(137g) Engineering Novel Therapies for Immune Modulation

Over 52 million people in the Unites States have an immune-related disease, predominantly an autoimmune disease or cancer. These diseases cost the medical care system over $350 billion dollars each year. The therapies themselves are often costly, but a majority of the cost is used to manage and troubleshoot toxic side effects and drug resistance. Many autoimmune therapies depend on broad immunosuppression to manage the disease, leading to a high risk of developing infections and malignancies. Whereas cancer immunotherapies can often cause broad immunostimulation leading to toxic side effects resulting from unregulated immune activation. In both cases, there is a need for targeted therapies to enhance drug efficacy, lessen toxic side effects, and reduce the risk of drug resistance. For the past ten years, I have been privileged enough to work under excellent principal investigators on immune-related diseases. Specifically, pulling concepts from drug delivery and immunoengineering to develop novel therapeutic platforms that might be applied to multiple diseases. Although I present here a snippet of the research that has been conducted, the following independently driven projects provide a strong basis for understanding my future research goals with a strong emphasis on increased efficacy without toxicity.

Immune Stimulants: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that can stimulate anti-tumor properties of the immune system. GM-CSF activates dendritic cells to recruit and present antigen to T-cells and is most effective as a cancer therapy when it activates cells in the tumor microenvironment. Additionally, off-tumor toxicity can occur with high fevers and muscle/joint pain being common side effects. Several GM-CSF fusion polypeptides were designed that bind to the extracellular matrix in the tumor microenvironment of solid tumors. Multiple different tumor binding peptides were tested including those binding to hyaluronic acid, collagen, integrin, and fibronectin. All tested peptides showed increased retention of GM-CSF in the tumor microenvironment tending toward higher efficacy and lower toxicity.

Insoluble yeast β-glucan has been shown to illicit trained immunity which allows cells to have faster and stronger pro-inflammatory responses when delivered orally. The goal of this study was to determine if the soluble β-glucan peptide derived from trametes versicolor could induce trained immunity and increase inflammation in the tumor microenvironment. β-glucan peptide was found to induce both in vitro and in vivo trained immunity. Treatment of mice inoculated with tumors with β-glucan peptide showed a significant reduction in tumor growth rate and increased survival. Flow analysis of the tumor microenvironment revealed significant reductions in the percentage of macrophages and myeloid derived suppressor cells in treated mice. Lastly, it was found that β-glucan peptide significantly increased checkpoint inhibitor effectiveness in a tumor model normally resistant.

Clickable Antibody Fragments: In general, antibodies contain an antigen binding Fab fragment and a crystallizable Fc fragment which interacts with Fc receptors on immune cells. Antibody sequences were engineered to contain a HRV-3C protease cut site in their hinge region. After transfection and purification of antibodies, HRV-3C protease was used to cut the antibody into the Fc and Fab region. Through strategic purification techniques using Protein A, G, or L the final product resulted in purified antibody fragments. Additional gene editing of the antibody included inserting a carboxyl-terminal sorting signal. The sortase enzyme was used to target this sequence and fuse the Fc or Fab region with GGG-azide. Purified Fab-azide or purified Fc-azide was then used for click chemistry with any molecule containing an alkyne or DBCO. These fragments were used for a wide range of applications including Fc fusion proteins, disease-specific therapies for autoimmune diseases, and viral neutralization.

mRNA-LNP Vaccines: Cancer vaccines can increase and diversify T-cell responses in tumors. However, clinically there remains the problem of low immunogenicity of mRNA-lipid nanoparticle (LNP) cancer vaccines, specifically the inability to mount strong T-cell responses. Several adjuvants have been tested in mRNA-LNP vaccines to increase immunogenicity, but they often have variable responses and introduce significant toxicity. The goal of this study was to determine of co-stimulatory molecules could act as adjuvants in mRNA-LNP cancer vaccines to increase anti-tumor T-cell responses while maintaining low toxicity. Seven co-stimulatory molecules were screened with a tumor-specific antigen and one optimal combination of three co-stimulatory molecules significantly enhanced antigen-specific T-cell responses. Extensive toxicity studies revealed mouse body weight and liver enzymes did not change even after repeated doses, indicating low toxicity of co-stimulatory molecules. In tumor models, mice receiving co-stimulatory molecule adjuvanted vaccine show increased T-cell infiltration and a reduction in tumor growth.

In order to mount strong T-cell responses, cancer vaccines are more effective when targeting antigen presenting cells located in the spleen. This project utilized a naturally-derived ligand believed to bind to a receptor primarily on immune cells. A thiol was introduced onto the ligand via an end-group modification. LNPs were then formulated with maleimide functionalized PEG lipids in order to click the ligand onto the LNP. LNP formulations were confirmed to bind the desired receptor and maintained endosomal escape even after modification. Using a Cre Lox mouse model, it was confirmed that the modified LNPs effectively targeted antigen presenting cells in the spleen. The targeted LNP showed superior vaccine capabilities by quantifying T-cell responses and showing therapeutic benefit in a tumor model.

Several of the above studies have gained the interest of clinicians and physician-scientists at Weill Cornell Medicine, Toronto SickKids, and Houston Methodist Hospital. We have been able to easily interchange components of these platforms to fit their specific purposes, which has allowed these therapies to be tested in clinically relevant models and potentially inform better therapies for patients. I will discuss what I expect my future research to entail, but in brief it will have a specific focus on targeted therapies, take inspiration from nature by using naturally derived materials, and emphasize immune material interactions for the treatment of immune-related diseases.