Repeated administration of dapirolizumab pegol in a randomised phase I study is well tolerated and accompanied by improvements in several composite measures of systemic lupus erythematosus disease activity and changes in whole blood transcriptomic profiles.

OBJECTIVES: Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease associated with diffuse immune cell dysfunction. CD40-CD40 ligand (CD40L) interaction activates B cells, antigen-presenting cells and platelets. CD40L blockade might provide an innovative treatment for systemic autoimmune disorders. We investigated the safety and clinical activity of dapirolizumab pegol, a polyethylene glycol conjugated anti-CD40L Fab' fragment, in patients with SLE. METHODS: This 32-week randomised, double-blind, multicentre study (NCT01764594) evaluated repeated intravenous administration of dapirolizumab pegol in patients with SLE who were positive for/had history of antidouble stranded DNA/antinuclear antibodies and were on stable doses of immunomodulatory therapies (if applicable). Sixteen patients were randomised to 30 mg/kg dapirolizumab pegol followed by 15 mg/kg every 2 weeks for 10 weeks; eight patients received a matched placebo regimen. Randomisation was stratified by evidence of antiphospholipid antibodies. Patients were followed for 18 weeks after the final dose. RESULTS: No serious treatment-emergent adverse events, thromboembolic events or deaths occurred. Adverse events were mild or moderate, transient and resolved without intervention. One patient withdrew due to infection.Efficacy assessments were conducted only in patients with high disease activity at baseline. Five of 11 (46%) dapirolizumab pegol-treated patients achieved British Isles Lupus Assessment Group-based Composite Lupus Assessment response (vs 1/7; 14% placebo) and 5/12 (42%) evaluable for SLE Responder Index-4 responded by week 12 (vs 1/7; 14% placebo). Mechanism-related gene expression changes were observed in blood RNA samples. CONCLUSIONS: Dapirolizumab pegol could be an effective biological treatment for SLE. Further studies are required to address efficacy and safety. TRIAL REGISTRATION NUMBER: NCT01764594.

Pristane-Accelerated Autoimmune Disease in (SWR X NZB) F1 Mice Leads to Prominent Tubulointerstitial Inflammation and Human Lupus Nephritis-Like Fibrosis.

Mouse models lupus nephritis (LN) have provided important insights into disease pathogenesis, although none have been able to recapitulate all features of the human disease. Using comprehensive longitudinal analyses, we characterized a novel accelerated mouse model of lupus using pristane treatment in SNF1 (SWR X NZB F1) lupus prone mice (pristane-SNF1 mice). Pristane treatment in SNF1 mice accelerated the onset and progression of proteinuria, autoantibody production, immune complex deposition and development of renal lesions. At week 14, the pristane-SNF1 model recapitulated kidney disease parameters and molecular signatures seen in spontaneous disease in 36 week-old SNF1 mice and in a traditional IFNα-accelerated NZB X NZW F1 (BWF1) model. Blood transcriptome analysis revealed interferon, plasma cell, neutrophil, T-cell and protein synthesis signatures in the pristane-SNF1 model, all known to be present in the human disease. The pristane-SNF1 model appears to be particularly useful for preclinical research, robustly exhibiting many characteristics reminiscent of human disease. These include i) a stronger upregulation of the cytosolic nucleic acid sensing pathway, which is thought to be key component of the pathogenesis of the human disease, and ii) more prominent kidney interstitial inflammation and fibrosis, which have been both associated with poor prognosis in human LN. To our knowledge, this is the only accelerated model of LN that exhibits a robust tubulointerstitial inflammatory and fibrosis response. Taken together our data show that the pristane-SNF1 model is a novel accelerated model of LN with key features similar to human disease.

BAFF (B cell activating factor) transcript level in peripheral blood of patients with SLE is associated with same-day disease activity as well as global activity over the next year.

OBJECTIVES: Quantitating gene expression is a potential method of developing biomarkers in systemic lupus erythematosus (SLE). Because of the known pathological role of B cell activating factor (BAFF) in SLE, we explored the association between BAFF gene expression and clinical activity in SLE. METHODS: A total of 275 patients with SLE completed this phase of a prospective observational study. At entry into the study, the BAFF gene expression levels were determined in peripheral blood RNA. Serum concentration of BAFF protein was also measured. We then determined clinical associations with SLE disease history, SLE activity on the same day and SLE activity over the course of the next year. RESULTS: Elevated BAFF gene expression was associated with a history of more leucopenia and serologically with more autoantibodies (anti-dsDNA, anti-Sm, anti-Ro, anti-La and anti-RNP) and low complement. Patients with higher amounts of BAFF transcript had higher measured levels of clinical disease activity. Initial high levels of BAFF gene expression also predicted increased disease activity over the course of the next year. In contrast, serum concentration of BAFF protein was not strongly associated with same-day global disease activity or with future disease activity. CONCLUSIONS: BAFF gene expression level is associated with clinical and serological SLE activity on the same day and predictive of clinical activity over the next year. BAFF gene expression is a better measure and predictor of SLE disease activity than the serum BAFF protein level.

Anti-BDCA2 monoclonal antibody inhibits plasmacytoid dendritic cell activation through Fc-dependent and Fc-independent mechanisms.

Type I interferons (IFN-I) are implicated in the pathogenesis of systemic lupus erythematosus (SLE). In SLE, immune complexes bind to the CD32a (FcγRIIa) receptor on the surface of plasmacytoid dendritic cells (pDCs) and stimulate the secretion of IFN-I from pDCs. BDCA2 is a pDC-specific receptor that, when engaged, inhibits the production of IFN-I in human pDCs. BDCA2 engagement, therefore, represents an attractive therapeutic target for inhibiting pDC-derived IFN-I and may be an effective therapy for the treatment of SLE. In this study, we show that 24F4A, a humanized monoclonal antibody (mAb) against BDCA2, engages BDCA2 and leads to its internalization and the consequent inhibition of TLR-induced IFN-I by pDCs in vitro using blood from both healthy and SLE donors. These effects were confirmed in vivo using a single injection of 24F4A in cynomolgus monkeys. 24F4A also inhibited pDC activation by SLE-associated immune complexes (IC). In addition to the inhibitory effect of 24F4A through engagement of BDCA2, the Fc region of 24F4A was critical for potent inhibition of IC-induced IFN-I production through internalization of CD32a. This study highlights the novel therapeutic potential of an effector-competent anti-BDCA2 mAb that demonstrates a dual mechanism to dampen pDC responses for enhanced clinical efficacy in SLE.

BAD is a pro-survival factor prior to activation of its pro-apoptotic function.

The mammalian BAD protein belongs to the BH3-only subgroup of the BCL-2 family. In contrast to its known pro-apoptotic function, we found that endogenous and overexpressed BAD(L) can inhibit cell death in neurons and other cell types. Several mechanisms regulate the conversion of BAD from an anti-death to a pro-death factor, including alternative splicing that produces the N-terminally truncated BAD(S). In addition, caspases convert BAD(L) into a pro-death fragment that resembles the short splice variant. The caspase site that is selectively cleaved during cell death following growth factor (interleukin-3) withdrawal is conserved between human and murine BAD. A second cleavage site that is required for murine BAD to promote death following Sindbis virus infection, gamma-irradiation, and staurosporine treatment is not conserved in human BAD, consistent with the inability of human BAD to promote death with these stimuli. However, loss of the BAD N terminus by any mechanism is not always sufficient to activate its pro-death activity, suggesting that the N terminus is a regulatory domain rather than an anti-death domain. These findings suggest that BAD is more than an inert death factor in healthy cells; it is also a pro-survival factor, prior to its role in promoting cell death.

BAD and glucokinase reside in a mitochondrial complex that integrates glycolysis and apoptosis.

Glycolysis and apoptosis are considered major but independent pathways that are critical for cell survival. The activity of BAD, a pro-apoptotic BCL-2 family member, is regulated by phosphorylation in response to growth/survival factors. Here we undertook a proteomic analysis to assess whether BAD might also participate in mitochondrial physiology. In liver mitochondria, BAD resides in a functional holoenzyme complex together with protein kinase A and protein phosphatase 1 (PP1) catalytic units, Wiskott-Aldrich family member WAVE-1 as an A kinase anchoring protein, and glucokinase (hexokinase IV). BAD is required to assemble the complex in that Bad-deficient hepatocytes lack this complex, resulting in diminished mitochondria-based glucokinase activity and blunted mitochondrial respiration in response to glucose. Glucose deprivation results in dephosphorylation of BAD, and BAD-dependent cell death. Moreover, the phosphorylation status of BAD helps regulate glucokinase activity. Mice deficient for BAD or bearing a non-phosphorylatable BAD(3SA) mutant display abnormal glucose homeostasis including profound defects in glucose tolerance. This combination of proteomics, genetics and physiology indicates an unanticipated role for BAD in integrating pathways of glucose metabolism and apoptosis.

Bad-deficient mice develop diffuse large B cell lymphoma.

The proapoptotic activity of the "BH3-only" molecule BAD can be differentially regulated by survival factor signaling. Bad-deficient mice lacking both BAD long and BAD short proteins proved viable, and most cell types appeared to develop normally. BAD did not exclusively account for cell death after withdrawal of survival factors, but it was an intermediate for epidermal growth factor- or insulin-like growth factor I-countered apoptosis, consistent with a "sensitizing" BH3-only molecule. Lymphocytes developed normally with no premalignant hyperplasia, but they displayed subtle abnormalities in proliferation and IgG production. Despite the minimal phenotype, Bad-deficient mice progressed, with aging, to diffuse large B cell lymphoma of germinal center origin. Exposure of Bad-null mice to sublethal gamma-irradiation resulted in an increased incidence of pre-T cell and pro-/pre-B cell lymphoblastic leukemia/lymphoma. Thus, proapoptotic BAD suppresses tumorigenesis in the lymphocyte lineage.

Survival factor-mediated BAD phosphorylation raises the mitochondrial threshold for apoptosis.

Growth factor suppression of apoptosis correlates with the phosphorylation and inactivation of multiple proapoptotic proteins, including the BCL-2 family member BAD. However, the physiological events required for growth factors to block cell death are not well characterized. To assess the contribution of BAD inactivation to cell survival, we generated mice with point mutations in the BAD gene that abolish BAD phosphorylation at specific sites. We show that BAD phosphorylation protects cells from the deleterious effects of apoptotic stimuli and attenuates death pathway signaling by raising the threshold at which mitochondria release cytochrome c to induce cell death. These findings establish a function for endogenous BAD phosphorylation, and elucidate a mechanism by which survival kinases block apoptosis in vivo.

Activation of BAD by therapeutic inhibition of epidermal growth factor receptor and transactivation by insulin-like growth factor receptor.

Novel cancer chemotherapeutics are required to induce apoptosis by activating pro-apoptotic proteins. Both epidermal growth factor (EGF) and insulin-like growth factor (IGF) provide potent survival stimuli in many epithelia, and activation of their receptors is commonly observed in solid human tumors. Here we demonstrate that blockade of the EGF receptor by a new drug in phase III clinical trails for cancer, ZD1839, potently induces apoptosis in mammary epithelial cell lines and primary cultures, as well as in a primary pleural effusion from a breast cancer patient. We identified the mechanism of apoptosis induction by ZD1839. We showed that it prevents cell survival by activating the pro-apoptotic protein BAD. Moreover, we demonstrate that IGF transactivates the EGF receptor and that ZD1839 blocks IGF-mediated phosphorylation of MAPK and BAD. Many cancer therapies kill tumor cells by inducing apoptosis as a consequence of targeting DNA; however, the threshold at which apoptosis can be triggered through DNA damage is often different from that in normal cells. Our results indicate that by targeting a growth factor-mediated survival signaling pathway, BAD phosphorylation can be manipulated therapeutically to induce apoptosis.