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EP0505566A1 - Anticorps biospecifique des cellules cancereuses et enzyme presentant des caracteristiques d'activation promedicamenteuse - Google Patents

Anticorps biospecifique des cellules cancereuses et enzyme presentant des caracteristiques d'activation promedicamenteuse

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Publication number
EP0505566A1
EP0505566A1 EP91900329A EP91900329A EP0505566A1 EP 0505566 A1 EP0505566 A1 EP 0505566A1 EP 91900329 A EP91900329 A EP 91900329A EP 91900329 A EP91900329 A EP 91900329A EP 0505566 A1 EP0505566 A1 EP 0505566A1
Authority
EP
European Patent Office
Prior art keywords
antibody
indications
ifo
prodrug
european patent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP91900329A
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German (de)
English (en)
Inventor
Susumu Iwasa
Kayoko Okamoto
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Takeda Pharmaceutical Co Ltd
Original Assignee
Takeda Chemical Industries Ltd
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Filing date
Publication date
Application filed by Takeda Chemical Industries Ltd filed Critical Takeda Chemical Industries Ltd
Publication of EP0505566A1 publication Critical patent/EP0505566A1/fr
Ceased legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • 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
    • A61K47/6875—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 being a hybrid immunoglobulin
    • A61K47/6879—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 being a hybrid immunoglobulin the immunoglobulin having two or more different antigen-binding sites, e.g. bispecific or multispecific immunoglobulin
    • 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
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6899—Antibody-Directed Enzyme Prodrug Therapy [ADEPT]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies

Definitions

  • the present invention relates to a bispecific antibody-enzyme complex that serves well as an anticancer therapeutic drug. More specifically, the present invention relates to a hybrid monoclonal antibody (hereinafter also referred to as hybrid MoAb) wherein one of the two specificities is to human cancer cells and the other is to a prodrug-activating enzyme, and a polydoma that produces said antibody.
  • hybrid MoAb a hybrid monoclonal antibody
  • the present invention also relates to an anti-human-cancer-protein complex obtained by immunologically binding the above-mentioned enzyme to the above-mentioned hybrid MoAb.
  • antibody missile therapy drugs antitumor immunocomplexes prepared by binding an antitumor antibody to a chemotherapeutic agent or a biotoxin which aims at selective destruction of cancer cells; some successes have been achieved in blood-related cancers such as leukemia and lymphoma. However, no satisfactory results have been obtained in actual clinical application. Particularly with respect to solid cancers, much remains unsolved, including the problem of serious side effects.
  • the present inventors investigated a recently developed bispecific antibody and developed the present invention. Accordingly, the inventors prepared a bispecific antibody capable of binding to both an enzyme that converts an inactive anticancer prodrug into active type and to a human cancer cell, and administered an immunocomplex comprising said antibody and said enzyme, as well as an inactive prodrug, to cancer patients, thus developing an anti-human-cancer- protein complex exibiting a cytotoxic effect selectively on cancer cells, regardless of their diversity.
  • the prodrug itself is present in blood and other organs and tissues in an inactive form; only in the vicinity of the target cancer tissue is the prodrug decomposed and activated by the anti-human-cancer-protein complex of the present invention, as bound to human cancer cells, to exhibit its anticancer activity.
  • the prodrug has almost no side effects in the administration method using the anti-human-cancer-protein complex of the present invention.
  • Administration using the anti-human-cancer-protein complex of the present invention is also characterized in that anticancer activities are exhibited against cancer cells which are present in the vicinity of the target cancer tissue but are free of target antigens, cytotoxic effect being exhibited regardless of the diversity of cancer cells.
  • one object of the present invention is to provide a bispecific hybrid monoclonal antibody wherein one of the two specificities is to human cancer cells and the other is to a prodrug-activating enzyme, and a polydoma that produces said antibody.
  • Another object of the present invention is to provide an anti-human- cancer-protein complex comprising the bispecific hybrid MoAb described above and a prodrug-activating enzyme immunologically coupled thereto.
  • Fig. 1 shows the cytotoxicity of a tripeptidated drug (Boc-Gly-Gly-Arg- ADR; - • -) and its activated body (ADR; - o -) on gastric cancer cell line NUGC4 (see Example 4).
  • Fig. 2 shows the cytotoxicity of a tripeptidated drug (Boc-Pro-Gly-Arg- TAN-1120; - • -) and its activated body (TAN-1120; - o -) on renal cancer cell line AM-RC-6 (see Example 4).
  • Fig. 3 shows the cytotoxicity of a tripeptidated drug (Boc-Gly-Gly-Arg- PM; - • -) and its activated body (PM; - o -) on renal cancer cell line AM-RC-6 (see Example 4).
  • Fig. 4 shows a chromatographic pattern of the trypsin-hydrolyzed product of Boc-Gly-Gly-Arg-ADR (see Example 6).
  • Fig. 5 shows a chromatographic pattern of the UK-hydrolyzed product of Boc-Gly-Gly-Arg-PM (see Example 6).
  • Fig. 6 shows the antibody dilution curve of the culture supernatant of anti-hTfR-anti-UK bispecific antibody producing mouse tetraoma UTF 20-7. (see Example 9)
  • the above-mentioned polydoma that produces a bispecific hybrid MoAb s prepared, for example, by fusing a hybridoma that produces an anti- human-cancer antibody with another hybridoma that produces an antibody against a prodrug-activating enzyme.
  • Any anti-human-cancer-antibody- producing hybridoma can serve for this purpose, as long as it produces an antibody capable of specifically binding to human cancer cells.
  • Examples of such hybridomas include mouse hybridoma 22C6 UFO 50172, FERM BP- 2054] [cf. Japanese Unexamined Patent Publication No.
  • target antigens for the anti-human-cancer antibodies produced by these hybridomas include cancer cell membrane surface antigens such as tumor-related antigens, immunocompetent cell surface receptors and virus-infected cell surface antigens.
  • hTfR a tumor-related antigen
  • target antigens include carcinoembryonic antigen (CEA), ⁇ - fetoprotein, some cancer-related sugar chain antigens such as CA19-9 [S. Hakomori: Cancer Research, 45, 2405 (1985)], the B-cell lymphoma membrane immunoglobulin idio-type [R. A. Miller et al.: New England Journal of Medicine, 306 * , 517 (1982)] and the T-cell lymphoma membrane immunoglobulin idio-type [L. L. Lanier et al.: Journal of Immunology, 137, 2286 (1986)].
  • CEA carcinoembryonic antigen
  • ⁇ - fetoprotein some cancer-related sugar chain antigens such as CA19-9 [S. Hakomori: Cancer Research, 45, 2405 (1985)]
  • B-cell lymphoma membrane immunoglobulin idio-type R. A. Miller et al.: New England Journal of Medicine, 306 * , 517 (1982)
  • hybridoma that produces an antibody against a prodrug- activating enzyme
  • an ordinary hybridoma preparation method is used [G. Kohleretal.: Nature, 256, 495 (1975)].
  • animals are immunized with the enzyme in accordance with a standard method, and the resulting antibody-producing cells are fused with myeloma cells etc.
  • mice examples include rabbits, rats, mice and guinea pigs, with preference given to mice in the case of MoAb preparation. Inoculation can be achieved by an ordinary method.
  • the mouse receives subcutaneous or intraperitoneal inoculation of the enzyme at the back or abdomen at a dose of 1 to 100 ⁇ g, preferably 10 to 25 ⁇ g, in emulsion in an equal volume (0.1 m €) of saline, in the presence of Freund's complete adjuvant, 3 to 6 times once every 2 to 3 weeks.
  • mice individuals with high antibody titer are selected.
  • spleens and/or lymph nodes are collected, and antibody-producing cells contained therein are fused with myeloma cells. Fusion can be achieved in accordance with a known method.
  • fusogens include polyethylene glycol (hereinafter also referred to as PEG) and Sendai virus, with preference given to PEG.
  • Example myeloma cell lines include NS-1, P3U1 and SP2/0, with preference given to P3U1.
  • a preferred ratio of, for example, splenocytes and myeloma cells, is 1:1 to 10:1. It is recommended that this cell mixture be incubated at 20 to 37°C, preferably 30 to 37°C, in the presence of a PEG with a molecular weight of about 1,000 to 9,000 at a concentration of 10 to 80% for 3 to 10 minutes.
  • Various methods are available for screening the antibody-producing hybridomas described above. For example, human cancer cells or enzyme proteins are adsorbed to a microplate to prepare an antigen-sensitized plate, to which is added the culture supernatant of the hybridomas obtained by cell fusion. This is followed by determination of antibody titer in the culture supernatant by enzyme immunoassay (hereinafter also referred to as EIA) for detection of plate-bound specific antibody.
  • EIA enzyme immunoassay
  • Hybridomas positive for antibody activity are selected, cultured in HAT (hypoxanthine-aminopterin- thymidine) medium etc. and immediately subjected to cloning, which can be done easily by the limiting dilution method.
  • the antibody titer of the culture supernatant of the cloned hybridomas is also determined by the EIA procedure described above; monoclonal hybridomas that stably produce a potent antibody can thus be selected and cultured.
  • a hybridoma that produces a neutralizing antibody against a prodrug-activating enzyme, such as urokinase can also be used as a parent cell in polydoma preparation.
  • an HAT- resistant hybridoma that produces an anti-human-cancer specific antibody is made resistant to 8-azaguanine (hereinafter also referred to as 8-AZG), and a hypoxanthine-guanine-phosphoribosyl transferase-deficient strain is cloned to make it HAT-sensitive.
  • 8-AZG 8-azaguanine
  • a hypoxanthine-guanine-phosphoribosyl transferase-deficient strain is cloned to make it HAT-sensitive.
  • a hybridoma that produces an anti-human-cancer-cell specific antibody is labeled with fluorescein isothiocyanate (hereinafter also referred to as FTTC), and another hybridoma that produces an antibody against a prodrug-activating enzyme is labeled with tetramethyl rhodamine isothiocyanate (hereinafter also referred to as TRTTC), followed by fusion of these two in accordance with a standard method.
  • the resulting cell suspension is applied to a fluorescence-activated cell sorter (hereinafter also referred to as FACS), and a tetraoma that shows both the green fluorescence of PTTC and the red fluorescence of TRTTC is selected and cloned.
  • FACS fluorescence-activated cell sorter
  • Polydoma selection can be carried out using the HAT medium described above and other means.
  • 8-AZG, 6-thioguanine (6- TG) or 5-BrdU is used for drug acclimation to obtain corresponding drug- resistant strains.
  • various selection media are used to introduce a new marker into fused cells. Examples of such selection media include media supplemented with neomycin or hygromycin B [B. Sugden et al.: Molecular and Cellular Biology, 5, 410 (1985)].
  • the polydoma positive for bispecific antibody activity is immediately subjected to cloning, easily be achieved by the limiting dilution method etc.
  • the antibody titer of the culture supernatant of the cloned polydoma is determined by the method described above, and a polydoma that stably produces a potent antibody is selected, whereby the desired polydoma (e.g., mouse tetraoma UTF20-7(IFO 50260, FERM BP-3156) obtained in following Example, or other methods) that produces the monoclonal bispecific antibody can be obtained.
  • the desired polydoma e.g., mouse tetraoma UTF20-7(IFO 50260, FERM BP-3156) obtained in following Example, or other methods
  • the polydoma of the present invention described above can be cultivated normally in liquid medium, or in the abdominal cavity of animals (e.g., mammalians such as mice) by a known method.
  • Purification of the antibody in the culture broth or ascites can be achieved using a combination of known biochemical techniques. For example, the cell culture broth or ascites fluid is centrifuged, and the resulting supernatant separated and subjected to salting-out (normally with ammonium sulfate or sodium sulfate). The resulting protein precipitate is dissolved in an appropriate solution, followed by dialysis.
  • the solution is then subjected to column chromatography (using an ion exchange column, gel filtration column, protein A column, hydroxyapatite column etc.) to separate and purify the desired antibody.
  • column chromatography using an ion exchange column, gel filtration column, protein A column, hydroxyapatite column etc.
  • the separation and purification procedures described above yield about 1 to 10 mg of a bispecific MoAb of a purity not less than 90% by protein weight.
  • the same MoAb is obtained in an amount of about 2 to 20 mg.
  • the bispecific MoAb thus obtained is uniform as a protein and, for example, F(ab')2 fragments etc. capable of binding to both human cancer cells and a prodrug-activating en2yme can be obtained, for example, by treatment with protease (e.g., pepsin). These fragments can be used for the same purpose as the bispecific MoAb of the present invention.
  • protease e.g., pepsin
  • a tetraoma formed between a hybridoma that produces an anti-human- cancer-cell MoAb and another hybridoma that produces an antibody against a prodrug-activating enzyme is included in the polydoma that produces the hybrid MoAb of the present invention, but any trioma formed between a hybridoma that produces one MoAb and a cell that produces the other MoAb, or any hybridoma obtained by immortalizing two kinds of cells that produce respective MoAbs using Epstein-Barr virus or other means, and then fusing them, can be used for the same purpose as the above-mentioned tetraoma, as long as it produces the bispecific MoAb of the present invention.
  • mice-human chimeric antibody When these polydomas produce mouse IgG MoAb, it is possible to prepare a mouse-human chimeric antibody by deriving a DNA that encodes a variable or hypervariable region containing the antigen recognition site for said bispecific hybrid MoAb and binding thereto a gene that encodes the constant region of human IgG, using a gene manipulation technique [Z. Steplewski et al.: Proceedings of National Academy of Science, 85, 4852 (1988)]. Such a chimeric antibody serves well in administration to humans, due to its low antigenicity.
  • bispecific MoAb of the present invention In anticancer therapy using the bispecific MoAb of the present invention or a selective anti-human-cancer-protein complex prepared from a prodrug-activating enzyme and said bispecific MoAb, several methods are available, including (1) the method in which the bispecific MoAb of the present invention is administered to the cancer patient, and after sufficient time has elapsed for it to bind to cancer tissues and cells, the enzyme and then the prodrug are administered, (2) the method in which the bispecific MoAb and the enzyme are administered simultaneously, followed by prodrug administration, and (3) the method in which the hybrid MoAb is reacted with the enzyme, and after separation of the unreacted portion of the enzyme, the resulting anti-human-cancer-protein complex is administered to the cancer patient, followed by prodrug administration.
  • the bispecific MoAb or prodrug-activating enzyme of the present invention can be used for the treatment of various cancerous diseases in the form of a preparation such as an injection, with or without being formulated with an appropriate pharmacologically acceptable carrier, excipient, diluent or other additive, after germ removal by filtration using, for example, a membrane filter, as desired.
  • Dose volume varies depending upon the type of target cancer, symptoms, route of administration and other aspects; but, for example, in intravenous administration to an adult human patient, it is normally about 0.02 to 1.0 mg/kg, preferably about 0.04 to 0.4 mg/kg, daily, as the bispecific antibody, or about 0.01 to 0.5 mg kg daily, as the prodrug- activating enzyme.
  • Any prodrug-activating enzyme can serve for the present invention, as long as it shows prodrug-activating action, but it is preferable to use a protease (e.g., urokinase (UK), trypsin), which cleaves peptide bonds, or a glycosidase (e.g., glucuronidase), which cleaves sugar chain bonds.
  • a protease e.g., urokinase (UK), trypsin
  • a glycosidase e.g., glucuronidase
  • the enzyme be a human-derived enzyme whose blood level is low or which is not present in blood, and that the enzyme be produced by cancer cells [J. C.
  • prodrugs comprising a drug active body and an appropriate peptide (e.g., Gly-Gly-Arg, Pro-Gly- Arg, Pyr-Gly-Arg) bound thereto can be used.
  • an appropriate peptide e.g., Gly-Gly-Arg, Pro-Gly- Arg, Pyr-Gly-Arg
  • glucuronidase glucuronidated drugs can be used as prodrugs.
  • the prodrug is a peptidated drug or a glucuronidated drug
  • its toxicity is expected to be extremely lower than that of the original drug active body, or even nontoxic; therefore, it is capable of being activated in the vicinity of the cancer tissue and selectively destroying the cancer tissue when used in combination with an anti-human-cancer-protein complex comprising the bispecific antibody of the present invention and a prodrug-activating enzyme immunologically bound thereto.
  • any anticancer agent can be used as the original drug for the above- mentioned prodrug, but preference is given to those in clinical application such as adriamycin, cisplatin, melphalan, methotrexate, mitomycin C, vincristine, puromycin and phenylenediamine mustard.
  • highly cytotoxic ansamitocins , TAN-1120 represented by the following formula (II) wherein X represents OH
  • related compounds may be used as anticancer agents. Examples of such compounds include compounds represented by the following formula (I) [cf. Japanese Patent Application No. 18560/1989, European Patent Publication No. 376176], their 4,5-deoxy bodies, and compounds represented by the following formula (IE) [cf.
  • any ansamitocin or TAN-1120 related compound can be used, as long as it possesses anticancer activity.
  • the drug is administered to the patient in the form of a nontoxic or weakly toxic prodrug, and is decomposed by the anti-human-cancer-protein complex of the present invention in the vicinity of the cancer tissue to exhibit its pharmacological activities.
  • Ansamitocins include compounds represented by the following formula:
  • R represents a hydrogen atom or a carboxylic acid-derived acyl group
  • Q represents a hydroxyl group (OH) or a mercapto group (SH)
  • X represents a chlorine atom or a hydrogen atom
  • Y represents a hydrogen atom, a lower alkylsulfonyl group, an alkyl group or an aralkyl group which may have a substituent.
  • X represents a hydroxyl group or a hydrogen atom.
  • amino acids and peptides are represented by the abbreviation system adopted by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN).
  • CBN Biochemical Nomenclature
  • adherent cells were dispensed to a 60-well microplate (produced by Nunc Intermed) at 500 cells per well and cultured for 24 to 48 hours.
  • Non-adherent cells were suspended in a serum-free culture medium and dispensed to a plate at the same ratio as above, followed by centrifugation at 400 X g for 5 minutes to adsorb them to the plate, on the day of examination.
  • sheep red blood cells were washed three times with phosphate buffered saline (20mM bisodiumphosphate, 0.15M NaCl; pH7.5) (hereinafter also referred to as PBS) and suspended in PBS to obtain a 2% suspension.
  • PBS phosphate buffered saline
  • This suspension was mixed with an equal amount of mouse anti-sheep-red-blood-cell antibody (produced by Ortho Co.), diluted with PBS to a concentration 2.5 times the maximum agglutination value, followed by reaction at 37°C for 30 minutes.
  • mouse anti-sheep-red-blood-cell antibody produced by Ortho Co.
  • sample solution containing a mouse anti-human-cancer antibody was dispensed to each well, and the plate was kept standing at room temperature for 1 hour.
  • indicator blood cells in 0.2% dilution in 5% FCS-VBS, were dispensed to each well, and the plate was kept standing at room temperature for 40 minutes.
  • the unreacted blood cells were washed away with VBS, and the plate was observed microscopically.
  • a rosette was found in not greater than 1% of the cells.
  • a "positive" test was definded as a rosette formed by not less than 25% of the target cells.
  • the subject cells were suspended in a 0.02% EDTA- PBS solution. This suspension was washed with a serum-free culture medium, and a solution containing a mouse anti-human-cancer antibody was added, followed by reaction at 4°C for 1 hour. After washing with culture medium, fluorescein-labeled anti-mouse-IgG antibody was added, followed by reaction at 4°C for 1 hour. After washing with PBS, the reaction product was observed using a fluorescent microscope.
  • Target tumor cells were seeded to a Nunc Intermed 96-well microplate at 10,000 to 40,000 cells per well, followed by incubation in a carbon dioxide incubator at 37°C for 1 day. After culture supernatant removal, a solution containing a mouse anti-human-cancer antibody was added, followed by reaction at room temperature for 2 hours. The plate was then washed with a medium supplemented with 0.2% bovine serum albumin (hereinafter also referred to as BSA); a rabbit anti-mouse-IgG antibody labeled with horseradish peroxidase (hereinafter also referred to as HRP) was then added, followed by reaction at room temperature for 2 hours.
  • BSA bovine serum albumin
  • HRP horseradish peroxidase
  • mice showing a high antibody titer against renal cancer cell line AM-RC-7 were subjected to the following experiment.
  • the immune mouse splenocytes obtained in (1) were fused with mouse myeloma cell line NS-1 by a standard method, followed by selection culture using HAT medium.
  • the hybridomas grown were subjected to screening by the MHA method described in Reference Example 1, and the group of hybridomas showing high antibody titer were further cloned to yield the desired mouse hybridoma RCS-1 (FERM BP-2333, IFO 50184), which produces an anti-human-renal-cell-cancer MoAb.
  • the RCS-1 antibody produced by the mouse hybridoma RCS-1 proved to belong to the IgGi subclass.
  • mice 5 X 106 cells of mouse hybridoma RCS-1 were intraperitoneally administered to MCH(AF)-nu mice. About 4 weeks later, 5 to 10 m£ of ascites fluid was collected. After salting-out with ammonium sulfate, the ascites fluid was purified using a DEAE-cellulose column. About 200 mg of purified mouse anti-human-renal-cell-cancer MoAb RCS-1 was obtained from 50 ⁇ a£ of ascites fluid.
  • RCS- 1 antibody was found to be positive for renal cancer cell lines AM-RC-3, AM- RC-6 and AM-RC-7, bladder cancer cell line T24 and lung cancer cell lines Luci-10 and PC-10, and negative for other cancers, namely gastric cancer, intestinal cancer, breast cancer and leukemia cancer cell lines; it was also negative for normal renal tissues.
  • a 5 ⁇ g/m € UK solution was dispensed to a 96-well microplate at 100 ⁇ € per well. After the microplate was kept standing at 4°C overnight, 150 ⁇ € of PBS containing 2% casein and 0.01% thimerosal was added, to prepare a sensitized plate. After removing the added solution, the plate was washed with PBS containing 0.05% Tween 20 (hereinafter also referred to as PBS- Tw), and 100 ⁇ € of the subject mouse antibody solution was added, followed by reaction at room temperature for 2 hours. Similarly, after the plate was thoroughly washed with PBS-Tw, an HRP-labeled rabbit anti-mouse-IgG antibody was added, followed by reaction for 2 hours.
  • PBS- Tw PBS containing 0.05% Tween 20
  • Spleens were excised 3 days after final immunization, and a splenocyte suspension was prepared by a standard method (about 108 cells). Then, 2
  • HAT medium containing hypoxanthine, aminopterin and thymidine, followed by cultivation for 10 days.
  • HAT medium was replaced with HT medium of the same composition as
  • the antibody titer of the hybridoma culture supernatant was determined by the EIA method described in Reference Example 5, using a
  • hybridomas began to appear, along with an antibody that specifically binds to UK.
  • the hybridomas showing particularly high affinity were subjected to cloning by the limiting dilution method.
  • the culture supernatant of the cloned hybridoma was subjected to screening in the same manner; those having high UK affinity were selected.
  • Hybridoma selection and cloning Hybridoma screening was carried out by the EIA method, described in
  • An antigen-sensitized plate was prepared in the same manner as in Reference Example 5 except that ⁇ -glucuronidase (produced by Sigma Co.) was used in place of the UK described therein. EIA was then carried out in the same manner as in Reference Example 5, to determine the anti- glucuronidase antibody titer.
  • the hTfR fraction eluted with a 0.02 M glycine buffer solution (pH 10.0) containing 0.5 M NaCl and 0.5% Triton X-100 was applied to an hTf-coupled column. After the column was washed with PB containing 1 M NaCl, elution was conducted using a 0.05 M glycine buffer solution (pH 10.0) containing 1 M NaCl and 1% Triton X-100 to yield about 1.5 mg of a purified sample of hTfR.
  • splenocyte suspension was prepared by a conventional method (approximately 108 cells). To this suspension was added 2 X 107 mouse myeloma cells (P3U1), followed by cell fusion in accordance with the method described in Reference Example 8-(2). After selection of parent cells in HAT medium, cultivation was continued using HT medium which had the same composition as that of HAT medium, but not including aminopterin.
  • a commercially available anti-mouse IgG rabbit antibody solution (20 ⁇ g/m €) was dispensed to a 96-well microplate at 100 ⁇ € per well. After this microplate was allowed to stand at 4°C overnight, PBS (pH 7.3) containing 2% BSA was added to prepare a sensitized plate.
  • the purified sample of hTfR obtained in (1) after being labeled with HRP in accordance with a conventional method, was used for EIA [T. Kitagawa: Yuki Gosei Kagaku, 42, 283 (1984)]. Accordingly, the culture supernatant of hybridomas was added to the above second antibody-sensitized plate, and reaction was carried out at room temperature for 2 hours. After the plate was washed with PBS, HRP-labeled hTfR was added, followed by reaction at room temperature for 2 hours. Enzyme reaction was then carried out by the method described in Reference Example 3, to determine the antibody titer.
  • hybridoma showing especially high binding activity was subjected to cloning by limiting dilution method to yield anti-hTfR-antibody-producing hybridoma 22C6.
  • the present antibody was identified as the IgGi ( chain) subclass, exhibiting high affinity to human leukemia cell strain K562 and human epidermoid carcinoma cell line A431.
  • Example 1 Preparation of hybridoma that produces anti-glucuronidase monoclonal antibody
  • Spleens were excised from mice that showed high serum antibody titer, as determined by the EIA method described in Reference Example 10; cell fusion was carried out in accordance with the method described in Reference Example 8-(2).
  • Fused cells that appeared at 10 to 20 days following fusion were screened by the EIA method described in Reference Example 10; the hybridomas showing particularly high affinity were subjected to cloning by the limited dilution method.
  • the cloned hybridomas were selected in the same EIA method to yield BG1-5 [FERM BP-2688, IFO 50219], a mouse hybridoma that produces an MoAb that specifically binds to glucuronidase.
  • the antibody produced by this hybridoma was identified as IgGi.
  • the neutralization experiment described in Reference Example 11 revealed that this antibody does not neutralize glucuronidase enzyme activity.
  • FACS Fluorescein-rhodamine double stained cells were separated and seeded, at 10 cells per well, to a 96-well microplate seeded with 5 X 105 cells/well mouse thymocytes as feeders, and cultivated.
  • the culture supernatant from each well in which cell growth occurred 1 to 2 weeks after fusion was subjected to Cell-EIA to determine the bispecific antibody titer.
  • the subject hybrid hybridoma culture supernatant was added, followed by reaction at room temperature for 2 hours.
  • biotin-labeled UK was added, followed by reaction at room temperature for 2 hours.
  • HRP- labeled avidin reaction at room temperature for 1 hour the plate was washed and the enzyme activity bonded to the solid phase was determined by the method described in Reference Example 3.
  • the cells in wells showing high bispecific antibody titer were subjected to cloning by the limiting dilution method, yielding the desired bispecific- antibody-producing tetraoma.
  • mice pretreated by intraperitoneal administration of 0.5 m € mineral oil, mouse hybrid hybridomas (tetraomas) were inoculated intraperitoneally at 5 X 106/mouse.
  • Ascites fluid whose retention occurred about 10 to 20 days after inoculation, was collected and subjected to salting- out with 50% saturated ammonium sulfate to yield an IgG fraction.
  • the IgG fraction was applied to a UK- coupled Cellulofine column, followed by elution with 0.2 M glycine-HCl buffer at pH 2.9.
  • the acid-eluted fraction was applied to a hydroxyapatite column to purify the desired bispecific anti-human-cancer- cell-anti-UK antibody.
  • This oily substance was dissolved in 170 m € of methanol (MeOH) containing 20 m € of 1 N HC €, followed by catalytic reduction in a hydrogen stream in the presence of palladium black (500 mg). After stirring in the hydrogen stream for 4.5 hours, the catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was stored in DMF (30 m €) solution for use for the next reaction.
  • MeOH methanol
  • Example 3 Boc-Gly-Gly-Arg-ADR, Boc-Pro-Gly-Arg-TAN-1120 and Boc-Gly- Gly-Arg-PM, respectively
  • gastric cancer cell line NUGC4 or renal cancer cell line AM-RC-6 were compared with those of their activated bodies (ADR, TAN-1120 and PM, respectively) [Figs, 1 through 3; - o -].
  • each drug was added at various concentrations to a microplate seeded with cultured human cancer cells at 5 X 103 cells/well, followed by cultivation for 4 days.
  • viable cells were counted using 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) [H. Tada et al.: Jounal of Immunological Methods, 93, 157 (1986)].
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide
  • the trypsin decomposition product of Boc-Gly-Gly-Arg-ADR and the UK decomposition product of Boc-Gly-Gly-Arg-PM obtained in Example 5 were subjected to reversed phase high performance liquid chromatography using an ODS column (YMC A-302 ODS 120A column, 4.6 X 150 mm, commercially available from YMC KK), and their chromatographic patterns were compared with those of their activated bodies ADR and PM. Elution was carried out with an eluent of 30% acetonitrile/0.01 M phosphate buffer (pH 3.0) at a flow rate of 1.0 m €/min; the ultraviolet absorbance of the column effluent was determined at 254 nm.
  • Figs.4 and 5 The results are shown in Figs.4 and 5.
  • Fig.4 reveals that Boc-Gly-Gly- Arg-ADR (peak B; eluted at 10.2 minutes) was decomposed and activated into ADR (peak A; eluted at 3.6 minutes) by trypsin.
  • Fig. 5 reveals that Boc-Gly- Gly-Arg-PM (peak B; eluted at 3.2 minutes) was decomposed and activated into PM (peak A; eluted at 1.8 minutes) by UK.
  • Phenylenediamine mustard (PDM) was synthesized in accordance with a known method [W. C. J. Ross: Journal of Chemical Society, 183 (1949) and J. L. Everett et al.: Journal of Chemical Society, 1972 (1949)].
  • Npys-Val-ADR as an orange-red crystal (27 mg).
  • 0.1 m € of a 1 N aqueous hydrochloric acid was added, followed by stirring at room temperature for 40 minutes.
  • Aqueous sodium bicarbonate was then added to neutralize the solution, followed by n-butanol extraction. After the extract was washed with water and dried, the solvent was distilled off under reduced pressure.
  • the starting material 6-(9-carboxynonyl-2,3-dimethoxy-5-methyl-l,4- benzoquinone (QS-10) was treated in the same manner as in (3) above and crystallized from ethyl acetate-n-hexane to yield 10-(2,3-dimethoxy-5-methyl- l,4-methyl-benzoquinone-6-yl)decanoylglycine ethyl ester (QS-10-Gly-OEt) in the form of an orange-yellow needle (mp.77 to 77.5°C).
  • ADR, QS-4-Gly-Gly-Arg-PM, QS-10-Gly-Gly-Arg-PM and Bzr-Gly-Ala-Pro- Gly-Arg-PM obtained in Example 7 were added, followed by addition of UK and cultivation at 37°C.
  • the prodrug activating reaction of UK was then determined by the method described in Example 5.
  • Hybridoma 22C6, which produces an anti-hTfR MoAb, obtained in Reference Example 12, and hybridoma UK1-6, which produces an anti-UK MoAb, obtained in Reference Example 9, were each incubated in Iskove-Ham F-12 mixed medium containing 0.5 ⁇ g/m € FTTC and 1.5 ⁇ g/m € TRTTC at 37°C for 30 minutes for fluorescent staining.
  • An LSM solution (commercially available from Wako Pure Chemical Industries Ltd.) was then added, and the dead cells were removed; the two hybridomas were then mixed at a ratio of 1 to 1 for cell fusion using PEG 6000 by the method described in Reference Example 8-(2).
  • the cell mixture was applied to FACS, and 25000 fluorescein-rhodamine double stained cells were separated and seeded, at 10 cells per well, to a 96-well microplate seeded with 5 X 105 cells/well mouse thymocytes as feeders, and cultivated.
  • mice pretreated by intraperitoneal administration of 0.5 m € mineral oil, mouse hybrid hybridomas (tetraomas) were inoculated intraperitoneally at 5 X 106/mouse.
  • Ascites fluid whose retention occurred about 10 to 20 days after inoculation, was collected and subjected to salting- out with 50% saturated ammonium sulfate to yield an IgG fraction.
  • the IgG fraction was applied to a UK- coupled Cellulofine column, followed by elution with 0.2 M glycine-HCl buffer at pH 2.9.
  • the acid-eluted fraction was applied to a hydroxyapatite column, the desired bispecific anti-hTfR-anti-UK antibody was purified.
  • Example 10 Prodrug activating reaction by bispecific antibody
  • the immunocomplex comprising the purified bispecific antibody obtained in Example 9 and UK (1:1) was added, followed by reaction at 5°C for 30 minutes. After cells were washed at a low temperature, the prodrug Boc-Gly- Gly-Arg-Val-ADR, described in Example 7-(2) or the prodrug QS-10-Gly-Gly- Arg-PM described in Example 7-(4) was added at a final concentration of 5.0 ⁇ g/ml and 0.2 ⁇ g/ml, respectively.
  • the prodrug activating reaction of the immunocomplex comprising UK and the bispecific antibody, bound to cell surface was then determined by the method described in Example 5. The results are shown in Table 4. All prodrugs were activated by the immunocomplex comprising UK and the bispecific antibody and showed strong cytotoxicity against the target cell line A431. On the other hand, they showed no cytotoxicity against the non-target cell line P388.

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Abstract

La présente invention décrit un anticorps monoclonal hybride possédant des spécificités contre une cellule cancéreuse humaine et une enzyme d'activation promédicamenteuse, un polydome qui produit ledit anticorps et un complexe protéique anticancéreux renfermant ledit anticorps, et une enzyme d'activation promédicamenteuse qui y est immunologiquement couplée, ainsi que des procédés pour utiliser ledit anticorps en combinaison avec un promédicament anticancéreux destiné à la thérapie du cancer.
EP91900329A 1989-12-15 1990-12-14 Anticorps biospecifique des cellules cancereuses et enzyme presentant des caracteristiques d'activation promedicamenteuse Ceased EP0505566A1 (fr)

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WO1992019973A1 (fr) * 1991-04-26 1992-11-12 Surface Active Limited Nouveaux anticorps et procedes d'utilisation de ces anticorps
US6787153B1 (en) 1991-06-28 2004-09-07 Mitsubishi Chemical Corporation Human monoclonal antibody specifically binding to surface antigen of cancer cell membrane
CA2072249C (fr) * 1991-06-28 2003-06-17 Saiko Hosokawa Anticorps monoclonal humain liant specifiquement a la surface d'un antigene des membranes des cellules cancereuses
US5679777A (en) * 1991-11-08 1997-10-21 Somatogen, Inc. Hemoglobins as drug delivery agents
GB9200417D0 (en) * 1992-01-09 1992-02-26 Bagshawe Kenneth D Cytotoxic drug therapy
GB9200415D0 (en) * 1992-01-09 1992-02-26 Bagshawe Kenneth D Inactivation of cytotoxic drugs
GB9819411D0 (en) * 1998-09-04 1998-10-28 Ks Biomedix Ltd Antibodies
US6361774B1 (en) 1999-09-17 2002-03-26 Immunomedics, Inc. Methods and compositions for increasing the target-specific toxicity of a chemotherapy drug
ATE499115T1 (de) * 1998-09-18 2011-03-15 Immunomedics Inc Antikörper verwiesene enzymprodrugtherapie mit glukuronidase
US6573074B2 (en) 2000-04-12 2003-06-03 Smithkline Beecham Plc Methods for ansamitocin production
US7445802B2 (en) 2000-12-26 2008-11-04 Yeda Research And Development Co. Ltd Site-specific in situ generation of allicin using a targeted alliinase delivery system for the treatment of cancers, tumors, infectious diseases and other allicin-sensitive diseases
WO2010019257A2 (fr) 2008-08-13 2010-02-18 The Johns Hopkins University Activation de promédicament dans des cellules cancéreuses à l'aide de commutateurs moléculaires
CN104717980A (zh) 2012-08-14 2015-06-17 米纳瓦生物技术公司 干细胞增强疗法
JP6895890B2 (ja) 2015-02-10 2021-06-30 ミネルバ バイオテクノロジーズ コーポレーション ヒト化抗ｍｕｃ１＊ 抗体
CN106554375B (zh) * 2016-06-08 2019-10-18 浙江海正药业股份有限公司 一种蒽环类化合物、其制备方法及其用途

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US4671958A (en) * 1982-03-09 1987-06-09 Cytogen Corporation Antibody conjugates for the delivery of compounds to target sites
EP0142905A2 (fr) * 1983-03-30 1985-05-29 Bio-Response Inc. Substance thérapeutique pour le traitement de maladies telles que le cancer
WO1986001720A1 (fr) * 1984-09-13 1986-03-27 Cytogen Corporation Conjugues d'agents therapeutiques-anticorps
GB8528761D0 (en) * 1985-11-22 1985-12-24 Axon Healthcare Ltd Enzyme-coupled antibodies
GB8705477D0 (en) * 1987-03-09 1987-04-15 Carlton Med Prod Drug delivery systems
NZ225599A (en) * 1987-08-04 1991-09-25 Bristol Myers Co Antibody-enzyme conjugates and combinations with prodrugs for the treatment of tumour cells
GB8809616D0 (en) * 1988-04-22 1988-05-25 Cancer Res Campaign Tech Further improvements relating to drug delivery systems

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