EP0042428A1 - Gereinigtes menschliches prostat-antigen - Google Patents

Gereinigtes menschliches prostat-antigen

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Publication number
EP0042428A1
EP0042428A1 EP81900373A EP81900373A EP0042428A1 EP 0042428 A1 EP0042428 A1 EP 0042428A1 EP 81900373 A EP81900373 A EP 81900373A EP 81900373 A EP81900373 A EP 81900373A EP 0042428 A1 EP0042428 A1 EP 0042428A1
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EP
European Patent Office
Prior art keywords
antigen
prostate
antibodies
prostatic
tissue
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EP81900373A
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English (en)
French (fr)
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EP0042428A4 (de
Inventor
Tsann Ming Chu
Ming Chang Wang
Lawrence Papsidero
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Research Corp
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Research Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001193—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; PAP or PSGR
    • A61K39/001194—Prostate specific antigen [PSA]
    • 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
    • C07K16/3038—Kidney, bladder

Definitions

  • This invention relates to a diagnostic reagent and method for the immunochemical detection of a human prostate antigen which is distinct from prostatic acid phosphatase. More particularly, this invention relates to a novel purified human prostate antigen and antibodies specific thereto which are suitable for use in prostatic cancer detection by laboratory methods.
  • Prostate cancer is very prevalent in old age, with approximately one half of all males over age 70 having been shown to develop prostatic cancer. This high incidence of prostate malignancy has led to the search for markers which may be used for its detection.
  • the elevation of serum acid phosphatase activity in patients having metastasized prostate carcinoma was first reported by Gutman et al. in J. Clin. Invest. 17: 473 (1938).
  • prostatic acid phosphatase is released from the cancer tissue into the blood stream with the result that the total serum acid phosphatase level greatly increases above normal values. Numerous studies of this enzyme and its relation to prostatic cancer have been made since that time, e.g., see the review by Yam in Amer. J. Med. 56: 604 (1974).
  • serum acid phosphatase by conventional spectrophotometric methods often fails to detect prostatic cancer in its early stages.
  • the activity of serum acid phosphatase is elevated in about 65-90 percent of patients having carcinoma of the prostate with bone metastasis; in about 30 percent of patients without roentgenological evidence of bone metastasis; and in about only 5-10 percent of patients lacking clinically demonstrable metastasis.
  • the prostate antigen of the present invention is present in all prostate tissue, (normal, benign or malignant) in almost equal amounts. Further, it is detectable in prostatic fluid and cultured human prostatic malignant cells and its medium as well.
  • the Ablin articles describe absorption of antisera to the antigens described therein with prostatic fluid, after which a precipitin line was still detected. Absorption of antibodies against the present antigen with prostatic fluid gives no precipitin line, indicating that the present antigen is present in prostatic fluid while that of Ablin et al. is not.
  • Another object of the present invention is to provide rapid and simple, yet highly specific and sensitive, immunochemical techniques and reagents useful in the early detection of prostatic cancer.
  • a further object of this invention is to provide a new marker for monitoring prostatic cancer and the effectiveness of curative therapy therefor.
  • An additional object of this invention is to provide useful monoclonal antibodies to human prostate antigen.
  • the above and other objects, features and advantages of the present invention are attained in one aspect thereof by providing a purified human prostate antigen which is distinct from prostatic acid phosphatase.
  • the present invention provides antisera which are highly specific to a purified human prostate antigen and which do not immunochemically cross-react with prostatic acid phosphatases or with acid phosphatases originating from other tissues.
  • an immunochemical method for the detection of prostatic cancer which exhibits high sensitivity, good specificity and substantially no false positive results for tumor other than the prostate.
  • the present invention provides specific marker antibodies against human prostate antigen which are useful, e.g., as carriers for in vivo radioimmunodetection of prostate cancer and immunospecific chemotherapy of prostatic cancer.
  • antigenic preparations from either normal or cancerous human prostate tissue, prostatic fluid, cultured human prostatic malignant cells or their media are purified to obtain a preparation consisting essentially of a human prostate antigen free of prostatic acid phosphatase.
  • These antigenic preparations are employed for immunological vaccination and diagnostic procedures, particularly for immunoprecipitin testing.
  • prostate tissue is first extracted in aqueous media at 4°C.
  • EDTA-PBS solution is conveniently used, saline, 3M KCl or 0.01 percent (v/v) Tween 80 (but not 1M perchloric acid) also can be used for extraction of the prostate antigen.
  • the antigen is thus also distinguished from carcinoembryonic antigens by its sensitivity to perchloric acid.
  • the supernatant from the crude tissue extract is subjected to ammonium sulfate fractionation.
  • Ammonium sulfate concentration at 20-80 percent saturation almost quantitatively recovers the prostatic antigen from EDTA-PBS extracts, with the highest yield at 45-50 percent saturation.
  • the precipitate is preferably collected between 35-55 percent saturation of ammonium sulfate, which contains approximately 70 percent of the total prostate antigen in the crude extract.
  • PA purified from seminal plasma and from prostatic tissue possesses an identical mobility and isoelectric point (pI 6.87 + 0.09) as shown by polyacrylamide gel electrophoresis and isoelectric focusing, respectively. Both purified PA preparations exhibit a molecular weight of 33-34,000 as shown by Sephadex G-75 gel filtration. In addition, a line of identity was obtained in immunodiffusion when purified PA preparations reacted with anti-PA serum.
  • seminal plasma is more readily available than prostatic tissues and contains abundance of PA, it appears an ideal source for isolating PA.
  • Using seminal plasma as the source of PA isolation also appears to have an advantage over the use of prostatic tissue. Firstly, the initial extraction step, which requires at least 4 hours, is eliminated. Secondly, at the initial stage of purification, handling a large volume of the tissue extract is avoided since 20 to 30 ml of seminal plasma is equivalent to 100 g of tissue in terms of PA content. Thirdly, seminal plasma contains less contaminating proteins and makes purification easier.
  • hemoglobin in the tissue extract is precipitated concurrently with PA by ammonium sulfate at concentration greater than 50 percent saturation, and the removal of this hemoglobin in later steps of purification results in a reduction of the yield of final purified PA.
  • seminal plasma contains less contaminating proteins, it is possible during fractional precipitation steps to recover a greater amount of PA by increasing the upper cut-off point of ammonium sulfate concentration to 75 percent saturation. A better recovery of PA in purified form is also achieved from seminal plasma.
  • DEAE-BioGel A anion exchange column effectively retains the prostate antigen and washing the column with tris-HCl buffer at pH 8.0 does not dissociate the antigen from the column. Elution of the antigen can be achieved with 18-78 mM of NaCl in the same buffer, followed by further purification by gel chromatography.
  • the bulk of contaminating proteins in the PA preparation eluted from the DEAE column have a molecular weight greater than 45,000 and can accordingly be separated from PA (molecular weight: 33,000 to 34,000) by gel filtration on a Sephadex G-100 column.
  • An additional step can be carried out with anion exchange chromatography on a DEAE column using a pH gradient solution as the eluant.
  • Two protein peaks were detected between fractions in the pH range of 7.6 to 6.7, which were shown to contain PA.
  • polyacrylamide gel electrophoresis protein heterogeneity was seen in the first peak fractions, while the second peak fractions gave a single protein band. Therefore, for obtaining homogeneous PA, only second peak fractions were collected.
  • approximately 20 percent of total PA subjected to chromatography was still bound to the DEAE column. This can be recovered quantitatively by elution with 0.08M NaCl, but protein heterogeneity in the eluted PA preparation was observed.
  • Preparative polyacrylamide gel electrophoresis of the partially purified antigen is effective in achieving a pure antigen preparation.
  • This purified antigen is shown to be homogeneous by polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. It has a molecular weight of 33,000-34,000 with no subunit, and exhibits a single pI of 6.9.
  • PA has been shown to be a prostate gland epithelial marker protein.
  • PA is localized in the epithelial lining of prostatic glands and ducts as well as in prostatic secretions and concretions, but not in epithelia of periurethral glands, seminal vesicles, vas deferens, urinary bladder transitional epithelium, prostatic urethra, glandular lining of vonBrunn's nests, or in testes.
  • the prostate antigen from prostate tissue extracts was characterized by gel filtration chromatography (m.w. 30-40,000), isoelectric focusing (pI 6.9) and agarose electrophoresis (M R 0.2 relative to bovine serum albumin).
  • concentration of the prostate antigen was not significantly different among extracts prepared from normal, benign hypertrophic and malignant prostatic tissues and the antigen exhibited no acid phosphatase enzyme activity as determined by histochemical staining procedures. Furthermore, its approximate molecular weight (30-40,000) differs significantly from that of prostatic acid phosphatase (100,000).
  • prostate antigen in circulation is immunologically identical to the human prostate antigen in prostate tissue and prostatic fluid.
  • the serum-borne antigen may be bound to a plasma protein.
  • plasma protein binding of a variety of antigens and hormonal substances is well known and has been reported by T. Peters, Jr. "Serum Albumin” In: F.W. Putman (ed.). The Plasma Proteins. Vol. 1: pp. 133-153, New York, N.Y. Academic Press, 1975.
  • SPD-PAGE revealed a molecular weight of 36,000, similar to that in prostate tissue and seminal plasma.
  • monoclonal antibodies against PA can be prepared using known hybridoma cell culture techniques.
  • this method involves preparing an antibody- producing fused cell line, e.g. of primary spleen cells fused with a compatible continuous line of myeloma cells, and growing the fused cells either in mass culture or in an animal species from which the myeloma cell line used was derived or is compatible.
  • fused cell line e.g. of primary spleen cells fused with a compatible continuous line of myeloma cells
  • this prostatic antigen in clinical applications is not known at present, as it does not appear to exhibit any tumor specificity or pathological association.
  • this prostate antigen can provide useful clinical information upon serum monitoring.
  • Recent experiments in our laboratory have shown that the prostate antigen is detected, by rocket i ⁇ rnur.oelectrophoresis and enzyme-linked immunoassay techniques, in the sera of some prostate cancer patients but not in the sera of normal healthy persons or of patients with other cancers. Further experiments have shown that PA is histologically detectible in both prostate tissue and established continuous tissue culture cell lines of prostate origin, and that it is released by prostate tumor cells, both in vivo and in vitro.
  • the PA level in serum of patients suffering from prostatic cancer is unrelated to the serum levels of prostatic acid phosphatase, so that simultaneous determination of circulating PA and circulating prostatic acid phosphatase may well provide an improved means of diagnosing prostatic cancer.
  • the human prostate antigen although an eutopic component of the prostate, can thus play a major role in the detection of prostate cancer.
  • a non- antigenic adjuvant e.g. alum, Freund's complete adjuvant, saponin, a quaternary ammonium surfactant, an alkyl amine, etc.
  • a suitable immunologically acceptable, non-antigenic carrier e.g. by filtration.
  • the vaccine can be administered parenterally following regimens already known for immunization with other proteins to stimulate the formation of immunoprecipitating antibodies, with the primary inoculation being preferably followed up by at least one additional injection one to ten weeks later. Good results have been obtained in rabbits using four booster injections at two week intervals one month after the primary immunization.
  • the protein content per injection in rabbits, goats and other mammals can be varied, but is generally about 50 micrograms of protein per kg. of body weight.
  • the antibodies can be collected and worked up using methods .well known to those skilled in the art of immunochemistry, and provide a useful reagent for the immunological detection of prostate specific antigen in a variety of immunochemical procedures, e.g., immunoprecipitin, fluorescent antibody, serum neutralization, etc. Such antibodies are useful as a control reagent in the diagnostic test for prostatic cancer described more particularly below.
  • the simplest immunoprecipitin test involves capillary tube precipitin testing, wherein separate antibody and antigen solutions are allowed to react at a common interface in a capillary tube and a positive reaction is indicated by the formation of a precipitate at the interface.
  • This method is relatively insensitive and inaccurate due, inter alia, to unavoidable diffusion of the two solutions across the interface, and furthermore the final test results cannot be preserved.
  • Agar gel diffusion is the simplest method which avoids these drawbacks.
  • a solution of the antigen (or serum sample) is placed in a central well punched in a continuous agar gel and appropriate dilutions of the serum containing antibodies (or, correspondingly, the antigen) thereto are placed in wells concentrically surrounding the center well.
  • a positive reaction is noted by the formation of the precipitin line between one or more of the concentric wells and the central well. This method is relatively insensitive and fairly slow, requiring 1 to 4 days to read the test results.
  • Radioimmunoassay e.g. radioimmunoprecipitin tests
  • RIA Radioimmunoassay
  • Countercurrent immunoelectrophoresis is a widely used immunoprecipitin method which takes only about an hour to perform and which is considerably more sensitive than agar gel diffusion. Reactive components are placed in opposing wells cut into an agar gel and a small electrical current applied thereto, causing both the antigen and the antibody to migrate towards each other. A positive reaction is indicated by the formation of a precipitate at the antigen-antibody interface. Since this test method is reasonably reliable, readily available and inexpensive, it represents a preferred embodiment of this aspect of the present invention.
  • the diagnostic antibody preparation of the present invention when used without purification is generally diluted with phosphate buffered saline in a volume ratio of 1:10 to 1:500, depending on the antibody titer thereof.
  • the limiting factor at the lower end of the range is the degree of distinction achieved in the precipitin lines, which is a function of the antibody content in the total protein present.
  • Purified antibody preparations can of course have lower total protein concentrations, and the protein content of even the unpurified preparations can be varied to suit the particular immunochemical test to be employed, the optimal amounts being determined, e.g. by testing simple serial dilutions.
  • circulating human prostate antigen can now be detected by immunochemical techniques, preferably by protein staining of the antibody-antigen precipitin complex.
  • immunochemical techniques preferably by protein staining of the antibody-antigen precipitin complex.
  • the antigen-antibody complex can be stained by a number of known histochemical staining techniques, e.g. fluorescent antibody, etc., to increase the sensitivity of this method.
  • a radioactive antibody for the assay, which not only provides a better quantitative value but may also further increase the sensitivity of the assay.
  • an enzyme e.g. ⁇ -galactosidase or peroxidase
  • an enzyme e.g. ⁇ -galactosidase or peroxidase
  • purified antibodies for use in an enzyme-linked immunoassay, e.g. using techniques analagous to those described by Kato et al . in J. Immunol. 116: 1554 (1976), the contents of which are incorporated by reference herein.
  • Especially preferred is the method described by M. Kuriyama et al. in Cancer Res. 40: 4658 (1980), the contents of which are incorporated by reference herein.
  • a water-insoluble support for use in the enzyme assay.
  • Many suitable such supports and techniques for binding proteins thereto are well known in the art and include inorganic as well as organic supports.
  • Presently preferred are those water-insoluble supports which can be activated with a cyanogen halide, preferably cyanogen bromide, prior to the covalent bonding of the antibodies thereto, e.g. as taught by Axen et al. in U.S. Patent 3,645,852, the contents of which are incorporated by reference herein.
  • Such supports are commercially available, e.g. the Enzymobeads available from Bio-Rad Laboratories.
  • Protein concentration was determined by the method of Lowry et al. described in J. Biol. Chem. 193: 265 (1951), using bovine serum albumin as the standard. Acid phosphatase activity was measured by the method of Babson and Phillips using ⁇ -naphthyl phosphate as the substrate. Immunoprecipitation techniques were modified from B. Weeke, Scand. J. Immunol. (Supplement 1) 2: 37-46 (1973).
  • Rocket-IEP was performed on cellulose acetate membranes using 0.83 percent agarose (Sigma low electro-endo-osmosis) in 0.08 M tris-0.024 M tricine-0.024M sodium barbital containing 1.3 mM calcium lactate and 0.02 percent sodium azide (TTB buffer). Antiserum at various final concentrations (0.5-2.0 percent) was incorporated into the agarose at 55°C prior to plating. Rocket-IEP samples were applied to circular wells (5.0 mm) and electrophoresed at 5 volts/cm overnight at 4°C using TTB running buffer.
  • TTB buffer sodium azide
  • Crossed-immunoelectrofocusing was performed in the second dimension as for rocket-IEP.
  • Isofocused acrylamide gels (5 x 100 mm) were molded into the antibody-agarose prior to immunoelectrophoresis modified from J. Soderholm et al., Scand. J. Immunol. (Supplement 2) 4: 107-113 (1975).
  • the first dimension isofocusing was done in 7.5 percent polyacrylamide containing 2 percent ampholytes, pH range 4-8 at 200 volts overnight at 4*C.
  • isofocused- gels were laid on cellulose acetate membrances and covered with 1 percent agrose. Troughs were cut adjacent to the embedded acrylamide gel, the antiserum placed in the parallel troughs was allowed to diffuse for 24-48 hours at 25°C and the resulting immunoprecipitin lines were recorded.
  • Cross-IEP Crossed-immunoelectrophoresis
  • PB-NaCl 0.05 M phosphate buffer containing 0.15 M NaCl, pH 7.2
  • IEP immunoelectrophoresis
  • TTB Tris-Tricine-Barbital (0.08M, 0.042M, 0.024M) buffer containing 0.3 mM calcium lactate and 0.02 percent sodium azide, pH 8.8
  • MR relative immunoelectrophoretic mobility
  • pI isoelectric point
  • m.w. molecular weight.
  • tissue extract Approximately 10 g of tissues were minced and washed three times with 30 ml of physiological saline, then mixed with 30 ml of 0.02 percent (w/v) disodium ethylenediamine tetraacetate (EDTA)-O.IM phosphate buffered saline (PBS), pH 6.8. The mixture was placed in an ice-water-chilled blending chamber of a Sorvall Omnimixer and subjected to three 5-minute blendings at a blade speed of 25,000 rpm with intermittent cooling time of 3 minutes. The homogenate was stirred overnight at 4°C, then centrifuged at 25,000 g for 30 minutes. The resultant supernatant constituted the crude tissue extract.
  • EDTA disodium ethylenediamine tetraacetate
  • Example 2 Ammonium Sulfate Fractionation Pooled prostate tissues, 100 g, were extracted with 300 ml of EDTA-PBS as described above. Ammonium sulfate (60 g) was added to 285 ml of crude extract (35 percent saturation), mixed for 30 minutes and centrifuged (26,000 g, 30 minutes). 38 g of ammonium sulfate were added to 295 ml of the resulting supernatant, (55 percent saturation), mixed for 30 minutes and centrifuged. The precipitate was dispersed in 200 ml of 55 percent saturated ammonium sulfate solution and centrifuged.
  • Example 3 Ion Exchange Chromography of Prostate Antigen The supernatant from Example 2, 28 ml, was applied onto a DEAE-BioGel A column (2.5 x 93 cm) pre-equilibrated with 0.01M tris-HCl buffer, pH 8.0.
  • the column was first washed with 420 ml of buffer, then eluted with 0-0.2M NaCl gradient (1 liter 0.01M tris-HCl buffer, pH 8.0 in mixer and 1 liter of 0.2M NaCl 0.01M tris-HCl, pH 8.0 in the reservoir) at a flow rate of 6.5 ml/hr/cm 2 .
  • the effluent was collected (10 ml /fraction) and monitored for the prostate antigen.
  • Gel diffusion according to the method of T. M. Chu et al. in Cancer Treat. Reports 61: 193 (1977) was used to measure the presence of prostate antigen in various chromatographic fractions. The entire procedure was carried out at 4°C.
  • the prostate antigen-containing fractions were pooled and concentrated with an Amicon concentrator fitted with a UM2 ultrafiltration membrane to 4.5 ml.
  • Example 6 Analytical Polyacrylamide Electrophoresis Acrylamide gel (7.5 percent) columns (50 x 60mm) were made according to the company's instruction manual (Shandon Southern Instruments, Ltd., Camberley, Surrey, England), and 50 ⁇ l of sample (10-40 ⁇ g protein in 25 percent sucrose solution) were applied onto each gel column. Ten percent sucrose solution was then layered carefully above the sample solution, followed by layering 0.05 M tris-glycine buffer, pH 8.5 to the top of the gel-containing tube. Tris-glycine buffer was used in preparing the sucrose solutions and used also as the electrolyte. After electrophoresis with a constant current of 5 mA per tube for 40 minutes, the gels were stained for protein by Coomassie brilliant blue G-250-perchloric acid solution.
  • Example 7 Sodium Dodecyl Sulfate (SDS) Polyacrylamide Gel Electrophoresis The method was essentially the procedure of Weber and Osborn described in J. Biol. Chem. 244: 4406 (1969). A sample (10-20 ⁇ g protein) in 50 ⁇ l of 0.05M tris-glycine buffer, pH 8.5, containing 250 ⁇ l. SDS and 2-mercaptoethanol each, was incubated at 37°C for 2 hours. After incubation, the sample was mixed with an equal volume of 50 percent sucrose, and 50 ⁇ l were subjected to the polyacrylamide gel (cpntaining 0.1 percent SDS) electrophoresis as described before.
  • SDS sodium Dodecyl Sulfate
  • Example 8 Preparative Polyacrylamide Gel Electrophoresis Although Sephadex G-75 chromatography produced a symmetrical protein peak with exhibited immunological reactivity of the prostate antigen, the analytical polyacrylamide gel electrophoresis of Examples 6 and 7 revealed several protein components in this preparation. Therefore, a preparative polyacrylamide gel electrophoresis (PAGE) was further used in the purification of prostate antigen.
  • PAGE preparative polyacrylamide gel electrophoresis
  • prostate antigen preparation was of high purity was indicated by a single protein band without any contaminating components in polyacrylamide ge,l electrophoresis in the absence and the presence of sodium dodecyl sulfate.
  • the purified prostate antigen was shown to have a molecular weight of 33,000 by gel filtration on Sephadex G-75, and 34,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis with no subunit component. Isoelectric focusing revealed a single pI of 6.9.
  • the procedure for the purification of PA reported in the preceding Examples takes at least two weeks and yields less than 0.5 mg of purified PA per 100 g of prostatic tissue. Furthermore, for the final step of purification, a painstaking construction of the polyacrylamide gel column as well as a special electrophoretic apparatus are necessary, and problems such as clogging of the elution channel during operation often developed. In this simplified procedure, in addition to the elimination of Sephadex G-75 chromatography and the application of short DEAE columns, the tedious preparative gel electrophoresis has been replaced by a simple pH gradient elution of the DEAE column. The yield of purified PA has increased to 1 to 2 mg per 100 g of prostatic tissue and the entire procedure has been shortened to 8 to 9 days.
  • Benign hypertrophic prostatic tissues (approximately 100 g) were extracted with disodium tetraacetate-phosphate buffered slaine as described in Example 1 except that the homogenate was stirred for only 2 hours rather than overnight.
  • the crude extract was then subjected to fractional precipitation by ammonium sulfate (30 to 50 percent saturation).
  • the precipitate was dissolved in 10 to 15 ml of 0.01M tris-HCl buffer, pH 7.8, and dialyzed against 4 ml of the same buffer for
  • the column was washed with 10 ml of the same buffer, followed by elution with pH gradient solution (mixer: 0.01M tris-HCl, pH 7.0, 300 ml; reservoir: 0.01M tris-HCl, pH 6.0, 500 ml) at a flow rate of 5 ml per hour per cm 2 .
  • pH gradient solution mixtureer: 0.01M tris-HCl, pH 7.0, 300 ml; reservoir: 0.01M tris-HCl, pH 6.0, 500 ml
  • Second peak fractions which contained PA were pooled and concentrated.
  • Table 1 summarizes the results of a typical purification of PA from prostatic tissue by this simplified procedure. Although modification at the salt precipitation step led to lesser recovery of PA, a large quantity of contaminating proteins such as hemoglobin were removed, thereby facilitating further purification of PA.
  • Radial immunodiffusion used in this study was the technique of Mancini et al described in Imnunochem. 2:
  • Anti-PA serum 100 ⁇ l was mixed with
  • Example 10 Purification of Antigen from Fluid PA was also purified from 20 ml of seminal plasma according to the procedure of Example 9 except that the extraction step was omitted and that, in the second step, PA was precipitated by ammonium sulfate at a concentration of 30 to 75 percent saturation. The results are shown in Table 2.
  • Example 11 Molecular Weight Determination
  • Serum samples 0.5 ml
  • prostate tissue extracts 0.5 ml containing 8 mg protein
  • Example 12 Preparation of Antisera
  • Female rabbits were immunized as described previously by T.M. Chu et al. in Investigative Urology 15: 319-323 (1978) with the crude extract of normal human prostatic tissue (for antiserum P 8 ), or with a purified prostate antigen (for antiserum P 1 7 ) obtained at the Sephadex G-75 step described above.
  • Sera were collected, heat inactivated and stored at -20 o C until use.
  • Absorption of the antiserum with normal female serum (NFS) or tissue extracts (10 mg protein/ml) was carried out as described by T. M. Chu et al. in Cancer Treatment Reports 61: 193-200 (1977).
  • Example 13 Specificity of Antiserum Raised Against Crude Prostate Antigen Immunoelectrophoresis was performed on a 9.5 x 10.2 cm agarose (0.65 percent, w/v) plate.
  • Barbital buffer, (pH 8.2, ionic strength 0.04) was used as the electrolyte and a constant voltage of 90 V. was applied for one hour.
  • the plate was washed with 0.154 M saline for 2 days and stained first for acid phosphatase with a solution of ⁇ -naphthyl phosphate-fast garnet GBC salt in 0.1M ammonium acetate, pH 5.0, and then for protein with Coomassie brilliant blue G-250-perchloric acid solution.
  • Immunoelectrophoresis of the crude extract prepared from normal prostatic tissues and antiserum P 8 an antiserum raised against the crude extract of normal prostate, resulted in three precipitin arcs.
  • One of these arcs was formed by a normal human serum component, as it disappeared after the antiserum was absorbed with normal female serum (NFS-P 8 ).
  • Example 15 Immunological Identity of Tissue and Serum Prostate Antigen
  • PB-NaCl diluting buffer
  • prostate antigen as detected in tissue, apply to extracts prepared from normal, benign hypertrophic and malignant prostate specimens; no physical differences in prostate antigen (i.e., M R , pI or m.w.) were observed among these antigen sources, and extracts prepared from benign and malignant prostate specimens exhibited immuno-precipitation lines of- identity with normal prostate when tested against anti-prostate antigen antiserum by double immunodiffusion.
  • the relative level of prostate antigen was compared among extracts prepared from normal, benign hypertrophic and malignant prostate tissues. As determined by rocket-IEP (Table 3) no statistically significant difference of antigen level was observed between these antigen sources and a wide variation in antigen level (indicated by the large standard deviations calculated) was also noted to occur for each category of tissue extract examined.
  • tissue extract was adjusted to 1 mg protein per ml prior to analysis for prostate antigen by rocket immunoelectrophoresis. b) All tissue specimens were histopathologically confirmed. c) For this experiment, one unit was arbitrarily chosen to represent one percent of the antigen level of a reference prostate extract. The reference extract was assayed in a dilution series and run on the same plates as experimental extracts. d) As analyzed using Student's t-test, no statistically significant difference was found among these groups.
  • Example 17 Immunological Identity of Fluid and Serum Prostate Antigen Since prostatic acid phosphatase has been reported in both prostatic tissue and seminal fluid, we wished to determine whether PA also exists in the seminal fluid.
  • Anti-PA serum raised against PA isolated from prostatic tissue, reacted in immunodiffusion with crude extract of prostatic tissue, as well as with seminal plasma, and formed a fused line, indicating immunologic identity of the PA in prostatic tissue with that in seminal plasma.
  • the PA concentration in seminal plasma was found to be 0.4 to 1.8 mg/ml, while 1 g. of prostatic tissue contained 0.15 to 0.45 mg of PA extractable with disodium ethylenediamine tetraacetate-phosphate buffered saline (EDTA-PBS).
  • tissue sections were further incubated with peroxidase- labelled goat anti-rabbit ⁇ -globulin. Excessive enzyme-label led second antibody was then removed by washing, and the tissue sections were stained for peroxidase activity.
  • the substrate alone, substrate plus conjugate, pre-immune rabbit serum, and anti-PA preabsorbed with specific antigen were used. It was observed that the staining was restricted to epithelial cells comprising the prostatic ductal elements. An intense staining was shown in. the apical cytoplasma of these cells, but no staining was seen in the nuclei. Within several ductal elements, positively staining secretory material was observed in the section examined.
  • PA has been demonstrated in all primary and metastatic prostatic tumors tested, but not in non-prostatic cancer tissues.
  • Immobilized anti-PA was prepared using CNBr-activated Sepharose IB (Pharmacia Fine Chemicals).
  • the reaction mixture consisted of 5 g of CNBr-activated Sepharose and 130 mg of IgG (anti-PA) in 0.1 M boratebuffer (pH 8.5) containing 0.5 M NaCl. After incubation at 4°C for 18 hours, the beads were washed with borate buffer and post-treated with a 1-M ethanolamine solution (pH 9.0) to block unreacted groups. Covalently coupled beads were further washed and stored in PBS at 4°C.
  • EIA 100 ⁇ l of an antigen sample was mixed with 300 ⁇ l of a fifty fold-diluted immobilized antibody and incubated for 3 hours at room temperature. After the addition of 1 ml of assay buffer (PBS containing 1 percent BSA), the mixture was centrifuged at l,000g to wash the beads. This procedure was repeated twice. To the washed beads was added 100 ⁇ l of peroxidase-IgG (anti-PA) conjugate in assay buffer. After a further incubation for 18 hours at room temperature, the beads were again washed as described above and assayed for the amount of bound peroxidase activity present.
  • assay buffer PBS containing 1 percent BSA
  • the reaction mixture contained 0.08 percent dianisidine and 0.003 percent H 2 O 2 in 0.01 M sodium phosphate buffer (pH 6.0) and was allowed to react with the beads for 90 minutes.
  • the enzyme reaction was then stopped with 100 ⁇ l of 1 N HC1, and the absorbance at 403 nm was determined.
  • Each run included standards of known prostate antigen concentration. Using this assay procedure, linearity of of the dose-response curve was achieved between 1 and 20 ng PA/ml. Before reexamination, samples containing higher levels of antigen were serially diluted with assay buffer.
  • F(ab') 2 fragments were accomplished with the use of solid-phase lactoperoxidase-glucose oxidase (Enzymobeads; Bio-Rad Laboratories). To. 1 mg of F(ab') 2 were added 50 ⁇ l of Enzymobeads, 25 ⁇ l of 1 percent ⁇ -D-glucose, 200 ⁇ l of 0.2 M sodium phosphate (pH 7.0), and 1 mCi of carrier-free 131 I or 125 I. The reaction was allowed to proceed for 30 minutes at room temperature. Unreacted iodide was separated from labeled protein by gel filtration over Sephadex G-25.
  • Preferential uptake of a specific radioactive nuclide was calculated as a localization ratio: ( 125 I cell bound 131 I cell bound) : ( 125 I added 1 31 I added).
  • a specific localization ratio was calculated for each cell type, and the results indicated a significantly higher antibody uptake by the LNCaP cells of prostate origin than for the HT-29 or MCF-7 cells.
  • Example 21 Release of Prostate Antigen by in vivo Tumor Cells When established as tumor cell xenografts in nude mice, LNCaP cells released detectable levels of PA into the circulation of these animals.
  • Congenitally athymic nude mice homozygous for the nu/nu allele were bred at Roswell Park Memorial Institute from matings of BABL/c nu/nu homozygous males and BALB/c +/nu heterozygous females.
  • Human tumors were established subcutaneously in the nude mice before sera were obtained for PA analysis by the injection of cultured cell suspensions. For this, the cells were washed in PBS, counted for viability, and adjusted to the desired concentration in sterile 0.9 percent NaCl solutions.
  • nude mouse serum was collected by severing the retro-orbital plexus and stored at -20°C.
  • Cell cultures used for the establishment of human tumor grafts included LNCaP (adenocarcinoma of the prostate gland), RT-4 (transitional cell carcinoma of bladder), Palarmo (malignant melanoma), and AsPC-1 (pancreatic carcinoma).
  • Example 22 Antiserum Specificity The optimal concentration of adsorbed antiserum required in the rocket-IEP procedure was determined by examining the migration of prostate antigen in gels containing varying concentrations of anti-prostate antigen antiserum. The reactivity of various tissues was examined by rocket-IEP. As shown in Table 4, all extracts prepared from normal prostate, benign hypertrophic and malignant prostate tissues showed reactivity, producing a single immunoprecipitin reaction. Extracts prepared from the non-prostate ti ssues, whether normal or neoplastic in nature, gave no immunologic reactivity.
  • Example 23 Diagnostic Specificity To examine the potential diagnostic value of the prostate antigen, serum samples were examined for its presence by the method of rocket-IEP, using anti-prostate antigen antiserum treated with normal human plasma to remove antibodies against normal plasma proteins (Table 5). Serum samples obtained from 20 normal healthy adults and 20 male volunteers over the age of 55 years showed no reactivity against the antiserum with this assay. Also, serum was drawn from a total of 175 patients with various advanced malignancies, including patients with malignancies of lung, colon, rectum, stomach, pancreas, thyroid, breast and with mye.loma. All sera obtained from patients with non-prostatic malignancies were prostate antigen-negative when assayed by the rocket-IEP procedure.
  • Example 24 Enzyme-Linked Immunoassay Tests Using IgG antibodies against the purified prostate-specific antigen with horseradish peroxidase and CNBr-activated Sepharose 4B as reagents, a sensitive sandwich-type (Sepharose 4B-anti-prostate antigen IgG: prostate antigen: anti-prostate antigen IgG-peroxidase) enzyme-linked immunoassay capable of detecting 0.1 ng of prostate antigen/ml. was evaluated. Of the various normal and tumor tissues examined, only human prostate tissue was shown to contain the prostate antigen (normal prostate 10 ⁇ 21.9 ⁇ g prostate antigen/mg.
  • the prostate antigen from the serum of prostate cancer patients was purified and shown to be immunochemical ly identical to the prostate antigen of normal prostate tissue. This data, therefore, demonstrate that the prostate antigen described herein, although a histotype-specific antigen of the normal prostate, can be used in the immunological detection of prostatic cancer.
  • Example 25 Prognostic Value of Enzyme-Linked Immunoassay Tests Using the sensitive enzyme immunoassay reported in Cancer Res. 40: 4658(1980), the circulating PA in prostatic cancer patients has been evaluated clinically.
  • the pretreatment serum PA levels were found to be of prognostic value in regard to patients' survival.
  • Example 26 Establishment of a Hybridoma Cell Line
  • P3X63Ag8.653 is derived from the ⁇ -1 K-type producing P3X63Ag8.
  • P3X63Ag8.653 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 15 percent heat-inactivated fetal bovine serum (FBS), 2mM glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin in a 10 percent CO 2 /air humidified incubator at 37°C.
  • DMEM Dulbecco's modified Eagle's medium
  • mice Female Balb/c mice (8-10 weeks old) were intraperitoneally injected on days 1 and 30 with 10 ⁇ g Prostate Antigen (PA) purified as described in Investigative Urology 17:159-163 (1979). Three to six weeks later, the mice received an intravenous injection of 10 yg PA in sterile saline. All mice used for fusion experiments showed the presence of serum antibodies against PA, as detected using double immunodiffusion against purified PA.
  • PA Prostate Antigen
  • Spleens were aseptically removed from immune mice after the last immunization, placed in 10 ml DMEM-15 percent FBS, and cell suspensions made by teasing with curved forceps. Clumps and membrane fragments were allowed to settle and the resulting single cells were washed once by centrifugation at 600g for 10 min. Red cells were lysed by incubation in 0.84 percent NH 4 Cl, and the cells were washed and resuspended in DMEM at a concentration of 2 X 10 7 cells/ml. The myeloma cells were similarly washed and adjusted to 2 X 10 7 cells/ml of DMEM.
  • spleen cells Three ml of spleen cells were added, to 3 ml of myeloma cells and co-pelleted at 600g for 10 min., and the medium was completely removed.
  • the cells were gently resuspended in 1 ml of 30 percent (w/v) polyethylene glycol (PEG), average molecular weight 1,000, in DMEM at pH 8.0.
  • PEG polyethylene glycol
  • the tube was rocked for 1 min. and centrifuged for 6 min. at 600g. After a total exposure time to the PEG of 8 minutes, the supernatant was removed and 10 ml DMEM slowly added with continuous circular agitation of the tube. Cells were then centrifuged for 10 min.
  • HAT selection medium DMEM-15 percent FBS containing 1.6 X 10 -5 M thymidine, 1 X 10 -4 M hypoxanthine, and 1 X 10 -7 M aminopterin.
  • HAT selection medium DMEM-15 percent FBS containing 1.6 X 10 -5 M thymidine, 1 X 10 -4 M hypoxanthine, and 1 X 10 -7 M aminopterin.
  • One ml amounts were dispensed into 48 wells of multiwell dishes and plates were incubated at 37°C in a humidified atmosphere of 10 percent C0 2 in air.
  • HAT medium was replenished every 4 days until day 21 when hypoxanthine/thymidine (HT) medium was added. As wells turned acid
  • Example 27 Selection of Hybridoma Cells Fusion supernatants were screened for anti-PA antibodies by a solid-phase enzyme immunoassay. Disposable 96-well microtiter plates were used as the solid adsorbing surface. The wells were filled with 100 ml poly-L-lysine succinate (0.25 mg/ml H 2 0) and incubated at 23°C for 15 min. Following 3 washes with PB-NaCl pH 7.2 (50 mM sodium phosphate:140 mM sodium chloride), 100 ml of purified PA were added at a concentration of 50 ⁇ g/ml 10 mM carbonate buffer, pH 9.6. After incubation for 24 hr. at 37°C, 100 ml of 1 percent (w/v) bovine serum albumin solution in PB-NaCl buffer were added for a further 3 hrs. at 37oC.
  • PB-NaCl pH 7.2 50 mM sodium phosphate:140 mM sodium chlor
  • the microtiter wells Prior to performing the antibody screening assay, the microtiter wells were washed 3 times using PB-NaCl buffer (200 ⁇ l). Culture fluids (100 ⁇ l) to be tested were applied to individual wells and allowed to incubate at 37°C for 3 hrs., followed by 3 washes with PB-NaCl buffer. Subsequently, 100 ⁇ l of peroxidase-conjugated antiserum to mouse Ig prepared according to the procedure described in J. Immunol. Methods 15: 305-310 (1977) were added to each well and incubated at 37°C for 3 hrs.
  • Hybridoma cells Desired myeloma hybrids were- cloned by limiting dilution in the presence of peritoneal macrophages to increase cloning efficiency.
  • Hybrid cells were plated in 96-well culture dishes at a density of 0.5 cells/well in DMEM-15 percent FBS complete medium. Vigorous growth was observed after 8-12 days, at which time supernatants were tested from wells showing single colonies. Cultures exhibiting antibody activity were re-cloned by this procedure to ensure population homogeneity.
  • Peritoneal macrophages were prepared by flushing the peritoneal cavity of Balb/c mice with 5 ml of ice cold, sterile 0.34M sucrose. To attain high yields of macrophages (5-15x10 /mouse), animals received an intraperitoneal injection of 0.5 ml sterile thioglycolate medium 4 days prior to harvesting the cells. Macrophages were washed once by centrifugation at 600g for 5 min. and were resuspended at 10 /ml in tissue culture medium. Each tissue culture well received 100 ⁇ l of macrophage suspension.
  • the hybridoma described herein has been deposited with the American Type Culture Collection, Rockville, Maryland 20850, U.S.A. and is designated ATCC No. HB 8051.
  • Example 29 Purification of Monoclonal Antibodies
  • Monoclonal antibodies from spent culture fluids were purified using immuno-affinity chromatography with rabbit-antimouse Ig: Sepharose 4B gel matrix (R ⁇ MIg:Sepharose). Twenty ml of R- ⁇ MIg:Sepharose were packed into a chromatographic column (1.5 X 30 cm) and equilibrated with PB-NaCl buffer. Samples of culture fluids were slowly passed through the column (5 ml/hr), followed by removal of non-specific reactants by elution with 100 ml of 0.1M glycine: 1M NaCl, pH 9.0. Reactive proteins were then eluted with 3 bed volumes of 4M KSCN: 0.01M sodium phosphate, pH 7.2. Dialyzed materials were concentrated using positive-pressure ultrafiltration.
  • An affinity matrix was prepared by coupling 200 mg Ig (R- ⁇ MIg) to 5 grams of cyanogen bromide-activated Sepharose 4B; an IgG fraction of rabbit antisera to mouse IgG (H + L) was prepared by a Rivanol procedure described in J. Immunol. Methods 15: 305-310 (1977). Coupling was performed at pH 8.0 in bicarbonate buffer according to directions provided by the manufacturer (Pharmacia; Piscataway, N.J.).
  • the class and subclass of isolated anti-PA antibodies were determined by double immunodiffusion in 0.6 percent agarose using rabbit anti-mouse IgGl, IgG2a, IgG2b, IgG3, IgM, anti-K and anti- ⁇ chain.
  • Supernatants from 96 of the most vigorously growing cultures were screened in duplicate for antibody activity against PA, using a solid phase enzyme-immunoassay. Results indicated that approximately 4 percent (4/96) of cultures were initially antibody-positive. Upon re-assay of these cultures, one hybridoma (F5-A-1/22) remained positive.
  • This culture was cloned by limiting dilution over peritoneal macrophages and yielded 30 cultures which macroscopically exhibited single colonies of growth.
  • One positive culture was re-cloned and all cultures derived therefrom were antibody-positive. These cells (clone F5-A-1/22.8.13, termed Cl. 8.13) were expanded to mass culture and examined for antibody content (Table 7).
  • culture fluids from P3X63Ag8 cells known to produce IgGl kappa-type immunoglobulen, were used.
  • Cells of the P3X63Ag8 strain produced approximately 12 ⁇ g/ml of isolated immunoglobulin which was reactive against specific antisera to IgGl and K-chain (Table 8). No precipitation occurred with other immunoglobulen subclass antiserum reagents.
  • Culture fluids from Cl. 8.13 showing anti-PA activity contained about 10 ⁇ g/ml of isolated material using R- ⁇ MIg: Sepharose purification. This isolate was identified as mouse immunoglobulin of the IgM K-type subclass (Table 8). This preparation did not precipitate against other subclass of antisera, indicating monoclonal ity of the isolated immunoglobulin preparation.
  • Clarified culture fluids were passed through R- ⁇ MIg: Sepharose 4B adsorbent as described. The gel was washed with IM NaCl pH 9.0 until absorbance at 280 nm reached baseline (less than 0.0200.D. units).
  • prostate antigen is an eutopic product, it appears to be a useful tumor marker such as other differentiated cell products, e.g., prostatic acid phosphatase and thyrocalcitonin.
  • the specificity of prostate antigen antiserum can allow for the identification of neoplastic cells in isolated metastases with unknown primary origin.
  • Anti-prostate antigen antiserum can also be used as a vector to localize and/or carry cytotoxic substances to neoplastic prostatic tissue. Further, the study of variations in the expression of normal differentiation between antigens of prostatic tissue during development of the gland and in prostatic lesions can provide insight into the phenomenon of growth regulation and metastatic ability of prostate cells.

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GB2139645A (en) * 1983-04-08 1984-11-14 Kureha Chemical Ind Co Ltd Antibody to human prostrate cancer
US4690890A (en) * 1984-04-04 1987-09-01 Cetus Corporation Process for simultaneously detecting multiple antigens using dual sandwich immunometric assay
US4708930A (en) * 1984-11-09 1987-11-24 Coulter Corporation Monoclonal antibody to a human carcinoma tumor associated antigen
SE9002480D0 (sv) * 1990-07-23 1990-07-23 Hans Lilja Assay of free and complexed prostate-specific antigen
JPH05142228A (ja) * 1992-01-17 1993-06-08 Kureha Chem Ind Co Ltd ヒト前立腺癌の分類および同定用試薬
US5489525A (en) * 1992-10-08 1996-02-06 The United States Of America As Represented By The Department Of Health And Human Services Monoclonal antibodies to prostate cells
EP0635575A1 (de) * 1993-07-22 1995-01-25 Wallac Oy Monoklonale Antikörper gegen Epitope in freiem uber nicht in Alpha-1-antichymotrypsin komplexiertem Prostata spezifisches Antigen
EP0725139A3 (de) * 1995-02-03 1997-11-26 Bayer Corporation Rekombinantes prostat-spezifisches Antigen und seiner Verwendung als Immunoassay-Kalibrator
US5807978A (en) * 1995-06-07 1998-09-15 Kokolus; William J. Immunogenic peptides of prostate specific antigen

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