FI94419B - Method for preparing a therapeutically useful membrane anchor-drug conjugate - Google Patents

Abstract

Conjugates are described in which the active ingredient is covalently bonded to the anchoring membrane, which is a compound of the formulae <IMAGE> in which A can be: sulphur, oxygen, disulphide (-S-S-), methylene (-CH2-) or -NH-; n = 0 to 5; m = 1 or 2; C* = an asymmetric carbon atom of R or S configuration, R, R' and R'' are identical or different and are an alkyl, alkenyl or alkynyl group having 7 to 25 carbon atoms or are hydrogen which can optionally be substituted by hydroxyl, amino, oxo, acyl, alkyl or cycloalkyl groups and R1 and R2 are identical or different and are defined as R, R' or R'' or can be -OR, -OCOR, -COOR, -NHCOR or -CONHR and X is an active ingredient or a spacer active ingredient group. The anchoring membrane active ingredient conjugates increase antibody formation.

Description

1 94419

Method for preparing a therapeutically useful membrane anchor-ri active ingredient conjugate

The invention relates to a process for the preparation of membrane anchor-active substance conjugates comprising at least one active substance covalently bound to a membrane anchor compound or compounds, to a process for their preparation and to their use.

Membrane anchor compounds are compounds that are sealable to biological and artificial membranes.

Such membrane anchor compounds can be, for example, natural merabran lipoproteins that have already been isolated from the outer membrane of Escherichia coli and are now being synthesized. The E. coli membrane anchor compound consists in the N-terminal region of three fatty acids associated with S-glyceryl L-cysteine (G. Jung et al., "Peptides, Structure and Function", V. J. Hruby and D. H.

Rich, pp. 179-182, Pierce Chem. Co., Rockford, Illinois, 20 1983).

Conformation-stabilized α-helix polypeptides have been described as a model for studies of biological membranes, including alamethicin, an α-helical amphiphilic eicosapeptide antibiotic that forms voltage-gated ion-conducting systems on the lipid-25 membrane (Boheim, G., Hanke, Hanke, Hanke). , G., Biophys. Struct.

Mech. 9, pages 181-191 (1983); Schmitt, H. and Jung, G., Liebigs Ann. Chem., Pp. 321-344 and 345-364 (1985)).

EP-A-330 discloses that lipopept-30 tides, which have been analogues of the E. coli lipo protein known since 1973, are immunopotentiating. Subsequent EP-A-114 787 deals with the ability of such lipopeptides to activate rat and mouse alveolar macrophages in vitro, so that after 24 hours of incubation with the substance they can eliminate tumor cells and in particular significantly increase the production of antibodies, e.g. against.

EP-A-114 787 proposes the use of these lipoprotein derivatives as adjuvants for immunization, i.e. the use of lipoprotein derivatives of E. coli membrane protein in admixture with antigens to enhance the immune response.

There is a great need for agents that stimulate and enhance the immune response, especially as purified antigens are often only available in very small amounts; in addition, when new batches of antigen are used, it is always possible to introduce new impurities or degradation products.

In addition, it is desirable that the test animal should not be vaccinated frequently, but that, where possible, a single administration of the immunogenic material would elicit the desired immune response.

It is therefore an object of the present invention to seek to increase the formation of antibodies against antigens or hapten and thus to obtain a specific immunopotentiating effect.

A solution to this object is a novel membrane anchor active substance conjugate prepared according to the invention, comprising at least one membrane anchor compound and at least one active substance covalently bound to the membrane anchor compound or compounds.

FI patent publication 66878 discloses a conjugate of an antigen and an adjuvant (muramyl peptide derivative). However, said adjuvant is not a membrane anchor.

FI patent publication 83527 and FI patent applications 781241: and 781933 relate to the use of lipoprotein derivatives of E. coli coat protein as adjuvants or immunostimulants, respectively; however, it cannot be concluded from the data presented that these compounds could be part of the structure of the membrane anchor-drug conjugate.

> · I 3 94419

The novel membrane anchor-drug conjugates prepared according to the present invention lead to a new principle of action, which, in simple terms, is that the organism is exposed to a particular antigen for a longer period of time under the action of the membrane-5 anchor, leading to specific antibody formation.

The invention thus relates to a process for the preparation of membrane anchor compounds, which process is characterized in that a peptide whose functions are protected in a manner known per se by protecting groups which must not undergo any reaction is synthesized by a known coupling method on a solid or soluble support, e.g. resin) that the carrier-bound peptide thus synthesized is covalently linked via the N-terminus or side function of the peptide to a membrane anchor-active substance conjugate having the general formula: R -C0-O-CH R-O-CH , R - O-CO-CH, I | z I 2 20 R'-C0-0-CH * R * -0-CH * R'- O-CO-CH ·

i i I

(CH,) (CH,) (CH,) n l 2 n | 2 n i 2 n

A A A

(ch,) (ch, j (ch,) I 2 Π * | 2 H »I 2 n

R "-C0-NH-CH * -C0-X R" -C0-NH-CH * -C0-X R "-C0-NH-CH" -C0-X

25 I. II. III.

R -NH-CO- ^ H2 R- co-nh-ch2 R * -NH-CO-CH * R'-CO-NH-CH * (CH,) (CH,)? I 2 n l 2 n i A f? (CH,) (CH-) _ (CH,) - i 2 a 2 m | 2 m

JU R * * -CO-NH-CH1 · -CO-X R * • -C0-NH-CH - C0-X R-NH-C0-CH * -CC-X

IV. V. VI.

RrCH2 R, -CH *

4 I

TR (CH,)

Jo I 2 n

A

(CH,)., I 2 ra

R-C0-NH-CH "-C0-X

VII.

4,54419 wherein A is sulfur, oxygen, disulfide (-S-5-), methylene (CH 2 -) or -NH-; B is a group -S (CH 2) n - (substituted alkyl), wherein substituted alkyl is any substituted alkyl of the formula - (CH 2) n - represented by formulas I to V; n 5 = 0 - 5; m is 1 or 2; C * is an asymmetric carbon atom having the configuration R or S; R, R ', R "are the same or different and denote a hydrogen atom or an alkyl, alkenyl or alkynyl group having 7 to 25 carbon atoms, which may be substituted by hydroxy, amino, oxo, acyl, alkyl or cycloalkyl groups, and R 1 and R 2 are the same or different and may represent the same as R, R 'or R "or the groups -OR, -OCOR, -COOR, -NHCOR, -CONHR, and X is an active ingredient or a spacer active ingredient group , except for compounds wherein X is an amino acid sequence of 2 to 10 natural amino acids, that the peptide conjugate thus prepared is isolated by deprotection and peptide / carrier bond in a manner known per se to give a membrane anchor peptide or a membrane anchor-active agent conjugate, the active substance is an antigen, such as a lower molecular weight subsequence of a protein or protein, for example a glycoprotein, a viral envelope protein, a bacterial cell wall protein or a protozoal protein gene determinant, epitope), bacterial membrane component such as muramyl 25 dipeptide, lipopolysaccharide, natural or synthetic hapten, antibiotic, hormone, nucleoside, nucleotide, nucleic acid, enzyme, enzyme substrate, enzyme substrate, enzyme, enzyme inhibitor, enzyme inhibitor , such as tuftsin, polylysine, fluorescent marker, FITC, RITC, dansyl, luminol, or ··. coumarin, bioluminescence marker, spin marker, alkaloid, steroid, biogenic amine, vitamin, toxin such as digoxin, phalloidin, amanitine, tetrodoxin and the like, complexing agent or drug.

• · 5 94419

The compounds of the invention can be used to prepare conventional and monoclonal antibodies in vivo and in vitro; however, they can also be advantageously used in genetic engineering to facilitate cell fusion, in the preparation of synthetic vaccines, in the preparation of cell markers with fluorescent markers, spin markers, radiolabels and other markers, as animal feedstocks, in addition to microbial culture media and cell cultures in general. The compounds according to the invention can then optionally be used for human and veterinary use in combination with carriers known per se in solutions, creams, adsorbed on solid carriers, emulsions or spray preparations.

As the membrane active substance conjugate, a compound of the general formula R4 R3-NH-CH-CO-X (VIII) can also be advantageously used. wherein R 1 is an α-acyl fatty acid residue having 7 to 25 carbon atoms, preferably 10 to 25 carbon atoms and particularly preferably 14 to 18 carbon atoms; an α-alkyl-β-hydroxy fatty acid residue or a β-hydroxyester thereof, wherein the ester moiety is preferably straight-chain or branched and contains more than 8 carbon atoms, preferably 10 to 20 carbon atoms and particularly preferably 14 to 18 carbon atoms; preferably the compound of formula VIII is an active ingredient conjugate wherein the anchor compound is one of the following: Ν, Ν'-diacylalysine; N, N'-diacyl ornithine; glutamic acid di (monoalkyl) amide or ester; aspartic acid di (monoalkyl) amide or ester; 6 · 54419 N, O-diacyl derivative of serine, homoserine or threonine or serine or homoserine of N, S-diacyl derivative of cysteine or homocysteine; R 4 is an amino acid side chain or a hydrogen atom; and X is an active ingredient or a spacer-5 active ingredient group, wherein when R4 is a side chain of lysine, ornithine, glutamic acid, aspartic acid or a derivative thereof, it may be attached at the α- or β-position by both an ester and amide bond to the same R4 molecule .

Coupling of the peptide and membrane anchor compound can be accomplished by a condensation, addition, substitution, or oxidation reaction (e.g., disulfide formation). As the membrane anchor compounds, conformationally stabilizing α-alkyl amino acid helices with alternating amino acid sequences are preferably used, so that the α-helix must not be destabilized by other amino acids, such as those of type X- (Ala-Aib-Ala-Aib-Ala) nY, where n = and X and Y are protecting groups known per se or -H, -OH or -NH2.

It may be advantageous for the active ingredient to be covalently bound to two, possibly different, membrane anchor compounds.

In addition, the active ingredient may also be covalently bound to an adjuvant used for immunization purposes known per se, such as, for example, muramyl dipeptide and / or lipopolysaccharide.

Depending on the nature of the active substance, completely new fields of application of the substances prepared according to the invention are obtained.

It may also be useful to crosslink several membrane anchor-drug conjugate compounds together in the lipid and / or drug component.

The membrane anchor compounds and the active substance can also be linked to one another via a crosslinking agent, as a result of which the active substance is further away from the membrane to which it is attached via the membrane anchor.

• «7 £ 4419

Suitable crosslinking agents are, for example, dicarboxylic acids or dicarboxylic acid derivatives, diols, diamines, polyethylene glycols, epoxides, maleic acid derivatives and the like.

According to the invention, an unambiguously defined, lower molecular weight conjugate is thus prepared which is suitable, inter alia, for immunization and which covalently links the carrier-antigen-adjuvant principles. The carrier and adjuvant may be mitogenically active lipopeptides, such as tripalmitoyl-S-glyceryl cysteine (Pam3Cys), or analogs thereof, or also lipophilic conformationally stabilized α-helixes, and combinations thereof, such as the Pam3Cys antigen helix, α-helix. antigenic helix or also only Pam3Cys antigen or antigen-15-N3-Pam3Cys (N- or C-terminal junction) and antigenic helix or helix-antigen (N- or C-terminal or junction of the helix Glu, Lys or the like side chain). Thus, the novel compounds differ substantially in all of the higher molecular weight conjugates of antigens used to date with higher molecular weight carriers, for example a protein such as serum albumin, globulin or polylysine, or a generally defined higher molecular weight, linear or crosslinked.

However, in particular, the new compounds also differ from all hitherto known adjuvants which are merely mixed and thus do not allow the directed presence of the antigen on the cell surface. The use of hitherto known adjuvants required considerably more immunizations, and inflammatory reactions were also obtained in animal experiments. A particular advantage of the invention is the possibility of reproducibly preparing pyrogen-free, pure, chemically unambiguously defined compounds which - in contrast to conventional compounds or Thus, the compounds of the invention have specific applications in the fields of antibody formation, genetic engineering, synthetic vaccines, veterinary and human medicine, diagnostics and therapy, as the new conjugates have, for the first time, been shown to provide better reproducibility of antibody formation. stimulating effect of a specific immune response when the adjuvants used heretofore only non-specifically stimulate the immune response. weakly immunogenic compounds can also be converted to highly immunogenic. Thus, the invention is of particular importance in that it makes it possible to save on animal experiments as well as on the cost of producing antibodies, since the new immunogens are also highly active in vitro. When the immunization method does not cause inflammation, the animal can also be used multiple times to generate various antibodies.

In addition, novel immunogens can also be used to prepare multi-potent vaccines, i.e., multiple antigens or haptens can be attached to the side chains of the membrane anchor so that a variety of active antibodies can be prepared during immunization.

The water-soluble, mitogenic lipid anchor group is, for example, Pam3Cys-Ser (Lys) n-OH, which is particularly suitable for the production of new immunogens, but also for the production of fluorescent, radioactive and biologically active cell markers. A particularly desirable property of the membrane anchor active agent conjugates prepared according to the invention is their amphiphilicity, i.

30 partial water solubility, since biological animal experiments and studies with living cells can then be performed substantially more simply. Also, artificial lipid bilayer membranes, liposomes, and vesicles, which are required in some experiments, can be prepared (and are stable) only in aqueous media.

• «9. 94419

A suitable axially specific, biologically active membrane anchor is, for example, Pam3Cys-Ser (Lys) n-CH. The serine residue associated with Pam3Cys-ket favors immunogenic properties and the polar, protonated ε-amino groups of the lysine residue form the hydrophilic portion of the molecule. This type of compound also has other interesting properties due to its multiple charges. It can be used as a fusion activator in the production of hybridoma cells by induction of cell interaction, especially in the case of a long lysine chain, for coupling to polyethylene glycol or for covalent attachment of a biotin / avidin system.

Furthermore, the compounds according to the invention can advantageously be used for the preparation of novel liposomes by crosslinking, which crosslinking takes place in either the fatty acid or peptide moiety.

Membrane anchors (Pam3Cys and analogs and helices) are also suitable for enhancing cell / cell interactions when covalently linked to, for example, biotin / avidin systems. In addition, advantageous properties of the compounds of the invention include their ability to facilitate cell fusions, which are required, for example, in genetic engineering work. In this case, the new immunogens can also be used in ELISA, RIA and bioluminescence experiments.

Pam3Cys derivatives are lipid and water soluble and strongly mitogenic in vivo and in vitro. They are also quite well suited for labeling cells with FITC and other markers such as RITC, dansyl and coumarin. In particular, they can also be used in fluorescence microscopy and FACS (Fluorescence Activated Cell Sorting).

A low-cost membrane anchor having an effect analogous to Pam3Cys is S- (1,2-diox-35-tadecyloxycarbonylethyl) cysteine, the preparation of which is described in more detail in the examples.

• <ίο £ 4419

The specific coupling of mitogenically active lipid anchors to antigens can also take place via a crosslinking agent, such as a dicarboxylic acid monohydrazide derivative of the general formula:

X-NH-NH-CO-A-CO-B-Y

or also X-NH-NH-CO-A-COOH, in which formulas A and B are amino acids or (CH2) n, and X and Y are protecting groups known per se.

Any other suitable crosslinking agent or spacer may also be used in the preparation of the novel substances, the principle (low molecular weight strain 15 and adjuvant) - (antigen) representing a particularly preferred form of the invention, provided that it contains lipopeptide structures with lipid membrane function and / or or conformationally stabilized helices.

Particularly advantageous effects of the use of the compounds according to the invention are obtained in affinity chromatography, in which lipopeptide-antigen (hapten) conjugates are particularly suitable. These are remarkably well suited for filling available reverse phase HPLC columns (or also preparative reverse phase columns) whereby, for example, the tripalmitoyl compound contained in the organic-aqueous system is anchored to the apolar alkyl layer. The antigen remains in the mobile aqueous phase on the cell surfaces and is thus ready to adsorb the antibodies. Thus, from the diluted serum, antibodies can be directly enriched or isolated on such an Affinity column. The elution of antibodies takes place as in other affinity columns, for example by adjusting the pH.

i · t

The invention is further illustrated by the following examples, in which the abbreviations used herein have the following meanings: 11 £ 4419

Aib = 2-methylalanine TFE = trifluoroacetic acid EGF R = epidemic growth factor receptor Pam = palmitoyl residue 5 DOC = dicyclohexylcarbodiimide DMF = dimethylformamide FITC = fluorescein-isothiocyanate Fmoc = fluorenylmethoxycarbonyl copolymer with 4- (hydroxymethyl) phenoxymethyl anchor groups HOBt = 1-hydroxybenzotriazole RITC = rhodamine isothiocyanate

Hu IFN- (Ly) 11-20 = antigenic ether-15 minant of human interferon DCH = Ν, Ν'-dicyclohexylurea EE = ethyl acetate

The accompanying figures, which illustrate the invention, show: Figure 1:

Preparation scheme for Pam-Cys (C18) 2-Ser-Ser-Asn-Ala-OH Figure 2: Table of 13 C-NMR spectra Figure 3: 25 Pam3Cys-Ser- (Lys) 4-0H x 3 TFA I3C- NMR spectrum in CDCl 3

Figure 4:

13 C-NMR spectrum of Pam-Cys (Pam) -OBu * in CDCl 3 Figure 5:

13 C-NMR spectrum of Pam-Cys (Pam) -OH in CDCl 3 / CD 3 OD 1: 1. Figure 6: 13 C-NMR spectrum (J-modulated spin-echo spectrum) of Pam (a-Pam) Cys-OBu '

Figure 7: 13 C-NMR spectrum (100 MHz) of α-helix 12,94419

Figure 8:

CD spectrum of the α-helix of HuIFN- (ot-Ly) -11-20 Figure 9:

Preparation of antibodies with Pam3Cys-Ser-EFG-R (516-529) 5 Figure 10:

In vivo immunization test Figure 11:

Comparison of in vivo / in vitro immunization experiments Figure 12: 10 Mitogenic activation of Balb / c mouse spleen cells by Pam3Cys

With Ser- (Lys) 4FITC.

The following is a description of some of the methods for preparing the substances of the invention and these precursors.

I. Preparation of Pam3Cys-EGF-R (516-529)

By a conventional stepwise construction method (Merrif ield synthesis, nitrogen shielding gas, aFmoc / (Bu ') protection, DCC / HOBt and symmetrical anhydrides), the EGF-R segment (526-529) was assembled and 20 Fmoc-Ser (Bul) was added as the last amino acid. OH. After cleavage of the Fmoc group (piperidine / DMF 1: 1, 15 min), the resin-bound pentadecapeptide EGF-RH: Ser (Bul) -Asn-Leu-Leu-Glu- (OBu *) -Gly-Glu (OBu1) - Pro-Arg- (H +) -Glu (OBu ') -Phe-Val-Glu (OBu *) -Asn-Ser (Bu') -Op-alkoxy-benzyl copoly (divinyl-benzene styrene) (1 g, batch 2 5 0 , 5 mmol / g) was combined with Pam-Cys- (CH 2 -CH (OPam) CH 2 - (OPam) (2 mmol, DMF / CH 2 Cl 2, 1: 1) and DCC / HOBt (2 mmol, 0 ° C, 20 min preactivated) (16 h), then post-coupling (4 h) The lipohexadecapeptide was cleaved over 2 h with trifluoroacetic acid (5 mL) with the addition of thioanisole: 30 (0.25 mL).

Yield: 960 mg (76%) of Pam-Cys (CH2-CH (OPam) CH2 (OPam)) Ser-

Asn-Leu-Leu-Glu-Gly-Glu-Pro-Arg-Glu-Phe-Val-Glu-Asn-Ser-OH x CF3COOH (correct amino acid analysis, no racemization).

• <94419 13 II. Preparation of S- (1,2-dioctadecyloxycarbonylethyl) -N-palmitoyl-L (or D) -cysteine tert-butyl ester

Maleic acid dioctadecyl ester is obtained by general esterification methods of maleic acid (H. Klostergaard, J. Org. Chem. 23 (1958), 108).

13 C-NMR spectrum:

See Figure 2.

1.2 mmol (500 mg) of N-palmitoyl-L-cysteine tert-10 butyl ester and 1.2 mmol (745 mg) of maleic acid dioctadecyl ether are dissolved in 20 ml of THF. 20 mmol (3 ml) of Ν, Ν, Ν1, N'-tetramethylenediamine are added and the mixture is refluxed under nitrogen for 12 h. 100 ml of methanol and 5 ml of water are added, the colorless precipitate is filtered off with suction, washed with water and methanol and dried in vacuo over P2O5.

Yield: 1 g (83%). Melting point: 51 ° C.

Thin layer chromatography:

Rf = 0.80 (solvent: CHCl 3 / ethyl acetate 14: 1) 13 C-NMR: See Figure 2. Molecular weight: C 63 H 11 NO 7 S (1035.7).

Elemental analysis:

Calculated: C 72.99, H 11.76, N 1.35, S 3.09%

Found: C 73.08, H 11.92, N 1.27, S 3.27% • III. Preparation of S- (1,2-dioctadecyloxycarbonylethyl) -N-palmitoyl cysteine 0.48 mmol (500 mg) of the tert-butyl ester described in II and 65.3 mmol (7.45 g, 5 ml) of trifluoroacetic acid are mixed at room temperature in a sealed vessel for 1.30 h. The mixture is evaporated on a rotary evaporator under high vacuum, the residue is dissolved in 1 ml of chloroform, the product is precipitated by adding 50 ml of petroleum ether at -20 ° C and dried in vacuo over P 2 O 5.

Yield: 420 mg (89%). Melting point: 64 ° C.

35 Thin layer chromatography on silica gel plates:

Rf = 0.73 (solvent: CHCl 3 / MeOH / H 2 O * 65: 25: 4).

14. £ 4419, 3 C-NMR: See Table 1. Molecular Weight: C59Hn3NO7S (980.6) Elemental Analysis:

Calculated: C 72.27, H 11.62, N 1.43, S 3.27%

Found: C 72.46, H 11.75, N 1.36, S 3.50% The new cysteine derivative and its tert-butyl ester can be separated into diastereomers by silica gel and reverse phase chromatography. Thus, both diastereomeric pairs of the L- and D-cysteine derivative can be prepared.

IV. Preparation of S- (1,2-dioctadecyloxycarbonylethyl) -N-pal-10 mitoyl-Cys-Ser (Bul) -Ser (Bul) -Asn-Ala-OBu '0.2 mmol (196 mg) of S- (1 , 2-Dioctadecyloxycarbonylethyl) -N-palmitoyl-cysteine is dissolved in 5 mL of dichloromethane and preactivated with HOBt (0.2 mmol, 27 mg) in 0.5 mL of DMF and DCC (0.2 mmol, 41 mg) with stirring at 150 ° C for 30 min. 0.2 mmol (109 mg) of H-Ser (Bu *) Ser- (Bu ') - Asn-Ala-OBu' in 3 ml of dichloromethane are added and the mixture is stirred for 12 h at room temperature. 40 ml of methanol are added to the reaction mixture without further treatment. After 3 h, the colorless product is filtered off with suction. It is dissolved in a small amount of dichloro-20 rimethane and reprecipitated with methanol, the precipitate is washed with methanol and dried in vacuo over P 2 O 5.

Yield: 260 mg (86%). Melting point: 194 ° C.

Thin layer chromatography: RP = 0.95 (solvent: CHCl 3 / MeOH / H 2 O = 65: 25: 4) 25 RF = 0.70 (solvent: CHCl 3 / MeOH / glacial acetic acid = 90: 10: 1) 13 C-NMR: See Figure 2. Molecular weight: CMH15gN6014S (1508.3). Elemental analysis:

Calculated: C 66.89, H 10.56, N 5.57%

Found: C 67.10, H 10.41, N 5.52%. 30 V. Preparation of S- (1,2-dioctadecyloxycarbonylethyl) -N-pal-: · mitoyl-Cys-Ser-Ser-Asn-Ala

The protected lipopeptide (IV) (53 μmol, 80 mg) is stirred with trifluoroacetic acid (13 mmol, 1.5 g, 1 mL) in a sealed vessel at room temperature for 1 h. The mixture is evaporated under high vacuum and the residue is dissolved in dichloromethane (£ 4419). 10 ml) and evaporated on a rotary evaporator, the same is repeated. The residue is dissolved in chloroform (3 ml) and the product is precipitated with methanol (5 ml) at 4 ° C over 12 h.

The product is filtered off with suction, washed with methanol and dried in a desiccator over P2O5.

Yield: 63 mg (87%). Melting point: 208 ° C (decomposes). Thin layer chromatography:

Rf = 0.63 (solvent: CHCl 3 / MeOH / glacial acetic acid / H 2 O = 64: 25: 3: 4)

Rf = 0.55 (solvent CHCl 3 / MeOH / H 2 O = 64: 25: 4) 10 RF = 0.06 (solvent: CHCl 3 / MeOH / glacial acetic acid = 90: 10: 1) Amino acid analysis:

Cysteic acid 0.6; aspartic acid 0.93; serine 1.8; alanine 1.0.

Molecular weight: C72H134N6014S (1340) 15 VI. Preparation of Pam3Cys-Ser- (Lys) 4-OH

Pam3Cys-Serp (Lys) 4-OH was assembled by the solid phase method (Merrifield) using p-alkoxybenzyl alcohol-PS-DVB (1%) copolymer with N-Fmoc amino acids and acid labile side chain protection (for serine tBu and lysine Boc). . Symmetrical anhydrides of Fmoc amino acids were used. Coupling to Pam3Cys-OH was performed by the DCC / HOBt method and repeated to obtain the most quantitative reaction possible. To cleave the lipopeptide from the support resin and to remove side chain protection, the resin was treated twice with 1.5 g of trifluoroacetic acid, which was then removed on a rotary evaporator under high vacuum. The product was recrystallized from acetone.

Elemental analysis as well as the 13 C-NMR spectrum show that the lipopeptide is in the form of trifluoroacetate. Assuming that: Pam3Cys-Ser- (Lys) 4-OH is a zwitterion, there are still three α-amino groups that can be protonated by 3 trifluoroacetic acid molecules.

The I3CNMR spectrum of Pam3Cys-Ser- (Lys) 4-OH x 3TFE shows that the compound is as trifluoroacetate (CF3 quaternary at 110-120 ppm and carbonyl signal 161-162

• I

16 in S4419 ppxn). Due to the aggregation of the polar moiety, the lines of the Lys and Ser-C atoms widen strongly. The carbonyl signal at 206.9 ppm is due to the product-associated acetone used in the recrystallization.

Molecular weight: 1510.4.

Elemental analysis:

Calculated: C 56.40, H 8.70, N 7.56, S 1.73%

Found: C 55.58, H 9.33, N 6.54, S 2.61% 10 Amino acid analysis:

Amino acid analysis showed a serine to lysine ratio of 1: 4.2. Typical degradation products from the hydrolysis of S-glyceryl cysteine (6-xn HCl, 110 ° C, 18 h) were present (comparison to a known standard). The peptide-15 diosa was calculated to be 83%. 3 TFE molecules per lipopeptide correspond to an 80.2% peptide fraction, which corresponds well to the analytical result.

VII. Preparation of Pam3Cys-Ser- (Lys) 4-OH x 3TFE VII.1. Coupling of Fmoc-Lys (Boc) -OH to carrier resin

To Fmoc-Lys (Boc) -OH (4.5 g, 9.6 mmol) in 15-20 mL of DMF / CH 2 Cl 2 (1: 1, v / v) is added DCC at 0 ° C. (0.99 g, 4.8 mmol). After 30 min, the precipitated urea is filtered and the filtrate is collected directly on a shaker with 25 p-benzyloxybenzyl alcohol resin (2.5 g, 1.6 mmol OH groups). Add pyridine (0.39 mL, 4.8 mmol) and shake for 18 h at room temperature. The solvent is filtered off and the resin is washed with DMF / CH 2 Cl 2 (3 x 20 mL). To the resin is added 20 mL of CH 2 Cl 2, then pyridine (28.8 mmol, 6 eq) and then benzoyl chloride (28.8 mmol, 6 · 1 eq). Shake at room temperature for 1 h. The solvent is removed by filtration and the resin is washed with CH 2 Cl 2 (3 x 20 mL), DMF (3 x 20 mL), isopropanol (3 x 20 mL) and PE-30/50 (3 x 20 ml).

17 - 94419 VII.2. Symmetrical Fmoc amino acid anhydride

Fmoc-Lys (Boc) -OH (4.5 g, 9.6 mmol, 3 eq) is dissolved in 15 mL of CH 2 Cl 2 / DMF, and DCC (4.8 mmol, 1.5 eq). After 30 min at 0 ° C, the urea is filtered and the filtrate is collected directly in the reactor, where it is further processed according to the following table.

The following instructions apply to 1/5 of the amount of resin used (0.5 g, 0.32 mmol OH groups).

Fmoc-O-butylserine (0.74 g, 1.92 mmol) is dissolved in 4 any CH 2 Cl 2 / DMF and 0 ° C is added.

DC in DCC (0.96 mmol).

• Λ 1 «ie 94419

Table

Assembly of the peptide sequence from symmetric Fmorc amino acid anhydride

Action Reagent Time Performance _ (min) _ number of 1 CH2C12 2 2 2 DMF 2 2 3 55% piperidine / DMF 5 1 (v / v) 4 55% piperidine / DMF 10 1 (v / v) 5 DMF 2 3 6 Isopropanol 5 2 7 DMF 2 3 8 CH2C12 2 3 9 DMF 2 2 10 Coupling symmetr. 90 1

Fmoc-amino acid anhydride (3 eq) in DMF / CH 2 Cl 2 (1: 1, v / v); After 15 min NMM addition (3 eq) 11 DMF 2 3 12 CH 2 Cl 2 2 3 13 Coupling completeness test (Kai-ser test); if necessary, repeat steps 10-12 14 Acetylation: 2 eq 15 1

Ac 2 O and 0.5 eq NMM

In 20 ml of CH2Cl2 15 CH2Cl2 2 3 16 Isopropanol 2 3 17 CH2Cl2 2 3 • «« After 130 min, the urea is filtered off at 0 [deg.] C., the filtrate is discharged directly into the reactor and worked up as usual.

19 · 94419 VII.3. Coupling to Pam3Cys-OH

Pam3Cys-OH (0.58 g, 0.64 mmol) is dissolved in 5 mL of CH2Cl2 / DMF (1: 1, v / v) and HOBt (93 mg, 0, 64 mmol) and DCC (0.64 mmol). After 30 min at 0 ° C, the mixture is introduced directly into the reactor. After shaking for 16 h, post-coupling is performed for 4 h at the same molar ratio. The solvent is filtered off and the resin is washed with DMF / CH 2 Cl 2 (3 x 20 mL).

VII. 4. Cleavage of the hexapeptide from the polymer 10 The Boc-protected peptide-polymer resin compound prepared in VII.3 (about 1 g) is washed well with CH 2 Cl 2 and shaken for 2 x 1.5 h with TFE (5 mL) and anisole (0.5 mL). with the mixture. The filtrate is evaporated in vacuo and the residue is dissolved in 5 ml of CHCl3. Pam3Cys-Ser (Lys) 4-OH x 3TFE 15 crystallizes on addition of 50 ml of acetone at -20 ° C, is centrifuged and dried under high vacuum.

Yield: 0.41 g (85%). Melting point: 205 ° C (decomposes). Thin layer chromatography on silica gel plates:

Rf = 0.42 (solvent: n-BuOH / pyridine / H 2 O / glacial acetic acid = 20 4: 1: 1: 2)

Rf = 0.82 (solvent: n-BuOH / MeOH / H 2 O / glacial acetic acid = 10: 4: 10: 6) Amino acid analysis: Ser 0.95 (1), Lys 4 (4)

Molecular weight: C87H159N10O19SF9 (1852.6).

‘25 Elemental analysis:

Calculated: C 56.40, H 8.70, S 1.73%

Found: C 55.58, H 9.33, N 6.94, S 2.61% VIII. Preparation of Pam3Cys-Ser- (Lys) 4-OH-FITC x 2 TFE

Fluorescein isothiocyanate (3.9 mg, 10 μιηοΐ) is dissolved in 2 ml of chloroform and the solution is added to a solution of Pam3Cys-Ser- (Lys) 4-0H x 3TFE (18.5 mg, 10 μιηοΐ) in 2 ml. of chloroform. Add 4-methylmorpholine (10 μΐ, 10 μιηοΐ) and stir for 1 h, then evaporate the solvent on a rotary evaporator. The residue is dissolved in 10 ml of a 1: 1 mixture of chloro-35 form / acetone. At -20 ° C a thick yellow precipitate precipitates, which is separated by centrifugation and dried under high heat.

Yield: 16 mg after purification with Sephadex LH20.

The product is in the form of trifluoroacetate and fluoresces very strongly with UV excitation at a wavelength of 366 nm. It differs from the starting material in that one ε-amino group is covalently attached to FITC. This results in the gross formula Pam3Cys-Ser- (Lys) 4-CH-FITC x 2TFE, where it is assumed that the zwitterionic structure is preserved.

Molecular weight: C106H, 69NnO22S2F6 (2127.68).

Thin layer chromatography on silica gel plate: RF = 0.72 (solvent: n-butanol / pyridine / water / glacial acetic acid = 4: 1: 1: 2)

Rf = 0.73 (solvent: n-butanol / formic acid / water = 7: 4: 2) Amino acid analysis: Ser 1.11 (1.00), Lys 4.00 (4.00) Glyceryl cysteine hydrolysis products are present.

IX. Preparation of Pam3Cys-Ser- (Lys) 4-OH x 3HCl Pam3Cys-Ser- (Lys) 4-OH x 3TFE (185.2 mg, 0.1 mmol) is dissolved in an amount of chloroform in which it is just soluble, and an equal volume of ethereal HCl solution is added to the solution. Shake well, precipitating some of the product but leaving most of it in solution. The mixture is evaporated to dryness on a rotary evaporator and HCl-ether solution is again added to the residue. The same is repeated several times, then the residue is dissolved in a small amount of chloroform, and acetone is added to the solution until turbidity begins.

The product crystallizes as a colorless powder at -20 ° C, is filtered off with suction and dried under high vacuum.

Yield: 153 mg. Molecular weight: CgiH159N.0.0.3SCl3 (1619.63).

30 Elemental analysis:

Calculated: C 60.07, H 9.89, N 8.65%

Found: C 57.64, H '11, 20, N 8.39%

Excess HCl remains in the product. Field desorption mass spectrometry: the 35 M + peak is at m / e 1510 and at M ++ l and

11 1 10.:1 ällll Iti UI I

21 - 94419 M ++ 2. Typical are the protonated fragments Pam3Cys-NH (908.5) at m / e 909, 910, 911 and 912.

X. N, S-Dipalmitoyl-cysteine tert-butyl ester Palmitic acid (2.5 g, 9.6 mmol), dimethylaminopyridine (130 mg, 0.9 mmol) and dicyclohexylcarbodiimide (9.6 mmol) are dissolved. To 100 ml of chloroform. The solution is stirred for 0.5 h and a pre-prepared solution of N-palmitoyl cysteine tert-butyl ester (2 g, 4.8 mmol) in 50 mL of chloroform is added dropwise. After 1.5 h, the solvent is evaporated off on a rotary evaporator and the residue is dissolved in 100 ml of a 1: 5 mixture of chloroform / methanol. The product precipitates as a thick precipitate at -20 ° C. It is filtered off with suction and dried under high vacuum.

Yield: 2.3 g (73%). Molecular weight: (mass spectrometer) 15 C39H75NO4S (655.20).

Elemental analysis:

Calculated: C 71.48, H 11.71, N 2.13, S 4.89%

Found: C 71.72, H 12.14, N 2.12, S 4.77%

Thin layer chromatography on silica gel plates: 20 RF = 0.67 (solvent: chloroform / ethyl acetate 95: 5)

Rf = 0.73 (solvent: chloroform / cyclohexane / MeOH 10: 7: 1) 13 C-NMR: See Figure 4.

XI. N- (α-tetradecyl-5-hydroxyoctadecanoyl) -cysteine-tert-butyl ester N- (α-palmitoyl-palmitoyl) -cysteine-tert-butyl ester (1.5 g, 2.3 mmol) is dissolved in 10 ml of: isopropanol and 1.5 times the molar amount of sodium borohydride is added to the solution. After stirring for 2 h, N2-saturated 1M hydrochloric acid is added to the mixture until hydrogen evolution ceases. In this case, the product precipitates as a thick precipitate. It is filtered off with suction, washed several times with saturated water and dried under high vacuum.

Yield: 1.4 g (93%). Molecular weight (mass spectral determination): 35 = 656.11.

22 94419

Thin layer chromatography on silica gel plates:

Rf = 0.84 (solvent: chloroform / ethyl acetate 95: 5). Elemental analysis:

Calculated: C 71.39, H 11.83, N 2.13, S 4.89% Found: C 71.32, H 12.39, N 2.04, S 5.33% XII. N- (α-tetradecyl-β-hydroxyoctadecanoyl) cysteine N- (α-tetradecyl-β-hydroxyoctadecanoyl) cysteine tert-butyl ester (1 g, 1.5 mmol) is treated with anhydrous trifluoroacetic acid for 0.5 h, then The TFE is immediately removed on a rotary evaporator under high vacuum. The residue is dissolved in tert-butanol and lyophilized.

Yield: 0.7 g (78%). Molecular weight (mass spectral determination): C35H69NO4S = 600.0.

15 Elemental analysis:

Calculated: C 70.06, H 10.92, N 2.33, S 5.34%

Found: C 70.36, H 10.44, N 2.45, S 5.01%

Thin layer chromatography on silica gel plate: RF = 0.43 (solvent: chloroform / methanol / water 65: 25: 4). N, S-dipalmitoyl-cysteine N, S-dipalmitoyl-cysteine tert-butyl ester (1 g, 1.5 mmol) is treated with anhydrous trifluoroacetic acid for 1 h. The TFE is removed on a rotary evaporator under high vacuum, the residue is dissolved in tert-butanol and lyophilized.

* 25 Yield: 0.8 g (39%). Molecular weight (mass spectral determination): C35H67NO4S (598.00).

Elemental analysis:

Calculated: C 70.18, H 11.44, N 2.34, S 5.34%

Found: C 69.97, H 11.31, N 2.50, S 5.17% 30 TLC: RF = 0.30 (solvent: chloroform / methanol / glacial acetic acid 90: 10: 1) RF = 0, 75 (solvent: chloroform / methanol / water 65: 25: 4)

Rf = 0.81 (solvent: chloroform / methanol / ammonia (25%) / water 65: 25: 3: 4).

35, 3 C-NMR: See Figure 5.

23 9 4 4 1 9 XIV. N- (α-palmitoyl-palmitoyl) -N7-palmitoyl-cysteine-di-tert-butyl ester

Palmitoyl chloride (8 g, 30 mmol) is dissolved in 40 mL of N2-saturated dimethylformamide and triethylarinoine (60 mL, 60 mmol) is added to the solution. The mixture is refluxed under nitrogen for 3 h, whereupon the triethylammonium chloride formed when the tetradecyl ketene dira is formed precipitates as a colorless salt. The reflux condenser is then replaced with a dropping funnel and a solution of cysteine di-tert-butyl ester (4.9 g, 15 mmol) in 20 mL of dimethylformamide is slowly added dropwise to the mixture. After 6 h, the solvent is removed on a rotary evaporator, the residue is dissolved in chloroform and washed with 5% potassium hydrogen sulfate solution (2 x 100 mL) and water (1 x 1200 mL). The organic phase is dried over anhydrous sodium sulfate and the solvent is removed again. At -20 ° C, a mixture of N- (α-palmitoyl-palmitoyl) -N'-palmitoyl-cysteine-tert-butyl ester and N, N7-dipalmitoyl-cysteine-di-tert-butyl ester crystallizes, which is separated by gel filtration on Sephadex LH -20 in chloro-20 form / methanol 1: 1.

Yield: 6.4 g (40%). Molecular weight (mass spectrum): C62H18N2O7S (1067.76).

. Thin layer chromatography on silica gel plates: RF = 0.69 (solvent: chloroform / ethyl acetate 91: 5).

25 XV. N- (α-palmitoyl-palmitoyl) -cysteine-tert-butyl ester N- (α-palmitoyl-palmitoyl) -N7-palmitoyl-cysteine-di-tert-butyl ester (3.2 g, 3 mmol) is dissolved in a small amount. methylene chloride and 100 ml of 9.1 M HCl-methanol solution are added to the solution. The solution is transferred to an electrolytic cell having a silver electrode as the anode and mercury as the cathode and reducing it at a constant voltage of -1.1 V. At the end of the electrochemical reduction, the current decreases from about 200 mA to almost 0. The solvent 35 is then removed on a rotary evaporator and the product mixture of N- (α-palmitoyl palmitic) -cysteine tert-butyl ester and N-palmitoylcysteine tert-butyl ester is precipitated from methanol at -20 ° C. Both compounds are separated by gel filtration on Sephadex LH-20 in chloroform / methanol-5 1: 1.

Yield: 1.5 g (76%). Molecular weight (mass spectral determination) C39H75NO4S (654.09).

Thin layer chromatography on a silica gel plate:

Rf = 0.75 (solvent: chloroform / ethyl acetate 95: 5) 10 Elemental analysis:

Calculated: C 71.48, H 11.71, N 2.13, S 4.89%

Found: C 71.16, H 11.31, N 2.00, S 4.65% XVI. Preparation of Boc-Asn-Arg (NO 2) -Arg (NO 2) -OH XVI. 1. Boc-Arg (NO 2) -OMe Boc-Arg (NO 2) -OMe (15.97 g, 50 mmol), HOBt (6.6 g, 50 mmol) and DMF (100 mL). ) is added at -10 ° C HCl x H-Arg (NO 2) -OMe (13.49 g, 50 mmol), NMM (5.5 mL, 50 mmol) and CH 2 Cl 2 (12 mL). ), the mixture is stirred at -10 ° C for 30 min, at 0 ° C for 1 h and at room temperature for 3 h.

20 The reaction is then stopped by adding a few drops of glacial acetic acid. The precipitated DCH is filtered off and the solvent is evaporated off under high vacuum. The oily residue is dissolved in ethyl acetate to which some has been added. n-butanol. The organic phase is washed with 5% KHSO 4 solution, 5% KHCO 3 solution and saturated NaCl solution, dried over Na 2 SO 4, and the product is precipitated in the cold by adding petroleum ether (30-50).

Yield: 2.30 g (76%). Melting point: 130 ° C (decomposes). Thin layer chromatography: RF (I) = 0.69 RF (II) = 0.87 RF (III) = 0.81 Rf (IV) = 0.32 Rf (V) = 0.42 35 Molecular weight determination: C18H54N10O9 (534 , 5) for IU 1 IM: 25 Il 25 - 94419

Elemental analysis:

Calculated: C 40.45, H 6.41, N 26.20%

Found: C 40.39, H 6.55, N 26.11% XVI. 2. HCl x H-Arg (NO 2) -Arg (NO 2) -OMe To Boc-Arg- (NO 2) -Arg (NO 2) -OMe (20.00 g, 37.42 mmol) was added 1,2- m HCl / acetic acid (110 ml) and after 30 minutes the mixture was poured into ether (600 ml) with stirring. Pure HCl x H-Arg (NO 2) -Arg (NO 2) -OMe precipitated by thin layer chromatography.

Yield: 17.3 g (98%).

Thin layer chromatography on silica gel:

Rf (I) = 0.37 Rf (II) = 0.29 RF (III) = 0.44 RP (IV) = 0.07 RF (V) = 0.10 XVI. 3. Boc-Asn-Arg (NO 2) -Arg (NO 2) -OMe

Boc-Asn-OH (8.39 g, 36.10 mmol) and HOBt (4.89 g, 36.10 mmol) in DMF (75 mL) are added at -10 ° C HCl x H-20 Arg ( To a solution of NO 2) -Arg (NO 2) -OMe (17.00 g, 36.10 mmol) and NMM (3.98 mmol) in DMF (75 mL). DCC (7.35 g, 36.50 mmol) in CH 2 Cl 2 (10 mL) is added and the mixture is stirred for 30 min at -10 ° C, 1 h at 0 ° C and 3 h at room temperature. mode. The reaction is quenched by the addition of a few drops of glacial acetic acid, the solvent is evaporated in vacuo and the residue is dissolved in a small amount of methanol. The solution is added dropwise to anhydrous ether with stirring. The precipitate is filtered and dissolved in methanol. A pure product precipitates in the cold.

Yield: 18.25 g (78%). Melting point: 170 ° C.

3 0 Thin layer chromatography: RF (I) = 0.59 RP (II) = 0.67 Rf (III) = 0.66 RF (IV) = 0.45 35 RF (V) = 0.65 26 9 4 4 1 9

Amino acid analysis:

Asn 1.00 (1), Arg 1.85 (2)

Molecular weight determination C22H40N120, (648.6)

Elemental analysis: δ Calculated: C 40.74, H 6.22, N 25.91%

Found: C 40.70, H 6.40, N 25.79%

XVI. 4. Boc-Asn-Arg (NO 2) -Arg (NO 2) -OH

Boc-Asn-Arg (NO 2) -OMe (18.00 g, 27.75 mmol) is saponified in methanol (180 mL) with 1 M NaOH (80 mL) at room temperature. After 2 h, neutralize with dilute HCl and evaporate the methanol in vacuo. The product is extracted exhaustively at pH 3 with ethyl acetate. The organic phases are washed with a small amount of saturated NaCl solution, dried over Na2SO4 and the product is crystallized from a methanol solution at -20 ° C.

Yield: 15.84 (90%). Melting point: 228 ° C (decomposes). Thin layer chromatography: RF (I) = 0.49; Rf (II) = 0.21; RF (III) = 0.26; RF (IV) = 0.05; Rf (V) = 0.21 20 Test materials and methods

chemicals

Solvents for pro analysis were provided by Merck, otherwise the solvents were dried and distilled in the usual manner. N-methylmorpholine (Merck) was distilled with ninhydrin 25 to remove sec amines. 1-Hydroxybenzotriazole and dicyclohexylcarbodiimide were also obtained from Merck. All L-amino acid derivatives were supplied by Bachem. Boc-Aib-OH and H-Aib-OMe x HCl were synthesized according to literature guidelines.

3 0 Thin layer chromatography:: Finished silica gel plates 60 F254 (Merck) and the following solvents were used.

(I) 1-butanol / glacial acetic acid / water 3: 1: 1 (II) chloroform / methanol / glacial acetic acid / water 65: 25: 3: 4 35 (III) chloroform / methanol / concentrated ammonia / water 65: 35: 3: 4 27 944 ': 9 (IV) chloroform / methanol / water 65: 25: 4 (V) chloroform / roethanol 1: 1

The following were used as spray reagents: ninhydrin reagent, chloro / 4,4'-bis (dimethylamino) diphenyl-5-methane (TDM reagent) and Sakaguchi reagent. The comparison was dicyclohexylurea with the following values:

Rf (I) 0.91, RF (II) 0.82, RP (III) 0.92, RF (IV) 0.81, RF (V) 0.83.

Amino acid analyzes: To identify intermediates, the protected peptide (approximately 200 was hydrolyzed in 6-m HCl for 24 h at 110 ° C.

The hexapeptide segment intermediates and final sequence containing the Leu-Leu bond were otherwise hydrolyzed under the same conditions for 72 h. Amino acid analyzes were performed on a 15 Biotronik amino acid analyzer LC 6000 E using standard programming.

Rasemaattimääritys:

The hydrolyzed amino acids were converted to pentafluoropropionylamino acid n-propyl ester derivatives, and the enantiomers were separated by gas chromatography on glass capillary columns with Chirasil-Val. The reported percentages of D-amino acids have not been corrected for hydrolysis-induced racemization.

Elemental analyzes: 25 C, H, N individual assays were performed on Elemental

Analyzer with model 1104 (Carlo Erba, Milan).

The melting points:

Melting points were determined on a Tottoli apparatus and are uncorrected.

Spectral assays: 13 C-NMR spectra: 30 mg of the protected eicosapeptide was dissolved in 400 ph of a 1 Cl 2 HCl / 1 2 C 2 H 3 O 2 H (1: 1) (Merck) and measured with a WM 400 NMR spectrometer (Firma Bruker) for 12 h at 30 ° C. Circular dichroism spectra: solutions of free eicosapeptide (c = 1-1.7 mg / ml) in ethanol, 28-94419 in trifluoroethanol, methanol, 1,1,1,3,3,3-hexafluoropropanol- (2) , in water and in ethanol / water mixtures were measured with a Dichrograph II (Jouhan-Roussel).

Chromatographic purification: After completion of the coupling reaction and removal of the solvent (oil-pump vacuum), the protected peptide intermediates were dissolved in an equal volume of CHCl 3 / MeOH (1: 1), the dicyclohexylurea was separated by centrifugation and the solution was chromatographed on Sephadex LH 20: pyl. -10 tired 3 x 115 cm; eluent CHCl 3 / MeOH 1: 1; addition volume 35 ml; flow rate 8.40 ml / 10 min; 3 ml fractions were examined by thin layer chromatography in System II (TDM reagent). Peptides appeared in an elution volume of 165-190 ml. The fractions were combined, the solvent was evaporated in vacuo, and the residue was dried over P 2 O 5. Amino acid analysis gave the expected values and a peptide content of 92-96%.

Immunointikokeet:

First, the B-cell mitogen, which is both an excellent carrier and a potent adjuvant, was covalently coupled to synthetic antigenic determinants. For this, the synthetic lipopeptide S- (2,3-bis (palmitoyloxy) propyl) -N-palmitoyl-cysteinyl-serine (Pam3Cys-Ser), corresponding to the N-terminus of the outer membrane lipoprotein of Escherichia coli, was used. On the other hand, the particularly pronounced amphiphilic properties due to the hard association with the antigen ensure the stable anchoring of the S-glyceryl compound containing three fatty acid residues in the lipid layer of the cell membrane. On the other hand, it pre-sensitizes the most polar antigen (or hapten) of the outer hydro-30 layer of the membrane. Because the activating effect of a lipoprotein is determined solely by its N-terminal portion, the immunostimulatory effect is maintained in all conjugates containing the Pam3Cys-Ser group or an analog thereof.

29 - 94419

As an example of this use, Figure 9 shows the production of specific antibodies against the epidermal growth factor receptor (GF-R). In this case, an extracytoplas-5 ma region 516-529 was selected by computer epitope search, assembled by Merrifield synthesis, and finally Fmoc-Ser (Bu ') - OH and then Pam3Cys-OH were added. After removal from the resin, the conjugate found to be analytically homogeneous was administered without other additives as a single dose to mice i.p. After ELISA experiments, a high titer of specific antibodies against tetradecapeptide was detected as early as 2 weeks later. Essentially, in control experiments, the apparently weakly immunogenic tetradecapeptide did not cause any antibody formation.

Because Pam3Cys conjugates are also highly immunogenic in cell cultures, in vitro immunization can rapidly and elegantly elicit conventional and monoclonal antibodies against weakly immunogenic compounds as well.

The advantages of this plan when used in cell cultures are: simple preparation of a chemically unambiguously defined antigen-adjuvant conjugate in arbitrary amounts; in contrast to other conjugates, a single administration without multiple administrations; high potency in vivo and in vitro. It is clear that this results in significant savings for experimental animals - often not needed at all for in vivo immunization - and significant time savings, especially in genetic engineering work. The experiments can also be performed in human cell culture systems.

Example of In Vivo Immunization 6-10 week old Balb / c mice were immunized by once i.p. 50 μg and 500 μq (0.2 ml of a solution of covalently coupled antigen-containing adjuvant 10β-10β) Pam3-35 Cys-Ser- (EGF-R 515-529). The control was antigen, adjuvant and a mixture of antigen and adjuvant, each given in a comparable molar amount, and medium. 2 weeks after injection, mice were bled from the anterior well venous plexus to obtain serum 5 for antibody titer determination by ELISA.

Similarly, immunization can also be performed using other routes of administration, such as iv, oral, rectal Π6Ωg x · m · f S ♦ O · 10 The formation of specific antibodies without Freund's adjuvant against the tetradecapeptide EGF-R 516-529 after in vivo immunization was also studied. .

Balb / c mice (see Figure 10) were immunized with I. conjugate Pam3Cys-Ser (EGF-R 516-526) II. with free tetradecapeptide alone, (EGF-R 516-529) III. with Pam3Cys-Ser alone IV. with free tetradecapeptide, (EGF-R 516-529) in admixture with Pam3Cys-Ser; once i.p. 0.2 μιηοοίίΐΐβ conjugate. Antibody titer 20 was determined by ELISA (ordinate OD at 405 nm) (Figure 10).

After 14 days of immunization, blood was drawn from the ocular vein of the mice and the obtained serum was examined by ELISA.

Values are obtained from the mean difference between the ELISA values of 25 of the PEP 14-BSA conjugate and BSA (3-5 mice) (Figure 10).

It can be seen that only by using the membrane anchor-active substance conjugate prepared according to the invention, dramatically increased antibody concentrations are obtained which exceed the efficiency obtained by the methods hitherto many times over.

Example of in vitro immunization

Mouse spleen cells were cultured in the presence of the conjugate Pam3Cys-Ser- (EGF-R 515-529), the adjuvant Pam3Cys-Ser, the tetradecapeptide EGF-R 516-529, and the antigen-adjuvant mixture for 5 days.

3i. 94419

Lymphocytes (cell density 2.5 x 106 / ml) were cultured for 48 h in 0.2 ml of RPM1-1640 medium supplemented with 10% heat-inactivated FCS, glutamine (2 mmol), penicillin (100 units / ml). ), sterptomycin (100 pg / ml) and 5-mercaptoethanol (5 x 10 mol).

The liquid on top of each experiment was examined for specific antibody formation by ELISA.

Mitogenic activation of mouse spleen cells

Mitogenic activation of Balb / c spleen cells 10 Pam3Cys-Ser- (Lys) 4-FITC (circles), Pam3Cys-Ser- (Lys) 4-0H x 3HCl (triangles) and Pam3Cys-Ser- (Lys) 4 The effect of -OH x 2TFE (squares) is shown in Figure 12. Cell culture conditions are described (Z. Immunoforsch. 153, 1977, p. 11 ff and Eur. J. Biochem. 115, 1981). The drawing shows the stimulation index (incorporation cpm value / mitogen-free control cpm value) of the incorporation of 3 H-thymidine into DNA with or-15 dinate as a function of the concentration of active substance used.

Comparison in vivo / in vitro Figure 11 compares the above in vivo test and the in vitro test:

The in vitro experiment was performed in microtiter plates: cell density: 2.5 x 10 6 cells / ml; substance concentration: 5 x 10 "7 mmol; incubation conditions: 37 ° C, 5% CO 2 for 5 days.

The abbreviations used in Figure 11 have the following meanings:

Conj.: Conjugate Pam3Cys-Ser- (EGF-R 515-529)

Pep: tetradecapeptide EGF-R 516-529 30 Adj. Pam3Cys-Ser

Mix: a mixture of the free tetradecapeptide EGF-R 516-529 and Pam3Cys-Ser.

A considerable increase in antibody concentration is also obtained in vitro, which considerably expands the use of cell cultures, especially in the production of antibodies.

♦

Claims (9)

32 94419 A process for the preparation of a therapeutically useful membrane anchor-active substance conjugate, which conjugate comprises at least one membrane anchor compound and at least one active substance covalently bound to the membrane anchor compound or compounds, characterized in that the peptide protected protecting groups, in which no reaction is allowed to occur, are synthesized by a known coupling method on a solid or soluble carrier such as a polymer (e.g. Merrifield resin) so that the thus-synthesized, carrier-bound peptide is bound via the peptide N-terminus or side function. covalently to form a membrane anchor-active substance conjugate having the general formula one of the following: R -CO-O-CH R -O-CH, R-O-CO-CH- I | * I z R'-CO-O-CH * R'-O-CH * R'- O-CO-CH * I i is IcH,) n (CH.) (CH,) n 20 I 2 ni 2 n, 2 n AAA (CH.) (CH,) (CH,) I lm, 2 ml 2 n R "-C0-NH-CH * -C0-X R" -C0-NH-CH * -C0-X R'e-C0-NH-CH * -C0-X I. II. III. '· 25 R -NH-CO- ^ H2 R- co-nh-ch2 R'-NH-CO-CH * R'-CO-NH-CH *' 4H2> n "fH2'n? * Ff« jV. R · 1-CO-NH-CH "-C0-X R ·· -CO-NH-CH'-CO-X R-NH-CO-CH * -CC-X 30 IV. v. VI. R1-CH2 R, -CH * iCHj) I 2 n A (CH,) I 2 n R-CO-NH-CH * -C0-X »VII. 33,94419 wherein A is sulfur, oxygen, disulfide (-S-S-), methylene (CH2-) or -NH-; B is a group -S (CH 2) D - (substituted alkyl), wherein substituted alkyl is any substituted alkyl associated with - (CH 2) n - in formulas I-V; n 5 = 0 - 5; m is 1 or 2; C * is an asymmetric carbon atom having the configuration R or S; R, R ', R "are the same or different and denote a hydrogen atom or an alkyl, alkenyl or alkynyl group having 7 to 25 carbon atoms, which may be substituted by hydroxy, amino, oxo, acyl, alkyl or cycloalkyl groups, and R 1 and R 2 are the same or different and may represent the same as R, R 'or R "or the groups -OR, -OCOR, -COOR, -NHCOR, -CONHR, and X is an active ingredient or a spacer active ingredient group, except for compounds wherein X is an amino acid sequence of 2 to 10 natural amino acids, that the peptide conjugate thus prepared is isolated by deprotection and peptide / carrier bond in a manner known per se to give a membrane anchor peptide or a membrane anchor-active substance conjugate, wherein the active ingredient is antigen, such as a lower molecular weight subsequence of a protein or protein, e.g., a glycoprotein, a viral envelope protein, a bacterial cell wall protein, or a protozoal protein tigenic determinant, epitope), bacterial membrane component such as muramyl dipeptide, lipopolysaccharide, natural or synthetic hapten, antibiotic, hormone, nucleoside, nucleotide, nucleic acid, enzyme, enzyme substrate, enzyme, enzyme, enzyme peptide active ingredient such as tuftsin, polylysine, fluorescent marker, FITC, RITC, dansyl, luminol or coumarin, bioluminescent marker, spin marker, alkaloid, steroid, biogenic amine, vitamin, toxin such as digoxin, fallin , amine nitin, tetrodoxin or the like, a complexing agent or a drug. 34 94419 Method according to claim 1, characterized in that the membrane anchor compound is Pam3Cys or Pam3Cys-Ser or a Pam3Cys peptide containing 1 to 10 amino acids. Process according to Claim 1, characterized in that the membrane anchor compound is PamCys (Pam) -OH or Pam (a-Pam) -Cys [Pam (a-Pam)] -OH. A method according to claim 1, characterized in that the membrane anchor compound is a 10 α-alkyl-β-hydroxyacyl peptide (mycolyl peptide) having 1 to 10 amino acids in the peptide chain. Process according to Claim 1, characterized in that the membrane anchor compound is S- (1,2-dioctadecyloxycarbonylethyl) cysteine or a homologue of this compound. Method according to one of the preceding claims, characterized in that the active substance is covalently bound to two, possibly different, membrane anchor compounds. Method according to one of the preceding claims, characterized in that the active ingredient is additionally covalently bound to an adjuvant used for immunization purposes known per se, e.g. muramyl dipeptide and / or lipopolysaccharide. i ‘25 Method according to one of the preceding claims, characterized in that the several membrane anchor-active substance conjugate compounds are crosslinked with one another in the lipid and / or active substance component. Process according to one of the preceding claims, characterized in that the membrane anchor compound and the active ingredient are covalently bonded to one another via a crosslinking agent, for example via a dicarboxylic acid derivative, a diol, a diamine, a polyethylene glycol, an epoxide, a maleic acid derivative or the like. t 35 94419