Antibody-mediated activation of the FGFR1/Klothoß complex corrects metabolic dysfunction and alters food preference in obese humans.

Fibroblast growth factor 21 (FGF21) controls metabolic organ homeostasis and eating/drinking behavior via FGF receptor 1/Klothoß (FGFR1/KLB) complexes expressed in adipocytes, pancreatic acinar cells, and the nervous system in mice. Chronic administration of recombinant FGF21 or engineered variants improves metabolic health in rodents, nonhuman primates, and humans; however, the rapid turnover of these molecules limits therapeutic utility. Here we show that the bispecific anti-FGFR1/KLB agonist antibody BFKB8488A induced marked weight loss in obese cynomolgus monkeys while elevating serum adiponectin and the adipose expression of FGFR1 target genes, demonstrating its action as an FGF21 mimetic. In a randomized, placebo-controlled, single ascending-dose study in overweight/obese human participants, subcutaneous BFKB8488A injection caused transient body weight reduction, sustained improvement in cardiometabolic parameters, and a trend toward reduction in preference for sweet taste and carbohydrate intake. These data suggest that specific activation of the FGFR1/KLB complex in humans can be used as therapy for obesity-related metabolic defects.

Effect of pirfenidone on gastric emptying in a rat model.

INTRODUCTION: Gastrointestinal (GI) adverse events (AEs) are commonly reported in patients with idiopathic pulmonary fibrosis who are treated with pirfenidone. Taking pirfenidone with a substantial amount of food or dividing the dose over the course of a meal has been reported to reduce the frequency of GI AEs in clinical practice. In humans, the maximum plasma concentration (Cmax) of pirfenidone was reduced when the drug was taken with food compared with the fasting state, and the lower Cmax was associated with a reduction in GI AE rates. In this study, the effects of the divided-dose approach and timing of pirfenidone administration relative to meal intake on gastric emptying were assessed using a rat model. The aim of this study was to investigate whether modification of dosing regimens could minimize pirfenidone's effect on inhibition of gastric emptying. METHODS: Gastric emptying was assessed in male Sprague-Dawley rats after administration of a test meal by weighing stomach contents at various time points up to 120 min after the meal. Pirfenidone was administered via oral gavage either as a single-bolus dose of 30 mg/kg or as divided doses of 3 × 10 mg/kg at intervals ranging from 10 to 30 min for a total duration of 30 to 90 min. In addition, the test meal was given either at 30 min before, coincident with, or 30 min following pirfenidone oral administration. RESULTS: Administration of an oral 30-mg/kg single-bolus dose of pirfenidone with a meal resulted in a statistically significant decrease in gastric emptying in a rat model. The effect of pirfenidone on decreasing gastric emptying was lessened when the same total dose (i.e., 30 mg/kg) was administered as 3 divided doses (i.e., 3 × 10 mg/kg) over intervals up to 30 min in between each divided dose. Pharmacokinetic simulation suggested that a divided-dosing regimen would decrease pirfenidone Cmax relative to single-bolus administration. When the same single-bolus dose of 30 mg/kg was administered 30 min following a meal rather than coincident with a meal, pirfenidone's effect on decreasing gastric emptying was reduced to the same extent as when the dose was divided as 3 × 10 mg/kg over a 90-min period. CONCLUSIONS: Administration of pirfenidone 30 min after a meal as a single-bolus dose or a divided dose over a 90-min period blunted pirfenidone's effect on inhibition of gastric emptying in rats compared with pirfenidone administration as a single-bolus dose coincident with a meal. Decreased gastric emptying, which is associated with pirfenidone administration, may be one of the contributing factors leading to GI tolerability issues associated with pirfenidone use in humans. Modification of the dosing regimen diminished this impact and may provide insight into possible mitigation strategies to minimize GI-related toxicities in the clinic.

Latest generation estrogen receptor degraders for the treatment of hormone receptor-positive breast cancer.

INTRODUCTION: The selective estrogen receptor degrader (SERD) and full receptor antagonist provides an important therapeutic option for hormone receptor (HR)-positive breast cancer. Endocrine therapies include tamoxifen, a selective estrogen receptor modulator (SERM), that exhibits receptor agonist and antagonist activity, and aromatase inhibitors that block estrogen biosynthesis but which demonstrate acquired resistance. Fulvestrant, the only currently approved SERD, is limited by poor drug-like properties. A key focus for improving disease management has been development of oral SERDs with optimized target occupancy and potency and superior clinical efficacy. AREAS COVERED: Using PubMed, clinicaltrials.gov, and congress websites, this review explored the preclinical development and clinical pharmacokinetics from early phase clinical studies (2015 or later) of novel oral SERDs, including giredestrant, amcenestrant, camizestrant, elacestrant, and rintodestrant. EXPERT OPINION: Numerous oral SERDs are in clinical development, aiming to form the core endocrine therapy for HR-positive breast cancer. Through property- and structure-based drug design of estrogen receptor-binding, antagonism, degradation, anti-proliferation, and pharmacokinetic properties, these SERDs have distinct profiles which impact clinical dosing, efficacy, and safety. Assuming preliminary safety and activity data are confirmed in phase 3 trials, these promising agents could further improve the management, outcomes, and quality of life in HR-positive breast cancer.

GDC-9545 (Giredestrant): A Potent and Orally Bioavailable Selective Estrogen Receptor Antagonist and Degrader with an Exceptional Preclinical Profile for ER+ Breast Cancer.

Breast cancer remains a leading cause of cancer death in women, representing a significant unmet medical need. Here, we disclose our discovery efforts culminating in a clinical candidate, 35 (GDC-9545 or giredestrant). 35 is an efficient and potent selective estrogen receptor degrader (SERD) and a full antagonist, which translates into better antiproliferation activity than known SERDs (1, 6, 7, and 9) across multiple cell lines. Fine-tuning the physiochemical properties enabled once daily oral dosing of 35 in preclinical species and humans. 35 exhibits low drug-drug interaction liability and demonstrates excellent in vitro and in vivo safety profiles. At low doses, 35 induces tumor regressions either as a single agent or in combination with a CDK4/6 inhibitor in an ESR1Y537S mutant PDX or a wild-type ERα tumor model. Currently, 35 is being evaluated in Phase III clinical trials.

Immunogenicity of biologically-derived therapeutics: assessment and interpretation of nonclinical safety studies.

An evaluation of potential antibody formation to biologic therapeutics during the course of nonclinical safety studies and its impact on the toxicity profile is expected under current regulatory guidance and is accepted standard practice. However, approaches for incorporating this information in the interpretation of nonclinical safety studies are not clearly established. Described here are the immunological basis of anti-drug antibody formation to biopharmaceuticals (immunogenicity) in laboratory animals, and approaches for generating and interpreting immunogenicity data from nonclinical safety studies of biotechnology-derived therapeutics to support their progression to clinical evaluation. We subscribe that immunogenicity testing strategies should be adapted to the specific needs of each therapeutic development program, and data generated from such analyses should be integrated with available clinical and anatomic pathology, pharmacokinetic, and pharmacodynamic data to properly interpret nonclinical studies.

A pharmacology guided approach for setting limits on product-related impurities for bispecific antibody manufacturing.

INTRODUCTION: bFKB1 is a humanized bispecific IgG1 antibody, created by conjoining an anti-Fibroblast Growth Factor Receptor 1 (FGFR1) half-antibody to an anti-Klothoß (KLB) half-antibody, using the knobs-into-holes strategy. bFKB1 acts as a highly selective agonist for the FGFR1/KLB receptor complex and is intended to ameliorate obesity-associated metabolic defects by mimicking the activity of the hormone FGF21. An important aspect of the biologics product manufacturing process is to establish meaningful product specifications regarding the tolerable levels of impurities that copurify with the drug product. The aim of the current study was to determine acceptable levels of product-related impurities for bFKB1. METHODS: To determine the tolerable levels of these impurities, we dosed obese mice with bFKB1 enriched with various levels of either HMW impurities or anti-FGFR1-related impurities, and measured biomarkers for KLB-independent FGFR1 signaling. RESULTS: Here, we show that product-related impurities of bFKB1, in particular, high molecular weight (HMW) impurities and anti-FGFR1-related impurities, when purposefully enriched, stimulate FGFR1 in a KLB-independent manner. By taking this approach, the tolerable levels of product-related impurities were successfully determined. DISCUSSION: Our study demonstrates a general pharmacology-guided approach to setting a product specification for a bispecific antibody whose homomultimer-related impurities could lead to undesired biological effects.

Preclinical pharmacokinetic characterization of an adipose tissue-targeting monoclonal antibody in obese and non-obese animals.

Target receptor levels can influence pharmacokinetics (PK) or pharmacodynamics (PD) of monoclonal antibodies (mAbs), and can affect drug development of this class of molecules. We generated an effector-less humanized bispecific antibody that selectively activates fibroblast growth factor receptor (FGFR)1 and ßKlotho receptor, a FGF21 receptor complex highly expressed in both white and brown adipocytes. The molecule shows cross-species binding with comparable equilibrium binding affinity (Kd) for human, cynomolgus monkey, and mouse FGFR1/ßKlotho. To understand the PK/PD relationship in non-obese and obese animals, we evaluated the adipose tissue distribution of the antibody, serum exposures, and an associated PD marker (high-molecular-weight adiponectin), in both non-obese and obese mice and monkeys. Antibody uptake into fat tissue was found to be higher on a per gram basis in non-obese animals compared to obese animals. Since obesity has been reported to be associated with reduced expression of FGFR1 and ßKlotho receptor in white adipose tissues in mice, our results suggest that the distribution in adipose tissues was influenced by target expression levels. Even so, the overall dose-normalized serum exposures were comparable between non-obese and obese mice and monkeys, suggesting that adipose tissue uptake plays a limited role in overall systemic PK determination. It remains to be determined if and how obesity and receptor expression in humans influence the PK and PD profile of this novel therapeutic candidate.

Effects of RG7652, a Monoclonal Antibody Against PCSK9, on LDL-C, LDL-C Subfractions, and Inflammatory Biomarkers in Patients at High Risk of or With Established Coronary Heart Disease (from the Phase 2 EQUATOR Study).

RG7652 (MPSK3169A), a fully human immunoglobulin G1 (IgG1) monoclonal antibody directed against proprotein convertase subtilisin/kexin type 9 (PCSK9), blocks the interaction between PCSK9 and low-density lipoprotein (LDL) receptor. EQUATOR (ClinicalTrials.govNCT01609140), a randomized, double-blind, and dose-ranging phase 2 study, evaluated RG7652 in patients (1) at high risk for or (2) with coronary heart disease (CHD). The primary end point was change in LDL cholesterol (LDL-C) from baseline to day 169. Patients (n = 248; median age, 64 years; 57% men; 52% with established CHD; 82% on statins) with baseline LDL-C levels of 90 to 250 mg/dl (mean, 126 mg/dl) continuing on standard-of-care therapy were randomized to receive 1 of 5 RG7652 doses or placebo, subcutaneously every 4, 8, or 12 weeks for 24 weeks. Significant dose-dependent reductions in LDL-C levels from baseline to nadir (56 to 74 mg/dl [48% to 60%]) were observed in all RG7652-dosed patients; effects persisted to day 169 with the highest doses (reduction vs placebo up to 62 mg/dl [51%]) with no unexpected safety signals. RG7652 reduced apolipoprotein B and lipoprotein(a) levels. LDL-C subfraction analysis by nuclear magnetic resonance spectroscopy revealed a prominent decrease in large LDL-C and some decrease in small LDL particles. There was significant reduction in mean particle size of LDL-C on day 169 but no significant reductions in systemic markers of inflammation (high-sensitivity C-reactive protein, interleukin-6, and tumor necrosis factor-alpha). RG7652 reduced LDL-C levels and was well tolerated in patients at high risk for or with CHD on standard-of-care therapy. In conclusion, RG7562 treatment affected large LDL-C and, to a lesser extent, small LDL-C particles; RG7562 did not affect systemic circulating pro-inflammatory cytokines or high-sensitivity C-reactive protein.

Preclinical pharmacokinetics, pharmacodynamics, and activity of a humanized anti-CD40 antibody (SGN-40) in rodents and non-human primates.

1. Cell-surface expression of CD40 in B-cell malignancies and multiple solid tumors has raised interest in its potential use as a target for antibody-based cancer therapy. SGN-40, a humanized monoclonal anti-CD40 antibody, mediates antibody-dependent cytotoxicity and inhibits B-cell tumor growth in vitro, properties of interest for the treatment of cancers, and is currently in Phase I clinical trials for B-cell malignancies. In this study, we determined in vivo activity and pharmacokinetics properties of SGN-40. 2. Effect of SGN-40 in xenograft model of CD40-expressing B-cell lymphoma in severe-combined immune deficiency mice and its in vivo pharmacokinetics properties in normal mice, rats and cynomolgus monkeys were studied. 3. Treatment with SGN-40 significantly increased the survival of mice xenografted with human B-cell lymphoma cell line. SGN-40 exhibited nearly 100% bioavailability in mice and it cleared faster when given at a low dose. In monkeys, clearance of SGN-40 was also much faster at low dose, suggesting nonlinear pharmacokinetics in these species. In rats, however, SGN-40 clearance at all tested doses was similar, suggesting that pharmacokinetics were linear in this dose range in rats. Administration of SGN-40 to monkeys also produced marked, dose-dependent, and persistent depletion of peripheral CD20(+) B lymphocytes. 4. Data presented in this report suggest that SGN-40 is active in in vivo, and based upon interspecies scaling, SGN-40 clearance in humans is predicted to be similar to observed SGN-40 clearance in monkeys. These data suggest that SGN-40 has appropriate pharmacokinetic properties that support its clinical use.

Assessment of placental transfer and the effect on embryo-fetal development of a humanized monoclonal antibody targeting lymphotoxin-alpha in non-human primates.

An enhanced embryo-fetal development study was conducted in cynomolgus monkeys using pateclizumab, a humanized IgG1 monoclonal antibody (mAb) targeting lymphotoxin-alpha. Pateclizumab administration between gestation days (GD) 20 and 132 did not induce maternal or developmental toxicities. The ratio of fetal-to-maternal serum concentration of pateclizumab was 0.73% on GD 50 and 61% by GD 139. Decreased fetal inguinal lymph node-to-body weight ratio was present in the high-dose group without microscopic abnormalities, a change attributable to inhibition of lymphocyte recruitment, which is a pharmacologic effect of pateclizumab during late lymph node development. The effect was observed in inguinal but not submandibular or mesenteric lymph nodes; this was attributed to differential susceptibility related to sequential lymph node development. Placental transfer of therapeutic IgG1 antibodies; thus, begins during the first trimester in non-human primates. Depending on the potency and dose levels administered, antibody levels in the fetus may be pharmacologically or toxicologically relevant.

Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/ßKlotho Complex.

Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/ßKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/ßKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/ßKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/ßKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/ßKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin.

Immunomodulation and lymphoma in humans.

Observational and clinical studies have associated increased cancer risks with primary or acquired immunodeficiencies, autoimmunity, and use of immunotherapies to treat chronic inflammation (e.g. autoimmunity) or support organ engraftment. Understanding of the relationship between immune status and cancer risk is generally grounded in two juxtaposing paradigms: that the immune system protects the host via surveillance of tumors and oncogenic viruses (e.g. immunosurveillance model) and that chronic inflammation can augment tumor growth and metastasis (inflammation model). Whereas these models support a role of immune status in many cancers, they are insufficient to explain the disproportionate increase in B-cell lymphoma risk observed across patient populations with either chronic immunosuppression or inflammation. Evaluation for the presence of Epstein-Barr virus (EBV) in lymphomas obtained from various populations demonstrates a variable role for the virus in lymphomagenesis across patient populations. An evaluation of the DNA alterations found in lymphomas and an understanding of B-cell ontogeny help to provide insight into the unique susceptibility of lymphocytes, primarily B-cells, to oncogenic transformation. EBV-independent B-cell oncogenic transformation is driven by chronic antigenic stimulation due to either inflammation (as seen in patients with autoimmune disease or a tissue allograft) or to unresolved infection (as seen in immunosuppressed patients), and the transformation arises as a result of DNA damage from genomic recombination and mutation during class switching and somatic hypermutation. This model explains the increased background rate of lymphoma in some patients with autoimmunity, and highlights the challenge of resolving the confounding that occurs between disease severity and use of targeted immunotherapies to treat chronic inflammation. The ability to distinguish between disease- and treatment-related risk of lymphoma and an appreciation of the etiology of B-cell transformation is central to an improved risk assessment by scientists, clinicians and regulators, including the approval, labeling, and chronic use of immunotherapies.

Combined administration of RG7652, a recombinant human monoclonal antibody against PCSK9, and atorvastatin does not result in reduction of immune function.

RG7652 is a human IgG1 monoclonal antibody designed to inhibit proprotein convertase subtilisin/kexin type 9 (PCSK9) binding to hepatic low density lipoprotein receptor (LDL-r), thereby blocking PCSK9-mediated degradation of LDL-r. This therapeutic candidate is under development for the prevention of cardiovascular mortality and morbidity in dyslipidemic patients. The primary objective of this study was to evaluate the potential immunotoxicological effects of RG7652 when given to cynomolgus monkeys either alone or in combination with a daily oral dose of atorvastatin. Administration of RG7652 via subcutaneous injection every other week for 12 weeks (a total of seven doses), daily oral doses of atorvastatin (total of 85 doses), and combinations of each up to 15 and 20 mg/kg/dose, respectively, were well tolerated and there was no evidence of alteration in immune function. Administration of pharmacologically relevant doses of RG7652 in combination with atorvastatin to healthy monkeys does not result in clinically meaningful immunosuppression as measured by T-cell dependent antibody responses, natural killer cell activity, immunophenotype, or delayed type hypersensitivity. The only pharmacologically mediated changes observed during the dosing period were the anticipated changes in circulating cholesterol.

Nonclinical evaluation of the serum pharmacodynamic biomarkers HGF and shed MET following dosing with the anti-MET monovalent monoclonal antibody onartuzumab.

Onartuzumab, a humanized, monovalent monoclonal anti-MET antibody, antagonizes MET signaling by inhibiting binding of its ligand, hepatocyte growth factor (HGF). We investigated the effects of onartuzumab on cell-associated and circulating (shed) MET (sMET) and circulating HGF in vitro and nonclinically to determine their utility as pharmacodynamic biomarkers for onartuzumab. Effects of onartuzumab on cell-associated MET were assessed by flow cytometry and immunofluorescence. sMET and HGF were measured in cell supernatants and in serum or plasma from multiple species (mouse, cynomolgus monkey, and human) using plate-based immunoassays. Unlike bivalent anti-MET antibodies, onartuzumab stably associates with MET on the surface of cells without inducing MET internalization or shedding. Onartuzumab delayed the clearance of human xenograft tumor-produced sMET from the circulation of mice, and endogenous sMET in cynomolgus monkeys. In mice harboring MET-expressing xenograft tumors, in the absence of onartuzumab, levels of human sMET correlated with tumor size, and may be predictive of MET-expressing tumor burden. Because binding of sMET to onartuzumab in circulation resulted in increasing sMET serum concentrations due to reduced clearance, this likely renders sMET unsuitable as a pharmacodynamic biomarker for onartuzumab. There was no observed effect of onartuzumab on circulating HGF levels in xenograft tumor-bearing mice or endogenous HGF in cynomolgus monkeys. Although sMET and HGF may serve as predictive biomarkers for MET therapeutics, these data do not support their use as pharmacodynamic biomarkers for onartuzumab.

Effect of antigen binding affinity and effector function on the pharmacokinetics and pharmacodynamics of anti-IgE monoclonal antibodies.

Modulating the binding affinities to IgE or changing the FcγR binding properties of anti-IgE antibodies offers an opportunity to enhance the therapeutic potential of anti-IgE antibodies, but the influence of increased affinity to IgE or reduced Fc effector function on the pharmacological properties of anti-IgE therapies remains unclear. Our studies were designed to characterize the pharmacokinetics, pharmacodynamics and immune-complex distribution of two high-affinity anti-IgE monoclonal antibodies, high-affinity anti-IgE antibody (HAE) 1 and 2, in mice and monkeys. HAE1, also known as PRO98498, is structurally similar to omalizumab (Xolair®), a humanized anti-IgE IgG1 marketed for the treatment of asthma, but differs by 9 amino acid changes in the complementarity-determining region resulting in a 23-fold improvement in affinity. HAE2 is similar to HAE1, but its Fc region was altered to reduce binding to Fcγ receptors. As expected given the decreased binding to Fcγ receptors, systemic exposure to pre-formed HAE2:IgE complexes in mice was greater (six-fold) and distribution to the liver lower (four-fold) compared with HAE1:IgE complexes. In monkeys, systemic exposure to HAE1 was similar to that previously observed for omalizumab in this species, but required comparatively lower serum drug concentrations to suppress free IgE levels. HAE2 treatment resulted in greater exposure and greater increase of total IgE, relative to HAE1, because of decreased clearance of HAE2:IgE complexes. Overall, these data suggest that increased binding affinity to IgE may provide a more effective therapeutic for asthma patients, and that retaining FcγR binding of the anti-IgE antibody is important for elimination of anti-IgE:IgE complexes.

Anti-BR3 antibodies: a new class of B-cell immunotherapy combining cellular depletion and survival blockade.

Removal of pathogenic B lymphocytes by depletion of monoclonal antibodies (mAbs) or deprivation of B-cell survival factors has demonstrated clinical benefit in both oncologic and immunologic diseases. Partial clinical responses and emerging data demonstrating incomplete B-cell depletion after immunotherapy fuels the need for improved therapeutic modalities. Lessons from the first generation of therapeutics directed against B-cell-specific antigens (CD20, CD22) are being applied to develop novel antibodies with additional functional attributes. We describe the generation of a novel class of B-cell-directed therapy (anti-BR3 mAbs) that combines the depleting capacity of a therapeutic mAb and blockade of B-cell-activating factor (BAFF)-BR3 B-cell survival. In mice, treatment with antagonistic anti-BR3 antibodies results in quantitatively greater reduction in some B-cell subsets and qualitatively different effects on bone marrow plasma cells compared with BR3-Fc BAFF blockade or with anti-CD20 treatment. Comparative analysis of BR3-Fc and anti-BR3 mAb reveals a lower B-cell dependence for BAFF-mediated survival in nonhuman primates than in mice. This novel class of B-cell-targeted therapies shows species characteristics in mice and primates that will guide translation to treatment of human disease.

A soluble BAFF antagonist, BR3-Fc, decreases peripheral blood B cells and lymphoid tissue marginal zone and follicular B cells in cynomolgus monkeys.

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.