WO2003031655A1

WO2003031655A1 - Method for diagnosis of multiple sclerosis by genetic analysis of the lag3 gene

Info

Publication number: WO2003031655A1
Application number: PCT/SE2002/001859
Authority: WO
Inventors: Dennis Hellgren; Zhiping Zhang
Original assignee: Astrazeneca Ab
Priority date: 2001-10-12
Filing date: 2002-10-11
Publication date: 2003-04-17
Also published as: SE0103423D0

Abstract

This invention relates to a method for the diagnosis of multiple sclerosis by genetic analysis. The method includes determining the nucleic acid sequence of LAG3 in a subject at position 1364 in the coding region of the LAG3 gene as defined by the position in SEQ ID NO: 1, wherein the presence of a T at position 1364 is diagnostic of multiple sclerosis or a predisposition thereto.

Description

Method for diagnosis of multiple sclerosis by genetic analysis ofthe LAG3 gene.

Field of the invention

This invention relates to a method for the diagnosis of multiple sclerosis by genetic analysis.

^' Background of the invention

Multiple sclerosis (MS) is the most common chronic neurological disease of young adults. The highest prevalence of MS is among Caucasians of Northern European origin, where the lifetime risk of the disease can be as high as 0.2-0.3%. The disease is characterized by widespread demyelination within the central nervous system leading to a range of neurological disabilities over time. The clinical phenotype is well defined, particularly due to the development of magnetic resonance scanning. The disease has a substantial genetic component showing 30% concordance in identical twins and an increased risk of between 20 and 40 fold in siblings compared to the general population (Dyment et al Human molecular Genetics. 6 (10): 1693-1698, 1997).

While several groups have completed systematic genome searches for linkage in MS ■ (Sawcer et al. Nature Genetics 13: 464-468, 1996; Ebers at al. Nature Genetics 13: 472- 476, 1996), none of these studies have so far been able to identify a major locus for determining MS disease susceptibility. Similar to most multifactorial diseases

(arteriosclerosis, type-I diabetes, schizophrenia, Parkinson), multiple sclerosis is thought the result from an interaction of multiple genes and environmental factors.

So far all the mapped loci for MS are extremely rough, at least 20cM on individual chromosomes (Sawcer et al. (1996) Nature Genetics 13: 464-468).. It is not possible to narrow down these regions by using microsatellite markers due to small genetic contribution of each individual gene, heterogeneity and the microsatellite marker instability. In contrast to microsatellite marker, the SNPs are stable, and distributed along the human genome in high density (average 1 SNP/lOOObp). This makes it possible to trace small effect of the individual gene by comparison of the allele frequency in affected and normal individuals (case/control study). By identifying linkage disequilibrium ofthe specific genotype (haplotype) in a region, SNPs can indicate the most localisation for a susceptibility gene for MS.

Summary of the invention The present invention provides novel methods and kits for determining whether a subject has or is predisposed to develop multiple sclerosis. Specifically, a polymorphism that occurs in the coding sequence of the LAG3 gene can be used to predict whether a subject has or is predisposed to multiple sclerosis. The polymorphism identified in the LAG3 gene results in a base subsitution from a C to a T at position 1364 of the wild type LAG3 gene (accession number X51985). The LAG3 gene sequence which includes the polymorphism is represented in SEQ ID NO:l. This polymorphism leads to a change in the sequence of the expressed LAG3 protein, specifically from a threonine to an isoleucine at position 455 of wild type human LAG3 protein (accession number X51985). The LAG3 protein sequence which includes the polymorphism is represented in SEQ ID NO:2.

In accordance with this finding there is provided a method for the diagnosis of multiple sclerosis or a predisposition thereto in a subject, which method includes determining the nucleic acid sequence of LAG3 in a subject at position 1364 in the coding region of the LAG3 gene as defined by the position in SEQ ID NO: 1, wherein the presence of a T at position 1364 is diagnostic of multiple sclerosis or a predisposition thereto. The method can further include determining the sequence at one or more of the following positions: position 14175 in the 3'UTR region of LAG3 wherein position 14175 is relative to +1 at ATG of LAG3 as derived using AC079387, wherein the presence of a G at position 14175 is diagnostic of multiple sclerosis or a predisposition thereto; position 14314 in the 3'UTR region of the LAG3 wherein position 14314 is relative to

+1 at ATG of LAG3 as derived using AC079387, and wherein the presence of an A at position 14314 is diagnostic of multiple sclerosis or a predisposition thereto.

The invention further features a method for the diagnosis of multiple sclerosis or a predisposition thereto in a human, which method includes determining the sequence of ^' LAG3 in a subject at position 455 in the LAG3 polypeptide as defined by the position in SEQ ID NO: 2; wherein the presence of isoleucine at position 455 is diagnostic of multiple sclerosis or a predisposition thereto.

The present invention can also be applied to a subject without any symptoms of multiple sclerosis, but who is found to possesss the genetic predisposition for multiple sclerosis, such that the subject can be treated prophylactically

treated to minimize his or her exposure to environmental risk factors in order to prevent or hinder the development of multiple sclerosis.

In another aspect of the invention, the diagnostic method can be used alone to determine if a subject has or is predisposed to muliple sclerosis, or may be used in conjuction with other available diagnostic tests in subjects suspected of having multiple sclerosis, e.g., in conjunction with MRI.

The diagnostic method can be performed by any known method in the art, e.g., allele specific oligonucleotide hybridisation, sequencing, primer specific extension, oligonύcleotide ligation assay and hybridization. In a preferred embodiment the nucleic acid is subject to an amplification step.

The invention also includes a primer set for diagnosing multiple sclerosis or a predisposition thereto in a subject. In one embodiment, the invention features a genetic testing kit for diagnosing multiple sclerosis or a predisposition thereto in a subject which includes the primer set.

In another aspect, the invention includes a method of identifying a test compound that modulates the variant LAG3 activity which includes contacting a cell capable of expressing variant LAG3 with a test compound; and determining the level of expression of variant LAG3 in the presence of the test compound, wherein a decrease or an increase in variant LAG3 expression, as compared to the level of expression of variant LAG3 in the absence of the compound, is indicative that the test compound modulates variant LAG3 activity. In one embodiment, the invention includes the step of preparing a pharmaceutical composition from the test compound. In another aspect, the invention as defined herein can also be used to identify compounds which assess the pharmacogenetics of a drug acting at LAG3. The invention also includes a method of treating a subject in need of treatment with a drug acting at LAG3 in which the method includes:

(i) diagnosing a polymorphism in LAG3 in a subject, which diagnosis includes determining the sequence at position 1364 in LAG3 as defined in SEQ ID NO:l, wherein the presence of a T at position 1364 is diagnostic of multiple sclerosis or a predisposition thereto; and (ii) administering an effective amount of the drug which modulates, e.g., decreases, the expression of the.LAG3 gene.

In another embodiment, the invention features treating a subject in need of treatment with a drug acting at LAG3 in which the method includes: (i) diagnosing a polymorphism in LAG3 in a subject, which diagnosis includes determining the sequence of LAG3 at position 455 in the LAG3 polypeptide as defined by the position in SEQ ID NO: 2; wherein the presence of isoleucine at position 455 is diagnostic of multiple sclerosis or a predisposition thereto; and

(ii) administering an effective amount of the drug which modulates, e.g., decreases, the production of LAG3 protein.

The invention also features an antibody, which is specific for the allelic variant of the human LAG3 polypeptide as described herein.

Therapeutic methods for treating multiple sclerosis and kits for exploiting the inventive methods of the present invention are also provided.

The term "polymorphism" includes single nucleotide substitution, nucleotide insertion and nucleotide deletion which in the case of insertion and deletion includes insertion or deletion of one or more nucleotides at a position of a gene and corresponding alterations in expressed protein. The term "nucleic acid" as used herein refers to DNA or RNA. Thus the methods of the ^' present invention may be performed, for example, by using genomic DNA as well as cDNA or mRNA.

By the term "isolated" nucleic acid molecule refers to a nucleic acid which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated LAG3 nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.

Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the LAG3 protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of LAG3 protein in which the protein is separated from cellular components of the cells from which it. is isolated or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes , preparations of LAG3 protein having less than about 30% (by dry weight) of non-LAG3 protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-LAG3 protein, still more preferably less than about 10% of non-LAG3 protein, and most preferably less than about 5% non-LAG3 protein. When the LAG3 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

The term "subject" is meant a human and includes both a human having or suspected of having multiple sclerosis and an asymptomatic human who may be tested for predisposition or susceptibility to multiple sclerosis.

The term "haplotype" as used herein is intended to refer to a set of alleles that are inherited together as a group (are in linkage disequilibrium) at statistically- significant levels (p.sub.corr <0.05).

The term "predisposition" to multiple sclerosis or any similar phrase, means that certain alleles are hereby discovered to be associated with or predictive of a subject's incidence of developing multiple sclerosis. The alleles are thus over-represented in frequency in individuals with disease as compared to healthy individuals. Thus, these alleles can be used to predict disease even in pre-symptomatic, pre-diseased individuals and disease individuals.

Point mutations in polypeptides will be referred to as follows: natural amino acid (using 1 or 3 letter nomenclature) the position in the sequence, and the new amino acid. For (a hypothetical) example "T455I" or "Thr455Ile" means that at position 455 a threonine (T) has been changed to isoleucine (I).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All U.S. patents and all publications mentioned herein are incorporated in their entirety by reference thereto.

Various features and advantages of the invention will be apparent from the following detailed description and from the claims. Brief description of the Figures

Fig. 1 sets forth a representative genomic DNA sequence of human LAG3 (in consecutive order reading 5' to 3'). The polymorphism is indicated by an arrow and it occurs in exon 7 of human LAG3 (PI). Polymorphisms occurring in the 3'UTR of LAG3 are also represented as P2 and P3.

Fig. 2 sets forth a cDNA sequence encoding human LAG3 (SEQ ID NO:l).

Fig. 3 sets forth the polypeptide sequence of LAG3 (SEQ ID NO:2).

Detailed description of the invention The wildtype LAG3 gene has eight exons and encodes a 498 amino acid membrane protein

• which is characterized as having 4 extracellular Ig-like domains, a transmembrane region and a short cytoplasmic tail (Triebel, F. et.al.J. Exp. Med. Vol.171, 1393-1405, 1990).

Variants of LAG3 have also been identified (WO 9858059). There is speculation that these variants might be useful in pharmaceutical compositions for the treatment of MS.

However, the biological function for Lag3 is at present unknown. Analysis of expression patterns show that its expression is very restricted and is undetectable in normal resting peripheral blood lymphocytes. The upregulation of LAG3 mRNA can be detected in activated T and NK cells (PHA treatment) (Triebel et al., supra). LAG3 seems also to be associated with a yet to be identified 45 kD protein on the surface of activated T and NK cells (Hannier, S. et.al. J. immunology Vol.161, 4058-4065, 1998).

While the definitive biological function for LAG3 has not been identified, various biological observations for LAG3 have been reported. LAG3 is reported to interact with MHC class II molecules (2, 3, 4,10), apparently with a higher avidity than CD4 (Huard et.al, Eur. J. Immunology Vol.26, 1180-1186, 1996). LAG3 has been found to expressed in Tumor Infiltrating Lymphocytes (TILs). Results point to a function for TIL-expressed LAG3 in the engagement of class II molecules on APCs, thereby contributing to APC activation and Thl/Tcl commitment (El Mir et al., J. Immunol. Vol. 164, 5583-5589, 2001; Demeure etal. Eur. J. Cancer. Vol. 37, 1709-1718, 2001).

The present invention is based, at least in part, on the finding of a linkage between a particular polymorphism in LAG3 and multiple sclerosis. The polymorphism identified arises in the coding sequence of the LAG3 gene resulting in a base subsitution from a C to a T at position 1364 of wild type LAG3 gene or in exon 7 of LAG3. This polymorphism leads to a change in the sequence ofthe expressed protein, specifically from a threonine to an isoleucine at position 455 of wild type LAG3. The polymorphism as described above provides the basis for convenient and reliable methods of screening for multiple sclerosis, providing physicians with valuable information in the diagnosis of multiple sclerosis and the determination of susceptibility to multiple sclerosis. This polymorphism can also be exploited in therapeutic treatments of multiple sclerosis.

In one embodiment of the invention, the presence or absence of a T at position 1364 of the LAG3 gene of SEQ ID NO: 1 in a subject's nucleic acid can be detected simply by starting with a test sample, e.g., any nucleated cell sample obtained from a subject, from which genomic DNA, for example, can be isolated in sufficient quantities for analysis. The presence or absence of the polymorphism can be determined by sequence analysis of the genomic DNA, e.g., as accomplished via Maxam-Gilbert or another conventional technique.

The test sample of nucleic acid is conveniently a body sample, e.g., a blood sample from a subject. It will be appreciated that the test sample may equally be a nucleic acid sequence corresponding to the sequence in the test sample, that is to say that all or a part of the region in the sample nucleic acid may firstly be amplified using any convenient technique e.g., PCR, before analysis of allelic variation.

It will be apparent to the person skilled in the art that there are a large number of analytical procedures which may be used to detect the presence or absence of variant nucleotides at the polymorphic position of the invention. In general, the detection of allelic variation requires a mutation discrimination technique, optionally an amplification reaction and optionally a signal generation system. Table 1 lists a number of mutation detection techniques, some based on PCR. These may be used in combination with a number of signal generation systems, a selection of which is listed in Table 2. Further amplification techniques are listed in Table 3. Many current methods for the detection of allelic variation are reviewed by NoUau et al., Clin. Chem. 43, 1114-1120, 1997; and in standard textbooks, for example "Laboratory Protocols for Mutation Detection", Ed. by U. Landegren, Oxford University Press, 1996 and "PCR", 2^nd Edition by Newton & Graham, BIOS Scientific Publishers Limited, 1997.

Abbreviations:

TGGE Temperature gradient gel electrophoresis

Table 1 - Mutation Detection Techniques

Table 2 - Signal Generation or Detection Systems

Table 3 - Further Amplification Methods

SSR,

NASBA

LCR

SDA b-DNA

Table 4- Protein variation detection methods

Immunoassay

Immunohistology

Peptide sequencing

Preferred mutation detection techniques include ARMS™, ALEX™, COPS, Taqman, Molecular Beacons, RFLP, and restriction site based PCR and FRET techniques. Immunoassay techniques are known in the art e.g. A Practical Guide to ELISA by D M Kemeny, Pergamon Press 1991; Principles and Practice of Immunoassay, 2^nd edition, C P Price & D J Newman, 1997, published by Stockton Press in USA & Canada and by Macmillan Reference in the United Kingdom.

Particularly preferred methods include ARMS™ and RFLP based methods. ARMS™ is an especially preferred method.

In another aspect of the invention, the polymorphism of this invention may be used as a genetic marker in linkage studies. The LAG3 gene is positioned on chromosome.3 and the LAG3 polymorphism described herein is particularly useful forhaplotyping as described below. A haplotype is a set of alleles found at linked polymorphic sites (such as within a gene) on a single (paternal or maternal) chromosome. In the present invention, the identification of a particular haplotype pattern and the association of this pattern with multiple sclerosis has been established. In particular, the LAG3 polymorphism described herein and two other single nucleotide polymorphisms (SNP) located downstream of the LAG3 gene have been shown to have significant association in MS patients (P2 and P3, respectively, in Fig. 1). One of the SNP's is at position 14175 in the 3'UTR region of LAG3 wherein position 14175 is relative to +1 at ATG of LAG3 as derived using AC079387 (i.e., A in ATG is at position 123430 in AC079387), wherein the presence of a G at position 14175 is diagnostic of multiple sclerosis or a predisposition thereto. The other SNP is at position 14314 in the 3'UTR region of the LAG3 wherein position 14314 is relative to +1 at ATG of LAG3 and derived using AC079387 (i.e., A in ATG is at position 123430 in AC079387), and wherein the presence of an A at position 14314 is diagnostic of multiple sclerosis or a predisposition thereto. The three SNPs are in linkage disequilibrium. These three SNPs form a haplotype TGA and are present in almost all of the MS patients having the initial LAG3 polymorphism (see Table 5). The distance between the different polymorphisms is around 8.5 Kb and this 8.5 Kb region does not contain any genes when BLAST:ed against LifeSeqGoldX (see Fig. 1). ENSEMBL do not predict any coding gene or exon in this region.

In addition to the allelic patterns described above, one of skill in the art can readily identigy other alleles (including polymorphisms and mutations) that are in linkage disequilibrium with the LAG3 allele associated with multiple sclerosis.

According to another aspect of the present invention there is provided a computer readable medium comprising the LAG3 polymorphism as described herein stored on the medium. The computer readable medium may be used, for example, in homology searching, mapping, haplotyping, genotyping or pharmacogenetic analysis.

The invention provides a method for identifying modulators, i.e., test compounds (e.g., peptides, peptidomimetics, small molecules or other drugs) which modulate the activity of the variant LAG3 as described herein, e.g., bind to the variant LAG3 protein or have a stimulatory or inhibitory effect on, for example, variant LAG3 expression. In one example, the invention provides methods for screening for test compounds which modulate the activity of the variant LAG3 protein, or a portion thereof which includes the polymorphism. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries. The method can be a cell-based method or a cell free method. For example, in a cell based method a cell expresses a variant LAG3 as described herein, is contacted with a test compound and the ability of the test compound to bind to the variant LAG3 protein determined. Determining the ability of the test compound to bind to the LAG3 protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the LAG3 protein or biologically active portion thereof can be determined by detecting the labeled compound in a complex.

The invention also includes a cell-based assay comprising contacting a cell expressing the variant LAG3, or a biologically active portion thereof which includes the polymorphism, with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the variant LAG3. Determining the ability of the test compound to modulate the activity of variant LAG3 or a biologically active portion thereof can be accomplished, for example, by determining the ability of the variant LAG3 protein to bind to or interact with a LAG3 target molecule. As used herein, a "target molecule" is a molecule with which a wild type LAG3 protein binds or interacts in nature.

Modulators of variant LAG3 expression can also be identified using a method wherein^' a cell is contacted with a test compound and the expression of variant LAG3 mRNA or protein in the cell is determined. The level of expression of variant LAG3 mRNA or protein in the presence of the test compound is compared to the level of expression of LAG3 mRNA or protein in the absence of the test compound. The test compound can be identified as a modulator based on this comparison. This method can also be used to determine if the test compound is specific for the variant LAG3. For example, by comparing if the test compound also modulates the level of expression of wild type LAG3 mRNA or protein. If the compound affects (statistically significantly effects) only the level of expression of the variant LAG3 mRNA or protein and not the level of expression of wild type LAG3 mRNA or protein, the test compound is said to be specific for the variant LAG3.

In a further aspect, the diagnostic methods of the invention are used to assess the pharmacogenetics of a drug acting at LAG3. It is generally known by those in the art that knowledge of polymorphisms can be used to help identify patients most suited to therapy with particular pharmaceutical agents (this is often termed "pharmacogenetics"). Pharmacogenetics can also be used in pharmaceutical research to assist the drug selection process. Polymorphisms are used in mapping the human genome and to elucidate the genetic component of diseases. The reader is directed to the following references for background details on pharmacogenetics and other uses of polymorphism detection: Linder et al. (1997), Clinical Chemistry, 43, 254; Marshall (1997), Nature Biotechnology, 15, 1249; International Patent Application WO 97/40462, Spectra Biomedical; and Schafer et al. (1998), Nature Biotechnology, 16, 33.

Assays, for example reporter-based assays, may be devised to detect whether the above polymorphisms affect transcription levels and/or message stability. Individuals who carry the particular allelic variant of the LAG3 gene may therefore exhibit differences in their ability to regulate protein biosynthesis under different physiological conditions and will display altered abilities to react to different diseases. In addition, differences arising as a result of allelic variation may have a direct effect on the response of an individual to drug therapy. The diagnostic methods of the invention may be useful both to predict the clinical response to such agents and to determine therapeutic dose.

In a further aspect, the diagnostic methods of the invention are used in the development of new drug therapies which selectively target the allelic variant of the LAG3 gene. Drugs may be designed to regulate the biological activity of the LAG3 variant implicated in the disease process. According to another aspect of the present invention there is provided a vector which includes the variant human LAG3 gene sequence, or a fragment thereof, of at least 20 bases including the polymorphism described herein.

The invention further provides a nucleotide primer which can detect a polymoφhism of the invention or an allele specific primer capable of detecting a LAG3 gene polymoφhism. An allele specific primer is used, generally together with a constant primer, in an amplification reaction such as a PCR reaction, which provides the discrimination between alleles through selective amplification of one allele at a particular sequence position e.g. as used for ARMS™ assays. The allele specific primer is preferably 17- 50 nucleotides, more preferably about 17-35 nucleotides, more preferably about 17-30 nucleotides. An allele specific primer preferably corresponds exactly with the allele to be detected but derivatives thereof are also contemplated wherein about 6-8 of the nucleotides at the 3' terminus correspond with the allele to be detected^" and wherein up to 10, such as up to 8, 6, 4, 2, or 1 of the remaining nucleotides may be varied without significantly affecting the properties of the primer. One skilled in the art would be able to design primers suitable for the diagnostic pmpose described herein.

Primers may be manufactured using any convenient method of synthesis. Examples of such methods may be found in standard textbooks, for example "Protocols for

Oligonucleotides and Analogues; Synthesis and Properties," Methods in Molecular Biology Series; Volume 20; Ed; Sudhir Agrawal, Humana ISBN: 0-89603-247-7; 1993; 1^st Edition. If required the primer(s) may be labelled to facilitate detection.

According to another aspect of the present invention there is provided an allele-specific oligonucleotide probe capable of detecting a LAG3 gene polymoφhism. The allele- specific oligonucleotide probe is preferably 17- 50 nucleotides, more preferably about 17- 35 nucleotides, more preferably about 17-30 nucleotides. The design of such probes will be apparent to the molecular biologist of ordinary skill. Such probes are of any convenient length such as up to 50 bases, up to 40 bases, more conveniently up to 30 bases in length, such as for example 8-25 or 8-15 bases in length. In general such probes will comprise base sequences entirely complementary to the corresponding wild type or variant locus in the gene. However, if required one or more mismatches may be introduced, provided that the discriminatory power of the oligonucleotide probe is not unduly affected. The probes ofthe invention may carry one or more labels to facilitate detection.

According to another aspect of the present invention there is provided a diagnostic kit comprising an allele specific oligonucleotide probe of the invention and/or an allele- specific primer of the invention. The diagnostic kits may comprise appropriate packaging and instructions for use in the methods of the invention. Such kits may further comprise appropriate buffer(s) and polymerase(s) such as thermostable polymerases, for example taq polymerase.

According to another aspect of the present invention there is provided an antibody specific for an allelic variant of human LAG3 polypeptide as described herein. Antibodies can be prepared using any suitable method. For example, purified polypeptide may be utilized to prepare specific antibodies. The term "antibodies" is meant to include polyclonal antibodies, monoclonal antibodies, and the various types of antibody constructs such as for example F(ab')₂, Fab and single chain Fv. Antibodies are defined to be specifically binding if they bind the allelic variant of LAG3 with a K_a of greater than or equal to about 10⁷ M^"1. Affinity of binding can be determined using conventional techniques, for example those described by Scatchard et al., Ann. N.Y. Acad. ScL, 5_1:660 (1949).

Polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, goats, sheep, dogs, chickens, rabbits, mice or rats, using procedures that are well-known in the art. In general, antigen is administered to the host animal typically through parenteral injection. The immunogenicity of antigen may be enhanced through the use of an adjuvant, for example, Freund's complete or incomplete adjuvant. Following booster immunizations, small samples of serum are collected and tested for reactivity to antigen. Examples of various assays useful for such determination include those described in: Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; as well as procedures such as countercurrent irnmuno- electrophoresis (CIEP), radioimmunoassay, radioimmunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA), dot blot assays, and sandwich assays, see U.S. Patent Nos. 4,376,110 and 4,486,530.

Monoclonal antibodies may be readily prepared using well-known procedures, see for example, the procedures described in U.S. Patent Nos. RE 32,011, 4,902,614, 4,543,439 and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), (1980).

The monoclonal antibodies of the invention can be produced using alternative techniques, such as those described by Alting-Mees et al., "Monoclonal Antibody Expression

Libraries: A Rapid Alternative to Hybridomas", Strategies in Molecular Biology 3: 1-9 (1990) which is incoφorated herein by reference. Similarly, binding partners can be constructed using recombinant DNA techniques to incoφorate the variable regions of a gene that encodes a specific binding antibody. Such a technique is described in Larrick et al., Biotechnology, 7: 394 (1989).

Once isolated and purified, the antibodies may be used to detect the presence of antigen in a sample using established assay protocols, see for example "A Practical Guide to ELISA" by D. M. Kemeny, Pergamon Press, Oxford, England.

According to another aspect of the invention there is provided a diagnostic kit comprising an antibody of the invention.

According to another aspect of the present invention there is provided a human LAG3 polypeptide comprising an isoleucine at position 455 of SEQ ID NO: 2. According to another aspect of the present invention there is provided a polynucleotide which encodes the human LAG3 polypeptide. Examples

The following examples are non-limiting and are given by way of illustration only. It will be appreciated by those skilled in the art that the examples are to be looked upon as guidelines, and the invention is not restricted to the exemplified compositions.

Example 1: Polymoφhisms in LAG3.

Polymoφhisms were identified by BLAST search with LAG3 cDNA against The SNP Consortium (TSC) data. Likewise, genomic region containing the whole LAG3 gene was used for BLAST search of TSC data for finding polymoφhisms in noncoding regions.

Example 2: Linkage of a polymoφhism in LAG3 and multiple sclerosis The polymoφhism C/T at 1364 (TSC0138680, Thr455Ee (Thr422He without signal peptide), SNP 1594) in exon 7 of the Lag-3 gene were used for association study. Details of the DNA samples used for the study are as follows. Swedish MS DNA samples (n=672) and part of Swedish normal controls (n=288) were provided by the Division of Neurology, Huddinge University hospital. The clinical characterisation was performed at Division of Neurology. The rest of the normal controls (n=384) are from Swedish Twin Register.

Genotyping of samples were performed as follows: lOng genomic DNA of each sample were used for PCR amplification. PCR reaction is performed in total 25 ul of reaction including 10pm each PCR primer. Forward primer was GGCCACCTCCTGCTGTTTCT (SEQ ID NO:3) and Reverse primer was GTCCCTGGCTCACCTGTCTT (SEQ ID NO:4) and 45 PCR cycles (95°C 20 sec, 55-60°C 20sec and 72°C 30sec) were run. The PCR products (96-format PCR plate) were analysed using Pyrosequencing technology for genotyping according to the standard protocol as described by Pyrosequencing. Sequencing primer was AGAAAGGACACCAAGG (SEQ ID NO:5)

The results of this analysis showed that individuals homozygous for the TT are elevated in the MS group compared to the control population (see Table 5). The relative risk for TT genotype is 1.6 with significant P value; 0.05. A similar significant association were found for SNPs 1254 (TSC0051254) and 8943 (TSC0978943), about 8kb downstream of the LAG3 gene. Haplotype analysis indicated these three SNPs are in linkage disequilibrium. In order to exclude the possibility that another gene situated downstream of the LAG3 gene may co-segregate with LAG3 gene, the genomic fragment downstream of LAG3 (lOkb) was BLAST:ed against transcribed sequence databases. No matching EST or predicated gene sequences were identified. Since the PAC clone containing LAG3 gene is not completed yet (unordered pieces), the DNA sequence further downstream, i.e., below 8 kb, is not defined. The association was not observed for the heterozygous individuals. This suggests that the effect of LAG3 (Thr455Ηe) on MS susceptibility is recessive, which could relate to functional structure of LAG3 on the cell membrane (oligomer on the cell surface).

Accession number for the cDNA of LAG3 is X51985. The accession number AC079387 provides for LAG3 genomic sequence. Note that the position of the 3'-UTR SNPs are relative to +1 at ATG of LAG3 and derived using AC079387 as template. The A of the start codon ATG of LAG3 is at position 123430 in AC079387. Chi-square statistics were calculated as described in Snedecor and Cochran, "Statistical Methods". Iowa State University Press. M = MS patients, and N= normal patients

Claims

Claims:

1. A method for the diagnosis of multiple sclerosis or a predisposition thereto in a subject comprising determining the nucleic acid sequence of LAG3 in a subject at position 1364 in the coding region of the LAG3 gene as defined by the position in SEQ LO NO: 1, wherein the presence of a T at position 1364 is diagnostic of multiple sclerosis or a predisposition thereto.

2. The method of claim 1, wherein the method further comprises determining the sequence at one or more of the following positions: position 14175 in the 3'UTR region of LAG3 wherein the position is relative to +1 at

ATG of LAG3 as derived using AC079387, wherein the presence of a G at position 14175 is diagnostic of multiple sclerosis or a predisposition thereto; or position 14314 in the 3'UTR region of the LAG3 wherein position 143^14 is relative to +1 at ATG of LAG3 as derived using AC079387 and wherein the .presence of an A at position 14314 is diagnostic of multiple sclerosis or a predisposition thereto.

3. The method of claim 1 , wherein the determining step is selected from the group consisting of: a) allele specific oligonucleotide hybridisation; b) sequencing; c) primer specific extension; d) oligonucleotide ligation assay; and e) hybridization.

4. The method of claim 1 , wherein the nucleic acid is subject to an amplification step.

5. A method for the diagnosis of multiple sclerosis or a predisposition thereto in a human, which method comprises determining the sequence of a LAG3 protein in a subject at position 455 as defined by the position in SEQ ID NO: 2; wherein the presence of an isoleucine at position 455 is diagnostic of multiple sclerosis or a predisposition thereto.

6. A method of identifying a test compound that modulates the LAG3 activity comprising: contacting a cell capable of expressing LAG3 with a test compound; and determining the level of expression of LAG3 in the presence of the test compound, wherein a decrease or an. increase in LAG3 expression, as compared to the level of expression of LAG3 in the absence of the compound, is indicative that the test compound modulates LAG3 activity.

7. The method of claim 6, Which additionally comprises the step of preparing a pharmaceutical composition from the test compound.

8. A method of treating a subject with a drug acting at LAG3 comprising:

(i) diagnosing a polymorphism in LAG3 in the subject, which diagnosis includes determining the sequence at position 1364 in LAG3, wherein the presence of a T at position 1363 is diagnostic of multiple sclerosis or a predisposition thereto; and

(ii) administering an effective amount of the drug which modulates, e.g., decreases the expression of the LAG3 gene.

9. A method of treating a subject with a drug acting at LAG3 in which the method comprises:

(i) diagnosing a polymorphism in LAG3 in the subject, which diagnosis includes determining the sequence of LAG3 in a subject at position 455 in the LAG3 polypeptide as defined by the position in SEQ ID NO: 2; wherein presence of isoleucine at position 455 is diagnostic of multiple sclerosis or a predisposition thereto; and

10. A nucleotide primer or an allele-specific oligonucleotide probe which can detect a polymoφhism in the LAG3 gene, wherein the polymoφhism is a T occuring at position 1364 in the coding region of the LAG3 gene as defined by the position in SEQ ED NO: 1.

11. A genetic testing kit for diagnosing multiple sclerosis or a predisposition thereto in a . subject comprising the primer or probe of claim 10.

12. An antibody which is specific for an allelic variant of the human LAG3 polypeptide, wherein the polypeptide comprises an isoleucine at position 455.