EP1373314A2

EP1373314A2 - Human arginine-rich protein-related compositions

Info

Publication number: EP1373314A2
Application number: EP02722272A
Authority: EP
Inventors: Lydie Bougueleret; Anne Niknejad; Amos Bairoch
Original assignee: Geneprot Inc
Current assignee: Geneprot Inc
Priority date: 2001-03-30
Filing date: 2002-03-26
Publication date: 2004-01-02
Also published as: WO2002079246A3; CA2441327A1; WO2002079246A2

Abstract

The invention provides arginine-rich protein-related compositions including polypeptides, polynucleotides, and antibodies. Such compounds are useful for detecting and/or monitoring conditions involving aberrant or uncontrolled growth of tissues, such as cancer.

Description

HUMAN ARGININE-RICH PROTEIN-RELATED COMPOSITIONS

Field of the Invention The invention relates generally to secreted low molecular weight human proteins, and more particularly, to polypeptides and other compositions related to human arginine-rich protein (ARP), nucleic acids encoding such polypeptides, and uses thereof. Nucleic acids encoding two forms of the ARP-related proteins, including an isoform found to be expressed in a tissue-specific manner in skeletal muscle.

BACKGROUND

Many low molecular weight secreted proteins have profound effects both in health and disease, either by growth stimulating roles, growth inhibitory roles, or the regulation of critical metabolic pathways. Such molecules include growth factors, cytokines, peptide hormones, and like compounds. Growth factors are proteins that bind to receptors on cell surfaces, with the primary result of activating cellular proliferation or differentiation. Many growth factors are pleiotropic, stimulating cell division or other effects in numerous different cell types; while others are specific to a particular cell type or tissue. Cytokines are a family of growth factors that are secreted primarily by leukocytes and play key roles in the humoral and cellular immune responses. Many growth factors or products derived from them have become important medicines, such as erythropoietin (EPO), interferon- (ocINF), and granulocyte macrophage colony stimulating factor (GM-CSF); while others, e.g. insulin-like growth factor- 1 (1GF- 1), tumor growth factor-α (TGF-oc), and others, have been associated with a variety of diseases, particularly cancer, e.g. Jameson, pp. 73-82, in Jameson, ed., Principles of Molecular Medicine (Humana Press, Totowa, NJ, 1998). Recently, a gene that apparently encodes an arginine-rich protein (ARP) has been discovered which, in mutated form, is associated with several different cancers, Shridhar et al, Oncogene, 12: 1931- 1939 (1996); Shridhar et al, Cancer Research, 56: 5576-5578 (1996); Shridhar et al, Oncogene, 14: 2213-2216 (1997), including renal cell carcinomas, and lung, breast, prostate, and pancreatic cancers. The mutations associated with the cancers all appear to be centered in an imperfect trinucleotide repeat that putatively encodes a stretch of 15 to 18 arginines. The widespread association of the mutations with cancer suggests that the putative protein may be a growth factor that plays an important role in the regulation of cell growth and development.

The availability of isolated or purified ARP and related compounds having putative growth factor activity could satisfy a need in the art by providing new therapeutic strategies for treating cancers. Further discovery and development of such compounds would have a beneficial affect on medical services and healthcare. SUMMARY OF THE INVENTION The present invention is directed to compositions related to human arginine-rich protein (ARP- related, or " ARPR" compositions), ARPR peptides, ARPR antibodies, and methods of making and using these compositions. It includes nucleic acids coding for ARPR polypeptides, antisense oligonucleotides complementary to ARPR gene sequences, oligonucleotides complementary to ARPR gene sequences useful in diagnostic and analytical assays, such as primers for polymerase chain reactions (PCRs), and vectors for expressing ARPR polypeptides. The invention further includes methods of using ARPR compositions, including antisense and antibody compounds, to treat disorders associated aberrant expression of ARPR in an individual, and methods of using ARPR compositions, including primers complementary to ARPR genes and/or messenger RNA and anti-ARPR antibodies, for detecting and measuring quantities of ARPR in tissues and biological fluids.

The inventors have identified two isoforms of the human chromosome 10 ARPR protein. One isoform includes coding sequence from exons 1, 2 and 3 of the ARPR gene, resulting in a 187-amino acid residue protein containing a 24 amino acid residue signal seqeunce. The second chlO-ARPR isoform contains the coding sequence from exons 1 and 3 but lacks exon 2 of the chlO-ARPR gene, resulting in a 54 amino acid residue protein (containing a 26 residue signal sequence). mRNA encoding the latter ch 10- ARPR isoform has been found expressed specifically in muscle tissue. In one aspect, the invention includes an isolated, purified or recombinant mature polypeptide having a sequence selected from SEQ ID NO: 1, 13 and 27. Preferably, such mature polypeptide has a sequence selected from the group consisting of SEQ ID NO: 2, 14 and 28, and most preferably, such mature polypeptide has the sequence listed in SEQ ID NO: 2 and 28. The mature polypeptide having the amino acid sequence of SEQ ID NO: 2 and 27 shall be referred to herein occasionally as "chlO-ARPR" for " chromosome 10 ARPR," and the mature polypeptide having the amino acid sequence of SEQ ID

NO: 14 is referred to herein occasionally as "ch3-ARPR" for "chromosome 3 ARPR." Polypeptides of the present invention also include polypeptides having a sequence which is at least 90 percent identical to SEQ ID NO: 1 , 2, 27 or 28. Preferably, the invention includes polypeptides having at least 95 percent, and more preferably at least 98 percent, and still more preferably at least 99 percent, identity with any one of the sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO: 13, and SEQ ID NO: 14. Most preferably, the invention includes polypeptides having a sequence at least 99 percent identical to SEQ ID NO: 2 or SEQ ID NO 28.

In another aspect, the invention includes an isolated, purified or recombinant peptide fragment of ch 10- ARPR having a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. Isolated peptide fragments of the present invention also include peptides having a sequence that is at least 90 percent identical to any one of the peptides having a sequence selected from SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. More preferably, the invention includes isolated peptides having at least 95 percent, and still more preferably at least 98 percent, and most preferably at least 99 percent, identity with any one of the sequences selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

In another aspect, the invention includes an isolated, purified or recombinant peptide fragment of ch3-ARPR having a sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21. Isolated peptide fragments of the present invention also include peptides having a sequence that is at least 90 percent identical to any one of the peptides of this paragraph. More preferably, the invention includes isolated peptides having at least 95 percent, and still more preferably at least 98 percent, identity with any one of the sequences selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

In another aspect, the invention includes a composition comprising a plurality of different peptide fragments of ch 10- ARPR each having a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. More preferably, such composition comprises a pair of different peptide fragments each having a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. Still more preferably, such composition consists of a pair of peptide fragments having amino acids sequences described by SEQ ID NO: 3 and SEQ ID NO: 5, respectively.

In another aspect, the invention includes an isolated, purified or recombinant peptide consisting of 6 to 40 amino acids whose sequence is identical to a subsequence of consecutive amino acids in a mature ch 10- ARPR polypeptide having the sequence of SEQ ID NOS 1 or 27. More preferably, the invention includes an isolated peptide consisting of 6 to 40 amino acids whose sequence is identical to a subsequence of consecutive amino acids in the mature chlO-ARPR of SEQ ID NOS: 2 or 28. Such peptides are useful intermediates in the production of antigenic compositions used in the production of peptide antibodies specific for chlO-ARPR.

In another aspect, the invention includes isolated, purified or recombinant antibodies specific for any of the polypeptides, peptide fragments, or peptides described above. Preferably, the antibodies of the invention are monoclonal antibodies. Such antibodies have diagnostic and therapeutic applications, particularly in treating ARPR-related disorders. Treatment methods include, but are not limited to, those that employ antibodies or antibody-derived compositions specific for an ARPR antigen. Diagnostic methods for detecting an ARPR in specific tissue samples, and for detecting levels of expression of an ARPR in tissues, also form part of the invention. Preferably, the invention includes a monoclonal antibody specific for a polypeptide or peptide having a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO 21, SEQ ID NO 27 and SEQ ID NO: 28. More preferably, the invention includes a monoclonal antibody specific for ch 10- ARPR or any peptide fragment thereof having a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. In one aspect, an antibody composition specifically binds an ARPR isoform of SEQ ID NOS 27 or 28 but does not substantially bind an ARPR isoform of SEQ ID NOS 1 or 2. Also encompassed is a method of binding an ARPR protein comprising: a) providing an antibody composition according to the invention and b) bringing said antibody composition into contact with a composition comprising an ARPR protein. In one aspect, said composition comprising an ARPR protein is a biological sample obtained from an individual. In another aspect, the monoclonal antibody is admnistered to a mammal with a pharmaceutically acceptable carrier.

In another aspect, the invention includes an isolated, purified or recombinant polynucleotide having a sequence at least 90 percent indentical to a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO 29 and any sequence of 100 to 1000 consecutive nucleotides of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8 or SEQ ID NO: 29, or any complementary sequence of the foregoing. More preferably, the invention includes an isolated polynucleotide having at least 95 percent, and still more preferably at least 98 percent, and most preferably at least 99 percent, identity with any one of the sequences selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO 29, and any sequence of 100 to 1000 consecutive nucleotides of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8 or SEQ ID-NO: 29, or any complementary sequence of the foregoing.

In another aspect, the invention includes an isolated, purified or recombinant polynucleotide that forms a detectable duplex under stringent hybridization conditions with a polynucleotide having a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,. SEQ ID NO: 12, SEQ ID NO 29, and any sequence of 100 to 1000 consecutive nucleotides of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO 29, or any complementary sequence of the foregoing. and a complementary sequence thereof.

In another aspect, the invention includes an isolated, purified or recombinant polynucleotide that encodes a mature polypeptide of ch 10- ARPR or a peptide fragment having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. More preferably, the invention includes an isolated polynucleotide that encodes a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

Also encompassed are isolated polynucleotide having a nucleotide sequence that is at least 95 percent identical to SEQ ID NOS: 8 or 29, or to nucleotides 600 to 1700 or 10,100 to 11,200 or 12,800 to 13,900 of SEQ ID NO: 6, or to nucleotides 2600 to 3800 of SEQ ID NO: 7. In further aspects the invention embodies an isolated polynucleotide having a nucleotide sequence that is at least 95 percent identical to a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 29, any complementary sequences thereof, and any sequence of 100 to 1000 consecutive nucleotides of SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, SEQ ID NO 29 and any complementary sequences thereof. Also encompassed are isolated polynucleotide having a length of at least 15 nucleotides, the polynucleotide forming a detectable duplex with a sense or antisense strand of a second polynucleotide under the following hybridization conditions: for 1-15 ng/μL of isolated polynucleotide probe hybridizing to said second nucleic acid bound to a nitrocellulose filter, hybridization in 25 mM KP0₄ (pH 7.4), 5X SSC, 5X Denhart's solution, 50 mg/mL denatured sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate at 42°C, with washes at 65°C in 2X SSC and 0.1% SDS for 30 minutes, and wherein said second polynucleotide having a sequence as set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 29, or any sequence of 100 to 1000 consecutive nucleotides of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO 29.

Also encompassed are any of the nucleic acids comprising a nucleic acid of the invention operably linked to a promoter. Further included is an expression cassette comprising said nucleic acids, as well as host cells comprising said expression cassette. Also encompassed is a method of making an ARPR polypeptide, said method comprising: 1) providing a population of host cells comprising a recombinant nucleic acid encoding an ARPR protein of the invention; and 2) culturing said population of host cells under conditions conducive to the expression of said recombinant nucleic acid, whereby said polypeptide is produced within said population of host cells. The resulting polypeptide is preferably purifyied from the population of cells.

The invention further includes a method of determining whether an ARPR polypeptide is expressed within a biological sample, said method comprising the steps of : (a) contacting a biological sample with (i) a polynucleotide that hybridizes under stringent conditions to a nucleic acid of an ARPR nucleic acid of the invention or (ii) a detectable polypeptide that selectively binds to an ARPR polypeptide of the invention; and (b) detecting the presence or absence of hybridization between said polynucleotide and an RNA species within said sample, or the presence or absence of binding of said detectable polypeptide to a polypeptide within said sample; wherein a detection of said hybridization or of said binding indicates that said ARPR-family polypeptide is expressed within said sample.

In another aspect, the invention provides method for detecting a polypeptide or nucleic acid derived from a muscle cell, or a method for typing a cell. In one example, the invention provides method of determining whether a biological sample contains a polypeptide of polynucleotide originating from a muscle cell, said method comprising the steps of : (a) contacting a biological sample with (i) a polynucleotide that hybridizes to a nucleic acid of SEQ ID NO: 29 but that does not hybridize to a nucleic acid of Claim SEQ ID NO 8 under stringent conditions; or (ii) a detectable polypeptide that selectively binds to a polypeptide of SEQ ID NOS 27 or 28 and does not substantially bind to a polypeptide of SEQ ID NOS 1 or 2; and (b) detecting the presence or absence of hybridization between said polynucleotide and an RNA species within said sample, or the presence or absence of binding of said detectable polypeptide to a polypeptide within said sample; wherein a detection of said hybridization or of said binding indicates that said sample contains a polypeptide or polynucleotide originating from a muscle cell.Preferably hybridization is detected by detecting the presence of an amplification product comprising said primer sequence. A preferred detectable polypeptide is an antibody.

In another aspect, the invention provides polypeptides capable of directing the secretion of a polypeptide. Encompassed is an isolated or purified nucleic acid encoding a chlO-ARPR signal or fragment thereof comprising an amino acid sequence of at least 8 contiguous amino acids of amino acid residues 1 to 24 of SEQ ID NO 1 or 1 to 26 of SEQ ID NO 27. Also encompassed is a nucleic acid comprising said nucleic acid operably linked to a nucleic acid encoding a protein of interest. Said protein of interest is a preferably an ARPR polypeptide according to the invetion, or may be any suitable heterologous polypeptide of interest. In preferred aspects, said nucleic acid is operably linked to a promoter. Also encompassed is an expression cassette comprising said nucleic acids, as well as a host cell comprising said expression cassette.

In another aspect, the invention includes primer pairs for carrying out a PCR to amplify a segment of a polynucleotide of the invention. Each primer of a pair is an oligonucleoti.de having a length of between 15 and 30 nucleotides such that i) one primer of the pair forms a perfectly matched duplex with one strand of a polynucleotide of the invention and the other primer of the pair form a perfectly match duplex with the complementary strand of the same polynucleotide, and ii) the primers of a pair form such perfectly matched duplexes at sites on the polynucleotide that separated by a distance of between 10 and 2500 nucleotides. Preferably, the annealing temperature of each primer of a pair with its respective complementary sequence is substantially the same. In another aspect, the invention includes natural variants of the chlO-ARPR polypeptide having a frequency in a selected population of at least two percent. More preferably, such natural variant has a frequency in a selected population of at least five percent, and still more preferably, at least ten percent. Most preferably, such natural variant has a frequency in a selected population of at least twenty percent. In another aspect, the invention provides a vector comprising DNA encoding a ch3-ARPR or a chlO-ARPR polypeptide. The invention also includes host cells comprising such a vector. A process for producing a ch3-ARPR or a chlO-ARPR is also provided which comprises culturing the host cells under conditions suitable for expression of such ARPR and its recovery from the cell culture materials.

In still a further aspect, the invention includes pharmaceutical compositions and formulations comprising a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO 21, SEQ ID NO 27 and SEQ ID NO: 28, and a pharmaceutically acceptable carrier compound. Preferably, such pharmaceutical compositions comprise chlO-ARPR or one or more peptide fragments thereof having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. Still more preferably, such pharmaceutical composition comprises ch 10- ARPR of the amino acid sequence described in SEQ ID NO: 2 or SEQ ID NO 28.

Brief Description of the Figures Figure 1 is a listing of a cDNA and deduced amino acid sequence of chlO-ARPR.

Figure 2 is a listing of the amino acid sequence of the preferred mature chlO-ARPR.

Figure 3 is a listing of the amino acid sequence of the preferred mature ch3-ARPR.

Figure 4 shows the genomic structure of the chlO-ARPR gene.

Figure 5 is a graph showing expression levels of chlO-ARPR mRNA in various monkey organs during real-time PCR monitoring.

Brief Description of the Sequence Listing

SEQ ID NO 1 is an amino acid sequence of the human chlO-ARPR protein. SEQ ID NO 2 is an amino acid sequence of a preferred mature form of the human chlO-ARPR protein.

SEQ ID NO 3 is an amino acid sequence of an active peptide form of the human chlO-ARPR protein.

SEQ ID NO 4 is an amino acid sequence of an active peptide form of the human chlO-ARPR protein.

SEQ ID NO 5 is an amino acid sequence of an active peptide form of the human ch 10- ARPR protein.

SEQ ID NO 6 is a genomic DNA sequence of part 1 (exon 1 to 3) of the gene encoding the human chlO-ARPR protein. SEQ ID NO 7 is a genomic DNA sequence of part 2 (exon 4) of the gene encoding the human chlO-

ARPR protein.

SEQ ID NO 8 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO 1. SEQ ID NO 9 is a nucleic acid sequence of Exon 1 of the gene encoding the human ch 10- ARPR protein of SEQ ID NO 1.

SEQ ID NO 10 is a nucleic acid sequence of Exon 2 of the gene encoding the human chlO-ARPR protein of SEQ ID NO 1. SEQ ID NO 11 is a nucleic acid sequence of Exon 3 of the gene encoding the human chlO-ARPR protein of SEQ ID NO 1.

SEQ ID NO 12 is a nucleic acid sequence of Exon 4 of the gene encoding the human ch 10- ARPR protein of SEQ ID NO 1.

SEQ ID NO 13 is an amino acid sequence of the human ch3-ARPR protein. SEQ ID NO 14 is an amino acid sequence of a preferred mature human ch3-ARPR protein.

SEQ ID NO 15 is an amino acid sequence of an active peptide form of the human ch3-ARPR protein.

SEQ ID NO 16 is an amino acid sequence of an active peptide form of the human ch3-ARPR protein. SEQ ID NO 17 is an amino acid sequence of an active peptide form of the human ch3-ARPR protein.

SEQ ID NO 18 is an amino acid sequence of an active peptide form of the human ch3-ARPR protein.

SEQ ID NO 19 is an amino acid sequence of an active peptide form of the human ch3-ARPR protein.

SEQ ID NO 20 is an amino acid sequence of an active peptide form of the human ch3-ARPR protein.

SEQ ID NO 21 is an amino acid sequence of an active peptide form of the human ch3-ARPR protein. SEQ ID NO 22 is a nucleic acid sequence of Exon 1 of the gene encoding the human ch3-ARPR protein of SEQ ID NO 13.

SEQ ID NOS 23 to 26 are amino acid sequences of respective fragments of the human ch 10- ARPR protein for chemical synthesis methods of Example 1.

SEQ ID NO 27 is an amino acid sequence of a chlO-ARPR isoform expressed in muscle. SEQ ID NO 28 is an amino acid sequence of a mature chlO-ARPR protein isoform of SEQ ID NO

27.

SEQ ID NO 29 is a nucleic acid sequence encoding the chlO-ARPR isoform of SEQ ID NOS 27 and

28.

SEQ ID NOS 30 to 35 are oligonucleotide primers used in ARPR expression analysis.

DEFINITIONS

The terms "polypeptide" or "peptide" or "peptide fragment" as used herein refers to a compound made up of a single unbranched chain of amino acid residues linked by peptide bonds. The number of amino acid residues in such compounds varies widely; however, preferably, peptides referred to herein usually have from six to forty amino acid residues. Polypeptides and peptide fragments referred to herein usually have from a few tens of amino acid residues, e.g. 20, to up to a few hundred amino acid residues, e.g. 200, or more. Generally, polypeptides are manufactured more conveniently by recombinant DNA methods. The term "protein" as used herein may be used synonymously with the term "polypeptide" or may refer to, in addition, a complex of two or more polypeptides which may be linked by bonds other than peptide bonds, for example, such polypeptides making up the protein may be linked by disulfide bonds. The term "protein" may also comprehend a family of polypeptides having identical amino acid sequences but different post-translational modifications, such as phosphorylations, acylations, glycosylations, and the like, particularly as may be added when such proteins are expressed in eukaryotic hosts.

Amino acid residues are referred to herein by their standard single-letter or three-letter notations: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, Isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, ryrosine.

As used interchangeably herein, the terms "ARPR", or an "ARPR protein" or "ARPR polypeptide" may be any polypeptide encoded by an ARPR nucleic acid, preferably a nucleic acid derived from a ARPR gene or transcript produced therefrom. The terms "ARPR", "ARPR protein" or "ARPR polypeptide" as used herein also encompass polypeptides and proteins, including mature polypeptides, having an amino acid sequence derived from the polypeptides whose sequences are selected from the group consisting of SEQ ID NOS: 1 to 5, 27 and 28 and SEQ ID NOS: 13 to 21. The term "ch3-ARPR" encompasses the polypeptides or proteins having the amino acid sequence of SEQ ID NOS: 13 to 21, including natural variants thereof, whether prepared by recombinant or synthetic methods. The term "chlO-ARPR" encompasses the polypeptides or proteins having the amino acid sequence of SEQ ID NOS: 1 to 5, 27 and 28, including natural variants thereof, whether prepared by purification, or by recombinant or synthetic methods.

"Perfectly matched" in reference to a duplex means that the poly- or oligonucleotide strands making up the duplex form a double stranded structure with one other such that every nucleotide in each strand undergoes Watson-Crick basepairing with a nucleotide in the other strand. The term also comprehends the pairing of nucleoside analogs, such as deoxyinosine, nucleosides with 2-aminopurine bases, and the like, that may be employed. In reference to a triplex, the term means that the triplex consists of a perfectly matched duplex and a third strand in which every nucleotide undergoes Hoogsteen or reverse Hoogsteen association with a basepair of the perfectly matched duplex. Conversely, a "mismatch" in a duplex between a tag and an oligonucleotide means that a pair or triplet of nucleotides in the duplex or triplex fails to undergo Watson-Crick and/or Hoogsteen and/or reverse Hoogsteen bonding.

The term "percent identical," or like term, used in respect of the comparison of a reference sequence and another sequence (i.e. a "candidate" sequence, means that in an optimal alignment between the two sequences, the candidate sequence is identical to the reference sequence in a number of subunit positions equivalent to the indicated percentage, the subunits being nucleotides for polynucleotide comparisons or amino acids for polypeptide comparisons. As used herein, an "optimal alignment" of sequences being compared is one that maximizes matches between subunits and minimizes the number of gaps employed in constructing an alignment. Percent identities may be determined with commercially available implementations of algorithms described by Needleman and Wunsch, J. Mol. Biol., 48: 443-453 (1970)("GAP" program of Wisconsin Sequence Analysis Package, Genetics Computer Group, Madison, WI). Other software packages in the art for constructing alignments and calculating percentage identity or other measures of similarity include the "BestFit" program, based on the algorithm of Smith and Waterman, Advances in Applied Mathematics, 2: 482-489 (1981) (Wisconsin Sequence Analysis Package, Genetics Computer Group, Madison, WI). In other words, for example, to obtain a polypeptide having an amino acid sequence at least 95 percent identical to a reference amino acid sequence, up to five percent of the amino acid residues in the reference sequence many be deleted or substituted with another amino acid, or a number of amino acids up to five percent of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence many occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence of in one or more contiguous groups with in the references sequence. It is understood that in making comparisons with reference sequences of the invention that candidate sequence may be a component or segment of a larger polypeptide or polynucleotide and that such comparisons for the purpose computing percentage identity is to be carried out with respect to the relevant component or segment.

The term " isolated" in reference to a polypeptide or polynucleotide of the invention means substantially separated from the components of its natural environment. Preferably, an isolated polypeptide or polynucleotide is a composition that consists of at least eighty percent of the polypeptide or polynucleotide identified by sequence on a weight basis as compared to components of its natural environment; more preferably, such composition consists of at least ninety-five percent of the polypeptide or polynucleotide identified by sequence on a weight basis as compared to components of its natural environment; and still more preferably, such composition consists of at least ninety-nine percent of the polypeptide or polynucleotide identified by sequence on a weight basis as compared to components of its natural environment. Most preferably, an isolated polypeptide or polynucleotide is a homogeneous composition that can be resolved as a single spot after conventional separation by two- dimensional gel electrophoresis based on molecular weight and isoelectric point. Protocols for such analysis by conventional two-dimensional gel electrophoresis are well known to one of ordinary skill in the art, e.g. Hames and Rickwood, Editors, Gel Electrophoresis of Proteins: A Practical Approach (IRL Press, Oxford, 1981); Scopes, Protein Purification (Springer- Verlag, New York, 1982); Rabilloud,

Editor, Proteome Research: Two-Dimensional Gel Electrophoresis and Identification Methods (Springer- Verlag, Berlin, 2000).

The term "oligonucleotide" as used herein means linear oligomers of natural or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, anomeric forms thereof, peptide nucleic acids (PNAs), and die like, capable of specifically binding to a polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like. Usually, monomers are linked by phosphodiester bonds, or analogs thereof, to form oligonucleotides ranging in size from a few monomeric units, e.g. 3-4, to several tens of monomeric units, e.g. 40-60. Whenever an oligonucleotide or polynucleotide is represented by a sequence of letters, such as "ATGCCTG, " or the lower case equivalent, it will be understood that the nucleotides are in 5'-»3' order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, "T" denotes thymidine, and "U" denotes uridine, unless otherwise noted or understood for their context. Usually oligonucleotides of the invention comprise the four natural nucleotides, and they are joined to one another by natural phosphodiester linkages; however, they may also comprise non-natural nucleotide analogs and may also contain non-natural inter-nucleosidic linkages, particularly when employed as antisense or diagnostic compositions. It is clear to those skilled in the art when oligonucleotides having natural or non-natural nucleotides may be employed in accordance with the invention, e.g. where processing by enzymes is called for, usually oligonucleotides consisting of natural nucleotides are required.

As used herein, "nucleoside" includes the natural nucleosides, including 2'-deoxy and 2'- hydroxyl forms, e.g. as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). "Analogs" in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g. described by Scheit, Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990), or the like, with the only proviso that they are capable of specific hybridization. Such analogs include synthetic nucleosides designed to enhance binding properties, reduce complexity, increase specificity, and the like.

As used herein, " detectable duplex" in reference to a hybridization assay means that for any type of signal generating means used the signal-to-noise ratio is at least two. Preferably, such signal- to-noise ratio is at least three, and more preferably, such signal-to-noise ratio is at least five.

DETAILED DESCRIPTION OF THE INVENTION The present invention encompasses ARPR polypeptides and related compositions of matter including, but not limited to, polynucleotides encoding ARPR polypeptides or fragments thereof, antibodies specific for ARPR polypeptides or fragments thereof, antisense compounds having nucleotide sequences complementary to the polynucleotides of the invention, oligonucleotides having sequences complementary to ARPR genes and gene transcripts that are useful in diagnostic and analytical methods, recombinant DNA constructs and vectors comprising polynucleotides of the invention as well as host cells containing such constructs or vectors used for replicating ARPR transcripts or for expressing ARPR polypeptides or proteins. The invention also encompasses pharmaceutical compositions comprising ARPR polypeptides, proteins, or peptide fragments, and agonists and antagonists thereof, particularly antagonists derived from monoclonal antibodies specific for ARPR compositions.

ARPR polypeptides and peptide fragments of the invention include natural and man-made variants whose amino acid sequences differ from the reference amino acid sequences of the Sequence Listing by one or more substitutions, insertions, or deletions. Such variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the ARPR polypeptide or peptide fragment, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture, as described more fully below. Variant ARPR polypeptides and peptide fragment may also be synthesize chemically using conventional peptide synthesis techniques or convergent synthesis techniques as described below.

The terms "ARPR protein" and "ARPR polypeptides" are used herein to embrace all of the proteins and polypeptides of the present invention. Also forming part of the invention are polypeptides encoded by the polynucleotides of the invention, as well as fusion polypeptides comprising such polypeptides. The invention embodies polypeptides encoded by the nucleic acid sequences of the ARPR gene, particularly by nucleic acid sequences of SEQ ID NOS 6 to 12, as well as the ARPR proteins from humans, including isolated or purified ARPR proteins consisting of, consisting essentially of, or comprising the sequence of SEQ ID NOS 1 to 5, 13 to 21, 27 and 28.

The invention concerns the polypeptide encoded by a nucleotide sequence of SEQ ID NOS 6 to 12, 22 and 29, a complementary sequence thereof or a fragment thereto.

The present invention embodies isolated, purified, and recombinant polypeptides comprising, consisting of or consisting essentially of a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID NOS 1 to 5, 27, 28 or 13 to 21, to the extent that said contiguous span is consistent with the SEQ ID NO. In one aspect, said contiguous span is selected from the group of amino acid positions 1 to 24 of SEQ ID NO 1, 1 to 26 of SEQ ID NO 27, and 1 to 21 of SEQ ID NO 13.

Provided are also polypeptides having amino acid sequences capable of directing secretion of a polypeptide from a cell, comprising the amino acids at positions 1 to 24 of SEQ ID NO 1, 1 to 26 of SEQ ED NO 27, or positions 1 to 21 of SEQ ID NO 13. Also encompassed are thus polypeptides comprising, consisting of or consisting essentially of an amino acid residues 1 to 24 of SEQ ID NO 1, 1 to 26 of SEQ ID NO 27, or positions 1 to 21 of SEQ ED NO 13, or a biologically active fragment thereof. Preferably said polypeptide is operably linked to a heterologous polypeptide which is to be secreted from a cell. Preferably said biologically active fragment is capable of directing the secretion of a polypeptide from a cell. Also embodied are isolated, purified, and recombinant polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of a polypeptide sequence encoded by a nucleic acid sequence of SEQ ID NOS 6 to 12, 22 or 29. In other preferred embodiments the contiguous stretch of amino acids comprises the site of a mutation or functional mutation, including a deletion, addition, swap or truncation of the amino acids in the ARPR protein sequence. The invention also concerns the polypeptide encoded by the ARPR nucleotide sequences of the invention, or a complementary sequence thereof or a fragment thereof.

A biologically active ARPR protein may, for example, comprise at least 1, 2, 3, 5, 10, 20 or 30 amino acid changes from the sequence of SEQ ID NOS 1 to 5, 27 and 28 or 13 to 2 lor fragment thereof, or may encode a biologically active ARPR protein comprising at least 1 %, 2%, 3%, 5%, 8%, 10 % or 15% changes in amino acids from the sequence of SEQ ID NOS 1 to 5, 27 and 28 or 13 to 21.

Natural variants of the polypeptides of the invention are obtained by conventional screening of individuals of a selected population using analysis techniques employing oligonucleotides of the invention. Preferably, genomic regions containing all or a portion of a genomic region is amplified using PCR or like technique, after which the amplified sequence is sequenced using conventional methods, or otherwise analyzed at specific loci using conventional techniques, e.g., Taylor, editor, Laboratory Methods for the Detection of Mutations and Polymorphisms in DNA (CRC Press, 1997); Landegren, editor, Laboratory Protocols for Mutation Detection (Oxford University Press, 1996); Shi, Clinical Chem., 47: 164-172 (2001); Pastinen et al, Genome Res., 10: 1031-1042 (2000); Armstrong et al, Cytometry, 40: 102-108 (2000); Mein et al, Genome Res., 10: 330-343 (2000); Li et al,

Electrophoresis, 20: 1258-1265 (1999); and the like. The sequence is then compared to polynucleotides of the invention to determine whether a variation affecting the encoded protein is present. Preferably, natural variants of the ch 10- ARPR polypeptide having a frequency in a selected population of at least two percent. More preferably, such natural variant has a frequency in a selected population of at least five percent, and still more preferably, at least ten percent. Most preferably, such natural variant has a frequency in a selected population of at least twenty percent. The selected population may be any recognized population of study in the field of population genetics. Preferably, the selected population is Caucasian, Negroid, or Asian. More preferably, the selected population is French, German, English, Spanish, Swiss, Japanese, Chinese, Irish, Korean, Singaporean, Icelandic, North American, Israeli, Arab, Turkish, Greek, Italian, Polish, Pacific Islander, Finnish, Norwegian, Swedish, Estonian, Austrian, or Indian. More preferably, the selected population is Icelandic, Saami, Finnish, French of Caucasian ancestry, Swiss, Singaporean of Chinese ancestry, Korean, Japanese, Quebecian, North American Pima Indians, Pennsylvanian Amish and Amish Mennonite, Newfoundlander, or Polynesian. Preferably, a selected population consists of a sample of at least 50, 100 or 500 individuals. More preferably, a selected population consists of a sample of at least 1000 individuals, and most preferably, a sample of at least 2000 individuals.

The nucleic acid sequence of the human chlO-ARPR gene is shown in SEQ ID NOS 6 and 1, while exons 1 to 4 are shown in SEQ ID NOS 9 to 12 respectively.

Two isoforms of the chlO-ARPR protein have been identified, as further described herein. A first isoform encoded by an mRNA containing exons 1, 2 and 3 of the chlO-ARPR gene is provided in SEQ ID NOS 1 and 2, while a second isoform expressed specifically in muscle containing exons 1 and 3 but not 2 of the chlO-ARPR gene is provided in SEQ ID NOS 27 and 28. A cDNA sequence encoding said first chlO-ARPR protein is shown in SEQ ID NO 8, and a cDNA sequence encoding said second chlO-ARPR protein is shown in SEQ ID 29. One aspect of the invention thus pertains to purified or isolated nucleic acid molecules that encode ARPR proteins or biologically active portions thereof, as well as nucleic acid fragments thereof.

One object of the invention is a purified, isolated, or recombinant nucleic acid comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NOS 6 to 12, 22 or 29, complementary sequences thereto, and fragments thereof. The invention also pertains to a purified or isolated nucleic acid comprising a polynucleotide having at least 95% nucleotide identity with a polynucleotide of SEQ ID NOS 6 to 12, 22 or 29, advantageously 99 % nucleotide identity, preferably 99.5% nucleotide identity and most preferably 99.8% nucleotide identity with a polynucleotide of SEQ ID NOS 6 to 12, 22 or 29, or a sequence complementary thereto or a fragment thereof. Another object of the invention relates to purified, isolated or recombinant nucleic acids comprising a polynucleotide that hybridizes, under the stringent hybridization conditions defined herein, with a polynucleotide of SEQ ID NOS 6 to 12, 22 or 29, or a sequence complementary thereto or a variant thereof or a fragment thereof. In further embodiments, nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500 or 1000 nucleotides, to the extent that said span is consistent with the length of the SEQ ID, of SEQ ID NOS 6 to 12, 22 or 29, or the complements thereof.

Also encompassed is a purified, isolated, or recombinant nucleic acid polynucleotide encoding a ARPR polypeptide of the invention, as further described herein. In another preferred aspect, the invention pertains to purified or isolated nucleic acid molecules that encode a portion or variant of a ARPR protein, wherein the portion or variant displays an a biological activity. In other embodiments, the invention relates to a polynucleotide encoding a ARPR portion consisting of at least 8-20, 20-50, 50-70, 60-100, 100, 120, 130, 140, 150 or 160 amino acids, to the extent that said portion is consistent with the length of the SEQ ID NO, of SEQ ID NOS 1 to 5, 13 to 21, 27 or 28 or a variant thereof, wherein said ARPR portion displays a biological activity.

The invention further encompasses nucleic acid molecules that differ from the ARPR nucleotide sequences of the invention due to degeneracy of the genetic code and encode the same ARPR proteins and fragment of the invention. It will be appreciated that the invention comprises polypeptides having an amino acid sequence encoded by any of the polynucleotides of the invention.

Preferred polynucleotides of the invention have sequences as listed in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO 22, SEQ ID NO 29 and any sequence of 100 to 1000 consecutive nucleotides of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 22 or SEQ ID NO: 29, or any complementary sequence of the foregoing, and further include polynucleotides having sequences that are at least 95 percent, and more preferably at least 98 percent, and most preferably 99 percent identical thereto. The invention also includes polynucleotides that form detectable duplexes under stringent hybridization conditions with polynucleotides having sequences listed in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO 22, SEQ ID NO 29 and any sequence of 100 to 1000 consecutive nucleotides of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8, SEQ ID NO 22 or SEQ ID NO: 29, or any complementary sequence of the foregoing.

The stringency of a hybridization reaction can be controlled using well know techniques, e.g. by varying temperature, salt concentration, concentration of organic solvents, such as formamide, and the like, Britten et al, Meth. in Enzymol., 29: 363-418.1974; Breslauer et al, Proc. Natl. Acad. Sci., 83 : 3746-3750.1986; Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26: 227-259.1991; Keller et alDNA Probes, Second Edition (Stockton Press, New York, 1993). The following exemplary hybridization stringency conditions are preferably used to identify polynucleotides within the scope of the invention: (1) probe DNA (1-15 ng/μL) hybridizes to target DNA at about 65°C in about 5xSSPE and is washed under conditions of about 65°C in about O.lxSSPE (see Sambrook, et al., MOLECULAR

CLONING: A LABORATORY MANUAL, 2d Edition (Cold Spring Harbor Laboratory, New York, 1989), and Ausubel, F. M., et al., Eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, (John Wiley & Sons, Inc., New York, 1990)); and more preferably, (2) for 1-15 ng/μL of isolated nucleic acid probe hybridizing to a target polynucleotide bound to a nitrocellulose filter, hybridization in 25 mM KP0₄ (pH 7.4), 5X SSC, 5X Denhart's solution, 50 mg/mL denatured sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate at 42°C, with washes at 65°C in 2X SSC and 0.1 % SDS. Preferably, the wash times in the above assays are 15 minutes, and more preferably, 30 minutes, and most preferably, 1 hour.

Recombinant Manufacture of ARPR Compositions

The polynucleotide sequences described herein can be used in recombinant DNA molecules that direct the expression of the corresponding polypeptides in appropriate host cells. Because of the degeneracy in the genetic code, other DNA sequences may encode the equivalent amino acid sequence, and may be used to clone and express the ARPR polypeptides. Codons preferred by a particular host cell may be selected and substituted into the naturally occurring nucleotide sequences, to increase the rate and/or efficiency of expression. The nucleic acid (e.g., cDNA or genomic DNA) encoding the desired ARPR polypeptide may be inserted into a replicable vector for cloning (amplification of the DNA), or for expression. The polypeptide can be expressed recombinantly in any of a number of expression systems according to methods known in the art (Ausubel, et al., editors, Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1990). Appropriate host cells include yeast, bacteria, archebacteria, fungi, and insect and animal cells, including mammalian cells, for example primary cells, including stem cells, including, but not limited to bone marrow stem cells. More specifically, these include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors, and yeast transformed with yeast expression vectors. Also included, are insect cells infected with a recombinant insect virus (such as baculovirus), and mammalian expression systems. The nucleic acid sequence to be expressed may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan. The ARPR proteins of the present invention are produced by culturing a host cell transformed with an expression vector containing a nucleic acid encoding a ARPR, under the appropriate conditions to induce or cause expression of the protein. The conditions appropriate for ARPR expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation. For example, the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction. In addition, in some embodiments, the timing of the harvest is important. For example, the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.

A host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the protein include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing, which cleaves a "prepro" form of the protein, may also be important for correct insertion, folding and/or function. By way of example, host cells such as CHO, HeLa, BHK, MDCK, 293, W138, etc. have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein. Of particular interest are Drosophila melangastev cells, Sacchavomyces cevevisiae and other yeasts, E. coli, Bacillus subtilis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells, fibroblasts, Schwanoma cell lines, immortalized mammalian myeloid and lymphoid cell lines, Jukat cells, human cells and other primary cells. The nucleic acid encoding an ARPR must be "operably linked" by placing it into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" DNA sequences are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. Promoter sequences encode either constitutive or inducible promoters. The promoters may be either naturally occurring promoters or hybrid promoters.. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention. The expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification. Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2: plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Further, for integrating expression vectors, the expression vector contains at least one sequence homologous to the host cell genome, and preferably, two homologous sequences which flank the expression construct. The integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.

Preferably, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used. Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, mefhotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available for from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.

Host cells transformed with a nucleotide sequence encoding a ARPR polypeptide may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The protein produced by a recombinant cell may be secreted, membrane-bound, 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 encoding the ARPR can be designed with signal sequences which direct secretion of the ARPR through a prokaryotic or eukaryotic cell membrane. The desired ARPR polypeptide may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the ARPR-encoding DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces a-factor leaders, the latter described in U.S. Pat. No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published Apr. 4, 1990), or the signal described in WO 90113646 published Nov. 15, 1990. In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders. According to the expression system selected, the coding sequence is inserted into an appropriate vector, which in turn may require the presence of certain characteristic "control elements" or "regulatory sequences." Appropriate constructs are known generally in the art (Ausubel, et al., 1990) and, in many cases, are available from commercial suppliers such as Invitrogen (San Diego, Calif.), Stratagene (La Jolla, Calif.), Gibco BRL (Rockville, Md.) or Clontech (Palo Alto, Calif).

Expression in Bacterial Systems. Transformation of bacterial cells may be achieved using an inducible promoter such as the hybrid lacZ promoter of the " BLUESCRIPT" Phagemid (Stratagene) or "pSPORTl" (Gibco BRL). In addition, a number of expression vectors may be selected for use in bacterial cells to produce cleavable fusion proteins that can be easily detected and/or purified, including, but not limited to "BLUESCRIPT" (a-galactosidase; Stratagene) or pGEX (glutathione S-transferase; Promega, Madison, Wis.). A suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of the coding sequence of the ARPR gene into mRNA. A bacterial promoter has a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan. Promoters from bacteriophage may also be used and are known in the art. In addition, synthetic promoters and hybrid promoters are also useful; for example, the tat promoter is a hybrid of the tip and lac promoter sequences. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. An efficient ribosome binding site is also desirable. The expression vector may also include a signal peptide sequence that provides for secretion of the ARPR polypeptide in bacteria. The signal sequence typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell, as is well known in the art. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria). The bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed. Suitable selection genes include drug resistance genes such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways. When large quantities of ARPR polypeptides are needed, e.g., for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be desirable. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the ARPR polypeptide coding sequence may be ligated into the vector in-frame with sequences for the amino-terminal Met and the subsequent 7 residues of beta-galactosidase so that a hybrid protein is produced; PIN vectors [Van Heeke & Schuster JBiol

Chem 264:5503-5509 1989)]; PET vectors (Novagen, Madison Wis.); and the like. Expression vectors for bacteria include the various components set forth above, and are well known in the art. Examples include vectors for Bacillus subtilis, E. coli, Streptococcus cvemovis, and Streptococcus lividans, among others. Bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride mediated transfection, electroporation, and others.

Expression in Yeast. Yeast expression systems are well known in the art, and include expression vectors for Sacchavomyces cevevisiae, Candida albicans and C. maltosa, Hansenula polymovpha, Kluyvevomyces fvagilis and K. lactis, Pichia guillevimondii and Ppastoris, Schizosaccha- vomyces pombe, and Yavvowia lipolytica. Examples of suitable promoters for use in yeast hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman et al., 7. Biol. Chem. 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg. 7:149 (1968); Holland, Biochemistry 17:4900 (1978)], such as enolase, glyceraldehyde-3- phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose- 6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, tri osephosphate isomerase, phosphoglucose isomerase, alpha factor, the ADH2IGAPDH promoter, glucokinase alcohol oxidase, and PGH. [See, for example, Ausubel, et al., 1990; Grant et al., Methods in Enzymology 153:516-544, (1987)]. Other yeast promoters, which are inducible have the additional advantage of transcription controlled by growth conditions, include the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors andpromoters for use in yeast expression are further described in EP 73,657. Yeast selectable markers include ADE2. HIS4. LEU2. TRP1. and ALG7, which confers resistance'to tunicamycin; the neomycin phosphotransferase gene, which confers resistance to G418; and the CUP1 gene, which allows yeast to grow in the presence of copper ions. Yeast expression vectors can be constructed for intracellular production or secretion of a ARPR from the DNA encoding the ARPR of interest. For example, a selected signal peptide and the appropriate constitutive or inducible promoter may be inserted into suitable restriction sites in the selected plasmid for direct intracellular expression of the ARPR polypeptide. For secretion of the ARPR, DNA encoding the ARPR polypeptide can be cloned into the selected plasmid, together with DNA encoding the promoter, the yeast alpha-factor secretory signal/leader sequence, and linker sequences (as needed), for expression of the ARPR polypeptide. Yeast cells, can then be transformed with the expression plasmids described above, and cultured in an appropriate fermentation media. The protein produced by such transformed yeast can then be concentrated by precipitation with 10% trichloroacetic acid and analyzed following separation by SDS-PAGE and staining of the gels with Coomassie Blue stain. The recombinant ARPR can subsequently be isolated and purified from the fermentation medium by techniques known to those of skill in the art.

Expression in Mammalian Systems. The ARPR proteins may be expressed in mammalian cells. Mammalian expression systems are known in the art, and include retroviral vector mediated expression systems. Mammalian host cells may be transformed with any of a number of different viral-based expression systems, such as adenovirus, where the coding region can be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a nonessential El or E3 region of the viral genome results in a viable virus capable of expression of the polypeptide of interest in infected host cells. A preferred expression vector system is a retroviral vector system such as is generally described in PCT/US97/01019 and PCT/US97/101048. Suitable mammalian expression vectors contain a mammalian promoter which is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence for ARPR protein into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, using a located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A mammalian promoter will also contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation. Of particular use as mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211, 504 published Jul. 5,1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems. Transcription of a DNA encoding a ARPR polypeptide by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer is preferably located at a site 5' from the promoter. In general, the transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3' terminus of the mature mRNA is formed by site-specific post- translational cleavage and polyadenylation. Examples of transcription terminator and polyadenylation signals include those derived from SV40. Long term, high-yield production of recombinant proteins can be effected in a stable expression system. Expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene may be used for this purpose. Appropriate vectors containing selectable markers for use in mammalian cells are readily available commercially and are known to persons skilled in the art. Examples of such selectable markers include, but are not limited to herpes simplex virus thymi-dine kinase and adenine phosphoribosyltransferase for use in tk- or hprt- cells, respectively. The methods of introducing exogenous nucleic acid into mammalian hosts, as well as other hosts, is well known in the art, and will vary with the host cell used. Techniques include dextran- mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

Expression in Insect Cells. ARPR polypeptides may also be produced in insect cells. Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art. In one such system, the ARPR-encoding DNA is fused upstream of an epitope tag contained within a baculovirus expression vector. Autographa calif ovnica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptevu fingipevdu Sf9 cells or in

Trichoplusia larvae. The ARPR-encoding sequence is cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of a ARPR-encoding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat. The recombinant viruses are then used to infect S. fingipevdu cells or Trichoplusia larvae in which the ARPR is expressed [Smith et al., J. Wol. 46:584 (1994); Engelhard E K et al., Pvoc. Nat. Acad. Sci. 91:3224-3227 (1994)]. Suitable epitope tags for fusion to the ARPR- encoding DNA include poly-his tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including commercially available plasmids such as pVL1393 (Novagen). Briefly, the ARPR-encoding DNA or the desired portion of the ARPR-encoding DNA is amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer may incorporate flanking restriction sites. The PCR product is then digested with the selected restriction enzymes and subcloned into an expression vector. Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGoldTM virus DNA (Pharmingen) into Spodopteva fvugipevda ("Sf9") cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL), or other methods known to those of skill in the art. Virus is produced by day 4-5 of culture in Sf9 cells at 28°C, and used for further amplifications. Procedures are performed as further described in O'Reilley et al., BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL, Oxford University Press (1994). Extracts may be prepared from recombinant virus-infected Sf9 cells as described in Rupert et al., Nature 362:175-179 (1993). Alternatively, expressed epitope-tagged ARPR polypeptides can be purified by affinity chromatography, or for example, purification of an IgG tagged (or Fc tagged) ARPR polypeptide can be performed using chromatography techniques, including Protein A or protein G column chromatography.

Evaluation of Gene Expression. Gene expression may be evaluated in a sample directly, for example, by standard techniques known to those of skill in the art, e.g., Southern blotting for DNA detection, Northern blotting to determine the transcription of mRNA, dot blotting (DNA or RNA), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be used in assays for detection of nucleic acids, such as specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Such antibodies may be labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected. Gene expression, alternatively, may be measured by immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to directly evaluate the expression of ARPRs. Antibodies useful for such immunological assays may be either monoclonal or polyclonal, and may be prepared against a native sequence ARPR based on the DNA sequences provided herein. Purification of Expressed Protein. Expressed ARPR polypeptides may be purified or isolated after expression, using any of a variety of methods known to those skilled in the art. The appropriate technique will vary depending upon what other components are present in the sample. Contaminant components that are removed by isolation or purification are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other solutes. The purification step(s) selected will depend, for example, on the nature of the production process used and the particular ARPR polypeptide produced. An ARPR polypeptide or protein may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage. Alternatively, cells employed in expression of ARPR polypeptides can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or by use of cell lysing agents. Exemplary purification methods include, but are not limited to, ion-exchange column chromatography; chromatography using silica gel or a cation-exchange resin such as DEAE; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; chromatography using metal chelating columns to bind epitope-tagged forms of the ARPR polypeptide; ethanol precipitation; reverse phase HPLC; chromatofocusing; SDS-PAGE; and ammonium sulfate precipitation. Ordinarily, an isolated ARPR polypeptide will be prepared by at least one purification step. For example, the ARPR protein may be purified using a standard anti-ARPR antibody column. Ultrafiltration and dialysis techniques, in conjunction with protein concentration, are also useful (see, for example, Scopes, R., PROTEIN PURIFICATION, Springer- Verlag, New York, N.Y., 1982). The degree of purification necessary will vary depending on the use of the ARPR. In some instances no purification will be necessary. Once expressed and purified as needed, the ARPR proteins and nucleic acids of the present invention are useful in a number of applications, as detailed below.

Labeling of Expressed Protein. The nucleic acids, proteins and antibodies of the invention may be labeled. By labeled herein is meant that a compound has at least one element, isotope or chemical compound attached to enable the detection of the compound. In general, labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies or antigens; and c) colored or fluorescent dyes. The labels may be incorporated into the compound at any position that does not interfere with the biological activity or characteristic of the compound which is being detected. ARPR Fusion Proteins. The ARPR of the present invention may also be modified in a way to form chimeric molecules comprising a ARPR fused to another, heterologous polypeptide or amino acid sequence. The term " fusion protein" used herein refers to a chimeric polypeptide comprising a ARPR polypeptide, or domain sequence thereof, fused to a "targeting polypeptide" . The targeting polypeptide has enough residues to facilitate targeting to a particular cell type or receptor, yet is short enough such that it does not interfere with the biological function of the ARPR polypeptide. The targeting polypeptide preferably is also fairly unique so that the fusion protein does not substantially cross-react with other cell types or receptors. Suitable targeting polypeptides generally have at least about 10 amino acid residues and usually between from about 10 to about 500 amino acid residues. Preferred targeting polypeptides have from about 20 to about 200 amino acid residues. The fusion protein may also comprises a fusion of a ARPR with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino-or carboxyl-terminus of the ARPR. Such epitope-tagged forms of an ARPR can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the ARPR to be readily purified by using an anti- tag antibody or another type of affinity matrix that binds to the epitope tag. Alternatively, the fusion protein may comprise a fusion of a ARPR with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule, such a fusion could be to the Fc region of an IgG molecule or, for example, GM-CSF. Preferred fusion proteins include, but are not limited to, molecules that facilitate immune taϊgeting of the ARPR. The ARPR fusion protein may be made for various other purposes using techniques well known in the art. For example, for the creation of antibodies, if the desired epitope is small, a partial or complete ARPR protein may be fused to a carrier protein to form an immunogen. Alternatively, the ARPR protein may be made as a fusion protein to increase the ability of the antigen to stimulate cellular and/or humoral (antibody-based) immune responses, or for other reasons. Synthetic Genes for ARPRs. Once nucleic acid sequence and/or amino acid sequence information is available for a native protein a variety of techniques become available for producing virtually any mutation in the native sequence, e.g.Shortle, in Science, Vol. 229, pgs. 1193-1201 (1985); Zoller and Smith, Methods in Enzymology, Vol. 100, pgs. 468-500 (1983); Mark et al., U.S. Patent 4,518,584; Wells et al., in Gene, Vol. 34, pgs. 315-323 (1985); Estell et al., Science, Vol. 233, pgs. 659-663 (1986); Mullenbach et 20 al., J. Biol. Chem., Vol. 261, pgs. 719-722 (1986), and Feretti et al., Proc. Natl. Acad. Sci., Vol. 83, pgs.. 597-603 (1986). Accordingly, these references are incorporated by reference.

Variants of the natural polypeptide (sometime referred to as "muteins") may be desirable in a variety of circumstances. For example, undesirable side effects might be reduced by certain variants, particularly if the side effect activity is associated with a different part of the polypeptide from that of the desired activity. In some expression systems, the native polypeptide may be susceptible to degradation by proteases. In such cases, selected substitutions and/or deletions of amino acids which change the susceptible sequences can significantly enhance yields, e.g. British patent application 2173- 804-A where Arg at position 275 of human tissue plasminogen activator is replaced by Gly or Glu. Variants may also increase yields in purification procedures and/or increase shelf lives of proteins by eliminating amino acids susceptible to oxidation, acylation, alkylation, or other chemical modifications. For example, methionines readily undergo oxidation to form sulfoxides, which in many proteins is associated with loss of biological activity, e.g. Brot and Weissbach, Arch. Biochem. Biophys., Vol. 223, pg. 271 (1983). Often methionines can be replaced by more inert amino acids with little or no loss of biological activity, e.g. Australian patent application AU-A-52451/86. In bacterial expression systems, yields can sometimes be increased by eliminating or replacing conformationally inessential cystiene residues, e.g. Mark et al., U.S. Patent 4,518,584.

Preferably cassette mutagenesis is employed to generate mutant proteins. A synthetic gene is constructed with a sequence of unique (when inserted in an appropriate vector) restriction endonuclease sites spaced approximately uniformly along the gene. The unique restriction sites allow segments of the gene to be conveniently excised and replaced with synthetic oligonucleotides (i.e. "cassettes") which code for desired mutations. Determination of the number and distribution of unique restriction sites entails the consideration of several factors including (1) preexisting restriction sites in the vector to be employed in expression, (2) whether species or genera-specific codon usage is desired, (3) the number of different non-vector-cutting restriction endonucleases available (and their multiplicities within the synthetic gene), and (4) the convenience and reliability of synthesizing and/or sequencing the segments between the unique restriction sites.

The above technique is a convenient way to effect conservative amino acid substitutions, and the like, in the native protein sequence. "Conservative" as used herein means (i) that the alterations are as conformationally neutral as possible, that is, designed to produce minimal changes in the tertiary structure of the mutant polypeptides as compared to the native protein, and (ii) that the alterations are as antigenically neutral as possible, that is, designed to produce minimal changes in the antigenic determinants of the mutant polypeptides as compared to the native protein. The following is a preferred categorization of amino acids into similarity classes: aromatic (phe, trp, tyr), hydrophobic (leu, ile, val), polar (gin, asn), basic (arg, lys, his), acidic (asp, glu), small (ala, ser, thr, met, gly).

Conformational neutrality is desirable for preserving biological activity, and antigenic neutrality is desirable for avoiding the triggering of immunogenic responses in patients or animals treated with the compounds of the invention. While it is difficult to select with absolute certainty which alternatives will be conformationally and antigenically neutral, rules exist which can guide those skilled in the art to make alterations that have high probabilities of being conformationally and antigenically neutral, e.g. Anfisen (cited above); Berzofsky, Science, Vol. 229, pgs. 932-940 (1985); and Bowie et al, Science, Vol. 247, pgs. 1306-1310 (1990). Some of the more important rules include (1) substitution of hydrophobic residues are less likely to produce changes in antigenicity because they are likely to be located in the protein's interior, e.g. Berzofsky (cited above) and Bowie et al (cited above); (2) substitution of physiochemically similar, i.e. synonymous, residues are less likely to produce conformational changes because the replacement amino acid can play the same structural role as the substituted amino acid; and (3) alteration of evolutionarily conserved sequences is likely to produce deleterious conformational effects because evolutionary conservation suggests sequences may be functionally important. In addition to such basic rules for selecting variant sequences, assays are available to confirm the biological activity and conformation of the engineered molecules. Biological assays for the polypeptides of the invention are described more fully above. Changes in conformation can be tested by at least two well known assays: the microcomplement fixation method, e.g. Wasserman et al., J. Immunol., Vol. 87, pgs. 290- 295 (1961), or Levine et al. Methods in Enzymology, Vol. 11, pgs. 928-936 (1967) used widely in evolutionary studies of the tertiary structures of proteins; and affinities to sets of conformation-specific monoclonal antibodies, e.g. Lewis et al., Biochemistry, Vol. 22, pgs. 948-954 (1983).

Chemical Manufacture of ARPR Compositions Peptides of the invention are synthesized by standard techniques, e.g. Stewart and Young, Solid Phase Peptide Synthesis, 2nd Ed. (Pierce Chemical Company, Rockford, IL, 1984). Preferably, a commercial peptide synthesizer is used, e.g. Applied Biosystems, Inc. (Foster City, CA) model 430A, and polypeptides of the invention may be assembled from multiple, separately synthesized and purified, peptide in a convergent synthesis approach, e.g. Kent et al, U.S. patent 6,184,344 and Dawson and Kent, Annu. Rev. Biochem., 69: 923-960 (2000). Peptides of the invention may be assembled by solid phase synthesis on a cross-linked polystyrene support starting from the carboxyl terminal residue and adding amino acids in a stepwise fashion until the entire peptide has been formed. The following references are guides to the chemistry employed during synthesis: Schnolzer et al, Int. J. Peptide Protein Res., 40: 180-193 (1992); Merrifield, J. Amer. Chem. Soc, Vol. 85, pg. 2149 (1963); Kent et al., pg 185, in Peptides 1984, Ragnarsson, Ed. (Almquist and Weksell, Stockholm, 1984); Kent et al., pg. 217 in Peptide Chemistry 84, Izumiya, Ed. (Protein Research Foundation, B.H. Osaka, 1985); Merrifield, Science, Vol. 232, pgs. 341-347 (1986); Kent, Ann. Rev. Biochem., Vol. 57, pgs. 957-989 (1988), and references cited in these latter two references.

Preferably, chemical synthesis of polypeptides of the invention is carried out by the assembly of oligopeptides by native chemical ligation, as described by Dawson et al, Science, 266: 776-779 (1994) and Kent el al, U.S. patent 6,184,344. Briefly, in the approach a first oligopeptide is provided with an N-terminal cysteine having an unoxidized sulfhydryl side chain, and a second oligopeptide is provided with a C-terminal thioester. The unoxidized sulfhydryl side chain of the N-terminal cysteine is then condensed with the C-terminal thioester to produce an intermediate oligopeptide which links the first and second oligopeptides with a β-aminofhioester bond. The β-aminothioester bond of the intermediate oligopeptide then undergoes an intramolecular rearrangement to produce the oligopeptide product which links the first and second oligopeptides with an amide bond. Preferably, the N-terminal cysteine of internal fragments are protected from undesired cyclization and/ro concatenation reactions by a cyclic thiazolidine protecting group as described below. Preferably, such cyclic thiazolidine protecting group is a thioprolinyl group.

Oligopeptides having a C-terminal thioester may be produced as described in the following references, which are incorporated by reference: Kent et al, U.S. patent 6,184,344; Tam et al, Proc.

Natl. Acad. Sci., 92: 12485-12489 (1995); Blake, Int. J. Peptide Protein Res., 17: 273 (1981); Canne et al, Tetrahedron Letters, 36: 1217-1220 (1995); Hackeng et al, Proc. Natl. Acad. Sci., 94: 7845-7850 (1997); or Hackeng et al, Proc. Natl. Acad. Sci., 96: 10068-10073 (1999). Preferably, the method described by Hackeng et al (1999) is employed. Briefly, oligopeptides are synthesized on a solid phase support (described below) typically on a 0.25 mmol scale by using the in situ neutralization/HBTU activation procedure for Boc chemistry dislosed by Schnolzer et al, Int. J. Peptide Protein Res., 40: 180- 193 (1992), which reference is incorporated herein by reference. ( HBTU is 2-(lH-benzotriazol-l-yl)- 1,1,3,3-tetramefhyluronium hexafluorophosphate and Boc is tert-butoxycarbonyl). Each synthetic cycle consists of N^α-Boc removal by a 1- to 2- minute treatment with neat TFA, a 1 -minute DMF flow wash, a 10- to 20-minute coupling time with 1.0 mmol of preactivated Boc-amino acid in the presence of

DIEA, and a second DMF flow wash. (TFA is trifluoroacetic acid, DMF is N,N-dimethylformamide, and DIEA is N,N-diisopropylethylamine). N^α-Boc-amino acids (1.1 mmol) are preactivated for 3 minutes with 1.0 mmol of HBTU (0.5 M in DMF) in the presence of excess DIEA (3 mmol). After each coupling step, yields are determined by measuring residual free amine with a conventional quantitative ninhydrin assay, e.g. as disclosed in Sarin et al, Anal. Biochem., 117: 147-157 (1981). After coupling of Gin residues, a DCM flow wash is used before and after deprotection by using TFA, to prevent possible high-temperature (TFA/DMF)-catalyzed pyrrolidone formation. After chain assembly is completed, the oligopeptides are deprotected and cleaved from the resin by treatment with anhydrous HF for 1 hour at 0°C with 4% -cresol as a scavenger. The imidazole side-chain 2,4- dinitrophenyl (dnp) protecting groups remain on the His residues because the dnp-removal procedure is incompatible with C-terminal thioester groups. However, dnp is gradually removed by thiols during the ligation reaction. After cleavage, oligopeptides are precipitated with ice-cold diethylether, dissolved in aqueous acetonitrile, and lyophilized. Thioester oligopeptides described above are preferably synthesized on a trityl-associated

' mercaptopropionic acid-leucine (TAMPAL) resin, made as disclosed by Hackeng et al (1999), or comparable protocol. Briefly, N^α-Boc-Leu (4 mmol) is activated with 3.6 mmol of HBTU in the presence of 6 mmol of DIEA and coupled for 16 minutes to 2 mmol of p-mefhylbenzhydrylamine (MBHA) resin, or the equivalent. Next, 3 mmol of S-trityl mercaptopropionic acid is activated with 2.7 mmol of HBTU in the presence of 6 mmol of DIEA and coupled for 16 minutes to Leu-MBHA resin. The resulting TAMPAL resin can be used as a starting resin for polypeptide-chain assembly after removal of the trityl protecting group with two 1 -minute treatments with 3.5% triisopropylsilane and 2.5% H₂0 in TFA. The thioester bond can be formed with any desired amino acid by using standard in situ-neutralization peptide coupling protocols for 1 hour, as disclosed in Schnolzer et al (cited above). Treatment of the final oligopeptide with anhydrous HF yields the C-terminal activated mercaptopropionic acid-leucine (MPAL) thioester oligopeptides.

Preferably, thiazolidine-protected thioester oligopeptide intermediates are used in native chemical ligation under conditions as described by Hackeng et al (1999), or like conditions. Briefly, 0.1 M phosphate buffer (pH 8.5) containing 6 M guanidine, 4% (vol/vol) benzylmercaptan, and 4% (vol/vol) thiophenol is added to dry peptides to be ligated, to give a final peptide concentration of 1-3 mM at about pH 7, lowered because of the addition of thiols and TFA from the lyophilized peptide. Preferably, the ligation reaction is performed in a heating block at 37°C and is periodically vortexed to equilibrate the thiol additives. The reaction may be monitored for degree of completion by MALDI-MS or HPLC and electrospray ionization MS. After a native chemical ligation reaction is completed or stopped, the N-terminal thiazolidine ring of the product is opened by treatment with a cysteine deprotecting agent, such as O- methylhydroxylamine (0.5 M) at pH 3.5-4.5 for 2 hours at 37°C , after which a 10-fold excess of Tris- (2-carboxyethyl)-phosphine is added to the reaction mixture to completely reduce any oxidizing reaction constituents prior to purification of the product by conventional preparative HPLC. Preferably, fractions containing the ligation product are identified by electrospray MS, are pooled, and lyophilized. After the synthesis is completed and the final product purified, the final polypeptide product may be refolded by conventional techniques, e.g. Creighton, Meth. Enzymol., 107: 305-329 (1984); White, Meth. Enzymol., 11: 481-484 (1967); Wetlaufer, Meth. Enzymol., 107: 301-304 (1984); and the like. Preferably, a final product is refolded by air oxidation by the following, or like: The reduced lyophilized product is dissolved (at about 0.1 mg/mL) in 1 M guanidine hydrochloride (or like chaotropic agent) with 100 mM Tris, 10 mM mefhionine, at pH 8.6. After gentle overnight stirring, the re-folded product is isolated by reverse phase HPLC with conventional protocols.

Anti-ARPR Antibodies. The present invention further provides anti-ARPR antibodies. The antibodies of the present invention include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies.

One antibody composition of the invention is capable of specifically binding or specifically bind to an ARPR protein of SEQ ID NOS 1 to 5, 13 to 21, 27 or 28. In one example, an antibody for use in distinguishing among the chlO-ARPR isoforms of SEQ ID NOS 1 or 2 and SEQ ID NOS 27 and 28 is provided which selectively binds to one of the isoforms but not the other. Such as antibody is useful for example in detecting a muscle cell. For an antibody composition to specifically bind to one chlO-ARPR isoform over another, it must demonstrate at least a 5%, 10%, 15%, 20%, 25%, 50%, or 100% greater binding affinity for a first chlO-ARPR isoform protein than for the second chlO-ARPR isoform protein in an ELISA, RIA, or other antibody-based binding assay.

Polyclonal Antibodies. The anti-ARPR antibodies of the present invention may be polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Such polyclonal antibodies can be produced in a mammal, for example, following one or more injections of an immunizing agent, and preferably, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected into the mammal by a series of subcutaneous or intraperitoneal injections. The immunizing agent may include a ARPR or a fusion protein thereof. It may be useful to conjugate the antigen to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Adjuvants include, for example, Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicoryno-mycolate). The immunization protocol may be determined by one skilled in the art based on standard protocols or by routine experimentation. Monoclonal Antibodies. Alternatively, the anti-ARPR antibodies may be monoclonal antibodies. Monoclonal antibodies may be produced by hybridomas, wherein a mouse, hamster, or other appropriate host animal, is immunized with an immunizing agent to elicit lympho-cytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent [Kohler and Milstein, Nature 256:495 (1975)]. Alternatively, the lymphocytes may be immunized in vitro. The immunizing agent will typically include the ARPR or a fusion protein thereof. Generally, spleen cells or lymph node cells are used if non-human mammalian sources are desired, or peripheral blood lymphocytes (" PBLs" ) are used if cells of human origin. The lymphocytes are fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to produce a hybridoma cell [Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE, Academic Press, pp. 59- 103 (1986)]. In general, immortalized cell lines are transformed mammalian cells, for example, myeloma cells of rat, mouse, bovine or human origin. The hybridoma cells are cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT), substances which prevent the growth of HGPRT- deficient cells. Preferred immortalized cell lines are those that fuse efficiently, support stable high level production of antibody, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine or human myeloma lines, which can be obtained, for example, from the American Type Culture Collection (ATCC), Rockville, MD. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Zmmunol. 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63 (1987)]. The culture medium (supernatant) in which the hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies directed against an ARPR. Preferably, the binding specificity of monoclonal antibodies present in the hybridoma supernatant is determined by immunoprecipitation or by an in vitro binding assay, such as radio- immunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Appropriate techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem. 107:220 (1980). After the desired antibody-producing hybridoma cells are identified, the cells may be cloned by limiting dilution procedures and grown by standard methods [Goding, 1986]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal. The monoclonal antibodies secreted by selected clones may be isolated or purified from the culture medium or ascites fluid by immunoglobulin purification procedures routinely used by those of skill in the art such as, for example, protein A-Sepharose, hydroxyl-apatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be isolated from the ARPR-specific hybridoma cells and sequenced, e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies. Once isolated, the DNA may be inserted into an expression vector, which is then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for the human heavy and light chain constant domains for the homologous murine sequences [Morrison et al., Proc. Nat. Acad. Sci. 81:6851-6855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)], or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. The non- immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody. The antibodies may also be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, in vitro methods are suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.

Antibodies and antibody fragments characteristic of hybridomas of the invention can also be produced by recombinant means by extracting messenger RNA, constructing a cDNA library, and selecting clones which encode segments of the antibody molecule, e.g. Wall et al., Nucleic Acids

Research, Vol. 5, pgs. 3113-3128 (1978); Zakut et al., Nucleic Acids Research, Vol. 8, pgs. 3591-3601 (1980); Cabilly et al., Proc. Natl. Acad. Sci., Vol. 81, pgs. 3273-3277 (1984); Boss et al., Nucleic Acids Research, Vol. 12, pgs. 3791-3806 (1984); Amster et al., Nucleic Acids Research, Vol. 8, pgs. 2055-2065 (1980); Moore et al., U.S. Patent 4,642,334; Skerra et al, Science, Vol. 240, pgs. 1038- 1041(1988); and Huse et al, Science, Vol. 246, pgs. 1275-1281 (1989). In particular, such techniques can be used to produce interspecific monoclonal antibodies, wherein the binding region of one species is combined with non-binding region of the antibody of another species to reduce immunogenicity, e.g. Liu et al., Proc. Natl. Acad. Sci., Vol. 84, pgs. 3439-3443 (1987).

Both polyclonal and monoclonal antibodies can be screened by ELISA. As in other solid phase immunoassays, the test is based on the tendency of macromolecules to adsorb nonspecifically to plastic. The irreversibility of this reaction, without loss of immunological activity, allows the formation of antigen-antibody complexes with a simple separation of such complexes from unbound material. To titrate antipeptide serum, peptide conjugated to a carrier different from that used in immunization is adsorbed to the wells of a 96-well microtiter plate. The adsorbed antigen is then allowed to react in the wells with dilutions of anti-peptide serum. Unbound antibody is washed away, and the remaining antigen-antibody complexes are allowed to react with antibody specific for the IgG of the immunized animal, this second antibody is conjugated to an enzyme such as alkaline phosphatase. A visible colored reaction product produced when the enzyme substrate is added indicates which wells have bound antipeptide antibodies. The use of spectrophotometer readings allows better quantification of the amount of peptide-specific antibody bound. High-titer antisera yield a linear titration curve between 10^"3 and

10^"5 dilutions.

ARPR peptide antibodies. The invention includes peptides derived from ARPR, and immunogens comprising conjugates between carriers and peptides of the invention. The term immunogen as used herein refers to a substance which is capable of causing an immune response. The term carrier as used herein refers to any substance which when chemically conjugated to a peptide of the invention permits a host organism immunized with the resulting conjugate to generate antibodies specific for the conjugated peptide. Carriers include red blood cells, bacteriophages, proteins, or synthetic particles such as agarose beads. Preferably, carriers are proteins, such as serum albumin, gamma- globulin, keyhole limpet hemocyanin, thyroglobulin, ovalbumin, fibrinogen, or the like. The general technique of linking synthetic peptides to a carrier is described in several references, e.g. Walter and Doolittle, "Antibodies Against Synthetic Peptides," in Setlow et al., eds., Genetic Engineering, Vol. 5, pgs. 61-91 (Plenum Press, N.Y., 1983); Green et al. Cell, Vol. 28, pgs. 477-487 (1982); Lerner et al., Proc. Natl. Acad. Sci., Vol. 78, pgs. 3403-3407 (1981); Shimizu et al., U.S. Patent 4,474,754; and Ganfield et al., U.S. Patent 4,311,639. Accordingly, these references are incorporated by reference. Also, techniques employed to link haptens to carriers are essentially the same as the above-referenced techniques, e.g. chapter 20 in Tissue Practice and Theory of Enzyme Immunoassays (Elsevier, New York, 1985). The four most commonly used schemes for attaching a peptide to a carrier are (1) glutaraldehyde for amino coupling, e.g. as disclosed by Kagan and Glick, in Jaffe and Behrman, eds. Methods of Hormone Radioimmunoassay, pgs. 328-329 (Academic Press, N.Y., 1979), and Walter et al. Proc. Natl. Acad. Sci., Vol. 77, pgs. 5197-5200 (1980); (2) water- soluble carbodiimides for carboxyl to amino coupling, e.g. as disclosed by Hoare et al., J. Biol. Chem., Vol. 242, pgs. 2447-2453 (1967); (3) bis-diazobenzidine (DBD) for tyrosine to tyrosine sidechain coupling, e.g. as disclosed by Bassiri et al., pgs. 46-47, in Jaffe and Behrman, eds. (cited above), and Walter et al. (cited above); and (4) maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) for coupling cysteine (or other sulfhydryls) to amino groups, e.g. as disclosed by Kitagawa et al., J. Biochem.

(Tokyo), Vol. 79, pgs. 233-239 (1976), and Lerner et al. (cited above). A general rule for selecting an appropriate method for coupling a given peptide to a protein carrier can be stated as follows: the group involved in attachment should occur only once in the sequence, preferably at the appropriate end of the segment. For example, BDB should not be used if a tyrosine residue occurs in the main part of a sequence chosen for its potentially antigenic character. Similarly, centrally located lysines rale out the glutaraldehyde method, and the occurrences of aspartic and glutamic acids frequently exclude the carbodiimide approach. On die other hand, suitable residues can be positioned at either end of chosen sequence segment as attachment sites, whether or not they occur in the "native" protein sequence. Internal segments, unlike the amino and carboxy termini, will differ significantly at the "unattached end" from the same sequence as it is found in the native protein where the polypeptide backbone is continuous. The problem can be remedied, to a degree, by acetylating the α-amino group and then attaching the peptide by way of its carboxy terminus. The coupling efficiency to the carrier protein is conveniently measured by using a radioactively labeled peptide, prepared either by using a radioactive amino acid for one step of the synthesis or by labeling the completed peptide by the iodination of a tyrosine residue. The presence of tyrosine in the peptide also allows one to set up a sensitive radioimmune assay, if desirable. Therefore, tyrosine can be introduced as a terminal residue if it is not part of the peptide sequence defined by the native polypeptide.

Preferred carriers are proteins, and preferred protein carriers include bovine serum albumin, myoglobulin, ovalbumin (OVA), keyhole limpet hemocyanin (KLH), or the like. Peptides can be linked to KLH through cysteines by MBS as disclosed by Liu et al., Biochemistry, Vol. 18, pgs. 690-697 (1979). The peptides are dissolved in phosphate-buffered saline (pH 7.5), 0.1 M sodium borate buffer (pH 9.0) or 1.0 M sodium acetate buffer (pH 4.0). The pH for the dissolution of the peptide is chosen to optimize peptide solubility. The content of free cysteine for soluble peptides is determined by Ellman's method, Ellman, Arch. Biochem. Biophys., Vol. 82, pg. 7077 (1959). For each peptide, 4 mg KLH in 0.25 ml of 10 mM sodium phosphate buffer (pH 7.2) is reacted with 0.7 mg MBS (dissolved in dimethyl formamide) and stirred for 30 in at room temperature. The MBS is added dropwise to ensure that the local concentration of formamide is not too high, as KLH is insoluble in >30% formamide. The reaction product, KLH-MBS, is then passed through Sephadex G-25 equilibrated with 50 mM sodium phosphate buffer (pH 6.0) to remove free MBS, KLH recovery from peak fractions of the column eluate (monitored by OD280) is estimated to be approximately 80% . KLH-MBS is then reacted with 5 mg peptide dissolved 25 in 1 ml of the chosen buffer. The pH is adjusted to 7-7.5 and the reaction is stirred for 3 hr at room temperature. Coupling efficiency is monitored with radioactive peptide by dialysis of a sample of the conjugate against phosphate-buffered saline, and ranged from 8 % to 60% . Once the peptide-carrier conjugate is available polyclonal or monoclonal antibodies are produced by standard techniques, e.g. as disclosed by Campbell, Monoclonal Antibody Technology (Elsevier, New York, 1984); Hurrell, ed. Monoclonal Hybridoma Antibodies: Techniques and Applications (CRC Press, Boca Raton, FL, 1982); Schreier et al. Hybridoma Techniques (Cold Spring Harbor Laboratory, New York, 1980); U.S. Patent 4,562,003; or the like. In particular, U.S. Patent 4,562,003 is incorporated by reference.

Humanized Antibodies. The anti-ARPR antibodies of the invention may further comprise humanized antibodies or human antibodies. The term "humanized antibody" refers to humanized forms of non-human (e.g., murine) antibodies that are chimeric antibodies, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab'), or other antigen-binding partial sequences of antibodies) which contain some portion of the sequence derived from non-human antibody. Humanized antibodies include human immunoglobulins in which residues from a complementary determining region (CDR) of the human immunoglobulin are replaced by residues from a CDR of a non-human species such as mouse, rat or rabbit having the desired binding specificity, affinity and capacity. In general, the humanized antibody will comprise substantially all of at least one, and generally two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature 321:522-525 (1986) and Presta, Cuvv. Op. Stvuct. Biol. 2:593-596 (1992)]. Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acids introduced into it from a source which is non-human in order to more closely resemble a human antibody, while still retaining the original binding activity of the antibody. Methods for humanization of antibodies are further detailed in Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); and Verhoeyen et al., Science 239:1534-1536 (1988). Such "humanized" antibodies are chimeric antibodies in that substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.

Heteroconjugate Antibodies. Heteroconjugate antibodies which comprise two covalently joined antibodies, are also within the scope of the present invention. Heteroconjugate antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be prepared using a disulfide exchange reaction or by forming a thioether bond.

Bispecific Antibodies. Bispecific antibodies have binding specificities for at least two different antigens. Such antibodies are monoclonal, and preferably human or humanized. One of the binding specificities of a bispecific antibody of the present invention is for a ARPR, and the other one is preferably for a cell-surface protein or receptor or receptor subunit. Methods for making bispecific antibodies are known in the art, and in general, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs in hybridoma cells, where the two heavy chains have different specificities [Milstein and Cuello, Nature 305:537-539 (1983)]. Given that the random assortment of immunoglobulin heavy and light chains results in production of potentially ten different antibody molecules by the hybridomas, purification of the correct molecule usually requires some sort of affinity purification, e.g. affinity chromatography.

Antibody antagonists. Preferably, antagonists of the invention are derived from antibodies specific for ARPR. More preferably, the antagonists of the invention comprise fragments or binding compositions specific for ARPR. Antibodies comprise an assembly of polypeptide chains linked together by disulfide bridges. Two major polypeptide chains, referred to as the light chain and the heavy chain, make up all major structural classes (isotypes) of antibody. Both heavy chains and light chains are further divided into subregions referred to as variable regions and constant regions. Heavy chains comprise a single variable region and three different constant regions, and light chains comprise a single variable region (different from that of the heavy chain) and a single constant region (different from those of the heavy chain). The variable regions of the heavy chain and light chain are responsible for the antibody's binding specificity. As used herein, the term "heavy chain variable region" means a polypeptide (1) which is from 110 to 125 amino acids in length, and (2) whose amino acid sequence corresponds to that of a heavy chain of a monoclonal antibody of the invention, starting from the heavy chain's N-terminal amino acid. Likewise, the term "light chain variable region" means a polypeptide (1) which is from 95 to 115 amino acids in length, and (2) whose amino acid sequence corresponds to that of a light chain of a monoclonal antibody of the invention, starting from the light chain's N-terminal amino acid. As used herein the term "monoclonal antibody" refers to homogeneous populations of immunoglobulins which are capable of specifically binding to ARPR. As used herein the term "binding composition" means a composition comprising two polypeptide chains (1) which, when operationally associated, assume a conformation having high binding affinity for ARPR, and (2) which are derived from a hybridoma producing monoclonal antibodies specific for ARPR. The term "operationally associated" is meant to indicate that the two polypeptide chains can be positioned relative to one another for binding by a variety of means, including by association in a native antibody fragment, such as Fab or Fv, or by way of genetically engineered cysteine-containing peptide linkers at the carboxyl termini. Normally, the two polypeptide chains correspond to the light chain variable region and heavy chain variable region of a monoclonal antibody specific for ARPR. Preferably, antagonists of the invention are derived from monoclonal antibodies specific for ARPR. Monoclonal antibodies capable of blocking, or neutralizing, ARPR are selected by their ability to inhibit ARPR-induced effects.

The use and generation of fragments of antibodies is also well known, e.g. Fab fragments: Tijssen, Practice and Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985); and Fv fragments: Hochman et al. Biochemistry, Vol. 12, pgs. 1130-1135 (1973), Sharon et al., Biochemistry, Vol. 15, pgs. 1591-1594 (1976) and Ehrlich et al., U.S. Patent 4,355,023; and antibody half molecules: Auditore- Hargreaves, U.S. Patent 4,470,925.

Uses of ARPR Polynucleotides Polynucleotide sequences (or the complements thereof) which encode ARPR polypeptides have various applications, including uses as hybridization probes, in chromosome and gene mapping, in the generation of antisense RNA and DNA and in tissue or cell typing methods. In view of the tissue specificity of ARPR mRNA expression, it is envisioned that ARPR nucleic acids can be used in cell typing applications. In addition, ARPR-encoding nucleic acids are useful as targets for pharmaceutical intervention, e.g. for the development of DNA vaccines, and for the preparation of ARPR polypeptides by recombinant techniques, as described herein. The polynucleotides described herein, including sequence variants thereof, can be used in diagnostic assays. Accordingly, diagnostic methods based on detecting the presence of such polynucleotides in body fluids or tissue samples are a feature of the present invention. Examples of nucleic acid based diagnostic assays in accordance with the present invention include, but are not limited to, hybridization assays, e.g., in situ hybridization, and PCR- based assays. Polynucleotides, including extended length polynucleotides, sequence variants and fragments thereof, as described herein, may be used to generate hybridization probes or PCR primers for use in such assays. Such probes and primers will be capable of detecting polynucleotide sequences, including genomic sequences that are similar, or complementary to, the ARPR polynucleotides described herein.

The invention includes primer pairs for carrying out a PCR to amplify a segment of a polynucleotide of the invention. Each primer of a pair is an oligonucleotide having a length of between 15 and 30 nucleotides such that i) one primer of the pair forms a perfectly matched duplex with one strand of a polynucleotide of the invention and the other primer of the pair form a perfectly match duplex with the complementary strand of the same polynucleotide, and ii) the primers of a pair form such perfectly matched duplexes at sites on the polynucleotide that separated by a distance of between 10 and 2500 nucleotides. Preferably, the annealing temperature of each primer of a pair to its respective complementary sequence is substantially the same.

Hybridization probes derived from polynucleotides of the invention can be used, for example, in performing in situ hybridization on tissue samples, such as fixed or frozen tissue sections prepared on microscopic slides or suspended cells. Briefly, a labeled DNA or RNA probe is allowed to bind its DNA or RNA target sample in the tissue section on a prepared microscopic, under controlled conditions. Generally, dsDNA probes consisting of the DNA of interest cloned into a plasmid or bacteriophage

DNA vector are used for this purpose, although ssDNA or ssRNA probes may also be used. Probes are generally oligonucleotides between about 15 and 40 nucleotides in length. Alternatively, the probes can be polynucleotide probes generated by PCR random priming primer extension or in vitro transcription of RNA from plasmids (riboprobes). These latter probes are typically several hundred base pairs in length. The probes can be labeled by any of a number of methods, including fluorescent tags, enzymes or radioactive moieties, according to methods well known in the art. The particular detection method will correspond to the type of label utilized on the probe (e.g., autoradiography, X-ray detection, fluorescent or visual microscopic analysis, as appropriate). The reaction can be further amplified in situ using immunocytochemical techniques directed against the label of the detector molecule used, such antibodies directed to a fluorescein moiety present on a fluorescently labeled probe, or against avidin, or marker enzymes (peroxidase, alkaline phosphatase). Specific labeling and in situ detection methods can be found, for example, in Howard, G. C, Ed., Methods in Nonradioactive Detection, Appleton & Lange, Norwalk, Conn., (1993), herein incorporated by reference. One preferred assay for detecting nucleic acids encoding ARPR protein utilizes the subject polynucleotides, or fragments thereof, as primers in a PCR-based assay. According to the assay, nucleic acids present in a test tissue or cell sample are amplified by polymerase chain reaction (PCR) using two primers consisting of at least 15 nucleotides derived from one or more of a group consisting of SEQ ID NOs: 6, 7, and 8, including primers derived from variants and/or extensions of such sequences, as described herein. Amplification products are detected in the sample by a method that is appropriate to the particular label used to label the amplification products, according to methods as described in U.S. Pat. No. 4,683,195. For use in PCR detection methods, such as PCR in situ hybridization, PCR primers are selected to be at least 15 nucleotides in length, and preferably between about 15 and 30 nucleotides in length, and are selected from the DNA molecule of interest, according to methods known in the art. While such primers can be selected from within the sequences identified as SEQ ID NOs: 6, 7, 8, 22 or 29, herein, it may also be desirable to select sequences that encompass the longer nucleotide sequences. Preferably, the probes are selected such that the two hybridization sites are separated by between about 10 to 1,000 nucleotides (occasionally up to about 10,000 nucleotides). PCR in situ hybridization of tissue sections and/or cell samples provides a highly sensitive detection method for rare cell types in fixed cell or tissue samples. The PCR in situ hybridization detection method is carried out in accordance with methods that are known in the art, e.g., Nuovo, G. J., PCR IN SITU HYBRIDIZATION: PROTOCOLS AND APPLICATIONS, Raven Press, N.Y., 1992; U.S. Pat. No. 5,538,871, both of which are incorporated herein by reference.

Briefly, a cell sample (tissue on microscopic slide, pelleted cell suspension) is fixed using a common fixative preparation, such as buffered formalin, formaldehyde or the like. Proteinase or detergent treatment is favored following fixation, to increase cell permeability to reagents. The PCR reaction is carried out in situ by polymerase chain reaction (PCR) using two primers. As discussed above, the primers are designed to selectively amplify one or more of the nucleotide sequences described herein, and particularly sequences described as SEQ ID NOs: 6, 7, 8, 22 or 29. The amplification reaction mixture contains, in addition to the target nucleotide sample and the primers, a thermostable DNA polymerase, such as a polymerase derived from Tliemus aquaticus (Taq polymerase, U.S. Pat. 4,889,818), and a sufficient quantity of the four standard deoxyribonucleotides (dNTPs), one or more of which may be labeled to facilitate detection. The reaction mixture is subjected to several rounds of fhermocycling to produce multiple copies (amplification products) of the target nucleotide sequence. Amplification products are then detected in the sample, for example by detecting radioactively labeled amplification products. Hybridization probes and PCR primers may also be selected from the genomic sequences corresponding to the full-length proteins identified in accordance with the present invention, including promoter, enhancer elements and introns of the gene encoding the naturally occurring polypeptide. Nucleotide sequences encoding a ARPR polypeptide can also be used to construct hybridization probes for mapping the gene which encodes that ARPR and for the genetic analysis of individuals. The nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries. Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the ARPR cDNA. Computer analysis of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, which would complicate the amplification process. Individuals carrying variations of, or mutations in the gene encoding an ARPR of the present invention may be detected at the DNA level by a variety of techniques. Nucleic acids used for diagnosis may be obtained from a patient's cells, including, for example, tissue biopsy and autopsy material. Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR [Saiki, et al. Nature 324:163-166 (1986)] prior to analysis. RNA or cDNA may also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid of the present invention can be used to identify and analyze mutations in the gene of the present invention. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA of the invention or alternatively, radiolabeled antisense DNA sequences of the invention. Sequence changes at specific locations may also be revealed by nuclease protection assays, such RNase and SI protection or the chemical cleavage method [e.g. Cotton, et al., Pvoc. Natl. Acad. Sci. USA 85:4397-4401 (1985)], or by differences in melting temperatures. "Molecular beacons" [Kostrikis L. G. et al., Science 279:1228-1229 (1998)], hairpin- shaped, single-stranded synthetic oligonucleotides containing probe sequences which are complementary to the nucleic acid of the present invention, may also be used to detect point mutations or other sequence changes as well as monitor expression levels of ARPR.

Cell Typing. As further described in Example 4, real-time mRNA expression experiments in monkey organs indicates that ARPR is expressed specifically in skeletal muscle. Thus, in one aspect, the invention provides a marker for the specific identifcation of skeletal muscle cells. The method may involve detecting the ARPR protein or a ARPR nucleic acid using detection means known in the art. For example, ARPR nucleic acids can be used in a rapid and sensitive assay for confirming the presence of specific mRNA species which are present in a cell suspension. The assay and method of this invention utilizes whole cells as a starting material for a reverse transcriptase polymerase chain reaction ("RT- PCR") analysis. Primers are designed and used to amplify specific cDNA sequences conforming to the mRNA. Specific and positive identification of cell types present in a mixed population of cells can thus be confirmed with only minimal time and sample manipulation.

These methods of the invention provides significant advantages over examining the cell or tissue morphology. The disadvantage of this currently available method is that it requires an extensive amount of training, and even then it is highly subjective and not consistently reproducible. This can be improved upon by applying histological stains to the cells or tissues. In general, histology is time consuming, and the stains are not very specific. In addition, the staining and fixing of cells or tissues often involves hazardous chemicals and facilities for working with and disposing of these materials.

In one aspect, the present invention can be used in a method for determining cell type by looking for a ARPR protein marker known to be expressed specifically by skeletal muscle cells. Methods used in such an analysis include well known immunohistology or FACS technology.

Although typing cells based on ARPR protein detection is much more specific than examining morphology, these methods often rely on the availability of antibodies raised against the protein marker of interest. If antibodies are not readily available, or require conditions to be optimized on an individual basis, cell typing can be carried out by examining the mRNA transcribed by cells. While every cell type has a unique mRNA profile, the methods employed for working with RNA are independent of the specific mRNA being examined. Techniques are applicable to a broad range of cell sources with at most very minor modifications. Northern blotting and ribonuclease protection assays are two methods that can detect and quantify the presence of a specific mRNA. Thus, the ARPR nucleic acids of the invention may be used to specifically identify skeletal muscle cells.

In some cases both of the latter methods can have the disadvantage of requiring substantial amounts (1 to 20 micrograms) of RNA which generally needs to be purified first. In this case, methods such as In Situ hybridization and in situ PCR can be used, which require much less starting material. In a further method, an RNase protection assay can be carried out, which can be quantitative, requires very little starting material, and can be performed directly on whole cells without prior RNA purification

(Strauss and Jacobowitz, Brain Res. Mol Brain Res , 20, pages 229-239, (1993). In yet another method, a RT-PCR assay can be used, as discussed herein.

A further method provides a rapid and sensitive assay and method for confirming the presence of specific mRNA species which are present in a cell suspension, described in International Patent Publication No. WO 98/04742. The latter assay utilizes whole cells as a starting material for a reverse transcriptase polymerase chain reaction ("RT-PCR") analysis. Primers are designed and used to amplify specific cDNA sequences conforming to the mRNA. That is, the method involves choosing primer pairs that distinguish RNA signal from genomic DNA signal. Specific and positive identification of cell types present in a mixed population of cells can thus be confirmed with only minimal time and sample manipulation. In carrying out the assay, the cells are separated from the components of a liquid sample by any convenient means, such as centrifugation. Preferably, the cells are formed into pellets. A reaction mixture is formed by combining the pellets, RT-PCR medium, a cell lysing agent or detergent, such as Tween-20, and optionally, an RNase inhibitor to minimize degradation of the mRNA. The reaction is maintained at a sufficiently high temperature, i.e. from about 60 C to about 90 C, in order to reduce mRNA degradation by temporarily inactivating Rnases and to reduce false priming.

Thus, ARPR can be used as a molecular marker specifically expressed in skeletal muscle cells, preferably in a modified single-tube RT-PCR analysis is performed to amplify the signal for these markers. The method and assay can be used in any procedure where it is necessary to confirm the presence of specific cell types in a mixed population of cells. More generally, it can be used wherever a specific RNA is expected to be transcribed by some or all cells.

Antisense Compounds. Oligonucleotides of the invention, including per primers and antisense compounds, are synthesized by conventional means on a commercially available automated DNA synthesizer, e.g. an Applied Biosystems (Foster City, CA) model 380B, 392 or 394 DNA/RNA synthesizer, or like instrument. Preferably, phosphoramidite chemistry is employed, e.g. as disclosed in the following references: Beaucage and Iyer, Tetrahedron, 48: 2223-2311 (1992); Molko et al, U.S. patent 4,980,460; Koster et al, U.S. patent 4,725,677; Carathers et al, U.S. patents 4,415,732; 4,458,066; and 4,973,679; and the like. For therapeutic use, nuclease resistant backbones are preferred. Many types of modified oligonucleotides are available that confer nuclease resistance, e.g. phosphorothioate, phosphorodifhioate, phosphoramidate, or the like, described in many references, e.g. phosphorothioates: Stec et al, U.S. patent 5,151,510; Hirschbein, U.S. patent 5,166,387; Bergot, U.S. patent 5,183,885; phosphoramidates: Froehler et al, International application PCT/US90/03138; and for a review of additional applicable chemistries: Uhlmann and Peyman (cited above). Antisense compounds of the invention are selected so that they are complementary to a contiguous subsequence of SEQ ID NO: 8 or 29. The length of the antisense oligonucleotides, i.e. such contiguous subsequence, is sufficiently large to ensure that specific binding will take place only at the desired target polynucleotide and not at other fortuitous sites. The upper range of the length is determined by several factors, including the inconvenience and expense of synthesizing and purifying oligomers greater than about 30- 40 nucleotides in length, the greater tolerance of longer oligonucleotides for mismatches than shorter oligonucleotides, and the like. Preferably, the antisense oligonucleotides of the invention have lengths in the range of about 15 to 40 nucleotides. More preferably, the oligonucleotide moieties have lengths in the range of about 18 to 25 nucleotides.

Purification and Pharmaceutical Compositions When polypeptides of the present invention are expressed in soluble form, for example as a secreted product of transformed yeast or mammalian cells, they can be purified according to standard procedures of the art, including steps of ammonium sulfate precipitation, ion exchange chromatography, gel filtration, electrophoresis, affinity chromatography, and/or the like, e.g. "Enzyme Purification and Related Techniques," Methods in Enzymology, 22:233-577 (1977), and Scopes, R., Protein Purification: Principles and Practice (Springer- Verlag, New York, 1982) provide guidance in such purifications. Likewise, when polypeptides of the invention are expressed in insoluble form, for example as aggregates, inclusion bodies, or the like, they can be purified by standard procedures in the art, including separating the inclusion bodies from disrupted host cells by centrifugation, solublizing the inclusion bodies with chaotropic and reducing agents, diluting the solubilized mixture, and lowering the concentration of chaotropic agent and reducing agent so that the polypeptide takes on a biologically active conformation. The latter procedures are disclosed in the following references, which are incorporated by reference: Winkler et al, Biochemistry, 25: 4041-4045 (1986); Wi kler et al,

Biotechnology, 3: 992-998 (1985); Koths et al, U.S. patent 4,569,790; and European patent applications 86306917.5 and 86306353.3.

As used herein "effective amount" means an amount sufficient to ameliorate a symptom of an autoimmune condition. The effective amount for a particular patient may vary depending on such factors as the state of the condition being treated, the overall health of the patient, method of administration, the severity of side-effects, and the like. Generally, ARPR is administered as a pharmaceutical composition comprising an effective amount of ARPR and a pharmaceutical carrier. A pharmaceutical carrier can be any compatible, non-toxic substance suitable for delivering the compositions of the invention to a patient. Generally, compositions useful for parenteral administration of such drugs are well known, e.g. Remington's Pharmaceutical Science, 15th Ed. (Mack Publishing Company, Easton, PA 1980). Alternatively, compositions of the invention may be introduced into a patient's body by implantable or injectable drug delivery system, e.g. Urquhart et al., Ann. Rev. Pharmacol. Toxicol., Vol. 24, pgs. 199-236 (1984); Lewis, ed. Controlled Release of Pesticides and Pharmaceuticals (Plenum Press, New York, 1981); U.S. patent 3,773,919; U.S. patent 3,270,960; and the like.

When administered parenterally, the ARPR is formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutical carrier. Examples of such carriers are normal saline, Ringer's solution, dextrose solution, and Hank's solution. Nonaqueous carriers such as fixed oils and ethyl oleate may also be used. A preferred carrier is 5% dextrose/saline. The carrier may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The ARPR is preferably formulated in purified form substantially free of aggregates and other proteins at a concentration in the range of about 5 to 20 μg/ml. Preferably, ARPR is administered by continuous infusion so that an amount in the range of about 50-800 μg is delivered per day (i.e. about 1-16 μg/kg/day). The daily infusion rate may be varied based on monitoring of side effects, such as blood cell counts, body temperature, and the like. ARPR polypeptides may be useful for example in a method of modulating (e.g. stimulating or inhibiting), most preferably stimulating, the growth of a cell. ARPR can be purified from culture supernatants of mammalian cells transiently transfected or stably transformed by an expression vector carrying an ARPR gene. Preferably, ARPR is purified from culture supernatants of COS 7 cells transiently transfected by the pcD expression vector. Transfection of COS 7 cells with pcD proceeds as follows: One day prior to transfection, approximately 10° COS 7 monkey cells are seeded onto individual 100 mm plates in Dulbecco's modified Eagle medium (DME) containing 10% fetal calf serum and 2 mM glutamine. To perform the transfection, the medium is aspirated from each plate and replaced with 4 ml of DME containing 50 mM Tris.HCl pH 7.4, 400 mg/ml DEAE-Dextran and 50 μg of plasmid DNA. The plates are incubated for four hours at 37°C, then the DNA-containing medium is removed, and the plates are washed twice with 5 ml of serum-free DME. DME is added back to the plates which are then incubated for an additional 3 hrs at 37°C. The plates are washed once with DME, after which DME containing 4% fetal calf serum, 2 mM glutamine, penicillin (100 U/L) and streptomycin (100 μg/L) at standard concentrations is added. The cells are then incubated for 72 hrs at 37°C, after which the growth medium is collected for purification of ARPR. Alternatively, transfection can be accomplished by electroporation as described in the examples. Plasmid DNA for the transfections is obtained by growing pcD(SRα), or like expression vector, containing the ARPR cDNA insert in E. coli MC1061, described by Casadaban and Cohen, J. Mol.

Biol., Vol. 138, pgs. 179-207 (1980), or like organism. The plasmid DNA is isolated from the cultures by standard techniques, e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory, New York, 1989) or Ausubel et al (1990, cited above). When the antagonists of the inventions are derived from antibodies, they are normally administered parenterally, preferably intravenously. Since such protein or peptide antagonists may be immunogenic they are preferably administered slowly, either by a conventional IV administration set or from a subcutaneous depot, e.g. as taught by Tomasi et al, U.S. patent 4,732,863. When administered parenterally, the antibodies and/or fragments are formulated in a unit dosage injectable form in association with a pharmaceutical carrier, as described above. The antibody is preferably formulated in purified form substantially free of aggregates, other proteins, endotoxins, and the like, at concentrations of about 5 to 30 mg/ml, preferably 10 to 20 mg/ml. Preferably, the endotoxin levels are less than 2.5 EU/ml. Selecting an administration regimen for an antagonist depends on several factors, including the serum turnover rate of the antagonist, the serum level of ARPR associated with the disorder being treated, the immunogenicity of the antagonist, the accessibility of the target ARPR (e.g. if non-serum ARPR is to be blocked), the relative affinity of ARPR to its receptor(s) versus ARPR to the antagonist, and the like. Preferably, an administration regimen maximizes the amount of antagonist delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of antagonist delivered depends in part on the particular antagonist and the severity of the condition being treated. Guidance in selecting appropriate doses is found in the literature on therapeutic uses of antibodies, e.g. Bach et al., chapter 22, in Ferrone et al., eds., Handbook of Monoclonal Antibodies (Noges Publications, Park Ridge, NJ, 1985); and Russell, pgs. 303-357, and Smith et al., pgs. 365-389, in Haber et al., eds. Antibodies in Human Diagnosis and Therapy (Raven Press, New York, 1977). Preferably, whenever the antagonist comprises monoclonal antibodies or Fab-sized fragments thereof (including binding compositions), the dose is in the range of about 1-20 mg/kg per day. More preferably the dose is in the range of about 1-10 mg/kg per day.

Example 1

Chemical Synthesis of Mature ch 10- ARPR In this example, chlO-ARPR having the sequence of Fig. 2 (SEQ ID NO: 2) is synthesized with the method and materials of the invention. The full length polypeptide is assembled from the previously synthesized oligopeptide intermediates listed below (the superscripted numbers indicate the position of the fragments in the sequence of Fig. 2). Fragment 1 is initially coupled to fragment 2 to give a first product, then after preparative HPLC purification, the first product is coupled to fragment 3 to give a second product. Again, after preparative HPLC purification, the second product is coupled to fragment 4 to give the desired polypeptide, which is purified and refolded.

Thioester formation. Fragments 2, 3, 4 were synthesized on a thioester generating resin, as described above. For this purpose two solid phase supports were prepared: S-acetylthioglycolic acid pentafluorophenylester for one resin or S-trityl mercaptopropionic acid for another resin is coupled to a Leu-PAM resin under conditions essentially as described by Hackeng et al (1999). In the first case, the resulting resin is used as a starting resin for peptide chain elongation on a 0.2 mmol scale after removal of the acetyl protecting group with a 30 min treatment with 10% mercaptoethanol, 10% piperidine in DMF. The thioester is formed with Boc-Ile-OH for synthesis of fragment 3 and Boc-Phe-OH for fragment 4 using conventional in situ neutralization coupling for 1 hour, Schnolzer et al (cited above). In the second case, removal of the trityl protecting group is achieved with two l-rnin treatments with 2.5% triisopropylsilane and 2.5% H₂0 in TFA. The first amino acid (Boc-Ala-OH for fragment 2) is immediately coupled manually to the resin using the conventional in situ neutralization coupling protocol for lh. The N^α of the N-terminal Cys residues of fragments 2 and 3 were protected in accordance with the invention by coupling a Boc-thioproline (Boc-SPr, i.e. Boc-L-thioproline, ) to the terminus of the respective chains instead of a Cys having conventional N^αor S^β protection, e.g. Brik et al, J. Org. Chem., 65: 3829-3835 (2000).

Peptide synthesis. Solid-phase synthesis is performed on a custom-modified 433A peptide synthesizer from Applied Biosystems, using in situ neutralization/2-(lH-benzotriazol-l-yl)-l,l,l,3,3- tetramethyluronium hexafluoro-phosphate (HBTU) activation protocols for stepwise Boc chemistry chain elongation, as described by Schnolzer et al, Int. J. Peptide Protein Res., 40: 180-193 (1992). Each synthetic cycle consisted of N™-Boc -removal by a 1 to 2 min treatment with neat TFA, a 1-min DMF flow wash, a 10-min coupling time with 2.0 mmol of preactivated Boc-amino acid in the presence of excess DIEA and a second DMF flow wash. N -Boc-amino acids (2 mmol) were preactivated for 3min with 1.8mmol HBTU (0.5M in DMF) in the presence of excess DIEA (6mmol). After coupling of Gin residues, a dichloromethane flow wash is used before and after deprotection using TFA, to prevent possible high temperature (TFA/DMF)-catalyzed pyrrolidone carboxylic acid formation. Side-chain protected amino acids were Boc-Arg(p-toluenesulfonyl)-OH, Boc-Asn(xanthyl)-OH, Boc-Asp(0- cyclohexyl)-OH, Boc-Cys(4-mefhylbenzyl)-OH, Boc-Glu(0-cyclohexyl)-OH, Boc- His(dinitrophenylbenzyl)-OH, Boc-Lys(2-Cl-Z)-OH, Boc-Ser(benzyl)-OH, Boc-Thr(benzyl)-OH, Boc- Trp(formyl)-OH and Boc-Tyr(2-Br-Z)-OH (Orpagen Pharma, Heidelberg, Germany). Other amino acids were used without side chain protection. C-terminal Fragment 1 is synthesized on Boc-Leu-0-CH₂-Pam resin (0.71mmol/g of loaded resin), while for Fragments 2 and 4 machine-assisted synthesis is started on the Boc-Xaa-S-CH₂-CO-Leu-Pam resin and for fragment 3 on Boc-Xaa-S-(CH₂)₂-CO-Leu-Pam resin. These two later resins were obtained by the coupling of S-acetylfhioglycolic acid pentafluorophenylester or S-trityl mercaptopropionic acid to a Leu-PAM resin under standard conditions; in the first case the resulting resin is used as a starting resin for peptide chain elongation on a 0.2 mmol scale after removal of the acetyl protecting group with a 30min treatment with 10% mercaptoethanol, 10% piperidine in DMF. The thioester is formed with Boc-Ile-OH for synthesis of fragment 3 and Boc-Phe-OH for fragment 4 using the standard in situ neutralization coupling for lh. In the second case, removal of the trityl protecting group is achieved with two 1-min treatments with 2.5% triisopropyl silane and 2.5% H₂0 in TFA. The first amino acid (Boc-Ala-OH for fragment 2) is immediately coupled manually to the resin using the standard in situ neutralization coupling protocole for lh.

After chain assembly is completed, the peptides were deprotected and cleaved from the resin by treatment with anhydrous hydrogen fluoride for lhr at 0°C with 5% p-cresol as a scavenger. In all cases except Fragment 1, the imidazole side chain 2,4-dinitrophenyl (DNP) protecting groups remained on His residues because the DNP-removal procedure is incompatible with C-terminal thioester groups. However DNP is gradually removed by thiols during the ligation reaction, yielding unprotected His. After cleavage, both peptides were precipitated with ice-cold diethylether, dissolved in aqueous acetonitrile and lyophilized. The peptides were purified by RP-HPLC with a C18 column from Waters by using linear gradients of buffer B (acetonitile/0.1 % trifluoroacetic acid) in buffer A (H₂O/0.1 % trifluoroacetic acid) and UV detection at 214nm. Samples were analyzed by electrospray mass spectrometry (ESMS) using an Esquire instrument (Briicker, Bremen , Germany). Native chemical ligations. As described more fully below, the ligation of unprotected fragments is performed as follows: the dry peptides were dissolved in equimolar amounts in 6M , guanidine hydrochloride (GuHCl), 0.2M phosphate, pH 7.5 in order to get a final peptide concentration of 1-8 mM at a pH around 7, and 1 % benzylmercaptan, 1 % thiophenol is added. Usually, the reaction is carried out overnight and is monitored by HPLC and electrospray mass spectrometry. The ligation product is subsequently treated to remove protecting groups still present. The formyl group of Tip is cleaved by shifting the pH of the solution up to 9.0 with hydrazine and incubating for lh at 37°C. Opening of the N-terminal thiazolidine ring further required the addition of solid methoxamine to a 0.5M final concentration at pH3.5 and a further incubation for 2h at 37 °C. A 10-fold excess of Tris(2- carboxyethyl)phosphine is added before preparative HPLC purification. Fractions containing the polypeptide chain were identified by ESMS, pooled and lyophilized.

The ligation of SPr⁸⁷-(87-134) -αCOSR and Cys¹³⁵(135-161)-COOH peptides is performed at pH7.0 in 6 M GuHCl. The concentration of each reactant is 8mM, and 1 % benzylmercaptan and 1 % thiophenol were added to create a reducing environment and facilitate the ligation reaction. An almost quantitative ligation reaction is observed after overnight stirring at 37°C. ESMS confirmed the molecular mass for the SPr⁸⁷-(87-161)-COOH polypeptide chain of 8720.3Da, in good agreement with the calculated average isotopic mass of 8719.18Da. At this point in the reaction, CH₃-0-NH₂.HCl is added as a powder to a 0.1 M final concentration and hydrazine added to shift the pH to 9.0, for the removal of the formyl group of Trp^12S. After a lh incubation at 37°C, CH₃-0-NH₂.HCl is further added to the solution to get a 0.5M final concentration, and the pH adjusted to 3.5 in order to open the thiazolidine ring of SPr⁸⁷. After 2h incubation at 37°C, ESMS confirmed the removal of the two protecting groups (Table 1). The reaction mixture is subsequently treated with a 10-fold excess of Tris(2-carboxyethylphosphine) over the peptide and after 15min, the ligation product is purified using the preparative HPLC (C4, 20-60% CH₃CN, 0.5% per min), lyophilised and stored at -20°C. The same procedure is repeated for ligations 2 and 3 with some slight modifications. Since ligation reaction 2 involves an Ile-Cys ligation site, which is one of the less favorable ones according to a comparative study (7), the ligation reaction is carried over 48h. Furthermore only the formyl group involved in the N-terminal thiazolidine ring of SPr⁴⁵ has to be removed to make the peptide ready to be used in the final ligation reaction. According to the HPLC profile, only a 50% ligation could be obtained after 48h incubation. Ligation 3 didn't require any further work-up of the full length peptide, but the mass obtained for the final polypeptide (18274.6) is in agreement with the N-terminal Gin already cyclized into pGlu with a loss of NH₃(calculated value, 18275.39), which is a common reaction of N-terminal Gin in peptides or proteins when incubated at neutral or slightly basic pH, which happens also for biologically expressed proteins. Polypeptide Folding. The full length peptide is refolded by air oxidation by dissolving the reduced lyophilized protein (about 0.1 mg/mL) in IM GuHCl, lOOmM Tris, lOmM methionine, pH 8.6 After gentle stirring overnight, the protein solution is purified by RP-HPLC as described above.

Example 2 Monoclonal Antibodies Specific for ARPR

A male Lewis rat is immunized with semi-purified preparations of COS 7-cell expressed ARPR. The rat is first immunized with approximately 50 μg of ARPR in Freund's Complete Adjuvant, and boosted twice with the same amount of material in Freund's Incomplete Adjuvant. Test bleeds are taken. The animal is given a final boost of 25 μg in phosphate-buffered saline, and four days later the spleen is obtained for fusion.

Approximately 3 x 10⁸ rat splenocytes are fused with an equal number of P3X63-AG8.653 mouse myeloma cells (available from the ATCC under accession number CRL 1580). 3840 microtiter plate wells are seeded at 5.7 x 10⁴ parental myeloma cells per well. Standard protocols for the fusion and subsequent culturing of hybrids are followed, e.g. as described by Chretien et al, J. Immunol. Meth., Vol. 117, pgs. 67-81 (1989). 12 days after fusion supernatants are harvested and screened by indirect ELISA on PVC plates coated with COS 7-produced ARPR.

Example 3

Antibodies Specific for the CLDTKGKEN-Peptide 50 mg of ovalbumin (OVA) and 50 mg of myoglobulin (MYO) (e.g. available from Sigma) are each dissolved in 10 ml of 0.1 M sodium bicarbonate, and reacted with 1 ml of 0.12 iodoacetamide solution (88 mg of iodoacetamide dissolved in 4 ml 0.1 M sodium bicarbonate) for 1 hour at room temperature in a 15 ml Falcon tube (Falcon Plastics, Oxnard, CA), or the like. Each reaction mixture is dialyzed overnight against 4 liters of 0.1 M sodium bicarbonate at 4RC. Separately, 10 mg of CLDTKGKEN is dissolved in 2 ml of 0.1 M DTT (dithiotheitol) solution (containing 50 mM Tris and 2.5 mM EDTA at pH8) in a 4 ml tube, incubated at 37°C overnight; and then applied to a GF05 gel- filtration column (1.5 x 26.5 cm) (LKB, Bromma, Sweden) and eluted with a peptide elution buffer consisting of 0.015 M acetic acid and 0.005 M beta-mercaptoethanol. Three fractions of about 3.5 ml each which contained the reduced peptide are identified by optical density at 206 nm, collected, pooled, frozen in dry ice, and lyophilized overnight. Meanwhile OVA and MYO are recovered from dialysis, and clarified by filtration through 0.45 micrometer filters. OVA and MYO are activated by mixing each with 380 microliters of N-hydroxysuccinimide ester of iodoacetic acid (NHIA) (disclosed by Rector et al., in J. Immunol. Meth., Vol. 24, pg. 321 (1978)) dissolved in tetrahydrofuran (THF) (5 mg/ml); stirring for 30 minutes at room temperature, and dialyzing overnight against 4 liters PBS (1.8 g NaH₂P0₄-H₂0, 7.2 g Na₂HP04-H₂0; and 34g NaCl in 4 liters H₂0). Separately the lyophilized peptide is resuspended in 5 ml of borate reduction buffer (2 g Na₂B4θ7-10H₂O, 17.4 g NaCl, and 336 mg EDTA-Na₂ in liter H₂0 with pH adjusted to 8.5 with concentrated HC1, deoxygenated under nitrogen for 15 minutes, after which 178 mg ascorbate is added). The dialyzed iodoacetylated OVA and MYO are recovered, separately mixed with equal volumes (preferably 2 ml) of borate reduction buffer containing the peptide, and incubated overnight at room temperature. The resulting conjugates are analyzed by SDS-PAGE (12.5% gel). The conjugate containing solution is diluted with PBS to 1 mg/ml, sterile filtered, and aliquotted to convenient volumes (e.g. 500 microliters) for immunizations, and/or stored at 4°C. Polyclonal anti-sera against the MYO conjugate is produced in both rats and rabbits (New Zealand White). The immunization schedule for rabbits is as follows: Initially (week 0) a 10 ml sample of serum is extracted as a control. One week later (week 1) 0.5 ml of peptide-carrier conjugate is mixed with 0.5 ml Freund's Complete Adjuvant and injected I. P. Three weeks later (week 4) a booster is given consisting of 0.5 ml peptide-carrier conjugate mixed with 0.5 ml Freund's Incomplete Adjuvant. The following week (week 5) an additional booster is given, again consisting of 0.5 ml peptide-carrier conjugate mixed with 0.5 ml Freund's Incomplete Adjuvant, followed by yet another identical booster the next week (week 6). On week 7, 20 ml of serum is bled from the animal. After separating out the cellular fraction the serum assayed for positive anti-CLDTKGKEN titer by ELISA. Rat immunization proceed similarly except that the initial injection consists of 0.15 ml PBS and 0.1 ml peptide-carrier conjugate mixed with 0.75 ml Freund's Complete Adjuvant, boosters consisted of 0.15 ml PBS and 0.1 ml peptide-carrier conjugate mixed with 0.75 ml Freund's Incomplete Adjuvant, and only 2-3 ml of serum is bled from the rat. Again, a positive anti-CLDTKGKEN reaction is detected by ELISA.

The descriptions of the foregoing embodiments of the invention have been presented for purpose of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention to thereby enable others skilled in the art to best utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto.

Example 4

ARPR Expression

1) Real time PCR monitoring of chlO-ARPR mRNA using SyBR Green A Real-Time PCR run was performed using SyBR Green and primers designed in the human

ARPR coding sequence. Using the chlO-ARPR gene sequence, two ESTs were identified, AZ537498 and AQ412924. AZ537498 was identified in PC-3 human prostatic carcinoma library, Homo sapiens. Primers matching EST sequences were designed as follows: 5 'primer: 5'-AAAAACACTGGACTTGGCATCA-3' (SEQ ID NO 30) 3-primer: 5'-TATGCAGGATCTGCTTCAGCT-3' (SEQ ID NO 31)

Results from real-time PCR in monkey organs is shown in Figure 5. The expression values for the ARPR mRNA are lower than for Beta-actin (ratio < 1) except for parotid gland, thyroid with parathyroid, adipose tissue (mesenteric) and muscle (gastronemius).

2) Real time PCR using primers designed in 5' and 3' ends of ARPR

A real time PCR run was performed using SyBR Green with primers designed in the extreme 5' end of the coding sequence in the mRNA, 5'-GCATGTGGTGCGCGAG-3 ' (SEQ ID NO 32); and the transcribed but not translated region at the 3 ' end of the mRNA, 5 '-TGCTGGCATTGGAGATCAGA-3 ' (SEQ ID NO 33).

The PCR product was analysed by agarose gel electrophoresis, showing an abundant PCR product in muscle indicating expression of the ARPR gene in this tissue. Observed were PCR products of approximately 600bp and 450bp.

3) Alternative splicing Further PCR runs were carried out to test for alternative splicing of the sequence corresponding to exon 2, resulting in an ARPR polypeptide (SEQ ID NO 27, and SEQ ID NO 28 for mature protein) encoded by a nucleic acid containing exons 1 and 3 (SEQ ID NO 29).

Primers were designed in exons 1 and 3: Exon 1 primer (forward): 5'-TTGCTGTGGTGGCCTTTT-3' (SEQ ID NO 34)

Exon 3 primer (reverse): 5'-ATATGCACACTCATTGGGCGA-3' (SEQ ID NO 35)

The result of a 40-cycle PCR was analysed after 2% gel electophoresis. The main PCR product was observed at about 300bp in all tissues tested, including parotid, thyroid, spleen liver, and skeletal muscle. However, in skeletal muscle an additional 170bp band was observed corresponding to the expected size for an alternatively spliced mRNA form lacking exon 2.

The 170bp and 300bp PCR products were sequenced using protocols for cycle sequencing using BigDye terminator chemistry. Sequencing reactions were analysed using an ABI PRISM 3700 96- capillary sequencer. The nucleotide sequence of the 300bp product was found to correspond to an mRNA comprising exons 1 , 2 and 3 (corresponding to the cDNA sequence of SEQ ID NO 8). The sequence of the 170bp product was found to correspond to to an mRNA lacking exon 2, as shown in SEQ ID NO 29.

Several references have been cited in the present patent application. All referenced cited are incorporated herein by reference.

Claims

WE CLAIM:

1. An isolated mature protein of a polypeptide having a sequence selected from SEQ ID NO: 1, SEQ ID NO: 13 and SEQ ID NO: 27.

2. The protein of claim 1 having an amino acid sequence selected from SEQ ID NO: 2 and SEQ ID NO: 28.

3. The protein of claim 1 having an amino acid sequence of SEQ ID NO 14.

4. An isolated polypeptide having an amino acid sequence at least ninety percent identical to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 27 and SEQ ID NO: 28.

5. The isolated polypeptide of claim 4 having an amino acid sequence as least ninety percent identical to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

6. An isolated peptide composition consisting of a pair of peptide fragments, each peptide fragment of the pair having a sequence selected without replacement from the group of consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

7. An isolated polynucleotide having a nucleotide sequence that is at least 95 percent identical to SEQ ID NOS: 8 or 29, or to nucleotides 600 to 1700 or 10,100 to 11,200 or 12,800 to 13,900 of SEQ

ID NO: 6, or to nucleotides 2600 to 3800 of SEQ ID NO: 7.

8. An isolated polynucleotide having a nucleotide sequence that is at least 95 percent identical to a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 29, any complementary sequences thereof, and any sequence of 100 to 1000 consecutive nucleotides of SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, SEQ ID NO 29 and any complementary sequences thereof.

9. An isolated polynucleotide having a length of at least 15 nucleotides, the polynucleotide forming a detectable duplex with a sense or antisense strand of a second polynucleotide under the following hybridization conditions: for 1-15 ng/μL of isolated polynucleotide probe hybridizing to said second nucleic acid bound to a nitrocellulose filter, hybridization in 25 mM KP0 (pH 7.4), 5X SSC, 5X Denhart's solution, 50 mg/mL denatured sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate at 42°C, with washes at 65°C in 2X SSC and 0.1 % SDS for 30 minutes, and wherein said second polynucleotide having a sequence as set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 29, or any sequence of 100 to 1000 consecutive nucleotides of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO 29.

10. A purified or isolated nucleic acid selected from the group consisting of:

(i) a nucleic acid molecule encoding a ch 10- ARPR polypeptide according to claims 1 to 6; (ii) a nucleic acid molecule encoding a ch 10- ARPR polypeptide or a biologically active fragment thereof, comprising the nucleic acid sequence selected from the group consisting of SEQ ID NO 6 to 12 and 29, fragments thereof, and the sequences complementary thereto; and

(iii) a nucleic acid the sequence of which is degenerate as a result of the genetic code to the sequence of a nucleic acid as defined in (i) and (ii).

11. The isolated polynucleotide of any one of claims 7 to 10 having a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO 28.

12. A monoclonal antibody that specifically binds to an ARPR protein of SEQ ID NO: 2.

13. A monoclonal antibody that specifically binds to an ARPR protein of SEQ ID NO: 27.

14. A peptide consisting of 6 to 30 amino acids whose sequence is identical to a subsequence of a mature ARPR of SEQ ID NO: 2 or SEQ ID NO: 27.

15. The nucleic acid of any one of claims 7 to 10, wherein said nucleic acid is operably linked to a promoter.

16. An expression cassette comprising the nucleic acid of claim 15.

17. A host cell comprising the expression cassette of claim 16.

18. A method of making a chlO-ARPR polypeptide, said method comprising providing a population of host cells comprising a recombinant nucleic acid encoding a chlO- ARPR protein of any of Claims 1 to 6; and culturing said population of host cells under conditions conducive to the expression of said recombinant nucleic acid; whereby said polypeptide is produced within said population of host cells.

19. The method of claim 18, further comprising purifying said polypeptide from said population of cells.

20. An isolated chlO-ARPR polypeptide, wherein said polypeptide is encoded by a nucleic acid of SEQ ID NOS 8 or 29.

21. An isolated chlO-ARPR polypeptide or fragment thereof, said polypeptide comprising at least 12 contiguous amino acids of a sequence selected from the group consisting of SEQ JD NOS 1, 2, 27 or

28.

22. A method of determining whether an ARPR polypeptide is expressed within a biological sample, said method comprising the steps of : (a) contacting a biological sample with: a polynucleotide that hybridizes under stringent conditions to a nucleic acid of any one of claims 7 to 10 or a detectable polypeptide that selectively binds to the polypeptide of any one of claims 1 to 6; and (b) detecting the presence or absence of hybridization between said polynucleotide and an RNA species within said sample, or the presence or absence of binding of said detectable polypeptide to a polypeptide within said sample; wherein a detection of said hybridization or of said binding indicates that said ARPR-family polypeptide is expressed within said sample.

23. A method of determining whether a biological sample contains a polypeptide of polynucleotide originating from a muscle cell, said method comprising the steps of :

(a) contacting a biological sample with: a polynucleotide that hybridizes to a nucleic acid of SEQ ID NO: 29 but that does not hybridize to a nucleic acid of Claim SEQ ID NO 8 under stringent conditions; or a detectable polypeptide that selectively binds to a polypeptide of SEQ ID NOS 27 or 28 and does not substantially bind to a polypeptide of SEQ ID NOS 1 or 2; and

(b) detecting the presence or absence of hybridization between said polynucleotide and an RNA species within said sample, or the presence or absence of binding of said detectable polypeptide to a polypeptide within said sample; wherein a detection of said hybridization or of said binding indicates that said sample contains a polypeptide or polynucleotide originating from a muscle cell.

24. The method of claim 23, wherein said polynucleotide is a primer, and wherein said hybridization is detected by detecting the presence of an amplification product comprising said primer sequence.

25. The method of claim 23, wherein said detectable polypeptide is an antibody.

26. An isolated or purified nucleic acid encoding a chlO-ARPR signal or fragment thereof comprising an amino acid sequence of at least 8 contiguous amino acids of amino acid residues 1 to 24 of SEQ ID NO 1 or 1 to 26 of SEQ ID NO 27.

27. The nucleic acid of claim 26, wherein said nucleic acid is operably linked to a nucleic acid encoding a protein of interest.

28. The nucleic acid of claim 26, wherein said protein of interest is a polypeptide according to claims 1 to 6.

29. The nucleic acid of claims 27 or 28, wherein said nucleic acid is operably linked to a promoter.

30. An expression cassette comprising the nucleic acid of claim 29.

31. A host cell comprising the expression cassette of claim 30.

32. A method of binding an ARPR protein comprising: a) providing an antibody composition according to any of claims 12 or 13 and b) bringing said antibody composition into contact with a composition comprising an ARPR protein.

33. The method of claim 32 wherein said ARPR protein is the protein of SEQ ID NOS 27 or 28.

34. The method of claim 32 wherein said ARPR protein is the protein of SEQ ID NOS 1 or 2.