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The survival of pediatric patients with cancer entities including osteosarcoma and Ewing's sarcoma (ES), remains extremely low hence novel treatment approaches are urgently needed. Therefore, based on the concept of targeted therapy, numerous potential targets for the treatment of these cancers have been evaluated pre-clinically or in some cases even clinically during the last decade. In ES the CD99 protein is an attractive target antigen. In this respect, a new entry site for therapeutic intervention may derive from specific human antibodies against CD99. Human scFvC7 was isolated from a semi-synthetic ETH-2 antibody phage library panned on the extracellular portion of recombinant human CD99 protein. The scFvC7 was genetically sequenced, tested for CD99 recognition on an array of recombinant CD99 fragments and measured for binding affinity by ELISA. Finally, it was tested for staining CD99 antigen on a large panel of tumor and normal cells and tissues by cytofluorimetric and immunohistochemical assays. The new antibody scFvC7 recognizes the CD99 extracellular domain included between residues 50 and 74 with a binding affinity of 2.4 x 10(-8) M. In contrast with all other antibodies to CD99 so far isolated, scFvC7 shows a unique specificity in cancer cell recognition: It stained prevalently ES cells while no or weak reactivity was observed on the majority of the other tumor and normal cells and tissues. Thanks to its properties the new anti-CD99 antibody here described represents the first step towards the construction of new selective ES therapeutics.

Background: Severe acute respiratory syndrome (SARS)-CoV is a newly emerging virus that causes SARS with high mortality rate in infected people. Successful control of the global SARS epidemic will require rapid and sensitive diagnostic tests to monitor its spread, as well as, the development of vaccines and new antiviral compounds including neutralizing antibodies that effectively prevent or treat this disease., Methods: The human synthetic single-chain fragment variable (scFv) ETH-2 phage antibody library was used for the isolation of scFvs against the nucleocapsid (N) protein of SARS-CoV using a bio panning-based strategy. The selected scFvs were characterized under genetics-molecular aspects and for SARS-CoV N protein detection in ELISA, western blotting and immunocytochemistry., Results: Human scFv antibodies to N protein of SARS-CoV can be easily isolated by selecting the ETH-2 phage library on immunotubes coated with antigen. These in vitro selected human scFvs specifically recognize in ELISA and western blotting studies distinct epitopes in N protein domains and detect in immunohistochemistry investigations SARS-CoV particles in infected Vero cells., Conclusion: The human scFv antibodies isolated and described in this study represent useful reagents for rapid detection of N SARS-CoV protein and SARS virus particles in infected target cells.

It is not rare that controversial indications about the presence or the expression level of multidrug-resistant (MDR) proteins come out from different laboratories upon examination of identical tumor specimens. Distinct aspects, including the use of weakly discriminating monoclonal antibodies (MAbs) and/or unsuitable techniques and procedures, contribute in generating differences in the MDR phenotype evaluation of cancer cells. In this regard we describe here an innovative immunohistochemical approach for the determination of P-glycoprotein expression in cells and tissues. The method is based on the ability of phage-displayed peptides to mimic antibody epitopes. For this purpose we utilized the phage clone #55, which was affinity-purified from a phage-displayed random-peptide library using the MAb MM4.17 (specific for MDR1-P-glycoprotein) as previously described. This clone has been chosen since it clearly and undoubtedly reacts with its cognate MAb, as was determined by ELISA and dot blot tests. Inhibition of the MAb MM4.17 binding to MDR1-P-glycoprotein-expressing cells could be performed by adding a calibrated concentration of phage clone #55 particles, which mimic MDR1-P-glycoprotein antigen. This methodology can eliminate misleading interpretations concerning the presence and expression level of MDR1-P-glycoprotein and might well contribute in routine clinical determinations of MDR in tumor specimens, thus contributing to our understanding of the basis of the mechanisms of tumor cell resistance to drugs.

The MDR1-P-glycoprotein binding sites of three different murine monoclonal antibodies (MM4.17, MM6.15 and MC57), directed towards living, intact human multidrug-resistant cells were investigated in order to study P-glycoprotein topology. By using synthetic peptide scanning, we demonstrated that well-defined regions localized on the predicted first, fourth and sixth extracellular loops are external. On the basis of the structure of MM6.15 epitope, which is distributed on the above three different extracellular loops (and thus is discontinuous), P-glycoprotein molecules result to be differently organized in the lipid bilayer. Moreover, the outcome of the MC57 and MM4.17 epitopes localization experiments, obtained through the use of phage-displayed peptide libraries, represent an additional challenge to the classical 12-transmembrane domain model of P-glycoprotein, since they agree with the novel topography of the molecule (10-transmembrane domain), which was recently proposed on the basis of biochemical and expression studies.

To study the structural conformation of the MM4.17 monoclonal antibody (mAb) epitope, twenty-six mAb MM4.17-specific phage clones were affinity-isolated and their inserts characterized for amino acid composition and homology with MDR1 gene product (MDR1-P-glycoprotein). The resulting sequence alignment shows that a unique consensus sequence, which corresponds to the previously mapped TRIDDPET linear peptide identified through synthetic peptide scanning, could not be identified. However, similarities between the inserts of positive phage clones and P-glycoprotein primary structure, consisting in two or three amino acid-long sequences, were observed. An analysis of the over-represented amino acid residues in the inserts of positive clones, and their comparison with the sequence of the antigen was also performed. The two different procedures led to the identification of four regions in which these similarities are clustered, indicating that four different antigen regions, one of which includes the TRIDDPET linear amino acid sequence, might participate in forming the structure of monoclonal antibody MM4.17 epitope.

Three monoclonal antibodies (mAb), MRK16, MM4.17 and MC57, directed against distinct epitopes on the external domain of human P-glycoprotein (Pgp), were used to follow its expression on multidrug resistant (MDR)-cells. The linear MM4.17 epitope and conformational MRK16 epitope showed a 4-fold higher expression at 37 degrees C than at 4 degrees C, while the detection of the conformational MC57 epitope did not change. Inhibition of Pgp function, by a short pretreatment of the MDR-cells with resistance-modulating agents (RMA), such as SDZ PSC 833 and SDZ 280-446, could not be related to depletion of Pgp from the cell surface, since their expression of the MM4.17 and MRK16 epitopes was found unchanged. However, a substantially higher expression of MC57 epitopes was found on RMA-treated cells than on untreated ones. Since this effect correlated to the strength of different RMA in reversing the MDR phenotype, MC57 epitopes might be more efficiently expressed on inactivate(d) forms of the Pgp molecules, suggesting that RMA might inhibit Pgp function by disturbing the conformation of individual Pgp molecules, their topographical distribution or polymerization status in the membrane.

A new murine monoclonal antibody (MAb), MM4.17, to human multi-drug-resistant (MDR) cells was found to be reactive in an ELISA with a synthetic 16-amino acid peptide selected from the fourth loop of the P-glycoprotein extracellular domain. Immunohistochemistry indicated that this MAb reacted in human tissues in the same pattern as that previously found with other human-specific MAbs to P-glycoprotein. For a precise definition of the MM4.17 epitope, a peptide library consisting of overlapping 4- to 10-mer residues covering the entire P-glycoprotein-fragment was synthesized on polyethylene pins and tested for MAb binding. The results of this ELISA demonstrated that the MM4.17 epitope is constituted by the continuous-linear TRIDDPET amino-acid sequence (residues 750-757 of the human MDRI-P-glycoprotein). The MAb MM4.17 recognizes only the human MDRI-P-glycoprotein isoform, and excess TRIDDPET peptide blocks the binding of the MAb to MDR variants of CEM cells. These results demonstrate that the amino-acid sequence TRIDDPET from the human MDRI gene represents the first continuous-linear epitope identified in the P-glycoprotein extracellular domain.

We describe a murine IgG1 monoclonal antibody (MAb56), specific for a cell-surface protein structure (MC56 determinant) expressed by the human CEM cell line. A large band of approximately 90 kDa was identified as the main specific component of the MC56 determinant. Such a 90-kDa protein is significantly associated with the drug-sensitive phenotype, its expression being progressively reduced quantitatively in multi-drug-resistant (MDR) variants of CEM cells, according to the extent of drug resistance. In addition, the MC56 determinant is expressed de novo in drug-sensitive revertant cell lines derived from MDR cells and unreactive with the MAb56. The MAb56 shows a high affinity towards the immunizing drug-sensitive CEM cell line (Ka = 1.86 x 10(9) L/mole) while not binding to MDR cell variants. The expression of the MC56 molecule on a variety of human cells and tissues makes such a cellular determinant a candidate as a marker for studying the MDR phenomenon both in vivo and in vitro.

The characterization of epitope specificity of a panel of 15 monoclonal antibodies against human chorionic somatomammotropin (hCS), previously prepared in our laboratory, allowed us to distinguish 4 antigenic determinants on the hCS molecule. Experiments were carried out with competition and sandwich assays. The results allowed us to divide our MAbs into 4 groups, recognizing 4 different epitopes, and to select 4 MAbs each distinguishing a different epitope in order to pursue further studies on the antigenic structure of hCS.