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Small Amounts of Sub-Visible Aggregates Enhance the Immunogenic Potential of Monoclonal Antibody Therapeutics

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Abstract

Purpose

Determine the effect of minute quantities of sub-visible aggregates on the in vitro immunogenicity of clinically relevant protein therapeutics.

Methods

Monoclonal chimeric (rituximab) and humanized (trastuzumab) antibodies were subjected to fine-tuned stress conditions to achieve low levels (<3% of total protein) of sub-visible aggregates. The effect of stimulating human dendritic cells (DC) and CD4+ T cells with the aggregates was measured in vitro using cytokine secretion, proliferation and confocal microscopy.

Results

Due to its intrinsic high clinical immunogenicity, aggregation of rituximab had minimal effects on DC activation and T cell responses compared to monomeric rituximab. However, in the case of trastuzumab (low clinical immunogenicity) small quantities of aggregates led to potent CD4+ T cell proliferation as a result of strong cytokine and co-stimulatory signals derived from DC. Consistent with this, confocal studies showed that stir-stressed rituximab was rapidly internalised and associated with late endosomes of DC.

Conclusions

These data link minute amounts of aggregates with activation of the innate immune response, involving DC, resulting in T cell activation. Thus, when protein therapeutics with little or no clinical immunogenicity, such as trastuzumab, contain minute amounts of sub-visible aggregates, they are associated with significantly increased potential risk of clinical immunogenicity.

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Abbreviations

ADA:

Anti-drug antibody

BCR:

B cell receptor

CBA:

Cytometric bead array

DC:

Dendritic cell

DLS:

Dynamic light scattering

mAbs:

Monocolonal antibodies

MHC:

Major histocompatibility complex

MoDC:

Monocyte derived dendritic cell

Tfh:

Follicular helper T cell

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Conflict of interest

The authors declare no conflicts of interest.

Author information

Author notes

  1. Christine J. Bryson

    Present address: Department of Haematology, Cambridge University Hospitals, Hills Road, Cambridge, CB2 0QQ, UK

  2. Vasco Filipe

    Present address: Adocia, 115 Avenue Lacassagne, 69003, Lyon, France

Authors and Affiliations

  1. Antitope Ltd, Babraham Research Campus, Babraham, Cambridge, CB22 3AT, UK

    Maryam Ahmadi, Christine J. Bryson, Edward A. Cloake, Katie Welch, Matthew P. Baker & Mark H. Fogg

  2. Division of Drug Delivery Technology, Leiden Academic Centre for Drug Research, Leiden University, Leiden, The Netherlands

    Vasco Filipe, Stefan Romeijn & Wim Jiskoot

  3. Coriolis Pharma, Am Klopferspitz 19, 82152, Martinsried, Germany

    Andrea Hawe

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Corresponding author

Correspondence to Mark H. Fogg.

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Ahmadi, M., Bryson, C.J., Cloake, E.A. et al. Small Amounts of Sub-Visible Aggregates Enhance the Immunogenic Potential of Monoclonal Antibody Therapeutics. Pharm Res 32, 1383–1394 (2015). https://doi.org/10.1007/s11095-014-1541-x

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  • DOI: https://doi.org/10.1007/s11095-014-1541-x

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