CA2004810A1 - Synthetic antigens, a process for the preparation thereof and the use thereof - Google Patents

Abstract

BEHRINGWERRE ARTIENGESELLSCHAFT

ABSTRACT
Synthetic antigens, a process for the preparation thereof and the use thereof Conjugates which are composed of membrane proteins of Neisseria meningitidis and peptides and which can be used as pharmaceuticals are described.

They are furthermore suitable for preparing antibodies which can be used for therapy and diagnosis.

Description

~004810 BEHRINGWERRE AgTIENGESELLSCH~FT 88/B 041 - Ma 701 Dr. Ha/Sd Synthetic antigens, a process for the preparation thereof and the use thereof _ The invention relates to synthetic antigens which are S composed of a peptide and of a membrane protein from Neisseria meningitidi~ as molecule conferring antigeni-city, to a proce~s for the preparation thereof, to the u~e thereof for obtaining antibodies, and to the use of the antigens or antibodies as pharmaceutical~ and of the antibodies for diagnostic purpose~.

EP-A-0,161,188 describes the po~sibility of linking the membrane proteins from Neisseria meningitidis to polysac-charides in order to improve the antigenicity thereof.

In the state of the art peptide~ which are not antigenic thaptens) and against which an antibody i8 to be gener-ated are linked to a high molecular weight, usually soluble, molecule, and so-called con~ugates are gener-ated, and then animals, for example rabbits or mice, are immunized therewith. After a period of some day~ or weeks has elapsed and possibly after booster steps, blood ~amples are taken from the animal and the antibodies are obtained.

Protein~ are mostly u~ed as high molecular weight mole-cules, particular mention being made of keyhole limpet hemocyanin and albumins.

Reyhole limpet hemocyanin con~ugate~ have the dis-advantages that they are ~paringly soluble and that antibodie~ against keyhole limpet hemocyanin are induced.
These disadvantage~ have now been eliminated, ~urpris-ingly, by the present invention in which a ~embraneprotein from Neis~eria meningitidis is employed as protein conferring antigenicity on non-antigenic ~00~8~0 peptides, preferably oligo- or polypeptides having up to 100 amino acids.

Hence the invention relates to conjugates composed of a membrane protein from Neisseria meningitidi~ and of a peptide.

Meningococcal protein B2 is preferably used.

The peptide-protein con~ugates are prepared by methods known per ~e, for ex~mple by coupling via glutaraldehyde or by introducing maleimido group~ on the protein with subsequent linkage of a peptide via the free sulfhydryl functionality thereof.

The peptide-protein con~ugates according to the invention are prepared using glutaraldehyde by, for example, dissolving the meningococcal B protein in a buffer, preferably 1 mg of protein in 1 ml of PBS (phosphate buffered saline), adding the peptide, preferably 1 - 2 mg of peptide per mg of protein, and adding 5 - 20 ~1, preferably 10 ~1, of a 25% strength aqueous solution of glutaraldehyde per mg of protein. The solution is stirred at room temperature for 30 min to 5 hours, preferably 1 hour, and 4 mg of lysine are added per mg of protein to block reactive aldehyde groups. After a further 10 min have elap~ed, the mixture is dialyzed against distilled water and the con~ugate is preferably obtained by lyo-philization.

It i8 possible, for linking peptides containing thiolgroups to the protein, to link thiol groups via disulfide bridges or thioether~ to the meningococcal B protein, the con~ugate preferably being prepared via thioether linkage.

In a particular preferred embodiment of the con~ugation, the protein is initially derivatized in such a way that thiol groups can then be directly linked to the protein.

~00~810 For this purpose, the protein is dissolved in a buffer of pH 7 - 9, preferably in a sodium bicarbonate or phosphate buffer, and reacted with a substance containing N-male-imido groups, preferably N-maleimidobutyric acid hydroxy-succinimide eRter. After the protein activated" in thisway has been separated off, for example by gel permeation on Sephadex(R) G 50, 1 - 3 mg of peptide containing thiol groups is added per mg of protein, and the mixture is left to react for 0.5 - 10 hours, preferably 1 hour. The protein-peptide con~ugate can then be obtained as usual, preferably by dialysis and lyophilization.

Suitable animals are immunized with a conjugate prepared in this way, in which case antibodies against the syn-thetic peptide are formed. The preferred animal species for the immunization and obtaining of antibodies in this connection is the rabbit. The synthetic antigen can likewise be u~ed for immunizing mice and, by use of the hybridoma technique, for preparing monoclonal antibodies.

The antiserum obtained by immunization with the con~ugate react~ specifically with the protein from which the peptide sequence used for the immunization originates.

Besides the good solubility of the con~ugate, it has emerged, surprisingly, that the antiserum directed against the peptide shows, in double Lmmunodiffusion, predominantly no reactivity with the meningococcal protein. This is all the more ~urprising since antibodies are always also induced against other proteins conferring antigenicity when these are used.

The antibodies obtained according to the in~ention can be employed for a number of immunoassays with a variety of design~.

The examples explain the invention. The following abbre-viations are used in the examples:

~00~810 GMBS Gamma-maleimidobutyric acid N-hydroxysuccinimide e~ter Cys L-Cysteine Leu L-Leucine Phe L-Phenylalanine Ser L-Serine Pro L-Proline Glu L-Glutamic acid Lys L-Lysine Gly Glycine Val L-Valine Ala L-Alanine Arg L-Arginine Asp L-Aspartic acid Tyr L-Tyrosine A~n L-Asparagine RLH geyhole limpet hemocyanin AT III Antithrombin III

Examples Example 1 -~ a3 Derivatization of meningococcal B protein with maleimido group~

40 mg of meningococcal B protein were dissolved in 0.05 mM sodium phosphate buffer pH 8.0 and activated with 5 mg of ~MBS for 1 hour. The erude product was chromatographed on a Sephadex G 50 column (2 x 30 cm) in 0.1 M sodium phosphate~0.5 mM EDTA pH 6.0, The protein fraction is collected and concentrated to about 8 ml.

b) Preparation of the meningococcal B protein-(AT III
343-363) peptide-protein con~ugate 40 mg of the peptide of the structure Cys-Leu-Phe-Ser-Pro-Glu-Lys-Ser-~y~-Leu-Pro-Gly-Ile-Val-Ala-Ala-Glu-Gly-Arg-A~p-Asp-Leu-Tyr (AT III 343 - 363), ~,:0~4810 which was prepared u~ing the solid-phase peptide method, were added to the protein fraction (see Example la), and the mixture wa~ stirred with exclusion of oxygen for 1 hour. Dialysis and lyo-philization resulted in 48 mg of peptide-protein con~ugate.
Example 2 a) Derivati~ation of keyhole limpet hemocyanin with maleimido group~

40 mg of KLH were derivatized in analo~y to Example la).
b) Preparation of RLH-(AT III 343 - 363) 40 mg of AT III 343 - 363 peptide were coupled in analogy to Example lb) to RIH derivatized a~ in Example 2a). Yield 45 mg.
Example 3 Preparation of meningococcal B protein-(AT III 129 - 140) 40 mg of the peptide Cys-Arq-Leu-Tyr-Arg-Lys-Ala-Asn-Lys-Ser-Ser-Lys-Leu (AT III 129 - 140) were coupled as in Example 1 to ~activated" meningococcal B protein.
Yield 47 mg.
Example 4 Preparation of RLH-(AT III 129 - 140) The AT III 129-140 peptide was coupled in analoqy to Example 3 to GNBS-derivatized RLH. Yield 49 mg.

200~8 10 Example 5 Immunization of rabbits and testing of the antisera 5 rabbits were immunized with 2 mg portions of antigen for a period of 8 weeks, the con~ugates being admini~-tered subcutaneously and intravenously. The animals were then exsanguinated, and the resulting antisera were pooled and stabilized with preservatives.

Example 6 a) Testing of the antisera using the enzyme immunoassay technique (ELISA) The immune reaction of the resulting antisera was tested using the ELISA technique. Highly purified AT
III antigen wa~ diluted with Tris buffer ~olution (0.025 mol/l) to a concentration of 10 ~g/ml and immobilized by adsorption onto polystyrene tubes.
The antiserum to be tested wa diluted 1 + 1 with incubation buffer (0.01 mol/l Tris, 0.05% Tween, pH
7.6) and 200 ~1 portions in each tube were incubated at 37C for 30 min. The incubation solution was subsequently removed, and the tube was washed twice with 500 ~1 of washing solution each time (0.02 mol/l sodium phosphate, 0.05% Tween, pH 7.6).
200 ~1 of peroxidase-con~ugated anti-rabbit gamma-globulin antibodies were then added, and the tubes were incubated at 37C for 30 min. After removal of the con~ugate solution and two washes, 200 ~1 of substrate/chromogen solution (hydrogen peroxide, o-phenylenediamine) were added and the tubes were incubated at 18 - 22C. After 30 min, the peroxidase was inactivated with sulfuric acid, and the absorp-tion of the reaction solution st 492 nm wa~ deter-mined.

The ELISA technique was used for testing all the antisera 20C~810 obtained using the peptide-protein con~ugate~ described in sections lb), 2b), 3) and 4). The antisera were employed in different dilutions for this (1:10~ 105 dilution~.

The absorptions at 492 nm as a function of the antiserum dilution and in relation to the absorption of a tube containing buffer solution are shown in the table which follows.

The similarity of the measured absorptions at the parti-cular dilution steps demonstrates that both RLH and meningococcal protein are equally suitable as high molecular weight protein conferring antigenicity, al-though the antiserum obtained with meningococcal protein con~ugate does not react with meningococcal protein in double immunodiffusion (see b)).

200~810 Table 1 Antiserun against Dilution step aD 492 ~m~30 nin Peptide-oon~ugated meringp- 1 : 10 2.0 coccal B protein (AT III 1 : 100 1.68 343 - 363) 1 : 1000 1.30 1 : 10000 0.77 1 : 100000 0.63 E~F~ik~-oon~ugated nEningp- 1 : 10 1.28 coocal B probein (AT III 1 : 100 0.83 12g - 140) 1 : 1000 0.26 1: 10000 0.09 Pepeld3rc~n~ugated RLH 1 : 10 2.0 (AT III 343 - 363) 1 : 100 1.76 1 : 1000 1.32 1 : 10000 0.79 1 : 100000 0.72 EeQtide-ocn~ugated RLH- 1 . 10 1.03 (A~ III 129 - 140) 1 : 100 0.84 1 : 1000 0.34 1 : 10000 0.14 Euffer blank 0.03 ~:0~4~31Q

g b) Testing of the antisera by the double immuno-diffusion technique The antisera described in section 6a) were investi-gated using the double immunodiffusion technique for the formation of precipitates with the carrier protein.

The antisera obtained with RLH-peptide conjugate showed, in dilutions down to l : 16, a distinct precipitate line with RLH antigen in the agarose gel. By contrast, the antisera obtained with menin-gococcal-peptide conjugate yielded no immunoprecipi-tates with meningococcal B protein.

Claims (9)

1. A conjugate composed of a membrane protein from Neisseria meningitidis and of a peptide.

2. A conjugate as claimed in claim 1, wherein the protein is meningococcal protein B2.

3. A conjugate as claimed in claim 1, wherein the peptide is composed of up to 100 amino acids.

4. A conjugate as claimed in claim 1, wherein the peptide is a peptide from antithrombin III.

5. A conjugate as claimed in claim 1, as pharmaceutical.

6. A process for the preparation of a conjugate composed of a membrane protein from Neisseria meningitidis and of a peptide, which comprises producing the linkage between the protein and the peptide using a process of peptide chemistry.

7. The use of a conjugate as claimed in claim 1 for the preparation of antibodies.

8. The use of an antibody as claimed in claim 7 as a pharma-ceutical or diagnostic aid.

9. The conjugate as claimed in claim 1, and substantially as described herein.