EP0449998A1 - Conjugues d'anticorps-oxydase a substrats non systemiques - Google Patents

Conjugues d'anticorps-oxydase a substrats non systemiques

Info

Publication number
EP0449998A1
EP0449998A1 EP90908348A EP90908348A EP0449998A1 EP 0449998 A1 EP0449998 A1 EP 0449998A1 EP 90908348 A EP90908348 A EP 90908348A EP 90908348 A EP90908348 A EP 90908348A EP 0449998 A1 EP0449998 A1 EP 0449998A1
Authority
EP
European Patent Office
Prior art keywords
oxidase
cells
target cell
binding protein
agent
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.)
Withdrawn
Application number
EP90908348A
Other languages
German (de)
English (en)
Other versions
EP0449998A4 (en
Inventor
John D. Duncan
Frieder K. Hofmann
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.)
Brunswick Corp
Original Assignee
Brunswick Corp
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Brunswick Corp filed Critical Brunswick Corp
Publication of EP0449998A1 publication Critical patent/EP0449998A1/fr
Publication of EP0449998A4 publication Critical patent/EP0449998A4/en
Withdrawn legal-status Critical Current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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/66—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 a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A61K47/67—Enzyme prodrug therapy, e.g. gene directed enzyme drug therapy [GDEPT] or VDEPT
    • 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
    • A61K47/6893—Pre-targeting systems involving an antibody for targeting specific cells clearing therapy or enhanced clearance, i.e. using an antibody clearing agents in addition to T-A and D-M
    • 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]

Definitions

  • the technical field of the present application relates to conjugates of antibodies and enzymes, linked together by linker molecules, which are designed for therapeutic purposes.
  • enzyme-immunoconjugates composed of any antibody or fragment thereof linked to an oxidase for which the natural substrate is not normally present in the body fluids or otherwise available in the extracellular spaces of the body.
  • Monoclonal antibodies specific for epitopes unique to certain types of cancer cells, T cell, B cells, and the like have been identified and are proposed for delivery of drugs directly and specifically to the target cells. Specificity, and thus amelioration of side effects, depends upon two factors: the specificity and tenacity of attachment to the target; and the effects of freely circulating (not attached) agent on non-target tissues in the body. These two factors are interrelated due to the fact that any binding agent, regardless of how strong the bond is, is in some state of equilibrium with the lymph and blood. Also, during administration, either intraperitoneally or intravenously, there will be a high concentration of freely circulating toxic agent until it is bound to the target cells or cleared by the liver and/or kidneys or the lymphocytes. This clearing process relieves the burden of freely circulating toxic agent, but also concentrates the toxic agent in the organs involved.
  • the present invention relates to a novel approach to the problem of targeting toxic agents to cancer cells or other pathogenic cells without a significant degree of side effect on healthy tissue.
  • the present invention involves a class of conjugates for therapeutic use which consist of an enzyme attached to a target cell binding protein.
  • the enzyme can be any which produces a freely diffusible cytotoxic agent when positioned on the exterior of a target cell or organism.
  • the target cell binding protein can be an antibody, either mono- or polyclonal, a fragment of an antibody, or any other molecule with specificity for a specific type of cell, e.g. a tumor cell, or organism.
  • the enzyme can be attached either directly to the target cell binding protein or via a linker molecule designed to provide adequate spacing to prevent steric hindrance.
  • the attachment of the enzyme to the target cell binding protein is preferably stable to all conditions of administration to a patient and to conditions present in the microenvironment at the site of action.
  • the novelty of the present invention lies in the fact that each of the enzymes chosen acts on substrates which are not normal constituents of the environment of the site in the body in which it is expected to function nor through which it passes on its way to that site. Thus the enzyme will be exposed to no substrate and produce no cytotoxic products while circulating in the body enroute to its target.
  • the conjugates of the present invention utilize an oxidase enzyme, attached to tumor-specific monoclonal antibodies or other target binding proteins, to produce hydrogen peroxide as the toxic agent.
  • Hydrogen peroxide is a highly toxic compound capable of causing rapid cell death through membrane disruption. Hydrogen peroxide is freely diffusible in aqueous solution and will rapidly diffuse to the surface of cells adjacent to the enzyme. In addition to being highly water-soluble, hydrogen peroxide also has lipophilic characteristics which give it an affinity for cell membranes which is probably a significant factor in its high toxicity to cells.
  • Hydrogen peroxide has an advantage in therapy over other cytotoxic agents as it is fairly unstable and will be decomposed to water and oxygen by erythrocytes or catalase released from lysed target cells. Therefore, no side reactions from activated agents diffusing away from the generation site are expected. This is in sharp contrast to the pro-drug approach where a modified drug (pro-drug) is rendered active again by action of an enzyme bound to an antibody. Even after cell death, the cell membrane bound enzyme will continue to convert pro-drugs to drugs.
  • One such conjugate is an ethanol oxidase conjugate.
  • Ethanol oxidase is not found in mammals and its substrate, ethanol, is not normally present anywhere in the mammalian body. However, even 0.1% ethanol concentration is sufficient to give good enzymatic turnover.
  • the toxic product of the enzymatic reaction is hydrogen peroxide.
  • Another conjugate is a galactose oxidase conjugate.
  • Galactose oxidase is not found in mammals and its substrate, galactose, can easily be eliminated from normal circulation by normal metabolic pathways. Again, the toxic agent produced is hydrogen peroxide.
  • Another conjugate is a D-amino acid oxidase conjugate.
  • This enzyme and its substrates, the D-amino acids are not found in mammals.
  • the toxic agent produced is hydrogen peroxide.
  • Another conjugate is an ⁇ -glycerol phosphate oxidase conjugate. This enzyme is not found in mammals and its substrate, L- ⁇ -glycerol phosphate, is found only inside of cells, not outside them. This particular conjugate most likely functions by attachment to the exterior of a cell acting upon exogenously added substrate, which does not cross cell membranes.
  • Attachment of the various enzymes to the target cell binding protein may be achieved by a variety of means.
  • PCT Application No. PCT/GB86/00711, filed November 21, 1986, published June 4, 1987, Publication No. WO 87/03205, Starkie - inventor and European Patent No. 0 088 695, to Cytogen both disclose such means.
  • the preferred aspect of such attachment is that it is preferably stable to the physiological conditions present during administration to a patient and transport to the site of action.
  • the attachment is preferably stable to the conditions present at the site of action, particularly to the concentration of hydrogen peroxide present in the immediate vicinity when the enzyme is fully functional. Covalent attachment of the enzyme to a linker molecule is preferable for such stability.
  • linking reactions are preferably achieved at a position on the enzyme and under conditions which do not affect the function of the catalytic site of the enzyme and on the antibody at a site which maintains the specificity and affinity of the antigen binding site.
  • the linker molecule may be of a variety of types depending upon the specific sites of attachment to the enzyme and the antibody. The preferred features are that the bond formed is preferably stable to all conditions which the therapeutic agent encounters and that the linker molecule itself preferably does not have reactive groups which would result in its degradation in vivo . The order of reaction would be determined by the specific chemistries of bonding used and could either follow the sequence of attaching the linker to the enzyme and finally to the antibody, or attaching the linker to the antibody followed by attachment to the enzyme.
  • the enzyme-antibody ratio is preferably about 1:1. Excess enzyme is generally to be avoided.
  • An enzyme-linker-antibody conjugate meeting the requirements described above would produce hydrogen peroxide in close proximity to the targeted cell.
  • the hydrogen peroxide produced would be free to diffuse to all adjacent cells. Given the known heterogeneity of tumor cells, this would enhance the effectiveness of such a conjugate as a therapeutic agent.
  • Even highly efficacious, specifically targeted cytotoxic agents which act directly on cells have the shortcoming that they will kill only those cells to which they attach.
  • Surrounding cells which may be neoplastic, but lack the specific epitope targeted will not be killed.
  • the only solution to this problem using this specific targeting technique is to create a bank of antibodies which will locate all tumor cells. This is a more complex solution and may not be possible when small tumor cell populations are involved.
  • the present invention would solve the problem of tumor cell heterogeneity by killing all cell adjacent to targeted cells. Some normal cells may also be killed, but this would be a very minor side effect compared to thorough destruction of all tumor cells.
  • the conjugates are preferably targeted to sites which are not rapidly internalized, since this would not allow for killing of adjacent heterogenous tumor cells and would very likely result in inactivation of the oxidase.
  • These conjugates once attached on the exterior of the cells, preferably utilize substrates which are not normally found in the body fluids filling the extracellular spaces but can diffuse to the site of attachment of the conjugate.
  • substrates can include molecules normally found within cells, but not external to them, molecules not normally found in the body, or molecules found in the body in vivo but normally in low enough concentrations that no toxicity is produced by reaction with the oxidases.
  • a corollary to the requirement that the substrate be not normally found external to cells in the body is that the specific enzyme itself preferably does not occur naturally in humans.
  • the enzyme portion of the conjugate would not produce toxic agent (hydrogen peroxide) until desired.
  • the procedure would involve administering the conjugate into the blood, allowing ample time for the conjugate to seek and attach to its specific target and for excess or unattached conjugate to be cleared from the general circulation by action primarily of the liver and kidneys. After such time, a dose of the enzyme's substrate would be injected into the blood at a concentration adequate for enzyme activity without side effects (if any) in the body from the presence of the substrate.
  • Glycerol phosphate and galactose are normal metabolites and can be administered at a fairly high dose without showing side effects. Although ethanol is not a normal metabolite, it will be metabolized to fat and its side reactions are generally regarded as pleasant. D- amino acids do not normally occur in humans. If at all, only a slight inhibitory reaction of the normal human enzymes which act on the L-amino acid form of the D-amino acid might be expected. This inhibitory reaction could possibly be overcome by simultaneous administration of a higher dose of the equivalent L-amino acid. Treatment regimes can be determined based on the life of the enzyme conjugate, the concentration of the substrate, the reaction rate of the enzyme, and the like.
  • an enzyme-linker conjugate could be prepared as a general agent for attachment to any antibody or antibody fragment of the desired specificity.
  • Polyclonal antibodies to viruses or bacteria, for example, could also be attached in like manner.
  • Specific enzyme-linker conjugates could also be prepared for attachment to other types of target cell binding proteins, such as hormones, growth factors, binding proteins of various types, and the like It would therefore be possible to use the present invention for destroying a certain population of T cells or B cells in the body, for example, or to "purify" a culture of cells by killing a contaminating population having a specific affinity which can be exploited.
  • Such conjugates, using a variety of linkers for various purposes is also an aspect of the present invention.
  • SMCC Derivatization of Antibody To 75 nmol (11.3 mg) of antibody 9.2.27 in 1 ml phosphate buffered saline (pH 7.0) is added 15 ⁇ l of a 25 mM solution of SMCC (succinimidyl-4- (N-maleimidomethyl)- cyclohexane-1-carboxylate) . The reaction is allowed to proceed for one hour at room temperature. The mixture is centrifuged and then gel filtered on a PD-10 column equilibrated in 0.1 M potassium phosphate (pH 6.0). Preparation of conjugate
  • the conjugate mixture is gel filtered on a PD-10 column equilibrated in 10 mM MES (pH 6.0) .
  • the void volume eluate is centrifuged and applied to a column (4 x 1 cm) of S-Sepharose equilibrated in 10 mM MES (pH 6.0).
  • the column is washed with equilibration buffer and the conjugate is eluted with an appropriate eluent depending on the oxidase, e.g. 10 mM MES, 0.2 M NaCl (pH 6.0).
  • an appropriate eluent depending on the oxidase, e.g. 10 mM MES, 0.2 M NaCl (pH 6.0).
  • to the conjugate eluate is added 200 ⁇ l of a 1 mM solution of cofactor for the particular oxidase in the conjugate.
  • the conjugate is further purified by chromatography on a column (60 x 2.6 cm) of Sephacryl S-300 HR equilibrated in 0.1 M potassium phosphate, 0.05 M NaCl
  • each conjugate is assayed and found to be a 1:1 adduct of the enzyme and antibody.
  • Example 1 Each of the conjugates prepared in Example 1 is evaluated for in vitro cytotoxic activity using the following procedure.
  • the immunoconjugate and catalase containing nutrient is removed 30 minutes later and the wells are rinsed three times with the original medium and the cells are allowed to continue to grow in the original medium, to which 2mM of substrate for the conjugate oxidase is added. After 24 hours, 10 ⁇ l of 1 ⁇ Ci - -thymidine containing medium is added in order to measure thymidine uptake. Thymidine is incorporated into DNA and thymidine uptake is used to measure DNA synthesis which relates to cell viability. After another day of growth the plates are shock frozen, then thawed and the individual well contents passed through glass fiber filters. The radioactivity is determined and taken as a measure of cell viability.
  • Each of the conjugates prepared in Example 1 is evaluated for in vivo binding specificity and affinity by the following procedure.
  • Thymus deficient BALBc (nude/nude) mice are subcutaneously injected with 2x10 6 M21-UCLA melanoma cells. After two weeks, 3 ⁇ Ci of I 125 iodinated immunoconjugate is injected into the tail vein. After 48 hours the animals are sacrificed and the radioactivity in individual organs is determined. The immunoconjugates are formed using enzyme and 9.2.27 monoclonal antibody following the procedures discussed in Example 1.
  • the in vivo biodistribution data obtained with tumor bearing nude mice also shows that each conjugate has a high degree of binding specificity and affinity. Further, the data indicates that for each conjugate, any unbound conjugate is cleared from the body as indicated by the low levels of conjugate found in the blood, liver, kidney, spleen and intestine.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Public Health (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Nanotechnology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medical Informatics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Immunoconjugués d'enzymes pouvant être utilisés à des fins thérapeutiques sans effets secondaires significatifs chez les patients, dus à leur administration. Le type général d'immunoconjugué d'enzymes se compose de n'importe quel anticorps ou fragment de celui-ci lié à une oxydase dont le substrat naturel n'est pas normalement présent dans les fluides biologiques ou autrement disponibles dans des espaces extracellulaires du corps. L'action thérapeutique de telles oxydases repose dans leur capacité à produire l'agent de peroxyde d'hydrogène cytotoxique mais à dégradation biologique rapide. Lesdits immunoconjugués d'oxydases auraient spécifiquement pour cibles les tissus désirés, à l'aide du composant d'anticorps, mais ne seraient actifs qu'au moment ou un substrat de source exogène est administré au patient, après quoi le peroxyde d'hydrogène serait produit par le processus enzymatique partout ou l'immunoconjugué d'oxydase est présent.
EP19900908348 1989-06-30 1990-03-21 Antibody-oxidase conjugates with non-systemic substrates Withdrawn EP0449998A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US37404889A 1989-06-30 1989-06-30
US374048 1989-06-30

Publications (2)

Publication Number Publication Date
EP0449998A1 true EP0449998A1 (fr) 1991-10-09
EP0449998A4 EP0449998A4 (en) 1992-01-15

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ID=23475040

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19900908348 Withdrawn EP0449998A4 (en) 1989-06-30 1990-03-21 Antibody-oxidase conjugates with non-systemic substrates

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EP (1) EP0449998A4 (fr)
AU (1) AU5732290A (fr)
WO (1) WO1991000108A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9021671D0 (en) * 1990-10-05 1990-11-21 Unilever Plc Delivery of agents
DE4233152A1 (de) 1992-10-02 1994-04-07 Behringwerke Ag Antikörper-Enzym-Konjugate zur Prodrug-Aktivierung
EP1137760A1 (fr) 1998-12-11 2001-10-04 Unilever N.V. Enzymes de blanchiment et compositions detergentes les renfermant

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986001720A1 (fr) * 1984-09-13 1986-03-27 Cytogen Corporation Conjugues d'agents therapeutiques-anticorps
EP0361908A2 (fr) * 1988-09-28 1990-04-04 Ideon Corporation Immunothérapie à base d'une combinaison d'enzymes

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4496539A (en) * 1982-02-01 1985-01-29 Massachusetts Institute Of Technology Method for treating cancer using galactose-specific lectins
FR2523445A1 (fr) * 1982-03-17 1983-09-23 Sanofi Sa Nouveaux conjugues associant, par liaison covalente, une enzyme et un anticorps, et associations medicamenteuses utilisant lesdits conjugues
GB8528761D0 (en) * 1985-11-22 1985-12-24 Axon Healthcare Ltd Enzyme-coupled antibodies

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986001720A1 (fr) * 1984-09-13 1986-03-27 Cytogen Corporation Conjugues d'agents therapeutiques-anticorps
EP0361908A2 (fr) * 1988-09-28 1990-04-04 Ideon Corporation Immunothérapie à base d'une combinaison d'enzymes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO9100108A1 *

Also Published As

Publication number Publication date
AU5732290A (en) 1991-01-17
WO1991000108A1 (fr) 1991-01-10
EP0449998A4 (en) 1992-01-15

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