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

Glioblastoma multiforme: a multidisciplinary approach to overcome chemoresistance and find new therapeutic strategies

Objectives: Glioblastoma multiforme is the most frequent malignant brain tumor. Patients die within 15 months after diagnosis. The failure of current therapies is ascribed to a subpopulation of cells with stem-like properties, called glioma stem cells (GSCs). The aim of this study is to develop new effective therapies. Moreover, we want to better characterize the orthotopic xenograft model established by GSCs injection into NOD/SCID mice. Materials and methods: We tested Temolomide and Valproic acid treatments, alone and in combination, on seven GSC lines by MTT assay and we sequenced p53. Moreover, we characterized our xenograft model investigating the expression of stemness and differentiation markers by immunohistochemistry on FFPE tissues and by immunofluorescence on the correspondent cell line. Finally, we performed aCGH on the DNA extracted from the cell line and from FFPE tissues. Results: GSCs were resistant to Temozolomide and slightly sensitive to Valproic acid. The two drugs exerted a synergistic effect when combined performing a pre-conditioning with Valproic acid. Furthermore, several cell lines carry p53 mutations. IF and IHC showed a perfect correspondence for stemness markers expression, but discordant data for the others. aCGH analysis evidenced numerous alterations specific for the ex vivo sample, suggesting the presence of an in vivo clonal selection. Discussion: This work shows the importance of murine microenvironment in GSCs phenotype in vivo and suggests the possibility to use our combined treatment for therapeutic purposes. Conclusions: Orthotopic models from GSCs and in vitro grown cell lines represent good models for the development of GSC-targeted therapies

DNAM-1 chimeric receptor-engineered NK cells: a new frontier for CAR-NK cell-based immunotherapy

DNAM-1 is a major NK cell activating receptor and, together with NKG2D and NCRs, by binding specific ligands, strongly contributes to mediating the killing of tumor or virus-infected cells. DNAM-1 specifically recognizes PVR and Nectin-2 ligands that are expressed on some virus-infected cells and on a broad spectrum of tumor cells of both hematological and solid malignancies. So far, while NK cells engineered for different antigen chimeric receptors (CARs) or chimeric NKG2D receptor have been extensively tested in preclinical and clinical studies, the use of DNAM-1 chimeric receptor-engineered NK cells has been proposed only in our recent proof-of-concept study and deserves further development. The aim of this perspective study is to describe the rationale for using this novel tool as a new anti-cancer immunotherapy

Thyrospheres from B-CPAP cell line with BRAF and TERT promoter mutations have different functional and molecular features than parental cells

Human thyroid cancer derived cell lines are widely used to study the mechanisms involved in thyroid carcinogenesis. However, there is limited availability of non-cross-contaminated cancer cell lines derived from papillary thyroid carcinoma (PTC), and the B-CPAP cell line is one of the few such lines. B-CPAP cells have been genetically and cytogenetically well-characterized, but details of their stemness features remain uncertain. Considering that this cell line is extensively used for in vitro studies on thyroid tumorigenesis, we broaden its functional and molecular profiles as well as the tumorigenic capacity. We used functional assays (sphere-forming capacity and efficiency), assessed self-renewal and propagation efficiency and tested in vivo tumorigenicity in Hsd:Athymic Nude-Foxn1nu mice. Expression of markers of stemness, differentiation, and epithelial-mesenchymal transition were estimated at RNA and protein levels in adherent parental cells and sphere-forming cells. Functional aspects and stemness features were compared with normal thyrocytes. Protein expression of xenograft tumors was evaluated by immunohistochemistry. B-CPAP sphere-forming cells were able to form thyrospheres theoretically indefinitely in an appropriate serum-free medium, reverting to the adherent parental cell phenotype when cultured in differentiation medium. Different expression of ALDH1-A1 and CD44 stemness markers and TTF-1 and CK19 differentiation markers allowed discrimination between isolated sphere-forming cells and adherent parental cells, indicating that sphere-forming cells retained stem-like features. In keeping with these observations, tumorigenicity assays confirmed that, relative to parental adherent cells, thyrospheres had enhanced capacity to initiate xenograft tumors. Thyrospheres from normal cell line retained very low functional capacity, as well as different stemness markers expression compared to tumor thyrospheres. Our findings may constitute a useful background to develop an in vitro model for assessing the origin and progression of papillary thyroid carcinoma bearing BRAFV600E and TERT promoter mutations

Biliverdin Protects against Liver Ischemia Reperfusion Injury in Swine

Ischemia reperfusion injury (IRI) in organ transplantation remains a serious and unsolved problem. Organs that undergo significant damage during IRI, function less well immediately after reperfusion and tend to have more problems at later times when rejection can occur. Biliverdin has emerged as an agent that potently suppress IRI in rodent models. Since the use of biliverdin is being developed as a potential therapeutic modality for humans, we tested the efficacy for its effects on IRI of the liver in swine, an accepted and relevant pre-clinical animal model. Administration of biliverdin resulted in rapid appearance of bilirubin in the serum and significantly suppressed IRI-induced liver dysfunction as measured by multiple parameters including urea and ammonia clearance, neutrophil infiltration and tissue histopathology including hepatocyte cell death. Taken together, our findings, in a large animal model, provide strong support for the continued evaluation of biliverdin as a potential therapeutic in the clinical setting of transplantation of the liver and perhaps other organs

A functional biological network centered on XRCC3: a new possible marker of chemoradiotherapy resistance in rectal cancer patients

Preoperative chemoradiotherapy is widely used to improve local control of disease, sphincter preservation and to improve survival in patients with locally advanced rectal cancer. Patients enrolled in the present study underwent preoperative chemoradiotherapy, followed by surgical excision. Response to chemoradiotherapy was evaluated according to Mandard's Tumor Regression Grade (TRG). TRG 3, 4 and 5 were considered as partial or no response while TRG 1 and 2 as complete response. From pretherapeutic biopsies of 84 locally advanced rectal carcinomas available for the analysis, only 42 of them showed 70% cancer cellularity at least. By determining gene expression profiles, responders and non-responders showed significantly different expression levels for 19 genes (P < 0.001). We fitted a logistic model selected with a stepwise procedure optimizing the Akaike Information Criterion (AIC) and then validated by means of leave one out cross validation (LOOCV, accuracy = 95%). Four genes were retained in the achieved model: ZNF160, XRCC3, HFM1 and ASXL2. Real time PCR confirmed that XRCC3 is overexpressed in responders group and HFM1 and ASXL2 showed a positive trend. In vitro test on colon cancer resistant/susceptible to chemoradioterapy cells, finally prove that XRCC3 deregulation is extensively involved in the chemoresistance mechanisms. Protein-protein interactions (PPI) analysis involving the predictive classifier revealed a network of 45 interacting nodes (proteins) with TRAF6 gene playing a keystone role in the network. The present study confirmed the possibility that gene expression profiling combined with integrative computational biology is useful to predict complete responses to preoperative chemoradiotherapy in patients with advanced rectal cance

Characterizing the involvement of FaMADS9 in the regulation of strawberry fruit receptacle development

FaMADS9 is the strawberry (Fragaria x ananassa) gene that exhibits the highest homology to the tomato (Solanum lycopersicum) RIN gene. Transgenic lines were obtained in which FaMADS9 was silenced. The fruits of these lines did not show differences in basic parameters, such as fruit firmness or colour, but exhibited lower Brix values in three of the four independent lines. The gene ontology MapMan category that was most enriched among the differentially expressed genes in the receptacles at the white stage corresponded to the regulation of transcription, including a high percentage of transcription factors and regulatory proteins associated with auxin action. In contrast, the most enriched categories at the red stage were transport, lipid metabolism and cell wall. Metabolomic analysis of the receptacles of the transformed fruits identified significant changes in the content of maltose, galactonic acid-1,4-lactone, proanthocyanidins and flavonols at the green/white stage, while isomaltose, anthocyanins and cuticular wax metabolism were the most affected at the red stage. Among the regulatory genes that were differentially expressed in the transgenic receptacles were several genes previously linked to flavonoid metabolism, such as MYB10, DIV, ZFN1, ZFN2, GT2, and GT5, or associated with the action of hormones, such as abscisic acid, SHP, ASR, GTE7 and SnRK2.7. The inference of a gene regulatory network, based on a dynamic Bayesian approach, among the genes differentially expressed in the transgenic receptacles at the white and red stages, identified the genes KAN1, DIV, ZFN2 and GTE7 as putative targets of FaMADS9. A MADS9-specific CArG box was identified in the promoters of these genes

Genetic Background Can Result in a Marked or Minimal Effect of Gene Knockout (GPR55 and CB2 Receptor) in Experimental Autoimmune Encephalomyelitis Models of Multiple Sclerosis

PMCID: PMC379391

Konsensusprotokoll zur Standardisierung von Entnahme und Biobanking des Liquor cerebrospinalis

Die Erforschung von Biomarkern in Körperflüssigkeiten bei neurodegenerativen und neuroinflammatorischen Erkrankungen blickt auf eine langjährige Geschichte zurück. Dennoch werden nur wenige Liquor cerebrospinalis (Liquor)-Biomarker in der klinischen Praxis verwendet. Einer der problematischen Faktoren in der Liquorbiomarker-Forschung ist die eingeschränkte Aussagekraft von Studien aufgrund einer nicht ausreichend großer Anzahl von Proben, die in Studien von einzelnen Zentren akquiriert werden können. Deshalb ist die Kooperation zwischen mehreren Zentren erforderlich, um große Biobanken von definierten Proben zu etablieren. Standardisierte Protokolle für Biobanking sind unumgänglich, um die durch die größere Anzahl von Liquorproben gewonnene statistische Aussagekraft sicherzustellen und nicht durch mangelhafte Präanalytik einzuschränken. Hier wird ein Konsensusbericht über Leitlinien zu Liquorentnahme und Biobanking durch das BioMS-eu Netzwerk für Liquorbiomarker-Forschung in Multipler Sklerose präsentiert. Schwerpunkte des Berichts sind Liquorentnahme, präanalytische Faktoren und klinische sowie sonstige Informationen. Biobanking-Protokolle sind für Liquor-Biobanken im Rahmen der Erforschung jeder neurologischen Krankheit anwendba