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Abstract

Background and Aims: Antibodies targeting tumor necrosis factor-alpha [TNF-alpha] are a mainstay in the treatment of inflammatory bowel disease. However, they fail to demonstrate efficacy in a considerable proportion of patients. On the other hand, glycosylation of antibodies might influence not only their immunogenicity but also their structure and function. We investigated whether specific glycosylation patterns of the Fc-fragment would affect the immunogenicity of anti-TNF-alpha antibody in monocyte-derived dendritic cells.

Methods: The effect of a specific Fc-glycosylation pattern on antibody uptake by monocyte-derived dendritic cells [mo-DCs] and how this process shapes the immunologic profile of mo-DCs was investigated. Three N-glycoforms of the anti-TNF-alpha antibody adalimumab, that differed in the content of fucose or sialic acid, were tested: [1] mock treated Humira, abbreviated ‘Fuc-G0’, where the N-glycan mainly consist of fucose and N-acetylglucosamine [GlcNAc], without sialic acid; [2] ‘Fuc-G2S1/G2S2’ with fucose and alpha 2,6 linked sialic acid; and [3] ‘G2S1/G2S2’ with alpha 2,6 linked sialic acid, without fucose.

Results: Our data demonstrated that neither fucosylation nor sialylation of anti-TNF-Abs [Fuc-G0, FucG2S1/G2S2, G2S1/G2S2] influence their uptake by mo-DCs. Additionally, none of the differentially glycosylated antibodies altered CD80, CD86, CD273, CD274 levels on mo-DCs stimulated in with lipopolysaccharide in the presence of antibodies. Next, we evaluated the levels of cytokines in the supernatant of mo-DCs stimulated with lipopolysaccharide in the presence of Fuc-G0, Fuc-G2S1/G2S2 or G2S1/G2S2-glycosylated anti-TNF antibodies. Only IL-2 and IL-17 levels were downregulated, and IL-5 production was upregulated by uptake of Fuc-G0 antibodies, as compared to control without antibodies.

Conclusions: The specific modification in the Fc-glycosylation pattern of anti-TNF-alpha Abs does not affect their immunogenicity under the tested conditions. As this study was limited to mo-DCs, further investigation is required to clarify whether Ab uptake into mo-DCs might change the immunological profile of T- and B-cells, in order to ultimately reduce the formation of anti-drug antibodies and to improve the patient care.

Anti-drug antibodies, anti-TNF-alpha treatment, glycosylation

1. Introduction

Antibodies targeting tumour necrosis factor-alpha [TNF-alpha] are a mainstay in the treatment of inflammatory bowel disease [IBD]. However, they fail to demonstrate efficacy in a considerable proportion of patients. Such treatment failure is frequently due to the development of anti-drug antibodies [ADAs] that might cause loss of response and adverse events.1,2 ADAs develop in 73% of patients on infliximab treatment and in 35% of patients on adalimumab treatment.3,4 They directly bind to the pharmacologically active site of the drug, thereby restrict its ability to bind the target, and by forming immune complexes they lower drug serum levels. As a consequence, disease activity is indirectly influenced by ADAs, limiting both the pharmacokinetics and pharmacodynamic properties of the drugs.5,6

The production of ADAs occurs via both the T-cell-dependent and -independent process of B cell activation.6,7 Initially, ADAs are probably of IgM isotype with little clinical impact.7 However, the involvement of T-cells triggers isotype switching and leads to the formation of the IgG isotype with long-lasting, high-affinity ADA titres.7 In the T-cell-dependent pathway, the drug first has to be taken up by antigen-presenting cells [APCs] and presented to T-cells. As a consequence, activated T-cells stimulate B-cells to produce ADAs.

The formation of ADAs serves as a marker, indicating that therapeutic antibodies are immunogenic. The glycosylation of antibodies might influence not only their immunogenicity but also their structure and function.8 It has been demonstrated that sialylation of immunoglobulins is associated with anti-inflammatory activity9 via either DC-SIGN [dendritic-cell-specific ICAM-3 grabbing non-integrin] receptor activity10,11 or involvement of Siglec/CD22 and C-type lectin dendritic cell immunoreceptors.12,13 IgG fucosylation has been reported to reduce antibody-dependent cellular cytotoxicity [ADCC], while fucose removal increases its affinity to FcyRIII, the main receptor of natural killer [NK] cells mediating ADCC.14

Here, we investigated whether specific glycosylation patterns of the Fc-fragment would affect the immunogenicity of adalimumab. Therefore, we focused on the effect of a specific Fc-glycosylation pattern on antibody uptake by monocyte-derived dendritic cells [mo-DCs] and how this process might shape the immunological profile of mo-DCs.

2. Material and Methods

2.1. Human mononuclear cells

Peripheral blood mononuclear cells [PBMCs] were isolated from buffy coats obtained from Blood Bank of Blutspendezentrum Limmattal, Switzerland, according to established gradient separation protocols [Ficoll: Histopaque, Sigma]. Monocytes were isolated and cultured according to manufacturer’s instructions for human CD14 MicroBeads [Miltenyi Biotec] as described in detail in the Supplementary Methods. Monocytes were cultured in cRPMI medium [Gibco; with l-glutamine and phenol red without HEPES] supplemented with 1 mM sodium pyruvate solution [Sigma], MEM non-essential amino acid solution [100×] [Sigma], 10% fetal bovine serum [OmniLab], in the presence of GM-CSF and IL-4 [CytoBox Mo-DC – premium grade, human; Miltenyi Biotec] for 6 days. On day 6 cells were collected and experiments performed.

If indicated, mo-DCs were stimulated with 50 ng/mL lipopolysaccharide (LPS) and Fc receptors were blocked with Fc receptor blocking solution [Human TruStain FcX; BioLegend]. To test antibody uptake, antibodies were labelled with pH rodo dye (pHrodo iFL Red STP Ester [amine-reactive]; ThermoFisher) according to the manufacturer’s instructions.

To verify the in vitro cell culture model, disialoganglioside from bovine brain [Sigma, G2392] was used as a positive control [Supplementary Figure 5].

2.2. Antibodies

Antibodies Fuc-G0 [Humira; mock treated] and in vitro glycoengineered Fuc-G2S1/G2S2 and G2S1/G2S2 antibodies were provided by LimmaTech Biologics. A detailed description of the monocolonal anti-TNF antibodies is provided in Supplementary Figures 2–4 and Supplementary Methods].

2.3. Flow cytometry analyses

Expression of co-stimulatory and co-inhibitory molecules was assessed with flow cytometry using antibodies targeting HLA-DR, CD14, CD86, CD80, CD274 and CD273 [all obtained from Biolegend]. Cytokine concentration in cell culture supernatants was measured using a Bio-Plex Pro Human Cytokine 17-plex assay [Bio-Rad] and Bio-Plex 200 system [Bio-Rad]. Immune complexes formed between anti-TNF-alpha antibodies and TNF-alpha were prepared by mixing 1 µg/mL TNF-alpha with 5 µg/mL anti-TNF-alpha antibodies and the complex was used in experiments at a concentration of 10 µg/mL.

3. Results

Three N-glycoforms of the anti-TNF-alpha antibody adalimumab, which differed in the content of fucose or sialic acid, were engineered and tested: [1] mock-treated Humira, abbreviated ‘Fuc-G0’, where the N-glycan mainly consist of fucose and N-acetylglucosamine [GlcNAc], without sialic acid; [2] ‘Fuc-G2S1/G2S2’ with fucose and alpha 2,6-linked sialic acid; and [3] ‘G2S1/G2S2’ with alpha 2,6-linked sialic acid, without fucose [Figure 1A, Table 1]. As shown by the mean fluorescence intensity of pH Rodo-dye within the cells, an antibody [Ab] concentration of 10 µg/mL and uptake by mo-DCs for 6 h was shown to be optimal [Supplementary Figure 1A]. Interestingly, a concentration close to 10 µg/mL of anti-TNF-alpha Ab was previously observed in the blood of patients receiving anti-TNF-alpha therapy.1 We next assessed whether the presence of LPS might affect anti-TNF Ab uptake when added either before, after or together with the antibody [Supplementary Figure 1B]. LPS-stimulated monocyte-derived dendritic cells express transmembrane TNF-alpha [TmTNF] on the cell surface.15 Binding of anti-TNF-alpha Ab to TmTNF results in active, rapid complex internalization as an added means of antibody uptake.15 Since we did not detect a significant difference in uptake of antibodies in all tested conditions, we continued with antibody/LPS co-treatment of cells for 6 h. Given that receptors detecting the Fc region of antibodies have a crucial role in the clearance of immune complexes,16 we studied the impact of FcR receptor blocking and LPS stimulation on anti-TNF alpha Ab uptake [Figure 1B]. Fc receptor blocking did not influence uptake of anti-TNF-alpha antibodies by mo-DCs. In both, blocked or unblocked FcR receptor conditions, LPS stimulation slightly, but not significantly, increased the uptake of all Abs. These data demonstrated that neither fucosylation nor sialylation of anti-TNF-Abs [Fuc-G0, Fuc-G2S1/G2S2, G2S1/G2S2] influenced their uptake by mo-DCs.

Table 1.
Open in new tab

Quality control analyses of monoclonal antibodies [mAbs]

QC testHumira-derived materialCGP-derived material
mAbHumira [mock treated]Sia-HumiraHumira-pHrodoSia-Humira-pHrodoSia-AdalimumabSia-Adalimumab pHrodo
AbbreviationFuc-G0Fuc-G2S1/G2S2Fuc-G0Fuc-G2S1/G2S2G2S1/G2S2G2S1/G2S2
Concentration [A280], mg/mL110.720.7610.7
Appearance [4°C]Colourless liquidColourless liquidClear pink colourClear pink colourColourless liquidClear pink colour
pH 666666
BufferPBSPBSPBSPBSPBSPBS
Purity, aggregation [SE-HPLC]≥99.5% Monomer≥99.5% Monomer≥99.9% Monomer≥99.9% Monomer99.6% Monomern.d.
≤0.5% Dimer ≤0.5% Dimer ≤0.1% Dimer≤0.1% Dimer 0.4% Dimer
N-glycan [RF-MS]77% G0F [Fuc-G0]65% G2FS2 [Fuc-G2S2]n.d.n.d.65% G2S2 n.d.
17% G1F 15% G2FS1 [FucG2S1]35% G2S1
1% G2FOthers: 20%
Others [e.g. Man5, Man6] 6%
[total: 0% Sialylated][total: >80% Sialylated][total: >99% Sialylated]
Endotoxin [LAL], EU/mg0.075<0.050.17<0.050.1 0.075
Binding affinity to TNF-alpha, ×10−10 M 1.68–1.811.81–1.92.20 ± 0.282.63 ± 0.281.65 ± 0.092.30 ± 0.22
KD [SPR]
Purity [SDS-PAGE] 99.5% [reducing]98.8% [reducing]98% [reducing]98.5% [reducing]99.9% [reducing]99.9% [reducing]
99.5% [non-reducing] 99.3% [non-reducing] 97% [non-reducing] 98.5% [non-reducing] 98.8% [non-reducing] 98% [non-reducing]
Freeze thaw, purity / aggregation≥99.5% Monomer≥99.5% Monomer≥99.9% Monomer≥.9% Monomer99.4% Monomern.d.
[SE-HPLC]≤0.5% Dimer ≤0.5% Dimer ≤0.1% Dimer ≤0.1% Dimer 0.6% Dimer
Degree of labellingn.a.n.a.1.782.05n.a.1.9
QC testHumira-derived materialCGP-derived material
mAbHumira [mock treated]Sia-HumiraHumira-pHrodoSia-Humira-pHrodoSia-AdalimumabSia-Adalimumab pHrodo
AbbreviationFuc-G0Fuc-G2S1/G2S2Fuc-G0Fuc-G2S1/G2S2G2S1/G2S2G2S1/G2S2
Concentration [A280], mg/mL110.720.7610.7
Appearance [4°C]Colourless liquidColourless liquidClear pink colourClear pink colourColourless liquidClear pink colour
pH 666666
BufferPBSPBSPBSPBSPBSPBS
Purity, aggregation [SE-HPLC]≥99.5% Monomer≥99.5% Monomer≥99.9% Monomer≥99.9% Monomer99.6% Monomern.d.
≤0.5% Dimer ≤0.5% Dimer ≤0.1% Dimer≤0.1% Dimer 0.4% Dimer
N-glycan [RF-MS]77% G0F [Fuc-G0]65% G2FS2 [Fuc-G2S2]n.d.n.d.65% G2S2 n.d.
17% G1F 15% G2FS1 [FucG2S1]35% G2S1
1% G2FOthers: 20%
Others [e.g. Man5, Man6] 6%
[total: 0% Sialylated][total: >80% Sialylated][total: >99% Sialylated]
Endotoxin [LAL], EU/mg0.075<0.050.17<0.050.1 0.075
Binding affinity to TNF-alpha, ×10−10 M 1.68–1.811.81–1.92.20 ± 0.282.63 ± 0.281.65 ± 0.092.30 ± 0.22
KD [SPR]
Purity [SDS-PAGE] 99.5% [reducing]98.8% [reducing]98% [reducing]98.5% [reducing]99.9% [reducing]99.9% [reducing]
99.5% [non-reducing] 99.3% [non-reducing] 97% [non-reducing] 98.5% [non-reducing] 98.8% [non-reducing] 98% [non-reducing]
Freeze thaw, purity / aggregation≥99.5% Monomer≥99.5% Monomer≥99.9% Monomer≥.9% Monomer99.4% Monomern.d.
[SE-HPLC]≤0.5% Dimer ≤0.5% Dimer ≤0.1% Dimer ≤0.1% Dimer 0.6% Dimer
Degree of labellingn.a.n.a.1.782.05n.a.1.9

n.d., not determined; n.a., not applicable.

Table 1.
Open in new tab

Quality control analyses of monoclonal antibodies [mAbs]

QC testHumira-derived materialCGP-derived material
mAbHumira [mock treated]Sia-HumiraHumira-pHrodoSia-Humira-pHrodoSia-AdalimumabSia-Adalimumab pHrodo
AbbreviationFuc-G0Fuc-G2S1/G2S2Fuc-G0Fuc-G2S1/G2S2G2S1/G2S2G2S1/G2S2
Concentration [A280], mg/mL110.720.7610.7
Appearance [4°C]Colourless liquidColourless liquidClear pink colourClear pink colourColourless liquidClear pink colour
pH 666666
BufferPBSPBSPBSPBSPBSPBS
Purity, aggregation [SE-HPLC]≥99.5% Monomer≥99.5% Monomer≥99.9% Monomer≥99.9% Monomer99.6% Monomern.d.
≤0.5% Dimer ≤0.5% Dimer ≤0.1% Dimer≤0.1% Dimer 0.4% Dimer
N-glycan [RF-MS]77% G0F [Fuc-G0]65% G2FS2 [Fuc-G2S2]n.d.n.d.65% G2S2 n.d.
17% G1F 15% G2FS1 [FucG2S1]35% G2S1
1% G2FOthers: 20%
Others [e.g. Man5, Man6] 6%
[total: 0% Sialylated][total: >80% Sialylated][total: >99% Sialylated]
Endotoxin [LAL], EU/mg0.075<0.050.17<0.050.1 0.075
Binding affinity to TNF-alpha, ×10−10 M 1.68–1.811.81–1.92.20 ± 0.282.63 ± 0.281.65 ± 0.092.30 ± 0.22
KD [SPR]
Purity [SDS-PAGE] 99.5% [reducing]98.8% [reducing]98% [reducing]98.5% [reducing]99.9% [reducing]99.9% [reducing]
99.5% [non-reducing] 99.3% [non-reducing] 97% [non-reducing] 98.5% [non-reducing] 98.8% [non-reducing] 98% [non-reducing]
Freeze thaw, purity / aggregation≥99.5% Monomer≥99.5% Monomer≥99.9% Monomer≥.9% Monomer99.4% Monomern.d.
[SE-HPLC]≤0.5% Dimer ≤0.5% Dimer ≤0.1% Dimer ≤0.1% Dimer 0.6% Dimer
Degree of labellingn.a.n.a.1.782.05n.a.1.9
QC testHumira-derived materialCGP-derived material
mAbHumira [mock treated]Sia-HumiraHumira-pHrodoSia-Humira-pHrodoSia-AdalimumabSia-Adalimumab pHrodo
AbbreviationFuc-G0Fuc-G2S1/G2S2Fuc-G0Fuc-G2S1/G2S2G2S1/G2S2G2S1/G2S2
Concentration [A280], mg/mL110.720.7610.7
Appearance [4°C]Colourless liquidColourless liquidClear pink colourClear pink colourColourless liquidClear pink colour
pH 666666
BufferPBSPBSPBSPBSPBSPBS
Purity, aggregation [SE-HPLC]≥99.5% Monomer≥99.5% Monomer≥99.9% Monomer≥99.9% Monomer99.6% Monomern.d.
≤0.5% Dimer ≤0.5% Dimer ≤0.1% Dimer≤0.1% Dimer 0.4% Dimer
N-glycan [RF-MS]77% G0F [Fuc-G0]65% G2FS2 [Fuc-G2S2]n.d.n.d.65% G2S2 n.d.
17% G1F 15% G2FS1 [FucG2S1]35% G2S1
1% G2FOthers: 20%
Others [e.g. Man5, Man6] 6%
[total: 0% Sialylated][total: >80% Sialylated][total: >99% Sialylated]
Endotoxin [LAL], EU/mg0.075<0.050.17<0.050.1 0.075
Binding affinity to TNF-alpha, ×10−10 M 1.68–1.811.81–1.92.20 ± 0.282.63 ± 0.281.65 ± 0.092.30 ± 0.22
KD [SPR]
Purity [SDS-PAGE] 99.5% [reducing]98.8% [reducing]98% [reducing]98.5% [reducing]99.9% [reducing]99.9% [reducing]
99.5% [non-reducing] 99.3% [non-reducing] 97% [non-reducing] 98.5% [non-reducing] 98.8% [non-reducing] 98% [non-reducing]
Freeze thaw, purity / aggregation≥99.5% Monomer≥99.5% Monomer≥99.9% Monomer≥.9% Monomer99.4% Monomern.d.
[SE-HPLC]≤0.5% Dimer ≤0.5% Dimer ≤0.1% Dimer ≤0.1% Dimer 0.6% Dimer
Degree of labellingn.a.n.a.1.782.05n.a.1.9

n.d., not determined; n.a., not applicable.

Uptake of pH Rodo-labelled antibodies by monocyte-derived dendritic cells.
Figure 1.

Uptake of pH Rodo-labelled antibodies by monocyte-derived dendritic cells.

[A] Three different glyco-forms of anti-TNF-alpha antibodies were used: Fuc-G0 with fucose and GlcNAc, Fuc-G2S1/G2S2 with fucose and sialic acid, and G2S1/G2S2 with sialic acid as a main N-linked glycosylation modification. [B] Uptake of pHrodo labelled anti-TNF-alpha antibodies by monocyte-derived dendritic cells was measured as mean fluorescence intensity [n = 4]. To investigate the effect of LPS stimulation and FcR blocking on antibody uptake, four experimental conditions were tested [no FcR blocking and no LPS stimulation, no FcR blocking and LPS stimulation, FcR blocking and no LPS stimulation, FcR blocking and LPS stimulation].

Open in new tabDownload slide

Exposure of mo-DCs to antigens triggers signals via co-stimulatory and co-inhibitory molecules that modulate subsequent T-cell activation and differentiation. We studied the surface expression of co-stimulatory or co-inhibitory molecules CD80, CD86, CD273, CD274, ILT2 and ILT4 on mo-DCs stimulated with LPS in the presence of Fuc-G0, Fuc-G2S1/G2S2 or G2S1/G2S2 antibodies. To our surprise, none of the differentially glycosylated antibodies altered CD80, CD86, CD273 or CD274 levels. Interestingly, all tested antibodies decreased expression of ILT2, but increased expression of ILT4 on the surface of LPS-stimulated mo-DCs [Figure 2A].

Expression of co-stimulatory and co-inhibitory molecules, and cytokine production by monocyte-derived dendritic cells [mo-DCs] in the presence of Fuc-G0, Fuc-G2S1/G2S2 and G2S1/G2S2 antibodies.
Figure 2.

Expression of co-stimulatory and co-inhibitory molecules, and cytokine production by monocyte-derived dendritic cells [mo-DCs] in the presence of Fuc-G0, Fuc-G2S1/G2S2 and G2S1/G2S2 antibodies.

For all experiments, mo-DCs were stimulated with LPS and FcR was blocked. [A] Expression of co-stimulatory [CD80, CD86] and co-inhibitory molecules [CD273, CD274, ILT2, ILT4] on mo-DCs was measured by flow cytometry and is shown as mean fluorescence intensity [MFI] [n = 4]. [B] Effect of antibody uptake on cytokine production by mo-DCs [pg/mL] was measured with the Multiplex Immuno assay System [n = 3]. [C] Complexes of anti-TNF-alpha antibodies with TNF-alpha were added to mo-DCs and production of cytokines was assessed with the Multiplex Immunoassay System [n = 4] [*p<0.05, Kurskal–Walis test with multiple comparisons].

Open in new tabDownload slide

Since mo-DC-derived cytokines might also modulate activation and differentiation of T cells, we evaluated levels of IL-8, IL-2, IL-6, IL-10, GM-CSF, MCP-1, IL-5, IL-1beta, IL-4, IL-12[p70], IL-17, G-CSF and IFN-gamma in the supernatant of mo-DCs stimulated with LPS in the presence of Fuc-G0, Fuc-G2S1/G2S2 or G2S1/G2S2-glycosylated anti-TNF antibodies. Only IL-2 and IL-17 levels were downregulated, and IL-5 production was upregulated by uptake of Fuc-G0 antibodies, as compared to control without antibodies. For the other tested cytokines, no differences in cytokine production were observed [Figure 2B].

Under physiological conditions, IgG antibodies mediate their effector functions mainly via formation of immune complexes with antigens.16 Therefore, we assessed cytokine production by mo-DCs stimulated with LPS in the presence of Fuc-G0, Fuc-G2S1/G2S2 or G2S1/G2S2 antibodies in complexes with TNF-alpha. No differences in G-CSF, IL-1beta, IL-13, GM-CSF, MCP-1 or IL-8 levels were observed, when culturing mo-DCs stimulated with all three glycoforms of the anti-TNF Ab. However, addition of G2S1/G2S2 complexes enhanced IL-6 secretion from mo-DCs, as compared to Fuc-G0 complexes. Conversely, G2S1/G2S2 complexes reduced IL-4, IL-17 and IL-2 production as compared to unstimulated cells [Figure 2C].

4. Discussion

The immunogenicity of anti-TNF-alpha Abs has a major impact on patient care, since formation of ADAs has a tremendous impact on therapy response and side effects. Modulating and decreasing the immunogenicity of anti-TNF-alpha Abs would be a highly important means to improve IBD patient care. In our study, we investigated whether the specific glycosylation pattern of anti-TNF-alpha Abs might influence their uptake by mo-DCs as well as the immunological profile of the cells. Three different glycoforms of anti-TNF alpha Abs that differed in fucosylation and sialylation patterns at the canonical N-297 position were tested. mo-DCs do not fully represent the highly diverse populations of DCs that are found in tissues and have adapted expression of pattern recognition receptors to these environments. However, we found that the N-linked glycosylation of the tested anti-TNF Ab does neither influence its uptake, nor the expression of co-stimulatory and co-inhibitory molecules, or the overall profile of cytokine production by mo-DCs.

The initial hypothesis assumed that sialylation of anti-TNF-alpha Abs would induce a tolerogenic phenotype of mo-DCs. It has been shown that addition of sialic acid modifies the immunogenicity of antigens,17 since sialylated antigens induce regulatory properties in mo-DCs and play a role in the generation of regulatory T-cells.17 However, we found that sialylation did not significantly impact the immunogenicity of the tested anti-TNF-alpha Ab towards DCs.

The co-inhibitor molecules ILT2 and ILT4 belong to a group of Ig-like transcript receptors that play a central role in the maintenance of a tolerogenic state of DCs.18 It has been indicated that monocyte-derived DCs expressing high levels of ILT2 and ILT4 have the potential to induce T-cell energy.19 Nevertheless, the functional impact of differential ILT2 and ILT4 expression levels induced by anti-TNF-alpha Ab treatment remains to be elucidated.

Our analysis of cytokine production by mo-DCs suggests that when complexed with TNF-alpha, only G2S1/G2S2 Abs with sialic acid as the main N-linked glycosylation modification affect cytokine secretion. Interestingly, when not complexed with TNF-alpha, only Fuc-G0 Abs impact cytokine production. For further studies, it would be interesting to address whether anti-TNF-alpha Abs form complexes with TNF-alpha in the blood of patients with IBD and to determine which state [Ab complexed with TNF-alpha or free] triggers ADA production.

In conclusion, we found that a specific modification in the Fc-glycosylation pattern of anti-TNF-alpha Abs does not affect their immunogenicity under the tested conditions. As this study was limited to mo-DCs, further investigation is required to clarify whether Ab uptake into mo-DCs might change the immunological profile of T- and B-cells, in order to ultimately reduce the formation of ADAs and to improve patient care.

Funding

This research was supported by a grant from Innosuisse – Swiss Innovation Agency Innovation project 30025.1 IP-LS to M.S. and an adjunct research grant from LimmatTech AG to M.S.

Conflict of Interest

R.M., G.T., S.H., J.B., M.M. and A.F. are current employees, and Y.T. and J.S. are former employees, of LimmaTech Biologics AG, a company developing the proprietary CustomGlycan Platform [CGP], a technology platform for the production of glycoengineered therapeutics.

Data Availability Statement

The data underlying this article will be shared on reasonable request to the corresponding author.

Author Contributions

M.W. – the conception and design of the study, acquisition, analysis and interpretation of data, drafting the article; Y.M. – the conception and design of the study, acquisition, analysis and interpretation of data, revising the article for important intellectual content; R.M., G.T., Y.T., S.H. – acquisition, analysis and interpretation of data; J.S., J.B., M.M., A.F. – the conception and design of the study, interpretation of data, revising manuscript critically for important intellectual content; M.S. – the conception and design of the study, data interpretation, revising the article for important intellectual content, final approval of the version to be submitted.

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