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BTK inhibition prevents anti-FVIII memory response
limit the memory B-cell response towards therapeutic FVIII. We first demonstrated that the inhibition of BTK in primed mice limits the increase in anti-FVIII IgG after a single re-challenge with FVIII. Plasma cells do not express BCR at their membrane and are, therefore, insensitive to BTK inhibition.19,38 The continuous endogenous produc- tion of anti-FVIII IgG by resident plasma cells may have introduced a bias in our experimental setup. Indeed, the formation of immune complexes between circulating anti-FVIII IgG and the administered FVIII neutralizes FVIII and alters its immunogenicity.39 In addition, immune complexes between FVIII and anti-FVIII IgG facilitate FVIII internalization by dendritic cells40 and could skew the target antigen-presenting cell population from memo- ry B cells to dendritic cells or macrophages. In support of our initial observation, however, inhibition of BTK also prevented the in vivo re-activation of FVIII-specific mem- ory B cells following adoptive transfer to FVIII-naïve mice and administration of FVIII, as well as the ex vivo differen- tiation of memory B cells into antibody-secreting plasma cells. Immune responses to FVIII in FVIII-deficient mice are notoriously heterogenous.21,26,27 This is illustrated by the fact that 80% of the mice developed primary immune responses to FVIII, and that memory responses were het- erogenous in the three experimental models used in the present study. Of note, although the effect of the inhibi- tion of BTK on the anti-FVIII memory response was het- erogenous among mice, results from all three experimen- tal models showed a significant reduction in the intensity of the anti-FVIII memory response. Our finding that BTK inhibition was efficient in blocking anti-FVIII recall but not primary immune responses is reminiscent of recent observations. Chronic inhibition of BTK with an analog of PF-06250112 was shown to prevent immune responses to T-independent antigens or to T-dependent antigens only provided that BCR had a strong affinity for the anti- gen, and provided that antigen ligation induced a strong signaling of the BCR.14,19 Memory B cells generally express BCR of high affinity for their cognate antigen owing to affinity maturation in the germinal centers. In the case of the anti-FVIII B-cell response, the rare human monoclonal anti-FVIII IgG studied to date were obtained following immortalization of memory B cells from inhibitor-posi- tive patients. The affinity of the anti-C2 domain IgG4k BO2C11 for FVIII41 is 10-11 M-1 and that of the anti-C1
domain IgG4k LE2E9 is greater than the affinity of von Willebrand factor for FVIII (i.e., KD<0.1 nM).42 Likewise, the affinity of polyclonal anti-FVIII IgG from inhibitor- positive patients was described to be in the low nanomo- lar range.43 Indirectly, the efficacy of PF-06250112 at blocking the recall anti-FVIII immune response confirms that FVIII-specific memory B cells express high affinity BCR.
A strong predictor of ITI failure is the intensity of the inhibitory titer peak that is reached within the 2 weeks that follow ITI initiation.45 The molecular and cellular mechanisms that underlie the reduced efficiency of ITI when the inhibitor peak is high after initiation of ITI are unknown. In particular, it is not understood whether a high level of circulating FVIII inhibitor reflects the quanti- tative or qualitative properties of the circulating anti-FVIII IgG, the affinity for FVIII of the BCR of memory B cells or the number of FVIII-specific memory B cells. Although the experimental protocols employed in the present work do not reflect the clinical situation, and although no animal model of ITI has been validated yet, we propose here that the inhibition of BTK-dependent BCR signaling as an adjunct therapy in patients starting ITI could increase the chances of the ITI being a success by preventing the reac- tivation of FVIII-specific memory B cells. Furthermore, the potential toxicity of PF-06250112 could be reduced by tar- geting the compound to FVIII-specific B cells using CD22L/FVIII-coated nanoparticles,44 thus paving the way towards their use in hemophilia A.
Acknowledgments
This study was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie (UPMC) Paris 6, and by a grant from Pfizer (Aspire Hemophilia research award 2014 WI185623). PF-06250112 was a gift from Pfizer according to a ‘Compound Transfer Program’-grant. The monoclonal mouse FVIII heavy chain-specific IgG mAb6 was a kind gift from Prof Jean-Marie Saint-Remy (KUL, Leuven, Belgium). JR and MI were recipients of fellowships from Ministère de l'Enseignement Supérieur et de la Recherche. We would like to acknowledge Carole Lasne at the Centre d’Explorations Fonctionnelles (CEF) for technical support and the Centre d'Histologie, d'Imagerie et de Cytométrie (CHIC) (Centre de Recherche des Cordeliers, Paris, France).
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