In vitro and in vivo evaluation of streptavidin immunoconjugates of the second generation TAG-72 monoclonal antibody CC49

Nucl. Med. Bid. Vol. 22, No. I, pp. 77786, 1995

Pergamon

0969-8051(94)EOO70-Y

Copyright 0 1995Elsevier ScienceLtd Printed in Great Britain. All rights reserved 0969-8051!95 $9.50 +O.OO

In Vitro and In Vivo Evaluation of Streptavidin Immunoconjugates of the Second Generation TAG-72 Monoclonal Antibody CC49 WILSON M. NGAI’, Division

RAYMOND M. REILLY’x3*, JEANNIE POLIHRONIS’ and BARUCH SHPITZ2

of Nuclear Medicine’, The Toronto Hospital, Department and Faculty of Pharmacy, University of Toronto 3, Toronto,

of Surgery*, Mount Sinai Hospital Ontario, Canada MSG 2C4

(Accepted 6 April 1994) Streptavidin was conjugated to the second-generation TAG-72 monoclonal antibody CC49 at lysine amino acids, oxidized carbohydrates or reduced disulfides on the immunoglobulin. The streptavidin immunoand their immunological characteristics evaluconjugates were radiolabelled with “‘In-DTPA-biocytin ated in vitro and in ho. FPLC analysis showed a single peak (mol. wt > 350 kDa) for the lysine conjugate and sulfhydryl conjugate (mol. wt N 210 kDa) but multiple peaks (-210 to >350 kDa) for the carbohydrate conjugate. There were only small differences in immunoreactivity against bovine submaxillary mucin in vitro. However, in mice bearing subcutaneous LS174T turnouts, the lysine conjugate exhibited significantly lower tumour uptake (12% i.d./g) compared to the other streptavidinCC49 conjugates (l&40% i.d./g) or DTPA-CC49 (1418% i.d./g). Due to the monomeric nature and smaller molecular size of the sulphhydryl conjugate, and its similar in uitro and in viva characteristics compared with DTPA-CC49, this conjugate has been selected for future pretargeting studies with “‘In and 9oY biotin.

Introduction

hypothesis that tumour/normal tissue ratios can be increased and radiotoxicity minimized by (1) allowing sufficient time for uptake of the bifunctional MAb into the tumour and elimination from normal tissues and (2) attaching the radionuclide to a small, water-soluble hapten which penetrates the tumour readily, binds to a functional group on the tumourbound MAb and is rapidly eliminated from normal organs. The (strept)avidin-biotin system may be used to pretarget radioactivity to tumours using bifunctional MAb. Avidin is a 60 kDa glycoprotein found in egg whites which exhibits a very high affinity (Ka = 1015) for the 244 Da water-soluble vitamin, biotin (Green, 1975). Streptavidin which is isolated from the culture broth of Streptomyces avidinii also binds biotin but contains no carbohydrate residues. Both avidin and streptavidin bind four molecules of biotin (Chaiet and Wolf, 1964). Several clinical studies (Paganelli et al., 1990; Kalofonos et al., 1990) have recently been reported demonstrating the feasibility of imaging tumours using this system. Preliminary data also suggest that the use of the (strept)avidin-biotin system to treat cancer patients in a pretargeted approach results in much less toxicity to the bone marrow than

Radiolabelled anti-tumour monoclonal antibodies (MAb) have been investigated over the past decade for immunoscintigraphy and immunotherapy of cancer patients (Perkins and Pimm, 1991; Reilly, 1991). The clinical application of radiolabelled MAb has been limited however by relatively low tumour/ normal tissue ratios and persistently high levels of radioactivity in the blood pool. The persistent blood pool radioactivity is partially due to the large molecular size of immunoglobulins (Ig) and is the cause of moderate radiotoxicity to the bone marrow when these agents are used for therapy (Stewart et al., 1988). One possible strategy to facilitate elimination of radioactivity from the blood and also improve tumour/normal tissue ratios is the use of bifunctional MAb and radiolabelled small molecular weight haptens in a pretargeting approach (Bamias and Epenetos, 1992). This approach is based on the *All correspondence should be addressed to: Raymond M. Reilly, Division of Nuclear Medicine, The Toronto Hospital, 200 Elizabeth St., Toronto, Ontario, Canada, M5G 2C4. 77

WILSON M. NGAI

78

with direct radioimmunotherapy (Paganehi ef a/., 1993). The majority of immunoscintigraphy trials with the (strept)avidin---biotin system have utilized a 3-step targeting protocol which involves administration of a biotinylated MAb, followed by avidin followed finally by radiolabelled biotin. An alternative, simpler strategy is to utilize a 2-step targeting protocol involving administration of an avidin (or streptavidin) conjugated MAb followed by radiolabelled biotin. Our objective in this study was to evaluate three different approaches to conjugation of streptavidin to the second-generation high affinity MAb CC49 directed against the TAG-72 (tumourassociated glycoprotein-72) antigen expressed in colorectal cancer and several other adenocarcinomas (Muraro er al., 1988). Streptavidin was conjugated to CC49 through lysine amino acids, oxidized carbohydrates on the Fc region or free sulphhydryls generated by chemical reduction of some of the disulphide linkages at the hinge region of the immunoglobulin (Ig). The in uitro and in vivo characteristics of the three different immunoconjugates were then evaluated to determine the optimum streptavidin immunoconjugate for future studies of pretargeted radiolocalization of colon cancer xenografts in nude mice.

Materials and Methods Cell line and tumour xenografts LS174T human colon carcinoma cells were obtained from the American Type Culture Collection (Rockville, MD) and were grown in RPM1 1640 with 10% fetal calf serum (Flow Laboratories, McLean, VA). The cells were recovered with trypsin/EDTA (GIBCO, Grand Island, NY), centrifuged and resuspended in saline, then injected S.C. (l-2 x lo6 cells/ animal) in the lower abdomen into Swiss nujnu mice (Charles River Canada, St Constant, Que.). The LS174T xenografts were allowed to grow for 7-10 days to approx 0.25505 cm in diameter. Monoclonal antibody CC49 Mouse ascites containing the second-generation TAG-72 MAb CC49 (IgG,, Ka = 2.8 x 10”) was supplied by Dr J. Schlom, National Cancer Institute, National Institutes of Health, U.S.A.). The MAb was purified from the ascites by ammonium sulphate precipitation followed by affinity chromatography on a Protein-G Sepharose column (Pharmacia, Baie d’Urfe, Que.). Other materials Affinity purified streptavidin, bovine submaxillary mucin (BSM), DTPA-biocytin, bicyclic anhydride of DTPA, pronase-E and 2-mercaptoethylamine hydrochloride were purchased from Sigma Chemical Co. (St Louis, MO). Sulfo-succinimidyl-4-(N-maleimidomethyl)cycIohexane-I-carboxylate (sulfa-SMCC), 2-

e/ ol

iminothiolane. 2-4’-hydroxyazobenzene-benzoic acid (HABA) and biotin LC-hydrazidc wcrc purchased from Pierce Chemical Co. (Rockford. II,). Sodium 2,4-dinitrophenylhydrazinc and metaperiodate (DNPH) were obtained from Aldrich Chemical Co. (Milwaukee, WI). Disodium EDTA was obtained from Fisher Scientific (Unionville, Ont.). High concentration (> 2000 MBq/mL) “‘In chloride was purchased from Nordion (Ranata, Ont.). Chromatography supplies included Sephadex G-SO and Superose- 12HR columns (Pharmacia), Chelex100 cation exchange resin (Biorad, Richmond. CA) and avidin-agarose resin (Sigma). Centriconmicroconcentrators were obtained from Amicon Canada (Oakville, Ont.). Conjugation of MAb CC49 with streptavidin viu lysine amino acids Streptavidin was conjugated to lysine amino acids on the CC49 MAb by reaction of maleimidederivatized CC49 with thiolated streptavidin as previously described by Sheldon et al. (1992) with some modifications. Maleimide-derivatized CC49 was prepared by reacting the MAb (1 mg in 100 uL of 100mM phosphate buffered saline pH 7.2, PBS) with sulpho-SMCC (2.5 PL of a freshly prepared 10mM solution in PBS) at a molar ratio (sulphoSMCC:IgG) of 3.7: 1 for 1 h at room temperature. Thiolated streptavidin was prepared by reacting streptavidin (200 pg in 20 PL of PBS) with 2-iminothiolane (60 p L of a 10 mM solution in PBS) at a molar ratio (2-iminothiolane:streptavidin) of 225: 1 for 1 h at room temperature. The derivatized CC49 MAb and streptavidin were separated from excess reagents by size-exclusion chromatography on a Sephadex G-50 mini-column eluted with PBS. The purified maleimide-CC49 and thiolated streptavidin were then reacted overnight at 4°C. Conjugation of MAb oxidized carbohydrates

CC49 with streptavidin

viu

Streptavidin was conjugated to the oxidized carbohydrate moities on the Fc region of the CC49 MAb by reaction with biotin LC-hydrazide followed by streptavidin. The carbohydrates on the CC49 Tg were oxidized by incubation of the MAb (1 mg in 100 PL of PBS) with sodium metaperiodate (100 PL of a 20mM solution in 100 mM acetate buffer pH 5.5) at a molar ratio (metaperiodate : IgG) of 300: 1 for 30 min in the dark on ice. The reaction was stopped by purifying the MAb from excess metaperiodate on a Sephadex G-50 mini-column eluted with 100mM acetate buffer. The purified, oxidized MAb was then incubated with biotin LC-hydrazide (5OOpL of a 10mM solution in acetate buffer) at a molar ratio of 750: 1 for 1 h at room temperature. Excess biotin LC-hydrazide was then removed from the mixture and the biotinylated CC49 MAb was reconcentrated by transferring the mixture to a Centricon30 microconcentrator and centrifuging at 1OOOg for

Streptavidin conjugates of monoclonal antibody CC49 15-30 min. After centrifugation, the reconcentrated CC49 MAb was diluted again with 1 mL of acetate buffer directly in the Centriconmicroconcentrator and recentrifuged at 1OOOg for 15-30 minutes. This procedure was repeated three times. Finally, the reconcentrated CC49 MAb in a volume of 100 PL (10 mg/mL) was recovered from the microconcentrator and reacted with streptavidin (100 pg in 10 PL of PBS) at a molar ratio (1gG:streptavidin) of 5: 1. The number of oxidized carbohydrate moieties on the CC49 MAb after treatment with sodium metaperiodate was determined by a spectrophotometric assay using 2,4-dinitrophenylhydrazine (DNPH).* Briefly, 50 mmols of DNPH (in 500 NL of dimethylsulphoxide) was incubated with 0.5 mg of oxidized CC49 MAb in acetate buffer (molar ratio DNPH: IgG of 100: 1) for 2 h at room temperature. The CC49 MAb was then purified from excess DNPH on a Sephadex G-50 column eluted with PBS. The fractions containing the purified MAb were then pooled and the absorbance measured at 280 and 360 nm. The concentration of oxidized carbohydrates was determined by dividing the absorbance at 360 nm by the molar extinction coefficient for DNPH (E,,, = 1.37 x 104). The corresponding concentration of Ig was determined by correcting the absorbance at 280nm for the absorbance of DNPH at this wavelength and then dividing the corrected absorbance by the molar extinction coefficient for murine IgG (E,,, = 2.1 x 105). The number of biotin groups conjugated to the CC49 Ig after reaction with biotin LC-hydrazide was determined by a spectrophotometric assay which involved displacement of 2-4’-hydroxyazobenzenebenzoic acid (HABA) from avidin by biotin (Green, 1965). Briefly, 750nmols of HABA (in 75pL of 10 mM NaOH) was added to 1.5 mg of avidin in PBS (molar ratio of HABA:avidin of 33: 1). The avidin-HABA mixture was then titrated with biotinylated CC49 (previously digested with 1% pronase-E overnight to prevent steric hindrance of the avidinbiotin interaction) or with a standard biotin solution. The change in absorbance of the titrated avidin solution was measured at 500 nm. The concentration of biotin in the biotinylated CC49 solution was determined by comparing the change in absorbance of the avidin solution with that produced by titration with the standard biotin solution. The biotin substitution level was calculated by dividing the concentration of biotin by the corresponding concentration of CC49 Ig in the biotinylated CC49 solution. Conjugation qf MAb reduced disulphides

CC49 with streptavidin

via

Streptavidin was conjugated to the reduced disulphide moieties on the CC49 MAb by reaction with maleimide derivatized streptavidin. Some of the disul-

*Personal communication, NM’322.I -L>

Dr Qi Pei, University

of Alberta.

79

phide linkages on the Ig were reduced by incubation of the MAb (1 mg in 100 PL of PBS) with mercaptoethylamine (5OpL of a 100mM solution in PBS containing 5 mM disodium EDTA) at a molar ratio (mercaptoethylamine: IgG) of 750: 1 for 2 h at room temperature. Maleimide derivatized streptavidin was prepared by reacting streptavidin (200 pg in 20 p L of PBS) with sulpho-SMCC (60 /JL of a 10 mM solution in PBS) at a molar ratio of 222: 1 for 1 h at room temperature. The reduced Ig and maleimide derivatized streptavidin were then purified from excess reagents on a Sephadex G-50 mini-column eluted with PBS. The purified, reduced Ig and maleimide derivatized streptavidin were then reacted for 2 h at room temperature. The number of free sulphhydryl moieties generated by mercaptoethylamine reduction of the disulphide linkages on the CC49 MA\, was determined by a spectrophotometric assay using Ellman’s reagent as previously described by Garron et al. (1991). Briefly Ellman’s reagent in PBS was reacted with an aliquot of purified, 2-mercaptoethylamine treated CC49 MAb at a molar ratio (Ellman’s reagent:IgG) of 10: 1. The absorbance of the reaction mixture was measured at 412 nm and compared with that of known concentrations of cysteine assayed under identical conditions.

qf MAb CC49 with DTPA

Conjugation

MAb CC49 (10 mg/mL) in 50 mM NaHCO, buffer pH 7.5, containing 150mM NaCl) was conjugated with the bicyclic anhydride of DTPA at a molar ratio (chelate : IgG) of 4: 1 as previously described (Reilly et al., 1989). PuriJcation

of’ immunoconjugates

The streptavidinCC49 conjugates were purified by size-exclusion FPLC on a Superose-12HR column eluted with 50 mM phosphate buffer pH 6.5 at a flow rate of 0.4 mL/min. The DTPA-CC49 conjugate was purified on a Sephadex G-50 mini-column eluted with NaHCO, buffer. The purified immunoconjugates were then reconcentrated to 10 mg/mL by centrifugation in a Centriconmicroconcentrator. Radiolabelling

of biotin with “‘In

Biotin was labelled with “‘In to a specific activity of > 130 MBq/pg (1.2 x lo* MBq/mmol) by the addition of “‘In chloride to a solution of DTPAbiocytin (O.O5mg/mL) in 100mM citrate buffer pH 5.5. After a 30 min incubation, radiochemical purity was determined by cation exchange chromatography on a Chelex-100 column eluted with 150 mM NaCl. Reactivity with streptavidin was determined by incuwith an bating a sample of “‘In-DTPA-biocytin equimolar amount of streptavidin in vitro for 30 min. The reaction mixture was then analysed for the relative percentages of radiolabelled streptavidin by size-exclusion and free “‘In-DTPA-biocytin FPLC using a flow-through radioactivity detector

(Beckman Model 170, Fullerton, CA). Alternatively, reactivity of I’ ‘In-DTPA-biocytin was determined by measuring the percentage of radioactivity bound to avidin-agarose resin after a 2 h incubation.

ively, as determined by the amount 01‘ remaining streptavidin in the reaction mixture by FPLC‘ analysis. These conjugation eficiencies correspond to avcrage substitution of the CC49 Ig with 0.3. ! and 0.5 mol of strcptavidin respectively. The conjugation Radiolabelling of‘ immunoconjugates efficiency of CC49 with the bicyclic anhydride of Streptavidin-CC49 conjugates or native strep- DTPA was 25% resulting in a substitution of I mol tavidin were radiolabelled to a specific activity of DTPA/mol of CC49 Ig. There were 2.6 thiol groups generated per molecule of CC49 Ig following of approx 74MBq/mg by incubation with ‘“InDTPA-biocytin for 30min at room temperature. treatment with 2-mercaptoethylamine and 19 aldchyde moieties generated by treatment with sodium DTPA-CC49 was radiolabelled to a specific activity metaperiodate. Approximately 3 mol of biotin LCof 74-148 MBq~mg by incubation with “‘In-acetate pH 6.0 as previously described (Reilly et al., 1989). hydrazide were conjugated to oxidized carbohydrate The radiochemical purity of the ‘“In labelled im- groups on the CC49 Ig. munoconjugates was determined by size-exclusion The immunoconjugates were purified, radioFPLC. FPLC was also used to estimate the molecular labelled and analysed by FPLC. The FPLC chrosize of the radiolabelled conjugates by comparison matograms of the conjugates prepared by linking of their retention times to that of radiolabelled streptavidin to lysine amino acids [Fig. l(A)] compounds of known molecular weights. or reduced disulfides [Fig. l(B)] showed only a single monomeric peak. The approximate molecular immunoreactivity of conjugates weight of the streptavidin immunoconjugates was The in o&o immunoreactivity of the radiolabelled >350 kDa (the upper limit of separation for the Superose-12HR column) when conjugated streptavid~n~~49 and DTPA-CC49 immunoconjugates was determined in a binding assay using bovine through lysine amino acids (lysine conjugation) submaxillary mu& (BSM) as a source of the TAG- and - 2 10 kDa when conjugated through reduced 72 antigen as previously reported (Ngai and Reilly, disulfides (sulphhydryl conjugation). The FPLC 1993). chromatogram of streptavidin-CC49 prepared by conjugation to oxidized carbohydrates [carbohydrate Biodistribution of immunoconjugates conjugation, Fig. l(C)] showed multiple unresolved Swiss nu/nu mice bearing S.C. LS174T colon peaks corresponding to compounds with molecular cancer xenografts were administered radiolabelled weights ranging from m 2 10 to > 350 kDa. The FPLC [Fig. l(D)J streptavidin-CC49 or DTPA-CC49 conjugates or chromatogram of “‘In-DTPA-CC49 native streptavidin (5&100 pg, 1.85-3.7 MBq) by i.p. showed a major peak corresponding to Ig monomer injection. The mice were then sacrificed by cervical (150 kDa) and a minor peak corresponding to a small dislocation at 3 or 6 days p.i. Samples of biood, amount (< 10%) of Ig dimer (300 kDa). other normal tissues and the LS174T tumour were The immunoreactive fraction (IRF) of the radioobtained, weighed and the radioactivity associated labelled immunoconjugates as measured in a binding with each and with a sample of the injectate measured assay against BSM is shown in Table 1. The IRF of in an automatic gamma counter using a window streptavidin-CC49 was slightly but significantly (150-190 keV) to include the 172keV gamma photon lower when conjugated through lysine moieties than of “‘In. The biodistribution results were expressedas when conjugated to reduced disulfide moieties on percent injected dose/g (% i.d./g) of tissue. All animal the Ig. The IRF of the lysine streptavidin--CC49 biodistribution studies were conducted under an immunoconjugate was also slightly but significantly approved protocol from the Animal Research Com- lower than that of DTPA-CC49. There were no mittee at The Toronto Hospital following CCAC significant differences in IRF between any of the guidelines. other immunoconjugates. The ~mmunoreactive fraction and biodistribution data for the various streptavidin-CC49 and DTPACC49 immunoconjugates and native streptavidin were compared using Student’s t-test (P < 0.05).

Results Preparation and in vitro characterization conjugates

of immuno-

Streptavidin was conjugated to MAb CC49 via lysine amino acids, oxidized carbohydrates or reduced disulphides in 60, 20 or 95% yields respect-

The labelling efficiency of DTPA-biocytjn with “‘In was >98% as determined by cation exchange chromatography on a Chelex-100 mini-column. “‘In-DTPA-biocytin bound to streptavidin in vitro as determined by FPLC analysis and by incubation with avidin-agarose resin. When incubated with avidin-agarose resin, approx 90% of “‘In-DTPAbiocytin was bound to the resin. Biodistribution

of immunoconjugates

Streptavidin immunoconjugates of MAb CC49 or native streptavidin were radiolabelled with “‘In-

Streptavidinconjugatesof monoclonaiantibody CC49

81

(A)

3 Time (min)

Time (min)

Time (min)

Time (min)

(El

0

10

20

30

40

50

3 PC, I60 0

Time (min)

, 10

20

30

\ 40

50

60

Time (min)

Fig. I. Size-exclusionFPLC chromatogramof streptavidin-CC49labelled with “‘In-DTPA-biocytin, preparedthrough conjugation to (A) lysine amino acids, (8) reduced disu~phidesor (C) oxidized carbohydrates on the immunoglobulin molecule. Also shown are FPLC chromatograms of (D) “‘In-DTPA-CC49, (E) streptavidin labelled with “‘In-DTPA-biocytin and (F) “‘In-DTPA-biocytin.

DTPA-biocytin and their biodistribution in nude mice bearing S.C.LS174T human colon cancer xenografts determined at 3 and 6 days pi. (except for the carbohydrate conjugated streptavidin-CC49 which was only evaluated at 3 days p.i.). The biodistribution results at 3 days p.i. are shown in Table 2 and at 6 days p.i. in Table 3. There was much lower tumour uptake of radioactivity following injection of the lysine

streptavidin-CC49 conjugate than following injection of any of the other streptavidin-CC49 conjugates or DTPA-CC49 at both time points. There were no significant differences in tumour radioactivity levels at 3 days p.i. between CC49 conjugated with streptavidin through oxidized carbohydrates or through reduced disulphide moieties on the Ig. However, there was significantly lower tumour uptake of radioactivity following injection of DTPA-CC49 than

WILSON M. NGAI rt ul. Table 1, lmmunoreactivily

of streptavidiwCC49 ‘Streptavidin-CC49

Funcuonal group on immunoglobulin for coniugation

Lysine amino acids

Immunoreactive fraction (mean + SD, n = 3)

0.25 * 0.04

or DTPA-CC49 conjugates

con~ugalrs

IDTPA-

CC49 conjugate

Oxidized carbohydrates

Reduced dlsulphides

Lyunc amiw acids

0.31 * 0.03

0.36 f 0.03

0.14 * 0.01

*StreptavidiwCC49 conjugates were labelled with “‘In-DTPA-biocytin. tDTPA-CC49 was labelled with “‘In-acetate.

following injection of either of these streptavidinCC49 conjugates at 3 days pi. Tumour radioactivity was lower at 6 days p.i. with the lysine and sulphhydryl streptavidin-CC49 immunoconjugates than at 3 days p.i. At this time point, the difference in tumour uptake between the sulphhydryl streptavidinCC49 conjugate or DTPACC49 was not significant. Radioactivity in the tumour 3 days following injection of radiolabelled streptavidin was lower than any of the CC49 immunoconjugates with the exception of the lysine streptavidinCC49 conjugate and decreased by 2-fold at 6 days pi. The level of radioactivity in the blood 3 days following injection of the lysine conjugated streptavidin-CC49 was much lower than any of the other CC49 immunoconjugates and similar to that observed following injection of radiolabelled streptavidin. The blood radioactivity level decreasedat 6 days p.i. for the CC49 immunoconjugates. There were no significant differences in liver radioactivity levels following injection of any of the streptavidin-CC49 or DTPACC49 immunoconjugates at either 3 or 6 days p.i. Liver uptake of radioactivity remained relatively constant between the two time points. Although the spleen radioactivity level at 3 days p.i. with the sulphhydryl conjugated streptavidinCC49 appeared to be higher than that of the other immunoconjugates, the difference was not significant due to considerable variability. At 6 days p.i. however, the spleen uptake with the sulphhydryl conjugated streptavidin-CC49 was significantly higher than that of lysine conjugated Table 2. Biodistribution

of streptavidiwCC49

Discussion There is a complex interplay of several pharmacokinetic and physiological factors which can ultimately affect the successor failure of pretargeting strategies to localize radioactivity in tumours using MAb and the (strept)avidin-biotin system. One of the major factors is the distribution in vivo of the streptavidin immunoconjugate. In a 2-step targeting strategy, the distribution of the streptavidin immunoconjugate in

or DTPA-CC49 immunoconjugates at 3 days p.i. in nude mice bearing subcutaneous LS 174T human colon cancer xenoarafts *Streptavidin-CC49

Lysine amino acids

Oxidized carbohydrates

Tissue TlUlVJUI Blood Heart Lung Liver Spleen Stomach Intestine Kidney

2.25 0.26 0.50 I .03 13.00 4.10 2.80 1.40 4.60

29.592 11.15 4.13 i 2.32 3.85 k 3.08 1.45* 1.94 10.47 + 1.65 8.32 f 6.59 4.77 _+4.78 5.29 + 4.56 4.63 _+2.98

I .57 0.43 0.44 0.95 10.64 4.40 3.30 1.47 4.05

TDTPA-CC49 conjugate

conjugates

Functional group on immunoglobulin for conjugation

* + * + + f i + +

streptavidin-CC49 but not significantly higher than that of DTPA-CC49. Liver radioactivity levels following injection of radiolabelled streptavidin were slightly lower than with the CC49 immunoconjugates but spleen radioactivity levels were similar. Radioactivity in the kidney following injection of the sulphhydryl conjugated streptavidin-CC49 was significantly higher at 3 days p.i. than with any of the other immunoconjugates. However, the kidney radioactivity was much lower at 6 days p.i. than at 3 days p.i. for this conjugate. The kidney radioactivity levels for the other CC49 immunoconjugates remained relatively constant between 3 and 6 days p.i. At 3 days p.i., the kidney uptake of DTPAK49 was significantly higher than that of carbohydrate conjugated streptavidin-CC49. The highest radioactivity levels in the kidney were observed following injection of radiolabelled native streptavidin. The high kidney radioactivity level following injection of radiolabelled streptavidin also remained relatively constant from 3 to 6 days p.i.

Reduced disulphides

Lysine amino acids

~Percent injected dose/g 40.63 f 13.86 18.07+9.17 4.38 * 0.71 6.29 + 4.39 5.71 f 1.79 3.24 f 1.98 9.06 _+4.50 5.19 + 3.39 9.42 & 2.35 16.77 + 5.52 20.71 F 10.67 8.47 f 5.97 2.1 1 + 1.29 2.87 i 0.73 1.81 kO.31 2.20 f 0.46 15.51 + 3.16 9.64 _+2.45

YQreptavidiwCC49 conjugates were labelled with “‘In-DTPA-biocytin. tDTPA-CC49 conjugate was labelled with “‘In-acetate. $%eptavidin was labelled with “‘In-DTPA-biocytin. §Mean _+ SD of 3-6 animals.

:Streptavidin

9.38 & 4.69 0.37 + 0.09 I .46 + 0.77 2.07 i- 0.38 4.60 _+ 1.40 3.49+ I.18 I .05 * 0.40 1.45 + 0.35 35.20 + 10.32

Streptavidinconjugatesof monoclonalantibody CC49 Table 3. Biodistribution

83

of streptavidkCC49 or DTPA-CC49 immunoconjugates at 6 days pi. in nude mice bearing subcutaneous LS174T human colon cancer xenografts

l Streptavidin-CC49

TDTPA-CC49 conjugate

conjugates Functional group on immunoglobulin for conjugation

Lysine amino acids

Reduced disulphides

Tissue Tumour Blood Heart Lung Liver Spleen Stomach Intestine Kidney

1.63kO.15 0.12 + 0.04 0.48IO.11 0.70 f 0.26 18.00 rt- 9.89 5.40 +_ 1.60 2.40 + 1.79 1.30f0.31 6.10 + 1.76

§Percent injected dose/g 10.6 i 0.78 13.99 i 5.16 I .25 k 0.07 I .55 * 1.07 2.0 f 0.35 1.02 f 0.56 2.9 k 0.14 2.21 + 0.78 9.8 + 0.21 11.86i 1.61 11.7*0.14 9.05 +_4.19 9.2 +_0.35 1.61 f 0.91 1.4 + 0.21 I .04 f 0.32 0.89 f 0.09 5.8 + I .34

fstreptavidin

Lysine amino acids 4.57 & 0.54 0.29 f 0.12 1.46kO.31 2.12+0.56 4.61 + 0.50 5.34 f 1.95 0.56 f 0.23 0.94f0.17 40.91 f 6.32

*StreptavidiwCC49 conjugates were labelled with “‘In-DTPA-biocytin. Streptavidin-CC49 prepared through conjugation to oxidized carbohydrates on the immunoglobulin was not evaluated at this time point. tDTPA-CC49 conjugate was labelled with “‘In-acetate. fstreptavidin was labelled with “‘In-DTPA-biocytin. @Mean i SD of 3-5 animals

the first step is the limiting factor for the subsequent distribution of the radiolabelled biotin in the second step (van Osdol et al., 1993). A recently published pharmacokinetic model of pretargeting of tumours using a streptavidin immunoconjugate and radiolabelled biotin (van Osdol et al., 1993) predicts that the distribution of the streptavidin immunoconjugate will be more heterogeneous than that of the native Ig resulting in a lower absolute tumour uptake of the MAb. This prediction is based on the slower rate of diffusion from the vascular to the tissue compartment which would be expected for the relatively larger streptavidin immunoconjugate molecule compared to the smaller native Ig molecule. Substitution of the Ig with a single molecule of streptavidin (60 kDa) will result in an immunoconjugate with a molecular weight of 210 kDa compared to 150kDa for the native MAb. If streptavidin is conjugated to the MAb by non-covalent binding to a biotin functional group introduced onto the Ig, there is also the possibility of cross-linking of two or more Ig molecules through a single streptavidin molecule to produce an immunoconjugate with a molecular weight of >360 kDa. In practice, depending on the chemistry and stoichiometry of the conjugation reaction used to prepare the streptavidin immunoconjugate, the molecular weight may range from a minimum of 210 kDa to more than 660 kDa (Del Rosario and Wahl, 1989; Del Rosario et al., 1992; Kalofonos et al., 1990). Besides the molecular size considerations, another important factor is the actual site of attachment of a large molecule such as streptavidin to the MAb. Conjugation of streptavidin to a region of the Ig close to, or directly involved in antigen-binding may result in an immunoconjugate with significantly diminished immunoreactivity compared to the native MAb. It is therefore critical to evaluate streptavidin immunoconjugates intended for pretargeting strategies on the basis of their immunological integrity in vitro and distribution in uiuo relative to that of an Ig conjugate

with a molecular size similar to that of the native MAb. Using different conjugation chemistries, we have prepared streptavidin immunoconjugates of the second-generation TAG-12 MAb CC49, in which the streptavidin moiety was conjugated either to lysine amino acids, oxidized carbohydrates on the Fc region or reduced disulphides at the hinge region of the Ig. The streptavidinCC49 immunoconjugates exhibiting the highest molecular weight by FPLC analysis were produced either by (1) reaction of maleimide derivatized CC49 with thiolated streptavidin (lysine conjugation) or (2) non-covalent binding of streptavidin to biotin-hydrazide derivatized CC49 (carbohydrate conjugation). The streptavidinCC49 immunoconjugate exhibiting the lowest molecular weight was obtained by reaction of mercaptoethylamine-reduced CC49 with maleimidederivatized streptavidin (sulphhydryl conjugation). The average substitution level in the case of the lysine conjugate was only 0.3 mol of streptavidin/mol of Ig, however the production of a high molecular weight species (>350 kDa) was observed by FPLC analysis. This was most likely due to cross-linking of two Ig monomers by reaction of their maleimide groups with one multiple-thiolated streptavidin molecule, rather than conjugation of multiple streptavidin molecules to the Ig. Sheldon et al. (1992) first reported on this particular method to conjugate streptavidin to the ovarian carcinoma MAb lOB, producing an immunoconjugate which exhibited a major band by SDS-PAGE analysis corresponding to a species with molecular weight of * 165kDa and a minor band corresponding to a species with molecular weight of - 180kDa. Since streptavidin dissociates into its individual subunits (15 kDa each) when subjected to SDS-PAGE analysis (but not during FPLC analysis), these two species actually represent 10B Ig with substitution by one or two streptavidin molecules (intact molecular weights of

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M.

210 or 270 kDa respectively). There were no very high molecular weight immunoconjugate species observed by Sheldon et al. (1992). The absence of these species was probably due to the much lower ratio of 2-iminothiolane: streptavidin (6 : 1) utilized by this group compared to that used in our study (225 : 1). The higher ratio of 2-iminothiolane: streptavidin used in our study may have resulted in multiple thiolation of the streptavidin molecule. The multiple thiolated streptavidin may have subsequently reacted with more than one maleimide-derivatized CC49 Ig to produce the very high molecular weight immunoconjugates (> 350 kDa) observed by FPLC analysis. Data from our preliminary experiments indicated however that high ratios of 2-iminothiolane: streptavidin were required in order to achieve substitution of the CC49 Ig with streptavidin. Yoshikawa and Pardridge (1992) utilizing similar chemistry to conjugate avidin to the anti-transferrin MAb OX26 also reported high molecular weight aggregates by FPLC analysis. The substitution level was also low (1 mol of streptavidin per mole of Ig) for the carbohydrate conjugation method. In this case, the several unresolved peaks by FPLC analysis may represent (1) CC49 Ig monomer substituted by a single streptavidin molecule (mol. wt - 210 kDa) and (2) Ig dimers or polymers (mol. wt > 350 kDa) which are cross-linked through a single streptavidin molecule by the biotin functional groups introduced onto the CC49 Ig, as previously described. Other groups which have prepared streptavidin-MAb conjugates through non-covalent binding of streptavidin to biotin functional groups introduced onto the Ig (Hnatowich et al., 1987; Del Rosario and Wahl, 1989; Kalofonos et al., 1990) have also reported the presence of high molecular weight species. Del Rosario and Wahl (1989) conjugated streptavidin to the ovarian carcinoma MAb 566.4 by first reacting the dithiothreitolreduced Ig with a biotin reagent which reacts specifically with free sulfhydryl moieties, then reacting the biotinylated MAb with streptavidin. This method of conjugation resulted in an oligomeric mixture with the major species being a dimeric streptavidin immunoconjugate (mol. wt - 440 kDa) and polymeric conjugates with molecular weight > 660 kDa. Lower molecular weight streptavidin-5G6.4 conjugates (-2OG-300 kDa) could only be obtained by temporarily binding the biotinylated MAb to a Protein A column and passing a dilute solution of streptavidin through the column (Del Rosario et al., 1992). It therefore appears that due to the tetrameric nature of binding of biotin by streptavidin, such non-covalent streptavidin immunoconjugates are prone to polymerization in vitro resulting in high molecular weight species. The sulphhydryl conjugated streptavidin-CC49 also exhibited a relatively low substitution level (0.5 mol of streptavidin/mol of Ig). FPLC analysis demonstrated a single peak with molecular weight of

NGAI

BI ul

- 210 kDa suggesting monosubstitution of the CC49 MAb with streptavidin without any cross-linking 01 the Ig. FPLC analysis of the DTPA--CC49 conjugate also demonstrated primarily a single monomeric: species with molecular weight - 150 kDa with only a small amount of cross-linking of Ig molecule< (< 10%) through the bifunctional bicyclic anhydridc DTPA reagent. Despite the polymerization of the carbohydrate conjugated streptavidinCC49 the immunorcactivity in vitro was similar to that of the monomeric sulphhydryl conjugated streptavidin-CC49 or DTPA-m CC49. Only the polymeric lysine conjugated streptavidinCC49 showed slightly lower immunoreactivity. The IRF observed for the streptavidinCC49 conjugates and DTPA-CC49 (0.25-0.36) was lower than that previously reported by Ranadive et 111. (1993) for ““Tc-CC49 (0.6) but comparable to that previously reported by us for radioiodinated CC49 (0.40-0.43) (Ngai and Reilly, 1993). Polymeric streptavidin immunoconjugates of the 5G6.4 MAb have also been shown to have comparable in vitro immunoreactivity to their radioiodinated monomeric forms (Del Rosario and Wahl, 1989: Del Rosario et al., 1992). Although the various streptavidinCC49 and DTPA-CC49 immunoconjugates demonstrated comparable in vitro immunoreactivity, they exhibited significant differences in their biodistribution in viva. The much lower levels of radioactivity in the blood following administration of the lysine conjugated streptavidin-CC49 compared with the other streptavidinCC49 or DTPACC49 conjugates could be due to poor absorption from the injection site in the peritoneal cavity or sequestration from the blood by normal organs. Del Rosario et al. (1992) found higher radioactivity levels in liver, kidney and spleen and much lower blood radioactivity levels in mice injected with a polymeric high molecular weight streptavidin conjugate of MAb 566.4 than following injection of a low molecular weight streptavidin conjugate. In our study however. the levels of radioactivity in the normal organs examined were not significantly different between the lysine-conjugated streptavidinCC49 and any of the other streptavidin CC49 or DTPA-CC49 conjugates. The reason for the low blood radioactivity levels with the lysine conjugated streptavidin-CC49 is therefore not clear but may relate to poor absorption from the injection site in the peritoneal cavity. The poor absorption however may not be entirely due to the large molecular size of the streptavidin-CC49 conjugate, since one study (Nagy et al., 1989) showed that dextran molecules with sizes in the range of 335000 kDa have been found to exhibit similar absorption rates from the peritoneal cavity in mice. Although the reasons remain unclear, the low blood level of radioactivity with the lysine streptavidinCC49 conjugate may be responsible for the much lower tumour uptake observed with the

Streptavidin conjugates of monoclonal antibody CC49 lysine-conjugated streptavidinXC49 compared with the other streptavidin-CC49 or DTPA-CC49 conjugates. Tumour radioactivity was ~2% i.d./g for the lysine-conjugated streptavidin-CC49 at 3 days p.i. compared with up to 40% i.d./g for the other streptavidinXC49 conjugates and 18% i.d./g for DTPAE49. The tumour radioactivity levels observed with the carbohydrate or sulphhydryl conjugated streptavidin-CC49 or DTPA-CC49 at 3 days p.i. were in a similar range to that reported by Colcher et al. (1988) for radioiodinated CC49 at 4 days pi. (28% i.d./g) and by Ranadive et al. (1993) for 99mT~labelled CC49 (16% i.d./g) at 1 day p.i. The moderate tumour uptake of radioactivity at 3 days p.i. with radiolabelled streptavidin may be due to non-specific blood tlow effects which have been previously reported for streptavidin in the LSl74T nude mouse model (Hnatowich et al., 1993). Kidney radioactivity levels 3 days following injection of the sulphhydryl conjugated streptavidinCC49 were significantly higher than that observed with the other immunoconjugates. However much lower levels of radioactivity were observed at 6 days p.i. Kidney uptake with the sulphhydryl conjugated streptavidin-CC49 may be related to the chemical reduction of the disulphide moieties on the Ig. High kidney uptake has been previously observed when the 566.4 MAb was reduced by dithiothreitol in order to bind streptavidin non-covalently to a biotin reagent which reacts with free sulphhydryl moieties (Del Rosario and Wahl, 1990). Del Rosario and Wahl (1990) speculate that the high kidney uptake observed with streptavidin-5G6.4 may have been due to renal elimination of a streptavidin conjugated Ig fragment. Structural instability has also been previously reported for MAb reduced by p-mercaptoethanol for subsequent radiolabelling with 99mTc (Pimm et al., 1991). No Ig fragments of the CC49 MAb were detected however by FPLC analysis in our study. Other possible explanations for kidney uptake of radioactivity at early time points with the sulphydryl conjugated streptavidin-CC49 could include renal elimination of small amounts of free “‘In-DTPAbiocytin impurities in the preparation or crossreactivity of the streptavidin moiety of the immunoconjugate with kidney tissue. The possibility of cross-reactivity is suggested by the very high uptake of radiolabelled streptavidin by the kidneys observed in our study and by others (Del Rosario and Wahl, 1990; Hnatowich et al., 1993; Rosebrough, 1993). Based on our in vitro and in vivo evaluation of three different approaches to conjugating streptavidin to the second-generation TAG-72 MAb CC49, we conclude that conjugation through reduced disulphide moieties at the hinge region of the Ig is the optimum method for constructing streptavidin-CC49 immunoconjugates for future pretargeting studies. This method produces a streptavidin-CC49 immunoconjugate which is monomeric, has a molecular size only slightly greater than that of the native Ig, and which

85

exhibits comparable immunoreactivity in vitro and tumour uptake in vivo to that of DTPA-conjugated CC49. In future studies we plan to investigate this streptavidin-CC49 conjugate for targeting of “‘In or 9oY labelled biotin to LS174T human colon cancer xenografts hosted in nude mice in a 2-step immunoscintigraphy or immunotherapy protocol. Acknowledgements-This study was supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society. Baruch Shpitz was supported by the Lederman Cancer Research Foundation and an American Physician Fellowship. The CC49 ascites for this study was generously provided by Dr J. Schlom, National Cancer Institute, National Institutes of Health, U.S.A. Parts of this study were presented at the Society of Nuclear Medicine 40th Annual Meeting, Toronto, June 11, 1993.

References Bamias A. and Epenetos A. A. (1992) Two-step strategies for the diagnosis and treatment of cancer with bioconjugates. Antibody Immunoconj. Radiopharm. 5, 385S395. Chaiet L. and Wolf F. J. (1964) The properties of streptavidin, a biotin-binding protein produced by streptomycetes. Arch. Biochem. Biophys. 106, l-5. Colcher D., Minelli M. F., Roselli M. et al. (1988) Radioimmunolocalization of human carcinoma xenografts with B72.3 second generation monoclonal antibodies. Cancer Res. 48, 45974603. Del Rosario R. B. and Wahl R. L. (1989) Site-specific radiolabelling of monoclonal antibodies with biotin/ streptavidin. Nucl. Med. Biol. 16, 525-529.

Del Rosario R. B. and Wahl R. L. (1990) Disulfide bond-targeted radiolabelling: tumor specificity of a streptavidin-biotinylated monoclonal antibody complex. Cancer Res. 50, 804s-808s. Del Rosario R. B., Baron L. A., Lawton R. G. and Wahl R. L. (1992) Streptavidin-biotinylated IgG conjugates: a simple procedure for reducing polymer formation. Nucl. Med. Biol. 19, 417421. Garron J. Y., Moinereau M., Pasqualini R. and Saccavini J. C. (1991) Direct wmTc labelling of monoclonal antibodies: radiolabelling and in -vitro stability. Nucl. Med. Biol. 18, 695-703. Green N. (1965) A spectrophotometric assay for avidin and biotin based on binding of dyes by avidin. Biochem. J. 94, 23~. Green N. M. (1975) Avidin. Adv. Protein Chem. 29, 85-133. Hnatowich D. J., Virzi R. and Rusckowski M. (1987) Investigations of avidin and biotin for imaging applications. .I. Nucl. Med. 28, 12941302. Hnatowich D. J., Fritz B., Virzi F. et al. (1993) Improved tumor localization with strept(avidin) and labelled biotin as a substitute for antibody. Nucl. Med. Biol. 20, 189-195. Kalofonos H. P., Rusckowski M., Siebecker D. A. et al. (1990) Imaging of tumor in patients with indium-I I Ilabelled biotin and streptavidin conjugated antibodies: preliminary communication. J. Nucl. Med. 31, 1791-1796. Muraro R., Kuroki M., Wunderlich D. ef al. (1988) Generation and characterization of B72.3 second generation monoclonal antibodies reactive with the tumor-associated glycoprotein 72 antigen. Cancer Res. 48, 45884596. Nagy J. A., Herzberg K. T., Masse E. M. et al. (1989) Exchange of macromolecules between plasma and peritoneal cavity in ascites tumor-bearing, normal, and serotonin-injected mice. Cancer Res. 49, 5448-5458. Ngai W. M. and Reilly R. M. (1993) A simple method to determine the immunoreactivity of radiolabelled mono-

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clonal antibodies to the TAG-72 antigen. A&. &r&l. I.sot. 44, 1193-l 197. van Osdol W. W., Sung C., Dedrick R. L. and Weinstein J. N. (1993) A distributed pharmacokineti~ model of two-step imaging and treatment protocols: application to streptavidin-conjugated monoclonal antibodies and radiolabelled biotin. J. Nurl. Med. 34, 1552..1564. Paganelli G.. Pervez S., Siccardi A. G. el al. (1990) Intraperitoneal radiolocalization of tumors pre-targeted by biotinylated monoclonal antibodies. Inr. J. Cmcvr 45, 11841189. Paganeili Cr., Magnani P., Meares C. et al. (1993) Antibody guided therapy of CEA positive tumors using biotinylated monoclonal antibodies, avidin and mY-DOTA-biotin: initial evaluation. J. Nucl. Med. 34, 94P. Perkins A. C. and Pimm M. V. (1991) Clinical role of immunoscintigraphy. In: r~~unffs~intigr~ph~: Practical Aspects und C~i~jea~ Applications, pp. 129-162. WileyLiss, New York. Pimm M. V., Rajput R. S., Frier M. and Gribben S. J. (1991) Anomalies in reduction-mediated technetium-99m labelling ofmonoclonal antibodies. Eur. J. Nucl. Med. 18, 973.-976. Ranadive Cr. N., Rosenzweig 1-I. S., Epperly M. W. et al.

(1993) A new method of technetium-99m labelling crt monoclonal antibodies through sugar residues. A study with TAG-72 specific CC-49 antibody. Vuci. :Mrti. Kitsi. 20, 7 19-726. Reilly R.. Sheldon K.. Marks A. and floule S. (19X9) Labelling of monoclonal antibodies 1OB. Xc’ and MIA with indium-I I I. Aunl. Radiut. Lwr. 40. 279. 283. Reilly R. M. (1991) R:ddioimmunotherapy of malignancies. Clin. Pharm. 10, 359 375. Rosebrough S. F. (1993) Pharm~~cokineti~s and biodistribution of rad~ola~lled avidin, streptavidin and biotin. Nucl. Med. Bioi. 20, 663 66X. Sheldon K., Baumal R. and Marks A. (1992) Targeting of “‘In biocytin to cultured ovarian adenocdrcinoma cells using covalent monoclonal antibody-streptavidin conjugates. Appl. Radiat. isot. 43, 1399~-1402. Stewart J. S. W.. Hird V., Snook D. tf (II. (1988) Intraperitoneal “‘I- and MY-labelled lnono~lonal ~~ntibodies for ovarian cancer: p~drmacokinetics and normal tissue dosimetry. Inf. J. C&w 42, (Suppt. 3) 71 76. Yoshikawa T. and Pardridge W. M. (1992) Biotin delivery to brain with a covalent conjugate of avidin and a monoclonal antibody to the transferrin receptor. J. Phnrmacol. Exp. Ther. 263, X97-903,