scholarly journals A Novel and Potent Inhibitor of E-Selectin, GMI-1687, Attenuates Thrombus Formation and Augments Chemotherapeutic Intervention of AML in Preclinical Models Following Subcutaneous Administration

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4678-4678
Author(s):  
John Peterson ◽  
Myung-Gi Baek ◽  
Silvia Locatelli-Hoops ◽  
Ji-Won Lee ◽  
Lingquan Deng ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Tumor Model ◽  
Phase Iii ◽  
Therapeutic Activity ◽  
Preclinical Models ◽  
Employment Equity ◽  
Equity Ownership ◽  
High Bioavailability

Abstract Uproleselan (GMI-1271), an E-selectin antagonist, has been shown in preclinical models to disrupt activation of cell survival pathways in acute myeloid leukemia (AML), enhance chemotherapy efficacy, and improve survival. Uproleselan received FDA breakthrough therapy designation for adult relapsed/refractory AML in 2017 and Phase III studies are ongoing. In the present studies we report on the in vitro and in vivo comparative activities of an innovative high potency E-selectin antagonist, GMI-1687, a potential subcutaneously administered follow-on drug candidate to Uproleselan. The binding constant, association and dissociation rates of GMI-1687 to immobilized recombinant human (rh) E-selectin were determined by surface plasmon resonance (SPR) at 25oC. The KD of GMI-1687 was 2.4 nM, with Kon = 3 x 106 M-1s-1 and Koff = 1 x10-2 s-1. Under similar experimental conditions the KD of Uproleselan was 520 nM with Kon = 0.02 x 106 M-1s-1 and Koff = 1 x10-2 s-1. GMI-1687 was evaluated for its ability to inhibit binding of sialyl Lea to immobilized rh E-selectin. The median IC50 (n=6 independent assays) of GMI-1687 and Uproleselan in this assay was 15 and 550 nM, respectively. The in vitro activity of GMI-1687 to release adherent KG1a AML cells from E-selectin coated wells was also determined. GMI-1687 at 100 nM detached approximately 55% of adherent AML cells and was significantly different from Uproleselan at an identical concentration (38% detachment, P=0.0216). The percent bioavailability (%F) of GMI-1687 was evaluated in male Sprague-Dawley rats following intravenous (IV) and subcutaneous (SC) routes of administration at 5 mg/kg. The mean (+/- SD) SC %F for GMI-1687 was 126 +/- 3.8%. GMI-1687 also showed high bioavailability in CD-1 mice after SC administration of 0.58 mg/kg with %F = 132 +/-38. The in vivo therapeutic activity of GMI-1687 following SC administration was assessed in an acute model of inferior vena cava (IVC) thrombosis and a tumor model of AML.Immediately following the induction of a non-occlusive thrombosis via electrical stimulation (250 mAmp) of the IVC, cohorts of male C57BL/6J mice (n=5/group) were given a single SC injection of saline (0.1 mL); Uproleselan (40 mg/kg); or GMI-1687 (4 mg/kg, 0.4 mg/kg or 0.04 mg/kg), and twenty-four hrs post thrombus induction the IVC was harvested from all mice and thrombus weights were determined. Treatment with GMI-1687 decreased thrombus formation with significant inhibition at 0.04 mg/kg (92%, P<0.001 compared to saline control). The inhibition of thrombus formation with GMI-1687 dosed at 0.04 mg/kg was statistically indistinguishable from Uproleselan administered SC at 40 mg/kg (97% inhibition). The therapeutic activity of SC GMI-1687 was also observed in combination with chemotherapy in a U937 tumor model. Three days post IV injection of U937 tumor cells, bone marrow ablated, female NOD/SCID mice (n=10/group) were treated with saline (0.1 mL SC QDx14); GMI-1687 (0.04 mg/kg SC QDx14) alone; cytarabine (AraC 300 mg/kg IP QDx3) + daunorubicin (DNR 3 mg/kg IV QDx1), or the combination of GMI-1687 and AraC + DNR. All treatments were well tolerated. The median survival time (MST) of mice treated with AraC + DNR was 36 days and statistically different (P<0.001) to groups treated with saline (MST=22 days) or GMI-1687 alone (MST=23 days). Importantly, the therapeutic activity of AraC+DNR was significantly enhanced when combined with GMI-1687 (MST>47.5 days, P=0.0153 compared to AraC+DNR alone). In summary, a highly potent innovative antagonist of E-selectin, GMI-1687, has been produced that demonstrates high bioavailability following SC administration. SC injection of GMI-1687 shows significant activity in preclinical models previously reported for parenteral administration of Uproleselan, but at approximately 250-fold lower dose. GMI-1687 is therefore well-positioned for potential use in outpatient treatment settings where an E-selectin antagonist has therapeutic relevance. IND-enabling studies with GMI-1687 are currently underway. Disclosures Peterson: GlycoMimetics: Employment, Equity Ownership. Baek:GlycoMimetics: Employment, Equity Ownership. Locatelli-Hoops:GlycoMimetics: Employment, Equity Ownership. Lee:GlycoMimetics: Employment, Equity Ownership. Deng:GlycoMimetics: Employment, Equity Ownership. Stewart:GlycoMimetics: Employment, Equity Ownership. Smith:GlycoMimetics: Employment, Equity Ownership. Fogler:GlycoMimetics: Employment, Equity Ownership. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

ACY-1215, a First-In-Class Selective Inhibitor Of HDAC6, Demonstrates Significant Synergy With Immunomodulatory Drugs (IMiDs) In Preclinical Models Of Multiple Myeloma (MM)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1952-1952 ◽  
Author(s):  
Steven N Quayle ◽  
Simon S Jones
Keyword(s):  
Selective Inhibitor ◽  
Clinical Development ◽  
Clinical Activity ◽  
Preclinical Models ◽  
Employment Equity ◽  
Phase Ib ◽  
Equity Ownership ◽  
Ongoing Phase

Abstract Histone deacetylase (HDAC) enzymes represent attractive therapeutic targets in multiple myeloma, but unfortunately non-selective HDAC inhibitors have led to dose-limiting toxicities in patients. ACY-1215 is a first generation, orally available HDAC inhibitor that is 11-fold selective for HDAC6, and synergizes in vitro and in vivo with bortezomib in preclinical models of MM without inducing unfavorable toxicities (Blood, 20[210]: 4061). Ongoing Phase Ib clinical trials with ACY-1215 have thus far confirmed an exceptional safety and tolerability profile (Raje, et al, EHA, 2013). The IMiD class of drugs, including lenalidomide and pomalidomide, exhibit striking anti-myeloma properties in a variety of MM models, and have demonstrated significant clinical activity in MM patients. Prior studies have shown clinical activity of a combination of the non-selective HDAC inhibitor vorinostat with lenalidomide and dexamethasone in myeloma patients (Richter, et al, ASH, 2011). However, many patients experienced significant toxicities with this regimen that significantly limits its clinical utility. In support of our ongoing clinical development program for ACY-1215 in MM, we show here that combining ACY-1215 with either lenalidomide or pomalidomide leads to synergistic decreases in the viability of MM cells in vitro. The relevance of inhibition of HDAC6 to this synergistic effect was validated by demonstrating synergistic interactions of either IMiD molecule with ACY-775, which is more than 300-fold selective for HDAC6 over class I HDAC’s. Further, the combination of ACY-1215, lenalidomide, and dexamethasone was well tolerated in vivo with no overt evidence of toxicity, and combination efficacy studies with this combination are now ongoing in models of MM. By demonstrating that a selective inhibitor of HDAC6 synergizes with IMiD’s while maintaining an improved safety profile, these results provided a rational basis for the clinical development of the orally available combination of ACY-1215 and lenalidomide plus dexamethasone in an ongoing Phase Ib clinical trial (NCT01583283) for the treatment of MM. Disclosures: Quayle: Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership.

scholarly journals Elucidating the Excessive Pro-Coagulant Effect of a Sequence Identical Analogue to ACE910 in Combination with Bypassing Agents

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 90-90
Author(s):  
Rudolf Hartmann ◽  
Tjerk Feenstra ◽  
Sabine Knappe ◽  
Michael Dockal ◽  
Friedrich Scheiflinger
Keyword(s):  
Whole Blood ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Thrombus Formation ◽  
Phase Iii ◽  
Hek293 Cells ◽  
Employment Equity ◽  
Equity Ownership ◽  
Bypassing Agents

Abstract Introduction: Emicizumab (ACE910), an antibody to FIX(a) and FX(a), is currently under investigation for treatment of hemophilia with inhibitors. In a phase III trial, two thromboembolic complications and three cases of microangiopathy were reported in patients on ACE910 prophylaxis [Oldenburg et al. NEJM 2017], whose breakthrough bleeding was treated with activated prothrombin complex concentrate aPCC (FEIBA) or aPCC and rFVIIa. We generated a sequence identical analogue (SIA) to ACE910 and analyzed its synergistic interplay with bypassing agents. Aims: To monitor in vitro the pro-coagulant activity of SIA ACE910 in the presence of FEIBA and rFVIIa, and detect the source of excessive coagulation induced by SIA ACE910 combined with FEIBA. Methods: A sequence identical analogue (SIA) to ACE910 was expressed in HEK293 cells, purified as previously described [Sampei et al. PLoS One 2013], and analyzed in several global hemostatic assays at different concentrations and test conditions using plasma and whole blood assays. In thrombin generation (TG) experiments, platelet-poor plasma (PPP) from hemophilia A inhibitor patients and hemophilia A plasma reconstituted with platelets from 3 healthy donors (PRP) was used. A normal TG range was established in healthy donor plasma. Therapeutic concentrations of SIA ACE910 (20-600 nM) were tested alone and with FEIBA (0.05-1 U/mL) or rFVIIa (0.88-5.25 µg/mL). To measure FEIBA components' contribution to the synergistic effect with SIA ACE910, PPP was spiked with select FEIBA components at concentrations corresponding to 0.5 U/mL FEIBA in combination with the antibody. Thrombus formation was analyzed in FVIII-inhibited blood using rotational thromboelastometry (ROTEM) and Total Thrombus-formation Analysis System (T-TAS). Results: Normal peak thrombin was 47-144 nM for PPP and 88-231 nM for PRP. rFVIIa and FEIBA had an additive effect on TG in combination with SIA ACE910 in both plasma types. Combined with rFVIIa (0.88 µg/mL) or FEIBA (0.5 U/mL), SIA ACE910 (600 nM) induced a ~2- and ~16-fold increase over SIA ACE910 alone. SIA ACE910+rFVIIa did not reach the normal range, while SIA ACE910+FEIBA far exceeded it. Adding individual FEIBA components to PPP showed that FIX was, with a half-maximal effect, the main driver for enhanced TG, followed by FIXa. formation in FVIII-inhibited whole blood using ROTEM and T-TAS confirmed the excessive effect of SIA ACE910+FEIBA. In ROTEM, FEIBA and rFVIIa reduced clotting time to shorter than normal, whereas SIA ACE910 had only little effect. Moreover, adding SIA ACE910 to rFVIIa exerted no effect over rFVIIa alone. Conclusion: Combining SIA ACE910 at plasma concentrations observed in patients [Oldenburg et al. NEJM 2017] with FEIBA induced excessive thrombin generation and faster clot formation. In vitro, this effect is mainly mediated by FEIBA component FIX. ACE910 binds to FIX and FIXa to the same extent, and displays its pro-coagulant effect via an unregulated mechanism. Therefore, careful judgement is needed in treating breakthrough bleeds with FEIBA. Disclosures Hartmann: Shire: Employment. Feenstra: Shire: Employment. Knappe: Shire: Employment. Dockal: Baxalta: Patents & Royalties; Shire: Employment, Equity Ownership; Baxter: Equity Ownership, Patents & Royalties. Scheiflinger: Baxter: Equity Ownership; Shire: Employment, Equity Ownership.

scholarly journals Phenotype Does Not Always Equal Function: HDAC Inhibitors and UM171, but Not SR1, Lead to Rapid Upregulation of CD90 on Non-Engrafting CD34+CD90-Negative Human Cells

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 659-659
Author(s):  
Kevin A. Goncalves ◽  
Megan D. Hoban ◽  
Jennifer L. Proctor ◽  
Hillary L. Adams ◽  
Sharon L. Hyzy ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Small Molecule ◽  
Hdac Inhibitors ◽  
Employment Equity ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Equity Ownership ◽  
Human Hscs

Abstract Background. The ability to expand human hematopoietic stem cells (HSCs) has the potential to improve outcomes in HSC transplantation and increase the dose of gene-modified HSCs. While many approaches have been reported to expand HSCs, a direct comparison of the various methods to expand transplantable HSCs has not been published and clinical outcome data for the various methods is incomplete. In the present study, we compared several small molecule approaches reported to expand human HSCs including HDAC inhibitors, the aryl hydrocarbon antagonist, SR1, and UM171, a small molecule with unknown mechanism, for the ability to expand phenotypic HSC during in vitro culture and to expand cells that engraft NSG mice. Although all strategies increased the number of phenotypic HSC (CD34+CD90+CD45RA-) in vitro, SR1 was the most effective method to increase the number of NOD-SCID engrafting cells. Importantly, we found that HDAC inhibitors and UM171 upregulated phenotypic stem cell markers on downstream progenitors, suggesting that these compounds do not expand true HSCs. Methods. Small-molecules, SR1, HDAC inhibitors (BG45, CAY10398, CAY10433, CAY10603, Entinostat, HC Toxin, LMK235, PCI-34051, Pyroxamide, Romidepsin, SAHA, Scriptaid, TMP269, Trichostatin A, or Valproic Acid) and UM171 were titrated and then evaluated at their optimal concentrations in the presence of cytokines (TPO, SCF, FLT3L, and IL6) for the ability to expand human mobilized peripheral blood (mPB)-derived CD34+ cells ex vivo . Immunophenotype and cell numbers were assessed by flow cytometry following a 7-day expansion assay in 10-point dose-response (10 µM to 0.5 nM). HSC function was evaluated by enumeration of colony forming units in methylcellulose and a subset of the compounds were evaluated by transplanting expanded cells into sub-lethally irradiated NSG mice to assess engraftment potential in vivo . All cells expanded with compounds were compared to uncultured or vehicle-cultured cells. Results. Following 7 days of expansion, SR1 (5-fold), UM171 (4-fold), or HDAC inhibitors (&gt;3-35-fold) resulted in an increase in CD34+CD90+CD45RA- number relative to cells cultured with cytokines alone; however, only SR1 (18-fold) and UM171 (8-fold) demonstrated enhanced engraftment in NSG mice. Interestingly, while HDAC inhibitors and UM171 gave the most robust increase in the number and frequency of CD34+CD90+CD45RA- cells during in vitro culture, these methods were inferior to SR1 at increasing NSG engrafting cells. The increase in CD34+CD90+CD45RA- cells observed during in vitro culture suggested that these compounds may be generating a false phenotype by upregulating CD90 and down-regulating CD45RA on progenitors that were originally CD34+CD90-CD45RA+. We tested this hypothesis by sorting CD34+CD90-CD45RA+ cells and culturing these with the various compounds. These experiments confirmed that both HDAC inhibitors (33-100 fold) and UM171 (28-fold) led to upregulation of CD90 on CD34+CD90-CD45RA+ cells after 4 days in culture. Since approximately 90% of the starting CD34+ cells were CD90-, these data suggest that most of the CD34+CD90+CD45RA- cells in cultures with HDAC inhibitors and UM171 arise from upregulation of CD90 rather than expansion of true CD34+CD90+CD45RA- cells and may explain the disconnect between in vitro HSC phenotype and NSG engraftment in vivo . This was further confirmed by evaluation of colony forming unit frequency of CD34+CD90-CD45RA+ cells after culture with compounds. Conclusions. We have showed that AHR antagonism is optimal for expanding functional human HSCs using the NSG engraftment model. We also demonstrated that UM171 and HDAC inhibitors upregulate phenotypic HSC markers on downstream progenitors. This could explain the discrepancy between impressive in vitro phenotypic expansion and insufficient functional activity in the NSG mouse model. Therefore, these data suggest caution when interpreting in vitro expansion phenotypes without confirmatory functional transplantation data, especially as these approaches move into clinical trials in patients. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership. Hoban: Magenta Therapeutics: Employment, Equity Ownership. Proctor: Magenta Therapeutics: Employment, Equity Ownership. Adams: Magenta Therapeutics: Employment, Equity Ownership. Hyzy: Magenta Therapeutics: Employment, Equity Ownership. Boitano: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

scholarly journals Highly Differentiated Anti-CD47 Antibody, AO-176, Potently Inhibits Hematologic Malignancies Alone and in Combination

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1844-1844
Author(s):  
John Richards ◽  
Myriam N Bouchlaka ◽  
Robyn J Puro ◽  
Ben J Capoccia ◽  
Ronald R Hiebsch ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Combination Therapy ◽  
Tumor Growth ◽  
Growth Inhibition ◽  
Tumor Cells ◽  
Tumor Growth Inhibition ◽  
Employment Equity ◽  
Equity Ownership

AO-176 is a highly differentiated, humanized anti-CD47 IgG2 antibody that is unique among agents in this class of checkpoint inhibitors. AO-176 works by blocking the "don't eat me" signal, the standard mechanism of anti-CD47 antibodies, but also by directly killing tumor cells. Importantly, AO-176 binds preferentially to tumor cells, compared to normal cells, and binds even more potently to tumors in their acidic microenvironment (low pH). Hematological neoplasms are the fourth most frequently diagnosed cancers in both men and women and account for approximately 10% of all cancers. Here we describe AO-176, a highly differentiated anti-CD47 antibody that potently targets hematologic cancers in vitro and in vivo. As a single agent, AO-176 not only promotes phagocytosis (15-45%, EC50 = 0.33-4.1 µg/ml) of hematologic tumor cell lines (acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, and T cell leukemia) but also directly targets and kills tumor cells (18-46% Annexin V positivity, EC50 = 0.63-10 µg/ml) in a non-ADCC manner. In combination with agents targeting CD20 (rituximab) or CD38 (daratumumab), AO-176 mediates enhanced phagocytosis of lymphoma and multiple myeloma cell lines, respectively. In vivo, AO-176 mediates potent monotherapy tumor growth inhibition of hematologic tumors including Raji B cell lymphoma and RPMI-8226 multiple myeloma xenograft models in a dose-dependent manner. Concomitant with tumor growth inhibition, immune cell infiltrates were observed with elevated numbers of macrophage and dendritic cells, along with increased pro-inflammatory cytokine levels in AO-176 treated animals. When combined with bortezomib, AO-176 was able to elicit complete tumor regression (100% CR in 10/10 animals treated with either 10 or 25 mg/kg AO-176 + 1 mg/kg bortezomib) with no detectable tumor out to 100 days at study termination. Overall survival was also greatly improved following combination therapy compared to animals treated with bortezomib or AO-176 alone. These data show that AO-176 exhibits promising monotherapy and combination therapy activity, both in vitro and in vivo, against hematologic cancers. These findings also add to the previously reported anti-tumor efficacy exhibited by AO-176 in solid tumor xenografts representing ovarian, gastric and breast cancer. With AO-176's highly differentiated MOA and binding characteristics, it may have the potential to improve upon the safety and efficacy profiles relative to other agents in this class. AO-176 is currently being evaluated in a Phase 1 clinical trial (NCT03834948) for the treatment of patients with select solid tumors. Disclosures Richards: Arch Oncology Inc.: Employment, Equity Ownership, Other: Salary. Bouchlaka:Arch Oncology Inc.: Consultancy, Equity Ownership. Puro:Arch Oncology Inc.: Employment, Equity Ownership. Capoccia:Arch Oncology Inc.: Employment, Equity Ownership. Hiebsch:Arch Oncology Inc.: Employment, Equity Ownership. Donio:Arch Oncology Inc.: Employment, Equity Ownership. Wilson:Arch Oncology Inc.: Employment, Equity Ownership. Chakraborty:Arch Oncology Inc.: Employment, Equity Ownership. Sung:Arch Oncology Inc.: Employment, Equity Ownership. Pereira:Arch Oncology Inc.: Employment, Equity Ownership.

scholarly journals Clinical Pharmacodynamic Markers and Combinations with SY1425 (tamibarotene) in a Genomically-Defined Subset of Non-APL AML

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2898-2898
Author(s):  
Michael R McKeown ◽  
Christopher Fiore ◽  
Emily Lee ◽  
Matthew L Eaton ◽  
Christian C. Fritz
Keyword(s):  
Transcription Factor ◽  
Cell Lines ◽  
Binding Sites ◽  
Clinical Studies ◽  
High Cell ◽  
Preclinical Models ◽  
Employment Equity ◽  
Equity Ownership ◽  
Maximal Induction

Abstract SY-1425, a potent and selective agonist of the retinoic acid receptor RARα, is being investigated in a Ph2 trial in a novel genomically-defined subset of non-APL AML and MDS patients (clinicaltrials.gov NCT02807558). RARa is a nuclear hormone receptor and transcription factor that regulates genes involved in cell differentiation and proliferation. We identified a super-enhancer (SE) at the RARA locus, the gene encoding RARa, in a subset of primary non-APL AML blasts. Preclinical models demonstrated a correlation between the presence of a RARA SE and sensitivity to SY-1425, providing the rationale for clinical investigation. Further research has investigated pharmacodynamics (PD) markers and combinations of drugs to support clinical development of SY-1425. In this study we identified DHRS3mRNA induction as a measure of RARα target engagement with SY-1425. We also demonstrated synergy in preclinical models with SY-1425 and hypomethylating agents. Since RARα is a transcription factor that regulates target genes when bound by a retinoid, we characterized the dynamic expression changes of a panel of RARA enhancer- high and - low non-APL AML cell lines (hereafter referred to as RARA-high and -low) in response to SY-1425 treatment. DHRS3 showed the largest expression increase following treatment in 3 RARA-high cell lines, with a range of 29 to 115 fold. In contrast, there was a much lower DHRS3 induction in 3 RARA-low cell lines (range of 1.6 to 6.1 fold). Induction was found to be both time- and dose-dependent with maximal induction at approximately 6 hours and half maximal induction near the EC50 for the anti-proliferative effect in RARA-high cell lines. DHRS3 encodes dehydrogenase/reductase (SDR family) member 3, a metabolic enzyme involved in maintaining cellular retinol homeostasis and had previously been shown to be induced by retinoids. Thus, DHRS3induction in tumor cells represents a potentially useful PD marker for clinical studies of SY-1425. To better understand the mechanism of induction of DHRS3 by SY-1425 we examined the chromosomal localization of RARα as well as the epigenomic state of the DHRS3 locus by ChIP-seq for RARα and H3K27 acetylation, the latter being an indicator of active enhancers and promoters. In the untreated state, OCI-AML3 (a typical RARA-high AML cell line) was found to have multiple RARα binding sites both within and distal to the DHRS3 gene but minimal H3K27 acetylation. Following treatment with SY-1425, the level of H3K27 acetylation at DHRS3 increased, resulting in the formation of a SE. Moreover, the SE encompassed the RARα binding sites, consistent with the model in which SY-1425 converts RARα into an activator of DHRS3expression. Similar results were seen for the CD38 locus in which SY-1425 treatment increased expression, H3K27 acetylation, and RARα binding. CD38 is a cell surface antigen and marker of myeloid maturation readily analyzed by FACS analysis, suggesting it could be an additional PD marker to be used in clinical studies. Indeed, it was found that SY-1425 induced CD38 cell surface expression at similar levels in RARA-high AML cell lines and the NB-4 APL cell line, but not in RARA-low cell lines. We also investigated combinations of SY-1425 with approved or investigational AML and MDS agents in in vitro and in vivo models to inform future clinical studies and to further explore potential PD markers unique to the combined action of the drugs. Several standard of care agents and drugs in current development were found to have synergistic interactions with SY-1425 in RARA-high but not RARA-low cell lines. In particular, azacitidine and decitabine each showed strong in vitro synergy with SY-1425. Evaluation of SY-1425 plus azacitidine in a RARA-high PDX model of non-APL AML demonstrated a better response compared to either agent alone. Additional genome-wide ChIP-seq and expression studies of RARA-high cells treated with various combinations are being investigated to identify optimal PD markers for these combinations. These studies support the use of DHRS3 mRNA induction in tumor cells as a PD marker in the recently initiated Ph2 study of SY-1425 in genomically-defined non-APL AML and MDS patients (clinicaltrials.gov NCT02807558) and further exploration as a PD marker for future combination studies. Disclosures McKeown: Syros Pharmaceuticals: Employment, Equity Ownership. Fiore:Syros Pharmaceuticals: Employment, Equity Ownership. Lee:Syros Pharmaceuticals: Employment, Equity Ownership. Eaton:Syros Pharmaceuticals: Employment, Equity Ownership. Fritz:Syros Pharmaceuticals: Employment, Equity Ownership.

Differential Effects of Milatuzumab On Human Antigen-Presenting Cells in Comparison to Malignant B Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2744-2744
Author(s):  
Xiaochuan Chen ◽  
Rhona Stein ◽  
Chien-Hsing Chang ◽  
David M. Goldenberg
Keyword(s):  
T Cell ◽  
B Cells ◽  
Cell Lines ◽  
Hairy Cell ◽  
Employment Equity ◽  
Cross Presentation ◽  
Equity Ownership ◽  
Antigen Presenting

Abstract Abstract 2744 Poster Board II-720 Introduction: The humanized anti-CD74 monoclonal antibody (mAb), milatuzumab, is in clinical evaluation as a therapeutic mAb for non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and multiple myeloma after preclinical evidence of activity in these tumor types. In addition to its expression in malignant cells, CD74 is also expressed in normal B cells, monocytes, macrophages, Langerhans cells, follicular and blood dendritic cells. A question therefore arises whether milatuzumab is toxic to or affects the function of these immune cells. This has important implications, not only for safe therapeutic use of this mAb, but also for its potential application as a novel delivery modality for in-vivo targeted vaccination. Methods: We assessed the binding profiles and functional effects of milatuzumab on human antigen-presenting cell (APC) subsets. Studies on the effect of milatuzumab on antigen presentation and cross-presentation are included. In addition, binding and cytotoxicity on a panel of leukemia/lymphoma cell lines and CLL patient cells were tested to demonstrate the range of malignancies that can be treated with this mAb. Results: Milatuzumab bound efficiently to different subsets of blood dendritic cells, including BDCA-1+ myeloid DCs (MDC1), BDCA-2+ plasmacytoid DCs (PDC), BDCA-3+ myeloid DCs (MDC2), B lymphocytes, monocytes, and immature DCs derived from human monocytes in vitro, but not LPS-matured DCs, which correlated well with their CD74 expression levels. In the malignant B-cells tested, milatuzumab bound to the surface of 2/3 AML, 2/2 mantle cell (MCL), 4/4 ALL, 1/1 hairy cell leukemia, 2/2 CLL, 7/7 NHL, and 5/6 multiple myeloma cell lines, and cells of 4/6 CLL patient specimens. Significant cytotoxicity (P<0.05) was observed in 2/2 MCL, 2/2 CLL, 3/4 ALL, 1/1 hairy cell, 2/2 NHL, and 2/2 MM cell lines, and 3/4 CD74-positive CLL patient cells, but not in the AML cell lines following incubation with milatuzumab. In contrast, milatuzumab had minimal effects on the viability of DCs or B cells that normally express CD74. The DC maturation and DC-mediated T-cell functions were not altered by milatuzumab treatment, which include DC-induced T-cell proliferation, CD4+CD25+FoxP3+ Treg expansion, and CD4+ naïve T-cell polarization. Moreover, milatuzumab had little effect on CMV-specific CD8- and CD8+ T cell interferon-g responses of peripheral blood mononuclear cells stimulated in vitro with CMV pp65 peptides or protein, suggesting that milatuzumab does not influence antigen presentation or cross-presentation. Conclusion: These results demonstrate that milatuzumab is a highly specific therapeutic mAb against B-cell malignancies with potentially minimal side effects. It also suggests that milatuzumab may be a promising novel delivery mAb for in vivo targeted vaccinations, given its efficient binding, but lack of cytotoxicity and functional disruption on CD74-expressing normal APCs. (Supported in part by NIH grant PO1-CA103985.) Disclosures: Chang: Immunomedics Inc.: Employment, Equity Ownership, Patents & Royalties. Goldenberg:Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Optimized Fully-Synthetic Salicylamide Heparin Antagonists Have Greater Efficacy Versus LMWHs and Display Improved Hemodynamic Responses as Compared to Protamine

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 191-191
Author(s):  
Richard W. Scott ◽  
Michael J. Costanzo ◽  
Katie B. Freeman ◽  
Robert W. Kavash ◽  
Trevor M. Young ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Cell Line ◽  
Factor Xa ◽  
In Vivo Model ◽  
Hemodynamic Effects ◽  
Employment Equity ◽  
Human Liver Cell ◽  
Equity Ownership

Abstract Abstract 191 A series of salicylamides, fully synthetic cationic foldamers designed to disrupt the binding of the pentasaccharide unit of heparin to antithrombin III, were found to be potent neutralizers of the activity of unfractionated heparin (UFH) and low molecular weight heparins (LMWHs). A compound from this series, PMX-60056, is currently in human clinical trials for neutralization of UFH and LMWHs. PMX-60056 potently neutralizes UFH and LMWHs but is not as efficacious versus fondaparinux (FPX). The goal of the present research was to 1) identify back-up compounds to optimize activity against the LMWHs and FPX and 2) mitigate the hemodynamic effects commonly associated with protamine and observed clinically with PMX-60056 in the absence of heparin. Compounds were first tested for their ability to neutralize the anticoagulant activity of enoxaparin (ENX), tinzaparin or FPX in an in vitro amidolytic assay for factor Xa activity. While only minor improvements were observed in the neutralization of ENX and tinzaparin, compounds were identified which had 6 to 40 fold increase in activity against FPX (EC50s of 0.09 – 0.58 uM) in comparison to PMX-60056 (EC50 3.64 uM). Activated partial thromboplastin time (aPTT) assays demonstrated that these compounds maintained activity against heparin in a plasma based clotting assay. Rotation thromboelastometry (ROTEM) was used to show that these compounds are able to neutralize heparin and ENX in human whole blood, restoring normal coagulation profiles. As an initial test for safety, compounds were tested in hemolysis and cytotoxicity assays using isolated human erythrocytes, a transformed human liver cell line (HepG2 cells) and a mouse fibroblast cell line (NIH3T3). Lead back-up compounds were not cytotoxic (or hemolytic) at >100 fold concentrations over their EC50 concentrations in the anti-coagulation assays, indicating a high selectivity index between toxicity and efficacy. Five compounds were selected for further studies based on their in vitro profiles. The in vivo efficacy of these compounds was evaluated in a rat coagulation model for neutralization of ENX (2 mg/kg). Three minutes following IV dosing with ENX, either saline, protamine or one of the five salicylamide test compounds was administered. Blood was collected before dosing with ENX, and at 1, 3, 10, and 60 min after dosing, for aPTT and factor Xa analysis. Three of the five salicylamides (PMX640, PMX686 and PMX747) were more efficacious than protamine; with PMX640 and PMX686 neutralizing 91 – 100% and PMX747 neutralizing 78–100% of the ENX anti-factor Xa activity over the entire 60 minute time course. In a second in vivo model, PMX747 and PMX686 (2 mg/kg) completely neutralized the prolonged bleeding times in a rat tail bleeding model caused by treatment with 2 mg/kg ENX. Significantly, with protamine at a 5 mg/kg dosage, only partial restoration was obtained. Protamine routinely causes a transient decrease in blood pressure upon dosing, and hemodynamic effects have also been observed with PMX-60056 in human subjects in the absence of heparin. To address this issue, structural features that have successfully reduced hemodynamic liabilities in other cationic compounds were incorporated into the design of the back-up salicylamides. The effect of compounds on blood pressure and heart rate was measured via arterial catheters in rats following IV administration of protamine, PMX-60056, or test agents. As expected, in rats treated with a low dose of UFH (50 u/kg) and high dosages of antagonist, both protamine and PMX-60056 displayed transient or prolonged blood pressure reductions at 8 and 16 mg/kg, respectively. However, the lead back-up salicylamides, PMX640, PMX686 and PMX747 had little to no effect on blood pressure at these same dosages. In conclusion, we have discovered compounds in the salicylamide series that have greater efficacy versus LMWHs and that have significantly reduced hemodynamic liabilities in rats as compared to protamine. Furthermore, these compounds potently neutralize FPX activity in vitro; exceeding the activity of protamine and our clinical lead salicylamide, PMX-60056, by up to 40 fold. Thus we have been able to optimize the salicylamide series, identifying compounds that offer the potential to greatly improve upon the current clinical heparin antagonist, protamine, in respect to both activity against LMWHs and side effect profile. Disclosures: Scott: PolyMedix Inc.: Employment, Equity Ownership. Costanzo:PolyMedix Inc.: Employment, Equity Ownership. Freeman:PolyMedix Inc.: Employment, Equity Ownership. Kavash:PolyMedix Inc.: Employment, Equity Ownership. Young:PolyMedix, Inc.: Employment, Equity Ownership. DeGrado:PolyMedix, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Jeske:PolyMedix, Inc.: Research Funding.

Stroma-Free Ex Vivo Expanded, CD34+ Cord Blood-Derived NK Cells Retain Lytic Activity After Long-Term Engraftment in NSGS Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 580-580
Author(s):  
Mark Wunderlich ◽  
Mahesh Shrestha ◽  
Lin Kang ◽  
Eric Law ◽  
Vladimir Jankovic ◽  
...  
Keyword(s):  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
Employment Equity ◽  
Cell Product ◽  
Equity Ownership ◽  
Cellular Therapeutics

Abstract Abstract 580 Generating a large number of pure, functional immune cells that can be used in human patients has been a major challenge for NK cell-based immunotherapy. We have successfully established a cultivation method to generate human NK cells from CD34+ cells isolated from donor-matched cord blood and human placental derived stem cells, which were obtained from full-term human placenta. This cultivation method is feeder-free, based on progenitor expansion followed by NK differentiation supported by cytokines including thrombopoietin, stem cell factor, Flt3 ligand, IL-7, IL-15 and IL-2. A graded progression from CD34+ hematopoietic progenitor cells (HSC) to committed NK progenitor cells ultimately results in ∼90% CD3-CD56+ phenotype and is associated with an average 10,000-fold expansion achieved over 35 days. The resulting cells are CD16- and express low level of KIRs, indicating an immature NK cell phenotype, but show active in vitro cytotoxicity against a broad range of tumor cell line targets. The in vivo persistence, maturation and functional activity of HSC-derived NK cells was assessed in NSG mice engineered to express the human cytokines SCF, GM-CSF and IL-3 (NSGS mice). Human IL-2 or IL-15 was injected intraperitoneally three times per week to test the effect of cytokine supplementation on the in vivo transferred NK cells. The presence and detailed immunophenotype of NK cells was assessed in peripheral blood (PB), bone marrow (BM), spleen and liver samples at 7-day intervals up to 28 days post-transfer. Without cytokine supplementation, very few NK cells were detectable at any time-point. Administration of IL-2 resulted in a detectable but modest enhancement of human NK cell persistence. The effect of IL-15 supplementation was significantly greater, leading to the robust persistence of transferred NK cells in circulation, and likely specific homing and expansion in the liver of recipient mice. The discrete response to IL-15 versus IL-2, as well as the preferential accumulation in the liver have not been previously described following adoptive transfer of mature NK cells, and may be unique for the HSC-derived immature NK cell product. Following the in vivo transfer, a significant fraction of human CD56+ cells expressed CD16 and KIRs indicating full physiologic NK differentiation, which appears to be a unique potential of HSC-derived cells. Consistent with this, human CD56+ cells isolated ex vivo efficiently killed K562 targets in in vitro cytotoxicity assays. In contrast to PB, spleen and liver, BM contained a substantial portion of human cells that were CD56/CD16 double negative (DN) but positive for CD244 and CD117, indicating a residual progenitor function in the CD56- fraction of the CD34+ derived cell product. The BM engrafting population was higher in NK cultures at earlier stages of expansion, but was preserved in the day 35- cultured product. The frequency of these cells in the BM increased over time, and showed continued cycling based on in vivo BrdU labeling 28 days post-transfer, suggesting a significant progenitor potential in vivo. Interestingly, DN cells isolated from BM could be efficiently differentiated ex vivo to mature CD56+CD16+ NK cells with in vitro cytotoxic activity against K562. We speculate that under the optimal in vivo conditions these BM engrafting cells may provide a progenitor population to produce a mature NK cell pool in humans, and therefore could contribute to the therapeutic potential of the HSC-derived NK cell product. The in vivo activity of HSC-derived NK cells was further explored using a genetically engineered human AML xenograft model of minimal residual disease (MRD) and initial data indicates significant suppression of AML relapse in animals receiving NK cells following chemotherapy. Collectively, our data demonstrate the utility of humanized mice and in vivo xenograft models in characterizing the biodistribution, persistence, differentiation and functional assessment of human HSC-derived cell therapy products, and characterize the potential of HSC-derived NK cells to be developed as an effective off-the-shelf product for use in adoptive cell therapy approaches in AML. Disclosures: Wunderlich: Celgene Cellular Therapeutics: Research Funding. Shrestha:C: Research Funding. Kang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Law:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Jankovic:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zhang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Herzberg:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Abbot:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hariri:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Mulloy:Celgene Cellular Therapeutics: Research Funding.

Combination of the glycoengineered Type II CD20 antibody obinutuzumab (GA101) and The novel Bcl-2 selective Inhibitor GDC-0199 Results in superior In Vitro and In Vivo Anti-tumor activity in models Of B-Cell Malignancies

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4412-4412 ◽  
Author(s):  
Deepak Sampath ◽  
Sylvia Herter ◽  
Frank Herting ◽  
Ellen Ingalla ◽  
Michelle Nannini ◽  
...  
Keyword(s):  
Cell Death ◽  
Single Agent ◽  
Employment Equity ◽  
Direct Cell ◽  
Xenograft Models ◽  
Equity Ownership ◽  
Anti Cd20 ◽  
Agent Treatment

Introduction Obinutuzumab (GA101) is a novel glycoengineered type II, anti-CD20 monoclonal antibody induces a high level of direct cell death. As a result of glycoengineering, GA101 has increased affinity for FcgRIIIa on effector cells resulting in enhanced direct cell death and ADCC induction. GA101 is currently in pivotal clinical trials in CLL, indolent NHL and DLCBL. ABT-199 (GDC-0199) is a novel, orally bioavailable, selective Bcl-2 inhibitor that induces robust apoptosis in preclinical models of hematological malignancies and is currently in clinical trials for CLL, NHL and MM. Based on their complementary mechanisms of action involving increased apoptosis (GDC-0199) or direct cell death (GA101) the combination of anti-CD20 therapy with a Bcl-2 inhibitor has the potential for greater efficacy in treating B lymphoid malignancies. Experimental Methods The combination of GA101 or rituximab with GDC-0199 was studied in vitro utilizing assays that measure direct cell death induction/apoptosis (AxV/Pi positivity) on WSU-DLCL2, SU-DHL4 DLBCL and Z138 MCL cells by FACS and the impact of Bcl-2 inhibition on ADCC induction. In vivo efficacy of the combination of GA101 or rituximab and GDC-0199 was evaluated in SU-DHL4 and Z138 xenograft models. Results GA101 and rituximab enhanced cell death induction when combined with GDC-0199 in SU-DHL4, WSU-DLCL2 and Z138 cell lines. When combined at optimal doses an additive effect of the two drugs was observed. GDC-0199 did not negatively impact the capability of GA101 or rituximab to induce NK-cell mediated ADCC. Combination of GDC-0199 and GA101 induced a greater than additive anti-tumor effects in the SU-DHL4 and Z138 xenograft models resulting in tumor regressions and delay in tumor regrowth when compared to monotherapy. Moreover, continued single-agent treatment with GDC-0199 after combination with GA101 resulted in sustained in vivo efficacy in the SU-DHL4 model. Conclusions Our data demonstrate that the combination of GA101 with GDC-0199 results in enhanced cell death and robust anti-tumor efficacy in xenograft models representing NHL sub-types that is comparable to the combination of rituximab with GDC-0199. In addition, single-agent treatment with GDC-0199 following combination with GA101 sustains efficacy in vivo suggesting a potential benefit in continued maintenance therapy with GDC-0199. Collectively the preclinical data presented here supports clinical investigation of GA101 and GDC-0199 combination therapy, which is currently in a phase Ib clinical trial (clinical trial.gov identifier NCT01685892). Disclosures: Sampath: Genentech: Employment, Equity Ownership. Herter:Roche: Employment. Herting:Roche: Employment. Ingalla:Genentech: Employment. Nannini:Genentech: Employment. Bacac:Roche: Employment. Fairbrother:Genentech: Employment, Equity Ownership. Klein:Roche Glycart AG: Employment.

Phase 1 Study of CB-839, a First-in-Class, Glutaminase Inhibitor in Patients with Multiple Myeloma and Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3059-3059 ◽  
Author(s):  
Dan T. Vogl ◽  
Anas Younes ◽  
Keith Stewart ◽  
Keith William Orford ◽  
Mark Bennett ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Research Funding ◽  
Blood Platelets ◽  
Safety Data ◽  
Employment Equity ◽  
Efficacy Data ◽  
Trough Concentrations ◽  
Equity Ownership

Abstract Background: Malignant cells alter metabolism in order to enable their highly anabolic state. In addition to a massive increase in glycolysis, malignant cells frequently become dependent on glutamine to feed the TCA cycle and provide key building blocks for cell growth and proliferation. CB-839 is a first-in-class potent and selective inhibitor of glutaminase (GLS), the first step in glutamine metabolism, that has broad in vitro and in vivo anti-tumor activity in solid and heme malignancies, including multiple myeloma. GLS inhibition with CB-839 induces apoptosis and/or growth arrest in multiple myeloma and lymphoma cell lines and is synergistic with pomalidomide and lenalidomide in vitro and as well as in multiple myeloma xenograft models in vivo. Methods: CX-839-002 is an ongoing Ph1 evaluation of escalating doses of CB-839 in patients with relapsed/refractory multiple myeloma (MM) or non-Hodgkins lymphoma (NHL) with the primary objective of assessing the safety profile and selecting a recommended Phase 2 dose (RP2D). Pharmacokinetics (PK) was monitored on Days 1 and 15. Initially, CB-839 was given three times daily (TID) without food, but based on PK and safety data generated across three Ph1 studies in patients with solid and heme malignancies, the drug is now being given twice daily (BID) with meals. Results: Safety data are available for a total of 14 patients (9 MM, 4 follicular lymphoma, 1 diffuse large B cell lymphoma) that have enrolled to date during the dose escalation (100-400 mg TID and 600 mg BID). The patients have received a median of 7 prior lines of systemic therapy. CB-839 has been well tolerated with only three subjects experiencing a Gr3/4 AEs considered possibly related to study drug and there have been no discontinuations due to AEs. A similar tolerability profile has been observed across three Ph1 studies for CB-839. With a total of 119 pts treated with CB-839 across the three studies, Gr3/4 drug-related AEs have occurred in 16 subjects (13%) and 4.3% of discontinuations were due to AEs. Reversible, asymptomatic elevations in transaminases have been the primary Gr3 AEs, occurring primarily on the TID schedule in 6/59 (10.2%) pts; only one occurred among 60 pts (1.7%) receiving the BID regimen. BID dosing with 600 mg was determined to be the RP2D and combination studies with pomalidomide and dexamethasone have been initiated. The half-life of CB-839 is ~4 hr, exposure increases with dose, and trough concentrations generally remain above the target threshold of 200 ng/mL for patients receiving the RP2D. Six of 8 MM pts that received ≥ 400 mg TID achieved steady state (D15) trough concentrations above the PK target threshold while 0 of 5 pts that received ≤ 250 mg TID achieved the PK threshold. Pharmacodynamic assessment of GLS activity in MM patients was consistent with a broader PK/PD assessment (across all 3 Ph1 studies), which established clear exposure-dependent inhibition of the target in peripheral blood platelets 4 hr after the first dose of CB-839, with >90% inhibition being maintained for most patients at the RP2D. Preliminary efficacy data include confirmed stable disease in 4 of 9 evaluable MM patients. Updated efficacy data and correlative studies on clinical samples will also be presented. The first pt treated with the combination of CB-839 and pomalidomide/dexamethasone (Pd) during dose escalation received 400 mg CB-839 BID, pomalidomide at 4 mg/day (D1-21) and dexamethasone at 40 mg on Days 1, 8, 15 and 22 of each 28-day cycle. This pt had a 71% decreased in urine M-protein and an 83% reduction in serum free light chain after the first 2 cycles of treatment. This pt had 11 prior lines of therapy but not pomalidomide and had two stem cell transplants and was progressing rapidly prior to study entry. The pt has tolerated the combination well and is continuing on study. Conclusions: CB-839 has been well tolerated at and above doses that produced robust inhibition of GLS in blood platelets and in tumors. Dosing BID with food has improved the PK profile and mitigated the frequency and severity of LFT elevations, which was the primary safety signal using TID dosing. Strong preclinical combination data, an excellent clinical safety profile, and initial data with CB-839 combined with Pd provide a strong rationale for continued development of CB-839 this combination in pts with relapsed/refractory multiple myeloma. Disclosures Vogl: Constellation Pharmaceuticals: Research Funding; Calithera Biosciences: Research Funding; Celgene Corporation: Consultancy; Acetylon Pharmaceuticals, Inc.: Research Funding; Millennium Pharmaceuticals: Research Funding; GSK: Research Funding. Younes:Celgene: Honoraria; Curis: Research Funding; Sanofi-Aventis: Honoraria; Seattle Genetics: Honoraria, Research Funding; Novartis: Research Funding; Janssen: Honoraria; Takeda Millenium: Honoraria; Bristol Meyer Squibb: Honoraria; Bayer: Honoraria; Incyte: Honoraria; Johnson and Johnson: Research Funding. Orford:Calithera Biosciences: Employment, Equity Ownership. Bennett:Calithera Biosciences: Employment, Equity Ownership. Siegel:Celgene Corporation: Consultancy, Speakers Bureau; Amgen: Speakers Bureau; Takeda: Speakers Bureau; Novartis: Speakers Bureau; Merck: Speakers Bureau. Berdeja:Curis: Research Funding; Acetylon: Research Funding; Novartis: Research Funding; Janssen: Research Funding; Takeda: Research Funding; BMS: Research Funding; Array: Research Funding; MEI: Research Funding; Abbvie: Research Funding; Celgene: Research Funding; Onyx: Research Funding.

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