Low Immunogenicity to Romiplostim in Clinical Studies with ITP Subjects.

Romiplostim, a member of the thrombopoietin (TPO) mimetic class, is an Fc-peptide fusion protein (peptibody) that activates intracellular transcriptional pathways leading to increased platelet production via the TPO receptor (also known as cMpl). The peptibody molecule contains two identical single-chain subunits each consisting of human immunoglobulin IgG1 Fc domain, covalently linked at the C-terminus to a peptide containing two TPO receptor-binding domains. Due to the general concern regarding the immunogenic potential for all therapeutic proteins and the specific concern for monitoring antibodies capable of neutralizing thrombopoietin (TPO), an extensive immunogenicity assessment program was developed to support romiplostim. Romiplostim has been engineered to have no amino acid sequence homology to endogenous TPO. A low theoretical risk of developing conformational antibodies that cross-react against TPO exists. This risk was addressed by using an immunogenicity assessment strategy that relied upon a surface plasmon resonance based biosensor immunoassay using the Biacore 3000 capable of simultaneously monitoring antibodies that bind to romiplostim, TPO, or the active peptide portion of romiplostim (TMP). Samples that tested positive for binding antibodies in the Biacore immunoassay were then tested in the definitive functional biological assay to identify any antibodies capable of neutralizing the biological effect of romiplostim or TPO. Serum samples from 236 actively treated subjects were obtained both before and after exposure to romiplostim and were tested for romiplostim and TPO antibodies. In baseline samples, seventeen subjects (7.1%) tested romiplostim antibody positive and 12 subjects (5.1%) tested TPO antibody positive for pre-existing binding antibodies. After romiplostim exposure, twenty-five out of 236 (10.5%) subjects with ITP developed binding antibodies against romiplostim (inclusive of antibodies to both peptide and the whole molecule) and 12 out of 236 (5.1%) subjects with ITP developed binding antibodies against TPO. The antibodies that developed against romiplostim did not cross react with TPO and the antibodies that developed against TPO did not cross react with romiplostim. The incidence of anti-romiplostim neutralizing antibodies among 236 subjects with ITP who were treated with romiplostim across 10 clinical studies was 0.4% (1 out of 236). No cases of anti-TPO neutralizing antibodies were detected in romiplostim treated samples. In conclusion, after thorough immunogenicity assessment of all subjects treated with romiplostim using sensitive methods to detect binding and neutralizing antibodies, only one subject was found positive for the presence of antibodies capable of neutralizing romiplostim that was negative at the time of follow up 4 months later. As expected, none of the subjects treated were positive for antibodies capable of neutralizing TPO. No clinical sequelae were observed in association to the presence of antibodies.