Screening and identification of a renal carcinoma specific peptide from a phage display peptide library

Background

Specific peptide ligands to cell surface receptors have been extensively used in tumor research and clinical applications. Phage display technology is a powerful tool for the isolation of cell-specific peptide ligands. To screen and identify novel markers for renal cell carcinoma, we evaluated a peptide that had been identified by phage display technology.

Methods

A renal carcinoma cell line A498 and a normal renal cell line HK-2 were used to carry out subtractive screening in vitro with a phage display peptide library. After three rounds of panning, there was an obvious enrichment for the phages specifically binding to the A498 cells, and the output/input ratio of phages increased about 100 fold. A group of peptides capable of binding specifically to the renal carcinoma cells were obtained, and the affinity of these peptides to the targeting cells and tissues was studied.

Results

Through a cell-based ELISA, immunocytochemical staining, immunohistochemical staining, and immunofluorescence, the Phage ZT-2 and synthetic peptide ZT-2 were shown to specifically bind to the tumor cell surfaces of A498 and incision specimens, but not to normal renal tissue samples.

Conclusion

A peptide ZT-2, which binds specifically to the renal carcinoma cell line A498 was selected from phage display peptide libraries. Therefore, it provides a potential tool for early diagnosis of renal carcinoma or targeted drug delivery in chemotherapy.     

Isolation of lung tumor specific peptides from a random peptide library: generation of diagnostic and cell-targeting reagents

Discovery of ligands specific to receptor(s) on a surface of a cancer cell could impact clinical issues including functional diagnosis and cell-specific drug delivery. Using a phage display approach, we have isolated 20-mer peptide ligands that bind to 3 different human lung tumor cell lines, NCI-H1299, NCI-H2009, and A549. The panning protocol is unbiased with no selection pressure towards binding a particular cellular receptor. The isolated phage bind to their target cells 24-300 times better than a control phage. Furthermore, the isolated peptides display remarkable cell-specificities and are able to discriminate between normal and cancerous cells as well as different lung tumor cells. The cell-specificities are not coincident with tumor classes indicating that the peptides are able to recognize cell-surface features that are not represented within the classification of tumor type. The isolated peptides are functional outside of the context of the phage and multimerization of the peptide increases its affinity for its given cell type, thus expanding their utility in clinical situations.     

A new TAG-72 cancer marker peptide identified by phage display

Radiolabeled peptides as markers of cancer targets have demonstrated their value in diagnostic imaging and radiotherapy. The 16 mer f88-4/Cys6 phage display library was applied to affinity purified TAG-72 and three consensus peptides were identified: VHHSCTKLTHCCQNWH (A2-13), GGVSCMQTSPVCENNL (A2-6) and TKRDCSAQNYGCQKAI (A2-11). The A2-13 and A2-6 phages showed the highest percent binding to LS-174T cells by flow cytometry and were 3-fold higher than a control phage, while fluorescence microscopy showed that both A2-6 and A2-13 phages bound to the LS-174T cell membrane. However, only the A2-6 phage demonstrated specificity by low binding to the TAG-72 negative cell HT-29. Furthermore, the synthesized free A2-6 peptide demonstrated specific binding to LS-174T cells by flow cytometry and by immunohistochemical staining of xenograft tumor compared to normal colon. These data indicate that the A2-6 peptide is specific for the TAG-72 cancer target.     

Identification of a high affinity TAG-72 binding peptide by phage display selection

Purpose

Phage display was used to select novel peptides that specifically bind the TAG-72 antigen and with properties suitable for imaging TAG-72 positive cancers.

Results

After three rounds of selection against TAG-72 and using two different elution conditions including a long elution, the consensus sequences FRE RCD KHP QKC TKF L and DPR HCQ KRV LPC PAW L were expressed on phages G3-15 and T3-15 respectively. ELISA, fluorescence-activated cell sorting analysis and fluorescence microscopy provided evidence that both phages specifically bound TAG-72 in vitro. Both peptides are stable in 37°C serum. By a cell binding competition assay, the IC₅₀ for T3-15 was measured as 0.29 nM and therefore 36-fold higher affinity than G3-15 at 10.32 nM. The biodistribution in mice carrying LS-174T tumors in one thigh were similar for both ^99mTc-peptides at 30 min, but at 90 min the ^99mTc-T3-15 peptide accumulated almost three times higher in the tumor. The SPECT/CT images were consistent with the biodistribution results.

Procedures

The f88-4/Cys6 phage library and two different elution conditions were used to identify two new higher affinity binding peptides for the TAG-72 antigen. One, was a single brief elution with pH 2.2 glycine buffer and the second began with the glycine elution but was followed with a longer elution with Tris buffered saline (TBS) at pH 7.4. The phages that bound TAG-72 were evaluated by fluorescence-activated cell sorting analysis using TAG-72 positive LS-174T cells and confirmed by immunofluorescence imaging. The consensus peptides displayed on the selected phages were synthesized and conjugated with NHS-MAG3 for radiolabeling with ^99mTc. The IC₅₀ for TAG-72 binding was evaluated by cell binding competition in vitro while binding affinity was evaluated in vivo by necropsy and SPECT/CT imaging in a tumor mouse model.

Conclusion

We have identified a peptide with a sub nanomolar inhibition constant for the TAG-72 antigen that may have applications in cancer imaging.Key words: phage display, TAG-72 antigen, peptide, colon cancer tumor cell LS-174T     

体内噬菌体展示技术筛选人髓样乳腺癌Bcap-37细胞特异性结合肽及其性质、结合效果研究

目的探讨体内噬菌体展示技术筛选的人髓样乳腺癌Bcap-37细胞特异性结合肽的性质和结合效果,为乳腺癌早期诊断提供分子靶向探针。方法制备人髓样乳腺癌Bcap-37细胞荷瘤裸鼠模型,采用噬菌体环七肽库进行3轮体内筛选。免疫组织化学法检测筛选的噬菌体在肿瘤及正常组织中的分布情况。酶联免疫吸附试验(ELISA)鉴定单克隆噬菌体对Bcap-37细胞的亲合力。提取阳性单克隆噬菌体DNA并测序,选取重复率高的序列合成多肽,制备光学分子探针,应用荧光分子成像验证合成的多肽在荷瘤小鼠体内对乳腺移植瘤的特异性和靶向性。结果第3轮体内筛选的噬菌体回收率为第1轮的107.2倍。免疫组织化学结果显示,随筛选轮次增加,肿瘤组织中结合的噬菌体依次增加;肿瘤组织结合的噬菌体多于正常组织(肺脏、骨骼肌、肝脏、肾脏),肿瘤组织切片扫描图像的吸光度(A)值均较正常组织高,差异均有统计学意义(均P<0.05)。ELISA结果显示,随机选取的50个单克隆噬菌体中,22个为阳性(亲合力≥2)。阳性单克隆噬菌体DNA测序分析后,得到4条有重复的氨基酸序列,选择重复率最高的氨基酸序列CSPLNTRFC,化学合成异硫氰酸荧光素(FITC)标记的CSPLNTRFC多肽。Bcap-37细胞荷瘤裸鼠模型体内验证实验显示,FITC-CSPLNTRFC多肽能明显富集在乳腺移植瘤组织。结论利用体内噬菌体展示技术能够筛选出可与人髓样乳腺癌Bcap-37细胞特异性结合的多肽CSPLNTRFC,有助于进行乳腺癌早期诊断的体外研究。     

ZS-1多肽修饰载紫杉醇靶向脂质体抑制NCI-H1299肺癌细胞的研究

目的 以人源性肺癌细胞NCI-H 1299为模型,研究ZS-1多肽修饰的载紫杉醇脂质体(ZS-1 modified paclitaxel liposome,ZSLP-PTX)的体外抗肿瘤作用.方法 采用薄膜分散法制备ZSLP-PTX,并对其理化性质进行表征.MTT实验研究ZSLP-PTX对NCI-H1299肿瘤细胞的增殖抑制能力,用定性和定量的细胞摄取实验研究ZS-1修饰脂质体与肺癌细胞的亲和力.构建NCI-H1299细胞肿瘤球模型,研究不同脂质体对肿瘤球的穿透能力.结果 ZSLP-PTX的粒径为(125.7±18.5)nm,24小时内,在50％的血清中能保持稳定.NCI-H1299细胞和NCI-H1299体外肿瘤球对ZSLP的摄取能力大于LP-PTX(P ＜0.05);ZSLP-PTX对NCI-H1299细胞的增殖抑制作用显著强于LP-PTX(P ＜0.05).结论 与LP-PTX比较,ZSLP-PTX能够更有效地被NCI-H1299肿瘤细胞所摄取,抑制肿瘤细胞的增殖,具有更强的抗肿瘤作用.     

环7肽库筛选胃癌耐药细胞特异性结合短肽

目的获得与胃癌耐药细胞株SGC7901/VCR特异结合的抑制耐药短肽,作为提高胃癌化疗效果的先导化合物。方法以SGC7901/VCR细胞为靶细胞,胃粘膜永生化上皮细胞GES,胃癌药敏细胞SGC7901为吸附细胞对噬菌体7肽库进行消减筛选,用细胞ELISA鉴定阳性噬菌体克隆并测序,利用竞争抑制试验确定阳性克隆的结合部位是否为外源性肽段。结果经4轮筛选,从随机挑选的30个噬菌体克隆中得到14个能特异性与SGC7901/VCR结合而不与胃癌药敏细胞结合的阳性克隆,确定其氨基酸序列分别为SY1和SY2(筛选所得安基酸序列正在专利申请中),含SY1为阳性克隆。结论用噬菌体环7肽库成功筛选到能特异性结合胃癌耐药细胞株的短肽,为肿瘤治疗或药物靶向治疗奠定基础。     

Neuroblastoma tumor cell-binding peptides identified through random peptide phage display

Random peptide phage display libraries have been employed widely to identify protein-protein interactions, using as targets either purified proteins, intact cells, or organs. To isolate peptides that bind to human neuroblastoma cells, we have used a phage display approach with the neuroblastoma cell line WAC 2 as the target. In particular, two bacteriophages, t147 and t160, displaying peptides p147 and p160, respectively, were isolated by repeated display cycles. Binding of t147 and t160 to WAC 2 cells was abrogated by pretreatment with the peptides p147 and p160, respectively, which strongly support that cellular binding of both phages is dictated by their displayed peptides. Immunofluorescence analysis by confocal light microscopy revealed that the major proportion of t147 remains on the surface of WAC 2 cells and that only a fraction is taken up into the cells. In contrast, the vast majority of t160 is internalized. K(+) depletion reduced the number of the phages internalized by the cells to approximately 20% for t160 and to 10% for t147, indicating that the phage internalization was through receptor-mediated endocytosis. Phage t147 appears to bind to a range of tumor cell lines, including neuroblastoma, breast cancer, glioblastoma and C-cell carcinoma, but less so to non-tumor lines, such as erythrocytes, lymphocytes, monocytes and epithelial cells. Phage t160 bound to a range of neuroblastoma cell lines and a breast cancer cell line, but not to other tested cell lines. While neither of the displayed peptides conferred a narrow tissue specific binding ability, they do provide a basis for targeted drug delivery in selected experimental or natural tumor systems.     

Screening peptides binding specifically to colorectal cancer cells from a phage random peptide library

The aim of this study was to screen for polypeptides binding specifically to LoVo human colorectal cancer cells using a phage-displayed peptide library as a targeting vector for colorectal cancer therapy. Human normal colorectal mucous epithelial cells were applied as absorber cells for subtraction biopanning with a c7c phage display peptide library. Positive phage clones were identified by enzyme-linked immunosorbent assay and immunofluorescence detection; amino acid sequences were deduced by DNA sequencing. After 3 rounds of screening, 5 of 20 phage clones screened positive, showing specific binding to LoVo cells and a conserved RPM motif. Specific peptides against colorectal cancer cells could be obtained from a phage display peptide library and may be used as potential vectors for targeting therapy for colorectal cancer.     

肺癌新肿瘤标志物periplakin的筛选研究(英文)

Objective: The aim of this study was to use lung cancer targeting binding polypeptide ZS-9 to screen cDNA library of human lung cancer and obtain ZS-9 specific ligand to confirm tumor marker of non small-cell lung cancer. Methods: Artificially synthesize biotin labeled peptide ZS-9, anchored ZS-9 in the enzyme label plate coupled by avidin, used ZS-9 as probe to screen cDNA library of human lung cancer, after screening, obtained bacteriophage clone specifically binding with anchored polypeptide ZS-9. Extracted plasmid of bacteriophage and performed sequencing after amplified by PCR. Results: It was demonstrated by bioinformatic analysis on the sequence of ligand binded by lung cancer specific peptide ZS-9 that the ligand was the cytoskeletal protein periplakin on the surface of lung cancer cells, suggesting that periplakin might be a new marker for non-small-cell lung cancer in lung cancer. Conclusion: Use specific lung cancer binding peptide to screen new tumor marker periplakin in lung cancer and further studies on its biologic functions in genesis and development of lung cancer are still needed.     

Identification of peptides that bind to irradiated pancreatic tumor cells

PURPOSE: Peptides targeting tumor vascular cells or tumor cells themselves have the potential to be used as vectors for delivering either DNA in gene therapy or antitumor agents in chemotherapy. We wished to determine if peptides identified by phage display could be used to target irradiated pancreatic cancer cells. METHODS AND MATERIALS: Irradiated Capan-2 cells were incubated with 5 x 10(12) plaque-forming units of a phage display library. Internalized phage were recovered and absorbed against unirradiated cells. After five such cycles of enrichment, the recovered phage were subjected to DNA sequencing analysis and synthetic peptides made. The binding of both phage and synthetic peptides was evaluated by fluorescence staining and flow cytometry in vitro and in vivo. RESULTS: We identified one 12-mer peptide (PA1) that binds to irradiated Capan-2 pancreatic adenocarcinoma cells but not to unirradiated cells. The binding of peptide was significant after 48 h incubation with cells. In vivo experiments with Capan-2 xenografts in nude mice demonstrated that these small peptides are able to penetrate tumor tissue after intravenous injections and bind specifically to irradiated tumor cells. CONCLUSION: These data suggest that peptides can be identified that target tumors with radiation-induced cell markers and may be clinically useful.     

胃癌耐药细胞特异性结合小肽对多药耐药的影响

目的 研究环九肽（SY1）与胃癌耐药细胞（SGC7901／VCR）的结合特性及其对胃癌耐药性的逆转作用。方法 将细胞分为SGC7901和SGC7901／VCR2组培养。以无关噬菌体、阴性噬菌体为对照组,用免疫荧光技术检测含有SY1的阳性噬菌体与SGC7901／VCR的结合特性;通过体外药敏试验（MTT）分析含有SY1的阳性噬菌体和化学合成的SY1对SGC7901／VCR耐药性的改变;应用流式细胞仪检测在SY1作用下SGC7901／VCR细胞内阿霉素（ADM）的蓄积和储留。结果 （1）免疫荧光分析的结果显示,含有SY1的阳性噬菌体能够结合于SGC7901／VCR的膜表面,而不与亲本细胞SGC7901结合,无关噬菌体和阴性噬菌体均不与SGC7901／VCR结合,表明SY1可与SGC7901／VCR特异性结合。（2）MTT试验结果显示,在含有SY1的阳性噬菌体及化学合成的SY1的作用下,SGC7901／VCR的存活率显著降低（P〈0．05）,其对长春新碱的耐药性降低。（3）在化学合成的SY1作用下,SGC7901／VCR细胞内ADM的储留蓄积高于对照组（P〈0．05）。结论 利用环七肽库差减筛选得到的环九肽SY1不仅能与SGC7901／VCR特异性结合,而且可部分逆转其对长春新碱的耐药性。这可能为胃癌多药耐药的逆转提供新思路。     

Targeting acute myeloid leukemia with a proapoptotic peptide conjugated to a toll‐like receptor 2‐mediated cell‐penetrating peptide

Cell‐penetrating peptides provide a unique platform to create a new generation of cancer therapeutics with enhanced efficacy and diminished toxicity. In our study, enhanced expression of toll‐like receptor 2 (TLR2) was observed in acute myeloid leukemia (AML) cells. Screening of a phage display peptide library using Biopanning and Rapid Analysis of Selective Interactive Ligands (BRASIL) identified a TLR2‐binding peptide motif, Pep2. We show that the TLR2‐binding peptide motif targeted and penetrated into leukemia cells in a TLR2‐dependent manner. Moreover, a synthetic, chimeric peptide composed of the TLR2‐binding motif linked to a programmed cell death‐inducing sequence, D(KLAKLAK)2, induced apoptosis in AML cells with high TLR2 expression (TLR2^high) but not in chronic myeloid leukemia (CML) cells with low TLR2 expression (TLR2^low). The antileukemia activity of this chimeric peptide was confirmed in leukemia patient samples and an animal model of myeloid leukemia, as the development of leukemia was significantly delayed in mice with TLR2^high AML compared to TLR2^low CML NOD/SCID mice. TUNEL assays on bone marrow tissue slices revealed that the chimerical peptide induced leukemia cell apoptosis in a TLR2‐dependent manner. Together, our findings indicate that TLR2 is a potential therapeutic target for the prevention and treatment of AML, and the prototype, Pep2‐D(KLAKLAK)2, is a promising drug candidate in this setting.     

宫颈癌HeLa细胞特异性结合肽的筛选及鉴定

背景与目的:宫颈癌的分子靶向治疗具有很好的疗效,同时可以显著减少抗癌药物对人体自身的损伤,因此备受关注。本研究利用噬菌体体内展示技术筛选及鉴定宫颈癌特异性结合肽,将有可能成为化疗药物的靶向载体,为宫颈癌靶向药物治疗奠定基础。方法:体外培养宫颈癌HeLa细胞接种裸鼠,建立肿瘤动物模型。将随机肽库尾静脉注入裸鼠体内,循环15 min,心脏灌注后回收肿瘤组织噬菌体扩增、纯化并以此作为起始物进行第2轮的筛选,如此进行3轮体内筛选后挑取噬菌体克隆,进行免疫组化及ELISA实验,初步鉴定噬菌体克隆对宫颈癌细胞的亲和力及特异性,并将具有强亲和力的克隆进行测序。结果:ELISA结果显示,随机挑选10个噬菌体单克隆中8个克隆对HeLa细胞具有很强的亲和力,将这8个克隆进行测序,获得相同短肽序列LLRSTGF。结论:利用噬菌体展示技术筛选出与宫颈癌细胞HeLa特异性结合的短肽,进一步与化疗药物结合,为宫颈癌靶向治疗提供新的方法。     

A peptide selected by biopanning identifies the integrin alphavbeta6 as a prognostic biomarker for nonsmall cell lung cancer

The development of new modes of diagnosis and targeted therapy for lung cancer is dependent on the identification of unique cell surface features on cancer cells and isolation of reagents that bind with high affinity and specificity to these biomarkers. We recently isolated a 20-mer peptide which binds to the lung adenocarcinoma cell line, H2009, from a phage-displayed peptide library. We show here that the cellular receptor for this peptide, TP H2009.1, is the uniquely expressed integrin, alphavbeta6, and the peptide binding to lung cancer cell lines correlates to integrin expression. The peptide is able to mediate cell-specific uptake of a fluorescent nanoparticle via this receptor. Expression of alphavbeta6 was assessed on 311 human lung cancer samples. The expression of this integrin is widespread in early-stage nonsmall cell lung carcinoma (NSCLC). Log-rank test and Cox regression analyses show that expression of this integrin is significantly associated with poor patient outcome. Preferential expression is observed in the tumors compared with the surrounding normal lung tissue. Our data indicate that alphavbeta6 is a prognostic biomarker for NSCLC and may serve as a receptor for targeted therapies. Thus, cell-specific peptides isolated from phage biopanning can be used for the discovery of cell surface biomarkers, emphasizing the utility of peptide libraries to probe the surface of a cell.     

AH6809 antagonizes non-small cell lung cancer prostaglandin receptors

The effects of prostaglandin (PG)E2 on lung cancer cells were investigated. 3H-PGE2 bound with high affinity to membranes derived from small cell lung cancer (SCLC) and non-SCLC (NSCLC) cell lines. Using NSCLC NCI-H1299 membranes, specific 3H-PGE2 binding to NCI-H1299 membranes was inhibited with moderate affinity by PGE2, PGE1, PGF2alpha and 6-isopropoxy-9-xanthone-2-carboxylic acid (AH6809) but not PGD2, LTB4 or 5-HETE. By RT-PCR, EP2 receptor PCR products were detected in extracts derived from lung cancer cells. PGE2 caused cAMP elevation in a concentration-dependent manner using NCI-H1299 cells and the increase in cAMP caused by PGE2 was antagonized by AH6809. PGE2 had no effect on cytosolic Ca2+ but PGE2 caused increased c-fos mRNA in NCI-H1299 cells. AH6809 inhibited the proliferation of NCI-H1299 cells using MTT and clonogenic assays. These results indicate that functional PG receptors are present on NSCLC cells which are antagonized by AH6809.     

TMTP1, a novel tumor-homing peptide specifically targeting metastasis

PURPOSE: Tumor metastasis continues to be the major obstacle to cancer therapy and the leading cause of cancer-related death. Methods used to detect metastasis, especially occult metastases, have received a great deal attention. In this study, a novel selective peptide was assessed for its specific binding to metastasis. METHODS: The FliTrx bacterial peptide display system, an alternative to phage peptide display, was used to identify a 5-amino acid peptide termed TMTP1 (NVVRQ), which binds to the highly metastatic prostate cancer cell line PC-3M-1E8. The synthetic TMTP1 was tested in vitro for its binding specificity and affinity to highly metastatic cancer cells. The tumor targeting assays were done in vivo by i.v. injection of FITC-conjugated TMTP1 into tumor-bearing mice. RESULTS: TMTP1 specifically bound to a series of highly metastatic tumor cells, including prostate cancer PC-3M-1E8, breast cancer MDA-MB-435S, lung cancer PG-BE1, and gastric cancer MKN-45sci, in vitro and in vivo but not to the poorly metastatic or nonmetastatic cell line, including prostate cancer PC-3M-2B4, breast cancer MCF-7, lung cancer PG-LH7, or murine fibroblast cell NIH/3T3. FITC-TMTP1 strongly and specifically targeted the metastasis foci in tumor-bearing mice 24 h after i.v. peptide injection. Moreover, the occult metastases were specifically detected by FITC-TMTP1. CONCLUSION: Our results suggest that TMTP1 is a potential strategy for the development of new diagnostic tracers or alternative anticancer agents for tumor metastasis.     

Analysis of BAC<Subscript>5</Subscript> mcAb-related epitope using random peptide library

Objective To identify epitope relating to BAC₅ mcAb, a kind of monoclonal antibody (mcAb) located on the surface of nasopharyngeal carcinoma (NPC) cells. Methods Using BAC₅ mcAb as a selected target, the 3 rounds of biopanning to a 12 mer random peptide library (RPL) presented by M13 phages were carried out. The positive M13 phage clones were chosen and confirmed with sandwich ELISA for antibody capture and competitive assay. The exogenous DNA fragments in the positive/negative M13 phages were sequenced to deduce and compare the order of the amino acids of exogenous peptides among the phage clones. Results 77% (35/45) of the phages eluted from the 3rd round of biopnning could be captured by BAC₅ mcAb. The 3 kinds of the peptides were displayed by M13 phages from the 8 positive clones identified with competitive assay. The same character of “-P-V-” structure existed near N-terminus of the 3 different peptides, i. e.-H-Q-S-H-Y-P-Y-P-V-V-S-L- (4/8)-Q-N-Q-A-W-F-S-Q-P-V-R-M-(3/8) and T-Q-A-Y-K-G-F-P-V-L-P-S- (1/8) in comparison with the peptide “-N-H-Q-S-T-F-W-Q-K-W-T-A-” displayed by M13 phages from the negative clones (6/6). Conclusion BAC₅ mcAb can recognize the 3 kinds of the peptides with-P-V-structure near N-terminus. These peptides mimic the structure of the epitope on the surface of NPC cells recognized by BAC₅ mcAb. This project was founded by Health Foundation of Guangdong Province (Grant No. A1999212)     

Identification of high-affinity VEGFR3-binding peptides through a phage-displayed random peptide library

Objective

Vascular endothelial growth factor (VEGF) interaction with its receptor, VEGFR-3/Flt-4, regulates lymphangiogenesis. VEGFR-3/Flt-4 expression in cancer cells has been correlated with clinical stage, lymph node metastasis, and lymphatic invasion. The objective of this study is to identify a VEGFR-3/Flt-4-interacting peptide that could be used to inhibit VEGFR-3 for ovarian cancer therapy.

Methods

The extracellular fragment of recombinant human VEGFR-3/Flt-4 (rhVEGFR-3/Flt-4) fused with coat protein pIII was screened against a phage-displayed random peptide library. Using affinity enrichment and enzyme-linked immunosorbent assay (ELISA) screening, positive clones of phages were amplified. Three phage clones were selected after four rounds of biopanning, and the specific binding of the peptides to rhVEGFR-3 was detected by ELISA and compared with that of VEGF-D. Immunohistochemistry and immunofluorescence analyses of ovarian cancer tissue sections was undertaken to demonstrate the specificity of the peptides.

Results

After four rounds of biopanning, ELISA confirmed the specificity of the enriched bound phage clones for rhVEGFR-3. Sequencing and translation identified three different peptides. Non-competitive ELISA revealed that peptides I, II, and III had binding affinities for VEGFR-3 with K_aff (affinity constant) of 16.4±8.6 µg/mL (n=3), 9.2±2.1 µg/mL (n=3), and 174.8±31.1 µg/mL (n=3), respectively. In ovarian carcinoma tissue sections, peptide III (WHWLPNLRHYAS), which had the greatest binding affinity, also co-localized with VEGFR-3 in endothelial cells lining lymphatic vessels; its labeling of ovarian tumors in vivo was also confirmed.

Conclusion

These finding showed that peptide III has high specificity and activity and, therefore, may represent a potential therapeutic approach to target VEGF-VEGFR-3 signaling for the treatment or diagnosis of ovarian cancer.     

Identification of a rhabdomyosarcoma targeting peptide by phage display with sequence similarities to the tumour lymphatic‐homing peptide LyP‐1

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. To improve existing therapies and broaden the spectrum of cytotoxic agents that can be used in RMS treatment, we performed a phage‐display‐based screening for peptides that bind specifically to RMS cells. Two peptides binding to RMS and to other tumour cell lines, but not to normal skeletal muscle cells and fibroblasts, were isolated from phage‐displayed random peptide libraries. One peptide, named RMS‐I (CQQSNRGDRKRC) contained the integrin‐binding motif RGD and its binding was blocked by an antibody against α_vβ₃integrin, which is expressed on the RMS cell line RD. The isolation of RMS‐I confirmed the validity of our screening procedure. The second peptide, named RMS‐II (CMGNKRSAKRPC), shows sequence similarity to a previously identified peptide with tumour lymphatic specificity, LyP‐1. However, RMS‐II binds in vivo to RMS xenografts better than LyP‐1 and homes to the tumour blood and not to lymphatic vessels. Therefore, RMS‐II represents a promising peptide for the development of RMS‐specific targeting approaches. © 2008 Wiley‐Liss, Inc.