Bioconjugate Chem. 1991, 2, 96-101
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In Vitro Antitumor Activity of 2’-Deoxy-5 -fluorouridine-Monoclonal Antibody Conjugates Ada Goerlach, Kenia G. Krauer, Ian F. C. McKenzie, and Geoffrey A. Pietersz’ Research Centre for Cancer and Transplantation, Department of Pathology, The University of Melbourne, Parkville, Victoria 3052, Australia. Received February 27, 1990
5-Fluorouracil (5-FU) is an anticancer drug used in patients for the treatment of gastric and breast cancer and used either alone or in combination with methotrexate is one of the few drugs with some effect on colon cancer. 2’-Deoxy-5-fluorouridine (5-FUdr) (1) is an analogue based on 5-FU and can be covalently linked to a murine anti-Ly-2.1 monoclonal antibody (mAb) with the active ester derivative of 2’-deoxy-5-fluoro-3’-0-(carboxypropanoyl)uridine (5-FUdr-succ) (4). Such immunoconjugates can contain up to 42 residues of drug, although the most antibody activity was retained when substitution ratios were between 10 and 25 molecules of drug to mAb. In a cytotoxicity assay, 50% inhibition of [3H]deoxyuridineincorporation ( E m ) with a murine L ~ - 2 . 1 +thymoma ”~ cell line was 6 nM for 5-FUdranti-Ly-2.1, which is 12-fold more than that for free 5-FUdr (IC50 = 0.51 nM) but similar to that of 5-FUdr-succ (IC50 = 5.2 nM). The 5-FUdr-monoclonal antibody conjugates (5-FUdr-mAb) were 100fold more active on the Ly-2.1+”eE3 cell line than on the Ly-2.l’e BW5147 OU- cell line. The high in vitro activity and specificity of 5-FUdr-MoAb conjugates indicates that potent in vivo activity of these conjugates should be expected.
INTRODUCTION Most cytotoxic drugs used clinically have little selective toxic effect on tumors and although extensive structural modification has resulted in analogues with improved antitumor activity, dose-limited toxic side effects are still a major problem (1,2). However, the targeting of cytotoxic agents to tumors by covalently linking them to monoclonal antibodies confers a degree of specificity not found with the drugs alone (3). The specificity of the monoclonal antibody (mAb),’ together with the internalization and subsequent release of the coupled drug, can lead to the selective killing of tumor cells expressing the antigen (4). There are, however, some limitations to this approach, as access of immunoconjugates to the tumor is inefficient and results in only a fraction of the dose entering the tumor (5). Immunoconjugates of highly toxic drugs or toxins may overcomethe problem of inefficient delivery of a lethal dose to the tumor and one example of this was to use a more toxic folic acid antagonist, aminopterin, which was found to be more potent than the related but less toxic methotrexate when delivered as an immunoconjugate (69)* In this light it is appropriate to examine more cytotoxic compounds such as 5-fluorouracil. 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 (10). This drug is metabolized by several enzymes and transformed into toxic species which interfere with severalmetabolic pathways (11).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
* To whom all correspondence should be addressed.
1 Abbreviations used: 5-FU,5-Fluorouracil;5-FUdr,2’-deoxy5-fluorouridine;mAb(s),monoclonal antibody(ies); 5-FUdr-succ, 2’-deoxy-5-fluoro-3’-O-(3-carboxypropanoyl)uridine; ICW, 50% inhibition of [3H]deoxyuridineincorporation; E3,ITT(1)75NS E3;DMTr-CI, 4,4’-dimethoxytriphenylmethylchloride; DMAP, 4-(N,N-dimethylamino)pyridine; NHS, N-hydroxysuccinimide; DCC, dicyclohexylcarbodiimide;PBS, phosphate-bufferedsaline; TLC, thin-layer chromatography;BW, BW5147 OU-.
is transformed to 2‘-deoxy-5-fluorouridine monophosphate, which inhibits thymidylate synthetase, resulting in the inhibition of DNA synthesis. In vitro 2’-deoxy-5fluorouridine (5-FUdr, 1) (Figure 1)is more cytotoxic than 5-FU. We now report on the linkage of 5-FUDr to mAbs and the demonstration of the specific cytotoxic action of the compounds on tumor cells. EXPERIMENTAL PROCEDURES General Procedures. Melting points were determined in open capillary tubes on an Electrothermal digital melting point apparatus and are uncorrected. ‘H nuclear magnetic resonance (‘H NMR) spectra were recorded on a Bruker AM-300 wide-bore NMR spectrometer. Chemical shifts of compounds are reported in ppm downfield from internal standard tetramethylsilane. Mass spectra were obtained with a JOEL JMS-AX 505 H mass spectrometer. Thinlayer chromatography (TLC) was performed on silica gel coated plastic sheets (Kieselgel6OFz~plates) (E. Merck, Darmustadt, Germany), with products being visualized by exposure to UV light and/or 80% aqueous acetic acid. 2’-Deoxy-5’- 0[ b i s (4-met hoxypheny1)phenylmethyl]-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’-Dimethoxytriphenylmethylchloride (304.5 mg, 0.9 mmol) in pyridine (2 mL) was added dropwise while being cooled and stirred, and the reaction mixture was stirred at room temperature for 16 h. The solution was poured into 10 mL of ice containing 5% NaHC03, extracted with dichloromethane, dried over anhydrous Na~S04,and evaporated to give 340 mg of crude product. Column chromatography on silica gel (40 g) using CHzClz-MeOH (13: 1)as eluant yielded 223.6 mg (68%)of 2: TLC (CH2ClzMeOH, 13:l)R f 0.25; ‘H NMR (CDC13) 6 2.26 and 2.48 (2 m, 2 H, 2’-Hz), 3.42 (m, 2 H, 5’-Hz), 3.79 (s, 6 H, OCHs), 4.06 (dt, J 4 , 5 = J s ,=~4 Hz, 1 H, 4’-H), 4.53-4.57 (m, 1 H, 3’-H), 6.29 (m, 1 H, 1’-H), 6.85 and 7.28 (2 m, 13 H, arom H), 7.83 (d, JG-H,F = 9 Hz, 1 H, 6-H). 2’-Deoxy-5’- 0[ b i s (4-met h o x y p h e n y 1 ) p h e n y l methyll-5-fluoro-3’-0(3-carboxypropanoyl)uridine (3). 2’-Deoxy-5’-0-[bis(4-methoxyphenyl)phenylmethyl]0 1991 American Chemical Society
2‘-Deoxy-5-fluorourMine 0
Bioconjugate Chem., Vol. 2, No. 2, 1991 07
temperature for 3 h and then centrifuged (400g X 3 min) to remove any precipitate. The conjugate was purified by HI A f F 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 l25I-1abeledantibody (6)and the amount 1 Rl-H, R2-H R2O 2 R,-DMTr, R H ., of FUdr-succ bound was determined by absorbance spec3 R1-DMTr, R2-COCH2CH2COOH trophotometry at 260 nm using the extinction coefficients 4 R,-H, R,-CCCH,C&COOH = 7600 M-’ cm-’ (FUdr-succ) and E260 = 12 9300 M-l Figure 1. Structure of 5-FUdr (1) and other derivatives [(2) cm-l (mAb). Analysis of these conjugates by SDS-PAGE 2’-deoxy-5’-0- [bis(4-methoxyphenyl)phenylmethyl]-5-fluorouriand gel-permeationchromatography (TSK-SW2000,Phardine, (3) 2‘-deoxy-5‘-0- [bis(4-methoxyphenyl)phenylmethyl]-5macia) showed less than 5% aggregated antibody (not fluoro-3’-0-(3-carbonylpropanoyl)uridine, (4) 2’-deoxy-5-fluoroshown). 3’-0-(3-carbonylpropanoyl)uridine]. Drug Activity. Cytotoxicity Assays. (a) Cells (100 pL, 2 x lo5 cells) were added to a flat-bottom microtiter 5-fluorouridine (2) (191.8mg, 0.35 mmol) was dissolved in plate and incubated for 1 h at 37 “C. Free drug in PBS dichloromethane (3mL) and after the addition of 4-(N,Nand conjugate solutions were filtered (0.22pM) to sterilize dimethy1amino)pyridine(64.1 mg, 0.525 mmol, 1.5 equiv) them, various dilutions were made in PBS, and 100 pL and succinic anhydride (140 mg, 1.4 mmol, 4 equiv) the was added to the cells. Control wells received 100 pL of mixture was kept at room temperature for 16 h and then PBS. The plates were then kept at 37 “C, 7% COZfor 24 poured into 10mL of 5 5% NaHC03, acidified with 1N HC1 h and harvested (see below). to pH 3, and extracted several times with dichloromethane. (b) Cells (200 pL, 2 X lo5cells, E3 and BW) were added The extract was dried and evaporated to give 132.4 mg to sterile tubes, 100 pL of various dilutions of conjugate (77 $1) of 3: TLC (9070 aqueous CH3CN) Rf 0.68. or free drug in PBS was added, and the mixtures were 2’-Deoxy-5-fluoro-3’-0-(3-carboxypropanoy1)uridine (4). 2’-Deoxy-5’-0-[bis(4-methoxyphenyl)phenyl- kept at room temperature for 30 min; 500 pL of growth medium was then added, and the cells were centrifuged methyl]-5-fluoro-3’-0-(3-carboxypropanoyl)uridine (3) (400g X 5 min). After resuspension in 200 pL of medium, (132.0 mg, 0.2 mmol) was dissolved in 80% aqueous acetic 100-pL aliquots were added to a microtiter plate and acid (2 mL) and left at room temperature for 2-3 h. The incubated at 37 “C, 7% COZfor 16 h. reaction mixture was diluted with water and extracted After incubation 50 pL of growth medium containing 1 with chloroform. The aqueous solution was lyophilized and the residue passed through a column of 5 g of silica pCi of [3H]deoxyuridine (specific activity, 21 Ci/mmol; Amersham, Amersham, England) was added and the gel with 70 91 aqueous ethanol. Evaporation of the solvent mixture incubated for 4 h, when the cells were harvested gave 61 mg (86%) of 4 as a white hygroscopic powder: with a Dynatech automash cell harvestor (Dynatech, TLC (70% aqueous EtOH) Rf0.80;‘H NMR (DzO) 6 1.8England) onto glass fiber-filters and dried and samples 2.5 (m, 6 H, SUCC-CH2, 2’-H2), 3.52-3.70, 3.95-4.05, (2 m, counted on a p-scintillation counter (Packard Instrument 5’-H2, 4’-H), 5.09 (m, 1 H, 3’-H), 6.07 (m, 1 H, 1’-H), 7.84 Co., Downers Grove, IL). The viability of the cells was (d, J~-H,F = 8 Hz, 1 H, 6-H). expressed as a percentage inhibition of [3H]deoxyuridine Biological Testing. Tumor Cells. The cell lines used incorporation compared to that of the controls (we have in this study were the E3 variant of the L ~ - 2 . 1 murine +~~ thymomaITT(1)75NS (E3) (12)and theL~-2.l-~~BW5147 previously shown that [3H]deoxyuridineuptake is equivalent to cell viability using a dye exclusion assay (unpubOU- cells (BW) (13). The cells were maintained in vitro lished results). Duplicates did not show more than 10% in Dulbecco’s modified Eagle’s medium supplemented with variation. 10$1 heat-inactivated newborn calf serum (Flow LaboStability of Conjugate in Growth Medium. To ratories, Sydney, Australia), 2 mM glutamine (Commondetermine the stability of 5-FUdr-anti-Ly-2.1 in growth wealth Serum Laboratories (CSL),Sydney, Australia), 100 medium, a conjugate prepared with radiolabeled anti-Lypg/mL streptomycin (Glaxo, Melbourne, Australia), and 2.1 was mixed with an equal volume of fetal calf serum or 100 IU/mL penicillin (CSL). PBS and incubated at 37 “C for 24 h. A similar incubation Monoclonal Antibodies. Anti-Ly-2.1 (IgGza) (14) was performed with radiolabeled anti-Ly-2.1. After inreactive with the murine Ly-2.1 antigen was isolated from cubation, the samples were passed through a PD-10 column ascites fluid. The ascites was diluted in phosphateto remove any free drug and the peak fractions were pooled buffered saline (5 mM, 0.17 M NaC1, pH 7.2) (PBS) and and counted for radioactivity. The concentration of adsorbed onto protein A-Sepharose (Pharmacia Inc., Pisantibody was determined from the known specific activity cataway, NY), washed extensively with PBS, and eluted of 1251-labeledanti-Ly-2.1and the cytotoxicity of the peak with 0.2 M citrate buffer (pH = 4.0). Following neutralfraction was measured by using the [3H]deoxyuridineassay ization the mAb was concentrated by precipitation with as described above. 45% aqueous (NH4)2S04and dissolved in PBS, and alAntibody Activity by Flow Cytometry. (a) Indirect iquots (5 mg/mL) were stored at -70 “C. When tested Assay. E3 cells (2 X 105) in 500 pL of growth medium with sodium dodecyl sulfate polyacrylamide gel electrowere incubated with 100 pL of various concentrations of phoresis (SDS-PAGE),this mAb preparation was 90-95% unconjugated antibody or with the 5-FUdr-mAb conjugate pure. for 1 h on ice. The cells were centrifuged, washed twice Preparation and Quantitation of Conjugates. The with 500 pL of PBS containing 0.025% sodium azide and active ester derivative of 5-FUdr-succ (4)was prepared by 2 % newborn calf serum, and resuspended in 500 pL of the dissolving 5-FUdr-succ (1.73 mg, 5 mmol) in 70 pL of dry same medium. PBS (100 pL) containing 10 pg of fluoDMF. N-Hydroxysuccinimide (0.59 mg, 5.1 mmol) in 17 rescein isothiocyanate labeled rabbit anti-mouse immupL of DMF and dicyclohexylcarbodiimide (6.18 mg, 30 noglobulin F(ab’)Z fragment (Silenus Laboratories Pty mmol) in 50 pL of DMF were added, and the reaction Ltd., Hawthorn, Australia) was then added and after an mixture was kept at room temperature for 3 h and at 4 “C incubation period of 45 min on ice 500 pL of medium was overnight. An aliquot of the active ester was mixed with added, and the cells were centrifuged, washed, and rethe mAb (3-5 mg/mL) in PBS and maintained at room
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suspended. Fluorescence was measured with a Becton and Dickinson FACScan. For calculations mean fluorescence values were used. (b) Competition Assay. Various concentrations of unmodified anti-Ly-2.1 or conjugates with different substitution ratios were mixed with 5 pg of FITC-labeled antiLy-2.1, and 100 pL of the mixture was added to 2 X lo5 E3 cells in 500 p L of growth medium. After a 1-h incubation on ice, the cells were centrifuged, washed twice with 500 pL of PBS containing 0.025% sodium azide and 2% new born calf serum, resuspended in 500 pL of the same medium, and analyzed with the FACScan.
,
50 I
.!
o ! .
I . I . I . I . , . , . I . I . I 60 20 40 60 80 100120140160180200
0
Molar excess of actlve ester
RESULTS
Chemistry. According to procedures given by Thomas et al. (15)and Habener et al. (16) 5-FUdr (1) was reacted with 4,4'-dimethoxytriphenylmethylchloride (DMTr-C1) in pyridine to protect the 5'-hydroxyl group prior to succinylation. Succinylation of hydroxyl compounds (mainly steroids and related substances) is usually performed with 1-1.5 equiv of succinic anhydride in the presence of 4-(N,Ndimethy1amino)pyridine (DMAP). For example, Morvan et al. (17) performed the reaction in dichloromethane with 1.5 mmol of DMAP, 1.5 mmol of succinic anhydride, and 1.5 mmol of triethylamine per millimole of nucleoside. For compound 2 (Figure 1) the reaction was exceedingly slow, and these conditions were modified by increasing the amount of succinic anhydride to 4 mmol, and after 16 h at room temperature compound 3 was isolated with a 77% yield. Compound 3 was used for the deprotection step without further purification. Removal of the dimethoxytrityl group of 3 was accomplished with 80% acetic acid without cleavage of the hemisuccinate ester bond. The crude product was purified by column chromatographyon silica gel with 70% aqueous ethanol to yield 5-FUdr-succ (4) in 86% yield. Coupling of 5-FUdr-succ to Monoclonal Antibody. Compound 4 was conjugated to mAb with the succinimido ester derivative. To obtain the active ester of 4 with NHS, various ratios of the reactants were tested (data not shown). The optimal reaction conditions were found to be 1.1 equiv of NHS and 6 equiv of dicyclohexylcarbodiimide (DCC). We were unable to crystallize the active ester and it was therefore used without further purification. To optimize coupling, different molar excesses of 5-FUdrsucc active ester were added to mAb 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 nine 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 (Figure 2). The conjugates that were tested further in vitro had between 5 and 20 molecules of 5-FUdr incorporated per molecule of mAb. To determine the stability of the 5-FUdr-mAb 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 (not shown). Antibody Activity of 5-FUdr-mAbConjugates. The antibody activity of the immunoconjugates was quantitated by flow cytometry. The binding of the conjugates to L ~ - 2 . 1 +ITT(1)75NS "~ E3 (E3) cells was measured by fluorescence of the cells after incubating with immunoconjugate, washing, and exposing to fluorescein-labeled rabbit anti-mouse antibody. The concentration of the im-
90
Figure 2. Coupling of 5-FUdr-succ active ester to anti-Ly-2.1 mAb. The number of residues of 5-FUdr incorporated per molecule of anti-Ly-2.1 ( 0 )and protein recovery ( e )is shown as a function of the molar excess of 5-FUdr-succ active ester.
0
10
20
30
40
50
Number of residues
"
I
0
10
20
30
Number of residues
Figure 3. Antibody activity of 5-FUdr-anti-Ly-2.1 conjugates on E3 cells. The antibody activity is expressed as a percentage of the initial antibody activity determined by flow cytometry and is shown as a function of the number of 5-FUdr residues coupled to anti-Ly-2.1: (A) direct binding assay, (B)competition assay.
munoconjugate giving 50 76 of maximum fluorescence was calculated and compared with that of unconjugated mAb to give a relative activity of conjugated to nonconjugated 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) (Figure 3A). A highly conjugated sample (42 residues) showed considerable loss of antibody activity and was 21fold less active. The antibody activity of conjugates were also measured by using a competition assay. The antibody activity of conjugate measured by using the competition assay was
Bioconjugate Chem., Vol. 2, No. 2, 1991 9Q
z
-10
-9 -8 -7 Log Concentratlon of drug (M)
-6
Figure 4. The inhibitory effect (as percent inhibition of [3H]deoxyuridine uptake) of 5-FUdr (+) and 5-FUdr-succ (0) on Ly-2-'eBW5147 cells and of 5-FUdr (m) and 5-FUdr-succ (0) on Ly-2+vE E3 cells.
-10
-9
-8
-7
-6
Log concentration of 5-FUdr (M)
Figure 5. The inhibitory effect on Ly-2.1+" E3 cells of 5-FUdranti-Ly-2.1 conjugates containing different numbers of 5-FUdr residues: (0)11 residues, (+) 19 residues, (m) 26 residues. m
Table I. Effect of 5-FUdr and 5-FUdr-succ on Tumor Cells. mean ICw, M tumor cells FUdr FUdr-succ 5-FUdr-mAb 5.2 X 10-9(6) 6.0 X 104(30) E3 5.1 X 10-1°(3) 5.3 X 104(2) 2.4 X (6) BW5147 1.4 X (2) a The number of individual values is in parentheses.
similar to the activity measured by using the direct binding assay (Figure 3B). Cytotoxicity of 5-FUdr-mAb Conjugates in Vitro. The in vitro cytotoxicity of 1 and 5-FUdr-succ (4) on the murine L ~ - 2 . 1 +E3~ cell ~ line and the L ~ - 2 . 1 BW - ~ ~cell line was measured in an inhibition assay, and IC50 values were determined (Table I, Figure 4). With E3 the cytotoxicity of 5-FUdr-succ (4) (IC50 = 5.2 nM) was 10-fold less than that of 5-FUdr (1) (IC50 = 0.51 nM), 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. With 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-FUsucc (4) was the same so that data using the immunoconjugates can be compared (Figure 4). The cytotoxicity of anti-Ly-2.1 conjugates with 2-20 molecules of 5-FUdr bound per molecule antibody was tested (Table I) on Ly2.1+Ve E3 and L ~ - 2 . 1 BW - ~ ~cell lines, where it was found that the IC50 values for E3 were in the range of 5.0-9.0 X 10-9 M (average of 6.0 X M from 30 individual values), while the ICMfor the BW cell line was 2-6 X 10-8M (average of 2.4 X 10-8 M), the results indicating that 5-FUdr-mAb is 4 times more active on the L ~ - 2 +cell ~ e 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 (Figure 5). These data show that 5-FUdr-anti-Ly-2.1 conjugates are 1 2 times less toxic than 5-FUdr (1) on the E3 cell line but of similar toxicity to FUdr-succ (4). Specificity in Vitro. To show that the cytotoxicity of the conjugates was due t o specific targeting by the antibody, a cytotoxicity assay was carried out with both L ~ - 2 . 1 + E3 ~ e cells and L~-2.1-~8 BW cells. In this assay conjugates were exposed to antibody reactive (E3) and antibody nonreactive (BW) cells for 30 min and washed and then incubated for 24 h and pulsed with [3H]deoxyuridine. The IC50 of the anti-Ly-2.1 conjugate for E3 was 7.1 X M while the IC50for BW was >5.01 X lo-' M (Figure 6) and indicates that the 5-FUdr-anti-Ly-2.1
E
-9
-0 -7 -6 Log concentratlon of 5-FUdr (M)
-5
Figure 6. The inhibitory effect of 5-FUdr-anti-Ly-2.1 (11 residues) on Ly-2+" E3 (0)and Ly-2" BW5147 (m)in a shortexposure cytotoxicity assay. 100
so 60
40 20
0
Concentration of
anti-Ly-2.1
(mg/ml)
Figure 7. The inhibitory effect of 5-FUdr-anti-Ly-2.1 on E3 on day 1 ( O ) , after a 24-h incubation without fetal calf serum (m) and with fetal calf serum (0).
conjugates are selectively toxic to Ly-2.1fvecells. Stability of 5-FUdr-anti-Ly-2.1in Growth Medium. To ascertain the degree of dissociation of 5-FUdr-mAb in growth medium, conjugates prepared with radiolabeled anti-Ly-2.1 were incubated with fetal calf serum for 24 h as described in the Experimental Procedures. The cytotoxicity of the various samples are shown in Figure 7. The IC50 of the conjugate in terms of antibody concenmg/mL on day 1. The IC50 of the tration was 2.5 X conjugate after 24-h incubation without serum was identical with the initial IC50. However, when the conjugate was incubated with serum the IC50was increased to 4 X mg/mL. Therefore, in the presence of fetal calf serum the cytotoxicity of the conjugate was reduced 1.6-fold,
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indicating a loss of some 5-FUdr residues. Unconjugated radiolabeled anti-Ly-2.1 a t the same specific activity as conjugate did not inhibit [3H]deoxyuridine uptake in cells. DISCUSSION
Monoclonal antibodies reactive with tumor antigens have been shown to be suitable carriers for drugs and toxins to target them to tumor cells (3). However, one major limitation to the use of drug-monoclonal antibody conjugates is the degree of drug and monoclonal antibody activity retained after conjugation. One approach in overcoming these problems is the use of more cytotoxic drugs such as 5-FUdr (l),which inhibit growth of cells a t nanomolar concentrations (Figure 1)as reported herein. 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 1prior to succinylation. By protecting of the 5’-position with a 4,4’-dimethoxytriphenylmethyl group, succinylation of the 3’-hydroxylgroup occurred and deprotection gave the 3’-isomer as the only product, with a yield of 45% after the three 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 mAb (Figure 2). This significant protein recovery may well be due to the high hydrophilicity of 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 which could have good therapeutic effectiveness were obtained. Coupling a large number of drug molecules to monoclonal antibodies often causes a loss in antibody activity, and FACS tests demonstrated this to be the case with 5-FUdr as 5-FUdr-anti-Ly-2.1 conjugates with up to 25 molecules of 5-FUdr bound demonstrated 2 0 4 0 % of the antibody activity of unconjugated anti-Ly-2.1 (Figure 3A). It is possible that binding of FITC-bound sheep anti-mouse antibody maybe hindered by 5-FUdr groups linked to antiLy-2.1 and result in an inaccurate measure of antibody activity. With this in mind a competition assay was also performed with FITC-labeled anti-Ly-2.1. As shown in Figure 3B the results from the competition assay were comparable to those of the direct binding assay. It should be noted that the succinylation of 5-FUdr (1) in the 3’position could have an influence on the mechanisms of action of the drug, and it was therefore important to test the in vitro activity of both 5-FUdr (1) and its derivative 5-FUdr-succ (4). 5-FUdr-succ was 10-fold less toxic than 5-FUdr on E3 cells and 4-fold less toxic than 5-FUdr for BW cells (Table I, Figure 4). The lower cytotoxicity of 5-FUdr-succ may possibly be due to some difficulty in its entry into cells via the nucleoside-transport system or due to the presence of a charged carboxylic acid group. 5-FUdr-succ-mAb 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-FUdrsucc (4) with an average 12times lower toxicity than 5-FUdr (1) (Table I, Figure 5). This loss in drug activity is mainly due to the chemical modification, as the IC50 values for the 5-FUdr-anti-Ly-2.1 is similar to that of 5-FUdr-succ (4), indicating that coupling to mAb did not alter the cytotoxicity of the free drug. In a 30-min cytotoxicity assay the 5-FUdr-anti-Ly-2.1 conjugates were selectively toxic to only cells possessing the Ly-2.1 antigen (Figure 6). In a 24-h cytotoxicity assay 5-FUdr-anti-Ly-2.1 conjugates
were 4-fold less active on L ~ - 2 . 1 - “BW ~ cells. The exact mechanisms for the observed cytotoxicity is not clear. However, it is possible that it may be due to internalization of conjugates by nonspecific pinocytosis or Fc receptor binding. This nonspecific cytotoxicity has been observed with a number of drug-mAb conjugates (18,19). The cytotoxicity observed in the 24-h assays was demonstrated to be due to conjugate and not due to drug dissociated from the conjugate by investigating the stability in fetal calf serum. Following an incubation with fetal calf serum the cytotoxicity decreased 1.6-fold(Figure 7). The exact mechanism of action of these 5-FUdr-mAb conjugates is not known. Several possible mechanism can be suggested. Two of which are the conjugates bind to the antigen and are internalized and digested in the lysosomes to release free drug, and esterases present in the lysosome could hydrolyze the ester bond between 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, and currently a %-labeled 5-FUdr-succ is being prepared which will enable extensive stability studies and cellular processing studies to be carried out. Preliminary in vivo studies in mice demonstrate specific antitumor effects, and the results of these studies will be reported elsewhere. LITERATURE CITED (1) Chabner, B. A., Fine, R. L., Allegra, C. J., Yeh, G. W., and Curt, G. A. (1984) Cancer chemotherapy. Progress and expectations. Cancer 54, 2599-2608. (2) Cassady, J. M., and Douros, J. D. (Eds.) (1980) Anticancer agents based on natural product models. Medicinal Chemistry, Val. 16, Academic Press, New York. (3) Pietersz, G. A., Kanellos, J., Smyth, M. J., Zalcberg, J., and McKenzie, I. F. C. (1987) The use of monoclonal antibody conjugates for the diagnosis and treatment of cancer. Immunol. Cell Biol. 65, 111-125. (4) Smyth, M. J., Pietersz, G. A., and McKenzie, 1. F. C. (1987) The mode of action of methotrexate monoclonal antibody conjugates. Immunol. Cell Biol. 65, 189-200. ( 5 ) Epenetos, A. A., Snook, D., Durbin, H., Johnson, P. M., and Taylor-Papadimitriou, J. (1986) Limitations of radiolabeled monoclonal antibodies for localization of human neoplasms. Cancer Res. 46, 3188-3191. (6) Smyth, M. J., Pietersz, G. A., and McKenzie, 1. F. C. (1987) Use of vasoactive agents to increase tumor perfusion and the antitumor efficacy of drug-monoclonal antibody conjugates. J. Natl. Cancer Inst. 79, 1367-1373. (7) Smyth, M. J., Pietersz, G. A., and McKenzie, I. F. C. (1988) Increased antitumor effect of immunoconjugates and tumor necrosis factor. Cancer Res. 48, 3607-3612. (8) Kanellos, J., Pietersz, G. A., Cunningham, Z., and McKenzie, I. F. C. (1987) Anti-tumor activity of aminopterin monoclonal antibody conjugates; in vitro and in vivo comparison with methotrexate monoclonal antibody conjugates. Immunol. Cell Biol. 65,483-493. (9) Vitetta, E. S., and Uhr, J. W. (1985) Immunotoxins: Redirecting nature’s poisons. Annu. Reo. Immunol. 3, 197212. (10) Meyers, C. E., Diasio, R. B., Eliott, H. M., and Chabner, B. A. (1976) Cancer Treat. Rev. 3, 175. (11) Cheng, C. C., Ellis, G. P., and West, G. B. (Eds.) (1969) Progress in Medicinal Chemistry, Vol. 6 , Butterworths, London. (12) Smyth, M. J., Pietersz, G. A., Classon, B. J., and McKenzie, I. F. C. (1986) Specific targeting of chlorambucil to tumors with the use of monoclonal antibodies. J.Natl. Cancer Znst. 76, 503-510. (13) Hyman, R., and Stallings, V. (1974) Complementation patterns of Thy-1 variants and evidence that antigen loss
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variants "pre-exist" in the parental population. J.Natl. Cancer Znst. 52, 429-436. (14) Hogarth, P. M., Edwards, J., McKenzie, I. F. C., Goding, J. W., and Liew, F. Y. (1982) Monoclonal antibodies to murine Ly-2.1 cell surface antigen. Immunology 46, 135-144. (15) Thomas, H. J., and Montogomery, J. A. (1962) Complex esters of thioinosinic(5') acid. J. Med. Pharm. Chem. 5, 2432. (16) Habener, J. F., Vo, C. D., Le, D. B., Gryan, G. P., Ercolani, L., and Wang, A. H. (1988) 5-Fluorodeoxyuridine as an alternative to the synthesis of mixed hybridization probes for the detection of specific sequences. Proc. Natl. Acad. Sei. U.S.A.85, 1735-1739.
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(17) Morvan, F., Rayner, B., Leonetti, J.-P., and Imbach, J. L. (1988) a-DNAVII. Solid phase synthesis of a-anomeric oligodeoxyribonucleotides. Nucl. Acid. Res. 16, 833-847. (18) Hermentin, P., Doenges. R., Gronski, Po, Bosslet, K., Kraemer, H. P., Hoffmann, D., Zilg, H., Steinstraesser, A,, Schwarz, A., Kuhlmann, L., Luben, G., and Seiler, F. R. (1990) Attachment of Rhodosaminylanthracyclinone-typeanthracyclines to the hinge region of monoclonal antibodies. Bioconjugate Chem. 1 , 100-107. (19) Pietersz, G. A., Smyth, M. J., and McKenzie, I. F. C. (1988) Immunochemotherapy of a murine thymoma with the use of idarubicin-monoclonal antibody conjugates. Cancer Res. 48, 926-931.
