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Case Reports
corticoids (1 mg/kg/day for 48 hours [h] to reduce peri- articular inflammation). A minor increase in emicizumab concentrations (1.7 mg/mL) and reduction in APTT-ratio (1.44) was observed (Figure 1), suggesting a potential cor- tico-sensitivity of the anti-emicizumab antibody-produc- ing plasmocytes. Although no bleeds were observed dur- ing a 3-week period, emicizumab levels remained unde- tectable following a short corticosteroid therapy (2 mg/kg/day, conform to the management of children’s immunologic thrombocytopenic purpura). Corticosteroid-therapy was therefore halted. Since anti- emicizumab antibodies have been reported to be tran- sient in some patients,11 emicizumab therapy (1.5 mg/kg/week) was continued for 3 months. As no improvement was observed, emicizumab therapy was terminated.
In order to further characterize the anti-emicizumab antibodies, additional tests were performed. We next analyzed eventual inhibition of emicizumab activity using the chromogenic activity assay (Biophen FVIII:C activity assay [ref 221402]; Hyphen BioMed, Andresy, France). Surprisingly, no reduction in emicizumab activity was observed, irrespective of whether normal or patient serum was tested (Figure 2C). Similar data were obtained using a one-stage clotting assay, suggesting that the anti- emicizumab antibodies are essentially non-inhibitory.
This was further assessed by analyzing binding of biotinylated (bt)-emicizumab to immobilized purified recombinant FIX (Pfizer, Paris, France) or plasma-derived FX (Cryopep; Montpellier, France). Biotinylation was per- formed using the NHS-PEO4-biotin kit (ref: UPR2027B; Griener Bio-one; Frickenhausen, Germany) without loss of cofactor activity. No inhibition of emicizumab binding to FIX was observed with normal or patient serum (Figure 2D). However, a modest inhibition of emicizum- ab binding to FX was observed using patient serum (Figure 2E). Maximal inhibition was 35±20% (n=4, P=0.0124 compared to normal serum) using 5-fold dilut- ed serum. The absence of a dominant inhibitory effect on the interactions between emicizumab and FIX/FX is com- patible with the non-inhibitory nature of the patient’s anti-emicizumab antibodies in the activity assays. Indeed, a modest decrease in emicizumab-FX complex formation would still allow for sufficient ternary complex to be formed (555 pM vs. 851 pM in the absence of inhibitor, when calculated according to Kitazawa et al.).12 We previously showed that a threshold of about 300 pM ternary complex is needed to produce sufficient hemosta- tic activity in vivo.13
As low emicizumab levels in the patient can be explained by an increased clearance, we determined sur- vival of bt-emicizumab in the absence or presence of serum in immuno-deficient mice. Bt-emicizumab was added to 0.9% NaCl, 100 mL control serum or 100 mL patient serum (both 5 mg per 100 mL), and incubated for 30 minutes (min) at ambient temperature. Mixtures were then injected via the retro-orbital plexus (at a dose of 0.25 mg/kg bt-emicizumab) to male NOD.CB17- Prkdcscid/NCrHsd-mice (age 9 weeks; ENVIGO, Gannat, France). Housing and experiments of NOD.CB17- Prkdcscid/NCrHsd-mice were performed in accordance with French regulations and the experimental guidelines of the European Community. Experimentation was approved by the local ethical committee of the Université Paris-Sud (Comité d’Éthique en Experimentation Animale no. 26, protocol APAFIS#4400- 2016021716431023v5). Blood samples were taken at 15 min, 6 h, 24 h and 48 h after infusion. Plasma was used to determine residual bt-emicizumab levels in an
enzyme-linked immonosorbent assay (ELISA) in which streptavidin-coated microtiter wells were incubated with plasma samples, and bound bt-emicizumab was probed using peroxidase-labeled anti-human IgG4-Fc antibodies. No difference in the disappearance from the circulation was found between bt-emicizumab alone or the bt-emi- cizumab/normal serum combination (t1/2=8.5 h and 8.3 h, respectively; Figure 2F). In contrast, bt-emicizumab was eliminated significantly faster in the presence of patient serum (t1/2=2.2 h; P=0.0013; Figure 2F), with no bt-emi- cizumab detectable at 48 h after infusion (compared to 21% for both other conditions). It, thus, seems that the main effect of the anti-emicizumab antibodies is to accel- erate clearance of emicizumab.
Contrary to previously reported inhibitors of emi- cizumab, we here describe the presence of antibodies that leave emicizumab activity unaffected, but that pro- voke the rapid elimination of emicizumab in mice, pro- viding an explanation for the low emicizumab levels in the patient. Although the occurrence of anti-emicizumab antibodies is rare, their presence may severely diminish its clinical efficacy, resulting in the re-appearance of spon- taneous bleeds. Clinical monitoring will usually be suffi- cient for patients receiving emicizumab therapy. However, in case of bleeding in the absence of any com- pliance concerns, biological monitoring (APTT and emi- cizumab concentration) is therefore helpful to detect pos- sible anti-drug antibodies.
Annie Harroche,1,* Thibaud Sefiane,2,*
Maximilien Desvages,2,3 Delphine Borgel,2,3 Dominique Lasne,2,3 Caterina Casari,2 Ivan Peyron,2 Laurent Frenzel,1 Stéphanie Chhun,4 Peter J. Lenting2 and Cécile Bally1
1Centre de Traitement de l'Hémophilie, AP-HP, Hôpital Necker Enfants Malades, Paris; 2Laboratory for Hemostasis, Inflammation & Thrombosis, Unité Mixed de Recherche 1176, Institut National de la Santé et de la Recherche Médicale, Université Paris-Saclay, Le Kremlin-Bicêtre; 3Laboratoire d’Hématologie, AP-HP, Hôpital Necker Enfants Malades, Paris and 4Laboratoire d’Immunologie Biologique, AP-HP, Hôpital Necker Enfants Malades, Paris, France
*AH and TS contributed equally as co-first authors. Correspondence: PETER J. LENTING - peter.lenting@inserm.fr doi:10.3324/haematol.2021.278579
Received: February 15, 2021.
Accepted: May 3, 2021.
Pre-published: May 20, 2021.
Disclosures: AH received fees for consultancy and board expert membership from Roche, Takeda, LFB, CSL-Behring, Sobi and NovoNordisk; DL received consulting fees from Roche (with fees going to Association de Recherche en Hématologie à Necker-Enfants Malades; ARHNEM); PJL received speaker fees from Biotest, Chugai, CSL-Behring, LFB-Biomedicament, NovoNordisk, Roche, Sanofi, Takeda and research support from Sobi, Catalyst Biosciences. All other authors declare no conflicts of interest.
Contributions: AH, CB were in charge of the patients and provided the clinical samples; MD, DB and DL performed the coagulation tests; TS, CC, IP and PJL designed and performed the in vitro and in vivo experiments. All authors contributed to the interpretation of the data. AH, CB and PJL wrote the first draft of the manuscript, and all authors contributed to the editing of the final manuscript.
Acknowledgments: we thank Dr. Alyanakian MA who leads the biological Necker Biobank.
Data sharing statement: data are available upon reasonable request to the corresponding author.
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