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Epitope-specific affinity maturation improved stability of potent protease inhibitory antibodies.

ABSTRACT: Targeting effectual epitopes is essential for therapeutic antibodies to accomplish their desired biological functions. This study developed a competitive dual color fluorescence-activated cell sorting (FACS) to maturate a matrix metalloprotease 14 (MMP-14) inhibitory antibody. Epitope-specific screening was achieved by selection on MMP-14 during competition with N-terminal domain of tissue inhibitor of metalloproteinase-2 (TIMP-2) (nTIMP-2), a native inhibitor of MMP-14 binding strongly to its catalytic cleft. 3A2 variants with high potency, selectivity, and improved affinity and proteolytic stability were isolated from a random mutagenesis library. Binding kinetics indicated that the affinity improvements were mainly from slower dissociation rates. In vitro degradation tests suggested the isolated variants had half lives 6-11-fold longer than the wt. Inhibition kinetics suggested they were competitive inhibitors which showed excellent selectivity toward MMP-14 over highly homologous MMP-9. Alanine scanning revealed that they bound to the vicinity of MMP-14 catalytic cleft especially residues F204 and F260, suggesting that the desired epitope was maintained during maturation. When converted to immunoglobulin G, B3 showed 5.0?nM binding affinity and 6.5?nM inhibition potency with in vivo half-life of 4.6 days in mice. In addition to protease inhibitory antibodies, the competitive FACS described here can be applied for discovery and engineering biosimilars, and in general for other circumstances where epitope-specific modulation is needed.

SUBMITTER: Lopez T

PROVIDER: S-EPMC6202216 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Epitope-specific affinity maturation improved stability of potent protease inhibitory antibodies.

Lopez Tyler T Chuan Chen C Ramirez Aaron A Chen Kuan-Hui E KE Lorenson Mary Y MY Benitez Chris C Mustafa Zahid Z Pham Henry H Sanchez Ramon R Walker Ameae M AM Ge Xin X

Biotechnology and bioengineering 20180915 11

Targeting effectual epitopes is essential for therapeutic antibodies to accomplish their desired biological functions. This study developed a competitive dual color fluorescence-activated cell sorting (FACS) to maturate a matrix metalloprotease 14 (MMP-14) inhibitory antibody. Epitope-specific screening was achieved by selection on MMP-14 during competition with N-terminal domain of tissue inhibitor of metalloproteinase-2 (TIMP-2) (nTIMP-2), a native inhibitor of MMP-14 binding strongly to its c ...[more]

PMID: 30102763

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Project description:ObjectiveRheumatoid arthritis (RA) is characterized by the presence of anti-citrullinated protein antibodies (ACPAs); nevertheless, the origin, specificity, and functional properties of ACPAs remain poorly understood. The aim of this study was to characterize the evolution of ACPAs by sequencing the plasmablast antibody repertoire at serial time points in patients with established RA.MethodsBlood samples were obtained at up to 4 serial time points from 8 individuals with established RA who were positive for ACPAs by the anti-cyclic citrullinated peptide test. CD19+CD3-IgD-CD14-CD20-CD27+CD38++ plasmablasts were isolated by single-cell sorting and costained with citrullinated peptide tetramers to identify ACPA-expressing plasmablasts. Cell-specific oligonucleotide barcodes were utilized, followed by large-scale sequencing and bioinformatics analysis, to obtain error-corrected, paired heavy- and light-chain antibody gene sequences for each B cell.ResultsBioinformatics analysis revealed 170 persistent plasmablast lineages in the RA blood, of which 19% included multiple isotypes. Among IgG- and IgA-expressing plasmablasts, significantly more IgA-expressing than IgG-expressing persistent lineages were observed (P < 0.01). Shared complementarity-determining region 3 sequence motifs were identified across subjects. A subset of the plasmablast lineages included members derived from later time points with divergent somatic hypermutations that encoded antibodies that bind an expanded set of citrullinated antigens. Furthermore, these recombinant, differentially mutated plasmablast antibodies formed immune complexes that stimulated higher macrophage production of tumor necrosis factor (TNF) compared to antibodies representing earlier time point-derived lineage members that were less mutated.ConclusionThese findings demonstrate that established RA is characterized by a persistent IgA ACPA response that exhibits ongoing affinity maturation. This observation suggests the presence of a persistent mucosal antigen that continually promotes the production of IgA plasmablasts and their affinity maturation and epitope spreading, thus leading to the generation of ACPAs that bind additional citrullinated antigens and more potently stimulate macrophage production of TNF.

Project description:ABSTRACT Background: Proteases are one of the largest pharmaceutical targets for drug developments. Their dysregulations result in a wide variety of diseases. Because proteolytic networks usually consist of protease family members that share high structural and catalytic homology, distinguishing them using small molecule inhibitors is often challenging. To achieve specific inhibition, this study described a novel approach for the generation of protease inhibitory antibodies. As a proof of concept, we aimed to convert a matrix metalloproteinase (MMP)-14 specific inhibitor to MMP-9 specific inhibitory antibodies with high selectivity. Methods: An error-prone single-chain Fv (scFv) library of an MMP-14 inhibitor 3A2 was generated for yeast surface display. A dual-color competitive FACS was developed for selection on MMP-9 catalytic domain (cdMMP-9) and counter-selection on cdMMP-14 simultaneously, which were fused/conjugated with different fluorophores. Isolated MMP-9 inhibitory scFvs were biochemically characterized by inhibition assays on MMP-2/-9/-12/-14, proteolytic stability tests, inhibition mode determination, competitive ELISA with TIMP-2 (a native inhibitor of MMPs), and paratope mutagenesis assays. Results: We converted an MMP-14 specific inhibitor 3A2 into a panel of MMP-9 specific inhibitory antibodies with dramatic selectivity shifts of 690–4,500 folds. Isolated scFvs inhibited cdMMP-9 at nM potency with high selectivity over MMP-2/-12/-14 and exhibited decent proteolytic stability. Biochemical characterizations revealed that these scFvs were competitive inhibitors binding to cdMMP-9 near its reaction cleft via their CDR-H3s. Conclusions: This study developed a novel approach able to convert the selectivity of inhibitory antibodies among closely related protease family members. This methodology can be directly applied for mAbs inhibiting many proteases of biomedical importance. Statement of Significance To achieve high selectivity required for therapies, we developed a novel approach for the generation of protease inhibitory antibodies with nM potency and decent proteolytic stability. The methodology demonstrated here can be readily applied to many proteases of biomedical importance.

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Epitope-specific affinity maturation improved stability of potent protease inhibitory antibodies.

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Epitope-specific affinity maturation improved stability of potent protease inhibitory antibodies.

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