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Recently, antibody-based therapeutic agents are becoming most leading biologics for treating many diseases, especially for cancer. However, large-scale application of antibody drugs is still hampered by high cost and complex technical process. Endogenous expression of proteins or antibodies can be achieved by applying in vitro transcription (IVT) technique to produce mRNA and then deliver into body, which supplies opportunity to avoid many disadvantages in antibody production as well as clinical applications.Here, we designed the IVT-mRNA encoding the Pembrolizumab, as a commercial anti-PD-1 monoclonal antibody (mAb). The in vitro functional properties and in vivo antitumor activities of the Pembrolizumab expressed from mRNA were both assessed. Maximized expression level of the Pembrolizumab from IVT-mRNA was achieved via optimizing the usage of signal peptide and molar ratio of heavy/light chain. Then the mRNA was further formulated by lipid nanoparticle (LNP), which enable efficient in vivo delivery and protect mRNA from degradation. Intravenously delivered the single dose of mRNA-LNPs in mice resulted in duration of serum Pembrolizumab level over 25 μg/mL more than 35 days. Pharmacokinetic study exhibited significantly enhanced drug exposure of mRNA-encoded mAbs compared with direct injection of Pembrolizumab at same dose. Chronic treatment of the tumor-bearing mice with LNP-encapsulated Pembrolizumab mRNA effectively downregulated the growth of intestinal tumors and improved the animal survival. In brief, our present research demonstrated that the application of LNP-encapsulated IVT-mRNA can change the human body into a protein drug manufacturing site to express full-size mAbs for treating cancer and hold potential to be a novel alternative to protein-based therapies.

Background: Intra-abdominal infection after curative surgery for colorectal cancer is a serious complication associated with an increased risk of recurrence. Lipopolysaccharides (LPS) -an essential component of the cell wall of Gram-negative bacteria -were found to exert a protumorigenic effect by stimulating the in ammatory pathology and formation of neutrophil extracellular traps (NETs). This study was conducted to test whether LPS-induced formation of NETs promotes the development of cancer and metastasis.Methods: The clinical characteristics, incidence of relapse, and serum myeloperoxidase-DNA complexes of 40 patients with infection and 40 patients without infection after curative surgery were analyzed. The effects of LPS on the induction of NETs were evaluated in a mouse model of colorectal cancer and liver metastasis. The toll-like receptor 9 (TLR9) -a DNA receptor -was knocked down to assess its effect on the mitogen-activated protein kinase pathway and activities implicated in the formation of NETs.Results: Analysis of the clinical data obtained from these patients showed the signi cant relation of the formation of NETs and incidence of metastasis and survival rates. Subsequent in-vitro experiments revealed an increased level of citrullinated-histone H3 and myeloperoxidase-DNA in LPS-injected mice with colorectal cancer. In the mimic metastatic model, injection of LPS enhanced the metastatic capacity, which was then attenuated by DNase I. This suggested that the formation of NETs was activated by LPS.Injection of TLR9-knockdown HCT116 cells in mice, followed by induction through LPS, mitigated the level of citrullinated-histone H3 and myeloperoxidase-DNA. This nding implied that the formation of NETs was suppressed.Conclusion: These ndings shed light on the mechanism underlying the relationship between the elevated rate of colorectal cancer recurrence in patients who underwent surgery and the incidence of infection. This mechanism involves the protumorigenic activities of LPS-induced formation of NETs. The NETs which could be mediated by the TLR9 and the mitogen-activated protein kinase signaling pathway.

Hepatocarcinoma is one of the most lethal malignancy haunting the Chinese population, which is partially due to the difficulties in diagnosis at an early stage. The search for a biomarker that could signify the presence and progress of hepatocarcinoma is never ended. MicroRNAs are 22-nt RNAs that could bind to 3' UTR of target mRNAs, mediating degradation of mRNAs or inhibiting the translation. Although much has been investigated, the role of miR-124 in hepatocarcinoma remained elusive. We first detected aberrant expression level of miR-124 in HCC tissues of 112 patients. By exploring the clinical parameters, we found a significantly inverse correlation between miR-124 level and TNM stages. Consistent with this, the survival analysis indicated the association of low miR-124 with longer survival time. Subsequent forced expression miR-124 resulted in reduced cell viability of Hep3B and SMMC-7221, which cell lines have high and low background expression of miR-124, respectively. TargetScan prediction rendered a subset of target candidates, which were selected for experimental validation, KLF4 was subject to luciferase assay. Ectopic expression of KLF4 increased the sphere formation ability and CD44/133-positive cell numbers, which can be reversed by abundant expression of miR-124, suggesting that KLF4 is a functional target of miR-124 in tumourigenesis and cancer progression of HCC.

The potential of antibodies, especially for the bispecific antibodies, are limited by high cost and complex technical process of development and manufacturing. A cost-effective and rapid platform for the endogenous antibodies expression via using the in vitro transcription (IVT) technique to produce nucleoside-modified mRNA and then encapsulated into lipid nanoparticle (LNP) may turn the body to a manufactory. Coinhibitory pathway of programmed death ligand 1 (PD-L1) and programmed cell death protein 1 receptor (PD-1) could suppress the T-cell mediated immunity. We hypothesized that the coblocking of PD-L1 and PD-1 via bispecific antibodies may achieve more potential antitumor efficacies compare with the monospecific ones. Here, we described the application of mRNA to encode a bispecific antibody with ablated Fc immune effector functions that targets both human PD-L1 and PD-1, termed XA-1, which was further assessed the in vitro functional activities and in vivo antitumor efficacies. The in vitro mRNA-encoded XA-1 held comparable abilities to fully block the PD-1/PD-L1 pathway as well as to enhance functional T cell activation compared to XA-1 protein from CHO cell source. Pharmacokinetic tests showed enhanced area under curve (AUC) of mRNA-encoded XA-1 compared with XA-1 at same dose. Chronic treatment of LNP-encapsulated XA-1 mRNA in the mouse tumor models which were reconstituted with human immune cells effectively induced promising antitumor efficacies compared to XA-1 protein. Current results collectively demonstrated that LNP-encapsulated mRNA represents the viable delivery platform for treating cancer and hold potential to be applied in the treatment of many diseases. Abbreviations: IVT: in vitro transcription; LNP: lipid nanoparticle; hPD-1: human PD-1; hPD-L1: human PD-L1; ITS-G: Insulin-Transferrin-Selenium; Pen/Strep: penicillin-streptomycin; FBS: fetal bovine serum; TGI: tumor growth inhibition; IE1: cytomegalovirus immediate early 1; SP: signal peptide; hIgLC: human immunoglobulin kappa light chain; hIgHC: human IgG1 heavy chain; AUC: area under the curve; Cl: serum clearance; Vss: steady-state distributed volume; MLR: mixed lymphocyte reaction.