MXPA99005107A - Synthetic bi-epitope compounds useful as standard measure in biological dosage of troponin i - Google Patents

Abstract

The invention concerns synthetic bi-epitope compounds useful as standard measure in immunoassays, for measuring out troponin I, the method for preparing them, compositions and kits containing such compounds and the immunoassay methods using such compounds.

Description

BIEPITOPIC SYNTHETIC COMPOUNDS USEFUL AS PATTERN IN IA TROPONIN BIOLOGICAL MEASUREMENT I DESCRIPTION OF THE INVENTION The present invention relates to synthetic biepitope compounds useful as a standard in immunoassays for the measurement of troponin I, to the process of preparing the compositions and vehicles containing such compounds, as well as to the immunoassay methods to use such compounds. It is known that troponin is a protein complex of myofibrils consisting of three proteins, troponins I, T and C. This protein complex allows to contribute to the regulation of muscle contraction by 2-Ca ions by interacting with myosin and actin .

More precisely, it is known that when a nervous flux arrives at the level of the motor plate of a muscle, there is the generation of an action potential that is transmitted to the sarcoplasmic reticulum. Ca is released in the cytosol and binds to troponin C, which causes a reinforcement of the interaction between troponin I and troponin C and, in turn, a conformational change of the troponin I, T, C complex. Then there is the release of actin-myosin interaction sites, which allows the movement of muscle contraction. REF .: 30378 When the muscle is damaged, either the cardiac muscle in myocardial necrosis following an iacárdico infarct, or the skeletal muscle by prolonged physical efforts, troponins are apparently released more or less rapidly into the bloodstream. Therefore, the measurement of troponin has recently been proposed for the early diagnosis of myocardial infarctions, either Troponin T in Circulation (1991), 83 ^, pp. 902-912, or troponin I in Am Heart J. (1987), U O, pp. 1333-1344, and Molecular Immunology (1992), 29 (2), pp. 271-278. Likewise, the measurement of cardiac troponin T has been proposed to measure the success of thrombolytic therapy after myocardial infarction in Br. Heart J., (1994), 72, p. 242-248, as well as the measurement of skeletal troponin I to measure skeletal muscle damage (abstract No. 35 of the American Association for Clinical Chemistry, 46th National Meeting, New Orleans, June 17-21, 1994). It should be noted that the measurement of the different cardiac and skeletal troponins at present is a very useful means to diagnose human and animal pathologies. It is well known that immunoassays practiced in biological analysis laboratories require the manufacturer to provide them with a package of necessary reagents (ie, labeled or unlabeled antibodies, developer agents and solutions for dilution), a pattern of the compound to be determined which is works in conditions analogous to those of the sample that is being studied, to serve as a reference for the calculation of the results and / or as a positive control. To obtain the standard and / or control of the compound to be determined, said purified compound can be used in lyophilized form (accompanied by a solvent in which the compound will be dissolved for use in the test) or ready to use. Because the biological reagents are unstable, standard or control solutions prepared from a freeze-dried product are frozen in unit doses and stored at a temperature of -80 ° C. Furthermore, it has been found that these solutions are stable only for a few hours at + 4 ° C, even if protease inhibitors or antibacterial and stabilizing agents are added. This forces users to prepare their contrast solutions extemporaneously. The Patent Application Published with the Number FR-A-2 701 954, describes a stabilized composition of troponin I or T for immunoassay, characterized in that it is constituted by an aqueous solution containing troponin I or troponin T, mixed with troponin C and in proportions of 1 to 10 molar equivalents of troponin C for each equivalent of troponin I or T, and calcium chloride. This technique allows the preservation for several days at + 4 ° C of the standard solutions, more or less diluted, of troponin I or T. The Patent Application Published with the Number FR 2 734 267 describes troponin standard solutions composed of a triple complex formed by troponin I, troponin T and troponin C. The raw materials used to obtain these standards are of human or animal origin and the standards or controls thus obtained are stable for approximately one month at + 4 ° C. International Application WO 94/15217 discloses certain synthetic peptides useful as immunogens for the preparation of antibodies recognizing the N-terminal peptide of troponin I. Certain of these peptides can be used as a standard in immunoassays of troponin I, using the antibodies subject of the invention encompassed by the application WO 94/15217. Similarly, the WO Patent Application 94/27156 relates to a method of measuring cardiac troponin I, using antibodies specific for cardiac troponin I. These antibodies can be prepared from peptide fragments with an absent sequence of troponin I from skeletal muscle and specific for cardiac troponin I. However, this Application does not disclose or suggest the possibility of using certain peptide fragments as standards in troponin I immunoassays. From this, it is known that the International Application WO 96/27661 describes aqueous solutions for stabilizing proteins and peptides. These solutions have their application mainly in diagnostic tests of proteins or peptides. In accordance with the International Publication WO 97/27661, these aqueous solutions make it possible to increase the stability of the troponin I fragments, which are known to be less stable than the whole troponin I. European Application EP-A-752 426 published January 8, 1997, also discloses troponin I standards composed of one or a plurality of fixed peptides on a carrier molecule, such as molecular weight proteins (> 100 kD) or polymers. European Application EP-A-650 053 describes synthetic patterns containing active sites for one or a plurality of receptors, linked together with an arborescent structure. This Application more specifically describes synthetic patterns of troponin T that are not stable in solution for more than 3 weeks. However, it has been found that it is possible to obtain useful synthetic standards for the evaluation of troponin I, which are stable for several months. Indeed, the particular structure of the compounds of the present invention gives it excellent stability. The compounds of the present invention allow, then, to obtain highly standardized patterns, stable in solution for several months and the purification steps and complex extraction procedures necessary for the preparation of troponin I standards from animal organs are avoided. The compounds of the present invention comprise two epitopes of troponin I, linked together by a linking group ("linker") and corresponding to the General Formula I? -E? -Z-E2-? (I) wherein: - Ei, E2 are identical or different and represent a peptide sequence comprising either a minimal epitope of troponin I, or a broad epitope, - Z represents • a peptide sequence of 1 to 40 amino acids ( aa), provided that -E? ~ Z-E2- do not together form a part of the sequence of troponin I • a linear or branched aminoalkylcarbonyl chain of 1 to 10 carbon atoms, or • a mixed construction consisting of at least one peptide sequence of 1 to 10 amino acids and at least one aminoalkylcarbonyl chain of 1 to 10 carbon atoms straight or branched, -? represents • a hydrogen atom, an acetyl group, a peptide sequence of 1 to 10 amino acids, a peptide sequence of 1 to 10 Na-acetylated amino acids, a cysteinyl, biotinyl or biocytinyl group, a peptide sequence of 1 to 10 amino acids that carry a cysteinyl, biotinyl or biocytinyl residue, • a linear or branched aminoalkylcarbonyl chain of 1 to 10 carbon atomsandDr. • a straight-chain or branched aminoalkylcarbonyl chain of 1 to 10 carbon atoms, Na-acetylated, • a mixed construction composed of at least one peptide sequence of 1 to 10 amino acids and at least one aminoalkylcarbonyl chain of 1 to 10 carbon atoms. linear or branched carbon, or • a mixed construction consisting of at least one peptide sequence of 1 to 10 amino acids and at least one linear or branched aminoalkylcarbonyl chain of 1 to 10 carbon atoms, carrying a biotinyl, biocytinyl or cysteinyl residue, -? represents • a hxyl radical, an amino radical, a peptide sequence of 1 to 10 amino acids, a peptide sequence of 1 to 10 amino acids carrying an amino-terminal radical, • an aminoalkylcarbonyl chain of 1 to 10 carbon atoms, straight or branched • a linear or branched aminoalkylcarbonyl chain of 1 to 10 carbon atoms, carrying a hxyl radical or an amino radical, • a mixed construct consisting of at least one peptide sequence of 1 to 10 amino acids and at least one chain of aminoalkylcarbonyl of 1 to 10 carbon atoms, linear or branched, • a mixed construction consisting of at least one peptide sequence of 1 to 10 amino acids and at least one aminoalkylcarbonyl chain of 1 to 10 carbon atoms, linear or branched, carrier of a hxyl radical or an amino radical. It is considered that -E! -Z-E2- is not a part of the sequence of troponin I when -E? -Z-E2- differ from a given fragment of the troponin I sequence by substitution, deletion or insertion not conserved from at least one amino acid, preferably 2 amino acids, more particularly 5 amino acids. Z may represent mainly • a chain of Formula II: -NH- (CH2) m-CO- (II) where m represents an integer from 1 to 10, • or a mixed construction of Formulas III to V: - [pep? -NH- (CH2) m-CO] p- (III) - [pep? -NH- (CH2) m-CO-pep2] p- ( IV) - [NH- (CH2) m-CO-pep2] p- (V) where - m represents an integer from 1 to 10, - p represents an integer from 1 to 5, and ~ pePi Y PeP2 / ie they are the same or different, they represent a peptide chain comprising from 2 to 10 amino acids; ? can represent mainly: • a chain of the formula lia: -NH- (CH2) q-CO- (Ha) where q represents an integer from 1 to 10, • or a mixed construction of the Illa to Va formulas: - [pep3-NH- (CH2) q-CO] r- (Illa) - [pep3-NH- (CH2) q-CO-pep4jr- (IVa) - [NH- (CH2) q-CO-pep4] r- < Goes > wherein - q represents an integer from 1 to 10, - r represents an integer from 1 to 5, and - pep3 and pep, which are identical or different, represent a peptide chain comprising from 2 to 10 amino acids, • a chain of Formulas Ha to Va Na-acetylated or a chain of Formulas Ha to Va carriers of a biotinyl, biocytin or cysteinyl residue; Y ? can represent mainly • a chain of the Formula Hb: -NH- (CH2) s-CO- (IIb) where s represents an integer from 1 to 10, • or a mixed construction of the Formulas IHb a Vb: -tpep5-NH- (CH2) s-CO] t- (Hlb) - [pep5-NH- (CH2) s-CO-pep6] t- (? Vb > - [NH- (CH2) s- CO-pep6] t- (Vb) - li ¬ in which - s represents an integer from 1 to 10, - t represents an integer from 1 to 5, and - peps and pepß / that are identical or different, represent a peptide chain comprising from 2 to 10 amino acids, • a chain of Formulas IIb to Vb carrying a hydroxyl radical or an amino radical. Among the preferred compounds of the present invention, are the compounds in which? represents either a chain of the Formula Ha, a mixed construction of the Formulas Illa to Va, or a chain of Formulas Ha to Na-acetylated or a chain of Formulas Ha to Va carriers of a biotinyl, biocytin or cysteinyl residue . Among the preferred compounds of the present invention are also the compounds in which? represents a chain of Formula IIb, a mixed construction of Formulas 11 Ib to Vb, or a chain of Hb to Vb Formulas bearing a hydroxyl radical or an amino radical. In particular, the compounds of the present invention are preferred in which Z represents either a chain of Formula II, or a mixed construction of the Formulas III to V. Peptide sequences comprising a minimal epitope of troponin I are determined with the aid of monoclonal antibodies antitroponin I. Such antibodies are described by Larue et al., (Molec.

Immunology, (1992), vol. 20, No. 29, pp. 271-278); Bodor et al., (Clin. Chem. (1992), vol.38, No. 11, pp. 2203-2214); Granier et al., (Protein Science, (1997), vol.6, supplement 1, page 61) and in the WO International Application 94/15217. The majority of these antibodies are commercially available (Hytest LTD-Turku, Finland). To determine the peptide sequence comprising a minimal epitope of troponin I, the simultaneous synthesis of peptides of 10 amino acids whose sequence encompasses the sequence of the preceding peptide in 7 amino acids is proceeded according to the following scheme: - Peptide 1 = amino acids 1 - 10 - Peptide 2 = amino acids 4 - 13 - Peptide 3 = amino acids 7 - 16 - etc. In their assembly, the synthesized and tested peptides describe the entirety of the protein sequence (Vallins J. et al., FEBS Letters (1990), vol.270, No. 1-2, pp. 57-61).

Subsequently, we proceed to the identification of the peptides that react with some of the monoclonal antibodies used, by means of an enzyme-linked immunoenzymatic test of each peptide with each antibody. For example, for certain antitroponin I monoclonal antibodies, the peptide sequence comprising a minimum immunologically reactive epitope for each antibody and the position of this peptide sequence in the sequence of troponin I, as described in FEBS Letters, are indicated below. (1990), vol. 270, No. 1 -2, pp. 57-61.

* Peptide sequences are indicated using a single letter code. Peptide sequences derived from said peptide sequences by substitution, deletion or insertion of an amino acid, apparently also fall within the domain of the present invention, insofar as they have a binding affinity for the equivalent antibodies. The linear sequences Ei and / or E2 number 1 to 5 below are preferred. Sequence No. 1: -Thr Glu Pro His- or -TEPH- Sequence No. 2: -His Ala Lys Lys- or -HAKK- Sequence No. 3: -Pro Ala Pro He Arg Arg Arg- or -PAPIRRR -Sequence No. 4: -Leu Leu Gly Ala Arg- or -LLGAR- Sequence No. 5: -Arg Lys Asn Ile- or -RKNI- The above sequences are presented by way of non-limiting example. When the Z-linking component comprises a peptide sequence, it can form with the Ei and / or E2 sequences a broad epitope of troponin I. This broad epitope can have as sequence -TEPHAKK- or -QALLGAR- or -PTLRRVRISA- or - GKFKRPTLRRVR- or -LGFAELQD- or -NYRAYATEPH- or -AYATEPH- or -LGFAELQ-, etc. These broad epitopes, which amplify the immune response for antibodies, are determined with the aid of amino acids in the peptide sequence comprising a minimum epitope determined in the manner described above. As already mentioned, the peptide sequence of the Z-linking component can comprise from 1 to 40 amino acids. It is preferred that the number of amino acids that form the peptide sequence of Z be less than 30, more particularly less than 20. A particularly preferred Z peptide sequence includes a sequence that is selected from the group consisting of the following amino acid sequences: -Gln Lys Met Gln- or -QKMQ-, -Gly Pro Asp Asn- or -GPDN-, -Ala Met Met- or -AMM-, -Ala Lys Lys- or -AKK-, -Lys Ser Lys - or -KSK-, and -Pro Gly Asn Ser- or -PGNS-. This particularly preferred Z-peptide sequence may contain a repeated amino acid chain and an amino acid number of less than 30. By way of example, the following Z-peptide sequences are presented, in which n represents an integer from 1 to 5. Sequence No. 6: -Gln Lys Met Gln- or -QKMQ- Sequence No. 7: -Gln Gly Pro Asp Asn- or -QGPDN-Sequence No. 8: -Gly Pro Asp Asn- or -GPDN- Sequence No. -Ala Met Met Lys Ser Lys (Gln Lys Met Gln) n- or -AMMKSK- (QKMQ) n- Sequence No. 10: -Ala Lys Lys Ala Met Met Lys Ser Lys- (Gln Lys Met Gln) n- or -AKKAMMKSK- (QKMQ) n- Sequence No. 11 -Ala Met Met Lys Ser Lys- (Gln Lys Met Gln) n-Gln Ala- or -AMMKSK- (QKMQ) n ~ QA- Sequence No. 12 -Ala Lys Lys Ala Met Met Lys Ser Lys- (Gln Lys Met Gln) n-Gln Ala- or -AKKAMMKSK- (QKMQ) n-QA- Sequence No. 13: -Lys Ser Lys- (Gln Lys Met Gln) ) n-Ala Met Met- or -KSK- (QKMQ) n-AMM- Sequence No. 1 - Lys Lys Lys Ser Lys- (Gln Lys Met Gln) ) n-Met Met Met- or -AKKKSK- (QKMQ) n-AMM-Sequence No. 15: -Lys Ser Lys- (Gln Lys Met Gln) n-Ala Met Met Gln Ala- or -KSK- (QKMQ) ) n-AMMQA- Sequence No. 16: -Ala Lys Lys Lys Ser Lys- (Gln Lys Met Gln) n-Ala Met Met Gln Ala- or -AKKKSK- (QKMQ) n-AMMQA- Sequence No. 17 -Ala Met Met- or -AMM- Sequence No. 18: -Ala Lys Lys Ala Met Met- or -AKKAMM- Sequence No. 19: -Ala Met Met Gln Ala- or -AMMQA- Sequence No. 20: -Ala Lys Lys Wing Met Met Gln Ala- or -AKKAMMQA- Sequence No. 21: -Pro Gly Asn Ser- or -PGNS- Z can also represent a mixed construction of Formulas III to V, in which m is a number whole from 1 to 10. By way of non-limiting example, Z may also have the following sequences: Sequence No. 22: -Ala Lys-NH- (CH2) m-CO- or -AKK-NH- (CH2) m-CO-Sequence No. 23: -NH- (CH2) m-CO-Ala Met Met- or -NH- (CH2) m-CO-AMM-Sequence No. 24: -Ala Lys Lys-NH (CH2 ) m-CO-Ala Met Met- or -AKK-NH (CH2) m-CO- MM- S sequence No. 25: -NH- (CH2) m-CO-Ala Met Met Gln Ala- or -NH- (CH2) m-CO-AMMQA- Sequence No. 26: -Ala Lys Lys-NH- (CH2) m-CO-Ala Met Met Gln Ala- or -AKK-NH- (CH 2) m-CO-AMMQA- The preferred compounds of the present invention are biepitope compounds of Formula I, in which Z includes a sequence that is selected from the group consisting of sequences No. 6 to No. 26, such as those defined above. When ? I ? they comprise a peptide sequence, this sequence can form a broad epitope with Ei and / or E with which it is linked. Advantageously, the chemical structure of? it is such that it allows the binding of the biepitope compounds of the present invention to a natural carrier protein, a peptide construct or a solid phase. The synthetic biepitope peptides of Formulas la and Ib are part of the peptides of Formula I and are preferred peptides:? -Thr Glu Pro His-Z-Leu Leu Gly Ala Arg-? da)? Pro Pro Pro He Arg Arg Arg-Z-Thr Glu Pro His-? (Ib) In Formulas la and Ib: - Z includes or represents a sequence that is selected from the group consisting of the following sequences: S Seeccuueenncciyaa N Noo .. 6 6 :: -Gln Lys Met Gln- Sequence No. 7: -Gln Gly Pro Asp Asn- Sequence No. 8: -Gly Pro Asp Asn-Sequence No. 20: -Ala Lys Lys Wing Met Met Gln Wing-Sequence No. 26: -Ala Lys Lys-NH- (CH2) m- CO-Ala Met Met Gln Ala Sequence No. 12: -Ala Lys Lys Ala Met Met Lys Ser Lys- (Gln Lys Met Gln) n-Gln Ala- Sequence No. 16: -Ala Lys Lys Ser Lys- (Gln Lys Met Gln) n-Ala Met Met Gln Ala- and Sequence No. 21: -Pro Gly Asn Ser-, in which n represents an integer from 1 to 5 and m an integer from 1 to 10, -? represents an acetyl radical, a cysteinyl radical, a biotinyl or biocytinyl radical, optionally linked to a residue of a peptide sequence comprising a sequence selected from the group consisting of the following sequences: -Gly Asn Tyr Arg Ala Tyr Ala- - Gly Gly Asn Tyr Arg Ala Tyr Ala- -Asn Tyr Arg Ala Tyr Ala-, and -Arg Pro Ala- -? represents an amino radical or a peptide sequence comprising one of the following sequences: -Ala Lys Glu -Ala Lys Lys Lys Ser Lys It is preferred that the peptide includes one of sequences 27 to 33, which are the following: Sequence No. 27: -Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys 1 5 10 Ala Met Met Gln Ala Leu Leu Gly Ala Arg Ala Lys Glu- 15 20 25 Sequence No. 28: -Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys- 1 5 10 NH- (CH2) s-CO-Ala Met Met Gln Ala Leu Leu Gly Ala Arg Ala 15 17 20 25 Lys Glu- Sequence No. 29: -Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys Lys Ser 1 10 15 Lys Gln Lys Met Gln Wing Met Met Gln Wing Leu Leu Gly Wing Arg 20 25 30 Ala Lys Glu- Sequence No.30: -Gly Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys 1 5 10 Lys Lys Ser Lys Gln Lys Met Gln Ala Met Met Gln Ala Leu Leu 15 20 25 Gly Ala Arg Ala Lys Glu- 30 35 Sequence No. 31: -Gly Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys 1 5 10 Lys Lys Ser Lys Gln Lys Met Gln Gln Lys Met Gln Ala Met Met 15 20 25 Gln Ala Leu Leu Gly Ala Arg Ala Lys Glu- 30 35 Sequence No. 32: -Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys 1 5 10 Lys Ser Lys Gln Lys Met Gln Gln Lys Met Gln Ala Met Met 15 20 25 Gln Ala Leu Leu Gly Ala Arg Ala Lys Glu- 30 35 Sequence No. 33: -Arg Pro Ala Pro Ala Pro He Arg Arg Arg Pro Gly Asn Ser 1 5 10 Thr Glu Pro His Ala Lys Lys Lys Ser Lys- 15 20 Synthetic peptides Biepitópicos of Formulas la and Ib above, are particularly preferred peptides. In the two Tables presented below, the peptide sequences are given using a single letter code. = - TEPH-Z-LLGAR-? gives) ? - PAPIRRR- Z - TEPH-W db) In the formulas la and Ib, the underlined amino acid sequences correspond to Ei and E2, which represent a minimal epitope of troponin I. The underlined amino acid sequences of Z form with Ei or E2, to which they are linked, a broad epitope of troponin I. Among the above-mentioned compounds, compounds 5, 6, 10, 11, 12, 13, 14 and 15 are particularly preferred. These peptides have the following sequences: Peptide 5: Ac-Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys 1 5 10 Ala Met Met Gln Ala Leu Leu Gly Ala Arg Ala Lys Glu-NH2 15 20 25 Peptide 6: Ac-Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys- 1 5 10 NH- (CH2) 5-CO-Ala Met Met Gln Ala Leu Leu Gly Ala Arg Ala 15 20 25 Lys Glu-NH2 Peptide 10: Ac-Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys Lys 1 5 10 Ser Lys Gln Lys Met Gln Wing Met Met Gln Wing Leu Leu Gly Wing 15 20 25 Arg Ala Lys Glu-NH2 30 Peptide 11 Ac-Gly Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys 1 5 10 Lys Lys Ser Lys Gln Lys Met Gln Ala Met Met Gln Ala Leu Leu 15 20 25 Gly Ala Arg Ala Lys Glu-NH2 30 35 Peptide 12: Ac-Gly Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys 1 5 10 Lys Lys Ser Lys Gln Lys Met Gln Gln Lys Met Gln Ala Met Met 15 20 25 Gln Ala Leu Leu Gly Ala Arg Ala Lys Glu-NH2 30 35 Peptide 13: Ac-Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys 1 5 10 Lys Ser Lys Gln Lys Met Gln Gln Lys Met Gln Ala Met Met Gln 15 20 25 Ala Leu Leu Gly Ala Arg Ala Lys Glu-NH2 30 35 Peptide 14: Cys-Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys 1 5 10 Lys-NH- (CH2) 5-CO-Ala Met Met Gln Ala Leu Leu Gly Ala Arg 15 20 25 Ala Lys Glu-NH2 Peptide 15: Ac-Arg Pro Ala Pro Ala Pro He Arg Arg Arg Pro Gly Asn Ser 1 5 10 Thr Glu Pro His Ala Lys Lys Lys Ser Lys-NH2 15 20 Synthetic and biepitope compounds of Formula I , object of the present invention, are obtained by synthesis in solid phase according to the classical methods of RB Merrifield, J. Amer. Chem. Soc. (1963), 85, pp. 2149-2154.

R. C. Sheppard, in "Peptides 1971", Nesvadba H. (ed.) North Holland, Amsterdam, pp. 111; E. Atherton and R. L. Sheppard, in "Solid phase peptide synthesis, a practical approach", IRL PRESS, (1989), Oxford Universi ty Press, p. 25-34. You can use the automatic synthesizer "9050 Plus Pep Synthetizer" from Millipore or an equivalent synthesizer. The solid support used for the synthesis must be compatible with the technique and chemistry used. For example, for a synthesis in the synthesizer "9050 Plus Pep Synthetizer", it is recommended to use a resin adapted to the technique called "in continuous flow"; PEG PS resins meet these criteria. These supports are constituted by an arm ("spacer"), a base of polyethylene glycol (PEG) located between the functional group of polystyrene and the anchoring point of the first amino acid. The nature of this anchor point may vary in accordance with the selected C-terminal functional group. For example, for a peptide in the form of an amide, a resin of the PAL PEG PS type may be used. The initial resin and the amino acids used as raw material are commercially available products (PerSeptive-Biosystem). The following side chain protective groups were used: The temporary protection of the primary amine functional group at the amino acid position a was carried out with the help of the 9-fluorenylmethyloxycarbonyl radical (F oc). The deprotection was carried out with a solution of 20% piperidine in dimethylformamide. For coupling, an excess of diisopropylcarbodiimide (DIPCDI) and 1-hydroxybenzotriazole (HOBt) was preferably used. After the synthesis, the resin was washed with organic solvents (dimethylformamide and then dichloromethane), dried under vacuum and then treated with a solution based on trifluoroacetic acid cooled to 0 ° C and containing the appropriate "waste collectors" . One could use, for example, reagent K containing 82% trifluoroacetic acid, 5% phenol, 5% water, 5% thioanisole and 3% ethanedithiol. The biepitope synthetic peptides thus isolated are then precipitated and rinsed with ether. Then, the synthetic biepitope compounds are purified by reversed-phase liquid chromatography and their purity is determined by mass spectrometry. As a phase, for example, the Bondapak C-18 phase can be used. The peptides are eluted by performing a linear gradient between two regulatory solutions, the first essentially aqueous (for example water-0.1% TFA) and the second organic (for example a mixture containing 60% acetonitrile, 40% water and 0.08% TFA) . The collected pure fractions are combined and the combined is concentrated in vacuo and lyophilized. The determination of the biepitope character of the compounds of the Formula I was carried out using a packet (kit) of troponin I. For example, a packet (kit) allowing a sandwich-type immunoassay containing the monoclonal antibodies can be used. which react with the peptide sequences Ei and E2. To determine the biepitope character, the compounds of Formula I are diluted in the package diluent (kit), which can be for example normal human serum or a buffer, and are measured as a patient sample. The use of the compounds of Formula I as a standard for the immunological measurement of troponin I is also part of the present invention. The present invention also relates to compositions containing the compounds of Formula I. Preferably these are aqueous solutions or compositions constituted by the compounds of Formula I in a buffer solution. As a regulatory solution, for example, a phosphate buffer (KH2PO4 / K2HPO4, pH = 6.5-7.5) containing Kathon and bovine serum albumin (ASB) or Kathon, Régilait and AEDT (ethylenediaminetetraacetic acid), or Kathon, Plasmion can be used. and eventually AEDT, or Kathon, casein and AEDT. In the same way, succinate (pH = 5-6) or Tris-HCl (pH = 7.5- 8.5) buffer solutions containing Kathon and ASB or Kathon, Régilait and AEDT, or Kathon, Plasmion and eventually AEDT, or Kathon, casein and AEDT. Regulatory solutions containing glycine, Kathon, Régilait and AEDT can also be used. The succinate or phosphate buffer solutions containing Kathon, Régilait and AEDT are preferred. Kathon® is an antibacterial agent marketed by Rhom and Hass, consisting of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (1.5%) . The Régilait® (Régilait) is marketed by the company Régilait. Plasmion® is marketed by Bellon laboratories and consists of modified gelatin modified (30 g / 1), NaCl (5,382 g / 1), MgCl (143 mg / l), KCl (373 mg / l), sodium lactate (3.360 g / 1), in water. The following regulatory solutions are particularly preferred. - 0.1 M succinate buffer solution (pH = 6) containing Kathon (0.2%), Régilait (0.05-2%) and 2 mM EDTA, - 0.1 M succinate buffer solution (pH = 6) containing Kathon (0.2%), casein (0.01-0.5%) and 2 mM EDTA, - Phosphate buffer solution KH2PO4 / K2HPO4 0.1 M (pH = 7.5) containing Kathon (0.2%), Régilait (0.05-0.5%) and 2 mM EDTA, - phosphate buffer KH2PO4 / K2HPO4 0. 1 M (pH = 7.5) containing Kathon (0.2%), casein (0.01-0.1%) and 2 mM EDTA. Plasma-containing compositions and a compound of Formula I also form part of the present invention. Immunological methods that use the compounds of Formula I as a standard or control also form part of the present invention. The present invention also encompasses packets (kits) for practicing immunoassays that include a compound of Formula I or a composition containing a biepitope peptide of Formula I. The following Examples illustrate the invention and are not limiting. EXAMPLE 1 Preparation of a compound according to the present invention (peptide 10) Ac-Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys Lys 1 5 10 Ser Lys Gln Lys Met Gln Ala Met Met Gln Ala Leu Leu Gly Ala 15 20 25 Arg Ala Lys Glu-NH2 30 This peptide was synthesized in solid phase. The technique perfected in 1963 by Merrifield (J. Am. Chem.

Soc. (1963) 8_5, pp. 2149-2154) consists in fixing the first amino acid to a solid polymeric support (resin) through its acidic functional group and prolonging the peptide sequence from that first amino acid, wherein the peptide in the course of remaining synthesis is anchored on the resin. For the synthesis of compound 10, the synthesizer "9050 Plus Pep Syntetizer" and the PEG PS resin described above were used. The different stages of the synthesis are summarized in Table 1: TABLE I After concluding the synthesis, the resin was washed with dimethylformamide and then with dichloromethane and dried under vacuum. Subsequently, the resin was treated with reagent K (82% trifluoroacetic acid, 5% phenol, 5% water, 5% thioanisole, 3% ethanedithiol). Compound 10 was isolated by precipitation and then rinsed with diethyl oxide. In this manner, 0.529 g of compound 10 was obtained. In an equivalent manner, and using the appropriate amino acids, the other compounds of the present invention were synthesized.

The molecular weight evaluated by mass spectrometry of certain compounds of Formulas la and Ib, are presented below. Compound Molecular Weight (in Daltons) 1 1549.8 2 1545.5 5 3018 6 3132.5 7 4448 3934.5 9 3820.5 10 3820.5 11 3934.5 12 4448 13 4391 15 2721 EXAMPLE 2 Evaluation of the immunoreactivity of the peptides of the present invention The determination of the biepitope character of the aforementioned compounds was carried out using a troponin I package. This is the ERIA Troponin I Pasteur kit (code) 79691. Its foundation It is based on a sandwich-type immunoenzymatic method that allows a quantitative measurement of cardiac troponin I in human serum. The solid phase is constituted by polystyrene tubes coated with a monoclonal cardiac antitroponin I antibody (8E1). The disclosure was made with the help of a second monoclonal cardiac antitroponin I antibody (11E12) coupled to peroxidase. The practice of the test comprises the following steps: Samples and standards (Troponin I patterns of animal origin), as well as monoclonal antibodies coupled with peroxidase are incubated in the presence of the cardiac antitroponin monoclonal antibody I immobilized on the solid phase . After a series of washes, enzymatic revelation is carried out by the addition of the tetramethylbenzidine chromogen. After the disclosure is completed, the optical density reading a? = 450 nm. The absorbance obtained is directly related to the concentration of cardiac troponin I present in the tube.

Compounds 1, 2, 5, 6, 10, 11 and 12 were dissolved in water (C = 1 mg / ml or 2 mg / ml, as the case may be). These solutions were rediluted using 0.1 M succinate buffer, pH = 6, containing Régilait 0.05% and Kathon 0.2% and 2 mM EDTA. The "final solutions", subsequently, they were distributed as "samples" according to the protocol described above. In addition, compounds 1, 2, 10 and 11 were tested after dilution in human serum. In this case, the "final solutions" were obtained by diluting in normal human serum the aqueous "mother" solutions containing compounds 1, 2, 10 and 11, at a concentration of 2 mg / ml. All compounds are reactive at the dose of troponin I. EXAMPLE 3 Stability tests A compound according to the present invention (compound 10) was diluted in water (C = 2 mg / ml). From this "mother" solution, four "daughter" solutions Si, S2, S3 and S4 were obtained. To perform the different dilutions, Si (C = 0.5 ng / ml) and S2 (C = 1 ng / ml), a buffer solution of 0.1 M sodium succinate (pH = 6) containing Kathon (0.2%) was used for the solutions. ), Régilait (0.05%) and 2 mM EDTA. The solutions S3 (C = 0.5 ng / ml) and S4 (C = 1 ng / ml) were obtained by diluting the "mother" solution with a phosphate buffer solution KH2PO4 / K2HPO4, pH = 7.5) containing Kathon (0.2%), Régilait (0.05%) and 2 mM EDTA. A dose of solutions Si, S2, S3, and S4 according to the protocol described in Example 2, was carried out in the DO (day of preparation of the solutions). The values obtained served as a reference for monitoring stability. Subsequently, the solutions Si, S2, S3 and S4 were kept at + 4 ° C and were periodically measured. After each series of measurements, three "control solutions" were used. These are freeze-dried troponin I control sera distributed in the range of dose measurement. For these sera, it was preferably shown that the preservation of a lyophilized control serum is greater than 18 months. After each stability test, it was verified that each of the tested controls had a white concentration. This allows to compare the concentrations obtained by the peptides after the different tests. In particular, it was possible to compare the concentration of the solution of the compound according to the present invention in the OD (day on which the solution was made), with the concentration of the compound according to the present invention after the stability tests with the passage of time. The fact of obtaining a constant value for the controls in the course of the stability tests allows to validate the tests carried out. The results obtained after the stability tests are indicated in Tables II and III.

TABLE II TABLE III The results indicated in Tables II and III demonstrate the excellent stability of the solutions of compound 10 in accordance with the present invention. The tests carried out with other compounds of the Formula I showed that their stability is comparable to that of the compound 10. For example, the tests carried out with the compound 6 confirmed that this compound has an excellent stability for at least 9 months, when it is stored at + 4 ° C in solution in a buffer solution of 0.1 M sodium succinate (pH = 6) containing Kathon (0.2%), Régilait (0.2%) and 2 mM EDTA. Compound 6 was dissolved in the above-mentioned buffer to obtain concentrations of 0.25, 4.8 and 18 ng / ml. This stability study was validated with the aid of compound 6 preserved at -20 ° C and used as a reference. It was previously verified that compound 6 is stable when stored at -20 ° C for at least 18 months. The results of these tests are presented in Table IV. TABLE IV It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (16)

1. RESINS The invention having been described as an antecedent, the content of the following claims is claimed as property: 1. Biepitope compounds of Formula I:? -E? -Z-E2-? (i) where Ei, E2, which are identical or different, represent a peptide sequence comprising a minimum epitope of troponin I, - Z represents • a peptide sequence of 1 to 40 amino acids (aa), provided that - E? -Z-E2- do not together form a portion of the sequence of troponin I, • a linear or branched aminoalkylcarbonyl chain of 1 to 10 carbon atoms, or • a mixed construct consisting of at least one peptide sequence of 1? to 10 amino acids and at least one aminoalkylcarbonyl chain of 1 to 10 carbon atoms straight or branched, -? represents • a hydrogen atom, an acetyl group, a peptide sequence of 1 to 10 amino acids, a peptide sequence of 1 to 10 Na-acetylated amino acids, a cysteinyl, biotinyl or biocytinyl group, a peptide sequence of 1 to 10 amino acids that carry a cysteinyl, biotinyl or biocytinyl residue, • a linear or branched aminoalkylcarbonyl chain of 1 to 10 carbon atoms, • a linear or branched, aminoalkylcarbonyl chain of 1 to 10 carbon atoms, Na-acetylated, • a mixed construction composed of at least one peptide sequence of 1 to 10 amino acids and at least one aminoalkylcarbonyl chain of 1 to 10 carbon atoms straight or branched, or • a mixed construct constituted with at least one peptide sequence of 1 to 10 amino acids and at least one chain of linear or branched aminoalkylcarbonyl of 1 to 10 carbon atoms, carrying a biotinyl, biocytinyl or cysteinyl residue, -? represents • a hydroxyl radical, an amino radical, a peptide sequence of 1 to 10 amino acids, a peptide sequence of 1 to 10 amino acids carrying an amino-ter-inal radical, • an aminoalkylcarbonyl chain of 1 to 10 linear carbon atoms or branched, • a linear or branched aminoalkylcarbonyl chain of 1 to 10 carbon atoms, carrying a hydroxyl radical or an amino radical, • a mixed construct consisting of at least one peptide sequence of 1 to 10 amino acids and at least one chain aminoalkylcarbonyl of 1 to 10 carbon atoms, linear or branched, • a mixed construction consisting of at least one peptide sequence of 1 to 10 amino acids and at least one aminoalkylcarbonyl chain of 1 to 10 carbon atoms, linear or branched, carrier of a hydroxyl radical or an amino radical.
2. 2. The biepitope compounds of Formula I according to claim 1, characterized in that Z comprises a peptide sequence that forms a broad epitope of troponin I with sequences Ei and / or E2.
3. 3. The biepitope compounds of Formula I according to any of claims 1 or 2, characterized in that Ei and / or E2, which are identical or different, represent a peptide sequence that includes a sequence that is selected from the group consisting of the following sequences: Sequence No. 1: -Thr Glu Pro His- Sequence No. 2: -His Ala Lys Lys- Sequence No. 3: -Pro Ala Pro He Arg Arg Arg- Sequence No.
4. 4: -Leu Leu Gly Ala Arg- Sequence No 5: -Arg Lys Asn Ile-. The biepitope compounds of Formula I according to any one of claims 1 to 3, characterized in that Z includes a sequence that is selected from the group consisting of the following amino acid sequences: -Gln Lys Met Gln-, -Gly Pro Asp Asn-, -Ala Met Met-, -Ala Lys Lys-, -Lys Ser Lys-, and -Pro Gly Asn Ser-.
5. 5. The biepitope compounds of Formula I according to any of claims 1 to 4, characterized in that Z represents: • a chain of Formula II -NH- (CH2) m-CO- (H) wherein m represents an integer from 1 to 10, • or a mixed construction of Formulas III a V: - [pep? -NH- (CH2) m-CO] p- (IH) - [pep? -NH- (CH2) m-CO-pep2] p- (IV) ~ [NH- (CH2) m -CO-pep2] p- (V) in which - m represents an integer from 1 to 10, - p represents an integer from 1 to 5, - pepi and pep2, which are identical or different, represent a peptide chain comprising from 2 to 10 amino acids.
6. 6. The biepitope compounds of Formula I according to any of claims 1 to 5, characterized in that Z includes a sequence that is selected from the group consisting of the following sequences: Sequence No. 6: -Gln Lys Met Gln- Sequence No. 7: -Gln Gly Pro Asp Asn- Sequence No. 8: -Gly Pro Asp Asn- Sequence No. 9: -Ala Met Met Lys Ser Lys (Gln Lys Met Gln) n-Sequence No. 10: -Ala Lys Lys Ala Met Met Lys Ser Lys- (Gln Lys Met Gln) n-Sequence No. 11: -Ala Met Met Lys Ser Lys- (Gln Lys Met Gln) n-Gln Ala- Sequence No. 12: -Ala Lys Lys Ala Met Met Lys Ser Lys- (Gln Lys Met Gln) n-Gln Ala- Sequence No. 13: -Lys Ser Lys- (Gln Lys Met Gln) n-Ala Met Met- Sequence No. 14: -Ala Lys Lys Lys Ser Lys- (Gln Lys Met Gln) n-Ala Met Met- Sequence No. 15: -Lys Ser Lys- (Gln Lys Met Gln) n-Ala Met Met- Sequence No. 16: -Ala Lys Lys Lys Ser Lys- ( Gln Lys Met Gln) n-Ala Met Met Gln Ala- Sequence No. 17: -Ala Met Met- Sequence No. 18: -Ala Lys Lys Ala Met Met- Sec uence No. 19: -Ala Met Met Gln Ala- Sequence No. 20: -Ala Lys Lys Ala Met Met Gln Ala- Sequence No. 21: -Pro Gly Asn Ser- Sequence No. 22: -Ala Lys Lys-NH- (CH2) m-CO- Sequence No. 23: -NH- (CH2) m-CO-Ala Met Met- Sequence No. 24: -Ala Lys Lys-NH (CH2) m-CO-Ala Met Met- Sequence No 25: -NH- (CH2) m-CO-Ala Met Met Gln Ala- Sequence No. 26: -Ala Lys Lys-NH- (CH2) m-CO-Ala Met Met Gln Ala- in which n represents a integer from 1 to 5 and m represents an integer from 1 to 10.
7. 7. The biepitope compounds according to any of claims 1 to 6, of the formulas Ia or Ib:? -Thr Glu Pro His-Z-Leu Leu Gly Ala Arg-? da)? Pro Pro Pro He Arg Arg Arg-Z-Thr Glu Pro His-? (Ib) in which - Z includes a sequence that is selected from the group consisting of the following sequences: Sequence No. 6: -Gln Lys Met Gln- Sequence No. 7: -Gln Gly Pro Asp Asn- Sequence No. 8 : -Gly Pro Asp Asn-Sequence No. 20: -Ala Lys Lys Wing Met Met Gln Wing-Sequence No. 26: -Ala Lys Lys-NH- (CH2) m-C0-Ala Met Met Gln Ala Sequence No. 12 : -Ala Lys Lys Wing Met Met Lys Ser Lys- (Gln Lys Met Gln) n-Gln Wing-Sequence No. 16: -Ala Lys Lys Ser Lys- (Gln Lys Met Gln) n-Ala Met Met Gln Ala- Sequence No. 21: - Arg Pro Gly Asn Ser- in which n represents an integer from 1 to 5 and m represents an integer from 1 to 10, -? represents an acetyl radical, a cysteinyl radical, a biotinyl or biocytinyl radical, optionally linked to a residue of a peptide sequence comprising a sequence selected from the group consisting of the following sequences: -Gly Asn Tyr Arg Ala Tyr Ala- - Gly Gly Asn Tyr Arg Ala Tyr Ala- -Asn Tyr Arg Ala Tyr Ala-, and -Arg Pro Ala-Y-? represents an amino radical or a peptide sequence comprising a sequence that is selected from the group consisting of the following sequences: -Ala Lys Glu and -Ala Lys Lys Lys Ser Lys
8. 8. The biepitope compounds according to any of claims 1 to 7, characterized in that they comprise a sequence that is selected from the group consisting of the following sequences: Sequence No. 27: -Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys 1 5 10 Ala Met Met Gln Ala Leu Leu Gly Ala Arg Ala Lys Glu- 15 20 25 Sequence No. 28: -Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys- 1 5 10 NH- (CH2) 5-CO-Ala Met Met Gln Ala Leu Leu Gly Ala Arg Ala 15 17 20 25 Lys Glu- Sequence No. 29: -Asn Tyr Arg Ala Tyr Thr Glu Pro His Wing Lys Lys Lys Ser 1 5 10 15 Lys Gln Lys Met Gln Ala Met Met Gln Ala Leu Leu Gly Ala Arg 20 25 30 Ala Lys Glu- Sequence No.30: -Gly Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys 1 5 10 Lys Lys Ser Lys Gln Lys Met Gln Ala Met Met Gln Ala Leu Leu 15 20 25 Gly Ala Arg Ala Lys Glu- 30 35 Sequence No. 31: -Gly Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys 1 5 10 Lys Lys Ser Lys Gln Lys Met Gln Gln Lys Met Gln Ala Met Met 15 20 25 Gln Ala Leu Leu Gly Ala Arg Ala Lys Glu- 30 35 Sequence No. 32: -Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys 1 5 10 Lys Ser Lys Gln Lys Met Gln Gln Lys Met Gln Ala Met Met 15 20 25 Gln Ala Leu Leu Gly Ala Arg Ala Lys Glu- 30 35 Sequence No. 33: -Arg Pro Ala Pro Ala Pro He Arg Arg Arg Pro Gly Asn Ser 1 5 10 Thr Glu Pro His Wing Lys Lys Lys Ser Lys- 15 20
9. 9. A biepitope compound according to any of the claims 8., characterized in that it is selected from the group consisting of the following compounds: Compound 5: Ac-Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys 1 5 10 Ala Met Met Gln Ala Leu Leu Gly Ala Arg Ala Lys Glu NH2 15 20 25 Compound 6: Ac-Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys- 1 5 10 NH- (CH 2) 5-CO-Ala Met Met Gln Ala Leu Leu Gly Ala Arg Ala 15 20 25 Lys Glu-NH2 Compound 10: Ac-Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys Lys 1 5 10 Ser Lys Gln Lys Met Gln Ala Met Met Gln Ala Leu Leu Gly Ala 15 20 25 Arg Ala Lys Glu-NH2 30 Compound 11: Ac-Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys 1 5 10 Lys Lys Ser Lys Gln Lys Met Gln Wing Met Met Gln Ala Leu Leu 15 20 25 Gly Ala Arg Ala Lys Glu-NH2 30 35 Compound 12: Ac-Gly Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys 1 5 10 Lys Lys Ser Lys Gln Lys Met Gln Gln Lys Met Gln Ala Met Met 15 20 25 Gln Ala Leu Leu Gly Ala Arg Ala Lys Glu-NH2 30 35 Compound 13: Ac-Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys 1 5 10 Lys Ser Lys Gln Lys Met Gln Gln Lys Met Gln Ala Met Met Gln 15 20 25 Ala Leu Leu Gly Ala Arg Ala Lys Glu-NH2 30 35 Compound 14: Cys-Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys 1 5 10 Lys-NH- (CH2) 5-CO-Ala Met Met Gln Ala Leu Leu Gly Ala Arg 15 20 25 Ala Lys Glu-NH2 Compound 15: Ac-Arg Pro Ala Pro Ala Pro He Arg Arg Arg Pro Gly Asn Ser Thr Glu Pro His Ala Lys Lys Ser Lys-NH2 15 20
10. 10. The use of a biepitope compound in accordance with any of claims 1 to 9 as a standard in an immunoassay of troponin I.
11. 11. A composition containing a compound of Formula I according to any one of claims 1 to 9, characterized in that it is dissolved in water, in plasma or in a buffer solution.
12. 12. A composition according to claim 11, characterized in that the regulatory solution is selected from the group consisting of phosphate, succinate or Tris-HCl buffer solutions.
13. 13. A composition according to any of claims 11 and 12, characterized in that the regulatory solution is one of the following regulatory solutions: - succinate buffer solution (pH = 5-6) containing Kathon, Régilait and AEDT, regulatory solution of succinate (pH = 5-6) containing Kathon, casein and AEDT, phosphate buffer (KH2PO4 / K2HPO4, pH = 6.5-7.5) containing Kathon, Régilait and AEDT, phosphate buffer (KH2PO4 / K2HPO4 0.1 M, pH = 6.5-7.5) containing Kathon, casein and AEDT.
14. 14. An immunoassay method using as a standard or control a biepitope compound of Formula I according to any of claims 1 to 9.
15. 15. The method according to claim 14, characterized in that the immunoassay is a sandwich type.
16. 16. A package for practicing the immunoassay, characterized in that it includes a biepitope peptide of Formula I, according to any one of claims 1 to 9 or a composition containing a biepitope compound of the Formula I according to any of claims 11 to 13.