Immune evasion by CD200: New approaches to targeted therapies for chronic lymphocytic leukemia (CLL)

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 2519-2519
Author(s):  
A. Kretz-Rommel ◽  
N. Dakappagari ◽  
F. Qin ◽  
J. McWhirter ◽  
D. Oltean ◽  
...  
Keyword(s):  
Immune System ◽  
Tumor Growth ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Immune Evasion ◽  
Human Lymphocytes ◽  
Lymphocytic Leukemia ◽  
Tumor Growth Inhibition ◽  
Cell Injection ◽  
Cell Immunity

2519 Background: Although the human immune system is capable of raising an immune response against many cancer types, that response is insufficient to eradicate the cancer in most patients, possibly due to immune evasion through negative regulation of the immune system by the tumor. We identified the immune-suppressive molecule CD200 to be upregulated 1.5–5.4-fold on CLL cells in all 80 patients examined. Interaction of CD200 with its receptor alters cytokine profiles from Th1 to Th2 in mixed lymphocyte reactions, and results in the induction of regulatory T cells, which are thought to hamper tumor-specific effector T cell immunity. We addressed whether CD200 expression on tumor cells plays a role in immune evasion, thereby preventing elimination of tumor cells by the immune system in a xenograft hu/SCID mouse model, and whether treatment with an antagonistic anti-CD200 antibody affects tumor growth. Methods: The human non-Hodgkins lymphoma cell lines RAJI and Namalwa were transduced with human CD200 and injected subcutaneously together with human peripheral blood lymphocytes (PBL) into NOD/SCID mice. Tumor growth over time was compared among mice that either received CD200-expressing tumor cells or received tumor cells lacking CD200 expression. In subsequent experiments, mice were treated with chimeric or humanized anti-CD200 antibodies (doses ranged from 1 to 20 mg/kg) by intravenous injection. Treatment was either started immediately or 7 days after tumor cell injection. Results: As expected, PBLs reduced CD200-negative RAJI or Namalwa tumor growth by up to 75%. In contrast, growth of RAJI or Namalwa tumors expressing CD200 at levels comparable to that of CLL was not reduced by PBLs. Administration of anti-CD200 antibodies at 5 mg/kg resulted in nearly complete tumor growth inhibition (1/10 mice developed a small tumor) over the course of the study even when treatment was started 7 days after tumor cell injection. Conclusions: CD200 expression on tumor cells inhibits the ability of human lymphocytes to eradicate tumor cells. Treatment of CD200-expressing tumors with antagonistic anti-CD200 antibodies inhibits tumor growth, indicating the potential for anti-CD200 therapy as a promising approach for CLL. No significant financial relationships to disclose.

Metastatic Growth of a Murine Tumor: Evidence of Dissemination to the Lungs in the Absence of Subcutaneous Growth

1986 ◽  
Vol 72 (4) ◽  
pp. 345-350 ◽  
Author(s):  
Saverio Alberti ◽  
Stefania Filippeschi ◽  
Federico Spreafico ◽  
Elsa Alberti ◽  
Francesco Colotta
Keyword(s):  
Tumor Growth ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Primary Tumor ◽  
Lung Metastases ◽  
Colon Adenocarcinoma ◽  
Cell Injection ◽  
Metastatic Dissemination ◽  
Metastatic Growth ◽  
Cell Inoculation

Growth of MCA-38/B colon adenocarcinoma was detectable 30-33 days after subcutaneous (s.c.) tumor cell inoculation in mice. Seventy percent of the mice receiving 107 tumor cells, 50 % of those receiving 104, and 15% of the mice given 105 cells developed s.c. tumors (mean of 4 experiments, total of 80 mice per group). Metastases in the presence of a primary tumor were observed in 11% of 107 and in 10% of 106 tumor-cell injected animals. Lung metastases were detected in the absence of tumor growth at the site of s.c. cell injection in 19% of 107, in 8% of 106 and in 5% of 105 and 104 tumor-cell inoculated mice. In parallel experiments an intravenous (i.v.) inoculum of tumor cells produced lung colonies in 40% of 106 and in 14% of 105 tumor-cell injected animals. Smaller inocula did not give rise to lung colonies, thus making it unlikely that accidental i.v. inoculations of tumor cells during the s.c. injections caused the observed metastatic dissemination to the lungs.

scholarly journals Highly Differentiated Anti-CD47 Antibody, AO-176, Potently Inhibits Hematologic Malignancies Alone and in Combination

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1844-1844
Author(s):  
John Richards ◽  
Myriam N Bouchlaka ◽  
Robyn J Puro ◽  
Ben J Capoccia ◽  
Ronald R Hiebsch ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Combination Therapy ◽  
Tumor Growth ◽  
Growth Inhibition ◽  
Tumor Cells ◽  
Tumor Growth Inhibition ◽  
Employment Equity ◽  
Equity Ownership

AO-176 is a highly differentiated, humanized anti-CD47 IgG2 antibody that is unique among agents in this class of checkpoint inhibitors. AO-176 works by blocking the "don't eat me" signal, the standard mechanism of anti-CD47 antibodies, but also by directly killing tumor cells. Importantly, AO-176 binds preferentially to tumor cells, compared to normal cells, and binds even more potently to tumors in their acidic microenvironment (low pH). Hematological neoplasms are the fourth most frequently diagnosed cancers in both men and women and account for approximately 10% of all cancers. Here we describe AO-176, a highly differentiated anti-CD47 antibody that potently targets hematologic cancers in vitro and in vivo. As a single agent, AO-176 not only promotes phagocytosis (15-45%, EC50 = 0.33-4.1 µg/ml) of hematologic tumor cell lines (acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, and T cell leukemia) but also directly targets and kills tumor cells (18-46% Annexin V positivity, EC50 = 0.63-10 µg/ml) in a non-ADCC manner. In combination with agents targeting CD20 (rituximab) or CD38 (daratumumab), AO-176 mediates enhanced phagocytosis of lymphoma and multiple myeloma cell lines, respectively. In vivo, AO-176 mediates potent monotherapy tumor growth inhibition of hematologic tumors including Raji B cell lymphoma and RPMI-8226 multiple myeloma xenograft models in a dose-dependent manner. Concomitant with tumor growth inhibition, immune cell infiltrates were observed with elevated numbers of macrophage and dendritic cells, along with increased pro-inflammatory cytokine levels in AO-176 treated animals. When combined with bortezomib, AO-176 was able to elicit complete tumor regression (100% CR in 10/10 animals treated with either 10 or 25 mg/kg AO-176 + 1 mg/kg bortezomib) with no detectable tumor out to 100 days at study termination. Overall survival was also greatly improved following combination therapy compared to animals treated with bortezomib or AO-176 alone. These data show that AO-176 exhibits promising monotherapy and combination therapy activity, both in vitro and in vivo, against hematologic cancers. These findings also add to the previously reported anti-tumor efficacy exhibited by AO-176 in solid tumor xenografts representing ovarian, gastric and breast cancer. With AO-176's highly differentiated MOA and binding characteristics, it may have the potential to improve upon the safety and efficacy profiles relative to other agents in this class. AO-176 is currently being evaluated in a Phase 1 clinical trial (NCT03834948) for the treatment of patients with select solid tumors. Disclosures Richards: Arch Oncology Inc.: Employment, Equity Ownership, Other: Salary. Bouchlaka:Arch Oncology Inc.: Consultancy, Equity Ownership. Puro:Arch Oncology Inc.: Employment, Equity Ownership. Capoccia:Arch Oncology Inc.: Employment, Equity Ownership. Hiebsch:Arch Oncology Inc.: Employment, Equity Ownership. Donio:Arch Oncology Inc.: Employment, Equity Ownership. Wilson:Arch Oncology Inc.: Employment, Equity Ownership. Chakraborty:Arch Oncology Inc.: Employment, Equity Ownership. Sung:Arch Oncology Inc.: Employment, Equity Ownership. Pereira:Arch Oncology Inc.: Employment, Equity Ownership.

scholarly journals Tumor quiescence: elevating SOX2 in diverse tumor cell types downregulates a broad spectrum of the cell cycle machinery and inhibits tumor growth

BMC Cancer ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Ethan P. Metz ◽  
Erin L. Wuebben ◽  
Phillip J. Wilder ◽  
Jesse L. Cox ◽  
Kaustubh Datta ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Tumor Growth ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Cell Cycle Machinery

Abstract Background Quiescent tumor cells pose a major clinical challenge due to their ability to resist conventional chemotherapies and to drive tumor recurrence. Understanding the molecular mechanisms that promote quiescence of tumor cells could help identify therapies to eliminate these cells. Significantly, recent studies have determined that the function of SOX2 in cancer cells is highly dose dependent. Specifically, SOX2 levels in tumor cells are optimized to promote tumor growth: knocking down or elevating SOX2 inhibits proliferation. Furthermore, recent studies have shown that quiescent tumor cells express higher levels of SOX2 compared to adjacent proliferating cells. Currently, the mechanisms through which elevated levels of SOX2 restrict tumor cell proliferation have not been characterized. Methods To understand how elevated levels of SOX2 restrict the proliferation of tumor cells, we engineered diverse types of tumor cells for inducible overexpression of SOX2. Using these cells, we examined the effects of elevating SOX2 on their proliferation, both in vitro and in vivo. In addition, we examined how elevating SOX2 influences their expression of cyclins, cyclin-dependent kinases (CDKs), and p27Kip1. Results Elevating SOX2 in diverse tumor cell types led to growth inhibition in vitro. Significantly, elevating SOX2 in vivo in pancreatic ductal adenocarcinoma, medulloblastoma, and prostate cancer cells induced a reversible state of tumor growth arrest. In all three tumor types, elevation of SOX2 in vivo quickly halted tumor growth. Remarkably, tumor growth resumed rapidly when SOX2 returned to endogenous levels. We also determined that elevation of SOX2 in six tumor cell lines decreased the levels of cyclins and CDKs that control each phase of the cell cycle, while upregulating p27Kip1. Conclusions Our findings indicate that elevating SOX2 above endogenous levels in a diverse set of tumor cell types leads to growth inhibition both in vitro and in vivo. Moreover, our findings indicate that SOX2 can function as a master regulator by controlling the expression of a broad spectrum of cell cycle machinery. Importantly, our SOX2-inducible tumor studies provide a novel model system for investigating the molecular mechanisms by which elevated levels of SOX2 restrict cell proliferation and tumor growth.

The effect of monotherapy and combined therapy with NSC-631570 (Ukrain) on growth of low- and high-metastasizing B16 melanoma in mice

2010 ◽  
Vol 17 (4) ◽  
pp. 339-349 ◽  
Author(s):  
LM Skivka ◽  
YaM Susak ◽  
OO Trompak ◽  
YuI Kudryavets ◽  
N Bezdeneznikh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Combined Therapy ◽  
Tumor Growth Inhibition ◽  
Cell Cycle Distribution ◽  
Colorimetric Determination ◽  
Chelidonium Majus ◽  
Melanoma B16

Background. NSC-631570 (Ukrain) is a semisynthetic derivative of the Chelidonium majus alcaloids and the alkylans thiotepa. It exerts a selective cytotoxic effect on tumor cells in vitro and in vivo and shows the ability to modulate immunocyte functions. Purpose. The aim of our work was to carry out a comparative investigation of the effects of NSC-631570 alone or in combination with pathogen-associated molecules (PAM) on the growth of low- and high-metastasizing melanoma B16 in mice. Methods. NSC-631570 was administered intravenously and PAM intramuscularly to tumor-bearing mice seven times every third day, starting from the second day after the transplantation of tumor cells. The effect of monotherapy and combined therapy on tumor growth was evaluated by the indices of tumor growth inhibition in experimental animals. Cell cycle distribution of cancer cells was determined by flow cytometry. TAP1 and TAP2 expression was evaluated by RT-PCR. The metabolic activity of phagocytes was determined by NBT-test, phagocytosis was tested by flow cytometry, and arginase activity was estimated by colorimetric determination of urea. Results. Combined therapy and monotherapy with NSC-631570 resulted in significant inhibition of tumor growth in melanoma-bearing mice. Monotherapy with Ukrain was more effective in mice with high-metastasizing tumors. The therapeutic efficacy of NSC-631570 used in combination with PAM was more expressed in mice with low-metastasizing melanoma. Conclusion. The effectiveness of monotherapy and combined therapy with NSC-631570 in the treatment of melanoma B16 depends on the biological properties of the tumor and the immune state of the organism.

Tumor Cell Thrombin/PAR-1 Signaling Drives Pancreatic Ductal Adenocarcinoma Growth and Dissemination

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1070-1070
Author(s):  
Matthew J. Flick ◽  
Cheryl Rewerts ◽  
Carolina Cruz ◽  
Joseph S. Palumbo ◽  
James P. Luyendyk ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Tumor Growth ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Coagulation System ◽  
Ductal Adenocarcinoma ◽  
Parental Line ◽  
Experimental Metastasis ◽  
Primary Tumor Growth ◽  
Wild Type

Abstract Pancreatic ductal adenocarcinoma (PDAC) accounts for ~85% of diagnosed pancreatic cancers and is among the most lethal malignancies. The 5-year survival rate for pancreatic cancer patients has improved only marginally in the last 40 years (3% → 7%), with effectively no change in survival profile for patients with metastatic disease (2%). High mortality is linked to the aggressive and invasive nature of the malignancy and poor efficacy of limited treatment options, which collectively highlight the need for novel treatment strategies. Notably, analyses of pancreatic cancer in patients and animal models have demonstrated that PDAC is associated with robust coagulation system activity. Previous work has shown that patient PDAC tumor cells often express high levels of tissue factor (TF) and protease-activated receptor (PAR)-1. To determine the potential contribution of tumor cell derived-TF and PAR-1 to PDAC growth and metastasis, a novel tumor cell line (termed KPC2) was derived from mice in which PDAC tumorigenesis was induced by activation of two established pancreatic cancer alleles, KrasG12D and Trp53R172H. In transplant studies, tumor growth and experimental metastasis were evaluated using KPC2 cells in which TF or PAR-1 expression was suppressed by shRNA knockdown. In addition, the interplay of tumor-derived TF and PAR-1 with host factors in promoting tumor growth and experimental metastasis were evaluated in mice with genetically imposed deficits in coagulation system components. TF knockdown (to ~10% of the parental line) in KPC2 cells resulted in a significant diminution of both primary tumor growth and experimental metastasis. This reduction appeared to be linked to thrombin activity as primary tumor growth and experimental metastasis of parental KPC2 cells were significantly reduced in fIIlow mice (which constitutively express 10% of normal prothrombin) relative to wild-type mice. PAR-1 knockout mice displayed similar KPC2 growth and experimental metastasis to wild-type animals indicating that stromal cell-derived PAR-1 was not significant determinant. In stark contrast, shRNA-mediated knockdown of PAR-1 in KPC2 (to ~10% of the parental line) cells resulted in significantly diminished tumor growth and experimental metastasis. Diminished tumor growth was linked to reduced expression of the macrophage chemokine MCP-1 and the metalloproteinase MMP9 by the tumor cells as well as reduced thrombin-stimulated ERK phosphorylation. Our results suggest that a major mechanism of PDAC growth and dissemination is through TF/thrombin-driven PAR-1 signaling on tumor cells. Disclosures No relevant conflicts of interest to declare.

Combination of TLR9 agonists EnanDIM with checkpoint inhibitors for cancer immunotherapy.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 134-134
Author(s):  
Manuel Schmidt ◽  
Kerstin Kapp ◽  
Detlef Oswald ◽  
Burghardt Wittig ◽  
Barbara Volz
Keyword(s):  
Immune System ◽  
Tumor Growth ◽  
Colon Carcinoma ◽  
Mononuclear Cells ◽  
Checkpoint Inhibitors ◽  
Tumor Model ◽  
Tumor Growth Inhibition ◽  
Mouse Tumor ◽  
New Family ◽  
Ifn Alpha

134 Background: TLR9 agonists have shown anti-tumor effects by modulating the innate and adaptive immune system. Ongoing clinical studies support the use of TLR9 agonists for immunotherapeutic approaches. The new family of TLR9 agonists, EnanDIM, consists of linear single-stranded ODN synthesized using L-deoxyribonucleotides (natural enantiomers of D-deoxyribonucleotides) at their 3’-ends to prevent degradation. Therefore, EnanDIM does not own the off-target effects of PTO-modified CpG-ODN. Methods: EnanDIM with varying nucleotid sequences were compared for IFN-alpha response from human peripheral blood mononuclear cells and those molecules inducing the stronges response were selected. A maximum feasible dose (MFD) approach was employed to evaluate their acute toxicity and immunomodulatory properties. In addition, a combinatory approach with aPD-1 was evaluated in an syngeneic colon carcinoma CT26 mouse tumor model. Results: EnanDIM581 and EnanDIM532 were selected due to their pronounced activation of the IFN-alpha pathway in vitro. Safety assessments throughout and a gross necropsy at the end of the study revealed no signs of toxicity despite extremely high doses (300 - 1700 mg/kg). Dose-dependent increase of IP-10 levels in serum was observed between 6 and 24 hours after injection. In the colon carcinoma CT26 model subcutaneous injection of EnanDIM532 and intraperitoneal injection of aPD-1 had a moderate effect on the tumor growth when used in monotherapy (28.3% and 57.0% tumor growth inhibition, TGI). Notably, a combination of EnanDIM532 and aPD-1 further reduced tumor growth (74.7% TGI) and thus prolonged survival of the mice. Conclusions: In conclusion, EnanDIM, a new family of TLR9 agonists and immune surveillance reactivators (ISR), broadly activates the immune system, shows no toxicity in an MFD study and enhances the anti-tumor effects of the aPD-1 checkpoint inhibitor in a pilot study of a murine colon carcinoma tumor model. These data show the promising potential of EnanDIM not only for monotherapeutic but also combinatory approaches.

scholarly journals Combinations of TLR Ligands: A Promising Approach in Cancer Immunotherapy

2013 ◽  
Vol 2013 ◽  
pp. 1-14 ◽  
Author(s):  
Saskia Stier ◽  
Claudia Maletzki ◽  
Ulrike Klier ◽  
Michael Linnebacher
Keyword(s):  
Tumor Growth ◽  
Tumor Cells ◽  
Cell Lines ◽  
Immune Cells ◽  
Human Lymphocytes ◽  
Growth Control ◽  
Poly I:C ◽  
The Impact

Toll-like receptors (TLRs), a family of pattern recognition receptors recognizing molecules expressed by pathogens, are typically expressed by immune cells. However, several recent studies revealed functional TLR expression also on tumor cells. Their expression is a two-sided coin for tumor cells. Not only tumor-promoting effects of TLR ligands are described but also direct oncopathic and immunostimulatory effects. To clarify TLRs’ role in colorectal cancer (CRC), we tested the impact of the TLR ligands LPS, Poly I:C, R848, and Taxol on primary human CRC cell lines (HROC40, HROC60, and HROC69)in vitroandin vivo(CT26). Taxol, not only a potent tumor-apoptosis-inducing, but also TLR4-activating chemotherapeutic compound, inhibited growth and viability of all cell lines, whereas the remaining TLR ligands had only marginal effects (R848 > LPS > Poly I:C). Combinations of the substances here did not improve the results, whereas antitumoral effects were dramatically boosted when human lymphocytes were added. Here, combining the TLR ligands often diminished antitumoral effects.In vivo, best tumor growth control was achieved by the combination of Taxol and R848. However, when combined with LPS, Taxol accelerated tumor growth. These data generally prove the potential of TLR ligands to control tumor growth and activate immune cells, but they also demonstrate the importance of choosing the right combinations.

Fibrinogen is an important determinant of the metastatic potential of circulating tumor cells

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3302-3309 ◽  
Author(s):  
Joseph S. Palumbo ◽  
Keith W. Kombrinck ◽  
Angela F. Drew ◽  
Timothy S. Grimes ◽  
John H. Kiser ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Metastatic Potential ◽  
Thrombin Inhibitor ◽  
Tumor Dissemination ◽  
Hemostatic Factors ◽  
The Impact ◽  
Deficient Mice

Abstract Detailed studies of tumor cell–associated procoagulants and fibrinolytic factors have implied that local thrombin generation and fibrin deposition and dissolution may be important in tumor growth and dissemination. To directly determine whether fibrin(ogen) or plasmin(ogen) are determinants of the metastatic potential of circulating tumor cells, this study examined the impact of genetic deficits in each of these key hemostatic factors on the hematogenous pulmonary metastasis of 2 established murine tumors, Lewis lung carcinoma and the B16-BL6 melanoma. In both tumor models, fibrinogen deficiency strongly diminished, but did not prevent, the development of lung metastasis. The quantitative reduction in metastasis in fibrinogen-deficient mice was not due to any appreciable difference in tumor stroma formation or tumor growth. Rather, tumor cell fate studies indicated an important role for fibrin(ogen) in sustained adhesion and survival of tumor cells within the lung. The specific thrombin inhibitor, hirudin, further diminished the metastatic potential of circulating tumor cells in fibrinogen-deficient mice, although the inhibitor had no apparent effect on tumor cell proliferation in vitro. The absence of plasminogen and plasmin-mediated fibrinolysis had no significant impact on hematogenous metastasis. The authors concluded that fibrin(ogen) is a critical determinant of the metastatic potential of circulating tumor cells. Furthermore, thrombin appears to facilitate tumor dissemination through at least one fibrin(ogen)-independent mechanism. These findings suggest that therapeutic strategies focusing on multiple distinct hemostatic factors might be beneficial in the containment of tumor metastasis.

Daratumumab, a Novel Human Anti-CD38 Monoclonal Antibody for the Treatment of Chronic Lymphocytic Leukemia and B-Cell Non–Hodgkin Lymphoma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3935-3935 ◽  
Author(s):  
Alba Matas-Céspedes ◽  
Anna Vidal-Crespo ◽  
Vanina Rodriguez ◽  
Gael Roue ◽  
Elias Campo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Lymphocytic Leukemia ◽  
Tumor Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Lymphocytic Leukemia ◽  
Effector Cells ◽  
Non Hodgkin Lymphoma ◽  
Cd38 Expression

Abstract Abstract 3935 Daratumumab (DARA) is a human CD38 antibody with broad-spectrum killing activity. DARA induces killing of tumor cells, mainly via complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) (de Weers M, J Immunol 2011). DARA is currently being evaluated in phase I/II clinical trials in patients with multiple myeloma. In these clinical studies the adverse events have been manageable and marked reductions in paraprotein and bone marrow plasma cells have been observed. In the present study, we have analyzed the potential of targeting CD38 using DARA in two types of B-cell non-Hodgkin lymphoma (B-NHL) (follicular lymphoma (FL) and mantle cell lymphoma (MCL)), and in chronic lymphocytic leukemia (CLL). Flow cytometry analysis demonstrated that MCL and CLL tumor cells show heterogeneous expression of CD38, while FL cells showed invariable high CD38 levels. CD38 has attracted special attention in CLL where high CD38 expression is a marker of bad prognosis (Hamblin et al, Blood 1999 and 2002) and is expressed preferentially in the proliferating fraction of the tumor (Damle RN, Blood 2007). In addition, we have recently shown that high CD38 expression in MCL was associated with resistance to the proteasome inhibitor bortezomib (Pérez-Galán P, Blood 2011). Here, we tested the cytotoxic activity of DARA in tumor cell lines and in fresh tumor cells obtained from patients. DARA did not induce CDC in MCL cell lines (MINO, REC, HBL2, JEKO), irrespective of CD38 expression levels. Also, FL cell lines (WSU-FSCCL, RL) expressing relatively high CD38 levels were insensitive to DARA-induced CDC. This low CDC was associated with high expression of the complement inhibitors CD55 and CD59. In addition, the number of CD38 molecules per cell in these MCL and FL cell lines was lower than that found on the CDC-sensitive Daudi Burkitt lymphoma cell line, suggesting a threshold for CD38-targeted CDC lysis. In the presence of PBMC effector cells obtained from healthy donors, DARA showed significant levels of ADCC in cells from MCL, FL and CLL. In CLL primary cases (n=8) tested, DARA (14–43% lysis) was generally superior or at least equally effective (mean+/−SD=28,78 +/− 9,78) in inducing ADCC as compared to the anti-CD20 antibodies ofatumumab (mean+/−SD=21,35 +/− 15,71) and rituximab (mean+/−SD=29,30 +/− 15,90). The immunomodulatory agent lenalidomide shows considerable single agent activity in MCL, FL and CLL. Interestingly, it has been shown that lenalidomide may be able to increase ADCC, probably via activation of NK cells. We therefore tested whether the combination of lenalidomide and DARA could enhance ADCC. Noteworthy, DARA-induced ADCC in MCL, FL cell lines and primary CLL cells was significantly (p<0,05) enhanced when effector cells were pretreated with the immunomodulatory agent lenalidomide (3 μM, 72 h) with DARA doses ranging from 0,01–1 μg/ml. Finally, CD38 is important for cell migration and adhesion, especially for CXCR4-CXCL12-induced migration of tumor cells (Vaisitti et al, Leukemia 2010). Our preliminary results suggest that in the CLL subtype with high CD38 and more migratory capacity DARA (10–30 μg/ml) inhibits CXCL12/SDF1α mediated migration up to 70%. These results are of high importance because inhibition of tumor cell trafficking to tissue sites as bone marrow and lymph nodes, may be clinically relevant, as increasing evidence indicates that tumor–microenvironment interactions may play an important role in drug resistance and contribute to clinical failures. We are currently validating this in vitro results in a CLL mouse model. Taken together, these results suggest that DARA may be a promising therapeutic agent both for MCL, FL and CLL, in which DARA exerts its effects mainly via ADCC and for CLL also via inhibition of migration of tumor cells. Interestingly, the cytotoxic activity of DARA by ADCC could be further augmented by addition of lenalidomide in these three models. Disclosures: Lammerts van Bueren: Genmab: Employment. Bakker:genmab: Employment. Parren:genmab: Employment. Perez-Galan:Genmab: Research Funding.

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