anti-B7 Antibody Research Articles

Mechanism of spontaneous activation of B cells in patients with systemic lupus erythematosus. Analysis with anti-class II antibody.

The mechanism of spontaneous activation of B cells in patients with systemic lupus erythematosus (SLE) was analyzed by using anti-class II monoclonal antibodies in vitro. B cells from SLE patients showed enhanced proliferation and Ig production by in vitro culture without any stimulation. The number of Ig-producing cells increased during a 5-day culture period, but the addition of anti-class II antibodies such as anti-HLA-DR, DQ, or DP monoclonal antibodies inhibited these B cell responses in a dose-dependent manner. Anti-class I and anti-B1 antibody gave no effect. The inhibitory effect of anti-class II antibodies on B cell responses became more remarkable when B cells were cultured on a longer period. By a Percoll gradient density centrifugation, Ig-producing cells were enriched in the lower density fraction, but became depleted in the higher density fraction. However, B cells of the higher density fraction developed into Ig-producing cells after 5 days of culture and anti-class II antibodies inhibited this development. When mitomycin C- and cycloheximide-treated B cells were added to the in vitro culture of B cells as a stimulator, B cell responses were enhanced in a dose-dependent manner. T cells treated with mitomycin C and cycloheximide had no enhancing effect on B cell responses. Furthermore, the enhancing effect of the stimulator B cells was inhibited by the pretreatment of stimulator B cells with anti-class II antibodies. These results suggest that in patients with SLE the abnormality exists in B precursor cells which are easily activated by interacting with other B cells to differentiate into Ig-producing cells and anti-class II antibodies inhibit the B cell activation by interfering with this cellular interaction.

Prolonged disease-free survival after autologous bone marrow transplantation in patients with non-Hodgkin's lymphoma with a poor prognosis.

Despite advances in the primary treatment of non-Hodgkin's lymphoma, relapse is common and treatment after relapse is unsatisfactory. Autologous bone marrow transplantation, although sometimes successful, has generally had disappointing results. We conducted a trial of such transplantation in patients with relapsed non-Hodgkin's lymphoma, using strict criteria in selecting patients; we included only those in whom disease was minimal after conventional treatment (nodal disease less than 2 cm and bone marrow involvement less than or equal to 5 percent on histologic examination) and whose tumor cells expressed the B1 antigen. Forty-nine patients meeting these criteria received cyclophosphamide and whole-body irradiation supported by transplantation of autologous bone marrow that had been treated in vitro with anti-B1 monoclonal antibody and complement. All patients had features of a poor prognosis, including relapse from primary chemotherapy, histologic conversion to more aggressive disease, and extra-nodal dissemination. Thirty-three patients had a history of bone marrow involvement--16 at the time that marrow was obtained. Hematologic and immunologic engraftment was achieved in all patients. Only two treatment-related deaths occurred, from venoocclusive disease of the liver and intracerebral hemorrhage, respectively. Disease-free remission without maintenance therapy has lasted from greater than 2 to greater than 52 months in 34 patients (median follow-up, greater than 11 months). These results are similar to those obtained in patients with advanced, high-grade non-Hodgkin's lymphoma treated with primary combination chemotherapy. This study demonstrates that autologous bone marrow transplantation has tolerable toxicity and high efficacy in a subset of patients who are otherwise incurable but still responsive to cytoreductive therapy. The results suggest a role for such transplantation in the treatment of selected patients with newly diagnosed non-Hodgkin's lymphoma.

Immunologic classification of lymphoma and lymphoid leukemia

Important insights into lymphocyte differentiation and the cellular origins of lymphoma and lymphoid leukemia have been gained through the use of monoclonal antibodies that define cell surface antigens and molecular probes that identify immunoglobulin and T cell receptor genes. Results of these studies have been combined with markers such as surface membrane and cytoplasmic immunoglobulin on B lymphocytes, sheep erythrocyte receptors on T lymphocytes, and cytochemical stains. Utilising all of the above markers, it is now clear that acute lymphoblastic leukemia (ALL) is heterogeneous. Furthermore, monoclonal antibodies that identify B cells such as the anti-B1 and anti-B4 antibodies in cobination with studies of immunoglobulin gene rearrangement have demonstrated that virtually all cases of non-T-ALL invole B lymphocytes. At least six distinct subgroups of non-T-ALL can now be identified. T-ALL is subdivided by the anti-Leu-9, anti-Leu-1, and additional antibodies that separate T lymphocyte subsets into three primary subgroups. Monoclonal antibodies are also useful in the subclassification of non-Hodgkin's lymphoma, and certain distinct markers can be correlated with morphologic classification.

Hematologic Engraftment and Immune Reconstitution Posttransplantation With Anti-B1 Purged Autologous Bone Marrow

Hematologic engraftment and immune reconstitution were examined in patients who received cyclophosphamide and total body irradiation therapy followed by infusion of autologous bone marrow purged with anti-B1 monoclonal antibody (MoAb) and complement as therapy for non-Hodgkin's lymphoma. Hematologic engraftment was prompt with return of greater than or equal to 0.5 X 10(3)/microL granulocytes and greater than or equal to 2 X 10(4)/microL platelets at a median of 26 and 29 days posttransplant, respectively. Immunologic reconstitution, in contrast, was prolonged. Normal numbers of circulating B cells were consistently noted by five months posttransplant, whereas return of normal immunoglobulin levels in some patients did not occur for one year. Normal numbers of T cells were evident within the first month posttransplant, but a reversed T4:T8 ratio persisted in some patients up to three years. In vitro responses of either B cells to triggers of activation or of T cells to mitogens and antigens were not normal for at least three months posttransplant. Natural killer (NK) cells predominated early after transplant and may demonstrate cytotoxicity against tumor cells. Our studies demonstrate that transplantation with anti-B1 purged autologous bone marrow results in complete hematologic and delayed immunologic engraftment. No significant acute or chronic clinical toxicities have been observed.

Use of radiolabeled monoclonal anti-B1 antibody for B lymphocyte imaging in rhesus monkeys

Imaging tissues rich in B lymphocytes in man using a radiolabeled monoclonal anti-B cell antibody would be extremely useful in the clinical staging of non-Hodgkins lymphomas. Studies were done in rhesus monkeys using radiolabeled monoclonal anti-B1 antibody to determine the feasibility of such an approach. Immunohistologic studies demonstrated that infused monoclonal anti-B1 binds in vivo with specificity to B cells in lymph nodes and spleen. The kinetics of clearance of 131I-labeled anti-B1 were determined. The B lymphocyte-rich spleen could be readily visualized by gamma camera scanning without significant background and without the need for image intensification or blood background subtraction techniques. These data support the feasibility of using anti-B1 for staging B cell lymphomas in man.

Purified immunotoxins that are reactive with human lymphoid cells. Monoclonal antibodies conjugated to the ribosome-inactivating proteins gelonin and the pokeweed antiviral proteins.

Seven different monoclonal antibodies of the IgG class that are reactive with four different antigens on human lymphoid cells were utilized to form immunotoxins with the ribosome-inactivating proteins gelonin and the three known pokeweed antiviral proteins. Thirteen different immunotoxin combinations were prepared. The ribosome-inactivating proteins were modified with 2-iminothiolane. The sulfhydryl groups so introduced were reacted with maleimido groups or with dithiopyridyl groups that had been introduced into the antibodies. The toxin-antibody conjugates so formed were purified by affinity chromatography on protein A-Sepharose CL-4B, ion exchange chromatography, and by gel filtration and were characterized by polyacrylamide-dodecyl sulfate gel electrophoresis. The purified immunotoxins were free of nonconjugated monomeric proteins and aggregates of very high molecular weight. All the immunotoxins showed the specific binding of the component antibody as measured by indirect immunofluorescence binding assays. The activities of the ribosome-inactivating proteins were unaffected by conjugation where the cross-link to the antibody contained a disulfide bond and when assayed after reductive cleavage of the linker. Disulfide-linked immunotoxins with six of the antibodies were highly cytotoxic for the target cells. However, immunotoxins containing an anti-B1 antibody showed no cytotoxicity.

The B cell surface molecule B1 is functionally linked with B cell activation and differentiation.

The B1 molecule is a 32,000 m.w. phosphorylated cell surface protein expressed exclusively by B cells from the mid pre-B until the plasma cell stage of differentiation. Two monoclonal antibodies (gamma 2a and mu) reactive with this molecule were used to assess the role of B1 in B cell activation, proliferation, and differentiation. The anti-B1 antibodies at concentrations ranging from 0.1 to 100 micrograms/ml significantly inhibited B cell proliferation induced by anti-mu antibodies, Staphylococcus aureus Cowan strain 1, activated T cells, and Epstein Barr virus. Although capable of inhibiting proliferation, anti-B1 antibody in soluble form or coupled to beads did not activate B cells or induce proliferation. Antibodies of comparable isotypes or against other B cell-restricted antigens, including B2, B4, B5, and HB-5, did not inhibit activation. Pretreatment of B cells with anti-B1 antibody did not inhibit activation, indicating that B cells had to be cultured with anti-B1 antibody for anti-B1-mediated inhibition to occur. Maximum inhibition was obtained when anti-B1 antibody was added at the initiation of culture. In agreement with this, growth factor-dependent proliferation of preactivated B cells was not inhibited by anti-B1 antibodies. Comparable inhibition of B cell activation was noted with antibodies reactive with class II antigens of the major histocompatibility complex with the exception that anti-B1 antibody inhibited immunoglobulin secretion in pokeweed mitogen assays, whereas anti-DR antibody did not. These results suggest that the B1 molecule may serve a central role in the regulation of B cell activation and differentiation.

Generation of a cloned NK cell line derived from the "null cell" fraction of human peripheral blood.

The present studies were designed to determine the conditions for generation of human NK clones. First the "null cell" (NC) fraction of human peripheral blood mononuclear cells, which contains the NK effectors, was purified using negative selection with anti-T3, anti-T8, anti-B1, and anti-Mo2 monoclonal antibodies. Subsequently, NC were cloned by limiting dilution using a combination of phytohemagglutinin (PHA) and lymphocyte-conditioned medium (LCM) as an initial stimulus. Colonies could be easily obtained with this procedure, but the maintenance of long-term growth of the cultures represented a major problem. A cloned cell line termed JT1 was generated and has been proliferating continuously in culture for more than 6 mo. The phenotype of JT1 cells was analyzed several times with a series of monoclonal antibodies. These cells did not express surface markers related to thymic (T3-, T4-, T8-, T11-), B cells (B1-, J5-), or myelomonocytic (Mo1-, Mo2-, MY7-) differentiation. In contrast, 95% of JT1 cells reacted with the anti-T10 monoclonal antibody. T9, Ia, and J2 antigens were also present on JT1 cells, but their expression appeared variable from one determination to another. This cloned cell line maintains a strong cytotoxicity against common NK targets, such as K562 and Molt 4 cell lines, and a moderate ADCC activity. In addition, after several months in culture, JT1 cells are still capable of being regulated by interferon, because this lymphokine rapidly enhances cytotoxicity against K562 cells.

The Reaction Specificity of Various Monoclonal Antibodies and Cellular Origin and Differentiation of Cultured Cell Lines Derived from Human Leukemias and Lymphomas

The specificity of 12 monoclonal antibodies was assessed by indirect immunofluorescence assay using 25 cultured cell lines derived from various human hemopoietic tumor cells. The reactivity of these 12 monoclonal antibodies was highly specific. However, the titer of the antibody was found to differ among these cell lines. Therefore, titration of each monoclonal antibody requires the use of several cultured cell lines in order to determine an appropriate concentration for clinical use. Anti-Leu-1 and 10.2 (anti-human Lyt-2) antibodies reacted with all T-cell lines of various stages of differentiation. Therefore, anti-Leu-1 and 10.2 are considered to react with pan T-ccll antigen (the so-called human T-lymphocyte antigen). 9.6 (anti-human Lyt-3) antibody is considered to react with E-receptor-associated antigen. All T-cell lines reactive with OKT4, OKT6 and OKT8 antibodies are derived from thymic T-ALL, and they are considered to belong to the same stage of differentiation as normal cortical thymocytes. Anti-T monoclonal antibodies designated as the OKT-series are very useful for analysis of the differentiation of T-cell malignancies. J5 antibody, which was reported to have the same reaction specificity as CALLA, reacted with cultured cell lines derived from common ALL, C/T hybrid type ALL, pre-B ALL and Burkitt's lymphoma. Therefore, J5 is considered to be a specific marker of lymphoid malignancies derived from lymphoid stem cells, some pre-T cells, pre-B cells and some immature B-cells. Anti-B1 antibody is considered to be reactive with a part of J5-positive common ALL, immature B-cell malignancy and mature B-cell malignancy, but unreactive with immunoblast and plasma cell malignancy. OK11 antibody showed almost the same reaction pattern as the heteroantiserum against Ia-like antigen. OKM1 and anti-Mo1 antibodies were found to react only with the P31/Fujioka monocytoid cell line. The monoclonal antibodies specific for certain differentiation antigens will be very useful for elucidating the cellular origin, stage of differentiation and subset identification of human hemopoietic tumor cells.