KR20010072988A - Bifunctional Antibodies and Their Use In Targeting Anti-tumour Agents - Google Patents

Abstract

The present invention relates to a bifunctional antibody having an affinity for an organic molecule covalently bound to an enzyme or cytotoxic drug that can convert a prodrug into a cytotoxic form and an affinity for a target site. The antibody can be used for therapeutic or diagnostic purposes, in particular for the treatment of tumors.

Description

Bifunctional Antibodies and Their Use In Targeting Anti-tumour Agents}

It is well known to target therapeutic agents to specific sites in vivo. In particular, it is highly desirable to target an anticancer agent at the tumor site, to increase the concentration of the anticancer agent at the site, thereby improving the efficiency of neutralizing the tumor. Many agents for target tumors are known, many of which rely on the specificity of monoclonal antibodies to deliver diagnostic or therapeutic agents to the target site. Attempts have been made to use radionuclide-antibody conjugates that allow the radionuclide to concentrate on target tissues that may exert its cytotoxic effects.

However, the use of radionuclide-antibody conjugates has been found to have problems such as weak penetration of the conjugate into the target site due to the high molecular weight of the conjugate. In addition, the conjugate must be given time to focus on the target site in order to be therapeutically effective. Thus, radionuclides are present in the body for long periods of time, resulting in undesirable toxicity to non-target sites.

Thus, in order to ensure that the antibody is concentrated at the target site prior to administration of the cytotoxic radiolabelled agent, it is desirable to administer the antibody independently of the cytotoxic radiolabelled agent.

US Pat. No. 5,630,996 discloses a method using an antibody-streptavidin conjugate that targets a radionuclide-labeled biotin. Streptavidin has a high affinity for biotin and can concentrate radionuclides at target sites through biotin-streptavidin interactions. However, streptavidin is a protein that immunizes humans, and consequently is not suitable for repeated long-term treatment.

U. S. Patent No. 5,591, 828 discloses bispecific antibodies having affinity for specific protein antigen elements and metal chelates. However, the chelates, EDTA-Y ⁹⁰ , and DTPA-Y ⁹⁰ are preferably fos-peptides that interact with the jun-peptide on the antibody, in order to concentrate sufficient Y ⁹⁰ at the target site. peptide should be added covalently. In addition, dissociation may occur between the chelating agent (EDTA) and the radionuclide (Y ⁹⁰ ). Therefore, concentration at the tumor site may not be effective, and the process of producing phosph-peptide-metal chelate conjugates is long and not suitable for large scale manufacture.

The present invention relates to bifunctional antibodies and their use for targeting antitumor agents in vivo.

The present invention relates to a bifunctional antibody having affinity for both the therapeutic agent and the diagnostic agent as well as the antigen present in the tumor site, wherein the therapeutic or diagnostic agent is a radiolabeled organic molecule or an organic molecule covalently bound to a cytotoxic drug. In another embodiment, the organic molecule is bound to an enzyme capable of converting the prodrug into a cytotoxic form.

Accordingly, the present invention provides a means of continuously administering a bifunctional antibody and an organic molecule to increase the concentration of the therapeutic agent at a target site in a mammal. The invention may be used for therapeutic or diagnostic treatment.

Typically, organic molecules are organic molecules or biotin that exhibit good penetration at the target site or are otherwise neutral biodistribution in vivo.

One of the advantages of the present invention is that it is possible to administer cytotoxic drugs which, while interacting with the antibody, focus on the desired target but which are less toxic in non-target tissues as the unbound drug is efficiently removed from the human body.

In one embodiment, the cytotoxic drug is a radionuclide and is covalently bound to the organic molecule or is itself part of the organic molecule. The radionuclide may be selected to give a therapeutic effect, for example acting as an anti-tumor agent, or may be administered for diagnostic purposes such as tumor imaging.

In other embodiments, organic molecules are combined with enzymes that can be used to convert suitable prodrugs into active cytotoxic forms.

Antibodies according to the invention can be produced using general techniques such as hybridoma synthesis, recombinant DNA techniques, or phase display. Although antibodies derived from mammals such as sheep, goats, or cows that produce antibodies of higher affinity than rodents are preferred, antibodies can be derived from any species, including rodents.

Typically, the antibody will have an affinity of at least 10 ¹⁰ l / mol for each ligand, but preferably at least 10 ¹¹ l / mol, more preferably at least 10 ¹² l / mol, most preferably 10 ¹³ l / mol That's it.

The bifunctional antibody according to the invention may be all or part of them, such as f (ab) ₂ . In other embodiments, the antibody may consist of two single chain fv fragments. Methods of making bifunctional sFvs are well known. For example, Current Opinion in Biotechnology 1997, 8: 449-454 (Carter et al.) Discloses the production of bifunctional sFvs using a phase display library.

In addition, antibodies can be modified by recombinant DNA techniques to "humanize" antibodies, such as making them less immune when administered to a patient. Humanized antibodies should include at least hypervariable regions from both sides of the monoclonal antibody having an affinity for the target antigen and a monoclonal antibody having an affinity for organic molecules. The remainder of the antibody variable region can be human immunoglobulin. More portions of human immunoglobulins may be present in whole or in antibody fragments such as F (ab ') ₂ . When a single chain Fv fragment is used, the fragment may be composed of a variety of structures from the hypervariable region and optionally from human immunoglobulins.

The antibody will have an affinity for a particular target site. Typically, the target site will be a tumor and the antibody will have an affinity for the tumor-associated antigen. Examples of tumor-associated antigens are cancer embryonic antigens found in colorectal tumors and other adeno-carcinoma.

In a preferred embodiment, the antibody has a ligand-affinity for the organic molecule that is radiolabeled. The present invention includes both covalently bonding radionuclides to organic molecules separately and simply radiolabeling appropriate atoms to the organic molecules themselves. For example, the organic molecule may comprise phosphorus or iodine atoms that are radiolabelled to provide cytotoxic organic molecules. Dosing the molecule will concentrate the radionuclide at the site of the tumor through binding to the antibody to have a cytotoxic effect on the tumor. Preferred radionuclides that can be used in the present invention are iodine radioisotopes such as I ¹²³ , I ¹²⁴ , and I ^{125 that} can be used for diagnostic purposes and I ^{131 that} can be used for treatment. A more preferred radionuclide that can be used in the present invention is P ³² .

Cytotoxic drugs can also be cytotoxic drugs such as ricin or calicheamycin.

In another embodiment, the organic molecule is linked (conjugated) with the enzyme. Enzymes can convert appropriate prodrugs into active cytotoxic forms. The term "prodrug" is used herein to define an inactive form of a drug that can be cleaved by enzymatic activity to release a therapeutically active form. Suitable enzyme-prodrug systems are well known to those skilled in the art and include carboxypeptidase and modified mustard gas derivatives.

Organic molecules useful in the present invention should be capable of specific interactions with antibodies. Thus, the organic molecule is large enough to elicit an immune response to produce an antibody when conjugated to a protein carrier, or large enough to produce an antibody from an antibody library as it appears on a filamentous phage. Should be Preferably, organic molecules linked to cytotoxic drugs or enzymes can pass through the lining of the vasculature to reach the target site. It is preferable that the molecular weight of an organic molecule is less than 1500, More preferably, it is less than 1000. Organic molecules are preferably non-toxic when they are not radiolabeled. In addition, the labeled organic molecules should be neutrally biodistributed when introduced into the patient in the absence of a bispecific antibody, and it is preferable to select molecules which are rapidly removed through the kidneys and do not accumulate in the thyroid gland. If the molecule is easily derived from an unlabeled parent molecule, it is preferred that the inducible label molecule is stable after dosing. The conjugate is preferably water soluble in order to facilitate the formulation in a suitable excipient. Although molecules can be made cytotoxic by radiolabels, suitable organic molecules include nontoxic compounds.

In a preferred embodiment, the radiolabeled organic molecule is a radiolabeled biotin. In another preferred embodiment said molecule is of formula (I): 4,4-bis (4-hydroxy-3,5-diiodophenyl) pentanoic acid.

In another preferred embodiment, the molecule is of formula (II)

Wherein R ¹ and R ² are each independently a radiolabeled portion, such as a radiolabeled iodine, methyl group, or phenyl group, and X ¹ and X ² are each independently H or OH.

Preferred radiolabeled molecules are N- (4-hydroxy-3,5-diiobenzobenzoyl) -1,6-hexanediamine and N- (2-hydroxy-3,5-dioodobenzoyl) -1, 6-hexanediamine, wherein each iodine atom or one of which may be one of the radioisotopes I ¹²³ , I ¹²⁵ , I ¹²⁴ , and I ¹³¹ .

For use in the present invention, bispecific antibodies and cytotoxic drugs may be packaged separately or formulated in kits, including the two components in separate containers. Each component may be formulated with a suitable carrier or excipient depending on the route of administration, such as well known oral administration or intravenous injection.

The two components will usually be administered sequentially. The effective amount of each component can be readily determined by one skilled in the art and will depend on typical factors such as the location, depth, and extent of the tumor and the symptoms of the individual. Of course it is a feature of the present invention that the amount of cytotoxic drug required will be less than in the absence of antibodies.

Claims (20)

Bifunctional antibodies that have an affinity for a therapeutic or diagnostic agent that is an organic molecule and an affinity for a tumor. A bifunctional antibody having affinity for a therapeutic agent that is an organic molecule covalently bound to an enzyme and affinity for a tumor. The antibody of claim 1 or 2, wherein the ligand-affinity is at least 10 ¹⁰ l / mol or more. The antibody according to any one of claims 1 to 3, wherein the organic molecule has a molecular weight of less than 1500. 5. The antibody of claim 1, wherein the organic molecule is water soluble and neutrally biodistributed in vivo. 6. The antibody of claim 1, wherein the radiolabel of the antibody is I ¹²³ , I ¹²⁴ , I ¹²⁵ , P ³² or I ¹³¹ . The antibody of any one of claims 1 to 6, wherein the molecule is a radiolabeled 4,4-bis (4-hydroxy-3,5-diiodophenyl) pentanoic acid. The antibody according to any one of claims 1 to 6, wherein the molecule is represented by the following structural formula (II): Wherein R ¹ and R ² are each independently a radiolabeled portion such as a radiolabeled iodine, methyl group, or phenyl group, and X ¹ and X ² are each independently H or OH. The method of claim 8, wherein the molecule is a radiolabeled N- (4-hydroxy-3,5-diiodobenzoyl) -1,6-hexanediamine or N- (2-hydroxy-3,5-diio An antibody that is dobenzoyl) -1,6-hexanediamine. The antibody of claim 1 or 2, wherein the molecule is biotin. The antibody of any one of the preceding claims, wherein the affinity of the antibody for tumor is by tumor-associated antigen. The antibody of claim 11, wherein the antigen is a cancer embryonic antigen. The antibody of any one of the preceding claims, wherein the antibody consists of two single chain Fvs. The antibody according to any one of claims 1 to 13, wherein the antibody consists of a normal region derived from at least human immunoglobulin. 4,4-bis (4-hydroxy-3,5-diiodophenyl) pentanoic acid, N- (4-hydroxy-3,5-diiobenzobenzoyl) -1,6-hexanediamine, and N A radiolabeled compound selected from-(2-hydroxy-3,5-diiodobenzoyl) -1,6-hexanediamine. Radiolabel 4,4-bis (4-hydroxy-3,5-diiodophenyl) pentanoic acid, radiolabel N- (4-hydroxy-3,5-diio, for use in therapeutic or diagnostic methods Dobenzoyl) -1,6-hexanediamine, or radiolabeled N- (2-hydroxy-3,5-diiodobenzoyl) -1,6-hexanediamine. A cancer therapeutic agent containing the antibody according to any one of claims 1 to 14. A diagnostic agent containing the antibody according to any one of claims 1 to 14. Products consisting of bifunctional antibodies having affinity for tumors and organic molecules for use simultaneously, continuously or separately in the treatment of tumors, and therapeutic or diagnostic agents which are organic molecules or radiolabeled organic molecules covalently bound to cytotoxic drugs . (i) bifunctional antibodies having specificity for a therapeutic or diagnostic agent and for tumors; (ii) a therapeutic or diagnostic agent that is an organic molecule covalently bound to a cytotoxic drug or labeled with a radiolabel; A method of treating or diagnosing a tumor-related disease consisting of continuously administering a drug.