scholarly journals A Novel System for the Quantification of the ADCC Activity of Therapeutic Antibodies

2017 ◽  
Vol 2017 ◽  
pp. 1-19 ◽  
Author(s):  
Christophe Lallemand ◽  
Feifei Liang ◽  
Flore Staub ◽  
Maud Simansour ◽  
Benoit Vallette ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Matrix Effects ◽  
Specific Antigen ◽  
Firefly Luciferase ◽  
Cell Number ◽  
Target Cells ◽  
The Novel ◽  
Adcc Activity ◽  
Effector Cells ◽  
High Level

Novel ADCC effector cells expressing the V-variant or F-variant of FcγRIIIa (CD16a) and firefly luciferase under the control of a chimeric promoter incorporating recognition sequences for the principal transcription factors involved in FcγRIIIa signal transduction, together with novel target cells overexpressing a constant high level of the specific antigen recognized by rituximab, trastuzumab, cetuximab, infliximab, adalimumab, or etanercept, confer improved sensitivity, specificity, and dynamic range in an ADCC assay relative to effector cells expressing a NFAT-regulated reporter gene and wild-type target cells. The effector cells also contain a normalization gene rendering ADCC assays independent of cell number or serum matrix effects. The novel effector and target cells in a frozen thaw-and-use format exhibit low vial-to-vial and lot-to-lot variation in their performance characteristics reflected by CVs of 10% or less. Homologous control target cells in which the specific target gene has been invalidated by genome editing providing an ideal control and a means of correcting for nonspecific effects were observed with certain samples of human serum. The novel effector cells and target cells expressing noncleavable membrane-bound TNFα have been used to quantify ADCC activity in serum from patients with Crohn’s disease treated with infliximab and to relate ADCC activity to drug levels.

A Non-Internalizing Anti-CD40 Antibody, CHIR-12.12, Blocks CD40L-Induced Cytokine Production and Mediates Greater ADCC Than Rituximab in Primary CLL Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2964-2964
Author(s):  
Xia Tong ◽  
Sharon Lea Aukerman ◽  
Karen Lin ◽  
Natasha Aziz ◽  
Cheryl Goldbeck ◽  
...  
Keyword(s):  
Cell Surface ◽  
B Cell ◽  
Cytokine Production ◽  
Ex Vivo ◽  
Lymphocytic Leukemia ◽  
Target Cells ◽  
Adcc Activity ◽  
Effector Cells ◽  
Cells Cultured

Abstract CD40 is expressed on chronic lymphocytic leukemia (CLL) cells, and CD40 activation leads to signaling critical for cell survival and proliferation. We have previously described a novel, fully human IgG1 anti-CD40 antagonistic monoclonal antibody, CHIR-12.12, generated in XenoMouse® mice (Abgenix, Inc.), and have demonstrated that it inhibits normal human B cell proliferation and survival and mediates potent antibody-dependent cellular cytotoxicity (ADCC) against primary CLL and non-Hodgkin’s lymphoma cells. In this study, we examined the ability of CHIR-12.12 to modulate cytokine production by primary CLL cells and compared the ADCC activity of CHIR-12.12 with rituximab against primary CLL cells. Primary CLL cells stimulated with CD40L produced a variety of cytokines, including IL-10, TNF-α , IL-8, GM-CSF, IL-6, MCP-1, and MIP-1β. Addition of CHIR-12.12 to primary CLL cells inhibited CD40L-mediated production of these cytokines. Cytokine production by primary CLL cells cultured with CHIR-12.12 alone in the absence of CD40L did not exceed levels produced by CLL cells cultured in medium. These data suggest that CHIR-12.12 is a potent antagonist for CD40L-mediated cytokine production by primary CLL cells and shows no agonistic activity by itself. We next compared the relative ADCC activity of CHIR-12.12 and rituximab against ex vivo primary CLL cells from 8 patients. CHIR-12.12 exhibited greater ADCC than rituximab against CLL cells from all patients. The average percent of maximum lysis by CHIR-12.12 and rituximab were 49 ± 16% and 31 ± 14%, respectively. CHIR-12.12 was greater than 10-fold more potent than rituximab, as measured by ED50 values (14.1 pM versus 155.5 pM, respectively). Quantitative CD20 and CD40 density on CLL cells and the degree of antibody internalization were investigated as potential reasons for the difference in ADCC activity. The greater ADCC potency and efficacy of CHIR-12.12 was not dependent on a higher density of cell surface CD40 molecules, as there were 1.3 to 14-fold higher numbers of CD20 than CD40 molecules on the cell surface. Antibody internalization studies using primary CLL cells conducted by flow cytometry and confocal microscopy show that upon binding to CD40 at 37°C, CHIR-12.12 remains uniformly distributed on the cell surface, even after 3 hours. In contrast, after binding at 37°C, rituximab is redistributed into caps and internalized. These data suggest that the potent ADCC activity of CHIR-12.12 may be partly related to its ability to remain on the surface of target cells uniformly, allowing optimal interaction with effector cells. Taken together, these results suggest that CHIR-12.12 may be effective at mediating potent ADCC against CLL cells in vivo. CHIR-12.12 is currently in Phase I trials for B-cell malignancies.

Humanized Anti-HM1.24 Antibody Mediates Myeloma Cell Cytotoxicity That Is Enhanced by Cytokine Stimulation of Effector Cells

Blood ◽  
1999 ◽  
Vol 93 (11) ◽  
pp. 3922-3930 ◽  
Author(s):  
Shuji Ozaki ◽  
Masaaki Kosaka ◽  
Yuji Wakahara ◽  
Yasuko Ozaki ◽  
Masayuki Tsuchiya ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Severe Combined Immunodeficiency ◽  
Interleukin 2 ◽  
Specific Antigen ◽  
Myeloma Cell ◽  
Adcc Activity ◽  
Myeloma Cells ◽  
Effector Cells

To develop a new immunotherapy for multiple myeloma, we have generated a monoclonal antibody (MoAb) that detects a human plasma cell-specific antigen, HM1.24. Our previous study has shown that mouse anti-HM1.24 MoAb inhibits the proliferation of human myeloma cells implanted into severe combined immunodeficiency mice. In this report, we evaluated the antitumor activity of the humanized anti-HM1.24 MoAb (IgG1κ), which was constructed by grafting the complementarity-determining regions. In contrast to the parent mouse MoAb, humanized anti-HM1.24 MoAb mediated antibody-dependent cellular cytotoxicity (ADCC) against both myeloma cell lines and myeloma cells from patients in the presence of human peripheral blood mononuclear cells (PBMCs). The PBMCs from untreated myeloma patients exhibited ADCC activity as efficiently as those of healthy donors. Although decreased ADCC activity of PBMCs was observed in patients who responded poorly to conventional chemotherapy, it could be significantly augmented by the stimulation with interleukin-2 (IL-2), IL-12, or IL-15. There was a strong correlation between the percentage of CD16+ cells and ADCC activity in the PBMCs of myeloma patients. Moreover, peripheral blood stem cell collections from myeloma patients contained higher numbers of CD16+ cells than PBMCs and exhibited ADCC activity that was enhanced by IL-2. These results indicate that humanized anti-HM1.24 MoAb has potential as a new therapeutic strategy in multiple myeloma and that treatment of effector cells with immunomodulating cytokines can restore the effect of humanized anti-HM1.24 MoAb in patients with diminished ADCC activity.

Humanized Anti-HM1.24 Antibody Mediates Myeloma Cell Cytotoxicity That Is Enhanced by Cytokine Stimulation of Effector Cells

Blood ◽  
1999 ◽  
Vol 93 (11) ◽  
pp. 3922-3930 ◽  
Author(s):  
Shuji Ozaki ◽  
Masaaki Kosaka ◽  
Yuji Wakahara ◽  
Yasuko Ozaki ◽  
Masayuki Tsuchiya ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Severe Combined Immunodeficiency ◽  
Interleukin 2 ◽  
Specific Antigen ◽  
Myeloma Cell ◽  
Adcc Activity ◽  
Myeloma Cells ◽  
Effector Cells

Abstract To develop a new immunotherapy for multiple myeloma, we have generated a monoclonal antibody (MoAb) that detects a human plasma cell-specific antigen, HM1.24. Our previous study has shown that mouse anti-HM1.24 MoAb inhibits the proliferation of human myeloma cells implanted into severe combined immunodeficiency mice. In this report, we evaluated the antitumor activity of the humanized anti-HM1.24 MoAb (IgG1κ), which was constructed by grafting the complementarity-determining regions. In contrast to the parent mouse MoAb, humanized anti-HM1.24 MoAb mediated antibody-dependent cellular cytotoxicity (ADCC) against both myeloma cell lines and myeloma cells from patients in the presence of human peripheral blood mononuclear cells (PBMCs). The PBMCs from untreated myeloma patients exhibited ADCC activity as efficiently as those of healthy donors. Although decreased ADCC activity of PBMCs was observed in patients who responded poorly to conventional chemotherapy, it could be significantly augmented by the stimulation with interleukin-2 (IL-2), IL-12, or IL-15. There was a strong correlation between the percentage of CD16+ cells and ADCC activity in the PBMCs of myeloma patients. Moreover, peripheral blood stem cell collections from myeloma patients contained higher numbers of CD16+ cells than PBMCs and exhibited ADCC activity that was enhanced by IL-2. These results indicate that humanized anti-HM1.24 MoAb has potential as a new therapeutic strategy in multiple myeloma and that treatment of effector cells with immunomodulating cytokines can restore the effect of humanized anti-HM1.24 MoAb in patients with diminished ADCC activity.

scholarly journals A luciferase transgenic mouse model: visualization of prostate development and its androgen responsiveness in live animals

2005 ◽  
Vol 35 (2) ◽  
pp. 293-304 ◽  
Author(s):  
C-L Hsieh ◽  
Z Xie ◽  
Z-Y Liu ◽  
J E Green ◽  
W D Martin ◽  
...  
Keyword(s):  
Mouse Model ◽  
Luciferase Activity ◽  
Prostate Gland ◽  
Specific Antigen ◽  
Firefly Luciferase ◽  
Prostate Development ◽  
Highly Active ◽  
Mouse Prostate ◽  
Imaging Results ◽  
High Level

Numerous mouse models of prostate carcinogenesis have been developed, but hitherto there has been no model in which the prostate gland could be imaged in live animals. The transgenic model generated here targeted mouse prostate gland using a firefly luciferase enzyme under the control of a small but highly active and specific supra prostate-specific antigen (sPSA) promoter. We evaluated postnatal prostate development, involution and androgen-induced restoration of prostate growth in adult transgenic mice using bioluminescence imaging. Results of our study showed that: (i) the prostate gland of male offspring did not yield a significant bioluminescence signal until after sexual maturity. Luciferase was detected in the luminal epithelial cells of the ventral and dorsolateral lobes of the prostate gland and caput epididymis, with little or no activity in 18 other organs evaluated. (ii) While a constant high level of bioluminescence was detected in the mouse prostate from 5 to 35 weeks of age, a slight drop in bioluminescence was detected at 36 to 54 weeks. (iii) Upon castration, the luciferase activity signal associated with mouse prostate detected by a cooled charge-coupled device camera was dramatically reduced. This signal could be rapidly restored to pre-castration levels after androgen administration. Androgen-induced luciferase activity subsided to nearly basal levels 5 days following the last injection. These data demonstrate that a bioluminescent mouse model with luciferase activity restricted to the prostate gland under the control of a (sPSA) promoter can be used on a real-time basis in live animals to investigate the development and responsiveness of the prostate gland to exogenously administered androgen. This model can be extended to detect the responsiveness of the prostate gland to therapy and used as a founder strain to visualize tumors in hosts with different genetic backgrounds.

scholarly journals Immune Reactions Against Elongation Factor 2 Kinase: Specific Pathogenesis of Gastric Ulcer fromHelicobacter pyloriInfection

2009 ◽  
Vol 2009 ◽  
pp. 1-10 ◽  
Author(s):  
Kiyoshi Ayada ◽  
Kenji Yokota ◽  
Yoshiro Kawahara ◽  
Yumiko Yamamoto ◽  
Kazuyuki Hirai ◽  
...  
Keyword(s):  
Cytotoxic T Lymphocyte ◽  
Specific Antigen ◽  
Elongation Factor ◽  
Causative Factor ◽  
Gastric Ulcers ◽  
Target Cells ◽  
Effector Cells ◽  
Elongation Factor 2 ◽  
H Pylori

Helicobacter pylori(H. pylori) infection is a definite causative factor for gastric ulcers (GUs). In the present study we detected a specific antigen of gastric epithelial cells (HGC-27) using cell ELISA, which was recognized by the sera of GU patients (n=20) but not in patients with chronic gastritis (CG;n=20) or in healthy volunteers (HC;n=10). This antigen was over-expressed by a stressful (heat-stressed) environment, and was identified as elongation factor 2 kinase (EF-2K) by western blotting. The GU patients' lymphocytes stimulated byH. pylorispecifically disrupted heat-stressed HGC-27 cells in a cytotoxic assay. In flow cytometry, the effector cells (lymphocytes) from GU patients were significantly differentiated to T helper type 1 lymphocyte (Th1) and cytotoxic T lymphocyte (CTL) as opposed to those from CG patients. The target cells (HGC-27) expressed EF-2K and MHC-class I together with costimulatory molecules from heat stress. This antigen specific immune mechanism could have a prominent role in the pathogenesis of GU.

Damping characteristics of a magneto-rheological damper with dual independent controllable ducts

2021 ◽  
pp. 1045389X2110188
Author(s):  
Jianqiang Yu ◽  
Xiaomin Dong ◽  
Tao Wang ◽  
Zhengmu Zhou ◽  
Yaqin Zhou
Keyword(s):  
Dynamic Range ◽  
Fluid Flows ◽  
Mr Damper ◽  
Viscous Damping ◽  
The Novel ◽  
The Finite Element Method ◽  
Damping Force ◽  
Damping Characteristics ◽  
Magneto Rheological ◽  
The Mathematical Model

This paper presents the damping characteristics of a linear magneto-rheological (MR) damper with dual controllable ducts based on numerical and experimental analysis. The novel MR damper consisting of a dual-rod cylinder system and a MR valve is used to reduce the influences of viscous damping force and improve dynamic range. Driven by the dual-rod cylinder system, MR fluid flows in the MR valve. The pressure drop of the MR valve with dual independent controllable ducts can be controlled by tuning the current of two independent coils. Based on the mathematical model and the finite element method, the damping characteristics of the MR damper is simulated. A prototype is designed and tested on MTS machine to evaluate its damping characteristics. The results show that the working states and damping force of the MR damper can be controlled by the two independent coils.

scholarly journals NKG2D Natural Killer Cell Receptor—A Short Description and Potential Clinical Applications

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1420
Author(s):  
Jagoda Siemaszko ◽  
Aleksandra Marzec-Przyszlak ◽  
Katarzyna Bogunia-Kubik
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Bacterial Infections ◽  
Killer Cell ◽  
Cell Receptor ◽  
Target Cells ◽  
Effector Cells ◽  
Stressed Cells ◽  
The Right ◽  
Nkg2d Receptor

Natural Killer (NK) cells are natural cytotoxic, effector cells of the innate immune system. They can recognize transformed or infected cells. NK cells are armed with a set of activating and inhibitory receptors which are able to bind to their ligands on target cells. The right balance between expression and activation of those receptors is fundamental for the proper functionality of NK cells. One of the best known activating receptors is NKG2D, a member of the CD94/NKG2 family. Due to a specific NKG2D binding with its eight different ligands, which are overexpressed in transformed, infected and stressed cells, NK cells are able to recognize and attack their targets. The NKG2D receptor has an enormous significance in various, autoimmune diseases, viral and bacterial infections as well as for transplantation outcomes and complications. This review focuses on the NKG2D receptor, the mechanism of its action, clinical relevance of its gene polymorphisms and a potential application in various clinical settings.

Role of NKG2D signaling in the cytotoxicity of activated and expanded CD8+ T cells

Blood ◽  
2004 ◽  
Vol 103 (8) ◽  
pp. 3065-3072 ◽  
Author(s):  
Michael R. Verneris ◽  
Mobin Karami ◽  
Jeanette Baker ◽  
Anishka Jayaswal ◽  
Robert S. Negrin
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Malignant Cell ◽  
Leukemic Cells ◽  
Target Cells ◽  
High Dose ◽  
Effector Cells ◽  
Nkg2d Expression

Abstract Activating and expanding T cells using T-cell receptor (TCR) cross-linking antibodies and interleukin 2 (IL-2) results in potent cytotoxic effector cells capable of recognizing a broad range of malignant cell targets, including autologous leukemic cells. The mechanism of target cell recognition has previously been unknown. Recent studies show that ligation of NKG2D on natural killer (NK) cells directly induces cytotoxicity, whereas on T cells it costimulates TCR signaling. Here we demonstrate that NKG2D expression is up-regulated upon activation and expansion of human CD8+ T cells. Antibody blocking, redirected cytolysis, and small interfering RNA (siRNA) studies using purified CD8+ T cells demonstrate that cytotoxicity against malignant target cells occurs through NKG2D-mediated recognition and signaling and not through the TCR. Activated and expanded CD8+ T cells develop cytotoxicity after 10 to 14 days of culture, coincident with the expression of the adapter protein DAP10. T cells activated and expanded in low (30 U/mL) and high (300 U/mL) concentrations of IL-2 both up-regulated NKG2D expression equally, but only cells cultured in high-dose IL-2 expressed DAP10 and were cytotoxic. Collectively these results establish that NKG2D triggering accounts for the majority of major histocompatibility complex (MHC)–unrestricted cytotoxicity of activated and expanded CD8+ T cells, likely through DAP10-mediated signaling. (Blood. 2004;103: 3065-3072)

scholarly journals Extracellular cytolysis by activated macrophages and granulocytes. I. Pharmacologic triggering of effector cells and the release of hydrogen peroxide.

1979 ◽  
Vol 149 (1) ◽  
pp. 84-99 ◽  
Author(s):  
C F Nathan ◽  
L H Brukner ◽  
S C Silverstein ◽  
Z A Cohn
Keyword(s):  
Trypan Blue ◽  
Response Curve ◽  
Target Cells ◽  
Activated Macrophages ◽  
Lymphoma Cells ◽  
Effector Cells ◽  
Proteose Peptone ◽  
Standard Assay ◽  
Different Types ◽  
J774 Cells

Lymphoma cells were rapidly lysed by activated macrophages and granulocytes in the presence of PMA. Release of 51Cr from lymphoma cells correlated closely with their destruction as viewed by scanning electron microscopy, and with reduction in the number of trypan blue-excluding cells. The standard assay involved 51 Cr release measured at 4.5 h, but injury appeared to be complete in 1 h. Of eight different types of effector cells tested, only those releasing abundant H2O2 in response to PMA were effective, that, is BCG-, C. parvum-, or casein-activated macrophages, or thioglycollate-elicited granulocytes. Normal macrophages, J774 cells, or macrophages elicited with thioglycollate broth or proteose-peptone were ineffective. BCG-activated macrophages and granulocytes caused 50% specific release of 51Cr from P388 lymphoma cells at E:T ratios between 1.4 and 4.5, and from mouse erythrocytes at E:T ratios of 0.017 to 0.025. 10 types of target cells varied widely in their susceptibility to lysis by reagent H2O2, with one-half maximal lysis occurring at H2O2 concentrations ranging from 3.63 X 10(-6) M to 3.85 X 10(-5) M. Effector cells were expected to generate approximately that much H2O2 during the period of injury. Susceptibility of the target cells to lysis by PMA-triggered granulocytes correlated closely with their sensitivity to H2O2 (r = 0.98). The membrane-active agents LPS and digitonin, which did not trigger H2O2 release, did not trigger cytotoxicity. The dose-response curve for triggering of H2O2 release by PMA was identical to that for triggering cytotoxicity. These results provided strong circumstantial evidence for the importance of H2O2 in extracellular cytolysis by activated macrophages and granulocytes when pharmacologically triggered.

scholarly journals Cell-mediated lympholysis to H-2-matched target cells modified with a series of nitrophenyl compounds.

1976 ◽  
Vol 144 (4) ◽  
pp. 1134-1140 ◽  
Author(s):  
T G Rehn ◽  
J K Inman ◽  
G M Shearer
Keyword(s):  
T Cell ◽  
Target Cells ◽  
Cell Recognition ◽  
Receptor Model ◽  
Spleen Cells ◽  
Effector Cells ◽  
T Cell Recognition ◽  
Cytotoxic Effector

The specificity of C57BL/10 cytotoxic effector cells generated by in vitro sensitization with autologous spleen cells modified with a series of related nitrophenyl compounds was investigated. The failure of trinitrophenyl (TNP)-sensitized effector cells to lyse TNP-beta-alanylglycylglycyl(AGG)-modified target cells is presented as evidence contradicting the intimacy or dual receptor model or T-cell recognition in its simplest form. Data are also shown indicating that sensitization with N-(3-nitro-4-hydroxy-5-iodophenylacetyl)-AGG-modified stimulating cells generates noncross-reacting clones of cytotoxic effector cells.

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