Last week, we published the excellent results of the Affimer scaffold in immunogenicity assays, using human ex vivo samples. Three different versions of the Affimer scaffold were analysed alongside the marketed therapeutic antibody Avastin®, and all three Affimer scaffolds showed low immunogenicity, comparable to that of Avastin. This clear demonstration, through industry-standard assays, of a lack of any immunogenicity problems with the Affimer scaffold de-risks the technology for future therapeutic use.
An immunogenic response to a substance, such as a therapeutic protein, typically involves an antibody response raised against the protein and the downstream effects of the immune system flaring up to fight this apparent threat. Undesired immunogenic responses in a patient can neutralise the efficacy of the therapeutic and cause serious side effects, so are of obvious concern to clinicians, manufacturers and regulators. While preclinical animal models are routinely used and predictive of therapeutic efficacy, immune responses to therapeutic proteins in conventional animal models have not generally been predictive (link is external) of the response in humans. Computational modelling and assaying primary human immune cell samples in vitro prior to clinical trials are the accepted industry-standard assays for determining the potential immunogenicity of a candidate therapeutic, and show a strong correlation with clinical studies.
For any new therapeutic, it is important to be able to show low immunogenicity, assuring the safety and function of the potential therapeutic. We used an industry-standard primary T-cell proliferation and activation assay to assess any potential immunogenic effects of the Affimer scaffold when cultured with primary human peripheral blood mononuclear cells (PBMCs) over a period of seven days.
Three different versions of the Affimer scaffold were analysed: a Type I Affimer scaffold based on the human stefin A protein, a Type II scaffold based on the plant consensus cystatin sequence and a Type III, a next generation version of the Type I scaffold. Avastin a marketed antibody therapeutic for cancer treatment was used as a control, along with positive and negative controls for T-cell proliferation and activation. As all the test substrates were used at 50 µg/mL, the small molecular weight of the Affimer scaffold compared to a standard antibody means the effective concentration of the Affimer scaffold was five times higher than that of the Avastin antibody.
Following seven days of culture the T-cells were analysed by flow cytometry, for the incorporation of 5-ethynyl-2′-deoxyuridine (EdU) from the media into the cells’ DNA, indicating proliferation. The ratio of stimulated cells per treatment to the average of non-stimulated cells from the negative control were used to determine the stimulation index. Industry standards define a stimulation index greater than 2.0 with a p<0.05 to be classed as a responder to the substance.
All three of the Affimer scaffolds tested showed low immunogenic responses in this study, comparable to that of Avastin, highlighting that there are no fundamental immunogenicity issues with the Affimer scaffolds. Compared to the positive control responder rate of 84%, no responders were observed across the set of 50 ex vivo samples for Avastin, Type I or Type II Affimer scaffolds, and only one responder to the Type III Affimer scaffold, despite the Affimer samples being tested at five times the concentration of the Avastin antibody therapeutic. These results increase the surety of the Affimer technology as a viable therapeutic platform with regards to future development and entry into the clinic.
Affimer proteins are easier to engineer than standard antibodies, and as therapeutics they offer many potential advantages over antibody therapeutics; their smaller size could increase tissue penetration, while the ability to build Affimer multimer molecules allows us to target different active pathways and processes within a disease system with the administration of just one molecule. Last year, our initial in vivo preclinical studies showed that the Affimer inhibitor of the PD-1/PD-L1 immune checkpoint was effective in reducing tumour growth in a murine model. Combined with the latest data on the low immunogenicity of the Affimer scaffold, this ensures Affimer technology remains on course to enter clinical development in 2019.
Affimer scaffolds show low immunogenicity, comparable to Avastin, in industry-standard ex vivo assays.
For more information on these results download our datapack.