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 ◽  
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.

scholarly journals A Novel FBXO45-Gef-H1 Axis Controls Oncogenic Signaling in B-Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 711-711
Author(s):  
Anagh Anant Sahasrabuddhe ◽  
Xiaofei Chen ◽  
Kaiyu Ma ◽  
Rui Wu ◽  
Richa Kapoor ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
De Novo ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
In Vivo Studies

Abstract Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common form of malignant lymphoma and may arise de novo, or through transformation from a pre-existing low-grade B cell lymphoma such as follicular lymphoma (FL). However, the post-translational mechanisms and deregulated pathways underlying the pathogenesis of disease evolution are not fully understood. Methods: We employed integrated functional and structural genomics and mass spectrometry (MS)-driven proteomics which implicated a possible novel tumor suppressor role for a conserved E3 ubiquitin ligase FBXO45 in DLBCL pathogenesis. We generated conditional knockout mice targeting loss of Fbxo45 in germinal center (GC) B-cells using the Cg1-Cre-loxP system and an assortment of CRISPR-mediated knockouts of FBXO45 in B cell lymphoma cells (FL518, BJAB, U2932). We engineered B cell lines (BJAB, U2932) to inducibly express FLAG-tagged FBXO45 to identify candidate substrates of FBXO45 using liquid chromatography-tandem MS. In vitro biochemical and in vivo studies using a variety of genetically-modified lines in xenograft studies in immunodeficient mice were performed to validate observations from proteogenomic studies. Whole genome sequencing (WGS) and genomic copy number studies were interrogated to investigate structural alterations targeting FBXO45 in primary human lymphoma samples. Results: Conditional targeting of Fbxo45 in GCB-cells in transgenic mice resulted in abnormal germinal center formation with increased number and size of germinal centers. Strikingly, targeted deletion of Fbxo45 in GCB-cells resulted in spontaneous B cell lymphomas with (22/22);100%) penetrance and none of the wild-type (WT) littermates (0/20; 0%) developed lymphoma at 24 months. Macroscopic examination revealed large tumor masses, splenomegaly, and lymphadenopathy at different anatomic locations including ileocecal junction, mesenteric, retroperitoneal and cervical lymph nodes and thymus. Next generation sequencing of immunoglobulin heavy chain genes revealed monoclonal or oligoclonal B cell populations. Using proteomic analysis of affinity-purified FBXO45-immunocomplexes and differential whole proteome analysis from GCB-cells of Fbxo45 wt/wt vs Fbxo45 fl/fl mice, we discovered that FBXO45 targets the RHO guanine exchange factor GEF-H1 for ubiquitin-mediated proteasomal degradation. FBXO45 exclusively interacts with GEF H1 among 8 F-box proteins investigated and silencing of FBXO45 using three independent shRNA and CRISPR-Cas9-mediated knockouts in B-cell lymphoma cell lines promotes RHOA and MAPK activation, B cell growth and enhances proliferation. GEF-H1 is stabilized by FBXO45 depletion and GEF-H1 ubiquitination by FBXO45 requires phosphorylation of GEF-H1. Importantly, FBXO45 depletion and expression of a GEF-H1 mutant that is unable to bind FBXO45 results in GEF-H1 stabilization, promotes hyperactivated RHO and MAPK signaling and B-cell oncogenicity in vitro and in vivo. Notably, this phenotype is reverted by co-silencing of GEF-H1. Inducible ectopic expression of FBXO45 triggers accelerated turnover of GEF H1 and decreased RHOA signaling. Genomic analyses revealed recurrent loss targeting FBXO45 in transformed DLBCL (25%), de novo DLBCL (6.6%) and FL (2.3%). In keeping with our observation of prolonged hyperactivation of pERK1/2 consequent to FBXO45 ablation, in vitro and in vivo studies using B-cell lymphoma cell lines and xenografts demonstrated increased sensitivity to pharmacologic blockade with the MAP2K1/2 (ERK1/2) inhibitor Trametinib. Conclusions: Our findings define a novel FBXO45-GEF-H1-MAPK signalling axis, which plays an important role in DLBCL pathogenesis. Our studies carry implications for potential exploitation of this pathway for targeted therapies. Disclosures Siebert: AstraZeneca: Speakers Bureau. Lim: EUSA Pharma: Honoraria.

Novel BAFF-Receptor Antibody to Natively Folded and Glycosylated Recombinant Protein Eliminates Drug Resistant B Cell Malignancies In Vivo

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 468-468
Author(s):  
Hong Qin ◽  
Guowei Wei ◽  
Ippei Sakamaki ◽  
Zhenyuan Dong ◽  
Diane Lynne Smith ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Acquired Resistance ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
Drug Resistant ◽  
L Cells

Abstract Background: Targeted monoclonal antibodies (mAbs) such as the anti-CD20 rituximab, are proven therapies in lymphoma, yet these diseases remain incurable because of primary or acquired resistance. Using a eukaryotic expression system to produce antigen closely representing endogenous protein, we developed a new therapeutic antibody against an alternative lymphoma target. B cell activating factor receptor (BAFF-R/TNFRSF13C) is a tumor-necrosis factor receptor superfamily member specifically involved in B lymphocyte development and mature B cell survival. Although earlier attempts to target the BAFF/BAFF-R axis therapeutically for B cell tumors yielded limited success, BAFF-R remains an attractive target for B cell lymphoma therapeutic antibody development, particularly for rituximab-resistant tumors. Methods and Results: We generated 2 mAbs to human BAFF-R expressed as a natively folded, eukaryotically glycosylated cell-surface immunogen on engineered mouse fibroblast (L) cells. Both mAbs specifically bound BAFF-R-expressing L cells, but not the parental counterparts. Each of the complementarity determining regions (CDRs) of the 2 mAbs are unique, suggesting different binding epitopes. Both mAbs bound with high affinity to the human B cell lymphoma cell lines JeKo-1 (mantle cell lymphoma; MCL), SU-DHL6 (diffuse large B cell lymphoma; DLBCL), Raji (Burkitt's lymphoma) and RL (follicular lymphoma). Because our goal is to develop antibodies for clinical use, we substituted in human IgG1 Fc to generate the chimeric mAbs C55 and C90. The chimeric mAbs retained the binding specificity and affinity of the mouse antibodies to their target cells. By immunohistochemistry C55 and C90 staining was specific to the B cell-containing organs tonsil and spleen. No detectable staining was observed in heart, lung, brain, liver, and kidney. Using primary human natural killer (NK) cells as effectors, we demonstrated the chimeric antibodies induced potent antibody-dependent cellular cytotoxicity (ADCC) against BAFF-R-expressing L cells and the JeKo-1, SU-DHL6, Raji and RL human lymphoma cell lines. C55 and C90 were also able to elicit ADCC in primary human lymphomas; they efficiently killed tumor cells from patients with MCL, DLBCL, follicular lymphoma and chronic lymphocytic leukemia (CLL) (n=8). Notably, 5 of these primary lymphomas were from patients who had relapsed after rituximab treatment (2 MCL, 3 CLL). We next determined the activity of C55 and C90 in models of drug-resistant lymphoma. Both ibrutinib and rituximab are effective anti-lymphoma agents, however, primary or acquired resistance to these drugs is common. We derived a rituximab-resistant JeKo-1 variant (JeKo-1 CD20KO) using the CRISPR/HDR system to knock-out CD20 gene expression, and also used the naturally ibrutinib-resistant (Z-138) lymphoma cell line. We confirmed that the C55 and C90 anti-BAFF-R antibodies induced ADCC in both drug-resistant cell lines in vitro. Using xenogenic tumor models in NOD scid gamma (NSG) mice we observed remarkable in vivo anti-tumor effects of both the C55 and C90 chimeric antibodies. We found our antibodies significantly inhibited growth of implanted Z-138 and JeKo-1 CD20KO lymphomas (P<0.001; Figure). Conclusion: In contrast to previously reported BAFF-R antibodies, our in vitro and in vivo results strongly support the translational development of our novel BAFF-R-specific monoclonal antibodies, especially as an alternative immunotherapy against ribuximab- or ibrunitib-resisitant B cell maglinancies. Other preliminary data also suggest BAFF-R may be an effective target of CAR T cells. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Targeting B-Cell Malignancies With Anti-CD20-Interleukin-21 Fusokine

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 377-377 ◽  
Author(s):  
Shruti Bhatt ◽  
Daxing Zhu ◽  
Xiaoyu Jiang ◽  
Seung-uon Shin ◽  
John M Timmerman ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cd20 Antibody ◽  
Anti Cd20

Abstract The anti-CD20 antibody rituximab has revolutionized the treatment for B cell non-Hodgkin lymphomas (NHLs). However, rituximab has limited effectiveness as a single agent in some NHL subtypes and its clinical efficacy is compromised by acquired drug resistance. As a result, many patients still succumb to NHLs. Hence, strategies that enhance the activity of anti-CD20 antibody may improve patient outcome. Interleukin-21 (IL21), a member of the IL2 cytokine family, exerts diverse regulatory effects on natural killer (NK), T and B cells. IL21 has been reported to possess potent anti-tumor activity against a variety of cancers not expressing IL21 receptor (IL21R) through activation of the immune system and is in clinical trials for renal cell carcinoma and metastatic melanoma. We have recently reported that apart from immuno-stimulatory effects, IL21 exerts direct cytotoxicity on IL21R expressing diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cell lines and primary tumors both in vitro as well in vivo (Sarosiek et al Blood 2010; Bhatt et al AACR 2013). Herein we designed a fusion protein comprising IL21 linked to the N-terminus of anti-CD20 antibody (αCD20-IL21 fusokine) to improve efficacy of its individual components and prolong IL21 half-life. We have verified the expression of full length fusion protein and demonstrated that αCD20-IL21 fusokine retained binding ability to its individual components; CD20 and IL21R, as analyzed by immunofluorescence and flow-cytometry analyses. Similar to our previous study of IL21 in DLBCL, treatment of B cell lymphoma cell lines with fusokine lead to phosphorylation of STAT1 and STAT3, upregulation of cMYC and BAX and downregulation of BCL-2 and BCL-XL, implying the activation of IL21R dependent signaling to trigger cytotoxic effects. In vitro, direct cell death induced by αCD20-IL21 fusokine in DLBCL (RCK8, WSU and Farage) and MCL (Mino, HBL2 and SP53) cell lines was markedly increased compared to its individual components (IL21 and parent αCD20-IgG1 antibody). More importantly, fusokine treatment resulted in cell death of MCL cell lines (L128, G519 and UPN1) that were found to be resistant to IL21 alone treatment. Furthermore, treatment of freshly isolated primary NHL cells with the αCD20-IL21 fusokine also exhibited a 40-50% increase in direct cell death compared to its individual components. Previous studies reported that IL21 enhances antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies by activation of NK cells. ADCC assays using chromium release with purified human NK cells demonstrated that ADCC induced by the parent antibody was enhanced in the presence of IL21 while IL21 alone had minimal effect on the lysis of Raji, Daudi, and Jeko1 target cells. Notably, αCD20-IL21 fusokine demonstrated increased ADCC activity in comparison to parent antibody plus IL21 in Raji, Daudi and Jeko-1 cells (p<0.001, p<0.005 and p<0.001, respectively). Similar results were obtained in primary MCL tumor cells. Consistent with this finding, fusokine treatment resulted in enhanced activation of the NK cells as assessed by CD69 upregulation and CD16 downregulation using flow-cytometry. Complement dependent cytotoxicity (CDC) of the fusokine was similar to the parent antibody and rituximab in Raji cells. Studies analyzing in vivo effects of the fusokine are in progress and will be presented at the meeting. These data strongly suggest that together with direct apoptotic potential, an anti-CD20 IL21 fusokine retains the ability to trigger indirect cell killing mediated via activation of immune effector cells. These dual effects may give remarkable advantage to the fusokine over existing anti-CD20 antibodies for the treatment of NHL tumors. Collectively, our study demonstrates that anti-tumor effects of IL21 and anti-CD20 antibodies can be enhanced by conjugation of IL21 with anti-CD20 antibody that may serve as a novel anti-lymphoma therapy. Disclosures: Rosenblatt: Seattle Genetics, Inc.: Research Funding.

Antibody-Targeted Chemotherapy with Immunoconjugates of Calicheamicin: Differential Anti-Tumor Activity of Conjugated Calicheamicin Targeted to B-Cell Lymphoma Via B-Cell Lineage Specific Molecules CD19, CD20 and CD22.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2490-2490 ◽  
Author(s):  
John F. DiJoseph ◽  
Douglas C. Armellino ◽  
Maureen M. Dougher ◽  
Arthur Kunz ◽  
Erwin R. Boghaert ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Targeted Chemotherapy ◽  
Phase I Clinical Trials ◽  
Anti Cd20 ◽  
Anti Tumor Activity ◽  
Acid Labile

Abstract Antibody-targeted chemotherapy using tumor-targeted immunoconjugates of the cytotoxic agent, calicheamicin, is a clinically validated strategy for the treatment of acute myeloid leukemia. Calicheamicin is a potent cytotoxic natural product that binds DNA in the minor groove and causes double strand DNA breaks. B lymphoid lineage-specific antigens CD19, CD20, and CD22 have been studied extensively as potential targets for therapeutic applications of immunotoxins. In order to determine which one of these three antigens is most suitable for antibody-targeted calicheamicin therapy, we conjugated monoclonal antibodies, BU12 (murine anti-CD19 mAb), rituximab (chimeric anti-CD20 mAb), and m5/44 (murine anti-CD22 mAb) to a hindered disulfide derivative of N-acetyl gamma calicheamicin and evaluated the anti-tumor activity of these conjugates against three human B-cell lymphoma lines (BCL), Ramos, Raji and RL. Each of these three mAb bound to their respective antigens on the surface of BCL and was modulated, indicative of their potential internalization. Immunoconjugates of these mAbs, prepared by covalently linking calicheamicin via either acid-labile or acid-resistant linkers, caused a potent inhibition of BCL growth in vitro (IC50s ranged from 7 pM for the acid-labile linked m544 up to 6.8 nM for the acid-resistant linked anti-CD20 conjugates of calicheamicin). Immunoconjugates with acid-labile linkers were more potent than their counterparts with the acid-stable linker and conjugates targeted to either CD19 or CD22 were more potent than those targeted to CD20 in inhibiting BCL growth in vitro. In contrast, unconjugated mAb to CD19 or CD22 had no effect on BCL growth in vitro whereas anti-CD20 mAb, at concentrations >1 μg/ml, had an inhibitory effect of 30% on in vitro BCL growth. When examined for their effects on the growth of established subcutaneous BCL xenografts in nude mice, calicheamicin conjugated to anti-CD22 was by far the most efficacious conjugate against each of the three BCL xenografts studied. Calicheamicin conjugated to rituximab caused significant inhibition of BCL growth but was less effective than the conjugates of anti-CD22 or anti-CD19 mAb. Interestingly, anti-CD19 conjugates of calicheamicin, while effective in vitro against both Raji and Ramos BCL and effective against Raji BCL xenografts, had no effect on the growth of Ramos BCL xenografts in vivo. The reasons underlying the lack of anti-tumor activity of CD19-targeted calicheamicin conjugate against Ramos xenografts in vivo remain unknown. Based on a number of factors including the potent and consistent anti-tumor activity of the anti-CD22-conjugated calicheamicin, CD22 was selected as the molecular target for further development. A calicheamicin conjugate containing an acid-labile linker of humanized anti-CD22 mAb, CMC-544, is currently being evaluated in phase I clinical trials in non-Hodgkin’s B-cell lymphoma.

Characterization of two new high-grade B-cell lymphoma cell lines with MYC and BCL2 rearrangements that are suitable for in vitro drug sensitivity studies

2018 ◽  
Vol 60 (4) ◽  
pp. 1043-1052
Author(s):  
Marie-Sophie Dheur ◽  
Hélène A. Poirel ◽  
Geneviève Ameye ◽  
Gaëlle Tilman ◽  
Pascale Saussoy ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Drug Sensitivity ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
High Grade ◽  
Sensitivity Studies

scholarly journals Repurposing dasatinib for diffuse large B cell lymphoma

2019 ◽  
Vol 116 (34) ◽  
pp. 16981-16986 ◽  
Author(s):  
Claudio Scuoppo ◽  
Jiguang Wang ◽  
Mirjana Persaud ◽  
Sandeep K. Mittan ◽  
Katia Basso ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Multikinase Inhibitor ◽  
Large B Cell Lymphoma ◽  
Large B Cell

To repurpose compounds for diffuse large B cell lymphoma (DLBCL), we screened a library of drugs and other targeted compounds approved by the US Food and Drug Administration on 9 cell lines and validated the results on a panel of 32 genetically characterized DLBCL cell lines. Dasatinib, a multikinase inhibitor, was effective against 50% of DLBCL cell lines, as well as against in vivo xenografts. Dasatinib was more broadly active than the Bruton kinase inhibitor ibrutinib and overcame ibrutinib resistance. Tumors exhibiting dasatinib resistance were commonly characterized by activation of the PI3K pathway and loss of PTEN expression as a specific biomarker. PI3K suppression by mTORC2 inhibition synergized with dasatinib and abolished resistance in vitro and in vivo. These results provide a proof of concept for the repurposing approach in DLBCL, and point to dasatinib as an attractive strategy for further clinical development in lymphomas.

scholarly journals Establishment of B-Cell Lymphoma Cell Lines Persistently Infected with Hepatitis C Virus In Vivo and In Vitro: the Apoptotic Effects of Virus Infection

2003 ◽  
Vol 77 (3) ◽  
pp. 2134-2146 ◽  
Author(s):  
Vicky M.-H. Sung ◽  
Shigetaka Shimodaira ◽  
Alison L. Doughty ◽  
Gaston R. Picchio ◽  
Huong Can ◽  
...  
Keyword(s):  
Hepatitis C ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Culture System ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Hcv Rna

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Studies of HCV replication and pathogenesis have so far been hampered by the lack of an efficient tissue culture system for propagating HCV in vitro. Although HCV is primarily a hepatotropic virus, an increasing body of evidence suggests that HCV also replicates in extrahepatic tissues in natural infection. In this study, we established a B-cell line (SB) from an HCV-infected non-Hodgkin's B-cell lymphoma. HCV RNA and proteins were detectable by RNase protection assay and immunoblotting. The cell line continuously produces infectious HCV virions in culture. The virus particles produced from the culture had a buoyant density of 1.13 to 1.15 g/ml in sucrose and could infect primary human hepatocytes, peripheral blood mononuclear cells (PBMCs), and an established B-cell line (Raji cells) in vitro. The virus from SB cells belongs to genotype 2b. Single-stranded conformational polymorphism and sequence analysis of the viral RNA quasispecies indicated that the virus present in SB cells most likely originated from the patient's spleen and had an HCV RNA quasispecies pattern distinct from that in the serum. The virus production from the infected primary hepatocytes showed cyclic variations. In addition, we have succeeded in establishing several Epstein-Barr virus-immortalized B-cell lines from PBMCs of HCV-positive patients. Two of these cell lines are positive for HCV RNA as detected by reverse transcriptase PCR and for the nonstructural protein NS3 by immunofluorescence staining. These observations unequivocally establish that HCV infects B cells in vivo and in vitro. HCV-infected cell lines show significantly enhanced apoptosis. These B-cell lines provide a reproducible cell culture system for studying the complete replication cycle and biology of HCV infections.

Lenalidomide (Revlimid®) Enhances Monoclonal Antibody-Associated Anti-Tumor Activity Against Rituximab-Sensitive and Rituximab-Resistant B-Cell Lymphoma Cell Lines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2522-2522 ◽  
Author(s):  
Nishitha Reddy ◽  
Raymond Cruz ◽  
Francisco Hernandez-Ilizaliturri ◽  
Joy Knight ◽  
Myron S. Czuczman
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Scid Mouse ◽  
In Vitro Exposure ◽  
Human Lymphoma ◽  
Scid Mouse Model ◽  
Anti Cd20 ◽  
Anti Tumor Activity

Abstract Background: Lenalidomide is a potent thalidomide analogue shown to activate both the innate and adoptive immune system, inhibit angiogenesis, and modify the tumor microenvironment. While lenalidomide has received approval by the U.S. Federal Drug Administration (FDA) for the treatment of various hematological conditions, ongoing clinical trials are addressing its role in the treatment of B-cell lymphomas. There is a dire need to develop novel well-tolerated, therapies which combine various target-specific agents such as lenalidomide and monoclonal antibodies (mAbs). We previously demonstrated that lenalidomide is capable of expanding natural killer (NK) cells in a human-lymphoma-bearing SCID mouse model and improve rituximab anti-tumor activity in vivo. Methods: In our current work we studied the effects of lenalidomide on the biological activity of a panel of mAbs against various B-cell lymphomas, utilizing various rituximab-sensitive (RSCL) and rituximab-resistant cell lines (RRCL) generated in our laboratory from Raji and RL cell lines. Functional assays including antibody-dependant cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CMC) were performed to demonstrate changes in sensitivity to rituximab. RSCL and RRCL (1′105 cells/well) were exposed to either lenalidomide (5 μg/ml) or vehicle with or without mAb at a final concentration of 10μg/ml. The mAb panel consisted of two anti-CD20 mAbs: rituximab (Biogen IDEC, Inc.) and hA20, a humanized anti-CD20 mAb (Immunomedics, Inc.); an anti-CD80 mAb (galixumab, Biogen IDEC Inc.), and an anti-CD52 antibody (Alemtuzumab, Berlex Inc.). Changes in DNA synthesis and cell proliferation were determined at 24 and 48 hrs by [3H]-thymidine uptake. For ADCC/CMC studies, NHL cells were exposed to lenalidomide or vehicle for 24 hrs and then labeled with 51Cr prior to treatment with one of various mAbs (10 mg/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. Changes in antigen (CD20, CD80, and CD52) expression following in vitro exposure to lenalidomide were studied by multicolor flow cytometric analysis. Results: Concomitant in vitro exposure of various RSCL and RRCL cells to lenalidomide and either galixumab, hA20 or alemtuzumab for 24 hrs resulted in improved anti-tumor activity when compared to controls. In addition, pre-incubation of both RSCL and RRCL with lenalidomide rendered cells more susceptible to alemtuzumab-, hA20- and galixumab-mediated ADCC and CMC. No antigen modulation (i.e., upregulation) was observed following in vitro exposure of lenalidomide to NHL cell lines, suggesting an alternative mechanism involved in the improvement antitumor activity observed. Conclusions: Our data suggest that the augmented antitumor effect of lenalidomide is not limited to its combination with rituximab, but also that it augments the antiproliferative and biological activity of alemtuzumab, hA20 and galixumab. Furthermore, these interactions are observed even in our RRCL. Future studies will be directed towards evaluating whether similar activity will be seen in vivo using a human lymphoma-bearing SCID mouse model. (Supported by USPHS grant PO1-CA103985 from the National Cancer Institute.)

Acadesine Inhibits Proliferation and Induces Apoptosis In Multiple Myeloma Cells, and Synergizes with Standard of Care Antimyeloma Agents

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5023-5023
Author(s):  
Susana Hernández-García ◽  
Mercè de Frias ◽  
Clara Campàs ◽  
Bruno Paiva ◽  
Enrique M. Ocio ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Cell Lymphoma ◽  
Hematologic Malignancy ◽  
B Cell Lymphoma ◽  
In Vivo Studies ◽  
Mutational Status

Abstract Abstract 5023 Multiple myeloma (MM) is a malignancy characterized by the accumulation of plasma cells. The disease represents the second most common hematologic malignancy and remains incurable, despite recent advances in its treatment. Therefore, studies to develop new therapies are still necessary, particularly in patients with bad prognostic factors, such as 17p deleted/p53 mutated patients. In this study we describe the preclinical activity of 5-Aminoimidazole-4-carboxamide-1–4-ribofuranoside (AICAR or acadesine) in multiple myeloma. Acadesine is an analog of AMP that is widely used as an activator of AMP-kinase (AMPK), a protein that regulates the responses of the cell to energy changes. Acadesine induces apoptosis in different cell types including CLL, mantle cell lymphoma (MCL) and splenic marginal zone B-cell lymphoma (SMZL) cells and tumor cell lines, without affecting primary T lymphocytes. Thus, acadesine is a promising drug for the treatment of B-cell neoplasms. A clinical phase I/II study of acadesine is currently being performed in CLL patients. We studied the effects of acadesine on the MTT metabolization of several multiple myeloma cell lines (MM1S, MM1R, RPMI-8266, RPMI-LR5, U266, U266-LR7, U266 Dox4, MM144, MGG, SJR, OPM-2, NCIH-929). Acadesine inhibited MM cell growth and induced apoptosis, with IC50 values in the micromolar range, and independently of the p53 mutational status. Cancer treatment, including myeloma, is generally based on combinations of drugs with different mechanisms of action. Thus, we studied the effect of acadesine in double combinations with drugs used in myeloma therapy, such as dexamethasone, melphalan, doxorubicin, bortezomib, and lenalidomide. Analyses of these data using the Chou and Talalay method indicated that acadesine was synergistic with dexamethasone (CI values of 0.60), and particularly with lenalidomide (CI values of 0.42). These promising results with double combinations promoted the investigation of triple combinations in the MM1S cell line. Triple combination of acadesine plus dexamethasone plus lenalidomide or bortezomib notably improved the efficacy of the respective double combinations, being the combination of acadesine plus lenalidomide plus dexamethasone especially efficient. Further studies to determinate the mechanism of action, and in vivo studies in MM1S xenograph are ongoing. Disclosures: de Frias: Advancell: Employment. Campàs:Advancell: Employment.

Constitutively Active STAT6 Represses BCL6 in Primary Mediastinal B Cell Lymphoma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2417-2417
Author(s):  
Olga Ritz ◽  
Jochen K Lennerz ◽  
Karolin Rommel ◽  
Karola Dorsch ◽  
Elena Kelsch ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Ectopic Expression ◽  
B Cell Lymphoma ◽  
Proximal Promoter ◽  
Constitutively Active

Abstract Abstract 2417 Primary mediastinal B-cell lymphoma (PMBL) is a subtype of diffuse large B-cell lymphoma (DLBCL) that affects predominantly young women (Swerdlow et al. 2008). Despite improvements due to addition of rituximab, which has become state of the art treatment, 20% of PMBL patients succumb to disease progression or relapse. Notably, here are currently no registered trials that are actively recruiting PMBL-patients and a better understanding of the underlying pathobiology may identify novel therapeutic targets and provide an alternative to dose escalation (Steidl and Gascoyne 2011). BCL6 is a key germinal center B-cell transcription factor that suppresses genes involved in lymphocyte activation, differentiation, cell cycle arrest and DNA damage response gene. BCL6 is aberrantly expressed in certain DLBCL subgroups and BCL6 overexpression is sufficient for lymphomagenesis in mice (Cattoretti et al. 2005). In cellular- and murine DLBCL models, targeting of BCL6 via retroinverted BCL6 peptid inhibitor (RI-BPI) appears effective (Polo et al. 2004; Cerchietti et al. 2010). In conjunction with the relatively restricted expression pattern of BCL6, these data collectively suggest BCL6 as a candidate for targeted therapy in BCL6-positive lymphomas. Despite substantial work on BCL6 in lymphomas, the function of BCL6 in PMBL is unknown. To address the BCL6 function in PMBL, we performed BCL6 depletion by siRNA in all three available PMBL cell lines: K1106, U-2940 and MedB-1. We found that BCL6 acts pro-proliferative and anti-apoptotic; however, PMBL models were only partially dependent on and not addicted to BCL6. Given that BCL6 expression in all PMBL cell lines is variable with a notable fraction of BCL6-negative cells, we argued that increasing the fraction of BCL6-positive cells might increase the level of BCL6-dependence. Since IL-4/STAT6 signaling upregulates BCL6 in mouse lymphocytes (Schroder et al. 2002), we treated PMBL cell lines with IL-4 (or IL-13) and, as expected, observed increased phosphorylated (p)STAT6 levels. Surprisingly, the pSTAT6 increase was not associated with higher – but with drastically lower BCL6 protein levels. Moreover, in untreated cells, co-localization studies for pSTAT6- and BCL6 demonstrated staining in mutually exclusive subsets of cells (Figure 1A), suggesting negative interaction between BCL6 and pSTAT6. Other STAT family members were already shown to participate in the transcriptional regulation of BCL6. Thus, we examined binding of STAT6 to the proximal promoter of BCL6 in all PMBL cell lines using shift assay and chromatin immunoprecipitation. We found that STAT6 can bind all five GAS binding sites within the BCL6 promoter in vitro and in all PMBL cell lines STAT6 was bound to proximal BCL6 promoter in vivo. Furthermore, transient STAT6 depletion by siRNA and/or ectopic expression of constitutively active STAT6 confirms that pSTAT6 is sufficient for transcriptional repression of BCL6. Co-localization studies in primary patient samples demonstrated mutually exclusive BCL6/pSTAT6 distribution as a visual hallmark of the repression mechanism (Figure 1B, C). Thus, our data demonstrate for the first time that constitutively active STAT6 transcriptionally represses BCL6 in PMBL. In conjunction with functional data, the delineated repression mechanism may prevent addiction to one single oncogenic pathway (i.e. BCL6) in PMBL. Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Disclosures: No relevant conflicts of interest to declare.

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