Bringing the Heavy Chain to Light: Creating a Symmetric, Bivalent IgG-Like Bispecific
Abstract
:1. Introduction
2. Materials and Methods
2.1. Selection of VH Domains from the Literature and the Protein Databank (PDB)
2.2. Expression and Purification of VH Domains
2.3. Expression and Purification of 2xVH Fabs and IgG Molecules
2.4. SPR Analysis
2.5. Analytical SEC
2.6. NanoDSF
2.7. ELISA
2.8. 2xVH Fab Crystallization
3. Results
3.1. Identification of In Vitro and In Vivo-Derived Candidate VH Domains
3.2. Biophysical Characterization of sVH Candidates Shows Source-Dependent Differences in Expression, Purity and Thermal Stability
3.3. Candidate In Vitro- and In Vivo-Derived sVH Domains Show nM and Sub-nM Binding
3.4. Generation and Characterization of 2xVH Molecules with High Yield and Purity
3.5. 2xVH IgG and Fab Can Co-Engage Two Antigens: A Case Study of Anti-HA/Anti-hVEGF
3.6. VH Domain Placement Does Not Impact Binding to Target Antigen in 2xVH Format: A Case Study of Clinically Relevant Anti-hVEGF/Anti-hTNFR1
3.7. Anti-Her2/Anti-Lysozyme: VH/VH Interfaces Are Larger than Canonical VH/VL Interfaces
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Construct | PDB | Contact Surface VH (Å2) or VH1 | Contact Surface VL (Å2) or VH2 | Interface (Å2) |
---|---|---|---|---|
H3-23:L1-39 | 5I19 | 795 | 817 | 806 |
H3-23:L3-11 | 5I1A | 822 | 834 | 828 |
H3-23:L3-20 | 5I1C | 670 | 698 | 684 |
H3-23:L4-1 | 5I1D | 743 | 770 | 757 |
2xVH anti-Her2, Lysozyme | 7JKB | 1200 | 1143 | 1171 |
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Ramasubramanian, A.; Tennyson, R.; Magnay, M.; Kathuria, S.; Travaline, T.; Jain, A.; Lord, D.M.; Salemi, M.; Sullivan, C.; Magnay, T.; et al. Bringing the Heavy Chain to Light: Creating a Symmetric, Bivalent IgG-Like Bispecific. Antibodies 2020, 9, 62. https://doi.org/10.3390/antib9040062
Ramasubramanian A, Tennyson R, Magnay M, Kathuria S, Travaline T, Jain A, Lord DM, Salemi M, Sullivan C, Magnay T, et al. Bringing the Heavy Chain to Light: Creating a Symmetric, Bivalent IgG-Like Bispecific. Antibodies. 2020; 9(4):62. https://doi.org/10.3390/antib9040062
Chicago/Turabian StyleRamasubramanian, Anusuya, Rachel Tennyson, Maureen Magnay, Sagar Kathuria, Tara Travaline, Annu Jain, Dana M. Lord, Megan Salemi, Caitlin Sullivan, Tristan Magnay, and et al. 2020. "Bringing the Heavy Chain to Light: Creating a Symmetric, Bivalent IgG-Like Bispecific" Antibodies 9, no. 4: 62. https://doi.org/10.3390/antib9040062