Noncovalent antibody catenation on a target surface greatly increases the antigen-binding avidity
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Curated by eLife
eLife Assessment:
The authors sought to enhance antibody binding to target antigens via reversible catenation, as an alternative to affinity maturation, beginning by computationally establishing parameters under which this type of binding enhancement via avidity effects would occur, and then following up with proof-of-principle experiments. While computational predictions and experiments are in excellent agreement, some controls that would further strengthen data interpretation are lacking. If generally applicable, the approach would accelerate efforts to develop antibodies with enhanced binding potency relative to their progenitors, applicable to any area of research employing antibodies.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Immunoglobulin G (IgG) antibodies are widely used for diagnosis and therapy. Given the unique dimeric structure of IgG, we hypothesized that, by genetically fusing a homodimeric protein (catenator) to the C-terminus of IgG, reversible catenation of antibody molecules could be induced on a surface where target antigen molecules are abundant, and that it could be an effective way to greatly enhance the antigen-binding avidity. A thermodynamic simulation showed that quite low homodimerization affinity of a catenator, e.g . dissociation constant of 100 μM, can enhance nanomolar antigen-binding avidity to a picomolar level, and that the fold enhancement sharply depends on the density of the antigen. In a proof-of-concept experiment where antigen molecules are immobilized on a biosensor tip, the C-terminal fusion of a pair of weakly homodimerizing proteins to three different antibodies enhanced the antigen-binding avidity by at least 110 or 304 folds from the intrinsic binding avidity. Compared with the mother antibody, Obinutuzumab(Y101L) which targets CD20, the same antibody with fused catenators exhibited significantly enhanced binding to SU-DHL5 cells. Together, the homodimerization-induced antibody catenation would be a new powerful approach to improve antibody applications, including the detection of scarce biomarkers and targeted anticancer therapies.
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Author Response
Reviewer #1 (Public Review):
The authors have used computational models and protein design to enhance antibody binding, which should have broad applications pending a few additional controls. The authors' new method could have a broad and immediate impact on a variety of diagnostic procedures that use antibodies as sensitivity is often an issue in these kinds of experiments and the sensitivity enhancement achieved in the two test cases is substantial. Affinity maturation is a viable approach, but it is laborious and expensive. If the catenation method is generalizable, it will open up opportunities for antibody optimization for cases where affinity maturation is either not feasible or otherwise impractical. Less clear is how this method might enhance therapeutic potency. Issues that arise when using therapeutic …
Author Response
Reviewer #1 (Public Review):
The authors have used computational models and protein design to enhance antibody binding, which should have broad applications pending a few additional controls. The authors' new method could have a broad and immediate impact on a variety of diagnostic procedures that use antibodies as sensitivity is often an issue in these kinds of experiments and the sensitivity enhancement achieved in the two test cases is substantial. Affinity maturation is a viable approach, but it is laborious and expensive. If the catenation method is generalizable, it will open up opportunities for antibody optimization for cases where affinity maturation is either not feasible or otherwise impractical. Less clear is how this method might enhance therapeutic potency. Issues that arise when using therapeutic antibodies are often multifactorial and vary depending on the target and disease state. Many issues that occur with antibody-based therapies will not be rectified with affinity enhancement.
We agree with the limitation.
Reviewer #2 (Public Review):
The paper presents an interesting design approach to having homodimeric IgGs with higher binding affinity to the antigens on a surface by fusing a weakly homodimerizing protein (a catenator) to the C-terminus of IgG. Considering the homodimeric IgGs with likely enhanced antigen binding ability and their stabilization with a reversible catenation when bound to the surface is an interesting idea. With agent-based modeling - the simulations based on Markov Chain Monte Carlo (MCMC) sampling - and proof of concept experiments, it has been possible to show the enhanced antigen binding ability of the homodimer Igs for many folds, where the weakly homodimerizing ability of the catenator is indicated to have a central role, enabling proximity effect driven catenation on the antigen bound surfaces. While the results render the enhanced binding affinity of the catenated homodimeric IgGs, the study would benefit from a more elaborated interpretation and discussions of the results.
The following discussion is now stated in the revision (pages 19-20, in the revision); “While we demonstrated that dual catenator-fused heterodimeric IgGs can enhance binding avidity, the oligomer formation or potential intramolecular homodimerization of the catenator necessitates the development of a more robust catenator for application to conventional homodimeric IgGs. Specifically, the ideal catenator should geometrically disallow intramolecular homodimerization, exhibit fast association kinetics, and be able to withstand the standard low pH purification step. On the other hand, our demonstration indicates that this approach can be applied to bispecific antibodies employing a heterodimeric Fc.”
One interesting base of the discussion may include how the fusion of the catenator may likely affect the binding behavior, the intrinsic binding behavior, and/or on the global structural changes, of IgGs (monomeric and homodimeric (catenated) per se beyond its proximity-driven contribution. Would it lead to a more restricted structure in the mobility in the unbound states so as to decrease the entropic cost for the binding and thus increase the binding avidity/affinity (in addition to external proximity-driven association). In other words, what would be the role of entropy in the free energy of binding, given that the enthalpic contributions remain the same? Possible effects of the length of the catenator should also in parts be related to the entropy. For example, if a longer and more flexible catenator is considered, what would the resulting observation experimentally and computationally be?
The binding site occupancy depends on [catAb]/KD. Figure 4-figure supplement 2 shows the binding site occupancy and (KD)eff as a function of (KD)catenator. In this simulation, [catAb] was fixed (10-9 M) while KD was varied (from 10-8 to 10-6). In the figure legend and in the main text, we now explicitly state that KD was varied from 10-8 to 10-6 (page 30, in the revision). To address this comment, we set KD = 10 nM (as used for simulation in Figures 3 and 4), and varied [catAb] from 0.1 to 10 nM. The binding site occupancy and (KD)eff as a function of [catAb] are plotted for three different set values of (KD)catenator (1 μM, 10 μM and 100 μM). The new figures are now presented as Figure 4-figure supplement 3. This simulation shows that the enhancement of (KD)eff by increasing the concentration of catAb is much less dramatic than that by increasing the affinity for catenator homodimerization at [catAb] > 10 nM.
On the other side, simple simulation approaches have a high value with a level of abstraction while still keeping the physical and biological relevance. In the simulations, i.e. in the sampling of various states, three main terms/rules to govern the behavior are implemented. One is a term favoring an increase in the ability to bind (preventing to unbinding) to the surface upon the catenation of IgGs. This may need to be substantiated for the simulations not imposing a preassumed ability to increase the binding (or decrease the unbinding) ability upon the catenation.
We agree with the review in that the third rule favors the binding ability of catenated IgGs, because it assumes that catenated antibodies are not allowed to dissociate from the binding site. While this assumption is not exactly correct, we think that it is valid, considering the behavior of a multivalent ligand. When the IgG portion dissociates completely from the binding site, it is still anchored by the catenation arm, and thus it will rebind the same binding site immediately. This postulation agrees with the quantitative analysis showing that multivalent ligand exhibits orders of magnitude binding likelihood increase when the ligand size is comparable to the stretch length of a conjugating linker [Liese, S. & Netz, R. R., ACS Nano, 12, 4140 (2018)].
The weakly homodimerizing state of the catenator appears as one of the important aspects of the proposed design strategy. Would it also be possible that the experimental observations may readily also imply the higher binding ability of the catenator fused IfgG without the homodimerization on the surface (due to the reduced entropic cost for the binding)? The presentation of the evidence of the homodimerization of the catenator and the catenated IgGs on the surface would strengthen the findings and discussions.
To fully address this comment, we would need to consider the detailed molecular behavior of the IgG part, the catenator and the linker, probably using molecular dynamics simulation, which we think is outside the scope of the current work. We like to qualitatively describe what we think about the raised issues. Fused to the C-terminus of Fc, the catenator won’t affect the complementary determining region (CDR) of Fab which is located on the opposite side of the C-terminus of Fc. This notion is supported by the observation that the SDF-1α-fused antibodies exhibited association kinetics similar to those of the mother antibodies (Figure 5).
Regarding the mobility of the structure, we presume that the fused catenator would not interact with the antibody portion and thus it would not affect the intrinsic structural mobility of the antibody.
Since the catenator is fused to the C-terminus of Fc by a flexible linker, the homodimerization of catenator would decrease the entropy upon catenation. However, the enthalpic contribution would overcome the entropic loss, and result in negative free energy of the catenator homodimerization.
Figure 2-figure supplement 1 (in the revision) shows the simulation for five different values of the reach length (R), which is the sum of the linker length and half of the catenator length. The simulation results show that the likelihood of catenation decreases as the linker length increases over the distance (d) between the two adjacent catAb-2Ag complexes, while it is maximum when the reach length equals d. Since the catenator length is fixed, increasing the linker length (such that R > d) will lower the catenation effect.
Reviewer #3 (Public Review):
The authors proposed an antibody catenation strategy by fusing a homodimeric protein (catenator) to the C-terminus of IgG heavy chain and hypothesized that the catenated IgGs would enhance their overall antigen-binding strength (avidity) compared to individual IgGs. The thermodynamic simulations supported the hypothesis and indicated that the fold enhancement in antibody-antigen binding depended on the density of the antigen. The authors tested a catenator candidate, stromal cell-derived factor 1α (SDF-1α), on two purposely weakened antibodies, Trastuzumab(N30A/H91A), a weakened variant of the clinically used anti-HER2 antibody Trastuzumab, and glCV30, the germline version of a neutralizing antibody CV30 against SARS-CoV-2. Measured by a binding assay, the catenator-fused antibodies enhanced the two weak antibody-antigen binding by hundreds and thousands of folds, largely through slowing down the dissociation of the antibody-antigen interaction. Thus, the experimental data supported the catenation strategy and provided proof-of-concept for the enhanced overall antibody-antigen binding strength. Depending on specific applications, an enhanced antibody-antigen binding strength may improve an antibody's diagnostic sensitivity or therapeutic efficacy, thus holding clinical potential.
Thanks for the favorable comments.
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eLife Assessment:
The authors sought to enhance antibody binding to target antigens via reversible catenation, as an alternative to affinity maturation, beginning by computationally establishing parameters under which this type of binding enhancement via avidity effects would occur, and then following up with proof-of-principle experiments. While computational predictions and experiments are in excellent agreement, some controls that would further strengthen data interpretation are lacking. If generally applicable, the approach would accelerate efforts to develop antibodies with enhanced binding potency relative to their progenitors, applicable to any area of research employing antibodies.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with …
eLife Assessment:
The authors sought to enhance antibody binding to target antigens via reversible catenation, as an alternative to affinity maturation, beginning by computationally establishing parameters under which this type of binding enhancement via avidity effects would occur, and then following up with proof-of-principle experiments. While computational predictions and experiments are in excellent agreement, some controls that would further strengthen data interpretation are lacking. If generally applicable, the approach would accelerate efforts to develop antibodies with enhanced binding potency relative to their progenitors, applicable to any area of research employing antibodies.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
-
Reviewer #1 (Public Review):
The authors have used computational models and protein design to enhance antibody binding, which should have broad applications pending a few additional controls.
The authors' new method could have a broad and immediate impact on a variety of diagnostic procedures that use antibodies as sensitivity is often an issue in these kinds of experiments and the sensitivity enhancement achieved in the two test cases is substantial. Affinity maturation is a viable approach, but it is laborious and expensive. If the catenation method is generalizable, it will open up opportunities for antibody optimization for cases where affinity maturation is either not feasible or otherwise impractical. Less clear is how this method might enhance therapeutic potency. Issues that arise when using therapeutic antibodies are often …
Reviewer #1 (Public Review):
The authors have used computational models and protein design to enhance antibody binding, which should have broad applications pending a few additional controls.
The authors' new method could have a broad and immediate impact on a variety of diagnostic procedures that use antibodies as sensitivity is often an issue in these kinds of experiments and the sensitivity enhancement achieved in the two test cases is substantial. Affinity maturation is a viable approach, but it is laborious and expensive. If the catenation method is generalizable, it will open up opportunities for antibody optimization for cases where affinity maturation is either not feasible or otherwise impractical. Less clear is how this method might enhance therapeutic potency. Issues that arise when using therapeutic antibodies are often multifactorial and vary depending on the target and disease state. Many issues that occur with antibody-based therapies will not be rectified with affinity enhancement.
-
Reviewer #2 (Public Review):
The paper presents an interesting design approach to having homodimeric IgGs with higher binding affinity to the antigens on a surface by fusing a weakly homodimerizing protein (a catenator) to the C-terminus of IgG. Considering the homodimeric IgGs with likely enhanced antigen binding ability and their stabilization with a reversible catenation when bound to the surface is an interesting idea. With agent-based modeling - the simulations based on Markov Chain Monte Carlo (MCMC) sampling - and proof of concept experiments, it has been possible to show the enhanced antigen binding ability of the homodimer Igs for many folds, where the weakly homodimerizing ability of the catenator is indicated to have a central role, enabling proximity effect driven catenation on the antigen bound surfaces. While the results …
Reviewer #2 (Public Review):
The paper presents an interesting design approach to having homodimeric IgGs with higher binding affinity to the antigens on a surface by fusing a weakly homodimerizing protein (a catenator) to the C-terminus of IgG. Considering the homodimeric IgGs with likely enhanced antigen binding ability and their stabilization with a reversible catenation when bound to the surface is an interesting idea. With agent-based modeling - the simulations based on Markov Chain Monte Carlo (MCMC) sampling - and proof of concept experiments, it has been possible to show the enhanced antigen binding ability of the homodimer Igs for many folds, where the weakly homodimerizing ability of the catenator is indicated to have a central role, enabling proximity effect driven catenation on the antigen bound surfaces. While the results render the enhanced binding affinity of the catenated homodimeric IgGs, the study would benefit from a more elaborated interpretation and discussions of the results.
One interesting base of the discussion may include how the fusion of the catenator may likely affect the binding behavior, the intrinsic binding behavior, and/or on the global structural changes, of IgGs (monomeric and homodimeric (catenated) per se beyond its proximity-driven contribution. Would it lead to a more restricted structure in the mobility in the unbound states so as to decrease the entropic cost for the binding and thus increase the binding avidity/affinity (in addition to external proximity-driven association). In other words, what would be the role of entropy in the free energy of binding, given that the enthalpic contributions remain the same? Possible effects of the length of the catenator should also in parts be related to the entropy. For example, if a longer and more flexible catenator is considered, what would the resulting observation experimentally and computationally be?
On the other side, simple simulation approaches have a high value with a level of abstraction while still keeping the physical and biological relevance. In the simulations, i.e. in the sampling of various states, three main terms/rules to govern the behavior are implemented. One is a term favoring an increase in the ability to bind (preventing to unbinding) to the surface upon the catenation of IgGs. This may need to be substantiated for the simulations not imposing a preassumed ability to increase the binding (or decrease the unbinding) ability upon the catenation.
The weakly homodimerizing state of the catenator appears as one of the important aspects of the proposed design strategy. Would it also be possible that the experimental observations may readily also imply the higher binding ability of the catenator fused IfgG without the homodimerization on the surface (due to the reduced entropic cost for the binding)? The presentation of the evidence of the homodimerization of the catenator and the catenated IgGs on the surface would strengthen the findings and discussions.
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Reviewer #3 (Public Review):
The authors proposed an antibody catenation strategy by fusing a homodimeric protein (catenator) to the C-terminus of IgG heavy chain and hypothesized that the catenated IgGs would enhance their overall antigen-binding strength (avidity) compared to individual IgGs. The thermodynamic simulations supported the hypothesis and indicated that the fold enhancement in antibody-antigen binding depended on the density of the antigen. The authors tested a catenator candidate, stromal cell-derived factor 1α (SDF-1α), on two purposely weakened antibodies, Trastuzumab(N30A/H91A), a weakened variant of the clinically used anti-HER2 antibody Trastuzumab, and glCV30, the germline version of a neutralizing antibody CV30 against SARS-CoV-2. Measured by a binding assay, the catenator-fused antibodies enhanced the two weak …
Reviewer #3 (Public Review):
The authors proposed an antibody catenation strategy by fusing a homodimeric protein (catenator) to the C-terminus of IgG heavy chain and hypothesized that the catenated IgGs would enhance their overall antigen-binding strength (avidity) compared to individual IgGs. The thermodynamic simulations supported the hypothesis and indicated that the fold enhancement in antibody-antigen binding depended on the density of the antigen. The authors tested a catenator candidate, stromal cell-derived factor 1α (SDF-1α), on two purposely weakened antibodies, Trastuzumab(N30A/H91A), a weakened variant of the clinically used anti-HER2 antibody Trastuzumab, and glCV30, the germline version of a neutralizing antibody CV30 against SARS-CoV-2. Measured by a binding assay, the catenator-fused antibodies enhanced the two weak antibody-antigen binding by hundreds and thousands of folds, largely through slowing down the dissociation of the antibody-antigen interaction. Thus, the experimental data supported the catenation strategy and provided proof-of-concept for the enhanced overall antibody-antigen binding strength. Depending on specific applications, an enhanced antibody-antigen binding strength may improve an antibody's diagnostic sensitivity or therapeutic efficacy, thus holding clinical potential.
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