ANTIBODIES BINDING A NON NATURALLY OCCURRING ENANTIOMER (L-BIOTIN) AND THEIR USE AS TARGETING AGENTS

Abstract: An antibody that has affinity for a non-naturally-occurring enantiomer (NNOE) of a chiral molecule whose naturally-occurring enantiomer (NOE) is endogenous, and that has no affinity for the NOE, can be used for targeting, in therapy or diagnostic procedures. The antibody may be conjugated to a therapeutic or diagnostic agent, e.g. a cytotoxic agent.

Description: ANTIBODIES BINDING A NON NATURALLY OCCURRING ENANTIOMER (L-BIOTTN) AND THEIR USE AS TARGETING AGENTS Field of the Invention This invention relates to targeting agents, and in particular to antibodies and to their use in targeting anti-tumour agents in vivo. 5 Background to the Invention The targeting of therapeutic agents to particular sites in vivo, is well known. In particular, it is very desirable to target anti-cancer agents to a tumour site, to increase the concentration of the agent at the site and thereby improve its effectiveness in neutralising the tumour. Many agents that target tumours rely on the specificity of 10 monoclonal antibodies for delivering the diagnostic or therapeutic agent to the target site. One approach has been to use a radionuclide-antibody conjugate which localises at a target tissue where the radionuclide may exert its cytotoxic effect. There are problems with the utility of radionuclide-antibody conjugates, for example poor penetration of the conjugates to the target site owing to the high 15 molecular weight of the conjugate. In addition, for the conjugate to be therapeutically effective, it must be given time to localise at the target site. The radionuclide is therefore present in the body for prolonged periods, and this results in undesirable toxicity at non-target sites. US-A-5630996 describes the use of antibody-streptavidin conjugates to target 20 a radionuclide-labelled biotin. Streptavidin has high affinity for biotin and is able to localise the radionuclide at the target site through the biotin-streptavidin interaction. However, streptavidin is a protein that is immunogenic in humans, and consequently may not be suitable for repeated, long-term therapeutic use. Summary of the Invention 25 The present invention is based on the realisation that L-biotin, i.e. any of the three non-naturally-occurring enantiomers of biotin, is a suitable ligand for antibody- targeted therapy. More generally, this is true for any endogenous chiral compound, so that any non-naturally-occurring enantiomer (NNOE) can be used, according to this invention. Since neither an anti-NNOE antibody nor the NNOE itself will interact with 30 endogenous/naturally-occurring enantiomer (NOE) or the receptors or carrier proteins for NOEs, the NNOE can be used to target therapeutic agents to a desired locus in vivo. By way of example, L-biotin is biologically inactive and therefore, in general, naturally occurring molecules which bind to the NOE or active enantiomer, D-biotin, will not disrupt the targeting of the agent. According to a further aspect of the invention, the NNOE is covalently or otherwise linked to a therapeutic or diagnostic agent, and used to target the agent to a site in vivo. The agent may be a chemotherapeutic agent, e.g. a radiolabelled compound. Typically, the NNOE will target a site in vivo by interaction with an antibody localised at the target site. Accordingly, a further aspect of the invention is an antibody having affinity for the NNOE and not the NOE. The antibody may be a bifunctional antibody, of which one functionality is as described above and the other involves, for example, affinity for a tumour. Alternatively, a novel conjugate comprises an antibody and the NNOE. The conjugate may be administered to a patient to localise the NNOE at a target site. Subsequently, usually after the administration of a clearing agent, an antibody- therapeutic agent conjugate is administered, the antibody having affinity for the NNOE, to localise the agent at the target site. In another novel conjugate, the NNOE molecule is linked to an enzyme that can be used to convert a suitable prodrug into an active cytotoxic form. Description of the Invention By way of illustration only, the invention will now be described for a specific NNOE, i.e. L-biotin. This is an example of a preferred NNOE for use in the invention, i.e. is a relatively small, non-toxic organic molecule that exhibits neutral biodistribution in the body, is water/serum-soluble, and is readily excreted by the kidney. It is therefore a suitable molecule for use in targeting therapeutic or diagnostic agents in vivo. Antibodies for use in the present invention may be produced using conventional techniques, for example, hybridoma synthesis, recombinant DNA techniques, phage display or other library technology. The antibodies may be derived from any species, including rodent, although it is preferred that the antibodies are derived from mammals other than rodents, e.g. sheep, goats or cows, and have higher affinity. Typically, the antibodies will have an affinity of at least 10 10 l/mol, preferably at least 10 11 l/mol, more preferably at least 10 12 l/mol and most preferably at least 10 13 l/mol, e.g. up to 10 15 or 10 16 l/mol, for the respective ligands. A preferred embodiment of the invention is a bifunctional antibody. A bispecific antibody has affinity for the NNOE rather than the NOE, and may also have affinity for a target site. As part of a multi-step procedure, in which the antibody is administered and allowed to localise before the active agent is given, a bispecific antibody allows the use of more potent doses of the active agent, with reduced systemic side-effects. A bifunctional or other antibody according to the invention may be a whole antibody or may be an engineered antibody or a fragment, e.g. f(ab) 2 . In a further embodiment, the antibody may comprise two single chain fv fragments. The preparation of bifunctional sFvs is well known. For example, Carter era/., Current Opinion in Biotechnology (1997) 8:449-454, discloses the production of bifunctional sFvs using a phage display library. The antibodies may be fully human. Alternatively, antibodies for use in the invention may be modified by recombinant DNA techniques to "humanise" them, making them less immunogenic when administered to a patient. A humanised bifunctional antibody should comprise at least the hypervariable region from both a monoclonal antibody having affinity for target antigen, and a monoclonal antibody having affinity for the NNOE. The remainder of the antibody variable region may be of human immunoglobulin. A higher proportion of human immunoglobulin may be present in a whole antibody or a fragment, e.g. F(ab') 2 . When a single-chain Fv fragment is used, the fragment may comprise hypervariable regions as described above and, optionally, the variable framework from human immunoglobulin. In addition to having affinity for the NNOE, the bifunctional antibody will also have affinity for a particular target site. Typically, the target site will be a tumour and the antibody will have affinity for a tumour-associated antigen. An example of a tumour-associated antigen is the carcinoembryonic antigen (CEA) which is found on colorectal tumours and other adeno-carcinomas. In a preferred embodiment, the NNOE is covalently or otherwise linked to a therapeutic or diagnostic agent. The agent may, for example, be a radionuclide. Administration of the NNOE-radionuclide conjugate will localise the radionuclide at a tumour site through binding to the antibody, to exert a cytotoxic effect on the tumour. Radionuclides having a cytotoxic effect are well known. A preferred radionuclide that may be used in the invention is a radioisotope of phosphorus, yttrium or iodine, e.g. I 123 , I 124 and I 125 which may be used for diagnostic purposes, and l 13 \ Y 90 and P 32 which may be used in therapeutics. Another suitable agent is a cytotoxic drug, e.g. ric or calicheamycin. In an alternative embodiment, the NNOE is linked (conjugated) to an enzyme. The enzyme is capable of converting a suitable prodrug into an active cytotoxic form. The term "prodrug" is used herein to define an inactive form of a drug which may be cleaved by enzymic action to release the therapeutically-active form. Suitable enzyme- prodrug systems are known to those skilled in the art and include carboxypeptidases and modified mustard gas derivatives. The NNOE targets the enzyme to the target site and subsequent administration of the prodrug will result in the active agent being localised at the target. Methods for attaching therapeutic agents to the NNOE will be apparent to those skilled in the art. For example, procedures for conjugating D-biotin to drugs are known, and can be adopted for the purposes of this invention. For use in the invention, the antibody and the NNOE-cytotoxic agent may be formulated in a kit, e.g. comprising the two components separately packaged or in separate containers. Each component may be formulated with a suitable carrier or excipient, examples of which are well known, depending on the route of administration, e.g. oral or intravenous. These two components will usually be administered sequentially. The respective times and durations of administration, and the effective amount of each may readily be determined by the skilled person, and will depend on typical factors such as the location, severity and spread of the tumour, the condition of the subject etc. It is of course a feature of this invention that the amount of cytotoxic agent that is required will be less than in the absence of the antibody. In a preferred embodiment, a bispecific antibody is administered, then a clearing agent and then the effector molecule. This sequence, and suitable clearing agents, will be familiar to those skilled in the art, e.g. with reference to ADEPT technology; see, for example, Drugs of the Future 1996, 21(2): 167-181 , incorporated herein by reference. The following Example illustrates the invention. In particular, it demonstrates the specificity of an anti D-biotin antibody for D-biotin as compared to L-biotin. Example Nunc Maxisorb ELISA plates (Nunc, Denmark) were coated with cytochrome C-D-bt. Cytochrome C-D-bt (C2022, Sigma Chemical, Poole, UK) was diluted to 200 ng/ml in PBS at pH 7.2 and 100 μl of this solution added to each well of the plate and incubated for 1 hr at 37°C. Prior to the addition of test samples, the plates were blocked with low fat milk protein. For blocking, the plates were washed 3 times in PBS with 0.01% Tween 20, and 200 μl of 0.2% low fat milk protein was added to each well and incubated at 37 °C for 30 minutes. Samples of the high affinity sheep monoclonal antibody to D-biotin (1D10, KS Biomedix, UK) were prepared for assay of anti D-biotin activity as follows: 100 μl aliquots of the purified monoclonal antibody at a concentration of 20 ng/ml in 1 % BSA in PBS at pH 7.2 were pre-incubated with various concentrations of either D-biotin or a racemic mixture of D, L-biotin at 37°C for 1 hr prior to addition to the assay plate. After washing the assay plates as above, the pre-incubated samples were then added to the blocked ELISA plate wells and incubated for 1 hr at 37°C. After the sample incubation, the plates were washed again and a 100 μl of secondary conjugate was added for 1 hr at 37 °C. The conjugate used was a donkey- anti-sheep antibody conjugated to alkaline phosphatase (A5187, Sigma Chemical, Poole, UK) diluted at 1 :5,000 in 1% BSA in PBS at pH 7.2. After conjugate incubation, the plates were washed as above and 100 μl of PNPP substrate (N-2770, Sigma Chemical, Poole, UK) added to each well and incubated for 30 minutes at 37°C. The plates were then read at 405 nm in a microtitre plate reader. The results were as follows: It can be seen that the pure D-biotin is more effective at inhibiting the binding of the anti D-biotin monoclonal antibody to immobilised cytochrome C-D-biotin than the racemic mixture that contains both D and L biotins. This confirms that the binding of the antibody to L-biotin is reduced or absent in solution.

Claims: CLAIMS I . An antibody (or fragment thereof) having affinity for a non-naturally-occurring enantiomer (NNOE) of a chiral molecule whose naturally-occurring enantiomer (NOE) is endogenous, and which has substantially reduced affinity for the NOE. 2. An antibody according to claim 1, wherein the NNOE is L-biotin and the NOE is D-biotin. 3. An antibody according to claim 1 or claim 2, which is bifunctional. 4. An antibody according to claim 3, which has affinity for a tumour. 5. An antibody according to any preceding claim, wherein the antibody is a Fab fragment or a single-chain Fv. 6. A conjugate of a NNOE (as defined in claim 1 or claim 2) and, covalently linked thereto, a therapeutic or diagnostic agent. 7. A conjugate according to claim 6, wherein the agent is a chemotherapeutic agent. 8. A conjugate according to claim 6 or claim 7, wherein the agent is a radiolabel. 9. A conjugate of a NNOE (as defined in claim 1 or claim 2) and an antibody. 10. A conjugate according to claim 9, wherein the antibody has affinity for a tumour. I I. Use of an antibody or conjugate according to any of claims 1 to 10, for the manufacture of a medicament for use in the treatment of cancer. 12. A product comprising an antibody according to any of claims 1 to 5 and a conjugate according to any of claims 6 to 10, for simultaneous, sequential or separate use in the treatment of cancer.

Inventors: HARRISON PETER JOHN, HARRISON, PETER, JOHN
Applicants: HARRISON PETER JOHN, KS BIOMEDIX LTD, HARRISON, PETER, JOHN, KS BIOMEDIX LTD.
Application year: 2000
Main classification: C07K16
IPC codes: A61K39, A61K45, A61K47, A61K51, A61P35, C07K16