Immunogenicity assay development and validation for biological therapy as exemplified by ustekinumab.

Introduction of biotherapeutics has been a major milestone in the treatment of different chronic diseases. Nevertheless, the immune system can recognize the administered biological as non-self and respond with generation of anti-drug antibodies (ADA), including neutralizing ADA (nADA). Immunogenic responses may result in altered drug dynamics and kinetics leading to changes in safety and efficacy. However, there are several challenges with standard techniques for immunogenicity testing. Ustekinumab (UST), used in different inflammatory diseases, is a therapeutic antibody directed against the shared p40 subunit of interleukin (IL)-12 and IL-23, interfering in the pathogenically crucial T helper type 1 (Th1)/Th17 pathway. We established and validated different approaches for detection and quantitation of UST, UST-specific ADA and nADA. Addressing the obstacle of complex formation of UST with nADA, we developed an acidification assay to approach the total amount of nADA. Validated methods were based on surface plasmon resonance spectroscopy (SPR), enzyme-linked immunosorbent assay (ELISA) and a cell-based approach to characterize neutralizing capacity of nADA. Parameters assessed were determination and quantitation limits, linearity, range, precision, accuracy and selectivity. Quantitation of ADA and UST was feasible at lower concentrations using ELISA, whereas SPR showed a wider linear range for determination of ADA and UST. Accuracy, precision and linearity for quantitation were comparable using ELISA, SPR and the cell-based approach. All validated parameters fulfill the requirements of regulatory agencies. A combination of the testing approaches could address the increasing demand of precision medicine as it can be suitable for capturing the whole spectrum of immunogenicity and is transferable to other biologicals.

Nano-LC-MS/MS for the quantitation of prostanoids in immune cells.

Prostanoids, derivatives of arachidonic acid, are involved in inflammation and immune reactions. To understand the role of prostanoids produced by diverse immune cells, a highly sensitive quantitation method for prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), 6-keto prostaglandin F1α (6-keto PGF1α), prostaglandin F2α (PGF2α), and thromboxane B2 (TXB2) by means of nano-liquid chromatography-tandem mass spectrometry has been developed. It was validated according to the guidelines of the Food and Drug Administration (FDA) in terms of linearity, precision, accuracy, recovery, stability, and lower limit of quantitation (LLOQ). The LLOQ were 25 pg/mL in the injected solution (75 fg on column (o.c.)) for PGE2 and PGD2 and 37.5 pg/mL (112.5 fg on column) for 6-keto PGF1α, PGF2α, and TXB2, respectively. It was successfully applied to murine mast cells isolated from paws after zymosan injection and to CD4(+) and CD8(+) T lymphocytes from blood of sensitized versus non-sensitized mice in context of a delayed type hypersensitivity model. About 5,000 (T cells) to 40,000 (mast cells) cells were sufficient for quantitation. In the mast cells, the production of PGE2 increased at a significantly higher extent than the synthesis of the other prostanoids. The T lymphocytes did not show any difference in prostanoid production, no matter whether they were obtained from sensitized mice or non-sensitized mice.

Activation of protein kinase C alpha and/or epsilon enhances transcription of the human endothelial nitric oxide synthase gene.

In primary human umbilical vein endothelial cells (HUVECs), incubation with phorbol-12-myristate-13-acetate (PMA) enhanced basal and bradykinin-stimulated nitric oxide production. In the HUVEC-derived cell line EA.hy 926, PMA and phorbol-12,13-dibutyrate stimulated endothelial nitric oxide synthase (NOS III) mRNA expression in a concentration- and time-dependent manner. Maximal mRNA expression (3.3-fold increase) was observed after 18 hr. NOS III protein and activity were increased to a similar extent. The specific protein kinase C (PKC) inhibitors bisindolylmaleimide I (1 microM), Gö 6976 [12-(2 cyanoethyl)-6,7,12, 13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo-[3, 4-c]carbazole] (1 microM), Ro-31-8220 [3-[1-[3(amidinothio)propyl-1H-inoyl-3-yl]3-(1-methyl-1H- indoyl-3-yl) maleimide methane sulfonate] (1 microM), and chelerythrine (3 microM) did not change NOS III expression when applied alone, but they all prevented the up-regulation of NOS III mRNA produced by PMA. Of the PKC isoforms expressed in EA.hy 926 cells (alpha, beta I, delta, epsilon, eta, zeta, lambda, and mu), only PKC alpha and PKC epsilon showed changes in protein expression after PMA treatment. Incubation of EA.hy 926 cells with PMA for 2-6 hr resulted in a translocation of PKC alpha and PKC epsilon from the cytosol to the cell membrane, indicating activation of these isoforms. After 24 hr of PMA incubation, both isoforms were down-regulated. The time course of activation and down-regulation of these two PKC isoforms correlated well with the PMA-stimulated increase in NOS III expression. When human endothelial cells (ECV 304 or EA.hy 926) were transiently or stably transfected with a 3.5-kb fragment of the human NOS III promoter driving a luciferase reporter gene, PMA stimulated promoter activity up to 2.5-fold. On the other hand, PMA did not change the stability of the NOS III mRNA. These data indicate that stimulation of PKC alpha, PKC epsilon, or both by active phorbol esters represents an efficacious pathway activating the human NOS III promoter in human endothelium.

Color-coded Doppler sonography in the diagnosis of abdominal masses.

A man in his forties was operated on for testicular cancer on the right-side with subsequent radiotherapy. Two years later, another tumor was found on the opposite side and surgically removed, followed by polychemotherapy. In the follow-up period, CT scanning and ultrasound showed large abdominal masses which were suspected to be metastases. Before initiating four cycles of chemotherapy, we checked their nature by Doppler sonography and found them to be recently developed abdominal varices due to an alcoholic liver cirrhosis. We were able to demonstrate that Doppler sonography can provide further information and is easy to use.

Efficacy of antiestrogen treatment in a benign metastasizing leiomyoma with paraneoplastic estradiol production.

Benign metastasizing leiomyoma (BML) is a rare hormone-dependent disease which occurs predominantly in women during their child-bearing years. After our patient had refused ablative hormone therapy (bilateral ovarectomy), evidence of estrogen and progesteron receptors in tumor tissue taken from the lung sites, as well as extremely high estradiol serum levels, led us to conduct high-dosage antiestrogen therapy for 5 years; daily administration of 250 mg of Tamoxpuren(R) resulted in stable disease of the pulmonary sites without any side effects. This also significantly lowered estradiol serum levels, which improved clinical symptoms. Five years later, the patient's vision suddenly deteriorated due to bilateral macula degeneration. This forced us to stop the antiestrogen therapy and commence alternative treatment with LHRH analogue (3.6 mg Goreselin). We observed stable disease of the pulmonary metastases and low estradiol serum levels during the first 6 months of Goserelin treatment. The response to antiestrogen therapy in BML suggests that the muscular component of these disorders is responsive to estrogen ablation.

Glycosaminoglycan-mediated leuserpin-2/thrombin interaction. Structure-function relationships.

Two recently identified structural elements important for glycosaminoglycan-mediated activation of human leuserpin-2 (hLS2) were investigated in detail by functional analysis of variants secreted by transiently transfected COS cells. Highly specific requirements with respect to the nature of the involved amino acids as well as to their spatial arrangements were found to be crucial for efficient activation of hLS2 by dermatan sulfate. In contrast, binding and activation of hLS2 by heparin seem to be determined mainly by the positive charge density of the involved inhibitor segment. A dimeric repeat enriched in acidic amino acids turned out to exert a dual role with respect to structure and function of hLS2. First, in the absence of functional activators the negatively charged dimer interacts intramolecularly with the glycosaminoglycan-binding site. Second, the acidic dimer is instrumental in glycosaminoglycan-mediated activation of hLS2. The monomers constituting the acidic dimer are functionally not equivalent.

On the activation of human leuserpin-2, a thrombin inhibitor, by glycosaminoglycans.

Human leuserpin-2 (hLS2) cDNA variants generated by site-directed mutagenesis were expressed in a transient COS cell system. Functional analysis of the mutants revealed two regions in the NH2-terminal half of hLS2 which are essential for glycosaminoglycan-enhanced thrombin inhibition by hLS2. One of these regions, which encompasses a dimeric structure enriched in basic amino acids, is required for both glycosaminoglycan binding and glycosaminoglycan-mediated acceleration of thrombin inhibition. Deletion of another dimeric region, which spans a sequence with a high negative charge density, resulted in a strong reduction in the glycosaminoglycan-enhanced activity of hLS2. This polyanionic region displays structural and functional similarities to the COOH-terminal end of hirudin, another potent thrombin inhibitor, indicating that both inhibitors may have a common binding site on thrombin. Based on our observations we propose a model for the activation of hLS2 by glycosaminoglycans. The key feature of this model is the suggestion that the glycosaminoglycan-enhanced reaction between hLS2 and thrombin is mediated by at least two regions of contact, involving both the reactive center region and the acidic domain of hLS2. Binding of glycosaminoglycans to hLS2 is suggested to result first in the release of the acidic region from intramolecular interactions. Then, amino acid sequences in thrombin are proposed to interact with the acidic dimer of hLS2.

Regulation of plasma aldosterone during hemodialysis.

In order to clarify the influence of serum potassium, serum sodium and plasma angiotensin II concentrations on aldosterone release during hemodialysis (HD), six chronic hemodialysis patients were studied during HD with varying dialysate sodium concentrations and different buffers. Plasma aldosterone concentrations were higher during acetate than bicarbonate HD, during low sodium compared to high sodium HD, and were correlated inversely to serum sodium concentrations. The decline in plasma aldosterone concentrations during HD paralleled the decrease in serum potassium concentrations, and plasma aldosterone concentrations were correlated with serum potassium concentrations. In addition, plasma aldosterone and plasma angiotensin II concentrations were correlated significantly. It is proposed that serum potassium and the renin-angiotensin system are the main factors of aldosterone release during hemodialysis, while serum sodium per se seems to be of less importance. The dialysate buffer employed also plays a role in aldosterone regulation (via the renin-angiotensin system).

The renin-angiotensin-aldosterone system during haemodialysis with acetate or bicarbonate at different dialysate sodium concentrations.

The hormones of the renin angiotensin aldosterone system were measured during regular haemodialysis with acetate or bicarbonate at dialysate sodium concentrations of 135, 140, 145, and 150 mmol/l. Plasma renin activity and aldosterone concentration were higher during acetate haemodialysis than during bicarbonate haemodialysis. At lower dialysate sodium concentrations, plasma renin activity (acetate dialysis and bicarbonate dialysis) and aldosterone concentration (only acetate dialysis) were higher than they were at higher dialysate sodium concentrations. Plasma renin activity increased during acetate dialysis, but did not change during bicarbonate dialysis. Aldosterone and potassium concentrations were positively correlated. Aldosterone decreased during haemodialysis (increase to predialysis values at the end of haemodialysis (4 h) at lower dialysate sodium concentrations). It is concluded that the renin angiotensin aldosterone system is activated more during acetate dialysis than during bicarbonate dialysis. Aldosterone concentrations seem to be related more closely to serum potassium than to renin-angiotensin-aldosterone system and to serum sodium intradialytically.