T. Albert

Novel products for haemostasis – current status

Currently, new clotting factor concentrates are becoming available or are in advanced clinical studies that will significantly improve the treatment of patients with Haemophilia A or Haemophilia B. Various technologies are applied to extend half‐life and/or allow for alternative routes of administration, e.g. subcutaneous route. Today, the advances for recombinant factor IX are significantly with half‐life extensions to up to 100 h, allowing substitution intervals of 1–2 weeks. For recombinant factor VIII (FVIII) products the effect so far is only moderate, as the half‐life extension is limited to about 15–18 h by the clearance of FVIII through its binding to von Willebrand factor. However, novel products applying new technologies with significantly extended half‐life are already at the horizont, as a bispecific antibody that mimics FVIII. The pharmacokinetic improvements of the new products will lead to a revision of our current treatment regimens, with regard to intended trough levels, number of tolerated bleeds and likely will drive a greater individualization of regimens. Clearly, the potential of anti drug antibody response for these modified proteins must not be higher than with our current products. Another challenge are the increasingly diverse biochemical characteristics of the new products, that have to be considered when determining potencies and also when monitoring treatment in patients with the various available assays. Despite these challenges, the new products will significantly improve treatment and quality of life for our patients with haemophilia.

Significance of F8 missense mutations with respect to inhibitor formation

We have identified 1,135 haemophilia A patients with missense mutations associated with mild (46%), moderate (22%), severe (16%), and mixed haemophilia phenotypes (11%). Altogether, we detected 374 different missense mutations of which 195 are not listed in the HAMSTeRS database. While missense mutations are strongly underrepresented within the factor VIII (FVIII) B-domain, they are evenly distributed throughout the entire F8 cDNA sequence. Only 36 (5%) of 720 patients with missense mutations and known inhibitor status showed an association with inhibitor formation. Inhibitor prevalence was four-fold higher for severe haemophilia compared to mild/moderate phenotypes. Mutations associated with inhibitor formation were especially clustered within the C1/C2 domain compared to the other domains (8.7% C1/C2 domain vs. 3.6% non-C1/C2-domain; p-value: 0.01). Three different missense mutations (T314A [T295A], S2010P [S1991P], R2169H [R2150H]) were associated twice with inhibitor formation. Importantly, we found that the risk of inhibitor formation in association with FVIII missense mutations is significant higher if the amino acid substitution belongs to another physicochemical class than the original residue (p-value 0.039). For this purpose distinct classes of substitutions were grouped in association with side chains properties: class I, small/hydrophobic; class II, neutral; class III, acidic; class IV, basic. Thus, although missense mutations were associated with an overall lower risk of inhibitor formation compared to other F8 gene mutation types, different missense mutations correlate with specific risks for inhibitor formation. These differences have to be identified in assigning risk profiles to aid in choice of preventative treatments designed to prevent inhibitor formation.

Kinetic parameters of monoclonal antibodies ESH2, ESH4, ESH5, and ESH8 on coagulation factor VIII and their influence on factor VIII activity.

The murine monoclonal antibodies ESH2, ESH4, ESH5, and ESH8 specifically bind and inhibit the procoagulant activity of human coagulation factor VIII (FVIII). They are frequently used as a model of inhibitors which are raised against injected FVIII in about 25% of hemophiliacs as a serious side effect of substitution therapy. However, binding kinetics of the interaction of these antibodies with FVIII and their influence on FVIII activity (inhibition) have not yet been examined systematically. For this, we examined association and dissociation of protein:antibody interaction using surface plasmon resonance (SPR) and determined their ability to inhibit the FVIII activity in a one‐stage and a two‐stage assay. SPR‐analysis revealed that the equilibrium dissociation constants (KD) of ESH8 and ESH4 are low and in a similar range (ESH8: KD(ESH8) = 0.542 nM; ESH4: KD(ESH4) = 0.761 nM). A 5.7 times higher KD than for ESH4 was observed for ESH2 (4.33 nM), whereas ESH5 showed the highest KD of 28.8 nM. In accordance with the lowest KD, ESH8, and ESH4 reduced FVIII activity of normal human plasma almost completely in a one‐stage clot inhibition assay (ESH8: 91.9%; ESH4: 90.1%). However, ESH8 inhibited FVIII activity more efficiently as only 1.0 µg/ml ESH8 was sufficient to obtain maximum inhibition compared to up to 600 µg/ml of ESH4. Despite its attenuated KD, ESH2 inhibits FVIII:C still efficiently, reducing 61.3% of FVIII activity at a concentration of 9 µg/ml in the one‐stage clotting assay. However, a discrepancy of inhibitory efficiency was found depending on the method used to measure FVIII activity. These effects seem to be mainly caused by differences of activation time of FVIII during both FVIII activity assays. The systematic assessment of these results should support FVIII interaction studies, and can provide data to rationally test peptides/mimotopes to remove or neutralize inhibitors of FVIII activity. Copyright

Modified expression of coagulation factor VIII by addition of a glycosylation site at the N terminus of the protein

Recently, it was shown that glycoproteins with N-glycans close to the NH2 terminus can directly enter the calnexin/calreticulin cycle and bypass BiP binding. This should allow efficient secretion of glycoproteins such as factor VIII (FVIII) whose secretion is negatively affected by BiP interaction. Examination of the glycosylation pattern of the NH2 terminus of FV and FVIII revealed N-glycans at positions 23 and 27 in FV and at position 41 in FVIII. To improve FVIII secretion, a 14-amino-acid-long polypeptide with (G3) or without (G0; control) three N-linked glycosylation consensus sites was inserted upstream of the NH2 terminus of a B-domain deleted FVIII protein. Expression of G3- and G0-constructs in three different cell lines resulted in the same or even higher expression rate of protein as found for the B-domain deleted FVIII. However, as demonstrated by Western blot analysis, the G3- as well as the G0-protein variants were mainly retained inside the cells in similar amounts. Thus, glycosylation alone does not automatically lead to higher secretion rates, but must be in context to the normal structure of the FVIII protein.

Drei Patienten mit milder Hämophilie A der Variante T295A

Missense mutations are the most common F8 gene defects among the patients with non-severe haemophilia A. This type of mutation is typically associated with low (5%) inhibitor risk. In the present retrospective study we analysed the clinical data of 16 haemophiliacs with the T295A missense mutation treated at Bonn Haemophilia Centre. In total, three patients developed inhibitors: two patients experienced low-titer and one high-titer inhibitors. Both patients with low titer inhibitors underwent successful ITI. The third patient, at the age of 81, developed initially low-titer inhibitors (3 BU/ml) after rFVIII therapy because of knee surgery. He experienced spontaneous multiple large skin haematomas and haemarthrosis. Immunosuppressive therapy was not applicable because of the infectious origin of discitis (Th3-Th4). Immunoadsorption was performed, but the inhibitor titer increased up to 42 BU/ml nine weeks after termination. A successful treatment of discitis with antibiotics finally allowed a weekly therapy (four times) with rituximab (375 mg/m²). This resulted in a decrease of inhibitor titre to 0.7 BU/ml eight weeks after the fourth rituximab application. Patient had endogenous FVIII levels of 3–5%. Twelve months after rituximab therapy (after B cells recovery) he relapsed with low-titer inhibitors and therefore was treated with single rituximab dose (375 mg/m²) again. This resulted in his depletion of B cells, measurable endogenous FVIII levels and non measurable inhibitors. This study demonstrated T295A variant to be associated with significantly increased (3/16 patients, 17%) inhibitor development.Missense mutations are the most common F8 gene defects among the patients with non-severe haemophilia A. This type of mutation is typically associated with low (5%) inhibitor risk. In the present retrospective study we analysed the clinical data of 16 haemophiliacs with the T295A missense mutation treated at Bonn Haemophilia Centre. In total, three patients developed inhibitors: two patients experienced low-titer and one high-titer inhibitors. Both patients with low titer inhibitors underwent successful ITI. The third patient, at the age of 81, developed initially low-titer inhibitors (3 BU/ml) after rFVIII therapy because of knee surgery. He experienced spontaneous multiple large skin haematomas and haemarthrosis. Immunosuppressive therapy was not applicable because of the infectious origin of discitis (Th3-Th4). Immunoadsorption was performed, but the inhibitor titer increased up to 42 BU/ml nine weeks after termination. A successful treatment of discitis with antibiotics finally allowed a weekly therapy (four times) with rituximab (375 mg/m(2)). This resulted in a decrease of inhibitor titre to 0.7 BU/ml eight weeks after the fourth rituximab application. Patient had endogenous FVIII levels of 3-5%. Twelve months after rituximab therapy (after B cells recovery) he relapsed with low-titer inhibitors and therefore was treated with single rituximab dose (375 mg/m(2)) again. This resulted in his depletion of B cells, measurable endogenous FVIII levels and non measurable inhibitors. This study demonstrated T295A variant to be associated with significantly increased (3/16 patients, 17%) inhibitor development.

Inhibitor development and management in three non-severe haemophilia A patients with T295A variant: Hemmkörperentwicklung und Management

Missense mutations are the most common F8 gene defects among the patients with non-severe haemophilia A. This type of mutation is typically associated with low (5%) inhibitor risk. In the present retrospective study we analysed the clinical data of 16 haemophiliacs with the T295A missense mutation treated at Bonn Haemophilia Centre. In total, three patients developed inhibitors: two patients experienced low-titer and one high-titer inhibitors. Both patients with low titer inhibitors underwent successful ITI. The third patient, at the age of 81, developed initially low-titer inhibitors (3 BU/ml) after rFVIII therapy because of knee surgery. He experienced spontaneous multiple large skin haematomas and haemarthrosis. Immunosuppressive therapy was not applicable because of the infectious origin of discitis (Th3-Th4). Immunoadsorption was performed, but the inhibitor titer increased up to 42 BU/ml nine weeks after termination. A successful treatment of discitis with antibiotics finally allowed a weekly therapy (four times) with rituximab (375 mg/m²). This resulted in a decrease of inhibitor titre to 0.7 BU/ml eight weeks after the fourth rituximab application. Patient had endogenous FVIII levels of 3–5%. Twelve months after rituximab therapy (after B cells recovery) he relapsed with low-titer inhibitors and therefore was treated with single rituximab dose (375 mg/m²) again. This resulted in his depletion of B cells, measurable endogenous FVIII levels and non measurable inhibitors. This study demonstrated T295A variant to be associated with significantly increased (3/16 patients, 17%) inhibitor development.Missense mutations are the most common F8 gene defects among the patients with non-severe haemophilia A. This type of mutation is typically associated with low (5%) inhibitor risk. In the present retrospective study we analysed the clinical data of 16 haemophiliacs with the T295A missense mutation treated at Bonn Haemophilia Centre. In total, three patients developed inhibitors: two patients experienced low-titer and one high-titer inhibitors. Both patients with low titer inhibitors underwent successful ITI. The third patient, at the age of 81, developed initially low-titer inhibitors (3 BU/ml) after rFVIII therapy because of knee surgery. He experienced spontaneous multiple large skin haematomas and haemarthrosis. Immunosuppressive therapy was not applicable because of the infectious origin of discitis (Th3-Th4). Immunoadsorption was performed, but the inhibitor titer increased up to 42 BU/ml nine weeks after termination. A successful treatment of discitis with antibiotics finally allowed a weekly therapy (four times) with rituximab (375 mg/m(2)). This resulted in a decrease of inhibitor titre to 0.7 BU/ml eight weeks after the fourth rituximab application. Patient had endogenous FVIII levels of 3-5%. Twelve months after rituximab therapy (after B cells recovery) he relapsed with low-titer inhibitors and therefore was treated with single rituximab dose (375 mg/m(2)) again. This resulted in his depletion of B cells, measurable endogenous FVIII levels and non measurable inhibitors. This study demonstrated T295A variant to be associated with significantly increased (3/16 patients, 17%) inhibitor development.

Inhibitor development and management in three non-severe haemophilia A patients with T295A variant

Missense mutations are the most common F8 gene defects among the patients with non-severe haemophilia A. This type of mutation is typically associated with low (5%) inhibitor risk. In the present retrospective study we analysed the clinical data of 16 haemophiliacs with the T295A missense mutation treated at Bonn Haemophilia Centre. In total, three patients developed inhibitors: two patients experienced low-titer and one high-titer inhibitors. Both patients with low titer inhibitors underwent successful ITI. The third patient, at the age of 81, developed initially low-titer inhibitors (3 BU/ml) after rFVIII therapy because of knee surgery. He experienced spontaneous multiple large skin haematomas and haemarthrosis. Immunosuppressive therapy was not applicable because of the infectious origin of discitis (Th3-Th4). Immunoadsorption was performed, but the inhibitor titer increased up to 42 BU/ml nine weeks after termination. A successful treatment of discitis with antibiotics finally allowed a weekly therapy (four times) with rituximab (375 mg/m²). This resulted in a decrease of inhibitor titre to 0.7 BU/ml eight weeks after the fourth rituximab application. Patient had endogenous FVIII levels of 3–5%. Twelve months after rituximab therapy (after B cells recovery) he relapsed with low-titer inhibitors and therefore was treated with single rituximab dose (375 mg/m²) again. This resulted in his depletion of B cells, measurable endogenous FVIII levels and non measurable inhibitors. This study demonstrated T295A variant to be associated with significantly increased (3/16 patients, 17%) inhibitor development.Missense mutations are the most common F8 gene defects among the patients with non-severe haemophilia A. This type of mutation is typically associated with low (5%) inhibitor risk. In the present retrospective study we analysed the clinical data of 16 haemophiliacs with the T295A missense mutation treated at Bonn Haemophilia Centre. In total, three patients developed inhibitors: two patients experienced low-titer and one high-titer inhibitors. Both patients with low titer inhibitors underwent successful ITI. The third patient, at the age of 81, developed initially low-titer inhibitors (3 BU/ml) after rFVIII therapy because of knee surgery. He experienced spontaneous multiple large skin haematomas and haemarthrosis. Immunosuppressive therapy was not applicable because of the infectious origin of discitis (Th3-Th4). Immunoadsorption was performed, but the inhibitor titer increased up to 42 BU/ml nine weeks after termination. A successful treatment of discitis with antibiotics finally allowed a weekly therapy (four times) with rituximab (375 mg/m(2)). This resulted in a decrease of inhibitor titre to 0.7 BU/ml eight weeks after the fourth rituximab application. Patient had endogenous FVIII levels of 3-5%. Twelve months after rituximab therapy (after B cells recovery) he relapsed with low-titer inhibitors and therefore was treated with single rituximab dose (375 mg/m(2)) again. This resulted in his depletion of B cells, measurable endogenous FVIII levels and non measurable inhibitors. This study demonstrated T295A variant to be associated with significantly increased (3/16 patients, 17%) inhibitor development.