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Cancer Therapy: Preclinical |
Authors' Affiliations: 1 Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania; 2 Creative BioMolecules, Hopkinton, Massachusetts; 3 EMD Lexigen Research Center, Billerica, Massachusetts; 4 Boston Biomedical Research Institute, Watertown, Massachusetts; 5 Department of Anesthesiology, University of California, San Francisco, California; and 6 Chiron Corporation, Emeryville, California
Requests for reprints: Gregory P. Adams, Department of Medical Oncology, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111. Phone: 215-728-3890; Fax: 215-728-2741; E-mail: gp_adams{at}fccc.edu.
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Abstract |
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This investigation was based on our preparation of 741F8-1 sFv monomers, dimers, and bispecific heterodimers (4, 6) reactive with the tumor-associated antigen c-erbB-2/HER2/neu that is overexpressed in a number of cancers, including those of the breast (7–9), ovaries (10), and gastrointestinal tract (11–13). In mouse models, the small size of these molecules (Mr 
27,000 for monomers and Mr 54,000 for dimers) facilitates their rapid penetration into tumors (14) and their rapid elimination from blood and all tissues devoid of target antigen (4, 15, 16). We observed significantly greater tumor retention with radioiodinated 741F8-1 (sFv')2 dimer than with sFv monomer (13). We also report that the dimers exhibited better tumor retention than enzymatically produced 741F8 Fab fragments of similar size. However, the comparison to monovalent Fab fragments was an imperfect control because a Fab fragment has a rigid conformation comprising the Fv region fused to constant domains, which necessarily differ from the flexible back-to-back conjugation of two sFv regions within the (sFv')2. The present studies were designed to conclusively examine how size and valence affect the tumor targeting properties of engineered sFv monomers and dimers.
We previously developed the sFv with cysteinyl residues in its COOH-terminal tail, designated sFv' (4, 16, 17), and constructed divalent disulfide-bonded 741F8-1 (sFv')2 molecules, heterodimeric disulfide-bonded 741F8-1/26-10-1 (sFv')2 molecules (monovalent for HER2/neu), and control 26-10-1 (sFv')2 molecules, where the designations 741F8-1 and 26-10-1 refer to a COOH-terminal (Ser-Gly4-Cys) tail, with all molecules containing a 14-residue serine-rich linker (-Ser4-Gly-Ser4-Gly-Ser4) bridging the variable domains of the sFv' constructs (refs. 6, 17; Table 1). The Gly4-Cys tail used for making covalent back-to-back (sFv')2 dimers was also shown to be useful as the first genetically engineered chelation site for 99mTc. Gamma camera imaging studies were conducted on mice treated with the 741F8-1 sFv'-[99mTc] and displayed clear tumor localization at times as early as 6 hours following i.v. administration to the same mouse tumor model used in this investigation. The Gly4-Cys peptide formed a square planar chelate with oxotechnetium using a standard coordination kit with radiometal in the form that is routinely available in radiology departments (18). The 741F8-1 (sFv')2 molecule employed here was also conjugated to the 
-emitting radioisotope 111In and effectively employed to image established c-erbB-2-overexpressing human tumor xenografts growing in immunodeficient mice (19).
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Materials and Methods |
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The extracellular domain, with a COOH-terminal hexahistidine purification tag, was produced by mammalian cell culture and purified to homogeneity from the extracellular medium in two steps, by immobilized metal affinity chromatography and by size exclusion chromatography, as previously described (4).
Affinity and kinetic measurements. The binding and dissociation kinetics of extracellular domain association with 741F8-1 sFv', 741F8-1 (sFv')2, 26-10-1 (sFv')2, and 741F8-1/26-10-1 (sFv')2 were determined using a BIAcore surface plasmon resonance instrument (Pharmacia, Uppsala, Sweden) as described (22). Briefly, 1,400 RU of HER2/neu extracellular domain (25 µg/mL in 10 mmol/L sodium acetate, pH 4.5) were covalently bound through amino groups to a CM5 sensor chip. Association and dissociation of the sFv species (100-800 nmol/L) were measured under continuous flow (5 µL/min). The determination of kon was done by plotting (ln(dR / dt)) / t versus concentration whereas koff was determined from the dissociation part of the sensogram at the highest sFv concentration analyzed. Based on these kinetic measurements, the dissociation constant, Kd, and association constant, Ka, were calculated.
Radioiodinations. All samples were labeled with 125I using the chloramine-T method (4). The sFv or (sFv')2 (200 µg) was combined with 125I (17.4 mCi/µg; NEZ 033H, Dupont NEN, Wilmington, DE) at an iodine-to-protein molar ratio of 1:6 in a 12 x 75-mm plastic test tube. Ten microliters (1 mg/mL) of chloramine-T (Sigma, St. Louis, MO) per 100 µg of protein were added and the reaction was allowed to proceed for 3 minutes at room temperature. The reaction was quenched with the addition of 10 µL (1 mg/mL) of sodium metabisulfite (Sigma) per 100 µg of protein. The radiolabeled protein was immediately separated from remaining free radioiodine by the G-50-80 centrifuged column method (4). The specific activities of labeled products were 2 mCi/mg.
The quality of the radiolabeled sFv was evaluated by high-performance liquid chromatography, SDS-PAGE, and a live cell binding assay (4). High-performance liquid chromatography analysis was done using a Superdex-75 column (Pharmacia) on a Rainin Dynamax High-Performance Liquid Chromatography system. Eluted fractions (0.25 mL) were collected and counted on a gamma well counter (Beckman, Irvine, CA). High-performance liquid chromatography consistently showed that >99% of the eluted activity was associated with the protein peak. The radioiodinated sFv monomers and dimers were also evaluated by SDS-PAGE. Nonreduced forms were run on 12% gels (10 x 10 cm; Jule, New Haven, CT) and the migration of the proteins was detected by autoradiography (Kodak X-OMAT scientific imaging film with X-omatic regular intensifying screens; Rochester, NY). Of the nonreduced dimers, >95% migrated as Mr 54,000 bands whereas a similar percentage of the 741F8-1 sFv' monomer migrated as Mr 27,000 bands.
The immunoreactivity of the radioiodinated 741F8-based sFv species was determined in a live cell-binding assay using SK-OV-3 cells (HBT 77; American Type Culture Collection, Rockville, MD) that overexpress HER2/neu. Ten nanograms of labeled sFv in 100 µL of PBS (0.154 mol/L NaCl, 10 mmol/L phosphate, pH 7.2) were added in triplicate to 5 x 106 cells in 15-mL polypropylene centrifuge tubes. After incubating for 30 minutes at room temperature, the cells were washed with 2.0 mL of PBS and centrifuged for 5 minutes at 500 x g. The supernatants were separated from the pellets and both were counted in a gamma counter. The percentage of the activity associated with the cell pellets was usually >70% (>50% for the heterodimer in one assay). The radioiodinated 26-10-1 (sFv')2 did not bind significantly to the cells.
Cell-surface retention studies. To assess the effect of sFv valence on association with cell-bound HER2/neu, an in vitro cell-surface retention assay was done. In this assay, 460 ng of radioiodinated 741F8-1 sFv, 741F8-1 (sFv')2 homodimer, or 741F8/26-10 (sFv')2 heterodimer were incubated with 11.5 x 106 SK-OV-3 cells in a total volume of 4.6 mL of PBS (for a final concentration of 1.9 nmol/L for the sFv and 3.7 nmol/L for the homodimers and heterodimers) for 30 minutes on ice. These concentrations are approximately the Kd of the individual 741F8 sFv complexed to extracellular domain. The cells treated with the various sFv species were centrifuged at 500 x g for 5 minutes at 4°C, washed with 10 mL of ice-cold PBS twice, and then resuspended gently in 4.6 mL of PBS at 37°C. The cell suspensions were then incubated at 37°C in a rotating water bath. To decrease the rebinding of dissociated radioiodinated sFv species to the cells, the suspensions were pelleted at 500 x g for 5, 15, and 30 minutes after commencing the incubation; the supernatants were aspirated and the cells were gently resuspended in the same volume of fresh PBS at 37°C. Immediately after each round of pelleting and resuspension, 0.2-mL aliquots containing 5 x 105 cells were removed in triplicate (i.e., at 0, 5, 15, and 30 minutes) and immediately centrifuged at 500 x g for 5 minutes at 4°C. Cell pellets and supernatants were counted in a gamma well counter (Beckman) to determine the quantity of radiolabeled species retained on the cells over time.
Biodistribution studies. Animal studies were done in accordance with institutional animal welfare guidelines. Four- to six-week-old C.B17/Icr-scid mice were obtained from the Fox Chase Cancer Center Laboratory Animal Facility. SK-OV-3 cells (2.5 x 106) were implanted s.c. on the abdomens of the mice. Six to eight weeks later, when the tumors had achieved a size of 100 to 200 mg, Lugol's solution was added to the drinking water to block thyroid accumulation of radioiodine. The biodistribution studies were initiated 3 days later.
Radiolabeled sFv preparations were diluted in PBS to a concentration of 200 µg/mL and cohorts of five to six mice were given 100 µL of each radiopharmaceutical by tail vein injection. The injected dose was determined by counting each mouse immediately after injection on a Series 30 multichannel analyzer/probe system (Probe model 2007, Canaberra, Meridian, CT). Whole body counts and blood samples (retro-orbital) were obtained and the mice were sacrificed at 24 hours following the injections. The tumors and organs were removed, weighed, and counted in a gamma counter along with the blood samples and standards to determine the percent injected dose per gram of tissue (%ID/g; refs. 4, 23). The mean and SE for each group of data were calculated, tumor-to-organ ratios were determined, and the significance of the results was determined by Student's t test.
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Results |
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Discussion |
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The association constant of an antibody for its target antigen is a thermodynamic measure of the stability of the antigen-antibody complex. Finally, the avidity of the interaction is dependent on the intrinsic affinity of single binding sites, the number of binding sites present per molecule (valence), and the mobility or density of the target antigen on tumor cells as well as their shape (29). From the thermodynamic viewpoint, Jacqueline Reynolds has developed theoretical analyses for the different scenarios governing the interaction of monovalent or divalent antibody with mobile cell-surface antigens (29). Of particular importance for this study, it should be noted that a monovalent antibody fragment in solution has the same simple binding equation for association with a cell-bound monomeric antigen as it does if the monomeric antigen is in solution. Thus, a monovalent 741F8 sFv and corresponding monovalent 741F8/26-10 (sFv')2 heterodimer should have the same affinity for the monomeric HER2/neu antigen on an idealized cell surface. If one takes the BIAcore chip as a model for such a simplified system, these two species were experimentally observed to have the same binding affinity within experimental error. However, the surfaces of cells in tissue culture present a sufficiently nonideal system that this generalization starts to break down when measured in terms of cell-surface retention as a function of time (Fig. 2).
When a divalent antibody binds to a soluble, monomeric antigen, its first interaction has an effective microscopic binding constant, k1, which is 2Ka, where Ka is the association of the monovalent binding site. The second site binds with k2, which is 0.5Ka. When the divalent antibody species binds to monomeric antigen on a cell surface, the equations become quite complex and various assumptions must be made, including antigen density and the radius of curvature of the cell surface, which preclude a rigorous interpretation of the antigen-antibody association in tumor xenograft models. Analysis of tumor localization of a monovalent antibody species in serum may be considered from a simplified viewpoint, with the caveat that one is ignoring many complexities.
The three-dimensionality of the antigen matrix that constitutes tumor epitope space in a solid tumor imposes additional boundary conditions that lead to a reduced diffusion rate of antibody caused by the entrapment of antibody species within the interstices of cells that constitute a solid tumor. Furthermore, the slower diffusion out of solid tumors results in a markedly increased probability of rebinding if the antibody species is divalent. There are also physiologic factors that reduce the localization of divalent antibody species in solid tumors, including antigen density, the heterogeneity of antigen localization within the tumor, and intratumoral interstitial pressure (25, 30, 31). Overall, the binding of divalent antibody species within a solid tumor must be significantly higher than to the surface of cells dispersed in solution. Of course, the Law of Mass Action always governs binding phenomena; thus, increasing the sFv or (sFv')2 concentrations will increase their localization within tumors until all available antigenic sites are saturated or some other binding site effect becomes limiting (25, 30, 32).
We examined the tumor-targeting properties of three divalent (sFv')2 molecules with zero, one, or two binding sites specific for the targeted tumor antigen. As all three molecules were of the same covalent size, we could focus on differences in tumor retention that result from the number of specific binding sites present. In these studies, 3-fold greater tumor retention was achieved with the divalent 125I-741F8-1 (sFv')2 than with either the 741F8/26-10 heterodimer or the 741F8-1 sFv' monomer, both of which have monovalent binding sites for c-erbB-2/HER2/neu (3.6 %ID/g versus 1.13 and 1.25 %ID/g, respectively). Furthermore, observed differences in their tumor retention were not due to differential clearance and processing as the blood and normal organ retentions of the three species with molecular weights of Mr 54,000 [741F8-1 (sFv')2 homodimer, the 741F8/26-10 (sFv')2 heterodimer, and the 26-10 (sFv')2 control] were very similar. Clearly, increased tumor localization of the species that have monovalent binding sites for c-erbB-2 can be achieved by increasing the dose of antibody administered. However, increased tumor localization achieved in this manner would also be associated with increased localization to normal tissues (27). Our observations that the degree of tumor retention at 24 hours following administration correlated with valence, rather than with molecular weight, represents the first fully controlled analysis of how binding site valence directly affects the sFv targeting and tumor localization.
When examined by BIAcore surface plasmon resonance, the binding of the different anti-c-erbB-2/HER2/neu species to immobilized HER2/neu extracellular domain paralleled the in vivo setting. The divalent 741F8-1 (sFv')2 bound with the greatest affinity, Ka,app = 1.09 x 108 (mol/L)–1 (Kd = 9.2 x 10–9); the monovalent species bound with very similar affinities, 741F8-1 sFv' Ka,app = 0.2 x 108 (mol/L)–1 and 741F8/26-10 heterodimer Ka,app = 0.3 x 108 (mol/L)–1 (Kd = 4.8 x 10–8 and 3.5 x 10–8, respectively); whereas the 26-10 sFv' and 26-10 (sFv')2 negative controls failed to bind to the extracellular domain in BIAcore experiments. The greater effective affinity of the divalent 741F8-1 (sFv')2, compared with the monovalent binding of sFv and heterodimer, results from avidity effects rather than improved intrinsic affinity of a single binding site, but suggests contributions from both sites would be consistent with the observed level of binding enhancement. In contrast with the BIAcore results, the in vitro cell-surface retention study revealed similar retention patterns for the homodimeric 741F8-1 (sFv')2 and heterodimeric 741F8/26-10 (sFv')2, both greater than 741F8-1 sFv monomer. These results are consistent with our previously published observations on the production of monomeric and dimeric sFv species (4, 6, 33). When examined in solution at a low concentration, 741F8-1 (sFv')2 was capable of binding a maximum of two HER2/neu extracellular domain molecules, and each 741F8/26-10 homodimer and 741F8-1 sFv' monomer bound a maximum of one HER2/neu extracellular domain. Size exclusion chromatography at high concentrations of the 741F8-26-10 (sFv')2 heterodimer formed tetramers, each capable of binding to two HER2/neu extracellular domain molecules. Our in vivo studies were conducted well below the threshold for dimerization; the 20 µg i.v. injection of 741F8/26-10 (sFv')2 was diluted into an 2-mL blood volume of a mouse, which rapidly resulted in an uppper limit blood concentration of 200 nmol/L. Whereas we have observed very similar tumor retention for 741F8-1 sFv' and monovalent 741F8/26-10 heterodimer, both decrease beneath the renal threshold and are cleared very rapidly from circulation. This reinforces the conclusion that the heterodimer did not form tetramers in vivo and could only associate with one HER2/neu molecule on the tumor cell surface.
The importance of the rapid elimination of the molecules in evaluating the effect of avidity on tumor retention is illustrated by a similar study done by Van Dijk et al. (30) in which divalent and bispecific F(ab')2, which exceeds the renal threshold, and monovalent Fab, which is beneath the renal threshold, were compared in tumor-bearing mice. In their study, the divalent and bispecific F(ab')2 species had similar tumor retentions at 6, 24, and 48 hours postadministration whereas the monovalent Fab consistently exhibited 3- to 5-fold lower accumulation. These results support our findings that binding avidity has a substantial effect on the tumor retention of small rapidly cleared antibody-based molecules, and it is a factor that can now be considered to be distinct from size below the renal threshold as well as a function of molecular size and clearance rate. It is likely that through the use of flexible, divalent, high-affinity engineered antibody species, such as the (sFv')2, the therapeutic potential of this class of sFv-based molecules may be realized.
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Acknowledgments |
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: M-S. Tai is currently at Strykr Biotech, Hopkinton, MA; J.E. McCartney is currently at Point Therapeutics, Inc., Boston, MA; L.L. Houston is currently at Whimbrel, Inc., Bainbridge Island, WA; and J.S. Huston is currently at EMD Lexigen Research Center, Billerica, MA.
7 M-S. Tai, H. Oppermann, J.S. Huston, unpublished results. 
8 W.F. Stafford, unpublished results.
Received 10/10/05; revised 12/20/05; accepted 12/22/05.
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), 741F8/26-10 (sFv')2 (
), and 741F8-1 sFv (
) on the surface of SK-OV-3 cells overexpressing c-erbB-2/HER2/neu. Assays were done as described in the text. Points, mean of three samples; bars, SE.
), 741F8-1 sFv (
), or 26-10 (sFv')2 (
). The %ID/g retained in tumor was calculated as described in the text. Columns, mean values; bars, SE.