A bispecific antibody targeting HER2 and PD-L1 inhibits tumor growth with superior efficacy.

Activation of the programmed cell death protein 1 and programmed cell death ligand 1 (PD-1/PD-L1) signaling axis plays important roles in intrinsic or acquired resistance to human epidermal growth factor receptor 2 (HER2)-directed therapies in the clinic. Therefore, therapies simultaneously targeting both HER2 and PD-1/PD-L1 signaling pathways are of great significance. Here, aiming to direct the anti-PD-L1 responses toward HER2-expressing tumor cells, we constructed a humanized bispecific IgG1 subclass antibody targeting both HER2 and PD-L1 (HER2/PD-L1; BsAb), which displayed satisfactory purity, thermostability, and serum stability. We found that BsAb showed enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity in vitro. In the late phase of peripheral blood mononuclear cell (PBMC)-humanized HER2+ tumor xenograft models, BsAb showed superior therapeutic efficacies as compared with monoclonal antibodies (mAbs) or combination treatment strategies. In cynomolgus monkeys, BsAb showed favorable pharmacokinetics and toxicity profiles when administered at a 10 mg/kg dosage. Thus, HER2/PD-L1 BsAb was demonstrated as a potentially effective option for managing HER2+ and trastuzumab-resistant tumors in the clinic. We propose that the enhanced antitumor activities of BsAb in vivo may be due to direct inhibition of HER2 signaling or activation of T cells.

Chemically synthesized LYRM03 could inhibit the metastasis of human breast cancer MDA-MB-231 cells in vitro and in vivo.

Aminopeptidase N (APN) belongs to the aminopeptidase family, which is widely distributed throughout the animal and plant kingdoms. APN is thought to be a very important target for cancer therapy as it is linked to cancer progression and metastasis. However, bestatin (Ubenimex) is the only approved drug that targets various aminopeptidases for the treatment of acute myelocytic leukemia and lymphedema. A compound 3-amino-2-hydroxy-4-phenylbutanoylvalylisoleucine (also known as LYRM03), isolated from a Streptomyces strain HCCB10043, exhibited more potent inhibitory activity than bestatin. In this work, we applied a chemical synthesis strategy to generate LYRM03 to overcome the low yields typically achieved from fermentation. Finally, we explored a suite of experiments to determine the bioactivity of LYRM03 and revealed that the metastasis of MDA-MB-231 cells was significantly restrained with LYRM03 treatment or injection both in vitro and in vivo. Because of its anti-metastasis capacity, further structure modifications of LYRM03 will be of interest for its use alone or in combination as a therapy in cancer.

Purification and properties of a novel beta-glucosidase, hydrolyzing ginsenoside Rb1 to CK, from Paecilomyces Bainier.

A novel ginsenoside-hydrolyzing beta-glucosidase was purified from Paecilomyces Bainier sp. 229 by a combination of QSepharose FF, phenyl-Sepharose CL-4B, and CHT ceramic hydroxyapatite column chromatographies. The purified enzyme was a monomeric protein with a molecular mass estimated to be 115 kDa. The optimal enzyme activity was observed at pH 3.5 and 60oC. It was highly stable within pH 3-9 and at temperatures lower than 55oC. The enzyme was specific to beta-glucoside. The order of enzyme activities against different types of beta-glucosidic linkages was beta-(1- 6)>beta-(1-2)>beta-(1-4). The enzyme converted ginsenoside Rb1 to CK specifically and efficiently. An 84.3% amount of ginsenoside Rb1, with an initial concentration of 2 mM, was converted into CK in 24 h by the enzyme at 45 degrees and pH 3.5. The hydrolysis pathway of ginsenoside Rb1 by the enzyme was Rb1-->Rd-->F2-->CK. Five tryptic peptide fragments of the enzyme were identified by a newly developed de novo sequencing method of post-source decay (PSD) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. By comparing the five identified peptide sequences with the NCBI database, this purified beta-glucosidase proves to be a new protein that has not been reported before.

The impact of liposomal linolenic acid on gastrointestinal microbiota in mice.

BACKGROUND: The prevalence of Helicobacter pylori has long been a global health issue. Triple therapy, being the first-line treatment, has caused dysbiosis of the gastrointestinal tract that led to various complications. A novel nanomedicine - liposomal linolenic acid (LipoLLA) - has been proven to have great potential in eradicating H. pylori. However, the possible side effects of LipoLLA due to alteration of the gastrointestinal microbiota remain unknown. AIM: This study focused on the impact of LipoLLA on gastrointestinal microbiota in mice in comparison with triple therapy in order to assess the safety profile. METHODS: Mice were divided into five groups: blank control group; H. pylori control group; triple therapy group; low-dose LipoLLA group (25 mg/kg); and high-dose LipoLLA group (50 mg/kg). Fecal samples were collected before and after the intake of corresponding formulas. Gastric tissues were obtained after mice dissection. These samples were analyzed with high-throughput sequencing. RESULTS: The analysis revealed that LipoLLA resulted in minor gut microbiota alteration at different levels. The altered proportions in the high-dose group were higher than that of the low-dose group. On the other hand, the triple therapy group showed dramatic shifts in the major community composition. It displayed a notable boost in the relative abundance of Proteobacteria and Firmicutes along with a decrease in that of Verrucomicrobia and Bacteroidetes. All of them belonged to the major phyla in the microbiome. Triple therapy also led to the growth of the family Enterobacteriaceae, Enterococcaceae, and Clostridiaceae_1 that may be associated with clinical illnesses. Gastric microbiota analysis reached similar conclusions. CONCLUSION: Our findings indicated that LipoLLA causes minor gastrointestinal microbiota alterations while the triple therapy triggered dramatic changes. Thus, LipoLLA is not only promising but also a safe therapeutic medication to eradicate H. pylori infection.

[Preparation of nosiheptide liposomes and its inhibitory effect on hepatitics B virus in vitro].

AIM: To prepare liposomes of nosiheptide and study its ability to inhibit hepatitis B virus HBsAg and HBeAg secreted. METHODS: Liposomes of nosiheptide was prepared by sodium deoxycholate dialysis and sonication. Nosheptide was determined by HPLC and partical size was determined by using laser light scattering instrument. Transmission electron microscopy (TEM) was used to examine the morphology of liposomes. Its actions to inhibit hepatitis B virus HBsAg and HBeAg secreted was studied by a HBV-transfectted cell line (HepG2 2. 2. 15 ). RESULTS: Encapsulation efficiency of liposomes by chloroform:methanol (2:1, v/v) was higher than that by dioxane. With the increase of the ratio of nosiheptide: PC (W/W), the encapsulation efficiency of liposomes decreased with the increase of ratio of sodium deoxycholate: PC, the liposomes partical size decreased. The liposomes kept stable at -20 degrees C after 2 years. The drug concentrations of liposomes that inhibit HBsAg secreted by (46.9 +/- 2. 6) %, (55.4 +/- 1.2) %, (65 +/- 3) % and HBeAg secreted by (15.1 +/- 2.3) %, (36.2 +/- 1.7) %, (36.8 +/- 2.5) % were 1.25, 2.5, 5.0 microg x mL(-1), respectively. CONCLUSION: Liposomes of nosheptide can be prepared by sodium deoxycholate dialysis and sonication, which ability to inhibit hepatitis B virus HBsAg and HBeAg secreted is better than nosheptide.

Baicalin inhibits autophagy induced by influenza A virus H3N2.

Baicalin, a natural product isolated from Scutellariaradix, has been reported to have significant in vivo and in vitro anti-influenza virus activity, but the underlying mechanism remains poorly understood. In this study, we found that baicalin inhibited autophagy induced by influenza virus A3/Beijing/30/95 (H3N2) in both A549 and Ana-1 cells. The results showed that H3N2 induced autophagy by suppressing mTOR signaling pathway, which however could be significantly inhibited by baicalin. Baicalin could suppress the expression of Atg5-Atg12 complex and LC3-II, and attenuate autophagy induced by starvation. Thus, the inhibition of autophagy induced by virus may account for the antiviral activities of baicalin against H3N2. Autophagy may be a potential marker in developing novel anti-influenza drugs.

Biotransformation of ginsenoside Rb1 to ginsenoside Rd by highly substrate-tolerant Paecilomyces bainier 229-7.

Paecilomyces bainier 229-7 was obtained after UV irradiation for 8 min in the presence of 0.4% LiCl and selection on potato dextrose media containing 30 mg/mL saponin from Panax notoginseng leaves (SPNL). The mutant produces ginsenoside Rd from ginsenoside Rb1 with a bioconversion rate as high as 94.9% under optimized culture conditions in shake flasks when supplied with 20 mg/mL of SPNL. Scale-up in 10-L fermenter resulted in an 89% bioconversion rate. Ginsenoside Rd was purified from the culture medium by a macroporous resin with a chromatographic purity of 92.6%. These results suggest that P. bainier 229-7 could be useful for the preparation of ginsenoside Rd in the pharmaceutical industry.

High-level expression and purification of recombinant human catalase in Pichia pastoris.

Catalase is one of the antioxidant enzymes and is involved in many pathophysiologic processes and human diseases. This study focused on high-level expression and purification of recombinant catalase in Pichia pastoris. The cDNA encoding catalase was cloned by RT-PCR from Fetal liver of Homo sapiens. After PCR and construction of expression vector pPIC9K-CAT, human catalase was expressed highly in P. pastoris yeast SMD1168 and secreted into the culture medium. The secreted catalase was purified to a purity of 95% by ammonium sulfate fractionation, anionic exchange-chromatography, and Macro-prep Ceramic Hydroxyapatite with a overall yield of 60%. This study provides a new method for large-scale expression and purification of recombinant protein catalase.

High yield and secretion of recombinant human apolipoprotein AI in Pichia pastoris.

Apolipoprotein AI (ApoAI) is an important apolipoprotein in plasma and is known to have various physiological functions suitable for pharmaceutical applications. Human blood has been the only source of this protein for research and large-scale applications. To obtain large amounts of ApoAI a Pichia pastoris expression system was first used to obtain a high level of expression of secreted, recombinant protein. The human gene encoding ApoAI was inserted into the secretion vector pPIC9K and used to transform P. pastoris GS115. AP16, a high expression transformant with high G418 resistance, was obtained. After induction with methanol, the expression level of rhApoAI (recombinant human ApoAI) was 160 mg/L in a 14L fermentor. RhApoAI was purified by cold acetone precipitation followed by Q-Sepharose Fast Flow ion exchange column chromatography with 60% recovery. The N-terminal amino acid sequence and molecular weight (mass spec.) of rhApoAI are identical to native human ApoAI. Purified rhApoAI has specific binding activity with liver cells SMC7721 and binding can be inhibited by native human ApoAI.

[Compared D-amino acid oxidase expression in different Pichia pastoris host strains].

To compare the DAAO expression level in different Pichia pastoris host strains, the gene encoding DAAO from Trigonopsis variabilis was cloned into plasmid pPIC3.5k and then transformed into P. pastoris GS115 and KM71 respectively. The positive transformants PDK13 (MutS) and PD27 (Mut+) were obtained by PCR analysis. Their optimal and different expression conditions were investigated. To compare with PD27, PDK13 was determined to poss a slower consumption of methanol, a longer induction time, a lower oxygen request and apparently higher expression of DAAO. The highest expression levels were reached up to 2700, 2500 IU/L in shaking flask and 10140, 8463.5 IU/L in fermentor respectively. The over-expression of DAAO can meet its large demand for production of 7-ACA, alpha-keto acid and L-amino acid. In addition, the phenylpyruvate and L-phenylalanine were obtained by crude DAAO reacting with DL-phenylalanine at 37 degrees C for 3h.