Alteration of fcrn binding affinities or serum half-lives of antibodies by mutagenesis
An affinity, antibody technology, applied in the fields of immunology and protein engineering, which can solve the problem of undisclosed mutation changing the binding ability of FcRn
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Embodiment 1
[0228] This example describes the antibody expression vectors used in the present invention.
[0229] The following are components of the heavy chain expression plasmid pVAg2M3-OST577, a derivative of the M3 variant of pVg2.D.Tt (Cole et al., J. Immunol. 159:3613-3621 (1997)). As shown in Figure 5A, clockwise before the EcoRI site, the heavy chain unit begins with the human cytomegalovirus (hCMV) major immediate early (IE) promoter and enhancer as an EcoRI-XbaI fragment (Boshart et al., Cell 41: 521-530 (1985)). The hCMV region is followed by OST577 V as an XbaI fragment H region, including signal sequence, J segment, and splice donor sequence. V H The region is followed by a modified genomic DNA fragment (Cole et al., supra) containing the human γ-2M3 heavy chain constant region as the XbaI-BamHI fragment, including the C with intervening intron. H 1. Hinge region (H), C H 2 and C H 3 exons, some introns in C H 1, while the polyadenylation (polyA) signal for mRNA proce...
Embodiment 2
[0236] This example describes vectors useful in the present invention.
[0237] OST577 heavy and light chain cDNAs were cloned intact by PCR from a trioma cell line expressing the human monoclonal anti-HBV antibody OST-577 (Ehrlich et al., supra). The heavy and light chain variable regions were converted by PCR into small exons flanked by XbaI sites at both ends, including the signal sequence, V, (D) and J segments, splice donor sequences and corresponding introns Part of the sub (as outlined by Co et al., supra). Expression vector pVAg2M3-OST577 (see Figure 5A), a derivative of the M3 variant of pVg2.D.Tt (Cole et al., supra), was obtained by using OST577-V H Small exon substitution containing OKT3-V H The XbaI fragment of the small exon was constructed. The PciI-FspI fragment containing the bacterial origin of replication was then replaced with the corresponding PciI-FspI fragment from pUC18 (Yanisch-Perron et al., supra) to increase the copy number of the vector in the b...
Embodiment 3
[0246] This example describes mutagenesis of the Fc region of the human γ2M3 heavy chain gene.
[0247] Molecular model:
[0248]The initial model of the human Fc / FcRn complex was based on the low-resolution crystal structure of the rat Fc / FcRn complex (Burmeister et al., Nature 372:379-383 (1994); RCSB protein database code 1FRT). First, by adding the high-resolution crystal structure of the human histocompatibility antigen HLA-A2 (Saper et al., J. Mol. Biol. 219: 277-319 (1991) ); RCSB is coded as human β2m in 3HLA) to replace rat β2m in the complex. Then, by adding human α-chain in the same orientation as the rat α-chain in the complex (West and Bjorkman, Biochemistry 29:9698-9708 (2000); RCSB code 1EXU) taken from the high-resolution crystal structure of human FcRn chain to replace the α-chain of rat FcRn. The rat residues in the Fc of the complex were then substituted by the corresponding residues from human IgG1 Fc (Kabat et al., supra) and analyzed using the SEGMOD a...
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