EP1328632A2

EP1328632A2 - C1q-related factor, homologous polypeptides and therapeutic uses thereof

Info

Publication number: EP1328632A2
Application number: EP01958853A
Authority: EP
Inventors: Eric Wen Su
Original assignee: Eli Lilly and Co
Current assignee: Eli Lilly and Co
Priority date: 2000-08-04
Filing date: 2001-07-23
Publication date: 2003-07-23
Also published as: WO2002012475A3; WO2002012475A2; AU2001280464A1

Abstract

The present invention provides nucleic acid sequences encoding novel human CRF-like protein having some similarity to cerebellin. These novel nucleic acids are useful for constructing the claimed DNA vectors and host cells of the invention and for preparing the claimed recombinant proteins and antibodies.

Description

Clq-RELATED FACTOR, HOMOLOGOUS POLYPEPTIDES AND THERAPEUTIC USES THEREOF

FIELD OF THE INVENTION The present invention relates to the identification and isolation of novel DNA, therapeutic uses and the recombinant production of novel polypeptides having sequence homology to human cerebellum, designated herein as LP231 polypeptide. The present invention also relates to vectors, host cells, and antibodies directed to LP231 polypeptides.

• BACKGROUND OF THE INVENTION

Recently a human Clq-related factor (CRF) was cloned and characterized as a 258 amino acid polypeptide having a hydrophobic signal sequence, a collagenous region and globular C terminal domain (Berube NG, et al , Cloning and characterization of CRF, a novel Clq-related factor, expressed in areas of the brain involved in motor function. Brain Res Mol Brain Res . 63 (2) :233-40, 1999).^' This protein is highly similar to the corresponding mouse protein suggesting an important conserved role in neural function ( id. ) CRF is most prevalent in areas of the brain stem associated with motor function, such as Purkinje cells of the cerebellin ( id. )

The cerebellum contains a hexadecapeptide, termed cerebellin. Three independent, overlapping cDNA clones were isolated from a human cerebellum cDNA library that encode the cerebellin sequence. The longest clone codes for a protein of 193 amino acids that has been named precerebellin. Proc Natl Acad Sci USA: 88 (3 ): 1069-73 ; Feb 1(1991). This protein has a significant similarity (31.3% identity, 52.2% similarity) to the globular region of the B chain of human complement component Clq. The region of relatedness extends over approximately 145 amino acids located in the carboxyl terminus of both proteins.

The precerebellin amino terminus contains three N-linked glycosylation sites but does not show a classical signal- peptide motif. No other obvious membrane-spanning domains were predicted from the cDNA sequence. The cDNA predicts that cerebellin is flanked by Val-Arg and Glu-Pro residues. Therefore, cerebellin is liberated from precerebellin by some means other than the classical dibasic amino acid proteolytic- cleavage mechanism seen in many neuropeptide precursors.

Precerebellin transcripts are abundant in the cerebellum but are present at either very low or undetectable levels in other brain areas. During rat development, it was shown that precerebellin transcripts mirror the level of cerebellin. Low levels of precerebellin mRNA are seen at birth. Levels increase modestly from postpartum day 1 to 8, then increase more dramatically between day 5 and 15. Eventually, they reach peak values between day 21 and 56. Proc Natl Acad Sci USA: 88 (3) :1069-73; Feb 1(1991). Moreover, cerebellin-like immunoreactivity has been shown to be associated with Purkinje cell poεtsynaptic structures which suggests that the cerebellin precursor may be involved in synaptic physiology.

A murine homolog of precerebellin, Cblnl, and a closely related gene, Cbln2 have been cloned. Brain Res Mol Brain Res; 27(l):152-6 (1994). Amino acid comparison of Cblnl with Cbln2 showed that Cbln2 is 88% identical to the carboxy terminal region of Cblnl. Southern blot analysis and genome mapping confirmed that these are independent genes.

Recently, cerebellin-2 was described in W09942576-A1 and bears significant homology to LP231. Cerebellin-2 was indicated as useful in treating or preventing neurological disorders associated with the inappropriate expression of cerebellin-2 proteins and disruption of the synapse function. Owing to its homology to cerebellin-2 and CRF, LP231 is thought to be useful for treating neurologic disorders that include Parkinson's disease, Alzheimer's disease, bipolar and unipolar affective disorders, schizophrenia, olivopontocerebellar atrophy, Shy-Dager syndrome and other disorders caused by disruption of synapse function. LP231 is also useful as an antigen in vaccine and antibody production as well as in assays to identify agonists and antagonists of LP231 function.

In general, all novel proteins are of interest. Extracellular proteins play an important role in the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, e.g., proliferation, migration, differentiation, or interaction with other cells, is typically governed by information received from other cells and/or the immediate environment. This information is often transmitted by secreted polypeptides (for instance, mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones) which are, in turn, received and interpreted by diverse cell receptors or membrane-bound proteins . These secreted polypeptides or signaling molecules normally pass through the cellular secretory pathway to reach their site of action in the extracellular environment.

Secreted proteins have various industrial applications, including pharmaceuticals, diagnostics, biosensors and bioreactors. Most protein drugs available at present, such as thrombolytic agents, interferons, interleukins, erythropoietins, colony stimulating factors, and various other cytokines, are secretory proteins. Their receptors, which are membrane proteins, also have potential as therapeutic or diagnostic agents .

Efforts are being undertaken by both industry and academia to identify new, native secreted proteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted proteins . Examples of screening methods and techniques are described in the literature [see, for example, Klein et al . , Proc . Natl . Acad. Sci . , B: 7108-13 (1996); U.S. Patent No. 5,536,637)]. The results of such efforts are presented herein.

SUMMARY OF THE INVENTION The present invention provides nucleic acid sequences encoding the novel human LP231 polypeptide. These novel nucleic acids are useful for constructing the claimed DNA vectors and host cells of the invention and for preparing the claimed recombinant proteins and antibodies . In particular, a full length human EST cDNA clone is disclosed that contains a 675 nucleotide open reading frame encoding a 225 amino acid homologue of human CRF that bears substantial similarity to known cerebellins.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Applicants have identified a cDNA clone that encodes a novel polypeptide having sequence identity with human CRF and cerebellin, wherein the polypeptide is designated in the present application as "LP231" . In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding the LP231 polypeptide. One aspect of the invention concerns an isolated nucleic acid molecule encoding the LP231 polypeptide comprising DNA hybridizing to the complement of the nucleic acid between about base pairs: (a) 1 to about 982, inclusive, of SEQ ID NO:l and (b ) 55 to about 729, inclusive, of SEQ ID NO:l shown in bold.

CCGACTCCCTGGTTCAAAGTCAGCTATCTGACTCCGCACCCTCGCCATGTAGTCATGCAGACCTCAGCGCCA CCTCTCCTCCTCCTGTCATCCAGGTACCAAGGTCGATCTGGGCGACCTGGCAAGTCCAGTCCGCGGGGCTCC TCTGGAGAGACGGCCCGTCCTGGACCCTGGCGGCCTCCGGGAGAGAAGGGCGACTCGGCGCGGCCCGCGCTC CTAGGTCTGCAACTGACGGACGGCACGGCCAGCGGCGTCGGGGTGGTGGCGCGCGGGGCCGGGGTAGGTGGC GATTCCGAGGGTGAAGTGACCAGTGCGCTGAGCGCCACCTTCAGCGGCCCCAAGATCGCCTTCTATGTGGGT CTCAAGAGCCCCCACGAAGGCTATGAGGTGCTGAAGTTCGATGACGTGGTCACCAACCTCGGCAATCACTAT GACCCCACCACGGGCAAGTTCAGCTGCCAGGTACGCGGCATCTACTTCTTCACCTACCACATCCTCATGCGC GGCGGCGACGGCACCAGCATGTGGGCGGACCTCTGCAAGAACGGGCAGGTCCGGGCCAGCGCCATTGCACAG GACGCCGACCAGAACTACGACTACGCCAGTAACAGCGTGGTGCTGCACTTGGATTGAGGGGACGAAGTGTAT GTGAAGCTGGATGGCGGGAAGGCTCACGGAGGCAATAATAACAAGTACAGCACGTTCTCGGGCTTTCTTCTG TACCCGGATTAGGGGCGCGGGGGGTGCGAGGCGGGGTGGCTGCAGGCGCCCCGGTCTCCGCCCGGGCGCGGC TCCTTGGCAAAGGCCACTCTCGATTCATAACACTTCCTGACATCTCCTTTGGAAAAGACAAATCCCTGCGTC CTCCCTGCCCCGCTCCTGGCCTCAGTGCGTCTGCGACCCACCACGCTCAGGGCTGTGCTCCTGGTCTCCATC CCCATCCCGGCAAGGGAGGAAGGGACGCCCGAGCCCTTGAGGCGG

(SEQ ID NO: 1)

In another aspect, the isolated nucleic acid comprises DNA encoding the LP231 polypeptide having amino acid residues from about 1 through 225 of SEQ ID NO: 2, or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, high stringency conditions.

In another embodiment, the isolated nucleic acid comprises DNA having at least about 91% sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity, yet more preferably at least about 99% sequence identity to (a) a DNA molecule encoding an LP231 polypeptide comprising the sequence of amino acid residues from 1 or about 20 to 225, inclusive, of SEQ ID NO: 2 or (b) the complement of the DNA molecule of (a) . Alternatively, the isolated nucleic acid comprises DNA encoding the LP231 polypeptide having the sequence of amino acid residues from about 1 to about 225, inclusive, of SEQ ID NO: 2.

+1 Met Gin

1 CCG ACT CCC TGG TTC AAA GTC AGC TAT CTG ACT CCG CAC CCT CGC CAT GTA GTC ATG CAG GGC TGA GGG ACC AAG TTT CAG TCG ATA GAC TGA GGC GTG GGA GCG GTA CAT CAG TAC GTC

+₃ Thr Ser Ala Pro Pro Leu Leu Leu Leu Ser Ser Arg Tyr Gin Gly Arg Ser Gly Arg Pro

61 ACC TCA GCG CCA CCT CTC CTC CTC CTG TCA TCC AGG TAC CAA GGT CGA TCT GGG CGA CCT TGG AGT CGC GGT GGA GAG GAG GAG GAC AGT AGG TCC ATG GTT CCA GCT AGA CCC GCT GGA

+2₃ Gly Lys Ser Ser Pro Arg Gly Ser Ser Gly Glu Thr Ala Arg Pro Gly Pro Trp Arg Pro

121 GGC AAG TCC AGT CCG CGG GGC TCC TCT GGA GAG ACG GCC CGT CCT GGA CCC TGG CGG CCT

CCG TTC AGG TCA GGC GCC CCG AGG AGA CCT CTC TGC CGG GCA GGA CCT GGG ACC GCC GGA +43 Pro Gly Glu Lys Gly Asp Ser Ala Arg Pro Ala Leu Leu Gly Leu Gin Leu Thr Asp Gly

181 CCG GGA GAG AAG GGC GAC TCG GCG CGG CCC GCG CTC CTA GGT CTG CAA CTG ACG GAC GGC

GGC CCT CTC TTC CCG CTG AGC CGC GCC GGG CGC GAG GAT CCA GAC GTT GAC TGC CTG CCG

+63 Thr Ala Ser Gly Val Gly Val Val Ala Arg Gly Ala Gly Val Gly Gly Asp Ser Glu Gly

241 ACG GCC AGC GGC GTC GGG GTG GTG GCG CGC GGG GCC GGG GTA GGT GGC GAT TCC GAG GGT

TGC CGG TCG CCG CAG CCC CAC CAC CGC GCG CCC CGG CCC CAT CCA CCG CTA AGG CTC CCA

+83 Glu Val Thr Ser Ala Leu Ser Ala Thr Phe Ser Gly Pro Lys lie Ala Phe Tyr Val Gly

301 GAA GTG ACC AGT GCG CTG AGC GCC ACC TTC AGC GGC CCC AAG ATC GCC TTC TAT GTG GGT

CTT CAC TGG TCA CGC GAC TCG CGG TGG AAG TCG CCG GGG TTC TAG CGG AAG ATA CAC CCA

+103 Leu Lys Ser Pro His Glu Gly Tyr Glu Val Leu Lys Phe Asp Asp Val Val Thr Asn Leu

361 CTC AAG AGC CCC CAC GAA GGC TAT GAG GTG CTG AAG TTC GAT GAC GTG GTC ACC AAC CTC

GAG TTC TCG GGG GTG CTT CCG ATA CTC CAC GAC TTC AAG CTA CTG CAC CAG TGG TTG GAG

+123 Gly Asn His Tyr Asp Pro Thr Thr Gly Lys Phe Ser Cys Gin Val Arg Gly He Tyr Phe

421 GGC AAT CAC TAT GAC CCC ACC ACG GGC AAG TTC AGC TGC CAG GTA CGC GGC ATC TAC TTC

CCG TTA GTG ATA CTG GGG TGG TGC CCG TTC AAG TCG ACG GTC CAT GCG CCG TAG ATG AAG

+143 Phe Thr Tyr His He Leu Met Arg Gly Gly Asp Gly Thr Ser Met Trp Ala Asp Leu Cys

481 TTC ACC TAC CAC ATC CTC ATG CGC GGC GGC GAC GGC ACC AGC ATG TGG GCG GAC CTC TGC

AAG TGG ATG GTG TAG GAG TAC GCG CCG CCG CTG CCG TGG TCG TAC ACC CGC CTG GAG ACG

+163 Lys Asn Gly Gin Val Arg Ala Ser Ala He Ala Gin Asp Ala Asp Gin Asn Tyr Asp Tyr

541 AAG AAC GGG CAG GTC CGG GCC AGC GCC ATT GCA CAG GAC GCC GAC CAG AAC TAC GAC TAC

TTC TTG CCC GTC CAG GCC CGG TCG CGG TAA CGT GTC CTG CGG CTG GTC TTG ATG CTG ATG

+183 Ala Ser Asn Ser Val Val Leu His Leu Asp Ser Gly Asp Glu Val Tyr Val Lys Leu Asp

601 GCC AGT AAC AGC GTG GTG CTG CAC TTG GAT TCA GGG GAC GAA GTG TAT GTG AAG CTG GAT

CGG TCA TTG TCG CAC CAC GAC GTG AAC CTA AGT CCC CTG CTT CAC ATA CAC TTC GAC CTA

+203 Gly Gly Lys Ala His Gly Gly Asn Asn Asn Lys Tyr Ser Thr Phe Ser Gly Phe Leu Leu

661 GGC GGG AAG GCT CAC GGA GGC AAT AAT AAC AAG TAC AGC ACG TTC TCG GGC TTT CTT CTG

CCG CCC TTC CGA GTG CCT CCG TTA TTA TTG TTC ATG TCG TGC AAG AGC CCG AAA GAA GAC

+22₃ Tyr Pro Asp *** 721 TAC CCG GAT TAG GGG CGC GGG GGG TGC GAG GCG GGG TGG CTG CAG GCG CCC CGG TCT CCG ATG GGC CTA ATC CCC GCG CCC CCC ACG CTC CGC CCC ACC GAC GTC CGC GGG GCC AGA GGC

781 CCC GGG CGC GGC TCC TTG GCA AAG GCC ACT CTC GAT TCA TAA CAC TTC CTG ACA TCT CCT GGG CCC GCG CCG AGG AAC CGT TTC CGG TGA GAG CTA AGT ATT GTG AAG GAC TGT AGA GGA

841 TTG GAA AAG ACA AAT CCC TGC GTC CTC CCT GCC CCG CTC CTG GCC TCA GTG CGT CTG CGA AAC CTT TTC TGT TTA GGG ACG CAG GAG GGA CGG GGC GAG GAC CGG AGT CAC GCA GAC GCT

901 CCC ACC ACG CTC AGG GCT GTG CTC CTG GTC TCC ATC CCC ATC CCG GCA AGG GAG GAA GGG GGG TGG TGC GAG TCC CGA CAC GAG GAC CAG AGG TAG GGG TAG GGC CGT TCC CTC CTT CCC

961 ACG CCC GAG CCC TTG AGG CGG TGC GGG CTC GGG AAC TCC GCC

(SEQ ID NO: 2)

In another aspect, the invention concerns an isolated nucleic acid molecule encoding an active LP231 polypeptide comprising a nucleotide sequence that hybridizes to the complement of a nucleic acid sequence that encodes amino acids

(a) 1 or about 20 to about 225, inclusive, of SEQ ID NO: 2 or

(b) about 20 to about 225, inclusive, of SEQ ID NO: 2. Preferably, hybridization occurs under stringent hybridization and wash conditions. In a further aspect, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 91% sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity, yet more preferably at least about 99% sequence identity, to: (a) DNA molecule comprising the sequence of nucleotides from about 1 or about 20 to about 225, inclusive, of SEQ ID NO : 2 or (b) the complement of the DNA molecule of (a) .

In another aspect, the isolated nucleic acid molecule comprises: (a) the nucleotide sequence from about 1 or about 20 to about 225, inclusive, of SEQ ID NO: 2 or (b) the complement of the DNA molecule of (a) .

In a further aspect, the invention concerns an isolated nucleic acid molecule produced by hybridizing a test DNA molecule under stringent conditions with: (a) a DNA molecule encoding an LP231 polypeptide having the sequence of amino acid residues from about 1 or about 20 to about 225. inclusive, of SEQ ID NO : 2 , or (b) the complement of the DNA molecule of (a) , and if the DNA molecule has at least about 91% sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% εequence identity, yet more preferably at least about 99% sequence identity to (a) or (b) , and isolating the test DNA molecule .

In yet a further aspect, the invention concerns an isolated nucleic acid molecule comprising: (a) DNA encoding a polypeptide scoring at least about 91% positives, yet more preferably at least about 92% positives, yet more preferably at least about 93% positives, yet more preferably at least about 94% positives, yet more preferably at least about 95% positives, yet more preferably at least about 96% positives, yet more preferably at least about 97% positives, yet more preferably at least about 98% positives, yet more preferably at least about 99% positives, when compared with the amino acid sequence of residues about 20 to about 225 inclusive, of SEQ ID NO: 2, or (b) the complement of the DNA of (a) . In a specific aspect, the invention provides an isolated nucleic acid molecule comprising DNA encoding an LP231 polypeptide without the N-terminal signal sequence and/or initiating methionine, or is complementary to such encoding nucleic acid molecule. The signal peptide has been presumptively identified as extending from about amino acid residue 1 to about amino acid residue 20, inclusive, in SEQ ID NO: 2. It is noted, however, that the C-terminal boundary of the signal peptide may vary, but likely by no more than about 6 amino acids on either side of the signal peptide C-terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art [Nielsen et al., Prot. Engin . 10(1): 1-6 (1997); von Heijne et al . , Nucl . Acid Res 14(11): 4683-4690 (1986)]. Moreover, it is also recognized that, in some cases, cleavage of the signal sequence from a secreted polypeptide is not entirely uniform, resulting in more than one secreted species. These polypeptides, and the polynucleotides encoding them, are contemplated by the present invention.

Another embodiment is directed to fragments of a LP231- encoding sequence that may find use as, for example, hybridization probes or for encoding fragments of an LP231 polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-LP231 antibody. Such nucleic acids fragments are usually at least about 20 nucleotides in length, preferably at least about 30 nucleotides in length, more preferable at least about 40 nucleotides in length, yet more preferably at least about 50 nucleotides in length, yet more preferably at least about 60 nucleotides in length, yet more preferably at least about 70 nucleotides in length, yet more preferably at least about 80 nucleotides in length, yet more preferably at least about 90 nucleotides in length, yet more preferably at least about 100 nucleotides in length, yet more preferably at least about 110 nucleotides in length, yet more preferably at least about 120 nucleotides in length, yet more preferably at least about 130 nucleotides in length, yet more preferably at least about 140 nucleotides in length, yet more preferably at least about 150 nucleotides in length, yet more preferably at least about 160 nucleotides in length, yet more preferably at least about 170 nucleotides in length, yet more preferably at least about 180 nucleotides in length, yet more preferably at least about 190 nucleotides in length, yet more preferably at least about 200 nucleotides in length, yet more preferably at least about 250 nucleotides in length, yet more preferably at least about 300 nucleotides in length, yet more preferably at least about 350 nucleotides in length, yet more preferably at least about 400 nucleotides in length, yet more preferably at least about 450 nucleotides in length, yet more preferably at least about 500 nucleotides in length, yet more preferably at least about 600 nucleotides in length, yet more preferably at least about 700 nucleotides in length, yet more preferably at least about 800 nucleotides in length, yet more preferably at least about 900 nucleotides in length, yet more preferably at least about 1000 nucleotides in length, wherein in this context "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length. In a preferred embodiment, the nucleotide sequence fragment is derived from any coding region of the nucleotide sequence shown in SEQ ID NO:l.

In another embodiment, the invention provides a vector comprising DNA encoding an LP231 polypeptide or its variants. The vector may comprise any of the isolated nucleic acid molecules hereinabove described.

In another embodiment, the invention provides a host cell comprising the above vector. By way of example, the host cells may be CHO cells, Eεcherichia coli , or yeast. A process for producing LP231 polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of LP231 polypeptides and recovering LP231 polypeptides from the cell culture.

In another embodiment, the invention provides isolated LP231 polypeptides encoded by any of the isolated nucleic acid sequences hereinabove defined.

In another aspect, the invention concerns an isolated LP231 polypeptide, comprising an amino acid sequence having at least about 91% sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity, yet more preferably at least about 99% sequence identity to the sequence of amino acid residues about 1 or about 20 to about 225, inclusive, of SEQ ID NO:2. In a preferred aspect, the polypeptide comprises amino acid residues about 1 or about 20 to about 225 inclusive, of SEQ ID NO: 2.

In a further aspect, the invention concerns an isolated LP231 polypeptide comprising an amino acid sequence scoring at least about 91% positives, yet more preferably at least about 92% positives, yet more preferably at least about 93% positives, yet more preferably at least about 94% positives, yet more preferably at least about 95% positives, yet more preferably at least about 96% positives, yet more preferably at least about 97% positives, yet more preferably at least about 98% positives, yet more preferably at least about 99% positives, when compared with the amino acid sequence of residues from about 1 or about 20 to about 225, inclusive, of SEQ ID NO : 2.

In a specific aspect, the invention provides an isolated LP231 polypeptide without the N-terminal signal sequence and/or initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore described. Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of LP231 polypeptide and recovering the LP231 polypeptide from the cell culture.

In still a further aspect, the invention provides a polypeptide produced by: (1) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding an LP231 polypeptide having the sequence of amino acid residues from about 20 to about 225, inclusive, of SEQ ID NO: 2, or (b) the complement of the DNA molecule of (a) ; and if the test DNA molecule has at least about an 91% sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequen'ce identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity, yet more preferably at least about 99% sequence identity to (a) or (b) ; (2) culturing a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, and (3) recovering the polypeptide from the cell culture.

In yet another aspect, the invention concerns an isolated LP231 polypeptide comprising the sequence of amino acid residues from about 1 or about 20 to about 225, inclusive, of SEQ ID NO:l, or a fragment thereof which is biologically active or sufficient to provide a binding site for an anti-LP231 antibody, wherein the identification of an LP231 polypeptide or fragments thereof that possess biological activity or provide a binding site for an anti-LP231 antibody may be accomplished in a routine manner using techniques which are well known in the art.

In another embodiment, the invention provides chimeric molecules comprising an LP231 polypeptide fused to a heterologous polypeptide or amino acid sequence. An example of such a chimeric molecule comprises an LP231 polypeptide fused to an epitope tag sequence or a histidine purification handle, or an Fc region of an immunoglobulin. In another embodiment, the invention provides an antibody that specifically binds to an LP231 polypeptide or fragment thereof. Optionally, the antibody is a monoclonal antibody, an antibody fragment or a single chain antibody.

In yet another embodiment, the invention concerns agonists and antagonists of a native LP231 polypeptide. In a particular aspect, the agonist or antagonist is an anti-LP231 antibody or a small molecule.

In yet another embodiment, the invention concerns a method of identifying agonists or antagonists of a native LP231 polypeptide by contacting the native LP231 polypeptide with a candidate molecule and monitoring a biological activity mediated by said polypeptide.

In still a further embodiment, the invention concerns a composition comprising an LP231 polypeptide, or an agonist or antagonist as hereinabove defined, in combination with a carrier. Preferably, the carrier is pharmaceutically acceptable.

In still a further embodiment, the invention concerns the use of an LP231 polypeptide, or an agonist or antagonist thereof as hereinbefore described, or an anti-LP231 antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the LP231 polypeptide or an agonist or antagonist thereof (e.g., anti-LP231 antibody). In a particular aspect, the invention concerns the use of an LP231 polypeptide, or an agonist or antagonist thereof in a method for treating a neurologic disorder.

In still a further embodiment, the invention relates to a method of treating a neurologic disorder by administration of a therapeutically effective amount of an LP231 polypeptide, agonist, or antagonist thereof to a mammal suffering from said disorder. In still a further embodiment, the invention relates to a method of diagnosing a neurologic disorder by (1) culturing test cells or tissues expressing LP231; (2) administering a compound which can inhibit LP231-modulated signaling; and (3) measuring the LP231 mediated phenotypic effects in the test cells .

In .still a further embodiment, the invention relates to LP231 antagonists and/or agonist molecules. In one aspect, the invention provides a method of screening compounds that mimic LP231 (agonists) or diminish the effect of LP231 (antagonists) .

In still a further embodiment, the invention relates to a therapeutic composition comprising a therapeutically effective amount of LP231 polypeptide, antagonist or agonist thereof in combination with a pharmaceutically-acceptable carrier.

In still a further embodiment, the invention relates to an article of manufacture comprising a container, label and therapeutically effective amount of LP231 polypeptide, antagonist or agonist thereof in combination with a pharmaceutically-acceptable carrier .

I . Definitions

The terms "LP231 polypeptide" and "LP231" when used herein encompass native sequence LP231 polypeptide and polypeptide variants thereof (which are further defined herein) . The LP231 polypeptides may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods .

A "native sequence LP231 polypeptide" comprises a polypeptide having the same amino acid sequence as an LP231 polypeptide derived from nature. Such native sequence LP231 polypeptide can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence LP231 polypeptide" specifically encompasses naturally- occurring truncated or secreted forms of an LP231 polypeptide, (e.g., soluble forms containing, for instance, an extracellular domain sequence) , naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally- occurring allelic variants of an LP231 polypeptide.

In one embodiment of the invention, the native sequence LP231 polypeptide is a full-length or mature native sequence LP231 polypeptide comprising amino acids 1 or 20 through 225 of SEQ ID NO:2. Also, while the LP231 polypeptides disclosed in SEQ ID NO: 2 are shown to begin with a methionine residue designated as amino acid position 1, it is conceivable and possible that another methionine residue located either upstream or downstream from amino acid position 1 may be employed as the starting amino acid residue.

"LP231 variant" means an "active" LP231 polypeptide as defined below, having at least about 80% amino acid sequence identity with the LP231 polypeptide, having the deduced amino acid sequence of residues 1 or about 20 to about 225 shown in SEQ ID NO:l, for a full-length or mature native sequence LP231 polypeptide. Such LP231 polypeptide variants include, for instance, LP231, wherein one or more amino acid residues are added, substituted or deleted, at the N- or C-terminus or within the sequence of SEQ ID NO:2. Ordinarily, an LP231 polypeptide variant will have at least about 91% sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity, yet more preferably at least about 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO:l with or without the signal peptide (e.g., with signal peptide amino acid residues 1 to 20 of SEQ ID NO: 2, without signal peptide about 28 to 225 of SEQ ID NO: 2) . The variants provided herein exclude native sequence LP231 as well the polypeptides and nucleic acids described herein with which the LP231 polypeptides share 100% identity and/or which are already known in the art.

"Percent (%) amino acid sequence identity" with respect to the LP231 amino acid sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in an LP231 polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as ALIGN, ALIGN-2, Megalign (DNASTAR) or BLAST (e.g., Blast, Blast-2, U-Blast-2) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, the % identity values used herein are generated using WU-BLAST-2 [Altschul et al . , Methods in Enzymology 266: 460-480 (1996)]. Most of the WU-BLAST-2 search parameters are set to the default values . Those not set to default values, i.e., the adjustable parameters, are set with the following values: overlap span = 1; overlap fraction = 0.125; word threshold (T) = 11; and scoring matrix = BLOSUM 62. For purposes herein, a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acid residues between the amino acid sequence of the LP231 polypeptide of interest and the comparison amino acid sequence of interest (i.e., the sequence against which the LP231 polypeptide of interest is being compared) as determined by WU-BLAST-2, by (b) the total number of amino acid residues of the LP231 polypeptide of interest, respectively.

A "LP231 variant polynucleotide" or "LP231 variant nucleic acid sequence" means an active LP231 polypeptide- encoding nucleic acid molecule as defined below having at least about 85% nucleic acid sequence identity with the nucleotide acid sequence of nucleotides about 259 to about 1039 of the LP231-encoding nucleotide sequence shown in SEQ ID NO : 1. Ordinarily, an LP231 polypeptide will have at least about 85% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, yet more preferably at least about 91% nucleic acid sequence identity, yet more preferably at least about 92% nucleic acid sequence identity, yet more preferably at least about 93% nucleic acid sequence identity, yet more preferably at least about 94% nucleic acid sequence identity, yet more preferably at least about 95% nucleic acid sequence identity, yet more preferably at least about 96% nucleic acid sequence identity, yet more preferably at least about 97% nucleic acid sequence identity, yet more preferably at least about 98% nucleic acid sequence identity, yet more preferably at least about 99% nucleic acid sequence identity with the nucleic acid sequence of nucleotides about 178 or about 259 to about 1039 of the LP231- encoding nucleotide sequence shown in SEQ ID NO:l. Variants specifically exclude or do not encompass the native nucleotide sequence, as well as those prior art sequences that share 100% identity with the nucleotide sequences of the invention.

"Percent (%) nucleic acid sequence identity" with respect to the LP231 sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the LP231 sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as ALIGN, Align-2, Megalign (DNASTAR) , or BLAST (e.g., Blast, Blast-2) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % nucleic acid identity values are generated using the WU-BLAST-2 (BlastN module) program (Altschul et al., Methods in Enzymology 266: 460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to the default values. Those not set default values, i.e., the adjustable parameters, are set with the following values: overlap span = 1; overlap fraction = 0.125; word threshold (T) = 11; and scoring matrix = BLOSUM62. For purposes herein, a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the LP231 polypeptide- encoding nucleic acid molecule of interest and the comparison nucleic acid molecule of interest (i.e., the sequence against which the LP231 polypeptide-encoding nucleic acid molecule of interest is being compared) as determined by WU-BLAST-2, by (b) the total number of nucleotides of the LP231 polypeptide- encoding nucleic acid molecule of interest.

The term "positives", in the context of sequence comparison performed as described above, includes residues in the sequences compared that are not identical but have similar properties (e.g., as a result of conservative substitutions). The % identity value of positives is determined by the fraction of residues scoring a positive value in the BLOSUM 62 matrix. This value is determined by dividing (a) the number of amino acid residues scoring a positive value in the BLOSUM62 matrix of WU-BLAST-2 between the LP231 polypeptide amino acid sequence of interest and the comparison amino acid sequence (i.e., the amino acid sequence against which the LP231 polypeptide sequence is being compared) as determined by WU-BLAST-2, by (b) the total number of amino acid residues of the LP231 polypeptide of interest.

"Isolated, " when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non- reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes polypeptide in si tu within recombinant cells, since at least one component of the LP231 polypeptide natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

An "isolated" LP231 polypeptide-encoding nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the LP231 polypeptide-encoding nucleic acid. An isolated LP231 polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated LP231 polypeptide-encoding nucleic acid molecules therefore are distinguished from the LP231 polypeptide- encoding nucleic acid molecule as it exists in natural cells. However, an isolated LP231 polypeptide-encoding nucleic acid molecule includes LP231 polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express LP231 polypeptide where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells .

The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is "operably linked" when it is placed 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 riboεome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked 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.

"Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes required higher temperatures for proper annealing, while short probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reactions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al . , Current Protocols in Mol ecular Biology, Wiley Interscience Publishers, 1995. "Stringent conditions" or "high stringency conditions", as defined herein, may be identified by those that (1) employ low ionic strength and high temperature for washing, for example, 15 mM sodium chloride/1.5 mM sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride/75 mM sodium citrate at 42 °C; or (3) employ 50% formamide, 5X SSC (750 mM sodium chloride, 75 mM sodium citrate) , 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42°C with washes at 42°C in 0.2X SSC (30 mM sodium chloride/3 mM sodium citrate) and 50% formamide at 55°C, followed by a high-stringency wash consisting of 0. IX SSC containing EDTA at 55°C.

"Moderately stringent conditions" may be identified as described by Sambrook et al . [Mol ecular Cloning: A Laboratory Manual , New York: Cold Spring Harbor Press, (1989)], and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5X SSC (750 mM sodium chloride, 75 mM sodium citrate) , 50 mM sodium phosphate at pH 7.6 , 5X Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in IX SSC at about 37-50°C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc., as necessary to accommodate factors such as probe length and the like. The term "epitope tagged" where used herein refers to a chimeric polypeptide comprising an LP231 polypeptide, or domain sequence thereof, fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody may be made, or which can be identified by some other agent, yet is short enough such that it does not interfere with the activity of the LP231 polypeptide. The tag polypeptide preferably is also fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 to about 50 amino acid residues (preferably, between about 10 to about 20 residues) .

As used herein, the term "immunoadhesion" designates antibody-like molecules that combine the binding specificity of a heterologous protein (an "adhesion") with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesions comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.e., is "heterologous"), and an immunoglobulin constant domain sequence. The adhesion part of an immunoadhesion molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesion may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.

The term "antibody" is used in the broadest sense and specifically covers single anti-LP231 polypeptide monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies) , anti-LP231 antibody compositions with polyepitopic specificity, single-chain anti-LP231 antibodies, and fragments of anti-LP231 antibodies. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts .

"Active" or "activity" for the purposes herein refers to form(s) of LP231 which retain the biologic and/or immunologic activities of native or naturally-occurring LP231 polypeptide. Elaborating further, "biological" activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally-occurring LP231 polypeptide other than the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring LP231 polypeptide. An "immunological" activity refers only to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring LP231polypeptide .

The term "antagonist" is used in the broadest sense and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native LP231 polypeptide disclosed herein. In a similar manner, the term "agonist" is used in the broadest sense and includes any molecule that mimics a biological activity of a native LP231 polypeptide disclosed herein. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native LP231 polypeptides, peptides, small organic molecules, etc. Methods for identifying agonists or antagonists of an LP231 polypeptide may comprise contacting an LP231 polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the LP231 polypeptide.

"Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas. The term "antibody" is used in the broadest sense and specifically covers, without limitation, intact monoclonal antibodies, polyclonal antibodies, multiεpecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

The terms "treating", "treatment" and "therapy" as used herein refer to curative therapy, prophylactic therapy, and preventive therapy. An example of "preventive therapy" is the prevention or lessened targeted pathological condition or disorder. Those in need of treatment include those already with the diεorder as well as thoεe prone to have the diεorder or thoεe in whom the disorder is to be prevented.

"Chronic" administration refers to administration of the agent (s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. "Intermittent" administration is treatment that is not conεecutively done without interruption but, rather, is cyclic in nature.

The term "mammal" as used herein referε to any mammal claεεified as a mammal, including humans, domestic and farm animals, and zoo, sports or pet animals, such as cattle (e.g., cowε) , horses, dogs, sheep, pigε, rabbits, goats, cats, etc. In a preferred embodiment of the invention, the mammal is a human .

Administration "in combination with" one or more further therapeutic agents includes simultaneouε (concurrent) and conεecutive adminiεtration in any order.

A "therapeutically-effective amount" is the minimal amount of active agent (e.g., an LP231 polypeptide, antagonist or agonist thereof) which is necessary to impart therapeutic benefit to a mammal. For example, a "therapeutically- effective amount" to a mammal suffering or prone to εuffering or to prevent it from εuffering from a neurologic disorder is such an amount which induces, ameliorateε or otherwiεe causes an improvement in the pathological symptomε, diεease progression, physiological conditions aεsociated with or resistance to succumbing to a disorder principally characterized by synaptic dysfunction

"Carriers" as used herein include pharmaceutically- acceptable carrierε, excipientε, or εtabilizerε which are nontoxic to the cell or mammal being expoεed thereto at the dosages and concentrations employed. Often the physiologically-acceptable carrier is an aqueous pH buffered solution. Exampleε of phyεiologically acceptable carriers include bufferε such aε phoεphate, citrate, and other organic acids; antioxidants including aεcorbic acid; low molecule weight (leεε than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, aεparagine, arginine or lysine; onoεaccharideε, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; εugar alcoholε εuch as mannitol or sorbitol; salt- forming counterions such as sodium; and/or nonionic surfactantε εuch as TWEEN™, polyethylene glycol (PEG) , and PLURONIC™ .

"Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')l and Fv fragments; diabodieε; linear antibodieε (Zapata et al . , Protein Engin . 8 (10): 1057-1062 (1995) ) ; εingle-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodieε produceε two identical antigen-binding fragmentε, called "Fab" fragments, each with a εingle antigen-binding εite, and a reεidual "Fc" fragment, a deεignation reflecting the ability to cryεtallize readily. Pepεin treatment yieldε an F(ab')₂ fragment that haε two antigen-combining εiteε and is still capable of cross-linking antigen.

"Fv" is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen- binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDR specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab fragmentε differ from Fv fragments by the addition of a few reεidueε at the carboxy terminuε of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH iε the deεignation herein for Fab' in which the cysteine residue (s) of the constant domains bear a free thiol group. F(ab')₂ antibody fragmentε originally were produced as pairε of Fab' fragmentε which have hinge cyεteines between them. Other chemical couplingε of antibody fragmentε are also known.

The "light chainε" of antibodies (immunoglobulins) from any vertebrate εpecieε can be aεεigned to one of two clearly diεtinct types, called kappa and lambda, baεed on the amino acid sequences of their constant domains.

Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be aεεigned to different claεseε . There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of theεe may be further divided into subclaεεeε (iεotypeε) , e.g., IgGl, IgG2, IgG3 , IgG4 , IgA and IgA2.

"Single-chain Fv" or "εFv" antibody fragmentε compriεe the V_H and V_L domainε of antibody, wherein theεe domainε are preεent in a εingle polypeptide chain. Preferably, the Fv polypeptide further compriεeε a polypeptide linker between the V_H and V_L domain, which enableε the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994) .

The term "diabodieε" refers to small antibody fragments with two antigen-binding siteε, which fragments comprise a heavy-chain variable domain (V_H) connected to a light-chain variable domain (V) in the same polypeptide chain (V_H-V_L) • By using a linker that is too short to allow pairing between the two domainε on the εame chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are deεcribed more fully in, for example, EP 404.097, WO 93/11161; and Hollinger et al . , Proc . Natl . Acad. Sci . USA 90: 6444-6448 (1993).

An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non- proteinaceouε εolutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 reεidueε of N-terminal or internal amino acid εequence by uεe of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditionε uεing Coomaεεie blue, or preferably, εilver εtain. Iεolated antibody includeε the antibody in εitu within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

The word "label" when uεed herein refers to a detectable compound or compoεition which iε conjugated directly or indirectly to the antibody so aε to generate a "labeled" antibody. The label may be detectable by itself (e.g., radioisotope labelε or fluoreεcent labelε) or, in the case of an enzymatic label, may catalyze chemical alternation of a subεtrate compound or compoεition which is detectable. "Solid phase" iε meant to be a non-aqueous matrix to which the antibody of the present invention can adhere. Examples of solid phaseε encompaεεed herein include thoεe formed partially or entirely of glaεε (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamideε, polystyrene, polyvinyl alcohol and εilicones. In certain embodimentε, depending on the context, the εolid phaεe can compriεe the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term alεo includeε a diεcontinuouε εolid phase of discrete particleε, εuch aε those deεcribed in U.S. Patent No. 4,275,149.

A "liposome" is a small vesicle composed of various types of lipids, phoεpholipids and/or surfactant which is useful for delivery of a drug (such as an LP231 polypeptide or antibody thereto) to a mammal. The components of the lipoεome are commonly arranged in a bilayer formation, εimilar to the lipid arrangement of biological membranes .

A "small molecule" iε defined herein to have a molecule weight below about 500 daltonε .

The term "modulate" meanε to affect (e.g., either upregulate, downregulate or otherwise control) the level of a signaling pathway. Cellular processes under the control of signal transduction include, but are not limited to, transcription of specific genes, normal cellular functions, such as metabolism, proliferation, differentiation, adhesion, apoptosis and survival, as well as abnormal proceεεeε, εuch aε tranεformation, blocking of differentiation and metaεtasis.

Of course, a polynucleotide which hybridizes only to polyA⁺ sequences (such as any 3' terminal polyA⁺ tract of a cDNA εhown in the sequence listing) , or to a complementary stretch of T (or U) residues, would not be included in the definition of "polynucleotide," εince such a polynucleotide would hybridize to any nucleic acid molecule containing a polyA⁴ stretch or the complement thereof (e.g., practically any double-stranded cDNA clone) .

The LP231 polynucleotide can be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, the LP231 polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of εingle- and double-εtranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-εtranded regionε. In addition, the LP231 polynucleotides can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. LP231 polynucleotides may alεo contain one or more modified baεeε or DNA or RNA backboneε modified for stability or for other reaεonε . "Modified" baεes include, for example, tritylated baεeε and unuεual baεes such as inosine. A variety of modificationε can be made to DNA and RNA; thuε, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.

LP231 polypeptides can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isostereε, and may contain amino acidε other than the gene-encoded amino acidε. The LP231 polypeptides may be modified by either natural processes, such aε poεttranεlational processing, or by chemical modification techniques which are well known in the art. Such modifications are well deεcribed in baεic texts and in more detailed monographε , aε well aε in a voluminous reεearch literature. Modifications can occur anywhere in the LP231 polypeptides, including the peptide backbone, the amino acid εide-chainε and the amino or carboxyl termini. It will be appreciated that the εame type of modification may be preεent in the εame or varying degreeε at εeveral sites in a given LP231 polypeptide. Also, a given LP231 polypeptide may contain many types of modifications. LP231 polypeptides may be branched, for example, aε a reεult of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic LP231 polypeptides may result from posttranslation natural processeε or may be made by εynthetic methodε . Modifications include acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinoεitol, croεε-linking, cyclization, diεulfide bond formation, demethylation, formation of covalent crosslinks, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic procesεing, phosphorylation, prenylation, racemization, selenoylation, εulfation, tranεfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. See, for instance, Creighton, Proteins - Structure and Molecular Properties, 2nd Ed . , W. H. Freeman and Company, New York (1993) ; Johnεon, Post transational Covalent Modification of Proteins, Academic Press, New York, pp. 1-12 (1983); Seifter et al . , Meth . Enzymol . 182: 626-46 (1990); Rattan et al., Ann . NY Acad. Sci . 663: 48-62 (1992). II . Compoεitionε and Methodε of the Invention

A. Full-length LP231 polynucleotide of the preεent invention provideε newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as LP231. In particular, applicants have identified and isolated cDNA encoding an LP231 (e.g., LP231-A, SEQ ID NO:l) polypeptide, as disclosed in further detail in the Examples below. Uεing BLAST ^' and FaεtA sequence alignment computer programs, Applicantε found that various portionε of the LP231 polypeptide have sequence identity with human cerebellin. Accordingly, it is preεently believed that LP231 polypeptide diεcloεed in the preεent application are newly identified members of the cerebellin family and, thus, may be involved in synaptic function.

B. LP231 Variants

In addition to the full-length native sequence LP231 polypeptides deεcribed herein, it iε contemplated that LP231 variants can be prepared. LP231 variants can be prepared by introducing appropriate nucleotide changes into the LP231- encoding DNA or by εyntheεiε of the deεired LP231 polypeptide. Those skilled in the art will appreciate that amino acid changeε may alter poεt-translational processes of the LP231 polypeptide, such as changing the number or position of glycosylation εites or altering the membrane anchoring characteristics .

Variations in the native full-length sequence LP231 or in various domainε of the LP231 polypeptide described herein can be made, for example, using any of the techniques and guidelines for conservative and non-conεervative mutations set forth, for instance, in U.S. Patent NO. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding LP231 polypeptide that resultε in a change in the amino acid εequence of the LP231 polypeptide as compared with the native sequence LP231 polypeptide. Optionally the variation is by substitution of at least one amino, acid with any other amino acid in one or more of the domains of the LP231 polypeptide Guidance in determining which amino acid residue may be inserted, subεtituted or deleted without adversely affecting the desired activity may be. found by comparing the sequence of the LP231 polypeptide with that of homologous known protein molecules and minimizing the number of amino acid sequence changeε made in regions of high homology. Amino acid substitutions can be the reεult of replacing one amino acid with another amino acid having εimilar structural and/or chemical properties, such as the replacement of a leucine with a εerine, i.e., conservative amino acid replacements. Inεertionε or deletions may optionally be in the range of 1 to 5 amino acids. The variation allowed may be determined by εyεtematically making insertionε, deletionε or εubεtitutionε of amino acidε in the εequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.

LP231 polypeptide fragments are provided herein. Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal reεidueε, for example, when compared with a full length or native protein. Certain fragmentε lack amino acid reεidueε that are not eεεential for a desired biological activity of the LP231 polypeptide.

LP231 fragments may be prepared by any of a number of conventional techniqueε. Desired peptide fragments may be chemically synthesized. An alternative approach involves generating LP231 fragments by enzymatic digestion, e.g., by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residueε, or by digeεting the DNA with εuitable reεtriction enzymes and isolating the deεired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a deεired polypeptide fragment by polymeraεe chain reaction (PCR) . Oligonucleotideε that define the deεired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR. Preferably, LP231 polypeptide fragmentε share at leaεt one biological and/or immunological activity with the native LP231 polypeptide shown in SEQ ID NO:l.

In particular embodiments, conservative subεtitutionε of intereεt are εhown in Table 1 under the heading of preferred subεtitutionε. If εuch εubstitutions reεult in a change in biological activity, then more εubεtantial changeε, denominated exemplary substitutions in Table 1, or as further described below in reference to amino acid classes, are introduced and the products screened.

Table 1 Conservative Substitutions

Subεtantial modifications in function or immunological identity of the LP231 polypeptide are accomplished by εelecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the εubεtitution, for example, as a εheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target εite, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups baεed on common εide-chain propertie : (1) hydrophobic: syε, ser, thr;

(2) neutral hydrophilic: cyε, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) reεidues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe

Non-conservative substitutionε will entail exchanging a member of one of these claεseε for another claεs . Such εubεtituted reεidueε also may be introduced into the conεervative εubstitution sites, or more preferably, into the remaining (non-conserved) sites. The variations can be made using methodε known in the art such aε oligonucleotides-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesiε. Site-directed mutageneεiε [Carter et al . , Nucl . Acids Res . 13(12): 4331-43 (1985); Zoller et al . , Nucl . Acidε Res . 10(20): 6487-500 (1982)], caεεette mutageneεiε [Wells et al . , Gene 34(2-3): 315-23 (1985)], restriction selection mutagenesis [Wells et al . , Philos . Trans . R . Soc . London Ser. A 317: 415 (1986)] or other known techniqueε can be performed on the cloned DNA to produce the LP231-encoding variant DNA.

Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, εerine, and cysteine. Alanine iε typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant. Alanine is also typically preferred because it iε the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins, W.H. Free an & Co., NY; Chothia, J. Mol . Biol .105(1) : 1-12 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be uεed.

C. Modificationε of LP231

Covalent modificationε of LP231 polypeptideε are included within the εcope of thiε invention. One type of covalent modification includeε reacting targeted amino acid reεidues of an LP231 polypeptide with an organic derivatizing agent that iε capable of reacting with selected side chains or the N- or C-terminal residueε of an LP231 polypeptide. Derivatization with bifunctional agentε iε uεeful, for inεtance, for cross- linking LP231 to a water-inεoluble εupport matrix or surface for use in the method for purifying anti-LP231 antibodies, and vice-verεa. Commonly uεed croεε-linking agentε include, e.g., 1, 1-biε (diazo-acetyl) -2-phenylethane, glutaraldehyde, N-hydroxy-εuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesterε, including disuccinimidyl esters such aε 3 , 3'-dithiobis- (succinimidylproprionate) , bifunctional maleimides εuch aε bis-N-maleimido-1, 8-octane and agents εuch as methyl-3- [ (p- azidophenyl) -dithiolproprioimidate.

Other modifications include deamidation of glutaminyl and aεparaginyl reεidueε to the correεponding glutamyl and aεpartyl reεidueε, reεpectively, hydroxylation of proline and lyεine, phosphorylation of hydroxyl groups of seryl or threonyl reεidueε, methylation of the alpha-amino groupε of lyεine, arginine, and histidine side chains [T.E. Creighton. Proteins : Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group . Another type of covalent modification of the LP231 polypeptide included within the εcope of this invention compriseε altering the native glycosylation pattern of the polypeptide. "Altering the native glycosylation pattern" iε intended for purpoεeε herein to mean deleting one or more carbohydrate moietieε found in native εequence LP231 polypeptide and/or adding one or more glycoεylation εiteε that are not preεent in the native εequence LP231 polypeptide. Additionally, the phraεe includeε qualitative changeε in the glycoεylation of the native proteinε, involving a change in the nature and proportionε of the variouε carbohydrate moieties present.

Addition of glycoεylation εiteε to LP231 polypeptideε may be accompliεhed by altering the amino acid sequence thereof. The alteration may be made, for example, by the addition of, or subεtitution by, one or more serine or threonine residues to the native sequence LP231 polypeptide (for 0-linked glycosylation εiteε) . The LP231 amino acid εequence may optionally be altered through changeε at the DNA level, particularly by mutating the DNA encoding the LP231 polypeptide at preεelected bases such that codons are generated that will translate into the desired amino acids.

Another means of increaεing the number of carbohydrate moietieε on the LP231 polypeptide iε by chemical or enzymatic coupling of glycoεideε to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330, publiεhed 11 September 1987, and in Aplin and Wriεton, CRC Cri t . Rev. Biochem. , pp. 259-306 (1981).

Removal of carbohydrate moietieε preεent on the LP231 polypeptide may be accomplished chemically or enzymatically or by utational substitution of codons encoding for amino acid residueε that εerve as targets for glycosylation. Chemical deglycoεylation techniqueε are known in the art and described, for instance, by Sojar et al . , Arch . Biochem . Biophys . 259: 52-7 (1987) and by Edge et al . , Anal. Biochem . 118: 131-7 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of. endo- and exo-glycoεidases aε described by Thotakura et al . , Meth . Enzymol . 138: 350-9 (1987).

Another type of covalent modification of LP231 compriεes linking the LP231 polypeptide to one of a variety of nonproteinaceouε 20 polymerε, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkyleneε, in the manner εet forth in U.S. Patent No . 4640,835; 4,496,689; 4,301,144; 4.670,417; 4.791,192 or 4,179,337.

LP231 polypeptideε of the preεent invention may also be modified in a way to form chimeric molecules comprising an LP231 polypeptide fused to another heterologous polypeptide or amino acid sequence. In one embodiment, εuch a chimeric molecule comprises a fusion of an LP231 polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag iε generally placed at the amino- or carboxyl- terminuε of the LP231 polypeptide. The preεence of such epitope-tagged forms of an LP231 polypeptide can be detected using an antibody against the tag polypeptide. Also, proviεion of the epitope tag enableε the LP231 polypeptide to be readily purified by affinity purification uεing an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.

Various tag polypeptides and their respective antibodies are well known in the art. Exampleε include poly-hiεtidine (poly-hiε) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA.5 [Field et al . , Mol . Cell . Biol . 8(5): 2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4 , B7 and 9E10 antibodies thereto [Evan et al., Mol . Cell . Biol . 5(12): 3610-16 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al . , Prot. Engin . 3(6): 547-53 (1990)]. Other tag polypeptideε include the Flag-peptide [Hopp et al . , Bio/Technology, 6:120410 (1988)]; the KT3 epitope peptide [Martin et al . , Science 255(5041): 192-4 (1992)]; a cr-tubulin epitope peptide [Skinner et al . , J. Biol . Chem . , 266(22): 14163-6 (1991)]; and the T7 gene 10 protein peptide tag [Lutz- Freyermuth et al., Proc. Natl . Acad. Sci . USA 87(16): 6393-7 (1990)] .

In an alternative embodiment, the chimeric molecule may compriεe a fuεion of an LP231 polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule, such a fuεion could be to the Fc region of an IgG molecule. The Ig fuεions preferably include the εubεtitution of a εoluble tranεmembrane domain deleted or inactivated form of an LP231 polypeptide in place of at leaεt one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fuεion includeε the hinge, CH2 and CH3 or the hinge, CHI, CH2 and CH3 regions of an IgGl molecule. For the production of immunoglobulin fusions, see also U.S. Patent 5,428,130, iεεued June 27, 1995.

In yet a further embodiment, the LP231 polypeptides of the present invention may also be modified in a way to form a chimeric molecule compriεing an LP231 polypeptide fuεed to a leucine zipper. Variouε leucine zipper polypeptides have been deεcribed in the art. See, e.g., Landεchulz et al . , Science 240(4860): 1759-64 (1988); WO 94/10308; Hoppe et al . , FEBS Letters 344(2-3): 191-5 (1994); Abel et al . , Nature 341(6237): 24-5 (1989) . It iε believed that use of a leucine zipper fuεed to an LP231 polypeptide may be deεirable to aεεiεt in dimerizing or trimerizing εoluble LP231 polypeptide in εolution. Thoεe εkilled in the art will appreciate that zipper may be fuεed at either the N- or C-terminal end of the LP231 molecule.

D. Preparation of LP231

The deεcription below relateε primarily to production of LP231 by culturing cells transformed or transfected with a vector containing LP231 polypeptide encoding nucleic acid. It iε, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare LP231 polypeptideε . For instance, the LP231 sequence, or portions thereof, may be produced by direct peptide syntheεiε uεing solid-phase techniques [see, e.g., Stewart et al . , Solid-Phase Peptide Synthesis , W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J. Am . Chem . Soc . 85: 2149-2154 (1963)]. In vi tro protein synthesis may be performed uεing manual techniques or by automation. Automated εynthesiε may be accomplished, for instance, uεing an Applied Bioεyεtemε Peptide Syntheεizer (Foεter City, CA) using manufacturer's instructions. Various portionε of LP231 polypeptideε may be chemically synthesized separately and combined using chemical or enzymatic methods to produce a full-length LP231 polypeptide.

1. Isolation of DNA Encoding LP231 DNA encoding an LP231 polypeptide may be obtained from a cDNA library prepared from tissue believed to possess the LP231 mRNA and to expresε it at a detectable level. Accordingly, human LP231-encoding DNA can be conveniently obtained from a cDNA library prepared from human tiεεue, such as described in the Examples. The LP231-encoding gene may also be obtained from a genomic library or by known synthetic procedureε (e.g., automated εynthetic procedureε, oligonucleotide εyntheεiε) .

Librarieε can be εcreened with probeε (such as antibodies to an LP231 polypeptide or oligonucleotides of at least about 20-80 bases) designed to identify the gene of intereεt or the protein encoded by it. Screening the cDNA or genomic library with the εelected probe may be conducted using standard procedures, such as described in Sambrook et al . , Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Presε, NY (1989) . An alternative means to isolate the gene encoding LP231 iε to use PCR methodology [Sambrook et al . , εupra; Dieffenbach et al . , PCR Primer: A Laboratory Manual , Cold Spring Harbor Laboratory Press, NY (1995)] .

The Examples below deεcribe techniqueε for εcreening a cDNA library. The oligonucleotide sequences selected as probes should be of εufficient length and εufficiently unambiguouε that falεe poεitiveε are minimized. The oligonucleotide iε preferably labeled εuch that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art and include the use of radiolabels like ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high εtringency, are provided in Sambrook et al . , εupra.

Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databaseε εuch as GenBank or other private sequence databaεe . Sequence identity (at either the amino acid or nucleotide level) within defined regionε of the molecule or across the full-length sequence can be determined using methods known in the art and as described herein (e.g., through sequence alignment using computer software programs εuch aε ALIGN, DNAεtar, BLAST, BLAST-2 , INHERIT and ALIGN-2 which employ variouε algorith ε to measure homology) .

Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid εequence diεcloεed herein for the firεt time and, if neceεεary, uεing conventional primer extension procedures aε deεcribed in Sambrook et al . , εupra, to detect precursors and procesεing intermediates of mRNA that may not have been reverse-tranεcribed into cDNA.

2. Selection and Tranεformation of Host Cells

Hoεt cellε are tranεfected or tranεformed with expreεεion or cloning vectors deεcribed herein for LP231 polypeptide production and cultured in conventional nutrient media modified aε appropriate for inducing promoterε, selecting transformants , or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the εkilled artisan without undue experimentation. In general, principles, protocols, and practical techniqueε for maximizing the productivity of cell cultureε can be found in Mammalian Cell Biotechnology: A Practi cal Approach, M. Butler, ed. (IRL Preεε, 1991) and Sambrook et al . , εupra.

Methods of transfection are known to the ordinarily skilled artisan, for example, CaP0 and electroporation. Depending on the hoεt cell uεed, tranεformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes or other cells that contain substantial cell- wall barriers. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al . Gene 23(3): 315-30 (1983) and WO 89/05859 published 29 June 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology 52(2): 456-67 (1973) can be employed. General aεpectε of mammalian cell hoεt syεtem tranεformations have been deεcribed in U.S. Patent No. 4,399,216. Transformations into yeast are typically carried out according to the method of van Solingen et al . , J Bact . 130(2): 946-7 (1977) and Hsiao et al . , Proc . Natl . Acad. Sci . USA 76(8): 3829-33 (1979) . However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene or polyomithine, may alεo be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology 185: 527-37 (1990) and Mansour et al . , Nature 336(6197): 348-52 (1988) .

Suitable host cells for cloning or expressing the nucleic acid (e.g., DNA) in the vectors herein include prokaryote, yeaεt, or higher eukaryote cellε. Suitable prokaryotes include but are not limited to eubacteria, such as Gram- negative or Gram-positive organisms, for example, Enterobacteriacea such as E. coli. Various E. coli εtrainε are publicly available, such as E. coli K12 strain MM294 (ATCC 3 1.446); E. coli XI 776 (ATCC 3 1.537); E. coli strain W3 110 (ATCC 27.325) and K5 772 (ATCC 53.635). Other εuitable prokaryotic hoεt cells include Enterobacteriaceae such as Escherichia, e.g.. E. coli, Enterobacter, Erwinia, Klebisella, Proteuε, Salmonella, e.g., Salmonella typhimuriu , Serratia, e.g., Serratia marceεcanε, and Shigeila, aε well aε Bacilli εuch as B. subtilis and B. lichentformis (e.g., B. licheniformis 4 1 P disclosed in DD266,7 10, published 12 April 1989) , Pseudomonas such as P. aeruginosa, and Streptomyceε . Theεe exampleε are illustrative rather than limiting. Strain W3110 is one particularly preferred hoεt or parent hoεt becauεe it is a common hoεt εtrain for recombinant DNA product fermentations. Preferably, the host cell secreteε minimal amounts of proteolytic enzymes. For example, strain W3 110 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with exampleε of εuch hoεtε including E. coli W3110 strain 1A2 , which haε the complete genotype ronA; E. coli W3 110 strain 9E4, which has the complete genotype ton4 ptr3 ; E. coli W3110 strain 27C7 (ATCC 55,244), which haε the complete genotype tonA, ptr3 phoA E15 (argF-lac) 169 degP ompT /can'; E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin reεiεtant degP deletion mutation; and an E. coli εtrain having mutant periplaεmic proteaεe diεcloεed in U.S. Patent No. 4,946,783 iεεued 7 Auguεt 1990. Alternatively, in vivo methodε of cloning, e.g., PCR or other nucleic acid polymeraεe reactionε, are εuitable.

In addition to prokaryoteε, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expresεion hoεts for LP231 vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic hoεt microorganiεm. Otherε include Schizoεaccharomyces pombe [Beach and Nurse, Nature 290: 140-3 (1981); EP 139,383 published 2 May 1995]; Muyveromyces hosts [U.S. Patent No. 4,943,529; Fleer et al . , Bio/Technology 9 (10) : 968-75 (1991)] such as, e.g., K lactis (MW98-8C, CBS683, CBS4574) [de Louvencourt et al . , J. Bacteriol . 154(2): 737-42 (1983)]; K. fiagilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K wickeramii (ATCC 24,178), K waltii (ATCC 56,500), K. droεophilarum (ATCC 36.906) [Van den Berg et al . , Bio/Technology 8(2): 135-9 (1990)]; K. thermotoieranε, and K. marxianuε; yarrowia (EP 402,226); Pichia pastoris (EP 183,070) [Sreekrishna et al . , J. Basic Microbiol . 28(4): 265-78 (1988)]; Candid; Trichoderma reeεia (EP 244,234); Neuroεpora crassa [Caεe et al . , Proc . Natl . Acad Sci . USA 76(10): 5259-63 (1979)]; Schwanniomyces εuch aε Schwanniomyces occidentulis (EP 394,538 published 31 October 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 publiεhed 10 January 1991) , and Aεpergilluε hoεtε εuch aε A. nidulans [Ballance et al., Biochem . Biophys . Res . Comm. 112(1): 284-9 (1983)]; Tilburn et al . , Gene 26(2-3): 205-21 (1983); Yelton et al . , Proc . Natl . Acad. Sci . USA 81(5): 1470-4 (1984)] and A. niger [Kelly and Hynes, EMBO J. 4(2): 475-9 (1985)]. Methylotropic yeastε are εelected from the genera conεiεting of Hanεenula, Candida, Kloeckera, Pichia, Saccharomyceε , Torulopεiε, and Rhodotoruia . A list of specific εpecieε that are exemplary of this class of yeast may be found in C. Antony, The Biochemistry of Methylotrophs 269 (1982) .

Suitable hoεt cells for the expression of glycoεylated LP231 are derived from multicellular organiεms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sp, Spodoptera high5 as well aε plant cells. Examples of useful mammalian host cell lines include Chineεe hamεter ovary (CHO) and COS cells. More specific exampleε include monkey kidney CVl line tranεformed by SV40 (COS-7, ATCC CRL 1651) ; human embryonic kidney line [293 or 293 cells εubcloned for growth in εuεpenεion culture, Graham et al . , J. Gen Virol . , 36(1): 59-74 (1977)]; Chinese hamster ovary cellε/-DHFR [CHO, Urlaub and Chaεin, Proc . Natl . Acad. Sci . USA, 77(7): 4216-20 (1980)]; mouεe εertoli cells [TM4, Mather, Biol . Reprod. 23(l):243-52 (1980)]; human lung cells (W138. ATCC CCL 75); human liver cells (Hep G2 , HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL51) . The selection of the appropriate host cell is deemed to be within the skill in the ^' art .

3. Selection and Use of a Replicable Vector The nucleic acid (e.g., cDNA or genomic DNA) encoding the desired LP231 polypeptide may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Various vectorε are publicly available. The vector may, for example, be in the form of a plasmid, coεmid, viral particle, or phage. The appropriate nucleic acid εequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate reεtriction endonuclease site(ε) uεing techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal εequence, an origin of replication, one or more marker geneε, an enhancer element, a promoter, and a tranεcription termination εequence. Conεtruction of εuitable vectorε containing one or more of theεe components employs standard ligation techniques which are known to the skilled artisan.

The LP231 polypeptide may be produced recombinantly not only directly, but also aε a fuεion 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 LP231-encoding DNA that is inserted into the vector. The signal εequence may be a prokaryotic signal εequence selected, for example, from_, the group of the alkaline phosphatase, penicillinase, lpp, or heat-εtable enterotoxin II leaderε. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharo yceε and Kluyveromyces cc-factor leaders, the latter described in U.S. Patent No. 5,010,182), or acid phosphataεe leader, the C. albicanε glucoamylase leader (EP 362,179 published 4 April 1990), or the signal described in WO 90/13646 publiεhed 15 November 1990. In mammalian cell expresεion, mammalian εignal εequences may be used to direct secretion of the protein, such as εignal sequences from secreted polypeptides of the same or related specieε aε well aε viral secretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more εelected hoεt cellε. Such εequenceε are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 iε εuitable for moεt Gram- negative bacteria, the 2u plasmid origin iε εuitable for yeaεt, and variouε viral origins (SV40, polyoma, adenoviruε, VSV or BPV) are uεeful for cloning vectorε in mammalian cellε.

Expreεεion and cloning vectorε will typically contain a selection gene, also termed a selectable marker. Typical selection geneε encode proteinε that (a) confer reεistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement autotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli .

An example of εuitable εelectable markers for mammalian cells are those that enable the identification of cellε competent to take up the LP231-encoding nucleic acid, such aε DHFR or thymidine kinaεe. An appropriate hoεt cell when wild- type DHFR is employed iε the CHO cell line deficient in DHFR activity, prepared and propagated aε deεcribed Urlaub and Chaεin, Proc . Natl . Acad. Sci . USA, 77(7): 4216-20 (1980). A εuitable εelection gene for use in yeast is the trpl gene present in the yeaεt plaεmid YRp7 [Stinchcomb et al., Na ture 282(5734): 39-43 (1979); Kingsman et al . , Gene 7(2): 141-52 .

(1979); Tschu per et al . , Gene 10(2): 157-66 (1980)]. The trpl gene provides a εelection marker for a mutant εtrain of yeaεt lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEPC1 [Jones, Genetics 85: 23-33 (1977)].

Expression and cloning vectors usually contain a promoter operably linked to the LP231-encoding nucleic acid sequence to direct mRNA εyntheεiε. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for uεe with prokaryotic hoεtε include the P-lactamase and lactose promoter εyεtemε [Chang et al . , Nature 275(5681): 617-24

(1978); Goeddel et al . , Nature 281(5732): 544-8 (1979)], alkaline phoεphatase, a tryptophan (up) promoter system

[Goeddel, Nucl eic Acids Res . 8(18): 4057-74 (1980); EP 36,776 publiεhed 30 September 1981] , and hybrid promoters such as the tat promoter [deBoer et al . , Proc . Natl . Acad. Sci . USA 80(1): 21-5 (1983)] . Promoters for uεe in bacterial εystems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the LP231 polypeptide.

Examples of suitable promoting .sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinaεe [Hitze an et al . , J. Biol . Chem. 255(24): 12073-80

(1980)] or other gl^'ycolytic enzymes [Hess et al . , J^". Adv. Enzyme Reg. 7: 149 (1968); Holland, Biochemistry 11 (23 ) : 4900- 7 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenaεe, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinaεe, trioεephoεphate iεomeraεe, phoεphoglucoεe isomerase, and glucokinase. Other yeaεt promoters, which are inducible promoters having the additional advantage of tranεcription controlled by growth conditionε, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phoεphataεe, degradative enzymes aεεociated with nitrogen metaboliεm, metallothionein, glyceraldehyde-3-phoεphate dehydrogenaεe, and enzymes responεible for maltoεe and galactoεe utilization. Suitable vectors and promoters for use in yeaεt expression are further described in EP 73,657. LP231 transcription from vectors in mammalian hoεt cells iε controlled, for example, by promoters obtained from the genomes of viruses such aε polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenoviruε (εuch aε Adenoviruε 2), bovine papilloma virus, avian εarcoma viruε, cytomegalovirus, a retrovirus, hepatitis- B viruε and Simian Viruε 40 (SV40), from heterologouε 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 syεtem .

Tranεcription of a DNA encoding an LP231 polypeptide by higher eukaryoteε may be increaεed by inεerting an enhancer εequence 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 εequenceε are now known from mammalian geneε (globin, elastase, albumin, a-ketoprotein, and insulin) . Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270) , the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenoviruε enhancerε . The enhancer may be εpliced into the vector at a poεition 5' or 3' to the LP231 coding εequence but iε preferably located at a εite 5' from the promoter .

Expreεεion vectorε uεed in eukaryotic hoεt cellε (yeaεt, fungi, inεect, plant, animal, human, or nucleated cellε from other multicellular organiεms) will also contain sequences necesεary for the termination of tranεcription and for stabilizing the mRNA. Such sequenceε are commonly available from the 5' and occasionally 3' untranslated regions of eukaryotic or viral DNAs or cDNAε . Theεe regionε contain nucleotide εegmentε transcribed aε polyadenylated fragments in the untranslated portion of the mRNA encoding LP231 polypeptide .

Still other methodε , vectors, and host cellε suitable for adaptation to the syntheεiε of LP231 polypeptideε in recombinant vertebrate cell culture are deεcribed in Gething et al., Nature 293(5834): 620-5 (1981): Mantei et al . , Nature 281(5726): 40-6 (1979); EP 117,060; and EP 117,058.

4. Detecting Gene Amplification/Expreεεion Gene amplification and/or expreεεion may be meaεured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the tranεcription of mRNA [Thomaε, Proc . Natl . Acad. Sci . USA 77(9): 5201-5 (1980)], dot blotting (DNA analyεis) , or in situ hybridization, using an appropriately labeled probe, based on the εequenceε provided herein. Alternatively, antibodies may be employed that can recognize εpecific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the aεεay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the preεence of antibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical εtaining of cells or tisεue sections and asεay of cell culture or body fluidε, to quantitate directly the expreεεion of gene product. Antibodieε uεeful for immunohiεtochemical staining and/or asεay of εample fluidε may be either monoclonal or polyclonal and may be prepared in any mammal. Conveniently, the antibodieε may be prepared againεt a native provided herein or againεt exogenouε εequence fuεed to LP231-encoding DNA and encoding a εpecific antibody epitope.

5. Purification of Polypeptide

Forms of LP231 may be recovered from culture medium or from host cell lyεates . If membrane-bound, it can be released from the membrane using a suitable detergent εolution (e.g., Triton-X 100) or by enzymatic cleavage. Cellε employed in expreεεion of LP231 polypeptides can be disrupted by variouε phyεical or chemical meanε, εuch aε freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agentε.

It may be deεired to purify LP231 from recombinant cell proteins or polypeptideε. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reversed-phase HPLC; chromatography on silica or on a cation-exchange resin εuch aε DEAE; chromatofocuεing; SDS-PAGE; ammonium εulfate precipitation; gel filtration uεing, for example, Sephadex G-75; protein A Sepharoεe columnε to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of the LP231 polypeptide. Variouε methodε of protein purification may be employed and such methods are known in the art and described, • for example, in Deutscher, Methods in Enzymology 182: 83-9 (1990) and Scopeε, Protein Purification : Principles and Practice, Springer-Verlag, NY (1982). The purification εtep(s) εelected will depend, for example, on the nature of the production process used and the particular LP231 polypeptide produced.

E. Useε for LP231

Nucleotide sequences (or their complement) encoding LP231 polypeptides have various applications in the art of molecular biology, including useε as hybridization probes, in chromosome and gene mapping and in the generation of antisense RNA and DNA. LP231-encoding nucleic acid will also be useful for the preparation of LP231 polypeptides by the recombinant techniques described herein.

The full-length LP231 nucleotide sequence (SEQ ID N0:1) or the full-length native sequence LP231 nucleotide-encoding sequence, or portions thereof, may be used as hybridization probes for a cDNA library to isolate the full-length LP231 gene or to isolate still other genes (for instance, those encoding naturally-occurring variants of LP231, or the εame from other εpecieε) which have a deεired εequence identity to the LP231 nucleotide εequence diεcloεed in SEQ ID N0:1. Optionally, the length of the probeε will be about 20 to about 50 baεeε. The hybridization probeε may be derived from the sequence of SEQ ID NO:l, or from genomic sequences including promoters, enhancer elements and introns of native sequence LP231-encoding DNA. By way of example, a screening method will compriεe isolating the coding region of the LP231 gene using the known DNA sequence to syntheεize a εelected probe of about 40 baεeε. Hybridization probes may be labeled by a variety of labels, including radionucleotides such as ³²P or enzymatic labels εuch aε alkaline phosphatase coupled to the probe via avidin/biotin coupling εyεtems . Labeled probeε having a εequence complementary to that of the LP231 gene of the preεent invention can be uεed to εcreen librarieε of human cDNA, genomic DNA or mRNA to determine members of εuch librarieε the probe hybridizes. Hybridization techniqueε are deεcribed in further detail in the Exampleε below.

Any EST εequence (or fragment thereof) diεcloεed in the present application may similarly be employed as probes, using the methods disclosed herein. Other useful fragmentε of the LP231 nucleic acidε include antisenεe or εense oligonucleotideε compriεing a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target LP231 mRNA (εenεe) of LP231 DNA (antiεenεe) sequences. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment of the coding region of LP231 DNA. Such a fragment generally comprises at least about 14 nucleotideε. preferably from about 14 to 30 nucleotideε. The ability to derive an antiεenεe or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is deεcribed in, for example, Stein and Cohen, Cancer Res 48(10): 2659-68 (1988) and van der Krol et al . , Bio/Techniques 6(10): 958-76 (1988).

Binding of antisense or senεe oligonucleotideε to target nucleic acid sequences resultε in the formation of duplexes that block transcription or tranεlation of the target εequence by one of εeveral means, including enhanced degradation of the duplexes, premature termination of transcription or tranεlation, or by other meanε . The antisense oligonucleotides thus may be used to block expresεion of LP231 proteinε . Antisense or senεe oligonucleotides further compriεe oligonucleotideε having modified sugar-phosphodieεter backboneε (or other sugar linkageε, εuch as those described in WO 91/06629) and wherein such εugar linkageε are reεiεtant to endogenous nucleaseε. Such oligonucleotideε with reεiεtant εugar linkages are εtable in vivo (i.e., capable of reεiεting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences .

Other examples of senεe or antiεense oligonucleotides include those oligonucleotideε which are covalently linked to organic moietieε, such as those described in WO 90/10448, and other moieties that increase affinity of the oligonucleotide for a target nucleic acid sequence, εuch poly-L-lyεine . Further still, intercalating agents, such as ellipticme, and alkylating agents or metal complexes may be attached to senεe or antiεenεe oligonucleotideε to modify binding εpecificitieε of the antiεenεe or εenεe oligonucleotide for the target nucleotide εequence.

Antiεenεe or εenεe oligonucleotideε may be introduced into a cell containing the target nucleic acid εequence by any gene tranεfer method, including, for example, CaP0-mediated DNA tranεfection, electroporation, or by using gene transfer vectors such as Epstein-Barr viruε. In a preferred procedure, an antiεenεe or εenεe oligonucleotide iε inεerted into a εuitable retroviral vector. A cell containing the target nucleic acid εequence iε contacted with the recombinant retroviral vector, either in vivo or ex vivo . Suitable retroviral vectorε include, but are not limited to, thoεe derived from the murine retroviruε M-MSV, N2 (a retroviruε derived from M-MuLV) , or the double copy vectors designated CDTSA, CTSB and DCTSC (see WO 90/13641) .

Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antiεenεe oligonucleotide or itε conjugated verεion into the cell.

Alternatively, a εenεe or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, aε deεcribed in WO 90/10448. The εenεe or antisenεe oligonucleotide-lipid complex iε preferably diεεociated within the cell by an endogenouε lipaεe.

The probeε may alεo be employed in PCR techniqueε to generate a pool of sequences for identification of closely related LP231 sequences.

Nucleotide sequences encoding an LP231 polypeptide can also be uεed to construct hybridization probes for mapping the gene which encodes that LP231 polypeptide and for the genetic analyεiε of individualε with genetic diεorderε. The nucleotide εequenceε 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.

When the coding sequences for LP231 encode a protein which binds to another protein (for example, where the LP231 polypeptide functions as a receptor) , the LP231 polypeptide can be uεed in aεεayε to identify the other proteins or molecules involved in the binding interaction. By such methods, inhibitors of the receptor/ligand binding interaction can be identified. Proteins involved in such binding interactions can also be used to screen for peptide or small molecule inhibitors or agonistε of the binding interaction. Alεo, the receptor LP231 polypeptide can be uεed to iεolate correlative ligand (ε). Screening assays can be designed to find lead compounds that mimic the biological activity of a native LP231 or a receptor for LP231. Such screening aεεayε will include aεεays amenable to high-throughput screening of chemical librarieε, making them particularly εuitable for identifying εmall molecule drug candidateε. Small moleculeε contemplated include synthetic organic or inorganic compounds. The asεays can be performed in a variety of formats, including protein-protein binding asεayε, biochemical εcreening aεεayε, immunoasεays and cell based asεays, which are well characterized in the art.

Nucleic acids which encode LP231 polypeptide or any of its modified forms can also be used to generate either transgenic animalε or "knock out" animalε which, in turn, are uεeful in the development and εcreening of therapeutically uεeful reagentε . A tranεgenic animal (e.g., a mouεe or rat) iε an animal having cells that contain a transgene, which tranεgene waε introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A tranεgene is a DNA which is integrated into the genome of a cell from which a tranεgenic animal developε . In one embodiment, cDNA encoding LP231 polypeptide can be uεed to clone genomic DNA encoding LP231 in accordance with eεtabliεhed techniques and the genomic sequenceε used to generate tranεgenic animals that contain cells which express DNA encoding LP231. Methods for generating transgenic animalε, particularly animalε εuch as mice or rats, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009. Typically, particular cells would be targeted for LP231 transgene incorporation with tisεue-εpecific enhancers . Transgenic animalε that include a copy of a tranεgene encoding LP231 introduced into the germ line of the animal at an embryonic εtage can be uεed to examine the effect of increased expression of DNA encoding LP231 polypeptide. Such animals can be uεed aε teεter animalε for reagentε thought to confer protection from, for example, pathological conditionε aεεociated with itε overexpreεεion . In accordance with this facet of the invention, an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animalε bearing the tranεgene, would indicate a potential therapeutic intervention for the pathological condition.

Alternatively, non-human homologueε of LP231 can be uεed to construct an LP231 "knock out" animal which has a defective or altered gene encoding LP231 polypeptide as a result of homologous recombination between the endogenous gene encoding LP231 polypeptide and altered genomic DNA encoding LP231 polypeptide introduced into an embryonic cell of the animal. For example. cDNA encoding LP231 polypeptide can be uεed to clone genomic DNA encoding LP231 polypeptide in accordance with eεtabliεhed techniqueε. A portion of the genomic DNA encoding LP231 polypeptide can be deleted or replaced with another gene, εuch aε a gene encoding a selectable marker which can be used to monitor integration. Typically, several kilobaεeε of unaltered flanking DNA (both at the 5' and 3' endε) are included in the vector [see, e.g., Thomaε and Capecchi, Cell 51(3): 503-12 (1987) for a deεcription of homologouε recombination vectors] . The vector is introduced into an embryonic stem cell line (e.g., by electroporation), and cells in which the introduced DNA has homologously recombined with the endogenouε DNA are εelected [see, e.g., Li et al., Cell 69(6): 915-26 (1992)]. The εelected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see, e.g., Bradley, Teratocarcinomas and Embryonic Stem Cells : A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113- 152] . A chimeric embryo can then be implanted into a εuitable pεeudopregnant female foster animal and the embryo brought to term to create a "knock out" animal. Progeny harboring the homologouεly recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animalε can be characterized, for inεtance, for their ability to defend againεt certain pathological conditionε and for their development of pathological conditionε due to abεence of the LP231 polypeptide .

"Gene therapy" includeε both conventional gene therapy, where a lasting effect is achieved by a εingle treatment, and the adminiεtration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA. Antisenεe RNAs and DNAs can be used aε therapeutic agentε for blocking the expreεεion of certain geneε in vivo . It has already been shown that εhort antiεenεe oligonucleotides can be imported into cellε where they act aε inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane [Zamecnik et al . , Proc . Natl . Acad Sci . USA 83(12): 4143-6 (1986)]. The oligonucleotides can be modified to enhance their uptake, e.g., by substituting their negatively charged phosphodiester groups with uncharged groups. There are a variety of techniques available for introducing nucleic acids into viable cellε. Trie techniqueε vary depending upon whether the nucleic acid iε transferred into cultured cell in vi tro or in vivo in the cells of the intended host. Techniques εuitable for the transfer of nucleic acid into mammalian cellε in vi tro include the uεe of liposomes, electroporation, microinjection, cell fusion, DEAE- dextran, the calcium phosphate precipitation method, etc. The currently preferred in vivo gene transfer techniqueε include tranεfection with viral (typically, retroviral) vectorε and viral coat protein-lipoεome mediated tranεfection [Dzau et al., Trends in Biotechnology 11(5): 205-10 (1993)]. In some situations it is desirable to provide the nucleic acid εource with an agent that targetε the target cellε, εuch aε an antibody specific for a cell εurface membrane protein or the target cell, a ligand for a receptor on the target cellε, etc. Where lipoεomeε are employed, proteinε which bind to a cell εurface membrane protein aεεociated with endocytoεiε may by uεed for targeting and/or to facilitate uptake, e.g., capεid proteinε or fragments thereof for a particular cell type, antibodieε for proteinε which undergo internalization in cycling, proteinε that target intracellular localization and enhance intracellular half-life. The technique of receptor- mediated endocytoεiε iε described, for example by Wu et al . , J. Biol . Chem. 262(10): 4429-32 (1987); and Wagner et al . , Proc . Natl . Acad. Sci . USA 87(9): 3410-4 (1990). For a review of gene marking and gene therapy protocols, see Anderεon, Science 256(5058) : 808-13 (1992).

The LP231 polypeptides deεcribed herein may alεo be employed aε molecular weight markers for protein electrophoreεiε purposes . The nucleic acid molecule encoding the LP231 polypeptideε or fragmentε thereof deεcribed herein are uεeful for chromoεome identification. In thiε regard, there exiεtε an ongoing need to idenfity new chromoεome markers, since relatively few chromosome marking reagents, based upon actual sequence data, are presently available. Each LP231 nucleic acid molecule of the preεent invention can be uεed as a chromosome marker .

The LP231 polypeptideε and nucleic acid molecules of the present invention may also be used for tisεue typing, wherein the LP231 polypeptideε of the preεent invention may be differentially expreεεed in one tiεεue aε compared to another. LP231 nucleic acid molecules will find use for generating probes for PCR, Northern analysiε, Southern analysis and Western analysiε.

LP231 polypeptideε of the preεent invention which possess biological activity related to that of cerebellin may be employed both in vivo for therapeutic purpoεeε and in vi tro . Thoεe of ordinary skill in the art will well know how to employ the LP231 polypeptideε of the preεent invention for εuch purposes .

F. Anti-LP231 Antibodies

The present invention further provides anti-LP231 polypeptide antibodies. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies.

1. Polyclonal Antibodies The anti-LP231 antibodies of the present invention may comprise polyclonai antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodieε can be raiεed in a mammal, for example, by one or more injectionε of an immunizing agent and, if deεired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injectionε. The immunizing agent may include the LP231 polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of εuch immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and εoybean trypεin inhibitor. Exampleε of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, εynthetic trehaloεe dicorynomycolate) . The immunization protocol may be εelected by one εkilled in the art without undue experimentation.

2. Monoclonal Antibodieε

The anti-LP231 antibodieε may, alternatively, be monoclonal antibodieε. Monoclonal antibodies may be prepared using ^'hybridoma methodε, εuch aε thoεe described by Kohler and Milstein, Na ture 256(5517): 495-7 (1975). In a hybridoma method, a mouse, hamεter, or other appropriate host animal iε typically immunized with an immunizing agent to elicit lymphocyteε that produce or are capable of producing antibodieε that will εpecifically bind to the immunizing agent. Alternatively, the lymphocyteε may be immunized in vi tro .

The immunizing agent will typically include the LP231 polypeptide or a fuεion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used, if cells of human origin are desired, or spleen cells or lymph node cells are used, if non-human mammalian εourceε are desired. The lymphocyteε are then fused with an immortalized cell line uεing a suitable fusing agent, such aε polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodi es : Principl es and Practice, Academic Press, (1986) pp. 59-103] . Immortalized cell lines are uεually tranεformed mammalian cellε, particularly myeloma cellε of rodent, bovine and human origin. Uεually, rat or mouεe myeloma cell lines are employed. The hybridoma cellε may be cultured in a εuitable culture medium that preferably contains one or more εubεtanceε that inhibit the growth or εurvival of the unfused, immortalized cellε. For example, if the parental cellε lack the enzyme hypoxanthine guanine phosphoriboεyl transferase (HGPRT or HPRT) , the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which prevents the growth of HGPRT- deficient cellε.

Preferred immortalized cell lineε are thoεe that fuεe efficiently, εupport εtable high level expression of antibody by the selected antibody-producing cellε, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lineε are murine myeloma lineε, which can be obtained, for inεtance, from the Salk Institute Cell Distribution Center, San Diego, California, and the American Type Culture Collection, Rockville, Maryland. Human myeloma and mouse- human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol . 133(6): 3001-5 (1984); Brodeur et al . , Monoclonal Antibody Production Techniques and Applica tions, Marcel Dekker, Inc., NY, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodieε directed againεt an LP231 polypeptide. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cellε iε determined by immunoprecipitation or by an in vi tro binding aεεay, εuch aε radioimmunoassay (RIA) or enzyme-linked immunoabεorbent assay (ELISA) . Such 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 Rodbard, Anal . Biochem. 107(1) : 220-39 ( 1980) .

After the desired hybridoma cellε are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, Monoclonal Antibodi es : Principles and Practice, Academic Preεs, (1986) pp. 59-103]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle 'ε Medium and RPMI-1640 medium. Alternatively, the hybridoma cellε may be grown in vivo as ascites in a mammal.

The monoclonal antibodieε εecreted by the εubcloneε may be iεolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography .

The monoclonal antibodies may also be made by recombinant DNA methodε, εuch as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies) . The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectorε, which are then tranεfected into hoεt cellε εuch aε simian COS cellε, Chineεe hamεter ovary (CHO) cellε, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesiε of monoclonal antibodieε in the recombinant hoεt cellε. The DNA also may be modified, for example, by subεtituting the coding εequence for human heavy and light chain conεtant domainε in place of the homologous murine sequenceε [U.S. Patent No. 4,816,567; Morrison et al . , Proc . Na tl . Acad. Sci . USA 81(21): 6851-5 (1984)] or by covalently joining to the immunoglobulin coding εequence all or part of the coding εequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be εubεtituted for the conεtant domainε of an antibody of the invention or can be εubεtituted for the variable domainε of one antigen-combining εite of an antibody of the invention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodieε. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain cross-linking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent cross-linking.

In vi tro methods are also εuitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using routine techniques known in the art. 3. Humanized Antibodies

The anti-LP231 antibodieε of the invention may further compriεe humanized antibodieε or human antibodieε. Humanized formε of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (εuch aε Fv, Fab, Fab', F(ab')₂ or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodieε include human immunoglobulinε (recipient antibody) in which reεidueε from a complementary-determining region (CDR) of the recipient are replaced by reεidues from a CDR of a non-human specieε (donor antibody) εuch as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residueε of the human immunoglobulin are replaced by correεponding non-human reεidues. Humanized antibodieε may alεo compriεe reεidueε which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise subεtantially all of at least one, and typically two, variable domains, in which all or subεtantially 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(6069): 522-5 (1986); Riechmann et al . , Nature 332(6162): 323-7 (1988); and Presta, Curr. Op. Struct . Biol . 2: 593-6 (1992)].

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid reεidues are often referred to as "import" residueε, which are typically taken from an "import" variable domain. Humanization can be eεεentially performed following the method of Winter and co- workers [Jones et al . , Na ture 321(6069): 522-5 (1986); Riechmann et al . , Nature 332(6162): 323-7 (1988); Verhoeyen et al., Science 239(4847): 1534-6 (1988)], by εubstituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) wherein substantially less than an intact human variable domain has been subεtituted by the correεponding εequence from a non- human εpecieε. In practice, humanized antibodieε are typically human antibodieε in which εome CDR residueε and poεεibly εome FR reεidueε are εubεtituted by reεidues from analogous siteε in rodent antibodieε.

Human antibodies can also be produced using various techniques known in the art, including phage display libraries

[Hoogenboom and Winter, J^". Mol . Biol . 227(2): 381-8 (1992); Markε et al . , J^". Mol . Biol . 222(3): 581-97 (1991)]. The techniqueε of Cole et al . and Boerner et al . are alεo available for the preparation of human monoclonal antibodieε

(Cole et al . , Monoclonal Antibodieε and Cancer Therapy, Alan R. Liεε, p. 77 (1985) and Boerner et al . , J. Immunol . 147(1): 86-95 (1991)] . Similarly, human antibodieε can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or complete inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respectε, including gene rearrangement, assembly and antibody repertoire. Thiε approach is deεcribed, for example, in U.S. Patent Noε . 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following εcientific publications: Marks et al . , Biotechnology 10(7): 779-83 (1992); Lonberg et al . , Nature 368(6474): 856-9 (1994); Morrison, Nature 368(6474): 812-3 (1994); Fishwild et al., Na ture Biotechnology 14 (7) : 845-51 (1996); Νeuberger, Nature Biotechnology 14(7): 826 (1996); Lonberg and Huεzar, Int. .Rev. Immunol . 13(1): 65-93 (1995).

4. Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)

The antibodieε of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO 81/01145) to an active anti- cancer drug. See, for example, WO 88/07378 and U. S. Patent No. 4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such as way so as to convert it into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but are not limited to, glycosidaεe, glucoεe oxidaεe, human lyεozyme, human glucuronidaεe, alkaline phosphatase useful for converting phoεphate-containing prodrugs into free drugs; arylsulfataεe uεeful for converting εulfate-containing prodrugε into free drugs; cytosine deaminaεe uεeful for converting non-toxic 5-fluorocytoεine into the anti-cancer drug 5-fluorouracil; proteaεeε, εuch as serratia protease, thermolysin, εubtiliεin, carboxypeptidaεeε (e.g., carboxypeptidaεe G2 and carboxypeptidaεe A) and cathepεinε (εuch aε cathepεins B and L) . that are useful for converting peptide-containing prodrugs into free drugε; D- alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as alpha-galactosidase and neura inidaεe useful for converting glycosylated prodrugs into free drugs; p-lactamase useful for converting drugε derivatized with p-lactams into free drugs; and penicillin amidaseε, εuch aε penicillin Vamidaεe or penicillin G amidaεe, useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugε. Alternatively, antibodieε with enzymatic activity, alεo known in the art aε "abzymeε" can be uεed to convert the prodrugs of the invention into free active drugs [see, e.g., Masεey, Nature 328(6129): 457-8 (1987)]. Antibody-abzyme conjugateε can be prepared aε deεcribed herein for delivery of the abzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the anti-LP231 antibodies by techniqueε well known in the art εuch as the use of the heterobifunctional croεs-linking agents discuεεed above. Alternatively, fuεion proteins comprising at least the antigen binding region of the antibody of the invention linked to at leaεt a functionally active portion of an enzyme of the invention can be conεtructed using recombinant DNA techniques well known in the art [εee, e.g., Neuberger et al . , Nature 312(5995): 604-8 (1984)].

5. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an LP231 polypeptide, the other one is for any other antigen, and preferably for a cell- surface protein or receptor or receptor εubunit.

Methodε for making biεpecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expresεion of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificitieε [Milεtein and Cuello, Nature 305 (5934) :537-9 (1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one haε the correct biεpecific εtructure. The purification of the correct molecule iε uεually accompliεhed by affinity chromatography εtepε. Similar procedureε are diεclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al . , EMBO J 10(12) : 3655-9 (1991) .

Antibody variable domainε with the deεired binding specificities (antibody-antigen combining sites) can be fuεed to immunoglobulin constant domain sequenceε. The fuεion preferably iε with an immunoglobulin heavy-chain conεtant domain, comprising at least part of the hinge, CH2 , and CH3 regions. It iε preferred to have the firεt heavy-chain conεtant region (CHI) containing the site necessary for light- chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fuεionε and, if deεired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a εuitable hoεt organiεm. For further details of generating bispecific antibodies see, for example, Suresh et al . , Methodε in Enzymology 121: 210-28 (1986) .

According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In thiε method, one or more εmall amino acid εide chainε from the interface of the firεt antibody molecule are replaced with larger εide chains (e.g., tyrosine or tryptophan) . Compenεatory "cavitieε" of identical or εimilar εize to the large εide chain (ε) are created on the interface of the εecond antibody molecule by replacing large amino acid εide chainε with smaller ones (e.g., alanine or threonine) . This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers .

Bispecific antibodies can be prepared as full length antibodieε or antibody fragmentε (e.g., F(ab') biεpecific antibodies). Techniqueε for generating bispecific antibodieε from antibody fragmentε have been deεcribed in the literature. For example, biεpecific antibodieε can be prepared can be prepared uεing chemical linkage. Brennan et al . [ Science 229(4708): 81-3 (1985)] deεcribe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') fragments. Theεe fragments are reduced in the presence of the dithiol complexing agent εodium arεenite to stabilize vicinal dithiols and prevent intermolecular diεulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab' thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the biεpecific antibody. The biεpecific antibodieε produced can be used as agents for the selective immobilization of enzymes. Fab' fragments may be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al. [ J. Exp . Med. 175(1): 217-25 (1992)] describe the production of a fully humanized biεpecific antibody F(ab')2 molecule. Each Fab' fragment waε εeparately εecreted from E. coli and subjected to directed chemical coupling in vi tro to form the bispecific antibody. The biεpecific antibody thuε formed was able to bind to cells overexpresεing the ErbB2 receptor and normal human T cellε, aε well aε trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have alεo been deεcribed. For example, biεpecific antibodieε have been produced uεing leucine zipperε [Koεtelny et al., J. Immunol . 148(5): 1547-53 (1992)], wherein the leucine zipper peptideε from the Foε and Jun proteinε were linked to the Fab' portionε of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Holliger et al, [ Proc . Natl . Acad Sci . USA 90(14): 6444-8 (1993)] has provided an alternative mechanism for making bispecific antibody fragmentε. The fragments comprise a heavy-chain variable domain (V_H) connected to a light-chain variable domain (V_L) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V_H and V_L domains of one fragment are forced to pair with the complementary V_H and V_L domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the uεe of single-chain Fv (sFv) dimerε haε alεo been reported. See Gruber et al . , J. Immunol . 152(11): 5368-74 (1994) .

Antibodieε with more than two valencieε are contemplated. For example, triεpecific antibodieε can be prepared [Tutt et al., J Immunol . 147(1): 60-9 (1991)].

Exemplary biεpecific antibodieε may bind to two different epitopeε on a given "LP" protein herein. Alternatively, an anti-"LP" protein arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g., CD2 , CD3 , CD28, or B7), or Fc receptors for IgG (FcyR) , εuch aε FcyRI (CD64), FcyRII (CD32) and FcyRIIl (CD16) so as to focuε cellular defenεe mechaniεmε to the cell expressing the particular "LP" protein. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular "LP" polypeptide. These antibodies posεeεε an "LP" -binding arm and an arm which bindε a cytotoxic agent or a radionuclide chelator, εuch aε EOTUBE, DPTA. DOTA, or TETA. Another bispecific antibody of interest binds the "LP231" polypeptide and further bindε tiεsue factor (TF) .

6. Heteroconiugate Antibodieε

Heteroconjugate antibodies are also within the εcope of the preεent invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cellε to unwanted cellε [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/20373]. It iε contemplated that the antibodies may be prepared in vi tro using known methods in synthetic protein chemistry, including those involving crosεlinking agentε. For example, immunotoxinε may be conεtructed using a disulfide exchange reaction or by forming a thioether bond. Examples of εuitable reagentε for thiε purpose include iminothiolate and methyl- - mercaptobutyrimidate and those discloεed, for example, in U.S. Patent No. 4,676,980.

7. Effector Function Engineering

It may be deεirable to modify the antibody of the invention with reεpect to effector function, so as to enhance the effectiveneεε of the antibody. For example, cyεteine reεidue(ε) may be introduced in the Fc region, thereby allowing interchain diεulfide bond formation in thiε region. The homodimeric antibody thuε generated may have improved internal!zation capability and/or increaεed complement- mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) . See Caron et al . , J. Exp Med. 176(4): 1191-5 (1992) and Shopes, J. Immunol . 148(9): 2918-22 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared uεing heterobifunctional cross-linkerε aε deεcribed in Wolff et al . , Cancer Res . 53(11): 2560-5 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lyεiε and ADCC capabilitie . See Stevenεon et al . , AntiCancer Drug Design 3 ( 4 ) : 219-30 (1989).

8. Immunoconjugateε

The invention alεo pertainε to immunoconjugateε compriεing an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof, or a small molecule toxin) , or a radioactive isotope (i.e., a radioconjugate) . Chemotherapeutic agentε uεeful in the generation of such immunoconjugates have been deεcribed above. Enzymatically active protein toxinε and fragmentε thereof which can be uεed include diphtheria A chain, nonbinding active fragments of diphtheria toxin, cholera toxin, botulinus toxin, exotoxin A chain (from Pεeudomonaε aertfginosa) , ricin A chain, abrin A chain, modeccin A chain, alpha-εarcin, Aleurites jbrdii proteins, dianthin proteins, Phytolaca americana proteins . (PAPI. PAPII, and PAP-S) , momordica charantia inhibitor, curcin, cretin, sapaonaria ofticinaliε inhibitor, gelonin, εaporin, mitogellin, reεtrictocin, phenomycin, enomycin and the tricotheceneε . Small molecule toxins include, for example, calicheamicins, maytanεinoidε, palytoxin and CC 1065. A variety of radionuclides are available for the production of radioconjugated antibodieε.

Conjugateε of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such aε N-εuccinimidyl-3- (2-pyridyldithiol) propionate (SPDP) , iminothiolane (IT) , bifunctional derivatives of imidoesters (εuch aε dimethyl adipimidate HCL) , active esters (such as disuccinimidyl εuberate) , aldehydeε (εuch as glutaraldehyde) , bis-azido compoundε, hexanediamine) , and biε-diazonium derivativeε . For example, a ricin immunotoxin can be prepared aε deεcribed in Vitetta et al . , Science 238(4830): 1098-104 (1987) . Carbon-14-labeled l-iεothiocyanatobenzyl-3- ethyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO 94/11026.

In another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent, and then administration of a "ligand" (e.g., avidin) which iε conjugated to a cytotoxic agent (e.g., a radionucleotide) .

9. Immunoliposomes

The antibodies diεcloεed herein may alεo be formulated aε immuno1iposomes . Liposomes containing the antibody are prepared by methods known in the art, εuch aε described in Eppstein et al . , Proc . Na tl . Acad. Sci . USA 82: 3688-92 (1985); Hwang et al . , Proc . Natl . Acad. Sci . USA 77(7): 4030-4 (1980); and U.S. Pat. Noε . 4,485,045 and 4,544,545. Lipoεomeε with enhanced circulation time are diεcloεed in U.S. Patent No. 5,013,556.

Particularly uεeful lipoεomes can be generated by the reverse phaεe evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG- derivatized phoεphatidylethanolamine (PEG-PE) . Lipoεomeε are extruded through filterε of defined pore εize to yield lipoεomeε with the deεired diameter. Fab' fragmentε of the antibody of the preεent invention can be conjugated to the lipoεomes as described in Martin et al . , J. Biol . Chem. 257(1): 286-8 (1982) via a disulfide interchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome. See Gabizon et al., J. Na tional Cancer Inst . 81(19): 484-8 ( 1989).

10. Pharmaceutical Compoεitionε of Antibodieε Antibodieε εpecifically binding an. LP231 polypeptide identified herein, aε well aε other molecules identified by the screening aεεayε diεclosed hereinbefore, can be adminiεtered for the treatment of variouε diεorders in the form of pharmaceutical compoεitionε .

If an LP231 polypeptide iε intracellular and whole antibodieε are uεed aε inhibitorε, internalizing antibodieε are preferred. However, lipofectionε or lipoεomeε can alεo be uεed to deliver the antibody, or an antibody fragment into cells. Where antibody fragmentε are uεed, the smallest inhibitory fragment that specifically bindε to the binding domain of the target protein iε preferred. For example, based upon the variable-region εequenceε of an antibody, peptide molecules can be deεigned that retain the ability to bind the target protein εequence. Such peptideε can be synthesized chemically and/or produced by recombinant DNA technology. See, e.g., Marasco et al., Proc . Natl . Acad. Sci . USA 90(16): 7889- 93 (1993) .

The formulation herein may alεo contain more than one active compound aε neceεεary for the particular indication being treated, preferably thoεe with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise an agent that enhances itε function, εuch aε, for example, a cytotoxic agent, cytokineε, chemotherapeutic agent, or growth-inhibitory agent. Such moleculeε are εuitable present in combination in amounts that are effective for the purpose intended. The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or felatin-microcapsules and poly- (methylmethactylate) microcapsules, respectively, in colloidal drug delivery syεterns (for example, liposomes, albumin microspheres , microemulsionε, nano-particleε, and nanocapεules) or in macroemulεions . Such techniques are diεcloεed in Remin on' Pharmaceu tical Sci enceε, supra.

The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes .

Sustained-release preparations may be prepared. Suitable exampleε of suεtained-releaεe preparationε include εemipermeable matriceε of εolid hydrophobic polymerε containing the antibody, which matriceε are in the form of εhaped articleε, e.g., films, or microcapsuleε . Examples of sustained-releaεe matrices include polyesters, hydrogels (for example, poly (2-hydroxyethyl-methacrylate) , or poly(vinylalcohol) ) , polylactideε (U.S. Pat. No. 3,773,919), copolymerε of L-glutamic acid y-ethyl-L-glutamate, non- degradable ethylene-vinylacetate, degradable lactic acid- glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D- (-) 3-hydroxylbutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid- glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, reεulting in a loεs of biological activity and posεible changes in immunogenicity. Rational strategies can be devised for εtabilization depending on the mechaniεmε involved. For example, if the aggregation mechaniεm is diεcovered to be intermolecular S-S bond formation through thioεuifide interchange, εtabilization may be achieved by modifying sulfhydryl residueε, lyophilizing from acidic εolutionε, controlling moiεture content, using appropriate additiveε, and developing εpecific polymer matrix co poεitionε .

G . Useε for anti-LP231 Antibodieε

The anti-LP231 antibodies of the present invention have variouε utilitieε. For example, anti-LP231 antibodieε may be uεed in diagnoεtic aεεayε for LP231 polypeptideε, e.g., detecting expression in specific cellε, tissueε, or serum. Various diagnostic assay techniques known in the art may be used, such as competitive binding assayε, direct or indirect εandwich asεays and immunoprecipitation aεεayε conducted in either heterogeneous or homogeneous phaεes [Zola, Monoclonal Antibodieε : A Manual of Techniqueε, CRC Presε, Inc. (1987) pp. 147-158] . The antibodieε uεed in the aεεayε can be labeled with a detectable moiety. The detectable moiety εhould be capable of producing, either directly or indirectly, a detectable εignal. For example, the detectable moiety may be a radioiεotope, εuch as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent or che iluminescent compound, such as fluorescein iεothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidaεe or horseradiεh peroxidaεe. Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including thoεe methodε deεcribed by Hunter et al . , Nature 144: 945 (1962); David et al . , Biochemistry 13 (5) : 1014-21 ( 1974); Pain et al., J Immunol . Meth . , 40(2): 219-30 (1981); and Nygren, J. Histochem. Cytochem. 30(5): 407-12 (1982).

Anti-LP231 antibodies also are useful for the affinity purification of LP231 polypeptideε from recombinant cell culture or natural sources. In this process, the antibodies against an LP231 polypeptide are immobilized on a suitable support, εuch a Sephadex reεin or filter paper, using methods well known in the art. The immobilized antibody is then contacted with a sample containing the LP231 polypeptide to be purified, and thereafter the support iε waεhed with a εuitable solvent that will remove substantially all the material in the sample except the LP231 polypeptide, which is bound to the immobilized antibody. Finally, the εupport iε waεhed with another εuitable solvent that will release the LP231 polypeptide from the antibody.

H. LP231 and Cerebellin Antagonistε/Agonists This invention encompasses methods of screening compounds to identity those that mimic the LP231 or cerebellin (agonists) or prevent the effect of the LP231 or cerebellin (antagonists) . Screening assays for antagonist drug candidates are designed to identity compounds that bind or complex with the LP231 or cerebellins encoded by the genes identified herein or otherwise interfere with the interaction of the encoded polypeptideε with other cellular proteinε. Such εcreening aεεayε will include aεεayε amenable to high- throughput εcreening of chemical libraries, making them particularly suitable for identifying small molecule drug candidateε .

The aεεayε can be performed in a variety of formats including protein-protein binding assays, biochemical screening aεsays, immunoassays, and cell-baεed assays, which are well characterized in the art. In binding assays, the interaction is binding, and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, the LP231 or cerebellin encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non- covalent attachments. Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the LP231 or cerebellin and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the LP231 or cerebellin to be immobilized can be uεed to anchor it to solid surface. The asεay iε performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated εurface containing the anchored component. When the reaction iε complete, the non-reacted components are removed, e.g.,- by washing, and complexes anchored on the solid surface are detected. When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the εurface indicateε that complexing occurred. Where the originally non-immobilized component doeε not carry a label, complexing can be detected, for example, by uεing a labeled antibody εpecifically binding the immobilized complex.

If the candidate compound interacts with but does not bind to a particular LP231 or cerebellin encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions. Such assays include traditional approaches, such as, e.g., cross-linking, co-immunoprecipitation, and co- purification through gradients or chromatographic columns. In addition, protein-protein interactions can be monitored through gradients or chromatographic columns. In addition, protein-protein interactionε can be monitored by uεing a yeaεt-baεed genetic εyste described by Fields and co-workers [Fields and Song, Nature 340(6230): 245-6 (1989); Chien et al., Proc . Natl . Acad. Sci . USA 88(21): 9578-82 (1991); Chevray and Nathans, Proc . Natl . Acad. Sci . USA 89(13): 5789- 93 (1992)]. Many transcriptional activators, such as yeast GAL4, consist of two physically diεcrete modular domainε, one acting as the DNA-binding domain, while the other functions aε the transcription-activation domain. The yeaεt expreεsion syεtem described in the foregoing publicationε (generally referred to aε the "two-hybrid εyεte ") takeε advantage of thiε property, and employε two hybrid proteinε, one in which the target protein iε fused to the DNA-binding domain of GAL4 , and another in which candidate activating proteins are fuεed to the activation domain. The expreεεion of GALl-lacZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonieε containing interacting polypeptideε are detected with chromogenic εubεtrate for β-galactoεidaεe. A complete kit (MATCHMAKER™) for identifying protein-protein interactions between two εpecific proteins using the two- hybrid technique is commercially available from Clontech. This system can also be extended to map protein domainε involved in specific protein interactions as well as to pinpoint amino acid residueε that are crucial for theεe interactionε .

Compounds that interfere with the interaction of a gene encoding an LP231 or cerebellin identified herein_. and other intra- or extracellular components can be teεted as follows. Typically, a reaction mixture is prepared containing the product of the gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products . To test the ability of a candidate compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound. In addition, a placebo may be added to a third reaction mixture to serve as a poεitive control. The binding (complex formation) between the test compound and the intra- or extracellular component preεent in the mixture iε monitored aε described hereinabove. The formation of a complex in the control reaction (ε) but not in the reaction mixture containing the teεt compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner .

Antagonistε may be detected by combining the LP231 or cerebellin and a potential antagoniεt with membrane-bound LP231 or cerebellin receptorε or recombinant receptors under appropriate conditions for a competitive inhibition assay. The LP231 or cerebellin can be labeled, such aε by radioactivity, εuch that the number of LP231 or cerebellin molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist. The gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting. See Coligan et al . , Current Protocols in Immunology 1(2): Ch. 5 (1991). Preferably, expression cloning is employed wherein polyadenylated RNA iε prepared from a cell responsive to the LP231 polypeptide, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responεive to the LP231 or cerebellin. Tranεfected cellε that are grown on glaεε εlides are exposed to labeled LP231 or cerebellin. The LP231 or cerebellin can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-εpecific protein kinaεe. Following fixation and incubation, the εlideε are εubj ected to autoradiographic analyεiε. Poεitive pools are identified and sub-poolε are prepared and re-transfected using an interactive sub-pooling and re-screening procesε, eventually yielding a single clone that encodes the putative receptor. Aε an alternative approach for receptor identification, labeled LP231 or cerebellin can be photoaffinity-linked with cell membrane or extract preparationε that expreεs the receptor molecule. Crosε-linked material iε reεolved by PAGE and exposed to X-ray film. The labeled complex containing the receptor can be exciεed, reεolved into peptide fragmentε, and εubj ected to protein micro-εequencing. The amino acid εequence obtained from micro-εequencing would be uεed to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor .

In another assay for antagonists, mammalian cells or a membrane preparation expresεing the receptor would be incubated with labeled LP231 or cerebellin in the presence of the candidate compound. The ability of the compound to enhance or block this interaction could then be removed.

More specific examples of potential antagonists include an oligonucleotide that binds to the fusionε of immunoglobulin with LP231 or cerebellin, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodieε and antibody fragmentε, εingle-chain antibodieε, anti- idiotypic antibodieε, and chimeric or humanized verεionε of such antibodies or fragmentε, aε well aε human antibodies and antibody fragments. Alternatively, a potential antagonist may be a closely related protein, for example, a mutated form of the LP231 or cerebellin that recognizeε the receptor but imparts no effect, thereby competitively inhibiting the action of the LP231 or cerebellin.

Another potential LP231 or cerebellin antagonist is an antisenεe RNA or DNA construct prepared uεing antiεenεe technology, where, e.g., an antiεense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to targeted mRNA and prevent its translation into protein. Antisense technology can be used to control gene expreεεion through triple-helix formation or antiεenεe DNA or RNA, both of which methodε are baεed on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide εequence, which encodes the mature LP231 or cerebellins herein, is used to design an antisenεe RNA oligonucleotide εequence of about 10 to 40 baεe pairε in length. A DNA oligonucleotide iε deεigned to be complementary to a region of the gene involved in transcription [triple helix; see Lee et al . , Nucl . Acidε Res 6(9): 3073-91 (1979); Cooney et al . , Science 241(4864): 456-9 (1988); Beal and Dervan, Science 251 (4999) : 1360-3 (1991)], thereby preventing transcription and the production of the LP231 polypeptide. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the LP231 or cerebellin [antisenεe; εee Okano, J. Neurochem. 56(2): 560-7 (1991); Oligodeoxynucleotides aε Antisense Inhibi tors of Gene Expression (CRC Preεε: Boca Raton, FL 1988)]. The oligonucleotideε deεcribed. above can alεo be delivered to cellε εuch that the antiεenεe RNA or DNA may be expreεεed in vivo to inhibit production of the LP231 polypeptide. When antiεense DNA is used, oligodeoxyribonucleotides derived from the translation- initiation site, e.g., between about -10 and +10 positions of the target gene nucleotide sequence, are preferred.

Potential antagonists include small molecules that bind to the active site, the receptor binding site, or growth factor or other relevant binding site of the LP231 polypeptide, thereby blocking the normal biological activity of the LP231 or cerebellin. Examples of small molecules include, but are not limited to, small peptides or peptide- like molecules, preferably soluble peptides, and εynthetic non-peptidyl organic or inorganic compounds.

Ribozymes are enzymatic RNA moleculeε capable of catalyzing the εpecific cleavage of RNA. Ribozymes act by εequence-εpecific hybridization to the complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage εites within a potential RNA target can be identified by known techniques. For further details, see, e.g., Rossi, Current Biology 4(5): 469-71 (1994) and PCT publication No. WO 97/33551 (published September 18, 1997).

Nucleic acid moleculeε in triple-helix formation uεed to inhibit transcription εhould be εingle-εtranded and composed of deoxynucleotides . The base composition of these oligonucleotides is designed such that it promotes triple- helix formation via Hoogsteen baεe-pairing rules, which generally require sizeable stretcheε of purines or pyrimidineε on one εtrand of a duplex. For further detailε see, e.g., PCT publication No. WO 97/33551, supra.

I. Diagnostic Uεeε

Another uεe of the compoundε of the invention (e.g., LP231 variants and anti-LP231 antibodies) described herein is to help diagnoεe whether a diεorder iε driven to εome extent by LP231 modulated signaling.

A diagnostic asεay to determine whether a particular diεorder iε driven by LP231 signaling can be carried out using the following εtepε : (1) culturing teεt cells or tiεεueε expreεεing LP231 ; (2) adminiεtering a compound which can inhibit LP231 modulated εignaling; and (3) meaεuring the LP231 mediated phenotypic effectε in the teεt cells. The steps can be carried out using standard techniques in light of the present discloεure-. For example, εtandard techniqueε can be used to isolate cells or tisεueε and to culture them in vivo . Compounds of varying degrees of selectivity are useful for diagnosing the role of LP231. For example, compounds which can inhibit LP231 modulated signaling, in addition to another form of adaptor molecule, can be uεed as an initial test compound to determine if one of several adaptor molecules drive the disorder. The selective compounds can then be used to further eliminate the posεible role of the other adaptor proteins in driving the disorder. Test compounds should be more potent in inhibiting intracellular signaling activity than in exerting a cytotoxic effect (e.g., an IC50 and LD₅₀ of greater than one) . The IC₅₀ and LD50 can be measured by standard techniqueε, such as an MTT assay or by measuring the amount of LDH releaεed. The degree of IC₅₀ and LD₅₀ of a compound should be taken into account in evaluating the diagnostic asεay. Generally, the larger the ratio, the more relative the information. Appropriate controlε take into account the poεεible cytotoxic effect of a compound, εuch as treating cells not associated with a cell proliferative disorder (e.g., control cells) with a test compound and can also be used as part of the diagnoεtic assay. The diagnostic methods of the invention involve the screening for agents that modulate the effects of LP231 upon synaptic diεorders. Exemplary detection techniques include radioactive labeling and immunoprecipitating (U. S. Patent No. 5,385,915).

For example, antibodies, including antibody fragments, can be used to qualitatively or quantitatively detect the expression of proteins encoded by the diseaεe-related genes ("marker gene products"). The antibody preferably is equipped with a detectable, e.g., fluorescent label, and binding can be monitored by light microscopy, flow cytometry, fluorimetry, or other techniqueε known in the art. In si tu detection of antibody binding to the marker gene products can be performed, for example, by immunofluorescence or immunoelectron microεcopy. For this purpose, a histological specimen iε removed from the patient, and a labeled antibody is applied to it, preferably by overlaying the antibody on a biological sample. This procedure also allows for determining the distribution of the marker gene product in the tissue examined. It will be apparent for those skilled in the art that a wide variety of histological methods are readily available for in si tu detection.

J. Pharmaceutical Compositions

The LP231 antagonistε or agonists thereof (e.g., antibodies) , as well as other molecules identified by the screening assays discloεed hereinbefore, can be employed as therapeutic agents. Such therapeutic agents are formulated according to known methods to prepare pharmaceutically useful compositions, whereby the LP231 antagonist or agonist thereof is combined in a mixture with a pharmaceutically acceptable carrier.

In the case of LP231 antagonist or agonist antibodies, if the protein encoded by the amplified gene is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, lipofections or lipoεomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment which specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable region sequences of an antibody, peptide molecules can be designed which retain the ability to bind the target protein sequence. Such peptides can be syntheεized chemically and/or produced by recombinant DNA technology [see, e.g., Marasco et al . , Proc . Natl . Acad. Sci . USA 90 ( 16 ) : 7889-93 (1993)].

Therapeutic formulations are prepared for storage by mixing the active ingredient having the deεired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizerε [Remington 's Pharmaceutical Sciences 16th edition (1980)], in the form of lyophilized formulationε or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosageε and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants include ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3- pentanol, and m-creεol; low molecular weight (less than about 10 residues) polypeptides; proteins, εuch as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucoεe, mannose, or dextrinε; chelating agentε such as EDTA; sugarε such as sucroεe, mannitol, trehaloεe or εorbitol; εalt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes) ; and/or non-ionic surfactants such as TWEEN™, PLURONIC™ or polyethylene glycol (PEG) .

The formulation herein may also contain more than one active compound as necesεary for the particular indication being treated, preferably thoεe with complementary activities that do not adverεely affect each other. Alternatively, or in addition, the compoεition may compriεe a cytotoxic agent, cytokine or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpoεe intended.

The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapεuleε and poly- (methylmethacylate) icrocapεuleε, reεpectively, in colloidal drug delivery εyεtemε (for example, lipoεomeε, albumin microεpheres, microemulεionε, nano-particles and nanocapsuleε) or in macroemulsions . Such techniqueε are diεcloεed in Remington 's Pharmaceutical Sciences 16th edition (1980).

The formulationε to be uεed for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

Therapeutic compositionε herein generally are placed into a container having a εterile access port, for example, and intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

Suεtained-releaεe preparations may be prepared. Suitable examples of sustained-releaεe preparations include semipermeable matriceε of εolid hydrophobic polymerε containing the antibody, which matrices are in the form of shaped articles, e.g., filmε, or microcapεuleε. Examples of sustained-release matrices include polyesters, hydrogels [for example, poly (2-hydroxyethyl-methacrylate) , or poly(vinylalcohol) ] , polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid- glycolic acid copolymerε εuch as those used in the LUPRON DEPOT™ (injectable microspheres composed of lactic acid- glycolic acid copolymer and leuprolide acetate) , and poly-D- (-) -3-hydroxybutyric acid. Microencapsulation of recombinant proteins for sustained release has been successfully performed with human growth hormone (rhGH) , interferon, interleukin-2, and MN rpg 120. Johnson et al . , Nat . Med.^'2(7): 795-9 (1996); Yasuda et al . , Biomed. Ther. 27: 1221-3 (1993); Hora et al . , Bio/Technology 8(8) : 755-8 (1990); Cleland, "Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Syεtemε" in Vaccine Design : The Subuni t and Adjuvant Approach, Powell and Newman, Edε . , Plenum Preεs, NY, 1995, pp. 439-462 WO 97/03692; WO 96/40072; WO 96/07399; and U.S. Pat. No. 5,654,010.

The suεtained-releaεe formulationε of theεe proteins may be developed using polylactic-coglycolic acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties. The degradation products of PLGA, lactic and glycolic acids, can be cleared quickly within the human body. Moreover, the degradability of this polymer can be adjusted from months to years depending on its molecular weight and compoεition. See Lewis, "Controlled release of bioactive agents from lactide/glycolide polymer" in Biodegradable Polymers aε Drug Delivery Systemε (Marcel Dekker; New York, 1990), M. Chasin and R. Langer (Eds.) pp. 1-41.

While polymers εuch as ethylene-vinyl acetate and lactic acid-glycolic acid enable releaεe of moleculeε for over 100 dayε, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, εtabilization may be achieved by modifying εulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions .

K. Methods of Treatment

It is contemplated that the compounds of the present invention may be uεed to treat various conditions including those characterized by overexpression and/or activation of the diseaεe-aεεociated geneε identified herein. Exemplary conditions or disorderε to be treated with εuch antibodies and other compounds, including, but not limited to, small organic and inorganic molecules, peptideε, antiεense molecules, etc., include Parkinson 'ε diεeaεe, Alzheimer 'ε diεease, bipolar and unipolar affective diεorders, schizophrenia, olivopontocerebellar atrophy, and Shy-Dager syndrome, especially those characterized by diεruption of εynapse function..

The active agents of the present invention, e.g., antibodies, are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration aε a bolus or by continuouε infusion over a period of time, by intramuscular, intraperitoneal, intracerebral, intracerobrospinal, εubcutaneouε, intra- articular, intrasynovial, intrathecal, intraoccular, intralesional, oral, topical, inhalation or through sustained release.

Other therapeutic regimens may be combined with the administration of the LP231 antagonists or antagonists, anti- cancer agents, e.g., antibodies of the instant invention. For the prevention or treatment of diεeaεe, the appropriate dosage of an active agent, (e.g., an antibody) will depend on the type of diseaεe to be treated, as defined above, the severity and course of the diεeaεe, whether the agent is adminiεtered for preventive or therapeutic purpoεeε, previous therapy, the patient 'ε clinical history and responεe to the agent, and the diεcretion of the attending physician. The agent is εuitably administered to the patient at one time or over a series of treatments.

Dosages and desired drug concentration of pharmaceutical compoεitionε of the preεent invention may vary depending on the particular uεe enviεioned. The determination of the appropriate doεage or route of administration is well within the εkill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective does for human therapy. Interspecieε εcaling of effective doεes can be performed following the principles laid down by Mordenti and Chappell, "The Use of Interspecieε Scaling in Toxicokineticε, " in Toxicokinetics and New Drug Development, Yacobi et al . , Eds., Pergamon Press, NY 1989, pp.4246.

When in vivo administration of an LP231 polypeptide or agonist or antagonist thereof is employed, normal dosage amounts may vary from about 10 ng/kg up to 100 mg/kg of mammal body weight or more per day, preferably about 1 pg/kg/day up to 100 mg/kg of mammal body weight or more per day, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature; εee, for example, U.S. Pat. Noε . 4,657,760, 5,206,344 or 5,225,212. It is within the scope of the invention that different formulations will be effective for different treatment compounds and different disorders, that administration targeting one organ or tisεue, for example, may necessitate delivery in a manner different from that to another organ or tiεsue. Moreover, dosageε may be administered by one or more separate administrations or by continuouε infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is suεtained until a deεired εuppression of diseaεe εymptomε occurε. However, other doεage regimens may be useful. Conventional techniques and assayε easily monitor the progress of therapy.

L. Articles of Manufacture

In another embodiment of the invention, an article of manufacture containing materials useful for the diagnosis or treatment of the disorders described above is provided. The article of manufacture compriseε a container and a label. Suitable containers include, for example, bottles, vials, syringeε, and test tubeε . The containers may be formed from a variety of materials such as glasε or plastic. The container holds a composition which is effective for diagnosing or treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) . The active agent in the composition is typically an LP231 polypeptide, antagonist or agonist thereof. The label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, εuch as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needleε, εyringeε, and package inserts with instructionε for uεe.

The following exampleε are offered for illuεtrative purpoεes only, and are not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

Example 1 Expreεεion and Purification of LP231 in E. coli The bacterial expreεεion vector pQE60 is used for bacterial expression in this example. (QIAGEN, Inc., Chatsworth, CA) . pQE60 encodes ampicillin antibiotic resiεtance ("Ampr") and containε a bacterial origin of replication ("ori"), an IPTG inducible promoter, a riboεome binding εite ("RBS"), εix codonε encoding hiεtidine reεidues that allow affinity purification using nickel-nitrilo-tri- acetic acid ("Ni-NTA") affinity resin sold by QIAGEN, Inc., and suitable single restriction enzyme cleavage sites. These elements are arranged such that a DNA fragment encoding a polypeptide can be inεerted in εuch a way as to produce that polypeptide with the six His residues (i.e., a "6 X His tag") covalently linked to the carboxyl terminus of that polypeptide. However, a polypeptide coding sequence can optionally be inserted such that translation of the six His codons is prevented and, therefore, a polypeptide is produced with no 6 X His tag.

The nucleic acid sequence encoding the desired portion of the LP231 lacking the hydrophobic leader sequence is amplified from a cDNA clone using PCR oligonucleotide primers (based on the sequenceε preεented, e.g., as preεented in SEQ ID NO:l), which anneal to the amino terminal encoding DNA εequences of the desired portion of the LP231 and to sequences in the construct 3 ' to the cDNA coding sequence. Additional nucleotideε containing reεtriction sites to facilitate cloning in the pQE60 vector are added to the 5' and 3' sequences, respectively.

For cloning a LP231, the 5' and 3' primers have nucleotides corresponding or complementary to a portion of the coding sequence of LP231, e.g., as presented in SEQ ID N0:1, according to known method εtepε. One of ordinary skill in the art would appreciate, of course, that the point in a polypeptide coding sequence where the 5 ' primer begins can be varied to- amplify a desired portion of the complete polypeptide shorter or longer than the mature form.

The amplified LP231 nucleic acid fragments and the vector pQE60 are' digested with appropriate restriction enzymes and the digested DNAs are then ligated together. Inεertion of the LP231 DNA into the reεtricted pQE60 vector places the LP231 polypeptide coding region including its associated stop codon downstream from the IPTG-inducible promoter and in-frame with an initiating AUG codon. The associated stop codon prevents translation of, the six histidine codons downstream of the insertion point.

The ligation mixture is tranεformed into competent E. coli cellε using standard procedures such as those described in Sambrook, et al . , 1989; Ausubel, 1987-1998. E. coli εtrain Ml5/rep4, containing multiple copies of the plasmid pREP4, which expresses the lac repressor and confers kanamycin resistance ("Kanr"), is used in carrying out the illustrative example described herein. This strain, which is only one of many that are suitable for expreεsing LP231 polypeptide, is available commercially from QIAGEN, Inc. Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA iε isolated from resiεtant colonieε and the identity of the cloned DNA confirmed by restriction analyεis, PCR and DNA sequencing.

Clones containing the desired constructε are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 μg/ml) and kanamycin (25 μg/ml) . The O/N culture is used to inoculate a large culture, at a dilution of approximately 1:25 to 1:250. The cells are grown to an optical density at 600 nm ("OD600") of between 0.4 and 0.6. Iεopropyl-b-D-thiogalactopyranoεide ("IPTG") iε then added to a final concentration of 1 mM to induce tranεcription from the lac repreεsor sensitive promoter, by inactivating the lad represεor. Cellε subsequently are incubated further for 3 to 4 hours. Cells then are harvested by centrifugation.

The cells are then stirred for 3-4 hours at 4°C in 6M guanidine-HCl, pH8. The cell debris is removed by centrifugation, and the supernatant containing the LP231 is dialyzed against 50 mM Na-acetate buffer pH6, supplemented with 200 mM NaCl. Alternatively, a polypeptide can be succeεsfully refolded by dialyzing it against 500 mM NaCl, 20% glycerol, 25 mM^' Tris/HCl pH7.4, containing protease inhibitors.

If insoluble protein is generated, the protein is made soluble according to known method steps. After renaturation, the polypeptide is purified by ion exchange, hydrophobic interaction, and size exclusion chromatography. Alternatively, an affinity chromatography step such as an antibody column is used to obtain pure LP231. The purified polypeptide is stored at 4°C or frozen at -40°C to -120°C. Example 2

Cloning and Expreεεion of LP231 in a

Baculovirus Expresεion Syεtem

In this example, the plasmid εhuttle vector pA2 GP iε uεed to inεert the cloned DNA encoding the mature polypeptide into a baculoviruε to expreεε LP231, uεing a baculovirus leader and standard methods as described in Summers, et al . , A Manual of Methodε for Baculovirus Vectorε and Inεect Cell Cul ture Procedureε, Texas Agricultural Experimental Station Bulletin No. 1555 (1987) . This expression vector contains the strong polyhedrin promoter of the Autographa califomica nuclear polyhedrosis virus (AcMNPV) followed by the secretory signal peptide (leader) of the baculovirus gp67 polypeptide and convenient restriction εiteε εuch as BamHI, Xba I, and Asp718. The polyadenylation εite of the simian virus 40 ("SV40") is used for efficient polyadenylation. For easy selection of recombinant viruε, the plaεmid containε the beta- galactosidase gene from E. coli under control of a weak Drosophila promoter in the same -orientation, followed by the polyadenylation signal of the polyhedrin gene. The inserted genes are flanked on both sides by viral sequences for cell- mediated homologous recombination with wild-type viral DNA to generate viable virus that expresεes the cloned polynucleotide .

Other baculovirus vectors are used in place of the vector above, such as pAc373, pVL941 and pAcIMl, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required. Such vectorε are deεcribed, for instance, in Luckow, et al . , Virology 170:31-39.

The cDNA εequence encoding the mature LP231 polypeptide in a clone, lacking the AUG initiation codon and the naturally aεsociated nucleotide binding site, is amplified using PCR oligonucleotide primers corresponding to the 5 ' and 3' sequences of the gene. Non-limiting examples include 5 ' and 3 ' primers having nucleotides correεponding or complementary to a portion of the coding εequence of a LP231 polypeptide, e.g., aε preεented in SEQ ID N0:1, according to known method εteps .

The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (e.g., "Geneclean, " BIO 101 Inc., La Jolla, CA) . The fragment then is then digested with the appropriate restriction enzyme and again is purified on a 1% agarose gel. This fragment is designated herein "Fl".

The plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated uεing calf inteεtinal phoεphataεe, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit ( "Geneclean" BIO 101 Inc., La Jolla, CA) . Thiε vector DNA is designated herein "VI" .

Fragment Fl and the dephosphorylated plasmid VI are ligated together with T4 DNA ligase. E. coli HB101 or other εuitable E. coli. hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, CA) cells are transformed with the ligation mixture and spread on culture plates. Bacteria are identified that contain the plasmid bearing the human LP231 gene using the PCR method, in which one of the primers that iε uεed to amplify the gene and the second primer is from well within the vector so that only thoεe bacterial colonies containing the LP231 gene fragment will show amplification of the DNA. The sequence of the cloned fragment is confirmed by DNA sequencing. Thiε plaεmid is designated herein pBac LP231 .

Five μg of the plaεmid pBacLP231 iε co-tranεfected with 1.0 μg of a commercially available linearized baculovirus DNA ( "BaculoGold™ baculovirus DNA", Pharmingen, San Diego, CA) , using the lipofection method described by Feigner, et al . , Proc . Natl . Acad. Sci . USA 84:7413-7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBac LP231 are mixed in a sterile well of a microtiter plate containing

50 μl of serum-free Grace's medium (Life Technologies, Inc., Rockville, MD) . Afterwards, 10 μl Lipofectin pluε 90 μl Grace ' ε medium are added, mixed and incubated for 15 minuteε at room temperature. Then the tranεfection mixture is added drop-wise to Sf9 insect cellε (ATCC CRL 1711) εeeded in a 35mm tiεεue culture plate with 1 ml Grace's medium without serum. The plate is rocked back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27°C. After 5 hours the transfection solution is removed from the plate and 1 ml of Grace ' s insect medium supplemented with 10% fetal calf serum is added. The plate is put back into an incubator and cultivation is continued at 27°C for four days.

After four days the supernatant is collected and a plaque assay is performed. An agarose gel with "Blue Gal" (Life Technologies, Inc., Rockville, MD) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a "plaque asεay" of thiε type can alεo be found in the user's guide for insect cell culture and baculovirology diεtributed by Life Technologies, Inc., Rockville, MD, page 9-10) . After appropriate incubation, blue stained plaques are picked with a micropipettor tip (e.g., Eppendorf). The agar containing the recombinant viruseε iε then reεuspended in a microcentrifuge tube containing 200 μl of Grace's medium and the suεpenεion containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35mm disheε. Four days later the supernatants of these culture disheε are harveεted and then they are εtored at 4°C. The recombinant virus is called V-LP231.

To verify the expression of the LP231 gene, Sf9 cells are grown in Grace ' s medium εupplemented with 10% heat- inactivated FBS. The cellε are infected with the recombinant baculovirus V-LP231 at a multiplicity of infection ("MOI") of about 2. Six hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available, e.g., from Life Technologies, Inc., Rockville, MD) . If radiolabeled polypeptides are desired, 42 hours later, 5 mCi of ³⁵S- methionine and 5 mCi ³⁵S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then they are harvested by centrifugation. The polypeptides in the supernatant as well as the intracellular polypeptides are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled) . Microsequencing of the amino acid sequence of the amino terminus of purified polypeptide can be used to determine the amino terminal sequence of the mature polypeptide and thus the cleavage point and length of the secretory signal peptide. Example 3 Cloning and Expreεεion of LP231 in Mammalian Cellε

A typical mammalian expreεsion vector contains at least one promoter element, which mediates the initiation of transcription of mRNA, the polypeptide coding εequence, and εignals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequenceε and intervening εequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late pro oterε from SV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV) . However, cellular elements can also be used (e.g., the human actin promoter) . Suitable expresεion vectorε for use in practicing the present invention include, for example, vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, CA) , pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or pcDNA3.1/Hygro (+/-) (Invitrogen) , PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109) . Other suitable mammalian host cells include human Hela 293, H9 , Jurkat cells, mouse NIH3T3 , C127 cellε, Cos 1, Cos 7 and CV 1, quail QCl-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

Alternatively, the gene can be expresεed in εtable cell lines that contain the gene integrated into a chromoεome. The co-transfection with a εelectable marker such as dhfr, gpt, neomycin, or hygromycin allows the identification and isolation of the transfected cells. The transfected gene can also be amplified to express large amounts of the encoded polypeptide. The DHFR (dihydrofolate reductase) marker is useful to develop cell lines that carry several hundred or even several thouεand copieε of the gene of interest. Another useful selection marker is the enzyme glutamine synthaεe (GS) (Murphy, et al . , Biochem . J. 227:277-279 (1991); Bebbington, et al . , Bio /Technology 10:169-175 (1992)). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest reεiεtance are εelected. These cell lines contain the amplified gene(s) integrated into a chromoεome. Chineεe hamεter ovary (CHO) and NSO cells are often uεed for the production of polypeptideε.

The expreεεion vectors pel and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen, et al . , Molec . Cell . Biol . 5:438-447 (1985)) plus a fragment of the CMV-enhancer (Boshart, et al . , Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, Xbal and Asp718, facilitate the cloning of the gene of intereεt. The vectors contain in addition the 3 ' intron, the polyadenylation and termination signal of the rat preproinsulin gene.

Example 3 (a) Cloning and Expression in COS Cells

The expresεion plaεmid, pLP231 HA, iε made by cloning a cDNA encoding LP231 into the expression vector pcDNAI/Amp or pcDNAIII (which can be obtained from Invitrogen, Inc.).

The expreεεion vector pcDNAI/amp contains: (1) an E. coli origin of replication effective for propagation in E. coli and other prokaryotic cells; (2) an ampicillin reεistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron; (5) several codons encoding a hemagglutinin fragment (i.e., an "HA" tag to facilitate purification) or HIS tag (see, e.g, Ausubel, εupra) followed by a termination codon and polyadenylation signal arranged so that a cDNA can be conveniently placed under expresεion control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation εignal by means of restriction εiteε in the polylinker. The HA tag correεpondε to an epitope derived from the influenza hemagglutinin polypeptide deεcribed by Wilεon, et al . , Cell 37:767-778 (1984) . The fusion of the HA tag to the target polypeptide allows easy detection and recovery of the recombinant polypeptide with an antibody that recognizeε the HA epitope. pcDNAIII containε, in addition, the εelectable neomycin marker .

A DNA fragment encoding the LP231 iε cloned into the polylinker region of the vector so that recombinant polypeptide expresεion iε directed by the CMV promoter. The plaεmid construction strategy is as follows. The LP231 cDNA of a clone is amplified using primers that contain convenient restriction siteε, much aε described above for construction of vectors for expresεion of LP231 in E. coli . Non-limiting exampleε of suitable primers include those based on the coding sequence presented in SEQ ID NO: 2, as they encode LP231 as described herein.

The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with suitable restriction enzyme (s) and then ligated. The ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037), and the transformed culture is plated on ampicillin media plateε which then are incubated to allow growth of ampicillin reεistant colonies. Plasmid DNA is isolated from resiεtant colonieε and examined by reεtriction analysis or other means for the presence of the LP231-encoding fragment.

For expression of recombinant LP231, COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in Sambrook, et al . , Molecular Cloning: a Laboratory Manual , Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989) . Cells are incubated under conditions for expression of LP231 by the vector.

Expresεion of the LP231-HA fuεion polypeptide is detected by radiolabeling and immunoprecipitation, using methods described in, for example Harlow, et al . , Antibodies : A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988) . To this end, two days after transfection, the cells are labeled by incubation in media containing ³⁵S-cysteine for 8 hours. The cells and the media are collected, and the cells are washed and lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by^' Wilson, et al . cited above. Proteins are precipitated from the cell lysate and from the culture media uεing an HA-specific monoclonal antibody. The precipitated polypeptides then are analyzed by SDS-PAGE and autoradiography . An expression product of the expected size is seen in the cell lysate, which is not seen in negative controls.

Example 3 (b) Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of LP231 polypeptide. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146) . The plasmid contains the mouse DHFR gene under control of the SV40 early promoter. Chinese hamster ovary cells or other cells lacking dihydrofolate activity that are tranεfected with theεe plaεmidε can be εelected by growing the cells in a εelective medium (alpha inuε MEM, Life Technologieε) εupplemented with methotrexate. The amplification of the DHFR genes in cells resistant to methotrexate (MTX) has been well documented (see, e.g., F. W. Alt, et al . , J. Biol . Chem. 253:1357-1370 (1978); J. L. Hamlin and C. Ma, Biochem. et Biophys . Acta 1097:107-143 (1990); and M. J. Page and M. A. Sydenham, Biotechnology 9:64-68 (1991)). Cells grown in increasing concentrations of MTX develop resistance to the drug by overproducing the target enzyme, DHFR, as a result of amplification of the DHFR gene. If a εecond gene iε linked to the DHFR gene, it is usually co-amplified and over-expresεed. It iε known in the art that this approach can be used to develop cell lines carrying more than 1,000 copies of the amplified gene(s). Subsequently, when the methotrexate is withdrawn, cell lines are obtained which contain the amplified gene integrated into one or more chromosome (s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strong promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen, et al . , Molec. Cell . Biol . 5:438-447 (1985)) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart, et al . , Cell 41:521-530 (1985)). Downstream of the promoter are BamHI, Xbal, and Asp718 restriction enzyme cleavage sites that allow integration of the genes. Behind these cloning sites the plasmid contains the 3 ' intron and polyadenylation εite of the rat preproinsulin gene. Other high efficiency promoters can also be uεed for the expreεεion, e.g., the human b-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI . Clontech's Tet-Off and Tet-On gene expression syεtemε and similar systems can be used to express the LP231 in a regulated way in mammalian cells (M. Gossen, and H. Bujard, Proc . Natl . Acad. Sci . USA 89: 5547-5551 (1992)). For the polyadenylation of the mRNA other signals, e.g., from the human growth hormone or globin genes can be used as well. Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate .

The plasmid pC4 is digested with restriction enzymes and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The vector is then isolated from a 1% agarose gel.

The DNA εequence encoding the complete LP231 polypeptide iε amplified uεing PCR oligonucleotide primers corresponding to the 5' and 3' sequenceε of the gene. Non- limiting examples include 5' and 3' primers having nucleotides corresponding or complementary to a portion of the coding sequence of LP231, e.g., aε presented in SEQ ID NO:2, according to known method steps.

The amplified fragment is digested with suitable endonucleases and then purified again on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC4 using, for instance, restriction enzyme analysiε.

Chineεe hamster ovary (CHO) cells lacking an active DHFR gene are uεed for tranεfection. 5 μg of the expreεεion plaεmid pC4 is cotransfected with 0.5 μg of the plaεmid pSV2-neo uεing lipofectin. The plaεmid pSV2neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 μg/ml G418. After 2 days, the cells are trypsinized and εeeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 μg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM) . Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 mM, 2 mM, 5 mM, 10 mM, 20 mM) . The same procedure is repeated until clones are obtained which grow at a concentration of 100 - 200 mM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reverse phase HPLC analysis.

Example 4 Tisεue Distribution of LP231 mRNA Expression

Northern blot analysis is carried out to examine LP231 gene expression in human tissues, using methods described by, among others, Sambrook, et al . , cited above. A cDNA probe containing the entire nucleotide sequence of LP231 polypeptide (SEQ ID NO: 2) is labeled with ³ P using the Rediprime™ DNA labeling system (Amersham Life Science) , -Ill-

according to the manufacturer's instruction . After labeling, the probe iε purified uεing a CHROMA SPIN-100™ column (Clontech Laboratorieε, Inc.), according to the manufacturer ' ε protocol number PT1200-1. The purified and labeled probe iε uεed to examine variouε human tiεεueε for LP231 mRNA.

Multiple Tiεεue Northern (MTN) blotε containing variouε human tiεεueε (H) or human immune εystem tiεεues (IM) are obtained from Clontech and are examined with the labeled probe using ExpreεεHyb hybridization εolution (Clontech) according to manufacturer's protocol number PT1190-1. Following hybridization and washing, the blots are mounted and expoεed to film at -70°C overnight, and developed according to εtandard procedures. The results show LP231 mRNA and protein to be selectively expresεed in certain neural tiεεueε.

Claims

WHAT IS CLAIMED IS:

1. Isolated nucleic acid comprising DNA having at least a 91% sequence identity to (a) a DNA molecule encoding an LP231 polypeptide comprising the sequence of amino acid residues 1 or 20 through 225, inclusive, of SEQ ID NO: 1, and (b) the complement of the DNA molecule of (a) .

2. The nucleic acid of Claim 1, wherein said DNA comprises the sequence of corresponding nucleotide positions 55 or 115 through 729, inclusive, of SEQ ID NO:l .

3. The nucleic acid of Claim 1, wherein said DNA compriseε the nucleotide εequence of εequence of SEQ ID NO:l.

4. The iεolated nucleic acid molecule of Claim 1 compriεing a nucleotide εequence that encodeε the sequence of amino acid residues from 1 or about 20 to about 225 of SEQ ID N0:1.

5. An isolated nucleic acid molecule encoding a LP231 polypeptide comprising DNA that hybridizes to the complement of the nucleic acid sequence that encodes amino acids 1 or about 20 to about 225 of SEQ ID NO: 1.

6. The isolated nucleic acid molecule of Claim 5, wherein the nucleic acid sequence that encodes amino acids 1 or about 20 to about 225, inclusive, of SEQ ID NO: 2 or comprises nucleotides 55 or about 115 to about 729, inclusive, of SEQ ID NO:l.

7. The isolated nucleic acid molecule of Claim 5, wherein hybridization occurs under stringent hybridization and wash conditions.

8. An isolated nucleic acid molecule comprising (a) DNA encoding a polypeptide scoring at least 91% positives when compared to the sequence of amino acid residues selected from the group consisting of: (a) from 1 or about 20 to about 225, inclusive, of SEQ ID NO: 1; or (b) the complement of the DNA of (a) .

9. An isolated nucleic acid molecule compriεing at least about 250 nucleotides in length and which is produced by hybridizing a test DNA under εtringent hybridization conditions with (a) a DNA molecule which encodes an LP231 polypeptide comprising a sequence of amino acid residues from 1 or about 20 to about 225, inclusive, of SEQ ID NO: 1, or (b) the complement of the DNA molecule of (a) .

10. The isolated nucleic acid molecule of Claim 9, which has at least about 91% sequence identity to (a) or (b) .

11. A vector comprising the nucleic acid molecule of any of Claims 1 to 10.

12. The vector of Claim 11, wherein said nucleic acid molecule is operably linked to control sequences recognized by a host cell tranεformed with a the vector.

13. A host cell comprising the vector of Claim 12.

14. The host cell of Claim 13, wherein said cell is a CHO cell.

15. The host cell of Claim 13, wherein εaid cell iε an E. coli cell.

16. The host cell of Claim 13, wherein said cell is a yeast cell.

17. A process for procuring an LP231 polypeptide comprising culturing the host cell of Claim 13 under conditions suitable for expresεion of εaid LP231 polypeptide and recovering εaid LP231 polypeptide from the cell culture.

18. An iεolated polypeptide compriεing an amino acid εequence compriεing at leaεt about 91% εequence identity to the εequence compriεing reεidueε 1 or about 20 to about 225 of SEQ ID NO: 1.

19. The iεolated LP231 polypeptide of Claim 18 compriεing amino acid reεidueε 1 or about 20 to about 225 of SEQ IDNO: 1.

20. An iεolated LP231 polypeptide εcoring at leaεt 91% positives when compared to the sequence of amino acids from about 1 or about 20 to about 225 of SEQ ID NO: 1.

21. An isolated LP231 polypeptide comprising the sequence of amino acid residueε from 1 or about 20 to about 225 of SEQ ID NO:l, or a fragment thereof sufficient to provide a binding site for an anti-LP231 antibody, respectively.

22. An isolated polypeptide produced by (i) hybridizing a test DNA^" molecule under stringent conditions with (a) a DNA molecule encoding an LP231 polypeptide comprising the εequence of amino acid reεidues from 1 or about 20 to about 225 of SEQ ID NO: 2; or (b) the complement of the DNA molecule of (a); (ii) culturing a host cell comprising the said test DNA molecule under conditions suitable for the expression of said polypeptide, and (iii) recovering said polypeptide from the cell culture.

23. The isolated polypeptide of Claim 22, wherein εaid' test DNA has at least about 91% εequence identity to (a) or (b) .

24. A chimeric molecule compriεing an LP231 polypeptide fused to a heterologous amino acid sequence.

25. The chimeric molecule of Claim 24, wherein εaid heterologous amino acid sequence is an epitope tag sequence.

26. The chimeric molecule of Claim 24, wherein said heterologous amino acid sequence is an Fc region of an immunoglobulin.

27. An antibody which specifically binds to an LP231 polypeptide .

28. The antibody of Claim 20, where said antibody is a monoclonal antibody.

29. The antibody of Claim 20, wherein said antibody is selected from the group consisting of a humanized antibody and a human antibody.

30. An agonist to LP231.

31. An antagonist to LP231.

32. A composition comprising a therapeutically effective amount of an active agent selected from the group consisting of: (a) an LP231 polypeptide, (b) an agonist to an LP231 polypeptide, (c) an antagonist to an LP231 polypeptide, and

(d) an anti-LP231 antibody; in combination with a pharmaceutically acceptable carrier.

33. A method of treating neurological disorders comprising administering a therapeutically effective amount of an LP231 agonist, or antagonist to a mammal εuffering from said disorder.

34. A method of diagnosing a neurological disorder by: (1) culturing test cells or tissues expressing LP231 ; (2) administering a compound which can inhibit LP231 modulated signaling; and (3) measuring the LP231 mediated phenotypic effects in the test cells or tissues.

35. An article of manufacture comprising a container, label and therapeutically effective amount of LP231 agonist or antagonist thereof in combination with a pharmaceutically effective carrier.