaging experiments using the Dylight755-labeled heterodimeric affibody revealed that strongly high-contrast tumor retention of the heterodimers occurred in both HPV16- and HPV18-derived tumors of nude mice 0.5 h post-injection. The accumulation of Dylight755-labeled heterodimers in tumors was achieved over 48 h. Therefore, we believe that this novel heterodimeric affibody molecule has great potential utility in molecular imaging in vivo and diagnosis of HPV-associated cervical cancers.

Generation of novel affibody molecules targeting the EBV LMP2A N-terminal domain with inhibiting effects on the proliferation of nasopharyngeal carcinoma cells

“…Kinetic BIAcore analysis indicated that each of the Z LMP2A-N affibodies bound to LMP2A-NCD with affinities approximately 10 5 times higher than that of Z WT and reached the μM level, in accordance with previous studies of Z HPV16E7 384 25 , Z HPV16E7 384-Z HPV18E7 228 29 , and Z HPV16E7 affitoxin384 30 in our laboratory. Furthermore, according to IFA, the Z LMP2A-N affibodies could specifically bind to EBV-positive cell lines, and LMP2A-NCD and Z LMP2A-N affibodies were also co-localised in the juxtamembrane region.…”

Novel EBV LMP-2-affibody and affitoxin in molecular imaging and targeted therapy of nasopharyngeal carcinoma

“…Because of their small size, affibody can be synthesized or recombinantly expressed. Since the first construct HER2-targeting affibody molecule Z HER2:342 was produced and confirmed to bind to HER2-positive cancer cell lines [24], several similar affibody molecules targeting EGFR [23,24], HER3 [27,28], IGF-1R [42], HIV-1-gp120 [43] and HPV16E7 [30,31] have been reported and also applied to image several tumour-associated molecular targets, such as HER2 [25,44], EGFR [45,46], HER3 [47]. The first clinical imaging study on patients with breast cancer was conducted using HER2-specific affibody, which Mice bearing C666-1tumor grafts were intravenously injected with 100 nmol/kg Z142X, Z142, or an equal amount of control agents or the same volume of PBS every two days for 15 times via tail vein.…”

“…To evaluate the target-binding of the selected Z EBVLMP-2 affibodies to EBV LMP-2, surface plasmon resonance (SPR) was performed on a ProteOn XPR36 system (Bio-rad, California, USA). The LMP-2 B-epitope fusion protein (1 nM) served as the ligand was immobilized onto the surface of carboxylate glucans in HTG sensor chip (Bio-rad), as described previously [30,31]. Subsequently, five or six concentrations of each affibody sample were prepared and injected over the chip surface to record sample binding to the surface.…”

“…To determine whether the affibody or affitoxin molecules could bind to the LMP-2 native proteins, IFA was performed as previously described with minor modifications [30,31]. Briefly, EBV + cells, including B95-8, C666-1, CNE-2Z cells and EBV-negative melanoma cells of A375 were seeded in a 24-well plate in 5% CO 2 incubator at 37˚C.…”

“…To date, over 400 published studies show that affibody molecules have been selected for targeting more than 40 different proteins and served as affinity moieties in a variety of applications [22]. The affibody-targeted proteins include epidermal growth factor receptor (EGFR) [23,24], human epidermal growth factor receptor 2 (HER2) [25,26], human epidermal growth factor receptor 3 (HER3) [27,28], vascular endothelial growth factor (VEGF) [29], and human papillomavirus type 16 E7 (HPV16E7) [30,31]. In particularly, affibody molecules attached with a cytotoxin appear to be a straightforward and efficient way to direct the action of the toxin to desired cell targets [22].…”