Rituximab-mediated Antibody-dependent Cellular Cytotoxicity Research Articles

A single bout of vigorous intensity exercise enhances the efficacy of rituximab against human chronic lymphocytic leukaemia B-cells ex vivo

Chronic lymphocytic leukaemia (CLL) is characterised by the clonal proliferation and accumulation of mature B-cells and is often treated with rituximab, an anti-CD20 monoclonal antibody immunotherapy. Rituximab often fails to induce stringent disease eradication, due in part to failure of antibody-dependent cellular cytotoxicity (ADCC) which relies on natural killer (NK)-cells binding to rituximab-bound CD20 on B-cells. CLL cells are diffusely spread across lymphoid and other bodily tissues, and ADCC resistance in survival niches may be due to several factors including low NK-cell frequency and a suppressive stromal environment that promotes CLL cell survival. It is well established that exercise bouts induce a transient relocation of NK-cells and B-cells into peripheral blood, which could be harnessed to enhance the efficacy of rituximab in CLL by relocating both target and effector cells together with rituximab in blood. In this pilot study, n = 20 patients with treatment-naïve CLL completed a bout of cycling 15 % above anaerobic threshold for ∼ 30-minutes, with blood samples collected pre-, immediately post-, and 1-hour post-exercise. Flow cytometry revealed that exercise evoked a 254 % increase in effector (CD3−CD56+CD16+) NK-cells in blood, and a 67 % increase in CD5+CD19+CD20+ CLL cells in blood (all p < 0.005). NK-cells were isolated from blood samples pre-, and immediately post-exercise and incubated with primary isolated CLL cells with or without the presence of rituximab to determine specific lysis using a calcein-release assay. Rituximab-mediated cell lysis increased by 129 % following exercise (p < 0.001). Direct NK-cell lysis of CLL cells – independent of rituximab – was unchanged following exercise (p = 0.25). We conclude that exercise improved the efficacy of rituximab-mediated ADCC against autologous CLL cells ex vivo and propose that exercise should be explored as a means of enhancing clinical responses in patients receiving anti-CD20 immunotherapy.

T-cell help in the tumor microenvironment enhances rituximab-mediated NK-cell ADCC

AbstractRituximab (RTX) and other monoclonal antibodies (mAbs) that bind directly to malignant cells are of great clinical value but are not effective for all patients. A major mechanism of action of RTX is antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells. Prior in vitro studies in our laboratory demonstrated that T cells contribute to maintaining the viability and cytotoxic potential of NK cells activated by anti-CD20–coated target B cells. Here, we conducted studies using a novel mouse model and clinical correlative analysis to assess whether T-cell help contribute to RTX-mediated NK-cell ADCC in the tumor microenvironment (TME) in vivo. A humanized mouse model was developed using Raji lymphoma cells and normal donor peripheral blood mononuclear cells that allows for control of T-cell numbers in the lymphoma TME. In this model, NK-cell viability and CD16 and CD25 expression dropped after RTX in the absence of T cells but increased in the presence of T cells. RTX therapy was more effective when T cells were present and was ineffective when NK cells were depleted. In patients with indolent lymphoma, fine needle aspirates were obtained before and ∼1 week after treatment with a RTX-containing regimen. There was a strong correlation between CD4+ T cells as well as total T cells in the pretherapy TME and an increase in NK-cell CD16 and CD25 expression after RTX. We conclude that T-cell help in the TME enhances RTX-mediated NK-cell viability and ADCC.

The KIR-HLA-CD16a Immunogenetic Profile Influences NK Cell-Mediated ADCC and Response to Rituximab Therapy in B-Cell Lymphomas

Rituximab-based chemoimmunotherapy (R-CHOP) is the standard of care in most B-cell lymphomas, however there is marked interpatient heterogeneity in treatment response. An important mechanism of Rituximab (anti-CD20) action is through natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), but the factors influencing this are poorly understood. Here, we present findings from a preclinical study investigating the role of NK cell receptor polymorphisms and HLA allelic variation on ADCC against aggressive B-cell lymphoma cell lines. Genotyping was conducted using Luminex-based SSO and Sanger sequencing. Multicolor flow cytometry was used to assess the response of activated NK cells from healthy donors against 8 lymphoma cell lines. Individuals carrying the GG or GT allele in CD16a were found to have a 3-8 fold greater NK cell response compared to individuals with a TT allele and rituximab-mediated ADCC was significantly greater in individuals with a KIR-3DL1 allele when tested against B-cell lymphomas lacking the HLA-Bw4 allele. An additive effect was observed when considering both CD16a and KIR-HLA together. As expected, CD20 expression was a strong correlate of NK cell response. In summary, NK cell-mediated ADCC in response to ADCC appears to be influenced by the KIR-HLA-CD16a genotype. These findings suggest a potential role for immunogenotyping in B-cell lymphomas treated with Rituximab. Additionally, the presence of the shared F c receptor domain opens the possibility of applying this framework towards predicting NK cell responses to antibody therapies in other diseases.

Abstract 4094: The TLR4 agonist G100 enhances rituximab-mediated ADCC in vitro and has synergistic anti-tumor effects with anti-CD20 mAb in vivo

Abstract Background: G100, which contains the synthetic TLR4 agonist Glucopyranosyl lipid A (GLA) formulated in a stable oil-in-water emulsion (SE) for intratumoral therapy, is currently in clinical development in combination with pembrolizumab for the treatment of B-cell follicular lymphoma (FL). The current study was undertaken to investigate the interaction of G100 with rituximab, the anti-CD20 mAb that is standard-of-care treatment for FL. Methods: In vitro studies were performed using peripheral blood mononuclear cells (PBMC) from healthy human donors to investigate the effect of GLA-AF (an aqueous formulation of GLA) on natural killer (NK) cell activation. IFNγ production in NK cells after activation with plate-bound anti-CD16 or target K562 leukemia cells was measured by intracellular cytokine staining (ICS). Rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC) was measured using pretreated or untreated PBMC and rituximab-coated Raji Burkitt’s lymphoma cells as target cells. In vivo studies were carried out in Balb/c mice with bilateral A20 lymphoma. Mice received G100 alone (10μg, intratumoral, 3 injections/week), anti-CD20 alone (clone 5D2, provided by Genentech, 200μg, intraperitoneal, 1 injection/week), or G100+anti-CD20 for a total of 3 weeks. Results: Incubation of PBMC from 3 different donors with GLA-AF (1 μg/mL) resulted in NK cell activation as demonstrated by increased IFNγ production, as well as enhanced response to the K562 target cells and anti-CD16. When PBMC from 5 normal donors were used in rituximab-mediated ADCC assay, pretreated PBMCs had significantly enhanced lysis of target Raji cells compared to untreated PBMC (p&amp;lt;0.05). In mice with bilateral A20 tumors, the combination of G100 with anti-CD20 resulted in significantly better tumor control and prolonged survival (n=5 per group, p&amp;lt;0.05). Conclusions: G100 enhances NK cell activity and rituximab-mediated ADCC in human PBMC. Combination of G100 and anti-CD20 mAb has synergistic anti-tumor effects in a mouse lymphoma model. These data support clinical testing of G100 with rituximab in patients with FL. Citation Format: Hailing Lu, Alec Betancur, Jan ter Meulen. The TLR4 agonist G100 enhances rituximab-mediated ADCC in vitro and has synergistic anti-tumor effects with anti-CD20 mAb in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4094.

Slan+ Monocytes and Macrophages Mediate CD20-Dependent B-cell Lymphoma Elimination via ADCC and ADCP.

Terminal tissue differentiation and function of slan+ monocytes in cancer is largely unexplored. Our recent studies demonstrated that slan+ monocytes differentiate into a distinct subset of dendritic cells (DC) in human tonsils and that slan+ cells colonize metastatic carcinoma-draining lymph nodes. Herein, we report by retrospective analysis of multi-institutional cohorts that slan+ cells infiltrate various types of non-Hodgkin lymphomas (NHL), particularly the diffuse large B-cell lymphoma (DLBCL) group, including the most aggressive, nodal and extranodal, forms. Nodal slan+ cells displayed features of either immature DC or macrophages, in the latter case ingesting tumor cells and apoptotic bodies. We also found in patients with DLBCL that peripheral blood slan+ monocytes, but not CD14+ monocytes, increased in number and displayed highly efficient rituximab-mediated antibody-dependent cellular cytotoxicity, almost equivalent to that exerted by NK cells. Notably, slan+ monocytes cultured in conditioned medium from nodal DLBCL (DCM) acquired a macrophage-like phenotype, retained CD16 expression, and became very efficient in rituximab-mediated antibody-dependent cellular phagocytosis (ADCP). Macrophages derived from DCM-treated CD14+ monocytes performed very efficient rituximab-mediated ADCP, however, using different FcγRs from those used by slan+ macrophages. Our observations shed new light on the complexity of the immune microenvironment of DLBCL and demonstrate plasticity of slan+ monocytes homing to cancer tissues. Altogether, data identify slan+ monocytes and macrophages as prominent effectors of antibody-mediated tumor cell targeting in patients with DLBCL.Significance: slan+ monocytes differentiate into macrophages that function as prominent effectors of antibody-mediated tumor cell targeting in lymphoma.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3544/F1.large.jpg Cancer Res; 78(13); 3544-59. ©2018 AACR.

Hepatitis C virus-induced NK cell activation causes metzincin-mediated CD16 cleavage and impaired antibody-dependent cytotoxicity

The Fc receptor family for immunoglobulin (Ig)G type III (FcγRIII, CD16) is an activating receptor on natural killer (NK) cells and an essential mediator of antibody-dependent cellular cytotoxicity (ADCC). There is only limited information on its role during chronic hepatitis C virus (HCV) infection. We studied CD16 expression in relation to NK cell functional activity in HCV-infected patients and sought mechanistic insights into virus-induced modulation. NK cell CD16 expression and activation status were evaluated ex vivo by flow cytometry in HCV-infected patients and healthy controls (HC) as well as in vitro after co-culture with HCV-infected HuH7.5 cells. Rituximab-mediated ADCC was assessed in HC and HCV-infected patients using Daudi cells as a target. The role of metzincins in CD16 down-modulation was assessed using specific inhibitory molecules and by evaluating intracellular mRNA levels. HCV-infected patients exhibited increased frequencies of ex vivo activated NK cells and a concomitantly decreased NK CD16 expression, which resulted in impaired ADCC activity. Moreover, exposure of NK cells to culture-derived HCV recapitulated the ex vivo findings of decreased CD16 expression and increased NK cell activation. Importantly, blockade of metzincin-mediated shedding activity, including selective a disintegrin and metalloproteinase 17 (ADAM-17) inhibition, restored NK CD16 expression. Successful treatment with direct-acting antivirals partially improved NK ADCC function despite delayed CD16 reconstitution. Chronic HCV infection induces NK cell activation resulting in ADAM-17-dependent CD16 shedding and consequent impaired ADCC function. Altered ADCC may contribute to failure to eradicate HCV-infected hepatocytes. We show here that hepatitis C virus (HCV) activates natural killer (NK) lymphocytes which, as a consequence, loose their Fc receptor for IgG (CD16), an essential molecule for antibody binding. We show that this occurs through the action of enzymes named metzincins, resulting in altered NK-mediated antibody-dependent killing (ADCC) of target cells. This mechanism may contribute to HCV persistence and may represent a general phenomenon whereby some viruses can escape host's immune responses.

Optimizing 25-OH-Vitamin D3 Serum Levels for Rituximab- and Obinutuzumab-Mediated Antibody-Dependent Cellular Cytotoxicity

Background: Vitamin D3 deficiency impairs the rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC) and the outcome of elderly patients with diffuse large B-cell lymphoma (Bittenbring et al., J Clin Oncol. 2014 Oct 10; 32(29):3242-8) treated with R-CHOP and of patients with follicular lymphoma (Kelly et al., J Clin Oncol. 2015 May 1; 33(1):1482-90. The aim of this study was to determine the optimal 25-OH-vitamin D3 (VD3) level for rituximab- and obinutuzumab-mediated ADCC.Methods: Ten individuals (5 males, 5 females; mean age: 67.7 years, range 41-79) without malignant disease or immunosuppression were included in this study after written informed consent. PBMC were isolated by density gradient centrifugation. CD16+ NK cells were separated by depleting all non-NK-cells magnetically. ADCC activity of the NK cells was tested against CD20-expressing Daudi cells without or after anti-CD20-antibody treatment with rituximab and obinutuzumab, respectively. Cytotoxic activity was assessed by LDH release by the target cells. NK cells were studied at four different VD3 serum levels: 1 (insufficient supply: < 20 ng/ml); 2 (lower normal range; 30 ng/ml), 3 (mid normal range; 65 ng/ml), and 4 (high normal range; 90 ng/ml). To achieve these levels the probands were substituted with cholecalciferol.Results: The median VD3 serum level before substitution was 10 ng/ml. The only formula for achieving predefined VD3 serum levels published by van Groningen et al. (Eur J Endocrinol. 2010; 162:805-11) failed to achieve the predetermined VD3 levels by far. Modifying this formula by using a multiplication factor of 1.8-2.2 we achieved the preset dose levels with high precision with median levels of 32.6 ng/ml, 66.4 ng/ml and 92.3 ng/ml aiming at dose levels of 30, 65 and 90 ng/ml, respectively. 2/10 test persons had starting VD3 levels above 30 ng/ml, but 6 of the remaining 8 individuals showed a significantly increased ADCC after VD3 substitution to level 2 (ranging from + 10 % to + 147 % increased Daudi lysis, p < 0.05). Further substitution to 65 ng/ml significantly increased ADCC activity in all 10 persons compared to the ADCC at 30 ng/ml (ranging from + 18.4 % to + 89.2 % increased Daudi lysis, p < 0.05). 8/10 individuals were further substituted to achieve 90 ng/ml. However, in 7/8 of these probands the NK-cell-mediated ADCC was significantly reduced compared to their values at 65 ng/ml (ranging from - 23.1 % to - 58.1 % decreased Daudi lysis, p < 0.05). The extent of the substitution-induced ADCC improvement varied individually and depended on the antibody concentration used to treat the target cells. Rituximab-mediated ADCC was significantly more affected by VD3 levels than obinutuzumab.Conclusion: Our study demonstrates for the first time that the ADCC of NK cells is optimal at median VD3 serum levels around 65 ng/ml. Our data strongly argue for a rapid vitamin D3 substitution before/at the beginning of R-CHOP treatment using the modified van Groningen formula. 65 ng/ml was chosen as the target VD3 level in the ongoing OPTIMAL>60 study of the German High-Grade Non-Hodgkin Lymphoma Study Group (DSHNHL) in elderly patients with DLBCL. This study is supported by the Eva Mayr-Stihl-Stiftung (Waiblingen, Germany). DisclosuresPfreundschuh:Roche, Janssen, Celgene: Honoraria, Research Funding.

Oncolytic reovirus enhances rituximab-mediated antibody-dependent cellular cytotoxicity against chronic lymphocytic leukaemia.

The naturally occurring oncolytic virus (OV), reovirus, replicates in cancer cells causing direct cytotoxicity, and can activate innate and adaptive immune responses to facilitate tumour clearance. Reovirus is safe, well tolerated and currently in clinical testing for the treatment of multiple myeloma, in combination with dexamethasone/carfilzomib. Activation of natural killer (NK) cells has been observed after systemic delivery of reovirus to cancer patients; however, the ability of OV to potentiate NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) is unexplored. This study elucidates the potential of oncolytic reovirus for the treatment of chronic lymphocytic leukaemia (CLL), both as a direct cytotoxic agent and as an immunomodulator. We demonstrate that reovirus: (i) is directly cytotoxic against CLL, which requires replication-competent virus; (ii) phenotypically and functionally activates patient NK cells via a monocyte-derived interferon-α (IFNα)-dependent mechanism; and (iii) enhances ADCC-mediated killing of CLL in combination with anti-CD20 antibodies. Our data provide strong preclinical evidence to support the use of reovirus in combination with anti-CD20 immunotherapy for the treatment of CLL.

Abstract 148: Release of the TNF-family member BAFF by NK cells contributes to the resistance of chronic lymphoid leukemia cells to direct and Rituximab-induced NK reactivity

Abstract NK cells are cytotoxic lymphocytes that are particularly important for the immunosurveillance of leukemia. Due to their ability to mediate antibody-dependent cellular cytotoxicity (ADCC), NK cells substantially contribute to the therapeutic benefit of antitumor antibodies like Rituximab, which is a crucial component in the treatment of B cell malignancies. Available data indicate that the ability of NK cells to target malignant cells and to mediate ADCC is compromised in Chronic Lymphoid Leukemia (CLL), but the underlying mechanisms are still unclear (Jaglowski et al., Blood 2010). With the TNF family member B cell activating factor (BAFF) that regulates, among others, the proliferation and survival of B cells, we believe to have identified a factor which contributes to these clinical observations. We report that NK cells express BAFF on the mRNA level and release the protein in a soluble form with the levels depending on activation state. Notably, relevant BAFF surface expression was neither detected on resting nor activated NK cells. Using a tetrazolium salt based colorimetric assay we found that NK cell-derived BAFF enhances the metabolic activity of primary CLL cells. In addition, BAFF protects CLL cells from chemotherapy-induced cell death e.g. upon treatment with Fludarabine and Cyclophosphamide. Notably, the BAFF-specific antibody Belimumab, which is approved for the treatment of systemic lupus erythematosus, was found to block the leukemia cell-protective effects of BAFF and to restore the sensitivity of the leukemia cells to chemotherapy. Furthermore, BAFF was found to substantially reduce direct NK cell lysis and also Rituximab-mediated ADCC in cultures with primary CLL cells as measured by Europium cytotoxicity assays. Our data on the capacity of BAFF to enhance the metabolic activity of B-CLL cells and their resistance to NK cell cytotoxicity suggest its involvement in the compromised ability of NK cells to combat lymphoid as compared to myeloid leukemias (Pende et al., Blood 2005) and the reportedly impaired ability of NK cells to mediate ADCC upon Rituximab treatment in CLL. Furthermore, our findings point to a possible benefit of combinatory approaches employing Rituximab and Belimumab for immunotherapy of B cell malignancies. Citation Format: Julia Wild, Benjamin J. Schmiedel, Andreas Maurer, Stefanie Raab, Pascal Schneider, Helmut R. Salih. Release of the TNF-family member BAFF by NK cells contributes to the resistance of chronic lymphoid leukemia cells to direct and Rituximab-induced NK reactivity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 148. doi:10.1158/1538-7445.AM2014-148

CALGB 150905 (Alliance): rituximab broadens the antilymphoma response by activating unlicensed NK cells.

Natural killer (NK) cells contribute to clinical responses in patients treated with rituximab, but the rules determining NK-cell responsiveness to mAb therapies are poorly defined. A deeper understanding of the mechanisms responsible for antibody-dependent cellular cytotoxicity (ADCC) could yield useful biomarkers for predicting clinical responses in patients. Unlicensed NK cells, defined as NK cells lacking expression of an inhibitory KIR for self-HLA class I ligands, are hyporesponsive in steady state, but are potent effectors in inflammatory conditions. We hypothesized that antitumor antibodies such as rituximab can overcome NK-cell dependence on licensing, making unlicensed NK cells important for clinical responses. Here, we examined the influences of variations in KIR and HLA class I alleles on in vitro responses to rituximab. We tested the clinical significance in a cohort of patients with follicular lymphoma treated with rituximab-containing mAb combinations, and show that rituximab triggers responses from all NK-cell populations regardless of licensing. Neither IL2 nor accessory cells are required for activating unlicensed NK cells, but both can augment rituximab-mediated ADCC. Moreover, in 101 patients with follicular lymphoma treated with rituximab-containing mAb combinations, a "missing ligand" genotype (predictive of unlicensed NK cells) is associated with a higher rate of progression-free survival. Our data suggest that the clinical efficacy of rituximab may be driven, in part, by its ability to broaden the NK-cell repertoire to include previously hyporesponsive, unlicensed NK cells. A "missing ligand" KIR and HLA class I genotype may be predictive of this benefit and useful for personalizing treatment decisions in lymphomas and other tumors.

Correlative Analysis and Clinical Update Of a Phase II Study Using Lenalidomide and Rituximab In Patients With Indolent Non-Hodgkin Lymphoma

BackgroundAlthough indolent non-Hodgkin lymphomas (iNHLs) are responsive to initial therapy, patients have a relapsing and progressive disease course and most remain incurable with current treatments. Lenalidomide is an immunomodulatory agent that enhances rituximab-mediated antibody-dependent cellular cytotoxicity. In lymphoma cell lines, animal models, and in several phase II trials, combining lenalidomide and rituximab (R2) resulted in improved antitumor activity relative to either agent alone. However, the immunological mechanisms remain unclear. Here we provide updated results from a phase II trial investigating the clinical activity of R2 in previously untreated and relapsed/refractory (R/R) patients with iNHL. We also present an analysis of immunologic correlative data that may provide insight into the therapy's mechanism-of-action and may also predict response. AimsStudy aims were to assess: the clinical activity of the R2 regimen in previously untreated and R/R iNHL patients and, assess the potential of serum cytokine levels to predict response. MethodsPatients had previously untreated (n=15) or R/R (n=30) iNHL with measurable disease, and ECOG Performance Status ² 2. Lenalidomide (20 mg/day) was administered on days 1–21 of a 28-day cycle and continued until disease progression, rituximab 375 mg/m2 IV was administered on day 15 of cycle 1 and repeated weekly for a total of 4 doses. The primary clinical endpoint was overall response rate (ORR). Secondary endpoints included response duration (DR), overall survival, progression-free survival (PFS), and safety. The correlative serum cytokine samples were obtained from patients at baseline, D15 (prior to treatment with rituximab), D30 and D60. IL-1, 2, 6, 8, 10, 12, GM-CSF, IFN-γ, TNF-α , CXCL-10/IP-10 levels were measured at each time point using a Meso Scale Discovery multi-plex cytokine assay. The cytokine levels at each time point were then correlated with response using a two-sided T test. ResultsOf the 30 R/R patients, 22 had follicular lymphoma (FL), the median age was 60 years, median number of prior therapies was 3 (range 1–11), and 50% were refractory to rituximab. Of the 15 previously untreated patients, 12 had FL and the median age was 58. For the 27 evaluable R/R patients, the ORR was 74%, including 12 patients (44%) with a CR. At a median follow-up of 34 months, median PFS was 12.4 months, the median DR and time to next therapy (TTNT) were 15.4 and 37.4 months respectively with 7 remissions that are ongoing including three that have lasted more than 48 months. Of the 15 previously untreated patients 13 are evaluable for response; the ORR was 92% with 6 of 13 (42%) having a CR. At a median follow-up of 18 months the median PFS is 14.5 months; the median DR has not been reached.The correlative analyses showed that there was a significant increase in IFN-γ, GM-CSF, CXCL-10 and IL-2 at Day 15 with a 652, 494, 737, and 242% increase respectively above baseline which correlated with patients who had a CR (p < 0.05), Table. IL-1, 6, 8, 10, 12, and TNF-α were also measured, but did not predict CR at any time point. Phenotypic and functional analyses are ongoing. There was no significant difference in the percent increase in cytokine levels in previously untreated versus R/R patients.TableAbsolute cytokine values in pg/ml and (%) above baseline;** p=<0.05Day 15Day 30Day 60IFN-γ**21.1 (652)0.6 (19)-0.1 (-4)GM-CSF**2.3 (494)0.2 (45)0.1 (16)CXCL-10**5755 (737)608 (78)25 (3)IL-2**3.5 (242)-0.6 (-40)-0.6 (-45)TNF-α24.5 (51)-28.0 (-59)-34.0 (-72)IL-6-3.9 (-23)-12.0 (-70)-12.0 (-70)IL-1016.4 (53)-20.1 (-66)-20.2 (-66)IL-10.4 (51)0.0 (4)-0.2 (-21)IL-820.9 (128)-1.3 (-7)0.3 (2)IL-12-0.34 (-42)0.06 (-8)-0.15 (-12) ConclusionThe R2 regimen has considerable activity in patients with both untreated or R/R iNHL, particularly those with FL. A significant increase in the serum levels of IFN-γ, GM-CSF, IL-2 and CXCL-10/IP-10 after 2 weeks of lenalidomide alone correlates with achievement of CR. Disclosures:Off Label Use:The use of lenalidomide for the treatment of indolent NHL will be discussed which is not an FDA approved indication. Tuscano:Celgene: Honoraria, Research Funding.

HLA-C Matching Status Does Not Affect Rituximab-Mediated Antibody-Dependent Cellular Cytotoxicity by Allogeneic Natural Killer Cells

Risk of leukemia relapse after T cell-depleted hematopoietic stem cell transplantation is lower in the “HLA-C mismatched” recipient-donor combinations. This might be attributable to increased natural killing by allogeneic NK cells carrying a KIR that does not bind to HLA-C on target cells (HLA-C-uncoupled KIR). Considering a new strategy of allogeneic NK cell transfer with rituximab to treat B-cell lymphomas, however, it is unknown whether the HLA-C matching status also affects rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC). To address this issue, we investigated the levels of ADCC by purified NK cells carrying an HLA-C-uncoupled KIR, where the NK cell donors had either matched or mismatched HLA-C combination with target cells. Purified NK cells carrying an HLA-C-uncoupled KIR consistently showed enhanced ADCC against target cells when NK cell donors had an HLA-C-mismatch. When NK cell donors did not have an HLA-C mismatch, it was inconsistent whether HLA-C-uncoupled KIR caused ADCC enhancement. When the levels of ADCC by whole NK cells were compared, there were substantial differences among the donors regardless of the HLA-C matching status. Subjects with HLA-C mismatch may not have an advantage when cytoimmunotherapy using allogeneic NK cells is considered in combination with rituximab.

Prenyl Transferases Are Involved in the Regulation of CD20 Levels and Influence Anti-CD20 Monoclonal Antibody-Mediated Activation of Complement-Dependent Cytotoxicity,

Abstract 3722Anti-CD20 monoclonal antibodies (mAbs) (rituximab or ofatumumab) are being successfully used in the treatment of non-Hodgkin's lymphomas (NHL) and B-cell chronic lymphocytic leukemia (B-CLL). They exert antitumor effects by triggering indirect effector mechanisms of the immune system, such as activation of complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. Moreover, upon crosslinking with secondary antibodies, anti-CD20 mAbs can induce cell death. It is frequently underscored that CD20 expression levels in various B-cell tumors is relatively constant. However, accumulating evidence indicates that CD20 can be modulated at transcriptional, posttranscriptional, and even posttranslational levels. Moreover, it has been clearly shown in vitro that CDC efficacy of anti-CD20 mAbs clearly depends on CD20 expression. We have previously observed that statins impair detection of CD20 in non-Hodgkin lymphoma cells and impair rituximab-mediated CDC and ADCC (Winiarska et al. PLoS Med 2008). Statins are inhibitors of cholesterol synthesis and decrease production of prenyl groups (farnesyl and geranylgeranyl PPi), which are necessary for posttranslational modification of approximately 1% of cellular proteins. In experiments aimed at elucidation of the molecular mechanisms of statin-mediated modulation of CD20 we observed that neither geranylgeranyl transferase (GGT) nor farnesyl transferase (FNT) inhibitors could mimic statins effects. On the contrary, prenyltransferase inhibitors improved rituximab-mediated CDC. Therefore, we decided to investigate in more detail the interaction of prenyltransferase inhibitors and anti-CD20 mAbs. In the initial experiments we evaluated the effects of three different farnesyl transferase inhibitors as well as three different geranylgeranyl transferase inhibitors. Among all FNT and GGT inhibitors, L-744,832 seemed to produce the most significant influence on both rituximab-mediated CDC and CD20 levels (Figure). Moreover, L-744,832 significantly increased rituximab-mediated CDC in 3 out of 5 primary tumor cell cultures isolated from patients with NHL or CLL. Therefore, L-744,832 was selected for further more systematic studies. Interestingly, in Raji cells L-744,832 did not improve rituximab-mediated ADCC and only at the highest non-toxic concentrations it sensitized to rituximab+crosslinking antibody-mediated cytotoxicity. In 10 out of 17 (58.8%) primary lymphoma/leukemia cells L-744,832 increased CD20 expression by at least 20% as measured with flow cytometry. Moreover, we observed that upon L-744,832 treatment CD20 is up-regulated in Raji cells at both mRNA as well as protein level. Experiments aimed at investigation of FTI influence on proteasome activity as well as CD20 endocytosis and shedding revealed that L-744,832 influences CD20 levels independently from its posttranslational regulation. To verify whether modulation of CD20 levels by L-744,832 results from specific inhibition of farnesyltransferase or is an off-target effect of this compound we performed FNT B subunit (FNTB) knock-down experiments that resulted in increased CD20 levels by almost 60%. Incubation of Raji cells with a transcription inhibitor cycloheximide completely prevented L-744,832-mediated increase of CD20 levels in WB. Therefore, a chromatin immunoprecipitation assay was performed to determine whether inhibition of FNT activity is associated with binding of transcription factors to the promoter of cd20 gene. These studies revealed that L-744,832 promotes binding of PU.1 and Oct2, but not TFE3 to target DNA sequences within cd20 promoter in Raji cells. To conclude, our studies indicate for the first time that CD20 expression can be modulated by prenyltransferase inhibitors. While inhibition of FNT activity significantly up-regulates expression of CD20, the influence of GGT inhibitors on this protein is more complex, and requires further studies. Furthermore, pre-incubation of NHL and CLL cells with L-744,832, a FNT inhibitor, potentiates anti-CD20 mAb-mediated activation of the complement-mediated cytotoxicity. Therefore, the combination seems to be promising and its efficacy should be determined in patients with NHL or CLL. [Display omitted] Disclosures:Winiarska:Genmab A/S: Research Funding. Golab:Genmab A/S: Research Funding.

Sorafenib Affects Membrane Complement Inhibitors and Improves Antitumor Activity of Rituximab,

Abstract 3723Monoclonal antibodies (mAbs) targeting CD20 antigen are now routinely used in the treatment of various types of non-Hodgkin's lymphomas (NHL) and B-cell chronic lymphocytic leukemia (B-CLL). Their antitumor action results from the ability to trigger complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. Moreover, direct cell death can be induced upon crosslinking with secondary antibodies. Previous studies have demonstrated a sigmoidal correlation between CD20 expression level and rituximab-mediated CDC but not ADCC. Next to CD20 expression level also other potential mechanisms of anti-CD20-mediated cytotoxicity have been observed. Among them an increased expression of complement regulatory proteins (CRP), such as CD46, CD55 or CD59 has been shown to contribute to resistance to mAb-mediated CDC. Antibodies blocking CRP or siRNA that knocks-down CRP expression facilitate rituximab-mediated cytotoxicity. Also in xenograft in vivo models it was shown that antibodies blocking the activity of CD55 and CD59 or a recombinant adenoviral fiber knob protein that cross-links CD46 molecules enhance therapeutic effects of rituximab. Fludarabine, the nucleoside analogue clinically active against CLL and indolent NHL has been shown to act synergistically with rituximab and to down-regulate the membrane expression of CD55 without significantly altering CD20 levels.The aim of this study was to investigate the influence of sorafenib, a multikinase inhibitor on the expression of CRPs in human CD20+ B-cell lymphomas and to evaluate its ability to trigger CDC in combination with rituximab. A 48-h incubation of four different human non-Hodgkin lymphoma cells DoHH2 (Fig. 1A), Daudi, Raji and Ramos with sorafenib resulted in a marked concentration-dependent drop in the surface expression of CD46, CD55 and CD59, but did not affect the levels of CD20. Analysis of primary cells isolated from 5 consecutive patients with CD20+ CLL also revealed a significant drop in the levels of CD46 (5 of 5 patients), CD55 (4 of 5 patients), and CD55 (5 of 5 patients) after a 48 h incubation of tumor cells with sorafenib. Accordingly, with previous reports, sorafenib significantly decreased phosphorylation of STAT3 transcription factors that are involved in the regulation of CRP expression. Incubation of tumor cells with sorafenib lowered the levels of mRNA for all three CRPs, but not that of CD20 indicating that sorafenib-mediated downregulation of CRPs occurs at transcriptional level. Pre-incubation of all four lymphoma cell lines cells with sorafenib at concentrations that down-modulate CRPs levels also significantly sensitized tumor cells to rituximab-mediated CDC (Fig. 1B). Moreover, sorafenib led to increased incorporation of membrane-attack complex (C5b-9) into the membranes of DoHH2 cells. On the other hand, sorafenib did not affect rituximab-mediated ADCC nor apoptosis in DoHH2 cells. Sunitinib, another multi-kinase inhibitor, did not affect rituximab-mediated complement-dependent cytolysis. [Display omitted] Altogether, these results indicate that sorafenib, which undergoes clinical trials in patients with NHL and B-CLL might be effectively used in combination with anti-CD20 mAbs. Disclosures:No relevant conflicts of interest to declare.

The impact of Fc-γ receptor polymorphisms in elderly patients with diffuse large B-cell lymphoma treated with CHOP with or without rituximab

AbstractFcγ receptor (FcγR) polymorphisms have been shown to affect rituximab-mediated antibody-dependent cellular cytotoxicity. Of 512 patients with diffuse large B-cell lymphoma treated in the RICOVER-60 trial, carriers of FcγRIII 158 valine homozygous receptors (V/V) presented with a slightly decreased incidence of B-symptoms (158 V/V: 26%, V/F: 35%, phenylalanine receptors [F/F]: 42%; P = .037). Survival curves of all FcγR single nucleotide polymorphisms were superimposable after cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP); but after CHOP with rituximab (R-CHOP), event-free survival (EFS) and progression-free survival (PFS), but not overall survival, of FcγRIIIa 158 F/F had a trend to be lower than those of 158 V/F and 158 V/V: 3-year EFS: FcγRIIIa 158 F/F: 64.5%, 158 V/F: 70.2%, 158 V/V: 76.9% (log-rank test: P = .224 F/F vs V/V; P = .285 F/F vs V/F + V/V); 3-year PFS: FcγRIIIa 158 F/F: 68.3%, V/F: 76.1%, V/V: 80.5% (log-rank test: P = .233 for F/F vs V/V; P = .185 for F/F vs V/F + V/V). By multivariate analysis adjusting for International Prognostic Index factors, relative risk of F/F compared with V/F plus V/V was 1.80 (P = .052) for PFS and 1.55 (P = .120) for EFS. The interaction of R-CHOP, but not CHOP with FcγRIIIa polymorphisms, indicates a window of opportunity for CD20 antibodies designed to mediate enhanced antibody-dependent cellular cytotoxicity.

Interleukin-15 enhances rituximab-dependent cytotoxicity against chronic lymphocytic leukemia cells and overcomes transforming growth factor beta-mediated immunosuppression

Chemoimmunotherapy with anti-CD20 monoclonal antibody rituximab is increasingly used for the treatment of patients with chronic lymphocytic leukemia (CLL). Antibody-dependent cytotoxicity (ADCC) is one of the most important mechanisms of action of rituximab against B-cell malignancies. We studied ways to increase the cytotoxic effect of rituximab on CLL cells by enhancing ADCC. Peripheral blood mononuclear cell (PBMC) or purified natural killer (NK) cells from healthy donors were activated with interleukin-15 (IL-15) and cultured with rituximab-coated CLL cells, and ADCC was evaluated using a (51)chromium release assay. The IL-15 significantly enhanced invitro ADCC against CLL cells, and this effect was mainly mediated by NK cells. The IL-15 treated effector cells with the low affinity FcγRIIIA receptor (158FF) had an ADCC comparable to those with the high affinity FcγRIIIA form (158VF). Inaddition, IL-15 enhanced rituximab-mediated ADCC of CLL cells in the presence of transforming growth factor-beta. The IL-15 increases rituximab-mediated ADCC against CLL, and supports the use of such agents with the goal of improving clinical response to chemoimmunotherapy in patients with CLL.

Lenalidomide Overcomes FcγRIIIa-Mediated Resistance to Rituximab In Patients with Relapsed/Refractory Indolent Non-Hodgkin's Lymphoma (NHL): A Correlative Analysis of a Phase 2 Study

AbstractAbstract 3967 Background:One of several mechanisms attributed to the antitumor activity of rituximab is antibody-dependent cellular cytotoxicity (ADCC), which depends on the affinity between the FcγRIIIa receptor (CD16) and human immunoglobulin G1 (IgG1). A genetic polymorphism at position 158 of the FcγRIIIa gene, which places either a phenylalanine (F) or a valine (V), results in an Fc receptor with high or low affinity. It has recently been reported that FL patients with the high affinity 158V/V genotype appear to have significantly higher objective response rates (ORR) and progression-free survival (PFS) compared to patients with at least one low affinity 158F allele when treated with rituximab monotherapy: ORR was 75% for patients with V/V compared with 23% and 30% for patients with V/F and F/F, respectively (Weng et al JCO 2003). Lenalidomide is an immunomodulatory agent that has been shown to enhance rituximab-mediated ADCC and to induce CD16 expression on NK cells (Zhang et al Am J Hematol 2009). In lymphoma cell lines and animal models, combining lenalidomide and rituximab (R2) resulted in improved antitumor activity relative to treatment with either agent alone. In a clinical setting, we previously reported on the activity and safety profile of the R2 regimen in patients with relapsed or refractory (rel/ref) indolent NHL. In this abstract, we update the efficacy data and report on whether the FcγRIIIa polymorphism affects the clinical outcome in the subset of patients with follicular lymphoma (FL) treated with R2. Methods:Enrolled patients had rel/ref indolent NHL with measurable disease, ≥1 prior therapy, and ECOG Performance Status ≤2. Lenalidomide 25 mg/day was initially administered on days 1–21 of a 28-day cycle and continued until disease progression, while rituximab 375 mg/m2 IV was administered on day 15 of cycle 1, and repeated weekly for a total of 4 doses. A second 4-weekly course of rituximab was permitted after cycle 2 for patients achieving less than a CR. After 2 of the first 4 patients developed Gr. 3 tumor lysis syndrome, the protocol was amended to reduce the lenalidomide starting dose to 20 mg, and prophylaxis with allopurinol was initiated. The primary endpoint was ORR. Secondary endpoints included response duration, overall survival, PFS, and safety. The FcγRIIIa genotype was determined in DNA isolated from peripheral blood cells by PCR amplification followed by allele-specific restriction enzyme digestion. Results:Of the 18 patients enrolled on study, 14 had FL, 2 had marginal zone lymphoma and 2 had chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) (1 patient with CLL/SLL was taken off study during cycle 1 due to transformation to Hodgkin's lymphoma). Median patients age was 60 years (range 50–91), median number of prior therapies was 3 (range 1–11), and 7 out of 16 (44%) patients were considered refractory to rituximab (defined as no response to, or progression within 6 months of receiving rituximab). The median number of cycles received was 10 (range 2-–24). The most common grade 3 and 4 toxicities were lymphopenia (23%), neutropenia (23%), fatigue (17%) and hyponatremia (11%). FcγRIIIa genotype data was available for 11 patients with FL: 1 patient had V/V, 6 patients were F/V heterozygotes, and 4 patients had F/F. For the 17 evaluable patients, the ORR (CR+PR) was 76%, which included 7 patients (41%) with a CR, and 6 patients (35%) with a PR, and additionally 2 patients (11%) had stable disease. At median follow-up of 21 months, median PFS was 12.4 months. Since all 7 patients with a CR had FL we investigated their polymorphism status. When grouped according to the genotype, the ORR was 100% in the V/V type, 100% in the V/F type, and 50% in the F/F type. Median PFS could not be calculated in the V/V type due to small number of patients, but it was 14.85 months in the V/F type and 8.31 months in the F/F type. Conclusion:The R2 regimen has considerable activity in patients with rel/ref indolent NHL, and particularly in patients with FL. In addition, the number of higher responses and longer PFS among patients with the F/V heterozygotes suggest that lenalidomide can potentially overcome the previously reported lower activity of single-agent rituximab in patients with the low affinity type FcγRIIIa receptor. This signal is currently being confirmed with additional patients receiving R2 and is anticipated for presentation at the meeting. Disclosures:Off Label Use: Lenalidomide use in NHL is off-label.

Toll-like receptor-3 as a target to enhance bioactivity of cancer immunotherapy

The purpose of this study was to explore the potential of toll-like receptor-3 stimulation, with polyI:C(12)U (poly[l].poly[C(12),U]; rintatolimod [Ampligen; Hemispherx Biopharma, Philadelphia, PA]) to enhance bioactivity of cancer immunotherapies. Several models of immune activation were assessed with polyI:C(12)U at concentrations that were achieved clinically. Dendritic cell maturation and antigen-specific immune responses were evaluated in vitro and in a murine model. The potential for polyI:C(12)U to enhance antibody-dependent cellular cytotoxicity against tumor was also evaluated. Dendritic cells are matured and T-cell stimulation is enhanced in the presence of polyI:C(12)U. In addition, polyI:C(12)U induced the release of proinflammatory chemokines and cytokines. Prostate-specific antigen-specific T-cell and antibody responses were enhanced significantly in a BALB/c prostate-specific antigen transgenic mouse model. Finally, rituximab-mediated antibody-dependent cellular cytotoxicity against tumor targets was improved significantly by the addition of polyI:C(12)U. PolyI:C(12)U shows promise as a potential agent for selective enhancement of effect with currently available and future cancer immunotherapies.

Macrophage Polarization and Acquired Resistance to Rituximab in CNS Lymphoma.

Abstract 3749Poster Board III-685Macrophages are a critical component of anti-tumor immunity but may be subverted from the classically-activated, or M1 phenotype, which mediates tumor elimination, to an alternatively-activated, M2 phenotype, which promotes tumor progression. Interleukin-4 (IL-4) signaling is a pivotal regulator of macrophage polarization to the M2 phenotype.The efficacy of rituximab appears to be mediated predominantly by antibody-dependent cellular cytotoxicity (ADCC) with tumor-associated macrophages being the dominant effector cell. We hypothesize that the phenotype of tumor-associated macrophages (M1 vs. M2) may be a potential determinant of rituximab efficacy or resistance. Relevant to this hypothesis is our demonstration of the intratumoral expression of IL-4 by CNS lymphoma (Blood, 2006; Clin Cancer Res., 2009) as well as our observation that the M2 polarization of macrophages by IL-4 treatment in vitro (18 h) results in an 8-fold decrease in rituximab-mediated ADCC of Raji lymphoma cells compared to ADCC-mediated by naïve or M1 polarized macrophages (p<0.001).There is an extreme paucity of molecular information regarding the role and phenotype of tumor macrophages in non-Hodgkin lymphoma in general, including CNS lymphoma. While macrophages from the cerebrospinal fluid (CSF) are routinely quantified in cytospin analyses, to date there is no established information regarding the phenotype and state of differentiation of macrophages within the leptomeningeal compartment. We have developed a novel flow-cytometry-based protocol for the isolation and phenotypic characterization of macrophages from the CSF of patients with CNS lymphoma. We have used candidate markers of M2 differentiation based upon our gene expression studies of CD14+ activated CSF macrophages, including CD206 and Factor XIII (each of which have previously been shown to be induced in macrophages upon IL-4 stimulation). We have identified M1 differentiation of CSF CD14+ macrophages by high fluorescence when incubated with DAF-FM diacetate, a cell-permeable marker of nitric oxide synthase (iNOS). Using flow-cytometry to evaluate the relative expression of DAF-FM and CD206, we demonstrate the presence of at least four subpopulations of activated macrophages in the CSF of CNS lymphoma patients. M1 macrophages, which highly express iNOS are denoted as DAF-FM(+)/CD206(-). M2 macrophages, which weakly express iNOS but which highly express CD206 are denoted as DAF-FM (-)/CD206(+). In addition, we have detected a mixed phenotype with features of both M1 and M2 macrophages, DAF-FM(+)/CD206(+), which we have termed dual-activated. A fourth subpopulation of activated macrophages within the CSF do not express iNOS or CD206.Thus far, CSF macrophage subpopulations have been characterized and sorted from thirty-five subjects: 18 patients with CNS lymphoma and 17 control subjects with non-neoplastic conditions. We demonstrate for the first time the association of M2 macrophages within the CSF with the pathogenesis of CNS lymphoma: there was a greater than six-fold increase in the proportion of macrophages with M2 features in immunocompetent subjects with CNS lymphoma compared to controls (p<0.001). By contrast, the proportion of macrophages with M1 features was similar between lymphoma and controls.In addition, we reproducibly detected an increase in the ratio of M1:M2 macrophages which correlated with therapeutic response to intrathecal methotrexate or cytarabine (within 94 h) in CNS lymphoma patients. By contrast, the intraventricular administration of rituximab was reproducibly associated with a greater than three-fold increase in the relative ratio of M2:M1 macrophages which was sustained compared to macrophages analyzed pre-intrathecal rituximab (p<0.001). In each of four cases, increases in M2 macrophage polarization anticipated the onset of intrathecal rituximab resistance and tumor progression.We believe this to be the first application of flow-cytometry to define the polarization states of intratumoral macrophages in non-Hodgkin lymphoma as well as the first description of dynamic changes in macrophage phenotypes during the evolution of resistance to rituximab therapy. The elucidation of distinct macrophage subpopulations based upon the expression of candidate markers of M1 vs. M2 phenotype may provide insight into tumor pathogenesis and prognosis. Disclosures:Off Label Use: While we describe the use of intrathecal administration of rituximab in patients with recurrent CNS and intraocular lymphomaa, the focus of our study is on the relationship between M2 macrophage polarization and the pathogenesis of CNS lymphoma as well as the potential relationship of macrophage polarization to acquired resistance to rituximab in CNS lymphoma patients.

Lenalidomide down-regulates the CD20 antigen and antagonizes direct and antibody-dependent cellular cytotoxicity of rituximab on primary chronic lymphocytic leukemia cells

AbstractLenalidomide, an immunomodulatory agent that enhances antibody-dependent cellular cytotoxicity (ADCC), is currently being investigated as a therapy for chronic lymphocytic leukemia (CLL). The anti-CD20 antibody rituximab is active in CLL and represents a rational agent to combine with lenalidomide. We therefore examined whether lenalidomide combined with rituximab enhances direct apoptosis and ADCC in CLL cells. In contrast to previous reports using CD20-positive lymphoma cell lines, lenalidomide down-regulated CD20 surface antigen expression in CLL patient cells via enhanced internalization, without influencing transcription. The CD20 surface antigen internalization enhanced delivery of an oligonucleotide incorporated into anti-CD20 immunoliposomes. In addition, CD20 surface antigen down-modulation by lenalidomide in CLL was accompanied by diminished rituximab-mediated apoptosis and ADCC. These observations suggest a need for alternative sequencing strategies to avoid antagonism between lenalidomide and rituximab therapy in CLL. In addition, they suggest that lenalidomide therapy might be useful to enhance targeted delivery of RNAi-based therapies using CD20 immunoliposomes in B-cell malignancies.