| dct.abstract |
Checkpoint inhibitors have been regarded as a milestone in cancer therapy due to the imminent success in the clinics. Among such checkpoint inhibitors, PD-L1 antibodies have shown outstanding clinical efficacy and have been approved for the treatment of more than 14 different type of cancers. Despite the success, only 14% of patients are eligible for PD-L1 antibody therapy and from them only 44% respond. Therefore, a clear improvement of such therapy is required. Such antibodies are not able to elicit effector mechanisms due to point mutations in the IgG Fc-region removing binding to Fc- receptors. This is done because of safety concerns, since PD-L1 expression is not solely limited to the tumor but widely expressed on different types of healthy cells. Nevertheless, in vivo studies have shown that arming PD-L1 checkpoint inhibitors with Fc-regions able to elicit effector mechanism leads to increased tumor killing. In need of enhanced PD-L1 checkpoint inhibitors, in this thesis we developed two powerful PD-L1 checkpoint inhibitors able to elicit Fc-effectors mechanisms of both an IgG1 and IgA1. Moreover, to limit side effects we used oncolytic adenoviruses as biological carries to express and limit the secretion of the PD-L1 checkpoint inhibitors to the tumor.
Oncolytic adenoviruses have a specific tumor-tropism that can be utilized to express any desired gene of interest to the tumor microenvironment. Nevertheless, the current methods using homologous recombination to clone oncolytic adenoviruses are time-consuming and inefficient. In Study I, we designed and tested a novel cloning method, called GAMER-Ad, which utilizes the Gibson assembly method rather than homologous recombination for cloning. To test GAMER-Ad, we designed three oncolytic adenoviruses to express CXCL9, CXCL10 or IL-15. GAMER-Ad was shown to be a viable strategy to clone oncolytic adenoviruses in the period of 2-3 days. Also, the cloning method did not affect the oncolytic/replication fitness of the viruses and yielded functioning viruses able to express the corresponding gene. GAMER-Ad was then used in the following studies to clone all oncolytic adenoviruses.
In Study II, we developed an oncolytic adenovirus (Ad-Cab) able to secrete a PD-L1 checkpoint inhibitor able to elicit Fc-effector mechanisms of an IgG1 and IgA1. The expressed checkpoint inhibitor consisted of PD-1 ectodomain (able to bind to murine and human PD-L1) connected to a cross-hybrid IgGA Fc-region (contains heavy chain regions of an IgG1 and IgA1). The virally released cross-hybrid Fc-fusion peptide was able to activate PBMCs, PMNs, complement proteins and macrophages not usually done by either IgG1 or IgA antibodies solely. The engagement of multiple effector mechanisms did lead to an enhanced tumor killing by Ad-Cab compared to PD-L1 antibodies with an IgG1 or IgA Fc-region when all immune components were present. This enhancement was also translated in vivo since Ad-Cab outperformed conventional PD-L1 antibodies with various tumor models (4T1, CT26 and A549). This enhancement was attributed to an increased activation of NK cells and reduction of myeloid derived suppressor cells in vivo. Moreover, Ad-Cab was shown not to require CD8+ T cells for in vivo efficacy unlike the conventional PD-L1 antibodies used. As expected, no signs of toxicity were observed since no reduction in weight was observed in mice and the Fc-fusion peptide was limited to the tumor. Therefore, arming PD-L1 antibodies with Fc-effector mechanisms of an IgG1 and IgA1 leads to higher tumor-killing and safety concerns can be circumvented using oncolytic adenoviruses
Further building on Ad-Cab, in Study III we designed Ad-Cab FT which had the same Fc-fusion peptide designed in Study II but with four-point mutations in the IgG region. These point mutations increased the affinity towards activating Fc- receptors leading to higher NK cell activation. At higher concentrations, Ad-Cab FT had similar levels of tumor lysis as Ad-Cab when PBMCs were added. However, at lower concentrations Ad-Cab FT induced higher tumor killing than Ad-Cab with PBMCs. This enhancement was not shown with PMNs or complement activation. Due to the high activation of PBMCs at lower concentration, Ad-Cab FT outperformed Ad-Cab in vivo when low doses and reduced administrations of the virus was given. With Ad-Cab FT treated mice, a higher activation of NK cells in the tumor microenvironment was observed compared to Ad-Cab treated mice. Hence, Ad-Cab FT represents a potentiated therapy with potential use in the clinic.
Taken together, this thesis has highlighted the importance of eliciting multiple immune populations to enhanc tumor killing with PD-L1 checkpoint inhibitors and potentially with other therapies. The IgGA Fc-region may be used in other antibody-based therapies to further increase tumor killing and subsequently clinical efficacy. Finally, oncolytic adenoviruses have demonstrated in this thesis to be excellent biological carriers, limiting the toxicity of dangerous anti-tumor agents. |
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