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.

First Selective KIT D816V Inhibitor for Patients with Systemic Mastocytosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3217-3217 ◽  
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.

scholarly journals 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

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5123-5123 ◽  
Author(s):  
Mark Joseph Axelrod ◽  
Peter Fowles ◽  
Jeff Silverman ◽  
Astrid Clarke ◽  
Jennifer Tang ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Growth Inhibition ◽  
B Cell ◽  
Cell Lines ◽  
Xenograft Model ◽  
Lymphocytic Leukemia ◽  
Employment Equity ◽  
Equity Ownership

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.

Anti-Tumor Activity of the Aurora a Inhibitor MLN8237 in Diffuse Large B-Cell Lymphoma Preclinical Models.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1592-1592 ◽  
Author(s):  
Jessica J Huck ◽  
Mengkun Zhang ◽  
Marc L Hyer ◽  
Mark G Manfredi
Keyword(s):  
Tumor Growth ◽  
Growth Inhibition ◽  
Tumor Model ◽  
B Cell Lymphoma ◽  
Tumor Growth Inhibition ◽  
Aurora A ◽  
Hct 116 ◽  
Xenograft Models

Abstract Aurora A kinase is a serine/threonine protein kinase that is essential for normal transit of cells through mitosis. In many tumor types the Aurora A gene is amplified and/or the protein is over-expressed. The Aurora A small-molecule inhibitor MLN8237 demonstrated robust tumor growth inhibition in xenograft models of solid tumors grown subcutaneously (S.C.) in immunocompromised mice. Here we explored the antitumor activity of MLN8237 in models of diffuse large B-cell lymphoma (DLBCL) both in vitro and in vivo. In vivo three established DLBCL xenograft models (OCI-Ly7, OCI-Ly19, and WSU-DLCL2; all cells expressing luciferase) and a primary DLBCL tumor model PHTX-22-06 were tested using MLN8237 at different doses. Rituximab, an anti-CD20 monoclonal antibody that is active against CD20+ malignant B cells and is a standard of care agent was used for comparison. Using these model systems, tumor cells were injected either I.V. (to evaluate disseminated disease), or S.C. in severe combined immunodeficient mice (SCID). Animals were dosed orally for 21 days with MLN8237 (QD or BID) at various doses, or Rituximab dosed at 10mg/kg IV (once/week) and tumor growth inhibition was monitored using either bioluminescent imaging for the disseminated models or vernier calipers for the S.C. models. Tumor growth inhibition by MLN8237 was dose dependent with 20 mg/kg bid being the most efficacious dose (TGI&gt;100% in both disseminated OCI-Ly19 and WSU models). All animals in the OCI-Ly19 disseminated model 20 mg/kg BID treatment group demonstrated regressions and remained disease free until the end of the study, day 65. In this study the Rituximab treated animals were euthanized on day 31 due to a high level of tumor burden. In the primary tumor model, PHTX-22-06, MLN8237 dosed at 20 mg/kg BID was also the most efficacious with a TGI of 95%. Moreover, tumor growth inhibition was durable as determined by prolonged tumor growth delay (&gt;50 days). Significant efficacy was achieved in all models tested, whether grown as disseminated or subcutaneous models. A noted increase in durability of response was observed with MLN8237 treatment when compared with previous data from solid tumor models. In vitro, MLN8237 treatment increased levels of apoptosis in the OCI-Ly19 cells in comparison to the solid tumor cell line HCT-116 (colon). Greater Annexin V positive cells and greater cleaved PARP and Caspase-3 signals were detected in the MLN8237 treated OCI-Ly19 cells when compared to HCT-116 cells. The demonstration of robust and durable anti-tumor activity in preclinical models treated with MLN8237 provides the basis for its clinical evaluation as a treatment option for DLBCL. MLN8237 is currently in multiple Phase I clinical trials.

Blockade of XBP1 Splicing by Inhibition of IRE1α Is a Promising Therapeutic Option in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 133-133 ◽  
Author(s):  
Naoya Mimura ◽  
Mariateresa Fulciniti ◽  
Gullu Gorgun ◽  
Yu-Tzu Tai ◽  
Diana D. Cirstea ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Er Stress ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Therapeutic Option ◽  
Rt Pcr ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Abstract 133 Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response (UPR). Therefore blockade of UPR could provide a novel therapeutic option in MM. Upon UPR, inositol-requiring enzyme 1α (IRE1α) is activated by auto-phosphorylation, resulting in activation of its endoribonuclease domain to cleave XBP1 mRNA from XBP1 unspliced form (XBP1u: inactive) to generate the XBP1 spliced form (XBP1s: active). XBP1s protein in turn regulates genes responsible for protein folding and degradation, playing a pro-survival signaling role in the UPR. In this study, we specifically examined whether IRE1α-XBP1 pathway is a potential therapeutic target in MM. We first examined the biologic significance of IRE1α by knockdown using lentiviral shRNA and observed significant growth inhibition in IRE1α knockdown cells. We next examined the impact of inhibition of XBP1 splicing using a novel small molecule IRE1α endoribonuclease domain inhibitor MKC-3946 (MannKind, Valencia CA). MKC-3946 blocked not only the basal level, but also inducible (by tunicamycin) XBP1s, evidenced by RT-PCR analysis in RPMI8226 cells, without affecting phosphorylation of IRE1α. Importantly, MKC-3946 also inhibited XBP1s in primary tumor cells from MM patients. We also confirmed functional inhibition of XBP1s, with target genes including SEC61A1, p58IPK, and ERdj4 downregulated by MKC-3946 treatment. Importantly, MKC-3946 triggered growth inhibition in MM cell lines, without toxicity in normal mononuclear cells. Furthermore, it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG in RPMI8226 and INA6 cells, as well as primary tumor cells from MM patients. Both bortezomib and 17-AAG induced ER stress with XBP1s, which was markedly blocked by MKC-3946. Moreover, apoptosis induced by bortezomib or 17-AAG was enhanced by MKC-3946, associated with increased CHOP mRNA and protein, a proapoptotic factor triggered by ER stress. We next demonstrated that XBP1s was induced by bortezomib in INA6 cells co-cultured with bone marrow (BM) stromal cells, which was inhibited by MKC-3946, associated with enhanced cytotoxicity induced by the combination. Finally, MKC-3946 inhibited XBP1s in a model of in vivo ER stress induced by tunicamycin. To evaluate the anti-MM effect of MKC-3946, we used the subcutaneous RPMI8226 xenograft model in mice. MKC-3946 significantly reduced MM tumor growth in the treatment versus control group, associated with prolonged overall survival. We also confirmed that MKC-3946 treatment significantly inhibited XBP1s in excised tumors, assessed by RT-PCR. In order to examine the activity of MKC-3946 on MM cell growth in the context of the human BM microenvironment in vivo, we used the SCID-hu model, in which INA6 cells are directly injected into a human bone chip implanted subcutaneously in SCID-mice. MKC-3946 treatment significantly inhibited tumor growth compared with vehicle control. Moreover, XBP1s in excised tumor cells was inhibited, evidenced by RT-PCR. In conclusion, these data demonstrate that blockade of XBP1s by MKC-3946 triggers MM cell growth inhibition in vivo and prolongs host survival. Taken together, our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1α endoribonuclease domain is a potential novel therapeutic option in MM. Disclosures: Tam: MannKind Corporation: Employment, Equity Ownership. Zeng:MannKind Corporation: Employment, Equity Ownership. Patterson:MannKind Corporation: Employment, Equity Ownership. Richardson:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; MannKind: Membership on an entity's Board of Directors or advisory committees.

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.

scholarly journals Development of Multi-Engineered iPSC-Derived CAR-NK Cells for the Treatment of B-Cell Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1729-1729
Author(s):  
Luis Borges ◽  
Mark A Wallet ◽  
Chiamin-Liao Bullaughey ◽  
Michael F Naso ◽  
Buddha Gurung ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Growth Inhibition ◽  
B Cell ◽  
Nk Cells ◽  
Stock Options ◽  
Tumor Growth Inhibition ◽  
B Cell Malignancies ◽  
Privately Held

Abstract Induced-pluripotent stem cells (iPSCs) can be differentiated into various somatic cells, including different immune cell types. We have engineered iPSC-derived NK cells with multiple features to generate therapeutic candidates designed to eliminate cancer cells while avoiding recognition by the host immune system. The unlimited replication capacity of iPSCs facilitates the engineering of several genetic modifications without the risk of driving cells to exhaustion as in the case of cell products derived from fully differentiated immune cells. Once all edits are completed, our cells are single-cell cloned and each clone is genetically characterized to select clones without off-target insertions or deletions. Following the genetic characterization, selected clones are differentiated and tested in vitro and in vivo to identify the final clinical candidate. The use of a single-cell iPSC clone enables the generation of a master cell bank producing a highly uniform cell product that can be made available off-the-shelf at any clinical site. CNTY-101 is an iPSC-derived CAR-NK clinical candidate for the treatment of B-cell malignancies. It incorporates six gene edits designed to improve persistence and functionality as well as safety. These modifications include edits to reduce graft rejection due to alloreactivity, the expression of a homeostatic cytokine to improve functionality and persistence, the introduction of a chimeric antigen receptor (CAR) targeting CD19 to mediate tumor cell engagement and killing, as well a safety switch to eliminate the cells, if ever necessary. To prevent rejection by the patient's CD8 T cells, the beta-2-microbulin (ß2M) gene was disrupted with simultaneous insertion of a transgene encoding the HLA-E protein tethered with ß2M and a peptide. HLA-E was introduced to prevent NK cell cytotoxicity against the engineered cells, which lack HLA-I. For resistance to CD4 T cell-mediated allogenic immune rejection, the class II major histocompatibility complex transactivator (CIITA) gene was disrupted with simultaneous insertion of a transgene encoding the extra-cellular and transmembrane domains of EGFR, and the NK cell growth factor IL-15. EGFR provides an elimination tag that can be engaged by clinically approved anti-EGFR antibodies, such as cetuximab. Finally, the CAR transgene targeting the CD19 antigen was inserted into the AAVS1 safe harbor locus. Our data indicates that CNTY-101 iNK cells have strong antitumor activity against lymphoma cell lines both in vitro and in vivo. In vitro, CNTY-101 eliminates lymphoma cell lines through multiple rounds of killing without reaching exhaustion. Clones expressing higher levels of IL-15 tend to have better persistence and functionality, with some clones showing robust cytotoxicity for over fifteen rounds of serial killing. In vivo, the clones that demonstrated better in vitro serial killing tend to mediate the best anti-tumor activity in lymphoma xenograft models. Upon 3 weekly doses, the most active candidate clone demonstrated significant tumor growth inhibition after administration of fresh (91 % tumor growth inhibition) or cryopreserved cells (76 % tumor growth inhibition). The efficacy of the EGFR-safety switch was also investigated both in vitro and in vivo. In vitro, addition of cetuximab to co-cultures of IL-2-activated PBMC and cells mediated antibody-dependent cellular cytotoxicity (ADCC) in a concentration-dependent fashion, with an EC50 of 2 ng/ml. In vivo, there was a 96% reduction in the number of iPSC-derived CAR-NK cells in the lungs and a 95% reduction in the number of CAR-NK cells in the blood of mice that received cetuximab versus PBS-treated mice. In summary, CNTY-101 is a novel, multi-engineered, allogeneic CAR-iNK product candidate for the treatment of B-cell malignancies. It includes multiple immune evasion features to prevent recognition by the patient's immune system and expression of IL-15 to facilitate persistence and functionality. We have initiated GMP manufacturing of CNTY-101 and plan to enter clinical trials in 2022. Disclosures Borges: Century Therapeutics: Current Employment, Current equity holder in publicly-traded company. Wallet: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Bullaughey: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Naso: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Gurung: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Keating: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Carton: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Wheeler: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Campion: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Mendonca: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Jessup: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Beqiri: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Chin: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Millar Quinn: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Morse: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company.

scholarly journals NKG2D-CAR Transduced Primary Natural Killer Cells Efficiently Target Multiple Myeloma Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 590-590 ◽  
Author(s):  
Alejandra Leivas ◽  
Paula Rio ◽  
Rebeca Mateos ◽  
Mari Liz Paciello ◽  
Almudena Garcia-Ortiz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Cell Therapy ◽  
Nk Cells ◽  
Tumor Cells ◽  
Research Funding ◽  
Car T ◽  
Equity Ownership

Abstract Introduction Immunotherapy represents a new weapon in the fight against multiple myeloma. Current clinical outcomes using CAR-T cell therapy against multiple myeloma show promise in the eradication of the disease. However, these CARs observe relapse as a common phenomenon after treatment due to the reemergence of neoantigens or negative cells. CARs can also be targeted using non-antibody approaches, including the use of receptors, as NKG2D with a wider range of ligands, and ligands to provide target specificity. Different cell types have been used to improve CAR cell therapy. CAR-T cells are the most commonly used. However, despite its effectiveness, there are still problems to face. The toxicity of the cytokine release syndrome is well known, that is why memory CD45RA- T cells are used to avoid collateral effects, although having lower efficacy. However, CAR-NK cells may have less toxicity and provide a method to redirect these cells specifically to refractory cancer. The objective of this work was to compare the anti-tumor activity of CAR-T, NKAEs and CAR-NK cells from multiple myeloma patients. Methods The activated and expanded NK cells (NKAE) were generated by coculture of peripheral blood mononuclear cells with the previously irradiated CSTX002 cell line. The CD45RA- T cells were obtained by depletion with CD45RA magnetic beads and subsequent culture. The NKAE and T were transduced with an NKG2D-CAR with signaling domains of 4-1BB and CD3z. The expansion of NKAE and the expression of NKG2D-CAR were evaluated by flow cytometry based on the percentage of NK cell population and transduction efficiency by the expression of NKG2D. Europium-TDA release assays (2-4 hours) were performed to evaluate in vitro cytotoxic activity. The antitumor activity of the NKAE (n=4) and CD45RA- (n=4) cells against MM U-266 cells was studied. Methylcellulose cultures were performed to assess the activity against the clonogenic tumor cell. In vivo studies were carried out in NSG mice receiving 5.106 of U266-luc MM cells i.v. injected at day 1. At day 4, mice received 15.106 i.v. injected of either CAR-NKAE or untransduced NKAE cells. Results In vitro. The killing activity of primary NKAE cells (n=4) was 86.6% (± 13.9%), considerably higher than that of CD45RA- lymphocytes (16.7% ± 13.6%) from the same patient (n=4). Even CD45RA- T cells from healthy donors (n=4) exhibit lower anti tumoral capacity (28.2% ± 9.7%) than NKAE cells. The transduction with an NKG2D CAR (MOI=5) improved the activity of autologous NKAE cells by 10% (96.4% ± 19%) leading to a nearly complete destruction of U-266 MM cells, and that of CD45RA- allogenic healthy cells in 19% (47.4% ± 12.6%). Nevertheless, CD45RA- autologous T cells transduced with NKG2D-CAR minimally improved their activity by 5.8% (22.5% ± 10.6%). Additionally, the CAR-NKAE cells were able to destroy the clonogenic tumor cell responsible for the progression of the MM from RPMI-8226 cell line. At an 8:1 ratio the CAR-NKAE cells were able to destroy 71.2% ± 2.5% of the clonogenic tumor cells, while the NKAE reached 56.5% ± 2.6% at a maximum ratio of 32: 1. The toxicity of the CAR-NKAE cells on healthy tissue from the same patient was assessed, and no activity against autologous PBMCs was observed, 1,8% at a maximun ratio of 32:1 (effector:target). In vivo. NKAE cells and CAR-NKAE cells were efficient in abrogating MM growth. However, CAR-NKAE cells treatment showed higher efficiency 14 days after tumor cells injection. Forty-two days after tumor cells injection, only animals receiving CAR-NKAE cells treatment remain free of disease (Figure 1). Conclusions It is feasible to modify primary NKAE cells and CD45RA- T cells from primary MM cells to safely express an NKG2D-CAR. Our data show that CD45RA- T cells from patients are not effective in vitro against MM even once transduced with our CAR. The resulting CAR-NKG2D NKAE cells are the most appropriate strategy for the destruction of MM in vitro and in vivo in our model. These results form the basis for the development of an NKG2D-CAR NK cell therapy in MM. Disclosures Rio: Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Lee:Merck, Sharp, and Dohme: Consultancy; Courier Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CytoSen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Martinez-Lopez:Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Vivia: Honoraria; Pfizer: Research Funding; BMS: Research Funding; Novartis: Research Funding.

scholarly journals Dual-Function Anti-CD47mAbs Induce Tumor Cell Death and Promote Phagocytosis Resulting in Enhanced in Vivo Efficacy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 991-991
Author(s):  
Pamela T. Manning ◽  
Benjamin J. Capoccia ◽  
Michael P. Rettig ◽  
Ronald R. Hiebsch ◽  
Robert W. Karr ◽  
...  
Keyword(s):  
Cell Death ◽  
Cancer Cells ◽  
Tumor Cells ◽  
Tumor Burden ◽  
Dual Function ◽  
Employment Equity ◽  
Hematologic Cancer ◽  
Equity Ownership

Abstract Recent success in immunomodulation of cancer has targeted immune checkpoints such as CTLA-4, PD-1 and PDL-1 to enhance adaptive immunity by stimulating production of tumor-selective, cytotoxic T cells. Anti-CD47mAbs enhance innate immunity by increasing the phagocytosis of tumor cells by macrophages leading to processing and presentation of tumor antigens to prime the adaptive T cell response. Many cancers, including hematologic cancers, up-regulate the expression of CD47 presumably to avoid immune destruction. Increased CD47 expression protects cancer cells from phagocytosis by sending a “don't eat me” signal to macrophages via SIRPalpha, an inhibitory receptor that prevents phagocytosis of CD47-bearing cells. CD47mAbs that block the CD47/SIRPalpha interaction (“blocking-only” mAbs) enhance phagocytosis of cancer cells in vitro. We have identified two CD47mAbs, Vx-1000 and Vx-1004, both of which block the CD47/SIRPalpha interaction and promote phagocytosis of tumor cells by macrophages equally well. However, Vx-1004 also has the unique property of killing cancer cells, but not normal blood cells, via a direct, cell-autonomous, cytotoxic mechanism. Therefore, Vx-1004 is a dual-function antibody. Vx-1004 selectively kills a variety of hematologic cancer cells in vitro, while Vx-1000, the blocking-only mAb, does not as assessed by annexin V staining and flow cytometry (Figure 1). In dose-response studies, cell death in leukemia cells was induced in 2 hrs by <1 ug="" vx-1004="" whereas="" normal="" peripheral="" blood="" mononuclear="" cells="" are="" resistant="" to="" the="" induction="" of="" cell="" death="" by="" following="" incubation="" with="" 10="" for="" 24="" hrs="" both="" these="" cd47mabs="" bind="" many="" species="" cd47="" including="" mouse="" and="" human="" p=""> To determine if the tumor-toxic activity of Vx-1004 confers enhanced efficacy in vivo compared to Vx-1000, we compared them in two mouse hematologic cancer models: murine acute promyelocytic leukemia (APL) and B cell lymphoma (BCL). Briefly, 1x106 GFP-labeled C57BL/6 APL cells were injected IV into wild-type C57BL/6 mice that were then treated IP with 0.4 mg/kg of either Vx-1000 or Vx-1004 on the day of tumor injection and on days 3 and 6 following tumor injection, a very low dose and limited dosing regimen. On day 25, the blood of these mice was analyzed for the number of circulating APL cells. As shown in Figure 2, Vx-1000 did not significantly reduce tumor burden compared to the control group. In contrast, Vx-1004 significantly reduced tumor burden compared to controls, demonstrating greater efficacy of the dual-function CD47mAb. In addition, enhanced efficacy of Vx-1004 compared to Vx-1000 was demonstrated in BCL (Figure 3). In this model, NSG mice were injected with 1x106 murine A20 lymphoma cells subcutaneously and then treated with 0.4mg/kg/day of the CD47mAbs IP for the first five days following tumor injection. In this model, Vx-1000 also failed to inhibit tumor growth compared to controls while Vx-1004 significantly reduced tumor burden at 35 days compared to both the control and Vx-1000 groups, nearly four weeks after treatment was stopped. These data demonstrate increased anti-cancer efficacy with a dual-function CD47mAb that not only blocks the CD47/SIRPalpha interaction to increase phagocytosis of cancer cells, but also selectively kills cancer cells. These studies indicate that dual-function CD47mAbs may have better anti-tumor activity in vivo and support their use in human clinical trials. Figure 1 Figure 1. Disclosures Manning: Corvus Pharmaceutical: Employment, Equity Ownership. Capoccia:Corvus Pharmaceutical: Employment, Equity Ownership. Hiebsch:Corvus Pharmaceutical: Employment, Equity Ownership. Karr:Corvus Pharmaceutical: Employment, Equity Ownership. Frazier:Corvus Pharmaceutical: Consultancy, Equity Ownership.

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