Effect of Storage on the Ability of 2-Mercaptoethanol to Inactivate γM Antibody

Pike, Robert M.; Chandler, Cleo H.

doi:10.1128/iai.5.3.416-417.1972

Cited by 4 publications

(2 citation statements)

References 7 publications

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“…4B]. Although it is unclear whether higher-order species in the 2 years AS sample occurred due to self-association or to disulfide bonding between monomers as BME became inactive (63), the reduction in binding affinity to VEGFR2 matched the loss of monomer in the sample. As a result, in this investigation we see the flow-on effects from the focus on thermostability alone to a broader set of biophysical features and a link between the concepts of evolvability (27,64) and developability (16).…”

Section: Discussionmentioning

confidence: 99%

Mutational and biophysical robustness in a prestabilized monobody

Chandler

Tan

Porebski

et al. 2021

Journal of Biological Chemistry

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Section: Discussionmentioning

confidence: 99%

Mutational and biophysical robustness in a prestabilized monobody

Chandler

Tan

Porebski

et al. 2021

Journal of Biological Chemistry

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“…The improved thermal stability of this construct then led to favourable features of accelerated stability (AS) [ Figure 4B]. Although it is unclear whether higher-order species in the 2 year AS sample occurred due to self-association or to disulfidebonding between monomers as BME became inactive (58), the reduction in binding affinity to VEGFR2 matched the loss of monomer in the sample. As a result, in this investigation we see the flow-on effects from the focus on thermostability alone to a broader set of biophysical features, and a link between the concepts of evolvability (59) and developability (16).…”

mentioning

confidence: 99%

Mutational and biophysical robustness in a pre-stabilized monobody

Chandler

Tan

Porebski

et al. 2020

Preprint

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The fibronectin type III (FN3) monobody domain is a promising non-antibody scaffold which features a less complex architecture than an antibody while maintaining analogous binding loops. We previously developed FN3Con, a hyper-stable monobody derivative with diagnostic and therapeutic potential. Pre-stabilization of the scaffold mitigates the stability-function trade-off commonly associated with evolving a protein domain towards biological activity. Here, we aimed to examine if the FN3Con monobody could take on antibody-like binding to therapeutic targets, while retaining its extreme stability. We targeted the first of the Adnectin derivative of monobodies to reach clinical trials, which was engineered by directed evolution for binding to the therapeutic target VEGFR2; however, this function was gained at the expense of large losses in thermostability and increased oligomerisation. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the pre-stabilized FN3Con scaffold to produce a domain that successfully bound with high affinity to the therapeutic target VEGFR2. This FN3Con-anti-VEGFR2 construct also maintains high thermostability, including remarkable long-term stability, retaining binding activity after 2 years of storage at 36 °C. Further investigations into buffer excipients doubled the presence of monomeric monobody in accelerated stability trials. These data suggest that loop grafting onto a pre-stabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics, and is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics.

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