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Design of next-generation therapeutic IgG4 with improved manufacturability and bioanalytical characteristics.

ABSTRACT: Manufacturability of immunoglobulin G4 (IgG4) antibodies from the Chemistry, Manufacture, and Controls (CMC) perspective has received little attention during early drug discovery. Despite the success of protein engineering in improving antibody biophysical properties, a clear gap still exists between rational design of IgG4 candidates and their manufacturing suitability. Here, we illustrate that undesirable two-peak elution profiles in cation-exchange chromatography are attributed to the S228P mutation (in IgG4 core-hinge region) intentionally designed to prevent Fab-arm exchange. A new scaffolding platform for engineering IgG4 antibodies amenable to bioprocessing and bioanalysis is proposed by introducing an "IgG1-like" single-point mutation in the hinge or CH1 region of IgG4S228P. This work offers insight into the design, discovery, and development of innovative therapeutic antibodies that are well suited for robust biomanufacturing and quality control.

SUBMITTER: Chen Z

PROVIDER: S-EPMC7577236 | biostudies-literature | 2020 Jan-Dec

REPOSITORIES: biostudies-literature

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Design of next-generation therapeutic IgG4 with improved manufacturability and bioanalytical characteristics.

Chen Zhiqiang Z Qian Yueming Y Song Yuanli Y Xu Xuankuo X Tao Li L Mussa Nesredin N Ghose Sanchayita S Li Zheng Jian ZJ

mAbs 20200101 1

Manufacturability of immunoglobulin G4 (IgG4) antibodies from the Chemistry, Manufacture, and Controls (CMC) perspective has received little attention during early drug discovery. Despite the success of protein engineering in improving antibody biophysical properties, a clear gap still exists between rational design of IgG4 candidates and their manufacturing suitability. Here, we illustrate that undesirable two-peak elution profiles in cation-exchange chromatography are attributed to the S228P m ...[more]

PMID: 33044887

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Project description:Inflammatory myofibroblastic tumor (IMT), a locally aggressive neoplasm capable of metastasis, may show an immunoglobulin (Ig)G4-rich lymphoplasmacytic infiltrate. Prior reports suggest that storiform-fibrosis and obliterative phlebitis aid in the distinction of IMT from IgG4-related diseases. Herein, we highlight the morphologic overlap between the 2 diseases, and emphasize the importance of a multiplex fusion assay in the distinction of IgG4-related disease (IgG4-RD) from IMT. We identified 7 IMTs with morphologic and immunohistochemical features of IgG4-RD; 3 patients were originally diagnosed with IgG4-RD. Demographic, clinical and morphologic data was recorded. We also reevaluated 56 patients with IgG4-RD. We performed immunohistochemistry for IgG4, IgG, ALK, and ROS1. In situ hybridization for IgG4 and IgG was performed in selected cases. A multiplex next-generation sequencing-based RNA assay for gene fusions was performed to detect all known IMT-related gene fusions. All 7 IMTs showed a dense lymphoplasmacytic infiltrate and storiform-type fibrosis, with obliterative phlebitis noted in 3 cases. The neoplastic stromal cells constituted <5% of overall cellularity and stromal atypia was either absent or focal and mild. Elevated numbers of IgG4 positive cells and increased IgG4 to IgG ratio was identified in all cases. Four cases showed ALK related abnormalities: 3 fusions and one alternative transcription initiation; while 2 patients showed ROS1 and NTRK3 fusions. One tumor was negative for known IMT-related gene fusions. All 56 IgG4-RD cases were negative for ALK and ROS1 on immunohistochemistry; 6 cases were negative on the fusion assay. Highly inflamed IMTs are indistinguishable from IgG4-RD both histologically and on immunohistochemistry for IgG4. We advocate scrutinizing patients with presumptive single organ IgG4-RD for IMT and the diagnostic algorithm should include ALK and ROS1 immunohistochemistry and, in selected cases, a next-generation sequencing-based fusion assay that covers known IMT-associated gene fusions.

Project description:BackgroundThe soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world's most economically depressed locations. With growing emphasis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays.Methodology/principal findingsUtilizing next-generation sequencing and the Galaxy-based RepeatExplorer web server, we performed repeat DNA analysis on five species of soil transmitted helminths (Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis). Employing high copy-number, non-coding repeat DNA sequences as targets, novel real-time PCR assays were designed, and assays were tested against established molecular detection methods. Each assay provided consistent detection of genomic DNA at quantities of 2 fg or less, demonstrated species-specificity, and showed an improved limit of detection over the existing, proven PCR-based assay.Conclusions/significanceThe utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other eukaryotic pathogens.

Dataset Information

Design of next-generation therapeutic IgG4 with improved manufacturability and bioanalytical characteristics.

Publications

Design of next-generation therapeutic IgG4 with improved manufacturability and bioanalytical characteristics.

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