Bivalent Hapten-Bearing Peptides Designed for Iodine-131

Jun 15, 1997 - considered for radioimmunotherapy. The advantages of bivalent haptens have been demonstrated in this context. A series of bivalent ...
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Bioconjugate Chem. 1997, 8, 526−533

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Bivalent Hapten-Bearing Peptides Designed for Iodine-131 Pretargeted Radioimmunotherapy E. Janevik-Ivanovska,†,‡ E. Gautherot,§ M. Hillairet de Boisferon,† M. Cohen,† G. Milhaud,‡ A. Tartar,| W. Rostene,† J. Barbet,§ and A. Gruaz-Guyon*,† INSERM U.339 and Service de Biophysique, Faculte´ de Me´decine Saint Antoine, 27 Rue Chaligny, 75012 Paris, France; IMMUNOTECH S.A., 130 Avenue de Lattre de Tassigny, B.P. 176, 13276 Marseille Cedex 9, France; and URA 1309 CNRS, Institut Pasteur de Lille, 1 Rue du Pr. Calmette, B.P. 245, 59019 Lille Cedex, France. Received November 15, 1996X

Pretargeting with bispecific antibodies has been used successfully for tumor detection and is now considered for radioimmunotherapy. The advantages of bivalent haptens have been demonstrated in this context. A series of bivalent molecules allowing efficient labeling with radioactive iodine has been designed for use with this new technology. They were based on the histamine-hemisuccinate hapten and prepared by solid phase peptide synthesis. Simultaneous binding of two antibody molecules to one bivalent hapten was possible with low steric hindrance when the two hapten groups were attached to the lateral chains of lysine residues separated by a single amino acid. Bispecific antibodies to the hapten and to carcinoembryonic antigen were shown to mediate specific binding of the haptens to tumor cells in vitro. These experiments demonstrated that the bivalent hapten AG3.0, with a lysylD-tyrosyl-lysine connecting chain, possessed the best binding properties. This peptide was used to target iodine-125 to human colon cancer xenografts in nude mice. High tumor uptake and tumor to normal tissue ratios were observed. This peptide thus appears as a good candidate for further development. Asymmetric bivalent haptens, with one histamine-hemisuccinate and one diethylenetriaminepentaacetic acid group, have also been prepared and shown to be capable of binding simultaneously two specific antibody molecules. These peptides should be useful to target radioiodine to cells characterized by the expression of two different antigenic markers.

INTRODUCTION

During the past 15 years, radionuclides have been targeted to tumors by means of monoclonal antibodies for diagnostic imaging and radioimmunotherapy [for reviews see Mach et al. (1991) and Goldenberg (1993)]. To increase the targeting specificity, two- and three-step pretargeting techniques have been proposed (Goodwin et al., 1986, 1988; Stickney et al., 1989, 1991; Lollo et al., 1994; Santos et al., 1995). We have worked on an improved two-step pretargeting technique (Le Doussal et al., 1989), which we refer to as the “affinity enhancement system” (AES). The AES uses a bispecific antibody (BsmAb) (anti-tumor antigen × anti-hapten) to target a radiolabeled bivalent hapten. The bivalent hapten exhibits preferential binding to cell-bound BsmAb, as opposed to excess circulating BsmAb, due to the formation of stable cyclic complexes at the cell surface (cell antigen-BsmAb-bivalent hapten-BsmAb-cell antigen). This technique affords high tumor to normal tissue ratios in animal models (Le Doussal et al., 1990). The advantages of bivalent haptens in this context have also been independently recognized by Goodwin et al. (1992). The efficacy of the AES technique has been established for the detection of tumors expressing the carcinoembryonic antigen (CEA) such as colon carcinoma (Le Doussal et al., 1993; Chetanneau et al., 1994) and medullary thyroid * Author to whom correspondence should be addressed [telephone (33) 01 40 01 14 66; fax (33) 01 43 43 89 46; e-mail [email protected]]. † INSERM. ‡ Service de Biophysique. § IMMUNOTECH. | URA 1309 CNRS. X Abstract published in Advance ACS Abstracts, June 15, 1997.

S1043-1802(97)00083-9 CCC: $14.00

carcinoma (MTC) (Peltier et al., 1993) in the clinic. Very small MTC occult metastases (2 mm in diameter) have been localized by peroperative detection and resected, demonstrating the accuracy and specificity of the method (de Labriolle-Vaylet et al., 1993). In clinical trials the diethylenetriaminepentaacetic acid (DTPA)-indium complex has been used as a hapten because indium-111 is well suited to tumor scintigraphy and peroperative detection. Radioimmunotherapy should also be markedly improved by targeting β-emitting isotopes to tumors using BsmAb and a bivalent hapten. We introduced a tyrosine residue in the peptide sequence of the bivalent hapten designed for 111Inlabeling to allow also iodine-125 or iodine-131 labeling (Gruaz-Guyon et al., 1991). Nevertheless, new bivalent haptens, with improved targeting efficiency, specifically designed for use in radioimmunotherapy with iodine131 would be of major interest. Here we report the synthesis of a series of bivalent haptens in which the histamine-hemisuccinate hapten (Morel et al., 1990) has been coupled to peptide connecting chains of various lengths and structures. The resulting bivalent haptens have been investigated for their ability to simultaneously bind, with high affinity, two anti-histaminehemisuccinate antibody molecules. One bivalent hapten has been selected on this basis and studied for its ability to bind to BsmAb-pretargeted tumor cells in vitro and in vivo. MATERIALS AND METHODS

Haptens and Antibodies. The hapten 3-[[[2-(4imidazolyl)ethyl]amino]carbonyl]propionylglycine has been coupled to a series of peptide chains. This hapten will be referred to as histamine-succinyl-glycyl (HSG). The anti-HSG monoclonal antibody (mAb) 679.1MC7 (IgG1,κ) has already been described (Morel et al., 1990). © 1997 American Chemical Society

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Table 1. Synthesized Peptides and Equilibrium Affinity Constants of HSG-DTPA and HSG Haptens for Anti-HSG Antibody Fab′ Fragmenta

a

HSG, histamine-succinyl-glycyl; GABA, γ-aminobutyric acid; DTPA, diethylenetriaminepentaacetic acid.

DTPA was also coupled to a series of peptides bearing the HSG hapten. The 734 monoclonal antibody (IgG1,λ), with specificity to the DTPA-indium complex, has been described previously (Le Doussal et al., 1990). F6 is a mouse IgG1,κ antibody specific for human CEA. The antibodies were produced in tissue culture, purified, and fragmented by pepsin digestion according to standard procedures. The F(ab′)2 fragments were reduced with 2-mercaptoethylamine for 1 h at 37 °C and alkylated with N-ethylmaleimide to prepare the monovalent tracers or with maleimidobiotin to prepare the antibody-coated solid phases, using avidin-coated tubes (Immunotech S.A., Marseille, France). The anti-CEA × anti-HSG BsmAb was prepared by chemical coupling of the two reduced Fab′ fragment using o-phenylenedimaleimide according to the procedure of Glennie et al. (1987). Peptides. We synthesized peptides bearing one HSG hapten (mono-HSG peptides, the AG4 series), two HSG haptens (di-HSG peptides, the AG3 series), and one DTPA and one HSG hapten (DTPA-HSG peptides, the AG5 series) as listed in Table 1. N-R-DTPA-tyrosyl-N-DTPA-lysine dipeptide (di-DTPA-TL) was synthesized as described previously (Le Doussal et al., 1990). Synthesis of Mono-HSG and Di-HSG Peptides. MonoHSG and di-HSG peptides were synthesized manually by the stepwise solid phase method. The protected amino acids (1.57 mmol) (Bachem, Switzerland) were sequentially coupled to p-methylbenzhydrylamine resin (0.63 mmol of active groups) after activation with dicyclohexylcarbodiimide and 1-hydroxybenzotriazole (HOBT) to synthesize the backbone. The tyrosine side chain was protected with the 2,6-dichlorobenzyl group. tert-Butyloxycarbonyl (tBoc), fluorenylmethyloxycarbonyl (Fmoc), and 2-chlorobenzyloxycarbonyl (2-ClZ) were used to protect either R or  amino groups, as shown in Figure 1, depending on in which position the hapten HSG was to be synthesized. We used for AG3.0 and AG4.0 the same protections as for AG3.1 and AG4.1. Each coupling step was repeated twice. After synthesis of the backbone, the desired NH2 groups were deprotected and N-Rprotected glycine (tBoc-Gly or Fmoc-Gly) was coupled under the conditions described above for backbone synthesis. After deprotection, a 3-fold molar excess of succinic anhydride was coupled in the presence of diiso-

propylethylamine (DIEA) (3-fold molar excess) (Aldrich, France). Histamine dihydrochloride (3-fold molar excess) (Aldrich) in solution in dimethyl sulfoxide/dimethylformamide (5:2, v/v) was coupled using benzotriazol-1yloxytris(dimethylamino)phosphonium (BOP) and HOBT (3-fold molar excess) in the presence of DIEA (15-fold molar excess). Then 600 mg of the dried peptide resin was treated by liquid hydrogen fluoride (6 mL) in the presence of p-cresol (750 µL) for 1 h at 4 °C. After evaporation in vacuo, the crude peptide was precipitated in cold diethyl ether and extracted with 10% acetic acid. The peptide was purified by gel permeation chromatography and C18 reversed phase chromatography (Nucleosil, Shandon, France) with a gradient (A ) 0.5‰ trifluoroacetic acid in water and B ) 0.5‰ trifluoroacetic acid in water/acetonitrile, 50:50, v/v) from 0% B to 40% B in 70 min. The purity of each peptide (g95%) was checked by C18 reversed phase HPLC (Nucleosil) in two different solvent systems: system 1 (A ) 0.5‰ trifluoroacetic acid in water; B ) acetonitrile; gradient ) isocratic 0% B for 5 min, to 30% B in 30 min, then to 60% in 10 min) and system 2 (A ) heptafluorobutyric acid 0.5‰ in water; B ) acetonitrile; gradient ) isocratic 0% B for 5 min, to 50% B in 30 min, then to 60% in 10 min). UV absorbance was monitored at 210 and 280 nm. The amino acid ratios determined after total acid hydrolysis for the HSG and DTPA-HSG series were consistent with theoretical results. We further characterized the peptides by plasma desorption mass spectrometry using a Bio-Ion mass spectrometer (Uppsala, Sweden). AG4.1 (M + H)+: 899.8 found, 899.8 calcd. AG4.0 (M + H)+: 729.6 found, 729.6 calcd. AG4.2 (M + H)+: 559.4 found, 559.6 calcd. AG4.3 (M + H)+: 559.4 found, 559.4 calcd. AG3.1 (M + H)+: 1149.6 found, 1149.11 calcd. AG3.0 (M + H)+: 980.1 found, 979.7 calcd. AG3.2 (M + H)+: 809.6 found, 809.5 calcd. AG3.3 (M + H)+: 809.9 found, 809.5 calcd. 2D NMR spectrometry (in DMSO at 313 K with a Bruker 400 MHz AMX spectrometer) confirmed the structure of the compound AG3.0 (Figure 2) selected for in vivo studies: 1H NMR (DMSO-d6) δ CH3CO 1.90; Lys1 (NH 8.00, HR 3.66, Hβ 1.40, Ηβ′ 1.43, Hγ 0.95, Hγ′ 1.00, Hδδ′ 1.45, H′ 3.05, NH 7.80); Tyr2 (NH 8.17, HR 4.46,

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Figure 1. Synthesis of mono-HSG and di-HSG haptens. Resin, p-methylbenzhydrylamine resin; tBoc, tert-butyloxycarbonyl; Fmoc, fluorenylmethyloxycarbonyl; 2-chloro-Z, 2-chlorobenzyloxycarbonyl; 2,6-dichloroBzl, 2,6-dichlorobenzyl.

Figure 2. Structure of the AG3.0 bivalent hapten.

Hβ 3.02, Ηβ′ 2.74, H-3,5 6.70, H-2,6 7.10); Lys3 (NH 7.95, HR 4.20, Hβ 1.72, Ηβ′ 1.57, Hγγ′ 1.25, Hδδ′ 1.45, H′ 3.10, NH 7.80); CONH2 7.05, 7.25; Gly (NH 8.03, HR 3.02); succinyl(CH2)2 2.43; histamine (NH 7.96, N-CH2 3.38, CH2-Im 2.75, imidazole H-2 8.45, H-4 7.10). Synthesis of DTPA-HSG Peptides. DTPA was coupled to the mono-HSG peptide as follows. Briefly, 25 µmol of a peptide solution (5 mM) in HEPES buffer (1 M, pH 8.2) was added with stirring to 150 µmol of DTPA dianhydride (Aldrich) in solution in DMSO (150 mM). The pH was maintained at 8.2 until the reaction was completed. After evaporation of the solvents, the crude product was dissolved in water and trifluoroacetic acid added to reach pH 2. The solution was filtered through Chelex 100 (BioRad, France). The resulting peptide was purified by C18 reversed phase preparative HPLC (Nucleosil) as described above for di-HSG peptides. The solvents were filtered through Chelex 100 (Bio-Rad). The purity g95% was checked by HPLC in systems 1 and 2. AG5.1 (M + H)+: 1275.7 found, 1275.0 calcd. AG5.0 (M + H)+: 1104.9 found, 1104.6 calcd. AG5.2 (M + H)+: 934.7 found, 934.4 calcd. AG5.3 (M + H)+: 934.4 found, 934.4 calcd.

The measured UV molar absorbance at 280 nm (pH 7) for a solution of each purified peptide was consistent with the peptide concentration (determined after total acid hydrolysis and amino acid analysis) and the tyrosine content (molar extinction coefficient ) 1300). This value was used to determine the concentration of the peptides in all experiments. Radiolabeling. Iodination of AG3.0 (2 nmol) was performed using Na125I (18.5 MBq) and chloramine T (10 µg) for 2 min at room temperature and stopped with 100 µg of sodium disulfide. The monoiodinated peptide was purified by reversed phase C18 HPLC. Antibodies (25 µg) were iodinated with Na125I (18.5 MBq) using iodogen (Salacinsky et al., 1981). Labeling with indium-111 (37 MBq InCl3) of DTPA peptides (250 pmol) was performed in citrate buffer (pH 5.0) for 24 h, and then 10 nmol of unlabeled InCl3 was added to saturate free DTPA groups. Free indium-111 was determined after chromatography on a Sep-Pak C18 cartridge (Waters, Milford, MA), and maximal immunoreactivity (g95%) was evaluated from binding experiments of trace amounts of 111In-labeled peptide to 734 antibody-coated tubes. Binding Experiments. Equilibrium Affinity Constant Determination. For Ka determination of mono-HSG and DTPA-HSG peptide binding to anti-HSG mAb, trace amounts of 111In-labeled AG5.1 were incubated for 2.5 h under shaking in 679.1MC7 mAb-coated tubes in the presence of increasing amounts of competitors at 37 °C (pH 7.4) in 1 mL (final volume) of PBS-0.2% BSA (four experiments in triplicate). Nonspecific binding was evaluated in the presence of excess unlabeled AG5.1 (2 × 10-7 M). Ka values were fitted from four experimental competition curves in triplicate (Barbet et al., 1993).

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For Ka determination of (In)DTPA-bearing peptide binding to anti-(In)DTPA, mAb was determined in similar experiments using 734 mAb-coated tubes and competition between 111In labeled AG5.0 and unlabeled AG5.1. Sandwich experiments with DTPA-HSG peptides were performed in 679.1MC7 antibody-coated tubes at 37 °C and pH 7.4. The coated antibody concentration (about 3 × 10-9 M) was determined from competition binding experiments as described above with unlabeled AG5.1 as competitor. Increasing concentrations of unlabeled DTPA-HSG peptides were incubated in the presence of trace amounts of labeled 125I-734 Fab′ (alkylated with N-ethylmaleimide) for 2.5 h under shaking in a final volume of 1 mL of PBS-0.2% BSA. Nonspecific binding (1.2%) was determined in the absence of bivalent hapten. Di-HSG peptides sandwich experiments were performed as for DTPA-HSG peptides, except for the antibodycoated tube concentration (about 5 × 10-10 M), and the use of 125I-labeled 679.1MC7 Fab′ antibody fragment. Cell binding experiments were performed on LS 174 T colorectal carcinoma strain cell (ATCC). One hundred microliters of suspension (2 × 107 cells/mL), 100 µL of bispecific anti-CEA × anti-histamine BsmAb (F6 × 6791MC7) dilutions, 150 µL of 125I-labeled AG3.0 (4.6 × 10-10 M final, specific activity ) 2.3 × 1018 cpm/mol) were incubated together in PBS-0.2% BSA supplemented with NaN3 (0.02%). After 2.5 h under shaking, 100 µL of the suspensions was centrifuged in triplicate tubes for 30 s through a phthalate mixture (Dower et al., 1981). Aliquots of supernatants and the bottom of each tube (containing the cell pellet) were counted. Binding studies of trace amounts of 125I-labeled BsmAb in the presence of increasing concentrations of unlabeled BsmAb were performed according to the same procedure. In Vivo Experiments. Animals. Female BALB/c-nu/ nu mice, 6-8 weeks old (Iffa-Credo, France) were grafted by sc injection in the flank with 2 × 106 LS174T human colorectal carcinoma cells. In some experiments a control tumor (2 × 106 A375 human melanoma cells) was grafted in the other flank. Immunoscintigraphy and biodistribution studies were performed 2 weeks later. Biodistribution. Triplicate mice were given 2 µg (in 50 µL of PBS) of F6 × 679.1MC7 (anti-CEA × anti-HSG) BsmAb by iv injection under light ether anesthesia. Seventeen hours later 125I-labeled AG3.0 (1 pmol, 2.7 × 1018 cpm/mol was injected iv. Mice were weighed and sacrificed with ether at selected time intervals (1-168 h). Blood was collected on heparin after heart puncture. Actual injected doses were estimated by subtraction of noninjected and sc-injected material from the total dose. Data from mice injected with 90%. 111In labeling of DTPA peptides led to 95% chelation of 111In (60%. Equilibrium Affinity Constant Determination. The equilibrium affinity constants (Ka) for the binding of the mono-HSG (AG4 series) and the DTPA-HSG (AG5 series) peptides (Table 1) were determined from competition binding curves with 111In-labeled AG5.1, run in triplicate. To allow comparisons with cell binding and in vivo experiments, all measurements were performed at 37 °C with physiological salt concentration. Nonlinear least-squares regression (Barbet et al., 1993) was used to identify the equilibrium parameters (affinity constant, number of binding sites). The affinity of the HSG hapten depends on its position on the peptide chain. The highest Ka value is observed for HSG coupled to the lateral chain of lysine [AG4.0 Ka ) (6.8 ( 0.2) × 109 M-1]. The Ka value for the homologous peptide in the DTPA-HSG series is lower [AG5.0 Ka ) (2.9 ( 0.1) × 109 M-1]. The presence of a γ-aminobutyric acid (GABA) next to the lysine also decreases Ka in the HSG series [AG4.1 Ka ) (3.8 ( 0.1) × 109 M-1]. The affinity constant [Ka ) (1.8 ( 0.2) × 109 M-1] of the anti-DTPA indium antibody to (In)DTPA coupled to the -NH2 of a lysine was determined from similar competition binding experiments, using 111In-labeled and unlabeled AG5.0. Sandwich Experiments. DTPA-HSG peptides were tested for simultaneous binding to two anti-hapten antibodies. Then, increasing concentrations of peptides were incubated in anti-HSG antibody-coated tubes (about 3 × 10-9 M) in the presence of trace amounts of 125Ilabeled anti-DTPA antibody. The results of two experiments (in triplicate tubes) were fitted to the calculated equilibrium isotherms by nonlinear least-squares regression (Barbet et al., 1993). A coefficient (σ) was introduced in the calculation to express the steric hindrance for the simultaneous binding of two antibodies to the bivalent hapten: if Ka is the affinity constant for the first binding event, then the affinity for the binding of a second antibody to the bivalent hapten is σKa. The σ parameter and the immunoreactivity of the labeled antibody were highly correlated, and as a consequence they could not be adjusted simultaneously. However, assuming 70% immunoreactivity in all calculations, comparison of σ values showed that the hapten accessibility depends on the length of the peptide chain. Very similar σ values

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Figure 3. di-HSG hapten mediated binding of anti-HSG 125I-Fab′ to anti-HSG Fab′-coated solid phase: the tracer antibody was incubated for 2.5 h at 37 °C in anti-HSG-coated tubes in the presence of increasing concentrations of di-HSG hapten. Then aliquots of supernatant were counted (F), and the tubes were emptied, washed, and counted again (B). Mean B/F ( SD bars are given unless smaller than point plotted (n ) 6).

were evaluated for the two longest peptides AG5.1 and AG5.0, 0.6 and 0.5 respectively. For the shortest peptide AG 5.2, σ was found lower than 0.1. With bivalent di-HSG haptens (the AG3 series), crosslinking of two antibodies coated on the tube must be avoided to allow binding analysis. Thus, a 6 × 10-10 M concentration of coated antibody was determined as appropriate to minimize this cross-linking phenomenon (data not shown). The accessibility of bivalent HSG haptens to two anti-HSG antibodies was then evaluated using 6 × 10-10 M anti-HSG antibody-coated tubes (Figure 3). Assuming that the immunoreactivity of the labeled antibody was 70%, the accessibility coefficients σ for AG3.1 and AG3.0 were 0.4 and 0.3, respectively. Equilibrium Binding to Tumor Cells in Vitro. The ability of the anti-CEA × anti-HSG BsmAb to target 125I-labeled AG3.0 to CEA-expressing cells was studied in binding experiments using LS174T human colorectal carcinoma cells. The binding of the labeled hapten to the target cells was monitored in the presence of increasing concentrations of BsmAb. At the optimal BsmAb concentration (3.2 × 10-9 M), 59% of the hapten was bound. The binding parameters of the BsmAb (immunoreactivity ) 50%), Ka ) (2.1 ( 0.3) × 108 M-1 and binding site concentration ) (3.3 ( 0.4) × 105 sites per cell, were calculated from binding studies of trace amounts of 125I-labeled BsmAb in the presence of increasing concentrations of unlabeled BsmAb in a parallel experiment. In Vivo Targeting of AG3.0 to Human Colon Carcinoma Grafted in Nude Mice. Tumor-bearing (from 0.15 to 1.3 g) mice were given an injection of BsmAb (anti-CEA × anti-HSG) and, 17 h later, 125I-labeled AG3.0. The targeted activity at 3 h was 15.6 ( 1.0 of the injected dose per gram of tumor (% ID/g) and remained stable until 6 h. Then it decreased slowly to reach 6% ID/g at 24 h and 2% ID/g at 96 h (T1/2 ) 54.2 ( 0.2 h, Figure 4). High tumor to normal tissue contrast ratios were observed as soon as 3 h after tracer injection: tumor/plasma ) 1.7 ( 0.2, tumor/liver ) 7.9 ( 2.5, tumor/kidney ) 4.0 ( 1.1 as shown in Figure 4, tumor/ heart ) 14.1 ( 3.8, tumor/gastrointestinal tract ) 12.7 ( 0.8, tumor/lung ) 6.0 ( 0.5, tumor/spleen ) 11.7 ( 2.8. They all increased with time. Similar tumor uptake

was observed with the directly labeled F(ab′)2, but maximum uptake was reached around 48 h. Tumor wash-out was slow and approximately parallel to that of pretargeted AG3.0. Tumor to plasma ratios were always significantly higher with the pretargeted hapten, whereas in other tissues the contrast ratios were higher with the pretargeted hapten for 24 h (kidneys) to 48 h (liver) and then plateaued at longer time intervals (Figure 4). Control experiments in mice bearing two grafted tumors showed that hapten pretargeting was specific: as soon as 1 h after tracer injection, the contrast between the CEA positive target tumor (LS174T) and the control tumor (A375) (CEA negative) was 2.0 ( 0.2; it was 7.3 ( 1.6 at 3 h and reached 31.2 ( 8.0 at 24 h. When compared to in vivo targeting of the 111In-labeled hapten, the 125I-labeled AG3.0 exhibited comparable tumor uptake but higher tumor to blood contrast ratios and tumor to other organs contrast ratios 24 h after tracer injection (Figure 5). Analysis of Plasma and Urine Samples. Mice were injected with BsmAb and then 17 h later with the labeled bivalent hapten. Plasma samples were collected 1 h later. The samples were submitted to gel filtration chromatography. Two peaks of activity were observed at 13 and 16 mL corresponding to 200 and 100 kDa (Figure 6), respectively. This demonstrated that most hapten remained bivalent and circulated bound to one (100 kDa) or two BsmAb (200 kDa) (the free hapten would elute around 22 mL under these conditions). In addition, urine samples were tested for immunoreactivity, which was found better than 90% up to 24 h after injection. DISCUSSION

We have demonstrated in previous studies that pretargeted AES immunodetection affords improved tumor detection sensitivity as compared to other imaging techniques such as magnetic resonance imaging, ultrasonography, computed tomography, and classical immunoscintigraphy with directly labeled antibodies. This technique is based on the formation of cyclic complexes at the cell surface between two BsmAb molecules and one bivalent hapten-bearing peptide. Our previous work

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Figure 4. Tumor localization (A), tumor/organ ratios (B, C, D) time course: nude mice bearing LS174T tumor (CEA positive) in the flank were injected in triplicate with 20 µg of 125I-labeled F(ab′)2 (dashed lines) or with 2 µg of anti-CEA × anti-HSG BsmAb and 17 h later with 125I-labeled AG3.0 (solid lines). Groups of three mice were sacrificed at selected time intervals, and dissected organs were counted. Mean ( SD are plotted. In each panel an insert shows the kinetics over the first 12 h after activity injection.

Figure 5. Biodistribution of 125I- and 111In-labeled bivalent haptens: nude mice bearing LS174T tumor (CEA-positive) in one flank and A375 (CEA-negative) tumor in the other were injected with 2 µg of the BsmAb and 17 h later with the labeled bivalent hapten, anti-ACE × anti-HSG BsmAb and 125I-labeled AG3.0, or anti-ACE × anti-indium-DTPA BsmAb and 111In-diDTPA. Groups of three mice were sacrificed and dissected at 1 and 24 h, and the organs were counted.

concerning tumor and metastasis detection has been performed with the 111In-labeled N-R-DTPA-tyrosyl-N-DTPA-lysine dipeptide. The high tumor to normal tissue ratios and the amount of injected dose localized in tumors, observed in animals and in the clinic, should allow radioimmunotherapy with this technique, and experiments are in progress to demonstrate this possibility. However, to use iodine-131, a β-emitting isotope useful for therapy, the DTPA-indium hapten required the development of specific labeling and purification techniques because indium must be complexed to DTPA before or after labeling with iodine. In addition, chemical syntheses with DTPA, a pentavalent carboxylic acid, are difficult and usually afford complex mixtures containing polymeric contaminants. The aim of this work was thus to synthesize new bivalent hapten-bearing peptides, suitable for radioimmunotherapy using the AES technique, with improved antibody-binding and targeting

Figure 6. Gel filtration radioactivity elution profile of plasma: the sample was collected 1 h after 125I-labeled AG3.0 injection to a mouse preinjected 17 h before with BsmAb.

efficiency characteristics. These hapten-bearing peptides were also designed to allow fast and easy radiolabeling with iodine-131. The other characteristics of an ideal hapten are low toxicity, high-affinity binding to available antibodies, and no cross-reactivity or nonspecific binding with body components. The histamine-succinyl-glycine hapten was chosen for these reasons. A first bivalent HSG peptide was synthesized. Although able to target radioactivity to tumors, it was rapidly degraded in vivo (Gruaz-Guyon et al., 1991). We therefore synthesized more stable compounds (GruazGuyon et al., 1993). A D-tyrosine was introduced in the backbone for iodine labeling. The C-terminal end was amidated, the N-terminal was acetylated, and lysine side chains were substituted. As a result, the bivalent HSG peptide AG 3.0 should be extremely resistant to proteases. In plasma most of the activity recovered 1 h after injection circulated bound to two bispecific antibody molecules (Figure 6), and in the urine 90% of the radioactivity is able to bind to anti-HSG antibody 24 h after injection. In addition, the stability of this hapten-

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bearing peptide after internalization by target cells in vitro has recently been demonstrated (Manetti et al., 1995). To determine the peptide chain providing the optimal distance between the two haptens, we synthesized a series of bivalent haptens listed in Table 1 and studied in vitro their ability to cross-link two antibodies. The HSG hapten equilibrium affinity constants have been measured for monohistamine derivatives and, as expected, the Ka depends on the hapten position in the peptide chain. Coupling the hapten to the lateral chain of lysine affords the highest affinity. This is consistent with the observation of Morel et al. (1990) that optimum binding affinity is obtained only when the hapten closely mimics the immunogen. We then studied the binding of a labeled anti-hapten Fab fragment to a solid phase coated with anti-hapten Fab fragment in the presence of increasing concentrations of bivalent haptens. As a model, we studied several peptides bearing two different haptens: DTPA-indium and HSG (Table 1). The equilibrium binding curves of 125I-labeled anti-DTPA-indium antibody to anti-HSG-coated tubes in the presence of increasing concentrations of peptide were fitted using a simple computer program (Barbet et al., 1993). As expected, the bivalent haptens AG5.0 and AG5.1 with the longest connecting peptide chain exhibited the best accessibility to two antibodies. The accessibilities for the bivalent HSG haptens AG3.0 and AG3.1 were found very close. It is remarkable that maximum cross-linking ability is obtained with rather short peptide chains (2 to 3 residues), in agreement with early work by Valentine and Green (1967). As the hapten affinity was higher for the AG3.0 peptide, it was selected for further studies as the most appropriate hapten-bearing peptide in this series. As expected, 125I-labeled AG 3.0 can be efficiently targeted to cultured colorectal cells (LS174T) in the presence of anti-CEA × anti-HSG BsmAb. In vivo, the BsmAb was used to target 125I-labeled AG 3.0 to LS174T tumor cells grafted in nude mice. The fraction of dose localized per gram of tumor was similar to the one observed with the directly labeled F(ab′)2 (Figure 4) or the 111In-labeled bivalent hapten (Figure 5), and it decreased very slowly with time (T1/2 ) 54.2 ( 0.2 h, Figure 4). The targeting of AG 3.0 was specific as high tumor to control tumor ratios were observed. Tumor uptake was equivalent to what was observed with a directly labeled F(ab′)2, but maximum uptake was obtained more rapidly and tumor to nontumor contrast ratios were higher in plasma at all times and in other tissues in the first 24-48 h after hapten injection (Figure 4). The iodine-labeled hapten also afforded higher contrasts than the 111In-labeled DTPA hapten. As a result, the more favorable biodistribution of 131I-labeled AG3.0 should translate into better dosimetry: it is indeed very important to minimize bone marrow stem cell exposure by circulating activity and to rapidly deliver the activity to the tumor. Further studies are in progress to evaluate the respective effects of the radioisotope and of the hapten-antibody system to explain the observed differences. The asymmetric haptens should be useful in designing more specific targeting reagents when no tumor-specific marker is available, as for example in the treatment of lymphomas. We have already shown, in a lymphocyte model, that tracers bearing two different haptens (DNP and DTPA), in combination with two different BsmAb, each directed to a different cellular antigen (mouse CD22 or MHC class II I-Ek) and to one of the haptens, provide a very efficient way of targeting with high specificity cells bearing simultaneously the two antigens (Le Doussal,

Janevik-Ivanovska et al.

1991). The peptides AG5.1 and AG5.0 of the DTPAHSG series should be especially well suited to this approach since they do not contain the hydrophobic hapten DNP. They will now be tested in a tumor model in which target cells will be differentiated from normal cells by the simultaneous expression of two differentiation antigens otherwise expressed independently in different normal cell types. CONCLUSION

AES immunoscintigraphy and radioimmunoguided surgery enable the detection of very small pathologic lymph nodes and their resection (de Labriolle-Vaylet et al., 1993). However, detecting lymph nodes smaller than 2 mm in diameter and micrometastases remains impractical. The management of these extremely small tumors should benefit from radioimmunotherapy with R- or β-emitting isotopes. The specificity of 125I-labeled AG 3.0 targeting, the low background, and the persistence of the tumor-bound activity for extended periods of time are very strong arguments in favor of the use of AG3.0 as a tracer for targeting iodine-131 tumor cells. Further studies are now in progress to determine the doses of radiation delivered to tumors and normal organs using this molecule (Manetti et al., 1997). In addition, new hapten-bearing peptides derived from AG3.0 have been synthesized with attached chelating agents capable of binding other radioisotopes of interest for scintigraphy (technetium-99m) or therapy (rhenium-186 or rhenium188). ACKNOWLEDGMENT

The chemical syntheses were performed by A.G.G. in the U.113 INSERM, Faculte´ de Me´decine Saint-Antoine, Paris, France, and URA 1309 CNRS, Institut Pasteur, Lille, France. We thank Dr. D. Marion for nuclear magnetic resonance spectroscopy (Institut de Biologie Structurale, Grenoble, France), Pr. G. C. Gesquiere and Pr. C. Sergherardt for helpful discussions, Pr. S. Askienazy for constant support, and Pr. P. Sautie`re for amino acid analysis. We are grateful to our colleagues at Immunotech E. Rouvier for fruitful discussions and to M. Martin and C. Manetti for the generous supply of indispensable antibody reagents. This work was supported in part by the Association pour la Recherche contre le Cancer through Grant 6073 allocated to G.M. We gratefully acknowledge Pr. M. Delaage, who pioneered the AES technology. LITERATURE CITED Barbet, J., Le Doussal, J. M., Gruaz-Guyon, A., Martin, M., Gautherot, E., and Delaage, M. (1993) Computer simulation of multiple binding equilibrium isotherms: application to the binding of bivalent ligands to antibodies interacting with cell surface Fc-receptors. J. Theor. Biol. 165, 321-340. Chetanneau, A., Barbet, J., Peltier, P., Le Doussal, J. M., GruazGuyon, A., Bernard, A. M., Resche, I., Rouvier, E., Bourguet, P., Delaage, M., and Chatal, J. F. (1994) Pretargeted imaging of colorectal cancer recurrences using an 111In-labeled bivalent hapten and a bispecific antibody conjugate. Nucl. Med. Commun. 15, 972-980. De Labriolle-Vaylet, C., Gruaz-Guyon, A., Wioland, M., Sarfati, E., Mensch, B., Delaage, M., Barbet, J., and Milhaud, G. (1993) Radioimmunodetection de me´tastases du cancer me´dullaire de la thyroide (CMT) a` l’aide d’une nouvelle me´thode en deux temps. J. Med. Nucl. 7/8, 318. Dower, S. K., DeLisi, C., Titus, J., and Segal, D. M. (1981) Mechanism of binding of multivalent immune complexes to Fc receptors. I. Equilibrium binding. Biochemistry 20, 63266334.

Bihaptens for

131I-Pretargeted

Bioconjugate Chem., Vol. 8, No. 4, 1997 533

Radioimmunotherapy

Glennie, M. J., McBride, H. M., Worth, A. T., and Stevenson, G. T. (1987) Preparation and performance of bispecific F(ab′γ)2 antibody containing thioether-linked Fab′γ fragments. J. Immunol. 139, 2367-2375. Goldenberg, D. M. (1993) Monoclonal antibodies in cancer detection and therapy. Am. J. Med. 94, 297-312. Goodwin, D. A., Meares, C. F., McTigue, M., and David, G. S. (1986) Monoclonal antibodies as reversible equilibrium carriers of radiopharmaceuticals. Int. J. Nucl. Med. Biol. 7, 569580. Goodwin, D. A., Meares, C. F., McCall, M. J., McTigue M., and Chaovapong, W. (1988) Pre-targeted immunoscintigraphy of murine tumors with indium-111-labelled bifunctional haptens. J. Nucl. Med. 29, 226-234. Goodwin, D. A., Meares, C. F., McTigue, M., Chaovapong, W., Diamanti, C. I., Ransone, C. H., and McCall, M. J. (1992) Pretargeted immunoscintigraphy: effect of hapten valency on murine tumour uptake. J. Nucl. Med. 33, 2006-2013. Gruaz-Guyon, A., Gras-Masse, H., and Le Doussal, J. M. (1991) Radiolabelled hapten derivatized peptides for tumour imaging with bispecific antibody conjugates. In Peptides 1990 (E. Giraldt and D. Andreu, Eds.) pp 822-825, ESCOM, Leiden, The Netherlands. Gruaz-Guyon, A., Gautherot, E., Martin, M., Le Doussal, J. M., Barbet, J., Delaage, M., and Milhaud, G. (1993) Hapte`nes bivalents marque´s a l’iode et au technetium pour l’immunoscintigraphie de tumeurs avec des anticorps bi-specifiques. J. Med. Nucl. 7/8, 328. Le Doussal, J. M., Martin, M., Gautherot, E., Delaage, M., and Barbet, J. (1989) In vitro and in vivo targeting of radiolabelled monovalent and divalent haptens with dual specificity monoclonal antibody conjugates: enhanced divalent hapten affinity for cell-bound antibody conjugate. J. Nucl. Med. 30, 13581366. Le Doussal, J. M., Gruaz-Guyon, A., Martin, M., Gautherot, E., Delaage, M., and Barbet, J. (1990) Targeting of indium-111labelled bivalent hapten to human melanoma mediated by bispecific monoclonal antibody conjugates: imaging of tumors hosted in nude mice. Cancer Res. 50, 3445-3452. Le Doussal, J. M., Gautherot, E., Martin, M., Barbet, J., and Delaage, M. (1991) Enhanced in vivo targeting of an asymmetric bivalent hapten to double-Ag-positive mouse B cells, using mAb conjugate cocktails. J. Immunol. 146, 169-175. Le Doussal, J. M., Chetanneau, A., Gruaz-Guyon, A., Martin, M., Gautherot, E., Lehur, P. A., Chatal, J. F., Delaage, M., and Barbet, J. (1993) Bispecific (anti-CEA, anti-In-DTPA)

monoclonal antibody-mediated targeting of an Indium-111labelled DTPA dimer to primary colorectal tumors: pharmacokinetics, biodistribution, scintigraphy and immune response. J. Nucl. Med. 34, 1662-1671. Lollo, C., Halpern, S., Bartholomew, R., David, G., and Hagan, P. (1994) Non-covalent antibody-mediated drug delivery. Nucl. Med. Commun. 15, 483-491. Mach, J. P., Pe`legrin, A., and Buchegger, F. (1991) Imaging and therapy with monoclonal antibodies in non-hematopoietic tumors. Curr. Opin. Immunol. 3, 685-693. Manetti, C., Le Doussal, J. M., Rouvier, E., Gruaz-Guyon, A., and Barbet, J. (1995) Intracellular uptake and catabolism of anti-IgM antibodies and bispecific-antibody-targeted hapten by B lymphoma cells. Int. J. Cancer 63, 250-256. Morel, A., Darmon, M., and Delaage, M. (1990) Recognition of imidazole and histamine derivatives by monoclonal antibodies. Mol. Immunol. 27, 995-1000. Peltier, P., Curtet, C., Chatal, J. F., Le Doussal, J. M., Daniel, G., Aillet, G., Gruaz-Guyon, A., Barbet, J., and Delaage M. (1993) Radioimmunodetection of medullary thyroid cancer using a bispecific anti-CEA/anti-Indium-DTPA antibody and an indium-111-labelled DTPA dimer. J. Nucl. Med. 34, 12671273. Salacinsky, P. R. P., McLean, P., Sykes, J. E. C., Clement-Jones, V. V., and Lowry, P. J. (1981) Iodination of proteins and peptides using a solid phase oxidizing agent 1,3,4,6-tetrachloro-3,6-diphenylglycoluryl (Iodogen). Anal. Biochem. 117, 136-141. Santos, O., Payne, J. K., Domitrowsky, J. B., Berkeley C., and Mackensen D. G. (1995) A two-step delivery system utilizing a bi-specific monoclonal antibody for radioimmunotherapy. Antibody Immunoconjugate Radiopharm. 8, 93-109. Stickney, D. R., Slater, B., Kirk, A., Ahlem, C., Chang, C., and Frincke, J. M. (1989) Bifunctional antibody: ZCE/CHA 111In BLEDTA-IV clinical imaging in colorectal carcinoma. Antibody Immunoconjugate Radiopharm. 2, 1-13. Stickney, D. R., Anderson, L. D., Slater, J. B., Ahlem, C. N., Kirk, G. A., Schweighardt, S. A., and Frincke, J. M. (1991) Bifunctional antibody: a binary radiopharmaceutical delivery system for imaging colorectal carcinoma. Cancer Res. 51, 6650-6655. Valentine, R. C., and Green, N. M. (1967) Electron microscopy of an antibody-hapten complex. J. Mol. Biol. 27, 615-623.

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