Isolation and light chain shuffling of a Plasmodium falciparum AMA1-specific human monoclonal antibody with growth inhibitory activity.

BACKGROUND: Plasmodium falciparum, the parasite causing malaria, affects populations in many endemic countries threatening mainly individuals with low malaria immunity, especially children. Despite the approval of the first malaria vaccine Mosquirix™ and very promising data using cryopreserved P. falciparum sporozoites (PfSPZ), further research is needed to elucidate the mechanisms of humoral immunity for the development of next-generation vaccines and alternative malaria therapies including antibody therapy. A high prevalence of antibodies against AMA1 in immune individuals has made this antigen one of the major blood-stage vaccine candidates. MATERIAL AND METHODS: Using antibody phage display, an AMA1-specific growth inhibitory human monoclonal antibody from a malaria-immune Fab library using a set of three AMA1 diversity covering variants (DiCo 1-3), which represents a wide range of AMA1 antigen sequences, was selected. The functionality of the selected clone was tested in vitro using a growth inhibition assay with P. falciparum strain 3D7. To potentially improve affinity and functional activity of the isolated antibody, a phage display mediated light chain shuffling was employed. The parental light chain was replaced with a light chain repertoire derived from the same population of human V genes, these selected antibodies were tested in binding tests and in functionality assays. RESULTS: The selected parental antibody achieved a 50% effective concentration (EC50) of 1.25 mg/mL. The subsequent light chain shuffling led to the generation of four derivatives of the parental clone with higher expression levels, similar or increased affinity and improved EC50 against 3D7 of 0.29 mg/mL. Pairwise epitope mapping gave evidence for binding to AMA1 domain II without competing with RON2. CONCLUSION: We have thus shown that a compact immune human phage display library is sufficient for the isolation of potent inhibitory monoclonal antibodies and that minor sequence mutations dramatically increase expression levels in Nicotiana benthamiana. Interestingly, the antibody blocks parasite inhibition independently of binding to RON2, thus having a yet undescribed mode of action.

Isothiocyanate-containing mustard protects human cells against genotoxins in vitro and in vivo.

BACKGROUND: Human intervention trials in which cytogenetic biomarkers are used as intermediate endpoints in carcinogenesis are implicitly required to support the assumption of chemo-preventive efficacy. METHODS: To evaluate the genotoxic and anti-genotoxic properties of defined isothiocyanate-containing mustard, we first used a human liver cell-line and then conducted a controlled pilot human intervention trial. Blood from volunteers served as surrogate tissue for time-kinetic analysis of the chemo-preventive effect of mustard consumption. RESULTS: Mustard extracts displayed significant anti-genotoxicity against benzo(a)pyrene in human HepG2 hepatoma cells. At high concentrations, the extracts induced genotoxicity by themselves without compromising cell viability. The protective effect of mustard supplementation against DNA damage induced ex vivo was detected in blood of volunteers within 12h after the start of the intervention, and increased over time. No genotoxicity was induced in human peripheral mononuclear blood cells by mustard intake over the whole period of the study. Also, liver parameters remained within the normal range at all times. Although no change in total plasma GST activity was detected, plasma alpha-GST levels increased over time, peaking at 48 h. CONCLUSIONS: The results suggest the capacity of small amounts of isothiocyanate-containing food to protect cells from DNA damage, even with short-term application.

Physicians' judgement and comprehensive geriatric assessment (CGA) select different patients as fit for chemotherapy.

INTRODUCTION: Elderly cancer patients are a very heterogeneous population. A comprehensive geriatric assessment (CGA) shall help to identify more precisely those patients who are fit, compared to those who are vulnerable or frail, when deciding on chemotherapeutical treatment. METHODS: In a prospective trial, 200 cancer patients treated in an out-patient setting were judged by their physician for their fitness for chemotherapy as fit, vulnerable or frail. A CGA was performed thereafter. We determined the feasibility of a CGA in an out-patient setting and the frequency of changes within the different assessment tools and compared physicians' judgement with the CGA results. RESULTS: Physicians judged 64.3% of their patients as fit, 32.4% as vulnerable, and 3.2% as frail. A CGA was completed by 97.5% of patients and lasted 20min per patients (range: 9-47min). 26.5% of all patients had no deficits in the CGA. The CGA identified a mean of 1.7 problems per patient, 1.3 in patients judged as fit, 2.3 in those judged as vulnerable, and 4.2 in those judged as frail. A CGA is more sensitive in classifying patients as fit compared to vulnerable or frail than physicians' judgement. CONCLUSION: A CGA is feasible and detects more elderly cancer patients as being unfit for chemotherapy than physicians' judgement. Further trials including disease and treatment related end-points are needed.

High anti-lymphoma activity of bendamustine/mitoxantrone/rituximab in rituximab pretreated relapsed or refractory indolent lymphomas and mantle cell lymphomas. A multicenter phase II study of the German Low Grade Lymphoma Study Group (GLSG).

On the basis of a preceding phase I study, the current trial explored bendamustine in combination with mitoxantrone and rituximab (BMR) in patients with stage III/IV relapsed or refractory indolent lymphomas and mantle cell lymphoma (MCL) with or without prior rituximab containing chemo-immunotherapy (R-chemo) treatment. Therapy consisted of bendamustine 90 mg/m(2) days 1 + 2, mitoxantrone 10 mg/m(2) day 1, rituximab 375 mg/m(2) day 8. Treatment was repeated on day 29 for a total of four cycles. Between 3 April and 04 July, 57 patients were recruited from 24 participating institutions, 39% of whom had received prior R-chemo therapy. Median age was 66 years (40 - 83). Lymphoma subtypes were 29 follicular (FL), 18 MCL, and 10 other indolent lymphomas. The overall response rate (ORR) was 89% with 35% CR and 54% PR. ORR in R-chemo pretreated patients was 76% (38% CR, 38% PR). After a median observation time of 27 months (1 - 43), the estimated median progression free survival is 19 months. The 2 year overall survival is 60% for patients with FL and MCL. Treatment related toxicities of grade 3/4 comprised a reversible myelosuppression (10% anemia, 78% leukocytopenia, 46% granulocytopenia, 16% thrombocytopenia). However, unexpected hospitalisations were necessary after 4% of BMR-application only. BMR is a very effective new outpatient immuno-chemotherapy with low toxicity for patients with relapsed/refractory FL, MCL and other indolent lymphomas.