Biopharmaceuticals (BPs), such as monoclonal antibodies (mAbs) are widely used for the treatment of autoimmune disease, and cancer.

•A major concern regarding treatment with therapeutic proteins is the risk of provoking an unwanted immune response, such as the development of anti-drug antibodies (ADAs).

•ADAs can potentially decrease the efficacy of the BPs, modify clearance, induce hypersensitivity reactions or cause severe adverse events.

•Due to the safety issues associated with immunogenicity, it is of great importance to reduce the risk for immunogenicity in the clinic.

•A T cell dependent Ab response relies on T cell recognition of protein-derived epitopes that have been taken up, processed and displayed by HLA class II on antigen presenting cells (APCs). The HLA class II can differ between individuals and should be considered for those screenings to reflect different populations.

The new assay uses CD4+-enriched T cells and irradiated PBMCs comprising the APC population, and the effects of the biopharmaceuticals are tested by:

Cell prolifation (H3 incooperation), Cytokine secretion (IL-2 Elispot).

Test with 26 donors to the immune responst forKLH (30 µg/ml), CMV (2 µl/ml), infliximab, rituximab, adalimumab and natalizumab (all 45 µg/ml). 

Advantages to other immune cell assays:

•controlled number of specific T cells

•stonger response of enriched specific T cell fraction,

•no generation of DCs needed- hence allowing high througput analysis, cytokine contribution from non-specific cells can be limited.

•2 independent read outs of the immune response and activation.

Results: a novel T cell:PBMC assay that can evaluate the immunogenicity potential of biopharmaceuticals / capability of detecting low frequencies of BP-specific T cells / demonstrates a high in vitro immunogenicity to several BPs with a documented high clinical immunogenicity / can be used in early drug development to select drug candidates with low immunogenicity potential to ultimately increase patient safety.

Details:

Red blood cells were lysed using RBC lysis buffer  and the PBMCs washed twice in PBS. A fraction of the PBMCs was ?-irradiated at 3000 rads to prevent cell division to
ensure that the responses seen solely are CD4+ T cell-mediated. From the remaining fraction, CD4+ T cells were isolated using a CD4+ T cell enrichment kit (Easysep). CD4+ T cell purity was assessed by flow cytometry and was within the range of 93.0±4.8%. The CD4+ T cells were co-cultured at 37ÊC in 5% CO2 in serum-free Optimizer
medium  supplemented with Optimizer T-cell expansion supplement, 2mMGlutaMAX , 50 U/ml Penicillin and 50 µg/ml Streptomycin at a ratio 1:2 with the autologous irradiated PBMCs. After six-eight days, proliferation and IL-2 secretion were determined.