InVivoMAb anti-human/monkey CD64 (FCGR1A)

The properties of the mononuclear phagocyte (Mph) high-affinity Fc receptor, FcRI, were investigated using a novel monoclonal antibody (mAb) designated 10.1. This receptor was shown to be a protein of 71 kDa, presented chiefly on monocytes and the myeloid cell lines U937 and HL60. mAb 10.1 inhibited the binding to Mph of erythrocytes opsonized with rabbit IgG or human IgG3. It also blocked T cell proliferation induced by murine CD3 mAb of the IgG2a but not the IgG1 subclass. These results suggest that rabbit IgG, human IgG3 and murine IgG2a all bind to FcRI in a similar manner and that mAb 10.1 reacts with an epitope on FcRI near to the binding site for the Fc region of IgG. In addition, although it is well known that FcRI has a high affinity for both monomeric human IgG1 and IgG3, we show in this study that while erythrocytes opsonized with human IgG3 bind to Mph, equivalent cells opsonized with IgG1 surprisingly do not. These results define further the nature of the constraints on the interaction between Mph FcRI and particular IgGs.

Objective: To evaluate Fcgamma receptor (FcgammaR) expression on synovial macrophages from rheumatoid arthritis (RA) patients and to determine whether this expression correlates with the production of the proinflammatory cytokines tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), IL-12, and matrix metalloproteinase 1 (MMP-1). We also sought to determine whether mature macrophages from RA patients express aberrant levels of FcgammaRI, FcgammaRII, and FcgammaRIII, and to determine the production of inflammatory mediators after immune complex (IC) stimulation. Methods: Immunohistochemistry was performed on cryostat sections of synovial biopsy specimens obtained from 27 RA patients and 5 controls. FcgammaR I, II, and III were detected, as well as the proinflammatory mediators IL-1, TNFalpha, IL-12, and MMP-1. Monocytes were isolated from the blood of 10 RA patients and 10 healthy controls and cultured for 7 days with macrophage colony-stimulating factor to obtain macrophages. Using fluorescence-activated cell sorting, the expression of FcgammaRI, FcgammaRII, and FcgammaRIII was determined. On day 7, macrophages were stimulated with heat-aggregated gamma globulins (HAGGs) for 24 hours. Production of cytokines was measured using enzyme-linked immunosorbent assay, and production of gelatinases/collagenases was measured by degradation of fluorescent gelatin. Results: Immunohistochemistry showed higher FcgammaRII and FcgammaRIII expression in RA synovium than in controls. FcgammaRII and FcgammaRIII, but not FcgammaRI, were highly correlated with the number of synovial macrophages. Consistent with this, TNFalpha expression correlated positively with FcgammaRIII expression. Moreover, MMP-1 expression strongly correlated with FcgammaR I, II, and III expression. Mature macrophages from RA patients showed significantly enhanced expression of FcgammaRII and FcgammaRIII compared with controls. Twenty-four hours after stimulation of RA macrophages with HAGGs, significantly higher production of TNFalpha and gelatinase/collagenase was measured. Conclusion: RA synovium and mature RA macrophages express significantly elevated levels of FcgammaRII and FcgammaRIII, resulting in much higher production of TNFalpha and gelatinase/collagenase after IC stimulation. These data suggest that disturbed expression of FcgammaR on mature synovial macrophages is involved in the pathology of RA.

Background: Rheumatoid arthritis (RA) is one of the most prevalent and debilitating joint diseases worldwide. RA is characterized by synovial inflammation (synovitis), which is linked to the development of joint destruction. Magnetic resonance imaging and ultrasonography are widely being used to detect the presence and extent of synovitis. However, these techniques do not reveal the activation status of inflammatory cells such as macrophages that play a crucial role in synovitis and express CD64 (Fc gamma receptor (FcγR)I) which is considered as macrophage activation marker. Objectives: We aimed to investigate CD64 expression and its correlation with pro-inflammatory cytokines and pro-damaging factors in human-derived RA synovium. Furthermore, we aimed to set up a molecular imaging modality using a radiolabeled CD64-specific antibody as a novel imaging tracer that could be used to determine the extent and phenotype of synovitis using optical and nuclear imaging. Methods: First, we investigated CD64 expression in synovium of early- and late-stage RA patients and studied its correlation with the expression of pro-inflammatory and tissue-damaging factors. Next, we conjugated an anti-CD64 antibody with IRDye 800CW and diethylenetriamine penta-acetic acid (DTPA; used for ¹¹¹In labeling) and tested its binding on cultured THP1 cells, ex vivo RA synovium explants and its imaging potential in SCID mice implanted with human RA synovium explants obtained from RA patients who underwent total joint replacement. Results: We showed that CD64 is expressed in synovium of early and late-stage RA patients and that FCGR1A/CD64 expression is strongly correlated with factors known to be involved in RA progression. Combined, this makes CD64 a useful marker for imaging the extent and phenotype of synovitis. We reported higher binding of the [¹¹¹In]In-DTPA-IRDye 800CW anti-CD64 antibody to in vitro cultured THP1 monocytes and ex vivo RA synovium compared to isotype control. In human RA synovial explants implanted in SCID mice, the ratio of uptake of the antibody in synovium over blood was significantly higher when injected with anti-CD64 compared to isotype and injecting an excess of unlabeled antibody significantly reduced the antibody-binding associated signal, both indicating specific receptor binding. Conclusion: Taken together, we successfully developed an optical and nuclear imaging modality to detect CD64 in human RA synovium in vivo.

The success of passive immunization suggests that antibody-based therapies will be effective at controlling malaria. We describe the development of fully human antibodies specific for Plasmodium falciparum by antibody repertoire cloning from phage display libraries generated from immune Gambian adults. Although these novel reagents bind with strong affinity to malaria parasites, it remains unclear if in vitro assays are predictive of functional immunity in humans, due to the lack of suitable animal models permissive for P. falciparum. A potentially useful solution described herein allows the antimalarial efficacy of human antibodies to be determined using rodent malaria parasites transgenic for P. falciparum antigens in mice also transgenic for human Fc-receptors. These human IgG1s cured animals of an otherwise lethal malaria infection, and protection was crucially dependent on human FcgammaRI. This important finding documents the capacity of FcgammaRI to mediate potent antimalaria immunity and supports the development of FcgammaRI-directed therapy for human malaria.