Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance
Ontology highlight
ABSTRACT:
SUBMITTER:
PROVIDER: S-EPMC11240353 | biostudies-literature | 2024 Jun
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Recent clinical data indicate a synergistic therapeutic effect between trastuzumab and taxanes in neoadjuvantly treated HER2-positive breast cancer (BC) patients. In HER2+ BC experimental models and patients, we investigated whether this synergy depends on the ability of drug-induced stress to improve NK cell effectiveness and thus trastuzumab-mediated ADCC. HER2+ BC cell lines BT474 and MDAMB361 treated with docetaxel showed up-modulation of NK activator ligands both in vitro and in vivo, accompanied by a 15-40% increase in in vitro trastuzumab-mediated ADCC; antibodies blocking the NKG2D receptor significantly reduced this enhancement. NKG2D receptor expression was increased by docetaxel treatment in circulating and splenic NK cells from mice xenografted with tumor cells, an increase related to expansion of the CD11b+Ly6G+ cell population. Accordingly, NK cells derived from HER2+ BC patients after treatment with taxane-containing therapy expressed higher levels of NKG2D receptor than before treatment. Moreover, plasma obtained from these patients recapitulated the modulation of NKG2D on healthy donors' NK cells, improving their trastuzumab-mediated activity in vitro. This enhancement occurred mainly using plasma from patients with low NKG2D basal expression. Our results indicate that taxanes increase tumor susceptibility to ADCC by acting on tumor and NK cells, and suggest that taxanes concomitantly administered with trastuzumab could maximize the antibody effect, especially in patients with low basal immune effector cytotoxic activity.
| S-EPMC4807996 | biostudies-literature
Project description:Freshly isolated PBMC are broadly used as effector cells in functional assays that evaluate antibody-dependent cell mediated cytotoxicity (ADCC) and NK activity; however, they introduce natural-individual donor-to-donor variability. Cryopreserved PBMC provide a more consistent source of effectors than fresh cells in cytotoxicity assays. Our objective was to determine the effects of cryopreservation of effector PBMC on cell frequency, and on the magnitude and specificity of ADCC and NK activity. Fresh, frozen/overnight rested and frozen/not rested PBMC were used as effector cells in (51)Cr-release and CD107a degranulation assays. Frozen/overnight rested PBMC had higher ADCC and NK activity in both assays when compared to fresh PBMC; however, when using frozen/not rested PBMC, ADCC and NK activities were significantly lower than fresh PBMC. Background CD107a degranulation in the absence of target cell stimulation was greater in PBMC that were frozen/not rested when compared to fresh PBMC or PBMC that were frozen overnight and rested. The percentages of CD16(+)CD56(dim) NK cells and CD14(+) monocytes were lower in PBMC that were frozen and rested overnight than in fresh PBMC. CD16 expression on CD56(dim) NK cells was similar for all PBMC treatments. PBMC that were frozen and rested overnight were comparable to fresh PBMC effectors. PBMC that were frozen and used immediately when evaluating ADCC or NK activity using either a (51)Cr-release assay or a CD107a degranulation assay had the lowest activity. Clinical studies of antibodies that mediate ADCC would benefit from using effector cells that have been frozen, thawed and rested overnight prior to assay.
| S-EPMC4029863 | biostudies-literature
Project description:Cell monolayers underpin the discovery and screening of new drugs and allow for fundamental studies of cell biology and disease. However, current cryopreservation technologies do not allow cells to be stored frozen while attached to tissue culture plastic. Hence, cells must be thawed from suspension, cultured for several days or weeks, and finally transferred into multiwell plates for the desired application. This inefficient process consumes significant time handling cells, rather than conducting biomedical research or other value-adding activities. Here, we demonstrate that a synthetic macromolecular cryoprotectant enables the routine, reproducible, and robust cryopreservation of biomedically important cell monolayers, within industry-standard tissue culture multiwell plates. The cells are simply thawed with media and placed in an incubator ready to use within 24 h. Post-thaw cell recovery values were >80% across three cell lines with low well-to-well variance. The cryopreserved cells retained healthy morphology, membrane integrity, proliferative capacity, and metabolic activity; showed marginal increases in apoptotic cells; and responded well to a toxicological challenge using doxorubicin. These discoveries confirm that the cells are "assay-ready" 24 h after thaw. Overall, we show that macromolecular cryoprotectants can address a long-standing cryobiological challenge and offers the potential to transform routine cell culture for biomedical discovery.
| S-EPMC9472225 | biostudies-literature
Project description:A biosimilar product needs to demonstrate biosimilarity to the originator reference product, and the quality profile of the latter should be monitored throughout the period of the biosimilar's development to match the quality attributes of the 2 products that relate to efficacy and safety. For the development of a biosimilar version of trastuzumab, the reference product, Herceptin®, was extensively characterized for the main physicochemical and biologic properties by standard or state-of-the-art analytical methods, using multiple lots expiring between March 2015 and December 2019. For lots with expiry dates up to July 2018, a high degree of consistency was observed for all the tested properties. However, among the lots expiring in August 2018 or later, a downward drift was observed in %afucose (G0+G1+G2). Furthermore, the upward drift of %high mannose (M5+M6) was observed in the lots with expiry dates from June 2019 to December 2019. As a result, the combination of %afucose and %high mannose showed 2 marked drifts in the lots with expiry dates from August 2018 to December 2019, which was supported by the similar trend of biologic data, such as FcγRIIIa binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity. Considering that ADCC is one of the clinically relevant mechanisms of action for trastuzumab, the levels of %afucose and %high mannose should be tightly monitored as critical quality attributes for biosimilar development of trastuzumab.
| S-EPMC5419076 | biostudies-literature
Project description:Ebolaviruses are highly virulent pathogens that cause Ebola viral disease (EVD). Data from non-human primate (NHP) models and from human survivors of EVD suggest that anti-Ebola antibodies play an integral role in protection. Antibody-dependent cell-mediated cytotoxicity (ADCC) is a potential mechanism through which anti-Ebola antibodies may mediate protection. We developed a robust Ebola-specific ADCC assay for use in ongoing trials of Ebola vaccines. Stable cell lines for inducible Zaire ebolavirus glycoprotein (EBOV GP) expression were developed to provide a uniform source of target cells in the assay, and were combined with an existing human natural killer (NK) cell line as the effector cell. When applied to commercially available anti-EBOV GP monoclonal antibodies, the assay clearly differentiated antibody with high ADCC activity from those with low or no ADCC activity. Anti-EBOV ADCC activity was also detected in plasma samples from rhesus macaques immunized with a candidate Ebola vaccine. The Ebola ADCC assay reported here will be a useful tool in studying the functionality of anti-EBOV GP antibodies elicited by Ebola vaccines in ongoing and future clinical trials.
| S-EPMC8451526 | biostudies-literature
Project description:ObjectivesSynovial mesenchymal stem cells (MSCs) have high freeze-thaw tolerance, whereas human umbilical vein endothelial cells (HUVECs) have low freezing tolerance. The differences in cell type-specific freeze-thaw tolerance and the mechanisms involved are unclear. This study thus aimed to identify the biological and physical factors involved in the differences in freeze-thaw tolerance between MSCs and HUVECs.Materials and methodsFor biological analysis, MSC and HUVEC viability after freeze-thawing and alteration of gene expression in response to dimethyl sulfoxide (DMSO, a cryoprotectant) were quantitatively evaluated. For physical analysis, the cell membrane fluidity of MSCs and HUVECs before and after DMSO addition was assessed using a histogram for generalized polarization frequency.ResultsHUVECs showed lower live cell rates and higher gene expression alteration related to extracellular vesicles in response to DMSO than MSCs. Fluidity measurements revealed that the HUVEC membrane was highly fluidic and sensitive to DMSO compared to that of MSCs. Addition of CAY10566, an inhibitor of stearoyl-coA desaturase (SCD1) that produces highly fluidic desaturated fatty acids, decreased the fluidity of HUVECs and increased their tolerance to DMSO. The combination of CAY10566 and antioxidant glutathione (GSH) treatment improved HUVEC viability from 57 to 69%. Membrane fluidity alteration may thus contribute to pore-induced DMSO influx into the cytoplasm and reactive oxygen species production, leading to greater cytotoxicity in HUVECs, which have low antioxidant capacity.ConclusionsDifferences in freeze-thaw tolerance originate from differences in the cell membranes with respect to fluidity and antioxidant capacity. These findings provide a basis for analyzing cell biology and membrane-physics to establish appropriate long-term preservation methods aimed at promoting transplantation therapies.
| S-EPMC9066911 | biostudies-literature
Project description:BackgroundTranscriptional enhancers are essential for gene regulation, but how these regulatory elements are best defined remains a significant unresolved question. Traditional definitions rely on activity-based criteria such as reporter gene assays, while more recently, biochemical assays based on chromatin-level phenomena such as chromatin accessibility, histone modifications, and localized RNA transcription have gained prominence.ResultsWe examine here whether these two types of definitions, activity-based and chromatin-based, effectively identify the same sets of sequences. We find that, concerningly, the overlap between the two groups is strikingly limited. Few of the data sets we compared displayed statistically significant overlap, and even for those, the degree of overlap was typically small (below 40% of sequences). Moreover, a substantial batch effect was observed in which experiment set rather than experimental method was a primary driver of whether or not chromatin-defined enhancers showed a strong overlap with reporter gene-defined enhancers.ConclusionsOur results raise important questions as to the appropriateness of both old and new enhancer definitions, and suggest that new approaches are required to reconcile the poor agreement among existing methods for defining enhancers.
| S-EPMC9837977 | biostudies-literature
Project description:Trastuzumab has improved survival rates in human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC), but drug resistance leads to treatment failure. Natural killer (NK) cell-mediated antibody-dependent cell cytotoxicity (ADCC) represents an essential antitumor immune mechanism of trastuzumab. Traditional Chinese medicine (TCM) has been used for centuries to treat diseases because of its capacity to improve immune responses. Xianling Lianxia formula (XLLXF), based on the principle of "strengthening body and eliminating toxin", exhibits a synergistic effect in the trastuzumab treatment of patients with HER2-positive BC. Notably, this synergistic effect of XLLXF was executed by enhancing NK cells and ADCC, as demonstrated through in vitro co-culture of NK cells and BC cells and in vivo intervention experiments. Mechanistically, the augmented impact of XLLXF on NK cells is linked to a decrease in cytokine inducible Src homology 2 (SH2) containing protein (CISH) expression, which in turn activates the Janus kinase 1 (JAK1)/signal transducer and activator of transcription 5 (STAT5) pathway. Collectively, these findings suggested that XLLXF holds promise for enhancing NK cell function and sensitizing patients with HER2-positive BC to trastuzumab.
| S-EPMC11531627 | biostudies-literature
Project description:Ovarian cancer is the deadliest gynecologic cancer. Although human epidermal growth factor receptor-2 (HER2) overexpression, a poor prognostic molecular marker in ovarian cancer, is found in almost 30% of ovarian cancer cases, there are no established therapies for HER2-overexpressing ovarian cancer. In this study, we investigated the efficacy of combined samfenet, a biosimilar compound of trastuzumab, and natural killer (NK) cells in preclinical model of HER2-overexpressing ovarian cancer. Firstly, we screened the HER2 expression in three ovarian cancer cell lines and eight ovarian cancer patient-derived tumor xenograft (PDTX) samples. Then, immunohistochemistry and silver in situ hybridization (SISH) were performed following clinical criteria. HER2-overexpressing cells exhibited the highest sensitivity to samfenet compared with low-HER2-expressing cells. In addition, the combination of samfenet with natural killer (NK) cells resulted in significantly enhanced sensitivity to HER2-overexpressing cells and showed a significant antitumor effect on PDTX mice compared with monotherapy. It is known that anti-HER2-humanized IgG1 monoclonal antibodies, including trastuzumab, induce antibody-dependent cellular cytotoxicity (ADCC). Consequently, the combination of samfenet with NK cells demonstrated NK cell-mediated ADCC, as confirmed using an in vitro NK cytotoxicity assay and in vivo antitumor efficacy. A transferase dUTP nick end labeling (TUNEL) assay using xenografted tumors further supported the ADCC effects based on the increase in the number of apoptotic cells in the combination group. Furthermore, high HER2 expression was associated with shorter progression-free survival and overall survival based on public mRNA expression data. In this study, we demonstrated that the combination of samfenet and NK cell therapy could be a promising treatment strategy for patients with HER2-overexpressing ovarian cancer, through ADCC effects. Therefore, this study supports a rationale for further clinical studies of the combination of samfenet and NK cells as a therapy for patients with HER2-overexpressing ovarian cancer.
| S-EPMC11545925 | biostudies-literature
Project description:Flow focusing is an important hydrodynamic technique for cytometric analysis, enabling the rapid study of cellular samples to identify a variety of biological processes. To date, the majority of flow-focusing devices are fabricated using conventional photolithography or flame processing of glass capillaries. This article presents a suite of low-cost, millifluidic, flow-focusing devices that were fabricated using a desktop sterolithgraphy (SLA) 3D printer. The suite of SLA printing strategies consists of a monolithic SLA method and a hybrid molding process. In the monolithic SLA approach, 1.3 mm square millifluidic channels were printed as a single piece. The printed device does not require any post processing, such as bonding or surface polishing for optical access. The hybrid molding approach consists of printing a mold using the SLA 3D printer. The mold is treated to an extended UV exposure and oven baked before using PDMS as the molding material for the channel. To demonstrate the viability of these channels, we performed a series of experiments using several flow-rate ratios to show the range of focusing widths that can be achieved in these devices. The experiments are validated using a numerical model developed in ANSYS.
| S-EPMC10383660 | biostudies-literature