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

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

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

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

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

The lower expression of CD20 antigen molecules on the B cell membrane is the primary characteristic of B-chronic lymphocytic leukemia (B-CLL). In this paper, we combined laser scanning confocal microscopy (LSCM) and quantum dots labeling to detect the expression and distribution of CD20 molecules on CD20+B lymphocyte surface. Simultaneously, we investigated the morphology and ultrastructure of the B lymphocytes that belonged to the normal persons and B-CLL patients through utilizing the atomic force microscope (AFM). In addition, we measured the force spectroscopy of CD20 antigen-antibody binding using the AFM tips modified with CD20 antibody. The fluorescent images indicated that the density of CD20 of normal CD20+B lymphocytes was much higher than that of B-CLL CD20+B cells. The AFM data show that ultrastructure of B-CLL CD20+B lymphocytes became more complicated. Moreover, the single molecular force spectroscopy data show that the special force of CD20 antigen-antibody was four times bigger than the nonspecific force between the naked AFM tip and cell surface. The force map showed that CD20 molecules distributed homogeneously on the normal CD20+B lymphocytes, whereas, the CD20 molecules distributed heterogenous on B-CLL CD20+B lymphocytes. Our data provide visualized evidence for the phenomenon of low-response to rituximab therapy on clinical. Meanwhile, AFM is possible to be a powerful tool for development and screening of drugs for pharmacology use.
Antigen-Antibody Reactions ; immunology ; Antigens, CD20 ; immunology ; B-Lymphocytes ; immunology ; ultrastructure ; Binding Sites, Antibody ; Cell Membrane ; immunology ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell ; immunology ; Microscopy, Atomic Force ; Microscopy, Confocal ; Quantum Dots

Animals ; Antigens, CD20 ; metabolism ; Brain Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Disease Models, Animal ; Female ; Humans ; Ki-67 Antigen ; metabolism ; Leukocyte Common Antigens ; metabolism ; Lymphoma, Large B-Cell, Diffuse ; immunology ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation

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