Epratuzumab (humanised anti-CD22 antibody) in primary Sjögren's syndrome: an open-label phase I/II study

This open-label, phase I/II study investigated the safety and efficacy of epratuzumab, a humanised anti-CD22 monoclonal antibody, in the treatment of patients with active primary Sjögren's syndrome (pSS). Sixteen Caucasian patients (14 females/2 males, 33–72 years) were to receive 4 infusions of 360 mg/m² epratuzumab once every 2 weeks, with 6 months of follow-up. A composite endpoint involving the Schirmer-I test, unstimulated whole salivary flow, fatigue, erythrocyte sedimentation rate (ESR), and immunoglobulin G (IgG) was devised to provide a clinically meaningful assessment of response, defined as a ≥20% improvement in at least two of the aforementioned parameters, with ≥20% reduction in ESR and/or IgG considered as a single combined criterion. Fourteen patients received all infusions without significant reactions, 1 patient received 3, and another was discontinued due to a mild acute reaction after receiving a partial infusion. Three patients showed moderately elevated levels of Human anti-human (epratuzumab) antibody not associated with clinical manifestations. B-cell levels had mean reductions of 54% and 39% at 6 and 18 weeks, respectively, but T-cell levels, immunoglobulins, and routine safety laboratory tests did not change significantly. Fifty-three percent achieved a clinical response (at ≥20% improvement level) at 6 weeks, with 53%, 47%, and 67% responding at 10, 18, and 32 weeks, respectively. Approximately 40%–50% responded at the ≥30% level, while 10%–45% responded at the ≥50% level for 10–32 weeks. Additionally, statistically significant improvements were observed in fatigue, and patient and physician global assessments. Further, we determined that pSS patients have a CD22 over-expression in their peripheral B cells, which was downregulated by epratuzumab for at least 12 weeks after the therapy. Thus, epratuzumab appears to be a promising therapy in active pSS, suggesting that further studies be conducted.     

Epratuzumab (humanised anti-CD22 antibody) in primary Sjögren's syndrome: an open-label phase I/II study

This open-label, phase I/II study investigated the safety and efficacy of epratuzumab, a humanised anti-CD22 monoclonal antibody, in the treatment of patients with active primary Sj?gren's syndrome (pSS). Sixteen Caucasian patients (14 females/2 males, 33-72 years) were to receive 4 infusions of 360 mg/m2 epratuzumab once every 2 weeks, with 6 months of follow-up. A composite endpoint involving the Schirmer-I test, unstimulated whole salivary flow, fatigue, erythrocyte sedimentation rate (ESR), and immunoglobulin G (IgG) was devised to provide a clinically meaningful assessment of response, defined as a > or = 20% improvement in at least two of the aforementioned parameters, with > or = 20% reduction in ESR and/or IgG considered as a single combined criterion. Fourteen patients received all infusions without significant reactions, 1 patient received 3, and another was discontinued due to a mild acute reaction after receiving a partial infusion. Three patients showed moderately elevated levels of Human anti-human (epratuzumab) antibody not associated with clinical manifestations. B-cell levels had mean reductions of 54% and 39% at 6 and 18 weeks, respectively, but T-cell levels, immunoglobulins, and routine safety laboratory tests did not change significantly. Fifty-three percent achieved a clinical response (at > or = 20% improvement level) at 6 weeks, with 53%, 47%, and 67% responding at 10, 18, and 32 weeks, respectively. Approximately 40%-50% responded at the > or = 30% level, while 10%-45% responded at the > or = 50% level for 10-32 weeks. Additionally, statistically significant improvements were observed in fatigue, and patient and physician global assessments. Further, we determined that pSS patients have a CD22 over-expression in their peripheral B cells, which was downregulated by epratuzumab for at least 12 weeks after the therapy. Thus, epratuzumab appears to be a promising therapy in active pSS, suggesting that further studies be conducted.     

Epratuzumab inhibits the production of the proinflammatory cytokines IL-6 and TNF-α, but not the regulatory cytokine IL-10, by B cells from healthy donors and SLE patients

IntroductionCytokines produced by B cells are believed to play important roles in autoimmune diseases. CD22 targeting by epratuzumab has been demonstrated to inhibit phosphorylation of B cell receptor (BCR) downstream signaling in B cells. It has been shown that other sialoadhesin molecules related to CD22 have immunoregulatory functions; therefore, in the present study, we addressed the role of epratuzumab on the production of key cytokines by B cells of patients with systemic lupus erythematosus (SLE) and of healthy donors (HD).MethodsPeripheral blood B cells were purified and activated by BCR with or without Toll-like receptor 9 (TLR9) stimulation in the presence or absence of epratuzumab. Cytokine production by B cells (interleukin [IL]-6, tumor necrosis factor [TNF]-α and IL-10) in the supernatant and the induction of IL-10⁺ B cells from patients with SLE and HD were analyzed.ResultsThe secretion of the proinflammatory cytokines TNF-α and IL-6 by anti-BCR and BCR- and/or TLR9-activated B cells from HD and patients with SLE was inhibited by epratuzumab. In contrast, the production of IL-10 by B cells was not affected by epratuzumab under either stimulation condition. Consistently, the induction of IL-10–producing B cells in culture was not affected by epratuzumab.ConclusionsEpratuzumab, by targeting CD22, was able to inhibit the production of the proinflammatory cytokines IL-6 and TNF-α by B cells, in contrast to IL-10, in vitro. These data suggest that targeting CD22 alters the balance between proinflammatory cytokines (TNF-α, IL-6) and the regulatory cytokine IL-10 as another B cell effector mechanism.     

Pharmacokinetics and tolerability of human mouse chimeric anti-CD22 monoclonal antibody in Chinese patients with CD22-positive non-Hodgkin lymphoma

The safety and pharmacokinetics assessment of antibodies targeting CD22 (e.g., epratuzumab) have been established in western Caucasian populations, but there are no reports of the effects in Chinese populations. This dose-escalation study examines the safety, pharmacokinetics and biologic effects of multiple doses of anti-CD22 human-murine chimeric monoclonal antibody SM03 in 21 Chinese patients with CD22-positive non-Hodgkin lymphoma. Most of drug-related adverse events (AEs) were mild and reversible. Two patients experienced serious AEs (hemorrhage); one patient had grade 4 neutropenia; one patient had asymptomatic grade III prolongation of activated partial thromboplastin time (APTT). Major AEs included fever (71%), prolongation of APTT (42.8%), leukocytopenia (44.4%), alanine transaminase elevation (28.6%), elevated serum creatinine (23.8%) and injection site skin redness (14.3%). Circulating B cells transiently decreased without significant effects on T cells or immunoglobulin levels. Pharmacokinetic data revealed that mean maximum observed SM03 concentration and mean AUC from time zero to infinity increased in a dose-dependent manner up to 360 mg/m² SM03. Mean clearance was similar at doses ≤ 360 mg/m² and decreased significantly at dose 480 mg/m², supporting saturation of B-cell binding at 360 mg/m². Across all dose levels and histologies, one patient achieved partial response at 480 mg/m² dose; 14 patients had stable disease as best response and four patients progressed. Overall, SM03 was tolerated at doses ranging from 60–480 mg/m² and had potential efficacy in Chinese patients with follicular lymphoma.     

Pharmacokinetics and tolerability of human mouse chimeric anti-CD22 monoclonal antibody in Chinese patients with CD22-positive non-Hodgkin lymphoma

The safety and pharmacokinetics assessment of antibodies targeting CD22 (e.g., epratuzumab) have been established in western Caucasian populations, but there are no reports of the effects in Chinese populations. This dose-escalation study examines the safety, pharmacokinetics and biologic effects of multiple doses of anti-CD22 human-murine chimeric monoclonal antibody SM03 in 21 Chinese patients with CD22-positive non-Hodgkin lymphoma. Most of drug-related adverse events (AEs) were mild and reversible. Two patients experienced serious AEs (hemorrhage); one patient had grade 4 neutropenia; one patient had asymptomatic grade III prolongation of activated partial thromboplastin time (APTT). Major AEs included fever (71%), prolongation of APTT (42.8%), leukocytopenia (44.4%), alanine transaminase elevation (28.6%), elevated serum creatinine (23.8%) and injection site skin redness (14.3%). Circulating B cells transiently decreased without significant effects on T cells or immunoglobulin levels. Pharmacokinetic data revealed that mean maximum observed SM03 concentration and mean AUC from time zero to infinity increased in a dose-dependent manner up to 360 mg/m² SM03. Mean clearance was similar at doses ≤360 mg/m² and decreased significantly at dose 480 mg/m², supporting saturation of B-cell binding at 360 mg/m². Across all dose levels and histologies, one patient achieved partial response at 480 mg/m² dose; 14 patients had stable disease as best response and four patients progressed. Overall, SM03 was tolerated at doses ranging from 60–480 mg/m² and had potential efficacy in Chinese patients with follicular lymphoma.     

Epratuzumab targeting of CD22 affects adhesion molecule expression and migration of B-cells in systemic lupus erythematosus

Introduction  

Epratuzumab, a humanized anti-CD22 monoclonal antibody, is under investigation as a therapeutic antibody in non-Hodgkin's lymphoma and systemic lupus erythematosus (SLE), but its mechanism of action on B-cells remains elusive. Treatment of SLE patients with epratuzumab leads to a reduction of circulating CD27^negative B-cells, although epratuzumab is weakly cytotoxic to B-cells in vitro. Therefore, potential effects of epratuzumab on adhesion molecule expression and the migration of B-cells have been evaluated.     

Anti-CD22/CD20 Bispecific Antibody with Enhanced Trogocytosis for Treatment of Lupus

The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma, leukemia and autoimmune diseases, treating currently over 1500 cases of non-Hodgkin lymphoma, acute lymphoblastic leukemias, Waldenström’s macroglobulinemia, Sjögren’s syndrome, and systemic lupus erythematosus. Because epratuzumab reduces on average only 35% of circulating B cells in patients, and has minimal antibody-dependent cellular cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, its therapeutic activity may not result completely from B-cell depletion. We reported recently that epratuzumab mediates Fc/FcR-dependent membrane transfer from B cells to effector cells via trogocytosis, resulting in a substantial reduction of multiple BCR modulators, including CD22, CD19, CD21, and CD79b, as well as key cell adhesion molecules, including CD44, CD62L, and β7 integrin, on the surface of B cells in peripheral blood mononuclear cells obtained from normal donors or SLE patients. Rituximab has clinical activity in lupus, but failed to achieve primary endpoints in a Phase III trial. This is the first study of trogocytosis mediated by bispecific antibodies targeting neighboring cell-surface proteins, CD22, CD20, and CD19, as demonstrated by flow cytometry and immunofluorescence microscopy. We show that, compared to epratuzumab, a bispecific hexavalent antibody comprising epratuzumab and veltuzumab (humanized anti-CD20 mAb) exhibits enhanced trogocytosis resulting in major reductions in B-cell surface levels of CD19, CD20, CD21, CD22, CD79b, CD44, CD62L and β7-integrin, and with considerably less immunocompromising B-cell depletion that would result with anti-CD20 mAbs such as veltuzumab or rituximab, given either alone or in combination with epratuzumab. A CD22/CD19 bispecific hexavalent antibody, which exhibited enhanced trogocytosis of some antigens and minimal B-cell depletion, may also be therapeutically useful. The bispecific antibody is a candidate for improved treatment of lupus and other autoimmune diseases, offering advantages over administration of the two parental antibodies in combination.     

Umbilical cord mesenchymal stem cell transplantation in active and refractory systemic lupus erythematosus: a multicenter clinical study

Introduction

In our present single-center pilot study, umbilical cord (UC)–derived mesenchymal stem cells (MSCs) had a good safety profile and therapeutic effect in severe and refractory systemic lupus erythematosus (SLE). The present multicenter clinical trial was undertaken to assess the safety and efficacy of allogeneic UC MSC transplantation (MSCT) in patients with active and refractory SLE.

Methods

Forty patients with active SLE were recruited from four clinical centers in China. Allogeneic UC MSCs were infused intravenously on days 0 and 7. The primary endpoints were safety profiles. The secondary endpoints included major clinical response (MCR), partial clinical response (PCR) and relapse. Clinical indices, including Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score, British Isles Lupus Assessment Group (BILAG) score and renal functional indices, were also taken into account.

Results

The overall survival rate was 92.5% (37 of 40 patients). UC-MSCT was well tolerated, and no transplantation-related adverse events were observed. Thirteen and eleven patients achieved MCR (13 of 40, 32.5%) and PCR (11 of 40, 27.5%), respectively, during 12 months of follow up. Three and four patients experienced disease relapse at 9 months (12.5%) and 12 months (16.7%) of follow-up, respectively, after a prior clinical response. SLEDAI scores significantly decreased at 3, 6, 9 and 12 months follow-up. Total BILAG scores markedly decreased at 3 months and continued to decrease at subsequent follow-up visits. BILAG scores for renal, hematopoietic and cutaneous systems significantly improved. Among those patients with lupus nephritis, 24-hour proteinuria declined after transplantation, with statistically differences at 9 and 12 months. Serum creatinine and urea nitrogen decreased to the lowest level at 6 months, but these values slightly increased at 9 and 12 months in seven relapse cases. In addition, serum levels of albumin and complement 3 increased after MSCT, peaked at 6 months and then slightly declined by the 9- and 12-month follow-up examinations. Serum antinuclear antibody and anti-double-stranded DNA antibody decreased after MSCT, with statistically significant differences at 3-month follow-up examinations.

Conclusion

UC-MSCT results in satisfactory clinical response in SLE patients. However, in our present study, several patients experienced disease relapse after 6 months, indicating the necessity to repeat MSCT after 6 months.

Trial registry

ClinicalTrials.gov identifier: {"type":"clinical-trial","attrs":{"text":"NCT01741857","term_id":"NCT01741857"}}NCT01741857. Registered 26 September 2012.     

Ophiopogonin D attenuates the progression of murine systemic lupus erythematosus by reducing B cell numbers

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by immune abnormalities leading to multi-organ damage. The activation of autoreactive B cell differentiation will lead to the production of pathogenic autoantibodies, contributing to the development of SLE. However, the effects of Ophiopogonin D (OP-D) on B cell activation and autoantibody production as well as renal injury in the pathogenesis of SLE remain unclear. MRL/lpr mice, one of the most commonly used animal models of SLE, were intragastrically administered with 5 mg/kg/d OP-D at 17 weeks of age for 3 weeks. The survival rates of mice in each group were monitored for 6 weeks until 23 weeks of age. Proteinuria and serum creatinine levels were measured. Serum levels of immunoglobulin (Ig)G, IgM, and anti-dsDNA autoantibodies were detected by enzyme-linked immunosorbent assay. Numbers of CD19⁺ B cells in the blood, spleen and bone marrow and numbers of splenic germinal center (GC) B cells were calculated by using flow cytometry. OP-D treatment prolonged survival in MRL/lpr mice. OP-D treatment reduced proteinuria and serum creatinine levels as well as mitigated renal pathological alternation in MRL/lpr mice. Furthermore, serum levels of IgG, IgM, and anti-dsDNA autoantibodies were reduced by OP-D treatment. OP-D lessened not only CD19⁺ B cells in the spleen and bone marrow but also plasma cells that secreted anti-dsDNA autoantibodies, IgG and IgM in the spleen and bone marrow. OP-D ameliorated the progression of SLE by inhibiting the secretion of autoantibodies though reducing B cell numbers.     

Restoration of regulatory and effector T cell balance and B cell homeostasis in systemic lupus erythematosus patients through vitamin D supplementation

Introduction

Systemic lupus erythematosus (SLE) is a T and B cell-dependent autoimmune disease characterized by the appearance of autoantibodies, a global regulatory T cells (Tregs) depletion and an increase in Th17 cells. Recent studies have shown the multifaceted immunomodulatory effects of vitamin D, notably the expansion of Tregs and the decrease of Th1 and Th17 cells. A significant correlation between higher disease activity and lower serum 25-hydroxyvitamin D levels [25(OH)D] was also shown.

Methods

In this prospective study, we evaluated the safety and the immunological effects of vitamin D supplementation (100 000 IU of cholecalciferol per week for 4 weeks, followed by 100 000 IU of cholecalciferol per month for 6 months.) in 20 SLE patients with hypovitaminosis D.

Results

Serum 25(OH)D levels dramatically increased under vitamin D supplementation from 18.7±6.7 at day 0 to 51.4±14.1 (p<0.001) at 2 months and 41.5±10.1 ng/mL (p<0.001) at 6 months. Vitamin D was well tolerated and induced a preferential increase of naïve CD4⁺ T cells, an increase of regulatory T cells and a decrease of effector Th1 and Th17 cells. Vitamin D also induced a decrease of memory B cells and anti-DNA antibodies. No modification of the prednisone dosage or initiation of new immunosuppressant agents was needed in all patients. We did not observe SLE flare during the 6 months follow-up period.

Conclusions

This preliminary study suggests the beneficial role of vitamin D in SLE patients and needs to be confirmed in randomized controlled trials.     

Engagement of CD22 on B cells with the monoclonal antibody epratuzumab stimulates the phosphorylation of upstream inhibitory signals of the B cell receptor

The binding of antigen to the B cell receptor (BCR) results in a cascade of signalling events that ultimately drive B cell activation. Uncontrolled B cell activation is regulated by negative feedback loops that involve inhibitory co-receptors such as CD22 and CD32B that exert their functions following phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). The CD22-targeted antibody epratuzumab has previously been shown to inhibit BCR-driven signalling events, but its effects on ITIM phosphorylation of CD22 and CD32B have not been properly evaluated. The present study therefore employed both immunoprecipitation and flow cytometry approaches to elucidate the effects of epratuzumab on direct phosphorylation of key tyrosine (Tyr) residues on both these proteins, using both transformed B cell lines and primary human B cells. Epratuzumab induced the phosphorylation of Tyr⁸²² on CD22 and enhanced its co-localisation with SHP-1. Additionally, in spite of high basal phosphorylation of other key ITIMs on CD22, in primary human B cells epratuzumab also enhanced phosphorylation of Tyr⁸⁰⁷, a residue involved in the recruitment of Grb2. Such initiation events could explain the effects of epratuzumab on downstream signalling in B cells. Finally, we were able to demonstrate that epratuzumab stimulated the phosphorylation of Tyr²⁹² on the low affinity inhibitory Fc receptor CD32B which would further attenuate BCR-induced signalling. Together, these data demonstrate that engagement of CD22 with epratuzumab leads to the direct phosphorylation of key upstream inhibitory receptors of BCR signalling and may help to explain how this antibody modulates B cell function.     

Removal and inactivation of human immunodeficiency virus (HIV-1) by cold ethanol fractionation and pasteurization during the manufacturing of albumin and immunoglobulins from human plasma

Viral safety is a prerequisite for manufacturing clinical albumin and immunoglobulins from human plasma pools. This study was designed to evaluate the efficacy of cold ethanol fractionation and pasteurization (60°C heat treatment for 10 h) for the removal inactivation of human immunodeficiency virus type 1 (HIV-1) during the manufacturing of albumin and immunoglobulins. Samples from the relevant stages of the production process were spiked with HIV-1, and the amount of virus in each fraction was quantified by the 50% tissue culture infectious dose (TCID₅₀). Both fraction IV fractionation and pasteurization steps during albumin processing were robust and effective in inactivating HIV-1, titers of which were reduced from an initial 8.5 log₁₀ TCID₅₀ to undetectable levels. The log reduction factors achieved were ≥4.5 and ≥6.5, respectively. In addition, fraction III fractionation and pasteurization during immunoglobulins processing were robust and effective in eliminating HIV-1. HIV-1 titers were reduced from an initial 7.3 log₁₀ TCID₅₀ to undetectable levels. The log reduction factors achieved in this case were ≥4.9 and ≥5.3, respectively. These results indicate that the process investigated for the production of albumin and immunoglobulins have sufficient HIV-1 reducing capacity to achieve a high margin of safety.     

Open label phase II trial of single,ascending doses of MRA in Caucasian children with severe systemic juvenile idiopathic arthritis: proof of principle of the efficacy of IL-6 receptor blockade in this type of arthritis and demonstration of prolonged clinical improvement

Eighteen Caucasian (white, Middle East and Asian) children diagnosed by paediatric rheumatologists in the UK and France as having systemic juvenile idiopathic arthritis (sJIA) were enrolled in this open label, single dose trial. All patients had evidence of continued symptoms and disease activity for at least three months while receiving >0.2 mg/kg/day of prednisolone, or its equivalent, prior to recruitment. Twelve patients also received methotrexate (≤20 mg/m²/week). The patients were divided into three groups receiving 2, 4 or 8 mg/kg of MRA (tocilizumab) by intravenous infusion. No evidence of dose-limiting toxicity was observed and there were no dose-limiting safety issues. MRA appeared to be dramatically effective, with clinical and laboratory responses observed by 48 h post infusion, and these improvements continued well after serum MRA was undetectable. Eleven patients achieved the JIA definition of improvement (at least 3 of 6 core set criteria with a 30% improvement and no more than one worsened by 30%) and eight achieved ≥50% improvement. There were no observable differences with age. Clinical improvement in these children was observed for up to eight weeks, supporting the hypothesis that IL-6 is a key cytokine in the upregulation of genes crucial in the inflammation processes of sJIA, and the possibility of sequestration of MRA in the extra-vascular compartment needs to be considered.     

Murine model of BCG lung infection: Dynamics of lymphocyte subpopulations in lung interstitium and tracheal lymph nodes

C57B1/6 female mice were infected with an intrapulmonary dose of 2.5 × 10⁴ BCG(Mycobacterium bovis Bacillus Calmette-Guerin). Lymphocyte populations in lung interstitium and lung-associated tracheal lymph nodes (LN) were examined at 1,2, 4, 5, 6, 8 and 12 weeks after infection. BCG load in lungs peaked between 4–6 weeks post-infection and declined to very low levels by the 12th week of infection. Lung leukocytes were obtained over the course of infection by enzyme digestion of lung tissue followed by centrifugation over Percoll discontinuous density gradients. By 4 to 6 weeks after infection, numbers of lung leukocytes had more than doubled but the proportions of lymphocytes (about 70%), macrophages (about 18%) and granulocytes (about 12%) remained essentially unaltered. Flow cytometric studies indicated: (i) the total number of CD3⁺ T cells in lungs increased by 3-fold relative to uninfected controls at 5 to 6 weeks post-infection, but the relative proportions of CD4 and CD8 cells within the T cell compartment remained unaltered; (ii) relative proportion of NK cells in lungs declined by 30% but the total number of NK cells (NK1.1⁺) per lung increased by about 50%, 5–6 weeks post infection; (iii) tracheal LN underwent marked increase in size and cell recoveries (6-10-fold increase) beginning 4 weeks after infection. While both T and B cells contributed to the increase in cell recoveries from infected tracheal LNs, the T/B ratio declined significantly but CD4/CD8 ratio remained unaltered. In control mice, IFNγ producing non-T cells outnumbered T cells producing IFNγ. However, as the adaptive response to infection evolves, marked increase occur in the number of IFNγ producing T cells, but not NK cells in the lungs. Thus, T cells are the primary cell type responsible for the adaptive IFNγ response to pulmonary BCG infection. Few T cells in tracheal LN of BCG infected mice produce IFNγ, suggesting that maturational changes associated with migration to the lungs or residence in the lungs enhance the capability of some T cells to produce this cytokine     

Renal expression and serum levels of high mobility group box 1 protein in lupus nephritis

Introduction

High mobility group box 1 protein (HMGB1) is a nuclear DNA binding protein acting as a pro-inflammatory mediator following extracellular release. HMGB1 has been increasingly recognized as a pathogenic mediator in several inflammatory diseases. Elevated serum levels of HMGB1 have been detected in autoimmune diseases including Systemic lupus erythematosus (SLE). However, the local expression of HMGB1 in active lupus nephritis (LN) is not known. Here we aimed to study both tissue expression and serum levels of HMGB1 in LN patients with active disease and after induction therapy.

Methods

Thirty-five patients with active LN were included. Renal biopsies were performed at baseline and after standard induction therapy; corticosteroids combined with immunosuppressive drugs. The biopsies were evaluated according to the World Health Organization (WHO) classification and renal disease activity was estimated using the British Isles lupus assessment group (BILAG) index. Serum levels of HMGB1 were analysed by western blot. HMGB1 expression in renal tissue was assessed by immunohistochemistry at baseline and follow-up biopsies in 25 patients.

Results

Baseline biopsies showed WHO class III, IV or V and all patients had high renal disease activity (BILAG A/B). Follow-up biopsies showed WHO I to II (n = 14), III (n = 6), IV (n = 3) or V (n = 12), and 15/35 patients were regarded as renal responders (BILAG C/D). At baseline HMGB1 was significantly elevated in serum compared to healthy controls (P < 0.0001). In all patients, serum levels decreased only slightly; however, in patients with baseline WHO class IV a significant decrease was observed (P = 0.03). Immunostaining revealed a pronounced extranuclear HMGB1 expression predominantly outlining the glomerular endothelium and in the mesangium. There was no clear difference in HMGB1 expression comparing baseline and follow-up biopsies or any apparent association to histopathological classification or clinical outcome.

Conclusions

Renal tissue expression and serum levels of HMGB1 were increased in LN. The lack of decrease of HMGB1 in serum and tissue after immunosuppressive therapy in the current study may reflect persistent inflammatory activity. This study clearly indicates a role for HMGB1 in LN.     

Interleukin 6 Accelerates Mortality by Promoting the Progression of the Systemic Lupus Erythematosus-Like Disease of BXSB.Yaa Mice

IL6 is a multifunctional cytokine that drives terminal B cell differentiation and secretion of immunoglobulins. IL6 also cooperates with IL21 to promote differentiation of CD4⁺ T follicular helper cells (T_FH). Elevated serum levels of IL6 correlate with disease flares in patients with systemic lupus erythematosus (SLE). We previously reported that IL21 produced by T_FH plays a critical role in the development of the SLE-like disease of BXSB.Yaa mice. To examine the possible contributions of IL6 to disease, we compared disease parameters in IL6-deficient and IL6-competent BXSB.Yaa mice. We report that survival of IL6-deficient BXSB.Yaa mice was significantly prolonged in association with significant reductions in a variety of autoimmune manifestations. Moreover, B cells stimulated by co-engagement of TLR7 and B cell receptor (BCR) produced high levels of IL6 that was further augmented by stimulation with Type I interferon (IFN1). Importantly, the frequencies of T_FH and serum levels of IL21 were significantly reduced in IL6-deficient mice. These findings suggest that high-level production of IL6 by B cells induced by integrated signaling from the IFN1 receptor, TLR7 and BCR promotes the differentiation of IL21-secreting T_FH in a signaling sequence that drives the lethal autoimmune disease of BXSB.Yaa mice.     

C4A gene deletion and HLA associations in black Americans with systemic lupus erythematosus

In North America and European Caucasoids with systemic lupus erythematosus (SLE) there is an increased frequency of aC4A, CYP21A gene deletion, largely associated with theHLA-B8,DR3,C4A ^* QO extended haplotype. There have been no consistent HLA associations reported for SLE in blacks, although an increased frequency of serologically determinedC4A null alleles has been reported in two studies. We studied 79 black American SLE patients and 68 black controls by restriction fragment length polymorphism analysis to dermine if aC4A gene deletion was a genetic risk factor for SLE. Moreover, the nature of the deletion and any HLA phenotypic associations were sought. Nineteen of 79 (24%) patients compared to 5 of 68 (7.4%) controls had a phenotypicC4A,CYP21A gene deletion (P=.005; RR=4). A homozygous deletion in four patients gave a genotypic frequency of 23/158 (14.5%) SLE patients vs 5/136 (3.7%) controls (P=.001; RR=4.5). The deletion was associated with HLA-DR2 (P=.03) and HLA-DR3 (P=.03). Moreover, all subjects with the deletion had HLA-DR2 or DR3 (P=7.7×10⁻⁶). HLA-B44 was also associated with the deletion (P=.02), and eight of the nine HLA-B44 positives also carried HLA-DR2. HLA-B8 approached significance (P=.08) and was always accompanied by HLA-DR3. Finally, this black population demonstrated a uniqueC4B gene size polymorphism with 80% C4B “short” as compared to the 40% C4B “short” frequency reported in whites. We conclude that a largeC4A,CYP21A gene deletion, particularly associated with theHLA-B44,-DR2, and-DR3 alleles, is the strongest genetic risk factor thus far identified for SLE susceptibility in black Americans. Furthermore, the unique preponderance of theC4B “short” gene form may be a factor in the actual formation of the deletion.     

B lymphocyte stimulator (BLyS) isoforms in systemic lupus erythematosus: disease activity correlates better with blood leukocyte BLyS mRNA levels than with plasma BLyS protein levels

Considerable evidence points to a role for B lymphocyte stimulator (BLyS) overproduction in murine and human systemic lupus erythematosus (SLE). Nevertheless, the correlation between circulating levels of BLyS protein and disease activity in human SLE is modest at best. This may be due to an inadequacy of the former to reflect endogenous BLyS overproduction faithfully, in that steady-state protein levels are affected not just by production rates but also by rates of peripheral utilization and excretion. Increased levels of BLyS mRNA may better reflect increased in vivo BLyS production, and therefore they may correlate better with biologic and clinical sequelae of BLyS overexpression than do circulating levels of BLyS protein. Accordingly, we assessed peripheral blood leukocyte levels of BLyS mRNA isoforms (full-length BLyS and ΔBLyS) and plasma BLyS protein levels in patients with SLE, and correlated these levels with laboratory and clinical features. BLyS protein, full-length BLyS mRNA, and ΔBLyS mRNA levels were greater in SLE patients (n = 60) than in rheumatoid arthritis patients (n = 60) or normal control individuals (n = 30). Although full-length BLyS and ΔBLyS mRNA levels correlated significantly with BLyS protein levels in the SLE cohort, BLyS mRNA levels were more closely associated with serum immunoglobulin levels and SLE Disease Activity Index scores than were BLyS protein levels. Moreover, changes in SLE Disease Activity Index scores were more closely associated with changes in BLyS mRNA levels than with changes in BLyS protein levels among the 37 SLE patients from whom repeat blood samples were obtained. Thus, full-length BLyS and ΔBLyS mRNA levels are elevated in SLE and are more closely associated with disease activity than are BLyS protein levels. BLyS mRNA levels may be a helpful biomarker in the clinical monitoring of SLE patients.     

Detection of cross reactive anti-DNA antibody idiotypes on tissue-bound immunoglobulins from skin biopsies of lupus patients

Cross-reactive anti-DNA antibody idiotypes have been identified on tissue-bound immunoglobulins from skin biopsies of patients with systemic lupus erythematosus (SLE) and discoid lupus erythematosus (DLE). Four polyclonal and two monoclonal anti-idiotypic reagents were used to screen biopsies from 24 patients with SLE, 23 patients with DLE, and 15 other patients with IgM-positive skin biopsies. Up to 46% of the SLE patients and 30% of the DLE patients were found to share idiotypes present on immunoglobulins deposited at the dermal-epidermal junction. Inhibition studies in four patients indicated that the idiotypes were on anti-DNA antibodies. In contrast, none of the anti-idiotypic antibodies bound to any of the control biopsies. These findings imply that some tissue-bound autoantibodies are derived from related families of high-frequency germ-line genes that are expressed in both SLE and DLE.     

A phase-I trial of the epidermal growth factor receptor directed bispecific antibody MDX-447 without and with recombinant human granulocyte-colony stimulating factor in patients with advanced solid tumors

Introduction MDX-447 is a bispecific antibody directed against the epidermal growth factor receptor (EGFR) and the high affinity Fc receptor (FcγRI). Preclinical data suggest that co-administration of granulocyte-colony stimulating factor (G-CSF) may enhance the tumor cytotoxicity of bispecific antibodies. Methods In group 1, patients received MDX-447 intravenously (IV) weekly. Dose levels of MDX-447 evaluated in group 1 were 1, 3.5, 7, 10, 15, 20, 30, and 40 mg/m². In group 2, patients received MDX-447 IV weekly with G-CSF (3 mcg/kg/day) subcutaneously (days −3 to +2, 5–9, 12–16, etc.). Dose levels of MDX-447 evaluated in group 2 were 1, 3.5, 7, 10, and 15 mg/m². Results Sixty-four patients with advanced solid tumors were treated. Forty-one patients received MDX-447 alone (group 1); 23 patients received MDX-447 + G-CSF (group 2). Hypotension was the predominant dose-limiting toxicity (DLT) in both treatment groups, with seven patients experiencing ≥grade 3 events. MDX-447 half-life (T_1/2) ranged from 1.9 to 8.4 h, with no obvious differences between the two treatment groups. MDX-447 binding to neutrophils and peak levels of circulating tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) were higher in group 2. The MTD for MDX-447 alone was 30 mg/m². When G-CSF was given with MDX-447, treatment was not well tolerated and group 2 was closed early because of safety concerns, with the last patient being treated at the 7 mg/m² dose level. There were no objective complete or partial responses in either group. Conclusion MDX-447 alone was generally well tolerated, but did not achieve objective tumor responses. The MTD for MDX-447 alone was 30 mg/m² weekly. Co-administration of G-CSF with MDX-447 precluded meaningful dose escalation.