Improved pharmacokinetics of HIV-neutralizing VRC01-class antibodies achieved by reduction of net positive charge on variable domain

ABSTRACT

The amino-acid composition of the immunoglobulin variable region has been observed to impact antibody pharmacokinetics (PK). Here, we sought to improve the PK of the broad HIV−1-neutralizing VRC01-class antibodies, VRC07-523LS and N6LS, by reducing the net positive charge in their variable domains. We used a structure-guided approach to generate a panel of antibody variants incorporating select Arg or Lys substituted to Asp, Gln, Glu, or Ser. The engineered variants exhibited reduced affinity to heparin, reduced polyreactivity, and improved PK in human FcRn-transgenic mice. One variant, VRC07-523LS.v34, with three charge substitutions, had an observed in vivo half-life and an estimated human half-life of 10.8 and 60 days, respectively (versus 5.4 and 38 days for VRC07-523LS) and retained functionality, neutralizing 92% of a 208-strain panel at a geometric mean IC₈₀ <1 µg/mL. Another variant, N6LS.C49, with two charge substitutions, had an observed in vivo half-life and an estimated human half-life of 14.5 and 80 days (versus 9.0 and 44 days for N6LS) and neutralized ~80% of 208 strains at a geometric mean IC₈₀ <1 µg/mL. Since Arg and Lys residues are prevalent in human antibodies, we propose substitution of select Arg or Lys with Asp, Gln, Glu, or Ser in the framework region as a general means to improve PK of therapeutic antibodies.

KEYWORDS:

• Antibody-mediated prevention
• net positive charge reduction
• pharmacokinetics
• serum half-life
• surface charge
• variable region
• VRC01-class antibody

Acknowledgments

We thank J. Stuckey for assistance with figures and members of the Structural Biology Section and Structural Bioinformatics Core Vaccine Research Center for discussions or comments on the manuscript. We thank J. Baalwa, D. Ellenberger, F. Gao, B. Hahn, K. Hong, J. Kim, F. McCutchan, D. Montefiori, L. Morris, E. Sanders-Buell, G. Shaw, R. Swanstrom, M. Thomson, S. Tovanabutra, C. Williamson, and L. Zhang for contributing the HIV-1 envelope plasmids used in the 208-strain panel. We thank R. Carroll, N. Jean-Baptiste, C. Moore, S. O’Dell, G. Padilla, S.D. Schmidt, C. Whittaker, and A.B. McDermott for their assistance with neutralization assessments on the 208-strain panel. We thank the VRC Production Program for providing manufacturability and biophysical risk assessment. The VRC Production Program includes Nadia Amharref, Nathan Barefoot, Christopher Barry, Elizabeth Carey, Ria Caringal, Naga Chalamalsetty, Adam Charlton, Rajoshi Chaudhuri, Mingzhong Chen, Peifeng Chen, Nicole Cibelli, Jonathan W. Cooper, Hussain Dahodwala, Marianna Fleischman, Julia C. Frederick, Haley Fuller, Jason Gall, Isaac Godfroy, Daniel Gowetski, Krishana Gulla, Vera Ivleva, Lisa Kueltzo, Venkata Mangalampalli, Sarah O’Connell, Aakash Patel, Erwin Rosales-Zavala, Elizabeth Scheideman, Nicole A. Schneck, Zachary Schneiderman, Andrew Shaddeau, William Shadrick, Alison Vinitsky, Sara Witter, Yanhong Yang, and Yaqiu Zhang. Support for this work was provided by the Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Disclosure statement

Y.D.K., A.P., E.S.Y., K.M., N.A.D-R., and P.D.K. are inventors on a PCT-patent application describing VRC07-523LS and N6LS antibody variants (filed March 29, 2023). All other authors have no competing interests.

Author Contributions

Y.D.K. conceived the project designed the antibody variants and performed BLI and SPR analyses; A.P. and E.S.Y. performed a pharmacokinetic study; B.Z. and T.L. produced antibody variants; Y.D.K. and M.F.B. purified the antibody variants performed heparin chromatography and prepared antibodies for PK study and neutralization assay; M.A. performed antibody polyreactivity analysis; Q.L. and P.L. provided critical insight into 03FR3 insertion. K.M., N.A.D.-R., and J.R.M., provided or oversaw HIV−1 neutralization assessments; B.C.L, M.K.L, N.A.D.-R, and J.R.M. provided or oversaw 208-panel neutralization assessment; R.R., M.R., and A.J.S. computed estimated serum half-lives in humans; C.-H.S. analyzed the relative frequency of Arg and Lys in human antibodies; Y.D.K. and P.D.K. wrote the manuscript with all authors providing comments. P.D.K. oversaw the project.

Abbreviations

IgG	=	Immunoglobulin G
BLI	=	biolayer interferometry
KD	=	dissociation constant
Fab	=	antigen-binding fragment
Fc region	=	the fragment crystallizable region
PK	=	Pharmacokinetics
SPR	=	surface plasmon resonance
FcRn	=	neonatal Fc receptor
ELISA	=	enzyme-linked immunoassay
IV	=	intravenous

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2023.2223350

Additional information

Funding

The work was supported by the Vaccine Research Center [ZIA AI005022-20].