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• W2149278865 abstract "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. 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) 1The 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-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.-based therapeutics represent a promising new class of anticancer agents because of their exclusive specificity toward defined antigens (1.Glennie M.J. Johnson P.W.M. Immunol. Today. 2000; 21: 403-410Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 2.Ross J.S. Gray K. Gray G.S. Worland P.J. Rolfe M. Am. J. Clin. Pathol. 2003; 119: 472-485Crossref PubMed Scopus (98) Google Scholar). 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.Czuczman M.S. Grillo-Lopez A.J. White C.A. Saleh M. Gordon L. LoBuglio A.F. Jonas C. Klippenstein D. Dallaire B. Varns C. J. Clin. Oncol. 1999; 17: 268-276Crossref PubMed Google Scholar) and Herceptin® plus Taxol in metastatic breast cancer (4.Baselga J. Oncology. 2001; 61: 14-21Crossref PubMed Scopus (161) Google Scholar), or as conjugates to other cytotoxic moieties, such as Zevalin® (a yttrium 90-labeled anti-CD20 mAb) (5.Grillo-Lopez A.J. Exp. Rev. Anticancer Ther. 2002; 2: 485-493Crossref PubMed Scopus (81) Google Scholar) and Bexxar® (an iodine 131-labeled anti-CD20 mAb) (6.Cheson B. Curr. Opin. Investig. Drugs. 2002; 3: 165-170PubMed Google Scholar) in non-Hodgkins lymphoma, and Mylotarg® (an anti-CD33 antibody linked to calicheamicin) in acute myeloid leukemia (7.Sievers E.L. Linenberger M. Curr. Opin. Oncol. 2001; 13: 522-527Crossref PubMed Scopus (121) Google Scholar). 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 tumor-associated antigens simultaneously may offer a novel and promising solution. Both epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor (IGFR) have been implicated in the tumorigenesis of a variety of human cancers (8.Baserga R. Exp. Cell Res. 1999; 253: 1-6Crossref PubMed Scopus (267) Google Scholar, 9.Werner H. Le Roith D. Crit. Rev. Oncol. 1997; 8: 71-92Crossref Scopus (131) Google Scholar, 10.Rubin R. Baserga R. Lab. Invest. 1995; 73: 311-331PubMed Google Scholar, 11.Arteaga C. Semin. Oncol. 2002; 29: 3-9Crossref PubMed Google Scholar, 12.Yarden Y. Eur. J. Cancer. 2001; 37: S3-S8Abstract Full Text Full Text PDF PubMed Google Scholar, 13.Wells A. Int. J. Biochem. Cell Biol. 1999; 31: 637-643Crossref PubMed Scopus (899) Google Scholar). 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.Waksal H.W. Cancer Metastasis Rev. 1999; 18: 427-436Crossref PubMed Scopus (121) Google Scholar, 15.Mendelsohn J. Cancer Immunol. Immunother. 2003; 52: 342-346Crossref PubMed Scopus (54) Google Scholar, 16.Mendelsohn J. Baselga J. J. Clin. Oncol. 2003; 21: 2787-2799Crossref PubMed Scopus (1191) Google Scholar, 17.Arteaga C. Semin. Oncol. 2003; 30: 3-14Crossref Scopus (228) Google Scholar, 18.Khalil M.Y. Grandis J.R. Shin D.M. Exp. Rev. Anticancer Ther. 2003; 3: 367-380Crossref PubMed Scopus (86) Google Scholar, 19.Laird A.D. Cherrington J.M. Exp. Opin. Investig. Drugs. 2003; 12: 51-64Crossref PubMed Scopus (126) Google Scholar, 20.Manegold C. Adv. Exp. Med. Biol. 2003; 532: 247-252Crossref PubMed Scopus (8) Google Scholar, 21.Grunwald V. Hidalgo M. Adv. Exp. Med. Biol. 2003; 532: 235-246Crossref PubMed Scopus (52) Google Scholar). For example, Erbitux™ (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.Saltz L. Rubin M. Hochster H. Tchekmeydian N.S. Waksal H. Needle M. LoBuglio A. Proc. Am. Soc. Clin. Oncol. 2001; 20 (Abstr. 7)Google Scholar, 23.Cunningham D. Humblet Y. Siena S. Khayat D. Bleiberg H. Santoro A. Bets D. Mueser M. Harstrick A. Van Cutsem E. Proc. Am. Soc. Clin. Oncol. 2003; 22 (Abstr. 1012)Google Scholar), 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.Cohen M.H. Williams G.A. Sridhara R. Chen G. Pazdur R. Oncologist. 2003; 8: 303-306Crossref PubMed Scopus (509) Google Scholar). Similarly, significant tumor inhibition has also been achieved in animal models with several IGFR targeting strategies including antisense oligonucleotides (25.Lee C.T. Wu S. Gabrilovich D. Chen H. Nadaf-Rahrov S. Ciernik I.F. Carbone D.P. Cancer Res. 1996; 56: 3038-3041PubMed Google Scholar), dominate-negative receptor mutants (26.Reinmuth N. Liu W. Fan F. Jung Y.D. Ahmad S.A. Stoeltzing O. Bucana C.D. Radinsky R. Ellis L.M. Clin. Cancer Res. 2002; 8: 3259-3269PubMed Google Scholar), and neutralizing mAb (27.Zia F. Jacobs S. Kull Jr., F. Cuttitta F. Mulshine J.L. Moody T.W. J. Cell Biochem. Suppl. 1996; 24: 269-275Crossref PubMed Scopus (45) Google Scholar, 28.Sachdev D. Li S.L. Hartell J.S. Fujita-Yamaguchi Y. Miller J.S. Yee D. Cancer Res. 2003; 63: 627-635PubMed Google Scholar) (for review, see Ref. 29.Wang Y. Sun Y. Curr. Cancer Drug Targets. 2002; 2: 191-207Crossref PubMed Scopus (120) Google Scholar). 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.Chakravarti A. Loeffler J.S. Dyson N.J. Cancer Res. 2002; 62: 200-207PubMed Google Scholar). 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.van Spriel A.B. van Ojik H.H. van de Winkel J.G. Immunol. Today. 2000; 21: 391-397Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 32.Weiner L.M. Alpaugh R.K. von Mehren M. Cancer Immunol. Immunother. 1997; 45: 190-192Crossref PubMed Scopus (11) Google Scholar, 33.Segal D.M. Weiner G.J. Weiner L.M. J. Immunol. Methods. 2001; 248: 1-6Crossref PubMed Scopus (32) Google Scholar). 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. 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% CO2. 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 125I-IGF-I and 125I-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 × 1010 unique clones (34.De Haard H.J. van Neer N. Reurs A. Hufton S.E. Roovers R.C. Henderikx P. de Bruine A.P. Arends J-W. Hoogenboom H.R. J. Biol. Chem. 1999; 274: 18218-18230Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar) following protocols previously described (35.Lu D. Jimenez X. Zhang H. Bohlen P. Witte L. Zhu Z. Int. J. Cancer. 2002; 97: 393-399Crossref PubMed Scopus (137) Google Scholar). 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. 2M. Liu, D. Hicklin, and Z. Zhu, manuscript in preparation. 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.Lu D. Shen J. Vil M.D. Jimenez X. Zhang H. Bohlen P. Witte L. Zhu Z. J. Biol. Chem. 2003; 278: 43496-43507Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar) led to the identification of A12, a clone with significantly improved binding affinity and neutralizing activity (37.Burtrum, D., Zhu, Z., Lu, D., Anderson, D. M., Prewett, M., Pereira, D. S., Bassi, R., Abdullah, R., Hooper, A., Finnerty, B., Koo, H., Jimenez, X., Johnson, D., Apblett, R., Kussie, P., Bohlen, P., Witte, L., Hicklin, D. J., and Ludwig, D. L. (2003) Cancer Res., in pressGoogle Scholar). 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.Bebbington C.R. Renner G. Thomson S. King D. Abrams D. Yarranton G.T. Bio/Technology. 1992; 10: 169-175Crossref PubMed Scopus (405) Google Scholar), 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.Lu D. Jimenez X. Zhang H. Wu Y. Bohlen P. Witte L. Zhu Z. Cancer Res. 2001; 61: 7002-7008PubMed Google Scholar). 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.Zuo Z. Jimenez X. Witte L. Zhu Z. Protein Eng. 2000; 13: 361-367Crossref PubMed Scopus (39) Google Scholar). 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.Lu D. Jimenez J. Zhang H. Atkins A. Brennan L. Balderes P. Bohlen P. Witte L. Zhu Z. J. Immunol. Methods. 2003; 279: 219-232Crossref PubMed Scopus (42) Google Scholar). 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 μl of a rabbit anti-human IgG Fc-HRP conjugate (Jackson ImmunoResearch Laboratory Inc., West Grove, PA). The plates were washed and developed following a procedure previously described (35.Lu D. Jimenez X. Zhang H. Bohlen P. Witte L. Zhu Z. Int. J. Cancer. 2002; 97: 393-399Crossref PubMed Scopus (137) Google Scholar, 39.Lu D. Jimenez X. Zhang H. Wu Y. Bohlen P. Witte L. Zhu Z. Cancer Res. 2001; 61: 7002-7008PubMed Google Scholar). 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. 125I-EGF or 125I-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 μl of 0.5 n NaOH and radioactivity was counted in a γ-counter. FACS Analysis—DiFi, MCF-7, BxPC3, A431, and HT29 cells were incubated with various antibodies (10 μg/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 × 104 DiFi or BxPC3 cells in 100 μl 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 μl 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 μl of HCl/isopropyl alcohol (40 mm) at room temperature for 10 min, followed by optical density reading at 570 nm. 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 Na3VO4, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 μg/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 μl of Protein A/GSepharose 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 anti-phospho-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 ∼200,000 (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 ∼200 kDa (representing the properly assembled BsAb), and one minor band at ∼100 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 ∼150 kDa (Fig. 1B, lane 2 and 5, respectively). Under reducing conditions, both BsAb yielded two major bands, one represents the scFv-CH1CH2CH3 fusion (∼62.5 kDa) and the other the scFv-CL fusion (∼37.5 kDa) (Fig. 1C, lanes 3 and 4). As expected, both IMC-A12 and IMC-11F8 showed two major bands: the IgG heavy chain (∼50 kDa) and IgG light chain (∼25 kDa) (Fig. 1C, lanes 2 and 5, respectively). Under size exclusion chromatography, both BsAb preparations yielded a single major peak (>90%) with estimated molecular weight of ∼200,000 (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 ED50 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 IC50 values, i.e. the antibody concentrations required to inhibit 50% of ligand binding, were ∼1.5 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 IC50 of ∼10 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 ∼5-fold less potent than IMC-11F8. The IC50 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: incubatio" @default.
• W2149278865 created "2016-06-24" @default.
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• W2149278865 date "2004-01-01" @default.
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• W2149278865 title "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" @default.
• W2149278865 cites W1041926633 @default.
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