The Combination of Entospletinib and Vincristine Demonstrates Synergistic Activity in a Broad Panel of Hematological Cancer Cell Lines and Anti-Tumor Efficacy in a DLBCL Xenograft Model

Abstract Background Entospletinib (GS-9973) selectively inhibits spleen tyrosine kinase (SYK), a critical signaling component of the BCR pathway that is expressed primarily in cells of hematopoietic lineage including normal and malignant B-lymphocytes. Entospletinib is currently in phase II clinical trials, where it has demonstrated both a high degree of safety as well as efficacy against chronic lymphocytic leukemia (Sharman, J., et al. Blood, 2015) and other B cell malignancies. Despite these successes, new therapeutic options, including combinations with standard of care agents, are needed in order to achieve the goal of curing disease through finite treatment. We show here that the combination of entospletinib and vincristine causes synergistic apoptosis in vitro in a broad panel of cell lines derived from hematological cancers including diffuse large B cell lymphoma (DLBCL), acute lymphocytic leukemia, follicular lymphom), multiple myeloma, and acute myelogenous leukemia. We also evaluated and compared the in vivo efficacy of entospletinib and vincristine as singe agents and in combination in a DLBCL tumor xenograft model using the SU-DHL-10 cell line. Methods In vitro growth inhibition of a panel of malignant hematological cell lines was assessed using CellTiter-Glo™ Assay (Promega) after 72h incubation with entospletinib or vincristine alone or in combination. Synergy was evaluated using the Bliss model of independence (Meletiadis, J., et al., Med Mycol, 2005). In vivo, SU-DHL-10 cells (5 x 106 cells) were implanted subcutaneously in the axilla in male SCID beige mice. All mice were sorted into study groups on Day 16 such that each group's mean tumor volume fell within 10% of the overall mean (197mm3). Dosing was initiated on Day 16 and animals were dosed for 17 days. Plasma concentrations of entospletinib and vincristine were assessed on Day 19, and the entospletinib 75 mg/kg dose was lowered on Day 22 to 50 mg/kg to approximate the human achievable SYK target coverage of EC80. Efficacy and tolerability were evaluated by tumor measurements and body weight monitored three times weekly. Tumor burden data were analyzed by the application of a two-way analysis of variance (ANOVA), with post-hoc analysis. Results In vitro combinations of entospletinib with low concentrations of vincristine resulted in marked inhibition of cell proliferation and induction of apoptosis in a broad panel of 19 tumor cell lines representing major B cell malignancies including DLBCL. The combination of entospletinib with vincristine had a profound inhibitory effect on proliferation in all subtypes of DLBCL. Entospletinib was evaluated at a concentration equivalent to the Cminof the clinical dose and vincristine was used at concentrations (≤ 10 nM) that had little to no significant single agent effect in these cell lines. In vivo in a SU-DHL-10 xenograft model, entospletinib dosed alone at 25 or 75/50 mg/kg significantly inhibited tumor growth, causing 39% and 20% tumor growth inhibition (TGI), respectively, compared to the vehicle-treated control group. Vincristine administered at either 0.15 and 0.5 mg/kg Q7D x 3 also resulted in significant TGI (42% and 85% TGI, respectively). The addition of entospletinib (75/50 mg/kg) to 0.5 mg/kg or 0.15 mg/kg vincristine resulted in a significant increase in TGI from 85% to 96% (p= 0.001) and 42% to 71% (p< 0.0001), respectively. The addition of entospletinib (25 mg/kg) to vincristine did not significantly increase the tumor growth inhibition. While the groups receiving either entospletinib or vincristine as single agents had no complete or partial tumor regression, 50% of the mice receiving the combination of 75/50 mg/kg entospletinib with 0.5 mg/kg vincristine had partial responses, 8% had complete regression and 8% were tumor free at the end of study (Figure 1). Conclusion Entospletinib and vincristine demonstrated efficacy and tolerability both alone and in combination in the SU-DHL-10 DLBCL cell line xenograft model in SCID beige mice. Vincristine combinations with entospletinib showed significantly greater efficacy than vincristine alone. These data support the further clinical development of entospletinib in combination with vincristine for the treatment of DLBCL. a ENTO: PO: Q12H x 2 (Day 16-32) b VCR: IV: Q7D x 3 (Days 18, 25, 32) Figure 1. Tumor Regressions in an Entospletinib/ Vincristine Treated Murine DLBCL Xenograft Figure 1. Tumor Regressions in an Entospletinib/ Vincristine Treated Murine DLBCL Xenograft Disclosures Axelrod: Gilead Sciences: Employment, Equity Ownership. Fowles:Gilead Sciences: Employment, Equity Ownership. Silverman:Gilead Sciences: Employment, Equity Ownership. Clarke:Gilead Sciences: Employment, Equity Ownership. Tang:Gilead Sciences: Employment, Equity Ownership. Rousseau:Gilead Sciences: Employment, Equity Ownership. Webb:Gilead Sciences: Employment, Equity Ownership. Di Paolo:Gilead Sciences: Employment, Equity Ownership.

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Pre-Clinical Development of Adct-402, a Novel Pyrrolobenzodiazepine (PBD)-Based Antibody Drug Conjugate (ADC) Targeting CD19-Expressing B-Cell Malignancies

Blood ◽

10.1182/blood.v126.23.1564.1564 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1564-1564 ◽

Cited By ~ 7

Author(s):

Francesca Zammarchi ◽

David G. Williams ◽

Lauren Adams ◽

Karin Havenith ◽

Simon Chivers ◽

...

Keyword(s):

Single Dose ◽

B Cell ◽

Cell Lines ◽

Xenograft Model ◽

B Cell Malignancies ◽

Employment Equity ◽

Equity Ownership ◽

Anti Tumor Activity

Abstract Human CD19 antigen is a 95 kilodalton type I transmembrane glycoprotein belonging to the immunoglobulin superfamily (Wang, Wei, & Liu, 2012). The role of CD19, both in health and disease, is well studied, and the therapeutic efficacy and safety of CD19 modulation have been well defined over several decades (Scheuermann & Racila, 1995). In normal human tissue, expression of CD19 is limited to the various stages of B-cell development and differentiation (except plasma cells) and its expression is maintained on the majority of B-cell malignancies, including B-cell leukemia and non-Hodgkin lymphomas of B-cell origin. CD19 has rapid internalization kinetics and it is not shed into the circulation (Blanc et al., 2011; Gerber et al., 2009). All these features make CD19 an attractive target for the development of an ADC to treat B-cell malignancies. ADCT-402 is an ADC composed of a humanized antibody directed against human CD19, stochastically conjugated via a valine-alanine cleavable, maleimide linker to a PBD dimer cytotoxin. PBD dimers are highly efficient anticancer drugs that covalently bind in the minor groove of DNA and form cytotoxic DNA interstrand cross-links. The average drug to antibody ratio of ADCT-402 is 2.3 ± 0.3, as shown by hydrophobic interaction chromatography and reverse-phase HPLC. In vitro, ADCT-402 demonstrated potent cytotoxicity in a panel of human-derived cell lines of differing levels of CD19, while its potency was strongly reduced in CD19-negative cell lines. In vivo, ADCT-402 demonstrated dose-dependent anti-tumor activity in a subcutaneously implanted human Burkitt's lymphoma-derived Ramos xenograft model, where a single dose at 0.33 mg/kg induced significantly delayed tumor growth compared to the vehicle-treated mice and at 0.66 mg/kg and 1 mg/kg gave 4/10 and 10/10 tumor-free survivors, respectively. In the same model, ADCT-402 showed remarkably superior anti-tumor activity compared to both maytansinoid- and auristatin-based CD19-targeting ADCs, when they were tested at the same dose and schedule (1 mg/kg, single dose). Moreover, ADCT-402 mediated an impressive increase in survival compared to both vehicle-treated and isotype control ADC-treated mice in the disseminated Ramos xenograft model when tested as a single dose at 0.33 mg/kg or 1 mg/kg. For example, a single dose of ADCT-402 at 1 mg/kg resulted in 10/10 survivors at day 91, while there were 0/10 survivors at day 19 in the group of animals treated with either the vehicle control or with a single dose of the non-binding, control ADC at 1 mg/kg. In rat, a single dose of ADCT-402 at 2 mg/kg was well tolerated with no adverse signs or hematologic effects. Altogether, these data show the potent and specific anti-tumor activity of ADCT-402 against CD19-expressing B-cell malignancies, both in vitro and in vivo, and warrant further development of this ADC into the clinic. Disclosures Zammarchi: ADC Therapeutics: Employment. Williams:Spirogen/Medimmune: Employment. Adams:Spirogen/Medimmune: Employment, Equity Ownership. Havenith:ADC Therapeutics: Employment. Chivers:ADC Therapeutics: Employment. D'Hooge:Spirogen/Medimmune: Employment, Equity Ownership. Howard:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Patents & Royalties. Hartley:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. van Berkel:ADC Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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

Blood ◽

10.1182/blood-2019-126298 ◽

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.

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The Bruton's Tyrosine Kinase (BTK) Inhibitor PCI-32765 Inhibits Growth of ABC DLBCL Tumors In Vivo and in Vitro by Preventing Activation of Pro-Survival NF-κB pathways

Blood ◽

10.1182/blood.v118.21.4969.4969 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4969-4969 ◽

Cited By ~ 1

Author(s):

Sriram Balasubramanian ◽

Richard Crowley ◽

Mint Sirisawad ◽

Patti Thiemann ◽

Jun Chen ◽

...

Keyword(s):

Tyrosine Kinase ◽

B Cell ◽

Signaling Pathways ◽

Cell Lines ◽

Active Site ◽

Employment Equity ◽

Equity Ownership ◽

Nuclear Relocation

Abstract Abstract 4969 Bruton's tyrosine kinase (BTK), a Tec family kinase essential to signaling via the B-cell Receptor (BCR) has emerged as a very promising drug target in lymphoma. PCI-32765, a potent and selective covalently-bound small molecule inhibitor of BTK (Honigberg, Proc. Natl Acad. Sci. USA 107:13075, 2010), has demonstrated promising clinical activity in a number of Phase I and II clinical trials in patients with chronic lymphocytic leukemia (CLL) (Byrd et al., ASCO 2011 Annual Meeting Abstracts, JCO29 (15): 8012, 2010) and several subtypes of B-cell Non-Hodgkin's Lymphoma (NHL), including diffuse large B-cell lymphoma (DLBCL). Many cell lines derived from B-cell malignancies do not show in vitro growth inhibition by BTK inhibitors, perhaps because most established B-cell lines no longer depend on BCR stimulation for survival. However, some cell lines derived from the aggressive ABC (activated B-cell) subtype of DLBCL such as OCI-Ly10 and TMD8 are very sensitive to BTK inhibition by PCI-32765 (Davis et al, Nature463: 88, 2010). We show here that both of these cell lines are potently and irreversibly inhibited by PCI-32765 (EC50 continuous exposure < 10 nM and EC50 following a 1-hr pulse < 50 nM). Interestingly, 2 other ABC-DLBCL cell lines, OCI-Ly3 and HBL-1, in which the NF-κB pathway is activated by a mutation in CARD11 which is downstream of BTK, are not inhibited by PCI-32765. Both sensitive and resistant cells express Btk at similar levels, and the active site of BTK is fully occupied in the presence of low concentrations of the inhibitor as shown using a fluorescently-labeled affinity probe. We show that the sensitive TMD8 and OCI-Ly10 cells have chronically active BCR signaling to NF-κB which is dose-dependently inhibited by PCI-32765, as measured by comparing the phosphorylation and nuclear relocation of NF-κB p65 subunit. These cells also show inhibition of other major signaling pathways downstream of the BCR, including p-ERK, p-AKT, p-PLCγ and intracellular calcium flux, ultimately leading to NF-κB activation. The non-responsive OCI-Ly3 cells do not show inhibition of NF-κB p65 phosphorylation or nuclear relocation, although Btk is present and active, as shown by the probe assay and inhibition of p-AKT by PCI-32765. Furthermore, whole genome expression analysis reveals inhibition of mRNA expression of several NF-κB target genes in OCI-Ly10, particularly c-Myc, NF-κB subunits and several chemokines and cytokines, leading to downregulation of STAT3 signaling, NFAT signaling, cell cycle and cytokine-chemokine signaling pathways, none of which was observed in OCI-Ly3 cells. In vivo, PCI-32765 dose-dependently inhibited tumor growth in mice carrying OCI-Ly10 (and TMD8, ongoing) xenografts. Analysis of these tumors exhibited dose-dependent occupancy of the BTK active site as well as inhibition of signaling pathways. These results demonstrate the mechanistic basis of PCI-32765 inhibitory activity in ABC-DLBCL and support the ongoing clinical investigation of the therapeutic use of PCI-32765 in patients with this devastating disease. Disclosures: Balasubramanian: Pharmacyclics: Employment, Equity Ownership. Crowley:Pharmacyclics: Employment, Equity Ownership. Sirisawad:Pharmacyclics: Employment, Equity Ownership. Thiemann:Pharmacyclics: Employment, Equity Ownership. Chen:Pharmacyclics: Employment, Equity Ownership. Buggy:Pharmacyclics, Inc.: Employment, Equity Ownership.

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Preclinical Development of LNA Antimir-155 (MRG-106) in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v126.23.3802.3802 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3802-3802 ◽

Cited By ~ 4

Author(s):

Nina C. Zitzer ◽

Parvathi Ranganathan ◽

Brent A Dickinson ◽

Aimee L Jackson ◽

David M Rodman ◽

...

Keyword(s):

Cell Lines ◽

Cellular Proliferation ◽

Phase 1 ◽

Xenograft Model ◽

Fold Increase ◽

Employment Equity ◽

Biodistribution Studies ◽

Equity Ownership

Abstract Introduction MicroRNAs (miRs) are deregulated in AML and play a key role in leukemogenesis. MiR-155 is one of the most frequently overexpressed miRs in AML. Higher expression of miR-155 is associated with FLT3 internal tandem duplication (FLT3-ITD) and is associated with worse outcome, independent of FLT3-ITD status. Preliminary data shows that silencing of miR-155 induces strong antileukemic effects in AML cell lines. Altogether these data support a therapeutic role for miR-155 antagonism in AML. Here, we show the in vitro and in vivo activity of MRG-106, a novel LNA antimiR-155 compound that we are developing as a potential treatment for hematological malignancies. Methods Unconjugated, LNA-modified oligonucleotide against miR-155 (MRG-106) was developed by miRagen Therapeutics, Inc. MRG-106 was evaluated in FLT3-ITD+ AML cell lines and primary FLT3-ITD+ AML samples for impact on apoptosis and cellular proliferation using Annexin V and MTS assays. Predicted and validated targets of miR-155 were measured by qPCR and Western Blotting to assess the efficacy of miR-155 silencing. The in vivo antileukemic effect of MRG-106 was evaluated in NOD/SCID gamma mice engrafted with MV4-11 AML cells that have elevated miR-155 expression. One week after leukemic cell inoculation, the mice were separated in 3 cohorts and received either MRG-106 (n=12); LNA-scramble control (n=12); or saline (n=6). Results Inhibition of miR-155 decreased cell proliferation in MV-4-11 and MOLM-13 cells at 48hrs (Absorbance 450 nM (A450nM): 0.5 and 0.4 vs controls 2.4 and 2.5, respectively, p<0.01). These effects were associated with increased apoptosis (2-fold increase in Annexin staining) and re-expression of the validated miR-155 target WEE1 protein. MRG-106 produced a 4-6 fold increase in apoptosis in primary samples from 3 newly diagnosed AML patients with FLT3 -ITD cytogenetically normal AML (p<0.01). In addition, inhibition of miR-155 reduced the colony forming ability of the primary blasts (average # of colonies; controls=90 vs. MRG-106=55,p<0.01). In an MV4-11 xenograft model, MRG-106 significantly increased survival compared to controls (p<0.01). Biodistribution studies confirmed adequate MRG-106 distribution to bone marrow. GLP preclinical safety studies have been completed in rats and non-human primates, demonstrating an acceptable safety profile for MRG-106. Conclusions Inhibition of miR-155 in AML cell lines and primary AML samples in vitro and in vivo induces significant antileukemic effects. These studies validate miR-155 as a therapeutic target in AML and support the testing of MRG-106 in AML patients in the context of a phase 1 clinical trial. Disclosures Dickinson: miRagen Therapeutics: Employment, Equity Ownership. Jackson:miRagen Therapeutics: Employment, Equity Ownership. Rodman:miRagen Therapeutics: Employment, Equity Ownership.

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Targeted Nanoparticles for the Delivery of Antagomir17: New Approach for the Treatment of Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v126.23.5293.5293 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5293-5293

Author(s):

Sara Capolla ◽

Riccardo Bomben ◽

Sonia Zorzet ◽

Ruben Spretz ◽

Tiziana D'Agaro ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Cell Lines ◽

Therapeutic Approach ◽

Lymphocytic Leukemia ◽

Employment Equity ◽

Tumor Mass ◽

Equity Ownership

Abstract MicroRNAs are associated with prognosis, progression and drug resistance of Chronic Lymphocytic Leukemia (CLL). Among them, microRNA from the miR-17~92 family were demonstrated to affect CLL cells' growth and survival through the inhibition of the tumor suppressor PTEN and the proapoptotic protein Bim. Previous studies demonstrated the effect of an antagomiR17 in the reduction of leukemia cells' growth both in vitro and in vivo. However, despite these results, the in vivo clearance of antagomiR17 is strictly related to its small dimension which induced a rapid renal elimination preventing significant tumor accumulation and, as a consequence, antagomiR17 efficacy. To overcome this efficacy problem, antagomiR17 was loaded inside biodegradable and biocompatible nanoparticles (BNPs) composed on polyethylene glycol (PEG), polycaprolactone (PCL) and polylactic acid (PLA). The advantages of using BNPs include the significant reduction of toxicity; the potential to load different types of drugs; the improved bioavailability of the payload and cell internalization. Moreover, to ensure the specific binding to cancer B cells, BNPs were conjugated to the antiCD20 antibody Rituximab, targeting leukemia B-cells. Thus, antagomiR17-loaded BNPs efficacy was evaluated by in vitro and in vivo assays.Initially, studies made by quantitative PCR demonstrated that antagomiR17 was able to reduce miR-17 expression in two leukemia cell lines, EHEB and MEC1 cells. Comprehensively, EHEB represents p53wild type patient's derived cells, which developed an indolent model of CLL, while MEC1 represents p53mut/del cells, which usually develop an aggressive model of the disease. Both cell lines were incubated with antagomiR17-loaded BNPs and a significant reduction of miR-17 expression was detected after 24 hours with a complete depletion of miR-17 after 72 hours. Cells growth was also evaluated in vitro affirming that antagomiR17 decreased the proliferation of both MEC1 and EHEB cells in comparison to non-treated cells. Our data also demonstrated that antagomiR17 maintained activity even after encapsulation inside BNPs. For what concerns in vivo studies, a localized model of B-cell malignancy was induced injecting MEC1 cells subcutaneously into SCID mice, causing the formation of a localized tumor mass at the site of cell injection after 3-4 weeks. When the tumor mass reached 300mg in weight, animals were treated once with an intraperitoneal injection of BNPs containing 26μg of antagomiR17 with or without antiCD20 antibody on the BNP surface. The tumor mass dimension and mice's survival were evaluated. Specifically, the decreased in vivo growth of MEC1 cells was more evident after a treatment with antiCD20-conjugated BNPs in respect to unconjugated BNPs (2500 vs 3500 mg), confirming again the importance of the antiCD20 antibody in BNPs homing on B cells. Furthermore, the evaluation of mice survival also confirmed the data; mice treated with antiCD20-BNPs showed an increased survival in respect to mice injected with unconjugated BNPs (37 vs 21 days). In conclusion, our data confirms the efficacy of antiCD20 polymeric nanoparticles in the delivery of antagomiR17 into tumor B-cells. This new therapeutic approach allows to selectively restore miR17 levels and to reduce tumor cell growth, both in vitro and in vivo. Altogether, these results provide a new path for the specific delivery of small nucleic acids like miRNA, antagomiR or siRNA into tumor B-cells and offers the development of future drug combinations with standard therapeutic approach in the same targeted nanostructure. Disclosures Spretz: LNK Chemosolutions LLC: Employment, Equity Ownership. Larsen:LNK Chemosolutions LLC, Biotarget Inc.: Employment, Equity Ownership. Nunez:LNK Chemosolutions LLC, Biotarget Inc.: Employment, Equity Ownership.

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Targeted Degradation of IRAK4 Protein Via Heterobifunctional Small Molecules for Treatment of MYD88 Mutant Lymphoma

Blood ◽

10.1182/blood-2018-99-120148 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2953-2953

Author(s):

Joseph Kelleher ◽

Laurent Audoly ◽

Veronica Campbell ◽

Jesse Chen ◽

Nan Ji ◽

...

Keyword(s):

Tumor Growth ◽

B Cell ◽

Xenograft Model ◽

B Cell Lymphoma ◽

Scaffolding Protein ◽

Tumor Growth Rate ◽

Employment Equity ◽

Recurrent Mutations ◽

Equity Ownership

Abstract Recurrent mutations in the scaffolding protein MYD88 are present in 30-40% of activated B cell diffuse lymphocytic B cell lymphoma (ABC-DLBCL)(Ngo et al. Nature 2011). MYD88 links activated interleukin 1 receptor (IL1R) and Toll-like receptors (TLRs) to downstream effectors by nucleating assembly of the Myddosome, a multi-protein complex containing MYD88, the protein kinases IRAK4 and IRAK1 and the pseudokinase IRAK2, via oligomerization of the N-terminal Death Domains in each of these proteins (Motshwene et al. JBC 2009; Lin, Lo and Wu. Nature 2010). The most prevalent MYD88 mutation, L265P, constitutively activates assembly of the Myddosome, causing IRAK4-dependent NFκB and MAP kinase signaling and leading to lymphoma survival and proliferation (Ngo et al. Nature 2011). Constitutive activation of the Myddosome has proven to be difficult to drug. MYD88 and IRAK2 lack enzymatic activity and cannot be targeted by conventional small molecule catalytic inhibitors. There are no IRAK1 inhibitors in clinical trials. Specific inhibitors of the kinase activity of IRAK4 have shown limited activity as single agents in preclinical models of MYD88 mutant lymphoma (Lim et al. Blood 2012; Booher et al. Blood 2014). This is consistent with the observations that there is kinase-independent activity of IRAK4 in mediating downstream signals, such as those leading to induction of NFκB (Qin et al. JBC 2004; Fraczek et al. JBC 2008; Sun et al. Sci Signal. 2016). IRAK4 kinase inhibitors thus might not provide optimal blockade of NFκB-dependent tumor survival signals driven by MYD88 mutations in ABC-DLBCL. Kymera Therapeutics is advancing a new class of drugs based on targeted protein degradation: heterobifunctional molecules that recruit disease-causing proteins to specific E3 ubiquitin ligases, resulting in their ubiquitination and subsequent degradation. We have developed a portfolio of potent and specific degraders of IRAK4 with drug-like properties Our lead molecules cause potent and specific degradation of IRAK4 in the ABC-DLBCL cell lines OCI-LY10 (MYD88 L265P) and U2932 (MYD88 WT) while showing selective cytotoxic effects on OCI-LY10 vs U2932. The apoptotic response is triggered within 24-48 h in OCI-LY10, demonstrated by appearance of cleaved caspase 3 and PARP. Quantitative proteomic studies show selective degradation of IRAK4 vs five to six thousand other proteins detected in OCI-LY10. IRAK4 degraders were tested in an OCI-LY10 xenograft model with daily dosing for 28 days, to determine the level of IRAK4 degradation and impact of that degradation on tumor growth in vivo. We observed that degraders mediated decrease in IRAK4 in vivo and produced a statistically significant decrease in tumor growth rate. Here we will present in vitro and in vivo activity and mechanism of action of our most advanced IRAK4 degraders. These IRAK4 degrader molecules represent a new therapeutic modality and provide an exciting opportunity to treat MYD88 mutation-driven ABC-DLBCL. Disclosures Kelleher: Kymera Therapeutics: Employment, Equity Ownership. Audoly:Kymera Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Campbell:Kymera Therapeutics: Employment, Equity Ownership. Chen:Kymera Therapeutics: Employment, Equity Ownership. Ji:Kymera Therapeutics: Employment, Equity Ownership. Kamadurai:Kymera Therapeutics: Employment, Equity Ownership. Li:Kymera Therapeutics: Employment, Equity Ownership. Loh:Kymera Therapeutics: Employment, Equity Ownership. Rong:Kymera Therapeutics: Employment, Equity Ownership. Vigil:Kymera Therapeutics: Employment, Equity Ownership. Weiss:Kymera Therapeutics: Employment, Equity Ownership. Yuan:Kymera Therapeutics: Employment, Equity Ownership. Zhang:Kymera Therapeutics: Employment, Equity Ownership. Mainolfi:Kymera Therapeutics: Employment, Equity Ownership.

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First Selective KIT D816V Inhibitor for Patients with Systemic Mastocytosis

Blood ◽

10.1182/blood.v124.21.3217.3217 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3217-3217 ◽

Cited By ~ 7

Author(s):

Erica K. Evans ◽

Brian L. Hodous ◽

Alexandra Gardino ◽

Julia Zhu ◽

Adam Shutes ◽

...

Keyword(s):

Mast Cells ◽

Tumor Growth ◽

Growth Inhibition ◽

Systemic Mastocytosis ◽

Tumor Growth Inhibition ◽

Employment Equity ◽

Equity Ownership ◽

Kit D816v

Abstract Systemic mastocytosis is a disease characterized by the abnormal proliferation and accumulation of mast cells. In aggressive cases, these mast cells accumulate in organs such as bone marrow, liver and spleen and result in compromised organ function with average patient survival only 3 to 5 years after diagnosis. The mast cells of nearly all systemic mastocytosis patients harbor a heterozygous D816V mutation in the activation loop of KIT conferring constitutive, ligand-independent activation of this receptor tyrosine kinase, suggesting this mutation is a driver of disease. While KIT D816V can be targeted by small molecules such as dasatinib and midostaurin, these agents have activity against many human kinases resulting in dose limiting toxicities in the clinic that prevent complete suppression of KIT D816V activity in vivo. In vitro, their potent activity against multiple kinases leads to uncertainties regarding their mechanism of action. Thus far, selective inhibition of the KIT D816V mutation has not been achieved. However starting with a novel chemical library optimized for kinase selectivity, we have identified BLU-285, a small molecule inhibitor targeting KIT exon 17 mutants including the activated KIT D816V kinase. BLU-285 potently disrupts KIT D816V oncogenic signaling as measured by inhibition of both KIT D816V autophosphorylation and phosphorylation of the downstream substrates Akt and Stat3 in the human mast cell leukemia cell line HMC1.2. In vitro, BLU-285 inhibits proliferation and induces apoptosis in the mouse mastocytoma cell line P815. In vivo, BLU-285 is a well-tolerated, orally bioavailable agent that achieves dose dependent tumor growth inhibition in a P815 mouse xenograft model with tumor regression observed at 30 mg/kg once daily dosing. Tumor growth inhibition correlates with inhibition of KIT autophosphorylation; greater than 80% target suppression throughout the 24-hour dosing period is required for effective tumor growth inhibition. Prolonged target suppression is achievable with BLU-285 but not dasatinib, even when dosed at the MTD in mouse. Furthermore, to more closely mimic the nature of systemic mastocytosis, we have developed a disseminated model of disease whereby the in vivo growth of P815-luciferase expressing cells inoculated intravenously can be measured by whole body bioluminescence. Treatment of mice with systemic disease leads to dose dependent inhibition of disease, with a 3-fold increase in survival time when dosed 30 mg/kg QD. In addition, as anticipated by its selectivity profile, BLU-285 is very well tolerated in vivo with no impact on body weight at efficacious doses. Our data demonstrate that selective inhibition of KIT D816V with BLU-285 achieves complete and prolonged inactivation of the disease-driving kinase and suggests that BLU-285 may provide a compelling new therapy for patients with systemic mastocytosis. Disclosures Evans: Blueprint Medicines: Employment, Equity Ownership. Hodous:Blueprint Medicines: Employment, Equity Ownership. Gardino:Blueprint Medicines: Employment, Equity Ownership. Zhu:Blueprint Medicines: Employment, Equity Ownership. Shutes:Blueprint Medicines: Employment, Equity Ownership. Davis:Blueprint Medicines: Employment, Equity Ownership. Kim:Blueprint Medicines: Employment, Equity Ownership. Wilson:Blueprint Medicines: Employment, Equity Ownership. Wilson:Blueprint Medicines: Employment, Equity Ownership. Zhang:Blueprint Medicines: Employment, Equity Ownership. Kohl:Blueprint Medicines: Employment, Equity Ownership. Guzi:Blueprint Medicines: Employment, Equity Ownership. Lengauer:Blueprint Medicines: Employment, Equity Ownership.

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

Blood ◽

10.1182/blood.v114.22.2744.2744 ◽

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.

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The Novel mTOR Kinase Inhibitor CC-223 Demonstrates Significant Activity In In Vitro Models Of Multiple Myeloma (MM), Both As a Single Agent and In Combination With The Approved Agents, Dexamethasone, Lenalidomide and Pomalidomide

Blood ◽

10.1182/blood.v122.21.3160.3160 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3160-3160 ◽

Cited By ~ 1

Author(s):

Emily Rychak ◽

Derek Mendy ◽

Karen Miller ◽

Jim Leisten ◽

Rama Krishna Narla ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Death ◽

Tumor Growth ◽

Cell Lines ◽

Combination Index ◽

Synergistic Effects ◽

Standard Of Care ◽

Employment Equity ◽

Equity Ownership

Abstract Over expression of the PI3 kinase/mTOR/AKT pathway has been well documented in MM patient biopsies and human MM cell lines, suggesting this pathway plays a key role in the survival and proliferation of malignant plasma cells. Rapamycin and the rapalogs are allosteric inhibitors of the mTORC1 complex (consisting of mTOR, raptor, mLST8 and PRAS40), inducing mainly cytostatic effects but not cell death. Inhibition of mTORC1 prevents a negative feedback loop to the mTORC2 complex (consisting of mTOR, Rictor, mLST8 and Sin 1) leading to the phosphorylation of AKT. Phosphorylated AKT is a key inducer of anti-apoptosis mechanisms and cell cycle progression, which may explain the limited results of the rapalogs in the clinic. Recently developed mTOR kinase inhibitors (i.e., CC-223) target both mTORC1 and mTORC2 complexes in order to inhibit tumor growth and importantly, induce cell death. Here we evaluate the effects of CC-223 on a panel of MM cell lines, in combination with current standard of care agents in MM (the corticosteroid, dexamethasone [DEX] and the IMiD® immunomodulatory drugs, lenalidomide [LEN] and pomalidomide [POM]), as well as in the context of LEN resistance. Single agent CC-223 was shown to inhibit cell proliferation in a panel of 10 MM cell lines achieving IC50 values between 0.1-1 µM following 5 days of treatment. CC-223 also reduced cell viability reaching IC50 values between 0.4-1 µM in 5 out of 10 MM cell lines tested. CC-223 induced concentration-dependent G1 phase arrest within 24h of treatment followed by an induction of cell death by 48h. The anti-MM tumor activity of CC-223 (0.3-10 mg/kg) was further tested in SCID mice with xenotransplants of NCI-H929 grown to approximately 100-150 mm3 in size. A dose-dependent tumor growth inhibition and tumor growth delay was seen with once daily dosing of CC-223. Combination of CC-223 with standard of care therapy compounds was also evaluated in vitro. The combination of CC-223 and DEX demonstrated synergistic effects on the inhibition of cell proliferation in 6 MM cell lines (combination index: 0.0002-0.38) tested over 5 days. CC-223 also had synergistic effects on the same panel of MM cell lines when combined with LEN (combination index: 0.05-0.8). Acquisition of drug resistance in patients receiving standard of care therapies is still one of the major clinical problems in MM. POM, the next generation of IMiD® immunomodulatory agents, has shown clinically meaningful results in patients that are resistant or have relapsed to their drug regimens, including LEN. We have recently developed in vitro cellular models of LEN-resistance using the H929 MM cell line. H929 cells with acquired resistance to LEN (H929 R10-1, R10-2, R10-3 and R10-4) were shown to have one copy number loss of cereblon compared to their matched LEN-sensitive control (H929 D1). In addition to this, protein expression analysis identified that these resistant cell lines also gained the activation of signaling pathways such as PI3K/AKT/mTOR, MEK/MAPK as well as anti-apoptotic factors. For example, S473 AKT phosphorylation was highly elevated in LEN-resistant cell lines which correlated with loss of PTEN protein expression (H929 R10-3 and R10-4). Interestingly, regardless of PI3K/AKT/mTOR pathway status, all LEN-sensitive and resistant H929 cells responded to CC-223 treatment with a strong inhibition of cell proliferation (H929 D1 IC50 0.2 µM, and H929 R10 1-4 IC50 0.2-0.35 µM) and to a lesser effect, induction of cell death, over a 5 day period. Similar to the panel of MM cell lines, G1 arrest occurred after 24h treatment and cell death (Sub-G1) was increased by 72h of treatment. CC-223 treatment reduced S473 pAKT and p-4EBP1 after 1h while total AKT and 4EBP1 remained unchanged in both the sensitive and resistant MM cell lines. Combination treatment of LEN-sensitive and resistant H929 cells with CC-223 and POM had synergistic inhibitory effects on cell proliferation (combination index: 0.35-0.7) and cell viability (combination index: 0.15-0.42). In conclusion, the mTOR kinase inhibitor, CC-223 potently inhibited MM cell proliferation by inducing G1 arrest and cell death in a panel of MM cell lines and reduction of tumor volume in vivo. The combination of LEN, POM or DEX with CC-223 had synergistic effects on MM cell proliferation and viability. Therefore, CC-223 in combination with other standard of care agents could become an important clinical tool for the treatment of MM in the future. Disclosures: Rychak: Celgene Corporation: Employment, Equity Ownership. Mendy:Celgene: Employment, Equity Ownership. Miller:Celgene Corporation: Employment, Equity Ownership. Leisten:Celgene Corporation: Employment, Equity Ownership. Narla:Celgene Corporation: Employment, Equity Ownership. Raymon:Celgene Corporation: Employment, Equity Ownership. Chopra:Celgene: Employment, Equity Ownership. Lopez-Girona:Celgene: Employment, Equity Ownership.

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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 ◽

10.1182/blood.v126.23.5125.5125 ◽

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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