Leveraging immune memory against measles virus as an antitumor strategy in a preclinical model of aggressive squamous cell carcinoma.

Viral antigens are among the strongest elicitors of immune responses. A significant proportion of the human population already carries pre-existing immunity against several childhood viruses, which could potentially be leveraged to fight cancer. We sought to provide proof of concept in mouse models that a pre-existing measles virus (MeV) immunity can be redirected to inhibit tumor growth by directly forcing expression of cognate antigens in the tumor. To this end, we designed DNA vaccines against known MeV cytotoxic and helper T epitopes, and administered these intradermally to mice that were subsequently challenged with syngeneic squamous cancer cells engineered to either express the cognate antigens or not. Alternatively, established wild-type tumors in vaccinated animals were treated intratumorally with in vitro transcribed mRNA encoding the cognate epitopes. Vaccination generated MeV cytotoxic T lymphocyte (CTL) immunity in mice as demonstrated by enhanced interferon gamma production, antigen-specific T cell proliferation, and CTL-mediated specific killing of antigen-pulsed target cells. When challenged with syngeneic tumor cells engineered to express the cognate antigens, 77% of MeV-vaccinated mice rejected the tumor versus 21% in control cohorts. Antitumor responses were largely dependent on the presence of CD8+ cells. Significant protection was observed even when only 25% of the tumor bulk expressed cognate antigens. We therefore tested the strategy therapeutically, allowing tumors to develop in vaccinated mice before intratumoral injection with Viromer nanoparticles complexed with mRNA encoding the cognate antigens. Treatment significantly enhanced overall survival compared with controls, including complete tumor regression in 25% of mice. Our results indicate that redirecting pre-existing viral immunity to fight cancer is a viable alternative that could meaningfully complement current cancer immune therapies such as personalized cancer vaccines and checkpoint inhibitor blockade.

Viromers as carriers for mRNA-mediated expression of therapeutic molecules under inflammatory conditions.

Therapeutic mRNA delivery has been described for several treatment options, such as vaccination and cancer immunotherapy. However, mRNA delivery has to be accompanied by the development and testing of suitable carrier materials due to the instability of mRNAs in human body fluids. In the present study, we investigated the ability of recently developed Viromers to deliver mRNAs in a classical inflammatory setting. We tested mRNAs coding for active components of preclinical (7ND) and approved (sTNF-RII) biologics, in vitro and in vivo. 7ND is an established blocker of the CCR2 axis, whereas sTNF-RII is the active component of the approved drug Etanercept. Viromer/mRNA complexes were transfected into murine macrophages in vitro. Protein expression was analysed using Luciferase reporter expression and mainly identified in spleen, blood and bone marrow in vivo. 7ND-mRNA delivery led to efficient blockage of monocytes infiltration in thioglycolate-induced peritonitis in mice, underlining the ability of Viromers to deliver a therapeutic mRNA cargo without overt toxicity. Therefore, we propose Viromer-based mRNA delivery as a suitable option for the treatment of inflammatory disorders beyond infusion of biological molecules.

A novel, lactase-based selection and strain improvement strategy for recombinant protein expression in Kluyveromyces lactis.

BACKGROUND: The Crabtree-negative yeast species Kluyveromyces lactis has been established as an attractive microbial expression system for recombinant proteins at industrial scale. Its LAC genes allow for utilization of the inexpensive sugar lactose as a sole source of carbon and energy. Lactose efficiently induces the LAC4 promoter, which can be used to drive regulated expression of heterologous genes. So far, strain manipulation of K. lactis by homologous recombination was hampered by the high rate of non-homologous end-joining. RESULTS: Selection for growth on lactose was applied to target the insertion of heterologous genes downstream of the LAC4 promoter into the K. lactis genome and found to yield high numbers of positive transformants. Concurrent reconstitution of the ß-galactosidase gene indicated the desired integration event of the expression cassette, and ß-galactosidase activity measurements were used to monitor gene expression for strain improvement and fermentation optimization. The system was particularly improved by usage of a cell lysis resistant strain, VAK367-D4, which allowed for protein accumulation in long-term fermentation. Further optimization was achieved by increased gene dosage of KlGAL4 encoding the activator of lactose and galactose metabolic genes that led to elevated transcription rates. Pilot experiments were performed with strains expressing a single-chain antibody fragment (scFvox) and a viral envelope protein (BVDV-E2), respectively. scFvox was shown to be secreted into the culture medium in an active, epitope-binding form indicating correct processing and protein folding; the E2 protein could be expressed intracellularly. Further data on the influence of protein toxicity on batch fermentation and potential post-transcriptional bottlenecks in protein accumulation were obtained. CONCLUSIONS: A novel Kluyveromyces lactis host-vector system was developed that places heterologous genes under the control of the chromosomal LAC4 promoter and that allows monitoring of its transcription rates by ß-galactosidase measurement. The procedure is rapid and efficient, and the resulting recombinant strains contain no foreign genes other than the gene of interest. The recombinant strains can be grown non-selectively in rich medium and stably maintained even when the gene product exerts protein toxicity.