WO1993006858A1 - Proteine associee au cancer du poumon - Google Patents

Proteine associee au cancer du poumon Download PDF

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Publication number
WO1993006858A1
WO1993006858A1 PCT/US1991/007585 US9107585W WO9306858A1 WO 1993006858 A1 WO1993006858 A1 WO 1993006858A1 US 9107585 W US9107585 W US 9107585W WO 9306858 A1 WO9306858 A1 WO 9306858A1
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lcap
antibody
levels
patients
monoclonal antibody
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PCT/US1991/007585
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English (en)
Inventor
Donald Kufe
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Dana Farber Cancer Institute, Inc.
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Priority to PCT/US1991/007585 priority Critical patent/WO1993006858A1/fr
Priority to CA002120363A priority patent/CA2120363A1/fr
Priority claimed from CA002120363A external-priority patent/CA2120363A1/fr
Publication of WO1993006858A1 publication Critical patent/WO1993006858A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6857—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from lung cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
    • A61K51/1054—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from lung
    • 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
    • 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
    • C07K16/3023—Lung
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo

Definitions

  • the field of the invention is immunoassays for cancer-specific antigens.
  • markers termed DF3 antigen Abe and Kufe, J. Immunol. 139:257- 61, 1987
  • carcinoembryonic antigen or CEA (Shively and Beatty, CRC Crt. Rev. Oncol/Hematol. 2:344-399, 1985)
  • SCC sguamous cell carcinoma-associated
  • Lung cancers can be divided into two groups: small-cell carcinomas and non-small-cell carcinomas, the latter category including epidermoid or squamous cell carcinomas, adenocarcinomas, and large cell carcinomas.
  • circulating levels of an appropriate biochemical marker are useful for monitoring the clinical course of a given cancer patient's disease.
  • the invention features an immunoassay for an antigen termed Lung Cancer-associated Protein, or LCAP.
  • LCAP Lung Cancer-associated Protein
  • the immunoassay of the invention provides a method for detecting LCAP in a biological sample (e.g., blood, serum, urine, sputum, mucosal scrapings, or biopsied tissue from a human or another mammal), which method includes the steps of contacting the biological sample with an aliquot or sample containing a monoclonal antibody (MAb) specific for LCAP, and detecting immune complex formation between the antibody and a constituent of the biological sample (for example, by ELISA), such immune complex formation being indicative of the presence of LCAP in the
  • a biological sample e.g., blood, serum, urine, sputum, mucosal scrapings, or biopsied tissue from a human or another mammal
  • MAb monoclonal antibody
  • the method of the invention may include the additional steps of providing a control sample containing a standard amount of LCAP (either purified or in a mixture, such as a sample of serum containing a known amount of LCAP); contacting the control sample with a second aliquot containing the monoclonal antibody; and comparing the amount of immune complex formation in the biological sample to the amount of immune complex formation in the control sample.
  • a monoclonal antibody specific for LCAP is herein defined as an antibody which (1) forms an immune complex with LCAP antigen purified from the supernatant of cultured CALU-3 cells (American Type Culture Collection accession no.
  • ATCC HTB 55 detects circulating antigen at elevated levels (i.e., above the normal cutoff level of 23 units of LCAP/ml) with serum samples from at least 70% of patients with either adenocarcinoma or squamous cell carcinoma of the lung; one such MAb is that produced by the hybridoma DF-L1.
  • an immunoassay kit including (1) a first reagent including a first monoclonal antibody specific for LCAP; (2) a second reagent including an enzyme conjugated to a second monoclonal antibody specific for LCAP (which second antibody may be identical to the first antibody, or at least capable of binding to the same type of determinant as that bound by the first antibody); a third reagent which includes a substrate for the enzyme; and
  • One or both of the monoclonal antibodies may optionally be that produced by the hybridoma DF-L1, or may be a MAb which binds to the same determinant as that bound by the MAb produced by the hybridoma DF-L1.
  • the enzyme and substrate are preferably horseradish peroxidase and hydrogen peroxide,
  • the kit may also include a fourth reagent that includes LCAP, to be used, for example, as a
  • This fourth reagent may be, e.g., a sample of human serum containing a
  • LCAP predetermined amount of LCAP, or may contain essentially purified LCAP in dry form or in solution.
  • LCAP essentially purified preparation of LCAP
  • the antigen may be extracted from membranes of human cells (e.g., primary tumor cells or a cell line expressing LCAP), or isolated from a bodily fluid (such as blood) or the medium bathing an in vitro-cultured cell which secretes LCAP.
  • An essentially purified preparation of LCAP may be made by (1) providing a population of cells capable of expressing LCAP (e.g., on their membranes and/or secreted into the medium bathing the cells); (2) culturing the population of cells in a medium under conditions which permit the population of cells to express LCAP; and isolating LCAP from the membranes of the cells, or from the medium [for example, by contacting the membranes, an extract of the membranes, or the spent medium with an immunoaffinity matrix having an antibody specific for LCAP (such as the MAb produced by the hybridoma DF-L1) affixed to a matrix material].
  • an immunoaffinity matrix having an antibody specific for LCAP such as the MAb produced by the hybridoma DF-L1
  • the population of cells is preferably descended from a CALU-3 cell (ATCC HTB 55), and is preferably cultured in a medium containing at least 50 ⁇ g/ml (more preferably 100 to 300 ⁇ g/ml, and most preferably 150 ⁇ g/ml) galactosamine.
  • the invention also includes a monoclonal antibody specific for LCAP, a hybridoma cell which expresses such an antibody, and a method of producing the antibody, including the steps of culturing the hybridoma cell in a medium and isolating the antibody from the medium.
  • the invention includes an immunotoxin in which the antibody portion of the
  • immunotoxin is an LCAP-specific monoclonal antibody
  • toxin e.g., the MAb produced by hybridoma DF-L1
  • LCAP-binding portion of the toxin conjugated to a toxin molecule.
  • conjugation may be accomplished by known chemical methodology, or, if the toxin is a protein, by means of genetically engineering a hybrid DNA molecule encoding both the toxin and an LCAP-binding portion of the
  • antibody as a single polypeptide: expression of this recombinant DNA molecule would result in an immunotoxin in which the antibody portion is linked to the toxin portion by a peptide bond.
  • proteinaceous toxins that could be incorporated into the immunotoxin of the invention include diphtheria toxin; Pseudomonas exotoxin A; ricin and other plant toxins such as abrin, modeccin, volkensin, and viscumin; cholera toxin (produced by Vibrio cholerae bacteria);
  • Shiga toxin produced by various strains of Shigella bacteria
  • Shiga toxin produced by various strains of Shigella bacteria
  • Shiga toxin produced by various strains of Shigella bacteria
  • Shiga toxin produced by various strains of Shigella bacteria
  • Shiga-like toxins produced by E. coli and other enteric bacteria
  • Salmonella heat- labile enterotoxin produced by E. coli and other enteric bacteria
  • Salmonella heat- labile enterotoxin produced by E. coli and other enteric bacteria
  • Salmonella heat- labile enterotoxin produced by E. coli and other enteric bacteria
  • Salmonella heat- labile enterotoxin produced by E. coli and other enteric bacteria
  • Salmonella heat- labile enterotoxin produced by E. coli and other enteric bacteria
  • Salmonella heat- labile enterotoxin produced by E. coli and other enteric bacteria
  • Salmonella heat- labile enterotoxin
  • anticancer agents such as doxorubicin, as well as ⁇ - emitting radionuclides such as astatine and ⁇ -emitting nuclides such as yttrium.
  • invention would be useful for targeting and killing tumor cells which express LCAP on their surfaces.
  • an imaging agent in which an LCAP-specific monoclonal antibody, or an LCAP- binding fragment thereof, is linked to a detectable label such as a radionuclide (for example, 25 I, 131 I,
  • Such an imaging agent would be useful for detecting tumors in situ by a method including the steps of identifying an animal suspected of having a tumor; introducing the imaging agent into the animal; and detecting (e.g., by radioimaging, using scintigraphy) the presence of the detectable label bound to a tissue (e.g., lung tissue) of the animal, a high level of such label bound to a given site being indicative of a tumor at that site.
  • a tissue e.g., lung tissue
  • Using such an imaging method permits a non- invasive determination of the presence, location, or absence of an LCAP-expressing tumor in such animal, which would be particularly useful for monitoring the condition of a patient being treated for a tumor known to express LCAP.
  • the invention features a vaccine for immunizing a human against tumors which express LCAP, which vaccine would include the protein portion of LCAP, or an antigenic fragment thereof, in a pharmaceutically-acceptable carrier.
  • the vaccine would preferentially also include an adjuvant such as Freund's, to enhance the recipient's immune response to the
  • Fig. 1 is a set of graphs illustrating the results of indirect immunofluorescence of human lung carcinoma cell lines with MAbs DF-L1 and DF-L2.
  • Suspensions of CALU-3, SK-MES, A-549, and ZR-75-1 cells were incubated with MAb DF-L1 (heavy solid line), MAb DF-L2 (dotted line), or an isotype-identical control antibody (thin solid line). After a second incubation with fluorescein- conjugated goat anti-mouse IgG1, the cells were analyzed by flow cytometry.
  • Fig. 2 is an immunoblot analysis of human tumor cell lines with MAb DF-L1.
  • A human lung carcinomas
  • B other human tumors. Extracts of the indicated cells were subjected to 3-10% gradient SDS-PAGE. The proteins were then transferred to nitrocellulose paper and monitored for reactivity with MAb DF-L1 and 125 I-labeled sheep anti- mouse immunoglobulin. Kd, M r in thousands.
  • Fig. 3 is a set of photographs showing
  • A adenocarcinoma
  • B squamous cell carcinoma
  • C normal alveolar lining cells
  • D normal bronchus (arrow, terminal web of brush border).
  • Fig. 4 is an autoradiogram showing
  • CALU-3 cells were labeled with [ 3 H]proline for 48 h.
  • Fig. 5 is an immunoblot illustrating the effects of tunicamycin on DF-L1 antigen.
  • CALU-3 cells were grown in the presence of 10 ⁇ g/ml tunicamycin for 24 h and then subjected to immunoblot analysis with MAb DF-L1. Kd, M r in thousands.
  • Fig. 6 is an immunoblot analysis of human plasma samples analyzed with MAb DF-L1.
  • Plasma specimens (3 ⁇ l) from normal subjects (lanes 1-4 ) and patients with lung carcinomas (lanes 5-8 ) were subjected to immunoblot analysis with MAb DF-L1.
  • Lane 9 1 ⁇ g of purified CALU- 3 antigen.
  • Fig. 7 is a graph showing the reproducibility of LCAP calibrator curves.
  • LCAP calibrators containing 0, 20, 100, and 200 units/ml were assayed in duplicate on 4 consecutive days. Absorbances (ABS) at 490 nm were determined for each calibrator. Open circles, Day 1;
  • Fig. 8 is a graph showing the effect of dilution on LCAP levels. Plasma samples from normal subjects and patients with lung cancer were assayed for LCAP levels as a function of dilution. Open circles and squares denote normal subjects; closed circles and squares denote patients with metastatic lung cancer. Points , mean;
  • Fig. 9 is a histogram illustrating the
  • Fig. 12 is a set of graphs showing LCAP spikes during effective treatment in patients with lung cancer.
  • Serial LCAP levels from a patient with Stage IIIA is a set of graphs showing LCAP spikes during effective treatment in patients with lung cancer.
  • Fig 15 is a graph illustrating a typical
  • MAb Production was generated by techniques analogous to those described previously (Kufe et al., Hybridoma 3:223-232, 1984). Briefly, BALB/c mice were immunized with an extract of a primary human
  • Mouse spleen cells were fused with P3X63-Ag8.653 myeloma cells (ATCC CRL 1580) and hybridomas cloned three times by limiting dilution in Dulbecco's modified Eagle's medium with 4.5 g/liter glucose, 10% fetal bovine serum, 10% NCTC-109 medium supplement (Sigma), 1% sodium pyruvate, 1% nonessential amino acids, 200 mM L-glutamine, 1% tylosin (Sigma
  • Hybridoma cells were injected into pristane-primed BALB/c mice and the MAbs purified from ascites using a Protein A-Sepharose column (BioRad, Richmond, VA). Yields ranged from 0.5-9.0 mg purified antibody/ml ascites fluid.
  • the human lung adenocarcinoma cell line CALU-3 (ATCC HTB 55), the
  • squamous cell carcinoma SK-MES ATCC HTB 58
  • undifferentiated lung carcinoma A-549 ATCC CCL 185
  • the squamous cell carcinoma cell line CALU-1 ATCC
  • HTB 54 was grown in McCoy's 5A medium (GIBCO)
  • Indirect ELISA was performed by coating 96-well polyvinyl microtiter plates with either 20 ⁇ g of crude antigen extract or 1 ⁇ g of purified antigen in 50 ml PBS. Nonspecific binding sites were blocked with 5% BSA, and the wells were then incubated with MAb for 1 h. After washing, the plates were incubated. with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin
  • a double-determinant ELISA was performed by coating 96-well microtiter plates with 2.5 ⁇ g MAb/well in 0.1 M sodium bicarbonate-0.5 M NaCl buffer at pH 8.7.
  • the wells were blocked with 5% BSA and incubated with antigen in PBS for 1 h. After washing, the wells were incubated with horseradish peroxidase-conjugated MAb in 0.1% Tween 20/PBS for 1 h, washed, developed with o-phenylenediamine, and monitored for absorbance at 490 nm. All ELISA incubations were performed at room temperature.
  • nitrocellulose membrane was blocked with 5% BSA
  • Immunoperoxidase staining Four- ⁇ m sections of formalin-fixed paraffin-embedded tissue were stained using an avidin-biotin-peroxidase staining technique (Vectastain ABC kit, Vector Laboratories, Burlingame, CA). Primary antibody was used at a concentration of 0.1 ⁇ g/ml. Reactivity was assessed on a 0-3+ visual scale for cytoplasmic and membrane-staining patterns.
  • CALU-3 cells were incubated in proline-free medium supplemented with dialyzed FBS for 6 h. The cells were then incubated for 48-72 h with fresh medium with 150 ⁇ Ci [ 3 H]proline (130 Ci/mmol;
  • CALU-3 cells were also incubated in complete medium containing 150 ⁇ Ci [ 3 H]glucosamine (40 Ci/mmol; Amersham). The cells were lysed with a 1% Nonidet-40/50 mM Tris-HCl lysis buffer (pH 8.0) in the presence of protease inhibitors (5 mM EDTA/20 mM phenylmethylsulfonyl fluoride/20 mM phenanthroline) and the cell extract was centrifuged at 10,000 ⁇ g for 10 min.
  • protease inhibitors 5 mM EDTA/20 mM phenylmethylsulfonyl fluoride/20 mM phenanthroline
  • IP immunoprecipitation
  • the supernatant of the cell extract was pre-cleared by incubating with the IP complex and an irrelevant mouse IgGl MAb.
  • the precleared extract was then incubated overnight at 4°C with MAb followed by incubation for 1 h with IP complex, washed extensively, subjected to SDS-PAGE under reducing conditions, and monitored by fluorography.
  • High molecular weight antigen was purified from spent culture medium of CALU-3 cells. Cells were grown for 3 days past confluence, and spent medium was collected, pooled, centrifuged at 10,000 ⁇ g for 20 min to remove debris, and concentrated
  • DF-L1 and DF-L2 were chosen for further study. The specificity of these MAbs was first
  • Fig. 2A MAb DF-L1 also reacted with a heterogeneous group of high molecular weight antigens in the breast and ovarian carcinoma cell lines (Fig. 2B). In concert with the findings by ELISA, there was no detectable reactivity of this MAb with exeracts of the U-937 or HL-60 leukemia lines (Fig. 2B). Reactivity with Human Tissues. Formalin-fixed paraffin-embedded sections of tumor and normal tissue were evaluated for reactivity with MAbs DF-L1 and DF-L2 using an immunoperoxidase-staining technique.
  • Fig. 3B membrane-predominant staining
  • MAb DF-L1 Several normal tissues reacted with MAb DF-L1 including kidney and lung. In the kidney, only the distal collecting ducts reacted with the antigen, while the glomeruli were negative. In the lung, normal alveolar lining cells adjacent to tumor tissue stained for this antigen but primarily with an apical pattern (Fig. 3C). Certain areas of the bronchus also were positive for this epitope with staining localized
  • Characterization of the epitopes recognized by these antibodies was performed using antigen purified from the culture supernatant of CALU-3 cells.
  • the antigen was fractionated on a Sepharose CL-4B column and then further purified by MAb DF-L1 immunoaffinity.
  • the purified antigen was analyzed by gel electrophoresis. No detectable contaminating proteins or carbohydrates were detected by Coomassie blue and PAS staining (data not shown).
  • the purified antigen was subjected to treatment with various agents that alter carbohydrate or protein structure. Reactivity of MAbs DF-L1 and DF-L2 was determined by dot immunoblotting. Pronase, but not trypsin, treatment was associated with loss of reactivity for both antibodies (Table 4). In contrast, while alkaline/borohydride and periodate had little effect on MAb DF-L1 reactivity, exposure of the antigen to these agents was associated with loss of MAb DF-L2 binding (Table 4). Similar findings were obtained by double- determinant ELISA. Neuraminidase had little effect on binding of either MAb, while periodate and alkaline- borohydride treatments predominantly decreased that for MAb DF-L2 (Table 4).
  • Plasma samples from normal individuals and patients with lung cancer were monitored by immunoblotting for the presence of this high molecular weight antigen. Low but detectable levels of antigen were present in samples from four normal individuals (Fig. 6). In contrast, reactivity with MAb DF-L1 was clearly greater in plasma samples from four patients with lung cancer (Fig. 6) .
  • the electrophoretic mobility of the antigen varied among individuals and up to three reactive species were detectable in certain patients (Fig. 6).
  • SPDP horseradish peroxidase
  • MAbs were conjugated to SMCC in dimethyl formamide. The two protein derivatives were then
  • MAbs 50 ⁇ g/ml were adsorbed to 96- well microtiter culture plates in a 0.1 M NaHCO 3 /0.5 M NaCl buffer (pH 8.5) for 1h. This concentration was chosen following antibody titration to optimize the signal:noise ratio of the high 200-unit/ml calibrator (see "Assay Calibrator"). The solution was aspirated, and the wells were incubated for 1 h with 5% BSA in 0.01 M PBS to block nonspecific protein binding sites on the plastic. The BSA was removed, and samples containing antigen were added to the wells.
  • Spent tissue culture medium of the human lung carcinoma cell line CALU-3 was pooled, concentrated, and used as a source of LCAP for
  • Plasma Samples Plasma samples from normal subjects were obtained from the American Red Cross
  • Plasma samples were collected from patients at the Dana-Farber Cancer Institute, Boston, MA, according to protocols approved by the Institutional Review Board. Samples were collected in evacuated tubes containing disodium EDTA. Plasma was separated by centrifugation at 100 ⁇ g for 15 min, aliquoted, and stored at -70°C.
  • a panel of 10 MAbs was generated on the basis of reactivity with purified LCAP derived from CALU-3 cells. As determined by direct immunoassay, 3 of the 10 MAbs retained reactivity with purified LCAP after conjugation to HRP. Moreover, 4 of the MAbs were found to adsorb reliably to microtiter plates, retaining activity. These MAbs were evaluated in various combinations to detect LCAP levels in solid-phase ELISAs. The five "sandwich" immunoassay combinations that detected soluble LCAP from cultured cell supernatants were then evaluated for
  • Spent tissue culture medium from CALU-3 human lung adenocarcinoma cells containing high concentrations of LCAP as determined by Western blot analysis, was pooled and diluted to make arbitrarily-defined calibrator solutions, ranging from 0 to 200 units/ml.
  • the LCAP assay was quite reproducible, as demonstrated in a series of calibration curves run on different days (Fig. 7). Serial dilutions of plasma samples from normal subjects and from lung cancer patients were evaluated using the DF-L1/DF-L1-HRP assay (Fig. 8). In undiluted plasma, absorbance was at the upper limits of the assay for both normal and cancer samples.
  • the LCAP assay was optimized for routine use and then characterized for inter- and intraassay variation, antigen recovery, interference effects, and sample handling.
  • Intra- and interassay reproducibility were assessed for the calibrators and three serum specimens containing different concentrations of LCAP.
  • the intraassay reproducibility was determined by one individual running the calibrators and serum specimens in replicates of 12 , calculating the mean absorbance value for each and determining the percentage of the coefficient of variation.
  • rheumatoid factor (XL: 160), or triglycerides (up to 1026 mg/dl)
  • XL rheumatoid factor
  • triglycerides up to 1026 mg/dl
  • Serum and EDTA plasma samples were obtained from 156 volunteer donors and assayed for LCAP levels. The values obtained for the plasma samples were regressed on the serum values. The correlation coefficient obtained was 0.966, and the slope, 0.979. The overall concordance using 23 units/ml as a reference cut-off (see following section) was 94%. Distribution of Circulating LCAP Levels in Normal
  • Plasma samples from 341 normal blood bank donors were evaluated for LCAP levels (Fig. 9).
  • the mean value of the normals was 7 units/ml (SD + 8) with a median value of 5 units/ml.
  • the levels ranged from a low of 0 units/ml to a high of 43 units/ml. Thirty-five percent of the samples had LCAP levels of 2 units/ml or less, while only 13.8% of the samples had levels above 15 units/ml.
  • metastatic lung cancer was screened to determine
  • LCAP levels were elevated in 27 of 35 (77.1%) lung cancer patients with a mean of 127 units/ml and a range of 8 units/ml to >1000 units/ml (Fig. 10). The difference in LCAP levels from lung cancer patients and those from normal controls was highly statistically significant (P ⁇ 0.001). Moreover, LCAP levels were elevated in patients with each histological type of lung cancer: adenocarcinoma, 16 of 19 (84.2%); squamous cell carcinoma, 4 of 7 (57.1%); other
  • LCAP levels were monitored in selected patients with lung cancer during treatment for metastatic disease. For example, one patient with adenocarcinoma was
  • Plasma samples from a small group of patients with cancers other than lung cancer were also screened to determine the specificity of this assay. Elevated levels of LCAP were detected in 58% of patients with pancreatic cancer, 50% of patients with breast cancer, 33% of
  • a small panel of patients with benign lung disease was screened for the determination of LCAP levels.
  • COPD chronic obstructive pulmonary disease
  • pneumonia had elevated LCAP levels.
  • One patient with tuberculosis had a normal LCAP level.
  • the means of the LCAP levels were 23.8 and 23.9 units/ml for the COPD and pneumonia patients, respectively, barely above the normal cut-off value.
  • Plasma samples collected in EDTA-treated tubes or serum samples were obtained from patients with lung cancer, other malignancies, and a variety of benign disorders. Samples from patients with malignancies were collected from patients at the Dana-Farber Cancer Institute and were stored at
  • Progression was defined as the appearance of any new malignant lesion documented by histologic, clinical, or radiographic criteria, or a doubling in the largest dimension of any existing tumor.
  • Response was defined as a decrease in size of a measurable lesion by at least 50%, lasting for at least 30 days, and complete response was defined as complete disappearance of known measurable disease.
  • Stable disease was defined as neither
  • LCAP or CEA Percent change in tumor marker
  • L f represents the level at the time of first documented clinical change
  • L i represents the initial level
  • LCAP ELISA Circulating LCAP levels were assayed using LCAP ELISA kits as described in detail below, using the method set forth below. The kits are manufactured by Terumo Medical Corporation, Elkton, MD. Briefly,
  • monoclonal antibody DF-L1 (50 ⁇ g/ml) was adsorbed to 96 well microtiter culture plates (0.1 M NaHClO 3 /0.5 M NaCl buffer at pH 8.5). The wells were blocked with 5% bovine serum albumin (BSA) in 0.1 M phosphate buffered saline (PBS) for 1 h, washed, and samples containing antigen (1:51) were added to the wells. After a 1 h incubation, the plates were washed and incubated with horseradish peroxidase-conjugated MAb DF-L1 for 1 h. The plates were developed with o-phenylene diamine and, after 30 min, stopped with 2 N H 2 SO 4 ; adsorbance was determined at a wavelength of 490 nm. LCAP levels were determined by comparison with a curve generated from standards
  • CEA levels were determined according to the manufacturer's instructions using a microparticle enzyme immunoassay (IMx CEA, Abbott, North Chicago, IL). CEA levels were determined on the same freshly thawed samples used for LCAP determinations.
  • IMx CEA microparticle enzyme immunoassay
  • Squamous Cell Carcinoma Antigen Determinations Squamous Cell Carcinoma antigen (SCC antigen) levels were determined according to the manufacturer's instructions using a microparticle enzyme immunoassay (IMx SCC,
  • SCC antigen levels were determined on the same freshly thawed samples used for LCAP determinations.
  • LCAP levels were elevated above 23 U/ml in 28 of 33 (85%) patients with adenocarcinoma, 11 of 13 (85%) patients with squamous cell carcinoma, and 6 of 9 (67%) patients with large cell carcinoma of the lung. Of 58 patients with all types of non-small cell lung carcinoma (NSCLC), 46 (79%) had LCAP levels >23 U/ml. Furthermore, 9 of 13 (69%) patients with metastatic small cell carcinoma (SCLC) of the lung also had LCAP values >23 U/ml. LCAP levels were
  • LCAP levels were >23 U/ml in 15 of 29 (52%) and >40 U/ml in 11 of 29 (38%) patients with all
  • LCAP levels in patients with lung cancer were compared with CEA levels in these same patients.
  • the reported distribution of CEA in normal subjects (Abbott Package Insert: IMX: CEA. 1990) is compared with that of LCAP in Table 12.
  • IMX Abbott Package Insert
  • the two assays complement one another in patients with metastatic lung cancer, although LCAP is generally the more sensitive of the two.
  • LCAP levels were more commonly elevated than CEA levels in patients who had any evidence of visceral metastases, including pulmonary, bone, or liver (79% vs. 63%)
  • LCAP and SCC antigen levels were compared with SCC antigen.
  • An SCC antigen level of 3 ng/ml was chosen as a cut-off, since previous studies have demonstrated that 95% of a normal population have SCC antigen levels below this level (Body et al., Cancer 65:1552-6, 1990).
  • SCC antigen levels >3 ng/ml were compared to 77% who had elevated LCAP levels (data not shown) (p ⁇ 0.001).
  • LCAP levels did not change by ⁇ 25%.
  • LCAP levels increased by >25% in 4 patients who were found to have progressive disease at the next clinical evaluation.
  • serial LCAP levels exhibited a spike, defined as a dramatic increase followed by a decrease to, or nearly to, baseline (Fig. 12).
  • One of these patients had Stage IIIA adenocarcinoma and was treated with three cycles of combination chemotherapy followed by radiation therapy to the primary site, resulting in a partial response (Fig. 12A).
  • LCAP levels rose from a baseline of 21 U/ml to a peak of 59 U/ml and then fell to levels slightly above 30 U/ml.
  • LCAP levels returned to baseline following completion of therapy, and the patients were found to be free of detectable disease at that time.
  • LCAP levels were monitored in 9 patients with newly diagnosed primary NSCLC who underwent complete resection of tumor (Fig. 13). In 7 of these patients, LCAP levels were elevated prior to complete resection of their tumor. Post-operatively, LCAP levels fell on a daily basis. Four patients had pre-operative levels ⁇ 50 U/ml, and serial LCAP values fell below the cut-off of 23 U/ml within 2 days. Three other patients had pre-operative levels between 80 and 100 U/ml, and none of these fell within the normal range within 4 post- operative days. The mean ( ⁇ SD) circulating half-life, which was calculated during the postoperative decline to baseline, was 4.4 ⁇ 3 days.
  • Circulating LCAP levels were also studied in patients with metastatic malignancies of non-lung epithelial tissues (Fig. 14). LCAP levels were >23 U/ml in 31 of 104 (30%) patients with colon cancer, 8 of 14 (57%) patients with breast cancer, 26 of 60 (43%) patients with ovarian cancer, 14 of 26 (54%) patients with pancreatic cancer and 3 of 17 (18%) patients with prostate cancer. Of 8 patients with gastric cancer, none had elevated LCAP levels. LCAP levels were also >23 U/ml in 53% (66/125) of patients with benign pulmonary disease (Table 15).
  • LCAP levels ranged as high as 233 U/ml, although generally levels were ⁇ 50 U/ml.
  • LCAP levels were >23 U/ml in 18 of 33 (55%) patients with chronic obstructive pulmonary disease (COPD) (range 9-64 U/ml), and in 5 of 11 (45%) patients with chronic bronchitis (range 4-65).
  • COPD chronic obstructive pulmonary disease
  • Highest LCAP levels in patients without evidence of lung cancer were found in patients with pulmonary cancer.
  • the reaction is stopped and the color developed by the addition of 2N H 2 SO 4 .
  • the absorbance of the calibrators and unknowns is determined in a suitable spectrophotometer at a wavelength of 492 nm.
  • absorbance is directly proportional to the concentration of LCAP.
  • a point-to-point calibrator curve is
  • Blood should be drawn using standard venipuncture technique and the serum should be removed as soon as possible. Handle all samples as if capable of
  • infectious diseases such as hepatitis B virus or HIV (AIDS).
  • the aseptic sample can be stored in a refrigerator (2 - 8°C).
  • the sample should be stored frozen (at least -20°C).
  • the wells were prepared as follows: purified DF-L1 antibody (15 ⁇ g/ml, 0.15 ml/well) in 0.05M aspartic acid coating buffer, pH 3.8, was coated on micro titer strips (Nunc), followed by post-coating with 1% bovine serum albumin (BSA) in 0.01M phosphate buffer, pH 7.4, and drying under nitrogen.
  • BSA bovine serum albumin
  • Each tablet contains 15 mg o-phenylenediamine dihydrochloride and 140 mg excipient. Keep bottle tightly closed. Avoid contact with skin.
  • Plate Washer Capable of washing an 8 well strip or 96 well plate.
  • Plate Reader A suitable microtiter plate colorimeter or spectrophotometer that can measure absorbance at 492 nm.
  • SAMPLE and CONTROL PREPARATION (1:51): Dilute each sample and control 1:51 with SAMPLE DILUENT by mixing 10 ⁇ L of sample or control with 0.5 mL of diluent.
  • the diluted wash buffer may be stored at room temperature.
  • microtubes by adding 10 ⁇ L of sample to 0.5 mL of SAMPLE DILUENT. Vortex briefly to thoroughly mix.
  • Washing Fill all wells needed for the run with diluted WASH BUFFER. Allow to soak for 15 ⁇ 5 minutes. Aspirate the plate and wash three (3) times with a suitable microtiter plate washer (1 ⁇ 8, or 96 well) or washing manifold (8 or 12 channels). Invert the plate and tap on absorbent pad to remove any excess wash solution.
  • Table 16 shows typical data obtained with this assay.
  • the absorbance of the 200 unit calibrator has been set at 22°C.
  • Ambient temperatures above or below 22°C may affect the absorbance.
  • the sensitivity of the LCAP assay was determined by running the calibration curve six times, calculating the mean and standard deviations for the calibrators, adding two standard deviations to the mean absorbance for the 0 Unit CALIBRATOR, and interpolating the LCAP value from the same calibrator curve.
  • the LCAP value obtained was consistently less than or equal to 1.60 Units/ml.
  • LCAP-specific monoclonal antibodies may be prepared by immunization of mice or other animals with extracts from lung carcinoma cells, as described above, or with preparations of purified or semi-purified LCAP. These antibodies may bind to the same or a different determinant or epitope on LCAP as is bound by the MAb produced by the hybridoma DF-L1 (as determined using standard competitive assays).
  • LCAP may be isolated from the membranes of cells (primary or tissue culture) on which it occurs in relative abundance, or from the bodily fluids of lung cancer patients or other individuals with high levels of the antigen, or from the spent medium of any cell line which, like CALU-3, secretes significant amounts of the antigen.
  • the LCAP immunoassay of the invention can utilize any standard immunoassay procedure known to those who practice the art of immunoassays, including but not limited to ELISA, radioimmunoassay, fluoroimmunoassay, luminescent immunoassay, and
  • both antibody molecules can be specific for the same type of determinant on LCAP (there being at least two of such determinant type on each molecule of LCAP), or can bind to different types of determinants on the antigen.
  • the immunotoxin of the invention can be prepared by chemically conjugating a monoclonal antibody specific for LCAP to any of a number of known toxic entities.
  • a typical way of conjugating antibodies to protein toxins is by crosslinking through a disulfide bond (e.g., Chang et al., J. Biol. Chem. 252:1515-1522, 1977) or a
  • the immunotoxin can be prepared by expression of a hybrid DNA engineered to encode both the toxin (or a toxic portion thereof) and the antibody (or an LCAP-binding portion thereof), using technology available to those of ordinary skill in the art of making such hybrids (see, e.g., Murphy, U.S. Pat. No. 4,675,382, and Chaudhary et al., Proc. Natl. Acad. Sci. USA 84:4538-4542, 1987; each of which is herein incorporated by reference).
  • the DNA sequence encoding the LCAP-binding portion of the immunotoxin would be based upon the variable light-chain (V L ) amino acid sequence and the variable heavy-chain (V H ) sequence of an LCAP-specific antibody of the invention; using the method of Bird et al., Science 242:423-426, 1988, a DNA sequence encoding the V L joined to the V H by a linker peptide would be constructed and linked to a DNA sequence encoding the protein toxin (or a toxic portion thereof, as taught by, for example, Murphy U.S. Pat. No. 4,675,382). Such manipulations would be routine to one of ordinary skill in the art of genetic engineering, given the disclosures set forth herein.
  • the resulting immunotoxin could be formulated for use as an anti-cancer agent, following procedures standard to the field of pharmacology.
  • An LCAP-specific monoclonal antibody can be any LCAP-specific monoclonal antibody.
  • the potential usefulness of such an agent can be assayed, for example, by implanting LCAP-specific tumor cells into an immunocompromised host (such as a nude mouse) and determining whether or not the imaging agent of the invention detectably labels the tumor produced by such implanted cells.
  • an immunocompromised host such as a nude mouse
  • the vaccine of the invention includes the LCAP protein core from which some or all of the carbohydrate has been removed, or an antigenic fragment of the LCAP protein core, dissolved or suspended in an appropriate vehicle for injection into a person.
  • LCAP is a human glycoprotein found (at relatively low levels) in the circulation of most normal individuals and thus is not inherently immunogenic in humans, removing some or all of the carbohydrate from the LCAP protein core uncovers antigenic sites which are hidden in the
  • LCAP core protein could be produced enzymatically, chemically, or by genetic engineering, using standard methods.
  • DNA encoding the LCAP core protein could be cloned and sequenced using methods similar to those described in Siddiqui et al., Proc. Natl. Acad. Sci. USA 85:2320-2323, 1988; Merlo et al.. Cancer Res. 49:6966-6971, 1989; and Abe and Kufe, Biochem. Biophys. Res.
  • NSCLC 4 4(100) 3 (75) 3 (75) 3 (75) 3 (75) 2 (50) 2 (50) 35.5 ⁇ 33
  • antigen levels increased by >25%.
  • antigen levels decreased by >25%.
  • antigen levels did not increase or decrease by ⁇ 25%.
  • Pulm. Embolus 4 (100) 42.5 ⁇ 7 35.0 - 52.0
  • Tuberculosis 17 8 (47) 31.0 ⁇ 20 4.0 - 78.0
  • Misc. 11 5 (45) 93.9 ⁇ 102 3.0 - 233.0 includes Acute Respiratory Insufficiency, Alveolitis, Atelectasis, Bronchial Polyp, Empyema, Hemoptysis, Rheumatoid Lung, Silicosis, Pulmonary Edema, Subcutaneous Emphysema.

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Abstract

L'invention se rapporte à une préparation essentiellement purifiée d'une protéine associée au cancer du poumon chez l'homme (PACP), d'un anticorps monoclonal spécifique à cette protéine, et d'un procédé pour détecter ladite protéine dans un échantillon biologique. Ce procédé consiste à (1) mettre en contact l'échantillon biologique avec une aliquote contenant l'anticorps monoclonal spécifique de la protéine PACP, et (2) à détecter une formation de complexe immun entre l'anticorps et un composant de l'échantillon biologique, cette formation de complexe immun indiquant la présence de la protéine PACP dans ledit échantillon biologique.
PCT/US1991/007585 1991-10-09 1991-10-09 Proteine associee au cancer du poumon WO1993006858A1 (fr)

Priority Applications (2)

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PCT/US1991/007585 WO1993006858A1 (fr) 1991-10-09 1991-10-09 Proteine associee au cancer du poumon
CA002120363A CA2120363A1 (fr) 1991-10-09 1991-10-09 Proteine associee au cancer pulmonaire

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PCT/US1991/007585 WO1993006858A1 (fr) 1991-10-09 1991-10-09 Proteine associee au cancer du poumon
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0695760A1 (fr) * 1994-08-05 1996-02-07 F. Hoffmann-La Roche Ag Marqueur tumorale pour détecter le cancer du poumon
US5989552A (en) * 1993-12-24 1999-11-23 Austin Research Institute Antigen carbohydrate compounds and their use in immunotherapy
WO2000043508A2 (fr) * 1999-01-22 2000-07-27 Incyte Pharmaceuticals, Inc. Proteines associees a certains cancers
US6548643B1 (en) 1994-11-16 2003-04-15 Austin Research Institute Antigen carbohydrate compounds and their use in immunotherapy
US8021667B2 (en) 1994-11-16 2011-09-20 Macfarlane Burnet Institute For Medical Research And Public Health Ltd Compositions for immunotherapy and uses thereof
US8771701B2 (en) 1997-09-29 2014-07-08 Macfarlane Burnet Institute For Medical Research And Public Health Ltd Compositions for immunotherapy and uses thereof
CN110438088A (zh) * 2019-08-16 2019-11-12 中国医学科学院肿瘤医院 肺癌相关蛋白1单克隆抗体的制备以及纯化方法

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* Cited by examiner, † Cited by third party
Title
CANCER RESEARCH, Volume 44, issued May 1984, ROSEN et al., "Analysis of Human Small Cell Lung Cancer Differentiation Antigens Using a Panel of Rat Monoclonal Antibodies", pages 2052-2061. *
CANCER RESEARCH, Volume 44, issued November 1984, OKABE et al., "Monoclonal Antibodies to Surface Antigens of Small Cell Carcinoma of the Lung", pages 5273-5278. *
CANCER RESEARCH, Volume 50, issued 15 October 1990, MAIMONIS et al., "Detection and Characterization of a High Molecular Weight Human Lung Carcinoma-Associated Glycoprotein", pages 6738-6743. *
CANCER, Volume 65, issued 15 March 1990, CHESTER et al., "An Analysis of Immunocomplexes for the Detection of the Early Stages of Colon Cancer", pages 1338-1344. *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5989552A (en) * 1993-12-24 1999-11-23 Austin Research Institute Antigen carbohydrate compounds and their use in immunotherapy
EP0695760A1 (fr) * 1994-08-05 1996-02-07 F. Hoffmann-La Roche Ag Marqueur tumorale pour détecter le cancer du poumon
WO1996004302A1 (fr) * 1994-08-05 1996-02-15 F. Hoffmann-La Roche Ag Nouveau marqueur tumoral du cancer du poumon
US6548643B1 (en) 1994-11-16 2003-04-15 Austin Research Institute Antigen carbohydrate compounds and their use in immunotherapy
US8021667B2 (en) 1994-11-16 2011-09-20 Macfarlane Burnet Institute For Medical Research And Public Health Ltd Compositions for immunotherapy and uses thereof
US8771701B2 (en) 1997-09-29 2014-07-08 Macfarlane Burnet Institute For Medical Research And Public Health Ltd Compositions for immunotherapy and uses thereof
WO2000043508A2 (fr) * 1999-01-22 2000-07-27 Incyte Pharmaceuticals, Inc. Proteines associees a certains cancers
WO2000043508A3 (fr) * 1999-01-22 2000-11-16 Incyte Pharma Inc Proteines associees a certains cancers
CN110438088A (zh) * 2019-08-16 2019-11-12 中国医学科学院肿瘤医院 肺癌相关蛋白1单克隆抗体的制备以及纯化方法

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