R-Etodolac and a Novel Indole-Pyran Structural Analog, SDX-308, Induce Cytotoxicity and Overcome Drug Resistance in Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1580-1580
Author(s):  
Hiroshi Yasui ◽  
Teru Hideshima ◽  
Paola Neri ◽  
Janice Jin ◽  
Tanyel Kiziltepe ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Clinical Trials ◽  
Cell Lines ◽  
Oral Treatment ◽  
Structural Analog ◽  
Scid Mice ◽  
Lymphocytic Leukemia ◽  
Parp Cleavage

Abstract SDX-101, the less toxic R-isomer of the commercially available non-steroidal inflammatory drug R,S-etodolac (Lodine®), lacks COX inhibitory activity and is being investigated in Phase II clinical trials in chronic lymphocytic leukemia. Recently, we reported that R-etodolac, at clinically relevant concentrations, induces potent in vitro cytotoxicity in drug-sensitive and conventional drug-resistant multiple myeloma (MM) cell lines, as well as in primary tumor cells from MM patients. R-etodolac triggers caspase/poly-ADP-ribose polymerase (PARP) cleavage and downregulates of cyclin D1 expression (Yasui et al. Blood 2005). Importantly, R-etodolac at sub-cytotoxic doses upregulates Mcl-1s and synergistically enhances dexamethasone (Dex)-induced caspase-dependent apoptosis in Dex-sensitive MM.1S cells. Combination of R-etodolac with Dex enhances cytotoxicity in Dex resistant OPM1 MM cells and in Dex-resistant patient MM cells in vitro. We further studied the in vivo anti-tumor effect of combined R-etodolac and Dex in SCID mice injected subcutaneously with OPM1 human MM cells. While oral treatment of SCID mice with R-etodolac alone (250 mg/kg/d) or Dex alone (1 mg/kg/d) did not induce any significant reduction of tumor volume compared with control (PBS), the combination of R-etodolac and Dex inhibited tumor growth synergistically (synergism quotient = 1.6) and significantly (p = 0.023), suggesting that R-etodolac may reverse Dex resistance in MM. Finally, we demonstrated that racemic SDX-308, a novel indole-pyran structural analog of etodolac, has 10-fold more potent cytotoxicity than R-etodolac in MM cell lines both sensitive and resistant to conventional therapies, as well as in patient’s MM cells. Moreover, SDX-308, like R-etodolac, can overcome the viability and proliferative enhancing effects of exogeneous IL-6, IGF-1, or bone marrow stroma cells. These combined observations indicate that SDX-308 is a promising more potent second generation analog of R-etodolac for MM therapy. Our data suggest that R-etodolac and its novel analog SDX-308 overcome resistance to some conventional therapeutics used for MM, and provide preclinical rationale to conduct clinical trials of R-etodolac and SDX-308 to treat MM.

The Multi-Targeted Receptor Tyrosine Kinase Inhibitor, ABT-869, Induces Apoptosis of AML Cells Both In Vitro and In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 616-616 ◽  
Author(s):  
Deepa B. Shankar ◽  
Jenny C. Chang ◽  
Bertrand Parcells ◽  
Salemiz Sandoval ◽  
Junling Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Scid Mice ◽  
Parp Cleavage

Abstract Children with acute myeloid leukemia (AML) have less than 60% overall survival despite aggressive chemotherapy and bone marrow transplantation. Only one third of the adult patients diagnosed with AML will be cured. AML blast cells from up to 30% of patients express a constitutively active receptor tyrosine kinase, FLT3-ITD, which contains an internal tandem duplication in the juxtamembrane domain. Patients with FLT3-ITD have a worse prognosis. ABT-869 is a novel multi-targeted small molecule inhibitor of receptor tyrosine kinases and is a potent inhibitor of FLT3, c-Kit, and all members of the VEGF and PDGF receptor families. To determine the effects of ABT-896 on AML cells, we treated AML cell lines, primary cells, and tumors in xenograft models with varying concentrations of the drug. In vitro viability assays showed that ABT-869 inhibited the growth of two different cell lines, MV-4-11 (human AML cell line that expresses FLT3-ITD) and BAF3-ITD (murine B-cell line stably transfected with the FLT3-ITD) at an IC50 of 10nM. ABT-869 was also effective against another mutation of FLT3, D835V, but at higher concentrations (IC50 of 100nM). Phosphorylation of FLT3 and activation of downstream signaling molecules, STAT5 and ERK, were inhibited by ABT-869 in a concentration-dependent manner. Cells were also stained with Annexin V-FITC and Propidium Iodide, and analyzed using FACS. ABT-869 induced apoptosis, caspase-3 activation, and PARP cleavage after 48 hours. To examine the in vitro effects of ABT-869 on normal hematopoietic progenitor cells, we performed methylcellulose-based colony assays with human bone marrow. No significant difference was observed in the number and type of colonies formed using BM cells treated with ABT-869 or control, up to a concentration of 1 micromolar. These results suggest that ABT-869 is not toxic to normal bone marrow progenitor cells at concentrations that are effective against AML cells. To examine the effects of ABT-869 in vivo, we treated SCID mice injected with MV-4-11, Baf3-ITD, Baf3-D835V, or Baf3-WT cells, with oral preparations of ABT-869. Complete regression of MV-4-11 tumors was observed in mice treated with ABT-869 at 20 and 40 mg/kg/day. No adverse effects were detected in the peripheral blood counts, bone marrow, spleen or liver. Histology of the tumors from the control-treated group showed a high degree of proliferation by Ki-67 staining, increased mitotic figures, and a well-defined tumor mass. In contrast, the tumors from mice treated with ABT-869 showed a number of apoptotic bodies by TUNEL staining and the presence of reactive, inflammatory cells. Interestingly, we also observed that mice that received ABT-869 the day after injection of AML cells remained tumor-free for over 2 months in contrast to the mice receiving the vehicle alone. Inhibition of FLT3 phosphorylation was demonstrated in the tumors from mice treated with ABT-869. We are evaluating the activity of ABT-869 treatment of SCID mice injected with Baf3-ITD, Baf3-D835V, or Baf3-WT cells. NOD-SCID mouse models are currently being used to analyze the effects of ABT-869 on primary AML cells in vivo. Our preclinical studies demonstrate that ABT-869 is effective and nontoxic, and provide rationale for the treatment and prevention of relapse in AML patients.

A Humanized Anti-Ganglioside GM2 Antibody, BIW-8962, Exhibits ADCC/CDC Activity against Multiple Myeloma Cells and Potent Anti- Tumor Activity in Mouse Xenograft Models.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1718-1718 ◽  
Author(s):  
Toshihiko Ishii ◽  
Asher Alban Chanan-Khan ◽  
Jazur Jafferjee ◽  
Noreen Ersing ◽  
Takeshi Takahashi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Phase 1 ◽  
Xenograft Model ◽  
Surface Expression ◽  
Scid Mice ◽  
Subcutaneous Xenograft ◽  
Anti Tumor Activity

Abstract BIW-8962 is a humanized anti-ganglioside GM2 (GM2) monoclonal antibody, produced by Poteligent technology to enhance ADCC activity. GM2 is expressed on many cancer cells including multiple myeloma (MM), small cell lung cancer and glioma cells. In this study, we evaluated the anti-myeloma activity of BIW-8962 in preclinical myeloma models both in vitro and in vivo. Expression of GM2 was analyzed in 15 human MM cell lines by FCM. Eleven out of 15 MM cell lines had positive surface expression of GM2. GM2 as a potential target was then verified in primary MM samples obtained from patients. Eleven out of 15 samples were positive for GM2. We then used two GM2 positive MM cell lines (U266B1 and KMS-11) and evaluated ADCC and CDC activity of BIW-8962 in vitro. BIW-8962 exhibited a potent ADCC and less potent CDC activity. In vivo anti-tumor activity of BIW-8962 was then examined using the standard subcutaneous xenograft model; KMS-11 was inoculated in the flank of SCID mice. BIW-8962 (intravenously administered biweekly for 3 weeks) exhibited a potent anti-tumor activity from as low a dose level as 0.1 mg/kg. Furthermore, in a more clinically relevant model, in which OPM-2/GFP (GM2 positive MM cell line) cells were intravenously inoculated into SCID mice with preferentially tumor growth within the bone marrow microenvironment, BIW-8962 (intravenously administered biweekly for 4 weeks, 10 mg/kg) suppressed OPM-2/GFP cell growth and serum M protein elevation, demonstrating in vivo anti-myeloma effect of BIW-8962. Our preclinical investigations rationalize clinical evaluation of BIW-8962 in patients with MM. Currently BIW-8962 is being investigated in a Phase 1 study in patients with multiple myeloma.

Mcl-1 Is Overexpressed in Multiple Myeloma and Correlates with Disease Severity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1419-1419
Author(s):  
Soraya Wuilleme-Toumi ◽  
Nelly Robillard ◽  
Patricia Gomez-Bougie ◽  
Philippe Moreau ◽  
Steven Le Gouill ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Disease Severity ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Lymphocytic Leukemia ◽  
Myeloma Cells

Abstract Multiple Myeloma (MM) is a fatal malignancy of B-cell origin characterized by the accumulation of plasma cells within the bone marrow. The expression of the pro-survival members of the Bcl-2 family has been shown to be a key process in the survival of myeloma cells. More particularly, Mcl-1 expression turned out to be critical for their survival. Indeed, knockdown of Mcl-1 by antisenses induces apoptosis in myeloma cells. Finally, Mcl-1 was found to be the only anti-apoptotic Bcl-2 family member which level of expression was modified by cytokine treatment of myeloma cells. For these reasons, we have evaluated the expression of Mcl-1 in vivo in normal, reactive and malignant plasma cells (PC) i.e., myeloma cells from 55 patients with MM and 20 human myeloma cell lines using flow cytometry. We show that Mcl-1 is overexpressed in MM in comparison with normal bone marrow PC. Forty-seven percent of patients with MM at diagnosis (p=.017) and 80% at relapse (p=.014 for comparison with diagnosis) overexpress Mcl-1. Of note, only myeloma cell lines but not reactive plasmocytoses have abnormal Mcl-1 expression, although both plasmocyte expansion entities share similar high proliferation rates (>20%). Of interest, Bcl-2 as opposed to Mcl-1, does not discriminate malignant from normal PC. This shows that the overexpression of Mcl-1 is clearly related to malignancy rather than to proliferation. It will be important to know whether the overexpression of Mcl-1 is related to an abnormal response to cytokines like Interleukin-6 or to mutations of the promoter of the Mcl-1 gene as already described in B chronic lymphocytic leukemia. Finally, level of Mcl-1 expression is related to disease severity, the highest values being correlated with the shortest event-free survival (p=.01). In conclusion, Mcl-1 which has been shown to be essential for the survival of human myeloma cells in vitro is overexpressed in vivo in MM and correlates with disease severity. Mcl-1 represents a major therapeutical target in MM.

Toll-Like Receptor 7-Targeting of Human B-Lineage Acute Lymphocytic Leukemia Induces Immunogeneicity and Apoptosis of Leukemia Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 706-706 ◽  
Author(s):  
Xueqing Liang ◽  
Jakub Tolar ◽  
Jeffery S. Miller ◽  
Tucker W. LeBien ◽  
Bruce R. Blazar ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Acute Lymphocytic Leukemia ◽  
Scid Mice ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Tlr7 Agonist ◽  
B Lineage

Abstract Acute lymphocytic leukemia (ALL) is the most common childhood leukemia and remains a difficult disease with poor survival in patients who have failed standard therapy. New therapeutic strategies are needed to achieve longer survival and improved cure rates in both pediatric and adult ALL patients. In this study, we show human B-lineage acute lymphocytic leukemia (B-ALL) cell lines (6/6 tested) and CD19+CD10+ primary B-ALL cells from patients (8/8 tested) express TLR7 mRNA by real-time RT-PCR and TLR7 proteins by Western blot. Triggering TLR7 on B-ALL cells with a TLR7 agonist (imiquimod) significantly increases the cell surface expression of molecules essential for T cell activation (CD40, CD54, CD80, CD86, and HLA-DR), the ligands for NKG2D and ligands for natural cytotoxicity receptors (NKp30, NKp44, and NKp46) which regulate NK-mediate killing. Thus, TLR7 signaling enhances the immunogenicity of B-ALL cells and makes them more suitable targets for T cell and NK cell mediated attack. Most importantly, TLR7 agonists strongly suppress in vitro growth of B-ALL cell lines (RS4;11, BLIN-1) and induces profound apoptosis of primary B-ALL cells from patients in culture in a TLR7 agonist dose-dependent manner. Both t(4;11)-positive RS4;11 cells and t(4;11)-negative BLIN-1 cells proliferate rapidly in culture with a 30–40 fold increases of leukemia cell number in 7 days. The addition of TLR7 agonist at 10 ug/ml fully inhibit the growth of RS4;11 and BLIN-1 cells in culture. Furthermore, TLR7 agonist treatment dramatically induces apoptosis of primary B-ALL cells isolated from the patients (2/2 with t(9;22), 6/6 without t(9;22)) with 0.4%–13.3% leukemia cells left at day 5 of culture. The TLR7 agonist-mediated apoptotic death of B-ALL cells was conformed by viable cell counts, TMRE staining, and, Western blots of the activation and cleavage of caspases. To study the in vivo therapeutic effects of TLR7 agonist against human B-ALL, RS4;11 and BLIN-1 cells were luciferase labeled and injected into NOD/SCID mice. Both RS4;11 and BLIN-1 leukemia cells engrafted in multiple organs (BM, spleen, liver, lymph nodes, kidney) resulting in uniform lethality of RS4;11 mice in 8 weeks and BLIN-1 mice in 12 weeks, respectively. Flow cytometry and tissue staining results confirmed that these organs were massively infiltrated with human CD45+19+ leukemia cells. To determine whether TLR7 preincubation of RS4;11 or BLIN-1 cells would prolong survival due to an apoptotic effect, cohorts of mice were injected with a lethal dose of RS4;11 or BLIN-1 cells with or without pre-incubation with TLR7 agonist. Mice receiving TLR7 agonist pre-pretreated B-ALL cells had a significant increase in long-term survival rate and significant reduction in tumor burden at the time points evaluated. These in vivo results confirm previous in vitro findings and suggest that TLR agonist-treated B-ALL cells are programmed to die. The antitumor efficacy of systemic administration of TLR7 agonist in NOD/SCID mice with established B-ALL is being investigated using these xenograft mouse models. These results form the basis for a clinical trial of systemic TLR7 agonist administration for treating patients with B-ALL. In summary, we have shown that TLR7 targeting increases B-ALL immunogenicity and directly induces B-ALL apoptosis, providing new insights into the biology and therapy of B-ALL.

Novel Etodolac Analog SDX-308 (CEP-18082) Induces Cytotoxicity in Multiple Myeloma Cells Associated with Inhibition of Wnt/β-Catenin Pathway.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5005-5005 ◽  
Author(s):  
Hiroshi Yasui ◽  
Teru Hideshima ◽  
Hiroshi Ikeda ◽  
Enrique M. Ocio ◽  
Tanyel Kiziltepe ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Clinical Trials ◽  
Tumor Cells ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Significant Inhibition ◽  
Lymphocytic Leukemia ◽  
Parp Cleavage ◽  
Target Proteins

Abstract In a previous study we reported that R-enantiomer of etodolac (R-etodolac) which is under investigation in Phase II clinical trials in chronic lymphocytic leukemia, induces potent cytotoxicity at clinically relevant concentrations in both drug-sensitive and drug-resistant multiple myeloma (MM) cell lines, as well as in patient MM tumor cells. In this study, we demonstrated that SDX-308 (CEP-18082), a novel analog of etodolac, has more potent cytotoxicity than R-etodolac against MM cell lines and tumor cells from patients with refractory MM. It also induces cytotoxicity against bortezomib-resistant tumor cells. The molecular mechanisms whereby SDX-308 triggers MM cell cytotoxicity were next delineated. SDX-308 induces apoptosis via caspase-8/-9/-3 activation and poly(ADP-ribose) polymerase PARP cleavage. It strongly inhibits Wnt/β-catenin pathway by blockade of nuclear translocation of β-catenin, followed by significant inhibition of transcription and expression of target proteins. These target proteins include cell cycle regulatory c-myc molecules and anti-apoptotic survivin molecules. Neither interleukin-6 nor insulin-like growth factor-1, which induce MM cell growth and abrogate Dex-induced apoptosis, protect against growth inhibition triggered by SDX-308. Importantly, growth of MM cells adherent to bone marrow (BM) stromal cells is also significantly inhibited by SDX-308. Our data therefore indicate that the novel etodolac analog SDX-308 has more potent cytotoxicity in MM cells than R-etodolac even in the context with BM microenvironment, providing the preclinical rationale to conduct clinical trials of this agent to improve patient outcome in MM.

Cytotoxic activity of the maytansinoid immunoconjugate B-B4–DM1 against CD138+ multiple myeloma cells

Blood ◽  
2004 ◽  
Vol 104 (12) ◽  
pp. 3688-3696 ◽  
Author(s):  
Pierfrancesco Tassone ◽  
Victor S. Goldmacher ◽  
Paola Neri ◽  
Antonella Gozzini ◽  
Masood A. Shammas ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Antitumor Activity ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Scid Mice ◽  
Dependent Manner ◽  
In Vivo Antitumor Activity ◽  
Mouse Tissues

We tested the in vitro and in vivo antitumor activity of the maytansinoid DM1 (N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine), a potent antimicrotubule agent, covalently linked to the murine monoclonal antibody (mAb) B-B4 targeting syndecan-1 (CD138). We evaluated the in vitro activity of B-B4–DM1 against a panel of CD138+ and CD138- cell lines, as well as CD138+ patient multiple myeloma (MM) cells. Treatment with B-B4–DM1 selectively decreased growth and survival of MM cell lines, patient MM cells, and MM cells adherent to bone marrow stromal cells. We further examined the activity of B-B4–DM1 in 3 human MM models in mice: (1) severe combined immunodeficient (SCID) mice bearing subcutaneous xenografts; (2) SCID mice bearing green fluorescent protein–positive (GFP+) xenografts; and (3) SCID mice implanted with human fetal bone (SCID-hu) and subsequently injected with patient MM cells. Tumor regression and inhibition of tumor growth, improvement in overall survival, and reduction in levels of circulating human paraprotein were observed in mice treated with B-B4–DM1. Although immunohistochemical analysis demonstrates restricted CD138 expression in human tissues, the lack of B-B4 reactivity with mouse tissues precludes evaluation of its toxicity in these models. In conclusion, B-B4–DM1 is a potent anti-MM agent that kills cells in an antigen-dependent manner in vitro and mediates in vivo antitumor activity at doses that are well tolerated, providing the rationale for clinical trials of this immunoconjugate in MM.

scholarly journals Effects of Proteasome Inhibition in the Anti-Tumor Activity of Venetoclax in Mantle Cell Lymphoma Pre-Clinical Models

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2942-2942
Author(s):  
Shalin K. Kothari ◽  
Cory Mavis ◽  
Juan Gu ◽  
Francisco J. Hernandez-Ilizaliturri
Keyword(s):  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Scid Mice ◽  
Parp Cleavage ◽  
Synergistic Activity ◽  
Clinical Models ◽  
Anti Tumor Activity

Abstract Background: At the molecular level, mantle cell lymphoma (MCL) is characterized by the deregulation of Bcl-2 family members (Mcl-1, BIM) and cell cycle (cyclin D1) regulatory proteins. Perhaps related to this, the clinical outcome of MCL continues to be poor specially for those patients with disease progression after high dose chemotherapy and autologous stem cell rescue and/or BTK inhibitors, stressing the need to develop novel therapeutic strategies or optimize current available options. Venetoclax (V), a highly selective Bcl-2 inhibitor, has shown modest activity against relapsed/refractory MCL. Over-expression of Mcl-1 has been postulated to be a mechanism of resistance to V limiting its anti-tumor activity in subtypes of lymphoma including MCL. The lethality by proteasome inhibitors (PIs) has been associated with changes in the Bcl-2 family members (Bax, Noxa, Mcl-1 and Bcl-XL) in lymphoma pre-clinical models, making them ideal agents to combine with V. To this end, we studied the anti-tumor activity of combining PIs with V in MCL pre-clinical models. Materials and Methods: A panel cytarabine sensitive (Rec-1, Jeko, Granta, HBL-2, Z-138 and Mino) and resistant (araC) cell lines (Jeko araC, HBL-2 araC, and Mino araC) were exposed to V, Bortezomib (BTZ), carfilzomib (CFZ), or ixazomib (IXZ) for 24, 48 and 72 hours. Cell viability was calculated measuring the ATP content. IC50 drug concentrations were calculated for each agent. Subsequently, MCL cell lines were exposed to escalating doses of V (0.001uM-5uM) and CFZ (1.5625nM-50nM), BTZ (3.125nM-100nM) or IXZ (3.125nM-100nM). In addition, primary tumor cells isolated from B-cell lymphoma patients (N=21) including MCL patients were exposed to V +/- BTZ or CFZ for 48 hrs. Cell viability was determined by Cell Titerglo. Coefficient of synergy were calculated using CalcuSyn software program. Induction of apoptosis was detected by Annexin V/Propidium iodine staining and PARP cleavage. Changes in Bcl-2 and cell cycle regulatory proteins were evaluated by Western blotting in HBL-2 cells. For in vivo experiments, 6-8 weeks old severe combined immunodeficiency (SCID) mice were inoculated with 10x106 HBL-2 cells via tail vein injection (IV). Subsequently, SCID mice were treated with V (100mg/kg/dose via gastric lavage on days 3-7, 10-14 and 17-21) or IXZ (6mg/kg/dose IV days 3, 6, 10, 13, 17 and 20) or combination of both agents. A group of untreated animals was used as a control. Differences in survival were evaluated between treatment groups. Results: In vitro exposure of MCL cell lines to either V, BTZ, CFZ, and IXZ induced cell death in a dose- and time-dependent manner. Significant synergistic activity was observed by combining both V with CFZ or IXZ at known sub-therapeutic and therapeutic doses of individual agents measured by ATP content and apoptosis potential. Anti-tumor activity was observed in cytarabine sensitive and resistant cell lines. Similar findings were observed in primary tumor cells isolated from B-cell lymphoma patients. In vitro exposure of MCL cell lines with the lowest IC50 (HBL-2) to V and PIs (BTZ, CFZ, or IXZ) resulted in the upregulation of Noxa, BIM, Mcl-1 cleavage form (pro-apoptotic) and downregulation of Bcl-XL leading to PARP cleavage and apoptosis. In vivo treatment of MCL bearing SCID mice with V resulted in significant anti-tumor activity when compared to single agent IXZ treated or control animals. Of interest, MCL bearing SCID animals treated with V and IXZ exhibited a better disease control and the survival was longer than SCID animals treated with V or IXZ single agent (P<0.05). Conclusion: Our data suggests that V exhibits strong synergistic activity with PIs, especially with CFZ (in vitro) or IXZ (in vitro and in vivo). Together, our data supports the evaluation of V in combination with readily available novel PIs (IXZ or CFZ) in relapsed/refractory MCL. (Supported by LRF grant 555463, an NIH grant R01 CA136907-01A1 and a grant from The Roswell Park Cancer Institute Alliance Foundation) Disclosures No relevant conflicts of interest to declare.

Targeting CD20 and CD22 with Rituximab in Combination with CMC-544 Results in Improved Anti-Tumor Activity Against Non-Hodgkin’s Lymphoma (NHL) Pre-Clinical Models.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1473-1473 ◽  
Author(s):  
Francisco J. Hernandez-Ilizaliturri ◽  
Suresh Devineni ◽  
Sarika Arora ◽  
Joy Knight ◽  
Myron S. Czuczman
Keyword(s):  
Cell Lines ◽  
Median Survival ◽  
Flow Cytometric Analysis ◽  
Scid Mice ◽  
In Vivo Studies ◽  
Parp Cleavage ◽  
Rank Test ◽  
Anti Tumor Activity

Abstract CMC-544 is an IgG4 CD22-humanized targeted immunoconjugate of calicheamicin. Upon CD22 biding, CMC-544 internalizes inducing cell apoptosis in vitro/in vivo against NHL with minimal modulation of the immune system. In addition to direct cell-death, rituximab, anti-CD20 monoclonal antibody (mAb), can induce complement-dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC). Combination of mAbs directed against unique tumor-associated targets can potentially enhance anti-tumor activity. Objectives: To study the effects of targeting CD20 and CD22 using rituximab in combination with CMC-544. Material and Methods: For in vitro studies, a panel of NHL cell lines [Raji, SUDHL-4, SU-DHL-10, Ramos, and two rituximab resistant cell lines (RRCL), [2R and 4RH] were exposed to CMC-544 (0.4pg/ml to 0.4μg/ml) +/− Rituximab (10μg/ml) or appropriate controls for 24 or 48 hrs. DNA synthesis was quantified by [H3]Thymidine incorporation and apoptosis was detected by multiparameter flow cytometric analysis and Poly(ADP-ribose) polymerase (PARP) cleavage. For ADCC/CMC studies, 51Cr-labeled NHL cells were exposed to CMC-544 (0.4μg/ml) prior to treatment with rituximab (10mg/ml) and peripheral blood mononuclear cells (Effector: Target ratio 40:1) or human serum, respectively. 51Cr-release was measured and the percentage of lysis was calculated. Statistical analysis of results was performed using the Chi-square test. For in vivo studies, 6–8-week old SCID mice were inoculated via tail vein injection (iv) with Raji cells (1 × 106 on day 0). Animals were assigned to receive 8 doses of either saline, CMC-544 (40μg/kg/dose), rituximab (10mg/kg/dose iv), or rituximab alternating with CMC-544. Antibodies were administered iv every other day starting on day +5. Survival analysis was performed by Kaplan-Meier curves and p values calculated using log rank test. Results: Exposure of various NHL cells to CMC-544 resulted in a dose-dependent significant decrease in cell proliferation and apoptosis. Rituximab-associated CMC or ADCC was not further enhanced by CMC-544 in vitro. In vivo studies demonstrated that the mean survival for animals treated with CMC-544 increased when compared to control mice [64 days (95% C.I. 48–80; 22 days (95% C.I. 21–23), respectively, P=0.001]. To a lesser degree rituximab monotherapy resulted in significant anti-tumor effects and the median survival reached 44 days (95% C.I. 31–56). Notably, animals treated with rituximab in combination with CMC-544 had the longest median survival (not reached at +90 days, log rank P=0.007). Conclusions: Our data suggest targeting CD20 and CD22 by rituximab and CMC-544 resulted in a longer survival in lymphoma-bearing SCID mice. Clinical trials with this combination will be needed to confirm these results for the treatment of lymphoma patients

An Fc-Engineered Humanized Anti-CD40 Monoclonal Antibody, XmAb5485, Exhibits Potent Activity in Vitro and in Vivo against Non-Hodgkin Lymphoma, Chronic Lymphocytic Leukemia and Multiple Myeloma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 881-881 ◽  
Author(s):  
Eugene A. Zhukovsky ◽  
Holly Horton ◽  
Matthias Peipp ◽  
Erik Pong ◽  
Matthew Bernett ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Line ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Lymphocytic Leukemia ◽  
Non Hodgkin Lymphoma

Abstract CD40, a transmembrane glycoprotein belonging to the tumor necrosis factor receptor family, is an attractive target for cancers of lymphoid origin since it is expressed on most mature B-cell malignancies, some early B-cell acute lymphocytic leukemias, and multiple myeloma. Finding efficient therapies for multiple myeloma (MM), chronic lymphocytic leukemia (CLL) and rituximab-refractory Non-Hodgkin Lymphoma (NHL) represents an unmet need. Several anti-CD40 antibodies, both agonistic and antagonistic, have demonstrated objective responses in early clinical NHL trials and thus validated this antigen as a target for lymphoproliferative diseases. Here we present the characterization of a novel Fc-engineered and humanized anti-CD40 antibody, XmAb®5485, that was generated using our XmAb antibody engineering technology. This antibody is highly cytotoxic against lymphoma, leukemia and multiple myeloma cell lines as well as primary cancer cells. XmAb5485 is characterized by: i) increased affinity for Fc gamma receptors (FcgR), ii) improved effector function, and iii) significantly increased antitumor potency. We investigated several direct and indirect (Fc-mediated) mechanisms of antibody-mediated cytotoxicity in vitro. The potency (EC50) of XmAb5485 in antibody-dependent cell-mediated cytotoxicity (ADCC) increased up to 150-fold relative to the native non Fc-engineered version (anti-CD40 IgG1) of the antibody in a screen of Burkitt’s lymphoma [BL], CLL and MM-derived cell lines. In the same cell lines, ADCC potency and maximal efficacy (% lysis) of XmAb5485 were also superior to that of rituximab: 74- and 1.3-fold higher in CLL, 12.5- and 1.4-fold higher in BL, and 190- and 1.9-fold higher in MM. In a MM cell line with low density of CD40 expression (~3500 per cell) XmAb5485 facilitated efficient ADCC whereas anti-CD40 IgG1 was virtually ineffective. Furthermore, using a BL cell line (Ramos) XmAb5485 displayed antibody-dependent cellular phagocytosis (ADCP) with potency and efficacy increased relative to rituximab (15- and 1.6-fold) and anti-CD40 IgG1 (5- and 1.2-fold). XmAb5485 also exhibited anti-proliferative apoptotic activity that was similar to that of rituximab. Ex vivo, XmAb5485 mediated potent ADCC of multiple primary patient-derived CLL, MCL, and plasma cell leukemia (PCL, an aggressive form of MM) cells, with substantially increased potency and efficacy relative to rituximab; in contrast, anti-CD40 IgG1 displayed minimal or no activity in these primary tumor cells. In vivo, in an established large (210–350 mm3) sc Ramos tumor xenograft model, 6 mg/kg XmAb5485 cured 80% of mice of detectable tumors and displayed statistically significant superiority over anti-CD40 IgG1. In contrast, only 7% of animals in the rituximab cohort were cured. In summary, our data suggest that XmAb5485, an anti-CD40 Fc variant antibody engineered for increased effector function, is a promising next-generation immunotherapeutic for leukemias, lymphomas, and multiple myeloma.

Hypoxia Induces Pgp-Mediated Carfilzomib Resistance in Multiple Myeloma Cells and HIF Inhibition Significantly Enhances Sensitivity and Response to Carfilzomib In Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1286-1286
Author(s):  
Barbara Muz ◽  
Joseph Abraham ◽  
Feda Azab ◽  
Pilar De La Puente ◽  
Nicholas Potter ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Progression ◽  
Cell Lines ◽  
Induced Resistance ◽  
Tumor Size ◽  
Research Funding ◽  
Scid Mice ◽  
Tumor Initiation

Abstract Introduction: A plasma cell malignancy called multiple myeloma (MM) is an incurable cancer in which a majority of patients with refractory disease become resistant to therapy. The introduction of carfilzomib, a proteasome inhibitor, significantly improved the clinical outcome of the MM treatment. However, MM patients develop resistance to carfilzomib and relapse. In this study, we investigated the role of hypoxia and P-gp in the carfilzomib-resistance in MM cells in vitro and in vivo, and its therapeutic translational potential using a HIF inhibitor, PX-478. Methods: In vitro, MM cell lines were treated with carfilzomib under normoxic (21% O2) or hypoxic (1% O2) conditions and cell survival was analyzed by MTT assay. The activity of P-gp was assessed by testing the efflux of a known P-gp substrate, RhodamineB (RhoB), in the hypoxic and normoxic conditions by measuring the intracellular RhoB content using flow cytometry. Moreover, we have tested the effect of HIF inhibition using PX-478 on the P-gp activity as well as the response to carfilzomib in hypoxic and normoxic cells. In vivo, we tested the effect of HIF inhibition on tumor initiation, where MM1s-Luc-GFP cells were injected intravenously (IV) into SCID mice, which were treated instantaneously with PX-478 (10mg/kg) three times a week. Furthermore, we tested the effect of PX-478 on MM tumor response to carfilzomib (measuring tumor size and mice survival). MM1s-Luc-GFP cells were injected IV into SCID mice, tumors grew for 3 weeks and the mice were then randomly divided into 4 groups treated with (1) vehicle (Captisol), (2) PX-478 (10mg/kg) alone, (3) carfilzomib (5mg/kg) alone, or (4) a combination of PX-478 (10mg/kg) and carfilzomib (5mg/kg). PX-478 or vehicle were administered by oral gavage three times a week (day 1, day 3, day 5), while vehicle and carfilzomib were injected IV twice a week (day 4 and day 5). Tumor size was imaged using bioluminescence imaging (BLI) and mice survival was followed for 70 days. Results: In vitro, we found that hypoxia induced resistance to carfilzomib in five MM cell lines. Moreover, hypoxia also increased activity of P-gp by causing decreased intracellular RhoB content in hypoxic MM cells. The HIF inhibitor, PX-478, as well as the P-gp inhibitor, tariquidar, reversed the activation of P-gp in hypoxic cells, while the combination of PX-478 and tariquidar did not induce further inhibition of P-gp activity. Furthermore, the combination of PX-478 or tariquidar with carfilzomib reversed the hypoxia-induced resistance in MM. However, tariquidar and other P-gp inhibitors have shown low selectivity and high toxicity in clinical trials; therefore, for our in vivo experiments we chose to inhibit HIF in order to reverse the hypoxia-induced P-gp-mediated resistance to carfilzomib in MM cells. In vivo, in the MM tumor-initiation model, our study revealed that the HIF inhibitor, PX-478, significantly delayed the tumor progression and extended survival in which all control mice died between 42-52 days, while the experiment was stopped at 70 days, with all PX-478-treated mice still alive. In the established in vivo tumor model, low dose carfilzomib alone delayed the progression by BLI but did not improve survival (vehicle and carfilzomib-treated mice died between 21-28 days after treatment). Despite the fact that PX-478 did not decrease tumor progression as shown by BLI compared to the vehicle-treated mice, it significantly extended the survival of the mice (animals died between 38-48 days). The combination of carfilzomib and PX-478 significantly decreased the proliferation of tumor shown by BLI (less than 5% of the growth at day 28), as well as considerable increase in survival (the experiment was stopped at 70 days with 100% of the group alive). Conclusions: We identified a novel resistance mechanism to carfilzomib in MM, in which hypoxia induces P-gp-mediated resistance to carfilzomib. Inhibition of the hypoxic response in MM cells by the HIF inhibitor reduced hypoxia-induced P-gp-mediated resistance to carfilzomib in MM cells in vitro, and delayed tumor progression significantly improving survival and response to carfilzomib in MM-bearing mice in vivo. Disclosures Azab: Verastem: Research Funding; Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner.

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