WO1992005200A1

WO1992005200A1 - Xenobiotic-antibody conjugates

Info

Publication number: WO1992005200A1
Application number: PCT/US1990/005297
Authority: WO
Inventors: Charles G. Smith; Bernard Loev
Original assignee: Chemex Pharmaceuticals, Inc.
Priority date: 1990-09-18
Filing date: 1990-09-18
Publication date: 1992-04-02

Abstract

The present invention relates to xenobiotic antibody conjugates and method of its use in vivo. The conjugates are obtained by covalent coupling of xenobiotic compound such as tubercidin, toyocasmycin, sangivamycin and analogs with antibody or antibody fragments via a linker which may or not be cleavable by serum components.

Description

XENOBIOTIC-ANTIBODY CONJUGATES

INTRODUCTION

FIELD OF THE INVENTION

This invention relates to the general area of antibody conjugates capable of in vivo delivery of compounds to target sites. More specifically it relates to a method of delivery of potent cytotoxins to diseased cells while sparing healthy cells. Finally, it describes delivery systems for unique cytotoxic compounds that are trapped within target cells.

BACKGROUND OF THE INVENTION

The development of monoclonal antibody technology has enabled the production of specific antibodies against a variety of malignant cells. The ability of such antibodies to discriminate between diseased cells and healthy cells has led to their use as a mechanism for delivering drugs which are attached to them, to cells to which they bind. Such so called "antibody conjugates" provide several advantages. Because they provide a mechanism for delivery and concentration of a drug at the site of the malignant cells, lower doses of the drug can be used. In addition the side effects resulting from action of a drug on healthy cells are reduced. For a review on the advantages of monoclonal antibody conjugates in the diagnosis and treatment of cancer see Pietersz et al, (1987) Immunology and Cell Biology 65: 111-125.

Considerable literature exists which describes linkage of various cytotoxic agents to antibodies for the so- called "immunotoxin" approach to killing tumor tissue. Although such conjugates have shown great promise in animal tumor models, the results to date in humans involving toxins such as the A chain of the naturally occurring toxin ricin, the anthracyclinone xenobiotics, adriamycin, daunomycin and other agents have been at best marginally successful. It has been suggested that the effectiveness of antibody conjugates may be enhanced if the antibody and its associated toxin are cleaved once the antibody has bound to the target cell. To this end, Rodwell et al. disclosed, in U.S. 4,671,958, a method of conjugating compounds to antibodies such that the conjugate, when delivered to the cell surface, is subsequently cleaved by complement to yield free drug at the target cell surface. The Rodwell disclosure, while providing a useful system of delivery, fails to disclose any compounds having unique metabolic characteristics that would insure that the conjugates would be any more successful than those heretofore suggested. Thus the problem of identifying compounds resistant to metabolic degradation in vivo, yet capable of adequate permeation and retention by target cells remains, to date, unsolved.

Among the desirable characteristics for a cytotoxic agent to be considered a good candidate for target directed immunotoxic therapy are the following: 1) highly potent cytotoxicity to human tumor cells when the agent is absorbed into the tumor cell; 2) sufficient chemical stability to allow chemical coupling to the antibody? 3) sufficient hydrophylicity to allow adequate solubility in the serum while being delivered to the tumor site; and 4) adequate "metabolic stability" to allow penetration of the drug into the cells (e.g., little or no enzymatic degradation at the surface of the tumor cell or in the serum, and adequate residence time within the tumor cell) .

Tubercidin is a cytotoxic analog of adenosine that has been shown to simultaneously inhibit RNA, DNA and protein synthesis in essentially every living mammalian cell line studied in which macromolecule synthesis is taking place, once the compound has been absorbed into the cells. This finding is quite contrary to that of most inhibitors of macromolecule synthesis as the others show a preference for either RNA synthesis, DNA synthesis or protein synthesis but do not inhibit all three simultaneously. In addition, tubercidin is incorporated into both RNA and DNA in multiplying cells. This is most unusual if not totally unique among substances that can be incorporated into nucleic acids.

Because tubercidin is indiscriminate in its ability to kill living cells, it has been studied topically as well as systemically. For example, Klein et al. (Cancer 34: 250-253) in 1974 reported on the treatment of basal cell carcinoma and actinic keratosis with topical tubercidin. The use of nitrobenzylthioinosinate has been reported to reduce tubercidin toxicity when it is applied to basal cell tumors or warts. DeClercq and his co-workers [Antibiotic Agents and Chemotherapy 30: 719-724 (1986)] reported activity of xylo-tubercidin against herpetic skin lesions in the hairless mouse.

Tubercidin has also been used for the treatment, in animals, of parasitic diseases. Jaffe and co-worker (J. Parasitol 62: 910-913 (1976) reported on the treatment of liver flukes in female mice with tubercidin. The same investigators reported treatment of schistosomiasis (both S_^ mansonii and S^ japonicum) through a single dose of tubercidin by iv drip to iv baboons. El Kouni et al. Biochem. Pharmcrol 36: 3816-3821 (1987) reported that a combination of tubercidin and nitrobenzylthioinosine provided a highly selective combination therapy for treatment of S^ mansonii in mice.

The major problem limiting the use of tubercidin in human (or animal) medicine is its toxicity to normal tissue, particularly the veins and the kidneys. In an attempt to ameliorate the toxicity to the host veins and kidneys, tubercidin was administered to human cancer patients after it had been taken up in vitro into the red blood cells of the patient being treated (it does not kill red blood cells because they are not synthesizing RNA, DNA or protein) and the red blood cells were reinfused into the patient [Cancer Research 30: 79-81 (1970)]. Unfortunately the degree of selectivity was inadequate to achieve the goal of adequate antitumor activity although venous and nephro- toxicities were reduced. Thus, tubercidin has not, to date, been found to be useful as a therapeutic composition.

Similarly, a number of other potent cytotoxins that could be effective anti-cancer agents cannot be used therapeutically due to their toxicity to healthy cells.

It is, therefore, an object of this invention to provide a form whereby tubercidin and other potent cytotoxins can be therapeutically useful in the treatment of malignant disease.

It is a further object of the invention to provide a mechanism for delivering to specific, metabolically active cells, a unique toxic compound that is capable of becoming trapped within specific targeted cells.

It is a further object of the invention to provide methods for the conjugation of these toxins to antibodies.

Further objects will be evident from the description of the invention which follows.

SUMMARY OF THE INVENTION

This invention describes therapeutically useful antibody conjugates of tubercidin or other xenobiotic agents for delivery of drug compounds to target cells.

This invention involves the chemical coupling of cytotoxic agents to appropriate antibodies using linkers that are sufficiently resistant to biological degradation in vivo so as to deliver the agents directly to the surface of the target cells in the human body. Most preferably, a monoclonal antibody produced against a tumor cell antigen is attached to a cytotoxic agent. Once the antibody has affixed itself to the specific receptor sites on the human tumor for which it has been designed and then produced in an appropriate laboratory system, the tumor will engulf the cytotoxic agent or the cytotoxic agent linked to the antibody by the usual processes of endo- and pino-cytosis, thereby delivering the cytotoxic agent into the cytoplasm of the malignant cells. Once present within the cytoplasm of these cells, the cytotoxic agents, which have been shown to have a broad spectrum of cytotoxic effects in vitro and shown to be able to kill tumors growing in experimental animals, and, in the case of tubercidin, in humans as well, will kill the tumor tissue in a highly selective way that will lead to far better antitumor activity than is possible upon administration of the non-conjugated compound to an animal. It is postulated that, should the tumor killing effect take place on a subacute or chronic basis, the whole animal may be exposed to products of tumor cell disintegration in such a manner that these products will induce an immunologic response on the part of the host that will aid the host in rejecting tumor tissue and preventing metastatic spread or growth of cells that have already lodged in a site distal to the original tumor. Furthermore, a very clear advantage of the conjugation of the cytotoxic agent with an appropriate antibody is that the antibody will prevent these compounds from entering normal cells as they are capable of doing when administered in free form into the blood stream of animals, thereby reducing whole animal toxicity and markedly increasing selective kill of tumor tissue.

The attachment of tubercidin and other xenobiotic compounds to antibodies is conceived to be of considerable value in the treatment of the cancer patient since these potent compounds have, heretofore, been unavailable for use in the treatment of disease. The selectivity of the antibody will assure the delivery of these highly cytotoxic compounds directly and specifically to the tumor site and will minimize its absorption into other normal tissues, including the local veins and the kidneys. Each of the cytotoxic agents, after coupling to antibodies that are selective for attachment to the surfaces of various tumors, will be administered by the intravenous, intracavitary, topical or intratumoral routes to patients suffering malignant disease and the MoAb's will affix the cytotoxic agents to the surface of the tumor and they will be absorbed in a highly concentrated and highly selective manner. Once they can enter the cytoplasm of the tumor, each of these unique agents will exert its selective and distinctive biochemical mechanisms that will result in the death of the tumor cells, thereby σuring the patient or offering significant amelioration of the course of the disease to give relief and to prolong life and well being of the treated individual.

Although the conjugates disclosed herein are particularly useful for the treatment of malignant disease, it will be recognized by those skilled in the art that these compounds may be useful in the treatment of other diseases, where the object is the destruction of macromolecule-synthesizing entities within the body. For example, a tubercidin-containing conjugate could be used against certain viruses, parasites, bacteria, fungi, non-malignant hyperproliferative disorders such as fibromatosis, certain brain tumors, psoriasis, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot showing the effect of tubercidin on KB cells.

DESCRIPTION OF THE INVENTION

According to the present invention, potent cytotoxic agents, most preferably, tubercidin, are linked to antibodies that are directed against particular constituents of target cells.

SELECTION OF CYTOTOXIC COMPOUNDS

The cytotoxic compounds contemplated by the present invention have unique biochemical properties and modes of action that make them particularly suitable for cancer therapy.

Although not intended to be limiting, the following compounds are contemplated by the present invention: 1. The xenobiotics tubercidin, methyl ester of tubercidin-5,-phosphate (MepTu) , xylotubercidin, toyoca ycin and sangivamycin or analogs with substantially the same structure or xenobiotic activity. These compounds are all deaza cytotoxic analogs of adenosine that have been shown to inhibit RNA, DNA, or protein synthesis. Tubercidin is most preferred, as it inhibits RNA, DNA and protein synthesis simultaneously. Thus, tubercidin kills mammalian cells that are synthesizing macromolecules and especially kills reproducing cells. Tubercidin is less cytotoxic to resting cells, although it also eventually kills them.

Although it is not intended to be a limitation to the invention, it is believed that the biochemical mechanism whereby tubercidin kills the tumor cell involves its phosphorylation through normal metabolic pathways, using enzyme kinases and ATP in exactly the same way that normal nucleosides are metabolized. If circulating kinases modify tubercidin to give the mono-, di- and triphosphates in the blood stream, the phosphoryl groups will nevertheless be hydrolyzed at cell surfaces (as are all phosphorylated nucleosides) to liberate free tubercidin. On the other hand, if one administers the methyl ester of the 5,- phosphate, this compound is more refractory to degradation in the human body and more likely to be absorbed and to deliver intact tubercidin inside the tumor cell even if it is not conjugated to an antibody.

An extremely interesting property of tubercidin is the resistance of its 4- amino group to deamination by nucleoside or nucleotide deaminases and the resistance of its nucleoside linkage to cleavage by nucleoside phosphorylase, the enzymes that normally convert nucleosides to the corresponding hydroxy analogues or to simple purines or pyrimidines, respectively. Since the preponderence of evidence indicates that tubercidin cannot be degraded by nucleoside phosphorylase and it is known not to be deaminated by adenosine deaminase, it provides a kind of residually active form of cytotoxic compound to the tumor cell. In addition, since tubercidin is rapidly and completely converted to its phosphorylated forms (nucleotides) as soon as it is adsorbed into mammalian cells, it cannot exit from the cells because nucleotides do not cross cell membranes. As a result, tubercidin remains trapped inside the cell in its undegraded nuclectide form such that its killing impact is maximized. The cytotoxicity of tubercidin is not reversed in mammalian cells by added purine or pyrimidine compounds and it is not cross-resistant with 6-MP. Furthermore, if one were to administer either the 5'-phosphonate isostere, CH_Tu,

7DzAd

or the methyl ester of pCH_Tu (MepCH₂Tu) , these compounds cannot be hydrolyzed by body or cellular phosphatases to liberate free tubercidin because they are not phosphates, but rather, phosphonates. The latter compound will be hydrolyzed by body esterases to liberate pCH_Tu which, in turn, will be further phosphorylated to the di- and tri- phosphates of the tubercidin phosphonate analog that will, in turn, exert full biological and cytotoxic activity inside the tumor cell.

By focusing these tubercidin related cytotoxic agents on the tumor cell by means of specific antibodies, the compounds can show enhanced effects due to their unique properties, which include 1) high degree of cytotoxicity to all cells into which they penetrate that are synthesizing macromolecules, 2) good chemical stability, 3) refractoriness to reversal of cytotoxicity by normal purine and pyrimidine compounds 4) lack of cross- resistance with 6-MP, 5) resistance to the key catabolic enzymes in the body (e.g., nucleoside phosphorylase and adenosine deaminase) , 6) "metabolic entrapment" within cells once it gains entry by virtue of being phosphorylated by cellular kinases, and 7) unique, and still unidentified mechanism of action.

2. The xenobiotics nogalamycin, nogalarol, and nogalarose or analogues with substantially the same structure or xenobiotic activity. Nogalamycin is an anthracyclinone, as are adriamycin and daunomycin.

Although chemically related to the latter compounds, its mechanism of action is quite unique in that it intercalates with DNA with a particular base specificity (namely, it intercalates only where "runs" of dAT bases occur in the chain) . As a result of this unique mechanism of intercalation, nogalamycin represents a very unique xenobiotic that differs from other available agents in its ability to kill tumor cells.

3. The xenobiotics porfiromycin, sparsomycin, pactamycin and streptozotocin or analogues with substantially the same structure or xenobiotic activity. Porfiromycin is a xenobiotic chemically related to mitomycin C. The latter antibiotic has been coupled with monoclonal antibodies and reported to show some selective antitumor activity in certain animal systems. Porfiromycin is conceived to be a good candidate for coupling with antibodies because of its unique chemical structure.

Sparsomycin is a very unique antibiotic, having as its mechanism of action a very unusual and specific inhibition of one of the sites of elongation of the peptide chain during protein biosynthesis in animal cells. In early studies, it showed a very broad spectrum of cytotoxicity against a variety of animal cells grown in vitro and a broad spectrum of antitumor activity in experimental animal models. When studied in human beings, sparsomycin caused a most unusual toxic reaction that limited its further investigation in man; namely, the production of so-called "ring scatoma" in the human retina [Cancer Chemo. Repts. 43:29-31

(1964)]. In spite of extensive studies in experimental animals attempting to reproduce this effect, it could not be shown conclusively to occur in any species except homo sapiens. According, and with great regret at the time, the scientists developing this compound were forced to abandon it as a clinical candidate. The above conception is directed toward the ability to prevent the uptake of sparsomycin by human retinal cells (and other normal cells) by virtue of conjugating it to selective antibodies that will, as noted above, bind the compound directly and specifically to the tumor. Its extremely broad base of cytotoxic activity and unique mechanism of action is expected to be most useful in killing a wide variety of tumor cells with the selectivity provided by the antibody directed against various surface antigens.

Pactamycin, like sparsomycin, is a very unique molecule unlike any other of its kind or any other under investigation in cancer research today. It inhibits, as does sparmocycin, protein synthesis in animal cells but by a mechanism different from that of sparsomycin. Most of the clinically useful antitumor agents (e.g., alkylating agents, adria ycin, daunomycin, pyrimidine antagonists, purine antagonists, etc.) inhibit nucleic acid, rather than protein, synthesis (either RNA or DNA synthesis) . No anitumor agent proven to be useful in the treatment of human cancer is available today for direct administration to human beings that has, as its primary mechanism of action, the inhibition of protein synthesis.

Streptozotocin is a very unique antibiotic/xenobiotic that alkylates various sensitive centers in macro olecules. It was discovered some years ago as an antibacterial agent but its development as an antibiotic was curtailed because I) it was not absorbed orally and 2) in experimental rodent systems the substance, when injected intravenously, produced permanent diabetes by killing the beta cells of the pancreas after a single dose. It was next screened broadly in animal systems against a variety of tumors and shown to have antileukemic activity in a mouse model. In clinical studies involving the treatment of human leukemia, it was found to be inactive as an antitumor agent but, surprisingly, it was also shown not to affect the pancreas of patients suffering leukemic disease. It was postulated that streptozotocin might possess an organotropic toxicity that was expressed in normal rodent pancreas and that might be expressed in human carcinoma of the pancreas cells, an extremely serious illness that, at the time streptozotocin was being studied, was not treatable in a meaningful way by the chemotherapeutic agents then available. When streptozotocin was studied in patients suffering from inoperable islet cell carcinoma (a uniformly fatal disease) , useful antitumor activity was demonstrated. In 1984, streptozotocin was approved by the FDA for the treatment of human islet cell carcinoma when administered by the intravenous route. Clearly, conjugation of streptozotocin with antibodies that will target this compound to tumor cells will markedly increase the therapeutic index and permit a much qreater exposure of tumor tissue to this drug. METHODS OF PREPARATION

The production of antibodies and methods for their isolation are well known to those skilled in the art. Monoclonal antibodies, as produced by hybridoma cells using recognized methodology, are most preferred due to their specificity to a particular target antigen. Alternatively, polyclonal antibodies produced by exposure of an immunocompetent organism to target cells can be purified to homogeneity as regards specific antigenic moieties.

The antibody and xenobiotic agent are conjugated or "linked" together in such a manner that they are not directly linked to each other, so as to interfere with each other,s activity; and linked in a manner which is pharmaceutically acceptable. The attachment is accomplished via chemical bonds (e.g. ester or amide linkages) and preferably via linkers which are susceptible to cleavage by one or more activated complement enzymes.

In certain applications, release of the compound is not desirable. Therefore, in an alternate embodiment, the conjugate may be designed so that the compound is delivered to the target but not released.

The compound may be attached to antibody molecules via linkers which are not susceptible to cleavage by complement enzymes. These linkers may include amino acids, peptides, or other organic compounds which may be modified to include functional groups that can subsequently be utilized in attachment to antibody molecules or antibody fragments by the methods described herein.

The compound may be attached to the linker before or after the linker is attached to the antibody molecule. If the compound contains functional groups that would interfere with attachment to the linker, these interfering functional groups can be blocked before attachment of the compound and deblocked once the conjugate is made (as in Example 1) .

The linker molecule, before conjugation with the antibody or xenobiotic compound, must contain a plurality of functional groups such as carboxylic acid, aldehyde, halide, ester, mercaptan, disulfide, hydroxyl, sulfonyl, amino or hydrazine groups. In certain instances, the linker molecule will not directly react with the functional groups of the cytotoxic agent or of the antibody, e.g., when the linker group contains carboxyl groups. In such instances, the functional groups of the agent, the antibody, or the union group must first be activated such that they will then undergo reaction.

Such activation can be carried out by methods well known to the art. For example, carboxyl groups can be activiated by first reacting with a carbodiimide, or converted to a hydroxylamine ester. Carbodiimides are a well-known class of compounds having the general formula: R-N=C-N=R wherein R and R^/ in the general carbodiimide formula represent any alkyl, aryl or allyl group which is essentially non-reactive.

A carbodiimide that may be used in this invention, although it is not in any sense critical, is l-ethyl- 3(3-dimethylaminopropyl) carbodiimide, hereinafter abbreviated "EDC".

The reaction between carbodiimide and a linker molecule containing carboxylic acid groups is carried out at room temperature and is a simple addition reaction wherein the carbodiimide adds to the carboxyl group. The carbodiimide "activates" the carboxyl group of the carboxylic acid such that it is now a reactive site for the cytotoxic agent. The carbodiimide is an intermediate activating composition which is a "leaving group". The reaction with cytotoxic a ine- containing agents is a direct addition reaction which can be carried out at room temperature and results in a carboxyamide bond involving the free carboxyl groups of the linker acid and the free amino groups of the cytotoxic agent. The reaction occurs in a short period of time, and has been noted to be substantially complete in as short as five minutes, for small reaction quantities. The reaction does not appear to be process critical.

In the next step the composition, which in its present state may be referred to as the "intermediate conjugate", now has the cytotoxic amine-containing agent attached to it. It now needs to have the tumor specific antibody attached to it. It will be recalled that the linker has multiple functional groups, such as carboxylic acids, only some of which have reacted with the cytotoxic agent. Thus, there are additional reactive sites which remain on the carrier molecule for reaction with the antibody.

In the reaction between the intermediate conjugate and the tumor specific antibody, it may again be necessary to assure that the remaining carboxylic acid groups are sufficiently reactive to react with the antibody, so carbodiimide activator may again be added. Carbodiimide functions in the exact manner as described above. After the carbodiimide is allowed to react for about five minutes, the reaction mixture may be diluted with, for example, phosphate buffered saline solution (PBS) and purified antibody is added. Again, the reaction does not appear to be time or temperature dependent. The only important process criteria in the antibody addition reaction is that the reaction mixture be allowed reactive contact for a sufficient period of time to allow carbodiimide groups to leave and be replaced by the linkage between free amine or mercaptan moieties of the antibody and. carboxyl groups of the acid. In laboratory experiments, three hours at room temperature will usually be sufficient.

After the reaction is complete, the excess carbodiimide may be quenched with sodium acetate, and the mixture dialyzed to separate low molecular weight reactants from the linked product with has bound to it both the antibody and the xenobiotic compound without either of them being directly bonded to the other.

Free sulfhydryl groups may be present in the antibodies or can be generated from the disulfide bonds of the immunoglobulin molecule. This is accomplished by mild reduction of the antibody molecule. The disulfide bonds of IgG which are generally most susceptible to reduction are those that link the two heavy chains. The disulfide bonds located near the antigen binding region of the antibody molecule remain relatively unaffected. Such reduction results in the loss of ability to fix complement but does not interfere with antibody-antigen binding ability (Karush et al., 1979, Biochem. 18: 2226-2232) . The free sulfhydryl groups generated in the intra-heavy chain region can then react with iodoalkyl derivatives of any linker compound containing carboxy or amino groups (e.g., iodoalkyl derivatives of linker groups which are attached to a compound) to form a covalent linkage.

A linker carboxylic acid may be activated by conversion to a hydroxylamine ester, for example to an O-acyl-N- hydroxysuccinimide. The preparation of such esters are well known, and may be prepared, for example by adding a carbodiimide to a solution containing equivalent amounts of the carboxylic acid and the N-hydroxy compound in an inert solvent such as methylene chloride. After storing at room temperature until the by-product urea has all precipitated (usually 2-14 hours) , the urea is filtered and the activated ester is isolated usually by evaporation of the solvent. The activated ester is then reacted with the amino, hydroxy or mercaptan group in the antibody or the cytotoxic compound in dimethylformamide or other suitable solvent.

The xenobiotic compound is attached to the linker molecule through covalent bonds such as those present in the carbonyl, alkyl, thiocarbonyl, sulfonyl, carbamyl, sulfide, disulfide, alkoxy, or alkenyl groups, before or after the linker is covalently bonded to an antibody or fragment thereof by chemical reactions well known to the art. The linker group is attached to the antibodies through covalent bonds such as those present in sulfonyl, carbamyl, sulfide, disulfide, amino, carbonyl, alkyl, hydrazino, hydrazano, and alkene groups by chemical reactions well known to the art and in such a manner that the pharmacological activity of the xenobiotic composition is approximately the same as in the non-substituted form.

METHOD OF USE

The linked product is concentrated and prepared for use. The compounds can be administered by intravenous, intra- cavitary r intratumoral routes or they can be applied topically. In either case the product must be sufficiently soluble so as to allow injection, or in the case of topical application, to allow the product to permeate the skin. The dosage, in all cases, will vary with the size of the subject and the size and nature of the tumor, or the nature of the viral or parasitic infection.

EXAMPLE 1

Tubercidin Conjugated to a Monoclonal Antibody Via a Succinate Linker

A. Synthesis of Hemisuccinoyltubercidin Acetonide A solution of 3.2 g (0.001 moles) 2 ' , 3' -0- isopropylidenetubercidin [Pike, et al, J. Heterocyclic Chem. 1 , 159 (1964)] in 25 ml of dry pyridine is cooled in an ice bath and stirred as a solution of 10 g (0.001 moles) of succinic anhydride in 20 ml pyridine is added dropwise. The reaction mixture is stirred at 0° for 2 hours and then allowed to come to room temperature. The solution is diluted with water and extracted with choloroform. The extracts are dried and the solvent removed. The product is chromatographed on silica gel using 95% ethyl acetate, 95% ethanol, water (94:4:2) to give hemisuccinoyltubercidin -2', 3 ' - acetonide.

B. Synthesis of Hemisuccinoyltubercidin

A solution of the acetonide (2.0 g) in 10 ml trifluroacetic acid is allowed to stand for ten minutes, then the solvent is removed in vacuo. The residue is stirred with aqueous sodium bicarbonate solution, filtered, and treated with dilute hydrochloric acid to precipitate the solid product.

C. Conjugation of Hemisuccinoyltubercidin with Monoclonal Antibodies

(1) Monoclonal anti-carcinoembryonic antigen (CEA) antibody, 11.285.14, has been shown to have strong anti-CEA binding activity, low reactivity with non¬ specific cross reactive antigen and a high degree of gastrointestinal tumour selectivity in comparison with a wide range of normal tissues. It was isolated from ascites fluids using a Protein A affinity column. Ascites fluids were diluted with one volume of 0.1 M phosphate buffer pH 8.0, filtered and adsorbed onto Protein A- Agarose (Sigma Chemical Comp., Poole, England), equilibrated with the pH 8.0 buffer and maintained at 4^βC. Unadsorbed material was removed by washing with the pH 8.0 buffer, and bound immunoglobulin was then eluted using 0.1 M citrate/phosphate buffer pH 3.5. The eluates were neutralised, concentrated to ca. 25 mg/ml by ultrafiltration, and dialysed against 0.34 M borate buffer, pH 8.6.

Hemisuccinoyltubercidin (20 mg) is dissolved in 0.8 ml of dry dimethylformamide (DMF) and a mixture of 3.4 mg of N-hydroxysuccinimide (Sigma) and 9.9 mg of 1- cyclohexyl-3-(2- morpholinoethyl) - carbodiimide metho- p-toluene-sulphonate (Sigma) in 0.2 ml of dry DMF is added. After 48 h at room temperature, a 0.25 ml aliquot is added to a stirred 1 ml aliquot of a 20 mg/ml solution of CEA antibody in 0.34 borate buffer, pH 8.6. The conjugation reaction is allowed to proceed at room temperature for 4 h, after which time it is adjusted to pH 7.2, clarified by centrifugation if necessary, and chromatoqraphed on a 1.5 x 110 cm column of Sephadex G- 200 (Pharmacia Milton Keynes, England) (IgG conjugates) or Sephadex G-100 (Pharmacia) (Fab conjugates) equilibrated with 0.01 M phosphate, 0.15 M sodium chloride, pH 7.2 (PBS). Fractions corresponding to conjugate are combined. Drug and protein content of the conjugate may be determined by spectrophotometry at 270 and 280 nm, relating the absorbances obtained to predetermined extinction coefficients for free drug and unconjugated antibody at the two wavelengths.

(2) 13 mg hemisuccinoyltubercidin, 3.5 mg N- hydroxysuccinimide and 6.9 mg 1,3- dicyclohexylcarbodiimide are dissolved in 1 ml methylene chloride and stirred for 12 hours. The precipitated dicyclohexyl urea is filtered and the filtrate concentrated in vaσuo. This activated ester is dissolved in dry dimethylformamide and added to a solution of KS1/4 monoclonal antibody in 3 ml of 0.34 M sodium borate containing 25% DMF at 4°C. The supernatant liquid is applied to Sephadex G-50 gel and the conjugate eluted with phosphate buffered saline (pH 7.4).

EXAMPLE 2

Tubercidin Conjugated to a Monoclonal Antibody Via the "Cis-aconityl" Link

A) Cis-aconityltubercidin

12 mg of cis-aconitic anhydride in 3 ml dioxane is added dropwise to an ice-cold solution of tubercidin (12 mg) in 6 ml water while maintaining the pH at 9 by addition of 0.5N sodium hydroxide as required. After 15 minutes the pH is adjusted to 7 with 0.5N hydrochloric acid and the solution is held for 1 hour.

B) Conjugation of Aconityltubercidin with a Monoclonal Antibody To the above solution is added six milligrams of 1- ethyl-3-(3-dimethylaminopropyl carbodiimide) (Sigma) . After 30 min to it is added a solution of 6 mg of monoclonal antibody 9.2.27 at a concentration of 10 mg/ml in isotonic saline at pH 7. (This antibody recognizes melanoma-associated chondroitin sulfate proteoglycan expressed preferentially on the cell surface of human melanoma cells.) The reaction proceeds at pH 7.0 for 3 hr at 25°C. The immunoconjugates are purified with Sephadex G-25 Fine (Pharmacia) and dialyzed against isotonic saline at 4^βC. The immunoconjugate is lyophilized in the presence of 10% lactose and stored at -20°C until use. This conjugate remains stable at the intravascular pH of 7, but once endocytosed by the target cells the tubercidin is cleaved in the intralysosomal acid environment and enters into the cytoplasm.

EXAMPLE 3 Tubercidin Conjugated to a Monoclonal Antibody Via a Dextran Linker

Dextran T-40 is oxidized to polyaldehyde dextran (PAD) using sodium periodate, as described by Manabe, et al, Bioche . and Biophys. Res. Co m. 1009 (1983). 60 mg of the polyaldehyde is incubated with 20 mg of monoclonal antibody H-l, an anti-HLA IgG produced by a hybridoma cell line (Manabe, v.s.), in 10 ml of 0.15 M phosphate buffered saline. HLA are antigens of the major histocompatibility antigen system A, B and C. The condensation is allowed to proceed for 24 h at pH 7.2 at 4°C.

10 mg of tubercidin in 5 ml of 0.15M phosphate buffered saline is added and the solution stored for another 24 h. The Schiff bases thus formed may be purified by equilibration with 0.15M phosphate buffered saline on a Sephadex G-200 column.

Alternately, the Schiff base solution is reduced by treating for 2 h with 0.3 ml of a sodium borohydride solution containing 10 mg borohydride in 10 ml of the phosphate buffer. The resulting mixture may be purified over the Sephadex column as described above.

EXAMPLE 4

Tubercidin Conjugated to a Monoclonal Antibody Via Human Serum Albumin (HSA) as the Link HSA (Sigma Chemical Co., St. Louis, MO) (111 mg of protein/ml, 9 ml) was first freed of its dimer by gel filtration on Sephadex G-150 in PBS, and then reduced with lOmM dithiothreitol and dialyzed against PBS to obtain a preparation with 1.65 mole of thiol group per mole of HSA. The HSA was kept at 4"C to allow the thiol group:HSA molar ratio to decrease to 0.72 ( 6 days) . The thiol group was determined with 5, 5'-dithiobis(2- nitrobenzoic acid) .

To the resulting solution of HSA with free thiol groups (20.9 mg/ml, 1.0 ml) is added dropwise 104.4 mM of the tubercidin succinoyl activated ester prepared (as described in Example I,C) in DMF (0.2 ml). The solution is adjusted to pH 8.0 with 0.2 N NaOH, stirred at 4°C for 16 h, and dialyzed against PBS to obtain a tubercidin:HSA conjugate with free thiol groups.

Murine monoclonal antibody aMM46, prepared as in Endo, et al, Cancer Research, 47_, 1076 (1987) , is an antibody to an antigen on syngeneic ascitic C3H/He mouse mammary tumor MM46 cells. 74.3 mM of the maleimidosuccinyl derivative of this antibody in phosphate buffered saline is added to the solution above in the same buffer. The mixture is kept at 4°C for 18 hours. Any unreacted maleimide groups are blocked by treating the solution with 71 mM of L-cysteine in 30 ml of the buffer for 1 hour and then subjecting to gel filtration on a Sephadex G-150 column to give the conjugated product.

EXAMPLE 5

Evaluation of Tubercidin Conjugated to Monoclonal Antibodies

In vitro assays 1 1)) 110000 mmll ooff cells (1-5x10⁶/^~-l) are added to a 96-2311 flatbottom microtiter plate and incubated for 2-3 hours at 37^βC. Drug-antibody conjugates are filtered through a 0.2-mm Millipore filter to ensure sterility and dilutions are performed in sterile PBS; 50 ml of conjugate are added to the cells with the use of duplicate wells per sample; control wells receive 50 ml of medium or PBS and the cells are cultured at 37°C in a 7% CO- atmosphere for 24 hours.

2) 200 ml of cells (1-5x106/ml) are collected in sterile plastic centrifuge tubes, resuspended in sterile conjugate and mixed for 30 minutes at 37°C. The cells are centrifuged (1,500 rpm for 5 min) and then resuspended in growth medium; 100 ml of cells are then seeded into a microtiter plate with the use of duplicate wells per sample and incubated for 16-24 hours; duplicate samples are performed at each concentration chosen.

After the incubation period 50 ml of medium containing 1 mCi of [ 3H] thymi.dm. e (specifi.c act.vι.ty=15 Ci/mmol) is added and the plates are incubated for 3-6 hours; cells are then harvested onto a glass fiber filter paper with the use of a cell harvester and dried for 10 minutes at

80°C, and individual samples are separated and counted

3 . . . on a beta counter. Incorporation of [ H] thymidme is expressed as a percentage inhibition in incorporation of controls. (Growth inhibition is approximately similar to cell cytoxicity as measured by the in vitro uptake of trypan blue.) The standard error for any given point is generated by duplicate determinations.

In vivo experiments

In a survival study, tumor cells are injected into B6CF1 mice and 6 hours later a series of ip treatments are begun. The percentage survival of mice in each group is plotted as a function of time. In a tumor growth study, tumor cells are injected sc into the abdominal wall of the mouse and allowed to develop into palpable tumors measuring 0.5 cm in diameter before treatment is begun. Mice are then subjected to a series of ip treatments and the size of the tumors are measured daily with a caliper square; the data is recorded as mean tumor size (product of two diameters + standard error of the mean) .

EXAMPLE 6 Antitumor properties of tubercidin Cytotoxicity was determined according to the method of Smith, et al, Cancer Res. 19, 843 (1959) .

Tubercidin inhibited the growth of KB carcinoma cells 50% at a concentration of 0.0075 mg/ml (range 0.003 to 0.013 mg/ml in 33 separate assays). Tubercidin inhibition was determined at daily intervals by the effect of various concentrations of the growth of KB cells. The results show that cytotoxicity was observed only in the first 24-hour period with tubercidin concentrations of 0.005 mg/ml or fewer (Fig. 1). At drug concentration of 0.0075 mg/ml and 0.01 mg/ml, cytotoxic effects, as measured by inhibition of protein synthesis, were present at the end 48 hours; at drug concentrations of 0.015 mg/ml and greater, tubercidin completely inhibited KB cell multiplication over a 3-day period.

Two systems were used to measure cytotoxicity to mammalian cells in culture. (1) In the 3-day assay with KB human epidermoid carcinoma cells, cell growth was determined by measuring protein content after 3 days, incubation in the presence of the agents under study and the test substances were added at zero time. (2) A modified agar plate assay with KB cells similar to those reported by Miyamura S.A. Antibiotics and Chemotherapy 6:280-282 (1956) and S.A. DiPaolo and Moore, G.E. Antibiotics and Chemotherapy 9:497-500 (1959) was also used; dye reduction by resting cells in agar was the end point measured. In this test, compounds in solution were pipetted onto paper discs which were incubated overnight prior to the measurement of dye reduction. The effect of the ccmpounds on uptake of radioactive precursors on plasma, urine and other biological fluids were carried out with the KB assay.

The cytotoxicities of tubercidin and certain analogues to KB cells under growing and resting conditions are shown in Table 1.

In vivo antitumor activity was done according to the methods described by Stock, Current Res. Cancer Chemother. 3-55: 3-19 (1955). The result is shown in Table 2.

TABLE 1 Cytotoxicities of Compounds

KB Test System

* ug per 12.7 mm disc resulting in a 20 mm zone of inhibition.

SUBSTITUTESHEET TABLE 2 The in vivo antitumor activities of tubercidin*

*Based on tumor volume When druq was injected mtraperitoneally, in DMF-analine. "Grade" is set by Sloan-Kettering Institute.

SUBSTITUTESHEET

Claims

1. A composition comprising an antibody or fragment thereof capable of binding to a target cell and, attached thereto, a pharmacologically active xenobiotic compound selected from the group comprising tubercidin, tubercidin-5'-phosphate, the methyl ester of tubercidin, xylotubercidin toyoca ycin, sangivamycin, nogalamycin, nogalarol, nogalarose, porfiromycin, sparsomycin, pactamycin, streptozotocin, and analogues with substantially the same structure or xenobiotic activity.

2. The composition of claim 1 wherein said antibody is a monoclonal antibody.

3. The composition of claim 1 wherein said antibody or fragment and said xenobiotic compound are attached through a linker, said linker being covalently bound to said antibody or fragment and said xenobiotic compound.

4. The composition of claim 3 wherein said linker is covalently attached to the antibody or fragment thereof via a first substituent group, said first substituent being selected from the group comprising sulfide, disulfide, amino, carbonyl, alkyl, hydrazino, hydrazono, and alkene groups.

5. The composition of claim 3 wherein said linker is covalently attached to the xenobiotic compound via a second substituent bound to the xenobiotic compound, said second substituent being selected from the group comprising carbonyl, alkyl, thiocarbonyl, sulfide, disulfide, alkoxy, and carbenyl groups.

6. The composition of claim 3 wherein the linker

SUBSTITUTESHEET is non-cleavable by serum components.

7. The composition of claim 3 wherein the linker is cleavable by serum components.

8. The composition of claim 7 wherein the linker is cleavable by serum complement upon binding of the antibody or antibody fragment to the target cell.

9. A composition for treatment of tumors comprising a monoclonal antibody or fragment thereof capable of binding to a tumor and, attached thereto, a pharmaceutically active amount of tubercidin.

10. A process for preparation of an anti-tumor composition comprising attaching a xenobiotic compound selected from the group comprising tubercidin, tubercidin-5^/-phosphate, methyl ester of tubercidin, toyocamycin, sangivamycin, xylotubercidin, nogalamycin, nogalarol, nogalarose, porfiromycin, sparsomycin, pactomycin, streptozotocin, and analogues with substantially the same structure or xenobiotic activity, to a linker molecule through the covalent attachment of a carbonyl, alkyl, thiocarbonyl, sulfide, disulfide, alkoxy, or alkene group before or after the linker is covalently bonded to an antibody or fragment thereof through sulfonyl, carbanyl, sulfide, disulfide, amino, carbonyl, alkyl, hydrazino, hydrazono, or carbene groups, wherein the binding capability of said antibody or fragment and the pharmacological activity of the xenobiotic composition are retained.

11. The process of claim 10 wherein said linker is non-cleavable by serum components.

12. The process of claim 10 wherein said linker is cleavable by serum components.

13. The process of claim 12 wherein said linker is cleavable by serum complement upon attachment of said antibody to the target cell.

14. A method for in vivo delivery of a compound to a target cell comprising administering an effective dose of a composition of sufficient solubility to be suitable for in vivo administration comprising an antibody or fragment thereof capable of binding the target cell and, attached thereto, a pharmacologically active xenobiotic compound selected from the group comprising tubercidin, tubercidin-5^/-phosphate, the methyl ester of tubercidin, toyocamycin, sangivamycin, xylotubercidin, nogalamycin, nogalarol, nogalarose, porfiromycin, sparsomycin, pactomycin, streptozotocin, and analogues having the same structure or xenobiotic activity.

15. The method of claim 14 wherein said administration is by topical application.

16. The method of claim 14 wherein said administration is by intravenous, intracavitary, or intratumoral routes.

17. A method for treating tumors comprising administering an effective dose of a composition of sufficient solubility to be suitable for in vivo administration comprising an antibody or fragment thereof capable of binding the target cell and, attached thereto, a pharmacologically active xenobiotic compound selected from the group comprising tubercidin, tubercidin-5^/-phosphate, the methyl ester of tubercidin, toyocamycin, sangivamycin, xylotubercidin, nogalamycin, nogalarol, nogalarose, porfiromycin, sparsomycin, pactomycin, streptozotocin, and analogues having the same structure or xenobiotic activity.

18. The method of claim 17 wherein said antibody is a monoclonal antibody and said xenobiotic compound is tubercidin.

SUBSTITUTE SHEET