Preparation of Pichia pastoris expression plasmids

When planning any heterologous expression experiment, the very first critical step is related to the design of the overall strategy, hence to the selection of the most adapted expression vector. The very flexible Pichia pastoris system offers a broad range of possibilities for the production of secreted, endogenous or membrane proteins thanks to a combination of various plasmid backbones, selection markers, promoters and fusion sequences introduced into dedicated host strains. The present chapter provides some guidelines on the choice of expression vectors and expression strategies. It also brings the reader a complete toolbox from which plasmids and fusion sequences can be picked and assembled to set up appropriate expression vectors. Finally, it provides standard starting protocols for the preparation of the selected plasmids and their use for host strain transformation.journal article2012importe

Safety and efficacy of subcutaneous iscalimab (CFZ533) in two distinct populations of patients with Sj\uf6gren\u27s disease (TWINSS): week 24 results of a randomised, double-blind, placebo-controlled, phase 2b dose-ranging study

\ua9 2024 Elsevier LtdBackground: Sj\uf6gren\u27s disease is a chronic autoimmune disease with an unmet need for targeted therapies. The aim of the TWINSS study is to evaluate the safety and efficacy of iscalimab, a monoclonal antibody against CD40, in patients with active Sj\uf6gren\u27s disease. Methods: This randomised, double-blind, placebo-controlled, phase 2b study, conducted at 71 sites in 23 countries, enrolled patients aged 18 years or older fulfilling the American College of Rheumatology/European Alliance of Associations for Rheumatology (EULAR) 2016 criteria. In the dose-ranging cohort 1, patients with a EULAR Sj\uf6gren\u27s Syndrome Disease Activity Index (ESSDAI) score of 5 or higher and a EULAR Sj\uf6gren\u27s Syndrome Patient Reported Index (ESSPRI) score of 5 or higher were randomly assigned (1:1:1:1) to subcutaneous iscalimab 150 mg, 300 mg, 600 mg, or placebo. In the proof-of-concept cohort 2, patients with an ESSDAI score of less than 5, ESSPRI (dryness or fatigue) score of 5 or higher, and Impact of Dry Eye on Everyday Life score of 30 or higher were randomly assigned (1:1) to iscalimab 600 mg or placebo. The sponsor, investigator, site personnel, and patients were masked to the treatment assignment. The primary objectives were to demonstrate a dose–response relationship of iscalimab based on the change in ESSDAI from baseline to week 24 in cohort 1 by Multiple Comparison Procedure—Modelling (MCP-Mod), and to assess the effect of iscalimab 600 mg on ESSPRI at week 24 in cohort 2. All the efficacy analyses included all patients who were randomly assigned, and safety analysis included all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov (NCT03905525), and is complete. Findings: Between Oct 1, 2019, and Feb 28, 2022, 460 patients were screened; 173 patients were assigned to cohort 1 (44 to iscalimab 150 mg, 43 to 300 mg, 43 to 600 mg, and 43 to placebo) and 100 to cohort 2 (50 to each group). In cohort 1, the MCP step showed a significant dose–response relationship for placebo-adjusted ESSDAI change from baseline in one of four models (Linlog model, one-sided p=0\ub70041). ESSDAI decreased from baseline to week 24 with all three doses of iscalimab; 150 mg and 600 mg doses showed statistically significant improvement (placebo-adjusted least squares [LS] mean difference –3\ub70 [95% CI –4\ub79 to –1\ub71]; p=0\ub70025 for 150 mg and –2\ub79 [–4\ub79 to –1\ub70]; p=0\ub70037 for 600 mg). In cohort 2, ESSPRI showed a trend towards improvement with iscalimab 600 mg (placebo-adjusted LS mean change from baseline –0\ub757 points [95% CI –1\ub730 to 0\ub715]; p=0\ub712). Serious adverse events were reported in nine patients in cohort 1 (one [2%] of 43 in the placebo group, one [2%] of 44 in the iscalimab 150 mg group, three [7%] of 42 in the 300 mg group, four [9%] of 44 in the 600 mg group) and four patients in cohort 2 (two [4%] of 50 in each group). No deaths occurred over the 24-week period. Interpretation: The study met the primary objective of demonstrating a significant dose–response relationship with iscalimab in terms of disease activity at week 24. Iscalimab was well tolerated and showed initial clinical benefit over placebo in two distinct populations of patients with Sj\uf6gren\u27s disease, to be confirmed in larger trials. Funding: Novartis Pharma