WO1992014758A1

WO1992014758A1 - Conjugate molecules

Info

Publication number: WO1992014758A1
Application number: PCT/AU1992/000047
Authority: WO
Inventors: Geoffrey Pietersz
Original assignee: The Austin Research Institute
Priority date: 1991-02-13
Filing date: 1992-02-13
Publication date: 1992-09-03

Abstract

Nucleoside-polypeptide conjugates, wherein the polypeptide is selected from an antibody or antigen binding fragment of an antibody, hormone, growth factor or biologically active peptide, characterised in that the nucleoside is coupled to the polypeptide through a 3' ester linkage are described. Nucleoside-polypeptide conjugates may be used in the treatment of tumours or viral diseases.

Description

CONJUGATE MOLECULES

This invention relates to conjugate molecules, particularly conjugates between drug molecules and polypeptides, and methods involving the same. Most cytotoxic drugs used clinically have little selective toxic effect on tumours and although extensive structural modification has resulted in analogues with improved antitumour activity, dose limiting toxic side effects are still a major problem. The same problems exist for antiviral agents, particularly those which interfere with DNA or RNA synthesis.

The targeting of cytotoxic agents to tumours by covalently linking them to monoclonal antibodies confers a degree of specificity not found with the drugs alone. The specificity of the monoclonal antibody (MoAb), together with the internalization and subsequent release of the coupled drug can lead to the selective killing of tumour cells expressing the antigen. However, the conjugation of a drug to monoclonal antibodies usually results in some loss of both drug and antibody activity. This, in combination with the relatively inefficient uptake of drug antibody conjugates, as a result of limited tumour access, has prompted the use of more potent drugs where relatively small doses inhibit tumour growth.

5-Fluorouracil (5-FU) is used in the treatment of carcinoma of the breast and intestine, but its use is limited by toxicity to bone marrow and intestinal epithelium. This drug is metabolized by several enzymes and transformed into toxic species which interfere with several metabolic pathways. 5-FU enters the pyrimidine biosynthetic pathway at the orotic acid step, to form 5- fluorouridine diphosphate which can be incorporated into RNA. 5-Fluorouridine diphosphate is transformed to 2'- deoxy-5-fluorouridinemonophosphate which inhibits thymidylate synthetase resulting in the inhibition of DNA synthesis. 2-Deoxy-5-fluorouridine (5-FUdr) is a more potent derivative of 5-fluorouracil. Other nucleoside drugs, such as bromodeoxyuridine and azidothymidine, are widely used in the treatment of tumours and viral infection. The effectiveness of these drugs is also somewhat limited due to their toxicity.

The coupling of nucleoside drug molecules to antibodies usually results in some loss of both drug and antibody activity. .This is particularly the case for nucleoside drugs, such as 2'-deoxy-5-fluorouridine (5- FUdr), which contains a sugar moiety (pentose), and a nitrogenous heterocyclic base. In such molecules, the sugar moiety is often oxidised in coupling reactions with antibodies, causing a loss in drug activity.

Similar considerations are also involved in coupling drugs to other proteins apart from antibodies, such as hormones (for example EGF) and growth factors.

In accordance with a first aspect of this invention there is provided a nucleoside-polypeptide conjugate, wherein said polypeptide is selected from an antibody or fragment thereof, growth factor, hormone or biologically active peptide; characterised in that the nucleoside is coupled to said polypeptide through a 3' ester linkage. The coupling takes place via an active ester, giving an ester linkage at the 3' position of the nucleoside and an amide linkage with the polypeptide. The precise chemical structure of the active ester employed in the conjugation is not of importance, and may be derived, from N- hydroxysuccinimide, N-hydroxysulphosuccinimide, 1- hydroxybenzotriazole, pentafluorophenol, nitrophenol, and other active esters as are well known in the art.

Preferably, said conjugate is represented by the formula:

X-NHCO-RCOO-Nu I where:

X is a monoclonal or polyclonal antibody or antigen binding fragment thereof; hormone; growth factor or other biologically active peptide; Nu is a nucleoside drug attached to the group RCOO through the 3' carbon of the sugar moiety; R is Cι_i5 alkyl optionally substituted with halogen (I, Br, Cl, F), hydroxy, cyano, phenyl, amino, carboxy, alkyl, alkoxy and the like; said alkyl chain optionally being interrupted by one or more groups selected from -0-, -S-, NH, -CH=CH, phenyl, -S0₂- and C3_s cycloalkyl; aryl or C^_₁5 aralkyl, such as phenyl, cumenyl, mesityl, tolyl, xylyl, benzyl, phenethyl, and fused polycyclic hydrocarbons, optionally substituted with halogen, hydroxy, amino, cyano and the like; or a heterocyclic compound (containing one or more of N, S, 0 and P) of 3 to 8 members, such as furan, pyran, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, morpholine, and the like, optionally substituted with halogen, hydroxy, amino, cyano and the like.

The nature of the group R is not essential to this invention as this group acts as a spacer between the ester and amide linkages of compounds of the formula I. Hormones which may form part of the conjugates of this invention include calcitonin, thyrotropin, melanotropin, insulin, gonadotropin and relaxin. Growth factors may include EGF, TGFα and β, IGF and FGF. Cytokines which may form part of the conjugates of this invention include colony stimulating factors (such as G- CSF and GM-CSF), interleukins and other cytokines as are well known in the art.

Preferably, Nu is 5-FUdr or a derivative thereof, bromodeoxyuridine, lododeoxyuridine, bromovinyluridine, azidothymidine (AZT), or any other nucleoside drug as are well known in the art (Merck Index, 11th Edition, Merck & Co., Inc., 1989). By derivative of 5-FUdr is meant substitutional variants thereof, wherein: i) The F substitution at position 5 on the nitrogenous base is substituted with I, Cl or Br; ii) one or more of the carbon groups of the sugar moiety or nitrogenous base are substituted with halogen (F, I, Br, Cl), hydroxy, C1-C3 lower alkyl or the like; and/or iii) one or more of the carbon atoms in the sugar moiety, and nitrogen and carbon atoms in the base moiety are replaced with different atoms selected from S, 0, N or C. The group R as defined above may be straight or branched chain alkyl, aryl, or a heterocyclic group, and may be unsaturated, saturated, or contain one or more single or double bonds. The alkyl chain may be optionally substituted with halogen (I, Br, Cl or F), hydroxy, cyano, phenyl, amino, carboxy, alkyl, alkoxy or other substituents as are well known in the art. The alkyl chain may be interrupted with a heteroatom (such as 0, N, S or P) which may be optionally substituted with substituents as are well known in the art.

As previously mentioned, antibodies may be polyclonal or monoclonal tumour reactive antibodies. Monoclonal antibodies are preferred, particularly in the light of their high degree of antigen specificity. As used reference to antibodies includes fragments of antibodies containing an antigen binding site such as Fab, or F(ab')2 fragments, chimeric antibodies, humanized antibodies (derived from an animal antibody, such as a murine antibody, wherein human amino acid sequences replace animal sequences), Fv antibodies, single chain antigen binding proteins, Dab, or any antibody fragment or derivative capable of binding antigen. Polypeptides are generally coupled to nucleoside drugs such as 5-FUdr through nucleophilic groups of amino acids, particularly the amino group of lysine.

Polypeptide conjugates in accordance with this invention generally comprise 1 or more FUdr molecule conjugated to each polypeptide. For example, conjugates may contain from 1 to 100 molecules of 5-FUdr per antibody molecule.

The extent of "labelling" of a polypeptide, such as an antibody molecule, would generally depend on factors such as the excess of nucleoside/nucleotide drug employed in the coupling reaction, and other reaction conditions as described hereinafter.

By virtue of the antigen binding specificity provided by antibodies or antigen binding fragments thereof, the antibody conjugates of this invention are capable of binding to selected targets, such as tumours, for example breast tumours or colon tumours, or virally infected cells. Any tissue or cell type expressing antigenic determinants reactive with the antibodies, which form part of the conjugate of this invention, may be selectively killed through action of the nucleotide/- nucleoside drug molecule. Similarly, conjugation of nucleoside drugs to hormones, growth factors and cytokines allows specific targeting to sites having receptors or affinity for these reagents.

In accordance with a further aspect of this invention there is provided a method for the production of nucleoside-polypeptide conjugates which comprises reacting an active ester of said nucleoside (linked through the 3' carbon) drug with a polypeptide. By 3' active ester is meant an ester with an easily displaceable (under nucleophilic attack) OR group, where R is as previously defined. Active esters may, for example, be derived from N-hydroxysuccinimide, N- hydroxysulphosuccinimide, 1-hydroxybenzotriazole, pentafluorophenol, nitrophenol and the like. The polypeptide couples to the active ester through nucleophilic groups such as the amino group of lysine.

Active esters of nucleoside drugs generally comprise the formula LCO-R-COO-Nu; wherein:

L is any leaving group, such as nitrophenol, N- hydroxysuccinimide, chloro, fluoro, hydroxysuccinimide, N-hydroxysulphosuccinimide, 1-hydroxybenzotriazole, pentafluorophenol, nitrophenol and the like; and R and Nu are as previously defined.

The reaction conditions between the activated ester and polypeptide, such as an antibody, are generally unimportant, and may for example, be carried out at temperatures between about 4°C an ambient temperature (such as 17-25°C), or higher temperatures as desired, although such temperatures are generally below about 60"C. The active ester is generally in excess to enable multiple labelling of each polypeptide molecule. Preferably said nucleoside drug is AZT or 5-FUdr, or a derivative thereof.

In accordance with a further aspect of this invention, there is provided an active ester of a nucleoside/nucleotide drug of the formula LCO-R-COO-Nu; wherein: L, R and Nu are as previously defined. In accordance with a yet further aspect of this invention, there is provided a method for the treatment of tumours or viral diseases, which comprises administering to a subject in need of such treatment an effective amount of a conjugate as hereinbefore defined optionally in association with one or more pharmaceutically acceptable carriers or excipients. In this aspect of the invention, the polypeptide portion of the conjugate would be selected to be reactive with tumour cells or virally infected cells.

Tumours which may be treated in accordance with this invention include lymphoma, Hodgkin's disease, leukemia, melanoma, endothelial carcinomas, adenocarcinomas, and cancers of the breast, lung, liver, pancreas, prostate, ovary, stomach, kidney, testes, head and neck.

Viral diseases (i.e. cells infected with virus) which may be treated in accordance with this aspect of the invention include HIV, herpes virus, hepatitis A-E virus, cytomegalovirus and papilloma virus.

The specificity provided by the polypeptide portion of the conjugates of this invention (such as where the polypeptide portion is an antibody or fragment thereof having antigen binding activity, or a hormone or growth factor) enables precise targeting of the nucleoside drug to its site of action. It is believed that the conjugates of this invention would be internalised into cells on binding to cellular receptors, and the nucleoside drug component liberated from the conjugate within the cell, such as in lysosomes. The nucleoside drug would then be free to act as a cytotoxic agent. Antibodies or antigen binding fragments thereof which may be used in this invention include antibodies reactive with MUC 1-3, CEA, CD3, CD4, CD5, CD7, CD8, CDll, CD18, CD19, CD25, EGF receptor, and the transferrin receptor (which antibodies are described in the 4th International Workshop on Lymphocyte Antigens citation reguire ) .

In accordance with a still further aspect of this invention, there is provided a method for the treatment of autoimmune disease or tissue rejection associated with transplantation, which comprises administering to a subject in need of such treatment an effective amount of a conjugate as hereinbefore defied optionally in association with one or more pharmaceutically acceptable carriers or excipients. Autoimmune diseases which may be treated in accordance with this aspect of the invention include rheumatoid arthritis, SLE, biliary cirrhosis, chronic active hepatitis and the like.

In respect of the treatment of transplantation rejection and autoimmune disease, conjugates of this invention are generally directed against lymphocytes as lymphocytes play a key role in tissue rejection.

In a still further aspect of this invention, there is provided a method for clearing bone marrow of tumour cells, which comprises treating bone marrow removed from a patient with a conjugate as previously described so as to kill tumour cells. This aspect of this invention is particularly relevant to cancer therapy where bone marrow is removed from a patient suffering from cancer and the patient treated with cytotoxic reagents, such as chemotherapeutic reagents and/or radiation treatment. Treatment of this type generally destroys a patient's bone marrow which is then supplemented with the removed bone marrow. However, such bone marrow may contain resident tumour cells, which can be removed using the conjugates of this invention. In this respect, the conjugates of this invention would be targeted against tumour cells such as utilising antibodies directed to tumour cell antigens. Conjugates may be administered to patients by any convenient mode of administration, such as subcutaneous, intramuscular, intratumour, or intravenous injection; intravenous drip; transdermal delivery system; orally, parenterally and the like.

The term "effective amount" refers to the amount of conjugate of this invention which is effective to ameliorate or treat tumourogenic, viral or other disease. What constitutes an effective amount will depend upon the nature and site of the tumour or viral disease being treated, physiological status of the patient, and judgement of the prescribing physician. In general terms, an effective amount of conjugate will comprise from .001 micrograms to 10 grams. A preferred dosage range is between 1 μg/kg to 100 mg/kg body weight.

The conjugate of this invention may be in the form of a sterile injectable form; tabletted for oral or rectal administration; in the form of a syrup or other aqueous solution of suspension; or in the form of a cream for tropical administration. In each of the aforementioned cases, the conjugate of this invention is generally combined with one or more pharmaceutically acceptable carriers or excipients, such as saline, glycerol, physiological buffers having a pKa around 7 to 8; magnesium stearate; and/or other well known excipients/carriers to give a pharmaceutically acceptable composition (see Remingtons Pharmaceutical Sciences, Osol. et al., 17th Edition, Mack Publishing Co., Eastern Pennsylvania, which is incorporated herein by reference).

Pharmaceutical compositions comprising the conjugates of this invention may contain protease inhibitors, and antibacterial agents. Dispersants may also be provided to prevent aggregation, as may albumin or other protein sources to guard against proteolysis.

This invention will now be described with reference to the following non-limiting examples which focus on 2'- deoxy-5-fluoro-3'-O-succinoyluridine conjugates to monoclonal antibodies. These results are merely illustrative of the broad scope of this invention.

It is evident that the active ester 2'-deoxy-5- fluoro-3'-O-succinoyluridine may be substituted with any other active ester of a nucleotide/nucleoside drug having anti-tumour/antiviral properties. Similarly, monoclonal antibodies may be clearly substituted with hormones, growth factors, or other polypeptides.

The succinyl active ester is exemplary of a wide range of ester groupings as defined herein.

Insofar as antibodies are concerned, monoclonal antibodies are particularly exemplified. Clearly, such antibodies may be substituted with polyclonal antibodies, human antibodies, humanised antibodies, or fragments of antibodies having antigen binding capacity.

The following abbreviations are used herein:

5-FU 5-Fluorouracil 5-FUdr 2'-Deoxy-5-fluorouridine MoAb(s) Monoclonal antibody(ies) 5-FUdr-succ 2'-Deoxy-5-fluoro-3'-O-succinoyluridine

I^C ₅₀ 50% inhibition of [^H]-deoxyuridine incorporation

E3 ITT(1)75NS E3

DMTr-Cl 4,4'-Dimethoxytriphenylmethyl chloride

DMAP N,N-Dimethylaminopyridine

NHS N-Hydroxysuccinimide

DOC Dicyclohexylcarbodiimide

PBS Phosphate buffered saline

TLC Thin layer chromatography

BW BW5147 0U-

TABLE 1 Effect of 5-FUdr and 5-FUdr-succ on tumour cells

Tumour cells mean IC50 mean IC50 m meeaann I iCL,ςn FUdr-succ 5-FUdr-MOAb

E3 5. 1xl0^{- 10}M( 3 ) 5 .2xl0^_9M( 6 ) 6 .0xlO^{- 9}M( 30 ) BW5147 1.4xl0⁹M( 2 ) 5 .3xlO^_9M( 2 ) 2. 4xlO^_8M( 6 )

(in brackets: No. of individual values) EXAMPLE 1

SYNTHESIS OF 5-FUdr SUCCINYL DERIVATIVES:

2'-Deoxy-5'-)-(4, '-dimethoxytriphenylmethyl)-5- fluorouridine (2): 2'-Deoxy-5-fluorouridine (1) (147.5 mg, 0.6 mmol) was evaporated three times with 2 ml of pyridine and then redissolved in 2 ml of pyridine. 4.4'-

Dimethoxytriphenylmethyl chloride (304.5mg, 0.9 mmol) in pyridine (2 ml) was added dropwise while cooling and stirring and the reaction mixture was stirred at room temperature for 16 hr. The solution was poured into 10 ml of ice containing 5% NaHCO , extracted with dichloromethane, dried over anhydrous Na2S0 and evaporated to give 340 mg of crude product. Column chromatography on silica gel (40 g) using CH2CI2 - MeOH, 13:1 as eluant yielded 223.6 mg (68%) of (2): TLC CH₂C1₂- MeOH, 13:1 R_f 0.25. ¹H-NMR (CDCI3):62.26 and 2.48 (2m, 2H, 2'-H₂), 3.42 (m, 2H, 5'-H₂), 3.79 (s, 6H, OCH3), 4.06 (dt, J4,5=J3,4=4Hz, 1H, 4'-H), 4.53-4.57 (m, 1H, 3' -H), 6.29 (m, 1H, 1-H'), 6.85 and 7.28 (2m, 13H, arom. H), 7.83 (d, J=9Hz, 1H, 6H).

2'-Deoxy-5'-O-(4,4'dimethoxytriphenylmethyl)-5-fluoro-3'- O-succinoyluridine (3): 2'-Deoxy-(4,4'-dimethoxytriphenylmethyl)-5- fluorouridine (2) (191.8 mg, 0.35 mmol) was dissolved in dichloromethane (3ml) and after the addition of N,N- dimethylaminopyridine (64.1mg, 0.525 mmol, 1.5eq) and succinic anhydride (140mg, 1.4 mmol, 4eq) the mixture was kept at room temperature for 16 hr and then poured into 10 ml 5% NaHC0₃, acidified with IN HCl to pH3 and extracted several times with dichloromethane. The extract was dried and evaporated to give 132.4 mg (77%) of (3); TLC 90% aqueous CH3CN, Rf 0.68.

2'-Deoxy-5-fluoro-3'-O-succinoyluridine (4) :

2'-Deoxy-(4,4'-dimethoxytriphenylmethyl)-5-fluoro- 3'-O-succinoyluridine (3) (132.4mg, 0.204 mmol) was dissolved in 80% aqueous acetic acid (2ml) and left at room temperature for 2-3 hours. The reaction mixture was diluted with water and extracted with chloroform. The aqueous solution was lyophilized and the residue passed through a column of 5g silica gel with 70% aqueous ethanol. Evaporation of the solvent gave 61 mg (86%) of (4) as a white hygroscopic powder. TLC 70% aqueous EtOh R_f 0.80; ^-H-NMR (D₂0):δl.8 - 2.5 (m, 6H, succ-CH₂, 2'- H₂), 3.52-3.70, 3.95-4.05, (2m, 5'-H₂, 3'-H, 4'-H), 5.09 (m, IH, 3'-H), 6.07 (m, IH, 1-H), 7.84 (d, J=8Hz, IH, 6- H).

EXAMPLE 2

PRODUCTION OF ANTIBODY CONJUGATES: Monoclonal Antibodies:

Anti-Ly-2.1 (lgG_2a) reactive with the murine Ly-2.1 antigen was isolated from murine ascites fluid. The ascites was diluted in phosphate buffered saline (5mM, 0.17M NaCl, pH 7.2) (PBS) and adsorbed onto Protein A- Sepharose (Pharmacia Inc., Piscataway, NY), washed extensively with PBS and eluted with 0.2M citrate buffer (pH=4.0). Following neutralization the MoAb was concentrated by precipitation with 45% aqueous (NH4)₂S04, dissolved in PBS and aliquots (5mg/ml) were stored at - 70^βC. When tested using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) this MoAb preparation was 90-95% pure.

Preparation and Quantitation of Conjugates: The active ester derivative of 2'-deoxy-5-fluoro-3'- O-succinoyluridine (5-FUdr-succ) (4) was prepared by dissolving 5-FUdr-succ (1.73 mg, 5 mmol) in 70 μl of dry DMF. N-Hydroxysuccinimide (0.59 mg, 5.1 mmol) in 17 μl of DMF and dicyclohexylcarbodiimide (6.18 mg, 30 mmol) in 50μl of DMF were added and the reaction mixture was kept at room temperature for 3 hr and at 4°C overnight to give the active ester. An aliquot of the active ester was mixed with the MoAb 3-5 mg/ml in PBS and maintained at room temperature for 3 hr and then centrifugated (400g x 3 min) to remove any precipitate. The conjugate was purified by gel filtration chromatography using a- Sephadex G-25 column (PD-10, Pharmacia ) , with PBS as eluant.

Antibody concentration was determined by using a standardized amount of ¹²⁵I-labelled antibody and the amount of FUdr-succ bound by absorbance spectrometry at 260nm using the extinction coefficients e₂6o⁼⁷600 ^{M_1 cm~} ¹ (FUdr-succ) and 6₂₆₀=129300 M~¹cm^"1 (MoAb). Analysis of these conjugates using SDS-PAGE or gel permeation chromatography (TSK-SW2000, Pharmacia) showed less than 5% aggregated antibody. The optimal reaction conditions were found to be l.leq NHS and 6eq dicyclohexylcarbodiimide (DCC). We were unable to crystallize the active ester and it was therefore used without further purification. To optimise coupling, different molar excesses of 5-FUdr-succ active ester were added to MoAb to yield conjugates with different degrees of substitution. It was found that the addition of a 20 fold molar excess of the active ester of (4) led to an incorporation of 9 molecules of 5-FUdr per antibody molecule with a protein recovery of 80%; a 60 fold excess yielded 21 incorporated 5-FUdr molecules (residues) with a 72% recovery of protein. The conjugates that were tested further in vitro had between 5 and 20 molecules of 5-FUdr incorporated per molecule of MoAb.

To determine the stability of the 5-FUdr-MoAb conjugate it was kept in phosphate buffered saline at 4°C and was passed through a Sephadex G-25 column to remove any dissociated 5-FUdr-succ and requantitated after 2 and 7 days; no loss of drug occurred.

5-FUdr (1) had to be chemically modified for conjugation and this was done by the introduction of a carboxyl group by succinylation of 5-FUdr. However, to obtain a single product it was necessary to protect one of the two available hydroxyl groups of (1) prior to succinylation. By protecting of the 5'-position with a 4,4*-dimethoxytriphenylmethyl group, succinylation of the 3'-hydroxyl group occurred and deprotection gave the 3'- isomer as the only product, with a yield of 45% after the 3 steps. Subsequently, the carboxyl group was activated by forming an ester with N-hydroxysuccinimide for coupling with monoclonal antibody. Conjugates were obtained with protein recoveries in excess of 70%, even when up to 40 residues of 5-FUdr were coupled to MoAb. This significant protein recovery may well be due to the high hydrophilicity of the 5-FUdr-succ (4) which prevents precipitation of the antibody due to a loss of charged amino groups on the antibody. Thus, highly conjugated antibodies were obtained.

EXAMPLE 3

ANTIBODY CONJUGATE CHARACTERISATION AND ACTIVITY IN-VITRO

(I) Stability of Conjugate in Growth Medium: To determine the stability of 5-FUdr-anti-Ly-2.1 in growth medium, a conjugate prepared using radiolabelled anti-Ly-2.1 was mixed with an equal volume of feotal calf serum or PBS and incubated at 37°C for 24 hrs. A similar incubation was performed with radiolabelled anti-Ly-2.1. After incubation, the samples were passed through a PD-10 column to remove any free drug and the peak fractions were pooled and counted for radioactivity. The concentration of antibody was determined from the known specific activity of ¹²⁵I-labelled anti-Ly=2.1 and the cytotoxicity of the peak fraction was measured using the [^H]-deoxyuridine assay as described hereinafter.

The IC50 of the conjugate in terms of antibody concentration was 2.5x10""* mg/ml on day 1. The IC50 of the conjugate after 24 hr incubation without serum was identical to the initial IC50. However, when the conjugate was incubated with serum the IC50 was increased to 4x10"* mg/ml. Therefore, in the presence of feotal calf serum the cytotoxicity of the conjugate was reduced 1.6 fold indicating a loss of some 5-FUdr residues.

(II) Biological Assays: Cell lines used for biological assay were the E3 variant of the Ly-2.1^+ve positive murine thymoma ITT(1)75NS, and the Ly-2.1^_ve BW5147 OU^" cells (BW). The cells were maintained in vitro in Dulbecco's modified Eagles medium supplemented with 10% heat-inactivated newborn calf serum (Flow Laboratories (CSL), Sydney, Australia), 2mM glutamine (Commonwealth Serum Laboratories, Sydney, Australia), lOOμg/ml streptomycin (Glaxo, Melbourne, Australia) and lOOlU/ml penicillin (CSL).

Cytotoxicity Assays:

(a) lOOμl of cells (2 x 10^ cells) were added to a flat bottom microtiter plate and incubated for lhr at

37°C. Free drug in PBS and conjugate solutions were filtered (0.22μM) to sterilize and various dilutions were made in PBS and lOOμl added to the cells. Control wells received lOOμl of PBS. The plates were then kept at 37°C, 7% C0₂ for 24hr, and harvested (see below).

(b) 200μl of cells (2 x 10⁵ cells, E3 and BW) were added to sterile tubes and lOOμl of various dilutions of conjugate or free drug in PBS were added and the mixtures kept at room temperature for 30 min; 500μl of growth medium was then added and the cells centrifuged (400g x 5 min). After resuspending in 200μl of medium lOOμl aliquots were added to a microtiter plate and incubated at 37°C, 7% C0₂ for 16hr.

After incubation 50μl of growth medium containing lμCi of [^Hj-deoxyuridine (specific activity, 21 Ci/mmol; Amersham, England) was added. After incubation for 4h, the cells were harvested using a Dynatech automash cell harvester (Dynatech, England) onto glass fibre filter, dried, and samples counted on a beta scintillation counter (Packard Instrument Company, IL). The viability of the cells was expressed as a percentage inhibition of [^H]-deoxyuridine incorporation compared to the controls (we have previously shown that [^Hj-deoxyuridine uptake is equivalent to cell viability using a dye exclusion assay (unpublished results). Duplicates did not show more than 10% variation.

Antibody Activity of 5-FUdr-MoAb Conjugates: The antibody activity of the immunoconjugates was quantitated by flow cytometry after cytotoxic assay (a) or (b). The binding of the conjugates to Ly-2.1^+ve ITT(1)75NS E3 (E3) cells was measured by fluorescence of the cells after incubating with immunoconjugate, washing and exposure to fluorescein labelled rabbit-anti-mouse-antibody. The concentration of the immunoconjugate giving 50% of maximum fluorescence was calculated and compared with unconjugated MoAb to give a relative activity of conjugated to non-conjugated antibody. It was found that low numbers of 5-FUdr residues (1-10) slightly reduced the antibody activity (conjugates were 1.5-2 fold less active than the unconjugated antibody) while higher numbers of residues led to a further reduction (10-25 residues, 3-5 fold less active).

The antibody activity of conjugates were also measured using a competition assay. The antibody activity of conjugate measured using the competition assay was similar to the activity measured using the direct binding assay.

Cytotoxicity of 5-FUdr-MoAb Conjugates In Vitro:

The in vitro cytotoxicity of (1) and 5-FUdr-succ (4) on the murine Ly-2.1^+ve E3 cell line and the Ly-2.1^_ve BW cell line was measured in an inhibition assay and IC50 values determined (Table 1). On E3 the cytotoxicity of 5-FUdr-succ (4) (IC50 = 5.2nM) was 10 fold less than 5- FUdr (1) (IC50 = 0.51nM), suggesting that succinylation of the 3' group of 5-FUdr (1) results in some loss of activity, possibly due to less efficient uptake into the cell by the nucleoside transport system. On BW a 4 fold loss of activity was observed. It was also noted that E3 was 3 fold more sensitive to 5-FUdr than BW. However, the sensitivity of both cell lines to 5-FU-succ (4) was the same so that data using the immunoconjugates can be compared. The cytotoxicity of anti-Ly-2.1 conjugates with 2-20 molecules of 5-FUdr bound per molecule antibody were tested (Table 1) on Ly-2.1^+ve E3 and Ly-2.1^_ve BW cell lines where it was found that the IC50 values on E3 were in the range of 5.0 - 9.0 x 10"⁹M (average of 6.0xl0^"9M from 30 individual values), while the IC50 on the BW cell line was 2 - 6xlO^"8M (average of 2.4xlO^_8M). These results, indicate that 5-FUdr-MoAb is 4 times more active on the Ly-2^+ve cell line than on the Ly-2"^ve cell line. In both cases, no significant differences were found when using 5-FUdr-anti-Ly-2.1 conjugates with different degrees of 5-FUdr substitution. This data shows that 5-FUdr-anti-Ly-2.1 conjugates are 12 times less toxic than 5-FUdr (1) on the E3 cell line but 'of similar toxicity to FU-dr-succ (4).

5-FUdr-succ-MoAb containing 11, 19 or 26 drug residues showed no significant variation of their IC50 values in a cytotoxicity assay based on the concentration of 5-FUdr-succ (4) and on average were 12 times less toxic than 5-FUdr (1) (Table 1).

This loss in drug activity is mainly due to chemical modification as the IC50 value for the 5-FUdr-anti-Ly-2.1 is similar to that of 5-FUdr-succ (4) indicating that coupling to MoAb did not alter the cytotoxicity of the free drug.

Specificity In Vitro:

To show that the cytotoxicity of the conjugates was due to specific targeting by the antibody, a cytotoxicity assay was carried out using both Ly-2.1+ve _E3 _cen_{s ancj} Ly-2.1^~ve BW cells. In this assay conjugates were exposed to antibody reactive (E3) and antibody non- reactive (BW) cells for 30 min and washed and then incubated for 24 h and pulsed with [^H]-deoxyuridine. The IC50 for the anti-Ly-2.1 conjugate on E3 was 7.1x20^" ⁸M while the IC50 on BW was >5.01xlO"⁷M and indicates that 5-FUdr-anti-Ly-2.1 conjugates are selectively toxic to Ly-2.1^+ve cells. The exact mechanism of action of these 5-FUdr-MoAb conjugates is not known. Several possible mechanisms can be suggested, two of which are: i) the conjugates bind to the antigen, are internalised and digested in the lysosomes to release free drug, and ii) esterases present in the lysosome could hydrolyze the ester bond between the 5-FUdr (1) and the succinyl group to release 5-FUdr which is likely to be metabolized to produce the active metabolites. However, the exact mechanism is unknown.

EXAMPLE 4

ANTIBODY CONJUGATE ACTIVITY IN-V VO

In Vivo Tumour Growth Studies: ITT(1)75NS E3 or Colo 205 cells were injected s.c. into the abdominal wall of CBF1 or swiss nude mice, respectively. Treatment (i.p. or i.v. ) was begun once palpable tumours (0.4cm² - 0.6cm²) developed. Liml899 tumour implants of 0.1cm² were transplanted s.c. into the right flank of nude mice. Treatment was begun once tumours were established and growth was evident. Daily measurements with a calliper square measuring along two perpendicular axes of the tumour were carried out and data recorded as mean tumour size (cm²) ± Standard Error (SE). Groups of 6-8 mice of the same age and sex were used within an experiment. The three mouse models used within this study were; the E3 tumour growing in CBF_j mice which is a congenic tumour model, and Colo 205 and Liml899 which are human tumours grown in nude mice.

In vivo Activity: ITT(1)75NS E3 Tumour Model:

Groups of 8 CBF-L mice injected s.c. with 5xl0⁶ E3 cells, developed tumours 7 days after inoculation and were injected i.p. with increasing doses of 5-FUdr-succ conjugated to anti-Ly 2.1 (48 μg, 70 μg, 85μg or 100 μg). The highest dose administered, lOOμg conjugated 5-FUdr- succ, showed marked tumour inhibition, where tumours were 15.0% of control tumours on day 15 and 100% survival of mice was seen within this group. However, minor weight loss and some diarrhoea was observed. 50% tumour inhibition, compared with control tumours, was produced by the lowest dose of conjugated 5-FUdr-succ (48μg) administered, by day 15. This demonstrates the tumour inhibiting potential of these conjugates at low doses. The immuno-conjugates are potent inhibitors of tumour growth. 5-FUdr-succ in an unconjugated form produced no tumour inhibition at all. Increased dosages of 5-FUdr-succ conjugates produced increased tumour inhibition with minor signs of systemic toxicity in the mice at the highest dose only. Few drugs or drug conjugates have shown such tumour inhibiting potential at such low quantities.

Initial in vivo experiments where sequential doses of 5-FUdr-succ conjugates were administered (data not shown) resulted in death of some mice, at a total dose of 120μg of FUdr succ, administered over 6 days. It was therefore necessary to establish the appropriate dose schedule for the most effective treatment of tumours without producing toxicity. Initially two protocols were studied, (a) low dose (total dose administered 30μg) and (b) high dose (total dose administered 71.73μg) of 5-FUdr-succ conjugated to anti-Ly-2.1. Five days after tumour inoculation of 5x10^ E3 cell s.c, i.v. administration of drug conjugates was begun, where mice received five sequential doses of conjugates. By day 13, significant tumour inhibition of 41.0% and 57.0% compared to controls resulted, with low and high dosages respectively, and all signs of systemic toxicity abolished. Further dose scheduling experiments showed four doses of 20μg conjugated 5-FUdr-succ administered on alternative days, to be safe, in terms of toxic effects, and thus an effective protocol.

To establish the specificity of FUdr-succ conjugates and the contribution antibody or free drug alone has on tumour inhibition, the effects of (a) FUdr-succ conjugated to αTFR (anti-transferrin receptor - E3 non- reactive), (b) a mixture of unconjugated FUdr-succ and αLy-2.1 MoAb and (c) FUdr-succ conjugated to αLy-2.1 (E3 reactive) were compared. Individual treatments consisting of a total of 80μgs of 5-FUdr-succ in either the conjugated or unconjugated form was administered on 4 alternate days beginning 4 days after tumour inoculation of 5x10^ E3 cells s.c. The 5-FUdr-succ-αTFR conjugate which is unreactive with the E3 thymoma showed 7.0% tumour inhibition, while the specific conjugate, 5-FUdr- succ-αLy-2.1, showed 60% tumour inhibition thus demonstrating the specificity of 5-FUdr MoAb conjugates. The free drug and MoAb mixture (with doses of each equivalent to the an.ti-Ly-2.1 conjugate) showed 25% tumour inhibition, yet free 5-FUdr-succ at these low doses has been shown to have no anti-tumour effect (data not shown). The MoAb αLy-2.1 is an IgG2a and may therefore be able to participate in ADCC to inhibit tumour growth. Previous experiments using this antibody have shown similar results. The in vivo effectiveness and specificity of 5-FUdr- succ conjugates illustrated in this model prompted further studies using colon carcinoma tumour models, (using the colon tumour cell line Liml899), since 5FU and its derivatives have been shown to be one of the drugs effective against colon carcinomas.

Effect of 5-FUdr-MoAb on Colon Tumour Xenografts:

Nude mice bearing the Liml899 tumour as a xenograft, were injected i.p. 21 days after implantation of tumour pieces, with a total of 66μg of 5-FUdr-succ conjugated to 1-1 MoAb (PBS was injected as a control). The i.p. administration of FUdr-succ-Ly-2.1 conjugates reduced tumours by 53.3% compared with controls. The individual tumour growth curves for control mice and conjugate receiving mice clearly demonstrated the in vivo efficacy of FUdr succ conjugates at doses as low as 66 μg against the Liml899 colon carcinoma.

As an alternative human colon xenograft, Colo 205 (5-FUdr resistant) was chosen. Groups of 6 Swiss nude mice were injected s.c. with 5x10° Colo 205 cells. Seven days later i.p. treatment was initiated where mice received either (a) PBS, (b) 5FUdrsucc-250-30.6 (Colo 205 reactive) or (c) 5FUdrsucc-anti-Ly-2.1 (Colo 205 non- reactive), every third day. Conjugate groups received four doses of 5FUdrsucc total does 122μg, (Day 7 - 31 μg, Day 10 - 30μg, Day 13 - 25 μg, Day 16 - 36 μg). No significant differences in tumour growth between the various groups was observed.

All references referred to herein are incorporated by reference.

Claims

CLAIMS :

1. A nucleoside-polypeptide conjugate, wherein said polypeptide is selected from an antibody or fragment thereof, hormone, growth factor or biologically active peptide; characterised in that the nucleoside is coupled to said polypeptide through a 3' ester linkage.

2. A conjugate according to claim 1 of the formula

X-NHCO-RCOO-Nu I where:

X is a monoclonal or polyclonal antibody or antigen binding fragment thereof; hormone; growth factor or other biologically active peptide;

Nu is a nucleoside drug attached to the group RCOO through the 3' carbon of the sugar moiety;

R is Cι_ 5 alkyl optionally substituted with halogen (I, Br, Cl, F), hydroxy, cyano, phenyl, amino, carboxy, alkyl, alkoxy and the like; said alkyl chain optionally being interrupted by one or more groups selected from -0-, -S-, NH, -CH=CH, phenyl and -S0 -; aryl or C^_^5 aralkyl; or a heterocyclic compound of 3 to 8 members.

3. A conjugate according to claim 1 or 2 wherein the monoclonal antibody is a chimeric antibody, human antibody, or humanised antibody.

4. A conjugate according to claims 1 or 2 wherein the antigen binding fragment of said antibody is selected from Fab, F(ab')₂ fragment Fvs, Dab or single chain antigen binding proteins.

5. A conjugate according to claim 1 or 2 wherein the hormone is selected from calcitonin, thyrotropin, melanotropin, insulin and gonadotropi .

6. A conjugate according to claim 1 or 2 wherein said growth factor is selected from EGF, TGFα, TGFβ, IGF and FGF.

7. A conjugate according to claim 1 or 2 wherein said biologically active peptide is a cytokine selected from G-CSF, GM-CSF and interleukins.

8. A conjugate according to claim 1 or 2 wherein said nucleoside drug is 4-FUdr or a derivative thereof, bromodeoxyuridine, iododeoxyuridine, bromovinyluridine and azidothymidine.

9. A method for the production of a nucleoside- polypeptide conjugate which comprises reacting a 3' active ester of a nucleoside drug with a polypeptide.

10. A method according to claim 9 wherein said active ester of a nucleoside drug comprises the formula

L-CO-R-COO-Nu wherein:

L is any leaving group; and R and Nu are as defined in claim 2.

11. An active ester of a nucleoside drug of the formula:

L-CO-R-COO-Nu wherein L, R and Nu are as defined in claim 10.

12. An active ester according to claim 11 which is the N-hydroxysuccinimide ester of 2'-deoxy-5-fluoro-3'-0- succinoyluridine.

13. A method for the treatment of tumours or viral diseases, which comprises administering to a subject in need of such treatment an effective amount of a conjugate according to claim 1 or 2 optionally in association with one or more pharmaceutically acceptable carriers or excipients.

14. A method according to claim 13 wherein said viral disease is selected from HIV, herpes virus, hepatitis A-E virus, cytomegalovirus and papilloma virus.

15. A method according to claim 13 wherein said tumours are selected from lymphoma, Hodgkin's disease, leukemia, melanoma, endothelial carcinomas, adenocarcinomas and cancers of the breast, lung, liver, pancreas, prostate, ovary, stomach, kidney, testes, head and neck.

16. A method for the treatment of transplantation rejection or autoimmune disease which comprises administering to a subject in need of such treatment an effective amount of a conjugate according to claims 1 or 2 optionally in association with one or more pharmaceutically acceptable carriers or excipients, wherein said polypeptide portion of said conjugate is reactive with lymphocytes.

17. A method for clearing bone marrow of tumour cells, which method comprises removing bone marrow from a patient, optionally freezing and thawing the bone marrow, and thereafter reacting bone marrow with a conjugate according to claims 1 or 2; wherein the polypeptide portion of said conjugate is reactive with tumour cells.

18. A pharmaceutical composition which comprises a conjugate according to claim 1 or 2 in association with one or more pharmaceutically acceptable carriers or excipients.