Figure 1.
Humanization of D9 Fv by CDR-grafting.
Residues are numbered according to Kabat. CDRs are marked with boxes and FRs are between boxes. Key FR residues are marked with *. Two key FR residues in D9 VH, which were kept in hD9 VH are marked with green.
Figure 2.
FRs in VH and VL are shown in green and orange. CDRs in VH and VL are shown in red and blue.
Table 1.
Sequence alignment of CDRs 1–2 and FRs 1–3 from D9 VH with human germline HC V genes.
Table 2.
Sequence alignment of CDRs 1–2 and FRs 1–3 from D9 VL with human germline LC V genes.
Table 3.
Sequence alignment of CDR3 and FR4 from D9 VH with human JH genes.
Table 4.
Sequence alignment of CDR3 and FR4 from D9 VL with human JK genes.
Table 5.
Different key residues of FRs between human donors and D9.
Figure 3.
VH44-N interaction with four VL residues in VH-VL interface.
VH44-N was an interchain packing residue, located in the VH-VL interface, interacting with VL87-Y, VL98-F, VL99-G, and VL 100-A. VH and VL are shown in green and orange.
Figure 4.
VL82a-L interaction with VH65-D of VH CDR2.
VH82a-L was a Vernier Zone residue to support VH CDR2 by interacting with VH65-D of VH CDR2. VH and VL are shown in green and orange.
Figure 5.
SDS-PAGE analysis of recombinant hD9.
Lane 1 is a molecular marker. Lanes 2 and 4 are control human IgG1 and hD9 in non-reducing condition. Lanes 3 and 5 are control human IgG1 and hD9 in reducing condition.
Figure 6.
In vitro binding affinity analysis by SPR.
SPR sensorgram of the kinetics of association and dissociation of a range of concentrations from 0 to 500 nm of hD9 (A) or D9 (B) to immobilized ricin.
Figure 7.
In vivo post-exposure therapeutic protection assay.
Ricin was given at the dose of 5×LD50 to mice by i.p route. hD9 or D9 at the dose of 5 µg was administered i.p. at 2, 4, or 6 hours after ricin challenge and then mouse survival rate was monitored for 7 days.
Figure 8.
hD9 or D9 at the dose of 5 µg was administered by the i.p. route into mice. Sera were collected at different time points to calculate hD9 or D9 concentrations using an immunoassay. The hD9 or D9 remaining in sera is expressed as percentages plotted against time in days on the figure.