WO2002074803A2

WO2002074803A2 - Regulation of human icos v protein

Info

Publication number: WO2002074803A2
Application number: PCT/EP2002/000158
Authority: WO
Inventors: Jeffrey Encinas; Eri Tanabe; Shinichi Watanabe
Original assignee: Bayer Aktiengesellschaft
Priority date: 2001-01-16
Filing date: 2002-01-10
Publication date: 2002-09-26
Also published as: WO2002074803A3; AU2002308267A1

Abstract

Reagents which regulate human ICOS and reagents which bind to human ICOS gene products can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to, allergic diseases, such as respiratory allergies, food allergies, asthma, and atopic dermatitis, as well as in the treatment of intracellular bacterial infections, such as tuberculosis, leprosy, listeriosis, and salmonellosis; and autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and type I diabetes, as well as in the treatment of helminth and extracellular microbial infections.

Description

REGULATION OF HUMAN ICOS V PROTEIN

TECHNICAL FIELD OF THE INVENTION

The invention relates to nucleotide and amino acid sequences of human ICOS splice variants, or biologically active derivative thereof (ICOS V) and to the regulation of the same.

BACKGROUND OF THE INVENTION

B7 family ligands, expressed on antigen presenting cells, are the counter-ligands for several receptors expressed on T lymphocytes. Costimulatory interactions between the B7 family ligands and their receptors play critical roles in the growth, differentia- tion, and death of T cells.

Recent studies show several new members of the B7-CD28 family, such as B7-H1 (B7 homolog 1) and ICOS (B7 homolog 2), that participate in the regulation of cellular and humoral immune responses. ICOS binds an inducible costimulator (ICOS).

ICOS (inducible co-stimulator) was originally identified with monoclonal antibodies generated against activated T cells (Hutloff A. et al, "ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28." Nature. 1999 Jan 21;397(6716):263-6.). After the protein was partially sequenced and the full-length

ICOS complementary DNA cloned, the molecule was found to have homology to the T cell co-stimulatory molecule CD28 (Hutloff A. et al., "ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28." Nature. 1999 Jan 21;397(6716):263-6). Further studies showed that ICOS to be a costimulatory receptor whose expression is upregulated on CD4+ and CD8+ T cells after T cell receptor stimulation ((Hutloff A et al., "ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28." Nature. 1999 Jan 21;397(6716):263- 6.), (Yoshinaga SK et al., "G. T-cell co-stimulation through B7RP-1 and ICOS." Nature. 1999 Dec 16;402(6763):827-32.), (Coyle AJ et al, "The CD28-related molecule ICOS is required for effective T cell-dependent immune responses." Immunity. 2000 Jul;13(l):95-105.), (McAdam AJ et al, "Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4(+) T cells." J Immunol. 2000 Nov 1;165(9):5035- 40.)). Stimulation of ICOS is thought to induce the production of IL-10 cytokine production, and to a lesser extent to increase production of IL-4, IL-5, EFN-γ, TNF-α, and GM-CSF, as well as to promote the function of activated Th2 helper cells

((Yoshinaga SK et al., "G. T-cell co-stimulation through B7RP-1 and ICOS." Nature. 1999 Dec 16;402(6763):827-32.), (Coyle AJ et al, "The CD28-related molecule ICOS is required for effective T cell-dependent immune responses." Immunity. 2000 Jul;13(l):95-105.), (McAdam AJ et al, "Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4(+) T cells." J Immunol. 2000 Nov l;165(9):5035-40.)).

The ICOS gene has been reported to be expressed predominantly in primary and secondary lymphoid tissues (Mages HW, Hutloff A, Heuck C, Buchner K, Himmelbauer H, Oliveri F, Kroczek RA. Molecular cloning and characterization of murine ICOS and identification of B7h as ICOS ligand. Eur J Immunol. 2000 Apr;30(4): 1040-7).

There is a need in the art to identify novel variants of ICOS proteins which can be regulated and provide therapeutic options.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a novel polynucleotide which encodes a novel polypeptide of a ICOS splice variants, or biologically active derivative thereof (ICOS N). The polynucleotide of the present invention is selected from the group consisting of:

a) a polynucleotide encoding a protein that comprises the amino acid sequence of SEQ JX> ΝO: 4;

b) a polynucleotide which comprises the sequence of SEQ ID NO 1, 2 or 3;

c) a polynucleotide which hybridizes under stringent conditions to a polynucleo- tide specified in (a) and (b) and encodes a ICOS V polypeptide;

d) a polynucleotide the nucleic acid sequence of which deviates from the nucleic aid sequences specified in (a) to (c) due to the degeneration of the genetic code and encodes a ICOS V polypeptide; and

e) a polynucleotide, which represents a fragment, derivative or allelic variation of a nucleic acid sequence specified in (a) to (d) and encodes a ICOS V polypeptide.

The polypeptide of the present invention is encoded by a polynucleotide defined above.

It is also an object of the present invention to provide reagents and methods of regulating a human ICOS N. This and other objects of the invention are provided by one or more of the embodiments described below.

One embodiment of the invention is a method for producing an isolated protein exhibiting biological properties of ICOS N. The host cell transfected by the vector containing the polynucleotide of the present invention is cultured and then the polypeptide from the host cell culture is recovered. Another embodiment of the invention is the method for the detection of poly- nucleotides encoding ICOS N or a protein exhibiting biological properties of ICOS N in a biological sample. Any of the polynucleotide of the present invention is hybridized to nucleic acid material of the biological sample, thereby forming a hybridization complex. The complex is then detected.

Yet another embodiment of the invention is a method of screening for agents which can regulate the activity of a human ICOS N. A test compound is contacted with a polypeptide encoded by any of the polynucleotide of the present invention. Binding of the test compound to the polypeptide is detected. A test compound which binds to the polypeptide is thereby identified as a potential therapeutic agent for regulating activity of the human ICOS N.

Another embodiment of the invention is a method of screening for agents that regulate an activity of a human ICOS N. A test compound is contacted with a polypeptide encoded by any of the polynucleotide of the present invention. An ICOS N activity of the polypeptide is detected. A test compound that decreases the ICOS N activity is thereby identified as a potential therapeutic agent for decreasing the activity of the human ICOS N. A test compound which increases the ICOS N activity of the polypeptide is thereby identified as a potential therapeutic agent for increasing the activity of the human ICOS.

Yet another embodiment of the invention is a method of screening for agents which regulate an activity of a human ICOS N. A test compound is contacted with any of the polynucleotide of the present invention. Binding of the test compound to the polynucleotide is detected. A test compound which binds to the product is thereby identified as a potential therapeutic agent for regulating the activity of the human ICOS N.

Even another embodiment of the invention is a method of modulating the activity of a human ICOS N. A cell is contacted with a reagent which specifically binds to any of the polynucleotide of the present invention or protein encoded by the polynucleotide of the present invention. The activity of the human ICOS N is thereby modulated.

Still another embodiment of the invention is a pharmaceutical composition comprising a reagent which modulate the activity of ICOS N polypeptide or polynucleotide of the present invention and a pharmaceutically acceptable carrier.

Even another embodiment of the invention is a method for treating diseases comprising administering to a subject in need of such treatment an effective amount of the reagent of the pharmaceutical composition of the present invention.

The invention thus provides a human ICOS N which can be used to identify test compounds which may act, for example, as enhancers or inhibitors of formation of the receptor complex. Human ICOS N and fragments thereof also are useful in raising specific antibodies which can block the protein and effectively reduce its activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the alignment of human ICOS alternative splice variants, nucleotide sequences (SEQ ID NO: 1, SEQ ED NO: 2, or SEQ ID NO: 3) of the present invention with other variant of ICOS V (SEQ ID NO: 9).

FIG. 2 shows the alignment of human ICOS alternative splice variants, amino acid sequence (SEQ ID NO: 4) of the present invention against other variant of ICOS V (SEQ ID NO: 10).

FIG. 3 shows the expression profiling of ICOS transcript 1 mRNA. DETAILED DESCRIPTION OF THE INVENTION

A novel human ICOS polynucleotide depicted in SEQ ID NO: 1 (transcript 1), a polynucleotide depicted in SEQ ID NO: 2 (transcript 2), a polynucleotide depicted in SEQ ID NO: 3 (transcript 3), and a protein encoded by the amino acid depicted in

SEQ JD NO: 4 is a discovery of the present invention.

The sequence of transcript 1 (SEQ ID NO: 1) has a coding region 513 bp in length and has an additional 234bp inserted between bases 83 and 84 of the original ICOS sequence (GenBank accession number AB0231353: SEQ ID NO: 9) reported for this gene. The additional sequence contains a stop codon (at base 93) in frame with the presumed start codon of ICOS (at base 26 of ICOS, base 9 of transcript 1), which changes the starting point for translation in this transcript to base 330. In addition, the T nucleotide at position 2272 of ICOS is changed to a G in the corresponding position 2489 of transcript 1.

The sequence of transcript 2 (SEQ ID NO: 2) has a coding region 513 bp in length. Compared with the original ICOS mRNA sequence (SEQ ED NO: 9), transcript 2 has an additional 154 bp inserted between bases 83 and 84 of the ICOS sequence. The additional sequence contains a stop codon (at base 147) in frame with the presumed start codon of ICOS (at base 26 of ICOS, base 9 of transcript 2), which changes the starting point for translation in this transcript to base 150. In addition, the A nucleotide at position 2194 of ICOS is changed to a G in the corresponding position 2331 of transcript 2, and the T nucleotide at position 2272 of ICOS is changed to a G in the corresponding position 2409 of transcript 2.

The sequence of transcript 3 (SEQ ID NO: 3) has a coding region 513 bp in length. Compared with the original ICOS mRNA sequence (SEQ ED NO: 9), transcript 3 has an additional 234 bp inserted between bases 83 and 84 of the ICOS sequence. The additional sequence contains a stop codon (at base 93) in frame with the presumed start codon of ICOS (at base 26 of ICOS, base 9 of transcript 1), which changes the starting point for translation in this transcript to base 330. En addition, the A nucleotide at position 1933 of ICOS is changed to a T in the corresponding position 2150 of transcript 3, the T nucleotide at position 1967 of ICOS is changed to a C in the corresponding position 2184 of transcript 3, the T nucleotide at position 2272 of ICOS is changed to a G in the corresponding position 2489 of transcript 3, and the T nucleotide at position 2313 of ICOS is changed to a C in the corresponding position 2530 of transcript 3.

The present inventors found ICOS expression to be high in lymphoid tissues such as the thymus and spleen, but also noted high levels of expression in the lung and gastrointestinal tissues, which is consistent with a role for ICOS in local immune responses in mucosal tissues.

The amino acid sequence translations of the three new ICOS transcripts give identical proteins of 170 amino acids in length that differ from the published ICOS sequence

(SEQ ED NO: 10) only in their extracellular region. This resulting ICOS variant sequence lacks the first 29 amino acid residues of the published ICOS sequence. It has been shown experimentally that the first 20 residues of ICOS comprise a signal peptide that is cleaved from the mature protein (ref. 2). Also within the first 29 residues is a potential N-glycosylation site at residue 23 (ref. 1). Since both the signal peptide and a potential N-glycosylation site are missing from the ICOS variant, the variant may be sorted less efficiently or to a different compartment within the cell than the original ICOS, and may lack a glycosylation important in binding to its ligand. Either of these differences have potentially important consequences on the expression of ICOS on the cell surface. Since ICOS is known to be a dimer (refs. 1,

5), the expression of two different forms of the ICOS protein may allow the formation of heterodimers between the original ICOS and its variant or two different types of homodimers. Indeed, Hutloff et al. in their initial publication on the discovery of ICOS (ref.l) show in figure lb of their paper that reduction of ICOS dimers gives two distinct species: a 29 k protein and a 27 k protein. While they suggest that the two species probably differ only in their post-translational modification, they may also represent the original ICOS and its smaller variant.

The differences in the forms of ICOS expressed on a cell have important effects in immune responses to pathogens and in disease pathogenesis. En order for the body to defend itself against different pathogens, different types of immune responses are necessary. For some pathogens, such as intracellular bacteria, a predominantly Thl type of response is required to control infection, while for others, such as helminths or microbes present in the extracellular milieu, a Th2 type of response is required. Inappropriate polarization of immune responses can result in inadequate protection against infection, while unregulated overpolarization of responses can have harmful sequelae. En the case of intracellular bacterial infections, the counter-regulatory cytokine EL- 10 is secreted rapidly after infection to control Thl responses (ref. 6). Such a response relies on ICOS both to transmit signals into the T cells on which it is expressed and to stimulate its ICOS counter-receptor on antigen presenting cells.

Similarly, when a Th2 response is appropriate, the amplification of the response by signaling through ICOS and stimulation of ICOS is necessary for adequate defense against a pathogen. On the other hand, in autoimmune diseases and allergic diseases, uncontrolled activation of the immune response causes tissue distraction, suffering, and sometimes life-threatening complications. Upregulated expression of ICOS following Thl immune responses and downregulated expression of ICOS following Th2 immune responses is a method that the body can use to avoid autoimmunity and allergy after normal immune responses. The expression of different splice variants of ICOS also allows T cells to receive and transmit different signals according to the situation. Therefore a cell's decision on which ICOS variant to express and at what level may be crucial to the development and control of an appropriate immune response.

The inhibition of the function of the ICOS is therefore useful for treatment of allergic diseases, such as respiratory allergies, food allergies, asthma, and atopic dermatitis, as well as in the treatment of intracellular bacterial infections, such as tuberculosis, leprosy, listeriosis, and salmonellosis, where a downregulation of the Th2 response and a repolarization towards a Thl response would be beneficial. The enhancement of the function of ICOS N is useful in the treatment of autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and type I diabetes, as well as in the treatment of helminth and extracellular microbial infections, where a repolarization towards a Th2 response would be beneficial.

Polypeptides

Human ICOS N polypeptides according to the invention comprise at least 6, 10, 15,

20, 25, 50, 75, 100, 125, 150, or 170 contiguous amino acids selected from the amino acid sequence shown in SEQ ED NO: 4 or a biologically active variant thereof, as defined below. A human ICOS V polypeptide of the invention therefore can be a portion of a human ICOS, a full-length human ICOS V, or a fusion protein comprising all or a portion of a human ICOS .

Biologically Active Variants

Human ICOS V polypeptide variants that are biologically active, e.g., retain an ICOS V binding activity, also are human ICOS V polypeptides. Preferably, naturally or non-naturally occurring human ICOS V polypeptide variants have amino acid sequences which are at least about 31, 35, 40, 45, 50, 55, 60, 65, or 70, preferably about 75, 80, 85, 90, 96, 96, or 98% identical to the amino acid sequence shown in

SEQ ED NO: 4 or a fragment thereof. Percent identity between a putative human ICOS V polypeptide variant and an amino acid sequence of SEQ ED NO: 4 is determined by conventional methods. See, for example, Altschul et al., Bull. Math.

Bio. 48:603 (1986), and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA

89:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff and Henikoff (ibid.). Those skilled in the art appreciate that there are many established algorithms available to align two amino acid sequences. The "FASTA"similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant. The FASTA algorithm is described y Pearson and Lipman, Proc. Nat'l Acad. Sci. USA 85:2444(1988), and by Pearson, Meth. Enzymol. 183:63

(1990) .Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g. SEQ ED NO: 2) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score. If there are several regions with scores greater than the "cutoff value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to for man approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman- Wunsch- Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SEAM J. Appl. Math. 26:787 (1974)), which allows for amino acid insertions and deletions. Preferred parameters for

FASTA analysis are: ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file ("SMATREX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default.

Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.

Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of a human ICOS N polypeptide can be found using computer programs well known in the art, such as DΝASTAR software.

Whether an amino acid change results in a biologically active human ICOS N polypeptide can readily be determined by assaying for Shh-binding activity, as described for example, in Carpenter, et al, PROC. NATL. ACAD. SCI. U.S.A. 95, 13630-34 (1998).

Fusion Proteins

Fusion proteins are useful for generating antibodies against human ICOS N polypeptide amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins that interact with portions of a human

ICOS N polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.

A human ICOS V polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond. The first polypeptide segment comprises at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, or 170 contiguous amino acids of SEQ ED NO: 4 or of a biologically active variant, such as those described above. The second polypeptide segment can be a full-length protein or a protein fragment. Proteins commonly used in fusion protein construction include β-galactosidase, β- glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horse- radish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Additionally, epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSN-G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DΝA binding domain (DBD) fusions, GAL4 DΝA binding domain fusions, and herpes simplex virus (HSN) BP16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the human ECOS polypeptide-encoding sequence and the heterologous protein sequence, so that the human ICOS polypeptide can be cleaved and purified away from the heterologous moiety.

A fusion protein can be synthesized chemically, as is known in the art. Preferably, a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology. Recombinant DΝA methods can be used to prepare fusion proteins, for example, by making a DΝA construct which comprises coding sequences selected from the complement of SEQ ED NO: 1, 2 or 3 in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art. Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA), Santa Cruz Biotechnology (Santa Cruz, CA), MBL International

Corporation (MIC; Watertown, MA), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS). Identification of Species Homoloss

Species homologs of human ICOS N polypeptide can be obtained using human ICOS polypeptide polynucleotides (described below) to make suitable probes or primers for screening cDΝA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDΝAs which encode homologs of human ICOS V polypeptide, and expressing the cDΝAs as is known in the art.

Polynucleotides

A human ICOS polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for a human ICOS N polypeptide. A coding sequence for human ICOS N is shown in SEQ ED NO: 1, 2, or 3.

Degenerate nucleotide sequences encoding human ICOS polypeptides, as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, or 98% identical to the nucleotide sequence shown in SEQ ED NO: 1, 2, or 3 or their complement also are human ECOS V polynucleotides.

Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2. Complementary DNA (cDNA) molecules, species ho- mologs, and variants of human ICOS V polynucleotides that encode biologically active human ICOS V polypeptides also are human ICOS V polynucleotides. Fragments comprising 8, 10, 12, 15, 20, or 25 contiguous nucleotides of SEQ ED NO: 1, 2, or 3 or their complement also are human ICOS V polynucleotides. Such polynucleotides can be used, for example, as antisense oligonucleotides or as hybridiza- tion probes. Identification of Polynucleotide Variants and Homologs

Variants and homologs of the human ICOS N polynucleotides described above also are human ICOS polynucleotides. Typically, homologous human ICOS V poly- nucleotide sequences can be identified by hybridization of candidate polynucleotides to known human ICOS V polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions— 2X SSC (0.3 M ΝaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2X SSC, 0.1% SDS, 50°C once, 30 minutes; then 2X SSC, room temperature twice, 10 minutes each—homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.

Species homologs of the human ICOS V polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDΝA expression libraries from other species, such as mice, monkeys, or yeast. Human variants of human ICOS V polynucleotides can be identified, for example, by screening human cDΝA expression libraries. It is well known that the T_m of a double-stranded DΝA decreases by 1-1.5°C with every 1% decrease in homology (Bonner et al, J. Mol.

Biol. 81, 123 (1973). Variants of human ICOS V polynucleotides or human ICOS V polynucleotides of other species can therefore be identified by hybridizing a putative homologous human ICOS V polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ED NO: 1, 2, or 3 or the complement thereof to form a test hybrid. The melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.

Nucleotide sequences which hybridize to human ICOS V polynucleotides or their complements following stringent hybridization and/or wash conditions also are human ICOS V polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.

Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20°C below the calculated T_m of the hybrid under study. The T_m of a hybrid between a human ICOS polynucleotide having a nucleotide sequence shown in SEQ ED NO: 1, 2, or 3, or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):

T_m = 81.5°C - 16.6(log₁₀[Na⁺]) + 0.41 (%G + C) - 0.63(%formamide) - 600/ ), where / = the length of the hybrid in basepairs.

Stringent wash conditions include, for example, 4X SSC at 65°C, or 50% formamide, 4X SSC at 42°C, or 0.5X SSC, 0.1% SDS at 65°C. Highly stringent wash conditions include, for example, 0.2X SSC at 65°C.

Preparation of Polynucleotides

A human ICOS V polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids. Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated human ICOS V polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments which comprises ICOS nucleotide sequences. Isolated polynucleotides are in preparations which are free or at least 70, 80, or 90% free of other molecules.

Human ICOS V cDNA molecules can be made with standard molecular biology techniques, using human ICOS V mRNA as a template. Human ICOS V cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.

Alternatively, synthetic chemistry techniques can be used to synthesizes human ICOS V polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a human ICOS V polypeptide having, for example, an amino acid sequence shown in SEQ ED NO: 4 or a biologically active variant thereof.

Extending Polynucleotides

Various PCR-based methods can be used to extend the nucleic acid sequences disclosed herein to detect upstream sequences such as promoters and regulatory elements. For example, restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al, Nucleic Acids Res. 16, 8186, 1988). Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72°C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.

Another method which can be used is capture PCR, which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al, PCR Methods Applic. 1, 111-119, 1991). In this method, multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.

Another method which can be used to retrieve unknown sequences is that of Parker et al, Nucleic Acids Res. 19, 3055-3060, 1991). Additionally, PCR, nested primers, and PROMOTERFENDER libraries (CLONTECH, Palo Alto, Calif.) can be used to walk genomic DNA (CLONTECH, Palo Alto, Calif.). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.

Commercially available capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products. For example, capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera. Output/light intensity can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled. Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA that might be present in limited amounts in a particular sample.

Obtaining Polypeptides

Human ICOS V polypeptides can be obtained, for example, by purification from human cells, by expression of human ICOS V polynucleotides, or by direct chemical synthesis.

Protein Purification

Human ICOS V polypeptides can be purified from any cell which expresses the molecule, including host cells which have been transfected with human ICOS V expression constructs. A purified human ICOS V polypeptide is separated from other compounds which normally associate with the human ICOS V polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatogr- aphy, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis. A preparation of purified human ICOS V polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%o, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis. Expression of Polynucleotides

To express a human ICOS V polynucleotide, the polynucleotide can be inserted into an expression vector that contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding human ICOS V polypeptides and appropriate transcriptional and transla- tional control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al, CURRENT

PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1989.

A variety of expression vector/host systems can be utilized to contain and express sequences encoding a human ICOS V polypeptide. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.

The control elements or regulatory sequences are those non-translated regions of the vector — enhancers, promoters, 5¹ and 3' untranslated regions ~ which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCREPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g, heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding a human ICOS polypeptide, vectors based on SV40 or

EBV can be used with an appropriate selectable marker.

Bacterial and Yeast Expression Systems

In bacterial systems, a number of expression vectors can be selected depending upon the use intended for the human ICOS V polypeptide. For example, when a large quantity of a human ICOS polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUES CREPT (Stratagene). En a

BLUESCR PT vector, a sequence encoding the human ICOS V polypeptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β-galactosidase so that a hybrid protein is produced. pEN vectors (Van Heeke & Schuster, J. Biol. Chem. 264, 5503-5509, 1989) or pGEX vectors (Promega, Madison, Wis.) also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.

In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For reviews, see Ausubel et al. (1989) and Grant et al, Methods Enzymol. 153, 516-544,

1987. Plant and Insect Expression Systems

If plant expression vectors are used, the expression of sequences encoding human ICOS polypeptides can be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6, 307-311, 1987). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al, EMBO J. 3, 1671-1680, 1984; Broglie et al, Science 224, 838-843, 1984; Winter et al, Results

Probl Cell Differ. 17, 85-105, 1991). These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (e.g., Hobbs or Murray, in MCGRAW HILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y., pp. 191-196, 1992).

An insect system also can be used to express a human ICOS V polypeptide. For example, in one such system Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. Sequences encoding human ICOS V polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of human ICOS V polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which human ICOS polypeptides can be expressed (Engelhard et al, Proc. Nat. Acad. Sci. 91, 3224-3227, 1994).

Mammalian Expression Systems

A number of viral-based expression systems can be used to express human ICOS V polypeptides in mammalian host cells. For example, if an adenovirus is used as an expression vector, sequences encoding human ICOS V polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing a human ICOS V polypeptide in infected host cells (Logan & Sherik, Proc. Natl. Acad.

Sci. 81, 3655-3659, 1984). If desired, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) also can be used to deliver larger fragments of DNA than can be contained and expressed in a plasmid. HACs of 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).

Specific initiation signals also can be used to achieve more efficient translation of sequences encoding human ICOS V polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding a human ICOS V polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al, Results Probl Cell Differ. 20, 125-162, 1994). Host Cells

A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed human ICOS V polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and

WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.

Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, cell lines which stably express human ICOS V polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced human ICOS sequences. Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, ANIMAL CELL CULTURE, R.I. Freshney, ed., 1986.

Any number of selection systems can be used to recover transformed cell lines.

These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al, Cell 11, 223-32, 1977) and adenine phosphoribosyltransferase (Lowy et al, Cell 22, 817-23, 1980) genes which can be employed in tk^' or aprt cells, respectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate (Wigler et al, Proc. Natl. Acad. Sci. 77, 3567-70, 1980), npt confers resistance to the amin- glycosides, neomycin and G-418 (Colbere-Garapin et al, J. Mol. Biol. 150, 1-14, 1981), and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murray, 1992, supra). Additional selectable genes have been described. For example, trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. 85, 8047-51, 1988). Visible markers such as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al, Methods Mol. Biol. 55, 121-131, 1995).

Detecting Expression

Although the presence of marker gene expression suggests that the human ICOS V polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding a human ICOS V polypeptide is inserted within a marker gene sequence, transformed cells containing sequences which encode a human ICOS V polypeptide can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a human ICOS V polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the human ICOS V polynucleotide.

Alternatively, host cells which contain a human ICOS V polynucleotide and which express a human ICOS V polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein. For example, the presence of a polynucleotide sequence encoding a human ICOS V polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding a human ICOS V polypeptide. Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding a human ICOS V polypeptide to detect transfonnants which contain a human ICOS V polynucleotide.

A variety of protocols for detecting and measuring the expression of a human ICOS V polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RLA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on a human ICOS V polypeptide can be used, or a competitive binding assay can be employed. These and other assays are described in Hampton et al, SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St. Paul, Minn., 1990) and Maddox et al, J. Exp. Med. 158, 1211-1216, 1983).

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding human ICOS V polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, sequences encoding a human ICOS V polypeptide can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical).

Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

Expression and Purification of Polypeptides

Host cells transformed with nucleotide sequences encoding a human ICOS V polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode human ICOS V polypeptides can be designed to contain signal sequences which direct secretion of soluble human ECOS V polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound human ICOS V polypeptide.

As discussed above, other constructions can be used to join a sequence encoding a human ICOS V polypeptide to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine- tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). Inclusion of cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the human ICOS V polypeptide also can be used to facilitate purification.

One such expression vector provides for expression of a fusion protein containing a human ICOS V polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilized metal ion affinity chromatography, as described in Porath et al, Prot. Exp. Purifi 3, 263-281, 1992), while the enterokinase cleavage site provides a means for purifying the _human ICOS V polypeptide from the fusion protein. Vectors which contain fusion proteins are disclosed in Kroll et al, DNA Cell Biol. 12, 441-453, 1993.

Chemical Synthesis

Sequences encoding a human ICOS V polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Carathers et al, Nucl. Acids Res. Symp. Ser. 215-223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225-232, 1980). Alternatively, a human ICOS V polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al, Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of human ICOS polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.

The newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N.Y., 1983). The composition of a synthetic human ICOS polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence of the human ICOS V polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.

Production of Altered Polypeptides

As will be understood by those of skill in the art, it may be advantageous to produce human ICOS V polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.

The nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter human ICOS V polypeptide-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences. For example, site-directed mutagenesis can be used to insert new restriction sites, alter glycosyla- tion patterns, change codon preference, produce splice variants, introduce mutations, and so forth.

Antibodies

Any type of antibody known in the art can be generated to bind specifically to an epitope of a human ICOS V polypeptide. "Antibody" as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab')₂, and

Fv, which are capable of binding an epitope of a human ICOS V polypeptide. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.

An antibody which specifically binds to an epitope of a human ICOS V polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immuno- precipitations, or other immunochemical assays known in the art. Various immuno- assays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the immunogen.

Typically, an antibody which specifically binds to a human ICOS V polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay. Preferably, antibodies which specifically bind to human ICOS V like polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a human ICOS V polypeptide from solution.

Human ICOS Vpolypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, a human ICOS V polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol). Among adjuvants used in humans, BCG (bacilli Calmette-

Gueriή) and Corynebacterium parvum are especially useful.

Monoclonal antibodies which specifically bind to a human ICOS V polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al, Nature 256, 495-497, 1985; Kozbor et al, J. Immunol. Methods 81, 31-42, 1985; Cote et al, Proc. Natl. Acad. Sci. 80, 2026-2030, 1983; Cole et al, Mol. Cell Biol. 62, 109-120, 1984). In addition, techniques developed for the production of "chimeric antibodies," the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al, Proc. Natl. Acad. Sci. 81, 6851-6855, 1984; Neuberger et al, Nature 312, 604-608, 1984; Takeda et al, Nature 314, 452-454, 1985). Monoclonal and other antibodies also can be "humanized" to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions. Alternatively, humanized antibodies can be produced using recombinant methods, as described in GB2188638B. Antibodies which specifically bind to a human ICOS V polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.

Alternatively, techniques described for the production of single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to human ICOS V polypeptides. Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci. 88, 11120-23, 1991).

Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al, 1996, Eur. J. Cancer Prev. 5, 507-11). Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in

Mallender & Voss, 1994, J. Biol. Chem. 269, 199-206. A nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below. Alternatively, single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et al, 1995, Int. J. Cancer 61, 497-501; Nicholls et al, 1993, J. Immunol. Meth. 165, 81-91).

Antibodies which specifically bind to human ICOS V polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al, Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter et al, Nature 349, 293-299, 1991).

Other types of antibodies can be constracted and used therapeutically in methods of the invention. For example, chimeric antibodies can be constracted as disclosed in WO 93/03151. Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94/13804, also can be prepared.

Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which a human ICOS polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.

Antisense Oligonucleotides

Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA constract and introduced into a cell as described above to decrease the level of human ICOS V gene products in the cell.

Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkyl- phosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al, Chem. Rev. 90, 543-583, 1990.

Modifications of human ICOS V gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of the human ICOS V gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred.

Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (e.g., Gee et al, in Huber & Carr, MOLECULAR AND IMMUNOLOGIC

APPROACHES, Futura Publishing Co., Mt. Kisco, N.Y., 1994). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of a human ICOS V polynucleotide. Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a human ICOS polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent human ICOS V nucleotides, can provide sufficient targeting specificity for human ICOS V mRNA. Preferably, each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular human

ICOS V polynucleotide sequence.

Antisense oligonucleotides can be modified without affecting their ability to hybridize to a human ICOS V polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule. For example, internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose. Modified bases and/or sugars, such as arabinose instead of ribose, or a 3', 5'-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al, Trends Biotechnol 10, 152-158, 1992; Uhlmann et al, Chem. Rev. 90, 543-584, 1990; Uhlmann et al, Tetrahedron. Lett. 215, 3539-3542, 1987.

Ribozymes

Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al, U.S. Patent 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.

The coding sequence of a human ICOS V polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from the human ICOS V polynucleotide. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al, EP 321,201).

Specific ribozyme cleavage sites within a human ICOS V RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate human ICOS V RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.

Ribozymes can be introduced into cells as part of a DNA constract. Mechanical methods, such as micromjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA constract into cells in which it is desired to decrease human ICOS V expression. Alternatively, if it is desired that the cells stably retain the DNA constract, the constract can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding DNA constract can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.

As taught in Haseloff et al, U.S. Patent 5,641,673, ribozymes can be engineered so that ribozyme expression will occur in response to factors which induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.

Differentially Expressed Genes

Described herein are methods for the identification of genes whose products interact with human ICOS V. Such genes may represent genes which are differentially expressed in disorders including, but not limited to, autoimmune diseases, allergic diseases, bacterial infections, and type I diabetes. Further, such genes may represent genes which are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human ICOS V gene or gene product may itself be tested for differential expression.

The degree to which expression differs in a normal versus a diseased state need only be large enough to be visualized via standard characterization techniques such as differential display techniques. Other such standard characterization techniques by which expression differences may be visualized include but are not limited to, quantitative RT (reverse transcriptase), PCR, and Northern analysis.

Identification of Differentially Expressed Genes

To identify differentially expressed genes total RNA or, preferably, mRNA is isolated from tissues of interest. For example, RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects. Any RNA isolation technique which does not select against the isolation of mRNA may be utilized for the purification of such RNA samples. See, for example, Ausubel et al, ed.„ CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Enc. New York, 1987-1993. Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, U.S. Patent 4,843,155.

Transcripts within the collected RNA samples which represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al, Proc. Natl. Acad. Sci. U.S.A. 85, 208-12, 1988), subtractive hybridization (Hedrick et al, Nature 308, 149-53; Lee et al, Proc. Natl. Acad. Sci. U.S.A. 88, 2825, 1984), and, preferably, differential display (Liang & Pardee, Science 257, 967-71, 1992; U.S. Patent 5,262,311).

The differential expression information may itself suggest relevant methods for the treatment of disorders involving the human ICOS V. For example, treatment may include a modulation of expression of the differentially expressed genes and/or the gene encoding the human ICOS V. The differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human ICOS gene or gene product are up-regulated or down- regulated. Screening Methods

The invention provides assays for screening test compounds that bind to or modulate the activity of a human ICOS V polypeptide or a human ICOS V polynucleotide. A test compound preferably binds to a human ICOS V polypeptide or polynucleotide.

More preferably, a test compound decreases or increases human ICOS V activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.

Test Compounds

Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound" library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.

Methods for the synthesis of molecular libraries are well known in the art (see, for example, DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al, J. Med. Chem. 37, 2678, 1994; Cho et al, Science 261, 1303, 1993; Carell et al, Angew. Chem. Int. Ed. Engl 33, 2059, 1994; Carell et al, Angew. Chem. Int. Ed. Engl 33, 2061; Gallop et al, J.

Med. Chem. 37, 1233, 1994). Libraries of compounds can be presented in solution (see, e.g., Houghten, BioTechniques 13, 412-421, 1992), or on beads (Lam, Nature 354, 82-84, 1991), chips (Fodor, Nature 364, 555-556, 1993), bacteria or spores (Ladner, U.S. Patent 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci. U.S.A. 89, 1865-1869, 1992), or phage (Scott & Smith, Science 249, 386-390, 1990; Devlin, Science 249, 404-406, 1990); Cwirla et al, Proc. Natl. Acad. Sci. 97, 6378-6382,

1990; Felici, J. Mol. Biol. 222, 301-310, 1991; and Ladner, U.S. Patent 5,223,409).

High Throughput Screening

Test compounds can be screened for the ability to bind to human ICOS V polypeptides or polynucleotides or to affect human ICOS V activity or human ICOS V gene expression using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.

Alternatively, "free format assays," or assays that have no physical barrier between samples, can be used. For example, an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al, Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose. The combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.

Another example of a free format assay is described by Chelsky, "Strategies for

Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995). Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying combi- natorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.

Yet another example is described by Salmon et al, Molecular Diversity 2, 57-63 (1996). En this example, combinatorial libraries were screened for compounds that had cytotoxic effects on cancer cells growing in agar.

Another high throughput screening method is described in Beutel et al, U.S. Patent 5,976,813. En this method, test samples are placed in a porous matrix. One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support. When samples are introduced to the porous matrix they diffuse sufficiently slowly, such that the assays can be performed without the test samples running together.

Binding Assays

For binding assays, the test compound is preferably a small molecule which binds to and occupies, for example, the active site of the human ICOS V polypeptide, such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules.

In binding assays, either the test compound or the human ICOS V polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound which is bound to the human ICOS V polypeptide can then be accomplished, for example, by direct counting of radio- emmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.

Alternatively, binding of a test compound to a human ICOS V polypeptide can be determined without labeling either of the interactants. For example, a microphysio- meter can be used to detect binding of a test compound with a human ICOS V polypeptide. A microphysiometer (e.g., Cytosensor™) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a human ICOS V polypeptide (McConnell et al, Science 257, 1906-1912, 1992).

Determining the ability of a test compound to bind to a human ICOS V polypeptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem. 63, 2338-2345,

1991, and Szabo et al, Curr. Opin. Struct. Biol. 5, 699-705, 1995). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

In yet another aspect of the invention, a human ICOS V polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et al, Cell 72, 223-232, 1993; Madura et al, J. Biol. Chem. 268, 12046-12054, 1993; Barrel et al, BioTechniques 14, 920-924, 1993; Iwabuchi et al,

Oncogene 8, 1693-1696, 1993; and Brent W094/10300), to identify other proteins which bind to or interact with the human ICOS V polypeptide and modulate its activity.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constracts. For example, in one construct, polynucleotide encoding a human ICOS V polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct a DNA sequence that encodes an unidentified protein ("prey" or "sample") can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact in vivo to form an protein-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcrip- tional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with the human ICOS polypeptide.

It may be desirable to immobilize either the human ICOS V polypeptide (or polynucleotide) or the test compound to facilitate separation of bound from unbound forms of one or both of the interactants, as well as to accommodate automation of the assay. Thus, either the human ICOS V polypeptide (or polynucleotide) or the test compound can be bound to a solid support. Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach the enzyme polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support. Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a human ICOS V polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes. Ln one embodiment, the human ICOS V polypeptide is a fusion protein comprising a domain that allows the human ICOS V polypeptide to be bound to a solid support. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed human ICOS V polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.

Other techniques for immobilizing proteins or polynucleotides on a solid support also can be used in the screening assays of the invention. For example, either a human ICOS polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated human ICOS V polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS(N- hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies which specifically bind to a human ICOS V polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of the human ICOS V polypeptide, can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.

Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies which specifically bind to the human ICOS V polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the human ICOS V polypeptide, and SDS gel electrophoresis under non-reducing conditions. Screening for test compounds which bind to a human ICOS V polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a human ICOS V polypeptide or polynucleotide can be used in a cell-based assay system. A human ICOS V polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a human ICOS V polypeptide or polynucleotide is determined as described above.

Gene Expression

In another embodiment, test compounds which increase or decrease human ICOS V gene expression are identified. A human ICOS V polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the human ICOS V polynucleotide is determined. The level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.

The level of human ICOS V mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used. The presence of polypeptide products of a human ICOS V polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry. Alternatively, polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a human ICOS V polypeptide.

Such screening can be carried out either in a cell-free assay system or in an intact cell. Any cell which expresses a human ICOS V polynucleotide can be used in a cell-based assay system. The human ICOS V polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used.

Pharmaceutical Compositions

The invention also provides pharmaceutical compositions that can be administered to a patient to achieve a therapeutic effect. Pharmaceutical compositions of the invention can comprise, for example, a human ICOS V polypeptide, human ICOS V polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to a human ICOS V polypeptide, or mimetics, activators, or inhibitors of a human ICOS V polypeptide activity. The compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drags or hormones.

h addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically. Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, , topical, sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspen- sions, and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyesruffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

Phaπnaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers. Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers also can be used for delivery. Optionally, the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms, h other cases, the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.

Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co., Easton, Pa.). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.

Therapeutic Indications and Methods

Human ICOS V protein may be regulated to treat autoimmune diseases, allergic diseases, bacterial infections, and type I diabetes.

The differences in the forms of ICOS V expressed on a cell could have important effects in immune responses to pathogens and in disease pathogenesis. In order for the body to defend itself against different pathogens, different types of immune responses are necessary. For some pathogens, such as intracellular bacteria, a predominantly Thl type of response is required to control infection, while for others, such as helminths or microbes presesnt in the extracellular milieu, a Th2 type of response is required. Inappropriate polaraization of immune responses can result in inadequate protection against infection, while unregulated overpolarization of responses can have harmful sequelae. In the case of intracellular bacterial infections, the counterregulatory cytokine EL- 10 is secreted rapidly after infection to control Thl responses (ref. 6). Such a response rely on ICOS both to transmit signals into the T cells on which it is expressed and to stimulate its ICOS counterreceptor on antigen presenting cells. Similarly, when a Th2 response is appropriate, the amplification of the response by signaling through ICOS and stimulation of ICOS is necessary for adequate defense against a pathogen. On the other hand, in autoimmune diseases and allergic diseases, uncontrolled activation of the immune response causes tissue distraction, suffering, and sometimes life-threatening complications. Upregulated expression of ICOS following Thl immune responses and downregulated expression of ICOS following Th2 immune responses is a possible method that the body can use to avoid autoimmunity and allergy after normal immune responses. The expression of different splice variants of ICOS also allow T cells to receive and transmit different signals according to the situation. Therefore a cell's decision on which ICOS variant to express and at what level may be cracial to the development and control of an appropriate immune response.

The development of inhibitors to block the function of the ICOS V would be expected to be useful in the treatment of allergic diseases, such as respiratory allergies, food allergies, asthma, and atopic dermatitis, as well as in the treatment of intracellular bacterial infections, such as tuberculosis, leprosy, listeriosis, and salmonellosis, where a downregulation of the Th2 response and a repolarization towards a Thl response would be beneficial. The development of molecules to enhance the functions of ICOS V would be expected to be useful in the treament of autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and type I diabetes, as well as in the treatment of helminth and extracellular microbial infections, where a repolarization towards a Th2 response would be beneficial.

This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a human ICOS V polypeptide binding molecule) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

A reagent which affects human ICOS V activity can be administered to a human cell, either in vitro or in vivo, to reduce human ICOS V activity. The reagent preferably binds to an expression product of a human ICOS V gene. If the expression product is a protein, the reagent is preferably an antibody. For treatment of human cells ex vivo, an antibody can be added to a preparation of stem cells that have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.

In one embodiment, the reagent is delivered using a liposome. Preferably, the lipo- some is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours. A liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human. Preferably, the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin.

A liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell. Preferably, the transfection efficiency of a liposome is about

0.5 μg of DNA per 16 nmole of liposome delivered to about 10⁶ cells, more preferably about 1.0 μg of DNA per 16 nmole of liposome delivered to about 10⁶ cells, and even more preferably about 2.0 μg of DNA per 16 nmol of liposome delivered to about 10⁶ cells. Preferably, a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 run, and even more preferably between about 200 and 400 nm in diameter.

Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More prefeπed liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol. Optionally, a liposome comprises a compound capable of targeting the liposome to a particular cell type, such as a cell-specific ligand exposed on the outer surface of the liposome. Complexing a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods that are standard in the art (see, for example, U.S. Patent 5,705,151). Preferably, from about 0.1 μg to about 10 μg of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 μg to about 5 μg of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 μg of polynucleotides is combined with about 8 nmol liposomes.

In another embodiment, antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery. Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol 11, 202-05 (1993);

Chiou et al, GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE

TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol Chem. 263, 621-24 (1988);

Wu et al, J. Biol. Chem. 269, 542-46 (1994); Zenke et al, Proc. Natl. Acad. Sci. USA. 87, 3655-59 (1990); Wu et al, J. Biol. Chem. 266, 338-42 (1991).

Determination of a Therapeutically Effective Dose

The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient that increases or decreases human ICOS V activity relative to the human ICOS V activity which occurs in the absence of the therapeutically effective dose.

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic efficacy and toxicity, e.g., ED s (the dose therapeutically effective in 50% of the population) and LD5₀ (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD₅₀/ED₅₀.

Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect.

Factors that can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drag combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.

Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. If the reagent is a single-chain antibody, polynucleotides encoding the antibody can be constracted and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun," and DEAE- or calcium phosphate-mediated transfection.

Effective in vivo dosages of an antibody are in the range of about 5 μg to about 50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about 200 to about 250 μg/kg of patient body weight. For administration of polynucleotides encoding single-chain antibodies, effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA.

If the expression product is mRNA, the reagent is preferably an antisense oligonucleotide or a ribozyme. Polynucleotides that express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.

Preferably, a reagent reduces expression of a human ICOS V gene or the activity of a human ICOS V polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100%o relative to the absence of the reagent. The effectiveness of the mechanism chosen to decrease the level of expression of a human ICOS V gene or the activity of a human ICOS V polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to human ICOS V -specific mRNA, quantitative RT-PCR, immunologic detection of a human ICOS V polypeptide, or measurement of human ICOS V activity.

In any of the embodiments described above, any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents can act synergis- tically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

Diagnostic Methods

Human ICOS V also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences that encode the enzyme. For example, differences can be determined between the cDNA or genomic sequence encoding human ICOS V in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.

Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method. In addition, cloned DNA segments can be employed as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags. Genetic testing based on DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al, Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et al, Proc. Natl. Acad. Sci. USA 85, 4397-

4401, 1985). Thus, the detection of a specific DNA sequence can be perfonned by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA. In addition to direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

Altered levels of a human ICOS V also can be detected in various tissues. Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and

ELISA assays.

All patents and patent applications cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided for purposes of illustration only and are not intended to limit the scope of the invention. EXAMPLE 1

Determination of the human ICOS transcripts lto3mRNA sequence

The predicted open reading frame of the ICOS V gene was cloned for analysis.

Primers flanking the open reading frame were designed using the computer program Primer 3.0 (Steve Rozen, Helen J. Skaletsky (1998) Primer3. Code available at http://www-genome.wi.mit.edu/genome_software/other/primer3.html.). Primers AEL- Ll (SEQ ED NO: 5) and AEL-R1 (SEQ ED NO: 6) were used to amplify the open reading by polymerase chain reaction using human peripheral blood leukocyte cDNA as the template in the reaction. The template cDNA was previously synthesized with the SMART RACE cDNA amplification kit (Clontech, Palo Alto, CA, USA) according to the manufacturer's protocol using human peripheral blood leukocyte- derived poly-A RNA as the starting material for cDNA synthesis. Successfully amplified fragments were cloned into the pCREE-TOPO vector (Envitrogen, Carlsbad,

CA, USA) and were sequenced on a ABI Prism 377 DNA sequencer (PE Biosystems) according to the manufacturer's standard sequencing protocol using primers complemetary to the SP6 and T7 promoter regions flanking the insert on each vector. Primers were then designed at approximately 400 base pair intervals to sequence the internal portions of each clone. After sequencing, the DNA sequences of eight selected clones (clones 1, 2, 3, 4, 5, 8, 9, and 10) were compared with the published sequence for ICOS using the computer program Sequencher (Gene Codes Corporation, Ann Arbor, MI, USA).

(Properties of the obtained cDNA)

We have cloned three new splice variants of the ICOS gene transcript.

1. The sequence of transcript 1 (from clone 1) has a coding region 513 bp in length. 2. Compared with the original ICOS mRNA sequence (GenBank accession number AB0231353) reported for this gene, transcript 1 has an additional 234 bp inserted between bases 83 and 84 of the ICOS sequence. The additional sequence contains a stop codon (at base 93) in frame with the presumed start codon of ICOS (at base 26 of ICOS, base 9 of transcript 1), which changes the starting point for translation in this transcript to base 330. En addition, the T nucleotide at position 2272 of ICOS is changed to a G in the corresponding position 2489 of transcript 1.

3. The sequence of transcript 2 (from clone 4) has a coding region 513 bp in length.

4. Compared with the original ICOS mRNA sequence, transcript 2 has an additional 154 bp inserted between bases 83 and 84 of the ICOS sequence. The additional sequence contains a stop codon (at base 147) in frame with the presumed start codon of ICOS (at base 26 of ICOS, base 9 of transcript 2), which changes the starting point for translation in this transcript to base 150. In addition, the A nucleotide at position 2194 of ICOS is changed to a G in the coπesponding position 2331 of transcript 2, and the T nucleotide at position 2272 of ICOS is changed to a G in the corresponding position 2409 of transcript 2.

5. The sequence of transcript 3 (from clone 9) has a coding region 513 bp in length.

6. Compared with the original ICOS mRNA sequence, transcript 3 has an additional 234 bp inserted between bases 83 and 84 of the ICOS sequence. The additional sequence contains a stop codon (at base 93) in frame with the presumed start codon of ICOS (at base 26 of ICOS, base 9 of transcript 1), which changes the starting point for translation in this transcript to base 330.

In addition, the A nucleotide at position 1933 of ICOS is changed to a T in the corresponding position 2150 of transcript 3, the T nucleotide at position 1967 of ICOS is changed to a C in the corresponding position 2184 of transcript 3, the T nucleotide at position 2272 of ICOS is changed to a G in the conesponding position 2489 of transcript 3, and the T nucleotide at position 2313 of ICOS is changed to a C in the conesponding position 2530 of transcript 3.

(Properties of the amino acid sequences encoded by the obtained cDNA)

1. Conceptual translations of transcripts 1, 2, and 3 give an identical amino acid sequence 170 residues in length (ICOS VI).

2. The translations of the transcripts 1, 2, and 3 sequences differ from the conceptual translation of ICOS (GenBank accession number BAA82129) by lacking the first 29 residues. The amino acid sequences are otherwise identical.

EXAMPLE 2

Tissue distribution of human ICOS V

Expression profiling is based on a quantitative polymerase chain reaction (PCR) analysis, also called kinetic analysis, first described in Higuchi et al., 1992 and Higuchi et al., 1993. The principle is that at any given cycle within the exponential phase of PCR, the amount of product is proportional to the initial number of template copies. Using this technique, the expression levels of particular genes, which are transcribed from the chromosomes as messenger RNA (mRNA), are measured by first making a DNA copy (cDNA) of the mRNA, and then performing quantitative PCR on the cDNA, a method called quantitative reverse transcription-polymerase chain reaction (quantitative RT-PCR) . Quantitative RT-PCR analysis of RNA from different human tissues was performed to investigate the tissue distribution of RCK007 ICOS mRNA. 25 .mu.g of total RNA from various tissues (Human Total RNA Panel I-V, Clontech Laboratories, Palo Alto, CA, USA) was used as a template to synthsize first-strand cDNA using the SUPERSCRIPT™ First-Strand Synthesis System for RT-PCR (Life Technologies,

Rockville , MD, USA). First-strand cDNA synthesis was carried out according to the manufacturer's protocol using oligo (dT) to hybridize to the 3' poly A tails of mRNA and prime the synthesis reaction. 10 ng of the first-strand cDNA was then used as template in a polymerase chain reaction. The polymerase chain reaction was performed in a LightCycler (Roche Molecular Biochemicals, Indianapolis, EN, USA), in the presence of the DNA-binding fluorescent dye S YBR Green I which binds to the minor groove of the DNA double helix, produced only when double-stranded DNA is successfully synthesized in the reaction (Morrison et al, 1998). Upon binding to double-stranded DNA, SYBR Green I emits light that can be quantita- tively measured by the LightCycler machine. The polymerase chain reaction was carried out using oligonucleotide primers AEL-L6 (SEQ ED NO: 7,) and AEL-R6 (SEQ ED NO: 8) and measurements of the intensity of emitted light were taken following each cycle of the reaction when the reaction had reached a temperature of 87 degrees C. Intensities of emitted light were converted into copy numbers of the gene transcript per nanogram of template cDNA by comparison with simultaneously reacted standards of known concentration.

To conect for differences in mRNA transcription levels per cell in the various tissue types, a normalization procedure was performed using similarly calculated expres- sion levels in the various tissues of five different housekeeping genes: glyceraldehyde-3-phosphatase (G3PDH), hypoxanthine guanine phophoribosyl transferase (HPRT), beta-actin, porphobilinogen deaminase (PBGD), and beta-2- microglobulin. The level of housekeeping gene expression is considered to be relatively constant for all tissues (Adams et al., 1993, Adams et al., 1995, Liew et al., 1994) and therefore can be used as a gauge to approximate relative numbers of cells per .mu.g of total RNA used in the cDNA synthesis step. Except for the use of a slightly different set of housekeeping genes and the use of the LightCycler system to measure expression levels, the normalization procedure was essentially the same as that described in the RNA Master Blot User Manual, Apendix C (1997, Clontech Laboratories, Palo Alto, CA, USA). In brief, expression levels of the five housekeeping genes in all tissue samples were measured in three independent reactions per gene using the LightCycler and a constant amount (25 .mu.g) of starting RNA. The calculated copy numbers for each gene, derived from comparison with simultaneously reacted standards of known concentrations, were recorded and converted into a percentage of the sum of the copy numbers of the gene in all tissue samples. Then for each tissue sample, the sum of the percentage values for each gene was calculated, and a normalization factor was calculated by dividing the sum percentage value for each tissue by the sum percentage value of one of the tissues arbitrarily selected as a standard. To normalize an experimentally obtained value for the expression of a particular gene in a tissue sample, the obtained value was multiplied by the normalization factor for the tissue tested.

Results are given in FIG.3, showing the experimentally obtained copy numbers of mRNA per 10 ng of first-strand cDNA on the left and the noπnalized values on the right. RNAs used for the cDNA synthesis, along with their supplier and catalog numbers are shown in table 1.

Tablel: Whole-body-screen tissues

As shown in Fig. 3, ICOS V are broadly expressed in all tissue types so far tested, with highest expression seen in thymus, spleen, stomach and lung.

References

Higuchi, R., Dollinger, G., Walsh, P.S. and Griffith, R. (1992) Simultaneous amplification and detection of specific DNA sequences. BioTechnology 10:413-417.

Higuchi, R., Fockler, C, Dollinger, G. and Watson, R. (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. BioTechnology 11:1026-1030.

T.B. Morrison, J.J. Weis & C.T. Wittwer .(1998) Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques 24:954-962.

Adams, M. D., Kerlavage, A. R., Fields, C. & Venter, C. (1993) 3,400 new expressed sequence tags identify diversity of transcripts in human brain. Nature Genet. 4:256- 265.

Adams, M. D., et al. (1995) Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence. Nature 377 supp:3-174.

Liew, C. C, Hwang, D. M., Fung, Y. W., Laurenson, C, Cukerman, E., Tsui, S. & Lee, C. Y. (1994) A catalog of genes in the cardiovascular system as identified by expressed sequence tags. Proc. Natl. Acad. Sci. USA 91:10145-10649. EXAMPLE 3

Expression of human ICOS V

The expression vector pcDNA 3.1 vector (Invitrogen, Carlsbad, CA) is used to produce large quantities of recombinant human ICOS V polypeptides in Chinese hamster ovary (CHO) cells. The human ICOS-encoding DNA sequence is derived from SEQ ED NO: 1, 2, or 3. Before insertion into vector pcDNA 3.1, the DNA sequence is modified by well known methods in such a way that it contains ICOS V and Ig fusion gene by fusing the cDNA of the extracellular domain of ICOS V in frame to the CH2-CH3 portion of human IgGl. Moreover, at both termini recognition sequences for restriction endonucleases are added and after digestion of the multiple cloning site of pcDNA 3.1 with the corresponding restriction enzymes the modified DNA sequence is ligated into pcDNA3.L The resulting phlCOS Ig vector is used to transfect the CHO cell, a ICOS negative cell line.

The cells are cultivated under usual conditions in 5 liter shake flasks and the secreted recombinantly produced protein (ICOS Ig) is purified and used in the next example.

EXAMPLE 4

B cell proliferation with the costimulation of ICOS V

The expression vector pcDNA 3.1 vector (Invitrogen, Carlsbad, CA) is used to produce recombinant human ICOS V polypeptides in B-cells. The human ICOS V

DNA sequence is derived from the sequence of GehBank accession number AB014553.

Before insertion into vector pcDNA 3.1, each of the DNA sequences is modified by well known methods in such a way that it contains at its 5 '-end an initiation codon and at its 3 '-end a termination codon. Moreover, at both termini recognition sequences for restriction endonucleases are added and after digestion of the multiple cloning site of pcDNA 3.1 with the conesponding restriction enzymes the modified DNA sequence is ligated into pcDNA3.1. The resulting phlCOS is used to transfect the B cell purified from human PBMC of healthy donors.

The cells are cultivated under usual conditions in 5 liter shake flasks and the transfectants with recombinantly produced protein are obtained. B cells so obtained are then stimulated with ICOS Ig obtained in Example 3 in the presence of suboptimal doses of an anti-CD3 mAb. B-cell proliferation is determined by incorporation of ³H-TdR after 3-day culture. ICOS Ig enhances B-cell proliferation compared to the control Ig in the presence of immobilized anti-CD3 mAb.

EXAMPLE 5

Cytokine secretion by ICOS V costimulation

The level of Cytokine e.g., EL-2, EL-4, and EL- 10 in the transfectant B-cell culture supernatants by the stimulation of ICOS Ig and an optimal dose of an anti-CD3 mAb are determined by sandwich ELISA. B-cells costimulated by ICOSIg in the presence of an optimal dose of an anti-CD3 mAb change levels of some cytokines.

EXAMPLE 6

Expression of human ICOS V

The expression vector pcDNA 3.1 vector (Invitrogen, Carlsbad, CA) is used to produce large quantities of recombinant human ICOS polypeptides in Chinese hamster ovary (CHO) cells. The human ICOS-encoding DNA sequence is derived from the sequence of GenBank accession number AB014553. Before insertion into vector pcDNA 3.1, the DNA sequence is modified by well known methods in such a way that it contains ICOS and Ig fusion gene by fusing the cDNA of the extracellular domain of ICOS in frame to the CH2-CH3 portion of human IgGl. Moreover, at both termini recognition sequences for restriction endonucleases are added and after digestion of the multiple cloning site of pcDNA

3.1 with the conesponding restriction enzymes the modified DNA sequence is ligated into pcDNA3.1. The resulting phlCOS Ig vector is used to transfect the CHO cell.

The cells are cultivated under usual conditions in 5 liter shake flasks and the secreted recombinantly produced protein (ICOSIg) is purified and used in the next example.

EXAMPLE 7

T cell proliferation with the costimulation of ICOS V

T cells are purified from human PBMC of healthy donors and then stimulated with ICOS Ig obtained in Example 6 in the presence of suboptimal doses of an anti-CD3 mAb. T-cell proliferation is determined by incorporation of ³H-TdR after 3-day culture. ICOS Ig enhances T-cell proliferation compared to the control Ig in the presence of immobilized anti-CD3 mAb.

EXAMPLE 8

Cytokine secretion by ICOS V costimulation

The level of Cytokine e.g., EL-2, EL-4, and EL- 10 in the T-cell culture supernatants by the stimulation of ICOSIg and an optimal dose of an anti-CD3 mAb are determined by sandwich ELISA. T-cells costimulated by ICOSIg in the presence of an optimal dose of anti-CD 3 mAb increase levels of EL-4 and EL- 10. EXAMPLE 9

Identification of test compounds that bind to human ICOS V polypeptides

Purified human ICOS polypeptides comprising a glutathione-S-transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution. Human ICOS polypeptides comprise the amino acid sequence shown in SEQ ED NO: 4. The test compounds comprise a fluorescent tag.

The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.

The buffer solution containing the test compounds is washed from the wells. Bind- ing of a test compound to a human ICOS V polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound that increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to a human ICOS polypeptide.

EXAMPLE 10

Identification of a test compound which modulates human ICOS V gene expression

A test compound is administered to a culture of human cells transfected with a human ICOS V expression construct and incubated at 37°C for 10 to 45 minutes. A culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control.

RNA is isolated from the two cultures as described in Chirgwin et al, Biochem. 18,

5294-99, 1979). Northern blots are prepared using 20 to 30 μg total RNA and hybridized with a ³²P-labeled human ICOS-specific probe at 65°C in Express-hyb (CLONTECH). The probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ED NO: 1, 2, or 3. A test compound that decreases the human ICOS-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of human ICOS gene expression.

EXAMPLE 11

In vivo testing of compounds/target validation

1. Tests for activity of T cells

Costimulatory molecules-cytokines, cytokine receptors, signalling molecules, any molecule involved in T cell activation

Mouse anti-CD3 induced cytokine production model

BALB/c mice were injected with a single intravenous injection of 10 μg of 145-2C11 (purified hamster anti-mouse CD3 ε monoclonal antibodies, PHARMENGEN). Compound was administered intraperitoneally 60 min prior to the anti-CD3 mAb injection. Blood was collected 90 min after the antibody injection. Serum was obtained by centrifiigation at 3000 r.p.m. for 10 min. EL- 2 and EL-4 levels in the serum was determined by an ELIS A.

Tests for activity of B cells

B cell receptor, signalling molecules, any molecule involved in B cell activation/Eg class switching

Mouse anti-IgD induced IgE production model BALB/c mice were injected intravenously with 0.8 mg of purified goat anti- mouse IgD antibody or PBS (defined as day 0). Compound was administered intraperitoneally from day 0 to day 6. On day 7 blood was collected and serum was obtained by centrifugation at 3000 r.p.m. for 10 min. Serum total levels of IgE were determined by YAM AS A' s ELISA kit and their Ig subtypes were done by an Ig ELISA KIT (Rougier Bio-tech's, Montreal, Canada).

3. Tests for activity of monocytes/macrophages, signalling molecules, Transcription factors

Mouse LPS-induced TNF-α production model BALB/c mice were injected intraperitoneally with LPS (200 μg/mouse).

Compound was administered intraperitoneally 1 hr before the LPS injection. Blood was collected at 90 min post-LPS injection and plasma was obtained. TNF-α concentration in the sample was determined using an ELISA kit.

4. Tests eosinophil activation

Eotaxin-eotaxin receptor (GPCR)

Signalling molecules, Cytoskeletal molecules, adhision molecules

Mouse eotaxin-induced eosinophilia model BALB/c mice were injected intradermally with a 2.5 ml of air on days -6 and

-3 to prepare airpouch. On day 0 compound was administered intraperitoneally 60 min before eotaxin injection (3 μg/mouse, i.d.). EL-5 (300 ng/mouse) was injected intravenously 30 min before the eotaxin injection. After 4 hr of the eotaxin injection leukocytes in exudate was collected and the number of total cells was counted. The differential cell counts in the exudate were performed by staining with May-Grunwald Gimsa solution. 5. Tests activation of Th2 cells

Molecules involved in antigen presentation, costimulatory molecules, signaling molecules, transcription factors

Mouse D10 cell transfer model

D10.G4.1 cells (1 x 107 cells/mouse) containing 2 mg of conalbumin in saline was administered i.v. to AKR mice. After 6 hr blood was collected and seram was obtained by centifugation at 3000 r.p.m. for lOmin. EL-4 and EL-5 level in seram were determined by ELISA kits. Compound was admimintered intraperitoneally at -4 and +1 hr after these cells injection.

6. Passive cutaneous anaphylaxis (PCA) test in rats

6 Weeks old male Wistar rats are sensitized intradermally (i.d.) on their shaved backs with 50 μl of 0.1 μg/ml mouse anti-DNP IgE monoclonal antibody (SPE-7) under a light anesthesia. After 24 hours, the rats are challenged intravenously with 1 ml of saline containing 0.6 mg DNP-BSA (30) (LSL CO., LTD) and 0.005 g of Evans blue. Compounds are injected intraperitoneally (i.p.) 0.5 hr prior to antigen injection. Rats without the sensitization, challenge, and compound treatment are used for a blank (control) and rats with sensitization, challenge and vehicle treatment are used to determine a value without inhibition. Thirty min after the challenge, the rats are killed, and the skin of the back is removed. Evans blue dye in the skin is extracted in formamide overnight at 63°C. Then an absorbance at 620 nm is measured to obtain the optical density of the leaked dye. Percent inhibition of PCA with a compound is calculated as follows: % inhibition = {(mean vehicle value - sample value)/(mean vehicle value — mean control value)} x 100 7. Anaphylactic bronchoconstriction in rats

6 Weeks old male Wistar rats are sensitized intravenously (i.v.) with 10 μg mouse anti-DNP IgE, SPE-7, and 1 days later, the rats are challenged intravenously with 0.3 ml of saline containing 1.5 mg DNP-BSA (30) under anesthesia with urethan (1000 mg/kg, i.p.) and gallamine (50 mg/kg, i.v.). The trachea is cannulated for artifical respiration (2 ml / stroke, 70 strokes / min). Pulmonary inflation pressure (PEP) is recorded thruogh a side-arm of cannula connected to pressure transducer. Change in PEP reflects change of both resistance and compliance of the lungs. To evaluate the drags, each drug is given i.v. 5 min before challenge.

REFERENCES

1. Hutloff A, Dittrich AM, Beier KC, Eljaschewitsch B, Kraft R,

Anagnostopoulos I, Kroczek RA. ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28. Nature. 1999 Jan 21;397(6716):263-6.

2. Yoshinaga SK, Whoriskey JS, Khare SD, Sarmiento U, Guo J, Horan T, Shih

G, Zhang M, Coccia MA, Kohno T, Tafuri-Bladt A, Brankow D, Campbell P, Chang D, Chiu L, Dai T, Duncan G, Elliott GS, Hui A, McCabe SM, Scully S, Shahinian A, Shaklee CL, Van G, Mak TW, Senaldi, G. T-cell co- stimulation through B7RP-1 and ICOS. Nature. 1999 Dec 16;402(6763):827- 32.

3. Coyle AJ, Lehar S, Lloyd C, Tian J, Delaney T, Manning S, Nguyen T,

Burwell T, Schneider H, Gonzalo JA, Gosselin M, Owen LR, Rudd CE,

Gutieπez-Ramos JC. The CD28-related molecule ICOS is required for effective T cell-dependent immune responses. Immunity. 2000 Jul;13(l):95-

105. 4. McAdam AJ, Chang TT, Lumelsky AE, Greenfield EA, Boussiotis VA, Duke-Cohan JS, Chernova T, Malenkovich N, Jabs C, Kuchroo VK, Ling V, Collins M, Sharpe AH, Freeman GJ. Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4(+) T cells. J Immunol. 2000 Nov l;165(9):5035-40.

5. Mages HW, Hutloff A, Heuck C, Buchner K, Himmelbauer H, Oliveri F, Kroczek RA. Molecular cloning and characterization of murine ICOS and identification of B7h as ICOS ligand. Eur J Lmmunol. 2000 Apr; 30(4) :l 040-7.

6. Tripp CS, Wolf SF, Unanue ER. Interleukin 12 and tumor necrosis factor alpha are costimulators of interferon gamma production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist. Proc Natl Acad Sci U S A. 1993 Apr

15;90(8):3725-9.

7. Higuchi, R., Dollinger, G., Walsh, P.S. and Griffith, R. (1992) Simultaneous amplification and detection of specific DNA sequences. BioTechnology 10:413-417.

8. Higuchi, R., Fockler, C, Dollinger, G. and Watson, R. (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. BioTechnology 11:1026-1030.

T.B. Morrison, J.J. Weis & C.T. Wittwer .(1998) Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques 24:954-962. 10. Adams, M. D., Keriavage, A. R., Fields, C. & Venter, C. (1993) 3,400 new expressed sequence tags identify diversity of transcripts in human brain. Nature Genet. 4:256-265.

11. Adams, M. D., et al. (1995) Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence. Nature 377 supp:3-174.

12. Liew, C. C, Hwang, D. M., Fung, Y. W., Laurenson, C, Cukerman, E., Tsui, S. & Lee, C. Y. (1994) A catalog of genes in the cardiovascular system as identified by expressed sequence tags. Proc. Natl. Acad. Sci. USA 91:10145— 10649f

Claims

1. An isolated polynucleotide being selected from the group consisting of:

a polynucleotide encoding a ICOS V polypeptide comprising an amino acid sequence selected form the group consisting of:

amino acid sequences which are at least about 90% identical to the amino acid sequence shown in SEQ ID NO: 4; and the amino acid sequence shown in SEQ ID NO: 4.

a) a polynucleotide comprising the sequence of SEQ ED NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3;

b) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b) and encodes a ICOS V polypeptide;

c) a polynucleotide the sequence of which deviates from the poly- nucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code and encodes a ICOS V polypeptide; and

d) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d) and encodes a ICOS V polypeptide.

2. An expression vector containing any polynucleotide of claim 1.

3. A host cell containing the expression vector of claim 2.

4. A substantially purified ICOS V polypeptide encoded by a polynucleotide of claim 1.

5. A method for producing a ICOS V polypeptide, wherein the method comprises the steps of:

a) culturing the host cell of claim 3 under conditions suitable for the expression of the ICOS V polypeptide; and

b) recovering the ICOS V polypeptide from the host cell culture.

6. A method for the detection of polynucleotides encoding a ICOS V polypeptide in a biological sample comprising the steps of:

a) hybrizing a polynucleotide of claim 1 to nucleic acid material of a biological sample, thereby forming a hybridization complex; and

b) detecting said hybridization complex; wherein the presence of said complex conelates with the presence of a polynucleotide encoding the ICOS V in said biological sample.

7. The method of claim 6, wherein before hybridization, the nucleic acid material of the biological sample is amplified.

8. A method for the detection of a polynucleotide of claim 1 or a ICOS V polypeptide of claim 4 comprising the steps of contacting a biological sample with a reagent which specifically interacts with the polynucleotide of the

ICOS V polypeptide.

9. A diagnostic kit for conducting the method of any one of claim 6 to 8.

10. A method of screening for agents which decrease the activity of ICOS V, comprising the steps of: contacting a test compound with any ICOS V polypeptide encoded by any polynucleotide of claim 1; and

detecting binding of the test compound to the ICOS V polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential therapeutic agent for decreasing the activity of ICOS V.

11. A method of screening for agents which regulate the activity of ICOS V, comprising the steps of:

contacting a test compound with any ICOS V polypeptide encoded by any polynucleotide of claim 1; and

detecting ICOS V activity of the polypeptide, wherein a test compound which increases the ICOS V activity is identified as a potential therapeutic agent for increasing the activity of ICOS V, and wherein a test compound which decreases the ICOS V activity of the polypeptide is identified as a potential therapeutic agent for decreasing the activity of ICOS V.

12. A method of screening for agents which decrease the activity of ICOS V, comprising the steps of:

contacting a test compound with any ICOS V polynucleotide of claim 1; and

detecting binding of the test compound to the polynucleotide, wherein a test compound which binds to the polynucleotide is identified as a potential therapeutic agent for decreasing the activity of ICOS V.

13. A method of reducing the activity of ICOS V, comprising the steps of:

contacting a cell with a reagent which specifically binds to any polynucleotide of claim 1 ; or any polypeptide of claim 4, whereby the activity of ICOS V is reduced.

14. A reagent that modulates the activity of ICOS V polypeptide or a poly- nucleotide wherein said reagent is identified by the method of any of the claim 10 to 12.

15. A phaπnaceutical composition, comprising the reagent of claim 14 and a phaπnaceutically acceptable carrier.

16. Use of the reagent of claim 14 in the preparation of a medicament for modulating the activity of ICOS V in a disease.

17. Use of claim 16 wherein the disease is an infectious disease, asthma, an allergic or inflammatory disease.

18. A method of screening for agents which can regulate the activity of ICOS V protein, comprising the steps of:

contacting a test compound with a polypeptide comprising an amino acid sequence which is at least about 90% identical to the amino acid sequence shown in SEQ ID NO: 4; or the sequence shown in SEQ ID NO: 4;

and detecting binding of the test compound to the polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential agent for regulating activity of the ICOS V protein.

19. A method of claim 18 wherein the step of contacting is in a cell.

20. The method of claim 18 wherein the cell is in vitro.

21. The method of claim 18 wherein the step of contacting is in a cell-free system.

22. The method of claim 18 wherein the polypeptide comprises a detectable label.

23. The method of claim 18 wherein the test compound comprises a detectable label.

24. The method of claim 18 wherein the test compound displaces a labeled ligand which is bound to the polypeptide.

25. The method of claim 18 wherein the polypeptide is bound to a solid support.

26. The method of claim 18 wherein the test compound is bound to a solid support.

27. A method of screening for agents which regulate the activity of ICOS V protein, comprising the steps of:

contacting a test compound with a polypeptide comprising an amino acid sequence which is at least about 90%) identical to the amino acid sequence shown in SEQ ID NO: 4; or the sequence shown in SEQ ED NO: 4; and

detecting an activity of the polypeptide, wherein a test compound which increases the activity of the polypeptide is identified as a potential agent for increasing the activity of the human ICOS V protein, and wherein a test compound which decreases the activity of the polypeptide is identified as a potential agent for decreasing the activity of the human ICOS V protein.

28. The method of claim 27 wherein the step of contacting is in a cell.

29. The method of claim 27 wherein the cell is in vitro.

30. The method of claim 27 wherein the step of contacting is in a cell-free system.

31. A method of screening for agents which regulate ICOS V protein, comprising the steps of:

contacting a test compound with a product encoded by a polynucleotide which comprises the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; and

detecting binding of the test compound to the product, wherein a test compound which binds to the product is identified as a potential agent for regulating the activity of human ICOS V protein.

32. The method of claim 31 wherein the product is a polypeptide.

33. The method of claim 31 wherein the product is RNA.

34. A method of reducing activity of a human ICOS V protein, comprising the step of:

contacting a cell with a reagent which specifically binds to a product encoded by a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, whereby the activity of a human

ICOS V protein is reduced.

35. The method of claim 34 wherein the product is a polypeptide.

36. The method of claim 35 wherein the reagent is an antibody.

37. The method of claim 35 wherein the product is RNA.

38. The method of claim 34 wherein the reagent is an antisense oligonucleotide.

39. The method of claim 34 wherein the reagent is a ribozyme.

40. The method of claim 34 wherein the cell is in vitro.

41. The method of claim 34 wherein the cell is in vivo.

42. A pharmaceutical composition, comprising:

a reagent which specifically binds to a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4; and

a pharmaceutically acceptable carrier.

43. The pharmaceutical composition of claim 42 wherein the reagent is an antibody.

44. A pharmaceutical composition, comprising:

a reagent which specifically binds to a product of a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1, SEQ TD NO: 2, or SEQ ID NO: 3; and

a pharmaceutically acceptable carrier.

45. The pharmaceutical composition of claim 44 wherein the reagent is a ribozyme.

46. The pharmaceutical composition of claim 44 wherein the reagent is an antisense oligonucleotide.

47. The pharmaceutical composition of claim 44 wherein the reagent is an antibody.

48. A method of treating ICOS V dysfunction related disease, wherein the disease is selected from an infectious disease, asthma, or an allergic or inflammatory disease comprising the step of:

administering to a patient in need thereof a therapeutically effective dose of a reagent that regulates the function of human ICOS V protein, whereby symptoms of the ICOS V dysfunction related disease are ameliorated.

49. The method of claim 48 wherein the reagent is identified by the method of claim 18.

50 The method of claim 48 wherein the reagent is identified by the method of claim 27.

51. The method of claim 48 wherein the reagent is identified by the method of claim 31.