Targeting of adenovirus vectors carrying a tumor cell-specific peptide: in vitro and in vivo studies

Previously, we have identified a tumor cell-specific peptide, HEW, by panning of phage display libraries on the human colorectal cancer cell line WiDr. In this report we demonstrate that this peptide can modify the infection properties of adenovirus vectors. Increased infectivity of replication-deficient adenovirus 5 vectors in WiDr cells was observed upon genetic insertion of the HEW peptide in the HI loop of the fiber knob. Moreover, whereas the coxsackie and adenovirus receptor (CAR)-ablating fiber mutation S408E abolished apparent infection in CAR-positive WiDr cells, the insertion of HEW completely restored infectivity toward these cells in vitro. To assess whether the de- and re-targeted infection profile was maintained in vivo, the fiber-modified adenovirus vectors were injected intratumorally or intravenously in WiDr tumor-bearing Swiss nu/nu mice. No significant differences in efficiency of infection could be observed suggesting alternative viral uptake mechanisms in vivo. Next, we have included the fiber shaft mutation S(*) in our studies, which was described to confer a de-targeted phenotype in vivo. Reduced gene transfer due to the S(*) mutation both in vitro and in vivo could be confirmed. Insertion of HEW in the HI knob loop of shaft-mutated fiber, however, did not rescue infectivity in target cells neither in vitro nor in vivo. We demonstrate the efficient ligand-mediated re-targeting of adenoviral vector infection to the human cancer cell line WiDr. The lack of apparent re-targeting in the in vivo situation is described.     

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.     

Identification of an oligopeptide binding to hepatocellular carcinoma

OBJECTIVES: We carried out identification of a small peptide binding to human hepatocellular carcinoma (HCC) cells with the aim of applying the peptide for future HCC-targeted therapy or imaging. METHODS: The biopanning technique using phage peptide display libraries was performed on HCC cells in vitro, and a phage clone expressing the HCC-binding peptide motif was selected. The binding activity of the selected phage was evaluated by plaque infection assay and immunofluorescence on cell lines. In addition, the binding activity of the peptide-expressing phage was investigated using HCC specimens derived from patients who had undergone hepatectomy for HCC. RESULTS: A heptapetide, Thr-Thr-Pro-Arg-Asp-Ala-Tyr (TTPRDAY), was identified as a motif binding to HCC. TTPRDAY bound specifically to HCC cells in comparison with other cancer cells, and the binding to HCC cells was also confirmed by immunofluorescence. In addition, the synthesized TTPRDAY peptide showed binding activity and a non-mitogenic effect on HCC cells in vitro. TTPRDAY-presenting phage showed more significant binding to HCC cells derived from specimens obtained from actual patients than to non-cancerous liver tissue. CONCLUSION: The motif TTPRDAY, identified by the biopanning technique, shows significant binding to HCC cells.     

Screening and Identification of Peptides Specifically Targeted to Gastric Cancer Cells from a Phage Display Peptide Library

Background: Gastric cancer is the second most common cancer among the malign cancer types. Inefficiency oftraditional techniques both in diagnosis and therapy of the disease makes the development of alternative and noveltechniques indispensable. As an alternative to traditional methods, tumor specific targeting small peptides can be usedto increase the efficiency of the treatment and reduce the side effects related to traditional techniques. The aim of thisstudy is screening and identification of individual peptides specifically targeted to human gastric cancer cells usinga phage-displayed peptide library and designing specific peptide sequences by using experimentally-eluted peptidesequences. Methods: Here, MKN-45 human gastric cancer cells and HFE-145 human normal gastric epithelial cellswere used as the target and control cells, respectively. 5 rounds of biopannning with a phage display 12-peptide librarywere applied following subtraction biopanning with HFE-145 control cells. The selected phage clones were establishedby enzyme-linked immunosorbent assay and immunofluorescence detection. We first obtain random phage clonesafter five biopanning rounds, determine the binding levels of each individual clone. Then, we analyze the frequenciesof each amino acid in best binding clones to determine positively overexpressed amino acids for designing novelpeptide sequences. Results: DE532 (VETSQYFRGTLS) phage clone was screened positive, showing specific bindingon MKN-45 gastric cancer cells. DE-Obs (HNDLFPSWYHNY) peptide, which was designed by using amino acidfrequencies of experimentally selected peptides in the 5th round of biopanning, showed specific binding in MKN-45cells. Conclusion: Selection and characterization of individual clones may give us specifically binding peptides, butmore importantly, data extracted from eluted phage clones may be used to design theoretical peptides with betterbinding properties than even experimentally selected ones. Both peptides, experimental and designed, may be potentialcandidates to be developed as useful diagnostic or therapeutic ligand molecules in gastric cancer research.     

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。     

乳腺癌干细胞富集及其特异性多肽的筛选

目的：无血清培养法富集乳腺癌干细胞（breast cancer stem cell,BCSC）并采用噬菌体展示技术,筛选能特异性结合乳腺癌干细胞的噬菌体多肽。方法：无血清培养法富集乳腺癌MDA—MB-231细胞株中干细胞并以此为靶标,以hs578bst人正常乳腺细胞及普通培养的MDA—MB-231细胞为减性筛选细胞,对噬菌体随机肽库进行双重减性筛选,选取富集后的阳性噬菌体单克隆,ELISA及DAB染色鉴定阳性噬菌体特异性并测序。结果：经过3轮筛选,噬菌体得到约500倍的富集,随机挑选10株单克隆噬菌体。ELISA显示,6号噬菌体单克隆对乳腺癌干细胞的亲和力是对照的6．14倍;DAB鉴定亦显示,其对乳腺癌干细胞的特异性及亲和力最高,对阳性噬菌体DNA测序翻译得到十二肽氨基酸为GYSASRsTIPGK。结论：通过干细胞富集及噬菌体展示技术,成功筛选出能够特异性结合乳腺癌干细胞的特异性噬菌体多肽,为乳腺癌的干细胞靶向治疗和深入研究奠定基础。     

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.     

Heparan sulfate enhances invasion by human colon carcinoma cell lines through expression of CD44 variant exon 3.

CD44 variant exon (CD44v) 3 is a heparan sulfate-binding isoform of CD44. The role of CD44v3 in invasion and metastasis associated with heparan sulfate in colon cancer cell lines and cases of colon cancer was examined. Expression of CD44v3 mRNA and protein was observed in five of six human colorectal cancer cell lines. Colo320 and WiDr cells expressed CD44v3 at high levels. Heparan sulfate treatment increased the invasive activity of Colo320 and WiDr cells to rates 14.3 and 12.6 times higher, respectively, than that of untreated cells. However, heparan sulfate treatment did not affect cell growth. Repression of CD44v3 protein production by antisense S-oligodeoxynucleotide treatment reduced the binding affinities and capacities for heparan sulfate by Colo320 and WiDr cells in comparison with that of control cells, and it also reduced the invasiveness of both cell lines to one-fifth that of control cells. In heparan sulfate-treated Colo320 cells, the levels of CD44v3 protein in the Triton X-100-insoluble fraction and moesin-precipitated fraction were increased, suggesting that heparan sulfate treatment facilitates association of CD44 molecules with the cytoskeleton. Immunohistochemical analysis showed CD44v3 to be expressed in 21 of 37 (57%) colorectal cancer cases. Positive CD44v3 expression was associated with more advanced pathological stage and poorer prognosis than negative CD44v3 expression. These data support a role for CD44v3 in invasion and metastasis by colorectal carcinoma cells.     

MDA-MB-231乳腺癌细胞特异性多肽的初步筛选

目的 利用噬菌体展示技术,从噬菌体随机十二肽库中筛选出能够特异性结合MDA-MB-231乳腺癌细胞的噬菌体克隆.方法 以人正常乳腺细胞为减性筛选细胞、MDA-MB-231乳腺癌细胞为靶细胞,对噬菌体随机十二肽库进行筛选,挑取富集后的阳性单克隆噬菌体,酶联免疫吸附试验（ELISA）及DAB染色鉴定阳性噬菌体的特异性及亲和力.结果 经过3轮筛选,噬菌体得到约113倍的富集,随机挑选11株单克隆噬菌体,ELISA显示8号噬菌体单克隆对乳腺癌细胞的亲和力是对照的6.5倍,DAB鉴定亦显示其对乳腺癌细胞的特异性及亲和力最高,命名为LK-8.结论 利用噬菌体筛选技术成功筛选出能够特异性结合MDA-MB-231乳腺癌细胞的特异性噬菌体单克隆LK-8,可为进一步合成特异性多肽用于早期诊断和靶向治疗乳腺癌奠定基础.     

结肠癌单抗MC5的噬菌体呈现型单链可变区片段的制备

背景与目的：MC5是一种特异性良好的针对人结肠癌的鼠源性单克隆抗体,而将鼠源性抗体小型化可使其用于在体研究时引起人抗鼠抗体反应的可能性大大降低。本研究的目的是制备MC5的噬菌体呈现型单链可变区片段（ScFv)。方法：从分泌MC5的杂交瘤细胞分离mRNA,RT-PCR分别扩增抗体的重,轻链可变区DNA（VH和VL DNA）,两者经linker DNA连接形成ScFvDNA,将ScFvDNA与噬粒载体pCANTAB5E的连接产物转化于大肠杆菌TG1,经M13KO7辅助噬菌体感染后,获得重组噬菌体抗体ScFv,以高表达MC5结合抗原的细胞株SW480对重组噬菌体抗体ScFv进行两轮筛选后,随机挑取克隆经ELISA筛选呈现MC5 ScFv的噬菌体单克隆,经竞争ELISA对阳性克隆结合抗原的能力进行鉴定。结果：VH,VL和ScFvDNA分别约为340bp,320bp和750bp,在随机筛检的25个克隆中得到10个呈现MC5ScFv的噬菌体单克隆,其中结合抗原能力强的克隆有3个,结论：用噬菌体呈现技术成功地制备了单抗MC5的ScFv,为拓展该抗体的应用范围奠定了基础。     

Peptide-mediated targeting to tumor blood vessels of lung cancer for drug delivery

Antiangiogenesis therapies for the treatment of cancers hold the promise of high efficacy and low toxicity. In vivo phage display was used to identify peptides specifically targeting tumor blood vessels. The peptide SP5-52 recognized tumor neovasculature but not normal blood vessels in severe combined immunodeficiency mice bearing human tumors. Synthetic peptide was shown to inhibit the binding of PC5-52 phage particles to the tumor mass in the competitive inhibition assay. Several selected phage clones displayed the consensus motif, proline-serine-proline, and this motif was crucial for peptide binding to the tumor neovasculature. SP5-52 peptides also bound vascular endothelial growth factor-stimulated human umbilical vein endothelial cells and blood vessels of human lung cancer surgical specimens. Furthermore, this targeting phage was shown to home to tumor tissues from eight different types of human tumor xenografts following in vivo phage display experiments. An SP5-52 peptide-linked liposome carrying doxorubicin enhanced the therapeutic efficacy of the drug, markedly decreased tumor blood vessels, and resulted in higher survival rates of human lung and oral cancer-bearing xenograft mice. The current study indicates that ligand-targeted therapy offers improved therapeutic effects over conventional anticancer drug therapy, and that the peptide SP5-52 specifically targets tumor neovasculature and is a good candidate for targeted drug delivery to solid tumors.     

Analysis of tissue factor and tissue factor pathway inhibitor expression in human colorectal carcinoma cell lines and metastatic sublines to the liver

To investigate the expression of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in human colorectal carcinomas, Northern blot analysis was performed in a series of human colorectal carcinoma cell lines and in normal or tumoral colorectal tissues. Of 16 human colorectal carcinoma cell lines examined, most expressed TF mRNA, though the levels of expression varied significantly. Considerably higher expression was observed in the cell line CaR-1, while lines established from metastatic lesions tended to express abundant TF mRNA. By contrast, TFPI mRNA levels were low in these high TF-expressing cell lines. TFPI was expressed abundantly in WiDr and in a few other cell lines which expressed a very low level of TF mRNA. Immunocytochemically, both proteins were stained predominantly on the cell surface; however, diffuse cytoplasmic staining for TF also was observed in CaR-1 cells. In addition, the cell surface TF activity was significantly higher in CaR-1 cells than in WiDr cells, confirming the results of mRNA analysis. The level of TF mRNA in colorectal carcinoma tissue in vivo and its ratio to the normal counterpart also varied significantly among the cases. To search for a possible role of TF/TFPI in metastasis of colorectal carcinoma cells, the expression of these genes was compared between a rectal adenocarcinoma cell line, RCM-1, and its highly metastatic subline, RCM-1 L-10. Compared with the parent line, RCM-1 L-10 expressed 7.5-fold higher levels of TF mRNA, whereas TFPI expression was not altered significantly or even decreased slightly. The higher cellular TF activity was confirmed in the metastatic subline in comparison with the parent line. Int. J. Cancer 72:878–884, 1997. © 1997 Wiley-Liss, Inc.     

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, characterization, and recovery of small peptide phage display epitopes selected against viable malignant glioma cells

Phage display techniques rely on nearly random oligonucleotide sequences inserted into the protein III filament binding protein of an Escherichia coli filamentous phage M13 to generate a library of phage that express more than 10(7) different peptides. Phage that expresses a sequence having high affinity for a specific molecule, cell, or tissue can then be isolated through selective binding and recovery. Selected phage cannot only be used as gene transfer vectors in themselves, but the small peptide epitopes can be sequenced and potentially recombined into the attachment proteins of viral vectors, or used by themselves to target other therapeutic agents and diagnostic imaging radiolabels. Most phage display selections are carried out against purified and/or fixed protein targets, raising concerns as to the relevance of the selected epitopes. We have selected phage from the CMTI library against viable U87-MG human malignant glioma cells using a derivation of biopanning. The library, which initially contained phage expressing 2x10(7) different epitope sequences, collapsed after four rounds of selection such that 42% of recovered clones expressed a consensus sequence. Selective binding to viable adherent U87-MG cells was subsequently demonstrated under physiologic conditions at 167% (+/-27%) unselected phage using a novel, viable enzyme-linked immunosorbent assay technique. In comparison, there was no difference in binding to control 9L rat gliosarcoma, PANC-1 human pancreatic adenocarcinoma, T98-MG human malignant glioma, or AST-4 human malignant glioma cells of selected compared to unselected phage. Using polymerase chain reaction, the epitope was recovered with flanking unique restriction sites for recombination into a herpes simplex virus type-1 vector. This study demonstrates and discusses optimized methodologies for using phage display to target viable cells.     

Identification of oligopeptides binding to peritoneal tumors of gastric cancer

This is a report of in vivo intraperitoneal biopanning, and we successfully identified a novel peptide to target the multiple peritoneal tumors of gastric cancer. A phage display library was injected directly into the abdominal cavity of mice bearing peritoneal tumors of human gastric cancer, and phages associated with the tumors were subsequently reclaimed from isolated samples. The tumor-associated phages were amplified and the biopanning cycle was repeated five times to enrich for high affinity tumor-selective binding peptides. Finally, a tri-peptide motif, KLP, which showed homology with laminin 5 (a ligand for alpha3beta1 integrin), was identified as a binding peptide for peritoneal tumors of gastric cancer. Phage clones displaying the sequence KLP showed 64-fold higher binding to peritoneal tumors than control phage and were preferentially distributed in tumors rather than in normal organs after intraperitoneal injection into mice. In addition, the KLP phages were more likely to bind to cancer cells in malignant ascites derived from a patient with recurrent gastric cancer. Synthesized peptide containing the motif KLP (SWKLPPS) also showed a strong binding activity to peritoneal tumors without cancer growth effect. Liposomes conjugated with SWKLPPS peptide appeared significantly more often in tumors than control liposomes after intraperitoneal injection into mice. Furthermore, modification of liposomes with SWKLPPS peptide enhanced the antitumor activity of adriamycin on gastric cancer cells. The peptide motif KLP seems a potential targeting ligand for the treatment of peritoneal metastasis of gastric cancer.     

Characterization of a specific phage-displayed Peptide binding to vasculature of human gastric cancer

Antivascular therapy provides a promising method for anticancer therapy. But targeting to gastric cancer vessels is nonselective due in part to the lack of specific cell-surface receptors identified on target vascular cells. Herein we used in vivo screening of phage displayed peptide library to identify some peptides that bind selectively to endothelial cells of human gastric cancer rather than nonendothelial cells. After four rounds of selection, one phage was obtained with a cyclic 7-mer peptide CGNSNPKSC homing to human gastric adenocarcinoma .There was a 4.6 approximately 137.26-fold increase in the number of the selected phage in gastric cancer xenograft in comparision with control organs brain, heart, liver, spleen and kidney. Immunohistochemistry in mouse and human tissue showed that this phage peptide only bind to the endothelial cells of human gastric cancer. This peptide was observed only specific binding to HUVEC not to SGC-7901, Eca-109, LoVo and Hep-G2 by ELISA. The competitive and inhibitory result between the synthetic CGNSNPKSC peptide and the phage displaying the peptide CGNSNPKSC on HUVEC and in vivo was also confirmed its specific binding effect. This peptide may be a possible candidate for targeted drug delivery in antivascular therapy.     

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。     

(E)-(2’)-脱氧-氟亚甲基胞苷的体外放射增敏作用

目的：观察一种新的核苷酸类拟物（E）－（2')－脱氧－氟亚甲基胞苷（FMdC)在体外的放射增殖作用。方法：在结肠癌（WiDr)和宫颈癌（C33－A和C4－I）细胞系进行克隆形成试验。常规照射剂量2Gy时的放射增敏比（SERSF2）定义为2Gy时对照组存活份数（SF）和药物处理组SF之比。结果MFdC增加WiDr细胞的放射敏感性,并呈时间依赖关系。照射前用30nmol/L FMdC处理WiDR细胞-72h,在6、12和24h未能观察到放射增敏作用,放射增敏作用仅在48和72h出现,其SERSF2分别为1．33和1．79。当高剂量FMdC(300nmol/L)处理WiDr细胞6、12和24h,各时间点均可见放射增敏效应。相应的放射增敏比SERSF2分别为1．19、173和3．50。FMDdC的放射增敏作用和药物剂量相关,20－50nmol/L FMdC处理细胞48h,SERSF2从1．15增加至2．28。用30nmol/L FMdC处理C33－A和C4－I细胞48h同样观察到放射增敏作用,SERSF2分别为1．32和2．08。结论FMdC对结肠癌和中颈癌细胞有明显的放射增敏作用,与时间和剂量相关。     

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 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.