A competitive immunoassay to detect a hapten using an enzyme-labelled peptide mimotope as tracer.

Mimotope peptides-peptides which mimic the binding of a hapten to its corresponding monoclonal antibody-were conjugated to peroxidase and used in competitive immunoassay. The established immunoassay was used to quantitatively determine the concentration of hapten. As model system in all the experiments described here, we used the binding of the monoclonal antibody B13-DE1 to fluorescein and the corresponding peptide mimotope.

Identification of peptide mimotopes for the fluorescein hapten binding of monoclonal antibody B13-DE1.

Using 6mer and 12mer phage peptide libraries three unique phage clones were identified which specifically bind to a monoclonal anti-FITC antibody, B13-DE1. The two 6mer and one 12mer peptide insert sequences are clearly related to each other and contain a high proportion of hydrophobic amino acids. The peptides are bound by the antibody combining site of B13-DE1 probably in a similar manner to FITC and represent therefore true peptidic mimics of the fluorescein hapten. No reactivity of the peptides could be demonstrated with another monoclonal anti-fluorescein antibody or with polyclonal anti-fluorescein antibodies. Immunization of mice with the peptides resulted in the production of antibodies cross-reacting with all peptides but not with fluorescein. The results show that phage peptide libraries can be used to isolate mimotope peptides which can mimic low molecular weight structures seen by a specific antibody and probably other recognition molecules.

Comparative study of the p53-mdm2 and p53-MDMX interfaces.

Mdm2 and MDMX are two structurally related p53-binding proteins which show the highest level of sequence similarity in the N-terminal p53-binding domains. Apart from its ability to inhibit p53 mediated transcription, a feature it shares with mdm2, very little is known about the physiological functions of MDMX. It is clearly distinct from mdm2 since its expression appears not to be regulated by p53 and it cannot compensate for lack of mdm2 in early development. We present data on the structural similarity between the p53 binding pockets of mdm2 and MDMX using p53- and phage-selected peptides. From the results we conclude that our recently devised innovative approach to reverse the mdm2-mediated inhibition of p53's transactivation function in vivo would probably target MDMX as well. Strategies for selectively targeting mdm2 and MDMX are suggested and a possible mechanism for regulating the p53-mdm2/MDMX interactions by protein phosphorylation is discussed.

Molecular characterization of the hdm2-p53 interaction.

A number of viral oncogenes target the tumour suppressor protein p53 and inactivate its function. This is an important step in tumourogenesis. The cellular oncogene hdm2 acts through a similar mechanism. It binds the N terminus of p53, thereby interfering with the ability of p53 transcriptionally to activate genes responsible for growth arrest or apoptosis after genotoxic insults. The disruption of the interaction of the two proteins therefore comprises a promising therapeutic target for treatment of the subset of human cancers in which this pathway is active. In this paper we attempt to characterize the p53-hdm2 interaction biochemically. We analyse the potential of a series of peptide inhibitors, derived from previously described mdm2 binding peptide display phage, to disrupt this interaction in ELISA assays. We conclude that F19, W23 and L26 of p53 are critical contact points for p53 binding to hdm2. Furthermore, we show the potential of the monoclonal antibody 3G5 to interfere with binding of p53 to hdm2 in ELISA assays. Consequently, we define the binding site of 3G5 on hdm2 using overlapping peptides derived from the N terminus of hdm2 and phage display libraries. The result indicates L66, Y67 and E69 on hdm2 as critical binding points for 3G5. In electrophoretic mobility shift assay we demonstrate the formation of hdm2-p53 complexes that can be disrupted in the presence of 3G5 or inhibitory peptides. Finally, we describe the effects of NEM and DTT on the interaction between the two molecules in ELISA assays. All our results are discussed in the light of the recently published crystal structure of the mdm2-p53 complex. A striking correspondence between our findings and the crystal structure is revealed.

Design of a synthetic Mdm2-binding mini protein that activates the p53 response in vivo.

BACKGROUND: The transcriptional activation function of the p53 tumour suppressor protein is induced by DNA damage and results in growth arrest and/or apoptotic responses. A key component of this response is the dramatic rise in p53 protein concentration resulting from an increase in the protein's stability. Very recently, it has been suggested that interaction with the Mdm2 protein may target p53 for rapid degradation. We have designed a gene encoding a small protein that binds tightly to the p53-binding pocket on the Mdm2 protein. We have constructed the gene by cloning a phage display optimised Mdm2-binding peptide into the active-site loop of thioredoxin. RESULTS: When introduced into cells containing low levels of wild-type p53, this protein causes a striking accumulation of the endogenous p53 protein, activation of a p53-responsive reporter gene, and cell cycle arrest mimicking the effects seen in these cells after exposure to UV or ionising radiation. Microinjection of a monoclonal antibody to the p53-binding site on Mdm2 achieves a similar effect, establishing its specificity. CONCLUSIONS: These results demonstrate that the p53 response is constitutively regulated in normal cells by Mdm2 and that disruption of the interaction alone is sufficient to stabilise the p53 protein and activate the p53 response. Our mini protein approach provides a powerful new method to activate p53 without causing DNA damage. More broadly, it establishes a powerful general method for determining the biological consequences of the specific disruption of protein-protein interactions in cells.

Identification of novel mdm2 binding peptides by phage display.

The oncogene mdm2 and its human homologue hdm2 bind to the tumour suppressor protein p53 and inactivate its function as a transcription factor. This has been implied as a possible mechanism for cancer development in several tumours including human sarcomas. The mdm2-p53 interaction is therefore a much persued target for the development of anti-cancer drugs. In order to find novel high affinity ligands for hdm2 which would interfere with its binding to p53 we screened phage display peptide libraries for mdm2 binding phage. We found a series of 12 and 15mer peptides which interact strongly with hdm2. The peptide sequences show striking homology with the previously established mdm2 binding site on p53, confirming that the peptide defined 18TFSDLW23 region is crucial for the interaction but that contact between the two molecules extends to position L26 on p53. Free synthetic peptides derived from the phage selected sequences proved to be up to 100 times stronger inhibitors of the p53-mdm2 interaction than the p53 derived wt-peptide in several ELISA-assays. This illustrates the potency of phage display libraries in the search for new peptide based lead structures designed to mimic or inhibit therapeutically important protein-protein interactions.

Epitope mapping of monoclonal antibodies to keratin 19 using keratin fragments, synthetic peptides and phage peptide libraries.

To generate tools for monitoring processing and folding in keratin intermediate filaments, a group of monoclonal antibodies reacting with the intermediate filament protein keratin 19 were studied using different approaches to define the structure and localization of their epitopes. The binding pattern to bacterially expressed human keratin 19 fragments allowed the definition of minimal amino acid sequences required for antibody binding. The screening of overlapping 15-residue peptides confirmed and further specified the epitope locations for a subset of the tested antibodies. In addition, the epitope of an antibody with apparent species-restricted specificity (LE64) was revealed by isolating and characterizing a full-length keratin 19 clone from a PtK2 cDNA library. Taken together with species cross-reactivity of individual antibodies and sequence information obtained by probing a phage display library, specific amino acid residues could be highlighted as likely to be involved in the antibody binding.

Comprehensive epitope analysis of monoclonal anti-proenkephalin antibodies using phage display libraries and synthetic peptides: revelation of antibody fine specificities caused by somatic mutations in the variable region genes.

Filamentous phage libraries, displaying 6, 12 or 20 amino acid residue peptides at the N terminus of coat protein pIII were used to define and localize the epitopes of 15 monoclonal antibodies raised against human proenkephalin, a neuropeptide precursor. Eight monoclonal antibodies (PE14 to PE19, PE23 and PE25), which inhibit each other's binding to proenkephalin, recognized phage clones selected by PE14, PE15, PE19, PE23 and PE25. With the peptide sequences DLL(X)(X)LL (12mer library) and DLL(X)(X)L (6mer library) shared by most of the phage clones it was possible to define the putative antibody epitope 155DLLKELL161 on human proenkephalin. For five antibodies (PE13, PE20 to PE22 and PE24) belonging to another inhibition group, a common consensus motif G(X)D(X)E(X)(X)V(X)(X)R could be defined with help of a 20mer library. The corresponding minimum epitope sequence has been found to be 175GSDNEEEVSKR185. Antibody PE1, raised in a separate fusion, was able to select phage clones from a 12mer and 20mer library, revealing that the sequence 187GGFMRG192 is probably the antibody epitope. The assumed localization of the epitopes was confirmed by screening a set of overlapping synthetic peptides, covering the region of human proenkephalin thought to contain all antibody binding sites. It was found that antibodies, although recognizing the same epitope, gave different binding patterns with the selected phage clones. By analysing the VH chain sequences of these antibodies it could be shown that a varying number of somatic mutations is likely to be the reason for the observed differences in antibody fine specificity.

Production of monoclonal antibodies against epitopes of the main coat protein of filamentous fd phages.

Three monoclonal antibodies (MAbs) were produced which react with epitopes of the main structural coat protein (pVIII) of filamentous fd phages as demonstrated by solid-phase fluorometric enzyme immunoassays and by immunoelectron microscopy. The antibodies are of the IgG1, IgG2a and IgG2b immunoglobulin subclasses. Since they also react with recombinant phages expressing antigen fragments in their pIII region they may be suitable reagents for the demonstration and isolation of filamentous phages used in recombinant protein technology.

Lung cancer-associated keratin 19 fragments: development and biochemical characterisation of the new serum assay Enzymun-Test CYFRA 21-1.

From a panel of 4 murine monoclonal antibodies directed against keratin 19 various antibody combinations were evaluated in solid-phase enzyme-linked sandwich immunoassays for detection of soluble keratin 19 fragments in patient sera. One of these antibody combinations, comprised of the monoclonal antibodies Ks 19.1 and BM 19.21, was selected for further development to a routine test (Enzymun-Test CYFRA 21-1) because of its high diagnostic sensitivity and specificity for non-small cell lung carcinoma (NSCLC). Both antibodies are specific for keratin 19, no reactivity could be observed with cytokeratin 8 or 18. The epitopes of the two antibodies were determined to be within helix 2B of the rod romain. The epitope sequences lie within the sequence 311-335 for the catcher antibody Ks 19.1 and 346-367 for the detector antibody BM 19.21. These sequences are unique, as could be confirmed from sequence databases. The standard material for the assay was prepared from a cytoskeleton fraction of cultivated MCF-7 cells. Subsequent digestion of this fraction with chymotrypsin yielded a soluble and stable standard material. Both the standard material and the serum analyte appeared as oligomers when analysed on gel chromatography: the serum analyte appeared exclusively at a M(r) of 100 +/- 10 kD, whereas the standard material eluted in fractions corresponding to 100 +/- 10 kD and 450 kD. Due to the precise definition of the antigen and the localisation of the antibody binding sequences, Enzymun-Test CYFRA 21-1 is one of the best characterised tumor markers so far.

A monoclonal antibody epitope on keratin 8 identified using a phage peptide library.

A bacteriophage random hexapeptide library was used to define the epitope of a monoclonal anti-keratin antibody. Phage selected by the keratin 8-specific antibody LE41 displayed highly related sequences on their pIII coat protein. The consensus sequence S(X)LNP allowed the precise localization of an LE41 epitope (SLLSP) within the head domain (H1 subdomain) of human keratin 8, known to be important for correct filament polymerisation. By sequencing the immunizing antigen, keratin 8 from Potorous tridactylis, it was shown that the natural epitope of LE41 is the pentapeptide SLLNP, which confirmed predictions from the phage library results. An SLL(X)P motif is found in the H1 region of all type II keratins (keratins 1 to 8) in different species, but mutational analysis revealed that LE41 can only bind to keratin 8 when Asn (N) or Ser (S) is found in the (X) position. Thus the monoclonal antibody LE41 retains its specificity for keratin 8, dependent on a single amino acid residue, even though it recognizes an epitope within the highly conserved H1 subdomain of the head region. Six other monoclonal antibodies tested on the phage library failed to select motifs.

Hybrid hybridomas producing bispecific antibodies to CEA and peroxidase isolated by a combination of HAT medium selection and fluorescence activated cell sorting.

A combination of fluorescence-activated cell sorting and HAT medium selection has been used to establish bispecific antibody (biAbs)-producing hybrid hybridomas. For this purpose hypoxanthine-guanine phosphoribosyl transferase (HGPRT)-deficient mutants were isolated from a hybridoma line (D11-DG2) producing anti-CEA antibodies by 8-azaguanine treatment. The resulting HAT-sensitive hybrid cells were stained with the fluorescence marker tetramethyl rhodamine isothiocyanate (TRITC) and fused by polyethylene glycol (PEG) with HAT-non-sensitive unstained hybrid cells producing antibodies to horseradish peroxidase (POD). Fluorescent fused hybrid hybridomas as well as non-fused stained anti-CEA cells were separated from the unstained anti-POD cells using a fluorescent activated cell sorter (FACS). Finally, non-fused enzyme-deficient anti-CEA cells were eliminated by cultivation in HAT selection medium which permits only an outgrowth of HAT-resistant hybrid hybridoma cells containing the genes for producing both antibodies.

Monoclonal antibodies to human chorionic gonadotropin (HCG) and their use in two-site binding enzyme immunoassays.

A panel of mouse monoclonal antibodies (MABs) was produced against human chorionic gonadotropin (HCG) and its isolated beta-subunit (beta-HCG). According to their binding specificities the antibodies could be divided into HCG-specific and cross-reactive MABs. The HCG-specific antibodies reacted with antigenic sites on holo-HCG or holo-HCG and beta-HCG, or exclusively with the non-associated beta-HCG chain. The cross-reactive antibodies reacted with either HCG and luteinizing hormone (LH) or with HCG, LH, follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH). According to the binding specificities of the MABs and their reciprocal inhibition detected in two-site binding enzyme immunoassays (EIA), altogether 13 epitopes (including the 3 hidden epitopes detectable only on free non-associated beta-HCG) were distinguished by the antibodies described here. Antibody combinations resulting in most effective and specific HCG- or beta-HCG-determination were used as clinical assays and proved their reliability and correctness for monitoring patients with HCG- and/or beta-HCG-producing tumors before and after therapy.

Production and characterization of monoclonal antibodies against carcinoembryonic antigen (CEA).

In the experiments presented here 22 monoclonal antibodies (MoAbs) were produced which reacted with the tumor marker carcinoembryonic antigen (CEA). Eleven of the MoAbs reacted neither with peripheral blood granulocytes nor with purified spleen NCA-60 kDa and were therefore regarded as "CEA-specific". Only three antibodies of this group reacted exclusively with CEA-180 kDa. Eight MoAbs reacted with CEA-180 kDa and with CEA-like substances of lower molecular mass (of 160 kDa and/or 120 kDa) present in colon carcinoma cells as determined by immunoblotting. These molecules seem to be different from the classical non-specific cross-reacting antigens (NCAs) present in peripheral blood granulocytes. In contrast to that, the other 11 anti-CEA MoAbs recognized in addition to CEA-180 kDa also NCAs on granulocytes. Six of them were reactive with a purified spleen NCA-60 kDa preparation. These MoAbs bound also to reduced and alkylated CEA-180 kDa (CEA r/a), i.e. they recognize sequential epitopes. All 22 MoAbs reacted with CEA expressed in different human tumor cell lines as determined by immunocytological analysis. But six of them did not bind to the surface of these cells when tested in a radioimmuno-binding assay. It was concluded that the epitope(s) recognized by these antibodies are involved in cell membrane anchoring of the CEA-molecules.

The production and radioimmunoassay application of monoclonal antibodies to fluorescein isothiocyanate (FITC).

Monoclonal antibodies (MoAbs) were produced against the fluorescence marker fluorescein isothiocyanate (FITC). FITC was used as a hapten to label different proteins and the anti-FITC MoAbs were used to identify these labelled proteins in a solid-phase radioimmunoassay and in cellular radioimmuno-binding assays for the demonstration of antigens and antibodies.

Differences of K562 leukemia cell clones in the pattern of monoclonal antibody binding and NK cell susceptibility.

Fifty-three K562-clones were tested against three monoclonal anti-K562-antibodies (MoAbs). The individual NK-cell sensitivity of thirty-one K562-clones was evaluated simultaneously. It was found that considerable differences exist in the antibody binding between single clones and the parental cell line indicating antigenic heterogeneity in the K562 line. Some K562-clones showed increased NK-cell sensitivity in comparison with the uncloned cell line. However, no correlation was observed between both investigated parameters.

Monoclonal antibodies against the human leukemia cell line K 562.

Three monoclonal antibodies raised against K 562, a cell line originally established from a patient with chronic myeloid leukemia (CML) in terminal blast crisis, were selected according to their distinct reaction pattern. Whereas two antibodies (ZIK-C1-A/C5 and ZIK-C1-A/H5 also designated C and H) recognized antigens, present on K 562 cells and other immature and mature hematopoietic cells (cell lines and normal blood and bone marrow cells), antibody ZIK-C1-A/D9 also designated Y showed an exclusive binding to K 562 cells. The results obtained (here and in the following paper) indicate, that antibody ZIK-C1-A/D9 defines an early differentiation antigen of hematopoiesis or a leukemia-associated antigen.

Reactivity of monoclonal anti-K 562 antibodies with cells of leukemia- and lymphoma-patients.

For further characterization, monoclonal anti-K 562 antibodies (1) were tested against blood or bone marrow cell samples of patients with various leukemias and lymphomas. One antibody, ZIK-C1-A/D9 (also designated Y) reactive in previous tests exclusively with K 562 cells, but not with normal blood cells, exhibited a selective binding to cells of most AML-patients and CML-patients in myeloid blast crisis. Cells of patients with other hematopoietic malignancies were negative, except three single cases (one lymphosarcoma, one AUL and one hairy cell leukemia). Antibody ZIK-C1-B/H5 (short name H) detected an antigenic determinant, preferentially expressed on cells of AML and CML patients, but also on normal granulocytes and some mononuclear cells. Two additional monoclonal anti-K 562 antibodies, ZIK-C1-A/F5 (short name C) and 2B7, yielded specificities shared by a variety of normal and malignant hematopoietic cells.