Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 4 de 4
Filter
Add more filters










Database
Language
Publication year range
1.
J Immunother ; 33(2): 146-54, 2010.
Article in English | MEDLINE | ID: mdl-20139776

ABSTRACT

The tumor suppressor gene p53 is mutated in more than half of human tumors. One important characteristic of p53 mutants is their accumulation in the nucleus of cancer cells. Thus, reactivation of mutant p53 proteins may trigger massive apoptosis in tumor cells. Pharmacologic methods are currently under development to induce mutant p53 proteins to resume their wild-type function. We have identified a human single-chain Fv fragment, designated as transcriptional transactivation and apoptosis restoring (TAR1), which specifically and with high affinity binds to mutant p53 and restores its wild-type active conformation. Binding of TAR1 to mutant p53 induced transcriptional transactivation of p53 target genes and down-regulation of mutant p53 transcriptional target genes. TAR1 treatment induced apoptosis in a variety of cell lines endogenously expressing p53 carrying different point mutations DNA contact or structural p53 mutants. Moreover, in an animal model of mice carrying human xenografts, TAR1 induced tumor regression with no apparent deleterious side effects. Thus, it may be considered as a potential candidate for anticancer treatment, targeting tumors with mutant p53.


Subject(s)
Mutant Proteins/immunology , Mutant Proteins/metabolism , Neoplasms, Experimental/immunology , Single-Chain Antibodies/administration & dosage , Tumor Suppressor Protein p53/immunology , Animals , Apoptosis/drug effects , Caspase 3/immunology , Caspase 3/metabolism , Caspase 7/immunology , Caspase 7/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Mice , Mice, Nude , Mutant Proteins/genetics , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Protein Binding , Signal Transduction/drug effects , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/isolation & purification , Single-Chain Antibodies/pharmacology , Transcriptional Activation , Tumor Burden/drug effects , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor Assays
2.
Cancer Immunol Immunother ; 56(2): 217-26, 2007 Feb.
Article in English | MEDLINE | ID: mdl-16738849

ABSTRACT

Conventional treatment of recurrent and metastasized prostate cancer (CaP) remains inadequate; this fact mandates development of alternative therapeutic modalities, such as specific active or passive immunotherapy. Previously, we reported the identification of a novel highly immunogenic HLA-A*0201-restricted Prostatic Acid Phosphatase-derived peptide (PAP-3) by a two-step in vivo screening in an HLA-transgenic (HHD) mouse system. In the present study we aimed at elucidating the efficiency of PAP-3-based vaccine upon active antitumor immunization. To this end we established preventive and therapeutic carcinoma models in HHD mice. The 3LL murine Lewis lung carcinoma clone D122 transduced to express HLA-A*0201 and PAP served as a platform for these models. The HLA-A*0201-PAP-3 complex specific recombinant single chain scFV-PAP-3 antibodies were generated and used to confirm an endogenous PAP processing resulting in PAP-3 presentation by HLA-A*0201. PAP-3 based vaccines significantly decreased tumor incidence in a preventive immunization setting. Therapeutic vaccination of HHD mice with PAP-3 led to rejection of early established tumors and to increase of mouse survival. These results strongly support a therapeutic relevance of the identified CTL epitope upon active antitumor immunization. The newly established carcinoma model presented herein might be a useful tool for cancer vaccine design and optimization.


Subject(s)
Cancer Vaccines/immunology , Immunotherapy/methods , Lymphokines/immunology , Prostatic Neoplasms/immunology , Protein Tyrosine Phosphatases/immunology , Sialoglycoproteins/immunology , Acid Phosphatase , Animals , Cancer Vaccines/therapeutic use , Carcinoma, Lewis Lung/immunology , Carcinoma, Lewis Lung/therapy , Epitopes, T-Lymphocyte/immunology , HLA-A Antigens/immunology , HLA-A2 Antigen , Humans , Lymphokines/therapeutic use , Male , Mice , Mice, Transgenic , Microscopy, Confocal , Polymerase Chain Reaction , Protein Tyrosine Phosphatases/metabolism , Protein Tyrosine Phosphatases/therapeutic use , Sialoglycoproteins/therapeutic use
3.
Proteomics ; 5(7): 1806-14, 2005 May.
Article in English | MEDLINE | ID: mdl-15825150

ABSTRACT

Non-DNA microarrays, such as protein, peptide and small molecule microarrays, can potentially revolutionize the high-throughput screening tools currently used in basic and pharmaceutical research. However, fundamental obstacles remain that limit their rapid and widespread implementation as an alternative bioanalytical approach. These include the prerequisite for numerous proteins in active and purified form, ineffectual immobilization strategies and inadequate means for quality control of the considerable numbers of multiple reagents. This study describes a simple yet efficient strategy for the production of non-DNA microarrays, based on the tenacious affinity of a carbohydrate-binding module (CBM) for its three-dimensional substrate, i.e., cellulose. Various microarray formats are described, e.g., conventional and single-chain antibody microarrays and peptide microarrays for serodiagnosis of human immunodeficiency virus patients. CBM-based microarray technology overcomes many of the previous obstacles that have hindered fabrication of non-DNA microarrays and provides a technically simple but effective alternative to conventional microarray technology.


Subject(s)
Carbohydrate Metabolism , Protein Array Analysis , Receptors, Cell Surface/metabolism , AIDS Serodiagnosis/methods , Carbohydrates/chemistry , HIV , Humans , Protein Binding , Protein Structure, Tertiary , Receptors, Cell Surface/chemistry
4.
J Mol Biol ; 335(1): 177-92, 2004 Jan 02.
Article in English | MEDLINE | ID: mdl-14659749

ABSTRACT

We describe a novel approach for high-throughput screening of recombinant antibodies, based on their immobilization on solid cellulose-based supports. We constructed a large human synthetic single-chain Fv antibody library where in vivo formed complementarity determining regions were shuffled combinatorially onto germline-derived human variable-region frameworks. The arraying of library-derived scFvs was facilitated by our unique display/expression system, where scFvs are expressed as fusion proteins with a cellulose-binding domain (CBD). Escherichia coli cells expressing library-derived scFv-CBDs are grown on a porous master filter on top of a second cellulose-based filter that captures the antibodies secreted by the bacteria. The cellulose filter is probed with labeled antigen allowing the identification of specific binders and the recovery of the original bacterial clones from the master filter. These filters may be simultaneously probed with a number of antigens allowing the isolation of a number of binding specificities and the validation of specificity of binders. We screened the library against a number of cancer-related peptides, proteins, and peptide-protein complexes and yielded antibody fragments exhibiting dissociation constants in the low nanomolar range. We expect our new antibody phage library to become a valuable source of antibodies to many different targets, and to play a vital role in facilitating high-throughput target discovery and validation in the area of functional cancer genomics.


Subject(s)
Antibodies/immunology , Complementarity Determining Regions/immunology , Gene Library , Immunoglobulin Fragments/immunology , Immunoglobulin Variable Region/immunology , Antibodies/genetics , Antigens, Neoplasm/immunology , Cellulose/chemistry , Combinatorial Chemistry Techniques , Complementarity Determining Regions/genetics , Drug Evaluation, Preclinical/methods , Escherichia coli/genetics , Humans , Immunoblotting , Immunoglobulin Fragments/genetics , Immunoglobulin Variable Region/genetics , Protein Binding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/immunology
SELECTION OF CITATIONS
SEARCH DETAIL
...