EP1169451A2

EP1169451A2 - Nucleic acids coding for novel truncated cart proteins, corresponding truncated cart polypeptides and their use for therapeutic and diagnostic purposes

Info

Publication number: EP1169451A2
Application number: EP00927399A
Authority: EP
Inventors: Alistair Dixon; James Hamer Murray
Original assignee: Warner Lambert Co LLC
Current assignee: Warner Lambert Co LLC
Priority date: 1999-04-15
Filing date: 2000-04-14
Publication date: 2002-01-09
Also published as: JP2002541842A; AU4580600A; WO2000063370A2; MXPA01010483A; BR0010594A; WO2000063370A3; CA2373359A1

Abstract

The present invention is directed to nucleic acids encoding novel truncated CART polypeptides, particularly CART proteins having 88 amino acids in length, as well as nucleic acid fragments thereof, the related recombinant vectors and recombinant host cells containing such recombinant vectors. The invention also deals with a truncated CART protein particularly a truncated CART protein having 88 amino acids in length, uses thereof in the identification of ligands, as well as antibodies specially directed against this protein, these antibodies being useful as diagnostic reagents. The invention also relates to the use of the nucleic acids encoding the truncated CART proteins of the invention or the truncated CART proteins themselves for therapeutic purposes and particularly to decrease or prevent neuronal degeneration and to promote neuronal regeneration and restoration of function, to treat addiction by aiding clearance of the polypeptide or its truncated forms in vitro and in vivo and also in order to regulate food intake.

Description

TITLE OF THE INVENTION

Nucleic acids coding for novel truncated CART proteins, corresponding truncated CART polypeptides and their use for therapeutic and diagnostic purposes.

FIELD OF THE INVENTION

The invention relates to new truncated CART polypeptides and to their use for diagnostic and therapeutic purposes.

BACKGROUND OF THE INVENTION

The cocaine and amphetamine regulated transcript encodes a polypeptide termed the cocaine and amphetamine regulated transcript protein (CART). The gene encoding CART has been localised to human chromosome 5ql3-14, a region identified as a human obesity locus (Echwald et al 1999).

Excluding hippocampal and cerebullar mRNAs, CART is the third most abundant mRNA in the hypothalamus (Gautvik et al, 1996). High concentrations of the CART transcript are found in the nucleus accumbens of the brain. Transcription of the CART locus increases in the striatum after administration of psychostimulants.

The mRNA encoding the CART polypeptide has been shown to be regionally localised in specific areas of the rat brain, including the nucleus accumbens, the hypothalamus, the dentate gyrus of the hippocampus, the Edinger-Westphall nucleus, the locus coeruleus, the nucleus of the solitary tract, and the inferior olive (Couceyro et al., 1997; Douglass et al., 1995). The mRNA has also been shown to be in the ganglia cells of the retina, lamina X of the spinal cord, mitral and tufted cells of the olfactory bulb, barrel field of neurones of the somatosensory cortex, the anterior pituitary, and the medulla of the adrenal cortex (Couceyro et al., 1997). Upon cocaine, amphetamine or food stimulus the level of this transcript is elevated in rat brain (Kristensen et al.,1998). When recombinant CART is injected intracerebroventricularly (icv) into rats the peptide inhibits both normal and starvation induced feeding, and completely blocks the feeding response induced by neuropeptide Y. An antiserum against CART increases feeding in normal rats, indicating that CART may be an endogenous inhibitor of food intake in normal animals (Kristensen et al. 1998). The longer form of the CART protein has been identified from rat and mouse brain cDNA, the shorter foim of 116 amino acids in length has been identified from human brain cDNA. The rat and mouse CART sequence differs from the human sequences at 19 residues (rat) and 17 residues (mouse), the differences being distributed throughout the sequence. Noticeably, the known rat and mouse CART protein include the presence of PRRQLRAPGA(V/M)LQ amino acid motif which is absent from the known human CART protein. Additionally, the human, rat and mouse CART proteins differ in five other amino acids at the N-terminus. There is a 80% sequence identity between the rat and human CART mRNA, with 92% similarity within the deduced protein coding region (Douglass and Daoud, 1996). The human and rat CART cDNA and translated amino acid sequences are also described in the PCT Application WO 96/34619.

Bioactive peptides derived from cocaine and amphetamine regulated transcript proteins have been isolated and are described in PCT Application WO 98/48824. These bioactive CART peptides consist of C-terminus portions of human or rat CART proteins. More particularly, this PCT Application discloses that CART protein fragment 89-103 reduced food intake but had no effect on locomotor activity. In addition, polyclonal sera raised against the CART 82-103 peptide produced a volume dependent increase in food intake after icv injection compared to injection of pre-immune sera. The finding that the icv effect of the sera is stimulatory and opposite to the effects of the peptides indicated that the observed effects are not nonspecific and reflect the activity of endogenous CART peptides.

On the other hand, it has been shown that the injection of antibodies specific to a 24 amino acid N-terminus portion of the CART polypeptide described in PCT Application WO 98/48824 reduces cocaine induced locomotor activity. It has also been described that inhibition of CART synthesis via icv administration of either anti sense oligonucleotides or antibodies specific to the CART 82-103 peptide results in a decrease in serum testosterone as well as a significant decrease in body weight. It is believed that the weight lost is due to an increase in the peripheral metabolic rate as a result of the icv immuno neutralisation of the 82-103 CART peptide.

SUMMARY OF THE INVENTION

Given the abundance of CART mRNAs in brain tissue and the fact that CART is a well studied molecule, the inventors unexpectedly discovered a novel CART variant. The present invention is directed to nucleic acids encoding novel truncated

CART polypeptides, particularly CART proteins having 88 amino acids in length as well as nucleic acid fragments thereof. The invention also pertains to a family of recombinant vectors containing a nucleic acid encoding such truncated CART polypeptides and to recombinant host cells containing such recombinant vectors. The invention also deals with a truncated CART protein, particularly a truncated CART protein having 88 amino acids in length (when the signal peptide sequence is included or 61 amino acids when the signal peptide sequence is not included) as well as fragments of the CART protein.

The invention also deals with antibodies specifically directed against this protein and fragments, these antibodies being useful as diagnostic and therapeutic reagents.

The invention also relates to the use of the nucleic acids encoding the truncated CART proteins of the invention or of the truncated CART proteins and fragments themselves for therapeutic purposes and particularly to decrease or prevent neuronal degeneration, to promote neuronal regeneration and restoration of function, to treat addiction by promoting more efficient clearance of the polypeptide or its truncated forms in vitro and in vivo, and also to regulate food intake and drug addiction.

The invention also relates to the use of nucleic acids encoding CART polypeptide and CART polypeptide and fragments thereof for use in vivo and in vitro functional screening assays for ligands that bind to the CART receptor and CART polypeptide and fragments thereof. The invention also relates to in vitro ligand binding screens for molecules that interact with CART receptor and CART polypeptide and fragments thereof..

Throughout this application, various publications, patents and published patent applications are cited. The disclosures of these publications, patents and published patents specifications are thereby incorporated by reference into the present disclosure to more fully described the state of the art to which this invention pertains.

The present invention will be described hereinafter more in details and is further illustrated by the following figures:

Fig.l : Amino acid sequence alignments of different human rat and mouse CART proteins.

The two upper lines (Genebank entries N°U 20325 (SEQ ID N° 1) and U16 826 (SEQ ID N° 2)) represent the human predicted CART protein amino acid sequences described in Douglass and Daoud (1996). The third line (Genebank entry U 10 071 (SEQ ID N° 3)) represents the rat CART protein predicted amino acid sequence that is described in Douglass (1995). The fourth line (Genebank entry P56388 (SEQ ID N° 4) represents the mouse CART protein predicted amino acid sequence that is described in figure 1.

Lines 5, 6 and 7 represent the predicted amino acid sequence of the truncated CART proteins of the invention: rCART V represents the predicted amino acid sequence of the truncated CART protein (SEQ ID N° 5) that is encoded by the novel rat CART cDNA of the invention; hCART V represents the predicted amino acid sequence of the human truncated CART protein (SEQ ID N° 6) of the invention. mCART V represents the predicted amino acid sequence of the mouse truncated CART protein (SEQ ID N° 7 of the invention. Bonded cysteins are indicated by number coding. Bold: represents points of deviation between the sequences.

*: represent potential cleavage sites and : represents the putative signal sequence.

Fig. 2: PCR amplification of heterogeneous rat and human hypothalamic cDNA pool prepared.

Conditions:

- M.J. tetrade DNA Engine

- Annealing temperatures = 55°C, 60°C and 65°C.

- Extension temperature = 72°C - Denaturing temperature = 92°C

- 35 cycles

- Solution per 20 μl reaction yielding to 5 μl of amplified cDNA at 10ng/5μl in 1 x PCR buffer (3.5 mM MgCl₂, pH8.8) containing 12.5% sucrose, 0.1 mM cresol red, 12 mM β-mercaptoethanol, 0.5 mM dNTP's, 0.6U AmpiTaq DNA polymerase (Applied Biosystems), and primers at 100 ng per reaction.

10 μl of amplified PCR mix have been electrophoresed on a 1.5% agarose gel.

Lanes 1, 8 and 15: lkb DNA markers

Lanes 2-7 are lOμl aliquots of PCR. Mix PCR amplification was carried out on a heterogeneous rat hypothalamic cDNA pool. Bands A, B and C were excised from the agarose gel.

Lanes 9-14 are lOμl aliquots of PCR. Mix PCR amplification was carried out on a heterogeneous human brain cDNA library.

Fig. 3 : PCR screening of E. coli colonies transformed with TOPO TA with rCART inserts A, B and C.

E. coli colonies harbouring TOPO TA cloning vector with rCART insert were picked from solid culture plastes and simultaneously spiked into LB (supplimented with 50 μg/μl Carbenicillin) in a 96 well titre plate, and PCR mix consisting of 20 μl of IxPCR buffer (above) and primers. PCR reactions were carried out in a similar manner than described in figure 2 and the E. coli was cultured under standard laboratory conditions. PCR positive colonies were further cultured in 3ml of LB (supplemented with carbenicillin).

E.coli colony harbouring plasmid was spiked into PCR mix and PCR products were generated. lOμl of PCR mixture was electrophoresed on a 1.5% agarose gel. The plasmid of PCR positive colonies was further characterised by restriction fragment analysis.

Fig.4: Restriction fragment analysis of PCR positive DNA plasmid preparations.

E. coli colonies were processed as described in the legend of figure 3.

Purified plasmid from colonies was restricted with BamHI and Ndel. 10 μl of restriction digest mixture was electrophoresed on a 1.5% agarose gel.

Fig 5: Identification of ~60kd and ~100kd CART peptide binding proteins in pig brain preparations. Electrophoresis of protein fractions (4-20% acrylamide SDS tris-glycine gel) from CART peptide chromatography column. The gel was visualised by silver staining Lane A: Eluate from CART peptide chromatography column; unbound protein fraction.

Lane B: Eluate from blank chromatography column; unbound protein fraction.

Lane C: Eluate from CART peptide chromatography column; CART peptide eluted fraction. Lane D: Eluate from blank chromatography column; buffer eluted fraction.

Lane E: Eluate from CART peptide chromatography column; acid eluted fraction.

Lane F: Eluate from blank chromatography column; acid eluted fraction.

Molecular weight markers are indicated.

DETAILED DESCRIPTION OF THE INVENTION

NUCLEOTIDE SEQUENCES ENCODING CART

The inventors have found a new mRNA splice variant encoding a truncated

CART protein of 88 amino acids in length.

The truncated CART protein of the invention differs from the two known forms of CART proteins (long forms: rat/mouse, short form; human) by the absence of a peptide fragment corresponding to amino acids 54 to 94 as represented in figure 1. This novel form of CART cDNA has been isolated from a rat brain mRNA. Taking into account that with the exception of nucleic acids encoding the PRRQLRAPGA(Vor M)LQ amino acid motif (amino acids 54 to 66, as shown in figure 1), the nucleic acid sequences of the rat and human CART mRNA's have 92% sequence identity (Douglass and Doud, 1996), the inventors believe that this novel variant is also present in the human and mammalian populations and have deduced both the nucleic acid sequences and the amino acid sequences of the human and mouse truncated CART protein counterparts. The inventors have also identified a cDNA derived from human cDNA library generated under identical conditions to the rat cDNA which is of similar size to that of the novel rat CART cDNA, figure 2. This finding further supports the claim of the existence of a human variant of the CART similar to that identified by the inventors in rat tissue.

The aim of the present invention is to provide nucleic acids encoding a truncated CART protein, particularly a truncated CART protein having 88 amino acids in length.

A first object of the invention consists of a purified or isolated nucleic acid comprising a polynucleotide encoding a truncated CART protein, said CART protein having 88 amino acids in length, or a complementary sequence thereof.

As used herein, the term " isolated " requires that the material be removed from its original environment (e.g. the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated. However, the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system where it is normally found, is isolated. Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or the composition is not a part of its natural environment.

As used herein, the term " purified " does not require absolute purity; rather, it is intended as a relative definition. Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. Throughout the present specification, the expression " nucleotide sequence " may be employed to designate indifferently a polynucleotide or a nucleic acid. More precisely, the expression " nucleotide sequence " encompasses the nucleic material itself and is not restricted to the sequence information (i.e. the succession of letters chosen among the four base letters) that biochemically characterises a specific DNA or RNA molecule. As used interchangeably herein, the terms " oligononucleotides ", " nucleic acids ", " polynucleotides " and " nucleotides " sequences include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single or duplex form. The nucleic acids of the invention may contain modified nucleotides which comprise at least one of the following modifications: (a) an alternative linking group, (b) an analogous form of purine, ( c) an analogous form of pyrimidine, or (d) an analogous sugar. For examples of analogous linking groups, purines, pyrimidines and sugars, see for example PCT publication WO 95/04064. The nucleic acids of the invention may be obtained by any known method, including synthetic, or recombinant methods, or a combination thereof, as well as through any purification method known in the art.

A second object of the invention consists of a purified or isolated nucleic acid comprising a polynucleotide encoding a CART protein having the amino acid sequence of SEQ ID N°5, or a complementary sequence thereof. For the purpose of the present invention, a first polynucleotide is deemed to be " complementary " to a second polynucleotide when each base in the first polynucleotide is paired with its complementary base and its orientation is reversed. Complementary bases are, generally. A and T (or A and U), or C and G.

A third object of the invention consists in a purified or isolated nucleic acid comprising a polynucleotide encoding a CART protein having the amino acid sequence of SEQ ID N°6, or a sequence complementary thereto.

A fourth object of the invention consists in a purified or isolated nucleic acid comprising a polynucleotide encoding a CART protein having the amino acid sequence of SEQ ID N°7, or a sequence complementary thereto.

The invention also concerns a purified or isolated nucleic acid comprising a polynucleotide having at least 80% nucleotide identity with the nucleotide sequence of SEQ ID N°8, preferably at least 90%, more preferably 95% and most preferably 98%o nucleotide identity with the nucleotide sequence of SEQ ID N°8, or a complementary sequence thereof. The invention also deals with a purified or isolated nucleic acid comprising a polynucleotide having at least 80% nucleotide identity with the nucleotide sequence of SEQ ID N°9, preferably at least 90%, more preferably 95%, and most preferably 98% nucleotide identity with the nucleotide sequence of SEQ ID N°9, or a complementary sequence thereof. The invention also deals with a purified or isolated nucleic acid comprising a polynucleotide having at least 80% nucleotide identity with the nucleotide sequence of SEQ ID N°10, preferably at least 90%, more preferably 95%, and most preferably 98% nucleotide identity with the nucleotide sequence of SEQ ID N°10, or a complementary sequence thereof. As used herein, the expression " percentage of sequence identity " for polynucleotides and polypeptides of the invention, is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e.gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimum alignment of the two sequences.

This percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by computerised implementations of known algorithms (e.g. GAP, BESTFIT. FASTA, and TFASTA) in the Wisconsin Genetics Software package, Genetics computer group (GCG), 575 Science Doctor, Madison, Wis., or BlastN and BlastX available from the National Center for Biotechnology Information, or by inspection.

The nucleic acids of the nucleotide sequences of SEQ ID N°8, SEQ ID N°9 and SEQ ID N°10 are the products of an alternative splicing event resulting in the absence of the sequence of exon 2 and exon 3 in the resulting rat mRNA product.

Consequently, the nucleotide in position 159 of any of the nucleotide sequences of SEQ ID N°8, SEQ ID N°9 or SEQ ID N°10 corresponds to the 3'-end nucleotide encoded by exon 1 and the nucleotide located in position 160 of any of the nucleotide sequences of SEQ ID N°8, SEQ ID N°9 and SEQ ID N°10 corresponds to the 5 '-end nucleotide encoded by exon 4.

A further object of the present invention consists of a purified or isolated nucleic acid comprising a polynucleotide, encoded by the nucleotide sequence of SEQ ED N°8. This polynucleotide consists of at least 8, preferably 12, preferably 15, more preferably 20 and most preferably 25 consecutive nucleotides of the nucleotide sequence of SEQ ID N°8, provided that said polynucleotide comprises the nucleotide sequence beginning at the nucleotide in position 157 and ending at the nucleotide in position 164 of SEQ ID N°8, or a complementary sequence thereof. The invention also relates to a purified or isolated nucleic acid comprising a polynucleotide, encoded by the nucleotide sequence of SEQ ID N°9. This polynucleotide consisting of consisting of at least 8, preferably 12, preferably, 15, more preferably 20 and most preferably 25 consecutive nucleotides of the nucleotide sequence of SEQ ID N°9, provided that said polynucleotide comprises the nucleotide sequence beginning at the nucleotide in position 157 and ending at the nucleotide in position 164 of SEQ ID N°9, or a complementary sequence thereof.

The invention also relates to a purified or isolated nucleic acid comprising a polynucleotide, encoded by the nucleotide sequence of SEQ ID N°10. This polynucleotide consisting of consisting of at least 8, preferably 12, preferably, 15, more preferably 20 and most preferably 25 consecutive nucleotides of the nucleotide sequence of SEQ ID N°10, provided that said polynucleotide comprises the nucleotide sequence beginning at the nucleotide in position 157 and ending at the nucleotide in position 164 of SEQ ID N°10, or a complementary sequence thereof.

These nucleic acids are useful as probes in order to detect the presence of at least one copy of a nucleotide sequence of SEQ ID N°8, SEQ ID N°9 or SEQ ID N°10 or a complementary sequence thereof or a fragment or a variant thereof in a test sample.

The invention also relates to nucleic acid probes characterised in that they hybridize specifically under stringent hybridization conditions defined, with the nucleic acids selected from the group consisting of:

(a) a purified or isolated nucleic acid comprising a polynucleotide consisting of at least 8, preferably 12, preferably 15, more preferably 20 and most preferably 25 consecutive nucleotides with the nucleotides sequence of SEQ ID N°8, provided that said polynucleotide comprises the nucleotide sequence beginning at the nucleotide in position 157 and ending at the nucleotide in position 164 of SEQ ID N°8 or a sequence complementary thereto;

(b) a purified or isolated nucleic acid comprising a polynucleotide consisting of at least 8, preferably 12, preferably 15, more preferably 20 and most preferably 25 consecutive nucleotides of the nucleotides sequence of SEQ ID N°9, provided that said polynucleotide comprises the nucleotide sequence beginning at the nucleotide in position 157 and ending at the nucleotide in position 164 of SEQ ID N°9, or a sequence complementary thereto.

(c) a purified or isolated nucleic acid comprising a polynucleotide consisting of at least 8, preferably 12, preferably 15, more preferably 20 and most preferably 25 consecutive nucleotides of the nucleotides sequence of SEQ ID N°10, provided that said polynucleotide comprises the nucleotide sequence beginning at the nucleotide in position 157 and ending at the nucleotide in position 164 of SEQ ID N°10, or a sequence complementary thereto.

As an illustrative embodiment, stringent hybridization conditions are defined as follows:

The hybridization step is realized at 65°C in the presence of 6 x SSC buffer, 5 x Denhardt's solution, 0.5% SDS and l OOμg/ml of salmon sperm DNA. The hybridization step is followed by four washing steps: • two washings during 5 min.. preferably at 65°C in a 2 x SSC and 0.1% SDS buffer; • one washing during 30 min, preferably at 65°C in a 2 x SSC and 0.1% SDS buffer,

• one washing during 10 min, preferably at 65°C in a 0.1 x SSC and 0.1% SDS buffer, it being understood that the hybridization conditions defined above are suitable for nucleic acids of approximately 20 nucleotides in length and that these conditions may be adapted for shorter or longer nucleic acids, according to techniques well known in the art, for example those described by Sambrook et al. (1989).

A probe according to the invention has between 8, preferably 12 and 250 nucleotides in length, and ranges preferably between 8, 10, 12, 14, 15 16, 18 or 20 to 25, 35, 40, 50, 60, 70, 80, 100, 250 contiguous nucleotides of anyone of the nucleotide sequences of SEQ ID N°8, SEQ ID N°9 or SEQ ID N°10 provided that this probe comprises a sequence beginning at the nucleotide in position 157 and ending at the nucleotide in position 164 of any of the nucleotide sequences of SEQ ID N°8, SEQ ID N°9 or SEQ ID N° 10 or a sequence complementary thereto. The appropriate length for probes under a particular set of assay conditions may be empirically determined by one of skill in the art. The probes can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as the phosphodiester method of Narang et al. (1979) , the phosphodiester method of Brown et al., (1979), the diethylphosphoran method of Beaucage et al. 1859-1862 and the solid support method described in n°EP-0 707 592. The disclosures of all these documents are incorporated herein by reference. Any of the nucleic acids of the present invention can be labelled, if desired, by incorporating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.

For example, useful labels include radioactive substances ( P, ⁵S, H, I), fluorescent dyes (5-bromodesoxyuridin, fluorescein, acetylaminofluoren, dioxygenin) or biotin. Examples of non-radioactive labelling of nucleic acid fragments are described in French Patent N°FR-78 10 975, by Urdea et al. (1988) or Sanchez Pescador et al. (1988). Advantageously, the probes according to the present invention may have structure and characteristics such that they allow signal amplification, such structural characteristics being, for example, branched DNA probes such as those described by Urdea et al. (1991).

Any of the nucleic acid probes of the invention can be conveniently immobilized on a solid support. Solid supports are known to those skilled in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitro-cellulose strips, membranes, microparticles such as latex particles, red blood cells, duracytes and others. The nucleic acids of the invention and particularly the nucleotide probes described above can thus be attached to or immobilised on a solid support individually or in groups of at least 2, 5, 8, 10, 12, 15, 20 or 25 distinct nucleic acids of the invention to a single solid support.

DETECTION OF CART

The invention also deals with a method for detecting the presence of a nucleic acid encoding a truncated CART protein, a fragment or a variant thereof or a complementary sequence thereto in a sample, said method comprising the following steps of:

(a) bringing into contact a nucleic acid probe or a plurality of nucleic acid probes of the invention which can hybridize with a nucleotide sequence included in any one of the nucleotide sequences of SEQ ID N°8, SEQ ID N°9 or SEQ ID N°10 or a fragment , a variant thereof or a complementary sequence thereto and a sample to be assayed;

(b) detecting the hybrid complex formed between the probe and a nucleic acid in the sample. In a preferred embodiment of this detection method, the nucleic acid probe or the plurality of nucleic acid probes are labelled with a detectable molecule.

In a second preferred embodiment of the method, the nucleic acid probe or the plurality of nucleic acid probes are immobilized on a substrate.

In a third preferred embodiment of the method, the nucleic acid contained in the sample is made available to hybridization before step (a), by any conventional procedure well known from the one skilled in the art. The invention further concerns a kit for detecting the presence of a nucleic acid comprising a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID N°8, SEQ ID N°9 and SEQ ID N°10, a fragment or a variant thereof or a complementary sequence thereto in a sample , said kit comprising :

(a) a nucleic acid probe or a plurality of a nucleic acid probes as described above; and

(b) optionally, the reagents necessary for performing the hybridisation reaction. In a first preferred embodiment of the detection kit, the nucleic acid probe or the plurality of nucleic acid probes are labelled with a detectable molecule.

In a third preferred embodiment of the detection kit, the nucleic acid probe or the plurality of nucleic acid probes are immobilised on a substrate.

RECOMBINANT EXPRESSION VECTORS

The present invention also encompasses a family of recombinant vectors comprising any one of the nucleic acids of the invention.

Thus, the invention further deals with a recombinant vector comprising a nucleic acid selected from the group consisting of :

(a) a purified or isolated nucleic acid comprising a polynucleotide encoding a truncated CART protein, said truncated CART protein having 88 amino acids in length, or a sequence complementary thereto;

(b) a purified or isolated nucleic acid comprising a polynucleotide encoding a truncated CART protein having the amino acid sequence of SEQ ID N°5, or a sequence complementary thereto;

(c) a purified or isolated nucleic acid comprising a polynucleotide encoding a truncated CART protein having the amino acid sequence SEQ ID N°6, or a sequence complementary thereto; (d) a purified or isolated nucleic acid comprising a polynucleotide encoding a truncated CART protein having the amino acid sequence SEQ ID N°7, or a sequence complementary thereto;

(e) a purified or isolated nucleic acid comprising a polynucleotide having at least 80%) nucleotide identity with the nucleotide sequence of SEQ ID N°8, or a sequence complementary thereto; (f) a purified or isolated nucleic acid comprising a polynucleotide having at least 80% nucleotide identity with the nucleotide sequence of SEQ ID N°9, or a sequence complementary thereto;

(g) a purified or isolated nucleic acid comprising a polynucleotide having at least 80%) nucleotide identity with the nucleotide sequence of SEQ ID N°10, or a sequence complementary thereto;

(h) a purified or isolated nucleic acid comprising a polynucleotide consisting of at least 8, preferably 12 consecutive nucleotides within the nucleotide sequence of

SEQ ID N°8, provided that said polynucleotide comprises the nucleotide sequence beginning at the nucleotide in position 157 and ending at the nucleotide in position 164 of SEQ ID N°8, or a sequence complementary thereto;

(i) a purified or isolated nucleic acid comprising a polynucleotide consisting of at least 8, preferably 12 consecutive nucleotides within the nucleotide sequence of

SEQ ID N°9, provided that said polynucleotide comprises the nucleotide sequence beginning at the nucleotide in position 157 and ending at the nucleotide in position 164 of SEQ ID N°9, or a sequence complementary thereto;

(j) a purified or isolated nucleic acid comprising a polynucleotide consisting of at least 8, preferably 12 consecutive nucleotides within the nucleotide sequence of

SEQ ID N°10, provided that said polynucleotide comprises the nucleotide sequence beginning at the nucleotide in position 157 and ending at the nucleotide in position 164 of SEQ ID N°10, or a sequence complementary thereto;

A first preferred embodiment, a recombinant vector of the invention is used to amplify the inserted polynucleotide derived from the nucleic acids encoding a truncated CART protein in a suitable host cell, this polynucleotide being amplified every time the recombinant vector replicates.

A second preferred embodiment of the recombinant vectors according to the invention consists of expression vectors comprising a nucleic acid encoding a truncated CART protein having 88 amino acids in length, preferably a nucleic acid comprising a polynucleotide encoding a CART protein having the amino acid sequence of any one of SEQ ID N° 5, 6 or 7, and most preferably a nucleic acid comprising a polynucleotide having at least 80% nucleotide identity with any one of the nucleotide sequences of SEQ LD N°8, 9 or 10.

Recombinant expression vectors comprising a nucleic acid encoding the peptide fragments of a truncated CART protein that are specified in the present specification are also part of the invention. Within certain embodiments, expression vectors are used to express a truncated CART polypeptide which can then be purified and, for example, used as an immunogen in order to raise specific antibodies directed against said truncated CART protein. On another embodiment, the expression vectors are used for constructing transgenic animals and also for gene therapy. Expression requires that appropriate signals are provided in the vectors, said signals including various regulatory elements such as enhancers/promoters from both viral and mammalian sources that drive the expression of the gene of interest in the host cell. The regulatory sequences of the expression vectors of the invention are operably linked to the nucleic acid encoding the truncated CART proteins. As used herein, the term " operably linked " refers to a linkage of polynucleotide elements in a functional relationship. For instance, a promoter or an enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. More precisely, two DNA molecules (such as a polynucleotide containing a promoter agent and a polynucleotide encoding a desired polypeptide or polynucleotide ) are said to be " operably linked " if the nature of the linkage between the two polynucleotides does not : (1) result in the introduction of a frame- shift mutation or (2) interfere with the ability of the polynucleotide containing the promoter to direct the transcription of the coding polynucleotide.

Generally, recombinant expression vectors will include origins or replication, selectable markers permitting transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence. The heterologous structural sequence is assembled in an appropriate fashion with translation, initiation and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein into the periplasmic space or the extracellular medium. In a specific embodiment wherein the vector is adapted for transfecting and expressing desired sequences in mammalian host cells, preferred vectors comprise an origin of replication in the desired host, a suitable promoter and an enhancer, and also any necessary ribosome binding sites, polyadenylation sites, transcriptional termination sequences, and optionally 5'- flanking non transcribed sequences. DNA sequences derived from the SV40 viral genome, for example SV40 origin, early promoter, enhancer, and polyadenylation sites may be used to provide the require non-transcribed genetic elements.

The suitable promoter regions used in the expression vectors according to the invention are chosen taking into account the host cell in which the heterologous nucleic acids are to be expressed. A suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively it can be endogenous to the native polynucleotide containing the coding sequence to be expressed.

Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted. Preferred bacterial promoters are the Lad, LacZ, T3 or T7 bacteriophage RNA polymerase promoters, the lambda PR, PL and tip promotors (EP 0 036 776), the polyhedrin promotor, or the plO protein promoter from baculovirus (Kit Novagen; Smith et al., 1983); O'Reilly et al.,1992, Baculovirus expression vectors: A Laboratory Manual. W.H. Freeman and Co., New York).

Preferred selectable marker genes contained in the expression recombinant vectors of the invention for selection of transformed host cells are preferably dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, TRP1 for S. cerevisiae or tetracyclin, rifampicin or ampicillin resistance in E. coli, or Levan saccharase for mycobacteria, this latter marker being a negative selection marker.

Preferred bacterial vectors of the invention are listed hereafter as illustrative but not limitative examples: pQE70, pQE60, pQE-9 (quiagen), pDIO, fephagescript, psiX174, p.Bluescript SK, pNH8A, pNH16A, pNH18A, pNH46A (stratagene); pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXTl, pSG (stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QIA express).

Preferred bacteriophage recombinant vectors of the invention are PI bacteriophage vectors such as described by Steinberg N.L. (1992; 1994).

A suitable vector for the expression of a truncated CART polypeptide of the invention or a fragment thereof, is a baculovirus vector that can be propagated in insect cells and in insect cell-lines. A specific suitable host vector system is the pVL

1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC N°CRL 1711) which is derived from spodoptera frugiperda.

The recombinant expression vectors of the invention may also be derived from an adenovirus such as those described by Feldman and Steg. (1996) or Ohno et al. (1994).

Another preferred recombinant adenovirus according to this specific embodiment of the present invention is the human adenovirus type two or five (Ad 2 or Ad 5) or an adenovirus of animal origin (French Patent Application n°FR 93 05 954). Particularly preferred retroviruses for the preparation or construction of retroviral in vitro or in vivo gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Mink-Cell Focus Inducing Virus, murine sarcoma virus, and Ross Sarcoma Virus. Other preferred retroviral vectors are those described in Roth et al. (1996), in PCT Application WO 93/25 234, in PCT Application WO 94/06920, and also in Roux et al. (1989), Man et al.(1992) and Nada et al. (1991).

Yet, another viral vector system that is contemplated by the invention consists in the adeno associated virus (AAV) such as those described by Flotte et al. (1992),

Samulski et al. (1989) and McLaughlin et al. (1996). Another object of the invention consists of a host cell that has been transformed or transfected with one of the nucleic acids described herein particularly nucleic acids encoding a truncated CART protein having 88 amino acids in length and most preferably nucleic acids encoding the truncated CART protein of any one of the amino acid sequences of SEQ ID N°5, SEQ ID N°6 or SEQ ID N°7

HOST CELLS EXPRESSING CART

Included are host cells that are transformed (prokaryotic cells) or are transfected (eukaryotic cells) with a recombinant vector such as one of those described above. Preferred host cells used as recipients for the expression vectors of the invention are the following:

(a) prokaryotic host cells: Escherichia coli, strains, (i.e. DH5- , strain) Bacillus subtilis, Salmonella typhimurium and strains from species like Vseudomonas, Streptomyces and Staphylococcus;

(b) eukaryotic host cells: HeLa cells (ATCC N°CCL2; N°CCL2.1; N°CCL2.2), Cv 1 cells (ATCC N°CCL70), COS cells (ATCC N°CRL 1650; N°CRL

1651), Sf-9 cells (ATCC N°CRL 1711), C127 cells (ATCC N°CRL-1804), 3T3 cells (ATCC N°CRL-6361), CHO cells (ATCC N°CCL-61), human kidney 293 cells (ATCC N° 45504; N°CRL-1573) and BHK (ECACC N°84100 501; N°84111301); PC12 (ATCC N° CRL-1721), NT2, SHSY5Y (ATCC N° CRL-2266), NG108 (ECACC N°88112302) and FI 1, SK-N-SH (ATCC N° CRL-HTB-11), SK-N-BE(2) (ATCC N° CRL-2271), IMR-32 (ATCC N° CCL-127). A preferred system to which the gene of the invention can be expressed are cell lines such as COS cells, 3T3 cells, HeLa cells, 292 cells and CHO cells. A preferred system for the efficient expression of CART involves the use of CHO and COS cell lines. The gene can be expressed through an endogenous promoter of native CHO or COS, or through an exogenous promoter. Suitable exogenous promoters include such as SV40 and CMV, or perhaps a eucaryotic promoter such as the tetracycline promoter. The preferred promoter being CMV.

CART POLYPEPTIDE AND FRAGMENTS Another object of the invention consists of a purified or isolated polypeptide comprising the amino acid sequence of the CART polypeptide, or a peptide fragment or a variant thereof.

In a first preferred embodiment, the polypeptide comprises the amino acids sequence of the CART polypeptide present in rat tissue or a peptide fragment or a variant thereof. A particularly preferred polypeptide is the polypeptide of SEQ ID N°5.

In a second preferred embodiment, the polypeptide comprise the amino acids sequence of a shortened form of the rat CART polypeptide such as the sequence of SEQ ID N°l 1 (CART polypeptide sequence from which the signal peptide has been removed) containing 61 amino acids.

In a third preferred embodiment, the polypeptide comprises the amino acids sequence of the CART polypeptide present in human tissue or a peptide fragment or a variant thereof. A particularly preferred polypeptide is the polypeptide of SEQ ID N°6.

In a fourth preferred embodiment, the polypeptide comprise the amino acids sequence of a shortened form of the human CART polypeptide such as the sequence of SEQ ID N°12 (CART polypeptide sequence from which the signal peptide has been removed) containing 61 amino acids. In a fifth preferred embodiment, the polypeptide comprises the amino acids sequence of the CART polypeptide present in mouse tissue or a peptide fragment or a variant thereof. A particularly preferred polypeptide is the polypeptide of SEQ ID N°7.

In a sixth preferred embodiment, the polypeptide comprise the amino acids sequence of a shortened form of the mouse CART polypeptide such as the sequence of SEQ ID N°13 (CART polypeptide sequence from which the signal peptide has been removed) containing 61 amino acids.

As shown in figure 1, the amino acids sequences of the rat (129 amino acids,

SEQ ID N°3), mouse (129 amino acids. SEQ ID N°4) and human (1 16 amino acids, SEQ ID N°l and 2) have a very strong sequence similarity, with only 19 non identical amino acids out of a total of 129 amino acids when the signal sequence is included (80% amino acid identity between the three polypeptides). Out of these non identical amino acids, 18 amino acids occur in identical positions in two of the three CART polypeptides. The sequence similarity between the three polypeptides is even higher (> 85% amino acid identity) if the 27 amino acids of the signal sequence are not included. Comparison of the rat CART variant polypeptide of SEQ LD N°5 containing 88 amino acids with SEQ LD N°l 2 and 4 indicates only 5 non identical amino acids out of a total of 88 when the signal sequence is included (> 95% amino acid identity). In the absence of the signal sequence (SEQ ID N°l 1) no non identical amino acids occur out of a total of 61 amino acids. Furthermore, the inventors have identified a similar cDNA band, corresponding to a CART polypeptide of 88 amino acids from human cDNA library, as shown in figure 2. Thus, the inventors believe that a CART polypeptide of 88 amino acids is present in human, such as that of SEQ ID N°6 and in other mammalian species. Moreover, the inventors believe that this CART polypeptide shares strong sequence similarity to the rat CART sequence of SEQ ID N°5.

The present invention also concerns a polypeptide comprising an amino acid sequence having at least 90%, preferably 95%, more preferably 98% and most preferably 99% amino acid identity with the amino acid sequences of SEQ ID N°5 or SEQ ID N°11.

Also within the scope of the present invention is a polypeptide comprising an amino acid sequence having at least 90%, preferably 95%, more preferably 98% and most preferably 99% amino acid identity with the amino acid sequences of SEQ ID N°6 or SEQ ID N°12.

Furthermore within the scope of the present invention is a polypeptide comprising an amino acid sequence having at least 90%, preferably 95%, more preferably 98% and most preferably 99% amino acid identity with the amino acid sequences of SEQ ID N°7 or SEQ ID N°13. The invention also relates to fragments of the CART polypeptide which can be useful for example in diagnostic and ligand screening applications.

Particularly, preferred fragments of interest have been selected from an analysis of the amino acid sequence of the CART protein. Figure 1 comprises annotations on the CART protein indicating regions of interest.

CART polypeptide is thought to form a linear polypeptide in the cell cytoplasm. The polypeptide is transported to the cell surface and secreted. Secretion is aided by the presence of the 27 amino acid N-terminal signal sequence, as shown in figure 1. Upon secretion the signal polypeptide is cleaved from the CART polypeptide sequence. This results in a CART polypeptide with a truncated N- terminus. Therefore, the inventors believe that the signal sequence of the CART polypeptide is a target for the development of therapeutics and diagnostics.

Preferred peptides which encode the rat CART signal sequence include the peptide of SEQ ID N°14 (amino acids 1-116, as shown in figure 1). Other preferred peptides which encode the rat CART signal sequence the peptide of SEQ ID N°15 (amino acids 9-104, as shown in figure 1).

Preferred peptides which encode the human CART signal sequence include the peptide of SEQ ID N°16 (amino acids 1-116, as shown in figure 1). Other preferred peptides which encode the human CART signal sequence include the peptide of SEQ ID N°17 (amino acids 9-104, as shown in figure 1).

Preferred peptides which encode the mouse CART signal sequence include the peptide of SEQ ID N°18 (amino acids 1-116, as shown in figure 1). Other preferred peptides which encode the mouse CART signal sequence include the peptide of SEQ ID N°19 (amino acids 9-104, as shown in figure 1).

Potential proteolytic cleavage sites have been identified in the rat CART polypeptide (Thim et al 1999), as shown in figure 1. These include amino acid 36; arginine (as indicated in figure 1 ), amino acid 51 ; lysine (as indicated in figure 1), amino acids 75-76; lysine (as indicated in figure 1), amino acid 78; lysine (as indicated in figure 1), amino acids 80-81; lysine-arginine (as indicated in figure 1), amino acids 87-88; lysine (as indicated in figure 1 ), amino acids 104-105; arginine-

SUBSTΓΓUTE SHEET (RULE 26) lysine (as indicated in figure 1), amino acid 108; arginine (as indicated in figure 1), amino acid 111; lysine (as indicated in figure 1) and amino acid 127; lysine (as indicated in figure 1). Identical amino acids are also present in the human and mouse CART polypeptide (as indicated in figure 1). The inventors believe that post translational modification of the CART protein plays an important role in the modulation of its functional activity. Therefore, the inventors believe that these cleavage sites are targets for the development of therapeutics and diagnostics. Preferred peptides which incorporate proteolytic cleavage sites include those of SEQ ID N°20 (amino acids 51-127, as shown in figure 1) and SEQ ID N°21 (amino acids, as shown in figure 1). Other preferred peptides include those of SEQ ID N°22 (amino acids 36-111, as shown in figure 1) and SEQ ID N°23 (amino acids 36-127, as shown in figure 1).

The present invention also concerns a polypeptide comprising an amino acid sequence having at least 90%, preferably 95%, more preferably 98% and most preferably 99% amino acid identity with the amino acid sequence of SEQ ID N°20, SEQ ID N°21, SEQ ID N°22 or SEQ ID N°23.

CART peptides have been shown to possess functional activity in mammals.

Functional activity has been attributed to the C-terminal portion of CART. lev injection of C-terminal CART peptides into rat has been shown to reduce food intake in the rats by up to 40% (Lambert et al 1998 and Lambert et al 1988). Furthermore, analysis of CART protein primary and secondary structure indicates that the C- terminus contains three disulphide bonds. The first disulphide bond is between C95- C113 (as shown in figure 1), the second between C115-C128 (as shown in figure 1) and the third between C101 and C121 (as shown in figure 1 ) (Thim et al 1998). N- terminal to C95 the CART peptide is devoid of cystein residues. Post translational modification of CART results in cleavage of the polypeptide N-terminal to C95 (Thim et al). The inventors believe that the discovery and analysis of the CART splice variant eludes to the true functionally active component of the CART peptide. The inventors believe that a common component of the C-terminus of the CART variant polypeptide of SEQ ID N°5, 6 and 7 and the large and short forms of the CART peptide of SEQ ID N°l, 2, 3 and 4 is a CDAGEQCAV motif between amino acids 95 and 103, as shown in figure 1. Moreover, due to the C-C bonds between C95-C113, C101-C121 and CI 15-C128 amino acids including and between C95 and VI 03 will be held in a more ridged conformation when compared with this peptide in "free" solution, thereby conferring structure to the peptide. The inventors believe that the elucidation of this peptide region in CART is important in the development of therapeutics and diagnostics. Preferred peptides which encode this region are those of SEQ ID N°24

(amino acids 95 to 103, as shown in figure 1) and SEQ ID N°25 (amino acids 95 to 101, as shown in figure 1). Other preferred peptides include those of SEQ ID N°26 (amino acids 51 to 101 as shown in figure 1), SEQ ID N°27 (amino acids 51 to 103, as shown in figure 1) and SEQ ID N°28 (amino acids 50 to 103, as shown in figure 1)

The present invention also concerns polypeptides comprising an amino acid sequence having at least 90%, preferably 95%, more preferably 98% and most preferably 99% amino acid identity with the amino acid sequence of SEQ ID N°24, SEQ ID N°25, SEQ ID N°26, SEQ ID N°27 or SEQ ID N°28.

Without wishing to be bound by any particular theory, the inventors believe that the amino acid sequence of SEQ ID N°24, SEQ ID N°25, SEQ ID N°26, SEQ ID N°27 or SEQ ID N°28 contained in any one of the truncated CART polypeptides of SEQ ID N°5, SEQ ID N°6 or SEQ ID N°7 are of functional relevance, and that the formation of a disulfide bridge between the two cysteine residues present in these peptide sequences could be important for the biological activity of the truncated CART proteins. The invention thus also deals with a purified or isolated polypeptide comprising at least 10 consecutive amino acids of any one of the amino acid sequences of SEQ ID N°5, 6 or 7, provided that said polypeptide comprise the amino acid sequence of SEQ ID N°24, SEQ ID N°25, SEQ ID N°26, SEQ ID N°27 or SEQ ID N°28.

Preferred peptide fragments of the truncated CART proteins of the invention include, but are not limited to, amino acid sequences located in the C-terminus end of anyone of the polypeptides of the invention and including the amino acid sequences of SEQ ID N°24, SEQ ID N°25, SEQ LD N°26, SEQ ID N°27 and SEQ ID N°28.

Likewise, other preferred peptides which encode this functionally active region are those of SEQ ID N°29 (amino acids 41-129, as shown in figure 1) and SEQ ID N°30 (amino acids 95-129, as shown in figure 1). The present invention also concerns polypeptides comprising an amino acid sequence having at least 90%, preferably 95%, more preferably 98% and most preferably 99% amino acid identity with the amino acid sequences of SEQ ID N°29 or SEQ ID N°30.

Without wishing to be bound by any particular theory, the inventors believe that the amino acid sequence of SEQ ID N°29 or SEQ ID N°30, contained in any one of the truncated CART polypeptides of SEQ ID N°5, SEQ ID N°6 or SEQ ID N°7 is of functional relevance, and that the formation of a disulfide bridge between the cysteine residues present in these peptide sequences could be important for the biological activity of the truncated CART proteins. The invention thus also deals with a purified or isolated polypeptide comprising at least 10 consecutive amino acids of any one of the amino acid sequences of SEQ ID N°5, 6 or 7, provided that said polypeptide comprises the amino acid sequence of SEQ ID N°29 or SEQ ID N°30.

The present invention also concerns a method for producing one of the truncated CART polypeptides described herein and especially a polypeptide selected from the group consisting of the amino acid sequences of SEQ ID N°5, SEQ ID N°6 or SEQ N°7 wherein said method comprises the steps of :

(a) inserting the nucleic acid encoding the desired truncated CART protein in an appropriate vector; (b) culturing, in an appropriate culture medium, a host cell previously transformed or transfected with the recombinant vector of step (a);

( c) harvesting the culture medium thus conditioned or lysing the host cell, for example by sonication or osmotic shock; (d) separating or purifying, from said culture medium, or from the pellet of the resultant host cell lysate, the thus produced truncated CART polypeptide of interest.

Preferred fragments such as defined herein have at least ten contiguous nucleotides of any one of the amino acid sequences of SEQ ID N°5, SEQ ID N°6 or SEQ N°7 preferably at least 12 or 15, more preferably at least 20 and most preferably at least 25 consecutive nucleotides of any one of the amino acid sequences of SEQ

ID N°5, 6 or 7.

In a first preferred embodiment of the above method, the nucleic acid to be inserted in the appropriate vector has previously undergone an amplification reaction, using a pair of primers. Preferred primers used for such an amplification reaction are the primers having the nucleotide sequences of SEQ ID N°33 and SEQ ID N°34.

Primers of the nucleotide sequences of SEQ ID N°33 and SEQ ID N°34 are also part of the invention. In a second preferred embodiment of the above method, the polypeptide thus produced is further characterized for example by binding onto an immuno affinity chromatography column on which polyclonal or monoclonal antibodies directed to a truncated CART polypeptide of the invention or a fragment thereof have been previously immobilised. Purification of the recombinant truncated CART proteins according to the present invention may be carried out by passage onto a nickel or copper affinity chromatography column.

In another embodiment, the truncated CART polypeptides thus obtained may be purified, for example, by high performance liquid chromatography, such as reverse phase and or cationic exchange HPLC, as described by Rougeot et al.(1994).

The reason to prefer this kind of peptide or protein purification is the lack of by-

SUBSTΓTUTE SHEET (RULE 26) products formed in the elution samples which renders the resultant purified protein or peptide more suitable for therapeutic use. Thus, another object of the present invention consists of a purified or isolated polypeptide comprising the amino acid sequence of a CART protein, said CART protein having 88 amino acids in length. The invention further deals with a purified or isolated polypeptide comprising the amino acid sequence of SEQ LD N°5.

The invention also relates to a purified or isolated polypeptide comprising the amino acid sequence of SEQ LD N°6.

The invention also relates to a purified or isolated polypeptide comprising the amino acid sequence of SEQ LD N°7.

The invention also pertains to a truncated CART polypeptide encoded by a nucleic acid having at least 80% nucleotide identity with any one of the nucleotide sequences of SEQ ID N°8, SEQ ID N°9 or SEQ ID N°10.

In a first embodiment an amino acid substitution in the amino acid sequence of a polypeptide according to the invention, one or several-consecutive or non- consecutive-amino acids are replaced by " equivalent " amino acids. The expression

" equivalent " amino acid is used herein to designate any amino acid that may be substituted for one of the amino acids belonging to the native protein structure without decreasing the binding properties of the corresponding peptides to the antibodies raised against the truncated CART polypeptides of the amino acid sequences of SEQ ID N° 5, 6 and 7. In other words, the " equivalent " amino acids are those which allow the generation or the synthesis of a polypeptide with a modified sequence when compared to the amino acid sequence of the native truncated CART protein, said modified polypeptide being able to bind to the antibodies raised against the truncated C.ΛRT protein of the amino acid sequences of

SEQ ID N° 5, 6 and 7 and/or to induce antibodies recognising the parent polypeptide.

These equivalent amino acids may be determined either by their structural homology with the initial amino acids to be replaced, by the similarity of their net charge or of their hydrophobicity. and optionally by the results of the cross- immunogenicity between the parent peptides and their modified counterparts.

The peptides containing one or several " equivalent " amino acids must retain their specificity and affinity properties to the biological targets of the parent protein. This can be assessed by a ligand binding assay or an ELISA assay.

For example, amino acids belong to specific classes such as Acidic (Asp, Glu), Basic (Lys, Arg, His), Non-polar (Ala, Val, Leu, He, Pro, Met, Phe, Trp) or uncharged Polar (Gly, Seu, Thr, lys, Tyr, Asn, Gin).

Preferably, a substitution of an amino acid in a truncated CART polypeptide of the invention, or in a peptide fragment thereof, consists in the replacement of an amino acid of a particular class for another amino acid belonging to the same class.

By an equivalent amino acid according to the present invention is also contemplated the replacement of a residue in the L-form by a residue in the D form or the replacement of a Glutamic acid (E) residue by a Pyro-glutamic acid compound. The synthesis of peptides containing at least one residue in the D-form is, for example, described by Koch (1977).

In a second embodiment of an amino acid substitution in the amino acid sequence of a polypeptide according to the invention, one or several consecutive or non consecutive amino acids are replaced by one or several other amino acids belonging to another specific class among the amino acid classes defined above.

These non-conservative changes in the amino acids sequence of a truncated CART polypeptide of the invention may consist in the replacement of a positively charged amino acid with a negatively charged amino acid or also in the replacement of a hydrophobic amino acid for a hydrophilic amino acid. Such non-conservative amino acid substitutions could lead to a peptide which is still capable of binding to its natural binding site but would be incapable of eliciting a physiological response. Therefore, these modified CART peptides might have a role as a partial or complete antagonist molecules in preventing the natural or endogenous CART polypeptide to bind to its natural binding site.

The antagonistic effect of these modified truncated CART peptides that have undergone non conservative amino acids substitutions may be assessed, for example by in vitro assays allowing the measure of the growth index or of the survival of different brain cells, such as rat brain cells, neonatal brain cells, spinal cord motor neurones and neurites, as described in PCT Application N° WO 96/34 619. The antagonistic effect of these modified CART peptides may also be tested in vivo, for example by observing the feeding behaviour in response to NPY neuropeptide of rats treated with the modified CART peptide of the invention, either orally, rectally, via mucosal route, via inhalation, or via systemic or local injection into given tissues.

The antagonistic effect of these modified CART peptides may also be measured by biochemical characterization at the sub-cellular, cell, tissue, organ and whole animal level.

Suitable techniques for in vivo experiments are for example those described by Lambert et al. (1998).

Thus, the invention also encompasses a truncated CART polypeptide, or a peptide fragment thereof comprising non-conservative amino acid changes ranging from 1, 2 , 3, 4, 5, 10 to 20 substitutions of one amino acid as regards to the truncated CART polypeptides of anyone of the amino acids sequences of SEQ ID N°5, 6 or 7. A specific embodiment of a modified peptide molecule of interest according to the present invention, includes, but is not limited to, a peptide molecule which is resistant to proteolysis. This is a peptide in which the -CONH- peptide bond is modified and replaced by a (CH₂NH) reduced bond, a (NHCO) retro inverso bond, a (CH₂-O) methylene-oxy bond, a (CH₂S) thiomethylene bond, a (CH₂CH₂) carbon bond, a (CO-CH₂) acetomethylene bond, a (CHOH-CH₂) hydroxyethylene bond), a (N-N) bound, a E-alcene bond or a -CH=CH-bond.

The invention also encompasses a truncated CART polypeptide or a fragment thereof in which at least one peptide bond has been modified as described above.

The polypeptides according to the invention may also be prepared by conventional methods of chemical synthesis, either in a homogenous solution or in solid phase. As an illustrative embodiment of such chemical polypeptide synthesis techniques, it may be cited the homogenous solution technique described by

Houbenweyl (1974).

The truncated polypeptide. or a fragment thereof may thus be prepared by chemical synthesis in liquid or solid phase by successive couplings of the different amino acid residues to be incorporated (from the N-terminal end to the C-terminal end in liquid phase, or from the C-terminal end to the N-terminal end in solid phase) wherein the N-terminal ends and the reactive side chains are previously blocked by conventional protecting groups.

For solid phase synthesis, the technique described by Merrifield (1965a; 1965b) may be used in particular.

The truncated CART polypeptides of the invention and their peptide fragments of interest can also be used for the preparation of polyclonal or monoclonal antibodies.

ANTLBODYS TO CART

Preferred peptide fragments used to prepare monoclonal or polyclonal antibodies include those which comprise the amino acid sequences of SEQ ID N°5, 6 and 7. Preferred peptide fragments include those which encode the CART polypeptide signal sequence, SEQ ID N°14, 15, 16, 17, 18 or 19.

Other preferred peptide fragments include those which encode the amino acid sequence of <CDAGEQC>. SEQ ID N°24, 25, 26, 27, 28, 29 or 30 encode this motif.

More preferred peptide fragments are those which comprise the proteolytic cleavage sites, SEQ ID N°20, 21 , 22 or 23.

Antibodies may be prepared from hybridomas according to the technique described by Kohler and Milstein (1975). The polyclonal antibodies may be prepared by immunization of a mammal, especially a mouse or a rabbit, with a polypeptide according to the invention that is combined with an adjuvant, and then by purifying the specific antibodies contained in the serum of the immunized animal on an affinity chromatography column on which has previously been immobilised the polypeptide that is used as the antigen.

The present invention also deals with antibodies directed against a truncated CART polypeptide of the invention or a fragment thereof produced by the trioma technique and by the human B cell hybridoma technique (Kozbor et al., 1983).

The present invention also includes chimeric single chain Fv antibody fragments (US Patent N°US 4,946,778; Martineau et al., 1998), antibody fragments obtained through phage display libraries (Ridder et al. 1995) and humanized antibodies (Leger et al, 1997). Other types of antibodies or antibody fragments which can be obtained by methods such as those described in PCT publications WO 94 18227, WO 95 08577, WO 95 15388, WO 98 01757, WO 93 11236, WO 93 06213, WO 92 20791, WO 92 01047, incorporated herein by reference, also fall within the scope of the present invention.

Antibody preparations obtained according to either protocol are useful in quantitative immunoassays for determining the presence or the concentrations of antigen bearing substances in biological samples. The antibodies may also be used in therapeutic compositions as it will be described hereafter.

DETECTION OF CART PEPTIDE AND FRAGMENTS

Consequently, the invention is also directed to a method for detecting the presence of a truncated CART protein according to the invention in a biological sample, said method comprising the following steps of :

(a) bringing into contact a biological sample with a polyclonal or a monoclonal antibody directed against a truncated CART protein or to a peptide fragment thereof;

(b) detecting the antigen antibody complex formed.

The invention also concerns a diagnostic kit for detecting the presence of a truncated CART protein according to the present invention in a biological sample, wherein the kit comprises a polyclonal or a monoclonal antibody directed against the truncated CART protein or a peptide fragment thereof.

In a preferred embodiment, the kit further comprises a reagent allowing the detection of the antigen-antibody complexes formed, said reagent optionally carrying a label, or being able to be recognised itself by a label reagent, particularly in the case when the above mentioned monoclonal or polyclonal antibody is not itself labelled.

The truncated CART polypeptides of the invention as well as the antibodies specifically directed against these polypeptides may be useful for therapeutic purposes.

The truncated CART polypeptides of the invention or a peptide fragments thereof may be included in compositions that are effective in order to decrease or prevent neuronal degeneration and in order to promote neuronal regeneration and restoration of function.

In a second embodiment, the truncated CART polypeptides of the invention or a peptide fragment thereof may be included in a composition for reducing food intake, for modulating pain behaviour and for treating congenital conditions or neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, epilepsy and also for treating substance abuse or addiction (such as alcoholism, cocaine, heroin and amphetamine dependency).

Additionally, compositions containing antibodies directed against the truncated CART polypeptides of the invention or peptide fragments thereof may be used in order to antagonize the effect of the endogenous CART polypeptides. For example, such composition including antibodies directed against truncated CART polypeptides of the invention or a fragment thereof may be used to reduce cocaine induced locomotor activity and thus be used in the treatment of substance abuse.

The therapeutic composition described above may be administered via different routes, such as systemic routes, but preferably via local routes and more preferably via intracranal route and most preferably by intra-cerebroventricular (ICV) route. The concentrations of the truncated CART polypeptides or its fragment thereof in a such pharmaceutical composition is adapted in order to allow a suitable administration within the body. These pharmaceutical compositions containing a therapeutically effective amount of a truncated CART polypeptide of the invention or a peptide fragment thereof or also a modified CART polypeptide as defined above, are preferably administered orally, by injection or infusion by intravenous, intraperitoneal, intracerebral, intracerebroventricular. intramuscular, intraarterial roots or by sustained release systems.

Preferably the daily dosage of the truncated CART protein or one peptide fragment thereof will range from O.Olμg/ kg to up to 100 mg/kg and will more preferably range from 0,1 μg/kg to up 300μg/kg.

Pharmaceutical compositions of the invention also contain physiologically and/or pharmaceutically acceptable carriers, excipients, or stabilizers and may be under the form either of a lyophilised preparation or an aqueous solution. For a detailed description of further embodiments of suitable pharmaceutical compositions according to the present invention, it may also be referred to PCT Application WO 96/34619 which is herein incorporated by reference in its entirety.

SCREENING FOR CART LIGANDS AND CART RECEPTOR LIGANDS

The present invention also concerns methods for screening ligand substances or molecules that are able to modulate the biological activity of the CART protein, peptide fragments thereof or of the CART receptor. Studies on the CART polypeptide and CART peptide fragments suggest that

CART has powerful anorectic and anti addiction properties. However, the precise mechanism(s) of the mode of action of CART polypeptide or peptide fragments thereof and the receptor is not fully elucidated. The inventors believe that therapeutics which potentiate or negate the action of CART can play a pivotal role in the treatment of conditions such as obesity, heart disease and drug addiction. The inventors believe there are at least two possible scenarios for the development of therapeutics. In the first scenario ligands are developed which bind to the CART receptor or CART polypeptide or CART peptide fragments and potentiate the effect of CART. These therapeutic molecules can be used to treat addiction and obesity and diseases associated with drug addiction and obesity. In a second scenario ligands are developed which bind to the CART receptor or CART polypeptide or CART peptide fragments and suppress the action of CART. There molecules can be used to treat eating disorders such as anorexia and cancer (cancer patients often suffer from lack of appetite) and other conditions which are associated with lack of appetite. Furthermore the inventors believe that the CART receptor is a leptin receptor or a leptin like recepotor. Strong evidence for this conclusion comes from their research as shown in figure 5 and example 3. Chromatography of pig brain tissue on a CART peptide affinity matrix produced a specific major protein band of approximately 60kd in size when protein samples were run on a gradient SDS PAGE gel and two minor bands of approximately lOOkd. These bands were not apparent when protein samples were fractionated on chromatography matrix which did not contain CART peptide. The predicted molecular weigh of the isoforms of leptin receptor are 37.5 to 125kd. However, there weight do not take into account glycosolation of the receptor. The inventors believe that the mechanism of action of CART peptide is either at the cell surface or intracellular. Both leptin and CART have been shown to modulate the metabolism of glucose and the regulation of c-Fos. The inventors conclude that CART either interacts directly with the leptin receptor, thereby modulating the action of leptin or interacts with a leptin like receptor which in-tum modulates the action of leptin.

Thus, the invention also relates to a method for the screening of compounds useful in the treatment of drug addiction and obesity. This assay involves screening for compounds which potentiate the action of CART.

The invention also concerns a method for the screening of compounds useful in the treatment of eating disorders. This assay involves screening for compounds which suppress the action of CART.

Production of CART or a fragment thereof

The CART polypeptide or fragments thereof can be prepared using recombinant technology, cell lines or chemical synthesis. Recombinant technology and chemical synthesis of the CART polypeptide or fragments thereof can allow the modification of the gene encoding the CART polypeptide or fragment to include such features as recognition tags, cleavage sites and modifications of the CART polypeptide or fragments thereof. For efficient polypeptide production, the recombinant expression system should allow the CART polypeptide or fragment to be expressed, transported at the cell surface and secreted in a functional form or allow production of CART polypeptide and fragments which can be purified. Preferred cell lines are those which allow high levels of expression of CART polypeptide or fragments thereof. Such cell lines include common mammalian cell lines such as CHO cells and COS cells, etc or more specific neuronal cell lines such as PC 12. However, other cell types which are commonly used for recombinant protein production such as insect cells, amphibian cells such as oocytes, yeast and procaryotic cell lines such as E.coli can also be considered.

The CART polypeptide or fragments thereof can be utilised in a ligand screen either as a purified protein, as a protein chimera such as those described above or in in phage display or as a component of a cell based assay or in vivo assay. The CART polypeptide or fragment thereof can be utilised in a functional screen format or ligand binding screen format. Examples of both screening formats are provided below.

Ligand binding screening methods

(A) A typical embodiment of a ligand binding screen comprises of the following steps;

(a) contacting the ligand with the CART polypeptide or a fragment thereof. The CART polypeptide or fragment can be bound to a matrix or in solution. The ligand can be for example a peptide or protein or antibody or a chemical entity. The principal property the ligand must have is that it must recognise and bind to a binding site determined by the CART polypeptide or fragments thereof.

Optionally, excess non CART bound ligand can be removed by separation. Separation can take the form of washing, filtration or centrifugation (to pellet the CART polypeptide or fragment). In this latter case, the supernatant can then be removed and the CART polypeptide or fragment thereof re-suspended in buffer.

(b) contact the medium containing the ligand and the CART polypeptide or a fragment thereof with a substrate and allow binding to occur.

A property of the substrate must be that it is detectable and quantifiable. To achieve this the substrate can be a chromophore or radio, fluorescent, phosphorescent, enzymatically or antibody labelled. If the substrate is not directly detectable it must be amenable to detection and quantification by secondary detection, which may employ the above technologies. Another property of the substrate must be that its binding characteristics are determined by the amino acids sequence or the CART polypeptide or fragment thereof and that the binding characteristics are modified upon ligand binding to the CART. The substrate can be a peptide, a protein, a antibody or chemical entity.

Optionally, unbound substrate can be removed from the mixture as described above.

(c) measurement of substrate binding

Binding of the ligand modifies affinity for the substrate with the CART binding site. The difference between the observed amount of substrate bound relative to the theoretical maximum amount of substrate bound is a reflection of the amount and affinity of ligand bound to the substrate-binding site. The mechanism of detection of substrate is determined by it properties.

Alternatively, the amount of ligand bound to the CART polypeptide or a fragment thereof can be determined by a combination of chromatography and mass spectroscopy.

The amount of ligand bound to the CART polypeptide or a fragment thereof can also be determined by direct measurement of the change in mass upon ligand or substrate binding to the CART polypeptide or a fragment thereof. This could be achieved with technologies such as Biocore (Amersham Pharmacia). Alternatively, the CART polypeptide or a fragment thereof, the substrate or the ligand can be fluorescently labeled and association of CART polypeptide or fragment with the ligand can be followed by changes in Fluorescence Energy

Transfer (FRET).

(B) A typical embodiment of a ligand binding screen utilising the CART receptor and CART polypeptide or fragment comprises the following steps; (a) contacting the ligand with the CART receptor.

Cell types expressing the CART receptor can be obtained by dissociation of brain tissue and subsequent culture. Receptor identification can be facilitated using labelled CART polypeptide or fragments thereof (as described above) to identify neuronal cells which express the receptor. Once identified these cell types can be immortalised using standard laboratory techniques. Quiescent or non quiescent cells can be used as a basis of a ligand binding screen. These cells can be used intact or as a membrane preparation. The ligand can be for example a peptide, a protein, a antibody or a chemical entity. The principal property the ligand must have is that it must recognise and bind to a binding site determined by the CART receptors amino acid sequence. Optional: excess non CART receptor bound ligand can be removed by separation. Separation can take the form of washing, filtration or centrifugation (to pellet the cells or membranes containing the CART receptor).

(b) contact the medium containing the ligand and the CART receptor with substrate (CART polypeptide or fragment thereof).

A property of the substrate must be that it is detectable and quantifiable. To achieve this the CART peptide or fragment can be labelled radio label or a chromophore. Alternatively, the CART polypeptide or fragment can be labelled with a compound that is fluorescent, phosphorescent, an enzyme or antibody. If the CART polypeptide or fragment is not directly detectable it must be amenable to detection and quantification by secondary detection, which may employ the above technologies.

Optionally, unbound CART polypeptide or fragment can be removed from the mixture as described above.

(c) measurement of CART polypeptide or fragment binding Binding of the ligand modifies affinity of the CART polypeptide or fragment for the CART receptor. The difference between the observed amount of CART polypeptide or fragment bound relative to the theoretical maximum amount of CART polypeptide or fragment bound is a reflection of the amount and affinity of ligand bound to the CART receptor binding site.

Alternatively, the amount of ligand bound to the CART receptor can be determined by a combination of chromatography and mass spectroscopy.

The amount of ligand bound to the CART receptor can also be determined by direct measurement of the change in mass upon ligand or substrate binding to CART receptor. This could be achieved with technologies such as Biocore (Amersham Pharmacia). Alternatively, the CART receptor and CART polypeptide or fragment could be fluorescently labelled and association the ligand can be followed by changes in Fluorescence Energy Transfer (FRET)

Functional screening methods Both the in vitro and in vivo functional screening assays, would allow the skilled artisan to distinguish between potentiators and suppressors of CART activity. Biochemical studies indicate that the CART polypeptide co-localises with the Leptin receptor (Kristensen et al 1998, Nature 393 72-76). Moreover, the leptin and neuropeptide-Y response in cells is thought to have regulatory input from the CART polypeptide (Kristensen et al 1998, Nature 393 72-76). Leptin is thought to regulate the flux and/or the metabolism of glucose in cells (Liu et al 1998, J. Biol. Chem, 273 47 31160-7). Administration of CART polypeptide to rats (ref) has been shown to induce c-Fos expression within 6o minutes of CART administration Vrang et al 1999 brain research 818 499-509). The inventors believe that the mode of action of the CART receptor is through the modulation of glucose flux in cells, which in-tum modulates the expression of c-Fos. The induction of c-Fos and modulation of glucose allows two mechanisms by which functional screens for CART ligands can be developed. An illustrative embodiment of a reporter assay would be a c-Fos promoter-reporter assay. However, other reporter assays based on characterisation of glucose metabolism and protein activity are also envisaged. The criteria for developing a c-Fos promoter-reporter assay are follows:

(1) Identification of CART receptor expressing cell lines CART polypeptide or a peptide thereof can be labelled with a reporter molecule and used to identify and characterise cell types obtained from brain tissue or cell lines derived from brain tissue. Cell types containing the CART receptor have been identified as "neuronal" in nature and are present in areas of the brain such as the hypothalamus. Once characterised, cells can be immortalised by introducing an immortalising gene such as that described by Thi et al and cell lines made.

(2) Development of a reporter assay

A reporter assay can be developed by splicing a gene encoding a reporter protein to the nucleic acids encoding the c-Fos reporter. Reporter molecules can include but are not limited to the genes encoding green fluorescent protein (GFP), leuciferase and β-lactamase. A property of these molecules is that they are easily detected or their substrates are easily detected.

The criteria for splicing the nucleic acids encoding the reporter to the nucleic acids encoding the promoter would be that the the reporter be spliced in a sense orientation 3' to a region of promoter nucleic acid encoding the TATA box. A preferred point of splicing would be the ATG codon of methionine-1 of the c-Fos gene. In addition it is desirable that the promoter-reporter nucleic acid variant contain the following: (a) sufficient nucleic acid encoding a functional promoter.

(b) Sufficient nucleic acid to encode a functional reporter. The nucleic acid encoding the reporter must also contain a start codon at the 5' end of the reporter gene and a stop codon at the 3 'end. The nucleic acid must also contain a polyadenylation sequence 3 ' to the stop codon. (c) The nucleic acid encoding the known c-Fos gene.

Therefore, c-Fos sequence information can be used to identify nucleic acids encoding the promoter elements in a human genomic library. The promoter-reporter can be introduced into the cell line using technologies such as virus technology or transformed into cells using technologies such as electroporation, lipids or detergents and calcium phosphate. The promoter-reporter construct can be stabily intergrated into the cell lines or transiently integrated. Selection of cell lines containing the promoter-reporter can be achieved using technologies such as FACS or selection of cell lines through the use of antibiotics (if an antibiotic marker was included). Clonal cell lines can then be made expressing the CART receptor and harbouring the promoter-reporter.

(3) Assay

(a) contact the ligand with the cell line expressing the CART receptor. The ligand can be a protein, a peptide, a antibody or chemical entity. Allow sufficient time for binding to occur. This can be determined by those skilled in the art. If the ligand is CART polypeptide or a fragment thereof c-Fos induction can be measured after 1 hour.

(b) contact CART polypeptide or a fragment thereof with the cell line/ligand and reporter substrate (if necessary). Allow sufficient time for signal transduction to occur, approximately 1 hour and measure the change in the induction or repression of the expression of the promoter.

(C) In vivo ligand screen

An in vivo ligand screen incorporating the CART vaπant or fragment may be used as a basis of a ligand screen. This screen would have several advantages over convention screens in that the researcher make directly correlate the effect of ligand on the behaviour of an animal. (a) Assay; The effect of endogenous CART peptide on an animal is first reduced by the icv administration of antibodies specific to CART. A property of these antibodies must be that they are able to recognise active CART polypeptide but not able to recognise an active peptide of CART which is administered by icv. This is followed after an appropriate time by icv administration of ligand and then CART peptide. The effect of ligand and CART peptide on locomotion, feeding and addiction can then be determined EXAMPLES:

EXAMPLE i: Cloning of the CART polypeptide gene.

Oligonucleotides were designed to the sense and anti sense DNA strands of the CART polypeptide gene. These oligonucleotides encompassed the N-terminus start codon ( SEQLD N°31 5'CAT ATG GAG AGC TCC CGC CTG CGG C) and C- terminus stop codon (SEQ ID N°32 5'GGA TCC TCA CAA GCA CTT CAA GAG GAA AG). Oligonucleotides also encoded an Ndel or BamHl restriction endonuclease site. These restrictions endonuclease sites were introduced to allow directional cloning of the CART polypeptide gene into appropriate E. coli expression vectors.

PCR amplification of a heterogeneous rat hypothalamic cDNA pool resulted in 3 major DNA bands of approximately 400 bp (band A), 375 bp (band B) and 300 bp (band C) and a minor band at approximately 325 bp when a PCR aliquote was electrophoresed in a 1.5% agarose gel (figure 2). A similar banding pattern was observed when these oligonucleotides were used to selectively amplify nucleic acid from a human library as shown in figure 2.

Major DNA bands were purified from agarose using the Qiagen Qiaex II system and spliced into the TOPO TA cloning vector (according to the manufacturers protocol (In vitrogen) prior to transformation into TOP 10 E. coli (In vitrogen).

EXAMPLE 2: Identification of a novel rCART variant.

Twenty white E. coli colonies corresponding to TOPO TA spliced with DNA band A & B and 13 white colonies corresponding to band C were selected from solid culture media and screened for the presence of CART DNA insert by PCR screening with the above oligonucleotides as primers and Ndel/BamHI restriction endonuclease fragment analysis. Electrophoresed samples of the PCR screened colonies in a 1.5% agarose gel indicated that PCR generated DNA bands of approximately 400 bp, 350 bp, 290bp and 250 bp were present in the PCR mix (figure 3). Plasmid DNA samples of representative bands were prepared from cultures derived from the picked E.coli colonies using a Qiagen plasmid mini kit (12125) and DNA restricted with Ndel and BamHI restriction endonuclease 's . Electrophoresis of the restricted DNA in a 2% agarose gel indicated the presence of DNA fragments of approximately 415 bp, 400bp, 380 bp and 300 bp figure 4). The DNA coding sequence of each representative DNA size was determined by DNA sequencing.

The code of DNA inserts in TOPO TA plasmid was determined by sequence analysis. Aliquots of DNA (approximately 0.5 μg) were mixed with ABI PRISM Big Dye termination cycle sequencing ready reaction (4303152) and T7 primer (3.2pmol) and processed according to the manufactures instructions using a Perkin Elmer Gene Amp PCR system 2400. Sequence was analysed using an ABI PRISM 310 Genetic analyser and the output stored as electronic data. Data was examined and base misscalls manually corrected.

Analysis of the sequence data indicated that the generated clones fell into three groups. The first group represented examples of known published rCART DNA sequences. The second group represented examples of truncated CART DNA where the N-terminus primer has miss primed in the initial PCR reaction. These were identified by amino acid frame shift at the junction between the oligonucleotide sequence and CART DNA sequence. The third group and the subject of this patent is a splice variant of the known CART sequence, the sequences of which is disclosed as the nucleotide sequence of SEQ ID N°5. 6 and 7.

This novel form differs from the two known forms by excision of DNA encoding amino acids 54 to 94. This novel form encodes 88 amino acids with 267 bp (including stop codon). Amino acid sequence alignment of this novel form against the known variants is shown in figure 1.

EXAMPLE 3: Identification of novel CART peptide binding proteins

Two chromatography matrix were prepared (Amersham Pharmacia); the first affinity matrix was prepared with covalently linked CART peptide encoding a

CDAGEQC motif (test). The second chromatography matrix was prepared in a similar manner but did not contain peptide(control).

Membrane protein were extracted from 50g of pig hypothalamus brain tissue using standard molecular biology techniques, and the protein solution passed over the test and control matrix for 16 hours at 4°C. The chromatography matrix was then washed with buffer until no further protein eluted off the matrix. The test matrix was further washed first with buffer containing "free" CART peptide lmg/ml and then with an acidified buffer solution. Aliquots of eluate were kept for analysis by SDS PAGE electrophoresis as shown in figure 5. Similarly the control matrix was first washed with buffer solution then acidified buffer solution and aliquotes of eluate kept for analysis as shown in figure 5.

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Claims

CLAIMS:WHAT IS CLAIMED IS:

1. A purified or isolated nucleic acid comprising a polynucleotide encoding a CART protein, said CART protein having 88 amino acids in length, or a sequence complementary thereto.

2. A purified or isolated nucleic acid comprising a polynucleotide encoding a CART protein having the amino acids sequence of SEQ ID N°5, or a sequence complementary thereto.

3. A purified or isolated nucleic acid comprising a polynucleotide encoding a CART protein having the amino acid sequence of SEQ ID N°6, or a sequence complementary thereto.

4. A purified or isolated nucleic acid comprising a polynucleotide encoding a CART protein having the amino acid sequence of SEQ ID N°7, or a sequence complementary thereto.

5. A purified or isolated nucleic acid comprising a polynucleotide encoding a CART polypeptide without a signal sequence having the amino acid sequence of SEQ ID N°11 , 12 or 13, or a sequence complementary thereto.

6 A purified or isolated nucleic acid comprising a polynucleotide encoding a CART polypeptide signal sequence having the amino acid sequence of SEQ ID N°14, 15, 16, 17, 18 or 19, or a sequence complementary thereto.

7. A purified or isolated nucleic acid comprising a polynucleotide encoding CART polypeptides proteolytic cleavage sites having the amino acid sequence of SEQ ID N°20, 21 , 22 or 23, or a sequence complementary thereto.

8. A purified or isolated nucleic acid comprising a polynucleotide encoding a functionally active CART polypeptide having the amino acid sequence of SEQ ID N°24, 25, 26, 27, 28, 29 or 30, or a sequence complementary thereto.

9. A purified or isolated nucleic acid comprising a polynucleotide having at least 80% nucleotide identity with the nucleotide sequence of SEQ ID N°8, or a sequence complementary thereto.

10. A purified or isolated nucleic acid comprising a polynucleotide having at least 80% nucleotide identity with the nucleotide sequence of SEQ

ID N°9, or a sequence complementary thereto.

11. A purified or isolated nucleic acid comprising a polynucleotide having at least 80% nucleotide identity with the nucleotide sequence of SEQ ID N°10, or a sequence complementary thereto.

12. A purified or isolated nucleic acid comprising a polynucleotide consisting of at least 8 consecutive nucleotides of the nucleotide sequence of SEQ ID N°8, provided that said polynucleotide comprises the nucleotide sequence beginning at the nucleotide in position 157 and ending at the nucleotide in position 164 of SEQ ID N°8, or a sequence complementary thereto.

13. A purified or isolated nucleic acid comprising a polynucleotide consisting of at least 8 consecutive nucleotides of the nucleotide sequence of SEQ ID N°9, provided that said polynucleotide comprises the nucleotide sequence beginning at the nucleotide in position 157 and ending at the nucleotide in position 164 of SEQ ID N°9, or a sequence complementary thereto.

14. A purified or isolated nucleic acid comprising a polynucleotide consisting of at least 8 consecutive nucleotides of the nucleotide sequence of SEQ ID N°10, provided that said polynucleotide comprises the nucleotide sequence beginning at the nucleotide in position 157 and ending at the nucleotide in position 164 of SEQ ID N°10, or a sequence complementary thereto.

15. A purified or isolated nucleic acid selected from the group consisting of the nucleotide sequences of SEQ ID N° 31 and 32.

16. The nucleic acid according to any one of claims 12 to 14 which is detectably labelled.

17. A recombinant vector comprising a nucleic acid according to any one of claims 1 to 14.

18. The recombinant vector of claim 17 which is eukaryotic.

19. The recombinant vector of claim 17 which is prokaryotic .

20. The recombinant vector of claim 17 which is viral or retroviral .

21. A recombinant host cell comprising a nucleic acid according to any one of claims 1 to 15 or a recombinant vector according to any one of claims 16 to 20.

22. A purified or isolated polypeptide comprising the amino acid sequence of a CART protein, said CART protein having 88 amino acids in length.

23. A purified or isolated polypeptide comprising the amino acid sequence of SEQ ID N°5.

24. A purified or isolated polypeptide comprising the amino acid sequence of SEQ ID N°6.

25. A purified or isolated polypeptide comprising the amino acid sequence of SEQ ID N°7.

26. A purified or isolated polypeptide encoded by a nucleic acid according to any one of claims 1 to 8.

27. A purified or isolated polypeptide comprising at least 10 consecutive amino acids of any one of the amino acid sequences of SEQ ID N°5, 6 or 7, provided that said polypeptide comprises the amino acid sequence CDAGEQC.

28. A purified or isolated polypeptide comprising at least 10 consecutive amino acids of anyone of the amino acid sequences of SEQ ID

N°5, 6 or 7, provided that said polypeptide comprises the amino acid sequence KELCDAGEQC.

29. A purified or isolated polypeptide comprising, as regards to the amino acid sequence of a polypeptide according to anyone of claims 22 to 28, amino acid changes ranging from 1 , 2, 3, 4, 5, 10 to 20 substitutions, additions or deletions of one amino acid.

30. A polyclonal or monoclonal antibody directed against a polypeptide according to any one of claims 22 to 29.

31. The antibody of claim 30 which is detectably labelled.

32. A method for detecting the presence of a nucleic acid encoding a truncated CART protein or a fragment thereof or a sequence complementary thereto in a sample, said method comprising the steps of :

(a) bringing into contact a nucleic acid probe or a plurality of nucleic acid probes comprising a nucleotide sequence according to any one of claims 12, 13 or 14 and a sample to be assayed; (b) detecting the hybrid complex formed between the probe and a nuclic acid in the sample.

33. The method of claim 32, wherein before step a) the nucleic acid of the sample is made available to hybridization.

34. A kit for detecting a nucleic acid encoding a CART polypeptide in a sample wherein said kit comprises: a) a nucleic acid probe or a plurality of nucleic acid probes comprising a nucleotide sequence according to anyone of claims 12, 13 or 14; and b) optionally, reagents necessary to perform a hybridization reaction.

35. A method for detecting a CART polypeptide in a sample, wherein said method comprises the steps of: a) bringing into contact a biological sample with an antibody or according to any one of claims 30 or 31 ; b) detecting the antigen-antibody complex formed.

36. A kit for detecting a CART polypeptide in a sample, wherein said kit comprises an antibody according to any one of claims 30 or 31.

37. A pharmaceutical composition comprising a polypeptide according to any one of claims 22 to 29.

38. A pharmaceutical composition comprising an antibody according to claim 30.

39. A screening method for ligands that interact with CART polypeptides having the amino acid sequence of claims 2 to 8 or 22 to 29, or a sequence complementary thereto, said method comprising the following steps:

(a) contacting the ligand with the CART polypeptide or a fragment thereof,

(b) contact the medium containing the ligand and the CART polypeptide or a fragment thereof with a substrate and allow binding to occur,

(c) measurement of substrate binding.