Coagulation factor VII variants resistant to inhibitory antibodies

Summary

Replacement therapy is currently used to prevent and treat bleeding episodes in coagulation factor deficiencies. However, structural differences between the endogenous and therapeutic proteins might increase the risk for immune complications. This study was aimed at identifying factor (F)VII variants resistant to inhibitory antibodies developed after treatment with recombinant activated factor VII (rFVIIa) in a FVII-deficient patient homozygous for the p.A354V-p.P464Hfs mutation, which predicts trace levels of an elongated FVII variant in plasma. We performed fluorescent bead-based binding, ELISA-based competition as well as fluorogenic functional (activated FX and thrombin generation) assays in plasma and with recombinant proteins. We found that antibodies displayed higher affinity for the active than for the zymogen FVII (half-maximal binding at 0.54 ± 0.04 and 0.78 ± 0.07 BU/ml, respectively), and inhibited the coagulation initiation phase with a second-order kinetics. Isotypic analysis showed a polyclonal response with a large predominance of IgG1. We hypothesised that structural differences in the carboxyl-terminus between the inherited FVII and the therapeutic molecules contributed to the immune response. Intriguingly, a naturally-occurring, poorly secreted and 5-residue truncated FVII (FVII-462X) escaped inhibition. Among a series of truncated rFVII molecules, we identified a well-secreted and catalytically competent variant (rFVII-464X) with reduced binding to antibodies (half-maximal binding at 0.198 ± 0.003 BU/ml) as compared to the rFVII-wt (0.032 ± 0.002 BU/ml), which led to a 40-time reduced inhibition in activated FX generation assays. Taken together our results provide a paradigmatic example of mutation-related inhibitory antibodies, strongly support the FVII carboxyl-terminus as their main target and identify inhibitor-resistant FVII variants.

Keywords

Inhibitory antibodies - replacement therapy - factor VII deficiency - carboxyl-terminus - recombinant proteins

^* A. Branchini and M. Baroni contributed equally to this study.

• References

• 1 Kreuz W, Becker S, Lenz E. et al. Factor VIII inhibitors in patients with haemophilia A: epidemiology of inhibitor development and induction of immune tolerance for factor VIII. Semin Thromb Haemost 1995; 21: 382-389.
• 2 DiMichele D. Inhibitor development in haemophilia B: an orphan disease in need of attention. Br J Haematol 2007; 138: 305-315.
• 3 Mariani G, Konkle B, Kessler CM. Inhibitors in Haemophilia A and B. In Hematology: Basic Principles and Practice. Philadelphia, PA: Elsevier Saunders; 2012. pp 1961-1970.
• 4 Gouw SC, van den Berg HM, Oldenburg J. et al. F8 gene mutation type and inhibitor development in patients with severe haemophilia A: systematic review and meta-analysis. Blood 2012; 119: 2922-2934.
• 5 Gouw SC, van den Berg HM, le Cessie S. et al. Treatment characteristics and the risk of inhibitor development: a multicenter cohort study among previously untreated patients with severe haemophilia A. J Thromb Haemost 2007; 5: 1383-1390.
• 6 Ingerslev J, Christiansen K, Sorensen B. International Registry on Factor VIIDSC Inhibitor to factor VII in severe factor VII deficiency: detection and course of the inhibitory response. J Thromb Haemost 2005; 3: 799-800.
• 7 Pruthi RK, Rodriguez V, Allen C. et al. Molecular analysis in a patient with severe factor VII deficiency and an inhibitor: report of a novel mutation (S103G). Eur J Haematol 2007; 79: 354-359.
• 8 Tokgoz H, Caliskan U, Lavigne-Lissalde G. et al. Successful prophylactic use of recombinant activated factor VII (rFVIIa) in a patient with congenital FVII deficiency and inhibitors to FVII. Haemophilia 2012; 18: e25-27.
• 9 Batorova A, Mariani G, Kavakli K. et al., on behalf of the STER Study Group. Inhibitors to factor VII and in congenital factor VII deficiency. Haemophilia 2014; 20: e188-191.
• 10 Greer J. Comparative modeling methods: application to the family of the mammalian serine proteases. Proteins 1990; 7: 317-334.
• 11 Furie B, Furie BC. Molecular and cellular biology of blood coagulation. N Engl J Med 1992; 326: 800-806.
• 12 Mariani G, Herrmann FH, Dolce A, et al. International Factor VIIDSG. Clinical phenotypes and factor VII genotype in congenital factor VII deficiency. Thromb Haemost 2005; 93: 481-487.
• 13 Rosen ED, Chan JC, Idusogie E. et al. Mice lacking factor VII develop normally but suffer fatal perinatal bleeding. Nature 1997; 390: 290-294.
• 14 Hay CR, Ollier W, Pepper L. et al. HLA class II profile: a weak determinant of factor VIII inhibitor development in severe haemophilia A. UKHCDO Inhibitor Working Party. Thromb Haemost 1997; 77: 234-237.
• 15 Oldenburg J, Pavlova A. Genetic risk factors for inhibitors to factors VIII and IX. Haemophilia 2006; 12 (Suppl. 06) 15-22.
• 16 Astermark J, Donfield SM, Gomperts ED. et al., Haemophilia Inhibitor Genetics Study Combined C. The polygenic nature of inhibitors in haemophilia A: results from the Haemophilia Inhibitor Genetics Study (HIGS) Combined Cohort. Blood 2013; 121: 1446-1454.
• 17 Pavlova A, Delev D, Lacroix-Desmazes S. et al. Impact of polymorphisms of the major histocompatibility complex class II, interleukin-10, tumor necrosis factor-alpha and cytotoxic T-lymphocyte antigen-4 genes on inhibitor development in severe haemophilia A. J Thromb Haemost 2009; 7: 2006-2015.
• 18 Bowen DJ. Haemophilia A and haemophilia B: molecular insights. Mol Pathol 2002; 55: 1-18.
• 19 Schwaab R, Pavlova A, Albert T. et al. Significance of F8 missense mutations with respect to inhibitor formation. Thromb Haemost 2013; 109: 464-470.
• 20 Sharathkumar A, Lillicrap D, Blanchette VS. et al. Intensive exposure to factor VIII is a risk factor for inhibitor development in mild haemophilia A. J Thromb Haemost 2003; 1: 1228-1236.
• 21 Arbini AA, Bodkin D, Lopaciuk S. et al. Molecular analysis of Polish patients with factor VII deficiency. Blood 1994; 84: 2214-2220.
• 22 Wulff K, Herrmann FH. Twenty two novel mutations of the factor VII gene in factor VII deficiency. Hum Mutat 2000; 15: 489-496.
• 23 Giles AR, Verbruggen B, Rivard GE. et al. A detailed comparison of the performance of the standard versus the Nijmegen modification of the Bethesda assay in detecting factor VIII:C inhibitors in the haemophilia A population of Canada. Association of Haemophilia Centre Directors of Canada. Factor VIII/ IX Subcommittee of Scientific and Standardisation Committee of International Society on Thrombosis and Haemostasis. Thromb Haemost 1998; 79: 872-875.
• 24 Branchini A, Rizzotto L, Mariani G. et al. Natural and engineered carboxy-terminal variants: decreased secretion and gain-of-function result in asymptomatic coagulation factor VII deficiency. Haematologica 2012; 97: 705-709.
• 25 Baroni M, Mazzola G, Kaabache T. et al. Molecular bases of type II protein S deficiency: the I203-D204 deletion in the EGF4 domain alters GLA domain function. J Thromb Haemost 2006; 4: 186-191.
• 26 Lavigne-Lissalde G, Rothschild C, Pouplard C. et al. Characteristics, mechanisms of action, and epitope mapping of anti-factor VIII antibodies. Clin Rev Allergy Immunol 2009; 37: 67-79.
• 27 Miller CH, Platt SJ, Rice AS. et al. Haemophilia Inhibitor Research Study I. Validation of Nijmegen-Bethesda assay modifications to allow inhibitor measurement during replacement therapy and facilitate inhibitor surveillance. J Thromb Haemost 2012; 10: 1055-1061.
• 28 Al Dieri R, Peyvandi F, Santagostino E. et al. The thrombogram in rare inherited coagulation disorders: its relation to clinical bleeding. Thromb Haemost 2002; 88: 576-582.
• 29 Castoldi E, Rosing J. Thrombin generation tests. Thromb Res 2011; 127 (Suppl. 03) S21-25.
• 30 Olivieri O, Martinelli N, Baroni M. et al. Factor II activity is similarly increased in patients with elevated apolipoprotein CIII and in carriers of the factor II 20210A allele. J Am Heart Assoc 2013; 2: e000440.
• 31 Reding MT. Immunological aspects of inhibitor development. Haemophilia 2006; 12 (Suppl. 06) 30-36.
• 32 van Helden PM, van den Berg HM, Gouw SC. et al. IgG subclasses of anti-FVIII antibodies during immune tolerance induction in patients with haemophilia A. Br Journal Haematol 2008; 142: 644-652.
• 33 Eigenbrot C, Kirchhofer D, Dennis MS. et al. The factor VII zymogen structure reveals reregistration of beta strands during activation. Structure 2001; 9: 627-636.
• 34 Larsen KS, Østergaard H, Bjelke JR. et al. Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa. Biochem J 2007; 405: 429-438.
• 35 Gray LD, Hussey MA, Larson BM. et al. Recombinant factor VIIa analog NN1731 (V158D/E296V/M298Q-FVIIa) enhances fibrin formation, structure and stability in lipidated haemophilic plasma. Thromb Res 2011; 128: 570-576.
• 36 Weimer T, Wormsbacher W, Kronthaler U. et al. Prolonged in-vivo half-life of factor VIIa by fusion to albumin. Thromb Haemost 2008; 99: 659-667.
• 37 Persson E, Bolt G, Steenstrup TD. et al. Recombinant coagulation factor VIIa--from molecular to clinical aspects of a versatile haemostatic agent. Thromb Res 2010; 125: 483-489.
• 38 Collins PW. Treatment of acquired haemophilia A. J Thromb Haemost 2007; 5: 893-900.
• 39 Abshire T, Kenet G. Recombinant factor VIIa: review of efficacy, dosing regimens and safety in patients with congenital and acquired factor VIII or IX inhibitors. J Thromb Haemost 2004; 2: 899-909.
• 40 James EA, van Haren SD, Ettinger RA. et al. T-cell responses in two unrelated haemophilia A inhibitor subjects include an epitope at the factor VIII R593C missense site. J Thromb Haemost 2011; 9: 689-699.
• 41 Eckhardt CL, Menke LA, van Ommen CH. et al. Intensive peri-operative use of factor VIII and the Arg593-->Cys mutation are risk factors for inhibitor development in mild/moderate haemophilia A. J Thromb Haemost 2009; 7: 930-937.