AU774697B2 - DNA encoding the prostate-specific membrane antigen-like gene and uses thereof - Google Patents

Description

DNA ENCODING THE PROSTATE-SPECIFIC MEMBRANE ANTIGEN-LIKE GENE AND USES THEREOF BACKGROUND OF THE INVENTION Cross-reference to Related Application This patent application is related to provisional patent application U.S. Serial number 60/128,839, filed April 9, 1999.

Field of the Invention The present invention relates generally to the field of cell biology. More specifically, the present invention relates to the prostate-specific membrane antigen-like gene and uses thereof.

Description of the Related Art Prostate cancer is the leading cause of cancer and second leading cause of cancer death among American males. Although oo oo ooooo -1- 11/12/03,sw 12328spa, I WO 00/61605 PCT/US00/09417 prostate tumors in the initial stages are slow growing and can be treated by radical prostatectomy and hormone deprivation, once the tumor is hormone refractory and/or has metastasized, there are few options for the patient. The major current biomarker for this disease is prostate specific antigen (PSA), however PSA is of limited value for assessing patients with disseminated disease as it is downregulated under conditions of low androgens, and these patients undergo androgen-ablative therapy. More markers for prostate cancer are needed that have increased effectiveness over those currently used for clinical diagnosis and patient management, as well as for future therapeutic targets of this disease.

Prostate specific membrane antigen (PSMA) is an ideal potential target for use in determining patient management, and therapeutic strategies against prostate cancer. The prostate specific membrane antigen is highly expressed in virtually 100% of prostate cancers and, in contrast to PSA, the prostate specific membrane antigen is further upregulated under conditions of androgen deprivation. Furthermore, in the normal prostate, alternative splicing of prostate specific membrane antigen mRNA produces a truncated form of the protein (which has been designated PSM') that is missing the intracellular and transmembrane domains, and as such, this form is localized to the cytosol At some stage during tumor initiation or progression, there is a change in the mRNA splicing that leads to the majority of prostate specific membrane antigen transcripts comprising the transmembrane domain, thereby producing a 750 amino acid membrane-bound protein (unlike PSA, which is secreted into the circulatory system), the majority of which is located extracellularly and is readily available for therapeutic WO 00/61605 PCT/US00/09417 targeting, clinical imaging or other diagnostic-type assays Prostate specific membrane antigen is already used clinically as the target of the imaging agent ProstaScint, and is the focus of a number of therapeutic strategies in development.

The known functions of the prostate specific membrane antigen carboxypeptidase are as an NAALadase and folate hydrolase.

Expression of prostate specific membrane antigen is largely confined to the prostate gland, although expression can also be detected in the duodenum, brain, salivary gland, kidney, and colon In prostate cancer, enhanced expression of prostate specific membrane antigen correlates with increasing grade of tumor Additionally, it now seems that therapeutic targeting of the prostate specific membrane antigen molecule may have additional advantages, since prostate specific membrane antigen expression has been found in the endothelial cells of tumor neovasculature of almost all types of tumors examined to date, including bladder, renal, breast and lung carcinomas No prostate specific membrane antigen expression has been found in any kind of normal established non-neovasculature. As such, a therapeutic approach targeted at prostate specific membrane antigen could have broad implications for the treatment of many types of solid tumors, and several groups are now attempting to utilize prostate specific membrane antigen as a treatment target.

However, although prostate specific membrane antigen is very highly expressed in normal prostate (PSM'; the cytosolic form) and in cancer of the prostate (PSMA; the membrane bound form), there are other tissues in the body that express low levels of prostate specific membrane antigen or a similar mRNA, including kidney, WO 00/61605 PCT/US00/09417 proximal small intestine and brain This mRNA could either be due to expression of the prostate specific membrane antigen gene, or another related gene such as the PSMA-like gene. Furthermore, one of the major enzymes involved in neurotransmission in the brain is NAALADase, which has the same enzymatic characteristics as prostate specific membrane antigen Accordingly, it is important to be able to distinguish between prostate- or cancerderived prostate specific membrane antigen and PSMA-like mRNA from other tissues if prostate specific membrane antigen is going to be used as a clinical marker via techniques like RT-PCR or for therapeutic strategies, which, for example, may use antibodies.

Fluorescent in situ hybridization (FISH) mapping using prostate specific membrane antigen cDNA as a probe indicates that there may be two very similar genes both residing on chromosome 11 Both genes have been mapped against a human-hamster radiation hybrid panel and determined that one of the genes resides on chromosome llpll.2, while the other gene resides on chromosome 11q14.3 It was recently determined that the gene on chromosome 1 lpll.2 is the PSMA gene originally cloned from the prostatic cancer cell line LNCaP while a highly conserved duplication of the PSMA gene, including at least some intronic sequences, is located on chromosome lq, a region which is known to have been duplicated to chromosome lip an estimated 22 million years ago [16,17]. Therefore, the so-called non-prostatic expression of the prostate specific membrane antigen gene is due to expression of another highly similar, but distinct gene, herein designated the PSMA-like gene, arising from the aforementioned gene duplication.

Tumor targeting for therapeutic approaches or clinical assays relies WO 00/61605 PCT/US00/09417 on the specificity of the marker targeted. As the prostate specific membrane antigen and PSMA-like genes have a common ancestral gene and only diverged from each other 22 million years ago it is likely that the two genes are extremely similar to each other both in sequence and in function.

The prior art is deficient in means of distinguishing between the prostate specific membrane antigen gene and the PSMA-like gene, and their respective protein products. The present invention fulfills this long-standing need and desire in the art.

SUMMARY OF THE INVENTION Prostate specific membrane antigen is a 100 kD type II transmembrane protein with folate hydrolase and NAALADase activity. Prostate specific membrane antigen is highly expressed in prostate cancer and the vasculature of most solid tumors, and is currently the target of a number of diagnostic and therapeutic strategies. PSMA is also expressed in the brain, and is involved in conversion of the major neurotransmitter, NAAG (n-acetyl-aspartyl glutamate) to NAA and free glutamate, the levels of which are disrupted in several neurological disorders including multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease and schizophrenia.

The prostate specific membrane antigen gene (having the nucleotide sequence shown in SEQ ID No. 3) encoding prostate specific membrane antigen was recently mapped to 1lpl11.2, and a gene homologous (PSMA-like), but not identical, to prostate specific membrane antigen was mapped to chromosome 11q14.3, which was subsequently mapped to the schizophrenia disorder type II locus. The mRNA tissue distribution pattern of the prostate specific membrane antigen gene and PSMA-like gene was examined using assays that specifically distinguish between the two genes by exploiting single base coding differences. Results indicate that the PSMA-like gene is expressed, as determined by RT-PCR, RNase protection assay, or using specific primers, and has a tissue distribution differing from that of the PSMA gene.

The present invention characterizes the differences between the prostatic and non-prostatic forms of prostate specific membrane antigen at the nucleic acid level, the protein level and functional level. The ability to distinguish between the PSMA and PSMA-like genes is essential for the utility of prostate specific membrane antigen, both as a prostate cancer marker and as a therapeutic target.

In one embodiment of the present invention, there is provided an isolated DNA fragment encoding a mammalian PSMA-like protein or fragments thereof having the amino acid sequence shown in SEQ ID No. 2 or fragment thereof.

In another embodiment of the present invention, there is provided an isolated and purified PSMA-like protein coded for by DNA having the nucleic acid sequence shown in SEQ ID No. 1 or fragments thereof.

i In still another embodiment of the present invention, there is provided a method of distinguishing PSMA gene expression from PSMA-like gene expression, comprising the steps of. contacting a sample with one or more oligonucleotide primer(s) selected from the group consisting of SEQ ID Nos. 5 to 38 under hybridizing conditions, wherein the sample comprises RNA; performing RT- PCR on the sample, thereby producing RT-PCR products; contacting the RTi PCR products with an appropriate restriction enzyme, thereby producing digested RT-PCR products; and analyzing the digested RT-PCR products, wherein prostate specific membrane antigen gene expression is distinguished from PSMA- 11/12/03,sw12328spa,6 like gene expression by detection of fragment size(s) in the digested RT-PCR products, wherein digested PSMA-specific RT-PCR products comprise different predicted fragment size(s) compared with digested PSMA-like-specific RT-PCR products.

In yet another embodiment of the present invention, there is provided a method of distinguishing prostate specific membrane antigen protein having the amino acid sequence as set forth in SEQ ID No. 4 from PSMA-like protein having the amino acid sequence as set forth in SEQ ID No. 2 in a sample, comprising the steps of: contacting the sample with at least one antibody specific for a PSMA protein and/or at least one antibody specific for a PSMA-like protein under appropriate conditions; and detecting binding of the antibody or antibodies. The specificity of binding is indicative of the presence of PSMA and/or PSMA-like proteins in the sample.

In yet another embodiment of the present invention, there is provided a vector for targeted gene therapy, comprising: a promoter/enhancer region from a PSMA gene or a PSMA-like gene; and a therapeutic gene. PSMA gene promoter/enhancer targets the therapeutic gene to prostate tissues and tumor neovasculature of solid tumors; whereas PSMA-like gene promoter/enhancer targets to non-prostate tissues.

In still yet another embodiment of the present invention, there is provided a method of screening for prostate specific membrane antigen or PSMA-like ligands, comprising the steps of contacting a PSMA-like protein, or fragment thereof having the amino acid sequence set forth in SEQ ID No. 2, with potential ligands under conditions that permit protein-protein binding removing non-specific proteino protein binding; and eluting protein bound to the PSMA or PSMA-like protein.

Typically, the eluted protein is a ligand for the PSMA or PSMA-like protein.

-7- 11/12/03,sw12328spa,7 Also provided in another embodiment of the present invention is a method of imaging cells expressing a prostate specific membrane antigen or PSMA-like protein, comprising the steps of: administering to the cells at least one compound, wherein the compound is specifically directed towards a PSMA-like protein having the amino acid sequence set forth in SEQ ID No. 2 and labeled with an imaging agent; and detecting the imaging agent in the cells.

Further provided in an embodiment of the present invention is a cytotoxic composition, comprising: a compound specific for a PSMA-like protein or fragment thereof having the amino acid sequence set forth in SEQ ID No. 2; and a cytotoxic agent.

Further provided is a pharmaceutical composition comprising an antibody directed against a prostate specific membrane antigen protein and does not recognize a PSMA-like protein. Such a composition can be used for diagnosing a cancer or a neurological disorder such as schizophrenia in an individual.

Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of

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So •disclosure.

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0 0 0 0 -8- 11/12/03,sw12328spa,8 BRIEF DESCRIPTION OF THE DRAWINGS The appended drawings have been included herein so that the aboverecited features, advantages and objects of the invention will become clear and can be understood in detail. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and should not be considered to limit the scope of the invention.

*o -9- Sl/12/03,sw 23 2 8spa,9 WO 00/61605 PCT/US00/09417 Figure 1 shows mapping of the prostate specific membrane antigen gene to chromosome llp. Figure 1A shows PCR amplification of the PSMA promoter region reported by Figure 1B shows amplification using primers to exon 16 of the PSMA gene.

Figure 1C shows amplification using primers to intron 6 of the PSMA gene. Genomic is normal human DNA, the subsequent 3 lanes used human-hamster hybrid DNA containing the indicated chromosomes. Hamster refers to the parental DNA. Panels A-C clearly show exonic and intronic duplication of the PSMA gene on l1p and llq, but only llp contains the prostate specific membrane antigen promoter region.

Figure 2 shows specific amplification of the 11q PSMAlike gene using primers designed by sequence analysis of the 1lq gene.

Figure 3 shows amplification of the 3' end of prostate specific membrane antigen or PSMA-like mRNA using cDNA-specific primers with the cDNA derived from the indicated tissues. Note the lower band (splice variant) that is only present in LNCaP and prostate cells.

Figure 4 shows the alignment between prostate specific membrane antigen protein (SEQ ID No. 4) and PSMA-like protein (SEQ ID No. 2).

Figure 5 shows the NAALADase enzymatic activity of PSMA-like protein.

DETAILED DESCRIPTION OF THE INVENTION WO 00/61605 PCTI/US00/09417 Prostate specific membrane antigen is expressed on the cell surface making it a useful target for both clinical and therapeutic strategies. While prostate specific membrane antigen appears to be an ideal prostate cancer marker and potential therapeutic target, there have been reports of prostate specific membrane antigen expression in non-prostatic tissues, including brain, kidney and proximal small intestine. Such expression of prostate specific membrane antigen could weaken the potential of this gene as a prostate cancer marker, or at least, produce confusing and conflicting data. However, there is reason to believe that the socalled non-prostatic expression of the prostate specific membrane antigen gene-is, in fact, due to expression of a highly similar, but distinct, gene, which is designated as "PSMA-like" gene. The prostate specific membrane antigen gene has recently been mapped to human chromosome llpll.2, and the "PSMA-like" gene to chromosome llq14.3. Characterization of the differences between the prostatic and non-prostatic forms of prostate specific membrane antigen at the nucleic acid level, the protein level and functional level is essential for the future utility of prostate specific membrane antigen, both as a prostate cancer marker and as a therapeutic target.

The differences unique to prostate specific membrane antigen can be used to generate specific antibodies for clinical imaging or immunotherapeutic approaches, RT-PCR analysis of bodily fluids specifically for prostate- or prostate cancer-derived cells. It is also possible that the two proteins differ in their enzymatic activity in such a way that prodrugs could specifically target PSMA-expressing tissues. The present invention also provides WO 00/61605 PCT/US00/09417 for analysis of the sequences in the prostate specific membrane antigen gene responsible for expression in the prostate and in prostate cancer. Comparison of the promoter and enhancer sequences from the prostate specific membrane antigen gene with the corresponding regions in the PSMA-like gene (which is not expressed in the prostate) allows elucidation of those sequences responsible for prostate-specific expression. These sequences can be used to generate tissue-specific constructs for use in gene therapy against prostate cancer.

In one embodiment of the present invention, there is provided an isolated DNA fragment encoding a mammalian PSMAlike protein selected from the group consisting of an isolated DNA fragment which encodes a PSMA-like protein; an isolated DNA fragment which hybridizes to the isolated DNA fragment of (a) and which encodes a PSMA-like protein; and an isolated DNA fragment differing from the isolated DNA fragments of and in codon sequence due to the degeneracy of the genetic code, and which encodes a PSMA-like protein. Preferably, the DNA fragment has the sequence shown in SEQ ID No. 1 or fragments thereof, and the PSMA-like protein has the amino acid sequence shown in SEQ ID No. 2 or fragment thereof.

In a preferred embodiment, there is provided a vector and/or a host cell comprising the above-disclosed DNA fragment.

Further preferably, the host cell can be a bacterial cell, a mammalian cell, a plant cell or an insect cell.

In another embodiment of the present invention, there is provided an isolated and purified PSMA-like protein coded for by DNA selected from the group consisting of isolated DNA which WO 00/61605 PCT/US00/09417 encodes a PSMA-like protein; isolated DNA which hybridizes to the isolated DNA of and which encodes a PSMA-like protein; and isolated DNA differing from the isolated DNAs from and (b) in codon sequence due to the degeneracy of the genetic code, and which encodes a PSMA-like protein. Preferably, the PSMA-like protein has an amino acid sequence shown in SEQ ID No. 2 or fragments thereof.

In a preferred embodiment, there is provided an antibody directed against the PSMA-like protein disclosed herein.

In still another embodiment of the present invention, there is provided a method of distinguishing prostate specific membrane antigen gene expression from prostate specific membrane antigen-like gene expression, comprising the steps of: (a) contacting a sample with one or more oligonucleotide primer(s) under hybridizing conditions, wherein the sample comprises RNA; performing RT-PCR on the sample, thereby producing RT-PCR products; contacting the RT-PCR products with an appropriate restriction enzyme, thereby producing digested RT-PCR products; and analyzing the digested RT-PCR products, wherein PSMA gene expression is distinguished from PSMA-like gene expression by detection of fragment size(s) in the digested RT-PCR products, wherein digested PSMA-specific RT-PCR products comprise different predicted fragment size(s) compared with digested PSMA-likespecific RT-PCR products. Preferably, the oligonucleotide primer is selected from the group consisting of SEQ ID Nos. 5-38.

Representative restriction enzymes are EcoRI and AccI. Additionally, restriction enzymes such as Bsp1286I, Sse9I, Tsp509I, TspEI, TspRI, Bstll07I, AciI, MspAI, NspBII, RsaI, HaeIII or SspI may be utilized.

WO 00/61605 PCT/US00/09417 Representative samples are blood cells, cells growing in culture, biopsied cells, epithelial cells, endothelial cells, urine and seminal fluid.

For example, when the oligonucleotide primers are SEQ ID No. 37 and SEQ ID No. 38, and the restriction enzyme is EcoRI, presence of fragment sizes of 348 nucleotides and 207 nucleotides indicates PSMA gene expression in the sample, while presence of fragment size of 555 nucleotides indicates PSMA-like gene expression in the sample. Alternatively, when the restriction enzyme is AccI, presence of fragment sizes of 506 nucleotides and 49 nucleotides indicates prostate specific membrane antigen gene expression in the sample and presence of fragment sizes of 319 nucleotides, 187 nucleotides and 49 nucleotides indicates PSMA-like gene expression in the sample.

In yet another embodiment of the present invention, there is provided a method of distinguishing prostate specific membrane antigen protein from prostate specific membrane antigen-like protein in a sample, comprising the steps of: (a) contacting the sample with at least one antibody specific for a PSMA protein and/or at least one antibody specific for a PSMA-like protein under appropriate conditions; and detecting binding of the antibody or antibodies. The specificity of binding is indicative of the presence of prostate specific membrane antigen and/or PSMAlike proteins in the sample. Preferably, the antibody specific for a prostate specific membrane antigen protein is specific for a region of the PSMA protein and does not cross-react with a PSMA-like protein, or alternatively, the antibody specific for a PSMA-like protein is specific for a region of the PSMA-like protein and does not WO 00/61605 PCT/US00/09417 cross-react with a prostate specific membrane antigen protein.

Representative means of detection are colorimetric assay, fluorescence, radioautography, nuclear medicine detection, electron microscopy, enzymatic assays, enzyme-linked immunoassays and

MRI.

In yet another embodiment of the present invention, there is provided a vector for targeted gene therapy, comprising: a promoter/enhancer region from a PSMA gene or a PSMA-like gene; and a therapeutic gene. PSMA gene promoter/enhancer targets the therapeutic gene to prostate tissues and tumor neovasculature of solid tumors, whereas PSMA-like gene promoter/enhancer targets to non-prostate tissues.

In still yet another embodiment of the present invention, there is provided a method of screening for prostate specific membrane antigen or prostate specific membrane antigen-like ligands, comprising the steps of contacting a PSMA or PSMA-like protein, or fragment thereof, with potential ligands under conditions that permit protein-protein binding; removing nonspecific protein-protein binding; and eluting protein bound to the PSMA or PSMA-like protein. Typically, the eluted protein is a ligand for the PSMA or PSMA-like protein.

Also provided in another embodiment of the present invention is a method of imaging cells expressing a prostate specific membrane antigen or prostate specific membrane antigen-like protein, comprising the steps of: administering to the cells at least one compound, wherein the compound is specifically directed towards a PSMA or PSMA-like protein and labeled with an imaging agent; and detecting the imaging agent in the cells. Preferably, the WO 00/61605 PCTIUS00/09417 compound directed towards a PSMA or PSMA-like protein is an antibody or a ligand.

Still provided in an embodiment of the present invention is a cytotoxic composition, comprising: a compound specific for either a prostate specific membrane antigen protein or fragment thereof, or a prostate specific membrane antigen-like protein or fragment thereof; and a cytotoxic agent. Preferably, the compound directed towards a PSMA or PSMA-like protein is an antibody or a ligand. Preferably, the cytotoxic agent is a radioisotope or a toxin.

The antibody may be linked to the cytotoxic agent either chemically or genetically. For example, the gene encoding the antibody may be fused to the gene encoding the cytotoxic agent.

Further provided is a pharmaceutical composition comprising an antibody directed against a PSMA protein and does not recognize a PSMA-like protein. Such composition can be used for diagnosing a cancer or a neurological disorder in an individual by detecting the localization of the antibody, wherein the detection of the antibody indicates a possibility of having a cancer or a neurological disorder. Representative examples of cancer include a prostate cancer, a bladder cancer, a pancreatic cancer, a sarcoma, a melanoma, a lung cancer and a kidney cancer. A representative example of a neurological disorder is schizophrenia.

In accordance with the present invention, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, Sambrook, Fritsch Maniatis, "Molecular Cloning: A Laboratory Manual (1982); "DNA Cloning: A Practical Approach," Volumes I and II Glover WO 00/61605 PCT/US00/09417 ed. 1985); "Oligonucleotide Synthesis" Gait ed. 1984); "Nucleic Acid Hybridization" Hames S.J. Higgins eds. (1985)]; "Transcription and Translation" Hames S.J. Higgins eds.

(1984)]; "Animal Cell Culture" Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning'. _j1984). Therefore, if appearing herein, the following terms shall have the definitions set out below.

A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA the strand having a sequence homologous to the mRNA).

A "vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. A "replicon" is any genetic element plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control. An "origin of replication" refers to those DNA sequences that participate in DNA synthesis. An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another WO 00/61605 PCTIUS00/09417 DNA sequence. A coding sequence is "operably linked" and "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.

Expression vectors containing promoter sequences which facilitate the efficient transcription and translation of the inserted DNA fragment are used in connection with the host. The expression vector typically contains an origin of replication, promoter(s), terminator(s), as well as specific genes which are capable of providing phenotypic selection in transformed cells. The transformed hosts can be fermented and cultured according to means known in the art to achieve optimal cell growth.

A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic DNA, and even synthetic DNA sequences.

A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence. A "cDNA" is defined as copy-DNA or complementary-DNA, and is a product of a reverse transcription reaction from an mRNA transcript. An "exon" is an expressed sequence transcribed from the gene locus, whereas an "intron" is a non-expressed sequence that is from the gene locus.

As used in the present invention, "exons" of PSMA-like gene are WO 00/61605 PCT/US00/09417 referred to regions of genomic DNA in the PSMA-like gene that are homologous to known exons in the PSMA gene; and "introns" of PSMA-like gene are referred to the regions of genomic DNA in the PSMA-like gene that are homologous to known introns in the PSMA gene.

Transcriptional_ and translational_- contr.ol-. sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell. As used herein, an "enhancer", "enhancer element" or "enhancer region" is a region separate from, or included with, a promoter element that typically enhances transcription or provides specific elements necessary from proper transcription. Enhancers typically can act at a distance, and often at either the 3' or 5' end of a gene. A "cis-element" is a nucleotide sequence, also termed a "consensus sequence" or "motif', that interacts with other proteins which can upregulate or downregulate expression of a specific gene locus. A "signal sequence" can also be included with the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell and directs the polypeptide to the appropriate cellular location. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and WO 00/61605 PCT/US00/09417 extends upstream direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters often contain ."TATA'. boxes and "CAAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences. As used herein, an enhancer element or region may be included with the minimal promoter elements required for transcription, to thereby create an expression pattern very similar to the native gene(s).

The term "oligonucleotide" is defined as a molecule comprised of two or more deoxyribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide. The term "primer" as used herein refers to an oligonucleotide, whether occurring naturally, as in a purified restriction digest, or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, WO 00/61605 PCT/US00/09417 source of primer and the method used. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

Primers are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a noncomplementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence or hybridize therewith and thereby form the template for the synthesis of the extension product. Additionally, a single base difference in a primer, particularly at the 3' end from which extension occurs, is sufficient to allow differential hybridization of the two primers, thereby allowing selected amplification based upon a single site difference. As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to enzymes which cut double-stranded DNA at or near a specific nucleotide sequence.

"Recombinant DNA technology" refers to techniques for uniting two heterologous DNA molecules, usually as a result of in vitro ligation of DNAs from different organisms. Recombinant DNA molecules are commonly produced by experiments in genetic engineering. Synonymous terms include "gene splicing", "molecular WO 00/61605 PCT/US00/09417 cloning" and "genetic engineering". The product of these manipulations results in a "recombinant" or "recombinant molecule".

A cell has been "transformed" or "transfected" with exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a vector or plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

As used herein, the term "host" is meant to include not only prokaryotes but also eukaryotes such as yeast, plant and animal cells. A recombinant DNA molecule or gene can be used to transform a host using any of the techniques commonly known to those of ordinary skill in the art. Prokaryotic hosts may include E.

coli, S. tymphimurium, Serratia marcescens and Bacillus subtilis.

Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cells and insect cells, and more preferentially, plant cells, such as Arabidopsis thaliana and Tobaccum nicotiana.

WO 00/61605 PCT/US00/09417 Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90% or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art.

See, Maniatis et al., supra; DNA Cloning, Vols. I II, supra; Nucleic Acid Hybridization, supra.

A "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. In another example, the coding sequence is a construct where the coding sequence itself is not found in nature a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.

As used herein, "fragment", as applied to a polypeptide, will ordinarily be at least 10residues, more typically at least residues, and preferably at least 30 50)residues in length, but less than the entire, intact sequence. Fragments can be generated by methods known to those skilled in the art, by enzymatic WO 00/61605 PCT/US00/09417 digestion of naturally occurring or recombinant protein, by recombinant DNA techniques using an expression vector that encodes a defined fragment, or by chemical synthesis. The ability of a candidate fragment to exhibit characteristics of a particular enzyme binding to a specific antibody, or exhibiting partial enzymatic or catalytic activity) can be assessed by methods described herein. Purified fragments or antigenic fragments can be used to generate new regulatory enzymes using multiple functional fragments from different enzymes, as well as to generate antibodies, by employing standard protocols known to those skilled in the art.

A standard Northern blot assay can be used to ascertain the relative amounts of mRNA in a cell or tissue obtained from transgenic tissue, in accordance with conventional Northern hybridization techniques known to those persons of ordinary skill in the art. Similarly, a dot blot procedure or an RNAse protection assay can be used to evaluate the levels of mRNA expression.

Alternatively, a standard Southern blot assay may be used to confirm the presence and the copy number of the gene in transgenic systems, in accordance with conventional Southern hybridization techniques known to those of ordinary skill in the art. The Northern blot, dot blot and Southern blot use a hybridization probe, e.g.

radiolabelled cDNA, either containing the full-length, single stranded DNA or a fragment of the DNA sequence at least (preferably at least 30, more preferably at least 50, and most preferably at least 100 consecutive nucleotides in length). The DNA hybridization probe can be labelled by any of the many different methods known to those skilled in this art.

WO 00/61605 PCT/US00/09417 The labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others. A number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate. Proteins can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures. The preferred isotope may be selected from 3 H, 14C, 32p, 35S, 36C1, 5 1 Cr, 7 Co, 5 8 Co, 5 9 Fe, 90Y, 1251, 1311, and 1 8 6 Re.

Enzyme labels are likewise useful, and can be detected by any of the presently utilized immunoenzymatic, colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques. Furthermore, the PSMA or PSMA-like enzymes can be labeled and the endogenous activities assayed. The enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, P-glucuronidase, P-D-glucosidase, p-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase.

U.S. Patent Nos. 3,654,090, 3,850,752, and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.

WO 00/61605 PCT/US00/09417 The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion: EXAMPLE 1 Differentiating the PSMA gene from the PSMA-like gene To map the human PSMA gene and resolve the controversy regarding its true location (lip versus lq), a number of primer pairs were designed with homology to various regions of the PSMA gene, including introns. These primers were then used to amplify DNA from the NIGMS somatic cell hybrid mapping panel which consists of a hybrid containing chromosome 11, one containing chromosome lip, one containing lq and a hamster parental line. While the amplified regions of exon 16, intron n-o (primers used correspond to nt 54278-54536 in the PSMA genomic sequence and encompass exon 15 of the PSMA gene) and intron 6 are found on both chromosome lip and lq, the promoter region of the PSMA gene is only amplified from the hybrid containing chromosome lip (see Figure The fact that intron sequences are present also confirms that the gene on chromosome lq is not a pseudogene, but in fact, a gene duplication.

Intron-based primers were then used to amplify and subsequently clone regions from the lip and llq genes. The existence of sequence differences between the two genes was confirmed by analysis of the corresponding regions in four normal DNA samples. Based on the number of single base differences between non-coding regions of the two genes, it is estimated that the gene duplication occurred 22 million years ago, after the divergence WO 00/61605 PCT/US00/09417 of man and mouse. Taken together with data from the mouse model only one gene corresponding to the PSMA gene family is present and maps to a region that corresponds to human chromosome lq), it is expected that the lq gene (the PSMA-like gene) is the ancestral gene, and therefore, is likely to be functional. The fact that the promoter region of the PSMA-like gene was not subject to duplication implies at least a differential expression pattern for the two genes, which is also supported by the fact that the mouse homologue of PSMA-like gene is not expressed in the prostate.

Based on sequence differences between the human PSMA and PSMA-like genes, primer sequences (sense, GCCTTCATTTTCAGAACATCTCATGCAT-3', SEQ ID No. 5; antisense, 5'-GTCCATATAAACTTTCAAGAATGTG-3', SEQ ID No. 6) were designed that only amplify the first intron of the PSMA-like gene on chromosome lq (see Figure These primers are used to screen a human PAC library for the PSMA-like genomic clone.

EXAMPLE 2 Evidence for a novel PSMA splice variant As analysis of the brain and prostate PSMA and PSMAlike genes was being carried out, RT-PCR of the terminal region of the PSMA gene detected an alternate splice form of PSMA present in LNCaP cells and normal prostate that is not present in normal brain (see Figure This appears to be a novel splice variant of PSMA and the expression pattern of this variant is evaluated in prostate and other tissues.

WO 00/61605 PCT/US00/09417 EXAMPLE 3 Screening for ligands of PSMA/PSM' It has previously been reported that mitochrondrial aspartate-aminotransferase (mAAST) binds to PSMA, since it coelutes with PSMA from affinity columns made with the 7E11C5.3 antibody Using a similar isolation method, co-elution with PSMA of a protein of the size expected for mAAST from LNCaP cells was demonstrated. However, this protein is apparently not a PSMA ligand, as it is also eluted from the same 7E11C5.3 affinity column that has been treated with protein lysate from non-PSMA expressing PC3 cells.

The yeast-two-hybrid system is also being used to screen for PSMA ligands. To date, six million clones have been screened from a prostate library and six different, consistently interacting clones have been identified. Significantly, one of the positive clones corresponds to Survivin, a recently cloned apoptosis inhibitor which is highly expressed in prostate tumors, but is not typically expressed in terminally differentiated adult tissues. A second clone identified in the screen corresponds to a gene whose sequence has been put in Genbank as part of the chromosome 22 sequencing project. All six clones will be subcloned into appropriate vectors for re-confirming the protein-protein interaction with PSMA in the mammalian-twohybrid system.

EXAMPLE 4 Sequencing the PSMA-like gene For prostate specific membrane antigen to be useful as a therapeutic and clinical target for prostate cancers, it is necessary to WO 00/61605 PCT[USOO/09417 be able to readily distinguish the various transcript and/or proteins from one another PSM', PSMA and PSMA-like). Primers that specifically amplify the chromosome lq PSMA-like gene are used to screen a human PAC library by PCR. The general insert size of a PAC is around 100 kb, and the PAC library is considered to have a threefold coverage of the human genome. Sequence is obtained directly from the PAC using at least two primers to PCR amplify each of the 19 exons. The primers are designed using the PSMA cDNA exonic sequences. This approach ensures that every intron-exon boundary is examined.

EXAMPLE Nucleotide differences between the PSMA and PSMA-like genes Of the 19 exons in the PSMA-like gene, 18 have been sequenced. Oligonucleotide primers based upon intronic sequences of the PSMA genomic clone (GenBank Accession No. AF007544) (Table 1) were used to amplify the corresponding regions of the PSMA-like gene from somatic cell hybrids containing human chromosome llq Hybrids GM11936 and GM07298 from Coriell Cell Repositories, NJ). The amplified PCR products were then purified, sequenced and compared to the cDNA sequence of the PSMA gene (GenBank Accession No. M99487). The nucleotide and amino acid differences obtained for exons 2-19 are described in Table 2. In the case of exon 19, the sequence was confirmed by 3' RACE, which also confirmed that the mRNA transcript of the PSMAlike gene ends in the same place as that of the PSMA gene.

TABLE I PCi' Sense Sense Primer Anti- Antisense product Exon Bases Primer Sequence sense Primer size sequence exeted 1 2488- 2863__ 2 4994- 4870 ctcacctaat 5254 agtatagtcctcctcag 384 5099 gtcagaggta atg (SEQ ID No. 8) (SEQ ID No. 7) 3 10726 10630 caaagtactt 11082 cataggaaagtagt 452 -ttgtgtaactctgc tgacacgg ____10912 ID No. 9) (SEQ ID No. 10) 4 18275 18157 cctgaaggat 18457 gaccctttaa 300 -tcattcaccctc ttatcggctgaaca 18376 (SEQ ID No. 11) (SEQ ID No. 12) 5-6 24400 24323 atgtccaaca 25593 gacatgctta 1270 -gtccccatgcag gtccattgtacc _____25500 (SEQ ID No. 13) (SEQ ID No. 14) 7 27927 27871 gaaccgtttg 28058 ttacccaaat 187 -aatgaaactgag agccatccatgg 28020 (SEQ ID No. 15) (SEQ ID No. 8-9 35216 35127 gcagatgctc 36334 ccagcacata 1207 -aataagtgaatcc acagttacttgatc 36281 (SEQ ID No. 17) (SEQ ID No. 18) 37697 37619 tagatgctat 37867 aaactgagac 248.

-tgagtcgtttgc tcagataggctg _____37816 ID No. 19) (SEQ ID No. 20) 11 39896 39825 ctgggcttgg 40045 gcttggcaaa 220 -tagtgtcctggg caagtcctggctac 39978 ID No. 21) (SEQ ID No. 0 0 0 0%

-C

0% 0 Il 12 41911 41792 tgtcgttaat 42035 ttaactagac 243 -atgggtcagctc tgctgctcctag 41974 (SEQ ID No. 23) (SEQ ID No. 24) 13 46402 46317 tggtaggaat 46687 gatgctacta 370 -ttagcagtggtc atgggctacctc 46469 (SEQ ID No. 25) (SEQ ID No. 26) 14 53129 53053 cttctggtta 53264 caatcccaca 211 -atggacatctag ctgaattcagtg 53220 (EIDNo. 27) (SEQ ID No. 28) agaatggggt igagtcactttt ttagtttaatgg tggagtcag (SEQ ID No. 29) (SEQ ID No. 16-17 56661 56614 ttgtaagcta 57393 agttcagcaa 779 -tccctataagag cagtcatgttag ____57307 (SEQ ID No. 31) (SEQ ID No. 32) 1 8 62423 62305 gggtggtcct 62553 gtgatattac 248 -gaaaccaatccc agaaaggagtc ____62515 ID No. 33) (SEQ ID No. 34) 19 64209 64127 atccaggaat 64586 ttcagtttta 459 -tgcagagtgctc atccatagggag 8 ID No. 35) (SEQ ID No. 36) WO 00/61605 WO 0061605PCT/USOO/09417 Exon in PSMAgn 2 3 4 6 7 8 9 10 1 1 12 13 14 TABLE 2 Nucleotide changes PSMA-->PSMA-like No change nt 630 t-ma nt 584 V nt 594 ant 739 c-+t nt 777 c- t nt 787 t-)c nt 877 g---a nt 948 c---t nt 993 t4 nt 1023 g-At nt 1092 t-*c nt 1103 g---a nt 1150 a-4g nt 1237 c--t nt 1320 a-+g nt 1454 t---c No change nt 1572 g-*t nt 1665 g-+a nt 1684 c--4t No change Amino acid changes PSMA-*PSMA-like No change Thr- Thr Val-* Ala Ala-*Ala Pr o- S er Gly---Gly Tyr---His Gly--*Arg Ser- Ser Asp- Asp Gln-*His Tyr-*Tyr Arg-4 Gin Ile- Va1 Pro- Ser Thr- Thr Ile-*Thr No change Glu-4Asp Pr o-)Pro His-if yr No change WO 00/61605 WO 0061605PCT/USOO/09417 TABLE 2 (co nt.) Exon in PSMA g=n Nucleotide changes PSNIA-WSMA-like Amino acid changes PSMA--*PSMA-like No change nt 2099 g-*a nt 2140 g--*t nt 2172 g-ma nt 2202 t-4c nt 2239 t nt 2241 a->g nt 2314 g->a nt 2442 a-4t nt 2459 a- c nt 2531 a--4c nt 2534 c--+t nt 2562 AG is deleted in PSMA-like gene nt 2571 c-+a nt 2572 g-4a No change Ser-*Asn Val- Leu Lys-->Lys S er-->S er Va1- Leu Arg-*Arg Glu- Asp Ty Ser No change No change

UTR)

UTR)

UTR)

UTR)

UTR)

change change change WO 00/61605 PCT/US00/09417 EXAMPLE 6 Sequences of PSMA-like gene and protein PSMA-like gene was isolated from a liver library and sequenced. The complete sequence is shown in SEQ ID No. 1, whereas the predicted amino acid sequence of PSMA-like protein is shown in SQ ID No. 2. The alignment between PSMA and PSMA-like proteins are shown in Figure 4. It seems that the PSMA-like starts transcribing in the middle of intron 6 (compared to PSMA). It therefore results in a smaller protein, which is significantly different from PSMA. The similarity of the homologous regions of the two genes is around 98% at the amino acid level. PSMA-like protein will be tested for enzyme activity.

EXAMPLE 7 Tissue distribution of the PSMA-like gene PCR on cDNAs from various tissues was performed using the following primer sequences: Primer 1: 5' ACAGATATGTCATTCTGGGAGGTC 3' (SEQ ID No. 37) (sense; exonl0) Primer 2: 5' ACTGTGATACAGTGGATAGCCGCT 3' (SEQ ID No. 38) (anti-sense; exon 16) PCR was run at 94 0 C for 3.5 min, 94 0 C for 20 sec, 61 0

C

for 20 sec, and 72 0 C for 50 sec for 35 cycles. The expected size after PCR amplification from both PSMA and PSMA-like RNA is 555 base pairs.

One fifth of the reaction was then digested with EcoRI or AccI. After 1-3 hours of digestion, the product was electrophoresed WO 00/61605 PCT/US00/09417 and photographed. If the product was digested with EcoRI and fragments of 348 and 207 nucleotides are produced, then PSMA mRNA was present in the original sample. If an undigested, single band of 555 nucleotides is present following EcoRI digestion, PSMAlike RNA was present in the sample. If the product was digested with AccI, bands of 506 and 49 nucleotides are expected if the original sample expressed the PSMA gene, and 319, 187 and 49 nucleotides if the PSMA-like gene was expressed.

RT-PCR analysis has shown that the PSMA gene is expressed in the vasculature of almost all solid tumors examined so far including bladder cancer, pancreatic cancer, sarcomas, melanomas, lung cancer, kidney cancer, as well as the prostate. The PSMA-like gene is expressed in kidney and liver. Some tissues exhibit all bands expected, meaning that both the PSMA and PSMAlike genes are expressed.

This method can be used to amplify other regions of the PSMA and PMSA-like gene that differ in nucleotide sequence.

Numerous combinations of primers are acceptable, providing the primers hybridize to both the PSMA and PSMA-like genes and amplify a region that differs between the two genes such that restriction analysis of the product will differentiate between the genes. For example, in exon 8, Bspl286I restricts PSMA but not PSMA-like DNA; in exon 10, the PSMA gene, but not the PSMA-like gene, is digested by Sse9I, Tsp509I or TspEI; in exon 12, PSMA is digested by EcoRI, while PSMA-like is not; in exon 13, PSMA-like DNA, but not PSMA DNA, is digested by TspRI, AccI or Bstl107I, and PSMA DNA, but not PSMA-like DNA is digested by AciI, MspAI, NspBl or RsaI; in exon 18, PSMA is restricted by HaeIII, while PSMA-like WO 00/61605 PCT/US00/09417 DNA is digested by SspI. This list is not meant to be all inclusive, but provides numerous restriction sites specific to either the PSMA or PSMA-like gene for differential identification and analysis.

EXAMPLE 8 Differential genetic marker or restriction site polymorphism To confirm that the EcoRI restriction enzyme site actually differed between the two genes and was not due to a polymorphism of the PSMA gene within the population, DNA obtained from more than 15 different people was amplified using PCR primers spanning this restriction site and subsequently digested with EcoRI. The presence of 3 bands after digestion indicated that all the people tested had both the PSMA and PSMA-like genes, and that those genes could be distinguished by the EcoRI site. This result is evidence that the EcoRI site is not a polymorphism, but is instead a genetic marker for distinguishing the PSMA gene from the PSMA-like gene.

EXAMPLE 9 NAALADase enzymatic activity of PSMA-like protein The PSMA-like clone obtained from screening the liver cDNA library was excised and cloned into the pIRES-neo vector (Clontech). PC-3 cells, which do not express PSMA, PSMA-like or have NAALADase activity were then transfected with the PSMA-likeneo vector using Lipofectamine Plus (Gibco-BRL) Transfected cells stably expressing the PSMA-like gene were then selected for by growing them in 1000ug/ml Geneticin. Protein was isolated from the cell lines by lysing them in 50 mM Tris-HCl pH 7.4, 0.5% Triton WO 00/61605 PCT/US00/09417 X-100. 2 jgg of protein was incubated with 20 giM tritiated NAAG in a total volume of 100 gl of lysis buffer for one hour. The substrate and its cleaved by-products were separated via ion-exchange chromatography and tritiated glutamate was eluted from the column in 1 M formic acid and quantified by counting in a scintillation counter. Control experiments included C4-2 LNCaP cells (positive control) and PC-3 cells that had been transfected with the pIRES-neo vector alone.

The data shows that cells transfected with the PSMA-like vector have nearly 15 fold over that seen with the cells transfected with vector alone (background counts, Figure Thus PSMA-like does have NAALADase activity, and should be taken into account when designing prodrug strategies targeting PSMA. NAALADase enzymatic activity suggests that PSMA-like may be able to be secreted to the serum/urine/seminal fluid, and that differentiating PSMA-like and PSMA proteins may make all the difference in diagnosis. For example, PSMA-like protein may be used for diagnosing neurological disorders such as schizophrenia.

The following references were cited herein: D.A. Silver, et al, Clin. Cancer Res. 3 (1997) 81-85.

M. Kawakami, et al, Cancer Res. 57 (1997) 2321-2324.

G.L.Wright Jr., et al, Urol. Oncol. 1 (1995)18-28.

R.S. Israeli, et al, Cancer Res. 54 (1994)1807-1811.

S.L. Su, et al, Cancer Res. 55 (1995) 1441-1443.

H. Liu, et al, Cancer Res. 57 (1997) 3629-3634.

R.E. Carter, et al, Proc. Natl. Acad. Sci. 93 (1996) 749-753.

C.W. Rinker-Schaeffer, et al, Genomics. 30 (1995) 105-108.

WO 00/61605 PCT/US00/09417 D.S. O'Keefe, et al. Biochimica Biophysica Acta. (1998) 1443:113- 127.

Luthi-Carter, et al. J. Pharm. Exp. Ther. 286 (1998) 1020-1025.

[11] Y. Kimoto, Mol. Gen. Genet. 258 (1998) 233-239.

[12] J.L. Gala, et al. Clin. Chem. 44 (1998) 472-481.

[13] J.T. Pinto, et al. Clin. Cancer Res. 2 (1996) 1445-1451.

[14] W.D.W. Heston. Mol. Urol. 1 (1997) 11-20.

Liu H, et al Cancer Res. (1998) 58:4055-60.

Any patents or publications mentioned in this :10 specification are indicative of the levels of those skilled in the art to which the invention pertains. Further, these patents and Spublications are incorporated by reference herein to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain 0 the ends and advantages mentioned, as well as those objects, ends S and advantages inherent herein. The present examples, along with the methods, procedures, treatments, molecules, and specific 20 compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the s c o p e of t h e claims.

Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component or group thereof EDITORIAL NOTE APPLICATION NUMBER 42189/00 The following Sequence Listing pages 1 to 16 are part of the description. The claims pages follow on pages 39 to 42.

WO 00/61605 WO 0061605PCTIUSOO/09417 <110> <120> <130> <141> <150> <151> <160> SEQUENCE LISTING O'Keefe, Denise S.

Heston, Warren D.W.

DNA Encoding the Prostate-Specific Membrane Antigen-Like Gene and Uses Thereof D623OPCT 2000-04-06 US 60/128,839 1999-04-09 38 <210> <211> <212> <213> <223> <400> agcaaatact aggattttta gatgtcc ttt gtactcattt accaatttaa gcccagctgg ctactttgct gaggtggtgt cc tctcacac tgcagaggct atgatgcaca agc agc tgga tactggaaac atgaagtgac gaaccagaca tggtggtat t ggagctttgg ttgtttgcaa gtgggcagag t taatgc tga acaccactga ccctgatgaa aaagtcc ttc tctggaaatg c agagc acgg cactgtatca gatccaatgt ggtgtttgag atgc tgtagt aaacatccac ttctgcagta tccaggactt caactcatgt c agac cttt t atgcagggga agcaaagtgg tgttgcagcc cctaagagga 1 1992

DNA

Homo sapiens cDNA sequence of PSMA-like gene cac taccaca gagcttatag tttgtaggcc aagtgtaata aataaggaaa caggggccaa c ctggggtga ccagcg tgga caggttaccc gttggtcttc gaagctccta gaggaagtct ttttctacac gagaatttac gatatgtcat gaccctcaga aac actgaaa gctgggatgc gataattcaa ctcatctata tgtacagc tt ggc tttgaag cccagagttc attttgaggt tatac taaaa cagtgtctat ttaaatatca ctagccaatt t ttaagaaag aggaaatgaa aaaaatttta tgacaaaagc tt ctggaaag tataggcatg gtcattccca acccttccaa ttcacagtgc ttctttagag aataagaaca tagcaaaaag taatgacaaa ttgaaaattg gaaagacact aggagtcatt agtcctatcc aatatcctaa agcaaatgaa caagtattcc gaaaaaatgg caaagtgtcc aaaaagtcaa aatgtgatag tctgggaggt gtggagcagc aaggaagggt agaagaatt t gactccttca gaaggaaact ggtatacaac gcaaatctct agtggcatgc gttcttccaa at tgggaaac gaaacatatg cctcactgtg ccatagtgct tatgc tgaca gacatacagt cagaaattgc aacccaatat agcatt tat t tcatctatgc ggaatttatg ggcctgggga aggcagctgc actctgtatt tttccaaatc aaaagggaaa aggttgaaga atattaccaa gtgttttcta ctctactcag agacggttgg atctgaatgg tacgcttata tgttcatcca gtggctcagc tacaatgttg gatgcacatc gtactctcag caccgggact tgttgttcat ggagacctag ggtct tc ttg agagcgtggc acac tc tgag ctaacaaaag ttatgaaagt ccaggataag cgacttggaa aaacaaattc agttggtgga gcccaggttc cccttttgat aaatctacaa ttatcatttg ttccaagttc tgttaagaat gatccat tag tccaagcagc atgctctgtt gatgtgaaga agagactttg gaatttgtgt tgatgttctg ttctctctga taaagttcta atctggaaca ggttaaaaat accctgctga aatcttcctg tgc aggagac gg cat gg aat gttggatact accaccagat gacctggctt cactctacca aggagcagtg catgggtgtt gaaactgtga aagaacaatt gttctactga gtggcttata agttgattgt agc tgaaaag tggac taaaa caaattggga ttgcttcagg agcggctatc aaagttttat gaggagggat tgtcgagat t tatttctatg attcactttt agcgagagac gatgaatgat ggttaccaga cacaacaagt tgatattgaa gacagatttc agtgaagtag ggtatgtc ac 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 SEQ I WO 00/61605 PCT/USOO/09417 tcaaagaata ataatgggta tattgataaa ttttaaaatt ggtatatttg 1950 aaataaagtt gaatattata tataaaaaaa aaaaaaaaaa aa 1992 Met Lys Thr Arg Asp Gly Val Arg <210> <211> <212> <213> <220> <223> <400> Gly Gly Ser Val Ser Tyr Gin Lys Val Ile Tyr Asn Arg Tyr Val Gly Ile Asp Arg Ser Phe Thr Ile Leu Ala Pro Pro Asn Vai Lys Met Val Ilie Ile Leu Pro Gin Gly Thr Phe Ala 110 Gly His Gly Gly Ser Leu Ser Ala Ile Asp Giu Glu Pro Asp Ser Ser Trp Arg 10 Pro Gly Phe Thr Gly 25 Ile His Ser Thr Asn 40 Thr Leu Arg Gly Ala 55 Gly His Arg Asp Ser 70 Gly Ala Ala Val Val 85 Lys Lys Giu Gly Trp 100 Trp Asp Ala Giu Glu 115 Giu Asp Asn Ser Arg 130 Asn Ala Asp Ser Ser 145 Cys Thr Pro Leu Met 160 Leu Lys Ser Pro Asp 175 Ser Trp Thr Lys Lys 190 Arg Ile Ser Lys Leu Gly Asn Giu Val Trp His Arg Phe Leu Ile Tyr Giu Ser Gly 2 442

PRT

Homo sapiens deduced amino acid sequence of PSMA-iike protein 2 Ser Phe Val Giu Val Giu Pro Gly Leu Giu Ser Gly Pro S er Leu Ser Thr Pro Phe Thr Arg 105 Leu 120 Gin 135 Giy 150 Leu 165 Phe 180 Ser 195 Gly 210 Leu Gly Ser Thr Glu.

125 Giu Arg Gly Val Ala 140 Asn Tyr Thr Leu Arg 155 Val Tyr Asn Leu Thr 170 Glu Gly Lys Ser Leu 185 Pro Giu Phe Ser Gly 200 Asn Asp Phe Glu Val 215 Trp Tyr Val Lys Tyr Met 205 Phe Phe Gin Arg Leu 220 Gly Ile Ala Ser Gly 225 SEQ2.

WO 00/61605 Arg Ala Arg Tyr Pro Leu Lys Phe Tyr Val Arg Gly Pro Phe Asp Asp Lys Ile Thr Tyr Ser Phe Thr Glu Asp Lys Ser Met Phe Leu Arg Pro Phe Lys Tyr Ala Asp Ile Glu Lys Arg Gin Glu Thr Leu <210> <211> <212> <213> <220> <223> <300> <308> <400> Tyr Thr 230 Tyr His 245 Asp Pro 260 Gly Met 275 Cys Arg 290 Tyr Asn 305 Leu Ser 320 Ile Ala 335 Asn Pro 350 Glu Arg 365 Tyr Arg 380 Gly Glu 395 Ser Lys 410 Ile Ser 425 Ser Glu 440 PCT/US00/09417 Lys Asn Trp Glu Thr Asn Lys Phe Ser Gly 235 240 Ser Val Tyr Glu Thr Tyr Glu Leu Val Glu 250 255 Met Phe Lys Tyr His Leu Thr Val Ala Gin 265 270 Val Phe Glu Leu Ala Asn Ser Ile Val Leu 280 285 Asp Tyr Ala Val Val Leu Arg Lys Tyr Ala 295 300 Ile Ser Met Lys His Pro Gin Glu Met Lys 310 315 Phe Asp Ser Leu Phe Ser Ala Val Lys Asn 325 330 Ser Lys Phe Ser Glu Arg Leu Gin Asp Phe 340 345 Ile Leu Leu Arg Met Met Asn Asp Gin Leu 355 360 Ala Phe Ile Asp Pro Leu Gly Leu Pro Asp 370 375 His Val Ile Tyr Ala Pro Ser Ser His Asn 385 390 Ser Phe Pro Gly Ile Tyr Asp Ala Leu Phe 400 405 Val Asp Pro Ser Lys Ala Trp Gly Asp Val 415 420 Val Ala Ala Phe Thr Val Gin Ala Ala Ala 430 435 Val Ala 2653

DNA

Homo sapiens nucleotide sequence of human PSMA gene GenBank Accession No. M99487 3 SEQ 3 WO 00/61605 c tcaaaaggg tctcgctcgg aactggaccc aggcattagt gcgcgcagta ctcgcgccga accgcgcgcc tggcttcttt atgaagctac gaattgaaag accacattta aatcccagtg gatgtcctgt aattaatgaa ctcctccagg ttctctcctc acgaactgaa c tgggaaaat gttaaaaatg ccctgctgac atcttcctgg gcaggagacc gcgtggaatt ttggatacta ccaccagata acctggcttt actctaccaa ggagcagtgg atgggtgttt aaattgtgag agaacaat tt ttctactgag tggcttatat gttgattgta gctgaaaagc ggac taaaaa aaattgggat tgcttcaggc gcggctatcc aagttttatg aggagggatg gtcgagatta atttctatga ttcacttttt gtgagagac t atgaatgatc gttaccagac acaacaagta gatat tgaaa acagatttat gtgaagtagc gtatgtcact tggtatattt aaa PCT/USOO/0941 7 gccggatttc attggttcag caggtc tgga gagattgaga gagcagcagc gatgtggaat gcccgcgctg c tc ctcggc t taacattact c tgagaacat gcaggaacag gaaagaattt tgtcctaccc gatggaaatg atatgaaaat aaggaatgcc gacttcttta tgtaattgcc cccagctggc tactttgctc aggtggtgtc ctctcacacc gcagaggctg tgatgcacag gcagctggag actggaaac t tgaagtgaca aaccagacag ggtggtattg gagctttgga tgtttgcaag tgggcagagg taatgc tgac caccgc tgat cctgatgaag aagtccttcc ctggaaatga agagcacggt ac tgtatcac atccaatgtt gtgtttgagc tgctgtagtt aacatccaca tctgcagtaa ccaggacttt aactcatgtt aggccttttt tgcaggggag gcaaagtgga gttgcagcct c taagaggat cagaaagaat gaaataaagt cttctcctgg aggcagatgt tgcactctag aaacactgct gcgaattcca gcctgcaggg gagactttac cccgccgtgg acaggcgcgg gtcccgggag ctccttcacg aaaccgactc gctgtgcgct ggggcgc tgg tcctcttcgg gtggtttata ccaaagcata atatgaaagc caagaagttc ttatataatt aacaaaactt tcagcttgca ggcctggatt ctgttgagct aaataagact catcccaact agattttcaa cacatcatta gtttcggata ttgtaccacc agagggcgat ctagtgtatg aattggaacg ggacatgaaa agatatggga aagttttcag aggggccaaa ggagtcattc ctggggtgaa gtcctatcca cagcgtggaa atatcctaaa aggttaccca gcaaatgaat ttggtcttcc aagtattcct aagctcctag aaaaaatggg aggaagtctc aaagtgccct tttctacaca aaaagtcaag agaatttaca atgtgatagg atatgtcatt ctgggaqgtc accctcagag tggagcagct acactgaaaa aggaagggtg ctgggatgca gaagaatttg agaattcaag actccttcaa tcatctatag aaggaaacta gtacagcttg gtacacaacc gctttgaagg caaatctctt ccagagttca gtggcatgcc ttttgaggtg ttcttccaac atactaaaaa ttgggaaaca agtgtctatg aaacatatga taaatatcac ctcactgtgg tagccaattc catagtgctc ttaagaaagt atgctgacaa ggaaatgaag acatacagtg agaattttac agaaattgct gacaaaagca acccaatagt tctggaaaga gcatttattg ataggcatgt catctatgct tcattcccag gaatttatga cccttccaag gcctggggag tcacagtgca ggcagctgca tctttagaga atccgtattg cgtaatgggt atattgataa tgaatattat atataaaaaa tgcctctctc gtggtggaga 100 ctgataagcg 150 tggttggagg 200 gccggctctg 250 ggctgtggcc 300 tgctggcggg 350 aaatcctcca 400 atttttggat 450 ttacacagat 500 aagcaaattc 550 agcacattat 600 acatctcaat 650 tttgaaccac 700 tttcagtgct 750 ttaactatgc 800 atcaattgct 850 aggaaataag 900 tctactccga 950 gatggttgga 1000 tctgaatggt 1050 atgcttatag 1100 gttcatccaa 1150 tggctcagca 1200 acaatgttgg 1250 atgcacatcc 1300 tactctcaga 1350 accgggactc 1400 gttgttcatg 1450 gagacctaga 1500 gtcttcttgg 1550 gagcgtggcg 1600 cactctgaga 1650 taacaaaaga 1700 tatgaaagtt 1750 caggataagc 1800 gacttggaat 1850 aacaaattca 1900 gttggtggaa 1950 cccaggttcg 2000 ccttttgatt 2050 aatctacagt 2100 tatcatttga 2150 tccaagttca 2200 attaagaatg 2250 atccattagg 2300 ccaagcagcc 2350 tgctctgttt 2400 aagtgaagag 2450 gagactttga 2500 aatttgtgtg 2550 attttaaaat 2600 aaaaaaaaaa 2650 2653 SEQ 4 WO 00/61605 <210> <211> <212> <213> <220> <223> <400> Met Trp Asn Arg Arg Pro Gly Phe Phe Ser Asn Glu Phe Leu Asp Asn Phe Thr Gin Leu Ala Asp Ser Val Asn Lys Thr Asn Glu Ile Tyr Glu Asn Pro Gin Gly Arg Thr Glu PCT/USOO/09417 4 750

PRT

Homo sapiens deduced amino acid sequence of PSMA protein Leu Arg Leu Ala Glu Gin Lys Glu His Phe Val Met Asp Leu His Glu Thr Asp Ser Ala Val Ala Thr Ala Trp Leu Leu Gly Thr Asn Leu Lys Ile Pro Gin Ile Leu Ala 110 Pro Asn 125 Asn Thr 140 Ser Asp 155 Pro Glu 170 Phe Phe 185 Ile Val 200 Lys Asn 215 Asp Pro 230 Gly Trp 10 Cys Ala Gly Ala 25 Phe Leu Phe Gly 40 Ile Thr Pro Lys 55 Ala Glu Asn Ile 70 His Leu Ala Gly 85 Gin Ser Gin Trp 100 His Tyr Asp Val 115 Tyr Ile Ser Ile 130 Ser Leu Phe Glu 145 Ile Val Pro Pro 160 Gly Asp Leu Val 175 Lys Leu Glu Arg 190 Ile Ala Arg Tyr 205 Ala Gin Leu Ala 220 Ala Asp Tyr Phe 235 Asn Leu Pro Gly Leu Val Trp Phe His Asn Lys Lys Thr Glu Lys Glu Leu Leu Ile Asn Pro Pro Phe Ser Tyr Val Asp Met Gly Lys Gly Ala Ala Pro Leu Ala Ile Lys Met Lys Phe Leu Gin Asn Phe Gly Ser Tyr Glu Asp Pro Pro Ala Phe Asn Tyr Lys Ile Val Phe Lys Gly Gly Val Gly Ser Ala Tyr Phe Leu 105 Pro 120 Gly 135 Gly 150 Ser 165 Ala 180 Asn 195 Arg 210 Val 225 Lys 240 Cys Gly Ile Ser Ser Gly Lys Asn Lys Val Leu Tyr Ser Tyr Pro Asp Gly Gly Val Gin Arg SEQ WO 00/61605 245 250 Gly Asn Ile Leu Asn Leu Asn Gly Ala Gly Asp Pro Let 260 Gly Ala Asp Asp Pro Ile Thr Gly Gly Lys Trp Glu Asn Cys Leu Ser Arg Gin Tyr Pro Ala Asn Glu Tyr 275 Val Gly Leu Pro Ser Ile 290 Ala Gin Lys Leu Leu Glu 305 Ser Ser Trp Arg Gly Ser 320 Gly Phe Thr Gly Asn Phe 335 His Ser Thr Asn Glu Val 350 Leu Arg Gly Ala Val Glu 365 His Arg Asp Ser Trp Val 380 Ala Ala Val Val His Glu 395 Lys Glu Gly Trp Arg Pro 410 Asp Ala Glu Glu Phe Gly 425 Glu Asn Ser Arg Leu Leu 440 Ala Asp Ser Ser Ile Glu 455 Thr Pro Leu Met Tyr Ser 470 Lys Ser Pro Asp Glu Gly 485 Trp Thr Lys Lys Ser Pro 500 Ile Ser Lys Leu Gly Ser 515 Arg Leu Gly Ile Ala Ser 265 Ala Tyr Arg 280 Pro Val His 295 Lys Met Gly 310 Leu Lys Val 325 Ser Thr Gin 340 Thr Arg Ile 355 Pro Asp Arg 370 Phe Gly Gly 385 Ile Val Arg 400 Arg Arg Thr 415 Leu Leu Gly 430 Gin Glu Arg 445 Gly Asn Tyr 460 Leu Val His 475 Phe Glu Gly 490 Ser Pro Glu 505 Gly Asn Asp 520 Gly Arg Ala SEQ 6 Arg Gly Pro Ile Gly Ser Pro Tyr Lys Val Tyr Asn Tyr Val Ile Asp Ser Phe Ile Leu Ser Thr Gly Val Thr Leu Ile Gly Ala Asr Lys Val Ile Prc Gly Phe Glu Ala Arc PCT/US00/09417 255 i Thr Pro 270 SAla Glu 285 STyr Tyr 300 SPro Pro 315 i Val Gly 330 SMet His 345 Ile Gly.

360 Leu Gly 375 Gin Ser 390 SThr Leu 405 SAla Ser 420 1 Trp Ala 435 STyr Ile 450 g Val Asp Asn Leu Thr Lys Ser Leu Phe Ser Gly Phe Glu Val Arg Tyr Thr 465 Lys Glu 480 Tyr Glu 495 Met Pro 510 Phe Phe 525 Lys Asn WO 00/61605 530 Trp Glu Thr Asn Lys Phe Ser Gly Tyr 545 Tyr Lys Glu Thr Tyr Glu Leu Val 560 Tyr His Leu Thr Val Ala 575 Glu Ala Met Ser Phe Leu Ile Ile Pro Pro Ala Leu Ala Asn Ser 590 Val Val Leu Arg 605 Lys His Pro Gin 620 Leu Phe Ser Ala 635 Ser Glu Arg Leu 650 Arg Met Met Asn 665 Asp Pro Leu Gly 680 Tyr Ala Pro Ser 695 Gly Ile Tyr Asp 710 Ser Lys Ala Trp 725 Phe Thr Val Gin 740 Ile Val Lys Tyr Glu Met Val Lys Gin Asp Asp Gin Leu Pro Ser His Ala Leu Gly Glu Glu Lys Gin Val Leu Pro Ala Asp Lys Thr Asn Phe Phe Asp Leu Met Asp Arg Asn Lys Phe Asp Val Lys PCT/US00/09417 535 540 Pro Leu Tyr His Ser Val 550 555 Phe Tyr ALp Pro Met Phe 565 570 Arg Gly Gly Met Val Phe 580 585 Phe Asp Cys Arg Asp Tyr 595 600 Lys Ile Tyr Ser Ile Ser 610 615 Tyr Ser Val Ser Phe Asp 625 630 Thr Glu Ile Ala Ser Lys 640 645 Lys Ser Asn Pro Ile Val 655 660 Phe Leu Glu Arg Ala Phe 670 675 Pro Phe Tyr Arg His Val 685 690 Tyr Ala Gly Glu Ser Phe 700 705 Ile Glu Ser Lys Val Asp 715 720 Arg Gin Ile Tyr Val Ala 730 735 Thr Leu Ser Glu Val Ala 745 750 Ala Ala Ala Glu <210> <211> <212> <213> <220> <221> <223> <400> gccttcattt 28

DNA

Artificial sequence primer_bind sense primer designed for only amplifying the first intron of the PSMA-like gene on chromosome llq tcagaacatc tcatgcat 28 SEQ 7 WO 00/61605 PCT/US00/09417 <210> <211> <212> <213> <220> <221> <223> <400> gtccatataa <210> <211> <212> <213> <220> <221> <223> <400> ctcacctaat

DNA

Artificial sequence primer_bind antisense primer designed for only amplifying the first intron of the PSMAlike gene on chromosome llq 6 actttcaaga atgtg 7

DNA

Artificial sequence primer_bind sense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 2) 7 gtcagaggta 8

DNA

Artificial sequence primer_bind antisense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 2) 8 <210> <211> <212> <213> <220> <221> <223> <400> agtatagtcc tcctcagatg <210> <211> <212> <213> <220> <221> <223> <400> caaagtactt 9 24

DNA

Artificial sequence primer_bind sense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 3) 9 ttgtgtaact ctgc 24 SEQ 8 WO 00/61605 PCT/US00/09417 <210> <211> <212> <213> <220> <221> <223> <400> cataggaaag <210> <211> <212> <213> <220> <221> <223> <400> cctgaaggat <210> <211> <212> <213> <220> <221> <223> <400> gaccctttaa <210> <211> <212> <213> <220> <221> <223> <400> atgtccaaca 22

DNA

Artificial sequence primer_bind antisense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 3) tagttgacac gg 22 11 22

DNA

Artificial sequence primer_bind sense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 4) 11 tcattcaccc tc 22 12 24

DNA

Artificial sequence primer_bind antisense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 4) 12 ttatcggctg aaca 24 13 22

DNA

Artificial sequence primer_bind sense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exons 5-6) 13 gtccccatgc ag 22 SEQ 9 WO 00/61605 <210> <211> <212> <213> <220> <221> <223> <400> gacatgctta <210> <211> <212> <213> <220> <221> <223> <400> gaaccgtttg <210> <211> <212> <213> <220> <221> <223> <400> ttacccaaat <210> <211> <212> <213> <220> <221> <223> <400> gcagatgctc PCT/USOO/09417 14 22

DNA

Artificial sequence primer_bind antisense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exons 5-6) 14 gtccattgta cc 22 22

DNA

Artificial sequence primerbind sense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 7) aatgaaactg ag 22 16 22

DNA

Artificial sequence primer_bind antisense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 7) 16 agccatccat gg 22 17 23

DNA

Artificial sequence primer_bind sense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exons 8-9) 17 aataagtgaa tcc 23 SEQ WO 00/61605 <210> <211> <212> <213> <220> <221> <223> <400> ccagcacata <210> <211> <212> <213> <220> <221> <223> <400> tagatgctat <210> <211> <212> <213> <220> <221> <223> <400> aaactgagac <210> <211> <212> <213> <220> <221> <223> <400> ctgggcttgg PCT/US00/09417 18 24

DNA

Artificial sequence primerbind antisense oligonucleotide primer based upon intronic sequences of the PSMA.genomic clone used to amplify the corresponding regions of the PSMA-like gene (exons 8-9) 18 acagttactt gate 24 19 22

DNA

Artificial sequence primer_bind sense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 19 tgagtcgttt gc 22 22

DNA

Artificial sequence primer_bind antisense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon tcagataggc tg 22 21 22

DNA

Artificial sequence primer_bind sense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 11) 21 tagtgtcctg gg 22 SEQ 11 WO 00/61605 <210> <211> <212> <213> <220> <221> <223> <400> gcttggcaaa <210> <211> <212> <213> <220> <221> <223> <400> tgtcgttaat <210> <211> <212> <213> <220> <221> <223> <400> ttaactagac <210> <211> <212> <213> <220> <221> <223> <400> tggtaggaat PCT/US00/09417 22 24

DNA

Artificial sequence primerbind antisense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 11) 22 caagtcctgg ctac 24 23 22

DNA

Artificial sequence primer_bind sense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 12) 23 atgggtcagc tc 22 24 22

DNA

Artificial sequence primer_bind antisense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 12) 24 tgctgctcct ag 22 22

DNA

Artificial sequence primer_bind sense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 13) ttagcagtgg tc 22 SEQ 12.

rt WO 00/61605 <210> <211> <212> <213> <220> <221> <223> <400> gatgctacta <210> <211> <212> <213> <220> <221> <223> <400> cttctggtta <210> <211> <212> <213> <220> <221> <223> <400> caatcccaca <210> <211> <212> <213> <220> <221> <223> <400> agaatggggt PCT/USOO/09417 26 22

DNA

Artificial sequence primer_bind antisense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 13) 26 atgggctacc tc 22 27 22

DNA

Artificial sequence primerbind sense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 14) 27 atggacatct ag 22 28 22

DNA

Artificial sequence primer_bind antisense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 14) 28 ctgaattcag tg 22 29 22

DNA

Artificial sequence primer_bind sense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 29 ttagtttaat gg 22 SEQ 13 WO 00/61605 <210> <211> <212> <213> <220> <221> <223> <400> tgagtcactt <210> <211> <212> <213> <220> <221> <223> <400> ttgtaagcta <210> <211> <212> <213> <220> <221> <223> <400> agttcagcaa <210> <211> <212> <213> <220> <221> <223> <400> gggtggtcct PCT/USOO/09417 21

DNA

Artificial sequence primerbind antisense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon tttggagtca g 21 31 22

DNA

Artificial sequence primer_bind sense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exons 16-17) 31 tccctataag ag 22 32 22

DNA

Artificial sequence primer_bind antisense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exons 16-17) 32 cagtcatgtt ag 22 33 22

DNA

Artificial sequence primerbind sense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 18) 33 gaaaccaatc cc 22 SEQ 14 1 WO 00/61605 <210> <211> <212> <213> <220> <221> <223> <400> gtgatattac <210> <211> <212> <213> <220> <221> <223> <400> atccaggaat <210> <211> <212> <213> <220> <221> <223> <400> ttcagtttta <210> <211> <212> <213> <220> <221> <223> <400> acagatatgt <210> PCT/USOO/09417 34 22

DNA

Artificial sequence primer_bind antisense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 18) 34 agaaaggagt c 21 22

DNA

Artificial sequence primerbind sense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 19) tgcagagtgc tc 22 36 22

DNA

Artificial sequence primer_bind antisense oligonucleotide primer based upon intronic sequences of the PSMA genomic clone used to amplify the corresponding regions of the PSMA-like gene (exon 19) 36 atccataggg ag 22 37 24

DNA

Artificial sequence primerbind sense primer (exon 10) used for performing PCR on cDNAs from various tissues 37 cattctggga ggtc 24 SEQ I) 9' WO 00/61605 PCT/USOO/09417 <211> 24 <212> DNA <213> Artificial sequence <220> <221> primer_bind <223> antisense primer (exon 16) used for performing PCR on cDNAs from various tissues <400> 38 actgtgatac.agtggatagc cgct 24 SEQ 16

Claims (19)

1. A DNA fragment which encodes a mammalian prostate specific membrane antigen-like protein or fragments thereof having the amino acid sequence as set forth in SEQ ID No. 2.

2. The DNA fragment of claim 1, wherein said DNA fragment has the sequence shown in SEQ ID No. 1 or fragments thereof.

3. A vector comprising the DNA fragment of claim 1 or claim 2 and regulatory elements necessary for expression of the DNA in a cell.

4. A host cell transfected with the vector of claim 3, wherein said vector expresses a prostate specific membrane antigen-like protein.

5. The host cell of claim 4, wherein said cell is selected from the group consisting of a bacterial cell, a mammalian cell, a plant cell and an insect cell.

6. An isolated and purified prostate specific membrane antigen-like protein coded for by DNA having the nucleic acid sequence as set forth in SEQ ID No. 1 or fragments thereof.

7. The isolated and purified prostate specific membrane antigen-like protein of claim 6, wherein said prostate specific membrane antigen-like protein has an amino acid sequence shown in SEQ ID No. 2 or fragments thereof. 2

8. An antibody directed against the prostate specific membrane antigen-like protein of claim 6.

9. A method of distinguishing prostate specific membrane antigen gene S 30 expression from prostate specific membrane antigen-like gene expression in a sample, i comprising the steps of: contacting the sample with one or more oligonucleotide primer(s) selected from the group consisting of SEQ ID Nos. 5 to 38 under hybridizing conditions, wherein said sample comprises RNA; -39- 11/12/03,sw 12328spa39 performing RT-PCR on said sample, thereby producing RT-PCR products; contacting said RT-PCR products with an appropriate restriction enzyme, thereby producing digested RT-PCR products; and analyzing said digested RT-PCR products, wherein prostate specific membrane antigen gene expression is distinguished from prostate specific membrane antigen-like gene expression by detection of fragment size(s) in the digested RT-PCR products, wherein digested prostate specific membrane antigen-specific RT-PCR products comprise different predicted fragment size(s) compared with digested prostate specific membrane antigen-like specific RT-PCR products.

The method of claim 9, wherein said sample is selected from the group consisting of blood cells, cells growing in culture, biopsied cells, epithelial cells, endothelial cells, urine and seminal fluid.

11. The method of claim 9, wherein said restriction enzyme is selected from the group consisting of EcoRI, AccI Bsp1286I, Sse9I, Tsp509I, TspEI, TspRI, Bstl 1071, AciI, MspAI, NspBII, RsaI, HaeIII and SspL

12. The method of claim 9, wherein when said oligonucleotide primers are SEQ ID No. 37 and SEQ ID No. 38, and said restriction enzyme is EcoR, presence of fragment sizes of 348 nucleotides and 207 nucleotides indicates PSMA gene expression in said 5 x sample, while presence of fragment size of 555 nucleotides indicates PSMA-like gene o *expression in said sample. 2

13. The method of claim 9, wherein when said oligonucleotide primers are SEQ ID No. 37 and SEQ ID No. 38, and said restriction enzyme is Acc, presence of fragment sizes of 506 nucleotides and 49 nucleotides indicates prostate specific membrane antigen gene expression in said sample, while presence of fragment sizes of 319 nucleotides, 187 30 nucleotides and 49 nucleotides indicates prostate specific membrane antigen-like gene expression in said sample.

14. A method of distinguishing prostate specific membrane antigen protein having the amino acid sequence set forth in SEQ ID No. 4 from prostate specific Sll/12/03,sw12328spa,40 membrane antigen-like protein having the amino acid sequence set forth in SEQ ID No. 2 in a sample, comprising the steps of: contacting a sample with at least one antibody specific for a prostate specific membrane antigen-like protein under appropriate conditions; and detecting binding of said antibody or antibodies, wherein binding is indicative of the presence of prostate specific membrane antigen-like proteins in said sample.

The method of claim 14, wherein said sample is selected from the group consisting of blood cells, cells growing in culture, biopsied cells, epithelial cells, endothelial cells, urine and seminal fluid.

16. The method of claim 14, wherein said antibody specific for a PSMA-like protein is specific for a region of said PSMA-like protein and does not cross-react with a PSMA protein.

17. The method of claim 14, wherein upon binding, said detecting is by means S 20 selected from the group consisting of a colorimetric assay, fluorescence, radioautography, nuclear medicine detection, electron microscopy, enzymatic assays, enzyme-linked S: immunoassays and MRI.

18. A method of screening for prostate specific membrane antigen-like ligands, 0 25 comprising the steps of: contacting a prostate specific membrane antigen-like protein or fragment thereof having the amino acid sequence set forth in SEQ ID No.2 with potential ligands under conditions that permit protein-protein binding; removing non-specific protein-protein binding; and eluting protein bound to said prostate specific membrane antigen- like protein or fragment thereof, wherein said protein bound to said prostate specific 27/04/04,atl 2328.specipgs.doc,4 -41- membrane antigen-like protein or fragment thereof is a ligand for said prostate specific membrane antigen-like protein.

19. The DNA fragment of claim 1, substantially as hereinbefore described with reference to any one of the Examples. DATED this 27 th day of April, 2004 SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH By their Patent Attorneys: CALLINAN LAWRIE 9* 27/04/04,atl 2328.specipgs.doc,42 -42-