Heat Shock Protein Hsp70 Fusion Protein Vaccine, The Anti-tumor Effect And The Role Of Hsp70 In The Regulation Of Immune

HSP70s are a family of ATP-dependent chaperones of relative molecular masses around 70 kDa. Immunization of mice with HSP70 isolated from tumor tissues has been proved to elicit specific protective immunity against the original tumor. Recent researches have demonstrated that the ATPase domain of HSP70 and the tumor antigenic peptide that binds to Hsp70 were the crucial parts eliciting tumor-specific immunity. These findings suggested that a recombinant protein expressed in Escherichia coli, comprising a covalently fused fragment of tumor rejection antigen to ATPase domain of HSP70, could be used as a tumor vaccine. The present thesis focused on HSP70 fusion proteins and managed to delay tumor emergence and inhibit tumor growth in mice by vaccination of them.In the first chapter, we expressed, purified a fusion protein comprising murine heat shock cognate protein 70 (Hsc70) N-terminal ATPase domain (Hsc70NTD) and a portion of TRP2 (aa153-417) as a model protein. The fusion protein was refolded via gel-filtration chromatography. The refolding effectivities were assessed according to their ATPase activities; the vaccine function was assessed according to immunization effect in inducing antigen-specific CTLs and to in vivo tumor protection against B16, which expresses protein TRP2. The results showed that the fusion protein refolded via gel-filtration chromatography exhibited ATPase activity, succeeded in eliciting antigen-specific CTL in vivo and delayed tumor growth on tumor-bearing mice.In the second chapter, we found that HSP70-TRP2 fusion protein can elicit strong cellular immune responses against murine tumor B16. The Hsp70 peptide-binding domain deletion mutant of the fusion protein was sufficient for inducing TRP2-specific CTL but was not for engendering potent anti-tumor immunity against B16. We also found that host NK cells were stimulated in vivo by C-terminal domain of HSP70. We thus presume that HSP70 fusion proteins suppress tumor growth via at least two distinct pathways; one is covalence-accompanying antigen dependent, another is antigen independent. The C-terminal domain of HSP70 seemed to be the crucial part in eliciting antigen-independent responses, including NK cell stimulation, against tumor challenges.In the third chapter, we constructed other three sorts of HSP70 fusion proteins, containing Mela, Pmel17 and MART-1 respectively. All the three proteins were defined as tumor associated antigens of B16. We found that immunization with multiple Hsp70 fusion proteins (Hsp70-Mela, Hsp70-Pmel17, Hsp70-MART-l and Hsp70-TRP2) resulted a better anti-tumor effect than single fusion protein (Hsp70-Mela). The further analysis on mRNA abundance demonstrated that tumor cell down regulated antigen expression in order to escape from immnuological surveillance.