Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

• the invention relates generally to detecting diseases of the pancreas. More particularly, the present invention relates to reagents such as polynucleotide sequences; polypeptide sequences encoded thereby, as well as methods which utilize these sequences, all of which are useful for detecting, diagnosing, staging, monitoring, prognosticating, in vivo imaging, preventing or treating, or determining predisposition to diseases or conditions of the pancreas such as pancreatic cancer.
• reagents such as polynucleotide sequences; polypeptide sequences encoded thereby, as well as methods which utilize these sequences, all of which are useful for detecting, diagnosing, staging, monitoring, prognosticating, in vivo imaging, preventing or treating, or determining predisposition to diseases or conditions of the pancreas such as pancreatic cancer.
• Pancreatic cancer has the fifth highest mortality among cancers in the U.S. with a projected 28,900 deaths during 1998. The number of pancreatic cancer- related deaths is about the same as the incidence of this disease which is estimated to be 29, 000 new cases during 1998. (American Cancer Society statistics). Pancreatic cancer is, therefore, almost always fatal.
• pancreatic cancer Improved procedures for detecting, diagnosing, staging, monitoring, prognosticating, in vivo imaging, preventing or treating, or determining predisposition to diseases or conditions of the pancreas, especially pancreatic cancer, are of critical importance to improving the outcome of the patient. For example, patients diagnosed with regional or distantly metastasized pancreatic cancers have only 5% and 2% five year survival rates, respectively. (American Cancer Society statistics). Greater than 80% of pancreatic cancers have progressed beyond the pancreas at diagnosis and at least half of these involve visceral metastases. M. F. Brennan, et al. In: Cancer: Principles and Practice of Oncology. Fourth Edition, pp. 849-882, Philadelphia, PA: J/B. Lippincott Co.
• pancreatic cancer has potential prognostic value and provides criteria for designing optimal therapy.
• clinical staging of pancreatic cancer depends on imaging modalities such as CT and endoscopic retrograde cholangiopancreatography. If clinical staging indicates the tumor is unresectable, histologic confirmation should be obtained. M. F. Brennan, et al.. supra.
• Disadvantages of imaging methods include technical problems which can limit the sensitivity of tumor detection, positioning and cooperation of the patient, and interpretation of the image by the radiologist. Histologic evaluation requires invasive procedures and is also subjective in nature. Staging of pancreatic cancer could be improved by detecting new markers in serum or other bodily fluids which could differentiate between different stages of invasion. Such markers could be mRNA or protein markers expressed by cells originating from the primary tumor in the pancreas but residing in blood, bone marrow or lymph nodes and could serve as sensitive indicators for metastasis to these distal organs.
• colorectal cancer for example, specific protein antigens and mRNA, associated with colorectal epithelial cells, have been detected by immunohistochemical techniques and RT-PCR, respectively, in bone marrow and lymph nodes of colorectal cancer patients suggesting metastasis.
• Pancreatic cancer patients are monitored following initial therapy and during adjuvant therapy to determine the patient's response, and to detect persistent or recurrent disease, or early distant metastasis.
• One monitoring method is the measurement of serum CA 19-9 which is an antigen found to be elevated in the serum of patients having pancreatic cancer.
• Diagnosis of diseases of the pancreas could be improved by discovering markers that appear in test samples obtained by minimally invasive procedures such as blood, plasma, serum, or urine, and are subsequently detected by immunological methods. Tests based on such markers would provide information to aid the physician in managing the patient with disease of the pancreas at low cost to the patient. Markers such as prostate specific antigen (PSA) and human chorionic gonadotropin (hCG) exist and are used clinically for screening patients for prostate cancer and testicular cancer, respectively. For example, PSA normally is secreted by the prostate at high levels into the seminal fluid, but is present in very low levels in the blood of men with normal prostates.
• PSA prostate specific antigen
• hCG human chorionic gonadotropin
• Elevated levels of PSA protein in serum are used in the early detection of prostate cancer in asymptomatic men. G.E. Hanks, et al., In: Cancer: Principles and Practice of Oncology. Fourth Edition, pp. 1073-1113, Philadelphia, PA: J.B. Lippincott Co. 1993. M. K. Schwartz, et al.. supra.
• the management of diseases of the pancreas could be improved by the use of new markers, normally expressed in the pancreas, but found in elevated amounts in an inappropriate body compartment as a result of disease of the pancreas.
• Such methods would include assaying a test sample for products of a gene which are overexpressed in diseases and conditions associated with the pancreas, including cancer. Such methods may also include assaying a test sample for products of a gene which have been altered by the disease or condition associated with the pancreas, including cancer. Such methods may further include assaying a test sample for products of a gene whose distribution among the various tissues and compartments of the body has been altered by a pancreas-associated disease or condition, including cancer.
• Such methods would comprise making cDNA from mRNA in the test sample, amplifying, when necessary, portions of the cDNA corresponding to the gene or a fragment thereof, and detecting the cDNA product as an indication of the presence of the disease or condition, including cancer, or detecting translation products of the mRNAs comprising gene sequences as an indication of the presence of the disease.
• Useful reagents include polynucleotide(s), or fragment(s) thereof, which may be used in diagnostic methods such as reverse transcriptase-polymerase chain reaction (RT-PCR), PCR, or hybridization assays of mRNA extracted from biopsied tissue, blood or other test samples; or proteins which are the translation products of such mRNAs; or antibodies directed against these proteins.
• Such assays would include, for example, methods for assaying a sample for product(s) of the gene and detecting the product(s) as an indication of disease of the pancreas.
• Drug treatment or gene therapy for diseases and conditions of the pancreas, including cancer can be based on these identified gene sequences or their expressed proteins, and efficacy of any particular therapy can be monitored.
• the present invention provides a method of detecting a target PA153 polynucleotide in a test sample which comprises contacting the test sample with at least one PA153-specific polynucleotide and detecting the presence of the target PA153 polynucleotide in the test sample.
• the PA153-specific polynucleotide has at least 80% identity with a polynucleotide selected from the group consisting of SEQUENCE ID NO 1, SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4, SEQUENCE ID NO 5, SEQUENCE ID NO 6, SEQUENCE ID NO 7, SEQUENCE ID NO 8, SEQUENCE ID NO 9, SEQUENCE ID NO 10, SEQUENCE ID NO 11 (SEQUENCE ID NOS 1-11), and fragments or complements thereof.
• the PA153-specific polynucleotide may be attached to a solid phase prior to performing the method.
• the present invention also provides a method for detecting PAl 53 mRNA in a test sample, which comprises performing reverse transcription (RT) with at least one primer in order to produce cDNA, amplifying the cDNA so obtained using PAl 53 oligonucleotides as sense and antisense primers to obtain PAl 53 amplicon, and detecting the presence of the PAl 53 amplicon as an indication of the presence of PAl 53 mRNA in the test sample, wherein the PAl 53 oligonucleotides have at least 80% identity with a sequence selected from the group consisting of SEQUENCE ID NOS 1-11, and fragments or complements thereof.
• Amplification can be performed by the polymerase chain reaction.
• the test sample can be reacted with a solid phase prior to performing the method, prior to amplification or prior to detection.
• This reaction can be a direct or an indirect reaction.
• the detection step can comprise utilizing a detectable label capable of generating a measurable signal.
• the detectable label can be attached to a solid phase.
• the present invention further provides a method of detecting a target PAl 53 polynucleotide in a test sample suspected of containing target PAl 53 polynucleotides, which comprises (a) contacting the test sample with at least one PAl 53 oligonucleotide as a sense primer and at least one PAl 53 oligonucleotide as an antisense primer, and amplifying same to obtain a first stage reaction product; (b) contacting the first stage reaction product with at least one other PAl 53 oligonucleotide to obtain a second stage reaction product, with the proviso that the other PAl 53 oligonucleotide is located 3 1 to the PAl 53 oligonucleotides utilized in step (a) and is complementary to the first stage reaction product; and (c) detecting the second stage reaction product as an indication of the presence of a target PAl 53 polynucleotide in the test sample.
• the PAl 53 oligonucleotides selected as reagents in the method have at least 80% identity with a sequence selected from the group consisting of SEQUENCE ID NOS 1-11, and fragments or complements thereof.
• Amplification may be performed by the polymerase chain reaction.
• the test sample can be reacted either directly or indirectly with a solid phase prior to performing the method, or prior to amplification, or prior to detection.
• the detection step also comprises utilizing a detectable label capable of generating a measurable signal; further, the detectable label can be attached to a solid phase.
• Test kits useful for detecting target PAl 53 polynucleotide in a test sample comprise a container containing at least one PAl 53 specific polynucleotide selected from the group consisting of SEQUENCE ID NOS 1-11, and fragments or complements thereof.
• These test kits further comprise containers with tools useful for collecting test samples (such as, for example, blood, urine, saliva and stool).
• tools include lancets and absorbent paper or cloth for collecting and stabilizing blood; swabs for collecting and stabilizing saliva; and cups for collecting and stabilizing urine or stool samples.
• Collection materials such as papers, cloths, swabs, cups, and the like, may optionally be treated to avoid denaturation or irreversible adsorption of the sample.
• the collection materials also may be treated with or contain preservatives, stabilizers or antimicrobial agents to help maintain the integrity of the specimens.
• the present invention also provides a purified polynucleotide or fragment thereof derived from a PAl 53 gene.
• the purified polynucleotide is capable of selectively hybridizing to the nucleic acid of the PAl 53 gene, or a complement thereof.
• the polynucleotide has at least 80% identity with a polynucleotide selected from the group consisting of SEQUENCE ID NOS 1-11, and fragments or complements thereof.
• the purified polynucleotide can be produced by recombinant and/or synthetic techniques.
• the purified recombinant polynucleotide can be contained within a recombinant vector.
• the invention further comprises a host cell transfected with the recombinant vector.
• the present invention further provides a recombinant expression system comprising a nucleic acid sequence that includes an open reading frame derived from PAl 53.
• the nucleic acid sequence has at least 80% identity with a sequence selected from the group consisting of SEQUENCE ID NOS 1-11, and fragments or complements thereof.
• the nucleic acid sequence is operably linked to a control sequence compatible with a desired host. Also provided is a cell transfected with this recombinant expression system.
• the present invention also provides a polypeptide encoded by PAl 53.
• the polypeptide can be produced by recombinant technology, provided in purified form, or produced by synthetic techniques.
• the polypeptide comprises an amino acid sequence which has at least 80% identity with an amino acid sequence selected from the group consisting of SEQUENCE ID NO 28, SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31, SEQUENCE ID NO 32, and fragments thereof.
• a specific binding molecule such as an antibody, which specifically binds to at least one PAl 53 epitope.
• the antibody can be a polyclonal or monoclonal antibody.
• the epitope is derived from an amino acid sequence selected from the group consisting of SEQUENCE ID NO 28, SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31, SEQUENCE ID NO 32, and fragments thereof.
• Assay kits for determining the presence of PAl 53 antigen or anti- PAl 53 antibody in a test sample are also included.
• the assay kits comprise a container containing at least one PAl 53 polypeptide having at least 80% identity with an amino acid sequence selected from the group consisting of SEQUENCE ID NO 28, SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31, SEQUENCE ID NO 32, and fragments thereof.
• the test kit can comprise a container with tools useful for collecting test samples (such as blood, urine, saliva, and stool). Such tools include lancets and absorbent paper or cloth for collecting and stabilizing blood; swabs for collecting and stabilizing saliva; and cups for collecting and stabilizing urine or stool samples. Collection materials such as papers, cloths, swabs, cups, and the like, may optionally be treated to avoid denaturation or irreversible adsorption of the sample. These collection materials also may be treated with or contain preservatives, stabilizers or antimicrobial agents to help maintain the integrity of the specimens.
• the polypeptide can be attached to a solid phase.
• antibodies or fragments thereof against the PAl 53 antigen can be used to detect or image localization of the antigen in a patient for the purpose of detecting or diagnosing a disease or condition.
• Such antibodies can be polyclonal or monoclonal, or made by molecular biology techniques, and can be labeled with a variety of detectable labels, including but not limited to radioisotopes and paramagnetic metals.
• antibodies or fragments thereof, whether monoclonal, polyclonal, or made by molecular biology techniques can be used as therapeutic agents for the treatment of diseases characterized by expression of the PAl 53 antigen.
• the antibody may be used without derivitization, or it may be derivitized with a cytotoxic agent such as a radioisotope, enzyme, toxin, drug, prodrug, or the like.
• Another assay kit for determining the presence of PAl 53 antigen or anti- PAl 53 antibody in a test sample comprises a container containing a specific binding molecule, such as an antibody, which specifically binds to a PAl 53 antigen, wherein the PAl 53 antigen comprises at least one PA153-encoded epitope.
• the PAl 53 antigen has at least about 80% sequence identity to a sequence of a PA153-encoded antigen selected from the group consisting of SEQUENCE ID NO 28, SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31 , SEQUENCE ID NO 32, and fragments thereof.
• These test kits can further comprise containers with tools useful for collecting test samples (such as blood, urine, saliva, and stool).
• Such tools include lancets and absorbent paper or cloth for collecting and stabilizing blood; swabs for collecting and stabilizing saliva; cups for collecting and stabilizing urine or stool samples.
• Collection materials such as papers, cloths, swabs, cups and the like, may optionally be treated to avoid denaturation or irreversible adsorption of the sample. These collection materials also may be treated with, or contain, preservatives, stabilizers or antimicrobial agents to help maintain the integrity of the specimens.
• the antibody can be attached to a solid phase.
• PAl 53 is provided, which method comprises incubating host cells transfected with an expression vector.
• This vector comprises a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 80% identity with a PAl 53 amino acid sequence selected from the group consisting of SEQUENCE ID NO 28, SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31, SEQUENCE ID NO 32, and fragments thereof.
• SEQUENCE ID NO 28 SEQUENCE ID NO 29
• SEQUENCE ID NO 31 SEQUENCE ID NO 32
• the method comprises contacting the test sample with a specific binding molecule, such as an antibody or fragment thereof, which specifically binds to at least one epitope of PA153 antigen, for a time and under conditions sufficient for the formation of antibody/antigen complexes; and detecting the presence of such complexes containing the antibody as an indication of the presence of PAl 53 antigen in the test sample.
• a specific binding molecule such as an antibody or fragment thereof, which specifically binds to at least one epitope of PA153 antigen, for a time and under conditions sufficient for the formation of antibody/antigen complexes; and detecting the presence of such complexes containing the antibody as an indication of the presence of PAl 53 antigen in the test sample.
• the antibody can be attached to a solid phase and may be either a monoclonal or polyclonal antibody.
• the specific binding molecule specifically binds to at least one PAl 53 antigen selected from the group consisting of SEQUENCE ID NO 28, SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQU
• Another method which detects antibodies which specifically bind to PAl 53 antigen in a test sample suspected of containing these antibodies.
• the method comprises contacting the test sample with a polypeptide which contains at least one PAl 53 epitope, wherein the PAl 53 epitope comprises an amino acid sequence having at least 80% identity with an amino acid sequence encoded by a PAl 53 polynucleotide, or a fragment thereof. Contacting is performed for a time and under conditions sufficient to allow antigen/antibody complexes to form.
• the method further entails detecting complexes which contain the polypeptide.
• the polypeptide can be attached to a solid phase.
• polypeptide can be a recombinant protein or a synthetic peptide having at least 80% identity with an amino acid sequence selected from the group consisting of SEQUENCE ID NO 28, SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31, SEQUENCE ID NO 32, and fragments thereof.
• the present invention provides a cell transfected with a PAl 53 nucleic acid sequence that encodes at least one epitope of a PAl 53 antigen, or fragment thereof.
• the nucleic acid sequence is selected from the group consisting of SEQUENCE ID NOS 1-11, and fragments or complements thereof.
• a method for producing antibodies to PAl 53 antigen comprises administering to an individual an isolated immunogenic polypeptide or fragment thereof, wherein the isolated immunogenic polypeptide comprises at least one PAl 53 epitope.
• the immunogenic polypeptide is administered in an amount sufficient to produce an immune response.
• the isolated, immunogenic polypeptide comprises an amino acid sequence selected from the group consisting of SEQUENCE ID NO 28, SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31, SEQUENCE ID NO 32, and fragments thereof.
• Another method for producing antibodies which specifically bind to PAl 53 antigen comprises administering to an individual a plasmid comprising a nucleic acid sequence which encodes at least one PAl 53 epitope derived from an amino acid sequence selected from the group consisting of SEQUENCE ID NO 28, SEQUENCE ID NO 29, SEQUENCE ID NO 30,
• composition of matter that comprises a PA153 polynucleotide of at least about 10-12 nucleotides having at least 80% identity with a polynucleotide selected from the group consisting of SEQUENCE ID NOS 1-11, and fragments or complements thereof.
• the PAl 53 polynucleotide encodes an amino acid sequence having at least one PAl 53 epitope.
• Another composition of matter provided by the present invention comprises a polypeptide with at least one PA153 epitope of about 8-10 amino acids.
• the polypeptide comprises an amino acid sequence having at least 80% identity with an amino acid sequence selected from the group consisting of SEQUENCE ID NO 28, SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31, SEQUENCE ID NO 32, and fragments thereof. Also provided is a gene, or fragment thereof, coding for a PAl 53 polypeptide which has at least 80% identity with SEQUENCE ID NO 28, and a gene, or a fragment thereof comprising DNA having at least 80% identity with SEQUENCE ID NO 10 or SEQUENCE ID NO 11.
• Figures 1A-1D show the nucleotide alignment of clones 2075919H1
• SEQUENCE ID NO 1 2383634H1 (SEQUENCE ID NO 2), 5069724H1 (SEQUENCE ID NO 3), 5070712H1 (SEQUENCE ID NO 4), 2773816H1 (SEQUENCE ID NO 5), 2374806H1 (SEQUENCE ID NO 6), 5071731H1 (SEQUENCE ID NO 7), 883484H1 (SEQUENCE ID NO 8), 887213H1 (SEQUENCE ID NO 9), the full-length sequence of clone 2075919 [designated as
• FIG. 1 shows the contig map depicting the formation of the consensus nucleotide sequence (SEQUENCE ID NO 11) from the nucleotide alignment of overlapping clones 2075919H1 (SEQUENCE ID NO 1), 2383634H1 (SEQUENCE ID NO 2), 5069724H1 (SEQUENCE ID NO 3), 5070712H1 (SEQUENCE ID NO 4), 2773816H1 (SEQUENCE ID NO 5), 2374806H1 (SEQUENCE ID NO 6),
• Figure 3 is a scan of a SYBR® Green stained agarose gel of PAl 53 RNA specific RT-PCR amplification products using various normal and cancer tissue RNAs as templates.
• Figure 4 shows the results of the Western blot performed on a panel of tissue protein extracts probed with antiserum against PA153 synthetic peptide (SEQUENCE ID NO 32).
• the present invention provides a gene, or a fragment thereof, which codes for a PAl 53 polypeptide having at least about 80% identity with SEQUENCE ID NO 28.
• the present invention further encompasses a PAl 53 gene, or a fragment thereof, comprising DNA which has at least about 80% identity with SEQUENCE ID NO 10 or SEQUENCE ID NO 11.
• the present invention also provides methods for assaying a test sample for products of a pancreatic tissue gene designated as PAl 53, which comprises making cDNA from mRNA in the test sample, and detecting the cDNA as an indication of the presence of pancreatic tissue gene PA153.
• the method may include an amplification step, wherein one or more portions of the mRNA from PAl 53 corresponding to the gene or fragments thereof, is amplified.
• Methods also are provided for assaying for the translation products of PAl 53.
• Test samples which may be assayed by the methods provided herein include tissues, cells, body fluids and secretions.
• the present invention also provides reagents such as oligonucleotide primers and polypeptides which are useful in performing these methods.
• nucleic acid sequences disclosed herein are useful as primers for the reverse transcription of RNA or for the amplification of cDNA; or as probes to determine the presence of certain mRNA sequences in test samples. Also disclosed are nucleic acid sequences which permit the production of encoded polypeptide sequences which are useful as standards or reagents in diagnostic immunoassays, as targets for pharmaceutical screening assays and/or as components or as target sites for various therapies. Monoclonal and polyclonal antibodies directed against at least one epitope contained within these polypeptide sequences are useful as delivery agents for therapeutic agents as well as for diagnostic tests and for screening for diseases or conditions associated with PAl 53, especially pancreatic cancer.
• Isolation of sequences of other portions of the gene of interest can be accomplished utilizing probes or PCR primers derived from these nucleic acid sequences. This allows additional probes of the mRNA or cDNA of interest to be established, as well as corresponding encoded polypeptide sequences. These additional molecules are useful in detecting, diagnosing, staging, monitoring, prognosticating, in vivo imaging, preventing or treating, or determining the predisposition to diseases and conditions of the pancreas, such as pancreatic cancer, characterized by PAl 53, as disclosed herein.
• compositions and methods described herein will enable the identification of certain markers as indicative of a pancreatic tissue disease or condition; the information obtained therefrom will aid in the detecting, diagnosing, staging, monitoring, prognosticating, in vivo imaging, preventing or treating, or determining diseases or conditions associated with PAl 53, especially pancreatic cancer.
• Test methods include, for example, probe assays which utilize the sequence(s) provided herein and which also may utilize nucleic acid amplification methods such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), and hybridization.
• PCR polymerase chain reaction
• LCR ligase chain reaction
• the nucleotide sequences provided herein contain open reading frames from which an immunogenic epitope may be found.
• This epitope is believed to be unique to the disease state or condition associated with PAl 53. It also is thought that the polynucleotides or polypeptides and protein encoded by the PAl 53 gene are useful as a marker. This marker is either elevated in disease such as pancreatic cancer, altered in disease such as pancreatic cancer, or present as a normal protein but appearing in an inappropriate body compartment.
• the uniqueness of the epitope may be determined by (i) its immunological reactivity and specificity with antibodies directed against proteins and polypeptides encoded by the PAl 53 gene, and (ii) its nonreactivity with any other tissue markers.
• RIA radioimmunoassay
• ELISA enzyme-linked immunoabsorbent assay
• HA hemagglutination
• FPIA fluorescence polarization immunoassay
• CLIA chemiluminescent immunoassay
• a polynucleotide "derived from” or “specific for” a designated sequence refers to a polynucleotide sequence which comprises a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding, i.e., identical or complementary to, a region of the designated nucleotide sequence.
• the sequence may be complementary or identical to a sequence which is unique to a particular polynucleotide sequence as determined by techniques known in the art. Comparisons to sequences in databanks, for example, can be used as a method to determine the uniqueness of a designated sequence. Regions from which sequences may be derived, include but are not limited to, regions encoding specific epitopes, as well as non-translated and/or non- transcribed regions.
• the derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of interest under study, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide. In addition, combinations of regions corresponding to that of the designated sequence may be modified in ways known in the art to be consistent with the intended use.
• a "fragment" of a specified polynucleotide refers to a polynucleotide sequence which comprises a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10- 12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding, i.e., identical or complementary to, a region of the specified nucleotide sequence.
• primer denotes a specific oligonucleotide sequence which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence.
• a primer serves as an initiation point for nucleotide polymerization catalyzed by either DNA polymerase, RNA polymerase or reverse transcriptase.
• probe denotes a defined nucleic acid segment (or nucleotide analog segment, e.g., PNA as defined hereinbelow) which can be used to identify a specific polynucleotide present in samples bearing the complementary sequence.
• Encoded by refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence. Also encompassed are polypeptide sequences which are immunologically identifiable with a polypeptide encoded by the sequence. Thus, a "polypeptide,” “protein,” or “amino acid” sequence has at least about 50% identity, preferably about 60% identity, more preferably about 80-85% identity, and most preferably about 90-95% or more identity with a PAl 53 amino acid sequence.
• PAl 53 "polypeptide,” “protein,” or “ amino acid” sequence may have at least about 60% similarity, preferably at least about 75% similarity, more preferably about 85% similarity, and most preferably about 95% or more similarity to a polypeptide or amino acid sequence of PA153.
• This amino acid sequence can be selected from the group consisting of SEQUENCE ID NO 28, SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31, SEQUENCE ID NO 32, and fragments thereof.
• a recombinant or encoded polypeptide or protein is not necessarily translated from a designated nucleic acid sequence. It also may be generated in any manner, including chemical synthesis or expression of a recombinant expression system.
• synthetic peptide as used herein means a polymeric form of amino acids of any length, which may be chemically synthesized by methods well- known to the routineer. These synthetic peptides are useful in various applications.
• polynucleotide as used herein means a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleo tides. This term refers only to the primary structure of the molecule. Thus, the term includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modifications, such as methylation or capping and unmodified forms of the polynucleotide.
• polynucleotide oligomer
• oligonucleotide and “ oligo” are used interchangeably herein.
• a sequence corresponding to a cDNA means that the sequence contains a polynucleotide sequence that is identical or complementary to a sequence in the designated DNA.
• the degree (or " percent” ) of identity or complementarity to the cDNA will be approximately 50% or greater, preferably at least about 70% or greater, and more preferably at least about 80% or greater, and most preferably at least about 90% or greater.
• the sequence that corresponds to the identified cDNA will be at least about 50 nucleotides in length, preferably at least about 60 nucleotides in length, and more preferably at least about 70 nucleotides in length.
• the correspondence between the gene or gene fragment of interest and the cDNA can be determined by methods known in the art and include, for example, a direct comparison of the sequenced material with the cDNAs described, or hybridization and digestion with single strand nucleases, followed by size determination of the digested fragments.
• Techniques for determining amino acid sequence "similarity" are well-known in the art. In general, “ similarity” means the exact amino acid to amino acid comparison of two or more polypeptides at the appropriate place, where amino acids are identical or possess similar chemical and/or physical properties such as charge or hydrophobicity. A so-termed "percent similarity" then can be determined between the compared polypeptide sequences.
• nucleic acid and amino acid sequence identity also are well known in the art and include determining the nucleotide sequence of the mRNA for that gene (usually via a cDNA intermediate) and determining the amino acid sequence encoded thereby, and comparing this to a second amino acid sequence.
• identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.
• Two or more polynucleotide sequences can be compared by determining their "percent identity.”
• Two or more amino acid sequences likewise can be compared by determining their "percent identity.”
• the percent identity of two sequences, whether nucleic acid or peptide sequences is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100.
• An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be extended to use with peptide sequences using the scoring matrix developed by Davhoff. Atlas of Protein Sequences and Structure. M.O. Dayhoff ed., 5 suppl.
• Polynucleotide refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about 90%, of the protein with which the polynucleotide is naturally associated.
• Techniques for purifying polynucleotides of interest include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.
• Purified polypeptide or “purified protein” means a polypeptide of interest or fragment thereof which is essentially free of, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about 90%, cellular components with which the polypeptide of interest is naturally associated. Methods for purifying polypeptides of interest are known in the art.
• isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
• a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, which is separated from some or all of the coexisting materials in the natural system, is isolated.
• Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment.
• Polypeptide and “protein” are used interchangeably herein and indicate at least one molecular chain of amino acids linked through covalent and/or non-covalent bonds. The terms do not refer to a specific length of the product. Thus peptides, oligopeptides and proteins are included within the definition of polypeptide. The terms include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. In addition, protein fragments, analogs, mutated or variant proteins, fusion proteins and the like are included within the meaning of polypeptide.
• a “fragment" of a specified polypeptide refers to an amino acid sequence which comprises at least about 3-5 amino acids, more preferably at least about 8-10 amino acids, and even more preferably at least about 15-20 amino acids derived from the specified polypeptide.
• Recombinant host cells refer to cells which can be, or have been, used as recipients for recombinant vector or other transferred DNA, and include the original progeny of the original cell which has been transfected.
• replicon means any genetic element, such as a plasmid, a chromosome or a virus, that behaves as an autonomous unit of polynucleotide replication within a cell.
• a “vector” is a replicon in which another polynucleotide segment is attached, such as to bring about the replication and/or expression of the attached segment.
• control sequence refers to a polynucleotide sequence which is necessary to effect the expression of a coding sequence to which it is ligated. The nature of such control sequences differs depending upon the host organism. In prokaryotes, such control sequences generally include a promoter, a ribosomal binding site and terminators; in eukaryotes, such control sequences generally include promoters, terminators and, in some instances, enhancers.
• control sequence thus is intended to include at a minimum all components whose presence is necessary for expression, and also may include additional components whose presence is advantageous, for example, leader sequences.
• Operably linked refers to a situation wherein the components described are in a relationship permitting them to function in their intended manner.
• a control sequence "operably linked" to a coding sequence is ligated in such a manner that expression of the coding sequence is achieved under conditions compatible with the control sequence.
• open reading frame or " ORF” refers to a region of a polynucleotide sequence which encodes a polypeptide. This region may represent a portion of a coding sequence or a total coding sequence.
• a " coding sequence” is a polynucleotide sequence which is transcribed into mRNA and translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5' -terminus and a translation stop codon at the 3' - terminus.
• a coding sequence can include, but is not limited to, mRNA, cDNA and recombinant polynucleotide sequences.
• immunologically identifiable with/as refers to the presence of epitope(s) and polypeptide(s) which also are present in and are unique to the designated polypeptide(s). Immunological identity may be determined by antibody binding and/or competition in binding. These techniques are known to the routineer and also are described herein. The uniqueness of an epitope also can be determined by computer searches of known data banks, such as GenBank, for the polynucleotide sequence which encodes the epitope and by amino acid sequence comparisons with other known proteins. As used herein, "epitope” means an antigenic determinant of a polypeptide or protein. Conceivably, an epitope can comprise three amino acids in a spatial conformation which is unique to the epitope.
• an epitope consists of at least five such amino acids and more usually, it consists of at least eight to ten amino acids.
• Methods of examining spatial conformation include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance.
• a “ conformational epitope” is an epitope that is comprised of a specific juxtaposition of amino acids in an immunologically recognizable structure, such amino acids being present on the same polypeptide in a contiguous or non-contiguous order or present on different polypeptides.
• a polypeptide is " immunologically reactive" with an antibody when it binds to an antibody due to antibody recognition of a specific epitope contained within the polypeptide. Immunological reactivity may be determined by antibody binding, more particularly, by the kinetics of antibody binding, and/or by competition in binding using as competitor(s) a known polypeptide(s) containing an epitope against which the antibody is directed. The methods for determining whether a polypeptide is immunologically reactive with an antibody are known in the art.
• immunogenic polypeptide containing an epitope of interest means naturally occurring polypeptides of interest or fragments thereof, as well as polypeptides prepared by other means, for example, by chemical synthesis or the expression of the polypeptide in a recombinant organism.
• transfection refers to the introduction of an exogenous polynucleotide into a prokaryotic or eucaryotic host cell, irrespective of the method used for the introduction.
• transfection refers to both stable and transient introduction of the polynucleotide, and encompasses direct uptake of polynucleotides, transformation, transduction, and f-mating.
• the exogenous polynucleotide may be maintained as a non-integrated replicon, for example, a plasmid, or alternatively, may be integrated into the host genome.
• Treatment refers to prophylaxis and/or therapy.
• individual as used herein refers to vertebrates, particularly members of the mammalian species and includes, but is not limited to, domestic animals, sports animals, primates and humans; more particularly, the term refers to humans.
• sense strand or "plus strand” (or “+) as used herein denotes a nucleic acid that contains the sequence that encodes the polypeptide.
• antisense strand or "minus strand” (or “-) denotes a nucleic acid that contains a sequence that is complementary to that of the "plus” strand.
• test sample refers to a component of an individual's body which is the source of the analyte (such as antibodies of interest or antigens of interest). These components are well known in the art.
• a test sample is typically anything suspected of containing a target sequence.
• Test samples can be prepared using methodologies well known in the art such as by obtaining a specimen from an individual and, if necessary, disrupting any cells contained thereby to release target nucleic acids.
• test samples include biological samples which can be tested by the methods of the present invention described herein and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, sputum, bronchial washing, bronchial aspirates, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supernatants; tissue specimens which may be fixed; and cell specimens which may be fixed.
• “Purified product” refers to a preparation of the product which has been isolated from the cellular constituents with which the product is normally associated and from other types of cells which may be present in the sample of interest.
• PNA denotes a "peptide nucleic acid analog” which may be utilized in a procedure such as an assay described herein to determine the presence of a target.
• MA denotes a "morpholino analog” which may be utilized in a procedure such as an assay described herein to determine the presence of a target. See, for example, U.S. Patent No. 5,378,841.
• PNAs are neutrally charged moieties which can be directed against RNA targets or DNA.
• PNAs can be labeled with (" attached to” ) such signal generating compounds as fluorescein, radionucleotides, chemiluminescent compounds and the like. PNAs or other nucleic acid analogs such as MAs thus can be used in assay methods in place of DNA or RNA. Although assays are described herein utilizing DNA probes, it is within the scope of the routineer that PNAs or MAs can be substituted for RNA or DNA with appropriate changes if and as needed in assay reagents.
• Analyte is the substance to be detected which may be present in the test sample.
• the analyte can be any substance for which there exists a naturally occurring specific binding member (such as an antibody), or for which a specific binding member can be prepared.
• an analyte is a substance that can bind to one or more specific binding members in an assay.
• “Analyte” also includes any antigenic substances, haptens, antibodies and combinations thereof.
• the analyte can be detected by means of naturally occurring specific binding partners (pairs) such as the use of intrinsic factor protein as a member of a specific binding pair for the determination of Vitamin B12, the use of folate- binding protein to determine folic acid, or the use of a lectin as a member of a specific binding pair for the determination of a carbohydrate.
• the analyte can include a protein, a polypeptide, an amino acid, a nucleotide target and the like.
• the analyte can be soluble in a body fluid such as blood, blood plasma or serum, urine or the like.
• the analyte can be in a tissue, either on a cell surface or within a cell.
• the analyte can be on or in a cell dispersed in a body fluid such as blood or urine, or obtained as a biopsy sample.
• diabetes a pancreatic disease
• condition of the pancreas refers to any disease or condition of the pancreas including, but not limited to, diabetes, pancreatitis, and cancer.
• Pantencreatic cancer refers to any malignant disease of the pancreas including, but not limited to, pancreatic ductal adenocarcinoma, ampullary carcinoma, duodenal carcinoma, cystadenocarcinoma, islet cell carcinoma; endocrine tumors of the pancreas such as insulinoma, gastrinoma (Zollinger-Ellison syndrome), glucagonoma, and vipoma (Verner-Morrison syndrome); and other pancreatic tumors such as somatostatinomas, carcinoids-islet cell tumors, and pancreatic polypeptide- producing tumors.
• An "Expressed Sequence Tag” or “EST” refers to the partial sequence of a cDNA insert which has been made by reverse transcription of mRNA extracted from a tissue followed by insertion into a vector.
• a "transcript image” refers to a table or list giving the quantitative distribution of ESTs in a library and represents the genes active in the tissue from which the library was made.
• a "specific binding member,” as used herein, is a member of a specific binding pair. That is, two different molecules where one of the molecules, through chemical or physical means, specifically binds to the second molecule. Therefore, in addition to antigen and antibody specific binding pairs of common immunoassays, other specific binding pairs can include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors, and enzymes and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding members, for example, an analyte-analog. Immunoreactive specific binding members include antigens, antigen fragments, antibodies and antibody fragments, both monoclonal and polyclonal and complexes thereof, including those formed by recombinant DNA molecules.
• Specific binding members include “specific binding molecules.”
• a "specific binding molecule” intends any specific binding member, particularly an immunoreactive specific binding member.
• the term “specific binding molecule” encompasses antibody molecules (obtained from both polyclonal and monoclonal preparations), as well as, the following: hybrid (chimeric) antibody molecules (see, for example, Winter, et al., Nature 349:293-299 (1991), and U.S. Patent No. 4,816,567); F(ab') 2 and F(ab) fragments; Fv molecules (non-covalent heterodimers, see, for example, Inbar, et al., Proc. Natl. Acad. Sci.
• hapten refers to a partial antigen or non-protein binding member which is capable of binding to an antibody, but which is not capable of eliciting antibody formation unless coupled to a carrier protein.
• a " capture reagent,” as used herein, refers to an unlabeled specific binding member which is specific either for the analyte as in a sandwich assay, for the indicator reagent or analyte as in a competitive assay, or for an ancillary specific binding member, which itself is specific for the analyte, as in an indirect assay.
• the capture reagent can be directly or indirectly bound to a solid phase material before the performance of the assay or during the performance of the assay, thereby enabling the separation of immobilized complexes from the test sample.
• the “ indicator reagent” comprises a " signal-generating compound” (" label” ) which is capable of generating and generates a measurable signal detectable by external means, conjugated (“ attached") to a specific binding member.
• label a signal-generating compound
• the indicator reagent also can be a member of any specific binding pair, including either hapten-anti-hapten systems such as biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme and the like.
• An immunoreactive specific binding member can be an antibody, an antigen, or an antibody/antigen complex that is capable of binding either to the polypeptide of interest as in a sandwich assay, to the capture reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay.
• reporter molecule comprises a signal generating compound as described hereinabove conjugated to a specific binding member of a specific binding pair, such as carbazole or adamantane.
• the various " signal-generating compounds" contemplated include chromagens, catalysts such as enzymes, luminescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as dioxetanes, acridiniums, phenanthridiniums and luminol, radioactive elements and direct visual labels.
• luminescent compounds such as fluorescein and rhodamine
• chemiluminescent compounds such as dioxetanes, acridiniums, phenanthridiniums and luminol
• radioactive elements include alkaline phosphatase, horseradish peroxidase, beta- galactosidase and the like.
• the selection of a particular label is not critical, but it must be capable of producing a signal either by itself or in conjunction with one or more additional substances.
• Solid phases are known to those in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic or nonmagnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells and Duracytes ® (red blood cells
• solid phase refers to any material which is insoluble, or can be made insoluble by a subsequent reaction.
• the solid phase can be chosen for its intrinsic ability to attract and immobilize the capture reagent.
• the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent.
• the additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent.
• the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid phase and which has the ability to immobilize the capture reagent through a specific binding reaction. The receptor molecule enables the indirect binding of the capture reagent to a solid phase material before the performance of the assay or during the performance of the assay.
• the solid phase thus can be a plastic, derivatized plastic, magnetic or non- magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal's) red blood cells, Duracytes ® and other configurations known to those of ordinary skill in the art. It is contemplated and within the scope of the present invention that the solid phase also can comprise any suitable porous material with sufficient porosity to allow access by detection antibodies and a suitable surface affinity to bind antigens. Microporous structures generally are preferred, but materials with a gel structure in the hydrated state may be used as well. Such useful solid supports include, but are not limited to, nitrocellulose and nylon.
• porous solid supports described herein preferably are in the form of sheets of thickness from about 0.01 to 0.5 mm, preferably about 0.1 mm.
• the pore size may vary within wide limits and preferably is from about 0.025 to 15 microns, especially from about 0.15 to 15 microns.
• the surface of such supports may be activated by chemical processes which cause covalent linkage of the antigen or antibody to the support. The irreversible binding of the antigen or antibody is obtained, however, in general, by adsorption on the porous material by poorly understood hydrophobic forces.
• Other suitable solid supports are known in the art. Reagents.
• the present invention provides reagents such as polynucleotide sequences derived from a pancreatic tissue of interest and designated as PAl 53, polypeptides encoded thereby and antibodies specific for these polypeptides.
• the present invention also provides reagents such as oligonucleotide fragments derived from the disclosed polynucleotides and nucleic acid sequences complementary to these polynucleotides.
• the polynucleotides, polypeptides, or antibodies of the present invention may be used to provide information leading to the detecting, diagnosing, staging, monitoring, prognosticating, in vivo imaging, preventing or treating of, or determining the predisposition to, diseases and conditions of the pancreas, such as pancreatic cancer.
• the sequences disclosed herein represent unique polynucleotides which can be used in assays or for producing a specific profile of gene transcription activity. Such assays are disclosed in European Patent Number 0373203B1 and International Publication No. WO
• Selected PA153-derived polynucleotides can be used in the methods described herein for the detection of normal or altered gene expression. Such methods may employ PAl 53 polynucleotides or oligonucleotides, fragments or derivatives thereof, or nucleic acid sequences complementary thereto.
• the polynucleotides disclosed herein, their complementary sequences, or fragments of either, can be used in assays to detect, amplify or quantify genes, nucleic acids, cDNAs or mRNAs relating to pancreatic tissue disease and conditions associated therewith. They also can be used to identify an entire or partial coding region of a PAl 53 polypeptide. They further can be provided in individual containers in the form of a kit for assays, or provided as individual compositions. If provided in a kit for assays, other suitable reagents such as buffers, conjugates and the like may be included.
• the polynucleotide may be in the form of RNA or DNA.
• Polynucleotides in the form of DNA, cDNA, genomic DNA, nucleic acid analogs and synthetic DNA are within the scope of the present invention.
• the DNA may be double-stranded or single-stranded, and if single stranded, may be the coding (sense) strand or non- coding (anti-sense) strand.
• the coding sequence which encodes the polypeptide may be identical to the coding sequence provided herein or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptide as the DNA provided herein.
• This polynucleotide may include only the coding sequence for the polypeptide, or the coding sequence for the polypeptide and an additional coding sequence such as a leader or secretory sequence or a proprotein sequence, or the coding sequence for the polypeptide (and optionally an additional coding sequence) and non-coding sequence, such as a non-coding sequence 5' and/or 3' of the coding sequence for the polypeptide.
• the invention includes variant polynucleotides containing modifications such as polynucleotide deletions, substitutions or additions; and any polypeptide modification resulting from the variant polynucleotide sequence.
• a polynucleotide of the present invention also may have a coding sequence which is a naturally occurring allelic variant of the coding sequence provided herein.
• the coding sequence for the polypeptide may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell.
• the polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the polypeptide.
• the polynucleotides may also encode for a proprotein which is the protein plus additional 5' amino acid residues.
• a protein having a prosequence is a proprotein and may, in some cases, be an inactive form of the protein.
• the polynucleotide of the present invention may encode for a protein, or for a protein having a prosequence, or for a protein having both a presequence (leader sequence) and a prosequence.
• the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention.
• the marker sequence may be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. a COS-7 cell line, is used.
• the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein. See, for example, I. Wilson et al., Cell 37:767 (1984).
• polynucleotides will be considered to hybridize to the sequences provided herein if there is at least 50%, preferably at least 70% to 80%, and more preferably at least 90% identity between the polynucleotide and the sequence.
• the degree of sequence identity between two nucleic acid molecules greatly affects the efficiency and strength of hybridization events between such molecules.
• a partially identical nucleic acid sequence is one that will at least partially inhibit a completely identical sequence from hybridizing to a target molecule.
• Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern blot, Northern blot, solution hybridization, in situ hybridization, or the like, see Sambrook, et al., Molecular Cloning: A Laboratory Manual. Second Edition, (1989) Cold Spring Harbor, N.Y.).
• Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency.
• the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.
• a partial degree of sequence identity for example, a probe having less than about 30% sequence identity with the target molecule
• a nucleic acid probe When utilizing a hybridization-based detection system, a nucleic acid probe is chosen that is complementary to a target nucleic acid sequence, and then by selection of appropriate conditions the probe and the target sequence " selectively hybridize," or bind, to each other to form a hybrid molecule.
• a nucleic acid molecule is capable of hybridizing selectively to a target sequence under moderately stringent hybridization conditions.
• moderately stringent hybridization conditions allow detection of a target nucleic acid sequence of at least 14 nucleotides in length having at least approximately 70% sequence identity with the sequence of the selected nucleic acid probe.
• such selective hybridization is performed under stringent hybridization conditions.
• Hybridization conditions allow detection of target nucleic acid sequences of at least 14 nucleotides in length having a sequence identity of greater than 90% with the sequence of the selected nucleic acid probe.
• Hybridization conditions useful for probe/target hybridization where the probe and target have a specific degree of sequence identity can be determined as is known in the art (see, for example, Nucleic Acid Hybridization: A Practical Approach, editors B.D. Hames and S J. Higgins, (1985) Oxford; Washington, DC; IRL Press).
• Hybrid molecules can be formed, for example, on a solid support, in solution, and in tissue sections. The formation of hybrids can be monitored by inclusion of a reporter molecule, typically, in the probe.
• Such reporter molecules, or detectable elements include, but are not limited to, radioactive elements, fluorescent markers, and molecules to which an enzyme-conjugated ligand can bind.
• stringency conditions for hybridization it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of probe and target sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., formamide, dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions.
• the selection of a particular set of hybridization conditions is well within the skill of the routineer in the art (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual. Second Edition, (1989) Cold Spring Harbor, N.Y.).
• the present invention also provides an antibody produced by using a purified PAl 53 polypeptide of which at least a portion of the polypeptide is encoded by a PAl 53 polynucleotide selected from the polynucleotides provided herein.
• These antibodies may be used in the methods provided herein for the detection of PAl 53 antigen in test samples. The presence of PAl 53 antigen in the test samples is indicative of the presence of a pancreatic disease or condition.
• the antibody also may be used for therapeutic purposes, for example, in neutralizing the activity of PAl 53 polypeptide in conditions associated with altered or abnormal expression.
• the present invention further relates to a PAl 53 polypeptide which has the deduced amino acid sequence as provided herein, as well as fragments, analogs and derivatives of such polypeptide.
• the polypeptide of the present invention may be a recombinant polypeptide, a natural purified polypeptide or a synthetic polypeptide.
• the fragment, derivative or analog of the PAl 53 polypeptide may be one in which one or more of the amino acid residues is substituted with a conserved or non- conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code; or it may be one in which one or more of the amino acid residues includes a substituent group; or it may be one in which the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol); or it may be one in which the additional amino acids are fused to the polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the polypeptide or a proprotein sequence.
• Such fragments, derivatives and analogs are within the scope of the present invention.
• the polypeptides and polynucleotides of the present invention are provided preferably in an isolated form and preferably purified.
• a polypeptide of the present invention may have an amino acid sequence that is identical to that of the naturally occurring polypeptide or that is different by minor variations due to one or more amino acid substitutions.
• the variation may be a " conservative change" typically in the range of about 1 to 5 amino acids, wherein the substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine or threonine with serine.
• variations may include nonconservative changes, e.g., replacement of a glycine with a tryptophan.
• Similar minor variations may also include amino acid deletions or insertions, or both.
• Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without changing biological or immunological activity may be found using computer programs well known in the art, for example, DNASTAR software (DNASTAR Inc., Madison WI).
• Probes constructed according to the polynucleotide sequences of the present invention can be used in various assay methods to provide various types of analysis.
• such probes can be used in fluorescent in situ hybridization (FISH) technology to perform chromosomal analysis, and used to identify cancer-specific structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR-generated and or allele specific oligonucleotides probes, allele specific amplification or by direct sequencing.
• FISH fluorescent in situ hybridization
• Probes also can be labeled with radioisotopes, directly- or indirectly- detectable haptens, or fluorescent molecules, and utilized for in situ hybridization studies to evaluate the mRNA expression of the gene comprising the polynucleotide in tissue specimens or cells.
• This invention also provides teachings as to the production of the polynucleotides and polypeptides provided herein. Probe Assays
• the sequences provided herein may be used to produce probes which can be used in assays for the detection of nucleic acids in test samples.
• the probes may be designed from conserved nucleotide regions of the polynucleotides of interest or from non-conserved nucleotide regions of the polynucleotide of interest. The design of such probes for optimization in assays is within the skill of the routineer. Generally, nucleic acid probes are developed from non-conserved or unique regions when maximum specificity is desired, and nucleic acid probes are developed from conserved regions when assaying for nucleotide regions that are closely related to, for example, different members of a multi-gene family or in related species like mouse and man.
• PCR polymerase chain reaction
• a pair of primers are employed in excess to hybridize to the complementary strands of the target nucleic acid.
• the primers are each extended by a polymerase using the target nucleic acid as a template.
• the extension products become target sequences themselves, following dissociation from the original target strand.
• New primers then are hybridized and extended by a polymerase, and the cycle is repeated to geometrically increase the number of target sequence molecules.
• PCR is disclosed in U.S. Patents 4,683,195 and 4,683,202.
• the Ligase Chain Reaction is an alternate method for nucleic acid amplification.
• probe pairs which include two primary (first and second) and two secondary (third and fourth) probes, all of which are employed in molar excess to target.
• the first probe hybridizes to a first segment of the target strand
• the second probe hybridizes to a second segment of the target strand, the first and second segments being contiguous so that the primary probes abut one another in 5' phosphate-3' hydroxyl relationship, and so that a ligase can covalently fuse or ligate the two probes into a fused product.
• a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybridize to a portion of the second probe in a similar abutting fashion.
• the secondary probes also will hybridize to the target complement in the first instance. Once the ligated strand of primary probes is separated from the target strand, it will hybridize with the third and fourth probes which can be ligated to form a complementary, secondary ligated product. It is important to realize that the ligated products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. This technique is described more completely in EP-A- 320 308 to K. Backman published June 16, 1989 and EP-A-439 182 to K. Backman et al., published July 31, 1991.
• RT-PCR polymerase chain reaction
• RT-AGLCR asymmetric gap ligase chain reaction
• amplification methods which can be utilized herein include but are not limited to the so-called "NASBA” or "3SR" technique described by J.C. Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878 (1990) and also described by J. Compton, Nature 350 (No. 6313):91-92 (1991); Q-beta amplification as described in published European Patent Application (EPA) No. 4544610; strand displacement amplification (as described in G.T. Walker et al., Clin. Chem. 42:9-13 [1996]) and European Patent Application No. 684315; and target mediated amplification, as described in International Publication No. WO 93/22461.
• Detection of PA153 may be accomplished using any suitable detection method, including those detection methods which are currently well known in the art, as well as detection strategies which may evolve later. See, for example, Caskey et al., U.S. Patent No. 5,582,989, Gelfand et al., U.S. Patent No. 5,210,015. Examples of such detection methods include target amplification methods as well as signal amplification technologies. An example of presently known detection methods would include the nucleic acid amplification technologies referred to as PCR, LCR, NASBA, SDA, RCR and TMA. See, for example, Caskey et al., U.S. Patent No. 5,582,989, Gelfand et al, U.S. Patent No.
• Detection may also be accomplished using signal amplification such as that disclosed in Snitman et al., U.S. Patent No. 5,273,882. While the amplification of target or signal is preferred at present, it is contemplated and within the scope of the present invention that ultrasensitive detection methods which do not require amplification can be utilized herein. Detection, both amplified and non-amplified, may be performed using a variety of heterogeneous and homogeneous detection formats. Examples of heterogeneous detection formats are disclosed in Snitman et al., U.S. Patent No. 5,273,882, Albarella et al., in EP-84114441.9, Urdea et al., U.S. Patent No.
• the present invention generally comprises the steps of contacting a test sample suspected of containing a target polynucleotide sequence with amplification reaction reagents comprising an amplification primer, and a detection probe that can hybridize with an internal region of the amplicon sequences.
• Probes and primers employed according to the method provided herein are labeled with capture and detection labels, wherein probes are labeled with one type of label and primers are labeled with another type of label. Additionally, the primers and probes are selected such that the probe sequence has a lower melt temperature than the primer sequences.
• the amplification reagents, detection reagents and test sample are placed under amplification conditions whereby, in the presence of target sequence, copies of the target sequence (an amplicon) are produced.
• the amplicon is double stranded because primers are provided to amplify a target sequence and its complementary strand.
• the double stranded amplicon then is thermally denatured to produce single stranded amplicon members.
• the mixture is cooled to allow the formation of complexes between the probes and single stranded amplicon members.
• the probe sequences preferentially bind the single stranded amplicon members.
• the probe sequences generally are selected to be shorter than the primer sequences and therefore have a lower melt temperature than the primers. Accordingly, the melt temperature of the amplicon produced by the primers should also have a higher melt temperature than the probes.
• the probes are found to preferentially bind the single stranded amplicon members. Moreover, this preference of probe/single stranded amplicon binding exists even when the primer sequences are added in excess of the probes.
• the probe/single stranded amplicon member hybrids are formed, they are detected.
• Standard heterogeneous assay formats are suitable for detecting the hybrids using the detection labels and capture labels present on the primers and probes.
• the hybrids can be bound to a solid phase reagent by virtue of the capture label and detected by virtue of the detection label.
• the detection label is directly detectable
• the presence of the hybrids on the solid phase can be detected by causing the label to produce a detectable signal, if necessary, and detecting the signal.
• the captured hybrids can be contacted with a conjugate, which generally comprises a binding member attached to a directly detectable label.
• the conjugate becomes bound to the complexes and the conjugate's presence on the complexes can be detected with the directly detectable label.
• the presence of the hybrids on the solid phase reagent can be determined.
• wash steps may be employed to wash away unhybridized amplicon or probe as well as unbound conjugate.
• the heterogeneous assays can be conveniently performed using a solid phase support that carries an array of nucleic acid molecules.
• Such arrays are useful for high-throughput and/or multiplexed assay formats.
• Various methods for forming such arrays from pre- formed nucleic acid molecules, or methods for generating the array using in situ synthesis techniques, are generally known in the art. (See, for example, Dattagupta, et al., EP Publication No. 0 234, 726A3; Southern, U.S. Patent No. 5,700,637; Pirrung, et al., U.S. Patent No. 5,143,854; PCT International Publication No. WO 92/10092; and, Fodor, et al., Science 251 -.161-111 (1991)).
• the target sequence is described as single stranded, it also is contemplated to include the case where the target sequence is actually double stranded but is merely separated from its complement prior to hybridization with the amplification primer sequences.
• the ends of the target sequences are usually known.
• the entire target sequence is usually known.
• the target sequence is a nucleic acid sequence such as, for example, RNA or DNA.
• the method provided herein can be used in well-known amplification reactions that include thermal cycle reaction mixtures, particularly in PCR and gap LCR (GLCR).
• Amplification reactions typically employ primers to repeatedly generate copies of a target nucleic acid sequence, which target sequence is usually a small region of a much larger nucleic acid sequence.
• Primers are themselves nucleic acid sequences that are complementary to regions of a target sequence. Under amplification conditions, these primers hybridize or bind to the complementary regions of the target sequence. Copies of the target sequence typically are generated by the process of primer extension and/or ligation which utilizes enzymes with polymerase or ligase activity, separately or in combination, to add nucleotides to the hybridized primers and/or ligate adjacent probe pairs.
• the nucleotides that are added to the primers or probes, as monomers or preformed oligomers, are also complementary to the target sequence.
• primers or probes Once the primers or probes have been sufficiently extended and or ligated, they are separated from the target sequence, for example, by heating the reaction mixture to a "melt temperature" which is one in which complementary nucleic acid strands dissociate. Thus, a sequence complementary to the target sequence is formed. A new amplification cycle then can take place to further amplify the number of target sequences by separating any double stranded sequences, allowing primers or probes to hybridize to their respective targets, extending and/or ligating the hybridized primers or probes and re-separating.
• the complementary sequences that are generated by amplification cycles can serve as templates for primer extension or filling the gap of two probes to further amplify the number of target sequences.
• a reaction mixture is cycled between 20 and 100 times, more typically, a reaction mixture is cycled between 25 and 50 times.
• the numbers of cycles can be determined by the routineer. In this manner, multiple copies of the target sequence and its complementary sequence are produced.
• primers initiate amplification of the target sequence when it is present under amplification conditions.
• two primers which are complementary to a portion of a target strand and its complement are employed in PCR.
• four probes, two of which are complementary to a target sequence and two of which are similarly complementary to the target's complement, are generally employed.
• a nucleic acid amplification reaction mixture may also comprise other reagents which are well known and include but are not limited to: enzyme cofactors such as manganese; magnesium; salts; nicotinamide adenine dinucleotide (NAD); and deoxynucleotide triphosphates (dNTPs) such as, for example, deoxyadenine triphosphate, deoxyguanine triphosphate, deoxycytosine triphosphate and deoxythymine triphosphate.
• enzyme cofactors such as manganese; magnesium; salts; nicotinamide adenine dinucleotide (NAD); and deoxynucleotide triphosphates (dNTPs) such as, for example, deoxyadenine triphosphate, deoxyguanine triphosphate, deoxycytosine triphosphate and deoxythymine triphosphate.
• Detection probes are generally nucleic acid sequences or uncharged nucleic acid analogs such as, for example, peptide nucleic acids which are disclosed in International Publication No. WO 92/20702; morpholino analogs which are described in U.S. Patents Nos 5,185,444, 5,034,506 and 5,142,047; and the like.
• the probe is employed to capture or detect the amplicon generated by the amplification reaction.
• the probe is not involved in amplification of the target sequence and therefore may have to be rendered "non-extendible" in that additional dNTPs cannot be added to the probe.
• analogs usually are non-extendible and nucleic acid probes can be rendered non-extendible by modifying the 3' end of the probe such that the hydroxyl group is no longer capable of participating in elongation.
• the 3' end of the probe can be functionalized with the capture or detection label to thereby consume or otherwise block the hydroxyl group.
• the 3' hydroxyl group simply can be cleaved, replaced or modified.
• U.S. Patent Application Serial No. 07/049,061 filed April 19, 1993 describes modifications which can be used to render a probe non- extendible.
• primers to probes are not important.
• either the probes or primers can be added to the reaction mixture in excess whereby the concentration of one would be greater than the concentration of the other.
• primers and probes can be employed in equivalent concentrations.
• the primers are added to the reaction mixture in excess of the probes.
• primer to probe ratios of, for example, 5:1 and 20:1, are preferred.
• the probe sequences are selected such that they have a lower melt temperature than the primer sequences. Hence, the primer sequences are generally longer than the probe sequences.
• the primer sequences are in the range of between 20 and 50 nucleotides long, more typically in the range of between 20 and 30 nucleotides long.
• the typical probe is in the range of between 10 and 25 nucleotides long.
• a primary amine can be attached to a 3' oligo terminus using 3'-Amine-ON CPGTM (Clontech, Palo Alto, CA).
• a primary amine can be attached to a 5' oligo terminus using Aminomodifier II ® (Clontech).
• the amines can be reacted to various haptens using conventional activation and linking chemistries.
• copending applications U.S. Serial Nos. 625,566, filed December 11, 1990 and 630,908, filed December 20, 1990 teach methods for labeling probes at their 5' and 3' termini, respectively.
• WO 92/10505 published 25 June 1992
• WO 92/11388 published 9 July 1992
• a label-phosphoramidite reagent is prepared and used to add the label to the oligonucleotide during its synthesis. See, for example, N.T. Thuong et al., Tet. Letters 29(46):5905-5908 (1988); or J.S. Cohen et al., published U.S. Patent Application 07/246,688 (NTIS ORDER No. PAT-APPL-7-246,688) (1989).
• probes are labeled at their 3' and 5' ends.
• a capture label is attached to the primers or probes and can be a specific binding member which forms a binding pair with the solid phase reagent's specific binding member.
• the primer or probe itself may serve as the capture label.
• a solid phase reagent's binding member is a nucleic acid sequence
• it may be selected such that it binds a complementary portion of the primer or probe to thereby immobilize the primer or probe to the solid phase.
• the probe itself serves as the binding member
• the probe will contain a sequence or "tail" that is not complementary to the single stranded amplicon members.
• the primer itself serves as the capture label
• at least a portion of the primer will be free to hybridize with a nucleic acid on a solid phase because the probe is selected such that it is not fully complementary to the primer sequence.
• probe/single stranded amplicon member complexes can be detected using techniques commonly employed to perform heterogeneous immunoassays.
• detection is performed according to the protocols used by the commercially available Abbott LCx ® instrumentation (Abbott Laboratories, Abbott Park, IL).
• primers and probes disclosed herein are useful in typical PCR assays, wherein the test sample is contacted with a pair of primers, amplification is performed, the hybridization probe is added, and detection is performed.
• Another method provided by the present invention comprises contacting a test sample with a plurality of polynucleotides, wherein at least one polynucleotide is a PAl 53 molecule as described herein, hybridizing the test sample with the plurality of polynucleotides and detecting hybridization complexes.
• Hybridization complexes are identified and quantitated to compile a profile which is indicative of pancreatic tissue disease, such as pancreatic cancer.
• Expressed RNA sequences may further be detected by reverse transcription and amplification of the DNA product by procedures well-known in the art, including polymerase chain reaction (PCR).
• the present invention also encompasses the use of gene therapy methods for the introduction of anti-sense PAl 53 derived molecules, such as polynucleotides or oligonucleotides of the present invention, into patients with conditions associated with abnormal expression of polynucleotides related to a pancreatic tissue disease or condition especially pancreatic cancer.
• anti-sense PAl 53 derived molecules such as polynucleotides or oligonucleotides of the present invention
• These molecules including antisense RNA and DNA fragments and ribozymes, are designed to inhibit the translation of PAl 53 mRNA, and may be used therapeutically in the treatment of conditions associated with altered or abnormal expression of PAl 53 polynucleotide.
• the oligonucleotides described above can be delivered to cells by procedures known in the art such that the anti-sense RNA or DNA may be expressed in vivo to inhibit production of a PAl 53 polypeptide in the manner described above.
• Antisense constructs to a PAl 53 polynucleotide therefore, reverse the action of PAl 53 transcripts and may be used for treating pancreatic tissue disease conditions, such as pancreatic cancer. These antisense constructs may also be used to treat tumor metastases.
• the present invention also provides a method of screening a plurality of compounds for specific binding to PAl 53 polypeptide(s), or any fragment thereof, to identify at least one compound which specifically binds the PAl 53 polypeptide.
• a method comprises the steps of providing at least one compound; combining the PAl 53 polypeptide with each compound under suitable conditions for a time sufficient to allow binding; and detecting the PAl 53 polypeptide binding to each compound.
• the polypeptide or peptide fragment employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.
• One method of screening utilizes eukaryotic or prokaryotic host cells which are stably transfected with recombinant nucleic acids which can express the polypeptide or peptide fragment.
• a drug, compound, or any other agent may be screened against such transfected cells in competitive binding assays. For example, the formation of complexes between a polypeptide and the agent being tested can be measured in either viable or fixed cells.
• the present invention thus provides methods of screening for drugs, compounds, or any other agent which can be used to treat diseases associated with PAl 53. These methods comprise contacting the agent with a polypeptide or fragment thereof and assaying for either the presence of a complex between the agent and the polypeptide, or for the presence of a complex between the polypeptide and the cell. In competitive binding assays, the polypeptide typically is labeled. After suitable incubation, free (or uncomplexed) polypeptide or fragment thereof is separated from that present in bound form, and the amount of free or uncomplexed label is used as a measure of the ability of the particular agent to bind to the polypeptide or to interfere with the polypeptide/cell complex.
• the present invention also encompasses the use of competitive screening assays in which neutralizing antibodies capable of binding polypeptide specifically compete with a test agent for binding to the polypeptide or fragment thereof. In this manner, the antibodies can be used to detect the presence of any polypeptide in the test sample which shares one or more antigenic determinants with a PAl 53 polypeptide as provided herein.
• Another technique for screening provides high throughput screening for compounds having suitable binding affinity to at least one polypeptide of PAl 53 disclosed herein. Briefly, large numbers of different small peptide test compounds are synthesized on a solid phase, such as plastic pins or some other surface. The peptide test compounds are reacted with polypeptide and washed. Polypeptide thus bound to the solid phase is detected by methods well-known in the art. Purified polypeptide can also be coated directly onto plates for use in the screening techniques described herein. In addition, non-neutralizing antibodies can be used to capture the polypeptide and immobilize it on the solid support. See, for example, EP 84/03564, published on September 13, 1984.
• the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of the small molecules including agonists, antagonists, or inhibitors with which they interact.
• Such structural analogs can be used to design drugs which are more active or stable forms of the polypeptide or which enhance or interfere with the function of a polypeptide in vivo. J. Hodgson, Bio/Technology 9:19-21 (1991).
• the three-dimensional structure of a polypeptide, or of a polypeptide-inhibitor complex is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shape and charges of the polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of a polypeptide may be gained by modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design analogous polypeptide-like molecules or to identify efficient inhibitors Useful examples of rational drug design may include molecules which have improved activity or stability as shown by S.
• the binding site of the anti-id is an analog of the original receptor.
• the anti-id then can be used to identify and isolate peptides from banks of chemically or biologically produced peptides.
• the isolated peptides then can act as the pharmacophore (that is, a prototype pharmaceutical drug).
• a sufficient amount of a recombinant polypeptide of the present invention may be made available to perform analytical studies such as X-ray crystallography.
• knowledge of the polypeptide amino acid sequence which is derivable from the nucleic acid sequence provided herein will provide guidance to those employing computer modeling techniques in place of, or in addition to, x-ray crystallography.
• Antibodies specific to a PAl 53 polypeptide e.g., anti-PA153 antibodies
• the antibodies may be used in therapy, for example, to treat pancreatic tissue diseases including pancreatic cancer and its metastases.
• Such antibodies can detect the presence or absence of a PAl 53 polypeptide in a test sample and, therefore, are useful as diagnostic markers for the diagnosis of a pancreatic tissue disease or condition especially pancreatic cancer.
• Such antibodies may also function as a diagnostic marker for pancreatic tissue disease conditions, such as pancreatic cancer.
• the present invention also is directed to antagonists and inhibitors of the polypeptides of the present invention.
• the antagonists and inhibitors are those which inhibit or eliminate the function of the polypeptide.
• an antagonist may bind to a polypeptide of the present invention and inhibit or eliminate its function.
• the antagonist for example, could be an antibody against the polypeptide which eliminates the activity of a PAl 53 polypeptide by binding a PAl 53 polypeptide, or in some cases the antagonist may be an oligonucleotide.
• small molecule inhibitors include, but are not limited to, small peptides or peptide- like molecules.
• the antagonists and inhibitors may be employed as a composition with a pharmaceutically acceptable carrier including, but not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof.
• a pharmaceutically acceptable carrier including, but not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof.
• Administration of PAl 53 polypeptide inhibitors is preferably systemic.
• the present invention also provides an antibody which inhibits the action of such a polypeptide.
• Antisense technology can be used to reduce gene expression through triple- helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
• the 5' coding portion of the polynucleotide sequence which encodes for the polypeptide of the present invention, is used to design an antisense RNA oligonucleotide of from 10 to 40 base pairs in length.
• a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription, thereby preventing transcription and the production of the PAl 53 polypeptide.
• triple helix see, for example, Lee et al., Nuc. Acids Res.
• RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of a mRNA molecule into the PAl 53 polypeptide.
• antisense see, for example, Okano, J. Neurochem. 56:560 (1991); and Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton, Fla. (1988).
• Antisense oligonucleotides act with greater efficacy when modified to contain artificial intemucleotide linkages which render the molecule resistant to nucleolytic cleavage.
• artificial intemucleotide linkages include, but are not limited to, methylphosphonate, phosphorothiolate and phosphoroamydate intemucleotide linkages.
• the present invention provides host cells and expression vectors comprising
• PA153 polynucleotides of the present invention and methods for the production of the polypeptide(s) they encode. Such methods comprise culturing the host cells under conditions suitable for the expression of the PAl 53 polynucleotide and recovering the PAl 53 polypeptide from the cell culture.
• the present invention also provides vectors which include PAl 53 polynucleotides of the present invention, host cells which are genetically engineered with vectors of the present invention and the production of polypeptides of the present invention by recombinant techniques.
• Host cells are genetically engineered (transfected, transduced or transformed) with the vectors of this invention which may be cloning vectors or expression vectors.
• the vector may be in the form of a plasmid, a viral particle, a phage, etc.
• the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transfected cells, or amplifying PAl 53 gene(s).
• the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
• the polynucleotides of the present invention may be employed for producing a polypeptide by recombinant techniques.
• the polynucleotide sequence may be included in any one of a variety of expression vehicles, in particular, vectors or plasmids for expressing a polypeptide.
• vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus and pseudorabies.
• any other plasmid or vector may be used so long as it is replicable and viable in the host.
• the appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into appropriate restriction endonuclease sites by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
• the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
• promoters include, but are not limited to, the LTR or the SV40 promoter, the R coli lac or trp, the phage lambda P sub L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
• the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
• the vector may also include appropriate sequences for amplifying expression.
• the expression vectors preferably contain a gene to provide a phenotypic trait for selection of transfected host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in R coli.
• the vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transfect an appropriate host to permit the host to express the protein.
• appropriate hosts there may be mentioned: bacterial cells, such as R coli. Salmonella tvphimurium: Streptomyces sp. fungal cells, such as yeast; insect cells, such as Drosophila and Sf9; animal cells, such as CHO, COS or Bowes melanoma; plant cells, etc.
• the selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings provided herein. More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
• the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
• the construct further comprises regulatory sequences including, for example, a promoter, operably linked to the sequence.
• a promoter operably linked to the sequence.
• Bacterial pINCY (Incyte Pharmaceuticals Inc., Palo Alto, CA), pSPORTl (Life Technologies, Gaithersburg, MD), pQE70, pQE60, pQE-9 (Qiagen) pBs, phagescript, psiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, ⁇ NH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic: pWLneo, pSV2cat, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
• Plasmid pINCY is generally identical to the plasmid pSPORTl (available from Life Technologies, Gaithersburg, MD) with the exception that it has two modifications in the poly linker (multiple cloning site). These modifications are (1) it lacks a Hindlll restriction site and (2) its EcoRI restriction site lies at a different location.
• pINCY is created from pSPORTl by cleaving pSPORTl with both Hindlll and EcoRI and replacing the excised fragment of the poly linker with synthetic DNA fragments (SEQUENCE ID NO 12 and SEQUENCE ID NO 13). This replacement may be made in any manner known to those of ordinary skill in the art.
• the two nucleotide sequences may be generated synthetically with 5' terminal phosphates, mixed together, and then ligated under standard conditions for performing staggered end ligations into the pSPORTl plasmid cut with Hindlll and EcoRI.
• Suitable host cells such as R coli DH5 ⁇ cells
• Plasmid DNA then is prepared from individual clones and subjected to restriction enzyme analysis or DNA sequencing in order to confirm the presence of insert sequences in the proper orientation.
• Other cloning strategies known to the ordinary artisan also may be employed.
• Promoter regions can be selected from any desired gene using CAT
• bacterial promoters include lad, lacZ, T3, SP6, T7, gpt, lambda P sub R, P sub L and trp.
• Eukaryotic promoters include cytomegalovirus (CMV) immediate early, herpes simplex virus (HS V) thymidine kinase, early and late SV40, LTRs from retroviruses and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
• the present invention provides host cells containing the above-described construct.
• the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
• Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation [L. Davis et al., Basic Methods in Molecular Biology. 2nd edition, Appleton and Lang, Paramount Publishing, East Norwalk, CT (1994)].
• the constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
• the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
• Recombinant proteins can be expressed in mammalian cells, yeast, bacteria, or other cells, under the control of appropriate promoters.
• Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
• Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al., Molecular Cloning: A Laboratory Manual. Second Edition, (Cold Spring Harbor, NY, 1989). Transcription of a DNA encoding the polypeptide(s) of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector.
• Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin (bp 100 to 270), a cytomegalo virus early promoter enhancer, a polyoma enhancer on the late side of the replication origin and adeno virus enhancers.
• recombinant expression vectors will include origins of replication and selectable markers permitting transfection of the host cell, e.g., the ampicillin resistance gene of R coli and S cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
• Such promoters can be derived from operons encoding glycolytic enzymes such as 3- phosphoglycerate kinase (PGK), alpha factor, acid phosphatase, or heat shock proteins, among others.
• PGK 3- phosphoglycerate kinase
• the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.
• the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
• Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.
• the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
• Suitable prokaryotic hosts for transfection include R coli. Bacillus subtilis. Salmonella typhimurium and various species within the genera Pseudomonas. Streptomyces and Staphylococcus. although others may also be employed as a routine matter of choice.
• Useful expression vectors for bacterial use comprise a selectable marker and bacterial origin of replication derived from plasmids comprising genetic elements of the well-known cloning vector pBR322 (ATCC 37017).
• Other vectors include but are not limited to PKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMl (Promega Biotec, Madison, WI). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
• the selected promoter is derepressed by appropriate means (e.g., temperature shift or chemical induction), and cells are cultured for an additional period.
• Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
• Microbial cells employed in expression of proteins can be disrupted by any convenient method including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well-known to the ordinary artisan.
• Various mammalian cell culture systems can also be employed to express recombinant protein.
• mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts described by Gluzman, Cell 23:175 (1981), and other cell lines capable of expressing a compatible vector, such as the C127, HEK- 293, 3T3, CHO, HeLa and BHK cell lines.
• Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences and 5' flanking nontranscribed sequences.
• DNA sequences derived from the SV40 viral genome may be used to provide the required nontranscribed genetic elements.
• useful vectors include pRc/CMV and pcDNA3 (available from Invitrogen, San Diego, CA).
• PAl 53 polypeptides are recovered and purified from recombinant cell cultures by known methods including affinity chromatography, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography or lectin chromatography.
• polypeptides of the present invention may be naturally purified products expressed from a high expressing cell line, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture).
• polypeptides of the present invention may be glycosylated with mammalian or other eukaryotic carbohydrates or may be non-glycosylated.
• the polypeptides of the invention may also include an initial methionine amino acid residue.
• the starting plasmids can be constructed from available plasmids in accord with published, known procedures.
• equivalent plasmids to those described are known in the art and will be apparent to one of ordinary skill in the art.
• the following is the general procedure for the isolation and analysis of cDNA clones.
• mRNA is isolated from pancreatic tissue and used to generate the cDNA library.
• Pancreatic tissue is obtained from patients by surgical resection and is classified as tumor or non-tumor tissue by a pathologist.
• the cDNA inserts from random isolates of the pancreatic tissue libraries are sequenced in part, analyzed in detail as set forth in the Examples, and are disclosed in the Sequence Listing as SEQUENCE ID NOS 1-9. Also analyzed in detail as set forth in the Examples, and disclosed in the Sequence Listing, is the full-length sequence of clone 2075919 [referred to herein as 2075919inh (SEQUENCE ID NO 10)]. The consensus sequence of these inserts is presented as SEQUENCE ID NO 11. These polynucleotides may contain an entire open reading frame with or without associated regulatory sequences for a particular gene, or they may encode only a portion of the gene of interest.
• the chain termination reaction products may be electrophoresed on urea/polyacrylamide gels and detected either by autoradiography (for radionucleotide labeled precursors) or by fluorescence (for fluorescent-labeled precursors). Recent improvements in mechanized reaction preparation, sequencing and analysis using the fluorescent detection method have permitted expansion in the number of sequences that can be determined per day using machines such as the Applied Biosystems 377 DNA Sequencers (Applied Biosystems, Foster City, CA).
• the reading frame of the nucleotide sequence can be ascertained by several types of analyses. First, reading frames contained within the coding sequence can be analyzed for the presence of start codon ATG and stop codons TGA, TAA or TAG. Typically, one reading frame will continue throughout the major portion of a cDNA sequence while other reading frames tend to contain numerous stop codons. In such cases, reading frame determination is straightforward. In other more difficult cases, further analysis is required.
• Coding DNA for particular organisms tends to contain certain nucleotides within certain triplet periodicities, such as a significant preference for pyrimidines in the third codon position.
• Coding DNA for particular organisms (bacteria, plants and animals) tends to contain certain nucleotides within certain triplet periodicities, such as a significant preference for pyrimidines in the third codon position.
• These preferences have been incorporated into widely available software which can be used to determine coding potential (and frame) of a given stretch of DNA.
• the algorithm- derived information combined with start/stop codon information can be used to determine proper frame with a high degree of certainty. This, in turn, readily permits cloning of the sequence in the correct reading frame into appropriate expression vectors.
• vectors of interest include cloning vectors, such as plasmids, cosmids, phage derivatives, phagemids, as well as sequencing, replication and expression vectors, and the like.
• vectors contain an origin of replication functional in at least one organism, convenient restriction endonuclease digestion sites and selectable markers appropriate for particular host cells.
• the vectors can be transferred by a variety of means known to those of skill in the art into suitable host cells which then produce the desired DNA, RNA or polypeptides.
• nucleotide sequences provided herein have been prepared by current, state-of-the-art, automated methods and, as such, may contain unidentified nucleotides. These will not present a problem to those skilled in the art who wish to practice the invention.
• Several methods employing standard recombinant techniques, described in J. Sambrook (supra) or periodic updates thereof, may be used to complete the missing sequence information.
• the same techniques used for obtaining a full length sequence, as described herein, may be used to obtain nucleotide sequences.
• Expression of a particular cDNA may be accomplished by subcloning the cDNA into an appropriate expression vector and transfecting this vector into an appropriate expression host.
• the cloning vector used for the generation of the pancreatic tissue cDNA library can be used for transcribing mRNA of a particular cDNA and contains a promoter for beta-galactosidase, an amino-terminal met and the subsequent seven amino acid residues of beta-galactosidase. Immediately following these eight residues is an engineered bacteriophage promoter useful for artificial priming and transcription, as well as a number of unique restriction sites, including EcoRI, for cloning.
• the vector can be transfected into an appropriate host strain of R coli.
• IPTG isopropylthiogalactoside
• the cDNA can be shuttled into other vectors known to be useful for expression of protein in specific hosts.
• Oligonucleotide primers containing cloning sites and segments of DNA sufficient to hybridize to stretches at both ends of the target cDNA can be synthesized chemically by standard methods. These primers can then be used to amplify the desired gene segments by PCR. The resulting new gene segments can be digested with appropriate restriction enzymes under standard conditions and isolated by gel electrophoresis. Alternately, similar gene segments can be produced by digestion of the cDNA with appropriate restriction enzymes and filling in the missing gene segments with chemically synthesized oligonucleotides.
• Segments of the coding sequence from more than one gene can be ligated together and cloned in appropriate vectors to optimize expression of recombinant sequence.
• Suitable expression hosts for such chimeric molecules include, but are not limited to, mammalian cells, such as Chinese Hamster Ovary (CHO) and human embryonic kidney (HEK) 293 cells, insect cells, such as Sf9 cells, yeast cells, such as Saccharomyces cerevisiae and bacteria, such as R coli.
• a useful expression vector may also include an origin of replication to allow propagation in bacteria and a selectable marker such as the beta-lactamase antibiotic resistance gene to allow selection in bacteria.
• the vectors may include a second selectable marker, such as the neomycin phosphotransferase gene, to allow selection in transfected eukaryotic host cells.
• a second selectable marker such as the neomycin phosphotransferase gene
• Vectors for use in eukaryotic expression hosts may require the addition of 3' poly A tail if the sequence of interest lacks poly A.
• the vector may contain promoters or enhancers which increase gene expression.
• promoters are host specific and include, but are not limited to, MMTV, SV40, or metallothionine promoters for CHO cells; trp, lac, tac or T7 promoters for bacterial hosts; or alpha factor, alcohol oxidase or PGH promoters for yeast.
• Adenoviral vectors with or without transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to drive protein expression in mammalian cell lines. Once homogeneous cultures of recombinant cells are obtained, large quantities of recombinantly produced protein can be recovered from the conditioned medium and analyzed using chromatographic methods well known in the art.
• RSV Rous sarcoma virus
• An alternative method for the production of large amounts of secreted protein involves the transfection of mammalian embryos and the recovery of the recombinant protein from milk produced by transgenic cows, goats, sheep, etc. Polypeptides and closely related molecules may be expressed recombinantly in such a way as to facilitate protein purification.
• One approach involves expression of a chimeric protein which includes one or more additional polypeptide domains not naturally present on human polypeptides.
• Such purification-facilitating domains include, but are not limited to, metal-chelating peptides such as histidine-tryptophan domains that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle, WA).
• metal-chelating peptides such as histidine-tryptophan domains that allow purification on immobilized metals
• protein A domains that allow purification on immobilized immunoglobulin
• the domain utilized in the FLAGS extension/affinity purification system Immunex Corp, Seattle, WA.
• the inclusion of a cleavable linker sequence such as Factor XA or enterokinase from Invitrogen (San Diego, CA) between the polypeptide sequence and the purification domain may be useful for recovering the polypeptide.
• PAl 53 polypeptides including fragments, derivatives, and analogs thereof, or cells expressing such polypeptides, can be utilized in a variety of assays, many of which are described herein, for the detection of antibodies to pancreatic tissue. They also can be used as immunogens to produce antibodies. These antibodies can be, for example, polyclonal or monoclonal antibodies, chimeric, single chain and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
• antibodies generated against a polypeptide comprising a sequence of the present invention can be obtained by direct injection of the polypeptide into an animal or by administering the polypeptide to an animal such as a mouse, rabbit, goat or human. A mouse, rabbit or goat is preferred.
• the polypeptide is selected from the group consisting of SEQUENCE ID NO 28, SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31, SEQUENCE ID NO 32, and fragments thereof.
• the antibody so obtained then will bind the polypeptide itself. In this manner, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies that bind the native polypeptide.
• Such antibodies then can be used to isolate the polypeptide from test samples such as tissue suspected of containing that polypeptide.
• any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique as described by Kohler and Milstein, Nature 256:495-497 (1975), the trioma technique, the human B-cell hybridoma technique as described by Kozbor et al., Immun. Today 4:72 (1983) and the EBV- hybridoma technique to produce human monoclonal antibodies as described by Cole et al., in Monoclonal Antibodies and Cancer Therapy. Alan R. Liss, Inc, New York, NY, pp. 77-96 (1985). Techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. See, for example, U.S.
• Various assay formats may utilize the antibodies of the present invention, including " sandwich" immunoassays and probe assays.
• the antibodies of the present invention, or fragments thereof can be employed in various assay systems to determine the presence, if any, of PA153 antigen in a test sample.
• a polyclonal or monoclonal antibody or fragment thereof, or a combination of these antibodies which has been coated on a solid phase, is contacted with a test sample, to form a first mixture. This first mixture is incubated for a time and under conditions sufficient to form antigen/antibody complexes.
• an indicator reagent comprising a monoclonal or a polyclonal antibody or a fragment thereof, or a combination of these antibodies, to which a signal generating compound has been attached, is contacted with the antigen/antibody complexes to form a second mixture.
• This second mixture then is incubated for a time and under conditions sufficient to form antibody/antigen/antibody complexes.
• the presence of PAl 53 antigen in the test sample and captured on the solid phase, if any, is determined by detecting the measurable signal generated by the signal generating compound.
• the amount of PA153 antigen present in the test sample is proportional to the signal generated.
• a mixture is formed by contacting: (1) a polyclonal antibody, monoclonal antibody, or fragment thereof, which specifically binds to PAl 53 antigen, or a combination of such antibodies bound to a solid support; (2) the test sample; and (3) an indicator reagent comprising a monoclonal antibody, polyclonal antibody, or fragment thereof, which specifically binds to a different PAl 53 antigen (or a combination of these antibodies) to which a signal generating compound is attached.
• This mixture is incubated for a time and under conditions sufficient to form antibody/antigen/antibody complexes.
• PAl 53 antigen present in the test sample and captured on the solid phase is determined by detecting the measurable signal generated by the signal generating compound.
• the amount of PAl 53 antigen present in the test sample is proportional to the signal generated.
• one or a combination of at least two monoclonal antibodies of the invention can be employed as a competitive probe for the detection of antibodies to PAl 53 antigen.
• PAl 53 polypeptides such as the recombinant antigens disclosed herein, either alone or in combination, are coated on a solid phase.
• a test sample suspected of containing antibody to PAl 53 antigen then is incubated with an indicator reagent comprising a signal generating compound and at least one monoclonal antibody of the invention for a time and under conditions sufficient to form antigen/antibody complexes of either the test sample and indicator reagent bound to the solid phase or the indicator reagent bound to the solid phase.
• the reduction in binding of the monoclonal antibody to the solid phase can be quantitatively measured.
• each of the monoclonal or polyclonal antibodies of the present invention can be employed in the detection of PAl 53 antigens in tissue sections, as well as in cells, by immunohistochemical analysis.
• the tissue sections can be cut from either frozen or chemically fixed samples of tissue.
• the cells can be isolated from blood, urine, or other bodily fluids.
• the cells may be obtained by biopsy, either surgical or by needle.
• the cells can be isolated by centrifugation or magnetic attraction after labeling with magnetic particles or ferrofluids so as to enrich a particular fraction of cells for staining with the antibodies of the present invention.
• these monoclonal antibodies can be bound to matrices similar to CNBr-activated Sepharose and used for the affinity purification of specific PAl 53 polypeptides from cell cultures or biological tissues such as to purify recombinant and native PAl 53 proteins.
• the monoclonal antibodies of the invention also can be used for the generation of chimeric antibodies for therapeutic use, or other similar applications.
• the monoclonal antibodies or fragments thereof can be provided individually to detect PAl 53 antigens.
• Combinations of the monoclonal antibodies (and fragments thereof) provided herein also may be used together as components in a mixture or "cocktail" of at least one PAl 53 antibody of the invention, along with antibodies which specifically bind to other PAl 53 regions, each antibody having different binding specificities.
• this cocktail can include the monoclonal antibodies of the invention which are directed to PAl 53 polypeptides disclosed herein and other monoclonal antibodies specific to other antigenic determinants of PA153 antigens or other related proteins.
• the polyclonal antibody or fragment thereof which can be used in the assay formats should specifically bind to a PAl 53 polypeptide or other PAl 53 polypeptides additionally used in the assay.
• the polyclonal antibody used preferably is of mammalian origin such as, human, goat, rabbit or sheep polyclonal antibody which binds PAl 53 polypeptide. Most preferably, the polyclonal antibody is of rabbit origin.
• the polyclonal antibodies used in the assays can be used either alone or as a cocktail of polyclonal antibodies.
• the cocktails used in the assay formats are comprised of either monoclonal antibodies or polyclonal antibodies having different binding specificity to PAl 53 polypeptides, they are useful for the detecting, diagnosing, staging, monitoring, prognosticating, in vivo imaging, preventing or treating, or determining the predisposition to, diseases and conditions of the pancreas, such as pancreatic cancer.
• PAl 53 antigen may be detectable in assays by use of a recombinant antigen as well as by use of a synthetic peptide or purified peptide, which peptide comprises an amino acid sequence of PAl 53.
• the amino acid sequence of such a polypeptide is selected from the group consisting of SEQUENCE ID NO 28, SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31, SEQUENCE ID NO 32, and fragments thereof.
• different synthetic, recombinant or purified peptides, identifying different epitopes of PAl 53 can be used in combination in an assay for the detecting, diagnosing, staging, monitoring, prognosticating, in vivo imaging, preventing or treating, or determining the predisposition to diseases and conditions of the pancreas, such as pancreatic cancer.
• all of these peptides can be coated onto one solid phase; or each separate peptide may be coated onto separate solid phases, such as microparticles, and then combined to form a mixture of peptides which can be later used in assays.
• multiple peptides which define epitopes from different antigens may be used for the detection, diagnosis, staging, monitoring, prognosis, prevention or treatment of, or determining the predisposition to, diseases and conditions of the pancreas, such as pancreatic cancer.
• Peptides coated on solid phases or labeled with detectable labels are then allowed to compete with those present in a patient sample (if any) for a limited amount of antibody.
• a reduction in binding of the synthetic, recombinant, or purified peptides to the antibody (or antibodies) is an indication of the presence of PAl 53 antigen in the patient sample.
• the presence of PA153 antigen indicates the presence of pancreatic tissue disease, especially pancreatic cancer, in the patient.
• the presence of anti-PA153 antibody and/or PAl 53 antigen can be detected in a simultaneous assay, as follows.
• a test sample is simultaneously contacted with a capture reagent of a first analyte, wherein said capture reagent comprises a first binding member specific for a first analyte attached to a solid phase and a capture reagent for a second analyte, wherein said capture reagent comprises a first binding member for a second analyte attached to a second solid phase, to thereby form a mixture.
• This mixture is incubated for a time and under conditions sufficient to form capture reagent/first analyte and capture reagent/second analyte complexes.
• These so-formed complexes then are contacted with an indicator reagent comprising a member of a binding pair specific for the first analyte labeled with a signal generating compound and an indicator reagent comprising a member of a binding pair specific for the second analyte labeled with a signal generating compound to form a second mixture.
• This second mixture is incubated for a time and under conditions sufficient to form capture reagent first analyte/indicator reagent complexes and capture reagent/second analyte/indicator reagent complexes.
• the presence ofone or more analytes is determined by detecting a signal generated in connection with the complexes formed on either or both solid phases as an indication of the presence ofone or more analytes in the test sample.
• recombinant antigens derived from the expression systems disclosed herein may be utilized, as well as monoclonal antibodies produced from the proteins derived from the expression systems as disclosed herein.
• PAl 53 antigen can be the first analyte.
• the polypeptides disclosed herein may be utilized to detect the presence of antibody against PAl 53 antigen in test samples.
• a test sample is incubated with a solid phase to which at least one polypeptide such as a recombinant protein or synthetic peptide has been attached.
• the polypeptide is selected from the group consisting of SEQUENCE ID NO 28, SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31, SEQUENCE ID NO 32, and fragments thereof. These are reacted for a time and under conditions sufficient to form antigen/antibody complexes. Following incubation, the antigen/antibody complex is detected.
• Indicator reagents may be used to facilitate detection, depending upon the assay system chosen.
• a test sample is contacted with a solid phase to which a recombinant protein produced as described herein is attached, and also is contacted with a monoclonal or polyclonal antibody specific for the protein, which preferably has been labeled with an indicator reagent. After incubation for a time and under conditions sufficient for antibody/antigen complexes to form, the solid phase is separated from the free phase, and the label is detected in either the solid or free phase as an indication of the presence of antibody against PAl 53 antigen.
• Other assay formats utilizing the recombinant antigens disclosed herein are contemplated.
• test sample with a solid phase to which at least one antigen from a first source has been attached, incubating the solid phase and test sample for a time and under conditions sufficient to form antigen/antibody complexes, and then contacting the solid phase with a labeled antigen, which antigen is derived from a second source different from the first source.
• a recombinant protein derived from a first source such as R coli is used as a capture antigen on a solid phase, a test sample is added to the so-prepared solid phase, and following standard incubation and washing steps as deemed or required, a recombinant protein derived from a different source (i.e., non-R coli) is utilized as a part of an indicator reagent which subsequently is detected.
• a recombinant antigen on a solid phase and synthetic peptide in the indicator phase also are possible.
• Any assay format which utilizes an antigen specific for PAl 53 produced or derived from a first source as the capture antigen and an antigen specific for PAl 53 from a different second source is contemplated.
• ion capture procedures for immobilizing an immobilizable reaction complex with a negatively charged polymer can be employed according to the present invention to effect a fast solution-phase immunochemical reaction.
• An immobilizable immune complex is separated from the rest of the reaction mixture by ionic interactions between the negatively charged poly-anion/immune complex and the previously treated, positively charged porous matrix and detected by using various signal generating systems previously described, including those described in chemiluminescent signal measurements as described in EPO Publication No. 0 273,115.
• the methods of the present invention can be adapted for use in systems which utilize microparticle technology including automated and semi-automated systems wherein the solid phase comprises a microparticle (magnetic or nonmagnetic).
• Such systems include those described in, for example, published EPO applications Nos. EP 0 425 633 and EP 0424 634, respectively.
• SPM scanning probe microscopy
• the use of scanning probe microscopy (SPM) for immunoassays also is a technology to which the monoclonal antibodies of the present invention are easily adaptable.
• the capture phase for example, at least one of the monoclonal antibodies of the invention
• a scanning probe microscope is utilized to detect antigen/antibody complexes which may be present on the surface of the solid phase.
• the use of scanning tunneling microscopy eliminates the need for labels which normally must be utilized in many immunoassay systems to detect antigen/antibody complexes.
• the use of SPM to monitor specific binding reactions can occur in many ways.
• one member of a specific binding partner analyte specific substance which is the monoclonal antibody of the invention
• the attachment of the analyte specific substance may be by adsorption to a test piece which comprises a solid phase of a plastic or metal surface, following methods known to those of ordinary skill in the art.
• covalent attachment of a specific binding partner (analyte specific substance) to a test piece which test piece comprises a solid phase of derivatized plastic, metal, silicon, or glass may be utilized.
• Covalent attachment methods are known to those skilled in the art and include a variety of means to irreversibly link specific binding partners to the test piece. If the test piece is silicon or glass, the surface must be activated prior to attaching the specific binding partner.
• polyelectrolyte interactions may be used to immobilize a specific binding partner on a surface of a test piece by using techniques and chemistries.
• the preferred method of attachment is by covalent means.
• the surface may be further treated with materials such as serum, proteins, or other blocking agents to minimize non-specific binding.
• the surface also may be scanned either at the site of manufacture or point of use to verify its suitability for assay purposes. The scanning process is not anticipated to alter the specific binding properties of the test piece.
• the present invention discloses the preference for the use of solid phases, it is contemplated that the reagents such as antibodies, proteins and peptides of the present invention can be utilized in non-solid phase assay systems. These assay systems are known to those skilled in the art, and are considered to be within the scope of the present invention.
• the reagent employed for the assay can be provided in the form of a test kit with one or more containers such as vials or bottles, with each container containing a separate reagent such as a probe, primer, monoclonal antibody or a cocktail of monoclonal antibodies, or a polypeptide (e.g. recombinantly, synthetically produced or purified) employed in the assay.
• the polypeptide is selected from the group consisting of SEQUENCE ID NO 28, SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31, SEQUENCE ID NO 32, and fragments thereof.
• Other components such as buffers, controls and the like, known to those of ordinary skill in art, may be included in such test kits.
• test kits which have means for collecting test samples comprising accessible body fluids, e.g., blood, urine, saliva and stool.
• Such tools useful for collection include lancets and absorbent paper or cloth for collecting and stabilizing blood; swabs for collecting and stabilizing saliva; cups f collecting and stabilizing urine or stool samples.
• Collection materials, papers, clo swabs, cups and the like may optionally be treated to avoid denaturation or irreversible adsorption of the sample.
• the collection materials also may be treated 5 5 with or contain preservatives, stabilizers or antimicrobial agents to help maintain t integrity of the specimens.
• kits designed for the collection, stabilization and preservation of test specimens obtained by surgery or needle biopsy are also usefu is contemplated that all kits may be configured in two components which can be provided separately; one component for collection and transport of the specimen a 0 10 the other component for the analysis of the specimen.
• the collection component can be provided to the open market user while the components for analy can be provided to others such as laboratory personnel for determination of the presence, absence or amount of analyte.
• kits for the collection, stabilizati and preservation of test specimens may be configured for use by untrained person 5 15 and may be available in the open market for use at home with subsequent transportation to a laboratory for analysis of the test sample.
• Antibodies of the present invention can be used in vivo: that is. they can b injected into patients suspected of having or having diseases of the pancreas for diagnostic or therapeutic uses.
• the use of antibodies for in vivo diagnosis is well known in the art. Sumerdon et al., Nucl. Med. Biol 17:247-254 (1990) have described an optimized antibody-chelator for the radioirmnunoscintographic imagi f 25 of carcinoembryonic antigen (CEA) expressing tumors using Indium- 111 as the la
• CEA carcinoembryonic antigen
• Radioactive labels such as Indium- 111, Technetium-99m, or Iodine-131 can be used for planar scans or single photon emission computed tomography (SPECT).
• Positron emitting labels such as Fluorine- 19 can also be used for positron emission tomography (PET).
• PET positron emission tomography
• paramagnetic ions such as Gadolinium (III) or Manganese (II) can be used. Localization of the label within the pancreas or external to the pancreas may allow determination of spread of the disease. The amount of label within the pancreas may allow determination of the presence or absence of cancer of the pancreas.
• an antibody directed against PAl 53 antigen may have therapeutic benefit.
• the antibody may exert its effect without the use of attached agents by binding to PAl 53 antigen expressed on or in the tissue or organ.
• the antibody may be conjugated to cytotoxic agents such as drugs, toxins, or radionuclides to enhance its therapeutic effect.
• Garnett and Baldwin, Cancer Research 46:2407-2412 (1986) have described the preparation of a drug-monoclonal antibody conjugate.
• Pastan et al., Cell 47:641 - 648 (1986) have reviewed the use of toxins conjugated to monoclonal antibodies for the therapy of various cancers.
• cytotoxic radionuclides include Copper-67, Iodine- 131, and Rhenium- 186 all of which can be used to label monoclonal antibodies directed against PAl 53 antigen for the treatment of cancer of the pancreas.
• R coli bacteria (clone 2075919) was deposited on March 9, 1998 with the American Type Culture Collection (A.T.C.C), 10801 University Boulevard., Manassas, VA. The deposit was made under the terms of the Budapest Treaty and will be maintained for a period of thirty (30) years from the date of deposit, or for five (5) years after the last request for the deposit, or for the enforceable period of the U.S. patent, whichever is longer. The deposit and any other deposited material described herein are provided for convenience only, and are not required to practice the present invention in view of the teachings provided herein. The cDNA sequence in all of the deposited material is incorporated herein by reference. Clone 2075919 was accorded A.T.C.C. Deposit No. 98681. The present invention will now be described by way of examples, which are meant to illustrate, but not to limit, the scope of the present invention.
• EST's Library Comparison of Expressed Sequence Tags (EST's) or Transcript Images. Partial sequences of cDNA clone inserts, so-called “expressed sequence tags" (EST's), were derived from cDNA libraries made from pancreatic tumor tissues, pancreatic non-tumor tissues and numerous other tissues, both tumor and non- tumor and entered into a database (LIFESEQTM database, available from Incyte Pharmaceuticals, Palo Alto, CA) as gene transcript images. See International Publication No. WO 95/20681. (A transcript image is a listing of the number of EST's for each of the represented genes in a given tissue library. EST's sharing regions of mutual sequence overlap are classified into clusters.
• a cluster is assigned a clone number from a representative 5' EST. Often, a cluster of interest can be extended by comparing its consensus sequence with sequences of other EST's which did not meet the criteria for automated clustering. The alignment of all available clusters and single EST's represent a contig from which a consensus sequence is derived.) The transcript images then were evaluated to identify EST sequences that were representative primarily of the pancreatic tissue libraries. These target clones then were ranked according to their abundance (occurrence) in the target libraries and their absence from background libraries. Higher abundance clones with low background occurrence were given higher study priority. EST's corresponding to the consensus sequence of PA153 were found in 55.0% (11 of 20) of pancreatic tissue libraries.
• Overlapping clones 2075919H1 (SEQUENCE ID NO 1), 2383634H1 (SEQUENCE ID NO 2), 5069724H1 (SEQUENCE ID NO 3), 5070712H1 (SEQUENCE ID NO 4), 2773816H1 (SEQUENCE ID NO 5), 2374806H1 (SEQUENCE ID NO 6), 5071731H1 (SEQUENCE ID NO 7), 883484H1 (SEQUENCE ID NO 8), 887213H1 (SEQUENCE ID NO 9), respectively, were identified for further study.
• Figures 1 A- ID show the nucleotide sequence alignment of these clones and their resultant nucleotide consensus sequence (SEQUENCE ID NO 11).
• Figure 2 presents the contig map depicting the clones 2075919H1 (SEQUENCE ID NO 1), 2383634H1 (SEQUENCE ID NO 2), 5069724H1 (SEQUENCE ID NO 3), 5070712H1 (SEQUENCE ID NO 4), 2773816H1 (SEQUENCE ID NO 5),
• the 607 residue amino acid sequence depicted in SEQUENCE ID NO 28 was compared with published sequences using software and techniques known to those skilled in the art.
• the amino acid sequence of a murine protein precursor was identified as having partial homology with the PAl 53 polypeptide of SEQUENCE ID NO 28.
• the sequence for this murine protein precursor is deposited with GenBank under Accession No. U69699.
• the polypeptide of SEQUENCE ID NO 28 also displayed partial homology to rat Ebnerin, a protein secreted from the von Ebner's gland of rat and described in International Publication No. WO 96/39513.
• Example 2 Sequencing of PAl 53 EST-Specific Clones The DNA sequence of clone 2075919inh of the PA153 gene contig was determined (SEQUENCE ID NO 10) using dideoxy termination sequencing with dye terminators following known methods [F. Sanger et al., PNAS U.S.A. 74:5463 (1977)].
• vectors such as pSPORTl (Life Technologies, Gaithersburg, MD) and pINCY (available from Incyte Pharmaceuticals, Inc., Palo Alto, CA) contain universal priming sites just adjacent to the 3' and 5' ligation junctions of the inserts
• the inserts were sequenced in both directions using universal primers, SEQUENCE ID NO 14 and SEQUENCE ID NO 15, respectively ( New England Biolabs, Beverly, MA and Applied Biosystems Inc, Foster City, CA).
• the sequencing reactions were run on a polyacrylamide denaturing gel, and the sequences were determined by an Applied Biosystems 377 Sequencer (available from Applied Biosystems, Foster City, CA).
• Additional sequencing primers, SEQUENCE ID NOS 16 -25 were designed from sequence information of the consensus sequence, SEQUENCE ID NO 11. These primers then were used to determine the remaining DNA sequence of the cloned insert from each DNA strand, as previously described.
• RNA Extraction from Tissue was isolated from pancreatic tissues and from non-pancreatic tissues. Various methods were utilized, including but not limited to the lithium chloride/urea technique, known in the art and described by Kato et al., (J. Virol. 61:2182-2191, 1987), and TRIzolTM (Gibco-BRL, Grand Island, NY). Briefly, tissue was placed in a sterile conical tube on ice and 10-15 volumes of 3 M LiCl, 6 M urea, 5 mM EDTA, 0.1 M ⁇ -mercaptoethanol, 50 mM Tris-HCl (pH 7.5), 0.1%) sarcosyl were added.
• the tissue was homogenized with an Omni TH homogenizer (Omni International, Inc., Warrenton, VA) for 30-50 sec on ice.
• the solution was transferred to a 15 ml plastic centrifuge tube and placed overnight at - 20°C.
• the tube was centrifuged for 90 min at 9,000 x g at 0-4°C and the supernatant was immediately decanted.
• Ten ml of 3 M LiCl were added and the tube was vortexed for 5 sec. The tube was centrifuged for 45 min at 9,000 x g at 0-4°C.
• the PCI extraction was repeated and followed by two similar extractions with chloroform/isoamyl alcohol (CI).
• CI chloroform/isoamyl alcohol
• the final aqueous solution was transferred to a prechilled 15 ml Corex glass tube containing 6.5 ml of 95% ethanol, the tube was covered with parafilm, and placed at -20°C overnight.
• the tube was centrifuged for 30 min at 10,000 x g at 0-4°C and the ethanol supernatant was decanted immediately.
• the RNA pellet was washed four times with 10 ml of 75% ice-cold ethanol and the final pellet was air dried for 15 min at room temperature.
• RNA samples were aliquoted and stored at -70°C in 100% FORMAzol ® .
• RNA samples that did not contain intact rRNAs were excluded from the study.
• RNA samples were aliquoted and stored at -70°C as ethanol precipitates. The sample was centrifuged at 12,000 x g for 30 min at 4°C, and the supernatant discarded. The remaining pellet was washed twice with cold 75% ethanol, resuspended by vortexing, and the resuspended material was then pelleted by centrifugation at 12,000 x g for 10 min at 4°C. Finally, the RNA pellet was dried in a Speedvac (Savant, Farmingdale, NY) for 5 min and reconstituted in RNase-free water.
• Speedvac Savant, Farmingdale, NY
• RNA Extraction from Blood Mononuclear Cells Mononuclear cells are isolated from blood samples from patients by centrifugation using Ficoll-Hypaque as follows. A 10 ml volume of whole blood is mixed with an equal volume of RPMI Medium (Gibco-BRL, Grand Island, NY). This mixture is then underlayed with 10 ml of Ficoll-Hypaque (Pharmacia, Piscataway, NJ) and centrifuged for 30 minutes at 200 x g.
• RNA is prepared from the isolated mononuclear cells as described by N. Kato et al.. J. Virology 61 : 2182-2191 (1987).
• the pelleted mononuclear cells are brought to a final volume of 1 ml and then are resuspended in 250 ⁇ L of PBS and mixed with 2.5 ml of 3M LiCl, 6M urea, 5mM EDTA, 0.1M 2-mercaptoethanol, 50mM Tris-HCl (pH 7.5).
• the resulting mixture is homogenized and incubated at - 20°C overnight.
• the homogenate is centrifuged at 8,000 RPM in a Beckman J2-21M rotor for 90 minutes at 0-4°C.
• the pellet is resuspended in 10 ml of 3M LiCl by vortexing and then centrifuged at 10,000 RPM in a Beckman J2-21M rotor centrifuge for 45 minutes at 0-4°C. The resuspending and pelleting steps then are repeated.
• the pellet is resuspended in 2 ml of 1 mM EDTA, 0.5% SDS, 10 mM Tris (pH 7.5) and 400 ⁇ g Proteinase K with vortexing and then it is incubated at 37°C for 30 minutes with shaking. One-tenth volume of 3M NaCI then is added and the mixture is vortexed.
• RNA is precipitated by the addition of 6 ml of absolute ethanol followed by overnight incubation at -20°C. After the precipitated RNA is collected by centrifugation, the pellet is washed 4 times in 75% ethanol. The pelleted RNA is then dissolved in solution containing ImM EDTA, lOmM Tris-HCl (pH 7.5). Non-pancreatic tissues are used as negative controls.
• the mRNA can be further purified from total RNA by using commercially available kits such as oligo dT cellulose spin columns (RediColTM from Pharmacia, Uppsala, Sweden) for the isolation of poly-adenylated RNA.
• Total RNA or mRNA can be dissolved in lysis buffer (5M guanidine thiocyanate, 0. IM EDTA, pH 7.0) for analysis in the ribonuclease protection assay.
• RNA Extraction from Polvsomes Tissue is minced in saline at 4°C and mixed with 2.5 volumes of 0.8 M sucrose in a TK 150 M (150 mM KCl, 5 mM MgCl 2 , 50 mM Tris-HCl, pH 7.4) solution containing 6 mM 2-mercaptoethanol.
• the tissue is homogenized in a Teflon-glass Potter homogenizer with five strokes at 100-200 rpm followed by six strokes in a Dounce homogenizer, as described by B. Mechler, Methods in Enzymology 152:241-248 (1987). The homogenate then is centrifuged at 12,000 x g for 15 min at 4°C to sediment the nuclei.
• the polysomes are isolated by mixing 2 ml of the supernatant with 6 ml of 2.5 M sucrose in TK 150 M and layering this mixture over 4 ml of 2.5 M sucrose in TK 150 M in a 38 ml polyallomer tube. Two additional sucrose TK, 50 M solutions are successively layered onto the extract fraction; a first layer of 13 ml 2.05 M sucrose followed by a second layer of 6 ml of 1.3 M sucrose. The polysomes are isolated by centrifuging the gradient at 90,000 x g for 5 hr at 4°C.
• the fraction then is taken from the 1.3 M sucrose/2.05 M sucrose interface with a siliconized pasteur pipette and diluted in an equal volume of TE (10 mM Tris- HCl, pH 7.4, 1 mM EDTA).
• TE 10 mM Tris- HCl, pH 7.4, 1 mM EDTA.
• An equal volume of 90°C SDS buffer 1% SDS, 200 mM NaCI, 20 mM Tris-HCl, pH 7.4
• Proteins next are digested with a Proteinase K digestion (50 mg/ml) for 15 min at 37°C.
• the mRNA is purified with 3 equal volumes of phenol-chloroform extractions followed by precipitation with 0.1 volume of 2 M sodium acetate (pH 5.2) and 2 volumes of 100% ethanol at -20°C overnight.
• the precipitated RNA is recovered by centrifugation at 12,000 x g for 10 min at 4°C.
• the RNA is dried and resuspended in TE (pH 7.4) or distilled water. The resuspended RNA then can be used in a slot blot or dot blot hybridization assay to check for the presence of PAl 53 mRNA (see Example 6).
• nucleic acid and proteins are dependent on the method of preparation used. Each sample may require a different preparation technique to maximize isolation efficiency of the target molecule. These preparation techniques are within the skill of the ordinary artisan.
• Example 4 Ribonuclease Protection Assay A. Synthesis of Labeled Complementary RNA (cRNA) Hybridization Probe and Unlabeled Sense Strand.
• Labeled antisense and unlabeled sense riboprobes are transcribed from the PAl 53 gene cDNA sequence which contains a 5' RNA polymerase promoter such as SP6 or T7.
• the sequence may be from a vector containing the appropriate PAl 53 cDNA insert, or from a PCR-generated product of the insert using PCR primers which incorporate a 5' RNA polymerase promoter sequence.
• the described plasmid, clone 2075919 or another comparable clone, containing the PAl 53 gene cDNA sequence, flanked by opposed SP6 and T7 or other RNA polymerase promoters is purified using a Qiagen Plasmid Purification Kit (Qiagen, Chatsworth, CA). Then 10 ⁇ g of the plasmid DNA are linearized by cutting with an appropriate restriction enzyme such as Dde I for 1 hr at 37°C.
• the linearized plasmid DNA is purified using the QIAprep Kit (Qiagen, Chatsworth, CA) and used for the synthesis of antisense transcript from the appropriate promoter using the Riboprobe ® in vitro Transcription System (Promega Corporation, Madison, WI), as described by the supplier's instructions, incorporating either (alpha 32 P) CTP (Amersham Life Sciences, Inc. Arlington Heights, IL) or biotinylated CTP as a label.
• restriction enzymes such as Xba I and Not I, and transcribed as above from the appropriate promoter. Both sense and antisense strands are isolated by spin column chromatography.
• Unlabeled sense strand is quantitated by UV absorption at 260 nm.
• B. Hybridization of Labeled Probe to Target Frozen tissue is pulverized to powder under liquid nitrogen and 100-500 mg are dissolved in 1 ml of lysis buffer, available as a component of the Direct ProtectTM Lysate RNase Protection Kit (Ambion, Inc., Austin, TX). Further dissolution can be achieved using a tissue homogenizer. In addition, a dilution series of a known amount of sense strand in mouse liver lysate is made for use as a positive control.
• solubilized tissue or diluted sense strand is mixed directly with either ; 1) 1 xlO 5 cpm of radioactively labeled probe, or 2) 250 pg of non-isotopically labeled probe in 5 ⁇ l of lysis buffer. Hybridization is allowed to proceed overnight at 37°C. See, T. Kaabache et al., Anal. Biochem. 232:225-230 (1995).
• RNA that is not hybridized to probe is removed from the reaction as per the Direct ProtectTM protocol using a solution of RNase A and RNase Tl for 30 min at 37°C, followed by removal of RNase by Proteinase K digestion in the presence of sodium sarcosyl. Hybridized fragments protected from digestion are then precipitated by the addition of an equal volume of isopropanol and placed at -70°C for 3 hr. The precipitates are collected by centrifugation at 12,000 x g for 20 min. D. Fragment Analysis.
• the precipitates are dissolved in denaturing gel loading dye (80% formamide, 10 mM EDTA (pH 8.0), 1 mg/ml xylene cyanol, 1 mg/ml bromophenol blue), heat denatured, and electrophoresed in 6% polyacrylamide TBE, 8 M urea denaturing gels.
• denaturing gel loading dye 80% formamide, 10 mM EDTA (pH 8.0), 1 mg/ml xylene cyanol, 1 mg/ml bromophenol blue
• the gels are imaged and analyzed using the STORMTM storage phosphor autoradiography system (Molecular Dynamics, Sunnyvale, CA). Quantitation of protected fragment bands, expressed in femtograms (fg), is achieved by comparing the peak areas obtained from the test samples to those from the known dilutions of the positive control sense strand (see Section B, supra).
• hybrids are transferred from the gels to membranes (nylon or nitrocellulose) by blotting and then analyzed using detection systems that employ streptavidin alkaline phosphatase conjugates and chemiluminesence or chemifluoresence reagents.
• Detection of a product comprising a sequence selected from the group consisting of SEQUENCE ID NOS 1-11, and fragments or complements thereof, is indicative of the presence of PAl 53 mRNA(s), suggesting a diagnosis of a pancreatic tissue disease or condition, such as pancreatic cancer.
• Example 5 Northern Blotting The Northern blot technique is used to identify a specific size RNA fragment from a complex population of RNA using gel electrophoresis and nucleic acid hybridization.
• Northern blotting is well-known technique in the art. Briefly, 5-10 ⁇ g of total RNA (see Example 3) are incubated in 15 ⁇ l of a solution containing 40 mM morpholinopropanesulfonic acid (MOPS) (pH 7.0), 10 mM sodium acetate, 1 mM EDTA, 2.2 M formaldehyde, 50% v/v formamide for 15 min at 65°C.
• MOPS morpholinopropanesulfonic acid
• RNA is transferred from the gel onto nylon membranes (Brightstar-Plus, Ambion, Inc., Austin, TX) for 1.5 hours using the downward alkaline capillary transfer method (Chomczynski. Anal. Biochem. 201:134-139. 1992).
• the filter is rinsed with IX SSC, and RNA is crosslinked to the filter using a StratalinkerTM
• a 32 P-labeled random-primed probe is generated using the PAl 53 insert fragment (obtained by digesting clone 2075919 or another comparable clone with Xbal and NotI) using Random Primer DNA Labeling System (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer's instructions.
• Half of the probe is boiled for 10 min, quick chilled on ice and added to the hybridization tube.
• Hybridization is carried out at 42°C for at least 12 hr.
• the hybridization solution is discarded and the filter is washed in 30 ml of 3X SSC, 0.1% SDS at 42°C for 15 min, followed by 30 ml of 3X SSC, 0.1% SDS at 42°C for 15 min.
• the filter is wrapped in Saran Wrap, exposed to Kodak XAR-Omat film for 8- 96 hr, and the film is developed for analysis.
• High level of expression of mRNA corresponding to a sequence selected from the group consisting of SEQUENCE ID NOS 1-11, and fragments or complements thereof, is an indication of the presence of PAl 53 mRNA, suggesting a diagnosis of a pancreatic tissue disease or condition, such as pancreatic cancer.
• Dot and slot blot assays are quick methods to evaluate the presence of a specific nucleic acid sequence in a complex mix of nucleic acid.
• RNA are mixed in 50 ⁇ l of 50% formamide, 7% formaldehyde, IX SSC, incubated 15 min at 68°C, and then cooled on ice. Then, 100 ⁇ l of 20X SSC are added to the RNA mixture and loaded under vacuum onto a manifold apparatus that has a prepared nitrocellulose or nylon membrane.
• the membrane is soaked in water, 20X SSC for 1 hour, placed on two sheets of 20X SSC prewet Whatman #3 filter paper, and loaded into a slot blot or dot blot vacuum manifold apparatus.
• the slot blot is analyzed with probes prepared and labeled as described in Example 4, supra. Detection of mRNA corresponding to a sequence selected from the group consisting of SEQUENCE ID NOS 1-11, and fragments or complements thereof, is an indication of the presence of PAl 53, suggesting a diagnosis of a pancreatic tissue disease or condition, such as pancreatic cancer.
• Example 7 In situ Hybridization This method is useful to directly detect specific target nucleic acid sequences in cells using detectable nucleic acid hybridization probes.
• Tissues are prepared with cross-linking fixative agents such as paraformaldehyde or glutaraldehyde for maximum cellular RNA retention. See, L. Angerer et al., Methods in Cell Biol. 35:37-71 (1991). Briefly, the tissue is placed in greater than 5 volumes of 1% glutaraldehyde in 50 mM sodium phosphate, pH 7.5 at 4°C for 30 min. The solution is changed with fresh glutaraldehyde solution (1% glutaraldehyde in 50mM sodium phosphate, pH 7.5) for a further 30 min fixing. The fixing solution should have an osmolality of approximately 0.375% NaCI. The tissue is washed once in isotonic NaCI to remove the phosphate.
• cross-linking fixative agents such as paraformaldehyde or glutaraldehyde for maximum cellular RNA retention. See, L. Angerer et al., Methods in Cell Biol. 35:37-71 (1991
• the fixed tissues then are embedded in paraffin as follows.
• the tissue is dehydrated though a series of increasing ethanol concentrations for 15 min each: 50% (twice), 70% (twice), 85%, 90% and then 100% (twice).
• the tissue is soaked in two changes of xylene for 20 min each at room temperature.
• the tissue is then soaked in two changes of a 1 : 1 mixture of xylene and paraffin for 20 min each at 60°C; and then in three final changes of paraffin for 15 min each.
• the tissue is cut in 5 ⁇ m sections using a standard microtome and placed on a slide previously treated with a tissue adhesive such as 3- aminopropyltriethoxysilane.
• Paraffin is removed from the tissue by two 10 min xylene soaks and rehydrated in a series of decreasing ethanol concentrations: 99% twice, 95%, 85%, 70%, 50%), 30%, and then distilled water twice.
• the sections are pre-treated with 0.2 M HCl for 10 min and permeabilized with 2 ⁇ g/ml Proteinase K at 37°C for 15 min.
• Labeled riboprobes transcribed from the PAl 53 gene plasmid are hybridized to the prepared tissue sections and incubated overnight at 56°C in 3X standard saline extract and 50% formamide. Excess probe is removed by washing in 2X standard saline citrate and 50% formamide followed by digestion with 100 ⁇ g/ml RNase A at 37°C for 30 min. Fluorescence probe is visualized by illumination with ultraviolet (UV) light under a microscope. Fluorescence in the cytoplasm is indicative of PAl 53 mRNA. Alternatively, the sections can be visualized by autoradiography.
• UV ultraviolet
• Example 8 Reverse Transcription PCR A.
• One Step RT-PCR Assay Target-specific primers are designed to detect the above-described target sequences by reverse transcription PCR using methods known in the art.
• One step RT-PCR is a sequential procedure that performs both RT and PCR in a single reaction mixture.
• the procedure is performed in a 200 ⁇ l reaction mixture containing 50 mM (N,N,-bis[2-Hydroxyethyl]glycine), pH 8.15, 81.7 mM KOAc, 33.33 mM KOH, 0.01 mg/ml bovine serum albumin, 0.1 mM ethylene diaminetetraacetic acid, 0.02 mg/ml NaN 3 8% w/v glycerol, 150 ⁇ M each of dNTP, 0.25 ⁇ M each primer, 5U rTth polymerase, 3.25 mM Mn(OAc) 2 and 5 ⁇ l of target RNA (see Example 3).
• RNA and the rTth polymerase enzyme are unstable in the presence of Mn(OAc) 2 , the Mn(OAc) 2 should be added just before target addition.
• Optimal conditions for cDNA synthesis and thermal cycling readily can be determined by those skilled in the art.
• the reaction is incubated in a Perkin- Elmer Thermal Cycler 480. Conditions which may be found useful include cDNA synthesis at 60°-70°C for 15-45 min and 30-45 amplification cycles at 94°C, 1 min; 55°-70°C, 1 min; 72°C, 2 min.
• RT-PCR also may be performed by using a dual enzyme procedure with Taq polymerase and a reverse transcriptase enzyme, such as MMLV (Moloney murine leukemia virus) or AMV (avian myeloblastosis virus) RT (reverse transcriptase) enzymes.
• MMLV Microloney murine leukemia virus
• AMV avian myeloblastosis virus
• RT reverse transcriptase
• Reverse transcription was performed at room temperature for 10 min, 42°C for 30 min in a PE-480 thermal cycler (Perkin-Elmer), followed by further incubation at 95°C for 5 min to inactivate the RT.
• PCR was performed using 2 ⁇ l of the cDNA reaction in a final PCR reaction volume of 50 ⁇ l containing IX PCR II buffer (Perkin-Elmer), 50 mM KCl, 1.5 mM MgC12, 200 ⁇ M dNTPs, 0.5 ⁇ M of each sense and antisense primer, SEQUENCE ID NO 26 and SEQUENCE ID NO 27, respectively, and 2.5 U of Taq Gold polymerase.
• the reaction was incubated in a PE-480 thermal cycler (Perkin-Elmer), as follows: 35 cycles of amplification (94°C, 45 sec; 58°C, 45 sec; 70°C, 2 min.); a final extension (72°C, 7 min); and a soak at 4°C.
• a PE-480 thermal cycler Perkin-Elmer
• Detection of high levels of a product comprising a sequence selected from the group consisting of SEQUENCE ID NOS 1-11, and fragments or complements thereof, is indicative of the presence of PAl 53 mRNA(s), suggesting a diagnosis of a pancreatic tissue disease or condition, such as pancreatic cancer.
• Example 9 OH-PCR A. Probe selection and Labeling.
• Target-specific primers and probes are designed to detect the above-described target sequences by oligonucleotide hybridization PCR.
• a label-phosphoramidite reagent is prepared and used to add the label to the oligonucleotide during its synthesis. For example, see N. T. Thuong et al., Tet.
• probes are labeled at their 3' end to prevent participation in PCR and the formation of undesired extension products.
• the probe should have a TM at least 15°C below the T of the primers.
• the primers and probes are utilized as specific binding members, with or without detectable labels, using standard phosphoramidite chemistry and/or post-synthetic labeling methods which are well-known to one skilled in the art.
• OH-PCR is performed on a 200 ⁇ l reaction containing 50 mM (N,N,-bis[2-Hydroxyethyl]glycine), pH 8.15, 81.7 mM KOAc, 33.33 mM KOH, 0.01 mg/ml bovine serum albumin, 0.1 mM ethylene diaminetetraacetic acid, 0.02 mg/ml NaN 3j 8% w/v glycerol, 150 ⁇ M each of dNTP, 0.25 ⁇ M each primer, 3.75 nM probe, 5U rTth polymerase, 3.25 mM Mn(OAc) 2 and 5 ⁇ l blood equivalents of target (see Example 3).
• RNA and the rTth polymerase enzyme are unstable in the presence of Mn(OAc) 2 , the Mn(OAc) 2 should be added just before target addition.
• the reaction is incubated in a Perkin-Elmer Thermal Cycler 480.
• Optimal conditions for cDNA synthesis and thermal cycling can be readily determined by those skilled in the art. Conditions which may be found useful include cDNA synthesis (60°C, 30 min), 30-45 amplification cycles (94°C, 40 sec; 55-70°C, 60 sec), oligo-hybridization (97°C, 5 min; 15°C, 5 min; 15°C soak).
• the correct reaction product contains at least one of the strands of the PCR product and an internally hybridized probe.
• PAl 53- derived nucleic acid sequences including, but not limited to, ligase chain reaction (LCR, Abbott Laboratories, Abbott Park, IL); Q-beta replicase (Gene-TrakTM,
• Example 10 Synthetic Peptide Production Synthetic peptides were modeled and then prepared based upon the predicted amino acid sequence of the PAl 53 polypeptide consensus sequence (see Example 1). In particular, a number of PAl 53 peptides derived from SEQUENCE ID NO 28 were prepared, including the peptides of SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31, and SEQUENCE ID NO 32. All peptides were synthesized on a Symphony Peptide Synthesizer (available from Rainin Instrument Co, Emeryville, CA) using FMOC chemistry, standard cycles and in-situ HBTU activation.
• a Symphony Peptide Synthesizer available from Rainin Instrument Co, Emeryville, CA
• Cleavage and deprotection conditions were as follows: a volume of 2.5 ml of cleavage reagent (77.5% v/v trifluoroacetic acid, 15% v/v ethanedithiol, 2.5% v/v water, 5% v/v thioanisole, 1-2% w/v phenol) were added to the resin, and agitated at room temperature for 2-4 hours. The filtrate was then removed and the peptide was precipitated from the cleavage reagent with cold diethyl ether. Each peptide was filtered, purified via reverse-phase preparative HPLC using a water/acetonitrile/O.1% TFA gradient, and lyophilized. The product was confirmed by mass spectrometry. The purified peptides were used to immunize animals (see Example 14).
• Plasmid 577 described in U.S. patent application Serial No. 08/478,073, filed June 7, 1995, has been constructed for the expression of secreted antigens in a permanent cell line.
• This plasmid contains the following DNA segments: (a) a 2.3 kb fragment of pBR322 containing bacterial beta-lactamase and origin of DNA replication; (b) a 1.8 kb cassette directing expression of a neomycin resistance gene under control of HSV-1 thymidine kinase promoter and poly-A addition signals; (c) a 1.9 kb cassette directing expression of a dihydrofolate reductase gene under the control of an Simian Virus 40 (SV40) promoter and poly-A addition signals; (d) a 3.5 kb cassette directing expression of a rabbit immunoglobulin heavy chain signal sequence fused to a modified hepatitis C virus (HCV) E2 protein under the
• Plasmids for the expression of secretable PAl 53 proteins are constructed by replacing the hepatitis C virus E2 protein coding sequence in plasmid 577 with that of a PAl 53 polynucleotide sequence selected from the group consisting of SEQUENCE ID NOS 1-11, and fragments or complements thereof, as follows. Digestion of plasmid 577 with Xbal releases the hepatitis C virus E2 gene fragment. The resulting plasmid backbone allows insertion of the PAl 53 cDNA insert downstream of the rabbit immunoglobulin heavy chain signal sequence which directs the expressed proteins into the secretory pathway of the cell. The PAl 53 cDNA fragment is generated by PCR using standard procedures.
• Encoded in the sense PCR primer sequence is an Xbal site, immediately followed by a 12 nucleotide sequence that encodes the amino acid sequence Ser-Asn-Glu-Leu (" SNEL") to promote signal protease processing, efficient secretion and final product stability in culture fluids.
• the primer contains nucleotides complementary to template sequences encoding amino acids of the PA153 gene .
• the antisense primer incorporates a sequence encoding the following eight amino acids just before the stop codons: Asp-Tyr-Lys- Asp- Asp-Asp- Asp-Lys (SEQUENCE ID NO 33).
• a recognition site to aid in analysis and purification of the PA153 protein product.
• a recognition site (termed “FLAG”) that is recognized by a commercially available monoclonal antibody designated anti- FLAG M2 (Eastman Kodak, Co., New Haven, CT) can be utilized, as well as other comparable sequences and their co ⁇ esponding antibodies.
• PCR is performed using GeneAmp ® reagents obtained from Perkin-Elmer-Cetus, as directed by the supplier's instructions. PCR primers are used at a final concentration of 0.5 ⁇ M. PCR is performed on the PA153 plasmid template in a 100 ⁇ l reaction for 35 cycles (94°C, 30 seconds; 55°C, 30 seconds; 72°C, 90 seconds) followed by an extension cycle of 72°C for 10 min.
• CHO/dhfr- cells are cultured in Ham's F-12 media supplemented with 10% fetal calf serum, L-glutamine (1 mM) and freshly seeded into a flask at a density of 5 - 8 x 10 5 cells per flask.
• the cells are grown to a confluency of between 60 and 80% for transfection.
• Twenty micrograms (20 ⁇ g) of plasmid DNA are added to 1.5 ml of Opti-MEM I medium and 100 ⁇ l of Lipofectin Reagent (Gibco-BRL; Grand Island, NY) are added to a second 1.5 ml portion of Opti-MEM I media. The two solutions are mixed and incubated at room temperature for 20 min.
• the cells are rinsed 3 times with 5 ml of Opti-MEM I medium.
• the Opti-MEM I-Lipofection-plasmid DNA solution then is overlaid onto the cells.
• the cells are incubated for 3 hr at 37°C, after which time the Opti-MEM I-Lipofectin-DNA solution is replaced with culture medium for an additional 24 hr prior to selection.
• F-12 minus medium G dhfr/G418 selection medium
• Selection medium is Ham's F-12 with L-glutamine and without hypoxanthine, thymidine and glycine (JRH Biosciences, Lenexa, Kansas) and 300 ⁇ g per ml G418 (Gibco-BRL; Grand Island, NY). Media volume-to-surface area ratios of 5 ml per 25
• DHFR G418 cells are expanded to allow passage and continuous maintenance in F-12 minus medium G.
• Amplification of each of the transfected PA153 cDNA sequences is achieved by stepwise selection of DHFR , G418 cells with methotrexate (reviewed by R. Schimke, CeU 37:705-713 [1984]). Cells are incubated with F-12 minus medium G containing 150 nM methotrexate (MTX) (Sigma, St. Louis, MO) for approximately two weeks until resistant colonies appear. Further gene amplification is achieved by selection of 150 nM adapted cells with 5 ⁇ M MTX.
• methotrexate 150 nM methotrexate
• F-12 minus medium G supplemented with 5 ⁇ M MTX is overlaid onto just confluent monolayers for 12 to 24 hr at 37°C in 5% CO 2 .
• the growth medium is removed and the cells are rinsed 3 times with Dulbecco's phosphate buffered saline (PBS) (with calcium and magnesium) (Gibco-BRL; Grand Island, NY) to remove the remaining media/serum which may be present.
• PBS Dulbecco's phosphate buffered saline
• Gabco-BRL Grand Island, NY
• VAS custom medium VAS custom formulation with L-glutamine with HEPES without phenol red, available from JRH Bioscience; Lenexa, KS, product number 52-08678P
• VAS custom medium VAS custom formulation with L-glutamine with HEPES without phenol red, available from JRH Bioscience; Lenexa, KS, product number 52-08678P
• Cells then are overlaid with VAS for production at 5 ml per T flask. Medium is removed after seven days of incubation, retained, and then frozen to await purification with harvests 2, 3 and 4.
• the monolayers are overlaid with VAS for 3 more seven day harvests.
• PAl 53 Antigen Expression Aliquots of VAS supernatants from the cells expressing the PAl 53 protein construct are analyzed, either by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using standard methods and reagents known in the art (Laemmli discontinuous gels), or by mass spectrometry.
• SDS-PAGE SDS-polyacrylamide gel electrophoresis
• F. Purification Purification of the PAl 53 protein containing the FLAG sequence is performed by immunoaffinity chromatography using an affinity matrix comprising anti-FLAG M2 monoclonal antibody covalently attached to agarose by hydrazide linkage (Eastman Kodak Co., New Haven, CT).
• protein in pooled VAS medium harvests from roller bottles is exchanged into 50 mM Tris-HCl (pH 7.5), 150 mM NaCI buffer using a Sephadex G-25 (Pharmacia Biotech Inc., Uppsala, Sweden) column. Protein in this buffer is applied to the anti-FLAG M2 antibody affinity column. Non-binding protein is eluted by washing the column with 50 mM Tris-HCl (pH 7.5), 150 mM NaCI buffer. Bound protein is eluted using an excess of FLAG peptide in 50 mM Tris-HCl (pH 7.5), 150 mM NaCI. The excess FLAG peptide can be removed from the purified PAl 53 protein by gel electrophoresis or HPLC.
• plasmid 577 is utilized in this example, it is known to those skilled in the art that other comparable expression systems, such as CMV, can be utilized herein with appropriate modifications in reagent and/or techniques and are within the skill of the ordinary artisan.
• the largest cloned insert containing the coding region of the PAl 53 gene is then sub-cloned into either (i) a eukaryotic expression vector which may contain, for example, a cytomegalovirus (CMV) promoter and/or protein fusible sequences which aid in protein expression and detection, or (ii) a bacterial expression vector containing a superoxide-dismutase (SOD) and CMP-KDO synthetase (CKS) or other protein fusion gene for expression of the protein sequence.
• CMV cytomegalovirus
• SOD superoxide-dismutase
• CKS CMP-KDO synthetase
• This so-purified protein can be used in a variety of techniques, including, but not limited to animal immunization studies, solid phase immunoassays, etc.
• Example 1 lb Expression of Protein in a Cell Line Using pcDNA3.1/Mvc-His
• Plasmid pcDN A3.1/Myc- His (Cat.# V855-20, Invitrogen, Carlsbad, CA) has been constructed, in the past, for the expression of secreted antigens by most mammalian cell lines. Expressed protein inserts are fused to a myc-his peptide tag.
• the myc-his tag (SEQUENCE ID NO 34) comprises a c-myc oncoprotein epitope and a polyhistidine sequence which are useful for the purification of an expressed fusion protein by using either anti-myc or anti-his affinity columns, or metalloprotein binding columns.
• Plasmids for the expression of secretable PAl 53 proteins are constructed by inserting a PAl 53 polynucleotide sequence selected from the group consisting of SEQUENCE ID NOS 1-11, and fragments or complements thereof. Prior to construction of a PAl 53 expression plasmid, the PAl 53 cDNA sequence is first cloned into a pCR ® -Blunt vector as follows:
• the PAl 53 cDNA fragment is generated by PCR using standard procedures. For example, PCR is performed procedures and reagents from Stratagene ® , Inc. (La Jolla, CA), as directed by the manufacturer. PCR primers are used at a final concentration of 0.5 ⁇ M. PCR using 5 U of pfu polymerase (Stratagene, La Jolla, CA) is performed on the PAl 53 plasmid template (see Example 2) in a 50 ⁇ l reaction for 30 cycles (94°C, 1 min; 65°C, 1.5 min; 72°C, 3 min) followed by an extension cycle of 72°C for 8 min.
• the sense PCR primer sequence comprises nucleotides which are either complementary to the pINCY vector directly upstream of the PAl 53 gene insert or which incorporate a 5' EcoRI restriction site, an adjacent downstream protein translation consensus initiator, and a 3' nucleic acid sequence which is the same sense as the 5'-most end of the PAl 53 cDNA insert.
• the antisense PCR primer incorporates a 5' NotI restriction sequence and a sequence complementary to the 3' end of the PAl 53 cDNA insert just upstream of the 3'-most, in- frame stop codon.)
• Five micro liters (5 ⁇ l) of the resulting blunted-ended PCR product are ligated into 25 ng of linearized pCR ® -Blunt vector (Invitrogen, Carlsbad, CA) interrupting the lethal ccdB gene of the vector.
• the resulting ligated vector is transformed into TOP 10 R coli (Invitrogen, Carlsbad, CA) using a One ShotTM Transformation Kit (Invitrogen, Carlsbad, CA) following manufacturer's instructions.
• the transformed cells are grown on LB-Kan (50 ⁇ g/ml kanamycin) selection plates at 37°C Only cells containing a plasmid with an interrupted ccdB gene will grow after transformation rGrant. S.G.N.. Proc. Natl. Acad. Sci. USA 87:4645-4649 (1990)]. Transformed colonies are picked and grown up in 3 ml of LB-Kan broth at 37°C Plasmid DNA is isolated by using a QIAprep ® (Qiagen Inc., Santa Clarita, CA) procedure, as directed by the manufacturer. The DNA is cut with EcoRI or SnaBI, and NotI restriction enzymes to release the PAl 53 insert fragment.
• QIAprep ® Qiagen Inc., Santa Clarita, CA
• the fragment is run on 1% Seakem ® LE agarose/0.5 ⁇ g/ml ethidium bromide/TE gel, visualized by UV irradiation, excised and purified using QIAquickTM (Qiagen Inc., Santa Clarita, CA) procedures, as directed by the supplier's instructions.
• QIAquickTM Qiagen Inc., Santa Clarita, CA
• the pcDNA3.1/Myc-His plasmid DNA is linearized by digestion with EcoRI or SnaBI, and NotI in the polylinker region of the plasmid DNA.
• the resulting plasmid DNA backbone allows insertion of the PAl 53 purified cDNA fragment, supra, downstream of a CMV promoter which directs expression of the proteins in mammalian cells.
• the ligated plasmid is transformed into DH5 alphaTM cells (GibcoBRL Grand Island, NY), as directed by the manufacturer. Briefly, 10 ng of pcDNA3.1/Myc-His containing a PAl 53 insert are added to 50 ⁇ l of competent DH5 alpha cells, and the contents are mixed gently.
• the mixture is incubated on ice for 30 min, heat shocked for 20 sec at 37°C, and placed on ice for an additional 2 min.
• Upon addition of 0.95 ml of LB medium the mixture is incubated for 1 hr at 37°C while shaking at 225 rpm.
• the transformed cells then are plated onto 100 mm LB/ Amp (50 ⁇ g/ml ampicillin) plates and grown at 37°C Colonies are picked and grown in 3 ml of LB/Amp broth. Plasmid DNA is purified using a QIAprep Kit. The presence of the insert is confirmed using techniques known to those skilled in the art, including, but not limited to restriction digestion and gel analysis. (J. Sambrook et al., supra.)
• the PAl 53 expression plasmid described in section A, supra is retransformed into DH5 alpha cells, plated onto LB/ampicillin agar, and grown up in 10 ml of LB/ampicillin broth, as described hereinabove.
• the plasmid is purified using a QIAfilterTM Maxi Kit (Qiagen, Chatsworth, CA) and is transfected into HEK293 cells [F.L. Graham et al., Gen. Vir. 36:59-72 (1977)1. These cells are available from the A.T.C.C, 12301 Parklawn Drive, Rockville, MD 20852, under Accession No. CRL 1573.
• Transfection is carried out using the cationic lipofectamine-mediated procedure described by P. Hawley-Nelson et al., Focus 15.73 (1993). Particularly, HEK293 cells are cultured in 10 ml DMEM media supplemented with 10% fetal bovine serum (FBS), L-glutamine (2 mM) and freshly seeded into 100 mm culture plates at a density of 9 x 10 6 cells per plate. The cells are grown at 37 °C to a confluency of between 70% and 80% for transfection.
• FBS fetal bovine serum
• L-glutamine 2 mM
• Opti-MEM I ® medium (Gibco-BRL, Grand Island, NY)
• 48-96 ⁇ l of LipofectamineTM Reagent (Gibco-BRL, Grand Island, NY) are added to a second 800 ⁇ l portion of Opti-MEM I media.
• the two solutions are mixed and incubated at room temperature for 15-30 min. After the culture medium is removed from the cells, the cells are washed once with 10 ml of serum-free DMEM.
• the Opti- MEM I-Lipofectamine-plasmid DNA solution is diluted with 6.4 ml of serum-free DMEM and then overlaid onto the cells.
• the cells are incubated for 5 hr at 37°C, after which time, an additional 8 ml of DMEM with 20% FBS are added. After 18-24 hr, the old medium is aspirated, and the cells are overlaid with 5 ml of fresh DMEM with 5% FBS. Supernatants and cell extracts are analyzed for PAl 53 gene activity 72 hr after transfection.
• the expressed PAl 53 recombinant protein can be analyzed by mass spectrometry (see Example 12).
• D. Purification Purification of the PA153 recombinant protein containing the myc-his sequence is performed using the Xpress ® affinity chromatography system (Invitrogen, Carlsbad, CA) containing a nickel-charged agarose resin which specifically binds polyhistidine residues. Supernatants from 10 x 100 mm plates, prepared as described supra, are pooled and passed over the nickel-charged column. Non-binding protein is eluted by washing the column with 50 mM Tris-HCl (pH 7.5)/l 50 mM NaCI buffer, leaving only the myc-his fusion proteins.
• Bound PAl 53 recombinant protein then is eluted from the column using either an excess of imidazole or histidine, or a low pH buffer.
• the recombinant protein can also be purified by binding at the myc-his sequence to an affinity column consisting of either anti-myc or anti-histidine monoclonal antibodies conjugated through a hydrazide or other linkage to an agarose resin and eluting with an excess of myc peptide or histidine, respectively.
• the purified recombinant protein can then be covalently cross-linked to a solid phase, such as N-hydroxysuccinimide-activated sepharose columns (Pharmacia Biotech, Piscataway, NJ), as directed by supplier's instructions. These columns containing covalently linked PAl 53 recombinant protein, can then be used to purify anti-PA153 antibodies from rabbit or mouse sera (see Examples 13 and 14).
• a solid phase such as N-hydroxysuccinimide-activated sepharose columns (Pharmacia Biotech, Piscataway, NJ)
• pcDNA3.1/Myc-His is utilized in this example, it is known to those skilled in the art that other comparable expression systems can be utilized herein with appropriate modifications in reagent and/or techniques and are within the skill ofone of ordinary skill in the art.
• the largest cloned insert containing the coding region of the PAl 53 gene is sub-cloned into either (i) a eukaryotic expression vector which may contain, for example, a cytomegalovirus (CMV) promoter and/or protein fusible sequences which aid in protein expression and detection, or (ii) a bacterial expression vector containing a superoxide-dismutase (SOD) and CMP-KDO synthetase (CKS) or other protein fusion gene for expression of the protein sequence.
• CMV cytomegalovirus
• SOD superoxide-dismutase
• CKS CMP-KDO synthetase
• Example 12 Chemical Analysis of Pancreatic Tissue Proteins A. Analysis of Tryptic Peptide Fragments Using MS. Sera from patients with pancreatic disease, such as pancreatic cancer, sera from patients with no pancreatic disease, extracts of pancreatic tissues or cells from patients with pancreatic disease, such as pancreatic cancer, extracts of pancreatic tissues or cells from patients with no pancreatic disease, and extracts of tissues or cells from other non-diseased or diseased organs of patients are run on a polyacrylamide gel using standard procedures and stained with Coomassie Blue. Sections of the gel suspected of containing the unknown polypeptide are excised and subjected to an in-gel reduction, acetamidation and tryptic digestion. P.
• the supernatant is aspirated and replaced with 5 to 10 ⁇ l of digestion buffer without trypsin and allowed to incubate overnight at 37°C
• Peptides are extracted with 3 changes of 5% formic acid and acetonitrile and evaporated to dryness.
• the peptides are adsorbed to approximately 0.1 ⁇ l of POROS R2 sorbent (Perseptive Biosystems, Framingham, Massachusetts) trapped in the tip of a drawn gas chromatography capillary tube by dissolving them in 10 ⁇ l of 5% formic acid and passing it through the capillary.
• the adsorbed peptides are washed with water and eluted with 5% formic acid in 60% methanol.
• the eluant is passed directly into the spraying capillary of an API III mass spectrometer (Perkin-Elmer Sciex, Thornhill, Ontario, Canada) for analysis by nano-electrospray mass spectrometry.
• API III mass spectrometer Perkin-Elmer Sciex, Thornhill, Ontario, Canada
• the masses of the tryptic peptides are determined from the mass spectrum obtained off the first quadrupole. Masses corresponding to predicted peptides can be further analyzed in MS/MS mode to give the amino acid sequence of the peptide.
• the serum specimen or tumor extract from the patient is denatured with SDS and reduced with dithiothreitol (1.5 mg/ml) for 30 min at 90°C followed by alkylation with iodoacetamide (4 mg/ml) for 15 min at 25°C Following acrylamide electrophoresis, the polypeptides are electroblotted to a cationic membrane and stained with Coomassie Blue. Following staining, the membranes are washed and sections thought to contain the unknown polypeptides are cut out and dissected into small pieces.
• the membranes are placed in 500 ⁇ l microcentrifuge tubes and immersed in 10 to 20 ⁇ l of proteolytic digestion buffer (100 mM Tris-HCl, pH 8.2, containing 0.1 M NaCI, 10% acetonitrile, 2 mM CaCl 2 and 5 ⁇ g/ml trypsin) (Sigma, St. Louis, MO). After 15 hr at 37°C, 3 ⁇ l of saturated urea and 1 ⁇ l of 100 ⁇ g/ml trypsin are added and incubated for an additional 5 hr at 37°C The digestion mixture is acidified with 3 ⁇ l of 10% trifluoroacetic acid and centrifuged to separate supernatant from membrane.
• proteolytic digestion buffer 100 mM Tris-HCl, pH 8.2, containing 0.1 M NaCI, 10% acetonitrile, 2 mM CaCl 2 and 5 ⁇ g/ml trypsin
• the supernatant is injected directly onto a microbore, reverse phase HPLC column and eluted with a linear gradient of acetonitrile in 0.05% trifluoroacetic acid.
• the eluate is fed directly into an electrospray mass spectrometer, after passing though a stream splitter if necessary to adjust the volume of material.
• the data is analyzed following the procedures set forth in Example 12, Section A.
• Example 13 Gene Immunization Protocol A. In Vivo Antigen Expression. Gene immunization circumvents protein purification steps by directly expressing an antigen in vivo after inoculation of the appropriate expression vector. Also, production of antigen by this method may allow correct protein folding and glycosylation since the protein is produced in mammalian tissue.
• the method utilizes insertion of the gene sequence into a plasmid which contains a CMV promoter, expansion and purification of the plasmid and injection of the plasmid DNA into the muscle tissue of an animal. Preferred animals include mice and rabbits. See, for example, H. Davis et al., Human Molecular Genetics 2:1847- 1851 (1993). After one or two booster immunizations, the animal can then be bled, ascites fluid collected, or the animal's spleen can be harvested for production of hybridomas.
• PAl 53 cDNA sequences are generated from the PAl 53 cDNA-containing vector using appropriate PCR primers containing suitable 5' restriction sites following the procedures described in Example 11.
• the PCR product is cut with appropriate restriction enzymes and inserted into a vector which contains the CMV promoter (for example, pRc/CMV or pcDNA3 vectors from Invitrogen, San Diego, CA).
• This plasmid then is expanded in the appropriate bacterial strain and purified from the cell lysate using a CsCl gradient or a Qiagen plasmid DNA purification column. All these techniques are familiar to one of ordinary skill in the art of molecular biology.
• Antiserum against PAl 53 was prepared by injecting rabbits with peptides whose sequences were derived from that of the predicted amino acid sequence of the PAl 53 consensus nucleotide sequence (SEQUENCE ID NO 11). The synthesis of peptides (SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31, and SEQUENCE ID NO 32) is described in Example 10. Peptides used as immunogens were not conjugated to a carrier such as keyhole limpet hemocyanine, KLH, (i.e., they were unconjugated.). Animal Immunization. Female white New Zealand rabbits weighing 2 kg or more were used for raising polyclonal antiserum.
• KLH keyhole limpet hemocyanine
• One animal was immunized per unconjugated peptide (SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31, and SEQUENCE ID NO 32).
• SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31, and SEQUENCE ID NO 32 One week prior to the first immunization, 5 to 10 ml of blood were obtained from the animal to serve as a non-immune prebleed sample.
• Unconjugated peptides SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31, and SEQUENCE ID NO 32, were used to prepare the primary immunogen by emulsifying 0.5 ml of the peptide at a concentration of 2 mg/ml in PBS (pH 7.2) which contained 0.5 ml of complete Freund's adjuvant (CFA) (Difco, Detroit, MI).
• CFA complete Freund's adjuvant
• the immunogen was injected into several sites of the animal via subcutaneous, intraperitoneal, and intramuscular routes of administration. Four weeks following the primary immunization, a booster immunization was administered.
• the immunogen used for the booster immunization dose was prepared by emulsifying 0.5 ml of the same unconjugated peptide used for the primary immunogen, except that the peptide now was diluted to 1 mg/ml with 0.5 ml of incomplete Freund's adjuvant (IF A) (Difco, Detroit, MI). Again, the booster dose was administered into several sites via subcutaneous, intraperitoneal and intramuscular types of injections. The animals were bled (5 ml) two weeks after the booster immunizations and each serum was tested for immunoreactivity to the peptide as described below. The booster and bleed schedule was repeated at 4 week intervals until an adequate titer was obtained.
• IF A incomplete Freund's adjuvant
• the titer or concentration of antiserum was determined by using unconjugated peptides in a microtiter EIA as described in Example 17, below. An antibody titer of 1 :500 or greater was considered an adequate titer for further use and study. Table 1. Titer of rabbit anti-PA153 peptide antisera (11 week bleed)
• mice are immunized using peptides which can either be conjugated to a carrier such as KLH [prepared as described hereinbelow, or unconjugated (i.e., not conjugated to a carrier such as KLH)] except that the amount of the unconjugated or conjugated peptide for monoclonal antibody production in mice is one-tenth the amount used to produce polyclonal antisera in rabbits.
• a carrier such as KLH [prepared as described hereinbelow, or unconjugated (i.e., not conjugated to a carrier such as KLH)] except that the amount of the unconjugated or conjugated peptide for monoclonal antibody production in mice is one-tenth the amount used to produce polyclonal antisera in rabbits.
• the primary immunogen consists of 100 ⁇ g of unconjugated or conjugated peptide in 0.1 ml of CFA emulsion while the immunogen used for booster immunizations consists of 50 ⁇ g of unconjugated or conjugated peptide in 0.1 ml of IF A.
• Hybr ⁇ domas for the generation of monoclonal antibodies are prepared and screened using standard techniques. The methods used for monoclonal antibody development follow procedures known in the art such as those detailed in Kohler and Milstein, Nature 256:494 (1975) and reviewed in J.G.R. Hurrel, ed., Monoclonal Hybridoma Antibodies: Techniques and Applications. CRC Press, Inc., Boca Raton, FL (1982).
• the immunization regimen (per mouse) consists of a primary immunization with additional booster immunizations.
• the primary immunogen used for the primary immunization consists of 100 ⁇ g of unconjugated or conjugated peptide in 50 ⁇ l of PBS (pH 7.2) previously emulsified in 50 ⁇ l of CFA.
• Booster immunizations performed at approximately two weeks and four weeks post primary immunization consist of 50 ⁇ g of unconjugated or conjugated peptide in 50 ⁇ l of PBS (pH 7.2) emulsified with 50 ⁇ l IFA. A total of 100 ⁇ l of this immunogen are inoculated intraperitoneally and subcutaneously into each mouse. Individual mice are screened for immune response by microtiter plate enzyme immunoassay (EIA) as described in Example 17 approximately four weeks after the third immunization.
• EIA microtiter plate enzyme immunoassay
• mice are inoculated either intravenously, intrasplenically or intraperitoneally with 50 ⁇ g of unconjugated or conjugated peptide in PBS (pH 7.2) approximately fifteen weeks after the third immunization.
• PBS PEG
• splenocytes are fused with, for example, Sp2/0-Agl4 myeloma cells (Milstein Laboratories, England) using the polyethylene glycol (PEG) method.
• PEG polyethylene glycol
• the fusions are cultured in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% fetal calf serum (FCS), plus 1% hypoxanthine, aminopterin and thymidine (HAT).
• IMDM Iscove's Modified Dulbecco's Medium
• FCS fetal calf serum
• HAT hypoxanthine
• HAT thymidine
• Imject ® contains about 250 moles of reactive maleimide groups per mole of hemocyanine.
• the activated KLH is dissolved in phosphate buffered saline (PBS, pH 8.4) at a concentration of about 7.7 mg/ml.
• PBS phosphate buffered saline
• the peptide is conjugated through cysteines occurring in the peptide sequence, or to a cysteine previously added to the synthesized peptide in order to provide a point of attachment.
• the peptide is dissolved in DMSO (Sigma Chemical Company, St.
• conjugation reaction described hereinbelow is based on obtaining 3 mg of KLH peptide conjugate ("conjugated peptide"), which contains about 0.77 ⁇ moles of reactive maleimide groups. This quantity of peptide conjugate usually is adequate for one primary injection and four booster injections for production of polyclonal antisera in a rabbit.
• conjugated peptide usually is adequate for one primary injection and four booster injections for production of polyclonal antisera in a rabbit.
• peptide is dissolved in DMSO at a concentration of 1.16 ⁇ moles/100 ⁇ l of DMSO.
• One hundred microliters (100 ⁇ l) of the DMSO solution are added to 380 ⁇ l of the activated KLH solution prepared as described hereinabove, and 20 ⁇ l of PBS (pH 8.4) are added to bring the volume to 500 ⁇ l.
• the reaction is incubated overnight at room temperature with stirring.
• the extent of reaction is determined by measuring the amount of unreacted thiol in the reaction mixture.
• the difference between the starting concentration of thiol and the final concentration is assumed to be the concentration of peptide which has coupled to the activated KLH.
• the amount of remaining thiol is measured using Ellman's reagent (5,5'-dithiobis(2- nitrobenzoic acid), Pierce Chemical Company, Rockford, IL).
• Cysteine standards are made at a concentration of 0, 0.1, 0.5, 2, 5 and 20 mM by dissolving 35 mg of cysteine HCl (Pierce Chemical Company, Rockford, IL) in 10 ml of PBS (pH 7.2) and diluting the stock solution to the desired concentration(s).
• the photometric determination of the concentration of thiol is accomplished by placing 200 ⁇ l of PBS (pH 8.4) in each well of an Immulon 2 ® microwell plate (Dynex Technologies, Chantilly, VA). Next, 10 ⁇ l of standard or reaction mixture are added to each well. Finally, 20 ⁇ l of Ellman's reagent at a concentration of 1 mg/ml in PBS (pH 8.4) are added to each well.
• the wells are incubated for 10 minutes at room temperature, and the absorbance of all wells is read at 415 nm with a microplate reader (such as the BioRad Model 3550, BioRad, Richmond, CA).
• the absorbance of the standards is used to construct a standard curve and the thiol concentration of the reaction mixture is determined from the standard curve. A decrease in the concentration of free thiol is indicative of a successful conjugation reaction.
• Unreacted peptide is removed by dialysis against PBS (pH 7.2) at room temperature for 6 hours.
• the conjugate is stored at 2-8°C if it is to be used immediately; otherwise, it is stored at -20°C or colder. 3. Production of Ascites Fluid Containing Monoclonal Antibodies.
• Frozen hybridoma cells prepared as described hereinabove are thawed and placed into expansion culture.
• Viable hybridoma cells are inoculated intraperitoneally into Pristane treated mice.
• Ascitic fluid is removed from the mice, pooled, filtered through a 0.2 ⁇ filter and subjected to an immunoglobulin class G (IgG) analysis to determine the volume of the Protein A column required for the purification.
• IgG immunoglobulin class G
• the immunoreactivity of the purified monoclonal antibody is confirmed by determining its ability to specifically bind to the peptide used as the immunogen by use of the EIA microtiter plate assay procedure of Example 17.
• the specificity of the purified monoclonal antibody is confirmed by determining its lack of binding to i ⁇ elevant peptides such as peptides of PAl 53 not used as the immunogen.
• the purified anti-PA153 monoclonal thus prepared and characterized is placed at either 2-8°C for short term storage or at -80°C for long term storage. 5. Further Characterization of Monoclonal Antibody.
• the isotype and subtype of the monoclonal antibody produced as described hereinabove can be determined using commercially available kits (available from Amersham.
• Stability testing also can be performed on the monoclonal antibody by placing an aliquot of the monoclonal antibody in continuous storage at 2- 8°C and assaying optical density (OD) readings throughout the course of a given period of time.
• OD optical density
• Immune sera obtained as described hereinabove in Examples 13 and/or 14, is affinity purified using immobilized synthetic peptides prepared as described in Example 10, or recombinant proteins prepared as described in Example 11.
• An IgG fraction of the antiserum is obtained by passing the diluted, crude antiserum over a Protein A column (Affi-Gel protein A, Bio-Rad, Hercules, CA). Elution with a buffer (Binding Buffer, supplied by the manufacturer) removes substantially all proteins that are not immunoglobulins. Elution with 0.1M buffered glycine (pH 3) gives an immunoglobulin preparation that is substantially free of albumin and other serum proteins.
• Immunoaffinity chromatography is performed to obtain a preparation with a higher fraction of specific antigen-binding antibody.
• the peptide used to raise the antiserum is immobilized on a chromatography resin, and the specific antibodies directed against its epitopes are adsorbed to the resin. After washing away non- binding components, the specific antibodies are eluted with 0.1 M glycine buffer, pH 2.3. Antibody fractions are immediately neutralized with 1.0M Tris buffer (pH 8.0) to preserve immunoreactivity.
• the chromatography resin chosen depends on the reactive groups present in the peptide.
• a resin such as Affi-Gel 10 or Affi-Gel 15 is used (Bio-Rad, Hercules, CA). If coupling through a carboxy group on the peptide is desired, Affi-Gel 102 can be used (Bio-Rad, Hercules, CA). If the peptide has a free sulfhydryl group, an organomercurial resin such as Affi-Gel 501 can be used (Bio-Rad, Hercules, CA). Alternatively, spleens can be harvested and used in the production of hybridomas to produce monoclonal antibodies following routine methods known in the art as described hereinabove.
• Example 16 Western Blotting of Tissue Samples Protein extracts were prepared by homogenizing tissue samples in a solution containing 0.1M Tris-HCl (pH 7.5), 15% (w/v) glycerol, 0.2 mM EDTA, 1.0 mM 1,4- dithiothreitol, 10 ⁇ g/ml leupeptin, 1.0 mM phenylmethylsulfonylfluoride and 0.1% Triton X-100 (S. R. Kain et al., Biotechniques 17:982 (1994). Following homogenization, the homogenates were centrifuged at 4°C for 5 minutes to separate supernatant from debris. For protein quantitation, 3-10 ⁇ l of supernatant were added to 1.5 ml of bicinchoninic acid reagent (Sigma, St. Louis, MO), and the resulting absorbance at 562 nm was measured.
• Tris-HCl pH 7.5
• 0.2 mM EDTA
• the bands were visualized directly on the membranes by the addition and development of chromogenic substrate 5-bromo-4- chloro-3-indolyl phosphate (BCIP).
• BCIP chromogenic substrate 5-bromo-4- chloro-3-indolyl phosphate
• This chromogenic solution contains 0.016% BCIP in a solution containing 100 mM NaCI, 5 mM MgCl2 and 100 mM Tris-HCl, pH 9.5.
• the filter was incubated in this solution at room temperature until the bands developed to the desired intensity.
• Molecular mass determination was made based upon the mobility of pre-stained molecular weight standards (Novex, San Diego, CA) and biotinylated molecular weight standards (Tropix, Bedford, MA).
• FIG 4 shows the results of the Western blot performed on a panel of tissue extracts using antiserum against synthetic peptide, SEQUENCE ID NO 32 (see Example 14).
• Each lane of Figure 4 represents a different tissue protein extract: 1, lung cancer; 2, ovarian cancer; 3, normal bladder; 4, normal colon; 5, breast cancer; 6, normal pancreas; 7, pancreatic cancer, 8 and 9, normal pancreas; 10, molecular weight markers (kD).
• kD molecular weight markers
• Broad, high molecular weight bands between 116 kD and 200 kD were observed with the pancreatic cancer protein extract (arrows, lane 7). These high molecular weight bands were not observed with any of the other tissue protein extracts (lanes 1-6, 8-9).
• Example 17 EIA Microtiter Plate Assay The immunoreactivity of antiserum preferably obtained from rabbits as described in Example 14 was determined by means of a microtiter plate EIA, as follows. Briefly, synthetic peptides, SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31, and SEQUENCE ID NO 32, prepared as described in Example 10, were dissolved in carbonate buffer (50 mM, pH 9.6) to a final concentration of 2 ⁇ g/ml. Next, 100 ⁇ l of the peptide or protein solution were placed in each well of an Immulon 2 ® microtiter plate (Dynex Technologies, Chantilly, VA).
• the plate was incubated overnight at room temperature and then washed four times with deionized water.
• the wells were blocked by adding 125 ⁇ l of a suitable protein blocking agent, such as Superblock ® (Pierce Chemical Company, Rockford, IL), to each well and then immediately discarding the solution. This blocking procedure was performed three times.
• Antiserum obtained from immunized rabbits or mice, prepared as previously described, was diluted in a protein blocking agent (e.g., a 3% Superblock ® solution) in PBS containing 0.05% Tween-20 ® (monolaurate polyoxyethylene ether) (Sigma Chemical Company, St.
• Example 18 Coating of Solid Phase Particles A. Coating of Microparticles with Antibodies Which Specifically Bind to
• PAl 53 Antigen Affinity purified antibodies which specifically bind to PAl 53 protein (see Example 15) are coated onto microparticles of polystyrene, carboxylated polystyrene, polymethylacrylate or similar particles having a radius in the range of about 0.1 to 20 ⁇ m. Microparticles may be either passively or actively coated.
• One coating method comprises coating ED AC (l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (Aldrich Chemical Co., Milwaukee, WI) activated carboxylated latex microparticles with antibodies which specifically bind to PAl 53 protein, as follows.
• a final 0.375% solid suspension of resin washed carboxylated latex microparticles (available from Bangs Laboratories, Carmel, IN or Serodyn, Indianapolis, IN) are mixed in a solution containing 50 mM MES buffer, pH 4.0 and 150 mg/1 of affinity purified anti-PA153 antibody (see Example 14) for 15 min in an appropriate container.
• ED AC coupling agent is added to a final concentration of 5.5 ⁇ g/ml to the mixture and mixed for 2.5 hr at room temperature.
• the microparticles then are washed with 8 volumes of a Tween 20 ® /sodium phosphate wash buffer (pH 7.2) by tangential flow filtration using a 0.2 ⁇ m Microgon Filtration module. Washed microparticles are stored in an appropriate buffer which usually contains a dilute surfactant and irrelevant protein as a blocking agent, until needed.
• Antibodies which specifically bind to PAl 53- antigen also may be coated on the surface of 1/4 inch polystyrene beads by routine methods known in the art (Snitman et al., US Patent 5,273,882) and used in competitive binding or EIA sandwich assays.
• Polystyrene beads first are cleaned by ultrasonicating them for about 15 seconds in 10 mM NaHCO3 buffer at pH 8.0. The beads then are washed in deionized water until all fines are removed. Beads then are immersed in an antibody solution in 10 mM carbonate buffer, pH 8 to 9.5.
• the antibody solution can be as dilute as 1 ⁇ g/ml in the case of high affinity monoclonal antibodies or as concentrated as about 500 ⁇ g/ml for polyclonal antibodies which have not been affinity purified.
• Beads are coated for at least 12 hours at room temperature, and then they are washed with deionized water. Beads may be air dried or stored wet (in PBS, pH 7.4). They also may be overcoated with protein stabilizers (such as sucrose) or protein blocking agents used as non-specific binding blockers (such as irrelevant proteins, Carnation skim milk, Superblock ® , or the like).
• Example 19 Microparticle Enzyme Immunoassay (MEIA) PAl 53 antigens are detected in patient test samples by performing a standard antigen competition EIA or antibody sandwich EIA and utilizing a solid phase such as microparticles (MEIA). The assay can be performed on an automated analyzer such as the IMx ® Analyzer (Abbott Laboratories, Abbott Park, IL).
• A. Antibody Sandwich EIA Briefly, samples suspected of containing PAl 53 antigen are incubated in the presence of anti-PA153 antibody-coated microparticles (prepared as described in Example 17) in order to form antigen/antibody complexes.
• microparticles then are washed and an indicator reagent comprising an antibody conjugated to a signal generating compound (i.e., enzymes such as alkaline phosphatase or horseradish peroxide) is added to the antigen/antibody complexes or the microparticles and incubated.
• a signal generating compound i.e., enzymes such as alkaline phosphatase or horseradish peroxide
• the microparticles are washed and the bound antibody/antigen/antibody complexes are detected by adding a substrate (e.g., 4-methyl umbelliferyl phosphate (MUP), or OPD/peroxide, respectively), that reacts with the signal generating compound to generate a measurable signal.
• MUP 4-methyl umbelliferyl phosphate
• OPD/peroxide respectively
• the presence of PA153 antigen in the test sample is indicative of a diagnosis of a pancreatic disease or condition, such as pancreatic cancer.
• B. Competitive Binding Assay uses a peptide or protein that generates a measurable signal when the labeled peptide is contacted with an anti-peptide antibody coated microparticle. This assay can be performed on the IMx ® Analyzer (available from Abbott Laboratories, Abbott Park, IL).
• the labeled peptide is added to the PAl 53 antibody-coated microparticles (prepared as described in Example 17) in the presence of a test sample suspected of containing PAl 53 antigen, and incubated for a time and under conditions sufficient to form labeled PAl 53 peptide (or labeled protein) / bound antibody complexes and/or patient PAl 53 antigen / bound antibody complexes.
• the PAl 53 antigen in the test sample competes with the labeled PAl 53 peptide (or PAl 53 protein) for binding sites on the microparticle.
• PAl 53 antigen in the test sample results in a lowered binding of labeled peptide and antibody coated microparticles in the assay since antigen in the test sample and the PAl 53 peptide or PAl 53 protein compete for antibody binding sites.
• a lowered signal indicates the presence of PA153 antigen in the test sample.
• the presence of PAl 53 antigen suggests the diagnosis of a pancreatic disease or condition, such as pancreatic cancer.
• the PAl 53 polynucleotides and the proteins encoded thereby which are provided and discussed hereinabove are useful as markers of pancreatic tissue disease, especially pancreatic cancer. Tests based upon the appearance of this marker in a test sample such as blood, plasma or serum can provide low cost, non-invasive, diagnostic information to aid the physician to make a diagnosis of cancer, to help select a therapy protocol, or to monitor the success of a chosen therapy.
• This marker may appear in readily accessible body fluids such as blood, urine or stool as antigens derived from the diseased tissue which are detectable by immunological methods. This marker may be elevated in a disease state, altered in a disease state, or be a normal protein of the pancreatic which appears in an inappropriate body compartment.
• Antiserum against a PAl 53 synthetic peptide derived from the consensus peptide sequence (SEQUENCE ID NO 28) described in Example 14, above, is used to immunohistochemically stain a variety of normal and diseased tissues using standard proceedures. Briefly, frozen blocks of tissue are cut into 6 micron sections, and placed on microscope slides. After fixation in cold acetone, the sections are dried at room temperature, then washed with phosphate buffered saline and blocked.
• the slides are incubated with the antiserum against a synthetic peptide derived from the consensus PAl 53 peptide sequence (SEQUENCE ID NO 28) at a dilution of 1:500, washed, incubated with biotinylated goat anti-rabbit antibody, washed again, and incubated with avidin labeled with horseradish peroxidase. After a final wash, the slides are incubated with 3-amino-9-ethylcarbazole substrate which gives a red stain. The slides are counterstained with hematoxylin, mounted, and examined under a microscope by a pathologist.

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