Tumor-targeting engineered probiotic Escherichia coli Nissle 1917 inhibits colorectal tumorigenesis and modulates gut microbiota homeostasis in mice.

AIMS: Preliminary studies have identified the use of probiotics as a potential treatment strategy against colorectal cancer (CRC). However, natural probiotics lack direct tumor-targeting and tumor-killing activity in the intestine. This study aimed to construct a tumor-targeting engineered probiotic to combat CRC. MAIN METHODS: Standard adhesion assay was performed to analyze the adherence ability of tumor-binding protein HlpA to CT26 cells. CCK-8 assay, Hoechst 33258 staining and flow cytometry analysis were used for examining cytotoxicity of tumoricidal protein azurin toward CT26 cells. An engineered probiotic Ep-AH harboring azurin and hlpA genes was developed using Escherichia coli Nissle 1917 (EcN) chassis. Antitumor effects of Ep-AH were evaluated in the azoxymethane (AOM) and dextran sodium sulfate salt (DSS)-induced CRC mice. Moreover, analysis of gut microbiota was conducted via fecal 16S rRNA gene sequencing and shotgun metagenomic sequencing. KEY FINDINGS: Azurin caused a dose-dependent increase of apoptosis in CT26 cells. Ep-AH treatment reversed weight loss (p < 0.001), fecal occult blood (p < 0.01), and shortening of colon length (p < 0.001) than model group, as well as reducing tumorigenesis by 36 % (p < 0.001). Both Ep-H and Ep-A (EcN expressing HlpA or azurin) were less effective than Ep-AH. Furthermore, Ep-AH enriched the members of beneficial bacteria (e.g., Blautia and Bifidobacterium) and reversed abnormal changes of genes associated with several metabolic pathways (e.g., lipopolysaccharide biosynthesis). SIGNIFICANCE: These results demonstrated that Ep-AH had excellent therapeutic benefits on cancer remission and gut microbiota modulation. Our study provides an effective strategy for anti-CRC treatment.

A first-in-class, first-in-human, phase I trial of p28, a non-HDM2-mediated peptide inhibitor of p53 ubiquitination in patients with advanced solid tumours.

BACKGROUND: This first-in-human, phase I clinical trial of p28 (NSC745104), a 28-amino-acid fragment of the cupredoxin azurin, investigated the safety, tolerability, pharmacokinetics and preliminary activity of p28 in patients with p53(+) metastatic solid tumours. METHODS: A total of 15 patients were administered p28 i.v. as a short infusion three times per week for 4 weeks followed by a 2-week rest under an accelerated titration 3+3 dose escalation design until either a grade 3-related adverse event occurred or the maximum tolerated dose (MTD) was reached. Single-dose and steady-state serum pharmacokinetics were characterised. Assessments included toxicity, best objective response by RECIST 1.1 Criteria, and overall survival. RESULTS: No patients exhibited any dose-limiting toxicities (DLTs), significant adverse events or exhibited an immune response (IgG) to the peptide. The No Observed Adverse Effect Level (NOAEL) and MTD were not reached. Seven patients demonstrated stable disease for 7-61 weeks, three a partial response for 44-125 weeks, and one a complete response for 139 weeks. Three patients are still alive at 158, 140, and 110 weeks post therapy completion. CONCLUSION: p28 was tolerated with no significant adverse events. An MTD was not reached. Evidence of anti-tumour activity indicates a highly favourable therapeutic index and demonstrates proof of concept for this new class of non-HDM2-mediated peptide inhibitors of p53 ubiquitination.

Preclinical pharmacokinetics, metabolism, and toxicity of azurin-p28 (NSC745104) a peptide inhibitor of p53 ubiquitination.

PURPOSE: Characterize the preclinical pharmacokinetics, metabolic profile, multi-species toxicology, and antitumor efficacy of azurin-p28 (NSC 745104), an amphipathic, 28 amino acid fragment (aa 50-77) of the copper containing redox protein azurin that preferentially enters cancer cells and is currently under development for treatment of p53-positive solid tumors. METHODS: An LC/MS/MS assay was developed, validated, and applied to liver microsomes, serum, and tumor cells to assess cellular uptake and metabolic stability. Pharmacokinetics was established after administration of a single intravenous dose of p28 in preclinical species undergoing chronic toxicity testing. Antitumor efficacy was assessed on human tumor xenografts. A human therapeutic dose was predicted based on efficacy and pharmacokinetic parameters. RESULTS: p28 is stable, showed tumor penetration consistent with selective entry into tumor cells and significantly inhibited p53-positive tumor growth. Renal clearance, volume of distribution, and metabolic profile of p28 was relatively similar among species. p28 was non-immunogenic and non-toxic in mice and non-human primates (NHP). The no observed adverse effect level (NOAEL) was 120 mg/kg iv in female mice. A NOAEL was not established for male mice due to decreased heart and thymus weights that was reversible and did not result in limiting toxicity. In contrast, the NOAEL for p28 in NHP was defined as the highest dose (120 mg/kg/dose; 1,440 mg/m(2)/dose) studied. The maximum-tolerated dose (MTD) for subchronic administration of p28 to mice is >240 mg/kg/dose (720 mg/m(2)/dose), while the MTD for subchronic administration of p28 to Cynomolgous sp. is >120 mg/kg (1,440 mg/m(2)/dose). The efficacious (murine) dose of p28 was 10 mg/kg ip per day. CONCLUSIONS: p28 does not exhibit preclinical immunogenicity or toxicity, has a similar metabolic profile among species, and is therapeutic in xenograft models.