Delicate balance among thermal stability, binding affinity, and conformational space explored by single-domain VHH antibodies

Ikeuchi, Emina; Kuroda, Daisuke; Nakakido, Makoto; Murakami, Akikazu; Tsumoto, Kouhei

doi:10.1038/s41598-021-98977-8

Cited by 15 publications

(12 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is possible that the spatial structure of B2-3 was reconstructed due to heat-induced denaturation that exceeded its melting point, which transformed the conformational space of a CDR, opened the hidden side of its binding to the antigen, and increased the binding affinity. 22 In a word, the AaLS could be used as a promising candidate for multifunctional delivery nanoplatform. Its advantages will further increase its application in disease diagnosis, prevention, and treatment.…”

Section: Discussionmentioning

confidence: 99%

A Multivalent and Thermostable Nanobody Neutralizing SARS-CoV-2 Omicron (B.1.1.529)

Lu¹,

Li²,

Fan³

et al. 2023

IJN

View full text Add to dashboard Cite

Background The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants have risen to dominance, which contains far more mutations in the spike protein in comparison to previously reported variants, compromising the efficacy of most existing vaccines or therapeutic monoclonal antibodies. Nanobody screened from high-throughput naïve libraries is a potential candidate for developing preventive and therapeutic antibodies. Methods Four nanobodies specific to the SARS-CoV-2 wild-type receptor-binding domain (RBD) were screened from a naïve phage display library. Their affinity and neutralizing activity were evaluated by surface plasmon resonance assays, surrogate virus neutralization tests, and pseudovirus neutralization assays. Preliminary identification of the binding epitopes of nanobodies by peptide-based ELISA and competition assay. Then four multivalent nanobodies were engineered by attaching the monovalent nanobodies to an antibody-binding nanoplatform constructed based on the lumazine synthase protein cage nanoparticles isolated from the Aquifex aeolicus (AaLS). Finally, the differences in potency between the monovalent and multivalent nanobodies were compared using the same methods. Results Three of the four specific nanobodies could maintain substantial inhibitory activity against the Omicron (B.1.1.529), of them, B-B2 had the best neutralizing activity against the Omicron (B.1.1.529) pseudovirus (IC 50 = 1.658 μg/mL). The antiviral ability of multivalent nanobody LS-B-B2 was improved in the Omicron (B.1.1.529) pseudovirus assays (IC 50 = 0.653 μg/mL). The results of peptide-based ELISA indicated that LS-B-B2 might react with the linear epitopes in the SARS-CoV-2 RBD conserved regions, which would clarify the mechanisms for the maintenance of potent neutralization of Omicron (B.1.1.529) preliminary. Conclusion Our study indicated that the AaLS could be used as an antibody-binding nanoplatform to present nanobodies on its surface and improve the potency of nanobodies. The multivalent nanobody LS-B-B2 may serve as a potential agent for the neutralization of SARS-CoV-2 variants.

show abstract

Section: Discussionmentioning

confidence: 99%

A Multivalent and Thermostable Nanobody Neutralizing SARS-CoV-2 Omicron (B.1.1.529)

Lu¹,

Li²,

Fan³

et al. 2023

IJN

View full text Add to dashboard Cite

show abstract

“…The development of safe and cost-e cient ligands effective for large-scale protein processing is critical for a nity puri cation. VHH-based a nity chromatography ful ls the needs of large-scale and 'one-step' puri cation, which is attributed to its unique characteristics such as small size (15KD), high a nity, speci city, structural stability, easy preparation via prokaryotic expression, and low cost of use [20] .…”

Section: Discussionmentioning

confidence: 99%

Purification of SARS-CoV-2 RBD in Affinity Chromatography Using a Novel Nanobody Ligand

Zhu

Lin²,

Qiu³

et al. 2023

Preprint

View full text Add to dashboard Cite

In the spike protein of SARS-CoV-2, the receptor-binding domain (RBD) contains multiple dominant neutralizing epitopes and can be used as an antigen for developing COVID-19 vaccines and neutral antibodies. Affinity chromatography is one of the most extensively used methods for rapid one-step protein purification. However, there is a lack of commercially available affinity ligands for RBD purification. Here, we report the rapid isolation of a nanobody suitable for purifying RBD as an affinity ligand from immune phage display libraries. After bio-panning, the enriched clones were sequenced on next-generation sequencing (NGS) platforms and classified into four groups based on the CDRH3 amino acid sequence. The representative sequences with high nanomolar affinities to RBD were further categorized into two groups via epitope binning analysis. Finally, from the two epitope bins, we found that SS3 showed easy elution under a mild eluting condition and could be used as a functional affinity ligand to purify RBD. These results also indicate that categorizing the bio-panned sequences via high-throughput sequencing (HTS) techniques followed by epitope binning represents a fast workflow to select specific binders with desired properties.

show abstract

“…The expression and purification of the recombinant VHHs ware carried out based on a previous report. 35 Briefly, Escherichia coli BL21(DE3) cells (Merck; Darmstadt, Germany) were transformed with the plasmid constructs. The recombinant VHHs were extracted from a periplasmic fraction of E. coli using ultrasonication, and purified using Ni‐NTA agarose (QIAGEN; Düsseldorf, Germany) equilibrated with the binding buffer (500 mM NaCl, 50 mM Tris–HCl, pH 8.0, containing 5 mM imidazole).…”

Section: Methodsmentioning

confidence: 99%

“…To generate 7D12‐Vob or introduce mutations, a standard inverse PCR method was performed using primers containing each CDR sequence or each of the mutations. The expression and purification of the recombinant VHHs ware carried out based on a previous report 35 . Briefly, Escherichia coli BL21(DE3) cells (Merck; Darmstadt, Germany) were transformed with the plasmid constructs.…”

Section: Methodsmentioning

confidence: 99%

Molecular basis for thermal stability and affinity in a VHH: Contribution of the framework region and its influence in the conformation of the CDR3

et al. 2022

Self Cite

View full text Add to dashboard Cite

The camelid single domain antibody, referred to VHH or Nanobody, is considered a versatile tool for various biotechnological and clinical applications because of its favorable biophysical properties. To take advantage of these characteristics and for its application in biotechnology and therapy, research on VHH engineering is currently vigorously conducted. To humanize a camelid VHH, we performed complementarity determining region (CDR) grafting using a humanized VHH currently in clinical trials, and investigated the effects of these changes on the biophysical properties of the resulting VHH. The chimeric VHH exhibited a significant decrease in affinity and thermal stability and a large conformational change in the CDR3. To elucidate the molecular basis for these changes, we performed mutational analyses on the framework regions revealing the contribution of individual residues within the framework region. It is demonstrated that the mutations resulted in the loss of affinity and lower thermal stability, revealing the significance of bulky residues in the vicinity of the CDR3, and the importance of intramolecular interactions between the CDR3 and the framework‐2 region. Subsequently, we performed back‐mutational analyses on the chimeric VHH. Back‐mutations resulted in an increase of the thermal stability and affinity. These data suggested that back‐mutations restored the intramolecular interactions, and proper positioning and/or dynamics of the CDR3, resulting in the gain of thermal stability and affinity. These observations revealed the molecular contribution of the framework region on VHHs and further designability of the framework region of VHHs without modifying the CDRs.

show abstract

Delicate balance among thermal stability, binding affinity, and conformational space explored by single-domain VHH antibodies

Cited by 15 publications

References 68 publications

A Multivalent and Thermostable Nanobody Neutralizing SARS-CoV-2 Omicron (B.1.1.529)

A Multivalent and Thermostable Nanobody Neutralizing SARS-CoV-2 Omicron (B.1.1.529)

Purification of SARS-CoV-2 RBD in Affinity Chromatography Using a Novel Nanobody Ligand

Molecular basis for thermal stability and affinity in a VHH: Contribution of the framework region and its influence in the conformation of the CDR3

Contact Info

Product

Resources

About