Effects of Bypassing Hemostatic Agents in the Co-Presence of NXT007, Emicizumab-Based Engineered Bispecific Antibody to Fixa/Fx, Using in Vitro Global Assays: Rough Indicators for Their Concomitant Use

Abstract Background: NXT007, emicizumab (Emi)-based engineered therapeutic bispecific antibody which increases tissue factor (TF)-triggered thrombin generation (TG) potential of factor(F) VIII-deficient plasma to non-hemophiliac ranges at around 5-30 μg/mL (Yamaguchi, ASH 2020), is currently in a phase 1/2 clinical study. In the clinical settings under NXT007-prophylaxis, bypassing hemostatic agents (BPAs), such as activated prothrombin complex concentrates (aPCC) and recombinant(r) FVIIa, may be concomitantly administered. Under Emi-prophylaxis, repeated doses of aPCC impose a thrombotic risk. Against the risk, NHF's Medical and Scientific Advisory Council (MASAC) recommends ≤50 U/kg and ≤100 U/kg of aPCC as initial dose and one day dosage, respectively. In case of NXT007-prophylaxis, concomitant-use with BPAs should be also carefully managed and thus basic non-clinical combination data are needed. Objectives: To examine in vitro effects of BPAs in the co-presence of NXT007 for providing rough indicators for determining their safe doses. Methods: First, TF-triggered TG assay was performed using commercial FVIII-deficient plasma. rFVIII, rFVIIa or aPCC was spiked in the co-presence of NXT007 or Emi (0.1-50 μg/mL). Second, the TG assay and whole blood clot viscosity test (ROTEM) using Ca 2+-trigger was performed using healthy volunteer's blood incubated with anti-FVIII antibodies (HA model), where aPCC or rFVIIa was spiked in the co-presence of NXT007. Third, ROTEM was performed as above using whole blood from persons with hemophilia A (PwHA) under Emi- or FVIII-prophylaxis. Results: Peak height of TF-triggered TG assay using FVIII-deficient plasma was increased by spiking each of the agents (rFVIII, rFVIIa or aPCC) in the co-presence of NXT007 (0.1-50 μg/mL). Peak height increase by rFVIII under NXT007 was roughly additive. Peak height increase by each BPA under NXT007 was synergistic. Synergistic effect by aPCC was more intensive than that by rFVIIa. The combination effect of 0.1 U/mL aPCC and 0.1-50 μg/mL NXT007 on peak height did not exceed that of 0.5 U/mL aPCC and 50 μg/mL Emi. Peak height at 2 μg/mL NXT007 alone was comparable to that at 50 μg/mL Emi alone. When adding BPAs to these two settings, similar synergistic effects were observed. It suggested that NXT007's combination actions with BPAs were qualitatively similar to Emi's. In the TG assay using the HA model plasma (n=2), the combined effect of aPCC and NXT007 was similarly confirmed. In ROTEM using the same HA model whole blood (n=2), clotting time (ROTEM-CT, 7380±605 sec) was shortened by spiking 5 μg/mL NXT007 (791±148 sec) to yield normal levels (1521±110 sec). Spiked aPCC (0.13, 0.65 U/mL in whole blood, equivalent to ~10, ~50 U/kg infusion) shortened ROTEM-CT (1228±549, 435±54 sec) and further shortened it in the co-presence of 5 or 15 μg/mL NXT007 (171±11, 113±1* or 143±4, 90±5* sec [* significantly ( p<0.05) shorter than that of the Emi-treated PwHA blood spiking 0.65 U/mL aPCC, 162±21 sec)]. In PwHA without Emi-prophylaxis (n=2), ROTEM-CT (5148±1290 sec) was shortened by spiking 5 or 15 μg/mL NXT007 (1598±482 or 1116±14 sec) to non-hemophiliac levels (20 IU/dL rFVIII, 1569±222 sec). Spiked aPCC (0.13, 0.65 U/mL) shortened ROTEM-CT (1383±322, 680±84 sec) and further shortened it in the co-presence of 5 or 15 μg/mL NXT007 (230±35, 144±4 or 193±11, 121±0* sec). Spiked rFVIIa also shortened ROTEM-CT in the co-presence of NXT007, but the intensity was less than spiked aPCC. ROTEM using blood from PwHA under Emi-prophylaxis (n=3) were also performed and demonstrated that the effects by co-spiking BPAs and NXT007 were roughly consistent with those using PwHA blood without Emi-prophylaxis. These ROTEM data indicated that the combined effect of 0.13 U/mL aPCC and 5-15 μg/mL NXT007 was less intensive than that of 0.65 U/mL aPCC spiked to the Emi-treated PwHA blood, while that of 0.65 U/mL aPCC and 5-15 μg/mL NXT007 had more intensive effect (Table). Conclusion: In considering concomitant use of BPA under NXT007-prophylaxis, dose of aPCC should be more carefully determined than that of rFVIIa. In this non-clinical study, the combined effect of ~0.13 U/mL aPCC (equivalent to ~10 U/kg infusion) and ~15 μg/mL NXT007 did not exceed that of 0.65 U/mL aPCC (50 U/kg infusion) under Emi-prophylaxis situation corresponding to the upper limit of initial concomitant dose recommended by MASAC, which might be rough indicators in future clinical settings. Figure 1 Figure 1. Disclosures Ogiwara: Chugai Pharmaceutical Co., Ltd.: Consultancy, Research Funding. Furukawa: Chugai Pharmaceutical Co., Ltd.: Research Funding. Sasai: Chugai Pharmaceutical Co., Ltd.: Research Funding. Inaba: Chugai Pharmaceutical Co., Ltd.: Current Employment. Kitazawa: Chugai Pharmaceutical Co., Ltd.: Current Employment, Current equity holder in publicly-traded company. Nogami: Chugai Pharmaceutical Co., Ltd.: Consultancy, Research Funding.

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Crystal Structure Of Tissue Factor Pathway Inhibitor (TFPI) Kunitz Domain 1 and 2 In Complex With An Inhibitory Cyclic Peptide,

Blood ◽

10.1182/blood.v122.21.453.453 ◽

2013 ◽

Vol 122 (21) ◽

pp. 453-453

Author(s):

Michael Dockal ◽

Rudolf Hartmann ◽

Markus Fries ◽

Katerina Prohaska ◽

Robert Pachlinger ◽

...

Keyword(s):

Whole Blood ◽

Research Funding ◽

Cyclic Peptide ◽

Intramolecular Interactions ◽

Chip Surface ◽

Activation Assay ◽

Kunitz Type ◽

Β Sheet ◽

Interaction Surface

TFPI is a Kunitz-type protease inhibitor which efficiently regulates the extrinsic coagulation pathway. It is composed of three flexible linked Kunitz-type domains (KD) where KD1 and KD2 are involved in efficient inhibition of TF/FVIIa and FXa. TFPI inhibition has been shown to improve coagulation and hemostasis in hemophilia models in vitro and in vivo. Recombinant KD1-KD2 (residues 22-150) produced by E. coli and complexed to JBT-B5, a cyclic peptide composed of 23 amino acids, was co-crystallized in 20% w/v PEG6000 and 50mM imidazole, pH8.0. JBT-B5 binding to TFPI was verified by BiaCore experiments with TFPI immobilized on a chip surface. Functional inhibition of TFPI by JBT-B5 was tested in model assays including TFPI inhibition of FXa, FX activation by TF/FVIIa, inhibition of TFPI released upon platelet activation, and by global hemostatic assays including calibrated automated thrombography in FVIII-inhibited plasma and rotational thomboelastometry (ROTEM) using FVIII-inhibited whole blood. Inhibition of cell surface TFPI was analyzed in an FX activation assay performed on HUVECs. TFPI KD1-KD2 bound to JBT-B5 formed a crystal containing two independent complexes in the asymmetric unit. The complexes belong to the orthorhombic spacegroup P212121 and diffracted to a maximum resolution of 1.95 Å. To our knowledge, this is the first TFPI structure consisting of KD1, KD2 and their linker. The KD1-KD2 structure is fully defined in the electron density. Both domains show a Kunitz-type structure, where only ∼1/3 of the structure is engaged in secondary structure elements. These are two short α-helical elements at Ser24-Ala27(KD1)/Asp95-Phe98(KD2) (α1/α3) and Leu69-Met75/Leu140-Glu148 (α2/α4) and a two-stranded β sheet comprising Met39-Asn45/Ile110-Asn116 (β1/β3) and Arg49-Ile55/Lys120-Lys126 (β2/β4). These elements form the topological framework that is stabilized by the three canonical disulfide bonds involving Cys26-Cys76, Cys35-Cys59, and Cys51-Cys72 in KD1 and Cys95-Cys147, Cys106-Cys130, and Cys122-Cys143 in KD2. The 23mer TFPI inhibitor, JBT-B5, is sandwiched between the two Kunitz domains of TFPI and assumes a β-hairpin-like structure. It can be segmented into (i) a two-stranded β sheet comprising Tyr2-Ala8 and Thr17-Phe23; (ii) and a long β-turn loop comprising Met9-Met16. The β-sheet is stabilized by a disulfide bridge (Cys7 and Cys18) and a hydrophobic zipper comprising the side chains of Tyr3, Trp5 and Trp20. Strikingly, JBT-B5 locks KD1-KD2 in a distinct conformational state in which both Kunitz-domains are related via a two-fold symmetry and reactive center loops (RCL) from each KD are forced to opposite sides. The interactions between KD1-KD2 and JBT-B5 are extensive, as are the intramolecular interactions within JBT-B5. Analyzing the interactions between KD1-KD2 and JBT-B5 with the PISA server resulted in a total interaction surface of 1340Å2. More than 2/3 of the interaction surface consists of a hydrophobic anchor in JBT-B5 which interact with residues spread all over TFPI including KD1, KD2 and their linker. In addition, several polar interactions stabilize the KD1-KD2/JBT-B5 complex, explaining JBT-B5´s exlusive binding to human TFPI. Interaction studies revealed high affinity binding to TFPI (KD: 0.5nM). The highly complex and extensive interaction of JBT-B5 with TFPI translates to a highly efficient inhibition of recombinant TFPI and;TFPI released from activated platelets which is demonstrated in model assays on FXa (EC50: 1.3nM) and FXa generation by TF/FVIIa (EC50: 0.2nM). HUVE cell-based FX activation and global hemostasis assays such as thrombin generation in hemophilia plasma (EC50: 4nM) and ROTEM in FVIII-inhibited whole blood confirm the inhibition of all physiologic TFPI forms. For the first time, a TFPI structure comprising KD1, KD2 and their linker in complex with a TFPI- inhibitory cyclic peptide was solved. This structure provides atomic details explaining the inhibitory mode of action of this efficient TFPI antagonist, and will guide the design of efficient inhibitors for use in hemophilia treatment. Disclosures: Dockal: Baxter Innovations GmbH, Vienna, Austria: Employment. Hartmann:Baxter Innovations GmbH, Vienna, Austria: Employment. Fries:Baxter Innovations GmbH, Vienna, Austria: Employment. Prohaska: Baxter Innovations GmbH, Vienna, Austria: Employment. Pachlinger:Baxter Innovations GmbH, Vienna, Austria: Employment. Polakowski:3B Pharmaceuticals, Berlin, Germany: Employment. Brandstetter: Baxter Innovations GmbH, Vienna, Austria: Research Funding. Rosing:Baxter Innovations GmbH, Vienna, Austria: Consultancy, Research Funding. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.

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Cord Blood Derived Natural Killer Cells Loaded with a Tetravalent Bispecific Antibody Construct (AFM13) As Off-the-Shelf Cell Therapy for CD30+ Malignancies

Blood ◽

10.1182/blood-2018-99-114954 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 341-341

Author(s):

Lucila Kerbauy ◽

Mecit Kaplan ◽

Pinaki P Banerjee ◽

Francesca Lorraine Wei Inng Lim ◽

Ana Karen Nunes Cortes ◽

...

Keyword(s):

T Cell ◽

Cord Blood ◽

Nk Cells ◽

Natural Killer ◽

Research Funding ◽

Bispecific Antibody ◽

Nk Cell ◽

Ex Vivo

Abstract Chimeric antigen receptors to redirect T cell specificity against tumor antigens have shown remarkable clinical responses against CD19+ malignancies. However, the manufacture of an engineered autologous T cell product is expensive and cumbersome. Natural killer (NK) cells provide an alternative source of immune effectors for the treatment of cancer. NK cell cytolytic function can be directed towards specific targets by exploiting their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) through the NK cell Fc receptor, CD16 (FcγRIIIa). AFM13 is a tetravalent bispecific antibody construct based on Affimed's ROCK™ platform. AFM13 is bispecific for CD30 and CD16A, designed for the treatment of CD30 expressing malignancies. It binds CD16A on the surface of NK cells, thus activating and recruiting them to CD30 expressing tumor cells and mediating subsequent tumor cell killing. Since autologous NK effector function is impaired in many patients with malignancies, we propose to overcome this by the use of allogeneic NK cells in combination with AFM13. Cord blood (CB) is a readily available ("off-the-shelf") source of allogeneic NK cells that can be expanded to large, highly functional therapeutic doses. The feasibility and safety of therapy with allogeneic ex vivo expanded CB-derived NK cells have been shown by our group and others. In this study, we hypothesized that we can redirect the specificity of NK cells against CD30+ malignancies by preloading ex vivo activated and expanded CB-derived NK cells with AFM13 prior to adoptive infusion. Briefly, mononuclear cells were isolated from fresh or frozen CB units by ficoll density gradient centrifugation. CD56+ NK cells were cultured with rhIL-12, rhIL-18 and rhIL-15 for 16 hrs, followed by ex vivo expansion with rhIL-2 and irradiated (100 Gy) K562-based feeder cells expressing membrane-bound IL-21 and CD137-ligand (2:1 feeder cell:NK ratio). After 14 days, NK cells were loaded with serial dilutions of AFM13 (0.1, 1, 10 and 100 mg/ml). After washing twice with PBS, we tested the effector function of AFM13-loaded NK-cells (AFM13-NK) compared to expanded CB-NK cells without AFM13 against Karpas-299 (CD30 positive) and Daudi (CD30 negative) lymphoma cell lines by 51Cr release and intracellular cytokine production assays. AFM13-NK cells killed Karpas-299 cells more effectively at all effector:target ratios tested than unloaded NK cells (Figure 1) and produced statistically more INFγ and CD107a (P=0.0034; P=0.0031 respectively, n=4). In contrast, AFM13-NK cells and unloaded NK cells exerted similar cytotoxicity against Daudi cells. Next, we established the optimal concentration of AFM13 for loading (determined to be 100 μg/ml) and the optimal incubation time to obtain maximal activity (1 h) in a series of in vitro experiments. We also confirmed that the activity of AFM13-NK cells against Karpas-299 cells remains stable for at least 72h post-wash (Figure 2). Additionally, we characterized the phenotype of AFM13-NK vs. unloaded NK cells by flow cytometry using monoclonal antibodies against 22 markers, including markers of activation, inhibitory receptors, exhaustion markers and transcription factors. Compared to unloaded NK cells, AFM13-NK cells expressed higher levels of CD25, CD69, TRAIL, NKp44, granzyme B and CD57, consistent with an activated phenotype. We next tested the in vivo anti-tumor efficacy of AFM13-NK cells in an immunodeficient mouse model of FFluc-Karpas-299. Briefly, six groups of NOD/SCID/IL2Rγc null mice (n=5 per group) were transplanted by tail-vein injection with 1 x 10e5 FFluc-transduced Karpas cells. Group 1 and 6 received tumor alone or tumor + AFM13 and served as a control. Groups 2-4 receive Karpas FFLuc with either expanded NK cells or AFM13-NK cells (NK cells loaded with AFM13) or expanded NK cells and AFM13 injected separately. Group 5 received AFM13-NK cells without tumor. Initial studies confirm the antitumor activity of AFM13-NK cells. In summary, we have developed a novel premixed product, comprised of expanded CB-NK cells loaded with AFM13 to 'redirect' their specificity against CD30+ malignancies. The encouraging in vitro and in vivo data observed in this study, provide a strong rationale for a clinical trial to test the strategy of an off-the-shelf adoptive immunotherapy with AFM13-loaded CB-NK cells in patients with relapsed/refractory CD30+ malignancies. Disclosures Champlin: Sanofi: Research Funding; Otsuka: Research Funding. Koch:Affimed GmbH: Employment. Treder:Affimed GmbH: Employment. Shpall:Affirmed GmbH: Research Funding. Rezvani:Affirmed GmbH: Research Funding.

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Exploiting an Anti-CD3/CD33 Bispecific Antibody to Redirect Donor T Cells Against HLA Loss Leukemia Relapses

Blood ◽

10.1182/blood-2019-126641 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 513-513

Author(s):

Pier Edoardo Rovatti ◽

Laura Zito ◽

Eleonora Draghi ◽

Monika Herrmann ◽

Anetta Marcinek ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Research Funding ◽

Cell Activation ◽

Bispecific Antibody ◽

Leukemic Cells ◽

Donor T Cells ◽

Hla Loss

Background Genomic loss of mismatched HLAs ("HLA loss") represents a frequent modality by which acute myeloid leukemia (AML) evades immune recognition from donor T cells after partially HLA-incompatible allogeneic hematopoietic cell transplantation (allo-HCT). One important consequence of this post-transplantation relapse mechanism is that infusions of lymphocytes from the original donor become ineffectual, prompting the search for alternative therapeutic options. Here, to circumvent the loss of physiological T cell receptor-HLA interactions in these patients, we tested the ability of an anti-CD3/CD33 bispecific antibody (BsAb) to re-target donor T cells towards HLA loss relapses. Methods For short-term in vitro experiments, T cells were co-cultured with the MOLM-13 AML cell line or with primary patient blasts for 96 hours in presence or absence of an anti-CD3/CD33 BsAb. As readouts, we measured T cell activation (as surface expression of CD25 and CD69) and the absolute counts and relative proportion of effectors and targets. For long-term in vitro experiments, we established mixed lymphocyte cultures (MLCs) of T cells purified from two patients after haploidentical HCT and primary AML blasts obtained from the same patients at the time of diagnosis. After sequential stimulations, the co-cultures were tested against targets of interest, with or without addition of the BsAb. Functional readouts were T cell degranulation (measured as CD107a expression), antigen-specific activation (as CD137/41-BB expression) and target-specific cytotoxicity (measured by time-lapse live cell imaging over a 48 hour time span). For in vivo experiments, human leukemic cells were infused intravenously into non-irradiated NSG mice, followed by intraperitoneal infusion of T cells and daily administration of the BiTE compound. Results First, we retrospectively analyzed immunophenotypic data of 36 AML patients who experienced HLA loss relapses at our Institution, documenting robust expression of CD33 on the surface of the relapsed leukemia in 35 of them (97%; Figure 1A). By short-term co-culture experiments we titrated the BsAb concentration to be used for subsequent in vitro assays to 100 ng/ml, and the most informative effector:target ratio to 1:3. Then, we established MLCs by stimulating T cells collected from two patients after partially HLA-incompatible allo-HCT with AML blasts collected from the same patients at the time of diagnosis. In both cases, donor-derived T cells robustly responded against the patient blasts both in term of degranulation (Figure 1B) and of antigen-specific activation (Figure 1C). As expected, when we tested the same T cells against the patient leukemia at time of HLA loss relapse, we detected no T cell-mediated responses. Noticeably, when the BsAb was added, in both cases we detected a strong response not only against the diagnosis but also against the HLA loss variants, indicating that T cells were effectively re-targeted towards leukemic cells. Similar results were obtained also by live cell imaging, measuring target cell apoptosis over 48 hours of recording: also in this assay, in fact, donor T cells recognized and killed leukemia at diagnosis (45% of detection area positive for apoptosis dye) and failed to recognize its HLA loss relapse counterpart (32% of area positive for apoptosis dye). Addition of the BsAb to the co-cultures had a minor effect on recognition of the original disease (45% of area positive for apoptosis dye) but drove dramatic cell death of HLA loss blasts (80% of area positive for apoptosis dye), demonstrating that the BsAb induced not only T cell activation but also and most importantly target cell killing (Figure 1D). Finally, we modeled the BsAb activity in vivo, showing that, whereas the sole infusion of human T cells is not able to prevent the outgrowth of leukemia in the bone marrow of NSG mice, addition of the bispecific antibody leads to effective disease clearance (Figure 1E). Conclusions Our results demonstrate that anti-CD3/CD33 BsAbs can effectively redirect donor T cells against HLA loss leukemia variants, resulting in their rapid and effective killing. Taken together, these promising findings strongly support translation of this approach to ad hoc designed early-phase clinical trials, to provide a rational therapy for this increasingly recognized but still treatment-orphan modality of post-transplantation relapse. Figure 1 Disclosures Subklewe: Janssen: Consultancy; Miltenyi: Research Funding; Pfizer: Consultancy, Honoraria; Oxford Biotherapeutics: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Morphosys: Research Funding; Roche: Consultancy, Research Funding; AMGEN: Consultancy, Honoraria, Research Funding. Vago:Moderna Therapeutics: Research Funding; GenDx: Research Funding.

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Identification of Specific Hnrnps As Novel Therapeutic Targets and Responsive Indicators of KPT330 (selinexor) in Leukemia

Blood ◽

10.1182/blood.v128.22.1657.1657 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1657-1657 ◽

Cited By ~ 2

Author(s):

Adam Cloe ◽

Li Chen ◽

Yuan Li ◽

Hongtao Liu ◽

Jason X. Cheng

Keyword(s):

Bone Marrow ◽

Research Funding ◽

Nuclear Accumulation ◽

Protein Accumulation ◽

Clinical Settings ◽

Bone Marrow Blast ◽

Protein Levels ◽

Small Cohort ◽

Pre Treatment

Abstract Background: Activenuclear-cytoplasmic shuttling of proteins and RNAs, such as heterogeneous ribonucleoproteins (hnRNPs), is essential for the normal function and survival of eukaryotic cells and tumorigenesis (Dreyfuss et al. 1993 Annu Rev Biochem 62, 289; Gorlich and Mattaj 1996 Science 271, 1513). Up-regulation of exportin 1 (XPO1)/chromosomal maintenance 1 (CRM1), a member of the karyopherin-β family of nuclear export receptor proteins, has been implicated in solid and hematologic malignancies (Kau Kau et al. 2004).Selinexor (KPT-330) has been shown to be able block in vitro and in vivo XPO1/CRM1 functions and is currently in phase-II/IIb clinical trials for treatment of hematologic and solid tumors (Senapedis et al., 2014 Nat Rev Cancer 4, 106). However, the mechanisms underlying the selectivity and efficacy of selinexor are incompletely understood, and no biomarkers are currently available to predict clinical responses to selinexor in clinical settings. In this study, we focus on determining the effects of selinexor on the nuclear-cytoplasmic shuttling of hnRNPs, particularly hnRNPK and hnRNPA1, to elucidate the roles of the hnRNPs in the regulation of selectivity and efficacy of selinexor in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Method:We performed growth inhibition/killing assays, histopathologic evaluations, immunohistochemical studies, subcellular fraction western blotting, super-resolution stimulated emission depletion (STED) confocal microcopy and siRNA knockdown experiments. Results: Our in vitro experiments demonstrate a marked increase in XPO1/CRM1 protein and decrease in TP53 in our azacitidine-resistant MDS/AML cell lines compared to our azacitidine-sensitive MDS/AML cell lines. Selinexor treatment efficiently blocks export of hnRNP K from nuclei and increased nuclear accumulation of hnRNPK and inhibits MDS/AML cell growth, while the protein levels of XPO1/CRM1 and TP53 remain unchanged. Our experiments using clinical bone marrow specimens show no significant difference in the total protein level or nuclear accumulation of XPO1/CRM1 between the normal control and MDS or AML bone marrow specimens. In contrast, a strong positive correlation between MDS/AML disease progression and hnRNPK protein accumulation is observed in those clinical specimens. We have extended our experiments to clinical bone marrow specimens from a small cohort in a clinical trial for selinexor in AML at the University of Chicago (NCT02573363). In our small cohort, 5 patients responded to selinexor, 4 patients did not respond and 1 had a partial response. All 5 responders show a striking decrease in their bone marrow blast percentage from their pre-treatment marrows (average blast percentage 37.4%) to their post-treatment (average blast percentage 1.8%). Non-responders show no such difference in pre and post-treatment blast percentage (56.3 and 57.1%, respectively). Importantly, our experiments demonstrate a marked difference in the protein accumulation and subcellular localization of hnRNPK and hnRNPA1, another member of the hnRNP family, between selinexor-responder and selinexor-non-responder bone marrow specimens. Specifically, selinexor responders had much higher levels of hnRNPK and hnRNPA1 proteins in their pre-treatment bone marrows than non-responders, despite the fact that the latter had higher bone marrow blast percentages on average. There is markedly reduced accumulation of hnRNPK and hnRNPA1 in the post-selinexor treatment bone marrow specimens from the responders, but not the non-responders, suggesting these hnRNPs as key therapeutic targets for selinexor in MDS and AML. In contrast, no significant change in XPO1/CRM1 protein levels is observed in the selinexor-responder vs. selinexor-non-responder bone marrow specimens. Conclusion:Our data have revealed a novel drug-action mechanism by which selinexor impairs the nuclear-cytoplasmic shuttling of hnRNPK and hnRNPA1 in MDS and AML cells. Differential expression and localization of these hnRNPs in normal vs. MDS vs. AML cells may provide the rationale for the preferential killing of leukemia cells by selinexor. Our data also suggest the possibility to develop novel hnRNP-based biomarkers to predict the response to selinexor in clinical settings. Disclosures Liu: Karyopharm: Research Funding; BMS: Research Funding.

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Procoagulant Assessment of Topical Battlefield Hemostats Using a Novel Semi-Automated Whole Blood Clotting Assay System.

Blood ◽

10.1182/blood.v110.11.3161.3161 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3161-3161 ◽

Cited By ~ 1

Author(s):

Keith A. Moskowitz

Keyword(s):

High Pressure ◽

Whole Blood ◽

Blood Clotting ◽

Complete Blood Count ◽

Clotting Time ◽

Contact Phase ◽

Hemostatic Agents ◽

Platelet Counts ◽

Arterial Bleeding

A new granular smectite hemostatic agent (WoundStat™ [WS]) is capable of producing hemostasis during high pressure arterial bleeding (Ward et al., J Trauma, 63:276–284, 2007). The mechanism by which WS and other topical hemostatic agents help achieve clotting has not been investigated. In this study, the procoagulant and hemo-compatibility functions of WS were assessed using a novel whole blood clotting time assay, and its performance compared to current hemostatic agents QuikClot® zeolite granules (QC); CELOX™ marine biopolymer hemostatic granules (CX); sterile gauze bandage (SG); and to glass beads (GB). A modified whole blood recalcified clotting time assay was developed using the Actalyke® Mini II activated clotting time (ACT) analyzer and empty G-ACT tubes devoid of contact phase activating reagents. Whole citrated blood recalcified to 10 mM with CaCl2 yielded clot times (CT) of 217+/−6 sec, 212+/−11 sec, and 236+/−5 sec, respectively, for three normal donors. Addition of WS resulted in a time and dose-dependent biphasic decrease in CT. At 0.5 mg/ml, WS lowered CT by over 40% to 124+/−7 sec, 133+/− 9 sec, and 126+/−5 sec, p <0.001. Equal concentrations of QC also diminished CT ∼ 30% to 146+/−15 sec, 139+/−18 sec, and 184+/−22 sec, p< 0.02. Differences in CT between WS and QC were not significant. Surprisingly, 0.5 mg/ml CX did not exhibit procoagulant activity, as CT were on average 8% higher than controls (234+/−4 sec, 227+/−8 sec and 256+/−5 sec). SG and GB gave results within 5% of untreated samples. WS at ≥1.5 mg/ml clotted all samples in <90 sec, (the ACT instrument detection limit) and also significantly diminished CT of heparinized blood from 790 to 135 sec. All agents were hemocompatible as judged by the lack of change in hematocrit and hemoglobin complete blood count values after 45 min incubation. However, citrated blood incubated for <45 min with QC consistently demonstrated a drop in baseline platelet counts by >50,000 /μl while platelet counts in WS-treated specimens were similar to untreated controls (214,000/μl versus 219,000/μl) and clumped granules, presumably platelet-mediated, were visually apparent in the QC-treated bloods. Blood incubated with CX for up to 45 min also exhibited a drop in platelets from 204,000/μl to 150,000/μl. When QC, CX and WS were incubated in blood for 3 hours, corresponding to the maximal times recommended during field application prior to reapplication or surgical debridement, all hemostats resulted in an ∼ 50% reduction in platelet counts from 258,000/μl to 132,000/μl; 158,000/μl and 155,000 /μl, respectively. In summary, the data suggest that WS is hemocompatible, is moderately capable of inducing in vitro platelet aggregates, and may assist in the arrest of high-pressure arterial bleeding, at least in part by promoting the intrinsic contact phase of blood coagulation. Additional studies are required to further elucidate the potential platelet- and plasma-mediated mechanisms by which WS may help achieve hemostasis during severe arterial traumatic injury.

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In Vivo Platelet Activation and Its In Vitro Inhibition by Prasugrel's Active Metabolite In Adolescents with Sickle Cell Disease

Blood ◽

10.1182/blood.v118.21.2141.2141 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2141-2141

Author(s):

Andrew L. Frelinger ◽

Joseph A. Jakubowski ◽

Julie K. Brooks ◽

Anu Nigam ◽

Michelle A. Berny-Lang ◽

...

Keyword(s):

Platelet Activation ◽

Platelet Function ◽

Whole Blood ◽

Research Funding ◽

Normal Subjects ◽

Cell Disease ◽

Platelet Surface ◽

Dependent Inhibition

Abstract Abstract 2141 Introduction: In patients with sickle cell disease (SCD), erythrocytes contribute to microvessel occlusion resulting in tissue damage and platelet activation. Platelet activation, aggregation, local thrombus formation and platelet activation-dependent leukocyte recruitment potentially amplify tissue ischemia. Antiplatelet therapy may therefore be useful in SCD. Here we evaluate levels of platelet activation markers in adolescents with SCD vs. normal controls and the effect of in vitro blockade of the platelet ADP receptor P2Y12 by prasugrel's active metabolite, R-138727. Methods: Blood was obtained from adolescents (10 – 18 yr) with SCD and healthy adult subjects. Platelet function was evaluated by: light transmission aggregation (LTA) in platelet-rich plasma with 20 μM ADP and in whole blood by VerifyNow P2Y12; Multiple Electrode Aggregometry (MEA) with 6.5 μM ADP; vasodilator stimulated phosphoprotein (VASP) P2Y12 assay; and whole blood flow cytometric analysis of basal and in vitro ADP-stimulated levels of platelet surface activated GPIIb-IIIa (reported by monoclonal antibody PAC1) and P-selectin, platelet-monocyte aggregates (PMA) and platelet-neutrophil aggregates (PNA). These endpoints were also evaluated after in vitro incubation of whole blood with R-138727 (0.1 – 10 μM). Results: In SCD patients compared with normal subjects, circulating PMA and PNA levels were significantly higher (76.5 ± 20.3% and 55.1 ± 21.8% vs. 20.1 ± 7% and 13.9 ± 4.2% [mean ± SD], respectively, p<0.0001 for both), and in vitro ADP-stimulated platelet surface activated GPIIb-IIIa and P-selectin levels (mean fluorescence, MFI) were significantly lower (128.7 ± 66.2 and 78.1 ± 11.5 vs. 257.3 ± 50.8 and 91.6 ± 5.8, p<0.05 for both). ADP-stimulated platelet aggregation by LTA, VerifyNow and MEA, did not significantly differ between SCD and normal subjects, although whole blood platelet aggregation by MEA and VerifyNow tended to be greater in blood from SCD patients (92.5 vs. 70.4 AU, p=0.064 and 362.9 vs. 314.8 PRU, p=0.488, respectively). Treatment of whole blood in vitro with R-138727 resulted in a concentration-dependent inhibition of platelet function in both SCD patients and normal subjects. However, compared with normal subjects, the IC50 in SCD subjects was significantly lower for LTA but significantly higher for VerifyNow and VASP (Table). R-138727 inhibition of platelet function in SCD patients was similar to normal subjects as judged by MEA, whole blood flow cytometry for ADP-stimulated platelet surface P-selectin and activated GPIIb-IIIa expression, and % PMAs (Table). Sensitivity to R-138727 inhibition in SCD patient blood was greatest as measured by ADP-stimulated platelet surface P-selectin MFI, LTA, and MEA, less with ADP-stimulated platelet surface activated GPIIb-IIIa, and least with VASP, VerifyNow P2Y12 and % P-selectin-positive platelets (Table). Conclusions: 1) The markedly higher circulating PMA and PNA levels in SCD patients relative to normal donors demonstrates increased in vivo platelet activation in SCD patients and suggests that PMA and PNA may be useful markers of the in vivo pharmacodynamic effects of antiplatelet therapy in SCD patients. 2) Blockade of platelet P2Y12 with R-138727 produces dose-dependent inhibition of platelet function in SCD platelets. 3) Assay-dependent differences in IC50 values between SCD patients and normal donors suggest the presence of additional variables that affect these measures of platelet function. Further studies are needed to determine the relationship between platelet inhibition measured by these assays and clinical events in SCD patients. Disclosures: Frelinger: GLSynthesis: Research Funding; Lilly/Daiichi Sankyo: Consultancy, Research Funding; Takeda: Research Funding. Jakubowski:Eli Lilly and Company: Employment, Equity Ownership. Heeney:Lilly: Consultancy. Michelson:GLSynthesis: Research Funding; Lilly/Daiichi Sankyo: Data Monitoring Committee for clinical trial, Research Funding; Takeda: Research Funding.

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Enhanced Global Hemostatic Potentials with a Bispecific Antibody to Factors IXa and X (ACE910) in the Whole Blood By Rotation Thromboelastometry (ROTEM)

Blood ◽

10.1182/blood.v126.23.3503.3503 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3503-3503 ◽

Cited By ~ 4

Author(s):

Koji Yada ◽

Keiji Nogami ◽

Yasuaki Shida ◽

Masahiro Takeyama ◽

Ryu Kasai ◽

...

Keyword(s):

Clinical Study ◽

Board Of Directors ◽

Whole Blood ◽

Research Funding ◽

Bispecific Antibody ◽

Hemophilia A ◽

Advisory Committees ◽

Group A ◽

Group B ◽

Group D

Abstract Background: ACE910, a humanized bispecific antibody to factor (F) IXa and FX mimicking the functions of FVIII, exerts tenase activities without FVIII (a) (Kitazawa et al. Nature Medicine. 2012;18, 1570). In primate hemophilia A (HA) models, the hemostatic enhancing effect of ACE910 has been reported and the clinical study investigating the effect and safety of ACE910 for human HA patients is on-going. However, the hemostatic effect of ACE910 remains unquantified and difficult to be evaluated. Objectives: In this study, we evaluated the viscoelastmetric parameters in the whole blood obtained from HA patients under long-term treatment of ACE910 in phase 1 and its extension studies, utilizing rotation thromboelastometry (ROTEM), in order to investigate global hemostatic function of HA patients under treatment with ACE910. Methods: Ca2+-triggered hemostatic functions were assessed by ROTEM in the citrated whole blood samples obtained and stabilized at the indicated points from five severe HA cases with inhibitors (Case 1 : 39 BU/ml, 2 : 41 BU/ml, 3 : 17 BU/ml, 4 : 11 BU/ml and 5 : 111 BU/ml ) and two severe cases without inhibitors (Case 6 and 7) under subcutaneous administration of ACE910, with the different types of dosing regimen (group A with 0.3 mg/kg/week subcutaneous injection (loading dose of 1 mg/kg) for Case 1, 2, group B with 1 mg/kg/week (loading dose of 3 mg/kg) for Case 3, 4, group C with 3 mg/kg/week for Case 6 and group D changing from group A to group C for Case 5, 7) in a course of clinical study. The samples from the subjects prior to administration of ACE910 were spiked ex vivo with ACE910 and the hemostatic functions of them were also evaluated by ROTEM. The parameters of clot time (CT), clot formation time (CFT), maximum clot formation (MCF) and alpha angle were evaluated. The hemostatic potentials in the samples from twenty healthy volunteers and other ten HA patients with the various FVIII:C were evaluated by ROTEM as controls and the correlation between FVIII:C and CT was obtained. Annualized bleeding rates (ABR) of each case were calculated. The studies were approved by local ethics committee and the informed consent was obtained from each patient. Results: Addition of ACE910 (f.c. 10 or 30 μg/ml) into the sample from each case prior to administration of ACE910 shortened CT from 5,562 ± 374 sec (median 5,924 sec) to 1,475 ± 138 or 1,131 ± 82 sec, equivalent to FVIII:C 3.3 or 12.2 IU/dL, respectively, in an ACE910 concentration dependent manner. In group A, the plasma concentration of ACE910 got to 12 ± 5 μg/ml at 12 week. CT was shortened to 1,443 ± 24 sec, equivalent to FVIII:C 3.7 IU/dL maximally at 47 week consistent with the result of the spiked result. ABR decreased from 38.6 ± 25.8 to 1.1 ± 0.8, showing 97% decrease. As for Case 3 in group B (ACE910: 31 μg/ml at 12 week), CT was shortened to 1,164 sec, equivalent to FVIII:C 11.9 IU/dL maximally at 47 week consistent with the spiked data. ABR apparently decreased from 38.6 to 3.6. In Case 4 who dropped out the clinical study at 4 week, CT was shortened to 977 sec, equivalent to FVIII:C 22 IU/dL, maximally at 3 week, and continued to be shortened till 29 week (1,758 sec, equivalent to FVIII:C 1.1 IU/dL). In group C, the shortening of CT from 1,594 ± 103 sec, equivalent to FVIII:C 2.0 ± 1.0 (median 3.6) IU/dL (before administration of ACE910), to 1,226 ± 71 sec (8.5 ± 2.5 (median 10.4) IU/dL) (at steady state) was observed in consistent with the decrease of ABR from 8.1 to 1.1. In group D, CT was shortened from 3,405 ± 386 sec to 1,409 ± 85 sec, equivalent to FVIII:C 4.2 IU/dL after middle to high dose escalation in consistent with decrease of ABR from 22.3 ± 9.6 to 3.8 ± 2.6. Among all cases except Case 4, CT and ABR were of clinically relevant difference between before and after administration of ACE910. Conclusions: The comprehensive hemostatic function evaluated by ROTEM in hemophilia A patients irrespective of presence of inhibitors was improved after administration of ACE910 in a dose dependent fashion, resulting in the reduction of ABR, which suggested the hemostatic effectiveness of ACE910 for HA patients. Disclosures Yada: Chugai Pharmaceutical Co., Ltd: Research Funding. Nogami:Bayer, Novo Nordisk, Baxalta. Biogen: Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees; Bayer, NovoNordisk, Baxalta, Chugai, Kaketsuken, Pfizer, Biogen: Honoraria. Shida:Chugai Pharmacoceutical Co. Ltd.: Research Funding. Takeyama:Chugai Pharmaceutical Co., Ltd.: Research Funding. Kasai:Chugai Pharmaceutical Co., Ltd.: Employment. Shima:Chugai Pharmaceutical Co., Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Baxalta: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Kaketsuken: Honoraria; Bayer: Honoraria, Research Funding.

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A Novel Clot Waveform Analysis to Measure the Plasma Coagulation Potency in the Presence of Anti-Fixa/Fx Bispecific Antibody, Emicizumab

Blood ◽

10.1182/blood.v128.22.1388.1388 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1388-1388 ◽

Cited By ~ 1

Author(s):

Keiji Nogami ◽

Tomoko Matsumoto ◽

Yuka Tabuchi ◽

Tetsuhiro Soeda ◽

Nobuo Arai ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Bispecific Antibody ◽

Waveform Analysis ◽

Fibrinogen Concentration ◽

Assay Condition ◽

Employment Equity ◽

Advisory Committees ◽

Chugai Pharmaceutical ◽

Equity Ownership

Abstract Emicizumab (also termed ACE910) is a humanized anti-factor (F)IXa/FX bispecific antibody with FVIIIa cofactor function. A clinical phase 3 study was initiated in 2015 for hemophilia A patients (HA-pts) with FVIII inhibitors. Since emicizumab, unlike FVIII, does not require activation by thrombin, its APTT-shortening effect is much greater than that of FVIII. Thus, APTT, a conventional assay to assess whole coagulation potency, would have limited utility in emicizumab-administered HA-pts, because emicizumab would mask the effect of residual FVIII or a FVIII agent on APTT. Clot waveform analysis (CWA) can provide multidimensional coagulation potencies by monitoring the process of plasma clot formation with an automated coagulation analyzer. We considered the possibility, therefore, that this assay system would overcome the above issue on APTT. In this study, we aimed to optimize concentrations of tissue factor (TF) and ellagic acid (Elg) in a trigger reagent for CWA as well as CWA parameters to provide precise evaluation of coagulation potency even in the presence of emicizumab with neither masking nor being masked by FVIII or bypassing agents. First, we determined an optimal concentration of TF/Elg trigger reagent. Various concentrations (10, 30, 100, and 300 μg/mL) of emicizumab were spiked into commercially available FVIII-deficient plasmas (George King) for testing. Recombinant (r)FVIII (Kogenate FS; Bayer)-spiked samples were also tested as a reference. PT reagent (under development; Sysmex) and APTT reagent (Thrombocheck APTT-SLA; Sysmex), used as a source of TF and Elg, respectively, were mixed in various ratios. The optimized mixture ratio (PT:APTT:buffer=1:15:135) was chosen to ensure that the maximum coagulation velocity (|min1|) in the presence of emicizumab would be in agreement with the animal study-based estimated conversion rate "0.2-0.4 IU/dL of equivalent FVIII per 1 μg/mL of emicizumab" (Muto. J Thromb Haemost. 2014). When evaluating several lots of FVIII-deficient plasmas, however, we observed large variations in transmittance depending on fibrinogen concentration of each plasma, which resulted in large variations of |min1| between donor plasmas. To decrease the bias due to fibrinogen concentration, % transmittance of clot waveform (CW) was adjusted to 100% and 0% at the pre- and post-coagulation phase, respectively. By using |min1| from the adjusted CW (adjusted-|min1|), we successfully reduced the inter-donor variations and chose it as a main parameter. Next, we evaluated adjusted-|min1| using plasmas from HA-pts without inhibitors (severe; n=2, moderate; n=2) and HA-pts with inhibitors (<10 BU/mL; n=2, >10 BU/mL; n=2) by adding emicizumab (30, 100, and 300 μg/mL) in vitro. After the addition of emicizumab, concentration-dependent increases in adjusted-|min1| were observed in all plasmas with rather small individual variations. Finally, we examined whether adjusted-|min1| reflected the effects of FVIII or bypassing agent that was added to plasmas containing emicizumab. Additive effects of a plasma-derived FVIII agent (CROSS EIGHT M; Japan Blood Products Organization), a rFVIII agent (ADVATE; Baxalta) and activated prothrombin complex concentrate (FEIBA; Baxalta) were confirmed by the increase of adjusted-|min1|. As for rFVIIa agent (NovoSeven; Novo Nordisk), its additive effect on adjusted-|min1| was not clear enough in this assay condition, but its additive effects were confirmed by the clot time. In conclusion, we established the Elg/TF-triggered CWA assay condition and parameters for measuring coagulation potency in plasmas from HA-pts even in the presence of emicizumab and a FVIII/bypassing agent without masking each other. Disclosures Nogami: Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees. Matsumoto:Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties, Research Funding. Tabuchi:Sysmex Corporation: Employment, Patents & Royalties; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties. Soeda:Sysmex Corporation: Patents & Royalties; Chugai Pharmaceutical Co., Ltd.: Employment, Patents & Royalties. Arai:Sysmex Corporation: Employment. Kitazawa:Chugai Pharmaceutical Co.: Employment, Equity Ownership, Patents & Royalties; Sysmex Corporation: Patents & Royalties. Takaoka:Sysmex Corporation: Employment. Hattori:Chugai Pharmaceutical Co.: Employment, Equity Ownership, Patents & Royalties. Shima:F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Sysmex Corporation: Patents & Royalties, Research Funding.

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A Modified Thrombin Generation Analysis to Measure the Plasma Coagulation Potency in the Presence of Anti-FIXa/FX Bispecific Antibody, Emicizumab

Blood ◽

10.1182/blood-2018-99-118504 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1200-1200

Author(s):

Kenichi Ogiwara ◽

Keiji Nogami ◽

Tomoko Matsumoto ◽

Mariko Noguchi-Sasaki ◽

Tetsuhiro Soeda ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Bispecific Antibody ◽

Peak Height ◽

Waveform Analysis ◽

High Dose ◽

Advisory Committees ◽

Plasma Coagulation ◽

Dose Dependent Increase ◽

Dose Dependent

Abstract 【Introduction】 Emicizumab is a humanized anti-factor (F)IXa/FX bispecific antibody with FVIIIa cofactor function. Since emicizumab, unlike FVIII, does not require activation by thrombin, its aPTT-shortening effect is much greater than that of FVIII. Thus, aPTT, a conventional assay to assess intrinsic coagulation potency, would have limited utility in emicizumab-administered hemophilia A-patients (HA-pts), because emicizumab would mask the effect of residual FVIII or a FVIII agent on aPTT. Also, aPTT cannot differentiate between different levels of emicizumab. Recently we reported that clot waveform analysis (CWA) using a trigger reagent comprising a balanced mixture of ellagic acid (Elg) and tissue factor (TF) to reflect both intrinsic and extrinsic coagulation activities could provide a useful means of assessing plasma coagulation potential in HA-pts treated with emicizumab with enhanced activity neither masking nor being masked by FVIII or bypassing agents (BPAs)1). Thrombin generation assay (TGA) can provide multidimensional plasma coagulation potencies as well as CWA. We considered the possibility, therefore, that the TGA would also overcome the above issue on aPTT. In this study, we aimed to assess TGA using Elg/TF trigger and two reference triggers (FXIa, TF) to evaluate the coagulation potency of patients with HA receiving emicizumab. 【Method】 TGA was assayed using Elg/TF trigger consisted of TF (0.5 pM) and Elg (0.3 μM) with phospholipids vesicles (PL, 4 μM), FXIa trigger consisted of FXIa (0.47 nM) with PL (20 μM), and TF trigger (PPP-Reagent LOW®). Various concentrations of emicizumab, FVIII agent, or BPAs (rFVIIa, aPCC) in clinical dosages were spiked into commercially available FVIII-deficient plasmas (George King) for testing. Then, FVIII or BPA was added to FVIII-deficient plasmas containing emicizumab. 【Result】 Emicizumab or FVIII showed dose-dependent increase in thrombin peak height in Elg/TF trigger as well as FXIa and TF triggers, but TF trigger had weak sensitivity to emicizumab and low range FVIII (1-10 IU/dl). Spiking both rFVIIa or aPCC showed dose dependent increase in thrombin peak height under Elg/TF trigger and TF trigger conditions, but the values were lower than the level of normal peak height. On the other hand, rFVIIa and aPCC resulted in little increase in peak height under FXIa trigger condition, suggesting that the FXIa trigger is not suitable for assessment of BPAs. These results indicated that Elg/TF trigger system was the most useful to evaluate the single-spiked effect of these agents. Then, we examined the additional effects of FVIII or BPAs in the presence of emicizumab by Elg/TF trigger system. The combination of FVIII and emicizumab showed additive increase in peak thrombin height, but this effect was saturated at high dose of FVIII more than 100 IU/dl. rFVIIa and emicizumab showed additive effect on increased peak thrombin height, achieving normal level even at very low dose rFVIIa (0.67 μg/ml), but remains within normal ranges at high dose (6.0 μg/ml). aPCC and emicizumab showed increased peak thrombin height, but in this case, exceeding the normal level even at clinical dosage of aPCC (0.5-1.0 U/ml). 【Conclusion】 TGA with Elg/TF trigger could provide a useful monitoring tool of assessing global coagulation potential during emicizumab prophyaxis including concomitant therapy with FVIII or BPAs. 1) Nogami K, et al. Modified clot waveform analysis to measure plasma coagulation potential in the presence of the anti-factor IXa/factor X bispecific antibody emicizumab. J Thromb Haemost. 2018 Jun;16(6):1078-1088. Disclosures Ogiwara: CSL Behring: Research Funding. Nogami:Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau. Matsumoto:Shire Japan Co. Ltd: Research Funding. Noguchi-Sasaki:Chugai: Employment. Soeda:Chugai: Employment, Patents & Royalties: Patents related to emicizumab. Matsumoto:Chugai Pharmaceutical Co., Ltd: Employment, Equity Ownership. Hirata:Chugai Pharmaceutical Co., Ltd: Employment, Equity Ownership. Shima:Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees.

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Chronic Lymphocytic Leukaemia Is Associated with a Broad Platelet-Function Defect Which Is Exacerbated By Ibrutinib and Acalabrutinib

Blood ◽

10.1182/blood-2018-99-120234 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3756-3756

Author(s):

Scott R McGregor ◽

Grace Gilmore ◽

Elizabeth E. Gardiner ◽

Sarah Hicks ◽

Shilpa Rakesh ◽

...

Keyword(s):

Board Of Directors ◽

Platelet Function ◽

Whole Blood ◽

Research Funding ◽

Blood Platelet ◽

Advisory Committees ◽

Function Defect ◽

Btk Inhibitors ◽

Platelet Function Assays

Abstract INTRODUCTION Bleeding from thrombocytopathy is a common complication of advanced chronic lymphocytic leukaemia (CLL). In addition to disease-related thrombocytopenia, the presence of the CLL clone and/or therapeutic interventions may further impair platelet function. In particular, the BTK inhibitors ibrutinib and acalabrutinib are known to inhibit platelet glycoprotein VI (GPVI)-mediated platelet aggregation. We compared platelet function and markers of GPVI activation between untreated CLL patients, ibrutinib-treated CLL patients and healthy controls, and studied the in vitro effects of ibrutinib and acalabrutinib on clinically utilised platelet function assays to assess their impact on GPVI-mediated as well as non-GPVI-mediated platelet activation pathways. METHODS Blood samples from 17 healthy volunteers and 8 untreated CLL patients were spiked with vehicle or comparable plasma concentrations of ibrutinib (0.3µM, 1.0µM) and acalabrutinib (1.8µM, 6.0µM) attainable during the treatment of CLL. Additional samples were obtained from 5 CLL patients undergoing ibrutinib treatment. Platelet function was evaluated using whole blood multiple electrode aggregometry (MEA - Multiplate®) and light transmission aggregometry (LTA - AggRAM®) in response to varying concentrations of aggregation-inducing reagents (collagen, CRP-XL, ADP, TRAP, ristocetin, arachidonic acid, and adrenaline). Shear-induced platelet adhesion was assessed using PFA-100®. Soluble GPVI plasma levels were assessed by ELISA. RESULTS In the absence of treatment, CLL patients exhibited significant platelet defects on whole-blood platelet function analyses in response to various agonists including ADP, ristocetin, TRAP and collagen (MEA) and prolongation of PFA-100® collagen/epinephrine closure time. This impairment was not replicated in assays using platelet-rich plasma (LTA). Ibrutinib-treated CLL patients demonstrated an additive impairment of platelet function, especially in regards to collagen-mediated activation by MEA or PFA-100®. There was no significant difference in soluble GPVI levels between normal, untreated or ibrutinib treated CLL patients. Addition of clinically-attainable concentrations of ibrutinib and acalabrutinib in vitro produced similar concentration-dependent inhibition of platelet function in healthy controls, with inhibition of aggregation evident in response to various agonists including collagen, CRP-XL, ristocetin and ADP but not arachidonic acid or TRAP. Ibrutinib also impaired aggregation in response to epinephrine, and caused selective prolongation of the PFA-100® collagen/epinephrine closure time, an effect not observed with acalabrutinib. MEA appears more sensitive and reproducible than LTA to describe the various inhibitory effects on platelet aggregation. Similar concentration-dependent inhibition of platelet function was observed by adding ibrutinib and acalabrutinib in vitro to blood samples from untreated CLL patients. CONCLUSIONS CLL is associated with a broad platelet function defect, which can be exacerbated by BTK inhibitors. Acalabrutinib induces a platelet function defect similar but less potent to that observed with ibrutinib, with the exception of shear-induced platelet adhesion (PFA-100®) which was only abnormal with ibrutinib. Routine platelet function assays are capable of quantifying BTK inhibitor-induced platelet dysfunction in CLL patients, with the most sensitive and reproducible measure being collagen-induced aggregation by MEA. There was no evidence for BTK-dependent platelet GPVI cleavage. Whole-blood platelet function assays may have utility in managing CLL patients presenting with bleeding or requiring urgent surgery during therapy with BTK inhibitors. Disclosures McGregor: Pfizer: Other: Conference travel support; Bristol-Myers Squibb: Other: Conference travel support; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria. Baker:CSL Behring: Research Funding; Biogen Idec: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Research Funding; Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support, Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support; Roche: Other: Conference travel support; Novo Nordisk: Other: Conference travel support.

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