Development of Mutant Transferrin-Based Molecular Conjugates and Polypeptide-Based Gold Nanoshells as Targeted Drug Carriers for Cancer Therapy
- Author(s): Chen, Kevin Yihao
- Advisor(s): Kamei, Daniel T
- et al.
Currently, cancer is the second leading cause of death in the United States, only behind heart disease. Current treatments of cancer include radiation therapy and chemotherapy, and they are often nonspecific, leading to undesired short and long term side effects. In order to improve current treatments and to reduce side effects, researchers have been investigating alternative methods that can increase the specificity of treatments towards tumor cells to reduce the damage to normal cells. This thesis presents two alternative therapeutic formulations that have increased specificity towards tumor cells. These two therapeutic formulations show potential as alternative methods for cancer therapy in the future.
Human glycoprotein transferrin (Tf), which is responsible for transporting iron into cells, has been extensively studied as a tumor-selective targeting ligand since the tumor cells overexpress the transferrin receptor (TfR) in order to support its rapid proliferation rate. However, the short duration of the Tf-TfR trafficking pathway limits the window of opportunity for Tf to function as a drug carrier. In order to increase the cellular association of Tf, our laboratory previously demonstrated that two Tf mutants (K206E/R632A Tf and K206E/R534A Tf) exhibit significant increases in cellular association compared to wild-type Tf. Subsequently, our laboratory showed that these Tf mutants conjugated with diphtheria toxin (DT) have improved drug carrier efficacy relative to the wild-type Tf-DT conjugate in HeLa and glioma cells. However, due to DT’s nonspecific toxicity at off-target sites, DT is not suitable for clinical applications. In Chapter 2 of this thesis, DT was substituted with cross-reacting material 107 (CRM107), a DT mutant with significantly decreased nonspecific toxicity. In vitro cytotoxicity experiments were conducted where the Tf-CRM107 conjugates were incubated with HeLa and glioma cells. These experiments demonstrated that the improvement in efficacy was greater between the mutant Tf-CRM107 conjugates and the wild-type Tf-CRM107 conjugate when compared to similar experiments performed with their DT counterparts. Moreover, in vitro cytotoxicity experiments with non-neoplastic cells demonstrated that cancer selectivity can be achieved with these new mutant and wild-type Tf-CRM107 conjugates due to the IC50 values being significantly higher for the normal cells. These results suggest that CRM107 appears to be a more suitable therapeutic agent in combination with the K206E/R632A and K206E/R534A mutant Tf ligands for future in vivo and clinical studies.
In addition to using molecular conjugates to kill cancer cells, laser-induced photothermal therapy has shown promise as an alternative method for cancer therapy. Photothermal therapy requires the use of a photosensitizer, i.e., a material that has the ability to convert electromagnetic energy into thermal energy, and a molecular targeting ligand for cancer therapy. In Chapter 3 of this thesis, we describe how we combined an engineered prostate cancer-specific targeting ligand, the A11 minibody, with a novel photothermal therapy agent, polypeptide-based gold nanoshells that generate heat in response to near infrared light. Our work demonstrated that the A11 minibody binds strongly to the prostate stem cell antigen (PSCA) that is overexpressed on the surfaces of metastatic prostate cancer cells. Compared to non-conjugated gold nanoshells, the A11 minibody-conjugated gold nanoshell exhibited significant laser-induced, localized killing of prostate cancer cells in vitro. In addition, we improved upon a comprehensive heat transfer mathematical model that was previously developed by our laboratory. By relaxing some of the assumptions of our earlier model, we were able to generate more accurate predictions. In the future, this model can be used to predict the effects of varying parameters in order to design the next generation of gold nanoshells for photothermal therapy. Our experimental and theoretical results demonstrate the potential of our novel minibody-conjugated gold nanoshells for metastatic prostate cancer therapy.