Modified recombinant human erythropoietin with potentially reduced immunogenicity

Abstract

Recombinant human erythropoietin (rHuEPO) is a biopharmaceutical drug given to patients who have a low hemoglobin related to chronic kidney disease, cancer or anemia. However, some patients repeatedly receiving rHuEPO develop anti-rHuEPO neutralizing antibodies leading to the development of pure red cell aplasia (PRCA). The immunogenic antibody response activated by rHuEPO is believed to be triggered by T-cells recognizing EPO epitopes bound to MHC molecules displayed on the cell surface of APCs. Previous studies have reported an association between the development of anti-rHuEpo-associated PRCA and the HLA-DRB1*09 gene, which is reported to be entrenched in the Thai population. In this study, we used computational design to screen for immunogenic hotspots recognized by HLA-DRB1*09, and predicted seventeen mutants having anywhere between one through four mutations that reduce affinity for the allele, without disrupting the structural integrity and bioactivity. Five out of seventeen mutants were less immunogenic in vitro while retaining similar or slightly reduced bioactivity than rHuEPO. These engineered proteins could be the potential candidates to treat patients who are rHuEpo-dependent and express the HLA-DRB1*09 allele.

HLA-B*46:01:01:01 and HLA-DRB1*09:01:02:01 are associated with anti-rHuEPO-induced pure red cell aplasia

“…2 ). These results are consistent with those reported in a previous study 25 . Consequently, the favorable interactions observed between rHuEPO and the mentioned HLAs could potentially stimulate the immune defense system.…”

Generation of bispecific antibodies by structure-guided redesign of IgG constant regions

“…Our structure-guided design process introduces mutations in the conserved regions of an IgG antibody. In order to assess the potential impact of the engineered mutations on increasing immunogenicity risk, we employed NetMHCIIpan version 4.0 ( 24 ) to predict MHC II binding sites on the WT and modified constant region sequences for common HLA class II alleles: DRB1_0101, DRB1_0701, DRB1_0901, DRB1_1101, DRB1_1201, DRB1_1501, DRB1_0401, DRB1_0405, DRB1_0301, DRB1_1302, DRB3_0101, DRB3_0202, DRB4_0101, and DRB5_0101 ( 25 ). The software was run using default settings.…”

Untoward immune effects of modern medication

“…Likewise, recombinant human erythropoietin (EPO) serves as an outstanding therapeutic peptide medicine for patients with low hemoglobin caused by certain serious illnesses, such as chronic renal disease and cancer. However, a long-term application of exogenous EPO may activate specific T cells, and thus stimulate the neutralizing ADAs that cross-react with endogenous EPO, leading to pure red cell aplasia consequently [ 20 ] . While the biological medication with recombinant factor Ⅷ confers a specific efficacy for the patients with hemophilia A, the neutralizing antibodies were stimulated in 20%–30% of the treated patients, thus resulting in the replacement therapy inefficient and even increasing mortality [ 21 ] .…”

“…In terms of mitigating measures for the above-mentioned peptide drugs and beyond, one possibility is to stop the antigenic agents, shifting to alternative medical options if available in the clinic [ 3 ] . Regarding pharmaceutical optimization in parallel, it has been insightfully explored to de-immunize the therapeutic proteins through advanced formulations, post-translational modifications, or/and a selective point mutation strategy to remove those crucial sites binding to human leukocyte antigen in the sequence of amino acid residues with preservation of due biologic effects [ 18 , 20 – 21 ] .…”