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Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding.

ABSTRACT: Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo.

SUBMITTER: Koenig P

PROVIDER: S-EPMC5278476 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding.

Koenig Patrick P Lee Chingwei V CV Walters Benjamin T BT Janakiraman Vasantharajan V Stinson Jeremy J Patapoff Thomas W TW Fuh Germaine G

Proceedings of the National Academy of Sciences of the United States of America 20170105 4

Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domain ...[more]

PMID: 28057863

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Project description:The identification of mutations that enhance antibody affinity while maintaining high antibody specificity and stability is a time-consuming and laborious process. Here, we report an efficient methodology for systematically and rapidly enhancing the affinity of antibody variable domains while maximizing specificity and stability using novel synthetic antibody libraries. Our approach first uses computational and experimental alanine scanning mutagenesis to identify sites in the complementarity-determining regions (CDRs) that are permissive to mutagenesis while maintaining antigen binding. Next, we mutagenize the most permissive CDR positions using degenerate codons to encode wild-type residues and a small number of the most frequently occurring residues at each CDR position based on natural antibody diversity. This mutagenesis approach results in antibody libraries with variants that have a wide range of numbers of CDR mutations, including antibody domains with single mutations and others with tens of mutations. Finally, we sort the modest size libraries (~10 million variants) displayed on the surface of yeast to identify CDR mutations with the greatest increases in affinity. Importantly, we find that single-domain (VHH) antibodies specific for the α-synuclein protein (whose aggregation is associated with Parkinson's disease) with the greatest gains in affinity (>5-fold) have several (four to six) CDR mutations. This finding highlights the importance of sampling combinations of CDR mutations during the first step of affinity maturation to maximize the efficiency of the process. Interestingly, we find that some natural diversity mutations simultaneously enhance all three key antibody properties (affinity, specificity, and stability) while other mutations enhance some of these properties (e.g., increased specificity) and display trade-offs in others (e.g., reduced affinity and/or stability). Computational modeling reveals that improvements in affinity are generally not due to direct interactions involving CDR mutations but rather due to indirect effects that enhance existing interactions and/or promote new interactions between the antigen and wild-type CDR residues. We expect that natural diversity mutagenesis will be useful for efficient affinity maturation of a wide range of antibody fragments and full-length antibodies.

Project description:Introduction: Protein conformational switches are often constructed by fusing an input domain, which recognizes a target ligand, to an output domain that establishes a biological response. Prior designs have employed binding-induced folding of the input domain to drive a conformational change in the output domain. Adding a second input domain can in principle harvest additional binding energy for performing useful work. It is not obvious, however, how to fuse two binding domains to a single output domain such that folding of both binding domains combine to effect conformational change in the output domain. Methods: Here, we converted the ribonuclease barnase (Bn) to a switchable enzyme by duplicating a C-terminal portion of its sequence and appending it to its N-terminus, thereby establishing a native fold (OFF state) and a circularly permuted fold (ON state) that competed for the shared core in a mutually exclusive fashion. Two copies of FK506 binding protein (FKBP), both made unstable by the V24A mutation and one that had been circularly permuted, were inserted into the engineered barnase at the junctions between the shared and duplicated sequences. Results: Rapamycin-induced folding of FK506 binding protein stretched and unfolded the native fold of barnase via the mutually exclusive folding effect, and rapamycin-induced folding of permuted FK506 binding protein stabilized the permuted fold of barnase by the loop-closure entropy principle. These folding events complemented each other to turn on RNase function. The cytotoxic switching mechanism was validated in yeast and human cells, and in vitro with purified protein. Discussion: Thermodynamic modeling and experimental results revealed that the dual action of loop-closure entropy and mutually exclusive folding is analogous to an engine transmission in which loop-closure entropy acts as the low gear, providing efficient switching at low ligand concentrations, and mutually exclusive folding acts as the high gear to allow the switch to reach its maximum response at high ligand concentrations.

Dataset Information

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding.

Publications

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding.

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