Assessment of immunogenicity of romiplostim in clinical studies with ITP subjects

Abstract

Romiplostim is an Fc-peptide fusion protein that activates intracellular transcriptional pathways via the thrombopoietin (TPO) receptor leading to increased platelet production. Romiplostim has been engineered to have no amino acid sequence homology to endogenous TPO. Recombinant protein therapeutics can be at a risk of development of an antibody response that can impact efficacy and safety. Hence, a strategy to detect potential antibody formation to the drug and to related endogenous molecules can be useful. The immunogenicity assessment strategy involved both the detection and characterization of binding and neutralizing antibodies. The method for detection was based on a surface plasmon resonance biosensor platform using the Biacore 3000. Samples that tested positive for binding antibodies in the Biacore immunoassay were then tested in a neutralization assay. Serum samples from 225 subjects with immune thrombocytopenic purpura (ITP) dosed with romiplostim and 45 ITP subjects dosed with placebo were tested for romiplostim and TPO antibodies. Prior to romiplostim treatment, 17 subjects (7%) tested romiplostim antibody positive and 12 subjects (5%) tested TPO antibody positive for pre-existing binding antibodies. After romiplostim exposure, 11% of the subjects exhibited binding antibodies against romiplostim and 5% of the subjects with ITP showed binding antibodies against TPO. The antibodies against romiplostim did not cross-react with TPO and vice versa. No cases of anti-TPO neutralizing antibodies were detected in romiplostim-treated subjects. The incidence of anti-romiplostim neutralizing antibodies to romiplostim was 0.4% (one subject); this subject tested negative at the time of follow-up 4 months later. No impact on platelet profiles were apparent in subjects that had antibodies to romiplostim to date. In summary, administration of romiplostim in ITP subjects resulted in the development of a binding antibody response against romiplostim and TPO ligand. One subject developed a neutralizing antibody response to romiplostim that impacted the platelet counts of this subject. No neutralizing antibodies to endogenous TPO were observed.

Fig. 1

Process for assessment of immunogenicity in the romiplostim clinical trial program. The strategy for immunogenicity assessment involved a screening step where the serum samples were assessed for their ability to bind to TPO, romiplostim, and peptide component of romiplostim. If the sample showed binding above the validated assay threshold, it was further confirmed in a specificity test. Based on the reactivity observed, excess of the relevant TPO or romiplostim was added to the reactive sample and assessed for neutralization of the reactive response. If the sample exhibited more than 50% depletion of signal in drug specificity analysis, the sample was then confirmed for its ability to neutralize romiplostim or TPO in a biological functional assay

Fig. 2

Antibody incidence in ITP patients and healthy subjects across clinical studies. a Pre-existing antibodies to romiplostim or TPO include subjects who had binding antibodies prior to administration of romiplostim. Some of these antibodies continued to persist even after romiplostim dosing and were considered pre-existing. b Post-exposure antibodies to romiplostim or TPO included subjects who had binding antibodies following dosing with romiplostim. Serum samples from healthy subjects that were a part of two initial Phase 1 studies were evaluated for pre-existing and post-exposure binding antibodies to romiplostim and TPO. The antibody incidence was compared to that observed for ITP patients enrolled across ten clinical studies. ITP patients had a higher incidence of pre-existing and post-exposure antibodies to romiplostim as compared to the healthy subjects. Similarly, ITP patients had a higher post-exposure incidence of anti-TPO binding antibodies compared to healthy subjects

Fig. 3

a Platelet count by study week for subject that developed neutralizing antibodies to romiplostim. Platelet profiles and antibody status of a subject from the roll-over extension study that developed neutralizing antibodies to romiplostim are provided. Blood samples obtained at week 1 and week 12 were negative for binding antibodies to romiplostim. At week 36, week 60, and at week 66, blood samples were positive for anti-AMG 531 binding antibodies and negative for anti-AMG 531 neutralizing antibodies, and yielded negative results. The subject discontinued study at week 79 and the blood sample obtained at week 79/end of study (EOS) was positive for anti-AMG 531 binding antibodies and neutralizing antibodies. Single asterisks indicate negative for anti-romiplostim binding antibodies. Down arrows indicate positive for anti-romiplostim binding antibodies; negative for anti-romiplostim neutralizing antibodies. Double down arrows indicate positive for anti-romiplostim binding antibodies; positive for anti-romiplostim neutralizing antibodies. b Platelet count by study week for subject that developed neutralizing antibodies to TPO. Platelet profiles and antibody status of a subject from the roll-over extension study that had pre-existing antibodies to TPO. Blood samples obtained at week 1, week 9, week 17, and week 25 were assessed for antibodies to romiplostim and TPO. Samples were negative for binding antibodies to romiplostim at all time-points tested. The sample collected at pre-dose time-point prior to exposure to romiplostim was positive for both binding antibodies to TPO. Following dosing with romiplostim, samples obtained at week 9 and week 17 were negative for antibodies to TPO and positive for binding antibodies to TPO at week 25. No obvious impact on platelets could be observed due to presence of these pre-exposure neutralizing antibodies to TPO. Single asterisks indicate negative for anti-romiplostim binding antibodies. Down arrows indicate positive for anti-TPO binding antibodies; negative for anti-TPO neutralizing antibodies. Double down arrows indicate positive for anti-TPO binding antibodies; positive for anti-TPO neutralizing antibodies. Delta indicates negative for anti-TPO binding antibodies; negative for anti-TPO neutralizing antibodies