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.

Arylamides: Discovery of a Novel Class of Fully-Synthetic Small Molecule Heparin and LMWH Antagonists.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1096-1096
Author(s):  
Richard W. Scott ◽  
Michael J. Costanzo ◽  
Katie B. Freeman ◽  
Robert W. Kavash ◽  
Trevor M. Young ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Small Molecule ◽  
Factor Xa ◽  
Lead Compound ◽  
Hemodynamic Effects ◽  
Employment Equity ◽  
Equity Ownership ◽  
Synthetic Small Molecule

Abstract Abstract 1096 Structural studies of the binding interaction between the negatively charged heparin pentasaccharide binding site and antithrombin III suggested that an amphiphilic structure resembling features designed into compounds prepared for antimicrobial activity might also provide an efficient design basis for the development of heparin antagonists. A key design element of our antimicrobial approach at PolyMedix involves compounds exhibiting polycationic arrays which are appended to a diverse set of rigid backbones. A broad screening of the antimicrobial compound library in an enoxaparin (ENX) anti-factor Xa assay, coupled with subsequent secondary testing, identified compounds with potent and broad neutralizing activity. A lead compound (PMX-60056) was identified in one structural series, the salicylamides, and this compound has successfully completed Phase 1 human clinical studies. Recently, a new series of antagonists identified in the initial screen, the arylamides, was investigated for heparin neutralizing activities. Compounds were first tested for their ability to neutralize the anticoagulant activity of ENX, tinzaparin (TZP) or fondaparinux (FPX) in an in vitro amidolytic assay for factor Xa activity. Activated partial thromboplastin time (aPTT) assays demonstrated that the compounds were also active against unfractionated heparin (UFH) in a plasma-based clotting assay. The lead compound PMX150 was comparable to or better than PMX-60056 versus all anticoagulants tested (EC50s: UFH 0.6 uM, ENX 0.1 uM, TZP 0.1 uM, FPX 0.7 uM). Early compounds have shown an interesting structure activity relationship (SAR) which has identified specific regions of the molecule that are important for activity. Additionally, rotation thromboelastometry (ROTEM) shows that compounds are able to neutralize heparin and ENX in human whole blood, restoring normal coagulation profiles. As an initial test for safety, compounds were evaluated in hemolysis and cytotoxicity assays using isolated human erythrocytes, a transformed human liver cell line (HepG2 cells) and a mouse fibroblast cell line (NIH3T3). PMX150 was not cytotoxic (or hemolytic) at concentrations 180 fold greater than the EC50 concentrations in the anti-coagulation assays, indicating a high selectivity index between toxicity and efficacy. Furthermore, the SAR has identified synthetic strategies that should further improve this safety margin. The in vivo efficacy of PMX150 was evaluated in a rat coagulation model using ENX (2 mg/kg) administered by IV injection followed 3 minutes later by saline, protamine or PMX150. Blood was collected before dosing with ENX, and at 1, 3, 10, and 60 min after dosing, for aPTT and factor Xa analysis. PMX150 was more efficacious than protamine; neutralizing 89–100% of the ENX anti-factor Xa activity over the entire 60 minute time course. The in vivo assays concur with the in vitro studies, showing that compounds have been identified with high efficacy versus low molecular weight heparin (LMWH). Protamine, the heparin antagonist currently used in the clinic, routinely causes a transient decrease in blood pressure upon dosing. Hemodynamic effects with PMX-60056 have also been observed in human subjects in the absence of heparin. As PMX150 represents a new chemical series, investigations into potential adverse hemodynamic effects were performed. Blood pressure and heart rate were measured via arterial catheters in rats administered protamine or PMX150 by a 10 minute IV infusion. As expected, protamine displayed transient and prolonged blood pressure reductions at 8 and 16 mg/kg dosages, respectively. However, PMX150 had little to no effect on blood pressure at 8 mg/kg and only a minor transient reduction at 16 mg/kg. In conclusion, we have identified a new series of fully synthetic small molecule antagonists that display potent in vitro activity against UFH, the LMWHs, and fondaparinux. The lead compound in this series, PMX150 is able to neutralize ENX in vivo with more efficacy than protamine and, furthermore, has an improved hemodynamic profile with respect to protamine. 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.

scholarly journals Providing a Homing Receptor for CAR Engineered NK Cells - Improving Cellular Immunotherapy for B-Cell Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4547-4547
Author(s):  
Nathan Thomas Schomer ◽  
Laurent Boissel ◽  
Karen Jiang ◽  
Hans Klingemann ◽  
John H. Lee ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
K562 Cells ◽  
Clinical Grade ◽  
Employment Equity ◽  
Effector Cells ◽  
High Affinity ◽  
Equity Ownership

Abstract Background Despite some robust initial responses, anti-CD19 Chimeric Antigen Receptor (CAR) T-cell therapy can be associated with significant short-term (cytokine release syndrome) and long-term (B-cell deficiency) toxicities. CAR-engineered natural killer (NK) cells potentially provide a safer alternative while maintaining efficacy. Activated Natural Killer (aNKTM) cells are a clinical grade cell line derived from the NK-92R cell line that has demonstrated potent cytotoxicity towards a broad spectrum of malignant cell lines as well as safety and efficacy in phase I trials. Variants of the aNKcell line are currently in Phase I/II clinical trials: a CAR-expressing aNK cell line and the haNKTM cell line, which have been engineered to carry a high-affinity version of the CD16/FcγRIII receptor to allow for combination therapy with monoclonal antibodies. haNK cells have also been genetically modified to express an endoplasmic reticulum-retained version of IL-2 (ERIL-2), which provides IL-2 independence and limits IL-2 secretion to sub-physiological, safe levels. A key factor for the efficacy of cellular immunotherapies against a given target is biodistribution, which affects the local effector to target ratio. Inability to reach the tumor cells, either by lack of homing or by the accumulation of extracellular matrix (ECM) surrounding a tumor, can be responsible for the clinical failure of even the most effective CAR. The chemokines CCL19 and CCL21 drive recruitment of CCR7-expressing immune cells to secondary lymphoid organs. Engineering aNK cells to express the CCR7 receptor is likely to improve their efficacy by increasing their targeted migration to lymphoma tumor sites. Methods and Results Clinical grade aNK cells were electroporated with a non-viral vector containing the CCR7 receptor, an anti-CD19 CAR, and a high affinity CD16 receptor. To assay the migration of these engineered cell lines, a modified Boyden Chamber assay was performed using Matrigel coated Transwells. K562 cells or modified K562 cells engineered to express CCL19 (K-19) were placed in the destination chamber and CFSE-stained effector cells were placed in the top well. After 24 hours, cells in the bottom well were analyzed by flow cytometry to measure the number of effectors which had migrated through the Matrigel (Fig 1a). The excellent activity of the CAR in stably transfected cells was confirmed against SUP-B15 cells (aNK-resistant), while the ADCC activity was tested against a SUP-B15 variant expressing CD20, but engineered to lack the CD19 antigen (Sup-B15 CD19-, CD20+). Migration towards human lymph node chemokine CCL19 was also tested in vivo in NSG mice with bilateral subcutaneous tumors - with parental K562 in one flank and K-19 tumors on the contralateral flank. CFSE-stained effector cells were delivered via tail vein injection once average tumor size reached 100mm3 and following randomization. Tumors were then harvested at multiple time points, dissociated, and the number of infiltrating effectors in each tumor compared by flow cytometric analysis. (Fig 1b). In testing, monoclonal cell lines expressing all components of the polycistronic system displayed preferential migration towards CCR7 chemokines both in vitro and in vivo, as well as robust cytotoxicity vs. K562 (92.4% +/- 2.4% at 5:1 E:T), Sup-B15(97% +/- 0.6% at 5:1 E:T), and Sup-B15(CD19-, CD20+) when pre-incubated with Rituximab(83.2% +/- 2.8% at 5:1 E:T) but not with control antibody Trastuzumab (22.3% +/- 1.1% at 5:1 E:T) in standard cytotoxicity and ADCC assays. Conclusion We show here that the incorporation of a CCR7 receptor into an off the shelf CAR engineered NK cell line improves their homing towards lymph node chemokines both in vitro and in vivo. This improved homing should result in a greater ratio of effector to target in lymphoid tissue, and maximize the immunogenic cell death. Disclosures Schomer: NantKwest, Inc.: Employment, Equity Ownership. Boissel:NantKwest, Inc.: Employment, Equity Ownership. Jiang:NantKwest, Inc.: Employment, Equity Ownership. Klingemann:NantKwest, INc.: Employment, Equity Ownership, Patents & Royalties. Lee:NantKwest, Inc.: Employment, Equity Ownership. Soon-Shiong:NantKwest, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

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 (>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.

Distinction of NPY receptors in vitro and in vivo. II. Differential effects of NPY and NPY-(18-36)

1990 ◽  
Vol 259 (1) ◽  
pp. H174-H180 ◽  
Author(s):  
N. A. Scott ◽  
M. C. Michel ◽  
J. H. Boublik ◽  
J. E. Rivier ◽  
S. Motomura ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Receptor Subtypes ◽  
Base Line ◽  
Hemodynamic Effects ◽  
Vascular Effects ◽  
Inotropic Effects ◽  
Npy Receptor ◽  
Negative Inotropic Effects

We have studied the hemodynamic effects of neuropeptide Y (NPY) and its COOH-terminal fragment NPY-(18–36) in conscious rats. Intra-arterial injection of NPY rapidly elevated systemic vascular resistance (SVR), which remained high for greater than 30 min. Cardiac output (CO) decreased, and it remained low for greater than 30 min. Accordingly, blood pressure rose only transiently and returned to base-line values within 5 min. The reduction of CO could be attributed to a decreased stroke volume with an only marginal reduction of heart rate. Thus a direct cardiodepressive effect of NPY rather than baroreflex activation appears to be the major cause of the reduced CO. In vitro experiments excluded the possibility that NPY has direct negative inotropic effects and suggest that its cardiodepressive action is caused by coronary vasoconstriction or by presynaptic inhibition of norepinephrine release. Intra-arterial injections of NPY-(18-36) caused different hemodynamic effects. NPY-(18–36) decreased CO in a manner similar to that seen with NPY but initially did not elevate SVR, resulting overall in a reduced blood pressure. Only later, when blood pressure was reduced, was an elevation of SVR observed, which could be associated with increased plasma levels of catecholamines, angiotensin II, vasopressin, and NPY. Thus NPY-(18–36) mimics the cardiac effects of NPY but does not elicit its vascular effects. As NPY-(18–36) discriminates between NPY receptor subtypes in vitro, we conclude that the cardiac and vascular effects of NPY are mediated by distinct receptor subtypes.

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.

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.

Coltuximab Ravtansine (SAR3419) Demonstrates Enhanced Activity in Combination with Bendamustine, Gemcitabine and Novel Targeted Agents Such As PI3K Inhibitors in Pre-Clinical Models of Relapsed and/or Refractory Non-Hodgkins Lymphoma (NHL)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5125-5125
Author(s):  
Callum M Sloss ◽  
Katie O'Callaghan ◽  
Jutta Deckert ◽  
Jenny Tsui ◽  
Leanne Lanieri ◽  
...  
Keyword(s):  
Cell Lines ◽  
Single Agent ◽  
Targeted Agents ◽  
Employment Equity ◽  
Pi3k Inhibitors ◽  
Xenograft Models ◽  
Equity Ownership ◽  
Chemotherapy Agents

Abstract Introduction: Relapsed/refractory B-cell NHL remains an area of significant medical need. CD19 is broadly expressed on B-cell malignancies making it an ideal target for antibody-drug conjugate (ADC) based therapy. Coltuximab ravtansine is a CD19-targeting ADC consisting of a CD19-targeting antibody conjugated to the maytansinoid anti-mitotic DM4. In preclinical studies, coltuximab ravtansine has shown potent, targeted activity against NHL cell lines and xenograft models. In early clinical trials, it has been well tolerated and has shown promising signs of efficacy as both a single agent and in combination with rituximab. In the STARLYTE Phase 2 trial coltuximab ravtansine monotherapy resulted in an ORR of 44% in R/R-DLBCL that included an ORR of 21% in hard-to-treat primary refractory patients (NCT01472887). Here we describe studies aimed at the identification of combination partners for coltuximab ravtansine to further optimize clinical benefit to R/R-NHL patients. We are employing a dual approach where we investigate combination of coltuximab ravtansine with multiple, novel targeted therapy partners whilst in parallel also investigating the combination of coltuximab ravtansine with chemotherapies commonly used in the late stage R/R-NHL setting. Methods: Coltuximab ravtansine and the DM4 payload were evaluated in a high throughput screen both as single agents and in combination with a selection of novel, emerging targeted agents across a panel of twenty NHL cell lines. The combinations were evaluated in a dose-response matrix and a statistical method was used to identify combination synergies significantly superseding baseline additivity values. The in vivo efficacy of coltuximab ravtansine was additionally assessed in combination with various clinically relevant chemotherapy agents in subcutaneous xenograft models of NHL. Results: Coltuximab ravtansine and DM4 both showed potent single agent activity against the entire panel of NHL cell lines with median GI50's of 770pM and 100pM, respectively. We observed a significant correlation in the cell line sensitivity of the two compounds suggesting that sensitivity to coltuximab ravtansine is driven, at least in part, by inherent sensitivity of cells to the cytotoxic effects of the DM4 payload. In vitro combination studies for coltuximab ravtansine were performed to identify targets or pathways that result in the most prominent combination effects across the cell line panel. Analysis of the in vitro combination dose-matrix revealed particularly strong synergy between coltuximab ravtansine and various inhibitors of the PI3K/AKT/mTOR axis. Studies to examine the synergism between coltuximab ravtansine and PI3K inhibitors in in vivo models of NHL are ongoing. In order to further determine the utility of coltuximab ravtansine as part of a potential combination regimen for the treatment of R/R-NHL, we assessed the combination of coltuximab ravtansine with the chemotherapy agents bendamustine and gemcitabine in vivo. As gemcitabine is typically used in combination we assessed the efficacy of a coltuximab ravtansine with rituximab and gemcitabine in vivo. In both cases the combination with coltuximab ravtansine was significantly more efficacious than the standard-of-care alone arms. Conclusions: Coltuximab ravtansine demonstrates synergistic activity in combination with multiple PI3K pathway inhibitors across a large panel of NHL cell lines. Additionally, we have shown that combination of coltuximab ravtansine with clinically relevant late stage treatments such as bendamustine and rituximab + gemcitabine is more efficacious than the chemotherapy regimens alone. These results support the continued development of coltuximab ravtansine in R/R-NHL in combination with chemotherapy regimens and suggest that a combination of coltuximab ravtansine with PI3K inhibitors may also be of interest in the clinical setting. Disclosures Sloss: ImmunoGen, Inc.: Employment, Equity Ownership. O'Callaghan:ImmunoGen, Inc.: Employment, Equity Ownership. Deckert:ImmunoGen, Inc.: Employment, Equity Ownership. Tsui:ImmunoGen, Inc.: Employment, Equity Ownership. Lanieri:ImmunoGen, Inc.: Employment, Equity Ownership. Romanelli:ImmunoGen, Inc.: Employment, Equity Ownership.

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