Enhanced Targeting Specificity to Tumor Cells by Simultaneous

Marc Hillairet de Boisferon,† Olivier Raguin,† Monique Dussaillant,† William ... This approach was evaluated using human Burkitt lymphoma cells ...
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Bioconjugate Chem. 2000, 11, 452−460

Enhanced Targeting Specificity to Tumor Cells by Simultaneous Recognition of Two Antigens Marc Hillairet de Boisferon,† Olivier Raguin,† Monique Dussaillant,† William Roste`ne,† Jacques Barbet,‡ and Anne Gruaz-Guyon*,† INSERM U.339, Faculte´ de Me´decine et Hoˆpital Saint-Antoine, Paris, France, and UPRES 2059, Faculte´ de Me´decine, Universite´ de la Me´diterrane´e, Marseille, France. Received August 17, 1999; Revised Manuscript Received January 27, 2000

Radioimmunotherapy recently afforded convincing results for B-cell non-Hodgkin’s lymphoma treatment with antibody specific for B-cell differentiation antigens. High doses of unlabeled or labeled antibodies are necessary to saturate specific sites on normal B-cells. We thus developed a new targeting strategy, taking advantage of dual binding cooperativity, to enhance the specificity of the radioactive uptake by tumor cells. This approach was evaluated using human Burkitt lymphoma cells (Ramos) which express both CD10 and CD20 antigens. Most normal cells express at most one of these two differentiation antigens but many hematological tumors, including most human B type acute lymphoblastic leukemia cells, express both. Cells pretargeted with two bispecific antibodies, one recognizing CD10 and a histamine derivative (HSG), the other recognizing CD20 and the DTPAindium complex, bind cooperatively radiolabeled mixed-haptens (DTPA-HSG). Increased binding (about 5-fold compared to binding to only one of CD10 or CD20 antigens) is observed at 37 °C, demonstrating the feasibility of the technique. This binding enhancement is a slow process, not observed at 4 °C. Such a binding enhancement will increase specificity for targeting isotopes to double antigen positive tumor cells compared to nontumor tissue cells bearing only one of them. This approach might be used to increase tumor irradiation with minimal irradiation of normal cells.

INTRODUCTION

Radioimmunotherapy, a systemic radiotherapy using labeled monoclonal antibodies (mAb) to deliver radioisotopes to malignant cells, recently afforded convincing results for non-Hodgkin’s lymphoma therapy (Corcoran et al., 1997; Wilder et al., 1996). These tumor cells, distributed in blood, bone marrow, lymph nodes and spleen, are readily accessible to labeled monoclonal antibodies. This and the relative radiosensitivity of lymphoma cells account for the efficacy of this blood borne therapy. The most encouraging results have been obtained using CD20 (Kaminski et al., 1993, 1996; Knox et al., 1996; Liu et al., 1998; Press et al., 1995), CD22 (Goldenberg et al., 1991; Juweid et al., 1995), CD37 (Kaminski et al., 1992; Press et al., 1993), and HLA-DR (DeNardo and DeNardo, 1995) as target antigens. Radioimmunotherapy has achieved complete response in almost half of the patients included in the numerous clinical studies concerning B-cell non-Hodgkin’s lymphoma (Wilder et al., 1996). Results obtained in leukemia radioimmunotherapy, using antibodies directed to the interleukin-2 receptor (Waldman et al., 1994), HLA-DR (DeNardo et al., 1994), CD33 (Appelbaum et al., 1992; Jurcic et al., 1995; Scheinberg et al., 1991), and CD45 (Matthews et al., 1995), suggest that this technique may intensify anti-leukemia therapy before bone marrow transplant. The most impressive results were obtained when large amounts of labeled antibodies were used or * To whom correspondence should be addressed. Phone: (33) 1 40 01 14 66. Fax: (33) 1 43 43 89 46. E-mail: [email protected]. † INSERM U.339. ‡ UPRES 2059.

when large amounts of unlabeled antibody were injected prior to a labeled antibody dose. The requirement for high doses or predosing with unlabeled antibody has been attributed to the necessary saturation of specific sites on normal cells. Indeed, in these approaches, target antigens are merely differentiation antigens, not specific for tumor cells. Pretargeting has afforded very encouraging results in radioisotope targeting to solid tumors in animal models and in the clinic. The “Affinity Enhancement System” (AES), which uses unlabeled bispecific antibody, directed to a tumor antigen and to a hapten, and a labeled bivalent hapten (Le Doussal et al., 1990), is now recognized as a good candidate for the detection (Barbet et al., 1998; Chetanneau et al., 1994; De Labriolle-Vaylet et al., 2000; Le Doussal et al., 1993; Manetti et al., 1997; Vuillez et al., 1997) and treatment (Barbet et al., 1999; Gautherot et al., 1997; Hillairet de Boisferon et al., 1997) of neoplasms and particularly solid tumors. This technique is based on the enhanced affinity of the labeled hapten provided by the cooperativity of its simultaneous binding to two unlabeled bispecific antibodies at the surface of the target cell. Scintigraphy studies demonstrated improved tumor to normal tissue ratio compared to classical immunoscintigraphy using directly labeled antibodies (Le Doussal et al., 1990; Le Doussal et al., 1993). Encouraging radioimmunotherapy results have also been obtained in animal models of mouse lymphoma (Hillairet de Boisferon et al., 1997) and human colorectal carcinoma cell line grafted to nude mice (Gautherot et al., 1997) and in the clinic (Barbet et al., 1999). AES could be considered for the treatment of hematological diseases. However, the huge amounts of bispecific antibodies that would be necessary to saturate specific and nonspecific

10.1021/bc9901090 CCC: $19.00 © 2000 American Chemical Society Published on Web 05/03/2000

Enhanced Specificity by Targeting Two Antigens

binding sites on normal cells would make this approach too costly to be practical. The scope of this work is to take advantage of the cooperativity of dual binding to enhance the specificity of the radioactivity uptake by the target tumor cell. The feasibility of enhanced binding to double antigen positive cells has been demonstrated in a model of mouse B-cells, using mixed 2,4-dinitrophenyl (DNP) × diethylenetriaminepentaacetic acid-indium [DTPA(In)] haptens. These hydrophobic haptens were efficiently targeted to normal mouse spleen cells using anti-Lyb-8.2 × anti-DTPA(In) and anti-IEk × anti-DNP bispecific antibodies (Le Doussal et al., 1991). Subsequent studies proved that hydrophobic haptens were poorly delivered to target cells with more limited blood supply, such as tumor cells. Thus, we have designed and synthesized hydrophilic mixed-haptens, associating DTPA(In) and histamine-hemisuccinate coupled to glycine (HSG) (Janevik-Ivanovska et al., 1997). In this study, we first evaluated, in a macrophage model, the simultaneous binding to two distinct antibodies of a series of hydrophilic peptides, of various length and structure, bearing these two different haptens. Then, we investigated the ability of two of these mixed-haptens to simultaneously bind to two different bispecific antibodies [anti-CD10 × anti-HSG and anti-CD20 × anti-DTPA(In)] pretargeted to Ramos cells (human Burkitt lymphoma) which bear at their surface CD10 and CD20 antigens. The effect of cooperativity was studied by computer simulation of equilibrium binding isotherms. MATERIAL AND METHODS

Cells. P388D1 cells (ATCC, Rockville) are mouse macrophages which express receptors (Fc gamma RI and RII) for the IgG Fc fragment on the cell surface (Koren et al., 1975). These cells were grown in DMEM medium supplemented with 10% fetal calf serum (FCS), 1% glutamine, 1% nonessential amino acid, 1% sodium pyruvate, and 0.1% gentamycine at 37 °C in 5% CO2. For binding experiments, the cells were harvested with trypsin-EDTA (0.05 and 0.02%, respectively) at 37 °C during 5 min. The cells were resuspended and washed once with culture medium and twice with binding medium [DMEM medium containing 0.2% of bovine serum albumin (BSA)]. The Ramos cell line (ATCC) is derived from a human Burkitt lymphoma known to express CD10 and CD20 at its surface. These cells were cultured in RPMI 1640 with 10% FCS, 1% glutamine, 1% nonessential amino acid, 1% sodium pyruvate, and 0.1% gentamycine at 37 °C in 5% CO2. For binding experiments, the cells were washed twice with the binding medium (RPMI 1640 medium containing 0.2% of BSA). Before experiments, the viability of the cells was assessed by trypan blue exclusion. In all cases, viability was greater than 90%. Mixed-Haptens. Peptides bearing one glycyl-succinylhistamine hapten (HSG) and one diethylenetriaminepentaacetic acid (DTPA) were synthesized. The DTPAHSG peptides (mixed-haptens) are listed in Table 1. As already described (Janevik-Ivanovska et al., 1997), monoHSG peptides were synthesized by the step-wise solidphase method using tert-butyloxycarbonyl, fluorenylmethyloxycarbonyl, and 2-chlorobenzyloxycarbonyl groups to protect R- or -amino groups depending on the position of the HSG hapten. The DTPA-HSG peptides were obtained by reacting DTPA dianhydride with the monoHSG peptide. All peptides were purified by C18 reversedphase chromatography. Antibodies. All antibodies used in this work are mouse monoclonal immunoglobulins (IgG) kindly pro-

Bioconjugate Chem., Vol. 11, No. 4, 2000 453 Table 1. Synthesized Mixed-Haptena

a HSG, histamine-succinyl-glycyl; DTPA, diethylenetriaminepentaacetic acid; GABA, γ-amino-butyric acid.

vided by Immunotech S. A. (France). The anti-HSG antibody (679.1MC7) is an IgG1,κ monoclonal antibody (Morel et al., 1990) and the antibody directed to the DTPA(In) complex (734) (Le Doussal et al., 1990) is an IgG1,λ monoclonal antibody. The anti-CD10 monoclonal antibody (ALB1 IOB5) is an IgG2a,κ, and the monoclonal antibody (B9E9, HRC20), specific to the CD20 antigen, an IgG2a,κ. Anti-CD20 × anti-DTPA(In) and anti-CD10 × antiHSG bispecific antibodies were prepared by chemical coupling of the two reduced Fab′ fragments using ophenylene-dimaleimide (Sigma) according to Glennie et al. (Glennie et al., 1987). Briefly, IgG was digested by pepsin (Sigma) (5% in 0.15 M acetate buffer, pH 4.0 for 2 h at 37 °C). The F(ab′)2 fragments were purified by preparative gel filtration on Superdex 200 (16 × 60 column, Pharmacia Biotech, France) in PBS pH 7.4. Fab′ fragments were obtained by reduction with 10 mM cysteamine (Sigma) in PBS (EDTA 10 mM, pH 7.4) during 1 h at 37 °C. Excess cysteamine was removed by gel filtration on a PD10 column (Pharmacia). The Fab′ fragment directed to the hapten was incubated with excess o-phenylene-dimaleimide. Excess dimaleimide was removed by gel filtration on a PD10 column. The other Fab′ fragment directed to the cell antigen was incubated with the Fab-dimaleimide during 24 h at 4 °C. The conjugate (100 kDa) was separated from higher molecular weight material and nonreacted Fab′ fragments by preparative gel filtration on Superdex 200 (16 × 60 column) in PBS buffer. Anti-DTPA(In) and anti-HSG Fab′ fragments were biotinylated and immobilized on avidin coated tubes (Immunotech S. A., France) to determine mixed-hapten immunoreactivity or equilibrium affinity constant. Radiolabeling. The mixed-haptens (0.5 nmol) were labeled with indium-111 (111InCl3, 11.1 MBq, Cis Bio International, France) in 100 mM acetate-10 mM citrate buffer, pH 5.0, during 24 h (room temperature). Then, since the anti-DTPA(In) antibody is specific to the DTPA-indium complex, free DTPA groups were saturated with nonradioactive InCl3 (5 nmol) in the same buffer. Excess free indium was removed on a Sep-Pak cartridge (Waters Milford) in acetate-citrate buffer, followed by elution of the labeled mixed-hapten in acetatecitrate buffer/methanol (50:50, v/v). Radioiodination was performed by the chloramine T method. Before labeling, the free DTPA groups of each peptide were saturated with nonradioactive InCl3 in 100 mM acetate-10 mM citrate buffer, pH 5.0. The solution of peptide (2 nmol, 100 µL) in 0.1 M phosphate buffer, pH 7.2, was incubated with Na125I (37 MBq, Amersham, France). The iodination was started by addition of 10 µL of chloramine T (1 mg/mL) in phosphate buffer. Reaction was stopped after 45 s by addition of 10 µL of sodium bisulfite (2 mg/mL) in water. Then the iodinated derivatives were separated by C18 reversed-phase HPLC with

454 Bioconjugate Chem., Vol. 11, No. 4, 2000

H2O/TFA 0.05% (A) and acetonitrile (B) with a linear gradient from 0 to 30% B during 40 min. Antibodies (0.5 nmol) were labeled with iodine-125 (18.5 MBq) by the iodogen method (Salacinsky et al., 1981) and purified by gel filtration chromatography on a PD 10 column in PBS. Quality Control. The fraction of each 125I- or 111Inlabeled mixed-hapten able to bind to the anti-hapten antibodies (e.g., immunoreactivity) was determined by incubating trace amounts of the labeled peptide in antiHSG or anti-DTPA(In) antibody-coated tubes (0.2 µg) for 1 h at 37 °C under shaking. Tubes were then washed twice with 0.9% NaCl, 0.02% Tween 20 and counted. DTPA(In)- and HSG-coated tubes were prepared by incubating DTPA(In)-substituted biotinylated BSA or biotinylated mono-HSG peptide on avidin-coated tubes. Anti-hapten immunoreactivity was determined by incubating trace amounts of labeled bispecific antibody on HSG- or DTPA(In)-coated tubes. Anti-CD10 or anti-CD20 immunoreactivity of each labeled antibody was determined from binding of trace amounts of the labeled antibody to increasing numbers of Ramos cells. 125I-labeled antibodies (150 µL, 1 × 10-12 M) and 100 µL of cell suspension (1 × 105 to 1 × 107 cells) in a 350 µL final volume of RPMI/0.2% BSA were incubated during 1.5 h at 37 °C under shaking. Then, 100 µL of the suspension were centrifuged in triplicate for 2.5 min through a phthalate oil mixture (Dower et al., 1981). The radioactivity of the bottom of each tube containing the cell pellet and of 50 µL of the supernatant was then counted. Determination of Antibody Binding Affinity. The equilibrium affinity constants (Ka) of the binding of antibodies to Ramos cells were determined by competition (three experiments in triplicate). The cell suspension (100 µL, 2 × 106 cells), 125I-labeled antibodies (150 µL, 2.3 × 10-10 M), and 100 µL of increasing concentrations of unlabeled antibodies (3 × 10-6 to 3 × 10-10 M) were incubated together in RPMI/0.2% BSA. After 1.5 h at 37 °C under shaking, 100 µL of the suspension was centrifuged in triplicate for 2.5 min through a phthalate oil mixture as described above. For the affinity constant determination of the binding of mixed-haptens to anti-hapten antibodies, trace amounts of 111In-labeled mixed-haptens (1 × 10-10 M) were incubated in anti-DTPA(In)- or anti-HSG-coated tubes (0.01 µg) for 2 h at 37 °C with increasing concentrations of unlabeled mixed-haptens (1 × 10-6 to 1 × 10-10 M) in a final volume of 1 mL of PBS/0.2% BSA under shaking. Then, 100 µL of supernatant were taken off and counted. Tubes were washed twice with 0.9% NaCl and 0.02% Tween 20 and counted. Demonstration of Antibody-Mediated Cooperative Binding of Mixed-Haptens. Binding experiments were performed on P388D1 macrophage cells (1 × 105) in a final volume of 350 µL of binding medium. Radiolabeled mixed-haptens (2 × 10-10 M final concentration) were incubated with cells during 2.5 h at 37 °C under shaking in the presence of unlabeled anti-DTPA(In) IgG or anti-HSG IgG or an equimolar mixture of both IgGs (4 × 10-7 to 2.4 × 10-12 M final concentrations). Nonspecific binding was evaluated in the absence of antibodies. Then, 100 µL aliquots were centrifuged in triplicate through phthalate oil mixture as described above. Binding experiments on Ramos cells were performed under the same conditions with a suspension of 2 × 106 cells in the presence of unlabeled anti-CD20 × antiDTPA(In) or anti-CD10 × anti-HSG or an equimolar

Hillairet de Boisferon et al.

mixture of both bispecific antibodies (1 × 10-7 to 1 × 10-10 M final concentrations). Binding and Internalization Kinetics. Ramos cells (2 × 106 cells) were incubated at 37 °C under shaking in 350 µL of culture medium containing 20 mM Hepes (pH 7.4) with iodinated mixed-hapten AG 5.0 and AG 5.2 (2 × 10-10 M) in the presence of a bispecific antibody concentration of 5.3 × 10-9 M and 1.6 × 10-8 M, respectively (final concentrations). At selected time intervals, 100 µL of cell suspension were applied to phthalate tubes and centrifuged to determine the total amount of radioactive peptide bound to the cells. Three different protocols were performed. Simultaneous incubation of antibodies and hapten was used in most experiments. When stated, antibodies were added to the cells first, incubated for 2 h, and the hapten was added without washing or after washing the cells free of unbound antibodies. To determine the amount of tracer internalized in parallel, 350 µL of cell suspension was pelleted, resuspended in 350 µL binding buffer adjusted to pH 2.0 with 1 M HCl, incubated at 4 °C for 15 min under shaking and 100 µL of suspension were applied to phthalate tubes. This last procedure dissociated surface-bound ligands. Internalization was expressed as the percentage of the total incubated activity that remained bound to cells after acid wash. Computer Simulations. The methodology introduced by Delaage and co-workers (Barbet et al., 1993; Bellon et al., 1973) is well suited to calculate binding equilibrium under complex situations. Basically, one adds up the concentrations of all interacting species, expressed as functions of free monomer concentrations, to form a partition function. For instance, if CA, CB, and CC represent the free concentrations of species A, B, and C, respectively, and KAB and KBC are the affinity constants for the interactions of A with B and B with C, then one gets CA + CB + CC + KABCACB + KBCCBCC. If C can bind to the AB complex, KABKBCCACBCC is added to the partition function. If binding involves cooperativity (or steric hindrance), a coefficient PC is included and PCKABKBCCACBCC is added instead. This is what was done for the complex involving the mixed-hapten, one molecule of each bispecific antibodies and one molecule of each membrane antigens and the term “cooperativity factor” refers to PC. For simulation, the partition function [completed by subtracting the logarithmic terms, Ao ln(CA), Bo ln(CB), Co ln(CC), Ao, Bo, Co being the total concentrations of each species] is minimized with respect to CA, CB, and CC. The program can deal with any reasonable number of interacting species and possible complexes. Unknown parameters may be estimated by minimizing the sum of variance-weighted squared differences between calculated and experimental data. The program was used to estimate the affinity constants, number of binding sites per cell, and immunoreactivity from competition experiments. Then, the values determined for the affinity constants were used as fixed parameters, but binding site concentrations and the cooperativity factor were adjustable parameters in fitting the binding curves obtained in the experiments with mixed-hapten binding to Ramos cells in the presence of bispecific antibodies. All these calculations were performed by the program which uses Microsoft Excel 97 for input and output on a PC. This program is available on request to Jacques Barbet (UPRES 2059, Faculte´ de Me´decine, Universite´ de la Me´diterrane´e, 27 Bd Jean Moulin, 13385, Marseille, France; e-mail: jacques.barbet@ medecine.univ-mrs.fr).

Enhanced Specificity by Targeting Two Antigens

Bioconjugate Chem., Vol. 11, No. 4, 2000 455

Table 2. Equilibrium Affinity Constants and Immunoreactivity of Monoclonal IgGs and Bispecific Antibodies for Binding to CD10 or CD20 Antigens antibody IgG anti-CD10 IgG anti-CD20 anti-CD10 × anti-HSG anti-CD20 × anti-DTPA(In)

affinity constantb Ka (M-1)

immunoreactivity to Ramos cellsa,b (%)

(3.6 ( 0.4) × 109 (1.7 ( 0.2) × 109 (4.0 ( 0.1) × 108

25.9 ( 5.6 57.0 ( 8.2 23.3 ( 2.3

(3.0 ( 0.2) × 108

16.2 ( 0.6

a To determine immunoreactivity trace amounts of 125I-labeled antibodies were incubated with increasing concentration of cells. b For each antibody, the immunoreactivity and the equilibrium binding constant were estimated simultaneously by nonlinear least-squares regression.

RESULTS

Determination of Antibody Equilibrium Affinity Constants. Equilibrium binding parameters of antiCD10 and anti-CD20 IgGs and anti-CD20 × anti-DTPA(In) and anti-CD10 × anti-HSG bispecific antibodies binding to Ramos cells were determined from three competition experiments run in triplicate. Nonspecific binding was evaluated with excess unlabeled anti-CD10 or anti-CD20 IgGs. For each antibody, the immunoreactivity was determined from binding experiments of the labeled antibody to increasing numbers of Ramos cells. Equilibrium binding constants and immunoreactivity were then determined from experimental results by fitting calculated to experimental bound-to-total ratios for all available data (Table 2). This was performed using an add-in to Microsoft Excel 97 developed by one of us (J.B.). Basically, this add-in uses variance-weighted nonlinear least-squares regression to estimate adjustable parameters and a description of multiple binding equilibria derived from Bellon et al. (1973) and Barbet et al. (1993). The immunoreactivity of 125I-labeled bispecific antibody against CD20 was found rather low (Table 2), whereas that of the corresponding 125I-labeled IgG was significantly higher, the immunoreactivity of bispecific antibody against CD10 was similar to that of the corresponding IgG (Table 2). This was taken into account to determine the affinity constants. The affinity of intact IgGs was 5-9-fold higher than that of the respective monovalent bispecific antibodies (Table 2). They were all in the nanomolar range, which is suitable for in vivo targeting applications. The immunoreactivity of 125I-labeled bispecific antibodies against haptens was similar: 14% for anti-CD20 × anti-DTPA(In) and 13% for anti-CD10 × anti-HSG (data not shown). Expression of the target antigens on Ramos cells varied from experiment to experiment in the 2.1 × 104 to 7.5 × 104 sites/cell range for CD10 and 1.6 × 104 to 1.5 × 105 for CD20.

Equilibrium Affinity Constant Determination of Mixed-Haptens Binding to Anti-Hapten Antibodies. Equilibrium affinity constants for the binding of mixedhaptens to anti-hapten antibodies were determined by competition (three experiments in triplicate). Binding of 111In-labeled AG 5.1 mixed-hapten to anti-DTPA(In) or anti-HSG coated tubes was competed with increasing concentrations of unlabeled mixed-hapten. Binding constants were estimated by nonlinear least-squares regression as above. All measurements were performed at 37 °C with physiological salt concentrations to allow the use of these Ka values to analyze the binding of the mixedhaptens to Ramos cells in the presence of the bispecific antibodies (Table 3). The highest Ka binding value to anti-HSG antibody was observed for HSG coupled to the epsilon-NH2 of a lysine (1.9 ( 0.2 and 2.7 ( 0.3 × 109 M-1 for AG 5.1 and AG 5.0, respectively). The anti-HSG Ka value decreased when HSG was coupled to the R-NH2 of a lysine (3.7 ( 1.5 × 108 M-1 for AG 5.2) and was 2.2 ( 0.1 × 107 M-1 for AG 5.3 when HSG was on the R-NH2 of a tyrosine. The highest anti-DTPA(In) Ka value is observed for the mixedhaptens AG 5.3 and AG 5.1 (1.9 ( 0.2 × 109 M-1 and 1.9 ( 0.3 × 109 M-1, respectively). The affinity constants are 1.5 ( 0.2 × 109 M-1 for AG 5.0 and 1.1 ( 0.1 × 109 M-1 for AG 5.2. Immunoreactivity for binding to the antiDTPA(In) or the anti-HSG antibodies were similar for 111 In- or 125I-labeled mixed-haptens AG 5.0, AG 5.1, and AG 5.2 and ranged from 79 to 94% (Table 3). Due to its low affinity, the immunoreactivity of AG 5.3 for binding to the anti-HSG antibody could not be determined. Binding to Macrophages of 111In-Labeled MixedHaptens in the Presence of Anti-Hapten IgGs. The P388D1 macrophage cells express receptors for the IgG Fc fragment at their surface (Koren et al., 1975). These receptors have a very low affinity for mouse IgG1 (Haeffner-Cavaillon et al., 1979). The binding of 111In-labeled mixed-haptens to P388D1 cells incubated in the presence of increasing concentrations of each anti-DTPA(In) or anti-HSG or a mixture of both IgGs was studied. The experiment was repeated three times with similar results. Figure 1 shows typical curves. Mixed-hapten binding to P388D1 cells pretargeted with only one antihapten IgG [anti-HSG or anti-DTPA(In)] was hardly detectable. In the presence of an equimolar mixture of the two anti-hapten antibodies, binding was dramatically increased for mixed-haptens AG 5.0, AG 5.1, and AG 5.2. Binding increased with increasing concentrations of the antibody mixture to reach a maximum. Then at higher antibody concentration, binding decreased as the labeled hapten was trapped by excess antibody in the supernatant. At the optimal IgG concentration (2.0 × 10-10 M), 13% of the mixed-hapten AG 5.0 and 11% of AG 5.1 were bound. The optimal IgG concentration for AG 5.2 binding was 1.8 × 10-9 M, with 12% of the mixed-hapten bound to the cells. In the presence of only one of each anti-

Table 3. Equilibrium Affinity Constants and Immunoreactivity of Mixed-Haptens for Anti-DTPA(In) and Anti-HSG Antibodies Coated Tubes

peptides AG 5.1 AG 5.0 AG 5.2 AG 5.3

binding to anti-DTPA(In) mAb affinity constant (M-1)

binding to anti-HSG mAb affinity constant (M-1)

immunoreactivity to anti-DTPA(In) coated tubes (%)

immunoreactivity to anti-HSG coated tubes (%)

(1.9 ( 0.3) × 109 (1.5 ( 0.2) × 109 (1.1 ( 0.1) × 109 (1.9 ( 0.2) × 109

(1.9 ( 0.2) × 109 (2.7 ( 0.3) × 109 (3.7 ( 1.5) × 108 (2.2 ( 0.1) × 107

89.4 ( 2.5 88.0 ( 6.1 84.8 ( 7.1 90.6 ( 2.7

82.7 ( 0.5 93.9 ( 3.7 79.0 ( 1.2 a

a Due to its low affinity, the immunoreactivity of AG 5.3 for binding to the anti-HSG antibody could not be determined from maximal binding to tubes coated with excess anti-HSG.

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Figure 1. In vitro equilibrium binding of mixed-haptens to P388D1 cells mediated by anti-hapten antibodies: P388D1 cells were incubated for 2.5 h at 37 °C with 111In-labeled mixed haptens AG 5.0 (a), AG 5.1 (b), AG 5.2 (c), or AG 5.3 (d) in the presence of increasing concentrations of anti-DTPA(In) IgG (dotted line), anti-HSG IgG (broken line) or an equimolar mixture of both IgGs (solid line). Cell suspension was centrifuged through phthalate oil, the supernatant and the cell pellet were counted. Mean bound/total labeled mixed-hapten (B/T %) ( sd unless smaller than point plotted (triplicate).

hapten IgG at the same concentration less than 1% of the mixed-haptens was bound. Interestingly, no binding increase was observed with AG 5.3. The observed binding of this peptide with the antibody mixture was roughly the sum of the binding in the presence of either the anti-DTPA(In) or the anti-HSG IgG. In contrast, with AG 5.0, AG 5.1, and AG 5.2, the simultaneous binding to both IgGs was roughly 14-17fold the sum of the binding to one IgG, suggesting a cooperativity of the binding. Binding to Ramos Cells of 111In- and 125I-Labeled Mixed-Haptens Mediated by Bispecific Antibodies. The ability of the anti-CD10 × anti-HSG and anti-CD20 × anti-DTPA(In) bispecific antibodies to target 111Inlabeled AG 5.0 and AG 5.2 to CD10 and CD20 expressing tumor cells was studied in binding experiments (at 37 °C, repeated twice) using human Burkitt lymphoma cells (Figure 2, panels a and b). Controls mimicking nontumor cells bearing only one antigen were performed by incubating Ramos cells with only one bispecific antibody. No more than 8% of AG 5.0 or AG 5.2 was bound in the presence of anti-CD10 × anti-HSG bispecific antibody only. Even less binding was observed with anti-CD20 × anti-DTPA(In) antibody alone. In the presence of an equimolar mixture of the two bispecific antibodies, increased binding was observed. At the optimal bispecific antibody concentrations (5.3 × 10-9 M for AG 5.0 and 1.6 × 10-8 M for AG 5.2), 32 and 18% of 111In-labeled AG 5.0 and AG 5.2, respectively, were bound. The 125I-labeled mixed-haptens AG 5.0 and AG 5.2 were targeted similarly to Ramos cells by bispecific antibody mixture under the same conditions with a maximal binding of 41 and 33%, respectively (Figure 2, panels c and d). Binding of 125I- and 111In-labeled mixed-haptens at 37 °C in bivalent conditions (i.e., simultaneous binding to both antigens) was, respectively, about 5- and 4-fold higher than the sum of monovalent bindings (i.e., binding to only one antigen) in a wide range of bispecific antibody concentration, suggesting a cooperativity of the binding.

Hillairet de Boisferon et al.

Figure 2. In vitro equilibrium binding of mixed-haptens to Ramos cells mediated by bispecific antibodies: Ramos cells were incubated for 2.5 h at 37 °C with 111In-labeled mixed haptens AG 5.0 (a), AG 5.2 (b), or 125I-labeled mixed-haptens AG 5.0 (c), AG 5.2 (d) in the presence of increasing concentrations of antiCD20 × anti-DTPA(In) (dotted line), anti-CD10 × anti-HSG (broken line) or an equimolar mixture of both bispecific antibodies (solid line). Cell suspension was centrifuged through phthalate oil, the supernatant and the cell pellet were counted. Mean bound/total labeled mixed-hapten (B/T %) ( sd unless smaller than point plotted (triplicate).

This cooperativity was evaluated by computer simulation of the binding curves and nonlinear least-squares regression to estimate a cooperativity factor (see Materials and Methods). Affinity constants calculated in independent experiments were used to analyze experimental results. As the antigen expression on Ramos cells varied from experiment to experiment, the number of CD10 and CD20 sites per cell were fitted for each experiment. Typical curve fits are presented in Figure 2. Cooperativity factors were estimated to (6.7 ( 0.2) × 103 (mean ( sd) for 125I-labeled 5.0, (2.1 ( 0.7) × 103 (mean ( sd) for 111 In-labeled 5.0, (2.5 ( 0.1) × 103 (mean ( sd) for 125I-labeled AG 5.2, and (1.6 ( 1.3) × 103 (mean ( sd) for 111In-labeled 5.2. As expected from the curves, the mixed-hapten 125I-labeled AG 5.0 exhibited the highest cooperativity. Influence of the Temperature on the Binding of 125I-Labeled Mixed-Haptens to Ramos Cells. The binding of 125I-labeled AG 5.0 to Ramos cells in the presence of optimal and suboptimal concentrations of bispecific antibodies was studied at 37 and 4 °C. In contrast to the increased targeting of 125I-labeled AG 5.0 observed at 37 °C, in the presence of an equimolar mixture of bispecific antibodies at 4 °C, a simple additivity of the binding was observed at 3 different antibody concentrations (data not shown). Kinetic experiments performed at 4 °C with a 5.3 × 10-9 M concentration of one of each or a mixture of both bispecific antibodies showed that maximal binding of AG 5.0 (Figure 3a) reached a plateau after about 5 h of incubation in monovalent or bivalent conditions (half-life: about 53 min for binding to CD20, 18 min for binding to CD10, and 67 min for bivalent binding). No cooperativity was observed at 4 °C. Similar results were obtained with AG 5.2. These results suggest that low-temperature prevents a process involved in the observed binding enhancement at 37 °C.

Enhanced Specificity by Targeting Two Antigens

Figure 3. Kinetics of binding at 4 °C (a), 37 °C (b), and endocytosis at 37 °C (c) of 125I-labeled mixed-hapten AG 5.0. Experiments were performed for various periods of time in the presence of the bispecific antibody concentration providing the maximal binding (5.3 × 10-9 M), anti-CD20 × anti-DTPA(In) (dotted line), anti-CD10 × anti-HSG (broken line) or an equimolar mixture of both bispecific antibodies (solid line). Fraction of radioactivity associated to the cells (B/T %) (a) and (b) or inside the cells (I/T %) (c) were counted (mean ( sd unless smaller than point plotted; triplicate).

Kinetics of 125I-Labeled Mixed-Hapten Binding and Internalization to Ramos Cells at 37 °C. Binding and internalization experiments were performed on Ramos cells during 24 h with a 5.3 × 10-9 M concentration of one of each or a mixture of both bispecific antibodies and 125I-labeled AG 5.0. Maximum binding of AG 5.0 was reached after 2.5 h incubation in the presence of an equimolar mixture of antibodies (half-life: about 25 min), whereas in monovalent binding conditions, binding was maximum after 15 min (half-life: about 4 min) (Figure 3b). The amount of internalized 125I-labeled AG 5.0 was measured in both monovalent and bivalent conditions (Figure 3c). Over 24 h, only a small fraction of tracer was internalized, but internalization was increased in the presence of the antibody mixture (5%) compared to monovalent conditions (less than 1.5%). Similar results were obtained with the AG 5.2 mixed-hapten. Comparison between Mixing Reagents and Antibody Pretargeting. Almost identical 125I-labeled AG 5.0 binding curves were obtained by mixing the labeled hapten and the antibodies (Figure 4a) or by preincubation of the antibodies for 2 h without washing before addition of the hapten (Figure 4b). Figure 4c shows the results obtained when cells were incubated first with the bispecific antibodies for 2 h and washed. Overall binding of the hapten was decreased under these conditions but cooperativity was maintained.

Bioconjugate Chem., Vol. 11, No. 4, 2000 457

Figure 4. Binding of 125I-labeled mixed-hapten AG 5.0 with pretargeting of antibodies. AG 5.0 (2 × 10-10 M) was incubated to Ramos cells together with a (5.3 × 10-9 M) bispecific antibody concentration (a) or after a preincubation of 2 h of the bispecific antibody without washing (b), or with a washing of the cells after the antibody preincubation (c). Binding was performed in the presence of anti-CD20 × anti-DTPA(In) (dotted line), antiCD10 × anti-HSG (broken line), or an equimolar mixture of both bispecific antibodies (solid line). Mean bound/total labeled mixed-hapten (B/T %) ( sd unless smaller than point plotted (triplicate). DISCUSSION

Numerous antibodies with high specificity to tumor antigens have been obtained since the introduction of monoclonal antibodies by Ko¨hler and Milstein (1975). Nevertheless, despite the encouraging results of radioimmunotherapy in lymphomas, the treatment of solid tumors is limited by the irradiation of normal tissues. Unfavorable antibody pharmacokinetics, cross-reactions with normal tissues, and nonspecific binding sites account for the limited specificity of irradiation. The Affinity Enhancement System, which uses bispecific antibodies and mixed-haptens, has proven useful to enhance tumor to normal tissue uptake ratio (Chetanneau et al., 1994; Le Doussal et al., 1990), and preliminary results in radioimmunotherapy look promising (Barbet et al., 1999; Gautherot et al., 1997; Hillairet de Boisferon et al., 1997). The feasibility of targeting two antigens simultaneously expressed at the surface of the target cell has been demonstrated in a normal mouse B-cell model using 2,4-dinitrophenyl-derived haptens to target antigens (Le Doussal et al., 1991). In this system, antigen modulation precluded an accurate determination of the binding enhancement. In addition, the high hydrophobicity of the DNP hapten made it disappointing in tumor targeting experiments. We demonstrated earlier that the histamine-hemisuccinate and the DTPA haptens are suitable to carry radioactive isotopes to tumor cells pretargeted with bispecific antibodies (Janevik-Ivanovska et al., 1997; Le Doussal et al., 1990). Both have low toxicity, high affinity to available antibodies and no cross-reactivity or non-

458 Bioconjugate Chem., Vol. 11, No. 4, 2000

specific binding to body components, mostly because they are highly hydrophilic. For these reasons, HSG and DTPA were coupled to peptide connecting chains of various length and structure. A D-tyrosine residue was introduced in the backbone to allow iodine labeling, the C-terminal end of the peptide chain was amidated, the N-terminal was acetylated, and lysine side chains were substituted. This conferred the products extreme proteolysis resistance, as 6 h after injection to a mouse, 65% of AG 5.0 recovered in urine is immunoreactive (data not shown). The series of synthesized compounds is listed in Table 1. The binding affinity constants of mixed-hapten AG 5.1, AG 5.0 and AG 5.2 to anti-HSG [(1.9 ( 0.2) × 109 M-1, (2.7 ( 0.3) × 109 M-1, (3.7 ( 1.5) × 108 M-1] and to anti-DTPA(In) antibodies [(1.9 ( 0.3) × 109 M-1, (1.5 ( 0.2) × 109 M-1, (1.1 ( 0.1) × 109 M-1] are sufficiently high to perform two-step targeting with bispecific antibodies. The haptens may be labeled to a high specific activity with 111In or 125I without impairing their immunoreactivity (range 79-94). Recently, similar haptens have been labeled to a high specific activity (79 MBq/nmol) with iodine-131 (Barbet et al., 1999; Gautherot et al.,1997), a radioisotope suitable for therapeutic applications. We used macrophages (P388D1 cells) to investigate the ability of the mixed-haptens AG 5.0, AG 5.1, AG 5.2, and AG 5.3 to cross-link two different anti-hapten antibodies bound at the cell surface. P388D1 macrophage cells express at their surface receptors for IgG1 Fc fragment. These receptors have low affinity for monomeric mouse IgG1 (Haeffner-Cavaillon et al., 1979). As a consequence, labeled mixed-haptens in the presence of increasing concentrations of anti-DTPA(In) or anti-HSG antibody show very limited binding. In the presence of IgG mixture, a dramatic increase of the binding of the mixedhaptens AG 5.0, AG 5.1, and AG 5.2 is observed, which is not simply additive, it is roughly 14-17-fold the sum of the monovalent binding. By contrast, AG 5.3 showed no significant binding cooperativity in the presence of the two IgGs. This results from the low affinity of this peptide for the anti-HSG antibody, attributed to an interaction between the histamine and the DTPA residues. As AG 5.1 structure was more complicated than that of the other mixed-haptens and the results obtained with this hapten were very similar to those obtained with AG 5.0, this peptide was not used for the following studies. The Ramos cell line derived from a human Burkitt lymphoma, as do most human B type acute lymphoblastic leukemia cells, bears at its surface both CD10 and CD20 antigens. Most normal cells express at most one of these two differentiation antigens. Thus, an enhanced binding to cells bearing simultaneously both CD10 and CD20 would result in an enhanced specificity to these tumor cells. Binding experiments of labeled mixed-haptens to Ramos cells were performed in the presence of an equimolar mixture of both antibodies. Controls with only one antibody were run in parallel in order to mimic the binding to nontumor cells bearing only one antigen. We demonstrated in previous studies that comparable tumor uptake was obtained with F(ab′)2-Fab′ and Fab′Fab′ antibody conjugates (Le Doussal et al., 1990). In addition, Fab′-Fab′, which is cleared more rapidly from plasma than F(ab′)2-Fab′, allowed higher tumor to nontumor tissues ratios because of lower trapping of the bivalent hapten in plasma. For these reasons anti-CD20 × anti-DTPA(In) and anti-CD10 × anti-HSG Fab′-Fab′ were preferred to F(ab′)2-Fab′ antibody conjugates though these latter, as do IgGs, would have higher affinity for tumor cells.

Hillairet de Boisferon et al.

The affinity of bispecific antibodies to the targeted antigens are higher than that of IgG1 for Fc receptors. Consistently, the observed monovalent binding of AG 5.0 and AG 5.2 were higher on Ramos cells in the presence of bispecific antibodies than on macrophages pretargeted with a single IgG. Similar binding curves were observed at 37 °C for 111In- and 125I-labeled mixed-haptens in the presence of a mixture of anti-CD10 × anti-HSG and antiCD20 × anti-DTPA(In) bispecific antibodies. Hapten binding to target cells was higher in the presence of both antibodies, and the binding curves obtained at 37 °C could not be explained by a simple additivity. Under bivalent binding conditions at 37 °C, binding was maximum after 2.5 h incubation, whereas monovalent binding reaches a maximum much quicker (about 15 min), suggesting that a slower process is involved in binding enhancement. Computer simulations showed that the simple hypothesis of cooperativity in the formation of complexes between the mixed-haptens and two bispecific antibody molecules bound to the target cell surface can account for the observed binding enhancement. By contrast, when the cooperativity factor is forced to 1.0 (no cooperativity) the calculated curves cannot fit satisfactorily the experimental data. Interestingly, the binding of mixed-haptens in the presence of the two bispecific antibodies was strongly dependent on the incubation temperature. No significant difference was observed for maximal binding in monovalent conditions at 4 or 37 °C. In contrast to the enhanced binding observed at 37 °C under bivalent conditions, binding at 4 °C was simply additive. Internalization cannot account for the increased binding under bivalent conditions at 37 °C as only 5% of the incubated radioactivity was internalized after 24 h. The binding enhancement also appears to be a slow process, suggesting that it involves the diffusion of the target antigens in membranes. We can hypothesize that antigens diffuse freely driven by thermal diffusion and that bridging of two bispecific antibodies can occur statistically when they are closely located on the cell surface. Then, the enhanced stability of the complex would result from the restricted diffusion of the antigens, due to bivalent binding. More complex phenomenon could occur including capping in specialized areas on the cell surface. These dual targeting reagents have been designed to enhance the specificity of pretargeting when tumorspecific antibodies are not available and antibodies to differentiation antigens are used instead. In pretargeting approaches, unlabeled bispecific antibodies are injected first, and the labeled hapten is injected a few days later when part of the circulating antibody has cleared. This situation is not easily reproduced in vitro. In all previous animal and clinical studies, the hapten was administered when significant concentrations of bispecific antibodies remained in the circulation. In some cases, both reagents have been administered simultaneously (Le Doussal et al., 1989, Manetti et al., 1997). This is the reason why in vitro binding experiments have been performed by simultaneous incubation of the antibodies and the hapten with the target cells. When the bispecific antibodies are preincubated with the cells before addition of the hapten, but not washed, hapten binding to target cells is essentially unchanged (Figure 4, panels a and b). When excess antibody is removed by washing the cells after preincubation, then hapten binding is reduced, because a fraction of the antibodies dissociates from the cells, but hapten binding cooperativity is clearly maintained (Figure 4c). In addition, the hydrophilicity of the labeled hapten should not be a drawback to the cooperative dual

Enhanced Specificity by Targeting Two Antigens

binding, which is a slow process (maximum binding after 2.5 h), as excess antibody, in the plasma, will trap the hapten and reduce its clearance, as it was demonstrated earlier for haptens bearing two DTPA (Le Doussal et al., 1990). This shows that the mixed haptens should also be able to bind more tightly to cells expressing both target antigens in vivo in the context of pretargeting. As a first step in the investigation of the potential of dual targeting for radioimmunotherapy, in vivo studies are now in progress in a model of SCID mice, i.v. grafted with Ramos cells which express CD10 and CD20 in this model as shown by autoradiographic studies. Such a binding enhancement will increase specificity for targeting isotopes to double antigen positive tumor cells and may be beneficial for targeting isotopes to B type acute lymphoblastic leukemia and Burkitt lymphoma, as well as other tumors coexpressing two markers of low specificity, and might increase tumor irradiation with minimal irradiation of normal cells. ACKNOWLEDGMENT

We thank Pr. B. P. Roques for fruitful discussions and Immunotech for the generous supply of indispensable antibody reagent. We are grateful to Pr. S. Askienazy for research facilities. This work was supported in part by the Association pour la Recherche contre le Cancer through Grant 4042 allocated to A.G.G. LITERATURE CITED (1) Appelbaum, F. R., Matthews, D. C., Eary, J. F., Badger, C. C., Kellog, M., Press, O. W., Martin, P. J., Fisher, D. R., Nelp, W. B., Donnal Thomas, E., and Bernstein, I. D. (1992) The use of radiolabeled anti-CD33 antibody to augment marrow irradiation prior to marrow transplantation for acute myelogenous leukemia. Transplantation 54 (5), 829-833. (2) 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. (3) Barbet, J., Peltier, P., Bardet, S., Vuillez, J. P., Bachelot, I., Denet, S., Olivier, P., Leccia, F., Corcuff, B., Huglo, D., Proye, C., Rouvier, E., Meyer, P., and Chatal, J. F. (1998) Radioimmunodetection of medullary thyroid carcinoma using indium-111 bivalent hapten and anti-CEA × anti-DTPA indium bispecific antibody. J. Nucl. Med. 39, 1172-1178. (4) Barbet, J., Kraeber-Bode´re´, F., Vuillez, J. P., Gautherot, E., Rouvier, E., and Chatal, J. F. (1999) Pretargeting with the Affinity Enhancement System for radioimmunotherapy. Cancer. Biother. Radiopharm. 14, 153-166. (5) Bellon, B., Jelsch, J., and Delaage, M. (1973) The´orie des e´quilibres multiples pour les polyme`res. Biochimie 55, 11591162. (6) Chetanneau, A., Barbet, J., Peltier, P., Le Doussal, J. M., Gruaz-Guyon, A., Bernard, A. M., Resche, I., Rouvier, E., Bourguet, P., Delaage, M., and Chatal, J. F. (1994) Pretargetted imaging of colorectal cancer recurrences using an 111In-labeled bivalent hapten and a bispecific antibody conjugate. Nucl. Med. Commun. 15, 972-980. (7) Corcoran, M. C., Eary, J., Bernstein, I., and Press, O. W. (1997) Radioimmunotherapy strategies for non-Hodgkin’s lymphoma. Ann. Oncol. 8 (Suppl. 1), 133-138. (8) De Labriolle-Vaylet, C., Cattan, P., Sarfati, E., Wioland, M., Billotey, C., Brocheriou, C., Rouvier, E., De Rocquancourt, A., Roste`ne, W., Askienazy, S., Barbet, J., Milhaud, G., and Gruaz-Guyon, A. (2000) Successful surgical removal of occult metastases of medullary thyroı¨d carcinoma recurrences with the help of immunoscintigraphy and radioimmunoguided surgery. Clin. Canc. Res. 6 (2), 363-371. (9) DeNardo, G. L., Lewis, J. P., DeNardo, S. J., and O’Grady, L. F. (1994) Effect of Lym-1 radioimmunoconjugate on refractory chronic lymphocytic leukemia. Cancer 73 (5), 1425-1432.

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