ALBERT

Description: Introduction Concizumab is an anti-tissue factor pathway inhibitor (TFPI) monoclonal antibody in clinical development for the subcutaneous prophylactic treatment of hemophilia patients. We present results from the main part (at least 24 weeks) of the concizumab explorer4 phase 2 trial (NCT03196284) in hemophilia A/B with inhibitor (HAwI/HBwI) patients. Methods The primary objective was to assess the efficacy of once-daily subcutaneous concizumab in preventing bleeds in HAwI/HBwI patients. Secondary objectives were the assessment of safety, including concomitant use of recombinant activated factor VII (rFVIIa), and immunogenicity. Patients were randomized 2:1 to concizumab prophylaxis or rFVIIa on-demand treatment via an interactive web-response system. A concizumab loading dose (0.5 mg/kg) was administered, followed by 0.15 mg/kg daily with potential dose escalation to 0.20 and 0.25 mg/kg. Efficacy was evaluated as the number of bleeding episodes (annualized bleeding rate [ABR]) at last dose level. The number of adverse events (AEs) and the occurrence of anti-drug antibodies (ADAs), as well as coagulation-related parameters were evaluated. Concizumab and free TFPI plasma levels were measured by ELISA, and peak thrombin generation (TG) potential using a standardized assay. Results 26 patients were randomized; 9 HAwI and 8 HBwI patients were exposed to concizumab, and 9 patients to rFVIIa (7 with HAwI and 2 with HBwI). All 25 patients who completed the main 24-week part of the trial chose to continue to the extension part. The estimated ABR at the last dose level for concizumab prophylaxis was 4.5 (95% CI: 3.2−6.4) and for rFVIIa on demand, 20.4 [95% CI: 14.4−29.1] (Figure 1). There was a 78, 88 and 79% reduction in all treated bleeds and in spontaneous and joint bleeds, respectively, with concizumab prophylaxis compared with on-demand treatment (Figure 1). Concizumab concentration varied considerably between patients on the same dose level. Increasing concizumab dose was associated with lower free TFPI and normalized TG potential (Figure 2). No deaths, thromboembolic events or AE-related withdrawals occurred. No safety concerns with concomitant use of concizumab and rFVIIa were identified. Three patients had positive (very-low to medium-titer) ADA tests (titer range: 1 to 128), but with no apparent clinical effect. As expected, elevated prothrombin fragment 1+2 and D-dimers were observed across all concizumab dose levels, reflecting the hemostatic effect of concizumab. Conclusions In the phase 2 explorer4 trial, concizumab was efficacious and safe as a subcutaneous prophylactic treatment in HAwI patients, as well as in HBwI patients for whom there is currently no prophylactic regimen available. There was no difference in safety and efficacy across hemophilia subtypes, including with the concomitant use of concizumab and the bypassing agent rFVIIa. The phase 2 trial results, which include the explorer5 trial in HA without inhibitors, support further development of concizumab as a prophylactic treatment for all hemophilia patients and have guided selection of the phase 3 dosing regimen. Disclosures Shapiro: Sangamo Biosciences Inc: Consultancy, Other: Clinical Research Protocol with the company; Genentech: Membership on an entity's Board of Directors or advisory committees, Other: Clinical Research Protocol with the company; Novo Nordisk Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Clinical Research Protocol with the company; Bioverativ: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Clinical Research Protocol with the company; OPKO: Other: Clinical Research Protocol with the company; Octapharma: Other: Clinical Research Protocol with the company; Prometic Life Sciences: Consultancy; Shire/Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Clinical Research Protocol with the company, Research Funding; Bayer: Other: Clinical Research Protocol with the company; Kedrion Biopharma: Other: Clinical Research Protocol with the company; Agios: Other: Clinical Research Protocol with the company; Prometic Bio Therapeutics: Other: Clinical Research Protocol with the company; BioMarin: Other: Clinical Research Protocol with the company; Daiichi Sankyo: Other: Clinical Research Protocol with the company; Glover Blood Therapeutics: Other: Clinical Research Protocol with the company; Novartis: Other: Clinical Research Protocol with the company; Pfizer: Other: Clinical Research Protocol with the company; American Thrombosis and Hemostasis Network: Membership on an entity's Board of Directors or advisory committees. Castaman:Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kedrion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Research Funding; Sobi: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Uniqure: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Werfen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda (SHIRE): Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Cepo:Novo Nordisk A/S: Employment. Hvitfeldt Poulsen:Novo Nordisk: Other: Clinical trials - investigator, Funding meetings and congresses; Bayer Health Care: Other: Clinical trials - investigator, Funding meetings and congresses; Pfizer: Other: Funding meetings and congresses; Sobi: Other: Funding meetings and congresses. Hollensen:Novo Nordisk: Employment. Matsushita:Bioverative: Research Funding; Pfizer: Consultancy, Honoraria; KM biologists: Consultancy, Honoraria, Research Funding; Novo Nordisk: Consultancy, Honoraria; CSL: Consultancy, Honoraria; uniQure: Consultancy, Honoraria. Young:Bioverativ/Sanofi: Consultancy, Honoraria; CSL Behring: Consultancy, Honoraria; Freeline: Consultancy, Honoraria; Genentech/Roche: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria; Kedrion: Consultancy, Honoraria; Novo Nordisk: Consultancy, Honoraria; Spark: Consultancy, Honoraria; Shire/Takeda: Consultancy, Honoraria; Uniqure: Consultancy, Honoraria. Zupancic-Salek:Novo Nordisk: Consultancy, Honoraria, Speakers Bureau; Biogen: Consultancy, Honoraria, Speakers Bureau; Sobi: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau. Jimenez-Yuste:Bayer, CSL Behring, Grifols, Novo Nordisk, Octapharma, Pfizer, Roche, Sobi, Shire: Consultancy, Honoraria, Other: reimbursement for attending symposia/congresses , Research Funding, Speakers Bureau.

Description: Introduction: Recombinant factor IX Fc fusion protein (rFIXFc) is an extended half-life therapy for severe hemophilia B. The safety, efficacy, and prolonged half-life of rFIXFc were demonstrated in previously treated pediatric, adolescent, and adult subjects with severe hemophilia B in the Phase 3 B-LONG and Kids B-LONG trials (NCT01027364 and NCT01440946, respectively) (Fischer et al, Lancet Haematol, 2017; Powell et al, N Engl J Med, 2013). Here, the final results are reported from B-YOND (NCT01425723), the long-term extension of those 2 studies. Methods: This was an open-label, multicenter, long-term trial of previously treated subjects of all ages with severe hemophilia B. Dosing regimens included weekly prophylaxis (WP; 20-100 IU/kg every 7 days), individualized prophylaxis (IP; 100 IU/kg every 8-16 days or twice monthly), modified prophylaxis (MP; for subjects not achieving optimal dosing on IP or WP), or episodic treatment (ET; on-demand dosing based on type and severity of bleeding episodes). Subjects 1 group for analyses. The primary endpoint was development of inhibitors. Other endpoints included annualized bleeding rates (ABRs), joint ABRs, spontaneous joint ABRs, exposure days (ED), and factor consumption. Descriptive statistics were used for analysis. Analyses were performed separately based on parent study. Results: A total of 120 subjects (93 from B-LONG and 27 from Kids B-LONG) enrolled in B-YOND, and 98 subjects (75 from B-LONG and 23 from Kids B-LONG) completed the study. Of the 93 subjects from B-LONG, the median (range) age was 29 (13‒63) years and most were prescribed WP (WP, n=51; IP, n=31; MP, n=17; ET, n=15). Subjects from Kids B-LONG had a median (range) age of 7 (3‒12) years. Among subjects

Description: Key Points Nonacog beta pegol, a recombinant glycoPEGylated FIX with extended half-life, was developed to improve care for patients with hemophilia B. Weekly prophylaxis with nonacog beta pegol was well tolerated and was associated with low bleeding rates and an improved quality of life.

Description: Key Points BMI1 overexpression is one of the second hit partner genes of RUNX1 mutations that contribute to the development of MDSs.

Description: Key Points CD19-deficient donors augmented Scl-cGVHD. Donor regulatory B cells suppressed Scl-cGVHD.

Description: Mechanical tension is a critical determinant of vascular cell growth, differentiation, apoptosis, migration and development. Integrins have been implicated in sensing force but little is known about how forces are transduced to biochemical signals. We now show that mechanical strain stimulates conformational activation of integrin αvβ3 in NIH3T3 cells by using conformation specific antibody WOW-1. LM609, an antibody against αvβ3 integrin that is insensitive to integrin conformation, showed no change in binding after stretch, excluding changes in surface expression. Stretch also induced an increase of LIBS-6 binding, evidence that conformational activation leads to new binding of integrins to ECM. PI3K and Akt are both rapidly activated by strain, and PI3K inhibition decreases integrin activation. These data strongly suggest that PI3K mediates αvβ3 integrin activation. The blocking anti-fibronectin antibody 16G3 strongly inhibited the stretch-induced activation of JNK under conditions where existing adhesions did not appear to be disrupted, indicating that new integrin-ECM binding is required for JNK activation. These data define a pathway by which early activation of PI3K, through induction of integrin activation and ECM binding, stimulates a cytoplasmic signaling pathway implicated in cellular responses.

Description: Junctional adhesion molecule-A (JAM-A/JAM-1/F11R) is a cell adhesion molecule expressed in epithelial cells, endothelial cells and also hematopoietic cells such as leukocytes, platelets and erythrocytes. However, the expression of JAM-A in hematopoietic stem cell (HSC) had not been known. Here we show that JAM-A is expressed at a high level in the enriched HSC fraction, i.e. CD34+ c-Kit+ cells in embryonic day 11.5 (E11.5) aorta-gonod-mesonephros (AGM) and E11.5 fetal liver (FL) as well as c-Kit+ Sca-1+ Lineage- (KSL) cells in E14.5 FL, E18.5FL and adult bone marrow (BM). While the percentage of JAM-A+ cells in those tissues decreases during the development, the expression in HSC fraction is maintained throughout life. In fact, c-Kit+Sca-1+ cells are enriched approximately 200-fold from whole BM cells by anti-JAM-A antibody alone, i.e. the c-Kit+Sca-1+ cells in whole BM was about 0.15% and that in JAM-A+ cells was about 30%. Colony forming assays reveal that multi-lineage colony forming activity (CFU-mix) in JAM-A+ cells is higher than that in JAM-A- cells in the enriched HSC fraction in all those tissues. HSC transplantation assay revealed that long-term repopulating HSC (LTR-HSC) activity is present in the mice received 100 JAM-A+ KSL (7/9) cells and 300 KSL cells (5/6). Since the ratio of JAM-A+ cells to JAM-A- cells in the KSL fraction is about 6.5:3.5, 100 JAM-A- KSL cells are equivalent to 285 total KSL cells. In contrast to JAM-A+ cells, the mice received 1000 JAM-A- KSL cells, which are equivalent to more than 2850 total KSL cells, failed to engraft for long-term (0/6). These data revealed that long-term repopulating HSC (LTR-HSC) activity is present exclusively in the JAM-A+ cells, but not in JAM-A- cells. Moreover only 100 JAM-A+ cells isolated from whole BM cells by anti-JAM-A antibody alone reconstituted the hematopoietic system for long-term (4/7). Together these results indicate that JAM-A is expressed on hematopoietic precursors in various hematopoietic tissues and is an excellent and convenient marker to enrich LTR-HSC from BM cells.

Description: Many studies have shown the presence of minimal residual disease (MRD) following therapy for childhood acute lymphoblastic leukemia (ALL) to be an important prognostic marker. We have also shown a significant relationship between survival outcomes in patients enrolled in the previous ALL 911 study and molecular MRD levels 5 weeks (time point 1, TP1) and 12 weeks (TP2) following the initiation of chemotherapy (Leukaemia and Lymphoma2002; 43: 1001). The aim of this study was to evaluate if polymerase chain reaction (PCR)-based MRD assay is sufficiently dependable for tailoring therapy, and if augmented therapy can reduce MRD levels to those associated with a favourable outcome. The subjects were under 18 years of age, and had newly diagnosed precursor B or T-cell ALL. Patients below one year old and those with t(9;22) were excluded. Written informed consent was obtained from patients or their legal guardians. The ALL 941-based protocol (45thASH, San Diego, 2003) utilized PCR-based MRD assay using immunoglobulin & T-cell receptor gene rearrangements. MRD was detected by nested PCR, with screening of rearrangements using multiplex PCR primers as described previously (Leukaemia and Lymphoma2002; 43: 1001). Patients were initially stratified into 3 risk groups (in ascending order: SR, HR, and HHR) according to leukocyte count and age at time of diagnosis. The MRD+/+ patients with levels ≥ 10−3 at both TP1 and TP2 received augmented therapy 14 weeks after initiation, and the remainder continued to receive the initial risk-adapted protocols. A total of 311 patients with a median age of 5.3 years (range 1.0–16.8) were eligible for this study. There were 4 (1.3%) non-responders and no deaths in induction. Of the 307 patients stratified, 169 (55%) were SR, 107 (35%) were HR, and 31 (10%) were HHR. The 2nd stratification by MRD level at TP2 was possible for 72.3% (222/307; insufficient DNA=28; missing time-points=25; no marker=32). Out of the 222 patients stratified, 125 (56.3%) were MRD−/−, 58 (26.1%) were MRD+/−, and 38 (17.4%) were MRD+/+. At the point of analysis, the median follow-up time was 63 months (range 33–89). The overall 5-year event–free survival (EFS) rate of the 307 patients was 80.1% (SE 2.5), higher than the EFS of the ALL941 study, which was 76.2% (SE 2.1) (p=0.167). The 5-year EFS rates according to the 1st stratification were 85.5% (SE 4) for SR, 76.1% (SE 4.5) for HR, and 64.6% (SE 9.2) for HHR, while the equivalent rates for the 2nd stratification were 87.0% (SE 3.1) for MRD−/−, 75.5% (SE 7.7) for MRD+/−, and 75.3% (SE 6.4) for MRD+/+. From the 95 patients whose MRD levels were measured at 5 consecutive points from TP1 to TP5 (5, 12, 18, 24, and 30 weeks after the start of therapy), 21 subjects with MRD+/+ received an augmented chemotherapy, and MRD levels became undetectable in 9 patients at TP3, 5 patients at TP4, and 4 patients at TP5. The corresponding cumulative 5-year relapse rates of those patients were 11%, 50%, and 50%, respectively. Thus, negative MRD status at TP3, but not at TP4 or TP5, seems to be associated with a favourable outcome. Our results confirm the strong performance of MRD-based treatment interaction in a multi-institutional study without adversely affecting the outcome in childhood ALL. Moreover, present findings suggest that an augmented therapy could reduce MRD to levels associated with a favourable outcome. To improve the applicability and accuracy of MRD assay, new MRD-PCR targets and RQ-PCR-based MRD detection are needed in subsequent studies. [Acknowledgment: This study was partly supported by grants from the Children’s Cancer Association of Japan (CCAJ)].

Description: Ionizing radiation is known to induce remodeling of stromal microenvironment and enhance cancer progression. In this study, we investigated the molecular alterations of low-dose ionizing radiation (LDIR) induced non-targeted/bystander responses which affect a complex interplay of stromal cells and pre-leukemic cells in the bone marrow (BM) microenvironment. As a model of BM stromal cells and pre-leukemic cells, we utilized primary BM-derived stromal cells (MSCs) and the Epstein- Barr virus (EBV) infected and immortalized pre-leukemic B-lymphocyte cell line (EBV-B). LDIR (100 mGy, 4MV X ray from a LINAC) caused cell growth inhibition and moderate apoptosis induction in MSCs (viable cells % of control; 75.8 ± 2.4, specific SubG1 % 7.1 ± 0.8 at 24 h) but not in EBV-B cells. We further observed persistent upregulation of p21 mRNA (p〈 0.001, RQ-PCR) after acute low-dose irradiation in MSCs but not in EBV-B cells, suggesting radiation induced senescence-like changes in MSCs. In EBV-B cells co-cultured with MSCs, low-dose irradiation induced moderate cell growth inhibition (viable cells % of control; 81.3 ± 6.5) without significant apoptosis induction. To gain insights into the molecular changes induced by LDIR in both, MSC and EBV-B cells we utilized genomic and proteomic analyses. To exclude possible contamination of MSCs, we confirmed negative expression of CD90 mRNA in the tested EBV-B samples. We first screened up to 28,869 genes by cDNA microarray (Affymetrix) and performed functional network analysis by MetaCore (GeneGo). LDIR induced upregulation of 48 genes and the downregulation of 45 (i.e., 〉 1.3-fold regulation) with prominent stimulation of cell adhesion pathways in MSCs. Of note, 31 of 48 up-regulated genes were small nucleolar RNAs. In EBV-B cells, LDIR upregulated 69 genes/downregulated 130 genes with significant stimulation of the TGFbeta dependent induction of epithelial-mesenchymal transition (EMT) pathways. In EBV-B cells co-cultured with MSCs, LDIR induced immune response signaling along with integrin-mediated cell-matrix adhesion pathway with 42 genes upregulation / 34 genes downregulation. Up-regulation of inflammatory IL8 mRNA (2.0±0.03 fold, p

Description: Abstract 4076 Purpose: Despite recent advances in the use of newly developed drugs, high-risk multiple myeloma (MM) patients harboring del13q, t(4;14) or del17p revealed significantly shorter survival. To overcome the limitation, we have screened forty synthetic anilinoquinazoline (AQ) derivatives, and found a novel compound, Q15, which significantly inhibited the growth of MM cell lines with high-risk chromosomal abnormalities. The purpose of this study is to examine anti-tumor and anti-osteoclastogenic activities of Q15 and to clarify the possibility of development of new drug effective for high-risk MM and bone diseases. Methods and Results: Forty AQ derivatives were synthesized and screened for anti-proliferative effect on KMS34 cells. Q15 strongly inhibited growth of t(4;14)-positive KMS34 cells and induced apoptosis in much lower concentration (IC50=78nM) compared with gefitinib (IC50=2500nM), a representative AQ. Q15 also inhibited growth of other MM cell lines harboring high-risk chromosomal abnormalities. It was also found that Q15 did not inhibit intracellular tyrosine phosphorylation induced by EGF, FGF-2, HGF and IL-6, suggesting that Q15 showed anti-tumor activity in a different mechanism from that of gefitinib. In vivo anti-myeloma activity was evaluated by intraperitoneal injection of Q15 into KMS34-bearing lcr/SCID mice. Twenty mg/kg Q15 significantly delayed the tumor growth in these mice. Histopathological examinations revealed apoptosis of MM cells in Q15-treated mice. Growth of colony-forming cells was not suppressed by much higher concentrations (25μ M) of Q15 than IC50, suggesting low hematopoietic toxicity of Q15. In pharmacokinetic study using high-performance liquid chromatography (HPLC), the plasma concentration of Q15 in mice reached a maximum (Cmax=4.5μ M) at 1.5hr after injection, and its half life (T1/2) was 4.5hr. In addition, anti-osteoclastogenic activity was also examined by adding Q15 to M-CSF/RANK ligand-induced osteoclastogenic culture of bone marrow mononuclear cells from C57BL/6JJcl mice. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts was reduced in the presence of Q15. Conclusion: Q15, a novel AQ derivative, has anti-MM activity in vivo and is a potentially safe and effective drug for high-risk MM with bone lesions. Disclosures: No relevant conflicts of interest to declare.