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Clinical Cancer Research 13, 5564s, September 15, 2007. doi: 10.1158/1078-0432.CCR-07-1204
© 2007 American Association for Cancer Research
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Cancer Therapy with Antibodies and Immunoconjugates

A Divalent Hapten-Peptide Induces Apoptosis in Human Non–Hodgkin Lymphoma Cell Lines Targeted by Anti-CD20 x Anti-Hapten Bispecific Antibodies

Pierre-Yves Brard1, Habibe Karacay1, Rhona Stein1, Robert M. Sharkey1, M. Jules Mattes1, Chien-Hsing Chang2, Edmund A. Rossi2, William J. McBride3 and David M. Goldenberg1

Authors' Affiliations: 1 Center for Molecular Medicine and Immunology, Belleville, New Jersey, 2 IBC Pharmaceuticals, Inc., and 3 Immunomedics, Inc., Morris Plains, New Jersey

Requests for reprints: Robert M. Sharkey, Center for Molecular Medicine and Immunology, 520 Belleville Avenue, Belleville, NJ 07109. Phone: 973-844-7121; Fax: 973-844-7020; E-mail: rmsharkey{at}gscancer.org.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
Purpose: Bispecific antibody (bsMAb) pretargeting procedures use divalent hapten-peptides to stabilize the binding of the hapten-peptide on tumor cells by a process known as the affinity enhancement system. The goal of this study was to determine if a divalent hapten-peptide could induce apoptosis by cross-linking bsMAb bound to CD20.

Methods: Three forms of bsMAbs were prepared by coupling the IgG, F(ab')2, or Fab' of a humanized anti-CD20 antibody to a Fab' of a murine antibody directed against the hapten histamine-succinyl-glycine (HSG). A recombinant bsMAb with divalent binding to CD20 and monovalent binding to HSG was also examined. Induction of apoptosis on SU-DHL-6, RL, and Ramos cells was examined by propidium iodide staining, caspase-3 activation, and mitochondrial membrane potential collapse, and compared with induction by cross-linking an anti-CD20 IgG with an antispecies antibody.

Results: The various forms of bsMAb had differing baseline levels of apoptosis in the absence of the divalent HSG peptide. The addition of the divalent HSG peptide significantly increased the level of apoptosis seen with the Fab' x Fab' bsMAb by 2.2- to 3.9-fold, as well as the F(ab')2 x Fab', IgG x Fab', and the recombinant bsMAbs by ~1.5-fold.

Conclusions: The addition of a divalent HSG peptide to various forms of bispecific anti-CD20 MAbs could enhance apoptotic signaling in several lymphoma cells. This effect was more consistently measured when the orientation of the anti–hapten-binding arm of the bsMAb was well defined, such as in the Fab' x Fab' and recombinant forms of bsMAb.

Antibodies alone, such as rituximab, trastuzumab, and cetuximab, have all been shown to initiate signals that lead to apoptosis, a mechanism that can kill cells, or at least make the cell more susceptible to the cytotoxic activity of other agents (chemotherapy or radiation; refs. 1–6). In the case of the anti-CD20, rituximab, the apoptotic signal is greatly enhanced when the antibody is cross-linked on the cell surface by an anti-antibody or through the use of rituximab complexes (3, 4).

Bispecific antibody pretargeting systems that use divalent haptens to enhance uptake and retention of the radiolabeled hapten-peptide, through a process known as the affinity enhancement system (7), can cross-link surface antigens, which may in turn result in the induction of apoptosis. In order to evaluate this possibility, we evaluated a pretargeting system that uses a humanized anti-CD20 bsMAb (8). In vitro studies have shown that humanized anti-CD20 IgG, designated hA20 or IMMU-106 (veltuzumab), had a similar antibody-dependent cell cytotoxicity and complement-mediated cytotoxicity properties as rituximab, and like rituximab, it could also induce apoptosis (9). Our goal in this series of studies was to determine whether apoptosis could be induced by the binding of an anti-CD20 bsMAb, and then further enhanced when cross-linked by a divalent hapten-peptide. To this end, three chemically conjugated bsMAbs and a recombinant bsMAb were tested in concert with a divalent hapten-peptide. The chemically conjugated bsMAb composed of the IgG and F(ab')2 of the hA20 MAb, as well as the recombinant bsMAb, which was prepared by a technique recently described by Rossi et al. (10), all have the capability of binding divalently to CD20, whereas the bsMAb chemical conjugate prepared with the Fab' of hA20 would only be monovalently bound to CD20. Each conjugate/construct contains a single Fab' fragment of the MAb specific for the histamine-succinyl-glycine (HSG) hapten (11, 12). Determinations of apoptotic induction were made in vitro primarily using the SU-DHL-6 cell line because our previous results indicated that apoptosis induction with anti-CD20 antibodies was strongest with this cell line (9), but the major positive findings were confirmed in two other B-cell lymphoma lines.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 Conclusions
 References
 
Cell lines. The Burkitt lymphoma line, Ramos, was purchased from the American Type Culture Collection. The non–Burkitt lymphoma cell lines, SU-DHL-6 and RL, which contains the chromosomal translocation t(14;18), were obtained from Dr. Alan Epstein (University of Southern California, Los Angeles, CA) and Dr. John Gribben (Dana-Farber Cancer Institute, Boston, MA), respectively. Cells were grown in suspension cultures in RPMI 1640 (Irvine Scientific Inc.), supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mL of penicillin, 100 µg/mL of streptomycin, and 2 mmol/L of L-glutamine (complete medium). When the cell count was ~106 cells/mL, they were harvested. For all studies, viability was >95%.

Preparation of antibodies. The humanized anti-CD20 IgG1, hA20 (veltuzumab) or IMMU-106 (9), and an isotype-matched control humanized anti–carcinoembryonic antigen (CEA) IgG1, hMN-14 (13), were provided by Immunomedics, Inc. The murine 679 anti-HSG antibody (12) and a novel recombinant anti-CD20 x anti-HSG bsMAb, designated TF4, were provided by IBC Pharmaceuticals, Inc. TF4 is a ~157 kDa protein that combines two of the humanized anti-CD20 Fabs with a single Fab of the humanized version of the 679 anti-HSG antibody using a unique "dock and lock" system (10). A similar recombinant construct, designated TF2, a divalent anti-CEA x monovalent anti-HSG bsMAb (10), was used as a control for the TF4. The chimeric anti-CD20 IgG, rituximab (IDEC Pharmaceuticals Corp.) was purchased.

Bispecific antibodies were prepared by conjugating the whole IgG, F(ab')2, or Fab' fragments of hA20 anti-CD20 antibody or the control hMN-14 Fab' to a Fab' fragment of the murine anti-HSG MAb using methods described previously (ref. 14; Fig. 1 ). The coupling conditions for the IgG and F(ab')2 bsMAbs were adjusted so that on average, the resulting conjugate contained 1 mol of the anti-HSG Fab' per mole of the IgG or F(ab')2 [presumed molecular weights were 200 and 150 kDa for the bispecific IMMU-106 IgG and F(ab')2, respectively]. The coupling methodology used for the formation of the Fab' bsMAb results in only the Fab' x Fab' configuration (presumed molecular weight, 100 kDa). The purity of bispecific conjugates and their binding properties to a radiolabeled di-HSG peptide were analyzed by size-exclusion high-performance liquid chromatography. The bsMAbs were radioiodinated with Na125I (Perkin-Elmer) using a chloramine T method (15) with excess tyrosine added to quench the reaction, followed by chromatographic purification. The immunoreactive fraction was determined by incubating the bsMAbs with an excess of an anti-id antibody developed against the hA20 IgG, and examining the shift to a higher molecular weight fraction by size-exclusion high-performance liquid chromatography. When incubating the radiolabeled HSG peptide with a mole excess of the bsMAb, the molecular profile of the radiolabeled peptide shifted quantitatively to peaks corresponding to monovalently and divalently bound bsMAbs. These peaks could be shifted to a larger molecular size by the addition of an anti-hA20 id antibody, indicating their bispecific binding capability. All studies indicated that >95% of the purified bispecific hA20 antibodies (bshA20) were reactive with CD20 and HSG.


Figure 1
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  		Fig. 1. Schematic representation of the various bsMAb conjugates/constructs examined. Humanized hA20 anti-CD20 IgG, F(ab')2, or Fab' were modified with N,N'-O-phenylenedimaleimide (PDM) which couples to sulfhydryl groups to form thio-ether bonds (*). Anti-HSG Fab'-SH was then added to the derivatized hA20 IgG, F(ab')2, or Fab', with the final products purified by size-exclusion high-performance liquid chromatography. The hA20 Fab'/anti–HSG Fab' conjugate forms in a very specific orientation, whereas the anti–HSG Fab' can be coupled to any number of locations on the derivatized hA20 IgG and F(ab')2, resulting in conjugates with the anti-HSG Fab' oriented differently. The recombinant TF4 will also have a well-defined orientation of the anti–HSG binding site. These bsMAbs can bind to CD20 present on a B cell and then be subsequently cross-linked by the divalent HSG peptide.

 
Peptide haptens. The DOTA-di-HSG peptide, IMP-241, [DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2] and the mono-HSG peptide, IMP-252 (DOTA-D-Phe-D-Tyr-Lys(HSG)-NH2) were provided by Immunomedics, Inc., and used as the specific HSG-binding peptide (12). IMP-156, a divalent diethylenetriaminepentaacetic acid [DTPA; Ac-Phe-Lys(DTPA)-Tyr-Lys(DTPA)-NH2], also provided by Immunomedics, Inc., was used as a nonbinding peptide control (16). The hapten-peptides were radiolabeled with 111InCl3 or 90YCl3 (IsoTex or Perkin-Elmer) as previously described (12).

Cell binding assays. Indirect immunofluorescence assay was done using SU-DHL-6 and Ramos cells incubated with the different anti-CD20 or control anti-CEA bsMAbs followed by the addition of FITC goat anti-human (GAH) IgG and FITC goat anti-mouse (GAM) IgG (The Jackson Laboratory; ref. 9). Cells were analyzed by flow cytometry using a FACSCalibur (Becton Dickinson).

A competitive binding assay was used to determine the concentration of each bsMAb required to saturate CD20 binding to SU-DHL-6 cells. Increasing concentrations (0.5-50 µg/mL) of the unlabeled bsMAb was mixed in the presence of 2.5 x 106 cpm of each 125I-labeled bsMAb [125I-bshA20 IgG, 15.3 µCi/µg; 125I-bshA20 F(ab')2, 14.5 µCi/µg; 125I-bshA20 Fab', 14.6 µCi/µg] with 106 SU-DHL-6 cells in 0.5 mL of complete medium for 1 h at 37°C. Controls included cells incubated in medium with no blocking bsMAb (i.e., maximum binding of each radiolabeled bsMAb), as well as cells that were preblocked by unlabeled hA20 IgG (100 µg/mL) prior to the addition of the radiolabeled bsMAbs (i.e., nonspecific binding of radiolabeled bsMAbs). For this latter control, cells were first incubated for 1 h at 37°C in a volume of 0.25 mL of complete medium followed by the addition of 0.25 mL of medium ± radiolabeled bsMAb. After incubation with the radiolabeled bsMAb, cells were washed twice in PBS containing 1% horse serum and counted. Data are expressed as the specific binding [(total binding) – (nonspecific binding)], and the results expressed as the nmol/mL required for saturation.

In vitro binding properties of di-HSG peptide. SU-DHL-6 cells (6.66 x 107; 500 µL) were dispensed in duplicate to glass tubes and incubated for 1 h at 37°C with 10 µg of bshA20 IgG, F(ab')2, Fab' MAbs, or the anti-CEA Fab' bsMAb as a control. Cells were washed once with 8 mL of PBS containing 1% horse serum before incubation with 3.2 pmol of 111In-IMP-241 di-HSG peptide or 111In-IMP-156 di-DTPA peptide (specific activity, 0.58 µCi/pmol) for 40 min at 37°C in 500 µL of complete medium. Cells were washed once and radioactivity in the cell pellet was determined. Results were expressed as the number of moles of peptide bound per cell.

Flow cytometric analysis of apoptosis using propidium iodide staining. The DNA hypodiploid peak (sub-G0/G1 DNA fragment) revealed by propidium iodide (PI) was used to analyze apoptosis (3). Using the procedure found previously to illustrate the apoptotic activity of anti-CD20 IgG (9), SU-DHL-6 cells were placed in 24-well plates (3.3 x 105 cells/well in 1.0 mL complete medium) and incubated with 5 µg (25 µL) of rituximab, hA20 IgG, bshA20 IgG, F(ab')2, Fab', or the nonbinding, bispecific anti-CEA bsMAb in addition to media control (i.e., no MAb or bsMAb added) for 20 min at 37°C in a CO2 (5%) incubator. After this initial incubation, the following reagents were added to cells in duplicate or triplicate samples: Fc{gamma}-specific GAH IgG or (Fab')2-specific GAM IgG second antibodies (11-15 µL, final concentration of 20 µg/mL), the di-HSG (IMP-241), mono-HSG (IMP-252), or control di-DTPA (IMP-156) peptide-haptens (25 µL, final concentration of 0.5 nmol/mL), or no subsequent additions (i.e., medium alone). Under these conditions, the amount of peptide was in 10-fold (i.e., for the Fab' x Fab' bsMAb) to 20-fold (i.e., for the IgG x Fab' bsMAb) molar excess of the bsMAbs in the incubation mixture. Several other incubation conditions were tested. In one, the cells were washed and media replaced to remove excess MAb or bsMAb prior to the addition of the secondary antibodies or hapten-peptides. In another, preformed complexes between the secondary antibody and MAb or the hapten-peptide with the bsMAb were added to the cells. In all of these additional assays, the initial incubation period was increased to 2 h at 37°C. Additional assays were done using two different bsMAb concentrations during an initial incubation of 30 min, and then the hapten-peptide was added at varying concentrations, thereby changing the hapten-peptide to bsMAb mole ratios. In all assays, the cells were subsequently incubated in the medium alone or in the medium containing the test materials for 48 h (37°C; 5% CO2). Finally, SU-DHL-6, Ramos, and RL cell lines were studied and incubated with bshA20 Fab' or TF4 for 30 min then the di-HSG peptide-hapten (IMP-241), GAH, or GAM were added for a total incubation time of 30 h. At the conclusion of this latter incubation period, the cells were washed with PBS, and then resuspended in hypotonic PI solution (50 mg/mL PI in 0.1% sodium citrate, 0.1% Triton X-100), and analyzed by flow cytometry. The percentage of apoptotic cells was defined as the percentage of cells with DNA staining before G1/G0 peak (hypodiploid). Comparisons of the percentage of apoptotic cells were determined by a Student's t test.

Mitochondrial membrane potential assays. Mitochondrial membrane potential was measured by flow cytometry with the lipophilic cationic probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol-carbocyanine iodide (JC-1; BD Biosciences; refs. 17, 18). SU-DHL-6 cells were placed in 24-well plates (3.3 x 105 cells/well in 1.0 mL complete medium) and incubated with 5 µg (0.025 mL) of rituximab, bispecific anti-CD20 hA20 IgG, Fab', or the nonbinding, bispecific anti-CEA bsMAb in addition to media control (i.e., no MAb or bsMAb added). After a 20-min incubation with the primary MAbs (37°C, 5% CO2), one set of samples received 25 µL of the di-HSG peptide IMP-241, at a final adjusted concentration of 0.5 nmol/mL, and another set received the control peptide, IMP-156, at the same final adjusted concentration. A final set had only complete medium added (i.e., primary MAb or bsMAb alone, no secondary antibody or peptide-hapten). After a 48-h incubation (37°C, 5% CO2), cells were transferred to test tubes, and assays were done by flow cytometry using the BD Mitoscreen kit, following the instructions of the manufacturer (BD Biosciences).

Caspase-3 activity assays. SU-DHL-6 cells were handled in an identical manner as described above, except that after the final 48-h incubation, the cells were analyzed for caspase-3 activity using the FITC-conjugated monoclonal active caspase-3 antibody apoptosis kit I (BD Biosciences), according to the instructions of the manufacturer.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 Conclusions
 References
 
Characterization of bispecific hA20 antibodies. An evaluation of apoptosis associated with an antibody should first show that the antibody preparation does not contain a sizeable portion of aggregates that could otherwise trigger apoptosis in the absence of cross-linking agents. Size-exclusion high-performance liquid chromatography was used to analyze the purity of the hA20 IgG-SH, F(ab')2-SH, and Fab'-SH prior to and after their coupling to the murine 679 Fab' to form the bsMAb conjugates. The three bispecific conjugates eluted at the expected times based on their higher molecular weights as compared with the IgG and fragments from which they were derived (Fig. 2A ). The binding properties of each hA20 x m679 Fab' bsMAb to 90Y-IMP-241 di-HSG peptide is illustrated in Fig. 2B. When IgG, F(ab')2, and Fab' bsMAb constructs were mixed with the peptide, the radiolabeled peptide was shifted to elution times of 8, 8.3, and 8.7 min, respectively, with a shoulder on the descending side. The major peak was indicative of two bsMAbs bound with a single, divalent peptide, whereas the shoulder corresponded to single bsMAb bound to a single peptide. Adding an excess of the anti-hA20 id antibody to each shifted the profiles to a higher molecular weight complex, indicating that 100% of the radiolabeled IMP-241 bound by the bsMAbs was also associated with anti–CD20-reactive bsMAb. Similar analyses done with TF4 and TF2 showed the antibody to be fully immunoreactive with their respective anti-id antibody and IMP-241 (data not shown).


Figure 2
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  		Fig. 2. Characteristics of bispecific hA20 conjugates. A, size-exclusion high-performance liquid chromatography chromatograms using an in-line UV detection of hA20 IgG, F(ab')2, and Fab' and their respective bispecific constructs after conjugation to m679 Fab'. Elution times are indicated. B, binding properties of the three bispecific hA20 conjugates first with 90Y-labeled IMP241 divalent HSG-DOTA peptide and then after incubating this complex with an anti-hA20 id antibody, monitoring 90Y-radioactivity using an in-line radiation detector. Elution times for the primary peaks are given.

 
In vitro binding of bispecific conjugates on SU-DHL-6 cells. Binding of each bispecific hA20 conjugate to SU-DHL-6 was confirmed by flow cytometry, with a mean channel fluorescence of ~250 with each of the anti-CD20 bsMAb compared with ~3.5 for the controls. Binding of each bispecific conjugate to Ramos cells was lower with a mean channel fluorescence of ~25 compared with ~2.5 for controls. Competitive binding studies on SU-DHL-6 cells indicated that antigen saturation was reached at a concentration of 30 µg/mL (0.15 nmol/mL) for bshA20 IgG, compared with 20 µg/mL (0.13 nmol/mL) for bshA20 F(ab')2 and 50 µg/mL (0.5 nmol/mL) for bshA20 Fab'.

In vitro binding of di-HSG peptide on bispecific conjugates previously bound to SU-DHL-6 cells. The binding capacity of di-HSG and di-DTPA peptides on bshA20 IgG, F(ab')2, and Fab' previously bound to SU-DHL-6 cells was studied by incubating cells with 111In-IMP-241 peptide or 111In-IMP-156 peptide. A similar amount of IMP-241 binding to the SU-DHL-6 cells was observed irrespective of the hA20 bsMAb used (data not shown). No binding was seen with the control IMP-156 and no binding of either peptide when cells were first incubated with the nonspecific anti-CEA bsMAb.

Induction of apoptosis with bispecific hA20 conjugates and di-HSG peptide. The potential ability of the divalent HSG peptide to induce apoptosis on SU-DHL-6 cells was determined by PI staining (Table 1 ), by JC-1 fluorescence determination (Fig. 3 ), and by FITC anti–caspase-3 tests (Fig. 4 ). As shown with PI staining (Table 1), compared with untreated cells, hA20 IgG had an apoptotic effect alone (22.5 ± 0.4%), which was 2-fold higher than the background signal (11.0 ± 0.8%), and was similar to rituximab (21.7 ± 0.8%). Cross-linking these IgGs with Fc{gamma}-specific GAH enhanced apoptosis (increasing the signal to 38.8 ± 2.1% and 45.4 ± 5.0% for the hA20 and rituximab, respectively), but the apoptotic signal was not significantly increased when either di-HSG peptide (P = 0.71 and P = 0.26 for the hA20 and rituximab, respectively) or GAM (P = 0.29 and P = 0.13 for the hA20 and rituximab, respectively) were used as a secondary agent when compared with the appropriate control conditions. The divalent bispecific anti-CD20 antibodies [i.e., IgG and F(ab')2] also had an apoptotic effect by themselves (18.8 ± 1.5% and 16.9 ± 2.2%, respectively) as compared with the media control. This effect was somewhat lower than that observed for the hA20 IgG, but the difference was not significant [P = 0.08 for bshA20 IgG and P = 0.07 for bshA20 F(ab')2]. The percentage of apoptotic cells did not increase when the divalent anti-CD20 bsMAbs were incubated with the di-HSG peptide, but when GAM was used as a secondary agent, an enhanced signal was observed for both bsMAbs [31.5 ± 1.5% and 29.7 ± 3.7% for the IgG and F(ab')2 bsMAb, respectively]. The addition of the GAH as a secondary agent did not have as appreciable an effect on apoptosis with the F(ab')2 bsMAb as the IgG bsMAb because the GAH was specific for Fc{gamma}, which is largely removed by pepsin digestion.


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  		Table 1. Apoptotic effect of bispecific hA20 conjugates and di-HSG peptide on SU-DHL-6 cells, as shown by PI staining

 

Figure 3
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  		Fig. 3. Apoptotic effect of bshA20 conjugates cross-linked with di-HSG peptide on SU-DHL-6 cells, shown by mitochondrial membrane collapse with JC-1. Percentages represent cells with mitochondrial membrane collapse after 48 h of incubation with bshA20 IgG and Fab' antibodies alone or with the di-HSG or di-DTPA control peptides. High red/low green fluorescence, nonapoptotic cells; low red/high green fluorescence, cells with disruption of the mitochondrial membrane (i.e., apoptosis). bs hMN14 was used as a negative control and rituximab was used as a positive control.

 

Figure 4
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  		Fig. 4. Flow cytometric analysis of caspase-3 activation of SU-DHL-6 cells after 48 h of incubation with bshA20 IgG and Fab' antibodies (values in boldface and underlined, percentage of caspase-3–activated cells) with di-HSG (open line) or di-DTPA control peptide (filled line). The bshMN14 anti-CEA was used as a negative control.

 
In contrast to the inability to enhance apoptosis when the divalent forms of the bsMAbs were evaluated under these conditions, the addition of the di-HSG peptide to cells preincubated with the bshA20 Fab' showed a significant enhancement in apoptosis, increasing from 10.3 ± 1.0% in the absence of the peptide to 23.1 ± 0.3% (P < 0.001). The addition of the control di-DTPA peptide had no effect on apoptosis. The level of apoptosis observed with the peptide cross-linked bshA20 was similar to that observed for the hA20 IgG alone (22.5 ± 0.4%), but it was significantly higher than that seen with the hA20 IgG or F(ab')2 bsMAbs alone (P = 0.015 and P = 0.014, respectively) or when these divalent anti-CD20 bsMAbs were incubated with di-HSG peptide (P = 0.003 and P = 0.027, respectively). The secondary GAM antibody enhanced the apoptotic signal to a higher level than that of the di-HSG peptide, but the GAH was ineffective.

Two additional assays were done on SU-DHL-6 cells to confirm the initial observations using the PI assay, and to determine the pathways of apoptosis: mitochondria-dependant and caspase-dependant pathways. By the mitochondrial membrane collapse assay with JC-1 fluorescence (Fig. 3), the amount of cells with mitochondria depolarization was increased ~4-fold when cells were preincubated with the bshA20 Fab' cross-linked with di-HSG peptide as compared with the controls. This rate was similar to the effect obtained with rituximab alone. However, no enhancement of apoptosis was observed with the bshA20 IgG by the addition of the di-HSG peptide. Caspase activation in SU-DHL-6 cells was determined with an anti-active caspase-3 FITC conjugate (Fig. 4). Interestingly, compared with the proportion of cells that activated their caspase-3 with control peptide, the bshA20 Fab' cross-linked with di-HSG peptide allows a caspase-3 activation in 53.3% of cells, which was higher than the 36.9% of the cells found with the hA20 IgG alone (data not shown). Again, compared with control peptides, no enhancement of apoptotic signals was observed with the divalent bshA20 IgG cross-linked with di-HSG peptide. These additional assays indicate that the pathways used to induce apoptosis are caspase-3- and mitochondria-dependant.

In addition to examining a divalent HSG peptide, a monovalent HSG peptide (IMP-252) was tested, which showed no ability to enhance apoptosis of the bshA20 Fab', further indicating the importance of having a divalent hapten-peptide to cross-link two bsMAbs for apoptosis induction to occur (Fig. 5 ). A recombinant bsMAb, TF4, with divalent CD20 binding and monovalent HSG binding was also examined. This construct, like the bshA20 IgG and F(ab')2 chemical conjugates, had an innate ability to induce apoptosis at a low level, but unlike chemical conjugates, apoptosis induction was increased when incubated in the presence of divalent IMP-241 (Fig. 5). Both TF4 and the bshA20 Fab' bsMAb have well-defined molecular configurations, whereas the bshA20 IgG and F(ab')2 bsMAb could have a more random placement of the anti-HSG Fab'. This may explain the failure of these latter two constructs to be affected by the divalent HSG peptide.


Figure 5
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  		Fig. 5. Apoptotic effect, as shown by PI staining, on SU-DHL-6 cells first incubated with rituximab, bshA20 Fab', recombinant bsMAb TF4, and control TF2 for 2 h, then incubated with mono-HSG (IMP-252), di-HSG (IMP-241), and no peptide for 48 h.

 
In the basic PI assay configuration, after the initial incubation with the antibody, the cross-linking agent is added to the medium, and the incubation continued for 2 more days. These conditions create an environment in which the excess antibody in the medium can be complexed with the cross-linking agent in addition to the potential for the cross-linking agent to bind to already cell-bound antibodies. In an in vivo pretargeting setting, the hapten-peptide (cross-linking agent) would not be administered until the bsMAb had cleared from the blood. Thus, additional assays were done in which the excess bsMAb was washed from the cells and fresh medium was added prior to the addition of the hapten-peptide. Under these conditions, the percentage of apoptotic cells was not significantly different from the levels observed in the control cells (data not shown). However, if the assay was done in the same manner with rituximab and hA20 IgG, there was also no evidence of apoptosis induction. This suggests that continuous exposure of the cells to the antibody and secondary cross-linking agent is required to induce a detectable enhancement in the apoptotic signal. If the bshA20 Fab' and IMP-241 or the hA20 and rituximab IgGs were premixed with GAH and then added to the cells, a similar level of apoptosis was measured as when the secondary agents were added after the primary agents had time to bind to cells (data not shown). Interestingly, premixing IMP-241 with the bshA20 IgG or F(ab')2 did not enhance the percentage of apoptotic cells over the level seen with these bsMAb alone.

Additional studies were undertaken to better understand why the divalent chemically conjugated bsMAb were not as effective in inducing apoptosis. In all of the earlier assays, the same amount of the bsMAbs (i.e., 5 µg/mL) was mixed with a molar excess of the peptide, ranging from 10- to 20-fold based on the molecular size of the conjugate. These conditions could have favored monovalent binding of the hapten-peptide to the bsMAb. In these additional assays, different amounts of bsMAb were used, but more importantly, the ratio of the amount of di-HSG peptide to the amount of bsMAb in the reaction mixture was varied so that the IMP-241/bshA20 IgG ratio changed from 1 to 10:1. As shown in Fig. 6 , apoptosis induction was enhanced with divalent bshA20 conjugates in the presence of IMP-241 [e.g., 500 nmol/L bshA20 IgG ± peptide at 1:1 ratio, P = 0.046; and 50 nmol/L bshA20 F(ab')2 ± peptide at a 1:1 ratio, P = 0.016]. Increasing the amount of peptide did not enhance the apoptotic signal. With bshA20 Fab', apoptotic enhancement was observed at 50 and 500 nmol/L of bsMAb and peptide, with the maximum effect occurring at a concentration of 500 nmol/L and a peptide/bsMAb ratio of 3:1. With the recombinant TF4, an apoptotic enhancement was also observed at both a 50 and 500 nmol/L concentration of the bsMAb with a 1:1 molar ratios, but at the 500 nmol/L concentration, increasing the amount of peptide seemed to further enhance apoptosis. Thus, the level of apoptosis induced is affected by the amount of bsMAb and molar ratio with the peptide.


Figure 6
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  		Fig. 6. Apoptotic effect of bispecific hA20 antibodies and TF4 concentration and peptide/antibody molar ratio variations as checked with PI staining of SU-DHL-6 cells incubated with bsMAb for 30 min and di-HSG peptide for 48 h.

 
The ability to enhance apoptosis by cross-linking the bshA20 Fab' and TF4 with di-HSG peptide observed on SU-DHL-6 cells was tested in two other cell lines (RL and Ramos). A comparison was made on the effect of bshA20 Fab' incubated with F(ab')2-specific GAH IgG. On Ramos and RL cells, the level of apoptosis obtained with bshA20 Fab' incubated with GAH was lower than the effect observed with these antibodies on SU-DHL-6 cells (4.1 ± 0.4% and 9.0 ± 1.2% for Ramos and RL, respectively, compared with 42.8 ± 3.7% with SU-DHL-6). This likely reflects the lower antigen levels in these cells, as was observed previously (9). The di-HSG peptide significantly enhanced (~2-fold) the apoptosis of cells incubated with bshA20 Fab' from 1.1 ± 0.2% to 2.1 ± 0.1% (P = 0.0011) on Ramos cells and from 5.8 ± 0.3% to 9.3 ± 0.3% (P = 0.0001) on RL cells. Moreover, the di-HSG peptide enhanced the apoptosis of cells incubated with TF4 from 1.5 ± 0.2% to 3.3 ± 0.2% (P = 0.002) on Ramos and from 8.2 ± 0.3% to 13.2 ± 0.4% (P < 0.0001) on RL cells. Di-HSG peptide had no effect on cells incubated with bshA20 IgG and control bsMAb TF2 on both cell lines (data not shown).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
Conclusions
 References
 
The successful application of radioimmunotherapy in non–Hodgkin lymphoma was an important achievement, but it has been far more challenging to translate this success to solid tumors. The primary difficulty most likely lies in the inherent differences in the radiosensitivity of solid tumors as compared with lymphomas because dosimetry studies have repeatedly shown solid tumor receiving as much, if not more, radiation delivered as to lymphoma, yet objective responses are rarely reported (19). In lymphoma, the anti-CD20 IgGs used to target the radioactivity have considerable antitumor activity alone. This antitumor activity has been attributed to antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, but these antibodies are also able to affect direct signaling, leading to apoptosis (5, 20). Cells undergoing apoptosis are more sensitive to radiation and chemotherapy, and therefore, this mechanism could have an important role in the success of radioimmunotherapy of lymphomas. Because most of the evidence for these mechanisms of action have been derived from in vitro studies, the precise extent to which any one of these mechanisms is responsible for the therapeutic effect remains largely speculative. Most studies that show apoptosis have revealed a relatively low percentage of cells affected, unless the anti-CD20 antibody is cross-linked in some manner (3, 4, 9). Because no cross-linking agent is used in anti-CD20 IgG therapy, this mechanism may play a minor role; however, because immune cells will bind the Fc-receptor of the anti-CD20 IgG bound to the target lymphoma cells, effective cross-linking can be initiated. Thus, apoptosis may work in concert with these other mechanisms.

Pretargeting radionuclides has become as promising an approach for improving therapy as compared with directly radiolabeled IgG or fragments, with the ability to increase the total radiation dose delivered to tumors, as well as increasing the dose rate (21). Whether pretargeted radionuclides alone will allow for greater success in solid tumors awaits clinical testing, but there are promising therapeutic results in medullary thyroid cancer, as well as promising dosimetry in colorectal cancer (22, 23). Pretargeting has also been shown in preclinical models to be a more effective therapeutic in non–Hodgkin lymphoma than directly radiolabeled anti-CD20 IgG, without having to resort to doses that would lead to serious myelosuppression (8, 24, 25). Bispecific antibody pretargeting methods are unique from the avidin-biotin systems because they commonly employ a bivalent hapten to enhance the binding avidity of the radiolabeled hapten-peptide locally at the tumor (26). Because this type of configuration could conceivably lead to the cross-linking of bsMAbs bound to the cell surface, a determination was made if apoptosis could be induced is an anti-CD20 bsMAb pretargeting system that uses a divalent hapten-peptide.

The in vitro assays we did showed that a divalent hapten-peptide can induce apoptosis, but it remains unclear whether this mechanism will have a role in vivo. A salient, but interesting aspect of the PI assay system is the requirement that the cells be exposed to the test articles for >2 h (our standard conditions used a 48-h exposure) in order for specific apoptotic induction to be detected. Washing the cells prior to the incubation with the hapten-peptide was used to simulate what would most likely occur in vivo in a pretargeting setting, where the radiolabeled hapten-peptide is not given until most of the bsMAb has cleared from the blood. Under these conditions, induction of apoptosis was not observed, not only with the bsMAb conjugates, but also with hA20 or rituximab IgG later exposed to the cross-linking agents. This raises the question of whether apoptosis would be induced in a standard pretargeting setting, where the primary goal is the efficient targeting of a radiolabeled hapten-peptide. Future in vivo studies will need to include control groups of the nonradiolabeled hapten-peptide to assess what, if any, antitumor activity might be attributed to this effect. However, if the induction of apoptosis alone was linked to enhanced efficacy, bsMAb could be cross-linked with compounds bearing multiple haptens to make it a more effective cross-linking agent. Indeed, the dock and lock technique could be used to prepare additional bsMAb structures with the capacity to bind to multiple tumor targets as well as multiple haptens. The bsMAb construct could be engineered to include a human Fc domain that would give the additional capacity to elicit antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Because the apoptotic effect observed with the divalent hapten never exceeded that of the divalent anti-CD20 IgG alone, the level of apoptosis induced by the current constructs, even the TF4 recombinant bsMAb, in combination with the divalent hapten-peptide, is limited. However, these studies illustrate the potential of this technique to present opportunities to develop new bsMAb and haptenated compounds that would likely enhance the therapeutic capability of this method.

In the current studies, the three bsMAb configurations were tested. Although anticipated to have a more robust ability to induce apoptosis in the presence of the di-HSG peptide, apoptosis was enhanced only ~1.5-fold with the divalent hA20 IgG and F(ab')2, and only under a narrow range of bsMAb and peptide concentrations. In contrast, apoptosis was enhanced 2.2- to 3.9-fold when di-HSG peptide was incubated with the bshA20 Fab'. Showing a significant apoptotic effect using the di-HSG peptide with the divalent bsMAbs may have been more difficult because of the higher baseline level of apoptosis observed with divalent bsMAbs alone than with monovalent bshA20 (refer to Table 1), but it is likely that the conformation of the bsMAb played an important role. The conjugates studied herein were prepared in a manner to limit the number of anti-HSG Fab' on the hA20 F(ab')2 and IgG to only one, but the anti-HSG Fab' could have been coupled to any number of sites on the hA20. Therefore, the orientation of anti-HSG Fab' on these bsMAb molecules would be more disordered as compared with the Fab' x Fab'. This very likely impeded the cross-linking of IgG and F(ab')2 bsMAb in a manner that would have allowed these conjugates, even when preformed, to enhance apoptosis. In addition to orientation, differences in molecular spacing might also have had an effect. The fact that the polyclonal anti-antibodies, which can create larger, more complex aggregates than the di-HSG peptide with the bsMAb carrying a single anti-HSG, were effective cross-linking agents also speaks to the more restricted orientation given to the bshA20 IgG and F(ab')2 complexes by the di-HSG peptide. In contrast to the chemical conjugates, the recombinant TF4 bsMAb (divalent anti-CD20 x monovalent anti-HSG), was able to orient the di-HSG peptide complexes in a manner that enhanced apoptosis. The apoptotic enhancement of the di-HSG peptide was also observed in two other NHL cell lines, but the level of apoptosis was lower than that seen with SU-DHL-6. This observation could be explained by the lower CD20 expression of the other NHL cell line, which is 3-fold lower in RL and 4-fold lower in Ramos than SU-DHL-6 cells (9).


   Conclusions
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 Conclusions
References
 
A divalent peptide-hapten can enhance the apoptosis of anti-CD20 bsMAb bound to lymphoma cells. This effect might further radiosensitize these cells, thereby adding to the therapeutic effect induced by pretargeted radionuclides. These results also suggest that hapten-peptide polymers may be used to increase cross-linking of bsMAb to improve the effect of anti-CD20 immunotherapy. In vivo studies using this divalent peptide-hapten system are now needed to verify whether this may have clinical prospects.


   Acknowledgments
 
We thank S. Chen, J. Jebsen, and D. Yeldell for their technical assistance.


   Footnotes
 
Grant support: National Cancer Institute grant P01-CA103985 and New Jersey Department of Health and Senior Services grant 06-1853-FS-N0. La Fondation de France, the French Ministry of Foreign Affairs (programme Lavoisier), the Isère Comity of La Ligue Contre le Cancer, and Amersham Health (P-Y. Brard).

Presented at the Eleventh Conference on Cancer Therapy with Antibodies and Immunoconjugates, Parsippany, New Jersey, USA, October 12-14, 2006.

Received 5/16/07; accepted 5/29/07.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 Conclusions
 References
 

  1. Harris M. Monoclonal antibodies as therapeutic agents for cancer. Lancet Oncol 2004;5:292–302.[CrossRef][Medline]
  2. Prewett M, Rockwell P, Rockwell RF, et al. The biologic effects of C225, a chimeric monoclonal antibody to the EGFR, on human prostate carcinoma. J Immunother Emphasis Tumor Immunol 1996;19:419–27.[Medline]
  3. Shan D, Ledbetter JA, Press OW. Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies. Blood 1998;91:1644–52.[Abstract/Free Full Text]
  4. Zhang N, Khawli LA, Hu P, Epstein AL. Generation of rituximab polymer may cause hyper-cross-linking-induced apoptosis in non-Hodgkin's lymphomas. Clin Cancer Res 2005;11:5971–80.[Abstract/Free Full Text]
  5. Jazirehi AR, Bonavida B. Cellular and molecular signal transduction pathways modulated by rituximab (rituxan, anti-CD20 mAb) in non-Hodgkin's lymphoma: implications in chemosensitization and therapeutic intervention. Oncogene 2005;24:2121–43.[CrossRef][Medline]
  6. Carnahan J, Stein R, Qu Z, et al. Epratuzumab, a CD22-targeting recombinant humanized antibody with a different mode of action from rituximab. Mol Immunol 2007;44:1331–41.[CrossRef][Medline]
  7. Le Doussal JM, Martin M, Gautherot E, Delaage M, Barbet J. In vitro and in vivo targeting of radiolabeled monovalent and divalent haptens with dual specificity monoclonal antibody conjugates: enhanced divalent hapten affinity for cell-bound antibody conjugate. J Nucl Med 1989;30:1358–66.[Abstract/Free Full Text]
  8. Sharkey RM, Karacay H, Chang CH, McBride WJ, Horak ID, Goldenberg DM. Improved therapy of non-Hodgkin's lymphoma xenografts using radionuclides pretargeted with a new anti-CD20 bispecific antibody. Leukemia 2005;19:1064–9.[CrossRef][Medline]
  9. Stein R, Qu Z, Chen S, et al. Characterization of a new humanized anti-CD20 monoclonal antibody, IMMU-106, and its use in combination with the humanized antiCD22 antibody, epratuzumab, for the therapy of non-Hodgkin's lymphoma. Clin Cancer Res 2004;10:2868–78.[Abstract/Free Full Text]
  10. Rossi EA, Goldenberg DM, Cardillo TM, McBride WJ, Sharkey RM, Chang CH. Stably tethered multifunctional structures of defined composition made by the dock and lock method for use in cancer targeting. Proc Natl Acad Sci U S A 2006;103:6841–6.[Abstract/Free Full Text]
  11. Janevik-Ivanovska E, Gautherot E, Hillairet de Boisferon M, et al. Bivalent hapten-bearing peptides designed for iodine-131 pretargeted radioimmunotherapy. Bioconjug Chem 1997;8:526–33.[CrossRef][Medline]
  12. Sharkey RM, McBride WJ, Karacay H, et al. A universal pretargeting system for cancer detection and therapy using bispecific antibody. Cancer Res 2003;63:354–63.[Abstract/Free Full Text]
  13. Sharkey RM, Juweid M, Shevitz J, et al. Evaluation of a complementarity-determining region-grafted (humanized) anti-carcinoembryonic antigen monoclonal antibody in preclinical and clinical studies. Cancer Res 1995;55:5935–45s.
  14. Karacay H, Sharkey RM, McBride WJ, et al. Pretargeting for cancer radioimmunotherapy with bispecific antibodies: role of the bispecific antibody's valency for the tumor target antigen. Bioconjug Chem 2002;13:1054–70.[CrossRef][Medline]
  15. Greenwood FC, Hunter WM, Glover JS. The preparation of I-131-labelled human growth hormone of high specific radioactivity. Biochem J 1963;89:114–23.[Medline]
  16. Karacay H, McBride WJ, Griffiths GL, et al. Experimental pretargeting studies of cancer with a humanized anti-CEA x murine anti-[In-DTPA] bispecific antibody construct and a 99mTc-/188Re-labeled peptide. Bioconjug Chem 2000;11:842–54.[CrossRef][Medline]
  17. Wakabayashi T, Karbowski M. Structural changes of mitochondria related to apoptosis. Biol Signals Recept 2001;10:26–56.[CrossRef][Medline]
  18. Salvioli S, Ardizzoni A, Franceschi C, Cossarizza A. JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett 1997;411:77–82.[CrossRef][Medline]
  19. Sharkey RM, Goldenberg DM. Perspectives on cancer therapy with radiolabeled monoclonal antibodies. J Nucl Med 2005;46 Suppl 1:115–27S.
  20. Sharkey RM, Burton J, Goldenberg DM. Radioimmunotherapy of non-Hodgkin's lymphoma: a critical appraisal. Expert Rev Clin Immunol 2005;1:47–67.[CrossRef]
  21. Sharkey RM, Karacay H, Cardillo TM, et al. Improving the delivery of radionuclides for imaging and therapy of cancer using pretargeting methods. Clin Cancer Res 2005;11:7109–21s.[CrossRef]
  22. Chatal JF, Campion L, Kraeber-Bodere F, et al. Survival improvement in patients with medullary thyroid carcinoma who undergo pretargeted anti-carcinoembryonic-antigen radioimmunotherapy: a collaborative study with the French Endocrine Tumor Group. J Clin Oncol 2006;24:1705–11.[Abstract/Free Full Text]
  23. Shen S, Forero A, LoBuglio AF, et al. Patient-specific dosimetry of pretargeted radioimmunotherapy using CC49 fusion protein in patients with gastrointestinal malignancies. J Nucl Med 2005;46:642–51.[Abstract/Free Full Text]
  24. Lin Y, Pagel JM, Axworthy D, Pantelias A, Hedin N, Press OW. A genetically engineered anti-CD45 single-chain antibody-streptavidin fusion protein for pretargeted radioimmunotherapy of hematologic malignancies. Cancer Res 2006;66:3884–92.[Abstract/Free Full Text]
  25. Press OW, Corcoran M, Subbiah K, et al. A comparative evaluation of conventional and pretargeted radioimmunotherapy of CD20-expressing lymphoma xenografts. Blood 2001;98:2535–43.[Abstract/Free Full Text]
  26. Chang CH, Sharkey RM, Rossi EA, et al. Molecular advances in pretargeting radioimmunotherapy with bispecific antibodies. Mol Cancer Ther 2002;1:553–63.[Abstract/Free Full Text]




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