Georg Fertig

Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II anti-CD20 antibody with enhanced direct and immune effector cell-mediated B-cell cytotoxicity

CD20 is an important target for the treatment of B-cell malignancies, including non-Hodgkin lymphoma as well as autoimmune disorders. B-cell depletion therapy using monoclonal antibodies against CD20, such as rituximab, has revolutionized the treatment of these disorders, greatly improving overall survival in patients. Here, we report the development of GA101 as the first Fc-engineered, type II humanized IgG1 antibody against CD20. Relative to rituximab, GA101 has increased direct and immune effector cell-mediated cytotoxicity and exhibits superior activity in cellular assays and whole blood B-cell depletion assays. In human lymphoma xenograft models, GA101 exhibits superior antitumor activity, resulting in the induction of complete tumor remission and increased overall survival. In nonhuman primates, GA101 demonstrates superior B cell-depleting activity in lymphoid tissue, including in lymph nodes and spleen. Taken together, these results provide compelling evidence for the development of GA101 as a promising new therapy for the treatment of B-cell disorders.

Nucleosomes in serum of patients with benign and malignant diseases.

High quantities of mono‐ and oligonucleosomes circulate in the blood of patients with malignant tumors. For their direct quantification in serum, we modified the Cell Death Detectionplus‐ELISA for its application in liquid materials. We examined sera samples from 590 persons, including 418 patients with malignant tumors, 109 patients with benign diseases and 63 healthy persons. We also observed the kinetics of the concentration of nucleosomes in serum samples from 20 patients undergoing chemotherapy and from 16 patients undergoing radiotherapy. Sera of patients with malignant tumors contained considerably higher concentrations of nucleosomes (mean = 350 arbitrary units [AU], median = 190 AU) compared with those of healthy persons (mean = 36 AU, median = 24 AU; p = 0.0001) and patients with benign diseases (mean = 264 AU, median = 146 AU; p = 0.072). Concerning the follow‐up investigations, the concentration of nucleosomes in serum increased 24–72 hr after the first application of chemotherapy and 6–24 hr after the start of radiotherapy. A subsequent decrease was often correlated with regression of the tumor. In patients undergoing chemotherapy, an increase in the baseline values of circulating nucleosomes >50%, which were determined before each new therapeutic cycle, was correlated with progression of disease; all patients with disease regression showed a decrease >50% of the baseline values. In patients undergoing radiotherapy, an early decrease of the nucleosomal concentration (≤1 day after the initial peak during therapy) to low minimum levels (≤100 AU) correlated with good clinical outcome; a late decrease (>1 day) to higher minimum levels (>100 AU) was associated with a worse clinical outcome. Thus, the concentration of nucleosomes in serum might be a useful tool for monitoring the biochemical response during antitumor therapy, especially for the early estimation of therapeutic efficacy.

Nucleosomes in serum as a marker for cell death.

Abstract The concentration of nucleosomes is elevated in blood of patients with diseases which are associated with enhanced cell death. In order to detect these circulating nucleosomes, we used the Cell Death Detection-ELISAplus (CDDE) from Roche Diagnostics (Mannheim, Germany) (details at http:\\biochem.roche.com). For its application in liquid materials we performed various modifications: we introduced a standard curve with nucleosome-rich material, which enabled direct quantification and improved comparability of the values within (CVintraassay:3.0–4.1%) and between several runs (CVinterassay:8.6–13.5%), and tested the analytical specificity of the ELISA. Because of the fast elimination of nucleosomes from circulation and their limited stability, we compared plasma and serum matrix and investigated in detail the pre-analytical handling of serum samples which can considerably influence the test results. Careless venipuncture producing hemolysis, delayed centrifugation and bacterial contamination of the blood samples led to false-positive results; delayed stabilization with EDTA and insufficient storage conditions resulted in false-negative values. At temperatures of −20 °C, serum samples which were treated with 10 mM EDTA were stable for at least 6 months. In order to avoid possible interfering factors, we recommend a schedule for the pre-analytical handling of the samples. As the first stage, the possible clinical application was investigated in the sera of 310 persons. Patients with solid tumors (n=220; mean=361 Arbitrary Units (AU)) had considerably higher values than healthy persons (n=50; mean=30 AU; p=0.0001) and patients with inflammatory diseases (n=40; mean= 296 AU; p=0.096). Within the group of patients with tumors, those in advanced stages (UICC 4) showed significantly higher values than those in early stages (UICC 1–3) (p=0.0004).

Prevention of hepatitis C virus infection and spread in human liver chimeric mice by an anti‐CD81 monoclonal antibody

CD81 is a required receptor for hepatitis C virus (HCV) infection of human hepatocytes in vitro. We generated several high‐affinity anti‐human CD81 monoclonal antibodies (mAbs) that demonstrated potent, specific, and cross‐genotype inhibition of HCV entry. One of these mAbs, K04, was administered to human liver chimeric mice before or after HCV infection to determine its ability to prevent HCV infection or spread of HCV infection, respectively. All vehicle control mice established HCV infection, reaching steady‐state levels of serum HCV RNA by day 21. Pretreatment of mice with K04 prevented HCV infection in all mice (n = 5). Treatment of mice with mAb K04 every 3 days for 21 days, starting at 6 hours postinfection, resulted in effective inhibition of virus spread. In 3 mice that were sacrificed on day 24, serum HCV levels remained detectable, below the limit of quantification (LOQ), indicating that infection was established, but virus spread was blocked, by the anti‐CD81 mAb. In 5 additional mice that were followed for a longer time, virus remained detectable, below LOQ, until days 24 and 30 in 4 of 5 mice. In the fifth mouse, viral load was quantifiable, but reduced to 64‐fold below the mean viral load in vehicle control at day 24. In addition, 2 of 5 mice cleared the infection by day 30 and 1 mouse had undetectable virus load from day 6 onward. Conclusion: These results demonstrate that CD81 is required for HCV infection and virus spread in vivo, and that anti‐CD81 antibodies such as K04 may have potential as broad‐spectrum antiviral agents for prevention and treatment of HCV infection. (Hepatology 2015;61:1136–1144)