Thomas Emrich

Molecular diagnosis of sepsis in neutropenic patients with haematological malignancies.

The rapid diagnosis of an infectious cause in the course of fever of unknown origin plays a pivotal role in the correct management of neutropenic patients. In this study, blood samples from febrile oncohaematological patients were tested using a novel commercial real-time PCR assay (LightCycler SeptiFast; Roche Molecular Systems) and blood culture (BacT/Alert 3D; bioMérieux). Twenty-one (20.4 %) and 34 (33 %) of the 103 samples under study tested positive by blood culture and PCR, respectively. The analysis of concordance evidenced a low correlation between the two approaches (83 %), mainly due to samples that tested negative by culture but positive using the molecular approach. Among 14 discordant cases negative by culture but positive by PCR, 12 were observed in sequential samples of patients with initial concordant results on samples drawn before the administration of a specific antimicrobial therapy. Moreover, DNA of a fastidious organism, Aspergillus fumigatus, not easily detectable by the cultural approach was rapidly detected in the two remaining discordant cases. Overall, the characteristics featured by the molecular method could be of interest in the development of new algorithms for the diagnosis of sepsis in critical patients.

Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity.

Charge-mediated influence of the antibody variable domain on FcRn-dependent pharmacokinetics

Significance Therapeutic antibodies of the immunoglobulin G (IgG) isotype show a pharmacokinetic (PK) profile that is strongly mediated by the interaction with the neonatal Fc receptor (FcRn). Therefore, modulating the FcRn–IgG interaction allows altering PK characteristics of therapeutic antibodies. So far, engineering the crystallizable fragment (Fc) is known to affect PK, and, although the influence of the antigen binding fragment (Fab) on FcRn interactions has been reported, the underlying mechanism remains unknown. Here, we demonstrate that the charge distribution in the distal variable fragment (Fv) of IgGs is involved in excessive binding to the FcRn, thereby reducing FcRn-dependent terminal half-lives in vivo. These findings contribute to a better understanding of the FcRn–IgG interaction. Here, we investigated the influence of the variable fragment (Fv) of IgG antibodies on the binding to the neonatal Fc receptor (FcRn) as well as on FcRn-dependent pharmacokinetics (PK). FcRn plays a key role in IgG homeostasis, and specific manipulation in the crystallizable fragment (Fc) is known to affect FcRn-dependent PK. Although the influence of the antigen-binding fragment (Fab) on FcRn interactions has been reported, the underlying mechanism is hitherto only poorly understood. Therefore, we analyzed the two IgG1 antibodies, briakinumab and ustekinumab, that have similar Fc parts but different terminal half-lives in human and systematically engineered variants of them with cross-over exchanges and varied charge distribution. Using FcRn affinity chromatography, molecular dynamics simulation, and in vivo PK studies in human FcRn transgenic mice, we provide evidence that the charge distribution on the Fv domain is involved in excessive FcRn binding. This excessive binding prevents efficient FcRn–IgG dissociation at physiological pH, thereby reducing FcRn-dependent terminal half-lives. Furthermore, we observed a linear correlation between FcRn column retention times of the antibody variants and the terminal half-lives in vivo. Taken together, our study contributes to a better understanding of the FcRn–IgG interaction, and it could also provide profound potential in FcRn-dependent antibody engineering of the variable Fab region.

A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies

Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.

Determination of critical quality attributes for monoclonal antibodies using quality by design principles

Quality by design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and their related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody. This chapter describes the identification of critical quality attributes (CQAs) as an important first step for QbD development of biopharmaceuticals. A systematic scientific based risk ranking and filtering approach allows a thorough understanding of quality attributes and an assignment of criticality for their impact on drug safety and efficacy. To illustrate the application of the approach and tools, a few examples from monoclonal antibodies are shown. The identification of CQAs is a continuous process and will further drive the structure and function characterization of therapeutic proteins.

Quantitative evaluation of telomerase subunits in urine as biomarkers for noninvasive detection of bladder cancer

The aim of our study was to prospectively evaluate the potential diagnostic value and clinical applicability of quantitative analysis of telomerase subunits gene expression in urine for noninvasive detection of bladder cancer. Expression levels of human telomerase reverse transcriptase (hTERT) and human telomerase RNA (hTR) were analyzed by real‐time reverse transcriptase polymerase chain reaction (RT‐PCR) in urine samples from 163 subjects with bladder cancer and 237 controls (163 individuals with benign genitourinary diseases; 74 healthy subjects). The sensitivity, specificity and optimal cutoffs were determined and compared to the corresponding values obtained by voided urine cytology. Quantitative urinary hTR analysis detects bladder cancer with an overall sensitivity of 77.0%, whereas hTERT analysis reached a sensitivity of 55.2%. The majority of undetected tumors were small, low‐grade pTa lesions. Both hTR and hTERT proved to be significantly more sensitive than cytology (34.5%; p < 0.001). Specificities for hTR, hTERT and cytology were 72.1%, 85.0% and 92.7%, respectively, in the total study population and 96.9%, 89.2% and 100%, respectively, in healthy subjects. Higher diagnostic accuracy was achieved by hTR than by hTERT analysis (p < 0.05). The specificity of hTR increased to 85.0% in the total population if urinary leukocyte contamination was excluded. These data suggest that quantitative hTR analysis is the most accurate telomerase‐based test for bladder cancer detection and has the potential to replace cytology as a noninvasive biomarker for disease diagnosis and follow‐up.

Real-time quantitative PCR of telomerase mRNA is useful for the differentiation of benign and malignant pancreatic disorders.

The presence of telomerase activity has been proposed as a specific and sensitive marker for malignant tissue, and positivity rates of up to 95% have been reported in pancreatic cancer. In the present study telomerase activity analysis was reevaluated in 29 pancreatic cancer tissues compared with 36 chronic pancreatitis tissues and 21 normal controls, and a study was made of whether malignant and benign pancreatic disorders can be better differentiated using a novel technique–real-time quantitative PCR analysis–analyzing telomerase mRNA expression. Telomerase activity was present in 35% (10 of 29) of pancreatic cancer samples, 3% (one of 36) of chronic pancreatitis samples, and none of the normal pancreatic tissue samples in the TRAP assay. Real-time quantitative PCR analysis revealed the presence of telomerase mRNA expression in 50% (10 of 20) of normal, 86% (31 of 36) of chronic pancreatitis, and 90% (26 of 29) of pancreatic cancer samples. However, quantification of the expression data revealed that the relative increase above normal was 5.5 (range, 3.5–8.6) for chronic pancreatitis and 23.9 (range, 18.6–30.7) for pancreatic cancer samples (p < 0.01). No relationship was found between telomerase activity and the fold increase of telomerase mRNA above normal and gender, patient age, tumor stage, or tumor grade. These data indicate that detection of telomerase activity using the TRAP assay has limitations in differentiating benign and malignant pancreatic disorders. However, telomerase mRNA analysis by real-time quantitative PCR analysis allows a highly sensitive detection and differentiation of pancreatic cancer from normal pancreas and chronic pancreatitis and thereby may serve as a new reliable, easy, and effective diagnostic tool for cancer diagnosis.

Analysis of human telomerase reverse transcriptase mRNA (hTERT) expression in myxoid liposarcomas using LightCycler real-time quantitative reverse transcriptase-polymerase chain reaction

We describe a convenient, nonradioactive reverse transcription – polymerase chain reaktion (RT‐PCR) method for the rapid and accurate quantitative detection of the human telomerase catalytic subunit human telomerase reverse transcriptase (hTERT) mRNA. The LightCycler TeloTAGGG hTERT Quantification Kit (Roche Molecular Biochemicals) was designed to be used for the highly sensitive and quantitative detection of hTERT mRNA relative to the house‐keeping gene porphobilinogen deaminase (PBGD). As a tumor progression model, we investigated 26 myxoid liposarcomas (11 pure myxoid grade I, 15 myxoid/round cell grade II/III) for the hTERT expression level and compared the results of the new method with former measurements performed in silver‐stained polyacrylamide gels. Both methods revealed similar results, with real‐time RT‐PCR being the more accurate quantification technique, which also saves time and material. Elevated hTERT expression (cut‐off ratio×100 at 1.3) was an indicator of round cell components and hence for tumor progression in myxoid liposarcoma. The new method is capable of differentiating between pure myxoid and myxoid/round cell liposarcomas for hTERT‐expression more accurately.

Comparison of the lateral tail vein and the retro-orbital venous sinus routes of antibody administration in pharmacokinetic studies.

In pharmacokinetic studies, intravenous (i.v.) administration of antibodies to mice is usually done via the lateral tail vein. This approach can cause stress to the mice and has a high rate of failure because it is challenging to perform correctly. Administration via the retro-orbital venous sinus has been suggested as a good alternative to tail vein i.v. administration of antibodies. Evidence is still needed, however, to determine whether the route of administration has an effect on the absorption or the pharmacokinetic activity of the injected antibody. The authors compared serum concentrations and pharmacokinetic parameters of a therapeutic antibody administered via tail vein injection or via retro-orbital injection. The findings suggest that there is no difference in the absorption or pharmacokinetic activity of therapeutic antibodies when administered via the lateral tail vein versus the retro-orbital venous sinus.

A two-pronged binding mechanism of IgG to the neonatal Fc receptor controls complex stability and IgG serum half-life

The success of recombinant monoclonal immunoglobulins (IgG) is rooted in their ability to target distinct antigens with high affinity combined with an extraordinarily long serum half-life, typically around 3 weeks. The pharmacokinetics of IgGs is intimately linked to the recycling mechanism of the neonatal Fc receptor (FcRn). For long serum half-life of therapeutic IgGs, the highly pH-dependent interaction with FcRn needs to be balanced to allow efficient FcRn binding and release at slightly acidic pH and physiological pH, respectively. Some IgGs, like the antibody briakinumab has an unusually short half-life of ∼8 days. Here we dissect the molecular origins of excessive FcRn binding in therapeutic IgGs using a combination of hydrogen/deuterium exchange mass spectrometry and FcRn affinity chromatography. We provide experimental evidence for a two-pronged IgG-FcRn binding mechanism involving direct FcRn interactions with both the Fc region and the Fab regions of briakinumab, and correlate the occurrence of excessive FcRn binding to an unusually strong Fab-FcRn interaction.