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. 2023 Jan-Dec;15(1):2261149.
doi: 10.1080/19420862.2023.2261149. Epub 2023 Sep 27.

A library approach for the de novo high-throughput isolation of humanized VHH domains with favorable developability properties following camelid immunization

Paul Arras  1   2 , Han Byul Yoo  1   3 , Lukas Pekar  1 , Christian Schröter  4 , Thomas Clarke  5 , Simon Krah  1 , Daniel Klewinghaus  3 , Vanessa Siegmund  3 , Andreas Evers  1 , Stefan Zielonka  1   2
Affiliations

A library approach for the de novo high-throughput isolation of humanized VHH domains with favorable developability properties following camelid immunization

Paul Arras et al. MAbs. 2023 Jan-Dec.

Erratum in

  • Correction.
    [No authors listed] [No authors listed] MAbs. 2023 Jan-Dec;15(1):2285575. doi: 10.1080/19420862.2023.2285575. Epub 2023 Nov 27. MAbs. 2023. PMID: 38010730 Free PMC article. No abstract available.

Abstract

In this study, we generated a novel library approach for high throughput de novo identification of humanized single-domain antibodies following camelid immunization. To achieve this, VHH-derived complementarity-determining regions-3 (CDR3s) obtained from an immunized llama (Lama glama) were grafted onto humanized VHH backbones comprising moderately sequence-diversified CDR1 and CDR2 regions similar to natural immunized and naïve antibody repertoires. Importantly, these CDRs were tailored toward favorable in silico developability properties, by considering human-likeness as well as excluding potential sequence liabilities and predicted immunogenic motifs. Target-specific humanized single-domain antibodies (sdAbs) were readily obtained by yeast surface display. We demonstrate that, by exploiting this approach, high affinity sdAbs with an optimized in silico developability profile can be generated. These sdAbs display favorable biophysical, biochemical, and functional attributes and do not require any further sequence optimization. This approach is generally applicable to any antigen upon camelid immunization and has the potential to significantly accelerate candidate selection and reduce risks and attrition rates in sdAb development.

Keywords: Antibody display; NGS; VHH; antibody engineering; humanization; in silico developability; library design; single domain antibody; yeast surface display.

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Conflict of interest statement

PA, HY, LP, SK, DK, VS, CS, TC, AE, and SZ are or were employees at Merck Healthcare KGaA or EMD Serono. Besides, this work was conducted in the absence of any further commercial interest.

Figures

Figure 1.
Figure 1.
Overview of the library construction process.
Figure 2.
Figure 2.
Amino acid distribution of CDR1 and CDR2 of non-immunized llamas compared with artificially designed diversities and observed compositions in humanized sdAb libraries.
Figure 3.
Figure 3.
Yeast surface display enables the isolation of (rh) NKp46-targeting sdAbs from both humanized libraries.
Figure 4.
Figure 4.
Binding capacities of humanized sdAbs and WT VHHs reformatted as one-armed SEEDbodies as determined by BLI.
Figure 5.
Figure 5.
NK cell engagers (NKCEs) harboring de novo humanized sdAbs from the FERF design trigger significant NK cell mediated killing of EGFR overexpressing tumor cells.
See this image and copyright information in PMC

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