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


Abstract

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 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 TGFβ1. 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 TGFβ1 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.

Submission Details

ID: aibGh5TN4

Submitter: Shu-Ching Ou

Submission Date: March 21, 2019, 5:13 p.m.

Version: 1

Publication Details
Lord DM;Bird JJ;Honey DM;Best A;Park A;Wei RR;Qiu H,MAbs (2018) Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody. PMID:29333938
Additional Information

Study Summary

Number of data points 135
Proteins Metelimumab
Unique complexes 24
Assays/Quantities/Protocols Experimental Assay: ka/kon ; Experimental Assay: kd/koff ; Experimental Assay: Kd
Libraries Variants for Metelimumab-TGFβ1

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
7CZU 2020-09-09T00:00:00+0000 3.4 S protein of SARS-CoV-2 in complex bound with P5A-1B6_2B
7CZV 2020-09-09T00:00:00+0000 3.3 S protein of SARS-CoV-2 in complex bound with P5A-1B6_3B
2IG2 1989-04-18T00:00:00+0000 3.0 DIR PRIMAERSTRUKTUR DES KRISTALLISIERBAREN MONOKLONALEN IMMUNOGLOBULINS IGG1 KOL. II. AMINOSAEURESEQUENZ DER L-KETTE, LAMBDA-TYP, SUBGRUPPE I (GERMAN)
4HAG 2012-09-26T00:00:00+0000 3.4 Crystal structure of fc-fragment of human IgG2 antibody (centered crystal form)
4HAF 2012-09-26T00:00:00+0000 2.04 Crystal structure of fc-fragment of human IgG2 antibody (primitive crystal form)
4L4J 2013-06-07T00:00:00+0000 1.92 Crystal structure of fc-fragment of human IgG2-Sigma antibody
2FB4 1989-04-18T00:00:00+0000 1.9 DIR PRIMAERSTRUKTUR DES KRISTALLISIERBAREN MONOKLONALEN IMMUNOGLOBULINS IGG1 KOL. II. AMINOSAEURESEQUENZ DER L-KETTE, LAMBDA-TYP, SUBGRUPPE I (GERMAN)
7CZT 2020-09-09T00:00:00+0000 2.7 S protein of SARS-CoV-2 in complex bound with P5A-2G9
5W38 2017-06-07T00:00:00+0000 1.8 1.80A resolution structure of human IgG3 Fc (N392K)
6D58 2018-04-19T00:00:00+0000 2.39 Crystal structure of a Fc fragment of Human IgG3

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
200.0 A,L Metelimumab P01834 IGKC_HUMAN
183.6 B,H Metelimumab P01857 IGHG1_HUMAN
191.8 B,H Metelimumab P01860 IGHG3_HUMAN
191.8 B,H Metelimumab P01859 IGHG2_HUMAN
191.8 B,H Metelimumab P01772 HV333_HUMAN
191.8 B,H Metelimumab P0DP02 HVC33_HUMAN
200.0 B,H Metelimumab P01861 IGHG4_HUMAN
191.8 B,H Metelimumab P0DP03 HV335_HUMAN
191.8 B,H Metelimumab P01768 HV330_HUMAN