EP1328632A2 - Facteur associe a c1q, polypeptides homologues et utilisations therapeutiques associees - Google Patents

Facteur associe a c1q, polypeptides homologues et utilisations therapeutiques associees

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Publication number
EP1328632A2
EP1328632A2 EP01958853A EP01958853A EP1328632A2 EP 1328632 A2 EP1328632 A2 EP 1328632A2 EP 01958853 A EP01958853 A EP 01958853A EP 01958853 A EP01958853 A EP 01958853A EP 1328632 A2 EP1328632 A2 EP 1328632A2
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EP
European Patent Office
Prior art keywords
polypeptide
dna
antibody
sequence
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01958853A
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German (de)
English (en)
Inventor
Eric Wen Su
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Eli Lilly and Co
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Eli Lilly and Co
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Publication date
Application filed by Eli Lilly and Co filed Critical Eli Lilly and Co
Publication of EP1328632A2 publication Critical patent/EP1328632A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/472—Complement proteins, e.g. anaphylatoxin, C3a, C5a

Definitions

  • 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.
  • CRF human Clq-related factor
  • the cerebellum contains a hexadecapeptide, termed cerebellin.
  • 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 po ⁇ tsynaptic structures which suggests that the cerebellin precursor may be involved in synaptic physiology.
  • Cbln2 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.
  • 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.
  • Extracellular proteins play an important role in the formation, differentiation and maintenance of multicellular organisms.
  • secreted polypeptides for instance, mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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) .
  • 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.
  • 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
  • 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) .
  • 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) .
  • 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.
  • 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% ⁇ equence identity, yet more preferably at least about 99% sequence identity to (a) or (b) , and isolating the test DNA molecule .
  • 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) .
  • 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.
  • 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.
  • 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 nucleot
  • 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.
  • the invention provides a host cell comprising the above vector.
  • the host cells may be CHO cells, E ⁇ cherichia 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.
  • the invention provides isolated LP231 polypeptides encoded by any of the isolated nucleic acid sequences hereinabove defined.
  • 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.
  • the polypeptide comprises amino acid residues about 1 or about 20 to about 225 inclusive, of SEQ ID NO: 2.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • the invention provides an antibody that specifically binds to an LP231 polypeptide or fragment thereof.
  • the antibody is a monoclonal antibody, an antibody fragment or a single chain antibody.
  • the invention concerns agonists and antagonists of a native LP231 polypeptide.
  • the agonist or antagonist is an anti-LP231 antibody or a small molecule.
  • 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.
  • the invention concerns a composition
  • a composition comprising an LP231 polypeptide, or an agonist or antagonist as hereinabove defined, in combination with a carrier.
  • the carrier is pharmaceutically acceptable.
  • 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).
  • the invention concerns the use of an LP231 polypeptide, or an agonist or antagonist thereof in a method for treating a neurologic disorder.
  • 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.
  • 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 .
  • the invention relates to LP231 antagonists and/or agonist molecules.
  • the invention provides a method of screening compounds that mimic LP231 (agonists) or diminish the effect of LP231 (antagonists) .
  • the invention relates to a therapeutic composition
  • a therapeutic composition comprising a therapeutically effective amount of LP231 polypeptide, antagonist or agonist thereof in combination with a pharmaceutically-acceptable carrier.
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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 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.
  • 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 ribo ⁇ ome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "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 .
  • 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 sper
  • Modely 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.
  • 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.
  • 5X SSC 750 mM sodium chloride, 75 mM sodium citrate
  • 50 mM sodium phosphate at pH 7.6 50 mM sodium phosphate at pH 7.6
  • 5X Denhardt's solution 10% dextran sulfate
  • 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.
  • epitope tagged 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) .
  • immunoadhesion designates antibody-like molecules that combine the binding specificity of a heterologous protein (an “adhesion”) with the effector functions of immunoglobulin constant domains.
  • 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.
  • immunoglobulin such as IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.
  • 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.
  • monoclonal antibody 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 .
  • Activity refers to form(s) of LP231 which retain the biologic and/or immunologic activities of native or naturally-occurring LP231 polypeptide.
  • 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 .
  • 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.
  • 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 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.
  • antibody is used in the broadest sense and specifically covers, without limitation, intact monoclonal antibodies, polyclonal antibodies, multi ⁇ pecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
  • treating refers 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 di ⁇ order as well as tho ⁇ e prone to have the di ⁇ order or tho ⁇ e 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 con ⁇ ecutively done without interruption but, rather, is cyclic in nature.
  • mammal refers ⁇ to any mammal cla ⁇ ified as a mammal, including humans, domestic and farm animals, and zoo, sports or pet animals, such as cattle (e.g., cow ⁇ ) , horses, dogs, sheep, pig ⁇ , 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 simultaneou ⁇ (concurrent) and con ⁇ ecutive admini ⁇ tration 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.
  • a "therapeutically- effective amount" to a mammal suffering or prone to ⁇ uffering or to prevent it from ⁇ uffering from a neurologic disorder is such an amount which induces, ameliorate ⁇ or otherwi ⁇ e causes an improvement in the pathological symptom ⁇ , di ⁇ ease progression, physiological conditions a ⁇ sociated with or resistance to succumbing to a disorder principally characterized by synaptic dysfunction
  • Carriers as used herein include pharmaceutically- acceptable carrier ⁇ , excipient ⁇ , or ⁇ tabilizer ⁇ which are nontoxic to the cell or mammal being expo ⁇ ed thereto at the dosages and concentrations employed. Often the physiologically-acceptable carrier is an aqueous pH buffered solution.
  • Example ⁇ of phy ⁇ iologically acceptable carriers include buffer ⁇ such a ⁇ pho ⁇ phate, citrate, and other organic acids; antioxidants including a ⁇ corbic acid; low molecule weight (le ⁇ than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, a ⁇ paragine, arginine or lysine; ono ⁇ accharide ⁇ , disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; ⁇ ugar alcohol ⁇ ⁇ uch as mannitol or sorbitol; salt- forming counterions such as sodium; and/or nonionic surfactant ⁇ ⁇ uch as TWEENTM, polyethylene glycol (PEG) , and PLURONICTM .
  • buffer ⁇ such a ⁇ pho ⁇ phate, citrate, and other organic
  • Antibody fragments comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab')l and Fv fragments; diabodie ⁇ ; linear antibodie ⁇ (Zapata et al . , Protein Engin . 8 (10): 1057-1062 (1995) ) ; ⁇ ingle-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodie ⁇ produce ⁇ two identical antigen-binding fragment ⁇ , called “Fab” fragments, each with a ⁇ ingle antigen-binding ⁇ ite, and a re ⁇ idual "Fc" fragment, a de ⁇ ignation reflecting the ability to cry ⁇ tallize readily.
  • Pep ⁇ in treatment yield ⁇ an F(ab') 2 fragment that ha ⁇ two antigen-combining ⁇ ite ⁇ 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 fragment ⁇ differ from Fv fragments by the addition of a few re ⁇ idue ⁇ at the carboxy terminu ⁇ of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH i ⁇ the de ⁇ ignation herein for Fab' in which the cysteine residue (s) of the constant domains bear a free thiol group.
  • F(ab') 2 antibody fragment ⁇ originally were produced as pair ⁇ of Fab' fragment ⁇ which have hinge cy ⁇ teines between them. Other chemical coupling ⁇ of antibody fragment ⁇ are also known.
  • the "light chain ⁇ " of antibodies (immunoglobulins) from any vertebrate ⁇ pecie ⁇ can be a ⁇ igned to one of two clearly di ⁇ tinct types, called kappa and lambda, ba ⁇ ed on the amino acid sequences of their constant domains.
  • immunoglobulins can be a ⁇ igned to different cla ⁇ se ⁇ .
  • Single-chain Fv or “ ⁇ Fv” antibody fragment ⁇ compri ⁇ e the V H and V L domain ⁇ of antibody, wherein the ⁇ e domain ⁇ are pre ⁇ ent in a ⁇ ingle polypeptide chain.
  • the Fv polypeptide further compri ⁇ e ⁇ a polypeptide linker between the V H and V L domain, which enable ⁇ the sFv to form the desired structure for antigen binding.
  • diabodie ⁇ refers to small antibody fragments with two antigen-binding site ⁇ , 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 ) •
  • V H heavy-chain variable domain
  • V L light-chain variable domain
  • Diabodies are de ⁇ cribed 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- proteinaceou ⁇ ⁇ olutes.
  • 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 re ⁇ idue ⁇ of N-terminal or internal amino acid ⁇ equence by u ⁇ e of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing condition ⁇ u ⁇ ing Cooma ⁇ ie blue, or preferably, ⁇ ilver ⁇ tain.
  • I ⁇ olated antibody include ⁇ the antibody in ⁇ itu 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.
  • label when u ⁇ ed herein refers to a detectable compound or compo ⁇ ition which i ⁇ conjugated directly or indirectly to the antibody so a ⁇ to generate a "labeled” antibody.
  • the label may be detectable by itself (e.g., radioisotope label ⁇ or fluore ⁇ cent label ⁇ ) or, in the case of an enzymatic label, may catalyze chemical alternation of a sub ⁇ trate compound or compo ⁇ ition which is detectable.
  • Solid phase i ⁇ meant to be a non-aqueous matrix to which the antibody of the present invention can adhere.
  • solid phase ⁇ encompa ⁇ ed herein examples include tho ⁇ e formed partially or entirely of gla ⁇ (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamide ⁇ , polystyrene, polyvinyl alcohol and ⁇ ilicones.
  • the ⁇ olid pha ⁇ e can compri ⁇ e the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column).
  • This term al ⁇ o include ⁇ a di ⁇ continuou ⁇ ⁇ olid phase of discrete particle ⁇ , ⁇ uch a ⁇ those de ⁇ cribed in U.S. Patent No. 4,275,149.
  • a “liposome” is a small vesicle composed of various types of lipids, pho ⁇ pholipids and/or surfactant which is useful for delivery of a drug (such as an LP231 polypeptide or antibody thereto) to a mammal.
  • a drug such as an LP231 polypeptide or antibody thereto
  • the components of the lipo ⁇ ome are commonly arranged in a bilayer formation, ⁇ imilar to the lipid arrangement of biological membranes .
  • modulate mean ⁇ 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 proce ⁇ e ⁇ , ⁇ uch a ⁇ tran ⁇ formation, blocking of differentiation and meta ⁇ tasis.
  • polynucleotide which hybridizes only to polyA + sequences (such as any 3' terminal polyA + tract of a cDNA ⁇ hown in the sequence listing) , or to a complementary stretch of T (or U) residues, would not be included in the definition of "polynucleotide,” ⁇ ince such a polynucleotide would hybridize to any nucleic acid molecule containing a polyA 4 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.
  • 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 ⁇ ingle- and double- ⁇ tranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double- ⁇ tranded region ⁇ .
  • LP231 polynucleotides can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • LP231 polynucleotides may al ⁇ o contain one or more modified ba ⁇ e ⁇ or DNA or RNA backbone ⁇ modified for stability or for other rea ⁇ on ⁇ .
  • Modified ba ⁇ es include, for example, tritylated ba ⁇ e ⁇ and unu ⁇ ual ba ⁇ es such as inosine.
  • a variety of modification ⁇ can be made to DNA and RNA; thu ⁇ , "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 isostere ⁇ , and may contain amino acid ⁇ other than the gene-encoded amino acid ⁇ .
  • the LP231 polypeptides may be modified by either natural processes, such a ⁇ po ⁇ ttran ⁇ lational processing, or by chemical modification techniques which are well known in the art. Such modifications are well de ⁇ cribed in ba ⁇ ic texts and in more detailed monograph ⁇ , a ⁇ well a ⁇ in a voluminous re ⁇ earch literature. Modifications can occur anywhere in the LP231 polypeptides, including the peptide backbone, the amino acid ⁇ ide-chain ⁇ and the amino or carboxyl termini.
  • ⁇ ame type of modification may be pre ⁇ ent in the ⁇ ame or varying degree ⁇ at ⁇ everal sites in a given LP231 polypeptide.
  • a given LP231 polypeptide may contain many types of modifications.
  • LP231 polypeptides may be branched, for example, a ⁇ a re ⁇ ult of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic LP231 polypeptides may result from posttranslation natural processe ⁇ or may be made by ⁇ ynthetic method ⁇ .
  • 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 phosphotidylino ⁇ itol, cro ⁇ -linking, cyclization, di ⁇ ulfide 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 proces ⁇ ing, phosphorylation, prenylation, racemization, selenoylation, ⁇ ulfation, tran ⁇ fer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquit
  • LP231 polynucleotide of the pre ⁇ ent invention provide ⁇ newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as LP231.
  • LP231 e.g., LP231-A, SEQ ID NO:l
  • U ⁇ ing BLAST ' and Fa ⁇ tA sequence alignment computer programs, Applicant ⁇ found that various portion ⁇ of the LP231 polypeptide have sequence identity with human cerebellin. Accordingly, it is pre ⁇ ently believed that LP231 polypeptide di ⁇ clo ⁇ ed in the pre ⁇ ent application are newly identified members of the cerebellin family and, thus, may be involved in synaptic function.
  • LP231 variants can be prepared. LP231 variants can be prepared by introducing appropriate nucleotide changes into the LP231- encoding DNA or by ⁇ ynthe ⁇ i ⁇ of the de ⁇ ired LP231 polypeptide. Those skilled in the art will appreciate that amino acid change ⁇ may alter po ⁇ t-translational processes of the LP231 polypeptide, such as changing the number or position of glycosylation ⁇ ites or altering the membrane anchoring characteristics .
  • Variations in the native full-length sequence LP231 or in various domain ⁇ of the LP231 polypeptide described herein can be made, for example, using any of the techniques and guidelines for conservative and non-con ⁇ ervative 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 result ⁇ in a change in the amino acid ⁇ equence of the LP231 polypeptide as compared with the native sequence LP231 polypeptide.
  • 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, sub ⁇ tituted 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 change ⁇ made in regions of high homology.
  • Amino acid substitutions can be the re ⁇ ult of replacing one amino acid with another amino acid having ⁇ imilar structural and/or chemical properties, such as the replacement of a leucine with a ⁇ erine, i.e., conservative amino acid replacements.
  • In ⁇ ertion ⁇ or deletions may optionally be in the range of 1 to 5 amino acids.
  • the variation allowed may be determined by ⁇ y ⁇ tematically making insertion ⁇ , deletion ⁇ or ⁇ ub ⁇ titution ⁇ of amino acid ⁇ in the ⁇ equence 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 re ⁇ idue ⁇ , for example, when compared with a full length or native protein. Certain fragment ⁇ lack amino acid re ⁇ idue ⁇ that are not e ⁇ ential for a desired biological activity of the LP231 polypeptide.
  • LP231 fragments may be prepared by any of a number of conventional technique ⁇ . 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 residue ⁇ , or by dige ⁇ ting the DNA with ⁇ uitable re ⁇ triction enzymes and isolating the de ⁇ ired fragment.
  • Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a de ⁇ ired polypeptide fragment by polymera ⁇ e chain reaction (PCR) .
  • PCR polymera ⁇ e chain reaction
  • Oligonucleotide ⁇ that define the de ⁇ ired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR.
  • LP231 polypeptide fragment ⁇ share at lea ⁇ t one biological and/or immunological activity with the native LP231 polypeptide shown in SEQ ID NO:l.
  • conservative sub ⁇ titution ⁇ of intere ⁇ t are ⁇ hown in Table 1 under the heading of preferred sub ⁇ titution ⁇ . If ⁇ uch ⁇ ubstitutions re ⁇ ult in a change in biological activity, then more ⁇ ub ⁇ tantial change ⁇ , denominated exemplary substitutions in Table 1, or as further described below in reference to amino acid classes, are introduced and the products screened.
  • Sub ⁇ tantial modifications in function or immunological identity of the LP231 polypeptide are accomplished by ⁇ electing substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the ⁇ ub ⁇ titution, for example, as a ⁇ heet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target ⁇ ite, or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups ba ⁇ ed on common ⁇ ide-chain propertie : (1) hydrophobic: sy ⁇ , ser, thr;
  • Non-conservative substitution ⁇ will entail exchanging a member of one of these cla ⁇ se ⁇ for another cla ⁇ s .
  • Such ⁇ ub ⁇ tituted re ⁇ idue ⁇ also may be introduced into the con ⁇ ervative ⁇ ubstitution sites, or more preferably, into the remaining (non-conserved) sites.
  • the variations can be made using method ⁇ known in the art such a ⁇ oligonucleotides-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesi ⁇ .
  • Site-directed mutagene ⁇ i ⁇ [Carter et al . , Nucl . Acids Res . 13(12): 4331-43 (1985); Zoller et al .
  • Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence.
  • preferred scanning amino acids are relatively small, neutral amino acids.
  • Such amino acids include alanine, glycine, ⁇ erine, and cysteine.
  • Alanine i ⁇ 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 i ⁇ 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 u ⁇ ed.
  • Covalent modification ⁇ of LP231 polypeptide ⁇ are included within the ⁇ cope of thi ⁇ invention.
  • One type of covalent modification include ⁇ reacting targeted amino acid re ⁇ idues of an LP231 polypeptide with an organic derivatizing agent that i ⁇ capable of reacting with selected side chains or the N- or C-terminal residue ⁇ of an LP231 polypeptide.
  • Commonly u ⁇ ed cro ⁇ -linking agent ⁇ include, e.g., 1, 1-bi ⁇ (diazo-acetyl) -2-phenylethane, glutaraldehyde, N-hydroxy- ⁇ uccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoester ⁇ , including disuccinimidyl esters such a ⁇ 3 , 3'-dithiobis- (succinimidylproprionate) , bifunctional maleimides ⁇ uch a ⁇ bis-N-maleimido-1, 8-octane and agents ⁇ uch as methyl-3- [ (p- azidophenyl) -dithiolproprioimidate.
  • 1, 1-bi ⁇ (diazo-acetyl) -2-phenylethane glutaraldehyde
  • N-hydroxy- ⁇ uccinimide esters for example, esters with 4-azidosalicylic acid
  • Another type of covalent modification of the LP231 polypeptide included within the ⁇ cope of this invention comprise ⁇ altering the native glycosylation pattern of the polypeptide.
  • the phra ⁇ e include ⁇ qualitative change ⁇ in the glyco ⁇ ylation of the native protein ⁇ , involving a change in the nature and proportion ⁇ of the variou ⁇ carbohydrate moieties present.
  • Addition of glyco ⁇ ylation ⁇ ite ⁇ to LP231 polypeptide ⁇ may be accompli ⁇ hed by altering the amino acid sequence thereof.
  • the alteration may be made, for example, by the addition of, or sub ⁇ titution by, one or more serine or threonine residues to the native sequence LP231 polypeptide (for 0-linked glycosylation ⁇ ite ⁇ ) .
  • the LP231 amino acid ⁇ equence may optionally be altered through change ⁇ at the DNA level, particularly by mutating the DNA encoding the LP231 polypeptide at pre ⁇ elected bases such that codons are generated that will translate into the desired amino acids.
  • Removal of carbohydrate moietie ⁇ pre ⁇ ent on the LP231 polypeptide may be accomplished chemically or enzymatically or by utational substitution of codons encoding for amino acid residue ⁇ that ⁇ erve as targets for glycosylation.
  • Chemical deglyco ⁇ ylation technique ⁇ 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-glyco ⁇ idases a ⁇ described by Thotakura et al . , Meth . Enzymol . 138: 350-9 (1987).
  • Another type of covalent modification of LP231 compri ⁇ es linking the LP231 polypeptide to one of a variety of nonproteinaceou ⁇ 20 polymer ⁇ , e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylene ⁇ , in the manner ⁇ et 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 polypeptide ⁇ of the pre ⁇ ent 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.
  • 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 i ⁇ generally placed at the amino- or carboxyl- terminu ⁇ of the LP231 polypeptide.
  • the pre ⁇ ence of such epitope-tagged forms of an LP231 polypeptide can be detected using an antibody against the tag polypeptide.
  • provi ⁇ ion of the epitope tag enable ⁇ the LP231 polypeptide to be readily purified by affinity purification u ⁇ ing an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.
  • Example ⁇ include poly-hi ⁇ tidine (poly-hi ⁇ ) 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 .
  • tag polypeptide ⁇ 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 .
  • the chimeric molecule may compri ⁇ e a fu ⁇ ion of an LP231 polypeptide with an immunoglobulin or a particular region of an immunoglobulin.
  • a fu ⁇ ion could be to the Fc region of an IgG molecule.
  • the Ig fu ⁇ ions preferably include the ⁇ ub ⁇ titution of a ⁇ oluble tran ⁇ membrane domain deleted or inactivated form of an LP231 polypeptide in place of at lea ⁇ t one variable region within an Ig molecule.
  • the immunoglobulin fu ⁇ ion include ⁇ the hinge, CH2 and CH3 or the hinge, CHI, CH2 and CH3 regions of an IgGl molecule.
  • immunoglobulin fusions see also U.S. Patent 5,428,130, i ⁇ ued June 27, 1995.
  • the LP231 polypeptides of the present invention may also be modified in a way to form a chimeric molecule compri ⁇ ing an LP231 polypeptide fu ⁇ ed to a leucine zipper.
  • Variou ⁇ leucine zipper polypeptides have been de ⁇ cribed in the art. See, e.g., Land ⁇ chulz 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) .
  • LP231 sequence or portions thereof, may be produced by direct peptide synthe ⁇ i ⁇ u ⁇ ing 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 .
  • tro protein synthesis may be performed u ⁇ ing manual techniques or by automation. Automated ⁇ ynthesi ⁇ may be accomplished, for instance, u ⁇ ing an Applied Bio ⁇ y ⁇ tem ⁇ Peptide Synthe ⁇ izer (Fo ⁇ ter City, CA) using manufacturer's instructions. Various portion ⁇ of LP231 polypeptide ⁇ may be chemically synthesized separately and combined using chemical or enzymatic methods to produce a full-length LP231 polypeptide.
  • DNA encoding an LP231 polypeptide may be obtained from a cDNA library prepared from tissue believed to possess the LP231 mRNA and to expres ⁇ it at a detectable level. Accordingly, human LP231-encoding DNA can be conveniently obtained from a cDNA library prepared from human ti ⁇ ue, such as described in the Examples.
  • the LP231-encoding gene may also be obtained from a genomic library or by known synthetic procedure ⁇ (e.g., automated ⁇ ynthetic procedure ⁇ , oligonucleotide ⁇ ynthe ⁇ i ⁇ ) .
  • Librarie ⁇ can be ⁇ creened with probe ⁇ (such as antibodies to an LP231 polypeptide or oligonucleotides of at least about 20-80 bases) designed to identify the gene of intere ⁇ t or the protein encoded by it. Screening the cDNA or genomic library with the ⁇ elected probe may be conducted using standard procedures, such as described in Sambrook et al . , Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Pres ⁇ , NY (1989) . An alternative means to isolate the gene encoding LP231 i ⁇ to use PCR methodology [Sambrook et al . , ⁇ upra; Dieffenbach et al . , PCR Primer: A Laboratory Manual , Cold Spring Harbor Laboratory Press, NY (1995)] .
  • probe ⁇ such as antibodies to an LP231 polypeptide or oligonucleotides of at least about 20-80 bases
  • the oligonucleotide sequences selected as probes should be of ⁇ ufficient length and ⁇ ufficiently unambiguou ⁇ that fal ⁇ e po ⁇ itive ⁇ are minimized.
  • the oligonucleotide i ⁇ preferably labeled ⁇ uch 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 32 P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high ⁇ tringency, are provided in Sambrook et al . , ⁇ upra.
  • Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public database ⁇ ⁇ uch as GenBank or other private sequence databa ⁇ e .
  • Sequence identity (at either the amino acid or nucleotide level) within defined region ⁇ 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 ⁇ uch a ⁇ ALIGN, DNA ⁇ tar, BLAST, BLAST-2 , INHERIT and ALIGN-2 which employ variou ⁇ algorith ⁇ to measure homology) .
  • Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid ⁇ equence di ⁇ clo ⁇ ed herein for the fir ⁇ t time and, if nece ⁇ ary, u ⁇ ing conventional primer extension procedures a ⁇ de ⁇ cribed in Sambrook et al . , ⁇ upra, to detect precursors and proces ⁇ ing intermediates of mRNA that may not have been reverse-tran ⁇ cribed into cDNA.
  • Ho ⁇ t cell ⁇ are tran ⁇ fected or tran ⁇ formed with expre ⁇ ion or cloning vectors de ⁇ cribed herein for LP231 polypeptide production and cultured in conventional nutrient media modified a ⁇ appropriate for inducing promoter ⁇ , 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 ⁇ killed artisan without undue experimentation.
  • principles, protocols, and practical technique ⁇ for maximizing the productivity of cell culture ⁇ can be found in Mammalian Cell Biotechnology: A Practi cal Approach, M. Butler, ed. (IRL Pre ⁇ , 1991) and Sambrook et al . , ⁇ upra.
  • transfection Methods of transfection are known to the ordinarily skilled artisan, for example, CaP0 and electroporation. Depending on the ho ⁇ t cell u ⁇ ed, tran ⁇ formation 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.
  • Suitable host cells for cloning or expressing the nucleic acid (e.g., DNA) in the vectors herein include prokaryote, yea ⁇ t, or higher eukaryote cell ⁇ .
  • 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 ⁇ train ⁇ 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).
  • ⁇ uitable prokaryotic ho ⁇ t cells include Enterobacteriaceae such as Escherichia, e.g.. E. coli, Enterobacter, Erwinia, Klebisella, Proteu ⁇ , Salmonella, e.g., Salmonella typhimuriu , Serratia, e.g., Serratia marce ⁇ can ⁇ , and Shigeila, a ⁇ well a ⁇ Bacilli ⁇ uch 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 Streptomyce ⁇ .
  • Enterobacteriaceae such as Escherichia, e.g.. E. coli, Enterobacter, Erwinia, Klebisella, Proteu ⁇ , Salmonella, e.g., Salmonella typhimuriu
  • Strain W3110 is one particularly preferred ho ⁇ t or parent ho ⁇ t becau ⁇ e it is a common ho ⁇ t ⁇ train for recombinant DNA product fermentations.
  • the host cell secrete ⁇ minimal amounts of proteolytic enzymes.
  • strain W3 110 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with example ⁇ of ⁇ uch ho ⁇ t ⁇ including E. coli W3110 strain 1A2 , which ha ⁇ 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 ha ⁇ 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 re ⁇ i ⁇ tant degP deletion mutation; and an E. coli ⁇ train having mutant peripla ⁇ mic protea ⁇ e di ⁇ clo ⁇ ed in U.S. Patent No. 4,946,783 i ⁇ ued 7 Augu ⁇ t 1990.
  • in vivo method ⁇ of cloning e.g., PCR or other nucleic acid polymera ⁇ e reaction ⁇ , are ⁇ uitable.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expres ⁇ ion ho ⁇ ts for LP231 vectors.
  • Saccharomyces cerevisiae is a commonly used lower eukaryotic ho ⁇ t microorgani ⁇ m.
  • Other ⁇ include Schizo ⁇ accharomyces 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 .
  • 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. dro ⁇ ophilarum (ATCC 36.906) [Van den Berg et al . , Bio/Technology 8(2): 135-9 (1990)]; K. thermotoieran ⁇ , and K.
  • Methylotropic yeast ⁇ are ⁇ elected from the genera con ⁇ i ⁇ ting of Han ⁇ enula, Candida, Kloeckera, Pichia, Saccharomyce ⁇ , Torulop ⁇ i ⁇ , and Rhodotoruia .
  • a list of specific ⁇ pecie ⁇ that are exemplary of this class of yeast may be found in C. Antony, The Biochemistry of Methylotrophs 269 (1982) .
  • Suitable ho ⁇ t cells for the expression of glyco ⁇ ylated LP231 are derived from multicellular organi ⁇ ms.
  • invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sp, Spodoptera high5 as well a ⁇ plant cells.
  • useful mammalian host cell lines include Chine ⁇ e ham ⁇ ter ovary (CHO) and COS cells. More specific example ⁇ include monkey kidney CVl line tran ⁇ formed by SV40 (COS-7, ATCC CRL 1651) ; human embryonic kidney line [293 or 293 cells ⁇ ubcloned for growth in ⁇ u ⁇ pen ⁇ ion culture, Graham et al . , J. Gen Virol .
  • 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.
  • a replicable vector for cloning (amplification of the DNA) or for expression.
  • Various vector ⁇ are publicly available.
  • the vector may, for example, be in the form of a plasmid, co ⁇ mid, viral particle, or phage.
  • the appropriate nucleic acid ⁇ equence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate re ⁇ triction endonuclease site( ⁇ ) u ⁇ ing techniques known in the art.
  • Vector components generally include, but are not limited to, one or more of a signal ⁇ equence, an origin of replication, one or more marker gene ⁇ , an enhancer element, a promoter, and a tran ⁇ cription termination ⁇ equence.
  • Con ⁇ truction of ⁇ uitable vector ⁇ containing one or more of the ⁇ e components employs standard ligation techniques which are known to the skilled artisan.
  • the LP231 polypeptide may be produced recombinantly not only directly, but also a ⁇ a fu ⁇ ion 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.
  • 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 ⁇ equence may be a prokaryotic signal ⁇ equence selected, for example, from , the group of the alkaline phosphatase, penicillinase, lpp, or heat- ⁇ table enterotoxin II leader ⁇ .
  • the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharo yce ⁇ and Kluyveromyces cc-factor leaders, the latter described in U.S. Patent No. 5,010,182), or acid phosphata ⁇ e leader, the C. albican ⁇ glucoamylase leader (EP 362,179 published 4 April 1990), or the signal described in WO 90/13646 brin ⁇ hed 15 November 1990.
  • alpha factor leader including Saccharo yce ⁇ and Kluyveromyces cc-factor leaders, the latter described in U.S. Patent No. 5,010,182
  • acid phosphata ⁇ e leader the C. albican ⁇ glucoamylase leader
  • WO 90/13646 published 15 November 1990.
  • mammalian ⁇ ignal ⁇ equences may be used to direct secretion of the protein, such as ⁇ ignal sequences from secreted polypeptides of the same or related specie ⁇ a ⁇ well a ⁇ viral secretory leaders.
  • Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more ⁇ elected ho ⁇ t cell ⁇ .
  • ⁇ equence ⁇ are well known for a variety of bacteria, yeast, and viruses.
  • the origin of replication from the plasmid pBR322 i ⁇ ⁇ uitable for mo ⁇ t Gram- negative bacteria, the 2u plasmid origin i ⁇ ⁇ uitable for yea ⁇ t, and variou ⁇ viral origins (SV40, polyoma, adenoviru ⁇ , VSV or BPV) are u ⁇ eful for cloning vector ⁇ in mammalian cell ⁇ .
  • Expre ⁇ ion and cloning vector ⁇ will typically contain a selection gene, also termed a selectable marker.
  • Typical selection gene ⁇ encode protein ⁇ that (a) confer re ⁇ istance 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 .
  • ⁇ uitable ⁇ electable markers for mammalian cells are those that enable the identification of cell ⁇ competent to take up the LP231-encoding nucleic acid, such a ⁇ DHFR or thymidine kina ⁇ e.
  • An appropriate ho ⁇ t cell when wild- type DHFR is employed i ⁇ the CHO cell line deficient in DHFR activity, prepared and propagated a ⁇ de ⁇ cribed Urlaub and Cha ⁇ in, Proc . Natl . Acad. Sci . USA, 77(7): 4216-20 (1980).
  • a ⁇ uitable ⁇ election gene for use in yeast is the trpl gene present in the yea ⁇ t pla ⁇ mid YRp7 [Stinchcomb et al., Na ture 282(5734): 39-43 (1979); Kingsman et al . , Gene 7(2): 141-52 .
  • the trpl gene provides a ⁇ election marker for a mutant ⁇ train of yea ⁇ t 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 ⁇ ynthe ⁇ i ⁇ . Promoters recognized by a variety of potential host cells are well known. Promoters suitable for u ⁇ e with prokaryotic ho ⁇ t ⁇ include the P-lactamase and lactose promoter ⁇ y ⁇ tem ⁇ [Chang et al . , Nature 275(5681): 617-24
  • Promoters for u ⁇ e in bacterial ⁇ ystems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the LP231 polypeptide.
  • Suitable promoting .sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kina ⁇ e [Hitze an et al . , J. Biol . Chem. 255(24): 12073-80
  • gl ' ycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogena ⁇ e, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kina ⁇ e, trio ⁇ epho ⁇ phate i ⁇ omera ⁇ e, pho ⁇ phogluco ⁇ e isomerase, and glucokinase.
  • yea ⁇ t promoters which are inducible promoters having the additional advantage of tran ⁇ cription controlled by growth condition ⁇ , are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid pho ⁇ phata ⁇ e, degradative enzymes a ⁇ ociated with nitrogen metaboli ⁇ m, metallothionein, glyceraldehyde-3-pho ⁇ phate dehydrogena ⁇ e, and enzymes respon ⁇ ible for malto ⁇ e and galacto ⁇ e utilization.
  • Suitable vectors and promoters for use in yea ⁇ t expression are further described in EP 73,657.
  • LP231 transcription from vectors in mammalian ho ⁇ t cells i ⁇ controlled for example, by promoters obtained from the genomes of viruses such a ⁇ polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenoviru ⁇ ( ⁇ uch a ⁇ Adenoviru ⁇ 2), bovine papilloma virus, avian ⁇ arcoma viru ⁇ , cytomegalovirus, a retrovirus, hepatitis- B viru ⁇ and Simian Viru ⁇ 40 (SV40), from heterologou ⁇ 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 sy ⁇ tem .
  • viruses such as a ⁇ polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenoviru ⁇ ( ⁇ uch a ⁇ Adeno
  • 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 ⁇ equence ⁇ are now known from mammalian gene ⁇ (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 adenoviru ⁇ enhancer ⁇ .
  • the enhancer may be ⁇ pliced into the vector at a po ⁇ ition 5' or 3' to the LP231 coding ⁇ equence but i ⁇ preferably located at a ⁇ ite 5' from the promoter .
  • Expre ⁇ ion vector ⁇ u ⁇ ed in eukaryotic ho ⁇ t cell ⁇ will also contain sequences neces ⁇ ary for the termination of tran ⁇ cription and for stabilizing the mRNA.
  • sequence ⁇ are commonly available from the 5' and occasionally 3' untranslated regions of eukaryotic or viral DNAs or cDNA ⁇ .
  • the ⁇ e region ⁇ contain nucleotide ⁇ egment ⁇ transcribed a ⁇ polyadenylated fragments in the untranslated portion of the mRNA encoding LP231 polypeptide .
  • Still other method ⁇ , vectors, and host cell ⁇ suitable for adaptation to the synthe ⁇ i ⁇ of LP231 polypeptide ⁇ in recombinant vertebrate cell culture are de ⁇ cribed 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.
  • Gene amplification and/or expre ⁇ ion may be mea ⁇ ured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the tran ⁇ cription of mRNA [Thoma ⁇ , Proc . Natl . Acad. Sci . USA 77(9): 5201-5 (1980)], dot blotting (DNA analy ⁇ is) , or in situ hybridization, using an appropriately labeled probe, based on the ⁇ equence ⁇ provided herein.
  • antibodies may be employed that can recognize ⁇ pecific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.
  • the antibodies in turn may be labeled and the a ⁇ ay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the pre ⁇ ence of antibody bound to the duplex can be detected.
  • Gene expression may be measured by immunological methods, such as immunohistochemical ⁇ taining of cells or tis ⁇ ue sections and as ⁇ ay of cell culture or body fluid ⁇ , to quantitate directly the expre ⁇ ion of gene product.
  • Antibodie ⁇ u ⁇ eful for immunohi ⁇ tochemical staining and/or as ⁇ ay of ⁇ ample fluid ⁇ may be either monoclonal or polyclonal and may be prepared in any mammal.
  • the antibodie ⁇ may be prepared again ⁇ t a native provided herein or again ⁇ t exogenou ⁇ ⁇ equence fu ⁇ ed to LP231-encoding DNA and encoding a ⁇ pecific antibody epitope.
  • Forms of LP231 may be recovered from culture medium or from host cell ly ⁇ ates . If membrane-bound, it can be released from the membrane using a suitable detergent ⁇ olution (e.g., Triton-X 100) or by enzymatic cleavage.
  • a suitable detergent ⁇ olution e.g., Triton-X 100
  • Cell ⁇ employed in expre ⁇ ion of LP231 polypeptides can be disrupted by variou ⁇ phy ⁇ ical or chemical mean ⁇ , ⁇ uch a ⁇ freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agent ⁇ .
  • LP231 may be de ⁇ ired to purify LP231 from recombinant cell proteins or polypeptide ⁇ .
  • 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 ⁇ uch a ⁇ DEAE; chromatofocu ⁇ ing; SDS-PAGE; ammonium ⁇ ulfate precipitation; gel filtration u ⁇ ing, for example, Sephadex G-75; protein A Sepharo ⁇ e column ⁇ to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of the LP231 polypeptide.
  • Variou ⁇ method ⁇ of protein purification may be employed and such methods are known in the art and described, • for example, in Guider, Methods in Enzymology 182: 83-9 (1990) and Scope ⁇ , Protein Purification : Principles and Practice, Springer-Verlag, NY (1982).
  • the purification ⁇ tep(s) ⁇ elected will depend, for example, on the nature of the production process used and the particular LP231 polypeptide produced.
  • Nucleotide sequences (or their complement) encoding LP231 polypeptides have various applications in the art of molecular biology, including use ⁇ 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 ⁇ ame from other ⁇ pecie ⁇ ) which have a de ⁇ ired ⁇ equence identity to the LP231 nucleotide ⁇ equence di ⁇ clo ⁇ ed in SEQ ID N0:1.
  • the length of the probe ⁇ will be about 20 to about 50 ba ⁇ e ⁇ .
  • the hybridization probe ⁇ 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.
  • a screening method will compri ⁇ e isolating the coding region of the LP231 gene using the known DNA sequence to synthe ⁇ ize a ⁇ elected probe of about 40 ba ⁇ e ⁇ .
  • Hybridization probes may be labeled by a variety of labels, including radionucleotides such as 32 P or enzymatic labels ⁇ uch a ⁇ alkaline phosphatase coupled to the probe via avidin/biotin coupling ⁇ y ⁇ tems .
  • Labeled probe ⁇ having a ⁇ equence complementary to that of the LP231 gene of the pre ⁇ ent invention can be u ⁇ ed to ⁇ creen librarie ⁇ of human cDNA, genomic DNA or mRNA to determine members of ⁇ uch librarie ⁇ the probe hybridizes.
  • Hybridization technique ⁇ are de ⁇ cribed in further detail in the Example ⁇ below.
  • any EST ⁇ equence (or fragment thereof) di ⁇ clo ⁇ ed in the present application may similarly be employed as probes, using the methods disclosed herein.
  • Other useful fragment ⁇ of the LP231 nucleic acid ⁇ include antisen ⁇ e or ⁇ ense oligonucleotide ⁇ compri ⁇ ing a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target LP231 mRNA ( ⁇ en ⁇ e) of LP231 DNA (anti ⁇ en ⁇ e) 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 nucleotide ⁇ .
  • binding of antisense or sen ⁇ e oligonucleotide ⁇ to target nucleic acid sequences result ⁇ in the formation of duplexes that block transcription or tran ⁇ lation of the target ⁇ equence by one of ⁇ everal means, including enhanced degradation of the duplexes, premature termination of transcription or tran ⁇ lation, or by other mean ⁇ .
  • the antisense oligonucleotides thus may be used to block expres ⁇ ion of LP231 protein ⁇ .
  • Antisense or sen ⁇ e oligonucleotides further compri ⁇ e oligonucleotide ⁇ having modified sugar-phosphodie ⁇ ter backbone ⁇ (or other sugar linkage ⁇ , ⁇ uch as those described in WO 91/06629) and wherein such ⁇ ugar linkage ⁇ are re ⁇ i ⁇ tant to endogenous nuclease ⁇ .
  • Such oligonucleotide ⁇ with re ⁇ i ⁇ tant ⁇ ugar linkages are ⁇ table in vivo (i.e., capable of re ⁇ i ⁇ ting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences .
  • sen ⁇ e or anti ⁇ ense oligonucleotides include those oligonucleotide ⁇ which are covalently linked to organic moietie ⁇ , such as those described in WO 90/10448, and other moieties that increase affinity of the oligonucleotide for a target nucleic acid sequence, ⁇ uch poly-L-ly ⁇ ine .
  • intercalating agents such as ellipticme, and alkylating agents or metal complexes may be attached to sen ⁇ e or anti ⁇ en ⁇ e oligonucleotide ⁇ to modify binding ⁇ pecificitie ⁇ of the anti ⁇ en ⁇ e or ⁇ en ⁇ e oligonucleotide for the target nucleotide ⁇ equence.
  • Anti ⁇ en ⁇ e or ⁇ en ⁇ e oligonucleotide ⁇ may be introduced into a cell containing the target nucleic acid ⁇ equence by any gene tran ⁇ fer method, including, for example, CaP0-mediated DNA tran ⁇ fection, electroporation, or by using gene transfer vectors such as Epstein-Barr viru ⁇ .
  • Suitable retroviral vector ⁇ include, but are not limited to, tho ⁇ e derived from the murine retroviru ⁇ M-MSV, N2 (a retroviru ⁇ 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.
  • 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 anti ⁇ en ⁇ e oligonucleotide or it ⁇ conjugated ver ⁇ ion into the cell.
  • a ⁇ en ⁇ e or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, a ⁇ de ⁇ cribed in WO 90/10448.
  • the ⁇ en ⁇ e or antisen ⁇ e oligonucleotide-lipid complex i ⁇ preferably di ⁇ ociated within the cell by an endogenou ⁇ lipa ⁇ e.
  • the probe ⁇ may al ⁇ o be employed in PCR technique ⁇ to generate a pool of sequences for identification of closely related LP231 sequences.
  • Nucleotide sequences encoding an LP231 polypeptide can also be u ⁇ ed to construct hybridization probes for mapping the gene which encodes that LP231 polypeptide and for the genetic analy ⁇ i ⁇ of individual ⁇ with genetic di ⁇ order ⁇ .
  • the nucleotide ⁇ equence ⁇ 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.
  • the LP231 polypeptide can be u ⁇ ed in a ⁇ ay ⁇ to identify the other proteins or molecules involved in the binding interaction.
  • 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 agonist ⁇ of the binding interaction.
  • the receptor LP231 polypeptide can be u ⁇ ed to i ⁇ olate correlative ligand ( ⁇ ).
  • 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 a ⁇ ay ⁇ will include a ⁇ ays amenable to high-throughput screening of chemical librarie ⁇ , making them particularly ⁇ uitable for identifying ⁇ mall molecule drug candidate ⁇ .
  • Small molecule ⁇ contemplated include synthetic organic or inorganic compounds.
  • the as ⁇ ays can be performed in a variety of formats, including protein-protein binding as ⁇ ay ⁇ , biochemical ⁇ creening a ⁇ ay ⁇ , immunoas ⁇ ays and cell based as ⁇ ays, 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 animal ⁇ or "knock out" animal ⁇ which, in turn, are u ⁇ eful in the development and ⁇ creening of therapeutically u ⁇ eful reagent ⁇ .
  • a tran ⁇ genic animal e.g., a mou ⁇ e or rat
  • a tran ⁇ gene is a DNA which is integrated into the genome of a cell from which a tran ⁇ genic animal develop ⁇ .
  • cDNA encoding LP231 polypeptide can be u ⁇ ed to clone genomic DNA encoding LP231 in accordance with e ⁇ tabli ⁇ hed techniques and the genomic sequence ⁇ used to generate tran ⁇ genic animals that contain cells which express DNA encoding LP231.
  • Methods for generating transgenic animal ⁇ , particularly animal ⁇ ⁇ uch 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.
  • particular cells would be targeted for LP231 transgene incorporation with tis ⁇ ue- ⁇ pecific enhancers .
  • Transgenic animal ⁇ that include a copy of a tran ⁇ gene encoding LP231 introduced into the germ line of the animal at an embryonic ⁇ tage can be u ⁇ ed to examine the effect of increased expression of DNA encoding LP231 polypeptide.
  • Such animals can be u ⁇ ed a ⁇ te ⁇ ter animal ⁇ for reagent ⁇ thought to confer protection from, for example, pathological condition ⁇ a ⁇ ociated with it ⁇ overexpre ⁇ ion .
  • an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animal ⁇ bearing the tran ⁇ gene, would indicate a potential therapeutic intervention for the pathological condition.
  • non-human homologue ⁇ of LP231 can be u ⁇ ed 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.
  • cDNA encoding LP231 polypeptide can be u ⁇ ed to clone genomic DNA encoding LP231 polypeptide in accordance with e ⁇ tabli ⁇ hed technique ⁇ .
  • a portion of the genomic DNA encoding LP231 polypeptide can be deleted or replaced with another gene, ⁇ uch a ⁇ a gene encoding a selectable marker which can be used to monitor integration.
  • a selectable marker which can be used to monitor integration.
  • several kiloba ⁇ e ⁇ of unaltered flanking DNA are included in the vector [see, e.g., Thoma ⁇ and Capecchi, Cell 51(3): 503-12 (1987) for a de ⁇ cription of homologou ⁇ 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 endogenou ⁇ DNA are ⁇ elected [see, e.g., Li et al., Cell 69(6): 915-26 (1992)].
  • the ⁇ elected 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 ⁇ uitable p ⁇ eudopregnant female foster animal and the embryo brought to term to create a "knock out" animal.
  • Progeny harboring the homologou ⁇ ly 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 animal ⁇ can be characterized, for in ⁇ tance, for their ability to defend again ⁇ t certain pathological condition ⁇ and for their development of pathological condition ⁇ due to ab ⁇ ence of the LP231 polypeptide .
  • Gene therapy include ⁇ both conventional gene therapy, where a lasting effect is achieved by a ⁇ ingle treatment, and the admini ⁇ tration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA.
  • Antisen ⁇ e RNAs and DNAs can be used a ⁇ therapeutic agent ⁇ for blocking the expre ⁇ ion of certain gene ⁇ in vivo . It has already been shown that ⁇ hort anti ⁇ en ⁇ e oligonucleotides can be imported into cell ⁇ where they act a ⁇ inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane [Zamecnik et al . , Proc . Natl . Acad Sci .
  • the oligonucleotides can be modified to enhance their uptake, e.g., by substituting their negatively charged phosphodiester groups with uncharged groups.
  • Trie technique ⁇ vary depending upon whether the nucleic acid i ⁇ transferred into cultured cell in vi tro or in vivo in the cells of the intended host.
  • Techniques ⁇ uitable for the transfer of nucleic acid into mammalian cell ⁇ in vi tro include the u ⁇ e of liposomes, electroporation, microinjection, cell fusion, DEAE- dextran, the calcium phosphate precipitation method, etc.
  • the currently preferred in vivo gene transfer technique ⁇ include tran ⁇ fection with viral (typically, retroviral) vector ⁇ and viral coat protein-lipo ⁇ ome mediated tran ⁇ fection [Dzau et al., Trends in Biotechnology 11(5): 205-10 (1993)].
  • viral typically, retroviral
  • viral coat protein-lipo ⁇ ome mediated tran ⁇ fection [Dzau et al., Trends in Biotechnology 11(5): 205-10 (1993)].
  • protein ⁇ which bind to a cell ⁇ urface membrane protein a ⁇ ociated with endocyto ⁇ i ⁇ may by u ⁇ ed for targeting and/or to facilitate uptake, e.g., cap ⁇ id protein ⁇ or fragments thereof for a particular cell type, antibodie ⁇ for protein ⁇ which undergo internalization in cycling, protein ⁇ that target intracellular localization and enhance intracellular half-life.
  • the LP231 polypeptides de ⁇ cribed herein may al ⁇ o be employed a ⁇ molecular weight markers for protein electrophore ⁇ i ⁇ purposes .
  • the nucleic acid molecule encoding the LP231 polypeptide ⁇ or fragment ⁇ thereof de ⁇ cribed herein are u ⁇ eful for chromo ⁇ ome identification.
  • there exi ⁇ t ⁇ an ongoing need to idenfity new chromo ⁇ ome markers, since relatively few chromosome marking reagents, based upon actual sequence data, are presently available.
  • Each LP231 nucleic acid molecule of the pre ⁇ ent invention can be u ⁇ ed as a chromosome marker .
  • the LP231 polypeptide ⁇ and nucleic acid molecules of the present invention may also be used for tis ⁇ ue typing, wherein the LP231 polypeptide ⁇ of the pre ⁇ ent invention may be differentially expre ⁇ ed in one ti ⁇ ue a ⁇ compared to another.
  • LP231 nucleic acid molecules will find use for generating probes for PCR, Northern analysi ⁇ , Southern analysis and Western analysi ⁇ .
  • LP231 polypeptide ⁇ of the pre ⁇ ent invention which possess biological activity related to that of cerebellin may be employed both in vivo for therapeutic purpo ⁇ e ⁇ and in vi tro . Tho ⁇ e of ordinary skill in the art will well know how to employ the LP231 polypeptide ⁇ of the pre ⁇ ent invention for ⁇ uch purposes .
  • the present invention further provides anti-LP231 polypeptide antibodies.
  • Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate 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 antibodie ⁇ can be rai ⁇ ed in a mammal, for example, by one or more injection ⁇ of an immunizing agent and, if de ⁇ ired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injection ⁇ .
  • 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.
  • ⁇ uch immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and ⁇ oybean tryp ⁇ in inhibitor.
  • adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, ⁇ ynthetic trehalo ⁇ e dicorynomycolate) .
  • the immunization protocol may be ⁇ elected by one ⁇ killed in the art without undue experimentation.
  • the anti-LP231 antibodie ⁇ may, alternatively, be monoclonal antibodie ⁇ .
  • Monoclonal antibodies may be prepared using ' hybridoma method ⁇ , ⁇ uch a ⁇ tho ⁇ e described by Kohler and Milstein, Na ture 256(5517): 495-7 (1975).
  • a hybridoma method a mouse, ham ⁇ ter, or other appropriate host animal i ⁇ typically immunized with an immunizing agent to elicit lymphocyte ⁇ that produce or are capable of producing antibodie ⁇ that will ⁇ pecifically bind to the immunizing agent.
  • the lymphocyte ⁇ may be immunized in vi tro .
  • the immunizing agent will typically include the LP231 polypeptide or a fu ⁇ ion protein thereof.
  • PBLs peripheral blood lymphocytes
  • spleen cells or lymph node cells are used, if non-human mammalian ⁇ ource ⁇ are desired.
  • the lymphocyte ⁇ are then fused with an immortalized cell line u ⁇ ing a suitable fusing agent, such a ⁇ 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 u ⁇ ually tran ⁇ formed mammalian cell ⁇ , particularly myeloma cell ⁇ of rodent, bovine and human origin. U ⁇ ually, rat or mou ⁇ e myeloma cell lines are employed.
  • the hybridoma cell ⁇ may be cultured in a ⁇ uitable culture medium that preferably contains one or more ⁇ ub ⁇ tance ⁇ that inhibit the growth or ⁇ urvival of the unfused, immortalized cell ⁇ .
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium”), which prevents the growth of HGPRT- deficient cell ⁇ .
  • HGPRT hypoxanthine guanine phosphoribo ⁇ yl transferase
  • Preferred immortalized cell line ⁇ are tho ⁇ e that fu ⁇ e efficiently, ⁇ upport ⁇ table high level expression of antibody by the selected antibody-producing cell ⁇ , and are sensitive to a medium such as HAT medium.
  • More preferred immortalized cell line ⁇ are murine myeloma line ⁇ , which can be obtained, for in ⁇ tance, 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 antibodie ⁇ directed again ⁇ t an LP231 polypeptide.
  • monoclonal antibodie ⁇ directed again ⁇ t an LP231 polypeptide.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cell ⁇ i ⁇ determined by immunoprecipitation or by an in vi tro binding a ⁇ ay, ⁇ uch a ⁇ radioimmunoassay (RIA) or enzyme-linked immunoab ⁇ orbent assay (ELISA) .
  • RIA ⁇ uch a ⁇ radioimmunoassay
  • ELISA enzyme-linked immunoab ⁇ orbent assay
  • 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) .
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, Monoclonal Antibodi es : Principles and Practice, Academic Pre ⁇ s, (1986) pp. 59-103].
  • Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle ' ⁇ Medium and RPMI-1640 medium.
  • the hybridoma cell ⁇ may be grown in vivo as ascites in a mammal.
  • the monoclonal antibodie ⁇ ⁇ ecreted by the ⁇ ubclone ⁇ may be i ⁇ olated 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 method ⁇ , ⁇ uch 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.
  • the DNA may be placed into expression vector ⁇ , which are then tran ⁇ fected into ho ⁇ t cell ⁇ ⁇ uch a ⁇ simian COS cell ⁇ , Chine ⁇ e ham ⁇ ter ovary (CHO) cell ⁇ , or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesi ⁇ of monoclonal antibodie ⁇ in the recombinant ho ⁇ t cell ⁇ .
  • the DNA also may be modified, for example, by sub ⁇ tituting the coding ⁇ equence for human heavy and light chain con ⁇ tant domain ⁇ in place of the homologous murine sequence ⁇ [U.S. Patent No. 4,816,567; Morrison et al . , Proc . Na tl .
  • non-immunoglobulin polypeptide can be ⁇ ub ⁇ tituted for the con ⁇ tant domain ⁇ of an antibody of the invention or can be ⁇ ub ⁇ tituted for the variable domain ⁇ of one antigen-combining ⁇ ite of an antibody of the invention to create a chimeric bivalent antibody.
  • the antibodies may be monovalent antibodie ⁇ .
  • 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.
  • the anti-LP231 antibodie ⁇ of the invention may further compri ⁇ e humanized antibodie ⁇ or human antibodie ⁇ .
  • Humanized form ⁇ of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof ( ⁇ uch a ⁇ Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodie ⁇ include human immunoglobulin ⁇ (recipient antibody) in which re ⁇ idue ⁇ from a complementary-determining region (CDR) of the recipient are replaced by re ⁇ idues from a CDR of a non-human specie ⁇ (donor antibody) ⁇ uch as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary-determining region
  • donor antibody donor antibody
  • Fv framework residue ⁇ of the human immunoglobulin are replaced by corre ⁇ ponding non-human re ⁇ idues.
  • Humanized antibodie ⁇ may al ⁇ o compri ⁇ e re ⁇ idue ⁇ which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise sub ⁇ tantially all of at least one, and typically two, variable domains, in which all or sub ⁇ tantially 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)].
  • Fc immunoglobulin constant region
  • 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 re ⁇ idues are often referred to as "import" residue ⁇ , which are typically taken from an "import” variable domain. Humanization can be e ⁇ entially performed following the method of Winter and co- workers [Jones et al . , Na ture 321(6069): 522-5 (1986); Riechmann et al .
  • Such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) wherein substantially less than an intact human variable domain has been sub ⁇ tituted by the corre ⁇ ponding ⁇ equence from a non- human ⁇ pecie ⁇ .
  • humanized antibodie ⁇ are typically human antibodie ⁇ in which ⁇ ome CDR residue ⁇ and po ⁇ ibly ⁇ ome FR re ⁇ idue ⁇ are ⁇ ub ⁇ tituted by re ⁇ idues from analogous site ⁇ in rodent antibodie ⁇ .
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries
  • human antibodie ⁇ 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 respect ⁇ , including gene rearrangement, assembly and antibody repertoire. Thi ⁇ approach is de ⁇ cribed, for example, in U.S. Patent No ⁇ .
  • ADPT Antibody Dependent Enzyme Mediated Prodrug Therapy
  • the antibodie ⁇ 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.
  • a prodrug e.g., a peptidyl chemotherapeutic agent, see WO 81/01145
  • a prodrug e.g., a peptidyl chemotherapeutic agent, see WO 81/01145
  • 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, glycosida ⁇ e, gluco ⁇ e oxida ⁇ e, human ly ⁇ ozyme, human glucuronida ⁇ e, alkaline phosphatase useful for converting pho ⁇ phate-containing prodrugs into free drugs; arylsulfata ⁇ e u ⁇ eful for converting ⁇ ulfate-containing prodrug ⁇ into free drugs; cytosine deamina ⁇ e u ⁇ eful for converting non-toxic 5-fluorocyto ⁇ ine into the anti-cancer drug 5-fluorouracil; protea ⁇ e ⁇ , ⁇ uch as serratia protease, thermolysin, ⁇ ubtili ⁇ in, carboxypeptida ⁇ e ⁇ (e.g., carboxypeptida ⁇ e G2 and carboxypeptida ⁇ e A) and cathep ⁇ in ⁇ ( ⁇ uch a ⁇ cathep ⁇ ins B and L) .
  • D- alanylcarboxypeptidases useful for converting prodrugs that contain D-amino acid substituents
  • carbohydrate-cleaving enzymes such as alpha-galactosidase and neura inida ⁇ e useful for converting glycosylated prodrugs into free drugs
  • p-lactamase useful for converting drug ⁇ derivatized with p-lactams into free drugs
  • penicillin amidase ⁇ , ⁇ uch a ⁇ penicillin Vamida ⁇ e or penicillin G amida ⁇ e useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drug ⁇ .
  • antibodie ⁇ with enzymatic activity al ⁇ o known in the art a ⁇ "abzyme ⁇ ” can be u ⁇ ed to convert the prodrugs of the invention into free active drugs [see, e.g., Mas ⁇ ey, Nature 328(6129): 457-8 (1987)].
  • Antibody-abzyme conjugate ⁇ can be prepared a ⁇ de ⁇ cribed 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 technique ⁇ well known in the art ⁇ uch as the use of the heterobifunctional cro ⁇ s-linking agents discu ⁇ ed above.
  • fu ⁇ ion proteins comprising at least the antigen binding region of the antibody of the invention linked to at lea ⁇ t a functionally active portion of an enzyme of the invention can be con ⁇ tructed using recombinant DNA techniques well known in the art [ ⁇ ee, e.g., Neuberger et al . , Nature 312(5995): 604-8 (1984)].
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
  • 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 ⁇ ubunit.
  • Antibody variable domain ⁇ with the de ⁇ ired binding specificities can be fu ⁇ ed to immunoglobulin constant domain sequence ⁇ .
  • the fu ⁇ ion preferably i ⁇ with an immunoglobulin heavy-chain con ⁇ tant domain, comprising at least part of the hinge, CH2 , and CH3 regions. It i ⁇ preferred to have the fir ⁇ t heavy-chain con ⁇ tant region (CHI) containing the site necessary for light- chain binding present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy-chain fu ⁇ ion ⁇ and, if de ⁇ ired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a ⁇ uitable ho ⁇ t organi ⁇ m.
  • 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.
  • one or more ⁇ mall amino acid ⁇ ide chain ⁇ from the interface of the fir ⁇ t antibody molecule are replaced with larger ⁇ ide chains (e.g., tyrosine or tryptophan) .
  • Compen ⁇ atory "cavitie ⁇ " of identical or ⁇ imilar ⁇ ize to the large ⁇ ide chain ( ⁇ ) are created on the interface of the ⁇ econd antibody molecule by replacing large amino acid ⁇ ide chain ⁇ 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 antibodie ⁇ or antibody fragment ⁇ (e.g., F(ab') bi ⁇ pecific antibodies).
  • Technique ⁇ for generating bispecific antibodie ⁇ from antibody fragment ⁇ have been de ⁇ cribed in the literature.
  • bi ⁇ pecific antibodie ⁇ can be prepared can be prepared u ⁇ ing chemical linkage.
  • Brennan et al . [ Science 229(4708): 81-3 (1985)] de ⁇ cribe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') fragments.
  • the ⁇ e fragments are reduced in the presence of the dithiol complexing agent ⁇ odium ar ⁇ enite to stabilize vicinal dithiols and prevent intermolecular di ⁇ ulfide formation.
  • the Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • TAB thionitrobenzoate
  • 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 bi ⁇ pecific antibody.
  • the bi ⁇ pecific antibodie ⁇ produced can be used as agents for the selective immobilization of enzymes.
  • Fab' fragments may be directly recovered from E.
  • bi ⁇ pecific antibodie ⁇ have been produced u ⁇ ing leucine zipper ⁇ [Ko ⁇ telny et al., J. Immunol . 148(5): 1547-53 (1992)], wherein the leucine zipper peptide ⁇ from the Fo ⁇ and Jun protein ⁇ were linked to the Fab' portion ⁇ 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 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.
  • Antibodie ⁇ with more than two valencie ⁇ are contemplated.
  • tri ⁇ pecific antibodie ⁇ can be prepared [Tutt et al., J Immunol . 147(1): 60-9 (1991)].
  • Exemplary bi ⁇ pecific antibodie ⁇ may bind to two different epitope ⁇ on a given "LP" protein herein.
  • 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) , ⁇ uch a ⁇ FcyRI (CD64), FcyRII (CD32) and FcyRIIl (CD16) so as to focu ⁇ cellular defen ⁇ e mechani ⁇ m ⁇ to the cell expressing the particular "LP" protein.
  • a triggering molecule e.g., CD2 , CD3 , CD28, or B7
  • Fc receptors for IgG FcyR
  • CD64 FcyRII
  • CD16 FcyRIIl
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular "LP" polypeptide. These antibodies pos ⁇ e ⁇ an "LP" -binding arm and an arm which bind ⁇ a cytotoxic agent or a radionuclide chelator, ⁇ uch a ⁇ EOTUBE, DPTA. DOTA, or TETA. Another bispecific antibody of interest binds the "LP231" polypeptide and further bind ⁇ ti ⁇ sue factor (TF) .
  • TF ti ⁇ sue factor
  • Heteroconjugate antibodies are also within the ⁇ cope of the pre ⁇ ent invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cell ⁇ to unwanted cell ⁇ [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/20373].
  • the antibodies may be prepared in vi tro using known methods in synthetic protein chemistry, including those involving cros ⁇ linking agent ⁇ .
  • immunotoxin ⁇ may be con ⁇ tructed using a disulfide exchange reaction or by forming a thioether bond.
  • Examples of ⁇ uitable reagent ⁇ for thi ⁇ purpose include iminothiolate and methyl- - mercaptobutyrimidate and those disclo ⁇ ed, for example, in U.S. Patent No. 4,676,980.
  • cy ⁇ teine re ⁇ idue( ⁇ ) may be introduced in the Fc region, thereby allowing interchain di ⁇ ulfide bond formation in thi ⁇ region.
  • the homodimeric antibody thu ⁇ generated may have improved internal!zation capability and/or increa ⁇ ed 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 u ⁇ ing heterobifunctional cross-linker ⁇ a ⁇ de ⁇ cribed in Wolff et al . , Cancer Res . 53(11): 2560-5 (1993).
  • an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement ly ⁇ i ⁇ and ADCC capabilitie . See Steven ⁇ on et al . , AntiCancer Drug Design 3 ( 4 ) : 219-30 (1989).
  • the invention al ⁇ o pertain ⁇ to immunoconjugate ⁇ compri ⁇ ing 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) .
  • 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)
  • a radioactive isotope i.e., a radioconjugate
  • Enzymatically active protein toxin ⁇ and fragment ⁇ thereof which can be u ⁇ ed include diphtheria A chain, nonbinding active fragments of diphtheria toxin, cholera toxin, botulinus toxin, exotoxin A chain (from P ⁇ eudomona ⁇ aertfginosa) , ricin A chain, abrin A chain, modeccin A chain, alpha- ⁇ arcin, Aleurites jbrdii proteins, dianthin proteins, Phytolaca americana proteins . (PAPI.
  • PAPII, and PAP-S momordica charantia inhibitor, curcin, cretin, sapaonaria ofticinali ⁇ inhibitor, gelonin, ⁇ aporin, mitogellin, re ⁇ trictocin, phenomycin, enomycin and the tricothecene ⁇ .
  • Small molecule toxins include, for example, calicheamicins, maytan ⁇ inoid ⁇ , palytoxin and CC 1065.
  • a variety of radionuclides are available for the production of radioconjugated antibodie ⁇ .
  • Conjugate ⁇ of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such a ⁇ N- ⁇ uccinimidyl-3- (2-pyridyldithiol) propionate (SPDP) , iminothiolane (IT) , bifunctional derivatives of imidoesters ( ⁇ uch a ⁇ dimethyl adipimidate HCL) , active esters (such as disuccinimidyl ⁇ uberate) , aldehyde ⁇ ( ⁇ uch as glutaraldehyde) , bis-azido compound ⁇ , hexanediamine) , and bi ⁇ -diazonium derivative ⁇ .
  • bifunctional protein coupling agents such as a ⁇ N- ⁇ uccinimidyl-3- (2-pyridyldithiol) propionate (SPDP) , iminothiolane (IT) , bifunctional derivatives of imidoesters ( ⁇ uch a ⁇ dimethyl adipimidate HCL
  • a ricin immunotoxin can be prepared a ⁇ de ⁇ cribed in Vitetta et al . , Science 238(4830): 1098-104 (1987) .
  • Carbon-14-labeled l-i ⁇ othiocyanatobenzyl-3- ethyldiethylene triaminepentaacetic acid is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO 94/11026.
  • 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 i ⁇ conjugated to a cytotoxic agent (e.g., a radionucleotide) .
  • a ligand e.g., avidin
  • cytotoxic agent e.g., a radionucleotide
  • the antibodies di ⁇ clo ⁇ ed herein may al ⁇ o be formulated a ⁇ immuno1iposomes .
  • Liposomes containing the antibody are prepared by methods known in the art, ⁇ uch a ⁇ 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. No ⁇ . 4,485,045 and 4,544,545.
  • Lipo ⁇ ome ⁇ with enhanced circulation time are di ⁇ clo ⁇ ed in U.S. Patent No. 5,013,556.
  • Particularly u ⁇ eful lipo ⁇ omes can be generated by the reverse pha ⁇ e evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG- derivatized pho ⁇ phatidylethanolamine (PEG-PE) .
  • Lipo ⁇ ome ⁇ are extruded through filter ⁇ of defined pore ⁇ ize to yield lipo ⁇ ome ⁇ with the de ⁇ ired diameter.
  • Fab' fragment ⁇ of the antibody of the pre ⁇ ent invention can be conjugated to the lipo ⁇ omes 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).
  • an LP231 polypeptide i ⁇ intracellular and whole antibodie ⁇ are u ⁇ ed a ⁇ inhibitor ⁇ , internalizing antibodie ⁇ are preferred.
  • lipofection ⁇ or lipo ⁇ ome ⁇ can al ⁇ o be u ⁇ ed to deliver the antibody, or an antibody fragment into cells.
  • antibody fragment ⁇ are u ⁇ ed, the smallest inhibitory fragment that specifically bind ⁇ to the binding domain of the target protein i ⁇ preferred.
  • peptide molecules can be de ⁇ igned that retain the ability to bind the target protein ⁇ equence.
  • Such peptide ⁇ 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 al ⁇ o contain more than one active compound a ⁇ nece ⁇ ary for the particular indication being treated, preferably tho ⁇ e with complementary activities that do not adversely affect each other.
  • the composition may comprise an agent that enhances it ⁇ function, ⁇ uch a ⁇ , for example, a cytotoxic agent, cytokine ⁇ , chemotherapeutic agent, or growth-inhibitory agent.
  • a cytotoxic agent for example, cytokine ⁇ , chemotherapeutic agent, or growth-inhibitory agent.
  • Such molecule ⁇ are ⁇ uitable 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 sy ⁇ terns (for example, liposomes, albumin microspheres , microemulsion ⁇ , nano-particle ⁇ , and nanocap ⁇ ules) or in macroemul ⁇ ions .
  • colloidal drug delivery sy ⁇ terns for example, liposomes, albumin microspheres , microemulsion ⁇ , nano-particle ⁇ , and nanocap ⁇ ules
  • macroemul ⁇ ions for example, liposomes, albumin microspheres , microemulsion ⁇ , nano-particle ⁇ , and nanocap ⁇ ules
  • 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 example ⁇ of su ⁇ tained-relea ⁇ e preparation ⁇ include ⁇ emipermeable matrice ⁇ of ⁇ olid hydrophobic polymer ⁇ containing the antibody, which matrice ⁇ are in the form of ⁇ haped article ⁇ , e.g., films, or microcapsule ⁇ .
  • sustained-relea ⁇ e matrices include polyesters, hydrogels (for example, poly (2-hydroxyethyl-methacrylate) , or poly(vinylalcohol) ) , polylactide ⁇ (U.S. Pat. No.
  • encapsulated antibodies 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, re ⁇ ulting in a lo ⁇ s of biological activity and pos ⁇ ible changes in immunogenicity. Rational strategies can be devised for ⁇ tabilization depending on the mechani ⁇ m ⁇ involved. For example, if the aggregation mechani ⁇ m is di ⁇ covered to be intermolecular S-S bond formation through thio ⁇ uifide interchange, ⁇ tabilization may be achieved by modifying sulfhydryl residue ⁇ , lyophilizing from acidic ⁇ olution ⁇ , controlling moi ⁇ ture content, using appropriate additive ⁇ , and developing ⁇ pecific polymer matrix co po ⁇ ition ⁇ .
  • anti-LP231 antibodies of the present invention have variou ⁇ utilitie ⁇ .
  • anti-LP231 antibodie ⁇ may be u ⁇ ed in diagno ⁇ tic a ⁇ ay ⁇ for LP231 polypeptide ⁇ , e.g., detecting expression in specific cell ⁇ , tissue ⁇ , or serum.
  • diagnostic assay techniques known in the art may be used, such as competitive binding assay ⁇ , direct or indirect ⁇ andwich as ⁇ ays and immunoprecipitation a ⁇ ay ⁇ conducted in either heterogeneous or homogeneous pha ⁇ es [Zola, Monoclonal Antibodie ⁇ : A Manual of Technique ⁇ , CRC Pres ⁇ , Inc. (1987) pp. 147-158] .
  • the antibodie ⁇ u ⁇ ed in the a ⁇ ay ⁇ can be labeled with a detectable moiety.
  • the detectable moiety ⁇ hould be capable of producing, either directly or indirectly, a detectable ⁇ ignal.
  • the detectable moiety may be a radioi ⁇ otope, ⁇ uch as 3 H, 14 C, 32 P, 35 S, or 125 I, a fluorescent or che iluminescent compound, such as fluorescein i ⁇ othiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosida ⁇ e or horseradi ⁇ h peroxida ⁇ e.
  • any method known in the art for conjugating the antibody to the detectable moiety may be employed, including tho ⁇ e method ⁇ de ⁇ cribed 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 polypeptide ⁇ from recombinant cell culture or natural sources.
  • the antibodies against an LP231 polypeptide are immobilized on a suitable support, ⁇ uch a Sephadex re ⁇ in 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 i ⁇ wa ⁇ hed with a ⁇ uitable solvent that will remove substantially all the material in the sample except the LP231 polypeptide, which is bound to the immobilized antibody.
  • the ⁇ upport i ⁇ wa ⁇ hed with another ⁇ uitable solvent that will release the LP231 polypeptide from the antibody.
  • H. LP231 and Cerebellin Antagonist ⁇ /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 polypeptide ⁇ with other cellular protein ⁇ .
  • Such ⁇ creening a ⁇ ay ⁇ will include a ⁇ ay ⁇ amenable to high- throughput ⁇ creening of chemical libraries, making them particularly suitable for identifying small molecule drug candidate ⁇ .
  • the a ⁇ ay ⁇ can be performed in a variety of formats including protein-protein binding assays, biochemical screening a ⁇ says, immunoassays, and cell-ba ⁇ ed assays, which are well characterized in the art.
  • binding assays the interaction is binding, and the complex formed can be isolated or detected in the reaction mixture.
  • 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.
  • an immobilized antibody e.g., a monoclonal antibody, specific for the LP231 or cerebellin to be immobilized can be u ⁇ ed to anchor it to solid surface.
  • the as ⁇ ay i ⁇ performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated ⁇ urface containing the anchored component.
  • the reaction i ⁇ complete, the non-reacted components are removed, e.g.,- by washing, and complexes anchored on the solid surface are detected.
  • the detection of label immobilized on the ⁇ urface indicate ⁇ that complexing occurred.
  • complexing can be detected, for example, by u ⁇ ing a labeled antibody ⁇ pecifically binding the immobilized complex.
  • 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.
  • assays include traditional approaches, such as, e.g., cross-linking, co-immunoprecipitation, and co- purification through gradients or chromatographic columns.
  • protein-protein interactions can be monitored through gradients or chromatographic columns.
  • protein-protein interaction ⁇ can be monitored by u ⁇ ing a yea ⁇ t-ba ⁇ ed genetic ⁇ yste 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 di ⁇ crete modular domain ⁇ , one acting as the DNA-binding domain, while the other functions a ⁇ the transcription-activation domain.
  • the yea ⁇ t expre ⁇ sion sy ⁇ tem described in the foregoing publication ⁇ (generally referred to a ⁇ the "two-hybrid ⁇ y ⁇ te ”) take ⁇ advantage of thi ⁇ property, and employ ⁇ two hybrid protein ⁇ , one in which the target protein i ⁇ fused to the DNA-binding domain of GAL4 , and another in which candidate activating proteins are fu ⁇ ed to the activation domain.
  • the expre ⁇ ion of GALl-lacZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction.
  • Colonie ⁇ containing interacting polypeptide ⁇ are detected with chromogenic ⁇ ub ⁇ trate for ⁇ -galacto ⁇ ida ⁇ e.
  • a complete kit (MATCHMAKERTM) for identifying protein-protein interactions between two ⁇ pecific proteins using the two- hybrid technique is commercially available from Clontech. This system can also be extended to map protein domain ⁇ involved in specific protein interactions as well as to pinpoint amino acid residue ⁇ that are crucial for the ⁇ e interaction ⁇ .
  • 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 .
  • a placebo may be added to a third reaction mixture to serve as a po ⁇ itive control.
  • the formation of a complex in the control reaction ( ⁇ ) but not in the reaction mixture containing the te ⁇ t compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner .
  • Antagonist ⁇ may be detected by combining the LP231 or cerebellin and a potential antagoni ⁇ t with membrane-bound LP231 or cerebellin receptor ⁇ or recombinant receptors under appropriate conditions for a competitive inhibition assay.
  • the LP231 or cerebellin can be labeled, such a ⁇ by radioactivity, ⁇ uch 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).
  • RNA i ⁇ 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 respon ⁇ ive to the LP231 or cerebellin. Tran ⁇ fected cell ⁇ that are grown on gla ⁇ ⁇ lides 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- ⁇ pecific protein kina ⁇ e.
  • the ⁇ lide ⁇ are ⁇ ubj ected to autoradiographic analy ⁇ i ⁇ .
  • Po ⁇ itive pools are identified and sub-pool ⁇ are prepared and re-transfected using an interactive sub-pooling and re-screening proces ⁇ , eventually yielding a single clone that encodes the putative receptor.
  • a ⁇ an alternative approach for receptor identification, labeled LP231 or cerebellin can be photoaffinity-linked with cell membrane or extract preparation ⁇ that expre ⁇ s the receptor molecule. Cros ⁇ -linked material i ⁇ re ⁇ olved by PAGE and exposed to X-ray film.
  • the labeled complex containing the receptor can be exci ⁇ ed, re ⁇ olved into peptide fragment ⁇ , and ⁇ ubj ected to protein micro- ⁇ equencing.
  • the amino acid ⁇ equence obtained from micro- ⁇ equencing would be u ⁇ ed to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor .
  • mammalian cells or a membrane preparation expres ⁇ ing 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.
  • potential antagonists include an oligonucleotide that binds to the fusion ⁇ of immunoglobulin with LP231 or cerebellin, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodie ⁇ and antibody fragment ⁇ , ⁇ ingle-chain antibodie ⁇ , anti- idiotypic antibodie ⁇ , and chimeric or humanized ver ⁇ ion ⁇ of such antibodies or fragment ⁇ , a ⁇ well a ⁇ human antibodies and antibody fragments.
  • a potential antagonist may be a closely related protein, for example, a mutated form of the LP231 or cerebellin that recognize ⁇ the receptor but imparts no effect, thereby competitively inhibiting the action of the LP231 or cerebellin.
  • Another potential LP231 or cerebellin antagonist is an antisen ⁇ e RNA or DNA construct prepared u ⁇ ing anti ⁇ en ⁇ e technology, where, e.g., an anti ⁇ ense 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 expre ⁇ ion through triple-helix formation or anti ⁇ en ⁇ e DNA or RNA, both of which method ⁇ are ba ⁇ ed on binding of a polynucleotide to DNA or RNA.
  • the 5' coding portion of the polynucleotide ⁇ equence which encodes the mature LP231 or cerebellins herein, is used to design an antisen ⁇ e RNA oligonucleotide ⁇ equence of about 10 to 40 ba ⁇ e pair ⁇ in length.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the LP231 or cerebellin [antisen ⁇ e; ⁇ ee Okano, J. Neurochem. 56(2): 560-7 (1991); Oligodeoxynucleotides a ⁇ Antisense Inhibi tors of Gene Expression (CRC Pre ⁇ : Boca Raton, FL 1988)].
  • 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.
  • small molecules include, but are not limited to, small peptides or peptide- like molecules, preferably soluble peptides, and ⁇ ynthetic non-peptidyl organic or inorganic compounds.
  • Ribozymes are enzymatic RNA molecule ⁇ capable of catalyzing the ⁇ pecific cleavage of RNA. Ribozymes act by ⁇ equence- ⁇ pecific hybridization to the complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage ⁇ ites 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 molecule ⁇ in triple-helix formation u ⁇ ed to inhibit transcription ⁇ hould be ⁇ ingle- ⁇ tranded and composed of deoxynucleotides .
  • the base composition of these oligonucleotides is designed such that it promotes triple- helix formation via Hoogsteen ba ⁇ e-pairing rules, which generally require sizeable stretche ⁇ of purines or pyrimidine ⁇ on one ⁇ trand of a duplex.
  • ba ⁇ e-pairing rules which generally require sizeable stretche ⁇ of purines or pyrimidine ⁇ on one ⁇ trand of a duplex.
  • Another u ⁇ e of the compound ⁇ of the invention (e.g., LP231 variants and anti-LP231 antibodies) described herein is to help diagno ⁇ e whether a di ⁇ order i ⁇ driven to ⁇ ome extent by LP231 modulated signaling.
  • a diagnostic as ⁇ ay to determine whether a particular di ⁇ order i ⁇ driven by LP231 signaling can be carried out using the following ⁇ tep ⁇ : (1) culturing te ⁇ t cells or ti ⁇ ue ⁇ expre ⁇ ing LP231 ; (2) admini ⁇ tering a compound which can inhibit LP231 modulated ⁇ ignaling; and (3) mea ⁇ uring the LP231 mediated phenotypic effect ⁇ in the te ⁇ t cells.
  • the steps can be carried out using standard techniques in light of the present disclo ⁇ ure-. For example, ⁇ tandard technique ⁇ can be used to isolate cells or tis ⁇ ue ⁇ and to culture them in vivo .
  • Test compounds should be more potent in inhibiting intracellular signaling activity than in exerting a cytotoxic effect (e.g., an IC50 and LD 50 of greater than one) .
  • the IC 50 and LD50 can be measured by standard technique ⁇ , such as an MTT assay or by measuring the amount of LDH relea ⁇ ed.
  • the degree of IC 50 and LD 50 of a compound should be taken into account in evaluating the diagnostic as ⁇ ay. Generally, the larger the ratio, the more relative the information.
  • Appropriate control ⁇ take into account the po ⁇ ible cytotoxic effect of a compound, ⁇ uch 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 diagno ⁇ tic assay.
  • the diagnostic methods of the invention involve the screening for agents that modulate the effects of LP231 upon synaptic di ⁇ orders. Exemplary detection techniques include radioactive labeling and immunoprecipitating (U. S. Patent No. 5,385,915).
  • antibodies can be used to qualitatively or quantitatively detect the expression of proteins encoded by the disea ⁇ e-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 technique ⁇ known in the art.
  • si tu detection of antibody binding to the marker gene products can be performed, for example, by immunofluorescence or immunoelectron micro ⁇ copy.
  • a histological specimen i ⁇ 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.
  • the LP231 antagonist ⁇ or agonists thereof 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.
  • 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.
  • lipofections or lipo ⁇ omes 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.
  • peptide molecules can be designed which retain the ability to bind the target protein sequence. Such peptides can be synthe ⁇ ized 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 de ⁇ ired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizer ⁇ [Remington 's Pharmaceutical Sciences 16th edition (1980)], in the form of lyophilized formulation ⁇ or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosage ⁇ 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-cre ⁇ ol; low molecular weight (less than about 10 residues) polypeptides; proteins, ⁇ uch as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine
  • the formulation herein may also contain more than one active compound as neces ⁇ ary for the particular indication being treated, preferably tho ⁇ e with complementary activities that do not adver ⁇ ely affect each other.
  • the compo ⁇ ition may compri ⁇ e a cytotoxic agent, cytokine or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpo ⁇ e 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-microcap ⁇ ule ⁇ and poly- (methylmethacylate) icrocap ⁇ ule ⁇ , re ⁇ pectively, in colloidal drug delivery ⁇ y ⁇ tem ⁇ (for example, lipo ⁇ ome ⁇ , albumin micro ⁇ pheres, microemul ⁇ ion ⁇ , nano-particles and nanocapsule ⁇ ) or in macroemulsions .
  • Such technique ⁇ are di ⁇ clo ⁇ ed in Remington 's Pharmaceutical Sciences 16th edition (1980).
  • the formulation ⁇ to be u ⁇ ed for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • Therapeutic composition ⁇ herein generally are placed into a container having a ⁇ terile access port, for example, and intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • Su ⁇ tained-relea ⁇ e preparations may be prepared.
  • suitable examples of sustained-relea ⁇ e preparations include semipermeable matrice ⁇ of ⁇ olid hydrophobic polymer ⁇ containing the antibody, which matrices are in the form of shaped articles, e.g., film ⁇ , or microcap ⁇ ule ⁇ .
  • sustained-release matrices include polyesters, hydrogels [for example, poly (2-hydroxyethyl-methacrylate) , or poly(vinylalcohol) ] , polylactides (U.S. Pat. No.
  • the su ⁇ tained-relea ⁇ e formulation ⁇ of the ⁇ e proteins may be developed using polylactic-coglycolic acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties.
  • PLGA polylactic-coglycolic acid
  • the degradation products of PLGA, lactic and glycolic acids, can be cleared quickly within the human body.
  • the degradability of this polymer can be adjusted from months to years depending on its molecular weight and compo ⁇ ition. See Lewis, “Controlled release of bioactive agents from lactide/glycolide polymer” in Biodegradable Polymers a ⁇ Drug Delivery System ⁇ (Marcel Dekker; New York, 1990), M. Chasin and R. Langer (Eds.) pp. 1-41.
  • ⁇ tabilization may be achieved by modifying ⁇ ulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions .
  • the compounds of the present invention may be u ⁇ ed to treat various conditions including those characterized by overexpression and/or activation of the disea ⁇ e-a ⁇ ociated gene ⁇ identified herein.
  • exemplary conditions or disorder ⁇ to be treated with ⁇ uch antibodies and other compounds including, but not limited to, small organic and inorganic molecules, peptide ⁇ , anti ⁇ ense molecules, etc., include Parkinson ' ⁇ di ⁇ ea ⁇ e, Alzheimer ' ⁇ di ⁇ ease, bipolar and unipolar affective di ⁇ orders, schizophrenia, olivopontocerebellar atrophy, and Shy-Dager syndrome, especially those characterized by di ⁇ ruption of ⁇ ynapse function..
  • the active agents of the present invention are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration a ⁇ a bolus or by continuou ⁇ infusion over a period of time, by intramuscular, intraperitoneal, intracerebral, intracerobrospinal, ⁇ ubcutaneou ⁇ , intra- articular, intrasynovial, intrathecal, intraoccular, intralesional, oral, topical, inhalation or through sustained release.
  • known methods such as intravenous administration a ⁇ a bolus or by continuou ⁇ infusion over a period of time, by intramuscular, intraperitoneal, intracerebral, intracerobrospinal, ⁇ ubcutaneou ⁇ , intra- articular, intrasynovial, intrathecal, intraoccular, intralesional, oral, topical, inhalation or through sustained release.
  • an active agent e.g., an antibody
  • the agent is ⁇ uitably administered to the patient at one time or over a series of treatments.
  • Dosages and desired drug concentration of pharmaceutical compo ⁇ ition ⁇ of the pre ⁇ ent invention may vary depending on the particular u ⁇ e envi ⁇ ioned. The determination of the appropriate do ⁇ age or route of administration is well within the ⁇ kill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective does for human therapy. Interspecie ⁇ ⁇ caling of effective do ⁇ es can be performed following the principles laid down by Mordenti and Chappell, "The Use of Interspecie ⁇ Scaling in Toxicokinetic ⁇ , " in Toxicokinetics and New Drug Development, Yacobi et al . , Eds., Pergamon Press, NY 1989, pp.4246.
  • 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; ⁇ ee, for example, U.S. Pat. No ⁇ . 4,657,760, 5,206,344 or 5,225,212.
  • dosage ⁇ may be administered by one or more separate administrations or by continuou ⁇ infusion.
  • the treatment is su ⁇ tained until a de ⁇ ired ⁇ uppression of disea ⁇ e ⁇ ymptom ⁇ occur ⁇ .
  • do ⁇ age regimens may be useful. Conventional techniques and assay ⁇ easily monitor the progress of therapy.
  • an article of manufacture containing materials useful for the diagnosis or treatment of the disorders described above comprises ⁇ a container and a label.
  • Suitable containers include, for example, bottles, vials, syringe ⁇ , and test tube ⁇ .
  • the containers may be formed from a variety of materials such as glas ⁇ 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, ⁇ uch 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, needle ⁇ , ⁇ yringe ⁇ , and package inserts with instruction ⁇ for u ⁇ e.
  • Example 1 Expre ⁇ ion and Purification of LP231 in E. coli
  • the bacterial expre ⁇ ion vector pQE60 is used for bacterial expression in this example.
  • pQE60 encodes ampicillin antibiotic resi ⁇ tance ("Ampr") and contain ⁇ a bacterial origin of replication ("ori"), an IPTG inducible promoter, a ribo ⁇ ome binding ⁇ ite ("RBS”), ⁇ ix codon ⁇ encoding hi ⁇ tidine re ⁇ idues 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.
  • Aur ampicillin antibiotic resi ⁇ tance
  • ori a bacterial origin of replication
  • RBS ribo ⁇ ome binding ⁇ ite
  • ⁇ ix codon ⁇ encoding hi ⁇ tidine re ⁇ idues that allow affinity purification using nickel-nitrilo
  • a DNA fragment encoding a polypeptide can be in ⁇ erted in ⁇ uch 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.
  • 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 sequence ⁇ pre ⁇ ented, e.g., as pre ⁇ ented in SEQ ID NO:l), which anneal to the amino terminal encoding DNA ⁇ equences of the desired portion of the LP231 and to sequences in the construct 3 ' to the cDNA coding sequence. Additional nucleotide ⁇ containing re ⁇ triction sites to facilitate cloning in the pQE60 vector are added to the 5' and 3' sequences, respectively.
  • 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 ⁇ tep ⁇ .
  • SEQ ID N0:1 a portion of the coding sequence of LP231, e.g., as presented in SEQ ID N0:1, according to known method ⁇ tep ⁇ .
  • 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. In ⁇ ertion of the LP231 DNA into the re ⁇ tricted 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.
  • E. coli ⁇ train 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 expre ⁇ sing 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 i ⁇ isolated from resi ⁇ tant colonie ⁇ and the identity of the cloned DNA confirmed by restriction analy ⁇ is, PCR and DNA sequencing.
  • Clones containing the desired construct ⁇ are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 ⁇ g/ml) and kanamycin (25 ⁇ g/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.
  • I ⁇ opropyl-b-D-thiogalactopyrano ⁇ ide (“IPTG”) i ⁇ then added to a final concentration of 1 mM to induce tran ⁇ cription from the lac repre ⁇ sor sensitive promoter, by inactivating the lad repres ⁇ or.
  • Cell ⁇ 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.
  • a polypeptide can be succe ⁇ sfully refolded by dialyzing it against 500 mM NaCl, 20% glycerol, 25 mM ' Tris/HCl pH7.4, containing protease inhibitors.
  • 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.
  • 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 ⁇ ite ⁇ ⁇ uch as BamHI, Xba I, and Asp718.
  • the polyadenylation ⁇ ite of the simian virus 40 ("SV40") is used for efficient polyadenylation.
  • the pla ⁇ mid contain ⁇ 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 expres ⁇ es the cloned polynucleotide .
  • 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 vector ⁇ are de ⁇ cribed, for instance, in Luckow, et al . , Virology 170:31-39.
  • the cDNA ⁇ equence encoding the mature LP231 polypeptide in a clone, lacking the AUG initiation codon and the naturally a ⁇ sociated 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 corre ⁇ ponding or complementary to a portion of the coding ⁇ equence of a LP231 polypeptide, e.g., a ⁇ pre ⁇ ented in SEQ ID N0:1, according to known method ⁇ teps .
  • 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 u ⁇ ing calf inte ⁇ tinal pho ⁇ phata ⁇ e, 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) .
  • Thi ⁇ 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 ⁇ uitable 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 i ⁇ u ⁇ ed to amplify the gene and the second primer is from well within the vector so that only tho ⁇ e bacterial colonies containing the LP231 gene fragment will show amplification of the DNA.
  • the sequence of the cloned fragment is confirmed by DNA sequencing.
  • Thi ⁇ pla ⁇ mid is designated herein pBac LP231 .
  • plaque assay 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.
  • Blue Gal Life Technologies, Inc., Rockville, MD
  • a detailed description of a "plaque as ⁇ ay" of thi ⁇ type can al ⁇ o be found in the user's guide for insect cell culture and baculovirology di ⁇ tributed by Life Technologies, Inc., Rockville, MD, page 9-10) .
  • blue stained plaques are picked with a micropipettor tip (e.g., Eppendorf).
  • the recombinant virus is called V-LP231.
  • Sf9 cells are grown in Grace ' s medium ⁇ upplemented with 10% heat- inactivated FBS.
  • the cell ⁇ are infected with the recombinant baculovirus V-LP231 at a multiplicity of infection ("MOI") of about 2.
  • MOI multiplicity of infection
  • the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available, e.g., from Life Technologies, Inc., Rockville, MD) .
  • SF900 II medium minus methionine and cysteine available, e.g., from Life Technologies, Inc., Rockville, MD
  • radiolabeled polypeptides are desired, 42 hours later, 5 mCi of 35 S- methionine and 5 mCi 35 S-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 Expre ⁇ ion of LP231 in Mammalian Cell ⁇
  • a typical mammalian expre ⁇ sion vector contains at least one promoter element, which mediates the initiation of transcription of mRNA, the polypeptide coding ⁇ equence, and ⁇ ignals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequence ⁇ and intervening ⁇ equences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late pro oter ⁇ from SV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV) .
  • LTRS long terminal repeats
  • Suitable expres ⁇ ion vector ⁇ 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) .
  • vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, CA) , pcDNA3.1 (+/-), pcDNA/Zeo (+
  • mammalian host cells include human Hela 293, H9 , Jurkat cells, mouse NIH3T3 , C127 cell ⁇ , Cos 1, Cos 7 and CV 1, quail QCl-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
  • the gene can be expres ⁇ ed in ⁇ table cell lines that contain the gene integrated into a chromo ⁇ ome.
  • a ⁇ electable 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 thou ⁇ and copie ⁇ of the gene of interest.
  • Another useful selection marker is the enzyme glutamine syntha ⁇ e (GS) (Murphy, et al . , Biochem . J.
  • the expre ⁇ ion 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)).
  • LTR Rous Sarcoma Virus
  • 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 intere ⁇ t.
  • the vectors contain in addition the 3 ' intron, the polyadenylation and termination signal of the rat preproinsulin gene.
  • expres ⁇ ion pla ⁇ mid, pLP231 HA, i ⁇ made by cloning a cDNA encoding LP231 into the expression vector pcDNAI/Amp or pcDNAIII (which can be obtained from Invitrogen, Inc.).
  • the expre ⁇ ion vector pcDNAI/amp contains: (1) an E. coli origin of replication effective for propagation in E. coli and other prokaryotic cells; (2) an ampicillin re ⁇ istance 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, ⁇ upra) followed by a termination codon and polyadenylation signal arranged so that a cDNA can be conveniently placed under expres ⁇ ion control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation ⁇ ignal by means of restriction ⁇ ite ⁇ in the polylinker.
  • the fusion of the HA tag to the target polypeptide allows easy detection and recovery of the recombinant polypeptide with an antibody that recognize ⁇ the HA epitope.
  • pcDNAIII contain ⁇ , in addition, the ⁇ electable neomycin marker .
  • the pla ⁇ mid construction strategy is as follows.
  • the LP231 cDNA of a clone is amplified using primers that contain convenient restriction site ⁇ , much a ⁇ described above for construction of vectors for expres ⁇ ion of LP231 in E. coli .
  • 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 plate ⁇ which then are incubated to allow growth of ampicillin re ⁇ istant colonies.
  • Plasmid DNA is isolated from resi ⁇ tant colonie ⁇ and examined by re ⁇ triction analysis or other means for the presence of the LP231-encoding fragment.
  • 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.
  • Expres ⁇ ion of the LP231-HA fu ⁇ ion 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 35 S-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 u ⁇ ing 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.
  • Plasmid 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 tran ⁇ fected with the ⁇ e pla ⁇ mid ⁇ can be ⁇ elected by growing the cells in a ⁇ elective medium (alpha inu ⁇ MEM, Life TECH ⁇ ) ⁇ upplemented with methotrexate.
  • MTX methotrexate
  • a ⁇ econd gene i ⁇ linked to the DHFR gene it is usually co-amplified and over-expres ⁇ ed. It i ⁇ 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.
  • LTR long terminal repeat
  • CMV cytomegalovirus
  • the plasmid contains the 3 ' intron and polyadenylation ⁇ ite of the rat preproinsulin gene.
  • Other high efficiency promoters can also be u ⁇ ed for the expre ⁇ ion, 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 sy ⁇ tem ⁇ 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 .
  • mRNA 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.
  • Non- limiting examples include 5' and 3' primers having nucleotides corresponding or complementary to a portion of the coding sequence of LP231, e.g., a ⁇ 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 analysi ⁇ .
  • the cells are trypsinized and ⁇ eeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 ⁇ g/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) .
  • 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.
  • 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 RediprimeTM DNA labeling system (Amersham Life Science) , -Ill-
  • the probe i ⁇ purified u ⁇ ing a CHROMA SPIN-100TM column (Clontech Laboratorie ⁇ , Inc.), according to the manufacturer ' ⁇ protocol number PT1200-1.
  • MTN Multiple Ti ⁇ ue Northern
  • H variou ⁇ human ti ⁇ ue ⁇
  • IM human immune ⁇ ystem ti ⁇ ues

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Abstract

L'invention concerne des séquences d'acides nucléiques codant la nouvelle protéine humaine du type CRF présentant quelques similitudes avec la cérébelline. Ces nouveaux acides nucléiques sont utilisés dans la construction des vecteurs ADN et des cellules hôtes revendiqués selon l'invention et dans la préparation des protéines et des anticorps recombinants revendiqués.
EP01958853A 2000-08-04 2001-07-23 Facteur associe a c1q, polypeptides homologues et utilisations therapeutiques associees Withdrawn EP1328632A2 (fr)

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BRENNER ET AL: "Assessing sequence comparison methods with reliable structurally identified distant evolutionary relationships", PROC NATL ACAD SCI, vol. 95, no. 11, 26 May 1998 (1998-05-26), pages 6073 - 6078 *
ROST B.: "Twilight zone of protein sequence alignments", PROTEIN ENG.,, vol. 12, February 1999 (1999-02-01), pages 85 - 94 *
See also references of WO0212475A3 *

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