https://orcid.org/0000-0002-3385-9191
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ABSTRACT
Over the past two decades, therapeutic antibodies have emerged as a rapidly expanding domain within the field of biologics. In silico tools that can streamline the process of antibody discovery and optimization are critical to support a pipeline that is growing more numerous and complex every year. High-quality structural information remains critical for the antibody optimization process, but antibody-antigen complex structures are often unavailable and in silico antibody docking methods are still unreliable. In this study, DeepAb, a deep learning model for predicting antibody Fv structure directly from sequence, was used in conjunction with single-point experimental deep mutational scanning (DMS) enrichment data to design 200 potentially optimized variants of an anti-hen egg lysozyme (HEL) antibody. We sought to determine whether DeepAb-designed variants containing combinations of beneficial mutations from the DMS exhibit enhanced thermostability and whether this optimization affected their developability profile. The 200 variants were produced through a robust high-throughput method and tested for thermal and colloidal stability (Tonset, Tm, Tagg), affinity (KD) relative to the parental antibody, and for developability parameters (nonspecific binding, aggregation propensity, self-association). Of the designed clones, 91% and 94% exhibited increased thermal and colloidal stability and affinity, respectively. Of these, 10% showed a significantly increased affinity for HEL (5- to 21-fold increase) and thermostability (>2.5C increase in Tm1), with most clones retaining the favorable developability profile of the parental antibody. Additional in silico tests suggest that these methods would enrich for binding affinity even without first collecting experimental DMS measurements. These data open the possibility of in silico antibody optimization without the need to predict the antibody–antigen interface, which is notoriously difficult in the absence of crystal structures.
Disclosure statement
J.J.G. is an unpaid board member of the Rosetta Commons. Under institutional participation agreements between the University of Washington, acting on behalf of the Rosetta Commons, Johns Hopkins University may be entitled to a portion of revenue received on commercial licensing of Rosetta software including programs used here. J.J.G. has a financial interest in Cyrus Biotechnology. Cyrus Biotechnology distributes the Rosetta software, which may include methods used in this paper. J.A.R. was supported by the Johns Hopkins-AstraZeneca Scholars Program and is currently employed at Profluent Bio and may or may not hold Profluent Bio stock. M.H., G.V., T.P., N.M, H.S., K.R., R.C.W, M.D., Y.F., A.D. and G.K. are all AstraZeneca employees and may or may not hold AstraZeneca stock. N.H. was an AstraZeneca employee and may or may not hold AstraZeneca stock and is currently at Horizon Therapeutics and may or may not hold Horizon Therapeutics stock. M.I. was an AstraZeneca employee and may or may not hold AstraZeneca stock and is currently at Honigman LLP and may or may not hold Honigman LLP stock.
Code availability
The DeepAb code including a script for design calculations based on (score_design.py) is available at https://github.com/RosettaCommons/DeepAb.
Structural visualization
Structural visualization was conducted using Molecular Operating Environment (MOE) software version 2023.12.
Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2024.2362775
Abbreviations
| AC | = | SINS- Affinity-capture self-interaction nanoparticle spectroscopy |
| BLI | = | Bio-Layer Interferometry |
| BSA | = | Bovine Serum Albumin |
| BVP | = | Baculovirus particles |
| CCE | = | Common Configuration Enumeration |
| CDRL/H | = | Complementarity-determining region Light/Heavy |
| Cryo-EM | = | Cryogenic electron microscopy |
| DMS | = | Deep Mutational Scanning |
| DMTA | = | design-make-test-analyze |
| DNA | = | deoxyribonucleic acid |
| DSF | = | Differential Scanning Fluorimetry |
| EDTA | = | Ethylenediamine-tetra-acetic acid |
| ELISA | = | enzyme-linked immunosorbent assay |
| FRWL/H | = | Framework Light/Heavy |
| HBS-EP | = | Hepes Buffered Saline EDTA and Surfactant P20 |
| HEK | = | Human Embryonic Kidney |
| HEL | = | Hen Egg Lysozyme |
| HPLC | = | high-performance liquid chromatography |
| HP-SEC | = | High-pressure Size exclusion chromatography |
| HT | = | High-Throughput |
| IgG | = | Immunoglobulin G |
| LIMS | = | laboratory information management system |
| mAbs | = | monoclonal antibodies |
| MW | = | Molecular Weight |
| NSB | = | Non-specific Binding |
| P/S | = | penicillin/streptomycin |
| PBS | = | Phosphate Buffered Saline |
| PCR | = | Polymerase chain reaction |
| PEI | = | polyethylenimine |
| QSOX1 | = | quiescin sulfhydryl oxidase 1 |
| RPM | = | Rotations per minute |
| RSA | = | reversible self-association |
| RT | = | Retention Time |
| SDS-PAGE | = | Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis |
| SEC | = | MALS- Size exclusion chromatography with multi-angle static light scattering |
| SLS | = | static light scattering |
| SPR | = | surface plasmon resonance |
| UPLC-SEC | = | Ultra High pressure liquid chromatography- Size exclusion chromatography |
| VCD | = | Viable Cell Density |
| VEGF | = | Vascular endothelial growth factor |
| VH | = | Variable Heavy |
| VL | = | Variable Light |
| WT | = | Wild Type |