Immunotherapy with LAK Cells and Anti-CD20 Monoclonal Antibodies for Follicular Lymphoma: Enhanced Antibody-Dependent Cell Cytotoxicity of LAK Cells in Association with GA101 Rather Than Rituximab

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4881-4881
Author(s):  
Carlos Panizo ◽  
Susana Inoges ◽  
Ascensión López Díaz de Cerio ◽  
Ricardo García Muñoz ◽  
Fernando Pastor ◽  
...  
Keyword(s):  
Synergistic Effect ◽  
Cell Line ◽  
Peripheral Blood ◽  
Peripheral Blood Lymphocytes ◽  
Lak Cells ◽  
Blood Samples ◽  
Effector Cells ◽  
Basal Cytotoxicity ◽  
Anti Cd20 ◽  
Cd20 Antibodies

Abstract Abstract 4881 Background The current gold standard treatment for follicular lymphoma (FL) consists in the association of rituximab, that is an anti-CD20 monoclonal antibody (mAb), and one among many possible chemotherapy regimens, followed by maintenance with the same mAb. However, LF is still considered incurable. Hence, it is necessary to keep investigating new strategies aiming at improving the overall clinical results. As an anti-tumor mechanism, antibody-dependent cellular cytotoxicity (ADCC) depends on both the immune system effector arm and the antibody structure. The culture of peripheral blood lymphocytes with IL-2 activates killer cell subpopulations (LAK cells), mainly consisting of natural killer (NK) cells and cytotoxic T lymphocytes (CTL), both with significant cytotoxic capacity. The combination of LAK cells with a mAb could achieve a synergistic effect by enhancing ADCC and potentially achieving clinical efficacy. As structural modifications in mABs may alter their biological activity, new anti-CD20 with structural changes in the Fc portion of the immunoglobulin have been synthesized in order to obtain a greater therapeutic effect. GA101 is a new anti-CD20 mAB that binds with higher affinity to the CD20 type II epitope, increasing the ADCC effect as compared to to rituximab. Currently, no data support the notion of a possible synergistic effect of GA101 and LAK cells. Aim The objective of this work was to assess the cytotoxic capacity of LAK cells generated from the peripheral blood of patients with FL. In addition, we aimed at ascertaining whether the combination of rituximab and LAK could improve the functional activity of LAK alone. Finally, we conducted a comparative study of two anti-CD20 antibodies structurally different, namely rituximab and GA101, used in combination with LAK cells. Material and methods LAK cells were expanded in vitro from 39 peripheral blood samples of patients with FL. Cytotoxicity studies were performed using chromium release assays in which LAK cells targeted K562 (as NK target), Daudi (as LAK cells target) and CRL-1596 (CD20 positive) cells. Overall, mononuclear cells were isolated and basal cytotoxicity was measured in 39 peripheral blood samples from patients with FL. The ability of two anti-CD20 antibody (rituximab and GA101) to enhance LAK cell activity was evaluated against the CRL-1596 cell line. As a control, an irrelevant antibody (cetuximab) was used. To perform these studies, target cells were incubated with effector cells and antibody at 10 mg/ml concentration. Results Results are expressed as mean percentages. Basal cytotoxicity against K562, Daudi and CRL-1596 cells was 19.4%, 11.9% and 1.5% respectively. After culture with IL-2, the cytotoxicity activity against the same cell lines was reassessed and in all cases a statistically significant increase was observed (p<0,05). In particular, cytotoxicity against K562, Daudi and CRL-1596 was 39.5%, 39.6% and 21.1%, respectively. Furthermore, we studied the rituximab effect on the cytotoxic capacity against CRL-1596. The observed cytotoxicity of LAK cells with rituximab was 39.9% vs 23.6% for LAK cells alone (p<0,001). In parallel, the GA101 effect on the cytotoxic capacity against CRL-1596 was also evaluated. The cytotoxicity of LAK cells with GA101 was 52.3% vs 23.6% for LAK cells alone (p<0,001). Vice versa, no difference was observed in the cytotoxic activity of LAK cells against CRL1596 cell line when the effector cells were incubated with the irrelevant antibody cetuximab (26.0% vs 23.6%). Finally, when comparing the ability of both anti-CD20 antibodies to enhance ADCC, we observed a significant difference in favor of GA101 (GA101: 52.3%, rituximab: 39.9%; p<0,001). Conclusions LAK cells generated from peripheral blood lymphocytes by culture with IL-2 in patients with LF show a higher cytotoxic activity than naive lymphocytes. The observed cytotoxic capacity of these LAK cells against a CD20 positive cell line (CRL-1596) is enhanced by the addition of anti-CD20 mAbs. Interestingly, GA101 was consistently more effective than rituximab in enhancing the cytotoxic capacity of LAK cells. Disclosures: No relevant conflicts of interest to declare.

Long-Term Follow-up of Patients with Recurrent Malignant Gliomas Treated with Adjuvant Adoptive Immunotherapy

Neurosurgery ◽  
1991 ◽  
Vol 28 (1) ◽  
pp. 16-23 ◽  
Author(s):  
Kevin O. Lillehei ◽  
Dawn H. Mitchell ◽  
Stephen D. Johnson ◽  
Larry E. McCleary ◽  
Carol A. Kruse
Keyword(s):  
Survival Time ◽  
Adoptive Immunotherapy ◽  
Peripheral Blood ◽  
Interleukin 2 ◽  
Peripheral Blood Lymphocytes ◽  
Lak Cells ◽  
Prior Chemotherapy ◽  
Blood Lymphocytes ◽  
Significant Difference

Abstract Between August 1986 and October 1987, the Denver Brain Tumor Research Group conducted a clinical trial using autologous human recombinant interleukin-2 (rIL-2)-activated lymphocytes to treat 20 patients with recurrent high-grade gliomas. The trial involved surgical resection and/or decompression followed by intracavitary implantation of lymphokine-activated killer (LAK) cells and autologous stimulated lymphocytes (ASL) along with rIL-2 in a plasma clot. One month later, stimulated lymphocytes and rIL-2 were infused through a Rickham reservoir attached to a catheter directed into the tumor bed. The LAK cells were rIL-2-activated peripheral blood lymphocytes cultured for 4 days; the ASL were lectin- and rIL-2-activated peripheral blood lymphocytes cultured for 10 days. Of the 20 patients treated, 11 were evaluated as a group (mean age, 44 years, range, 15-61 years; mean Karnofsky rating, 69, range, 50-100; mean Decadron dose at entry, 14 mg/d. range, 0-32). The average number of lymphocytes implanted was 7.6 × 109 (range, 1.9-27.5 × 109), together with 1 to 4 × 106 U of rIL-2. To date, 10 of the 11 patients died, all from recurrent tumor growth. The median overall survival time was 63 weeks (range, 36-201; mean, 86). The median survival time after immunotherapy was 18 weeks (range, 11-151; mean, 39). No significant difference in survival after immunotherapy was found between those patients who had received previous chemotherapy and those who had not. The use of steroids or prior chemotherapy did not influence the in vitro generation of ASL or LAK cells. Prior chemotherapy did correlate, however, with diminished in vitro cytotoxicity against the natural killer-sensitive (K562) target cell by LAK cells (P &lt; 0.05) but not that by ASL. There were no major adverse side effects. Although adoptive immunotherapy was safe and well tolerated, its therapeutic potential remains in question.

scholarly journals CYTOTOXIC EFFECTS OF ARIPIPRAZOLE ON MKN45 AND NIH3T3 CELL LINES AND GENOTOXIC EFFECTS ON HUMAN PERIPHERAL BLOOD LYMPHOCYTES

2019 ◽  
Vol 56 (2) ◽  
pp. 155-159 ◽  
Author(s):  
Mohammad SHOKRZADEH ◽  
Abbas MOHAMMADPOUR ◽  
Mona MODANLOO ◽  
Melika HASSANI ◽  
Nasrin Ghassemi BARGHI ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Line ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Normal Cell ◽  
Micronucleus Assay ◽  
Peripheral Blood Lymphocytes ◽  
Nih3t3 Cell ◽  
Dopamine Antagonists ◽  
Blood Lymphocytes

ABSTRACT BACKGROUND: Gastric cancer is known as the fourth most common cancer. Current treatments for cancer have damaged the sensitive tissues of the healthy body, and in many cases, cancer will be recurrent. Therefore, need for treatments that are more effective is well felt. Researchers have recently shifted their attention towards antipsychotic dopamine antagonists to treat cancer. The anticancer activities of aripiprazole remain unknown. OBJECTIVE: This study aimed to evaluate the efficacy and safety of aripiprazole on gastric cancer and normal cell lines. METHODS: In this regard, the cytotoxicity and genotoxicity of aripiprazole were investigated in MKN45 and NIH3T3 cell lines by methyl tetrazolium assay and on peripheral blood lymphocytes by micronucleus assay. For this purpose, cells were cultured in 96 wells plate. Stock solutions of aripiprazole and cisplatin were prepared. After cell incubation with different concentrations of aripiprazole (1, 10, 25, 50, 100 and 200 μL), methyl tetrazolium solution was added. For micronucleus assay fresh blood was added to RPMI culture medium 1640 supplemented, and different concentrations of aripiprazole (50, 100 and 200 μL) were added. RESULTS: The finding of present study showed that the IC50 of aripiprazole in the cancer cell line (21.36 μg/mL) was lower than that in the normal cell line (54.17 μg/mL). Moreover, the micronucleus assay showed that the frequency of micronuclei of aripiprazole at concentrations below 200 μM was much less than cisplatin. CONCLUSION: Aripiprazole can be a good cytotoxic compound and good candidate for further studies of cancer therapy.

Comparison of Cytotoxic Mechanisms of Anti-CD20 Antibodies GA101 and Rituximab Against Fresh Chronic Lymphocytic Leukemia Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2467-2467
Author(s):  
Lina Reslan ◽  
Stéphane Dalle ◽  
Cindy Tournebize ◽  
Stephanie Herveau ◽  
Emeline Cros ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Death ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Peripheral Blood ◽  
Lymphocytic Leukemia ◽  
Oxygen Species ◽  
Cytochrome C Release ◽  
Anti Cd20 ◽  
Cd20 Antibodies

Abstract Abstract 2467 GA101, a novel glycoengineered type II IgG1 antibody against CD20, has shown a direct and immune effector cell-mediated cytotoxicity in numerous B-cell disorders. Chronic Lymphocytic Leukemia (CLL) is the most common hematologic malignancy in the western world. Since circulating mature B-CLL cells express high levels of antiapoptotic proteins that are implicated in the survival mechanism, we investigated whether the effects of GA101 compared to rituximab, induces apoptosis in these cells and what mechanism underlies GA101-mediated cytotoxicity. CLL cells were isolated from peripheral blood samples by density gradient centrifugation and B lymphocytes were purified by a negative selection method using the EasySep® B Cell Enrichment Cocktail. Cell viability was measured flow cytometrically by annexinV binding. We assessed the mitochondrial transmembrane potential (ΔΨm) by staining with 3,3-dihexyloxacarbocyanine iodide (DiOC6[3]), the generation of reactive oxygen species by staining with Dihydroethidine (DHE) as well as cytochrome c release. Moreover, the expression of several apoptosis-regulating proteins, including the Bcl-2 family proteins (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak and Bad) and the activation of the caspase cascade were evaluated by immunoblotting on 34 fresh peripheral blood B-CLL specimens. We showed that GA101 initiates an early extensive cell death. The average decrease of viability of freshly isolated and purified CLL cells 24 hours post-treatment with 10μg/ml of anti CD20 antibodies were 37.6% for GA101 (n=11) and 28.8% for Rituximab (n=11). The GA101-induced cell death was paralleled by a rapid loss of mitochondrial membrane potential accompanied with the production of ROS and cytochrome c release that occurred significantly as early as 3 hours post-treatment. However, rituximab was unable to initiate a loss of ΔΨm and the production of ROS. The use of antioxidants such as N-acetyl cysteine and L-ascorbic acid were unable to circumvent either the GA101-induced cell death or the loss of ΔΨm. However, the preincubation of CLL cells with Z-VAD.fmk (N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone), a broad caspase inhibitor, abolished the exposure of phosphatidylserine residues, the generation of reactive oxygen species and reversed the loss of ΔΨm. Furthermore no change was observed in the expression level of Bcl2 pro-survival family members, while GA101 induced the pro-apoptotic proteins such as Bax and Bak and caused cleavage of the active form of caspase 9 and 3 and the proteolytic cleavage of PARP, in 5 out of 9 patients studied. Altogether, these data show that GA101 induced-cell death in B-CLL cells, unlike what has been observed in cell lines, is mediated by a caspase-dependent mechanism involving the loss of ΔΨm and the generation of ROS. Ongoing studies aim to analyze the role, the conformational changes and the cellular redistribution of Bax and Bak in response to GA101 and the modifications of other apoptosis-related proteins in CLL cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CXCL12 Inhibition By Nox-A12 (Olaptesed Pegol) Synergizes with the ADCC Activity of CD20 Antibodies By Increasing NK Cell Infiltration in a 3D Lymphoma Model

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3021-3021 ◽  
Author(s):  
Dirk Zboralski ◽  
Anna Kruschinski ◽  
Axel Vater
Keyword(s):  
Nk Cells ◽  
Peripheral Blood ◽  
Hematological Malignancies ◽  
Nk Cell ◽  
Cell Infiltration ◽  
Malignant Cells ◽  
Immune Effector ◽  
Effector Cells ◽  
Immune Effector Cells ◽  
Anti Cd20

Abstract Hematological malignancies are characterized by the expansion of malignant cells in the peripheral blood and in stroma-rich niches such as the bone marrow or lymphoid tissues. Anti-CD20 monoclonal antibodies (mAbs) are highly effective in eliminating malignant cells in the peripheral blood with the help of immune effector cells, e.g. NK cells mediating antibody-dependent cellular cytotoxicity (ADCC). However, residual malignant cells often continue to persist in protective stromal niches. These compartments have similarities to the solid tumor microenvironment (TME) where mAb therapy is restricted by poor tissue penetration and low effector cell infiltration. The CXCL12-neutralizing L-RNA aptamer NOX-A12 (olaptesed pegol) has been shown to mobilize malignant cells from the bone marrow into the peripheral blood, thereby sensitizing them to the action of standard therapy such as the anti-CD20 mAb rituximab (Blood. 2014;124(21):1996). In addition to malignant cells, CXCR4 expressing immune cells are effectively mobilized by NOX-A12 (Clin Pharmacol Ther. 2013;94(1):150-157). Recently we have shown that NOX-A12 increases lymphocyte infiltration into solid tumor-stroma spheroids, thereby synergizing with anti-PD-1 checkpoint blockade (Cancer Res 2016;76(14 Suppl): 1473). Here we established 3D lymphoid spheroidal microtissues mimicking the stroma-rich and CXCL12-abundant TME of lymphoid malignancies. We investigated the effect of NOX-A12 on NK effector cell infiltration into lymphoma spheroids and tested the combination with anti-CD20 mAbs. Spheroids were generated by co-culturing of CXCL12-expressing murine stromal MS-5 cells and CD20-expressing lymphoma cells in ultra-low attachment plates for 24 hours. Primary human NK cells, isolated from healthy donors, were added to the spheroids in the presence of various concentrations of NOX-A12 and an anti-CD20 mAb, either rituximab or obinutuzumab. The next day, spheroids were washed and dissociated for NK cell quantification and lymphoma cell viability determination by flow cytometry. We found that the ADCC efficacy of anti-CD20 mAbs is lower in 3D spheroids compared to conventional 2D assays due to low NK cell infiltration into the microtissues. Interestingly, NOX-A12 increased the amount of NK cells in the lymphoma-stroma spheroids up to 8-fold in a dose-dependent manner (Figure A), likely by forming de novo CXCL12 gradients into the dense microtissue due to the particular penetration characteristic of L-RNA aptamers. Of note, the NOX-A12-mediated increase of NK cells in the spheroids synergized with both anti-CD20 mAbs tested in terms of NK cell-mediated killing of lymphoma cells (Figure B). The present work complements the mechanism of action data of NOX-A12 by adding enhancement of NK cell infiltration into stroma-rich tumor compartments to the already established effects of mobilizing malignant and immune effector cells into the peripheral blood. These data as well as the good toxicity profile and the promising data in phase 2a clinical trials in patients with CLL and MM justify further clinical trials in patients with hematological malignancies to verify the greater efficacy of combination treatment using ADCC-inducing mAbs and NOX-A12. Figure Figure. Disclosures Zboralski: NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment. Vater:NOXXON Pharma AG: Employment.

Hyperactive ENaC identifies hypertensive individuals amenable to amiloride therapy

2001 ◽  
Vol 281 (5) ◽  
pp. C1413-C1421 ◽  
Author(s):  
Artensie R. Carter ◽  
Zhen Hong Zhou ◽  
David A. Calhoun ◽  
James K. Bubien
Keyword(s):  
Patch Clamp ◽  
Peripheral Blood ◽  
Peripheral Blood Lymphocytes ◽  
Refractory Hypertension ◽  
Blood Samples ◽  
Whole Cell ◽  
Blood Lymphocytes ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Hypertensive Therapy

Pathophysiological features of both primary aldosteronism and pseudohyperaldosteronism are hyperactive amiloride-sensitive epithelial Na+ channels (ENaC) and refractory hypertension. Peripheral blood lymphocytes express ENaC, which functions and is regulated similarly to ENaC expressed by renal principal cells. Thus it was hypothesized that individuals with either of these hypertensive etiologies could be identified by assessment of the function and regulation of peripheral blood lymphocyte ENaC, by whole cell patch clamp. We also tested the hypothesis that specific inhibition of hyperactive ENaC with amiloride could ameliorate the hypertension. To test these hypotheses, we solicited blood samples from normotensive, controlled hypertensive, and refractory hypertensive individuals. Lymphocytes were examined electrophysiologically to determine whether ENaC was hyperactive. All positive findings were from refractory hypertensive individuals. Nine refractory hypertensive patients had amiloride added to their hypertensive therapy. Amiloride normalized the blood pressure of four subjects. These individuals all had hyperactive ENaC. Amiloride had no effect on individuals with normal ENaC. These findings suggest that whole-cell patch clamp of peripheral blood lymphocytes can be used to identify accurately and rapidly hypertensive individuals who will respond to amiloride therapy.

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