Non-inhibitory antibodies inducing increased emicizumab clearance in a severe haemophilia A inhibitor patient

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A Novel Hemostatic Monitoring System Convertible to FVIII Activity Based on Non-Activated ROTEM (NATEM) for Hemophilia a Patients during Emicizumab Prophylaxis

Emicizumab is a factor (F) VIII(a) mimicking bispecific antibody to FIX(a) and FX, which is a new therapeutic agent for hemophilia A patients (PWHA) with inhibitor. Although the clinical effect and safety were confirmed in the clinical studies (NEJM 2016, NEJM 2017, Blood Adv. 2017), there exists difficulty in monitoring of emicizumab by APTT-based assay. In the present study, we challenged to establish a hemostatic monitoring system convertible to FVIII activity (FVIII:C) for PWHA under the emicizumab prophylaxis by utilizing non-activated rotational thromboelastometry (ROTEM). The studies were approved by local ethics committee and the informed consent was obtained from each patient. We firstly prepared the FVIII-deficient whole blood samples treated with a neutralizing anti-FVIII polyclonal IgG (30BU/mL), and compared the in vitro hemostatic response in the presence of emicizumab (0, 5 and 50µg/mL) spiked into these samples among three modes of ROTEM such as NATEM, EXTEM and INTEM. According to CaCl2 triggered-NATEM without any other activator, the sum of clot time and clot formation time (CT+CFT) was 1269 ± 197 sec (mean ± SD) for normal control and 6713 ± 855 sec for the samples without emicizumab. The value of CT+CFT was significantly shortened in the presence of emicizumab at 5 µg/ml and 50 µg/ml to 2157 ± 192 sec and 1521 ± 313 sec, respectively (p = 0.025). By contrast, measured by EXTEM utilizing tissue factor (0.5 pM) and CaCl2 as triggers, the values of CT+CFT for control, 0, 5 and 50 µg/mL of emicizumab were 552 ± 97 sec, 771 ± 186 sec, 658 ± 135 sec and 778 ± 90 sec, respectively. There was no significant difference (p = 0.44) among them. By INTEM, triggered by ellagic acid with CaCl2, the values of CT+CFT for control, 0, 5, and 50 µg/mL of emicizumab were 662 ± 202 sec, 5854 ± 705 sec, 951 ± 170 sec and 814 ± 216 sec, respectively. The value of CT+CFT was shortened in the presence of emicizumab, however, little difference was observed between two doses. The other parameters, maximum clot firmness and alpha angle, were not suitable for quantitative evaluation irrespective of the mode of measurement. These suggested that CT+CFT based on NATEM is a suitable parameter for the monitoring of emicizumab. In order to develop a NATEM-based hemostatic scale convertible to FVIII:C, we secondly measured CT+CFT in the samples (n = 81) from PWHA with various FVIII:C levels in our hospital. The samples were classified based on FVIII:C into three groups, such as Tertile (T)1 with undetectable FVIII:C (<0.2 IU/dL,n = 22), T2 (0.2≤ FVIII:C <12 IU/dL, median FVIII:C 1.7 IU/dL, interquartile range 1.1-5.6 IU/dL, n = 35) and T3 group (12≤ FVIII:C <60 IU/dL, 19 IU/dL, 13-39 IU/dL, n = 24). T2 and T3 were delimited by FVIII:C 12 IU/dL in which zero joint-bleeding can be expected (den Uijil, Haemophilia 2011). The value of CT+CFT in T1, T2 and T3 group was 6702 ± 1517 sec, 2747 ± 817 sec and 2024 ± 630 sec, respectively, with the significant difference (p < 0.01). By utilizing this NATEM scale, we investigated the hemostatic function of four PWHA with inhibitor (patient 1, 2, 3 and 5) and two without inhibitor (patient 4, 6) enrolled in the phase 1 study (ACE001JP) and its extension study (ACE002JP) with the different types of dosing-regimen (0.3, 1.0 and 3.0 mg/kg/week after loading dose) (Table). The baseline value of CT+CFT among all of the patients was 6502 ± 1297 sec in the range of T1-level classified as above. The value was shortened and maintained to T2-level at the concentration of ~10 μg/ml after emicizumab infusion at all three dosing. The value reached up to T3-level of ~20 μg/ml in 1.0 and 3.0 dosing groups. The hemostatic function reached up to T2- and T3-level during loading phase before achievement to the steady state. No joint bleeding was observed among the patients who achieved T3-level except for patient 5 and 6 who developed breakthrough bleedings in target joints. In conclusion, the global hemostatic function during emicizumab prophylaxis can be quantitatively evaluated by NATEM-based scaling before steady state among PWHA. Disclosures Yada: Shire Japan Co., Ltd.: Other: Teacher at a endowed course. Nogami:Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau. Kasai:Chugai Pharmaceutical Co., Ltd: Employment. Shima:Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees.

The burden of inhibitors in haemophilia patients

SummaryThe burden of disease in haemophilia patients has wide ranging implications for the family and to society. There is evidence that having a current inhibitor increases the risk of morbidity and mortality. Morbidity is increased by the inability to treat adequately and its consequent disabilities, which then equates to a poor quality of life compared with non-inhibitor patients. The societal cost of care, or `burden of inhibitors’, increases with the ongoing presence of an inhibitor. Therefore, it is clear that successful eradication of inhibitors by immune tolerance induction (ITI) is the single most important milestone one can achieve in an inhibitor patient. The type of factor VIII (FVIII) product used in ITI regimens varies worldwide. Despite ongoing debate, there is in vitro and retrospective clinical evidence to support the use of plasma-derived VWF-containing FVIII concentrates in ITI regimens in order to achieve early and high inhibitor eradication success rates.

Frequency and Epitope Specificity of Anti-Factor VIII C1 Domain Antibodies in Acquired and Hereditary Hemophilia Inhibitor Patient Plasma

Acquired hemophilia A (AHA) is a rare autoimmune disease caused by development of inhibitory anti-factor VIII (fVIII) antibodies (also called inhibitors) resulting in severe hemorrhages. In addition, inhibitor development is the most serious complication of today's replacement therapy in patients with hereditary X-linked hemophilia A (HA) disorder. Earlier studies showed that antibodies in AHA and HA inhibitor plasmas are both primarily directed to the A2 and C2 domains suggesting that these two domains are the predominant immunogenic fVIII regions (Fulcher et al, 1987; Prescott et al, 1997; Lollar, 2004). However, the C1 domain also makes a major contribution to the humoral anti-fVIII immune response in hemophilic mice (Healey et al, 2007), which motivated us to analyze the frequency and epitope specificity of anti-C1 antibodies in AHA and HA inhibitor patient plasma. The frequency of domain-specific antibodies were studied by antibody binding to human A2, C1 and C2 domains presented as (i) single human domain (SHD) human/porcine hybrid fVIII and (ii) HSA-fusion proteins. While similar frequencies of A2- and C2-specific antibodies were observed for both applied mapping strategies the use of isolated C1 domain resulted in much higher detection level of anti-C1 antibodies compared to the use of the human C1 domain human/porcine hybrid fVIII protein. As homologue-scanning mutagenesis relies on differences among human and porcine sequences these results suggest the presence of a large number of cross-reactive anti-C1 antibodies binding to species-conserved epitopes. Overall, anti-C1 antibodies were detected in 90 of 115 (78%) AHA and 36 of 63 (57%) HA inhibitor patients. Two well-characterized monoclonal C1 inhibitors, human LE2E9 (Jacquemin et al, 2000) and murine MAb 2A9 (ASH 2014 poster, Batsuli et al) were used for indirect epitope mapping of anti-C1 antibodies in AHA patients by competition binding studies. Our results for AHA patients with non-crossreactive anti-C1 antibodies only (n=11) show that antibody binding to human C1 domain human/porcine hybrid fVIII (HP53) protein was completely blocked in the presence of MAb 2A9. In contrast, antibody binding to the isolated C1 domain was only partially reduced in the presence of MAb 2A9 for a selected number of (high responding) AHA patients (n=10) suggesting the presence of a second population of crossreactive anti-C1 antibodies that exclusively bind to conserved amino acid residues. Competition binding to native and denatured fVIII and HP53 proteins revealed that MAb 2A9 and LE2E9 bind mutually exclusive to a conformational C1 epitope involving amino acid residues that are not conserved between humans and pigs. Consequently, essential binding residues were identified for both C1 inhibitors via the use of HP53 variants, in which surface exposed non-conserved amino acid residues on the human C1 domain were substituted for porcine residues. The results of this mutational analysis showed that despite their competitive binding different amino acid residues are essential for binding of MAb 2A9 and LE2E9. These findings are in agreement with the different specific inhibitory activities of the two C1 inhibitors (97 BU/mg vs 10000 BU/mg). Finally, HSA-C1 point mutants were used to directly map essential epitope residues of anti-C1 antibodies in AHA and HA inhibitor patient plasma. Our study demonstrates that a large number of AHA and HA inhibitor patients (126 of 178; 71%) have anti-C1 antibodies that comprise at least two different populations, crossreactive and non-crossreactive to porcine fVIII. Therefore, in addition to the A2 and C2 domains, the C1 domain seems to significantly contribute to the immune response to fVIII in these patients. As recent data point toward a functional role of the fVIII C1 domain for membrane-, fX-, and von Willebrand factor-binding (Lü et al, 2011) the clinical relevance of anti-C1 antibodies should be analyzed in further studies. Disclosures Tiede: Leo Pharma: Consultancy, Honoraria; Novo Nordisk: Consultancy, Honoraria, Research Funding; Coachrom: Research Funding; SOBI: Consultancy, Honoraria; Biogen Idec: Consultancy, Honoraria; CSL Behring: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Boehringer Ingelheim: Consultancy, Honoraria; Octapharma: Other: Investigator, Speakers Bureau; Biotest: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Investigator, Research Funding; Baxter: Consultancy, Honoraria, Research Funding. Königs:Bayer: Research Funding, Speakers Bureau; Biotest: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Sobi: Consultancy; CSL Behring: Research Funding, Speakers Bureau; Intersero: Research Funding; NovoNordisk: Speakers Bureau.