This work was supported by Contract No. toxin-neutralizing activity. However, the toxin-neutralizing activity of the V1C7G29R mutant was not correspondingly improved, indicating that in the V1C7 family binding affinity alone does not account for differences in antibody function. V1C7 and V5C1, as well as their respective point mutants, recognized indistinguishable epitopes on RTA, at least at the level of sensitivity afforded by hydrogen-deuterium mass spectrometry. The results of this study have implications for engineering therapeutic antibodies because they demonstrate that even subtle differences in epitope specificity can account for important differences in antibody function. Keywords: antibody engineering, structure-based protein modeling, epitope mapping, vaccine INTRODUCTION The eventual success of a given subunit vaccine for most infectious diseases and biothreat agents will depend on its capacity to elicit a robust antibody response against key protective epitopes on the target antigen and minimize off target interactions with non-neutralizing or decoy epitopes 1C3. Ricin, a toxic glycoprotein from the castor bean (and and application. We preserved the bound pose of the VHH relative to RTA from the crystal structure of the V1C7CRTA complex (PDB: 5J56), generating an ensemble of 5,000 energetically minimized conformations using the Rosetta application 48; this procedure searches for alternate side-chain rotamers on both RTA and VHH, and also includes gradient-based minimization of their backbones and side-chains, and the relative orientation between them. The disulfide bond between VHH residues Cys22 and Cys100 was enforced throughout, and the pwSHO term was included to describe polar solvation. This modeling approach yielded models for all three VHHs that were structurally very similar to the V1C7 template, as expected. The average VHHCRTA interaction energy over the ensemble of 5,000 low-energy models was then used as an estimate of the relative RTA binding affinity. The VHHCRTA interaction energy of each model was computed as the difference between the total energy of the complex Parsaclisib and the total energy of the two monomers taken in isolation. We selected a representative model of each VHHCRTA ensemble as the center of the largest cluster formed by the Rosetta application, where clustering (by RMSD) was limited to the 1,000 models of lowest VHHCRTA interaction energy. In the analysis of representative models, we identified the most favorable residue-pair interactions at the VHHCRTA interface using the Rosetta application. HX-MS Analysis Hydrogen exchange was Parsaclisib performed using an H/DX PAL robot (LEAP Technologies, Carrboro, North Carolina). MS measurements were conducted using a QTOF mass analyzer (Agilent 6530, Santa Clara, California) with Agilent 1260 Infinity LC System. For HX, 4 L of RiVax prepared at 20 M was incubated with 36 L deuterated buffer (20 mM sodium phosphate, 150 mM sodium chloride, pD 7.7) at 25 C for 13 s C 24 h. For experiments in the presence of VHH, RiVax was prepared at 20 M with a VHH Rabbit polyclonal to PELI1 concentration of 40 M. To account for differences in measuring pD with a pH meter, a value 0.4 units was added to the pH meter reading 49. Labeling at each time point was performed in triplicate. The exchange was quenched by a 1:1 dilution into quench buffer Parsaclisib (4 M Gdn-HCl, 0.2 M phosphate, pH 2.5) at 0 C for 60 s. Fifty five L of the quenched sample was then injected into the 100 L sample loop of the refrigerated compartment of the H/DX PAL, maintained at 0 C, containing a pepsin column (50 mm 2.1 mm), a C8 trap (Poroshell 120 SB-C8, 2.1 5 mm, 2.7 m particle diameter), and a reversed phase column (ZORBAX 300SB-C18, 2.1 50 mm, 1.8 m particle diameter). The immobilized pepsin column was prepared in-house as described elsewhere 50. The quenched sample was carried through the pepsin column using 0.1% formic.
