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The importance of tumour necrosis factor (TNF)alpha in rheumatoid arthritis (RA) was initially proposed on the basis of analysis of cytokine gene regulation at the local site of the disease, the synovium. This was then verified in animal models and established in an extensive series of clinical trials, culminating in now 250000 treated patients with either of two approved TNF inhibitors, antibody or fusion protein. The degree and magnitude of clinical benefit has enabled analyses of the mechanism by which anti-TNF benefits, and hence insights into important steps in the disease process. It was found that essentially all aspects of RA were ameliorated, and important mechanisms of benefit involved diminution of multiple pro-inflammatory cytokines, adhesion molecules and chemokines, leading to reduced cell trafficking, reduced angiogenesis and most importantly halting of joint destruction. What of the problems? Safety is better than prior drugs, but there is a small increase in severe infections, smaller than might have been anticipated. Cost is the major drawback limiting greater use. In view of the central pathological processes down-regulated, and their role in many diseases, the early clinical success of anti-TNF in RA led to subsequent successful trials and registration in Crohn's disease and juvenile rheumatoid arthritis, and successful trials in ankylosing spondylitis, psoriasis and psoriatic arthritis. The era of anti-cytokine therapeutics is just dawning.

Objective: To evaluate the efficacy, pharmacokinetics, immunogenicity, and safety of multiple infusions of a chimeric monoclonal anti-tumor necrosis factor alpha antibody (cA2) (infliximab; Remicade, Centocor, Malvern, PA) given alone or in combination with low-dose methotrexate (MTX) in rheumatoid arthritis (RA) patients., Methods: In a 26-week, double-blind, placebo-controlled, multicenter trial, 101 patients with active RA exhibiting an incomplete response or flare of disease activity while receiving low-dose MTX were randomized to 1 of 7 groups of 14-15 patients each. The patients received either intravenous cA2 at 1, 3, or 10 mg/kg, with or without MTX 7.5 mg/week, or intravenous placebo plus MTX 7.5 mg/week at weeks 0, 2, 6, 10, and 14 and were followed up through week 26., Results: Approximately 60% of patients receiving cA2 at 3 or 10 mg/kg with or without MTX achieved the 20% Paulus criteria for response to treatment, for a median duration of 10.4 to >18.1 weeks (P < 0.001 versus placebo). Patients receiving cA2 at 1 mg/kg without MTX became unresponsive to repeated infusions of cA2 (median duration 2.6 weeks; P=0.126 versus placebo). However, coadministration of cA2 at 1 mg/kg with MTX appeared to be synergistic, prolonging the duration of the 20% response in >60% of patients to a median of 16.5 weeks (P < 0.001 versus placebo; P=0.006 versus no MTX) and the 50% response to 12.2 weeks (P < 0.001 versus placebo; P=0.002 versus no MTX). Patients receiving placebo infusions plus suboptimal low-dose MTX continued to have active disease, with a Paulus response lasting a median of 0 weeks. A 70-90% reduction in the swollen joint count, tender joint count, and C-reactive protein level was maintained for the entire 26 weeks in patients receiving 10 mg/kg of cA2 with MTX. In general, treatment was well tolerated and stable blood levels of cA2 were achieved in all groups, except for the group receiving 1 mg/kg of cA2 alone, at which dosage antibodies to cA2 were observed in approximately 50% of the patients., Conclusion: Multiple infusions of cA2 were effective and well tolerated, with the best results occurring at 3 and 10 mg/kg either alone or in combination with MTX in approximately 60% of patients with active RA despite therapy with low-dose MTX. When cA2 at 1 mg/kg was given with low-dose MTX, synergy was observed. The results of the trial provide a strategy for further evaluation of the efficacy and safety of longer-term treatment with cA2.

Juvenile chronic arthritis (JCA) is the commonest chronic rheumatic disorder of childhood. Although conventional therapy of JCA continues to improve, many patients experience long-term ill health as a result of their disease or treatment. In adult rheumatoid arthritis (RA), similar concerns have led to the development of therapies designed to interfere in key disease processes. One such therapy is cA2, a chimeric neutralizing monoclonal antibody to the inflammatory cytokine, tumour necrosis factor-alpha (TNF-alpha). The administration of cA2 in adult RA has led to impressive short-term suppression of disease, with a good safety profile. Here, we report the first use of cA2 in childhood arthritis, choosing a patient with severe systemic-onset JCA, resistant to conventional therapies. The patient received two i.v. infusions of cA2, each at a dose of 10 mg/kg, separated by 1 week. The treatment was well tolerated and induced rapid control of fever, anorexia and serositis, together with downregulation of interleukin (IL)-6, soluble TNF receptors (sTNFR) and IL-1ra, and the acute-phase proteins C-reactive protein (CRP) and serum amyloid A (SAA). In contrast, we saw no significant improvement in joint pain or tenderness. Our findings suggest that TNF-alpha is a mediator of fever and other systemic aspects of disease in systemic JCA. TNF-alpha blockade as a treatment modality in JCA deserves further study.

This study investigates immunogenicity and in vivo effects on T-cells of long-term CD4 monoclonal antibody treatment of patients with rheumatoid arthritis. Patients were treated with several dosage regimens of a chimeric CD4 monoclonal antibody entitled cM-T412 over the course of 1 year. The circulating CD4+ T-cell count sharply decreased after the first cM-T412 injection and slowly recovered after the last injection. Within the CD4+ subset there was a selective depletion of CD45RA+ T cells, HLA-DR+, and CD25+ cells, providing evidence that activated/memory CD4+ cells resist the effect of CD4 monoclonal antibodies. Studies on cytokine production by peripheral blood mononuclear cells cultures in vitro revealed no differential effect on the production of interleukin-4 compared to interferon-gamma, indicating that a shift from a Th1 to a Th2 lymphokine production profile was not achieved. Human anti-monoclonal antibodies (HAMA) were induced in a minority of the patients predominantly after the first treatment course. All the sera containing HAMA specifically inhibited the binding of cM-T412 to T-cells. However, HAMA formation does not interfere with the biological effect of repeated cM-T412 administration since the degree of CD4 depletion following repeated administration of cM-T412 to patients with and without blocking antibodies was similar. We conclude that the currently available data are of critical importance in the interpretation of the obtained clinical experience and for further development of this therapeutic strategy.

There is evidence that treatment with an antibody to tumor necrosis factor alpha (TNF alpha) improves an animal model of multiple sclerosis (MS) and is beneficial in two systemic inflammatory disease in humans, but there are no reports about anti-TNF treatment of MS. Therefore, we treated two rapidly progressive MS patients with intravenous infusions of a humanized mouse monoclonal anti-TNF antibody (cA2) in an open-label phase I safety trial and monitored their clinical status, gadolinium-enhanced brain magnetic resonance imaging (MRI), and peripheral blood and cerebrospinal fluid (CSF) immunologic status. We did not notice any clinically significant neurologic changes in either patient. The number of gadolinium-enhancing lesions increased transiently after each treatment in both patients. CSF leukocyte counts and IgG index increased after each treatment. The transient increase in the number of gadolinium-enhancing lesions that followed each infusion of cA2 together with the increase in cells and immunoglobulin in the CSF of each patient suggest that the treatment caused immune activation and an increase in disease activity. These results suggest that further use of cA2 in MS is not warranted and that studies of other agents that antagonize TNF alpha should be carried out with frequent monitoring of gadolinium-enhanced MRIs.

Tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine known to stimulate human immunodeficiency virus type 1 (HIV-1) replication, has been implicated in the pathogenesis of HIV-1 infection. Inhibition of TNF-alpha by a chimeric humanized monoclonal antibody, cA2, was investigated in 6 HIV-1-infected patients with CD4 cell counts < 200/mm3. Two consecutive infusions of 10 mg/kg 14 days apart were well tolerated, and a prolonged serum half-life for cA2 (mean, 257 +/- 70 h) was demonstrated. Serum immunoreactive TNF-alpha concentrations fell from a mean prestudy value of 6.4 pg/mL (range, 4.2-7.9) to 1.1 pg/mL (range, 0.5-2.2) 24 h after the first infusion and returned to baseline within 7-14 days. A similar response was seen after the second infusion. No consistent changes in CD4 cell counts or plasma HIV RNA levels were observed over 42 days. Future studies evaluating the therapeutic utility of long-term TNF-alpha suppression using anti-TNF-alpha antibodies are feasible and warranted.

Objective: To assess whether monoclonal antibody to tumor necrosis factor alpha (TNF alpha) reduces endothelial activation in rheumatoid arthritis (RA)., Methods: Levels of serum E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1), and circulating leukocytes (differential counts) were measured in RA patients before and up to 4 weeks after infusion of either placebo or chimeric anti-TNF alpha antibody cA2 (1 or 10 mg/kg)., Results: Treatment with anti-TNF alpha decreased serum E-selectin and ICAM-1 levels, with the earliest detectable changes observed on days 1-3 after anti-TNF alpha infusion. No effect on VCAM-1 levels was detected. In parallel, there was a rapid and sustained increase in circulating lymphocytes. The extent of the decrease in serum E-selectin and ICAM-1 levels and the increase in lymphocyte counts was significantly higher (P < or = 0.05) in patients in whom a clinical benefit of anti-TNF alpha was observed ( > or = 20% response, by Paulus criteria, at week 4) compared with that in patients who failed to respond to anti-TNF alpha at this time point., Conclusion: We propose that decreased serum levels of adhesion molecules may reflect diminished activation of endothelial cells in the synovial microvasculature, leading to reduced migration of leukocytes into synovial joints, and thus prolonging the therapeutic effect of anti-TNF alpha in RA.

Due to the unknown etiology of RA, specific treatment is not available. Recently, in a double-blinded, placebo-controlled clinical trial, in vivo blockade of TNF-alpha by a single infusion of a chimeric TNF-alpha-blocking mAb, cA2, has proven to be highly effective in the treatment of RA. In parallel to this trial, we tested the consequences of cA2 infusion in ex vivo and in vitro experiments. In this paper, we describe an increase in CD4+ and CD8+ T lymphocyte counts on day 1 and a marked decrease in monocyte counts preferentially on day 7 after cA2 treatment, without major changes in B lymphocyte or NK cell counts. In addition, we found an increased responsiveness of PBMC to CD28 mAb/PMA, but not to CD3 mAb, superantigen staphylococcus enterotoxin B, or PHA on day 1 after infusion. The increase in DNA synthesis of PBMC was paralleled by increased IL-2 mRNA and IL-4 mRNA expression and IL-2 protein secretion in culture supernatants after in vitro stimulation of PBMC with CD28 mAb/PMA. In PBMC, we did not find any significant changes in mRNA or protein expression of CD28 Ag or CD28 ligands, B7-1 and B7-2. Serum concentrations of IL-1 beta, IL-6, and soluble CD14 were significantly diminished after in vivo TNF-alpha blockade. We did not see relevant changes in granulocyte function in vitro after cA2 infusion. Finally, we observed a statistically significant decrease in slCAM-1 molecules in the serum of patients treated with verum compared with that in the serum of subjects given placebo. This change in slCAM-1 concentration was evident on days 1 and 7 after the infusion of 10 mg/kg cA2, whereas it occurred only on day 7 in the serum of patients treated with the low dose (1 mg/kg) of cA2.

Objective: We assessed the effect of a daily dosing schedule of the chimeric anti-CD4 monoclonal antibody (MAb), cM-T412, in rheumatoid arthritis (RA) patients, and compared lymphocyte changes in the peripheral blood (PB) and synovial fluid (SF) of these patients., Methods: Twelve patients received 50 mg/day of cM-T412 for 5 days, followed by a maintenance treatment of 50 mg/week for 5 weeks (6 patients), or a retreatment course of 50 mg/day for 5 days after 5 weeks (6 patients). Paired PB and SF samples were obtained during treatment for analysis., Results: Changes in lymphocyte count and coating with the MAb in PB did not reflect changes in the SF. After 5 daily treatments, the percentage of cM-T412-coated CD4+ lymphocytes in SF correlated with the degree of clinical improvement seen in patients at 2 weeks after the initiation of therapy (r = 0.75, P < 0.05)., Conclusion: These results demonstrate the importance of antibody dosage and treatment regimen in determining clinical benefit. Our findings suggest that the percentage of cM-T412-coated CD4+ lymphocytes in SF may be a predictor of clinical outcome.

Objective: To evaluate the clinical response to and safety of single and repeat doses of a chimeric anti-CD4 monoclonal antibody, cM-T412, in patients with rheumatoid arthritis (RA) concomitantly treated with a stable regimen of low-dose methotrexate., Methods: Sixty-four patients with refractory RA, who were already receiving stable doses of methotrexate, were randomized into a multicenter, double-blind, placebo-controlled trial to receive 3 monthly treatments with either a placebo, or 5, 10, or 50 mg cM-T412, given intravenously., Results: Using > or = 50% improvement in swollen joint counts as a criterion for clinical response, 13%, 13%, 18%, and 13% of patients receiving 50, 10, or 5 mg cM-T412, or the placebo, respectively, exhibited a clinical response at 3 months of therapy. Using > or = 50% improvement in tender joint counts as a measure of clinical efficacy at 3 months, 19%, 13%, 12%, and 6% of patients receiving 50, 10, or 5 mg cM-T412, or the placebo, respectively, exhibited a clinical response. "Flu-like" symptoms (fever, chills, rigor) within 24 hours of the infusion occurred more frequently in the groups receiving 50-mg (29%) and 10-mg (31%) doses of cM-T412 than those receiving 5 mg cM-T412 (12%) or the placebo (13%). Significant CD4+ T cell depletion occurred in the 50-mg group (mean of 353 CD4+ T cells/mm3 at 6 months versus 856 CD4+ T cells/mm3 at baseline). All patients were followed up for 12 months after the final treatment; no opportunistic infectious complications occurred., Conclusion: Treatment with cM-T412 in this cohort of RA patients who were also taking methotrexate was not associated with clinical efficacy or enhanced toxicity from infectious complications, despite significant peripheral CD4+ T cell depletion.

Background & Aims: Increased concentrations of tumor necrosis factor (TNF), a potent proinflammatory cytokine, can be shown in the mucosa of patients with active Crohn's disease. Neutralization of TNF has been shown to decrease recruitment of inflammatory cells and granuloma formation in several animal models. The aim of this study was to investigate the safety and potential efficacy of an anti-TNF monoclonal antibody in the treatment of active Crohn's disease., Methods: Ten patients with active Crohn's disease that was unresponsive to therapy were administered a single infusion of an anti-TNF human/mouse chimeric monoclonal antibody (cA2) in an open-label treatment protocol while the baseline anti-inflammatory therapy was continued., Results: Eight patients showed normalization of Crohn's Disease Activity Index scores and healing of ulcerations as judged by colonoscopy within 4 weeks after treatment. One patient had a perforation after colonoscopy and recovered completely after surgery. One elderly patient showed a poor response. The average duration of response after a single infusion was 4 months. No adverse experiences related to cA2 were observed., Conclusions: The results support the hypothesis that TNF is of major importance in the pathogenesis of Crohn's disease. Treatment with cA2 was safe and may be useful in patients with Crohn's disease that is unresponsive to steroid treatment.

Our in-vitro, animal, and early clinical data suggest that tumour necrosis factor alpha (TNF alpha) is an important target for specific biological therapy in rheumatoid arthritis. We report the results of repeated treatment with a chimeric monoclonal antibody to TNF alpha (cA2) in patients having disease flares. 7 patients originally enrolled in an open-label trial completed two to four cycles, each of which was followed by a good clinical response, with median improvements in the swollen-joint count and C-reactive protein exceeding 80%. cA2 may be useful therapy in the control of acute disease flares in rheumatoid arthritis and treatment programmes including cA2 may be effective in the long-term management of this disease.

Human monoclonal IgM antibody HA-1A, which recognizes the lipid A component of bacterial lipopolysaccharide (LPS), has been shown to reduce mortality in Gram negative septicemia. The vascular endothelial lining of blood vessels, which controls leucocyte traffic and activation, as well as haemostatic balance, may be one of the primary targets of LPS action during sepsis. In earlier studies we have described HA-1A-induced immune adherence of LPS to complement receptors on erythrocytes, and showed that pre-incubation with HA-1A, in the presence of complement and red blood cells, markedly reduced LPS-induced cytokine production from peripheral blood mononuclear cells. In the present study, we measured the effect of immune adherence of LPS in the presence of HA-1A on the responses of cultured endothelial cells, and found that subsequent expression of adhesion molecules such as E-selectin, ICAM-1 and VCAM-1, and secretion of the cytokines interleukin-6 and granulocyte-macrophage colony stimulating factor were markedly reduced. Moreover, the ability of LPS to increase levels of tissue factor procoagulant activity on endothelial cells was markedly diminished by LPS immune adherence to HA-1A. This decrease in endothelial activation in response to LPS following immune adherence to HA-1A may play a significant role in the protective effect of HA-1A in vivo during the course of Gram negative sepsis.

HA-1A is a human monoclonal IgM antibody which recognizes the lipid A component of lipopolysaccharide (LPS). This antibody has reduced mortality in the septic shock syndrome resulting from Gram negative bacteria, in which many of the manifestations are considered to be due to cellular activation and secretion of cytokines, most notably TNF-alpha. However HA-1A does not directly neutralize LPS effectively in vitro, and studies reported to date have not defined its mechanism of action. Here we demonstrate that HA-1A, which in the presence of complement promotes immune adherence, may inhibit LPS action by facilitating its sequestration on red blood cells and clearance to an extent that cytokine production is reduced. Incubation of LPS at clinically significant (pg/ml) does with HA-1A at therapeutic levels (e.g. 10 micrograms/ml) and complement resulted in LPS association with erythrocyte CR1 receptors. This reduced the ability of the residual, free LPS by 50-70% to induce the secretion of TNF-alpha, IL-1 beta and IL-6 from normal blood mononuclear cells. This mechanism is likely to be operative in vivo, and could account for the protective effect of HA-1A, and its reduction of TNF-alpha production in vivo.

Human splenic lymphocytes were fused with a new mutant human myeloma cell line (HFB-1) to produce hybridomas that secrete human monoclonal antibodies. A cloned hybridoma line was selected that secretes IgG antibodies reactive with Haemophilus influenzae type b polyribosylribitolphosphate (PRP) capsular polysaccharide. This human monoclonal antibody was active in an in-vitro neutrophil-mediated bactericidal assay, and provided significant protection in an animal model of H. influenzae type b disease. Human monoclonal antibody to H. influenzae type b may have prophylactic value in man, particularly in infants who do not produce protective antibody after active immunisation with PRP.

Monoclonal antibodies (DA-2 and CA-2) and xenoantisera (rabbit anti-human, p23,30) specific for HLA-DR framework determinants were added to primary and secondary mixed lymphocyte cultures. Although such antisera were shown to inhibit primary MLC, primed lymphocytes were much less sensitive to the blocking effects of these antibodies. In the studies shown here, the concentration of antibody required to inhibit primary MLC reactions was 0.1-1.5 micrograms/ml).