BiOconJugateChem. W92, 3, 477-483
477
Site-Specific Conjugation of Chain-Terminal Chelating Polymers to Fab’ Fragments of Anti-CEA mAb: Effect of Linkage Type and Polymer Size on Conjugate Biodistribution in Nude Mice Bearing Human Colorectal Carcinoma M. A. Slinkin,’,’ C. Curtet,+ C. Sai-Maure1,t J. F. Gestin,+V. P. Torchilin,t and J. F. Chatalt Laboratoire Biophysique-Cancerologie,INSERM U.211, Plateau Technique CHR, Quai Moncousu, 44035 Nantes Cedex 01, France, and Center for Imaging and Pharmaceutical Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129. Received February 6, 1992
Polylysine-based chelating polymers were used for site-specific modification of anti-CEA mAb Fab’ fragments via their SH group distal to the antigen-binding site of the antibody molecule. Conjugation was performed using chain-terminal (pyridy1dithio)propionateor 4-@-maleimidophenyl)butyratemoieties to form reducible (S-S) or stable (S-C) bonds between a polymer and Fab’ molecule, respectively. One S-S conjugate (S-Sg) and two different S-C conjugates ( 5 x 3 and S-Cg) were prepared using 3- and 9-kDa molecular weight polymers. No significant loss of immunoreactivity was observed in solid-phase immunoassay, 90-95 % of lllIn-labeled conjugates being bound to CEA-coated Sepharose beads. After labeling with ll1In, the conjugates had a specific radioactivity of 90-120 pCi/pg. Injected in nude mice bearing LS 174T carcinoma, the conjugates produced different biodistribution patterns. S-Sg was practically unable to accumulate in tumor and produced very rapid blood clearance of radioactivity and high uptake of radioactivity in liver, spleen, and especially kidneys (225% ID/g 24 h postinjection). 5x3and S-Cg produced practically the same blood clearances (much slower than that of S-Sg) and significant tumor uptake (9-1074 ID/g at 24 h). S-C,gave significantly lower radioactivity in spleen, skin, and bones, and cleared more rapidly from liver and kidneys. Renal uptake for 5 x 3 and S-Cg was rather high (45% ID/g at 24 h), but much lower than for S-Sg.
INTRODUCTION
The use of chelating polymers for mAb modification was initially proposed as a means of increasing the number of heavy-metal ions which could be bound to mAb molecule without loss of its immunoreactivity (1-3). The main purpose was to use immunoreactive mAb molecules highly loaded with Gd atoms as an immunospecific contrast agent in magnetic resonance imaging. Subsequently, biodistribution studies using different animal models also showed some unexpected and very favorable effects of mAb coupling with chelating polymers in target visualization in immunoscintigraphy (4,5),thus enhancing the potential role of such polymers as mAb modifiers. We recently proposed the use of chain-terminal polylysine-based chelating polymers to reduce the risk of antibody inactivation during its modification with chelating polymer (6). Our approach allows every polymer chain to be coupled to an antibody molecule strictly via a single amino acid residue, thereby providing a high yield during the conjugation reaction. However, the choice of an ‘anchor” residue for polymer attachment to antibody remains to be determined. If it were adjacent to the antigen-binding site, this would result in some loss of antibody immunoreactivity upon its modification. Therefore, it would be advisable to ensure polymer binding to antibody molecule via the amino acid residue, which is undoubtedly located distal to the antibody hypervariable region (Le., near the hinge region), in order to ensure sitespecific binding of polymer to antibody. The conjugate could thus be prepared with exact knowledge of the molecular structure. ~
~
t
*
~~~~
~~~~~
~
~
~
Laboratoire Biophysique-Cancerologie. Center for Imaging and Pharmaceutical Research.
This approach was applied in the present work to modification of SH groups of anti-CEA Fab’ fragments by polymer chain-terminal groups. Reducible disulfide (SS) or stable thioether (S-C) bonds were used to test whether the type of Fab’-polymer molecule binding has an effect on conjugate biodistribution. The S-C bond was formed using a maleimide-terminal polymer not previously described. As we were also interested in the possible effect of polymer size, conjugates were prepared using two different maleimide-terminal polymers with 3- and 9-kDa molecular weights (MW). EXPERIMENTAL PROCEDURES
Anti-CEA mAb F6, and IgGl specific for human gastrointestinal tract adenocarcinomas, was kindly provided by the CIS Biointernational Co. (Saclay, France) (7). F(ab’)2 fragments were obtained by pepsin digestion. As starting polymers, e-N-(carbobenzoxy)poly(D,Llysine) with 3 and 9-kDa MW were used (PL-Z). PL-Z, N-succinimidyl 3-(pyridy1dithio)propionate (SPDP), 2(methylsulfony1)ethylN-succinimidyl carbonate (MSOCNHS), N-succinimidyl4- (p-maleimidophenyl)butyric acid (SMPB), diethylenetriamine-N,N,”,h”’,”‘-pentaacetic acid cyclic anhydryde (caDTPA),trinitrobenzenesulfonic acid (TNBS), dithitreitol (DTT), and 5,5’-dithiobis(2nitrobenzoic acid) (DTNB) were obtained from Sigma Chemical Co. (La Veprillihre, France). Buffer solution components and organic solvents were obtained from Aldrich (St. Quentin Fallavier, France) and carriers for gel chromatography from Pharmacia-LKB (St. Quentin en Yvelines, France). F(ab’)t Reduction and Activation with Ellman’s Reagent. Fab’ fragments were prepared from F(ab’)z as described in ref 8. Briefly, a portion of F(ab’)2was reduced by addition of 2-mercaptoethylamine to a concentration
1043-1802192129Q3-Q477$Q3.QQlQ 0 1992 American Chemical Society
Slinkln et ai.
Bloconlugete Chem., Vol. 3, No. 0, 1992
478
0
0
(CH2)4
I
0
(CH2)4
(PL-2)
(CH2)4
I
I
DTPA
1
(MSOC-NHS)
J
DTPA
SUC
1
+DTT
DTPA
SUC
+SMPB
U
0
0
I/
II
0
7-
HO-C-CH-NH-[C- H NH],-C-
I (CH2)4 I
(CH2)4
I
0
II
0
I1
H-NH-C-O-CHZCHZ-O-S-CH~
I‘F (CH2)4 I
DTPA
rH rH YH i, (i
I
HBr/CH$OOH
0 0 II I1 HO-C-CH-NH-[C- H NH]n-C- H-NH-C-O-CHZCHZ-O-S-CH~
0 II
SUC
DTPA
DTPA
+ Fab’-TNB
SUC
1
DTPA
+ Fab’-SH
0 I1
1
I
NH2
0
(CH2)4
I
NH2
0
II II . HO-C-CH-NH-[C- H NH],-[CI I NH Ic=o I
(CH2)4
(7H2)2 COOH
liF (CH2)4 I
-
(CH2)4
(CH2)4
I I c=o I
NH
DTPA
I
0
II
(PL-MSOC)
NH2
1 ) caDTPA
2) Succinic anhydride 3) NaOH 0 2M 5 min 4) HCI pH - 7 - 8
0
(iH.NH]i.IIc C H NH2
(CH2)4
I NH I c=o I (CH212 I
(CH2)4
1
’c=o YH
(PLsUc-DTPA-NH2)
I
DTPA
COOH
Figure 1. Preparation of amino chain-terminalchelating polymer
based on polylysine. Z is a benzyloxycarbonyl residue.
of 1mM, EDTA to 1mM, and N a A s 0 ~to 10 mM. After incubation of the solution a t room temperature for 18 h, solid Ellman’s reagent (DTNB) was added to 20 mM. After a further 3 h, excess reagents were removed by centrifugal gel filtration (9) on Sephadex G-25, preequilibrated with 0.1 M sodium phosphate and 1 mM EDTA, pH 6.8. Recovery of Fab’-thionitrobenzoate (Fab’-TNB) derivatives was 8O-85%. A small sample of each Fab’-TNB was analyzed by HPLC at 0.7 mL/min on a TSK 3000-SW gel filtration column fitted with a guard column packed with TSK 2000-SW. The column (7.5 mm X 30 cm) was equilibrated to 0.1 M sodium phosphate, pH 6.8. Absorbance was monitored at 254 and 330 nm. F(ab’)z emerged at 13 min and Fab’-TNB at 15 min in this system. Polymer Synthesis. PL,,,-DTPA-PDP. Chelating polymer PL-DTPA-PDP was prepared based on caDTPA and PL-Z with an average MW of 9 kDa as described in ref 6. This polymer was further modified to prepare succinylated polymer (PL,,,-DTPA-PDP) by addition of 10-20-fold molar excess of succinic anhydride (with respect to the polymer unit) in 0.1 M carbonate, pH 8 (concentrated NaOH solution being used to maintain the pH at 8-9 during the reaction). The absence of free NHz groups in PL,,,-DTPA-PDP was confirmed immediately after reaction by colorimetric assay with 2,4,6-trinitrobenzenesulfonic acid (IO). PLS,-DTPA-NH2. Amino chain-terminal polymer PLwDTPA-NH2 prepared according to Figure 1 was used as a precursor to introduce the MPB functional moiety at the polymer terminus. Twenty milligrams of PL-Z with 3- or 9-kDa MW was dissolved in 0.5 mL of DMSO, and
Figure2. Synthetic schemes for the preparation of bioconjugates with disulfide (a) or thioether (b) bonds.
a 2-fold molar excess of triethylamine was added with respect to the polymer molecule. An &fold molar excess of MSOC-NHS was then added and the mixture was incubated at room temperature overnight. The terminalmodified polymer was then separated, Z residues deprotected, and DTPA groups introduced as previously described (61,to obtain PL-DTPA-MSOC. The remaining free c-amino groups of lysine monomers were subsequently modified with succinic anhydride in the same way as indicated above for PL,,,-DTPA-PDP, producing PL,,,DTPA-MSOC, which gave no color reaction with TNBS reagent. To deprotect the MSOC group, solid NaOH was added to the solution of the latter polymer up to a concentration of 0.2 M. After 5 min of incubation, pH 7-8 was restored with concentrated HCl. The appearance of chain-terminal amino groups after the deprotection step was tested by TNBS reaction using aliquota from the solutions of both polymers at a concentration of 2 mg/mL (with respect to initial PL-MSOC). Briefly, 75 p L of polymer solution were added to 125 pL of water + 200 pL of 0.1 M borate, pH 9.2. To this mixture was added 100 pL of 5.7 mM TNBS solution. After 1 h of incubation, 100 pL of the solution of 2 M NaH2P04 + 20 mM NaZS03 were added, and optical densities at 420 nm were measured. After the deprotection step, Sephadex G-25 column centrifugation was performed to separate all low molecular weight compounds and to change the buffer to 0.1 M phosphate, pH 7.0. Conjugate Preparation. Fab‘-S-S-PL,,,-DTPA (SS,) (Figure 2a). Immediately before the conjugation reaction, the SH group was generated on the terminus of the polymer chain. PL,,,-DTPA-PDP was reduced with dithiothreitol (DTT) added to the polymer solution (0.2 mg, 2 mg/mL) to a final concentration of 25 mM. The mixture obtained was then incubated for 20 min. Reduced polymer was separated from low molecular weight impurities (including DTPA) by centrifugal gel filtration on Sephadex G-25 columns equilibrated with 0.1 M sodium phosphate, pH 6.5. Immediately after separation, SH polymer solution was added to 0.5 mg of anti-CEA Fab’TNB (3 mg/mL), and the mixture was then incubated for 18 h a t 4 “C. This reaction formed the disulfide bond between the polymer and Fab’ molecules. A small aliquot from the solution was then analyzed by HPLC as described above, with the resulting single broad peak a t 13 min indicating the absence of free Fab’ in the mixture. To
Fab'4heIatlng Polymer Conjugate
purify the conjugate from a free polymer, gel chromatography of the mixture was performed on a Sephadex G-100 column. Fab'-S-C-PL,,,-DTPA (BC3and S-C.) (Figure2b). A 0.4-mg sample of both of the PL,,,-DTPA-NHz prepared as described above were modified with a 3-fold molar excess (with respect to polymer molecule) of SMPB reagent. The reaction was performed for 2 h in 0.1 M phosphate, pH 7.0, under a polymer concentration of 2 mg/mL. The low molecular weight substances were then separated by Sephadex G-25 column centrifugation, and PL,,,-DTPAMPB polymers were obtained in 0.1 M phosphate/l mM EDTA, pH 6.8. Simultaneously, 1mg of Fab'-TNB in 0.1 M phosphate/ 1 mM EDTA (3 mg/mL) was reduced with 1 mM of 2-mercaptoethylamine for 1h, and the Fab'-SH produced was purified by Sephadex G-25 column centrifugation. Immediately after the polymer and Fab'-SH purification step, reactions were allowed to proceed between 0.3-0.4 mg of every PL,,,-DTPA-MPB and 0.5 mg of Fab'-SH in 0.1 M phosphate/l mM EDTA, pH 6.8, for 1h. They were then stopped by addition of 1 mg of DTNB. The conjugates were subsequently separated from nonreacted Fab' fragments and polymers by DEAE-Sephadex A-25 anion-exchange chromatography as described in ref 4. Briefly, nonreacted protein was washed from the column with the starting buffer (0.05M acetate, pH 6.0), conjugates were washed with a 0.45 M NaCl step gradient, and highly negatively charged polymers were washed only with a 0.95 M NaCl step gradient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis [4-16% (w/v) gel gradient] was done to control the purity, molecular weight, and stability of the conjugates prepared (see Figure 5 for details). lllIn Conjugate Labeling. To label polymeric conjugates with "'In, 10 pg of every conjugate was incubated for 2 h in 0.02 M citrate + 0.1 M acetate, pH 5, with 1.4 mCi "'indium chloride. Free 11lIn was then separated in an Amicon C-30 ultrafiltration cell, and bound and total radioactivities were counted in a Medi 202 y counter (Medisystem, France). Immunoreactivity Assay. Two parameters of conjugate immunoreactivity were studied: apparent affinity and the immunoreactive fraction. To estimate the apparent affinity of anti-CEA F(ab')z or Fab' preparations, tubes coated with anti-CEA (CIS Biointernation Co., Saclay, France) were used. First, the antigen solution in 0.05 M citrate, pH 5.0, was added to the tubes and incubated at 45 "C for 3 h. Each tube was then washed twice with 3 mL of 0.05% Tween-20 solution, and serial dilutions of the preparation were done in the presence of a constant amount of 1251-anti-CEA.The tubes were subsequently incubated overnight at room temperature, washed twice with 0.05% Tween, and counted in a Compugamma LKB y counter. The apparent affinity of conjugates was estimated by the half-binding point derived from the titration curves obtained. The immunoreactive fraction of llh-labeled conjugates was determined by a binding assay using CEA-coated Sepharose beads (11). Briefly, a 200-pL aliquot of "'Inlabeled conjugate, previously diluted with PBS to a protein concentration of 50 ng/mL, was added to 0.2 mL of a 50 7% (v/v) CEA-coated bead suspension, previously incubated with BSA for 1h, in an Eppendorf test tube. The test tube was counted in a y counter, and the contents were mixed under vortexing at room temperature for 1h. The tube was then centrifuged and the pellet of beads collected and washed twice by resuspension in 0.5 mL of
Bioconjugate Chem., Vol. 3, No. 6, 1992 470
PBS and recentrifugation. The final bead pellet was counted in a y counter, and the percentage of radiolabeled conjugate bound to the CEA-coated beads was determined by comparing the net cpm of test tube contents before and after washing. Biodistribution Studies. Nude mice (8-weeks-old) were injected subcutaneously in the right flank with 107 LS 174T cells. Twelve to fifteen days later, the mice were injected in the tail vein with 100 pL of 11'In-labeled conjugate in 0.9% NaCl(1pg of protein). The radioactivity injected was 3.44-5.18 MBq for polymeric conjugates. After anesthesia, mice were killed by cervical dislocation 4, 24 and 96 h after injection. All organs were removed from each animal, washed, dried, and weighed. Tumor weight at the time of removal ranged from 0.2 to 0.5 g. Radioactivity was measured on a y counter, and resulta were expressed as the percentage (mean f SEM) of injected dose per gram of tissue (5% ID/%)and in tumor-to-normal tissue ratios for an average of four or five mice injected at each time interval. RESULTS
Synthesis of PL,,,-DTPA-MPB Polymers. To introduce the MPB moiety at the terminus of the polylysine chain, a synthetic route (see Figure 1)somewhat longer than that used for the synthesis of PL,,-DTPA-PDP was chosen. Introduced in the first step (as in the case of the PDP group),the maleimide function would obviouslyreact with deprotected e-amino groups of lysine monomers under the conditions of polymer modification with caDTPA and succinic anhydride. As there is no simple way to conserve a maleimide group in the protected state (as in the case of the SH group), we decided to use the MSOC group well known in peptide chemistry (12,13) to protect the NH2terminal group of PL-Z at the first step. This protecting group could easily be eliminated after introduction in the polymer chain of DTPA and succinic acid residues, with release of the terminal NH2 group available for reaction with SMPB reagent. TNBS reagent analysis of the terminal amino groups in both PLs,,-DTPA-NH2 polymers prepared (based on PL-Z of 3- and 9-kDa MW) showed a 3-fold difference in their content which correlated well with the molecular weight differences of the corresponding PL-Z polymers. When the same TNBS reaction of NH2-terminal polymers was used after the column centrifugation step, recovery of polymers with initial MW of 3 and 9 kDa after separation was found to be 70% and 85%, respectively. Conjugate Preparation and Analysis. S-SS. Figure 3a,b shows the HPLC chromatograms of Fab'-TNB and the aliquot from the reaction mixture after Fab'-TNB conjugation with PLs,,-DTPA-SH, demonstrating the complete incorporation of Fab'-TNB in conjugate and the absence of any aggregate formation during the modification procedure. The shifting and broadening of the conjugate peak, easily apparent compared to that of Fab'-TNB alone, can be attributedto an increase in protein molecular weight after conjugation to a polymer with a rather broad molecular weight distribution. The absence of free Fab'-TNB in the mixture after conjugation was independently proven by DEAE-Sephadex ion-exchange chromatography under the conditions described above for S-C conjugates (see Experimental Procedures), which gave no protein peak washed with the starting buffer (0.05 M acetate, pH 5.5). This high reaction yield allowed purification of the conjugate to be carried out by Sephadex G-100 gel chromatography alone, which easily allowed separation of much smaller molecules of free polymer (see Figure 4).
480
chem.,Vd. 3,NO. 6, 1992
”te
Sllnkhr
1
I
10925
-m
94
.-m
67
c
v)
a al.
43 30 6680 1
I
I
20.1
9447
-mc .-CI)
14.41
L
v)
J
1
2
3
4
5
7
6
8
Figure 5. Analysis of the Conjugates by SDS-PAGE (4.516%) was done under reducing (lanes 2-4) and nonreducing (lanes 5-8) conditions: lanes 2 and 6, anti-CEA Fab’-TNB; lanes 3 and 7, S-Sg; lanes 4 and 8, SX9; lane 5, anti-CEA F(ab’)B. The gel was calibrated with the marker proteins (lane 1).
5536 7850
-mc
-*-
I
.-m v)
6632
2 -
-
7381
d)
-m C
.-cn v)
0.0
3 10
15
Fraction number
Figure 6. Ion-exchange chromatography on DEAESephadex 5
10
15
20
25
min
Figure 3. HPLC elution profiles of Fab’-TNB (a), S-S9 in the mixture after conjugationreaction (b), and purified S-CS (c) F d S-Cg (d),as detected by UV adsorption at 280nm. Sizeexclusion column chromatography (TSK G3OOOSW, 7.5 X 300 mm) was followed by elution with 0.1 M acetate + 0.1 M NaCl (pH 5.0) at a flow rate of 0.7 mL/min. A I
0 Y
.-s; .-> 0 .--0
-
c. 0
E
0.0
Fraction number
Figure 4. Gel chromatography of the reaction mixture after
conjugation of Fab’-TNB with PL,,,-DTPA-SH (a) and of ll1In-PLUc-DTPA-PDP(b) on a Sephadex G-100 column (1.0 X 20 cm) equilibrated with 0.01 M acetate + 0.02 M citrate + 0.08 M NaC1, pH 5.0. Fractions (0.6 mL) were collected, and optical density and y radioactivity were measured.
Molecular weight increase and the absenceof aggregates were also demonstrated by using SDS-polyacrylamide gel electrophoresis (see Figure 5). Under nonreducing conditions, Fab’-TNB migrated as a narrow band of apparent MW 50 OOO, whereas S-S9 migrated as a broad band of
A-25 (1 X 2 cm) of the reaction mixture after conjugation of Fab’-SH with PL,,-DTPA-MPB. Total salt concentrations are indicated above the gradient steps. Fraction volume is 1 mL.
apparent MW 55-70 OOO. Under reducing conditions, the complete splitting of S-S9 conjugate showed two bands of apparent MW 28 0oO and 30 OOO corresponding to fragments of light and heavy chains with no evidence of polymer coupled to these fragments. S-C3 and S-C* HPLC chromatographyona TSK 3000 column showed that the coupling effeciency of PL,,,DTPA-MPB polymers with Fab’-SH was not as high as that of PL,,,-DTPA-SH with Fab’-TNI3. For both polymers only 3 0 4 5 % of protein was incorporated into the conjugate. Therefore, these conjugates had to be purified using DEAE-Sephadex ion-exchange chromatography, which allowed the separation of unbound Fab’ fragmentsand polymers from the conjugate via application of different salt concentrations. Figure 6 illustrates such separation in the case of S+. The protein peak washed in 0.05 M acetate + 0.45 M NaCl, pH 5.5, was used for “‘In labeling, and lllIn-labeled SX9 gave a single peak on Sephadex G-100column chromatography,showing the absence of free polymer (data not shown). HPLC chromatography of purified S-C3 and S-C9 (Figure 3c,d) showed some molecular weight difference and the absence of aggregates in both preparations. Purified SX9was alsoanalyzed by SDS-polyacrylamide gel electrophoresis (Figure 5). While under nonreducing conditions it gave practically the same band as S-S9, reducingconditions revealed the difference between these two preparations. S-C9 then showed one narrow band of apparent MW 30 OOO corresponding to chain fragments and one broad band of MW 35-40 OOO obviously corre-
Fab’4helating Polymer Conjugate
Bloconlugere Chem., Vol. 3, No. 6, 1992 401
I2000
10000
P
a n v) (Y r
* F(ab’)P -
4000 2000 -
8000
with some slight increase in tumor radiactivity between 4and 24 h postinjection and a significant decrease between 24 and 96 h. Such biodistribution patterns resulted in significantly higher tumor:blood, tumor:liver, and tumor: x3as compared to S-Cg at 24 and 96 kidney ratios for 5 h postinjection (Table 11).
6WO
DISCUSSION
0 001
0 01
0 1
1
10
Ab added, u g h 1
Figure 7. Comparison of the apparent affinity of different antiCEA F(ab’)*-and Fab’-based preparations by solid-phase competitive radioimmunoassay.
sponding to polymer-modified chain fragments. Such splitting proves that there was indeed a single coupling point between the polymer and Fab’ molecules. When modified with a polymer carrying randomly distributed protein-reactive groups, Fab fragments do not split at all in reducing conditions due to their intramolecular crosslinking with the polymer molecule (14). Specific Conjugate Radioactivity and Immunoreactivity. The maximal specific radioactivity attained for lllIn-labeled conjugates was 90-120 pCi/pg of protein, which is much higher than that of Fab or F(ab92 fragments labeled via low molecular weight chelating agents (15,16), indicating that a large number of DTPA residues were incorporated into the conjugate. Figure 7 compares the apparent affinity of the conjugates prepared with those of anti-CEA F(ab’)2 and Fab’-TNB. There was some decrease in the immunoreactivity of Fab’ as compared to F(ab’)z (a well-known phenomenon in immunochemistry), but there was practically no further immunoreactivity loss for polymer-modified Fab’ fragments. The absence of adverse affection of Fab’ immunoreactivity was also confirmed by measurement of the immunoreactive fraction: the percentage of binding of l1’1n-labeled conjugates with CEA-coated microbeads in conditions of approximately infinite antigen excess was 90-95% , indicating excellent retention of initial Fab’ fragment immunoreactivity during conjugatepreparation. Biodistribution Studies. The results of biodistribution in human colorectalcarcinoma tumor-bearing athymic mice for lllIn-labeled S-Cs,S-Cg, and S-Sg are summarized in Table I. At 4 h postinjection, blood radioactivity for S-S9 was already much lower than for both of the thioether conjugates, obviously due to the breakdown of the disulfide bond in vivo. Such rapid blood clearance of radioactivity appears to be the main reason for the very low accumulation of S-S9 radioactivity in tumor, which was 3.5 and 9 times as low as for the other two conjugates at 4 and 24 h postinjection, respectively. At the same time, compared with S-C conjugates, S-Sg produced significantly higher radioactivities in liver, spleen, and especially kidneys, apparently reflecting the biodistribution of lllIn-labeled PL,,,-DTPA-SH released from S-59. When the biodistribution Of S-CSand S-Cg is compared, a difference can be noted in the kinetics of radioactivity clearance from liver, kidneys, spleen, skin, and bone. Whereas the concentrations of conjugates in these organs 4 h postinjection did not differ statistically, S-C3 radioactivity was significantly lower at 24 and especially 96 h compared to that of S-Cg. Conversely, tumor concentrations revealed the same kinetics for both conjugates,
The present work describes a simple approach for sitespecificconjugation of chain-terminal chelating polymers with Fab’ fragments of IgG antibody. This approach ensures a single binding site between the chelating polymer chain and Fab’ distal to the hypervariable region of the antibody molecule. It combines the advantages of chainterminal chelating polymer and site-specific antibody modification; i.e., it completely excludes the possibility of inter- and intramolecular cross-linking of the modified antibody and dramatically reduces the risk of its inactivation due to modification of amino acid residues essential to maintain the spatial organization of the antigen-binding site. A priori, it seems unlikely that antibody immunoreactivity would be changed by such modification unless there were some sort of unfavorable polymer-protein interaction not connected with the polymer-binding site. The method used for Fab’-TNB preparation allowed the generation of two or three protected SH groups thus enabling two depending on the subclass of IgG or three polymer chains to be coupled to an antibody molecule. Two different methods of Fab’ modification were performed in order to prepare conjugateswith two different linkages between Fab’ and polymer molecules. The simpler way of preparing the conjugate consisted of the formation of a disulfide bond using Fab’-TNB and PL,,,DTPA-SH. Under the conditions described, Fab’ fragments were completely incorporated into the conjugate without aggregate formation. Preparation of the conjugate with a thioether bond was more complicated, requiring in particular the synthesis of PL,,,-DTPA-MPB through the PL,,,-DTPA-MSOC and PL,,,-DTPA-NHz polymers. Furthermore, the previous reduction of Fab’-TNB and separation of the Fab’-SH obtained were indispensable to the conjugation reaction. The efficiencyof this reaction was not as high as for disulfide bond formation since only 35-40 96 of Fab’-SH was incorporated into the conjugate. The synthetic scheme elaborated for the preparation of PLsUc-DTPA-NH2 seems somewhatmore flexiblethan that for PL,,,-DTPA-PDP since it allows the introduction a t the polymer chain terminus of any functional moiety able to react with amino groups. The only prerequisite is to block all deprotected lysine monomer e-aminogroups. This can easily be performed with a suitable acylating agent selected according to the net electron polymer charge required. The choice of acetic instead of succinic anhydride allows the preparation of a polymer not carrying such a high negative charge (unpublished results). In keeping with the main idea behind the approach used in this study, the immunoreactivity of Fab’ fragmenta was completely preserved after their conjugation with polymers. Interestingly, the apparent affinity of Fab’ was tested using nonlabeled conjugates, whereas immunoconjugates highly labeled with lllIn were used to determine the immunoreactive fraction. The results suggest that DTPA residue saturation with a heavy metal had no influence on the immunoreactivity of the conjugates. The different stability of S-S and S-C linkages between Fab’ and polymer molecules was clearly demonstrated by SDS gel electrophoresis since the former was completely
(In,
482
Slinkln et ai.
Biocon)ugate Chem., Vol. 3, No. 6, 1992
Table I. Biodistribution of
SC3,
SCS,and S-Sg in Athymic Mice Bearing Human Colorectal Carcinoma Tumor.
SC3
organ tumor blood liver kidney spleen bone skin
4h 8.2 f 2.1 15.8f 2.8 8.0 f 1.2 48.4 f 12.5 4.6 f 0.6 2.7 f 1.2 2.9 f 0.8
24 h 9.7 f 1.9 1.3 f 0.3 6.2 f 0.9 43.5 f 5.7 2.2 f 0.2 1.2 f 0.1 2.7 f 0.1
5-ss 24 h
sc9
96 h 4.2 f 0.6 0.16f0.01 4.2 f 1.0 13.2 f 3.1 2.0 f 0.6 1.0 f 0.2 1.6 f 0.2
4h 8.9 f 1.6 16.0f 1.7 8.4 f 2.1 32.5 f 8.9 4.7 f 1.0 4.5 f 1.4 2.9 f 1.1
24 h 9.3 f 0.2 1.8 f 0.1 10.1 f 1.6 45.5 f 2.3 4.3 f 0.8 3.4 f 0.7 4.9 f 1.1
96 h 5.1 f 1.0 0.21 fO.06 9.1 f 0.9 24.0 f 5.7 5.9 f 1.0 4.1 f 0.8 4.1 f 0.8
4h 2.6 f 0.5 1.9f 0.4 25.4 f 4.2 121.2 f 9.8 9.8 f 1.7 1.5 f 0.2 1.4 f 0.6
1.0 f 0.1
1.2f0.4 18.7 f 3.1 223.4 f 9.4 17.3 f 2.4 3.9 f 1.5 4.3 f 2.2
96 h 0.6 f 0.2 0.04f0.01 11.8 f 1.4 62.6 f 4.3 6.5 f 1.0 3.0 & 0.4 2.6 f 0.8
a Organ levels of radioactivity are expressed as percentage of injected doseigram of tissue (mean f SD, n = 4-5) after iv injection of 1 pg of the corresponding conjugate.
Table 11. Tumor-to-Organ Ratios of
S C 3
and SC9 at 4,24, and 96 h Postinjection
scs
SC2 tumorlorean ratio . tumor/ blood tumorjliver tumor/ kidney tumorispleen tumoribone
4h 0.50 1.03 0.17 1.78 3.03
24 h 7.46 1.56 0.22 4.40 8.08
96 h 26.25 1.00 0.31 2.10 4.20
4h 0.55 1.06 0.27 1.89 1.98
24 h 5.17 0.92 0.20 2.16 2.73
96 h 24.28 0.56 0.21 0.86 1.24
decrease in blood clearance for the former. Instead of 3 % broken down under reducing conditions. The disulfide ID/$ of radioactivity remaining in blood 4 h after injection bond is also known to be labile and unstable in circulation of ll’In-Fab’, there was about 16% ID/g of lllIn-labeled (18, 19), apparently because of the presence of low S-C3 and S-Cg still circulating. Such a decrease is concentrations of reduced gluthathione which is continually being manufactured by the liver and is maintained obviously related to the increased molecular weight of the polymeric conjugates. in plasma at a level of about 24 pM (20). Our biodistribution data, in particular the very fast blood clearance of The differences in S-C3 and 5429 biodistribution could also be related to differences either in the conjugate radioactivity for S-59 as compared to S-C conjugates, also strongly suggest a breakdown of 5-S9 in vivo. High electron charges or molecular weight. The fact that S-C3 clears from kidneys and liver significantly faster than S - C g accumulation of radioactivity in kidneys can obviously be suggests that negatively charged lllIn-labeled digestion related to the biodistribution of released lllIn-PLsucproducts from S-C3 leave these organs more easily than DTPA-SH. Such accumulation seems strange in the light of the results reported for the highly negatively charged do those from S-Cg because of their lower molecular weight. However, the real mechanism accounting for these compound lllIn-antimyosin-Fab-succinimidyl-PLsucDTPA (4)which demonstrated low kidney concentration differences is still not clear. From a practical point of view, it would be of interest to decrease renal and liver in a canine model of myocardial infarction. The unexuptake of polymeric conjugates further by using chelating pected accumulation of this rather low molecular weight agents which provide more stable metal complexescapable negatively charged polymer could be due to the role of the of metabolism in liver and kidney (23). polymer terminal SH group available for some modifiIt may be concluded that the simple chemical approach cations in vivo. This could have changed the biodistridescribed in this paper could allow the preparation of a bution pattern of the polymer, which could also account number of immunoreactive conjugates highly charged with for the relatively high accumulation of 5-Sg radioactivity metal ions. Easily available S-S conjugate characterized in liver. Even S-C3 and S-Cg conjugates with stable thioether by a poor biodistribution pattern could be used for some in vitro applications, e.g., the enhancement of the sensilinkages demonstrated relatively high kidney accumulation tivity of time-resolved fluorescence immunoassay (after of radioactivity, with rather unfavorable tumoxkidney ratios for tumor imaging. However, renal uptake for both labeling with Eu3+ or Tb3+) (24). However, for in vivo applications only conjugates with a relatively stable linkage these conjugates in a nude mouse model was much lower than that of Fab’or Fab fragments labeled with lllIn using between a polymer and Fab’ fragments would seem caDTPA. Thus, the renal uptake of lllIn after injection suitable. Labeled with Gd3+, such conjugates could be used as immunospecific contrasts agents for magnetic of ll’In-labeled anti-CEA C198 Fab was approximately 60% of the whole body activity at 72 h postinjection (211, resonance imaging (251,or when labeled with a cytotoxic and lllIn-labeled MoAb OV-TL 3 Fab’ produced 150% radioactive isotope up to high specific radioactivity, they ID/g in kidneys 24 h postinjection (22). It is not absolutely could also be used in radioimmunotherapy to reduce the clear from this study whether decreased renal uptake of amount of administered xenoprotein and hence the risk polymeric as compared to DTPA-modified conjugates is of anti-murine antibody response (26). due to negative polymer charge or simply to the somewhat LITERATURE CITED higher molecular weight of the former. It is possible that (1) Shreve, P.,Aisen, A. E. (1986) Monoclonal antibodies labeled both effects are involved. In any event, liver uptake of with polymeric paramagnetic ion chelates. Magn. Res. Med. radioactivity for S-Ca and S-Cg was 2-3 times as great as 3, 336-340. that of lllIn-labeled Fab (or Fab’) fragments in the studies (2) Manabe, Y., Longley, C., and Furmanski, P. (1986) Highjust mentioned. This suggests that for Fab’-polymer level conjugation of chelating agents onto immunoglobulins: conjugates as opposed to Fab(Fab’)-DTPA the organ use of intermediary poly(L-lysine)-diethylenetriaminepencatabolizing the immunoconjugate shifts to some extent taacetic acid carrier. Biophys. Biochym. Acta 883,460-467. from kidneys to liver. It is also noteworthy that comparison (3) Torchilin, V. P., Klibanov, A. L., Nossiff, N. D., Slinkin, M. of the biodistribution of S-C polymeric conjugates with A., Strauss, W., Haber, E., Smirnov, V. N., and Khaw, B.A. that of l1’1n-OV-TL 3 Fab’-DTPA shows a significant (1987) Monoclonal antibody modification with chelate-linked
Fab’-Chelatlng Polymer Conjugate
high-molecular-weightpolymers: major increasesin polyvalent cation binding without loss of antigen binding. Hybridoma 6, 229-240. (4) Khaw,B. A.,Klibanov,A.,ODonnell,S. M., Saito,T., Nossiff, N., Slinkin, M. A., Newell, J. B., Strauss, H. W., and Torchilin, V. P. (1991) Gamma imaging with negatively charge-modified monoclonal antibody: Modification with synthetic polymers. J. Nucl. Med. 32, 1742-1751. (5) Wang, T. S. T., Fawwaz, R. A,, and Alderson, P. 0. (1992) Reduced hepatic accumulation of radiolabeled monoclonal antibodies with indium-111-thioether-poly-L-lysine-DTPAmonoclonal antibody-TP41.2F(ab’)~.J.Nucl. Med. 33,570574. (6) Slinkin, M. A., Klibanov, A. L., and Torchilin, V. P. (1991) Terminal-modified polylysine-basedchelating polymers: Highly efficient coupling to antibody with minimal loss in immunoreactivity. Bioconjugate Chem. 2, 342-348. (7) Accolla, R. S., Carrel, S., and Mach, J. P. (1980) Monoclonal antibodies specificfor carcinoembryonicantigen and produced by two hybrid cell lines. Proc. Natl. Acad. Sci. U.S.A. 77, 563-566. (8) Brennan, M. (1986)A chemicaltechnique for the preparation of bispecific antibodies from Fab fragments of mouse monoclonal IgGl. BioTechniques 4, 424-427. (9) Parkinson, A. J., Scott, E. N., and Muchmore, H. G. (1981) Purification of labeled antibody by minicolumn gel centrifugation. Anal. Biochem. 118,401-404. (10) Fields, R. (1972) The rapid determination of amino groups with TNBS. In Methods of Enzymology (Hirs C. H. W., and Timasheff, S. N., Eds.) Vol. XXV, Part B, pp 464-468, Academic Press, New York. (11) Sumerdon, G. A., Rogers,P. E.,Lombardo, C. M.,Scnobrich, K. E., Melvin, S. L., Hobart, E. D., Tribby, I. I. E., Stroupe, S. D., and Johnson, D. K. (1990) An optimized antibodychelator conjugate for imaging of carcinoembryonic antigen with indium-111. Nucl. Med. Biol. 17, 247-254. (12) Wolters,E.T. M.,Tesser,G., andNivard,R. (1974)Synthesis of the A 14-21 sequence of ovine insulin by the solid-phase technique. J. Org. Chem. 39, 3388-3392. (13) Tesser, G. I., and Balvert-Geers, I. C. (1975) The methylsulfonylethyloxycarbonyl group, a new and versatile amino protective function. Znt. J. Pept. Protein Res. 7, 295-305. (14) Slinkin, M. A,, Klibanov, A. L., Khaw, B. A., and Torchilin, V. P. (1990)Succinylated polylysine as a possible link between the antibody molecule and deferoxamine. Bioconjugate Chem. 1,291-295. (15) Khaw,B. A.,Yasuda,T.,Gold,H. K.,Leinbach,R. C.,Johns, J. A., Kanke, M.,Barlai-Kovach,M., Strauss, H. W., and Haber, E. (1987) Acute myocardial infarct imaging with indium-111 labeled monoclonal antimyosin Fab. J.Nucl. Med. 28,76-82.
Bioconlugate Chem., Vol. 3,
No. 6, l9g2 483
(16) Yemul, S., Leon, J. A., Seldin, D. M., Link, M. J., Kramer, P., Mesa-Tejada, R., and Estabrook, A. (1991) Tumor localization in nude mice bearing human breast carcinoma xenografts using lllIn-DTPA conjugates of monoclonal antibodies. Nucl. Med. Biol. 18, 295-304. (17) Svasti, J., and Milstein, C. (1972) The disulfide bridges of a mouse immunoglobulin G1 protein. Biochem. J. 126, 837850. (18) Paik, C. H., Quadri, S. M., and Reba, R. C. (1989) Interposition of different chemical linkages between antibody and 111In-DTPAto accelerate clearance from non-target organs and blood. Nucl. Med. Biol. 16, 475-481. (19) Blakey, D. C., Watson, G. L., Knowles, P. P., and Thorpe, P. E. (1987)Effect of chemical deglycosylationof ricin-A chain on the in vivo fate and cytotoxic activity of an immunotoxin compound of ricin-A chain and anti-Thy 1.1antybody. Cancer Res. 47, 947-952. (20) Thorpe, P. E., Wallace, P. M., Knowles, P. P., Relf, M. G., Brown, A. N. F., Watson, G. J., Knyba, R. E., Wawrzynczak, E. J., and Blakey, D. C. (1987) New coupling agents for the synthesis of immunotoxins containing a hindered disulfide bond with improved stability in vivo. Cancer Res. 47,59245931. (21) Andrew, S. M., Pimm, M. V., Perkins, A. C., and Baldwin, R. W. (1986)Comparative imaging and biodistribution studies with an anti-CEA monoclonal antibody and its F(ab’)z and Fab fragments in mice with colon carcinoma xenografts. Eur. J. Nucl. Med. 12, 168-175. (22) Massuger, L. F. A. G., Claessens, R. A. M. J., Pak, K. Y., Boerman, 0. C., Daddona, P. E., Koenders, E. B., Kenemans, P., and Corstens, F. H. M. (1991) Tissue distribution of hTc, 111In and 1231-OV-TL3 Fab’ in ovarian carcinoma bearing nude mice. Nucl. Med. Biol. 18, 77-83. (23) Paik, C. H., Yokoyama, K., Reynolds, J. C., Quadri, S. M., Min, C. Y., Shin, S. Y., Maloney, P. J., Larson, S. M., and Reba, R. C. (1989) Reduction of background activities by introduction of a diester linkagebetween antibody and a chelate in radioimmunodetection of tumor. J. Nucl. Med. 30, 16931701. (24) Hemmila, I. (1985) Fluoroimmunoassays and immunofluorometric assays. Clin. Chem. 318, 359-370. (25) Anderson-Berg, W. T., Strand, M., Lempert, T. E., Rasenbaum, A. E., and Joseph, P. M. (1986) Nuclear magnetic resonance and gamma camera tumor imaging using Gd-labeled monoclonal antibodies. J. Nucl. Med. 27, 829-833. (26) Pimm, M. V., Perkins, A. C., Armitage, N. C., and Baldwin, R. W. (1985)The characteristics of blood-born radiolabels and the effect of anti-mouse IgG antibodies on localization of radiolabeled monoclonalantibody in cancer patients. J.Nucl. Med. 26, 1011-1023.
