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WO1990012109A1 - Anticorps monoclonaux s'associant a des complexes de platine - Google Patents

Anticorps monoclonaux s'associant a des complexes de platine Download PDF

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Publication number
WO1990012109A1
WO1990012109A1 PCT/US1989/001327 US8901327W WO9012109A1 WO 1990012109 A1 WO1990012109 A1 WO 1990012109A1 US 8901327 W US8901327 W US 8901327W WO 9012109 A1 WO9012109 A1 WO 9012109A1
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WIPO (PCT)
Prior art keywords
platinum
complex
strain
monoclonal antibody
antibody
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Ceased
Application number
PCT/US1989/001327
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English (en)
Inventor
Michael G. Rosenblum
James L. Murray
Peter J. Kelleher
Robert A. Newman
Abdul R. Khokhar
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University of Texas System
University of Texas at Austin
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University of Texas System
University of Texas at Austin
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Priority to PCT/US1989/001327 priority Critical patent/WO1990012109A1/fr
Publication of WO1990012109A1 publication Critical patent/WO1990012109A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Definitions

  • the present invention relates to monoclonal antibodies directed toward platinum (II) complexes, their preparation and uses such as carrying platinum (II) complexes to tumor.
  • antigenic determinants expressed on the surface of human tumor cells have the potential to selectively localize in tumors after systemic administration (Goldenberg et al., Science 208:1284-1286, (1980); Goldenberg et al., Cancer 45:2500-2505, (1980); Larson et al., J. Nucl. Med.,
  • MIDP appears to have an advantage over the parent platinum (II) compound, i.e., retaining antitumor efficacy and potency with the diminution of toxic side effects.
  • the antitumor activity of platinum complexes arises as a consequence of their chemical nature, as does a high degree of non-specific reactivity. More than 90% of cisplatin in plasma is tightly bound to plasma protein, leading to a prolonged plasma half-life of bound, inactive drug (Hill et al., Cancer Chemother. Rep., 59:647-659 (1975). The remaining unbound, highly reactive drug can participate in a number of hydrolytic and degradative reactions in plasma; the metabolites formed have greater nephrotoxic potential and decreased antitumor activity (Daley-Yates et al., Biochem. Pharmacol., 33:3063-3070 (1984)).
  • the next logical goal in the development of effective platinum analogues and object of the present invention is to solve the problem of non-tumor associated reactivity and to increase deposition within the tumor.
  • an antitumor agent bound or coupled to a monoclonal antibody may not only exhibit increased tumor distribution but also altered clearance and systemic effects.
  • a purpose of the delivery system of the present invention is to utilize murine monoclonal antibodies for favorably modifying the pharmacology and, tissue
  • a tumor targeting antibody may be directly coupled to a drug, forming an irreversible covalent complex.
  • a monoclonal antibody capable of reversibly binding a drug may be covalently coupled to an antibody recognizing a tumor-associated antigen.
  • the direct coupling approach has been utilized for a number of therapeutic agents such as adriamycin, vindesine and methotrexate as well as plant and bacterial toxins, including ricin A chain, pseudomonas exotoxin, pokeweed antiviral protein and gelonin
  • antiproliferative activity of such covalently altered complexes may depend on: 1) extracellular hydrolytic or enzymatic release of the active agent from the conjugate at the tumor site (Ghose et al., J. Natl. Cane. Inst., 61:657-676 (1978); or 2) internalization and intracellular cleavage of the stable bond between the active agent and the antibody (Bjorn et al., Cancer Res., 45:1-8 (1985). Hydrolytic or enzymatic release of cytotoxic agents from covalent complexes at the tumor site may not be uniformly dependable. Degradation of the complex by enzymes present in plasma may provide premature release of the active agent and thereby compromise further the therapeutic efficacy of these covalent complexes.
  • An object of this invention is to describe a novel; tumor-targeted antibody drug delivery system consisting of a monoclonal antibody capable of reversibly binding a therapeutic agent in combination with an
  • the monoclonal antibody delivery system contains a reversible, drug- binding site, as schematically shown in Fig. 1. This may be accomplished by producing a carrier monoclonal antibody to the active agent and coupling this carrier to a tumor- targeting antibody. Such coupling may be accomplished, for example, by using a heterobifunctional cross-linking reagent to generate an antibody-antibody linkage (Bjorn et al., Cancer Res., 45:1-8 (1985); Carlsson et al., Biochem. J., 173:723-727 (1978).
  • the affinity of the complex for the active agent can be varied by selecting anti-drug antibodies with desired affinity constants (Ka), thereby subtly altering the exchange properties of the active agent with the targeting site. This should change the efficiency with which a complex delivers cytotoxic agents to target sites. Rapidly-reversible (lower affinity) complexes may be important, for quick release of low molecular weight cytotoxic agents at the tumor surface to allow more rapid transport of the agent across the cell membrane and interaction at its site of action without the hindrance of an attached, high molecular weight carrier protein. Higher affinity complexes which result in slow or minimal release of active agents may be important for the delivery of toxins such as gelonin.
  • This particular agent should remain coupled since the antibody complex acts as both a cell-targeting carrier and an internalization mechanism to allow the toxin to localize on tumor cells and to cross mammalian cell membranes for interaction with ribosomal structure, respectively (Stirpe et al., J. Biol. Chem., 255:6947-6953, (1980). 2)
  • the active agent itself is not subjected to chemical modification. This is of exceptional importance in attempting to target cytotoxic agents such as platinum complexes, with a definitive structure activity relationship (SAR) or chemical lability.
  • antibody:antibody tumor-specific cross-linking conditions have been well-defined, a large library of reversibly agent-binding tumor targeting antibody complexes may be produced.
  • New complexes may be generated, for example, by using tumor targeting antibodies directed to different epitopes of the same cell surface antigen, antibodies to different surface antigens present on the same cell type or using antibodies to target different tumor types of interest.
  • the reversible drug-binding delivery system approach may allow the specific delivery of reversibly-coupled, unmodified agents such as MIDP to tumors.
  • This type of delivery system is schematically shown in Figure 1.
  • Raso et al. Previous studies by Raso et al. have demonstrated that this non-covalent targeting approach should be effective (Raso et al., ed. Proc, 37:1350 (1978); Raso et al., Cancer Res., 41:2073-2078 (1981); Raso, Immunological Rev., 62:93-117 (1982); Raso et al., In Receptor-mediated Targeting of Drugs: NATO Advanced Studies Institute,
  • Neocarzinostatin, an antitumor antibiotic, and the A-chain of ricin were delivered to tumor cells in vitro by means of reversibly binding antibody:antibody complexes.
  • the ricin A chain-binding antibody:antibody complex was found to specifically deliver ricin to antigen-bearing target cells. This ricin-bearing complex was also shown to substantially inhibit cell growth and lead to cell death.
  • the ricin:antibody complex was found to be 5000 times more potent compared to ricin A chain alone. The in vivo
  • conjugates with various targeting antibodies may be made. This flexibility will allow a panel of antibody complexes to be prepared, all directed towards a specific tumor type or a number of tumor types.
  • the reversible drug-binding antibody complexes of the present invention may represent a first step in the
  • a broad object of the present invention is to utilize a novel, specific monoclonal antibody to reversibly bind therapeutic platinum (II) complex.
  • Other potential uses of this antibody also include but are not limited to:
  • composition in which an antibody specific for a Pt (II) complex itself or a specific Pt (II) complex is utilized in an enzyme-linked immune- absorbent (ELISA) assay for quantitation of platinum or platinum complex in urine, serum, other biological fluids or tissues.
  • ELISA enzyme-linked immune- absorbent
  • platinum (II) complex antibodies as specific probes to detect platinum complexes at the cellular or sub-cellular level.
  • The. present invention relates to monoclonal antibodies specifically binding platinum (II) complex, most preferably a DACH platinum II complex.
  • the monoclonal antibodies of the present invention are characteristically produced by antibody-producing cell lines such as
  • hybrid ⁇ mas These cell lines may result, for example, from fusion of a neoplastic cell with an antibody- producing animal cell which may be obtained from an animal immunized against a platinum (II) complex.
  • the cellular fusion products include cell lines forming monoclonal antibody specifically binding DACH platinum (II) complexes in competition with an antibody produced by: hybridoma strain 1C 1 H 2 A 5 , deposited with the American Type Culture Collection, Rockville, MD on May 1, 1987 (tested as viable May 7, 1987) and having ATCC accession number HB 9411;
  • the cell lines of the present invention are preferably continuous murine hybridoma cell lines which secrete recoverable quantities of monoclonal antibody, particularly a monoclonal antibody which is of an IgG or IgM isotype.
  • the antibodies may be those produced by the above-referenced hybridoma strains.
  • the cell line which produces monoclonal antibody specifically binding platinum (II) complex is produced by fusion of a neoplastic cell such as a myeloma cell with an antibody- producing animal cell such as a splenic cell.
  • an antibody-producing animal cell is obtained from an animal immunized against platinum (II) complex.
  • An animal such as a mouse, for example, may be immunized against a platinum (II) complex by a process involving immunization with a platinum (II) complex coupled to a carrier.
  • An immunized animal may be produced by a process involving immunization with a conjugate of a platinum (II) complex and a macromolecular species, for example, a protein or a polynucleotide such as DNA.
  • a preferred platinum (II) complex of the present invention is a 1,2- diaminocyclohexane (DACH)-platinum complex.
  • DACH 1,2- diaminocyclohexane
  • the platinum (II) complexes of the present invention are most commonly usable as antitumor agents but may have other biological or therapeutic activity.
  • platinum (II) complexes of the present invention are most commonly usable as antitumor
  • the cell lines of the present invention include hybridoma strain 1C 1 H 2 A 5 having ATCC accession number HB 9411, strain 3A 2 A 1 , strain 1A 6 A 2 , strain 3A 6 B 1 and strain 1B 1 .
  • a murine hybridoma cell line for example, one of the above strains, secreting recoverable quantities of a monoclonal antibody which specifically binds a platinum (II) complex exemplifies a central aspect of the present invention.
  • a composition of matter comprising a monoclonal antibody such as one described above which specifically binds a platinum (II) complex is within the scope of the present invention.
  • This composition of matter may be defined further as comprising a monoclonal antibody which binds a platinum (II) complex in competition with an antibody produced by one of the cell lines of the present invention.
  • the monoclonal antibody of this composition of matter may be, for example, of an IgG or IgM isotype and be one actually produced by a cell line of the present invention
  • An antibody which is competitive for binding to a platinum (II) complex with monoclonal antibody from a cell line of the present invention may readily be identified by methods well-known to those skilled in the relevant arts upon examination of the present application. For example, a platinum complex could first be immobilized upon a solid matrix as described elsewhere herein.
  • a monoclonal antibody from a cell line of the present invention may be labeled with a detectable label such as an enzyme,
  • the immobilized platinum complex would then be treated with an aqueous solution of the potentially competitive antibody or with a control aqueous solution devoid of potentially competitive antibody. After washing the treated immobilized platinum complex, the immobilized platinum complex would be exposed to a solution of the labeled monoclonal antibody of the present invention. A decrease in detectable label bound to the immobilized platinum complex which is attributable to treatment with the potentially competitive antibody would indicate that the potentially competitive antibody was indeed competitive for binding to the platinum
  • the present invention may involve a method for increasing the circulating half-life and therapeutic effectiveness of a platinum (II) complex parenterally administered to an animal. This method comprises
  • platinum (II) complex and the monoclonal antibody would combine in an antibody-antigen combinant. Formation of an antibody-antigen combinant of monoclonal antibody and platinum (II) complex should prevent the platinum (II) complex from being rapidly removed from circulating blood by ordinary modes of drug clearance.
  • the platinum (II) complex and the monoclonal antibody may be parenterally administered in a preformed combination or separately administered to form a combinant in vivo.
  • the present invention may involve a composition of matter which is specifically directed toward a tumor target site.
  • a composition of matter would comprise, for example, in addition to a first monoclonal antibody directed toward a platinum (II) complex, a second monoclonal antibody which specifically binds to a tissue target site.
  • the second monoclonal antibody is preferably covalently bound to the first monoclonal antibody.
  • the first monoclonal antibody may be a monoclonal antibody produced by a cell line of the present invention may be competitive with this monoclonal antibody for binding to a platinum (II) complex such as a DACH-platinum (II) complex.
  • the amount of a platinum (II) complex present in a biological sample may be determined by processes of the present invention.
  • One such process could comprise a series of steps which may be described as follows.
  • a first monoclonal antibody which has a first isotype and specifically binds platinum (II) complex would be
  • the immobilized antibody would then be contacted with the biological sample, for example in aqueous solution, whereupon any platinum (II) complex in the biological sample would bind to the immobilized antibody.
  • the contacted immobilized antibody would then be washed essentially free of unbound biological sample.
  • the immobilized first monoclonal antibody would then be treated with a second monoclonal antibody, the second monoclonal antibody having a second isotype and specifically binding the platinum (II) complex. After washing the immobilized first monoclonal antibody essentially free of unbound second monoclonal antibody, it would be treated with a labeled antibody having an affinity for antibody of the second isotype. Finally, the amount of labeled antibody bound to the immobilized antibody would be measured by observation of the label as an index of the amount of platinum (II) complex present in the biological sample.
  • Additional possible methods for determination of platinum (II) complex amounts in biological samples are provided within the scope of the present invention.
  • One such additional method would involve immobilizing a platinum (II) complex upon a solid matrix such as that of a bead or wall, for example.
  • the immobilized platinum (II) complex would then be contacted with fresh first monoclonal antibody or with the incubated first monoclonal antibody so that monoclonal antibody with an unoccupied binding site for platinum (II) complex binds to the immobilized platinum (II) complex. Unbound biological sample would then be removed from the immobilized platinum (II) complex, for example by washing. The washed
  • immobilized platinum (II) complex would then be contacted with a labeled antibody having an affinity for antibodies of the first animal species.
  • the amount of labeled antibody bound to the immobilized platinum (II) complex would finally be measured through its label. Any decrease in the amount of labeled antibody binding to the
  • platinum (II) complex b) incubating the immobilized first antibody with the biological sample; c) washing the immobilized first monoclonal antibody essentially free of unbound biological sample; d) contacting the washed immobilized first monoclonal antibody with a second monoclonal antibody specifically binding said platinum (II) complex and bearing a detectable label; e) washing the immobilized antibody essentially free of unbound second monoclonal antibody; and f) measuring the amount of detectable label bound to the immobilized antibody as an index of the amount of platinum (II) complex present in the biological sample.
  • the present invention further comprises methods for detecting the presence of platinum (II) complex in a biological sample at the cellular or subcellular level. These methods may frequently involve microscopic
  • One such method would involve: a) treating a biological sample with a first monoclonal antibody specifically binding platinum (II) complex and bearing a detectable label and b) determining the amount and location of detectable label bound to cellular and subcellular components of the biological sample.
  • Another method for detecting the presence of platinum (II) complex in a biological sample at the cellular or subcellular level would comprise: a) treating said
  • a platinum (II) complex in a biological sample at the cellular or sub-cellular level would be readily apparent to those skilled in the art upon examination of the present application.
  • One other variant would comprise: a) treating a biological sample with a first monoclonal antibody specifically binding platinum (II) complex and having a specific isotype; b) washing the biological sample essentially free of unbound antibody; c) treating the biological sample with an antibody bearing a detectable label and having a binding affinity for the specific isotype of the first monoclonal antibody; and d) determining the amount and location of detectable label bound to cellular and subcellular components of the biological sample, preferably by microscopic means.
  • the first monoclonal antibody is one of the present invention or competitive for binding to platinum (II) complex with a monoclonal antibody of the present invention.
  • the second monoclonal antibody when specific for platinum (II) complexes used in practicing methods of the present invention preferably is of the same description as the first monoclonal antibody.
  • platinum (II) complexes utilized in the practice of the methods of the present invention preferably are DACH-platinum (II) complexes.
  • the present invention involves methods for the production of monoclonal antibodies specifically binding to a platinum (II) complex,
  • One such method comprises the steps of: a) parenterally administering a conjugate comprising haptenic platinum (II) complex and a carrier to an animal to induce the production of antibody; b) fusing antibody-producing cells from the animal with neoplastic cells such as a murine mydoma; c) screening cellular fusion products to identify hybrid cells
  • platinum (II) complex e.g. by noting binding to immobilized platinum II complex; and d) cultivating the identified' cells to produce monoclonal antibody specifically binding to platinum (II) complex.
  • kits useful for the detection of platinum (II) complex in a biological sample such as a patient's blood, for example.
  • the form and components of such kits would be to enable practitioners, such as those involved with cancer chemotherapy, to readily monitor the amounts of platinum (II) complex present in a patient.
  • kits One kit prototype would comprise: a) a carrier such as a cardboard or plastic box, for example, compartmentalized to receive one or more container means such as vials in close confinement therein; b) a first container means comprising a platinum (II) complex which may be immobilized; and c) a second container means comprising a first monoclonal antibody, for example a murine monoclonal antibody, with a detectable label and specifically binding platinum (II) complex.
  • a carrier such as a cardboard or plastic box, for example, compartmentalized to receive one or more container means such as vials in close confinement therein
  • a first container means comprising a platinum (II) complex which may be immobilized
  • a second container means comprising a first monoclonal antibody, for example a murine monoclonal antibody, with a detectable label and specifically binding platinum (II) complex.
  • the first monoclonal antibody of the kits described herein is the monoclonal antibody of the present
  • the platinum (II) complex in a container means is preferably a may be a DACH-platinum (II) complex.
  • a carrier compartmentalized to receive one or more container means in close confinement therein comprises: a) a carrier compartmentalized to receive one or more container means in close confinement therein; b) a first container means comprising a first monoclonal antibody, for example a murine monoclonal antibody, which may be bound to a solid matrix and specifically binds platinum (II) complex; and c) a second container means comprising a platinum (II) complex bearing a detectable label.
  • a kit useful for the detection of platinum (II) complex in a biological sample may also comprise: a) a carrier compartmentalized to receive one or more container means in close confinement therein; b) a first container means comprising a first monoclonal antibody which specifically binds
  • platinum (II) complex and c) a second container means comprising a detectably labeled second monoclonal antibody which specifically binds platinum (II) complex.
  • detection of platinum (II) complex in a biological sample may also comprise: a) a carrier compartmentalized to receive one or more container means in close confinement therein; b) a first container means comprising a first monoclonal antibody of a first isotype which specifically binds platinum (II) complex; c) a second container means comprising a second monoclonal antibody of a second isotype which specifically binds platinum (II) complex; and d) a third container means comprising an antibody specifically binding antibody of the second isotype and bearing a detectable label.
  • a carrier compartmentalized to receive one or more container means in close confinement therein comprise: a) a carrier compartmentalized to receive one or more container means in close confinement therein; b) a first container means comprising a first monoclonal antibody specifically binding a platinum (II) complex and being from a first animal; c) a second container means comprising a second monoclonal antibody which specifically binds platinum (II) complex and being from a second animal; and d) a third container means comprising an antibody specifically binding antibody from the second animal and bearing a detectable label.
  • the present invention also involves methods for preparing and using a tumor-directed platinum (II) complex. These methods comprise a series of steps
  • a first monoclonal antibody having binding affinity for the platinum (II) complex is obtained, for example, by immunizing an animal against a platinum (II) complex, preferably against a DACH platinum (II) complex, and fusing antibody-producing cells from the animal with neoplastic cells to form hybridomas.
  • a second monoclonal antibody, preferably murine and having binding specificity for a surface antigen of the tumor may be obtained by standard methods comprising, for example, immunization of an animal against purified tumor surface antigens. The first monoclonal antibody would then be conjugated to the second monoclonal antibody to produce an antibody
  • conjugation is preferably covalent and may involve a bifunctional coupling agent, more preferably a heterobifunctional agent, such as N-succimidiyl-3-(2- pyridyldithio)propionate,m-maleidimido-N-hydroxy- succinimidyl ester, bromoacetyl-p-aminobenzoyl-N-hydroxy- succimidyl ester, bromoacetyl-p-aminobenzoyl-N- hydroxysuccimidyl ester and iodoacetyl-N-hydroxysuccimidyl ester (see e.g., Ghose et al. J. Nat'l. Cancer Inst.
  • a bifunctional coupling agent more preferably a heterobifunctional agent, such as N-succimidiyl-3-(2- pyridyldithio)propionate,m-maleidimido-N-hydroxy- succinimidyl
  • a quantity of the platinum (II) complex would then be added to the antibody conjugate to produce a tumor-directed platinum (II) complex.
  • a method for treatment of animals afflicted with a tumor sensitive to a platinum (II) complex would then comprise the
  • Figure 1 is a schematic representation of a hybrid drug-binding: target antigen binding antibody complex involved in delivery of a drug molecule to an antigen- bearing cell.
  • FIG. 2 schematically illustrates the structure of platinum complexes related to the present invention.
  • FIG 3 shows that antibody from hybridoma strain 1C 1 H 2 A 5 grown both in ascites and in culture media is capable of binding to radiolabeled platinum complex in solution. Equilibrium dialysis was performed with the antibody and Pt complex. Control monoclonal antibody (ABY96.5 CTR) was unrelated to platinum complex.
  • Figure 4 shown the gel permeation HPLC profile of a [ 3 H]Pt/MOAB complex. The absence of free platinum complex after equilibrium dialysis at 4'C is evident.
  • Figure 5 shows the HPLC gel permeation profile of free [ 3 H] Pt complex.
  • Figure 6 shows an in vitro dose response assay for MIDP toxicity (0) MIDP-MOAB complex toxicity (X) toward MCF-7 cells (72 hr exposure). Eight thousand MCF-7 cells were seeded per well and an MTT dye viability test
  • Figure 7 shows the relative levels of blood-bourne [ 3 H] DACH Pt when free [ 3 H] DACH Pt (X) was administered intravenously to a rat and blood-bourne [ 3 H] DACH Pt when a MOAB/[ 3 H] DACH Pt combinant (0) was intravenously administered to a rat.
  • Figure 8 shows the tissue to plasma distribution ratio of [ 3 H] DACH Pt for rat from small intestine, large intestine, lung, kidney or liver three hours after
  • Figure 9 shows the tissue to plasma distribution ratio of [ 3 H] DACH Pt found in rat skeletal muscle, heart, spleen, t :estes and brain three hours after administration of free [ 3 H] DACH Pt complex (M) or a MOAB [ 3 H] DACH combinant (M+A).
  • Figure 10 shows the competition of MIDP (X) and Pt complex A (MIDP in retrospect) (0) for binding to MOAB from hybridoma 1C 1 H 2 A 5 .
  • a DACH Pt complex was immobilized on the surface of microtiter wells.
  • Figure 11 shows the competition of MIDP (X) and DACH Pt (II)1,1-dicarboxylatocyclobutane (0) for binding to MOAB from hybridoma 1C 1 H 2 A 5 .
  • a DACH Pt complex was immobilized on the surface of microtiter wells.
  • Figure 12 shows the competition of MIDP (X) and diamine Pt(II)1,1-dicarboxylatocyclobutane (0) for binding to MOAB from hybridoma 1C 1 H 2 A 5 .
  • a DACH Pt complex was immobilized on the surface of microtiter wells, to which MOAB binding was measured.
  • Platinum (II) complexes are extremely active therapeutic agents highly toxic to both normal and tumor cells alike. Coupled to targeting monoclonal antibodies, drugs and toxins have potential for improved selectivity while simultaneously reduced toxic side effects. However, coupling platinum complexes to monoclonal antibodies by covalent modification of the platinum complexes leads to their inactivation.
  • An alternative means of coupling to an antibody has been developed which employs a monoclonal antibody which reversibly binds to platinum complex in a non-neutralizing manner. This reversible, drug carrier- antibody may be covalently coupled, for example, to a tumor-targeting antibody.
  • a central aim of the present invention involves the creation of a specific delivery system for platinum (II) complexes particularly DACH platinum (II) complexes, in biologically active form which results in specific transport of the complex to a tumor.
  • This system may be created, for example, by covalently conjugating an anti-Pt(II) monoclonal antibody to a monoclonal antibody having specific affinity for a tumor surface antigen.
  • the platinum complex will be maintained in stable active form for specific delivery to the
  • the present invention in one embodiment, involves a combination between a therapeuti- cally active platinum complex with a monoclonal antibody selected to bind the platinum complex in a manner which does not materially impair its therapeutic activity but which forms a combinant with the complex to confer upon the complex an in vivo serum half-life longer than that of the therapeutic agent alone.
  • the invention could comprise a similar
  • the therapeutic platinum complexes most useful in the invention are those which are or can be made immunogenic, i.e., those for which an immune response can be obtained either directly or, in the case of a hapten, by binding the agent to a molecule which is immunogenic.
  • Monoclonal antibodies against the therapeutic agent can be obtained by methods which are now well known to the art and which need not be described in detail. These methods generally involve immunization of a mouse or other animal species, usually mammalian or avian, with the immunogen. After an immune response is generated, spleen cells of the
  • immunized mouse are fused with cells of an established lympoid tumor line using known techniques to form
  • hydridomas which produce monoclonal antibodies. Clones of hydridomas are screened to select those which are
  • Monoclonal antibodies having the desired specificity are further screened to select those that form an antibody Pt complex combination in which the complex retains all, or substantially all, of its therapeutic activity. These combinations may be further screened to select those which have an extended serum half-life. In certain circumstances, it may be beneficial to use a mixture of two or more monoclonal antibodies. In some circumstances it may also be desirable to use a stoichiometric excess of antibody.
  • Polyclonal antibodies conceivably useful in the invention may be obtained by well known techniques as well. These include stimulating an immune response against a platinum (II) complex, or fragment thereof, in a suitable animal host such as a rabbit or other mammal.
  • a platinum (II) complex or fragment thereof, in a suitable animal host such as a rabbit or other mammal.
  • Chickens and other avian species may also be used. Serum taken from the host is subjected to affinity purification to isolate polyclonal antibodies against the therapeutic agent. These antibodies are subsequently fractionated, if necessary, to isolate a subpopulation which complexes with the therapeutic agent without materially impairing its desirable activity.
  • human antibodies against platinum (II) complex therapeutic agent are particularly preferred for future uses in this invention.
  • These human antibodies may be produced by hybridomas which, for example, are products of fusion between a B-lymphocyte obtained, for example, from a human appropriately immunized and an established
  • antibody includes fragments thereof such as Fab, Fab' and Fab'2 or mixtures thereof and including mixtures with whole antibody.
  • fractions may be less immunogenic in some patients and may also better allow better penetration of the agent to the target site.
  • monoclonal antibody may be a hybrid or more than one antibody having a dual specificity, one specificity for finding a therapeutic platinum (II) complex and the other specificity for binding against another antigen, for example, an antigen associated with site of disease where it is desired to optimize delivery of the agent.
  • these sites may involve tumor associated antigens such as carcinoembryonic antigen (CEA), prostatic acid phosphatase (PAP, ferritin and prostate specific antigen (PSA).
  • CEA carcinoembryonic antigen
  • PAP prostatic acid phosphatase
  • ferritin ferritin
  • PSA prostate specific antigen
  • the other specificity could be selected to bind with a platinum complex which has anti-tumor
  • Deoxyribonucleic acid-cellulose (DNA/cellulose, Sigma Chemical Co.) was obtained. The DNA/cellulose was washed thrice with distilled water and centrifuged to form a pellet. A disodium 1,2-diaminocyclohexane platinum sulfate complex (DACH/Pt) was dissolved in 1 ml of DACH/Pt.
  • DACH/Pt disodium 1,2-diaminocyclohexane platinum sulfate complex
  • the DACH/Pt solution was added to the pellet of DNA/cellulose, mixed and allowed to react overnight at 4' C.
  • the DACH/Pt-DNA/cellulose product was washed thrice with distilled water and pelleted by centrifugation. To the pellet was added 0.25 ml of H 2 O and 0.75 ml of adjuvant. Freund's complete adjuvant was used for the first immunization and incomplete Freund's adjuvant for all subsequent injections.
  • mice Females, 6-8 weeks old were used for the production of antibody against platinum complex.
  • Immunization doses were prepared for three animals.
  • the immunization dose per animal was 2 mg of DACH/Pt with 10 mg of DNA/cellulose.
  • the first immunization was given intraperitoneally and all follow-up injections were intramuscular. All animals were given one shot/week for 3 weeks, rested 2 weeks and given one booster shot before checking for antibody production. A few drops of blood were obtained for testing of antibody production. An antibody titer of 1:6,400 to 1:12,000 was obtained (tail vein blood sample) before a fusion was considered. Three days prior to fusion of spleen cells from the animal, DACH/Pt
  • DNA/cellulose (200 ug/ml) in saline was administered by tail vein injection. Fusion of Myeloma Cells to Splenic Lymphocytes
  • a mouse which produced high antibody titer was selected and the spleen removed in a sterile fashion.
  • the spleen was immediately transferred to a small sterile Petri dish containing 5 ml of fusion medium.
  • the medium for the fusion was Iscove's (Hazleton Research Products, Inc.) with 10% normal horse serum and antibiotics of penicillin/streptomycin/fungizone solution (5 ml/500 ml bottle).
  • the spleen was transferred to a second petri dish containing 5 ml of the Iscove's medium.
  • the spleen was minced by teasing with the use of two 18g needles (holding the spleen with one needle and teasing with the other).
  • the cell suspension was transferred to a 15 ml conical centrifuge tube and allowed to settle for 10 minutes. The cell suspension was then transferred to another 15 ml tube with care to avoid tissue or cell clumps. The cell suspension (20 ml) and mix was removed and treated with Trypan blue for counting and viability. The cell suspension was centrifuged for 10 minutes at 1200 rpm.
  • a non-secretor myeloma cell line (P 3 -NSl-Ag4-1) was obtained from a commercial source. These P 3 cells were maintained in log phase growth at low density. On the day prior to fusion, the P 3 cells were split and suspended at a density of 5 x 10 5 cell/ml. The P3 cells should have a growth rate resulting in overnight doubling.
  • Example: M 4 gms.
  • the PEG solution should remain for 4 days at 4'C before being used in the fusion but may be stored at room temperature.
  • the P 3 cells and spleen cell (at an adjusted ratio) were combined in a 50 ml tube, topped with media, washed and centrifuged at 1200 rpm for 10 minutes to form a pellet. A desired concentration of 600 cells/well was used for plating.
  • the most sensitive step was next: adding 40% PEG.
  • PEG apparently changes the membrane density and may enduce cells to fuse because their cell membrane is not intact.
  • the pellet supernatant was removed from the centrifuged cell mixture and the pellet resuspended.
  • Two ml of warm (37'C) PEG was added over a 30 second period while gently shaking the tube to resuspend the cells. The shaking was continued for 45 seconds at 37'C.
  • 10 ml of Iscove's medium was added drip-wise to the mixture. At the same time, gentle shaking of the tube was continued. This process very carefully diluted the PEG concentration without shocking the fused membranes.
  • the volume was raised to 50 ml, the tube inverted very carefully to mix and then the cells allowed to rest for a few minutes. After a 10 minute centrifugation at 1200 rpm the supernantant was discarded. To mix, the pellet was inverted slowly, washed with Iscove's medium and again inverted slowly. After a 10 minute centrifugation at 1200 rpm the supernantant was discarded. The pellet was resuspended to 50 or 100 ml in Iscove's medium + HAT.
  • HAT human spleen yielded sufficient cells to plate 100 ul/well on five 96-well plates - approximately 1 x 10 4 cells/well.
  • HAT was added only to the completed fusion before plating on a monocyte feeder layer. HAT media was used for feeding cultures until transferral to 6-well plates. The A-(aminopterin) was removed and feeding continued to HT (hypoxanthine and thymidine) for several more days before reversion to Iscove's media. After 2 or more clonings, cells were "weaned" off fetal calf serum and serum-free media added (Nutridoma-NU) Boehringer- Mannhaeim Biochemical Co.
  • the PEG-treated cells (100 ul) were plated on top of the monocyte feeder layer and returned to an incubator undisturbed for several days. Hybrids were usually not observed for at least seven days. Plates should not be fed until some colonies of hybrids are observed.
  • Plates were coated on day of test by adding 50 ul of PBS containing 10% goat serum for 15 minutes. From 50-100 ul of hybridoma culture supernatant was added to the wells of both DNA and DNA-DACH/Pt plates and incubated for 3 hours at 4'C. A control of spent media was added to each plate. After incubation, plates were washed 4 x in PBS, pH 7.2 + 1% goat serum. B-galactosidase - conjugated goat anti-mouse antibody 1:200 dilution in PBS containing 1% goat serum (BRL - reagent) 50 ul was added and the plates were incubated 1 hour at room temperature and washed 4 times with PBS.
  • PNPG chromophoric substrate p-nitrophenyl glucose
  • the first cloning should preferably be plated at 2, 1 and 0.5 cells/well. However, recloning should be done at 0.5 to 0.3 cells/well. This will help insure that clones will be only one clone/well and hope- fully only one resultant isotype.
  • Isotyping should be performed on clones selected for production of monoclonal antibody.
  • Isotyping was done by using the Boehringer Mannheim Biochemicals - mouse immunoglobulin subtype identification kit. Two antigens were used to coat the plates. Cappel's affinity purified goat anti-mouse IgG-heavy and light chain at a 1:50 dilution in H 2 O. The second antigen was the DACH/Pt at a concentration of 100 ul/ml, prepared by the same method as for the screening test.
  • hybridoma cells Once the hybridoma cells have been successfully cloned, they may be and were grown in bulk. Cultures were expanded gradually, especially in the early phases. Some hybrids were particularly intolerant of dilutions.
  • Typical antibody concentrations that may be achieved in culture supernatants were 10-100 ul/ml. At this point, the cells were usually converted to a serum free environment. The horse serum-containing medium was replaced with Nutridoma-NU (Boehringer-Mannheim). The cultures were checked weekly by ELISA to insure that they were
  • Antibody 1C 1 H 2 A 5 was prepared either from spent hybridoma culture media or as an ascites in Balb/C mice. The antibody was incubated with 800 x 10 3 CPM of [ 3 H] labeled platinum complex for 24 hrs and then dialyzed for 4 hrs against PBS and counted to determine [ 3 H] activity. As shown in Fig. 3, the 1C 1 H 2 A 5 monoclonal antibody, from either ascites fluid or ammonium-sulfate precipitated culture medium, contained 12-14 fold greater [ 3 H] label than control antibody. Analysis of the [ 3 H] label by HPLC showed that there was no free [ 3 H] platinum complex (Fig. 4). In addition, the [ 3 H] label was found at a molecular weight corresponding to 160,000 Daltons. The HPLC elution profile of free platinum complex is shown in Figure 5.
  • the antibody (2 mg) was incubated with 200 ug of MIDP platinum complex for 24 hrs.
  • the MOAB/MIDP solution was then dialyzed for 4 hrs at 4'C against phosphate- buffered saline (PBS).
  • PBS phosphate- buffered saline
  • the dialysate was then analyzed for total platinum by atomic absorption spectrometry.
  • the monoclonal antibody/MIDP complex was then added to human breast carcinoma cells (MCF-7) in concentrations between 0.01 and 10 ug Pt/ml.
  • MIDP was also added at equal platinum concentrations.
  • the wells were assessed for viable cells by MTT assay.
  • MIDP and MIDP/MOAB preparations were capable of inhibiting the growth of MCF-7 cells.
  • concentration of MIDP/MOAB complex required for 50% inhibition of cell growth was 4 fold higher than that required for MIDP alone (0.6 ug/ml for MIDP/ABY to 0.15 ug/ml for MIDP alone) suggesting a slight diminution of antiproliferative activity of the MIDP complex.
  • Figure 6 shows the dose and response profile of the MCF-7 cells to free MIDP and MIDP/MOAB.
  • **Cell survival was determined using the tetrazolium dye-bin ing method (MTT) which dye reacts only with living cells.
  • the area under the concentration curve (AUC) for Pt/MOAB complex was 7-fold higher than that found for free drug, suggesting reduced tissue distribution of the antibody/drug complex.
  • the tissue disposition data shown in Figure 8 and 9 confirm that the antibody inhibited the platinum from distributing to kidney, small intestine and liver as well as other organs. This study showed that the pharmacology of platinum (II) complex may be substantially. modified using a
  • Monoclonal antibody from hybridoma 1C 1 H 2 A 5 was added to microtiter wells having an immobilized DACH Pt complex.
  • the MOAB was added either with no free platinum complex
  • Compound B was a DACH-platinum compound with a different leaving group on the platinum, namely B was DACH Pt(II) 1,1-dicarboxylatocyclobutane.
  • Compound C was diamine Pt(II) 1,1-dicarboxylatocyclobutane. As shown in Figures 8-10, while the antibody recognized compound C, it took 100-200 times more of compound C to compete at a level with MIDP.
  • Compound B with the same leaving group as compound C (1,1-dicarboxylatocyclobutane), but with a DACH substituent instead of two amine groups , bound to the antibody about 50 fold more effectively than compound C. This relationship of chemical structure to binding
  • complexes particularly when the complexes have an organic stable ligand and/or an alkylamine substituent such as DACH, for example.

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Abstract

L'invention se rapporte à des anticorps monoclonaux s'associant spécifiquement à un complexe de platine (II). Les anticorps monoclonaux selon l'invention sont préprés, de façon caractéristique, par des lignées cellulaires productrices d'anticorps, telles que des hybridomes. Ces lignées cellulaires peuvent être issues, par exemple, de la fusion d'une cellule néoplastique avec une cellule animale productrice d'anticorps, provenant d'un animal immunisé vis-à-vis d'un complexe de platine (II). Les produits de la fusion cellulaire comprennent des lignées cellulaires formant un anticorps monoclonal s'associant spécifiquement à un complexe de platine (II), concurremment avec un anticorps produit par une souche d'hybridome 1C1H2A5, déposée auprès de la Collection de culture-type américaine, Rockville, MD, le 1er mai 1987, sous le numéro d'accession ATCC, HB 9411; une souche 3A¿2?A1, une souche 1A6A2; une souche 3A6B1 ou une souche 1B1. Les lignées cellulaires de la présente invention sont, de préférence, des lignées cellualires de murine hybridome continues qui sécrètent des quantités récupérables d'anticorps monoclonal, en particulier un anticorps qui est un isotype IgG ou IgM.
PCT/US1989/001327 1989-03-30 1989-03-30 Anticorps monoclonaux s'associant a des complexes de platine Ceased WO1990012109A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1983003679A1 (fr) * 1982-04-12 1983-10-27 Hybritech Inc Anticorps ayant des doubles specificites, leur preparation et utilisation
WO1986001407A1 (fr) * 1984-08-31 1986-03-13 Hybritech Incorporated Anticorps monoclonaux contre les chelates metalliques
EP0235457A2 (fr) * 1985-12-13 1987-09-09 Syngene International N.V. Anticorps monoclonaux
EP0263046A1 (fr) * 1986-09-19 1988-04-06 Immunotech Partners Immunoréactifs à affinité améliorée pour la détection et la destruction de cellules cibles spécifiques

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1983003679A1 (fr) * 1982-04-12 1983-10-27 Hybritech Inc Anticorps ayant des doubles specificites, leur preparation et utilisation
WO1986001407A1 (fr) * 1984-08-31 1986-03-13 Hybritech Incorporated Anticorps monoclonaux contre les chelates metalliques
EP0235457A2 (fr) * 1985-12-13 1987-09-09 Syngene International N.V. Anticorps monoclonaux
EP0263046A1 (fr) * 1986-09-19 1988-04-06 Immunotech Partners Immunoréactifs à affinité améliorée pour la détection et la destruction de cellules cibles spécifiques

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