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.     

Novel transmembrane GTPase of non-small cell lung cancer identified by mRNA differential display.

The technique of differential display was used previously to profile the gene expression patterns of non-small cell lung cancer, and several genes differentially expressed were thus identified. In this report, we demonstrate that a DNA fragment of 347-bp length, up-regulated in tumor tissues, showed 100% sequence similarity to human cDNA FLJ20693 for a 370-residue protein. The gene product of cDNA FLJ20693 was postulated to be a shorter isoform of transmembrane GTPase, termed TG370, based upon the results of searching for sequence homology. The nucleotide sequence alignment also indicated that the cDNA FLJ20693 and the cDNA for 741-residue human mitofusin 1 (TG741) possibly resulted from the event of alternative splicing from which a 127-bp region was retained in the latter. Analysis of the genome sequence confirmed the speculation that both cDNAs were mapped to the same chromosomal position composing of 18 exons, of which the 127-bp region of TG741 constituted exon 11. The alternative splicing in all lung cancer cell lines was also observed to occur nearly in all tissue specimens examined. The up-regulated expression of transmembrane GTPase was subsequently found in tumor tissues from at least five of seven non-small cell lung cancer patients. Also, a distinct PCR product was initially detected in cell line H520, and further sequence analysis identified the presence of the 86-bp region mapped to the genome sequence immediately followed by exon 10. To evaluate the retention of 86-bp region, it was found that, besides the predicted 486-bp product, an unexpected 332-bp product was concomitantly observed and identified as the result of exon 8 deletion. The expression and subcellular localization of the full-length TG741 and other shorter isoforms were detected by flow cytometry using three polyclonal antibodies. It was concluded that the full-length TG741 located at plasma membrane with its NH(2)-terminal domain exposed extracellularly and the shorter isoforms retained at cytosol. Finally, the up-regulation of transmembrane GTPase in tumor tissues was further illustrated using immunohistochemical staining.     

Conversion of a tumor-binding peptide identified by phage display to a functional chimeric T cell antigen receptor

Adoptive transfer of ex vivo expanded tumor-specific T cells is a promising therapeutic modality for promoting or augmenting antitumor immunity. Several groups, including ours, are developing antigen receptor gene transfer strategies as a means of generating effector cells for adoptive therapy. Chimeric antigen receptors (CARs) have been described that use single-chain antibodies or cytokine ligands as tumor targeting domains. Here, we describe the capacity of a tumor-binding peptide identified by phage display combinatorial library screening to serve as a CAR targeting domain. A phage library-selected high-affinity 12-mer peptide (Bpep) specific for alpha(v) beta(6) integrin (alpha v beta6) was chosen for these studies. Primary human T cells were genetically modified to express the Bpep-CAR consisting of an alpha v beta6-specific peptide and human IgG4 hinge-Fc extracellular domain fused to the cytoplasmic tail of CD3-zeta. T cell expression of the Bpep-CAR was assessed by Western blot analysis, and trafficking of the Bpep-CAR to the cell surface was demonstrated by flow cytometry. Functionally, Bpep-CAR redirected cytotoxic T lymphocytes specifically kill integrin alpha v beta6+ ovarian tumor targets, and are activated for interferon gamma secretion. Our data suggest that large new repertoires of tumor-specific T cell antigen receptor transgenes might be available through merging combinatorial peptide libraries with CAR construct design.     

Gefitinib (Iressa): a novel treatment for non-small cell lung cancer

Current standard therapy for advanced or metastatic non-small cell lung cancer (NSCLC) includes chemotherapy, which is often associated with limited efficacy and toxicity. Thus, there is an unmet need for novel anticancer agents that are both effective and well tolerated in patients with NSCLC. Gefitinib (Iressa) is an orally active epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor in advanced clinical development, and the first agent of its kind to receive approval. Extensive evidence indicates that gefitinib provides significant antitumor activity in previously treated advanced NSCLC, along with fast symptom improvement. This orally administered agent is generally well tolerated. Gefitinib has also shown promising efficacy in other solid tumors that rely on EGFR-related mechanisms for growth and survival. This article reviews the profile of gefitinib and the evidence supporting its use in the treatment of NSCLC.     

A novel molecular staging protocol for non-small cell lung cancer.

A molecular staging protocol using reliable markers is of importance in predicting the prognosis of patients with non-small cell lung cancer (NSCLC) and for instituting their appropriate post-surgical treatment. We analysed tumor tissues from 187 NSCLC patients. The DNA and mRNA were extracted from frozen specimens, and then polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and direct sequencing were performed to investigate mutations of p53 from exons 5-8, and mutations of K-ras at exon 1. To determine MRP-1/CD9 gene and KA11/CD82 gene expression, which have been postulated to be metastasis suppressor genes, we have applied quantitative RT-PCR. A Cox multivariate regression analysis showed that nodal status, MRP-1/CD9 and K-ras status were significant factors for prognosis (P<0.0001, P=0.0083 and P=0.0004, respectively). Based on these results, we classified the patients into three groups according to their MRP-1/ CD9 and K-ras status. Patients with both MRP-1/CD9 positive and wild K-ras tumors were defined as group A, patients with either reduced MRP-1/CD9 or mutant K-ras tumors were defined as group B and patients with both reduced MRP-1/CD9 and mutant K-ras tumors were designated as group C. This new classification was significantly correlated with the tumor status and pathological stage (P=0.0098 and P=0.0017, respectively). The overall survival rate of the group A patients was significantly better than the group B patients (59.6% vs 27.9%, P=0.0001) and also that of group B patients was better than the group C patients (27.9% vs 20.0%, P=0.0378). This tendency was also found in patients with 110 node-negative NSCLCs (A vs B vs C=75.8% vs 34.9% vs 0.0%, P<0.0001). A Cox multivariate regression analysis in NSCLC patients demonstrated that an evaluation for both MRP-1/CD9 expression and K-ras mutations had a significant prognostic effect as well as nodal status (P<0.0001).     

Gemcitabine (Gemzar) in non-small cell lung cancer

Of the new chemotherapeutic substances of the last decade, gemcitabine (Gemzar, Eli Lilly) is probably the most valuable for the treatment of early and advanced stage non-small cell lung cancer (NSCLC). When used as a single agent in both chemotherapeutically pretreated and chemotherapy-naive patients, gemcitabine shows an objective tumor regression rate of approximately 20%. Gemcitabine's unique mechanism of action and its lack of overlapping toxicity with other cytotoxic agents also define it as an ideal candidate for combination therapy. Early clinical development has included single-agent first- and second-line treatment, doublet combination regimens and incorporation into multimodality treatment strategies for operable and inoperable locally advanced nonmetastatic NSCLC. Gemcitabine/platinum-based combination chemotherapy has become the most attractive treatment standard for NSCLC patients in good clinical condition. The role of gemcitabine in the concurrent or sequential application of chemo- and radiotherapy for inoperable locally advanced NSCLC has also been addressed in several Phase I and II studies. Based on data available, gemcitabine can be safely administered in combination with radiotherapy. This review summarizes results from representative Phase I, II and III studies in order to underline gemcitabine's clinical importance for patients suffering from early and advanced NSCLC.     

ZD1839 (Iressa) in non-small cell lung cancer

Despite the advent of cisplatin-based combination chemotherapy for advanced non-small cell lung cancer (NSCLC), the prognosis for this patient population remains poor. Novel biologically targeted agents currently in development have the potential for greater efficacy against NSCLC, and possibly less toxicity than is associated with conventional cytotoxic chemotherapy. The epidermal growth factor receptor (EGFR) is recognized as a potentially useful target, and the small molecule, orally active EGFR-tyrosine kinase inhibitor ZD1839 (Iressa) is currently the furthest along in clinical development of the anti-EGFR agents. This review summarizes the currently available clinical data on the use of ZD1839 in the treatment of NSCLC.     

Evolution of non-small cell lung cancer chemotherapy (Review)

Non-small cell lung cancer (NSCLC) is a common malignancy which has been increasing in incidence over the last decades. In the past, these tumors were considered quite resistant to chemotherapy and until the development of cisplatin 30 years ago, the use of cytotoxic drugs was considered palliative. Cisplatin (CDDP) proved to be an active agent and when combined with other cytotoxic drugs, effectiveness in NSCLC increased. Many trials have taken place during the last 30 years with cisplatin combinations. Despite the effectiveness of cisplatin, the renal toxicity and neurotoxicity that are produced obliged researchers and clinicians to create other combinations without CDDP. Carboplatin, an analogue of CDDP and then taxanes, gemcitabine and vinorelbine, a Vinca alkaloid, that proved to be effective in NSCLC, led to substitute combinations for CDDP. In the present article we review the literature on NSCLC chemotherapy in order to visualize the effectiveness of older drug combinations vs. the newer ones. It seems that combinations with or without cisplatin are of similar effectiveness with respect to response rate and median and overall survival but the toxicity is different with a prevalence of myelotoxicity.     

Inhibition of non-small cell lung cancer (NSCLC) growth by a novel small molecular inhibitor of EGFR

The epidermal growth factor receptor (EGFR) is a therapeutic target (oncotarget) in NSCLC. Using in vitro EGFR kinase activity system, we identified a novel small molecule, WB-308, as an inhibitor of EGFR. WB-308 decreased NSCLC cell proliferation and colony formation, by causing G2/M arrest and apoptosis. Furthermore, WB-308 inhibited the engraft tumor growths in two animal models in vivo (lung orthotopic transplantation model and patient-derived engraft mouse model). WB-308 impaired the phosphorylation of EGFR, AKT, and ERK1/2 protein. WB-308 was less cytotoxic than Gefitinib. Our study suggests that WB-308 is a novel EGFR-TKI and may be considered to substitute for Gefitinib in clinical therapy for NSCLC.     

Gefitinib (Iressa) trials in non-small cell lung cancer

Gefitinib (Iressa) is a synthetic anilinoquinazoline capable of inhibiting the epidermal growth factor receptor tyrosine kinase in vitro at nanomolar concentrations. In phase I trials, gefitinib was well tolerated at doses above that required to induce antitumor effects in vitro. Notably, antitumor activity was observed in lung cancer patients. These findings resulted in the initiation of phase II trials employing gefitinib monotherapy in patients with recurrent non-small cell lung cancer (the so-called IDEAL trials). Study participants were randomized to 250 mg or 500 mg of gefitinib per day. Objective response rates between 10 and 20% were achieved with minimal host related toxicity (mainly acne like rash and mild diarrhea). Median survivals ranged between 6 and 8 months. Subsequently, phase III trials (the so-called INTACT trials) combined gefitinib and chemotherapy in chemonaive patients with advanced non-small cell lung cancer. These trials failed to demonstrate a survival advantage with the addition of gefitinib to standard platinum-based chemotherapy regimens. However, overall host related toxicities were not substantially worsened with the addition of gefitinib to chemotherapy. Further studies employing single agent gefitinib as well as regimens employing a different sequencing of chemotherapy and gefitinib are planned in recurrent and previously untreated lung cancer patients.     

Personalized peptide vaccination in patients with refractory non-small cell lung cancer

Since the prognosis of non-small cell lung cancer (NSCLC) remains poor, the development of novel therapeutic approaches, including cancer vaccines, is highly desirable. In the current study, we conducted a phase II study of personalized peptide vaccination (PPV), in which a maximum of 4 peptides were selected based on pre-existing humoral immune responses and administered subcutaneously (weekly for 6 consecutive weeks and bi-weekly thereafter) in refractory NSCLC patients. Forty-one refractory NSCLC patients (4 stage IIIb, 22 stage IV and 15 recurrent), who had failed to respond to chemotherapy and/or targeted therapy (median number of regimens, 3; median duration, 10 months), were enrolled. Median overall survival (OS) was 304 days with a one-year survival rate of 42% in the enrolled patients. The main toxicity of PPV was skin reactions at the injection sites, but no serious adverse events were observed. In order to identify potential biomarkers for predicting OS, pre-vaccination and post-vaccination clinical findings and laboratory data were retrospectively assessed and evaluated by multivariate Cox regression analysis. Among the pre-vaccination factors examined, high C-reactive protein (CRP) level was a significant predictor of unfavorable OS [hazard ratio (HR)=10.115, 95% confidence interval (CI)=2.447-41.806, P=0.001]. Among the post-vaccination factors, high CRP level and low frequency of CD3?CD26? cells were significant predictors of unfavorable OS (HR=23.127, 95% CI=2.919-183.233, P=0.003; HR=0.952, 95% CI=0.917-0.989, P=0.012). Taken together, our results suggest the feasibility of PPV for the treatment of refractory NSCLC. Evaluation of the identified factors before or at an early stage of vaccination could be potentially useful for selecting NSCLC patients who would likely have better prognosis following PPV.     

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.     

Targeting novel and established therapies for non-small cell lung cancer

The prognosis in advanced non-small cell cancer (NSCLC) remains poor despite the introduction of several new cytotoxic drugs in the past decade. New approaches are required, and an improved understanding of lung cancer biology is identifying molecular mechanisms that are potential targets for novel therapies. Antagonists of signalling via the erbB and VEGFR families of transmembrane receptors have promising activity in NSCLC, and survival benefit has already been demonstrated for both erlotinib and bevacizumab. Although some patients enjoy dramatic and sustained responses to some of the new targeted drugs, overall response rates in unselected NSCLC patient groups are modest. This reflects the molecular heterogeneity of the disease; further clinical progress will require improved patient selection for treatment with both novel agents and established chemotherapy drugs. Here, we review recent advances in NSCLC biology likely to provide insight into such selection strategies.     

新型抗血管生成药物在非小细胞肺癌中的作用

肿瘤血管生成(angiogenesis)在非小细胞肺癌(non-small cell lung cancer,NSCLC)的生长、侵袭和转移过程中有重要作用,以血管生成为靶点的治疗成为NSCLC靶向治疗的热点。作用于肿瘤血管生成不同环节的各种药物不断的被发现和合成,近期多项临床试验的结果使研究者对这些药物充满希望,但也发现很多问