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G-CSF is a hemopoietic growth factor that has a role in steady state granulopoiesis, as well as in mature neutrophil activation and function. G-CSF– and G-CSF receptor–deficient mice are profoundly protected in several models of rheumatoid arthritis, and Ab blockade of G-CSF also protects against disease. To further investigate the actions of blocking G-CSF/G-CSF receptor signaling in inflammatory disease, and as a prelude to human studies of the same approach, we developed a neutralizing mAb to the murine G-CSF receptor, which potently antagonizes binding of murine G-CSF and thereby inhibits STAT3 phosphorylation and G-CSF receptor signaling. Anti–G-CSF receptor rapidly halted the progression of established disease in collagen Ab-induced arthritis in mice. Neutrophil accumulation in joints was inhibited, without rendering animals neutropenic, suggesting an effect of G-CSF receptor blockade on neutrophil homing to inflammatory sites. Consistent with this, neutrophils in the blood and arthritic joints of anti–G-CSF receptor–treated mice showed alterations in cell adhesion receptors, with reduced CXCR2 and increased CD62L expression. Furthermore, blocking neutrophil trafficking with anti–G-CSF receptor suppressed local production of proinflammatory cytokines (IL-1β, IL-6) and chemokines (KC, MCP-1) known to drive tissue damage. Differential gene expression analysis of joint neutrophils showed a switch away from an inflammatory phenotype following anti–G-CSF receptor therapy in collagen Ab-induced arthritis. Importantly, G-CSF receptor blockade did not adversely affect viral clearance during influenza infection in mice. To our knowledge, we describe for the first time the effect of G-CSF receptor blockade in a therapeutic model of inflammatory joint disease and provide support for pursuing this therapeutic approach in treating neutrophil-associated inflammatory diseases.

Human complement receptor 1 (HuCR1) is a pivotal regulator of complement activity, acting on all three complement pathways as a membrane-bound receptor of C3b/C4b, C3/C5 convertase decay accelerator, and cofactor for factor I-mediated cleavage of C3b and C4b. In this study, we sought to identify a minimal soluble fragment of HuCR1, which retains the complement regulatory activity of the wildtype protein. To this end, we generated recombinant, soluble, and truncated versions of HuCR1 and compared their ability to inhibit complement activation in vitro using multiple assays. A soluble form of HuCR1, truncated at amino acid 1392 and designated CSL040, was found to be a more potent inhibitor than all other truncation variants tested. CSL040 retained its affinity to both C3b and C4b as well as its cleavage and decay acceleration activity and was found to be stable under a range of buffer conditions. Pharmacokinetic studies in mice demonstrated that the level of sialylation is a major determinant of CSL040 clearance in vivo. CSL040 also showed an improved pharmacokinetic profile compared with the full extracellular domain of HuCR1. The in vivo effects of CSL040 on acute complement-mediated kidney damage were tested in an attenuated passive antiglomerular basement membrane antibody-induced glomerulonephritis model. In this model, CSL040 at 20 and 60 mg/kg significantly attenuated kidney damage at 24 h, with significant reductions in cellular infiltrates and urine albumin, consistent with protection from kidney damage. CSL040 thus represents a potential therapeutic candidate for the treatment of complement-mediated disorders.

Activation of Fc receptors and complement by immune complexes is a common important pathogenic trigger in many autoimmune diseases and so blockade of these innate immune pathways may be an attractive target for treatment of immune complex-mediated pathomechanisms. High-dose IVIG is used to treat autoimmune and inflammatory diseases, and several studies demonstrate that the therapeutic effects of IVIG can be recapitulated with the Fc portion. Further, recent data indicate that recombinant multimerized Fc molecules exhibit potent anti-inflammatory properties. In this study, we investigated the biochemical and biological properties of an rFc hexamer (termed Fc-μTP-L309C) generated by fusion of the IgM μ-tailpiece to the C terminus of human IgG1 Fc. Fc-μTP-L309C bound FcγRs with high avidity and inhibited FcγR-mediated effector functions (Ab-dependent cell-mediated cytotoxicity, phagocytosis, respiratory burst) in vitro. In addition, Fc-μTP-L309C prevented full activation of the classical complement pathway by blocking C2 cleavage, avoiding generation of inflammatory downstream products (C5a or sC5b-9). In vivo, Fc-μTP-L309C suppressed inflammatory arthritis in mice when given therapeutically at approximately a 10-fold lower dose than IVIG, which was associated with reduced inflammatory cytokine production and complement activation. Likewise, administration of Fc-μTP-L309C restored platelet counts in a mouse model of immune thrombocytopenia. Our data demonstrate a potent anti-inflammatory effect of Fc-μTP-L309C in vitro and in vivo, likely mediated by blockade of FcγRs and its unique inhibition of complement activation.

Diabodies are noncovalent dimers of single-chain antibody fragments that retain the avidity of intact IgG but have more favorable blood clearance than intact IgG. Radiometals offer a wide range of half-lives and emissions for matching imaging and therapy requirements and provide facile labeling of chelate-antibody conjugates. However, because of their high retention and metabolism in the kidney, the use of radiometal-labeled diabodies can be problematic for both imaging and therapy. Methods: Having previously shown that 111In-DOTA-polyethylene glycol (PEG)3400-anti–carcinoembryonic antigen diabody has less than half the kidney uptake and retention of non-PEGylated diabody and that the two have similarly high tumor uptake and retention, we synthesized a similar derivative for an anti–tumor-associated glycoprotein 72 diabody. We also reduced the molecular size of the polydispersed PEG3400 to monodispersed PEG27 and PEG12 (nominal masses of 1,321 and 617, respectively). We performed biodistributions of their DOTA conjugates radiolabeled with 125I, 111In, or 64Cu in tumor-bearing athymic mice. Results: The addition of PEG3400 to the diabody reduced kidney uptake to a level (≈10 percentage injected dose/g) comparable to that obtained with radiometal-labeled intact IgG. The PEG27 and PEG12 diabody conjugates also demonstrated low kidney uptake without reduction of tumor uptake or tumor-to-blood ratios. When radiolabeled with 64Cu, the DOTA-PEG12 and -PEG27 diabody conjugates gave high-contrast PET images of colon cancer xenografts in athymic mice. Conclusion: PEGylated diabodies may be a valuable platform for delivery of radionuclides and other agents to tumors.

Summary Although vaccines have been highly successful in preventing and treating many infectious diseases (including smallpox, polio and diphtheria) diseases prevalent in the developing world such as malaria and HIV, that suppress the host immune system, require new, multiple strategies that will be defined by our growing understanding of specific immune activation. The definition of adjuvants, previously thought of as any substance that enhanced the immunogenicity of antigen, could now include soluble mediators and antigenic carriers that interact with surface molecules present on DC (e.g. LPS, Flt3L, heat shock protein) particulate antigens which are taken up by mechanisms available to APC but not other cell types (e.g. immunostimulatory complexes, latex, polystyrene particles) and viral/ bacterial vectors that infect antigen presenting cells (e.g. vaccinia, lentivirus, adenovirus). These approaches, summarized herein, have shown potential in vaccinating against disease in animal models, and in some cases in humans. Of these, particle-antigen conjugates provide rapid formulation of the vaccine, easy storage and wide application, with both carrier and adjuvant functions that activate DC. Combined vaccines of the future could use adjuvants such as virus-like particles and particles targeted towards a predominant cellular type or immune response, with target cell activation enhanced by growth factors or maturation signals prior to, or during immunization. Collectively, these new additions to adjuvant technology provide opportunities for more specific immune regulation than previously available.

Previous studies compared uptake by dendritic cells (DC) of 20, 40, 100, 200, 500, 1000, and 2000 nm beads in vivo. When beads were used as antigen carriers, bead size influenced antibody responses and induction of IFN-gamma-producing CD4 and CD8 T cells. Beads of 40-50 nm were taken up preferentially by DC and induced particularly strong immunity. Herein, we examine immunity induced by minute differences in nanobead size, specifically within a narrow viral-sized range (20, 40, 49, 67, 93, 101, and 123 nm), to see if bead carrier size influenced the induction of type 1 or type 2 cells as demonstrated by the production of IFN-gamma or IL-4. In vivo uptake by DC was assessed for selected sizes in this range. Responses to whole ovalbumin (OVA) or the OVA-derived CD8 T cell peptide epitope (SIINFEKL) were tested. After one immunization with beads-OVA, IFN-gamma responses to both OVA and SIINFEKL were significantly better with 40 and 49 nm beads than other sizes, while, in contrast, IL-4 responses to OVA were higher after immunization with OVA conjugated to larger beads (93, 101, and 123 nm). Thus IFN-gamma induction from CD8 T cells was limited to 40-49 nm beads, while CD4 T cell activation and IL-4 were induced by 93-123 nm beads-OVA. After two immunizations, there were comparable high levels of IFN-gamma produced with 40 and 49 beads and IL-4 reactivity was still higher for larger beads (93, 101, 123 nm). Production of IgG1 was seen across the full range of bead sizes, increasing after two immunizations. Since protection against respiratory syncytial virus (RSV) depends on strong IFN responses, while IL-4 responses are reported to cause asthma-like symptoms, immunization with RSV antigens on the 49 nm carrier beads could provide the basis for a suitable vaccine. When the 49 nm beads were conjugated to RSV proteins G88 (surface) or M2.1 (internal capsid), one immunization with G88 induced high levels of IFN-gamma and low levels of IL-4. IL-4 increased with two immunizations. Beads-M2.1 induced only moderate levels of IFN-gamma and low titer antibody after two immunizations. Mice vaccinated once with G88-conjugated 49 nm beads and challenged intranasally with RSV strain A2 subtype showed reduced viral titers and recovered from weight loss more rapidly than mice immunized with M2.1-conjugated 49 nm beads or naive control mice. These results show that precise selection of nanobead size for vaccination can influence the type 1/type 2 cytokine balance after one immunization, and this will be useful in the development of effective vaccines against common human pathogens such as RSV.

The ability and efficiency of various techniques to purifyadult stem cells from a heterogeneous cell population is animportant determinant for the successful characterizationand application of stem cells. Strategies employed to isolateor purify such cells are numerous,and these include sortingof cells according to specific cell surface markers usingeither fluorescence [ 1] (the current gold standard in purify-ing stem cells) or immunomagnetic technology [ 2],exploit-ing the differential plating efficiencies of stem cells on cul-ture plastic [3], and column-separation techniques [4].Adding to this, a strategy reported by Hung et al. [5] uses“double-decker culture plates” with “3- µm” pores to sepa-rate stem cells from a heterogeneous population.Hung et al. described plating Percoll gradient separatedbone marrow stem cells on double-decker culture plates(Transwell,Corning,NY) where the top plate was fabricatedwith 3- µm pores. Size-sieved (SS) postcultured cells on theupper plate were described to be fibroblastic in morphologyand large in size,while the lowere plate (LP) cells were smalland polygonal. A series of assays characterizing the differen-tiation potential, proliferation, and a selected group of sur-face markers was reported,but only on the SS cells. Hung etal. suggested that the 3-µm pores separated the stem cellsfrom the heterogeneous cell population. The true advantageand attractiveness of the proposed strategy would be its costeffectiveness, straightforward and user-friendly protocol.However, it would be more convincing to the scientificcommunities of stem cell and tissue engineering if the LPcells were also characterized and shown not to possess thesame differentiation potential and characteristics. Thiswould be important before one can arrive at the conclusionthat successful “sieving” of stem cells had occurred. The“stem-ness”of the separated cell population subsets (SS,LP)would directly correlate with the efficacy of the proposedstrategy. The greater the efficacy, the more localized stem-ness would be on SS cells and the less on LP cells. Conse-quently, this would mean that the SS to LP data justify andreflect the efficacy of the procedure. Reporting SS cells ashaving stem cell characteristics may not be adequate becausewithout sieve separation, the multipotency of bone mar-row–derived stem cells have already been reported [6–10],thus leading us to expect some degree of stem cell character-istics with the SS cells anyway. Confirming the stem-ness ofa stem cell population must precede its characterization.Without such confirmation,any conclusions arising from thecharacteristics of the purported stem cell population wouldbe incomplete, and the derived clinical applications wouldhave profound repercussion throughout the field. Ideally,research should assess stem-ness in terms of cellular self-renewal ability–the differentiation of in vitro and in vivo sin-gle-cell clones into cell types of the tissue of origin and atleast one other different tissue cell type [11].In addition, the characterization of LP cells would alsoprovide some insights to stem cell biology. The presupposi-tion of the proposed strategy is that stem cells in suspensionwould be larger than 3 µm and would thus remain in the upper

Granulocyte colony-stimulating factor (G-CSF) is a haemopoietic cytokine first identified for its role in steady state granulopoiesis but is now also recognised as a proinflammatory mediator of mature neutrophil activation and function. G-CSF binds to the G-CSF receptor (G-CSFR), which is present on neutrophils and haemopoietic progenitors. G-CSF-deficient mice are protected from disease in several models of rheumatoid arthritis, and blockade of G-CSF is also protective in those models. To further investigate the actions of blocking G-CSF/G-CSFR signalling in inflammatory disease, we developed a neutralising monoclonal antibody to mouse G-CSFR which prevents the binding of G-CSF and inhibits G-CSFR signalling. In the anti-collagen antibody induced arthritis model, anti-G-CSFR treatment rapidly halted the progression of established disease. This therapeutic effect was observed at low dose and was associated with transient suppression of peripheral blood neutrophilia. Anti-G-CSFR treatment significantly inhibited neutrophil accumulation in the joints without rendering animals neutropenic, suggesting an effect of G-CSFR blockade on neutrophil homing to inflammatory sites. Consistent with this, neutrophils in the blood and arthritic joints of anti-G-CSFR treated mice showed alterations in homing receptors, with reduced CXCR2 and increased CD62L expression. Microarray analysis of neutrophils from joints and peripheral blood showed a common up-regulation of 6 genes and down-regulation of 22 genes following anti-G-CSFR treatment. Blocking neutrophil traffic to joints with anti-G-CSFR suppressed local production of proinflammatory cytokines (IL-1b and IL-6) and chemokines (KC and MCP-1) known to drive tissue damage. These data show for the first time the effect of blockade of G-CSFR in a therapeutic model of inflammation and provide further support for utilising this approach in the treatment of inflammatory diseases such as rheumatoid arthritis.