WO1991000112A1

WO1991000112A1 - Antibody-lactate oxidase conjugates

Info

Publication number: WO1991000112A1
Application number: PCT/US1990/001056
Authority: WO
Inventors: John D. Duncan; Wolfgang J. Wrasidlo
Original assignee: Brunswick Corporation
Priority date: 1989-06-30
Filing date: 1990-02-22
Publication date: 1991-01-10
Also published as: AU5546090A

Abstract

Conjugates of antibodies and lactate oxidase, linked together by linker molecules, which are designed for therapeutic purposes. The bacterial lactate oxidase linked to an antibody produces the cytotoxic substance hydrogen peroxide at the site of action as dictated by antibody specificity.

Description

ANTIBODY-LACTATE OXIDASE CONJUGATES TECHNICAL FIELD

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.

More specifically, it relates to therapeutic preparations of bacterial lactate oxidase linked to an antibody for production of the cytotoxic substance hydrogen peroxide at the site of action as dictated by the antibody specificity.

BACKGROUND ART It has long been recognized that therapeutic agents which specifically target invading organisms or diseased cells are highly desirable. Such specific targeting by the biologically active agent allows lower doses to be given and reduces the side effects created by non-specific action of the agent. Specific targeting has previously been restricted to drugs which act by exploitation of biochemical differences between the host and the disease organism. However, the development of techniques for the selection of specific hybrido a cell lines which produce specific monoclonal antibodies, and the subsequent technologies developed for mass production of such monoclonal antibodies, has brought the therapeutic use of targeted drugs in situations where the biochemical properties of the diseased cells are nearly identical to those of healthy cells to the threshold of reality.

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. One of the possible means of attacking such target cells using monoclonal antibodies as the targeting mechanism involves the use of an enzyme attached to the antibody. The enzyme can be attached to the antibody, resulting in attachment to the cell in such a manner that the enzyme directly attacks cell membrane components and kills the cell. Examples of this type of enzyme would be the phospholipases. Alternatively, indirect attack on a cell may be accomplished by using an enzyme which utilizes substrate(s) present in its immediate environment (internal or external to the cell) to produce cytotoxic agents. Such enzymes may be attached to an antibody and then delivered to the target cell. Examples of this type of enzyme would be the oxidases. The glucose oxidase immunoconjugate has been described in the prior art, particularly in PCT Application No. PCT/GB86/00711, November 21, 1986, published June 4, 1987, Publication No. WO 87/03205, Starkie - inventor.

Attachment of an enzyme to an antibody will direct that enzyme to specific cells. Cancers, however, are known to be antigenically heterogeneous and therefore present problems for specific targeting by antibody methods. Production of cytotoxic agents just outside the targeted cells will result in diffusion of the cytotoxic agent to all adjacent cells and, therefore, will result in the death of these cells, solving the problem of epitope heterogeneity among the tumor cells. An inherent advantage of an enzyme for production of the cytotoxic agent is that it produces many toxic molecules for each attached enzyme molecule, thus reducing the amount of antibody-enzyme conjugate necessary for treatment and keeping side effects to a minimum. Another advantage is that the antibody-enzyme conjugate does not have to be internalized by the targeted cell to be effective and preferably is not internalized. DISCLOSURE OF INVENTION

The present invention involves a conjugate for therapeutic use which consists of an enzyme attached to a target cell binding protein. The enzyme is lactate oxidase 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. A specific embodiment of this therapeutic agent is the enzyme lactate oxidase attached to a monoclonal antibody using a heterobifunctional linker molecule, or combinations of such linkers.

Both lactate oxidase and glucose oxidase produce hydrogen peroxide as byproducts of the reactions they catalyze. However, as will be shown in the examples below, lactate oxidase has been shown to be superior to glucose oxidase in terms of its in vitro cytotoxicity and its greater tumor site concentration in in vivo studies. MODES FOR CARRYING OUT THE INVENTION

Lactate oxidase is a bacterial enzyme which is commercially available. The reaction which lactate oxidase catalyzes is the conversion of lactate to pyruvate. This reaction utilizes dissolved oxygen (O2) and produces hydrogen peroxide (H2O2) • 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.

One substrate, lactate, is a compound which is naturally found in the fluid surrounding mammalian cells, since it is a product of cellular metabolism. In the specific case of targeting a lactate oxidase therapeutic conjugate on cancer cells, the effectiveness may be enhanced due to the greater amount of lactate produced by tumor cells. Tumor cells generally utilize the anaerobic metabolic pathway and therefore produce more lactate than normal cells. It would also be possible to provi-^ lactate substrate exogenously to patients in doses which would enhance the production of hydrogen peroxide while having minimal side effects. The other substrate, oxygen, is also present dissolved in the interstitial fluid. Attachment of the lactate oxidase enzyme 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. However, 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. In addition, 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. Such 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 about 1:1, preferably ranging from 0.7:1 to 1.1:1. If there is not enough enzyme, then enzyme-free antibodies... can. compete, with the- enzyme-antibody conjugates and target sites are bound but with no enzyme to produce the desired localized cytotoxic effects. Excess enzyme is generally to be avoided, as well, because of problems such as steric hindrance and general interference with antibody binding and specificity.

An enzyme-linker-antibody conjugate meeting the requirements described above would produce hydrogen peroxide in close proximity to the targeted cell. In addition, 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 technique is to create a bank of antibodies which will locate all tumor cells. This is a more complex problem 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 cells 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. Further, should the enzyme be released in a healthy area of the body, the chances of destruction of normal cells is likely to be minimal because of the very low levels of substrate. Lactate is usually not produced except under conditions of extreme muscular activity and in most cases would not be present in substantial amounts in a healthy area of the body. The released lactate oxidase would, under normal conditions, merely be taken to the liver and then could either perform its normal functions on the lactate contained therein or be eliminated.

Since hydrogen peroxide is a general cytotoxic agent, meaning that it will kill all cell types, the generating enzyme could be linked to any target cell binding protein. Thus, 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. Example 1 Preparation of Lactate Oxidase-Antibody Conjugates Thiolation of Lactate Oxidase

To a solution containing 48 nmol of lactate oxidase in 0.75 ml of 10 M triethanolamine, 0.2 M NaCl (pH 8.0) was added 7.5 μl of 0.1 M 2-iminothiolane (in 0.5 M triethanolamine, pH 8.2). The reaction was run on ice under N₂ for two hours. The reaction mixture was gel filtered on a PD-10 column (Pharmacia) equilibrated in 0.1 M potassium phosphate, 1 mM EDTA (pH 7.3). The solution was kept on ice under N2 and was reacted with SMCC derivatized monoclonal antibody as described below. SMCC Derivatization of Antibody

To 75 nmol (11.3 mg) of antibody 9.2.27 in 1 ml phosphate buffered saline (pH 7.0) was added 15 μl of a 25 mM solution of SMCC (succinimidyl-4-(W-maleimidomethyl)- cyclohexane-1-carboxylate) . The reaction was allowed to proceed for one hour at room temperature. The mixture was centrifuged and then gel filtered on a PD-10 column equilibrated in 0.1 M potassium phosphate (pH 6.0). Preparation of conjugate To 34 nmol of the thiolated lactate oxidase was added 34 nmol of the SMCC-derivatized monoclonal antibody. The reaction was allowed to proceed on ice under N₂ for 2 hours and then overnight at 4°C. The reaction was stopped by the addition of excess (700 nmol) of 2-mercaptoethanol. The conjugate mixture was gel filtered on a PD-10 column equilibrated in 10 mM MES (pH 6.0). The void volume eluate was centrifuged and applied to a column (4 x 1 cm) of S-Sepharose equilibrated in 10 mM MES (pH 6.0). The column was washed with equilibration buffer and the conjugate was eluted with 10 mM MES, 0.2 M NaCl (pH 6.0). To the conjugate eluate was. added 200 μl of a 1 mM solution of flavin mononucleotide.

The conjugate was 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 (pH 7.0). Those fractions which contained 1:1 conjugate were pooled and concentrated by ultrafiltration to a concentration of 0.24 mg/ml antibody equivalents. The preparation contained 24 unit/ml lactate oxidase activity. Example 2 Preparation of Glucose Oxidase-Antibodv Conjugates Thiolation of Glucose Oxidase

To a solution containing 87 nmol of glucose oxidase in 1.4 ml of 10 mM triethanolamine, 0.2 M NaCl (pH 8.0) was added 14 μl of 0.1 M 2-iminothiolane (in 0.5 M triethanolamine, pH 8.2). The reaction was run on ice under N₂ for one hour. The reaction mixture was gel filtered on a PD-10 column (Pharmacia) equilibrated in 0.1 M potassium phosphate, 1 mM EDTA (pH 7.3). The solution was kept on ice under N2 and was reacted with SMCC derivatized monoclonal antibody as described below. SMCC Derivatization of Antibody

To 150 nmol (22.6 g) of antibody 9.2.27 in 2 ml phosphate buffered saline (pH 7.0) was added 30 μl of a 25 mM solution of SMCC (succinimidyl-4-(N-maleimidomethyl)- cyclohexane-1-carboxylate) . The reaction was allowed to proceed for one hour at room temperature. The mixture was centrifuged and then gel filtered on a PD-10 column equilibrated in 0.1 M potassium phosphate (pH 6.0). Preparation of conjugate

To 77 nmol of the thiolated glucose oxidase was added 77 nmol of the SMCC-derivatized monoclonal antibody. The reaction was allowed to proceed on ice under N₂ for 1 hour and then overnight at 4°C. The reaction was stopped by the addition of excess (500 nmol) of 2-mercaptoethanol.

The conjugate mixture was gel filtered in 2 ml aliquots on a PD-10 column equilibrated in 10 mM MES (pH 6.0). The void volume eluate was centrifuged and applied to a column (4 x 1 cm) of S-Sepharose equilibrated in 10 mM MES (pH 6.0). The column was washed with equilibration buffer and the conjugate was eluted with 10 mM MES, 0.2 M NaCl (pH 6.0) .

The conjugate was 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 (pH 7.0). Those fractions which contained 1:1 conjugate were pooled and concentrated by ultrafiltration to a concentration of 1.13 mg/ml antibody equivalents. The preparation contained 122 unit/ml glucose oxidase activity. Example 3 In a study done to compare the effectiveness of lactate oxidase immunoconjugates over glucose oxidase immunoconjugates, 10⁴ M21-UCLA melanoma cells per well were allowed to grow in 96-well plates in a 10% C0₂ atmosphere at 37°C in 100 μl of RPMI 1640, 10% FBS medium. After 24 hours, the nutrient was taken off and replaced by 100 μl of glucose and lactate free RPMI 1640 containing varying concentrations of the immunoconjugate, either lactate oxidase immunoconjugate or glucose oxidase immunoconjugate, and a 10-fold activity excess of catalase. Catalase is an enzyme which acts to break down hydrogen peroxide into oxygen and water and serves to prevent cytotoxic effects during the binding step. The immunoconjugates were formed using enzyme and 9.2.27 monoclonal antibody, as indicated in Examples 1 and 2. The immunoconjugate and catalase containing nutrient was removed 30 minutes later and the wells were rinsed three times with the original medium and the cells were allowed to continue to grow in the original medium, which contains glucose, to which 2mM lactate was added. After 24 hours, 10 μl of 1 μCi H-thymidme containing medium was added m order to measure thy idine 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 were shock frozen, then thawed and the individual well contents passed through glass fiber filters. The radioactivity was determined and taken as a measure of cell viability. Viability of 100% corresponds to the radioactivity of the cells that were treated with glucose free nutrient containing no immunoconjugate.

The in vitro cytotoxicity of a lactate oxidase immunoconjugate is about 10 times greater than that of the equivalent glucose oxidase as shown in Table I below.

TABLE I

cells. After two weeks, 3 μCi of I¹²⁵iodinated immunoconjugate, either lactate oxidase immunoconjugate or glucose oxidase immunoconjugate, was injected into the tail vein. After 48 hours the animals were sacrificed and the radioactivity in individual organs was determined. The immunoconjugates were formed using enzyme and 9.2.27 monoclonal antibody following the procedures discussed in Examples 1 and 2.

The in vivo biodistribution data obtained with tumor bearing nude mice also show that the lactate oxidase immunoconjugate is superior to the glucose oxidase one. Radioactivity in the tumor of the lactate oxidase immunoconjugate treated mouse, 48 hours after injection, is 6 times greater than in the tumor of the glucose oxidase immunoconjugate treated mouse as shown in Table II below. Further, the glucose oxidase conjugate is apparently much more rapidly cleared. This is indicated by the comparatively low blood and high liver and spleen values of the glucose oxidase immunoconjugate treated animals. TABLE II Numbers given as % injected dose/g tissue

Organ Glucose Oxidase Lactate Oxidase

-9.2.27 •9.2.27 Tumor 0.1 0.6

Blood 0.2 1.9

Liver 7.0 1.2

Spleen 8.5 0.6

Kidney 0.2 0.7 Intestine 0.2 0.2

INDUSTRIAL APPLICABILITY

The preparations of bacterial lactate oxidase linked to an antibody can be used for production of the cytotoxic substance hydrogen peroxide at the site of action as dictated by the antibody specificity, either in vitro or in vi vo .

Claims

1. An agent for producing localized cytotoxic effects on targeted cells comprising a conjugate of lactate oxidase bound to a target cell binding protein, said conjugate having binding specificity and binding affinity for said target cells and having lactate oxidase catalytic activity.

2. The agent of claim 1, wherein said target cell binding protein is a member of the group consisting of monoclonal antibodies, polyclonal antibodies, antibody fragments, hormones, growth factors, and binding proteins.

3. The agent of claim 1, wherein said target cell binding protein is a monoclonal antibody.

4. The agent of claim 1, wherein said target cell binding protein is fragment of a monoclonal antibody.

5. The agent of claim 1, wherein said lactate oxidase is bound to said target cell binding protein via a linker molecule.

6. A method for producing localized cytotoxic effects on targeted cells which comprises: combining an effective amount of an agent for producing localized cytotoxic effects on targeted cells, said agent comprising a conjugate of lactate oxidase bound to a target cell binding protein, said conjugate having binding specificity and binding affinity for said target cells and having lactate oxidase catalytic activity, said lactate oxidase catalyzing a reaction, utilizing lactate and oxygen as substrates, which results in the formation of hydrogen peroxide in the vicinity of said targeted cells, said hydrogen peroxide causing the destruction of said targeted cells and cells in the vicinity of said targeted cells, with an acceptable vehicle; and administering said combined agent and vehicle.

7. The method of claim 6, wherein said lactate oxidase reacts with endogenous lactate and oxygen present in the vicinity of said targeted cells.

8. The method of claim 6, wherein exogenous lactate is administered in the vicinity of said targeted cells to provide additional substrate for said lactate oxidase.

9. The method of claim 6, wherein said targeted cells are in vitro.

10. The method of claim 6, wherein said targeted cells are in vivo .

11. The method of claim 10, wherein said vehicle is pharmaceutically acceptable.

12. The method of claim 10, wherein said agent is administered topically.

13. The method of claim 10, wherein said agent is administered intravenously.

14. The method of claim 10, wherein said agent is administered subcutaneously.