Optimisation of small-scale coupling of A5B7 monoclonal antibody to carboxypeptidase G2

Journal of Immunological Methods, 158 (1993) 49-56 © 1993 Elsevier Science Publishers B.V. All rights reserved 0022-1759/93/$06.00

49

JIM 06555

Optimisation of small-scale coupling of A5B7 monoclonal antibody to carboxypeptidase G 2 R.G. Melton a, J.M.B. Boyle a, G.T. Rogers u, P. Burke b, K.D. Bagshawe b and R.F. Sherwood a Division of Biotechnology, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wilts SP4 0JG, UK, and b Cancer Research Campaign Laboratories, Department of Medical Oncology, Chafing Cross Hospital, Fulham Palace Road, London 14168RF, UK (Received 22 April 1992, revised received 13 July 1992, accepted 28 August 1992)

Conjugates of F(ab') 2 fragment of the monoclonal antibody ASB7 coupled to carboxypeptidase G 2 (CPG 2) have been produced using the heterobifunctional reagents 2-mercapto-[S-acetyl]acetic acid, N-hydroxysuccinimide ester (SATA) and m-maleimidobenzoyl-N-hydroxysuccinimideester (SMPB). The effect of various levels of modifying reagent on enzyme activity and antigen binding activity were determined, and it was shown that whilst CPG a is relatively sensitive to modification, insertion of three maleimide groups per CPG 2 resulted in the loss of 30% of enzyme activity; ASB7 F(ab') 2 was insensitive to modification, little or no activity being lost. The coupling efficiency of the reaction was shown to be fairly constant over a wide range of substitution levels. There was thus no advantage to be gained in using high substitution levels, which may result in loss of enzyme activity. The formation of undesired high molecular weight aggregates could be controlled by adjustment of the protein concentration during the final coupling step. Key words: Conjugate; Antibody-directed enzyme-prodrug therapy; Antibody-enzyme conjugate; Carboxypeptidase G2; Monoclonal antibody; Carcinoembryonic antigen

Introduction

The use of a monoclonal antibody (MAb) to target an enzyme capable of cleaving a relatively inactive prodrug to release an active, cytotoxic drug in vivo has been reported, and forms the basis of a promising new strategy for the treatment of cancer. It has been termed antibody-directed enzyme-prodrug therapy (ADEPT) (Bagshawe, 1989; Bagshawe et al., 1988; Senter et al., 1988, 1989). A folate-degrading enzyme, carCorrespondence to: R.G. Melton, Division of Biotechnology, PHLS-CAMR, Porton Down, Salisbury, Wilts SP4 0JG, UK. Tel.: 0980 610391, ext. 2430; Fax: 0980 610898.

boxypeptidase G2, (CPG2) , which cleaves the terminal glutamate residue from folic acid and a wide range of analogues (Sherwood et al., 1985), has been used to activate a glutamate-deactivated prodrug form of a benzoic acid mustard, 4-(bis(2chloroethyl)amino) benzoic acid (Springer et al., 1990). The success of ADEPT in preventing tumour development in a human tumour, mouse xenograft model (Bagshawe, 1989; Bagshawe et al., 1988) has led to a requirement to produce multigram quantities of antibody:enzyme conjugate sufficient for human clinical trials. Scale-up production of conjugate has meant a re-evaluation of the chemical coupling process used, in order to maximise protein yields and minimise

50 losses of the enzymic and antigen-binding activity of the respective components. In order to achieve optimum properties of tumour specific localisation and tissue penetration, we have elected in these studies to use F(ab') 2 fragment rather than intact MAb, and to aim to produce a conjugate composed of one F(ab') 2 fragment coupled to a single molecule of CPG 2. Initial clinical trials have used the A5B7 anticarcinoembryonic antigen (anti-CEA), an IgG1 monoclonal antibody (MAb), which binds selectively to CEA-expressing colorectal cancer cells, coupled to CPG 2 (Bagshawe et al., 1991). The preparation of conjugates of CPG 2 with W14, an IgGI MAb to human chorionic gonadotrophin, using the heterobifunctional reagent MBS for insertion of maleimide residues into CPG 2 and SPDP for the thiolation of MAb, has previously been reported (Searle et al., 1986) and the conditions used successfully in that instance have been used as a starting point for the study reported here. It has been necessary, however, to simplify the coupling process and provide a conjugate with greater separation between the antibody and enzyme moieties in order to reduce the possibility of steric hindrance of the enzyme active site or antigen-binding sites. These modifications involve the use of an alternative heterobifunctional reagent, SMPB (Martin and Papahadjopoulous, 1982) having an increased alkyl spacer between the active moieties, for the insertion of maleimide residues into CPG 2, and the use of SATA (Duncan et al., 1983) for the thiolation of MAb. Materials and methods

Materials 2-mercapto-[ S-acetyl]acetic acid,N-hydroxysuccinimide ester (SATA), m-maleimidobenzoyI-Nhydroxysuceinimide ester (SMPB), N-ethylmaleimide, orthophenylene diamine, dimethyi sulphoxide, hydroxylamine hydrochloride, dithiobis(2-nitrobenzoie) acid (DTNB) and goat antimouse Fab-peroxidase conjugate were purchased from Sigma Chemical Co., Poole, UK. Preparation of CPG 2 and A5B7 F(ab') 2 fragment CPG 2 was prepared by the Division of Biotechnology, CAMR, Porton Down, UK, as

previously described (Sherwood et al., 1985). Purified carcinoembryonic antigen (CEA) and immunopurified mouse monoclonal A5B7 anti-CEA antibody were supplied by the Department of Medical Oncology, Charing Cross Hospital, UK. F(ab') 2 fragments of A5B7 were produced by digestion of intact antibody with cysteineactivated bromelain (Boehringer) (Milenic et al., 1989) and were isolated by ion exchange chromatography using a column (0.5 x 5.0 cm) of Mono S (Pharmacia). The F(ab') 2 fragment was further purified by gel filtration chromatography on a column (1. 6×60 cm) of Superdex G200 (Pharmacia). 2.0 ml aliquots of F(ab') 2 preparation were loaded and eluted with phosphatebuffered saline (PBS) at a flow rate of 1.0 ml/min. 2.5 ml fractions were collected. Fractions containing F(ab') 2 fragment were concentrated using an Amicon model 8010 concentrator fitted with a PM10 membrane, filter sterilised and stored at 4°C. Sodium dodecyl sulphate-polyacrylamide gels run under non-reducing conditions of the F(ab') 2 fragments produced in this way showed no detectable differences from F(ab') 2 produced by pepsin digestion (data not shown). The immunoreactivity of F(ab') 2 preparations was assessed by an indirect ELISA against the CEA antigen. Antibody bound to CEA-coated 96-well plates (0.2 p.g CEA/well, Dynatech M129H plates) was measured by goat anti-mouse Fab and compared with an ASB7 F(ab') 2 standard using orthophenylene diaminc (OPD) as substrate. The developed plates wcrc read at 450 nm using a Titertek 'Multiscan' reader and the binding curves fitted using 'Titersoft II' software. Typically, conjugate showed greater than 90% retention of full antigen binding capacity. Determination of levels of maleimide incorporation &to CPG 2 Stock CPG 2 solution (12.5 mg/ml) was transferred to PBS (pH 7.2) using disposable columns of Sephadex G25 (PD10 columns, 10 ml bed volume, Pharmacia). 2.5 ml samples of CPG 2 were loaded onto the columns and eluted with 3.5 ml PBS. The recovered CPG 2 was then reassayed for protein content using the Coomassie Blue G250 mcthod (Bradford, 1976) with bovine serum albumin fraction V (Sigma) as standard. Aliquots

51 of CPG~ solution (1.0 ml containing 8.9 mg) were reacted with SMPB at various concentrations, added as a 0-14-fold molar excess with respect to CPG2 in the form of an 8.6 m g / m l solution in a total volume of 70 /xl dimethyl sulphoxide (DMSO). The reaction was allowed to proceed for 60 min and was then stopped by a further buffer exchange step using a NAP5 (2.7 ml bed volume) prepacked Sephadex G25 column (Pharmacia). The protein concentration was then remeasured and maleimide incorporation determined by measuring the decrease of the thiol content of a known amount of/3-mercaptoethanol upon addition of activated CPG 2 solution, after thc method of Sedlak and Lindsay (1968).

Determination of the effect of SMPB modification on CPG 2 activity Aliquots of CPG 2 were reacted with SMPB as described above and the reaction stopped at various times by diluting 150-fold with 0.05 M glycine. The samples were allowed to stand for 10 rain and were then assayed for CPG 2 activity using 0.06 mM methotrexate (Lederle), as previously described (Sherwood et al., 1985).

Determination of thiol incorporation into A5B7 F(ab 92 Aliquots of purified F(ab') 2 fragment (0.5 ml of 6.67 m g / m l in PBS) were reacted with SATA at various concentrations, added as a 0-10-fold molar excess in DMSO. The reaction was allowed to proceed for 40 min and was terminated by buffer exchange to PBS using a NAP5 column as above. Thioacetyl residues inserted into F(ab') z were deprotected by the addition of hydroxylamine (0.1 ml of 0.5 M in 0.2 M sodium phosphate buffer, pH 7.5) and incubation for 60 rain. The protein content of each sample was then determined using the Coomassie Blue method. Free thiol groups present on the F(ab') 2 preparation were then measured using Ellman's reagent, dithio-bis(2-nitrobenzoic) acid (Ellman, 1959).

Determination of effect of thiol incorporation on A5B7 F(ab')• antigen-binding capacity Aliquots of purified F(ab') 2 (0.2 ml of 10.3 m g / m l in PBS) were reacted with SATA at various concentrations, added as a 0-10-fold molar

excess in DMSO (50 ~tl) and made up to a total volume of 0.5 ml with PBS. The reaction was allowed to proceed for 30 min and was then terminated by buffer exchange to PBS using NAP 5 columns yielding 1.0 ml active fraction (activated F(ab')2). 0.2 ml hydroxylamine solution (0.5 M in 0.2 M sodium phosphate buffer containing 0.025 M E D T A ) was then added to liberate free thiol and the mixture allowed to stand for 40 min. 0.5 ml of 0.1 M N-ethylmaleimide was then added and allowed to react with the thiol residues for 30 min to prevent non-specific binding via these residues in subsequent ELISA experiments. The protein concentrations of the samples were then determined using the Coomassie Blue method as above, and the antigen binding activity of the modified F(ab') 2 determined against CEA as described above, using the 0-fold molar excess (reagent blank) as control.

Effect of protein concentration on aggregate formation CPG 2 (2.5 ml of 16.0 m g / m l ) and F(ab') 2 (2.5 ml of 16.0 m g / m l ) were reacted with a five-fold molar excess of SMPB and a four-fold molar excess of SATA respectively, added as a solution in 50 /xl DMSO. The reaction of SATA with F(ab')/ was terminated after 40 rain by buffer exchange into PBS, using a PDI0 column. Inserted thioacetyl residues were deacetylated by reaction with hydroxylamine, as described above, for 40 min. The reaction of SMPB with CPG 2 was started simultaneously with that of S A T A / F ( a b ' ) z and was similarly terminated by buffer exchange after 90 min. CPG z and F(ab')~ were then mixed in equimolar ratios in a range of total protein concentrations from 1.0 to 10 mg/ml, with dilution by PBS as appropriate. The coupling reaction was allowed to proceed for 16 h at room temperature, after which glycine (1 ml of 0.5 M) was added to block further reaction. The molecular weight distribution of the conjugate product was determined by gel filtration chromatography using a Superose 12 column (HR 10/30 column, Pharmacia). Peak data were collected using a Pharmacia LCC500plus integrator and used to calculate the relative areas of peaks corresponding to high molecular weight aggregate, conjugate, and uncoupled protein.

52

Effect of reagent substitution levels on conjugate formation The effect of different reagent substitution levels on the formation of 1:1 conjugate was investigated by reacting fixed amounts of CPG 2 (2.5 ml, 6.9 mg) and F(ab') 2 (2.5 ml, 8.5 mg) with a range of molar ratios of SMPB and SATA respectively, added as solutions of the appropriate concentration in 50/zl DMSO. The activation reactions were terminated by buffer exchange and protected thiol residues deactylated with hydroxylamine as previously described. The activated CPG 2 and F(ab') 2 were mixed at a final concentration of 2 m g / m l and allowed to react for 16 h at room temperature. Samples of the incubation mixtures were analysed by gel filtration chromatography using a Superose 12 column (HR 10/30). Relative peak areas were calculated from integrator data for 1 : 1, 1 : 1 plus 2:1 conjugates, and high molecular weight aggregates as percentage of total protein elutcd.

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Fig. 1. The relationship between molar excess of SMPB to CPG2 and the number of active maleimide residues inserted per CPGz molecule.

Results

Determination of the effect of SMPB modification on CPG e actit,ity

Determination of levels of maleimide incorporation into CPG 2

The effect of increasing substitution levels with SMPB on CPG 2 specific activity is illustrated in Fig. 2. As substitution levels increased, the specific activity of the enzyme decreased. At low

The effect of reacting a fixed quantity of CPG 2 with escalating amounts of SMPB is illustrated in Fig. l, in which the measured number of active maleimide residues incorporated into CPG 2 is plotted against the molar excess of SMPB reagent with respect to CPG 2. At 5-10-fold molar excess, the incorporation of active residues was virtually stoichiometric. At low concentrations of modifying reagent, however, although there was some evidence of saturation of the available sites at high substitution levels. At low substitution levels the incorporation of maleimide was less efficient. This was not due to a lack of sensitivity of the assay at lower levels of substitution. Under the conditions described the assay for maleimide was linear over the range 1 × 10-6-2.5 × 10 -4 M (M. Browne and R. Melton, unpublished data), with the maleimide concentration in the modified CPG 2 being about 1 × 10 -s M at 2-4-fold molar excess SMPB.

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53

levels of modification there was relatively little loss of enzyme activity. W h e n higher levels of coupling reagent were used there was pron o u n c e d loss of activity, but at very high levels (greater than ten-fold molar excess of reagent) the loss of activity b e c a m e less p r o n o u n c e d , resulting in a sigmoidal curve for activity versus reagent excess. However, when enzyme specific activity was plotted against m e a s u r e d maleimide incorporation (Fig. 3), a somewhat different curve was obtained, indicating that the most pron o u n c e d losses of enzyme activity werc incurred at relatively low ratios of reagent to enzyme, but that high levels of substitution did not result in further extensive damage. From these results it was c o n c l u d e d that an o p t i m u m ratio of S M P B : C P G 2 was 5 : 1, i.e., approximately three maleimide residues per C P G z molecule, with the loss of about 30% of enzyme activity. Higher substitution levels resulted in an increased loss of enzyme activity and increased aggregation, whilst the use of lower levels resulted in less efficient coupling. T h e effect of duration of incubation at different molar excesses of S M P B is presented in Fig. 4. T h e results of this experiment show that the activation reactions were rapid and were essentially complete within 10-15 min. With higher molar excesses of S M P B there was some further 500

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Determination ,of thiol incorporation into A5B7 F(ab')e The incorporation of thiol residues into F ( a b ' ) e (Fig. 5 ) w a s linear over the range 0-10-fold molar excess S A T A measured. In contrast to SMPB, the incorporation of thiol residues was not stoichiometric over the range measured, incorporation occurring at the rate of about 0.4 thiol group per IgG molecule per mole of S A T A used. T h e optimal level of S A T A : F ( a b ' ) 2 was assessed as 4 : 1, high levels presenting an increased risk of aggregate formation.

Effect of thiol incorporation on antigen-binding capacity of A5B7 F(ab') 2

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A comparison of the ability of thiolated F ( a b ' ) 2 to bind to antigen-coated plates was carried out, and the results of this experiment are presented in Fig. 6. These results suggest that there was an a p p a r e n t increase in binding activity due to thiol incorporation, which b e c a m e more p r o n o u n c e d as thiol substitution increased. It is unlikely that this effect was due to e n h a n c e d non-specific bind-

54

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ing to the plates via incorporated thiol residues, since these were blocked by reaction with N-ethylmaleimide prior to commencing the assay; simi-

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larly, the plates were blocked by incubation with casein after coating with antigen.

Effect of protein concentration on aggregate formation High molecular weight aggregates were present in some experiments as a peak which eluted at the void volume of the Superose 12 column, implying molecular weights in excess of 600,000. The effect of protein concentration on aggregate formation was studied at molar ratios of 5 : 1 for SMPB : CPG z and 4 : 1 for SATA : F(ab') 2. The results of this experiment (Fig. 7) showed that the formation of high molecular weight aggregates became pronounced as the protein concentration in the reaction mix increased, and that high concentrations were not appropriate for the formation of 1 : 1 conjugate in high yields.

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Effect of reagent substitution levels on conjugate formation The effect of varying the ratios of coupling reagent to proteins are presented in Table I, which shows the percentage of protein eluted from the Superose 12 analytical column as a mixture of 1:1 antibody:enzyme conjugate, the

55 TABLE I T H E EFFECT OF V A R I O U S MOLAR EXCESSES OF H E T E R O B I F U N C T I O N A L R E A G E N T S TO CPG 2 AND lgG ON T H E YIELDS OF 1:1 AND 2:1 C O N J U G A T E S AND F O R M A T I O N OF H I G H MOLECULAR W E I G H T A G G R E G A T E AS D E T E R M I N E D FROM PEAK AREAS OF GEL F I L T R A T I O N C H R O M A T O G R A M S RUN ON SUPEROSE 12 H R I 0 / 3 0 SATA : IgG

SMPB : CPG 2

2: 1 + 1 : 1 (% total)

1: 1 (% total)

Aggregate (% total)

15 10 5 3 1.5 5 5 2.5 2 1.5

8 5 5 5 5 10 1.5 7.5 2 1.5

53.5 46.3 37.3 36.2 25.8 54.4 45.7 51.0 32.9 34.4

26.8 22.0 16.5 21.4 18.8 30.1 26.5 25.9 20.8 22.7

16.2 37.3 8.7 n.m. n.m. 18.1) n.m. 6.1 2.0 n.m.

a Not measurable.

percentage of the desired 1:1 conjugate in the mixture, and the percentage of high molecular weight aggregates formed, estimated from peak areas. The relative yields obtained for the various components of the crude mix remained reasonably constant despite the various substitution ratios. With high levels of substitution there was an increase in total yield, but the proportion of 1 : 1 conjugate did not similarly increase, and there was also an increased tendency towards the formation of high molecular weight aggregates. At very low substitution levels there was little formation of aggregate, but the coupling efficiency was lower.

Discussion

The experiments described in this paper were designed to investigate the optimum protocol for the production of 1:1 a n t i b o d y : C P G 2 conjugate on a multigram scale for use in clinical studies of the A D E P T system. The coupling studies were, therefore, carried out using protein concentrations which were likely to be required for largescale manufacture, and with the aim of developing a rapid and easily scaled protocol.

It is clear from the results that the two proteins responded quite differently to substitution by what is effectively the same reactive group in each case, the N-hydroxysuccinimide ester moiety of SMPB or SATA. In the case of C P G 2 there was pronounced loss of enzyme activity at relatively low substitution levels, whereas F(ab') 2 showed little or no loss of activity even when treated with high levels of reagent. To some extent this was reflected in the differing levels of active moieties measurable as having been incorporated, with approximately 1 mol m a l e i m i d e / mol SMPB incorporated, compared with a corresponding ratio of 0.4 mol thiol per mol SATA. Equally, it was apparent that the use of high levels of substitution did not increase the yields of conjugate to any pronounced degree. These results lead, therefore to the conclusion that it is preferable to use relatively low levels of substitution, particulary if one of the proteins to be coupled - in this case C P G 2 - is sensitive to the modifying reagent. The yields typically obtained in these experiments are comparable with those reported by a number of other workers (Searle et al., 1986; Thorpe et al., 1984; Bernhard et al., 1983) for antibody:enzyme or antibody : toxin conjugates. The one p a r a m e t e r which did appear to be crucial was the concentration of the proteins at the mixing stage, where too high a concentration resulted in extensive aggregate formation, even when fairly low levels of substitution were employed. When higher levels of activation were used aggregate formation became more pronounced at lower concentrations. The maximum protein concentration in the incubation mix compatible with obtaining optimal yields was about 2 mg/ml. The optimised coupling conditions described in this paper have been directly scaled up for the production of A5B7 F(ab') 2 : CPG 2 conjugate on a 10-12 g batch size (6.5 g F(ab') 2 and 5 g CPG 2) with no problems (Melton et al., in preparation) and have proved to be effective in producing acceptable yields of 1 : 1 conjugate for these early studies, but for the A D E P T system to be generally viable more efficient ways of producing the conjugate will need to be developed. The first report in the literature describing the construc-

56 tion o f an a n t i b o d y ( F a b ) - e n z y m e c o n j u g a t e for use in A D E P T has a l r e a d y a p p e a r e d ( B o s s l e t et al., 1992) a n d this m a y be i n d i c a t i v e o f t h e f u t u r e a p p r o a c h to t h e c o n s t r u c t i o n o f c o n j u g a t e s for A D E P T , b u t it is n o t yet c l e a r w h e t h e r c o n j u g a t e s c o n s t r u c t e d f r o m F a b l o c a l i s e as e f f i c i e n t l y as t h o s e p r e p a r e d u s i n g F ( a b ' ) 2. If F ( a b ' ) 2 c o n j u g a t e s d o p r o v e to give t h e o p t i m u m c o n j u g a t e s for clinical use, c o n s t r u c t i o n by p r o t e i n e n g i n e e r ing t e c h n i q u e s m a y p r o v e m o r e difficult. T h e c h e m i c a l l i n k a g e t e c h n i q u e s d e s c r i b e d in this p a p e r m a y t h e r e f o r e c o n t i n u e to h a v e utility for t h e foreseeable future.

Acknowledgements T h e a u t h o r s wish to t h a n k t h e C a n c e r R e s e a r c h C a m p a i g n for t h e i r s u p p o r t o f this res e a r c h u n d e r G r a n t s SP1391 a n d SP2135.

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