No abstract

BAFF (also known as BLyS), a member of the tumor necrosis factor superfamily, plays a critical role in the maturation and development of B cells. BAFF has three receptors on B cells, the most crucial of which is BR3. In this study, we demonstrate the biological outcome of BAFF blockade in cynomolgus monkeys using a soluble fusion protein consisting of human BR3 and human IgG1 Fc. In vitro, BR3-Fc blocked BAFF-mediated survival and proliferation of cynomolgus monkey B cells. Weekly treatment of cynomolgus monkeys with BR3-Fc for 13 to 18 weeks resulted in significant B-cell reduction in the peripheral blood and in lymphoid organs. CD21(high) B cells in lymphoid tissues, a subset analogous to human marginal zone B cells, expressed nearly twofold higher BR3 levels than did CD21(med) B cells. Lymphoid tissue flow cytometric analysis showed that BR3-Fc reduced this CD21(high) B-cell subset to a greater extent than it reduced CD21(med) B cells. Dual-label immunohistochemistry and morphometric image analysis supported these results by demonstrating that BR3-Fc reduced a significant proportion of the B cells within the splenic inner and outer marginal zones. These findings should prove very useful in guiding the desired therapeutic use of BR3-Fc for autoimmune diseases in the clinic.

[1][2][3][4] Since approval of a depleting anti-CD20 monoclonal antibody (mAb) (rituximab [Genentech, South San Francisco, CA; Biogen-IDEC, Cambridge, MA; Roche, Basel, Switzerland)] in 1997 for the treatment of non-Hodgkin lymphoma (NHL), almost 1 million patients have been treated with rituximab as a first or second line therapy, either alone or in combination with chemotherapy. In addition, rituximab maintenance therapy significantly prolongs tumor remission and patient survival in patients with indolent B-cell NHL or chronic lymphocytic leukemia (CLL). 5,6 More recently, rituximab has demonstrated clinical benefit in a variety of autoimmune diseases including rheumatoid arthritis, pemphigus vulgaris, immune thrombocytopenia and autoimmune hemolytic anemia. 4,7,8 As a result, understanding the contribution of B lymphocytes to human autoimmune diseases received revived interest, and several mechanisms have been postulated to participate in disease pathogenesis, including autoantibody production, B-cell antigen presentation, cytokine generation, and lymphorganogenesis. 2,3,9,10 Inhibition of different combinations of these mechanisms is probably responsible for clinical benefit.The success of anti-CD20 B-cell immunotherapy spearheaded a large number of preclinical and clinical efforts to understand in vivo mechanisms of drug activity. Direct elimination of malignant B cells through antigen-dependent cell-mediated cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), and apoptosis have been demonstrated as the main mechanisms of action in a variety of systems including mouse xenotumors and normal mouse and nonhuman primate (NHP) B-cell subsets. 11-15 Two major determinants affecting normal mouse B-cell depletion have been identified. 13,16 First, the kinetics of B-cell recirculation determines the speed and magnitude of anti-CD20 mAb-mediated B-cell depletion. Cells with higher recirculatory kinetics from blood, lymph nodes, and spleen follicular areas are depleted faster and more completely than cells with lower recirculatory kinetics (eg, peritoneal cavity [PEC], marginal zone [MZ], germinal centers [GC]). Second, the local microenvironment influences the extent of B-cell depletion. Marginal zone, Peyer patches (PP), germinal center, memory, and peritoneal cavity B cells exhibit greater resistance to depletion in mice and nonhuman primates. Reduced recruitment of effector mechanisms in the peritoneal cavity as well as intrinsic B1 B cells properties appear to cause the slower kinetics and B-cell reduction after anti-CD20 mAb treatment. 16 Differences across mouse strains and epitopes recognized by anti-CD20 antibodies used for depletion might explain the differential effects seen on mouse splenic marginal zone B cells. 13,16 Small but consistent numbers of residual B cells can be detected in most lymphoid organs in mice and primates treated with anti-CD20 mAbs. One possibility of achieving a more complete B-cell reduction would be to block B-cell survival signals in addition to Submitted April 30, 2007...

Efalizumab (anti-CD11a) interferes with LFA-1/ICAM-1 binding and inhibits several key steps in psoriasis pathogenesis. This study characterizes the effects of efalizumab on T-cell activation responses and expression of surface markers on human circulating psoriatic T cells during a therapeutic trial. Our data suggest that efalizumab may induce a unique type of T-cell hyporesponsiveness, directly induced by LFA-1 binding, which is distinct from conventional anergy described in animal models. Direct activation of T cells through different activating receptors (CD2, CD3, CD3/28) is reduced, despite T cells being fully viable. This hyporesponsiveness was spontaneously reversible after withdrawal of the drug, and by IL-2 in vitro. In contrast to the state of anergy, Ca(+2) release is intact during efalizumab binding. Furthermore, lymphocyte function-associated antigen-1 (LFA-1) blockade resulted in an unexpected downregulation of a broad range of surface molecules, including the T-cell receptor complex, co-stimulatory molecules, and integrins unrelated to LFA-1, both in the peripheral circulation and in diseased skin tissue. These observations provide evidence for the mechanism of action of efalizumab. The nature of this T-cell hyporesponsiveness suggests that T-cell responses may be reduced during efalizumab therapy, but are reversible after ceasing efalizumab treatment.

No abstract

Background: Cynomolgus monkeys are widely used animal models in biomedical research. The differences between cynomolgus monkey and human B cells are not completely understood. However, these differences are of crucial importance for interpretation of data from studies on new therapeutic agents aimed at B-cell depletion, such as anti-CD20 monoclonal antibodies. Methods: Multicolor fluorescence-activated cell sorting analysis of peripheral blood B cells was performed on samples treated ex vivo with the anti-CD20 therapeutic monoclonal antibody, Rituxan, in a whole blood matrix. Results: In contrast to humans, cynomolgus monkeys had two distinct B-cell subsets, CD20 high CD40 low CD21 Ϫ and CD20 low CD40 high CD21 ϩ . These B-cell subsets had a 2.5-fold difference in the EC 50 for Rituxan binding and differed significantly in their in vitro susceptibility to Rituxan depletion. Human B cells were similar to the

PRO70769 is a humanized IgG1 monoclonal antibody against the CD20 molecule that is present on normal and malignant B cells. PRO70769 is being evaluated for treatment of B-cell-mediated diseases and is in a phase 1 trial for rheumatoid arthritis. As part of the preclinical toxicology evaluation, B-cell depletion profiles and safety of PRO70769 were assessed in cynomolgus monkeys. Animals were administered drug (IV) on days 1 and 15 with 10, 50, or 100 mg/kg PRO70769 and killed 2 weeks after the second dose and after a 3-month recovery period. In a parallel study, animals were not necropsied but instead were retreated with a second cycle of PRO70769 administered under an identical regimen. PRO70769 suppressed B cells in the blood to undetectable levels and significantly reduced B cells in lymphoid tissues. Splenic B cells were depleted to a greater extent compared with lymph node B cells. A second cycle of treatment resulted in a greater extent of depletion in lymphoid tissues compared with the depletion observed after one cycle of treatment; however, residual B cells in lymphoid tissues were still detectable, even at the highest dose. The rate of B-cell recovery in peripheral blood appeared similar between one and two cycles of treatment. Upon depletion there was a change in the profile of lymph node B-cell subsets. After recovery, B-cell subsets were reconstituted to normal levels. Depletion of CD20-expressing cells and lymphoid follicular atrophy were the only treatment-related effects.

Background: Cynomolgus monkeys (Macaca fascicularis) are widely used animal models in biomedical research. However, the phenotypic characteristics of cynomolgus monkey (CM) B cells in peripheral blood (PB) and lymphoid organs are poorly understood. Methods: FACS analyses of PB-, spleen-, lymph node (LN)-, and bone marrow (BM)-derived B cells were performed. Results: CM peripheral blood B cells have a smaller fraction of CD27Ϫ (naive) cells (ϳ40%), as compared to human blood samples (ϳ70%). Similar to humans, an early activation marker, CD23, is expressed more on CD27