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

BACKGROUND: Bone marrow biopsies are routinely performed for staging patients with B-cell non-Hodgkin lymphoma (NHL). In addition to histomorphological studies, ancillary tools may be needed for accurate diagnosis. We investigated the clinical utility of multiparameter flow cytometric examination of bone marrow aspirates. METHODS: A total of 248 bone marrow specimens from 232 patients diagnosed with B-cell NHL were examined. Monoclonal antibodies directed against CD19, CD20, CD10 (or CD5), and kappa and lambda immunoglobulins were used. Multi-stage sequential gating was performed to select specific cells of interest, and the results were compared with bone marrow histology. RESULTS: The concordance rate between histomorphology and flow cytometry was 91.5% (n=227). Eight cases (3.2%) were detected by flow cytometry alone and were missed by histomorphology analysis, and 6 of these 8 cases showed minimal bone marrow involvement (0.09-2.2%). The diagnosis in these cases included large cell lymphoma (n=3), mantle cell lymphoma (n=3), and mucosa-associated lymphoid tissue (MALT) lymphoma (n=2). Thirteen cases were histopathologically positive and immunophenotypically negative, and the diagnoses in these cases included diffuse large cell lymphoma (n=7), T-cell/histiocyte-rich large B-cell lymphoma (n=2), anaplastic lymphoma kinase (ALK)-positive large B-cell lymphoma (n=1), follicular lymphoma (n=1), MALT lymphoma (n=1), and unclassifiable lymphoma (n=1). CONCLUSIONS: Multi-color flow cytometry can be a useful method for assessing bone marrow in staging NHL and also plays a complementary role, especially in detecting small numbers of lymphoma cells.
Antibodies, Monoclonal/immunology ; Antigens, CD19/immunology/metabolism ; Antigens, CD20/immunology/metabolism ; Bone Marrow/*pathology ; Female ; Flow Cytometry ; Humans ; Immunophenotyping ; Lymphoma, B-Cell/*pathology ; Male ; Neoplasm Staging ; Neprilysin/immunology/metabolism

As targeted drugs to B-cell malignancies, anti-CD20 monoclonal antibodies have been proved to be important in therapeutic antibody field. With three generations in more than ten years' development, the structures of these drugs have been improved, and many new indications have been found. Nowadays, these kinds of antibodies are not only used in the treatment of lymphoid malignancies, but also been proved to be useful in some autoimmune diseases treatment, and their new indications are still being expanded. With the optimization of their clinical dosage regimens, drug reaction has been increased, thus, therapeutic and side effects of anti-CD20 monoclonal antibody have been further improved as well. However, the exact mechanism of action of their combination therapy with other chemical drugs is still unclear, which remains to be further studied. This article reviewed new development of anti-CD20 therapeutic monoclonal antibodies research in recent years.
Antibodies, Monoclonal ; therapeutic use ; Antibodies, Monoclonal, Humanized ; therapeutic use ; Antigens, CD20 ; immunology ; Antineoplastic Agents ; therapeutic use ; Arthritis, Rheumatoid ; drug therapy ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell ; drug therapy ; Lymphoma, Non-Hodgkin ; drug therapy ; Rituximab ; therapeutic use

OBJECTIVETo assess the prognostic value of CD20(+) tumor-infiltrating lymphocytes (TILs) in early-stage breast cancer.

METHODSParaffin sections were collected from 130 cases of stage I-III breast cancer undergoing surgery between January, 2000 and December, 2002 in our hospital. Immunohistochemistry was used to analyze mesenchymal CD20(+) TILs infiltration in the tumor and evaluate its association with the density of CD4(+) and CD8(+) TILs. The association of CD20(+) TILs was evaluated with the histopathologic features, overall survival (OS), distant disease-free survival (DDFS), and disease-free survival (DFS) of the patients.

RESULTSAggregations of CD20(+) lymphocytes were observed in 37.69% (49/130) of the cases. CD3(+) T cells were found to aggregate around CD20(+) B cell aggregations to form lymphoid follicle-like structures. The aggregations of CD20(+) TILs were positively correlated with the densities of mesenchymal CD8+ and CD4(+) TILs. Overall, CD20(+) TIL aggregations were not significantly correlated with the outcomes of the patients, but multivariate COX regressions suggested that CD20(+) TIL aggregations were positively correlated with DDFS (HR=0.251, 95% CI=0.071-0.894, P=0.033) and OS (HR=0.325, 95% CI=0.103-1.028, P=0.056) in hormone receptor-negative patients but not in the positive patients. Further analysis suggested that post-operative adjuvant endocrine therapy significantly improved the OS of patients positive for hormone receptors without CD20(+) TIL aggregations (P=0.001).

CONCLUSIONThe long-term therapeutic effects of adjuvant endocrine therapy are correlated with CD20(+) TIL aggregations to affect prognostic value of CD20(+) TIL aggregations in early-stage breast cancer patients.

Antigens, CD20 ; metabolism ; B-Lymphocytes ; cytology ; Breast Neoplasms ; immunology ; pathology ; Disease-Free Survival ; Female ; Humans ; Lymphocytes, Tumor-Infiltrating ; cytology ; Middle Aged ; Neoplasm Invasiveness ; Prognosis ; Survival Rate

This study was aimed to investigate clinical and prognostic significances of 4 target antigens (CD19, CD20, CD22 and CD33) for antibody-based immunotherapy and to evaluate the applications of these antibody-based target therapy to adult acute lymphoblastic leukemia (ALL). The immunophenotype of 220 adult patients with ALL were analyzed by four-color flow Cytometry, and cytogenetic and molecular parameters were detected by conventional cytogenetics, fluorescence in situ hybridization, real-time quantitative PCR, nested PCR and DNA sequencing. The results showed that CD19 positive (CD19(+)) cases were more in female (46.4% vs. 23.4%, P = 0.006), elderly patients aged > 60 years (14.4% vs. 2.1%, P = 0.022), CD33(+) co-expression cases (47.8% vs. 12.0%, P = 0.001) and genetic high-risk group (55.8% vs. 20.8%, P = 0.002) compared with CD19 negative (CD19(-)) cases; CD20(+) cases had lower co-expression of CD13 than CD20(-) cases (31.6% vs.67.1%, P = 0.000) and no significant prognostic indications for CD20(+) was observed; CD22(+) cases had higher relapse rate at 12-month than CD22(-) cases (93.9% vs.57.1%, P = 0.041) in B-ALL patients; CD33(+) cases had higher incidence of Ph(+) than CD33(-) cases (43.5% vs.19.4%, P = 0.007) and significantly correlated with Ph(+) (r = 0.261, P = 0.006). It is concluded that elucidation of the characteristics of the target antigens (CD19, CD20, CD22, CD33) used for antibody-based immunotherapy will help hematologists making the correct decision whether and when to use these antibody-based target therapies.
Adolescent ; Adult ; Aged ; Antigens, CD19 ; immunology ; Antigens, CD20 ; immunology ; Child ; Female ; Humans ; Immunophenotyping ; Male ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; immunology ; Sialic Acid Binding Ig-like Lectin 2 ; immunology ; Sialic Acid Binding Ig-like Lectin 3 ; immunology ; Young Adult

Antibodies ; immunology ; therapeutic use ; Antigens, CD20 ; immunology ; Antilymphocyte Serum ; therapeutic use ; CD3 Complex ; immunology ; Diabetes Mellitus, Type 1 ; drug therapy ; immunology ; Humans ; Immunotherapy ; methods

Castleman's disease is a slowly progressive and rare lymphoproliferative disorder. Here, we report a 55-year-old woman with superior mediastinal Castleman's disease being misdiagnosed for a long term. We found a 4.3 cm mass localized in the superior mediastinum accompanied with severe clinical symptoms. The patient underwent an exploratory laparotomy, but the mass failed to be totally excised. Pathologic examination revealed a mediastinal mass of Castleman's disease. After radiotherapy of 30 Gy by 15 fractions, the patient no longer presented previous symptoms. At 3 months after radiotherapy of 60 Gy by 30 fractions, Computed tomography of the chest showed significantly smaller mass, indicating partial remission. Upon a 10-month follow-up, the patient was alive and free of symptoms.
Antigens, CD20 ; metabolism ; Castleman Disease ; diagnosis ; immunology ; pathology ; radiotherapy ; surgery ; Female ; Follow-Up Studies ; Humans ; Mediastinal Diseases ; diagnosis ; immunology ; pathology ; radiotherapy ; surgery ; Mediastinum ; diagnostic imaging ; pathology ; Middle Aged ; Multimodal Imaging ; Positron-Emission Tomography ; Radiotherapy, Intensity-Modulated ; Tomography, X-Ray Computed

Monoclonal antibodies (mAbs) have become a major class of therapeutic agents providing effective alternatives to treating various human diseases. To date, 15 mAbs have been approved by regulatory agencies in the world for clinical use in oncology indications. The selectivity and specificity, the unique pharmacokinetics, and the ability to engage and activate the host immune system differentiate these biologics from traditional small molecule anticancer drugs. mAb-based regimens have brought clinical benefits, including improvements in overall survival, to patients with a variety of cancers. Many challenges still remain, however, to fully realize the potential of these new medicines. With our further understanding of cancer biology, mechanism of antibody action, and advancement of antibody engineering technologies, many novel antibody formats or antibody-derived molecules are emerging as promising new generation therapeutics. Carefully designed and engineered, they retain the advantage of specificity and selectivity of original antibodies, but in the meantime acquire additional special features such as improved pharmacokinetics, increased selectivity, and enhanced anticancer efficacy. Promising clinical results are being generated with these newly improved antibody-based therapeutics.
Antibodies, Monoclonal ; therapeutic use ; Antibodies, Monoclonal, Humanized ; therapeutic use ; Antigens, CD20 ; immunology ; Humans ; Immunoconjugates ; therapeutic use ; Neoplasms ; drug therapy ; immunology ; Protein Engineering ; RANK Ligand ; immunology ; Receptor, Epidermal Growth Factor ; immunology ; Receptor, ErbB-2 ; immunology ; Vascular Endothelial Growth Factor A ; immunology

To date, more than 30 antibodies have been approved worldwide for therapeutic use. While the monoclonal antibody market is rapidly growing, the clinical use of therapeutic antibodies is mostly limited to treatment of cancers and immunological disorders. Moreover, antibodies against only five targets (TNF-alpha, HER2, CD20, EGFR, and VEGF) account for more than 80 percent of the worldwide market of therapeutic antibodies. The shortage of novel, clinically proven targets has resulted in the development of many distinct therapeutic antibodies against a small number of proven targets, based on the premise that different antibody molecules against the same target antigen have distinct biological and clinical effects from one another. For example, four antibodies against TNF-alpha have been approved by the FDA -- infliximab, adalimumab, golimumab, and certolizumab pegol -- with many more in clinical and preclinical development. The situation is similar for HER2, CD20, EGFR, and VEGF, each having one or more approved antibodies and many more under development. This review discusses the different binding characteristics, mechanisms of action, and biological and clinical activities of multiple monoclonal antibodies against TNF-alpha, HER-2, CD20, and EGFR and provides insights into the development of therapeutic antibodies.
Animals ; Antibodies, Monoclonal/*pharmacology/therapeutic use ; Antigens, CD20/immunology ; Drug Discovery ; Humans ; Immune System Diseases/*drug therapy/immunology ; *Immunotherapy/trends ; *Molecular Targeted Therapy ; Neoplasms/*drug therapy/immunology ; Receptor, Epidermal Growth Factor/immunology ; Receptor, erbB-2/immunology ; Tumor Necrosis Factor-alpha/immunology ; United States ; United States Food and Drug Administration ; Vascular Endothelial Growth Factor A/immunology

To date, more than 30 antibodies have been approved worldwide for therapeutic use. While the monoclonal antibody market is rapidly growing, the clinical use of therapeutic antibodies is mostly limited to treatment of cancers and immunological disorders. Moreover, antibodies against only five targets (TNF-alpha, HER2, CD20, EGFR, and VEGF) account for more than 80 percent of the worldwide market of therapeutic antibodies. The shortage of novel, clinically proven targets has resulted in the development of many distinct therapeutic antibodies against a small number of proven targets, based on the premise that different antibody molecules against the same target antigen have distinct biological and clinical effects from one another. For example, four antibodies against TNF-alpha have been approved by the FDA -- infliximab, adalimumab, golimumab, and certolizumab pegol -- with many more in clinical and preclinical development. The situation is similar for HER2, CD20, EGFR, and VEGF, each having one or more approved antibodies and many more under development. This review discusses the different binding characteristics, mechanisms of action, and biological and clinical activities of multiple monoclonal antibodies against TNF-alpha, HER-2, CD20, and EGFR and provides insights into the development of therapeutic antibodies.
Animals ; Antibodies, Monoclonal/*pharmacology/therapeutic use ; Antigens, CD20/immunology ; Drug Discovery ; Humans ; Immune System Diseases/*drug therapy/immunology ; *Immunotherapy/trends ; *Molecular Targeted Therapy ; Neoplasms/*drug therapy/immunology ; Receptor, Epidermal Growth Factor/immunology ; Receptor, erbB-2/immunology ; Tumor Necrosis Factor-alpha/immunology ; United States ; United States Food and Drug Administration ; Vascular Endothelial Growth Factor A/immunology