WO2000050457A1 - Antigenes d'enveloppe prostatique specifique, ses procedes de production et d'utilisation - Google Patents

Antigenes d'enveloppe prostatique specifique, ses procedes de production et d'utilisation Download PDF

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Publication number
WO2000050457A1
WO2000050457A1 PCT/US1999/026844 US9926844W WO0050457A1 WO 2000050457 A1 WO2000050457 A1 WO 2000050457A1 US 9926844 W US9926844 W US 9926844W WO 0050457 A1 WO0050457 A1 WO 0050457A1
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Prior art keywords
monoclonal antibody
psm
psma
ppid
amount
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PCT/US1999/026844
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English (en)
Inventor
Lana S. Grauer
Kristine Kuus-Reichel
Roger Sokoloff
Mohammad S. Saedi
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Beckman Coulter, Inc.
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Priority claimed from PCT/US1999/003810 external-priority patent/WO1999043710A1/fr
Application filed by Beckman Coulter, Inc. filed Critical Beckman Coulter, Inc.
Publication of WO2000050457A1 publication Critical patent/WO2000050457A1/fr

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    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • the invention relates generally to prostate cancer-related proteins and specifically to the identification and purification of an alternatively spliced variant of prostate-specific membrane antigen (PSMA) called PSM' antigen.
  • PSMA prostate-specific membrane antigen
  • it relates to antibodies which recognize such antigens.
  • it relates to methods for producing such antibodies and diagnostic uses therefor.
  • Prostate cancer is the second leading cause of cancer death in men (1), being especially prevalent among men over the age of 50. There are approximately 73,000 new cases of prostate carcinoma each year, and approximately 23,000 deaths per year. The importance of early diagnosis is readily apparent by the statistic that the five-year survival rate in patients with localized prostate tumors is twice as high as those with disseminated cancer. Additionally, the detection of recurrences prior to the development of subjective symptoms or clinical manifestations of the disease, and the monitoring and evaluation of therapy, are clearly important to an improved prognosis. The identification of reliable prostate cancer markers, therefore, contributes significantly to early diagnosis as well as staging, assessing and monitoring the disease.
  • biomarkers have been used for screening, diagnosis and predicting disease progression (2, 3).
  • biomarkers include prostate specific antigen (PSA) and prostatic acid phosphatase (PAP).
  • PSA prostate specific antigen
  • PAP prostatic acid phosphatase
  • the relative amount of PSA and/or PAP in prostatic cancer is reduced as compared to normal or benign tissue. Therefore, the measurement of serum PSA is suggested as a potential screening method for prostatic cancer.
  • PSMA prostate specific membrane antigen
  • a radioimmunoconjugate of the 7E11-C5 antibody, designated CYT356, is currently being used as an imaging agent for prostate cancer (8).
  • the cDNA coding for PSMA was obtained from a LNCaP cDNA library (9). It coded for a putative type -3 transmembrane protein consisting of a short intracellular segment (amino acids 1-18), a transmembrane domain (amino acids 19-43) and an extensive extracellular domain (amino acids 44-750). The extracellular domain contains a region with 54% homology to the transferrin receptor.
  • PSMA and PSM' cDNAs are identical except for a 266-nucleotide region near the 5' end of PSMA cDNA (nucleotides 114-380) that is absent from PSM'.
  • the absent region includes the translation initiation codon and codons for the putative transmembrane domain of PSMA.
  • the protein product of PSM' would therefore lack the transmembrane domain of PSMA as well the signal sequence of PSMA. The location of the protein in the cell would probably be cytoplasmic because of the omission of the transmembrane domain.
  • PSMA is the dominant form. In normal human prostate, however, more PSM' is expressed than PSMA. Benign prostatic hypertrophy samples showed about equal expression of both variants (10).
  • the ratio of PSMA:PSM' level also called a tumor index
  • the index reflects the increased expression of PSMA over PSM' following the progression from normal to tumor state. This tumor index may be a useful indicator for the measurement of tumor progression.
  • a purified and isolated human PSM' protein which is substantially free of other human proteins.
  • the N- terminal amino acid sequence of the PSM' protein is two amino acids short compared to the N-terminal amino acid sequence of the putative human PSM' protein.
  • the present invention also provides a method of producing human PSM' from a cell line containing both PSMA and PSM'. The method includes the steps of: (a) preparing a cell lysate from the cell line, (b) removing PSMA from the cell lysate, (c) absorbing PSM' from the cell lysate onto a solid phase bound to a monoclonal antibody specific for PSMA and PSM', and (d) recovering PSM' from the solid phase.
  • a monoclonal antibody or a fragment thereof recognizing the PSM' protein of the present invention.
  • a polyclonal antibody that recognizes the PSM' protein of the present invention.
  • the present invention also provides a monoclonal antibody or a fragment thereof recognizing both PSM' and PSMA. Monoclonal antibodies that are specific for PSM' are also provided.
  • the present invention further provides a method for making a monoclonal antibody that is specific for PSM' protein.
  • the method comprises the steps of : (a) synthesizing a peptide composed of the first 15 to 20 amino acids of PSM' protein with an added Cys at the C-terminus,
  • step (c) immunizing a mouse or a suitable host with the conjugates of step (b), (d) fusing spleen cells of the immunized mouse or other suitable host with suitable myeloma cells, thereby obtaining a mixture of hybrid cell lines,
  • an immunoassay for determining the PSM' in a sample comprising the steps of:
  • Yet another aspect of the present invention provides a method for determining PSM' in a sample which contains both PSM' and PSMA. In accordance with one embodiment of the present invention, the method comprises the steps of:
  • a further aspect of the present invention provides a method for predicting prostate cancer progression.
  • the method comprises the steps of : (a) providing a first monoclonal antibody which recognizes both PSM' and
  • the first monoclonal antibody binds to either the PSM' or the PSMA of the sample
  • a method for predicting prostate cancer progression in a sample containing both PSM' and PSMA by using antibodies of the present invention that are specific for PSM' protein includes the steps of :
  • kits for determining the prostate cancer progression includes carrier means compartmentalized to receive in close confinement therein one or more containers comprising a container containing a monoclonal antibody that recognizes PSM'.
  • the kit includes another container which contains a monoclonal antibody that recognizes PSMA.
  • One aspect of the present invention provides a method for the detection of cancer in a suspected cancer patient. In accordance with one embodiment of the present invention, the method includes contacting a tissue specimen obtained from the patient with an antibody recognizing PSM', and determining the sites on the specimen to which the antibody is bound by immunohistochemical means.
  • Another aspect of the present invention provides a method for the in vivo diagnosis of prostate cancer in a suspected cancer patient.
  • the method comprises administering a predetermined diagnostic effective amount of an antibody recognizing the PSM' of the present invention and detecting the sites of localization of the antibody, the antibody being administered in a pharmaceutically acceptable carrier and labeled so as to permit detection.
  • a further aspect of the present invention provides a method of treating prostate cancer in a cancer patient, comprising administering a predetermined effective amount of an antibody recognizing the 'PSM protein of the present invention, the antibody being administered in a pharmaceutically acceptable carrier and conjugated with a suitable therapeutic agent.
  • FIG. 1 shows the mapping of PSMA monoclonal antibodies 7E11 and PEQ226 to PSMA and PSM'.
  • FIG. 2 is a western blot showing the reactivity of PSMA Mab PEQ226 with GST- PSMA fusion proteins.
  • FIG. 3 shows the western blot analysis of the enrichment of PSM' from LNCaP cell lysate using immunoaffinity resins.
  • FIG. 4 shows the N-terminal amino acid sequence of PSM' isolated from LNCaP cells.
  • FIG. 5 is a western blot which demonstrates that PSM' is located in the cytoplasm of
  • FIG. 6 is a diagram which shows a PSM' mammalian expression vector.
  • FIG. 7 shows the results of a western bot analysis of AN12-PSM' spent media.
  • FIGS. 8 is a diagrams which shows antigens that are used for screening PSM' specific antibody.
  • FIGS. 9(A) and (B) show the serum titers of PSM' peptide immunization.
  • FIG. 10 shows the results of silver stain (FIG. 10 (A)) and western blot (FIG. 10 (B)) of purified recombinant PSM'.
  • FIG. 11(A) shows the serum titers of recombinant PSM' immunization to PSM' peptide 1-15
  • FIG. 11(B) shows the serum titers of recombinant PSM' immunization to recombinant PSM'.
  • the present invention provides a purified and isolated human PSM' protein which is substantially free of other human proteins.
  • the PSM' protein is considered substantially free from other human proteins if the protein yields a single major band on a non-reducing polyacrylamide gel.
  • the purity of the PSM' protein can also be determined by amino- terminal amino acid sequence analysis, which analysis is well known in the art.
  • the human PSM' protein has an N-terminal sequence as indicated in FIG. 4. It is noted that the N-terminal sequence of the PSM' protein differs from the predicted N-terminal sequence of the putative PSM' protein. While the putative translation initiation site for PSM' was identified as residue
  • the human PSM' protein of the present invention can be prepared in accordance with a method of the present invention.
  • One embodiment of the present invention provides a method of producing human PSM' from a cell line containing both PSMA and PSM'. The method includes the steps of:
  • a cell line can be any human cell lines that contain both PSMA and PSM' proteins. Examples of such a cell line include, but are not limited to, LNCaP cell line, and the like. Cell lysates can be prepared from a cell line of the present invention using conventional procedures.
  • PSMA can be removed from a cell lysate by absorbing PSMA from the cell lysate onto a solid phase bound to a monoclonal antibody specific for PSMA.
  • monoclonal antibodies specific for PSMA include, but are not limited to, 7E11 monoclonal antibody, and the like.
  • monoclonal antibodies recognizing PSM' protein include monoclonal antibodies that are specific for PSM' or for both PSMA and PSM', as long as the monoclonal antibodies recognize PSM'.
  • the term "specific for PSM'” as used herein means that the monoclonal antibody only recognizes PSM', not PSMA.
  • the term "specific for both PSM' and PSMA” as used herein means that the monoclonal antibody can recognize both PSM' and PSMA.
  • the human PSM' protein of the present invention may also be prepared by recombinant expression technologies that are known in the art. Briefly, the DNA encoding PSM' may be cloned into an expression vector and expressed in a mammalian host cell.
  • the DNA is cloned downstream of a secretion signal contained in the expression vector so that the expressed protein may be secreted into the spent media.
  • One aspect of the present invention provides antibodies or fragments thereof that recognize PSMA and PSM'.
  • Antibodies which consist essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations, are provided.
  • Examples of monoclonal antibodies that recognize both PSMA and PSM' include, but are not limited to, PM1T485.5, PM2H043, PM2H207, PEQ226, PMlx310.5, PM2E343 and PM2E086.
  • Monoclonal antibodies that recognize both PSMA and PSM' proteins can be generated by methods known to one skilled in the art (Kohler et al., Nature, 256:495, 1975).
  • antigens that may be used to generate the monoclonal antibodies that recognize both PSMA and PSM' include, but are not limited to, plasma membranes from LNCaP cells or from prostate cancer patients, PSMA protein, and PSMA protein fused to a carrier protein such as complete Freunds adjuvant (CFA), PSMA synthetic peptides and PSMA fusion proteins.
  • CFA complete Freunds adjuvant
  • Another aspect of the present invention provides an antibody or a fragment thereof that binds to the PSM' protein of the present invention, but not to PSMA proteins.
  • Antibodies which consist essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations, are provided.
  • antibody as used in this invention is meant to include intact molecules as well as fragments thereof, such as, but not limited to, Fab, Fab', F(ab') 2 and Fv, etc., which are capable of binding an epitopic determinant on a PSMA or PSM' protein. These antibody fragments retain some ability to selectively bind with their antigens or receptors and are defined as follows:
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of the whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
  • Fab' the fragment of an antibody molecule
  • pepsin the fragment of an antibody molecule
  • F(ab') the fragment of an antibody, can be obtained by treating the whole antibody with the enzyme pepsin without subsequent reduction;
  • F(ab') 2 is a dimer of two Fab' fragments held together by two disulfide bonds;
  • Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains, can be obtained by genetic engineering methods.
  • Single chain antibody defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule, can be obtained by genetic engineering methods. Methods of making these fragments are known in the art. (See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York
  • epitopic determinants means any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of
  • Monoclonal antibodies that are specific for PSM' may be made from an antigen containing PSM' protein or fragments thereof by methods well known to those skilled in the art (Kohler et al., Nature, 256:495, 1975).
  • Antigens that may be used to generate monoclonal antibodies that recognize PSM' but not PSMA include, but are not limited to, PSM' protein, recombinant PSM' protein, and PSM' peptides or a fragment thereof fused to a carrier protein.
  • a synthesized peptide composed of the first 15-20 amino acids of PSM' may be used as an antigen for producing monoclonal antibodies that are specific for PSM'.
  • Cys is added to the peptide.
  • One example of such a synthesized peptide has an amino acid sequence of :
  • a mouse or other suitable host is immunized with the synthesized peptides of the present invention.
  • the peptides are conjugated to a carrier protein, such as KLH, prior to immunization.
  • the spleen cells of the immunized mouse are fused with the cells from a suitable mouse myeloma line to obtain a mixture of hybrid cell lines.
  • the hybrid cell lines are cultured in a suitable medium and, thereafter, hybrid cell lines producing an antibody having a specific reactivity with the PSM' protein of the present invention are selected and cloned, and the monoclonal antibodies thus produced are recovered.
  • a method of making monoclonal antibodies of the present invention also includes a step of screening for monoclonal antibodies that recognize PSM', but not PSMA.
  • a monoclonal antibody that is specific for PSM', not PSMA antibodies produced by hybrid cell lines will be tested on an identical peptide that is used as the antigen. Those antibodies that test positive will also be tested on the sample peptide but with the peptide' s N-terminus alanine blocked with His-Asn-Met.
  • the amino acid sequence of the peptide with a blocked N-terminus is:
  • Clones that fail to detect the N-terminus blocked peptide but are able to detect the PSM' peptide are saved and tested on PSMA and PSM' proteins to confirm their specificity to the PSM' protein, not the PSMA protein.
  • Monoclonal antibodies of the present invention may also be generated by immunizing a mouse or other suitable host with a recombinant PSM' protein using standard techniques.
  • the generated monoclonal antibodies are called PSM' recombinant monoclonal antibodies.
  • the generated monoclonal antibodies are then screened against PSM' protein, PSMA protein, and other suitable peptides to ensure their specificity to PSM' protein.
  • Examples of monoclonal antibodies that recognize PSM', but not PSMA include, but are not limited to, PP1D 329, PP1D 449, PP1D 603, PP1D 423 and PP1D 559.
  • monoclonal antibodies can be bound to many different solid phase carriers.
  • solid phase carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetites.
  • insoluble carriers are a bead and/or a microtiter plate. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such using routine experimentation.
  • the monoclonal antibodies of the present invention that are specific for PSM' can be used in any subject in which it is desirable to administer in vitro or in vivo immunodiagnosis or immunotherapy. Accordingly, the present invention also provides methods for in vitro expression in human samples, particularly in patients with prostate carcinoma.
  • PSM' expression can be detected in patient tissue samples by immunohistochemical assays and/or in patient fluid samples by in vitro immunoassay procedures. A determination of the expression of PSM' in patient specimens is of significant diagnostic utility and may be indicative of, or correlate with, the progression of a disease state.
  • Immunohistochemical methods for the detection of antigens in patient tissue specimens are well known to the art and need not be described in detail herein.
  • methods for the immunohistochemical detection of antigens are generally described in Taylor, Arch. Pathol. Lab. Med. 102:113 (1978).
  • a tissue specimen obtained from a patient suspected of having prostate cancer is contacted with an antibody, preferably a monoclonal antibody, specific for the PSM' protein.
  • the site at which the antibody is bound is thereafter determined by selective staining of the tissue specimen by standard immunohistochemical procedures.
  • an immunoassay for determining the PSM' protein in a sample comprises the steps of :
  • any body fluid that may contain the PSM' protein may be used in the immunoassay of the present invention.
  • body fluid samples include, but are not limited to, urine, saliva, serum and semen.
  • the antibody used in the immunoassay in accordance with the present invention can be any antibody that is specific to the PSM' protein. Both monoclonal antibodies and polyclonal antibodies may be used as long as such antibodies possess the requisite specificity for the antigen provided by the present invention. Preferably, monoclonal antibodies are used. Examples of monoclonal antibodies include, but are not limited to, monoclonal antibodies PP1D 329, PP1D 449, PP1D 603, PP1D 423 and PP1D 559.
  • antibodies that recognize both PSM' and PSMA may also be used in an immunoassay for determining PSM' in a biological fluid sample.
  • a method for determining the PSM' protein in a sample which contains both PSM' and PSMA includes the steps of:
  • monoclonal antibodies that recognize both PSMA and PSM' include, but are not limited to, PM1T485.5, PM2H043, PM2H207, PEQ226, PMlx310.5, PM2E343 and PM2E086.
  • the monoclonal antibody that recognizes only PSMA, not PSM' may be the monoclonal antibody 7E11.
  • Antibodies of the present invention may also be used in an in vitro immunoassay to predict cancer progression, particularly prostate cancer progression. Accordingly, one embodiment of the present invention provides a method for predicting prostate cancer progression in a sample containing both PSM' and PSMA by using antibodies of the present invention that are specific for PSM' protein. The method includes the steps of :
  • the mathematical combination is a ratio.
  • examples of monoclonal antibodies that are specific for PSM' include, but are not limited to, monoclonal antibodies PP1D 329, PP1D 449, PP1D 603, PP1D 423 and PP1D 559.
  • Another embodiment of the present invention provides a method for predicting prostate cancer progression in a sample containing both PSM' and PSMA by using antibodies of the present invention that are specific to both PSM' and PSMA proteins.
  • the method includes the steps of :
  • determining the ratio of the amount of PSMA to the amount of PSM' and PSMA The term "specific for PSMA" as used herein means that the monoclonal antibody only recognizes PSMA, not PSM'. As discussed above, the ratio of PSMA to PSM' in a sample can be a useful indicator for the measurement of tumor progression since there is an increased expression of PSMA over PSM' in a prostate cancer patient following the progression from normal to tumor state.
  • monoclonal antibodies can be utilized in liquid phase or bound to a solid phase carrier. Monoclonal antibodies can be bound to many different carriers and used to determine the PSM' protein contained in a sample.
  • Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetites.
  • the nature of the carrier can be either soluble or insoluble for purposes of the invention.
  • insoluble carriers examples include, but are not limited to, a bead and a microtiter plate. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such using routine experimentation.
  • the monoclonal antibodies in these immunoassays can be detectably labeled in various ways.
  • the monoclonal antibodies of the present invention can be coupled to low molecular weight haptens. These haptens can then be specifically detected by means of a second reaction.
  • it is common to use such haptens as biotin, which reacts with avidin, or dinitrophenyl, pyridoxal and fluorescein, which can react with specific antihapten antibodies.
  • monoclonal antibodies of the present invention can also be coupled with a detectable label such as an enzyme, radioactive isotope, fluorescent compound or metal, chemiluminescent compound or bioluminescent compound.
  • a detectable label such as an enzyme, radioactive isotope, fluorescent compound or metal, chemiluminescent compound or bioluminescent compound.
  • the binding of these labels to the desired molecule can be done using standard techniques common to those of ordinary skill in the art.
  • One of the ways in which the antibodies can be detectably labeled is by linking them to an enzyme.
  • This enzyme when later exposed to its substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected by, for example, spectrophotometric or fluorometric means (ELISA system).
  • ELISA system spectrophotometric or fluorometric means
  • enzymes that can be used as detectable labels are horseradish peroxidase, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-
  • glycerophosphate dehydrogenase triose phosphate isomerase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase and acetylcholine esterase.
  • the procedures described can be modified using a biotinylated antibody reacting with avidin-peroxidase conjugates.
  • the amount of antigen can also be determined by labeling the antibody with a radioactive isotope.
  • the presence of the radioactive isotope would then be determined by such means as the use of a gamma counter or a scintillation counter.
  • Isotopes which are particularly useful are 3 H, 125 I, 123 1, 32 P, 35 S, 14 C, 51 Cr, 36 C1, 57 Co, 58 Co, 59 Fe, 75 Se, l ⁇ n, "mTc,
  • Determination of the antigen is also possible by labeling the antibody with a fluorescent compound.
  • a fluorescent compound When the fluorescently labeled molecule is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence of the dye.
  • fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • Fluorescence-emitting metal atoms such as Eu (europium), and other lanthanides, can also be used. These can be attached to the desired molecule by means of metal-chelating groups, such as DTPA or EDTA.
  • Another way in which the antibody can be detectably labelled is by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged immunoglobulin is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labelling compounds are luminol, isoluminol, aromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • Bioluminescence is a special type of chemiluminescence which is found in biological systems and in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent molecule would be determined by detecting the presence of luminescence.
  • bioluminescent compounds for purposes of labelling are luciferin, luciferase and aequorin.
  • Qualitative and/or quantitative determinations of the PSM' in a sample may be accomplished by competitive or non-competitive immunoassay procedures in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay.
  • RIA radioimmunoassay
  • sandwich immunometric
  • immunometric assay or "sandwich immunoassay” includes a simultaneous sandwich, forward sandwich and reverse sandwich immunoassay. These terms are well understood by those skilled in the a ⁇ . Those of skill will also appreciate that antibodies according to the present invention will be useful in other variations and forms of assays which are presently known or which may be developed in the future. These are intended to be included within the scope of the present invention.
  • the present invention also provides for the in vivo diagnosis and therapy of cancer in humans, particularly of prostate carcinoma. Methods for in vivo tumor imaging and therapy are generally described in Holt, S. et al., Can. Med. Assoc. J. 129: 18 (1983) and Sfakianakis, G. et al., J.
  • methods for tumor localization and detection may be performed by administering to a suspected cancer patient a predetermined diagnostically effective amount of an antibody recognizing the targeted tumor-associated proteins, such as PSM' or PSMA, and thereafter detecting the sites of localization of the antibody by standard imaging techniques.
  • an antibody recognizing the targeted tumor-associated proteins such as PSM' or PSMA
  • antibodies preferably monoclonal antibodies used for the purpose of in vivo diagnosis, are labeled so as to permit detection. Examples of the types of labels and labeling techniques are described above and require no repetition here.
  • antibodies are labeled with a radionuclide emitting gamma radiation and administered to the patient in a pharmaceutically acceptable carrier, e.g., buffered saline and human serum albumin.
  • the detectably labeled antibody is given a dose which is diagnostically effective.
  • diagnostically effective means that the amount of detectably labeled monoclonal antibody is administered in sufficient quantity to enable detection of the site having the targeted antigens for which the monoclonal antibodies are specific.
  • the concentration of detectably labeled monoclonal antibodies which is administered should be sufficient such that the binding to those cells having the targeted antigens is detectable compared to the background. Further, it is desirable that the detectably labeled monoclonal antibody be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio.
  • the dosage of the detectably labeled monoclonal antibody for in vivo diagnosis will vary depending on such factors as age, sex, and the extent of disease of the individual. Such dosages may vary, for example, depending on whether multiple injections are given, the degree of antigenic burden, and other factors known to those of skill in the art.
  • the type of detection instrument available is a major factor in selecting a given radioisotope.
  • the radioisotope chosen must have a type of decay which is detectable for a given type of instrument.
  • Still another important factor in selecting a radioisotope for in vivo diagnosis is that deleterious radiation with respect to the host is minimized.
  • a radioisotope used for in vivo imaging will lack a particle emission but produce a large number of photons in the 140-250 keV range, which may readily be detected by conventional gamma cameras.
  • a predetermined effective amount of an antibody preferably a monoclonal antibody, recognizing the tumor-associated antigens characterized by the invention, is administered to a cancer patient.
  • the antibody is conjugated with a suitable therapeutic agent, e.g., radioisotopes, preferably emitters of beta particles, drugs, toxins or biological proteins selected for delivery to the tumor site, and administered to the cancer patient in a pharmaceutically acceptable carrier, e.g., buffered saline and human serum albumin.
  • a suitable therapeutic agent e.g., radioisotopes, preferably emitters of beta particles, drugs, toxins or biological proteins selected for delivery to the tumor site, and administered to the cancer patient in a pharmaceutically acceptable carrier, e.g., buffered saline and human serum albumin.
  • the "predetermined effective amount" of antibody suitable for use may vary with individual patients and will depend upon such factors as the disease state, the circulating antigen concentration, antibody specificity, antibody kinetics and biodistribution, and whether an antibody is administered alone or as an antibody cocktail.
  • Antibody preparations comprising mixtures of antibodies or fragments thereof, i.e., antibody cocktails, having specificity for the tumor-associated antigens of the invention, may be used in certain instances to enhance the detection, localization and treatment of tumors.
  • the term "antibody” includes fragments thereof such as Fab, Fab 1 and Fab 2 fragments or mixtures thereof, including mixtures with whole antibodies.
  • kits for determining PSM' in a biological sample includes carrier means compartmentalized to receive in close confinement therein one or more containers comprising a container containing a monoclonal antibody that recognizes PSM' in a sample.
  • the monoclonal antibody recognizes only the PSM' protein.
  • monoclonal antibodies that are specific for PSM' include, but are not limited to, monoclonal antibodies PP1D 329, PP1D 449, PP1D 603, PP1D 423 and PP1D 559.
  • the kit contains two containers, wherein one container contains a first monoclonal antibody recognizing both PSM' and PSMA, and another container contains a second monoclonal antibody recognizing only PSMA.
  • first monoclonal antibody include, but are not limited to, PM1T485.5, PM2H043, PM2H207, PEQ226, PMlx310.5, PM2E343 and PM2E086.
  • second monoclonal antibody is 7E11.
  • monoclonal antibodies to various regions of PSMA are first developed.
  • the monoclonal antibodies to PSMA are then selected for their ability to recognize the PSM' of the present invention.
  • the PM2M Mabs were obtained from A/J mice (Jackson Labs) which received the following immunization protocol. On day 1 the mice were injected i.p. with 50 ⁇ g of plasma membranes purified from LNCaP cells with alum. Two weeks later the mice received a second injection of 25 ⁇ g of membranes with alum. Those mice which responded with good titers were injected i.p. 3 weeks later with 10 ⁇ g of PSMA purified from LNCaP cells with alum.
  • mice were finally boosted with 10 ⁇ g of purified PSMA (i.v.) 3 days prior to fusion.
  • the mice were sacrificed and their spleens removed.
  • Cell fusion was carried out according to the procedure of Kohler and Milstein, Nature 256, 4495-497 (1975).
  • 1 x 10 8 splenocytes were fused in 1.0 ml of a fusion medium composed of 35% polyethylene glycol (PEG 1500) in an APMEM medium (Flow Laboratories, Inglewood, California) with 2.5 x 10 7 P3.653 myeloma cells.
  • PEG 1500 polyethylene glycol
  • APMEM medium Flow Laboratories, Inglewood, California
  • mice The PM2H and PM2J Mabs were obtained from Balb/C mice (Harlan Labs) which received the following immunization schedule. On day 1 the mice were injected i.p. with 25 ⁇ g PSMA purified from L ⁇ CaP cells with alum. Two weeks later the mice received a second injection of 10 :g purified PSMA with alum. The mice were finally boosted i.v. with
  • PM1X310.5 was obtained from an A/J mouse which received 50 ⁇ g of T7-134-437 PSMA fusion protein i.p. with CFA. This was followed two weeks later with 25 ⁇ g of fusion protein injected i.p. with IFA. A final boost of fusion protein (25 ⁇ g i.v.) was given three days prior to fusion. The clone was selected based on its strong reactivity with T7- 134-437 PSMA fusion protein as well as LNCaP membranes and minimal reactivity on DU145 membranes. PM1T485.5 was obtained from an A/J mouse which received 50 ⁇ g of T7-438-750
  • PSMA fusion protein i.p. with alum. This was followed two weeks later with 25 ⁇ g of fusion protein injected i.p. with alum. After a third injection of fusion protein (25 ⁇ g) the mice were bled and good titers were obtained on T7-438-750 PSMA. The mice were final boosted with 25 ⁇ g fusion protein i.v. 3 days prior to fusion. The clone was selected based on its strong reactivity with T7-438-750 PSMA as well as LNCaP membranes and lack of reactivity with DU145 membranes. The PM2B clones were developed in a similar manner except that the fusion protein, GST-438-750 PSMA, was used as the immunogen.
  • Monoclonal antibody PEQ226 was developed from a Balb/C mouse which was immunized with tumor plasma membranes obtained from an autopsy specimen of prostate carcinoma. The mice were injected 5 times i.p. at 14 day intervals with 200 ⁇ g of prostate carcinoma plasma membranes. Three days after the 5th immunization, a mouse was sacrificed and the spleen was harvested. The fusion technique has been previously described. Hybridomas were selected based on their binding to prostate carcinoma membranes and failure to bind to normal liver membranes by ELISA. PEQ226 was more recently shown to bind to purified PSMA as well as recombinant PSMA.
  • the resin/supernatant solution was centrifuged on a tabletop centrifuge to sediment the beads.
  • the beads were then washed 4X with 10 volumes (5mls) of extraction buffer.
  • the beads were transferred to a column with a fritted support and washed 5X with 10 column volumes of extraction buffer.
  • the beads were then washed 5X with 10 column volumes of 25 mM Tris-HCI pH 7.4, 150 mM NaCl, 1% reduced Triton X-100.
  • the PSMA protein was eluted with 100 mM giycine, 150 mM NaCl, 1% reduced Triton X-100, pH 2.5. The fractions were immediately neutralized by the addition of 1/10 volume 1 M Tris pH 8.0.
  • LNCaP cells or DU145 cells were harvested from tissue culture flasks by mild trypsinization. The cells were centrifuged at lOOx g for 5 min at 4° C and then diluted 5-fold in a homogenization buffer (30 mM NaCl, 1 mM EDTA, 1 mM PMSF, 10 mM tris-HCI pH 7.2) and disrupted by nitrogen cavitation (500 PSI for 20 min). All subsequent steps were performed at 4° C. The cell lysate was centrifuged at 1,000 x g for 5 min and the supernatant was removed and centrifuged at 100,000 x g for 1 h.
  • a homogenization buffer 30 mM NaCl, 1 mM EDTA, 1 mM PMSF, 10 mM tris-HCI pH 7.2
  • the supernatant (cytosol fraction) was removed and the pellet was resuspended in a 1.0 ml homogenization buffer with freshly added PMSF and layered onto an ultracentrifuge tube containing 16 mis of 40% sucrose and 16 mis of 20% sucrose in a homogenization buffer.
  • the gradient was spun in a Beckman L8 ultracentrifuge 100,000 x g for 16 h.
  • the turbid layer at the 20%/40% interface was collected, suspended in a homogenization buffer with fresh PMSF added and centrifuged at 100,000 x g for 1 h.
  • the membrane fraction (pellet) was collected and resuspended in a minimal volume (0.5-1.0 ml) homogenization buffer. Protein was quantitated using the BCA assay (Pierce, Rockville, IL).
  • PACGHISNTA-PSMA1.9 (Seattle) was used as the template to amplify fragments PSMA 134-437 and PSMA 438-750.
  • PDR2 plasmid (Seattle) was used as the template to amplify PSMA 1-173 fragments. These fragments were cloned into PGEX (Promega) or pET5a (Novagen) vectors to generate GST and T7 PSMA fusion proteins, respectively. The DNA of the resulting fusion proteins were transformed into E.coli BL21 (DE3) (for T7 fusion proteins) or E. coli DH 5a (for GST fusion proteins).
  • Single cell clones were propagated in an LB broth and protein production was induced by adding IPTG (0.4 M).
  • the cell pellets were collected after 2 hours of induction and resuspended in a sample buffer for analysis.
  • the PSMA 134-437 and 438-750 fusion protein induced cell pellets were lysed by sonication and the pellet containing the inclusion bodies was collected and fusion proteins were purified by HPLC.
  • PSMA 1- 173 the E. coli pellet was resuspended in a sample buffer and used without further purification
  • Plasma membrane and cytosol fractions were prepared according to a previously described and published procedure (12) with minor modifications. Approximately 5 X 10 8 LNCaP cells were harvested from tissue culture flasks by mild trypsinization. The cells were centrifuged at 100 x g for 5 min at 4°C and then diluted 5-fold in a homogenization buffer (30 mM NaCl, 1 mM EDTA, 1 mM PMSF, 10 mM tris-HCI pH 7.2) and disrupted by nitrogen cavitation (500 PSI for 20 min). All subsequent steps were performed at 4°C.
  • a homogenization buffer (30 mM NaCl, 1 mM EDTA, 1 mM PMSF, 10 mM tris-HCI pH 7.2
  • the cell lysate was centrifuged at 1,000 x g for 5 min and the supernatant was removed and centrifuged at 100,000 x g for 1 h.
  • the supernatant (cytosol fraction) was removed and the pellet was resuspended in a 1.0 ml homogenization buffer with freshly added PMSF (1 mM) and layered onto an ultracentrifuge tube containing 16 mis of 40% sucrose and 16 mis of 20%) sucrose in a homogenization buffer.
  • the gradient was spun in a Beckman L8 ultracentrifuge at 100,000 x g for 16 h.
  • the turbid layer at the 20%/40% interface was collected, suspended in homogenization buffer with fresh PMSF added and centrifuged at 100,000 x g for 1 h.
  • the membrane fraction (pellet) was collected and resuspended in a minimal volume (0.5-1.0 ml) homogenization buffer. Protein was quantitated using the BCA assay (Pierce, Rockville, IL
  • Table 1 summarizes monoclonal antibodies obtained based on the methods described above.
  • FIG. 1 shows the mapping of PSMA MAbs 7E11 and PEQ226 to PSMA and PSM'.
  • FIG. 2 is a western blot showing the reactivity of PSMA Mab PEQ226 with GST-PSMA fusion proteins.
  • FIG. 2 shows that PEQ226 binds to a T7 fusion protein of PSMA which spans the 134-437 domain of the protein.
  • Both antibodies 7E11 and PEQ226 were used to purify the PSM' of the present invention. They were bound to agarose beads (AminoLink resin, Pierce, Rockford, IL) and utlilized in tandem to first remove full length PSMA from the lysate (7E11 -agarose beads) and then capture the remaining PSM' on the second antibody resin (PEQ226-agarose beads). The details of the experiment are described below.
  • LNCaP cells obtained from American Type Culture Collection (Rockville, Maryland) were grown in RPMI media with 10% horse serum (Gibco, Grand Island, NY) and maintained in 5% CO 2 at 37°C. Cells were grown in a T160 flask until 80% cell confluency was obtained. Cells were lysed following the addition of 2 mis of 1% triton X-100, 10% glycerol, 15 mM MgCl 2 , 1 mM phenylmethylsulfonylfluoride, 1 mM sodium vanadate, 10 uM aprotinin, 50 mM Hepes pH 7.5.
  • the crude lysate was incubated with 1.0 ml of 7E11- AminoLink beads prepared according to manufacturer's instructions (Pierce, Rockford, Illinois) for 14 h at 4°C.
  • the unbound fraction was then incubated with 1 ml of PEQ226- AminoLink beads for 4 h at 4°C.
  • Beads were washed five times with 10 mis of 1% triton x- 100, 10% glycerol, 15 M MgCl, 50 mM Hepes pH 7.5 and then eluted with 1 ml of 2% sodium dodecyl sulfate in 10 mM sodium phosphate, 150 mM NaCl pH 7.5.
  • the eluents and unbound fractions were western blotted and probed with both the 7E11 and PEQ226 Mabs.
  • the eluent from the PEQ226-AminoLink beads was blotted to a PVDF membrane and stained with coomassie blue. The band was cut out of the blot and sequenced on a Procise Protein Sequencer (ABI, Foster City, CA).
  • FIG. 3 shows enrichment of PSM' from LNCaP cell lysate using immunoaffinity resins. Two sequential immunoaffinity resins were utilized to enrich specifically for PSM'. LNCaP cell lysate was incubated with 0.5 mis 7E1 1-
  • AminoLink resin The unbound fraction was incubated with PEQ226-AminoLink resin. The eluted fractions and unbound fractions were examined on western blots with antibodies at
  • FIG. 3 shows
  • the protein which was eluted from the PEQ226 column contained a molecule of approximately 95kDa which was detected with PEQ226 (lane 4) but not with 7E11 (lane 2).
  • the entire PEQ226 eluent was concentrated and loaded in a single lane and blotted to PNDF paper.
  • the blot was stained with coomassie blue and the 95kDa band was cut out and sequenced.
  • the sequence matched the predicted protein sequence for PSM' deduced from the cD ⁇ A sequence. The only deviation from the predicted sequence was the absense of residues 58 (Met) and 59 (Lys) at the ⁇ -terminus. While the putative translation initiation site for PSM' was identified at residue 58 (Met), the actual ⁇ -terminal amino acid by protein sequencing was alanine at residue 60 of PSMA.
  • the ⁇ -terminus sequence of PSM' is shown in FIG. 4. Unlike full length PSMA from L ⁇ CaP cells, PSM' was not ⁇ -terminally blocked.
  • PSM' has 25 potential phosphorylation sites, 10 ⁇ - myristoylation sites and 9 ⁇ -glycoslyation sites (Su SL, Huang I, Fair WR, Powell CT, Heston WD, "Alternatively spliced variants of prostate-specific membrane antigen R ⁇ A: Ratio of expression as a potential measurement of progression" Cancer Res 55: 1441-1443 1995.).
  • EXAMPLE III Localization of the PSM' in the Cytoplasm of LNCaP Cells Since PSM' lacks the transmembrane domain expressed by PSMA, PSM' may reside in the cytoplasm of the cell.
  • LNCaP crude cell lysate (lanes 1 and 4), LNCaP cytoplasm, (lanes 2 and 5) and LNCaP plasma membranes (lanes 3 and 6) were loaded at 0.5ug/lane and probed with the PSMA specific Mab, 7E11 at lOug/ml (lanes 1-3) or PEQ226 which recognizes both PSMA and PSM' at lOug/ml (lanes 4-6).
  • the reactivity in the cytosol fraction was detected only with
  • PEQ226 (lane 4) and not with 7E11 (lane 2). Both antibodies reacted with full length PSMA in the crude cell lysate (lanes 1 and 4) and in the plasma membrane fraction (lanes 3 and 6). The membrane staining in lane 6 with PEQ226 suggests that membranes may contain some PSM' activity. This may be due to minor contamination of the plasma membrane fraction with cytosol or it may represent a subset of PSM' possibly in the process of being secreted from the cell.
  • PSMA appears to be present in the LNCaP lysate at higher levels than PSM' since PEQ226 only detects the full length lOOkDa protein in whole cell lysates (Fig 5 lane 4). Longer exposure of this lane did reveal a weaker band comigrating with the PSM' band in lane 5. This result is in agreement with previous evaluations of PSMA and PSM' RNA levels (Su, S.L.; Huang, I.; Fair, W.R.; Powell, C.T.; Heston, W.D.W. Alternatively spliced variants of prostate-specific membrane antigen RNA: Ratio of expression as a potential measurement of progression.
  • the present invention is the first study outlining methods to purify PSM' from LNCaP cell lysates which are free of PSMA. N-terminal sequence analysis has confirmed the presence of PSM' and shown that the protein actually begins at Ala-60 in the PSMA sequence. The protein has been shown to reside in the cytoplasm as previously speculated. Ratios of PSMA/PSM' may be of diagnostic value in prostate cancer.
  • the peptide would be conjugated to a carrier molecule such as KLH or thyroglobulin through the C-terminal Cys prior to immunization into mice. Following fusion, the resulting hybridomas would be tested for reactivity with the PSM' peptide biotinylated at the C- terminus through the Cys. Unlike the immunogen, this peptide would not be conjugated to a carrier protein.
  • the ELISA screen would involve capture of the biotinylated peptide on streptavidin-coated microtiter plates followed by incubation with supernatant from each of the hybridomas wells.
  • This peptide would also be biotinylated at the C-terminus Cys and then captured on streptavidin-coated microtiter plates. Clones that fail to detect the N-terminus blocked peptide, but detect PSM' peptide, would be saved and tested on LNCAP purified PSMA and PSM' by western blot.
  • FIG.s 9(a) and (b) show the serum titers of PSM' peptide immunization.
  • FIG. 6 shows the structure of a PSM' mammalian expression vector.
  • PSM' cDNA coding for PSMA sequence starting from Ala 60, was cloned downstream of a secretion signal under the control of the GBMT promoter in the PGTD plasmid.
  • the secretion signal was used to facilitate secretion of the protein into the spent media.
  • the GBMT promoter is inducible by the adenovirus El a.
  • the PGTD vector carries the DHFR gene; thus, recombinant clones will be resistant to methotrexate.
  • a trypsin cleavage site (Arg-Ala) was also engineered in the plasmid to facilitate clipping of the signal sequence.
  • the PGTD-PSM' plasmid was transfected into the adenovirus-transformed hamster cell line, AV12.
  • a single cell clone (AV12-PSM') was selected in 400 nM methotre
  • FIG. 7 shows the western blot analysis of AV12-PSM' spent media.
  • AV12-PSM', AV12-PSMAd (PSMA cDNA lacking the transmembrane domain), and AV12-PGTD (empty vector) cells were grown in DMEM + 10% fetal clone. At about 70% confluency, cells were washed and media was replaced with serum-free HH4 media. Spent media was harvested after 4 days, concentrated -10 fold, and subjected to SDS/PAGE on a 12% gel. Proteins were electroblotted onto nitrocellulose membrane and subjected to western blot analysis using PM1X310 (oc-PSMA mAbs) and goat oc-mouse HRP (1 :5000) as primary and secondary
  • the blots were developed using the ECL system.
  • PSM' Immunogen AV12-PSM' was grown in DMEM + 10% fetal clone. At about 70% confluency, cells were washed and media was replaced with serum-free HH4 media. Spent media was harvested at 4 days. PSM' was purified to homogeneity by using a PEQ226.5 (anti PSMA mAb) affinity column.
  • FIG. 10 shows the results of silver stain and western blot of purified recombinant PSM'.
  • FIG. 10 (A) shows the silver stain results
  • FIG. 10(B) shows the western blot results.
  • Lane 1 starting from the left is starting material AVI 2 supernatant.
  • Lane 2 is the purified recombinant PSM' (rPSM').
  • Lane 3 is the molecular weight marker.
  • the results of FIG. 10 indicates that — . Production of Monoclonal Antibodies to PSM'
  • Fusion Code PP1D r-PSM' immune spleen cells were fused to P3.653 myeloma cells, using standard techniques. Briefly, spleens were aseptically removed from the immunized mice and a single cell suspension was prepared. The cell suspension was depleted of T cells with anti-thyl .2 plus complement. B cells were fused to P3.653 myelomas with PEG and distributed into 96 well plates at a concentration of 2 x 10 (e5) cells per well. Culture supernatants from each well were screened for reactivity to r-PSM' as well as other antigens as described below.
  • FIG. 11 shows serum titers of rPSM' immunization to either PSM' peptide 1 to 15 (FIG. 11 (A)) or to rPSM' (FIG. 11(B)).
  • FIG. 8 shows antigens that were used to screen PSM' specific antibodies. Clones were selected for reactivity to PSM', and not to PSMA, which is identical to PSM', except it lacks the first 43 amino acids or d-PSMA that has the same signal sequence attached to it as r- PSM'.
  • Clones also do not react to PSM' peptide (1-15) that corresponds to amino acids 43 to 57 of the PSMA molecule.
  • Table 3 summarizes the results. These data show that the Mabs react to a unique confor ational determinant on the N terminus of PSM'. Clones 329 and 449 were subcloned to assure monoclonality. Both Mabs are of the IgGl isotype.

Abstract

L'invention concerne une protéine d'enveloppe prostatique spécifique (PSM') purifiée et isolée, sensiblement exempte d'autres protéines humaines. La séquence d'amino-acides N-terminal de la protéine de PSM' possède deux acides aminés de moins que la séquence d'acides aminés N-terminal de la protéine PSM' humaine putative. Des procédés de purification de la protéine PSM', des anticorps dirigés contre la protéine PSM' de l'invention sont également décrits ainsi que des immunodosages et des kits d'immunodosage pour la détection du cancer de la prostate chez un patient.
PCT/US1999/026844 1999-02-22 1999-11-11 Antigenes d'enveloppe prostatique specifique, ses procedes de production et d'utilisation WO2000050457A1 (fr)

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EP1390523A2 (fr) * 2000-11-20 2004-02-25 Eastern Virginia Medical School Procedes et dispositifs permettant la detection quantitative des antigenes de membrane specifiques de la prostate (psma) et d'autres marqueurs prostatiques
WO2004067570A3 (fr) * 2003-01-28 2005-10-13 Proscan Rx Pharma Diagnostic et traitement du cancer de la prostate
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Cited By (14)

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US7163680B2 (en) 1996-05-06 2007-01-16 Cornell Research Foundation, Inc. Treatment and diagnosis of cancer
US7666425B1 (en) 1996-05-06 2010-02-23 Cornell Research Foundation, Inc. Treatment and diagnosis of prostate cancer
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US6770450B1 (en) 1996-05-06 2004-08-03 Cornell Research Foundation, Inc. Treatment and diagnosis of cancer
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US7112412B1 (en) 1996-05-06 2006-09-26 Cornell Research Foundation, Inc. Treatment and diagnosis of prostate cancer
EP1390523A2 (fr) * 2000-11-20 2004-02-25 Eastern Virginia Medical School Procedes et dispositifs permettant la detection quantitative des antigenes de membrane specifiques de la prostate (psma) et d'autres marqueurs prostatiques
EP1390523A4 (fr) * 2000-11-20 2004-03-31 Eastern Virginia Med School Procedes et dispositifs permettant la detection quantitative des antigenes de membrane specifiques de la prostate (psma) et d'autres marqueurs prostatiques
US7192586B2 (en) 2001-09-20 2007-03-20 Cornell Research Foundation, Inc. Methods and compositions for treating or preventing skin disorders using binding agents specific for prostate specific membrane antigen
US7771953B2 (en) 2003-01-10 2010-08-10 Millenium Pharmaceuticals, Inc. Methods of diagnosing and treating cancer
WO2004067570A3 (fr) * 2003-01-28 2005-10-13 Proscan Rx Pharma Diagnostic et traitement du cancer de la prostate
US7811564B2 (en) 2003-01-28 2010-10-12 Proscan Rx Pharma Prostate cancer diagnosis and treatment
US8101713B2 (en) 2003-01-28 2012-01-24 Proscan Rx Pharma Inc. Prostate cancer diagnosis and treatment
US8512702B2 (en) 2003-01-28 2013-08-20 Proscan Rx Pharma Inc. Prostate cancer diagnosis and treatment

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