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Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGF?1 antibody.

ABSTRACT: Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGF?1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGF?1 binding affinity was reduced by ?50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGF?1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGF?1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies.

SUBMITTER: Lord DM

PROVIDER: S-EPMC5916551 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody.

Lord Dana M DM Bird Julie J JJ Honey Denise M DM Best Annie A Park Anna A Wei Ronnie R RR Qiu Huawei H

mAbs 20180201 3

Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGFβ1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGFβ1 binding affinity was reduced by ∼50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be alt ...[more]

PMID: 29333938

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Project description:Development of biotherapeutics is hampered by the inherent risk of immunogenicity, which requires extensive clinical assessment and possible re-engineering efforts for mitigation. The focus in the pre-clinical phase is to determine the likelihood of developing treatment-emergent anti-drug antibodies (TE-ADA) and presence of pre-existing ADA in drug-naïve individuals as risk-profiling strategies. Pre-existing ADAs are routinely identified during clinical immunogenicity assessment, but their origin and impact on drug safety and efficacy have not been fully elucidated. One specific class of pre-existing ADAs has been described, which targets neoepitopes of antibody fragments, including Fabs, VH, or VHH domains in isolation from their IgG context. With the increasing number of antibody fragments and other small binding scaffolds entering the clinic, a widely applicable method to mitigate pre-existing reactivity against these molecules is desirable. Here is described a structure-based engineering approach to abrogate pre-existing ADA reactivity to the C-terminal neoepitope of VH(H)s. On the basis of 3D structures, small modifications applicable to any VH(H) are devised that would not impact developability or antigen binding. In-silico B cell epitope mapping algorithms were used to rank the modified VHH variants by antigenicity; however, the limited discriminating capacity of the computational methods prompted an experimental evaluation of the engineered molecules. The results identified numerous modifications capable of reducing pre-existing ADA binding. The most efficient consisted of the addition of two proline residues at the VHH C-terminus, which led to no detectable pre-existing ADA reactivity while maintaining favorable developability characteristics. The method described, and the modifications identified thereby, may provide a broadly applicable solution to mitigate immunogenicity risk of antibody-fragments in the clinic and increase safety and efficacy of this promising new class of biotherapeutics.

Dataset Information

Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGF?1 antibody.

Publications

Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody.

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