CANCER THERAPY THROUGH TLR-INDUCED LOCAL INNATE IMMUNITY ACTIVATION AND BLOCK OF IMMUNE CHECKPOINTS OR SUPPRESSIVE CELLS

The immunostimulatory ability of oligonucleotides containing CpG motif (CpG-ODN), agonists of TLR9, can be harnessed to promote antitumor immunity by their application at the tumor site to stimulate local activation of innate immunity; however, this is not always true for lung tumor where the immunosuppressive microenvironment, crucial to avoid tissue damage by inflammatory response to the continuous exposition to inhaled particles, limits the power of immunotherapy. Thus, the tumor microenvironment is a critical factor for successful use of these immunotherapeutics, and strategies to shift a tumor-supporting milieu to a host-friendly one might lead to improved anti-tumor activity of CpG-ODN. Since bronchial and bronchoalveolar tumors are accessible via the endobronchial space, inhalation of aerosolized immunotherapeutic agents might represent a convenient and simple approach to shape a tumor microenvironment more favorable to induce an immune response against lung primary cancer and/or metastases. Aim of this thesis is the evaluation of strategies to improve the antitumor activity of aerosolized TLR9 agonist in the lung. The thesis is divided in three tasks: 1) the evaluation of aerosol delivery CpG-ODN combined with Poly(I:C), a TLR3 agonist able to convert tumor-supporting macrophages to tumoricidal effectors, in the treatment of B16 melanoma lung metastases in C57BL/6 mice. 2) the investigation of the mechanism by which the two combined agonists could induce the activation of effector cells population. 3) the exploration of strategies to improve the therapeutic efficacy of TLR9/TLR3 agonists by including in the inhalant either an antibody directed to both Ly6G and Ly6C markers, to locally deplete myeloid-derived suppressive cells (MDSC), or IFNα, to directly activate innate immune cells in the lung. Aerosolization of CpG-ODN with Poly(I:C) into the bronchoalveolar space reduced the presence of M2-associated arginase- and IL-10-secreting macrophages in tumor-bearing lungs and increased the anti-tumor activity of aerosolized CpG-ODN alone against B16 lung metastases without apparent signs of toxicity or injury of the bronchial-bronchiolar structures and alveolar walls. Moreover, CpG-ODN/Poly(I:C) aerosol combined with dacarbazine, a therapeutic agent used in patients with inoperable metastatic melanoma able to exert immunostimulatory effects, led to a significant increase in anti-tumor activity as compared to treatments with aerosolized CpG-ODN/Poly(I:C) or dacarbazine alone. This effect was related to an enhanced recruitment and cytotoxic activity of tumor-infiltrating NK cells in the lung. Our in vitro and in vivo experiments to elucidate the mechanism of NK cells activation by TLR9/TLR3 stimulation revealed that the two agonists were able to directly induce IFN-γ secretion by NK cells, but the stimulation of their cytotoxic activity required the presence of alveolar macrophages. Interestingly, our experiments demonstrated that, reciprocally, NK cells were able to influence macrophages polarization, since alveolar macrophages incubated with activated NK cells from the lungs of melanoma metastases-bearing mice that were given aerosolized TLR9/TLR3 agonists, up-regulated M1- and down-regulated M2-related genes. Addition of nebulized anti-MDSC antibody RB6-8C5 to aerosolized CpG-ODN/Poly(I:C) resulted in reduced mRNA levels of immunosuppressive molecules (IL10, Arg-1 and Nos2), increased activation of resident NK cells and improved treatment outcome, with a significant reduction in established B16 melanoma lung metastases, compared to treatment with CpG-ODN/Poly(I:C) alone. Likewise, addition of aerosolized IFN-α led to increased mRNA levels of pro-inflammatory cytokines (IL15 and IFN-γ) in the lung and recruitment of highly activated NK cells, with no evident signs of toxicity and with a significantly improved anti-tumor effect, as compared with aerosolized CpG-ODN/Poly(I:C). Combining both IFN-α and RB6-8C5 with CpG-ODN/Poly(I:C) did not produce additive effects. In conclusion, the results of this thesis indicate that the pulmonary route is a feasible and non-invasive strategy to deliver immunodulatory molecules, including antibodies and cytokines, to reprogram the lung microenvironment shaping it more favorable to foster immune destruction of tumors.