Multi-peptides Loaded Nano-Vaccine For Immunotherapy Against Melanoma

Along with the development of tumor biology and immunology, the new method of anti-tumor has been applied. Peptide vaccine research has become a hot spot for cancer therapy because it is easier to prepare, store, and safe to administer. However, the vaccine constructed with simple peptides can only induce a weak immune response. Each peptide generally contains only one epitope for the immune system and offers the narrowest spectrum of tumor targets which is easy to cause immune escape. It is possible to overcome immune escape with multi-peptides by the increased number of epitopes and broadened response spectrum.The activation of anti-tumor immune response is induced with the uptake and presentation of antigen by the antigen presenting cells (APC). Dendritic cells (DC) are the most powerful professional antigen presenting cells with strong ability to activate the initial T cells and are the initiators of the adaptive immune response. Enhancing the uptake of antigens by DC, combined with the pattern recognition receptor agonists is a commonly used method to enhance the immunogenicity of antigens and break the immune tolerance. But these methods are often hampered by the co-delivery of multi-drugs. The nano delivery system can meet the requirements of the co-delivery of multiple antigens and immune adjuvant.In this study, we designed nanoparticles loaded with multi-peptides (hgp100, KVPRNQDWLC; p15E, KSPWFTTLC; TRP2, SVYDFFVWLC) and immune adjuvant (monophosphoryl lipid A, MPLA) together, to enhance the anti-tumor immune response, aiming for treatment of highly aggressive, heterogeneous melanoma. The construction of nanoparticles was according the following four steps: (1) peptides reacted with poly (lactic-co-glycolic acid) (PLGA) to obtain the PLGA-peptides with reduced hydrophobicity differences (2) the resulted PLGA-peptides were prepared nanoparticles by nanoprecipitation method (3) nanoparticles were extruded with the membrane of red blood cells (RBCm) to prepare RBCm-coated nanoparticles (4) the MPLA were loaded into RBCm-coated nanoparticles by ultrasonic methods.The structures of the synthesized PLGA-peptides were confirmed by nuclear magnetic resonance spectroscopy (’H-NMR), and the reductive response of the materials was determined under the condition of Dithiothreitol (DTT). Spherical particles with a diameter of 130-200nm were confirmed by transmission electron microscopy (TEM) and the core shell structure of RBCm coated nanoparticles was observed. After determination, the loading capacity of the mutip-peptides was close to 1:1 for two and 1:1:1 (wt) for three kinds of peptides. The character of redox sensitive release was verified by simulation of intracellular environment in vitro. No adverse reactions were induced in mice after injection, which satisfied the requirement of administration. The peptides loaded nanoparticles could stimulate DC maturation both in vitro and in vivo, but different combinations of peptides showed different potential in DC maturation. Although the three kinds of peptides are all tumor associated antigens, they derived from different protein molecules. For the different function of these proteins, the combination of different peptides has differences in effect of anti-tumor. hgp00 is a differentiation antigen in the early stage of the embryo, over expressed in the process of melanoma, which belongs to the H-2Db restricted epitope of melanoma associated antigen. p15E is an oncogenic retrovirus capsid protein epitope, which belongs to H-2Kb restricted epitope. Tyrosinase related protein 2 (TRP2) is expressed in normal melanocytes, but over expressed in melanoma, which belongs to the H-2Db restricted epitope. For the prophylactic and therapeutic experiment, we got the similar results. The combination of hgp100 and TRP2 showed the best anti-tumor effect. However, the results of nano-vaccine loaded with three peptides were not better than some certain combination of 2 peptides, which was out of forecast. The anti-tumor mechanism of the multi-peptide nano-vaccine was studied, and the experimental results were consistent with the studies on pharmacodynamics. But the compromised effects of combining three peptides have not been explained reasonably.To sum up, we constructed a polymer nano-vaccine with the feature of reductive sensitivity loaded with multi-peptides and immune adjuvant. The results of the experiments showed that different combination of peptides would produce different therapeutic effects. But the immune response is a complex physiological process, and the mechanism behind this process affects the outcome of therapeutic effect. When an immune response is induced, a negative regulation mechanism is also triggered to protect the body from damage. Studying the mechanism will provide theoretical support for the selection of effective antigens. With the identification of new tumor associated antigen peptides, the nano platform constructed in this paper would show a great potential in the combination of peptides for therapeutic evaluation. The results of this study provide a new thinking for the selection of antigens.