Abstract
Pharmaceutical manufacturing relies on rigorous methods of quality control of drugs and in particular of the physico-chemical and functional characterizations of monoclonal antibodies. To that end, robust bioassays are very often limited to reporter gene assays and the use of immortalized cell lines that are supposed to mimic immune cells such as natural killer (NK) cells to the detriment of primary materials, which are appreciated for their biological validity but are also difficult to exploit due to the great diversity between individuals. Here, we characterized the phenotype of the peripheral blood circulating cytotoxic cells of 30 healthy donors, in particular the repertoire of cytotoxic markers, using flow cytometry. In parallel, we characterized the antibody-dependent cellular cytotoxicity (ADCC) effector functions of these primary cells by measuring their cytolytic activity against a cancer cell-line expressing HER2 in the presence of trastuzumab and with regards to FCGR3A genotype. We could not establish a correlation or grouping of individuals using the data generated from whole peripheral blood mononuclear cells, however the isolation of the CD56-positive population, which is composed not only of NK cells but also of natural killer T (NKT) and γδ-T cells, as well as subsets of activated cytotoxic T cells, monocytes and dendritic cells, made it possible to standardize the parameters of the ADCC and enhance the overall functional avidity without however eliminating the inter-individual diversity. Finally, the use of primary CD56+ cells in ADCC experiments comparing glycoengineered variants of trastuzumab was conclusive to test the limits of this type of ex vivo system. Although the effector functions of CD56+ cells reflected to some extent the in vitro receptor binding properties and cytolytic activity data using NK92 cells, as previously published, reaching a functional avidity plateau could limit their use in a quality control framework.
Highlights
The development of therapeutic and preventive antibodies requires a high standard quality control of structural and functional characterization as well as defining specifications that are in line with regulatory guidance established by regulatory authorities and the International Council for Harmonization (ICH) [1, 2]
A first panel was designed for the analysis of Fc receptors (FcRs) and Fc receptor-like (FcRL) receptors, a second panel for the analysis of important markers of cytotoxicity, in particular the natural cytotoxicity receptors (NCRs), killer-cell immunoglobulin-like receptors (KIRs) and killer lectin like receptors (KLRs) markers, and a third panel based on OMIP-41 [61] for the most comprehensive possible immunophenotyping of the major lymphocyte and myeloid subsets (Table S1 and Figure S1)
The initial objective of this study was to identify biomarkers in the population of circulating cytotoxic cells in healthy donors that may be linked to their cytotoxic activity, namely antibody-dependent cellular cytotoxicity (ADCC), in an experimental system where a target tumor cell-line express strongly HER2 and in the presence of anti-HER2 antibody trastuzumab
Summary
The development of therapeutic and preventive antibodies requires a high standard quality control of structural and functional characterization as well as defining specifications that are in line with regulatory guidance established by regulatory authorities and the International Council for Harmonization (ICH) [1, 2]. The proper characterization of critical quality attributes and assessment of immunogenicity is important during the development process of biopharmaceuticals [3], but characterizing the biological functions at the cellular level is essential to guide manufacturing of the antibody-based drug [4]. In this good manufacturing practice (GMP) based context of quality control, the requirements for precision, accuracy and statistical power often necessitates neglecting the qualitative and quantitative evaluation of underlying and highly complex biological processes of the drug in parallel. Fine tuning and feature engineering such as modification of N-linked glycosylation [7] affect the binding affinity of the Fc domain to its cognate receptors [8], affect the ADCC activity in vitro and in vivo [9,10,11], leading to enhanced clinical responses [12, 13]
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