Framework Mutations of the 10-1074 bnAb Increase Conformational Stability, Manufacturability, and Stability While Preserving Full Neutralization Activity

• Published in: Journal of pharmaceutical sciences Vol. 109; no. 1; pp. 233 - 246
• Main Authors: Kerwin, Bruce A., Bennett, Chelsey, Brodsky, Yan, Clark, Rutilio, Floyd, J. Alaina, Gillespie, Alison, Mayer, Bryan T., McClure, Megan, Siska, Christine, Seaman, Michael S., Seaton, Kelly E., Shaver, Jeremy, Tomaras, Georgia D., Yates, Nicole L., Ketchem, Randal R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2020; Elsevier
• Subjects: analytical biochemistry; Animals; antibody(s); biopharmaceutical characterization; Broadly Neutralizing Antibodies - chemistry; Broadly Neutralizing Antibodies - genetics; Broadly Neutralizing Antibodies - metabolism; developability; fluorescence spectroscopy; HEK293 Cells; high throughput technology(s); HIV Antibodies - chemistry; HIV Antibodies - genetics; HIV Antibodies - metabolism; HIV/AIDS; Humans; Mice; Mutation - physiology; pharmacokinetics; physical stability; protein aggregation; Protein Conformation; protein folding; Protein Stability; protein structure; Protein Structure, Secondary; stability; stabilization; high throughput technology(s); stabilization; physical stability; protein structure; CDR; DSF; pharmacokinetics; protein folding; stability; bnAbs; HIV/AIDS; PBS; CDCA; antibody(s); developability; COGs; fluorescence spectroscopy; mAb; analytical biochemistry; HMW; PEG; biopharmaceutical characterization; HP-SEC; protein aggregation; Gdn-HCl; LC; HC; PK
• ISSN: 0022-3549; 1520-6017
• DOI: 10.1016/j.xphs.2019.07.009

The broadly neutralizing anti-HIV antibody, 10-1074, is a highly somatically hypermutated IgG1 being developed for prophylaxis in sub-Saharan Africa. A series of algorithms were applied to identify potentially destabilizing residues in the framework of the Fv region. Of 17 residues defined, a variant was identified encompassing 1 light and 3 heavy chain residues, with significantly increased conformational stability while maintaining full neutralization activity. Central to the stabilization was the replacement of the heavy chain residue T108 with R108 at the base of the CDR3 loop which allowed for the formation of a nascent salt bridge with heavy chain residue D137. Three additional mutations were necessary to confer increased conformational stability as evidenced by differential scanning fluorimetry and isothermal chemical unfolding. In addition, we observed increased stability during low pH incubation in which 40% of the parental monomer aggregated while the combinatorial variant showed no increase in aggregation. Incubation of the variant at 100 mg/mL for 6 weeks at 40°C showed a 9-fold decrease in subvisible particles ≥2 μm relative to the parental molecule. Stability-based designs have also translated to improved pharmacokinetics. Together, these data show that increasing conformational stability of the Fab can have profound effects on the manufacturability and long-term stability of a monoclonal antibody.