Infliximab (IFX) is a chimeric IgG1 monoclonal antibody specific for human tumor necrosis factor-alpha that is approved in the United States and Europe for the treatment of rheumatoid arthritis ( RA) and Crohn's disease ( CD). Approximately 10% of RA and CD patients receiving maintenance treatment with IFX will develop antibodies to IFX. The objective of this study was to develop a model to assess the in vivo formation, distribution, and elimination of immune complexes resulting from a low-level immune response in the presence of the excess concentration of a therapeutic antigen. In this model, cynomolgus monkeys were treated with a single intravenous injection of IFX, followed by injection of either radiolabeled, purified monkey anti-IFX IgG antibody (n = 3, test group) or radiolabeled monkey, nonimmune IgG ( n = 3, control group). High-performance liquid chromatography analysis of collected sera revealed a rapid formation of immune complexes comprised of IFX and radiolabeled anti-IFX IgG antibody immune complexes. The terminal half-life of the anti-IFX IgG antibody immune complex was approximately 38 h compared with 86 h for the nonimmune antibody. However, the pharmacokinetic profile of IFX, although slightly lower in concentration over time for the test group, was not notably different relative to the control group. There were no macroscopic or microscopic histological findings in either treatment group. These data confirm that immune complexes between IFX and anti-IFX IgG antibodies can form in vivo and that these immune complexes are eliminated more rapidly than nonimmune antibodies in the presence of excess IFX.

Background and Purpose We compared the current antithrombotic strategy of antiplatelet therapy with aspirin, and anticoagulant therapy with heparin, with a specific genetically engineered chimeric antibody (c7E3 Fab) directed against the human glycoprotein IIb/IIIa receptor in an animal model of arterial thrombosis.Methods Anesthetized cynomolgus monkeys (Macaca fascicularis) were instrumented for monitoring of arterial blood pressure, heart rate, and carotid artery flow velocity. Animals were treated with saline (n=6), aspirin (25 mg PO daily for 3 days; n=6), heparin (100 U/kg IV plus infusion adjusted to maintain activated partial thromboplastin time at 2 to 3 times baseline; n=6), aspirin plus heparin (as administered separately, n=6), or c7E3 Fab (0.10 mg/kg IV, n=7; 0.15 mg/kg IV, n=6; 0.20 mg/kg IV, n=6; 0.25 mg/kg IV, n=6). Thrombus formation via anodal electrolytic stimulation (100 mu A) to the intimal surface of the right carotid artery was initiated 15 minutes after drug administration and continued for 180 minutes. Electrolytic injury to the left carotid artery began 210 minutes after drug administration and continued for 180 minutes. Whole blood cell counts, glycoprotein IIb/lIIa receptor blockade, ex vivo platelet aggregation, template bleeding time, and activated partial thromboplastin time were assessed at various time points throughout the experimental protocol.Results Hemodynamic and hematologic parameters were comparable among groups at baseline. Treatment with c7E3 Fab inhibited ex vivo platelet aggregation, increased bleeding time, decreased thrombus weight, and increased time to occlusion in a dose-dependent manner in both vessels. Treatment with aspirin, heparin, or the combination of aspirin plus heparin was ineffective for the prevention of carotid artery thrombosis in this model.Conclusions Inhibition of the platelet glycoprotein IIb/IIIa receptor with c7E3 Fab was found to be safe and effective for the prevention of primary thrombus formation, whereas treatment with either aspirin or heparin or the combination of the two agents failed to protect against occlusive thrombus formation in cynomolgus monkeys.

Transforming growth factor beta 1 (TGF beta 1) inhibits the proliferative response of mink lung epithelial cells (CCL64) to serum and to epidermal growth factor (EGF). This response to TGF beta 1 can be inhibited by prior exposure of the cells to nanogram concentrations of pertussis toxin (PT), suggesting the involvement of a guanine-nucleotide-binding regulatory protein (G-protein) in mediating TGF beta 1-induced growth inhibition. To characterize further this G-protein dependence, we have isolated, by chemical mutagenesis, a CCL64 variant (CCL64-D1) that is resistant to TGF beta 1. Whereas in the parental CCL64 cells TGF beta 1 stimulates both GTP[35S] (guanosine 5'-[gamma-[35S]thio]triphosphate) binding and GTPase activity, in the CCL64-D1 variants TGF beta 1 is without effect. Quantitative immunoblotting with antisera for G-protein alpha- and beta-subunits, as well as PT-catalysed ADP-ribosylation analyses, revealed no appreciable changes in the level of G-protein expression in the CCL64-D1 variants compared with parental cells. In contrast with another TGF beta-resistant clone, MLE-M, which we show lacks detectable type I receptor protein, the CCL64-D1 cells retain all three TGF beta cell-surface binding proteins. On the basis of these studies, we propose that a necessary component of TGF beta 1-mediated growth inhibition in CCL64 epithelial cells is the coupling of TGF beta 1 receptor binding to G-protein activation.

The effect of pertussis toxin (PT) on transforming growth factor β1 (TGFβl)‐induced proto‐oncogene expression was investigated in AKR‐2B fibroblasts. PT substantially abolished c‐sis and c‐myc mRNA expression following TGFβl stimulation. This inhibitory effect was specific for TGFβ1‐stimulated proto‐oncogene expression and associated with the ADP‐ribosylation of a 41‐kDa substrate. Actinomycin D decay and nuclear run‐on experiments demonstrated that the inhibitory effects of PT are a result of decreased transcriptional activation and not to an increased decay of proto‐oncogene message. PT did not, however, affect TGFβl‐stimulated fibronectin and collagen mRNA accumulation nor did it have any inhibitory effect on TGFβl‐induced morphological transformation. These data indicate that TGFβl‐stimulated gene expression is coupled to multiple pathways distinguished by their sensitivity to PT.

Transfection of C3H/10T1/2 cells with either a c-myc or an activated c-Ha-ras gene decreased the cellular dependence for serum-derived factors to proliferate in monolayer. The c-myc-transfected cells did, however, require a high plasma concentration for significant growth, while the ras transfectants grew extremely well in either low or high concentrations of either plasma or serum. Stimulation of quiescent cultures with purified growth factors demonstrated that c-myc transfection did not alter qualitatively or quantitatively the requirement for both epidermal growth factor (EGF) and insulin to progress to DNA synthesis. Cells transfected with either a ras gene alone or a combination of ras plus c-myc lost their dependence on EGF for DNA synthesis; cultures became committed to S phase in serum-free medium supplemented with insulin alone. The ras transfectants arrested in mid-G1, 6 h prior to S phase. The EGF independence of the ras transfectants is consistent with the mid-G1 arrest of these cells at a point(s) distal to the primary action of EGF in early G0-G1.

Growth factor-stimulated mitogenicity in mouse embryo-derived AKR-2B cells was inhibited in a dose-dependent fashion by a mixture of alpha and beta mouse interferons (IFN). A 60% decrease in epidermal growth factor (EGF) and insulin-stimulated DNA synthesis was observed with 10 kU/ml IFN, and half-maximal inhibition was seen at 1 kU/ml. Likewise, the mitogenic effect of 5% fetal bovine serum (FBS) was inhibited by 60% with 10 kU/ml IFN and by 38% with 1 kU/ml IFN. IFN inhibition of DNA synthesis was paralleled by a decrease in monolayer growth of AKR-2B cells by 60% on the 3rd day of culture and by 40% on the 6th day of culture. Soft agar growth of two AKR-2B derived lines, AKR-MCA and AKR-2B (clone 84A), was also inhibited significantly with the addition of 1-10 kU/ml of IFN. The effect of IFN on EGF receptors was also examined. Treatment of AKR-2B cells with 10 kU/ml IFN resulted in a 35% decrease in EGF binding to cell surface receptors. The reduced binding of EGF to cells treated with IFN was due to a loss of EGF receptors as determined by Scatchard analysis. IFN treatment of AKR-2B cells neither altered the affinity of the EGF receptor for its ligand nor affected receptor internalization. Nuclear transcription and actinomycin D decay analysis indicated that within 2 hr, IFN reduced c-myc messenger RNA levels at the level of transcription with no affect on message decay.

The effects of cholera toxin (CT) on transforming growth factor beta 1-stimulated protooncogene expression, [gamma-35S]GTP binding, GTPase activity and growth under anchorage-independent and -dependent conditions were studied in AKR-2B fibroblast cells. CT was shown to inhibit TGF beta 1-stimulated c-sis and c-myc mRNA expression. Actinomycin D decay and nuclear runon experiments demonstrated that this inhibition was not due to an increased decay of protooncogene message, but to a decreased transcriptional activation. These inhibitory effects were not secondary to changes in the ability of TGF beta 1 to bind to its receptor(s) since radioreceptor assays and affinity labeling studies demonstrated that CT had no effect on TGF beta 1 binding. ADP ribosylation of AKR-2B plasma membranes with [alpha-32P]NAD+ revealed a Mr 45,000 protein as the major CT substrate. The labeling of this Mr 45,000 protein in membranes could be inhibited by prior pretreatment of the cells with increasing concentrations of CT. Treatment of membranes with nanogram concentrations of CT abolished the increase in [gamma-35S]GTP binding following addition of TGF beta 1 as well as decreased basal binding. Similarly, CT pretreatment of membranes inhibited TGF beta 1-stimulated GTPase activity. Unexpectedly however, the stimulatory effects of TGF beta 1 on anchorage-independent growth in soft agar were unaffected by CT. Only pertussis toxin was able to inhibit TGF beta 1-induced colony formation in soft agar in a dose-dependent manner. Furthermore, differential effects of both CT and pertussis toxin were observed on TGF beta 1-stimulated monolayer growth; CT was inhibitory, whereas pertussis toxin was without effect. These results suggest that the diverse biological effects of TGF beta 1 are mediated through multiple intracellular pathways distinguishable by their toxin sensitivities.