A single-valent long-acting human CD47 antagonist enhances antibody directed phagocytic activities.

Many cancer cells express CD47 as a 'don't eat me' signal to mask their presences from immune recognition and destruction. Such a signal is transmitted when CD47 binds to the signal regulatory protein-α (SIRPα) on macrophages to cut the phagocytic reaction. Most recent studies have focused on developing CD47 blocking agents with different affinities and avidities in order to optimize the therapeutic window between efficacy and toxicities involving normal cells expressing CD47. We described in this study a new design to fuse one CD47 binding domain of SIRPα with a pharmacokinetics modifying domain F8. The resulted single valent long-acting CD47 antagonist SIRPα-F8 was able to bind to CD47 and disrupt CD47-SIRPα axis. However, by itself it cannot trigger endocytosis and has no effect on tumor growth. Only when used in combination with the anti-CD20 mAbs, there were greatly improved phagocytic activities towards CD20 positive cancer cells. In vivo the combination also resulted in better tumor growth inhibition comparing to the vehicle control group. In addition, we showed that the F8 fusion bound to hFcRn only inside endosomes at pH 6.0, enabled hFcRn mediated recycling and thus greatly extended the circulation half-life in hFcRn knock-in mice. Taken together, the SIRPα-F8 design may suggest a new option to improve the therapeutic index of antibody treatment in clinical use towards tumors.

Single chain antibody fragments with pH dependent binding to FcRn enabled prolonged circulation of therapeutic peptide in vivo.

The neonatal Fc receptor for IgG (FcRn) is considered critical for the regulation of endogenous IgG and serum albumin (SA) and their circulation half-life in vivo. Both IgG and SA can bind to FcRn tightly at acidic pH but not so much at neutral pH. Here we reported a few novel single chain antibody fragments (scFv) obtained based on screening of a phage library. FnAb-8 and FnAb-12 can bind to human FcRn with higher affinities than IgG at acidic pH but similar or lower affinities than IgG at pH7.4. Fusion proteins consisted of the therapeutic peptide, GLP-1 (Glucagon-like peptide-1) connected to the N-terminus of FnAb-8 and FnAb-12, named as G8 and G12, were shown to retain the pH-dependent binding capabilities to FcRn while also bound to the GLP-1 receptor. In vivo efficacy studies in diet induced diabetes mice confirmed the GLP-1 receptor (GLP-1R) agonist activities and sustained blood sugar lowering effect. In vivo pharmacokinetics (PK) studies were performed in nonhuman primates and FnAb-8 was found to have circulation half-life several folds longer than what have been reported for scFvs. G8 may be developed into long acting GLP-1R agonists with great potentials in clinical applications.

Examining the interactome of huperzine A by magnetic biopanning.

Huperzine A is a bioactive compound derived from traditional Chinese medicine plant Qian Ceng Ta (Huperzia serrata), and was found to have multiple neuroprotective effects. In addition to being a potent acetylcholinesterase inhibitor, it was thought to act through other mechanisms such as antioxidation, antiapoptosis, etc. However, the molecular targets involved with these mechanisms were not identified. In this study, we attempted to exam the interactome of Huperzine A using a cDNA phage display library and also mammalian brain tissue extracts. The drugs were chemically linked on the surface of magnetic particles and the interactive phages or proteins were collected and analyzed. Among the various cDNA expressing phages selected, one was identified to encode the mitochondria NADH dehydrogenase subunit 1. Specific bindings between the drug and the target phages and target proteins were confirmed. Another enriched phage clone was identified as mitochondria ATP synthase, which was also panned out from the proteome of mouse brain tissue lysate. These data indicated the possible involvement of mitochondrial respiratory chain matrix enzymes in Huperzine A's pharmacological effects. Such involvement had been suggested by previous studies based on enzyme activity changes. Our data supported the new mechanism. Overall we demonstrated the feasibility of using magnetic biopanning as a simple and viable method for investigating the complex molecular mechanisms of bioactive molecules.

[Molecular cloning and expression of extremely thermostable and acid-stable amylase gene in Pichia pastoris].

The gene encoding a extremely thermostable and acid-stable alpha-Amylase was amplified by PCR using hyperthermophilic archaebacterium pyrococcus furiosus genomic DNA as template. Then the gene was cloned into the vector of pPIC9K. The recombinant vector pPIC9K-amy was then transformed into E. coli DH5alpha strain. Sequencing test showed that the a-amylase gene cloned consisted of 1305 base pairs and the mature protein encoded by the gene consisted of 435 amino acids. The recombinant vector was transformed into chromosome of methylotrophic yeast Pichia pastoris GS115 strain. Regulated by the alpha-Factor, promoter of AOX1 gene and termination signal of yeast genomic, the recombinant a-Amylase was expressed and excreted out of the cells. The expression of the recombinant alpha-amylase was strictly induced by methanol. As induction time increased, the activity of amylase per milliliter medium went up accordingly. After 7 days induction, the activity of the amylase reached the max. The recombinant alpha-amylase exhibited maximal activity at 90 to approximately 100 degrees C and at pHranging from 4.5 to 5.0. The enzyme is so thermostable that after disposed at 100 degrees C for 5 hours over 60% of activity was retained.