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【Objective】 To establish a method for determinating the antigen-dependent cell-mediated cytotoxicity (ADCC) of human immunoglobulin (pH4)for intravenous injection (IVIG) on luciferase reporter gene-modified cell assay. 【Methods】 As effector cells, Jurkat-NFAT-Luc-CD16 cells were used in the assay, and PLC/PRF/5 cells were used as target cells. After incubation of effector cells and target cells with IVIG, the method for determinating ADCC biological activity of IVIG was established by detecting luciferase released by activated T nuclear factor after binding of IVIG Fc fragment to effector cells. Meanwhile, the experimental assay conditions were optimized, and the methodology was verified subsequently. 【Results】 IVIG had a dose-response relationship in this method, which was consistent with four parameter logistic model. And the PLC/PRF/5 cells were finally determined as the target cells. The initial dilution concentration of antibody was 20 mg/mL, and the ratio dilution was 1∶2, and the effector to target ratio was 1∶3, and co-incubation time of two cells and IVIG was 24 hours. Within-run and between-run analysis including three independent tests, initial working concentration relative light unit (RLU) and the relative standard deviation (RSD) of the concentration for 50% of maximal effect(EC50) were less than 11%. The relative titers of the recovery samples of the two different dilution groups were (23.50±1.69)% and (49.30±2.97)%, respectively, and the corresponding recovery rates were (93.50±6.30)% and (96.24±5.43)%, respectively, with RSD less than 11%. 【Conclusion】 The method for determinating ADCC biological activity of IVIG based on luciferase reporter gene-modified cell assay was successfully established. It could be applied in determinating the ADCC biological activity of IVIG, and has the advantages of satisfactory linearity, accuracy, precision and specificity.
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Cryoablation (CRA) and microwave ablation (MWA) are two main local treatments for hepatocellular carcinoma (HCC). However, which one is more curative and suitable for combining with immunotherapy is still controversial. Herein, CRA induced higher tumoral PD-L1 expression and more T cells infiltration, but less PD-L1highCD11b+ myeloid cells infiltration than MWA in HCC. Furthermore, CRA had better curative effect than MWA for anti-PD-L1 combination therapy in mouse models. Mechanistically, anti-PD-L1 antibody facilitated infiltration of CD8+ T cells by enhancing the secretion of CXCL9 from cDC1 cells after CRA therapy. On the other hand, anti-PD-L1 antibody promoted the infiltration of NK cells to eliminate PD-L1highCD11b+ myeloid cells by antibody-dependent cell-mediated cytotoxicity (ADCC) effect after CRA therapy. Both aspects relieved the immunosuppressive microenvironment after CRA therapy. Notably, the wild-type PD-L1 Avelumab (Bavencio), compared to the mutant PD-L1 atezolizumab (Tecentriq), was better at inducing the ADCC effect to target PD-L1highCD11b+ myeloid cells. Collectively, our study uncovered the novel insights that CRA showed superior curative effect than MWA in combining with anti-PD-L1 antibody by strengthening CTL/NK cell immune responses, which provided a strong rationale for combining CRA and PD-L1 blockade in the clinical treatment for HCC.
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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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Therapeutic monoclonal antibodies are among the most effective biotherapeutics to date. An important aspect of antibodies is their ability to bind antigen while at the same time recruit immune effector functions. The majority of approved recombinant monoclonal antibody therapies are of the human IgG1 subclass, which can engage both humoral and cellular components of the immune system. The wealth of information generated about antibodies has afforded investigators the ability to molecularly engineer antibodies to modulate effector functions. Here, we review various antibody engineering efforts intended to improve efficacy and safety relative to the human IgG isotype. Further, we will discuss proposed mechanisms by which engineering approaches led to modified interactions with immune components and provide examples of clinical studies using next generation antibodies.
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Animals , Humans , Antibodies, Monoclonal , Metabolism , Antigens , Metabolism , Complement System Proteins , Metabolism , Immunoglobulin G , Metabolism , Protein Engineering , Receptors, Fc , MetabolismABSTRACT
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 N-glycan profile of therapeutic antibodies by UPLC-Qda MS. METHODS: The Nlinked glycan in Fc region was released by PNGase F digestion and labeled by RapiFluor-MS, and labeled glycans were analyzed by UPLC-Qda MS. RESULTS: The UPLC-Qda MS method showed good accuracy in N-glycan quantitaion and qualification, and the Δm/z value of individual N-glycan was in a range of ± 0.5. Three similar biotherapeutic products (SBP) showed a nearly same N-glycan profile with the reference biotherapeutic products (RBP) and the total percentage of fucosylated glycans was comparable, only one fraction of N-glycans had some difference. The major N-glycans of antibodies expressed by CHO cells, NS0 cells and SP2/0 cells were G0F-N, G0F, Man5, G1Fa, G1Fb and G2F, accounting for above 80% of total glycans. Eleven glycoforms were detected in CHO cell expressed antibodies, 22 and 26 glycoforms were detected in NS0 cell and SP2/0 cell expressed antibodies respectively. The N-glycans of NS0 cell and SP2/0 cell expressed antibodies contained more sialylated and galactosylated complex glycoforms, which was related to the antibody half-life in vivo and immunogenicity. CONCLUSION: The HILIC UPLC-Qda MS, as a fast and accurate analytical method, can be used in the quality control of N-glycan profile of antibody.
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Objective To establish a flow cytometry-based assay for the detection of monocyte-me-diated antibody-dependent cell-mediated cytotoxicity ( ADCC ) .Methods P815 cells double stained with PKH26 and carboxyfluorescein succinimidyl ester ( CFSE ) were used as target cells and coated with P 815 specific antibodies to form antigen-antibody complexes .The peripheral blood mononuclear cells were isolated as effector cells and co-cultured with the antigen-antibody complexes .The CD3-CD14+PKH26+CFSE-cell population were gated by flow cytometry .Optimized effector/target cell ratio and incubation time for killing assay were identified .Monocyte-mediated ADCC in 23 patients with chronic HCV infection and 22 healthy subjects were analyzed .Results The monocyte-mediated ADCC could be evaluated through analyzing the CD3-CD14+PKH26+CFSE-cells with flow cytometry .The optimized effector/target cell ratio was 10 ∶1 and the optimized time for incubation was 4 h.Monocyte-mediated ADCC was inhibited in patients with chronic HCV infection as compared with healthy subjects (P=0.009).Conclusion A flow cytometry-based assay for the detection of monocyte-mediated ADCC was established , which could be used as a fast , sensitive and safety method for the evaluation of monocyte-mediated ADCC during viral infections and the research and de-velopment of drugs .
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Rituximab,a chimeric monoclonal antibody targeted against the pan-B-cell marker CD20,comparing with chemotherapy has significantly improved the treatment outcome of all subgroups of B-cell lymphoma patients.However,not all patients respond to rituximab plus chemotherapy or relapse in a short time after response,which suggests the existence of resistance mechanisms.Antibody-dependent cell-mediated cytotoxicity (ADCC) is one of the main mechanisms of rituximab,so any abnormalities in ADCC pathway may affect the efficacy of rituximab.With the widely application of rituximab,its resistance mechanisms are attracting more attention.This review will summarize the factors within ADCC pathway that may affect the antitumor effect of rituximab.
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AIM: To observe the cytotoxicity on myeloma cells mediated by anti-CD20 monoclonal antibody-mabthera, after heightening level of CD20 expression on myeloma cells membrane by ?-interferon. METHODS: 10 untreated(UT) and 10 relapsed or refractory(RR) MM patients'myeloma cells were cultured with human recombinant ?-interferon (hr?-IFN) at concentrations of (0-800)?10~3 U/L to heighten level of CD20 expression, then complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) on myeloma cells mediated by mabthera were studied through MTT methods. RESULTS: When CD20 expression of UT MM and RR MM patients' myeloma cells increased after treated by hr?-IFN, 12 mg/L and 16 mg/L mabthera mediated ADCC and CDC (against) myeloma cells in group UT patients and group RR patients, respectively. CONCLUSION: After heightened level of CD20 expression on myeloma cells membrane by hr?-IFN, mabthera mediated ADCC and CDC against myeloma cells in vitro.
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BACKGROUND: The several forms of treatment of Graves disease-thyroidectomy, antithyroid drugs and radioiodide therapy-are in wide use now. But which therapy is best is a matter of debate. Some authors reported that in patients who underwent thyroidectomy, higher titers of serum antimicrosomal antibody were associated with 1) higher formation rates of germinal centers, 2) more lymphocyte infiltration in the thyroid tissue, 3) higher incidence of hypothyroidism, and 4) lower incidence of recurrence. We were interested in the relationship of thyroid autoantibody titers, ADCC(antibody-dependent cell-mediated cytotoxicity) activity and the clinical response to antithyroid medication. METHODS: We measured ADCC activities from patients in Graves disease(n-48), Hashimoto thyroiditis(n=17) and normal control(n=9). The patients of Graves disease were followed up for more than 1 year, and they were grouped into A(n=17, well responsed group to antithyroid medication) and B(n=31, poorly responsed group). We examined ADCC activities of patients' sera by chromium release assay. RESULTS: 1) Mean age of patients with Graves disease was 34.4210.4 years and 15 patients were male(31%). 2) Results of thyroid function tests of the Graves' patients were T 585.9 +/- 255.3 ng/dL, T4 21.3 +/- 12.2 mg/dL, TSH 0.11 +/- 0.06mIU/mL. Concentrations of antimicrosomal antibody, antithyroglobulin antibody and thyrotropin binding inhibitory immunoglobulin were 1279.1 +/- 1486.7 IU/mL, 488.1 +/- 751.1 IU/mL, and 38.5 +/- 33.4U/L respectively. 3) There was no significant difference between levels of thyroid hormones or concentrations of thyroid autoantibodies and ADCC activities in graves patients. 4) The ADCC activity of the Graves patient group(24.49%) was significantly higher than that of the normal control group(3.76%), and significantly lower than that of the Hashimotos thyroiditis group(36.34%). 5) There was no significant difference in ADCC activity between group A(18.24 +/- 13.44%) and B(27.91 +20.02%). CONCLUSION: From this results, we suggested that ADCC activity seems to be no value as a prognostic factor in predicting the response to antithyroid drugs in Graves disease patients. But, further studies, larger number of patients and long-term follow up, are needed.
Subject(s)
Humans , Antibody-Dependent Cell Cytotoxicity , Antithyroid Agents , Autoantibodies , Chromium , Follow-Up Studies , Germinal Center , Graves Disease , Hypothyroidism , Immunoglobulins , Incidence , Lymphocytes , Recurrence , Thyroid Function Tests , Thyroid Gland , Thyroid Hormones , Thyroidectomy , Thyroiditis , ThyrotropinABSTRACT
AIM: To further investigate the mechanism of bronchial asthma.METHODS: Experimental asthma mouse model was established with OVA, and immunocytes were isolated, including macrophages, dendritic cells, B cells, CD3 +T cells, CD4 +T cells and CD8 +T cells from spleen. Antibody dependent cell-mediated cytotoxicity(ADCC) effects of all these cells were detected with cell proliferation and improved MTT colorimetry methods.RESULTS: The ADCC functions of M, B cells, CD3 +T cells,CD4 +T cells and CD8 +T cells from asthmatic mice were weaker than that from control ( P