Novel BAFF-Receptor Antibody to Natively Folded Recombinant Protein Eliminates Drug-Resistant Human B-cell Malignancies In Vivo

2017 ◽  
Vol 24 (5) ◽  
pp. 1114-1123 ◽  
Author(s):  
Hong Qin ◽  
Guowei Wei ◽  
Ippei Sakamaki ◽  
Zhenyuan Dong ◽  
Wesley A. Cheng ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
B Cell ◽  
Drug Resistant ◽  
B Cell Malignancies ◽  
Receptor Antibody ◽  
Human B Cell

scholarly journals Cure of multidrug-resistant human B-cell lymphoma xenografts by combinations of anti-B4-blocked ricin and chemotherapeutic drugs

Blood ◽  
1996 ◽  
Vol 87 (9) ◽  
pp. 3892-3898 ◽  
Author(s):  
C Liu ◽  
JM Lambert ◽  
BA Teicher ◽  
WA Blattler ◽  
R O'Connor
Keyword(s):  
B Cell ◽  
Drug Combination ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Multidrug Resistant ◽  
Scid Mice ◽  
B Cell Malignancies ◽  
Mdr 1 ◽  
Human B Cell

The CD-19-directed immunotoxin anti-B4-blocked ricin (anti-B4-bR) is currently in clinical trials for the treatment of B-cell malignancies. To explore the potential of using anti-B4-bR with chemotherapy protocols we tested the in vivo efficacy of the immunotoxin in combination with two multi-drug chemotherapeutic regimens in severe combined immunodeficient (SCID) mice bearing disseminated tumors of the multidrug-resistant human B-cell lymphoma Namalwa/mdr-1. In cytotoxicity studies in vitro, combinations of the immunotoxin with cisplatin produced supra-additive killing effects on both Namalwa and Namalwa/mdr-1 cells, whereas anti-B4-bR combined with 4-hydroperoxy- cyclophosphamide caused additive killing of both cell lines. In vivo cyclophosphamide, cisplatin, vincristine, doxorubicin, and etoposide as single agents, were effective in prolonging the survival of SCID mice burdened with the Namalwa tumor, whereas only cyclophosphamide and cisplatin were effective on Namalwa/mdr-1 tumors. Treatment of Namalwa/mdr-1-bearing mice with anti-B4-bR alone or with the drug combination CHOE (consisting of cyclophosphamide, vincristine, doxorubicin, and etoposide) alone increased the lifespan of the tumor- burdened mice by 58% and 73%, respectively. However, treatment with five daily bolus intravenous injections of anti-B4-bR followed by CHOE increased the lifespan by 173%, and 20% of the mice were cured. The drug combination CCE (cyclophosphamide, cisplatin, and etoposide) alone could increase the lifespan of the Namalwa/mdr-1 tumor-burdened mice by 129% compared with untreated controls. Combination therapy with anti-B4- bR and CCE produced long-term cures in 50% of the tumor-burdened mice. These results suggest that anti-B4-bR in combination with current multidrug regimens may constitute a highly efficacious modality for the treatment of drug-resistant B-cell malignancies.

Phosphatidylinositol-3 Kinase Delta (PI3Kδ) Inhibitor AMG 319 Is a Potent, Selective and Orally Bioavailable Small Molecule Inhibitor That Suppresses PI3K-Mediated Signaling and Viability in Neoplastic B Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4964-4964 ◽  
Author(s):  
Angus Sinclair ◽  
Daniela Metz ◽  
Tim Cushing ◽  
Liqin Liu ◽  
Rachael Brake ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Small Molecule ◽  
Single Agent ◽  
Chemotherapeutic Agents ◽  
B Cell Malignancies ◽  
Molecule Inhibitor ◽  
Human B Cell

Abstract Abstract 4964 Immune receptors such as the B cell receptor (BCR) require key signaling intermediate phosphatidylinositol-3 kinase delta (PI3Kδ) for normal immune cell survival, development and function. PI3Kδ is a class IA lipid kinase, is expressed primarily within the hematopoietic system and is composed of a catalytic subunit p110δ and a regulatory subunit p85. Recently, deregulated BCR-PI3Kδ signaling has been reported to play a role in B-cell malignancies such as chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL) by mediating abnormal B-cell growth and survival. Indeed, the constitutive phosphorylation of downstream signaling intermediate AKT is associated with poor prognosis in several B cell malignancies. Here, we have investigated the potential of a novel small molecule inhibitor of PI3Kδ, AMG 319, to suppress PI3K signaling in human B cell lines and assessed the subsequent effects on viability as a single agent and in combination with chemotherapeutic drugs in preclinical models. Small molecule AMG 319 is a potent and selective inhibitor of PI3Kδ with excellent preclinical pharmacokinetic (PK) properties. AMG 319 was found to potently inhibit PI3Kδ in enzyme assays (IC50 <10 nM). AMG 319 also potently suppressed the phosphorylation of AKT (pAKTS473) in primary murine splenocytes (IC50<5 nM) after BCR cross linking and demonstrated a less than 10 fold shift in human whole blood B cells using a similar BCR pAKT assay in vitro. In a cell based selectivity screen, AMG 319 was selective for PI3Kδ against other PI3K class I isoforms (200 to >5000 fold). Furthermore, AMG 319 was considered inactive at 10 μM on non-PI3K class I kinases in a broader kinome screen of 402 kinases. In preclinical PK studies, AMG 319 had low systemic clearance, T1/2 range of 2–4 hr, oral bioavailability of >45% and unbound fractions in plasma of 5–19%. Here, we have investigated the potential for AMG 319 to inhibit constitutive PI3K mediated signaling and effects on human B cell line viability. In a broad screen of >20 cell lines derived from B cell malignancies, the majority of lines were found to express PI3Kδ protein, all cells lines expressed the PI3Kα and β isoforms and variable levels of constitutive pAKTS473 were detected. AMG 319 was found to potently suppress constitutive pAKTS473 in the cell lines with IC50 in the low single to double digit nM range. Cellular viability was inhibited by AMG 319 though lines were variably sensitive to drug (range low double digit nM to μM IC50). As cell lines were variably sensitive to AMG 319 as a single agent, we examined if AMG 319 could enhance the efficacy of chemotherapeutic agents in vitro and in vivo. These studies focused on a DLBCL cell line HT which was relatively insensitive to AMG 319 as a single agent (IC50 ∼10 μM) in viability assays even though pAKTS473 was potently suppressed (IC50 ∼ 0.030 μM). Treatment with AMG 319 was found to synergize with the effects of vincristine to reduce cell viability in vitro using a 72 hr viability assay. Next we examined whether the enhanced cytotoxicity using these drugs in combination could be observed in vivo. Using the human B-cell lymphoma HT xenograft model, we found that AMG 319 in combination with vincristine enhanced tumor growth inhibition above that observed with either agent alone. Taken together, these findings suggest that the inhibition of PI3Kδ with AMG 319 may enhance the effects of chemotherapeutic agents in B cell malignancies. In conclusion, AMG 319 is a potent and selective inhibitor of PI3Kδ with excellent PK properties. AMG 319 inhibited constitutive pAKTS473, reduced the viability of B cell lines and synergized with vincristine in vitro and in vivo. The safety, PK and preliminary efficacy of AMG 319 are currently being investigated in a Phase I trial in patients with relapsed or refractory lymphoid malignancies. Disclosures: Sinclair: Amgen: Employment, Stock and Options. Metz:Amgen, Inc: Employment, Stock and Options. Cushing:Amgen, Inc: Employment, Stock and Options. Liu:Amgen, Inc: Employment, Stock and Options. Brake:Amgen, Inc: Employment, Stock and Options. Starnes:Amgen, Inc: Employment, Stock and Options. Means:Amgen, Inc: Employment, Stock and Options. Henne:Amgen, Inc: Employment, Stock and Options. Archibeque:Amgen: Employment, Stock and Options. Mattson:Amgen, Inc: Employment, Stock and Options. Drew:Amgen, Inc: Employment, Stock and Options. Busse:Amgen, Inc: Employment, Stock and Options. Wang:Amgen, Inc: Employment, Stock and Options. Al-Assaad:Amgen, Inc: Employment, Stock and Options. Molineux:Amgen: Employment, Stock and Options.

scholarly journals Chimeric CLL-1 Antibody Fusion Proteins Containing Granulocyte-Macrophage Colony-Stimulating Factor or Interleukin-2 With Specificity for B-Cell Malignancies Exhibit Enhanced Effector Functions While Retaining Tumor Targeting Properties

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4437-4447 ◽  
Author(s):  
Jason L. Hornick ◽  
Leslie A. Khawli ◽  
Peisheng Hu ◽  
Maureen Lynch ◽  
Peter M. Anderson ◽  
...  
Keyword(s):  
B Cell ◽  
Fusion Proteins ◽  
Tumor Targeting ◽  
B Cell Malignancies ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Although monoclonal antibody (MoAb) therapy of the human malignant lymphomas has shown success in clinical trials, its full potential for the treatment of hematologic malignancies has yet to be realized. To expand the clinical potential of a promising human-mouse chimeric antihuman B-cell MoAb (chCLL-1) constructed using the variable domains cloned from the murine Lym-2 (muLym-2) hybridoma, fusion proteins containing granulocyte-macrophage colony-stimulating factor (GM-CSF) (chCLL-1/GM–CSF) or interleukin (IL)-2 (chCLL-1/IL–2) were generated and evaluated for in vitro cytotoxicity and in vivo tumor targeting. The glutamine synthetase gene amplification system was employed for high level expression of the recombinant fusion proteins. Antigenic specificity was confirmed by a competition radioimmunoassay against ARH-77 human myeloma cells. The activity of chCLL-1/GM–CSF was established by a colony formation assay, and the bioactivity of chCLL-1/IL–2 was confirmed by supporting the growth of an IL-2–dependent T-cell line. Antibody-dependent cellular cytotoxicity against ARH-77 target cells demonstrated that both fusion proteins mediate enhanced tumor cell lysis by human mononuclear cells. Finally, biodistribution and imaging studies in nude mice bearing ARH-77 xenografts indicated that the fusion proteins specifically target the tumors. These in vitro and in vivo data suggest that chCLL-1/GM–CSF and chCLL-1/IL–2 have potential as immunotherapeutic reagents for the treatment of B-cell malignancies.

In Vitro and In Vivo Effects of CT-011, a Humanized Anti–PD-1 Monoclonal Antibody, in Combination with Rituximab against Human B-Cell Lymphomas.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 724-724
Author(s):  
Fuliang Chu ◽  
Myriam Foglietta ◽  
Hong Qin ◽  
Rakesh Sharma ◽  
Qing Yi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
T Cells ◽  
B Cell ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
In Vivo Effects ◽  
Human B Cell

Abstract Abstract 724 Background: Programmed death (PD)–1 is an inhibitory receptor that impairs the function of activated T-cells and natural killer (NK) cells when engaged by its ligands PD-L1 or PD-L2. We have previously demonstrated that PD-1 is markedly up-regulated in intratumoral and peripheral blood CD4+ and CD8+ T cells in patients with follicular lymphoma (FL), a finding associated with impaired T-cell function, suggesting that PD-1 blockade may improve FL immune control. CT-011, a humanized anti PD-1 monoclonal antibody, was previously studied in a phase I clinical trial in patients with advanced hematological malignancies. CT-011 was well tolerated and induced sustained elevations of CD4+ T cells in the peripheral blood. More importantly, apparent clinical benefit was observed in six patients, including one patient with FL who had large tumor masses that achieved a durable complete remission lasting >14 months. Here, we studied the in vitro and in vivo effects of CT-011 on T-cell and/or NK-cell immune responses against human B-cell lymphoma and the hypothesis that CT-011 may improve tumor control when combined with rituximab, a chimeric anti-CD20 monoclonal antibody for the treatment of human FL. Materials and Methods: To determine the effects of CT-011 on antitumor T cells, intratumoral T cells were isolated from primary FL tumor samples, and cultured with or without autologous tumor cells in the presence or absence of CT-011 or isotype control antibody (50 μg/ml each) for 5 days, and tested for proliferation by 3H thymidine incorporation assay. To determine the effects of CT-011 on NK cells, peripheral blood mononuclear cells (PBMCs) derived from normal donors or patients with FL were cultured in the presence or absence of CT-011 (50 μg/ml) with or without IL-2 for 96 hours and analyzed for expression of various activating receptors including CD16, CD32, CD64, Fas ligand, NKG2D, NKp30, NKp44, and NKp46. The in vivo effects of CT-011 were tested in two B-cell lymphoma xenograft models. Ramos and RL lymphoma tumor cells were injected subcutaneously into nude and SCID mice, respectively, and CT-011 (10 μg/mouse) was injected weekly with or without rituximab starting approximately 7–10 days after tumor inoculation. Results: We observed that CT-011 significantly increased the proliferation of intratumoral T cells in response to autologous tumor cells compared with isotype control antibody. Treatment with CT-011 enhanced the expression of Fas ligand, CD32, CD64, and NKp30 on human NK cells in the presence of IL-2 as compared with PBMCs treated with IL-2 alone or media control. In the RL lymphoma xenograft model in SCID mice, treatment with CT-011 significantly delayed tumor growth (P≤0.05) and improved survival (P≤0.01) compared with control mice injected with saline. In a Ramos lymphoma xenograft model in nude mice, treatment with CT-011 and rituximab eradicated established tumors in a significant proportion of mice (P≤0.05) and markedly improved survival compared with rituximab alone or saline. Conclusions: Taken together, these studies suggest that blockade of PD-1 with CT-011 enhances the function of anti-tumor T-cells and augments the expression of activating receptors on NK cells. Treatment with CT-011 led to improved tumor control against human B-cell lymphoma in xenograft models and the combined use of CT-011 and rituximab was more effective that rituximab alone. These results provide the rationale to test the combination of CT-011 with rituximab in patients with B-cell lymphoma, given that the combination is likely to be complementary and may even be synergistic, leading to enhanced clinical efficacy without increasing toxicity. The development of such approaches that activate both the innate (NK-cells) and adaptive (T-cells) immune systems is likely to minimize the emergence of immune escape variants and improve clinical outcome in patients with lymphoma. A clinical trial evaluating CT-011 in combination with rituximab is planned in patients with relapsed FL. Disclosures: Rodionov: Cure Tech Ltd.: Employment. Rotem-Yehudar:Cure Tech Ltd.: Employment.

TRU-016, An Anti-CD37 SMIP™ Biologic, In Combination with Other Therapeutic Drugs In Models of Non-Hodgkin's Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3931-3931 ◽  
Author(s):  
Paul A. Algate ◽  
Jennifer Wiens ◽  
Christy Nilsson ◽  
Mien Sho ◽  
Debra T. Chao ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Cell Lymphoma ◽  
Combination Index ◽  
Growth Delay ◽  
Tumor Growth Delay ◽  
B Cell Malignancies

Abstract Abstract 3931 Background: CD37 is a 50–55 kDa heavily glycosylated member of the tetraspanin superfamily of molecules. This cell surface protein is expressed on normal and transformed B-cells, and has been implicated in diverse processes including cellular activation and proliferation, cell motility, and cell-cell adhesion. TRU-016 is a novel humanized anti-CD37 SMIP™ protein. Pre-clinical studies have demonstrated that anti-CD37 SMIP™ protein mediates caspase-independent direct killing of normal and malignant B-cells, a mechanism of action that appears to be different than CD20 therapies. In addition, TRU-016 results in indirect killing through NK cell mediated SMIP-protein directed cellular cytotoxicity (SDCC). The therapeutic potential of TRU-016 against several subsets of B-cell malignancies is currently being investigated in the clinic. Methods: The ability of TRU-016 to interact and increase cell killing with established therapeutics rituximab (anti-CD20 antibody), bendamustine (bi-functional alkylating agent/nucleoside analog), LY294002 (PI3K inhibitor) and temsirolimus (mTOR inhibitor) was investigated in vitro using the Rec-1 (mantle cell lymphoma) and SU-DHL-6 (diffuse large B cell lymphoma) cell lines. Individual drugs were tested in combination with TRU-016 as well as in a multiple drug cocktail. Combination index analyses were performed for drug combinations over the 20–90% effect levels. To determine whether in vitro synergy could be recapitulated in vivo, DoHH-2 (follicular lymphoma) xenografts were treated with TRU-016, bendamustine, and the combination of TRU-016 and bendamustine with or without rituximab. Furthermore, the effect of the dosing schedule with the combination of TRU-016 and rituximab was explored by comparing the treatment over a short time period to an extended (maintenance) dosing regimen. CD37 expression on the tumor xenografts was evaluated post different treatment by immunohistochemistry. Results: Combination index analyses determined that the killing effects of TRU-016 was synergistic with rituximab, bendamustine and temsirolimus in NHL models. Furthermore, TRU-016 provided additional efficacy when added to the combination of rituximab and bendamustine. In vivo results demonstrated that the in vitro synergy results were applicable to a more complex in vivo disease model. The combination of TRU-016 with bendamustine or rituximab resulted in increased tumor growth delay compared to that attained with the individual drugs. The addition of TRU-016 to the combination of bendamustine and rituximab resulted in increased tumor growth delay compared to the two drugs alone. The observed efficacy of the combination of TRU-016 and rituximab could be extended with repeated (maintenance) dosing with tumor free survival being observed beyond the 35 days of dosing. The combination of TRU-016 with temsirolimus also resulted in a reduction of tumor growth compared to either molecule alone. CD37 target expression was detected in the xenograft tumors post-treatment with all drugs tested. Conclusions: TRU-016 in combination with rituximab, bendamustine or temsirolimus increased cell killing of NHL cells in vitro over that observed for each agent alone. Furthermore, the triple combination of TRU-016 with rituximab, bendamustine or temsirolimus displayed greater anti-tumor activity in vivo than each of the agents alone against a follicular lymphoma tumor model. The addition of TRU-016 to a combination of rituximab and bendamustine resulted in increased killing in vitro and in vivo. The combinatorial activity of TRU-016 and rituximab in vivo was increased when the drugs were administered over a longer period. These results provide preclinical rationale for the potential different combinations of TRU-016 with several established therapeutics for the treatment of NHL and related B-cell malignancies. Disclosures: Algate: Trubion Pharmaceuticals: Employment. Wiens:Trubion Pharmaceuticals: Employment. Nilsson:Trubion Pharmaceuticals: Employment. Sho:Facet/Abbott: Employment. Chao:Facet/Abbott: Employment. Starling:Facet/Abbott: Employment. Gordon:Trubion Pharmaceuticals: Employment.

Anti-CD20-Interferon-α Fusion Protein Demonstrates Potent Direct Growth Inhibition, Antibody-Dependent Cellular Cytotoxicity, and Enhanced Complement-Dependent Cytotoxicity Against Human B Cell Lymphomas In Vitro, and Activity Against Multiple Human NHL Xenografts In Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2724-2724
Author(s):  
Reiko E Yamada ◽  
Kristopher K Steward ◽  
Gataree Ngarmchamnanrith ◽  
Sanjay Khare ◽  
Raj Sachdev ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Growth Inhibition ◽  
B Cell ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Complement Dependent Cytotoxicity ◽  
Direct Growth ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Human B Cell

Abstract Abstract 2724 The type 1 interferons (IFNα and IFNβ) are potent regulators of malignant cell growth. IFNα has anti-proliferative and pro-apoptotic effects against many cancers, including non-Hodgkin lymphomas (NHL), and immunostimulatory effects including activation of natural killer cells, dendritic cells, and T cell anti-tumor immunity. Until now however, the clinical use of these agents has been limited by the inability to achieve effective concentrations of IFN at sites of tumor without causing systemic toxicity. We recently reported the ability of an anti-CD20 antibody-IFNα fusion protein to induce apoptosis and promote in vivo eradication of CD20-expressing mouse and human B cell lymphomas (C. Xuan et al, Blood 115:2864, 2010). We now report on the preclinical anti-lymphoma activity of a recombinant anti-CD20-human IFNα (IgG1 anti-CD20-hIFNα) fusion protein derived from rituximab variable region sequences. Anti-CD20-hIFNα was evaluated against a large panel of human B cell NHL lines representing aggressive histologies including Burkitt (Daudi, Raji, Ramos), diffuse large B cell (SUDHL-4, OCI-Ly2, OCI-Ly3, OCI-Ly19, HBL-1, RC-K8), and mantle cell (Granta-519) lymphomas. Proliferation was measured by [3H]-thymidine incorporation in quadruplicate 48 hour cultures, apoptosis measured by Annexin-V and propidium iodide (PI) staining, complement-dependent cytotoxicity (CDC) measured by PI flow cytometry, and antibody-dependent cellular cytotoxicity (ADCC) measured by LDH release using peripheral blood mononuclear cell effectors. NHL xenografts Daudi, Raji, and Namalwa were grown in SCID mice. Equimolar doses of rituximab and fusion protein were compared in each assay. Against IFN-sensitive CD20-negative U266 tumor cells, anti-CD20-hIFNα fusion protein had 10–15% IFNα bioactivity when compared to conventional recombinant IFNα (rIFNα). However, when targeting CD20-positive Daudi cells the inhibitory growth activity is significantly enhanced over rIFNα. Anti-CD20-hIFNα fusion protein induced stronger direct growth inhibition than rituximab (23.3–93.1% vs. 0.0–39.8%); particularly against Burkitt (44.7–93.1% vs. 0.0–10.4%) and germinal center-type diffuse large B cell (59.0–88.8% vs. 10.5–39.8%) NHLs. Tumor growth inhibition by anti-CD20-hIFNα was associated with substantial apoptosis in some cell lines. The ADCC activity of fusion protein against Daudi, Ramos, and Raji cells was identical to that of rituximab. Against established human NHL xenografts (Daudi, Raji, and Namalwa), fusion protein achieved improved survival compared to rituximab. Surprisingly, anti-CD20-hIFNα exhibited superior CDC compared to rituximab against Daudi (62.0% vs. 28.3%), Ramos (81.0% vs. 57.3%), and Raji (78.0% vs. 54.6%) cells. We hypothesized that the increased CDC activity of the fusion protein might result from enhanced target cell avidity due to the IFNα moeity binding to IFNα receptors (IFNAR) on the tumor cell surface. However, this was not the case, as flow cytometric studies demonstrated no improvement of fusion protein binding over rituximab, and comparable Kd values. Also, antibody blockade of IFNAR-IFNα interactions, or pre-incubation with excess free IFNα did not prevent the increased CDC activity of anti-CD20-hIFNα fusion protein against Ramos cells. Moreover, the enhanced CDC depended upon linkage of IFNα to the anti-CD20 antibody, as CDC using rituximab plus equimolar free IFNα was equivalent to rituximab alone, suggesting superior complement fixation by anti-CD20-hIFNα. In conclusion, we have demonstrated that anti-CD20-hIFNα has substantially stronger direct anti-proliferative and CDC activities than rituximab against human lymphomas, while retaining potent ADCC activity. The greater effects of IFNα targeted to NHL via fusion protein suggest a broader therapeutic index than rIFNα. Anti-CD20-hIFNα fusion protein is also superior to rituximab in vivo against multiple human NHL xenografts. These results support the further development of anti-CD20-hIFNα fusion protein for the treatment of B cell malignancies. Disclosures: Khare: ImmunGene, Inc.: Employment. Sachdev:ImmunGene, Inc.: Employment. Grewal:ImmunGene, Inc.: Employment.

Overexpression of BCL9, a Wnt/β-Catenin Transcriptional Co-Activator, in Transgenic Mice Promotes Spontaneous Development of B-Cell Malignancies

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 441-441
Author(s):  
Tomasz Sewastianik ◽  
Jianjun Zhao ◽  
Meng Jiang ◽  
Peter S. Dennis ◽  
Myles Brown ◽  
...  
Keyword(s):  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
Signaling Pathway ◽  
Transcriptional Activity ◽  
Hematological Malignancies ◽  
B Cell Malignancies ◽  
Human Cancers ◽  
Stop Cassette

Abstract Dysregulation of the Wnt signaling pathway underlies the pathogenesis of a wide range of human cancers, including hematological malignancies such as multiple myeloma (MM). The terminal effector of this signaling pathway is a transcriptional complex formed by β-catenin and BCL9. This complex is of particular interest because the BCL9 locus resides on the frequently recurring 1q21 chromosomal amplification in MM, which has been linked to poor clinical prognosis and outcome. Our previous studies indicate that BCL9-mediated enhancement of β-catenin activity increases cells proliferation, migration, invasion, and the metastatic potential of MM cells. Therefore, in order to: (I) unequivocally determine the oncogenic role of BCL9, (II) better understand its mechanism of action, and (III) develop mouse preclinical model of cancer with dysregulated Wnt/β-catenin/BCL9 activity, we generated transgenic mouse models. To overcome problems inherently related to embryonic lethality, we generated BCL9fl/- conditional transgenic mice using site-specific transgene integration into the mouse ColA1 gene in embryonic stem cells. To remove the stop cassette and activate BCL9 expression in vivo, we generated AID-Cre+/-; BCL9fl/- and ERT2-Cre+/-; BCL9fl/- compound mice. Recombinase activity driven by AID (activation-induced cytidine deaminase) gene promoter or ER receptor after tamoxifen administration, caused removal of the stop cassette and expression of BCL9 in germinal center (GC) B cells or several tissues, respectively, as confirmed by immunoblot, immunohistochemical (IHC) and PCR analysis. Since BCL9 is a β-catenin co-activator, next we generated cohorts of AID-Cre+/-; BCL9fl/-; TCF/Lef1-lacZ+/- and ERT2-Cre+/-; BCL9fl/-; TCF/Lef1-lacZ+/- triple compound transgenic mice carrying the Wnt reporter system that expresses β-galactosidase (β-gal), to determine whether Wnt/β-catenin transcriptional activity is increased as a consequence of BCL9 overexpression in vivo. β-gal stain was increased in frequency and intensity in cells within GCs but not outside them in AID-Cre+/-; BCL9fl/-; TCF/Lef1-lacZ+/- compared to control mice. In ERT2-Cre+/-; BCL9fl/-; TCF/Lef1-lacZ+/- mice β-gal staining was primarily detected in cells outside the GCs, not within them. Overall, these results indicate that Wnt transcriptional activity is increased in B-cells as a consequence of Cre-induced expression of BCL9 and that AID-Cre+/- and ERT2-Cre+/- target expression of BCL9 to GC and non-GC B cells, respectively. Because BCL9 is involved in the pathogenesis of human cancers, we evaluated whether our transgenic mice develop hematological malignancies. Except for mild splenic enlargement, BCL9-transgenic mice were indistinguishable from control mice between 8 and 30 weeks of age as assessed by weight and posture. However, after 40 weeks of age and at variable times thereafter, 80% (32/40) of AID-Cre+/-; BCL9fl/- and 70% (28/40) of ERT2-Cre+/-; BCL9fl/- mice but none from control cohorts showed signs of disease. Gross pathologic examination of euthanized animals with BCL9 overexpression revealed enlargement of the spleen and LNs. Two distinct patterns of clonal hematological malignancies were identified after detailed histological, IHC and molecular examination. In AID-Cre+/-; BCL9fl/- mice tumors resembled human plasmacytomas (PCs), whereas in ERT2-Cre+/-; BCL9fl/- mice B-cell acute lymphoblastic leukemia (B-ALL). This later result is of particular interest, since BCL9 was first identified by cloning the t(1;14)(q21;q32) translocation from a patient with B-ALL. These findings indicate that BCL9 overexpression at different stages of B-cell development leads to distinct subtypes of B-cell malignancies. Finally, we investigated the BCL9 expression in human extramedullary plasmocytomas (EMP) and B-ALL. 32% of EMP cases analyzed by IHC expressed BCL9 at significant levels. Utilizing gene expression data available in the public domain we also showed that BCL9 is significantly overexpressed in ETV6-RUNX1 and TCF3-PBX1 subtypes of human B-ALL when compared to normal bone marrow counterparts, suggesting that BCL9 may play important roles in the pathogenesis of EMP as well as B-ALL in humans. Since BCL9 is highly expressed in tumors but not in the cells of origin and its interaction with β-catenin is specific, these results imply BCL9 as a promising candidate for targeted therapy. Disclosures No relevant conflicts of interest to declare.

Depletion of Normal and Malignant B cells with a CD37-specific SMIP molecule

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 3063-3063
Author(s):  
C. Cerveny ◽  
L. Grosmaire ◽  
E. Espling ◽  
R. Bader ◽  
C. Nilsson ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Target Cell ◽  
Growth Arrest ◽  
Xenograft Model ◽  
B Cell Malignancies ◽  
B Lymphoma ◽  
Anti Tumor Activity

3063 Background: CD37 is a member of the tetraspanin family expressed at high levels by normal mature B cells and by most B cell malignancies. Previously, an antibody to CD37 has been labeled with 131I and tested in clinical trials for therapy of NHL. Treatment with 131I-MB-1, resulted in durable tumor remissions in patients lasting from 4 to 11 months (Press OW, Eary JF, Badger CC, et al. Treatment of refractory non-Hodgkin’s lymphoma with radiolabeled MB-1 (anti-CD37) antibody. J Clin Oncol. 1989;7:1027–1038). Here we assess the functional properties and therapeutic potential of a small modular immunopharmaceutical (SMIP) targeting CD37. Methods: Growth arrest and apoptosis of B lymphoma cell lines was assessed. ADCC activity was evaluated using BJAB targets and human peripheral blood mononuclear cells (PBMC) effectors. Drug-drug interactions were assessed by the Combination Index method. In vivo studies were performed utilizing established human B cell tumor xenografts in nude mice. Results: A CD37-directed SMIP drug candidate mediated growth arrest, apoptosis and ADCC, but not CDC, towards B lymphoma cell lines. The protein showed significant anti-tumor activity in a mouse xenograft model, and selectively depleted normal human B cells in short term cultures of PBMC. When combined with rituximab, the molecule increased apoptosis, C1q binding, and C’ dependent target cell death in vitro, and increased anti-tumor activity in vivo in a xenograft model. Conclusions: In vitro and in vivo characterization of the CD37-targeted SMIP drug suggest a potent capacity to eliminate target cells through combined effects of direct target cell signaling and effector cell recruitment. CD37-mediated growth was synergistic with standard chemotherapies in vitro and showed additive in vivo activity with CD20-targeted therapy. On the basis of these data CD37-directed SMIP therapy is being developed for clinical evaluation against B cell malignancies. No significant financial relationships to disclose.

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