Antibodies and methods of treating cancer
An antibody, cancer technology, applied in the field of cancer biology
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Embodiment 1
[0159] Selection of human Fab specific for human GPR49 from a phage display library
[0160] Human antibodies that specifically recognize the extracellular domain of the human GPR49 receptor were isolated using phage display technology.
[0161] Part I: Phage display panning
[0162] Method: Using recombinant human GPR49-Fc ectodomain (GPR49-Fc) (SEQ ID NO: 3) to screen cells containing 3.5x10 10 A native human phagemid Fab library of unique clones (Hoet, R.M., et al. NatBiotechnol. 23(3):344-8 (2005)). Before incubation with the phage library, biotinylated anti-Fc antibodies were captured on magnetic beads, which then captured the GPR49-Fc fusion protein. Selection was performed as described by Hoet et al. After 3 rounds of panning, the 479 bp gene III stump was removed by MluI digestion, and the vector was used for soluble Fab expression in TGI cells.
[0163] Results: 61 unique clones were isolated in this panning. Unique clones were subsequently purified and binding...
Embodiment 2
[0174] Construction of full-length anti-GPR49 IgG
[0175] Methods: Three Fabs were converted into human IgG1 and expressed in CHO cells. DNA sequences encoding 3 different anti-GPR49 Fabs (76C12, 78F05 and 76B04) were isolated from a human antibody phage library (Dyax Corp) by biopanning against the recombinant human GPR49 ectodomain-Fc fusion protein. The Fab gene sequence was used to construct expression plasmids encoding full-length antibodies using the pV90AS expression vector system for antibody production in mammalian cells. pV90AS is a modified pV90 expression vector designed to generate two transcripts from a single promoter by altering the splicing pattern of the primary transcript (see: USPTO application WO2005 / 089285). The native CMV splice donor is spliced into a partially damaged splice acceptor to generate a transcript encoding the antibody light chain, or into a native CMV splice acceptor to generate a transcript encoding the antibody heavy chain. The par...
Embodiment 3
[0180] Construction of full-length anti-GPR49 IgG for increased expression in mammalian cells
[0181] In order to improve the antibody expression yield and product quality, the original VH gene sequences from anti-GPR49Fab76C12, 78F05, 76B04 were modified.
[0182] Methods: First, anti-GPR49 VH sequences were analyzed for sequences containing putative splice sites using public sequence recognition programs (www.tigr.org / tdb / GeneSplicer / gene_spl.html (The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, Md.20850), www.fruitfly.org / seq_tools / splice.html) (Martin G. Reese and Frank H. Eeckman, Lawrence Berkeley National Laboratory, Genome Informatics Group, 1 Cyclotron Road, Berkeley, Calif, 94720; see also, Reese M G, Eeckman, F H, Kulp, D, Haussler, D, 1997. "Improved Splice Site Detection in Genie". J Comp Biol 4(3), 311-23). Second, the heavy chain variable region codons of the anti-GPR49 Fab were replaced with codons corresponding to the same Kabat p...
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