EP0224540A1 - Produkte aus der kupplung eines makromolekularen trägers, eines haptens und eines muramylpeptids - Google Patents

Produkte aus der kupplung eines makromolekularen trägers, eines haptens und eines muramylpeptids

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Publication number
EP0224540A1
EP0224540A1 EP19860903429 EP86903429A EP0224540A1 EP 0224540 A1 EP0224540 A1 EP 0224540A1 EP 19860903429 EP19860903429 EP 19860903429 EP 86903429 A EP86903429 A EP 86903429A EP 0224540 A1 EP0224540 A1 EP 0224540A1
Authority
EP
European Patent Office
Prior art keywords
group
peptide
muramyl
hapten
product according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19860903429
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English (en)
French (fr)
Inventor
Michel Jolivet
Françoise Audibert
Louis Chedid
Pierre Lefrancier
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Bpifrance Financement SA
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Agence National de Valorisation de la Recherche ANVAR
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Publication of EP0224540A1 publication Critical patent/EP0224540A1/de
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6062—Muramyl peptides

Definitions

  • the invention relates to products resulting from the conjugation of a macromolecular support, a hapten and a muramyl-peptide via a group substituting the saccharide part of the muramyl-peptide, these products having selective immunogenic properties of the haptenic part which enter into their constitution. It relates more particularly still to immunogenic compositions containing such products in association with an acceptable pharmaceutical vehicle, compositions which can be used for vaccination against antigens having an antigenic or epitope site in common with hapten.
  • synthetic peptides generally in low molecular weight haptens, to constitute the active principle of vaccines capable of protecting the host against hapten or more often against a pathogenic antigen.
  • haptens do not exhibit any sensitive immunogenic power when they are presented to the host organism.
  • Various methods have already been proposed either to confer or reinforce the immunogenic properties of such haptens, or else to reinforce the immunogenicity of weak antigens, whether they are inherently weak antigens, or highly purified antigens of which a part of the immunogenic power has been lost, in particular during the operations necessary for their purification or of subunits derived from these antigens, these subunits however still carrying sites antigenic characteristics of these antigens.
  • hapten for the convenience of language.
  • the word will designate not only the haptens themselves, having for example a molecular weight at most equal to 5000, but also the weak antigens of which the question above is concerned.
  • One solution which has been envisaged and which leads to high immunogenicity consists in the fixing (conjugation or covalent coupling) of said haptens on macromolecular supports of high molecular weights.
  • the conjugation or coupling products thus obtained often often exhibit increased immunogenicity capable of inducing in vivo an effective production of antibodies against the hapten present in the conjugation product or even against higher molecular weight antigens having a site. antigenic or an epitope in common with this hapten.
  • the object of the invention is to remedy at least in large part the difficulties which may be encountered in the use of conjugation products involving a hapten and a macromolecular support of the above-mentioned type. More particularly, the invention aims to make these conjugation products such that, while having an immunogenic effect characteristic of hapten, they are at the same time substantially devoid of this effect vis-à-vis the macromolecular support itself. In other words, the invention is intended to provide conjugation products of this type, in which this immunogenic macromolecular anti-support activity is practically suppressed.
  • the object of the invention can be achieved by using carrier molecules resulting from the conjugation of the macromolecular support chosen with one or more muramyl-peptide groups, via a chemical group playing the role of an arm, substituting the glucopyranosyl nucleus of the muramyl-peptide, preferably at position 6 of this nucleus.
  • the products of the invention therefore consist of conjugation products between the carrier molecules thus formed and the hapten - or haptens - themselves carrying one or more epitopes contained in a molecule, in particular an antigen against which protection can be wanted.
  • the product of the invention can therefore be characterized as containing three principles coupled together via covalent bonds, these three principles consisting respectively of a macromolecular support, a muramyl-peptide linked to this support by a chemical group via its nucleus glucopyranosyl and at least one hapten carrying at least one epitope responsible for the desired immunogenic activity.
  • the arm is chosen so as to confer, if necessary, amphiphilic properties on the arm-muramyl-peptide assembly.
  • this tripleconjugation product benefits from a double amplification of the immunogenic activity of hapten, in other words amplifications respectively induced by the muramyl-peptide, the immuno-adjuvant activities of which are known, and by the carrier molecule, this triple conjugation product however being substantially, if not totally, devoid of immunogenic activity with respect to the macromolecular support or of the carrier molecule.
  • Muramyl peptides preferred for the desired modification of the macromolecular support are represented by the following general formula:
  • R 1 is an OH group or an OC 6 H 4 -NH 2 or O- (CL 2 ) m -R a group , m being an integer from 1 to 10, and R a being an amino, carboxyl or thiol function, hydroxyl or hydrogen,
  • - A is oxygen or an NH group
  • R 6 is a BR or CO-BR group in which B is a group or a linear or branched chain which can contain up to about 100 carbon atoms and R is a hydroxyl, carboxyl, amine, thiol or hydrogen, group B being optionally itself carrying one or more functional groups of the type of those designated above for R, or alternatively carbonyl, alkoxyl or acyl;
  • - R 2 is hydrogen or an alkyl group comprising from 1 to 4 carbon atoms, in particular methyl;
  • - X is an alanyl, arginyl, lysyl, asparagyle, aspartyle, cystéinyle, glutaminyle, glutamyle, glycyle, histidyle, hydroxyprolyle, isoleucyle, leucyle, casethionyle, phenylalanyl, prolyl, seryle, threonyle, tryptophanyle or valyle
  • Y is an OH, NH 2 group or, preferably, OR 7 or NHR 7 , R 7 being a hydrocarbon group which may have from 1 to 20 carbon atoms or which may be an amino acid as indicated for X, such as alanyl, seryl, valyl or glycyl, said amino acid being free, amidated or esterified, the esterification group possibly comprising up to 4 carbon atoms;
  • Z is a carboxyl, carboxamide, ester group containing from 1 to 10 carbon atoms, or an alkyl group comprising from 1 to 4 carbon atoms, preferably -CH 2 -CH 3 or - (CH 2 ) 2 -CH 3 ; it being understood that at least one of the groups R 1 and R 6 (and preferably R 6 ) has a reactive function allowing its conjugation with a reactive function mentary carried by another molecule, in particular the macromolecular support.
  • the muramyl-peptide on the one hand, the hapten on the other hand are coupled to the macromolecular support via distinct functional groups initially present on or carried by the support macromolecular.
  • the preferred conjugates of the invention are those in which the muramyl peptide and the hapten are grafted separately from each other on the macromolecular support.
  • a preferred group of muramyl-peptides used in the "triple conjugates" of the invention are characterized by the following formula:
  • R 1 is -OH or -OC 6 H 4 -NH 2 or O- (CH 2 ) m -R with m from 1 to
  • R 10 and R a being an amine, carboxyl, thiol, hydroxyl or hydrogen function;
  • - R 2 is -H or -CH 3 ;
  • R 6 is -CO (CH 2 ) n -R with n from 1 to 10 and R being an amine, carboxyl, thiol, hydroxyl or hydrogen function;
  • - X is Ala, Gly, Ser or Val;
  • Y is O (CH 2 ) p -H with p from 1 to 10;
  • - Z is CO-NH 2 , COO (CH 2 ) r -H or (CH 2 ) r -CH 3 with r from 1 to 4.
  • amino acid residues forming part of the group X are levogyres (except in the case where X is constituted by a glycyl residue).
  • the muramyl-peptides corresponding to derivatives of the 2- (2-acetamido-2-deoxy-3-OD-glucopyranosyl) - alkanoyl-peptides type are sufficiently known that it is unnecessary to insist on their methods of preparation.
  • the expression muramyl-peptides should be understood to also extend to muramyl-peptides of the 2- (2-acetamido-2-deoxy-) type 3-OD-glucopyranosyl) -alkanoyl-aminoacylnor Leucine or to derivatives of the latter muramyl-peptides.
  • Preferred muramyl peptides belonging to the latter type of preferred compounds are those in which Y is an OR 7 or NHR 7 group , R 7 having the above-mentioned meaning, and in which the O CH group is a derivative of a dextrorotatory nor Leucine group, more particularly of a D-nor Leucine group, in which
  • the groups R 6 contain, alone or in combination with the groups which have just been mentioned, linear or branched chains, the links of which consist of hydrocarbon chains, in which case where appropriate, some of the carbon atoms are replaced, from place to place, by nitrogen and / or oxygen atoms, with in particular the creation of ester, ether, amido, imido, imido, etc. functions.
  • aminoacyl residues in particular links containing from 1 to 3 successive aminoacyl residues, for example of the lysyl or alanyl type, (these being able moreover to be replaced by any other aminoacyl of the type indicated with respect to group X), or alternatively glyceryl elements: -O-CH 2 -CH (O) -CH 2 -O- or glycolyl: -O-CH 2 -CH 2 -O- or derived elements glycerol or glycol, glyceric or glycolic acids, for example those combining glyceryl or glycolyl elements, on the one hand, and aminocyl elements, on the other hand.
  • preferred groups B are formed by chains as defined above, themselves carrying ramifications, for example by acyl groups, in particular fatty acids, which respectively comprise in particular from 14 to 30 carbon atoms, by example palmitoyle groups.
  • preferred groups B contain in particular of the terminal elements represented by the formula:
  • R 8 and R 9 are acyl groups comprising from 14 to 30, in particular from 16 to 24, carbon atoms.
  • muramyl-peptides capable of being used in the context of the present invention, mention will be made of those which may be designated by the following short formulas (the designation "MurNAc” relating to the N-acetylmuramic group):
  • the preferred macromolecular supports capable of being used in the "triple conjugates" of the invention mention will be made of natural molecules of the human serum albumin type, toxoids or other carrier macromolecular supports having molecular weights of at least 50,000 , for example from 50,000 to 250,000 daltons.
  • Synthetic peptide polymers can also be used, of the polylysine type or of the kind described in European patent 13 651.
  • macromolecular supports consisting of peptide chains whose sequence contains a large number of lateral functional groups allowing covalent coupling, with the particular muramyl-peptides used in the context of this invention and / or with haptens whose immunogenicity must be induced.
  • tetanus and diphtheria toxins are particularly preferred.
  • the tetanus toxin which has a molecular weight of the order of 160,000, carries around fifty free terminal NH 2 groups making it possible to fix a sufficient number of hapten and muramyl-peptide groups.
  • toxins can, after fixing said muramyl peptides and / or haptens, then be easily detoxified, for example by a conventional treatment with formaldehyde.
  • Conditions under which these conjugations can be carried out, then the detoxified toxins (if necessary) are illustrated below, in particular in relation to the attachment to the tetanus toxin of a peptide containing a surface element of the surface protein of the sporozoite of Plasmodium knowlesi and 6-O-epsilon-aminocaproyl-MurNAc-L-Ala-D-Gln-OnBu.
  • the invention finally applies to the production of "triple conjugates” involving any hapten comprising at least one "antigenic site” usable for the induction of antibodies in vivo, provided that it is conjugated to an amplifying molecule of its immunogenicity.
  • group comprising at least one antigenic site is meant any group contained in an antigen in respect of which an in vivo production of antibodies is sought or any group equivalent on the immunogenic level.
  • haptens likely to be taken into account in the context of this patent application, mention will be made, for example, of those which meet the definition given by European patent application 89 290 filed by the National Agency Research Valuation (ANVAR). It is therefore not only haptens carrying an antigenic site which, when these haptens are coupled to a amplifying molecule, gives them the ability to induce protective antibodies against specific pathogens in vivo.
  • the invention further extends to the manufacture of triple conjugates involving other haptens, such as those envisaged in European patent application 89 290, for example hormones.
  • the luteotropin releasing factor known under the designation LH-RH, peptide fragments, or synthetic peptides endowed with analogous properties, for example - decapeptides of the Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 type or the corresponding free acid pGlu-His-Trp-Ser-Tyr-Gly- Leu-Arg-Pro-Gly; C-terminal peptides of human chorionic gonadotropin (hCG); - C-terminal peptides of the ⁇ subunit of hCG, in particular a triacontapeptide containing aminoacyl residues corresponding to those of hCG- ⁇ or of synthetic terminal derivatives of the genus described by S.
  • LH-RH luteotropin releasing factor
  • peptide fragments or synthetic peptides endowed with analogous properties, for example - decapeptides of the Glu-His
  • haptens which can be used in the triple conjugates according to the invention, those which involve different synthetic peptides having aminoacyl residue sequences in common with the main peptides constituting the HBsAg antigen, in particular those whose end aminoacyles correspond to those which, in the sequence of HBsAg, occupy the following positions: 48-81; 2-16; 22-35; 95-100; 117-137; 122-137; 117-135 (according to the structures given by Pasek et al. And reproduced in the article by Lerner et al. (1981), Proc. Natl. Acad. Sci. USA, 28., N ⁇ 6, 3403-3407. will also mention as suitable haptens the vaccinating peptides carrying antigenic sites characteristic of diphtheria toxin, influenza viruses, herpes, polio, etc.
  • Particularly preferred peptide haptens are one or more of those defined below:
  • haptens involved were as follows: 1 . ) SODP: octodecapeptide containing a sequence of the "diphtheria loop" and of formula: H 2 N-Ala-Ala-cys-Ala-Gly-Asn-Arg-Val-Arg-Arg-Ser-Val-Gly-Ser-Ser -Leu-Lys-Cys. 2 . ) SCB7: peptide containing an epitope characteristic of the streptococcus type M24 containing the sequence:
  • HBs (peptide 99-121); peptide comprising an antigenic site of the envelope of the virus containing the sequence: Asp-tyr-Gln-Gly-Met-Leu-Pro-Val-Cys-Pro-Leu-Ile-Pro-Gly- 100 110
  • DDP Mal peptide carrying an epitope characteristic of a protective antigen against Plasmodium knowlesi. agent of monkey malaria, described by Godson GN et al. (1983), Nature, 305 (29-33, constituted by the sequence:
  • the haptens that can be used cannot be limited to peptides. They are, for example, also saccharide or osidic haptens. Mention will be made, for example, of the blood groups A and B, the polysaccharide C of streptococcus C, the cellobiuronic acid (Pneumo type III), as described by WF Goebel, "J. Exp. Medicine", 1939, p. 353-363, or paraaminophenylcellobiuronic acid.
  • the haptens capable of being used can consist of synthetic or semi-synthetic polypeptides containing several antigenic sites which are distinct from each other and capable of providing "polyvaccines". Such polyvaccines have been described in French patent application 84 13989 filed on September 12, 1984.
  • a coupling reaction can be used via glutaraldehyde, insofar as the macromolecular support, the hapten and the muramyl-peptide considered all contain amino functions.
  • the conjugation between the macromolecular support and the muramyl-peptide one can have recourse to all forms of coupling involving determined positions on the glucopyranosyl cycle of the muramyl-peptide. In particular, these bonds will occur at the C 1 position, preferably that at C 6 of the glucopyranosyl ring of the muramyl peptide.
  • substitutions involving the R 6 group recourse can be had to the coupling methods commonly used in peptide synthesis between the appropriate functions carried, on the one hand, by the muramyl-peptide and, on the other hand, by the macromolecular support, one carrying for example an amino function and the other a carboxyl, hydroxyl or sulfhydrile function or vice versa.
  • any other type of link can be used, bringing into play the combinations which may occur between a sulfhydride function carried by one of the partners that constitute the macromolecular support and the muramyl-peptide and a maleimide group carried by the other reagent, for example by making use of the technique described by T. Kitagawa and T. Aikagawa in "J. Biochem.” 21 (1976), 233.
  • Conjugation can be done using conventional modes of diazotization or reaction involving an isothiocyanate, in particular when the muramylpeptide carries an amino aromatic function (in particular at the level of the R 1 group) and when the macromolecular support carries an amino function capable of to intervene in this reaction.
  • Such production techniques are described for example by BF Erlanger in "Pharmacol. Rev.”, 25 (1973), 271.
  • the coupling can also be carried out by the reduction of a Schiff base produced between an aldehyde function carried by the one of the reagents and an amino function carried by the other (GR Gray, "Arch. Biochem. Biophys. 163 (1974), 426).
  • the conjugations can be carried out by means of bridging, the bridging agent consisting for example of a bifunctional reagent.
  • the bridging group between the muramyl-peptide and the macromolecular support in the final conjugate will not exceed the length of a corresponding sequence ndant to that of a p-decylic hydrocarbon chain.
  • Such bridging agents are for example mentioned in patent 78 16792 filed on June 5, 1978. In the foregoing, it was mainly mentioned the case of the coupling between the muramyl-peptide chosen and the macromolecular support.
  • the relative proportions of hapten and muramylpeptides will be adjusted as a function of the number of free functional groups available on said macromolecular support.
  • Experience and examples below show that it is not necessary to "saturate" the macromolecular support with muramyl-peptide groups of the category envisaged. It is indeed observed that the masking or the suppression of the immunogenic reaction specific to the macromolecular support itself is rapidly annihilated.
  • it will generally be advantageous to fix on the macromolecular support - or on the chain - as many hapten groups as possible, in order to obtain a maximum immunogenic reaction specific to the hapten.
  • the number of muramylpeptide groups relative to the number of chains of the macromolecular support (numbers expressed in NH 2 equivalents used in the reaction) will be in a 1: 1 ratio and the number of hapten groups relative to the number of macromolecular support chains will also be in a ratio at least equal, however preferably greater than 1, for example from 1 to 5.
  • the coupling agent is glutaraldehyde used at the final concentration of 5.2 mM.
  • Glutaraldehyde serves as both a coupling and a detoxifying agent when the carrier molecule is the non-detoxified toxin, which allows the availability of enough NH 2 groups (fifty for a molecule having 160,000 molecular weight) to bind enough muramyl - adjuvant peptide and antigenic peptide (it goes without saying that "detoxification" is superfluous when the carrier molecule used is itself non-toxic).
  • the peptide is, in this example, a peptide copying a structural element of the surface protein of the sporozoite of Plasmodium knowlesi. Its structure is described in Godson et al. (Nature, 1983, 305. 29) and its immunogenicity was studied by gysin et al. (J. Exp. Mec, 1984, 160, 935).
  • the reaction is carried out in 0.1 m sodium carbonate-bicarbonate buffer, pH 8.5. Tetanus toxin (2.35 mg), sporozoite peptide (3.75 mg) and 6-O- ( ⁇ -amino-caproyl) -murabutide (2 mg) are dissolved and glutaraldehyde is added with stirring .
  • the reaction lasts one week and the product is filtered on a molecular sieve, in particular that sold under the brand SEPHADEX-G25 so as to eliminate all molecules with molecular weights less than 20,000.
  • the reaction product is removed by filtration; also, the only possible conjugate is the tryptic where the peptide (pep) and the adjuvant muramylpeptide (adj) are linked separately on the tetanus toxin (adj-support-pep).
  • the muramyl peptide alone can be reacted with the tetanus toxin for a certain time, before adding the peptide to the reaction medium, to promote the toxin binding tetanus adjuvant with respect to the peptide-adjuvant bond likely to come to conjugate in turn to the macromolecular support.
  • the conjugate finally obtained is then subjected to the following tests: a) HPLC.
  • the molecular weight of the conjugate sought corresponds to a monomer (between 150,000 and 180,000 daltons).
  • tetanus toxin and the conjugates are diluted so as to obtain 10 ⁇ g of tetanus antigen in 50 ⁇ l. Fifty microlitres of each dilution are deposited in the wells of the plate. After adequate treatment to obtain the fixation of the antigen on the plates, a fixed quantity of tetanus serum is calculated, calculated to have an optical density close to 1, after revelation by the anti-antibody system labeled with peroxidase. In the experiment implemented, the following figures were obtained:
  • the second test consisted of a pyrogenic test; it showed that 1.5 ⁇ g of muramyl-peptide conjugate did not induce, in any of the three rabbits tested, a rise in temperature greater than or equal to 0.5oC.
  • 0.8 ⁇ g of a conjugate of MDP-Lys and toxin tetanus obtained by coupling under the same conditions, caused temperature increases ranging from 1.6oC to 2oC. It will be noted that in this last test, the conjugation was, by necessity, carried out at the level of the peptide group of the muramyl-peptide!
  • triple conjugates were produced using variable initial proportions of the peptide, of the tetanus toxin and of 6-O - ⁇ -aminocaproyl-murabutide (conjugates D, E and F), or MDP-lysine (conjugates A, B and C).
  • the amounts of muramyl-peptide will be expressed in MDP- (N-acetyl-muramyl) -L-alanyl-D-isoglutamine equivalents).
  • NH 2 equivalents for the assessment of the ratios of the various constituents entering into the triple conjugates considered below.
  • Conjugates A and D were obtained from 2.35 mg of tetanus toxin, 3.75 mg of the malaric peptide and 2 mg of MDP; conjugates B and E were obtained from 2.35 mg of TT; 7.5 mg PEP and 1 mg MDP; finally, conjugates C and F were obtained from 11.7 mg of TT, 18.75 mg of PEP and 0.5 mg of MDP. Finally, in particular for the purposes of comparisons, a PEP-TT conjugate was obtained by proceeding as described above, by the conjugation of PEP and tetanus toxin. The toxin was used as a carrier because it contained more reactive groups than toxoid.
  • the detoxification of the conjugates obtained was carried out by adding sufficient quantities of glutaraldehyde, then by incubation for 5 days.
  • the conjugates obtained and the relative proportions of their constituents appear in Table I.
  • the complete detoxification of each of the conjugates was checked before use, by subcutaneous injection into 3 mice of a dose of conjugate corresponding to the double dose of TT resulting in the death of animals. Detoxification was noted due to the absence of any sign of paralysis in the mice used in this test or in those which were the subject of the tests which follow.
  • mice each comprising 6 adult BALB / c female mice were immunized subcutaneously with 50 ⁇ g of each of the AC conjugates (Table II) administered in the form of saline solution.
  • Another group received 50 ⁇ g of PEP-TT in a mixture with 1.00 ⁇ g of MDP-lysine in a saline solution.
  • 6 control mice received 50 ⁇ g of PEP-TT in saline solution in the absence of adjuvant. The animals were boosted with the same antigens 3 weeks later and then bled either 7 days or 14 days after the boost.
  • mice were immunized with 50 ⁇ g of the DF conjugates (table I) in a saline solution or with 50 ⁇ g of PEP-TT mixed with 100 ⁇ g of 6-O-aminocaproyl- murabutide in a saline solution.
  • Control mice received 50 ⁇ g of PEP-TT without the adjuvant.
  • the animals were bled 2 weeks after the primary immunizations. On day 25, they received a booster injection. Finally, blood samples were taken after delays of 7, 14 and 123 days respectively after the recall.
  • the sera collected from animals in each of the groups considered were pooled and tested for their ability to induce in vivo production of anti-peptide and anti-tetanus antibodies in diagnostic tests involving the ELISA method.
  • the native protein was recognized by anti-peptide antibodies in radioimmunoassays (from the radioimmunoassay: RIA) using sporozoite extracts fixed on plastic plates, according to the method of Vanderberg et al. ., 1969, and by tests involving antibodies and indirect fluorescence (indirect fluorescence antibody test: IFA) according to the method Gvadz et al., 1979.
  • the reaction of circumsporozoites (CSP) according to Nordin et al., 1979, was used to test the biological activity of anti-PEP antibodies. Antibody production in vitro.
  • Spleen cells were obtained from mice. These cells, obtained under aseptic conditions, were washed and suspended at a concentration of 10 7 cells / ml in the medium known under the designation RPMI 1640 manufactured by the company SEROMED, containing 2 g / 1 of NaHCO 3 of glutamine ( 2 mM), 100 units / ml of penicillin, 100 ⁇ g / ml of streptomycin, 5% fetal calf serum and 2-mercaptoethanol (2x10 -5M), the suspensions were divided into aliquots of 100 ⁇ l. These were placed in the wells of 96-well tissue culture plates (Costar).
  • mice splenic cells in contact with a PEP-TT-6-O- ( ⁇ -amino-caproyl) -murabutide conjugate.
  • PEP-TT-6-O- ( ⁇ -amino-caproyl) -murabutide conjugate The ability of mouse spleen cells to produce anti-TT antibodies in vitro following stimulation with PEP-TT or free TT was tested, due to the particularly low level of circulating anti-TT antibodies in these animals, when these had been treated with conjugates containing murabutide.
  • the results are shown in Table V.
  • the supernatants of these cultures were tested in the ELISA system. They reveal that only splenic cells of groups 4 and 5 produced anti-TT antibodies, after being stimulated either with TT or with PEP-TT in vitro.
  • 6-O- ( ⁇ -amino-caproyl) -murabutide with tetanus toxin may make the toxoid sufficiently hydrophobic for it to be slightly masked with respect to the immune system, while on the contrary the peptide is more exposed and sensitive to the adjuvant action of muramyl-peptide. It is perhaps because of the hydrophilic nature of MDP-lysine that the adjuvant effect with respect to the toxin is added to the observable adjuvant effect with respect to the peptide.
  • the triple conjugates according to the invention have an immunogenicity specifically directed against hapten.
  • the invention therefore also relates to pharmaceutical compositions in which the above-defined triple conjugates are associated with a physiologically acceptable vehicle.
  • compositions consist of injectable solutions containing an effective dose of at least one triple conjugate according to the invention.
  • these solutions are produced in an isotonic sterile aqueous phase, preferably saline or glucose.
  • the invention also relates to compositions in which these triple conjugates are encapsulated in liposomes capable of forming injectable suspensions.
  • the invention relates more particularly to such solutions or suspensions which are capable of being administered by intradermal, intramuscular or subcutaneous, intravenous injections or even by scarification.
  • the invention also relates to pharmaceutical compositions which can be administered by other routes, in particular by the oral or rectal route, or also in forms intended to come into contact with mucous membranes, in particular the ocular, nasal, pulmonary or vaginal mucous membranes.
  • compositions in which at least one of the triple conjugates according to the invention is associated with pharmaceutically acceptable excipients, solid or liquid, suitable for the constitution of oral, ocular or nasal administration forms, or with excipients adapted for the constitution of rectal administration forms. It also relates to compositions intended for the pulmonary route, in particular solutions prepared for administration by means of a conventional aerosol device.
  • doses which can be administered, in order to stimulate the immune responses of the host against determined antigens, mention will be made of doses, in micrograms per kg of body, for example from 50 to 1000, preferably from 100 300, ⁇ g / kg, when administered parenterally, or 0.5-10 mg / kg orally.
  • Preferred vaccine compositions according to the invention further contain a vehicle, such as polyvinylpyrrolidone, facilitating the administration of the vaccine.
  • a vehicle such as polyvinylpyrrolidone
  • polyvinylpyrrolidone any other type of adjuvant in the conventional sense that was formerly given to this expression, that is to say a substance allowing the easier absorption of a drug. or facilitating its action in the body.
  • adjuvants of the latter type mention will also be made of carboxymethylcellulose, aluminum hydroxides and phosphates or any other adjuvants of this type, well known to those skilled in the art.
  • the pharmaceutical compositions according to the invention are therefore essentially intended to induce in the host an immune reaction, allowing him to produce antibodies or cell-mediated immunity with respect to an antigen containing the antigenic site specifically carried by the hapten entering the triple conjugate according to the invention.
  • the invention also relates more particularly to another type of pharmaceutical composition useful in human or veterinary therapy, whenever a change in the evolution or the behavior of the human or animal organism is sought, for example at the level of fertility. of the species or of the directed orientation of its metabolism towards the suppression of certain hormones. In preferred cases of the latter type, the hapten entering the conjugate used consists of the hormone itself or a fragment thereof.
  • the subject of the invention is in particular veterinary compositions containing effective doses of the above triple conjugates, in which the hapten itself consists either of the hormone itself, or of a peptide sequence having a common part with the hormone , the hormone belonging to the class of peptide or glycopeptide hormones.
  • a particularly interesting hormone in this regard is the release factor of the luteostimulating hormone LH-RH.
  • Effective unit doses of these conjugates are, for example, between approximately 50 and 1000, preferably 100 to 300, ⁇ g / kg of body, when administered parenterally, and between approximately 0.5 and 10 mg / kg when administered orally.
  • the invention also also relates to biological reagents essentially formed from these triple conjugates.
  • the haptenic part can consist of an active principle of medicament or drug (for example morphine) or also the expression product, of an oncogene.
  • These biological reagents can then be used to make antibodies, in particular monovalent or monoclonal antibodies. These antibodies are then in their turn useful for the constitution of reagents allowing for example the study of the action of drugs in question on the living host. In particular, these antibodies will allow the detection of cellular receptors, or the study of the metabolism and the mode of action of the drugs in question.
  • the invention is in no way limited to those of its more specially indicated embodiments, on the contrary, it embraces all variants, in particular those where muramyl- peptide entering into the triple conjugates of the invention would carry substituents distinct from those which have been indicated, insofar as these substituents would not however cause any modification of the above-mentioned behavior of the muramyl-peptide thus modified inside the triple conjugates here considered.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
EP19860903429 1985-06-10 1986-06-10 Produkte aus der kupplung eines makromolekularen trägers, eines haptens und eines muramylpeptids Withdrawn EP0224540A1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR8508769A FR2583049B1 (fr) 1985-06-10 1985-06-10 Produits resultant de la conjugaison d'un support macromoleculaire, d'un haptene et d'un muramyl-peptide par l'intermediaire d'un groupement substituant la partie saccharidique du muramyl-peptide et compositions selectivement immunogenes contenant ces produits
FR8508769 1985-06-10

Publications (1)

Publication Number Publication Date
EP0224540A1 true EP0224540A1 (de) 1987-06-10

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EP19860903429 Withdrawn EP0224540A1 (de) 1985-06-10 1986-06-10 Produkte aus der kupplung eines makromolekularen trägers, eines haptens und eines muramylpeptids

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EP (1) EP0224540A1 (de)
JP (1) JPS63500515A (de)
FR (1) FR2583049B1 (de)
WO (1) WO1986007365A1 (de)

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2428050A1 (fr) * 1978-06-05 1980-01-04 Anvar Oligomeres de composes du type muramyl-peptide et medicaments les contenant
FR2446292A1 (fr) * 1979-01-12 1980-08-08 Anvar Muramyl-peptides fixes sur polymeres peptidiques et medicaments les contenant
US4284537A (en) * 1980-07-03 1981-08-18 The United States Of America As Represented By The Department Of Health And Human Services Conjugate of streptococcal M protein peptide vaccine
FR2569984B1 (fr) * 1984-09-12 1987-08-14 Anvar Molecule synthetique contenant une pluralite d'epitopes distincts, procede pour son obtention et application a la production de polyvaccins

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8607365A1 *

Also Published As

Publication number Publication date
JPS63500515A (ja) 1988-02-25
WO1986007365A1 (fr) 1986-12-18
FR2583049B1 (fr) 1988-08-05
FR2583049A1 (fr) 1986-12-12

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