Identification of their epitope reveals the structural basis for the mechanism of action of the immunosuppressive antibodies basiliximab and daclizumab

Interleukin-2 (IL-2) and its receptor (IL-2R) play a major role in cellular immunity. The monoclonal antibodies basiliximab and daclizumab directed against the IL-2R subunit CD25 are widely used to prevent graft or host rejection after allogeneic tissue transplantation. Although these antibodies have been used for this purpose for many years, their common epitope within the CD25 protein is unknown. We screened a random phage display library to isolate peptides specifically binding to basiliximab. A striking amino acid sequence motif was enriched. This motif is homologous to the peptide ERIYHFV comprising amino acid positions 116 to 122 within the extracellular domain of CD25, suggesting that this is the basiliximab epitope. Basiliximab and daclizumab binding of selected phage was specific, as no binding was observed to isotype antibody controls. Phage binding could be inhibited by the cognate peptide. In cells expressing mutant CD25, binding of basiliximab was abolished when two or more amino acids of the suspected epitope were changed. In contrast, basiliximab binding remained unaffected in cells expressing CD25 versions with mutations outside this epitope. We therefore conclude that the (116)ERIYHFV(122) string within CD25 is the epitope recognized by basiliximab and daclizumab. This epitope overlaps with the interaction site of CD25 and IL-2, thus revealing the structural basis for the inhibition of IL-2R binding by this class of immunosuppressive antibodies.     

利用体内噬菌体展示技术筛选肝癌组织特异性结合肽

目的 筛选人源肝癌组织特异性结合短肽。方法 体外培养人源肝癌细胞株BEL-7402,建立荷瘤裸鼠模型。尾静脉注射噬菌体12肽文库至荷瘤裸鼠体内,循环20min后回收肿瘤组织中噬菌体,同时取正常对照组织进行噬菌体效价测定和免疫绀化观察。将同收的噬菌体扩增、纯化,并以此作为起始物进行下一轮筛选。经过3轮体内筛选,得到与肝癌组织或细胞特异结合的肽段。随机挑选噬菌体单克隆进行测序,分析序列㈣源性后进行体外细胞酶联免嫂吸附试验（ELISA）和体内回输实验,验证噬菌体克隆的导向性。结果 经过3轮体内筛选,瘤组织中噬菌体的回收率逐步提高,回收量随着输入量的增加迅速增加,而每轮筛选肝组织中的噬菌体回收晕始终保持在一个恒定范围,并不随输入量的增加而增加。免疫组化结果硅示,第3轮筛选后,瘤组织中的噬菌体得到高水平富集,同时其他组织的非特异性结合降至最低,肝癌细胞特异性结合最强的是A54号单克降,A67、B2号单克隆次之。B2的导向效果最好,A54次之。通过对噬菌体单克隆的序列分析,初步确定了PSS／FTT基序。结论 利用体内噬菌体展示技术,可以成功筛选到与肝癌细胞或组织特异结合的噬菌体肽。     

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.     

Mild Heat Shock Induces Autophagic Growth Arrest,But Not Apoptosis in U251-MG and U87-MG Human Malignant Glioma Cells

Although hyperthermia has been used as a treatment of malignant brain tumors, it is not yet clear what is the mechanism of the cell growth inhibition by heat shock, especially by the temperature which has clinically been applied to tumor-brain border-zone, 42-43 degrees C. Therefore, we evaluated the change of U251-MG and U87-MG human malignant glioma cells after 43 degrees C-heat shock comparing with that of 45 degrees C. First, we observed that cell growth was transiently inhibited after 43 degrees C-heat shock for 3 or 5 days, in U251-MG or U87-MG cells, respectively, which was followed by regrowth. During the period of transient growth inhibition, mild G2/M arrest was observed. However, apoptosis was observed in only 2.7% or 1.5%, of 43 degrees C-heated cells, in U251-MG or U87-MG cells, respectively. Instead, transmission electron micrography showed the formation of vacuoles, degeneration of mitochondria, and autophagosomes. Moreover, in the both cell lines, flow-cytometric analysis with acridine orange revealed the induction of acidic vesicle organelles, which was blocked by 3-methyladenine (3-MA), suggesting the involvement of autophagy. Furthermore, while 3-MA did not increase the anti-tumor effect of 43 degrees C-heat shock, bafilomycin A1, another autophagy inhibitor, did significantly enhance the effect in U251-MG cells. Taken together, mild heat shock (43 degrees C for 2 h) causes autophagy and mild G2/M arrest, but does not induce apparent apoptosis in U251-MG and U87-MG glioma cells. Inhibition of autophagy with bafilomycin A1 may increase the anti-tumor efficacy of mild heat shock against some malignant glioma cells.     

Selection of tumor-targeting agents on freshly excised human breast tumors using a phage display library

The selective delivery of therapeutic agents to tumor site without harming rest of the body is a major challenge in clinical oncology today. Phage display approach has been used for searching ligands for cell-surface macromolecules on cancer cells so that they can be employed as drug targeting agents. Although basic protocols for biopanning cells are available, they are not as such suitable for screening highly complex and diverse target as whole tumor. Present study is an attempt to select peptide ligands specific to whole tumors. The cells from freshly collected human breast tumors were biopanned with phage displayed disulfide-constrained random heptapeptide library, following subtraction on normal human breast cells. Comparative analysis of amino acid frequencies in tumor-selected peptides and in random peptides from unselected library showed that selection was not random. The binding assessment of tumor-selected clones, using highly sensitive chemiluminescence ELISA, demonstrated that 47-75% of selected clones, depending on a tumor, bound to tumor cells they were panned on. Furthermore, several clones bound exclusively or preferentially to tumor cells in comparison to normal breast cells. It was interesting to note that insert sequences of tumor-binding clones from different tumors shared significant motifs. It shows the possibility of identifying ligands that may bind to tumor-specific targets common in certain tumors. The results of this investigation on seven human breast tumors demonstrated that, using procedures developed in the present study, whole tumors can be panned successfully with phage displayed library and tumor-binding ligands can be identified rapidly in high throughput manner. This is an important enabling step in identifying lead molecules for developing novel, specific, and effective agents that can be used for the diagnosis and treatment of cancer.     

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.     

Modifications of the fiber in adenovirus vectors increase tropism for malignant glioma models

Recombinant adenovirus (Ad) vectors provide a means of local, therapeutic gene delivery to a wide range of neoplasms. Ad-mediated gene therapy trials in malignant glioma models have been limited by the need for high viral titers and multiple dosages. In an attempt to improve Ad vector gene transfer, we studied human (U87, D54) and rodent (GL261, C6) malignant glioma cell lines transfected with various doses of unmodified Ad vectors (AdZ), Ad vectors that contain an alteration of the fiber-coat protein and that direct virus binding to heparan sulfate receptors (AdZ.F(pK7)), and Ad vectors with modifications of the fiber-coat protein that direct virus binding to alpha1, integrin cellular receptors (AdZ.F(RGD)). AdZ.F(pK7) increased the frequency of cells expressing the reporter gene, beta-galactosidase, and improved transduction by 2- to 20-fold compared with AdZ in U87, D54, and GL261 cells. In U87, D54, GL261, and C6 tumors, AdZ.F(pK7) increased gene transfer by 10- to 100-fold compared with AdZ. AdZ.F(RGD) increased gene expression in C6 xenografts compared with AdZ, but had reduced transduction compared with the C6 xenografts of AdZ in all other glioma tumors. These findings suggest that the increased tropisms resulting from alterations of the Ad vector fiber-coat protein as in AdZ.F(pK7) and AdZ.F(RGD) offer a feasible approach to improving in vitro and in vivo transduction efficiencies in certain malignant glioma cell lines.     

Impact of the coxsackie and adenovirus receptor (CAR) on glioma cell growth and invasion: requirement for the C-terminal domain

Expression of the coxsackie and adenovirus receptor (CAR) is downregulated in malignant glioma cell lines and is barely detectable in high-grade primary astrocytoma (glioblastoma multiforme). We determined the effect of forced CAR expression on the invasion and growth of the human glioma cell line U87-MG, which does not express any CAR. Although retrovirally mediated expression of full-length CAR in U87-MG cells did not affect monolayer growth in vitro, it did reduce glioma cell invasion in a 3-dimensional spheroid model. Furthermore, in xenograft experiments, intracerebral implantation of glioma cells expressing full-length CAR resulted in tumors with a significantly reduced volume compared to tumors generated by control vector-transduced U87-MG cells. In contrast, U87-MG cells expressing transmembrane CAR with a deletion of the entire cytoplasmic domain (except for the first 2 intracellular juxtamembrane cysteine amino acids) had rates of invasion and tumor growth that were similar to those of the control cells. This difference in behavior between the 2 forms of CAR was not due to improper cell surface localization of the cytoplasmically deleted CAR as determined by comparable immunostaining of unpermeabilized cells, equivalent adenoviral transduction of the cells and similar extent of fractionation into lipid-rich domains. Taken together, these results suggest that the decrease or loss of CAR expression in malignant glioma may confer a selective advantage in growth and invasion to these tumors.     

RNA interference targeting survivin exerts antitumoral effects in vitro and in established glioma xenografts in vivo

Malignant glioma represents the most common primary adult brain tumor in Western industrialized countries. Despite aggressive treatment modalities, the median survival duration for patients with glioblastoma multiforme (GBM), the highest grade malignant glioma, has not improved significantly over past decades. One promising approach to deal with GBM is the inactivation of proteins essential for survival or progression of glioma cells by means of RNA interference (RNAi) techniques. A likely candidate for an RNAi therapy of gliomas is the inhibitor of apoptosis protein survivin. Survivin is involved in 2 main cellular processes-cell division and inhibition of apoptosis. We show here that stable RNAi of survivin induced polyploidy, apoptosis, and impaired proliferation of human U343-MG, U373-MG, H4, and U87-MG cells and of primary glioblastoma cells. Proteome profiler arrays using U373-MG cells identified a novel set of differentially expressed genes upon RNAi-mediated survivin knockdown. In particular, the death receptor TRAIL R2/DR5 was strongly upregulated in survivin-depleted glioma cells, inducing an enhanced cytotoxic response of allogeneic human NK cells. Moreover, an experimental in vivo therapy using polyethylenimine (PEI)/siRNA complexes for survivin knockdown efficiently blocked tumor growth of established subcutaneous U373-MG tumors and enhanced survival of NMRI(nu/nu) mice orthopically transplanted with U87-MG cells. We conclude that survivin is functionally relevant in gliomas and that PEI-mediated exogenous delivery of siRNA targeting survivin is a promising strategy for glioblastoma therapy.     

Synergistic augmentation of rapamycin-induced autophagy in malignant glioma cells by phosphatidylinositol 3-kinase/protein kinase B inhibitors

The mammalian target of rapamycin (mTOR) is a downstream effector of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway and a central modulator of cell proliferation in malignant gliomas. Therefore, the targeting of mTOR signaling is considered a promising therapy for malignant gliomas. However, the mechanisms underlying the cytotoxic effects of a selective mTOR inhibitor, rapamycin, on malignant glioma cells are poorly understood. The purpose of this study was thus to elucidate how rapamycin exerts its cytotoxic effects on malignant glioma cells. We showed that rapamycin induced autophagy but not apoptosis in rapamycin-sensitive malignant glioma U87-MG and T98G cells by inhibiting the function of mTOR. In contrast, in rapamycin-resistant U373-MG cells, the inhibitory effect of rapamycin was minor, although the phosphorylation of p70S6 kinase, a molecule downstream of mTOR, was remarkably inhibited. Interestingly, a PI3K inhibitor, LY294002, and an Akt inhibitor, UCN-01 (7-hydroxystaurosporine), both synergistically sensitized U87-MG and T98G cells as well as U373-MG cells to rapamycin by stimulating the induction of autophagy. Enforced expression of active Akt in tumor cells suppressed the combined effects of LY294002 or UCN-01, whereas dominant-negative Akt expression was sufficient to increase the sensitivity of tumor cells to rapamycin. These results indicate that rapamycin exerts its antitumor effect on malignant glioma cells by inducing autophagy and suggest that in malignant glioma cells a disruption of the PI3K/Akt signaling pathway could greatly enhance the effectiveness of mTOR inhibitors.     

Distinct patterns of hypoxic expression of carbonic anhydrase IX (CA IX) in human malignant glioma cell lines

The hypoxia-inducible enzyme carbonic anhydrase IX (CA IX) has recently been discussed as a surrogate marker of tumor hypoxia, an indicator of prognosis and a potential therapeutic target in malignant glioma. To characterize patterns of expression of CA IX in human malignant glioma cells, we studied CA IX protein, CA9 mRNA and hypoxia-inducible factor-1α (HIF-1α) protein levels in U87-MG, U251, U373 and GaMG cells exposed to in vitro hypoxia (1, 6 or 24 h at 5%, 1% or 0.1% O₂). All cell lines displayed a strong hypoxic induction of CA9 mRNA in response to prolonged severe hypoxia with cell-line specific patterns at moderate to mild hypoxia and shorter treatment times. Only U87-MG exhibited a strong constitutive, normoxic expression of CA IX protein without a detectable change under hypoxia. In U251 and GaMG cell lines, a marked induction of CA IX protein in response to severe hypoxia was seen. CA IX changes under severe hypoxia and the inhibitory effect of the glycolysis inhibitor iodoacetate (IAA, 50 μM) on hypoxic CA IX overexpression were paralleled by the results for HIF-1α protein. Therefore, immunohistochemical CA IX staining in human malignant glioma specimens can result from low oxygen concentrations or constitutive, oncogene-related, overexpression both of which may be prognostically relevant.     

Quercetin promotes degradation of survivin and thereby enhances death-receptor-mediated apoptosis in glioma cells

The flavonoid quercetin has been reported to inhibit the proliferation of cancer cells, whereas it has no effect on nonneoplastic cells. U87-MG, U251, A172, LN229, and U373 malignant glioma cells were treated with quercetin (50-200 microM). Quercetin did not cause cytotoxicity 24 h after treatment. Combining quercetin with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) strongly augmented TRAIL-mediated apoptosis in U87-MG, U251, A172, and LN229 glioma cells; U373 cells could not be sensitized by quercetin to TRAIL-mediated apoptosis. TRAIL-induced apoptosis was enhanced by quercetin-induced reduction of survivin protein levels. Upon treatment with quercetin, the protein level of survivin was strongly suppressed in U87-MG, U251, and A172 but not in U373 glioma cells. Quercetin exposure resulted in proteasomal degradation of survivin. TRAIL-quercetin-induced apoptosis was markedly reduced by overexpression of survivin. In addition, upon treatment with quercetin, downregulation of survivin was also regulated by the Akt pathway. Taken together, the results of the present study suggest that quercetin sensitizes glioma cells to death-receptor-mediated apoptosis by suppression of inhibitor of the apoptosis protein survivin.     

Identification of cell proliferation-associated epitope on CD98 oncoprotein using phage display random peptide library

CD98 is known as a cell surface antigen expressed in proliferating normal tissues and in almost all tumor cells. Although the function of CD98 is not yet fully elucidated, it is suggested that CD98 is concerned functionally in lymphocyte activation, cell proliferation, and malignant transformation. Monoclonal antibody against human CD98 heavy chain (h.c.), termed HBJ127, shows inhibition of lymphocyte activation and tumor cell growth in vitro . These observations suggest that the epitope recognized by HBJ127 may be crucial for CD98 function. In the present study, the authors investigated the epitope recognized by HBJ127 using a phage display random heptapeptide library. Approximately 2.4 × 10⁴-fold amplification of eluted phage titer was obtained after three rounds of panning of the phage library against HBJ127. Seven different heptapeptide sequences were isolated from 30 randomly selected clones of the post-panning phage population. A homology search using ClustalW identified the peptide sequence corresponding to ⁴⁴²AFS⁴⁴⁴ of human CD98 h.c. It was also found that ⁴⁴³F is a human-specific amino acid by comparing sequences of human, rat, and mouse origin. Reduced reactivity of HBJ127 was detected against the phenylalanine-substituted peptide but not detected against the alanine or serine-substituted one. It has been identified that HBJ127 reacts only with human species and the HBJ127 epitope position is predicted in 418–529 of human CD98 h.c. From these results and observations, it was estimated that ⁴⁴²AFS⁴⁴⁴ of human CD98 h.c. may be the HBJ127 epitope. Moreover, ⁴⁴³F may be critical for the binding of HBJ127 against human CD98 h.c. ( Cancer Sci 2007; 98: 1696–1700)     

In Vivo Selection of Phage Sequences and Characterization of Peptide-specific Binding to Breast Cancer Cells

OBJECTIVE To screen specific polypeptide target binding to breast cancer xenogra s in vivo from a phage-displayed peptide library in order to provide peptide sequences for breast cancer tumor-targeting diagnosis and therapy. METHODS A mouse model for carrying breast cancer xenografts was established using Tientsin Albinao II mice(TA II).A 12-peptide library was biopanned through 4 rounds. Phages were recovered and titrated from tumor xenografts and control tissue(liver).The distribution of phages was detected by immunohistochemical staining. RESULTS Phage homing to breast cancer was enriched through 4 rounds of biopanning,being 14-fold of that recovered from liver tissue.A peptide sequence,ASANPFPTKALL was characterized by randomly picked-up clones which appeared most frequently. Immunohistochemical staining revealed phage localization in cancer xenografts 40 min after injection of the enriched phages. When a specific phage was tested individually,the phage reclaimed from breast cancer xenografts was 14 times as those from control tissues. CONCLUSION Tumor-specific homing peptides may provide an effective tool for breast cancer target therapy.The in vivo phage display selection technique employed in this study was feasible and applicable to screening peptides that home to breast cells.     

Identification of transforming growth factor-beta1-binding protein overexpression in carmustine-resistant glioma cells by MRNA differential display

BACKGROUND: The authors previously demonstrated the presence of cells in primary human malignant gliomas that intrinsically are resistant to carmustine (BCNU). Numerous studies have identified mechanisms of therapy resistance in these cells; however, the authors' work and that of others suggest that additional mechanisms of resistance exist. METHODS: The authors identified a glioma cell line that lacks detectable methylguanine methyltransferase expression and does not alter its expression of glutathione-S-transferase-pi in response to BCNU chemotherapy. This cell line was used in mRNA differential display experiments to identify genes involved in what to the authors' knowledge were previously undescribed mechanisms of resistance. RESULTS: The overexpression of the gene encoding the transforming growth factor latency binding protein was demonstrated in glioma cells selected for resistance to BCNU, compared with their parental unselected cells. CONCLUSIONS: Transforming growth factor-beta1 has pleiotropic functions in transformed and normal cells. Although activation of TGF-beta1 does not appear to be a causative factor in BCNU resistance in the current study, it may be involved in the growth of these resistant cells.     

Tumor cell-targeting by phage-displayed peptides

We isolated cancer cell-specific phages by subtracting and selecting complex peptide display phage libraries on cultured human cancer cells. The best candidate was selected by performing three rounds of subtraction before each of five selections on the human colorectal WiDr cell line. The phage showed more than 1000-fold higher binding efficiency for WiDr cells when compared to five other human cancer cell lines, including two of colorectal origin, and when compared to wild-type M13 phage. Fifty-fold higher binding efficiency was also seen for a human breast cancer cell line. We show that the WiDr cell binding of the selected phage was efficiently competed by the synthetic peptide HEWSYLAPYPWF, predicted from the phage sequence. This confirms that the specificity of the peptide is independent of the display by the phage coat proteins. The identified peptide may target biomarkers linked to colorectal cancer, and thus be useful for designing gene transfer vectors as well as diagnostic and prognostic tools for this disease.     

Identification of FGF receptor-binding peptides for cancer gene therapy

Linear FGF receptor-binding heptapeptides were identified by phage display using sequential rounds of biopanning against cells with displacement of phage by FGF2. The consensus motif MXXP was iterated after four to five rounds and the peptide MQLPLAT was studied in depth. Phage bearing MQLPLAT showed high levels of binding to FGF receptor positive cells, with over 90% of phage bound being eluted competitively by adding free FGF2. MQLPLAT phage showed only limited binding to Cos7 cells deficient in receptors for FGF. MQLPLAT phage bound to SKOV3 cells with a K(d) of 2.51 x 10(-10) M. Although binding could be blocked by preincubation with free FGF2, heparin could not displace the phage. Use of MQLPLAT to target polyelectrolyte gene delivery vectors in vitro in the presence of serum achieved up to 40-fold greater transgene transduction than nontargeted vectors. MQLPLAT phage were administered into gastric carcinomas via the tumor-feeding artery immediately following resection from patients. The phage showed up to 9-fold more accumulation in the tumor than in adjacent regions of normal tissue, whereas control phage showed less than 2-fold. These peptides should provide useful ligands for specific delivery of gene therapy vectors to clinically relevant targets.     

全人源抗乳腺癌噬菌体单链抗体库的构建

目的：利用噬菌体展示技术构建全人源性抗乳腺癌单链抗体库。方法：从临床获取未化疗乳腺癌病人外周血样30份,分离出单个核细胞（PBMC）,提取总RNA,用RT-PCR技术逆转录获得cDNA,并扩增出全套人抗体重链（VH）和轻链（VL）基因,经重叠延伸PCR（SOE-PCR）,在体外将两者连接成单链抗体（scFv）基因片段,将该片段用Sfi I和Not I酶切后克隆至pCantab5E噬菌体载体,电转化TG1感受态菌,收集培养后平板上的菌落,即构建初级噬菌体单链抗体库。结果：得到长度约为360bp和340bp的VH和VL,拼接后得到的scFv长度约为750bp;经PCR初步鉴定插入率约为80%,BstN 1多样性酶切检验,酶切图谱呈多样性。经测序验证,最终获得库容约为2.4×106pfu/ml初级单链抗体库。结论：本研究获得了全人源抗乳腺癌噬菌体单链抗体库,为下一步筛选抗人乳腺癌细胞特异性单链抗体奠定了基础。