Bioconjugate Chem. 1§95, 6, 179-1 86
179
A Cross-Linked Monoclonal Antibody Fragment for Improved Tumor Targeting Maria A. Stalteri a n d Stephen J. Mather” Imperial Cancer Research Fund, Department of Nuclear Medicine, St. Bartholomew’s Hospital, West Smithfield, London EClA 7BE, U.K. Received May 31, 1994@
Cross-linked F(ab’)z fragments derived from PRlA3, a murine monoclonal antibody used in radioimmunoscintigraphy of colorectal tumors, were produced using the bifunctional reagent bismaleimidohexane (BMH) as follows: Digestion of PRlA3 with pepsin gave F(ab’)z fragments which were purified by ion-exchange chromatography. Fab‘ was produced by reduction of F(ab‘)z with cysteine. Following reaction with BMH, cross-linked F(ab’)z fragments, XL-F(ab’)z, were isolated by preparative size-exclusion HPLC. Analysis by HPLC and SDS-PAGE demonstrated the presence of a molecule of -100 kDa containing a nonreducible 50 000 MWt chain. Competitive and direct radioligand binding assays demonstrated that the XL-F(ab’)z had a capacity to bind to antigen similar to that of unmodified F(ab’)z. The biodistribution of lZ5I-labeledXL-F(ab’)z and unmodified F(ab’)z was compared in a nude mouse human tumor xenograft model a t 4,24, and 48 h after injection. Differences between the two preparations were most significant after 24 or 48 h. Tumor uptake of the XL-F(ab’)z was greater and normal tissue retention less than with the unmodified fragment. Tumor to normal tissue ratios a t 48 h ranged from 6.2 to 35.2 for XL-F(ab’I2while for the normal F(ab’I2 they ranged from 1.5 to 14.2. These results suggest that cross-linked antibody fragments may produce better tumor targeting in clinical application.
INTRODUCTION
Antibodies labeled with various radioisotopes including iodine-123, iodine-131, indium-111,and technetium-99m have been used for radioimmunoscintigraphy and radioimmunotherapy of cancer in patients as well as tumor xenograft models (1,2). Although monoclonal antibodies are highly specific, only a very small fraction of the injected dose, generally less than 1%, actually binds to the tumor (3). There are a number of factors responsible for this low uptake, but among the most important are (i) limited access to antigen caused by poor tumor blood flow and high interstitial pressure (4) and (ii) nonantigen-mediated mechanisms of uptake in major organs such as liver (5). Intact IgG antibodies are relatively large proteins with a molecular weight of about 150 000. They are consequently slow to clear from the circulation and localize in the tumor. The Fc portion of the molecule is also responsible for a host of interactions with systems such as the RES. It has been proposed that antibody fragments such as F(ab’I2,Fab’, and recombinant sc-Fvl may improve targeting as they (a) are smaller and may therefore show better tumor penetration and (b) lack an Fc region which may reduce “nonspecific” interactions. In fact, most studies have shown that tumor uptake of radiolabeled fragments is often lower than that of intact antibodies but that a greatly enhanced rate of blood
* To whom correspondence should be addressed. Tel: 44-71-601-7153. Fax: 44-71-796-3907. E-mail:
[email protected]. Abstract published in Advance A C S Abstracts, January 1, 1995. Abbreviations: BMH, 1,6-dimaleimidohexane;BSA, bovine serum albumin; CEA, carcinoembryonic antigen; DEAE, (diethy1amino)ethyl; DMF, dimethylformamide; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; HPLC, highpressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; RES, reticuloendothelial system; RIA, radioimmunoassay; sc-Fv, singlechain F’v;SDS, sodium dodecyl sulfate; Tris, tris(hydroxymethy1)aminomethane; XL-F(ab’)z, cross-linked F(ab’)z fragments. @
clearance does indeed result in improved tumor to background ratios (6-8). One problem observed with the use of F(ab’)z and Fab’ fragments is high kidney activity. A high renal uptake of the smaller fragments can be explained by filtration a t the glomerulus followed by tubular reabsorption and metabolism. However, F(ab’)z fragments, which have a molecular weight of about 100 000, are generally considered to be too large to undergo glomerular filtration since they exceed the generally accepted limit of 50-60 kDa for filterable biomolecules (9, IO). Disulfide bonds which are accessible have been shown to be susceptible to reduction in-vivo (11). A likely cause of the kidney uptake of F(ab’)z, therefore, is in-vivo reduction of the disulfide bonds bridging the two arms of the F(ab’I2which have been exposed by pepsin digestion, followed by filtration of the resultant Fab’ (12). The relative merits of divalent (such as F(ab‘Iz)and monovalent (such as Fab) fragments have not been systematically independently explored. In many studies, F(ab’)z normally show a higher tumor uptake than Fab, but whether this is due to the larger molecular weight or the potential for divalent binding is not clear. Elucidation of the optimum properties of the ideal fragment is further hindered by the possibility of this in-vivo reduction. In the study described here we sought to improve the in-vivo stability of the F(ab’)z fragment by replacing the labile disulfide bridge with a more stable thioether linkage. Using a method adapted from that of Glennie (131,we have prepared a cross-linked F(ab’Iz fragment of the antiCEA antibody PRlA3, XL-F( ab')^, using dimaleimidohexane as a cross-linker. The ability of XL-F(ab’)z to bind to MKN-45 cells, a human gastric cancer cell line, was compared to unmodified F(ab’)z in both direct and indirect binding assays. The biodistribution of lz5I-labeledXLF(ab’)2 in nude mice bearing MKN-45 human tumor xenografts was compared with that of lZ5I-labeledunmodified F(ab’)2.
1043-1802/95/2906-0179$09.00/00 1995 American Chemical Society
180 Bioconjugate Chem., Vol. 6,No. 2, 1995 MATERIALS AND METHODS
Radioisotopes. Na[1251]Iwas obtained from ICN Radiochemicals, Imine, CA. Monoclonal Antibodies. PRlA3, a mouse IgGl monoclonal antibody used in the study of colorectal cancer, was obtained from the Hybridoma Development Unit, Imperial Cancer Research Fund, London. PRlA3 is used in radioimmunoscintigraphy of patients with colorectal cancers (14). The epitope recognized by this antibody is a cell-associated form of CEA which is lost when the molecule is shed (15). Cell Line. MKN-45 cells were obtained from the Director's Laboratory, ICRF, London. MKN-45 is a human gastric cancer cell line which expresses CEA on the surface (16). The cells were grown in vitro in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum. HPLC. Size exclusion HPLC was performed using a Beckman 114M pump with a Beckman 160 W detector a t 254 nm connected to a Spectra-Physics SP4290 integrator or a Beckman System Gold system with a Beckman 166 U V detector a t 280 nm. Analytical work was done using Du Pont Zorbax GF-250 (optimal separation range 10-250 kDa) or Beckman SEC 3000 (5-700 kDa) columns. A Du Pont Zorbax GF-250 XL column was used for preparative work. The mobile phases used were 0.2 M sodium phosphate pH 7.0, 2 mM EDTA or 0.1 M sodium phosphate pH 7.0,2 mM EDTA. Analytical work was performed using a 20 pL injection loop and a flow rate of 0.5 mL per min. For preparative work a 2.0 mL injection loop and a flow rate of 0.5 mL per min were used. Ion-exchange HPLC was performed using a Beckman System Gold dual pump system with a Beckman 166 W detector a t 280 nm, a 2.0 mL injection loop, and a TSK gel DEAE-5 PW column. Samples were eluted using a gradient system with 10 mM Tris-HC1 pH 7.5 and 10 mM Tris-HC1 pH 7.5, 0.5 M NaCl as the mobile phases a t a flow rate of 1.0 mL per min. SDS-PAGE. Antibodies and antibody fragments were analyzed by discontinuous SDS-PAGE according to the method developed by Laemmli (171, with and without reduction in 0.1 M DTT. Samples, containing 1-10 yg of protein, were run using a 5% acrylamide (w/ v) stacking gel and a 12.5% running gel. Gels were stained in Coomassie brilliant blue G-250 (BDH, Poole, England) in acetic acid/methanol/water (7%/30%/63%) and then destained in acetic acid/methanol/water (7%/ 30%/63%). Prestained molecular weight markers were obtained from Gibco BRL, Gaithersburg, MD. Preparation of F(ab')zFragments. Thirty mg of PRlA3 antibody was repeatedly centrifuged using Centriprep-30 microconcentrators (Amicon, Beverly, MA) in order to change the buffer to 20 mM sodium citrate, pH 3.5. The final antibody concentration was 5 mg/mL. Five hundred pL of immobilized pepsin slurry (Pierce, Rockford, IL) was added to 8 mL of 20 mM sodium citrate in a centrifuge tube, mixed well by shaking, and then centrifuged for 5 min at 2000 rpm. The supernatant was removed, and the pepsin was washed again with another 8 mL of sodium citrate. The antibody was then added to the immobilized pepsin and incubated a t 37 "C with continuous rotation for 5 h. The progress of the reaction was monitored by size exclusion HPLC. When the reaction was 70-90% complete, the pepsin was pelleted by centrifuging for 5 min a t 2000 rpm. The supernatant containing the F(ab')z fragments and the unreacted antibody was removed and the pH was adjusted to 7.08.0 by addition of 0.5 mL of 1.0 M Tris-HC1 pH 9.0. The digestion mixture was analyzed by SDS-PAGE.
Stalteri and Mather
Purification of F(ab')a Fragments. The digestion mixture was centrifuged in a Centriprep 30 concentrator (Amicon, Beverly, MA) to change the buffer to 10 mM Tris-HC1, pH 7.5. The solution was then filtered through a 0.2 pm syringe filter (Sterile Acrodisc, Gelman Sciences, Ann Arbor, MI) to remove any particles before injection onto the HPLC. The fragments were purified by anionexchange HPLC using a gradient system with 10 mM Tris-HC1 pH 7.5 as buffer A and 10 mM Tris-HC1 pH 7.5,0.5 M NaCl as buffer B, a t time = 0 min, 0% B; time 5-35 min, 0-30% B; time 35-40 min, 30-50% B. Using this system, PRlA3 F(ab'h fragments had a retention time of 23 min, while PRlA3 antibody eluted a t 33 min. The purified F(ab'Iz fragments were analyzed by size exclusion HPLC and by SDS-PAGE. Reduction of F(ab')z Fragments Using Cysteine. Fifty-one mg of cysteine was dissolved in 1.5 mL of 0.2 M trisodium phosphate to give a 200 mM solution of cysteine. One hundred pL of the cysteine solution was added, with stirring, to 1mL of a 3.0 mg/mL solution of PRlA3 F(ab')z fragments in 0.1 M sodium phosphate pH 8.0,0.5 mM EDTA. The reaction mixture was incubated a t 37 "C with continuous rotation for 1 h 15 min. The progress of the reaction was monitored by size exclusion HPLC. When the reaction had gone to completion the solution was applied onto a 30 x 1 cm column of Sephadex G-50 and eluted with 50 mM sodium acetate pH 5.3, 0.5 mM EDTA. Three mL fractions were collected, and the presence of antibody fragments was determined by measuring the absorbance a t 280 nm on a UVJvis spectrophotometer. The fractions containing antibody were pooled and analyzed by SDS-PAGE. Cross-Linkingof Reduced Fab Fragments. Thioether-linked PRlA3 fragments were produced using the bifunctional cross-linker BMH (Pierce, Rockford, IL), by a method similar to that of Glennie (13). All solutions were kept on ice throughout the experiment, and the sodium acetate buffer was purged with nitrogen. BMH was dissolved in anhydrous DMF (Aldrich, Milwaukee, WI) a t a concentration of 4 mg/mL. The reduced Fab' fragments were concentrated to 5 mg/mL in 50 mM sodium acetate buffer pH 5.3 containing 0.5 mM EDTA using a Centricon 30 microconcentrator. One hundred and fortypL of the BMH solution was added rapidly, with stirring, to 0.5 mL of Fab'SH fragments, and the reaction mixture was then incubated on ice for 30 min. Excess BMH was removed by passing the solution through a 30 x 1cm column of Sephadex G-50 and eluting the sample with 50 mM sodium acetate buffer pH 5.3 containing 0.5 mM EDTA. Two mL fractions were collected and the presence of antibody fragments was determined by measuring the absorbance a t 280 nm on a UV/visible spectrophotometer. The fractions (4 and 5) containing Fab'maleimide fragments (typically 80% recovery) were pooled, and 0.5 mL of Fab'SH fragments was added. The mixture was concentrated to a volume of 1 mL using a Centricon 30 microconcentrator and incubated overnight a t 4 "C. The reaction mixture was analysed by size exclusion HPLC and by SDS-PAGE. The cross-linked F(ab'h fragments were separated from unreacted Fab' and from higher molecular weight species by size exclusion HPLC using a DuPont Zorbax GF-250 XI, column. The purified cross-linked fragments were analyzed by SDS-PAGE, Radiolabeling. Antibody, F(ab'12 fragments, and cross-linked fragments were labeled with iodine-125 using Iodogen (18). Briefly, 100 pg of antibody or antibody fragment was pipetted into a test tube containing 20 pg of dried Iodogen. A 100-200 pCi portion of iodine-125 was added, and after mixing, the vial was
Bioconjugate Chem., Vol. 6,No. 2, 1995 181
Cross-Linked Monoclonal Antibody Fragment
incubated a t room temperature for 10 min. The reaction mixture was applied to the top of a PD-10 column (Pharmacia), and the labeled antibody was eluted in 1 mL fractions of 1%BSA in PBS. The radiochemical purity of the labeled proteins was assessed by thin-layer chromatography using ITLC (Gelman Sciences, Ann Arbor, MI) developed in 85% methanol. Measurement of Immunoreactivity. Two types of antigen binding assays were performed as follows. Direct Binding Assay for PRlA3 Fragments Using MKN-45 Cells. The ability of the cross-linked antibody fragments to bind to MKN-45 cells was measured by RIA, using a method similar to that of Lindmo (19). The cells from one culture flask of MKN-45 cells were harvested by treatment with 1 mL of versene in PBS and 1 mL of 0.25% trypsin in Tris saline a t 37 “C for 10 min. Eight mL of cold PBS containing 1%BSA was added, and the cells were centrifuged a t 900 rpm for 10 min. The cell pellet was resuspended in 6 mL of PBSBSA. The cell suspension was taken up several times into a syringe with a fine gauge needle in order to break up clumps. A 500 pL aliquot of the cell suspension was diluted to 10 mL with PBSBSA, and the cells were counted using a haemocytometer slide. The assay was performed in duplicate using seven 1 mL Eppendorf tubes. Five hundred pL of PBSBSA was added to tubes 2-5. Five hundred pL of the cell suspension was added to tubes 1,2, and 6. Tube 2 was vortexed, and 500 pL of the cell suspension was transferred to tube 3. The process was repeated so as to give a series of double dilutions of cells in tubes 1-5. Five hundred pL of the cell suspension in tube 5 was discarded. Two pg of PRlA3 antibody was then added to tube 6. Iodine-125 labeled cross-linked antibody fragments were diluted to 50 ng/mL with PBSBSA, and 250 pL of the labeled fragments was added to each of tubes 1-7. Tubes 1-6 were vortexed and then incubated for 2 h a t room temperature with continuous rotation. The tubes were then centrifuged a t 13 000 rpm for 5 min to pellet the cells. The supernatant was aspirated, taking care not to disturb the cell pellet. Five hundred pL of PBSBSA was added, and the tubes were vortexed until the cell pellets were resuspended. The tubes were again centrifuged a t 13 000 rpm for 5 min, the supernatant was removed, and the cell pellets were counted in a n LKB Wallac CompuGamma CS y counter. A measure of the immunoreactive fraction was obtained by calculating the proportion of radioactivity bound to the cells (counts in relevant tube divided by counts in tube 7) and plotting its reciprocal against the reciprocal of the cell concentration. Indirect Binding Assay for PRlA3 Fragments Using MKN-45 Cells. The ability of the cross-linked antibody fragments to compete with labeled PRlA3 antibody in a cell-binding assay was compared with that of PRlA3 F(ab’Iz fragments and unlabeled PRlA3 antibody. The cells from one confluent 250 mL flask were obtained and counted as described for the direct binding assay and diluted with PBSBSA to a concentration of 1.5 million cells/mL. The assay was done in duplicate, using 1mL Eppendorfvials. Increasing amounts (0,160, 640, 2560, 10 240, and 40 960 ng) of unlabeled PRlA3 antibody, PRlA3 F(ab’)z fragments, or cross-linked PRlA3-PRlA3 fragments were added to rows of six Eppendorf vials. Forty ng of iodine-125 labeled PRlA3 antibody was then added to each vial, followed by PBS/ BSA if necessary to give the same total volume in all the vials. The vials were vortexed, and 500 pL of the MKN45 cell suspension was added. After vortexing, the vials were incubated a t room temperature for 1 h with
continuous rotation. The vials were centrifuged for 10 min a t 13 000 rpm in a microcentrifuge, and the supernatants were aspirated. The cell pellets were resuspended in 500 pL of PBSBSA by vortexing for a few min. The vials were centrifuged for another 10 min at 13 000 rpm, the supernatants were aspirated, and the amount of radioactivity bound to the cells was determined using a y counter. Animal Biodistribution Studies. Three-month old nude mice were injected with transplanted MKN-45 human tumor samples (0.1 mL) subcutaneously in each flank. After 3-4 weeks, when the tumors were 0.3-0.5 cm in diameter, the animals’ drinking water was supplemented with potassium iodide (10 mg/100 mL) for 3 days prior to and during the biodistribution experiments. PRlA3 F(ab’)z fragments and PRlA3-PRlA3 crosslinked fragments were radiolabeled with iodine-125 a t a specific activity of 1 mCi per microgram. The labeled antibody fragments were diluted with PBS to a final concentration of 50 pg per mL. The mice were divided into two groups of 15. Group A was injected intravenously with 100 pL of the iodine-125 labeled F(ab’)2 fragments, and group B was injected with 100 pL of the labeled cross-linked fragments. The total injected dose was calculated by accurately weighing the injection syringes before and after injecting each animal. Five animals from each group were sacrificed a t 4,24, and 48 h after injection. Samples of blood were taken, and organs and tissues of interest were resected, rinsed in saline, blotted dry, and placed into preweighed tubes. All samples were counted in an LKB Wallac 1282 Compugamma CS y counter together with appropriate dilutions of the iodine-125 labeled F(ab’)z and XL-F(ab’)z. Results were analyzed using Student’s t-test. RESULTS
Preparation and Characterization of XL-F(ab)2. F(ab’)z fragments of PRlA3, a mouse monoclonal IgG1, were produced by digestion with pepsin beads a t pH 3.5. The rate of the reaction was quite sensitive to pH, becoming much slower when the pH was above 3.5. The reaction was monitored by size exclusion HPLC using a Beckman SEC 3000 column. When the digestion was allowed to go to completion lower molecular weight species, possibly due to further digestion of F(ab’Iz, were also produced. We therefore decided to stop the reaction when it was 70-90% complete and to purify the F(ab’Iz from undigested antibody by ion-exchange HPLC (20). Figure 1 shows a chromatogram of the purification of F(ab‘)2 using a DEAE HPLC column, while an analytical size exclusion HPLC chromatogram of the purified F(ab’), is shown in Figure 2. Various reagents have been used for the reduction of F(ab’)z to Fab’SH including organophosphines (21))DTT (221, mercaptoethanol(13), cysteamine (231, and cysteine (24, 25). We briefly investigated the use of mercaptoethanol, Reduce-Imm immobilized reducing agent (Pierce, Rockford, IL), tris(2-carboxyethy1)phosphine (26), and cysteine in order to determine which conditions would give complete reduction of F(ab’)z while keeping formation of free heavy and light chains to a minimum and without affecting the immunoreactivity of the antibody. We obtained good results using 20 mM cysteine in phosphate buffer pH 8.0, 0.5 mM EDTA a t 37 “C and subsequent large scale reductions were carried out using these conditions. An example of a HPLC chromatogram of this material, with a purity of 97.8% Fab’, 2.2% F(ab’)z is shown in Figure 3. Typical yields were of the order of 90%.
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182 Bioconjugate Chem., Val. 6,No. 2, 1995 0.25
0.20
F(ab‘)P A/
0.15 0
3 buffer salts
6
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Intact Ab
n W
> a
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-0.05 0
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Retention time, min
Figure 1. Purification of PRlA3 F(ab’)z from intact antibody by ion-exchange HPLC. Separation was achieved using a DEAE-5 PW column and a gradient system with 10 mM TrisHC1 pH 7.5 as mobile phase A and 10 mM Tris-HC1 pH 7.5, 0.5 M NaCl as mobile phase B, monitored at 280 nm. Key: flow rate, 1.0 mumin; retention times, F(ab’)z 23 min, intact antibody 33 min.
XL-F(ab’)z were prepared using the bifunctional crosslinking agent dimaleimidohexane under conditions similar to those reported by Glennie (13)for the preparation of bispecific F(ab’)z. During the development of our methodology, occasional low yields of 10-20% were obtained. However, optimization of the method by using longer Sephadex columns to purify Fab’SH from the reducing agents and Fab’maleimide from excess BMH and purging the cross-linking reaction buffer with nitrogen resulted in uniformly higher yields ranging from 40 to 50% with a mean of 46.5%. In addition to XL-F(ab’k small amounts of higher molecular weight species, possibly aggregates, but probably F(ab’)a as described by Glennie (13),were also obtained. The cross-linked fragments were purified from the reaction mixture by size exclusion HPLC using a preparative GF-250XL column and were analyzed by SDS-PAGE and analytical HPLC. Figures 4 and 5 show analytical HPLC chromatograms of the reaction mixture and the purified XL-F(ab’)z, respectively. A gel run under reducing conditions is shown in Figure 6. Lane 2 shows native PRlA3 F(ab’)z, while lanes 4-7 show different fractions from the preparative HPLC purification of the XL-F(ab’)z. Lanes 4 and 5 represent unreacted Fab’ recovered from the reaction mixture, while lanes 6 and 7 are XL-F(ab’)z. The nonreducible bands of MW -50 000 in lanes 6 and 7 demonstrate the presence of cross-linked H chains. Radiolabeling. Radioiodination yields typically ranged from 50 to 80%. Final purity as measured by ITLC was always greater than 95%. The specific activities of the fragment preparations used for the biodistribution study were 1.24 mCi/mg for the XL-F(ab’)z fragment and 1.09 mCi/mg for the F(ab’)z. Measurement of Immunoreactivity. The immunoreactivity of the cross-linked F(ab’)z fragments was
20
30
Retention time, min
Figure 2. Analytical size exclusion HPLC profile (Beckman SEC 3000 column) of purified PRlA3 F(ab’)z, monitored at 254 nm. Key: flow rate, 0.5 mlimin; mobile phase, 0.2 M sodium phosphate pH 7.0, 2 mM EDTA retention times, F(ab’)z 15.94 min, buffer salts 21.22 min. 0.25
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Figure 3. Analytical size exclusion HPLC profile of reduced Fab’, monitored a t 280 nm. Key: Beckman SEC 3000 column; flow rate, 0.5 mumin; mobile phase, 0.2 M sodium phosphate pH 7.0, 2 mM EDTA; retention times, F(ab’)z 15.16 min, Fab’ 17.14 min, buffer salts 20.62 min.
measured by both a direct and an indirect radioimmunoassay. In the indirect RIA, increasing amounts of
Bioconjugate Chem., Vol. 6, No. 2, 1995 183
Cross-Linked Monoclonal Antibody Fragment kba
1
2
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Figure 6. SDS-PAGE of cross-linked antibody fragments. The gel was run under reducing conditions (0.1 M DTT) using a 5%
0
10
acrylamide stacking gel and a 12.5% running gel. Protein bands were visualized by staining with Coomassie blue. Lanes 1 and 8: prestained molecular weight markers, apparent molecular weights 215, 105, 70,43,28, 18, and 15 kDa. Lane 2: PRlA3 F(ab’12. Lane 3: F(ab’):! fragments from an irrelevant antibody. Lanes 4 and 5: unreacted Fab’ and Fab’maleimide recovered from the preparative size exclusion HPLC purification of the cross-linked fragments. Lanes 6 and 7: purified XL-F(ab’j2.
30
20
Retention time, min
Figure 4. Analytical size exclusion HPLC profile of the cross-
Competition for binding to MKN-45 cells
linked F(ab’)z reaction mixture monitored at 254 nm. Key: Beckman SEC 3000 column; flow rate, 0.5 mumin; mobile phase, 0.2 M sodium phosphate pH 7.0,2 mM EDTA;retention times, XL-F(ab’)a or aggregates14.09 min, XL-F(ab’)2 14.89 min, unreacted Fab’ and Fab’maleimide 16.52 min, buffer salts 20.28 min.
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cross-linkedF(ab)2 PR1A3 F(ab’)2 PRlA3Ab
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o f
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I
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Figure 7. Competition for binding to MKN-45 cells between iodine-125 labeled PRlA3 antibody and unlabeled antibody or fragments. MKN-45 cells were incubated with 40 ng of lz51labeled intact PRlA3 antibody and 0, 160,640,2560, or 10240 ng of unlabeled intact PRlA3 Ab, F(ab’)z or XGF(ab’I2.
0
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20
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Retention time, min
Figure 5. Analytical size exclusion HPLC profile of purified XL-F(ab’)*, monitored a t 254 nm. Key: Du Pont Zorbax GF2SO column; flow rate, 0.5 mumin; mobile phase, 0.1 M sodium phosphate pH 7.0, 2 mM EDTA; retention times, XL-F(ab’)z 16.87 min, buffer salts 24.13 min, 27.43 min.
unlabeled antibody or fragments competed with a constant amount of iodinated intact antibody for binding to MKN-45 cells. Figure 7 shows that the XL-F(ab’I2 competed essentially as well as F(ab’12 and intact antibody. A direct binding assay, using a method similar to that described by Lindmo (191,was used to determine the immunoreactivefraction of 1251-labeledXEF(ab’12 and 1251-labeledF(ab’)2. A double reciprocal plot of T/B against the reciprocal of the number of cells produces a
straight line where the reciprocal of the y-intercept gives the immunoreactive fraction. The results are shown in Figure 8. The immunoreactive fraction of 1251-labeledXLF(ab’)2 was 64%compared with 69%for the 125”Ilabeled F(ab’I2. Biodistribution Studies. The biodistribution of 12!jIlabeled XL-F(ab’)z was compared to that of ‘“1-labeled F(ab’), in nude mice bearing MKN-45 human tumor xenografts. Table 1shows the percent injected dose per gram of tissue for l2!jII-F(ab’)2and 1251-XL-F(ab’)2a t 4,24, and 48 h postinjection. Differences between the biodistributions of cross-linked fragments and unmodified F(ab’)Z were not significant at 4 h. At 24 h there was significantly less 1251-XL-F(ab’)2in all tissues ( p < 0.01 except for muscle, p < 0.05) except tumor. At 48 h the amount of XL-F(ab’)z in tumor was significantly higher ( p < 0.005),while that in liver, spleen, and kidneys was
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184 Bioconjugate Chem., Vol. 6, No. 2, 1995 Direct binding to MKN-45 cells
“
I
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40
80
llno of cells x 10E7
Figure 8. Comparison of direct binding of lz5I-labeledPRlA3 F(ab’)z and 12jI-labeled XL-F(ab’)z to MKN-45 cells. 12.5 ng of ‘*bI-F(ab’)z or 125I-XL-F(ab’)zwere incubated with a series of double dilutions of MKN-45 cells. The figure shows a Lindmo plot of totaL’bound counts against l/cell number. The reciprocal of the y-intercept gives the immunoreactive fraction of the antibody fragments.
significantly lower ( p < 0.001) than that of F(ab’)z. Tumor to tissue ratios for 1251-XL-F(ab‘)z and 1251-F(ab’)~ are given in Table 2. Tumor to tissue ratios a t 48 h ranged from 1.5 to 14.2 for F(ab’)z and from 6.2 to 35.2 for XL-F(ab’j2. Ratios for tumor to blood, liver, spleen, and kidneys were significantly higher a t 24 ( p < 0.05) and 48 h ( p < 0.005) for XL-F(ab’h. DISCUSSION
The design of the optimum antibody-based molecule for tumor targetting is still a matter of debate. While
many antibodies have proved successful for imaging a variety of tumors, it seems likely that small molecules with rapid kinetics that mirror the short physical half lives of the best diagnostic isotopes will have advantages for imaging. For radiotherapeutic applications, the retention time of the antibody in the tumor and the degree of accretion of the radiolabel by normal tissues become increasingly important. In this arena, it seems likely that larger molecules, probably multivalent, will be preferred. The designer antibodies of the future will almost certainly be made using recombinant techniques; however, more information on the effects of changes in molecular structure needs to be acquired before the blueprints for such molecules can be finalized, and much of this data can be more easily obtained by the use of conventional protein chemistry. We have explored the use of cross-linking agents for linking Fab‘ fragments of various antibodies. The first result of this approach has been the generation of a homobifunctional XL-F(ab’j2 fragment of the antibody PRlA3. We have characterized this molecule using conventional chromatographic techniques and shown in in-vitrobinding assays that the extensive processing does not adversely affect the ability of the molecule to bind to its epitope. Separation techniques based on size alone are unable to distinguish between F(ab’j2 fragments linked by disulfide bridges or other cross-linkers. We therefore performed SDS-PAGE separation under reducing conditions to show that our XL-F(ab’)z fragment contained the nonreducible 50000 MW band of the thioether bridged chains (lanes 6 and 7) compared with the conventional F(ab’Iz(lane 2). This analysis does not exclude the possibility that thiol reoxidation may also occur simultaneously, resulting in the presence of a quantity of contaminating F(ab’12 in our XL-F(ab‘)z preparation. However, we took great pains to avoid
Table 1. Biodistribution Data for lz5I-LabeledPRlA3 F(ab)z and Cross-Linked F(ab’)zin Nude Mice Bearing MKN-45 Human Tumor Xenografts“
tissue
4h
PRlA3 F(ab’)z 24 h
tumor blood liver spleen kidneys lungs muscle femur stomach intestines whole body clearancec
6.40 f 0.85 13.82 f 1.93 3.60 f 0.48 2.70 f 0.39 7.26 f 0.96 5.83 f 1.60 1.00 f 0.34 2.19 i 0.80 5.49 i 1.08 2.81 i 0.42 70.13 i 7.04
4.02 f 0.44 2.54 & 0.38 0.93 i 0.12 0.92 i 0.20 2.33 i 0.42 1.49 i 0.18 0.38 i 0.08 0.61 f 0.13 3.20 & 1.21 0.58 k 0.11 21.28 f 5.40
a
percent injected dose per gram cross-linked PRlA3 F(ab’)z 48 h 4 hb 24 hb 1.16 f 0.22 0.38 f 0.11 0.30 i 0.05 0.26 f 0.05 0.77 i 0.16 0.30 f 0.10 0.09 f 0.03 0.14 i 0.04 0.45 i 0.38 0.12 i 0.05 4.99 i 1.74
6.50 f 0.41 12.30 i 1.65 3.32 f 0.35 2.85 f 0.22 7.90 f 0.34 5.95 f 0.98 1.12 f 0.10 1.78 f 0.05 4.53 i 1.51 1.82 i 0.21 63.34 f 5.69
4.20 f 1.52 1.44 i 0.16 0.46 f 0.05 0.47 i 0.12 1.00 f 0.27 0.87 f 0.15 0.24 i 0.06 0.29 i 0.08 0.82 i 0.35 0.27 i 0.06 12.00 z t 2.36
48 h
1.95 & 0.41 0.34 f 0.12 0.14 i 0.02 0.14 f 0.02 0.26 i 0.04 0.25 i 0.07 0.06 i 0.02 0.09 f 0.02 0.24 f 0.11 0.07 i 0.02 4.11 i 1.17
Mean i standard deviation, n = 5 . n = 4. Percent injected dose.
Table 2. Tumor to Tissue Ratios for 12SI-labeledPRlA3 F(ab)z and Cross-Linked F(ab’)z in Nude Mice Bearing MKN-45 Human Tumor Xenograftsa cross-linked F(ab’)z F(ab’)z tissue blood liver spleen kidneys lungs muscle femur stomach intestines a
4h
0.47 i 0.09 1.79 i 0.31 2.41 i 0.50 0.88 f 0.10 1.16 f 0.32 7.32 f 3.67 3.29 k 1.37 1.19 rt 0.24 2.30 i 0.40
24 h 1.58 i 0.15 4.31 i 0.34 4.44 k 0.58 1.73 i 0.20 2.71 i 0.47 10.93 i 2.38 6.65 i 0.78 1.35 f 0.36 6.96 i 0.85
48 h 3.09 i 0.35 3.83 i 0.32 4.49 i 0.30 1.51 f 0.17 4.01 i 0.63 14.20 f 3.88 8.67 f 1.52 4.28 f 2.79 10.81 f 2.99
4h 0.54 f 0.06 1.99 & 0.21 2.31 f 0.13 0.83 f 0.04 1.12 f 0.16 5.91 f 0.65 3.68 f 0.02 1.59 f 0.59 3.64 f 0.39
24 h 2.95 i 1.04 9.11 f 2.83 8.95 i 2.11 4.50 i 2.13 4.97 f 1.89 19.46 i 9.81 14.34 i 3.93 5.41 ?c 1.81 15.50 i 3.91
Percent injected dose per gram for tumor divided by percent injected dose per gram for normal tissues.
48 h 6.16 f 1.47 14.34 i 1.04 14.54 f 2.93 7.84 i 1.11 8.27 i 1.25 35.21 310.35 23.22 f 4.27 10.21 i 5.39 28.55 f 8.47
Cross-Linked Monoclonal Antibody Fragment
reoxidation by the use of low pH, nitrogen-purged solvents, low temperature, and EDTA to scavenge the metal ions which catalyze the process in order to keep such contaminants to a minimum. In biodistribution studies in tumor-bearing mice, the cross-linked and native molecules show significant differences in-vivo,resulting in greater retention of the radionuclide by the tumor and lower uptake by normal tissues, notably the liver and kidneys, of XT.,-F(ab’)z. There was no overall significant difference in the blood clearance of the two fragments. From a n imaging perspective the end result of this comparison is that tumor to normal tissue ratios are enhanced by a factor of up to 5. From a therapeutic point of view the lower radiation burden received by normal organs would permit an escalation of the administered dose of radioactivity with a subsequent increase in tumor radiation dose. Although we have performed these studies with only one antibody the combination of these results with those in other publications (12,25,27) suggest that this could be a general phenomenon. We have also used only one radionuclide, radioiodine, in this study. The use of other radiolabels, such as radiometals, is likely to produce conjugates which show differences in biodistribution. Since the iodine-protein bond itself shows evidence of poor in-vivostability (281,the use of more stable indium or technetium chelates may well result in a still greater differential between cross-linked and native F(ab’Iz fragments, but this remains to be substantiated by further experimentation. From a technical point of view, the methodology as described above is elaborate and the multiplicity of steps results in poor yields-of the order of 10%. It is hard to imagine, therefore, that this method could be recommended for the routine preparation of cross-linked fragments for clinical application. Our aim is more to identify those important aspects of molecular design which should be incorporated into recombinant antibody molecules. While this work was in progress two reports of biodistribution studies with maleimide-linked fragments in tumor xenograft models have appeared in the literature. Quadri et al. (12)prepared a series of three lllIn-labeled cross-linked F(ab’)z and found that cross-linked F(ab‘Iz showed significantly less kidney activity and higher tumor retention than that of unmodified F(ab’Iz in good agreement with our results. Bood clearance of the crosslinked fragments, however, was intermediate between that of IgG and F(ab’Iz. The difference between this finding and our results may perhaps be related to the use of a different radiolabel. Schott et al. (27) reported the synthesis of two new maleimide reagents which they used to prepare Io5Rh-labeled F(ab’)3 and F(ab‘I4 fragments. They reported that the biodistribution behavior of the F(ab’)3 fragments in both Balb/c and tumor-bearing nude mice was intermediate between that of IgG and unmodified F(ab’Ie, with lower kidney uptake than unmodified F(ab’)z. The larger F(ab‘)( fragments, however, were found to accumulate in the liver in Balb/c mice. We can, therefore, conclude that antibody fragments which are cross-linked with nonreduceable bridges have the potential to improve the current status of both immunoscintigraphy and radioimmunotherapy. ACKNOWLEDGMENT
We gratefully acknowledge the financial support of the Imperial Cancer Research Fund and the facilities of the Dominion House Centre for Clinical Research.
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