Journal of Immunological Methods, 134 (1990) 139-149 Elsevier

139

JIM 05743

Development of technology for linking photosensitizers to a model monoclonal antibody F r a n k N. Jiang 1, Shiyi Jiang 3, Daniel Liu 2, A n n a Richter 1 and Julia G. Levy 1 i Department of Microbiology, University of British Columbia, 300-6174 University Blvd, Vancouver, B.C. V6T 1 W5, Canada, 2 Quadra Logic Technologies, Inc., 520 W 6th A venue, Vancouver, B.C. V5Z 9tt5, Canada, and 3 Department of Chemistry, Shanghai Medical University, Shanghai, People's Republic of China

(Received 13 April 1990, revised received 12 July 1990, accepted 13 July 1990)

A procedure is described whereby the photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA) was covalently linked to a model monoclonal antibody in a manner which is reproducible, quantifiable, and retains both the biological activity of the antibody and the cytotoxicity of the photosensitizer. Preliminary steps involved the linkage of BPD-MA to a modified polyvinyl alcohol (PVA) backbone, followed by conjugation to the antibody using heterobifunctional linking technology. Briefly, polyvinyl alcohol (MW ca. 10,000) was modified with 2-fluoro-l-methyl pyridinium toluene-4-sulfonate and 1,6-hexanediamine to produce side chains containing free amino groups. The free carboxyl group of BPD-MA was utilized to conjugate photosensitizer molecules to modified PVA using a standard carbodiimide reaction. Final linkage of the PVA-BPD to a model monoclonal antibody involved further substitution of the carrier with 3-mercaptopropionic acid and carbodiimide to introduce 3-4 sulfhydryl residues per carrier molecule, and introduction of sulfo-m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester residues to the monoclonal (3-4 residues/molecule). Conjugation was effected by reaction of the two species at pH 5.5 for 18 h. Detailed methodology and tests for efficacy of the procedure are provided. Key words: Photosensitizer conjugation; Phototoxicity; Polyvinyl alcohol; Benzoporphyrin derivative; Monoclonal antibody

Introduction Correspondence to: J.G. Levy, Department of Microbiology, University of British Columbia, 300-6174 University Blvd., Vancouver, B.C. V6T 1W5, Canada. Abbreviations: BPD-MA, benzoporphyrin derivative monoacid; c.p.m., counts per minute; DMSO, dimethyl sulfoxide; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; FMP, 2,2-fluoro-l-methylpyridinium toluene-4sulfonate; HCG, human chorionogonadotropic hormone; HPLC, high performance liquid chromatography; MoAb, monoclonal antibody; 3-MPA, 3-mercaptopropionic acid; MPVA, modified polyvinyl alcohol; MTT, tetrazolium salt; PEG, polyethylene glycol; PDT, photodynamic therapy; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; SMBS, sulfo-m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester; TLC, thin layer chromatography.

Photodynamic therapy (PDT) is an experimental cancer treatment modality that selectively destroys cells by interaction between absorbed light and retained photosensitizing agent (Dougherty, 1987; Manyak et al., 1988). The treatment involves the administration of the photosensitizing drug following which visible light is focused on the tumor. Photosensitizer, when exposed to activating light, produces singlet oxygen - a reaction resulting in high levels of cytotoxicity. Although photosensitizers have been found in a number of studies to have a greater affinity for malignant cells than for normal cells (Spikes,

0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

140 1984), their selectivity for cancerous tissues is not optimal. They also accumulate in normal tissues and may be retained in the body for a considerable period of time. To achieve higher specificity of photosensitizers for cancerous and possibly other targeted tissues, improvement in their delivery to the intended target is worth pursuing. This can be achieved in a number of ways. Using monoclonal antibodies (MoAbs) as a delivery system is one of them. MoAbs which react with antigens associated with malignant cells offer an approach for targeting anti-cancer agents. Preliminary experiments in which photosensitizers were conjugated to MoAbs have established the feasibility of this approach (Mew et al., 1983, 1985; Steele et al., 1988). Early experiments usually involved conjugation of photosensitizers directly to MoAb molecules. This approach, while successful in many instances, can result in significant losses in biological activity of the antibody, due probably to interference in the antigen-binding region by photosensitizer molecules. In our hands, reproducibility using this approach has been poor, and loss of antibody activity was frequently significant. The approach described below involves the conjugation of a limited number (one to three) of carrier molecules to a model MoAb using a heterobifunctional reagent. The carrier molecule (modified polyvinyl alcohol) had been previously loaded with photosensitizer molecules at a ratio of 25 : 1 (Steele et al., 1989). This procedure was found to be reliable and reproducible and resulted in negligible loss of MoAb specificity and photosensitizer activity. The results are discussed in the context of potential applications of this technology.

Materials and methods

Preparation of modified P VA-BPD conjugates The procedure for this conjugation step has been described previously (Steele et al., 1989; Allison et al., 1990) Briefly, polyvinyl alcohol (PVA, MW = 10,000, Aldrich Chemical Co., Milwaukee, WI 53233, U.S.A.) was modified with 2-fluoro-1methyl pyridinium toluene-4-sulfonate (FMP, Aldrich Chemical Co.) (Ngo, 1986) and 1,6-hexanediamine (BDH Chemicals, Poole, England) to pro-

duce side chains containing terminal free amino groups. This reaction was carried out in dimethylsulfoxide (DMSO; Aldrich Chemical Co.) rather than other, possibly more suitable solvents because PVA is readily soluble in DMSO and essentially insoluble in reagents like dimethylformamide. Ratios were such that each molecule of PVA contained approximately 30-40% substitution of - O H groups, as determined by elemental analysis of the reaction product. Subsequent conjugation of the modified PVA (M-PVA) with BPD-MA (Chemistry Department, University of British Columbia, Vancouver, Canada) was effected by reacting M-PVA in DMSO with a 25-fold molar excess of BPD-MA. Carbodiimide was used as the coupling agent (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HC1 (EDCI, Sigma Chemical Co., St. Louis, MO 63178, U.S.A.) to bring about peptide bond formation (Kurzer et al., 1967) between the free amino groups of M-PVA and the free carboxyl group of BPD-MA. Analysis was carried out spectrophotometrically following dialysis of the product. Final preparations were lyophilized, and stored at 4°C in drierite in the dark. Carrier conjugates were analyzed by HPLC. The HPLC system used (column: ultrasphere 5/zm ODS, 250 x 4.6 mm; solvent system: solution A, 1% (NH4)2SO 4 500 ml, CH3CN 500 ml and CH3COOH 50 ml, pH 3.0; solution B, 1% (NH4)2SO 4 500 ml, C 4 H 8 0 500 ml and CH3COOH 50 ml; flow rate: 1.7 ml/min.; column temperature: 30°C with pressure between 2600 and 3000 psi; gradient: A : B = 6 0 % : 4 0 % for 5 rain, then starting gradient flow from 40% B to 70% B in 20 min and staying at 70% B for 5 min then returning to 40% B) effected elution of the M-PVA-BPD-MA conjugate at 8-10 min whereas the unconjugated BPD-MA isomers eluted at 18 and 19 rain. Efficiency of the conjugation reaction between M-PVA and BPD-MA was determined to be greater than 95% as determined by spectrophotometric and HPLC analyses. The reactants used and the resulting reactions are outlined in Fig. 1. The molecular weight of the conjugate was estimated to be approximately 28 kDa.

Introduction of thiol groups to M-PVA-BPD-MA The procedures described in this manuscript were based on plans to couple M-PVA-BPD-MA

141

to MoAbs using heterobifunctional reagents. In order to render the M-PVA-BPD-MA carrier complex reactive with such reagents, it was reacted with 3-mercaptopropionic acid (3-MPA, Aldrich Chemical Co.) in the presence of 0.8 mmol carbodiimide at molar ratios of 1:5, 1 : 1 0 and 1 : 20 (PVA : 3-MPA). Our goal was to introduce thiol ( - S H ) groups to the carrier system such that we would get suitable reactions with the heterobifunctional reagent selected (reaction is shown in Fig. 2). The - S H substituted carrier (M-PVA-

CH21 CHOH t CH2 CHOH

GT -I-

F

sO"

CHa Pyridine/DMSO

BPD-MA-SH) was dialysed and reacted with an excess of [2,3J4C]maleic anhydride (New England Nuclear; molar ratio was 1 : 10, SH : maleic anhydride) in order to quantify the actual number of thiol groups present on carrier molecules. The reaction (Fig. 3) was allowed to take place for 2 h with stirring at room temperature following which reacted material was dialysed exhaustively against acetate buffer (0.01 M, p H 5.5). Controls constituted M-PVA-BPD-MA preparations which had not been substituted with 3-MPA but which were exposed to aac-maleic anhydride and treated exactly as the substituted material. After dialysis, aliquots were transferred to 20 ml scintillation vials with 15 ml of Aquasol. Samples were counted in a Beckman liquid scintillation system (Beckman, LS 3801, Beckman Instruments, Fullerton, CA 92534, U.S.A.). All the reactions were carried out under argon and all the buffers were degassed with argon before use.

Selection of heterobifunctional cross-linking reagent

CHO--~,,.~

d:.= I

CHOH

'r ~,o CHa

1,6-Hexanediamine

CH3 I

CH-NH-CH2(CH2)4CH2NH2 I

CHOH

g, I

O

BPD-COOH/EDCI

I

CH-NH-CH2(CH2),=CH2-NH-CO-B PD I jCH= CHOH I CHz IM'PVA'BPD'MA I Fig. 1. Scheme for production of the M-PVA carrier system. PVA was modified with 1,6-hexanediamine (shown previously to yield 30-40% substitution) following which it was reacted with BPD-MA in the presence of carbodiimide to yield a 1 : 25 ratio of M-PVA-BPD-MA.

Since 3-MPA was to be the thiol group to be used in the reaction, it was necessary to determine the reactivity of this molecule with various crosslinkers commercially available. 3-MPA was added to: sulfo-m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (SMBS, Pierce, Rockford, IL, 61105, U.S.A.), sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate (S-SMCC, Pierce) and sulfo-succinimidyl 4-(p-maleimidophenyl) butyrate (S-SMPB, Pierce) at a molar ratio of 1 : 1. Reactions were qualitatively analyzed at 0, 15, 30, 60 and 120 min by monitoring the relative intensities of the fluorescent products, measured at 254 nm in a UV light box (Spectrolin, Westbury, NY, U.S.A.). SMBS was chosen as the cross-linking agent for further studies because of the efficiency of its reaction with 3-MPA.

Selection of model monoclonal antibody The monoclonal antibody (MoAb) selected for this study has a specificity for human chorionogonadotropic hormone (HCG). This MoAb (T48, Quadra Logic Technologies, Vancouver, Canada) was chosen because its binding properties had been well characterized. Also, its activity was easy to monitor by a standard enzyme linked immunosorbent assay (ELISA) making quantitative assess-

142 CH2 ~H-NH-(CH2)6-NH2

MoAb

CH2

CH-NH-(CH2)s-NH-CO-BPD CH2 CH-NH-(CH2 s-NH2

,CH2

CH-OH

6

I

MoAb-SMBS

I I HSCH2CH2COOH

i CHz ~H-NH-(CH2Is*NH-CO-CH2-CH2-SH

CH2

CH-NH-(CH2)s-NH-CO-BPD

,CH2

CH-NH-(CH2)s-NH2

,CH= CH-OH

6

M-PVA(SH)-BPD-MA I CH2 0 6H'NH'(CH2)s'NH-CO'CH2"CH2"S'I~ (~H2 IN,-('( CH-NH-(CH;~),-NH-CO-BPD ~ ~H2 O ~H-NH-(CH2)s-NH2

)) CO-NH-MoAb

cH~ CH-OH

Fig. 2. Scheme for conjugation of MoAb-SMBS with the M-PVA-BPD-MA-SH carrier system (see text for details).

ment of binding properties through all steps of the procedures easy to carry out.

S M B S labelling the T48 MoAb The T48 M o A b (0.5 m g in 50 #1) was reacted with SMBS at molar ratios of I : 5, 1 : 10 and 1 : 30 ( M o A b : S M B S ) for 45 min at 37°C in 0.01 M carbonate buffer, p H 8.5. In order to determine the actual n u m b e r of SMBS groups b o u n d to the M o A b , the preparations, after dialysis were mixed with L-[UJ4C]cysteine (Amersham Co., Arlington Heights, IL 60005, U.S.A.) at a molar ratio of 1 : 100 ( M o A b : cysteine) (Fig. 4). The mixtures as well as controls ( M o A b : c y s t e i n e mixtures which had not been previously reacted with SMBS) were

subjected to three rounds of dialysis with Centricon-30 (Amicon, M A 01923, U.S.A.) using 0.01 M acetate buffer, p H 5.5, and further dialysed against the same buffer. Samples were c o u n t e d on a Beckm a n liquid scintillation counter.

Preparation of M - P VA-BPD-MA-MoAb conjugates (.4) Introduction of thiol groups onto the M - P VABPD-MA. It was established that molar ratios of I : 10 ( M - P V A - B P D - M A : 3-MPA) introduced between three and four thiol groups per carrier molecule. Therefore, material to be conjugated to M o A b was reacted with 3 - M P A at this molar ratio in the presence of 0.8 m m o l E D C I in D M S O . The reaction (Fig. 2) was stirred for 4 h at r o o m

143 BPD-MA-M-PVA-SH

[2, 3 - 14C]Maleic Anhydride

BPD'MA'M'PVA'S~o

0

0

temperature under argon following which it was dialysed also under argon against 0.01 M acetate buffer (pH 5.5). (B) Reaction of T48 with SMBS. A molar ratio of 1 : 30 (MoAb : SMBS) was used since this procedure was found to introduce between two and three SMBS groups to the MoAb with no loss of MoAb activity (see below). T48 at a concentration of 9.615 m g / m l was dialysed overnight against 0.01 M carbonate buffer, pH 8.5. SMBS was added to this in the same buffer at a 30-fold molar excess. The mixture was stirred for 2 h after which it was washed through a Centricon-30 and the buffer was changed to 0.01 M acetate (pH 5.5).

(C) Conjugation of M-PVA-BPD-MA-SH to MoAb-SMBS. The final reaction (Fig. 2) be-

I BPD-MA-M-PVA-[2,3 - 14C]Maleic Anhydride Fig. 3. Scheme by which availability of reactive -SH residues was tested in the M-PVA-BPD-MA-SHcarrier. The carrier was reacted with 14C-labelled maleic anhydride following which it was dialysed and tested for bound 14C.

tween the carrier and T48 was carried out by mixing the two materials at equimolar concentrations in acetate buffer (0.01 M, pH 5.5) and stirring gently for 18 h at 4°C. Materials were concentrated by dialysis against polyethylene glycol (PEG).

Thin layer chromatography (TLC)

M°Ab-NH-COo~O

÷NI-Ia.~CH.~CO2" ,I CH2

+

I SH

L - [U-14C]Cysteine

In order to monitor rapidly whether the anticipated reactions had taken place, aliquots of test materials were routinely run in a silica TLC system using a solvent mixture of ethylacetate : ethanol : H 2 0 (2 : 1 : 1). Controls consisted of free M-PVA-BPD-MA and free MoAbs, as well as mixture of the various reactants. Plates were observed in a UV light box (254 nm).

Separation of conjugates by gel filtration

M°Ab-NH-CO O~

O

S *.ICH2 +NH~--CH C0£ i

•

I

MoAb.SMBS-L-[U°14C] Cysteine

Fig. 4. Scheme by which availability of SMBS residues was tested in the MoAb-SMBSconjugate. The conjugate was reacted with ~4C-labelled cysteine in a 100-fold molar excess. Bound cysteine was determined followingreaction and dialysis.

A Sepharose CL-4B (Pharmacia LKB, Uppsala, Sweden) was used to separate conjugated from unconjugated materials. The column had a bed volume of 68.4 ml and was equilibrated with acetate buffer (0.01 M, pH 5.5). In early studies we experienced some difficulties in developing a chromatographic procedure for separating PVAconjugated materials, since PVA and PVA-BPD conjugates (including MoAb-PVA conjugates) have a marked tendency to stick to essentially all cross-linked dextrans. Subsequently we found that the addition of 0.5% (final concentration) low molecular weight (2000) PVA to materials to be chromatographed, successfully blocked this reaction and permitted appropriate elution of the de-

144

sired product. Therefore all samples to be chromatographed were made up to 0.5% ( w / v ) PVA before addition to the column. Samples to be run were added to the column in volumes of 1.0 ml or less and eluted at room temperature at a flow rate of 0.43 m l / m i n . Fractions of 2 ml were collected and analyzed at both 280 nm and 688 nm using a spectrophotometer (LKB Ultrospectrophotometer 4050).

Analysis of materials by gel electrophoresis (SDSPAGE) In order to determine definitively whether the heterobifunctional cross-linkers had actually formed covalent bonds between the MoAb and carrier system, materials eluting from the Sepharose column were subjected to analysis by reducing and SDS-PAGE according to standard procedures using a 7.5% minigel. Gels were run at 30 MA for 45 min and silver stained as described (Oakley et al., 1980).

ELISA The specific activity of the M o A b was monitored at every step in order to determine whether any of the procedures resulted in loss of activity. A standard ELISA was used for this purpose. Immulon II (Dynatech Lab, Virginia, U.S.A.) ELISA plates were coated with the antigen ( H C G , Sigma Chemical Co.) at a concentration of 5 /~g/ml in a volume of 100 ttl/well in bicarbonate buffer (0.1 M, p H 9.6). Test M o A b preparations and controls were titrated over these plates. Plates were developed with alkaline phosphatase-labelled rabbit anti-mouse Ig (Jackson I m m u n o Research Lab) and the p-nitrophenyl phosphate substrate. Plates were read at 405 nm on a Titertek Multiskan plate reader (Flow Lab). Plates were always run with a standard preparation of T48 so that quantification of test materials could be carried out.

In vitro tests of photosensitizing activity M-1 tumor cells, in single cell suspension, obtained from a freshly prepared excised, s.c. grown tumor, were plated in 96-well Falcon plates in 200 /~1 D M E (Gibco, G r a n d Island, NY) containing 10% FCS (FCS, Sigma Chemical Co., St. Louis, MO) at a concentration of 105 cells/well. 24 h

later the culture medium was changed, and at 48 h the cytotoxicity test was performed as described in detail earlier (Richter et al., 1987). Briefly, the cells were washed and then incubated with various concentrations of BPD-MA, M - P V A - B P D - M A and conjugate for 1 h at 37°C in the dark in the absence of serum. Following the incubation the cells were exposed to light for 1 h (4 J / c m 2) and then incubated further in DME-5% FCS at 37°C in the dark, in a CO 2 humidified incubator for 18 h. At that time the viability of the cells was tested using M T T (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tertrazolium bromide, Sigma) as described by Mosmann et al. (1983), and % killing calculated for each tested material. LDs0 for the cell line was determined. Care was taken throughout the procedure to shield the porphyrin solutions and microtiter wells containing porphyrins from light, except as described.

Ab activity in conjugate To determine the antibody activity in final conjugates, it is essential to know the Ab concentration so that the amount of conjugate could be directly compared with standard Ab. However, Ab concentration can not be accurately measured by reading the conjugation product at 280 nm since PVA also absorbs at 280 nm. Therefore, it was measured by the Lowry method (Lowry et al., 1951). The concentration of Ab in the conjugate was usually below 800 # g / m l , at which level the concentration of PVA was below 50 # g / m l . At this concentration PVA causes negligible interference in Lowry method. A standard curve was made using Ab and M - P V A - B P D - M A mixed at molar ratio of 1 : 1, as in the conjugation mixture. 50 #1 of final conjugation product was measured by Lowry method and Ab concentration determined from the standard curve.

Results

Testing the M-PVA-BPD-MA-SH conjugates The M-PVA-BPD-MA work constituted material stituted at a molar ratio (Steele et al., 1989; Allison

conjugate used in this which had been subof 1 : 2 5 ( P V A : B P D ) et al., 1990) and shown

145 TABLE I RESULTS OF EXPERIMENTS TO DETERMINE THE PRESENCE OF AVAILABLE THIOL GROUPS ON MPVA-BPD-MA-SH CONJUGATES M-PVA-BPD-MA conjugates were reacted with 3-mercaptopropionic acid (3-MPA) in the presence of carbodiimide at molar ratios of 1:5, 1 : 10 and 1 : 20 (PVA : 3-MPA). These -SH substituted conjugates (M-PVA-BPD-MA-SH) were further reacted with a ten-fold molar excess of 14C-labelled maleic anhydride (-SH:maleic anhydride) to determine the actual number of thiol groups present on carrier molecules. Unsubstituted M-PVA-BPD-MA served as control. The actual readings for each preparation are presented. The results show that reaction with five-fold molar excess of 3-MPA produces a cartier system with one available -SH group per molecule while ten-fold and 20-fold molar excesses of 3-MPA produce 3-4 and 7-8 available -SH groups, respectively. Molar ratios of M-PVA-BPDMA : 3-MPA

c.p.m,of product

Calculated substitutions (molar equivalents)

1:5 1:10 1:20 1:0 (control)

109,410 253,760 457,020 51,017

1.08 3.83 7.50 -

to h a v e c o u p l e d at greater t h a n 95% efficiency. F u r t h e r m o d i f i c a t i o n of the M - P V A b a c k b o n e was effected by r e a c t i o n with 3 - m e r c a t o p r o p i o n i c acid ( 3 - M P A ) in o rd er to i n t r o d u c e thiol groups to the conjugate. T h e M - P V A - B P D - M A molecules were re a c t e d at m o l a r ratios of 1 : 5 , 1 : 1 0 a n d 1 : 2 0 ( P V A : 3-MPA). T h e n u m b e r of free - S H groups a va il a b l e for sulfide b o n d f o r m a t i o n on the carrier m o l e c u l e s was d e t e r m i n e d by reacting the c o m p l e t e d c o n j u g a t e with 14C-maleic a n h y d r i d e u n d e r m i l d acidic condition. T h e n u m b e r of 14C-maleic a n h y d r i d e residues associated w i t h the P V A on a m o l a r base was d e t e r m i n e d . T h e results showed that r e a c t i o n w i t h ten-fold m o l a r excesses of 3M P A p r o d u c e d a carrier system with b e t w e e n thr e e an d f o u r available - S H groups per m o l e c u l e ( T a b l e I). This ratio was used s u b s e q u e n t l y in conjugate preparations.

c o n d i t i o n s f a v o u r i n g alkylation. T h e ratios 1 : 5 a n d 1 : 1 0 y i el d ed c o n j u g a t e s in w h i c h b e t w e e n 0 - 2 S M B S residues were associated p er M o A b molecule, w h er eas b e t w e e n 2 - 3 were p r esen t w h e n S M B S was reacted at a 30-fold m o l a r excess. F u r t h e r increase of S M B S d i d n o t increase the substitutions ( T a b l e II). It was felt that this l i m i t ed n u m b e r of residues w o u l d p r o b a b l y be suitable for c o n j u g a t i o n with the carrier. T h e reactivity of T48 w i t h H C G was m o n i t o r e d t h r o u g h o u t these procedures. As sh o w n in Fig. 5, n o n e of the steps i n v o l v e d h ad any m e a s u r a b l e effect on the activity of the M o A b .

Heterobifunctional linkage between the P VA carrier and T48 T h e M - P V A - B P D - M A - S H carrier m o l ecu l es to be reacted with T48 were p r e p a r e d b y r e a c t i n g the M - P V A - B P D - M A m o l ecu l es with a t e n - f o l d m o l a r excess o f 3 - M P A . T h e T48 m a t e r i a l h a d b e e n r eact ed with a 30-fold m o l a r excess o f SMBS. T h e two reactants were m i x e d t o g e t h e r at m o l a r equivalence an d tested s u b s e q u e n t l y for effectiveness of

TABLE II RESULTS OF EXPERIMENTS TO DETERMINE THE PRESENCE OF AVAILABLE SMBS GROUPS ON MoAbSMBS CONJUGATES MoAb T48 was reacted with SMBS at molar ratios of 1:5, 1 : 30 and 1 : 50 (MoAb : SMBS). These resulting mixtures were tested for the efficiency of binding by reacting the MoAb-SMBS conjugates with an excess of 14C-labelled cysteine (MoAb : cysteine = 1 : 100) under conditions favouring alkylation. For MoAb-SMBS 1:5 and 1:10 preparations (experiment 1), 14C-labelledcysteine only was used. In experiment 2, (MoAb-SMBS 1:30 and 1:50) 20% of the cysteine added was I4C-labelled and 80% was unlabelled. 1 : 10,

Experiment I

AB : SMBS molar ratios

c.p.m,of Calculated product substitutions (molar equivalents)

1:5 1 : 10 MoAb

37,554 65,136 5,711