CA2383074A1 - Novel abc transporter and uses thereof - Google Patents

Abstract

The invention provides isolated nucleic acid molecules, designated ABCB12 transporter nucleic acid molecules, which encode novel ABC transporter famil y members. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing ABCB12 transported nucleic acid molecules, host cells into which the expression vectors have been introduced , and nonhuman transgenic animals in which an ABCB12 transported gene has been introduced or disrupted. The invention still further provides isolated ABCB1 2 transporter proteins, fusion proteins, antigenic peptides, anti-ABCB12 transporter antibodies, and screening assays for ABCB12 transporter modulators. Diagnostic and therapeutic methods utilizing compositions of the invention are also provided.

Description

NOVEL ABC TRANSPORTER AND USES THEREOF
RELATED INFORMATION
This application claims priority to U.S. Provisional Application Serial Nos.
60/161,724 and 60/151,473 both of which are entitled "NOVEL ABC TRANSPORTER AND USES
THEREOF", filed, respectively, on August 20, 1999 and August 30, 1999, the entire contents of which are hereby incorporated herein by reference. The contents of all patents, patent applications, and references cited throughout the specification are hereby incorporated by reference in their entireties.
BACKGROUND OF THE INVENTION
ABC transporter proteins represent a large superfamily of proteins with conserved features in both prokaryotes and eukaryotes. ABC transporters catalyze ATP-dependent transport of endogenous or exogenous substrates across biological membranes (Borst, P., ~5 (1997) Seminar in Cancer Biology 8:131-213) and/or allosterically modify the function of heterologous proteins (Higgins CF, 1995, Cell 82:693-696). Several ABC
transporters have been associated with clinically relevant phenotypes including the phenomenon of multidrug resistance (Ambudkar S.V. et al., (1999), Annu. Rev. Toxicol., 39:361-398), cystic fibrosis (Riordin JR et al., (1989) Science 245:1066-1073), atherosclerosis (Brooks-Wilson A et al., 20 (1999) Nature Genetics 22:336-345), hyperinsulinemic hypoglycemia (Thomas PM et al., (1995) Science 268:46-429), macular degeneration (Allikmets R et al., (1997) Science 277:1805-1807), and adrenoleukodystrophy (Mosser J et al., (1993) Nature 361:726-730), to name but a few.
While the genes associated with these disease phenotypes have been identified to some 25 degree, it is clear that a large number of putative ABC transporters exist in the human genome, as evidenced by the partial sequences noted in various EST databases (Allikmets et al., ( 1996) Hum Mol Genet 5:1649-1655). However, the utility of such information is compromised by the absence of the full-length nucleotide sequence of the coding region of the relevant gene and its translated protein product.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of a novel ATP Binding Cassette (ABC) transporter family member, referred to herein as ABCB12 transporter nucleic acid and protein molecules (also human UMAT (after the rat homolog termed ubiquitously expressed mammalian ABC half-transporter (UMAT)). The ABCB 12 transporter molecules of the present invention are useful as targets for developing modulating agents to regulate a variety of cellular processes, particularly the transport of neurotoxic molecules, e.g., (3-amyloid SUBSTITUTE SHEET (RULE 26) peptide, across cell membranes or, e.g., the blood-brain barrier (BBB).
Neurotoxic molecules such as ~i-amyloid peptide are involved in neurological disorders such as Alzheimer's disease (see, e.g., Goate et al. (1991) Nature 349:704; Games et al. (1995) Nature 373:523; and Suzuki et al. (1994) Science 264:1336). Other neurological disease involving toxic polypeptides include, e.g., prion diseases, Huntington's disease, Parkinson's disease, etc.
(for a review see Hardy et al. ( 1998) Science 282:1075-1079.
Accordingly, modulation of amyloid-(3 protein export with a modulator of human ABCB 12 transporter would be expected to modulate amyloid deposition and thus, Alzheimer's disease.
In addition, the ABCB 12 transporter molecules of the invention are useful as targets for developing modulating agents of multidrug resistance. Moreover, the molecules of the present invention are useful as diagnostic and therapeutic tools.
Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding ABCB 12 transporter proteins or biologically active portions thereof, as well as ~s nucleic acid fragments suitable as primers or hybridization probes for the detection of ABCB 12-encoding nucleic acids.
In one embodiment, an ABCB12 transporter nucleic acid molecule of the invention is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more identical to the nucleotide sequence (e.g., to the entire length of 2o the nucleotide sequence) shown in SEQ ID NO: 1 or 3, or a complement thereof.
In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO: 1 or 3, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO: 3 and nucleotides 1-164 of SEQ ID
NO: 1. In another embodiment, the nucleic acid molecule includes SEQ ID NO: 3 and nucleotides 2697-2s 2893 of SEQ ID NO: 1.
In another embodiment, an ABCB 12 transporter nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO: 2. In a preferred embodiment, an ABCB12 transporter nucleic acid molecule includes a nucleotide sequence encoding a protein 3o having an amino acid sequence at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more homologous to the entire length of the amino acid sequence of SEQ ID NO: 2.
In another preferred embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of human ABCB 12 transporter having the amino acid sequence of SEQ ID NO:
3s 2. In yet another preferred embodiment, the nucleic acid molecule is at least 2696 nucleotides in length. In a further preferred embodiment, the nucleic acid molecule is at least 2574 SUBSTITUTE SHEET (RULE 26) nucleotides in length and encodes a protein having ABCB 12 transporter activity (as described herein).
Another embodiment of the invention features nucleic acid molecules, preferably ABCB 12 transporter nucleic acid molecules, which specifically detect ABCB 12 transporter nucleic acid molecules relative to nucleic acid molecules encoding non-ABCB 12 transporter proteins. For example, in one embodiment, such a nucleic acid molecule is at least 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 500-1000, 1000-1500, 1500-2000, 2000-2500, or 2500-3000 or more nucleotides in length and/or hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO: 1, or a complement thereof. It should be understood that the nucleic acid molecule can be of a length within a range having one of the numbers listed above as a lower limit and another number as the upper limit for the number of nucleotides in length, e.g., molecules that are 60-80, 300-1000, or 150-400 nucleotides in length.
In preferred embodiments, the nucleic acid molecules are at least 15 (e.g., contiguous) nucleotides in length and hybridize under stringent conditions to nucleotides 165-2696 of SEQ
ID NO: 1. In other preferred embodiments, the nucleic acid molecules comprise nucleotides 165-2696 of SEQ ID NO: 1.
In other preferred embodiments, the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ff~ NO:
~, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID
NO: 1 or 3 under stringent conditions.
Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to an ABCB12 transporter nucleic acid molecule, e.g., the coding strand of an ABCB 12 transporter nucleic acid molecule.
Another aspect of the invention provides a vector comprising an ABCB 12 transporter nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector.
In another embodiment, the invention provides a host cell containing a vector of the invention.
In yet another embodiment, the invention provides a host cell containing a nucleic acid molecule of the invention. The invention also provides a method for producing a protein, 3o preferably an ABCB 12 transporter protein, by culturing in a suitable medium, a host cell, e.y., a mammalian host cell such as a non-human mammalian cell, of the invention containing a recombinant expression vector, such that the protein is produced.
Another aspect of this invention features isolated or recombinant ABCB 12 transporter proteins and polypeptides. In a preferred embodiment, the protein, preferably an ABCB 12 transporter protein, includes at least one transmembrane domain and has an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more homologous to the amino acid sequence of SUBSTITUTE SHEET (RULE 26) SEQ ID NO: 2. In yet another preferred embodiment, the protein, preferably an transporter protein, includes at least one transmembrane domain and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1.
In another embodiment, the invention features fragments of the protein having the amino acid sequence of SEQ ID NO: 2, wherein the fragment comprises at least 15 amino acids (e.g., contiguous amino acids) of the amino acid sequence of SEQ ID NO: 2. In another embodiment, the protein, preferably an ABCB 12 transporter protein, has the amino acid sequence of SEQ ID NO: 2.
In another embodiment, the invention features an isolated protein, preferably an ABCB 12 transporter protein, which is encoded by a nucleic acid molecule consisting of a nucleotide sequence at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more homologous to a nucleotide sequence of SEQ ID NO:
1 or 3, or a complement thereof. This invention further features an isolated protein, preferably an ~5 ABCB12 transporter protein, which is encoded by a nucleic acid molecule consisting of a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ )D NO: 1 or 3, or a complement thereof.
The proteins of the present invention or portions thereof, e.g., biologically active portions thereof, can be operatively linked to a non-ABCB 12 transporter polypeptide (e.g., 2o heterologous amino acid sequences) to form fusion proteins. The invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind proteins of the invention, preferabl y ABCB 12 transporter proteins. In addition, the ABCB 12 transporter proteins, biologically active portions thereof, or expressible nucleic acids encoding the foregoing, can be incorporated into pharmaceutical compositions, which optionally include 25 pharmaceutically acceptable carriers.
In another aspect, the present invention provides a method for detecting the presence of an ABCB 12 transporter nucleic acid molecule, protein or polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting an ABCB 12 transporter nucleic acid molecule, protein or polypeptide such that the presence of an ABCB 12 transporter so nucleic acid molecule, protein or polypeptide is detected in the biological sample.
In another aspect, the present invention provides a method for detecting the presence of ABCB 12 transporter activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of ABCB 12 transporter activity such that the presence of ABCB 12 transporter activity is detected in the biological sample.
35 In another aspect, the invention provides a method for modulating ABCB 12 transporter activity comprising contacting a cell capable of expressing an ABCB12 transporter with an agent that modulates ABCB12 transporter activity such that ABCB12 transporter activity in the SUBSTITUTE SHEET (RULE 26) cell is modulated. In one embodiment, the agent inhibits ABCB 12 transporter activity. In another embodiment, the agent stimulates ABCB 12 transporter activity. In one embodiment, the agent is an antibody that specifically binds to an ABCB 12 transporter protein. In another embodiment, the agent modulates the ability of the ABCB 12 transporter to allosterically modify the function of other membrane proteins. In another embodiment, the agent modulates expression of ABCB 12 transporter by modulating transcription of an ABCB 12 transporter gene or translation of an ABCB 12 transporter mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of an ABCB 12 transporter mRNA or an ABCB 12 transporter gene.
In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant or unwanted ABCB 12 transporter protein or nucleic acid expression or activity by administering an agent which is an ABCB
12 transporter modulator to the subject. In one embodiment, the ABCB 12 transporter modulator is an ABCB 12 transporter protein. In another embodiment the ABCB 12 transporter modulator is an ~5 ABCB 12 transporter nucleic acid molecule. In yet another embodiment, the transporter modulator is a peptide, peptidomimetic, or other small molecule, e.g., a molecule that is carbohydrate-based, lipid-based, nucleic-acid based, natural organic-based, or synthetically derived organic-based.
The present invention also provides a diagnostic assay for identifying the presence or 2o absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding an ABCB 12 transporter protein; (ii) mis-regulation of the gene;
and (iii) aberrant post-translational modification of an ABCB 12 transporter protein, wherein a wild-type form of the gene encodes a protein with an ABCB12 transporter activity.
In another aspect the invention provides a method for identifying a compound that 25 binds to or modulates the activity of an ABCB 12 transporter protein, by providing an indicator composition comprising an ABCB 12 transporter protein having ABCB 12 transporter activity, contacting the indicator composition with a test compound, and determining the effect of the test compound on ABCB 12 transporter activity in the indicator composition to identify a compound that modulates the activity of an ABCB 12 transporter protein (e.g., an ABCB 12 so transporter protein associated with a membrane).
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the cDNA sequence and predicted amino acid sequence of a human ABCB12 transporter. The nucleotide sequence corresponds to nucleic acids 1 to 2893 of SEQ
35 ID NO: 1. The coding region without the 5' and 3' untranslated regions of the human ABCB 12 transporter gene is shown in SEQ >D NO: 3.
-S-SUBSTITUTE SHEET (RULE 26) Figure 2 depicts the amino acid sequence of the ABCB 12 transporter molecule corresponding to amino acids 1 to 843 of SEQ 117 NO: 2.
Figure 3 depicts an amino acid sequence alignment of the human ABCB 12 transporter polypeptide and polypeptides found in various species.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based, at least in part, on the discovery of a novel, human, ATP
Binding Cassette (ABC) transporter family member, referred to herein as ABCB
12 transporter nucleic acid and protein molecules (or human UMAT (after the rat homolog termed "ubiquitously expressed mammalian ABC half-transporter" (LJMAT)). ABC
transporter molecules are transmembrane proteins which catalyze ATP-dependent transport of endogenous or exogenous substrates across biological membranes. ABC transporters have been associated with the transport of polypeptides, e.g., a neurotoxic polypeptide, such as (3-amyloid, which is involved in Alzheimer's disease. Other neurological diseases caused by neurotoxic ~5 polypeptides include prior diseases, Parkinson's disease, Huntington's disease, etc. (for a review, see Hardy et al. ( 1998) Science 282: 1075-1079). In particular, ABC
transporters are associated with the transport of substrates across the blood-brain-battier.
Thus, the transport of substances in cells of the brain and substances across the blood-brain-barrier is, at lease in pan, controlled by ABC transporter molecules.
2o In addition, ABC transporters are associated with multidrug resistance found in cells especially, e.g., cells that are refractory to cytotoxic anti-cancer drugs (Borst, P. (1997) Seni.
Cancer Bio. 8:131-134).
Accordingly, the ABCB12 transporter molecules of the invention are suitable targets for developing novel diagnostic targets and therapeutic agents to control cellular transport in cells 25 of the brain (e.g., neuronal cells) and transport across the blood-brain-barner. Moreover, the ABCB12 transporter molecules are suitable targets for developing diagnostic targets and therapeutic agents for detecting and/or treating cells or tissues having multidrug resistance, e.~., a cancer.
In particular, the novel human ABCB 12 transporter molecules described herein, can 3o have one or more of the following functions and/or applications:
-ABC transporters expressed in the brain are implicated in the transport of substrates through the blood brain barrier (Schinkel A.H., et al, (1994) Cell, 77, 491) and therefore identification of the sequence of the human ABCB 12 transporter described herein, affords the development of new strategies for altering the function of the blood brain barrier. Given that s5 many drugs of potential utility in treating diseases of the brain are discarded because they do not enter the brain at therapeutically relevant concentrations, the present invention allows for the development of strategies to assist in the delivery of drugs to the brain.

SUBSTITUTE SHEET (RULE 26) -ABC transporters expressed in the brain (as described in, e.g., U.S. Patent Application No. 08/847,616 and PCT/L1S98/08463, the text of which is incorporated herein) are potential transporters for the (3-amyloid peptide, a peptide whose deposition in senile plaques is a fundamental feature of Alzheimer's disease. Thus, identifying novel transporters that regulate ~ (3-amyloid deposition is crucial in developing therapeutic treatment for Alzheimer's disease.
-The human homologue of the Drosophila melanogasrer white gene has been reported to be associated with mood and panic disorders (Nakamura M et al., ( 1999), Mol. Psychiatry, 4, 155-162), and this gene is a member of the superfamily of ABC transporters.
The human ABCB12 transporter of the invention is also expressed in the brain, and, accordingly, may be involved in mood and panic disorders. Identification of the sequence of human transporter described herein allows for the development of new treatments for mood and panic disorders.
-ABC transporters have been shown to be involved in the phenomenon of multidrug resistance (Ling, V., (1997) Cancer Chernother Pharmacol 40:S3-S8; Naomi, M., et a1.,(1998) ~5 Cancer Research, 58:1332-1337). The present invention will allow a determination of the ability of ABCB12 to contribute to the multidrug resistance phenotype and the design of agents capable of ameliorating multidrug resistance using techniques similar to those described by Boer, R., et al. ((1996) European Journal of Cancer, 32A:857-861).
The term "family" when referring to the protein and nucleic acid molecules of the 2o invention is intended to mean two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin, as well as other, distinct proteins of human origin or alternatively, can contain 25 homologues of non-human origin. Members of a family may also have common functional characteristics.
For example, the family of ABC transporter proteins comprise at least one "transmembrane domain" and preferably two transmembrane domains. As used herein, the term "transmembrane domain" includes an amino acid sequence of about 18 amino acid so residues in length which spans the plasma membrane. More preferably, a transmembrane domain includes about at least 18, 20, 25, 30, 35, 40, or 45 residues or more and spans the plasma membrane. Transmembrane domains are described in, for example, Zagotta W.N. et al, ( 1996) Annual Rev. Neuronsci. 19: 235-63, the contents of which are incorporated herein by reference. Amino acid residues 27-50, 262-285, 304-326, 383-405, 503-521, and 530-548 of 35 the human ABCB 12 transporter protein comprise transmembrane domains. One or more of these transmembrane domains may associate to form a membrane-spanning domain.
_7_ SUBSTITUTE SHEET (RULE 26) Isolated proteins of the present invention, preferably ABCB12 transporter proteins, have an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ 1D
NO: 2 or are encoded by a nucleotide sequence sufficiently homologous to SEQ
ID NO: 1 or 3.
As used herein, the term "sufficiently homologous" refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity. For example, amino acid or nucleotide sequences which share common structural domains have at least 60% homology, more preferably 70%-80%, and even more preferably 90-95% homology across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as sufficiently homologous.
Furthermore, amino acid or nucleotide sequences which share at least 60%, more preferably 70-80%, or 90-95% homology and share a common functional activity are defined herein as ~5 sufficiently homologous.
As used interchangeably herein, an "ABCB12 transporter activity", "biological activity of ABCB12 transporter" or "functional activity of ABCB12 transporter ", refers to an activity exerted by an ABCB 12 transporter protein, polypeptide or nucleic acid molecule on an ABCB 12 transporter responsive cell or on an ABCB 12 transporter protein substrate, as 2o determined in vitro, or in vitro, according to standard techniques.
Preferably, an ABCB12 transporter activity has the ability to act as an energy-dependent (ATP) molecular pump.
In one embodiment, an ABCB12 activity is a direct activity, such as an association with membrane associated protein andlor the transport of an endogenous or exogenous substrate across a biological membrane. In another embodiment, the ABCB12 activity is the ability of 25 the polypeptide to allosterically modify the function of other membrane protein. For example, in some cells, modulation of p-glycoprotein by an ABCB 12 transporter modulator has been shown to alter the magnitude of volume-activated chloride currents (reviewed in Higgins, C. F.
Volume-activated chloride currents associated with the multidrug resistance P-glycoprotein, J.
Physiol. 482:31S-36S (1995)). Thus, in this model, p-glycoprotein and other ABC transporters so have multiple functions, one of which is to allosterically modify the function of the other membrane proteins. The present invention is consistent with a model in which the allostenc modification of other membrane proteins by e.g., an ABCB 12 transporter, is responsible for a change in the transport of a substrate, e.g., (3-amyloid, a cytotoxic drug, or other small molecule. Accordingly, an ABCB12 activity is at least one or more of the following activities:
35 (i) activation of an ABCB 12-dependent signal transduction pathway; (ii) modulation of the transport of a substrate (e.g., cytotoxic drug, ~3-amyloid) across a membrane;
(iii) interaction of an ABCB12 protein with a non-ABC2 membrane associated molecule; (iv) modulation of the _g_ SUBSTITUTE SHEET (RULE 26) development or differentiation of an ABCB 12-expressing cell; (v) modulation of the development or differentiation of a non- ABCB12-expressing cell; (vi) modulation of the homeostasis of an ABCB12-expressing cell; and (vii) modulation of the homeostasis of a non-ABCB 12-expressing cell.
Accordingly, another embodiment of the invention features isolated ABCB12 transporter proteins and polypeptides having an ABCB 12 transporter activity.
Preferred proteins are ABCB 12 transporter proteins having at least one transmembrane domain, preferably two transmembrane domains, and, preferably, an ABCB12 transporter activity.
The nucleotide sequence of the isolated human ABCB 12 transporter protein cDNA
and the predicted amino acid sequence of the human ABCB 12 transporter polypeptide are shown in Fig. 5 and in SEQ m NOs: 1 and 2, respectively.
The human ABCB 12 transporter gene, which is approximately 2893 nucleotides in length, encodes a protein having a molecular weight of approximately 94 kDa and which is approximately 843 amino acid residues in length.
~5 Various aspects of the invention are described in further detail in the following subsections:
I. Isolated Nucleic Acid Molecules One aspect of the invention pertains to isolated nucleic acid molecules that encode ABCB 12 transporter proteins or biologically active portions thereof, as well as nucleic acid 2o fragments sufficient for use as hybridization probes to identify ABCB 12-encoding nucleic acid molecules (e.g., ABCB 12 transporter mRNA) and fragments for use as PCR
primers for the amplification or mutation of ABCB 12 transporter nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA
or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using 25 nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
The term "isolated nucleic acid molecule" includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term "isolated"
includes nucleic so acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated ABCB 12 transporter nucleic acid molecule can contain less than 35 about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially SUBSTITUTE SHEET (RULE 26) free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 or 3, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or portion of the nucleic acid sequence of SEQ ID NO: 1 or 3 as a hybridization probe, ABCB 12 transporter nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID NO: 1 or 3 can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NO: 1 or 3.
A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, ~5 genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to ABCB 12 transporter nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
2o In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 1. The sequence of SEQ
ID NO: 1 corresponds to the human ABCB 12 cDNA. This cDNA comprises sequences encoding the human ABCB 12 protein (i.e., "the coding region", from nucleotides 165-2696), as well as 5' untranslated sequences (nucleotides 1-164) and 3' untranslated sequences (nucleotides 2697-25 2893). Alternatively, the nucleic acid molecule can comprise only the coding region of SEQ
ID NO: 1 (e.g., nucleotides 165-2696, corresponding to SEQ ID NO: 3).
In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO: 1 or 3, or a portion of any of these nucleotide sequences. A
nucleic acid molecule 3o which is complementary to the nucleotide sequence shown in SEQ ID NO: 1 or 3 is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 1 or 3 such that it can hybridize to the nucleotide sequence shown in SEQ ID NO: 1 or 3, thereby forming a stable duplex.
In still another preferred embodiment, an isolated nucleic acid molecule of the present 35 invention comprises a nucleotide sequence which is at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more homologous to SUBSTITUTE SHEET (RULE 26) the entire length of the nucleotide sequence shown in SEQ ID NO: 1 or 3, or a portion of any of these nucleotide sequences.
Moreover, the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO: 1 or 3, for example, a fragment which can be used as a probe or primer or a fragment encoding a portion of an ABCB 12 transporter protein, e.g., a biologically active portion of an ABCB 12 transporter protein. The nucleotide sequence determined from the cloning of the ABCB 12 transporter gene allows for the generation of probes and primers designed for use in identifying and/or cloning other ABCB
12 transporter family members, as well as ABCB 12 transporter homologues from other species.
The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ ID NO: 1 or 3 of an anti-sense sequence of SEQ ID NO: 1 or 3, or of a naturally occurring allelic variant or mutant of ~5 SEQ ID NO: 1 or 3. In an exemplary embodiment, a nucleic acid molecule of the present invention comprises a nucleotide sequence which is greater than 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500-1000, 1000-1500, 1500-2000, or 2000-2500, or 2500-3000, or more nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NO: 1 or 3.
2o Probes based on the ABCB 12 transporter nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which 25 misexpress an ABCB 12 transporter protein, such as by measuring a level of an ABCB 12-encoding nucleic acid in a sample of cells from a subject e.g., detecting ABCB
12 transporter mRNA levels or determining whether a genomic ABCB 12 transporter gene has been mutated or deleted.
A nucleic acid fragment encoding a "biologically active portion of an ABCB12 3o transporter protein" can be prepared by isolating a portion of the nucleotide sequence of SEQ
m NO: 1 or 3 which encodes a polypeptide having an ABCB12 transporter biological activity (the biological activities of the ABCB 12 transporter proteins are described herein), expressing the encoded portion of the ABCB 12 transporter protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the ABCB 12 transporter protein.
3s The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO: 1 or 3 due to degeneracy of the genetic code and thus encode the same ABCB 12 transporter proteins as those encoded by the nucleotide SUBSTITUTE SHEET (RULE 26) sequence shown in SEQ ID NO: 1 or 3. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO: 2.
In addition to the ABCB 12 transporter nucleotide sequences shown in SEQ ID
NO: 1 or s 3, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the ABCB 12 transporter proteins may exist within a population (e.g., the human population). Such genetic polymorphism in the transporter genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules which include an open reading frame encoding an ABCB12 transporter protein, preferably a mammalian ABCB 12 transporter protein, and can further include non-coding regulatory sequences, and introns.
Allelic variants of human ABCB 12 transporter include both functional and non-functional ABCB 12 transporter proteins. Functional allelic variants are naturally occurring ~s amino acid sequence variants of the human ABCB 12 transporter that maintain the ability to bind an ABCB12 transporter ligand. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO: 2 or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein.
Non-functional allelic variants are naturally occurring amino acid sequence variants of 2o the human ABCB 12 transporter protein that do not have the ability to either bind an ABCB 12 transporter ligand. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ
ID NO: 2 or a substitution, insertion or deletion in critical residues or critical regions.
The present invention further provides non-human orthologues of the human ABCB

25 transporter protein. Orthologues of the human ABCB 12 transporter protein are proteins that are isolated from non-human organisms and possess the same ABCB 12 transporter activity of the human ABCB 12 transporter protein. Orthologues of the human ABCB 12 protein can readily be identified as comprising an amino acid sequence that is substantially homologous to SEQ ID NO: 2.
3o Moreover, nucleic acid molecules encoding other ABC transporter family members and, thus, which have a nucleotide sequence which differs from the ABCB 12 transporter sequences of SEQ ID NO: 1 or 3 are intended to be within the scope of the invention. For example, another ABC transporter cDNA can be identified based on the nucleotide sequence of the human ABCB 12 transporter. Moreover, nucleic acid molecules encoding ABCB

3s transporter proteins from different species, e.g., mammals, and which, thus, have a nucleotide sequence which differs from the ABCB 12 transporter sequences of SEQ ID NO: 1 or 3 are SUBSTITUTE SHEET (R ULE 26) WO 01/14547 CA 02383074 2002-02-14 pCT/CA00/00964 intended to be within the scope of the invention. For example, a mouse ABCB 12 transporter cDNA can be identified based on the nucleotide sequence of the human ABCB12 transporter.
Nucleic acid molecules corresponding to natural allelic variants and homologues of the ABCB 12 transporter cDNAs of the invention can be isolated based on their homology to the s ABCB 12 transporter nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. Nucleic acid molecules corresponding to natural allelic variants and homologues of the ABCB 12 transporter cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the ABCB 12 transporter gene.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 15, 20, 25, 30 or more nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ m NO: 1 or 3. In other embodiment, the nucleic acid is at least 30, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2739 or more nucleotides in length. As ~5 used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 75%
homologous to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 80%, even more preferably at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% homologous to each other typically 2o remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50°C, preferably at 55°C, more preferably at 60°C, 2s and even more preferably at 65°C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID
NO: 1 or 3 corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
3o In addition to naturally-occurring allelic variants of the ABCB 12 transporter sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO: 1 or 3, thereby leading to changes in the amino acid sequence of the encoded ABCB 12 transporter proteins, without altering the functional ability of the ABCB 12 transporter proteins. For example, nucleotide 35 substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ m NO: 1 or 3. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of ABCB12 transporter (e.g., the sequence of SUBSTITUTE SHEET (R ULE 26) SEQ ID NO: 2) without altering the biological activity, whereas an "essential"
amino acid residue is required for biological activity.
Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding ABCB 12 transporter proteins that contain changes in amino acid residues that are not essential for activity. Such ABCB12 transporter proteins differ in amino acid sequence from SEQ >D NO: 2, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or more homologous to SEQ ID
NO:
2.
An isolated nucleic acid molecule encoding an ABCB 12 transporter protein homologous to the protein of SEQ ID NO: 2 can be created by introducing one or more nucleotide substitutions, additions, or deletions into the nucleotide sequence of SEQ >D NO: 1 or 3, such that one or more amino acid substitutions, additions or deletions are introduced into ~5 the encoded protein. Mutations can be introduced into SEQ ID NO: 1 or 3 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid 2o residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, 25 valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in an ABCB 12 transporter protein is preferably replaced with another amino acid residue from the same side chain family.
Alternatively, in another embodiment, mutations can be introduced randomly along all or part of an ABCB12 transporter coding sequence, such as by saturation mutagenesis, and the so resultant mutants can be screened for ABCB12 transporter biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO: 1 or 3 the encoded protein can be expressed recombinantly and the activity of the protein can be determined.
In a preferred embodiment, a mutant ABCB 12 transporter protein can be assayed for the ability to interact with a non-ABCB 12 transporter molecule, e.g., an ABCB
12 transporter 35 ligand, e.g., a polypeptide or a small molecule.
In addition to the nucleic acid molecules encoding ABCB 12 transporter proteins described above, another aspect of the invention pertains to isolated nucleic acid molecules SUBSTITUTE SHEET (RULE 26) which are anti sense thereto. An "antisense" nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA
sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
The antisense nucleic acid can be complementary to an entire ABCB 12 transporter coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding ABCB 12. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the coding region of human ABCB 12 corresponds to SEQ m NO: 3). In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding ABCB 12. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
~5 Given the coding strand sequences encoding ABCB 12 transporter disclosed herein (e.g., SEQ ID NO: 3), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of ABCB12 transporter mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of 2o ABCB12 transporter mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of ABCB 12 transporter mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides or more in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known 25 in the art. For example, an antisense nucleic acid (e.y., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides so which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 35 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-SUBSTITUTE SHEET (RULE 26) oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced s biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The anti sense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an ABCB I2 transporter protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific ~5 interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention include direct injection at a tissue site.
Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected 2o cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
2s In yet another embodiment, the antisense nucleic acid molecule of the invention is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual (3-units, the strands run parallel to each other (Gaultier et al. ( 1987) Nucleic Acids. Res.
15:6625-6641 ). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (moue et al.
30 ( 1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (moue et al.
( 1987) FEBS Lett. 215:327-330).
In still another embodiment, an antisense nucleic acid of the invention is a ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a ss complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave transporter mRNA transcripts to thereby inhibit translation of ABCB12 transporter mRNA. A

SUBSTITUTE SHEET (RULE 26) ribozyme having specificity for an ABCB 12-encoding nucleic acid can be designed based upon the nucleotide sequence of an ABCB 12 transporter cDNA disclosed herein (i.e., SEQ ID NO:
1). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be s cleaved in an ABCB12-encoding mRNA. See, e.g., Cech et al. U.S. Patent No.
4,987,071; and Cech et al. U.S. Patent No. 5,116,742. Alternatively, ABCB12 transporter mRNA
can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA
molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418.
Alternatively, ABCB12 transporter gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the ABCB12 transporter (e.g., the ABCB 12 transporter promoter and/or enhancers) to form triple helical structures that prevent transcription of the ABCB12 transporter gene in target cells. See generally, Helene, C.
( 1991 ) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. ( 1992) Ann. N.
Y. Acad. Sci.
660:27-36; and Maher, L.J. ( 1992) Bioassays 14( 12):807-15.
~5 In yet another embodiment, the ABCB12 transporter nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-20 23). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using 2s standard solid phase peptide synthesis protocols as described in Hyrup B.
et al. ( 1996) supra;
Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.
PNAs of ABCB 12 transporter nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or anti gene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or 3o translation arrest or inhibiting replication. PNAs of ABCB 12 transporter nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., S 1 nucleases (Hyrup B. ( 1996) supra)); or as probes or primers for DNA
sequencing or hybridization (Hyrup B. et al. ( 1996) supra; Perry-O'Keefe supra).
3s In another embodiment, PNAs of ABCB 12 transporter nucleic acid molecules can be modified, (e.g., to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or SUBSTITUTE SHEET (RULE 26) WO 01/14547 CA 02383074 2002-02-14 pCT/CA00/00964 other techniques of drug delivery known in the art. For example, PNA-DNA
chimeras of ABCB 12 transporter nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNA polymerases), to interact with the DNA portion while the PNA
s portion would provide high binding affinity and specificity. PNA-DNA
chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B. (1996) sccpra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. ( 1996) supra and Finn P.J. et al.
( 1996) Nucleic Acids Res. 24 ( 17): 3357-63. For example, a DNA chain can be synthesized on to a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used as a between the PNA and the 5' end of DNA (Mag, M. et al. ( 1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn P.J. et al. ( 1996) supra).
Alternatively, 1 s chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA
segment (Peterser, K.H. et al. (1975) Bioorganic Med. Chenc. Lett. 5: 1119-11124).
In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vitro), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci.
USA 86:6553-6556;
2o Lemaitre et al. ( 1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT
Publication No.
W088/09810) or the blood-brain bamer (see, e.g., PCT Publication No.
W089/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al. ( 1988) Bio-Techniques 6:958-976) or intercalating agents (See, e.g., Zon ( 1988) Phann. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another 2s molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).
II. Isolated ABCB 12 Transporter Proteins and Anti-ABCB 12 Transporter Antibodies One aspect of the invention pertains to isolated ABCB 12 transporter proteins, and 3o biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-ABCB 12 transporter antibodies. In one embodiment, native ABCB 12 transporter proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, ABCB 12 transporter proteins are produced by recombinant DNA techniques.
Alternative to 35 recombinant expression, an ABCB 12 transporter protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.

SUBSTITUTE SHEET (RULE 26) An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the ABCB12 transporter protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free s of cellular material" includes preparations of ABCB 12 transporter protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of ABCB12 transporter protein having less than about 30% (by dry weight) of non-ABCB 12 transporter protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-ABCB 12 transporter protein, still more preferably less than about 10% of non-ABCB 12 transporter protein, and most preferably less than about 5%
non-ABCB12 transporter protein. When the ABCB12 transporter protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about t5 10%, and most preferably less than about 5% of the volume of the protein preparation.
The language "substantially free of chemical precursors or other chemicals"
includes preparations of ABCB 12 transporter protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals"
2o includes preparations of ABCB 12 transporter protein having less than about 30% (by dry weight) of chemical precursors or non-ABCB 12 transporter chemicals, more preferably less than about 20% chemical precursors or non-ABCB 12 transporter chemicals, still more preferably less than about 10% chemical precursors or non-ABCB 12 transporter chemicals, and most preferably less than about 5% chemical precursors or non-ABCB 12 transporter chemicals.
2s As used herein, a "biologically active portion" of an ABCB12 transporter protein includes a fragment of an ABCB 12 transporter protein which participates in an interaction between an ABCB 12 transporter molecule and a non-ABCB 12 transporter molecule.
Biologically active portions of an ABCB12 transporter protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of 3o the ABCB 12 transporter protein, e.g., the amino acid sequence shown in SEQ
ID NO: 2, which include less amino acids than the full length ABCB 12 transporter proteins, and exhibit at least one activity of an ABCB12 transporter protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the ABCB 12 transporter protein. A
biologically active portion of an ABCB 12 transporter protein can be a polypeptide which is, for 3s example, 10, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800 or more amino acids in length.
Biologically active portions of an ABCB 12 transporter protein can be used as targets for developing agents which modulate an ABCB 12 transporter mediated activity.

SUBSTITUTE SHEET (RULE 26) In one embodiment, a biologically active portion of an ABCB 12 transporter protein comprises at least one transmembrane domain. It is to be understood that a preferred biologically active portion of an ABCB 12 transporter protein of the present invention may contain at least one transmembrane domain. Another preferred biologically active portion of s an ABCB 12 transporter protein may contain at least two transmembrane domains. One or more of these transmembrane domains may associate to form a membrane-spanning domain.
In addition, or alternatively, the biologically active portion of the ABCB12 transporter protein may include multiple clusters of conserved residues that define an ATP binding domain. In addition, or alternatively, the biologically active portion of the ABCB 12 transporter protein may comprise a Walker domain, e.g., a Walker A and/or Walker B domain (see Fig. 2.; Patel et al. (1998) Trends Cell Biol 8: 65-71). Identification of these domains may be facilitated using any of a number of art recognized molecular modeling techniques as described herein (see also Example 2). Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native ABCB 12 transporter protein.
In a preferred embodiment, the ABCB 12 transporter protein has an amino acid sequence shown in SEQ ID NO: 2. In other embodiments, the ABCB 12 transporter protein is substantially homologous to SEQ ID NO: 2, and retains the functional activity of the protein of SEQ ID NO: 2, yet differs in amino acid sequence due to natural allelic variation or 2o mutagenesis, as described in detail in subsection I above. Accordingly, in another embodiment, the ABCB 12 transporter protein is a protein which comprises an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more homologous to SEQ ID NO: 2.
To determine the percent identity of two amino acid sequences or of two nucleic acid 2s sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 70%, preferably 80%, 90% or 100% of the length of the so reference sequence (e.g., when aligning a second sequence to the ABCB12 transporter amino acid sequence of SEQ ID NO: 2 having 843 amino acid residues, at least 253, preferably at least 337, more preferably at least 422, even more preferably at least 506, and even more preferably at least 590, 674, 759, or 843 amino acid residues are aligned).
The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then 3s compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to SUBSTITUTE SHEET (RULE 26) WO 01/14547 CA 02383074 2002-02-14 pCT/CA00/00964 amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
A comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using standard art recognized comparison software using standard parameter settings. For example, the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been to incorporated into the GAP program in the GCG software package (available at http://www.gcg.com) can be employed using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at ~5 http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E.
Meyers and W. Miller (CABIOS, 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 20 12 and a gap penalty of 4.
The nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST
25 nucleotide searches can be performed with the NBLAST program, score = 100, wordlength =
12 to obtain nucleotide sequences homologous to ABCB12 transporter nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST
program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to ABCB 12 transporter protein molecules of the invention. To obtain gapped alignments for comparison purposes, 3o Gapped BLAST can be utilized as described in Altschul et al., ( 1997) Nucleic Acids Res.
25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
See http://www.ncbi.nlm.nih.gov.
The invention also provides ABCB 12 transporter chimeric or fusion proteins.
As used 35 herein, an ABCB 12 transporter "chimeric protein" or "fusion protein"
comprises an ABCB 12 transporter polypeptide operatively linked to a non-ABCB 12 transporter polypeptide. An "ABCB 12 transporter polypeptide" refers to a polypeptide having an amino acid sequence SUBSTITUTE SHEET (RULE 26) corresponding to ABCB 12, whereas a "non-ABCB 12 transporter polypeptide"
refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the ABCB 12 transporter protein, e.g., a protein which is different from the ABCB 12 transporter protein and which is derived from the same or a different organism. Within an ABCB 12 transporter fusion protein the ABCB 12 transporter polypeptide can correspond to all or a portion of an ABCB 12 transporter protein. In a preferred embodiment, an ABCB12 transporter fusion protein comprises at least one biologically active portion of an ABCB 12 transporter protein. In another preferred embodiment, an transporter fusion protein comprises at least two biologically active portions of an ABCB12 to transporter protein. Within the fusion protein, the term "operatively linked" is intended to indicate that the ABCB 12 transporter polypeptide and the non-ABCB 12 transporter polypeptide are fused in-frame to each other. The non-ABCB 12 transporter polypeptide can be fused to the N-terminus or C-terminus of the ABCB 12 transporter polypeptide.
For example, in one embodiment, the fusion protein is a GST-ABCB 12 transporter fusion protein in which the ABCB 12 transporter sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant ABCB12.
In another embodiment, the fusion protein is an ABCB 12 transporter protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of ABCB 12 transporter can be increased through use of a 2o heterologous signal sequence.
The ABCB 12 transporter fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vitro. The ABCB
12 transporter fusion proteins can be used to affect the bioavailability of an ABCB 12 transporter substrate.
Use of ABCB 12 transporter fusion proteins may be useful therapeutically for the treatment of 2s disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding an ABCB12 transporter protein; (ii) mis-regulation of the ABCB12 transporter gene: and (iii) aberrant post-translational modification of an ABCB 12 transporter protein.
Moreover, the ABCB 12-fusion proteins of the invention can be used as immunogens to produce anti-ABCB 12 transporter antibodies in a subject, to purify ABCB 12 transporter so ligands and in screening assays to identify molecules which inhibit the interaction of an ABCB 12 transporter with an ABCB 12 transporter substrate.
Preferably, an ABCB 12 transporter chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with ss conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic SUBSTITUTE SHEET (RULE 26) ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR
amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST
polypeptide). An ABCB 12 transporter-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the ABCB 12 transporter protein.
The present invention also pertains to variants of the ABCB 12 transporter proteins which function as either ABCB 12 transporter agonists or as ABCB 12 transporter antagonists.
Variants of the ABCB 12 transporter proteins can be generated by mutagenesis, e.g., discrete point mutation or truncation of an ABCB 12 transporter protein. An agonist of the ABCB 12 transporter proteins can retain substantially the same, or a subset, of the biological activities of the naturally occumng form of an ABCB 12 transporter protein. An antagonist of an ABCB 12 transporter protein can inhibit one or more of the activities of the naturally occurring form of the ABCB 12 transporter protein by, for example, competitively modulating an activity of an ABCB 12 transporter protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a 2o subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the ABCB12 transporter protein.
In one embodiment, variants of an ABCB 12 transporter protein which function as either ABCB 12 transporter agonists (mimetics) or as ABCB 12 transporter antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of an ABCB 12 transporter protein for ABCB 12 transporter protein agonist or antagonist activity. In one embodiment, a variegated library of ABCB 12 transporter variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
A variegated library of ABCB 12 transporter variants can be produced by, for example, 3o enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential ABCB 12 transporter sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of ABCB 12 transporter sequences therein. There are a variety of methods which can be used to produce libraries of potential ABCB 12 transporter variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one SUBSTITUTE SHEET (RULE 26) mixture, of all of the sequences encoding the desired set of potential ABCB 12 transporter sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev.
Biochent. 53:323;
Itakura et al. ( 1984) Science 198:1056; Ike et al. ( 1983) Nucleic Acid Res.
11:477.
In addition, libraries of fragments of an ABCB 12 transporter protein coding sequence can be used to generate a variegated population of ABCB 12 transporter fragments for screening and subsequent selection of variants of an ABCB 12 transporter protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an ABCB 12 transporter coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes ~5 N-terminal, C-terminal and internal fragments of various sizes of the ABCB12 transporter protein.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA
libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the 2o gene libraries generated by the combinatorial mutagenesis of ABCB 12 transporter proteins.
The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity 25 facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify ABCB 12 transporter variants (Arkin and Yourvan ( 1992) Proc. Natl. Acad. Sci.
USA 89:7811-7815: Delgrave et al. (1993) Protein Engineering 6(3):327-331).
so An isolated ABCB 12 transporter protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind ABCB 12 transporter using standard techniques for polyclonal and monoclonal antibody preparation. A full-length transporter protein can be used or, alternatively, the invention provides antigenic peptide fragments of ABCB 12 transporter for use as immunogens. Preferred epitopes encompassed by 35 the antigenic peptide are regions of ABCB 12 transporter that are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity.

SUBSTITUTE SHEET (RULE 26) An ABCB 12 transporter immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen. An appropriate immunogenic preparation can contain, for example, recombinantly expressed ABCB 12 transporter protein or a chemically synthesized ABCB 12 transporter polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent.
Immunization of a suitable subject with an immunogenic ABCB 12 transporter preparation induces a polyclonal anti-ABCB 12 transporter antibody response.
Accordingly, another aspect of the invention pertains to anti-ABCB12 transporter antibodies. The temp "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as ABCB12 transporter. Examples of immunologically active portions of immunoglobulin molecules include Flab) and F(ab~2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind ABCB12 transporter. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of ABCB12 transporter. A monoclonal antibody composition thus typically displays a single 2o binding affinity for a particular ABCB 12 transporter protein with which it immunoreacts.
Polyclonal anti-ABCB 12 transporter antibodies can be prepared as described above by immunizing a suitable subject with an ABCB 12 transporter immunogen. The anti-transporter antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized 25 ABCB 12 transporter. If desired, the antibody molecules directed against ABCB 12 transporter can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the anti-ABCB12 transporter antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal 3o antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein ( 1975) Nature 256:495-497) (see also, Brown et al. ( 1981 ) J. Immunol.
127:539-46; Brown et al. ( 1980) J. Biol. Chem .255:4980-83; Yeh et al. ( 1976) Proc. Natl.
Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) I»t. J. Cancer 29:269-75), the more recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol Todav 4:72), the EBV-35 hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing monoclonal antibody hybridomas is well known (see generally R. H. Kenneth, in Monoclonal Antibodies: A New SUBSTITUTE SHEET (RULE 26) Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York (1980); F!
A. Lerner ( 1981 ) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. ( 1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an ABCB 12 transporter immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds ABCB12 transporter.
Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-ABCB 12 transporter monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature 266:55052; Gefter et al.
Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. tLled., cited supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized ~5 with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. These myeloma 20 lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG"). Hybtidoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
Hybridoma cells producing a monoclonal antibody of the invention are detected by screening 25 the hybridoma culture supernatants for antibodies that bind ABCB12, e.g., using a standard ' ELISA assay.
Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-ABCB 12 transporter antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with ABCB12 3o transporter to thereby isolate immunoglobulin library members that bind ABCB12. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-O1; and the Stratagene SurfLAPTM Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can 35 be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. PCT
International Publication No. WO 92/18619; Dower et al. PCT International Publication No.
WO 91/17271; Winter et al. PCT International Publication WO 92/20791; Markland et al. PCT

SUBSTITUTE SHEET (RULE 26) International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO
93/01288; McCafferty et al. PCT International Publication No. WO 92/01047;
Garrard et al.
PCT International Publication No. WO 92/09690; Ladner et al. PCT International Publication No. WO 90/02809; Fuchs et al. (1991) BiolTechnology 9:1370-1372; Hay et al.
(1992) Hum.
s Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
Griffiths et al.
(1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896;
Clarkson et al.
(1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA
89:3576-3580;
Garrad et al. ( 1991 ) Bio/1'echnology 9:1373-1377; Hoogenboom et al. ( 1991 ) Nuc. Acid Res.
19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982;
and McCafferty et al. Nature ( 1990) 348:552-554.
Additionally, recombinant anti-ABCB 12 transporter antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA
~s techniques known in the art, for example using methods described in Robinson et al.
International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496: Morrison et al.
European Patent Application 173,494; Neuberger et al. PCT International Publication No.
WO 86/01533;
Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al. European Patent Application 125,023;
2o Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl.
Acad. Sci. USA
84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl.
Acad. Sci. USA 84:214-218; Nishimura et al. ( 1987) Canc. Res. 47:999-1005;
Wood et al.
( 1985) Nature 314:446-449; and Shaw et al. ( 1988) J. Natl. Cancer Inst.
80:1553-1559);
Motrison, S. L. ( 1985) Science 229:1202-1207; Oi et al. ( 1986) BioTechnigues 4:214; Winter 2s U.S. Patent 5.225,539; Jones et al. ( 1986) Nature 321:552-525; Verhoeyan et al. ( 1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060.
An anti-ABCB12 transporter antibody (e.g., monoclonal antibody) can be used to isolate ABCB 12 transporter by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-ABCB 12 transporter antibody can facilitate the purification of 3o natural ABCB 12 transporter from cells and of recombinantly produced ABCB
12 transporter expressed in host cells. Moreover, an anti-ABCB 12 transporter antibody can be used to detect ABCB 12 transporter protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the ABCB 12 transporter protein.
Anti-ABCB 12 transporter antibodies can be used diagnostically to monitor protein levels in tissue as part of a 3s clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic SUBSTITUTE SHEET (RULE 26) groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, -galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ~ZSI, '3'I, 355, 33P, 3zP or 3H.
III. Recombinant Expression Vectors and Host Cells Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding an ABCB 12 transporter protein (or a portion thereof). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a ~5 circular double stranded DNA loop into which additional DNA segments can be ligated.
Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are 2o integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be 2s used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the 3o invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequences) 3s in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other SUBSTITUTE SHEET (RULE 26) expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA ( 1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., ABCB
12 transporter proteins, mutant forms of ABCB12 transporter proteins, fusion proteins, and the like).
The recombinant expression vectors of the invention can be designed for expression of ABCB 12 transporter proteins in prokaryotic or eukaryotic cells. For example, transporter proteins can be expressed in bacterial cells such as E. coli, insect cells (using ~5 baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzvmology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
2o Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of 25 the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, so thrombin and enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc: Smith, D.B. and Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRITS (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
35 Purified fusion proteins can be utilized in ABCB12 transporter activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for ABCB 12 transporter proteins, for example. In a preferred embodiment, an SUBSTITUTE SHEET (RULE 26) transporter fusion protein expressed in a retroviral expression vector of the present invention can be utilized to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six (6) weeks).
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., ( 1988) Gene 69:301-315) and pET l 1d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California ( 1990) 60-89). Target gene expression from the pTrc vector relies on host RNA polymerise transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET l 1d vector relies on transcription from a T7 gnl0-lac fusion promoter mediated by a coexpressed viral RNA
polymerise (T7 gnl). This viral polymerise is supplied by host strains BL21(DE3) or HMS 174(DE3) from a resident prophage harboring a T7 gn 1 gene under the transcriptional control of the IacUV 5 promoter.
One strategy to maximize recombinant protein expression in E. coli is to express the ~5 protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in En, vmology 185, Academic Press, San Diego, California (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., ( 1992) 2o Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the ABCB 12 transporter expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec 1 (Baldari, et al., (1987) Ernbo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), 25 pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and picZ (InVitrogen Corp, San Diego, CA).
Alternatively, ABCB 12 transporter proteins can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.
( 1983) Mol. Cell Biol.
30 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al.
(1987) EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions 35 are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of SUBSTITUTE SHEET (RULE 26) WO 01/14547 CA 02383074 2002-02-14 pCT/CA00/00964 Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
In another embodiment, the recombinant mammalian expression vector is capable of s directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev.
1:268-277), lymphoid-specific promoters (Calame and Eaton ( 1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore ( 1989) EMBO J.
8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter;
Byrne and Ruddle ( 1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al.
(1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey t5 promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss ( 1990) Science 249:374-379) and the a-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
The invention further provides a recombinant expression vector comprising a DNA
2o molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to ABCB 12 transporter mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the 25 antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can 3o be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which an ABCB 12 transporter nucleic acid molecule of the invention is introduced, e.g., an ABCB 12 transporter nucleic acid ss molecule within a recombinant expression vector or an ABCB 12 transporter nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome. The terms "host cell" and "recombinant host cell"
are used SUBSTITUTE SHEET (RULE 26) interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, an ABCB12 transporter protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation. DEAE-dextran-mediated transfection, lipofection, or electroporation.
is Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al.
(Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the 2o expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.b~., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as 6418, hygromycin and methotrexate. Nucleic acid encoding a 2s selectable marker can be introduced into a host cell on the same vector as that encoding an ABCB 12 transporter protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can so be used to produce (i.e., express) an ABCB12 transporter protein.
Accordingly, the invention further provides methods for producing an ABCB 12 transporter protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding an ABCB 12 transporter protein has been introduced) in a suitable medium such that an ABCB 12 transporter protein is 35 produced. In another embodiment, the method further comprises isolating an transporter protein from the medium or the host cell.

SUBSTITUTE SHEET (RULE 26) The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which ABCB 12-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous ABCB 12 transporter sequences have been introduced into their genome or homologous recombinant animals in which endogenous ABCB12 transporter sequences have been altered.
Such animals are useful for studying the function and/or activity of an ABCB
12 transporter and for identifying and/or evaluating modulators of ABCB 12 transporter activity.
As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more ~5 cell types or tissues of the transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous ABCB 12 transporter gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
2o A transgenic animal of the invention can be created by introducing an encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. The ABCB 12 transporter cDNA sequence of SEQ ID NO: 1 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a nonhuman homologue of a 25 human ABCB 12 transporter gene, such as a mouse or rat ABCB 12 transporter gene, can be used as a transgene. Alternatively, an ABCB 12 transporter gene homologue, such as another ABCB12 transporter family member, can be isolated based on hybridization to the ABCB12 transporter cDNA sequences of SEQ ID NO: 1 or 3 (described further in subsection I above) and used as a transgene. Intronic sequences and polyadenylation signals can also be included so in the transgene to increase the efficiency of expression of the transgene.
A tissue-specific regulatory sequences) can be operably linked to an ABCB 12 transporter transgene to direct expression of an ABCB 12 transporter protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos.
35 4,736,866 and 4,870,009, both by Leder et al., U.S. Patent No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic SUBSTITUTE SHEET (RULE 26) animals. A transgenic founder animal can be identified based upon the presence of an ABCB 12 transporter transgene in its genome and/or expression of ABCB 12 transporter mRNA
in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene s encoding an ABCB 12 transporter protein can further be bred to other transgenic animals carrying other transgenes, for example, animals carrying a transgene encoding a neurotoxic polypeptide such as (3-amyloid.
To create a homologous recombinant animal, a vector is prepared which contains at least a portion of an ABCB 12 transporter gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the ABCB 12 transporter gene.
The ABCB12 transporter gene can be a human gene (e.g., the cDNA of SEQ ID NO:
3), but more preferably, is a non-human homologue of a human ABCB 12 transporter gene (e.g., a cDNA isolated by stringent hybridization with the nucleotide sequence of SEQ
ID NO: 1 ). For example, a mouse ABCB 12 transporter gene can be used to construct a homologous recombination nucleic acid molecule, e.g., a vector, suitable for altering an endogenous ABCB 12 transporter gene in the mouse genome. In a preferred embodiment, the homologous recombination nucleic acid molecule is designed such that, upon homologous recombination, the endogenous ABCB 12 transporter gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector). Alternatively, the homologous 2o recombination nucleic acid molecule can be designed such that, upon homologous recombination, the endogenous ABCB 12 transporter gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous ABCB 12 transporter protein). In the homologous recombination nucleic acid molecule, the altered portion of the ABCB 12 transporter gene is 2s flanked at its 5' and 3' ends by additional nucleic acid sequence of the ABCB 12 transporter gene to allow for homologous recombination to occur between the exogenous ABCB

transporter gene carried by the homologous recombination nucleic acid molecule and an endogenous ABCB12 transporter gene in a cell, e.g., an embryonic stem cell.
The additional flanking ABCB 12 transporter nucleic acid sequence is of sufficient length for successful 3o homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the homologous recombination nucleic acid molecule (see, e.g., Thomas, K.R. and Capecchi, M. R. (1987) Cell 51:503 for a description of homologous recombination vectors). The homologous recombination nucleic acid molecule is introduced into a cell, e.g., an embryonic stem cell line (e.g., by electroporation) and cells in 3s which the introduced ABCB12 transporter gene has homologously recombined with the endogenous ABCB 12 transporter gene are selected (see e.g., Li, E. et al. ( 1992) Cell 69:915).
The selected cells can then injected into a blastocyst of an animal (e.g., a mouse) to form SUBSTITUTE SHEET (RULE 26) aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embr-vt9nic Stem Cells: A
Practical Approach, E.J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A
chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination nucleic acid molecules, e.g., vectors, or homologous recombinant animals are described further in Bradley, A. ( 1991 ) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO
91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO 93/04169 by Berns et al.
In another embodiment, transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the crelloxP recombinase system of bacteriophage P1. For a description of the crelloxP recombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.
Sci. USA
~5 89:6232-6236. Another example of a recombinase system is the FLP
recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355. If a crelloxP
recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by 2o mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. ( 1997) Nature 385:810-813 and PCT
International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic 25 cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter Go phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster 3o animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
IV. Pharmaceutical Compositions The ABCB 12 transporter nucleic acid molecules, fragments of ABCB 12 transporter proteins, anti-ABCB12 transporter antibodies (also referred to herein as "active compounds"), 3s expressible nucleic acids encoding ABCB 12 transporters (or fragments thereof), or any compound identified as a modulator of an ABCB 12 transporter (as described herein) can be incorporated into pharmaceutical compositions suitable for administration.
Such compositions SUBSTITUTE SHEET (RULE 26) typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable Garner" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components:
a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, ~s propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;
chelating agents such as ethylenediaminetetraacetic acid: buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can 2o be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELT"'' (BASF, 2s Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene 3o glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and s5 the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
Prolonged SUBSTITUTE SHEET (RULE 26) absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a fragment of an ABCB 12 transporter protein or an anti-ABCB 12 transporter antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic ABCB 12 transporter dispersion medium and the required other ingredients from those enumerated above.
In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier.
They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic t5 administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and 2o the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide: a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, 25 or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For 3o transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into 35 ointments, salves, gels, or creams as generally known in the art.

SUBSTITUTE SHEET (RULE 26) The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect s the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
~5 It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
The specification for 2o the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
Toxicity and therapeutic efficacy of such compounds can be determined by standard 25 pharmaceutical procedures in cell cultures or experimental animals, e.g..
for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be 3o used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies 35 preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the SUBSTITUTE SHEET (RULE 26) WO 01/14547 CA 02383074 2002-02-14 pCT/CA00/00964 invention, the therapeutically effective dose can be estimated initially from cell culture assays.
A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be s used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or ~s more cells which produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
V. Uses and Methods of the Invention 2o The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.~., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic).
The isolated nucleic acid molecules of the invention can be used, for example, to 25 express ABCB 12 transporter protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect ABCB 12 transporter mRNA (e.g., in a biological sample) or a genetic alteration in an ABCB 12 transporter gene, and to modulate ABCB
12 transporter activity, as described further below. The ABCB 12 transporter proteins can be used to treat disorders characterized by insufficient or excessive production of an ABCB 12 transporter 3o substrate or production of ABCB 12 transporter inhibitors. In addition, the ABCB 12 transporter proteins can be used to screen for naturally occurring ABCB 12 transporter substrates, to screen for drugs or compounds which modulate ABCB 12 transporter activity, as well as to treat disorders characterized by insufficient or excessive production of ABCB 12 transporter protein or production of ABCB 12 transporter protein forms which have decreased, aberrant or 3s unwanted activity compared to ABCB12 transporter wild type protein.
Moreover, the anti-ABCB12 transporter antibodies of the invention can be used to detect and isolate ABCB12 SUBSTITUTE SHEET (RULE 26) transporter proteins, regulate the bioavailability of ABCB 12 transporter proteins, and modulate ABCB 12 transporter activity.
A. Screening Assays:
s The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to ABCB 12 transporter proteins, have a stimulatory or inhibitory effect on, for example, ABCB 12 transporter expression or ABCB 12 transporter activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of ABCB12 transporter substrate.
In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates of an ABCB 12 transporter protein or polypeptide or biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of an ABCB 12 1s transporter protein or polypeptide or biologically active portion thereof.
The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity 2o chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. ( 1997) Anticancer Drug Des. 12:145).
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) 2s Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. ( 1994). J. Med.
Chenc. 37:2678; Cho et al. ( 1993) Science 261:1303; Carrell et al. ( 1994) Angew. Chenc. Int. Ed.
Engl. 33:2059;
Carell et al. ( 1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. ( 1994) J. Med.
Chem. 37:1233.
Libraries of compounds may be presented in solution (e.b~., Houghten (1992) 3o Biotechniques 13:412-421 ), or on beads (Lam ( 1991 ) Nature 354:82-84), chips (Fodor ( 1993) Nature 364:555-556), bacteria (Ladner USP 5,223,409), spores (Ladner USP
'409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin ( 1990) Science 249:404-406); (Cwirla et al. ( 1990) Proc. Natl.
Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladner supra.).
35 In one embodiment, an assay is a cell-based assay in which a cell which expresses an ABCB12 transporter protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate ABCB12 transporter activity is SUBSTITUTE SHEET (RULE 26) determined. Determining the ability of the test compound to modulate ABCB 12 transporter activity can be accomplished by monitoring, for example, cellular transport of organic anions, organic cations, cytotoxic anti-cancer drugs, intracellular calcium, potassium, phosphatidylcholine, sodium concentration, neuronal membrane depolarization, a neurotoxic s polypeptide (e.g., (3-amyloid), or the activity of an ABCB 12 transporter-regulated transcription factor. The cell, for example, can be of mammalian origin, e.g., a neuronal cell. The ability of the test compound to modulate ABCB 12 transporter binding to a substrate or to bind to ABCB 12 transporter can also be determined. Determining the ability of the test compound to modulate ABCB 12 transporter binding to a substrate can be accomplished, for example, by coupling the ABCB 12 transporter substrate with a radioisotope or enzymatic label such that binding of the ABCB 12 transporter substrate to ABCB 12 transporter can be determined by detecting the labeled ABCB12 transporter substrate in a complex. Determining the ability of the test compound to bind ABCB 12 transporter can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to ~ 5 ABCB 12 transporter can be determined by detecting the labeled ABCB 12 transporter compound in a complex. For example, compounds (e.g., ABCB12 transporter substrates) can be labeled with 'zSI, 35S, ''~C, 33P, 3zP, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline 2o phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
In one embodiment, suitable compounds include, but are not limited to, verapamil, desmethoxyverapamil, chloroquine, quinine, chinchonidine, primaquine, tamoxifen, dihydrocyclosporin, yohimbine, corynanthine, reserpine, physostigmine, acridine, acridine 2s orange, quinacrine, trifluoroperazine chlorpromazine, propanolol, atropine, tryptamine, forskolin, 1,9-dideoxyforskolin, cyclosporin, (US Patent 4,117,118 (1978)), (cyclosporin D, 6-[(2S, 4R, 6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]-(9CI)), [US
Patent 5,525,590] [ACS 121584-18-7], Keller et al., "SDZ PSC 833, a non-immunosuppressive cylcosporine: its potency in overcoming p-glycoprotein-mediated multidrug resistance of 3o murine leukemia", Int J Cancer 50:593-597 (1992)), RU-486 (17(3-hydroxy-113-[4-dimethylaminophenyl]-17a prop-1-ynyl estra-4, 9-dien-3 one), RU-49953 (17~i-hydroxy-11(3, 17a-[4-dimethylaminophenyl] - 17a prop-1-ynyl estra-4, 9 dien-3 one), S9778 (6-{4-[2,2-di( )-ethylamino]-1-piperidinyl }-N,N', di-2-propenyl-1,3,5-triazine-2,4-diamine, bismethane sulfonate, [US patent 5,225,411; EP 466586] [ACS # 140945-O1-3]; Dhainaut et al., "New ss triazine derivatives as potent modulators of multidrug resistance," J
Medicinal Chemistry 35:2481-2496 (1992)), MS-209 (5-[3-[4-(2,2-diphenylacetyl)piperazin-1-yl]-2-hydroxypropoxy]quinoline sesquifumarate, [US patent 5,405,843 (continuation of 5,112,817)], SUBSTITUTE SHEET (R ULE 26) (ACS # 158681-49-3], Sato et al., "Reversal of multidrug resistance by a novel quinoline derivative, MS-209, Cancer Chemother Pharncacol 35:271-277 (1995)), MS-073 (Fukazawa et al., European Patent Application 0363212 (1989)), FK-506 (Tanaka et al., M.
Physicochemical properties of FK-506, a novel immunosuppressant isolated from Streptoncvces tsukubaensis" Transplantation Proceedings. 19(5 Suppl 6):11-6, ( 1987); Naito et al., "Reversal of multidrug resistance by an immunosuppressive agent FK-506," Cancer Chemother &
Pharmacol. 29:195-200 (1992); Pourtier-Manzanedo et al., "FK-506 (fujimycin) reverses the multidrug resistance of tumor cells in vitro," Anti-Cancer Drugs 2:279-83 ( 1991 ); Epand &
Epand, "The new potent immunosuppressant FK-506 reverses multidrug resistance in Chinese 1 o hamster ovary cells," Anti-Cancer Drug Design 6:189-93 ( 1991 )), VX-710 (2-peperidinecarboxylic acid, 1-[oxo(3,4,5-trimethoxyphenyl)acetyl]-3-(3-pyridinyl)-1-[3-(3-pyridinyl)propyl]butyl ester [ACS 159997-94-1] [US patent number 5,620,971]
Germann et al., "Chemosensitization and drug accumulation effects of VX-710, verapamil, cyclosporin A, MS-209 and GF120918 in multidrug resistance-associated protein MRP" Anti-Cancer Drugs 8, ~5 141-155 (1997) ; Germann et al., "Cellular and biochemical characterization of VX-710 as a chemosensitizer: reversal of P-glycoprotein-mediated multidrug resistance in vitro" Anti-cancer Drugs 8, 125-140 ( 1997)), VX-853 ([US patent number 5,543,423] [ACS #

1), AHC-52 (methyl 2-(N-benzyl-N-methylamino)ethyl-2, 6-dimethyl-4-(2-isopropylpyrazolo[1,5-a]pyridine-3-yl)-1,4-dihyropyridine-3,5-dicarboxylate;
[Japanese Patent 20 63-135381; European Patent 0270926] [ACS 119666-09-0] Shinoda et al., "In vivo circumvention of vincristine resistance in mice with P388 leukemia using a novel compound, AHC-52," Cancer Res 49:1722-6 (1989)), GF-120918 (9,10-dihydro-5-methoxy-9-oxo-N-[4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxyisoquinol-2-yl) ethyl]phenyl]-4 acridinecarboxamide,[US
patent 5.604.237] [ACS # 143664-11-3] Hyafil et al., "In vitro and in vivo reversal of 2s multidrug resistance by GF120918, an acridonecarboxamide derivative,"
Cancer Res 53:4595 4602 (1993)), and XR-9051 ( 3-[(3Z, 6Z)-6-Benzylidene-1-methyl-2,5-dioxopiperazin-3 ylidenemethyl]-N-[4-[2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl]phenyl]benzamide hydrochloride, (ACS#57-22-7]).
It is also within the scope of this invention to determine the ability of a compound (e.g., 3o an ABCB 12 transporter substrate) to interact with ABCB 12 transporter without the labeling of any of the interactants. For example, a microphysiometer can be used to detect the interaction of a compound with ABCB 12 transporter without the labeling of either the compound or the ABCB12. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a "microphysiometer" (e.g., Cytosensor) is an analytical instrument that measures the rate at 35 which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS).
Changes in this acidification rate can be used as an indicator of the interaction between a compound and ABCB12.

SUBSTITUTE SHEET (RULE 26) In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing an ABCB 12 transporter target molecule (e.g., an ABCB 12 transporter substrate) with a test compound and determining the ability of the test compound to modulate (e.g.
stimulate or inhibit) the activity of the ABCB 12 transporter target molecule.
Determining the ability of the test compound to modulate the activity of an ABCB 12 transporter target molecule can be accomplished, for example, by determining the ability of the ABCB 12 transporter protein to bind to or interact with the ABCB 12 transporter target molecule.
Determining the ability of the ABCB 12 transporter protein or a biologically active fragment thereof, to bind to or interact with an ABCB 12 transporter target molecule can be accomplished by one of the methods described above for determining direct binding. In a preferred embodiment, determining the ability of the ABCB12 transporter protein to bind to or interact with an ABCB 12 transporter target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i.e., 15 intracellular Ca2+, diacylglycerol, IP3, and the like), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), cellular transport of, e.g., a reference compound or, e.g., a neurotoxic polypeptide (e.g., (3-amyloid) or detecting a target-regulated cellular response.
2o In yet another embodiment, an assay of the present invention is a cell-free assay in which an ABCB 12 transporter protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the ABCB 12 transporter protein or biologically active portion thereof is determined. Preferred biologically active portions of the ABCB 12 transporter proteins to be used in assays of the present invention include 25 fragments which participate in interactions with non-ABCB12 transporter molecules, e.b~., fragments with high surface probability scores (see, for example, Figure 2).
Binding of the test compound to the ABCB 12 transporter protein can be determined either directly or indirectly as described above. In a preferred embodiment, the assay includes contacting the transporter protein or biologically active portion thereof with a known compound which binds so ABCB 12 transporter to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an transporter protein, wherein determining the ability of the test compound to interact with an ABCB 12 transporter protein comprises determining the ability of the test compound to preferentially bind to ABCB12 transporter or biologically active portion thereof as compared to 35 the known compound.
In another embodiment, the assay is a cell-free assay in which an ABCB 12 transporter protein or biologically active portion thereof is contacted with a test compound and the ability SUBSTITUTE SHEET (X ULE 26) of the test compound to modulate (e.g., stimulate or inhibit) the activity of the ABCB12 transporter protein or biologically active portion thereof is determined.
Determining the ability of the test compound to modulate the activity of an ABCB 12 transporter protein can be accomplished, for example, by determining the ability of the ABCB 12 transporter protein to s bind to an ABCB 12 transporter target molecule by one of the methods described above for determining direct binding. Alternatively, for example, ATP binding can be measured.
Determining the ability of the ABCB 12 transporter protein to bind to an ABCB
12 transporter target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991) Anal.
Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705. As used herein, "BIA" is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
In an alternative embodiment, determining the ability of the test compound to modulate ~5 the activity of an ABCB 12 transporter protein can be accomplished by determining the ability of the ABCB 12 transporter protein to further modulate the activity of a downstream effector of an ABCB 12 transporter target molecule. For example, the activity of the effector molecule on an appropriate target can be determined or the binding of the effector to an appropriate target can be determined as previously described.
2o In yet another embodiment, the cell-free assay involves contacting an transporter protein or biologically active portion thereof with a known compound which binds the ABCB 12 transporter protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the ABCB 12 transporter protein, wherein determining the ability of the test compound to interact with the 2s ABCB 12 transporter protein comprises determining the ability of the ABCB
12 transporter protein to preferentially bind to or modulate the activity of an ABCB 12 transporter target molecule.
The cell-free assays of the present invention are amenable to use of both soluble and/or membrane-bound forms of isolated proteins (e.g., ABCB 12 transporter proteins or biologically 3o active portions thereof ). In the case of cell-free assays in which a membrane-bound form of an isolated protein is used it may be desirable to utilize a solubilizing agent such that the membrane-bound form of the isolated protein is maintained in solution.
Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton~ X-35 100, Triton~ X-114, Thesit~, Isotridecypoly(ethylene glycol ether)n, 3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS), 3-[(3-SUBSTITUTE SHEET (RULE 26) cholamidopropyl)dimethylamminioJ-2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.
In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either ABCB 12 transporter or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to an ABCB12 transporter protein, or interaction of an ABCB 12 transporter protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/ABCB 12 transporter fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione Sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined ~5 with the test compound or the test compound and either the non-adsorbed target protein or ABCB 12 transporter protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for 2o example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of ABCB12 transporter binding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either an ABCB 12 transporter protein or an ABCB12 transporter target molecule can be immobilized utilizing conjugation of biotin and 25 streptavidin. Biotinylated ABCB 12 transporter protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with ABCB 12 transporter protein or target molecules but which do not interfere with binding of the 3o ABCB 12 transporter protein to its target molecule can be derivatized to the wells of the plate, and unbound target or ABCB 12 transporter protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the ABCB 12 transporter protein or target molecule, as well as enzyme-linked 35 assays which rely on detecting an enzymatic activity associated with the ABCB12 transporter protein or target molecule.

SUBSTITUTE SHEET (RULE 26) In another embodiment, modulators of ABCB 12 transporter expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of ABCB 12 transporter mRNA or protein in the cell is determined. The level of expression of ABCB 12 transporter mRNA or protein in the presence of the candidate compound is compared s to the level of expression of ABCB 12 transporter mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of ABCB 12 transporter expression based on this comparison. For example, when expression of ABCB 12 transporter mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of ABCB 12 transporter mRNA or protein expression.
Alternatively, when expression of ABCB 12 transporter mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of ABCB12 transporter mRNA or protein expression.
The level of ABCB 12 transporter mRNA or protein expression in the cells can be determined by methods t5 described herein for detecting ABCB12 transporter mRNA or protein.
In yet another aspect of the invention, the ABCB 12 transporter proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S.
Patent No.
5,283,317; Zervos et al. (1993) Cel172:223-232; Madura et al. (1993) J. Biol.
Chem.
268:12046-12054; Bartel et al. (1993) Biotechnigues 14:920-924; Iwabuchi et al. (1993) 2o Oncogene 8:1693-1696; and Brent W094/10300), to identify other proteins, which bind to or interact with ABCB 12 transporter ("ABCB 12-binding proteins" or "ABCB 12-by") and are involved in ABCB12 transporter activity. Such ABCB12-binding proteins are also likely to be involved in the propagation of signals by the ABCB 12 transporter proteins or transporter targets as, for example, downstream elements ef an ABCB 12-mediated signaling 25 pathway. Alternatively, such ABCB 12-binding proteins are likely to be ABCB
12 transporter inhibitors.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for an 3o transporter protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vitro, forming an ABCB 12-dependent complex, the 35 DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor.

SUBSTITUTE SHEET (RULE 26) Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the ABCB 12 transporter protein.
In another aspect, the invention pertains to a combination of two or more of the assays s described herein. For example, a modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of an ABCB
12 transporter protein can be confirmed in vitro, e.g., in an animal.
This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., an ABCB 12 transporter modulating agent, an antisense ABCB 12 transporter nucleic acid molecule, an ABCB 12-specific antibody, or an ABCB 12-binding partner) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used t5 in an animal model to determine the mechanism of action of such an agent.
Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
B. Detection Assays:
2o Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome;
and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological 25 sample. These applications are described in the subsections below.
1. Chromosome Mapp Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called so chromosome mapping. Accordingly, portions or fragments of the ABCB 12 transporter nucleotide sequences, described herein, can be used to map the location of the transporter genes on a chromosome. The mapping of the ABCB 12 transporter sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
3s Briefly, ABCB12 transporter genes can be mapped to chromosomes by preparing PCR
primers (preferably 15-25 by in length) from the ABCB 12 transporter nucleotide sequences.
Computer analysis of the ABCB 12 transporter sequences can be used to predict primers that do SUBSTITUTE SHEET (RULE 26) not span more than one exon in the genomic DNA, thus complicating the amplification process.
These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the ABCB 12 transporter sequences will yield an amplified fragment.
Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but human cells can, the one human chromosome that contains the gene encoding the needed to enzyme, will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D~ustachio P. et al.
(1983) Science 220:919-924). Somatic cell hybrids containing only fragments of human chromosomes can ~5 also be produced by using human chromosomes with translocations and deletions.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the ABCB 12 transporter nucleotide sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from 2o specific chromosomes. Other mapping strategies which can similarly be used to map an ABCB 12 transporter sequence to its chromosome include in situ hybridization (described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA
libraries.
25 Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step.
Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical such as colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands 3o develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases.
However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a 35 review of this technique, see Verma et al., Human Chromosomes: A Manual of transporter Techniques (Pergamon Press, New York 1988).

SUBSTITUTE SHEET (RULE 26) Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al. ( 1987) Nature, 325:783-787.
Moreover, differences in the DNA sequences between individuals affected and ~5 unaffected with a disease associated with the ABCB12 transporter gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease.
Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible 2o from chromosome spreads or detectable using PCR based on that DNA sequence.
Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
~. Tissue Typing 25 The ABCB 12 transporter sequences of the present invention can also be used to identify individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification.
3o This method does not suffer from the current limitations of "Dog Tags"
which can be lost, switched, or stolen, making positive identification difficult. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S.
Patent 5.272,057).
Furthermore, the sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected 35 portions of an individual's genome. Thus, the ABCB 12 transporter nucleotide sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the SUBSTITUTE SHEET (RULE 26) WO 01/14547 CA 02383074 2002-02-14 pCT/CA00/00964 sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such s DNA sequences due to allelic differences. The sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue. The transporter nucleotide sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases.
Each of the sequences described herein can, to some degree, be used as a standard against which DNA
from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO: 1 can comfortably provide positive ~s individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO: 3 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
If a panel of reagents from ABCB 12 transporter nucleotide sequences described herein 2o is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.
25 3. Use of Partial ABCB 12 transporter Sequences in Forensic Biology DNA-based identification techniques can also be used in forensic biology.
Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime. To make such an identification, PCR technology can be used to amplify DNA sequences taken from 3o very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.
The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the 3s reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual).
As mentioned above, actual base sequence information can be used for identification as an SUBSTITUTE SHEET (RULE 26) accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions of SEQ ID NO: 1 are particularly appropriate for this use as greater numbers of polymorphisms occur in the noncoding regions, making it easier to differentiate individuals using this technique. Examples of polynucleotide reagents include the ABCB12 transporter nucleotide sequences or portions thereof, e.g., fragments derived from the noncoding regions of SEQ ID NO: 1 having a length of at least 20 bases, preferably at least 30 bases.
The ABCB 12 transporter nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such ABCB 12 transporter probes can be used to identify tissue by species andlor by organ type.
In a similar fashion, these reagents, e.g., ABCB 12 transporter primers or probes can be ~5 used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).
C. Predictive Medicine:
The present invention also pertains to the field of predictive medicine in which 2o diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically.
Accordingly, one aspect of the present invention relates to diagnostic assays for determining ABCB 12 transporter protein and/or nucleic acid expression as well as ABCB 12 transporter activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an 25 individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant or unwanted ABCB 12 transporter expression or activity. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with ABCB 12 transporter protein, nucleic acid expression or activity. For example, mutations in an ABCB 12 transporter gene 3o can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with ABCB 12 transporter protein, nucleic acid expression or activity.
Another aspect of the invention pertains to monitoring the influence of agents (e.g., 35 drugs, compounds) on the expression or activity of ABCB 12 transporter in clinical trials.
These and other agents are described in further detail in the following sections.

SUBSTITUTE SHEET (RULE 26) 1. Diagnostic Assay An exemplary method for detecting the presence or absence of ABCB 12 transporter protein or nucleic acid in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting s ABCB 12 transporter protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes ABCB 12 transporter protein such that the presence of ABCB 12 transporter protein or nucleic acid is detected in the biological sample. A preferred agent for detecting ABCB12 transporter mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to transporter mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length ABCB 12 transporter nucleic acid, such as the nucleic acid of SEQ ID NO: 1, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to ABCB 12 transporter mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.
~s A preferred agent for detecting ABCB12 transporter protein is an antibody capable of binding to ABCB12 transporter protein, preferably an antibody with a detectable label.
Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab~2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by 2o coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is 2s intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect ABCB 12 transporter mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vitro. For example, in vitro techniques for detection of ABCB 12 transporter mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for 3o detection of ABCB 12 transporter protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. Irz vitro techniques for detection of ABCB 12 transporter genomic DNA include Southern hybridizations.
Furthermore, in vitro techniques for detection of ABCB 12 transporter protein include introducing into a subject a labeled anti-ABCB 12 transporter antibody. For example, the 3s antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

SUBSTITUTE SHEET (RULE 26) In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a serum sample isolated by conventional means from a subject.
In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting ABCB 12 transporter protein, mRNA, or genomic DNA, such that the presence of ABCB 12 transporter protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of ABCB 12 transporter protein, mRNA or genomic DNA in the control sample with the presence of ABCB 12 transporter protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of ABCB 12 transporter in a biological sample. For example, the kit can comprise a labeled compound or agent capable of detecting ABCB 12 transporter protein or mRNA in a biological sample; means for t5 determining the amount of ABCB12 transporter in the sample; and means for comparing the amount of ABCB 12 transporter in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect an ABCB 12 transporter protein or nucleic acid.
20 2. Prognostic Assays The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant or unwanted ABCB 12 transporter expression or activity. As used herein, the term "aberrant"
includes an ABCB 12 transporter expression or activity which deviates from the wild type 25 ABCB 12 transporter expression or activity. Aberrant expression or activity includes increased or decreased expression or activity, as well as expression or activity which does not follow the wild type developmental pattern of expression or the subcellular pattern of expression. For example, aberrant ABCB 12 transporter expression or activity is intended to include the cases in which a mutation in the ABCB 12 transporter gene causes the ABCB 12 transporter gene to be so under-expressed or over-expressed and situations in which such mutations result in a non-functional ABCB 12 transporter protein or a protein which does not function in a wild-type fashion, e.g., a protein which does not interact with an ABCB 12 transporter ligand or one which interacts with a non-ABCB 12 transporter ligand. As used herein, the term "unwanted"
includes an unwanted phenomenon involved in a biological response. For example, the term s5 unwanted includes an ABCB 12 transporter expression or activity which is undesirable in a subject.

SUBSTITUTE SHEET (RULE 26) WO 01/14547 PCTlCA00/00964 The assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with a misregulation in ABCB 12 transporter protein activity or nucleic acid expression.
Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for s developing a disorder associated with a misregulation in ABCB 12 transporter protein activity or nucleic acid expression. Thus, the present invention provides a method for identifying a disease or disorder associated with aberrant or unwanted ABCB 12 transporter expression or activity in which a test sample is obtained from a subject and ABCB 12 transporter protein or nucleic acid (e.g., mRNA or genomic DNA) is detected, wherein the presence of transporter protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted ABCB 12 transporter expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
15 Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant or unwanted ABCB 12 transporter expression or activity, e.g., a cancer where the cells of the cancer have developed multidrug resistance. Thus, the present invention 2o provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant or unwanted ABCB 12 transporter expression or activity in which a test sample is obtained and ABCB 12 transporter protein or nucleic acid expression or activity is detected (e.g., wherein the abundance of ABCB 12 transporter protein or nucleic acid expression or activity is diagnostic for a subject that can be administered the agent to treat a 25 disorder associated with aberrant or unwanted ABCB 12 transporter expression or activity).
The methods of the invention can also be used to detect genetic alterations in an ABCB12 transporter gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in ABCB 12 transporter protein activity or nucleic acid expression. In preferred embodiments, the methods include detecting, in a sample of cells from 3o the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding an ABCB 12-protein, or the mis-expression of the ABCB 12 transporter gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from an ABCB 12 transporter gene: 2) an addition of one or more nucleotides to an ABCB
12 transporter 3s gene; 3) a substitution of one or more nucleotides of an ABCB 12 transporter gene, 4) a chromosomal rearrangement of an ABCB12 transporter gene; 5) an alteration in the level of a messenger RNA transcript of an ABCB 12 transporter gene, 6) aberrant modification of an SUBSTITUTE SHEET (RULE 26) ABCB 12 transporter gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of an ABCB12 transporter gene, 8) a non-wild type level of an ABCB 12-protein, 9) allelic loss of an ABCB 12 transporter gene, and 10) inappropriate post-translational modification of an ABCB 12-protein.
As described herein, there are a large number of assays known in the art which can be used for detecting alterations in an ABCB 12 transporter gene. A preferred biological sample is a tissue or serum sample isolated by conventional means from a subject.
In certain embodiments, detection of the alteration involves the use of a probe/primer in a polymerise chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acid.
Sci. USA 91:360-364), the latter of which can be particularly useful for detecting point mutations in the ABCB 12-gene (see Abravaya et al. ( 1995) Nucleic Acids Res .23:675-682).
This method can include the steps of collecting a sample of cells from a subject, isolating t5 nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to an ABCB
12 transporter gene under conditions such that hybridization and amplification of the ABCB 12-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is 2o anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
Alternative amplification methods include: self sustained sequence replication (Guatelli, J.C. et al., (1990) Proc. Natl. Acid. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D.Y. et al., ( 1989) Proc. Natl. Acid. Sci. USA
86:1173-1177), Q-25 Beta Replicase (Lizardi, P.M. et al. ( 1988) Bio-Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
In an alternative embodiment, mutations in an ABCB12 transporter gene from a sample so cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA
indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for 35 example, U.S. Patent No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
-SS-SUBSTITUTE SHEET (RULE 26) In other embodiments, genetic mutations in ABCB 12 transporter can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin, M.T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. et al. ( 1996) Nature Medicine 2: 753-759). For example, genetic mutations in ABCB 12 transporter can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, M.T. et al.
supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe aiTays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the ABCB 12 transporter gene and detect mutations by comparing the sequence of the sample ABCB12 transporter with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger (( 1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT
International Publication No. WO 94/16101; Cohen et al. (1996) Adv.
Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).
Other methods for detecting mutations in the ABCB 12 transporter gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242). In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type ABCB 12 transporter sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded so duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to base pair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. ( 1988) Proc.
Natl Acad Sci USA

SUBSTITUTE SHEET (RULE 26) WO 01/14547 CA 02383074 2002-02-14 pCT/CA00/00964 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295. In a preferred embodiment, the control DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA
s mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in ABCB 12 transporter cDNAs obtained from samples of cells. For example, the mutt enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).
According to an exemplary embodiment, a probe based on an ABCB12 transporter sequence, e.g., a wild-type ABCB 12 transporter sequence, is hybridized to a cDNA or other DNA
product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to identify ~5 mutations in ABCB 12 transporter genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal.
Tech. Appl.
9:73-79). Single-stranded DNA fragments of sample and control ABCB 12 transporter nucleic 2o acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA
(rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
2s In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. ( 1991 ) Trends Genet 7:5).
In yet another embodiment the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient 3o gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE
is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 by of high-melting GC-rich DNA by PCR.
In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner 35 ( 1987) Biophys Chem 265:12753).
Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer SUBSTITUTE SHEET (RULE 26) extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. ( 1986) Nature 324:163); Saiki et al.
(1989) Proc. Natl Acad. Sci USA 86:6230). Such allele specific oligonucleotides are s hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology which depends on selective PCR
amplification may be used in conjunction with the instant invention.
Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerise extension (Prossner ( 1993) Tibtech 11:238). In addition it may be desirable to introduce a novel restriction site in ~5 the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1 ). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acid. Sci USA
88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the 2o presence or absence of amplification.
The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g.., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an ABCB12 transporter 25 gene.
Furthermore, any cell type or tissue in which ABCB 12 transporter is expressed may be utilized in the prognostic assays described herein.
3. Monitoring of Effects Durino Clinical Trials 3o Monitoring the influence of agents (e.~., drugs) on the expression or activity of an ABCB 12 transporter protein can be applied not only in ABCB 12 transporter drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase ABCB 12 transporter gene expression, protein levels, or upregulate ABCB 12 transporter activity, can be monitored in clinical trials of subjects 3s exhibiting decreased ABCB 12 transporter gene expression, protein levels, or downregulated ABCB12 transporter activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease ABCB 12 transporter gene expression, protein levels, or SUBSTITUTE SHEET (RULE 26) downregulate ABCB12 transporter activity, can be monitored in clinical trials of subjects exhibiting increased ABCB 12 transporter gene expression, protein levels, or upregulated ABCB 12 transporter activity. In such clinical trials, the expression or activity of an ABCB 12 transporter gene, and preferably, other genes that have been implicated in, for example, an ABCB 12-associated disorder can be used as a "read out" or markers of the phenotype of a particular cell.
For example, and not by way of limitation, genes, including ABCB 12, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates ABCB 12 transporter activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on ABCB 12-associated disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of ABCB 12 transporter and other genes implicated in the associated disorder, respectively. The levels of gene expression (e.g., a gene expression pattern) can be quantified by northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of ABCB 12 transporter or other genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during treatment of the individual with the agent.
2o In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) including the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent;
(ii) detecting the 25 level of expression of an ABCB 12 transporter protein, mRNA, or genomic DNA
in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the ABCB 12 transporter protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the ABCB 12 transporter protein, mRNA, or genomic DNA in the pre-3o administration sample with the ABCB 12 transporter protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of ABCB 12 transporter to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent 35 may be desirable to decrease expression or activity of ABCB12 transporter to lower levels than detected, i.e. to decrease the effectiveness of the agent. According to such an embodiment, SUBSTITUTE SHEET (RULE 26) WO 01/14547 CA 02383074 2002-02-14 pCT/CA00/00964 ABCB 12 transporter expression or activity may be used as an indicator of the effectiveness of an agent, even in the absence of an observable phenotypic response.
D. Methods of Treatment:
The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted ABCB 12 transporter expression or activity. With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
"Pharmacogenomics", as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or "drug response genotype".) Thus, another aspect of the invention provides methods for t5 tailoring an individual's prophylactic or therapeutic treatment with either the ABCB12 transporter molecules of the present invention or ABCB 12 transporter modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.
1. Prop>~lactic Methods In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted ABCB12 transporter expression or activity, by administering to the subject an ABCB 12 transporter or an agent which modulates ABCB 12 transporter expression or at least one ABCB 12 transporter activity. Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted ABCB 12 transporter expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the ABCB 12 transporter aberrancy, such so that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of ABCB 12 transporter aberrancy, for example, an ABCB 12, ABCB 12 transporter agonist or ABCB 12 transporter antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.
2. Therapeutic Methods Another aspect of the invention pertains to methods of modulating ABCB 12 transporter expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the SUBSTITUTE SHEET (RULE 26) modulatory method of the invention involves contacting a cell with an ABCB
1'_~transporter or agent that modulates one or more of the activities of ABCB12 transporter protein activity associated with the cell. An agent that modulates ABCB 12 transporter protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of an ABCB 12 transporter protein (e.g., an ABCB 12 transporter substrate), an ABCB 12 transporter antibody, an ABCB 12 transporter agonist or antagonist, a peptidomimetic of an ABCB 12 transporter agonist or antagonist, or other small molecule. In one embodiment, the agent stimulates one or more ABCB 12 transporter activities. Examples of such stimulatory agents include active ABCB 12 transporter protein and a nucleic acid molecule encoding ABCB 12 transporter that has been introduced into the cell. In another embodiment, the agent inhibits one or more ABCB 12 transporter activities. Examples of such inhibitory agents include antisense ABCB 12 transporter nucleic acid molecules, anti-ABCB 12 transporter antibodies, and ABCB 12 transporter inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vitro (e.g., by ~5 administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of an ABCB12 transporter protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or 2o downregulates) ABCB12 transporter expression or activity. In another embodiment, the method involves administering an ABCB 12 transporter protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted ABCB 12 transporter expression or activity.
Stimulation of ABCB 12 transporter activity is desirable in situations in which ABCB 12 25 transporter is abnormally downregulated and/or in which increased ABCB12 transporter activity is likely to have a beneficial effect. For example, stimulation of ABCB12 transporter activity is desirable in situations in which an ABCB 12 transporter is downregulated and/or in which increased ABCB 12 transporter activity is likely to have a beneficial effect. Likewise, inhibition of ABCB 12 transporter activity is desirable in situations in which 3o transporter is abnormally upregulated and/or in which decreased ABCB12 transporter activity is likely to have a beneficial effect.
In one embodiment, an agent found to inhibit ABCB12 transporter activity is used in combination with another therapy such that the targeting of that therapy across the blood-brain-barner is achieved.

SUBSTITUTE SHEET (RULE 26) WO 01/14547 CA 02383074 2002-02-14 pCT/CA00/00964 3. Pharmacoaenomics The ABCB 12 transporter molecules of the present invention, as well as agents, or modulators which have a stimulatory or inhibitory effect on ABCB 12 transporter activity (e.g., ABCB 12 transporter gene expression) as identified by a screening assay described herein can s be administered to individuals to treat (prophylactically or therapeutically) ABCB 12-associated disorders associated with aberrant or unwanted ABCB 12 transporter activity.
In conjunction with such treatment, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic to failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer an transporter molecule or ABCB12 transporter modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with an ABCB 12 transporter molecule or ABCB

~5 transporter modulator.
Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11) :983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types of 2o pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism).
These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency 2s (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
One pharmacogenomics approach to identifying genes that predict drug response, known as "a genome-wide association", relies primarily on a high-resolution map of the human 3o genome consisting of already known gene-related markers (e.g., a "bi-allelic" gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.) Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking pan in a Phase II/III drug trial to identify markers associated with a particular observed drug response or side effect.
3s Alternatively, such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of SUBSTITUTE SHEET (RULE 26) DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease-associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.
Alternatively, a method termed the "candidate gene approach", can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drugs target is known (e.g., an ABCB 12 transporter protein of the present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic ~5 polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C 19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizes (EM) and poor 2o metabolizes (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no 25 therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
Alternatively, a method termed the "gene expression profiling", can be utilized to 3o identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g., an ABCB 12 transporter molecule or ABCB 12 transporter modulator of the present invention) can give an indication whether gene pathways related to toxicity have been turned on.
Information generated from more than one of the above pharmacogenomics approaches 35 can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic SUBSTITUTE SHEET (R ULE 26) efficiency when treating a subject with an ABCB 12 transporter molecule or transporter modulator, such as a modulator identified by one of the exemplary screening assays described herein.
This invention is further illustrated by the following examples which should not be construed as limiting.
EXEMPLIFICATION

~o ISOLATION AND CLONING OF THE HUMAN ABCB12 TRANSPORTER cDNA
In this example, the isolation and cloning of the gene encoding a novel human transporter is described.
The human ABCB 12 transporter cDNA was isolated using polymerise chain reaction t5 (PCR) and primers designed (described below) from a rat nucleotide sequence (Genbank Accession No. AJ003004; Hirsch-Ernst et al., (1998) Biochem Biophy Res Chem 249:151-155) and two partial human nucleotide sequences (Genbank Accession Nos. AF070598 and U66673;
Yu et al. (1997) Genome Res 7:353-358; Andersson et al., (1996) Anal Biochem 5:107-113;
Allikmets et al., (1996) Hum Mol Genet 5:1649-1655.
2o First, a double stranded (ds) cDNA library was constructed using 15 ~g of normal human total RNA (commercially available from Invitrogen, Order No. D6030-01 ) that was subjected to first and second strand synthesis, phenol-chloroform extraction, alcohol precipitation, and ligation to double-stranded cDNA adaptors using the Clontech MARATHON
cDNA Amplification Kit (Clontech, Order No. K1802-1) according to the manufacturer's 25 instructions.
Next, a polymerise chain reaction (PCR) reaction was carried out in a 20 p.1 final volume using 2 ~l of the ds cDNA synthesized (as described above), primers P1, P2, and P3 (as set forth below), and reagents provided by and according to the Clontech Advantage cDNA
PCR kit (Clontech, Order No. K1905-1). The cycling parameters were:
denaturation at 94° C
so for 1 min; denaturation at 94° C for 30 sec, annealing at 60°
C (P1 and P2) or 70° C (P3 and an adaptor specific primer) for 30 sec, elongation at 70° C for 2 min for 30 cycles; a final elongation step at 70° C for 5 min.

SUBSTITUTE SHEET (RULE 26) WO 01/14547 CA 02383074 2002-02-14 pCT/CA00/00964 Table 1. Oligonucleotide primers used for isolating the human ABCB12 transporter.
5'-GTGACTGTGGGCAACTACTGCGAGG-3' P1 nucleotide 158 to 182 of AJ003004 5'-CTGTGAAATGTCCTGCCCATCTATTCGG-3' P2 nucleotide 950 to 977 of AF070598 5'-TGGAGCTGCTCATCTTCTCCCACCTGCACG P3 nucleotide 43 to 72 of AF070598 5'-GCCTGAGTCCCTGCTTCTTCTTCACG-3' P4 nucleotide 229 to 254 of hsA14.3 5'-AACAGCCCTCCAGAGACCACATACCGC-3' P5 nucleotide 787 to 763 of hsA14.3 A second series of PCR reactions were carried out to isolate the 5' untranslated region (UTR) of the human ABCB12 transporter cDNA using the following methodology.
Briefly, a PCR reaction was carried out in a 25 ~tl final volume using 5 ~tl (masterplate) or 1 ~I (subplate) of a human brain cDNA library (Origene Technologies, Order No. LAB-1001), primers P4 and PS (Table 1), and appropriate reaction conditions (10 mM Tris-HC1 pH 8.3, 50 mM KC1, 1.5 mM MgCl2, 0.2 mM each dNTP, 0.1 ~M each primer, 2% DMSO, 1.25 unit of AmpliTaq 2o DNA Polymerise (Perkin Elmer)). The cycling parameters were: denaturation at 94°C for 2 min; denaturation at 94°C for 30 sec, annealing at 65°C for 30 sec, elongation at 72°C for 1 min for 30 cycles; and a final elongation step at 72°C for 5 min.
Using the above techniques, two cDNA fragments were isolated. The first cDNA
fragment (hsA14.2), obtained by polymerise chain reaction (PCR) from a human brain double-strand (ds) cDNA library using primers P1 and P2, was determined to be 1964 base pairs (bp).
A second cDNA fragment (hsA14.1) was obtained using rapid amplification of cDNA 3'-ends (3' RACE) using PCR, the human brain ds cDNA library, and primers P3 and an oligonucleotide specific to the adaptor linked to the 5' and 3' ends of the cDNA library, and the resultant fragment was determined to be 1725 bp. Each of the above cDNA
fragments were 3o subcloned and their nucleotide sequences were obtained using a automated DNA sequencing engine (LICOR). The fragments hsA14.1 and hsA14.2 overlap by 967 bp. The nucleic acid sequence of the isolated fragments was predicted to encode the human ortholog of the rat ubiquitously expressed mammalian ABC half-transporter termed UMAT. The hsAl4.l and hsA14.2 fragments were determined to encode the 3' and 5' ends of the gene, respectively.
Together, the above fragments allowed for the isolation of a 2893 by clone (hsA14.3) which contained the entire human ABCB 12 transporter gene coding sequence in addition to 5' and the 3' untranslated regions (UTR). The nucleotide sequence encoding the human ABCB 12 SUBSTITUTE SHEET (RULE 26) transporter protein is shown in Figure 1 and is set forth as SEQ ID NO: 1. The longest open reading frame of the human ABCB 12 transporter begins with the sequence GCCATGG which matches the consensus eukaryotic translation initiation motif at nucleotide position 165 (ATG) and ends with a TGA termination signal at position 2694. The coding region (open reading s frame) of SEQ >D NO: 1 is set forth as SEQ ID NO: 3. A putative polyadenylation signal (AATAAA) is located in the 3' untranslated region at nucleotide position 2844 (Fig. 1).
Starting from the first in-frame ATG, the open reading frame is predicted to encode a polypeptide of 843 amino acids with a molecular weight of 94 kDa. The amino acid sequence of the full length polypeptide is shown in Figure 2 and provided in SEQ ID NO:
2.

MOLECULES
In this example, the amino acid sequences of the human ABCB 12 transporter polypeptide was compared to amino acid sequences of known polypeptides and various motifs were identified.
In particular, by comparing the nucleic acid sequence (and predicted amino acid sequence) of the isolated clone to publicly available databases (e.g., GenBank), the isolated clone was determined to encode a novel human ABC transporter polypeptide (ABCB
12) with 2o homology (85% at the amino acid level (BLASTp program against non-redundant SwissProt sequences), 86% at the nucleotide level (BLASTn program against non-redundant GenBank +
EMBL + DDBJ + PDB sequences)) to the rat ubiquitously expressed mammalian ABC
half-transporter (UMAT) polypeptide (Accession No. AJ003004; Hirsch-Ernst et al., 1998, Biochent. Biophys. Res. Comm. 249:151-155). Accordingly, the novel human ABC
transporter 25 molecule of the invention (ABCB 12) is also referred to herein as the human UMAT
polypeptide.
The human ABCB12 transporter polypeptide of the invention belongs to the ATP-binding cassette (ABC) superfamily. Members of this family are structurally and functionally related in their ability to catalyze the translocation of substrates across biological membranes.
3o The deduced sequence of 843 amino acids comprises an N-terminal membrane anchor domain and a single conserved C-terminal nucleotide binding fold, specifying the human ABCB 12 polypeptide as an ABC half-transporter (see Fig. 2 and SEQ >D NO: 2). Using standard art recognized bioinformatic software, the novel human ABCB 12 transporter protein was determined to have no other immediate human homolog with > 34% identity (Pairwise Global 35 Clustal W Alignment). The closest human homolog is ABC7 (BLASTp program against non redundant SwissProt sequences). Applying the Pairwise Global Clustal W
Alignment reveals a 34% identity between the amino acid sequences of ABCB 12 and ABC7.

SUBSTITUTE SHEET (RULE 26) The ABCB 12 transporter polypeptide was also analyzed with respect to potential membrane spanning segments. Hydropathy plots and 2D fold models obtained using bioinformatic tools (PredictProtein server, Heidelberg, Germany, see, http://dodo.cpmc.columbia.edu/predictprotein/) defined ten putative membrane spanning regions in the N-terminal sequence of the polypeptide, six of them having a helical 2-D fold structure, which suggests that the human ABCB 12 transporter is a membrane anchored protein.
The N-terminal part of the protein (amino acids 1-550) represents a non-conserved feature of ABCB12; amino acids I-550 exhibit only 13% identity with ABC7, the closest human homolog to ABCB 12. However, the C-terminal sequence of ABCB 12 (amino acids 601-843) contains multiple clusters of conserved residues which define an ATP binding domain (see, e.g., Figs. 2 and 3).
Table 2. Percentage of amino acid similarity and identity between ABCB12 and the closest polypeptide orthologs (similarity/identity) using the Pairwise Global Alignment software program (MacVector 6.5) and standard parameter settings.
ABCB12 UMAT rat ATM1 yeast ABC7 HS HTM1 UMAT rat 93/88 100/100 ATM1 yeast44/29 45/29 100/100 HTM1 Sch 45/30 49/34 42/25 42/26 100/100 o Other features noted in the human ABCB 12 transporter were the presence of a Walker A motif, Walker B motif, and an ABC signature motif which implicates ATPase/ATP-binding 2o function (see Fig 2; Patel et al., 1998, Trends Cell Biol., 8:65-71).
Therefore, the human ABCB 12 transporter polypeptide exhibits the typical features conserved among ABC
transporters. ABC transporter proteins structural organization represents variations on common themes. One common theme is the basic structure of those transporters with twelve hydrophobic transmembrane segments and two hydrophilic ATP binding sites, either present in a single polypeptide chain or assembled from half or quarter molecules.
Therefore, the amino acid sequence of human ABCB 12 defines it as a novel hemitransporter. It is likely, but not necessary, that the human ABCB 12 transporter would function as pan of a dimeric transporter structure. To date, the few hemitransporters to be characterized in mammalian cells have been found to be localized to the membrane of 3o intracellular compartments. Art recognized bioinformatic modeling tools indicate that the N-terminal part as well as the ATP binding cassettes of the predicted protein is in an "outside"

SUBSTITUTE SHEET (RULE 26) W~ 01/14547 CA 02383074 2002-02-14 pCT/CA00/00964 conformation. Accordingly, it appears that human ABCB12 transporter can be associated with an intracellular membrane structure.

TISSUE DISTRIBUTION OF HUMAN ABCB12 TRANSPORTER mRNA
This example describes the tissue distribution of ABCB 12 transporter mRNA, as can be determined by Northern blot hybridization and irt situ hybridization.
Northern blot hybridizations with the various RNA samples are performed under standard conditions and washed under stringent conditions, i.e., 0.2XSSC at 65°C. The DNA
probe is radioactively labeled with 32P-dCTP using the Prime-It kit (Stratagene, La Jolla, CA) according to the instructions of the supplier. Filters containing human mRNA
(MultiTissue Northern I and MultiTissue Northern II from Clontech, Palo Alto, CA) are probed in ExpressHyb hybridization solution (Clontech) and washed at high stringency according to manufacturer's recommendations.
t5 For in .situ analysis, various tissues obtained from brains, e.g. rat or mouse brains, are first frozen on dry ice. Ten-micrometer-thick coronal sections of the tissues are postfixed with 4% formaldehyde in DEPC treated 1X phosphate- buffered saline at room temperature for 10 minutes before being rinsed twice in DEPC 1X phosphate-buffered saline and once in 0.1 M
triethanolamine-HCI (pH 8.0). Following incubation in 0.25% acetic anhydride-0.1 M
2o triethanolamine-HCI for 10 minutes, sections are rinsed in DEPC 2X SSC (1X
SSC is O.15M
NaCI plus O.O15M sodium citrate). Tissue is then dehydrated through a series of ethanol washes, incubated in 100% chloroform for 5 minutes, and then rinsed in 100%
ethanol for 1 minute and 95% ethanol for 1 minute and allowed to air dry.
Hybridizations are performed with 35S-radiolabeled (5 X 107 cpm/ml) cRNA
probes.
25 Probes are incubated in the presence of a solution containing 600 mM NaCI, 10 mM Tris (pH
7.5), 1 mM EDTA, 0.01% sheared salmon sperm DNA, 0.01% yeast tRNA, 0.05% yeast total RNA type X1, 1X Denhardt's solution, 50% formamide, 10% dextran sulfate, 100 mM
dithiothreitol, 0.1% sodium dodecyl sulfate (SDS), and 0.1% sodium thiosulfate for 18 hours at 55°C.
3o After hybridization, slides are washed with 2X SSC. Sections are then sequentially incubated at 37°C in THE (a solution containing 10 mM Tris-HC1 (pH
7.6), 500 mM NaCI, and 1 mM EDTA), for 10 minutes, in THE with 10 ~tg of RNase A per ml for 30 minutes, and finally in THE for 10 minutes. Slides are then rinsed with 2X SSC at room temperature, washed with 2X SSC at 50°C for 1 hour, washed with 0.2X SSC at 55°C for 1 hour, and 0.2X
35 SSC at 60°C for 1 hour. Sections are then dehydrated rapidly through serial ethanol-0.3 M
sodium acetate concentrations before being air dried and exposed to Kodak Biomax MR
scientific imaging film for 24 hours and subsequently dipped in NB-2 photoemulsion and SUBSTITUTE SHEET (RULE 26) exposed at 4°C for 7 days before being developed and counter stained.
Electronic Northern analysis has indicated that the ABCB 12 transporter mRNA is fairly neuron-specific.

POLYPEPTIDE IN BACTERIAL CELLS
In this example, human ABCB 12 transporter is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. Specifically, the human ABCB 12 transporter is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB 199. As the human ABCB
12 transporter polypeptide is predicted to be approximately 94 kDa and GST is predicted to be 26 kDa, the fusion polypeptide is predicted to be approximately 120 kDa in molecular weight. Expression of the GST-ABCB 12 transporter fusion protein in PEB 199 is induced with IPTG.
The recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB 199 ~s strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined. If desired, the polypeptide may be prepared in association with a membrane, such as, e.g., a micelle or membrane vesicle using art recognized techniques.

POLYPEPTIDE IN MAMMALIAN CELLS
To express the ABCB 12 transporter gene in a mammalian cell, e.g., COS cells, the 2s pcDNA/Amp vector by Invitrogen Corporation (San Diego, CA) is used. This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site.
A DNA fragment encoding the entire ABCB12 transporter protein and an HA tag (Wilson et al.
( 1984) Cell 37:767) or a FLAG tag fused in-frame to its 3' end of the fragment is cloned into 3o the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.
To construct the plasmid, the ABCB12 transporter DNA sequence (see, e.g., SEQ
ID
NO: 3) is amplified by PCR using two primers. The 5' primer contains the restriction site of interest followed by approximately twenty nucleotides of the ABCB 12 transporter coding ss sequence starting from the initiation codon; the 3' end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG
tag and the last 20 nucleotides of the ABCB 12 transporter coding sequence.
The PCR

SUBSTITUTE SHEET (RULE 26) amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, MA). Preferably the two restriction sites chosen are different so that the ABCB 12 transporter gene is inserted in the correct orientation. The ligation mixture is transformed into s E. coli cells (strains HB 101, DHSa, SURE, available from Stratagene Cloning Systems, La Jolla, CA, can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.
Eukaryotic cells, e.g., COS cells are subsequently transfected with the ABCB12 transporter-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran-mediated transfection, lipofection, or electroporation.
Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboraron~. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
The is expression of the ABCB12 transporter polypeptide is detected by radiolabelling (35S_ methionine or 35S-cysteine available from NEN, Boston, MA, can be used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988) using an HA specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with 35S-methionine (or 35S-cysteine). The 2o culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM
NaCI, 1 % NP-40, 0.1 % SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody.
Precipitated polypeptides are then analyzed by SDS-PAGE.
Alternatively, DNA containing the ABCB12 transporter coding sequence is cloned 2s directly into the polylinker of the pCDNA/Amp vector using the appropriate restriction sites.
The resulting plasmid is transfected into COS cells in the manner described above, and the expression of the ABCB 12 transporter polypeptide is detected by radiolabelling and immunoprecipitation using an ABCB12 transporter specific monoclonal antibody.
so EXAMPLE 6 IN VITRO AND IN VITRO SCREENING ASSAYS FOR MODULATORS OF A

In this example, in vitro and in vitro assays for screening modulators of the human ABCB 12 transporter molecule are described.
35 Briefly, the ABCB 12 transporter is expressed in a mammalian cell line as described above and a base line for the cellular transport of a reference molecule, e.g., ~i-amyloid or other small molecule, is established. Cells are then incubated with a test compound and a change in SUBSTITUTE SHEET (RULE 26) the level of the cellular transport of the reference molecule is determined using standards techniques. A test compound that changes the transport of the detectable reference molecule is then identified as a candidate compound that alters ABCB 12 transporter activity. Lead compounds may then be tested on eukaryotic cells, e.g., cells of neuronal origin or, s alternatively, on brain tissue from a test animal. Methods for conducting such an assay are known in the art. Preferably, lead test compounds that inhibit ABCB 12-mediated transport of, e.g., ~3-amyloid are further tested in vitro. Mouse models for various neurological diseases, e.g., amyloid disease, are also known in the art (see, e.g., Hardy et al., (1998) Science 282:1075-1079; Goate, et al., (1991) Nature 349: 704; Games, et al., (1995) Nature 373: 523;
and Suzuki, et al., (1994) Science 264: 1336).
Alternatively, a transgenic mouse model overexpressing an ABCB12 transporter polypeptide, e.g., the human ABCB 12 transporter polypeptide or corresponding mucine polypeptide is generated using the techniques described herein. This animal may then be tested directly or used as a cell or tissue source, e.g., brain tissue source, for testing modulators of the ~5 human ABCB12 transporter polypeptide. Preferably, the animal is directly tested with a test compound and monitored for a physiological result, e.g.., presence of (3-amyloid in the cerebral spinal fluid (CSF). In a related extension of this in vitro assay, the animal may be breed with an animal overexpressing a undesired polypeptide, e.g., an amyloid polypeptide, and a change in the transport of the polypeptide is measured (e.g., in the CSF) as compared to a control.
2o Ideally, these test animals are further monitored in the presence or absence of a modulator of the ABCB 12 transporter.
In a modification of the assay, the activity of the ABCB 12 transporter is determined regarding the ability of a detectable reference molecule to cross the blood-brain barrier. In yet another modification of the assay, the animal is examined for the prevalence of multidrug 2s resistant cells or tissues, and/or the sensitivity of the animal to cytotoxic drugs suitable for treating a neoplasm.
Accordingly, these animals represent in vivo assay systems in which to test the ability of an ABCB 12 transporter and/or ABCB 12 transporter modulator to prevent, treat, or delay the onset of disease.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

SUBSTITUTE SHEET (RULE 26) SEQUENCE LISTING
<110> Stephane Le Bihan and Catriona Wilson <120> NOVEL ABC TRANSPORTER AND USES THEREOF
<130> APZ-001 <140>
<141>
<150> 60/161,724 <151> 1999-OS-20 <150> 60/151,473 <151> 1999-08-30 <160> 3 <170> PatentIn Ver. 2.0 <210> 1 <211> 2893 <212> DNA
<213> Homo sapiens <220>
<221> CDS
<222> (165)..(2696) <400> 1 cggtagggct ggtccaagtg cgtcccttcc cgggaccccc gcagcttggc gcccagcggc 60 tacgtgagcc aaggcacccg gatgtccgcg cccctctccg agtgacaagt cccggcctcc 120 ggtcccgcag tgcccgcagc ctcggccggc gtccacgcat tgcc atg gtg act gtg 176 Met Val Thr Val ggc aac tac tgc gag gcc gaa ggg ccc gtg ggt ccg gcc tgg atg cag 224 Gly Asn Tyr Cys Glu Ala Glu Gly Pro Val Gly Pro Ala Trp Met Gln gat ggc ctg agt ccc tgc ttc ttc ttc acg ctc gtg ccc tcg acg cgg 272 Asp Gly Leu Ser Pro Cys Phe Phe Phe Thr Leu Val Pro Ser Thr Arg atg get ctg ggg act ctg gcc ttg gtg ctg get ctt ccc tgc aga cgc 320 Met Ala Leu Gly Thr Leu Ala Leu Val Leu Ala Leu Pro Cys Arg Arg SUBSTITUTE SHEET (RULE 26) cgg gag cgg ccc get ggt get gat tcg ctg tct tgg ggg gcc ggc cct 368 Arg Glu Arg Pro Ala Gly Ala Asp Ser Leu Ser Trp Gly Ala Gly Pro cgc atc tct ccc tac gtg ctg cag ctg ctt ctg gcc aca ctt cag gcg 416 Arg Ile Ser Pro Tyr Val Leu Gln Leu Leu Leu Ala Thr Leu Gln Ala gcg ctg ccc ctg gcc ggc ctg get ggc cgg gtg ggc act gcc cgg ggg 464 Ala Leu Pro Leu Ala Gly Leu Ala Gly Arg Val Gly Thr Ala Arg Gly gcc cca ctg cca agc tat cta ctt ctg gcc tcc gtg ctg gag agt ctg 512 Ala Pro Leu Pro Ser Tyr Leu Leu Leu Ala Ser Val Leu Glu Ser Leu gcc ggc gcc tgt ggc ctg tgg ctg ctt gtc gtg gag cgg agc cag gca 560 Ala Gly Ala Cys Gly Leu Trp Leu Leu Val Val Glu Arg Ser Gln Ala cgg cag cgt ctg gca atg ggc atc tgg atc aag ttc agg cac agc cct 608 Arg Gln Arg Leu Ala Met Gly Ile Trp Ile Lys Phe Arg His Ser Pro ggt ctc ctg ctc ctc tgg act gtg gcg ttt gca get gag aac ttg gcc 656 Gly Leu Leu Leu Leu Trp Thr Val Ala Phe Ala Ala Glu Asn Leu Ala ctg gtg tct tgg aac agc cca cag tgg tgg tgg gca agg gca gac ttg 704 Leu Val Ser Trp Asn Ser Pro Gln Trp Trp Trp Ala Arg Ala Asp Leu ggc cag cag gtt cag ttt agc ctg tgg gtg ctg cgg tat gtg gtc tct 752 Gly Gln Gln Val Gln Phe Ser Leu Trp Val Leu Arg Tyr Val Val Ser gga ggg ctg ttt gtc ctg ggt ctc tgg gcc cct gga ctt cgt ccc cag 800 Gly Gly Leu Phe Val Leu Gly Leu Trp Ala Pro Gly Leu Arg Pro Gln tcc tat aca ttg cag gtt cat gaa gag gac caa gat gtg gaa agg agc 848 Ser Tyr Thr Leu Gln Val His Glu Glu Asp Gln Asp Val Glu Arg Ser cag gtt cgg tca gca gcc caa cag tct acc tgg cga gat ttt ggc agg 896 Gln Val Arg Ser Ala Ala Gln Gln Ser Thr Trp Arg Asp Phe Gly Arg aag ctc cgc ctc ctg agt ggc tac ctg tgg cct cga ggg agt cca get 944 Lys Leu Arg Leu Leu Ser Gly Tyr Leu Trp Pro Arg Gly Ser Pro Ala ctg cag ctg gtg gtg ctc atc tgc ctg ggg ctc atg ggt ttg gaa cgg 992 Leu Gln Leu Val Val Leu Ile Cys Leu Gly Leu Met Gly Leu Glu Arg SUBSTITUTE SHEET (RULE 26) gca ctc aat gtg ttg gtg cct ata ttc tat agg gac att gtg aac ttg 1040 Ala Leu Asn Val Leu Val Pro Ile Phe Tyr Arg Asp Ile Val Asn Leu ctg act gag aag gca cct tgg aac tct ctg gcc tgg act gtt acc act 1088 Leu Thr Glu Lys Ala Pro Trp Asn Ser Leu Ala Trp Thr Val Thr Thr tat gtc ttc ctc aag ttc ctt cag ggg ggt ggc act ggc agt aca ggc 1136 Tyr Val Phe Leu Lys Phe Leu Gln Gly Gly Gly Thr Gly Ser Thr Gly ttc gtg agc aac ctg cgc acc ttc ctg tgg atc cgg gtg cag cag ttc 1184 Phe Val Ser Asn Leu Arg Thr Phe Leu Trp Ile Arg Val Gln Gln Phe acg tct cgg cgg gtg gag ctg ctc atc ttc tcc cac ctg cac gag ctc 1232 Thr Ser Arg Arg Val Glu Leu Leu Ile Phe Ser His Leu His Glu Leu tca ctg cgc tgg cac ctg ggg cgc cgc aca ggg gag gtg ctg cgg atc 1280 Ser Leu Arg Trp His Leu Gly Arg Arg Thr Gly Glu Val Leu Arg Ile gcg gat cgg ggc aca tcc agt gtc aca ggg ctg ctc agc tac ctg gtg 1328 Ala Asp Arg Gly Thr Ser Ser Val Thr Gly Leu Leu Ser Tyr Leu Val ttc aat gtc atc ccc acg ctg gcc gac atc atc att ggc atc atc tac 1376 Phe Asn Val Ile Pro Thr Leu Ala Asp Ile Ile Ile Gly Ile Ile Tyr ttc agc atg ttc ttc aac gcc tgg ttt ggc ctc att gtg ttc ctg tgc 1424 Phe Ser Met Phe Phe Asn Ala Trp Phe Gly Leu Ile Val Phe Leu Cys atg agt ctt tac ctc acc ctg acc att gtg gtc act gag tgg aga acc 1472 Met Ser Leu Tyr Leu Thr Leu Thr Ile Val Val Thr Glu Trp Arg Thr aag ttt cgt cgt get atg aac aca cag gag aac get acc cgg gca cga 1520 Lys Phe Arg Arg Ala Met Asn Thr Gln Glu Asn Ala Thr Arg Ala Arg gca gtg gac tct ctg cta aac ttc gag acg gtg aag tat tac aac gcc 1568 Ala Val Asp Ser Leu Leu Asn Phe Glu Thr Val Lys Tyr Tyr Asn Ala gag agt tac gaa gtg gaa cgc tat cga gag gcc atc atc aaa tat cag 1616 Glu Ser Tyr Glu Val Glu Arg Tyr Arg Glu Ala Ile Ile Lys Tyr Gln ggt ttg gag tgg aag tcg agc get tca ctg gtt tta cta aat cag acc 1664 Gly Leu Glu Trp Lys Ser Ser Ala Ser Leu Val Leu Leu Asn Gln Thr SUBSTITUTE SHEET (RULE 26) cag aac ctg gtg att ggg ctc ggg ctc ctc gcc ggc tcc ctg ctt tgc 1712 Gln Asn Leu Val Ile Gly Leu Gly Leu Leu Ala Gly Ser Leu Leu Cys gca tac ttt gtc act gag cag aag cta cag gtt ggg gac tat gtg ctc 1760 Ala Tyr Phe Val Thr Glu Gln Lys Leu Gln Val Gly Asp Tyr Val Leu ttt ggc acc tac att atc cag ctg tac atg ccc ctc aat tgg ttt ggc 1808 Phe Gly Thr Tyr Ile Ile Gln Leu Tyr Met Pro Leu Asn Trp Phe Gly acc tac tac agg atg atc cag acc aac ttc att gac atg gag aac atg 1856 Thr Tyr Tyr Arg Met Ile Gln Thr Asn Phe Ile Asp Met Glu Asn Met ttt gac ttg ctg aaa gag gag aca gaa gtg aag gac ctt cct gga gca 1904 Phe Asp Leu Leu Lys Glu Glu Thr Glu Val Lys Asp Leu Pro Gly Ala ggg ccc ctt cgc ttt cag aag ggc cgt att gag ttt gag aac gtg cac 1952 Gly Pro Leu Arg Phe Gln Lys Gly Arg Ile Glu Phe Glu Asn Val His ttc agc tat gcc gat ggg cgg gag act ctg cag gac gtg tct ttc act 2000 Phe Ser Tyr Ala Asp Gly Arg Glu Thr Leu Gln Asp Val Ser Phe Thr gtg atg cct gga cag aca ctt gcc ctg gtg ggc cca tct ggg gca ggg 2048 Val Met Pro Gly Gln Thr Leu Ala Leu Val Gly Pro Ser Gly Ala Gly aag agc aca att ttg cgc ctg ctg ttt cgc ttc tac gac atc agc tct 2096 Lys Ser Thr Ile Leu Arg Leu Leu Phe Arg Phe Tyr Asp Ile Ser Ser ggc tgc atc cga ata gat ggg cag gac att tca cag gtc acc cag gcc 2144 Gly Cys Ile Arg Ile Asp Gly Gln Asp Ile Ser Gln Val Thr Gln Ala tct ctc cgg tct cac att gga gtt gtg ccc caa gac act gtc ctc ttt 2192 Ser Leu Arg Ser His Ile Gly Val Val Pro Gln Asp Thr Val Leu Phe aat gac acc atc gcc gaa aat atc cgt tac ggc cgt gtc aca get ggg 2240 Asn Asp Thr Ile Ala Glu Asn Ile Arg Tyr Gly Arg Val Thr Ala Gly aat gat gag gtg gag get get get cag get gca ggc atc cat gat gcc 2288 Asn Asp Glu Val Glu Ala Ala Ala Gln Ala Ala Gly Ile His Asp Ala att atg get ttc cct gaa ggg tac agg aca cag gtg ggc gag cgg gga 2336 Ile Met Ala Phe Pro Glu Gly Tyr Arg Thr Gln Val Gly Glu Arg Gly SUBSTITUTE SHEET (RULE 26) ctg aag ctg agc ggc ggg gag aag cag ccc gtc gcc att gcc cgc acc 2384 Leu Lys Leu Ser Gly Gly Glu Lys Gln Pro Val Ala Ile Ala Arg Thr atc ctc aag get ccg ggc atc att ctg ctg gat gag gca acg tca gcg 2432 Ile Leu Lys Ala Pro Gly Ile Ile Leu Leu Asp Glu Ala Thr Ser Ala ctg gat aca tct aat gag agg gcc atc cag get tct ctg gcc aaa gtc 2480 Leu Asp Thr Ser Asn Glu Arg Ala Ile Gln Ala Ser Leu Ala Lys Val tgt gcc aac cgc acc acc atc gta gtg gca cac agg ctc tca act gtg 2528 Cys Ala Asn Arg Thr Thr Ile Val Val Ala His Arg Leu Ser Thr Val gtc aat get gac cag atc ctc gtc atc aag gat ggc tgc atc gtg gag 2576 Val Asn Ala Asp Gln Ile Leu Val Ile Lys Asp Gly Cys Ile Val Glu agg gga cga cac gag get ctg ttg tcc cga ggt ggg gtg tat get gac 2624 Arg Gly Arg His Glu Ala Leu Leu Ser Arg Gly Gly Val Tyr Ala Asp atg tgg cag ctg cag cag caa gga cag gaa gaa acc tct gaa gac act 2672 Met Trp Gln Leu Gln Gln Gln Gly Gln Glu Glu Thr Ser Glu Asp Thr aag cct cag acc atg gaa cgg tga caa aag ttt ggc cac ttc cct ctc 2720 Lys Pro Gln Thr Met Glu Arg aaa gac taa ccc aga agg gaa taa gatgtgtctc ctttccctgg cttatttcat 2774 Lys Asp cctggtcttg gggtatggtg ctagctatgg taagggaaag ggacctttcc gaaaaacatc 2834 ttttggggaa ataaaaatgt ggactgtgaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 2893 <210> 2 <211> 843 <212> PRT
<213> Homo sapiens <400> 2 Met Val Thr Val Gly Asn Tyr Cys Glu Ala Glu Gly Pro Val Gly Pro Ala Trp Met Gln Asp Gly Leu Ser Pro Cys Phe Phe Phe Thr Leu Val SUBSTITUTE SHEET (RULE 26) Pro Ser Thr Arg Met Ala Leu Gly Thr Leu Ala Leu Val Leu Ala Leu Pro Cys Arg Arg Arg Glu Arg Pro Ala Gly Ala Asp Ser Leu Ser Trp Gly Ala Gly Pro Arg Ile Ser Pro Tyr Val Leu Gln Leu Leu Leu Ala Thr Leu Gln Ala Ala Leu Pro Leu Ala Gly Leu Ala Gly Arg Val Gly Thr Ala Arg Gly Ala Pro Leu Pro Ser Tyr Leu Leu Leu Ala Ser Val Leu Glu Ser Leu Ala Gly Ala Cys Gly Leu Trp Leu Leu Val Val Glu Arg Ser Gln Ala Arg Gln Arg Leu Ala Met Gly Ile Trp Ile Lys Phe Arg His Ser Pro Gly Leu Leu Leu Leu Trp Thr Val Ala Phe Ala Ala Glu Asn Leu Ala Leu Val Ser Trp Asn Ser Pro Gln Trp Trp Trp Ala Arg Ala Asp Leu Gly Gln Gln Val Gln Phe Ser Leu Trp Val Leu Arg Tyr Val Val Ser Gly Gly Leu Phe Val Leu Gly Leu Trp Ala Pro Gly Leu Arg Pro Gln Ser Tyr Thr Leu Gln Val His Glu Glu Asp Gln Asp Val Glu Arg Ser Gln Val Arg Ser Ala Ala Gln Gln Ser Thr Trp Arg Asp Phe Gly Arg Lys Leu Arg Leu Leu Ser Gly Tyr Leu Trp Pro Arg Gly Ser Pro Ala Leu Gln Leu Val Val Leu Ile Cys Leu Gly Leu Met Gly Leu Glu Arg Ala Leu Asn Val Leu Val Pro Ile Phe Tyr Arg Asp Ile Val Asn Leu Leu Thr Glu Lys Ala Pro Trp Asn Ser Leu Ala Trp Thr Val Thr Thr Tyr Val Phe Leu Lys Phe Leu Gln Gly Gly Gly Thr SUBSTITUTE SHEET (RULE 26) Gly Ser Thr Gly Phe Val Ser Asn Leu Arg Thr Phe Leu Trp Ile Arg Val Gln Gln Phe Thr Ser Arg Arg Val Glu Leu Leu Ile Phe Ser His Leu His Glu Leu Ser Leu Arg Trp His Leu Gly Arg Arg Thr Gly Glu Val Leu Arg Ile Ala Asp Arg Gly Thr Ser Ser Val Thr Gly Leu Leu Ser Tyr Leu Val Phe Asn Val Ile Pro Thr Leu Ala Asp Ile Ile Ile Gly Ile Ile Tyr Phe Ser Met Phe Phe Asn Ala Trp Phe Gly Leu Ile Val Phe Leu Cys Met Ser Leu Tyr Leu Thr Leu Thr Ile Val Val Thr Glu Trp Arg Thr Lys Phe Arg Arg Ala Met Asn Thr Gln Glu Asn Ala Thr Arg Ala Arg Ala Val Asp Ser Leu Leu Asn Phe Glu Thr Val Lys Tyr Tyr Asn Ala Glu Ser Tyr Glu Val Glu Arg Tyr Arg Glu Ala Ile Ile Lys Tyr Gln Gly Leu Glu Trp Lys Ser Ser Ala Ser Leu Val Leu Leu Asn Gln Thr Gln Asn Leu Val Ile Gly Leu Gly Leu Leu Ala Gly Ser Leu Leu Cys Ala Tyr Phe Val Thr Glu Gln Lys Leu Gln Val Gly Asp Tyr Val Leu Phe Gly Thr Tyr Ile Ile Gln Leu Tyr Met Pro Leu Asn Trp Phe Gly Thr Tyr Tyr Arg Met Ile Gln Thr Asn Phe Ile Asp Met Glu Asn Met Phe Asp Leu Leu Lys Glu Glu Thr Glu Va1 Lys Asp Leu Pro Gly Ala Gly Pro Leu Arg Phe Gln Lys Gly Arg Ile Glu Phe Glu Asn Val His Phe Ser Tyr Ala Asp Gly Arg Glu Thr Leu Gln Asp SUBSTITUTE SHEET (RULE 26) Val Ser Phe Thr Val Met Pro Gly Gln Thr Leu Ala Leu Val Gly Pro Ser Gly Ala Gly Lys Ser Thr Ile Leu Arg Leu Leu Phe Arg Phe Tyr Asp Ile Ser Ser Gly Cys Ile Arg Ile Asp Gly Gln Asp Ile Ser Gln Val Thr Gln Ala Ser Leu Arg Ser His Ile Gly Val Val Pro Gln Asp Thr Val Leu Phe Asn Asp Thr Ile Ala Glu Asn Ile Arg Tyr Gly Arg Val Thr Ala Gly Asn Asp Glu Val Glu Ala Ala Ala Gln Ala Ala Gly Ile His Asp Ala Ile Met Ala Phe Pro Glu Gly Tyr Arg Thr Gln Val Gly Glu Arg Gly Leu Lys Leu Ser Gly Gly Glu Lys Gln Pro Val Ala Ile Ala Arg Thr Ile Leu Lys Ala Pro Gly Ile Ile Leu Leu Asp Glu Ala Thr Ser Ala Leu Asp Thr Ser Asn Glu Arg Ala Ile Gln Ala Ser Leu Ala Lys Val Cys Ala Asn Arg Thr Thr Ile Val Val Ala His Arg Leu Ser Thr Val Val Asn Ala Asp Gln Ile Leu Val Ile Lys Asp Gly Cys Ile Val Glu Arg Gly Arg His Glu Ala Leu Leu Ser Arg Gly Gly Val Tyr Ala Asp Met Trp Gln Leu Gln Gln Gln Gly Gln Glu Glu Thr Ser Glu Asp Thr Lys Pro Gln Thr Met Glu Arg <210> 3 <211> 2532 <212> DNA
<213> Homo sapiens <220>
<221> CDS
<222> (1)..(2532) SUBSTITUTE SHEET (RULE 26) <400> 3 atg gtg act gtg ggc aac tac tgc gag gcc gaa ggg ccc gtg ggt ccg 48 Met Val Thr Val Gly Asn Tyr Cys Glu Ala Glu Gly Pro Val Gly Pro gcc tgg atg cag gat ggc ctg agt ccc tgc ttc ttc ttc acg ctc gtg 96 Ala Trp Met Gln Asp Gly Leu Ser Pro Cys Phe Phe Phe Thr Leu Val ccc tcg acg cgg atg get ctg ggg act ctg gcc ttg gtg ctg get ctt 144 Pro Ser Thr Arg Met Ala Leu Gly Thr Leu Ala Leu Val Leu Ala Leu ccc tgc aga cgc cgg gag cgg ccc get ggt get gat tcg ctg tct tgg 192 Pro Cys Arg Arg Arg Glu Arg Pro Ala Gly Ala Asp Ser Leu Ser Trp ggg gcc ggc cct cgc atc tct ccc tac gtg ctg cag ctg ctt ctg gcc 240 Gly Ala Gly Pro Arg Ile Ser Pro Tyr Val Leu Gln Leu Leu Leu Ala aca ctt cag gcg gcg ctg ccc ctg gcc ggc ctg get ggc cgg gtg ggc 288 Thr Leu Gln Ala Ala Leu Pro Leu Ala Gly Leu Ala Gly Arg Val Gly act gcc cgg ggg gcc cca ctg cca agc tat cta ctt ctg gcc tcc gtg 336 Thr Ala Arg Gly Ala Pro Leu Pro Ser Tyr Leu Leu Leu Ala Ser Val ctg gag agt ctg gcc ggc gcc tgt ggc ctg tgg ctg ctt gtc gtg gag 384 Leu Glu Ser Leu Ala Gly Ala Cys Gly Leu Trp Leu Leu Val Val Glu cgg agc cag gca cgg cag cgt ctg gca atg ggc atc tgg atc aag ttc 432 Arg Ser Gln Ala Arg Gln Arg Leu Ala Met Gly Ile Trp Ile Lys Phe agg cac agc cct ggt ctc ctg ctc ctc tgg act gtg gcg ttt gca get 480 Arg His Ser Pro Gly Leu Leu Leu Leu Trp Thr Val Ala Phe Ala Ala gag aac ttg gcc ctg gtg tct tgg aac agc cca cag tgg tgg tgg gca 528 Glu Asn Leu Ala Leu Val Ser Trp Asn Ser Pro Gln Trp Trp Trp Ala agg gca gac ttg ggc cag cag gtt cag ttt agc ctg tgg gtg ctg cgg 576 Arg Ala Asp Leu Gly Gln Gln Val Gln Phe Ser Leu Trp Val Leu Arg tat gtg gtc tct gga ggg ctg ttt gtc ctg ggt ctc tgg gcc cct gga 624 Tyr Val Val Ser Gly Gly Leu Phe Val Leu Gly Leu Trp Ala Pro Gly SUBSTITUTE SHEET (RULE 26) ctt cgt.ccc cag tcc tat aca ttg cag gtt cat gaa gag gac caa gat 672 Leu Arg Pro Gln Ser Tyr Thr Leu Gln Val His Glu Glu Asp Gln Asp gtg gaa agg agc cag gtt cgg tca gca gcc caa cag tct acc tgg cga 720 Val Glu Arg Ser Gln Val Arg Ser Ala Ala Gln Gln Ser Thr Trp Arg gat ttt ggc agg aag ctc cgc ctc ctg agt ggc tac ctg tgg cct cga 768 Asp Phe Gly Arg Lys Leu Arg Leu Leu Ser Gly Tyr Leu Trp Pro Arg ggg agt cca get ctg cag ctg gtg gtg ctc atc tgc ctg ggg ctc atg 816 Gly Ser Pro Ala Leu Gln Leu Val Val Leu Ile Cys Leu Gly Leu Met ggt ttg gaa cgg gca ctc aat gtg ttg gtg cct ata ttc tat agg gac 864 Gly Leu Glu Arg Ala Leu Asn Val Leu Val Pro Ile Phe Tyr Arg Asp att gtg aac ttg ctg act gag aag gca cct tgg aac tct ctg gcc tgg 912 Ile Val Asn Leu Leu Thr Glu Lys Ala Pro Trp Asn Ser Leu Ala Trp act gtt acc act tat gtc ttc ctc aag ttc ctt cag ggg ggt ggc act 960 Thr Val Thr Thr Tyr Val Phe Leu Lys Phe Leu Gln Gly Gly Gly Thr ggc agt aca ggc ttc gtg agc aac ctg cgc acc ttc ctg tgg atc cgg 1008 Gly Ser Thr Gly Phe Val Ser Asn Leu Arg Thr Phe Leu Trp Ile Arg gtg cag cag ttc acg tct cgg cgg gtg gag ctg ctc atc ttc tcc cac 1056 Val Gln Gln Phe Thr Ser Arg Arg Val Glu Leu Leu Ile Phe Ser His ctg cac gag ctc tca ctg cgc tgg cac ctg ggg cgc cgc aca ggg gag 1104 Leu His Glu Leu Ser Leu Arg Trp His Leu Gly Arg Arg Thr Gly Glu gtg ctg cgg atc gcg gat cgg ggc aca tcc agt gtc aca ggg ctg ctc 1152 Val Leu Arg Ile Ala Asp Arg Gly Thr Ser Ser Val Thr Gly Leu Leu agc tac ctg gtg ttc aat gtc atc ccc acg ctg gcc gac atc atc att 1200 Ser Tyr Leu Val Phe Asn Val Ile Pro Thr Leu Ala Asp Ile Ile Ile ggc atc atc tac ttc agc atg ttc ttc aac gcc tgg ttt ggc ctc att 1248 Gly Ile Ile Tyr Phe Ser Met Phe Phe Asn Ala Trp Phe Gly Leu Ile gtg ttc ctg tgc atg agt ctt tac ctc acc ctg acc att gtg gtc act 1296 Val Phe Leu Cys Met Ser Leu Tyr Leu Thr Leu Thr Ile Val Val Thr SUBSTITUTE SHEET (RULE 26) gag tgg aga acc aag ttt cgt cgt get atg aac aca cag gag aac get 1344 Glu Trp Arg Thr Lys Phe Arg Arg Ala Met Asn Thr Gln Glu Asn Ala acc cgg gca cga gca gtg gac tct ctg cta aac ttc gag acg gtg aag 1392 Thr Arg Ala Arg Ala Val Asp Ser Leu Leu Asn Phe Glu Thr Val Lys tat tac aac gcc gag agt tac gaa gtg gaa cgc tat cga gag gcc atc 1440 Tyr Tyr Asn Ala Glu Ser Tyr Glu Val Glu Arg Tyr Arg Glu Ala Ile atc aaa tat cag ggt ttg gag tgg aag tcg agc get tca ctg gtt tta 1488 Ile Lys Tyr Gln Gly Leu Glu Trp Lys Ser Ser Ala Ser Leu Val Leu cta aat cag acc cag aac ctg gtg att ggg ctc ggg ctc ctc gcc ggc 1536 Leu Asn Gln Thr Gln Asn Leu Val Ile Gly Leu Gly Leu Leu Ala Gly tcc ctg ctt tgc gca tac ttt gtc act gag cag aag cta cag gtt ggg 1584 Ser Leu Leu Cys Ala Tyr Phe Val Thr Glu Gln Lys Leu Gln Val Gly gac tat gtg ctc ttt ggc acc tac att atc cag ctg tac atg ccc ctc 1632 Asp Tyr Val Leu Phe Gly Thr Tyr Ile Ile Gln Leu Tyr Met Pro Leu aat tgg ttt ggc acc tac tac agg atg atc cag acc aac ttc att gac 1680 Asn Trp Phe Gly Thr Tyr Tyr Arg Met Ile Gln Thr Asn Phe Ile Asp atg gag aac atg ttt gac ttg ctg aaa gag gag aca gaa gtg aag gac 1728 Met Glu Asn Met Phe Asp Leu Leu Lys Glu Glu Thr Glu Val Lys Asp ctt cct gga gca ggg ccc ctt cgc ttt cag aag ggc cgt att gag ttt 1776 Leu Pro Gly Ala Gly Pro Leu Arg Phe Gln Lys Gly Arg Ile Glu Phe gag aac gtg cac ttc agc tat gcc gat ggg cgg gag act ctg cag gac 1824 Glu Asn Val His Phe Ser Tyr Ala Asp Gly Arg Glu Thr Leu Gln Asp gtg tct ttc act gtg atg cct gga cag aca ctt gcc ctg gtg ggc cca 1872 Val Ser Phe Thr Val Met Pro Gly Gln Thr Leu Ala Leu Val Gly Pro tct ggg gca ggg aag agc aca att ttg cgc ctg ctg ttt cgc ttc tac 1920 Ser Gly Ala Gly Lys Ser Thr Ile Leu Arg Leu Leu Phe Arg Phe Tyr gac atc agc tct ggc tgc atc cga ata gat ggg cag gac att tca cag 1968 Asp Ile Ser Ser Gly Cys Ile Arg Ile Asp Gly Gln Asp Ile Ser Gln SUBSTITUTE SHEET (RULE 26) gtc acc cag gcc tct ctc cgg tct cac att gga gtt gtg ccc caa gac 2016 Val Thr Gln Ala Ser Leu Arg Ser His Ile Gly Val Val Pro Gln Asp act gtc ctc ttt aat gac acc atc gcc gaa aat atc cgt tac ggc cgt 2064 Thr Val Leu Phe Asn Asp Thr Ile Ala Glu Asn Ile Arg Tyr Gly Arg gtc aca get ggg aat gat gag gtg gag get get get cag get gca ggc 2112 Val Thr Ala Gly Asn Asp Glu Val Glu Ala Ala Ala Gln Ala Ala Gly atc cat gat gcc att atg get ttc cct gaa ggg tac agg aca cag gtg 2160 Ile His Asp Ala Ile Met Ala Phe Pro Glu Gly Tyr Arg Thr Gln Val ggc gag cgg gga ctg aag ctg agc ggc ggg gag aag cag ccc gtc gcc 2208 Gly Glu Arg Gly Leu Lys Leu Ser Gly Gly Glu Lys Gln Pro Val Ala att gcc cgc acc atc ctc aag get ccg ggc atc att ctg ctg gat gag 2256 Ile Ala Arg Thr Ile Leu Lys Ala Pro Gly Ile Ile Leu Leu Asp Glu gca acg tca gcg ctg gat aca tct aat gag agg gcc atc cag get tct 2304 Ala Thr Ser Ala Leu Asp.Thr Ser Asn Glu Arg Ala Ile Gln Ala Ser ctg gcc aaa gtc tgt gcc aac cgc acc acc atc gta gtg gca cac agg 2352 Leu Ala Lys Val Cys Ala Asn Arg Thr Thr Ile Val Val Ala His Arg ctc tca act gtg gtc aat get gac cag atc ctc gtc atc aag gat ggc 2400 Leu Ser Thr Val Val Asn Ala Asp Gln Ile Leu Val Ile Lys Asp Gly tgc atc gtg gag agg gga cga cac gag get ctg ttg tcc cga ggt ggg 2448 Cys Ile Val Glu Arg Gly Arg His Glu Ala Leu Leu Ser Arg Gly Gly gtg tat get gac atg tgg cag ctg cag cag caa gga cag gaa gaa acc 2496 Val Tyr Ala Asp Met Trp Gln Leu Gln Gln Gln Gly Gln Glu Glu Thr tct gaa gac act aag cct cag acc atg gaa cgg tga 2532 Ser Glu Asp Thr Lys Pro Gln Thr Met Glu Arg SUBSTITUTE SHEET (RULE 26)

Claims (25)

What is claimed is:

1. An isolated nucleic acid molecule selected from the group consisting of:
a) a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 1, or a complement thereof; and b) a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 3, or a complement thereof.

2. An isolated nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2.

3. An isolated nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence set forth in SEQ
ID NO: 2.

4. An isolated nucleic acid molecule selected from the group consisting of:
a) a nucleic acid molecule comprising a nucleotide sequence which is at least 87% homologous to the nucleotide sequence of SEQ ID NO: 1 or 3, or a complement thereof;
b) a nucleic acid molecule comprising a fragment of at least 150 nucleotides of a nucleic acid comprising the nucleotide sequence of SEQ ID NO:
1 or 3, or a complement thereof;
c) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least about 86% homologous to the amino acid sequence of SEQ ID
NO: 2: and d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, wherein the fragment comprises at least 50 contiguous amino acid residues of the amino acid sequence of SEQ ID NO: 2.

5. An isolated nucleic acid molecule which hybridizes to the nucleic acid molecule of any one of claims 1, 2, 3, or 4 under stringent conditions.

6. An isolated nucleic acid molecule comprising a nucleotide sequence which is complementary to the nucleotide sequence of the nucleic acid molecule of any one of claims 1, 2, 3, or 4.

7. An isolated nucleic acid molecule comprising the nucleic acid molecule of any one of claims 1, 2, 3, or 4, and a nucleotide sequence encoding a heterologous polypeptide.

8. A vector comprising the nucleic acid molecule of any one of claims 1, 2, 3, or 4.

9. The vector of claim 8, which is an expression vector.

10. A host cell transfected with the expression vector of claim 9.

11. A method of producing a polypeptide comprising culturing the host cell of claim in an appropriate culture medium to, thereby, produce the polypeptide.

12. An isolated polypeptide selected from the group consisting of:
a) a fragment of a polypeptide comprising the amino acid sequence of SEQ
ID NO: 2, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:
2;
b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule consisting of SEQ ID NO:
1 or 3 under stringent conditions;
c) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 50% homologous to a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 or 3;
d) a polypeptide comprising an amino acid sequence which is at least 50%
homologous to the amino acid sequence of SEQ ID NO: 2.

13. The isolated polypeptide of claim 12 comprising the amino acid sequence of SEQ ID NO: 2.

14. The polypeptide of claim 12, further comprising heterologous amino acid sequences.

15. An antibody which selectively binds to a polypeptide of claim 12.

16. A method for detecting the presence of a polypeptide of claim 12 in a sample comprising:

a) contacting the sample with a compound which selectively binds to the polypeptide: and b) determining whether the compound binds to the polypeptide in the sample to thereby detect the presence of a polypeptide of claim 12 in the sample.

17. The method of claim 16, wherein the compound which binds to the polypeptide is an antibody.

18. A kit comprising a compound which selectively binds to a polypeptide of claim 12 and instructions for use.

19. A method for detecting the presence of a nucleic acid molecule of any one of claims 1, 2, 3, or 4 in a sample comprising:
a) contacting the sample with a nucleic acid probe or primer which selectively hybridizes to the nucleic acid molecule; and b) determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample to thereby detect the presence of a nucleic acid molecule of any one of claims 1, 2, 3, or 4 in the sample.

20. The method of claim 19, wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe.

21. A kit comprising a compound which selectively hybridizes to a nucleic acid molecule of any one of claims 1, 2, 3, or 4 and instructions for use.

22. A method for identifying a compound which binds to a polypeptide of claim comprising:
a) contacting the polypeptide, or a cell expressing the polypeptide with a test compound; and b) determining whether the polypeptide binds to the test compound.

23. The method of claim 22, wherein the binding of the test compound to the polypeptide is detected by a method selected from the group consisting of:
a) detection of binding by direct detection of test compound/polypeptide binding;
b) detection of binding using a competition binding assay; and c) detection of binding using an assay for ABCB12 transporter activity.

24. A method for modulating the activity of a polypeptide of claim 12 comprising contacting the polypeptide or a cell expressing the polypeptide with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity of the polypeptide.

25. A method for identifying a compound which modulates the activity of a polypeptide of claim 12 comprising:
a) contacting a polypeptide of claim 12 with a test compound; and b) determining the effect of the test compound on the activity of the polypeptide to thereby identify a compound which modulates the activity of the polypeptide.