Understanding and predicting the consequences of aggregation for the immunogenicity of therapeutic protein products

Project Description: (maximum of 4,000 characters)
Please make sure this description clearly indicates how the project sits within the BBSRC remit, how it enables new ways of working and how it aligns with the DTP themes (World Class Underpinning Biosciences, Industrial Biotechnology and Bioenergy or Agriculture and Food Security). If you have been awarded an in vivo skills supplement, please outline the in vivo skills the student will learn during the project.

Repeated administration of therapeutic proteins can result in unwanted immunogenicity, in the form of anti-drug antibodies (ADA), which can impact on pharmacokinetics (PK), efficacy and safety. High affinity ADA responses, which are considered to be most likely to impact efficacy are believed to be triggered by T cell-dependent activation of B cells. Activation of T and B cells is driven by short engineered regions of non-self-sequences within the therapeutic protein. Activation of T cells requires antigen presenting cells (APCs), which internalise the therapeutic protein, process and present on their surface to T cells. Antigen presentation alone is insufficient for T cell activation, co-stimulatory signals are required in addition, in the form of upregulated surface stimulatory markers on the APC, and soluble cytokine signals, also coming from the APCs. Thus, in order for a therapeutic protein to initiate an ADA response, APCs must be activated to produce these co-stimulatory signals. There are multiple aspects of therapeutic protein drug treatment that have been identified as risk factors for unwanted immunogenicity, and several are proposed to promote activation of APCs, and thus provide these co-stimulatory signals to T cells. One of these risk factors is protein aggregates.
Protein aggregates can form during the manufacture and storage of therapeutic proteins, and range in size and solubility, from small, soluble, reversible aggregates consisting of dimers, trimers, to higher-order, irreversible aggregates that contain large numbers of molecules, and fall in the sub-visible size range of 100 nm - 50 um. Studies of therapeutic protein preparations containing low levels of these different types of aggregates have been demonstrated to cause weak activation of APCs in vitro, suggesting that they could function as adjuvants in vivo, and help initiate an adaptive immune response to a therapeutic protein.
Relevance to BBSRC DTP Themes - Immunogenicity of biologics is a major problem in the development of new therapeutic proteins. The project therefore relates to the Industrial Biotechnology theme.
Aims - To compare the response of APCs to preparations of therapeutic proteins of monomer versus those containing aggregates of different sizes.
Plan
1. Generation and characterization of aggregated preparations of selected therapeutic proteins to use as tool reagents (Manchester & Cambridge)
a) Check aggregation conditions
b) Further characterization will be by SEC MALLS and other methods, as appropriate. This may involve some work in Cambridge, as well as Manchester.
c) Label antibodies with fluorophore
2. Studies of uptake and activation in defined cell lines - The aim of this section is to quantify uptake quantity and kinetics of aggregated protein into APCs and differentiate behaviour from non-aggregated material. We will also seek to obtain some information about the different uptake pathways used. Methods used will be FACS and confocal microscopy.
3. Transcriptomic analysis - Once we have a well-defined set of experimental conditions with a particular combination of cell line, test molecules containing aggregates and defined stimulation time points, we will conduct a transcriptomic experiment, to identify genes up- or down-regulated in response to exposure to aggregated protein vs monomer
4. Translation to primary cells - Using the RT-PCR panel, combined with microscopy, we will compare uptake of aggregated and non-aggregated material in primary cells