Synthetic bi-epitope compound

Abstract

A bi-epitope compound of formula I: ##STR00001##
in which: E1 and E2, identical or different, each separately represents a peptide sequence including at least one epitope of an analyte; X and Y, identical or different, each separately represents a linking arm, the carrier molecule is soluble and Z represents an amino acid derivative bearing a thiol function prior to the bonding of same with the carrier molecule. The compound may be contained in a composition, used as a control or standard in an immunoassay and associated method, and/or provided in a kit for implementing an immunoassay.

Claims

1. An immunoassay system comprising one or more antibodies for binding an analyte and a bi-epitope compound of formula (I) as a control or standard: ##STR00005## wherein: E1 represents SEQ ID NO: 1 or SEQ ID NO: 3; E2 represents SEQ ID NO: 2; X and Y each represent a dimer of 8-amino-3,6-dioxaoctanoic acid; the carrier molecule is bovine serum albumin or immunoglobulin G; and Z represents a cysteine bonded with the carrier molecule.

2. The immunoassay system as claimed in claim 1, wherein the carrier molecule is bovine serum albumin.

3. The immunoassay system as claimed in claim 2, wherein E1 represents SEQ ID NO: 1.

4. The immunoassay system as claimed in claim 2, wherein E1 represents SEQ ID NO: 3.

5. The immunoassay system as claimed in claim 1, wherein the carrier molecule is immunoglobulin G and E1 represents SEQ ID NO: 1.

6. An immunoassay system comprising one or more antibodies for binding an analyte and a bi-epitope compound of formula (I) as a control or standard: ##STR00006## wherein: E1 represents SEQ ID NO: 1; E2 represents SEQ ID NO: 2; X represents SEQ ID NO: 25; Y represents SEQ ID NO: 26; the carrier molecule is bovine serum albumin; and Z represents a cysteine bonded with the carrier molecule.

7. An immunoassay system comprising one or more antibodies for binding an analyte and a bi-epitope compound of formula (I) as a control or standard: ##STR00007## wherein: E1 represents SEQ ID NO: 23; E2 represents SEQ ID NO: 5; X and Y each represent a dimer of 8-amino-3,6-dioxaoctanoic acid; the carrier molecule is bovine serum albumin; and Z represents a cysteine bonded with the carrier molecule.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) The invention will be understood more clearly by means of the following examples which are given by way of nonlimiting illustration, and also by means of the figures, in which:

(2) FIG. 1 is a graph giving the fluorescence signal RFV, determined by the VIDAS® automated device, emitted by a bi-epitope compound according to the prior art (REF Compound), a bi-epitope compound according to the invention (Compound 1) and a bi-epitope peptide corresponding to the bi-epitope compound 1 of the invention, but not coupled to a carrier molecule (noncoupled Peptide 1), as a function of their concentration;

(3) FIG. 2 is a graph giving the fluorescence signal RFV, determined by the VIDAS® automated device, emitted by the bi-epitope compound according to the prior art (REF Compound) and bi-epitope compounds according to the invention (Compounds 1 and 3), as a function of their concentration;

(4) FIG. 3 is a graph giving the fluorescence signal RFV, determined by the VIDAS® automated device, emitted by the bi-epitope compound according to the prior art (REF Compound) and bi-epitope compounds according to the invention (Compounds 1 and 4), as a function of their concentration.

EXAMPLES

Example 1: Peptide Synthesis

(5) The peptide syntheses were carried out using either the ABI 433A synthesizer from Applied Biosystems (Foster City, Calif., United States), or the Liberty synthesizer from CEM Corporation (Matthews, N.C., United States). The Rink Amide MBHA resin (Cat. No. 855003, Novabiochem®, Merck Millipore, Molsheim, France) was used as polymeric solid support.

(6) At the end of the chemical synthesis, the peptides were deprotected and cleaved from the polymer in the presence of a mixture of trifluoroacetic acid-ethanedithiol-triisopropylsilane-water (94/2.5/1/2.5 V/V/V/V) for approximately 2 hours. After elimination of the polymer by filtration, the peptides were isolated by precipitation from diethyl ether at 0° C.

(7) In order to increase their degree of purity, the peptides were purified by reverse-phase preparative high performance liquid chromatography (HPLC) on a Vynac Denali™ 120 Å C18, 10 μm column (Mandel Scientific Company Inc., Guelph, Ontario, Canada). Each peptide was eluted with a stepwise gradient of acetonitrile (from 0 to 95%) in aqueous solution containing 0.1% of trifluoroacetic acid, the percentage of acetonitrile of the steps having been chosen so as to optimize the isolation of the peak which corresponds to the peptide of interest. After this final step, two different analysis techniques were carried out in order to verify and characterize the peptides obtained.

(8) For each peptide, an analytical HPLC profile was generated on a Chromolith® High Resolution RP-18 encapped reverse-phase column (Merck Millipore, Molsheim, France). The elution was carried out by means of a linear gradient of acetonitrile (from 0 to 100%) in aqueous solution containing 0.1% of trifluoroacetic acid and monitored by measuring the absorbance at 214 nm. This analysis makes it possible to determine the level of purity of the peptide.

(9) Each peptide was also analyzed by liquid chromatography-mass spectrometry (LC/MS) on a Zorbax Eclipse Plus C18 RRHD 2.1×50 mm column, particle size 1.8 μm (Agilent Technologies, Santa Clara, Calif., United States) coupled to an Accurate-Mass Q-TOF LC/MS 6540UHD mass spectrometer (Agilent Technologies). This analysis makes it possible to determine the molar mass of the peptide.

(10) The sequences of the peptides synthesized and also the characterization results are presented in Table 1.

(11) TABLE-US-00003 TABLE 1 Sequences and characteristics of the peptides synthesized. Molar Amount mass obtained measured Identifier Analyte Sequence (mg) Purity (Daltons) Peptide 1 TnI ATEPHAKKK-Ado.sub.2-C-Ado.sub.2- 78.8 97% 2806.47 AGLGFAELQDL-NH.sub.2 (SEQ ID NOS: 1 and 2) Peptide 2 TnI ATEPHAKKKC-NH.sub.2 48.0 97% 1110.60 (SEQ ID NO: 29) Peptide 3 TnI AGLGFAELQDLC-NH.sub.2 50.0 95% 1234.60 (SEQ ID NO: 30) Peptide 4 TnI KISASRKLQLKT-Ado.sub.2-C- 59.7 99% 3169.75 Ado.sub.2-AGLGFAELQDL-NH.sub.2 (SEQ ID NOS: 2 and 3) Peptide 5 TnI ATEPHAKKKGGGSCSGGG 10.0 89% 2741.36 AGLGFAELQDL-NH.sub.2 (SEQ ID NO: 24) Peptide 6 PDEF- QAEDDPLQAK-Ado.sub.2-C- 32.0 99% 3215.50 A6 Ado.sub.2-WTGVLSPTQEYR-NH.sub.2 (SEQ ID NOS: 5 and 23)

(12) The abbreviation TnI corresponds to cardiac troponin I and PDEF-A6 to prodefensin-A6. The abbreviation Ado corresponds to 8-amino-3,6-dioxaoctanoic acid (CAS No.: 134978-97-5).

(13) TABLE-US-00004 TABLE 2 Summary of the bi-epitope compounds according to the invention obtained (formula I) and tested in terms of immunoreactivity Arm Arm Carrier Identifier Analyte Epitope E1 Epitope E2 X Y molecule Compound TnI ATEPHAKKK AGLGFAELQDL (Ado).sub.2 (Ado).sub.2 BSA 1 (SEQ ID NO:  (SEQ ID NO: 2) 1) Compound TnI KISASRKLQLKT AGLGFAELQDL (Ado).sub.2 (Ado).sub.2 BSA 2 (SEQ ID NO:  (SEQ ID NO: 2) 3) Compound TnI ATEPHAKKK AGLGFAELQDL (Ado).sub.2 (Ado).sub.2 IgG 3 (SEQ ID NO:  (SEQ ID NO: 2) 1) Compound TnI ATEPHAKKK AGLGFAELQDL GGGS SGGG BSA 4 (SEQ ID NO:  (SEQ ID NO: 2) (SEQ ID (SEQ ID 1) ID NO: ID NO: 25) 26) Compound PDEF- QAEDDPLQAK WTGVLSPTQEYR (Ado).sub.2 (Ado).sub.2 BSA 5 A6 (SEQ ID NO:  (SEQ ID NO: 5) 23)

(14) The abbreviation TnI corresponds to cardiac troponin 1 and PDEFA6 to prodefensin-A6. The abbreviation Ado corresponds to 8-amino-3,6-dioxaoctanoic acid (CAS No.: 134978-97-5). The abbreviation BSA corresponds to bovine serum albumin. The IgG is rabbit immunoglobulin G.

Example 2: Preparation of the Bi-Epitope Compounds

(15) The bi-epitope compounds were obtained by carrying out covalent couplings between, on the one hand, the peptides obtained in Example 1 and, on the other hand, carrier molecules. Table 2 presents in detail the various bi-epitope compounds according to the invention that were prepared. All these compounds correspond to formula I. Table 3, for its part, summarizes all of the couplings performed, while specifying the peptide and carrier-molecular pairs.

(16) The procedure for the couplings is the following:

(17) Firstly, the protein chosen as carrier molecule was activated in the presence of an excess of Sulfo-SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, CAS No.: 92921-24-9, Cat. No. 22322, Pierce, Thermo Scientific, Villebon sur Yvette, France). For the bovine serum albumin (BSA, Proliant Health & Biologicals, Ankeny, Iowa, United States), a 1/20 BSA/SMCC molar ratio was chosen. Thus, the BSA was diluted to 10 mg/ml in PBS (phosphate buffered saline), pH 7.2, and 53 μl of a solution of sulfo-SMCC at 25 mg/ml in water, prepared extemporaneously, was added dropwise. After incubation for 1 hour±5 minutes at 30° C.±2° C. in a water bath, with gentle magnetic stirring, the BSA-SMCC was dialyzed against a 50 mM phosphate buffer containing 150 mM NaCl, pH 6.8, in dialysis tubing having a cut-off threshold of 12 to 14 kDa. The dialysis was performed at ambient temperature and the dialysis bath was changed every hour, 3 times. After the dialysis, the protein concentration of the BSA-SMCC solution was determined by measuring the absorbance at 280 nm and this concentration was adjusted to 5 mg/ml in 50 mM phosphate buffer containing 150 mM NaCl, pH 6.8. This step makes it possible to modify the surface of the carrier molecule which from then on bears several reactive groups of maleimide type.

(18) The peptide to be coupled was dissolved at 5 mg/ml in 50 mM phosphate buffer containing 150 mM NaCl and 5 mM EDTA, pH 6.8, taking into account the purity. A 1/10 BSA/peptide molar ratio was chosen. Thus, 2.35 mg of BSA-SMCC at a concentration of 5 mg/ml (0.47 ml) were added to 1 mg of peptide at a concentration of 5 mg/ml (200 μl). This mixture was incubated for a minimum of 16 hours at 2/8° C. on a wheel. The reaction was then blocked by adding 0.1 M of 2-mercaptoethylamine (CAS No. 60-23-1, cysteamine) in 50 mM phosphate buffer containing 150 mM NaCl, pH 6.8, prepared extemporaneously. After incubation for 20±5 minutes on a wheel at the laboratory temperature, the peptide-BSA conjugate was dialyzed against a PBS buffer, pH 7.2, in dialysis tubing having a cut-off threshold of 12 to 14 kDa. The dialysis was continued for a minimum of 16 hours at 2/8° C. After the dialysis, the protein concentration was adjusted to a theoretical concentration of 2 mg/ml of BSA in PBS buffer, pH 7.2. The concentration of the peptide-BSA conjugate was then determined by measuring the absorbance at 280 nm. This step allows the reaction between the maleimide groups and the sulfhydril groups (—SH) of the peptide, at the level of the terminal or median cysteine depending on the peptide sequence to be coupled, in order to form thioether bonds.

(19) The compounds 1, 2, 4 and 5 were all obtained by applying the procedure described above. For the REF compound, which corresponds to the bi-epitope compound as described in patent U.S. Pat. No. 6,114,180, the same procedure was also applied, except that 2 peptides (peptide 2 and peptide 3) were placed in the presence of BSA-SMCC simultaneously and each peptide was coupled at a theoretical BSA/peptide molar ratio of 1/10. For the compound 3, peptide 1 was coupled to the rabbit polyclonal immunoglobulin G (bioMérieux). The procedure was identical, except that the BSA was replaced with another carrier molecule. The theoretical carrier molecule/peptide molar ratio was 1/10.

(20) TABLE-US-00005 TABLE 3 Summary of the peptide-carrier molecule couplings Identifier Peptide Carrier molecule Bi-epitope Compound 1 Peptide 1 BSA (bovine compounds serum albumin) according to Compound 2 Peptide 4 BSA the invention Compound 3 Peptide 1 Rabbit immunoglobulin G Compound 4 Peptide 5 BSA Compound 5 Peptide 6 BSA Bi-epitope REF Peptide 2 and BSA compound compound Peptide 3 according to the prior art (U.S. Pat. No. 6,114,180)

Example 3: Study of the Immunoreactivity of the Bi-Epitope Compound 1 According to the Invention

(21) The study of the bi-epitope nature of the compounds was carried out by means of a cardiac troponin I immunoassay using the VIDAS® immunoanalysis automated device (bioMérieux). The single-use tip serves both as solid phase for the reaction and as pipetting system. The cartridge is composed of 10 wells (X0 to X9 covered with a sealed and labeled sheet of aluminum. The first well (X0) comprises a precut part so as to facilitate the introduction of the sample. The last well (X9) is an optical cuvette in which the fluorescence of the substrate is measured. The various reagents required for the analysis are contained in the intermediate wells. All the steps of the test are carried out automatically by the instrument. They consist of a succession of cycles of suctioning/blowing back of the reaction medium. The cardiac troponin I immunoassay was carried out by means of a single-step sandwich test.

(22) a) Sensitization and Passivation of the Tips

(23) The characteristics and the suppliers of the antibodies used are presented in Table 4. The tips were sensitized with 300 μl of a solution of the 19C7 and B90 monoclonal antibodies each diluted to 2.5 μg/ml in a PBS buffer, pH 6.2. After approximately 20 h of incubation at +18/25° C. with the sensitizing solution, the tips were emptied. 300 μl of this same solution containing 10 g/l of bovine serum albumin are then added. The passivation continues at +18/25° C. overnight. The tips are emptied, dried, and then stored at +4° C. until use, in a moisture-free environment.

(24) TABLE-US-00006 TABLE 4 Antibodies used for the cardiac troponin I immunoassay Antibody Sequence of Supplier name Target E1 or E2 (Cat. No.) 19C7 TnI KISASRKLQLKT Hytest (SEQ ID NO: 3) (4T21-19C7) B90 TnI ATEPHAKKK SDIX (SEQ ID NO: 1) (B9085MA06-MA) 3D5F7 TnI AGLGFAELQDL bioMérieux (SEQ ID NO: 2) (noncommercial) 7B9 TnC NA Hytest (4T27-7B9) NA: not available. The abbreviation TnI corresponds to cardiac troponin I and the abbreviation TnC corresponds to cardiac troponin C. The abbreviation Cat. No. corresponds to the catalog reference of the supplier.

(25) b) Immunoassay Procedure

(26) The test compounds were diluted in a PBS-BSA buffer at various concentrations and assayed as sample.

(27) As soon as the VIDAS® tip is in contact with the sample, the immunological reaction begins because the capture antibodies are immobilized on this tip. The automated device mixes the test sample (135 μl) with 270 μl of the solution of conjugate. This solution contains the 2 monoclonal antibodies, 3D5F7 and 7B9, in the form of Fab′ fragments coupled to alkaline phosphatase. These conjugates were diluted to approximately 0.75 μg/ml in a 100 mM phosphate buffer, pH 6.4, also containing 150 mM of NaCl and filler proteins.

(28) The incubation lasts 6.8 minutes at 37° C. and enables the specific binding of the cardiac troponin I, or of the cardiac TnI bi-epitope compounds, or the cardiac TnI peptides, on the one hand to the antibodies adsorbed onto the tip and, on the other hand, to the conjugates. The unbound components are then removed by 3 washes with a 200 mM Tris buffer, pH 7.8, containing 300 mM NaCl and 0.2% Triton X-100. During the final revealing step, the 4-methylumbelliferyl phosphate substrate is suctioned up and then blown back in the tip; the enzyme of the conjugate catalyzes the reaction for hydrolysis of this substrate to 4-methylumbelliferone, the emitted fluorescence of which is measured at 450 nm. The value of the fluorescence signal (RFV=relative fluorescence value) is proportional to the concentration of the antigen present in the sample.

(29) Table 5 summarizes the fluorescence signals (RFV=relative fluorescence value) determined by the VIDAS® automated device when the immunoreactivity of the REF bi-epitope compound (prior art), of the bi-epitope compound 1 according to the invention and of the noncoupled peptide 1 (Example 1) is compared. FIG. 1 represents these same data in graph form. As a reminder, peptide 1 comprises 2 cardiac TnI epitopes, one recognized by the B90 capture antibody of the immunoassay previously described, and the other by the 3D5F7 detection antibody. In compound 1 according to the invention, peptide 1 is coupled via the median cysteine on BSA in order to ensure better antigen presentation thereof and improved stability. The REF compound has the same two TnI epitopes also coupled to BSA. Unlike compound 1, each of the 2 epitopes is in the form of an individual peptide (peptides 2 and 3) which has been coupled to BSA at the level of the terminal cysteine. Compound 1, the REF compound, which corresponds to a bi-epitope compound as described in patent U.S. Pat. No. 6,114,180, and peptide 1, which corresponds to a synthetic bi-epitope compound as described in patent application WO 98/24816, are both reactive in the cardiac TnI immunoassay, but their levels of reactivity are very different. Thus, in order to obtain a signal of approximately 1000 RFV, 21 μM of compound 1 are necessary compared with 1118 μM of the REF compound, that is to say approximately 50 times less. Compound 1 thus exhibits much better immunoreactivity than the REF compound. Moreover, the good dynamics of the VIDAS® signal obtained with Compound 1 is not reproduced with the REF compound, even when testing much higher concentrations of this compound. The noncoupled peptide 1 is much less well recognized than the two bi-epitope compounds coupled to BSA.

(30) TABLE-US-00007 TABLE 5 Immunoreactivity of the bi-epitope compounds 1, 3, 4 and REF and of cardiac TnI peptide 1 Compound 1 Compound 3 Compound 4 [c] REF (94 kDa) Noncoupled (188 kDa) (93 kDa) peptide compound Peptide 1 - peptide 1 Peptide 1 - Peptide 5 - in (89 kDa) BSA (28 kDa) IgG BSA ng/mL [c] S [c] S [c] S [c] S [c] S 0.5 — — 5.3 270 — — — — — — 1 — — 11 451 — — 5.3 175 11 60 2 — — 21 935 — — 11 360 21 118 5 56 62 53 1903 — — 27 882 54 295 10 112 120 106 3606 — — 53 1688 107 545 20 224 223 213 5911 713 2 106 3276 214 1100 50 559 617 — — 1781 21 266 6209 535 2569 100 1118 997 — — 3562 69 — — — — 200 2236 1484 — — 7125 356 — — — — 500 5589 2102 — — 17811 1825 — — — — 1000 11179 2155 — — 35623 3798 — — — — 2000 — — — — 71245 6140 — — — — 4000 — — — — 142491 7542 — — — — The abbreviation [c] corresponds to the concentration in μM of the compound. The abbreviation S corresponds to the signal in RFV.

Example 4: Comparison of the Immunoreactivity of the Bi-Epitope Compounds According to the Invention Using Various Carrier Molecules

(31) In this example, peptide 1 which comprises 2 different epitopes of cardiac TnI was coupled to 2 different carrier molecules: BSA (compound 1) and rabbit immunoglobulin G (compound 3). The obtaining of these bi-epitope compounds is described in Example 2. The comparison of the immunoreactivity of these compounds in the cardiac TnI immunoassay was carried out as described in Example 3 and the results are presented in Tableau 5 above and FIG. 2, which represents a graph giving the RFV fluorescence signals emitted by the various compounds, bi-epitope compound according to the prior art (REF Compound) and bi-epitope compounds according to the invention (Compounds 1 and 3), as a function of their concentration. The results show that compounds 1 and 3 are both reactive in the cardiac TnI immunoassay and exhibit a comparable reactivity which is much greater than that observed for the REF compound.

Example 5: Comparison of the Immunoreactivity of the Bi-Epitope Compounds According to the Invention Using Various Spacer Arms

(32) In this example, the bi-epitope compounds compared differ only in terms of the spacer arm. In the case of compound 1, the two spacer arms are identical, it is a dimer of the Ado artificial amino acid. Compound 4, for its part, comprises the GGGS sequence as arm X and the SGGG sequence as arm Y. As a reminder, the two compounds have 2 cardiac TnI epitopes and the carrier molecule is BSA. The obtaining of these bi-epitope compounds is described in Example 2. The comparison of the immunoreactivity of these compounds in the cardiac TnI immunoassay was carried out as described in Example 3 and the results are presented in Table 5 above and FIG. 3, which represents a graph giving the RFV fluorescence signals emitted by the various compounds, bi-epitope compound according to the prior art (REF Compound) and bi-epitope compounds according to the invention (Compounds 1 and 4), as a function of their concentration. The results show that compound 4 comprising GGGS and SGGG arms is less well recognized than compound 1 comprising Ado arms, but is much greater than the REF compound.

Example 6: Stability Tests

(33) Compounds 1 and 2 were diluted to 3.75 ng/ml in the various buffers indicated in Table 6. A first assay was carried out on D0, the day on which the solutions were prepared. The values obtained served as a reference for monitoring the stabilities. The diluted solutions of the bi-epitope compounds were stored at +2/8° C. and assayed on D7 (7th day after preparation). Table 6 below presents the variation in the RFV signal of the immunoassay between D0 and D7 (Signal D7/Signal D0×100). Compounds 1 and 2 are stable and their antigenic properties are preserved when they are stored at +2/8° C. for 1 week.

(34) TABLE-US-00008 TABLE 6 Stability of compounds 1 and 2 at +2/8° C. for 7 days Dilution buffer Compound 1 Compound 2 Citrate pH 5, 150 mM NaCl, 50 g/l BSA 102% 98% Citrate pH 6, 150 mM NaCl, 50 g/l BSA 99% 95% PBS, pH 6.2, 50 g/l BSA 99% 89%

(35) In a second step, a stability study of longer duration was carried out for compound 1 only, diluted in PBS, pH 6.2, containing 50 g/l of BSA. When stored at +2/8° C., compound 1 is stable in dilute solution: 97% of the signal of the immunoassay is found at 1 month storage, 94% at 3 months and 90% at 6 months. When stored at +18/25° C., compound 1 is stable in dilute solution for approximately 1 month (88% of the signal is found). It is also important to note that compound 1 is capable of withstanding at least three freezing/thawing cycles at −20° C. without any degradation of its antigenic properties. A higher number of freezing/thawing cycles was not tested.

(36) All of these results demonstrate the excellent stability of the solutions of compound 1 according to the invention.

Example 7: Study of the Immunoreactivity of the Bi-Epitope Compound 5 According to the Invention Comprising a Mimotope

(37) The bi-epitope compound 5 was designed so as to operate as a control and/or standard and/or adjuster during a prodefensin A6 immunoassay. Compound 5 combines a linear epitope (QAEDDPLQAKL) and a mimotope (WTGVLSPTQEYR). The prodefensin A6 immunoassay was carried out using the VIDAS® automated immunoanalysis device (bioMérieux), according to the protocol described in application WO 2010/112777, namely using, as capture antibody, the 12H4E1 clone (bioMérieux), which recognizes the linear minimal epitope of sequence EDDPLQ, and, as detection antibody, the 1H8C9 clone (bioMérieux), the epitope of which is not linear but is a mimotope of sequence WTGVLSPTQEYR. These epitopes/mimotopes are those found in compound 5.

(38) Table 7 below summarizes the fluorescence signals (RFV=relative fluorescence value) determined by the VIDAS® automated device when various concentrations of compound 5 (molecular molar mass: 98 155 Daltons) are tested. Compound 5 is indeed reactive in the prodefensin A6 immunoassay.

(39) TABLE-US-00009 TABLE 7 Immunoreactivity of the bi-epitope compound 5 [c] peptide in ng/ml [c] in mM RFV signal    17.5 0.2 385    35 0.4 583   175 1.8 1306   1750 18 2637 17 500 178 3448

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