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WO1999043710A1 - Antigenes d'enveloppe specifiques a la prostate et procedes de preparation et d'utilisation desdits antigenes - Google Patents

Antigenes d'enveloppe specifiques a la prostate et procedes de preparation et d'utilisation desdits antigenes Download PDF

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Publication number
WO1999043710A1
WO1999043710A1 PCT/US1999/003810 US9903810W WO9943710A1 WO 1999043710 A1 WO1999043710 A1 WO 1999043710A1 US 9903810 W US9903810 W US 9903810W WO 9943710 A1 WO9943710 A1 WO 9943710A1
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Prior art keywords
monoclonal antibody
psm
psma
amount
protein
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PCT/US1999/003810
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English (en)
Inventor
Lana S. Grauer
Kristine Kuus-Reichel
Roger Sokoloff
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Beckman Coulter, Inc.
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Application filed by Beckman Coulter, Inc. filed Critical Beckman Coulter, Inc.
Priority to AU33056/99A priority Critical patent/AU3305699A/en
Publication of WO1999043710A1 publication Critical patent/WO1999043710A1/fr
Priority to PCT/US1999/026844 priority patent/WO2000050457A1/fr

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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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. Several biomarkers have been used for screening, diagnosis, and predicting disease progression (2, 3).
  • biomarkers examples include prostate specific antigen (PSA), and prostatic acid phosphatase (PAP).
  • PSA prostate specific antigen
  • PAP prostatic acid phosphatase
  • 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 II 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 Also provided is 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
  • the present invention further provides a method of making a monoclonal antibody that is specific for PSM' protein In accordance with one embodiment of the present invention, the method comprises the steps of
  • step (c) immunizing a mouse or a suitable host with the conjugates of step (b),
  • 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.
  • 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 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 : (a) providing a first monoclonal antibody specific for PSM' , (b) contacting the first monoclonal antibody with the sample under a condition that the first monoclonal antibody binds to the PSM' of the sample,
  • kits for determining 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 containing 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.
  • 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 comprise 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.
  • FIGURE 1 shows the mapping of PSMA monoclonal antibodies 7E11 and PEQ226 to PSMA and PSM'
  • FIGURE 2 is a western blot showing the reactivity of PSMA Mab PEQ226 with GST-PSMA fusion proteins l
  • FIGURE 3 shows the western blots analysis of the enrichment of PSM' from
  • FIGURE 4 shows the N-terminal amino acid sequence of PSM' isolated from LNCaP cells
  • FIGURE 5 is a western blot which demonstrates that PSM' is located in the cytoplasm of LNCaP cells
  • 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 Figure 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 58 (Met) of PSMA, the actual N-terminal amino acid by protein sequencing was alanine at residue 60 of PSMA. In addition, it has been observed that PSM' protein resides in the cytoplasm of a cell.
  • 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. 2 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, 7E1 1 monoclonal antibody, and the like.
  • monoclonal antibodies recognizing CD34 and CD34 CD34 and CD34.
  • 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.
  • One aspect of the present invention provides antibodies or fragments thereof that are specific for both PSMA and PSM'.
  • Antibodies which consist essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibodies preparations are provided. Examples of monoclonal antibodies that recognize both PSMA and PSM' include, but are not limited to, PM1T485.5,
  • 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, PSMA protein fused to a carrier protein such as complete Freunds adjuvate (CFA), PSMA synthetic peptides and PSMA fusion proteins.
  • CFA complete Freunds adjuvate
  • 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 antibodies preparations are provided
  • the term 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
  • Fab 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
  • (Fab')2 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 dimmer 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 genetical engineering methods, and
  • 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 genetical engineering methods
  • epitopic determinants means any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • 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).
  • 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 synthesized peptides has an amino acid sequence of : Ala-Phe-Leu-Asp-Glu-Leu-Lys-Ala-Glu-Asn-lle-Lys-Lys-Phe-Leu-Cys-
  • 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. In accordance with one embodiment of the present invention, to select a monoclonal
  • monoclonal antibodies can be bound to many different solid phase carriers.
  • examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetites.
  • insoluble carriers are used such as a microtiter plate.
  • 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 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.
  • 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
  • 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 comprising 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 4
  • 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
  • monoclonal antibodies are used
  • 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 (a) providing a first monoclonal antibody which recognizes both PSM' and
  • 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 monoclonal antibody 7E11
  • Antibodies of the present invention may also be used in an n 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
  • the method includes the steps of :
  • 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 : (a) providing a first monoclonal antibody which recognizes both PSM' and
  • PSMA protein styrene-maleic anhydride copolymer
  • PSM' 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.
  • 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. Examples of insoluble carriers 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
  • haptens as biotin, which reacts with avidin, or dinitrophenyl, pyridoxal, and fluorescein, which can react with specific anti-hapten 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 1 "mTc 7 Ga, a ⁇ d 90 Y.
  • Determination of the antigen is also possible by labeling the antibody with 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 are fluorescein
  • 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 labeled 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.
  • chemiluminescent labeling compounds are luminol, isoluminol, aromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.
  • bioluminescent compound may also be used as a label.
  • 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.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase, and aequorin.
  • Detection of the antigens using the monoclonal antibodies of the present invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.
  • 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 art. Those of skill will also appreciate
  • 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
  • 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
  • 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 radio nuclide 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
  • diagnosis 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 v vo 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
  • 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
  • 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
  • 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.
  • the kit contains two containers, wherein one container contains a first monoclonal antibody recognizing both PSM' and PSMA, and the another container contains a second monoclonal antibody recognizing only PSMA.
  • the first monoclonal antibody include, but are not limited to,
  • the 7E11 antibody was obtained from Dr. Gerry Murphy, Pacific Northwest Cancer Foundation, Seattle, WA. Its development and clinical use with ⁇ In 5 has been described previously (4). 7E11 recognizes the N-terminus portion of PSMA (residues 1-7) (13).
  • 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 ug of plasma membranes purified from LNCaP cells with alum Two weeks later the mice received a second injection of 25 ug of membranes with alum Those mice which responded with good titers were injected i p 3 weeks later with 10 ug of PSMA purified from LNCaP cells with alum The mice were finally boosted with 10 ug 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
  • 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 ug PSMA purified from LNCaP cells with alum Two weeks later the mice received a second injection of 10 ug purified PSMA with alum The mice were finally boosted i v with 10 ug of purified PSMA three days prior to fusion Fusions
  • PM1X310 5 was obtained from an A/J mouse which received 50 ug of T7-134- 437 PSMA fusion protein i p with CFA This was followed two weeks later with 25 ug of fusion protein injected i p with IFA A final boost of fusion protein (25 ug 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 ug of T7-438- 750 PSMA fusion protein i p with alum This was followed two weeks later with 25 ug of fusion protein injected i.p with alum After a third injection of fusion protein
  • mice were bled and good titers were obtained on T7-438-750 PSMA
  • the mice were finally boosted with 25 ug 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 ug 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
  • PSMA human transient expression of PSMA
  • Full-length PSMA cDNA was isolated from the plasmid PDR2 (Seattle) and was cloned into the plasmid pCDNA3 (Invitrogen) to yield the clones pCDNA3- PSMA#7 DNA sequencing was used to verify the identity and proper orientation of the insert This clone was transfected into COS-1 cells using lipofectamine (Life Technologies) and the transient expression of PSMA was monitored by western blot using the 7E1 1 monoclonal antibody Results showed that the PSMA (100 kDa) protein was expressed in the cell lysates 24-48 hours after transfection
  • 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
  • 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
  • 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
  • Plasma membrane and cytosol fractions were prepared according to a previously described and published procedure (12) with minor modifications
  • Table 1 summarizes monoclonal antibodies obtained based on the methods described above Table l 6
  • Antibodies that are positive to PSMA are further tested against PSM' protein of the present inveniton by using routine immuoassays. The results are summarized in table 2.
  • PSM' The schematic of full-length PSMA and alternatively spliced PSM' showing the transmembrane domain (TM) and transferrin receptor homologous region (TR). Binding regions for 7E11 and PEQ226 are indicated by arrows.
  • Figure 2 is a western blot showing the reactivity of PSMA Mabs, 7E1 1 (lanes 1,3,5,7) and PEQ226 (lanes 2,4,6,8) with GST-PSMA fusion proteins. Lanes 1-2, GST-PSMA 1 173; Lanes 3-4,
  • FIG. 1 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 utilized 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,
  • 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 lOug/mi Lane 1, 3 ul of 7E11- resin eluent detected with 7E11, Lane 2, 3 ul of PEQ226-resin eluent detected with 7E11, Lane 3, 3 ul of 7E11 -resin eluent detected with PEQ226, Lane 4, 3 ul of PEQ226-resin eluent detected with PEQ226, Lane 5, 6 ul of
  • the entire PEQ226 eluent was concentrated and loaded in a single lane and blotted to PVDF 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 cDNA sequence The only deviation from the predicted sequence was the absence of residues 58 (Met) and 59 (Lys) at the N-terminus While the putative
  • 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 SL, Huang I, Fair WR, Powell CT, Heston WDW Alternatively spliced variants of prostate-specific membrane antigen RNA Ratio of expression as a potential measurement of progression Cancer Res 55 1441-1443
  • the present invention is the first study outlining methods to purify PSM' from LNCaP cell lysates which is free of PSMA N-terminal sequence analysis have 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 thru 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 Following extensive washing, a secondary goat anti-mouse immunoglobulin conjugated to horseradish peroxidase would be added to each well Color development would be monitored at
  • 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

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Abstract

Protéine d'enveloppe purifiée et isolée humaine, spécifique à la prostate (PSM), qui est pratiquement exempte d'autres protéines humaines. La séquence d'acides aminés N-terminale de la protéine PSM compte deux acides aminés en moins par comparaison à la séquence d'acides aminés N-terminale de la protéine PSM humaine généralement admise. Des procédés de purification de la protéine PSM et des anticorps contre la protéine PSM selon la présente invention sont également décrits. Des dosages immunologiques et des kits de dosage destinés à la détection du cancer de la prostate chez un patient sont encore décrits.
PCT/US1999/003810 1998-02-26 1999-02-22 Antigenes d'enveloppe specifiques a la prostate et procedes de preparation et d'utilisation desdits antigenes WO1999043710A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000050457A1 (fr) * 1999-02-22 2000-08-31 Beckman Coulter, Inc. Antigenes d'enveloppe prostatique specifique, ses procedes de production et d'utilisation
US6649163B1 (en) 1996-05-06 2003-11-18 Cornell Research Foundation, Inc. Treatment and diagnosis of cancer
WO2004113571A3 (fr) * 2003-06-26 2005-04-28 Exonhit Therapeutics Sa Genes specifiques de la prostate et leur utilisation comme cibles dans le traitement et le diagnostic du cancer de la prostate
US7045605B2 (en) 2001-06-01 2006-05-16 Cornell Research Foundation, Inc. Modified antibodies to prostate-specific membrane antigen and uses thereof
US7112412B1 (en) 1996-05-06 2006-09-26 Cornell Research Foundation, Inc. Treatment and diagnosis of prostate cancer
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
WO2008003656A3 (fr) * 2006-07-03 2008-03-20 Exonhit Therapeutics Sa Produits de la transcription spécifiques de la prostate et leur utilisation pour des thérapeutiques et des diagnostics du cancer de la prostate
US7514078B2 (en) 2001-06-01 2009-04-07 Cornell Research Foundation, Inc. Methods of treating prostate cancer with anti-prostate specific membrane antigen antibodies
US7771953B2 (en) 2003-01-10 2010-08-10 Millenium Pharmaceuticals, Inc. Methods of diagnosing and treating cancer
US12257304B2 (en) 2023-03-03 2025-03-25 Arsenal Biosciences, Inc. Systems targeting PSMA and CA9

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996026272A1 (fr) * 1995-02-24 1996-08-29 Sloan-Kettering Institute For Cancer Research Antigene membranaire specifique de la prostate et ses utilisations
WO1997004802A1 (fr) * 1995-07-31 1997-02-13 Pacific Northwest Cancer Foundation Procede permettant d'isoler et/ou de conserver des cellules dendritiques pour l'immunotherapie du cancer de la prostate
WO1997035616A1 (fr) * 1996-03-25 1997-10-02 Pacific Northwest Cancer Foundation Anticorps monoclonaux de l'antigene membranaire specifique de la prostate

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996026272A1 (fr) * 1995-02-24 1996-08-29 Sloan-Kettering Institute For Cancer Research Antigene membranaire specifique de la prostate et ses utilisations
WO1997004802A1 (fr) * 1995-07-31 1997-02-13 Pacific Northwest Cancer Foundation Procede permettant d'isoler et/ou de conserver des cellules dendritiques pour l'immunotherapie du cancer de la prostate
WO1997035616A1 (fr) * 1996-03-25 1997-10-02 Pacific Northwest Cancer Foundation Anticorps monoclonaux de l'antigene membranaire specifique de la prostate

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ABDEL-NABI H ET AL: "MONOCLONAL ANTIBODIES AND RADIOIMMUNOCONJUGATES IN THE DIAGNOSIS AND TREATMENT OF PROSTATE CANCER", SEMINARS IN UROLOGY, vol. 10, no. 1, 1 February 1992 (1992-02-01), pages 45 - 54, XP000645163 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6649163B1 (en) 1996-05-06 2003-11-18 Cornell Research Foundation, Inc. Treatment and diagnosis of cancer
US6770450B1 (en) 1996-05-06 2004-08-03 Cornell Research Foundation, Inc. Treatment and diagnosis of cancer
US8951737B2 (en) 1996-05-06 2015-02-10 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
US7112412B1 (en) 1996-05-06 2006-09-26 Cornell Research Foundation, Inc. Treatment and diagnosis of prostate cancer
US7163680B2 (en) 1996-05-06 2007-01-16 Cornell Research Foundation, Inc. Treatment and diagnosis of cancer
WO2000050457A1 (fr) * 1999-02-22 2000-08-31 Beckman Coulter, Inc. Antigenes d'enveloppe prostatique specifique, ses procedes de production et d'utilisation
US7514078B2 (en) 2001-06-01 2009-04-07 Cornell Research Foundation, Inc. Methods of treating prostate cancer with anti-prostate specific membrane antigen antibodies
US7045605B2 (en) 2001-06-01 2006-05-16 Cornell Research Foundation, Inc. Modified antibodies to prostate-specific membrane antigen and uses thereof
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
US7834163B2 (en) 2003-06-26 2010-11-16 Exonhit Therapeutics S.A. Prostate specific genes and the use thereof as targets for prostate cancer therapy
WO2004113571A3 (fr) * 2003-06-26 2005-04-28 Exonhit Therapeutics Sa Genes specifiques de la prostate et leur utilisation comme cibles dans le traitement et le diagnostic du cancer de la prostate
WO2008003656A3 (fr) * 2006-07-03 2008-03-20 Exonhit Therapeutics Sa Produits de la transcription spécifiques de la prostate et leur utilisation pour des thérapeutiques et des diagnostics du cancer de la prostate
US12257304B2 (en) 2023-03-03 2025-03-25 Arsenal Biosciences, Inc. Systems targeting PSMA and CA9

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