Preparation and identification of an anti-idiotypic antibody antagonist (FG8) for EGFR that shows potential activity against liver cancer cells.

OBJECTIVE: Currently, there are two categories of epidermal growth factor receptor (EGFR) antagonists, small molecule antagonists and anti-EGFR antibodies. In the current study, we developed a new EGFR antagonist employing the anti-idiotypic antibodies strategy. RESULTS: First, using EGF as an antigen, through a series of immunological protocols and hybridoma technology, we obtained an anti-idiotypic antibody against EGF receptor-binding epitopes. On this basis, we screened and characterized the anti-idiotype antibodies against EGFR through competitive ELISA, co-localization analysis, competitive receptor binding analysis, and immunofluorescence. Finally, an internal image anti-idiotype antibody called FG8 was successfully prepared. Experiment result shows that FG8 inhibits EGFR-mediated signaling pathways in vitro. Additionally, FG8 inhibits liver tumor cell proliferation as well as induces tumor cell apoptosis. CONCLUSIONS: The present study suggests that FG8 is a potential therapeutic agent for liver cancer. In addition, this study provides a novel method for the preparation of EGFR antagonists.

Different forms of alpha-1 antitrypsin and neutrophil activation mediated by human anti-PR3 IgG antibodies.

BACKGROUND: One of characteristic findings in granulomatosis with polyangiitis (GPA) is the presence of proteinase-3 (anti-PR3) specific antibodies. These antibodies can cause neutrophil activation, degranulation and generation of reactive oxygen species (ROS). Each of these inflammatory events can be suppressed by circulating alpha-1 antitrypsin (A1AT). A1AT is an acute phase protein increasing during inflammation, however, it may circulate as an inactive polymeric protein. The aim was to analyze how different types of A1AT can affect anti-PR3 mediated neutrophil activation. METHODS: Granulocytes were obtained from the blood of healthy volunteers and purified by density gradient centrifugation. Effects of A1AT on IgG anti-PR3-mediated neutrophil activation were evaluated by stimulation of the cells with native IgG anti-PR3 antibodies in the presence of native or polymerized A1AT. Analyses of selected proinflammatory genes expression were performed using quantitative real-time. Flow cytometry was used to study the cell membrane PR3, its binding by anti-PR3 IgG, and production of ROS at presence A1AT. Neutrophil elastase complexes with A1AT were measured by ELISA. RESULTS: Native A1AT inhibited formation of the immune complex of PR3 with anti-PR3 and anti-PR3-mediated neutrophil activation/ROS production. Protective effect of polymerized A1AT against these events was diminished at least fivefold. CONCLUSIONS: Native A1AT can prevent pivotal events of neutrophils' activation by anti-PR3 IgG, the main autoantibody in anti-PR3 dependent vasculitis. Inhibitory properties of polymerized A1AT, decreased plausibly due to a loss of anti-protease function, can explain more severe course of the disease in subjects with deficiency of A1AT.

Survey of immuno-allergological ultra high dilution research.

BACKGROUND: Experiments about basic research in Immuno-allergology reported by M. Bastide and B. Poitevin in Ultra High Dilution (1994) have been appraised from a 20 year perspective. The numerous experiments published mainly focus on immunological regulation, inflammatory process and basophil activation. They are analyzed according to one essential criterion: repeatability. METHODS: The commentary reflects the research details made available in a recently published literature review, also published in French. RESULTS: The regulatory effect of high dilution of bursin on immune response has been observed in multiple experiments but not reproduced by independent teams. The immunomodulating effect of Thymulin has been confirmed in mice. Rhus toxicodendron has an anti-inflammatory activity on different models, from mother tincture (TM) to very high dilutions. The homeopathic complex Canova activates macrophages in vitro and in vivo, induces lymphocyte proliferation, and reduces the size of tumors and mortality of sarcoma-bearing mice. Some homeopathic medicines used in clinical inflammation modulate in vitro the neutrophil activation, with variability in the protocols used and in the medicines tested. In allergology, high dilution histamine has an inhibitory effect on basophil activation in multicenter trials and with independent teams, either with methods implying a change in basophil staining or with flow cytometry. However, high dilution histamine had no effect in some well-conducted experiments. The inhibitory effect of Apis mellifica has not been studied with the flow cytometry method, as well as the activation of basophil by anti-IgE high dilution, published in Nature. CONCLUSIONS: Despite considerable research activity in immuno-allergology and a great increase in the number of publications, there is still not in this domain a "gold standard" trial in basic research in homeopathy. The most studied system, the inhibitory effect of histamine high dilutions on basophil activation, requires clarifications of various factors, including individual sensitivity. For scientific and epistemological reasons, the same work should be carried out for independent reproduction of the experiments conducted with anti-IgE and Apis mel high dilution, in complement of the new axes of research in immunoallergology developed since 20 years.

Prognostic vs predictive molecular biomarkers in colorectal cancer: is KRAS and BRAF wild type status required for anti-EGFR therapy?

An important molecular target for metastatic CRC treatment is the epidermal growth factor receptor (EGFR). Many potential biomarkers predictive of response to anti-EGFR monoclonal antibodies (cetuximab and panitumumab) have been retrospectively evaluated, including EGFR activation markers and EGFR ligands activation markers. With regard to the "negative predictive factors" responsible for primary or intrinsic resistance to anti-EGFR antibodies a lot of data are now available. Among these, KRAS mutations have emerged as a major predictor of resistance to panitumumab or cetuximab in the clinical setting and several studies of patients receiving first and subsequent lines of treatment have shown that those with tumors carrying KRAS mutations do not respond to EGFR-targeted monoclonal antibodies or show any survival benefit from such treatments. The role of B-RAF mutations, mutually exclusive with KRAS mutations, in predicting resistance to anti-EGFR mAbs is not yet consolidated. It therefore appears that BRAF mutations may play a strong negative prognostic role and only a slight role in resistance to anti-EGFR Abs.

Stimulus-specific regulation of CD63 and CD203c membrane expression in human basophils by the flavonoid quercetin.

BACKGROUND: Flavonoids, such as quercetin, were reported to inhibit histamine release and cytokine production by basophils, but there is no evidence describing their action on membrane markers and intracellular biochemical pathways. OBJECTIVE: The aim of the study was to examine the effect of several quercetin doses on an in vitro human basophil activation system that evaluates up-regulation of membrane markers in response to agonists. METHODS: Leukocyte buffy coats from K(2)-EDTA anti-coagulated blood were treated with different concentrations of quercetin and triggered with anti-IgE ("allergy model") and with N-formyl-Met-Leu-Phe (fMLP) ("inflammation model"). Basophils were captured as CD123(bright)/HLA-DR(non-expressing) cells in a flow cytometry analysis and fluorescence values of CD63-FITC, CD203c-PE and CD123-PECy5 were used to produce dose response curves. RESULTS: Quercetin at a dose of 10 microg/ml strongly inhibited CD63 and CD203c membrane up-regulation triggered by both agonists, but it neither affected cell viability nor changed the expression of the phenotypic marker CD123. The anti-IgE model appeared highly sensitive to the effect of quercetin: a dose as low as 0.01 microg/ml was able to significantly decrease CD63 and CD203c membrane expression. In the fMLP model the dose response was different: quercetin doses from 0.01 to 0.1 microg/ml significantly increased up-regulation of membrane markers, achieving the highest effect with CD63. CONCLUSION: Very low doses of quercetin, within the pharmacological range, inhibit IgE-mediated membrane marker's up-regulation but prime the response to the chemotactic peptide fMLP; this stimulus specificity may have implications on the possible therapeutic action of the flavonoid in different pathologies.

Cyclophosphamide for anti-GAD antibody-positive refractory status epilepticus.

Glutamic acid decarboxylase (GAD) is the enzyme which catalyzes the production of gamma aminobutyric acid (GABA), the main inhibitory neurotransmitter in the central nervous system (CNS). There is increasing evidence that severe GAD autoimmunity may be associated with refractory epilepsy. Immunomodulation and GABAergic drugs have been suggested as treatment options. We report here for the first time on a patient with sudden onset of refractory status epilepticus in the presence of strong intrathecal anti-GAD antibody synthesis who was successfully treated with cyclophosphamide, and give an overview of available data on epilepsy associated with GAD autoimmunity.

Anti-fibroblast antibodies detected by cell-based ELISA in systemic sclerosis enhance the collagenolytic activity and matrix metalloproteinase-1 production in dermal fibroblasts.

OBJECTIVES: Antibodies binding to the surface of fibroblasts (anti-fibroblast antibodies: AFA) have been described in systemic sclerosis (SSc). We aimed to assess the effect of AFA on extracellular matrix (ECM) turnover and whether AFA were associated with anti-topoisomerase-I antibody. METHODS: IgG were purified from AFA-positive and AFA-negative sera selected within 20 SSc and 20 healthy individuals, and tested on normal dermal fibroblasts, at protein and mRNA level, for their capacity to induce collagen deposition or degradation. RESULTS: Fibroblasts stimulated with AFA-positive but not with AFA-negative and control IgG showed an increased capacity to digest collagen matrix and produce metalloproteinase-1 (MMP-1) while their production of total collagen, type I collagen and tissue inhibitor of metalloproteinase-1 (TIMP-1) was unaffected. The steady-state mRNA levels of MMP-1, COL1A1 and TIMP-1 paralleled the protein levels. AFA-positive IgG did not induce Smad 2/3 phosphorylation, indicating that this transforming growth factor-beta signalling pathway was not involved. IL-1 and tumour necrosis factor (TNF) neutralization did not reverse the enhanced production of MMP-1, suggesting a direct effect of AFA on fibroblasts. Finally, anti-topoisomerase-I antibodies were present in 11 of 12 AFA-negative IgG, and an anti-topoisomerase-I monoclonal antibody failed to enhance MMP-1 production, thus indicating a lack of correlation between AFA and anti-topoisomerase-I antibody. CONCLUSIONS: These results indicate that SSc antibodies binding to fibroblasts enhance matrix degradation and MMP production events that may favour inflammation but do not directly impact on fibrosis development.

Immune responses in advanced colorectal cancer following repeated intradermal vaccination with the anti-CEA murine monoclonal antibody, PR1A3: results of a phase I study.

BACKGROUND AND AIMS: The aim was to determine the toxicity, clinical and immune responses to the murine monoclonal anti-carcinoembryonic antigen (CEA) antibody, PR1A3, in patients with advanced colorectal cancer. MATERIALS AND METHODS: Fifteen patients with advanced colorectal cancer received either 0.5-, 1.0- or 5.0-mg doses of PR1A3 mixed with 10% w/v Alum adjuvant (Superfos Biosector, Denmark) intradermally at 4-week intervals for 3 months. Patient serum was assessed for anti-idiotypic (Ab2), anti-anti-idiotypic (Ab3) and human anti-mouse antibody (HAMA) reactivity. Peripheral blood mononuclear cell (PBMC) proliferation with phytohaemagglutinin (PHA), CEA and PR1A3, stimulated IL-2, IL-4 and IFN-gamma levels and PR1A3-stimulated IL-2 receptor expression during immunotherapy were determined. Comparisons were made with 16 age-matched controls without malignant disease. RESULTS: Hyperimmune sera from 12 of the 15 patients showed Ab2 reactivity with no detectable Ab3 responses. Strong HAMA reactivity was recorded in 7 of the 15 cases with no adverse clinical effect. Delayed-type hypersensitivity (DTH) responses developed in 12 of the 15 patients. Pre-treatment PBMC proliferation with PHA was subnormal in each patient compared with controls, becoming normal (or supranormal) in all patients during immunisation (P<0.001). PBMC proliferation with CEA and PR1A3 increased during immunotherapy (P<0.001) along with stimulated production of IL-2, IFN-gamma and IL-2 receptor expression. Progressive disease was observed in 14 of the 15 patients with minimal toxicity. CONCLUSION: PR1A3 generated limited idiotypic responses but robust DTH reactivity in most patients. In vitro PBMC proliferation with mitogens and recall antigens is greatly increased during the course of immunisation, with a shift in stimulated cytokine profile.

Decreased production of anti-mouse antibody after administration of human/mouse chimeric monoclonal antibody-neocarzinostatin conjugate to human.

BACKGROUND/AIMS: One problem that has been encountered with the use of murine Mabs is the development of a human anti-mouse antibody (HAMA) which lead to serious problems such as anaphylaxis. In order to evaluate the decreased HAMA production after administration of Fab fragments of chimeric monoclonal antibody, Fab fragments of chimeric Mab A7 (chA7Fab) was administered as neocarzinostatin (NCS) conjugate to seven patients with colonic cancer. METHODOLOGY: The in vivo pharmacokinetics of chA7Fab and HAMA production was examined. These patients received an infusion of 4,000 U: dose of NCS (18 mg: dose of chA7Fab). RESULTS: The plasma disappearance curves showed that chA7Fab-NCS was more rapidly cleared than A7-NCS. The chA7Fab-NCS did not elicit of HAMA in two of seven evaluable patients. The chA7Fab-NCS NCS elicited low levels of HAMA in five of seven evaluable patients. In contrast, A7-NCS elicited high levels of HAMA in all patients tested. Anti-isotype HAMA was not seen in seven evaluable patients tested with chA7Fab-NCS, while A7-NCS elicited high levels in all patients tested. CONCLUSIONS: This study demonstrates the reduced immunogenicity and shorter clearance time of chA7Fab-NCS compared to A7-NCS.

Intravenous immunoglobulin monotherapy in long-term treatment of myasthenia gravis.

OBJECTIVE: To investigate effectiveness of long-term treatment of myasthenia gravis (MG) with intravenous immunoglobulin (IVIG). BACKGROUND: There are no definitive studies showing effectiveness of IVIG therapy in long-term treatment of MG. Most studies have investigated the acute treatment of MG with IVIG. We describe our experience with long-term treatment of MG with IVIG in six patients. METHODS: Acute treatment of MG by IVIG therapy has been well established in the literature. We describe six patients who were treated on a long-term basis with IVIG therapy. All of these patients had positive acetylcholine receptor antibody titers. They all received initial infusion for 5 days of IVIG at a dose of 400 mg/kg/day followed by maintenance therapy of 400 mg/kg for 1 day every 3-4 months. These patients were followed for 2 years. All other medications, including prednisone and cholingeric drugs such as Mestinon, were gradually weaned. For the last years, each of these patients maintained better than functional class 2 on an average of 1.5-2.2+/-0.5 grades on the University of Virginia modification of Ossermann's classification scale for MG. They were solely treated with IVIG infusion every 3-4 months without any other concomitant medications. Three of the patients had previously undergone thymectomies. None of the patients noticed any worsening in their scores on the University of Virginia modification of Ossermann's classification worse than Grade II in the last 2 years. There were no complications related to IVIG therapy, and all patients tolerated a single infusion of IVIG every 3-4 months at 400 mg/kg for 1 day. RESULTS: Our study demonstrates that IVIG maintenance is effective treatment of MG in selected patients and it is well tolerated. CONCLUSIONS: IVIG therapy is a convenient, effective therapy when used selectively for treatment of MG on a long-term basis without any significant side effects.

Do early childhood immunizations influence the development of atopy and do they cause allergic reactions?

Concerns about allergic side-effects of vaccines and about a possible promotion of allergic diseases contribute to incomplete vaccination rates in childhood. This article reviews the current understanding of these issues. There is evidence that pertussis and diphtheria/tetanus antigens elicit immunoglobulin E (IgE) antibody formation as part of the immune response. In murine models, pertussis toxin is an effective adjuvant for IgE formation against simultaneously administered antigens. In children, however, sensitization to unrelated antigens or development of allergic diseases do not seem to be augmented. In contrast, bacille Calmette-Guérin (BCG) and measles vaccination have been proposed as suppressors of allergy because of their T helper 1 (Th1)-fostering properties. In the murine system, BCG inhibits allergic sensitization and airway hyper-reactivity. Some epidemiological studies in humans suggest an inhibitory effect of tuberculosis on allergy. BCG vaccination in children, however, has no or merely a marginal suppressive effect on atopy. Other vaccine components such as egg proteins, gelatin, and antibiotics are a potential hazard to children with severe clinical reactions to these allergens. These rare children should be vaccinated under special precautions. In conclusion, vaccination programs do not explain the increasing prevalence of allergic diseases, but individual children may uncommonly develop an allergic reaction to a vaccine. The risks of not vaccinating children, however, far outweigh the risk for allergy. Therefore, childhood vaccination remains an essential part of child health programs and should not be withheld, even from children predisposed for allergy.

Immobilization of antibodies on alginate-chitosan beads.

An anti-hapten IgG was covalently immobilized on glutaraldehyde-activated alginate-chitosan gel beads. The antibody immobilization efficiency was influenced by glutaraldehyde-bead reaction time, IgG concentration and pH. In addition, immobilization conditions such as glutaraldehyde and antibody concentrations influenced antibody hapten binding affinity. The immobilized IgG on the beads was stable and no reduction in the percent binding to hapten was noticed following 25 days of storage. It was concluded that antibodies could be successfully immobilized on alginate-chitosan gel beads. Such a system can be applied for the development of immunoaffinity purification and immunoassays.

Specific depletion of preformed IgM natural antibodies by administration of anti-mu monoclonal antibody suppresses hyperacute rejection of pig to baboon renal xenografts.

BACKGROUND: The elimination of circulating anti-porcine preformed antibodies is crucial for avoiding hyperacute vascular rejection (HAVR) of primarily vascularized xenograft in discordant pig to baboon model. Previously described methods used for eliminating natural antibodies, however, constantly removed both anti-porcine IgM and IgG antibodies, as well as often complement proteins. To study specifically the role of preformed anti-porcine IgM antibodies, a specific anti-IgM monoclonal antibody (mAb) has been designed and evaluated in vivo. METHODS: Iterative injections of anti-IgM mAb (LO-BM2) at high dose (20 mg/kg) depleted to undetectable level the circulating IgM and therefore anti-porcine IgM antibodies but did not change the concentration of anti-pig IgG antibodies. The serum concentration of IgM and IgG antibodies was assessed by ELISA and the level of anti-pig natural IgM and IgG antibodies by flow cytometry (FC). Anti-rat sensitization was assessed by specific ELISA as well as the serum concentration of LO-BM2. RESULTS: Iterative injections of LO-BM2 allowed to specifically eliminate the anti-porcine IgM antibodies to undetectable levels at ELISA. Despite a normal serum level of anti-porcine IgG and complement proteins, HAVR was avoided. Without immunosuppression, the specific elimination of preformed anti-porcine IgM prolonged the survival of a renal xenograft in baboon up to 6 days, whereas without IgM antibody elimination, the renal xenografts were hyperacutely rejected within hours. The lost of activity of LO-BM2 after 10 days was concomitant to an IgM and IgG antibody rebound, which caused an acute vascular rejection of the xenograft. CONCLUSION: Specific elimination of natural anti-porcine IgM antibodies allows to avoid HAVR of a pig to baboon renal xenograft, whereas anti-porcine IgG antibodies and complement proteins were present in the serum. This result confirms previous in vitro reports and demonstrates for the first time in vivo that preformed IgM antibodies alone are responsible for HAVR, while preformed anti-porcine IgG antibodies are unable alone to cause HAVR. Anti-IgM therapy appears as an important tool to transiently but completely eliminates xeno-IgM antibodies in vivo.

Aging affects oral tolerance induction but not its maintenance in mice.

B6D2F1 mice, which are very susceptible to tolerance induction by a single gavage with 20 mg of ovalbumin (Ova) at age 8 weeks, become less susceptible at age 25 weeks and totally refractory at age 70 weeks. However, 70-week-old mice may be rendered tolerant by repeated ingestion of Ova. Mice orally exposed to Ova at age 8 weeks remain tolerant at age 70 weeks. The isotypic pattern of anti-Ova antibodies formed by orally-tolerant and normal mice after immunization is similar and all isotypes are equally suppressed by oral tolerance. In old mice, oral exposures to Ova alone triggered an early transient antibody response; some of these responding animals were, nevertheless, tolerant to subsequent parenteral injection of Ova in adjuvant.

Olive pollen allergy: searching for immunodominant T-cell epitopes on the Ole e 1 molecule.

BACKGROUND: The amino-acid and nucleotide sequence of Ole e 1 (the major antigen of olive pollen) has been described and the IgE antibody response to this major allergen was associated with DR7/DQ2 antigens. With this previous data we try to define the T-cell epitopes implicated in Ole e 1 reactivity. OBJECTIVES: To study the recognition of T cells (derived from allergic and non-allergic Ole e 1 patients) to Ole e 1 synthetic peptides in order to define immunodominant T-cell epitopes. METHODS: We have compared the proliferative response of the peripheral blood mononuclear cells from Ole e 1 sensitized patients vs. non-sensitized controls, induced by 14 Ole e 1 synthetic peptides. Thirty subjects were classified in two groups: group 1 (non-responders against Ole e 1, n=16) and group 2 (Ole e 1 responders, n=14), according to their clinical parameters and the presence or not in their sera of the significant Ole e 1 IgE antibody levels. RESULTS: Our results shown that it is possible to find T cells reactive to Ole e 1 peptides in patients with and without significant levels of Ole e 1 IgE antibodies. However, the percentage of response was higher in patients with IgE antibodies 71.4% vs 25%), and the recognition profile was different: the control group showed a broad reactivity pattern, in contrast, the response by the 'Ole e 1 responders' group was mainly directed against three peptides of the carboxi-terminal region, peptides 10 (91-102), 12 (109-120) and 13 (119-130), with a response frequency of 35.7, 28.5 and 28.5%, respectively. By direct and inhibition test no antibody response was found against the synthetic peptides. CONCLUSIONS: Our data suggest that the regions between 91 and 102 and 109-130 aminoacids on the Ole e 1 molecule are immunodominant T-cell epitopes. These epitopes are not recognized by IgE antibodies.

Effect of 3 months vitamin E supplementation on indices of the cellular and humoral immune response in elderly subjects.

It has been suggested that decreased immune responsiveness in the elderly may be counteracted by the antioxidant vitamin E. In a 3-month double-blind placebo-controlled intervention trial among elderly subjects aged 65 years and over we studied the effects of a daily dose of 100 mg dl-alpha-tocopheryl acetate on the cellular immune responsiveness (n 52) measured by the in vitro response of peripheral blood mononuclear cells (PBMC) to the mitogens concanavalin A (ConA) and phytohaemagglutinin (PHA). Also effects on the humoral immune responsiveness (n 74) were investigated by measuring immunoglobulin (Ig)G, IgG4 and IgA antibody concentrations against various common antigens. In the vitamin E group plasma alpha-tocopherol increased by 51% (P = 0.0001) during intervention whereas no significant changes were observed in the control group. Initial proliferative PBMC responses differed between the vitamin E group and the control group whereas all other baseline characteristics were comparable. No significant changes were observed in cellular immune responsiveness when adjusted for initial values in either the control group or the vitamin E group and, after the trial period, responses in the two groups were not significantly different. Similarly, in the vitamin E group no significant changes were found in levels of IgG and IgA raised against Penicillium or IgG4 raised against egg, milk, or wheat proteins. In the control group small but significant increases in IgG anti-Penicillium (P < 0.05) and decreases in IgG4 against milk proteins (P < 0.05) were observed. Thus, the results of this study performed with the relatively low dose of 100 mg dl-alpha-tocopheryl acetate do not support the claims of a beneficial effect of vitamin E intake on the overall immune responsiveness of elderly subjects.

Repeated antigen inhalation-induced reproducible early and late asthma in guinea pigs.

To develop a model of chronic experimental asthma in guinea pigs, the animal was forced to inhale the mist of a low dose of ovalbumin (OA) adsorbed on fine Al(OH)3 for sensitization once every 4 weeks. The animal was challenged by inhalation with the mist of OA on day 14 after the respective sensitizations. Either the first or the second antigen challenge markedly induced an early asthmatic response (EAR), whereas there was hardly any late asthmatic response (LAR). At the 3rd challenge, LAR also emerged with some severity. These dual responses were consistently observed until the 10th challenge. On the other hand, repeated inhalation/challenge, once every 2 weeks, with OA alone at the same dose tended to lead to the desensitization of the EAR. In addition, LAR was hardly observed throughout the experiments. In both groups, gamma 1 and IgE levels in the serum were elevated by the repetitive antigen inhalations, yet no obvious relationship between these antibody levels and the intensity of either EAR or LAR was recognized. The present results indicate that the asthmatic model with reproducible EAR and LAR developed in this study appears to be very beneficial for the investigation of bronchial asthma and for the assessment of anti-asthma drugs.

Systemic interleukin-2 modulates the anti-idiotypic response to chimeric anti-GD2 antibody in patients with melanoma.

The induction of human antimouse antibodies (HAMA) and human anti-idiotypic (anti-Id) responses in cancer patients receiving therapeutic monoclonal antibody (mAb) may limit the effectiveness of the administered mAb. This report evaluates the influence of systemic interleukin-2 (IL-2) on the anti-Id response to anti-disialoganglioside (anti-GD2) antibody given as treatment for patients with melanoma. Twenty-eight patients with melanoma received combined immunotherapy with anti-GD2 antibody and IL-2 at 1.5 x 10(6) U/m2/day given 4 days/week. The anti-GD2 antibody [murine 14.G2a mAb; dose levels of 2-5 mg/m2/day (4 patients); or human-mouse chimeric 14.18 (ch14.18) antibody; dose levels of 2-10 mg/m2/day (24 patients)] was scheduled to be given for 5 days either before, during, or after initial systemic IL-2 treatment. All four patients who received murine 14.G2a developed HAMA anti-isotype antibodies (660-1,000 ng/ml) as well as measurable anti-Id antibodies. All three patients who received initial treatment with ch14.18 alone developed a strong anti-Id antibody response after IL-2 was started 1 week later. The serum level of anti-Id antibody decreased during subsequent ch14.18 infusions, suggesting that the anti-Id antibody may be binding the administered ch14.18. In contrast, measurable anti-Id antibody was detected in only 3 of 14 patients who received IL-2 before, during, and after initial ch14.18 administration. Two of four patients receiving systemic IL-2 before and during initial ch14.18 infusions, and two of three patients receiving systemic IL-2 concurrent with initial ch14.18 infusions developed anti-Id antibodies. These data suggest that the anti-Id response to chimeric anti-GD2 antibody is influenced by the timing of systemic IL-2 in relation to antibody administration and can be suppressed by systemic treatment with IL-2 given before, during, and after the antibody administration.