©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cross-reactivity of Antibodies to Retro-Inverso Peptidomimetics with the Parent Protein Histone H3 and Chromatin Core Particle
SPECIFICITY AND KINETIC RATE-CONSTANT MEASUREMENTS (*)

Nadia Benkirane , Gilles Guichard , Marc H. V. Van Regenmortel , Jean-Paul Briand , Sylviane Muller (§)

From the (1) Institut de Biologie Moléculaire et Cellulaire, UPR 9021 CNRS, 15 rue Descartes, 67084 Strasbourg Cedex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A series of monoclonal antibodies has been generated against an hexapeptide of sequence IRGERA corresponding to the C-terminal residues 130-135 of histone H3 and three analogues of this model peptide. The analogues correspond to the D-enantiomer, containing only D-residues, and two retro-peptides containing NH-CO bonds instead of natural amide peptide bonds. The chirality of each residue was maintained in the retro-peptide and inverted in the retro-inverso-peptide. Monoclonal antibodies were generated from mice immunized with the analogues coupled to neutral small unilamellar liposomes containing monophosphoryl lipid A as adjuvant. The reactivity of antibodies with the four analogues and with the parent protein H3 was studied in enzyme-linked immunosorbent assay and in a biosensor system. The equilibrium affinity constant (K) toward the retro-inverso-peptide of two out of three antibodies of IgG1 isotype induced against the L-hexapeptide was 7-75-fold higher than toward the homologous L-peptide. The range of Kvalues of four antibodies of IgG1 and IgG2a isotypes generated against the retro-inverso-peptide was 0.6-1.9 10M for both the retro-inverso- and L-peptides. Furthermore, antibodies to the L- and retro-inverso-peptides cross-reacted strongly (in some cases better than with the homologous peptide) with the parent histone H3 and with chromatin subunits containing H3. The results are thus promising in respect to the potential use of retro-inverso-analogues, which are particularly stable, in the design of much more potent synthetic vaccines or to generate antibody probes.


INTRODUCTION

Studies of the relationship between peptide conformation and activity have shown that the introduction of backbone modifications into biologically active peptides affects potency, enzymatic stability, and conformation properties, which, in some cases, leads to the production of analogues with advantageous properties (1) . For instance, pseudopeptides containing reduced peptide bonds -(CH-NH) or retro-inverso modifications -(NH-CO) have been particularly investigated both in biological and structural studies (2, 3, 4, 5, 6, 7) .

In view of recent progress toward the preparation of pseudopeptides and peptide mimetics for obtaining useful drugs structurally related to their parent peptides, the design of such structures to replace natural peptides in immunology may be promising. However, in contrast with intense research developed in the domain of peptide drug design, very few studies have been performed with peptidomimetics in immunology. Potential applications of pseudopeptides and peptidomimetics cover many aspects of basic immunology including synthetic vaccines, immunodiagnostics, and the development of new generations of immunomodulators. Recently, we have analyzed the antigenic and immunogenic properties of an all D-, a retro- and a retro-inverso-analogue of the model hexapeptide of sequence IRGERA corresponding to the COOH-terminal residues 130-135 of histone H3 (8, 9) . Both retroanalogues contained NH-CO bonds instead of the CO-NH peptide bonds; the chirality of each residue was maintained in the retro-peptide, and inverted in the retro-inverso-peptide. It was found that the three analogues induced in mice IgG antibodies of various subclasses. While IgG3 antibodies reacted similarly with the four analogues (L-, retro-inverso-, D-, and retro-peptides), antibodies of the IgG1, IgG2a, and IgG2b isotypes showed a strong conformational preference for certain analogues. Thus, regarding IgG1, IgG2a, and IgG2b antibodies, the retro-inverso-peptide was found to mimic the antigenic activity of the natural L-peptide but not of the all D- and retro-peptides. Conversely, the retro-peptide mimicked the D-peptide but not the L- and retro-inverso-peptides. The finding that a retro-inverso-peptide can mimic and thus replace a natural L-peptide was confirmed by the reactivity of a particular monoclonal antibody (mAb)() induced against the L-peptide (mAb 4x11), which recognized the retro-inverso-peptide (equilibrium affinity constant K, 2 10M) much better than the parent L-peptide (K, 3 10M). Furthermore, we have also shown that the retro-inverso-peptide was much more resistant to proteolysis than the L-peptide. Its half-life in the presence of trypsin was at least 7 times longer.

In order to assess the potential use of retro-inverso-peptides as immunogens useful for vaccination or for eliciting antibody probes for immunotherapy, it was necessary to analyze in more detail the capacity of antibodies generated against these peptide analogues to react with the parent protein and particularly with assembled complex structures. The model peptide studied in the present investigation is particularly interesting to study cross-reaction of anti-peptide antibodies since the region 130-135 of H3 is accessible at the surface of chromatin core particles, constituted by two copies of each core histone H2A, H2B, H3, and H4 and 145 base pairs of DNA. In the present study, we describe the properties of a set of murine mAbs against the L- and the all D-IRGERA peptides and, for the first time, against two types of analogue, namely the retro-inverso- and retro-peptides. These antibodies were generated from mice immunized with the analogues coupled to neutral small unilamellar vesicles containing a nontoxic derivative of lipid A, monophophoryl lipid A (MPLA), as adjuvant. We have measured the equilibrium affinity constants of these antibodies in the BIAcore toward the four analogues, the parent protein H3, and core particles. The results present the first unequivocal example for the potential application of retro-inverso peptidomimetics in the generation of strongly cross-reactive antibodies.


MATERIALS AND METHODS

Histone H3, Chromatin Core Particles, and Peptides

Core particles and histone H3 were isolated from chicken erythrocytes and purified as described previously (10) . The four peptide analogues were synthesized by the solid phase methodology on a multichannel peptide synthesizer (11) . For retro-inverso- and retro-peptides, the end group-modified retro- and retro-inverso-isomers were assembled in t-butyloxycarbonyl chemistry on a p-methyl benzhydrylamine resin (Applied Biosystem, Roissy, France). Protected amino acids were from Neosystem (Strasbourg, France). Assembly of the protected peptide chain was carried out on a 200-µmol scale using the in situ neutralization protocol described previously (11) . The (R,S)-2-methyl malonic acid monobenzyl ester obtained by alcoholysis of 2,2,5,-trimetyl-1,3-dioxane-4-6-dione (12) was incorporated into the peptide chain as a racemate. The coupling was monitored with the ninhidrin test. After this last coupling, the peptide resin was washed twice with ether and dried under vacuum in a dessicator. The peptides were cleaved from the resin by treatment with anhydrous HF containing 10% (v/v) anisol and 1% (v/v) 1,2-ethanedithiol. After removal of HF in vacuo, the peptides were extracted from the resin and lyophilized. The crude peptides were then purified on a C18 column using a middle pressure apparatus (Kronwald Separation Technology, Sinsheim, Germany) by elution with a linear gradient of 5-50% (v/v) acetonitrile in aqueous 0.06% trifluoroacetic acid. The purity of each fraction was assessed by analytical runs on a Novapack C18 column 5 µm (3.9 150 mm) using a linear gradient of 7-32% (v/v) acetonitrile in aqueous 0.1 M triethylammonium phosphate buffer. Runs were performed with a Waters apparatus (Waters Corp., Milford, MA). The fractions containing the pure diastereomeric mixture were pooled and lyophilized. Mass spectra were obtained on a VG analytical ZAB-2SE double focusing instrument and recorded on a VG 11-250 data system (VG Analytical, Manchester, United Kingdom) as described previously (13) .

Peptide-Carrier Conjugation

To allow the coating of peptides in a direct solid phase ELISA test, IRGERA analogues were conjugated to bovine serum albumin using N-succinimidyl 3-[2-pyridyldithio]propionate (SPDP) as described previously (14) . For immunization of mice, peptides were covalently coupled to preformed small unilamellar vesicles containing MPLA (8, 15) .

Generation of mAbs

For mAb production, BALB/c mice were injected intraperitoneally with the four small unilamellar vesicles preparations containing 1 µmol of lipid, 2 µg of MPLA, and 100 µg of peptide/injection/animal. Mice received three injections at intervals of 3 weeks. Booster injections were given at days 105, 106, 107, and 108, i.e. -4, -3, -2, and -1 before fusion. mAbs were prepared by standard fusion protocols (16) . Spleen lymphocytes were mixed at a 1:1 ratio with nonsecreting PAI myeloma cells (17) . The cells were distributed in 96-well plates at a concentration of 2 10 cells/ml. Starting 7 days after the fusion, culture supernatants were tested by ELISA for the presence of specific antibodies reacting with peptide analogues and H3. Positive cultures were recloned twice in agar in the presence of 10 macrophages/ml as feeder cells. Positive clones were amplified by in vitro culture in Dulbecco's modified Eagle's medium containing 5% fetal calf serum and glutamax (Life Technologies, Inc.). Antibodies (in 370-1000 ml of culture medium) were subsequently precipitated with ammonium sulfate at 50% saturation. Some of them were further purified by protein A-Sepharose chromatography. Their purity was checked by 12% SDS-polyacrylamide electrophoresis. The antibody concentration was determined by optical density measurement at 280 nm using an absorption coefficient of 1.4. Protein concentration was also determined using the Bio-Rad protein assay (Bio-Rad).

ELISA and Kinetic Analysis of mAb Binding

The ELISA procedures (direct binding experiments and determination of mAb isotypes) were as described previously (8) . For real time binding experiments, a BIAcore biosensor system (Pharmacia Biosensor, AB, Uppsala, Sweden) was used. Reagents including sensor chips CM5, surfactant P20, and coupling kit containing N-hydroxysuccinimide, N-ethyl-N`-(3-dimethylaminopropyl)carbodiimide (EDC), 2-(2-pyridinyldithio)ethane amine (PDEA) and ethanolamine HCl were obtained from Pharmacia Biosensor AB. To immobilize peptides to the sensor chip, the carboxyl-dextran matrix was first activated with 0.2 M EDC and 0.05 MN-hydroxysuccinimide. It was further modified by injecting PDEA thiol coupling reagent (15 µl of 80 mM PDEA in 0.1 M borate buffer, pH 8.5) allowing the thiol reactive group of peptide analogues to be coupled to the activated matrix. After the peptide immobilization run, remaining reactive groups on the sensor surface were deactivated by a 4-min pulse of 50 mM cysteine in 1 M NaCl, pH 4.3. Histone H3 was immobilized by the conventional procedure using EDC and N-hydroxysuccinimide-activated dextran matrix. Core particles were immobilized by trapping them on sensor chips containing covalently bound rabbit anti-mouse IgG Fc and a purified murine mAb directed against histone H2B (mAb LG11-2; a gift of M. Monestier, Philadelphia, PA) as a second layer. mAbs were then allowed to interact with sensor chips on which the four peptide analogues, H3, or core particles had been immobilized. 6-10 concentrations of each mAb ranging from 50 to 800 nM in HBS, pH 7.4 (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.05% BIAcore surfactant P20) were used in each test. The antibody preparation was injected at a constant flow rate of 5 µl/min during 7 min at 25 °C, and report points for calculation were taken every 10 s during 5 min, starting 1.5 min after the end of mAb injection. Antibody kinetic constants were measured as described previously (18) . Theory of kinetic measurementsusing the BIAcore biosensor system has been described previously (19, 20, 21) .

RESULTS

Generation of mAbs to the L-Parent Model Peptide IRGERA and IRGERA Analogues

Four groups of two BALB/c mice were injected with the L-, retro-inverso-, D-, and retro-IRGERA analogues coupled to small unilamellar vesicles containing MPLA as adjuvant, and four fusion experiments were performed with the spleen cells of these mice. Characteristics of retro-inverso- and retro-analogues, which contain reversed peptide bonds (i.e. NH-CO instead of CO-NH) have been described previously (Fig. 1) (9) . The four peptides contain a cysteine and two additional glycine residues at the NH-terminal end to allow selective conjugation of the peptides to bovine serum albumin and liposomes, and to enhance their accessibility when bound to carriers. ELISA reaction with peptide analogues and with H3 of polyclonal mouse antisera raised against these four peptides have been described previously (8, 9) . Hybridomas secreting antibodies to peptide analogues and H3 were screened by ELISA using antigens adsorbed to plastic of microtiter plates, and positive cultures were recloned twice in agar. A total of 67 stabilized specific hybridomas were obtained from the four fusions (). A number of these clones were of the IgG3 isotype. Since the reactivity of IgG3 antibodies in regard to their recognition of peptide analogues appeared quite different from that of antibodies of the IgG1, IgG2a, and IgG2b subclasses (8, 9) , data obtained with IgG3 mAbs will not be described here. Nine positive clones secreting IgG1, IgG2a, and IgG2b antibodies () were amplified in vitro, purified, and tested for their ability to cross-react with the parent IRGERA peptide, peptide analogues, the parent protein histone H3, and chromatin subunits. Between 35 and 63 mg of each mAb were available for these measurements.


Figure 1: Schematic structure of the natural L-peptide IRGERA and retro-inverso analogue.



Reactivity in ELISA and in the BIAcore of mAbs to IRGERA and IRGERA Analogues

The capacity of the nine mAbs to react in ELISA with IRGERA peptides and H3 is summarized in . In most cases, mAbs to the L- and retro-inverso-peptides cross-reacted more or less strongly with the L- and retro-inverso-peptides as well as with the parent protein H3, while mAbs generated from mice immunized with the D- and retro-peptides cross-reacted with the D- and retro-peptides but not with the L- and retro-inverso-peptide and with H3. These results are thus in complete agreement with those described previously with mouse antisera raised against the four IRGERA peptides (9) . The only exception was found with mAb 13x13 (anti-retro-inverso) which did not react with the parent L-peptide while, intriguingly, it recognized the whole histone H3 ().

The capacity of mAbs to recognize the four peptide analogues and H3 was measured in the BIAcore using antigens covalently linked to the dextran matrix through the free SH group introduced in peptides for this purpose or through amino groups of histone H3. Several concentrations of each mAb were allowed to react with the various immobilized antigens. Kinetic rate constants and equilibrium affinity constants of mAbs for the four peptide analogues and H3 are shown in I. As described above for ELISA, mAbs to the L- and retro-inverso-peptides bound preferentially the L- and retro-inverso-peptides in the BIAcore system. The only striking exceptions concerned mAb 13x13 which, as in ELISA (), did not bind the parent L-peptide and mAb 4x8, which, in addition to binding the L- and retro-inverso-peptide, also recognized the D- and retro-peptide. The latter cross-reaction was not seen in ELISA (). Interestingly, mAbs 4x8 and 4x11 (anti-L) both had a higher equilibrium affinity value Kfor the retro-inverso-analogue compared with Kvalues for the homologous peptide. In the case of mAb 4x11, this was due to a lower dissociation rate constant. Association and dissociation rate constants of mAbs 13x12, 13x14, and 13x18 with respect to the L- and retro-inverso-peptides were very similar for each mAb. mAbs 11x4 (anti-D-peptide) and 12x10 (anti-retro-peptide) presented similar kand kvalues with respect to the retro- and D-peptides.

The seven anti-L- and retro-inverso-peptide mAbs reacted with H3. Depending on the mAbs, equilibrium affinity values Kwere between 0.3 and 6.5 10M. mAbs 4x8 and 4x11 (anti-L-peptide) showed a much higher affinity for H3 than for the parent L-peptide (54 and 76 times, respectively). As expected, mAbs 11x4 (anti-D-peptide) and 12x10 (anti-retro-peptide) used as control antibodies did not recognize H3.

Reactivity of mAbs to IRGERA and IRGERA Analogues with H3 in Core Particles

Since several mAbs generated against IRGERA and the retro-inverso-analogue recognized the parent histone H3, their capacity to recognize H3 in more complex structures such as core particles was further studied. Core particles are the basic repeating structural subunits of eukaryotic chromatin and are composed of two molecules of each histone H2A, H2B, H3, and H4 surrounded by 145 base pairs of DNA. When adsorbed to a solid surface, a part of the conformation of these subunits may be affected. Likewise, the conformation of core particles may be considerably altered when they are covalently bound to the dextran matrix of the sensor chip via primary groups, particularly because histone tails, which are very basic, play an important role in the stabilization of the edifice. To overcome this problem, isolated core particles were captured by a first mAb directed against histone H2B, which was presented by rabbit anti-mouse IgG Fc immobilized on the sensor chip. Anti-H2B mouse mAb LG11-2 was shown in independent experiments to specifically recognize peptide 1-25 of H2B.() A typical immobilization plot of presenting antibodies (rabbit anti-mouse IgG and mAb LG 11-2) and the binding of core particles is shown in Fig. 2 . This figure also shows an example of interaction between mAb 4x11 and core particles. As shown in , six of the seven mAbs induced against the L- and retro-inverso-peptide reacted with core particles. The equilibrium affinity constants Kof mAbs 4x8, 4x10, and 4x11 (anti-L-peptide) for core particles were 1.2 10M, 5.9 10M, and 1.2 10M, respectively. Kvalues of mAbs 13x12, 13x14 and 13x18 (anti-retro-inverso-peptide) for core particles were 1.7 10M, 1.9 10M, and 2.2 10M, respectively. mAb 13x13 did not recognize core particles in the BIAcore system. As expected, the mAbs induced against D- and retro-peptides did not react with core particles.


Figure 2: Sensorgram showing the immobilization of chromatin core particles on the sensor chip and an example of mAb binding. The flow rate was 5 µl/min. A, activation of the carboxylated dextran matrix by the injection of 30 µl EDC/N-hydroxysuccinimide mixture; B, injection of 35 µl of a 100 µg/ml solution of rabbit anti-mouse IgG Fc in HBS, pH 7.4; C, inactivation of unreacted groups by 35 µl of ethanolamine; D, washing off noncovalently bound rabbit anti-mouse IgG Fc with 15 µl of 0.1 M HCl; E, injection of 10 µl of anti-H2B mAb LG11-2 (400 nM); F and G, injection (2) of a nonrelated ascitic fluid diluted 1:5; H, injection of 10 µl of core particles (200 nM) in HBS, pH 7.4; I, injection of 35 µl of mAb 4x11 (200 nM). mAbs were allowed to react 8 min with captured core particles at a flow rate of 5 µl/min. J, injection of 15 µl of 0.1 M HCl. Arrows indicate start and end of injections. Steps E-H were repeated for each new injection of anti-peptide mAb.



DISCUSSION

The IRGERA peptide provides an excellent model for the study of antigenic and immunogenic properties of pseudopeptides. It is known to be non-immunogenic in the absence of a carrier protein and particularly sensitive to proteases, and because this region of H3 is exposed at the surface of nucleosome in chromatin (22) , anti-peptide antibodies are interesting probes to study antibody interaction with complex nucleoprotein particles. In this study, we have investigated the specificity and affinity of mAbs induced against the natural L-hexapeptide and analogues containing reversed amide bonds. The retro-inverso transformation has been used by numerous bioorganic chemists to convert biologically active peptides into more stable compounds (2, 3, 6) . We have shown previously (9) that the retro-inverso IRGERA peptide was much more resistant to trypsin than the corresponding natural peptide.

The most important finding in this work was to show that three of the four mAbs to the retro-inverso IRGERA peptide bound equally well the retro-inverso and the natural peptide (with Kvalues ranging from 0.6 to 1.9 10M). The four mAbs reacted with the cognate protein (Kvalues, 0.3-2 10M), and three of them reacted with chromatin core particles presented toward the liquid phase by the N-terminal end of histone H2B (Kvalues, 0.2-2.2 10M). In contrast, mAbs induced against the all D-peptide and the retro-analogues, used as control antibodies in this series of experiments, did not recognize the natural peptide, histone H3 and H3 within isolated core particle.

In terms of structural and functional consequences in antibody-antigen reaction of specific modifications of peptidic bonds in a peptide, the results indicate that in our system, antibody specificity is not affected when the backbone of the IRGERA peptide is changed. In contrast, we show evidence that antibody interaction does not occur when, instead of presenting naturally-oriented side chains, the IRGERA peptide contains side chains with the same charge but a mirror orientation. The mechanism of IgG3 antibody interaction is apparently different. The fact that IgG3 Mabs induced against the D-enantiomer react with the L-peptide and H3 and, conversely, that IgG3 Mabs induced against the L-peptide react with the all D-peptide (8, 9) supposes that for this class of antibodies, the orientation of side chains is not important. Understanding the details of fine mechanisms of this interaction should be gained by further structural and dynamic studies by x-ray crystallography and NMR.

In the context of vaccine design, the demonstration that antibodies to a retro-inverso-peptide can cross-react particularly well with the cognate nucleoprotein structure is very important and promising for further development. The antibodies described in this study have been generated against the peptide analogue covalently bound to liposome containing the nontoxic adjuvant MPLA. Since the present study was performed with the C-terminal region of H3, which is known to be highly accessible and probably particularly mobile, the results should be confirmed with other exposed protein domains, which correspond to internal sequences in the primary structure of the protein. However, collectively, the strategy is encouraging in respect to the potential use of retro-inverso-peptides in the design of much more potent synthetic vaccines. We are currently investigating the immunogenicity of retro-inverso-peptides corresponding to neutralization epitopes, and other types of peptide bond modification are also under investigation (23) . Theoretically, a vaccine consisting of such modified peptides in combination with T-epitopes associated with immunosomes may be effective in producing neutralizing antibodies against complex infectious structures.

  
Table: Summary of results obtained in fusion experiments of splenocytes from mice immunized against the L-parent IRGERA hexapeptide and peptide analogues


  
Table: Reactivity in ELISA of mAbs induced against IRGERA and IRGERA analogues

Microtiter plates were coated with 2 µM peptide conjugated to bovine serum albumin (BSA) (carrier to peptide molar ratio 1:10) or with 100 ng/ml H3 and allowed to react with 4 µg/ml mAb. Anti-mouse IgG-peroxidase conjugate was diluted 1:5000. Results are expressed in terms of: -, absorbance values measured at 450 nm <0.4; +, 0.4-1.0; ++, >1.0->2.0; +++, >2.0->3.0. L, natural peptide; RI, retro-inverso-peptide; D, D-peptide; R, retro-peptide.


  
Table: Kinetic rate constants and equilibrium affinity constants of mAbs induced against IRGERA and IRGERA analogues

NB, no binding. Association (k) and dissociation (k) rate constants are the mean values obtained in two to four independent experiments. L, natural L-peptide; RI, retro-inverso-peptides; D, D-peptide; R, retro-peptide. mAbs 4 8, 4 10, and 4 11 are anti-L-peptide antibodies; mAbs 13 12, 13 13, 13 14, and 13 18 are anti-retro-inverso-peptides; mAb 11 4 is an anti-D-peptide and mAb 12 10 is an anti-retro-peptide.


  
Table: Kinetic rate constants and equilibrium affinity constants of mAbs induced against IRGERA and IRGERA analogues with respect to core particles

For details, see the legend of Table III.



FOOTNOTES

*
This work was supported by the CNRS (Groupe de Coordination Chimie-Biologie, Project 28D4). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33 88 41 70 27; Fax: 33 88 61 06 80.

The abbreviations used are: mAb, monoclonal antibody; MPLA, monophophoryl lipid A; ELISA, enzyme-linked immunosorbent assay; EDC, N-ethyl-N`-(3-dimethylaminopropyl)carbodiimide; PDEA, 2-(2-pyridinyldithio)ethane amine.

M. Monestier and S. Muller, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. M. Monestier for the gift of mAb LG11-2, Dr. M. Friede for the preparation of liposomes, and G. Sommermeyer for skilled technical assistance in the preparation of mAbs.


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