Comparability study of monocyte derived dendritic cells, primary monocytes, and THP1 cells for innate immune responses

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Abstract

Immunogenicity is one major challenge to the successful development of biotherapeutics because it could adversely affect PK/PD, safety, and efficacy. Preclinical immunogenicity risk assessment strategies and assays have been developed and implemented to screen and optimize discovery molecules. Internalization by antigen presenting cells (APC) and innate immune activation are initial prerequisite steps in eliciting immune responses to biotherapeutics. Dendritic cells (DC)- and monocyte-based assays are employed to interrogate such risks, and their value has been well documented in the literature. However, these assays have limited throughput, exhibit higher variability, and entail lengthy and complex procedures as they are based on primary cells such as peripheral blood mononuclear cells (PBMC) from individual donors. Herein, we investigated THP1 cells as surrogate cells to study APC internalization and innate immune activation. Comparability studies showed that THP1 cells could resemble innate immune responses of monocyte-derived DC and primary CD14+ monocytes using a panel of therapeutic antibodies. In addition, an automated high throughput THP1 internalization assay was qualified to enable risk assessment at pre‑lead stages. The results demonstrated that THP1 cells can be utilized to assess immunogenicity risk in a high throughput manner.

Introduction

Biotherapeutics have the potential to trigger undesired immune responses, i.e., immunogenicity (Mingozzi and High, 2013; Jawa et al., 2020; Nidetz et al., 2020). Both humoral and cellular responses can occur, with the latter usually associated with novel modalities, for instance recombinant adeno-associated virus (rAAV) based gene therapies (Mingozzi and High, 2013; Nidetz et al., 2020). Immunogenicity can negatively impact pharmacokinetics/pharmacodynamics (PK/PD), safety, and efficacy as documented in many reports. Some good examples include bococizumab (Ridker et al., 2017), anti-TNF antibodies (Bartelds et al., 2007; Radstake et al., 2009), epoetin (Casadevall et al., 2002; Bennett et al., 2004), and AAV-factor IX gene therapy (Mingozzi et al., 2007).

Immunogenicity is complicated in nature with many attributes and factors collectively contributing to it (Singh, 2011; van Beers and Bardor, 2012; FDA, 2014; Wen and Jawa, 2021). In the context of antibodies and protein therapeutics, a classical T cell-dependent pathway drives the production of anti-drug antibodies (ADA) (Rosenberg and Sauna, 2018; Tourdot and Hickling, 2019; Jawa et al., 2020). This pathway entails several critical steps, including internalization, processing, and presentation of proteins by antigen presenting cells (APC), activation of innate immunity, engagement and activation of CD4 T cells, and eventual activation and differentiation of B cells. To drive positive responses, the molecule and/or product must possess mechanisms and properties to gain entry to APCs and activate innate immune responses in addition to potent CD4 T and B cell epitopes.

APC internalization and activation are initial critical events in this pathway. Since dendritic cells (DC) are considered potent APCs, DC internalization and DC activation assays have been developed and implemented to assess immunogenicity risk preclinically (Xue et al., 2016; Deora et al., 2017; Walsh et al., 2020; Wen et al., 2020; Wickramarachchi et al., 2020; Liao et al., 2021). DC internalization assays involve the labeling of protein of interest directly (in most reported cases, mAb) or a secondary probe with fluorescent dyes, for instance, pHrodo, Alexa Fluor 647, and the TAMRA/QSY7 Förster Resonance Energy Transfer (FRET) pair (Xue et al., 2016; Deora et al., 2017; Wen et al., 2020). We had previously reported a DC internalization assay using a universal probe labeled with TAMRA/QSY7 FRET pair (Wen et al., 2020). The assay was systematically optimized using design of experiment (DoE) principles and was qualified using 29 antibodies with clinical immunogenicity data. The assay enables investigation of both internalization and processing because separation of the quencher QSY7 from the fluorophore TAMRA is required for positive fluorescent signals. When combined with MHC-associated Peptide Proteomics (MAPPs) and a human antibody repertoire database, a statistical model was developed to predict clinical immunogenicity with high sensitivity and predictive values (Higgs, 2021). Likewise, DC activation assays were used by different organizations to assess immunogenicity risk (Walsh et al., 2020; Wickramarachchi et al., 2020; Liao et al., 2021). Test samples could be directly added to immature DCs, and upregulation of activation markers (CD40, CD80, CD83, and CD86) and/or production of pro-inflammatory cytokines (IL-1β, IL-6, MCP-1, MIP-1α, MIP-1β, TNF-α, etc) are quantified. These reports demonstrate that DC internalization and activation assays, along with in silico tools, MAPPs, and T cell assays, are powerful tools for preclinical immunogenicity risk assessment.

While monocyte-derived dendritic cells (MDDC) represent the most practical way to generate immature DC for in vitro assays that best mimic primary DCs, alternatives are needed to overcome drawbacks associated with MDDC, including limited throughput, tedious and lengthy culture process, access to fresh blood donations, and donor-to-donor variability. Therefore, we intended to develop a platform that 1) would enable increased throughput as compared to the published MDDC based method and therefore the screening of a larger number of molecules at early discovery; 2) would not depend on availability of blood donation, in particular considering the restrictions that the COVID-19 pandemic has imposed on resourcing blood products; and 3) would ensure a more consistent evaluation over time by eliminating the variability.

THP1 is a monocytic leukemia cell line, which could differentiate to macrophages (Tsuchiya et al., 1980; Auwerx, 1991; Chanput et al., 2014; Schopohl and Melzig, 2014). It resembles human primary monocytes and provides the advantages of a cell line. High numbers of homogenous cells can be supplied consistently, which enables the implementation of high throughput in vitro assays (Chanput et al., 2014). THP1 cells and differentiated macrophages have been employed to investigate immune modulation of chemicals and drugs (Chanput et al., 2014), and more recently the immunogenicity risk associated with biotherapeutic quality attributes, including aggregates, host cell proteins, and microbial contaminants (Haile et al., 2015; Moussa et al., 2016; Hamamura-Yasuno et al., 2020; Yasuno et al., 2020). For instance, Hamamura-Yasuno et al. reported elevated proinflammatory cytokines and activation markers from THP1 cells when treated with enfuvirtide and glatiramer acetate, which are protein therapeutics with injection site reactions (Hamamura-Yasuno et al., 2020). These studies suggested THP1 cells may be a good alternative to MDDC for studying innate immune responses.

In this project, we sought to explore THP1 cells as surrogate cells for high throughput assays to study innate immune responses to therapeutic mAbs. We investigated the comparability of MDDC, primary CD14+ monocytes, and THP1 cells with regards to internalization and activation by therapeutic antibodies. We focused on a set of well characterized antibodies, namely, infliximab, bococizumab, mAb1, mAb2, mAb3, mAb4, and mAb5. mAb1–5 are internal antibodies that had been tested in clinical trials. mAb1 is a humanized IgG4 targeting a membrane protein that are expressed by various human cancer cells and immune cells. mAb2, mAb3, and mAb4 are humanized IgG1 antibodies that target non-membrane proteins. mAb5 is a humanized IgG4 that recognizes a non-membrane protein target. mAb1, mAb2, and mAb3 showed high ADA rate in clinical trials, while mAb4 and mAb5 were not immunogenic (≤ 1% ADA rate).

Section snippets

Materials

mAbs were either purchased commercially (adalimumab as Humira®, guselkumab as Tremfya®, infliximab as Remicade®, and secukinumab as Cosentyx®) or expressed and purified internally (all other antibodies). AffiniPure F(ab’)2 fragment goat anti-human IgG (Fcγ fragment specific) was obtained from Jackson ImmunoResearch. QSY7-NHS and TAMRA-SE were obtained from Molecular Probes. Sodium Bicarbonate was purchased from Fisher. Zeba Spin Desalting Column (MWCO 7 k Da) was purchased from Thermo Fisher

Comparison of MDCC, primary monocytes and THP1 cells

With the goal of implementing a fully automated THP1 internalization assay, we first sought to compare the internalization of a set of mAbs by MDDC, primary monocytes, THP1 cells from manual culture (manual THP1), and THP1 cells grown in the automated cell culture system SelecT™ (automated THP1). This was considered as intermediate bridging between the DC internalization assay and the fully automated THP1 internalization platform. These internalization experiments were performed manually as

Discussion

In this study, we extensively investigated THP1 as a cellular substrate in assays to study innate immune responses to therapeutic mAbs with the goal of overcoming some of the limitations in throughput and comparability posed by the adoption of CD14+ monocytes isolated from healthy donors' PBMC. Using a panel of well characterized antibodies, we performed internalization studies following a previously published method (Wen et al., 2020), and innate activation was assessed by measuring

Conclusions

Biotherapeutics have the potential to elicit unwanted immune responses, and these immune responses, i.e., immunogenicity, could be manifested as compromised PK/PD, safety, and efficacy profiles in the clinic. PBMC-based in-vitro assays have been integrated to investigate immunogenicity holistically, which has been demonstrated to provide a reliable strategy to guide molecule selection and design. However, the value of those assays has not been fully realized, largely due to limited throughput.

Declaration of Competing Interest

All authors are employees of Eli Lilly and Company at the time of the work described here.

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