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F Hudecz

Publications and source records attributed to F Hudecz.

At least 55 records · Page 3Linked to original sources
PubMed1995

Alteration of immunogenicity and antibody recognition of B-cell epitopes by synthetic branched chain polypeptide carriers with poly[L-lysine] backbone.

In order to elucidate structural and biological properties required for an optimal immunological carrier function and to provide a rational basis for its selection, two new groups of synthetic branched polypeptides with a general formula poly[Lys-(X(i)-DL-Ala(m))][XAK] or poly[Lys-(DL-Ala(m)-X(i))][AXK], where m approximately 3 and i < 1 were introduced by our laboratory. Here we review our recent results on the application of these polypeptides as biodegradable carriers for constructing synthetic immunogens/antigens with a well-known phenyl oxazolone hapten, peptide epitopes of epithelial mucin [MUCI] or herpes simplex virus [HSV 1] glycoprotein D. Observations collected during the last five years with the conjugates presented serve to illustrate the usefulness of branched polypeptides as carriers for the rational design of synthetic immunogens for the development of vaccines or clinically relevant immunodiagnostics. Furthermore, this polypeptide model system enables the analysis and potentially reliable interpretation of the correlation between chemical structure and immunogenic/antigenic features.

Amino Acid Sequence↗
PubMed1994

Characterisation of a recombinant Fv fragment of anti-MUC1 antibody HMFG1.

A recombinant Fv (variable fragment) has been produced for the murine monoclonal antibody HMFG1. This antibody was raised against human milk fat globules and reacts with an epitope (PDTR) in the protein core of MUC1 mucins, which are up-regulated in human breast and other carcinomas. Binding specificity of the Fv fragment has been demonstrated through immunoaffinity purification, and by radioimmunoassay. The affinity constants for this Fv fragment and for the proteolytically produced Fab (antigen binding fragment) of the related humanised antibody HuHMFG1 were determined by monitoring the fluorescence quenching of the antibody fragments whilst adding aliquots of MUC1 related antigenic peptides KAPDTRPAPG and VTSAPDTRPAPG. Using these techniques it has been demonstrated that the products of these different methods of antibody fragmentation are comparable, and suitable for solution structure analysis using nuclear magnetic resonance (NMR) spectroscopy.

Animals↗
PubMed1994

Chromatographic characterization of HSV-1 gD 268-284 and IL-6 179-185 synthetic oligopeptides by reversed-phase high-performance liquid chromatography, automated Edman degradation and mass spectrometric analysis.

Two groups of synthetic oligopeptides (namino acid = 7 and 17) were prepared by solid-phase peptide synthesis using the Boc-polystyrene strategy. After deprotection, cleavage and gel permeation, the crude products were analysed by conventional RP-HPLC methods. Separation and isolation of major components were performed on a semi-preparative RP-HPLC column. In order to clarify the primary structure of these products, amino acid analysis, Edman degradation sequence determination and analytical RP-HPLC characterization were applied. The isolated fractions were further assessed by direct molar mass investigation utilizing the fast atom bombardment and 252Cf plasma desorption mass spectrometry. The results with an interleukin-6 oligopeptide corresponding to the 179LRALRQM185 sequence indicate that the single peak product obtained by RP-HPLC separation contains only one component, as verified by amino acid analysis and mass spectrometry. In contrast, the analysis of 268LAPEDPEDSALLEDPVG284-NH2 from HSV-1 gD protein suggests that this large peptide amide showing a single peak after repeated purification by RP-HPLC contains microheterogeneities as revealed by mass spectrometry and sequencing, but not by amino acid analysis.

Amino Acid Sequence↗
PubMed1994

Antitumour effect of a gonadotropin-releasing-hormone antagonist (MI-1544) and its conjugate on human breast cancer cells and their xenografts.

Our gonadotropin-releasing hormone (GnRH) antagonist analogue MI-1544 ([Ac-D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10]GnRH) was developed as a potential contraceptive material, because it decreased the luteinizing hormone level without unfavourable side-effects. The antagonist was covalently bound to poly[Lys-(Ac-Glu0.96-DL-Ala3.1)] (AcEAK)-a branched polypeptide having a polylysine backbone--resulting in a MI-1544-AcEAK conjugate. According to our in vitro experiments the MI-1544 induced a 33%-35% decrease in cell numbers of MCF-7 and MDA-MB-231 human breast cancer cell lines at a dose of 30 microM. The biodegradable polymeric carrier, AcEAK, alone inhibited cell proliferation by only 13%-15%, while the MI-1544-AcEAK conjugate, applied at the same dose, was capable of producing 45%-50% inhibition of cell proliferation. Our in vivo experiments using immunosuppressed mice showed that MI-1544, applied twice daily s.c., inhibited the growth of oestrogensensitive and -insensitive xenografts by 65% and 30% respectively. This effect was potentiated (70%) in both types of xenografts by the presence of the polymeric carrier in the conjugate; however, the carrier by itself did not cause tumour growth inhibition. The polymeric polypeptide carrier is supposed to increase the stability of the GnRH antagonist and to prevent the rapid excretion of the covalently bound peptide molecule. The antagonist and its conjugate may have various direct and indirect effects on breast cancer cells and, as a consequence, the new GnRH antagonist conjugates are suitable for treating an extended range of breast cancers.

Amino Acid Sequence↗
PubMed1994

The influence of branched polypeptide carriers on the immunogenicity of predicted epitopes of HSV-1 glycoprotein D.

To investigate the role of synthetic polypeptide carriers in inducing an epitope-specific immune response relevant for vaccine design, peptides comprising two distinct regions of herpes simplex virus type I glycoprotein D (1-23 and 273-284) have been conjugated to the branched polypeptides with polylysine backbone, poly[L-Lys-(DL-Alam)] (AK), or poly[L-Lys-(Leui-DL-Alam)] (LAK) and to keyhole limpet haemocyanin (KLH). The magnitude, fine specificity and isotype distribution of the conjugate-, peptide-and carrier-specific antibody responses were characterized in immunized BALB/c and CBA mice. Conjugates containing the polypeptide carrier AK were the most effective in inducing HSV gD-peptide-specific antibody responses while KLH peptide conjugates resulted in conjugate-specific antibody responses without measurable peptide specificity. The efficacy of AK-peptide conjugates was verified by the dominant appearance of peptide-specific antibodies belonging to functionally efficient IgG isotopes, accompanied by low levels of carrier specific antibody responses. Preimmunization of BALB/or CBA mice with AK conjugates comprising the 1-23 or 276-284 HSV peptides resulted in prolonged survival of animals infected with a lethal dose of infectious HSV-1. The potency of these conjugates in eliciting a protective immune response shows a close correlation with the relative levels of conjugate-induced virus-specific antibodies and the neutralizing activity of sera as measured in preimmunized survivors.

Amino Acid Sequence↗
PubMed1994

Scintigraphic determination of the biodistribution of an 111In-labelled poly(L-lysine) backbone branched polypeptide drug carrier in tumour-bearing mice.

A branched polypeptide drug carrier, with a poly(L-lysine) backbone, has been labelled with 111In and its biodistribution imaged in mice with transplanted B16 melanoma. Levels of tracer in tumours were not great enough for tumours to be discerned on gamma-camera images, and this was confirmed by subsequent dissection analysis. Tumour levels of 111In from labelled polymer were about 3% of the dose g-1 two days after injection. Similar levels of tracer were found in tumour tissue of mice injected with mouse immunoglobulin or serum albumin labelled with 111In by DTPA chelation, or injected with free 111In-chloride to label serum transferrin. There was rapid excretion of a sub-component of the 111In-labelled polymer visible in the images. Gel permeation chromatography suggested that the polymer was heterogeneous, some components having Stoke's radii below that allowing renal clearance. Gel permeation chromatography was used to separate labelled polymer into fractions having high, intermediate and low renal clearance. The low-excretion fraction showed a two-fold increase in tumour levels, compared with native polymer, although as this fraction showed greater survival in the blood and body as a whole, discrimination between tumour and normal tissue was not increased.

Animals↗
PubMed1993

Measurement of antibody binding to antigenic peptides conjugated in situ to albumin-coated microtitre plates.

Monoclonal antibodies have been prepared against a synthetic peptide with a sequence corresponding to a repeated hydrophilic region of the protein core of the human MUC-2 gastrointestinal mucin. Peptide conjugates, prepared by glutaraldehyde cross-linking with keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA), were employed as the immunogen and target antigen (for screening by ELISA), respectively. However, for the measurement of antibody binding to peptide by an ELISA procedure, an alternative strategy was developed and is described in this report: peptides were conjugated directly to BSA immobilized by physical adsorption to the surface of microtitre plate wells. This procedure permits peptides to be tested as target antigens by ELISA without prior preparation of peptide-carrier conjugates.

Amino Acid Sequence↗
PubMed1993

Carrier design: conformational studies of amino acid (X) and oligopeptide (X-DL-Alam) substituted poly (L-lysine).

The present study was undertaken to examine the influence of the reversal of the side-chain sequential order on the conformation of branched polypeptides. At the same time, the influence of the optically active amino acid joined directly to the poly (L-Lys) backbone and the DL-Ala oligomer grafted as chain-terminating fragment were separately analyzed. Therefore two sets of polypeptides were synthesized corresponding to the general formula poly[Lys-(Xi)] (XK) and poly[Lys-(DL-Alam-Xi)] (AXK) when X = Ala, D-Ala, Leu, D-Leu, Phe, D-Phe, Ile, Pro, Glu, D-Glu, or His. For coupling amino acid X to polylysine, three types of active ester methods were compared: the use of pentafluorophenyl or pentachlorophenyl ester, and the effect of the addition of an equimolar amount of 1-hydroxy-benzotriazole. After cleavage of protecting groups, AXK polypeptides were synthesized by grafting short oligo(DL-Ala) chains to XK by using N-carboxy-DL-Ala anhydride. The CD measurements performed in water solutions of various pH values and ionic strengths were used for classification of the polypeptide conformations as either ordered (helical) or unordered. Different from what was observed with the unsubstituted poly (L-Lys), poly[Lys-(Xi)] type polypeptides can adopt ordered structure even under nearly physiological conditions (pH 7.3, 0.2M NaCl). These data suggest that the introduction of amino acid residue with either (ar) alkyl side chain (Ala, Leu, Phe) or negatively charged side chain (Glu) promotes markedly the formation of ordered structure. Comparison of chiroptical properties of poly[Lys-(DL-Alam-Xi)] and of poly[Lys-(Xi)] reveals that side-chain interactions play an important role in the stabilization of ordered solution conformation of AXK type branched polypeptides. The results give rather conclusive evidence that not only hydrophobic interactions, but also ionic attraction, can be involved in the formation and stabilization of helical conformation of branched polypeptides.

Alanine↗
PubMed1993

Suppression of antibody responses to ricin A chain (RTA) by monoclonal anti-RTA antibodies.

Balb/c mice treated with an immunotoxin constructed by conjugation of murine monoclonal antibody 791T/36 via a disulfide linker to ricin A chain generate a pronounced antibody response to peptide epitopes on ricin A chain. Monoclonal anti-RTA antibodies which recognize peptide epitopes have been developed and these have been used to down-regulate anti-RTA antibody responses in 791T/36-RTA immunotoxin-treated Balb/c mice. Of the five MAB tests, two (608/7 and 596/134) proved most effective, inhibiting anti-RTA antibody formation by up to 73%. MAB treatment was effective when initiated up to 3 days after immunotoxin treatment. Pharmacokinetic studies with 791T/36-RTA have shown that the immunotoxin is rapidly eliminated from the circulation, with no more than 4% remaining in blood after 24 hr. It is proposed that the down-regulation of anti-RTA antibodies is effected by MAB interfering with antigen processing.

Animals↗
PubMed1992

Monoclonal antibody binding to peptide epitopes conjugated to synthetic branched chain polypeptide carriers. Influence of the carrier upon antibody recognition.

The peptide C A P D T R P A P G has been linked covalently to defined branched polypeptides with a polylysine backbone and side chains of DL-alanine or D-leucine-DL-alanine oligopeptides. The peptide was coupled via its N terminal cysteine to the side chains of the macromolecular carrier to ensure uniform orientation. The compounds were subjected to compositional analyses to characterise the degrees of substitution and secondary structural studies were performed using circular dichroism spectroscopy. The peptide selected for investigation contains the immunodominant sequence P D T R P A P which is expressed in the protein core of epithelial mucins. It is to this region that many anti-mucin monoclonal antibodies bind (Burchell et al., 1989; Price et al., 1990a,b). With these characterised constructs, it has been possible to evaluate the influence of secondary structure upon the binding of monoclonal antibodies which recognise short linear sequences in the synthetic antigenic peptide. The findings are relevant to the design and construction of synthetic immunogens and vaccines as well as to the production of synthetic analogues of clinically relevant antigens (in this case, epithelial mucins associated with breast and ovarian carcinomas).

Amino Acid Sequence↗
PubMed1992

Scintigraphic evaluation of the pharmacokinetics of a soluble polymeric drug carrier.

There is a growing interest in the use of macro-molecular carriers for therapeutic agents. If these carriers can be labelled with an appropriate gamma-emitter, their biodistribution could be followed by scintigraphy. We have imaged the biodistribution of a synthetic branched polypeptide, based on a poly-L-lysine backbone (average molecular mass 45 kDa). The polymer was conjugated to diethylene triamine penta-acetic acid and labelled by chelation with indium-111. Mice were injected i.v. with labelled material and imaged with a gamma-camera with a pin-hole collimator. Images showed the majority of tracer remaining in the blood pool, but about 35% appeared in the urinary bladder within 1.5 h. When the 111In-polymer was fractionated by gel filtration chromatography on S-300, the imaging showed that the early eluting material was retained, the intermediate showed some renal clearance, and the late was rapidly excreted. These findings show the value of gamma-scintigraphy for biodistribution studies with such polymeric drug carriers and its potential for clinical pharmacokinetic studies.

Animals↗
PubMed1992

Side chain distribution and enantiomer composition of biodegradable branched polypeptides with polylysine backbone.

A detailed investigation is reported about the primary structure of biodegradable branched polypeptides suitable for carrier function. The side chain distribution of poly[Lys(DL-Alam)], m approximately 3.5, the common inside area of a branched polypeptide model system, was determined by automated Edman degradation. These data indicate that 65% of the branches consist of 2-4 Ala residues and 25% of the side chains are longer than 4 amino-acid residues. The enantiomer composition of poly[Lys(Xi-DL-Alam)], (X = Glu, D-Glu, Leu, D-Leu) and of poly[Lys(DL-Ala2.9-Leu0.79)] polypeptides was investigated by HPLC analysis of their hydrolysates following derivatization with N-(5-fluoro-2,4-dinitrophenyl)-L-alanine amide (Marfey's reagent). The results proved that the synthesis methods applied for the preparation of branched polypeptides produce compounds whose enantiomer compositions are in good agreement with calculated expectations. These findings could be useful for the reliable interpretation of various biological phenomena observed in connection with the presence of L- or D-amino-acid residues in the side chains.

Amino Acid Sequence↗
PubMed1990

Immunological and structural features of the protein core of human polymorphic epithelial mucin.

The protein core of high mol. wt polymorphic epithelial mucin (PEM--approximately 400 kDa glycoprotein) which is associated with breast carcinomas, consists of a repeating 20 amino acid peptide motif [Gendler et al. (1988) J. biol. Chem. 263, 12,820-12,823]. Monoclonal antibodies C595 (anti-urinary mucin) and NCRC-11 (anti-breast carcinoma cells), and other antibodies against human milk fat globule membranes, were found to recognize determinants present within this 20 amino acid peptide. A model of the peptide was developed based on hydropathicity and structure prediction calculations and these indicated that the repeated structure is dominated by a hydrophilic domain of seven amino acids, extending into two flanking beta turns. NMR analysis of the 20 amino acid peptide was undertaken to probe the secondary structure. Epitope mapping experiments involving solid phase synthesis of overlapping heptapeptides in the repeat unit identified the minimum structures for antibody binding as Arg-Pro-Ala-Pro and Arg-Pro-Ala for the C595 and NCRC-11 antibodies, respectively. These determinants were found within the predicted hydrophilic turn region domain of the peptide. The epitopes for six other PEM-reactive monoclonal antibodies were also determined to reside within the predicted hydrophilic turn domain. This evidence is in accord with the disposition of this region of the PEM peptide core being at the exterior of the glycoprotein where it would be accessible to antibody recognition and binding events.

Amino Acid Sequence↗
PubMed1990

C595--a monoclonal antibody against the protein core of human urinary epithelial mucin commonly expressed in breast carcinomas.

Urinary mucins which express determinants for the anti-breast carcinoma monoclonal antibody, NCRC-11 (IgM), closely resemble the mammary mucins found in milk fat globules and carcinomas. An IgG3 monoclonal antibody, C595, was prepared against urinary mucins isolated on a NCRC-11 antibody affinity column, and this 'second generation' antibody was shown to have a very similar pattern of reactivity to the original NCRC-11 antibody. By immunohistology, the profile of reactivity of both antibodies with tumour and normal tissue specimens was virtually identical. Both antibodies reacted with epithelial mucins isolated from breast tumours or normal urine using an NCRC-11 antibody affinity column, although the antibodies were unreactive with other antigen preparations. Heterologous immunoradiometric assays ('sandwich' tests) confirmed that NCRC-11 and C595 epitopes were co-expressed on the same molecule. C595 antibodies inhibited the binding of radiolabelled NCRC-11 antibodies to antigen, suggesting that the two epitopes were in close topographical proximity. The protein core of the mammary mucins has recently been shown to consist predominantly of a repeated 20 amino acid sequence (Gendler et al., 1988). Peptides with this complete sequence and small fragments were synthesised, and the C595 antibody was found to recognise an epitope within this repeat. The ability to identify and synthesise monoclonal antibody-defined determinants, as well as those in the adjacent or overlapping sequences within the protein core of epithelial mucins, is viewed as a strategy for facilitating the production of antibodies of new and novel specificity to complement the panels of existing anti-breast cancer reagents.

Antibodies, Monoclonal↗
PubMed1989

The influence of the side-chain sequence on the structure-activity correlations of immunomodulatory branched polypeptides. Synthesis and conformational analysis of new model polypeptides.

New branched polypeptides were synthesized for a detailed study of the influence of the side-chain structure on the conformation and biological properties. The first subset of polypeptides were prepared by coupling of tetrapeptides to poly[L-Lys]. These polymers contain either DL-Ala3-X [poly[Lys-(X-DL-Ala3)n]] or X-DL-Ala3 [poly[Lys-(DL-Ala3-X)n] (n less than or equal to 1)] tetrapeptide side chains. Another group of branched polymers comprise a mixture of DL-Alam and of DL-Alam-X oligomeric branches in a random distribution [poly[Lys-(DL-Alam-Xi)] (i less than 1, m approximately 3)]. In each subset the X = Leu or Phe derivatives were prepared. The N-protected tetrapeptides were synthesized by conventional liquid phase methods and were coupled as active esters. The degree of racemization was found relatively high both for active esters and coupled derivatives, when optically active amino acids were in the C-terminal position of the tetrapeptides. In the case of the poly[Lys-(Leu-DL-Ala3)n] derivative, comparative experiments were carried out using various methodical alterations. The highest stereochemical homogeniety could be achieved when the tetrapeptide active ester was synthesized by the "backing off" method. CD spectra of poly[Lys-(Xi-DL-Alam)] (i less than 1, m approximately 3) and of poly[Lys-(X-DL-Ala3)n] were analyzed and compared to those of poly[Lys-(DL-Alam-Xi)] and of poly[Lys-(DL-Ala3-X)n]. All measurements were performed in water solutions of varying pH values and ionic strengths. The data obtained suggest that branched polypeptides containing a mixture of two different types of oligomeric side chains (DL-Alam and DL-Alam-Xi or Xi-DL-Alam) distributed randomly adopt an almost identical conformation to those that comprise only the respective tetrapeptide (DL-Ala3-X or X-DL-Ala3) branches. The results also indicate that the tendency to form an ordered structure is determined by the identity and the position of the chiral amino acid X (Phe or Leu) in the side chain.

Adjuvants, Immunologic↗
PubMed1989

Structural characteristics influencing the carrier function of synthetic branched polypeptides based on poly[Lys-(DL-Ala)3)]backbone.

Effective carrier function of selected representatives of new branched polypeptides covalently coupled with the synthetic monovalent hapten, oxazolone was studied. The effectiveness of oxazolone-synthetic polypeptide conjugates in inducing oxazolone-as well as carrier-specific antibody responses in inbred mice was compared to that of bovine serum albumin (BSA)- and KLH-oxazolone conjugates. The synthetic polypeptides, poly[Lys-(D-Leui-DL-Alam)] (D-LAK), LAK and FAK, as well as the common poly[Lys-(DL-Alam)](AK) core covalently coupled to oxazolone (Ox) induced a T cell-dependent antibody response when repeatedly administered with or without Freund's adjuvant in mice. This was evidenced by: the increasing titer of oxazolone-specific IgG during the course of the memory response; the appearance of all IgG subclasses; the effective oxazolone-specific priming by the conjugates; and the induction of an intense oxazolone- and carrier-specific DTH reaction. Although the oxazolone-specific antibody response was 10-100 times lower than that induced by KLH- or BSA-oxazolone conjugates, it was accompanied by a lower level or no detectable carrier-specific antibody response despite an effective carrier-specific T cell-mediated response. Significant differences were observed between the effectiveness of synthetic polypeptides used as carrier: highest oxazolone-specific antibody titers were observed using the AK, LAK and FAK conjugates. The intensity and specificity of the DTH reaction and antibody response induced by the carrier-oxazolone conjugates suggested that the distinct effectiveness of L- and D-amino acid-containing conjugates (LAK vs D-LAK and FAK vs D-FAK) was dependent on altered B cell recognition of the haptenic group. Circular dichroism (CD) spectra indicating different local orientation of oxazolone, when coupled to L or D side chain-terminating amino acids, support this suggestion.

Amino Acids↗
PubMed1989

Biodegradability of synthetic branched polypeptide with poly(L-lysine) backbone.

A detailed investigation is reported about the biodegradation of poly[Lys(DL-Alam)], m approximately 3, (AK) the common inside area of a branched polypeptide model system developed by our group over the last decade. Enzymatic hydrolysis was carried out by the exopeptidase aminopeptidase M, or the endopeptidase trypsin, or their mixture. Ion-exchange column chromatography, paper electrophoresis and thin-layer chromatography were utilised to achieve separation of metabolites. Breakdown products were identified by the aid of synthetic oligopeptides representing the potential fragments (DL-Ala2, DL-Ala3, Lys(DL-Alam), m = 1-3). The kinetics and the degree of enzymatic degradation were determined. The ratio of peptide/amino acid amounts in the hydrolysate was found to be 1.07 after 24 h treatment with aminopeptidase M, 3.0 with trypsin and 1.3 with aminopeptidase - trypsin mixture. The overall results indicated that the proteolysis of AK by an aminopeptidase M and trypsin mixture proceeds stepwise at multiple sites on the polypeptide chain. The degradation is significantly retarded as compared to that of alpha- or epsilon-polylysine. A mechanism of degradation is suggested based on the experimental results.

Alanine↗