Design and synthesis of peptide antigens and small-molecule immunomodulators for cancer immunotherapy

Cancer is one of the leading causes of death worldwide, affecting one in eight men and one in ten women during their lifetime. Conventional cancer therapies, like surgery and radiation therapy, face challenges as they may fail to completely remove cancer cells in the entire body, resulting in recurrent disease and metastasis, which is one of the most common causes of cancer-related deaths. In contrast, efficient cancer immunotherapy provides a systemic and long-term treatment, by reviving the patient’s immune response against cancer cells and exploiting the unique specificity of the immune system. This allow for recognition and selective elimination of cancer in the entire body without systemic side effects. Furthermore, the immune cells have the ability to develop into long-lived memory cells, that prevent regrowth of cancer cells displaying the same antigenic profile and thereby protect against recurrent disease and metastasis. Despite the promising potential of cancer immunotherapy, there are still challenges to be resolved in order to establish immunotherapy as the standard of care for cancer patients and enhance its efficacy for a wider range of patients. This PhD thesis presents different approaches and strategies to improve current cancer immunotherapy and expand the knowledge of how to optimize immunotherapeutic tactics.
In the first project of this thesis, a versatile liposome-based cancer vaccine platform was explored, in which a toll-like receptor 7 (TLR7) agonist was co-formulated with tumor-antigen inserted into the liposome membrane by a lipid anchor via a reducible linker. Here, a CD4+ T-cell epitope as well as high- and low-affinity CD8+ T-cell epitopes were synthesized and incorporated in the liposomal platform. These liposomes induced successful in vitro antigen presentation to T-cells and/or stimulated T-cell proliferation. The liposomal platform was optimized by improving the synthesis process of lipidated antigens as well as by incorporating a new linker system for anchoring peptide antigens liposomes. Collectively, this led to a more facile and efficient production of disulfide-based lipidated antigens applicable for a wider range of peptides. The developments furthermore improved reduction-responsive antigen release and in vitro antigen presentation. This finally translated into potent tumor control in B16-OVA tumor-bearing mice when the liposomal platform was combined with adoptive T-cell transfer.
In the second project, the liposome-based cancer vaccine platform was further explored, but with a focus on developing a pH-sensitive linker within the lipidated antigen peptide to allow pH-responsive antigen release. Here, a hydrazone-based linker was employed for conjugating antigen peptide to its lipid anchor, allowing successful incorporation of antigen in liposomes. These liposomes did not release antigen in acidic conditions as intended, but the project established a useful synthesis and formulation strategy for hydrazone-based lipidated peptides and will serve as a template for future pH-sensitive lipidated antigens with anticipated improved pH-sensitivity.
In the third project, ligands targeting tumor-associated macrophages (TAMs) were conjugated to a TLR7/8 agonist resiquimod, to allow selective uptake and repolarization of immunosuppressive TAMs in the tumor microenvironment (TME). By employing a disulfide-based pro-drug strategy for conjugation, active resiquimod could be rapidly released from the targeting ligands in reductive conditions mimicking the intracellular environment. The disulfide-based pro-drug strategy was proven to be crucial in order to prevent disruption of resiquimod activity in vitro and allow potent cellular TLR7/8 activation. Finally, the TAMs targeted resiquimod conjugates were incorporated in in situ forming gel formulations to allow intratumoral sustained release of the compounds, in order to warrant a proper toxicity profile in future studies.
In the fourth project, a series of hypoxia activated pro-drugs (HAPs) of resiquimod were developed to allow tumor specific delivery of resiquimod with the purpose of reducing the systemic toxicity. Three lead candidates of the pro-drugs were selected and proven to be stable in buffer and serum while rapidly activated in conditions that mimic bioreduction in hypoxic tumor tissue. The resiquimod HAPs demonstrated markedly reduced cellular TLR7/8 activation compared to resiquimod in vitro, while pro-drug activation led to complete recovery of this activity. The first in vivo study confirmed potent TLR7/8 activation in tumor tissues of CT26 tumor bearing mice but did not provide proof of reduced systemic toxicity compared to resiquimod. However, the in vitro studies provide a proof-of-principle of using resiquimod HAPs for hypoxia selective activation. Further studies will be performed in vivo to fully elucidate the therapeutic potential of the strategy.
In the fifth project, an allosteric inhibitor of Src homology region 2 (SH2) containing protein tyrosine phosphatase 2 (SHP-2) called SHP099 was explored, with a focus on applying intratumoral drug delivery to exploit the ability of SHP099 to transform the immunosuppressive TME. SHP099 was synthesized in high yields and investigated in vitro, where it was confirmed to inhibit the RAS/extracellular signal-regulated kinases (ERK) pathway as well as inhibit the programmed death-1 (PD-1)/programmed death-ligand-1 (PD-L1) axis. SHP099 was formulated in in situ forming gels based on esterified sugar matrices to allow intratumoral sustained release. The drug demonstrated a sustained release from gels in vitro as well as good stability in formulation. An in vivo study with CT26 tumor bearing mice, did not prove any beneficial anti-tumor effects of using the intratumoral gel formulation of SHP099 compared to intratumoral injection of the free drug. Future studies will be conducted to fully elucidate the therapeutic potential of SHP099 intratumoral gel therapy.
Taken together, the projects of this thesis have presented multiple approaches and strategies to improve current cancer immunotherapy as well as expand the knowledge of how to optimize immunotherapeutic tactics. Several of the developed technologies are still in further in vivo testing to fully clarify their therapeutic potential in animal studies and it is hoped that this thesis will contribute directly or indirectly to improve cancer immunotherapy in the future.