GlycoVariant Anti-CD37 Small Modular Immuno-Pharmaceutical Exhibits Superior Natural Killer Cell Mediated Cytotoxicity Against Chronic Lymphocytic Leukemia Cells at Low Concentrations and Low Antigen Density

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1847-1847
Author(s):  
Sarwish Rafiq ◽  
Carolyn M Cheney ◽  
Gerard Lozanski ◽  
Rosa Lapalombella ◽  
Xiaokui Mo ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
B Cell Lymphoma ◽  
Leukemia Cells ◽  
Low Concentrations ◽  
New Variant ◽  
Direct Cytotoxicity

Abstract Abstract 1847 CD37 is a tetraspanin transmembrane family protein that is strongly expressed on the surface of mature human B cells and transformed mature B cell lymphoma and leukemia cells, including CLL cells. It is absent or minimally expressed on normal T cells, NK cells, monocytes, and granulocytes. Predominant expression of CD37 on CLL cells makes it an ideal candidate to target with potential agents for treatment of CLL. TRU-016, a small modular immunopharmaceutical protein (SMIP) that specifically binds to an extracellular region of CD37, is presently in clinical trials in CLL patients. TRU-016 includes humanized immunoglobulin variable regions (scFv) fused to a human IgG1 Fc region. We have previously reported that SMIP-016, the chimeric version of the humanized TRU-016, induced apoptosis in CLL B cells in the presence of goat anti-human Fc antibody cross-linker through a novel, caspase-independent pathway. Furthermore, SMIP-016 showed potent in vivo activity in a SCID xenograft mouse model. Aside from direct cytotoxicity, SMIP-016 mediates antibody-dependent cellular cytotoxicity (ADCC) by NK cells both in vitro and in vivo. In an attempt to enhance its ADCC function, a new variant of SMIP-016, SMIP-016GV, was designed with a modification of the glycosylation of the Fc portion of the molecule. SMIP-016GV exhibits enhanced binding to both low- and high-affinity molecular variants of human CD16 (FcγRIII) and augments ADCC potency when compared to SMIP-016. In this study, we compared SMIP-016GV and SMIP-016 in direct cytotoxicity and ADCC against CLL B cells. While SMIP-016 and SMIP-016GV mediated comparable direct cytotoxicity at 48 hrs in the presence of goat anti-human Fc crosslinker, the SMIP-016GV resulted in 2 to 4 fold increase in NK cell mediated ADCC function at all effector to target ratios tested. This increased ADCC with SMIP-016GV was observed using NK cell effectors derived from both normal as well as CLL-affected individuals. In addition, this enhanced cytotoxicity was sustained at concentrations of SMIP-016GV as low at 5E-6 μg/ml. These low concentrations of SMIP-016GV were also able to mediate superior ADCC in 697 cells expressing as few as 10,000 molecules of surface CD37 antigen. Furthermore, NK cells stimulated with the glycovariant were potently activated and released 3 to 4 fold more IFNγ compared to SMIP-016. Ongoing studies are aimed at defining other effector cells which may interact with SMIP-016GV via different Fcγ Receptors. Collectively, these results suggest potential use of the SMIP-016GV with enhanced ADCC function as an alternate for TRU-016 in B cell malignancies including CLL therapy. Disclosures: Siadak: Trubion Pharmaceuticals: Employment.

Glycovariant CD37 Small Modular Immuno-Pharmaceutical (TruADhanCe™ SMIP) Promotes Enhanced Natural Killer Cell Mediated Cytotoxicity against Primary Chronic Lymphocytic Leukemia Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1744-1744
Author(s):  
Sarwish Rafiq ◽  
Carolyn Cheney ◽  
Peter A Thompson ◽  
Tony Siadak ◽  
Paul Algate ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Natural Killer ◽  
B Cell Lymphoma ◽  
Leukemia Cells ◽  
New Variant ◽  
Direct Cytotoxicity ◽  
Cross Linker

Abstract Abstract 1744 Poster Board I-770 CD37 is a tetraspanin transmembrane family protein that is strongly expressed on the surface of mature human B-cells and transformed mature B-cell lymphoma and leukemia cells, including CLL cells. It is expressed minimally or is absent on normal T-cells, natural killer cells, monocytes, and granulocytes. Predominant expression of CD37 on CLL cells makes it an ideal candidate to target with potential agents for treatment of CLL. TRU-016, a Small Modular ImmunoPharmaceutical protein (SMIP) targeted towards the extracellular region of CD37, is presently in clinical trials in CLL patients. TRU-016 consists of variable regions (scFv) and engineered constant regions encoding the human IgG1 domains. We have previously reported that SMIP-016, the chimeric precursor of the fully humanized TRU-016, induced apoptosis in CLL B cells in the presence of goat anti-human Fc ab cross-linker through a novel, caspase-independent pathway. Furthermore, SMIP-016 showed potent in-vivo activity in a SCID xenograft mouse model. Aside from direct cytotoxicity, SMIP-016 mediates antibody-dependent cellular cytotoxicity (ADCC) by NK cells both in vitro and in vivo. Recently, in an attempt to enhance the ADCC function, a new variant of SMIP-016, Tru-ADhanCe SMIP-016, has been created with a modification of the glycosylation of the Fc portion of the molecule. TRU-ADhanCe SMIP-016 has been shown to exhibit enhanced binding to both low- and high-affinity molecular variants of human CD16 (FcRIII) and augmented ADCC potency when compared to SMIP-016. In this study, we compared TruADhanCe SMIP-016 and SMIP-016 in direct cytotoxicity and ADCC experiments using CLL B-cells. While SMIP-016, and TruADhanCe SMIP-016 mediated comparable direct cytotoxicity at 24, 48 and 72 hrs in the presence of anti-human Fc crosslinker, the TruADhanCe SMIP-016 resulted in 2 to 4 fold increased NK cell mediated ADCC function. Consistent with the comparable direct cytotoxic effects, the early phosphorylation patterns were similar in cells treated with TruADhanCe SMIP-016 or SMIP-016 in the presence of anti-human Fc cross linker. Ongoing studies are aimed to define the mechanistic basis of the enhanced ADCC function by TruADhanCe SMIP-016 and to determine if use of soluble CD16.Fc as a cross-linker, an in vitro model of in vivo Fc receptor binding, may reveal enhanced apoptotic-signaling of TruADhanCe SMIP-016. These results suggest potential use of TruADhanCe versions of TRU-016 with enhanced ADCC function as an alternate for TRU-016 in B cell malignancies including CLL therapy. [This work was supported by D. Warren Brown Foundation, Leukemia and Lymphoma Society and National Cancer Institute.] Disclosures Thompson: Trubion Pharmaceuticals: Employment. Siadak:Trubion Pharmaceuticals: Employment. Algate:Trubion Pharmaceuticals: Employment. Cerveny:Trubion Pharmaceuticals: Employment.

scholarly journals Suppressing Synthesis of the Long Isoform of the Prolactin Receptor Is a Targeted Strategy to Prevent and Treat B Cell Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1135-1135
Author(s):  
Adeleh Taghi Khani ◽  
Anil Kumar ◽  
Kelly Radecki ◽  
Sung June Lee ◽  
Mary Lorenson ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Nk Cells ◽  
Malignant Transformation ◽  
Nk Cell ◽  
B Cell Lymphoma ◽  
B Cell Malignancies ◽  
Autoreactive B Cells ◽  
Cell Numbers ◽  
Bcl2 Expression

Abstract Rationale B cell malignancies, including leukemia and lymphoma, are high-risk lymphoid neoplasms. B cell malignancies predispose to autoimmune diseases including systemic lupus erythematosus (SLE) which increase the risk of developing these malignancies by >5-fold. Increased prolactin (PRL) expression is known to exacerbate SLE and promote the survival of autoreactive B cells. Furthermore, PRL induces expression of the protooncogenes, MYC and BCL2, in lymphoid tissues. However, whether PRL drives the initiation and maintenance of B cell malignancies was not known. Results We first tested our hypothesis that PRL, specifically signaling through the pro-proliferative and anti-apoptotic long isoform (LF) of the PRL receptor (PRLR), drives the progression of SLE to B cell malignancies. To this end, we knocked down the LF PRLR in MRL-lpr mice predisposed to developing SLE using a splice-modulating oligomer (SMO) that blocks splicing to produce the LF PRLR without affecting the short isoforms. LF PRLR knockdown reduced splenic and circulating B cell numbers in MRL-lpr SLE mice (Fig.1a). Consistent with reduced B cell numbers, BCL2 expression in B cells of SLE mice was suppressed after LF PRLR knockdown, although MYC was unaltered (Fig.1b). By sequencing the immunoglobulin heavy chains (IGH), we compared the composition of the splenic B cell repertoire between control- and LF PRLR SMO-treated SLE mice. Control oligomer treated SLE mice accumulated splenic B cells with long complementary determining region 3 (CDR3) and B cells with non-functional IGH, characteristics of autoreactive B cells. Treatment with the LF PRLR SMO reduced both. We then measured the expression of enzymes known to induce malignant transformation of B cells, namely recombination activating genes 1/2 (RAG1/2) and activation-induced cytidine deaminase (AID), in B cells of SLE mice in controls versus LF PRLR knockdown. Importantly, LF PRLR knockdown significantly reduced RAG1 (Fig.1c) and AID expression in splenic B cells of SLE mice (Fig.1d,e). Our findings thus underscore a causal role for LF PRLR signaling in promoting of malignant transformation of B cells in SLE. Because PRL induces the expression of BCL2 and MYC in lymphocytes, we next determined whether LF PRLR promotes the survival of overt B cell malignancies that overexpress MYC and BCL2, including diffuse large B cell lymphoma (DLBCL) and B-cell acute lymphoblastic leukemia (B-ALL). We observed that B-lymphoblasts expressed significantly higher levels of PRL and the LF PRLR as compared to normal B cells (Fig.1f). We also found that higher expression of PRL at diagnosis predicts poor clinical outcome in DLBCL patients (P=0.0244), and that patients with MYC/BCL2-overexpressing ALLs with a poor prognosis had significantly higher expression of the LF PRLR compared to their MYC lowBCL2 low counterparts (P<0.0001). These observations suggested that LF PRLR may modulate MYC and BCL2 expression. Knockdown of the LF PRLR using the LF PRLR SMO in MYC/BCL2-driven human B cell malignancies killed lymphoblasts and reduced MYC and BCL2 protein levels (Fig.1g). Because we previously showed that MYC-driven lymphoid malignancies are sensitive to natural killer (NK) cell-mediated immune clearance, we also examined whether LF PRLR knockdown synergized with NK cells in killing DLBCL. We found that LF PRLR knockdown enhanced NK cell-mediated killing of B-lymphoblasts (Fig.1h). Of note, no reductions were observed in NK cell viability or MYC levels within NK cells upon LF PRLR knockdown, suggesting that LF PRLR selectively kills B-lymphoblasts without negatively impacting NK homeostasis. Conclusion Our studies identify the specific knockdown of LF PRLR as a potentially safe and targeted strategy to prevent the onset of B cell malignancies in SLE patients and to treat flagrant DLBCL and B-ALL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals The IL-15 Superagonist ALT-803 Enhances NK Cell ADCC and in Vivo Clearance of B Cell Lymphomas Directed By an Anti-CD20 Monoclonal Antibody

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 807-807 ◽  
Author(s):  
Maximillian Rosario ◽  
Bai Liu ◽  
Lin Kong ◽  
Stephanie E Schneider ◽  
Emily K Jeng ◽  
...  
Keyword(s):  
B Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Vehicle Treatment ◽  
Anti Cd20

Abstract Anti-CD20 monoclonal antibodies (mAbs) have provided an important therapeutic passive immunotherapy approach for B cell malignancies. Typically anti-CD20 mAbs are combined with traditional chemotherapy and/or radiation therapy that have the potential for serious long term adverse complications. Novel approaches are needed to improve anti-CD20 mAb anti-lymphoma efficacy with agents that do not result in long term toxicity. ALT-803 is a super agonist IL-15 variant bound to an IL-15Ralpha – Fc fusion and represents a novel immunostimulatory agent for NK and T cells. Notably, ALT-803 exhibits extended in vivo pharmacokinetics and altered biodistribution compared to recombinant human IL-15. We hypothesized that ALT-803 would enhance anti-CD20-mAb-(rituximab) directed NK cell antibody dependent cellular cytotoxicity (ADCC) against B cell lymphomas. ALT-803 at concentrations of 0.35-35 ng/mL potentiated rituximab-triggered NK cell ADCC against the Raji (27% vs. 74%, E:T 25:1, P<0.01) and Daudi (39% vs. 84%, E:T 2:1, P<0.05) B cell lymphoma lines in vitro. Moreover, the activation of NK cells with ALT-803 significantly increased ADCC against primary human follicular lymphoma cells in vitro (11% vs. 33% at a 2.5:1 E:T ratio, P<0.001, N=5 primary follicular lymphomas, N=15 NK cell donors). One mechanism whereby ALT-803 may be modulating human NK cells is via the induction of cytotoxic effector molecules. After 24-48 hour in vitro activation with ALT-803 (0.35-350 ng/mL) an increased expression of granzyme B and perforin were observed in primary human NK cells (P<0.05). The effectiveness of ALT-803 enhancement of NK cell ADCC against B cell lymphoma cell lines was assessed with two in vivo mouse models. First, Daudi cells were engrafted into SCID mice (that have an intact NK cell compartment), and groups were treated with vehicle, rituximab (10 mg/kg), ALT-803 (0.2 mg/kg), or ALT-803+rituximab at day 15 and 18, and assessed for lymphoma percentages in the bone marrow at day 22. Mice treated with ALT-803+rituximab had significantly reduced Daudi B cell burden, compared to rituximab, ALT-803, or vehicle treatment (vehicle versus ALT-803+rituximab, 38% vs. 5%, P<0.01). At doses of 0.2-0.02 mg/kg the effect of ALT-803 on rituximab mediated lymphoma clearance was demonstrated (P<0.02 compared to vehicle treatment). Further, the survival of mice treated with ALT-803+rituximab was significantly longer compared vehicle control, rituximab alone, and ALT-803 alone groups (see KM survival curves, Figure, P<0.05). In our second in vivo model, malignant Raji B cells expressing luciferase were injected into immunodeficient NOD-SCID-gamma-c-/- (NSG) mice (100,000/mouse, day 0) followed by primary human NK cells (4 million / mouse, day 3), and treated (initiated on day 3) with vehicle, ALT-803 (0.05 mg/kg q3-4 days), rituximab (10 mg/kg day 3), or ALT-803+rituximab. At day 16, ALT-803+rituximab exhibited a significant reduction in Raji signal compared to the control groups (P<0.05). These results were confirmed in a second approach where an increased Raji cell numbers were engrafted (1 million / mouse, day 0), primary human NK cells (4 million / mouse) were infused on day 3, and groups of mice were treated with rituximab (5 mg/kg) or ALT-803 (0.2 mg/kg q3-4 days)+rituximab (mean photons per second at day 34, 3.8x109 versus 3.1x108, respectively, P<0.05). ALT-803 was well tolerated at all of the administered dose levels in combination with rituximab. Thus, ALT-803 represents an effective immunostimulatory agent that augments NK cell cytotoxic potential and ADCC against malignant follicular lymphoma and B cell lines in vitro, and significantly increases rituximab-directed clearance of B cell lymphoma by NK cells in two in vivo models. Based on these findings, a phase 1/2 clinical trial of ALT-803 plus rituximab is planned for patients with relapsed/refractory indolent non-Hodgkin lymphomas. Figure 1 Figure 1. Disclosures Liu: Altor BioScience Corporation: Employment. Kong:Altor BioScience Corporation: Employment. Jeng:Altor BioScience Corporation: Employment. Rhode:Altor BioScience Corporation: Employment. Wong:Altor BioScience Corporation: Employment.

scholarly journals A Novel Anti-Lymphoma Immune Evasion Mediated By the Interaction Between PD-1 Enriched NK-Cells and CD163+PD-L1+PD-L2+ Tumor Associated Macrophages, That Is More Prominent in Hodgkin Lymphoma Than Diffuse Large B-Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 918-918 ◽  
Author(s):  
Maher K Gandhi ◽  
David Arpon ◽  
Colm Keane ◽  
Erica Han ◽  
Josh Tobin ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Nk Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
B Cell Lymphoma ◽  
Hla Class I ◽  
Cell Phenotype ◽  
Tumor Associated Macrophages ◽  
Large B Cell Lymphoma

Abstract PD-L1/PD-L2 are immunomodulatory molecules that engage with the PD-1 receptor on immune effector T and NK-cells to inhibit anti-lymphoma immunity. PD-1/PD-L1/PD-L2 axis molecules are prognostic in Hodgkin Lymphoma (HL, Roemer et al J Clin Oncol 2016) and Diffuse Large B-cell Lymphoma (DLBCL, Keane et al Lancet Haem 2015). Importantly, blockade of the axis is associated with particularly potent clinical responses in relapsed/refractory HL (Ansell et al NEJM 2015), as well as response in DLBCL (Armand et al J Clin Oncol 2013). Focus has been on the interaction of PD-L1 on malignant B-cells with PD-1 on HLA-class I restricted CD8+ effector T-cells. This is despite considerable evidence that: A) malignant B-cells in HL and DLBCL frequently lack the ability to present HLA-class I due to mutations in b2M and associated antigen presenting molecules (Challa-Malladi et al Cancer Cell 2013). This makes them insensitive to direct lysis by CD8+ T-cells (Zaretsky et al NEJM 2016) but potentially enhances their sensitivity to NK-cells; B) PD-L1/PD-L2 are expressed by inhibitory CD163+ monocytes/macrophages as well as by malignant B-cells (Chen et al CCR 2013). Here, we seek to establish the contribution of NK-cells and inhibitory CD163+ expressing monocytes/macrophages in the setting of HL and DLBCL. CD163/PD-1/PD-L1/PD-L2 gene expression was quantified by nanoString in 194 patients and was elevated in HL relative to DLBCL tissues (P<0.01, <0.01, <0.0001, <0.0001 respectively). By FACS, intratumoral tumor associated macrophages (TAMs) demonstrate pronounced protein expression of PD-L1/PD-L2 within HL and DLBCL diseased lymph nodes (Fig A). Pre-therapy blood was tested in 114 patients. Interestingly levels in each of total monocytes, CD14+HLA-DRlo monocytoid derived suppressor cells (moMDSC) and CD163+CD14+ monocytes were equivalent between lymphoma sub-types. However, consistent with tissue findings, there was marked increase in PD-L1 expression on CD14+ monocytes, moMDSC and CD163+CD14+ monocytes in HL compared to DLBCL patients (P<0.001, <0.0001 and 0.0086 respectively). The NK-cell marker CD56 were higher in HL compared to DLBCL tissues (P<0.0001). Levels of PD-1 on circulating NK-cells were 7-fold elevated in HL relative to DLBCL (P<0.0001), whereas CD4+ and CD8+ T-cell PD-1 levels were equivalent between lymphoma sub-types. NK-cells can be subdivided into CD3-CD56dimCD16+ and CD3-CD56hiCD16- subsets. The CD16- subset produces abundant cytokines but are only weakly cytotoxic before activation. Although CD16- NK-cells are typically <10% of all NK-cells in the healthy circulation, we show their relative proportion is markedly expanded by 3.5-fold in HL patients. This is of particular importance since CD16- NK-cells are enriched in secondary lymphoid tissues, i.e. the context in which lymphoma resides. Notably, CD3-CD56hiCD16- NK-cells had substantially higher PD-1 expression relative to CD3-CD56dimCD16+ cells (P<0.0001, Fig B). A similarly aberrant NK-cell phenotype was observed in DLBCL. An in-vitro functional model of TAM-like monocytes was developed to demonstrate the potential impact of inhibitory CD163+ expressing monocytes/macrophages on NK-cells in HL and DLBCL. Monocytes were cultured with the M6 TAM inducing cytokine cocktail of M-CSF and IL-6. Consistent with an inhibitory phenotype, M6 cultured monocytes were highly enriched for CD163 (P<0.001) and PD-L1 (P=0.0024). Critically, M6 cultured monocytes suppressed activation of primary NK-cells in direct cytotoxicity and ADCC assays against lymphoma targets. In line with these findings, depletion of circulating monocytes from the blood of pre-therapy HL and DLBCL patients enhanced NK-cell activation relative to monocyte intact PBMC, whereas this was not observed in age/gender-matched healthy control participants. Interestingly, the increase in NK-cell activation following monocyte depletion was most pronounced in the CD16-CD56hiCD3- NK-cell subset. We describe a hitherto unrecognised immune evasion strategy mediated via skewing towards an exhausted PD-1 enriched CD16-CD56hiCD3- NK-cell phenotype. In addition to inhibition of NK-cells by the malignant B-cell, suppression of NK-cells occurs by PD-L1/PD-L2 expressing tumor associated macrophages. This mechanism is more prominent in HL than DLBCL. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Pax5 determines B- versus T-cell fate and does not block early myeloid-lineage development

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4342-4346 ◽  
Author(s):  
Claudiu V. Cotta ◽  
Zheng Zhang ◽  
Hyung-Gyoon Kim ◽  
Christopher A. Klug
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Fate ◽  
Nk Cell ◽  
Cell Lineage ◽  
Blood Analysis ◽  
Hematopoietic Stem ◽  
Peripheral Blood Analysis

Abstract Progenitor B cells deficient in Pax5 are developmentally multipotent, suggesting that Pax5 is necessary to maintain commitment to the B-cell lineage. Commitment may be mediated, in part, by Pax5 repression of myeloid-specific genes. To determine whether Pax5 expression in multipotential cells is sufficient to restrict development to the B-cell lineage in vivo, we enforced expression of Pax5 in hematopoietic stem cells using a retroviral vector. Peripheral blood analysis of all animals reconstituted with Pax5-expressing cells indicated that more than 90% of Pax5-expressing cells were B220+ mature B cells that were not malignant. Further analysis showed that Pax5 completely blocked T-lineage development in the thymus but did not inhibit myelopoiesis or natural killer (NK) cell development in bone marrow. These results implicate Pax5 as a critical regulator of B- versus T-cell developmental fate and suggest that Pax5 may promote commitment to the B-cell lineage by mechanisms that are independent of myeloid gene repression.

scholarly journals Mouse model of Epstein–Barr virus LMP1- and LMP2A-driven germinal center B-cell lymphoproliferative disease

2017 ◽  
Vol 114 (18) ◽  
pp. 4751-4756 ◽  
Author(s):  
Takeharu Minamitani ◽  
Yijie Ma ◽  
Hufeng Zhou ◽  
Hiroshi Kida ◽  
Chao-Yuan Tsai ◽  
...  
Keyword(s):  
B Cells ◽  
Mouse Model ◽  
B Cell ◽  
Germinal Center ◽  
Epstein Barr Virus ◽  
Target Genes ◽  
Nk Cell ◽  
Barr Virus ◽  
Epstein Barr

Epstein–Barr virus (EBV) is a major cause of immunosuppression-related B-cell lymphomas and Hodgkin lymphoma (HL). In these malignancies, EBV latent membrane protein 1 (LMP1) and LMP2A provide infected B cells with surrogate CD40 and B-cell receptor growth and survival signals. To gain insights into their synergistic in vivo roles in germinal center (GC) B cells, from which most EBV-driven lymphomas arise, we generated a mouse model with conditional GC B-cell LMP1 and LMP2A coexpression. LMP1 and LMP2A had limited effects in immunocompetent mice. However, upon T- and NK-cell depletion, LMP1/2A caused massive plasmablast outgrowth, organ damage, and death. RNA-sequencing analyses identified EBV oncoprotein effects on GC B-cell target genes, including up-regulation of multiple proinflammatory chemokines and master regulators of plasma cell differentiation. LMP1/2A coexpression also up-regulated key HL markers, including CD30 and mixed hematopoietic lineage markers. Collectively, our results highlight synergistic EBV membrane oncoprotein effects on GC B cells and provide a model for studies of their roles in immunosuppression-related lymphoproliferative diseases.

CDDO and CDDO-Im Reduce Tumor Burden in a Transgenic Mouse Model of CLL.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3477-3477
Author(s):  
Juan M. Zapata ◽  
Christina L. Kress ◽  
Marina Konopleva ◽  
Maryla Krajewska ◽  
Mark Hyer ◽  
...  
Keyword(s):  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
B Cell Lymphoma ◽  
Chemotherapeutic Agents ◽  
Tumor Burden ◽  
Leukemic Cells ◽  
Medium Size ◽  
Control Group

Abstract Transgenic mice over-expressing in B lymphocytes both Bcl-2 and a TRAF2 mutant lacking the N-terminal RING and zinc finger domains (TRAF2DN), which mimics TRAF1, develop small B cell lymphoma and leukemia that have remarkably similar characteristics to human chronic lymphocytic leukemia (CLL). TRAF2DN/Bcl-2 mice develop over time leukemia, severe splenomegaly, and lymphadenopathy, which are associated with monoclonal and oligoclonal B cell neoplasms. The lifespan of TRAF2DN/Bcl-2 mice is markedly reduced compared to Bcl-2 and TRAF2DN single transgenics or wild-type littermates. The expanded B cell population in the blood of leukemic TRAF2DN/Bcl-2 double transgenic mice is primarily comprised of small-medium size, non-cycling B220M/IgMH/IgDL/CD21L/CD23−/CD11b+/CD5+ cells that were Bcl-6 negative, consistent with a B-1 phenotype, closely resembling their human CLL counterparts. Indeed, these B cells showed comparable proliferation rates to normal B-cells, but exhibited markedly increased survival and were resistant to apoptosis induced by chemotherapeutic agents and glucocorticoids. We studied the effects of synthetic triterpenoid 2-Cyano-3,12-Dioxooleana-1,9-Dien-28-Oic Acid (CDDO) and its imidazolide derivative (CDDO-Im) on cultured B-cells from the TRAF2DN/Bcl-2 transgenic mice. Both CDDO and CDDO-Im efficiently induced apoptosis of these cells in vitro, although CDDO-Im was approximately 10-times more potent than CDDO (LD50: 0.35μM CDDO-Im vs 3.8 μM CDDO). To study the effect of CDDO and CDDO-Im in vivo, groups of TRAF2DN/Bcl-2 mice that had developed leukemia were injected i.v. with liposomes alone or liposomes containing either CDDO or CDDO-Im, at a dose of 20 mg/kg/day. Each mouse received a total of nine injections administered over a period of 22 days. The concentration of B cells in the blood of these mice was monitored daily after each injection, using a mini-FACS (Guava Technologies, Inc.). CDDO-treated mice showed a steady reduction in the number of leukemic cells in blood during the treatment and this tendency was maintained 10 days after the last treatment. In contrast, CDDO-Im treated mice showed a striking increase in the concentration of B cells in blood (B220+ events) immediately after the first inoculation. One mouse of this group died after the first injection, and 2 more mice died after 5 injections. Only 2 mice treated with CDDO-Im survived the full treatment, showing a striking reduction of leukemic cells in blood by the end of the treatment. Administration of empty liposomes had no inhibitory effect on the leukemia, and mice in this control group had massive splenomegaly (1431±323 mg; n=3) and severe disseminated lymphadenopathy. In contrast, CDDO-treated mice had less severe splenomegaly (938±234; n=4) but still had severe lymphadenopathy. CDDO-Im treated mice showed a dramatic reduction in the spleen size that was evident also in those mice that died after 5 injections (474±185 mg; n=4) and had no signs of lymphadenopathy. Although preliminary, these results indicate that in vivo administration of CDDO and CDDO-Im reduced the tumor burden in a transgenic model of CLL, and illustrate the potential of triterpenoids as single agents for the treatment of CLL.

Pathologic Co-Expression and Physical Interaction of c-MYC and BCL6 in B-Cell Lymphomas.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2-2 ◽  
Author(s):  
Masumichi Saito ◽  
Ryan T. Phan ◽  
Herbert C. Morse ◽  
Laura Pasqualucci ◽  
Riccardo Dalla-Favera
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lymphoma ◽  
Somatic Hypermutation ◽  
B Cell Lymphoma ◽  
Transgenic Animals ◽  
Primary Lymphoma ◽  
Physical Interaction

Abstract Deregulated expression of the proto-oncogenes BCL6 and c-MYC caused by chromosomal translocation or somatic hypermutation is common in non-Hodgkin B cell lymphoma derived from germinal center (GC) B cells, including diffuse large cell lymphoma (DLBCL) and Burkitt lymphoma (BL). Normal GC B cells express BCL6, whereas, surprisingly, they do not express c-MYC, suggesting that the expression of this oncogene in BL and DLBCL (20% of cases) is ectopic (Klein, U. et al. Proc Natl Acad Sci U S A100, 2639–2644, 2003). Here we report that c-MYC is absent in proliferating GC B cells because it is transcriptionally suppressed by BCL6, as demonstrated by the presence of specific BCL6 binding sites in the c-MYC promoter region and by chromatin immunoprecipitation experiments showing that BCL6 is bound to these sites in vivo. Thus, c-MYC escapes BCL6-mediated suppression in lymphoma leading to the co-expression of the two transcription factors, an event never observed in immunohistochemical and gene expression profile analysis of normal GC B cells. Surprisingly, co-immunoprecipitation experiments and in vitro binding experiments indicate that, when co-expressed, BCL6 and c-MYC are physically bound in a novel complex detectable in DLBCL and BL cell lines as well as in primary lymphoma cases. The formation of the BCL6/c-MYC complex has several significant functional consequences on the function of both c-MYC and BCL6: 1) a two fold, BCL6-binding dependent increase in c-MYC half-life, an event that has been shown to contribute to its oncogenic activation; 2) a synergistic increase in the ability of both BCL6 and c-MYC to suppress MIZ1-activated transcription of the p21CIP cell cycle arrest gene; 3) MYC-dependent inhibition of BCL6 acetylation by p300, an event that physiologically inactivates BCL6 via c-MYC-mediated recruitment of HDAC. Notably, the pathologic co-expression of c-MYC and BCL6 was shown to have pathologic consequences in vivo, since double transgenic BCL6/c-MYC mice display accelerated lymphoma development and the appearance of a novel GC-derived tumor phenotype not recognizable in single transgenic animals and containing the pathologic c-MYC/BCL6 complex. Thus, the pathologic co-expression and illegitimate physical interaction of BCL6 and c-MYC leads to an increase in the constitutive activity of both oncogenes. These results identify a novel mechanism of oncogenic function for BCL6 and c-MYC and a novel tumor-specific protein complex of potential therapeutic interest.

Optimal NK Cell Expansion Depends on Accessory Cells, Synergy between Physiologic Concentrations of IL-2 and IL-15, and Umbilical Cord Blood (UCB) NK Cell Precursors Expand Better Than Adult NK Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3642-3642 ◽  
Author(s):  
Purvi Gada ◽  
Michelle Gleason ◽  
Valarie McCullar ◽  
Philip B. McGlave ◽  
Jeffrey S. Miller
Keyword(s):  
T Cells ◽  
B Cells ◽  
Nk Cells ◽  
Cell Expansion ◽  
Nk Cell ◽  
High Dose ◽  
Unexpected Result ◽  
Accessory Cells

Abstract Allogeneic NK cells may play a therapeutic role in treating patients with AML. We have previously shown that high dose cyclophosphamide (120 mg/kg × 1 day) and fludarabine (125 mg/m2 × 5 days) can clear lymphoid space and induce a surge of endogenous IL-15 to expand haploidentical NK cells obtained from CD3-depleted lymphapheresis products from adult donors. In this initial study, 5 of 19 patients achieved remissions and in vivo NK cell expansion. Limitations of this therapy includeinability of NK cells to expand in most patients,development of PTLD (in one patient) andinadequate disease control.We hypothesized that contaminating T cells could compete for NK cell expansion, that B-cells may contribute to PTLD, and that a 2-step NK cell purification method using CD3 depletion followed by CD56 selection (CliniMacs) may overcome these problems. We tested this in 9 patients with advanced AML. The purified NK cells, activated with 1000 U/ml IL-2 (16–20 hours), were infused 48 hours after the last fludarabine dose. Patients then received subcutaneous IL-2 (10 MU) every other day × 6 doses to expand NK cells in vivo. None of the 9 pts treated on this protocol achieved remission or exhibited evidence of in vivo expansion. Several studies were designed to investigate this unexpected result. First, we found that the more extensive processing resulted in approximately 1/3 the NK cell recovery compared to CD3 depletion alone (38±% viable NK cells vs. 91±2% respectively). In addition, we questioned whether the contaminating B cells and monocytes that were removed in the 2-step depletion strategy had served a critical role in NK cell activation or expansion. Cytotoxicity assays performed against K562 targets showed that the killing was about 3-fold higher with the purified (CD3-CD56+) product compared the CD3-depleted product alone (P=0.001 at E:T of 6.6:1). Proliferation, measured by a 6-day thymidine assay, was higher in proportion to the higher NK cell content. The only difference between the two NK products was their expansion after 14 days of culture, where the CD3-depleted product, with contaminating B-cells and monocytes, gave rise to greater NK cell expansion (14 ±3-fold) compared to the 2-step purified product (4.5±0.9, n=6, P=0.005). If this finding holds true in vivo, the co-infusion of accessory cells may be required for NK cell expansion. We next developed in vitro assays using very low concentrations (0.5 ng/ml) of IL-2 and IL-15 to understand their role in expansion. IL-2 or IL-15 alone induced low proliferation and the combination was synergistic. Lastly, UCB, a rich source of NK cell precursors, was compared to adult NK cells. In a short term proliferation assay, CD56+ NK cells stimulated with IL-2 + IL-15 expanded better from adult donors (61274±12999, n=6) than from UCB (20827± 6959, n=5, P=0.026) but there was no difference after 14 days in expansion culture suggesting that the only difference is in kinetics. However, UCB depleted of T-cells (enriching for NK cell precursors) exhibited higher fold expansion over 14 days under different culture conditions conducive to NK cell progenitors. In conclusion, NK cell expansion in vitro depends on cell source, IL-2 and IL-15 (increased in vivo after lymphoid depleting chemotherapy) as well as accessory cells. The role of these factors to enhance in vivo expansion is under clinical investigation to further exploit the NK cell alloreactivity against AML targets.

Allogeneic Donor NK Cell Infusion and IL-2 for Patients with Refractory B-Cell Non-Hodgkin Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3036-3036
Author(s):  
Veronika Bachanova ◽  
Linda J. Burns ◽  
David H. McKenna ◽  
Julie Curtsinger ◽  
Sarah Cooley ◽  
...  
Keyword(s):  
B Cell ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Cell Expansion ◽  
Nk Cell ◽  
Peripheral Blood Cells ◽  
Non Hodgkin Lymphoma ◽  
Pre Treatment

Abstract Abstract 3036 Poster Board II-1012 The potential role of allogeneic natural killer (NK) cells for therapy of refractory lymphoma is supported by the curative potential of allogeneic transplantation for lymphoid malignancies. Haploidentical donor derived NK cells may overcome Class I MHC Ag mediated inhibition and deliver an NK versus lymphoma effect. In a Phase II study we evaluated allogeneic NK cell infusions with Rituximab and IL-2 in a non-transplant setting to determine the expansion of NK cells in vivo and the clinical response in patients with refractory B-cell non-Hodgkin lymphoma (NHL). Six patients with advanced NHL received conditioning with Rituximab 375mg/m2 days -8,-1,+6,+15; Cyclophosphamide 60 mg/kg IV day -5; Fludarabine 25 mg/m2 IV days -6 through -2 as immunosupression to permit homeostatic expansion of allogeneic donor NK cells. Peripheral blood cells were obtained by lymphapheresis from unmobilized, HLA-haploidentical donors and selected for “killer immunoglobulin receptor” (KIR) ligand mismatch when available (3 out of 6 patients). Donor peripheral blood cells were enriched for NK cells with the Miltenyi CliniMACS device by depletion of T (CD3+) cells. The donor NK cells were then activated by overnight incubation with IL-2 (1,000 U/mL) and infused at a median nucleated cell dose of 2.27 ±0.4 × 107/kg. Subcutaneous IL-2 10×106 units (qod x 6 doses) was given to facilitate NK cell survival and expansion. All patients were evaluable for toxicity and efficacy. Patients tolerated the NK infusion well with only transient grade 1-2 toxicity and 5 received all 6 scheduled doses of IL-2. IL-2 activated donor NK cell products showed > 55% cytotoxicity against K562 targets. After IL-2 therapy, we observed a median absolute lymphocyte count of 980 ±440/μL. All cells were of recipient origin with no detectable donor NK cells. Importantly, in all patients the median number of host regulatory T cells (T regs phenotype CD4+Foxp3+CD127−) post treatment was significantly increased compared to pre-treatment (day 14 T regs: 134 ±141 cells/μL versus pre-treatment T regs: 24 ±12 cells/μL; P=0.06). To investigate the possibility of NK trafficking to affected lymph nodes, we performed fine needle aspiration of palpable tumor in 1 patient and demonstrated a low level of donor DNA by RFLP testing (2.5% donor chimerism). Simultaneous absence of NK cells in peripheral blood in the same patient suggested NK cell tissue homing to lymphoma-bearing nodes. Three patients achieved a partial remission (PR), one of whom proceeded to non-myeloablative cord blood allograft 2 month after NK cell infusion; two remain in partial remission after 1 and 4 months of follow-up. The trial failed to achieve prospective statistical parameters established to detect circulating NK cell expansion rate and will be modified. Conclusions This “proof of principle” study demonstrated lack of in vivo expansion of haploidentical NK cells in peripheral blood of patients with lymphoma. However, we identified host factors that interfered with NK cell expansion, including T reg proliferation and possibly inadequate immunosupression, and additionally, the finding of donor DNA in sites of tumor suggested donor NK cell localization to extravascular or tumor sites. Novel approaches to adoptive NK cell therapy trials should incorporate strategies to eliminate or prevent T reg expansion using alternate lymphodepleting regimens. Disclosures No relevant conflicts of interest to declare.

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