Here, we describe a method that offers a unique way to engineer plasmids with precision but without digestion using restriction enzymes for the insertion of DNA. The method allows the insertion of PCR fragments in between any two nucleotides within a target plasmid. The only requirement is that the amplified fragments must be embedded between DNA sequences homologous to the site in which the integration is planned. This method is an adaptation of the QuikChange Site-Directed Mutagenesis protocol. It is simpler than the existing cloning strategies and is suitable for multiparallel constructions of new plasmids. We have demonstrated its utility by constructing plasmids in which we have successfully integrated PCR fragments up to 1117 bp.
Background and objective
Anti‐IL‐17A IgG/κ monoclonal antibody CJM112 binds both IL‐17A and IL‐17AF. The purpose of this First‐in‐Human study was to assess CJM112 effects on safety and efficacy in patients with moderate to severe plaque psoriasis.
Methods
This study had two parts: single ascending doses of 5–450 mg subcutaneous (s.c.) CJM112 (SAD) and multi‐dose parallel groups of CJM112 15 mg, 50 mg and 150 mg s.c. low frequency or high frequency (MD). SAD/MD were double‐blind, randomized and placebo‐controlled; MD also included a secukinumab 150 mg s.c. arm as an active comparator. Patients 18–65 years with moderate to severe psoriasis were included in this study. The efficacy outcome was the change in Psoriasis Area Severity Index (PASI) from baseline to Week 4 in the SAD part of the study, and from baseline to Week 12 in the MD part.
Results
96 patients were enrolled in this study (SAD, n = 42; MD, n = 54). In SAD, CJM112 doses from 15 mg and above demonstrated higher PASI responses compared with placebo at Week 12. CJM112 450 mg did not add further efficacy, but efficacy duration was prolonged compared with CJM112 150 mg. CJM112 MD resulted in a dose‐dependent decrease in PASI over time to Week 12. CJM112 150 mg high frequency did not exceed the effect of CJM112 150 mg low frequency and had similar efficacy to secukinumab 150 mg. The safety profile of CJM112 was as expected for an antibody targeting IL‐17A/IL‐17AF.
Conclusions
CJM112 had clinical efficacy in moderate to severe psoriasis and was generally safe and well tolerated in the doses tested. Additional neutralization of IL‐17AF did not translate to increased clinical efficacy compared with secukinumab.
Opioid receptors, like many G protein-coupled receptors (GPCRs), are notoriously unstable in detergents. We have now developed a more stable variant of the mu-opioid receptor (MOR) and also a method for the immobilization of solubilized, functional opioid receptors on a solid phase (magnetic beads). Starting with the intrinsically more stable kappa-opioid receptor (KOR), we optimized the conditions (i.e. detergents and stabilizing ligands) for receptor extraction from lipid bilayers of HEK293T cells to obtain maximal amounts of functional, immobilized receptor. After immobilization, the ligand binding profile remains the same as observed for the membrane-embedded receptor. For the immobilized wild-type mu-opioid receptor, however, no conditions were found under which ligand binding capacity was retained. To solve this problem, we engineered the receptor chimera KKM where the N-terminus and the first transmembrane helix (TM1) of wild-type MOR is exchanged for the homologous receptor parts of the wild-type KOR. This hybrid receptor behaves exactly as the wild-type MOR in functional assays. Interestingly, the modified MOR is expressed at six times higher levels than wild-type MOR and is similarly stable as wild-type KOR after immobilization. Hence the immobilized MOR, represented by the chimera KKM, is now also amenable for biophysical characterization. These results are encouraging for future stability engineering of GPCRs.
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