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Wadih Arap

Publications and source records attributed to Wadih Arap.

At least 37 records · Page 2Linked to original sources
PubMed2004

Cell surface-associated Tat modulates HIV-1 infection and spreading through a specific interaction with gp120 viral envelope protein.

Human immunodeficiency virus-1 (HIV-1) Tat, a nuclear transactivator of viral gene expression, has the unusual property of being released by infected cells. Recent studies suggest that extracellular Tat is partially sequestered by heparan sulfate proteoglycans. As a consequence, Tat is concentrated on the cell surface and protected from proteolytic degradation, thus remaining in a biologically active form. We show that Tat binds the surfaces of both HIV-1-infected and surrounding uninfected cells. We provide evidence for a specific interaction between Tat and the HIV-1 glycoprotein 120 (gp120) envelope protein, which enhances virus attachment and entry into cells. We map the interacting sites of both Tat and gp120 and show that synthetic peptides mimicking the gp120 site inhibit HIV-1 infection. Our data demonstrate that membrane-associated Tat is a novel modulator of virus entry and suggest that the Tat-gp120 interaction represents a critical step in HIV-1 spreading during the course of infection.

Gene Products, tat↗
PubMed2004

An HSP90-mimic peptide revealed by fingerprinting the pool of antibodies from ovarian cancer patients.

To gain insight into the mechanisms of molecular recognition and humoral immune response in ovarian cancer, we used fingerprinting, a phage display-based combinatorial selection to isolate peptide ligands to tumor-related antibodies present in ascites from patients with advanced disease. First, we have isolated a consensus motif (sequence CVPELGHEC) in 86% of the peptides screened; this enriched motif was selected from a total of 10(8)-10(9) unique random sequences present in the library. Next, we identified the heat-shock protein 90 kDa (HSP90) as the native antigen mimicked by the motif. Finally, we evaluated the expression of HSP90 and the presence of antibodies against the HSP90-mimic peptide in a large panel of ovarian cancer patients and controls. In tissue microarrays, we show that the expression of HSP90 is ubiquitous. However, the corresponding humoral immune response against HSP90 is restricted to a subset of patients with stage IV disease. Together, these results show that screening humoral response can identify tumor antigens that may serve as molecular targets in ovarian cancer. Recognition of such relevant proteins in the immunobiology of malignant tumors may lead to the development of therapies

Amino Acid Motifs↗
PubMed2004

Reversal of obesity by targeted ablation of adipose tissue.

Obesity is an increasingly prevalent human condition in developed societies. Despite major progress in the understanding of the molecular mechanisms leading to obesity, no safe and effective treatment has yet been found. Here, we report an antiobesity therapy based on targeted induction of apoptosis in the vasculature of adipose tissue. We used in vivo phage display to isolate a peptide motif (sequence CKGGRAKDC) that homes to white fat vasculature. We show that the CKGGRAKDC peptide associates with prohibitin, a multifunctional membrane protein, and establish prohibitin as a vascular marker of adipose tissue. Targeting a proapoptotic peptide to prohibitin in the adipose vasculature caused ablation of white fat. Resorption of established white adipose tissue and normalization of metabolism resulted in rapid obesity reversal without detectable adverse effects. Because prohibitin is also expressed in blood vessels of human white fat, this work may lead to the development of targeted drugs for treatment of obese patients.

Adipose Tissue↗
PubMed2004

Combinatorial screenings in patients: the interleukin-11 receptor alpha as a candidate target in the progression of human prostate cancer.

Direct screening of combinatorial peptide libraries in patients may allow the identification of ligands that target biochemical differences in the endothelium of blood vessels. In a screening performed in a patient, we selected and isolated a mimic motif of interleukin 11 (IL-11) from prostate biopsies after an i.v. administration of a phage display peptide library. We also demonstrated that the IL-11 phage mimic (displaying the cyclic nonapeptide CGRRAGGSC) bound specifically to a corresponding IL-11 receptor (IL-11Ralpha). Here we show that IL-11Ralpha is a potential target for intervention in human prostate cancer through morphological and functional analyses. First, a comprehensive serial immunohistochemical analysis of primary and metastatic prostate cancer samples showed increased stage-specific expression of IL-11Ralpha during disease progression. Second, a proapoptotic peptide was specifically targeted and internalized through this functional IL-11Ralpha-based ligand-receptor pair: treatment of prostate cancer cells in vitro with a proapoptotic peptide guided by the CGRRAGGSC peptide to the IL-11Ralpha resulted in dose-dependent apoptosis. Together, these data indicate that the IL-11Ralpha is a candidate target for translational clinical trials against advanced and metastatic prostate cancer. Moreover, our results illustrate the ability of direct combinatorial screening systems in cancer patients for identification of relevant targets in the context of human disease.

Apoptosis↗
PubMed2004

Cell surface expression of the stress response chaperone GRP78 enables tumor targeting by circulating ligands.

We have recently identified glucose-regulated protein-78 (GRP78) as a relevant molecular target expressed in metastatic tumors by fingerprinting the circulating repertoire of antibodies from cancer patients. Here we design and evaluate a ligand-receptor system based on the tumor cell membrane expression of GRP78. We show that GRP78 binding peptide motifs target tumor cells specifically in vivo and in human cancer specimens ex vivo. Moreover, synthetic chimeric peptides composed of GRP78 binding motifs fused to a programmed cell death-inducing sequence can suppress tumor growth in xenograft and isogenic mouse models of prostate and breast cancer. Together, these preclinical data validate GRP78 on the tumor cell surface as a functional molecular target that may prove useful for translation into clinical applications.

Animals↗
PubMed2004

Design and validation of a bifunctional ligand display system for receptor targeting.

Here we developed a bacteriophage display particle designed to serve as a bifunctional entity that can target tumors while delivering an agent. We engineered a chimera phage vector containing a pIII-displayed alphav integrins-targeting moiety and a pVIII-displayed streptavidin binding adaptor moiety. By using the chimeric phage particle, targeting of alphav integrins on cells in culture and tumor-related blood vessels was shown through different applications, including luminescent quantum dots localization, surface plasmon resonance-based binding detection, and an in vivo tumor model. The strategy validated here will accelerate the discovery and characterization of receptor-ligand binding events in high throughput, and cell-specific delivery of diagnostics or therapeutics to organs of choice without the need for chemical conjugation.

Animals↗
PubMed2004

Model of unidirectional transluminal gene transfer.

Gene transfer assays in vitro are poor indicators of transduction efficacy observed in vivo. We designed and optimized an intermediate model for assessing and quantifying unidirectional transduction ex vivo. The model enables simultaneous transmucosal evaluation of up to 96 different variables under the same tissue conditions. We show that the model is versatile and suitable for use with different vectors (adenovirus and AAV), different reporter genes (beta-galactosidase and green fluorescent protein), and viscera with various tissue features such as peritoneum and urothelium. Ex vivo transduction assays may correlate better with in vivo gene transfer results. Because the experimental model described here can be performed in small samples, it may enable translational applications in tissues of human origin.

Animals↗
PubMed2004

Aminopeptidase A is a functional target in angiogenic blood vessels.

We show that a membrane-associated protease, aminopeptidase A (APA), is upregulated and enzymatically active in blood vessels of human tumors. To gain mechanistic insight, we evaluated angiogenesis in APA null mice. We found that, although these mice develop normally, they fail to mount the expected angiogenic response to hypoxia or growth factors. We then isolated peptide inhibitors of APA from a peptide library and show that they specifically bind to and inhibit APA, suppress migration and proliferation of endothelial cells, inhibit angiogenesis, and home to tumor blood vessels. Finally, we successfully treated tumor-bearing mice with APA binding peptides or anti-APA blocking monoclonal antibodies. These data show that APA is a regulator of blood vessel formation, and can serve as a functional vascular target.

Amino Acid Motifs↗
PubMed2004

Engineered embryonic endothelial progenitor cells as therapeutic Trojan horses.

While the hematogenic contribution of circulating endothelial cells to tumor angiogenesis is not entirely understood, one can exploit this phenomenon as a therapeutic strategy. In this issue of Cancer Cell, show that murine embryonic endothelial progenitor cells preferentially home to sites of lung metastases, evade immunological rejection, and can exert a bystander antitumor effect when modified to contain a suicide gene construct that activates a prodrug. Treatment with the prodrug led to improved survival in syngeneic and nonsyngeneic tumor-bearing mouse models. The conceptual advance put forward by this study might result in translational applications.

Animals↗
PubMed2003

Hybridoma-free generation of monoclonal antibodies.

Production of monoclonal antibodies requires immortalization of splenocytes by somatic fusion to a myeloma cell line partner (hybridomas). Although hybridomas can be immortal, they may depend on a feeder cell layer and may be genetically unstable. Since the inception of hybridoma technology, efforts to improve efficiency and stability of monoclonal antibody-producing cell lines have not brought about substantial progress. Moreover, suitable human multiple myeloma-derived cell lines for the production of human antibodies have been very difficult to develop. Here we report a strategy that greatly simplifies the generation of antibodies and eliminates the need for hybridomas. We show that splenocytes derived from transgenic mice harboring a mutant temperature-sensitive simian virus 40 large tumor antigen under the control of a mouse major histocompatibility promoter are conditionally immortal at permissive temperatures and produce monoclonal antibodies. This simple approach may become a method of choice for generation and production of both polyclonal and monoclonal antibodies with advantages in high-throughput discovery and antibody-based immunotherapy.

Animals↗
PubMed2003

Mapping tumor vascular diversity by screening phage display libraries.

The structural and molecular diversity of tumor-associated vasculature provides a basis for the development of targeted diagnostics and therapeutics. Novel organ and disease-specific proteins expressed on tumor vasculature can be identified by in vivo phage display technology. Strategies for targeting tumor vessels should provide the advantages of selectivity of action plus improved accessibility and efficacy. The development of such targeting agents relies on the identification of specific ligand-receptor pairs and on information as to their cellular distribution and location. To date, the in vivo phage display screening system has allowed the selection of a variety of peptides targeting normal tissues, cancer and other angiogenesis-related diseases. Many receptors for these peptides have also been identified. In addition, complementary imaging studies are helping in the functional characterization of abnormalities occurring in blood vessels during tumor progression. These efforts will lead to the delineation of a ligand-receptor-based map of the microvasculature in human cancer. Several clinical applications may follow.

Angiogenesis Inhibitors↗
PubMed2003

Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors.

Characterizing the molecular diversity of the cell surface is critical for targeting gene therapy. Cell type-specific binding ligands can be used to target gene therapy vectors. However, targeting systems in which optimum eukaryotic vectors can be selected on the cells of interest are not available. Here, we introduce and validate a random adeno-associated virus (AAV) peptide library in which each virus particle displays a random peptide at the capsid surface. This library was generated in a three-step system that ensures encoding of displayed peptides by the packaged DNA. As proof-of-concept, we screened AAV-libraries on human coronary artery endothelial cells. We observed selection of particular peptide motifs. The selected peptides enhanced transduction in coronary endothelial cells but not in control nonendothelial cells. This vector targeting strategy has advantages over other combinatorial approaches such as phage display because selection occurs within the context of the capsid and may have a broad range of applications in biotechnology and medicine.

Amino Acid Sequence↗
PubMed2003

An artificially designed pore-forming protein with anti-tumor effects.

Protein engineering is an emerging area that has expanded our understanding of protein folding and laid the groundwork for the creation of unprecedented structures with unique functions. We previously designed the first native-like pore-forming protein, small globular protein (SGP). We show here that this artificially engineered protein has membrane-disrupting properties and anti-tumor activity in several cancer animal models. We propose and validate a mechanism for the selectivity of SGP toward cell membranes in tumors. SGP is the prototype for a new class of artificial proteins designed for therapeutic applications.

Amino Acid Sequence↗
PubMed2003

Peptides targeting caspase inhibitors.

Here we report on the identification of peptides targeting the X-inhibitor of apoptosis protein (XIAP). XIAP functions as a caspase inhibitor and is a member of the inhibitors of apoptosis (IAP) family of proteins. IAPs are often overexpressed in cancers and leukemias and are associated with an unfavorable clinical prognosis. We have selected peptides from a phage library by using recombinant full-length human XIAP or a fragment containing only the baculovirus IAP repeat 2 (BIR2) domain. A consensus motif, C(D/E/P)(W/F/Y)-acid/basic-XC, was recovered from two independent screenings by using different libraries. Phage-displaying variations of the consensus sequence bound specifically to the BIR2 domain of XIAP but not to other IAPs. The interaction was specific as it could be blocked by the cognate synthetic peptides in a dose-dependent manner. Phage displaying the XIAP-binding motif CEFESC bound to the BIR2 domain of XIAP with an estimated dissociation constant of 1.8 nm as determined by surface plasmon resonance. Protein-protein interaction assays revealed that caspase-3 and caspase-7 (but not caspase-8) blocked the binding of the CEFESC phage to XIAP, indicating that this peptide targets a domain within XIAP that is related to the caspase-binding site. In fact, the sequence EFES is homologous to a loop unique to the executioner caspase-3 and caspase-7 that are targeted by XIAP. Finally, we demonstrated that an internalizing version of the XIAP-binding peptide identified in our screenings (PFKQ) can induce programmed cell death in leukemia cells. Peptides interacting with XIAP could serve as prototypes for the design of low molecular weight modulators of apoptosis.

Amino Acid Motifs↗
PubMed2003

Alpha v beta 5 integrin-dependent programmed cell death triggered by a peptide mimic of annexin V.

The diverse cytoplasmic domain sequences within the various integrin subunits are critical for integrin-mediated signaling into the cell (outside-in signaling) and for activation of ligand binding affinity (inside-out signaling). Here we introduce an approach based on phage display technology to identify molecules that specifically interact with the cytoplasmic domain of the beta 5 integrin subunit. We show that a peptide selected for binding specifically to the beta 5 cytoplasmic domain (VVISYSMPD) induces apoptosis upon internalization. The cell death process induced by VVISYSMPD is sensitive to modulation by growth factors and by protein kinase C (PKC), and it cannot be triggered in beta 5 null cells. Finally, we show that the VVISYSMPD peptide is a mimic of annexin V. Our results suggest a functional link between the alpha v beta 5 integrin, annexin V, and programmed cell death. We propose the term "endothanatos" to designate this phenomenon.

Amino Acid Sequence↗
PubMed2003

Targeting urothelium: ex vivo assay standardization and selection of internalizing ligands.

PURPOSE: With the goal of targeting the human bladder using phage display technology we designed and tested a tissue binding assay on intact urothelium ex vivo. This approach may form the molecular basis for clinical development of peptide or peptidomimetic guided intravesical compounds. MATERIALS AND METHODS: We screened 2 phage display random peptide libraries on human urothelium. Select peptides were tested for their binding ability to human urothelium, 2 human transitional cell carcinoma cell lines and a nontransitional cell carcinoma cell line. Next we standardized an ex vivo binding assay, validated binding of selected phage to whole urothelium, and evaluated whether receptor mediated internalization into urothelium derived cells occurred. Finally we tested if the presence of the glycosaminoglycan layer had any effect on the binding of the urothelium targeted phage. RESULTS: Phage selected and recovered in the screening were isolated and sequenced. Displayed peptide sequences were searched against online protein databases. Five classes of peptide motifs were characterized based on their ability to bind to normal urothelium but not to control cell lines. Remarkable consistency and reproducibility were observed in the ex vivo binding assays. Two classes of peptide motifs sharing the sequence Ile/Leu-Ser-Gly-Leu bound to normal urothelium and to 2 transitional cell carcinoma cells but not to nontransitional cell carcinoma cells in a glycosaminoglycan independent manner and mediated internalization into cells of urothelial origin. CONCLUSIONS: We introduce a strategy for screening combinatorial peptide libraries on bladder mucosa, a standard model for ex vivo intact urothelium binding assays and a panel of urothelium binding peptides that may be suitable for translation into targeted intravesical therapy applications.

Carcinoma, Transitional Cell↗
PubMed2002

Fingerprinting the circulating repertoire of antibodies from cancer patients.

Recognition of molecular diversity in disease is required for the development of targeted therapies. We have developed a screening method based on phage display to select peptides recognized by the repertoire of circulating tumor-associated antibodies. Here we isolated peptides recognized by antibodies purified from the serum of prostate cancer patients. We identified a consensus motif, NX(S/T)DK(S/T), that bound selectively to circulating antibodies from cancer patients over control antibodies from blood donors. We validated this motif by showing that positive serum reactivity to the peptide was specifically linked to disease progression and to shorter survival in a large patient population. Moreover, we identified the corresponding protein eliciting the immune response. Finally, we showed a strong and specific positive correlation between serum reactivity to the tumor antigen, development of metastatic androgen-independent disease, and shorter overall survival. Exploiting the differential humoral response to cancer through such an approach may identify molecular markers and targets for diagnostic and therapeutic intervention.

Antibodies, Neoplasm↗