Carbohydrate derived nanoplatforms as tools for lectin isolation, and delivery of cytotoxic T-cell peptide epitopes for cytotoxic T-cell mediated tumor immunotherapy.

摘要

Abstract: Cancer cells can have characteristic carbohydrate binding properties. Previously, it was shown that a highly metastatic melanoma cell line B16F10 bound to galactoside functionalized nanoparticles much stronger than the corresponding less metastatic B16F1 cells. The results presented in the second chapter of this dissertation documents the isolation and characterization of endogenous galactose binding proteins from B16F10 cells using magnetic glyconanoparticles. The galactose coated magnetic glyconanoparticles could bind with lectins present in the cells and be isolated through magnet mediated separation. Through Western blot and mass spectrometry, arginine/serine rich splicing factor Sfrs1 was identified as a galactose selective endogenous lectin overexpressed in B16F10 cells compared to B16F1 cells. In addition, Sfrs1 was found in higher amounts in B16F10 cells. Finally, the glyconanoparticles exhibited a superior efficiency in lectin isolation, from both protein mixtures as well as live cells, than the more traditional micro-particles functionalized with carbohydrates. The magnetic glyconanoparticles present a useful tool for discovery of endogenous lectins as well as binding partners of lectins without prior knowledge of protein identities.;On the other hand, Cytotoxic T lymphocyte (CTL) mediated cancer immunotherapy has clinically shown the potential to treat cancer patients. For CTL therapy to be successful, cancers cells must express antigens that are targets for specific CTLs. However, it has been established that due to genetic instability of cancer cells, sub-population of cancer cells may fail to express the target antigen, possibly leading to escape from CTL destruction, hence tumors grow progressively. These antigen-loss variant (ALV) cancer cells can be eliminated as bystanders by targeting tumor associated stromal cells, but only if the cancer cells express sufficient antigens to be effectively cross-presented by the stromal cells. In the third chapter of this dissertation, we present results that investigated whether acid responsive nanoparticles can be used to deliver CTL-specific antigen to the cancer microenvironment to enhance tumor eradication by activated antigen-specific CTLs in a mouse model. Our results show that model CTL antigen (OVA peptide, SIINFEKL) encapsulated in pH sensitive acetalated dextran nanoparticles (OVA(P)-Ac-Dx-NPs) could be successfully delivered to tumor cells in vitro and tumor microenvironment in vivo. The uptake and presentation of the peptide antigen by major histocompatibility molecules class I (MHC-I) in vitro and in vivo was confirmed by flow cytometry and confocal laser scanning microscopy through antibody staining. In addition, solid tumor bearing mice treated with OVA(P)-Ac-Dx-NPs showed much slower tumor growth compared to mice treated with free OVA(P), empty Ac-Dx-NPs, or PBS. Taken together, these findings offer a promising new direction for treating established solid tumors using CTL therapy.