To develop a method for determining the antibody-dependent cell-mediated phagocytosis (ADCP) activity of human epidermal growth factor receptor 2 (HER2)-targeted antibody drug conjugate (ADC) based on the reporter gene assay, we established an ADCP activity assay with Jurkat/NFAT/FcγRIIa cells as the effector cells and BT474 as the target cells. Then, the target cell density, the ratio of effector to target cells, the target cell adhesion time, the incubation time for drug administration, and the induction time after adding effector cells were optimized by the method of design of experiment (DOE). The method showed a significant dose-response relationship, which was complied with the four-parameter equation: y=(A-D)/[1+(x/C)^B]+D. The durability ranges of the target cell density, the ratio of effector to target cells, the target cell adhesion time, the incubation time for drug administration, and the induction time after adding effector cells were (2.5-4.0)×10⁵ cells/mL, 3-5, 1.0-2.0 h, 0 h, and 5.0-6.0 h, respectively. The results of the methodological validation showed that the linear equation was y=1.106 8x-0.011 6, r=0.969 2. The established method showed the relative accuracy ranging from -6.59% to 2.98% and the geometric coefficient of variation less than 11% in the intermediate precision test. Furthermore, the method was target-specific. The method was then applied to the determination of ADCP activity of HER2-targeted ADC, demonstrating the result of (103.5±5.7)%. We developed a reporter gene assay for determining the ADCP activity of HER2-targeted ADC and the assay demonstrated high accuracy and good reproducibility, which proposes a highly efficient and approache for evaluating ADCP effect of this HER2-targeted ADC, and also provides a referable technique for characterizing the Fc effector functions of ADCs with diverse targets.
Humans ; Receptor, ErbB-2/immunology* ; Phagocytosis/drug effects* ; Immunoconjugates/immunology* ; Genes, Reporter ; Antibody-Dependent Cell Cytotoxicity ; Jurkat Cells

OBJECTIVE: To investigate the injury effect of anti-donor specific antibody (DSA) on human umbilical vein enolothelial cells (HUVEC) in NK-mediated antibody-dependent cell cytotoxity (ADCC). METHODS: The peripheral blood of 10 healthy donors was colleced for allo-HSCT of AML patients diagnosed in Department of Hemology of the Tumor Hospital affiliated to Shanxi Medical University, then the peripheral blood NK cells were isolated and used as the effector cells; the HUVEC of passages 9-6 were selected and co-cultured with DSA, then the DSA-binding HUVEC were used as the target cells (CDH group), while the DSA-unbinding HUVEC were used as negative control (UDH group). After co-culture of effecor cells with target cells, the expression of IFN-γ was detected by flow cytometry and the HUVEC activity was detected by using MTT method, so as to indirectily reflect the injury effect of DSA-mediated ADCC on endothelial cells. RESULTS: With the increase of effector-target (E:T) ratio, the activity of HUVEC decreased, the expression level of IFN-γ increased. Under the some effector-target ratio (1∶1, 10∶1, 20∶1), the activity of HUVEC in CDH group was significantly lower than that of UDH group, and the expression of IFN-γ was significantly higher than that of the UDH group (P<0.05). CONCLUSION DSA can damage vascular endothelial cells through the ADCC effect mediated by NK cells.
Antibodies, Monoclonal ; Antibody-Dependent Cell Cytotoxicity ; Endothelial Cells ; Flow Cytometry ; Humans ; Killer Cells, Natural

BACKGROUND: Recent findings in molecular pathology suggest that genetic translocation and/or overexpression of oncoproteins is important in salivary gland tumorigenesis and diagnosis. We investigated PLAG1, SOX10, and Myb protein expression in various salivary gland neoplasm tissues. METHODS: A total of 113 cases of surgically resected salivary gland neoplasms at the National Cancer Center from January 2007 to March 2017 were identified. Immunohistochemical staining of PLAG1, SOX10, and Myb in tissue samples was performed using tissue microarrays. RESULTS: Among the 113 cases, 82 (72.6%) were benign and 31 (27.4%) were malignant. PLAG1 showed nuclear staining and normal parotid gland was not stained. Among 48 cases of pleomorphic adenoma, 29 (60.4%) were positive for PLAG1. All other benign and malignant salivary gland neoplasms were PLAG1-negative. SOX10 showed nuclear staining. In normal salivary gland tissues SOX10 was expressed in cells of acinus and intercalated ducts. In benign tumors, SOX10 expression was observed in all pleomorphic adenoma (48/48), and basal cell adenoma (3/3), but not in other benign tumors. SOX10 positivity was observed in nine of 31 (29.0%) malignant tumors. Myb showed nuclear staining but was not detected in normal parotid glands. Four of 31 (12.9%) malignant tumors showed Myb positivity: three adenoid cystic carcinomas (AdCC) and one myoepithelial carcinoma with focal AdCC-like histology. CONCLUSIONS: PLAG1 expression is specific to pleomorphic adenoma. SOX10 expression is helpful to rule out excretory duct origin tumor, but its diagnostic value is relatively low. Myb is useful for diagnosing AdCC when histology is unclear in the surgical specimen.
Adenoma ; Adenoma, Pleomorphic ; Antibody-Dependent Cell Cytotoxicity ; Carcinogenesis ; Carcinoma, Adenoid Cystic ; Diagnosis ; Immunohistochemistry ; Oncogene Proteins ; Oncogene Proteins v-myb ; Parotid Gland ; Pathology, Molecular ; Salivary Gland Neoplasms ; Salivary Glands ; SOX Transcription Factors ; Translocation, Genetic

Allogeneic natural killer (NK) cell therapy is a potential therapeutic approach for a variety of solid tumors. We established an expansion method for large-scale production of highly purified and functionally active NK cells, as well as a freezing medium for the expanded NK cells. In the present study, we assessed the effect of cryopreservation on the expanded NK cells in regards to viability, phenotype, and anti-tumor activity. NK cells were enormously expanded (about 15,000-fold expansion) with high viability and purity by stimulating CD³⁺ T cell-depleted peripheral blood mononuclear cells (PBMCs) with irradiated autologous PBMCs in the presence of IL-2 and OKT3 for 3 weeks. Cell viability was slightly reduced after freezing and thawing, but cytotoxicity and cytokine secretion were not significantly different. In a xenograft mouse model of hepatocellular carcinoma cells, cryopreserved NK cells had slightly lower anti-tumor efficacy than freshly expanded NK cells, but this was overcome by a 2-fold increased dose of cryopreserved NK cells. In vivo antibody-dependent cell cytotoxicity (ADCC) activity of cryopreserved NK cells was also demonstrated in a SCID mouse model injected with Raji cells with rituximab co-administration. Therefore, we demonstrated that expanded/frozen NK cells maintain viability, phenotype, and anti-tumor activity immediately after thawing, indicating that expanded/frozen NK cells can provide ‘ready-to-use’ cell therapy for cancer patients.
Animals ; Antibody-Dependent Cell Cytotoxicity ; Carcinoma, Hepatocellular ; Cell Survival ; Cell- and Tissue-Based Therapy ; Cryopreservation* ; Freezing ; Heterografts ; Humans* ; Interleukin-2 ; Killer Cells, Natural* ; Methods ; Mice ; Mice, SCID ; Muromonab-CD3 ; Phenotype ; Rituximab

Antibody-mediated rejection (AMR) is a major complication after ABO-incompatible liver transplantation. According to the 2016 Banff Working Group on Liver Allograft Criteria for the diagnosis of acute AMR, a positive serum donor specific antibody (DSA) is needed. On the other hand, the clinical significance of the histological findings of AMR in the absence of DSA is unclear. This paper describes a 57-year-old man (blood type, O+) who suffered from hepatitis B virus cirrhosis with hepatocellular carcinoma. Pre-operative DSA and cross-matching were negative. After transplantation, despite the improvement of the liver function, acute AMR was observed in the protocol biopsy on postoperative day 7; the cluster of differentiation 19+ (CD19+) count was 0% and anti-ABO antibody titers were 1:2. This paper presents the allograft injury like AMR in the absence of DSA after ABOi living donor liver transplantation with low titers of anti-ABO antibody and depleted serum CD19+ B cells.
Allografts ; Antibody-Dependent Cell Cytotoxicity ; B-Lymphocytes ; Biopsy ; Carcinoma, Hepatocellular ; Diagnosis ; Fibrosis ; Hand ; Hepatitis B virus ; HLA Antigens ; Humans ; Liver Transplantation* ; Liver* ; Living Donors ; Middle Aged ; Tissue Donors*

PURPOSE: The selective elimination of cancer stem cells (CSCs) in tumor patients is a crucial goal because CSCs cause drug refractory relapse. To improve the current conventional bispecific immune-engager platform, a 16133 bispecific natural killer (NK) cell engager (BiKE), consisting of scFvs binding FcγRIII (CD16) on NK cells and CD133 on carcinoma cells, was first synthesized and a modified interleukin (IL)-15 crosslinker capable of stimulating NK effector cells was introduced. MATERIALS AND METHODS: DNA shuffling and ligation techniques were used to assemble and synthesize the 1615133 trispecific NK cell engager (TriKE). The construct was tested for its specificity using flow cytometry, cytotoxic determinations using chromium release assays, and lytic degranulation. IL-15–mediated expansion was measured using flow-based proliferation assays. The level of interferon (IFN)-γ release was measured because of its importance in the anti-cancer response. RESULTS: 1615133 TriKE induced NK cell–mediated cytotoxicity and NK expansion far greater than that achieved with BiKE devoid of IL-15. The drug binding and induction of cytotoxic degranulation was CD133+ specific and the anti-cancer activity was improved by integrating the IL-15 cross linker. The NK cell–related cytokine release measured by IFN-γ detection was higher than that of BiKE. NK cytokine release studies showed that although the IFN-γ levels were elevated, they did not approach the levels achieved with IL-12/IL-18, indicating that release was not at the supraphysiologic level. CONCLUSION: 1615133 TriKE enhances the NK cell anti-cancer activity and provides a self-sustaining mechanism via IL-15 signaling. By improving the NK cell performance, the new TriKE represents a highly active drug against drug refractory relapse mediated by CSCs.
Antibody-Dependent Cell Cytotoxicity ; Chromium ; DNA Shuffling ; Flow Cytometry ; Humans ; Interferons ; Interleukin-15 ; Interleukins ; Killer Cells, Natural ; Ligation ; Neoplastic Stem Cells ; Recurrence ; Sensitivity and Specificity

Neuroblastoma, one of the most common solid tumors in early childhood, exhibits aberrant cell-surface glycosylation patterns. In neuroblastoma, disialoganglioside (GD2) is expressed homogeneously and abundantly on 100% of neuroblastoma cells. GD2 is a good tumor marker for developing an anti-tumor-monoclonal antibody (mAb) to neuroblastoma. Immunotherapy, using anti-GD2-mAb, has been tried since last 20 years to improve the prognosis of high risk neuroblastoma patients who show a 5-year survival rate of less than 30% regardless of an intense multimodal therapy. Since the first clinical trial of murine anti-GD2-mAb 3F8 had been performed, multiple clinical studies showed that anti-GD2-mAb might improve the prognosis of high risk neuroblastoma patients. Anti-GD2-mAb removes the neuroblastoma cells via apoptosis by antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity. To elicit a stronger ADCC response to antibody therapy, cytokines such as, GM-CSF and interleukin-2 are concomitantly administered, which stimulate the natural anti-tumor activity of the immune system. Children's Oncology Group performed a study of chimeric anti-GD2-mAb (ch14.18) administration with GM-CSF, IL-2 for high risk neuroblastoma patients and showed the improvement of overall survival rate. Based on this study US FDA approved the chimeric anti-GD2-mAb (commercially manufactured dinutuximab) for the treatment of high risk neuroblastoma. Dinutuximab is the the first mAb for use in combination of cytokines for the maintenance treatment of pediatric patients with high risk neuroblastoma who achieve at least a partial response to intensified multimodal therapy. The first anti-tumor-mAb used for children, dinutuximab, could be the base of further development of mAb against the cancers in childhood.
Antibody-Dependent Cell Cytotoxicity ; Apoptosis ; Child ; Cytokines ; Glycosylation ; Granulocyte-Macrophage Colony-Stimulating Factor ; Humans ; Immune System ; Immunotherapy ; Interleukin-2 ; Interleukins ; Neuroblastoma ; Prognosis ; Survival Rate

The biological activity of ADCC by anti-CD20 monoclonal antibody was determined by BioGlo™ Luciferase Assay System using Jurkat/NFAT-luc+FcγRIIIa cell line as effector cell and WIL2-S cell line as target cell. The developed method was verified for specificity, precision and accuracy. Anti-CD20 monoclonal antibody showed a dose-response mode by the developed method, and the determination result complied with the following four-parameter equation: y = (A-D)/[1 + (X/C)(B)] + D. The optimized parameters of the method were determined including the antibodies diluted concentration (18,000 ng·mL(-1)), dilution rate (1:5), the ratio of effector cell and target cell (6:1), and induction time (6 h). The values of eight independent tests have passed a statistical test for curve regression analysis, linear or parallelism, which showed the method possessed good specificity. Four different dilute groups of recovery rates sample were determined for 3 times, and the result showed mean relative potencies of (44.39±3.93)%, (72.74±2.78)%, (128.28±7.01)% and (168.19±2.70)% respectively, with a variation coefficient of less than 10%, and the recoveries of (88.78±7.85)%, (96.99±3.70)%, (102.63±5.61)% and (112.12±1.80)% respectively. A novel reporter gene method for determination of biological activity of ADCC by anti-CD20 monoclonal antibody was successfully developed, which showed strong specificity, good reproducibility and high accuracy, and might be used routinely.
Antibodies, Monoclonal, Murine-Derived ; pharmacology ; Antibody-Dependent Cell Cytotoxicity ; Antigens, CD20 ; immunology ; Genes, Reporter ; Humans ; Reproducibility of Results ; Rituximab

OBJECTIVETo investigate the effect of FCGR3A polymorphisms on the antibody-dependent cell-mediated cytotoxicity (ADCC) activity induced by cetuximab against A549 cells.

METHODSA549 cell line was used as target cells and NKTm cells as effector cells. FCGR3A polymorphisms were detected by direct sequencing. The ADCC activity mediated by cetuximab was assessed by CCK-8 assay.

RESULTSThree genotypes of FCGR3A were detected:V/V,V/F,and F/F. The ADCC activity of NKTm cells with these three different genotypes mediated by cetuximab were significantly different (P=0.0015). NKTm cells with FCGR3A-158V/V genotypes had significantly higher ADCC activity than FCGR3A-V/F or F/F genotypes (P<0.01),whereas the ADCC activity between V/F and F/F genotype showed no statistical significance(P>0.05).

CONCLUSIONFCGR3A polymorphisms have an impact on ADCC activity mediated by cetuximab in NKTm cells.

BACKGROUND: Cytologic diagnosis of pulmonary adenoid cystic carcinoma (AdCC) is frequently challenging and differential diagnosis with small cell carcinoma is often difficult. METHODS: Eleven cytologically diagnosed cases of pulmonary AdCC were collected and reviewed according to fifteen cytomorphologic characteristics: small cell size, cellular uniformity, coarse chromatin, hyperchromasia, distinct nucleolus, frequent nuclear molding, granular cytoplasm, organoid cluster, sheet formation, irregular border of cluster, hyaline globule, hyaline basement membrane material, individual cell necrosis or apoptotic body, and necrotic background. Twenty cases of small cell carcinoma and fifteen cases of non-pulmonary AdCC were also reviewed for the comparison. RESULTS: Statistically significant differences were identified between pulmonary AdCC and small cell carcinoma in fourteen of the fifteen cytomorphologic criteria (differences in sheet formation were not statistically significant). Cellular uniformity, distinct nucleolus, granular cytoplasm, distinct cell border, organoid cluster, hyaline globule, and hyaline basement membrane material were characteristic features of AdCC. Frequent nuclear molding, individual cell necrosis, and necrotic background were almost exclusively identified in small cell carcinoma. Although coarse chromatin and irregular cluster border were observed in both, they favored the diagnosis of small cell carcinoma. Hyaline globules were more frequently seen in non-pulmonary AdCC cases. CONCLUSIONS: Using the fifteen cytomorphologic criteria described by this study, pulmonary AdCC could be successfully distinguished from small cell carcinoma. Such a comprehensive approach to an individual case is recommended for the cytologic diagnosis of pulmonary AdCC.
Adenoids* ; Antibody-Dependent Cell Cytotoxicity ; Basement Membrane ; Carcinoma, Adenoid Cystic* ; Carcinoma, Small Cell* ; Cell Size ; Chromatin ; Cytoplasm ; Diagnosis ; Diagnosis, Differential ; Fungi ; Hyalin ; Lung ; Necrosis ; Organoids