Bispecific antibody targeting both B7-H3 and PD-L1 exhibits superior antitumor activities.

Clinical application of PD-1 and PD-L1 monoclonal antibodies (mAbs) is hindered by their relatively low response rates and the occurrence of drug resistance. Co-expression of B7-H3 with PD-L1 has been found in various solid tumors, and combination therapies that target both PD-1/PD-L1 and B7-H3 pathways may provide  additional therapeutic benefits. Up to today, however, no bispecific antibodies targeting both PD-1 and B7-H3 have reached the clinical development stage. In this study, we generated a stable B7-H3×PD-L1 bispecific antibody (BsAb) in IgG1-VHH format by coupling a humanized IgG1 mAb against PD-L1 with a humanized camelus variable domain of the heavy-chain of heavy-chain antibody (VHH) against human B7-H3. The BsAb exhibited favorable thermostability, efficient T cell activation, IFN-γ production, and antibody-dependent cell-mediated cytotoxicity (ADCC). In a PBMC humanized A375 xenogeneic tumor model, treatment with BsAb (10 mg/kg, i.p., twice a week for 6 weeks) showed enhanced antitumor activities compared to monotherapies and, to some degree, combination therapies. Our results suggest that targeting both PD-1 and B7-H3 with BsAbs increases their specificities to B7-H3 and PD-L1 double-positive tumors and induces a synergetic effect. We conclude that B7-H3×PD-L1 BsAb is favored over mAbs and possibly combination therapies in treating B7-H3 and PD-L1 double-positive tumors.

Thrombospondin 1 Promotes Endoplasmic Reticulum Stress and Apoptosis in HK-2 Cells by Upregulating ATF6-CHOP.

OBJECTIVE: The goal of this study is to investigate the role and mechanism of endoplasmic reticulum stress and apoptosis regulated by thrombospondin 1 (TSP1) in human renal tubular epithelial cells (HK-2 cells). METHODS: HK-2 cells were exposed to high concentrations of glucose (HG). The endoplasmic reticulum stress inhibitor 4-phenylbutyric acid (4-PBA) was administered by transfecting TSP1 or an empty vector to explore the mechanism of the endoplasmic reticulum response regulated by TSP1 and stress in renal cell apoptosis. The effects of TSP1 and 4-PBA on the proliferation and apoptosis of HK-2 cells under HG conditions were assessed using Cell counting kit-8 and flow cytometry. Western blotting was used to detect the apoptosis- and endoplasmic reticulum stress-related protein expression regulated by TSP1 and 4-PBA. RESULTS: HG treatment induced high cell apoptosis, abundantly expressed TSP1 level and restrained viability in HK-2 cells. Overexpression of TSP1 significantly inhibited the proliferation of and facilitated apoptosis of HK-2 cells under HG conditions. Administration of endoplasmic reticulum stress inhibitor 4-PBA after overexpression of TSP1 antagonized the inhibitory proliferation and promoted apoptosis rate in HG-triggered HK-2 cells induced by TSP1 overexpression. 4-PBA treatment significantly hindered the expression of endoplasmic reticulum stress markers, such as PERK, ATF4, ATF6, p-eIF2α, IRE1, CHOP and XBP1, suggesting that the administration of 4-PBA was successful. CONCLUSION: Overexpression of TSP1 activated endoplasmic reticulum stress by regulating the ATF6-CHOP axis. TSP1 restrained cell proliferation, and promoted apoptosis and endoplasmic reticulum stress by activating the ATF6-CHOP axis.

[Expression of a thermostable a-amylase mutant into Escherichia coli and Pichia pastoris].

Alpha-amylase are of considerable commercial value. It can be produced by a wide variety of microorganisma. The alpha-amylase gene (amyE) from Bacillus licheniformis, which is widely used for the industrial hydrolysis of starch, was mutated (amyEM), then amplified by PCR and inserted into pBV220 and pPIC9k to obtain the recombinant vector pBV220-amyEM and pPIC9k-amyEM. These recombinant vectors were transformed into corresponding competent cell E. coli DH5alpha and P. pastoris GS115 respectively. The resulting recombinant strains, DH5alpha/pBV220-amyEM and GS115/ pPIC9k-amyEM, were then screened by measuring the enzymatic activity and SDS-PAGE. DH5alpha/pBV220-amyEM was induced by temperature and GS115/pPIC9k-amyEM by methanol. In contrast to the parent cells, the a-amylases were expressed in both the recombinant strains. In E. coli the molecular weight was approximately 55kDa; optimal temperature and pH of the recombinant a-amylase were 80 degrees C - 90 degrees C and 6.0 respectively. The recombinant amylase had high activity in pH 5.0 - 5.5 compared to wild type. In Pichia pastoris, the recombinant amylase was secreted to the medium; molecular weight was 60kDa for the putative post-translational modifications; optimal pH shifted to 5.5. The specific activities of alpha-amylase produced by E. coli and P. pastoris were 8.1U/mg and 102U/mg respectively. This result indicated that the alpha-amylase were secreted into the culture medium with high efficiency in the recombinant P. pastoris High activity in high temperature and lower pH properties impart the recombinant amylase potential applications in industry.

[Purification and properties of recombinant extremely thermostable and acid-stable amylase].

Extremely thermostable and acid-stable a-amylase produced by Pichia pastoris GS115/pPIC9K-Amy-228 was purified to electrophoretic homogeneity by the steps of ultrafiltration and PAGE. Purification of about 11.7 fold was achieved with an overall yield of 29.8%. Its molecular weight was estimated to be about 55kD by SDS-PAGE. The isoelectric point was 5.0 (room temperature). Michaelis constant of the enzyme for soluble starch was 1.12g/L. The carbohydrate content was 15.4% by the phenol-sulfuric acid method. The optimum temperature and pH of the enzyme activity were 95 degrees C and 4.5 respectively. The enzyme activity was stable under room temperature in the pH rang of 4.0 - 7.0 for 48 hours. About 60% of the initial enzyme activity was measured after 1h of incubation at 110 degrees C. The activity was strongly inhibited by Fe2+, Cr2+ and Cu2+, While Ca2+ had no effect on it. DTT and EDTA had no effect on the activity.

Nano-gel containing thermo-responsive microspheres with fast response rate owing to hierarchical phase-transition mechanism.

A new strategy is developed in this study to achieve thermo-responsive microspheres with fast response rates by designing a hierarchical phase-transition mechanism. The proposed thermo-responsive microspheres are composed of poly(N-isopropylacrylamide-co-acrylic acid) (PNA) microsphere matrixes and embedded poly(N-isopropylacrylamide) (PNIPAM) nano-gels, which have different volume phase-transition temperatures (VPTTs). The VPTT of PNIPAM nano-gels (VPTT(1)) is lower than that of PNA microsphere matrixes (VPTT(2)). Upon heating-up, the temperature increases across the VPTT(1) first and then the VPTT(2), as a result the PNIPAM nano-gels shrink earlier than the PNA microsphere matrixes. Upon cooling-down, the temperature decreases across the VPTT(2) first and then the VPTT(1), as a result the PNA microsphere matrixes swell earlier than the PNIPAM nano-gels. Consequently, large amounts of voids and channels form around the nano-gels inside the microsphere matrixes when the temperature changes across the range between VPTT(1) and VPTT(2), which are beneficial to the enhancement of water transport rate inside the microsphere matrixes. The experimental results show that, compared with normal homogeneous PNA (N-PNA) microspheres, the nano-gel containing PNA (C-PNA) microspheres exhibit remarkably fast response rate due to the hierarchical phase-transition mechanism attributed to different VPTT values of the embedded nano-gels and the microsphere matrixes.

[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.