Selection of phage-displayed peptides mimicking an extracellular epitope of human MDR1-P-glycoprotein.

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.

A new immunohistochemical methodology for the specific detection of MDR1-P-glycoprotein in human tissues based on phage-displayed peptides mimicking the MM4.17 epitope.

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.

Isolation of antigenic mimics of MDR1-P-glycoprotein by phage-displayed peptide libraries.

To identify an MC57 epitope which is more efficiently expressed on inactivated forms of P-glycoprotein we utilized peptide libraries displayed on filamentous phage. Using this technology, we selected specific phage clones blocking the binding of the murine monoclonal (MAb) MC57 with live human multi-drug-resistant (MDR) cells, and sequenced and analyzed their DNA. The results we obtained indicate that MAb MC57 epitope could be formed by 2 regions localized on the predicted fourth and sixth extracellular loops of the current 12-transmembrane-domain model predicted for MDR1-P-glycoprotein. Surprisingly, a third region, defined by residues 800-807 of the MDR1-P-glycoprotein sequence and postulated to be intracellular, was also identified as a putative part of the MC57 epitope. This finding adds weight to the interesting hypothesis that a P-glycoprotein structure different from the current model may exist.

Topology of MDR1-P-glycoprotein as indicated by epitope mapping of monoclonal antibodies to human MDR cells.

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.

[Immunological parameters in subjects with essential arterial hypertension. I. Lymphocyte subpopulations]. / Osservazioni su alcuni parametri immunologici in soggetti con ipertensione arteriosa essenziale. I. Sottopopolazioni linfocitarie.

Peripheral venous blood lymphocytes were defined, in 21 subjects with essential hypertension and 20 controls, on the basis of reactivity with monoclonal antibodies OKT3, OKT4, OKT8, OK1Ia. Total lymphocyte count, OKT3+ and OKT4+ cells percentage were similar in normal and hypertensive. OKT8+ cells percentage was significantly lower only in hypertensive stage II and III patients (WHO classification).

P-glycoprotein epitope mapping. II. The murine monoclonal antibody MM6.15 to human multidrug-resistant cells binds with three distinct loops in the MDR1-P-glycoprotein extracellular domain.

A new murine monoclonal antibody (MAb), MM6.15, to human MDR1 P-glycoprotein was found to be reactive in ELISA with synthetic peptides selected from the predicted sequences of the first, fourth and sixth extracellular loop of MDR1-P-glycoprotein. In order to precisely define the MM6.15-binding site, a peptide library of overlapping 5- to 9-mer residues covering the entire sixth extracellular loop of both human and rodent class-1 P-glycoproteins was synthesized on polyethylene pins and tested for MAb binding. The results of this ELISA demonstrated that the MAb MM6.15 reacts only with human synthetic peptides and that the critical component of the MAb recognition is made up of the amino-acid sequence LVAHKL (residues 963-968 of the MDR1-P-glycoprotein) with histidine (H), lysine (K) and possibly leucine (L), key residues of this immunogenic domain.

Antibodies to cardiac Purkinje cells: further characterization in autoimmune diseases and atrioventricular heart block.

We confirmed the occurrence of IgG antibodies reacting with ox cardiac conducting tissue in the serum of some human subjects. These antibodies failed to react with all ox cardiac conducting tissue cells; they reacted only with the cells defined as Purkinje cells. Having checked 352 sera, we found that the prevalence of antibodies to Purkinje cells was 11% in normal subjects (no correlation with sex and age), 14% in systemic lupus erythematosus, 21% in rheumatoid arthritis, 18% in progressive systemic sclerosis, and 23% in Sjögren syndrome. In 50 patients with permanent pacemakers for chronic non-postinfarction atrioventricular (AV) block the prevalence was 30% (P = 0.008). In a selected set of 29 patients with clinically idiopathic AV block located at or below the level of the His bundle the prevalence was 34.5% (P = 0.006). The possible role of anti-Purkinje cell antibodies in autoimmune damage of cardiac conduction tissue is discussed.

Identification of a LFA-1 region involved in the HIV-1-induced syncytia formation through phage-display technology.

We have identified a peptide region on CD18 molecule (the beta subunit of the LFA-1 molecule) involved in syncytia formation of HIV-1-infected lymphocytes. Several phage clones mimicking an epitope of the CD18 cell-surface determinant were isolated from two 9-mer random peptide phage-displayed libraries via their binding to the CD18-specific monoclonal antibody (mAb) MHM23, which in in vitro assay inhibits syncytia formation in HIV-1-infected cells. The peptide sequences displayed on phages that blocked immunolabeling of this mAb on LFA-1-expressing cells were used to identify the epitope recognized by mAb MHM23 by sequence comparison. On the basis of this analysis, two peptides which inhibited syncytia formation in HIV-1-infected cells in vitro were synthesized, thus confirming that they mimic a CD18 domain that is involved in this phenomenon. The results here presented highlight the potential of phage-display technology for the study of biological processes at the basis of virus infection, but also suggest new approaches for the therapy of AIDS.

Epitope mapping of the monoclonal antibody MM12.10 to external MDR1 P-glycoprotein domain by synthetic peptide scanning and phage display technologies.

Epitope mapping of MDR1-P-glycoprotein using specific monoclonal antibodies (mAbs) may help in delineating P-glycoprotein topology and hence in elucidating the relationship between its structural organization and drug-efflux pump function. In this work, by using synthetic peptide scanning and phage display technologies, the binding sites of the mAb MM12.10, a novel antibody to intact human multidrug resistant (MDR) cells, were studied. The results we obtained confirm that two regions localized on the predicted fourth and sixth loops are indeed external and that MDR1 peptides covering the inner domain of the current 12 transmembrane segment (TMs) model of P-glycoprotein could form part of the MM12.10 epitope.