e supernatant exhibited binding activity for IGF-1R but not IR, was isolated and expanded in cultures to obtain the mouse antibody designated R1 or mR1. Novel Humanized Antibodies Targeting IGF-1R Generation of cR1 Chimerization of R1 to obtain cR1 was performed as follows. The VH and VK genes of R1 were cloned by 59-RACE. The DNA sequences of the cloned VH and VK genes were determined with the authenticity confirmed by N-terminal protein sequencing that showed an exact match of the first 15 N-terminal amino acids with the corresponding amino acids deduced from DNA sequences. The cloned VH and VK genes were inserted into the pdHL2 vector to generate cR1pdHL2, which was used to transfect SpE-26 cells, a variant ofSp2/0-Ag14 developed in-house. Transfectants were selected with 0.075 mM methotrexate, and screened by ELISA for human Fc binding activities. Higher producing clones were further expanded to obtain the two best clones, from which cR1 was produced in batch cultures and purified by Protein A chromatography. Competition Binding Studies Homogeneous polystyrene microsphere beads coated with rhIGF-1R to serve as surrogates of cells expressing IGF-1R were used in all competition binding studies. To compare the binding affinity, varying concentrations of unlabeled mR1, cR1, and hR1 were mixed with a constant amount of Alexa Fluor 532-labeled cR1. The coated beads were added to a final density of 26105 particles/mL and the mixtures were incubated at room temperature for 1 h with gentle rocking. The bound 532-cR1 was determined by measuring median fluorescence intensity of 2,000 beads on a Guava PCA System. To determine whether cR1 can block the binding of 
IGF-1 or IGF-2 to IGF-1R, varying concentrations of cR1, IGF-1, or IGF-2 were mixed with a constant amount of 125I-IGF-1 or 125I-IGF-2. The coated beads were then added, incubated at RT for 1 h with gentle rocking, washed, and counted for radioactivity. To probe the binding region of hR1 on IGF-1R, a panel of commercially available anti-IGF-1R mAbs, with their epitopes to IGF-1R mapped, were evaluated as competitors for blocking hR1 from binding to the rhIGF-1R-coated beads. In these experiments, R1, cR1, hR1, MAB391, 2460, and aIR-3 were each labeled with R-phycoerythrin and incubated with an unlabeled antibody of interest at varying concentrations. Generation of hR1 Humanization of cR1 to hR1 was achieved by grafting the CDRs onto the human framework regions of hMN-14. For certain framework positions, murine residues of R1 were retained, resulting in the amino acid sequences of hR1 VH and hR1 VK. Synthetic genes encoding hR1 VH and hR1 Vk were engineered into pdHL2 to obtain hR1pdHL2, the expression vector for hR1. Subsequent efforts to secure the production clone for hR1 were similar to those described above for cR1, except that the positive clones were selected for binding activities to both human Fab and rhIGF-1R. Binding to Cell Surface IGF-1R Each sample was prepared in duplicate to contain 26105 cells and 10 mg/mL of a test antibody in a final volume of 200 mL. After incubation at 4uC for 45 min, samples were washed twice with PBS-1% BSA, followed by the addition of FITC-GAH IgG,, and a further incubation at 4uC for 45 min in the dark. Samples were then washed twice with PBS-1% BSA, resuspended in 500 mL of PBS-buffered formalin, and analyzed on MedChemExpress PNU-100480 FACScan. Generation of Hex-hR1 by DNL CH1-DDD2-Fab-hR1 and CH3-AD2-IgG-hR1 were produced as fusion proteins in SpESF cells transfected
