Baseline plasma proinsulin response to glucose for predicting therapeutic response to otelixizumab in recent-onset type 1 diabetes.

AIMS: In recent-onset type 1 diabetes, clamp-derived C-peptide predicts good response to anti-CD3. Elevated proinsulin and proinsulin/C-peptide ratio (PI/CP) suggest increased metabolic/inflammatory beta cell burden. We reanalyzed trial data to compare the ability of baseline acutely glucose-stimulated proinsulin, C-peptide and PI/CP to predict functional outcome. METHODS: Eighty recent-onset type 1 diabetes patients participated in the placebo-controlled otelixizumab (GSK; NCT00627146) trial. Hyperglycemic clamps were performed at baseline, 6, 12 and 18 months, involving 3 h of induced euglycemia, followed by acutely raising and maintaining glycemia to ≥ 10 mmol/l for 140 min. Plasma proinsulin, C-peptide and PI/CP were determined after acute (minute 0 at 10 mmol/l; PI0, CP0, PI/CP0) and sustained glucose stimulation (AUC between minutes 60-140). Outcome was assessed as change in AUC60-140 C-peptide from baseline. RESULTS: In multiple linear regression, higher baseline (≥median [P50]) PI0 independently predicted preservation of beta cell function in response to anti-CD3 and interacted significantly with IAA. During follow-up, anti-CD3 tempered a further increase in PI/CP0, but not in PI0. CP0 outperformed PI0 and PI/CP0 for post-treatment monitoring. CONCLUSIONS: In recent-onset type 1 diabetes, elevated acutely glucose-stimulated proinsulin may complement or replace acutely or sustainedly stimulated C-peptide release for identifying good responders to anti-CD3, but not as outcome measure.

Development of Neutralizing Multimeric Nanobody Constructs Directed against IL-13: From Immunization to Lead Optimization.

IL-13 is a pleiotropic cytokine mainly secreted by Th2 cells. It reacts with many different types of cells involved in allergy, inflammation, and fibrosis, e.g., mastocytes, B cells, and fibroblasts. The role of IL-13 in conditions involving one or several of these phenotypes has therefore been extensively investigated. The inhibition of this cytokine in animal models for various pathologies yielded highly promising results. However, most human trials relying on anti-IL-13 conventional mAbs have failed to achieve a significant improvement of the envisaged disorders. Where some studies might have suffered from several weaknesses, the strategies themselves, such as targeting only IL-13 using conventional mAbs or employing a systemic administration, could be questioned. Nanobodies are recombinant Ag-binding fragments derived from the variable part of H chain-only Abs occurring in Camelidae. Thanks to their single-domain structure, small size (≈15 kDa), good stability, and solubility, they can be engineered into multispecific constructs for combined therapies or for use in new strategies such as formulations for local administration, e.g., pulmonary administration. In this study, we describe the generation of 38 nanobodies that can be subdivided into five CDR3 families. Nine nanobodies were found to have a good affinity profile (KD = 1-200 nM), but none were able to strongly inhibit IL-13 biological activity in vitro (IC50 > 50 µM: HEK-Blue IL-13/IL-4 cells). Multimeric constructs were therefore designed from these inhibitors and resulted in an up to 36-fold improvement in affinity and up to 300-fold enhancement of the biological activity while conserving a high specificity toward IL-13.

Correction: Prediction of Impending Type 1 Diabetes through Automated Dual-Label Measurement of Proinsulin:C-Peptide Ratio.

[This corrects the article DOI: 10.1371/journal.pone.0166702.].

Prediction of Impending Type 1 Diabetes through Automated Dual-Label Measurement of Proinsulin:C-Peptide Ratio.

BACKGROUND: The hyperglycemic clamp test, the gold standard of beta cell function, predicts impending type 1 diabetes in islet autoantibody-positive individuals, but the latter may benefit from less invasive function tests such as the proinsulin:C-peptide ratio (PI:C). The present study aims to optimize precision of PI:C measurements by automating a dual-label trefoil-type time-resolved fluorescence immunoassay (TT-TRFIA), and to compare its diagnostic performance for predicting type 1 diabetes with that of clamp-derived C-peptide release. METHODS: Between-day imprecision (n = 20) and split-sample analysis (n = 95) were used to compare TT-TRFIA (AutoDelfia, Perkin-Elmer) with separate methods for proinsulin (in-house TRFIA) and C-peptide (Elecsys, Roche). High-risk multiple autoantibody-positive first-degree relatives (n = 49; age 5-39) were tested for fasting PI:C, HOMA2-IR and hyperglycemic clamp and followed for 20-57 months (interquartile range). RESULTS: TT-TRFIA values for proinsulin, C-peptide and PI:C correlated significantly (r2 = 0.96-0.99; P<0.001) with results obtained with separate methods. TT-TRFIA achieved better between-day %CV for PI:C at three different levels (4.5-7.1 vs 6.7-9.5 for separate methods). In high-risk relatives fasting PI:C was significantly and inversely correlated (rs = -0.596; P<0.001) with first-phase C-peptide release during clamp (also with second phase release, only available for age 12-39 years; n = 31), but only after normalization for HOMA2-IR. In ROC- and Cox regression analysis, HOMA2-IR-corrected PI:C predicted 2-year progression to diabetes equally well as clamp-derived C-peptide release. CONCLUSIONS: The reproducibility of PI:C benefits from the automated simultaneous determination of both hormones. HOMA2-IR-corrected PI:C may serve as a minimally invasive alternative to the more tedious hyperglycemic clamp test.

Identification of Useful Nanobodies by Phage Display of Immune Single Domain Libraries Derived from Camelid Heavy Chain Antibodies.

BACKGROUND: The discovery of functional heavy chain-only antibodies devoid of light chains in sera of camelids and sharks in the early nineties provided access to the generation of minimal-sized, single-domain, in vivo affinity-matured, recombinant antigenbinding fragments, also known as Nanobodies. METHODS: Recombinant DNA technology and adaptation of phage display vectors form the basis to construct large naïve, synthetic or medium sized immune libraries from where multiple Nanobodies have been retrieved. Alternative selection methods (i.e. bacterial display, bacterial two-hybrid, Cis-display and ribosome display) have also been developed to identify Nanobodies. The antigen affinity, stability, expression yields and structural details of the Nanobodies have been determined by standard technology. Nanobodies were subsequently engineered for higher stability and affinity, to have a sequence closer to that of human immunoglobulin domains, or to add designed effector functions. RESULTS: Antigen specific Nanobodies recognizing with high affinity their cognate antigen were retrieved from various libraries. High expression yields are obtained from microorganisms, even when expressed in the cytoplasm. The purified Nanobodies are shown to possess beneficial biochemical and biophysical properties. The crystal structure of Nanobody::antigen complexes reveal the preference of Nanobodies for cavities on the antigen surface. CONCLUSION: Thanks to the properties described above, Nanobodies became a highly valued and versatile tool for biomolecular research. Moreover, numerous diagnostic and therapeutic Nanobody-based applications have been developed in the past decade.

Plasma GAD65, a Marker for Early ß-Cell Loss After Intraportal Islet Cell Transplantation in Diabetic Patients.

CONTEXT AND OBJECTIVE: Intraportal islet transplantation can restore insulin production in type 1 diabetes patients, but its effect is subject to several interfering processes. To assess the influence of ß-cell loss before and during engraftment, we searched for a real-time marker of ß-cell destruction. Previous studies showed that 65-kDa isoform of glutamate decarboxylase (GAD65) is discharged by chemically damaged rat ß-cells. We therefore examined the utility of the GAD65 assay to detect and quantify destruction of human ß-cells in vitro and in vivo. DESIGN AND PARTICIPANTS: A time-resolved fluorescence immunoassay was used to measure GAD65 discharge from ß-cells after administration of toxins or after intraportal transplantation. The study in patients involved type 1 diabetes recipients of 56 implants. RESULTS: GAD65 was discharged from cultured human ß-cells between 4 and 24 hours after acute insult and proportional to the number of dying cells. It was also detected in plasma during the first 24 hours after intraportal transplantation of human islet cell grafts. Diabetic nude rat recipients without hyperglycemic correction exhibited higher plasma GAD65 levels than those with normalization. In type 1 diabetes recipients of grafts with 2-5 × 10(6) ß-cells per kilogram of body weight, five of six with plasma GAD65 greater than 1 ng/mL failed to increase plasma C-peptide by greater than 0.5 ng/mL at posttransplant month 2, whereas five of six with undetectable plasma GAD 65 and 15 of 19 with intermediate levels did result in such increase. CONCLUSION: Plasma GAD65 qualifies as a marker for early ß-cell loss after intraportal transplantation. Further studies are needed to extend its clinical utility.

Nanoimmunoassay onto a screen printed electrode for HER2 breast cancer biomarker determination.

A chip format sandwich-type immunoassay based on Nanobodies(®) (Nbs) with the Human Epidermal Growth Factor Receptor (HER2) extracellular domain as antigen model has been developed. The HER2 is considered as an important biomarker because its overexpression causes an aggressive type of breast cancer. Nbs are single domain antigen-binding fragments derived from camelid heavy-chain antibodies. The strategy of the presently developed sandwich immunoassay takes advantage of the small size of Nbs for the detection of the electroactive redox tracer onto the screen printed electrode (SPE). A capture anti HER2 Nb was covalently immobilized onto the SPE, and the detection Nb, raised against another epitope of HER2, was labeled with horseradish peroxidase (HRP). The biosensor signal corresponded to the electroreduction of para-quinone generated at the SPE by the HRP in the presence of hydroquinone and hydrogen peroxide. The best performing and optimized immunoassay conditions consisted of 2 and 20 min for the first and the second incubation times, respectively. The amperometric signal obtained was proportional to the logarithm of HER2 concentration between 1 and 200 µg/mL and the modified SPE storage stability lasted for at least three weeks. Determination of HER2 in human cells has been realized.

Nanobodies and their potential applications.

Nanobodies are recombinant, antigen-specific, single-domain, variable fragments of camelid heavy chain-only antibodies. The innate supremacy of nanobodies as a renewable source of affinity reagents, together with their high production yield in a broad variety of expression systems, minimal size, great stability, reversible refolding and outstanding solubility in aqueous solutions, and ability to specifically recognize unique epitopes with subnanomolar affinity, have combined to make them a useful class of biomolecules for research and various medical diagnostic and therapeutic applications. This article speculates on a number of technological innovations that might be introduced in the nanobody identification platform to streamline the generation of more potent nanobodies and to expand their application range.

Development of a multipurpose time-resolved fluorescence immunoassay for rat insulin.

We present a time-resolved fluorescence immunoassay (TR-FIA) for the measurement of rat insulin in cell extracts and culture media. This assay is based on the binding of two monoclonal antibodies to different parts of the insulin molecule in a 96-well microtiter plate. For the detection, europium-labeled streptavidin that interacts with the second biotinylated antibody is used. Samples of 25 microl could be analyzed in less than 2 days with a measuring range between 5 and 1250 pg (0.2-50 microg/L or 34.4-8600 pM). The inter- and intraassay percentage coefficients of variation were less than 8.3 and 5.1, respectively. Recoveries of 0.48 to 40 microg/L rat insulin, added to culture medium, ranged between 94 and 107%. Results were significantly correlated with those of an in-house radioimmunoassay (RIA) for rodent insulin (P<0.0001, r(2)=0.99). The TR-FIA method had a similar detection limit (0.16 microg/L), but its working range was at least 5-fold larger. Additional advantages include the lower cost, the applicability to measurements in tissue and serum, and the quantification of insulin from other species.

Functional beta-cell mass and insulin sensitivity is decreased in insulin-independent pancreas-kidney recipients.

BACKGROUND: To compare functional beta-cell mass and insulin sensitivity in insulin-independent pancreas-kidney recipients with that in age- and body mass index-matched nondiabetic kidney recipients and normal controls. METHODS: All transplant recipients were on maintenance immunosuppression with mycophenolate mofetil and tacrolimus since more than 2.7 years (2.2-3.8 years). Their C-peptide release was measured during a 170-min hyperglycemic clamp, first in absence and then in presence of glucagon. Data were compared with those after glucose stimulation alone. Insulin sensitivity under basal and stimulated conditions was calculated using homeostasis model assessment of insulin resistance and insulin sensitivity index, respectively. RESULTS: Functional beta-cell mass in pancreas-kidney recipients with systemic venous drainage was reduced, representing, respectively, 63% and 80% of that in healthy controls and kidney recipients. Pancreas-kidney recipients exhibited lower insulin sensitivity than healthy controls (homeostasis model assessment of insulin resistance was 0.8, 0.7-1.1 vs. 0.4, 0.3-0.8; P=0.02 and insulin sensitivity index was 17, 12-24 mg/kg/min per 100 microU/mL vs. 31, 20-38 mg/kg/min per 100 microU/mL; P=0.04). CONCLUSIONS: Using a hyperglycemic clamp, the functional beta-cell mass in insulin-independent pancreas-kidney recipients was found to be 37% and 20% lower than in healthy controls and nondiabetic kidney recipients.

Simultaneous measurement of plasma concentrations of proinsulin and C-peptide and their ratio with a trefoil-type time-resolved fluorescence immunoassay.

BACKGROUND: When the concentrations of 2 or more substances are measured separately, their molar ratios are subject to the additive imprecisions of the different assays. We hypothesized that the cumulative error for concentration ratios of peptides containing a common sequence might be minimized by measuring the peptides simultaneously with a "trefoil-type" immunoassay. METHODS: As a model of this approach, we developed a dual-label time-resolved fluorescence immunoassay (TRFIA) to simultaneously measure proinsulin, C-peptide, and the proinsulin-C-peptide ratio (PI/C). A monoclonal antibody captures all C-peptide-containing molecules, and 2 differently labeled antibodies distinguish between proinsulin-like molecules and true C-peptide. RESULTS: The trefoil-type TRFIA was capable of measuring plasma C-peptide and proinsulin simultaneously without mutual interference at limits of quantification of 48 and 8125 pmol/L, and 2.1 and 197 pmol/L, respectively. Within-laboratory imprecision values for the trefoil-type TRFIA ranged between 8.4% and 12% for the hormone concentrations. Unlike the hormone results obtained with separate assays, imprecision did not increase when PI/C was calculated from trefoil assay results (P < 0.05). Peptide concentrations were highly correlated with results obtained in individual comparison assays (r(2) > or = 0.965; P < 0.0001). The total error for PI/C obtained with the trefoil-type TRFIA remained < or = 25% over a broader C-peptide range than with separate hormone assays (79-7200 pmol/L vs 590-4300 pmol/L C-peptide). Preliminary data indicate little or no interference by heterophile antibodies. CONCLUSIONS: The developed trefoil-type TRFIA is a reliable method for simultaneous measurement of proinsulin, C-peptide, and PI/C and provides proof of principle for the development of other trefoil-type multiple-label immunoassays.

Species and epitope specificity of two 65 kDa glutamate decarboxylase time-resolved fluorometric immunoassays.

The 65 kDa isoform of human glutamate decarboxylase (GAD65) is a major autoantigen in type 1 diabetes (T1D). In the present study, we have developed a sensitive sandwich time-resolved fluorescence immunoassay (TRFIA) for the quantification of GAD65 in cell extracts, cell media and serum. The monoclonal antibody GAD-6 is used to selectively capture GAD65 but not the slightly larger isoform GAD67, and the utilization of different detecting antibodies with distinct GAD65 epitope specificity allows modulating the specificity of the assay. To this effect we have biotinylated a recombinant antigen-binding fragment (rFab) with epitope specificity for the N-terminal region of rat and human GAD65 (rFab N-GAD65) and another rFab that selectively binds to the middle part of human GAD65 (rFab b96.11). In the assay the biotinylated rFabs are recognized by Europium labeled streptavidin. The obtained time-resolved fluorescence (TRF) is directly proportional to the concentration of GAD65 over a large measuring range (0.1 to >100 ng/mL). Based on total error estimation including both bias and imprecision, the lower limit of quantitation (LLOQ) of GAD65 in cell extracts is 0.33 ng/mL with the N-GAD65 TRFIA, and 0.10 ng/mL with the b96.11 TRFIA, but the latter is suitable for human GAD65 only, whereas the N-GAD65 TRFIA has equal sensitivity with rat and human GAD65. Specificity was further checked with GAD65/67 fusion proteins, confirming that the presence of intact capture as well as detection epitope on the analyte is a prerequisite for recognition in both assays. We show that the beta cell-specific marker GAD65 can be quantified in pancreatic cell extracts and in serum, allowing studies on discharge during cell death in vitro as well as in vivo.

Insulin needs after CD3-antibody therapy in new-onset type 1 diabetes.

BACKGROUND: Type 1 diabetes mellitus is a T-cell-mediated autoimmune disease that leads to a major loss of insulin-secreting beta cells. The further decline of beta-cell function after clinical onset might be prevented by treatment with CD3 monoclonal antibodies, as suggested by the results of a phase 1 study. To provide proof of this therapeutic principle at the metabolic level, we initiated a phase 2 placebo-controlled trial with a humanized antibody, an aglycosylated human IgG1 antibody directed against CD3 (ChAglyCD3). METHODS: In a multicenter study, 80 patients with new-onset type 1 diabetes were randomly assigned to receive placebo or ChAglyCD3 for six consecutive days. Patients were followed for 18 months, during which their daily insulin needs and residual beta-cell function were assessed according to glucose-clamp-induced C-peptide release before and after the administration of glucagon. RESULTS: At 6, 12, and 18 months, residual beta-cell function was better maintained with ChAglyCD3 than with placebo. The insulin dose increased in the placebo group but not in the ChAglyCD3 group. This effect of ChAglyCD3 was most pronounced among patients with initial residual beta-cell function at or above the 50th percentile of the 80 patients. In this subgroup, the mean insulin dose at 18 months was 0.22 IU per kilogram of body weight per day with ChAglyCD3, as compared with 0.61 IU per kilogram with placebo (P<0.001). In this subgroup, 12 of 16 patients who received ChAglyCD3 (75 percent) received minimal doses of insulin (< or =0.25 IU per kilogram per day) as compared with none of the 21 patients who received placebo. Administration of ChAglyCD3 was associated with a moderate "flu-like" syndrome and transient symptoms of Epstein-Barr viral mononucleosis. CONCLUSIONS: Short-term treatment with CD3 antibody preserves residual beta-cell function for at least 18 months in patients with recent-onset type 1 diabetes.

Relation between disease phenotype and HLA-DQ genotype in diabetic patients diagnosed in early adulthood.

We investigated inaugural disease phenotype in relation to the presence or absence of diabetes-associated autoantibodies and human leukocyte antigen (HLA) DQ risk genotypes in adult-onset diabetic patients. Blood samples and questionnaires were obtained from 1584 recent-onset Belgian Caucasian patients (age 15-39 yr at diagnosis of primary diabetes) who were recruited by the Belgian Diabetes Registry over an 11-yr period. At clinical diagnosis, antibody-positive patients (n = 1198) were on average younger and had more symptoms, a more acute disease onset, lower body mass index, and random C-peptide levels, but higher insulin needs, glycemia, and prevalence of ketonuria, HLA-DQ, and 5' insulin gene susceptibility genotypes (P < 0.001 vs. antibody-negative patients; n = 386). In antibody-positive patients, these characteristics did not differ according to HLA-DQ genotype. However, in antibody-negative subjects, we found that patients were younger (P = 0.001); had a lower body mass index (P < 0.001), higher insulin needs (P = 0.014), and amylasemia (P = 0.001); and tended to have a higher glycemia and lower C-peptide in the presence of susceptible HLA-DQ genotypes. Differences according to HLA-DQ genotype subsisted after careful age-matching. In conclusion, we found no relation between initial disease phenotype and HLA-DQ genotype in antibody-positive diabetic young adults. In contrast, antibody-negative patients displayed more type 1-like features when carrying susceptible HLA-DQ genotypes known to promote the development of antibody-positive diabetes. The overrepresentation of these susceptibility genotypes in antibody-negative patients suggests the existence of an immune-mediated disease process with as yet unidentified immune markers in a subgroup of seronegative patients.