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.

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.

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.

scholarly journals CXCR4 Blockade By BL-8040 in T Cell Acute Lymphoblastic Leukemia Decreases Mitochondrial Mass and Induces Non-Apoptotic Cell Death

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2745-2745
Author(s):  
Jun Xia ◽  
Stephanie Sun ◽  
Matthew RM Jotte ◽  
Geoffrey L. Uy ◽  
Osnat Bohana-Kashtan ◽  
...  
Keyword(s):  
Cell Death ◽  
Apoptotic Cell ◽  
Lymphoblastic Leukemia ◽  
Apoptotic Cell Death ◽  
Employment Equity ◽  
Cxcr4 Signaling ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Equity Ownership

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that accounts for 10-15% of pediatric and 25% of adult ALL cases. CXCL12 is a CXC chemokine that is constitutively expressed at high levels in the bone marrow. CXCR4 is the major receptor for CXCL12 and is by far the most highly expressed chemokine receptor on T-ALL cells. Two groups recently showed that genetic loss of CXCR4 signaling in murine or human T-ALL cells markedly suppressed their growth in vivo. We previously reported that BL-8040, a potent new CXCR4 antagonist with sustained receptor occupancy, is active as monotherapy against T-ALL in mice. Indeed, a 2-week course of daily BL-8040 resulted in a median reduction in tumor burden of 32.1-fold (range 6.8 to 176) across 5 different T-ALL xenografts. Preliminary data from a clinical trial of BL-8040 plus nelarabine for relapsed T-ALL also suggest therapeutic activity, with a complete remission rate observed in 4/8 patients (50%), which compares favorably to published response rates of approximately 30% with single agent nelarabine. Here, we explore molecular mechanisms by which CXCR4 blockade induces T-ALL death. NOD-scid IL2Rgammanull (NSG) mice were injected with P12-Ichikawa cells, a T-ALL cell line modified to express click beetle red luciferase and GFP. Following T-ALL engraftment, mice were treated with a single dose of BL-8040, and then leukemic cells in the bone marrow harvested 24-48 hours later. Treatment with BL-8040 resulted in a marked suppression of Akt and Erk1/2 phosphorylation, suggesting that signaling through CXCR4 is the major source of PI3 kinase pathway activation in T-ALL cells. Surprisingly, treatment with BL-8040 did not affect cellular proliferation, as measured by Ki67/FxCycle Violet staining or by EdU labeling. Moreover, no increase in apoptosis, as measured by annexin V or activated caspase 3 expression, was observed. These data suggest that CXCR4 blockade induces a non-apoptotic cell death. To explore this possibility further, we performed transcriptome sequencing on T-ALL cells recovered from mice 24 hours after 1 dose of BL-8040. A total of 151 differentially expressed genes (FDR of < 0.05% and ≥ 2-fold change) were identified. Gene set enrichment analysis was strongly positive for alterations in oxidative phosphorylation, ribosome biogenesis, and carbohydrate metabolism. Ribosome function was assessed using O-propargyl-puromycin (OPP), which monitors global protein translation. No difference in global protein synthesis in T-ALL cells was observed after CXCR4 blockade in vivo. T-ALL cells are dependent on glutamine as a source of carbon, and PI3 kinase signaling positively regulates glutaminolysis. Thus, we hypothesized that CXCR4 blockade may induce T-ALL cell death by reducing glutamine metabolism. However, treatment of T-ALL cells in vitro with BL-8040 did not alter the cellular levels of glutamine or glutamate, as measured using a commercial bioluminescent assay. Confirmatory metabolic tracing studies using 13C-labeled glutamine and glucose are in progress. Finally, to explore the reduction in oxidative phosphorylation, we examined mitochondria function using Mitotracker Green. Treatment of T-ALL cells in vitro with BL-8040 for 24-48 hours induced a significant decrease in mitochondria number, suggesting induction of mitophagy. Collectively, these data suggest that T-ALL cells are addicted to CXCR4 signaling in vivo. CXCR4 blockade with BL-8040 induces a non-apoptotic cell death that is characterized by a loss of mitochondria. Disclosures Uy: Astellas: Consultancy; Pfizer: Consultancy; Curis: Consultancy; GlycoMimetics: Consultancy. Bohana-Kashtan:BiolineRx: Employment, Equity Ownership. Sorani:BiolineRx: Employment, Equity Ownership. Vainstein:BiolineRx: Employment, Equity Ownership.

TNB-486, a Novel Fully Human Bispecific CD19 x CD3 Antibody That Kills CD19-Positive Tumor Cells with Minimal Cytokine Secretion

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4070-4070
Author(s):  
Harbani Malik ◽  
Ben Buelow ◽  
Udaya Rangaswamy ◽  
Aarti Balasubramani ◽  
Andrew Boudreau ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Cytokine Secretion ◽  
Employment Equity ◽  
Equity Ownership

Introduction The restricted expression of CD19 in the B-cell lineage makes it an attractive target for the therapeutic treatment of B-cell malignancies. Many monoclonal antibodies and antibody drug conjugates targeting CD19 have been developed, including bispecific T-cell redirecting antibodies (T-BsAbs). In addition, anti-CD19 chimeric antigen receptor T-cells (CAR-T) have been approved to treat leukemia and lymphoma. However, despite the impressive depth of responses achieved by T-cell redirecting approaches such as T-BsAbs and CAR-T cells, toxicity from over-activation of T-cells remains a substantial limitation for this type of therapy, in particular neurotoxicity. In designing TNB-486, a novel CD19 x CD3 T-BsAb, we endeavored to retain activity against CD19-positive tumor cells while limiting the cytokine secretion thought to underlie toxicity from T-cell redirecting therapies. Utilizing TeneoSeek, a next generation sequencing (NGS)-based discovery pipeline that leverages in silico analysis of heavy chain only/fixed light chain antibody (HCA/Flic, respectively) sequences to enrich for antigen specific antibodies, we made a high affinity αCD19 HCA and a library of αCD3 Flic antibodies that showed a >2 log range of EC50s for T cell activation in vitro. Of note, the library contained a low-activating αCD3 that induced minimal cytokine secretion even at concentrations that mediated saturating T-cell dependent lysis of lymphoma cells (when paired with an αCD19 HCA). We characterized the relative efficacy and potential therapeutic window of this unique molecule, TNB-486, in vitro and in vivo and compared it to two strongly activating bispecific CD19 x CD3 antibodies similar to those currently available and in clinical development. Methods Affinity measurements of the αCD19 moiety were made via Biacore (protein) and flow cytometry (cell surface). Stability measurements were made by subjecting the molecule to thermal stress and the %aggregation was measured by Size Exclusion Chromatography. T-cell activation was measured via flow cytometry (CD69 and CD25 expression) and cytokine was measured by ELISA (IL-2, IL-6, IL-10, INF-ɣ, and TNFα) in vitro. Lysis of B-cell tumor cell lines (Raji, RI-1, and Nalm6) was measured via flow cytometry in vitro. In vivo, NOG mice were engrafted subcutaneously with NALM-6 or SUDHL-10 cells and intravenously with human peripheral blood mononuclear cells (huPBMC), and the mice treated with multiple doses of TNB-486 or negative or positive control antibody. Tumor burden was evaluated via caliper measurement. Pharmacodynamic/Pharmacokinetic (PK/PD) studies were performed in NOG mice. A pharmacokinetic (PK) study was performed in BALB/c mice, and a tolerability and PK study are ongoing in cynomolgus monkeys. Results TNB-486 bound to cell surface CD19 with single digit nanomolar affinity (~3nM). EC50s for cytotoxicity were in the single-digit nanomolar range for TNB-486, and sub-nanomolar for the strongly activating controls; TNB-486 maximum achievable lysis was identical to the positive controls. TNB-486 induced significantly less cytokine release for all cytokines tested compared to the positive controls even at doses saturating for tumor lysis. No off-target activation was observed in the absence of CD19 expressing target cells. In vivo, TNB-486 eradicated all CD19-positive tumors tested (NALM-6 and SUDHL10) at doses as little as 1µg administered every four days after tumors had reached ~200mm3. TNB-486 showed a PK profile consistent with other IgG molecules in mice (T1/2 ~6 days in mice). Conclusions TNB-486 induced comparable lysis of CD19-positive tumor cells as the strongly activating control bispecific antibodies while inducing significantly reduced cytokine secretion, even at doses saturating for tumor lysis in vitro. In vivo TNB-486 eradicated all tested CD19 positive tumor cell lines in established tumor models. No off-target binding was observed. In summary, TNB-486 shows promise as a lymphoma therapeutic differentiated from T-cell targeted therapies currently in the clinic and in clinical trials. Disclosures Malik: Teneobio, Inc.: Employment, Equity Ownership. Buelow:Teneobio, Inc.: Employment, Equity Ownership. Rangaswamy:Teneobio, Inc.: Employment, Equity Ownership. Balasubramani:Teneobio, Inc.: Employment, Equity Ownership. Boudreau:Teneobio, Inc.: Employment, Equity Ownership. Dang:Teneobio, Inc.: Employment, Equity Ownership. Davison:Teneobio, Inc.: Employment, Equity Ownership. Force Aldred:Teneobio, Inc.: Equity Ownership. Iyer:Teneobio, Inc.: Employment, Equity Ownership. Jorgensen:Teneobio, Inc.: Employment, Equity Ownership. Pham:Teneobio, Inc.: Employment, Equity Ownership. Prabhakar:Teneobio, Inc.: Employment, Equity Ownership. Schellenberger:Teneobio, Inc.: Employment, Equity Ownership. Ugamraj:Teneobio, Inc.: Employment, Equity Ownership. Trinklein:Teneobio, Inc.: Employment, Equity Ownership. Van Schooten:Teneobio, Inc.: Employment, Equity Ownership.

Expression of Bcl-2 Pro-Survival Family Proteins Predicts Pharmacological Responses to ABT-199, a Novel and Selective Bcl-2 Antagonist, in Multiple Myeloma Models

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5028-5028 ◽  
Author(s):  
Deepak Sampath ◽  
Elizabeth Punnoose ◽  
Erwin R. Boghaert ◽  
Lisa Belmont ◽  
Jun Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Cell Lines ◽  
Single Agent ◽  
Employment Equity ◽  
Bone Marrow Biopsies ◽  
Xenograft Models ◽  
Equity Ownership

Abstract Abstract 5028 Multiple myeloma (MM) is a hematological malignancy of the bone marrow caused by the dysregulated proliferation of monoclonal antibody producing plasma cells. A hallmark feature of cancer is the ability to evade cell death signals induced by stress response cues. The Bcl-2 family of proteins regulates the intrinsic apoptosis pathways and consists of pro-apoptotic (Bax, Bak, Bad, Bim, Noxa, Puma) and pro-survival (Bcl-2, Bcl-xL, Mcl-1); the balance of which dictates the life or death status of MM tumor cells. Thus, there is a strong rationale to target members of the Bcl-2 proteins for the treatment of MM. ABT-199 is a potent BH3-only mimetic that selectively antagonizes Bcl-2 and is currently in phase I clinical trials for the treatment of hematological malignancies. Therefore, we evaluated the efficacy of ABT-199 as a single agent and in combination with standard of care drugs such as Velcade (bortezomib) in preclinical models of MM. A panel of 21 human MM cell lines was evaluated in vitro for to sensitivity to ABT-199. ABT-199 potently inhibited cell viability in a sub-set of MM cell lines (7/21) with EC50 values less than 1 μM. Expression of Bcl-2, Bcl-xL, Mcl-1, Bim and other Bcl-2 family proteins were evaluated by protein and mRNA. Cell line modeling identified thresholds for expression of Bcl-2, Bcl-xL and Mcl-1 that best predicted sensitivity and resistance to ABT-199 and the dual Bcl-2/Bcl-xL antagonist, navitoclax. Consistent with the target inhibition profile of these drugs, we found that MM lines that were Bcl-2high/Bcl-xLlow/Mcl-1low are the most sensitive to ABT-199 treatment. Whereas cell lines that are Bcl-xLhigh remain sensitive to navitoclax but not ABT-199. MM cell lines that are Mcl-1high are less sensitive to both ABT-199 and navitoclax, suggesting that Mcl-1 is a resistance factor to both drugs. Utilizing a novel Mesoscale Discovery based immunoassay we determined that levels of Bcl-2/Bim complexes also correlated with sensitivity of ABT-199 in the MM cell lines tested. In addition, the t(11;14) status in these cell lines associated with sensitivity to ABT-199. The clinical relevance of the Bcl-2 pro-survival expression pattern in MM cell lines, was determined by a collection of bone marrow biopsies and aspirates (n=27) from MM patients by immunohistochemistry for prevalence of Bcl-2 and Bcl-xL. Similar to our in vitro observations, the majority (75%) of the MM bone marrow biopsies and aspirates had high Bcl-2 levels whereas 50% had high Bcl-xL expression. Therefore, a subset of patient samples (33%) were identified with a favorable biomarker profile (Bcl-2high/Bcl-xLlow) that may predict ABT-199 single agent activity. ABT-199 synergized with bortezomib in decreasing cell viability in the majority of MM cell lines tested in vitro based on the Bliss model of independence analyses (Bliss score range = 10 to 40). However the window of combination activity was reduced due to high degree of sensitivity to bortezomib alone. Therefore, the combination efficacy of ABT-199 and bortezomib was further evaluated in vivo in MM xenograft models that expressed high levels of Bcl-2 protein (OPM-2, KMS-11, RPMI-8226, H929 and MM. 1s). Bortezomib treatment alone at a maximum tolerated dose resulted in tumor regressions or stasis in all xenograft models tested. ABT-199 at a maximum tolerated dose was moderately efficacious (defined by tumor growth delay) as a single agent in xenograft models that expressed high protein levels of Bcl-2 but relatively lower levels of Bcl-xL. However, the combination of ABT-199 with bortezomib significantly increased the overall response rate and durability of anti-tumor activity when compared to bortezomib, resulting in increased cell death in vivo. Treatment with bortezomib increased levels of the pro-apoptotic BH3-only protein, Noxa, in MM xenograft models that expressed high levels of Mcl-1. Given that the induction of Noxa by bortezomib results in neutralization of Mcl-1 pro-survival activity in MM models [Gomez-Bougie et al; Cancer Res. 67:5418–24 (2007)], greater efficacy may be achieved when Bcl-2 is antagonized by ABT-199 thereby inhibiting pro-survival activity occurring through either Bcl-2 or Mcl-1 and increasing cell death. Thus, our preclinical data support the clinical evaluation of ABT-199 in combination with bortezomib in MM patients in which relative expression of the Bcl-2 pro-survival proteins may serve as predictive biomarkers of drug activity. Disclosures: Sampath: Genentech: Employment, Equity Ownership. Punnoose:Genentech: Employment, Equity Ownership. Boghaert:Abbott Pharmaceuticals: Employment, Equity Ownership. Belmont:Genentech: Employment, Equity Ownership. Chen:Abbott Pharmaceuticals: Employment, Equity Ownership. Peale:Genentech: Employment, Equity Ownership. Tan:Genentech: Employment, Equity Ownership. Darbonne:Genentech: Employment, Equity Ownership. Yue:Genentech: Employment, Equity Ownership. Oeh:Genentech: Employment, Equity Ownership. Lee:Genentech: Employment, Equity Ownership. Fairbrother:Genentech: Employment, Equity Ownership. Souers:Abbott Pharmaceuticals: Employment, Equity Ownership. Elmore:Abbott Pharmaceuticals: Employment, Equity Ownership. Leverson:Abbott Pharmaceuticals: Employment, Equity Ownership.

A CD19/CD3 Bispecific Tandab, AFM11, Recruits T Cells To Potently and Safely Kill CD19+ Tumor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4405-4405
Author(s):  
Eugene Zhukovsky ◽  
Uwe Reusch ◽  
Carmen Burkhardt ◽  
Stefan Knackmuss ◽  
Ivica Fucek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cancer Cells ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Cross Reactivity ◽  
Equity Ownership

Abstract To harness the potent tumor-killing capacity of T cells for the treatment of CD19+ malignancies, we developed a humanized bispecific tetravalent antibody, with two binding sites for CD3 and CD19, the CD19/CD3 RECRUIT-TandAb AFM11. CD19 is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies such as Non Hodgkin Lymphoma. Since native antibodies cannot recruit T cells, we engineered a bispecific anti-CD19/anti-CD3 TandAb. The tumor-specific CD19 antigen module targets the TandAb to cancer cells, while simultaneously, the CD3 effector module recruits and activates T cells, leading to cancer cell lysis. The advantages of the TandAb technology, relative to other bi-functional fragment antibody scaffolds, include: improved pharmacokinetics (PK) enabling intravenous dosing, more drug-like properties, and avidity-enhanced efficacy for the targeting and killing of tumor cells. We evaluated in vitro efficacy and safety using CD19+ cell lines, and in vivo efficacy in a murine NOD/scid xenograft model reconstituted with human PBMC. Further, we used standard preclinical IND enabling assays to evaluate tissue cross reactivity, PK, and toxicological profile (local tolerance, hematocompatibility, effects on hematopoesis, etc). In vitro assays demonstrated the higher potency and efficacy of target cell lysis by AFM11 relative to a bispecific tandem scFv (that is currently in clinical evaluation). CD8+ T cells dominate early AFM11-mediated cytotoxicity (4 hrs) while after 24 hrs both CD4+ and CD8+ T cells equally contribute to tumor lysis with EC50 between 0.5 – 5 pM; cytotoxicity was independent of CD19 cell-surface density. AFM11 exhibited similar cytotoxicity over effector:target ratios ranging from 5:1 to 1:5, and facilitated serial T cell-killing of its targets. The advantage of AFM11 over the bispecific tandem scFv was most pronounced at lower effector:target ratios. AFM11 activated T cells only in the presence of CD19+ cells. In PBMC cultures, AFM11 induced CD69 and CD25 expression, T cell proliferation, and production of IFN-γ, TNF-α, IL-2, IL-6, and IL-10. Depletion of CD19+ cells from PBMC abrogated these effects, demonstrating that the T cell activation is strictly CD19+ target-dependent. Thus, AFM11 should not elicit the devastating cytokine release observed when full-length antibodies bind CD3. Up to one week co-incubation with AFM11 did not inhibit T cell cytotoxicity, suggesting that the TandAb does not induce anergy. In vivo, AFM11 induced dose-dependent growth inhibition of Raji tumors; a single 0.5 mg/kg dose exhibited efficacy similar to 5 daily injections. In the tissue cross reactivity study, only tissues containing CD19+ and CD3+ cells were stained by AFM11; all other tissues, including vital organs, displayed no cross reactivity. Similarly, no local intolerance was observed in rabbits, and no effect on myeloid and erythroid progenitors was observed in a colony-forming assay. Strong accumulation of 125I-labeled AFM11 was observed in the tumors of mice engrafted with CD19+ cancer cells, and no unspecific organ accumulation was observed. Finally, evaluated on the basis of Cmax and the area under the curve (AUC), AFM11 exhibited dose linearity (20 – 500 mg AFM11 dose range) upon single i.v. bolus administration in mice; half-life (T1/2) ranged from 18.4 to 22.9 hr. In summary, AFM11 is a highly efficacious novel drug candidate for the treatment of CD19+ malignancies with an advantageous safety profile and anticipated dosing regimen. Disclosures: Zhukovsky: Affimed Therapeutics AG: Employment, Equity Ownership. Reusch:Affimed Therapeutics AG: Employment. Burkhardt:Affimed Therapeutics AG: Employment. Knackmuss:Affimed Therapeutics AG: Employment. Fucek:Affimed Therapeutics AG: Employment. Eser:Affimed Therapeutics AG: Employment. McAleese:Affimed Therapeutics AG: Employment. Ellwanger:Affimed Therapeutics AG: Employment. Little:Affimed Therapeutics AG: Consultancy, Equity Ownership.

scholarly journals Lirilumab Enhances Anti-Tumor Efficacy of Elotuzumab

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4711-4711 ◽  
Author(s):  
Caroline Sola ◽  
Mathieu Blery ◽  
Cécile Bonnafous ◽  
Elodie Bonnet ◽  
Nicolas Fuseri ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Nk Cells ◽  
Tumor Cells ◽  
Therapeutic Efficacy ◽  
Nk Cell ◽  
Employment Equity ◽  
Equity Ownership ◽  
Checkpoint Receptors

Abstract Tumor cells that express reduced levels of Major Histocompatibility Complex (MHC) class I molecules may be recognized and killed by Natural Killer cells (NK cells), through a process known as “missing self” recognition. In humans, this is controlled by inhibitory receptors such as Killer Immunoglobulin-like Receptors (KIR) that recognize Human Leukocyte Antigen (HLA)-A, -B or –C. Engagement of KIR by HLA molecules results in inhibitory signaling that reduces NK cell-mediated natural killing and antibody-dependent cellular cytotoxicity (ADCC). Hence, antibodies that block interactions between inhibitory KIR and their HLA ligands are being evaluated as an anti-cancer therapeutic strategy. The anti-KIR2DL1/2/3-specific monoclonal antibody, lirilumab (BMS-986015 / IPH2102), is a fully human IgG4 that blocks binding of KIR to HLA-C, and is being developed for treating hematologic malignancies and solid tumors. Elotuzumab (BMS901608 / HuLuc63) is a humanized IgG1 anti-SLAMF7 (signaling lymphocyte activation molecule family member 7, CS-1) being developed for the treatment of Multiple Myeloma (MM). SLAMF7 is a cell surface glycoprotein highly expressed in myelomatous cells and only at low levels on normal cells. NK cell-mediated ADCC is one of the main mechanisms of action of elotuzumab, but ADCC is negatively regulated by KIR checkpoint receptors. Thus a combination of lirilumab and elotuzumab has strong scientific rationale. The aim of the present study was to assess whether lirilumab would enhance elotuzumab anti-MM activity in vitro with human peripheral blood NK cells and MM cell lines, and in vivo in a newly developed xenogenic mouse model. Two MM cell lines (OPM-2 and U266B1) were identified that express both HLA-C and SLAMF7. These MM cells were capable of activating peripheral blood NK cells from healthy donors in vitro, as assessed by three different endpoints (CD107 mobilization on NK cells surface and intracellular production of the cytokines IFN-g and TNF- a); each of these responses were significantly enhanced, in a dose-dependent manner, by both lirilumab and elotuzumab independently. Moreover, the elotuzumab-mediated functional activation of KIR2D+ NK cells could be further enhanced by the addition of increasing doses of lirilumab. The best combinatorial effect was observed in response to MM cells expressing low densities of SLAMF7. These data suggest that lirilumab treatment may increase the therapeutic efficacy of elotuzumab, particularly in MM patients with low SLAMF7 expression. In these experiments, it was not possible to clearly identify the impact of Fc receptor genotype or HLA-C genotype on the NK cell responses. To assess the therapeutic efficacy of lirilumab and elotuzumab in vivo, we generated a novel strain of double-transgenic mice expressing human KIR2DL3 as well as its ligand, HLA-cw3, on a Rag1-/- background (KIR-cw3-tgRAG mice), to allow engraftment of human MM tumor cells expressing SLAMF7. The OPM-2 MM cell line was subcutaneously engrafted in these mice and when high tumor volumes were reached, mice were treated with lirilumab, elotuzumab or a combination of both. As monotherapy, each of monoclonal antibody had some therapeutic effect while the combination of both resulted in a significantly stronger anti-tumor effect and increased survival of the mice. Median survival of mice treated with huIgG control was 38 days, 41 days with lirilumab, 42 days with elotuzumab and 51 days with both mAbs in combination (10 mice per group). In conclusion, we demonstrate that blockade of KIR checkpoint receptors with lirilumab was able to augment elotuzumab mediated ADCC in vitro and synergized with elotuzumab to mediate potent anti-MM activity in vivo. Taken together, these data provide a rationale for clinical trials to test combination treatment of lirilumab and elotuzumab in MM patients. Disclosures Sola: InnatePharma: Employment, Equity Ownership. Blery:Innate Pharma: Employment, Equity Ownership. Bonnafous:Innate Pharma: Employment, Equity Ownership. Bonnet:Innate Pharma: Employment, Equity Ownership. Fuseri:Innate Pharma: Employment, Equity Ownership. Graziano:Bristol-Myers Squibb: Employment; Bristol-Myers Squibb: Equity Ownership. Morel:Innate Pharma: Employment, Equity Ownership. André:Innate Pharma: Employment, Equity Ownership.

scholarly journals Combination Treatment with 5F9 and Azacitidine Enhances Phagocytic Elimination of Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2729-2729 ◽  
Author(s):  
Dongdong Feng ◽  
Phung Gip ◽  
Kelly Marie McKenna ◽  
Feifei Zhao ◽  
Ofelia Mata ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Bioluminescence Imaging ◽  
Employment Equity ◽  
Advisory Committees ◽  
Human Macrophages ◽  
Equity Ownership

Abstract CD47 is an anti-phagocytic signal and macrophage checkpoint that acute myeloid leukemia (AML) and other cancer cells utilize to evade innate immunity and establish disease. 5F9 is a humanized IgG4 monoclonal antibody (mAb) that binds to human CD47 and blocks its interaction with its macrophage receptor SIRPα, thereby promoting phagocytosis of cancer cells. We have found in numerous preclinical studies that anti-CD47 Abs synergize with targeted Abs (such as rituximab and cetuximab) by promoting phagocytosis, and also enable antigen cross-presentation and activation of cytotoxic T cells. These preclinical findings are being translated into clinical results as we have established in several clinical trials promising preliminary evidence of 5F9's therapeutic potential. In this study, we hypothesized that combining 5F9 with azacytidine (AZA) would enhance therapeutic efficacy against AML. AZA (Vidaza®) is a hypomethylating and chemotherapeutic agent indicated for AML. AZA's anti-cancer mechanism of action is believed to be twofold, the first being induction of DNA demethylation and the second being its anti-metabolite activity. Interestingly, it has also been found that AZA can increase the expression of the anti-phagocytic signal, CD47, and the pro-phagocytic signal, calreticulin, in myeloid malignancies. Based on these previous findings, we hypothesized that AML cells may be more efficaciously eliminated using a combination of AZA and 5F9 through enhancement of AML cell phagocytosis. We first tested this hypothesis using an in vitro phagocytosis assay. AML cells (i.e. GFP-expressing HL60 cells) were incubated for 24 hours with 3µM AZA and afterwards, the HL60-GFP cells were co-cultured for 2 hours with either human macrophages plus IgG4 control or 5F9 (10µg/ml). Phagocytosis of HL60 AML cells was calculated as a percentage of GFP-positive macrophages (i.e. the amount of macrophages that engulfed GFP-positive HL60 cells), compared to total number of macrophages. Results were normalized to a condition that produced the maximum amount of phagocytosis (100%). We found that the combination of AZA with 5F9 enhanced the human macrophage-mediated phagocytic elimination of HL60-GFP cells compared to either agent alone (Fig. 1). Next, we asked whether we could confirm our in vitro findings in vivo utilizing an aggressive AML xenograft mouse model. HL60-GFP cells (500,000 cells/per mouse) expressing luciferase were engrafted by intravenous injection into 6 - 8 week old immune-deficient NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. Three days post engraftment (PE), bioluminescence imaging was performed to assess AML engraftment based on total flux (photons/sec). Animals were randomized based on these values into 6 treatment cohorts with 8 animals per group. Treatment was performed as follows: (1) control (PBS) was initiated on day 4 PE and continued for 14 consecutive daily doses; (2) AZA (7.5 mg/kg) was initiated on day 4 PE and continued for 5 consecutive daily doses; (3) two cohorts of 5F9 (10mg/kg) were initiated at day 4 or day 7 PE and continued for 14 consecutive daily doses; and (4) two combination cohorts of AZA with 5F9 were initiated according to the 5F9 monotherapy dosing regimens. Routine bioluminescence imaging was performed during treatment and for several months after to assess AML burden and reoccurrence. Both combination cohorts inhibited AML growth as early as day 10 PE, and maintained elimination of growth and overall survival up to 255 days PE. In contrast, the AZA and 5F9 monotherapies initiated at day 7 PE (D7), decreased AML growth at day 10 PE, but failed to produce a durable response. Notably, as the AML expanded, all animals from the AZA cohort died by 46 days PE, and all animals from the 5F9 cohort died by 61 days PE. Of the 8 animals from the 5F9 cohort that received treatment on day 4 PE, only two animals demonstrated progressive disease and did not survive. The remaining animals from this cohort had no detectable AML cancer cells (Fig 2). In summary, the combination of 5F9 with AZA significantly enhanced the phagocytic elimination of AML cells by human macrophages in vitro, enhanced clearance of AML in vivo, and prolonged survival compared to single agent treatment with AZA or 5F9. These results support the rationale for investigating a combinatorial treatment of 5F9 and AZA in patients with AML. A clinical trial with this combination in patients with AML is currently ongoing (NCT03248479). Disclosures Feng: Forty Seven Inc: Employment, Equity Ownership. Gip:Forty Seven Inc: Equity Ownership. McKenna:Forty Seven Inc.: Equity Ownership. Zhao:Forty Seven Inc: Consultancy. Mata:Forty Seven Inc: Employment, Equity Ownership. Choi:Forty Seven Inc: Employment, Equity Ownership. Duan:Forty Seven Inc: Employment, Equity Ownership. Sompalli:Forty Seven Inc: Employment, Equity Ownership. Majeti:BioMarin: Consultancy; Forty Seven, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Weissman:Forty Seven, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Takimoto:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Chao:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Chen:Forty Seven Inc: Consultancy, Equity Ownership. Liu:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Volkmer:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties.

Bispecific SCORPION™ Molecules Effectively Redirect T-Cell Cytotoxicity Toward CD19-Expressing Tumor Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3722-3722
Author(s):  
Ruth A. Chenault ◽  
Rebecca Gottschalk ◽  
Gabriela Hernandez-Hoyos ◽  
Jennifer Wiens ◽  
Brian Gordon ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Mouse Serum ◽  
Cell Cytotoxicity ◽  
Employment Equity ◽  
Equity Ownership ◽  
T Cell Cytotoxicity

Abstract Abstract 3722 Background: Despite advances in treatments for B-cell leukemias and lymphomas, many patients ultimately relapse and succumb to disease following multiple courses of therapy. Bispecific antibody fragments that can simultaneously engage T cells and tumor cells have been shown, in the literature, to destroy tumor cells by effectively redirecting the cytotoxic function of T cells. T-cell engaging bispecific molecules linking anti-CD19 and anti-CD3 binding domains in the context of novel SCORPION™ (multi-specific protein therapeutic) proteins were evaluated both in vitro and in vivo for function and stability. Methods: Redirected T-cell cytotoxicity (RTCC) was measured by combining CD19 positive or negative cell lines with SCORPION proteins in the presence of human T cells. In a similar assay context, CFSE-labeled T cells were monitored for activation and proliferation. Functional RTCC assays were also used to analyze serum stability of SCORPION molecules in vitro and to complete an in vivo pharmacokinetic analysis. In vivo efficacy was assessed by monitoring the rate of tumor outgrowth of Ramos xenografts co-implanted with human peripheral blood mononuclear cells (PBMC) in NOD/SCID mice after treatment with SCORPION molecules. Results: SCORPION molecules potently mediate target-specific T-cell cytotoxicity toward tumor cell lines presenting cell surface CD19, with EC50 values for cytotoxicity at low pM concentrations. These molecules also demonstrate induction of T-cell activation and proliferation in the presence of target-bearing tumor cells but not in the absence of target expression. SCORPION molecules retain stable function following incubation at 37°C in mouse serum for up to a week in vitro, and pharmacokinetic analysis of SCORPION protein function in BALB/c mouse serum following intravenous administration resulted in half-life estimates of 69–84 hours. In efficacy studies conducted in NOD/SCID mice, SCORPION proteins significantly inhibited the outgrowth of Ramos tumor xenografts in the presence of human effector cells. Conclusion: SCORPION molecules targeting CD19 and CD3 effectively harness the cytotoxic activity of T cells to kill CD19 positive tumor cells both in vitro and in vivo and show potential for further investigation as possible therapeutic agents for B-cell malignancies. Disclosures: Chenault: Emergent BioSolutions: Employment. Gottschalk:Emergent BioSolutions: Employment. Hernandez-Hoyos:Emergent BioSolutions: Employment. Wiens:Emergent BioSolutions: Employment. Gordon:Emergent BioSolutions: Employment. Klee:Emergent BioSolutions: Employment, Equity Ownership. Bienvenue:Emergent BioSolutions: Employment. Dasovich:Emergent BioSolutions: Employment. Kumer:Emergent BioSolutions: Employment. Aguilar:Emergent BioSolutions: Employment. Bannink:Emergent BioSolutions: Employment, Equity Ownership. McMahan:Emergent BioSolutions: Employment, Equity Ownership. Natarajan:Emergent BioSolutions: Employment, Equity Ownership. Algate:Emergent BioSolutions: Employment, Equity Ownership. Blankenship:Emergent BioSolutions: Employment, Equity Ownership, Patents & Royalties.

scholarly journals Comparative Study of Obinutuzumab (GA101) Vs. Rituximab Against CD20+ rituximab-Sensitive and -Resistant Burkitt (BL) and Acute Lymphoblastic Leukemia (B-ALL): Potential Targeted Therapy in Patients with High Risk BL and Pre-B-ALL

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2251-2251 ◽  
Author(s):  
Aradhana Awasthi ◽  
Janet Ayello ◽  
Carmella van de Ven ◽  
Mona Elmacken ◽  
Christopher Reggio ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Children And Adolescents ◽  
Nk Cells ◽  
Tumor Cells ◽  
Lymphoblastic Leukemia ◽  
Tumor Burden

Abstract Background: Aggressive non-Hodgkin lymphoma (NHL) represents >90% of all NHL that occur in children and adolescents. Among all NHLs, Burkitt Lymphoma (BL) is the most common NHL in children and adolescents and has an excellent prognosis (≥80% 5 yrs, EFS) following short but intense multi-agent chemotherapy (Cairo et al. Blood, 2007). Patients who relapse with CD20+ B-NHL and B cell Acute lymphoblastic leukemia (B-ALL) have a dismal prognosis, often associated with chemotherapy resistance and may require alternative therapeutic strategies (Cairo et al. JCO, 2012, Barth/Cairo et al. BJH, 2013). Rituximab (RTX) in combination with FAB 96 chemotherapy is a safe and well-tolerated and is associated with >90% EFS in children with newly diagnosed and advanced mature B-Cell NHL (Goldman/Cairo et al. Leukemia, 2013). Resistance to RTX, however, may predispose patients with CD20+ B-NHL/ALL to an increase risk of relapse and/or disease progression (Barth/Cairo et al. BJH, 2012; Tsai et al. Cl. Can. Res, 2012,). Obinutuzumab, a novel glycoengineered type II CD20 antibody, has been shown to enhance cell death and ADCC vs. RTX (Herter et al, Clinc Canc Res, 2013), and was recently approved by FDA and EMA for first line treatment of CLL in combination with chlorambucil. Objective: To evaluate anti-tumor activity of obinutuzumab vs RTX against RTX resistant and sensitive BL and pre-B-ALL tumor targets in-vitro and in-vivo in xenografted NSG mice. Methods: Raji (CD20+) and Loucy (T-ALL, CD20-), (ATCC, Manhass, VA), U698-M (CD20+, DSMZ, Germany) and Raji-4RH (provided by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS. For in-vitro studies, tumor cells were incubated with 100 µg/ml obinutuzumab (supplied by Christian Klein, PhD, Roche Research & Early Development, Zurich), and/or RTX for 24 hrs. Cell death was evaluated by staining with AnnexinV/7AAD and analysis by flow-cytometry. Loucy cells (CD20-) were used as the negative control. ADCC were performed with K562-IL-15-41BBL expanded NK cells (Ayello/Cairo et al. ASH, 2010) at 20:1 effector: target ratio (E: T, n=3) using an europium release assay (Perkin-Elmer).The lentiviral construct, pSico PolII-eGFP-Luc2, was transfected into Raji, Raji 4RH (RTX resistant), U698M and Loucy for in vivo evaluation by BLI. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), mice, bred in-house under pathogen free conditions, were divided into 5 groups: PBS only (control), isotype control (IgG), obinutuzumab 10 mg/kg, obinutuzumab (30 mg/kg), and RTX (30 mg/kg). Mice were xenografted with intravenous injections of Luc+ Raji, Raji4RH, U698M and Loucy cells at 5x106 tumor cells/mouse. 6-8 days after tumor cell injection, mice were then injected every 7 days with the respective therapy for 8 weeks. Mice were monitored for tumor burden and survival for up to 12 weeks ( approx. 80 days) via bioluminescent imaging (BLI) using the IVIS Spectrum system. Results: Obinutuzumab compared to RTX (100 mg/ml, 24hrs), significantly enhanced cell death in Raji 45.1±3.3% vs 32.7±6.8%, (p=0.005), Raji4RH 15.8±2.2% vs 2.1±1.5% (p=0.001) and U698-M 40.5±2.9 % vs 26.36±2.6% (p=0.001) n=6. Obinutuzumab vs RTX also elicited a significant increase ADCC with K562-IL15-41BBL expanded NK cells, in Raji 73.8±8.1% vs 56.81±4.6% (p=0.001), Raji-4RH 40.0±1.6% vs 0.5±1.1%, (p=0.001), and U-698-M 70.0±6 % vs. 45.56± 0.1% (p=0.001) n=3. Further, we found that, in vivo, obinutuzumab was significantly more effective than RTX when administered at the same doses in BL (RTX resistant/sensitive) and pre-B-ALL xenografts. Overall survival in mice receiving 30 mg/kg of obinutuzumab was significantly increased when compared to mice receiving 30 mg/kg of RTX in BL; Raji (p=0.05), Raji4RH (p=0.024) and U698-M (p=0.03) (Figure1: A, B and C). Conclusion: Obinutuzumab significantly enhances cell death and NK mediates ADCC in sensitive and RTX resistant CD20+ B-NHL and B-ALL compared to RTX. These preliminary studies also demonstrate that RTX sensitive/resistant BL and pre-B-ALL xenografted mice display significantly increased survival when given 30 mg/kg of obinutuzumab and decreased tumor burden in BL and Pre-B-ALL xenografts compared to an equal dose of RTX. Obinutuzumab may be a novel agent to investigate as adjuvant therapy in patients with relapsed refractory CD20+ B-NHL and/or B-ALL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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