Treatment of acetaminophen-induced liver failure by blocking the death checkpoint protein TRAIL.

Acetaminophen (APAP) is one of the most commonly used drugs worldwide, and APAP-induced liver injury is the most frequent cause of acute liver failure in developed countries. However, the mechanisms of APAP-induced hepatotoxicity are not well understood, and treatment options for the disorder are very limited. Here, we show that TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a major mediator of APAP-induced liver injury in mice, and its blockade markedly ameliorates the liver failure. In APAP-treated mice, TRAIL was expressed in the liver, spleen, and peripheral blood primarily by CD11b+Gr1+ neutrophils. The concentration of soluble TRAIL in the blood, and the frequencies of TRAIL+ leukocytes in the spleen and liver positively correlated with the severity of liver injury. APAP sensitized hepatocytes to TRAIL-induced apoptosis by upregulating the expression of the TRAIL receptor DR5 (death receptor 5), presumably through its transcription factor CHOP (C/EBP homologous protein). Importantly, blocking TRAIL with a soluble DR5-Fc fusion protein (sDR5-Fc) significantly attenuated APAP-induced liver injury, the hepatic infiltration of leukocytes, the levels of inflammatory cytokines, and the mortality of mice. When administered alongside N-acetylcysteine, sDR5-Fc further protected against APAP-induced acute liver injury. Thus, the TRAIL-DR5 signaling pathway plays a key role in APAP-induced liver inflammation and failure, and its blockade represents an effective new strategy to treat the liver disease.

Preclinical studies of a death receptor 5 fusion protein that ameliorates acute liver failure.

Acute liver failure (ALF) is a life-threatening disease with a high mortality rate. There is an urgent need to develop new drugs with high efficacy and low toxicity. In this study, we produced a pharmaceutical-grade soluble death receptor 5 (sDR5)-Fc fusion protein for treating ALF and evaluated the pharmacology, safety, pharmacokinetics, efficacy, and mechanisms of sDR5-Fc in mice, rats, and cynomolgus monkeys. sDR5-Fc bound with high affinity to both human and monkey tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) effectively blocked TRAIL-induced apoptosis in vitro and significantly ameliorated ALF induced by concanavalin A (Con A) in mice. Mechanistically, sDR5-Fc inhibited hepatocyte death and reduced inflammation in vivo. Furthermore, sDR5-Fc attenuated the production of inflammatory cytokines by splenocytes activated with Con A or an anti-CD3 antibody in vitro. Consistent with these results, splenocytes from TRAIL-/- mice produced much lower levels of inflammatory cytokines than those from TRAIL+/+ mice. In cynomolgus monkeys, sDR5-Fc was safe and well tolerated when intravenously administered as a single dose of up to 1200 mg/kg or multiple doses of 100 mg/kg. After treatment with a single dose, linear pharmacokinetics with a mean half-life of > 1.9 days were observed. After 12 weekly doses, sDR5-Fc exposure increased in an approximately dose-proportional manner, and the mean accumulation ratio ranged from 1.82- to 2.11-fold. These results support further clinical development of our sDR5-Fc protein as the first TRAIL-targeting drug for ALF treatment. KEY MESSAGES: sDR5-Fc binds with high affinity to TRAIL to effectively block TRAIL-induced apoptosis. sDR5-Fc ameliorates Con A-induced acute liver failure in mice by inhibiting hepatocyte death and inflammation. sDR5-Fc or TRAIL knockout attenuates the production of inflammatory cytokines by activated splenocytes in vitro. sDR5-Fc is safe and well tolerated in acute or long-term toxicity study.

SYN023, a novel humanized monoclonal antibody cocktail, for post-exposure prophylaxis of rabies.

Rabies is a neglected zoonotic disease that is preventable in humans by appropriate post-exposure prophylaxis (PEP). However, current PEP relies on polyclonal immune globulin products purified from pooled human (HRIG) or equine (ERIG) plasma that are either in chronic shortage or in association with safety concerns. Here, we present the development of an antibody cocktail, SYN023, made of two novel monoclonal antibodies (MAb) CTB011 and CTB012 that could serve as safer and more cost-effective alternatives to the current RIG products. Both CTB011 and CTB012 are humanized MAbs that bind to non-overlapping epitopes on the rabies virus (RABV) glycoprotein (G) with sub-nanomolar affinities. Sequence analysis revealed that many of the critical residues in binding are highly conserved across different species of lyssaviruses. When combined at a 1:1 ratio, CTB011/CTB012 exhibited neutralization capabilities equivalent or superior to HRIG against 10 North American street RABV isolates in vitro and 15 prevalent Chinese RABV strains in animal models. Finally, SYN023, at a dosage of 0.03 mg/kg, was able to offer the same degree of protection as standard HRIG administration (20 IU/kg) in Syrian hamsters challenged with a highly virulent bat (Tadarida brasiliensis) RABV variant. Taken together, the high-potency and broad-spectrum neutralization demonstrated by SYN023 make it an effective candidate for human rabies PEP consideration.

NRG1-dependent activation of HER3 induces primary resistance to trastuzumab in HER2-overexpressing breast cancer cells.

This study was conducted to determine the role of neuregulin 1 (NRG1)-dependent human epidermal growth factor receptor 3 (HER3) activation in trastuzumab primary resistance, and to observe the inhibitory effect of HER3 monoclonal antibody on HER2-overexpressing breast cancer cells. BT474 cells (trastuzumab sensitive) and MDA-MB-453 cells (trastuzumab resistant) were first stimulated with NRG1 and then treated with either trastuzumab, HER3 antibody, or a combination of both. The expression of phospho human epidermal growth factor receptor 2 (p-HER2), phospho human epidermal growth factor receptor 3 (p-HER3), phospho protein kinase B (p-Akt) and phospho mitogen-activated protein kinase (p-MAPK) were detected by western blotting. Apoptosis was detected by flow cytometry. Cell viability was detected by MTT assay. Without NRG1 stimulation, trastuzumab treatment significantly down-regulated the expression of p-HER2, increased early apoptosis, and decreased cell viability in BT474 cells. After NRG1 stimulation, the aforementioned effects weakened or disappeared in the trastuzumab treatment group, whereas in the HER3 antibody treatment group, there was significant downregulation in p-HER3 expression and increase in early apoptosis of BT474 cells. In MDA-MB-453 cells, the HER3 antibody significantly downregulated both p-HER2 and p-HER3 and promoted early apoptosis after NRG1 stimulation, however, trastuzumab hardly played a role. p-Akt and p-MAPK were also significantly downregulated by the HER3 antibody after NRG1 stimulation. The expressions of p-HER2, p-HER3, p-Akt and p-MAPK were all downregulated after HER3 gene silencing, compared to the control. NRG1-dependent activation of HER3 induces primary resistance to trastuzumab in HER2-overexpressing breast cancer cells. HER3 monoclonal antibody combined with trastuzumab may serve as a treatment choice for patients with primary resistance to trastuzumab.

The anti-HER3 antibody in combination with trastuzumab exerts synergistic antitumor activity in HER2-positive gastric cancer.

The anti-HER2 monoclonal antibody trastuzumab is central to the treatment of HER2-positive gastric cancer (GC); however, its responses are limited. HER3 seems to be the preferred dimerization partner with HER2 and is emerging as a key target for complete blockade of downstream pathways and better clinical response. In this study, we report that novel anti-HER3 antibodies (1A5-3D4) that can neutralize multiple modes of HER3 activation, combined with trastuzumab, exhibited synergistic inhibitory effect on the cell proliferation in HER2-positive GC cell lines. Follow-up studies revealed that the combination treatment significantly inhibited phosphorylation of HER3 as well as AKT and ERK signals. In vivo experiments further showed that the anti-tumor effect of trastuzumab was enhanced by its combination with 1A5-3D4 in NCI-N87 xenograft and patient derived xenografts (PDX). Particularly in an HER2-negative whereas neuregulin1 (a ligand of HER3) positive PDX, the combination was also superior to monotherapy. 1A5-3D4 in combination with trastuzumab exhibits a synergistic inhibitory effect on tumor activity, suggesting that targeting both HER2 and HER3 resulted in an improved treatment effects on HER2-positive GC.

[Inhibitory effect of anti-type IV collagenase intrabody on invasiveness of human pulmonary giant cell carcinoma PG cells in vitro].

OBJECTIVE: To explore the inhibitory effects of endoplasmic reticulum-retained intrabody on the secretion of type IV collagenase and the invasion of human pulmonary giant cell carcinoma PG cells in vitro. METHODS: Two expression plasmids were constructed, pcDNA3.1-CP.scFv and pcDNA3.1-ER.scFv encoding cytoplasm-retained and endoplasmic reticulum-retained single chain antibodies against the type IV collagenase, respectively. The intracellular antibody genes were transfected into the human pulmonary giant cell carcinoma PG cells. Western blot was performed to detect the expression of pcDNA3.1-CP.scFv and pcDNA3.1-ER.scFv. Gelatin zymography was performed to detect seretion of type IV collagenase in PG cells and Matrigel assay was employed for determination of the cell invasiveness. RESULTS: Both of cytoplasm-retained and endoplasmic reticulum-retained introbodies, CP.scFv and ER.scFv, were expressed in PG cells. ER.scFv, significantly inhibited the secretion of type IV collegenase. As shown, matrix metalloproteinase 9 and matrix metalloproteinase 2 were inhibited by 85.7% and by 51.2%, respectively. However, CP.scFv did not show such inhibitory effect. The ER.scFv encoding gene-transfected PG cells were much less invasive than parental or vector control cells, the inhibition rate was 76.3% (P < 0.05), whereas CP.scFv encoding gene-transfected PG cells showed no reduction in invasiveness. CONCLUSION: Those findings demonstrate that endoplasmic reticulum (ER)-retained intracellular antibody technology may selectively abrogate the activity of type IV collagenase in the protein trafficking and secretory pathway and effectively inhibit tumor cell invasion in vitro. Anti-type IV collagenase intrabody may be further used in cancer gene therapy.

[Inhibitory effect of endoplasmic reticulum-retained anti-type IV collagenase intrabody on invasion and proliferation of cancer cell].

BACKGROUND & OBJECTIVE: Invasion and metastasis are significant characteristics of cancer cells, Type IV collagenase (matrix metalloproteinase-2, and-9) plays an important role in cancer invasion and metastasis. This study was designed to block the secretion of type IV collagenase via intracellular antibody (intrabody) methods, and inhibit cancer invasion and metastasis. METHODS: We constructed expression plasmid pcDNA3.1-ER.scFv coding for an endoplasmic reticulum (ER)- retained, single chain antibody (ER.scFv) against the type IV collagenase. The intrabody gene was transfected into human giant cell pulmonary carcinoma PG cells. Western blot was performed to detect the expression of pcDNA3.1-ER.scFv. Co-immunoprecipitation was used to analyse the interaction between ER. scFv and target protein in PG cells. The secretion of type IVcollagenase was detected by gelatin zymography. Cell behavior was examined by invasion and proliferation assay in vitro. RESULTS: ER. scFv expressed in PG cells, and can recognize and combine its cognate target protein matrix metalloproteinase-9. Intrabody gene transfection significantly blocked the function and activity of type IV collegenase. The ER.scFv-transfected PG cells were less invasive than parental or vector control cells, the suppression rate of ER.scFv-transfected PG cells was 76.3%(P< 0.05). ER. scFv showed anti-proliferative effect on PG cells cultured on Matrigel. CONCLUSIONS: ER-retained intrabody technology may restrain the activity of type IV collagenase in protein processing and secretory pathway, and inhibits cancer cell invasion and proliferation. Anti-type IV collagenase intrabody may be further used in cancer gene therapy.