scholarly journals Rituximab Treatment in Hepatitis C Infection: An In Vitro Model to Study the Impact of B Cell Depletion on Virus Infectivity

PLoS ONE ◽  
2011 ◽  
Vol 6 (9) ◽  
pp. e25789 ◽  
Author(s):  
Zania Stamataki ◽  
Samantha Tilakaratne ◽  
David H. Adams ◽  
Jane A. McKeating
Keyword(s):  
Hepatitis C ◽  
B Cell ◽  
In Vitro Model ◽  
Cell Depletion ◽  
Virus Infectivity ◽  
B Cell Depletion ◽  
Hepatitis C Infection ◽  
Vitro Model ◽  
The Impact

scholarly journals Obinutuzumab (GA101) Is Less Prone to Antagonism of Immune Effector Function By Ibrutinib Than Rituximab in Vitro and in Vivo

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1765-1765 ◽  
Author(s):  
Sylvia Herter ◽  
Idit Sagiv-Barfi ◽  
Cariad Chester ◽  
Mohith Sadaram ◽  
Jonathan Hebb ◽  
...  
Keyword(s):  
B Cell ◽  
Whole Blood ◽  
Dose Range ◽  
Xenograft Model ◽  
Cell Depletion ◽  
B Cell Depletion ◽  
Immune Effector ◽  
The Impact

Abstract Introduction: Kohrt et al., Blood, 2014 demonstrated that ibrutinib antagonizes ADCC function of rituximab in vitro in ADCC assays and in vivo in the DHL-4 xenograft model through inhibition of FcgammaR signaling in immune effector cells, possibly mediated by inhibition of ITK. Obinutuzumab (GA101) is a glycoengineered type II CD20 antibody that mediates higher direct cell death induction than rituximab, and by being glycoengineered mediates enhanced induction of ADCC and ADCP. Here we aimed to investigate the impact of ibrutinib on the immune effector function of obinutuzumab as compared to rituximab. Experimental methods: The impact of ibrutinib (dose range 30, 100, 300 ng/ml to cover Cmax and Ctrough in patients) on NK cell mediated ADCC induction by obinutuzumab and rituximab was investigated using SU-DHL4 and Z138 cells as targets in LDH and chromium release assays or measuring CD16 downmodulation and the degranulation marker CD107a. IFNg release as a surrogate for NK cell activation was investigated using DHL-4 target cells or an autologous in vitro system using leukemic cells derived from CLL/NHL patients. Depletion of CD19 positive B-cells was determined in whole blood from healthy volunteers in flow cytometry-based whole blood assay. In vivo the combination of obinutuzumab or rituximab (10 mg/kg once weekly for 3 weeks) with ibrutinib (25mg/kg BID days 14-28) was investigated in the DHL-4 xenograft model. Results: In ADCC assays, ibrutinib (dose range 30, 100, 300 ng/ml) resulted in a reduction of the ADCC potency of obinutuzumab and rituximab. However, at saturating antibody concentrations of 10 ug/ml, ADCC mediated by obinutuzumab was retained while ADCC mediated by rituximab was strongly reduced as measured by chromium release (Figure 1A). Interestingly, in the whole blood B cell depletion assay only little impact of ibrutinib on obinutuzumab-mediated B cell depletion in terms of EC50 and maximal killing was observed at clinically meaningful concentrations of ibrutinib (30, 100, 300 ng/ml), while the activity of rituximab could be completely abolished with 300 ng/ml ibrutinib (Figure 1B). Notably, control experiments using an effector dead version of obinutuzmab that cannot any longer mediate ADCC or ADCP demonstrate that the retained B cell depletion by obinutuzumab in presence of ibrutinib is not due to direct cell death induction, but also due to immune effector cell mediated function (ADCC and ADCP). In the DHL-4 xenograft model where ibrutinib as a single agent has no anti-tumoral efficacy, the combination resulted in a reduced anti-tumoral efficacy of rituximab, whereas efficacy of obinutuzumab was not affected (Figure 1C). Conclusions: Surprisingly, we found that the inhibitory effect of ibrutinib on the immune effector mediated activity of obinutuzumab is not observed when compared to rituximab. Most notably, ADCC at saturating antibody doses, whole blood B cell depletion and in vivo efficacy of obinutuzumab were retained in presence of clinically relevant concentrations of ibrutinib covering Cmax and Ctrough levels, whereas the activity of rituximab was almost completely abolished under these conditions. We hypothesize that the differential behavior of obinutuzumab and rituximab may be related to the enhanced FcgRIII affinity and stronger FcgRIII signaling activation mediated by obinutuzumab as a consequence of glycoengineering that may subsequently overwrite inhibitory effects of ibrutinib. While the clinical relevance of the observed preclinical antagonism for the combination of rituximab with ibrutinib still needs further clinical investigation, these preclinical data strongly support the clinical investigation of ibrutinib in combination with the glycoengineered Type II CD20 antibody obinutuzumab for the treatment of chronic lymphocytic leukemia and other B-cell malignancies. Figure 1 Figure 1. Disclosures Herter: Roche: Employment. Bacac:Roche: Employment. Umana:Roche: Employment. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

Effect of Prior Rituximab on Residence Time of I131 Tositumomab Consolidation Therapy in Patients (pts) with Non Hodgkin’s Lymphoma (NHL): Analysis of SWOG Clinical Trials.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2000-2000
Author(s):  
Julia Schaefer-Cutillo ◽  
Vaseem Chengazi ◽  
Derick R Peterson ◽  
David G. Maloney ◽  
Kevin Kibler ◽  
...  
Keyword(s):  
B Cell ◽  
Residence Time ◽  
Wilcoxon Test ◽  
Cell Depletion ◽  
Residence Times ◽  
B Cell Depletion ◽  
Consolidation Therapy ◽  
Iodine 131 ◽  
The Impact

Abstract Backround: Anti CD-20 radioimmunotherapy (RIT) is effective therapy for indolent B-cell NHL, and under investigation in more aggressive histologies. Most data on safety and efficacy of RIT is from the pre-rituximab era, and the effect of rituximab exposure on RIT in pts with NHL is unknown. Gopal et al recently demonstrated that exposure to rituximab correlated with inferior tumor response and alteration in tumor: organ dosimetry ratio both in vitro and in mouse models following therapy with iodine-131 tositumomab (Blood 112:830). Two ongoing SWOG trials evaluating RIT consolidation therapy provide a unique opportunity to evaluate the impact of prior rituximab on pharmacodynamics of iodine-131 tositumomab in humans. S0016 enrolls previously untreated pts with follicular NHL, and iodine-131 tositumomab consolidation is administered after 6 cycles of CHOP. S0433 enrolls previously untreated pts with DLBCL, and iodine-131 tositumomab is administered after 6 cycles of CHOP with rituximab, and 2 additional cycles of CHOP alone. As rituximab leads to B-cell depletion for 6 months or more, we hypothesized the residence time of iodine-131 tositumomab would differ in pts exposed recently to rituximab compared to no prior rituximab. Methods: Prospective pts at the University of Rochester enrolled in S0016 and S0433 were analyzed. Residence times of iodine-131 tositumomab were calculated using serial imaging on a Picker XP 2000 gamma camera. Rituximab levels were performed within one week prior to dosimetric iodine-131 tositumomab administration using ELISA. Medians were used to summarize the data, and the 2-tailed Mann-Whitney-Wilcoxon test was used for hypothesis testing. Results: 16 pts (6 female) on S0016 and 12 pts (6 female) on S0433, were identified, with median ages of 54.5 and 69.5 respectively. All pts had advanced stage disease, and median BMI and creatinine were similar for both groups. Pts on S0433 had a median time from rituximab to RIT of 78.6 days (range 58–98 days). Despite this, rituximab levels were present at time of iodine-131 tositumomab in all pts measured (N=9; median rituximab level 37.2 ug/ml, range 15.6–61.69). Median absolute lymphocyte count appeared lower in the S0433 group compared to the S0016 group (600 vs 1050 /ul), but this difference was not significant (p=0.12). Pts on S0433 (all had received rituximab prior to iodine-131 tositumomab consolidation) had significantly longer RIT residence times when compared to those on S0016, (not treated with prior rituximab): 115 hours vs. 107 hours; p=0.02. Therapeutic doses of iodine-131 tositumomab were not significantly different between the two studies (S0433: 72 mCi vs. S00016: 78 mCi p=0.59). Conclusions: Our results indicate that prior therapy with rituximab results in a longer residence time of iodine-131 tositumomab when used as consolidation after chemotherapy. Measurable rituximab levels at time of RIT suggest that rituximab-induced B-cell depletion decreases clearance of RIT, possibly allowing for longer exposure times. The significance of this longer residence time is unknown but it could be associated with greater toxicity to normal organs, and could be indicative of decreased tumor binding. If confirmed in larger studies, these findings could have profound implications on RIT administration in the context of rituximab. Rituximab-induced B-cell depletion could obligate the need for unlabeled antibody dosing prior to RIT, and may affect dosimetry of RIT. Prospective studies of RIT in the rituximab era should evaluate the impact of prior rituximab and RIT residence time on toxicities and outcomes in pts treated with RIT.

scholarly journals RNA-Seq Based Immunoglobulin Repertoire Analysis of Normal Plasma Cells Generated in an in Vitro B Cell Differentiation System

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1051-1051
Author(s):  
Renee C Tschumper ◽  
Collin A Osborne ◽  
Pritha Chanana ◽  
Jaime I Davila ◽  
Denise K Walters ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
In Vitro Model ◽  
Plasma Cells ◽  
B Cell Differentiation ◽  
Rna Seq ◽  
Repertoire Analysis ◽  
V Region ◽  
Vitro Model

Antibody secreting plasma cells (PCs) play an important role in effective humoral immune responses. The low frequency of bone marrow PCs in humans makes it challenging to obtain sufficient numbers of PCs for biologic studies. Previous studies have employed in vitro model systems to generate cells that morphologically, phenotypically, and functionally resemble normal polyclonal PCs. Gene expression profiles of in vitro generated PCs (IVPCs) mirror their normal counterparts, however to date extensive immunoglobulin (Ig) repertoire analysis of IVPCs is lacking. Here, we used a modified 3-step protocol to generate IVPCs and used RNA-seq to explore the transcriptome with emphasis on the Ig repertoire of plasmablasts and PCs. Total B cells were isolated from 3 normal donors and cultured with various cytokines and the B cell activators CpG ODN and CD40L. RNA was obtained from freshly isolated B cells (Day 0; D0) as well as from Day 4 (D4) plasmablasts, and Day 10 (D10) IVPCs. Morphologically, D10 cells exhibited typical PC morphology, including an eccentric nucleus and perinuclear hof. RNA-seq was performed on total RNA from all 3 donors and time points using the Standard TRuSeq v2 library prep and with paired end sequencing on the Illumina HiSeq 4000 platform. Principle component analysis of gene expression data showed that D0, D4 and D10 cells could be clearly segregated across all 3 normal donors. Of importance, transcripts previously described as distinguishing B cells from PCs were found to be differentially expressed including overexpression of CXCR5, CD19, EBF, CD83, PAX5, IRF8 in D0 B cells and overexpression of IRF4, Blimp-1, XBP1, BCMA, SLAMF7, Syndecan-1, CD38 and CD27 in IVPCs, thus validating our in vitro model for generating PCs. Furthermore, expression of cell cycle related transcripts such as CKS1, CDK1, and CCDN2 followed the pattern of low expression in resting B cells, increased expression in plasmablasts, and decreased expression in IVPCs confirming the cells are actively cycling in a manner comparable to cells in vivo. D10 IVPCs also overexpressed transcripts known to be upregulated during the unfolded protein response. As expected from Ig secreting cells, D10 IVPCs had an over-representation of Ig transcripts. At D0, resting B cells had high levels of IgD and IgM heavy chain (HC) transcripts. At D10, IgM transcripts modestly increased with Log2 fold change (FC) = 3 and as expected, IgD levels decreased significantly (Log2 FC = -2.2). IgA and IgG isotype transcripts significantly increased at D10 (Log2 FC > 6.0) with the IgG4 subtype having the greatest Log2 FC at 8.4. Next we focused on the Ig repertoire of D0, D4, and D10 cells. By aligning to known germline Ig sequences in IMGT/V-Quest (www.imgt.org) and then assembling the paired ends of D0, D4 and D10 Ig transcripts, we were able to analyze the Ig repertoire. Since the Ig HC variable (V) region is encoded by V, diversity (D) and joining (J) segments, only fragments that could be confidently determined were considered. All but 3 IGHV transcripts (IGHV3-35, IGHV3-47 and IGHV7-8) and 2 IGHD transcripts (IGHD4-4 and IGHD5-5) were found and all IGHJ segments were represented across the differentiation spectrum. In D0 cells, the number of unique VDJ combinations ranged from 643 to 863 across all 3 normal samples and increased to a range of 2524 to 2867 in D10 IVPCs. When looking at the differential expression of each VDJ combination from D0 to D10, a pairwise t-test for relative frequency showed that there was no significant change greater than 1%, suggesting the repertoire diversity was not skewed, thus proving the conditions for stimulation were not targeting any one starting B cell. Our data also allowed us to track clonal expansions during differentiation as defined by the increasing frequency of sequences with identical nucleotide sequence in the V region and CDR3 (including D and J regions). Hence, a single sequence could be tracked from D0 to D10. Of interest, in a small sampling of the total available sequences, only those B cells with a mutated IGHV region, characteristic of a memory B cell, went on to expand in this system whereas B cells with an unmutated IGHV did not. Our analysis of the Ig repertoire of IVPCs suggests this system provides a functional model to study Ig repertoire along the B cell differentiation process and further delineate the conditions that may result in a clonal expansion, a hallmark of many hematologic malignancies including multiple myeloma. Disclosures No relevant conflicts of interest to declare.

scholarly journals CAFs affect the proliferation and treatment response of head and neck cancer spheroids during co-culturing in a unique in vitro model

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Mustafa Magan ◽  
Emilia Wiechec ◽  
Karin Roberg
Keyword(s):  
Cell Proliferation ◽  
Tumor Cell ◽  
Head And Neck ◽  
Treatment Response ◽  
Tumor Cells ◽  
In Vitro Model ◽  
Tumor Spheroids ◽  
Vitro Model ◽  
The Impact

Abstract Background Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors for which the overall survival rate worldwide is around 60%. The tumor microenvironment, including cancer-associated fibroblasts (CAFs), is believed to affect the treatment response and migration of HNSCC. The aim of this study was to create a biologically relevant HNSCC in vitro model consisting of both tumor cells and CAFs cultured in 3D to establish predictive biomarkers for treatment response, as well as to investigate the impact of CAFs on phenotype, proliferation and treatment response in HNSCC cells. Methods Three different HNSCC patient-derived tumor cell lines were cultured with and without CAFs in a 3D model. Immunohistochemistry of the proliferation marker Ki67, epidermal growth factor receptor (EGFR) and fibronectin and a TUNEL-assay were performed to analyze the effect of CAFs on both tumor cell proliferation and response to cisplatin and cetuximab treatment in tumor spheroids (3D). mRNA expression of epithelial-mesenchymal transition (EMT) and cancer stem cells markers were analyzed using qRT-PCR. Results The results demonstrated increased cell proliferation within the tumor spheroids in the presence of CAFs, correlating with increased expression of EGFR. In spheroids with increased expression of EGFR, a potentiated response to cetuximab treatment was observed. Surprisingly, an increase in Ki67 expressing tumor cells were observed in spheroids treated with cisplatin for 3 days, correlating with increased expression of EGFR. Furthermore, tumor cells co-cultured with CAFs presented an increased EMT phenotype compared to tumor cells cultured alone in 3D. Conclusion Taken together, our results reveal increased cell proliferation and elevated expression of EGFR in HNSCC tumor spheroids in the presence of CAFs. These results, together with the altered EMT phenotype, may influence the response to cetuximab or cisplatin treatment.

Potent B-Cell Depletion by IMGN529, a CD37-Targeting Antibody-Maytansinoid Conjugate for the Treatment of B-Cell Malignancies,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3726-3726
Author(s):  
Jutta Deckert ◽  
Sharon Chicklas ◽  
Yong Yi ◽  
Min Li ◽  
Jan Pinkas ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Whole Blood ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Depletion ◽  
B Cell Depletion ◽  
B Cell Malignancies

Abstract Abstract 3726 CD37 is a B-cell surface antigen which is widely expressed on malignant B cells in non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL). In normal tissues CD37 expression is limited to blood cells and lymphoid tissues. This restricted expression profile makes CD37 an attractive therapeutic target for antibodies and antibody-drug conjugates. We developed a novel anti-CD37 antibody, K7153A, which provides a unique combination of functional properties: it demonstrated strong pro-apoptotic and direct cell killing activity against NHL cell lines and could mediate effector activity such as CDC and ADCC. The antibody-maytansinoid conjugate, IMGN529, was produced by conjugation of K7153A with the potent maytansinoid, DM1, via the non-cleavable linker, SMCC. The direct cytotoxic potency of the K7153A antibody was superior to that of the CD20-directed rituximab and was further enhanced with maytansinoid conjugation in IMGN529. In vivo, IMGN529 demonstrated better anti-tumor activity than the K7153A antibody in established subcutaneous follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), and CLL xenograft models in SCID mice. A single administration of IMGN529 showed similar or improved efficacy compared to anti-CD20 antibodies or standard chemotherapy where tested. Immunohistochemical (IHC) staining of formalin fixed paraffin-embedded (FFPE) NHL tissue sections was performed to evaluate CD37 expression. CD37 exhibited a similar prevalence to CD20 in subtypes of NHL such as FL, DLBCL, Burkitt's lymphoma (BL) and mantle cell lymphoma (MCL). B-cell depletion is an important measure of efficacy for targeted therapies, such as CD20-directed antibodies, in B-cell malignancies. CD37 expression in blood cells from healthy human donors was measured by quantitative flow cytometry in comparison to CD20. The greatest CD37 expression was found in B cells at approximately 77,000 antibodies bound per cell (ABC), which was similar to CD20 expression in B cells at 95,000 ABC. In other blood cell types CD37 staining was seen at low levels, about 2,000 – 5,000 ABC, in monocytes, NK cells and T cells. In vitro depletion experiments were performed with purified peripheral blood mononuclear cells (PBMCs) and with whole blood, both derived from several healthy donors. Cells were incubated for 1 hr with 10 μg/mL of either K7153A, IMGN529, CD37-targeting TRU-016, rituximab or the anti-CD52 antibody alemtuzumab, with cell depletion determined relative to counting beads by flow cytometry. The K7153A antibody and the IMGN529 conjugate efficiently and specifically depleted B-cells in a dose-dependent manner in the context of purified PBMCs and whole blood. With purified PBMCs, both K7153A and IMGN529 caused 50–60% depletion of B cells, with little to no depletion of T cells or monocytes. IMGN529 was more potent than rituximab, which led to 30–40% B-cell depletion, or TRU-016, which caused 20–30% B-cell depletion. IMGN529 also was more specific than alemtuzumab, which depleted T-cells and monocytes as well as B cells. With whole blood samples, both K7153A and IMGN529 resulted in 30–40% B-cell depletion with no effect on T cells, NK cells or monocytes. IMGN529 was again more potent than rituximab or TRU-016, which caused approximately 10% B-cell depletion, and was more specific than alemtuzumab, which depleted the majority of T cells in addition to 40% of B cells. IMGN529 embodies a unique B-cell targeted agent as it combines the intrinsic pro-apoptotic, CDC and ADCC activities of its anti-CD37 antibody component with the potent cytotoxic mechanism provided by the targeted delivery of its maytansinoid payload. It is highly active in vitro and in vivo against B-cell lymphoma and CLL cell lines. In addition, it mediates specific B-cell depletion in vitro that is greater than B-cell depletion by CD20-directed rituximab. Together, these findings indicate that IMGN529 is a promising therapeutic candidate for the treatment of B-cell malignancies. Disclosures: Deckert: ImmunoGen, Inc.: Employment. Chicklas:ImmunoGen, Inc.: Employment. Yi:ImmunoGen, Inc.: Employment. Li:ImmunoGen, Inc.: Employment. Pinkas:ImmunoGen, Inc.: Employment. Chittenden:ImmunoGen, Inc.: Employment. Lutz:ImmunoGen, Inc.: Employment. Park:ImmunoGen, Inc.: Employment.

Steady-state generation of mucosal IgA+ plasmablasts is not abrogated by B-cell depletion therapy with rituximab

Blood ◽  
2010 ◽  
Vol 116 (24) ◽  
pp. 5181-5190 ◽  
Author(s):  
Henrik E. Mei ◽  
Daniela Frölich ◽  
Claudia Giesecke ◽  
Christoph Loddenkemper ◽  
Karin Reiter ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Steady State ◽  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Plasma Cells ◽  
Cell Depletion ◽  
B Cell Depletion ◽  
Ki 67

AbstractThe anti-CD20 antibody rituximab depletes human B cells from peripheral blood, but it remains controversial to what extent tissue-resident B cells are affected. In representative patients with rheumatoid arthritis, we here demonstrate that recently activated presumably short-lived plasmablasts expressing HLA-DRhigh and Ki-67 continuously circulate in peripheral blood after B-cell depletion by rituximab at 26%-119% of their initial numbers. They circulate independent of splenectomy, express immunoglobulin A (IgA), β7 integrin, and C-C motif receptor 10 (CCR10) and migrate along CCL28 gradients in vitro, suggesting their mucosal origin. These plasmablasts express somatically hypermutated VH gene rearrangements and spontaneously secrete IgA, exhibiting binding to microbial antigens. Notably, IgA+ plasmablasts and plasma cells were identified in the lamina propria of patients treated with rituximab during peripheral B-cell depletion. Although a relation of these “steady state”–like plasmablasts with rheumatoid arthritis activity could not be found, their persistence during B-cell depletion indicates that their precursors, that is, B cells resident in the mucosa are not deleted by this treatment. These data suggest that a population of mucosal B cells is self-sufficient in adult humans and not replenished by CD20+ B cells immigrating from blood, lymphoid tissue, or bone marrow, that is, B cells depleted by rituximab.

Influence of Bacillus subtilis C-3102 on microbiota in a dynamic in vitro model of the gastrointestinal tract simulating human conditions

2012 ◽  
Vol 3 (3) ◽  
pp. 229-236 ◽  
Author(s):  
M. Hatanaka ◽  
Y. Nakamura ◽  
A.J.H. Maathuis ◽  
K. Venema ◽  
I. Murota ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Gastrointestinal Tract ◽  
In Vitro Model ◽  
Germination Rate ◽  
Human Colon ◽  
Micro Array ◽  
Vitro Model ◽  
Oral Doses ◽  
The Impact

Survival and germination rate of Bacillus subtilis C-3102 spores were investigated in a stomach and small intestine model (TIM-1), while the impact of C-3102 cells that had passed through TIM-1 on human colon microbiota was evaluated in a model of the large intestine (TIM-2). The survival of C-3102 spores in TIM-1 was 99%; 8% of the spores had germinated. Effluent of TIM-1 was subsequently introduced into TIM-2 and a micro-array platform was employed to assess changes in the microbiota composition. The effluent, which contained germinated C-3102 cells, increased some Bifidobacterium species and decreased some Clostridium groups. These changes were greater compared to those obtained by adding C-3102 spores directly to TIM-2. The present study suggests that oral doses of B. subtilis C-3102 spores have the potential to modulate the human colon microbiota. This effect may be caused by germination of the spores in the gastrointestinal tract.

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