FR2843395A1 - New synthetic peptide from apolipoprotein AIV related protein, useful for raising antibodies, used for diagnosis and treatment of disorders of lipid metabolism - Google Patents

Abstract

Pure synthetic peptide (I) is a 95 amino acid sequence given in the specification, or its derivative or immunogenic fragment, is new. Independent claims are also included for: (1) an antibody (Ab) specific for (I), and its fragments or derivatives with essentially the same antigenic specificity; (2) preparing (M1) specific anti-AA4RP (apolipoprotein AIV related protein) antibodies (Ab1) by immunization with (I); (3) detecting (M2) or quantifying AA4RP by complex formation with Ab or Ab1; and (4) identifying (M3) a predisposition or risk of developing disorders of lipid metabolism, and for monitoring treatment of these disorders, by in vitro/ex vivo quantification of AA4RP, using Ab or Ab1.

Description

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Compositions and methods for the determination of AA4RP
The present invention relates to compositions and methods for the assay or detection of Apolipoprotein AIV related protein (AA4RP). It relates in particular to a method allowing the direct detection and quantification of AA4RP. This invention also relates to synthetic products of AA4RP, the corresponding antibodies, kits comprising them, and their uses for detecting and quantifying AA4RP in a biological sample.

 The increase in plasma triglyceride levels is one of the major determinants in the development of cardiovascular diseases currently responsible for the majority of deaths worldwide (Hokanson and Austin 1996). Studies have shown that alterations in the level or structure of apolipoproteins are linked to abnormal lipid levels and an increased risk of atherosclerosis (Rubin, Krauss et al. 1991; Plump, Smith et al. 1992; Duverger , Tremp et al. 1996; Huang, Liu et al. 1998; Lusis 2000; and Tall 2000). A very relevant example of the involvement of apolipoproteins in plasma triglyceride homeostasis is that of AA4RP. This apolipoprotein was recently discovered and has been described as an important factor in the regulation of triglyceride metabolism in mice and humans (Pennacchio, Olivier et al. 2001).

Mice expressing the human AA4RP transgene show a decrease in the plasma triglyceride level by 3 times compared to the control mice.

Conversely, knockout mice lacking the gene encoding AA4RP have a plasma triglyceride concentration increased by 4 times compared to the control mice. The variations in the triglyceride level observed in the transgenic mice and in the AA4RP knockout mice go in the same direction as those of the levels of apolipoprotein CIII (Apo CIII), known to be a positive indicator of the levels of triglycerides, since this apolipoprotein inhibits the hydrolysis of triglycerides by lipoprotein lipase (LpL). The plasma concentration of Apo CIII is reduced in human AA4RP transgenic mice after a rich diet

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 in lipids as the concentration of Apo CIII in knockout mice is increased, indicating that one of the mechanisms of action of AA4RP could be the regulation of the expression of the gene coding for Apo CIII and thus confirming the positive role of AA4RP in the elimination of triglycerides.

 In humans, it has been shown that there is a very close correlation between the polymorphism of AA4RP and the level of triglycerides. Indeed, the SNPs1-3 minor alleles of AA4RP polymorphism are associated with high triglyceride levels. A representative result is the 30% increase in the level of triglycerides in individuals with one of these alleles compared to subjects homozygous for the wild allele (Pennacchio, Olivier et al. 2001).

 The results obtained in mice and in humans clearly show the importance of the role of AA4RP in triglyceride homeostasis and they suggest a possible use of AA4RP as a marker in the prognosis or diagnosis of hypertriglyceridemias. It is therefore particularly relevant to develop a method for detecting and / or quantifying AA4RP.

 For various reasons, measuring the plasma concentration of AA4RP is quite difficult. First, AA4RP shows 27% identity and 48% similarity to the apolipoprotein AIV (Apo AIV). Second, AA4RP is present in very low concentrations in the plasma. It was therefore essential to develop a specific and sensitive method to quantify and / or detect this apolipoprotein.

 This protein has the primary structure shown below, comprising 366 amino acids (SEQ ID NO: 1).

MASMAAVLTWALALLSAFSATQARKGFWDYFSQTSGDKGRVEQIHQQKMAR EPATLKDSLEQDLNNMNKFLEKLRPLSGSEAPRLPQDPVGMRRQLQEELEEV KARLQPYMAEAHELVGWNLEGLRQQLKPYTMDLMEQVALRVQELQEQLRVV GEDTKAQLLGGVDEAWALLQGLQSRWHHTGRFKELFHPYAESLVSGIGRHV

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 QELHRSVAPHAPASPARLSRCVQVLSRKLTLKAKALHARIQQNLDQLREELSR AFAGTGTEEGAGPDPQMLSEEVRQRLQAFRQDTYLQIAAFTRAIDQETEEVQ QQLAPPPPGHSAFAPEFQQTDSGKVLSKLQHLLDDL

 This protein corresponds to the sequence of the RAP3 protein previously mentioned as potentially involved in hepatic regeneration (Genbank access number: AF202889.1: Van der Vliet HN, Groenink M., Leegwater ACJ and Chamuleau RAFM Submitted (09- NOV- 1999) Experimental Hepatology, Academy Medical Center, Meibergdreef 9, Amsterdam 1105 AZ, Netherlands). It also corresponds to the protein AA4RP (Apolipoprotein AIV-Related Protein) of the international patent application W001 / 00803 A2 (SEQ ID NO: 3 of this application).

 The present invention provides a strategy for producing a synthetic peptide specific for AA4RP (which is used for the production of specific anti-AA4RP antibodies) and a new immunoenzymatic method for detecting and directly quantifying this apolipoprotein.

The quantification of AA4RP is advantageously carried out by the ELISA (Enzyme Linked Immuno-Sorbant Assay) technique of the sandwich type. The anti-AA4RP antibody is fixed on a solid substrate for the capture of the protein, then the same antibody, this time labeled with an enzyme, is used as detection antibody for AA4RP fixed by the first antibody. The detection phase is carried out in the presence of an enzyme substrate and the extent of the reaction developed between the enzyme and its substrate is directly proportional to the concentration of AA4RP. To detect, quantify or purify AA4RP using specific anti-AA4RP antibodies, either polyclonal antibodies, monoclonal antibodies or a mixture of the latter can be used.

 The invention also describes such antibodies, kits comprising them and their uses for detecting, quantifying or purifying AA4RP in samples, in particular serum or plasma. These antibodies provide

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 also a new approach to modulate the activity of AA4RP in vitro or in vivo, and to regulate lipid metabolism in a subject.

 A first particular subject of the invention relates to a synthetic peptide specific for substantially pure AA4RP, comprising the sequence SEQ ID NO: 2 or an immunogenic fragment or a derivative of this peptide.

 Another subject of the invention relates to a method for producing anti-AA4RP antibodies comprising a step of immunization using a synthetic peptide specific for AA4RP as defined above. This invention also includes the antibodies prepared according to this method, as well as, more generally, the antibodies capable of binding a peptide as defined above as well as the fragments or derivatives of such antibodies.

 Another aspect of this invention relates to a method of detecting or assaying AA4RP in biological samples (especially in prepared lipoparticles or in whole plasma or serum samples), using an antibody (including a fragment or a derivative thereof) as defined above.

 Another object of this invention relates to a method of detecting the presence of a predisposition or of a risk of developing a disorder linked to lipid metabolism in a subject, comprising the in vitro detection, in a sample of said subject, of the 'AA4RP using an antibody (including a fragment or a derivative thereof) as defined above.

 Another subject of this invention relates to a method for detecting or monitoring in a subject the cellular capture of lipoproteins rich in triglycerides and HDL, comprising the in vitro detection of particles containing AA4RP using an antibody (including a fragment or a derivative thereof) as defined above.

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 Another subject of the invention relates to a method for detecting and / or monitoring the formation, development or progression of atherosclerosis in a subject, comprising the detection in vitro, in a sample of said subject, of the amount or the level of AA4RP using an antibody (including a fragment or a derivative thereof) as described above.

 The subject is generally a mammal, preferably a human being, even more preferably a subject at risk of developing cardiovascular diseases linked to dyslipidemia (more preferably hypertriglyceridemia) such as coronary artery disease for example.

 Another object of this invention relates to a pharmaceutical composition comprising an antibody (including a fragment or a derivative thereof) as defined above and a pharmaceutically acceptable excipient or vehicle.

Specific Peptides
As indicated above, one aspect of the present invention relates to a synthetic peptide specific for substantially pure AA4RP comprising the sequence SEQ ID NO: 2 or an immunogenic fragment or a derivative of this peptide. The term substantially pure indicates that the peptide is essentially devoid of amino acid sequences present in Apo AIV and / or of contaminating proteins normally present or associated with lipoparticles, such as in particular Apo AI. The synthetic term indicates that the peptide is not a molecule obtained naturally but that it has been prepared by an artificial process (eg, chemical synthesis, assembly, etc.) in particular as described in the examples. In this context, the synthetic peptide specific for the AA4RP of the invention is preferably essentially non-glycosylated. A peptide according to the invention generally comprises less than 200 amino acids, more preferably

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 less than 150 amino acids, even more preferably less than 120 amino acids.

 The present invention now demonstrates that synthetic peptides specific to AA4RP can be produced and used to generate specific anti-AA4RP antibodies. The invention also demonstrates that such antibodies are capable of specifically binding to the AA4RP obtained either naturally or in the form of a soluble antigen, or included in lipoparticles. The invention also demonstrates that such antibodies can bind to different lipoparticles containing AA4RP (VLDL and HDL). These synthetic peptides specific to AA4RP and their corresponding antibodies thus represent new products which are particularly advantageous for detecting AA4RP and specifically quantifying this apolipoprotein.

 More particularly, a preferred synthetic peptide of the preferred AA4RP of the invention comprises the sequence SEQ ID NO: 2, as described below, or an immunogenic fragment or a derivative of this peptide.

SEQ ID NO: 2 ATQARKGFWD YFSQTSGDKG RVEQIHQQKM AREPATLKDS LEQDLNNMNK FLEKLRPLSG SEAPRLPQDP VGMRRQLQEE LEEVKARLQP YMAEA
This peptide fragment of 95 amino acids corresponds to residues 20 to 114 of the sequence of mature human AA4RP.

 As illustrated in the examples, this peptide (comprising the sequence SEQ ID NO: 2) can be prepared in a particularly advantageous manner by synthesis in solid phase, more particularly using a Boc / Bzl strategy (Merrifield 1963).

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 The term derivative includes peptides comprising one or more mutations, substitutions, deletions and / or additions of one or more amino acid residues and having substantially the same antigenic specificity. Typical examples of derivatives include sequence variations due to AA4RP polymorphism, splicing, etc. Particularly preferred derivatives include a sequence SEQ ID NO: 2 or a modified sequence comprising at most 5 acid residues amines distinct from those present in SEQ ID NO: 2. The additional residues may correspond to residues of transport or binding sequence, to protective groups, etc. In addition, the peptide may be modified, for example, by chemical, physical and / or enzymatic, so as to increase its stability, increase its immunogenicity or even incorporate a Tag or any other molecule trolley or a marker, etc. Examples of such modifications include glycosylation, the addition of a cart, marker (eg, radioactive or enzymatic labeling, etc.), etc.

 The term fragment designates any peptide comprising from 5 to 95 consecutive residues of the sequence SEQ ID NO: 2, preferably from 10 to 95.

The term fragment includes any portion of the sequence SEQ ID NO: 2 comprising an epitope.

 A particular subject of the invention relates to a synthetic peptide specific for AA4RP, characterized in that it comprises epitopes specific for AA4RP and in that it is devoid of epitopes specific for Apo AIV, and in what it includes the sequence SEQ ID NO: 2 or a fragment or derivative of these sequences.

 The peptides can be soluble, purified or complexed with a carrier molecule, such as KLH (Keyhole Limpet Hemocyanin) or serum albumin for example, or any other inert molecule (eg, synthetic) such as a bead, etc. a particular embodiment of the invention, the peptides are coupled to a carrier molecule. They are particularly so in the context of their use for producing antibodies. The coupling can be

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 carried out according to conventional methods known to those skilled in the art (Vaitukaitis, Robbins et al. 1971) (Bassiri 1979).

 The peptides can also be conjugated or coupled to a heterologous polypeptide molecule, such as a biologically active molecule for example. The heterologous character designates any peptide which does not originate from an AA4RP molecule.

 A particular object of the invention relates to a composition characterized in that it comprises a synthetic peptide comprising the sequence SEQ ID NO: 2, and in that it is devoid of other proteins and in particular apolipoproteins.

 The peptides can be used as part of screening methods for titration assays, as controls, standards or to calibrate assays. They can also be used to modulate the activity of AA4RP. They are also particularly useful for producing anti-AA4RP antibodies.

Antibody
In this regard, another object of the invention relates to any antibody capable of binding to a peptide as defined above. The antibody can be polyclonal or monoclonal. In addition, the term antibody also includes all fragments and derivatives of antibodies, in particular fragments and derivatives of said monoclonal or polyclonal antibodies having substantially the same antigenic specificity. The latter include antibody fragments (eg, Fab, F (ab ') 2, CDRs, etc.), humanized antibodies, polyfunctional antibodies, single-stranded antibodies (ScFv), etc. The antibodies of the invention can be produced using conventional methods, including immunizing an animal and recovering its serum (polyclonal) or spleen cells (so as to produce hybridomas by fusion with appropriate cell lines).

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 Methods of producing polyclonal antibodies from a variety of species including mice, rodents, primates, horses, pigs, rabbits, poultry, etc. are described for example in Vaitukaitis et al.

(Vaitukaitis, Robbins et al. 1971). The antigen is combined with an adjuvant (eg, Freund's adjuvant) and administered to an animal, typically by subcutaneous injection. Repeated injections can be given. Blood samples are collected and immunoglobulin or serum are separated.

 Methods of producing monoclonal antibodies from different species can be found for example in Harlow et al.

(Harlow 1988) or in Kohler et al. (Kohler and Milstein 1975). These methods include immunizing an animal with an antigen, followed by the recovery of spleen cells which are then fused with immortalized cells, such as myeloma cells. The resulting hybridomas produce monoclonal antibodies and can be selected by limiting dilutions so as to isolate the individual clones. Antibodies can also be produced by selection of combinatorial immunoglobulin banks, such as those disclosed for example in Ward et al.

(Ward, Gussow et al. 1989).

 Preferred antibodies of the invention are prepared by immunization using a synthetic peptide specific for substantially pure AA4RP as described above, preferably comprising the sequence SEQ ID NO: 2 or an immunogenic fragment or a derived from this peptide, eg, a subfragment comprising at least one epitope.

 The Fab or F (ab ') 2 fragments be produced by digestion using a protease according to conventional techniques. Humanized antibodies can be prepared according to one of the methods described, for example, in Riechmann et al. (Riechmann 1988) (Jones 1986).

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 The invention also relates to a method for producing anti-AA4RP antibodies, comprising injecting a peptide of sequence SEQ ID NO: 2 or an immunogenic fragment or a derivative of this peptide into a non-human animal and recovering it antibodies or antibody producing cells. The method advantageously allows the production of specific antibodies. The specificity can be verified by demonstrating the absence of a cross-reaction with other circulating proteins in the blood. More generally, the specificity indicates that the antibodies are capable of binding to AA4RP with a higher affinity than any other antigen. As illustrated in the examples, the polyclonal antibodies of the present invention can be used to detect and / or assay AA4RP with great efficiency.

 The antibodies can be coupled to heterologous fragments such as toxins, markers, drugs or any other therapeutic agent, covalently or not, either directly or through coupling agents. The markers can be chosen from radio-markers, enzymes, fluorescent agents, magnetic particles, etc. Preferred toxins are for example diphtheria toxin, botulinum toxin, etc. The drugs or the therapeutic agents are chosen in particular among lymphokines, antibiotics, antisense sequences, growth factors, etc. Methods making it possible to carry out the coupling of antibodies and such heterologous fragments are illustrated for example in US patent 4,277,149 and in US patent 3,996,345.

 The antibodies of the invention have numerous applications including therapeutic, prophylactic, diagnostic, purification, detection, etc. applications.

 In vitro, they can be used as screening agents or to purify antigens from various samples, including various biological samples (eg, blood samples). They can also be used to detect or quantify the presence or amount of AA4RP in

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 lipoparticles within a sample collected from a subject, typically, a blood sample from a mammal or, preferably, a human.

Detection / Dosing
In this regard, another subject of the invention relates to a method for detecting, measuring, assaying or quantifying AA4RP within a biological sample, comprising bringing the sample into contact with an antibody as defined above. above (including fragments or derivatives thereof) and the detection, measurement, assay or quantification (of the presence) of immune antigen-antibody complexes. Typically this method allows the determination of AA4RP levels in a sample, by comparison with standard conditions or with a calibration curve for example.

 The determination of the immune complexes can be carried out using conventional techniques such as direct or competitive immunological methods, for example the RIA technique (Radio Immuno Assay), EIA (Electro Immuno Assay), by nephelometry, turbidimetry, quantitative immunoblot, calorimetry, interferometry, surface resonance, force field measurement, other developing biosensors, etc. However, a particularly preferred object of the invention relates to a method involving the ELISA technique of the sandwich type. In fact, as indicated above, the antibodies are specific and can recognize AA4RP in a sample, thus making it possible to assay in a precise, sensitive and effective manner AA4RP with a polyclonal antibody (or a mixture of monoclonal antibodies) anti -AA4RP.

 A more preferred object of the invention therefore relates to a method for detecting, measuring, assaying or quantifying AA4RP within a biological sample, comprising bringing the sample into contact (or dilutions thereof) with a first antibody immobilized on a support, called capture antibody, this antibody being as defined above (including fragments or derivatives of the latter), and the revelation of the complexes

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 antigen-antibody immune agents optionally formed by means of a second labeled antibody, called the revealing antibody, this antibody being as defined above (including fragments or derivatives of the latter).

 In a particularly preferred embodiment, the capture antibody and the revealing antibody are polyclonal antibodies. They can be the same polyclonal antibody. The capture antibody can be immobilized on any type of support, in particular plastic, such as a plate, ball, column, gel, etc. It is advantageously a multi-well plate. Immobilization is typically achieved by adsorption of antibodies on the surface of the wells. It is understood that any other deposition technique can be implemented, direct or indirect, covalent or not. The capture antibody is typically used in soluble form. It is previously marked to allow its detection and quantification. The labeling can be of a varied nature, such as radioactive, fluorescent, enzymatic, luminescent labeling, etc. In a particular embodiment, the labeling is an enzymatic labeling, and the immune complexes are revealed by measuring the activity enzyme. A specific example of enzymatic labeling is peroxidase, the activity of which can be revealed in the presence of the substrate OPD for example.

 A particularly preferred subject of the invention relates to a method for detecting, measuring, assaying or quantifying AA4RP within a biological sample, comprising bringing the sample (or dilutions thereof) into contact with a support on which is immobilized a polyclonal capture antibody as defined above, under conditions suitable for the production of specific immune complexes, and the revelation of the antigen-antibody immune complexes optionally formed by means of a polyclonal revealing antibody labeled, this polyclonal antibody being as defined above.

 The method can be performed on different biological samples, including plasma, serum, interstitial fluid, cell culture supernatant, etc. The sample can be collected from a subject (e.g., a subject

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 human) and used directly for testing. Alternatively, the sample can be diluted and / or stored (for example in frozen form) to be tested later. The invention also makes it possible to measure the concentrations of AA4RP contained in the lipoparticles by means of the use of specific anti-AA4RP antibodies, with very high sensitivity, reproducibility and repeatability.

 Detection can be carried out under different experimental, clinical, epidemiological, prognostic and diagnostic conditions. In particular, the method can be used to detect the predisposition of certain individuals to develop disorders linked to lipid metabolism.

 A particular object of the invention thus relates to a method for detecting the presence of a predisposition or of a risk of developing a disorder linked to lipid metabolism in a subject, comprising in vitro detection or in vitro measurement, from a sample taken from the subject, of lipoparticles comprising AA4RP, using an antibody as defined above (including fragments or derivatives of the latter). The levels of AA4RP (compared to an average value in normal subjects) may be characteristic of the high risk of developing disorders linked to lipid metabolism.

 Another subject of the invention relates to a method for detecting or monitoring the cellular uptake of HDL and lipoproteins rich in triglycerides in a subject, comprising the in vitro detection of the amounts of AA4RP present in lipoparticles, using an antibody as defined above (including fragments or derivatives thereof).

 Another subject of the invention relates to a method of monitoring a treatment intended to correct disorders linked to lipid metabolism in a subject, comprising the detection of AA4RP levels in vitro, in a sample originating from said subject, at the using an antibody as defined above (including fragments or derivatives thereof), after administration

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 of said treatment to said subject. The efficacy of the treatment is correlated with the level of AA4RP in the subject. These results may be correlated with the ability of the treatment to regulate the levels or activity of AA4RP or the ability to restore normal activity or level of AA4RP in a subject.

 Another subject of the invention relates to a method for evaluating the physiological state of a subject, eg the level of lipid metabolism in a subject, comprising the detection of the levels of AA4RP in vitro or ex vivo in a sample from said subject, using antibodies as defined above (including fragments and derivatives thereof).

 As indicated above, these methods can be performed on different samples (typically plasma or serum) and by RIA, EIA, nephelometry, quantitative immuonblot turbidimetry, calorimetry, interferometry, surface resonance, force field measurement, other biosensors in development, etc ..., or preferably thanks to an ELISA sandwich type test.

 The invention further comprises a pharmaceutical composition comprising an antibody as described above and optionally a pharmaceutically acceptable excipient or vehicle.

 This invention also relates to a kit comprising a peptide or an antibody as described above. The kit can be used to detect or quantify AA4RP in any sample.

 Other aspects and advantages of the invention will be described in the examples which follow, which should be considered as illustrative and do not limit the scope of the invention.

 Legend of figures

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Specificity of antibodies.

Control in SDS PAGE of the specificity of anti-AA4RP antibodies and demonstration of the distribution of AA4RP in VLDLs and
HDL.

Figure 2:
ELISA test on AA4RP
The calibration curve is constructed from the DOs as a function of the concentrations of peptide 20-114 (#), a plasma of normolipid subjects (#) and the HDLs prepared from plasma of normolipid subjects (#) according to the protocol. shown in Example 3.

Figure 3:
Plasma concentrations of AA4RP calculated from a peptide 20-114 range or from a plasma pool of normolipid subjects according to the ELISA method
Figure 4:
ELISA assay for AA4RP in C57 BL / 6 mice, non-transgenic FVB mice, AA4RP knockout mice (KO) with FVB genetic background and transgenic mice for human AA4RP (genetic background FVB).

EXAMPLES
Example 1: Peptide sequence
1. Choice of the appropriate peptide sequence

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The following peptide fragment was synthesized. This fragment was determined using different algorithms to predict flexibility, hydrophilicity, antigenicity, and secondary structures. This fragment of 95 amino acids corresponds to residues 20 to 114 of the sequence SEQ ID NO: 1.

ATQARKGFWD YFSQTSGDKG RVEQIHQQK AREPATLKDS LEQDLNNMNK FLEKLRPLSG SEAPRLPQDP VGMRRQLQEE LEEVKARLQP YMAEA
2. Peptide synthesis.

 The peptide was synthesized by the solid phase synthesis method (Merrifield 1963) on an automatic synthesizer model ABI 431 A (Applied Biosystems Inc., California, USA) using a Boc / Bzl strategy on a 0.5 mmol MBHA resin. (0.57 mmol / g). Each amino acid was coupled twice in the presence of dicyclohexylcarbodiimide / hydroxybenzotriazole without capping. The side chain protecting groups are: Arg (Ts), Asp (Ochex), Glu (Ochex), Lys (2-CI-Z), His (Dnp), Ser (Bzl), Thr (Bzl), Met (O), Trp (formy) and Tyr (Br-Z).

 The Dnp group on the Histidine residue was removed from the peptide before cleavage from the support by treatment in the presence of 10% p-mercaptoethanol, 5% iiso pro pylethylamine in DCM medium for 2 hours and then in NMP medium for 2 hours . The peptidyl resin was treated with 50% TFA in DCM medium for 20 minutes to remove the terminal amino acid Boc. The peptide was cleaved from the resin and deprotected simultaneously according to a slow and fast HF procedure: the resin (1 g) was treated with anhydrous HF (2.5 ml) in the presence of p-cresol (0.75 g ), p-thiocresol (0.25 g) and dimethyl sulfide (0.5 ml) at 0 C.

 3 hours later, the hydrogen fluoride and dimethylsulfide were removed by evaporation in vacuo and the residual scavengers and side products were extracted with diethyl ether. The devices of the

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 The reaction were recharged with p-cresol (0.75 g), p-thiocresol (0.25 g) and 10 ml of anhydrous HF, then the mixture was incubated at 0 ° C. for 1.5 hours. The hydrogen fluoride was removed by evaporation and the residue was mixed in the presence of diethyl ether. The residue was filtered, washed with diethyl ether and extracted with 200 ml of a 10% aqueous acetic acid solution and then lyophilized.

3. Mass spectrometry
The molecular weight was determined using an ion electrospray mass spectrometer. The spectrum of the electrospray was obtained using an API device (Perkin-Elmer-Sciex) on a mass spectrometer by single quadrupole ion electrospray, equipped with an ion spray (electrospray assisted by a nebulizer) (Sciex, Toronto, Canada).

4. immunization
The peptide was emulsified in Freund's complete adjuvant and injected subcutaneously in rabbits using 0.5 mg of peptide per injection for the first two injections, followed at 15 days interval of booster injections in the same adjuvant but using 0.25 mg of peptide only.

Example 2: Isolation and characterization of specific anti-AA4RP rabbit antibodies.

1. Isolation of anti-AA4RP antibodies
The polyclonal antibodies were isolated by precipitation with 27% sodium sulphate and then purified by affinity chromatography on Sepharose gel 4B activated (pharmacia, Uppsala, Sweden), coupled with the peptide of AA4RP residue 20 to 114 AA ( Axen, Porath et al. 1967). The proteins not retained on the antigen gel were eliminated by washing with a phosphate buffered saline solution (PBS: Phospate 50 mmol / L, pH 7.2, NaCl 150

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 mmol / L). Fractions not specifically bound on the AA4RP gel were removed with 25 mmol / L of PBS. Elution of AA4RP specific polyclonal IgGs was accomplished using 0.2 M glycine at pH 2.8. The purified antibodies were dialyzed immediately against 10 mmol / L of PBS and then concentrated by ultrafiltration using an Amicon system (cutoff threshold 100 kD) (Amicon, Bervely, USA), tested in terms of protein content (Lowry OH 1951) , then stored in 1 ml (1 mg) aliquots at - 30 C.

2. Western Blot Analysis
2. 1- Protocol:
The purity and specificity of the antibody were tested in Western Blot (Towbin, Staehelin et al. 1979). Human VLDL, LDL and HDL particles were subjected to denaturing electrophoresis on SDS-PAGE gel (5 to 24%) then transferred to a nitrocellulose membrane and reacted with the purified human anti-AA4RP antibody. The immunoreactive proteins were visualized with an anti-IgG polyclonal antibody conjugated to horseradish peroxidase (Sanofi-Diagnostics Pasteur, Mares-la-Coquette, France). The development of the reaction is carried out by chemiluminescence (Amersham, Pharmacia, Biotec).

2. 2- Results
The results obtained are shown in Figure 1. It appears in this figure that the specific band which is revealed by the anti-AA4RP antibody is located between the molecular weight markers 32.5 and 47.5 kDa.

2. 3- Interpretation
The result of the immunoblot on the various human lipoproteins, shows a localization of AA4RP at the level of VLDL and HDL, with a very majority distribution at the level of HDL.

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 The presence of AA4RP at the VLDL level would explain the role of this apolipoprotein in regulating the metabolism of these lipoproteins rich in triglycerides, and therefore the modulation of the concentration of these atherogenic lipids.

 The localization of AA4RP at the HDL level is certainly related to the role of these particles in the reverse transport of cholesterol.

In fact, most of the apolipoproteins located at the HDL level are promoters of the capture of cholesterol from peripheral cells by the HDLs and of its return to the liver where it is eliminated.

Example 3: ELISA assay of AA4RP (sandwich type)
1. Reagents and materials used in the ELISA test
1.1- Capture antibodies The stock solution of anti-AA4RP is concentrated to 1 mg / ml, the coating (fixing of the antibody in the wells of a 96-well plate) is carried out at 10 g / ml, the dilution of the antibody is produced in a saline phosphate solution (0.1 M PBS, 0.15 M NaCl, pH 7.2 to 7.4).

 1.2- Detection antibodies The same antibody used for the capture is labeled with peroxidase. It is diluted 1/5000 in 0.1M PBS buffer containing 1% BSA.

 1. 3- Standard The peptide 20-114 is used for the construction of the standard range. The concentration of the peptide is 1 mg / ml.

For the construction of a calibration curve, the standard must be diluted in a 0.1 M PBS / 1% BSA solution according to the table below:

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<tb> Points <SEP> Concentration <SEP> (ng / ml)
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<tb> 1 <SEP> 125.00 <SEP>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<tb> 2 <SEP> 62.50 <SEP>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<tb> 3 <SEP> 31.25 <SEP>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<tb> 4 <SEP> 15.62 <SEP>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<tb> 5 <SEP> 7.81 <SEP>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<tb> 6 <SEP> 3.90 <SEP>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<tb> 7 <SEP> 1.95 <SEP>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<tb> 8 <SEP> 0.97 <SEP>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<tb> 9 <SEP> 0.48 <SEP>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<tb> 10 <SEP> 0.24 <SEP>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<Tb>
<tb> 11 <SEP> 0.12 <SEP>
<Tb>

1.4- Preparation of samples
Fresh samples are recommended for analysis. Plasmas should be collected through established procedures and used in clinical laboratories. If necessary, the plasma can be stored between 2 and 8 C for up to a week. Samples stored frozen can be used for a longer period.

The samples are diluted according to their origin: - Plasma of normolipid subjects: from 1.3 to 4 times.

 - Plasma of hypertriglyceridemic subjects: from 2 to 16 times.

 - HDL prepared from plasma by ultracentrifugation: from 4 to 128 times.

 Dilutions are made in a 1% PBS / BSA solution.

 2. Protocol

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 - Coating: the unlabeled anti-AA4RP capture antibody solution is incubated overnight at room temperature at a rate of 100 l / well (96-well microtiter plate).

- Washing of the plate: the antibodies which are not fixed on the plate are eliminated by
4 washes of the microtiter plate with 0.1 M PBS.

- Deposit of samples and standard range: The preparations of the peptide range, human plasma or HDL are deposited at the rate of 100 l per well, then are incubated at 37 ° C. for 2 hours.

- Washing: the excess of the peptide, of the AA4RP of the biological samples which is not fixed by the anti-AA4RP of capture is eliminated by 4 successive washes of the microtitration plate.

- Detection: the detection is carried out by anti-AA4RP labeled with peroxidase diluted to 1/5000 in PBS / BSA 1%. 100 l of the preparation are deposited per well of the microtiter plate. The incubation is carried out at 37 ° C. for 2 hours.

- Washing: the excess of anti-AA4RP detection antibody is eliminated by 4 successive washes of the plate with 0.1 M PBS.

- Development of the enzymo-colorimetric reaction: carried out by adding
100 l of o-Phenylenediamine dihydrochloride (OPD) substrate per well, then the reaction is developed for 30 minutes in the dark.

- Stop the reaction: by adding 100 l of 1N HCl per well.

- Absorbance reading: is performed by a spectrophotometer at the wavelength 492 nm.

3. Results and interpretations
3. 1- Calibration curve
The calibration curve is constructed according to a function with four unknowns representing the growth of the optical density as a function of the concentration of the different dilutions of the standard range. It shows a sigmoid appearance characteristic of the enzyme-linked immunosorbent assay ELISA (Figure
2).

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 The curves representing the different dilutions of peptide 20-114, of normolipid plasma and of human HDL are confused, indicating that the anti-AA4RP recognizes with the same affinity the synthetic peptide and the native AA4RP in plasma or in human lipoproteins. . In addition, the assay of AA4RP in several subjects using a calibration range constructed either from the peptide or from a pool of human plasmas, gives significantly similar results for the concentration of AA4RP (Figure 3). .

3. 2- Work interval and detection threshold
It is approximately 2 Log, between 1 and 62 ng / ml, which makes it possible to assay samples whose AA4RP concentrations are very variable. The detection threshold is around 1 ng / ml, which indicates that the assay is very sensitive.

3. 3- Normal values
The dosage of AA4RP in a population of normolipid subjects whose triglyceride level does not exceed 1 mg / ml gives values of the concentration of apolipoprotein between 8.94 and 29.44 ng / ml.

EXAMPLE 4 Correlation Between the Concentration of AA4RP in the Different Plasma Compartments and the Levels of Total Triglycerides
1. Protocol
Correlations of plasma triglyceride levels with plasma AA4RP or AA4RP levels in non-HDL lipoproteins (VLDL, IDL and LDL) or AA4RP in HDL lipoproteins are carried out in a population whose triglyceride levels are between 0.5 and 4 mg / ml.

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1. 1- Preparation of HDL and non-HDL lipoproteins
HDL and non-HDL lipoproteins are prepared by precipitation of the latter by adding phosphotungstic acid and Mg2 + to the plasmas of the various subjects (selective precipitation of lipoproteins containing Apo B, namely VLDL, IDL and LDL) (Burstein, Scholnick et al. 1970; Lopes-Virella, Stone et al. 1977). Briefly, the plasma is incubated for 10 minutes at room temperature with a phosphotungstate solution, then centrifuged at 2500 rpm for 15 minutes and finally the supernatant containing the HDL is separated from the pellet containing the non-HDL lipoproteins.

1. 2- Determination of triglycerides
Plasma triglycerides are dosed by the enzymo-colorimetric method, against a standard range, prepared using the CFAS lipid calibrator Ref. N 759350 (Boehringer Mannheim GmbH, Germany). The standard range was built from 16 to 500 g / ml. 100 l of each dilution of sample or standard range are deposited per well of a titration plate (96 wells). Then 200 l of triglyceride reagents Ref. 701912 (Boehringer Mannheim GmbH, Germany) are added to each well, and the entire plate is incubated for 30 min. at 37 C. The reading of the Optical Densities (OD) is carried out at 492 nm on the spectrophotometer. The triglyceride concentrations of each sample are calculated after construction of the standard curve according to a linear function y = ax + b, where y represents the OD and x the triglyceride concentrations.

1. 3- Determination of total AA4RP, HDL lipoproteins and non-HDL lipoproteins:
The concentrations of total AA4RP in plasma and that of HDL lipoproteins are determined according to the protocol of Example 3. The concentration of AA4RP in non-HDL lipoproteins is calculated by difference between the concentration of total plasma AA4RP and that HDL lipoproteins.

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2. - Results and interpretations:
We did not observe any correlation between the plasma levels of AA4RP and those of total triglycerides or those of triglycerides of non-HDL lipoproteins. On the other hand, there is a significant negative correlation between the concentration of the protein and that of the triglycerides of the HDL lipoproteins (r = 0.5, p <0.001). This indicates that the higher the level of triglycerides, the more the amount of AA4RP in HDL decreases, thus confirming the anti-atherogenic role of HDL and therefore that of AA4RP present in this type of lipoproteins.

EXAMPLE 5 Quantification of AA4RP in Transgenic Mice for Human AA4RP
1.- Protocol:
The level of AA4RP was assayed in normal C57BL / 6 mice, non-transgenic FVB mice, AA4RP knockout mice (K4) with FVB genetic background and transgenic mice for human AA4RP (genetic background FVB) . The assay protocol is identical to that described in Example 3.

 2. - Results and interpretations: Figure 4 shows that our assay detects AA4RP only in human AA4RP transgenic mice indicating the specificity of the method between humans and mice. This is very interesting, since it is possible to quantify exogenous AA4RP in modified animal models without contamination by endogenous one, which makes it possible to dissociate between the effect of the animal's own AA4RP and that of origin exogenous.

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Claims (18)

 CLAIMS 1. Substantially pure synthetic peptide, characterized in that it comprises the amino acid sequence SEQ ID NO: 2 or an immunogenic derivative or fragment thereof. 2. Peptide according to claim 1, characterized in that the peptide is soluble or complexed with a carrier molecule such as KLH, serum albumin or a bead. 3. Antibody specific for a peptide according to one of claims 1 or 2, or fragment or derivative of said antibody having substantially the same antigenic specificity. 4. Antibody according to claim 3, characterized in that it is produced by immunization of a non-human animal with a peptide according to one of claims 1 or 2, and recovery of the antibodies or of the cells producing the antibodies. 5. Antibody according to one of claims 3 or 4, characterized in that it is a polyclonal antibody. 6. Antibody according to one of claims 3 or 4, characterized in that it is a monoclonal antibody. 7. A method of producing a specific anti-AA4RP antibody, comprising administering a peptide according to one of claims 1 or 2 to a non-human animal and recovering the antibodies or antibody-producing cells. 8. Method for detecting AA4RP in a biological sample comprising bringing the sample into contact with an antibody according to one of claims 3 to 6 or with an antibody produced <Desc / Clms Page number 28>  using a method according to claim 7, and detecting the presence of antigen-antibody immune complexes. 9. Method according to claim 8, characterized in that the presence of an antigen-antibody immune complex is determined by EIA, RIA, nephelometry, turbidimetry, quantitative immunoblot, calorimetry, interferometry, surface resonance or force field measurement. 10. Method according to claim 8, characterized in that the presence of an antigen-antibody immune complex is determined by a test ELISA. 11. Method for quantifying AA4RP in a biological sample, comprising bringing said sample into contact with an antibody according to one of claims 3 to 6 or with an antibody produced by a method according to claim 7, and quantifying the AA4RP-antibody immune complexes. 12. Method according to claim 11, characterized in that the quantification is carried out by ELISA method. 13. Method according to one of claims 8 to 12, characterized in that the biological sample is a sample of blood, serum, an interstitial fluid or a tissue or cell extract. 14. Method for detecting the presence of a predisposition or of a risk of developing a disorder linked to lipid metabolism in a subject, comprising the in vitro or ex vivo quantification of the levels of AA4RP in a sample taken from a subject, with an antibody according to one of claims 3 to 6, or with an antibody produced by a method according to claim 7. <Desc / Clms Page number 29>  15. Method for monitoring a treatment intended to correct disorders linked to lipid metabolism in a subject, comprising measuring the amounts of AA4RP in vitro or ex vivo, in a sample from said subject, using 'an antibody according to one of claims 3 to 6 or an antibody produced by a method according to claim 7. 16. Antibody according to one of claims 3 to 6 or produced by a method according to claim 7, characterized in that the antibody is coupled to a heterologous fragment such as a toxin, a marker, a medicament or any other agent therapeutic. 17. A pharmaceutical composition comprising an antibody according to one of claims 3 to 6 and 16 or produced by a method according to claim 7 and a pharmaceutically acceptable excipient or vehicle. 18. Kit comprising a peptide according to one of claims 1 or 2 or an antibody according to one of claims 3 to 6 and 16 or produced by a method according to claim 7.