Antagonistic Inflammatory Phenotypes Dictate Tumor Fate and Response to Immune Checkpoint Blockade.

Inflammation can support or restrain cancer progression and the response to therapy. Here, we searched for primary regulators of cancer-inhibitory inflammation through deep profiling of inflammatory tumor microenvironments (TMEs) linked to immune-dependent control in mice. We found that early intratumoral accumulation of interferon gamma (IFN-γ)-producing natural killer (NK) cells induced a profound remodeling of the TME and unleashed cytotoxic T cell (CTL)-mediated tumor eradication. Mechanistically, tumor-derived prostaglandin E2 (PGE2) acted selectively on EP2 and EP4 receptors on NK cells, hampered the TME switch, and enabled immune evasion. Analysis of patient datasets across human cancers revealed distinct inflammatory TME phenotypes resembling those associated with cancer immune control versus escape in mice. This allowed us to generate a gene-expression signature that integrated opposing inflammatory factors and predicted patient survival and response to immune checkpoint blockade. Our findings identify features of the tumor inflammatory milieu associated with immune control of cancer and establish a strategy to predict immunotherapy outcomes.

Enhanced gene expression conferred by stepwise modification of a nonprimate lentiviral vector.

The practical application of gene transfer as a treatment for genetic diseases such as cystic fibrosis or hemophilia has been hindered, in part, by low efficiencies of vector delivery and transgene expression. We demonstrated that a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the envelope glycoprotein from the baculovirus Autographa californica (GP64) efficiently transduces and persistently expresses a reporter gene in respiratory epithelium in the absence of agents that disrupt cellular tight junction integrity. GP64-pseudotyped FIV also efficiently transduced murine hepatocytes after tail vein delivery. To improve the FIV-based vector, we tested the contribution of a series of modifications to luciferase expression in vitro and in vivo. These modifications included the addition of spleen necrosis virus U5 (SNV U5) and mutation of the major splice donor and gag start codon located in the packaging region of the FIV transgene plasmid. After vector modification, we observed significantly enhanced expression of luciferase in respiratory epithelia after nasal application and in the liver after tail vein delivery. In addition, we observed significantly enhanced human factor VIII production after tail vein delivery. These sequential modifications provide an improved FIV lentivirus platform for gene therapy applications and may be applied to other retroviral vectors.

Inhibition of murine K-BALB and CT26 tumour growth using a Semliki Forest virus vector with enhanced expression of IL-18.

The enhanced Semliki Forest virus vector (SFV10-E), an RNA-based suicide expression vector system, expresses foreign genes at levels up to 10x higher than the original SFV10 vector. This vector has been used previously to express interleukin-12 for a tumour treatment study in a BALB/c murine model. Interleukin-18, an IFN-gamma-inducing cytokine, plays a key role in the early induction of T helper1 (Th1) cell-mediated immune responses in addition to anti-angiogenic activity. In this study, the murine IL-18 gene along with an Ig-kappa leader sequence was cloned into the SFV10-E vector. The pSFV10-E-IL-18 construct was characterised in vitro for levels of expression and secretion, and the production of biologically active IL-18 was confirmed. An in vivo tumour treatment study using high titre rSFV10-E-IL-18 virus-like particles to treat subcutaneous K-BALB and CT26 tumours in BALB/c mice demonstrated therapeutic efficacy including the disappearance of tumour cells in a minority of treated animals. Tumour regression was associated with induction of avascular and suppurative necrosis.

CEACAM6 attenuates adenovirus infection by antagonizing viral trafficking in cancer cells.

The changes in cancer cell surface molecules and intracellular signaling pathways during tumorigenesis make delivery of adenovirus-based cancer therapies inefficient. Here we have identified carcinoembryonic antigen- related cell adhesion molecule 6 (CEACAM6) as a cellular protein that restricts the ability of adenoviral vectors to infect cancer cells. We have demonstrated that CEACAM6 can antagonize the Src signaling pathway, downregulate cancer cell cytoskeleton proteins, and block adenovirus trafficking to the nucleus of human pancreatic cancer cells. Similar to CEACAM6 overexpression, treatment with a Src-selective inhibitor significantly reduced adenovirus replication in these cancer cells and normal human epithelial cells. In a mouse xenograft tumor model, siRNA-mediated knockdown of CEACAM6 also significantly enhanced the antitumor effect of an oncolytic adenovirus. We propose that CEACAM6-associated signaling pathways could be potential targets for the development of biomarkers to predict the response of patients to adenovirus-based therapies, as well as for the development of more potent adenovirus-based therapeutics.

Regression of mouse tumours and inhibition of metastases following administration of a Semliki Forest virus vector with enhanced expression of IL-12.

The Semliki Forest virus (SFV) vector is an RNA-based suicide expression vector that has been used experimentally for tumour therapy. Recently, a new enhanced vector pSFV10-E has been developed that expresses foreign genes at levels up to 10 times higher than the original vector. Interleukin-12 (IL-12), an immunomodulatory cytokine, plays a key role in the induction of T-helper1 responses. The two IL-12 gene subunits were cloned from mouse splenocytes and inserted into the pSFV10-E and pSFV10 (non-enhanced) vectors. Both constructs expressed and secreted biologically active murine IL-12. Administration of high titre rSFV10-E-IL12 particles intratumourally to treat implanted K-BALB tumours in BALB/c mice demonstrated complete tumour regression in comparison to control or rSFV10-IL12 treated groups. High titre rSFV10-E-IL12 particles were also effective in the CT26 tumour model. Histological and immunohistochemical studies revealed tumour necrosis in addition to aggressive influx of CD4+ and CD8+ T cells and other immune cells. Furthermore, inhibition of primary tumour growth and lung metastases of a metastatic (4T1) tumour model indicated the potential of high titres of rSFV10-E-IL12 particles as an efficient antitumour therapeutic agent.