A general approach for stabilizing nanobodies for intracellular expression
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eLife assessment
Dingus et al. have developed an innovative approach for improving the intracellular stability of nanobodies. Working with a set of 75 nanobodies, the authors have identified key amino acid changes that can improve the stability of nanobodies expressed within the cell that might be generalized to other nanobodies.
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Abstract
Conventional antibodies and their derived fragments are difficult to deploy against intracellular targets in live cells, due to their bulk and structural complexity. Nanobodies provide an alternative modality, with well-documented examples of intracellular expression. Despite their promise as intracellular reagents, there has not been a systematic study of nanobody intracellular expression. Here, we examined intracellular expression of 75 nanobodies from the Protein Data Bank. Surprisingly, a majority of these nanobodies were unstable in cells, illustrated by aggregation and clearance. Using comparative analysis and framework mutagenesis, we developed a general approach that stabilized a great majority of nanobodies that were originally unstable intracellularly, without significantly compromising target binding. This approach led to the identification of distinct sequence features that impacted the intracellular stability of tested nanobodies. Mutationally stabilized nanobody expression was found to extend to in vivo contexts, in the murine retina and in E. coli . These data provide for improvements in nanobody engineering for intracellular applications, potentiating a growing field of intracellular interrogation and intervention.
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Author Response
Reviewer #3 (Public Review):
Dingus et al. have developed an innovative and powerful approach for improving the intracellular stability of nanobodies. Nanobodies are single chain antibodies that are typically generated in select species such as llamas or alpacas. Because nanobodies are secreted and are present in general in the extracellular environment, they often become unstable when expressed in the reduced intracellular environment. Dingus et al. investigated 75 nanobodies from the Protein Data Bank and found that 42 were unstable when expressed intracellularly. In order to improve stability of these nanobodies, they first determined consensus residues that were present within the framework region, which does not include the CDR regions, in over 80% of the stable nanobodies. Mutating residues within the framework …
Author Response
Reviewer #3 (Public Review):
Dingus et al. have developed an innovative and powerful approach for improving the intracellular stability of nanobodies. Nanobodies are single chain antibodies that are typically generated in select species such as llamas or alpacas. Because nanobodies are secreted and are present in general in the extracellular environment, they often become unstable when expressed in the reduced intracellular environment. Dingus et al. investigated 75 nanobodies from the Protein Data Bank and found that 42 were unstable when expressed intracellularly. In order to improve stability of these nanobodies, they first determined consensus residues that were present within the framework region, which does not include the CDR regions, in over 80% of the stable nanobodies. Mutating residues within the framework of unstable nanobodies to match consensus residues in the stable nanobodies stabilized 26 of 42 nanobodies. Mutating consensus unstable residues stabilized another 11. Thus 37/42 unstable nanobodies were stabilized using this mutational approach. Further experiments provided evidence that some of the stabilized nanobodies still had some affinity for their targets. Furthermore, one stabilized nanobody was stable when expressed in the retina in vivo and 3 of 5 were stable when expressed in bacteria.
- This study provides a straightforward approach to improving the intracellular stability of nanobodies that could prove to be very useful for solving a common and vexing problem.
Thanks!
- From the data provided, it was difficult to determine whether the binding affinity of the mutated nanobodies had been diminished by the mutations that increased stability, and if so, by how much. Furthermore, target binding affinity was assessed for just 5 nanobodies, which calls into question whether this strategy will be useful.
It is the case that we are unable to guarantee that any nanobody stabilized by our consensus-based approach will retain full target-binding affinity. It is additionally not guaranteed that a given nanobody will be able to bind its target in cells in the absence of any mutagenesis, as paratope structure may be influenced/compromised in the intracellular environment. We are additionally limited in what we can test intracellularly, as the majority of current nanobodies target extracellular factors that cannot be effectively expressed intracellularly. What we provide is a rationale for limited impact on target binding via partial consensus mutagenesis, which excludes highly variable framework positions, most likely to contribute directly to binding, from mutagenesis. While our approach to generalizable intracellular stabilization may not be perfect for every nanobody, we believe it is likely to be a simple and useful approach in a variety of cases, and likely the majority of cases.
- Ultimately, the goal of expressing most nanobodies intracellularly is to bind to endogenous targets. It is difficult to assess how useful the stabilization strategy will be since it was not determined whether any of the stabilized nanobodies could bind their endogenous targets intracellularly.
We are limited in the number of intracellular targets we are currently able to test, as most current nanobodies target extracellular antigens. Endogenous intracellular targets are even more limited. However, we agree that targeting endogenous targets is ultimately the goal. We have included an in vivo experiment against the endogenous target GFAP in our revised manuscript, where we show that binding is preserved following mutagenesis (Figure 6).
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eLife assessment
Dingus et al. have developed an innovative approach for improving the intracellular stability of nanobodies. Working with a set of 75 nanobodies, the authors have identified key amino acid changes that can improve the stability of nanobodies expressed within the cell that might be generalized to other nanobodies.
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Reviewer #1 (Public Review):
Dingus J et al investigated an important technical issue with the use of single domain antibodies (nanobodies) as intracellularly expressed proteins to probe cellular biology. Over the past decade, the relative simplicity and stability of nanobodies compared to conventional antibodies has led to many interesting uses of these molecules as either sensors or means to perturb intracellular protein function. Many have generally assumed that the increased stability of nanobodies enables them to be expressed in a functional form within the reducing environment of the cytoplasm. With an observation that many nanobodies are actually not stable within the cytoplasm, the authors aimed to determine the sequence determinants of what drives stability/instability, and then devised strategies to rescue folding of unstable …
Reviewer #1 (Public Review):
Dingus J et al investigated an important technical issue with the use of single domain antibodies (nanobodies) as intracellularly expressed proteins to probe cellular biology. Over the past decade, the relative simplicity and stability of nanobodies compared to conventional antibodies has led to many interesting uses of these molecules as either sensors or means to perturb intracellular protein function. Many have generally assumed that the increased stability of nanobodies enables them to be expressed in a functional form within the reducing environment of the cytoplasm. With an observation that many nanobodies are actually not stable within the cytoplasm, the authors aimed to determine the sequence determinants of what drives stability/instability, and then devised strategies to rescue folding of unstable nanobodies in the cytosol. They first looked at 75 nanobodies and use a fluorescence based metric to determine which nanobodies are stable and unstable. This revealed a set of residues that are enriched in either category. With a further strategy to determine consensus changes for stability, the authors rescued the stability of a large fraction of unstable nanobodies. Further analysis allowed the authors to whittle down to a few mutations that are important for stability, with some structural considerations in mind. In further important experiments, the authors show that these rescuing mutations generally do not destroy antigen binding. Importantly, they clearly highlight a few examples where the stability rescue strategy impairs antigen binding. Finally, experiments in retinal cells and bacteria support the success of this strategy.
The overall manuscript is well presented with clear data and appropriate caveats included throughout the work.
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Reviewer #2 (Public Review):
This study investigated a substantial set of camelid nanobodies for their characteristics when expressed in mammalian cells as intrabodies. Intrabodies have a variety of important research, diagnostic and therapeutic uses, and nanobodies have several inherent characteristics that make them amenable for use as intrabodies. While a substantial number of nanobodies have been developed that are effective as intrabodies, a systematic study of the suitability of a set of otherwise unrelated nanobodies for this purpose has not been performed. As such, the molecular characteristics of what may make an nanobody suitable for use as an intrabody have not been defined. This study addresses this gap in knowledge by FP-tagging a set of 75 nanobodies selected from among those whose structure has been solved. The study uses …
Reviewer #2 (Public Review):
This study investigated a substantial set of camelid nanobodies for their characteristics when expressed in mammalian cells as intrabodies. Intrabodies have a variety of important research, diagnostic and therapeutic uses, and nanobodies have several inherent characteristics that make them amenable for use as intrabodies. While a substantial number of nanobodies have been developed that are effective as intrabodies, a systematic study of the suitability of a set of otherwise unrelated nanobodies for this purpose has not been performed. As such, the molecular characteristics of what may make an nanobody suitable for use as an intrabody have not been defined. This study addresses this gap in knowledge by FP-tagging a set of 75 nanobodies selected from among those whose structure has been solved. The study uses live cell imaging to evaluate expression of these nanobodies when expressed in mammalian cells. These results are used in bioinformatics analyses to define key amino acids positions in the nanobodies that distinguish those that have high level expression in diffuse cytoplasmic pattern that is consistent with expression in a stable, soluble form. These analyses inform mutagenesis to phenoconvert poorly expressed nanobodies into those with improved expression. The outcome is a set of rules that can be used by investigators to predict the likely characteristics of a nanobody with a given sequence when expressed in cells as an intrabody. The strengths of the study is the elegant and rational manner it is pursued by the iterative application of bioinformatics analyses of nanobody sequences, cell biological assays of expression as intrabodies and mutagenesis. This study has great value to the field as nanobodies gain increased use as intrabodies. The weakness is the lack of a quantitative analysis of expression levels and solubility, with all of the results based on a subjective visual determination of the appearance of the FP-tagged nanobody in expressing cells. Moreover, steady-state appearance is used to infer active processes of aggregation and clearance. Another weakness is that the study presumes that the steady-state expression levels of FP-tagged nanobodies are determined solely by posttranslational stability/solubility, and not by differences in transfection levels, transcription, and translation. Lastly, the study implies that the set studied here is representative of nanobodies in general and the results are transferable across all nanobodies. While the study still has substantial value in spite of these weaknesses, the manuscript would be greatly improved by explicitly stating these limitations of the study.
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Reviewer #3 (Public Review):
Dingus et al. have developed an innovative and powerful approach for improving the intracellular stability of nanobodies. Nanobodies are single chain antibodies that are typically generated in select species such as llamas or alpacas. Because nanobodies are secreted and are present in general in the extracellular environment, they often become unstable when expressed in the reduced intracellular environment. Dingus et al. investigated 75 nanobodies from the Protein Data Bank and found that 42 were unstable when expressed intracellularly. In order to improve stability of these nanobodies, they first determined consensus residues that were present within the framework region, which does not include the CDR regions, in over 80% of the stable nanobodies. Mutating residues within the framework of unstable …
Reviewer #3 (Public Review):
Dingus et al. have developed an innovative and powerful approach for improving the intracellular stability of nanobodies. Nanobodies are single chain antibodies that are typically generated in select species such as llamas or alpacas. Because nanobodies are secreted and are present in general in the extracellular environment, they often become unstable when expressed in the reduced intracellular environment. Dingus et al. investigated 75 nanobodies from the Protein Data Bank and found that 42 were unstable when expressed intracellularly. In order to improve stability of these nanobodies, they first determined consensus residues that were present within the framework region, which does not include the CDR regions, in over 80% of the stable nanobodies. Mutating residues within the framework of unstable nanobodies to match consensus residues in the stable nanobodies stabilized 26 of 42 nanobodies. Mutating consensus unstable residues stabilized another 11. Thus 37/42 unstable nanobodies were stabilized using this mutational approach. Further experiments provided evidence that some of the stabilized nanobodies still had some affinity for their targets. Furthermore, one stabilized nanobody was stable when expressed in the retina in vivo and 3 of 5 were stable when expressed in bacteria.
1. This study provides a straightforward approach to improving the intracellular stability of nanobodies that could prove to be very useful for solving a common and vexing problem.
2. From the data provided, it was difficult to determine whether the binding affinity of the mutated nanobodies had been diminished by the mutations that increased stability, and if so, by how much. Furthermore, target binding affinity was assessed for just 5 nanobodies, which calls into question whether this strategy will be useful.
3. Ultimately, the goal of expressing most nanobodies intracellularly is to bind to endogenous targets. It is difficult to assess how useful the stabilization strategy will be since it was not determined whether any of the stabilized nanobodies could bind their endogenous targets intracellularly.
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