Characterization of a novel bispecific antibody targeting tissue factor-positive tumors with T cell engagement.

T cell engaging bispecific antibody (TCB) is an effective immunotherapy for cancer treatment. Through co-targeting CD3 and tumor-associated antigen (TAA), TCB can redirect CD3+ T cells to eliminate tumor cells regardless of the specificity of T cell receptor. Tissue factor (TF) is a TAA that involved in tumor progression. Here, we designed and characterized a novel TCB targeting TF (TF-TCB) for the treatment of TF-positive tumors. In vitro, robust T cell activation, tumor cell lysis and T cell proliferation were induced by TF-TCB. The tumor cell lysis activity was dependent upon both CD3 and TF binding moieties of the TF-TCB, and was related to TF expression level of tumor cells. In vivo, in both tumor cell/human peripheral blood mononuclear cells (PBMC) co-grafting model and established tumor models with poor T cell infiltration, tumor growth was strongly inhibited by TF-TCB. T cell infiltration into tumors was induced during the treatment. Furthermore, efficacy of TF-TCB was further improved by combination with immune checkpoint inhibitors. For the first time, our results validated the feasibility of using TF as a target for TCB and highlighted the potential for TF-TCB to demonstrate efficacy in solid tumor treatment.

A Novel Bispecific Antibody Targeting CD3 and Lewis Y with Potent Therapeutic Efficacy against Gastric Cancer.

Lewis Y antigen, a glycan highly expressed on most epithelial cancers, was targeted for cancer treatment but lacked satisfactory results in some intractable and refractory cancers. Thus, it is highly desirable to develop an effective therapy against these cancers, hopefully based on this target. In this work, we constructed a novel T cell-engaging bispecific antibody targeting Lewis Y and CD3 (m3s193 BsAb) with the IgG-[L]-scfv format. In vitro activity of m3s193 BsAb was evaluated by affinity assay to target cells, cytotoxicity assay, cytokines releasing assay, and T cells proliferation and recruiting assays. Anti-tumor activity against gastric cancer was evaluated in vivo by subcutaneous huPBMCs/tumor cells co-grafting model and huPBMCs intravenous injecting model. In vitro, m3s193 BsAb appeared to have a high binding affinity to Lewis Y positive cells and Jurkat cells. The BsAb showed stronger activity than its parent mAb in T cell recruiting, activation, proliferation, cytokine release, and cytotoxicity. In vivo, m3s193 BsAb not only demonstrated higher therapeutic efficacy in the huPBMCs/tumor co-grafting gastric carcinoma model than the parent mAb but also eliminated tumors in the model of intravenous injection with huPBMCs. Strong anti-tumor activity of m3s193 BsAb revealed that Lewis Y could be targeted in T cell-engaging BsAb for gastric cancer therapy.

A Rational Designed Novel Bispecific Antibody for the Treatment of GBM.

Epidermal growth factor receptor variant III (EGFRvIII) is highly and specifically expressed in a subset of lethal glioblastoma (GBM), making the receptor a unique therapeutic target for GBM. Recently, bispecific antibodies (BsAbs) have shown exciting clinical benefits in cancer immunotherapy. Here, we report remarkable results for GBM treatment with a BsAb constructed by the "BAPTS" method. The BsAb was characterized through LC/MS, SEC-HPLC, and SPR. Furthermore, the BsAb was evaluated in vitro for bioactivities through FACS, antigen-dependent T-cell-mediated cytotoxicity, and a cytokine secretion assay, as well as in vivo for antitumor activity and pharmacokinetic (PK) parameters through immunodeficient NOD/SCID and BALB/c mouse models. The results indicated that the EGFRvIII-BsAb eliminated EGFRvIII-positive GBM cells by recruiting and stimulating effector T cells secreting cytotoxic cytokines that killed GBM cells in vitro. The results demonstrated the antitumor potential and long circulation time of EGFRvIII-BsAb in NOD/SCID mice bearing de2-7 subcutaneously heterotopic transplantation tumors and BALB/c mice. In conclusion, our experiments in both in vitro and in vivo have shown the remarkable antitumor activities of EGFRvIII-BsAb, highlighting its potential in clinical applications for the treatment of GBM. Additional merits, including a long circulation time and low immunogenicity, have also made the novel BsAb a promising therapeutic candidate.

A novel bispecific antibody targeting CD3 and prolactin receptor (PRLR) against PRLR-expression breast cancer.

BACKGROUND: Prolactin receptor (PRLR) is highly expressed in a subset of human breast cancer and prostate cancer, which makes it a potential target for cancer treatment. In clinical trials, the blockade of PRLR was shown to be safe but with poor efficacy. It is therefore urgent to develop new therapies against PRLR target. Bispecific antibodies (BsAbs) could guide immune cells toward tumor cells, and produced remarkable effects in some cancers. METHODS: In this study, a bispecific antibody targeting both tumor antigen PRLR and T cell surface CD3 antigen (PRLR-DbsAb) was constructed by split intein mediated protein transsplicing (BAPTS) system for the first time. Its binding activity was determined by Biacore and Flow cytometry, and target-dependent T cell mediated cytotoxicity was detected using LDH release assay. ELISA was utilized to study the secretion of cytokines by immune cells. Subcutaneous tumor mouse models were used to analyze the in vivo anti-tumor effects of PRLR-DbsAb. RESULTS: PRLR-DbsAb in vitro could recruit and activate T cells to promote the release of Th1 cytokines IFN- γ and TNF- α, which could kill PRLR expressed breast cancer cells. In xenograft models with breast cancer cell line T47D, NOD/SCID mice intraperitoneally injected with PRLR-DbsAb exhibited significant inhibition of tumor growth and a longer survival compared to mice treated with PRLR monoclonal antibody (PRLR mAb). CONCLUSIONS: Both in vitro and in vivo experiments showed PRLR-DbsAb had a potential therapy of cancer treatment potential therapy for cancer. Immunotherapy may be a promising treatment against the tumor target of PRLR.

Naturally split intein Npu DnaE mediated rapid generation of bispecific IgG antibodies.

High product purity, preserving natural IgG architecture, and excellent production efficiency are highly desirable in bispecific antibody manufacturing. We have reported a platform called Bispecific Antibody by Protein Trans-Splicing (BAPTS) to synthesize BsAbs with natural human IgG structure and no chain mispairing. In the method, two antibody fragments carrying different target-specificities are separately expressed in mammalian cells and subsequently fused to form BsAbs by utilizing the trans-splicing property of the split intein Npu DnaE. The hinge region of antibody, a region with less functional impact, is selected for conjugating the two fragments. The method involves the following steps: (i) constructing five plasmids coding antibody components; (ii) separately expressing and purifying two antibody fragments A and B. Fragment A contains one Fab, "Knobs-into-Holes" mutations in the CH3 domain and NPU DnaEC. Fragment B contains another Fab and NPU DnaEN; (iii) mixing of fragments A and B under permissive reducing conditions in vitro to enable trans-splicing reaction; (iv) removing the reductant to allow re-oxidation of disulfide bonds; (v) isolating BsAb product from unreacted precursors by affinity chromatography. The method allows correct assembly of two heavy and two light chains to form bispecific IgG antibodies in natural structure with no synthetic linkers. No chain mispairing was observed in the product by UPLC-MASS. In addition, the observed kinetics and low reaction activation energy confirmed that the trans-splicing is thermodynamically favored reaction. The BAPTS technology is feasible for industrial applications.

Enhanced production of anti-PD1 antibody in CHO cells through transient co-transfection with anti-apoptotic genes Bcl-x L and Mcl-1.

Apoptosis has a negative impact on the cell survival state during cell cultivation. To optimize mammalian cell culture for production of biopharmaceuticals, one of the important approaches is to extend cell life through over-expression of anti-apoptotic genes. Here, we reported a cost-effective process to enhance cell survival and production of an antibody through transient co-transfection with anti-apoptotic genes Bcl-x L or Mcl-1 in Chinese hamster ovary (CHO) cells with polyethylenimine (PEI). Under the optimal conditions, it showed reduced levels of apoptosis and improved cell viability after co-transfected with Bcl-x L or Mcl-1. The overall production yield of the antibody anti-PD1 increased approximately 82% in CHO cells co-transfected with Bcl-x L , and 34% in CHO cells co-transfected with Mcl-1. This work provides an effective way to increase viability of host cells through delaying apoptosis onset, thus, raise production yield of biopharmaceuticals without the process of generating stable cell lines and subsequent screening.

The mTOR promotes oxidative stress-induced apoptosis of mesangial cells in diabetic nephropathy.

Glomerular mesangial cell (MC) apoptosis is one of the important mechanisms of glomerulosclerosis, which induces an increased severity of albuminuria and promotes the development of diabetic nephropathy (DN). However, the mechanism by which high glucose (HG) induces MCs apoptosis is not fully understood. In the present study, we investigated the effects of mTOR signalling on apoptosis in cultured MCs exposed to HG and in type I diabetes, and tried to clarify the specific mechanisms underlying these effects. In vitro, exposure of MCs to HG stimulated ROS production, decreased the antioxidant enzyme superoxide dismutase (SOD) activity and glutathione (GSH) level, increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, upregulated P53 expression and Bax/Bcl-2 ratio and enhanced cleavage of caspase 3, resulting in an increase in programmed cell death. Pretreatment of the cells with rapamycin ameliorated oxidative stress, reduced the number of apoptotic cells induced by HG and caused the downstream effects of mTOR activation. In vivo, compared with control rats, diabetic rats had more apoptotic cells in glomeruli. Induction of diabetes increased the level of MDA and NADPH oxidase activity, decreased the SOD activity and GSH level, elevated the Bax/Bcl ratio and P53 expression and activated caspase 3. mTOR inhibitor rapamycin treatment prevented these changes further alleviated albuminuria and improved renal function. Taken together, our data suggest that mTOR plays a key role in mediating ROS-induced MC apoptosis in diabetic nephropathy, and these effects have been associated with the promotion of ROS production by upregulating the antioxidant enzyme and downregulating the NADPH oxidase activity.

Up-regulation of Tight-Junction Proteins by p38 Mitogen-Activated Protein Kinase/p53 Inhibition Leads to a Reduction of Injury to the Intestinal Mucosal Barrier in Severe Acute Pancreatitis.

OBJECTIVES: The aim of this study was to explore the role of the p38 mitogen-activated protein kinase (p38MAPK)/p53 signaling pathway in injury to the intestinal mucosal barrier during severe acute pancreatitis (SAP). METHODS: Both sham operation and SAP groups had 3 subgroups analyzed 3, 6, or 12 hours after the SAP induction. The concentrations of amylase, endotoxin, diamine oxidase, tumor necrosis factor α, and phospho-p38MAPK, p53, and caspase-3 and the messenger RNA levels of zonula occludens protein-1 and occludin in the intestine were measured. Immunohistochemical staining was used to determine the expression of zonula occludens protein-1 and occludin. Pathological changes of the pancreas and intestine were also assessed. Then, rats were randomly assigned to 5 groups-sham operation group, SAP group, 3 groups treated with different concentrations of p38MAPK-inhibitor SB203580-and the abovementioned experiment was repeated and analyzed 6 hours after the SAP induction. RESULTS: The phospho-p38MAPK reached a peak value at 6 hours after the SAP induction with obvious pathological injury to the pancreas and intestine. Treatment with SB203580 led to a less damage to the pancreatic and intestinal tissues. CONCLUSIONS: These results suggest that SAP activates the p38MAPK/p53 signaling pathway and induces injury to the intestinal mucosal barrier, which can be alleviated by inhibiting the p38MAPK/p53 pathway.

Ethanolic Ginkgo biloba leaf extract prevents renal fibrosis through Akt/mTOR signaling in diabetic nephropathy.

BACKGROUND: Recently, extract of Ginkgo biloba leaves (GbE) have become widely known phytomedicines and have shown various pharmacological activities, including improvement of blood circulation, protection of oxidative cell damage, prevention of Alzheimer's disease, treatment of cardiovascular disease and diabetes complications. This study was designed to investigate the effects of an ethanolic GbE on renal fibrosis in diabetic nephropathy (DN) and to clarify the possible mechanism by which GbE prevents renal fibrosis. STUDY DESIGN: We investigated the protective effects of GbE on renal fibrosis in STZ-induced diabetic rats. Rats were randomized into six groups termed normal control, diabetes mellitus, low dose of GbE (50 mg/kg/d), intermediate dose of GbE (100 mg/kg/d), high dose of GbE (200 mg/kg/d) and rapamycin (1 mg/kg/d). METHODS: After 12 weeks, the rats were sacrificed and then fasting blood glucose (FBG), creatinine (Cr), blood urea nitrogen (BUN), urine protein, relative kidney weight, glycogen and collagen accumulation, and collagen IV and laminin expression were measured by different methods. The amounts of E-cadherin, α-SMA and snail, as well as the phosphorylation of Akt, mTOR and p70S6K in the renal cortex of rats, were examined by western blotting. RESULTS: Compared with diabetic rats, the levels of Cr, BUN, urine protein, relative kidney weight, accumulation of glycogen and collagen, and expression of collagen IV and laminin in the renal cortex were all decreased in GbE treated rats. In addition, GbE reduced the expression of E-cadherin, α-SMA, snail and the phosphorylation of Akt, mTOR and p70S6K in diabetic renal cortex. CONCLUSION: GbE can prevent renal fibrosis in rats with diabetic nephropathy, which is most likely to be associated with its abilities to inhibit the Akt/mTOR signaling pathway.

Quercetin inhibits the mTORC1/p70S6K signaling-mediated renal tubular epithelial-mesenchymal transition and renal fibrosis in diabetic nephropathy.

Quercetin is a classic flavonoid that inhibits the epithelial-mesenchymal transition (EMT) of tumor cells. However, the effects of quercetin on the EMT of renal tubular epithelial cells, a potential mechanism of renal fibrosis and important characteristic of diabetic nephropathy (DN), remain largely unknown. In the present study, we investigated the effects of quercetin on the EMT of two lines of renal tubular proximal epithelial cells (HK-2 and NRK-52E) induced with high glucose and renal fibrosis resulting from type 1 diabetes and tried to clarify the specific mechanisms underlying these effects. The in vitro results showed that the EMT of HK-2 and NRK-52E cells was induced by high glucose, and mTORC1/p70S6K was highly activated in these two cell lines cultured under high glucose. Quercetin effectively ameliorated the high glucose-induced EMT of HK-2 and NRK-52E cells and inhibited the activation of mTORC1/p70S6K. In vivo, diabetic rats showed a significant decline in renal function and severe renal fibrosis at 14 weeks after STZ injection. Furthermore, mTORC1/p70S6K was activated in the renal cortex of diabetic rats. Treatment with quercetin alleviated the decline in renal function, and the progression of renal fibrosis and inhibited mTORC1/p70S6K activation in the diabetic renal cortex. In addition, we examined the protein and mRNA levels of four transcriptional factors (snail, slug, twist and ZEB-1), which regulate E-cadherin expression at the transcriptional level both in vivo and in vitro. The results revealed that the elevated expression of snail and twist in HK-2 and NRK-52E cells cultured under high glucose and in the renal cortex of diabetic rats was inhibited by quercetin. These results demonstrated that quercetin ameliorates the EMT of HK-2 and NRK-52E cells induced by high glucose and renal fibrosis induced by diabetes, and these effects have been associated with the inhibition of the two transcriptional factors (snail and twist) and the activation of mTORC1/p70S6K.