Bispecific antibody CAP256.J3LS targets V2-apex and CD4-binding sites with high breadth and potency

ABSTRACT

Antibody CAP256-VRC26.25 targets the second hypervariable region (V2) at the apex of the HIV envelope (Env) trimer with extraordinary neutralization potency, although less than optimal breadth. To improve breadth, we linked the light chain of CAP256V2LS, an optimized version of CAP256-VRC26.25 currently under clinical evaluation, to the llama nanobody J3, which has broad CD4-binding site-directed neutralization. The J3-linked bispecific antibody exhibited improved breadth and potency over both J3 and CAP256V2LS, indicative of synergistic neutralization. The cryo-EM structure of the bispecific antibody in complex with a prefusion-closed Env trimer revealed simultaneous binding of J3 and CAP256V2LS. We further optimized the pharmacokinetics of the bispecific antibody by reducing the net positive charge of J3. The optimized bispecific antibody, which we named CAP256.J3LS, had a half-life similar to CAP256V2LS in human FcRn knock-in mice and exhibited suitable auto-reactivity, manufacturability, and biophysical risk. CAP256.J3LS neutralized over 97% of a multiclade 208-strain panel (geometric mean concentration for 80% inhibition (IC₈₀) 0.079 μg/ml) and 100% of a 100-virus clade C panel (geometric mean IC₈₀ of 0.05 μg/ml), suggesting its anti-HIV utility especially in regions where clade C dominates.

KEYWORDS:

• Antibody half-life
• antibody improvement
• bispecific antibody
• CAP256-VRC26.25
• CD4-binding site
• cryo-electron microscopy
• HIV-1
• J3 nanobody
• V2 apex

Abbreviations

AMP, antibody-mediated prevention; CDR, complementarity determining region; DLS, dynamic light scattering; Env, HIV-1 envelope glycoprotein; Fab, antigen-binding fragment; FcRn, neonatal Fc receptor; IC₅₀, half maximal inhibitory concentration; IC₈₀, 80% inhibitory concentration; IgG, immunoglobulin G; ITC, isothermal titration calorimetry; MPER, membrane-proximal external region; OD, optical density; RT, room temperature; scFv, single-chain variable region; V2, second hypervariable region

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 Chen-Hsiang Shen for calculating SHM for the antibodies. 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 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 BG505 DS-SOSIP.664 Env trimer. The VRC Production Program includes Nadia Amharref, Frank J. Arnold, 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.

Disclosure statement

B.Z., J.G., and P.D.K. have submitted a US Provisional Patent Application describing CAP256-J3 bispecific antibodies (filed March 26, 2022).

Data availability statement

Cryo-EM maps and fitted coordinates have been deposited with EMDB entry ID EMD-29209 (https://www.ebi.ac.uk/emdb/search/EMD-29209) and PDB entry ID 8FIS (https://www.rcsb.org/structure/unreleased/8FIS), respectively.

Author Contributions

B.Z. designed bispecific antibodies and prepared antibodies for neutralization, ITC and structural assays; J.G. determined and analyzed the cryo-EM structure; C.W.C and T.L. produced antibodies and antibody Fab for analysis; Y.D.K. and M.F.B. headed charge variant design and evaluation; A.P. and E.S.Y. preformed pharmacokinetic study; M.A. and C.L. preformed antibody autoreactivity analysis; T.B. and N.D.R. designed the 30-virus panel for neutralization assessment; A.S.O. produced HIV-1 trimer proteins for antibody-binding analysis; R.R. performed neutralization-fingerprinting analysis; A.S. performed ITC experiments, Y.Y. performed Anti-HIV-1 ENV trimer ELISA; M.S.S. evaluated antibodies on a 100-clade C virus panel; D.G., P.L., Y.L., and K.C. preformed manufacturability and biophysical risk assessment; L.D., M.K.L., and K.M. performed HIV-1 neutralization assays; L.S. oversaw neutralization fingerprinting analysis; J.R.M. and N.D.R. oversaw HIV-1 neutralization assays. P.D.K. oversaw the project and, with S.W. and B.Z., wrote the manuscript, with all authors providing comments or revisions.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2023.2165390

Additional information

Funding

This work was supported by the Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases [ZIA AI005022]. Some of this work was performed at the Simons Electron Microscopy Center and the National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center, supported by grants from the Simons 590 Foundation [SF349247], NYSTAR, and the NIH National Institute of General Medical Sciences [GM103310].