Anti Tumor Activity of Ethanolic extract column fraction of Crataeva magna Lour (DC) against Dalton's ascites lymphoma cell lines in Mice

Background: Celularity, Inc. is developing human placental hematopoietic stem cells-derived, cryopreserved, off-the-shelf, ex-vivo expanded and allogenic natural killer (PNK) cells for various hematological malignancies and solid tumors. NK cells play a central role in antibody dependent cell mediated cytotoxicity (ADCC) through Fc receptor CD16 in monoclonal antibody mediated anti-tumor therapies. Two allelic forms of CD16 have been identified. The 158Val/Val form has shown to have higher IgG binding affinity compared to the 158Phe/Phe form.1 The high IgG binding allele are found in about 10-20% of the normal population.2,3 In addition, activation of NK cells induces CD16 shedding by matrix metalloprotease ADAM17 at 197Ser, thus limiting ADCC responses. A single mutation (Ser197Pro) prevents CD16 shedding and increases ADCC activity in NK cells.4 Since the antibody binding affinity and CD16 expression of PNK could vary with different donors, we hypothesize that expressing a high affinity (158Val) and proteinase cleavage resistant (197Pro) CD16 variant (CD16VP) augments anti-tumor ADCC activity. Methods: Lentivirus expressing CD16VP was used to transduce human placental CD34+ cells. After transduction, the cells were cultured in the presence of cytokines including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2, for 35 days to generate PNK-CD16VP cells. Non-transduced PNK cells (NT) served as a control. Expression of CD16VP was evaluated by activating cells with PMA/ionomycin to induce CD16 cleavage (CD16 shedding assay) followed by immunostaining with CD16 antibody and analyzed using flowcytometry. ADCC of PNK-CD16VP cells was assessed against Daratumumab (anti-CD38) or Rituximab (anti-CD20) opsonized lymphoma cell lines at various effector to target (E:T) ratios. IgG was used as ADCC control. In vivo anti-tumor activity was assessed in a Daudi disseminated Xenograft model in NSG mice. Luciferase-expressing Daudi cells (3x106) were intravenously (IV) administered at day 0, followed by PNK-CD16VP cells (10x106) IV at day 1 and day 3, and Daratumumab at day 3. Tumor burden in mice was monitored by Bioluminescence Imaging (BLI). Statistical differences between the groups were calculated using paired t-test using Prism. Results: Lentiviral transduction of CD16VP achieved high expression efficiency in multiple placental CD34+ donors. These cells expanded [7095 ± 2998 folds (n=8)] and differentiated into PNK cells (>90% CD56+CD3-) at day 35. PNK-CD16VP expressed 64.6 ± 10.3% (n=8) of CD16, while the NT expressed 12.1 ± 3.3% (n=8) CD16. PMA/ionomycin induced >89% shedding of CD16 in NT cells, while significantly less (<11%) CD16 shedding was observed in PNK-CD16VP cells. These results indicated that CD16VP was expressed and maintained throughout the culture process. In vitro ADCC assay demonstrated improved anti-tumor activity of PNK-CD16VP cells over NT cells against Daratumumab or Rituximab opsonized lymphoma cell lines. At 10:1 E:T ratio PNK-CD16VP cells elicited higher cytotoxicity compared to NT: 47 ± 13% against Daratumumab opsonized Daudi cells versus 25 ± 5% (n=5; p<0.05); 30 ± 13% against Daratumumab opsonized HS-Sultan cells versus 21 ± 14% (n=3; p<0.05); 30 ± 7% against Daratumumab opsonized Sudhl6 cells versus 16 ± 10% (n=3; p<0.05). Improved ADCC activities in PNK-CD16VP were also observed in other cell lines including Raji and Sudhl4 with Daratumumab and Rituximab antibodies. PNK-CD16VP were used to test anti-tumor ADCC in vivo using a disseminated Daudi Xenograft model. The preliminary data demonstrated that PNK-CD16VP combined with Daratumumab reduced BLI signal (>50%) compared to vehicle or Daratumumab alone at day 10 after treatment. This observation suggested that PNK-CD16VP demonstrated in vivo ADCC anti-tumor activity. Conclusions: In this study, we genetically modified PNK to express high affinity and cleavage resistant CD16 variant using lentivirus. The PNK-CD16VP cells demonstrated enhanced ADCC function against lymphoma cell lines in vitro and in vivo. Further development of PNK-CD16VP for immune-oncology therapeutics is warranted. References: Wu J et al. J Clin Invest. 1997;100(5):1059-1070.Sugita N et al. Clin Exp Immunol. 1999;117(2):350-354.Koene HR et al. Blood. 1997;90(3):1109-1114.Jing Y et al. PLoS One. 2015;10(3):e0121788. Disclosures Guo: Celularity, Inc.: Employment. Somanchi:Celularity Inc: Employment. Mathur:Celularity Inc: Employment. He:Celularity Inc: Employment. Ye:Celularity Inc: Employment. Difiglia:Celularity Inc: Employment. Rotondo:Celularity Inc: Employment. Rana:Celularity Inc: Employment. Ling:Celularity Inc: Employment. Edinger:Celularity Inc: Employment. Hariri:Celularity Inc: Employment. Zhang:Celularity Inc: Employment.

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Epratuzumab (Humanized Anti-CD22 MAb) Conjugated with SN-38, a New Antibody-Drug Conjugate (ADC) for the Treatment of Hematologic Tumors: Preclinical Studies Alone and In Combination with Veltuzumab, a Humanized Anti-CD20 MAb

Blood ◽

10.1182/blood.v116.21.3941.3941 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3941-3941

Author(s):

David M Goldenberg ◽

Serengulam Govindan ◽

Tom M Cardillo ◽

Robert M Sharkey

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

In Vivo Studies ◽

Anti Cd20 ◽

Anti Tumor Activity

Abstract Abstract 3941 Background: Monoclonal antibody (MAb) therapy has had a significant impact on the management of B-cell malignancies, but is most often used in combination with chemotherapy. We developed an ADC that combines SN-38, the active component of irinotecan, a topoisomerase I inhibitor, with the internalizing, humanized, anti-CD22 IgG, epratuzumab, and determined its activity alone and in combination with an anti-CD20 antibody therapy (veltuzumab). Methods: Epratuzumab was conjugated with SN-38 (E-SN-38) at a mole ratio of ∼6:1. The conjugate is designed specifically to be released slowly in the presence of serum (50% released over ∼1.5 days), allowing liberation of the drug when internalized, but also being released locally after being bound to the tumor. In vitro and in vivo studies were performed to assess the activity of the conjugate against several subcutaneously- or intravenously-inoculated B-cell lymphoma cell lines. In vivo studies also examined combination therapy using E-SN-38 and the veltuzumab (V). Results: In vitro studies in 4 B-cell lymphoma cells lines (Daudi, Raji, Ramos, WSU-FSCCL) and 4 acute lymphoblastic lymphoma cell lines (697, REH, MN-60, and RS4;11) expressing varying amounts of CD22 showed an IC50 for E-SN-38 in the nanomolar range, confirming potent activity. Nude mice bearing SC Ramos human lymphoma had significant selective anti-tumor activity compared to a control, non-targeting, IgG-SN-38 conjugate, at a dosing regimen of 75 to 250 μg of the conjugates given twice-weekly for 4 weeks. Significant anti-tumor activity was also found in several other cell lines. When combined with veltuzumab, significant improvement in therapeutic activity was observed. For example, median survival in a WSU-FSCCL human follicular B-cell lymphoma IV model with treatment initiated 5 days after implantation was 42 d (0/10 surviving at 160 d) and 91 d (2/10 surviving) for untreated and veltuzumab-treated animals, respectively; 63d (0/10 surviving after 160 d) and >160 d (with 6/10 surviving) for E-SN-38 and E-SN-38 + V, respectively; and 63 d (0/10) and 91 d (2/10) for non-targeting IgG-SN-38 conjugate alone and combined with V). The E-SN-38 conjugate combined with V was significantly better than all treatment or control groups (P ≤ 0.05). Conclusion: E-SN-38 ADC is a potent therapeutic, even at non-toxic dose levels, and shows significantly enhanced efficacy when combined with anti-CD20 immunotherapy, representing an important new ADC treatment regimen. Disclosures: Goldenberg: Immunomedics, Inc.: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Govindan:Immunomedics, Inc.: Employment. Cardillo:Immunomedics, Inc.: Employment.

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Interleukin-21-Induced Apoptosis and Cell Death of Diffuse Large B-Cell Lymphoma (DLBCL) Cell Lines and Primary Tumors Are Associated with an Induction of Bim.

Blood ◽

10.1182/blood.v108.11.2503.2503 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2503-2503

Author(s):

Kristopher A. Sarosiek ◽

Jun Chen ◽

Dien G. Pham ◽

Hovav Nechushtan ◽

E. Avisar ◽

...

Keyword(s):

Cell Death ◽

Animal Models ◽

Cell Line ◽

Cell Lines ◽

Cellular Proliferation ◽

Lymphoma Cell ◽

Primary Tumors ◽

Protein Levels ◽

Induced Apoptosis ◽

Anti Tumor Activity

Abstract IL-21, a member of the IL-2 cytokine family, is reported to have an immune-mediated anti-tumor activity against renal cell carcinoma, malignant melanoma and Non-Hodgkin’s Lymphoma (NHL) cell lines in xenograph animal models. Whether IL-21 exhibits direct anti-tumor activity against NHL cell lines and primary tumors is presently unknown. We analyzed seven DLBCL and two Burkitt’s lymphoma cell lines for IL-21 receptor (IL-21R) expression. IL-21R was expressed at high levels in the two Burkitt’s lymphoma cell lines (RAJI and RAMOS). Out of the seven DLBCL lines, four expressed high levels of the receptor (OCI-LY-3, OCI-LY-7, OCI-LY-19, and RCK-8) while three had low receptor expression levels (OCI-LY-10, SU-DHL-4, and SU-DHL-6). IL-21 stimulation induced tyrosine phosphorylation of STAT-1, -3, and -5 as early as fifteen minutes post treatment in DLBCL lines expressing either high (OCI-LY-3 and RCK-8) or low (SU-DHL-6 and OCI-LY-10) levels of IL-21R. In six of the seven DLBCL lines tested, IL-21 dramatically inhibited or completely abolished cellular proliferation at 25 ng/mL and 100 ng/mL concentrations, respectively. In contrast, one DLBCL cell line (OCI-LY-3) exhibited a threefold increase in cellular proliferation after stimulation with 25 ng/mL IL-21, but proliferation was effectively inhibited by 100 ng/mL IL-21. Marked apoptosis and cell death were observed by flow cytometry at 72 hours post IL-21 exposure in all nine NHL cell lines tested with the exception of OCI-LY-3 which exhibited no significant change in cell viability. The IL-21-induced apoptosis was associated with an activation of caspases 3/7, 8, and 9, detected as early as 12 hours post IL-21 exposure. IL-21 also induced an increase in the levels of the pro-apoptotic protein Bim in all the cell lines exhibiting marked apoptosis. In contrast, Bim protein levels decreased in response to the IL-21 stimulation in the resistant OCI-LY-3 DLBCL cell line. IL-21 treatment led to a decrease in the protein levels of Bcl-2 in all the analyzed cell lines except OCI-LY-3. An increase in expression of Bcl-6 protein in three of the four tested DLBCL cell lines was observed in response to IL-21 exposure. In primary tumors, IL-21 induced apoptosis in two of two DLBCLs, two of three follicular lymphomas, and two of six chronic lymphocytic leukemias. No apoptosis or cell death was induced in normal peripheral B-lymphocytes or in the HeLa or 293T control cells. These results suggest that IL-21 exhibits a direct anti-tumor effect on the DLBCL cell lines and primary tumors and point to a potential applicability of IL-21 in anti-DLBCL therapy. Further work interrogating the molecular mechanism of the IL-21-induced apoptosis of DLBCL cells in vitro and in animal models is in progress.

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Evaluation of MLN4924, an investigational NEDD8-activating enzyme (NAE) inhibitor, as a novel targeted agent with single-agent antitumor activity in mantle cell lymphoma (MCL) and Hodgkin lymphoma preclinical models.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.e18539 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. e18539-e18539

Author(s):

Richa Dawar ◽

Matthew John Barth ◽

Cory Mavis ◽

Jospeh J. Skitzki ◽

Myron Stefan Czuczman ◽

...

Keyword(s):

B Cell ◽

Hodgkin Lymphoma ◽

Tumor Cells ◽

Cell Lines ◽

Primary Tumor ◽

Cell Lymphoma ◽

Resistant Cell ◽

Chemotherapeutic Agents ◽

Lymphoma Cell ◽

Anti Tumor Activity

e18539 Background: MLN4924 is a novel, potent, and selective inhibitor of NAE, an essential component of the ubiquitin-proteosome system (UPS). We have previously demonstrated that the UPS plays a pivotal role in the development of rituximab and chemotherapy resistance in B-cell lymphomas. There is a scientific need to target the UPS more efficiently in an attempt to reverse acquired resistance to biological and chemotherapeutic agents in relapsed/refractory aggressive lymphoma. To this end, we studied the anti-tumor activity of MLN4924 in rituximab-chemotherapy sensitive and resistant pre-clinical models. Methods: A panel of Burkitt (BL), diffuse large B-cell (DLBCL), MCL and HL lymphoma cell lines and primary tumor cells isolated from patients with non-Hodgkin lymphoma (NHL) (N=10) were exposed to escalating doses of MLN4924 alone or in combination with a panel of chemotherapeutic agents for up to 72 hrs. Cell viability was determined by alamar Blue reduction or CellTiter-glo assay. Apoptosis was determined by Western blotting. Cell cycle analysis was performed by flow cytometry. Results: MLN4924 demonstrated activity in all cell lines in a time-and dose-dependent manner, including the rituximab/chemotherapy-resistant cell lines. The most potent activity was noted in MCL and HL cell lines (IC50 doses were tenfold lower as compared to DLBCL or BL cell lines). A variable degree of anti-tumor activity was observed in primary tumor cells isolated from NHL patients. Induction of apoptosis was observed in rituximab-resistant cell lines. MLN4924 exhibited synergistic anti-tumor activity when combined with bortezomib, bendamustine, and cytarabine in MCL cell lines. Conclusions: MLN4924 exhibits potent in vitro cytotoxic activity against a variety of human B-cell lymphoma cell lines and primary tumor cells isolated from NHL patients. Significant activity was observed in MCL cell lines. Experiments investigating the in vivo activity of MLN4924 are ongoing. MLN4924 is a highly promising agent for the treatment of relapsed/refractory MCL or HL.

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The XIAP Inhibitor BMT-062789 Displays Anti-Tumor Activity in Cell Line Models of De Novo and Rituximab Relapse/Refractory Lymphoma

Blood ◽

10.1182/blood.v128.22.5375.5375 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5375-5375 ◽

Cited By ~ 1

Author(s):

Kyle L. Runckel ◽

Cory Mavis ◽

Juan J Gu ◽

Francisco J. Hernandez-Ilizaliturri

Keyword(s):

Cell Line ◽

Cell Lines ◽

De Novo ◽

Resistant Cell ◽

Resistant Cell Line ◽

Lymphoma Cell ◽

Lymphoma Cell Line ◽

Raji Cells ◽

Ic50 Values ◽

Anti Tumor Activity

Abstract The addition of rituximab to front line therapy for aggressive lymphomas has improved clinical outcomes, but it has also altered the biology of relapsed/refractory disease. To better understand the mechanisms responsible for rituximab associated chemotherapy cross-resistance our group developed several rituximab resistance cell lines (Raji 4RH and RL 4RH), which also display significant resistance to a wide range of chemotherapy agents. These rituximab resistant cell lines (RRCL)s exhibit multiple deregulations in the BCL-2 and inhibitor of apoptosis (IAP) protein families, including loss of the pro-apoptotic proteins Bax and Bak. We previously demonstrated that the X linked inhibitor of apoptosis protein (XIAP) is critically required for chemotherapy resistance in the RRCLs, and that an shRNA knockdown of XIAP increased chemotherapy response in both in vitro and in vivo models of rituximab resistant lymphoma. BMT-062789 is a heterodimeric mimetic of the second mitochondrial activator of caspases (SMAC) developed by Bristol-Myers Squibb, which can inhibit both the caspase 9 and caspase 3/7 binding domains of XIAP. BMT-062789 demonstrated dose and time dependent single agent anti-tumor effect (as measured by the Cell TiterGlo luminescent viability assay) in a panel of lymphoma cell lines, with IC50 values of less than 5uM for all cell lines tested except the rituximab resistant cell line Raji 4RH. The Burkitt's lymphoma cell line Daudi and diffuse large B-cell lymphoma cell line U2932 were particularly sensitive to BMT-062789 with IC50 values of 0.91uM and 0.76uM respectively. To investigate if the observed anti-tumor effect of BMT-062789 was due to increased apoptosis we exposed rituximab sensitive (Raji, RL) and rituximab resistant (Raji 4RH, RL 4RH) cells to escalating doses of BMT-062789 with or without the addition of 20uM etoposide for 48 hours. The induction of apoptosis was measured by flow cytometry with an Annexin-V:PE-Cy7 conjugate and Sytox blue (a DNA stain). 2uM BMT-062789 alone triggered apoptosis in 75% of Raji cells and 65% of RL cells. It is also worth adding that 2uM BMT-062789 induced higher rates of apoptosis than 20uM etoposide alone in both cell lines. The combination of 2uM BMT-062789 and 20uM etoposide together triggered apoptosis in 80% of Raji cells and 85% of RL cells, indicating that BMT-062789 may be able to augment the anti-tumor activity of conventional chemotherapy. More importantly, the combination of BMT-062789 and etoposide was also able to induce apoptosis in the rituximab resistant cell line models Raji 4RH and RL 4RH. 3uM BMT-062789 in combination with 20uM etoposide triggered apoptosis in 90% of Raji 4RH cells and 55% of the RL 4RH cells, which is a substantial improvement compared to 15% apoptosis with 20uM etoposide alone in each cell line. Additional studies are in progress to evaluate the anti-tumor effect of BMT-062789 in ex vivo samples from lymphoma patients with de novo and relapse/refractory disease. In summary, the novel heterodimeric XIAP inhibitor BMT-062789 has anti-tumor effect at low micromolar concentrations in lymphoma cell line models, including models of rituximab resistant disease. These results support earlier studies by our group indicating that XIAP is critical for survival in models of rituximab resistant lymphoma, and establish that XIAP inhibitors may have potential clinical value for the treatment of both de novo, and rituximab relapse/refractory lymphomas. Disclosures No relevant conflicts of interest to declare.

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