Generation of affibody molecules specific for HPV16 E7 recognition

Xue, Xiangyang; Wang, Bingbing; Du, Wangqi; Zhang, Chanqiong; Song, Yun‐Chun; Cai, Yiqi; Cen, Danwei; Wang, Ledan; Xiong, Yirong; Jiang, Pengfei; Zhu, Shanli; Zhao, Kong‐Nan; Zhang, Lifang

doi:10.18632/oncotarget.12174

Cited by 28 publications

(53 citation statements)

References 37 publications

(36 reference statements)

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“…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.…”

Section: Discussionsupporting

confidence: 90%

“…A combinatorial phage library of the Z domain was prepared as described previously 25 with random amino acid residues at positions 9, 10, 11, 13, 14, 17, 18, 24, 25, 27, 28, 32 and 35. Briefly, according to the amino acid sequence and structure of the Wild SPA-Z scaffold (Z WT ), random primers were designed to target the coding sequences corresponding to the three helix structural domains. An SPA sequence that could cause random amino acid changes was amplified by polymerase chain reaction (PCR) and named SPA-N.…”

Section: Construction Of a Z Domain Combinatorial Librarymentioning

confidence: 99%

“…Selection of potential affibodies binding to LMP2A-NCD with high affinity Highly purified LMP2A-NCD protein was used as a targeted protein during three rounds of phage display panning and enzyme-linked immunosorbent assay (ELISA) selections for target-binding activity. Screening for potential affibody molecules binding to LMP2A-NCD was performed as described in a previous study 25 . After panning, ELISA screening and DNA sequencing, the sequences of inserted fragments in selected phages acted as potential affibodies with high affinity and specifically bound to the recombinant protein.…”

Section: Production Of Recombinant Lmp2a-ncd Proteinmentioning

confidence: 99%

“…To date, more than 500 papers have been published on this topic (www.ncbi.nlm.nih.gov). As highaffinity ligands, affibody molecules specifically target more than 40 membrane molecules or viral oncoproteins, including human epidermal growth factor receptor 2 (HER2) 22 , epidermal growth factor receptor (EGFR) 23 , HIV-1 envelope glycoprotein gp120 (HIV-1-gp120) 24 , and human papillomavirus type 16 E7 (HPV16E7) 25 , showing great potential for in vivo molecular imaging, receptor signal blocking and biotechnology applications 20,21 .…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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Nasopharyngeal carcinoma (NPC) induced by latent infection with Epstein-Barr virus (EBV) remains the most common head and neck cancer in Southeast Asia, especially in the southern part of China. It is well known that persistent expression of two EBV latent membrane proteins (LMP1/LMP2A) plays a key role in nasopharyngeal carcinogenesis. Therefore, the therapeutic approach of targeting the LMP1/LMP2A protein and subsequently blocking the LMP1/ LMP2A-mediated signalling pathway has been considered for treating patients with NPC. Recently, affibody molecules, a new class of small (~6.5 kDa) affinity proteins, have been confirmed to be powerful generalisable tools for developing imaging or therapeutic agents by targeting specific molecules. In this study, three EBV LMP2A N-terminal domain-binding affibody molecules (Z LMP2A-N 85, Z LMP2A-N 110 and Z LMP2A-N 252) were identified by screening a phagedisplayed peptide library, and their high affinity and specificity for the EBV LMP2A N-terminal domain were confirmed by surface plasmon resonance (SPR), indirect immunofluorescence, co-immunoprecipitation and near-infrared small animal fluorescence imaging in vitro and in vivo. Moreover, affibody molecules targeting the EBV LMP2A N-terminal domain significantly reduced the viability of the EBV-positive cell lines C666-1, CNE-2Z and B95-8. Further investigations showed that affibody Z LMP2A-N 110 could inhibit the phosphorylation of AKT, GSK-3β and β-catenin signalling proteins, leading to suppression of β-catenin nuclear translocation and subsequent inhibition of c-Myc oncogene expression, which may be responsible for the reduced viability of NPC-derived cell lines. In conclusion, our findings provide a strong evidence that three novel EBV LMP2A N-terminal domain-binding affibody molecules have great potential for utilisation and development as agents for both molecular imaging and targeted therapy of EBVrelated NPC.

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Section: Discussionsupporting

confidence: 90%

Section: Construction Of a Z Domain Combinatorial Librarymentioning

confidence: 99%

Section: Production Of Recombinant Lmp2a-ncd Proteinmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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“…Therefore, detection of HPV E7 is essential for the diagnosis of cervical cancer in the early stage [21]. Recently, the HPV18 L1 ELISA detecting method and HPV16 E7-binding affibody molecules and nanobodies against the HPV16 E7 oncoprotein have been shown to have promising potential for the diagnosis of HPV-induced cancers [22][23][24]. The goal of this research is to establish a method with high sensitivity for quantitative detection of the HPV16 E7 oncoprotein in cervical cancer tissues.…”

Section: Discussionmentioning

confidence: 99%

Preparation, Characterization and Diagnostic Valuation of Two Novel Anti-HPV16 E7 Oncoprotein Monoclonal Antibodies

Dong

Zhang

et al. 2020

Viruses

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At present, the clinical detection method of human papillomavirus (HPV) is mainly based on the PCR method. However, this method can only be used to detect HPV DNA and HPV types, and cannot be used to accurately predict cervical cancer. HPV16 E7 is an oncoprotein selectively expressed in cervical cancers. In this study, we prepared an HPV16 E7-histidine (HIS) fusion oncoprotein by using a prokaryotic expression and gained several mouse anti-HPV16 E7-HIS fusion oncoprotein monoclonal antibodies (mAbs) by using hybridoma technology. Two mAbs, 69E2 (IgG2a) and 79A11 (IgM), were identified. Immunocytochemistry, immunofluorescence, immunohistochemistry, and Western blot were used to characterize the specificity of these mAbs. The sequences of the nucleotide bases and predicted amino acids of the 69E2 and 79A11 antibodies showed that they were novel antibodies. Indirect enzyme-linked immunosorbent assay (ELISA) with overlapping peptides, indirect competitive ELISA, and 3D structural modeling showed that mAbs 69E2 and 79A11 specifically bound to the three exposed peptides of the HPV16 E7 (HPV16 E7 49-66 , HPV16 E7 73-85 , and HPV16 E7 91-97 ). We used these two antibodies (79A11 as a capture antibody and 69E2 as a detection antibody) to establish a double-antibody sandwich ELISA based on a horseradish peroxidase (HRP)-labeled mAb and tetramethylbenzidine (TMB) detection system for quantitative detection of the HPV16 E7-HIS fusion oncoprotein, however, it was not ideal. Then we established a chemiluminescence immunoassay based on a labeled streptavidin-biotin (LSAB)-ELISA method and luminol detection system-this was sufficient for quantitative detection of the HPV16 E7-HIS fusion oncogenic protein in ng levels and was suitable for the detection of HPV16-positive cervical carcinoma tissues. Collectively, we obtained two novel mouse anti-HPV16 E7 oncoprotein mAbs and established an LSAB-lumino-dual-antibody sandwich ELISA method for the detection of the HPV16 E7-HIS fusion oncogenic protein, which might be a promising method for the diagnosis of HPV16-type cervical cancers in the early stage.Viruses 2020, 12, 333 3 of 23 carcinoma, and 5 cases of cervical cancer tissues derived from stage IIa HPV16-positive cervical cancers. All cancerous specimens were obtained by using the extensive hysterectomy, while normal specimens were obtained by using the panhysterectomy. A Mini Shaker MH-1 microplate oscillator (Kylin-Bell Lab Instruments, Haimen, Jiangsu, China), a 1510 MULTISKAN GO full-wavelength microplate reader (Thermo Fisher Scientific, Waltham, MA, USA), and an Infinite ® M200 PRO microplate reader (TECAN, Männedorf, Switzerland) were used for general experiments. Preparation of HPV16 E7-HIS Fusion OncoproteinAccording to the full-length HPV16 E7 gene, specific primers with 6 histidine (HIS) tags were designed. The sequences of primers F and R were as follows: F: 5 -GCCCATGGACCATCACCATC ACCATATGCATGGAGATACACCTAC-3 ; R: 5 -GCAAGCTTTTATGGTTTCTGYGAACAGATGG GGCACAC-3 . Using the total genome DNA of CaSki cells, which was pre-e...

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

Zhu

Song

et al. 2018

Appl Microbiol Biotechnol

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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.

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Generation of affibody molecules specific for HPV16 E7 recognition

Cited by 28 publications

References 37 publications

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

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

Preparation, Characterization and Diagnostic Valuation of Two Novel Anti-HPV16 E7 Oncoprotein Monoclonal Antibodies

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

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