A general method for the selection of high-level scFv and IgG antibody expression by stably transfected mammalian cells

The isolation of mammalian cell lines capable of high-yield expression of recombinant antibodies is typically performed by screening multiple individual clones by limiting dilution techniques. A number of experimental strategies have recently been devised to identify high-expressing clones, but protocols are often difficult to implement, time consuming, costly and limited in terms of number of clones which can be screened. In this article, we describe new vectors for the expression of recombinant antibodies in IgG format and in other formats, based on the single-chain Fv module, as well as a high-throughput screening procedure, based on the direct staining of antibodies transiting the membrane of a stably transfected cell, followed by preparative sorting using a high-speed cell sorter. This procedure allows, in one step, to deposit single cells into individual wells of a 96-well microtiter plate (thus facilitating cloning) and to preferentially recover those rare cell populations which express dramatically higher levels of recombinant antibody. Using cell cultures followed by affinity purification techniques, we could confirm that the new vectors and the new screening procedure reliably yield high-expression clones and homogenous protein preparations. We expect that these techniques should find broad applicability for both academic and industrial antibody engineering researc

Expression, engineering and characterization of the tumor-targeting heterodimeric immunocytokine F8-IL12

Proinflammatory cytokines have been used for several years in patients with advanced cancer but their administration is typically associated with severe toxicity hampering their application to therapeutically active regimens. This problem can be overcome by using immunocytokines (cytokines fused to antibody or antibody fragments) which selectively deliver the active cytokine to the tumor environment. Preclinical and recent clinical results confirmed that this approach is a very promising avenue to go. We designed an immunocytokine consisting of the scFv(F8) specific to extra-domain A of fibronectin and the very potent human cytokine interleukin-12 (IL12). The heterodimeric nature of IL12 allows the engineering of various immunocytokine formats, based on different combinations of the two subunits (p35 and p40) together with the scFv. In comparison to monomeric or homodimeric cytokines, the construction of a heterodimeric immunocytokine poses many challenges, e.g. gene dosing, stable high-yield expression as well as good manufacture practice (GMP) purification and characterization. In this paper, we describe the successful construction, characterization and production of the heterodimeric immunocytokine F8-IL12. The positive outcome of this feasibility study leads now to GMP production of F8-IL12, which will soon enter clinical trial

A multicenter real-world evidence study in the Swiss treatment landscape of chronic myeloid leukemia

Background: The real-world experience of Swiss chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors (TKIs) is largely unknown, in particular with regard to achievement of response per European Leukemia Net (ELN) criteria and adherence to ELN recommendations. Methods: This was a retrospective, non-interventional, multicenter chart review of patients with newly diagnosed CML who had received first-line TKI and were solely treated with TKIs between 2010 and 2015, with a minimum follow-up of 18 months, at six Swiss hospitals. Effectiveness was evaluated according to ELN 2013 milestone achievements at 3, 6, 12 and 18 months, and at last follow-up. Results: Data from 63 patients (56% men; median age at diagnosis 55 years) were collected (first-line imatinib [n = 27], nilotinib [n = 27], dasatinib [n = 8], or ponatinib [n = 1]). TKI switches (49 times) and dosing changes (165 times) due to intolerance or insufficient response were frequent. Compared with patients receiving first-line imatinib, a higher proportion of patients receiving first-line nilotinib or dasatinib achieved optimal response at all timepoints, irrespective of subsequent TKI therapy, and a higher proportion of patients treated with first-line nilotinib and dasatinib reached deep molecular response (BCR-ABL1IS ≤ 0.01%) at 18 months (42 and 38%, respectively, versus 27%). Patients who received nilotinib or dasatinib switched therapies less frequently than patients treated with imatinib, irrespective of subsequent TKI therapy. Conclusions: Although patient numbers were small, this real-world evidence study with patients with CML confirms that ELN guidelines are generally implemented in Swiss clinical practice, with a large proportion of patients achieving ELN 2013 milestones. While TKI use involved all inhibitors approved at the time of the study, an unexpectedly high number of TKI therapy switches suggests a clear difference in TKI use between registration trials and clinical practice. Keywords: Chronic myeloid leukemia; Deep molecular response; Real-world evidence; Tyrosine kinase inhibitors

A general method for the selection of high-level scFv and IgG antibody expression by stably transfected mammalian cells

ISSN:1741-0126ISSN:1741-0134ISSN:0269-2139ISSN:1460-213

The pywmi Framework and Toolbox for Probabilistic Inference using Weighted Model Integration

Weighted Model Integration (WMI) is a popular technique for probabilistic inference that extends Weighted Model Counting (WMC) -- the standard inference technique for inference in discrete domains -- to domains with both discrete and continuous variables. However, existing WMI solvers each have different interfaces and use different formats for representing WMI problems. Therefore, we introduce pywmi (http://pywmi.org), an open source framework and toolbox for probabilistic inference using WMI, to address these shortcomings. Crucially, pywmi fixes a common internal format for WMI problems and introduces a common interface for WMI solvers. To assist users in modeling WMI problems, pywmi introduces modeling languages based on SMT-LIB.v2 or MiniZinc and parsers for both. To assist users in comparing WMI solvers, pywmi includes implementations of several state-of-the-art solvers, a fast approximate WMI solver, and a command-line interface to solve WMI problems. Finally, to assist developers in implementing new solvers, pywmi provides Python implementations of commonly used subroutines.status: Published onlin

Expression, engineering and characterization of the tumor-targeting heterodimeric immunocytokine F8-IL12

ISSN:1741-0126ISSN:1741-0134ISSN:0269-2139ISSN:1460-213

Role of multidetector CT angiography and contrast-enhanced ultrasound in redefining follow-up protocols after endovascular abdominal aortic aneurysm repair.

Abstract PURPOSE: Contrast-enhanced ultrasonography (CEUS) is an appealing alternative to computed tomography angiography (CTA) for the follow-up of patients who underwent endovascular abdominal aortic aneurysm repair (EVAR). We sought to evaluate the accuracy of CEUS compared with a particularly tailored protocol of CTA performed with a 64-row multidetector CT. MATERIALS AND METHODS: The study prospectively enrolled 88 consecutive patients for CEUS and CTA imaging during follow-up after EVAR, yielding 142 paired examinations. The outcome is represented by three main goals: identification and characterisation of endoleaks, evaluation of graft patency and measurement of aneurysm diameter. Triple-phase CTA was the gold standard. RESULTS: Sensitivity and specificity of CEUS compared with CTA in endoleak and graft patency evaluation were 91.89% and 100% and 72% and 100%, respectively. A very high correlation between CTA and CEUS diameter measurements was established. CEUS did not appear superior to CTA in endoleak detection, probably because a tailored CTA protocol with a delayed phase (180 s) allows detection of low-flow endoleaks. CONCLUSIONS: Patient management was not different stafollowing CEUS and CTA results. CTA cannot yet be completely replaced, but several limitations (radiation exposure, contrast agent) encourage redefining the routine follow-up imaging modality. We suggest an algorithm of surveillance alternating CTA and CEUS

Using qualitative interviews to identify patient-reported clinical trial endpoints and analyses that are the most meaningful to patients with advanced breast cancer.

BackgroundDesigning clinical trials with the emphasis on the patient-centered approach and focusing on clinical outcomes that are meaningful to patients is viewed as a priority by drug developers, regulatory agencies, payers, clinicians, and patients. This study aimed to capture information on clinical trial endpoints that would be most important and relevant for patients with advanced breast cancer, based on patient-reported outcomes.MethodsPatients with either advanced triple-negative breast cancer [TNBC] and a maximum of two lines of systemic therapy or hormone receptor-positive/human epidermal growth factor receptor 2-negative [HR+/HER2-] breast cancer and a maximum of three lines of systemic therapy, participated in semi-structured concept elicitation interviews. Concept saturation was assessed. A sign, symptom, or impact was defined as "salient" if mentioned by ≥ 60% of participants, with an average bother rating of ≥ 5 (0-10 Scale). Participants were also asked about treatment priorities and to evaluate hypothetical scenarios showing different health-related functioning and quality-of-life treatment outcomes, using graphical representations.ResultsThirty-two participants (97% women; aged 29+ years) with TNBC (n = 17) or HR+/HER2- breast cancer (n = 15) provided generally similar reports on symptom experience, with fatigue and pain being most salient, though importance of certain treatment-related symptoms varied between the two groups. Patients reported consistent perspectives on the importance of treatment outcomes: when considering a new treatment, they prioritized efficacy of the therapy, acceptable tolerability, stability, predictability of symptoms over time, and the duration of preserved health-related quality of life and physical functioning. The meaningful difference in preserved physical functioning was 2-3 months for 46% of participants with TNBC, whereas for most participants with HR+/HER2- breast cancer it started from 6-7 months. Both groups of participants found it easier to accept some toxicity at the beginning of therapy if it was followed by improvement, as opposed to improvement followed by deterioration.ConclusionThe results may help to inform the design of patient-centered clinical trials, to interpret health-related quality of life and/or patient-reported outcomes, and to optimize care for patients with advanced breast cancer

Structured reporting using a shared indexed multilingual radiology lexicon

PURPOSE: A system for creating structured reports (SRs) using a standardized radiology lexicon was developed and tested to facilitate automated translation of content and multidisciplinary international communications. METHODS: A database of radiology terms, RadLex developed by the Radiological Society of North America, was used to create a shared indexed multilingual radiology lexicon. A diagnostic workstation for generating structured reports (OpenEye) was implemented with a "RadLex manager" function for adding new words to the lexicon in both English and Italian. Sample reports of examinations included in the Medical Imaging Resource Center (MIRC) radiology imaging database of clinical cases were prepared using this system. The system was evaluated for teaching purposes and scientific dissemination. RESULTS: The OpenEye system was able to manage the glossary to create new SRs and manually translate existing reports containing freely worded descriptions. The OpenEye system provides instant translation from Italian into English and enables clinical cases to be published in the MIRC, while making them accessible for consultation on an international scale. CONCLUSION: The SR is advantageous compared with a freely worded report in terms of clarity and completeness of the content. Creating SRs for each clinical case enables rapid and focused analysis at multidisciplinary meetings. All our cases have been included in the MIRC database as part of a broader-based European Project for research on soft tissues sarcomas (CONTICANET)