In vitro pharmacological profile of PHA-022121, a small molecule bradykinin B 2 receptor antagonist in clinical development
PHA-022121 is a novel small molecule bradykinin B2 receptor antagonist, in clinical development for the treatment and prevention of hereditary angioedema attacks. The present study describes the in vitro pharmacological characteristics of PHA-022121 and its active metabolite, PHA-022484 (M2-D). In mammalian cell lines, PHA-022121 and PHA-022484 show high affinity for the recombinant human bradykinin B2 receptor with Ki values of 0.47 and 0.70 nM, respectively, and potent antagonism of the human bradykinin B2 receptor with Kb values of 0.15 and 0.26 nM, respectively (calcium mobilization assay).
Antagonist potency at the recombinant cynomolgus monkey bradykinin B2 receptor is similarly high (Kb values of 1.42 and 1.12 nM for PHA-022121 and PHA-022484, respectively), however, potency at rat, mouse, rabbit and dog bradykinin B2 receptors is at least 100-fold lower than the potency at the human receptor for both compounds. In the human umbilical vein contractility assay, both PHA-022121 and PHA-022484 show a potent, surmountable and reversible B2 antagonist activity with pA2 values of 0.35 and 0.47 nM, respectively. The in vitro off-target profile of PHA-022121 and PHA-022484 demonstrates a high degree of selectivity over a wide range of molecular targets, including the bradykinin B1 receptor.
It is concluded that PHA-022121 is a novel, low-molecular weight, competitive antagonist of the human bradykinin B2 receptor with high affinity, high antagonist potency, and high selectivity. It is about 20-fold more potent than icatibant at the human bradykinin B2 receptor as https://joplink.net/mammalian-cell-recombinant/ assessed using recombinant or endogenously expressed receptors.
Designed SARS-CoV-2 receptor binding domain variants form stable monomers
The receptor binding domain (RBD) of the SARS-CoV-2 spike (S)-protein is a prime target of virus-neutralizing antibodies present in convalescent sera of COVID-19 patients and thus is considered a key antigen for immunosurveillance studies and vaccine development. Although recombinant expression of RBD has been achieved in several eukaryotic systems, mammalian cells have proven particularly useful. We aimed to optimize RBD produced in HEK293-6E cells towards a stable homogeneous preparation and addressed its O-glycosylation as well as the unpaired cysteine residue 538 in the widely used RBD (319-541) sequence.
We found that an intact O-glycosylation site at T323 is highly relevant for the expression and maintenance of RBD as a monomer. Furthermore, we show that deletion or substitution of the unpaired cysteine residue C538 reduces the intrinsic propensity of RBD to form oligomeric aggregates, concomitant with an increased yield of the monomeric form of the protein.
Bead-based and enzyme-linked immunosorbent assays utilizing these optimized RBD variants displayed excellent performance with respect to the specific detection of even low levels of SARS-CoV-2 antibodies in convalescent sera. Hence, these RBD variants could be instrumental for the further development of serological SARS-CoV-2 tests and inform the design of RBD-based vaccine candidates. This article is protected by copyright. All rights reserved.
Structure-based discovery of small molecules improving stability of human broadly-neutralizing anti-HIV antibody 2F5 in plant suspension cells
The production of biopharmaceuticals in engineered plant-based systems is a promising technology that has proven its suitability for the production of various recombinant glyco-proteins that are currently undergoing clinical trials. However, compared to mammalian cell lines, the productivity of plant-based systems still requires further improvement. A major obstacle is the proteolytic degradation of recombinant target proteins by endogenous plant proteases mainly from the subtilisin family of serine proteases.
- In the present study, we screened for putative small molecule inhibitors for subtilases that are secreted from tobacco BY-2 suspension cells using an in silico approach.
- The effectiveness of the substances identified in this screen was subsequently tested in degradation assays using the human broadly-neutralizing anti-HIV monoclonal antibody 2F5 (mAb2F5) and spent BY-2 culture medium as a model system.
- Among 16 putative inhibitors identified by studies, three naphthalene sulfonic acid derivatives showed inhibitory activity in degradation assays and are similar to or even more effective than phenylmethylsulfonyl fluoride (PMSF), a classical inhibitor of serine proteases, which served as a positive control.
- This article is protected by copyright. All rights reserved.
The effect of Ccnb1ip1 insulator on monoclonal antibody expression in Chinese hamster ovary cells
Background: The increasing need for therapeutic monoclonal antibodies (mAbs) entails the development of innovative and improved expression strategies. Chromatin insulators have been utilized for the enhancement of the heterologous proteins in mammalian cells.
Methods and results: In the current study the Ccnb1ip1 gene insulator element was utilized to construct a novel vector system for the expression of an anti-CD52 mAb in Chinese hamster ovary (CHO) cells. The insulator containing (pIns-mAb) and control (pmAb) vectors were generated and stable cell pools were established using these constructs. The expression level in the cells created with pIns-mAb vector was calculated to be 233 ng/mL, and the expression rate in the control vector was 210 ng/mL, which indicated a 10.9% increase in mAb expression in pIns-mAb pool. In addition, analysis of mAb expression in clonal cells established from each pool showed a 10% increase in antibody productivity in the highest mAb producing clone derived from the pIns-mAb pool compared to the clone isolated from pmAb pool.
Conclusions: More studies are needed to fully elucidate the effects of Ccnb1ip1 gene insulator on recombinant therapeutic protein expression in mammalian cells. The combination of this element with other chromatin-modifying elements might improve its augmentation effect which could pave the way for efficient and cost-effective production of therapeutic drugs.
Plant-Based Vaccines: Antigen Design, Diversity, and Strategies for High Level Production
Vaccines for human use have conventionally been developed by the production of (1) microbial pathogens in eggs or mammalian cells that are then inactivated, or (2) by the production of pathogen proteins in mammalian and insect cells that are purified for vaccine formulation, as well as, more recently, (3) by using RNA or DNA fragments from pathogens. Another approach for recombinant antigen production in the last three decades has been the use of plants as biofactories.
Only have few plant-produced vaccines been evaluated in clinical trials to fight against diseases, of which COVID-19 vaccines are the most recent to be FDA approved. In silico tools have accelerated vaccine design, which, combined with transitory antigen expression in plants, has led to the testing of promising prototypes in pre-clinical and clinical trials.
Therefore, this review deals with a description of immunoinformatic tools and plant genetic engineering technologies used for antigen design (virus-like particles (VLP), subunit vaccines, VLP chimeras) and the main strategies for high antigen production levels. These key topics for plant-made vaccine development are discussed and perspectives are provided.
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