The immunogenicity of an anti-EGFR single domain antibody (VHH) is enhanced by misfolded amorphous aggregation but not by heat-induced aggregation

Kibria, Golam; Akazawa-Ogawa, Yoko; Rahman, Nafsoon; Hagihara, Yoshihiro; Kuroda, Yutaka

doi:10.1016/j.ejpb.2020.05.006

Cited by 16 publications

(21 citation statements)

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“…The secondary structure content of V HH -7D12 variants was assessed by circular dichroism (CD) using a far-UV range of 205-260 nm. The CD spectrum of the untagged V HH -7D12 was typical of a β-sheeted protein, and according to BestSel [34], it contained 44% of β-sheets and 1.2% of α-helices (Figure 2C and Table S2), which is in line with our previous report and the crystal structure [32,33]. The secondary structure content of the tagged variants was very similar to that of the untagged V HH -7D12 (Figure 2C and Table S2).…”

Section: Biophysical and Functional Properties Of V Hh -7d12 Variantssupporting

confidence: 89%

“…BL21 (DE3) pLysS cell lines, transformed with V HH plasmid, were grown in Luria Bertani (LB) medium (1 L) at 37 • C. Protein expression was induced with 1 mM IPTG when the OD at 590 nm of the culture reached 0.6. After 6 h of IPTG induction, the E. coli cells were harvested by centrifugation at 4500 rpm for 20 min at 4 • C. After sonication, V HH was purified by Ni-NTA (Qiagen, Hilden, Germany), followed by ion-exchange chromatography (Gigacap-s-650 M, Tosoh Bioscience, Tokyo, Japan) according to our previously reported protocol [33]. Protein identities were confirmed by MALDI-TOF mass spectrometry (Autoflex III, Bruker Daltonics, MA, USA), and the purified proteins dissolved in Milli-Q (MQ) water was kept at −30 • C as a stock protein solution.…”

Section: Large Scale Protein Expression and Purificationmentioning

confidence: 99%

“…The binding affinity of the anti-EGFR V HH -7D12 variant was measured by surface plasmon resonance (SPR) (Biacore x100, GE Healthcare, MA, USA), as previously reported [33]. In short, the extracellular domain of human EGFR (Abcam, Cambridge, UK) was immobilized onto a CM5 sensor chip using amine coupling according to the manufacturer's guidance.…”

Section: Surface Plasmon Resonance (Spr)mentioning

confidence: 99%

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Anti-EGFR VHH Antibody under Thermal Stress Is Better Solubilized with a Lysine than with an Arginine SEP Tag

et al. 2021

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In this study, we assessed the potential of arginine and lysine solubility-enhancing peptide (SEP) tags to control the solubility of a model protein, anti-EGFR VHH-7D12, in a thermally denatured state at a high temperature. We produced VHH-7D12 antibodies attached with a C-terminal SEP tag made of either five or nine arginines or lysines (7D12-C5R, 7D12-C9R, 7D12-C5K and 7D12-C9K, respectively). The 5-arginine and 5-lysine SEP tags increased the E. coli expression of VHH-7D12 by over 80%. Biophysical and biochemical analysis confirmed the native-like secondary and tertiary structural properties and the monomeric nature of all VHH-7D12 variants. Moreover, all VHH-7D12 variants retained a full binding activity to the EGFR extracellular domain. Finally, thermal stress with 45-minute incubation at 60 and 75 °C, where VHH-7D12 variants are unfolded, showed that the untagged VHH-7D12 formed aggregates in all of the four buffers, and the supernatant protein concentration was reduced by up to 35%. 7D12-C5R and 7D12-C9R did not aggregate in Na-acetate (pH 4.7) and Tris-HCl (pH 8.5) but formed aggregates in phosphate buffer (PB, pH 7.4) and phosphate buffer saline (PBS, pH 7.4). The lysine tags (either C5K or C9K) had the strongest solubilization effect, and both 7D12-C5K and 7D12-C9K remained in the supernatant. Altogether, our results indicate that, under a thermal stress condition, the lysine SEP tags solubilization effect is more potent than that of an arginine SEP tags, and the SEP tags did not affect the structural and functional properties of the protein.

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Section: Biophysical and Functional Properties Of V Hh -7d12 Variantssupporting

confidence: 89%

Section: Large Scale Protein Expression and Purificationmentioning

confidence: 99%

Section: Surface Plasmon Resonance (Spr)mentioning

confidence: 99%

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Anti-EGFR VHH Antibody under Thermal Stress Is Better Solubilized with a Lysine than with an Arginine SEP Tag

et al. 2021

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“…ELISA was performed as previously reported [14,33]. In short, anti‐BPTI IgG levels were evaluated using untagged BPTI‐19A (2.5 µg·mL −1 in PBS) as coating antigen on the 96‐well microtiter plates (TPP microtiter plates, Japan).…”

Section: Methodsmentioning

confidence: 99%

A systematic mutational analysis identifies a 5‐residue proline tag that enhances the in vivo immunogenicity of a non‐immunogenic model protein

et al. 2020

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“…The challenges associated with assembly and purification of multispecific antibodies may also result in an increased risk of immunogenicity. Moreover, critical quality attributes like aggregation, particle size, and polyspecificity are also thought to increase the likelihood of immunogenicity [ 88 , 89 , 239 , 242 , 243 , 244 ]. Given the unique immunogenic liabilities of multispecific antibodies and their increasing numbers in clinical trials, new tools for the analysis and reduction of immunogenic liabilities will be of increasing importance.…”

Section: Polyspecificty and In Vivo Propertiesmentioning

confidence: 99%

Toward Drug-Like Multispecific Antibodies by Design

Sawant

Streu

et al. 2020

IJMS

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The success of antibody therapeutics is strongly influenced by their multifunctional nature that couples antigen recognition mediated by their variable regions with effector functions and half-life extension mediated by a subset of their constant regions. Nevertheless, the monospecific IgG format is not optimal for many therapeutic applications, and this has led to the design of a vast number of unique multispecific antibody formats that enable targeting of multiple antigens or multiple epitopes on the same antigen. Despite the diversity of these formats, a common challenge in generating multispecific antibodies is that they display suboptimal physical and chemical properties relative to conventional IgGs and are more difficult to develop into therapeutics. Here we review advances in the design and engineering of multispecific antibodies with drug-like properties, including favorable stability, solubility, viscosity, specificity and pharmacokinetic properties. We also highlight emerging experimental and computational methods for improving the next generation of multispecific antibodies, as well as their constituent antibody fragments, with natural IgG-like properties. Finally, we identify several outstanding challenges that need to be addressed to increase the success of multispecific antibodies in the clinic.

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The immunogenicity of an anti-EGFR single domain antibody (VHH) is enhanced by misfolded amorphous aggregation but not by heat-induced aggregation

Cited by 16 publications

References 50 publications

Anti-EGFR VHH Antibody under Thermal Stress Is Better Solubilized with a Lysine than with an Arginine SEP Tag

Anti-EGFR VHH Antibody under Thermal Stress Is Better Solubilized with a Lysine than with an Arginine SEP Tag

A systematic mutational analysis identifies a 5‐residue proline tag that enhances the in vivo immunogenicity of a non‐immunogenic model protein

Toward Drug-Like Multispecific Antibodies by Design

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