scholarly journals Elimination of FVIII-Specific B Cells By Immunotoxins Comprised of a Single FVIII Domain Fused to Pseudomonas Exotoxin a

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 561-561
Author(s):  
Kerstin Brettschneider ◽  
Anja Schmidt ◽  
Joerg Kahle ◽  
Aleksander Orlowski ◽  
Diana Stichel ◽  
...  
Keyword(s):  
B Cells ◽  
Knockout Mice ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Coagulation Factor ◽  
High Dose ◽  
Dependent Manner ◽  
C2 Domain ◽  
Exotoxin A

Abstract The development of inhibitory antibodies (inhibitors) against coagulation factor VIII (FVIII) is the most serious complication for patients with hemophilia A that undergo FVIII replacement therapy. In addition, healthy individuals can spontaneously develop inhibitory anti-FVIII auto-antibodies, which results in acquired hemophilia A. The current standard therapy for patients with hemophilia A and inhibitors, named immune tolerance induction (ITI), is based on frequent and mostly high dose administrations of FVIII. Unfortunately, the eradication of inhibitors can only be achieved in about 70% of patients. Alternative treatment of inhibitor patients with the monoclonal anti-CD20 antibody rituximab results in complete eradication of inhibitors; however, depletion of the entire CD20-positive B cell population is potentially accompanied by severe side effects. Recent studies in hemophilic FVIII knockout mice showed that the application of a FVIII-toxin conjugate resulted in (i) prevention of inhibitor development in naïve mice and (ii) long-term eradication of inhibitors in FVIII-immunized mice. As the use of FVIII for cell targeting of immunotoxins is presumably limited by its high molecular weight (250 kDa) and adhesiveness (off-target reactivity) we explored the potential use of alternative immunotoxins in the current study. The introduced immunotoxins are comprised of a single FVIII domain fused to the Exotoxin A (ETA) from Pseudomonas aeruginosa.The rationale for the use of a single domain instead of full length FVIII as cell-binding component is that immunodominant domains like A2 and C2 might still allow targeting of sufficient amounts of FVIII-specific B-cells by immunotoxins. For proof of concept studies, we generated a histidine-tagged C2 domain-ETA fusion protein (C2-ETA) that was bacterially expressed and purified by affinity chromatography. Purified C2-ETA was recognized by a panel of commercially available monoclonal anti-C2 antibodies in ELISA suggesting proper folding of the C2 domain in the bacterially expressed protein. To test the capacity of C2-ETA to eliminate FVIII-specific B-cells, splenocytes of FVIII-immunized FVIII knockout mice were re-stimulated with FVIII ex vivo in presence and absence of different concentrations of C2-ETA and ETA alone (as control). Re-stimulation of FVIII-specific memory B cells to FVIII- and C2-specific antibody secreting cells (ASC) was analyzed in anEnzyme linked immunospot (ELISPOT) assay using FVIII and C2 as antigens. While differentiation to FVIII-specific ASC was only partially inhibited by C2-ETA, differentiation to C2-specific ASC was completely blocked in a dose-dependent manner. In contrast, the use of ETA alone had no effect. Further analysis of the FVIII domain specificity of antibodies in plasma of FVIII-immunized FVIII knockout mice used for depletion studies revealed a strong contribution of C2-specific antibodies to the overall FVIII-specific immune response. In summary, our results show that the developed C2-ETA immunotoxin is able to specifically eliminate FVIII C2 domain-specific B cells ex vivo. Currently, C2-ETA is tested for its capacity to eliminate FVIII-specific B cells in FVIII knockout mice and additional FVIII domain-ETA immunotoxins are developed. Disclosures No relevant conflicts of interest to declare.

scholarly journals Coagulation Factor XII Plays an Important Role in Procoagulant Activity of Apoptotic Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2457-2457
Author(s):  
Aizhen Yang ◽  
Yi Wu
Keyword(s):  
Knockout Mice ◽  
Conflicts Of Interest ◽  
Coagulation Factor ◽  
Annexin V ◽  
Procoagulant Activity ◽  
Contact System ◽  
Coagulation System ◽  
Factor Xii ◽  
Apoptotic Cells ◽  
Dependent Manner

Abstract Apoptosis can be induced in a variety of pathological disorders, including inflammation, autoimmune diseases, and chemotherapy. When cells undergo apoptosis, they express phosphatidylserine (PS) on cell membrane surface and thus become procoagulant. Although it has been known that the procoagulant activity of apoptotic cells are tightly associated with thrombotic disorders, such as atherothrombosis and Trousseau syndrome, the mechanisms by which apoptotic cells activate the coagulation system and enhance blood clotting are largely unknown. In this study we investigated which coagulation factor(s) is involved in this process. Using western blotting and chromogenic substrate assay, we found that incubation with apoptotic cells induced by Dexamethasone (DXMS), but not with viable cells, resulted in rapid cleavage and activation of FXII. Moreover, apoptotic cells-mediated FXII activation was significantly increased in the presence of prekallikrein (PK) and high molecular weight kininogen (HK), other two components of plasma contact system. However, incubation of apoptotic cells did not cause dramatic changes of other coagulation factors, suggesting a selective association of FXII activation with apoptotic cells. Activation of FXII by apoptotic cells was markedly inhibited by a specific anti-kallikrein antibody, indicating the activation of the contact system by apoprotic cells. Flow cytometric measurement showed that FXII bound to apoptotic cells in a concentration-dependent manner, which was inhibited by annexin V and PS liposome. A surface plasmon resonance assay showed a direct binding of FXII to PS (KD=3.9E-9 M). When challenged by apoptotic cells, clotting time of plasma from FXII-knockout mice was significantly prolonged, which was reversed by replenishment with human FXII. Moreover, an inhibitory anti-FXII antibody completely prevented apoptotic cells-induced intrinsic tenase complex formation. Consistently, apoptotic cells significantly increased thrombin production in normal plasma, which were attenuated by PS blocker annexin V, an inhibitory anti-FXII antibody, and the deficiency of FXII, respectively. Addition of human FXII to XII-deficient plasma recovered thrombin generation. As evaluated by ELISA, the levels of thrombin-antithrombin complex in circulation were significantly increased when apoptotic cells were intravenously injected into wild-type mice, but not in FXII-knockout mice. In conclusion, FXII plays an important role in apoptotic cells-mediated procoagulant activity. Disclosures No relevant conflicts of interest to declare.

scholarly journals Optimized FVIII-Domain-Based Chimeric Antigen Receptors to Specifically Target FVIII Inhibitor-Producing B Cells in Hemophilia a

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2196-2196
Author(s):  
Vijay Bhoj ◽  
Lucy Li ◽  
Benjamin J. Samelson-Jones ◽  
Bhavya S Doshi ◽  
Christoph Ellebrecht ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
B Cells ◽  
Hemophilia A ◽  
Population Doubling ◽  
Target Cells ◽  
High Dose ◽  
C2 Domain ◽  
Hydrophobic Surfaces ◽  
Specific Lysis

Abstract Hemophilia A (HA) is a X-linked bleeding disorder caused by deficiency of coagulation factor VIII (FVIII). Optimal clinical management centers on FVIII protein concentrate replacement. However, up to 30% of patients with severe HA develop neutralizing antibodies to FVIII (inhibitors) upon exposure to therapeutic FVIII. Inhibitors neutralize the infused FVIII and, thus, pose a significant challenge in the management of these patients. Immune tolerance induction (ITI) using high-dose FVIII infusions can eliminate inhibitors but is not effective at generating long-term eradication in all patients. Thus, we developed an immunotherapy to more effectively eliminate FVIII-specific B cells to induce lasting eradication of inhibitors. Adoptive T cell immunotherapies using chimeric antigen receptor (CAR)-modified T cells (CARTs) are showing encouraging results in the treatment of cancer. We previously reported a proof-of-concept CART approach to eliminate autoantibodies to desmoglein-3, implicated in pemphigus vulgaris, by targeting non-malignant B cells. Here, using a similar strategy, we constructed two FVIII-based CARs using isolated FVIII A2 and C2 domains, which are commonly targeted by inhibitors Both FVIII fragments were used as the CAR extracellular domain followed by CD8a-derived hinge and transmembrane domains and intracellular signaling domains derived from 4-1BB and CD3z (BBz), as has been used in a recently FDA-approved CD19-targeted CAR (CART19) that have shown efficacy in patients. A2- and C2-CARs were introduced into primary human T cells using lentivirus vectors and were found to express on the cell surface. Primary expansion following anti-CD3/CD28 activation and lentivirus transduction yielded comparable population doublings of CART19 and A2-CARTs over 9-11 days (mean, 4.3 vs. 3.7; p=0.16). However, C2-CARTs consistently achieved fewer doublings compared to CART19 (mean 4.3 vs. 2.6; p=0.01). Additionally, C2-CARTs maintained larger cell volumes following initial activation compared to A2 and CART19, reminiscent of other CARs that demonstrate a high-level of ligand-independent basal activation. Consistent with hyper-activation and activation-induced cell death, flow cytometric analysis of C2-CART cultures showed prolonged expression of CD69, increased levels of cell death, and gradual loss of CAR+ cells compared to A2 and CART19 cultures. The FVIII C2 domain contains hydrophobic surfaces involved in binding to phospholipid membranes and von Willebrand Factor. We hypothesized that these hydrophobic regions may cause unfavorable interactions that result in CAR clustering and, in-turn, ligand-independent signaling. However, since these regions are also targeted by FVIII inhibitors, mutation of hydrophobic residues to improve CAR function would likely result in loss of binding to intended targets. Thus, we tested two independent strategies to improve function of C2-CARTs, both of which maintain native FVIII domain sequences. First, to block unfavorable interactions with C2 hydrophobic surfaces, we tested whether addition of C2 domain specific antibodies to the CART culture would improve expansion. Indeed, addition of anti-C2 antibodies improved C2-CART expansion by approximately one population doubling and reduced both cell volume and CD69 expression in C2-CARTs. As an alternate and perhaps more easily translatable approach, we replaced the BBz CAR signaling domain with a killer immunoglobulin receptor (KIR)-based multi-chain signaling system, which we previously utilized to improve function of a mesothelin-targeted CAR. KIR-based signaling effectively "rescued" the C2 CAR, and showed an expansion and activation profile comparable to CART19. Lastly, KIR-based FVIII CARs, demonstrated specific lysis of and cytokine response to target cells engineered to express surface anti-FVIII, even in the presence of soluble inhibitors. These data indicate that unfavorable interactions may be ameliorated directly by blocking agents or, indirectly, by altering intracellular signal transduction domains. These strategies have allowed us to construct optimized FVIII-based CARTs to target FVIII-specific B cells that are responsible for producing FVIII inhibitors in patients with HA. Disclosures Doshi: Bayer Hemophilia Awards Program: Research Funding. Milone:Novartis: Patents & Royalties.

Abstract 56: Coagulation Factor XI Promotes Distal Platelet Activation and Single Platelet Consumption in the Bloodstream Under Shear Flow

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Jenya Zilberman-Rudenko ◽  
Chantal Wiesenekker ◽  
Asako Itakura ◽  
Owen J McCarty
Keyword(s):  
Shear Flow ◽  
Platelet Activation ◽  
Platelet Adhesion ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Coagulation Factor ◽  
Thrombin Inhibitor ◽  
Dependent Manner ◽  
Factor Xi ◽  
Primate Model

Objective: Coagulation factor XI (FXI) has been shown to contribute to thrombus formation on collagen or tissue factor (TF)-coated surfaces in vitro and in vivo by enhancing thrombin generation. Whether the role of the intrinsic pathway of coagulation is restricted to the local site of thrombus formation is unknown. This study was designed to determine whether FXI could promote both proximal and distal platelet activation and aggregate formation in the bloodstream. Approach and Results: Pharmacological blockade of FXI activation or thrombin activity in blood did not affect local platelet adhesion, yet reduced local platelet aggregation, thrombin localization and fibrin formation on immobilized collagen and TF under shear flow, ex vivo . Downstream of the thrombus formed on immobilized collagen or collagen and 10 pM TF, platelet CD62P expression and microaggregate formation and progressive platelet consumption were significantly reduced in the presence of FXI-function blocking antibodies or a thrombin inhibitor in a shear rate- and time-dependent manner. In a non-human primate model of thrombus formation, we found that inhibition of FXI reduced single platelet consumption in the bloodstream distal to a site of thrombus formation. Conclusions: This study demonstrates that the FXI-thrombin axis contributes to distal platelet activation and procoagulant microaggregate formation in the blood flow downstream of the site of thrombus formation. Our data highlights FXI as a novel therapeutic target for inhibiting distal platelet activation without affecting proximal platelet adhesion.

scholarly journals Diagnosis and Treatment of Acquired Hemophilia: One Single-Center Experience from PR. China

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4250-4250
Author(s):  
Rong-Fu Zhou ◽  
Yueyi Xu ◽  
Wenjin Gao
Keyword(s):  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Clinical Manifestations ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Acquired Hemophilia A ◽  
Acquired Hemophilia ◽  
Coagulation Factor Vii ◽  
Prolonged Aptt ◽  
Inhibitor Titer

Abstract Objective: To deepen the understanding of the clinical manifestations of acquired hemophilia A for timely and correctly treatment. Methods: The clinical data of the acquired hemophilia A patients diagnosed in the hospital from Jan 2006 to Mar 2021 were retrospectively analyzed, and the relevant literature was reviewed. Results: 17 patients with acquired hemophilia A, male: female =10: 7, median age 61 years (19 to 78 years), were diagnosed and treated in the hospital with the median time from the onset to diagnosis 21 days (2 days to 6 months). Six patients had comorbidity, including hepatitis B carrying, chronic myelomonocytic leukemia, diabetes, hypertension and positive autoantibodies, pemphigoid and gastric cancer, respectively. Other 11 patients were healthy before the onset. All patients had large large ecchymosis of skin, and one case was combined with hematuria, and one case with retroperitoneal hematoma. All patients had APTT extension (45s-144.7s) and the prolonged APTT could not be corrected with normal mixed plasma with and without incubation at 37℃ for 2 hours. FVIII activity was 1% - 8.9% and inhibitor titer 2 - 128 Bu/ml. All patients with bleeding were with prothrombin complex/recombinant activated coagulation factor VII, some of them with pd-coagulation factor FVIII preparations. Inhibitors were removed with prednisone acetate (1 case) + chemotherapy (1 case), prednisone acetate / + CTX (11 cases) + chemotherapy (1 case), prednisone acetate/prednisolone + mabthera (2 cases) + CTX (1 case), respectively. The removal time of inhibitor was from 8 days to 4 years. During the treatment process, two patients developed lower extremity venous thrombosis, and one patient was complicated with lung infection. Conclusion: Patients with unexplained bleeding and prolonged APTT should be conducted normal mixed plasma correction test, coagulation factor activity and inhibitor titer examination. After correctly diagnosis, bypass agents /coagulation factor VIII preparations should be given timely for hemostasis, protocol based on glucocorticoid + CTX/mabthera to remove the inhibitor and symptomatic treatment for patients with primary comorbidity disease at the same time. Disclosures No relevant conflicts of interest to declare.

scholarly journals Sustained expression of therapeutic levels of human factor VIII in mice

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4671-4677 ◽  
Author(s):  
S Connelly ◽  
JM Gardner ◽  
RM Lyons ◽  
A McClelland ◽  
M Kaleko
Keyword(s):  
Factor Viii ◽  
Adenoviral Vector ◽  
Human Factor ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
High Dose ◽  
Specific Enzyme ◽  
Adult Mice ◽  
Human Factor Viii

Deficiency of coagulation factor VIII (FVIII) results in hemophilia A, a common hereditary bleeding disorder. Using a human FVIII-encoding adenoviral vector, Av1ALAPH81, we have demonstrated expression of therapeutic levels of human FVIII in mice sustained for more than 5 months after vector administration. Administration of a high dose (4 x 10(9) plaque-forming units [pfu]) of Av1ALAPH81 to mice resulted in a peak expression of 2,063 ng/mL of human FVIII in the mouse plasma, with levels decreasing to background by weeks 15 to 17. Normal FVIII levels in humans range from 100 to 200 ng/mL and therapeutic levels are as low as 10 ng/mL. Alternatively, administration of 8- to 80-fold lower vector doses (5 x 10(8) pfu to 5 x 10(7) pfu) to normal adult mice resulted in expression of FVIII at therapeutic levels sustained for at least 22 weeks. Detailed analysis of vector toxicity indicated that the high vector dose caused a dramatic elevation of liver-specific enzyme levels, whereas an eight-fold lower vector dose was significantly less hepatotoxic. The data presented here demonstrate that administration of lower, less toxic vector doses allow long-term persistence of FVIII expression.

A Subset of High Titer Acquired Hemophilia Patients May Benefit from Treatment with High Dose Factor VIII.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3476-3476
Author(s):  
Shannon Meeks ◽  
John F Healey ◽  
Ernest T Parker ◽  
Pete Lollar
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
High Titer ◽  
High Dose ◽  
Type I ◽  
C2 Domain ◽  
Type Ii ◽  
Acquired Hemophilia ◽  
Proteolytic Activation

Abstract Abstract 3476 Poster Board III-413 Approximately 30% of patients with severe hemophilia A will develop inhibitory antibodies (Abs) to factor VIII (fVIII inhibitors). The immune response to fVIII currently is the most significant complication in the management of patients with hemophilia A. In addition, autoimmune Abs to fVIII can develop in non-hemophiliacs, producing acquired hemophilia A, which frequently produces life- or limb-threatening bleeding. Patients with autoimmune hemophilia often have Abs with type II kinetics in which there is incomplete inactivation of fVIII at saturating concentrations of inhibitor. We have characterized the antibody response to the C2 domain of human fVIII in a murine hemophilia model and described 5 structural groups of Abs. Groups A, AB, and B are classical anti-C2 Abs that block fVIII and fVIIIa binding to phospholipid. Groups BC and C consist of non-classical anti-C2 Abs that inhibit the proteolytic activation of fVIII but do not block the binding of fVIII to phospholipid. Subsequently, we identified classical and non-classical anti-C2 Abs in human fVIII inhibitor plasmas. Most murine non-classical Abs have inhibitor titers greater than 10,000 Bethesda units/mg IgG. In a murine in vivo bleeding model, both type I classical C2 Abs, type II non-classical C2 Abs, and a type I anti-A2 Ab produced similar amounts of blood loss that were significantly greater than control mice injected with 180 U/kg of fVIII alone. Increasing the dose of fVIII to 360 U/kg overcame the bleeding diathesis produced by the type II MAbs, but not the type I Abs. These results were consistent with the in vitro Bethesda assay in which a type I anti-A2 Ab, 4A4, completely inhibited both 1 U/mL and 3 U/mL fVIII, while there was 40% residual activity at saturating concentrations of a type II anti-C2 Ab, 2-77, at either concentration of fVIII. To determine if similar in vitro characteristics exist in patients with acquired hemophilia, plasmas from 3 patients with high titer type II inhibitors were studied. All 3 plasmas primarily had C2 domain epitope specificity that included non-classical Abs. Plasma A7 additionally had detectable anti-A2 activity. Recovery of fVIII activity after a 2 h incubation at 37 °C at nominal added concentrations of 1 mL and 3 U/mL fVIII was compared (Table 1). At 3 U/mL added fVIII, recovery of activity in plasmas A4 and A5 was 1.1 U/mL and 0.51 U/mL, respectively, despite the presence of inhibitor titers of 18 and 11 Bethesda units (BU) per mL. The presence of anti-A2 Abs, which typically have type I kinetics, may have contributed to the overall lower recovery of activity in plasma A7. These results suggest that treatment with high-dose fVIII rather than bypassing agents may be warranted in patients with an inhibitor response dominated by non-classical anti-C2 Abs. Table 1 Patient Plasma Inhibitor Titer (BU/mL) Recovered Activity at 1U/mL FVIII (U/mL) Recovered Activity at 3 U/mL FVIII (U/mL) A4 18 0.31 1.1 A5 11 0.18 0.51 A7 62 0.07 0.12 Disclosures: No relevant conflicts of interest to declare.

Successful Long Term Therapeutic Expression of Factor VIII in Hemophilia A Dogs After Administration of AAV-cFVIII Using a Two-Chain or Single Chain Delivery Approach.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 546-546
Author(s):  
Denise E. Sabatino ◽  
Ekaterina Altynova ◽  
Amy M. Lange ◽  
Shangzhen Zhou ◽  
Elizabeth P. Merricks ◽  
...  
Keyword(s):  
Low Dose ◽  
Hemophilia A ◽  
High Dose ◽  
Dependent Manner ◽  
Igg Antibodies ◽  
Single Chain ◽  
Spontaneous Bleeding ◽  
Aav Vector ◽  
Bleeding Episodes

Abstract Abstract 546 While adeno-associated virus (AAV) is a promising gene delivery vector, it has been challenging to deliver FVIII due to the large size of the FVIII cDNA and the high frequency of FVIII antibody formation in hemophilia A (HA) patients. We used two approaches to overcome the size limitation of AAV for FVIII: (1) two-chain delivery in which the canine FVIII (cFVIII) heavy chain (HC) is delivered in one AAV vector and the cFVIII light chain (LC) is delivered in a second AAV vector and (2) single chain delivery in which the B-domain deleted cFVIII cDNA with minimal regulatory elements is within one AAV vector. In the two-chain approach AAV-HC (4.0 Kb) and AAV-LC (3.9 Kb) with a liver specific promoter was co-injected at a dose of 6×1012 vector genomes/vector/kg or 1.25×1013vg/vector/kg using AAV8 or AAV9 via hepatic infusion. Five hemophilia A dogs treated with AAV-HC and AAV-LC expressed 0.5-11% cFVIII in a dose-dependent manner. The mean cFVIII activity based on Coatest assay for the low dose was 1.3% (>1220d)(Linus)(AAV8) and 0.6% (>1770d)(H19)(AAV9), while for the high dose it was 5.2% (800d)(F24)(AAV8) and 2.4% (>1270d)(Woodstock)(AAV9). One dog (J60) had a splenectomy due to a complication at the time of surgery and has maintained high levels of expression (mean 11.0%; >820d). The WBCT consistently remained at a mean of 17.6 min for low dose dogs and 13.7 min for high dose dogs compared to 8-12 min in normal dogs. Using novel reagents that we generated specific to cFVIII, we developed assays to detect cFVIII antigen levels and IgG antibodies. Despite receiving equal doses of each vector, at day 85 the cFVIII-LC antigen levels (71.7 ± 19.2 ng/ml) were >10-fold higher than would be predicted based on activity while the cFVIII-HC antigen levels (14.6 ± 9.2 ng/ml) were >3-fold higher than activity. Since functional FVIII synthesis relies on the co-transduction of AAV-HC and AAV-LC in the same cell, this suggests that only a portion of the vector co-transduces and expresses cFVIII in the same cell and that the light chain is secreted more efficiently than the HC. No IgG antibodies to cFVIII were detected at any time point in these dogs. Three dogs have maintained FVIII expression for >3.5 years and two dogs for >2 years with ongoing observation. No spontaneous bleeding episodes have been observed in these dogs for a cumulative observation of >16 years while >80 bleeding episodes would be expected during this time period. The second approach, the single chain delivery, overcomes the co-transduction requirement of the two-chain approach by ensuring that each transduced cell expresses functional FVIII. However, it is difficult to efficiently package the large 5.2 Kb single chain construct into an AAV vector. Since no significant differences were observed between AAV8 and AAV9 using the two-chain approach, we used AAV8 to deliver the single chain cFVIII by peripheral vein infusion at 2×1013vg/kg or 4×1013vg/kg. The mean cFVIII activity was 0.7% (>430d) for the low dose dog (L51) and 6.8% (>290d) and 2.2% (>110d) for the high dose dogs (M06, M50). cFVIII HC and LC ELISA showed that cFVIII antigen levels correlated with activity. WBCT was a mean of 19.1 min for L51, 15.3 min for M06 and 11.6 min for M50. No spontaneous bleeding episodes have been observed in these dogs. The high dose dogs had no IgG antibodies to FVIII. L51 had transient IgG antibodies to FVIII until d52 in the absence of a Bethesda titer. A rise in FVIII expression in L51 coincided with the disappearance of anti-cFVIII antibodies. Comparison of single chain and two-chain delivery of FVIII reveals that (1) long term therapeutic levels of cFVIII in a dose-dependent manner can be obtained with both delivery approaches; (2) circulating cFVIII antigen levels are >10-fold higher than activity in the two-chain delivery in contrast to single chain delivery in which antigen correlates with activity; and (3) high antigen levels may facilitate tolerance to FVIII in the setting of liver-directed gene transfer, since a transient non-inhibitory antibody was observed in only one dog with very low FVIII levels. Notably, no cellular toxicity due to continuous expression of various forms of FVIII was found in these animals based on long-term sustained FVIII expression levels and normal liver enzymes in all eight HA dogs. Further studies to characterize the immune responses to the transgene will define the optimal vector approach. These data will form the basis for clinical studies in humans with severe HA. Disclosures: No relevant conflicts of interest to declare.

Trace FVIII Augments Rfviia Induced Thrombin Generation and Improves Hemostasis in Hemophilia A Patients with Inhibitors: A clinical Cohort Study

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 40-40
Author(s):  
Tami Livnat ◽  
Uri Martinowitz ◽  
Shirley Azar-Avivi ◽  
Ariela Zivelin ◽  
Gili Kenet
Keyword(s):  
Thrombin Generation ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Therapy Response ◽  
Response To Therapy ◽  
Individual Therapy ◽  
Severe Bleeding ◽  
Inhibitor Level

Abstract Abstract 40 Treatment of Hemophilia A patients with inhibitors is challenging, as correlation between inhibitor level and hemostatic response to therapy may be limited. Thrombin generation (TG) assays may be used to monitor hemostasis and/or predict patients' response to various bypass agents. Since combination of excess FVIII and bypassing agents may potentiate improved TG in inhibitor plasma tested in-vitro, we aimed to define the therapeutic feasibility of co-administration of rFVIIa and FVIII in hemophilia A patients with inhibitors. Patients and Methods: Following consent, blood was sampled from 15 hemophilia patients (age: 0.5–46y) with inhibitor (0.5–668 BU). Platelet poor plasma (PPP) was prepared, spiked and incubated with excess FVIII. Ex-vivo kinetics of FVIII neutralization over time was evaluated by sequential measurements of residual FVIII activity. We then used recalcification induced-TG (performed in PPP supplemented with 4μM phospholipids), to measure the ex-vivo response to increasing concentrations of FVIII (0–200%) and rFVIIa (0–6.8μg/ml), alone or in combination. Based upon these ex-vivo studies, an individually tailored therapeutic regimen of concomitant bolus doses of rFVIIa and FVIII was applied to nine hemophilia patients with inhibitors. Results: FVIII ex- vivo measurements post incubation detected either rapid or slow neutralization- not correlating with inhibitor level. Flat baseline TG curves were recorded for all inhibitor patients, with variable responses to FVIII and/or rFVIIa. Combined spiking with FVIII and rFVIIa dramatically increased rFVIIa induced ETP (762.7 ±305.7 as compared to 339.3±179.9 nM/min with rFVIIa only) and peak height (48.7±23.6 vs 23.7±16.6) in all patients' plasma samples. Based upon individual ex vivo assays, concomitant bolus doses of rFVIIa (120–200 mcg/kg) and FVIII (50–100 U/Kg), were applied to 9 patients, for a total of 333 episodes during study period (February 2010-Septemeber 2012). Patients during immune tolerance received rFVIIa prophylaxis with combined rFVIIa/FVIII dosing applied 3 times weekly. For most mild- moderate joint bleeds hemostasis was defined as satisfactory following a single combined dose. Severe bleeding episodes or target joint bleeds responded to 2–8 (median:3) combined doses, applied every 12 hours. During study period the median number of spontaneous joint bleeds decreased from 4 to 1 per month. Neither thrombosis nor any other complications evolved. Conclusions: Prediction of individual therapy response may be achieved by pre-analytical studies, assessing FVIII neutralization kinetics as well as ex-vivo TG responses to combined bypass/FVIII therapy. Such studies enabled treatment of inhibitor patients according to individually tailored regimens. We confirmed for the first time that the in- vitro advantage of combining FVIII and rFVIIa, indeed accounts for improved hemostasis and may safely be applied to inhibitor patients. Disclosures: No relevant conflicts of interest to declare.

Structural Studies Of The Factor VIII C2 Domain In a Ternary Complex With Two Inhibitory Antibodies Reveals Classical and Non-Classical Epitopes

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2358-2358
Author(s):  
Justin D Walter ◽  
Rachel A Werther ◽  
Caileen M Brison ◽  
John F. Healey ◽  
Shannon L. Meeks ◽  
...  
Keyword(s):  
Factor Viii ◽  
Ternary Complex ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Structural Basis ◽  
C2 Domain ◽  
Proteolytic Activation ◽  
X Ray ◽  
Binding Interface ◽  
Inhibitory Antibodies

Abstract The factor VIII C2 domain is a highly immunogenic domain, whereby inhibitory antibodies develop following factor VIII replacement therapy for congenital hemophilia A patients. Inhibitory antibodies also arise spontaneously in cases of acquired hemophilia A. The structural basis for molecular recognition by two classes of anti-C2 inhibitory antibodies that bind to factor VIII simultaneously has been investigated by small angle X-ray scattering and X-ray crystallography. The C2 domain/3E6 FAB/G99 FAB stable ternary complex, both in solution and in its crystalline state, illustrates that each antibody epitope resides on opposing faces of the factor VIII C2 domain. The 3E6 epitope is a classical antibody that forms direct contacts to the C2 domain at two loops consisting of Glu2181-Ala2188 and Thr2202-Arg2215, which inhibits the binding of the C2 domain to von Willebrand Factor and phospholipid surfaces. The G99 is a non-classical antibody that prevents proteolytic activation of factor VIII, and its epitope centers on Lys2227 and also makes direct contacts with loops Gln2222-Trp2229, Leu2261-Ser2263, His2269-Val2282 and Arg2307-Gln2311. Each binding interface is highly electrostatic, with positive charges present on both C2 epitopes and complementary negative charges on each antibody. A new model of phospholipid membrane association is also presented, where the 3E6 epitope faces the negatively charged membrane surface and Arg2320 is poised at the center of the binding interface. Furthermore, a 1.7 Å X-ray crystal structure of the porcine factor VIII C2 domain has also been determined, which supports the presented model for phospholipid binding. These results illustrate the complex nature of the polyclonal immune response against the factor VIII C2 domain, and further define the epitopes for both classical and non-classical inhibitory antibodies. Disclosures: No relevant conflicts of interest to declare.

CD138 Regulates Myeloma Cell Survival, Proliferation, BM Engraftment and Tumor Organization

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2974-2974
Author(s):  
David R Fooksman ◽  
Amitabha Mazumder ◽  
Mark McCarron
Keyword(s):  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Early Stage ◽  
Serum Starvation ◽  
High Dose ◽  
Rapid Reduction ◽  
Myeloma Cells

Abstract Multiple myeloma is the 2nd most common blood cancer in adults with a median survival time of 5 years despite high-dose chemotherapy and bone marrow transplantation interventions. Syndecan-1 or CD138, is a heparan-sulfate coated glycoprotein, which is highly expressed on the surface of plasma cells and myeloma cells, important for adhesion and accumulating survival signals. Expression of CD138 is heterogeneous in myeloma tumors, in vivo and in vitro leading some to speculate it may distinguish stem-like subpopulations. While this role is highly disputed, we investigated the effect of CD138 expression on tumor pathology in vivo. To characterize CD138neg and CD138high subpopulations, we used GFP+ Vk*myc myeloma model from Leif Bergsagel, which develops myeloma tumors in BM and spleen of C57Bl/6 mice. We found CD138high populations were more proliferative in vivo based on EdU incorporation experiments. We transferred equal numbers of sorted subpopulations into hosts and found that CD138high cells generated larger tumors in the BM than CD138neg cells after 12 weeks. Analysis of these tumor-bearing mice revealed that all tumors contained both subpopulations, indicating that these two subsets are hierarchically equivalent. We find that in mice with small tumors, the majority of cells (80% or more) are CD138high cells, while in large tumors, the level drops (to 30-50% of tumor) with higher composition of CD138neg cells. We also find lower CD138 levels on myeloma cells found in the blood compared to BM. Using intravital two-photon time-lapse imaging in the tibial BM, we find that tumor cells from smaller, early stage tumors are physically arrested within the BM parenchyma, while in larger, more advanced tumors, myeloma cells are more motile and active. CD138neg cells were more apoptotic based on ex vivo Annexin V staining following serum starvation. Interestingly, serum starvation led to rapid reduction in CD138 surface expression. Taken together, we propose a model where CD138 expression regulates localization and survival in the BM niche, but is downregulated from the plasma membrane when tumor size outgrows the necessary resources, allowing myeloma cells to migrate and metastasize to distant new locations. Disclosures No relevant conflicts of interest to declare.

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