"Redirecting an anti-IL-1ß antibody to bind a new, unrelated and computationally predicted epitope on hIL-17A".

Antibody engineering technology is at the forefront of therapeutic antibody development. The primary goal for engineering a therapeutic antibody is the generation of an antibody with a desired specificity, affinity, function, and developability profile. Mature antibodies are considered antigen specific, which may preclude their use as a starting point for antibody engineering. Here, we explore the plasticity of mature antibodies by engineering novel specificity and function to a pre-selected antibody template. Using a small, focused library, we engineered AAL160, an anti-IL-1ß antibody, to bind the unrelated antigen IL-17A, with the introduction of seven mutations. The final redesigned antibody, 11.003, retains favorable biophysical properties, binds IL-17A with sub-nanomolar affinity, inhibits IL-17A binding to its cognate receptor and is functional in a cell-based assay. The epitope of the engineered antibody can be computationally predicted based on the sequence of the template antibody, as is confirmed by the crystal structure of the 11.003/IL-17A complex. The structures of the 11.003/IL-17A and the AAL160/IL-1ß complexes highlight the contribution of germline residues to the paratopes of both the template and re-designed antibody. This case study suggests that the inherent plasticity of antibodies allows for re-engineering of mature antibodies to new targets, while maintaining desirable developability profiles.

Accurate profiling of full-length Fv in highly homologous antibody libraries using UMI tagged short reads.

Deep parallel sequencing (NGS) is a viable tool for monitoring scFv and Fab library dynamics in many antibody engineering high-throughput screening efforts. Although very useful, the commonly used Illumina NGS platform cannot handle the entire sequence of scFv or Fab in a single read, usually focusing on specific CDRs or resorting to sequencing VH and VL variable domains separately, thus limiting its utility in comprehensive monitoring of selection dynamics. Here we present a simple and robust method for deep sequencing repertoires of full length scFv, Fab and Fv antibody sequences. This process utilizes standard molecular procedures and unique molecular identifiers (UMI) to pair separately sequenced VH and VL. We show that UMI assisted VH-VL matching allows for a comprehensive and highly accurate mapping of full length Fv clonal dynamics in large highly homologous antibody libraries, as well as identification of rare variants. In addition to its utility in synthetic antibody discovery processes, our method can be instrumental in generating large datasets for machine learning (ML) applications, which in the field of antibody engineering has been hampered by conspicuous paucity of large scale full length Fv data.

Computational Design of Epitope-Specific Functional Antibodies.

The ultimate goal of protein design is to introduce new biological activity. We propose a computational approach for designing functional antibodies by focusing on functional epitopes, integrating large-scale statistical analysis with multiple structural models. Machine learning is used to analyze these models and predict specific residue-residue contacts. We use this approach to design a functional antibody to counter the proinflammatory effect of the cytokine interleukin-17A (IL-17A). X-ray crystallography confirms that the designed antibody binds the targeted epitope and the interaction is mediated by the designed contacts. Cell-based assays confirm that the antibody is functional. Importantly, this approach does not rely on a high-quality 3D model of the designed complex or even a solved structure of the target. As demonstrated here, this approach can be used to design biologically active antibodies, removing some of the main hurdles in antibody design and in drug discovery.

VB/MM protein landscapes: a study of the S(N)2 reaction in haloalkane dehalogenase.

QM/MM methods are widely used for studies of reaction mechanisms in water and protein environments. Recently, we have developed the VB/MM method in which the QM part is implemented by the ab initio valence bond (VB) method. Here, we report on further improvement of the VB/MM method which makes it possible to use the method for reactivity studies in systems where the QM and MM parts are connected by covalent bonds followed by first ab initio VB study of reactivity in proteins. We implemented a simple link atom scheme to treat the boundary interactions. We tested the performance of the link atom treatment in combination with the VB/MM method on an S(N)2 reaction and found it to be sufficiently accurate. We then used the VB/MM method to study the S(N)2 reaction in haloalkane dehalogenase (DhlA). We show that the predicted reaction barrier heights are in good agreement with estimated experimental values, thereby validating the method. Finally, we analyze the reaction energetics in terms of contributions of the VB configurations and conclude that such analysis is instrumental in pinpointing the essential features of the catalytic mechanism.

Studies of proton translocations in biological systems: simulating proton transport in carbonic anhydrase by EVB-based models.

Proton transport (PTR) processes play a major role in bioenergetics and thus it is important to gain a molecular understanding of these processes. At present the detailed description of PTR in proteins is somewhat unclear and it is important to examine different models by using well-defined experimental systems. One of the best benchmarks is provided by carbonic anhydrase III (CA III), because this is one of the few systems where we have a clear molecular knowledge of the rate constant of the PTR process and its variation upon mutations. Furthermore, this system transfers a proton between several water molecules, thus making it highly relevant to a careful examination of the "proton wire" concept. Obtaining a correlation between the structure of this protein and the rate of the PTR process should help to discriminate between alternative models and to give useful clues about PTR processes in other systems. Obviously, obtaining such a correlation requires a correct representation of the "chemistry" of PTR between different donors and acceptors, as well as the ability to evaluate the free energy barriers of charge transfer in proteins, and to simulate long-time kinetic processes. The microscopic empirical valence bond (Warshel, A., and R. M. Weiss. 1980. J. Am. Chem. Soc. 102:6218-6226; and Aqvist, J., and A. Warshel. 1993. Chem. Rev. 93:2523-2544) provides a powerful way for representing the chemistry and evaluating the free energy barriers, but it cannot be used with the currently available computer times in direct simulation of PTR with significant activation barriers. Alternatively, one can reduce the empirical valence bond (EVB) to the modified Marcus' relationship and use semimacroscopic electrostatic calculations plus a master equation to determine the PTR kinetics (Sham, Y., I. Muegge, and A. Warshel. 1999. Proteins. 36:484-500). However, such an approximation does not provide a rigorous multisite kinetic treatment. Here we combine the useful ingredients of both approaches and develop a simplified EVB effective potential that treats explicitly the chain of donors and acceptors while considering implicitly the rest of the protein/solvent system. This approach can be used in Langevin dynamics simulations of long-time PTR processes. The validity of our new simplified approach is demonstrated first by comparing its Langevin dynamics results for a PTR along a chain of water molecules in water to the corresponding molecular dynamics simulations of the fully microscopic EVB model. This study examines dynamics of both models in cases of low activation barriers and the dependence of the rate on the energetics for cases with moderate barriers. The study of the dependence on the activation barrier is next extended to the range of higher barriers, demonstrating a clear correlation between the barrier height and the rate constant. The simplified EVB model is then examined in studies of the PTR in carbonic anhydrase III, where it reproduces the relevant experimental results without the use of any parameter that is specifically adjusted to fit the energetics or dynamics of the reaction in the protein. We also validate the conclusions obtained previously from the EVB-based modified Marcus' relationship. It is concluded that this approach and the EVB-based model provide a reliable, effective, and general tool for studies of PTR in proteins. Finally in view of the behavior of the simulated result, in both water and the CA III, we conclude that the rate of PTR in proteins is determined by the electrostatic energy of the transferred proton as long as this energy is higher than a few kcal/mol.

Converting conformational changes to electrostatic energy in molecular motors: The energetics of ATP synthase.

F1-ATPase is the catalytic component of the ATP synthase molecular machine responsible for most of the uphill synthesis of ATP in living systems. The enormous advances in biochemical and structural studies of this machine provide an opportunity for detailed understanding of the nature of its rotary mechanism. However, further quantitative progress in this direction requires development of reliable ways of translating the observed structural changes to the corresponding energies. This requirement is particularly challenging because we are dealing with a large system that couples major structural changes with a chemical process. The present work provides such a structure-function correlation by using the linear response approximation to describe the rotary mechanism. This approach allows one to evaluate the energy of transitions between different conformational states by considering only the changes in the corresponding electrostatic energies of the ligands. The relevant energetics are also obtained by calculating the linear response approximation-based free energies of transferring the ligands from water to the different sites of F1-ATPase in their different conformational states. We also use the empirical valence bond approach to evaluate the actual free-energy profile for the ATP synthesis in the different conformational states of the system. Integrating the information from the different approaches provides a semiquantitative structure-function correlation for F1-ATPase. It is found that the conformational changes are converted to changes in the electrostatic interaction between the protein and its ligands, which drives the ATP synthesis.

Apparent NAC effect in chorismate mutase reflects electrostatic transition state stabilization.

The catalytic reaction of chorismate mutase (CM) has been the subject of major current attention. Nevertheless, the origin of the catalytic power of CM remains an open question. In particular, it has not been clear whether the enzyme works by providing electrostatic transition state stabilization (TSS), by applying steric strain, or by populating near attack conformation (NAC). The present work explores this issue by a systematic quantitative analysis. The overall catalytic effect is reproduced by the empirical valence bond (EVB) method. In addition, the binding free energy of the ground state and the transition state is evaluated, demonstrating that the enzyme works by TSS. Furthermore, the evaluation of the electrostatic contribution to the reduction of the activation energy establishes that the TSS results from electrostatic effects. It is also found that the apparent NAC effect is not the reason for the catalytic effect but the result of the TSS. It is concluded that in CM as in other enzymes the key catalytic effect is electrostatic TSS. However, since the charge distribution of the transition state and the reactant state is similar, the stabilization of the transition state leads to reduction in the distance between the reacting atoms in the reactant state.