| dc.description.abstract | Antibody-based therapeutics are a rapidly expanding class of biopharmaceuticals. The number of approved antibody-based therapeutics by the FDA and EMA has more than doubled in the last five years, and they are expanding into areas such as antibody-drug conjugates, Fc fusions, bispecific antibodies, and biosimilars. Most antibody-based therapeutics use the Immunoglobulin isotype G subclass 1 (IgG1) antibody. Some IgG1-based therapeutics obtain their therapeutic efficacy by modulating the immune system for the treatment of several disease-types including cancer, autoimmune disease, and organ transplant rejection. This work further explores our understanding of IgG1-mediated immunomodulation by utilizing the fragment crystallizable (Fc) region of IgG1 in three research projects. In the first project, an IgG1 Fc fusion was prepared as a potential treatment for multiple sclerosis, an autoimmune disease that affects 2.3 million people worldwide. This disease involves immune system attack and destruction of the myelin protein surrounding the neurons in the central nervous system. One promising class of compounds that selectively prevent the activation of immune cells involved in the destruction of myelin are Bifunctional Peptide Inhibitors (BPIs). In an effort to further improve the bioactivity of BPIs, the BPI peptides were conjugated to the termini of IgG1 Fc to prepare a BPI-Fc fusion. This fusion was tested in a mouse model of multiple sclerosis. Compared to the PBS-treated control, mice treated with the BPI-Fc fusion showed significantly reduced disease symptoms, did not experience weight loss, and showed reduced demyelination. These results demonstrated that the BPI peptides were highly active at suppressing the disease when prepared as an Fc fusion. In the second project, the N-glycosylation of IgG1 Fc was modified. The N-glycosylation of IgG1 can markedly affect its function, stability, pharmacokinetics, solubility, and immunogenicity. However, recombinant expression of IgG1 results in a mixture of N-glycoforms. This heterogeneity makes it difficult to understand how N-glycosylation affects IgG1 because different N-glycoforms can affect the antibody differently. To solve this problem, this work utilized IgG1 Fc as a model system to prepare homogenous IgG1 Fc N-glycoforms, and their effects on IgG1 Fc were studied individually. In-vitro enzymatic synthesis was used to prepare homogenous oligomannose, hybrid, and complex N-glycoforms. The effect of each non-fucosylated N-glycoform on IgG1 Fc stability was compared using differential scanning calorimetry. The results showed that the complex N-glycoform was more stable than the hybrid and oligomannose N-glycoforms. Additionally, the effect of each N-glycoform on IgG1 Fc function was compared in an in-vitro receptor-binding assay using FcγRIIIa, the receptor involved in activating Antibody-dependent Cellular Cytotoxicity (ADCC). Results showed that the binding affinity increased with increased N-glycan processing. Lastly, the hybrid and complex IgG1 Fc N-glycoforms were compared for their abilities to accept core-linked fucose. Results showed that the complex N-glycoform accepted fucose much more slowly compared to the hybrid N-glycoforms. In the third project, IgG1 Fc was assembled as a protein-polymer conjugate in order to increase its valency. Multivalent display of IgG1 is necessary in order to obtain an avidity effect strong enough to activate immune system effector functions, such as ADCC. IgG1 Fc-polymer conjugate was prepared using controlled polymerization to first synthesize a water soluble, linear poly(acrylamide-peptide) co-polymer. This co-polymer was then used as a scaffold onto which multiple IgG1 Fc proteins were site-specifically ligated. The IgG1 Fc-polymer conjugate was compared against IgG1 Fc in a receptor binding assay with FcγRIIIa. The results showed that the IgG1 Fc-polymer conjugate bound FcγRIIIa 800 times stronger compared to free IgG1 Fc. This large increase in binding strength was caused by the multimer having a much slower dissociation rate. The research presented in this dissertation improves our fundamental understanding IgG1-mediated immunomodulation and may also help in the development of improved antibody- based therapeutics. | |
