Simultaneous Blockade of Both the Epidermal Growth Factor Receptor and the Insulin-like Growth Factor Receptor Signaling Pathways in Cancer Cells with a Fully Human Recombinant Bispecific Antibody*

Both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR) have been implicated in the tumorigenesis of a variety of human cancers. Effective tumor inhibition has been achieved both experimentally and clinically with a number of strategies that antagonize either receptor activity. Here we constructed and produced two fully human recombinant bispecific antibodies (BsAb) that target both EGFR and IGFR, using two neutralizing human antibodies originally isolated from a phage display library. The BsAb not only retained the antigen binding capacity of each of the parent antibodies, but also were capable of binding to both targets simultaneously as demonstrated by a cross-linking enzyme-linked immunosorbent assay. Furthermore, the BsAb effectively blocked both ligands, EGF and IGF, from binding to their respective receptors, and inhibited tumor cell proliferation as potently as a combination of both the parent antibodies. More importantly, the BsAb were able to completely block activation of several major signal transduction molecules, including Akt and p44/p42 MAP kinases, by both EGF and IGF, whereas each individual parent antibody was only effective in inhibiting those signal molecules activated by the relevant single growth factor. The BsAb molecules retained good antigen binding activity after incubation with mouse serum at 37 °C for up to 6 days. Taken together, our results underscore the benefits of simultaneous targeting multiple growth factor receptor pathways for more efficacious cancer treatment. This report describes the first time use of a recombinant BsAb for targeting two tumor-associated molecules on either a single or adjacent tumor cells for enhanced antitumor activity.

One of the hallmarks in effective cancer treatment is the use of combinational therapeutic regimens comprising several cytotoxic agents, e.g. various chemotherapeutics and radiations, that target cancer cells via different mechanisms. Unfortunately, the difference between malignant and normal cells in regards to their sensitivity to these cytotoxic therapies is not sufficient to allow potentially curative doses of chemotherapeutic agents or radiations to be administered without unacceptable toxicity to normal cells, the administration of therapeutic doses of these cytotoxic agents during the treatment also kills or damages normal rapidly proliferating cells such as hematopoietic cells, hair follicles, and lining epithelium of the gastrointestinal tract. In this regard, monoclonal antibody (mAb) 1based therapeutics represent a promising new class of anticancer agents because of their exclusive specificity toward defined antigens (1,2). On the other hand, because of their limited intrinsic cytotoxic activity, antitumor antibodies are most therapeutically efficacious when used either in combination with conventional chemotherapy regimens, e.g. Rituxan® plus CHOP in non-Hodgkins lymphoma (3) and Herceptin® plus Taxol in metastatic breast cancer (4), or as conjugates to other cytotoxic moieties, such as Zevalin® (a yttrium 90-labeled anti-CD20 mAb) (5) and Bexxar® (an iodine 131-labeled anti-CD20 mAb) (6) in non-Hodgkins lymphoma, and Mylo-targ® (an anti-CD33 antibody linked to calicheamicin) in acute myeloid leukemia (7). The dose limiting toxicity of these combined, or conjugate therapies are usually associated with the cytotoxic components in the regimens. Based on these observations, it is plausible that the combination of antitumor antibodies directed against different tumor-associated targets may yield enhanced therapeutic activity without adding severe unwanted toxicities. Clinical application of combinational antibody therapy is, however, greatly hindered by a number of factors, including limited availability of antibody products, high cost of each product, and the FDA-associated regulatory issues (e.g. every antibody in the combination as well as the combination regimen itself may require a separate review and approval by the agency). To this end, the development of bispecific or multispecific antibodies that target two or more tumorassociated antigens simultaneously may offer a novel and promising solution.
Both epidermal growth factor receptor (EGFR) and insulin-* 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.
§ Both authors contributed equally to this study. ‡ ‡ To whom correspondence should be addressed: Dept. of Antibody Technology, ImClone Systems Inc., 180 Varick St., New York, NY 10014. Tel.: 646-638-5190; Fax: 212-645-2054; E-mail: Zhenping@ imclone.com. 1 The abbreviations used are: mAb, monoclonal antibody; BsAb, bispecific antibodies; CH1 and CL, the first constant domain of the antibody heavy chain and the constant domain of the antibody light chain, respectively; ECD, extracellular domain; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; IGF, insulin-like growth factor; IGFR, insulin-like growth factor receptor; MAPK, mitogen-activated protein kinase; scFv, single chain Fv; VEGFR, vascular endothelial growth factor receptor; VH and VL, the variable domains of antibody heavy and light chains, respectively; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; FACS, fluorescence-activated cell sorter; bis-Tris, 2- like growth factor receptor (IGFR) have been implicated in the tumorigenesis of a variety of human cancers (8 -13). Targeted inhibition of EGFR with mAb or small molecular kinase inhibitors has shown good anticancer activity in a number of animal models as well as in various clinical studies (for reviews, see Refs. 14 -21). For example, Erbitux TM (cetuximab, previously known as IMC-C225), an anti-EGFR mAb, has been proven to be effective in chemorefractory colorectal cancer patients in two independent phase II studies (22,23), and Iressa® (ZD1839), a small molecular EGFR kinase inhibitor, was approved in 2003 by the FDA for treatment of patients with non-small cell lung carcinoma (24). Similarly, significant tumor inhibition has also been achieved in animal models with several IGFR targeting strategies including antisense oligonucleotides (25), dominatenegative receptor mutants (26), and neutralizing mAb (27,28) (for review, see Ref. 29). A recent study demonstrated that tumor cells may gain resistance to anti-EGFR therapies without altering EGFR expression, but rather through up-regulation and activation of other proliferative and/or anti-apoptotic activities, e.g. IGFR and downstream signal transduction through the phosphatidylinositol 3-kinase/Akt pathway (30). Taken together, these observations suggest that a combinational regimen targeting both EGFR and other growth factor receptors, such as IGFR, simultaneously may yield greater anticancer activity than those approaches that address only a single receptor.
In past years, both laboratory and early clinical studies have demonstrated that BsAb may have significant potential application in cancer therapy by targeting tumor cells with cytotoxic agents including effector cells, radionuclides, drugs, and toxins (31)(32)(33). Here we explored a new concept of utilizing BsAb by constructing a novel antibody molecule that targets two relevant tumor targets, i.e. growth factor receptors, thus blocking simultaneously multiple receptor activation and downstream signal transduction pathways. Using two neutralizing antibodies, one directed against EGFR and the other against IGFR, as the "building blocks" we constructed and produced two different versions of an IgG-like tetravalent BsAb. The BsAb molecules bound to both EGFR and IGFR, and blocked the receptors from interacting with their respective ligands, as efficient as their parent monospecific IgG antibodies. Furthermore, whereas individual monospecific mAb was only able to inhibit single growth factor-stimulated receptor activation, the BsAb blocked both EGF and IGF stimulated activation of the receptors as well as the receptor-associated downstream signal transduction molecules.

EXPERIMENTAL PROCEDURES
Cell Lines and Proteins-Human tumor cell lines, DiFi and HT29 (colorectal carcinoma), MCF7 (breast carcinoma), A431 (epidermoid carcinoma), and BxPC3 (pancreatic carcinoma) were obtained from ATCC and maintained in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal calf serum (HyClone, Logan, UT) at 37°C in 5% CO 2 . Recombinant extracellular domain (ECD) of IGFR1 and its ligand, IGF-I, were purchased from R&D Systems Inc. (Minneapolis, MN). Recombinant EGFR ECD, and IMC-1121, a fully human antibody directed against vascular endothelial growth factor receptor 2 (VEGFR2) that does not cross-react with EGFR and IGFR, were produced at ImClone Systems Inc. Both 125 I-IGF-I and 125 I-EGF were purchased from Amersham Biosciences.
Generation of Fully Human Antibodies to IGFR and EGFR-Recombinant human IGFR1 ECD and A431 tumor cells were used to screen a human naïve phage display Fab library containing 3.7 ϫ 10 10 unique clones (34) following protocols previously described (35). 11F8, an anti-EGFR clone identified after 3 rounds of selection on A431 cells, binds to both recombinant and cell surface-expressed EGFR with high affinity and neutralizes EGF-stimulated receptor activation and cell proliferation. 2 Initial selection on IGFR1 ECD yielded a clone, 2F8, with modest binding affinity and neutralizing activity. Affinity maturation of 2F8 via a chain-shuffling approach (36) led to the identification of A12, a clone with significantly improved binding affinity and neutralizing activity (37). To produce full-length IgG antibodies, IMC-11F8 and IMC-A12, the DNA sequences encoding the heavy and light chain variable genes of 11F8 and A12 were amplified by PCR and cloned into an expression vector containing the human IgG1 constant domains (the glutamine synthetase expression system from Lonza Biologics Inc.). The expression vector was stably transfected into myeloma NS0 cells (38), followed by antibody production in serum-free media and purification via Protein A affinity chromatography.
Construction and Production of the Bispecific Anti-EGFR x Anti-IGFR Antibodies-Single chain Fv (scFv) molecules of both 11F8 and A12 were first constructed following a previously described protocol (39). These two scFv were then used as the building blocks to construct the bispecific anti-EGFR x anti-IGFR antibodies in the Bs(scFv)4-IgG format we previously described (40). Two different versions of the BsAb were constructed: in one version (BsALFH), the scFv encoding A12 was fused to the N terminus of the constant domain of the light chain (CL) and the scFv encoding 11F8 was linked to the N terminus of the first constant domain of the heavy chain (CH1), whereas in the other version (BsFLAH), the alternate orientation was used (for illustration see Fig.  1). Both genes encoding the scFv-CL and scFv-CH1CH2CH3 fusions were subcloned into the expression vector and expressed in NS0 cells, followed by antibody purification with Protein A chromatography. The purity of the BsAb was assayed via SDS-PAGE analysis under both reducing (Nupage 4 -12% bis-Tris gel, Invitrogen) and non-reducing (4 -20% Tris glycine gel, Invitrogen) conditions. The solution behavior of the BsAb preparations was examined via size exclusion chromatography as previously described (41). Briefly, the purified BsAb was applied to a Bio-Sep 3000 column (Phenomenex, Torrance, CA) linked to a high performance liquid chromatography system with UV and refractive index detectors (Agilent 1100, Agilent, Palo Alto, CA), and followed by a Mini-Dawn LS (Wyatt Technology, Santa Barbara, CA). The column was equilibrated in PBS (pH 7.0) and run at a flow rate of 0.5 ml/min.
Receptor Binding Assays-Two different assays were carried out to examine the binding specificity and efficiency of the BsAb. In the first assay, the cross-linking assay, the BsAb was tested for their capability in simultaneously binding two target antigens: the BsAb or the monospecific antibodies (5 nM) were first incubated with a biotin-labeled IGFR (100 ng) in solution and then transferred to a microtiter plate coated with EGFR (100 ng/well), followed by incubation with streptavidin-HRP to measure the plate-bound biotin activity. In the second assay, the direct binding assay, various amounts of antibodies were added to triplicate wells of 96-well plates (Nunc, Roskilde, Denmark) pre-coated with human IGFR1 or EGFR ECD (100 ng/well) and incubated at room temperature for 1 h, after which the plates were washed 3 times with PBS containing 0.1% Tween 20. The plates were then incubated at room temperature for 1 h with 100 l of a rabbit antihuman IgG Fc-HRP conjugate (Jackson ImmunoResearch Laboratory Inc., West Grove, PA). The plates were washed and developed following a procedure previously described (35,39).
Cell-based Competitive Blocking Assay-A431 or MCF-7 cells were seeded into 24-well plates and cultured overnight. The subconfluent cell monolayers were washed 3 times with binding buffer (Iscove's medium containing 0.1% bovine serum albumin) followed by incubation with various amounts of antibodies on ice for 15 min. 125 I-EGF or 125 I-IGF (40 pM) were added to each well and incubated for an additional 3 h with gentle agitation. After washed three times with ice-cold PBS, 0.1% bovine serum albumin, the cells were lysed with 200 l of 0.5 N NaOH and radioactivity was counted in a ␥-counter.
FACS Analysis-DiFi, MCF-7, BxPC3, A431, and HT29 cells were incubated with various antibodies (10 g/ml) at 4°C for 1 h, followed by incubation with an anti-human Fc antibody-fluorescein isothiocyanate conjugate (BIOSOURCE Int., Camarillo, CA) for an additional 1 h at 4°C. After several washes with cold PBS the cells were analyzed by a flow cytometer (model EPICS® ELITE, Coulter Corp., Edison, NJ).
Cell Proliferation Assays-1 ϫ 10 4 DiFi or BxPC3 cells in 100 l of complete medium were seeded in each well of 96-well plates and cultured overnight. Various amounts of the antibodies were added in triplicate wells and allowed to culture for 4 days, after which 10 l of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (5 mg/ml, Sigma) was added to each well and incubated for an additional 4 h. The plates were washed twice with PBS and incubated with 100 l of HCl/isopropyl alcohol (40 mM) at room temperature for 10 min, followed by optical density reading at 570 nm. 2 M. Liu, D. Hicklin, and Z. Zhu, manuscript in preparation.

FIG. 2. Bispecific and dose-dependent binding of the BsAb to EGFR and IGFR.
A, receptor cross-linking assay. Various antibody preparations were first incubated with a biotin-labeled IGFR in solution and then transferred to a microtiter plate coated with EGFR, followed by incubation with streptavidin-HRP to measure the platebound biotin activity. B and C, dose-dependent binding to immobilized EGFR and IGFR by the BsAb. Various amounts of antibodies were added to 96-well plates coated with human EGFR (B) or IGFR ECD (C) and incubated at room temperature for 1 h, after which the plates were washed 3 times with PBS containing 0.1% Tween 20. The plates were then incubated at room temperature for 1 h with a rabbit anti-human IgG Fc-HRP conjugate. The plates were washed, peroxidase substrate was added, and A 450 nm was read. Data shown represent the mean Ϯ S.D. of triplicate samples.
Western Blotting Analysis-Tumor cells were plated onto 75-mm dishes and grown to 70 -80% confluence, after which the cells were washed twice in PBS and cultured overnight in serum-free medium. The cells were first incubated with various antibodies at 37°C for 30 min, followed by stimulation with EGF, IGF, or both at 37°C for 20 min. The cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM Na 3 VO 4, 1 g/ml leupeptin, 1 g/ml pepstatin, and 1 g/ml aprotinin), followed by centrifugation of the lysate at 12,000 rpm for 10 min at 4°C. Both EGFR and IGFR1 were immunoprecipitated from the cell lysate supernatant by using a mixture of anti-EGFR and anti-IGFR antibodies, followed by addition of 20 l of Protein A/G-Sepharose beads (Santa Cruz Biotechnology, Santa Cruz, CA). The precipitated receptor proteins were resolved on a 4 -12% Nupage bis-Tris gel (Invitrogen) and transferred to a polyvinylidene difluoride membrane. Phospho-EGFR and phosphor-IGFR were detected on the blot using an anti-phosphotyrosine antibody-HRP conjugate (Santa Cruz Biotechnology). Total receptor proteins loaded on the gel were assayed with a mixture of an anti-EGFR and anti-IGFR antibody (Santa Cruz Biotechnology). For phosphorylation of Akt and p44/p42 MAPK, whole cell lysate was resolved by SDS-PAGE using a 10% acrylamide gel, and the phospho-Akt and phospho-p44/p42 were detected with an antibody mixture containing anti-phospho-Akt and antiphospho-p44/p42 antibodies (Cell Signaling), followed by an anti-mouse antibody-HRP conjugate. Total Akt and p44/p42 proteins were assayed with a mixture of an anti-Akt (Santa Cruz Biotechnology) and an anti-p44/p42 antibody (Cell Signaling). All signals were visualized with the ECL reagent (Amersham Biosciences).
Stability of the Antibodies in Mouse Serum-Various antibody preparations were added to 10% freshly isolated mouse serum (in PBS) and incubated at room temperature or 37°C. Aliquots of samples were removed at predefined intervals of incubation and assayed for efficiency for binding to both EGFR and IGFR using the enzyme-linked immunosorbent assay described above.

Construction and Production of the Anti-EGFR x Anti-IGFR
BsAb-Two scFv molecules, the anti-EGFR, 11F8 scFv, and the anti-IGFR, A12 scFv, were used as the building blocks to construct an IgG-like tetravalent BsAb. Two different versions of the BsAb were produced (Fig. 1A): in one construct (BsALFH), the A12 scFv was linked to the N terminus of CL (A12 scFv-CL) and the 11F8 scFv was linked to the N terminus of CH1 of an IgG1 molecule (11F8 scFv-CH1CH2CH3); whereas in the other construct (BsFLAH), the 11F8 scFv-CL and the A12 scFv-CH1CH2CH3 orientation was used (see Fig. 1 for details). Co-expression in mammalian cells of A12 scFv-CL along with 11F8 scFv-CH1CH2CH3, or A12 scFv-CH1CH2CH3 with 11F8 scFv-CL, resulted in an IgG-like tetravalent molecule with two binding specificities (Fig. 1). Both BsAb were produced by stably transfected NS0 cells in serum-free conditions and purified from the cell culture supernatant via a Protein A affinity column. Electrophoresis analysis of BsALFH under non-reducing conditions yielded a single protein band with molecular weight of ϳ200,000 (Fig. 1B, lane 3), the expected molecular mass of the tetravalent BsAb. Under the same conditions, BsFLAH gave rise to two bands: one major band at ϳ200 kDa (representing the properly assembled BsAb), and one minor band at ϳ100 kDa, suggesting the existence of A12 scFv-CH1CH2CH3 homodimer (without association with the 11F8 scFv-CL chain) (Fig. 1B, lane 4). As controls, both monospecific IgG, IMC-A12 and IMC-11F8, gave one major band with the expected mobility of ϳ150 kDa (Fig. 1B, lane 2 and 5, respectively). Under reducing conditions, both BsAb yielded two major bands, one represents the scFv-CH1CH2CH3 fusion (ϳ62.5 kDa) and the other the scFv-CL fusion (ϳ37.5 kDa) (Fig. 1C, lanes 3 and 4). As expected, both IMC-A12 and IMC-11F8 showed two major bands: the IgG heavy chain (ϳ50 kDa) and IgG light chain (ϳ25 kDa) (Fig. 1C, lanes 2 and 5, respectively). Under size exclusion chromatography, both BsAb preparations yielded a single major peak (Ͼ90%) with estimated molecular weight of ϳ200,000 (not shown), indicating that the majority of the proteins exist in solution as the expected tetravalent BsAb monomer.
The BsAb Binds to Both EGFR and IGFR-A number of assays were used to confirm that the BsAb molecules were capable of binding to both EGFR and IGFR. In the first assay, the cross-linking assay, we examined whether the BsAb could bind to both its targets simultaneously. The antibodies were first incubated with a biotin-labeled IGFR in solution and then transferred to a 96-well plate coated with EGFR, followed by incubation with streptavidin-HRP to measure the plate-bound biotin activity, i.e. the amount of IGFR that was cross-linked to the immobilized EGFR by the BsAb. As shown in Fig. 2A, both BsAb molecules, but not the monospecific IMC-A12 or IMC-11F8, were able to cross-link IGFR in solution with the immobilized EGFR, as demonstrated by the plate-associated biotin activity.
In the second assay, the BsAb were compared with their monospecific counterparts in antigen binding efficiency. Various amounts of antibodies were added to 96-well plates coated with EGFR or IGFR ECD and assayed for their efficiency in binding to the receptors. IMC-A12 and IMC-11F8 bound only to their respective targets, whereas the BsAb reacted to both immobilized EGFR and IGFR with similar efficiencies to their monospecific counterparts (Fig. 2, B and C). The ED 50 values, i.e. the antibody concentrations that yield 50% of maximum binding, to EGFR were 0.05 nM for IMC-11F8 and 0.1 nM for both BsALFH and BsFLAH, and to IGFR were 0.1 nM for IMC-A12 and 0.25 nM for both BsALFH and BsFLAH.
Finally, the BsAb were examined by FACS analysis for binding to tumor cell surface-expressed receptors. A431, HT-29, and BxPC3 express almost equal levels of EGFR and IGFR as demonstrated by fluorescence intensity when stained by IMC-11F8 and IMC-A12 (Fig. 3). On the other hand, DiFi cells express significantly higher levels of EGFR, whereas MCF-7 cells have significantly higher IGFR expression (Fig. 3). Both BsAb bound to all tumor cells with higher efficiency (as demonstrated by the mean fluorescence intensity) than did each individual antibody (except for IMC-A12 to MCF-7 cells), indicating additive binding to both EGFR and IGFR on the cell surface by the BsAb molecules (Fig. 3).
The BsAb Blocks Both IGF and EGF from Binding to Its Receptor-The BsAb was compared with their monospecific counterparts for efficacy in blocking ligand/receptor interaction. As shown in Fig. 4, whereas IMC-11F8 and IMC-A12 effectively blocked individual ligand, EGF and IGF, respectively, from binding to its receptor on tumor cell surface, the BsAb were able to compete with both EGF and IGF for binding to the receptors. The IC 50 values, i.e. the antibody concentrations required to inhibit 50% of ligand binding, were ϳ1.5 nM for IMC-11F8, 12 nM for BsALFH, and 20 nM for BsFLAH in EGFR binding (Fig. 4A), and 2 nM for IMC-A12, 40 nM for BsALFH, and 25 nM for BsFLAH in IGFR binding (Fig. 4B). As positive controls, the unlabeled ligands, EGF and IGF, competed efficiently with its radiolabeled counterpart, with IC 50 of ϳ10 nM for both EGF and IGF (Fig. 4).
Inhibition of Tumor Cell Proliferation in Vitro by the BsAb-We next examined the efficacy of the BsAb in inhibiting tumor cell proliferation in vitro in comparison to their monospecific counterparts. Two tumor cell lines were used in this study: DiFi cells that express significantly higher levels of EGFR, and BxPC3 cells that express high levels of both EGFR and IGFR. The anti-EGFR IMC-11F8 significantly inhibited the proliferation of DiFi cells, whereas the anti-IGFR IMC-A12, as well as the control antibody (the anti-VEGFR2 IMC-1211), had no effect on tumor cell growth (Fig. 5). Simple combinations of both IMC-11F8 and IMC-A12 yielded similar activity to that of IMC-11F8 alone. Both BsAb molecules demonstrated good anti-proliferative activity: BsALFH was equally potent to the combination of IMC-11F8 and IMC-A12 (both treatments were slightly more active than IMC-11F8), whereas BsFLAH was ϳ5-fold less potent than IMC-11F8. The IC 50 values, i.e. the antibody concentrations required for 50% tumor growth inhibition, were 1.8 nM for IMC-11F8, 1.2 nM for IMC-11F8 plus IMC-A12, 1.2 nM for BsALFH, and 10 nM for BsFLAH.
BxPC3 cells, which express high levels of both EGFR and IGFR, were much less sensitive to anti-EGFR therapy: incubation with IMC-11F8 alone, or with IMC-A12 alone, only resulted in ϳ25 to 35% of cell growth inhibition (Fig. 5B). Combination of both IMC-11F8 and IMC-A12 demonstrated significantly enhanced inhibitory activity (ϳ80 -85% growth inhibition, p Ͻ 0.05 compared with either IMC-11F8 or IMC- FIG. 5. Anti-proliferative activity of the BsAb and its parent mAb preparations. DiFi or BxPC3 cells in complete medium were seeded in 96-well plates and cultured overnight. Various amounts of antibodies were added into the culture and incubated with the cells for 4 days, after which 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added and incubated for additional 4 h. The plates were washed twice with PBS and incubated with HCl/isopropyl alcohol at room temperature for 10 min, followed by optical density reading at 570 nm. Data shown represent the mean Ϯ S.D. of triplicate samples. A12). Both BsAb were stronger inhibitors to BxPC3 cell proliferation (to ϳ50 -60% inhibition) than each of their monospecific counterparts, although they were not as effective as the combination of both antibodies (Fig. 5B).
The BsAb Block Signal Transduction Pathways Stimulated by Both EGF and IGF-The BsAb was examined on their efficacy in blocking EGF-and IGF-stimulated receptor phosphorylation and downstream signal transduction. Whereas incubation of MCF-7 cells with individual growth factors, EGF or IGF, resulted in significant levels of phosphorylation of the respective receptor, combination of both EGF and IGF yielded activation of both EGFR and IGFR (Fig. 6, lane 2-4). As expected, when the tumor cells were stimulated with both EGF and IGF, treatment with either IMC-11F8 or IMC-A12 only inhibited phosphorylation of the individual receptor (Fig. 6,  lanes 7 and 8). On the other hand, combination of both antibodies, either as a mixture (Fig. 6, lane 9) or as BsAb (Fig. 6,  lanes 5 and 6), significantly blocked activation of both receptors.
The effect of IMC-11F8, IMC-A12, and BsAb on the two major downstream signal transduction molecules associated with both EGFR and IGFR, Akt and p44/p42 MAP kinases, were also studied in MCF-7 cells (Fig. 6). Stimulation with IGF resulted in strong phosphorylation (ϳ3-fold increase) of Akt, and, to a much lesser extent, of p44/p42 MAPK (Fig. 6, lane 2), whereas EGF caused significant phosphorylation of both Akt and p44/p42 MAPK (Fig. 6, lane 3). Addition of IGF to EGF did not further increase Akt and p44/p42 MAPK phosphorylation (Fig. 6, lane 4). In the presence of both EGF and IGF, IMC-11F8 completely inhibited the activation of MAPK but only moderately reduced the activation of Akt (Fig. 6, lane 8), and interestingly, IMC-A12, whereas also moderately reduced Akt phosphorylation, failed to abolish the activation of p44/p42 MAPK (Fig. 6, lane 7). Similar to the status of receptors, combination of both IMC-11F8 and IMC-A12, either as a mixture or as BsAb, almost completely abolished phosphorylation of both Akt and p44/p42 MAPK induced by EGF and IGF (Fig.  6, lanes 5, 6, and 9).
The dual-receptor neutralizing activity of the BsAb was further confirmed on two other tumor cell lines, HT29 and BxPC3. Similar to the observation in MCF-7 cells, treatment with both IMC-11F8 and IMC-A12, either as a mixture or BsAb, significantly neutralized EGF/IGF-induced activation of both EGFR and IGFR, whereas each individual antibody only inhibited phosphorylation of its respective receptor (not shown).
The BsAb Retained Good Reactivity after Incubation in Mouse Serum-The stability of the BsAb molecules was examined after incubation in the presence of 10% mouse serum at 37°C for up to 6 days. The monospecific antibodies, IMC-11F8 and IMC-A12, retained full binding activity to their respective target antigen after incubation at 37°C for 6 days (Fig. 7, A and  D, respectively). Both BsAb molecules also demonstrated good stability. Among the two BsAb, BsALFH showed a moderately better stability than BsFLAH: the former retained Ͼ90% activity to EGFR (Fig. 7B) and ϳ53% activity to IGFR (Fig. 7E), compared with that of 58% to EGFR (Fig. 7C) and 50% to IGFR (Fig. 7F) for BsFLAH at the end of the 6-day incubation in mouse serum. DISCUSSION Both EGFR and IGFR have been implicated in tumorigenesis of a variety of human cancers, thus constituting excellent tar-gets for effective cancer intervention. Effective tumor inhibition has been achieved both experimentally and clinically with a number of strategies that antagonize either receptor activity. In this study, we hypothesize that a combinational regimen targeting both EGFR and IGFR simultaneously may yield greater antitumor activity than those approaches that address only a single receptor. To test this hypothesis, we constructed and produced a BsAb using two neutralizing mAb as the building blocks (one directed against EGFR and the other against IGFR), and examined the biological activities of the BsAb using a number of in vitro assays. Several novel observations are noteworthy in this report: first, BsAb was capable of binding to both EGFR and IGFR simultaneously and blocking the receptors from interacting to their respective ligands . In contrast to their monospecific counterparts that only inhibited single ligand-induced receptor activation, the BsAb effectively, as potent as the combination of the anti-EGFR (IMC-11F8) and the anti-IGFR (IMC-A12) antibodies, neutralized both EGFand IGF-stimulated receptor activation and downstream signal transduction molecules including Akt and p44/p42 MAPK (Fig.  6). These results demonstrated for the first time the use of a recombinant BsAb that targets two tumor-associated molecules on either single or adjacent tumor cells.
The second noteworthy observation of this study is that we demonstrated that simultaneous blockade of both EGFR and IGFR activation by a BsAb led to an enhanced antitumor activity. Tumor cells may gain their growth advantage and/or resistance to apoptosis by (over)expressing a number of growth factor receptors including EGFR and IGFR (8 -13). Binding of the ligands/growth factors, e.g. EGF and transforming growth factor ␣ to EGFR and IGF to IGFR, on the tumor cell surface leads to activation of these receptors and major downstream signal transduction molecules, including p44/p42 MAPK and phosphatidylinositol 3-kinase/Akt pathways, resulting in cell proliferation, invasion, and increased resistance to apoptosis FIG. 7. Stability of the BsAb incubated at 37°C in mouse serum. Various antibody preparations were added to 10% mouse serum (in PBS) and incubated at 37°C. Aliquots of samples were removed at predefined intervals of incubation and assayed for efficiency for binding to both EGFR (panels A-C) and IGFR (panels D-F) using an enzyme-linked immunosorbent assay. A, IMC-11F8; D, IMC-A12; B and E, BsALFH; C and F, BsFLAH. (8 -13). There is considerable debate on whether the expression level of any single growth factor receptor in tumor cells correlates with the response to individual anti-receptor therapy. A number of recent studies suggested that, however, at least in the case of EGFR, a simple measurement of tumor EGFR expression may not be adequate in predicting the outcome of the anti-EGFR therapy both in vitro and in patients (22,23,30). In certain tumor cells, inhibition of EGFR function could be effectively compensated by up-regulation of other growth factor receptor (e.g. IGFR) signaling pathways. For example, stimulation of IGFR has been shown to activate the phosphatidylinositol 3-kinase/Akt signal transduction pathway, leading to increased cell proliferation and resistance to apoptosis (10,42). To support this notion, a recent study has shown that malignant glioma cell lines expressing equivalent EGFR had significantly different sensitivity to EGFR inhibition depending on their capability in activating IGFR and its downstream signal transduction molecules (30). Other studies have also demonstrated that overexpression and/or activation of IGFR in tumor cells might contribute to their resistance to chemotherapeutic agents, radiations, and antibody therapy (43)(44)(45)(46), and consequently, inhibition of IGFR signaling has resulted in increased sensitivity of tumor cells to these therapeutic agents (47,48). Taken together, these observations suggest that a combinational regimen targeting both EGFR and IGFR simultaneously may yield greater anticancer activity than does individual anti-EGFR or anti-IGFR therapy. Here we showed that in MCF-7 cells, IGFR activation resulted in strong phosphorylation of Akt (and to much less extent, phosphorylation of p44/p42), whereas EGFR stimulation led to significant phosphorylation of both Akt and p44/p42. Incubation with the anti-EGFR x anti-IGFR BsAb almost completely abolished, as efficiently as the combination of IMC-11F8 and IMC-A12, the activation of not only EGFR and IGFR, but also the downstream signal transduction molecules, including both Akt and p44/p42, stimulated by EGF and IGF. In contrast, treatment with IMC-A12 or IMC-11F8 alone failed to block completely the phosphorylation of Akt and p44/p42. Furthermore, the BsAb were also more potent than each individual antibody in inhibiting proliferation of BxPC3 cells overexpressing both EGFR and IGFR. It is possible that the therapeutic responses to the BsAb or antibody combinations may vary among different tumor cells, depending on the expression level of the receptors (e.g. EGFR/IGFR expression ratio) and activation status of each receptor and its downstream signal transduction molecules (49,50). Our results nonetheless demonstrated that simultaneous targeting more than one growth factor receptor in tumor cells with BsAb or multispecific antibodies represents a novel and powerful approach to more effective cancer treatment.
The third noteworthy result of this study is that we designed and constructed via genetic engineering a novel BsAb molecule that could be efficiently expressed in mammalian cells. A major obstacle in the development of BsAb has been the difficulty in producing the materials via traditional methods, including the hybrid hybridoma and chemical conjugation (51). In contrast to rapid and significant progresses with various recombinant BsAb fragments (51)(52)(53), only limited success has been achieved in past years in both engineering and production of full-length IgG-like BsAb (Refs. 40 and 54 -56, for review, see Ref. 57). In this study, by fusing two scFv molecules of different specificities to the N terminus of the CL and the CH1 domains, we produced an IgG-like BsAb with an intact Fc region. Our BsAb format may offer several potential advantages over other approaches of BsAb construction. First, our BsAb possesses bivalent binding sites to each of their target antigens. Some therapeutic antibodies may require bivalency binding for their functions, e.g. cross-linking the receptors on the target cell surface to stimulate activation, to induce apoptosis, or to promote receptor internalization. In addition, the bivalency may yield higher binding avidity that is usually desirable and may be even necessary for each arm of a BsAb destined for human therapy (58). Second, compared with smaller recombinant BsAb fragments, such as diabody (59,60), Fab-scFv fusion (61), and miniantibody (62), our BsAb contains the intact Fc domain that confers long serum half-life and capability in supporting secondary immune function, such as antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity. This BsAb format provided an efficient method for production of homogenous IgG-like BsAb preparations and should be readily applicable to the construction of BsAb from antibodies recognizing any pairs of antigens.