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Objective:To investigate the antibody-dependent cell-mediated cytotoxicity (ADCC) in plasma samples from patients with SARS-CoV-2 infection and to evaluate its correlation with antibody titer and neutralizing activity.Methods:A simple method for ADCC detection was established using HEK293T cells expressing SARS-CoV-2 spike (S) protein as target cells and FcγRⅢa-V158-expressing Jurkat cells as effector cells. It was used to analyze the ADCC activity in 38 plasma samples after the ratio of effector cells to target cells was optimized. Plasma-specific antibody was detected by capturing ELISA, which was to capture the C-terminal-tagged recombinant SARS-CoV-2 S protein with an anti-tag antibody. The neutralizing activity in plasma samples was detected using a pseudovirus neutralization assay. Mann-Whitney U test was used for comparison between different groups and non-parametric Spearman correlation test was performed for correlation analysis. Results:The seroconversion rates for antibodies specific for S protein, S1 protein and RBD were all 97.4% (37/38), and the dynamic changes in antibody titers with recovery time showed that antibody titers peaked at 3-4 weeks. Among the plasma samples with neutralizing activity, those with antibody titers >1∶320 had stronger neutralizing activity than the plasma samples with antibody titers <1∶320 [IC 50: 749.6 (396.5-3 772.0) vs 81.4 (11.6-228.4), P<0.01]. ADCC activity was detectable in 86.8% (33/38) of the plasma samples, and its dynamic change with recovery time were consistent with that of specific antibody titer with a peak at 3-4 weeks. Correlation analysis showed ADCC was positively correlated with the titers of antibodies specific for S protein, S1 protein and RBD ( r=0.686, 0.535 and 0.471, all P<0.01). A positive correlation was also found between ADCC and neutralizing activity ( r=0.573, P<0.01). Conclusions:This study established a simple method for the detection of ADCC. Results of this study suggested that SARS-CoV-2 could induce specific ADCC in plasma and the ADCC might be associated with non-neutralizing antibodies. Besides, the activity of ADCC peaked at 3-4 weeks. These findings would be of reference value for clinical treatment with convalescent plasma.
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@#[Abstract] Objective: To construct and purify the recombinant bispecific antibody (BsAb) targeting PD-1 and CD19 and evaluate its activity. Methods: With pCAR1 plasmid as the vector, the eukaryotic expression vector of anti-PD-1/CD19 BsAb was constructed by molecular cloning technology, and then transfected into mammalian cell line CHO-S by PEI reagent for transiently expressing antibody. The BsAb was purified by Affinity chromatography and then identified by SDS-PAGE and WB. The blocking activity of BsAb on PD-1/PD-L1 in vitro was detected by Luciferase reporter gene assay. The activity of antibody (BsAb)-dependent cell (PBMC)-mediated cytotoxicity (ADCC) in vitro was evaluated by lactate dehydrogenase (LDH) cytotoxicity assay. Results: The double plasmid eukaryotic expression vector pCAR1-19X3 was successfully constructed, and anti-PD-1/CD19 BsAb was successfully expressed in CHO-S cells, named pCAR1-19X3-TY. pCAR1-19X3-TY could effectively block the binding of PD-1 to its ligand PD-L1 in vitro, and the EC50 based on the dose-response curve was 0.306 μg/ml. ADCC results showed that pCAR1-19X3-TY could mediate the cytotoxicity of PBMC against Raji cells, and the curve showed a linear upward trend; when the effect/target ratio was 50∶1, the target cell lysis rate of pCAR1-19X3-TY was (38.9±0.3)%, which was not significantly different from that of the positive treatment group (46.7±4.9)% (P>0.05), but significantly higher than that of the negative control group (1.2±0.1)% (P<0.05). Conclusion: The recombinant anti-PD-1/CD19 BsAb can effectively block the binding of PD-1 and PD-L1 and activate PBMC mediated cytotoxicity against Raji cells. pCAR1-19X3-TY has the potential application value in the treatment of B-cell malignant tumor.
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Antibody-mediated rejection (AMR), also known as humoral rejection, is an immune injury caused by rejection involved with multiple humoral immune effectors, such as antibodies and complements, etc. AMR plays a pivotal role in hyperacute, acute and chronic rejection. In this article, the basic definition of AMR, the research progress and major achievements on AMR pathology according to Banff classification on allograft pathology (Banff classification), and main pathological characteristics of AMR in renal allograft were reviewed, aiming to provide reference for accurate diagnosis and timely treatment of AMR, and guarantee the long-term survival of renal graft and recipients.
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Objective:To investigate the induction of specific T lymphocyte by bispecific monoclonal antibody in pancreatic cancer and its killing effects on KIF20A positive pancreatic cancer PANC1 cell line.Methods:CD 3/KIF20A bispecific monoclonal antibody was prepared and concentrated by chemical cross-linking method and purified by Sephrose-25 gel chromatography. Peripheral blood samples of healthy volunteers were collected, and monocytes were isolated using lymphocyte separation solution, and then cultured as dendritic cells (DC) and T cells respectively, and then co-cultured as DC-T cells. Meanwhile vitamin C was used to treat DC-T cells (vcDC-T cells). The levels of IFN-γ, IL-2, IL-4 and IL-12 in the supernatants and T cell subsets were detected by flow cytometry. About 1×10 5 T cells, DC-T cells, and vcDC-T cells with 10, 50, 100 and 300 ng CD 3/KIF20A antibody loaded were cocultured with PANC1 cells (20∶1) for 2, 6 and 10 hours to determine the highest killing rate dosage of CD 3/KIF20A antibody loaded cells. DC-T cells and DC-T cells, vcDC-T cells loaded with the highest killing rate dosage of CD 3/KIF20A antibody were cocultured with PANC1 cells (20∶1) for 2, 6, 8, 10 and 12 hours. The aggregation effect of effector cells on target cells was observed under inverted microscope, the killing rate of tumor cells was detected by LDH method. Results:The molecular weight of CD 3/KIF20A antibody was 130 000 measured and validated by SDS gel electrophoresis. The ratio of CD 8+ CD 28+ and CD40L subsets of vcDC-T cells was increased [(47.6±15.8)% vs (38.2±7.6)%, (52.1±4.9)% vs (44.7±3.2)% ] compared with that of DC-T cells, the ratio of negative regulatory cells (Treg) was decreased [(4.3±0.8)% vs (8.3±1.1)%]; the release of IL-2, IFN-γ and IL-12 was increased [(201.2±17.3) ng/L, (163.4±13.1)ng/L, (303.3±22.6)ng/L vs 221.8±17.6)ng/L, (190.4±11.7)ng/L vs (80.3±8.6)ng/L]. All the differences were statistically significant ( P<0.01). 100 ng CD 3/KIF20A loaded T cells were observed under microscope, which obviously targeted KIF20A + pancreatic cancer PANC1 cells and had a strongest killing power. At the killing cells to targeting cells ratio of 20∶1 with 4-hour coculture, the killing rate of CD 3/KIF20A-vcDC-T cells on PANC1 cells was (88.6±2.6)%, which was significantly higher than (68.4±3.4)% and (39.2±2.1)% in the CD3/KIF20A-DC-T group and (39.2±2.1)% in the DC-T group, increasing by 20% and at lease 45%, respectively. Conclusions:DC-T cells loaded with CD 3/KIF20A antibody can significantly increase the killing rate of KIF20A positive pancreatic cancer PANC1 cells, and vitamin C intervention can further enhance the killing ability of antibody loaded T cells.
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OBJECTIVE: To develop a novel optimization and validation method based on design of experiment(DOE) for the antibody-dependent cell-mediated cytotoxicity (ADCC) potency of anti-programmed cell death 1 and anti-programmed cell death-ligand 1 (PD-1/PD-L1) monoclonal antibodies using reporter genes. METHODS: Jurkat-hFcγRIIIa-NFAT transgenic cell line was used as effector cells, 293FT-PD-1 cell line and CHO-PD-L1 cell line were used as target cells, respectively. The ADCC potency for anti-PD-1/PD-L1 monoclonal antibodies was detected with Luciferase detection system (BrightGloTM Luciferase Assay system), then the method was optimized and validated based on DOE. RESULTS: The anti-PD-1/PD-L1 monoclonal antibodies showed a dose-response relationship and the determination result complied with the following four-parameter equation: y=(A-D)/+D. The method was optimized and the testing parameters were determined as follows: the working concentration of anti-PD-1 monoclonal antibody was 10 000 ng•mL-1 to 4.833 ng•mL-1 and that of anti-PD-L1 was 2 000 ng•mL-1 to 0.488 ng•mL-1, the ratio of effector cells and target cells for anti-PD-1/PD-L1 monoclonal antibodies were 6:1 and 3:1, and the induction time for both of these antibodies was 20 h. The method possessed good specificity. The recovery rate test samples in the four different dilution groups were determined for 3 times, and the results showed that the relative potencies of anti-PD-1 monoclonal antibody were (51.74±2.22)%, (77.12±3.14)%, (118.71±2.83)% and (156.20±12.99)%, and the recoveries of which were (103.49±4.44)%, (102.83±4.19)%, (94.96±2.26)% and (104.14±8.66)%, respectively. While as for anti-PD-L1 monoclonal antibody, the relative potencies were (54.32±4.75)%, (75.24±4.25)%, (127.40±2.43)%, (156.82±3.27)% and the recoveries were (108.64±9.51)%, (100.33±5.67)%, (101.92±1.94)% and (104.55±2.18)%, respectively. The RSDs of the above results were all less than 10%. CONCLUSION: A novel optimization and validation method based on DOE for detecting ADCC potency of anti-PD-1/PD-L1 mAb is successfully developed. This detecting method based on reporter gene shows high specificity, good reproducibility and high accuracy, and might be used in the evaluation of ADCC potency of anti-PD-1/PD-L1 mAb.
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A human immunodeficiency virus type-1 (HIV-1) vaccine which is able to effectively prevent infection would be the most powerful method of extinguishing pandemic of the acquired immunodeficiency syndrome (AIDS). Yet, achieving such vaccine remains great challenges. The membrane-proximal external region (MPER) is a highly conserved region of the envelope glycoprotein (Env) gp41 subunit near the viral envelope surface, and it plays a key role in membrane fusion. It is also the target of some reported broadly neutralizing antibodies (bNAbs). Thus, MPER is deemed to be one of the most attractive vaccine targets. However, no one can induce these bNAbs by immunization with immunogens containing the MPER sequence(s). The few attempts at developing a vaccine have only resulted in the induction of neutralizing antibodies with quite low potency and limited breadth. Thus far, vaccine failure can be attributed to various characteristics of MPER, such as those involving structure and immunology; therefore, we will focus on these and review the recent progress in the field from the following perspectives: (1) MPER structure and its role in membrane fusion, (2) the epitopes and neutralization mechanisms of MPER-specific bNAbs, as well as the limitations in eliciting neutralizing antibodies, and (3) different strategies for MPER vaccine design and current harvests.
Subject(s)
Humans , AIDS Vaccines , Chemistry , Allergy and Immunology , Antibodies, Neutralizing , Allergy and Immunology , HIV Antibodies , Allergy and Immunology , HIV Envelope Protein gp41 , Allergy and Immunology , HIV-1 , Chemistry , Allergy and ImmunologyABSTRACT
HER3 belongs to the human epidermal growth factor receptor (HER) family which also includes HER1/EGFR/erbB1, HER2/erbB2, and HER4/erbB4. As a unique member of the HER family, HER3 lacks or has little intrinsic tyrosine kinase activity. It frequently co-expresses and forms heterodimers with other receptor tyrosine kinases (RTKs) in cancer cells to activate oncogenic signaling, especially the PI-3K/Akt pathway and Src kinase. Elevated expression of HER3 has been observed in a wide variety of human cancers and associates with a worse survival in cancer patients with solid tumors. Studies on the underlying mechanism implicate HER3 expression as a major cause of treatment failure in cancer therapy. Activation of HER3 signaling has also been shown to promote cancer metastasis. These data strongly support the notion that therapeutic inactivation of HER3 and/or its downstream signaling is required to overcome treatment resistance and improve the outcomes of cancer patients.
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Objective To analyze the possibility of using intracellular cytokine staining (ICS) to evaluate NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) and to detect the changes in ADCC activity among patients with chronic HIV and/or HCV infection. Methods Flow cytometry was per-formed to determine the percentages of NK cells and the expression of NK cell receptors. ImageStreamX MarkⅡ system was used to identify the expression of CD3, CD56, CD16 and CD32 on CD56brightNK and CD56dimNK subsets. Degranulation process and cytokine production in NK cells were detected using an anti-gen-antibody complex model of P815/Ab in combination with ICS. Differences in NK cell-mediated ADCC were evaluated among patients infected with HIV and/or HIV and healthy subjects by flow cytometry. Re-sults The percentages of CD107a+and IFN-γ+NK cells were positively correlated with the decrease of mean fluorescence intensity (MFI) of CD16. ICS assay revealed a positive correlation between the secretion of CD107a and IFN-γ by NK cells. CD16 was highly expressed in CD56dimNK cells. The ADCC mediated by CD56dimNK cells was stronger than that mediated by CD56brightNK cells. The rate of target cell lysis detected by rapid fluorescence assay was positively correlated with the percentage of CD107a+/IFN-γ+NK cells meas-ured by ICS. NK cell-mediated ADCC was suppressed in patients with chronic HIV and/or HCV infection. Conclusion This study suggests that ICS assay could be used to evaluate NK cell-mediated ADCC. It also reveals that NK cell-mediated ADCC is suppressed in patients with chronic HIV and/or HCV infection.
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Objective To evaluate the biological activity of anti-Rh(D) antibodies in vitro. Methods The titres of plasma Anti-Rh (D) antibodies in the RH(-) patients uncorrectly infused RH(+) blood were detected by various methods. The anti-D antibodies were evaluated for their capacity to sensitize erythrocytes, trigger monocyte and K-cell mediated antibody-dependent cellular cytotoxicity (ADCC), mediate binding to monocyte and lymphocyte Fc gamma R, and stimulate phagocytosis by monocytes. The synergistic effect of anti-D antibodies in promoting hemolysis was detected by complement fixation test (CFT). Results The titre of plasma anti-D antibodies measured by micro-column gel indirect anti-globulin technique(MGIAT) was the highest compared with the other detection methods. When red cells were sensitized with anti-D antibodies, the binding and phagocytosis of red cells by monocytes and the lysis of red cells by monocytes or lymphocytes were great, and the hemolysis of red cells by alexin was great too. Conclusion Anti-Rh (D) antibodies had great biological activity in vitro, and MGIAT had the highest sensitivity for detecting anti-Rh(D) antibodies.
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PURPOSE: During differentiation of HL-60 cells by all-trans retinoic acid(ATRA), we analyzed the expression of Fcr receptors and Mac-1 by molecules of equivalent soluble fluorochromes(MESF) and functional studies. METHODS: HL-60 cells were induced to differentiate by adding 1micrometer ATRA. On the 4th and 7th day after stimulation as well as before stimulation, the cells were analyzed for phenotypic and functional differentiation. Phenotypic analysis was performed by flow cytometry after staining the cells with PE-conjugated anti-human CD64(FcrRI), CD32(FcrRII), CD16(FcrRIII), CD11b, CD18. The measured fluorescent intensity was transformed into MESF. Phagocytic activity was measured by flow cytometry after incubation of the cells with fluorochrome-conjugated beads. Respiratory burst was measured by chemiluminescence assay. ADCC was measured by hemoglobin release assay. Opsonophagocytic activity was measured by fungicidal assay. Correlation between MESF of FcrR and Mac-1 and function of HL-60 was measured. RESULTS: Percent positive cells and MESF of CD11b and FcrRI increased on the 4th day and decreased on the 7th day. Percent positive cells of CD18 was 99% regardless of differentiation. But MESF of CD18 was increased on the 4th day and decreased on the 7th day. Percent positive cells of FcrRII were above 90% regardless of differentiation. MESF of FcrRII showed no significant change. FcrRIII expression was not induced. Phagocytic activity of HL-60 cells was increased twofold. Chemiluminescence of HL-60 cells was increased up to 60-fold on the 7th day. ADCC of HL-60 cells was incerased up to 2.5-fold on the 7th day. Opsonophagocytic activity increased twice on the 4th day. ADCC and opsonophagocytic activity correlates with the expression of CD11b/CD18 and FcrRII. CONCLUSION: Differentiation of HL-60 cells with ATRA induces several functional maturations until 7 days with expression of FcrR and Mac-1.