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Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration.

Koenig P, Lee CV, Sanowar S, Wu P, Stinson J, Harris SF, Fuh G
J Biol Chem 290 21773-86 (2015)
Cited: 25 times
EuropePMC logo PMID: 26088137

Abstract

The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies.

Reviews - 4zfg mentioned but not cited (1)

  1. Deep sequencing methods for protein engineering and design. Wrenbeck EE, Faber MS, Whitehead TA. Curr. Opin. Struct. Biol. 45 36-44 (2017)

Articles - 4zfg mentioned but not cited (2)

  1. Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration. Koenig P, Lee CV, Sanowar S, Wu P, Stinson J, Harris SF, Fuh G. J Biol Chem 290 21773-21786 (2015)
  2. Tuning the specificity of a Two-in-One Fab against three angiogenic antigens by fully utilizing the information of deep mutational scanning. Koenig P, Sanowar S, Lee CV, Fuh G. MAbs 9 959-967 (2017)


Reviews citing this publication (4)

  1. Functional assays for transcription mechanisms in high-throughput. Qiu C, Kaplan CD. Methods 159-160 115-123 (2019)
  2. Hierarchical design of artificial proteins and complexes toward synthetic structural biology. Arai R. Biophys Rev 10 391-410 (2018)
  3. Protein engineering via sequence-performance mapping. McConnell A, Hackel BJ. Cell Syst 14 656-666 (2023)
  4. Understanding and Modulating Antibody Fine Specificity: Lessons from Combinatorial Biology. Rojas G. Antibodies (Basel) 11 48 (2022)

Articles citing this publication (18)

  1. Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding. Koenig P, Lee CV, Walters BT, Janakiraman V, Stinson J, Patapoff TW, Fuh G. Proc. Natl. Acad. Sci. U.S.A. 114 E486-E495 (2017)
  2. Learning the heterogeneous hypermutation landscape of immunoglobulins from high-throughput repertoire data. Spisak N, Walczak AM, Mora T. Nucleic Acids Res 48 10702-10712 (2020)
  3. Antibody design using LSTM based deep generative model from phage display library for affinity maturation. Saka K, Kakuzaki T, Metsugi S, Kashiwagi D, Yoshida K, Wada M, Tsunoda H, Teramoto R. Sci Rep 11 5852 (2021)
  4. Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning. van Rosmalen M, Janssen BM, Hendrikse NM, van der Linden AJ, Pieters PA, Wanders D, de Greef TF, Merkx M. J. Biol. Chem. 292 1477-1489 (2017)
  5. Understanding and overcoming trade-offs between antibody affinity, specificity, stability and solubility. Rabia LA, Desai AA, Jhajj HS, Tessier PM. Biochem. Eng. J. 137 365-374 (2018)
  6. A Generic Strategy to Generate Bifunctional Two-in-One Antibodies by Chicken Immunization. Harwardt J, Bogen JP, Carrara SC, Ulitzka M, Grzeschik J, Hock B, Kolmar H. Front Immunol 13 888838 (2022)
  7. A bispecific IgG format containing four independent antigen binding sites. Ljungars A, Schiött T, Mattson U, Steppa J, Hambe B, Semmrich M, Ohlin M, Tornberg UC, Mattsson M. Sci Rep 10 1546 (2020)
  8. In vitro evolution of antibody affinity via insertional scanning mutagenesis of an entire antibody variable region. Skamaki K, Emond S, Chodorge M, Andrews J, Rees DG, Cannon D, Popovic B, Buchanan A, Minter RR, Hollfelder F. Proc Natl Acad Sci U S A 117 27307-27318 (2020)
  9. A multispecific monoclonal antibody G2 recognizes at least three completely different epitope sequences with high affinity. Mahmud MN, Oda M, Usui D, Inoshima Y, Ishiguro N, Kamatari YO. Protein Sci. 26 2162-2169 (2017)
  10. Accurate profiling of full-length Fv in highly homologous antibody libraries using UMI tagged short reads. Levin I, Štrajbl M, Fastman Y, Baran D, Twito S, Mioduser J, Keren A, Fischman S, Zhenin M, Nimrod G, Levitin N, Mayor MB, Gadrich M, Ofran Y. Nucleic Acids Res 51 e61 (2023)
  11. Adaption of human antibody λ and κ light chain architectures to CDR repertoires. van der Kant R, Bauer J, Karow-Zwick AR, Kube S, Garidel P, Blech M, Rousseau F, Schymkowitz J. Protein Eng. Des. Sel. 32 109-127 (2019)
  12. An optimized thermodynamics integration protocol for identifying beneficial mutations in antibody design. Sheng Z, Bimela JS, Wang M, Li Z, Guo Y, Ho DD. Front Immunol 14 1190416 (2023)
  13. Circular permutation profiling by deep sequencing libraries created using transposon mutagenesis. Atkinson JT, Jones AM, Zhou Q, Silberg JJ. Nucleic Acids Res. 46 e76 (2018)
  14. Epistasis in a Fitness Landscape Defined by Antibody-Antigen Binding Free Energy. Adams RM, Kinney JB, Walczak AM, Mora T. Cell Syst 8 86-93.e3 (2019)
  15. Mapping protein selectivity landscapes using multi-target selective screening and next-generation sequencing of combinatorial libraries. Naftaly S, Cohen I, Shahar A, Hockla A, Radisky ES, Papo N. Nat Commun 9 3935 (2018)
  16. Next-generation sequencing-guided identification and reconstruction of antibody CDR combinations from phage selection outputs. Barreto K, Maruthachalam BV, Hill W, Hogan D, Sutherland AR, Kusalik A, Fonge H, DeCoteau JF, Geyer CR. Nucleic Acids Res. 47 e50 (2019)
  17. Position-Specific Enrichment Ratio Matrix scores predict antibody variant properties from deep sequencing data. Smith MD, Case MA, Makowski EK, Tessier PM. Bioinformatics 39 btad446 (2023)
  18. Quantitative mapping of binding specificity landscapes for homologous targets by using a high-throughput method. Aharon L, Aharoni SL, Radisky ES, Papo N. Biochem J 477 1701-1719 (2020)


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