BPC-1: a secreted brain-specific protein expressed and secreted by prostate and bladder cancer cells

Described is a novel gene and its encoded secreted tumor antigen, termed BPC-1, and to diagnostic and therapeutic methods and compositions useful in the management of various cancers which express BPC-1, particularly including prostate cancer and bladder cancer. In human normal tissues, BPC-1 is only expressed in certain tissues of the brain. However, BPC-1 is expressed at high levels in prostate cancer cells and is also expressed in bladder cancer cells. The structure of BPC-1 includes a signal sequence and a CUB domain. BPC-1 protein is secreted. Preliminary experimental evidence suggests that BPC-1 is directly involved in oncogenesis or maintenance of the transformed phenotype of cancer cells expressing BPC-1. BPC-1 also appears to bind specifically to a cellular protein expressed in prostate cancer cells and other cells.

FIELD OF THE INVENTION
 The invention described herein relates to a novel gene and its encoded
 secreted tumor antigen, termed BPC-1, and to diagnostic and therapeutic
 methods and compositions useful in the management of various cancers which
 express BPC-1, particularly including prostate cancer and bladder cancer.
 BACKGROUND OF THE INVENTION
 Cancer is the second leading cause of human death next to coronary disease.
 Worldwide, millions of people die from cancer every year. In the United
 States alone, cancer causes the death of well over a half-million people
 each year, with some 1.4 million new cases diagnosed per year. While
 deaths from heart disease have been declining significantly, those
 resulting from cancer generally are on the rise. In the early part of the
 next century, cancer is predicted to become the leading cause of death.
 Worldwide, several cancers stand out as the leading killers. In particular,
 carcinomas of the lung, prostate, breast, colon, pancreas, and ovary
 represent the primary causes of cancer death. These and virtually all
 other carcinomas share a common lethal feature. With very few exceptions,
 metastatic disease from a carcinoma is fatal. Moreover, even for those
 cancer patients who initially survive their primary cancers, common
 experience has shown that their lives are dramatically altered. Many
 cancer patients experience strong anxieties driven by the awareness of the
 potential for recurrence or treatment failure. Many cancer patients
 experience physical debilitations following treatment. Many cancer
 patients experience a recurrence.
 Generally speaking, the fundamental problem in the management of the
 deadliest cancers is the lack of effective and non-toxic systemic
 therapies. Molecular medicine, still very much in its infancy, promises to
 redefine the ways in which these cancers are managed. Unquestionably,
 there is an intensive worldwide effort aimed at the development of novel
 molecular approaches to cancer diagnosis and treatment. For example, there
 is a great interest in identifying truly tumor-specific genes and proteins
 that could be used as diagnostic and prognostic markers and/or therapeutic
 targets or agents. Research efforts in these areas are encouraging, and
 the increasing availability of useful molecular technologies has
 accelerated the acquisition of meaningful knowledge about cancer.
 Nevertheless, progress is slow and generally uneven.
 As discussed below, the management of prostate cancer serves as a good
 example of the limited extent to which molecular biology has translated
 into real progress in the clinic. With limited exceptions, the situation
 is more or less the same for the other major carcinomas mentioned above.
 Worldwide, prostate cancer is the fourth most prevalent cancer in men. In
 North America and Northern Europe, it is by far the most common male
 cancer and is the second leading cause of cancer death in men. In the
 United States alone, well over 40,000 men die annually of this
 disease--second only to lung cancer. Despite the magnitude of these
 figures, there is still no effective treatment for metastatic prostate
 cancer. Surgical prostatectomy, radiation therapy, hormone ablation
 therapy, and chemotherapy remain fixed as the main treatment modalities.
 Unfortunately, these treatments are ineffective for many and are often
 associated with significant undesirable consequences.
 On the diagnostic front, the lack of a prostate tumor marker that can
 accurately detect early-stage, localized tumors remains a significant
 limitation in the management of this disease. Although the serum PSA assay
 has been a very useful tool, its specificity and general utility is widely
 regarded as lacking in several important respects, as further discussed
 below. Most prostate cancers initially occur in the peripheral zone of the
 prostate gland, away from the urethra. Tumors within this zone may not
 produce any symptoms and, as a result, most men with early-stage prostate
 cancer will not present clinical symptoms of the disease until significant
 progression has occurred. Tumor progression into the transition zone of
 the prostate may lead to urethral obstruction, thus producing the first
 symptoms of the disease. However, these clinical symptoms are
 indistinguishable from the common non-malignant condition of benign
 prostatic hyperplasia (BPH). Early detection and diagnosis of prostate
 cancer currently relies on digital rectal examinations (DRE), prostate
 specific antigen (PSA) measurements, transrectal ultrasonography (TRUS),
 and transrectal needle biopsy (TRNB). At present, serum PSA measurement in
 combination with DRE represent the leading tool used to detect and
 diagnose prostate cancer. Both have major limitations which have fueled
 intensive research into finding better diagnostic markers of this disease.
 Similarly, there is no available marker that can predict the emergence of
 the typically fatal metastatic stage of prostate cancer. Diagnosis of
 metastatic stage is presently achieved by open surgical or laparoscopic
 pelvic lymphadenectomy, whole body radionuclide scans, skeletal
 radiography, and/or bone lesion biopsy analysis. Clearly, better imaging
 and other less invasive diagnostic methods offer the promise of easing the
 difficulty those procedures place on a patient, as well as improving
 diagnostic accuracy and opening therapeutic options. A similar problem is
 the lack of an effective prognostic marker for determining which cancers
 are indolent and which ones are or will be aggressive. PSA, for example,
 fails to discriminate accurately between indolent and aggressive cancers.
 Until there are prostate tumor markers capable of reliably identifying
 early-stage disease, predicting susceptibility to metastasis, and
 precisely imaging tumors, the management of prostate cancer will continue
 to be extremely difficult.
 PSA is the most widely used tumor marker for screening, diagnosis, and
 monitoring prostate cancer today. In particular, several immunoassays for
 the detection of serum PSA are in widespread clinical use. Recently, a
 reverse transcriptase-polymerase chain reaction (RT-PCR) assay for PSA
 mRNA in serum has been developed. However, PSA is not a disease-specific
 marker, as elevated levels of PSA are detectable in a large percentage of
 patients with BPH and prostatitis (25-86%)(Gao et al., 1997, Prostate 31:
 264-281), as well as in other nonmalignant disorders and in some normal
 men, a factor which significantly limits the diagnostic specificity of
 this marker. For example, elevations in serum PSA of between 4 to 10 ng/ml
 are observed in BPH, and even higher values are observed in prostatitis,
 particularly acute prostatitis. BPH is an extremely common condition in
 men. Further confusing the situation is the fact that serum PSA elevations
 may be observed without any indication of disease from DRE, and
 visa-versa. Moreover, it is now recognized that PSA is not
 prostate-specific (Gao et al., supra, for review).
 Various methods designed to improve the specificity of PSA-based detection
 have been described, such as measuring PSA density and the ratio of free
 vs. complexed PSA. However, none of these methodologies have been able to
 reproducibly distinguish benign from malignant prostate disease. In
 addition, PSA diagnostics have sensitivities of between 57-79% (Cupp &
 Osterling, 1993, Mayo Clin Proc 68:297-306), and thus miss identifying
 prostate cancer in a significant population of men with the disease.
 There are some known markers which are expressed predominantly in prostate,
 such as prostate specific membrane antigen (PSM), a hydrolase with 85%
 identity to a rat neuropeptidase (Carter et al., 1996, Proc. Natl. Acad.
 Sci. USA 93: 749; Bzdega et al., 1997, J. Neurochem. 69: 2270). However,
 the expression of PSM in small intestine and brain (Israeli et al., 1994,
 Cancer Res. 54: 1807), as well its potential role in neuropeptide
 catabolism in brain, raises concern of potential neurotoxicity with
 anti-PSM therapies. Preliminary results using an Indium-111 labeled,
 anti-PSM monoclonal antibody to image recurrent prostate cancer show some
 promise (Sodee et al., 1996, Clin Nuc Med 21: 759-766). More recently
 identified prostate cancer markers include

DETAILED DESCRIPTION OF THE INVENTION
 Unless otherwise defined, all terms of art, notations and other scientific
 terminology used herein are intended to have the meanings commonly
 understood by those of skill in the art to which this invention pertains.
 In some cases, terms with commonly understood meanings are defined herein
 for clarity and/or for ready reference, and the inclusion of such
 definitions herein should not necessarily be construed to represent a
 substantial difference over what is generally understood in the art. The
 techniques and procedures described or referenced herein are generally
 well understood and commonly employed using conventional methodology by
 those skilled in the art, such as, for example, the widely utilized
 molecular cloning methodologies described in Sambrook et al., Molecular
 Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor
 Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures
 involving the use of commercially available kits and reagents are
 generally carried out in accordance with manufacturer defined protocols
 and/or parameters unless otherwise noted.
 As used herein, the terms "advanced prostate cancer", "locally advanced
 prostate cancer", "advanced disease" and "locally advanced disease" mean
 prostate cancers which have extended through the prostate capsule, and are
 meant to include stage C disease under the American Urological Association
 (AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, and
 stage T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system.
 In general, surgery is not recommended for patients with locally advanced
 disease, and these patients have substantially less favorable outcomes
 compared to patients having clinically localized (organ-confined) prostate
 cancer. Locally advanced disease is clinically identified by palpable
 evidence of induration beyond the lateral border of the prostate, or
 asymmetry or induration above the prostate base. Locally advanced prostate
 cancer is presently diagnosed pathologically following radical
 prostatectomy if the tumor invades or penetrates the prostatic capsule,
 extends into the surgical margin, or invades the seminal vesicles.
 As used herein, the terms "metastatic prostate cancer" and "metastatic
 disease" mean prostate cancers which have spread to regional lymph nodes
 or to distant sites, and are meant to include stage D disease under the
 AUA system and stage T.times.N.times.M+ under the TNM system. As is the
 case with locally advanced prostate cancer, surgery is generally not
 indicated for patients with metastatic disease, and hormonal (androgen
 ablation) therapy is the preferred treatment modality. Patients with
 metastatic prostate cancer eventually develop an androgen-refractory state
 within 12 to 18 months of treatment initiation, and approximately half of
 these patients die within 6 months thereafter. The most common site for
 prostate cancer metastasis is bone. Prostate cancer bone metastases are,
 on balance, characteristically osteoblastic rather than osteolytic (i.e.,
 resulting in net bone formation). Bone metastases are found most
 frequently in the spine, followed by the femur, pelvis, rib cage, skull
 and humerus. Other common sites for metastasis include lymph nodes, lung,
 liver and brain. Metastatic prostate cancer is typically diagnosed by open
 or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans,
 skeletal radiography, and/or bone lesion biopsy.
 As used herein, the term "polynucleotide" means a polymeric form of
 nucleotides of at least 10 bases or base pairs in length, either
 ribonucleotides or deoxynucleotides or a modified form of either type of
 nucleotide, and is meant to include single and double stranded forms of
 DNA.
 As used herein, the term "polypeptide" means a polymer of at least 10 amino
 acids. Throughout the specification, standard three letter or single
 letter designations for amino acids are used.
 As used herein, the terms "hybridize", "hybridizing", "hybridizes" and the
 like, used in the context of polynucleotides, are meant to refer to
 conventional hybridization conditions, preferably such as hybridization in
 50% formamide/6.times.SSC/0.1% SDS/100 .mu.g/ml ssDNA, in which
 temperatures for hybridization are above 37 degrees C and temperatures for
 washing in 0.1.times.SSC/0.1% SDS are above 55 degrees C, and most
 preferably to stringent hybridization conditions.
 In the context of amino acid sequence comparisons, the term "identity" is
 used to express the percentage of amino acid residues at the same relative
 position which are the same. Also in this context, the term "homology" is
 used to express the percentage of amino acid residues at the same relative
 positions which are either identical or are similar, using the conserved
 amino acid criteria of BLAST analysis, as is generally understood in the
 art. Further details regarding amino acid substitutions, which are
 considered conservative under such criteria, are provided below.
 Additional definitions are provided throughout the subsections which
 follow.
 MOLECULAR AND BIOCHEMICAL FEATURES OF BPC-1
 As is further described in the Examples which follow, the BPC-1 gene and
 protein have been characterized using a number of analytical approaches.
 For example, analyses of nucleotide coding and amino acid sequences were
 conducted in order to identify potentially related molecules, as well as
 recognizable structural domains, topological features, and other elements
 within the BPC-1 mRNA and protein structure. RT-PCR and Northern blot
 analyses of BPC-1 mRNA expression were conducted in order to establish the
 range of normal and cancerous tissues expressing BPC-1 message. Western
 blot analyses of BPC-1 protein expression in experimentally transfected
 cells were conducted to determine cell localization and secretion of
 processed and unprocessed recombinant human BPC-1 protein. Functional
 assays designed to determine BPC-1 interaction with cellular binding
 partner(s) and activity were also conducted.
 BPC-1 is an oncogenic, secreted, CUB domain-containing protein which is
 expressed in prostate and bladder carcinoma cells and binds to a cellular
 protein. BPC-1 expression is exquisitely brain-specific in normal adult
 human tissues. In fetal tissues, BPC-1 expression is predominant in brain,
 but is also turned on in a number of other developing organs and tissues.
 BPC-1 gene expression is activated in human prostate cancer. In
 particular, BPC-1 is expressed at very high levels in androgen dependent
 human prostate tumor xenografts originally derived from a patient with
 high grade metastatic prostate cancer, and is expressed at lower but
 significant levels in other prostate cancer samples. BPC-1 is also
 expressed at high levels in at least some bladder carcinomas.
 The BPC-1 protein is initially translated into a 158 amino acid precursor
 containing a signal sequence. During post-translational processing, the
 signal sequence is cleaved to yield the mature 135 amino acid secreted
 protein. The 5' non-coding region of the BPC-1 gene is extremely G/C rich
 (approximately 72% G/C content, compared to 42% in the coding region and
 30% in the 3' non-coding region), implying that this region of the gene
 contains elements involved in transcriptional or translational control
 (FIG. 1) (SEQ ID NO. 1).
 The BPC-1 primary structure (SEQ ID NO. 2) contains a recognizable CUB
 domain (Complement subcomponents C1r/C1s, Uegf, Bmp1)(Borck and Beckmann,
 1993, J. Mol. Biol. 231: 539-545) which shares homology with other CUB
 domain proteins (FIG. 1; FIG. 3). CUB domains were originally found in
 complement subcomponents C1r and C1s, and were subsequently identified in
 Uegf (epidermal growth factor related sea urchin protein) and Bmp1 (bone
 morphogenetic protein 1), a protease involved in bone development.
 Functionally, CUB domains have been associated with protein interaction,
 receptor binding and other activities. Unlike other CUB domain proteins
 which have additional enzymatic functions, BPC-1 is unique in that it is
 essentially a secreted CUB domain with no other apparent functional
 domains. The CUB domain of BPC-1 could function as a protein-protein
 interaction domain, mediating interactions with other secreted molecules,
 extracellular matrix molecules and/or cell surface receptors. This would
 imply a potential growth-factor or cell stimulator function.
 The presence of a CUB domain in the BPC-1 structure further supports the
 conclusion that BPC-1 interacts with and probably binds to other proteins.
 The CUB domain, viewed as an extracellular domain involved in
 protein-protein interaction, occurs in many diverse secreted or cell
 surface proteins involved in a variety developmental processes (Borck and
 Beckmann, 1993, J. Mol. Biol. 231: 539-545). One family of proteins
 characterized by CUB domains, to which BPC-1 protein may bear some
 relation, are the Spermadhesins. The Spermadhesins are CUB domain
 containing secreted proteins produced by the seminal vesicles and are
 estimated to be about 15-18 kd in size (approx. 140 amino acids); these
 proteins function to inhibit sperm motility and are inactivated by
 proteolysis (Iwamoto et al., 1995, FEBBS Letters 368: 420-424).
 Preliminary experimental evidence suggests that BPC-1 is directly involved
 in oncogenesis or maintenance of the transformed phenotype of cancer cells
 expressing BPC-1. In this regard, BPC-1 shows transforming activity in
 soft agar assays and binds to a cellular protein expressed by cells
 including those expressing BPC-1. Taken together, this evidence indicates
 that BPC-1 is functionally involved in an oncogenic pathway, and that
 BPC-1 activity in this pathway may occur through interaction with a BPC-1
 binding partner or through binding to or association with other
 protein(s). As further described herein, this understanding leads to a
 number of potential approaches to the treatment of cancers expressing
 BPC-1, involving the inhibition of BPC-1 function.
 BPC-1 POLYNUCLEOTIDES
 One aspect of the invention provides polynucleotides corresponding or
 complementary to all or part of a BPC-1 gene, mRNA, and/or coding
 sequence, preferably in isolated form, including polynucleotides encoding
 a BPC-1 protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and
 related molecules, polynucleotides or oligonucleotides complementary to a
 BPC-1 gene or mRNA sequence or a part thereof, and polynucleotides or
 oligonucleotides which hybridize to a BPC-1 gene, mRNA, or to a
 BPC-1-encoding polynucleotide (collectively, "BPC-1 polynucleotides"). As
 used herein, the BPC-1 gene and protein is meant to include the BPC-1 gene
 and protein specifically described herein and the genes and proteins
 corresponding to other BPC-1 proteins and structurally similar variants of
 the foregoing. Such other BPC-1 proteins and variants will generally have
 coding sequences which are highly homologous to the BPC-1 and/or BPC-1-2
 coding sequences, and preferably will share at least about 50% amino acid
 identity and at least about 60% amino acid homology (using BLAST
 criteria), more preferably sharing 70% or greater homology (using BLAST
 criteria).
 A BPC-1 polynucleotide may comprise a polynucleotide having the nucleotide
 sequence of human BPC-1 as shown in FIG. 1 (SEQ ID NO. 1), a sequence
 complementary to the foregoing, or a polynucleotide fragment of any of the
 foregoing. Another embodiment comprises a polynucleotide which is capable
 of hybridizing under stringent hybridization conditions to the human BPC-1
 cDNA shown in FIG. 1 (SEQ ID NO. 1) or to a polynucleotide fragment
 thereof.
 Specifically contemplated are genomic DNA, cDNAs, ribozymes, and antisense
 molecules, as well as nucleic acid molecules based on an alternative
 backbone or including alternative bases, whether derived from natural
 sources or synthesized. For example, antisense molecules can be RNAs or
 other molecules, including peptide nucleic acids (PNAs) or non-nucleic
 acid molecules such as phosphorothioate derivatives, that specifically
 bind DNA or RNA in a base pair-dependent manner. A skilled artisan can
 readily obtain these classes of nucleic acid molecules using the BPC-1
 polynucleotides and polynucleotide sequences disclosed herein.
 Further specific embodiments of this aspect of the invention include
 primers and primer pairs, which allow the specific amplification of the
 polynucleotides of the invention or of any specific parts thereof, and
 probes that selectively or specifically hybridize to nucleic acid
 molecules of the invention or to any part thereof. Probes may be labeled
 with a detectable marker, such as, for example, a radioisotope,
 fluorescent compound, bioluminescent compound, a chemiluminescent
 compound, metal chelator or enzyme. Such probes and primers can be used to
 detect the presence of a BPC-1 polynucleotide in a sample and as a means
 for detecting a cell expressing a BPC-1 protein. Examples of such probes
 include polypeptides comprising all or part of the human BPC-1 cDNA
 sequence shown in FIG. 1 (SEQ ID NO. 1). Examples of primer pairs capable
 of specifically amplifying BPC-1 mRNAs are also described in the Examples
 which follow. As will be understood by the skilled artisan, a great many
 different primers and probes may be prepared based on the sequences
 provided in herein and used effectively to amplify and/or detect a BPC-1
 mRNA. As used herein, a polynucleotide is said to be "isolated" when it is
 substantially separated from contaminant polynucleotides which correspond
 or are complementary to genes other than the BPC-1 gene or which encode
 polypeptides other than BPC-1 gene product or fragments thereof. A skilled
 artisan can readily employ nucleic acid isolation procedures to obtain an
 isolated BPC-1 polynucleotide.
 The BPC-1 polynucleotides of the invention are useful for a variety of
 purposes, including but not limited to their use as probes and primers for
 the amplification and/or detection of the BPC-1 gene(s), mRNA(s), or
 fragments thereof; as reagents for the diagnosis and/or prognosis of
 prostate cancer and other cancers; as coding sequences capable of
 directing the expression of BPC-1 polypeptides; as tools for modulating or
 inhibiting the expression of the BPC-1 gene(s) and/or translation of the
 BPC-1 transcript(s); and as therapeutic agents.
 METHODS FOR ISOLATING BPC-1-ENCODING NUCLEIC ACID MOLECULES
 The BPC-1 cDNA sequences described herein enable the isolation of other
 polynucleotides encoding BPC-1 gene product(s), as well as the isolation
 of polynucleotides encoding BPC-1 gene product homologues, alternatively
 spliced isoforms, allelic variants, and mutant forms of the BPC-1 gene
 product. Various molecular cloning methods that can be employed to isolate
 full length cDNAs encoding a BPC-1 gene are well known (See, for example,
 Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2d edition.,
 Cold Spring Harbor Press, New York, 1989; Current Protocols in Molecular
 Biology. Ausubel et al., Eds., Wiley and Sons, 1995). For example, lambda
 phage cloning methodologies may be conveniently employed, using
 commercially available cloning systems (e.g., Lambda ZAP Express,
 Stratagene). Phage clones containing BPC-1 gene cDNAs may be identified by
 probing with a labeled BPC-1 cDNA or a fragment thereof. For example, in
 one embodiment, the BPC-1 cDNA (FIG. 1) (SEQ ID NO. 7) or a portion
 thereof can be synthesized and used as a probe to retrieve overlapping and
 full length cDNAs corresponding to a BPC-1 gene. The BPC-1 gene itself may
 be isolated by screening genomic DNA libraries, bacterial artificial
 chromosome libraries (BACs), yeast artificial chromosome libraries (YACs),
 and the like, with BPC-1 DNA probes or primers.
 RECOMBINANT DNA MOLECULES AND HOST-VECTOR SYSTEMS
 The invention also provides recombinant DNA or RNA molecules containing a
 BPC-1 polynucleotide, including but not limited to phages, plasmids,
 phagemids, cosmids, YACs, BACs, as well as various viral and non-viral
 vectors well known in the art, and cells transformed or transfected with
 such recombinant DNA or RNA molecules. As used herein, a recombinant DNA
 or RNA molecule is a DNA or RNA molecule that has been subjected to
 molecular manipulation in vitro. Methods for generating such molecules are
 well known (see, for example, Sambrook et al, 1989, supra).
 The invention further provides a host-vector system comprising a
 recombinant DNA molecule containing a BPC-1 polynucleotide within a
 suitable prokaryotic or eukaryotic host cell. Examples of suitable
 eukaryotic host cells include a yeast cell, a plant cell, or an animal
 cell, such as a mammalian cell or an insect cell (e.g., a
 baculovirus-infectible cell such as an Sf9 or HghFive cell). Examples of
 suitable mammalian cells include various prostate cancer cell lines such
 LnCaP, PC-3, DU145, LAPC-4, TsuPr1, other transfectable or transducible
 prostate cancer cell lines, as well as a number of mammalian cells
 routinely used for the expression of recombinant proteins (e.g., COS, CHO,
 293, 293T cells). More particularly, a polynucleotide comprising the
 coding sequence of a BPC-1 may be used to generate BPC-1 proteins or
 fragments thereof using any number of host-vector systems routinely used
 and widely known in the art.
 A wide range of host-vector systems suitable for the expression of BPC-1
 proteins or fragments thereof are available, see for example, Sambrook et
 al., 1989, supra; Current Protocols in Molecular Biology, 1995, supra).
 Preferred vectors for mammalian expression include but are not limited to
 pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector
 pSR.alpha.tkneo (Muller et al., 1991, MCB 11:1785). Using these expression
 vectors, BPC-1 may be preferably expressed in several prostate cancer and
 non-prostate cell lines, including for example 293, 293T, rat-1, 3T3,
 PC-3, LNCaP and TsuPr1. The host-vector systems of the invention are
 useful for the production of a BPC-1 protein or fragment thereof. Such
 host-vector systems may be employed to study the functional properties of
 BPC-1 and BPC-1 mutations.
 Mature recombinant human BPC-1 protein may be produced and secreted by
 mammalian cells transfected with a construct encoding precursor BPC-1. In
 a particular embodiment described in the Examples, 293T cells are
 transfected with an expression plasmid encoding the precursor form of
 BPC-1 (i.e., including the signal sequence) and mature BPC-1 protein is
 secreted into the cell culture medium where it may be conveniently
 isolated using standard purification methods. Mature recombinant human
 BPC-1 may also be produced by cells which process but do not secrete the
 mature protein. One example of such a system is a BPC-1 encoding
 baculovirus-infected cell. As described in the examples, such cells
 express and process high levels of BPC-1 intracellularly. The mature BPC-1
 protein may be recovered, in such cases, from cell lysates using standard
 procedures. Whether the mature BPC-1 is secreted or is retained
 intracellularly by the host cell, BPC-1 may be affinity purified from
 media or cell lysates using BPC-1 antibodies.
 Proteins encoded by the BPC-1 genes, or by fragments thereof, will have a
 variety of uses, including but not limited to generating antibodies and in
 methods for identifying ligands and other agents and cellular constituents
 that bind to a BPC-1 gene product Antibodies raised against a BPC-1
 protein or fragment thereof may be useful in diagnostic and prognostic
 assays, imaging methodologies (including, particularly, cancer imaging),
 and therapeutic methods in the management of human cancers characterized
 by expression of a BPC-1 protein, including but not limited to cancer of
 the prostate. Various immunological assays useful for the detection of
 BPC-1 proteins are contemplated, including but not limited to various
 types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA),
 enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical
 methods, and the like. Such antibodies may be labeled and used as
 immunological imaging reagents capable of detecting prostate cells (e.g.,
 in radioscintigraphic imaging methods). BPC-1 proteins may also be
 particularly useful in generating cancer vaccines, as further described
 below.
 BPC-1 Proteins
 Another aspect of the present invention provides BPC-1 proteins and
 polypeptide fragments thereof. The BPC-1 proteins of the invention include
 those specifically identified herein, as well as allelic variants,
 conservative substitution variants and homologs that can be
 isolated/generated and characterized without undue experimentation
 following the methods outlined below. Fusion proteins which combine parts
 of different BPC-1 proteins or fragments thereof, as well as fusion
 proteins of a BPC-1 protein and a heterologous polypeptide are also
 included. Such BPC-1 proteins will be collectively referred to as the
 BPC-1 proteins, the proteins of the invention, or BPC-1. As used herein,
 the term "BPC-1 polypeptide" refers to a polypeptide fragment or a BPC-1
 protein of at least 10 amino acids, preferably at least 15 amino acids.
 A specific embodiment of a BPC-1 protein comprises a polypeptide having the
 amino acid sequence of human BPC-1 as shown in FIG. 1 (SEQ ID NO. 2), from
 about amino acid residue number 1 through about amino acid residue number
 158 as shown therein. Another specific embodiment of a BPC-1 protein
 comprises a polypeptide having the amino acid sequence of human BPC-1 as
 shown in FIG. 1 (SEQ ID NO. 2), from about amino acid residue number 24
 through about amino acid residue number 158 as shown therein.
 In general, naturally occurring allelic variants of human BPC-1 will share
 a high degree of structural identity and homology (e.g., 90% or more
 identity). Typically, allelic variants of the BPC-1 proteins will contain
 conservative amino acid substitutions within the BPC-1 sequences described
 herein or will contain a substitution of an amino acid from a
 corresponding position in a BPC-1 homologue. One class of BPC-1 allelic
 variants will be proteins that share a high degree of homology with at
 least a small region of a particular BPC-1 amino acid sequence, but will
 further contain a radical departure form the sequence, such as a
 non-conservative substitution, truncation, insertion or frame shift.
 Conservative amino acid substitutions can frequently be made in a protein
 without altering either the conformation or the function of the protein.
 Such changes include substituting any of isoleucine (I), valine (V), and
 leucine (L) for any other of these hydrophobic amino acids; aspartic acid
 (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N)
 and vice versa; and serine (S) for threonine (T) and vice versa. Other
 substitutions can also be considered conservative, depending on the
 environment of the particular amino acid and its role in the
 three-dimensional structure of the protein. For example, glycine (G) and
 alanine (A) can frequently be interchangeable, as can alanine (A) and
 valine (V). Methionine (M), which is relatively hydrophobic, can
 frequently be interchanged with leucine and isoleucine, and sometimes with
 valine. Lysine (K) and arginine (R) are frequently interchangeable in
 locations in which the significant feature of the amino acid residue is
 its charge and the differing pK's of these two amino acid residues are not
 significant. Still other changes can be considered "conservative" in
 particular environments.
 BPC-1 proteins may be embodied in many forms, preferably in isolated form.
 As used herein, a protein is said to be "isolated" when physical,
 mechanical or chemical methods are employed to remove the BPC-1 protein
 from cellular constituents that are normally associated with the protein.
 A skilled artisan can readily employ standard purification methods to
 obtain an isolated BPC-1 protein. A purified BPC-1 protein molecule will
 be substantially free of other proteins or molecules which impair the
 binding of BPC-1 to antibody or other ligand. The nature and degree of
 isolation and purification will depend on the intended use. Embodiments of
 a BPC-1 protein include a purified BPC-1 protein and a functional, soluble
 BPC-1 protein. In one form, such functional, soluble BPC-1 proteins or
 fragments thereof retain the ability to bind antibody or other ligand.
 The invention also provides BPC-1 polypeptides comprising biologically
 active fragments of the BPC-1 amino acid sequence, such as a polypeptide
 corresponding to part of the amino acid sequences for BPC-1 as shown in
 FIG. 1 (SEQ ID NO. 2). Such polypeptides of the invention exhibit
 properties of the BPC-1 protein, such as the ability to elicit the
 generation of antibodies which specifically bind an epitope associated
 with the BPC-1 protein.
 BPC-1 polypeptides can be generated using standard peptide synthesis
 technology or using chemical cleavage methods well known in the art based
 on the amino acid sequences of the human BPC-1 proteins disclosed herein.
 Alternatively, recombinant methods can be used to generate nucleic acid
 molecules that encode a polypeptide fragment of a BPC-1 protein. In this
 regard, the BPC-1-encoding nucleic acid molecules described herein provide
 means for generating defined fragments of BPC-1 proteins. BPC-1
 polypeptides are particularly useful in generating and characterizing
 domain specific antibodies (e.g., antibodies recognizing an extracellular
 or intracellular epitope of a BPC-1 protein), in identifying agents or
 cellular factors that bind to BPC-1 or a particular structural domain
 thereof, and in various therapeutic contexts, including but not limited to
 cancer vaccines. BPC-1 polypeptides containing particularly interesting
 structures can be predicted and/or identified using various analytical
 techniques well known in the art, including, for example, the methods of
 Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or
 Jameson-Wolf analysis, or on the basis of immunogenicity. Fragments
 containing such structures are particularly useful in generating subunit
 specific anti-BPC-1 antibodies or in identifying cellular factors that
 bind to BPC-1.
 In a specific embodiment described in the examples which follow, mature
 secreted BPC-1 is conveniently expressed in 293T cells transfected with a
 CMV-driven expression vector encoding BPC-1 with a C-terminal 6.times.His
 and MYC tag (pcDNA3.1/mycHIS, Invitrogen). The secreted HIS-tagged BPC-1
 in the culture media may be purified using a nickel column using standard
 techniques.
 BPC-1 Antibodies
 Another aspect of the invention provides antibodies that bind to BPC-1
 proteins and polypeptides. The most preferred antibodies will selectively
 bind to a BPC-1 protein and will not bind (or will bind weakly) to
 non-BPC-1 proteins and polypeptides. Anti-BPC-1 antibodies that are
 particularly contemplated include monoclonal and polyclonal antibodies as
 well as fragments containing the antigen binding domain and/or one or more
 complementarity determining regions of these antibodies. As used herein,
 an antibody fragment is defined as at least a portion of the variable
 region of the immunoglobulin molecule which binds to its target, i.e., the
 antigen binding region.
 BPC-1 antibodies of the invention may be particularly useful in prostate
 cancer therapeutic strategies, diagnostic and prognostic assays, and
 imaging methodologies. Similarly, such antibodies may be useful in the
 treatment, diagnosis, and/or prognosis of other cancers, to the extent
 BPC-1 is also expressed or overexpressed in other types of cancer. One
 such cancer that expresses BPC-1 is bladder carcinoma.
 The invention also provides various immunological assays useful for the
 detection and quantification of BPC-1 and mutant BPC-1 proteins and
 polypeptides. Such assays generally comprise one or more BPC-1 antibodies
 capable of recognizing and binding a BPC-1 or mutant BPC-1 protein, as
 appropriate, and may be performed within various immunological assay
 formats well known in the art, including but not limited to various types
 of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA),
 enzyme-linked immunofluorescent assays (ELIFA), and the like. In addition,
 immunological imaging methods capable of detecting prostate cancer are
 also provided by the invention, including but limited to
 radioscintigraphic imaging methods using labeled BPC-1 antibodies. Such
 assays may be clinically useful in the detection, monitoring, and
 prognosis of prostate cancer, particularly advanced prostate cancer.
 BPC-1 antibodies may also be used in methods for purifying BPC-1 and mutant
 BPC-1 proteins and polypeptides and for isolating BPC-1 homologues and
 related molecules. For example, in one embodiment, the method of purifying
 a BPC-1 protein comprises incubating a BPC-1 antibody, which has been
 coupled to a solid matrix, with a lysate or other solution containing
 BPC-1 under conditions which permit the BPC-1 antibody to bind to BPC-1;
 washing the solid matrix to eliminate impurities; and eluting the BPC-1
 from the coupled antibody. Other uses of the BPC-1 antibodies of the
 invention include generating anti-idiotypic antibodies that mimic the
 BPC-1 protein.
 BPC-1 antibodies may also be used therapeutically by, for example,
 modulating or inhibiting the biological activity of a BPC-1 protein or
 targeting and destroying cancer cells expressing a BPC-1 protein or BPC-1
 binding partner. Because BPC-1 is a secreted protein which appears to bind
 to a cellular protein and because BPC-1 appears to have oncogenic
 activity, antibodies may be therapeutically useful for blocking BPC-1's
 ability to bind to its receptor or interact with other proteins through
 which it exerts its oncogenic biological activity. In a particular
 embodiment, a BPC-1 specific antibody or combination thereof (preferably a
 monoclonal antibody or combination thereof) is administered to a patient
 suffering from a BPC-1 expressing tumor such that the antibody binds to
 BPC-1 and inhibits its ability to execute its function. BPC-1 antibody
 therapy is more specifically described in the THERAPEUTIC METHODS AND
 COMPOSITIONS subsection below.
 Various methods for the preparation of antibodies are well known in the
 art. For example, antibodies may be prepared by immunizing a suitable
 mammalian host using a BPC-1 protein, peptide, or fragment, in isolated or
 immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds.,
 Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY
 (1989)). In addition, fusion proteins of BPC-1 may also be used, such as a
 BPC-1 GST-fusion protein. In a particular embodiment, a GST fusion protein
 comprising all or most of the open reading frame amino acid sequence of
 FIG. 1 may be produced and used as an immunogen to generate appropriate
 antibodies. Cells expressing or overexpressing BPC-1 may also be used for
 immunizations. Similarly, any cell engineered to express BPC-1 may be
 used. Such strategies may result in the production of monoclonal
 antibodies with enhanced capacities for recognizing endogenous BPC-1.
 Another useful immunogen comprises BPC-1 proteins linked to the plasma
 membrane of sheep red blood cells. In addition, naked DNA immunization
 techniques known in the art may be used (with or without purified BPC-1
 protein or BPC-1 expressing cells) to generate an immune response to the
 encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev.
 Immunol. 15:617-648).
 The amino acid sequence of BPC-1 as shown in FIG. 1 (SEQ ID NO. 2) may be
 used to select specific regions of the BPC-1 protein for generating
 antibodies. For example, hydrophobicity and hydrophilicity analyses of the
 BPC-1 amino acid sequence may be used to identify hydrophilic regions in
 the BPC-1 structure. Regions of the BPC-1 protein that show immunogenic
 structure, as well as other regions and domains, can readily be identified
 using various other methods known in the art, such as Chou-Fasman,
 Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf
 analysis.
 Methods for the generation of BPC-1 antibodies are further illustrated by
 way of the examples provided herein.
 Methods for preparing a protein or polypeptide for use as an immunogen and
 for preparing immunogenic conjugates of a protein with a carrier such as
 BSA, KLH, or other carrier proteins are well known in the art. In some
 circumstances, direct conjugation using, for example, carbodiimide
 reagents may be used; in other instances linking reagents such as those
 supplied by Pierce Chemical Co., Rockford, Ill., may be effective.
 Administration of a BPC-1 immunogen is conducted generally by injection
 over a suitable time period and with use of a suitable adjuvant, as is
 generally understood in the art. During the immunization schedule, titers
 of antibodies can be taken to determine adequacy of antibody formation.
 BPC-1 monoclonal antibodies are preferred and may be produced by various
 means well known in the art. For example, immortalized cell lines which
 secrete a desired monoclonal antibody may be prepared using the standard
 method of Kohler and Milstein or modifications which effect
 immortalization of lymphocytes or spleen cells, as is generally known. The
 immortalized cell lines secreting the desired antibodies are screened by
 immunoassay in which the antigen is the BPC-1 protein or BPC-1 fragment.
 When the appropriate immortalized cell culture secreting the desired
 antibody is identified, the cells may be expanded and antibodies produced
 either from in vitro cultures or from ascites fluid.
 The antibodies or fragments may also be produced, using current technology,
 by recombinant means. Regions that bind specifically to the desired
 regions of the BPC-1 protein can also be produced in the context of
 chimeric or CDR grafted antibodies of multiple species origin. Humanized
 or human BPC-1 antibodies may also be produced and are preferred for use
 in therapeutic contexts. Methods for humanizing murine and other non-human
 antibodies by substituting one or more of the non-human antibody CDRs for
 corresponding human antibody sequences are well known (see for example,
 Jones et al., 1986, Nature 321: 522-525; Riechmnan et al., 1988, Nature
 332: 323-327; Verhoeyen et al., 1988, Science 239: 1534-1536). See also,
 Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims et al.,
 1993, J. Immunol. 151: 2296. Methods for producing fully human monoclonal
 antibodies include phage display and transgenic methods (for review, see
 Vaughan et al., 1998, Nature Biotechnology 16: 535-539).
 Fully human BPC-1 monoclonal antibodies may be generated using cloning
 technologies employing large human Ig gene combinatorial libraries (i.e.,
 phage display) (Griffiths and Hoogenboom, Building an in vitro immune
 system: human antibodies from phage display libraries. In: Protein
 Engineering of Antibody Molecules for Prophylactic and Therapeutic
 Applications in Man. Clark, M. (Ed.), Nottingham Academic, pp 45-64
 (1993); Burton and Barbas, Human Antibodies from combinatorial libraries.
 Id., pp 65-82). Fully human BPC-1 monoclonal antibodies may also be
 produced using transgenic mice engineered to contain human immunoglobulin
 gene loci as described in PCT Patent Application WO98/24893, Kucherlapati
 and Jakobovits et al., published Dec. 3, 1997 (see also, Jakobovits, 1998,
 Exp. Opin. Invest. Drugs 7(4): 607-614). This method avoids the in vitro
 manipulation required with phage display technology and efficiently
 produces high affinity authentic human antibodies.
 Reactivity of BPC-1 antibodies with a BPC-1 protein may be established by a
 number of well known means, including Western blot, immunoprecipitation,
 ELISA, and FACS analyses using, as appropriate, BPC-1 proteins, peptides,
 BPC-1-expressing cells or extracts thereof.
 A BPC-1 antibody or fragment thereof of the invention may be labeled with a
 detectable marker or conjugated to a second molecule, such as a cytotoxic
 agent, and used for targeting the second molecule to a BPC-1 positive cell
 (Vitetta, E. S. et al., 1993, Immunotoxin therapy, in DeVita, Jr., V. T.
 et al., eds, Cancer: Principles and Practice of Oncology, 4th ed., J. B.
 Lippincott Co., Philadelphia, 2624-2636). Examples of cytotoxic agents
 include, but are not limited to ricin, ricin A-chain, doxorubicin,
 daunorubicin, taxol, ethiduim bromide, mitomycin, etoposide, tenoposide,
 vincristine, vinblastine, colchicine, dihydroxy anthracin dione,
 actinomycin D, diphteria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin,
 arbrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin,
 retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin,
 sapaonaria officinalis inhibitor, and glucocorticoid and other
 chemotherapeutic agents, as well as radioisotopes such as .sup.212 Bi,
 .sup.131 I, .sup.131 In, .sup.90 Y, and .sup.186 Re. Suitable detectable
 markers include, but are not limited to, a radioisotope, a fluorescent
 compound, a bioluminescent compound, chemiluminescent compound, a metal
 chelator or an enzyme. Antibodies may also be conjugated to an anti-cancer
 pro-drug activating enzyme capable of converting the pro-drug to its
 active form. See, for example, U.S. Pat. No. 4,975,287.
 Further, bi-specific antibodies specific for two or more BPC-1 epitopes may
 be generated using methods generally known in the art. Further, antibody
 effector functions may be modified so as to enhance the therapeutic effect
 of BPC-1 antibodies on the growth of cancer cells. Homodimeric antibodies
 may also be generated by cross-linking techniques known in the art (e.g.,
 Wolff et al., Cancer Res. 53: 2560-2565). Such antibodies may provide a
 means for achieving enhanced BPC-1 inhibition.
 METHODS FOR THE DETECTION OF BPC-1
 Another aspect of the present invention relates to methods for detecting
 BPC-1 polynucleotides and BPC-1 proteins, as well as methods for
 identifying a cell which expresses BPC-1.
 More particularly, the invention provides assays for the detection of BPC-1
 polynucleotides in a biological sample, such as serum, bone, prostate, and
 other tissues, urine, semen, cell preparations, and the like. Detectable
 BPC-1 polynucleotides include, for example, a BPC-1 gene or fragments
 thereof, BPC-1 mRNA, alternative splice variant BPC-1 mRNAs, and
 recombinant DNA or RNA molecules containing a BPC-1 polynucleotide. A
 number of methods for amplifying and/or detecting the presence of BPC-1
 polynucleotides are well known in the art and may be employed in the
 practice of this aspect of the invention.
 In one embodiment, a method for detecting a BPC-1 mRNA in a biological
 sample comprises producing cDNA from the sample by reverse transcription
 using at least one primer; amplifying the cDNA so produced using a BPC-1
 polynucleotides as sense and antisense primers to amplify BPC-1 cDNAs
 therein; and detecting the presence of the amplified BPC-1 cDNA. In
 another embodiment, a method of detecting a BPC-1 gene in a biological
 sample comprises first isolating genomic DNA from the sample; amplifying
 the isolated genomic DNA using BPC-1 polynucleotides as sense and
 antisense primers to amplify the BPC-1 gene therein; and detecting the
 presence of the amplified BPC-1 gene. Any number of appropriate sense and
 antisense probe combinations may be designed from the nucleotide sequences
 provided for BPC-1 (FIG. 1) (SEQ ID NO. 2) and used for this purpose. The
 invention also provides assays for detecting the presence of a BPC-1
 protein in a tissue of other biological sample such as serum, bone,
 prostate, and other tissues, urine, cell preparations, and the like.
 Methods for detecting a BPC-1 protein are also well known and include, for
 example, immunoprecipitation, immunohistochemical analysis, Western Blot
 analysis, molecular binding assays, ELISA, ELIFA and the like. For
 example, in one embodiment, a method of detecting the presence of a BPC-1
 protein in a biological sample comprises first contacting the sample with
 a BPC-1 antibody, a BPC-1-reactive fragment thereof, or a recombinant
 protein containing an antigen binding region of a BPC-1 antibody; and then
 detecting the binding of BPC-1 protein in the sample thereto.
 Methods for identifying a cell which expresses BPC-1 are also provided. In
 one embodiment, an assay for identifying a cell which expresses a BPC-1
 gene comprises detecting the presence of BPC-1 mRNA in the cell. Methods
 for the detection of particular mRNAs in cells are well known and include,
 for example, hybridization assays using complementary DNA probes (such as
 in situ hybridization using labeled BPC-1 riboprobes, Northern blot and
 related techniques) and various nucleic acid amplification assays (such as
 RT-PCR using complementary primers specific for BPC-1, and other
 amplification type detection methods, such as, for example, branched DNA,
 SISBA, TMA and the like). Alternatively, an assay for identifying a cell
 which expresses a BPC-1 gene comprises detecting the presence of BPC-1
 protein in the cell or secreted by the cell. Various methods for the
 detection of proteins are well known in the art and may be employed for
 the detection of BPC-1 proteins and BPC-1 expressing cells.
 BPC-1 expression analysis may also be useful as a tool for identifying and
 evaluating agents which modulate BPC-1 gene expression. For example, BPC-1
 expression is significantly upregulated in prostate cancer, and may also
 be expressed in other cancers. Identification of a molecule or biological
 agent that could inhibit BPC-1 expression or over-expression in cancer
 cells may be of therapeutic value. Such an agent may be identified by
 using a screen that quantifies BPC-1 expression by RT-PCR, nucleic acid
 hybridization or antibody binding.
 ASSAYS FOR DETERMINING BPC-1 EXPRESSION STATUS
 Determining the status of BPC-1 expression patterns in an individual may be
 used to diagnose cancer and may provide prognostic information useful in
 defining appropriate therapeutic options. Similarly, the expression status
 of BPC-1 may provide information useful for predicting susceptibility to
 particular disease stages, progression, and/or tumor aggressiveness. The
 invention provides methods and assays for determining BPC-1 expression
 status and diagnosing cancers which express BPC-1, such as prostate and
 bladder cancers.
 In one aspect, the invention provides assays useful in determining the
 presence of cancer in an individual, such as prostate and bladder cancers,
 comprising detecting a significant increase in BPC-1 mRNA or protein
 expression in a test cell or tissue sample relative to expression levels
 in the corresponding normal cell or tissue. The presence of BPC-1 mRNA
 may, for example, be evaluated in tissue samples of the colon, lung,
 prostate, pancreas, bladder, breast, ovary, cervix, testis, head and neck,
 brain, stomach, etc. The presence of significant BPC-1 expression in any
 of these tissues may be useful to indicate the emergence, presence and/or
 severity of these cancers, since the corresponding normal tissues do not
 express BPC-1 mRNA or express it at lower levels.
 In a related embodiment, BPC-1 expression status may be determined at the
 protein level rather than at the nucleic acid level. For example, such a
 method or assay would comprise determining the level of BPC-1 protein
 expressed by cells in a test tissue sample or in serum, semen or urine,
 and comparing the level so determined to the level of BPC-1 expressed in a
 corresponding normal sample. In one embodiment, the presence of BPC-1
 protein is evaluated, for example, using immunohistochemical methods.
 BPC-1 antibodies or binding partners capable of detecting BPC-1 protein
 expression may be used in a variety of assay formats well known in the art
 for this purpose. In another embodiment, the presence of secreted BPC-1
 protein in serum or urine or other body fluids is examined.
 Because BPC-1 is a secreted protein expressed in prostate, bladder, and
 possibly other cancers, assays for detecting and quantifying BPC-1 in
 blood or serum are expected to be useful for the detection, diagnosis,
 prognosis, and/or staging of a BPC-1 expressing tumor in an individual.
 For example, BPC-1 is not expressed in normal prostate, but is expressed
 in prostate and bladder cancers. Accordingly, detection of serum BPC-1 may
 provide an indication of the presence of a prostate or bladder tumor.
 Diagnosis of prostate or bladder cancer may be made on the basis of this
 information and/or other information. In respect of prostate cancer, for
 example, such other information may include serum PSA measurements, DRE
 and/or ultrasonography. Further, the level of BPC-1 detected in the serum
 may provide information useful in staging or prognosis. For example, very
 high levels of BPC-1 protein in serum may suggest a larger and/or more
 aggressive tumor.
 The brain-specific expression of BPC-1 in normal tissues is expected to
 provide an important advantage of this aspect of the invention, namely,
 very low to non-existent background levels of circulating BPC-1, resulting
 in a high correlation between the presence of serum BPC-1 protein and the
 presence of cancer. This advantage is expected to result from the
 characteristics of the blood-brain barrier, a system of tight junctions in
 capillaries of the central nervous system that resists the passage of
 cells, pathogens and macromolecules into and out of the subarachnoid
 space. Accordingly, BPC-1 expressed in the brain is not expected to be
 released into the vascular system. Since no other normal tissue tested has
 demonstrated significant expression of BPC-1, the presence of serum BPC-1
 would strongly suggest the presence of a BPC-1 expressing tumor.
 In addition, peripheral blood may be conveniently assayed for the presence
 of cancer cells, including but not limited to prostate cancer, using
 RT-PCR to detect BPC-1 expression. The presence of RT-PCR amplifiable
 BPC-1 mRNA provides an indication of the presence of prostate cancer.
 RT-PCR detection assays for tumor cells in peripheral blood are currently
 being evaluated for use in the diagnosis and management of a number of
 human solid tumors. In the prostate cancer field, these include RT-PCR
 assays for the detection of cells expressing PSA and PSM (Verkaik et al.,
 1997, Urol. Res. 25: 373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:
 1195-2000; Heston et al., 1995, Clin. Chem. 41: 1687-1688). RT-PCR assays
 are well known in the art.
 A related aspect of the invention is directed to predicting susceptibility
 to developing cancer in an individual. In one embodiment, a method for
 predicting susceptibility to cancer comprises detecting BPC-1 mRNA or
 BPC-1 protein in a tissue sample, its presence indicating susceptibility
 to cancer, wherein the degree of BPC-1 mRNA expression present is
 proportional to the degree of susceptibility. In a specific embodiment,
 the presence of BPC-1 in prostate tissue is examined, with the presence of
 BPC-1 in the sample providing an indication of prostate cancer
 susceptibility (or the emergence or existence of a prostate tumor). In
 another specific embodiment, the presence of BPC-1 in bladder tissue is
 examined, with the presence of BPC-1 in the sample providing an indication
 of bladder cancer susceptibility (or the emergence or existence of a
 bladder tumor). In yet another specific embodiment, the presence of BPC-1
 in serum is examined, with the presence of BPC-1 providing an indication
 of susceptibility to (or presence of) a BPC-1 expressing tumor, such as a
 bladder or prostate tumor. In another embodiment, the presence of BPC-1 in
 urine is examined, with the presence of BPC-1 therein providing an
 indication of susceptibility to (or presence of) a BPC-1 expressing
 bladder tumor.
 Yet another related aspect of the invention is directed to methods for
 gauging tumor aggressiveness. In one embodiment, a method for gauging
 aggressiveness of a tumor comprises determining the level of BPC-1 mRNA or
 BPC-1 protein expressed by cells in a sample of the tumor, comparing the
 level so determined to the level of BPC-1 mRNA or BPC-1 protein expressed
 in a corresponding normal tissue taken from the same individual or a
 normal tissue reference sample, wherein the degree of BPC-1 mRNA or BPC-1
 protein expression in the tumor sample relative to the normal sample
 indicates the degree of aggressiveness. In a specific embodiment,
 aggressiveness of prostate tumors is evaluated by determining the extent
 to which BPC-1 is expressed in the tumor cells, with higher expression
 levels indicating more aggressive tumors.
 In a related embodiment, serum levels of BPC-1 may be used to provide an
 indication of the extent and aggressiveness of a BPC-1 expressing tumor,
 wherein higher levels of serum BPC-1 may suggest a more advanced and more
 aggressive tumor. Serum BPC-1 measurements over time would be expected to
 provide further information, wherein an increase in BPC-1 would be
 expected to reflect progression and the rate of the increase would be
 expected to correlate with aggressiveness. Similarly, a decline in serum
 BPC-1 would be expected to reflect a slower growing or regressing tumor.
 The identification of BPC-1 in serum may be useful to detect tumor
 initiation and early stage disease, particularly since background BPC-1
 interference is expected to be minimal to non-existent in view of the
 BPC-1 brain specific expression profile in normal individuals. In patients
 who have undergone surgery or therapy, serum BPC-1 levels would be useful
 for monitoring treatment response and potential recurrence. As an
 alternative or adjunct to serum BPC-1 measurements, the presence and
 levels of BPC-1 secreted in urine may be useful in relation to bladder
 cancer.
 Methods for detecting and quantifying the expression of BPC-1 mRNA or
 protein are described herein and use standard nucleic acid and protein
 detection and quantification technologies well known in the art. Standard
 methods for the detection and quantification of BPC-1 mRNA include in situ
 hybridization using labeled BPC-1 riboprobes, Northern blot and related
 techniques using BPC-1 polynucleotide probes, RT-PCR analysis using
 primers specific for BPC-1, and other amplification type detection
 methods, such as, for example, branched DNA, SISBA, TMA and the like. In a
 specific embodiment, semi-quantitative RT-PCR may be used to detect and
 quantify BPC-1 mRNA expression as described in the Examples which follow.
 Any number of primers capable of amplifying BPC-1 may be used for this
 purpose, including but not limited to the various primer sets specifically
 described herein. Standard methods for the detection and quantification of
 protein may be used for this purpose. In a specific embodiment, polyclonal
 or monoclonal antibodies specifically reactive with the wild-type BPC-1
 protein may be used in an immunohistochemical assay of biopsied tissue.
 ASSAYS FOR CIRCULATING AND EXCRETED BPC-1
 The mature BPC-1 is a secreted protein. Tumors which express BPC-1 would be
 expected to secrete BPC-1 into the vasculature, and/or excreted in urine
 or semen, where the protein may be detected and quantified using assays
 and techniques well known in the molecular diagnostic art. Excreted BPC-1
 may also be detectable in urine and semen. Detecting and quantifying the
 levels of circulating or excreted BPC-1 is expected to have a number of
 uses in the diagnosis, staging, and prognosis of prostate, bladder and
 other such BPC-1 expressing tumors. A number of different technical
 approaches for the detection and quantification of serum proteins are well
 known in the art.
 Detecting BPC-1 protein in urine may indicate the presence of a bladder
 cancer secreting BPC-1. Normally, significant levels of protein are not
 detected in urine, provided that renal function is normal. However,
 proteins expressed and secreted by bladder cancer cells may enter urine in
 the bladder directly, permitting their detection in urine. Interestingly,
 the BPC-1 protein exhibits a relatively high degree of stability in
 recombinant cell culture media, suggesting that the protein may also
 remain stable in urine.
 In one embodiment, a capture ELISA is used to detect and quantify BPC-1 in
 serum, urine or semen. A capture ELISA for BPC-1 comprises, generally, at
 least two monoclonal antibodies of different isotypes that recognize
 distinct epitopes of the BPC-1 protein, or one anti-BPC-1 monoclonal
 antibody and a specific polyclonal serum derived from a different species
 (e.g., rabbit, goat, sheep, hamster, etc.). In this assay, one reagent
 serves as the capture (or coating) antibody and the other as the detection
 antibody (see Example 13 herein).
 THERAPEUTIC METHODS AND COMPOSITIONS
 The identification of BPC-1 as a secreted protein which is only expressed
 in tissues of the brain in normal individuals but which is highly
 expressed in prostate cancer (as well as expressed in bladder carcinoma
 and possibly other cancers), opens a number of therapeutic approaches to
 the treatment of prostate, bladder and potentially other cancers.
 Applicants' initial functional research suggests that BPC-1 has
 transformation activity and that this activity is initiated through the
 interaction of BPC-1 to a cellular protein, or through binding to or
 association with another protein. The protein's CUB domain may also
 function as a protein-protein interaction domain, mediating interactions
 with other secreted molecules, extracellular matrix molecules and/or cell
 surface receptors.
 Accordingly, therapeutic approaches aimed at inhibiting the activity of the
 BPC-1 protein are expected to be useful for patients suffering from
 prostate cancer, bladder cancer, and other cancers expressing BPC-1. These
 therapeutic approaches generally fall into two classes. One class
 comprises various methods for inhibiting the binding of the BPC-1 protein
 to its receptor, or inhibiting its binding to or association with another
 protein. Another class comprises a variety of methods for inhibiting the
 transcription of the BPC-1 gene or translation of BPC-1 mRNA.
 A. Therapeutic Methods Based on Inhibition of BPC-1 Protein Function
 Within the first class of therapeutic approaches, the invention includes
 various methods and compositions for inhibiting the binding of BPC-1 to
 its receptor or other binding partner or its association with other
 protein(s) as well as methods for inhibiting BPC-1 function.
 A.1. Therapeutic Inhibition of BPC-1 with BPC-1 Antibodies
 In one approach, antibodies which bind to BPC-1 and thereby inhibit the
 ability of BPC-1 to bind to its coordinate binding partner, or to bind to
 or associate with other protein(s), may be used to attenuate an
 oncogenic/transformation signal pathway involving BPC-1. To the extent
 BPC-1 is involved in initiating, promoting and/or sustaining tumor cell
 growth or other tumor cell properties through a binding partner-mediated
 signal, such antibodies are expected to be therapeutically useful.
 BPC-1 antibodies and fragments thereof which are capable of inhibiting
 BPC-1 function are expected to be useful in treating prostate, bladder,
 and possibly other cancers. Such antibodies may function to inhibit BPC-1
 activity in different ways. For example, a BPC-1 antibody may prevent
 BPC-1 binding to its receptor or binding to/associating with another
 protein.
 Alternatively, a BPC-1 antibody may bind to a biologically active domain of
 the BPC-1 protein, thereby inhibiting function. In this regard, antibodies
 specifically directed to the BPC-1 CUB domain (see FIG. 1) may be
 particularly effective in either inhibiting BPC-1 binding (if the CUB
 domain is functionally involved in binding) or in otherwise inhibiting the
 CUB domain's function. Such domain-specific BPC-1 antibodies may be
 generated as previously described. For example, the CUB domain amino acid
 sequence shown in FIG. 1 may be used to generate a CUB-domain immunogen
 for the generation of such antibodies.
 With respect to the treatment of cancer with BPC-1 antibodies, a number of
 factors may be considered, including but not limited to the following.
 First, monoclonal antibodies are generally preferred, particularly those
 with very high binding affinity for the secreted BPC-1 protein. Second,
 fully human or humanized monoclonal antibodies exhibiting low or no
 antigenicity in the patient are preferred. The use of murine or other
 non-human monoclonal antibodies and human/mouse chimeric mAbs may induce
 moderate to strong immune responses in some patients. Third, the method by
 which the antibodies are delivered to the patient may vary with the type
 of cancer being treated.
 Generally, where the therapeutic objective is the inhibition of BPC-1
 activity or signal transduction in the target tumor tissue, administration
 of BPC-1 antibodies directly to the tumor site may provide local
 elimination of BPC-1 function sufficient to generate a clinical response.
 Direct administration of BPC-1 Mabs is also possible and may have
 advantages in certain contexts. For example, for the treatment of bladder
 carcinoma, BPC-1 Mabs may be injected directly into the bladder.
 Alternatively, BPC-1 antibodies may be administered systemically, which may
 result in elimination of BPC-1 function in the primary tumor, in
 circulating micrometastasis, and/or in established metastasis. The degree
 of tumor vascularization may provide guidance on which delivery approach
 is recommended. Similarly, the grade and/or stage of disease would be
 expected to provide useful information in this regard. For example, a
 higher grade, more advanced tumor may be more likely to seed metastasis,
 suggesting systemic administration in order to treat or prevent the
 emergence of metastases.
 BPC-1 mAbs may be therapeutically useful either alone or as well as
 combinations, or "cocktails", of different mAbs such as those recognizing
 different epitopes. Such mAb cocktails may have certain advantages
 inasmuch as they contain mAbs which bind to different epitopes and enhance
 the functional inhibition of BPC-1. In addition, the administration of
 BPC-1 mAbs may be combined with other therapeutic agents, including but
 not limited to various chemotherapeutic agents, androgen-blockers, and
 immune modulators (e.g., IL-2, GM-CSF).
 Treatment of cancer with a BPC-1 antibody will generally involve the
 administration of the BPC-1 antibody preparation via an acceptable route
 of administration such as intravenous injection (IV) or bolus infusion,
 typically at a dose in the range of about 0.1 to about 200 mg/kg body
 weight. Doses in the range of 10-500 mg mAb per week (or more) may be
 effective and well tolerated. An initial loading dose followed by smaller
 weekly doses of the mAb preparation may be used. As one of skill in the
 art will understand, various factors will influence the ideal dose regimen
 in a particular case. Such factors may include, for example, the binding
 affinity and half life of the mAb or mAbs used, the degree of BPG-1
 expression in the patient, the desired steady-state antibody concentration
 level, frequency of treatment, and the influence of chemotherapeutic
 agents or other therapies used in combination with the therapeutic
 composition.
 A.2. Therapeutic Inhibition of BPC-1 with Intracellular Antibodies
 In another approach, recombinant vectors encoding single chain antibodies
 which specifically bind to BPC-1 may be introduced into BPC-1 expressing
 cells via gene transfer technologies, wherein the encoded single chain
 anti-BPC-1 antibody is expressed intracellularly, binds to BPC-1 protein,
 and thereby inhibits its function. Methods for engineering such
 intracellular single chain antibodies are well known. Such intracellular
 antibodies, also known as "intrabodies", may be specifically targeted to a
 particular compartment within the cell, providing a great deal of control
 over where the inhibitory activity of the treatment will be focused. This
 technology has been successfully applied in the art (for review, see
 Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodies have been
 shown to virtually eliminate the expression of otherwise abundant cell
 surface receptors. See, for example, Richardson et al., 1995, Proc. Natl.
 Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol. Chem. 289:
 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337.
 Single chain antibodies comprise the variable domains of the heavy and
 light chain joined by a flexible linker polypeptide, and is expressed as a
 single polypeptide. Optionally, single chain antibodies may be expressed
 as a single chain variable region fragment joined to the light chain
 constant region. Well known intracellular trafficking signals may be
 engineered into recombinant polynucleotide vectors encoding such single
 chain antibodies in order to precisely target the expressed intrabody to
 the desired intracellular compartment. For example, intrabodies targeted
 to the endoplasmic reticulum (ER) may be engineered to incorporate a
 leader peptide and, optionally, a C-terminal ER retention signal, such as
 the KDEL amino acid motif. Intrabodies intended to exert activity in the
 nucleus may be engineered to include a nuclear localization signal. Lipid
 moieties may be joined to intrabodies in order to tether the intrabody to
 the cytosolic side of the plasma membrane. Intrabodies may also be
 targeted to exert function in the cytosol. For example, cytosolic
 intrabodies may be used to sequester factors within the cytosol, thereby
 preventing them from being transported to their natural cellular
 destination.
 In one embodiment, intrabodies may be used to capture BPC-1 in the ER,
 thereby preventing its maturation and secretion outside of the cell.
 ER-targeting signals and/or leader peptides may be engineered into such
 BPC-1 intrabodies in order to achieve the desired targeting. Such
 intrabodies would be expected to capture BPC-1 as its is being processed
 by the ER, thereby inhibiting BPC-1 processing or transport through the
 plasma membrane of the cell. This method would essentially prevent the
 existence of secreted mature bioactive BPC-1 at the level of the ER. Such
 BPC-1 intrabodies may be designed to bind specifically to a particular
 BPC-1 domain, including the signal sequence of precursor BPC-1.
 Endoplasmic reticulum-targeted intrabodies reactive with the BPC-1 protein
 would be expected to capture BPC-1 in the endoplasmic reticulum. In
 another embodiment, intrabodies specifically reactive with the BPC-1 CUB
 domain may be used to block BPC-1 CUB domain function within the cytosol.
 A.3. Therapeutic Inhibition of BPC-1 with Recombinant Proteins
 In another approach, recombinant molecules which are capable of binding to
 BPC-1 thereby preventing BPC-1 from accessing/binding to its coordinate
 receptor or associating with another protein involved in transmitting an
 oncogenic signal may be used to inhibit BPC-1 function. Such recombinant
 molecules may, for example, contain the reactive part(s) of a BPC-1
 specific antibody molecule. In a particular embodiment, the BPC-1 ligand
 binding domain of a BPC-1 receptor or binding partner may be engineered
 into a dimeric fusion protein comprising two BPC-1 ligand binding domains
 linked to the Fc portion of a human IgG, such as human IgG1. Such IgG
 portion may contain, for example, the C.sub.H 2 and C.sub.H 3 domains and
 the hinge region, but not the C.sub.H 1 domain. Such dimeric fusion
 proteins may be administered in soluble form to patients suffering from a
 cancer associated with the expression of BPC-1, including but not limited
 to prostate and bladder cancers, where the dimeric fusion protein
 specifically binds to BPC-1 thereby blocking BPC-1 interaction with its
 receptor or other binding partner. Such dimeric fusion proteins may be
 further combined into multimeric proteins using known antibody linking
 technologies.
 B. Therapeutic Methods Based on Inhibition of BPC-1 Transcription or
 Translation
 Within the second class of therapeutic approaches, the invention provides
 various methods and compositions for inhibiting the transcription of the
 BPC-1 gene. Similarly, the invention also provides methods and
 compositions for inhibiting the translation of BPC-1 mRNA into protein.
 In one approach, a method of inhibiting the transcription of the BPC-1 gene
 comprises contacting the BPC-1 gene with a BPC-1 antisense polynucleotide.
 In another approach, a method of inhibiting BPC-1 mRNA translation
 comprises contacting the BPC-1 mRNA with an antisense polynucleotide. In
 another approach, a BPC-1 specific ribozyme may be used to cleave the
 BPC-1 message, thereby inhibiting translation. Such antisense and ribozyme
 based methods may also be directed to the regulatory regions of the BPC-1
 gene, such as the BPC-1 promoter and/or enhancer elements. Similarly,
 proteins capable of inhibiting a BPC-1 gene transcription factor may be
 used to inhibit BPC-1 mRNA transcription. The various polynucleotides and
 compositions useful in the aforementioned methods have been described
 above. The use of antisense and ribozyme molecules to inhibit
 transcription and translation is well known in the art.
 The 5' untranslated region (UTR) of the BPC-1 cDNA of FIG. 1 is an
 extremely GC rich sequence, strongly implying the presence of
 translational control elements within this part of the BPC-1 mRNA. This
 characteristic of the BPC-1 gene suggests that blocking accessibility of
 the 5' UTR may result in inhibition of BPC-1 translation. In one approach,
 an antisense molecule complementary to the 5' UTR of the BPC-1 mRNA is
 contacted with the 5' UTR of the BPC-1 message, thereby resulting in
 hybridization which prevents endogenous BPC-1 translation factors from
 accessing the necessary activation element(s) in the BPC-1 5' UTR. A
 modification of this approach uses an polynucleotide comprising a sequence
 complementary to the 5' UTR BPC-1 mRNA joined to a ribozyme or similarly
 active polynucleotide capable of splicing the BPC-1 mRNA, thereby adding a
 second layer of translational inhibition.
 Although, in the above method, a BPC-1 splicing ribozyme may be contacted
 with the BPC-1 mRNA separately, i.e., not joined to a heterologous
 sequence such as the anti-5' UTR mentioned above, delivering the ribozyme
 or similarly active polynucleotide as part of such a heterologous BPC-1
 hybridizing polynucleotide would be expected to result in placing the
 ribozyme in direct proximity to the target sequence and may result in a
 higher level of inhibitory activity.
 Other factors which inhibit the transcription of BPC-1 through interfering
 with BPC-1 transcriptional activation may also be useful for the treatment
 of cancers expressing BPC-1, and cancer treatment methods utilizing such
 factors are also within the scope of the invention. Similarly, factors
 which are capable of interfering with BPC-1 processing may be useful for
 the treatment of cancers expressing BPC-1.
 C. General Considerations
 Gene transfer and gene therapy technologies may be used for delivering
 therapeutic polynucleotide molecules to tumor cells synthesizing BPC-1
 (i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other
 BPC-1 inhibitory molecules). A number of gene therapy approaches are known
 in the art. Recombinant vectors encoding BPC-1 antisense polynucleotides,
 ribozymes, factors capable of interfering with BPC-1 transcription,
 factors which are capable of interfering with processing and/or secretion
 of mature BPC-1, and so forth, may be delivered to target tumor cells
 using such gene therapy approaches.
 The above therapeutic approaches may be combined with chemotherapy or
 radiation therapy regimens. These therapeutic approaches may also enable
 the use of reduced dosages of concomitant chemotherapy, particularly in
 patients that do not tolerate the toxicity of the chemotherapeutic agent
 well.
 The anti-tumor activity of a particular composition (e.g., antibody,
 ribozyme, recombinant fusion protein), or a combination of such
 compositions, may be evaluated using various in vitro and in vivo assay
 systems. In vitro assays for evaluating therapeutic potential include cell
 growth assays, soft agar assays and other assays indicative of tumor
 promoting activity, binding assays capable of determining the extent to
 which a therapeutic composition will inhibit the binding of BPC-1 to its
 coordinate receptor or other binding partner, cell adhesion assays, and
 the like. For example, antibody to HER2 inhibits binding of ligand to
 receptor and leads to the inhibition of tumor growth. Additionally, for
 example, antibodies to EGFR inhibit binding of EGF to receptor, leading to
 growth arrest and tumor inhibition. See, also, the Examples below.
 Various in vitro assays for determining binding affinity of the therapeutic
 composition for its target are also known. For example, binding affinities
 of BPC-1 antibodies may be determined using a number of techniques well
 known in the art (e.g., BIAcore technology). Higher affinity BPC-1
 antibodies are expected to provide greater levels of the desired
 inhibition and are therefore preferred.
 In vivo, the effect of a BPC-1 therapeutic composition may be evaluated in
 a suitable animal model. For example, xenogenic prostate cancer models
 wherein human prostate cancer explants or passaged xenograft tissues are
 introduced into immune compromised animals, such as nude or SCID mice, are
 appropriate in relation to prostate cancer and have been described (Klein
 et al., 1997, Nature Medicine 3: 402-408). For Example, PCT Patent
 Application WO98116628, Sawyers et al., published Apr. 23, 1998, describes
 various xenograft models of human prostate cancer capable of
 recapitulating the development of primary tumors, micrometastasis, and the
 formation of osteoblastic metastases characteristic of late stage disease.
 Various bladder carcinoma models are known (see, for example, Russell et
 al., 1986, Cancer Res. 46: 2035-2040; Raghavan et al., 1992, Semin. Surg.
 Oncol. 8: 279-284; Rieger et al., 1995, Br. J. Cancer 72: 683-690;
 Oshinsky et al., 1995, J. Urol. 154: 1925-1929). Efficacy may be predicted
 using assays which measure inhibition of tumor formation, tumor regression
 or metastasis, and the like. See, also, the Examples below.
 In vivo assays which qualify the promotion of apoptosis may also be useful
 in evaluating potential therapeutic compositions. In one embodiment,
 xenografts from bearing mice treated with the therapeutic composition may
 be examined for the presence of apoptotic foci and compared to untreated
 control xenograft-bearing mice. The extent to which apoptotic foci are
 found in the tumors of the treated mice would provide an indication of the
 therapeutic efficacy of the composition.
 The therapeutic compositions used in the practice of the foregoing methods
 may be formulated into pharmaceutical compositions comprising a carrier
 suitable for the desired delivery method. Suitable carriers include any
 material which when combined with the therapeutic composition retains the
 anti-tumor function of the therapeutic composition and is non-reactive
 with the patient's immune system. Examples include, but are not limited
 to, any of a number of standard pharmaceutical carriers such as sterile
 phosphate buffered saline solutions, bacteriostatic water, and the like
 (see, generally, Remington's Pharmaceutical Sciences 16.sup.th Edition, A.
 Osal., Ed., 1980).
 Therapeutic formulations may be solubilized and administered via any route
 capable of delivering the therapeutic composition to the tumor site.
 Potentially effective routes of administration include, but are not
 limited to, intravenous, parenteral, intraperitoneal, intramuscular,
 intratumor, intradermal, intraorgan, orthotopic, and the like. A preferred
 formulation for intravenous injection comprises the therapeutic
 composition (i.e., BPC-1 monoclonal antibody) in a solution of preserved
 bacteriostatic water, sterile unpreserved water, and/or diluted in
 polyvinylchloride or polyethylene bags containing 0.9% sterile Sodium
 Chloride for Injection, USP. The anti-BPC-1 mAb preparation may be
 lyophilized and stored as a sterile powder, preferably under vacuum, and
 then reconstituted in bacteriostatic water containing, for example, benzyl
 alcohol preservative, or in sterile water prior to injection.
 Dosages and administration protocols for the treatment of cancers using the
 foregoing methods will vary with the method and the target cancer and will
 generally depend on a number of other factors appreciated in the art.
 Cancer Vaccines
 The invention further provides prostate cancer vaccines comprising a BPC-1
 protein or fragment thereof. The use of a tumor antigen in a vaccine for
 generating humoral and cell-mediated immunity for use in anti-cancer
 therapy is well known in the art and has been employed in prostate cancer
 using human PSMA and rodent PAP immunogens (Hodge et al., 1995, Int. J.
 Cancer 63: 231-237; Fong et al., 1997, J. Immunol. 159: 3113-3117). Such
 methods can be readily practiced by employing a BPC-1 protein, or fragment
 thereof, or a BPC-1-encoding nucleic acid molecule and recombinant vectors
 capable of expressing and appropriately presenting the BPC-1 immunogen.
 For example, viral gene delivery systems may be used to deliver a
 BPC-1-encoding nucleic acid molecule. Various viral gene delivery systems
 which can be used in the practice of this aspect of the invention include,
 but are not limited to, vaccinia, fowlpox, canarypox, adenovirus,
 influenza, poliovirus, adeno-associated virus, lentivirus, and sindbus
 virus (Restifo, 1996, Curr. Opin. Immunol. 8: 658-663). Non-viral delivery
 systems may also be employed by using naked DNA encoding a BPC-1 protein
 or fragment thereof introduced into the patient (e.g., intramuscularly) to
 induce an anti-tumor response. In one embodiment, the full-length human
 BPC-1 cDNA may be employed. In another embodiment, BPC-1 nucleic acid
 molecules encoding specific cytotoxic T lymphocyte (CTL) epitopes may be
 employed. CTL epitopes can be determined using specific algorithms (e.g.,
 Epimer, Brown University) to identify peptides within a BPC-1 protein
 which are capable of optimally binding to specified HLA alleles.
 Various ex vivo strategies may also be employed. One approach involves the
 use of dendritic cells to present BPC-1 antigen to a patient's immune
 system. Dendritic cells express MHC class I and II, B7 costimulator, and
 IL-12, and are thus highly specialized antigen presenting cells. In
 prostate cancer, autologous dendritic cells pulsed with peptides of the
 prostate-specific membrane antigen (PSMA) are being used in a Phase I
 clinical trial to stimulate prostate cancer patients' immune systems (Tjoa
 et al., 1996, Prostate 28: 65-69; Murphy et al., 1996, Prostate 29:
 371-380). Dendritic cells can be used to present BPC-1 peptides to T cells
 in the context of MHC class I and II molecules. In one embodiment,
 autologous dendritic cells are pulsed with BPC-1 peptides capable of
 binding to MHC molecules. In another embodiment, dendritic cells are
 pulsed with the complete BPC-1 protein. Yet another embodiment involves
 engineering the overexpression of the BPC-1 gene in dendritic cells using
 various implementing vectors known in the art, such as adenovirus (Arthur
 et al., 1997, Cancer Gene Ther. 4: 17-25), retrovirus (Henderson et al.,
 1996, Cancer Res. 56: 3763-3770), lentivirus, adeno-associated virus, DNA
 transfection (Ribas et al., 1997, Cancer Res. 57: 2865-2869), and
 tumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med. 186:
 1177-1182). Cells expressing BPC-1 may also be engineered to express
 immune modulators, such as GM-CSF, and used as immunizing agents.
 Anti-idiotypic anti-BPC-1 antibodies can also be used in anti-cancer
 therapy as a vaccine for inducing an immune response to cells expressing a
 BPC-1 protein. Specifically, the generation of anti-idiotypic antibodies
 is well known in the art and can readily be adapted to generate
 anti-idiotypic anti-BPC-1 antibodies that mimic an epitope on a BPC-1
 protein (see, for example, Wagner et al., 1997, Hybridoma 16: 33-40; Foon
 et al., 1995, J Clin Invest 96: 334-342; Herlyn et al., 1996, Cancer
 Immunol Immunother 43: 65-76). Such an anti-idiotypic antibody can be used
 in cancer vaccine strategies.
 Genetic immunization methods may be employed to generate prophylactic or
 therapeutic humoral and cellular immune responses directed against cancer
 cells expressing BPC-1. Constructs comprising DNA encoding a BPC-1
 protein/immunogen and appropriate regulatory sequences may be injected
 directly into muscle or skin of an individual, such that the cells of the
 muscle or skin take-up the construct and express the encoded BPC-1
 protein/immunogen. Expression of the BPC-1 protein immunogen results in
 the generation of prophylactic or therapeutic humoral and cellular
 immunity against prostate cancer. Various prophylactic and therapeutic
 genetic immunization techniques known in the art may be used (for review,
 see information and references published at Internet address
 www.genweb.com).
 KITS
 For use in the diagnostic and therapeutic applications described or
 suggested above, kits are also provided by the invention. Such kits may
 comprise a carrier means being compartmentalized to receive in close
 confinement one or more container means such as vials, tubes, and the
 like, each of the container means comprising one of the separate elements
 to be used in the method. For example, one of the container means may
 comprise a probe which is or can be detectably labeled. Such probe may be
 an antibody or polynucleotide specific for a BPC-1 protein or a BPC-1 gene
 or message, respectively. Where the kit utilizes nucleic acid
 hybridization to detect the target nucleic acid, the kit may also have
 containers containing nucleotide(s) for amplification of the target
 nucleic acid sequence and/or a container comprising a reporter-means, such
 as a biotin-binding protein, such as avidin or streptavidin, bound to a
 reporter molecule, such as an enzymatic, florescent, or radioisotope
 label.
 EXAMPLES
 Various aspects of the invention are further described and illustrated by
 way of the several examples which follow, none of which are intended to
 limit the scope of the invention.
 Example 1
 Isolation of cDNA Fragment of BPC-1 Gene
 Materials and Methods
 LAPC Xenografts:
 LAPC xenografts were obtained from Dr. Charles Sawyers (UCLA) and generated
 as described (Klein et al, 1997, Nature Med. 3: 402-408). Androgen
 dependent and independent LAPC-4 xenografts LAPC-4 AD and AI,
 respectively) and LAPC-9 AD xenografts were grown in male SCID mice and
 were passaged as small tissue chunks in recipient males. LAPC-4 AI
 xenografts were derived from LAPC-4 AD tumors. Male mice bearing LAPC-4 AD
 tumors were castrated and maintained for 2-3 months. After the LAPC-4
 tumors re-grew, the tumors were harvested and passaged in castrated males
 or in female SCID mice.
 Cell Lines:
 Human cell lines (e.g., HeLa) were obtained from the ATCC and were
 maintained in DMEM with 10% fetal calf serum.
 RNA Isolation:
 Tumor tissue and cell lines were homogenized in Trizol reagent (Life
 Technologies, Gibco BRL) using 10 ml/g tissue or 10 ml/10.sup.8 cells to
 isolate total RNA. Poly A RNA was purified from total RNA using Qiagen's
 Oligotex mRNA Mini and Midi kits. Total and mRNA were quantified by
 spectrophotometric analysis (O.D. 260/280 nm) and analyzed by gel
 electrophoresis.
 Oligonucleotides:
 The following HPLC purified oligonucleotides were used.
 DPNCDN (cDNA synthesis primer):
 5'TTTTGATCAAGCTT.sub.30 3' (SEQ ID NO. 9)
 Adaptor 1:
 (SEQ ID NO. 10)
 5'CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3'
 3'GGCCCGTCCTAG5'
 Adaptor 2:
 (SEQ ID NO. 11)
 5'GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3'
 3'CGGCTCCTAG5'
 PCR primer 1:
 5'CTAATACGACTCACTATAGGGC3' (SEQ ID NO. 12)
 Nested primer (NP)1:
 5'TCGAGCGGCCGCCCGGGCAGGA3' (SEQ ID NO. 13)
 Nested primer (NP)2:
 5'AGCGTGGTCGCGGCCGAGGA3' (SEQ ID NO. 14)
 Suppression Subtractive Hybridization:
 Suppression Subtractive Hybridization (SSH) was used to identify cDNAs
 corresponding to genes which may be down-regulated in androgen independent
 prostate cancer compared to androgen dependent prostate cancer.
 Double stranded cDNAs corresponding to the LAPC-4 AD xenograft (tester) and
 the LAPC-4 AI xenograft (driver) were synthesized from 2 .mu.g of
 poly(A).sup.+ RNA isolated from xenograft tissue, as described above,
 using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of
 oligonucleotide DPNCDN as primer. First- and second-strand synthesis were
 carried out as described in the Kit's user manual protocol (CLONTECH
 Protocol No. PT117-1, Catalog No. K1804-1). The resulting cDNA was
 digested with Dpn II for 3 hrs. at 37.degree. C. Digested cDNA was
 extracted with phenol/chloroform (1:1) and ethanol precipitated.
 Driver cDNA (LAPC-4 AI) was generated by combining in a 1:1 ratio Dpn II
 digested LAPC-4 AI cDNA with a mix of digested cDNAs derived from human
 benign prostatic hyperplasia (BPH), the human cell lines HeLA, 293, A431,
 Colo205, and mouse liver.
 Tester cDNA (LAPC-4 AD) was generated by diluting 1 .mu.l of Dpn II
 digested LAPC-4 AD cDNA (400 ng) in 5 .mu.l of water. The diluted cDNA (2
 .mu.l, 160 ng) was then ligated to 2 .mu.l of adaptor 1 and adaptor 2 (10
 .mu.M), in separate ligation reactions, in a total volume of 10 .mu.l at
 16.degree. C. overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation
 was terminated with 1 .mu.l of 0.2 M EDTA and heating at 72.degree. C. for
 5 min.
 The first hybridization was performed by adding 1.5 .mu.l (600 ng) of
 driver cDNA to each of two tubes containing 1.5 .mu.l (20 ng) adaptor 1-
 and adaptor 2-ligated tester cDNA. In a final volume of 4 .mu.l, the
 samples were overlayed with mineral oil, denatured in an MJ Research
 thermal cycler at 98.degree. C. for 1.5 minutes, and then were allowed to
 hybridize for 8 hrs at 68.degree. C. The two hybridizations were then
 mixed together with an additional 1 .mu.l of fresh denatured driver cDNA
 and were allowed to hybridize overnight at 68.degree. C. The second
 hybridization was then diluted in 200 .mu.l of 20 mM Hepes, pH 8.3, 50 mM
 NaCl, 0.2 mM EDTA, heated at 70.degree. C. for 7 min. and stored at
 -20.degree. C.
 PCR Amplification, Cloning and Sequencing of Gene Fragments Generated from
 SSH:
 To amplify gene fragments resulting from SSH reactions, two PCR
 amplifications were performed. In the primary PCR reaction 1 .mu.l of the
 diluted final hybridization mix was added to 1 .mu.l of PCR primer 1 (10
 .mu.M), 0.5 .mu.l dNTP mix (10 .mu.M), 2.5 .mu.l 10.times.reaction buffer
 (CLONTECH) and 0.5 .mu.l 50.times.Advantage cDNA polymerase Mix (CLONTECH)
 in a final volume of 25 .mu.l. PCR 1 was conducted using the following
 conditions: 75.degree. C. for 5 min., 94.degree. C. for 25 sec., then 27
 cycles of 94.degree. C. for 10 sec, 66.degree. C. for 30 sec, 72.degree.
 C. for 1.5 min. Five separate primary PCR reactions were performed for
 each experiment. The products were pooled and diluted 1:10 with water. For
 the secondary PCR reaction, 1 .mu.l from the pooled and diluted primary
 PCR reaction was added to the same reaction mix as used for PCR 1, except
 that primers NP1 and NP2 (10 .mu.M) were used instead of PCR primer 1. PCR
 2 was performed using 10-12 cycles of 94.degree. C. for 10 sec, 68.degree.
 C. for 30 sec, 72.degree. C. for 1.5 minutes. The PCR products were
 analyzed using 2% agarose gel electrophoresis.
 The PCR products were inserted into pCR2.1 using the T/A vector cloning kit
 (Invitrogen). Transformed E. coli were subjected to blue/white and
 ampicillin selection. White colonies were picked and arrayed into 96 well
 plates and were grown in liquid culture overnight. To identify inserts,
 PCR amplification was performed on 1 ml of bacterial culture using the
 conditions of PCR1 and NP1 and NP2 as primers. PCR products were analyzed
 using 2% agarose gel electrophoresis.
 Bacterial clones were stored in 20% glycerol in a 96 well format. Plasmid
 DNA was prepared, sequenced, and subjected to nucleic acid homology
 searches of the GenBank, dBest, and NCI-CGAP databases.
 RT-PCR Expression Analysis:
 First strand cDNAs were generated from 1 .mu.g of mRNA with oligo (dT)12-18
 priming using the Gibco-BRL Superscript Preamplification system. The
 manufacturers protocol was used and included an incubation for 50 min at
 42.degree. C. with reverse transcriptase followed by RNAse H treatment at
 37.degree. C. for 20 min. After completing the reaction, the volume was
 increased to 200 .mu.l with water prior to normalization. First strand
 cDNAs from 16 different normal human tissues were obtained from Clontech.
 Normalization of the first strand cDNAs from multiple tissues was performed
 by using the primers 5'atatcgccgcgctcgtcgtcgacaa3' and
 5'agccacacgcagctcattgtagaagg 3' to amplify .beta.-actin. First strand cDNA
 (5 .mu.l) was amplified in a total volume of 50 .mu.l containing 0.4 .mu.M
 primers, 0.2 .mu.M each dNTPs, 1.times.PCR buffer (Clontech, 10 mM
 Tris-HCL, 1.5 mM MgCl.sub.2, 50 mM KCl, pH8.3) and 1.times.Klentaq DNA
 polymerase (Clontech). Five .mu.l of the PCR reaction was removed at 18,
 20, and 22 cycles and used for agarose gel electrophoresis. PCR was
 performed using an MJ Research thermal cycler under the following
 conditions: initial denaturation was at 94.degree. C. for 15 sec, followed
 by a 18, 20, and 22 cycles of 94.degree. C. for 15, 65.degree. C. for 2
 min, 72.degree. C. for 5 sec. A final extension at 72.degree. C. was
 carried out for 2 min. After agarose gel electrophoresis, the band
 intensities of the 283 bp .beta.-actin bands from multiple tissues were
 compared by visual inspection. Dilution factors for the first strand cDNAs
 were calculated to result in equal .beta.-actin band intensities in all
 tissues after 22 cycles of PCR. Three rounds of normalization were
 required to achieve equal band intensities in all tissues after 22 cycles
 of PCR.
 To determine expression levels of the 19P1E8 gene, 5 .mu.l of normalized
 first strand cDNA was analyzed by PCR using 25, 30, and 35 cycles of
 amplification using the following primer pairs, which were designed with
 the assistance of (MIT; for details, see, www.genome.wi.mit.edu):
 5'-TGC CGT ATG TCA CTG TCT CTA GGT-3' (SEQ ID NO. 15)
 5'-GAA ATC ATG GGT ATT TCA TGT GCT-3' (SEQ ID NO. 16)
 These primers were designed from the sequence of the SSH fragment of the
 initially isolated 19P1E8 gene. Use of the following primer pair, based on
 sequences within the open reading frame of the 19P1E8 gene, produced the
 same expression pattern.
 5'-CTC CCA ACT ATC CCA GCA AGT ATC-3' (SEQ ID NO. 17)
 5'-AAA TCC CAT AGA TTC CAG CTC TCC-3' (SEQ ID NO. 18)
 Semi quantitative expression analysis was achieved by comparing the PCR
 products at cycle numbers that give light band intensities.
 RESULTS:
 Several SSH experiments were conduced as described in the Materials and
 Methods, supra, and led to the isolation of numerous candidate gene
 fragment clones (SSH clones). All candidate clones were sequenced and
 subjected to homology analysis against all sequences in the major public
 gene and EST databases in order to provide information on the identity of
 the corresponding gene and to help guide the decision to analyze a
 particular gene for differential expression. In general, gene fragments
 which had no homology to any known sequence in any of the searched
 databases, and thus considered to represent novel genes, as well as gene
 fragments showing homology to previously sequenced expressed sequence tags
 (ESTs), were subjected to differential expression analysis by RT-PCR
 and/or Northern analysis.
 One of the SSH clones comprising about 700 bp, showed no homology to any
 known gene or EST sequence was designated 19P1E8. The nucleotide sequence
 of this SHH clone is shown in FIG. 1, approximately nucleotide residues
 1883-2583. Differential expression analysis by Northern blot showed that
 19P1E8 is expressed in LAPC-4 AD xenograft, and to a significantly lesser
 extent in LAPC-4 AI, LAPC-9 AD and LAPC-9 AI (FIG. 9). No expression was
 detected in normal prostate (FIG. 9). Three distinct transcripts are
 shown, with sizes of 3.5 kb, 8 kb, and greater than 9 kb.
 RT-PCR analysis of 19P1E8 expression produced essentially identical results
 (FIG. 5, Panel A). In addition, further RT-PCR expression analysis of
 first strand cDNAs from 16 normal tissues detected expression of the
 19P1E8 gene only in brain, spleen and testis tissue, and only at very low
 levels detectable at 35 and not 30 cycles of PCR amplification (FIG. 5,
 panels B and C). In comparison, substantial expression was detected in
 LAPC-4 AD with only 30 cycles (FIG. 5).
 Example 2
 Isolation of Full Length BPC-1 Encoding cDNA
 The 19P1E8 SHH clone above (Example 1) was used to isolate a full length
 19P1E8 cDNA. Briefly, a cDNA library generated from LAPC-4 mRNA was
 screened with a labeled probe generated from the SSH clone. Specifically,
 a full length 19P1E8 cDNA of 2639 base pairs (bp) was cloned from an
 LAPC-4 AD cDNA library generated in lambda ZAP Express (Stratagene).
 The cDNA encodes an open reading frame (ORF) of 158 amino acids containing
 a signal sequence and a CUB domain (Complement sub-components C1r/C1s,
 Uegf, Bmp1) (Borck and Beckmann, 1993, J. Mol. Biol. 231: 539-545). The 5'
 UTR (untranslated region) is very GC rich, suggesting that this region
 contains regulatory elements for translation. CUB domains were originally
 found in complement sub-components CIr and CIs, and were subsequently
 identified in Uegf (epidermal growth factor related sea urchin protein)
 and Bmp1 (bone morphogenetic protein 1) a protease involved in bone
 development.
 In view of the exclusive expression of this gene in brain and its
 up-regulation in prostate cancer xenografts, this gene was named BPC-1
 (Brain/Prostate cancer CUB protein). The nucleotide and deduced amino acid
 sequences of the isolated BPC-1 cDNA are shown in FIG. 1. A schematic
 representation of the BPC-1 structure is shown in FIG. 2. An amino acid
 alignment between the CUB domain of BPC-1 and the CUB domains of other
 proteins is shown in FIG. 3. Referring to FIG. 3, of particular interest
 is that the CUB domain of BPC-1 is 30-40% identical to the CUB domains in
 BMP-1.
 The full length BPC-1 cDNA (p19P1E8, clone 6.1) was deposited with the
 American Type Culture Collection, 10801 University Boulevard, Manassas,
 Va. 20110-2209, on Aug. 7, 1998 and has been accorded ATCC accession
 number 98833.
 Example 3
 BPC-1 Gene Expression Analysis--Brain Specific in Normal Tissues
 Initial analysis of BPC-1 mRNA expression in normal human tissues was
 conducted by Northern blotting two multiple tissue blots obtained from
 Clontech (Palo Alto, Calif.), comprising a total of 16 different normal
 human tissues, using labeled 19P1E8 SSH clone (Example 1) as a probe. RNA
 samples were quantitatively normalized with a .beta.-actin probe.
 The results are shown in FIG. 4. Expression was only detected in normal
 brain. The northern blots showed two transcripts of 3.5 kb and 8.0 kb
 (FIG. 5). The 3.5 kb transcript corresponds to the cDNA identified from
 LAPC-4 AD that encodes the BPC-1 ORF. The larger transcript may encode an
 un-processed message or an alternative isoform of the gene.
 To further explore BPC-1 expression in normal tissues, a multi-tissue RNA
 dot blot was probed with a BPC-1 probe. Out of 37 different normal tissues
 tested, only brain regions exhibited detectable levels of BPC-1 (FIG. 6).
 Interestingly, expression of BPC-1 was confined to cortical regions of the
 brain, such as the temporal, frontal and occipital lobes. Expression was
 also seen in hippocampus, amygdala, caudate nucleus and putamen. Other
 brain regions such as thalamus, sub-thalamic nucleus and substantia nigra
 did not express BPC-1. Similarly, no expression was detected in other
 central nervous system (CNS) structures such as cerebellum, spinal cord
 and medulla oblongata (mid-brain). The RNA dot blot results were confirmed
 with a northern blot containing RNA for different CNS tissues (FIG. 7).
 Example 4
 BPC-1 Expression in Fetal Tissues--Broader Expression in Development
 CUB domain proteins often are often developmentally regulated. To determine
 if BPC-1 is expressed in human fetal tissue, RT-PCR was performed on first
 strand cDNA derived from 8 different fetal tissues. The results show that
 BPC-1 is highly expressed in fetal brain, with lower levels detected in
 all other fetal tissues (FIG. 8). This suggests that expression in the
 adult is exclusive to brain, while expression in other tissues is turned
 off during development.
 Example 5
 High Level BPC-1 Expression in Prostate Cancer
 To analyze BPC-1 expression in cancer tissues and cell lines, Northern blot
 analysis was performed on RNA derived from the LAPC prostate cancer
 xenografts as well as a panel of prostate and bladder cancer cell lines.
 The results, shown in FIG. 9, reveal the highest levels of BPC-1
 expression in the LAPC-4 AD prostate cancer xenograft and in the LNCaP
 prostate cancer cell line, both of which originated from lymph-node
 metastasis of prostate cancer (Klein et a., 1997, Nature Med. 3:402;
 Horoszewicz et al., 1983, Cancer Res. 43:1809). Lower level expression of
 BPC-1 was detected in LAPC-4 AI, LAPC-9 AD and LAPC-9 AI (FIG. 9). Among
 the bladder cancer cell lines tested, one (5637) showed detectable BPC-1
 expression (FIG. 9). No expression was detected in PrEC cells (Clonetics),
 which represent the basal cell compartment of the prostate and normal
 prostate.
 Example 6
 Production of Secreted Recombinant BPC-1 in Vitro
 To express recombinant BPC-1 and analyze the subcellular localization of
 BPC-1 protein, the full length cDNA was cloned into an expression vector
 that provides a 6His tag at the carboxyl-terminus (pCDNA 3.1 myc-his,
 InVitrogen). The construct was transfected into 293T cells which were
 labeled for one hour with .sup.35 S-methionine. The cells were then washed
 and incubated in non-radioactive media to chase the labeled proteins for
 various time points. BPC-1-His tagged protein was immunoprecipitated using
 anti-His antibodies (Santa Cruz) from cell extracts and from cell
 supernatant (media) at various time points after the chase. The
 immunoprecipitates were analyzed by SDS-PAGE (sodium-dodecyl sulfate
 polyacrylamide-gel electrophoresis) with subsequent autoradiography to
 visualize .sup.35 S-methionine labeled protein.
 The results show that BPC-1 protein appears in the cell extract and the
 cell media immediately after the .sup.35 S-methionine labeling period
 (FIG. 10). Within two hours of the chase, nearly all BPC-1 protein is
 secreted into the media and remains stable in the media for several hours.
 The half-life of the protein is estimated to be longer than 24 hours.
 Vector transfected cells were also labeled and analyzed using the same
 protocol. Interestingly, a non-specific protein appears in both the vector
 and the BPC-1 transfected cells. This protein seems to have a very short
 half-life in the media compared to BPC-1, as it disappears after the 4
 hour time point. These results demonstrate that BPC-1 is indeed a secreted
 protein that appears to be very stable in cell culture media.
 Example 7
 Production of Recombinant BPC-1 Using Baculovirus System
 To generate recombinant BPC-1 protein in a baculovirus expression system,
 BPC-1 cDNA was cloned into the baculovirus transfer vector pMelBac
 (Invitrogen) which provides the honeybee mellitin signal sequence for
 secretion into the media of insect cells. pMelBac-BPC-1 was co-transfected
 with helper plasmid pBlueBac4.5 (Invitrogen) into SF9 (Spodoptera
 frugiperda) insect cells to generate recombinant baculovirus (see
 Invitrogen instruction manual for details). Baculovirus was collected from
 cell supernatant and was purified by plaque assay.
 Recombinant BPC-1 protein was generated by infection of HighFive insect
 cells (InVitrogen) with purified baculovirus. Recombinant BPC-1 protein
 was detected in both cell extract and cell supernatant using anti-BPC-1
 mouse polyclonal antibody (see Example 8, below). Interestingly, the cell
 extract contains two forms of BPC-1, signal sequence cleaved BPC-1 and
 unprocessed BPC-1 (FIG. 11). The supernatant only contained cleaved mature
 BPC-1. This recombinant BPC-1 protein may be purified and used in various
 cell based assays or as immunogen to generate polyclonal and monoclonal
 antibodies specific for BPC-1.
 Example 8
 Generation of BPC-1 Polyclonal Antibodies
 In order to generate antibody reagents that specifically bind to BPC-1, a
 glutathione-S-transferase (GST) fusion protein encompassing amino acids
 29-93 of the BPC-1 protein was synthesized to serve as immunogen. This
 fusion protein was generated by PCR-mediated amplification of nucleotides
 877-1,071 (AA 29-93) of the cDNA clone of BPC-1 with the following
 primers:
 (SEQ ID NO:17)
 5'PRIMER TTGAATTCCAAGCAAACCACCTCAGA
 EcoRI
 (SEQ ID NO:18)
 3'PRIMER AAGCTCGAGTCAGACGGTTCAATAGAGT
 XhoI
 The resultant product was cloned into the EcoR1 and Xhol restriction sites
 of the pGEX-2T GST-fusion vector (Pharmacia). Recombinant GST-BPC-1 fusion
 protein was purified to greater than 90% purity from induced bacteria by
 glutathione-sepaharose affinity chromatography.
 To generate polyclonal sera to BPC-1, the purified fusion protein was used
 as follows. A rabbit was initially immunized with 200 .mu.g of GST-BPC-1
 fusion protein mixed in complete Freund's adjuvant. The rabbit was
 injected every two weeks with 200 .mu.g of GST-BPC-1 protein in incomplete
 Freund's adjuvant. Test bleeds were taken approximately 7-10 days
 following each immunization. ELISA, Western blotting and
 immunoprecipitation analyses were used to determine specificity and titer
 of the rabbit serum to BPC-1. Cell lines that express BPC-1 endogenously
 such as LNCaP and cell lines engineered to overexpress BPC-1 by
 transfection (293T) and by retroviral infection (PC-3 and NIH3T3) were
 used for characterization of the antiserum. Antiserum representing
 specific high titer to BPC-1 protein is purified by a 3 step process: (1)
 removal of GST-reacUve antibody by depletion over a GST affinity column,
 (2) BPC-1 specific IgG antibody was isolated by passage over a GST-BPC-1
 affinity column, and (3) protein G chromatography.
 The mouse polyclonal antibody was successfully used for detecting
 recombinant BPC-1 expressed in a baculovirus expression system (see
 Example 7, above), affinity (nickel) purified MYC/HIS BPC-1 protein (FIG.
 13) and recombinant BPC-1 protein in tissue culture supernatants of cells
 expressing the BPC-1 gene (FIG. 13). Rabbit polyclonal serum was also
 generated and similarly was capable of detecting BPC-1 in tissue culture
 supernatants of cells expressing the BPC-1 gene.
 Example 9
 Generation of BPC-1 Monoclonal Antibodies
 To generate mAbs to BPC-1, 5 Balb C mice were initially immunized
 intraperitoneally with 200 .mu.g of GST-BPC-1 fusion protein mixed in
 complete Freund's adjuvant. Mice were subsequently immunized every 2 weeks
 with 75 .mu.g of GST-BPC-1 protein mixed in Freund's incomplete adjuvant
 for a total of 3 immunizations. Reactivity of serum from immunized mice to
 full length BPC-1 protein was monitored by ELISA using a partially
 purified preparation of HIS-tagged BPC-1 protein expressed from 293T
 cells. Two mice with strongest reactivity were rested for 3 weeks and
 given a final injection of fusion protein in PBS and then sacrificed 4
 days later. The spleens of the sacrificed mice were harvested and fused to
 SPO/2 myeloma cells using standard procedures (Harlow and Lane, 1988).
 Supernatants from growth wells following HAT selection are being screened
 by ELISA and Western blot to identify BPC-1 specific antibody producing
 clones.
 The binding affinity of a BPC-1 monoclonal antibody may be determined using
 standard technology. Affinity measurements quantify the strength of
 antibody to epitope binding and may be used to help define which BPC-1
 monoclonal antibodies are preferred for diagnostic or therapeutic use. The
 BIAcore system (Uppsala, Sweden) is a preferred method for determining
 binding affinity. The BIAcore system uses surface plasmon resonance (SPR,
 Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998, Methods
 in Enzymology 295: 268) to monitor biomolecular interactions in real time.
 BIAcore analysis conveniently generates association rate constants,
 dissociation rate constants, equilibrium dissociation constants, and
 affinity constants.
 Example 10
 Production and Purification of Recombinant BPC-1 Expressed in a Mammalian
 Expression System
 293T cells transiently transfected or 293 cells stably expressing a
 CMV-driven expression vector encoding BPC-1 with a C-terminal 6.times.His
 and MYC tag (pcDNA3.1/mycHIS, Invitrogen) serves as source of secreted
 soluble BPC-1 protein for purification (see Example 6, above). The
 HIS-tagged BPC-1 protein secreted into the conditioned media is purified
 using the following method. Conditioned media (500 ml) is concentrated 10
 fold and simultaneously buffer exchanged into a phosphate buffer (pH 8.0)
 containing 750 mM NaCl and 10 mM imidazole using an amicon ultrafiltration
 unit with a 10 kd MW cutoff membrane. The preparation is then passed over
 a nickel metal affinity resin with a 0.5 ml bed volume (Ni-NTA, Qiagen)
 and washed extensively with phosphate buffer (pH 6.0) containing 10%
 ethanol and 300 mM NaCl. The HIS-tagged BPC-1 protein is then eluted with
 phosphate buffer (pH 6.0) containing 250 mM imidizole. Higher purity
 preparations are obtained by repeating the above chromatography step with
 higher stringency of wash (phosphate buffer containing 75 mM imidizole) or
 by passage over an anti-HIS Ab immunoaffinity column. This method was
 successfully used to purify recombinant HIS-BPC-1. A western blot of the
 purified protein is shown in the far left hand lane of FIG. 13.
 Example 11
 Retrovirus Mediated Expression of Secreted Human BPC-1
 The BPC-1 coding region was subcloned into the retroviral SR.alpha.msvtkneo
 vector (Muller et al., 1991 MCB 11: 1785-1792). Retroviruses were made and
 used to generate cell lines expressing the BPC-1 gene. The cell lines
 generated are 3T3/BPC-1 and PC3/BPC-1. 3T3 cells acutely infected with the
 SR-.alpha.BPC-1 virus express a very high level of BPC-1 mRNA, as
 demonstrated by the Northern blot shown in FIG. 12. The PC3/BPC-1 lysate
 and supernatant were tested for BPC-1 expression by Western blot analysis
 using a polyclonal antibody against a GST fusion protein containing the
 N-terminal portion of the BPC-1 protein (aa29-93) as follows. PC3 and 3T3
 cells stably expressing either control(Neo) or BPC-1 encoding retrovirus
 and 3T3 cells acutely infected with BPC-1 retrovirus were cultured in 10
 cm tissue culture plates for 4 days. 25 .mu.l of neat supernatant from
 each line was subjected to Western blot analysis using a 1:500 dilution of
 murine anti-BPC-1 polyclonal serum. The blot was then incubated with
 anti-mouse-HRP conjugated secondary antibody and BPC-1 specific signals
 were visualized by enhanced chemiluminescent detection. The results of
 this Western blot analysis are shown in FIG. 13.
 Example 12
 BPC-1 Expression Analysis in Vitro and in Vivo
 Western and immunoprecipitation analyses of cell lysates and conditioned
 media with BPC-1 specific antibodies may be used to identify and
 characterize BPC-1 protein expression in cell lines and tissues, such as
 LAPC4 and LAPC9 xenografts, LNCaP prostate cancer cells, 5637 bladder
 carcinoma cells, normal human brain lysate, all of which express BPC-1
 mRNA, as well as a variety of other carcinoma cell lines, xenografts, and
 normal tissues. Due to the structural homology of BPC-1 to the porcine and
 bovine spermadhesin family of proteins (Topfer-Petersen et al. Andrologia,
 1998) human semen may also contain detectable levels of BPC-1 protein.
 Also, given its expression in bladder carcinoma, BPC-1 protein may also be
 detectable in the urine of bladder carcinoma patients. MYC-HIS BPC-1
 transfected 293T cells and retrovirally transduced PC3 and NIH3T3 cells
 serve as positive controls for BPC-1 protein expression (FIG. 13).
 Identification and quantitation of BPC-1 protein present in clinical
 samples of human serum, semen, and urine may be carried out by capture
 ELISA as described in the following example. Immunohistochemical analysis
 of BPC-1 protein in normal and cancerous tissues may be conducted on
 formalin-fixed, paraffin-embedded or frozen tissue sections using standard
 immunohistochemical methods well known in the art and the BPC-1 antibodies
 provided herein. Formalin-fixed, paraffin-embedded sections of LNCaP cells
 may used as a positive control.
 Example 13
 BPC-1 Capture ELISA
 Capture ELISA may be used to identify and quantify BPC-1 protein present in
 clinical samples of human serum, semen, and urine as follows. The capture
 ELISA for BPC-1 is dependent on the generation of at least 2 mAbs of
 different isotypes that recognize distinct epitopes of the BPC-1 protein
 or 1 mAb and a specific rabbit polyclonal serum. One reagent will serve as
 the capture (or coating) Ab and the other as the detection Ab. Captured
 BPC-1 is then visualized by the addition of a secondary Ab-HRP conjugate
 against the detection antibody followed by incubation with TMB substrate.
 Optical density of wells is then measured in a spectrophotometric plate
 reader at 450 nm. Purified MYC/HIS tagged BPC-1 protein serves as a
 standardization antigen for the ELISA.
 Example 14
 BPC-1 Expression Results in Anchorage Independent Colony Formation in Vitro
 Retrovirally-infected cells expressing BPC-1 were generated as described in
 EXAMPLE 11 and used along with the respective neo control cell lines to
 perform soft agar assays to evaluate the oncogenic potential of BPC-1. The
 agar assay was performed according to conditions previously described
 (Lugo, T. R and O. N. Witte, 1989, Molec. Cell. Biol. 9: 1263-1270).
 Briefly, cells were trypsinized and resuspended in Iscove medium
 containing 0.3% Noble agar and 20% fetal bovine serum. This cell agar
 suspension (10.sup.4 cells/60 mm plate) was plated between a bottom and
 top layer of medium containing 0.6% Noble agar and 20% fetal bovine serum.
 The plates were fed after 7 days, and colonies examined and scored 2 or 3
 weeks after the agar assay was set up, depending on the size of the
 colonies. The colonies were counted suing a software from Alphalmager 200.
 The results are tabulated below in Table 1.
 TABLE 1
 BPC-1 EXPRESSION INDUCES CELL TRANSFORMATION
 AVG. NO. COLONIES AVG. NO. COLONIES
 CELLS [acute infection] [G418 selection for 2 weeks]
 3T3CL7/neo 14 65
 3T3CL7/BPC-1 116 235
 3T3CL7 cells infected with retrovirus expressing BPC-1 or neo were used.
 Colonies were scored 3 weeks after the agar assays were set up. The
 3T3CL7/BPC-1 cells generated about 8 fold more colonies compared to the
 control plate for acutely infected cells. Using G418 selected cells, there
 are about 3.6 fold more colonies in the 3T3CL7/BPC-1 plates compared to
 the 3T3CL7/neo plates.
 The above results indicate that the BPC-1 protein induces anchorage
 independent growth in cells experimentally engineered to express and
 secrete BPC-1 and thus exerts a transforming effect on those cells.
 Example 15
 BPC-1 Binds to a Cellular Protein
 In order to establish whether BPC-1 binds to cellular proteins expressed in
 prostate cancer cells and other cancer cells or normal cells, two
 approaches were taken. In the first approach, in vitro assay for
 recombinant HIS-tagged BPC-1 (Example 6, above) binding to various cell
 lines are used. In another approach, a recombinant alkaline
 phosphatase-BPC-1 fusion protein are generated using the AP-TAG system
 from GenHunter Corporation (Nashville, Tenn., cat# Q202), and the AP-TAG
 fusion used to test BPC-1 binding to a variety of prostate cancer cell
 lines.
 A. HIS-TAGGED BPC-1 Cell Surface Binding Analysis
 PC-3 and NIH3T3 cells are incubated on ice at 4 degrees C for 2 hours with
 conditioned media containing HIS-tagged BPC-1 (from 293T transfected
 cells) or media containing purified HIS-tagged BPC-1 or control media.
 Cells are washed extensively with ice cold PBS with 0.5% FBS and then
 incubated with an excess of anti-HIS rabbit polyclonal antibody (5 ug/ml,
 PBS 0.5% FBS) at 4 degrees C for 1 hour. Cells are again washed and then
 incubated with anti-rabbit FITC conjugated secondary Ab (1:4,000 in
 PBS/0.5% FBS) for 30 minutes at 4 degrees C. Cell bound BPC-1 is then
 detected by fluorimetric analysis of cells in a Cytofluor 4000 fluorimeter
 (PE Biosystems) and/or by flow cytometry.
 As an alternative to the fluorescence-based assay used above, binding
 assays can be carried out with .sup.125 I-labeled BPC-1 protein.
 Determination of BPC-1 receptor number and affinity on cells and
 monitoring internalization of receptor bound BPC-1 protein is carried out
 using standard published procedures (Raitano and Korc, J. Biol. Chem.,
 1990, J. Biol. Chem. 265: 10466-10472).
 B. Alkaline Phosphatase Tagged BPC-1 Generates Cell Surface Staining in
 Prostate Cancer Cells
 Alkaline phosphatase-tagged BPC-1 was generated as follows. The sequence
 encoding mature BPC-1 (i.e., without the signal sequence) was cloned into
 pAPtag-5 (GenHunter Corp. Nashville, Tenn.). The BPC-1.HindIII and
 BPC1.BamH1 primers, below, were used to amplify the BPC-1 open reading
 frame between amino acids 23 and 58 from the plasmid template SRa-19P1E8
 clone 1. The HindIII and BamH1 digested PCR product was ligated into
 HindIII and BgIII digested pAPtag-5 while keeping the IgGK signal
 sequence, BPC-1 ORF, and alkaline phosphatase all in frame. The BPC-1-AP
 fusion protein contains an IgGK signal sequence to promote secretion along
 with myc/His tags at the carboxy terminus of alkaline phosphatase.
 BPC1.HINDIII PRIMER: (SEQ ID NO. 19)
 GTGTAAGCTTCCACCAAGAAAGGAACAGAA (SEQ ID NO. 20)
 BPC1.BAMHI PRIMER:
 CACAGGATCCCTTACCAGGTGTGAAATTG
 To detect whether BPC-1 binds with a cell surface receptor on prostate
 cancer cells, several prostate cancer cell lines and xenograft tissues are
 incubated with the BPC-1-AP fusion protein as described (Cheng and
 Flanagan, 1994, Cell 79:157-168). After washing the cells and adding the
 AP substrate BCIP, which forms an insoluble blue precipitate upon
 dephosphorylation, BPC-1 receptor binding is determined by identifying
 cells staining blue under the light microscope. Various cancer cell lines
 can be examined, including without limitation, various prostate cancer
 cell lines (e.g., LNCaP, PC-3, DU145, TSUPR, LAPC4) and bladder carcinoma
 cell lines. Other cell lines such as PREC prostate cell line, 293T, and
 NIH 3T3, etc. may also be examined. Additionally, the LAPC and other
 prostate cancer xenografts may be tested.
 Equilibrium dissociation rate constants may be calculated to evaluate the
 strength of the binding interaction. In addition, the number of cell
 surface receptors per cell can be determined. Cell lines or tissues with
 the highest binding capacity for BPC-1 would be preferred for cloning the
 BPC-1 receptor or other binding partner.
 The BPC-1-AP fusion protein was detected in the conditioned media of 293T
 cells transfected with the above construct by Western blot analysis.
 Western blot analysis using anti-alkaline phosphatase and anti-HIS
 antibodies detects the BPC-1-AP fusion protein running at approximately 90
 kDa (FIG. 14).
 Conditioned media containing this fusion protein was used to detect a 45
 kDa binding partner for BPC-1 (FIG. 15), as follows. A western blot
 procedure was used to identify a 45 kDa receptor interacting with BPC-1.
 Lysates from brain, testis, prostate, the xenografts LAPC4AD and LAPC9AD,
 and the cell lines 3T3, LAPC4, LNCaP, and PC-3 were used to generate two
 duplicate western blots. After blocking in 5% milk in PBS for 1 hr and
 washing twice with PBS-Tween for 7 minutes each, the blots were incubated
 with conditioned media from a 293T cell line producing only secreted
 alkaline phosphatase and with media containing BPC-1-AP fusion protein
 (see FIG. 14). Following 3 washes with PBS-Tween, the blot was developed
 using chemiluminescent alkaline phosphatase substrate (Immune-Star,
 BioRad, cat 170-5010). The results are shown in FIG. 15. The arrow (FIG.
 15) shows BPC-1-AP binding to a 45 kDa protein in 3T3, LAPC9AD, LNCaP,
 PC-3, and to a lesser extent in LAPC4AD and the LAPC-4 cell line. The 45
 kDa protein is not detected in brain, testis or prostate. The protein
 interaction is due to BPC-1 and not AP since the blot shown in FIG. 15
 (which was incubated with AP conditioned media) did not detect binding the
 45 kDa protein.
 Example 16
 Identification of Potential Signal Transduction Pathways
 To determine whether BPC-1 directly or indirectly activates known signal
 transduction pathways in cells, luciferase (luc) based transcriptional
 reporter assays are carried out in cells expressing BPC-1 or exposed to
 exogenously added BPC-1. These transcriptional reporters contain consensus
 binding sites for known transcription factors which lie downstream of well
 characterized signal transduction pathways. The reporters and examples of
 there associated transcription factors, signal transduction pathways, and
 activation stimuli are listed below.
 1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress
 2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation
 3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress
 4. ARE-luc, androgen receptor; steroids/MAPK;
 growth/differentiation/apoptosis
 5. p53-luc, p53; SAPK; growth/differentiation/apoptosis
 6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress
 Cells to be assayed for BPC-1-mediated effects include LAPC4, LNCaP, PC3,
 and NIH3T3. The luciferase reporter plasmids may be introduced by lipid
 mediated transfection (TFX-50, Promega). Luciferase activity, an indicator
 of relative transcriptional activity, is measured by incubation of cells
 extracts with luciferin substrate and luminescence of the reaction is
 monitored in a luminometer.
 Example 17
 In Vitro Assays of BPC-1 Function
 A. Cell Invasion/Migration/Chemoattraction Assay
 Cell lines expressing BPC-1 may be assayed for alteration of invasive and
 migratory properties by measuring passage of cells through a matrigel
 coated porous membrane chamber (Becton Dickinson). Passage of cells
 through the membrane to the opposite side is monitored using a fluorescent
 assay (Becton Dickinson Technical Bulletin #428) using calcein-Am
 (Molecular Probes) loaded indicator cells. Cell lines analyzed include
 parental and BPC-1 overexpressing PC3, 3T3 and LNCaP cells. To assay
 whether BPC-1 has chemoattractant properties, parental indicator cells are
 monitored for passage through the porous membrane toward a gradient of
 BPC-1 conditioned media compared to control media.
 This assay may also be used to qualify and quantify specific neutralization
 of the BPC-1 induced effect by candidate cancer therapeutic compositions.
 B. Cell Growth Assay
 To determine whether BPC-1 alters the growth rate of established prostate
 and non-prostate cell lines, growth curves are generated comparing
 parental cells transduced with a control retroviral vector to cells
 transduced with a retrovirus encoding the BPC-1 gene. Cell lines to assay
 include LNCaP, PC3, TsuPR prostate cell lines and murine NIH3T3
 fibroblasts and various other human non-prostate cell lines. In addition,
 the growth rate of parental cells is assayed in the presence and absence
 of exogenously added purified MYC-HIS BPC-1. As an alternative source of
 exogenous BPC-1, conditioned media from the respective BPC-1 retrovirally
 transduced cell line can be used. Growth of the cell lines is monitored in
 a 96 well format MTT colorimetric assay (Raitano and Korc, 1990, J. Biol.
 Chem. 265:10466-10472).
 Example 18
 In Vivo Models for Studying BPC-1 and Testing Prostate Cancer Therapeutic
 Compositions
 A. Determination of Serum BPC-1 Levels in Mice Bearing Xenogenic Tumors
 LNCaP prostate cancer cells and LAPC-4 AD xenograft cells express high
 levels of BPC-1 as determined by Northern blot analysis. To evaluate BPC-1
 as a serum diagnostic marker, SCID mice are injected SQ or orthotopically
 with either 1.times.10.sup.6 LNCaP or LAPC-4 AD cells. Mice are injected
 on each flank and tumor growth is monitored by caliper measurements to
 reflect length.times.width.times.height (L.times.W.times.H). The mice are
 bled at the initial appearance of palpable tumors and every week
 thereafter until tumors are 1,000 mm.sup.3 in size. Serial bleeds are
 screened for the presence of BPC-1 by an ELISA assay as described above.
 As a control, serum from the tumor-bearing mice is assessed for the
 secretion of PSA using a specific ELISA kit. To confirm BPC-1 expression,
 tumors are harvested from the mice and screened for BPC-1 expression by
 Western blot.
 In addition, the 5637 bladder cancer cell line has been shown to express
 BPC-1 by Northern blot analysis. To evaluate bladder cancer BPC-1
 expression, 5637 bladder tumor xenografts are established in SCID mice and
 serum collected and evaluated for BPC-1 protein by ELISA as described.
 Alternatively, prostate cancer cell lines that do not express endogenous
 BPC-1 and engineered to overexpress BPC-1 may be injected into SCID mice
 to confirm BPC-1 secretion. These include PC-3, TSUPR1, and DU145.
 Individual mice are injected SQ with either 1.times.10.sup.6 PC3, TSUPR1,
 and DU145 cells expressing an empty tkNeo vector (tkNeo) or a vector
 containing BPC-1. All mice are injected on each flank and tumor growth is
 monitored by caliper measurements as described above. The mice are bled at
 the initial appearance of palpable tumors and every week thereafter until
 tumors are 1,000 mm.sup.3 in size. Differences in tumor growth rate, if
 apparent, are noted and studied further (see below). Serial bleeds may be
 screened for the presence of BPC-1 by an ELISA assay. To confirm BPC-1
 expression, tumors may be harvested from the mice and screened for BPC-1
 expression by Western blots.
 B. in vivo Assay for BPC-1 Tumor Growth Promotion
 The effect of the BPC-1 protein on tumor cell growth may be evaluated in
 vivo either by gene overexpression or addition of soluble, purified BPC-1
 protein to tumor-bearing mice. In the first example, SCID mice are
 injected SQ on each flank with 1.times.10.sup.6 of either PC3, TSUPR1, or
 DU145 cells containing tkNeo empty vector or BPC-1. At least two
 strategies may be used: (1) Constitutive BPC-1 expression under regulation
 of an LTR promoter, and (2) Regulated expression under control of the
 ecdysone-inducible vector system. Tumor volume is then monitored at the
 appearance of palpable tumors and followed over time to determine if BPC-1
 expressing cells grow at a faster rate. Additionally, mice may be
 implanted with 1.times.10.sup.5 of the same cells orthotopically to
 determine if BPC-1 has an effect on local growth in the prostate or on the
 ability of the cells to metastasize, specifically to lungs, lymph nodes,
 and bone marrow.
 In the second example, purified BPC-1 protein is be evaluated for an effect
 on tumor cell growth in vivo. Mice are first divided into groups injected
 SQ with either 1.times.10.sup.6 LNCaP or LAPC-4 AD cells, which express
 BPC-1, or PC3 cells, which do not express BPC-1. On the same day as tumor
 cells are injected, groups are injected IV with a range of purified BPC-1
 protein (for example 100, 500, or 1,000 .mu.g). As a control, one group of
 each tumor type is injected with PBS only. Injections continue 2 times per
 week for 4 consecutive weeks until tumors grow and reach a size of 1,000
 mm.sup.3. Tumor volume is followed to determine if BPC-1 has a dose
 response effect on tumor growth.
 In a separate set of experiments to determine if BPC-1 accelerates tumor
 growth, LNCaP, LAPC-4 AD, and PC3 tumors may be allowed to establish SQ to
 a size of 100 mm.sup.3, at which time purified BPC-1 protein is injected
 IV in the doses and regimen indicated above. To determine if BPC-1
 promotes metastasis, the same tumors may also be implanted orthotopically,
 and after tumors have been established (determined by circulating PSA
 levels) purified BPC-1 can be administered as described and metastatic
 growth evaluated.
 The above assays are also useful to determine the BPC-1 inhibitory effect
 of candidate therapeutic compositions, such as for example, BPC-1
 antibodies and intrabodies, BPC-1 mRNA antisense molecules and ribozymes,
 and BPC-1 receptor compositions.
 C. in Vivo BPC-1 Antibody Tumor Inhibition Assay
 To study the effect of BPC-1 specific mAbs on the formation and growth of
 tumors, mice are divided in groups of either BPC-1 positive LNCaP, LAPC-4
 AD, and PC3-BPC-1 tumors, or PC3-tkNeo, which does not express BPC-1. To
 evaluate an effect on tumor formation, mice are injected with
 1.times.10.sup.6 tumor cells SQ and on the same day are injected IP with a
 range of BPC-1 specific mAb or control Ig (for example, 100, 500, or 1,000
 .mu.g). Injections of mAbs continue 2 times per week for 4 consecutive
 weeks. Tumor growth is followed as described above. Alternatively, to
 evaluate an effect on established tumors, mice are divided into groups
 bearing established tumors 100 mm.sup.3 in size and are injected IP with
 mAbs according to the doses and regimen described previously. Tumor volume
 is followed to determine the mAb's effect on growth of established tumors.
 To study effect on metastasis, 1.times.10.sup.5 of LAPC-4 AD cells are
 injected orthotopically into SCID mice. At the same time the mice are
 injected IP with a range of anti-BPC-1 mAb or control Ig as described
 above. Tumor growth is followed by weekly determinations of circulating
 PSA. At the end of the antibody administration, the mice are sacrificed
 and local tumor growth and metastasis to lungs, lymph nodes, and bone
 marrow are evaluated. To examine an effect on mice with established
 tumors, LAPC-4 AD are injected orthotopically and PSA levels are followed
 weekly. When PSA reaches measurable levels, the mice are injected with the
 same dose and regimen of mAbs described. The mice are sacrificed after the
 completion of antibody injections to evaluate local tumor growth as well
 as metastasis.
 Example 19
 Molecular Cloning of the BPC-1 Receptor or Binding Partner
 Expression cloning strategies such as described in Tartaglia et al., 1995,
 Cell 83:1263-1271 and Cheng and Flanagan, 1994, Cell 79:157-168 and others
 may be used to clone the receptor for BPC-1. An expression library is
 first constructed from cells showing BPC-1-AP binding. The library may be
 constructed in pools of approximately 1000 clones and then screened by a
 sib selectil on procedure. Transient transfections of COS cells with DNA
 from each pool and subsequent screening with BPC-1-AP binding, washing,
 and staining for AP activity identifies cells binding BPC-1 and
 consequently expression of BPC-1 receptor. After successive rounds of pool
 subdivision and screening, single colonies binding to BPC-1-AP can be
 identified.
 An alternative approach to cloning BPC-1 receptor/binding partner genes
 utilizes expression cloning in phage (Stone J. in Current Protocols in
 Molecular Biology (1997): 20.3.1-20.3.9). For example, a LAPC-9 AD phage
 expression library in Lambda Zap Express (Stratagene) may be used.
 Membrane lifts can be probed using BPC-1-AP and positive clones detected
 with an alkaline phosphatase chemiluminescent reagent (e.g., BioRad).
 Plaques binding BPC-1-AP and producing a blue precipitates are selected
 and plasmids isolated and evaluated for the receptor/binding partner
 sequences. This approach may also result in the identification of
 cytoplasmic or secreted proteins interacting with BPC-1.
 Throughout this application, various publications are referenced within
 parentheses. The disclosures of these publications are hereby incorporated
 by reference herein in their entireties.
 The present invention is not to be limited in scope by the embodiments
 disclosed herein, which are intended as single illustrations of individual
 aspects of the invention, and any which are functionally equivalent are
 within the scope of the invention. Various modifications to the models and
 methods of the invention, in addition to those described herein, will
 become apparent to those skilled in the art from the foregoing description
 and teachings, and are similarly intended to fall within the scope of the
 invention. Such modifications or other embodiments can be practiced
 without departing from the true scope and spirit of the invention.
 SEQUENCE LISTING
 &lt;100&gt; GENERAL INFORMATION:
 &lt;160&gt; NUMBER OF SEQ ID NOS: 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 1
 &lt;211&gt; LENGTH: 2639
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Homo sapiens
 &lt;400&gt; SEQUENCE: 1
 cagccccggg gcgccggccg cgcgcagcct cgctatccca cccaggctcc gggcttccag 60
 gagggtcgcg gagccccaag ccatgactaa ggagcccatt tgatagcaga ggtggcgcgc 120
 agcccggcga gccgatgacg gaccccttct tcctgccttc aatgcctcag cggaagatcc 180
 ccaagggctg gagcgaggag cgctgccgct ggacatcctc ccggggaggc tgctccgacc 240
 tgctgcgcgg cgcgtctgag actggggact gagccactcc gccgccgccg gcgccgccgc 300
 cgccgcccgc tccgtcgctg ccgtcggtct ggactggccc ccacctcgct gcgccctctc 360
 cccggccccg gccccggctc ggggcgtccc ggggctcgcc ctgcgaccgc cgcctcccgc 420
 gcgccgcgtc ctcccgaccc cgcggcggcg acgatgcccg ggaggagggt cctgacggcg 480
 gcggcgcgga tggtggcggc cggcgcccgg gtgtgatgcg agcgtcacgg tggggatgct 540
 gctggctgcg cggcgctgag ggccagcgag agcgagagcc cgcccggggc ggaggacgga 600
 ctcatccgga tctggctgca gcgtgggctc ggagctcccc cttcctctcg gtctccctct 660
 cggcccccct ttatttcctt cttgctttgc gtctttaaca cctctcgacc ctgtcctccc 720
 cccgccactg gaagtcttcc cgtctctaaa tggaattagt ggagcccgga gcctctggtg 780
 taacgcacag acatgatcca tgggcgcagc gtgcttcaca ttgtagcaag tttaatcatc 840
 ctccatttgt ctggggcaac caagaaagga acagaaaagc aaaccacctc agaaacacag 900
 aagtcagtgc agtgtggaac ttggacaaaa catgcagagg gaggtatctt tacctctccc 960
 aactatccca gcaagtatcc ccctgaccgg gaatgcatct acatcataga agccgctcca 1020
 agacagtgca ttgaacttta ctttgatgaa aagtactcta ttgaaccgtc ttgggagtgc 1080
 aaatttgatc atattgaagt tcgagatgga ccttttggct tttctccaat aattggacgt 1140
 ttctgtggac aacaaaatcc acctgtcata aaatccagtg gaagatttct atggattaaa 1200
 ttttttgctg atggagagct ggaatctatg ggattttcag ctcgatacaa tttcacacct 1260
 ggtaagtaag tacttaaaaa aaaaatttct ttttcttcct catttttcta tcttcatagt 1320
 acaaaatctt gtgtaagaca acattatact ttctcagaga atgttccagt tctatttaaa 1380
 accaaatcta cagtgctttt tcttttccct acacaaattc tgaaaggaaa agatgttttc 1440
 cttaaaacag cctatactag aggtaaagag tagtgactca aggctctaaa tgggcatcag 1500
 ccacatcatc aagtggactt ttgttatgat ggaatgtgta attggagaga cagtctgtga 1560
 taaggaaact atacatagga gctgaataaa cttgaaaaga caattgtagt attataaaat 1620
 atatccacca aaatgatctt tggggaactt gaatcaaaag tttatttgtt ctgaaaatta 1680
 ccgtgtttca atcaaataga tcctacttta ggaagtagtc tgctctcttt tcaggaaagc 1740
 aaattcttaa gagttttgat gaaaggaaaa ctgagacctg taacagccaa atactcattt 1800
 acaaggtctt gcagaaattg tgtgcaatta tcaaattatg caatctgtat caattttcct 1860
 tttaactcgc tagaattaaa aagatcctgt gttgttgcct ggcccacttg attaagagtt 1920
 accattcatt acaataaaaa taggttatca cattttttca ctgcaagaac actacatgca 1980
 ttaatttaaa tggaaaaatg attcaaatta cataaagccc attttttata tagtttgttt 2040
 tcagtttgta tgtattgttt tatttaagtt aggcaatagc ataatttcaa atatatgtaa 2100
 agttggttga agtttgtatt ccatgttaaa gaagtaacat ctaaatacag ctttgatact 2160
 cagttaaaaa actaaaattt taaaaattat taatataagt ttaatgatga ctttcattat 2220
 gacatcatgg ggtatgttaa atcaagtatt tactgtagca tatatattag ctttaagcat 2280
 taggaatgtt tttaataata tcactaaagg attgtggttt taattatgct ttgctgataa 2340
 tggattactc acagaaatca tgggtatttc atgtgctaca gtcgaactaa tttgaagtat 2400
 tcccaaaagg tacaaatgtt agcttaattt gtttgttcag attattagtg ctagagttgt 2460
 aaatggaaag gtaggtattt ttttcttaac tgataatttt gaatataacc tgtacctaga 2520
 gacagtgaca tacggcatgt tctaggtttc ataagttata ttttcattct gggtttggtg 2580
 atcatgaaaa taatgtcttg gatttaaaat tgtggtttca caaaaaaaaa aaaaaaaaa 2639
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 2
 &lt;211&gt; LENGTH: 158
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Homo sapiens
 &lt;400&gt; SEQUENCE: 2
 Met Ile His Gly Arg Ser Val Leu His Ile Val Ala Ser Leu Ile Ile
 1 5 10 15
 Leu His Leu Ser Gly Ala Thr Lys Lys Gly Thr Glu Lys Gln Thr Thr
 20 25 30
 Ser Glu Thr Gln Lys Ser Val Gln Cys Gly Thr Trp Thr Lys His Ala
 35 40 45
 Glu Gly Gly Ile Phe Thr Ser Pro Asn Tyr Pro Ser Lys Tyr Pro Pro
 50 55 60
 Asp Arg Glu Cys Ile Tyr Ile Ile Glu Ala Ala Pro Arg Gln Cys Ile
 65 70 75 80
 Glu Leu Tyr Phe Asp Glu Lys Tyr Ser Ile Glu Pro Ser Trp Glu Cys
 85 90 95
 Lys Phe Asp His Ile Glu Val Arg Asp Gly Pro Phe Gly Phe Ser Pro
 100 105 110
 Ile Ile Gly Arg Phe Cys Gly Gln Gln Asn Pro Pro Val Ile Lys Ser
 115 120 125
 Ser Gly Arg Phe Leu Trp Ile Lys Phe Phe Ala Asp Gly Glu Leu Glu
 130 135 140
 Ser Met Gly Phe Ser Ala Arg Tyr Asn Phe Thr Pro Gly Lys
 145 150 155
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 3
 &lt;211&gt; LENGTH: 115
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Caenorhabditis elegans
 &lt;400&gt; SEQUENCE: 3
 Ile Phe Thr Ser Pro Asn Phe Pro Asp Arg Tyr Pro Pro Asn Ile Asp
 1 5 10 15
 Cys Val Arg Val Ile His Ser Arg Pro Gln His Asp Val Val Val Lys
 20 25 30
 Phe His His Val Phe His Ile Glu Ser Thr Tyr Asp Lys Ile Asp Ala
 35 40 45
 Gly Glu Glu Cys Pro Asn Asp Phe Ile Glu Phe Arg Asp Gly Arg Tyr
 50 55 60
 Gly Phe Ser Pro Leu Ile Ala Arg Phe Cys Gly Asp Arg Met Pro Lys
 65 70 75 80
 Arg Glu Ile Arg Ala Val Ser Gly Phe Leu Trp Ile Arg Phe Arg Ser
 85 90 95
 Asp Ser Met Leu Glu Tyr Gln Gly Phe Ser Ala Glu Tyr Ala Ile Val
 100 105 110
 Pro Ser Lys
 115
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 4
 &lt;211&gt; LENGTH: 101
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Mouse
 &lt;400&gt; SEQUENCE: 4
 Gly Asn Phe Ser Ser Pro Glu Tyr Pro Asn Gly Tyr Ser Ala His Met
 1 5 10 15
 His Cys Val Trp Arg Ile Ser Val Thr Pro Gly Glu Lys Ile Ile Leu
 20 25 30
 Asn Phe Thr Ser Met Asp Leu Tyr Arg Ser Arg Leu Cys Trp Tyr Asp
 35 40 45
 Tyr Val Glu Val Arg Asp Gly Phe Trp Arg Lys Val Trp Val Arg Gly
 50 55 60
 Arg Phe Cys Gly Gly Lys Leu Pro Glu Pro Ile Val Ser Thr Asp Ser
 65 70 75 80
 Arg Leu Trp Val Glu Phe Arg Ser Ser Ser Asn Trp Val Gly Lys Gly
 85 90 95
 Phe Phe Ala Val Tyr
 100
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 5
 &lt;211&gt; LENGTH: 103
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Mouse
 &lt;400&gt; SEQUENCE: 5
 Asp Asn Gly His Ile Gln Ser Pro Asn Tyr Pro Asp Asp Tyr Arg Pro
 1 5 10 15
 Ser Lys Val Cys Ile Trp Arg Ile Gln Val Ser Glu Gly Phe His Val
 20 25 30
 Gly Leu Thr Phe Gln Ser Phe Glu Ile Glu Arg His Asp Ser Cys Ala
 35 40 45
 Tyr Asp Tyr Leu Glu Val Arg Asp Gly His Ser Glu Ser Ser Asn Leu
 50 55 60
 Ile Gly Arg Tyr Cys Gly Tyr Glu Asn Pro Asp Asp Ile Lys Ser Thr
 65 70 75 80
 Ser Ser Arg Leu Trp Leu Lys Phe Val Ser Asp Gly Ser Ile Asn Lys
 85 90 95
 Ala Gly Phe Ala Val Asn Phe
 100
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 6
 &lt;211&gt; LENGTH: 101
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Mouse
 &lt;400&gt; SEQUENCE: 6
 Gly Ser Ile Thr Ser Pro Gly Trp Pro Lys Glu Tyr Pro Pro Asn Lys
 1 5 10 15
 Asn Cys Ile Trp Gln Leu Val Ala Pro Thr Gln Tyr Arg Ile Ser Leu
 20 25 30
 Gln Phe Asp Phe Phe Glu Thr Glu Gly Asn Asp Val Cys Lys Tyr Asp
 35 40 45
 Phe Val Glu Val Arg Ser Gly Leu Thr Ala Asp Ser Lys Leu His Gly
 50 55 60
 Lys Phe Cys Gly Ser Glu Lys Pro Glu Val Ile Thr Ser Gln Tyr Asn
 65 70 75 80
 Asn Met Arg Val Glu Phe Lys Ser Asp Asn Thr Val Ser Lys Lys Gly
 85 90 95
 Phe Lys Ala His Phe
 100
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 7
 &lt;211&gt; LENGTH: 102
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Mouse
 &lt;400&gt; SEQUENCE: 7
 Gly Thr Ile Thr Ser Pro Asn Trp Pro Asp Lys Tyr Pro Ser Lys Lys
 1 5 10 15
 Glu Cys Thr Trp Ala Ile Ser Ser Thr Pro Gly His Arg Val Lys Leu
 20 25 30
 Thr Phe Val Glu Met Asp Ile Glu Ser Gln Pro Glu Cys Ala Tyr Asp
 35 40 45
 His Leu Glu Val Phe Asp Gly Arg Asp Ala Lys Ala Pro Val Leu Gly
 50 55 60
 Arg Phe Cys Gly Ser Lys Lys Pro Glu Pro Val Leu Ala Thr Gly Asn
 65 70 75 80
 Arg Met Phe Leu Arg Phe Tyr Ser Asp Asn Ser Val Gln Arg Lys Gly
 85 90 95
 Phe Gln Ala Ser His Ser
 100
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 8
 &lt;211&gt; LENGTH: 95
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Mouse
 &lt;400&gt; SEQUENCE: 8
 Asn Asn Tyr Pro Gly Gly Val Asp Cys Glu Trp Val Ile Val Ala Glu
 1 5 10 15
 Glu Gly Tyr Gly Val Glu Leu Val Phe Gln Thr Phe Glu Val Glu Glu
 20 25 30
 Glu Thr Asp Cys Gly Tyr Asp Tyr Ile Glu Leu Phe Asp Gly Tyr Asp
 35 40 45
 Ser Thr Ala Pro Arg Leu Gly Arg Tyr Cys Gly Ser Gly Pro Pro Glu
 50 55 60
 Glu Val Tyr Ser Ala Gly Asp Ser Val Leu Val Lys Phe His Ser Asp
 65 70 75 80
 Asp Thr Ile Ser Lys Lys Gly Phe His Leu Arg Tyr Thr Ser Thr
 85 90 95
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 9
 &lt;211&gt; LENGTH: 14
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence cDNA
 synthesis primer
 &lt;400&gt; SEQUENCE: 9
 ttttgatcaa gctt 14
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 10
 &lt;211&gt; LENGTH: 42
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence DNA Adaptor
 1
 &lt;400&gt; SEQUENCE: 10
 ctaatacgac tcactatagg gctcgagcgg ccgcccgggc ag 42
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 11
 &lt;211&gt; LENGTH: 40
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence DNA Adaptor
 2
 &lt;400&gt; SEQUENCE: 11
 gtaatacgac tcactatagg gcagcgtggt cgcggccgag 40
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 12
 &lt;211&gt; LENGTH: 22
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence PCR primer
 1
 &lt;400&gt; SEQUENCE: 12
 ctaatacgac tcactatagg gc 22
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 13
 &lt;211&gt; LENGTH: 22
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence Nested
 primer (NP)1
 &lt;400&gt; SEQUENCE: 13
 tcgagcggcc gcccgggcag ga 22
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 14
 &lt;211&gt; LENGTH: 20
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence Nested
 primer (NP)2
 &lt;400&gt; SEQUENCE: 14
 agcgtggtcg cggccgagga 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 15
 &lt;211&gt; LENGTH: 24
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence RT-PCR
 primer
 &lt;400&gt; SEQUENCE: 15
 tgccgtatgt cactgtctct aggt 24
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 16
 &lt;211&gt; LENGTH: 24
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence RT-PCR
 primer
 &lt;400&gt; SEQUENCE: 16
 gaaatcatgg gtatttcatg tgct 24
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 17
 &lt;211&gt; LENGTH: 26
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence RT-PCR
 primer
 &lt;400&gt; SEQUENCE: 17
 ttgaattcca agcaaaccac ctcaga 26
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 18
 &lt;211&gt; LENGTH: 28
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence RT-PCR
 primer
 &lt;400&gt; SEQUENCE: 18
 aagctcgagt cagacggttc aatagagt 28
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 19
 &lt;211&gt; LENGTH: 30
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence BPC1.
 HINDIII primer
 &lt;400&gt; SEQUENCE: 19
 gtgtaagctt ccaccaagaa aggaacagaa 30
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 20
 &lt;211&gt; LENGTH: 29
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence BPC1.BAMHI
 primer
 &lt;400&gt; SEQUENCE: 20
 cacaggatcc cttaccaggt gtgaaattg 29