Formation of antibodies against infliximab and adalimumab strongly correlates with functional drug levels and clinical responses in rheumatoid arthritis.

BACKGROUND: Tumour necrosis factor alpha (TNFalpha) neutralising antibody constructs are increasingly being used to treat rheumatoid arthritis (RA). OBJECTIVE: To determine potential differences in clinical responses, soluble drug levels and antibody formation between patients with RA receiving infliximab and adalimumab. METHODS: 69 patients with RA fulfilling the 1987 American College of Rheumatology criteria and about to start treatment with infliximab or adalimumab, were enrolled consecutively. All patients had active disease (28-joint count Disease Activity Score >3.2). Infliximab was given intravenously at 3 mg/kg at baseline and after 2, 6 and 14 weeks. Adalimumab was administered as 40 mg biweekly subcutaneously. Concomitant drug treatment was monitored and continued at constant dosage during the study. All serum samples were tested for infliximab/adalimumab levels and anti-infliximab/anti-adalimumab antibodies. RESULTS: 35 patients received infliximab, 34 received adalimumab. At 6 months, 15 (43%), 6 (17%) and 14 (40%) of the infliximab-treated patients fulfilled the EULAR criteria for good, moderate and non-responders, respectively, whereas the corresponding figures for adalimumab-treated patients were 16 (47%), 8 (24%) and 10 (29%). Clinical responses correlated with the levels of S-infliximab/adalimumab and the formation of anti-infliximab/anti-adalimumab antibodies. CONCLUSION: The clinical response to two anti-TNFalpha biological agents closely follows the trough drug levels and the presence of antibodies directed against the drugs. Further studies that focus on the underlying pathways leading to antibody formation are warranted to predict immunogenicity of these expensive biological agents and treatment outcomes.

Cytotoxicity of human pI 7 interleukin-1 for pancreatic islets of Langerhans.

Activated mononuclear cells appear to be important effector cells in autoimmune beta cell destruction leading to insulin-dependent (type 1) diabetes mellitus. Conditioned medium from activated mononuclear cells (from human blood) is cytotoxic to isolated rat and human islets of Langerhans. This cytotoxic activity was eliminated from crude cytokine preparations by adsorption with immobilized, purified antibody to interleukin-1 (IL-1). The islet-inhibitory activity and the IL-1 activity (determined by its comitogenic effect on thymocytes) were recovered by acid wash. Purified natural IL-1 and recombinant IL-1 derived from the predominant pI 7 form of human IL-1, consistently inhibited the insulin response. The pI 6 and pI 5 forms of natural IL-1 were ineffective. Natural and recombinant IL-1 exhibited similar dose responses in their islet-inhibitory effect and their thymocyte-stimulatory activity. Concentrations of IL-1 that inhibited islet activity were in the picomolar range. Hence, monocyte-derived pI 7 IL-1 may contribute to islet cell damage and therefore to the development of insulin-dependent diabetes mellitus.

Binding of cytokines to pharmaceutically prepared human immunoglobulin.

Pharmaceutically prepared IgG, pooled from sera of over 2,000 normal individuals, contained both monomeric and dimeric IgG. Each type of IgG bound 125I-labeled interleukin (IL)-1 alpha, IL-1 beta, IL-6, and tumor necrosis factor (TNF)-alpha. Increased binding to IgG was observed if 125I-IL-1 beta was denatured by heating to 39 degrees C. However, the binding of both nondenatured and denatured 125I-IL-1 beta was not inhibited by unlabeled IL-1 beta. In contrast, binding of 125I-IL-1 alpha, 125I-IL-6, and 125I-TNF alpha was inhibited by the corresponding unlabeled cytokine. Papain-digestion of IgG abolished binding of 125I-TNF alpha but failed to influence the displaceable binding of 125I-IL-1 alpha and 125I-IL-6. 125I-TNF alpha was a mixture of trimeric and monomeric forms, the latter being the predominant form at lower concentrations. The apparent saturability of 125I-TNF alpha was explained by a higher nonspecific binding of monomeric than of trimeric 125I-TNF alpha to IgG. The amounts of cytokine antibodies in IgG preparations would contribute approximately 2 micrograms anti-IL-1 alpha IgG and 1 microgram anti-IL-6 IgG per kg body wt during high dose immune globulin therapy. In conclusion, pharmaceutical preparations of human IgG contain specific and neutralizing, high affinity antibodies against IL-1 alpha and IL-6, but not against TNF alpha or IL-1 beta. There are significant methodological pitfalls that hamper detection of IgG autoantibodies against cytokines.

High avidity IFN-neutralizing antibodies in pharmaceutically prepared human IgG.

This paper demonstrates and characterizes naturally occurring antibodies to interferon (IFN) in human IgG preparations. In vitro neutralization of the antiviral effect of IFN alpha and IFN beta, but not IFN gamma, was observed in 12 of 15 normal IgG preparations. The neutralizing capacity was higher against rIFN alpha 2A and rIFN alpha 2C than against lymphoblastoid IFN alpha and IFN beta. Frühsommer meningoencephalitis hyperimmune IgG and hepatitis-B hyperimmune IgG showed potent neutralization, whereas anti-rhesus D-, anti-rabies-, and anti-tetanus IgG showed weak neutralization. Saturable binding of 125I-rIFN alpha 2A was demonstrated only in those IgG preparations found to neutralize the antiviral effect of IFN. Significant correlation between IFN binding and neutralization capacity was observed. The antibodies bound with Fab to rIFN alpha 2A with an avidity of approximately 30 pM; the majority was of the IgG1 subclass. Maximum binding capacity was 490 pg rIFN alpha 2A/mg IgG. Cross-binding of rIFN alpha 2C, lyIFN alpha N1 and IFN beta occurred with 10 and 100-200 times lower activities than that of rIFN alpha 2A. There was no cross-binding with rIFN gamma or rIL-6. IgG preparations containing anti-IFN antibodies blocked the binding of 125I-rIFN alpha 2A to A549 cells. In conclusion, pharmaceutically prepared human IgG preparations contain variable but significant levels of high-avidity IFN alpha and IFN beta neutralizing antibodies.

Monitoring patients treated with anti-TNF-alpha biopharmaceuticals: assessing serum infliximab and anti-infliximab antibodies.

OBJECTIVES: Infliximab is an anti-tumour necrosis factor-alpha (TNF-alpha) mouse-human IgG1/kappa antibody used to treat patients with rheumatoid arthritis (RA) and other inflammatory diseases. Unfortunately, response failure and side-effects due to immunogenicity of the drug are not rare. In this study, we have compared different methods of assessing drug levels and anti-infliximab antibodies (Abs) and analysed the character of these Abs in sera of RA patients treated with infliximab for 1.5-18 months. METHODS: Functional serum infliximab levels and anti-infliximab Abs were measured by fluid-phase RIAs using 125I-labelled ligands in combination with molecular size and affinity chromatography, and immune complex precipitation. RESULTS: Anti-infliximab Abs were predominantly IgG, 36% being IgG4, and half the immune complexes were lambda-light-chain-positive. Ab titres were associated with inhibition of TNF binding to the drug, and low trough levels of infliximab were most frequent in anti-infliximab Ab-positive sera. Cross-binding to two other anti-TNF drugs was not observed. Detection of anti-infliximab Abs by solid-phase RIA using cross-binding of plastic-fixed and soluble infliximab exhibited low sensitivity and the data were inconsistent with results obtained from binding of the Abs to soluble infliximab. CONCLUSIONS: Specific and neutralizing anti-infliximab antibodies develop in RA patients treated with infliximab, and that low trough levels of functional infliximab are associated with the presence of such antibodies. The most sensitive antibody assay involved binding to soluble and intact infliximab. Assessments of bioavailability and immunogenicity of anti-TNF biologicals may be used to optimize dose regimens and prevent prolonged use of inadequate therapy.

Induction of interleukin-6 (IL-6) autoantibodies through vaccination with an engineered IL-6 receptor antagonist.

Neutralization of cytokine activity by monoclonal antibodies or receptor antagonists is beneficial in the treatment of immune and neoplastic diseases, but the necessity for continuous parenteral delivery of these anticytokine agents poses considerable practical limitations. A viable alternative is to induce a neutralizing antibody response. Using transgenic mice with high circulating levels of human interleukin-6 (hIL-6), we show that injection of the hIL-6 receptor antagonist Sant1 (an IL-6 variant with seven amino-acid substitutions) induces a strong anti-hIL-6 antibody response. The elicited antibodies bind circulating hIL-6 with very high affinity, totally masking it, and neutralize hIL-6 bioactivity both in vitro and in vivo.

Interleukin-6 autoantibodies: possible biological and clinical significance.

The pleiotropic cytokines, interleukin (IL)-1 alpha, type I interferons and IL-6 also act on cells involved in antibody production. Somehow the immunologic tolerance to these cytokines is often spontaneously broken--even in healthy individuals. Thus, relatively high concentrations of high affinity IgG antibodies against IL-1 alpha and IL-6 frequently occur in the circulation of healthy adults. The autoantibodies specifically antagonize the respective cytokines in vitro. Thermodynamic estimations strongly suggest that autoimmunity can play a significant role in the regulation of certain cytokines. In the light of IL-6 autoantibodies the possible biological and clinical significance of cytokine autoimmunity is discussed.

Cloning and expression of an alternatively spliced mRNA encoding a soluble form of the human interleukin-6 signal transducer gp130.

The membrane-bound gp130 glycoprotein acts as an affinity converting and signal transducing receptor (R) for interleukin-6 and several other cytokines. In this work, we RT-PCR amplified gp130 cDNA using primers flanking the sequence encoding the transmembrane domain of gp130. We observed in blood mononuclear cells, in addition to the expected 333-bp length fragment, a second major band of 418 bp. Sequencing of the 418-bp fragment and its genomic counterpart showed a new 85-bp exon located in the sequence encoding the extracellular region of the gp130 protein. This exon is most likely due to alternative splicing and leads to a frame-shift resulting in a stop-codon 1 bp before the transmembrane coding region. Correspondingly, supernatants from chinese hamster ovary cells transfected with this cDNA contained 4-5 times more soluble (s) gp130 than supernatants from cells transfected with a cDNA encoding the membrane-bound gp130 protein. Both gp130 and alternatively spliced sgp130 were also transcribed by the myeloma cell lines XG-1, XG-2, XG-4, XG-4CNTF XG-6, XG-7, XG-9, XG-10, U266 and RPMI 8226. However, XG-4A cells derived from XG-4 cells, but growing independently of exogenous IL-6, did not transcribe sgp130 mRNA. A possible interference with intracrine stimulatory factors by alternatively spliced sgp130 needs to be further investigated.

Stimulation of the B9 hybridoma cell line by soluble interleukin-6 receptors.

Interleukin-6 (IL-6) exerts its effects by binding to specific receptors on the cell surface. The IL-6 receptor consists of at least two components, a ligand binding 80 kDa low-affinity component (IL-6R) and a signal-transducing non-ligand binding 130 kDa component (gp130). The presence of soluble forms of these components has been described in both conditioned media and biological fluids. The soluble (s) IL-6R has been shown to enhance the IL-6 sensitivity of several both murine and human IL-6 sensitive cell types. A sensitive and commonly used method for measuring biological IL-6 activity is based on the IL-6 dependent proliferation of the murine hybridoma cell line B9. In this paper, we demonstrate that recombinant (r) human (h) sIL-6R enhances the sensitivity of B9 cells in a dose-dependent manner. The rhsIL-6R enhanced the binding of 125I-rhIL-6 to B9 cells. The rhsIL-6R induced stimulation of B9 proliferation was maximal at 100 ng/ml, even without addition of rhIL-6 and in the presence of anti-hIL-6 antibodies. This may be due to endogenous IL-6 production by the B9 cells, low levels of IL-6 in the fetal calf serum used, or perhaps an IL-6 independent effect by the rhsIL-6R. In conclusion, this and other reports point to the necessity of confirming measured biological activities through the use of neutralizing specific antibodies or parallel measurements in immunochemical assays.

Cytokine vaccination: neutralising IL-1alpha autoantibodies induced by immunisation with homologous IL-1alpha.

High-affinity IgG autoantibodies (aAb) to IL-1alpha are among the most frequently found aAb to cytokines in humans. To establish an animal model with aAb to IL-1alpha, we immunised mice with recombinant murine IL-1alpha. Unprimed and Bacille Calmette-Guérin (BCG)-primed BALB/cA mice were vaccinated with IL-1alpha coupled to purified protein derivative of tuberculin (PPD). Both unprimed and primed animals developed IgG aAb to IL-1alpha. These aAb persisted at high levels more than 100 days after vaccination and did not cross-react with murine IL-1beta. The induced anti-IL-1alpha aAb inhibited binding of IL-1alpha to the murine T-cell line NOB-1 by simple competition and neutralised IL-1alpha, but not IL-1beta-induced IL-6 in vivo. The aAb did not induce visible discomfort in the animals. In conclusion, long-lasting and high levels of neutralising and specific IgG aAb to IL-1alpha can be induced in mice by vaccination with recombinant murine IL-1alpha conjugated to PPD. Studies of the effects of IL-1alpha aAb in such animals may help clarify the importance of naturally occurring IL-1alpha aAb in humans and permit the evaluation of future therapies with cytokine aAb in patients with immunoinflammatory diseases and cytokine-dependent tumours.

1 alpha,25-Dihydroxyvitamin D3 and a novel vitamin D analogue MC 903 are potent inhibitors of human interleukin 1 in vitro.

1 alpha,25-dihydroxycholecalciferol (1,25(OH)2D3) inhibits the lymphocyte growth hormone, interleukin 2. Since its production is dependent upon interleukin 1 (IL-1) produced by antigen-presenting cells, we tested five vitamin D3 analogues for effects on the production and function of human natural and recombinant IL-1. The production was not affected, but 1,25(OH)2D3 (greater than 10(-11) M) and a synthetic derivative MC 903 (greater than = 10(-10) M) inhibited the proliferation of mouse thymocytes to IL-1. The vitamins failed to affect the cytotoxic activity of tumor necrosis factor. 1,25(OH)2D3 may play a physiological immunomodulatory role as a selective inhibitor of the function of IL-1, and MC 903 may prove clinically useful in this regard because of its limited calcium metabolic activity.

Human anti-interleukin 1 alpha antibodies.

Autoantibodies to the cytokine interleukin (IL)-1 alpha are frequently found in sera of apparently healthy humans. We have developed a sensitive radioimmunoassay (RIA) and an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of human serum antibodies to IL-1 alpha at concentrations below 10 pmol/l. The RIA is based on coprecipitation of 125I-labelled human recombinant IL-1 alpha (rIL-1 alpha) by rabbit antibodies to human immunoglobulins. The ELISA is based on recovery of added rIL-1 alpha to serum samples and takes advantage of the fact that free human autoantibodies to IL-1 alpha in a dose dependent manner reduce recovery of added rIL-1 alpha. The assays correlate exceedingly well (r = 0.99, P less than 0.001). Their inter- and intraassay coefficients of variation were less than 30% and less than 5% (RIA) and less than 20% and less than 10% (ELISA). Both assays were used to measure the presence of anti-IL-1 alpha antibodies in sera of patients with various autoimmune diseases. Autoantibodies to IL-1 alpha were detectable in up to 75% of these sera, but the frequencies and titers varied considerably between individuals with different diseases.

Differential interleukin-6 (IL-6) responses of three established myeloma cell lines in the presence of soluble human IL-6 receptors.

We investigated the possible influence of recombinant (r) sIL-6R on the growth of three IL-6 non-responsive or weakly IL-6 responsive long-term myeloma cell lines. The three cell lines chosen for the study (U266, L363 and Fravel) all expressed gp130 but differed in their expression of IL-6R and IL-6. mRNA analysis by northern blot and reverse transcriptase polymerase reaction showed that the cell line U266 was the only one that expressed IL-6 mRNA. Only U266 and L363 expressed IL-6R mRNA. 125I-rIL-6 binding studies and FACS analysis, using biotinylated IL-6 and antibodies directed against the IL-6R and gp130, showed corresponding results on the protein level. Addition of rsIL-6R resulted in induction of IL-6 responsiveness in L363 cells, whereas the 3H-thymidine incorporation of the cell lines U266 and Fravel was unaffected by rsIL-6R addition. In conclusion, the IL-6 unresponsive growth of several long-term myeloma cell lines in vitro can in some, but not all cases, be due to a deficiency in exogenous sIL-6R.

IgG for intravenous use, autologous serum and plasma induce comparable interleukin-1 receptor antagonist liberation from human mononuclear cells: an in vitro phenomenon depending upon plastic adherence.

Immunoglobulin G (IgG) for intravenous use (IVIg) selectively stimulates production of interleukin-1 receptor antagonist protein (IL-1ra) by mononuclear cells in vitro and has been proposed to stimulate IL-1ra production in vivo as part of the therapeutic effect. We tested if IVIg differed from human IgG-containing media (i.e., autologous serum (HS) and plasma (HP)) in stimulating IL-1ra release by MNC in vitro and whether IVIg induced a delayed increase in serum levels of IL-1ra. IVIg, 0.01-0.5 mg/ml, increased the IL-1ra liberation from MNC 10-15 times over that of untreated controls. HP and HS (5% v/v) had comparable effects. However, the stimulated IL-1ra liberation was reduced to less than twice the background liberation when fetal calf serum (FCS)-precoated tubes were used. Three days of high-dose IgG infusion had no significant effect on the serum levels of IL-1ra. It is concluded that therapeutic effects of IVIg cannot be ascribed to significant stimulation of IL-1ra production in vivo, as previously suggested, and that the observed stimulation of IL-1ra production in vitro is an epiphenomenon strictly dependent upon adherence of human serum and plasma constituents.

Production of IL-1beta, IL-1 receptor antagonist and IL-10 by mononuclear cells from patients with SLE.

Depositions of immune-complexes are responsible for many of the pathological features of systemic lupus erythematosus (SLE). For example, immune-complex-induced tissue damage in glomerulonephritis has been shown to be mediated, at least in part, by interleukin (IL)-1. Inappropriate production or function of IL-1 may therefore contribute to disease manifestations in SLE. We investigated lipopolysaccharide (LPS)- and adherent IgG-stimulated release of IL-1beta, IL-1 receptor antagonist (IL-1ra) and IL-10, a potent modulator of IL-1, by blood mononuclear cells from patients with SLE. Mediator production was measured as ng cytokines/10(6) monocytes and compared with clinical parameters. Release of IL-1beta was only detectable in LPS-stimulated cultures and substantially reduced in patients with both active and inactive disease (P < 0.001). LPS-stimulated IL-1ra release was normal and the IL-1ra/IL-1beta ratio was therefore increased (P < 0.05) and correlated inversely to prednisolone dosage (P = 0.009). IgG-stimulated release of IL-1ra was reduced in patients with active disease compared to those with inactive disease and controls (P = 0.002). IL-10 release was similar in patients and controls. We conclude that monocytes from patients with active SLE are deficient in Fc gamma-R-mediated production of IL-1ra, whereas LPS-stimulated IL-1beta release by SLE monocytes is reduced regardless of disease activity. The former may contribute to immune-complex-mediated tissue damage in SLE.

Decreased B lymphocyte ecto-5'nucleotidase and increased adenosine deaminase in mononuclear cells from patients infected with human immunodeficiency virus.

The levels of purine enzyme activities were studied in 10 patients with acquired immunodeficiency syndrome (AIDS) or AIDS related complex (ARC) and in 6 healthy individuals with antibodies against human immunodeficiency virus (HIV). All AIDS/ARC patients studied had ecto-5'nucleotidase (ecto-5'NUC) activity in B lymphocytes below the normal range and 4 out of 6 clinically healthy HIV-positive likewise had reduced activity. Increased numbers of activated B lymphocytes were found both in the group of healthy HIV positive individuals and in AIDS/ARC patients. Further studies are needed to define whether the decrease in ecto-5'NUC activity on the B lymphocytes is a result of increased activation of the cells or of a B cell defect. No significant changes were found in ecto-5'NUC levels in T lymphocytes or mononuclear cells (MNC), neither in the group of AIDS/ARC patients nor in the healthy HIV-positive group. Both AIDS/ARC patients and healthy individuals with antibodies against HIV had increased levels of adenosine deaminase (ADA) activity in mononuclear cells, but only in the group of AIDS/ARC patients was the increase significant. No changes were found in purine nucleoside phosphorylase (PNP) activity in the two groups tested. From these investigations of purine enzyme levels and other markers of immune function in both sick and healthy HIV infected individuals we conclude that the observed changes in ecto-5'NUC and ADA activities in HIV infected patients are not a direct result of the HIV infection but develop early in the course of the disease.

Massive parallel gene expression profiling of RINm5F pancreatic islet beta-cells stimulated with interleukin-1beta.

Interleukin 1 (IL-1) is a pleiotropic cytokine with the potential to kill pancreatic beta-cells, and this unique property is thought to be involved in the pathogenesis of type I diabetes mellitus. We therefore determined the quantitative expression of 24,000 mRNAs of RINm5F, an insulinoma cell line derived from rat pancreatic beta-cells, before and after challenge with 30 and 1,000 pg/ml of recombinant human IL-1beta. The highest concentration resulted in decreased insulin production and cell death over a period of 4 days. Using three different time points, 2, 4 and 24 hours after challenge, we found that 146 full-length genes and a large number of expressed sequence tags were differentially regulated 3-fold or more. Most of the differentially regulated transcripts have not previously been described to be regulated by IL-1beta in beta-cells. We have analysed the expression data and sorted the genes into groups according to functional relations on the basis of knowledge of the structure or function ascribed to the individual genes. Many of the differentially regulated genes are known to play a role in immune- and stress-related pathways as well as in insulin secretion and vesicle trafficking, e.g. alpha-endosulfine and K+ channel Kir6.2 are differentially regulated. A number of transcripts in the biosynthesis pathway for cholesterol are also differentially regulated.

Low prevalence of antibodies and other plasma factors binding to CC chemokines and IL-2 in HIV-positive patients.

Neutralizing cytokine antibodies are found in healthy and diseased individuals, including patients treated with recombinant cytokines. Identification of CCR-5 as co-receptor for HIV has focused interest on CC chemokines and their potential therapeutic use. Chemokine-binding components in plasma of HIV-infected patients were therefore assessed by radioimmunoassay and radioreceptor assay. IgG from 4/505 HIV patients and 9/2000 healthy controls (p>0.05) bound rMIP-1alpha and rMIP-1beta, but not rRANTES. No other plasma factors bound the chemokines. The antibodies inhibited receptor binding of both chemokines. There was no association between presence of antibodies and disease stage or HIV progression rate. Three of 11 patients treated with rIL-2 developed IgG antibodies suppressing cellular binding and growth promotion of rIL-2. Hence, circulating factors, including antibodies MIP-1alpha/MIP-1beta, are uncommon in healthy individuals and HIV patients, and are apparently without prognostic significance. In contrast to earlier reports, IL-2 antibodies were found only in HIV patients treated with rIL-2.

Detection of autoantibodies to cytokines.

Autoantibodies to various cytokines have been reported in normal individuals and in patients with various infectious and immunoinflammatory disorders, and similar antibodies (Ab) may be induced in patients receiving human recombinant cytokines. The clinical relevance of these Ab is often difficult to evaluate. Not only are in vitro neutralizing cytokine Ab not necessarily neutralizing in vivo, but assays for binding and neutralizing Ab to cytokines are often difficult to interpret. For example, denaturation of immobilized cytokines in immunoblotting techniques and immunometric assays may leave Ab to the native forms of the mediators unrecognized. On the other hand, Ab may bind nonspecifically and/or with biologically irrelevant low affinities, leading to erroneous interpretations. This article describes in detail the use of radioimmunoassays that we have optimized and used successfully for the detection of high-affinity (auto) Ab to IL-1 alpha, IL-6, GM-CSF, and IFN alpha.

Increase in natural killer cell activity during diethylcarbamazine treatment of patients with filariasis.

Two patients, one with Bancroftian filariasis and the other with onchocerciasis, and two healthy controls were treated with diethylcarbamazine (DEC). The natural killer (NK) cell activity of the two patients increased during DEC treatment to 2.5 and 2.8 times, respectively, while that of the controls remained unchanged. We conclude that the augmentation of baseline NK cell activity, as well as interferon- and interleukin-2-enhanced NK cell activity seen in the patients, is not a direct effect of DEC, but is related to the reaction to DEC in lymphatic filariasis and onchocerciasis.