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WO1998010292A1 - Anticorps monoclonaux contre l'antigene prostatique specifique et procedes de detection de l'antigene prostatique specifique - Google Patents

Anticorps monoclonaux contre l'antigene prostatique specifique et procedes de detection de l'antigene prostatique specifique Download PDF

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Publication number
WO1998010292A1
WO1998010292A1 PCT/US1997/014909 US9714909W WO9810292A1 WO 1998010292 A1 WO1998010292 A1 WO 1998010292A1 US 9714909 W US9714909 W US 9714909W WO 9810292 A1 WO9810292 A1 WO 9810292A1
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Prior art keywords
monoclonal antibody
specific antigen
prostate specific
amino acids
seq
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PCT/US1997/014909
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English (en)
Inventor
George A. Heavner
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Centocor, Inc.
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Application filed by Centocor, Inc. filed Critical Centocor, Inc.
Priority to AU40863/97A priority Critical patent/AU4086397A/en
Publication of WO1998010292A1 publication Critical patent/WO1998010292A1/fr

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    • 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
    • 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

Definitions

  • Prostate specific antigen is a serine protease of 237 amino acids, with one N-linked and three O-linked carbohydrate side chains.
  • Prostate specific antigen is a 33-34 kD protein produced by prostatic epithelial cells.
  • the prostate Surrounding the neck of the bladder and the urethra in men, the prostate is part muscular and part glandular, with ducts opening into the prostatic portion of the urethra. The gland secretes a thin, opalescent, slightly alkaline fluid that forms part of the seminal fluid. PSA is a major protein component of this fluid.
  • PSA is a member of the kallikrein family along with pancreatic/renal kallikrein, and glandular kallikrein of the prostate (hGK-1 or hK-2).
  • PSA and hGK-1 have 78% protein sequence homology, and their genes are found in tandem, 12 kb apart on chromosome 19.
  • PSA functions as a chymotrypsin-like protease, and the identified targets are seminogelin I and II, and fibronectin. Cleavage of seminogelin allows liquefaction of seminal coagulum.
  • PSA is a marker to detect and monitor prostate cancer and benign prostatic hyperplasia. It is also found in small quantities (0-4 ng/ml) in serum, but if the gland is inflamed or cancerous, the serum levels increase. PSA is found in serum in both a free form and bound (or complexed) with alpha- 1 -antichymotrypsin (ACT), an endogenous serine protease inhibitor. The majority of serum PSA is found complexed to serine protease inhibitors alpha- 1 -antichymotrypsin (ACT; resulting in a Mr of 90 kD), or alpha- 2-macroglobulin (AMG; resulting in a Mr of 750 kD). PSA bound to AMG is not immunogenic probably because the AMG engulfs PSA.
  • ACT alpha- 1 -antichymotrypsin
  • AMG alpha- 2-macroglobulin
  • PSA was cloned from cDNA libraries by Lundwall and Lilja (1987) and a longer PSA sequence was cloned by Henttu and Vihko (1989). The disclosures of each of the references cited herein are incorporated herein by reference in their entirety. Total genomic DNA was searched and the PSA gene cloned by Lundwall (1989). hGK-1 was cloned from a genomic library by Schedlich et al. (1987).
  • Benign prostatic hyperplasia is common to nearly all men over 60, and occurs in younger men as well. Because of the location of the gland, symptoms include the need for frequent nocturnal urination.
  • Prostatic cancer (PCa) is the most common non-skin cancer in men, the fourth most common cause of death in the United States. In 1994, approximately 38,000 men died of PCa.
  • the combination of PSA serum level testing and digital rectal exam (DRE) is sufficient for diagnosis in many of the cases.
  • the treatment of PCa is radical prostatectomy in which the prostate gland, seminal vesicles, and narrow cuff of bladder neck are removed. The chance of impotence has decreased from 100% to 30%-50% in the last five years as surgery was developed to try to save the capsular and periprostatic nerves. There is also a chance of incontinence.
  • PSA levels There are 18 million men in the united States 50-70 years old that should be screened for PSA levels annually. Of these only 8-10 million men comply. Within this population, 10% (approximately 1 million men) will have PSA values between 4-10 ng/ml, a range where BPH and PCa are indistinguishable by total PSA testing. Currently this group is biopsied, but the inconvenience and expense is encouraging the search for better and different PSA testing. PSA levels should also be followed after radical prostatectomy to predict persistent disease.
  • PSA free/total ratio define the current anti-PSA antibodies' epitopes and any subsequently isolated antibodies, including anti-free PSA, anti-bound PSA, and anti-total PSA antibodies.
  • the present invention was developed using a series of 15-mers overlapping by 12 amino acids to define areas of reactivity for antibodies recognizing predominantly conformational epitopes.
  • synthetic peptides in an ELISA format epitopes were identified that were recognized by both monoclonal antibodies and polyclonal antisera to PSA.
  • homology modeling a structure of PSA was constructed which revealed a loop in the three dimensional structure of free PSA. The epitopes identified were mapped to the PSA model.
  • the present invention is directed to monoclonal antibodies that bind to determinants found in free and bound prostate specific antigen, hereinafter denoted anti-PSA antibodies.
  • a monoclonal antibody which binds free prostate specific antigen at a determinant comprising amino acids 82-87 of free prostate specific antigen is provided in the present invention.
  • the present invention is directed to monoclonal antibodies that bind to amino acids 139-144 or a fragment thereof of prostate specific antigen, a hybridoma producing a monoclonal antibody which binds amino acids 139-144 or a fragment thereof of prostate specific antigen, and a monoclonal antibody produced therefrom. Also embodied by the present invention are monoclonal antibodies that bind to amino acids 55-60 or a fragment thereof of prostate specific antigen, a hybridoma producing a monoclonal antibody which binds amino acids 55-60 or a fragment thereof of prostate specific antigen, and a monoclonal antibody produced therefrom.
  • the monoclonal anti-PSA antibodies of this invention are useful in a number of diagnostic and therapeutic applications, including methods for the detection of cancer in mammals.
  • the present invention also provides methods that permit the discovery of prostate specific antigen in a sample.
  • the prostate specific antigen may be free, bound, or total.
  • kits for the detection of free, bound, and/or total prostate specific antigen comprising an immunoabsorbant, labeled monoclonal antibody specific for free, bound, and/or total prostate specific antigen, and a control are also provided in the present invention.
  • Another embodiment of the present invention is a method of screening for monoclonal antibodies specific for amino acids 82-87 or a fragment thereof of free prostate specific antigen.
  • Figure 1 displays the three dimensional backbone structure of serine proteases thrombin, ⁇ -thrombin, trypsin, epsilon-thrombin, elastase, trypsinogen, tonin, chymotrypsinogen, kallikrenin, and rat mast cell protease.
  • the catalytic triad represented by three yellow circles, is found in the center of the structure.
  • Figures 2A and 2B display the epitope map of anti-PSA antibodies of monoclonal antibodies 10, 11, 15.2, 16, 22.2, 156, 225, and 365. A series of 75 15-mer peptide sequences covered the entire sequence of PSA.
  • Figure 3 displays an alignment of the protein sequences of PSA, SEQ ID NO: 1
  • Figure 4 displays the sequences of PSA peptides synthesized as 15-mers.
  • the present invention is directed to a monoclonal antibody that binds to amino acids 82-87, 169-171, and 223-228 of free prostate specific antigen (PSA).
  • PSA prostate specific antigen
  • the amino acids 82-87 are in a loop region evident in the tertiary structure of free prostate specific antigen, see Figure 1. While not intending to be bound by any particular theory of operation, it is believed that amino acids 82-87 of the loop region of prostate specific antigen are conformationally available when PSA is in the free form, that is, PSA not bound or complexed to ⁇ -1 -antichymotrypsin (ACT).
  • ACT ⁇ -1 -antichymotrypsin
  • the monoclonal antibody found to bind to amino acids 82-87 is designated herein as monoclonal antibody 365.
  • the antigenic/immunogenic peptide also identified herein as the epitope, target antigen, or determinant of PSA to which monoclonal antibody 365 binds comprises the amino acid sequence LKNRFL (SEQ ID NO. 1), amino acids 82-87.
  • the epitope may be contained within the six amino acid sequence of SEQ ID NO: 1.
  • fragment refers to part of the amino acid sequence of SEQ ID NO: 1 substantially similar thereto, to which PSA specifically or immunologically binds, which fragment thus has PSA specific activity.
  • the present invention also includes monoclonal antibodies having characteristics of monoclonal antibody 365.
  • Peptide 15-mers having the sequence of SEQ ID NO: 1 or a fragment thereof are peptides 24-29 set forth in Figure 4.
  • Monoclonal antibodies that bind the amino acid sequence 55-60, SLFHPE (SEQ ID NO: 2), or a fragment thereof of prostate specific antigen are also embodied by the present invention.
  • the monoclonal antibodies are monoclonal antibodies 15.2, 156, and 225, or monoclonal antibodies having the characteristics of any of these foregoing monoclonal antibodies.
  • Peptide 15-mers having the sequence of SEQ ID NO: 2 are peptides 16-19 of Figure 4.
  • Monoclonal antibodies 10, 11, 16, 22.2 or monoclonal antibodies having the characteristics of any of these foregoing monoclonal antibodies which bind amino acids 139-144, EELFLTP (SEQ ID NO: 3), or a fragment thereof of prostate specific antigen are within the scope of the present invention.
  • Peptides 44-47 of Figure 4 include the sequence of SEQ ID NO: 3.
  • Hybridomas capable of producing any of the monoclonal antibodies identified herein are also embodied by the present invention.
  • antibodies refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. Of these, IgM and IgG are particularly preferred.
  • the antibodies may be monoclonal or polyclonal and may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See, for example., Walker et al. (1989).
  • the antibodies may be recombinant monoclonal antibodies produced according to the methods disclosed in Reading, U.S. Patent No. 4,474,893, or Cabilly et al., U.S. Patent No. 4,816,567.
  • the antibodies may also be chemically constructed by specific antibodies made according to the method disclosed in Segel et al., U.S. Patent No. 4,676,980.
  • Antibodies other than the antibodies set forth above which bind the antigen bound by the above-identified antibodies may be obtained in accordance with known techniques. For example, cells identified as carrying the antigen having amino acids 82-87 bound by the 365 antibody can be washed with an aqueous solution containing a detergent to remove the antigen therefrom, the various fractions in the solution separated by chromatography (e.g., high performance liquid chromatography), and the fraction containing the antigen identified by its ability to bind the antibody.
  • chromatography e.g., high performance liquid chromatography
  • the antibody may be immobilized on a solid support to provide an affinity chromatography column, a sample, such as and not limited to a serum sample, from an appropriate patient (e.g., a patient diagnosed as carrying free prostate specific antigen) passed through the column, antigen bound to the antibody eluted from the column, and the antigen used to produce an antibody.
  • a purified antigen produced by the foregoing processes.
  • Such an antigen is recognized by monoclonal antibody.
  • the antigen may be provided in an aqueous carrier, or in lyophilized form.
  • the antigen may be used to immunize animals to produce polyclonal antibodies, or may be used as an assay standard.
  • the antibodies to an antigen having amino acids 55-60 or an antigen having amino acids 139-144 of prostate specific antigen may be prepared with known techniques similar to those set forth above.
  • Antibodies which bind to the epitope (i.e., the specific binding site) bound by any of the above-identified antibodies can be identified in accordance with known techniques, such as their ability to compete with labelled antibody in a competitive binding assay.
  • Antibody fragments included within the scope of the present invention include, for example, Fab, F(ab')2, and Fv fragments, and the corresponding fragments obtained from antibodies other than IgG. Such fragments can be produced by known techniques.
  • Polyclonal antibodies used to carry out the present invention may be produced by immunizing a suitable animal (e.g., rabbit, goat, etc.) with an antigen to which a monoclonal antibody binds, collecting immune serum from the animal, and separating the polyclonal antibodies from the immune serum, in accordance with known procedures.
  • a suitable animal e.g., rabbit, goat, etc.
  • the monoclonal anti-PSA antibodies are produced by antibody-producing cell lines.
  • the anti-PSA antibody-producing cell lines may be hybridoma cell lines commonly known as hybridomas, produced according to the technique of Kohler and Milstein (1975).
  • the hybrid cells are formed from the fusion of an anti-PSA antibody- producing cell and an immortalizing cell line, that is, a cell line which imparts long term tissue culture stability on the hybrid cell.
  • the first fusion partner, the anti-PSA antibody-producing cell may be a spleen cell of an animal immunized against PSA.
  • the anti-PSA antibody-producing cell may be an anti-PSA generating B lymphocyte obtained from the spleen, peripheral blood, lymph nodes or other tissue.
  • the second fusion partner, the immortal cell may be a lymphoblastoid cell or a plasmacytoma cell such as a myeloma cell, itself an antibody-producing cell but also malignant.
  • Murine hybridomas which produce monoclonal anti-PSA antibodies are formed by the fusion of mouse myeloma cells and spleen cells from mice immunized against PSA. To immunize the mice, a variety of different immunization protocols may be followed. For instance mice may receive primary and boosting immunizations of PSA. The fusions are accomplished by standard procedures such as those disclosed by Kohler and Milstein (1975) and Kennet (1980). The hybridomas are then screened for production of antibody reactive with
  • PSA in accordance with standard methods such as those disclosed by Tarn (1994). Those which secrete reactive antibodies are cloned.
  • Human hybridomas which produce monoclonal anti-PSA antibodies are formed from the fusion of spleen cells from an individual immunized against PSA and a human lymphoblastoid cell line.
  • the fusion partner for the myeloma cell may be a human peripheral blood antibody-producing lymphocyte sensitized against PSA.
  • the fusion and screening techniques are essentially the same as those used in the production and selection of murine anti-PSA generating hybridomas.
  • mouse and human hybridomas which produce human monoclonal anti- PSA antibody may be formed from the fusion of a human antibody-producing cell and a murine plasmacytoma cell.
  • the mouse plasmacytoma cell may be used as a fusion partner for other mammalian antibody-producing cells to form hybridomas which produce anti-PSA antibody of the particular mammal.
  • Another way of forming the anti-PSA antibody-producing cell line is by transformation of antibody-producing cells.
  • an anti-PSA antibody producing B lymphocyte obtained from an animal immunized against PSA, may be infected and transformed with a virus such as the Epstein-Barr virus in the case of human B lymphocytes to give an immortal anti-PSA antibody-producing cell. See, for example, Kozbor and Roder (1983).
  • the B lymphocyte may be alternatively transformed by a transforming gene or transforming gene product.
  • the monoclonal anti-PSA antibodies are produced in large quantities by injecting anti-PSA antibody-producing hybridomas into the peritoneal cavity of mice and, after an appropriate time, harvesting the ascites fluid which contains very high titer of homogenous antibody and isolating the monoclonal anti-PSA antibodies therefrom.
  • Xenogeneic hybridomas should be injected into irradiated or athymic nude mice.
  • the antibodies may be produced by culturing anti-PSA producing cells in vitro and isolating secreted monoclonal anti-PSA antibodies from the cell culture medium.
  • Monoclonal Fab fragments may be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, for example, Huse (1989).
  • the monoclonal anti-PSA antibodies of this invention have a number of important therapeutic uses. Because of their restricted specificity for PSA, the anti-PSA antibodies of this invention may be used to detect and measure free, bound, and total (free plus bound) PSA in a patient sample such as and not limited to fluid including blood, serum, urine, and a tear.
  • the antibodies may be used in conventional immunoassays such as radioimmunoassay or enzyme linked immunoadsorbent assay. For many of these assays an immunoadsorbent is formed by attaching anti-PSA antibody to a solid phase.
  • a sample of the biological fluid to be assayed is contacted with the immunoadsorbent.
  • the mixture is incubated after which a predetermined amount of labeled PSA is added.
  • the immunoadsorbent with the affixed PSA that is, the PSA anti-PSA complex
  • the label is separated from the PSA not affixed thereto and the activity of the label in the affixed PSA fraction is measured in order to determine the amount of PSA in the sample.
  • the label may be a radioisotope, an enzyme, or a fluorescent compound.
  • the anti-PSA antibodies can be provided as reagents in kits for radioimmunoscintigraphy in mammals.
  • kits for radioimmunoscintigraphy in mammals would include the labeled monoclonal anti-PSA antibody or fragments of the antibodies, labeled for example with one of the gamma-emitting radioisotopes.
  • a method of screening for cancer in a patient comprising contacting a patient sample with a monoclonal antibody specific for amino acids 82-87 or a fragment thereof of free prostate specific antigen under conditions permitting the monoclonal antibody to specifically bind an antigen in the sample to form a first antibody-antigen complex; contacting the sample with a monoclonal antibody specific for free and bound prostate specific antigen under conditions permitting the antibody to specifically bind an antigen in the sample to form a second antibody-antigen complex; determining the amount of the first antibody-antigen complex in the sample; determining the amount of the second antibody-antigen complex in the sample; determining the ratio of the first antibody-antigen complex to the second antibody-antigen complex.
  • the method of detecting cancer includes the detection of prostate cancer and breast cancer. In addition the method may be used to determine the presence of benign prostatic hyperplasia (BPH) in a patient.
  • Monoclonal antibodies useful for the detection of free PSA include monoclonal antibodies binding to SEQ ID NO: 1 , such as a monoclonal antibody having the characteristics of monoclonal antibody 365.
  • Monoclonal antibodies useful for the detection of total PSA include monoclonal antibodies binding to SEQ ID NOS: 2 and 3, such as a monoclonal antibody having the characteristics of a monoclonal antibody selected from the group consisting of monoclonal antibodies 10, 11, 15.2, 16, 22.2, 156, and 225.
  • the ratio of free to total PSA of a patient of about 4 to about 10 ng/ml currently require biopsy to determine if the patient has prostate cancer of BPH. Accordingly, the monoclonal antibodies of the present invention, particularly 365 which is specific for free PSA, provide a more accurate diagnosis of prostate cancer or BPH than currently available techniques.
  • the first and second monoclonal antibodies useful in the method of detecting cancer may be labeled such that the label of the first monoclonal antibody is distinguishable from the label for the second.
  • Diagnostic kits for performance of the assays described above include monoclonal anti-PSA antibody or labeled anti-PSA antibody or mixtures of labeled or unlabeled antibody, and a PSA control.
  • the assays described herein provide physicians with a quick and reliable method of ruling out prostate cancer or benign prostatic hype ⁇ lasia in patients. Current techniques for determining a clinical diagnosis of prostate cancer or benign prostatic hyperplasia are time consuming.
  • the monoclonal antibodies, kits, and methods of the present invention would avoid delaying treatment of a cancerous disease.
  • Assays of the present invention quickly rule out prostate cancer and avoid subjecting patients to painful biopsy and the attendant risk of complications, without any significant delay in treatment of those patients who have prostate cancer.
  • mice Five 12-13 week old female Balb/c mice obtained from Charles River Laboratories were immunized according to the method of Cianfriglia (1984). Mice were injected IP with 50 ⁇ g PSA (Scripps) emulsified with an equal volume of Complete Freunds adjuvant (Gibco) in a final volume of 250 ⁇ L on day 1 and day 8. All mice were boosted IP on day 13 with 50 ⁇ g PSA (Scripps) diluted in 250 ⁇ L in Dulbecco's phosphate buffered saline (PBS). On day 14, mice received 10 ⁇ g PSA in 250 ⁇ L PBS administered IP and 10 ⁇ g PSA in 100 ⁇ L PBS given IV.
  • mouse #3 was sacrificed by cervical dislocation on day 16 and the spleen was removed aseptically and immersed into 10 mL of cold PBS containing 100U penicillin, 0.1 mg streptomycin, and 0.25 ⁇ g amphotericin B (PSA) (Sigma).
  • PBS cold PBS containing 100U penicillin, 0.1 mg streptomycin, and 0.25 ⁇ g amphotericin B (PSA) (Sigma).
  • PSA amphotericin B
  • PSA-PBS PSA-PBS.
  • the cells were washed once in cold PSA-PBS, counted and diluted to 2xl0 7 cells/mL.
  • the cells were then layered into Lympholyte-M (Cedar Lane Laboratories Limited) and centrifuged at 500 g for 20 minutes at RT to separate out the lymphocytes.
  • the lymphocyte layer was carefully removed and the cells were washed in PSA-PBS.
  • the remaining mice were reimmunized IP on day 49 with 100 ⁇ g PSA emulsified in an equal volume of Incomplete Freunds adjuvant (Gibco) in a final volume of 250 ⁇ L.
  • Incomplete Freunds adjuvant Gabco
  • mice On day 126, all mice were again boosted IP with 50 ⁇ g PSA (Scripps) diluted in 250 ⁇ L PBS. Two weeks later, the mice were bled by retro-orbital stick without anti-coagulant. The blood was allowed to clot at room temperature and the serum collected. A solid phase EIA-HRP was run to titer the mouse immune serum for anti-PSA activity. On day 150, 2 mice (#1 and #2) were boosted IV with 25 ⁇ g PSA in 250 ⁇ L PBS. Three days after injection, mouse #2 with a titer > 1:200,000 was sacrificed by cervical dislocation and the spleen was prepared as described for fusion #1.
  • PSA Ses
  • Murine Myeloma Fusion Partner A cell bank of the non-secreting Balb/c mouse myeloma fusion partner, FO
  • Both fusions were carried out at a 2:1 ratio of murine myeloma cells to viable spleen cells according to the method of Fazekas de St. Groth. Briefly, approximately 2xl0 8 spleen cells and 4x10 8 myeloma cells were pelleted together. The pellet was gently resuspended over 1 minute in 2 mL of 50% PEG solution (5 g PEG 3,000 (Sigma), 5 mL dH 2 O (Baxter), 500 ⁇ L DMSO (Sigma) at 37°C. The PEG/cell mixture was then immersed in a 37'C water bath for 1.5 to 2 minutes with gentle agitation.
  • the fusion was stopped by slowly adding incremented volumes of PBS (37°C) over 2 minutes.
  • the fused cells were allowed to rest for 5 minutes at RT and pelleted.
  • the cells were washed once in HAT medium ( ⁇ MEM, 20% FBS, 1 mM sodium pyruvate, 0.1 mM NEAA, 2 mM L-glutamine, 25 ⁇ g/mL gentamicin (Sigma)), and HAT (100 ⁇ M hypoxanthine, 0.4 ⁇ M aminopterin, and 16 ⁇ M thymidine, (Sigma)) and then plated at 100 ⁇ L/well in 25 96-well flat bottom plates (Corning) which contained 50 ⁇ L/well PECs as feeder cells.
  • HAT medium ⁇ MEM, 20% FBS, 1 mM sodium pyruvate, 0.1 mM NEAA, 2 mM L-glutamine, 25 ⁇ g/mL gentamicin (Sigma)
  • PECs were prepared by lavaging the peritoneum of naive Balb/c mice with 10 mL of ice cold PSA-PBS. PECs were washed twice and plated in HAT media 2 days prior to fusion. The plates were then placed in a humidified 37°C incubator containing 5% CO 2 and 95% air for 7-10 days.
  • fusion #1 referred to as fP hybridomas
  • fusion #2 referred to as PSA hybridomas
  • Polyclonal sheep anti-human PSA IgG (The Binding Site) was coated into wells of 96-well flat bottom EIA plates (Corning) at 10 ⁇ g/ml in 10 mM carbonate- bicarbonate buffer, pH 9.6, at 100 ⁇ l/well for six hours, at room temperature. After washing with 0.05% tween 20 in H 2 0), plates were blocked with 1 % BSA (Intergen) in PBS, at 250 ⁇ l/well overnight at 4°C.
  • BSA Intergen
  • the blocker was removed and the dilution of either purified PSA (Scripps) or complexed PSA-ACT (present in prostate cancer patient serum) in Incubation buffer (Boehringer-Mannheim) were added to the plates at 2 ng/ml and 35.4 ng/ml, respectively and incubated for two hours, followed by washing as above.
  • the hybridoma supernatants or control antibody dilutions were added at 50 ⁇ l/well, and allowed to react for 3 hours at room temperature.
  • the plates were washed as above and 50 ⁇ l/well of HRP-conjugated sheep anti-mouse IgG (ICN) at 1 :500 dilution in 1 % BSA- PBS was added and incubated for one hour at room temperature. After washing, the reaction was visualized by adding 100 ⁇ l of OPD (Sigma) solution in 25 mM citric acid, 50 mM sodium phosphate, 10 mM 2-chloracetamide and 0.01 % H 2 0 2 and incubated for 30 minutes in the dark. The reaction was terminated by adding 50 ⁇ l of 4N H 2 SO 4 and the OD was determined at 490-650 nm using a Kinetic Microplate Reader (Molecular Devices).
  • OPD OPD
  • the antibodies from fusion #1 and fusion #2 that were reactive equally to both free PSA and complexed PSA (cancer serum) were considered as having anti-total PSA reactivity and those preferentially reactive to only free PSA were considered as having anti-free PSA reactivity.
  • the monoclonal antibodies 156, 15.2, and 22.2 were determined as anti-total PSA clones, the monoclonal antibodies 365, 225, 10, 11 , and 16 as anti-free PSA clones.
  • fP165 from fusion #1
  • PS A3 and PSA15 from fusion #2
  • Streptavidin coated microtiter plates were obtained from Labsyste s. HRP- conjugated goat anti-mouse IgG was purchased from Pierce. O-phenylene diamine was purchased from Sigma.
  • Peptides were synthesized on an ACT396 using FMOC chemistry on Rink resin and the standard double coupling DIC/HOBt protocol. 15-mers overlapping by 12 amino acids were synthesized covering the entire length of PSA. The sequence of PSA is that disclosed by Lundwall, 1989. All cystines were replace with ⁇ -amino-L-butyric acid. After completion of the 15th amino acid coupling, FMOC-L-Lys (biotin) was coupled to the peptide resin. Deprotection and subsequent acetylation gave the fully protected peptide amide resins.
  • Peptides were simultaneously cleaved from the resin and deprotected using 1 ml of a mixture of 22.5 g phenol, 15 g dithiothreitol, 15 ml thianisole, 15 ml water and 300 ml trifluoroacetic acid for 2 hours at ambient temperature.
  • the resin was removed by filtration and the resulting solution added to 15 ml methyl-t-butylether.
  • the precipitated peptide was isolated by centrifugation,, washed three times by resuspension in methyl-t- butylether with vigorous vortexing followed by centrifugation and dried under reduced pressure at ambient temperature. The peptides were used without further purification.
  • Fifty ⁇ l of peptide (concentration 3.5 ⁇ M in % BSA in PBS) was added to the wells and incubated at room temperature for three hours. After this, and every consecutive incubation step, the plates ere washed three times with deionized water containing 0.05% Tween-20. Fifty ⁇ l culture supernatant containing the test antibodies in 1 to 10 ⁇ g/ml concentrations was added to the wells and incubated overnight (16 hours) at 4 ° C.
  • reaction was detected by consecutive incubations with an HRP-conjugated goat anti-mouse IgG (H+L specific) reagent Pierce, 1 :10,000 dilution, 50 ⁇ l/well, 90 minutes at room temperature) and 100 ⁇ l substrate-chromogen reagent (1 mg/ml o-phenylene diamine (Sigma) in citrate buffer, 0.4 ⁇ g/ml hydrogen peroxide, 30 minutes).
  • substrate-chromogen reagent 1 mg/ml o-phenylene diamine (Sigma) in citrate buffer, 0.4 ⁇ g/ml hydrogen peroxide, 30 minutes.
  • the reaction was terminated with 50 ⁇ l 4 NH 2 SO 4 and the absorbance read at 490 ⁇ m-650 ⁇ m wavelength using a microtiter plate reader (Molecular Devices). Buffer without peptide was used as a negative antigen control and an unrelated mouse antibody was used as a negative control for the test antibodies.
  • the reaction of an antibody to a peptide sequence was considered significant only if the absorbance exceeded the respective background level by at least three fold and at least two consecutive peptide sequences reacted.
  • Amino acids 21 and 22 were inserted from the corresponding sequence of kallikrein.
  • the PSA sequence WQ...GG was excised and replaced with the WQ...GG kallikrein loop. Amino acids were mutated and side chains adjusted were necessary.
  • the 25 structures coalesced into four families of similar energy, one concatenated structure and three with large loops having no interactions with the rest of the structure.
  • Two of the families of structures of similar energy have the 83-88 sequence buried within the molecule. Since this sequence is an epitope recognized by some of the anti-PSA antibodies, these structures are inconsistent with the mapping data.
  • the remaining two families of structures have the 83-88 loop in distinctly different conformations.
  • the catalytic triad of His 57 , Asp 102 , and Ser 195 is well conserved and accessible for substrate interactions.
  • neither of these structures are the loops identical with the one in the theoretical structure PDB1PSA.ENT in the Brookhaven crystallographic database.
  • biotinylated peptides were selected from different regions of PSA to determine the optimum coating concentration.
  • Peptides were dissolved in 1% BSA in PBS. From an initial concentration of 70 ⁇ M, 5 fold serial dilutions were made to a concentration of 4.5 nM. Titration curves were generated and it was determined that peptide concentrations of 3.5 ⁇ M were sufficient to saturate all biotin binding sites on the streptavidin plates. Incubation times between 3 hours at ambient temperature and overnight at 4°C gave identical results. All subsequent studies were done using peptide concentrations of 3.5 ⁇ M.
  • the common sequence of peptides 17-19 is SLFHPEDTG, SEQ ID NO: 9 (55-63), a sequence that is surface exposed on the PSA model.
  • the common sequence to peptides 32-33 is LMLLRLSEPAEL, SEQ ID NO: 10 (97-108), but only SEPAEL, SEQ ID NO: 11 (103-108) is surface exposed.
  • the common sequence of peptides 44-47 is the surface exposed EEFLTP, SEQ ID NO: 12 (140-145).
  • Peptide 53, NDVCAQVHPQKVTKF, SEQ ID NO: 13 (158-172) is completely surface exposed.
  • Monoclonal 15.2 recognizes the surface exposed sequence SLFHPEDTG, SEQ ID NO: 9 (55-63) defined by peptides 17-19 and monoclonal 16 recognizes LTP (143-145), the sequence common to peptides 44-48. These are similar to the epitopes defined by the mouse #2 polyclonal antisera. Although not spatially adjacent, the distance between these two epitopes is such that antibodies recognizing one epitope could be experimentally competitive with antibodies recognizing the other. In addition to the binding clusters at peptides 55-57 and 72-74, the antibody also shows reproducible binding to peptide 14.
  • TKF TKF (170-172) is surface exposed and peptides 72-74 define the surface exposed sequence VHYRKW, SEQ ID NO: 14 (224-229). Although not sequentially contiguous, these two sequences are topologically adjacent and define a single conformational epitope. Although peptide 14 is totally surface exposed, its distance from the other two sequences is larger than the size of an antibody binding domain.
  • the anti-PSA monoclonal antibodies recognizing linear sequences bind to peptides identifying two epitopes. These fall within peptides 16-19 and 44-47. Although similar, the epitopes are not identical.
  • the epitope of monoclonal is 15.2 is defined by peptides 17-19 while that of monoclonal 225 is defined by peptides 16-18. Although these two antibodies would be expected to compete, the epitopes are not identical on the amino acid level.
  • the epitopes of the polyclonal antisera are similar to those of the monoclonals in that binding is observed to both the 16-19 and 44-47 peptide regions, although peptides 42 and 43 are also recognized by some antisera. There are, however, additional epitopes recognized as well. It cannot be determined if all epitopes are recognized by the polyclonal antisera are linear or conformational. The epitopes cover a significant portion of one face of the PSA molecule.
  • the biotin was attached to the epsilon amino group of an additional alpha-acetyl-lysine on the N-terminus.
  • the peptides were captured on a streptavidin lawn, followed by the anti-PSA antibodies. Quantitation of peptide-bound antibodies was achieved using HRP-conjugated antisera.
  • a summary of the binding of monoclonal antibodies and polyclonal antisera to PSA epitopes is shown in Figures 2A and 2B.
  • a model of PSA was constructed using homology modeling.
  • the serine proteases tonin and kallikrein were used, with tonin as the template and kallikrein serving as the source of a loop present in PSA but absent in tonin.
  • a loop search was done for PSA (83-88), a sequence for which no serine protease has a homologous region.
  • Two similar structures were generated, differing only in the 83-88 loop. These models are similar to a published PSA model, with the major difference bing the 83-88 loop.
  • Monoclonal antibody 365 recognizes the sequences TKFMLCAGR (SEQ ID NO: 15), PSA (169-177), and TKVVHYRKW (SEQ ID NO: 16), PSA (220-228) and also the flexible loop LKNRFL (SEQ ID NO: 1), PSA (82-87). Examination of the model shows that only TKF, PSA (160-171), and VHYRKW, SEQ ID NO: 17, PSA (223-228), are surface exposed. Although noncontinuous, these sequences are topologically adjacent, consistent with them being a conformational epitope. The binding of monoclonal antibody 365 to SEQ ID NO: 1 is significant since the loop region comprising SEQ ID NO: 1 is only conformational ly available in free PSA.
  • the polyclonal sera tested define additional epitopes in the 1-126 region.
  • PSA and HK2 share a 79% amino acid identity.
  • PSA epitopes of the anti-PSA antibodies are highly homologous or identical to corresponding sequences in HK2, suggesting the possibility of cross reactivity to HK2.
  • the fine mapping of anti-PSA antibodies allows for the selection of antibodies to unique PSA sequences, avoiding potential cross reactivity.
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
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  • SEQUENCE DESCRIPTION SEQ ID NO:24:
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  • HYPOTHETICAL NO
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  • SEQUENCE DESCRIPTION SEQ ID NO:26:
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  • HYPOTHETICAL NO
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  • SEQUENCE DESCRIPTION SEQ ID NO: 30:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
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  • HYPOTHETICAL NO
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  • SEQUENCE DESCRIPTION SEQ ID NO:36:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
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  • SEQUENCE DESCRIPTION SEQ ID NO:40:
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  • HYPOTHETICAL NO
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  • HYPOTHETICAL NO
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  • SEQUENCE DESCRIPTION SEQ ID NO:44:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
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  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
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  • HYPOTHETICAL NO
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  • SEQUENCE DESCRIPTION SEQ ID NO:50:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIP ⁇ ON SEQ ID NO:54:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:56:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIP ⁇ ON SEQ ID NO:58:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
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  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 64:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:66:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
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  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:68:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
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  • SEQUENCE DESCRIPTION SEQ ID NO:70:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
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  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
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  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ANTI-SENSE NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83: Leu Val Cys Asn Gly Val Leu Gin Gly He Thr Ser T ⁇ Gly Ser 1 5 10 15
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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Abstract

La présente invention concerne des anticorps monoclonaux utilisés dans la détection de l'antigène prostatique spécifique. Plusieurs anticorps monoclonaux lient l'antigène prostatique spécifique libre et lié, et un des anticorps monoclonaux lie l'antigène prostatique spécifique. Cette invention concerne également des procédés de détection du cancer tel que le cancer de la prostate.
PCT/US1997/014909 1996-09-06 1997-08-25 Anticorps monoclonaux contre l'antigene prostatique specifique et procedes de detection de l'antigene prostatique specifique WO1998010292A1 (fr)

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AU40863/97A AU4086397A (en) 1996-09-06 1997-08-25 Monoclonal antibodies specific for prostate specific antigen and methods of detecting prostate specific antigen

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0947832A1 (fr) * 1998-04-03 1999-10-06 Roche Diagnostics GmbH Procédé immunologique de détermination de PSA
WO1999065523A1 (fr) * 1998-06-15 1999-12-23 Altarex Corp. Composition et methode immunotherapeutiques de traitement du cancer de la prostate
WO2000066718A1 (fr) * 1999-04-30 2000-11-09 Hybritech Incorporated Formes de l'antigene prostatique specifique (aps) specifiques a l'hyperplasie prostatique benigne (hpb) et procedes de leur utilisation
FR2813394A1 (fr) * 2000-08-25 2002-03-01 Urogene Procede de dosage de l'antigene specifique de la prostate, procede de diagnostic in vitro du cancer de la prostate, et kits pour la mise en oeuvre de ces procedes
US11365234B2 (en) 2015-03-27 2022-06-21 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
AU2020256298B2 (en) * 2015-03-27 2022-07-14 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides for use in immunotherapy against various tumors
AU2021202060B2 (en) * 2015-08-05 2023-06-15 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides for use in immunotherapy against prostate cancer and other cancers

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992001936A1 (fr) * 1990-07-23 1992-02-06 Hans Lilja Analyse de l'antigene specifique de la prostate (psa) a l'etat libre et a l'etat formant complexe
WO1995003334A1 (fr) * 1993-07-22 1995-02-02 Mayo Foundation For Medical Education And Research Anticorps specifiques contre la kallicreine glandulaire humaine de la prostate

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1992001936A1 (fr) * 1990-07-23 1992-02-06 Hans Lilja Analyse de l'antigene specifique de la prostate (psa) a l'etat libre et a l'etat formant complexe
WO1995003334A1 (fr) * 1993-07-22 1995-02-02 Mayo Foundation For Medical Education And Research Anticorps specifiques contre la kallicreine glandulaire humaine de la prostate

Non-Patent Citations (3)

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Title
CLINICAL CHEMISTRY, April 1997, Vol. 43, No .4, COREY et al., "Prostate-Specific Antigen: Characterization of Epitopes by Synthetic Peptide Mapping and Inhibition Studies", pages 575-584. *
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1994, Vol. 220, CHRISTENSSON et al., "Complex Formation Between Protein C Inhibitor and Prostate-Specific Antigen In Vitro and in Human Semen", pages 45-53. *
PROTEIN SCIENCE, May 1996, Vol. 5, VILLOUTREIX et al., "Structural Investigation of the Alpha-I-Antichymotrypsin: Prostate-Specific Antigen Complex by Comparative Model Building", pages 836-851. *

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0947832A1 (fr) * 1998-04-03 1999-10-06 Roche Diagnostics GmbH Procédé immunologique de détermination de PSA
US6361955B1 (en) 1998-04-03 2002-03-26 Roche Diagnostics Gmbh Immunological process for PSA determination
WO1999065523A1 (fr) * 1998-06-15 1999-12-23 Altarex Corp. Composition et methode immunotherapeutiques de traitement du cancer de la prostate
US6881405B2 (en) 1998-06-15 2005-04-19 Altarex Medical Corp. Reagents and methods for inducing an immune response to prostate specific antigen
US7579445B2 (en) 1998-06-15 2009-08-25 Altarex Medical Corp. Reagents and methods for inducing an immune response to prostate specific antigen
WO2000066718A1 (fr) * 1999-04-30 2000-11-09 Hybritech Incorporated Formes de l'antigene prostatique specifique (aps) specifiques a l'hyperplasie prostatique benigne (hpb) et procedes de leur utilisation
FR2813394A1 (fr) * 2000-08-25 2002-03-01 Urogene Procede de dosage de l'antigene specifique de la prostate, procede de diagnostic in vitro du cancer de la prostate, et kits pour la mise en oeuvre de ces procedes
US11407810B2 (en) 2015-03-27 2022-08-09 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
US11440947B2 (en) 2015-03-27 2022-09-13 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
AU2020256298B2 (en) * 2015-03-27 2022-07-14 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides for use in immunotherapy against various tumors
US11407808B2 (en) 2015-03-27 2022-08-09 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
US11407809B2 (en) 2015-03-27 2022-08-09 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
US11365234B2 (en) 2015-03-27 2022-06-21 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
US11407807B2 (en) 2015-03-27 2022-08-09 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
US11434273B2 (en) 2015-03-27 2022-09-06 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
US11434274B2 (en) 2015-03-27 2022-09-06 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
US11365235B2 (en) 2015-03-27 2022-06-21 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
US11459371B2 (en) 2015-03-27 2022-10-04 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
US11466072B2 (en) 2015-03-27 2022-10-11 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
US12060406B2 (en) 2015-03-27 2024-08-13 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
US11702460B2 (en) 2015-03-27 2023-07-18 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
US11873329B2 (en) 2015-03-27 2024-01-16 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
US11897934B2 (en) 2015-03-27 2024-02-13 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
US11965013B2 (en) 2015-03-27 2024-04-23 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
US12006349B2 (en) 2015-03-27 2024-06-11 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
US12018064B2 (en) 2015-03-27 2024-06-25 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
AU2021202060B2 (en) * 2015-08-05 2023-06-15 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides for use in immunotherapy against prostate cancer and other cancers

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