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

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
• antibody engineering
• humanization
• in silico developability
• library design
• NGS
• single domain antibody
• VHH
• yeast surface display

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Acknowledgments

We thank Shira Warszawski, Satyendra Kumar, Sigrid Auth, Vanessa Lautenbach, and Dirk Müller-Pompalla for discussion and experimental and data support.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2023.2261149

Disclosure 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.

Additional information

Funding

The author(s) reported that there is no funding associated with the work featured in this article.