WO2003075010A2

WO2003075010A2 - Non-peptide immunologic tracer precursors comprising a tyrosyl-(x)n or lysine-(x)n tyrosine motif

Info

Publication number: WO2003075010A2
Application number: PCT/FR2003/000707
Authority: WO
Inventors: Anny Cupo; Cécile Le Saint; Jean-Pierre Vincent
Original assignee: Centre National De La Recherche Scientifique -Cnrs-
Priority date: 2002-03-05
Filing date: 2003-03-05
Publication date: 2003-09-12
Also published as: WO2003075010A3; FR2836996A1; AU2003227812A1; AU2003227812A8

Abstract

The invention concerns an immunologic tracer characterized in that it comprises a non-peptide hapten coupled with a TYR-(X)n-LYS or LYS-(X)n-TYR motif, wherein X is selected among a single bond, an amino acid, except for Lysine, Glutamine, Asparagine or Tyrosine, a succinyl group, a citrate group, a hydroxymethyl-(CH(OH)- group, a methylene group: -CH2-, an oxygen atom, a sulphur atom, an oxyethylene-CH2-O- group, or a methylamine -CHNH- group, and n is an integer ranging between 1 and 20, preferably between 1 and 10, more preferably between 1 and 2. The invention also concerns methods for preparing said precursors, the use thereof for preparing immunologic markers useful in competitive immunologic assays.

Description

PRECURSORS OF NON-PEPTIDE IMMUNOLOGICAL TRACERS

COMPRISING A TYROSYL- (X) _n -LYSINE, OR LYSINE- (X) „- PATTERN

TYROSINE, METHOD OF PREPARATION AND THEIR APPLICATIONS

The present invention relates to the field of analytical immunology and in particular the preparation of tracers useful for the immunological assays of haptens.

Among all the methods allowing quantification in biological media such as, for example, intracellular content, blood, cerebrospinal fluid (CSF), saliva, or urine, small molecules, such as those used in anti-HIV therapy, or in oncology, immunoassays have many advantages in terms of sensitivity, because they are suitable for measuring low intracellular concentrations of said molecules and also in terms of speed due to the time required to prepare the samples. The assays of haptens, molecules of small size, are carried out by means of specific immunological assays, hereinafter called “assays by competition” in which, the hapten to be assayed is placed in the presence of a specific antibody, the antibody anti-hapten. The bond between these two molecules, haptene and the anti-hapten antibody, is measured in the presence of a third molecule, analog of haptene, generally labeled, called "tracer".

These assays allow their realization with samples of reduced size, as much in volume as in number of cells, they can be carried out in complex mixture and are well adapted to the follow-up of patients in hospital environment. In the context of the present invention, the following terms have the meanings indicated below:

Haptene: A small molecule, generally having a molecular weight of less than 5000 daltons capable of having antigenic properties and of inducing an immune response only when it is coupled to a larger carrier molecule.

Carrier molecule: A molecule of high molecular weight, generally greater than 50,000 daltons and capable of inducing an immune response in an animal to which it is administered. When it is a protein, the carrier molecule must be phylogenetically distant from the species in which the immunization is carried out.

Antigen: A molecule against which the immune system is able to react. The term “antigen” also denotes a molecule which reacts with an antibody specific for said molecule. Immunogen: an antigen, in particular when the latter is obtained by coupling a hapten and a carrier molecule in order to allow the former to induce an immune response and in particular a humoral response with production of antibodies having an affinity for said hapten.

Tracer: molecule derived from a hapten analog comprising a marker.

Precursor of a tracer: molecule derived from a hapten analog capable of reacting with a marker to obtain said tracer.

Marker: A molecule or a particle having physicochemical properties, such as emission or the absorption of light, radioactive or other energy, allowing its detection, by any means.

Examples of markers that may be mentioned include: enzymes, radioactive isotopes, fluorochromes.

The use of "*" indicates a radioactively labeled compound, particularly with Iodine ¹²⁵

Immunological assays for small molecules which are of particular importance in public health are, for example, the assays of nucleoside and non-nucleoside analogs, often used during anti-viral treatments such as those used for the inhibition of the HIV protease or reverse transcriptase or in many anti-cancer treatments, such as methotrexate or vincristine or when it is a question of detecting traces of molecules during the screening of drugs such as narcotics, or during detection of non-peptide hormones, or even neuromediators.

In the context of the present invention, the following definitions are intended:

- natural nucleoside: a molecule formed by the association of a purine (adenine or guanine) or pyrimidine (cytosine, uracil and thymine) base with a pentose residue (beta-D-ribofuranose or beta-D- deoxyribofuranose).

- nucleoside analog (AN): any molecule composed of a modified base or not coupled to a ribose or an analog thereof.

Such analogues are for example described in the document: (Périgaud, C. et al. Nucleosides and Nucleotides 1992, vol. 11 (2-4), pages 903-945).

Thus, compared to a natural nucleoside, an analog may include modifications of ribose, such as for example those mentioned on page 607 of the aforementioned document, such as the substitution of one or more atoms, the displacement of the bond between the base and the sugar. , anomeric inversions (beta-alpha), the addition of various functions, the inversion, substitution or elimination of hydroxyl groups, the modification of the size of the cycle (pyranose), the inversion of the configuration (D— L) or alternatively the breaking of the cycle (acyclonucleosides) or modifications of the heterocyclic base such as for example those indicated on page 908 of said document, for the analogues: Fura, FdUrd, Thi-Gua, 6-MP, AraC or Fludarabine phosphate.

By way of example of such nucleoside analogs, mention may also be made of:

Adenosine, S-Adenosyl-L-methionine, 3'- Azido- 3 '-deoxythymidine, Carbovir, Cordycepine, Cytidine, Cytosine-b-D-arabinoside, Deoxycytidine,

Deoxytubercidine, 2 '-Deoxyuridine, Formycin A, Formycin B, Ganciclovir, Guanosine, Inosine, Puromycin, Ribavirin, Sangivamycin, Saquinavir, Thymidine, Tubercidine, Uridine.

Other haptens whose dosage is particularly interesting are, for example, non-nucleoside inhibitors of NNRTI reverse transcriptase. There are approximately 30 families of non-nucleoside reverse transcriptase inhibitors which will bind to a hydrophobic pocket of reverse transcriptase.

The first NNRTIs were a benzodiazepine (TIBO) and 1- (2 -hydroxyethoxymethy1) -9- (ρhenylthio) thymine (HEPT) and to date around thirty NNRTI classes have been developed.

Among the most used NNRTIs, the following may be mentioned: dipyridodiazepinones, such as Nevirapine, pyridinones, bis (heteroaryl) piperazines, TSAO derivatives, alpha-anilinophenylacetamide (alpha-APA), quinoxalines, benzoxaninone DMP266 (Efavirenz) . With regard to the development of tracers suitable for immunological tests, the visualization of binding proteins, such as receptors, antibodies, or even enzymes, was at the origin of the development of numerous tracers resulting from technological tricks.

Labeling techniques have favored the emergence of modified tracers in regions which do not affect their interaction with the target molecule, allowing this tracer to have the best affinity for the binding protein. This concept has applied to ligand-receptor interactions as well as antigen-antibody interactions.

With regard to the development of a radioactive or enzymatic tracer allowing the characterization of anti-hapten antibodies and the development of the corresponding immunological tests, several strategies have been adopted.

For radioactive labeling, more often than not, certain studies report the coupling to the molecule of interest, of one of the two amino acids capable of being radiolabelled with iodine, histidine and tyrosine. Tyrosine can be introduced using tyrosine methyl ester, tyramine or the dipeptide Glycyl- Tyrosine. (Boulenguez P et al .; J Neurochem 1992 Mar; 58 (3): 951-9 Biochemical and pharmacologi cal char ac te izat ion of serotonin-0-carboxymethylglycyl [ ¹²⁵ l] iodotyrosinamide, a new radioiodinated probe for 5-HT1B and 5-HTlD binding sites, and Garrigues AM. Et al. FEBS Lett 1987 Nov 30; 224 (2): 267-71, Ha l op e ri dol - succinylglycyl [ ¹²⁵ I] iodotyrosine, a novel iodinated ligand for dopamine D2 receptors ).

The introduction of an absorbent group into the ultraviolet (UV) range, such as the Tyr residue, also makes it possible to follow the formation and the purification of the tracer by ultraviolet spectrometry.

On the other hand, to obtain an enzyme tracer, the most frequently used strategy has been that of coupling the hapten on the amino groups of an enzyme, such as for example peroxidase, alkaline phosphatase, l acetylcholinesterase, etc.

In the case of ligands, the Bolton Hunter reagent (N-succinimidyl-3 (4-hydroxyρhenyl) propionate) has been most commonly used for iodine 125 radiolabelling. Radiolabelling can be performed either before or after coupling of the reagent to the target molecule. On the same principle, it is also possible to carry out labeling with ³ H using the tritiated N-hydroxysuccinimide propionate.

However, it was observed during the development of certain assays, such as for example the immunoassay of Saquinavir, that the antigen coupled with the dipeptide Glycyl-Tyrosine was not an adequate tracer. Indeed, the hemisuccinate of Saquinavir coupled to the dipeptide Glycyl-Tyrosine has a poor affinity for the antibody if compared to that of Saquinavir modified by a succinyl link (form hapten). One of the hypotheses which can account for this observation is that tyrosine, by hydrophobic interaction and / or by steric hindrance, interferes in the antigen-antibody interaction. One of the main requirements of these immunological tests is indeed that of having a good sensitivity with respect to the molecule which must be measured.

The sensitivity of the test is defined as the minimum quantity that can be reliably detected, generally obtained from 15 to 20% inhibition of the test.

The IC 50 is defined by the concentration of unlabeled antigen capable of inhibiting 50% of the antigen-antibody bond. Under the conditions of the immunological test, by using a dilution of antibodies giving 50% of binding in a test in competition and in lack of reagent, as many antibodies as of labeled antigen, the IC 50 is a good reflection of the 'affinity. The affinity is determined by the Scatchard method.

This characteristic of immunoassays depends on several factors, such as the affinity of the antibodies involved, obtained by immunization of an animal with an immunogen, obtained by coupling said hapten to a carrier molecule.

But in the particular case of immunological assays "by competition" used for the assays of small molecules, the sensitivity of the immunological test is also dependent on the quality of the "competition" between the antibody, the molecule to be assayed and the mimic tracer. said molecule to be assayed.

In other words, the sensitivity of the “competitive” immunoassay depends on the capacity of the tracer to move the molecule to be measured, the hapten, when the latter has already been recognized by the antibody directed against it, and is found therein.

In order for said tracer to be able to effect such a displacement, it must have the structure as close as possible to that of the hapten.

It should however be taken into account that the structure of the hapten has often been modified by the very fact of its coupling with the carrier molecule, in order to obtain the immunogen.

As a result, the structure of the hapten put in the presence of the immune system for the production of specific antibodies is somewhat different from that of the free hapten and consequently the antibodies induced often have a better affinity for the structure of the l 'haptene slightly modified only for the structure of the unmodified free form.

The Applicant has now designed immunological tracers which are closer to the structure of the hapten modified by its coupling to the carrier molecule than to the structure of the free hapten and which allow the development of immunological assays by competition having a increased sensitivity. The goal is to obtain a tracer whose structure is similar to that of the immunogenic form of the hapten used for the production of anti-hapten antibodies and against which the tracer must enter into competition. This characteristic is essential in terms of the sensitivity of the immunological test for measuring the hapten.

The present invention therefore relates to a precursor of an immunological tracer comprising a non-peptide hapten coupled to a TYR- (X) _n -LYS or LYS- (X) _n -TYR motif in which X is chosen from a single bond, an amino acid, with the exception of Lysine, Glutamine, Asparagine or Tyrosine , a succinyl group, a hydroxymethyl group —CH (OH) -, a methylene group - CH ₂ -, an oxyethylene group —CH ₂ -0- or a methylamine group -CHNH- and n is an integer between 1 and 20 , preferably between 1 and 10, most preferably between 1 and 2. According to a preferred embodiment, the non-peptide hapten is coupled to a TYR-LYS or LYS-TYR motif.

The introduction into the hapten of the Tyrosyl- (X) _n- Lysine motif makes it possible both to carry out easy radioactive labeling and to mimic the peptide link between the hapten and the carrier protein. The functional group of the Tyrosine residue, the phenol group, is in fact used to carry out a radiolabelling with iodine 125 and the Lysine residue mimics the link established between the hapten and the carrier protein via the amino functional group. primary of its side chain.

The use of a tracer uniting these two essential characteristics allows the implementation of radioimmunological tests of high sensitivity and consequently allows the quantification of the epitopes recognized by the antibody even when these are poorly represented in a biological medium.

The coupling is oriented by reacting the ε amino group of the lysine of the Tyrosyl- (X) _n - Lysine motif on a carboxylic acid residue of the modified hapten.

According to preferred modes of implementation, the precursor of immunological tracers of the invention include non-peptide aptens chosen from: nucleoside analogs, non-nucleoside inhibitors, anticancer compounds, antiviral compounds (antiproteases, entry inhibitors), narcotic drugs, drugs, hormones non-peptide, neuromediators.

When the hapten is a nucleoside or a nucleoside analog, it can be chosen from analogs of purine bases: or analogs of pyrimidine bases such as for example: Acyclovir, Adenosine, S-Adenosyl-L-methionine, 2 ′, 3'- dideoxyadénosione (ddA), 2 ', 3' -didehydro-2 ', 3' - dideoxythymidine (d4T), 2 ', 3' -dideoxy-3 '-thiacytidine (3TC), 3'- Azido-3' -deoxythymidine (AZT), Carbovir,

Cordycepin, Cytidine, Cytosine-b-D-arabinoside,

Deoxycytidine, Deoxytubercidine, 2 '-Deoxyuridine, Formycin A, Formycin B, Ganciclovir, Guanosine, Inosine, Puromycin, Ribavirin, Sangivamycin, Thymidine, Tubercidine, Uridine, Abacavir, 3-fluoro- 2', 3 '-dideoxythymidine Fura, FdUrd, Thi-Gua, 6-MP, AraC or Fludarabine phosphate.

When hapten is an anti-cancer compound, it can be chosen from all the classes of anti-cancer, such as antimetabolites, alkaloids and carcinostats.

Among the antimetabolic anticancer products, mention may be made of: analogues of purine bases: 6-mercaptoρurine, cladribine, fludarabine.

- analogues of pyrimidine bases: 5-fluorouracil, cytarabine, gemcitabine, Tenofovir.

When hapten is an antiviral compound, it can be chosen from the following classes: antiproteases, entry inhibitors, integrase inhibitors; it can be chosen from: Saquinavir (SON), Indinavir (IDV), Νelfinavir (ΝFV), Ritonavir (RTV), Amprenavir (APV), Lopinavir (LPV), Tipranavir, Atazanavir, T20 and T22.

Among the narcotics, mention may be made of psychotropic drugs such as cocaine, morphine, heroin and their related derivatives or tranquilizers. The subject of the invention is also a method for obtaining a precursor of an immunological tracer such as those defined above comprising the coupling between a carboxylic derivative of hapten and the ε-amine group of the Lysine of the motif Tyrosyl- (X) n- Lysine.

Sometimes, the haptens naturally have a carboxylic acid group, sometimes it is chemically introduced therein, according to methods known to those skilled in the art, to allow their coupling to a carrier protein, such as, for example, hemocyanin from Patelle or KLH. In such cases, the coupling is carried out by formation of a peptide bond between the carboxylic acid function of the hapten and the free amino functions of the side chains of the Lysine residues of the carrier protein.

The vast majority of haptens do not have a group capable of being engaged in the formation of a peptide bond with the free amino residues of a carrier protein of the KLH type. Consequently, the first step in implementing the tracers of the invention consists in modifying said hapten so as to introduce a free carboxylic acid function, when the latter is not present naturally, followed by coupling of the carboxylic derivative of said hapten with the motif Tyrosyl- (X) _n -Lysine.

Thus, more particularly, the coupling is carried out by activation of the carboxylic acid of the hapten or of the modified hapten in the presence of ethyl chloroformate followed by the reaction of said activated derivative with the ε-amine group of the Lysine of the motif. Tyrosyl- (X) _n -Lysine or alternatively by means of a carbodiimide in the presence of N-hydroxysuccinimide. The first step of the coupling between the carboxylic derivative of hapten and the Tyrosyl- (X) _n- Lysine motif is carried out by activation of the carboxylic acid function of the hapten modified by ethyl chloroformate, particularly suitable for obtaining tracer precursors useful for the detection of HIV protease inhibitors and other antivirals, both nucleoside and non-nucleoside, either by coupling with a carbodiimide in the presence of N- hydroxysuccinimi.de, particularly suitable for obtaining tracer precursors useful for the detection of nucleoside analogues, in order to obtain a reaction intermediate.

This reaction intermediate reacts with the free amino group ε of Lysine when coupling is carried out in a buffer medium at pH between

8 and 10, preferably at a pH between 8.5 and 9, so as to promote the reactivity of the ε amino group.

The invention also relates to an immunological tracer comprising the above precursor defined or obtained by one of the below mentioned methods coupled to a marker.

According to a first embodiment, the tracer of the invention comprises a marker coupled to the Tyrosine functional group.

According to a second embodiment, the tracer of the invention comprises a marker on the functional group of Lysine. According to a preferred implementation, the immunological tracers of the invention comprise markers chosen from an enzyme, a chromophore, a magnetic particle, a colored particle, a luorochrome, the green fluorescence protein (GFP), the f luorescein, rhodamine, red texas, a metallic particle, biotin, an avidin-biotin complex, a streptavidin-biotin complex, coupled on the free amino group of lysine of the Lys- (X) _n -Tyr motif.

According to a particular embodiment, the immunological tracer of the invention comprises a radioelement on the tyrosine residue and preferably said radioelement is chosen from radioactive atoms: ¹²⁵ I, ³ H.

Particularly preferred tracers are the radioactive immunological tracers: Saquinavir-HS-

KY (or YK) ¹²⁵ I, Saquinavir-Citrate-KY (or YK) ^{: 25} I, ddA-

HS-KY (or Y_K) ¹²⁵ I, ddA-Citrate-KY (or YK) ¹²⁵ I, NFV-Ac- βAla-KY (or YK) ¹²⁵ I, NFV-HS-KY (or YK) ¹²⁵ I, NFV- Citrate-

KY (or YK) ¹²⁵ I, Ritonavir-HS-KY (or YK) ^{1 5} I, Ritonavir- Citrate-KY (or YK) ¹²⁵ I, AZT-HS-KY (or YK) ¹²⁵ I, AZT-

Citrate-KY (or YK) ¹²⁵ I, d4T-HS-KY (or YK) ¹²⁵ I, d4T ~

Citrate-KY (or YK) ¹²⁵ I, 3TC-HS-KY (or YK) ¹²⁵ I, 3TC-

Citrate-KY (or YK) ¹²⁵ I, IDV-HS-KY (or YK) ¹²⁵ I, IDV-

Citrate-KY (or YK) ¹²⁵ I, and the enzymatic immunological tracers: Saquinavir-HS-KY (or YK) peroxidase,

Saquinavir-Citrate-K-Y (or Y-K) peroxidase, ddA-HS-K-Y

(or Y-K) peroxidase, ddA-Citrate-K-Y (or Y-K) peroxidase,

NFV-HS-KY (or YK) peroxidase, NFV-Citrate-KY (or YK) peroxidase, Ritonavir-HS-KY (or YK) peroxidase, Ritonavir-Citrate-KY (or YK) peroxidase, AZT-HS-KY (or YK) peroxidase, AZT-Citrate-KY (or YK) peroxidase, d4T-HS- KY (or YK) peroxidase, d4T-Citrate-KY (or YK) peroxidase, 3TC-HS-KY (or YK) peroxidase, 3TC-Citrate- KY (or YK) peroxidase, IDV-HS-KY (or YK) peroxidase , IDV-Citrate-KY (or YK) peroxidase.

The subject of the invention is also a process for obtaining an immunological tracer comprising a step of coupling a marker on the tyrosine residue or on the lysine residue of a precursor defined above or obtained by a process below. above mentioned.

According to a particular embodiment of said method, the motif TYR- (X) _n -LYS or LYS- (X) _n -TYR is previously marked before its coupling to the non-peptide hapten.

In particular, the prior labeling of the TYR- (X) _n -LYS motif is carried out either by means of a radioactive marker on the tyrosine residue or by means of an enzymatic marker on the lysine residue.

In all cases, the reaction is monitored by high pressure liquid chromatography. The coupling products are characterized by mass spectrometry (MALDI-TOF). For the preparation of radioactive tracers, after purification by chromatography, each derivative obtained is radiolabelled with iodine 125. The radiolabelling is carried out in the presence of radioactive sodium iodide and an oxidant in a phosphate buffer at pH 7.5. Depending on the nature of the derivatives and their sensitivity to oxidation, different oxidants can be used; by way of example, mention may be made of: chloramine T, iodogen, lactoperoxidase and glucose oxidase.

The radiolabelled derivatives are purified by high pressure liquid chromatography using a support, solvents and gradients adapted to each molecule.

The subject of the invention is very particularly the radiolabelled immunological tracers: ANN-Lys-Tyr- ¹²⁵ I, AN-Lys-Tyr- ¹²⁵ I.

Still according to another embodiment, the invention relates to an immunological tracer comprising an enzymatic marker on the lysine residue and preferably said enzymatic marker is chosen from the group comprising peroxidase, alkaline phosphatase, urease. The invention is illustrated below by way of nonlimiting examples in which the process for the preparation of tracer precursors with the motif Tyr- (X) n-Lys, immunological tracers derived from them, as well as described is described. that their use for immunological assays and by means of the appended figures in which:

- Figure 1 illustrates the comparative analysis of the specificity of anti-saquinavir antibody by means of an immunoassay using different tracers, among which the tracer SQV-HS-Y-K * of the invention.

- Figure 2 illustrates the comparative study, from a sampling of plasmas, between immunological assay techniques, ELISA and RIA using the tracer SQV-HS-KY * and chromatographic for Saquinavir and shows a good correlation between reference technique (HPLC) and the RIA assay implemented with the SQV-HS-KY * tracer. EXPERIMENTAL PART. EXAMPLES

1-Dipeptide coupling protocol.

The coupling of the dipeptide to the carboxylic acid function of the modified hapten can be carried out either on the terminal amino function of the dipeptide or on the ε amino group of the lysine. The monodirectional coupling on one or the other of these two amino functions can be favored by using conditions of pH and ratio of particular concentrations. The coupling product must then be isolated by purification by high pressure liquid chromatography (HPLC). The choice of experimental conditions, that is to say the orientation of the coupling, is dictated by the need to obtain the best match between the structure of the immunogenic form and that of the tracer so as to obtain the best affinity for immunoassay.

A) Protocol used for the modified antiproteases: For the modified antiproteases containing a free carboxylic acid function, the conditions for coupling to the dipeptide Tyrosyl-Lysine were as follows:

The coupling is carried out in two stages: a) the carboxylic acid function on the hapten (1 equivalent) is activated beforehand by ethyl chloroformate (2.2 equivalents) in dimethylformamide in the presence of triethylamine (2.5 equivalents) ), for 10 minutes cold (water and ice bath). b) the dipeptide Tyrosyl-Lysine (1.5 equivalents), previously dissolved in a buffer 50 mM borate phosphate, at pH 8 - 9, is added to the reaction medium.

The mixture is then placed under stirring at 4 ° C. The coupling is monitored by high pressure liquid chromatography.

The reaction is stopped by dilution in water and freezing, when the quantity of the desired coupling product no longer changes and / or when side products of the reaction appear. B) Protocol used for the modified nucleoside analogues:

The glycosidic bond of nucleoside analogs is sensitive in an acid medium. Therefore the use of ethyl chloroformate which releases hydrochloric acid upon activation of the carboxylic acid function is discarded and activation is carried out with dicyclohexylcarbodiimide (DCC) in the presence of N-hydroxysuccinimide.

The coupling takes place in two stages: a) After modification of the hydroxyl in 5 ′ of the nucleoside analogues of ribose by a hemisuccinate link, the free carboxylic acid function (1 equivalent) is activated in the presence of DCC (3.6 equivalents ) and N-hydroxysuccinimide (3.6 equivalents) in dimethylformamide for 3 hours at room temperature. b) the dipeptide tyrosyl-lysine _(1/5 equivalents) is predissolved in borate buffer 50 mM phosphate, pH 8.9 and then added to the reaction medium.

The mixture is stirred at room temperature. The monitoring of the coupling is carried out by chromatography high pressure liquid on a C18 reverse phase column. The elution gradient is indicated in table 1 below,

Table 1

and the reaction is stopped as described above.

2-Iodine 125 radiolabelling protocols.

The iodization conditions (nature of the oxidant and conditions of purification by HPLC) have been developed as a function of the sensitivity to oxidation and to the degradation of each molecule.

Generally, 1 equivalent of Tyrosine and 0.5 equivalent of ¹²⁵ INa are placed in a 250 mM Phosphate buffer, pH 7.5 in the presence of the oxidizing agent:

Chloramine T: 10 μg per equivalent of

Tyrosine.

Iodogen 100 μg per equivalent of

Tyrosine.

Lactoperoxidase: 50 μg in the presence of hydrogen peroxide, either added to the medium (at the rate of 3 times 10 μl at 0.01% by volume), or produced in situ by the addition of glucose oxidase (5 μg) and glucose (0.5 μmoles).

The iodization reaction, carried out for 1 minute, is stopped either by adding an excess of tyrosine (100 equivalents) or by adding metabisulfite of sodium or MBS (10 μg). The ¹²⁵ I / Tyr molar ratio is kept below one throughout the reaction to avoid the formation of diiodotyrosines.

The chromatography support and the elution conditions (nature of the eluent and gradient) used for the purification are defined for each of the molecules.

RESULTS

Concerning Saquinavir, the attachment of the dipeptide Glycyl-Tyrosine to the hemisuccinate of Saquinavir leads to a decrease in the affinity of a factor of 100 compared to the hemisuccinate of Saquinavir whereas the modified haptene (hemisuccinate of Saquinavir) and the derivative comprising the Tyrosyl-Lysine dipeptide are recognized with good affinity and in an equivalent manner.

This result indicates that, in the case of the anti-Saquinavir antibody, the choice of the dipeptide Tyrosyl-Lysine is more judicious than the dipeptide Glycyl-Tyrosine. Taking into account the results obtained for antiproteases, for nucleoside analogs and in particular ddA, the coupling of the dipeptide Tyrosyl-Lysine to the carboxylic acid group of the hapten modified by succinylation (in 5 'on ribose) was carried out directly .

1. Characterization of the antibodies.

Antiproteases (Saquinavir (SQV), Nelfinavir (NFV) and Ritonavir (RTV)) and nucleoside analogues, ddA, AZT, d4T and 3TC, were coupled to KLH via an arm interposing. Antibodies obtained in rabbits were characterized using a radiolabelled iodine tracer on the hapten previously modified with the dipeptide tyrosyl-lysine.

For Saquinavir, the antibodies produced have been characterized in different ways: in RIA with two tracers radiolabelled with iodine 125 and in ELISA with an immobilized form of the antigen. Initially, anti-SQV antibodies were characterized in an RIA test using the hemisuccinate of Saquinavir coupled with the dipeptide Glycyl-Tyrosine (SQV-HS-G-Y *) as a tracer. The affinity of these antibodies for SQV was found to be low (64 nM). These antibodies were then characterized in an ELISA test with the immobilized form of the antigen, SQV-HS-BSA on a solid phase. The characteristics of these anti-SQV antibodies, established in an ELISA test, are presented in Table 1 below.

TABLE 1

Amino acids are presented according to the international nomenclature.

* represents isotope ¹²⁵ I.

It is found that the derivative SQV-HS-GY is 30 times less well recognized than the hemisuccinate of Saquinavir (SQV-HS). Consequently, the latter was coupled to the lysine side chain of the tyrosyl-lysine dipeptide in order to mimic the link established between the haptene and the carrier protein. Anti-SQV antibodies were characterized in radioimmunoassay with SQV-HS-KY labeled with iodine 125. The characteristics of anti-SQV antibodies, established in an RIA test with the tracer SQV-HS-KY *, are presented in table 2 below .

TABLE 2

ni: not immunoreactive up to concentrations of 10 ^"

M. The characteristics of anti-SQV antibodies established in ELISA and in RIA with the tracer SQV-HS-K-Y * show that SQV-HS-G-Y is on average 30 times less well recognized than hemisuccinate of Saquinavir. On the other hand, SQV-HS-K-Y is as well recognized as Saquinavir hemisuccinate.

In order to generalize this approach to other antibodies, we have characterized the antibodies directed against another antiprotease, Nelfinavir, with both the tracers corresponding to the coupling products to the dipeptides Glycyl-Tyrosine and Tyrosyl-Lysine. The results obtained concerning the characteristics of the anti-NFV antibodies established in an RIA test with the tracers NFV-Ac-βAla-G-Y * and NFV-Ac-βAla-K-Y * are presented in Table 3 below. TABLE 3

12

The characteristics of the anti-NFV antibodies show that for this antibody, the immunoassays using the tracers NFV-Ac-βAla-G-Y * and NFV-Ac - βAla-K-Y * are equivalent in terms of sensitivity and specificity. In addition, these two molecules are recognized identically in each of the tests indicating that they are equivalent in terms of recognition by the antibody.

Subsequently, we directly used this dipeptide to produce a tracer in order to characterize the antibodies directed against ddA.

The characteristics of these anti-ddA antibodies established in an RIA test with the ddA-HS-K-Y * tracer are presented in Table 4 below.

Table 4

ni: not immunoreactive up to concentrations of 10 ^" M. As with the previous antibodies, ddA-HS-KY is as well recognized as hapten.

In an absolutely identical manner anti-RTV, anti-AZT and anti-d4T antibodies were prepared and their characteristics measured. Tables 5, 6 and 7 below give the results of these characterizations.

Table 5 (RTV)

ACE acetyl-cholinesterase enzyme tracer

Table 6 (AZT)

The specificity of anti-SQV, anti- NFV, anti-ddA, anti-RTV, anti-Azt and anti-d4T is excellent since no cross-reactivity is observed with other antivirals used in anti-HIV therapy. The sensitivity of the tests and the absence of interference with components of biological media such as plasma proteins, intracellular content, or the culture medium in in vitro tests, allows the crude assay without prior purification of the samples. The comparative study of the specificities shows that the affinity of the immunological test obtained with the tracer (KY or YK) * makes it possible to increase the sensitivity by a factor of 10.

2. Clinical applications.

It can be seen that the results of the RIA assays are comparable to those obtained by HPLC and allow the quantification of the plasma SQV.

The results of the ELISA assays are very different from those obtained by HPLC. The ELISA test being more sensitive to protein interference than the assay

RIA, one way to overcome this interference would be to extract the SQV from biological samples.

Also, since the study of the specificity of the antibodies shows that the hemisuccinate of Saquinavir is

100 times better recognized, the optimization of the test could go through the succinylation of the SQV of biological samples.

3.Conclusions. The experimental work carried out within the framework of the present invention and the results of the immunological tests mentioned in the example below allow to conclude that the tracer designed by the applicant, comprising the pattern KY allows the realization of an immunoassay having a good sensitivity. These results confirm that the immunological tracer designed in the context of the present invention, which takes account of the recognition of the epitope by the antibody, has the best match with the immunogen. Indeed, the comparison of the structures of the ddATP isostere and the ddA hemisuccinate shows analogies confirmed by molecular modeling. This structural analogy has been demonstrated experimentally by the fact that the ddA-HS-K- ¹²⁵ IY derivative is perfectly recognized by the anti-isostere antibodies of ddATP, as is the non-radiolabelled derivative.

Indeed, in the example, shown by the applicant to illustrate the invention, the hapten is coupled to the primary amino functions of the side chains of the lysines of the carrier protein. Therefore, by coupling the hapten on the lysine side chain of the tyrosyl-lysine dipeptide, we mimic the link established between the hapten and the carrier protein. The tracer thus obtained is structurally closest to the immunogenic form.

Taken as a whole, the study of the specificity of the different antibodies that the Applicant has characterized shows that a precursor of a tracer using the dipeptide Tyrosyl-Lysine leads to the production of a tracer making it possible to obtain a test with sufficient sensitivity to detect and quantify weakly represented molecules. IOGRAPHIC BIB REFERENCES

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Claims

1. A precursor of an immunological tracer, characterized in that it comprises a non-peptide hapten coupled to a TYR- (X) _n -LYS or LYS- (X) _n -TYR motif in which X is chosen from a single bond , an amino acid, with the exception of Lysine, Glutamine, Asparagine or Tyrosine, a succinyl group, a hydroxymethyl group —CH (OH) -, a methylene group: -CH ₂ -, an oxygen atom, a sulfur, an oxyethylene group —CH ₂ -0-, or a methylamine group -CHNH- and n is an integer between 1 and 20, preferably between 1 and 10, most preferably between 1 and 2.

2. A precursor according to claim 1, characterized in that it comprises a non-peptide hapten coupled to a TYR-LYS or LYS-TYR motif.

3. A precursor according to any one of claims 1 or 2, characterized in that the non-peptide hapten is chosen from: nucleosides, nucleoside analogs, non-nucleoside inhibitors, anticancer compounds, antiviral compounds ( antiproteases, entry inhibitors, integrase inhibitors), narcotics, drugs, non-peptide hormones and neuromediators.

4. A precursor according to claim 3, characterized in that the nucleoside analog is chosen from analogs of pyrimidine and purine bases.

5. A precursor according to claim 4, characterized in that the nucleoside analog is chosen from rAcyclovir, Adenosine, S-Adenosyl-L-methionine, 2 ', 3' -dideoxyadenosione (ddA), 2 ', 3' -didehydro -2 ', 3' - dideoxythymidine (d4T), 2 ', 3' -dideoxy-3 '-thiacytidine (3TC), 3'- Azido-3' -deoxythymidine (AZT), Carbovir, Cordycepine, Cytidine, Cytosine-bD -arabinoside, Deoxycytidine, Deoxytubercidine, 2 '-Deoxyuridine, Formycin A, Formycin B, Ganciclovir, Guanosine, Inosine, Puromycin, Ribavirin, Sangivamycin, Saquinavir, Thymidine, Tubercidine, Uridine, Abacavir, 3- fluoro-2', 3 '-didine ), Fura, FdUrd, Thi- Gua, 6-MP, AraC or Fludarabine phosphate.

6. A precursor according to claim 3, characterized in that the non-nucleoside inhibitor is chosen from: a dipyridodiazepinone, a pyridinone, a bis (heteroaryl) piperazine, a TSAO derivative, an alpha-anilinophenylacetamide (alpha-APA), quinoxaline, benzoxaninone DMP266 (Efavirenz).

7. A precursor according to claim 3, characterized in that the anti-cancer compound is chosen from: alkaloids, carcinostats and antimetabolites chosen from analogs of purine bases or analogs of pyrimidine bases.

8. A precursor according to claim 3, characterized in that the antiviral compound is chosen from Saquinavir (SQV), Indinavir (IDV), Nelfinavir (NFV), Ritonavir (RTV), Amprenavir (APV), Lopinavir (LPV), Tipranavir , Atazanavir, T20 and T22.

9. A precursor according to claim 3, characterized in that the narcotic is chosen from cocaine, morphine, heroin and their related derivatives.

10. Method for obtaining a precursor of an immunological tracer defined in any one of claims 1 to 9, characterized in that it comprises the coupling between a carboxylic derivative of hapten and the ε-amine group of Lysine from the Tyrosyl- (X) n- Lysine motif.

11. Method according to claim 10, characterized in that the coupling is carried out by activation of the carboxylic acid of the hapten in the presence of ethyl chloroformate followed by the reaction of said derivative activated with the ε-amine group of Lysine of the Tyrosyl- (X) n-Lysine motif.

12. Method according to claim 10, characterized in that the coupling is carried out by means of a carbodiimide in the presence of N-hydroxysuccinimide.

13. Method according to any one of claims 10 to 12, characterized in that the coupling is carried out in a buffer medium at a pH between 8 and 10, preferably at a pH between 8.5 and 9.

14. An immunological tracer characterized in that it comprises a precursor defined in any one of claims 1 to 9, or obtained by a process defined in any one of claims 10 to 13, coupled to a marker.

15. An immunological tracer according to claim 14, characterized in that the marker is chosen from the group comprising: a radioactive atom, an enzyme, a magnetic particle, a colored particle, a chromophore, a fluorophore, a metallic particle, biotin , an avidin-biotin complex, a streptavidin-biotin complex.

16. An immunological tracer according to any one of claims 14 or 15, characterized in that the marker is coupled to the functional group of Tyrosine

17. An immunological tracer according to any one of claims 14 or 15, characterized in that the marker is coupled to the functional group of Lysine.

18. An immunological tracer according to claim 16, characterized in that it comprises a radioactive marker on the tyrosine residue.

19. An immunological tracer according to claim 18, characterized in that the radioactive marker is chosen from radioactive atoms: I ¹²⁵ , H ³ .

20. An immunological tracer according to claim 19 chosen from: Saquinavir-HS-KY (or Y-K) ¹²⁵ I, Saquinavir-Citrate-KY (or YK) ¹²⁵ I, ddA-HS-KY (or Y_K) ¹²⁵ I, ddA-Citrate-KY (or YK) ¹²⁵ I, NFV-Ac-βAla-KY (or YK) ¹⁵ I, NFV-HS-KY (or YK) ¹²⁵ I, NFV-Citrate-KY (or Y- K) ¹²⁵ I, Ritonavir-HS-KY (or YK) ¹²⁵ I, Ritonavir-Citrate-KY (or YK) ¹²⁵ I, AZT-HS-KY (or YK) ¹²⁵ I, AZT-Citrate-KY (or YK) ¹²⁵ I, d4T-HS-KY (or YK) ¹²⁵ I, d4T-Citrate-KY (or Y- K) ¹²⁵ I, 3TC-HS-KY (or YK) ¹²⁵ 1, 3TC-Citrate-KY (or YK) ¹²⁵ I, IDV-HS-KY (OU YK) ¹²⁵ I, IDV-Citrate-KY (OU YK) ¹²⁵ I.

21. An immunological tracer according to claim 17, characterized in that it comprises an enzymatic marker on the lysine residue.

22. An immunological tracer according to claim 21, characterized in that the enzymatic marker is chosen from the group comprising peroxidase, alkaline phosphatase and urease.

23. An immunological tracer chosen from: Saquinavir-HS-KY (or YK) peroxidase, Saquinavir- Citrate-KY (or YK) peroxidase, ddA-HS-KY (or YK) peroxidase, ddA-Citrate-KY (or YK) peroxidase, NFV-HS- KY (or YK) peroxidase, NFV-Citrate-KY (or YK) peroxidase, Ritonavir-HS-KY (or YK) peroxidase, Ritonavir-Citrate-KY (or YK) peroxidase, AZT-HS- KY (or YK) peroxidase, AZT-Citrate-KY (or YK) peroxidase, d4T-HS-KY (or YK) peroxidase, d4T-Citrate-KY (or YK) peroxidase, 3TC-HS-KY (or YK) peroxidase, 3TC-Citrate- KY (or YK) peroxidase, IDV-HS-KY ( or YK) peroxidase, IDV-Citrate-KY (or YK) peroxidase.

24. Method for obtaining an immunological tracer, characterized in that it comprises a step of coupling a marker on the tyrosine residue or on the lysine residue of a precursor defined in any one of claims 1 to 9 .

25. Method for obtaining an immunological tracer, characterized in that it comprises a step of coupling a TYR- (X) _n -LYS or LYS- (X) _n -TYR motif previously labeled with a non-peptide hapten .

26. The method of claim 25, characterized in that the prior marking of the pattern TYR-

(X) _n -LYS is carried out using a radioactive marker on the tyrosine residue.

27. The method of claim 25, characterized in that the prior labeling of the pattern TYR- (X) _n -LYS is carried out by means of an enzymatic marker on the lysine residue.