WO1993014775A1 - Anticorps monoclonal pd41 et antigene associes aux carcinomes de la prostate - Google Patents

Anticorps monoclonal pd41 et antigene associes aux carcinomes de la prostate Download PDF

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Publication number
WO1993014775A1
WO1993014775A1 PCT/US1992/000852 US9200852W WO9314775A1 WO 1993014775 A1 WO1993014775 A1 WO 1993014775A1 US 9200852 W US9200852 W US 9200852W WO 9314775 A1 WO9314775 A1 WO 9314775A1
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Prior art keywords
prostate
antigen
monoclonal antibody
mab
carcinoma
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PCT/US1992/000852
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English (en)
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George L. Wright, Jr.
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Wright George L Jr
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Priority to PCT/US1992/000852 priority Critical patent/WO1993014775A1/fr
Priority to CA002106487A priority patent/CA2106487A1/fr
Priority claimed from CA002106487A external-priority patent/CA2106487A1/fr
Publication of WO1993014775A1 publication Critical patent/WO1993014775A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a novel monoclonal antibody that shows preferential binding to prostate carcinoma tissue with little or no cross-reactivity to benign prostatic hyperplasia or to normal prostate epithelium, as well as a hybridoma cell line and method for producing the antibody. Additionally, the present invention relates to a novel antigen with which the antibody reacts specifically.
  • MAbs monoclonal antibodies
  • PAP prostatic acid phosphatase
  • PSA prostate-specific antigen
  • Additional MAbs that have shown potential for detecting circulating prostate antigens in patient serum include TURP-27 (Starling et al., 1986, Cancer Res. 4_6:367-374) , 7E11-C5 (Horoszewicz et al., 1987, Anticancer Res. 2:927-936) and PR92 (Kim et al., 1988, Cancer Res. 8:4543-4548).
  • tumor antigens that are either prostate-organ specific, such that they react with normal and benign prostate antigens as well as carcinoma, i.e., PSA, PAP, 7E11-C5 and TURP-27, or they cross-react with non-prostate cells or carcinoma, i.e., PR92 cross- reacts with breast carcinomas.
  • the present invention encompasses hybridoma cell lines that produce novel monoclonal antibodies and the monoclonal antibodies (and fragments thereof) that show preferential specific binding to prostate carcinoma with little to no binding to benign prostatic hyperplasia or to normal prostate epithelium.
  • the invention is directed to a hybridoma cell line PD41, having ATCC
  • the present invention further encompasses other monoclonal antibodies that bind to or recognize the PD41 antigen, designated the prostate mucin antigen (PMA) , as well as monoclonal antibodies that competitively inhibit the binding of the PD41 monoclonal antibody produced by the hybridoma cell line ATCC Accession No. HB to PMA, as measured by an enzyme immunoassay, a radioimmunoas ⁇ ay or other competitive inhibition immunoassay.
  • PMA prostate mucin antigen
  • the present invention additionally encompasses a novel antigen, PMA, in isolated or substantially pure form, to which the monoclonal antibodies of the invention bind, as well as methods and kits for using the antigen and/or the antibodies for detection or treatment or prophylaxis of prostate carcinoma.
  • the present invention also encompasses kits for using the monoclonal antibodies and/or antigen for in vitro or n vivo applications for diagnosis, monitoring, prophylaxis or therapy of prostate carcinoma.
  • the invention encompasses compounds comprising the antigen binding region of the monoclonal antibodies of the invention or portions thereof, including Fv, F(ab*) 2 , Fab fragments, chimeric antibodies, humanized antibodies, single chain antibodies, complementarity determining regions (CDRs) , etc.
  • the invention encompasses the use of the hybridoma cell lines as a source of DNA or RNA encoding for the rearranged, activated immunoglobulin genes, which may be isolated, cloned by recombinant DNA techniques and transferred to other cells for the production of specific immunoglobulin specific for prostate carcinoma.
  • a sequence free of introns may be obtained.
  • the invention encompasses the nucleotide sequence encoding the PMA antigen of this invention.
  • MAb monoclonal antibody
  • PAP prostatic acid phosphatase
  • PSA prostate-specific antigen
  • PMA prostate mucin antigen
  • PBS phosphate-buffered saline
  • TBS Tris-buffered saline
  • BPH benign prostatic hyperplasia
  • CDR complementarity determining region
  • CaP prostate adenocarcinoma
  • NCA non-cross-reacting antigen
  • TAA tumor-associated antigen
  • TCC transitional cell carcinoma
  • BSM bovine submaxillary mucin
  • FIG. l(A-F) illustrates staining patterns of prostate carcinoma tissue sections with MAb PD41.
  • FIG. 1A well- to moderately-differentiated prostate adenocarcinoma showing cytoplasmic staining of epithelial cells of neoplastic ducts as well as luminal secretions (upper left) and no staining of benign and normal ducts (arrows)
  • FIG. IB well- to moderately-differentiated prostate adenocarcinoma showing intense staining of all tumor cells in a large cribriform neoplastic duct and little to no staining in smaller neoplastic ducts.
  • X 200 FIG.
  • FIG. ID poorly differentiated prostate carcinoma with intense 5 staining of tumor cells in most neoplastic ducts.
  • FIG. IE undifferentiated prostate carcinoma with very few tumor cells (arrows) staining.
  • X 200 undifferentiated prostate carcinoma with very few tumor cells (arrows) staining.
  • FIG. IF section from bone metastasis showing a large nest of intensely stained prostate tumor cells and no v staining in cartilage and non-tumor tissue.
  • FIG. 2 is a scattergram representing the percentage of cells staining with PD41 MAb in fixed or frozen well differentiated (WD) , moderately 5 differentiated (MD) , poorly differentiated (PD) , and undifferentiated (UD) prostate carcinomas. Symbols indicate, percent of positive cells in a given tumor sample.
  • FIG. 3 is a representative Western immunoblot of normal, benign and carcinoma tissues.
  • FIG. 3A is a blot of tissues or fluid samples reacted with MAb PD41: prostate carcinoma membrane extracts (Lanes l, 2 and 3) ; CaP seminal plasma (Lane 4) ; membrane extracts from breast carcinoma (Lane 5) ; colon carcinoma (Lane 6) ; normal prostate (Lanes 7 and 8) ; BPH (Lanes 9 and 10) ; and normal seminal plasma (Lane 11) , respectively.
  • FIG. 3B is a blot as in FIG. 3A, but reacted with an isotype-matched negative control antibody.
  • Blots were transferred from a 3-15% gradient SDS-PAGE gel (50 ⁇ g of protein loaded per Lane) onto immobilon-P transfer membrane and blotted. Blots were exposed to x-ray film for 72 hours. SG indicates top of separating gel. See text for details.
  • FIG. 4 is a graphic representation of a competitive binding assay of 1-125 labeled PD41 MAb against MAbs B72.3, anti-CEA, HMFG-2 and PD41. Each curve represents the mean cpm of triplicate determinations performed in duplicate experiments.
  • FIG. 5 is graphic representation of a double determinant immunoradiometric assay. See text for details and Table 9 for identification of the tumor- associated MAbs (competing MAbs) .
  • FIG. 6 is a graphic representation of a reciprocal blocking experiment. See text for details and Table 9 for identificati.on of the mucin tumor- associated MAbs. Symbols are as follows: where "Block” represents blocking MAb and "Trace” represents radiolabelled MAb: O—O Block: B72.3, Trace: B72.3; •—• Block: PD41, Trace: B72.3, CEA, M344, OC125; ⁇ — ⁇ Block: CEA, Trace: CEA; D—D Block: M344,
  • FIG. 7 is a graphic representation of a double determinant immunoradiometric assay to assess epitope co-expression. Symbols indicate: D Antigen 1; D Antigen 2. See text for details and Table 9 for identification of the tumor-associated MAbs.
  • the present invention is directed to hybridoma cell lines, that produce monoclonal antibodies, and monoclonal antibodies specific for prostate carcinoma that are advantageously useful for detection, diagnosis and/or monitoring and for prophylaxis or treatment of pathological disease associated with prostate carcinoma. More particularly, the present invention encompasses a novel hybridoma cell line, which produces a monoclonal . . . antibody that shows preferential binding to prostate carcinoma, with little or no specific binding to benign prostatic hyperplasia or to normal prostate epithelium. The invention further encompasses the monoclonal antibody specific for prostate carcinoma, . . . which shows little or no binding to benign hyperplasia or normal prostate. The invention also encompasses other prostate carcinoma specific monoclonal antibodies that bind specifically with the novel antigen of the invention as well as antibodies that competitively inhibit binding of the antibody to the novel antigen of the invention.
  • the present invention is directed to a novel antigen, PMA, with which the monoclonal antibodies of the invention react, as well as methods and kits for using the antigen and/or antibodies for detection, prophylaxis or treatment of prostate carcinoma.
  • the description of the invention is divided into the following sections: (a) preparation of hybridoma cell lines and monoclonal antibodies; (b) characterization of the monoclonal antibodies and the novel antigen; and (c) applications for which the hybridoma cell lines, monoclonal antibodies and the antigen are suited.
  • the epitope recognized by the antibody of this invention is present in primary prostate carcinomas; including poorly-, moderately- and well-differentiated tumors; in metastatic prostate carcinomas; in seminal plasma; split ejaculates; and in prostatic fluids of prostate carcinoma patients as well as in a cultured colorectal carcinoma cell line LS180, dialyzed spent culture medium and the glycopeptide fraction digest of the LS180 cell line and in bovine submaxillary gland.
  • such cells or fluids and/or membrane fractions or extracts thereof also represent potential "antigen" or sources of immunogen with which to immunize animals or cells to obtain somatic cells for fusion to generate antibodies of the invention.
  • Somatic cells with the potential for producing antibody and, in particular B lymphocytes, are suitable for fusion with a B-cell myeloma line.
  • Somatic cells may be derived from the lymph nodes, spleens and peripheral blood of primed animals and the lymphatic cells of choice depend to a large extent on their empirical usefulness in the particular fusion system.
  • Once-primed or hyperimmunized animals can be used as a source of antibody-producing lymphocytes.
  • Mouse lymphocytes give a higher percentage of stable fusions with the mouse myeloma lines described below. Of these, the BALB/c mouse is preferred. However, the use of rat, rabbit, sheep and frog cells is also possible. As reviewed by Goding (in Monoclonal Antibodies: Principles and Practice, 2d ed. , pp. 60- 61, Orlando, Fla, Academic Press, 1986) use of rat lymphocytes may provide several advantages.
  • human somatic cells capable of producing antibody are suitable for fusion with myeloma cell lines. While B lymphocytes from biopsied spleens, tonsils or lymph nodes of individual may be used, the more easily accessible peripheral blood B lymphocytes are preferred.
  • the lymphocytes may be derived from patients with diagnosed prostate carcinomas.
  • Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of the desired hybridomas.
  • myeloma cell lines may be used for the production of fused cell hybrids of the invention, including P3-X63/Ag8, X63-Ag8.653, NSl/l.Ag 4.1,
  • mice S194/5XX0 Bul, all derived from mice; R210.RCY3, Y3-Ag
  • IR983F and 4B210 derived from rats and ⁇ -266, GM1500-GRG2, LICR-LON-HMy2 , UC729-6, all derived from humans (Goding in Monoclonal Antibodies: Principles and Practice, 2d ed. , pp. 65-66, Orlando, Fla, Academic Press, 1986; Campbell, d-n Monoclonal Antibody 5 Technology, Laboratory Techniques in Biochemistry and Molecular Biology Vol. 13, Burden and Von Knippenberg, eds. pp. 75-83, Amsterdam, Elseview, 1984).
  • Methods for generating hybrids of antibody- producing spleen or lymph node cells and myeloma cells Q usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion as in the example below, (though the proportion may vary from about 20:1 to about 1:1), respectively, in the presence of an agent or agents (chemical or electrical) that promote the 5 fusion of cell membranes. It is often preferred that the same species of animal serve as the source of the somatic and myeloma cells used in the fusion procedure. Fusion methods have been described by Kohler and Milstein [Nature 256:495-497 (1975) and 0 Eur. J. Immunol. 6.:511-519 (1976)], and by Gefter et al. [Somatic Cell Genet.
  • the fusion-promotion agents used by those investigators were Sendai virus and polyethylene glycol (PEG) , respectively. Fusion methods reviewed by Goding (in 5 Monoclonal Antibodies: Principles and Practice, 2d ed. , pp. 71-74, Orlando, Fla, Academic Press, 1986) including the above as well as electrically induced fusion are also suitable to generate monoclonal antibodies of the invention. 0 Fusion procedures usually produce viable hybrids at very low frequency, about 1 x 10" 6 to 1 x
  • the fused cells are cultured in 5 selective media, for instance HAT medium containing hypoxanthine, aminopterin and thymidine.
  • HAT medium permits the proliferation of hybrid cells and prevents growth of unfused myeloma cells which normally would continue to divide indefinitely.
  • Aminopterin blocks I Q de novo purine and pyrimidine synthesis by inhibiting the production of tetrahydrofolate.
  • the addition of thymidine bypasses the block in pyrimidine synthesis, while hypoxanthine is included in the media so that inhibited cells synthesize purine using the nucleotide
  • the myeloma cells employed are mutants lacking hypoxanthine phosphoribosyl transferase (HPRT) and thus cannot utilize the salvage pathway.
  • HPRT hypoxanthine phosphoribosyl transferase
  • the B lymphocyte supplies genetic information for production of this 0 enzyme. Since B lymphocytes themselves have a limited life span in culture (approximately two weeks) , the only cells which can proliferate in HAT media are hybrids formed from myeloma and spleen cells.
  • the mixture of fused myeloma and B lymphocytes is diluted in HAT medium and cultured in multiple wells of microtiter plates.
  • the supernatant fluid of the individual wells containing hybrid clones is assayed for specific antibody.
  • the assay must be sensitive, simple and rapid. Assay techniques include radioimmunoassays, enzyme immunoassays, cytotoxicity 35 assays, plaque assays, dot immunobinding assays, and the like.
  • each cell line may be propagated in either of two standard ways.
  • a sample of the hybridoma can be injected into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion.
  • the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can be tapped to provide monoclonal antibodies in high concentration.
  • the individual cell lines may be propagated i-n vitro in laboratory culture vessels; the culture medium, also containing high concentrations of a single specific monoclonal antibody, can be harvested by decantation, filtration or centrifugation.
  • PD41 a new hybridoma cell line, that produces a novel MAb, PD41, that reacts selectively with prostate adenocarcinoma was generated.
  • the present invention encompasses not only the PD41 MAb, but also, other monoclonal antibodies that bind specifically to PMA as well as any MAbs that competitively inhibit the binding of the PD41 MAb to PMA as assessed in an enzyme immunoassay, a radioimmunoassay or any other competitive binding immunoassay.
  • the immunohistochemical reactivity of MAb PD41 is highly restricted to prostate carcinoma tissues, in particular ductal epithelia and secretions of prostate adenocarcinoma tissues. Sixty-five percent of the prostate tumor specimens examined stained with MAb
  • MAb PD41 reacts only minimally with normal prostate tissues, as less than 1% of the epithelial cells of normal tissue specimens appear to stain, and then, only weakly. Moreover, MAb 0 PD41 does not react with nonprostate carcinomas or a variety of normal non-prostate human tissues, although it is reactive with metastatic prostate carcinoma, e.g. , in lymph nodes.
  • MAb PD41 reacts with about 53% of prostatic intraepithelial neoplasia (PIN) lesions adjacent to PD41 positive staining tumor areas in primary prostate carcinoma.
  • PIN prostatic intraepithelial neoplasia
  • PIN-positive reactivity in areas of PIN could be predictive of disease potential.
  • MAb PD41 does not react with available cultured human prostate tumor cell lines, including DU145, PC3, PC3- 5 p. LNCaP and PPC-1, with human blood cells, or with purified antigens, including prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) , using both radioimmunoassay and im unofluorescence procedures.
  • PSA prostate-specific antigen
  • PAP prostatic acid phosphatase
  • MAb PD41 binds specifically to target
  • ⁇ - anti .gen present i .n seminal plasma obtained from prostate carcinoma patients, but not to seminal plasma from normal donors.
  • MAb PD41 can also be distinguished from other MAbs that bind specifically to prostate
  • MAb PD41 also can be shown to be distinct from the anti-prostate MAbs PR92 (Kim et al., 1988, Cancer Res. 8 . :4543-4548) and 7E11-C5 (Horoszewicz et al. , Anticancer Res. 2:927-936) on the basis of their respective antigens and cell-line reactivity.
  • the PR92 antigen is reported to be a glycoprotein with an approximate molecular weight of 470,000 (unreduced) and 44,000 (reduced), whereas the antigen defined by 7E11-C5 consists of a single M, 100,000 band (reduced) (Wright et al. , 1990, Radiopharm. 3_:89, Abst. 193).
  • PD41 also can be distinguished from the PR92 and 7E11- C5 MAbs, as well as several other prostate-directed MAbs, i.e., TURP-73 and TURP-27 (Starling et al. , 1986, Cancer Res. .46:367-374), MAbs 35 and 24 (Frankel et al., 1982, Proc. Nat'l Acad. Sci. USA 79:903-907) ,
  • the immunoblots of gel-separated components of prostate carcinoma tissue extracts indicate that the molecular weight of the proteins carrying the PD41 antigenic determinant differ among individual tumors, ranging from about M r 90,000 to greater than about 400,000.
  • the predominant component carrying the PD41 antigenic determinant is the diffuse M r > 400,000 band.
  • MAb PD41 is distinct from other mucin- directed MAbs and that PMA is distinct from other mucin antigens. Additional support is provided by the inability of MAbs to other mucin-like TAAs to block PD41 binding to its target antigen. (See Section 6.4, below) .
  • Monoclonal antibodies of the present invention can be used to detect potential prostate carcinoma cells in histological and cytological specimens, and, in particular, to distinguish malignant tumors from normal tissues and non-malignant tumors.
  • monoclonal antibodies of this invention stain (1) strongly to very strongly in well and moderately differentiated primary prostate carcinomas; (2) moderately to very strongly in poorly differentiated primary prostate carcinoma; and (3) moderately to strongly in undifferentiated primary prostate carcinomas.
  • strong staining was observed in metastatic prostate carcinoma in lymph node, bone, breast and lung metastases.
  • the PD41 MAb did not bind to fetal prostate tissues, frozen normal prostate tissues and fixed BPH tissues.
  • immunofluorescence techniques can use the monoclonal antibodies of the present invention to examine human specimens. In a typical protocol, slides containing cryostat sections of frozen, unfixed tissue biopsy samples or cytological smears are air dried, formalin or acetone fixed, and incubated with the monoclonal antibody preparation in a humidified chamber at room temperature.
  • the slides are then washed and further incubated with a preparation of antibody directed against the monoclonal antibody, usually some type of anti-mouse immunoglobulin if the monoclonal antibodies used are derived from the fusion of a mouse spleen lymphocyte and a mouse myeloma cell line.
  • This anti- mouse immunoglobulin is tagged with a compound, for instance rhodamine or fluorescein isothiocyanate, that fluoresces at a particular wavelength.
  • the staining pattern and intensities within the sample are then determined by fluorescent light microscopy and optionally photographically recorded.
  • computer enhanced fluorescence image analysis or flow cytometry can be used to examine tissue specimens or exfoliated cells, i.e., single cell preparations from aspiration biopsies of prostate tumors using the monoclonal antibodies of the invention.
  • MAb PD41 reacts with about 53% of prostatic intraepithelial neoplasia (PIN) lesions adjacent to PD41 positive staining areas.
  • PIN prostatic intraepithelial neoplasia
  • monoclonal antibodies of the invention can be used to examine PIN areas in histological and cytological specimens. Positive PD41 reactivity in such PIN areas may be useful to predict disease progression.
  • the monoclonal antibodies of the invention could be used in quantitation of the fluorescing tumor cells on tissue slides or exfoliated cells, i.e., single cell preparations from aspiration biopsies of prostate tumors by computer enhanced fluorescence image analyzer or with a flow cytometer.
  • Use of MAb PD41 in such assays would be valuable to differentiate benign from malignant prostate tumors since the PMA antigen to which the monoclonal antibody binds is expressed only by malignant tumors.
  • the percent PMA reactive cell population, alone or in conjunction with determination of the DNA ploidy of these cells, may, additionally, provide very useful prognostic information by providing an early indicator of disease progression. (See, Wright et al. , 1990, Cancer 65.:1242-1252, McGowan et al. , 1990, Amer. J. Surg. 159.:172-177) .
  • the monoclonal antibodies of the present invention can be used in combination with other known prostate MAbs to provide additional information regarding the malignant phenotype of a prostate carcinoma.
  • the monoclonal antibody of the invention can be used in immunohistological or immunocytological tests as part of a panel of MAbs including such as the P25 MAbs of Bazinet et al. (supra) to distinguish early evidence of neoplastic change (PD41 MAb staining pattern) from potentially aggressive tumors (P25 MAb staining pattern) .
  • human fluids such as prostatic fluid, seminal fluid, serum or urine can be taken from a patient and assayed for the specific epitope, either as released antigen or membrane-bound on cells in the sample fluid, using the anti-prostate carcinoma monoclonal antibodies in standard radioimmunoassays or enzyme-linked immunoassays known in the art, competitive binding enzyme-linked immunoassays, dot blot or Western blot, or other assays known in the art.
  • Kits containing the PD41 MAb or fragments of MAbs (as well as conjugates thereof) or PMA antigen of the invention can be prepared for .in vitro diagnosis, prognosis and/or monitoring prostate carcinoma by the immunohistological, immunocytological and immunoserological methods described above.
  • the components of the kits can be packaged, either in aqueous medium or in lyophilized form.
  • the monoclonal antibodies (or fragments thereof) are used in the kits in the form of conjugates in which a label moiety is attached, such as a radioactive metal ion, the components of such conjugates can be supplied either in fully conjugated form, in the form of intermediates or as separate moieties to be conjugated by the user of the kit.
  • a kit may comprise a carrier being compartmentalized to receive in close confinement therein one or more container means or series of container means such as test tubes, vials, flasks, bottles, syringes, or the like.
  • a first of said container means or series of container means may contain the monoclonal antibody (or fragment thereof) or the PMA antigen of the invention.
  • a second container means or series of container means may contain a label or linker-label intermediate capable of binding to the monoclonal (or fragment thereof) or PMA of the invention.
  • the monoclonal antibodies or fragments thereof of this invention are particularly useful for targeting carcinoma cells j-n vivo. Thus, they can be used for tumor localization for detection and monitoring (enhancing patient management) as well as for therapy of primary prostate carcinoma and metastases. For these in vivo applications, it is preferable to use purified monoclonal antibodies or purified fragments of the monoclonal antibodies having at least a portion of an antigen binding region, including such as Fv, F(ab*) 2 , Fab fragments, single chain antibodies, chimeric or humanized antibodies, CDRs, etc.
  • Purification of the antibodies or fragments can be accomplished by a variety of methods known to those of skill including, precipitation by ammonium sulfate or sodium sulfate followed by dialysis against saline, ion exchange chromatography, affinity or immunoaffinity chromatography as well as gel filtration, zone electrophoresis, etc. (see Goding in. Monoclonal Antibodies: Principles and Practice, 2d ed. , pp 104-126, Orlando, Fla, Academic Press).
  • the purified monoclonal antibodies can be covalently attached, either directly or via a linker, to a compound which serves as a reporter group to permit imaging of specific tissues or organs following administration and localization of the conjugates or complexes.
  • a variety of different types of substances can serve as the reporter group, including such as radiopaque dyes, radioactive metal and non-metal isotopes, fluorogenic compounds, fluorescent compounds, positron emitting isotopes, non- paramagnetic metals, etc.
  • the purified monoclonal antibodies can be covalently attached, either directly or via a linker, to a compound which serves as a therapeutic agent to kill and/or prevent proliferation of the malignant cells or tissues following administration and localization of the conjugates.
  • a linker to a compound which serves as a therapeutic agent to kill and/or prevent proliferation of the malignant cells or tissues following administration and localization of the conjugates.
  • a variety of different types of substances can serve as the therapeutic agent including radioactive metal and non- metal isotopes, chemotherapeutic drugs, toxins, etc.
  • Kits for use with such in vivo tumor localization and therapy methods containing the monoclonal antibodies (or fragments thereof) conjugated to any of the above types of substances can be prepared.
  • the components of the kits can be packaged either in aqueous medium or in lyophilized form.
  • the monoclonal antibodies (or fragments thereof) are used in the kits in the form of conjugates in which a label or a therapeutic moiety is attached, such as a radioactive metal ion or a therapeutic drug moiety the components of such conjugates can be supplied either in fully conjugated form, in the form of intermediates or as separate moieties to be conjugated by the user of the kit.
  • Other components of the kits can include such as those mentioned in Section 5.3.3 above.
  • the PMA antigen of the present invention is a unique antigen selectively expressed by prostate carcinomas. It is envisaged that PMA and the PD41 MAb will additionally be valuable to study the natural history, development, effect of hormone/drug manipulation, etc. of prostate carcinoma.
  • the PMA may be used to prepare a vaccine formulation for prostate carcinoma.
  • the hybridoma cell lines including, in particular, the PD41 hybridoma cell line, of the present invention may be used to produce compositions comprising an antigen binding site or antibody variants which combine the murine variable or hypervariable regions with the human constant region or constant and variable framework regions, i.e., chimeric or humanized antibodies as well as humanized antibodies that retain only the antigen-binding CDRs from the parent PD41 MAb in association with human framework regions (see, Waldmann, 1991, Sci. 252:1657. 1662, particularly 1658-59 and references cited therein) .
  • Such chimeric or humanized antibodies retaining binding specificity of the antibodies of the invention would be expected to have reduced immunogenicity when administered in vivo for diagnostic, prophylactic or therapeutic applications according to the invention.
  • the invention encompasses the use of the hybridoma cell lines as a source of DNA or mRNA encoding for the rearranged, activated immunoglobulin genes, which may be isolated, cloned by known recombinant DNA techniques and transferred to other cells for the production of antigen binding fragments specific for prostate carcinoma.
  • a sequence free of introns may be obtained.
  • an immunoexpression library can be prepared and screened for antibody binding fragments for PMA as follows (See. Huse et al. , 1989, Sci. 246:1275-1281; Mullinax et al., 1990, Proc. Nat'l Acad. Sci. USA 27:8045-8099).
  • Total RNA can be purified (e.g.. using commercially available kits) and converted to cDNA using an oligo (dT) primer for the light (L) chain and a specific primer for the heavy (H) chain using reverse transcriptase.
  • PCR Polymerase chain reaction
  • Upstream primers can be designed to hybridize to partially conserved sequences in the leader and/or framework regions of V H or V L and downstream primers can be designed to hybridize to constant domain sequences. Such primers would preserve full length L chain and provide H chains corresponding to the Fd of IgG and conserving the H-L disulfide bonds.
  • the PCR amplified L and H DNA fragments are then digested and separately ligated into H and L chain vectors.
  • Such vectors contain a pelB leader sequence, a ribosome binding site and stop codons.
  • coli can be prepared from commercially available vectors (ImmunoZAP L, ImmunoZAP H; Stratacyte, La Jolla, CA) .
  • the ligated recombinant phage DNA is incorporated into bacteriophage with in vitro packaging extract and used to infect E ⁇ _ coli.
  • the immunoexpression library thus created is screened for antigen binding fragments using PMA. Positive clones can be screened and identified as described by Mullinax et al. (supra) .
  • the invention encompasses the nucleotide sequence encoding the PMA antigen of this invention.
  • the PMA antigen of the invention may be isolated and purified using only methods known in the art based on binding to the PD41 MAb of the present invention.
  • PMA may be isolated from extracts of prostate carcinoma either by affinity chromatography, in which the PD41 MAb is bound to a solid support, or by preparative SDS-polyacrylamide gel electrophoresis, in which gel slices containing PMA are identified by allowing labeled PD41 MAb to bind to the antigen.
  • the PMA may require further purification and is subjected to amino acid sequencing using known techniques.
  • Oligonucleotide probes corresponding to the amino acid sequence thus obtained may be generated by standard techniques and then used to identify DNA or genomic clones encoding PMA using standard techniques including PCR (see generally, Sambrook et al., in Molecular Cloning: A Laboratory Manual, 2d. ed. , Cold Spring Harbor Laboratory Press, 1989) .
  • the gene encoding PMA is cloned, it can be produced in large quantity using standard expression systems.
  • the PMA gene can be cloned by a "shotgun" approach in which genomic DNA or, preferably, cDNA obtained from prostate carcinoma cells may be used to create an expression library in which clones expressing PMA are identified by binding to labelled PD41 MAb using standard techniques.
  • the following examples are intended as non- limiting illustrative examples of certain embodiments of the present invention.
  • FCA Freunds Complete Adjuvant
  • FICA Freunds Incomplete Adjuvant
  • PD41.84 One hybrid, designated PD41.84 (PD41) , was selected for further analysis, subcloned using a Coulter Epics 5 flow cytometer, and isotyped (IgGl,k) using an enzyme- linked immunosorbent assay and kit (Hyclone Laboratories, Logan, UT) .
  • hybridoma cell line PD41 was obtained.
  • the hybridoma cell line was cultured to produce monoclonal antibody PD41 in sufficient quantity for characterization and further analysis as described below.
  • Cancer 44:898-903) (prostate); Calu-1, A-427, Plano-1, SKLU-1, A549, SKMES-1, OH-1, and SKLC-2 (lung); VAMT-1, JMN, and NCI28 ( esothelioma) ; MCF-7, SKBr3, and ZR-75-1 (breast); LS174, CX-1, SW480, SW1463, and LS180 (colorectal) ; A375, WM56, and H1477 (melanoma); PANC-1 and MIA (pancreatic) ; CMVMJHEL-1 (cytomegalovirus transformed fibroblast) ; and CCRF-HSB2 (human T-cell lymphocytic leukemia) .
  • Tumor Tissue preparations were prepared from prostate carcinoma specimens or other tissues by first finely mincing the tissue in 10 ml of 1.0 mM NaHC0 3 buffer containing 200 ⁇ l of a 5OX protease cocktail (antipain, 3.4 mg; pepstatin, 10.0 mg; EDTA, 0.372 g (Sigma Chemical Co., St. Louis, MO) dissolved in 20.0 ml of DDH 2 0) . The minced tumor tissue was then homogenized in a Polytron (Brinkman Instruments, Westbury, NY) and further disrupted using Wheaton glass homogenizers.
  • a 5OX protease cocktail antipain, 3.4 mg; pepstatin, 10.0 mg; EDTA, 0.372 g
  • the minced tumor tissue was then homogenized in a Polytron (Brinkman Instruments, Westbury, NY) and further disrupted using Wheaton glass homogenizers.
  • the homogenate was centrifuged at 2000 X g for 5 min, and the supernatant resulting from this spin was further centrifuged (2 h, 138,000 X g, 4°C) .
  • the resulting pellet was resuspended in a minimum volume of PBS and stored at -70°C. Protein concentrations were determined using the Bicinchoninic Acid (BCA) Protein Assay (Pierce Chemical Co., Rockford, IL) .
  • BCA Bicinchoninic Acid
  • the staining reactivity on frozen or fixed tissues was evaluated by the avidin-biotin peroxidase assay using the ABC Elite Vectastain kit (Vector Laboratories, Burlingame, CA) as described previously. (Wright et al. , 1983, Cancer Res. 42:5509-5516; Wahab et al., 1985, Int. J. Cancer 3_6.:677-683) . Following development with the chro ogen 3,3 '-diaminobenzidine tetrahydrochloride (Sigma) , the tissues were counterstained with Mayer's hematoxylin and mounted in aquamount (Learner Laboratories, Pittsburgh, PA) .
  • Tissues were scored independently by 2 investigators for both intensity of reactivity, using a scale of 0
  • the cells were washed in PBS, they were incubated for 30 min at 4°C with 50 ⁇ l of fluorescein-conjugated goat anti-mouse IgGs (Organon Teknika-Cappel, Malvern, PA) at 50 ⁇ g/ml.
  • the cells were again washed and observed for fluorescent staining using an Olympus microscope equipped with a fluorescence vertical illuminator system. The percent of intact cells showing fluorescence and their staining intensity (scale 0 to +4) , out of a total of 300 cells analyzed, was determined.
  • Electrophoresis was carried out in polyacrylamide gels under reducing conditions [Laemmli et al., 1970, Nature (London) 227:680-6851. and protein migration in gels was determined using Rainbow protein molecular weight markers (Amersham, Arlington
  • TBS blocking buffer (1 h at 37°C) .
  • the membrane was incubated with PD41 MAb culture supernatant (20 ⁇ g/ml,
  • Membranes were washed in dilute TBS to remove unbound antibody, then incubated with 125 I-labeled rabbit anti- mouse IgG (secondary antibody, specific activity 1.25 ⁇ Ci/ ⁇ g) at 1 x 10 7 cpm/50 ml of TBS for an additional 2 h at 25°C. Following incubation with the secondary antibody, the membrane was further washed, air dried, and exposed to Kodak XAR X-ray film (-70°C, 48-72 h) . The membranes were gently agitated during all incubations.
  • anti.-carcm. oembryoni.c antigen (CEA) MAb Zymed, San Francisco, CA) that had been adsorbed to eliminate reactivity to non-cross- reacting antigen
  • anti-HMFG-2 MAb Unipath
  • Monoclonal antibody PD41 was screened against a panel of 44 cultured human tumor cell lines
  • LS180, a colorectal carcinoma As demonstrated in Table 1, only one cultured tumor cell line, LS180, a colorectal carcinoma, reacted with the PD41 MAb. PD41 reactivity was also detected in the concentrated, dialyzed spent culture medium and the glycopeptide fraction digest from the LS180 cell line (data not shown) . No other cell type, including the 5 prostate cell lines, i.e., DU145, PC3, PC3-P, LNCaP, and PPC-1, expressed the PD41 antigen.
  • tissue specificity of monoclonal antibody PD41 was determined using an avidin biotin complex immunoperoxidase assay (see Section 6.2.4 above) . The results are presented in Table 2 and FIGS. 1 (A-F) and 2.
  • Intensity and percent positive cells apply to both fixed or frozen tissues.
  • WD, MD, PD, UD well, moderately, poorly, and undifferentiated prostate carcinoma.
  • PD41 MAb reacts to both frozen and fixed primary prostate carcinoma.
  • the sensitivity for prostate cancer specimens is 47% (fixed tissues) and 65% (frozen tissues) .
  • both fixed and frozen specimens from the same case could be evaluated, 26% of the fixed specimens failed to stain, suggesting the possibility that some denaturation of the target antigen may occur during either the formalin fixation or deparaffinization procedures.
  • strong staining of the tumor cells was observed in the majority of the PD41-positive prostate carcinoma specimens (FIG. 1) .
  • the PD41 staining appears confined to an antigen expressed by prostatic epithelial cells, although staining of luminal secretions and the borders of the ductal epithelial cells was also observed (FIG. 1) .
  • the staining pattern was, however, very heterogeneous with the number of PD41-positive tumor cells ranging from 2 to 95% (Table 2) irrespective of specimen preparation (FIG. 2) , and this pattern of expression remained fairly constant for the differentiated carcinomas (i.e. well, moderately, and poorly differentiated)
  • PD41 MAb did not bind substantially to fetal prostate tissues, frozen normal prostate tissues, and fixed BPH tissues (Table 2) .
  • One of 68 frozen BPH specimens and 3 of 22 fixed normal prostate specimens were PD41-positive, however only weak staining was observed in less than 1% of the ductal epithelial cells.
  • the PD41 target antigen also was expressed in the tumor cells of 2 nude mouse prostate xenografts (Table 2) , and the staining pattern observed, especially in the PC-EW heterotransplant, was similar to that described above for human prostate surgical specimens.
  • a MAb B72.3 or HMFG 2 was used as a positive control MAb where appropriate.
  • Antibodies PD41, anti-PSA, and Anti-PAP in
  • Isotype- atched negative control MAb either IgGl or IgG2a.
  • Table 5 shows that the PMA antigen could be detected in three of eight CaP seminal plasma samples by RIA, although it was not detected in normal seminal plasma . Comparison of MAbs to PSA and PAP (generated in our laboratory) indicated that these antigens were detectable, as expected, in both types of seminal plasma specimens.
  • FIG. 3A is a representative immunoblot developed with the PD41 MAb.
  • the most prominent band observed, a large diffuse band (M r > 400,000) is seen in both the CaP tissue extracts (Fig. 3A, Lanes 1 and 3) and CaP seminal plasma (FIG. 3A, Lane 4) , although this band was not observed in all CaP tissue extracts FIG. 3A, Lane 2) .
  • Two other prominent components reactive with MAb PD41, a M r 166,000 and a M r 91,000 band, were usually observed in all the CaP extracts examined (FIG.3 A, Lanes 1-3), however, occasionally, additional minor bands were observed in some CaP tissues.
  • the pattern i.e., . . .
  • BSM bovi.ne submaxi.llary muci.n
  • Percent control binding of the PD41 antibody to its target antigen was determined by comparing the cpms of the untreated antigen with those obtained after various antigen treatments in a standard solid phase radioimmunoassay. Binding of the antibody to the untreated antigen is considered to be 100%.
  • Bovine submaxillary mucin As shown in Table 6, the PMA antigen is sensitive to treatment with various proteolytic enzymes. Additionally, the PMA antigen is sensitive to agents which affect carbohydrate moieties, including sodium borohydride, sodium meta-periodate, O-glycanase, and beta-galactosidase. Such results indicate that carbohydrate forms an important portion of the PD41 proteinaceous antigen.
  • PBS phosphate buffered saline
  • BPA Bauhimina purpurea agglutinin
  • Con A concanavalin A
  • DBA Dolichos biflourus agglutinin
  • GS-I Griffonia simplicifolia I agglutinin
  • GS-II Griffonia simplicifolia II agglutinin
  • LPA Limulus polyphemus agglutinin
  • MPA Maclura pomifora agglutinin
  • PNA Arachis hypogea agglutinin
  • UEA Ulex europaeus agglutinin
  • WGA Triticum vulgaris agglutinin
  • S-WGA succinyl-Triticum vulgaris agglutinin
  • SBA Glycine max.
  • Percent control binding was determined by comparing PD41 binding in the absence (100%) and presence of various lectins using a standard radioimmunoassay procedure.
  • sialic acid does not inhibit binding of the PD41 MAb to PMA, despite the fact that treatment of the antigen with neuraminidase appears to enhance PD41 MAb binding (Table 6) .
  • sialic acid does not appear to be a component of the antigenic determinant of the PMA antigen, although removal of sialic acid residues may expose more of the PD41 antigenic determinant to the PD41 MAb.
  • n-acetyl galactosamine and raffinose both blocked binding of PD41 MAb to the PMA antigen by about 30%.
  • HMFG-2 which are known to bind to high molecular weight TAA's, could compete with 1-125 labeled MAb PD41 for binding with PMA.
  • Double determinant immunoradiometric inhibition experiments were conducted as follows. A series of unlabelled MAbs, (listed in Table 9 together with antigens with which such MAbs react) were then added, at a concentration of 100 ⁇ g/ml to the target PMA bound to unlabeled "capture" PD41 MAb. Non- saturating amounts of the 125 I-labeled PD41 MAb were then added.
  • the percent inhibition or blocking of the labelled PD41 MAb to its target antigen was calculated as:
  • CEA GI GI, lung, breast, etc.
  • unlabeled PD41 MAb did not block the binding of the selected 1-125 labeled MAbs to their respective mucin tumor associated antigen targets. Blocking occurred only with the homologous MAb.
  • Epitope co-expression was determined using a radioimmunometric assay as described above.
  • the ability of the radiolabelled PD41 MAb to bind target antigen bound by a different capture mucin tumor associated MAb was determined.
  • a series of MAbs which bind to different TAAs were immobilized on the plates to serve as "capture” MAb.
  • Radiolabelled PD41 MAb was then added to determine whether it would bind to whatever antigen bound to the capture MAbs. Results are presented in FIG. 7.
  • radiolabelled PD41 MAb showed minimal to no binding to whatever antigen bound to the different tumor mucin "capture" Mabs.
  • the PMA epitope is not coexpressed on the same glycoproteins recognized by the other tumor Mabs.
  • a series of selected Mabs specific for tumor-associated antigen did not block Mab PD41 binding to the PMA target antigen (FIG. 5) ; and PD41 MAb did not block the tumor-associated MAbs in reciprocal blocking experiments (FIG. 6) .
  • MAb PD41 did not bind to target antigens bound to other MAbs which recognize TAA's antigens.
  • Radiolabelled PD41 bound only to the antigen bound to MAb PD41 as "capture" antibody.
  • Another double determinant immunoradiometric competitive inhibitor experiment was conducted using radiolabelled PD41 MAb against a series of MAbs specific for human blood group antigens listed in Table 9. All of the human blood group MAbs failed to block binding of PD41 MAb to the target PMA antigen (results not shown) .
  • PD41 MAb does not react with any of these T,TN and sialosyl-TN antigens. As can be seen from the data, neither antibody reacts with the "T" antigen. B72.3 reacts with both bovine (with approximately 50% of the carbohydrate chains consisting of sialosyl-Tn) and ovine (90% sialosyl-Tn) submaxillary mucin in the native state, but not after desialylation with neuraminidase, thus indicating B72.3 recognizes the sialosyl-Tn form. PD41 reactivity with bovine submaxillary mucin occurs in the native state and is enhanced following neuraminidase treatment.
  • PD41 does not react with ovine submaxillary mucin in the native state (sialosyl-Tn) or in the neuraminidase treated state (Tn) .
  • Immunoperoxidase staining of tissues showed that PMA was expressed by bovine (confirming the above finding) but not by ovine, porcine, monkey or human submaxillary tissues (data not shown) .
  • the PD41 monoclonal antibody reacts with a distinct and novel mucin antigen selectively expressed by human prostate carcinoma cells and by the bovine submaxillary gland, and is not related to any previously described ucin- associated tumor antigen.
  • the PMA antigen therefore, is a new and unique mucin that has not been previously described.
  • a cell line, PD41, as described herein has been deposited with the American Type Culture Collection, Rockville, Maryland and been assigned accession number ATCC No. .
  • the invention described and claimed herein is not to be limited in scope by the cell lines deposited since the deposited embodiment is intended as an illustration of one aspect of the invention and any equivalent cell lines which produce functionally equivalent monoclonal antibodies are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Abstract

Cette invention concerne des anticorps monoclonaux qui se lient de manière spécifique au carcinome de la prostate et qui ne se lient pratiquement pas à l'adénome prostatique ou à la prostate normale, ainsi que des lignées cellulaires d'hybridomes produisant les anticorps monoclonaux. Dans un mode de réalisation un anticorps monoclonal appelé Mab PD41 est décrit. Un nouvel antigène appelé antigène de muccine prostatique est présenté sous forme isolée et pratiquement pure. Cette invention concerne également des procédés d'utilisation des lignées cellulaires d'hybridomes, de l'anticorps monoclonal et/ou de l'antigène pour le diagnostic, la prophylaxie et/ou le traitement du carcinome de la prostate.
PCT/US1992/000852 1992-01-31 1992-01-31 Anticorps monoclonal pd41 et antigene associes aux carcinomes de la prostate WO1993014775A1 (fr)

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PCT/US1992/000852 WO1993014775A1 (fr) 1992-01-31 1992-01-31 Anticorps monoclonal pd41 et antigene associes aux carcinomes de la prostate
CA002106487A CA2106487A1 (fr) 1992-01-31 1992-01-31 Anticorps monoclonal pd41 et antigene associe aux adenocarcinomes de la prostate

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PCT/US1992/000852 WO1993014775A1 (fr) 1992-01-31 1992-01-31 Anticorps monoclonal pd41 et antigene associes aux carcinomes de la prostate
CA002106487A CA2106487A1 (fr) 1992-01-31 1992-01-31 Anticorps monoclonal pd41 et antigene associe aux adenocarcinomes de la prostate

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0804458A1 (fr) * 1995-01-11 1997-11-05 The Trustees of Columbia University in the City of New York Developpement de sondes d'adn et de reactifs immunologiques specifiques de molecules exprimees sur une surface cellulaire et genes associes a la transformation
WO1998037418A2 (fr) * 1997-02-25 1998-08-27 Corixa Corporation Composes servant au diagnostic immunologique de cancer de la prostate et leurs procedes d'utilisation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CANCER RESEARCH, Volume 48, issued 01 December 1988, BAZINET et al., "Immunobhistochemical Characterization of Two Monoclonal Antibodies, pages 25.48 and 25.91, which Define a New Prostate-Specific Antigen", pages 6938-6942. *
CANCER RESEARCH, Volume 51, issued 15 February 1991, BECKETT et al., "Monoclonal Antibody PD41 Recognizes an Antigen Restricted to Prostate Adenocarcinomas", pages 1326-1333. *
FEDERAL PROCEEDINGS, Volume 46, No. 3, issued 1987, WRIGHT et al., "Differentiation of Benign From Malignant Prostate Disease With Monoclonal Antibodies", page 1059. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0804458A1 (fr) * 1995-01-11 1997-11-05 The Trustees of Columbia University in the City of New York Developpement de sondes d'adn et de reactifs immunologiques specifiques de molecules exprimees sur une surface cellulaire et genes associes a la transformation
EP0804458A4 (fr) * 1995-01-11 2002-08-14 Univ Columbia Developpement de sondes d'adn et de reactifs immunologiques specifiques de molecules exprimees sur une surface cellulaire et genes associes a la transformation
US6811972B1 (en) 1995-01-11 2004-11-02 The Trustees Of Columbia University In The City Of New York Development of DNA probes and immunological reagents specific for cell surface-expressed molecules and transformation-associated genes
WO1998037418A2 (fr) * 1997-02-25 1998-08-27 Corixa Corporation Composes servant au diagnostic immunologique de cancer de la prostate et leurs procedes d'utilisation
WO1998037418A3 (fr) * 1997-02-25 1999-02-25 Corixa Corp Composes servant au diagnostic immunologique de cancer de la prostate et leurs procedes d'utilisation
EP1630235A2 (fr) * 1997-02-25 2006-03-01 Corixa Corporation Composés pour l'immunodiagnostic du cancer de la prostate et méthodes pour leurs utilisations
EP1630235A3 (fr) * 1997-02-25 2009-05-27 Corixa Corporation Composés pour l'immunodiagnostic du cancer de la prostate et méthodes pour leurs utilisations

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