Interleukin-12 message in a bottle

IL12 is a very potent cancer immunotherapy agent, but is difficult to harness safely if given systemically. Local gene transfer aims to confine the effects of IL12 to malignant tissues, thus avoiding toxicity. Lipid-nanoparticle mRNA achieves IL12 expression and efficacy in mouse models, opening the way to an ongoing trial

Intrahepatic injection of recombinant adeno-associated virus serotype 2 overcomes gender-related differences in liver transduction

The liver is an attractive organ for gene therapy because of its important role in many inherited and acquired diseases. Recombinant adeno-associated viruses (rAAVs) have been shown to be good candidates for liver gene delivery, leading to long-term gene expression. We evaluated the influence of the route of administration on rAAV-mediated liver transduction by comparing levels of luciferase expression in the livers of male and female mice after injection of rAAV serotype 2, using three different routes of administration: intravenous (IV), intraportal (IP), or direct intrahepatic (IH) injection. To determine transgene expression we used a noninvasive optical bioluminescence imaging system that allowed long-term in vivo analysis. After IV injection dramatic differences in liver transgene expression were observed, depending on gender. When IP injection was used the differences were reduced although they were still significant. Interestingly, direct intrahepatic injection of rAAV vectors was associated with the fastest and strongest onset of luciferase expression. Moreover, no gender differences in liver transduction were observed and luciferase expression was confined to the site of injection. Thus, direct intrahepatic injection of rAAV offers specific advantages, which support the potential of this route of administration for future clinical applications

Impact of prophylactic TNF blockade in the dual PD-1 and CTLA-4 immunotherapy efficacy and toxicity

The TNF blockade therapy is currently a well-established treatment option for a variety of autoimmune diseases such as rheumatoid arthritis (RA), psoriasis or Crohn's disease, given the proinflammatory role of TNF in the course of these diseases. Importantly, TNF neutralization is also used for the treatment of corticosteroid-refractory immune-related adverse events (irAEs) induced by the combined anti-PD-1 and anti-CTLA-4 immunotherapy. The manifestation of these toxicities is an important limiting factor for the successful implementation of the inhibitory checkpoint blockade therapy (ICB), restraining its anti-tumor efficacy. In our recent study (Perez-Ruiz et al., Nature 569(7756): 428-432.), we analyzed the potential impact of prophylactic TNF neutralization therapy in the anti-PD1/CTLA-4 efficacy. Through several mouse models, we demonstrated that TNF neutralization ameliorated ICB-exacerbated colitis in addition to improving ICB-dependent anti-tumor efficacy. Similar results were obtained after prophylactic TNF blockade in graft vs host xenografted mouse models with human immune cells, which showed a reduction in colitis and hepatitis. Importantly, there was a preservation of the immunotherapeutic control of xenografted tumors after ICB treatment. Moreover, TNF and TNF-dependent gene expression is upregulated in the colon mucosa from patients affected by colitis as a side effect of ipilimumab and nivolumab. Our results, thus, provide evidence of the successful combination of prophylactic TNF neutralization with ICB therapy strategy to ameliorate toxicities, while keeping or even ameliorating anti-tumor efficacy. The prophylactic TNF blockade strategy is clinically feasible since excellent TNF inhibitors have been approved for the treatment of autoimmunity and are used for the immune-related serious adverse events in immunotherapy

In vitro and in vivo comparative study of chimeric liver-specific promoters

Targeting therapeutic genes to the liver is essential to improve gene therapy protocols of hepatic diseases and of some hereditary disorders. Transcriptional targeting can be achieved using liver-specific promoters. In this study we have made chimeric constructs combining promoter and enhancer regions of the albumin, alpha 1-antitrypsin, hepatitis B virus core protein, and hemopexin genes. Tissue specificity, activity, and length of gene expression driven from these chimeric regulatory sequences have been analyzed in cultured cells from hepatic and nonhepatic origin as well as in mice livers and other organs. We have identified a collection of liver-specific promoters whose activities range from twofold to less than 1% of the CMV promoter in human hepatoma cells. We found that the best liver specificity was attained when both enhancer and promoter sequences of hepatic genes were combined. In vivo studies were performed to analyze promoter function during a period of 50 days after gene transfer to the mouse liver. We found that among the various chimeric constructs tested in this work, the alpha1-antitrypsin promoter alone or linked to the albumin or hepatitis B enhancers is the most potent in directing stable gene expression in liver cells

Upregulation of natural killer cells functions underlies the efficacy of intratumorally injected dendritic cells engineered to produce interleukin-12

OBJECTIVE: Injection of dendritic cells (DC) engineered with recombinant adenoviral vectors to produce interleukin-12 (IL-12) inside experimental murine tumors frequently achieves complete regressions. In such a system the function of CD8(+) T cells has been shown to be an absolute requirement, in contrast to observations made upon depletion of CD4(+) T cells, which minimally affected the outcome. The aim of this work was to study the possible involvement of natural killer (NK) cells in this setting. MATERIALS, METHODS, AND RESULTS: Depletions with anti-AsialoGM1 antiserum showed only a small decrease in the proportion of complete regressions obtained that correlated with induction of NK activities in lymphatic tissues into which DC migrate, whereas combined depletions of CD4(+) and NK cells completely eliminated the antitumor effects. Likewise in vivo neutralization of interferon-gamma (IFN-gamma) also eliminated those therapeutic effects. Trying to define the cellular role played by NK cells in vivo, it was observed that injection of cultured DC inside the spleen of T- and B-cell-deficient (Rag1(-/-)) mice induced upregulation of NK activity only if DC had been adenovirally engineered to produce IL-12. In addition, identically transfected fibroblasts also activated NK cells, indicating that IL-12 transfection was the unique requirement. Equivalent human DC only activated in vitro the cytolytic and cytokine-secreting functions of autologous NK cells if transfected to express human IL-12. CONCLUSIONS: Overall, these results point out an important role played by NK cell activation in the potent immunotherapeutic effects elicited by intratumoral injection of IL-12--secreting DC and that NK activation under these conditions is mainly, if not only, dependent on IL-12

Indirect Impact of PD-1/PD-L1 Blockade on a Murine Model of NK Cell Exhaustion

The induction of exhaustion on effector immune cells is an important limiting factor for cancer immunotherapy efficacy as these cells undergo a hierarchical loss of proliferation and cytolytic activity due to chronic stimulation. Targeting PD-1 has shown unprecedented clinical benefits for many cancers, which have been attributed to the prevention of immune suppression and exhaustion with enhanced anti-tumor responses. In this study, we sought to evaluate the role of the PD-1/PD-L1 pathway in murine natural killer (NK) cell activation, function, and exhaustion. In an in vivo IL-2-dependent exhaustion mouse model, neutralization of the PD-1/PD-L1 pathway improved NK cell activation after chronic stimulation when compared to control-treated mice. These cells displayed higher proliferative capabilities and enhanced granzyme B production. However, the blockade of these molecules during long-term in vitro IL-2 stimulation did not alter the progression of NK cell exhaustion (NCE), suggesting an indirect involvement of PD-1/PD-L1 on NCE. Given the expansion of CD8 T cells and regulatory T cells (Tregs) observed upon acute and chronic stimulation with IL-2, either of these two populations could influence NK cell homeostasis after PD-L1/PD-1 therapy. Importantly, CD8 T cell activation and functional phenotype were indeed enhanced by PD-1/PD-L1 therapy, particularly with anti-PD-1 treatment that resulted in the highest upregulation of CD25 during chronic stimulation and granted an advantage for IL-2 over NK cells. These results indicate a competition for resources between NK and CD8 T cells that arguably delays the onset of NCE rather than improving its activation during chronic stimulation. Supporting this notion, the depletion of CD8 T cells reversed the benefits of PD-1 therapy on chronically stimulated NK cells. These data suggest a bystander effect of anti-PD1 on NK cells, resulting from the global competition that exists between NK and CD8 T cells for IL-2 as a key regulator of these cells’ activation. Thus, achieving an equilibrium between these immune cells might be important to accomplish long-term efficacy during anti-PD-1/IL-2 therapy

Oxaliplatin in combination with liver-specific expression of interleukin 12 reduces the immunosuppressive microenvironment of tumours and eradicates metastatic colorectal cancer in mice

BACKGROUND AND AIMS: New options are needed for the management and prevention of colorectal cancer liver metastases. Interleukin 12 (IL-12) is an immunostimulatory cytokine with proven antitumour effect in animal models. Despite evidence indicating its biological effect in humans, neither the recombinant protein nor gene therapy vectors expressing IL-12 have shown a relevant benefit in patients with cancer. OBJECTIVE: To develop a new approach to overcome the difficulties in obtaining a suitable expression pattern and the immunosuppressive milieu in the tumours which contribute to this poor performance. METHODS: A high-capacity ('gutless') adenoviral vector carrying a liver-specific, mifepristone (Mif)-inducible system for the expression of IL-12 (HC-Ad/RUmIL-12) was used in combination with chemotherapy. Tumours were established in the liver of C57BL/6 mice by inoculation of MC38 colon cancer cells. RESULTS: Intrahepatic injection of HC-Ad/RUmIL-12 and tailored induction regimens allowed the maintenance of safe and efficient levels of IL-12 in vivo. An individualised, stepwise increase in the dose of Mif (125-4000 μg/kg) was needed to compensate for the progressive but transient downregulation of the inducible system. Repeated cycles of Mif induction (every 24 h for 10 days) were needed for optimal tumour eradication. However, complete protection against tumour rechallenge was seen in < 25% of the animals. The administration of oxaliplatin (5 mg/kg intraperitoneally) 3 days before starting the induction regimen achieved efficient elimination of liver metastases with a single cycle of IL-12 induction, and improved protection against tumour rechallenge. This was associated with a shift in the tumour microenvironment towards a more pro-immunogenic phenotype, with an increase in the CD8+/T regulatory cell ratio and a reduction in myeloid-derived suppressor cells. These effects were not seen with 5-fluorouracil, irinotecan or gemcitabine

Low surface expression of B7-1 (CD80) is an immunoescape mechanism of colon carcinoma

Artificially enforced expression of CD80 (B7-1) and CD86 (B7-2) on tumor cells renders them more immunogenic by triggering the CD28 receptor on T cells. The enigma is that such B7s interact with much higher affinity with CTLA-4 (CD152), an inhibitory receptor expressed by activated T cells. We show that unmutated CD80 is spontaneously expressed at low levels by mouse colon carcinoma cell lines and other transplantable tumor cell lines of various tissue origins. Silencing of CD80 by interfering RNA led to loss of tumorigenicity of CT26 colon carcinoma in immunocompetent mice, but not in immunodeficient Rag-/- mice. CT26 tumor cells bind CTLA-4Ig, but much more faintly with a similar CD28Ig chimeric protein, thus providing an explanation for the dominant inhibitory effects on tumor immunity displayed by CD80 at that expression level. Interestingly, CD80-negative tumor cell lines such as MC38 colon carcinoma and B16 melanoma express CD80 at dim levels during in vivo growth in syngeneic mice. Therefore, low CD80 surface expression seems to give an advantage to cancer cells against the immune system. Our findings are similar with the inhibitory role described for the dim CD80 expression on immature dendritic cells, providing an explanation for the low levels of CD80 expression described in various human malignancies

Induction of gp120-specific protective immune responses by genetic vaccination with linear polyethylenimine-plasmid complex

The induction of IFN-gamma-secreting CD8+ T cells and neutralizing antibodies to HIV-1 are both key requirements for prevention of viral transmission and clearance of pathogenic HIV. Although DNA vaccination has been shown to induce both humoral and cellular immune responses against HIV antigens, the magnitude of the immune responses has always been disappointing. In this report, we analyze the ability of polyethylenimine (PEI)-DNA complex expressing an HIV-glycoprotein 120 (gp120) antigen (PEI-pgp120) to induce systemic CD8+ T cell and humoral responses to the gp120 antigen. The administration of PEI-plasmid complex resulted in rapid elevation of serum levels of IL-12 and IFN-gamma. Furthermore, a single administration of PEI-pgp120 complex elicits a number of gp120-specific CD8+ T cells 20 times higher than that elicited by three intramuscular injections of naked DNA. Interestingly, we found that systemic vaccination with PEI-pgp120 induced protective immune responses against both systemic and mucosal challenges with a recombinant vaccinia virus expressing a gp120 antigen. The data also demonstrated that the depletion of macrophages with liposome-encapsulated clodronate completely abolished gp120-specific cellular response. Overall, our results showed that a single administration of PEI-pgp120 complexes, eliciting strong immune responses, is an effective vaccination approach to generate protection against systemic and mucosal viral infections