ositively charged His and the conformationally restricted Pro. In the second library, variation in the LCDRs was designed to mimic diversity of natural antibodies derived from the VH1 69 germline and paired with V�� light chains. We queried the PDB to identify antibodies with high homology to the VH1 69 germline segment that fulfilled three criteria, their three dimensional structures had been solved in complex with the antigen, the antibody represented a product or variant of natural rearrangement, the sequences were unique. We compiled se quences from 24 total antibodies and found that 18 of these contained VK light chains. These antibodies target a variety of antigens, and were isolated from phage display and other sources. In general, the LCDR loop lengths among these antibodies were similar to those found in D5.

We examined each of the crystal structures and assessed LCDR positions for their importance in the structural paratope as gauged by surface area buried upon complex formation. Anacetrapib We assigned a qualitative contact score at each position based on the extent to which the residue at that position participated in structural paratopes across the datasets. In general, those positions with high contact score contained side chains in which 80% of the surface area was buried upon binding in three or more complexes. We determined the amino acid distribution at each position and designed restricted diversity codons to allow composition that reflected the distribution at each position or, in some cases, residues that had similar physicochemical properties to the nat ural distribution.

At several positions, we allowed greater diversity than was observed in the structural dataset. For HCDR3, we allowed variation among the 12 residues encoded by the DVK codon, since HCDR3 has a high degree of variability among all antibody scaffolds. During synthesis of each library, we permitted WT D5 side chain identity in both HCDR3 and LCDR1 by using template DNA that contained WT D5 side chain identity at these positions. Our rationale for this ap proach was to examine whether WT D5 sequences in HCDR3 and LCDR1 would be preferred to library se quences, if so, then clones containing these WT se quences should be selected over clones that contain library sequences. Both libraries were produced in bi valent scFv format with 3 x 109 unique members each.

Analysis of selectants We screened both libraries for three rounds against 5 Helix. A large number of clones from the round 3 populations from both libraries were characterized by se quence analysis and monoclonal ELISA. Fifty five of the 276 clones from D5 Lib I R3 population contained library sequences and had positive but moderate binding signals for 5 Helix. Furthermore, these clones displayed moderate specificity for binding to 5 Helix. In contrast, selec tion of D5 Lib II resulted in a R3 population that was dom inated by library members that had strong positive ELISA signals for 5 Helix, and were highly specific.