All experiments were performed at 25 C

All experiments were performed at 25 C. structure was used to design a series of mutants with reduced affinity to investigate the correlation between the affinity for albumin and serum half-life. Reduction in the affinity for MSA by 144-fold from 2.2?nM to 316?nM had no effect on serum half-life. Strikingly, despite a FBW7 reduction in affinity to 62?M, an extension in serum half-life of 26.4?h was still obtained. CA645 Fab and the CA645 Fab-HSA complex have been deposited in the Protein Data Bank (PDB) with accession codes, 5FUZ and 5FUO, respectively. value of 21.14% and value of 25.13%. The structure of the complex was refined to 3.6 ? with a final value of 21.38% and value of 25.23%. Table 3. X-ray data collection and refinement statistics. Values in parentheses are for highest-resolution shell. (?)111.21, 111.21, 89.20217.68, 217.68, 78.68?, , ()90.00, 90.00, 120.0090.00, 90.00, 120.00Resolution (?)30.0C2.68 (2.82C2.68) *30.0C3.58 (3.79C3.58) */ by grafting the CDRs from antibody V-regions onto the V1 and VH3 human germline antibody V-region frameworks. The CDRs grafted from the donor to the acceptor sequence were as defined by Kabat em et?al. /em ,33 with the exception of CDR-H1 (residues 26C35) where the combined definitions of Kabat em et?al. /em , and loop structure was used.23 Where a framework residue differed between the donor rabbit sequence and the acceptor human sequence in a position that was considered to be important for retention of antigen binding, then the donor residue was included in the initial conservative graft. 21 The conservative graft genes were chemically synthesized by Entelechon, GmbH. Heavy chain graft genes (gH1) were cloned into 2 UCB expression vectors, one made up of human 1 CH1 domain name and another made up of the full human 1 constant region. Light chain graft genes (gL1) were cloned into a UCB expression containing human kappa constant region (Km3 allotype). These constructs were subsequently modified by oligonucleotide-directed mutagenesis to create a number of different variants of both the heavy and light chain grafts. Heavy and light chain vectors were co-transfected into HEK293 cells and the recombinant Fab or IgG molecules screened using a SPR binding assay to measure affinity for HSA, MSA, RSA, CSA, RbSA and bovine serum albumin. Antibody expression Antibodies were transiently expressed in either HEK-293 cells using 293Fectin Cucurbitacin B lipid transfection (Life Technologies, catalog #12347-019, according to the manufacturer’s instructions) or CHO-S XE cells, a CHO-K1 derived cell line,34 using electroporation. HEK-293 cells were used for small scale expression ( 100?ml) to prepare antibodies for SPR analysis. CHO-S XE cells were used for large scale expression (1 L) to prepare antibodies for crystallography Cucurbitacin B and in vivo pharmacokinetic studies. Protein purification Affinity chromatography was used to purify Fab protein from culture supernatants. Supernatants were passed over a HiTrap Protein G column (GE Healthcare) at a flow rate that gave a column contact time of 25?min. Following a washing step with PBS pH 7.4, the bound material was eluted with 0.1?M glycine pH 2.7 and neutralized with 2?m Tris-HCl (pH 8.5). Fractions made up of Fab were pooled, quantified by absorbance at 280?nm, and concentrated using Amicon Ultra centrifugal filters (Merck Millipore). To isolate the monomeric fraction, size exclusion Cucurbitacin B chromatography (SEC) over a HiLoad 16/60, Superdex 200 column (GE Healthcare) equilibrated with PBS, pH 7.4, was used. Fractions made up of monomeric Fab were pooled, quantified, concentrated and stored at 4C. Surface plasmon resonance The binding affinities and kinetic parameters for the interactions of antibodies were determined by SPR conducted on either a Biacore T200 or Biacore 3000 using CM5 sensor chips (GE Healthcare Bio-Sciences AB) and HBS-EP (10?mM HEPES, 150?mM NaCl, 3?mM EDTA, 0.05% v/v P20, pH7.4) running buffer. For analysis at pH 7.0, 6.0, 5.5 and 5.0, a running buffer of 40?mM citric acid, 80?mM sodium phosphate 50?mM NaCl, 3mM EDTA, 0.05% v/v P20 was used. The required pH was achieved by altering the ratio of citric acid to sodium phosphate. All experiments.