Vx3 Functionalized Alumina Nanoparticles Assisted Enrichment Of Ubiquitinated Proteins From Cancer Cells For Enhanced Cancer Immunotherapy

Cancer immunotherapy is growing to be the fourth most important cancer therapy,after surgery,chemotherapy,and radiation therapy.Tumor vaccine is one of the important means of tumor immunotherapy.Our previous studies have shown that Ubiquitinated Proteins（UPs）enriched from tumor cells was an important part of tumor antigens.UPs based tumor vaccine could induce tumor specific immune response and have anti-tumor effort.For the preparation of UPs based tumor vaccine the key step was the enrichment of UPs.Objective Previously,Vx3,a recombinant protein which contains three ubiquitin binding sites,was linked to Ni agarose beads and UPs was enriched.However,the method was cumbersome and inefficient,a better method for the enrichment of UPs was needed.In this study,UPs was enriched from cancer cells with the aid of α-Al₂O₃-Vx3 which was obtained by conjugating Vx3 with α-Al₂O₃ nanoparticles followed by a centrifugation（the asobtained nanocomposite was denoted as α-Al₂O₃-Vx3-UPs）.It should be noted that this strategy for enriching UPs was simple and highly efficient,and by this way the vaccine efficacy was much enhanced since the as-obtained α-Al₂O₃-Vx3-UPs could be employed as both adjuvants and antigens.Methods 1.Vx3 conjugated to 4-Hydroxybenzoic acid modified α-Al₂O₃（α-Al₂O₃-COOH-Vx3）for the enrichment of UPs and its anti-tumor effect.1)Preparation and identification of α-Al₂O₃-COOH-Vx3: α-Al₂O₃ nanoparticle was firstly reacted with 4-Thydroxybenzoic acid at 90°C,after blocked by 1-butanol the modified α-Al₂O₃ was catalyzed by EDC/NHS,products were tested by Fourier infrared spectrometer;The modified α-Al₂O₃ was reacted with Vx3 at 4°C overnight,fluorescence distribution on the surface of the α-Al₂O₃ was detected by fluorescence microscope after reaction;The amount of Vx3 modified on α-Al₂O₃ was evaluated by collecting and calculating the amount of Vx3 in the supernatant,to examine the stability of α-Al₂O₃-COOH-Vx3,it was dispersed in PBS for different times and Green fluorescence was detected by fluorescence microscope;2)α-Al₂O₃-COOH-Vx3 used for the enrichment of UPs（α-Al₂O₃-COOH-Vx3-UPs）and it’s identification: α-Al₂O₃-COOH-Vx3 was added to 4T1 cell lysate and stirred at 4°C overnight,UPs enriched were texted by western blot;By calculating concentration difference,the amount of UPs enriched by α-Al₂O₃-COOH-Vx3 was got;To examine the stability of α-Al₂O₃-COOHVx3-UPs,it was dispersed in PBS for different times and western blot was used;Transmission electron microscopy was used to detect the changes of α-Al₂O₃ after conjugated with proteins;3)Immune response induced by α-Al₂O₃-COOH-Vx3-UPs and it’s anti-tumor effort: BABL/c mice were subcutaneously injected with α-Al₂O₃-COOH-Vx3-UPs,the splenocytes were cocultured with inactivated 4T1 tumor cells and released IFN-γ was detected by ELISA;Tumor burden mice was injected with α-Al₂O₃-COOH-Vx3-UPs,anti-tumor effort was evaluated by tumor size and mice survive.2.Vx3 conjugated to Triethoxysilane modified α-Al₂O₃（α-Al₂O₃-NH2-Vx3）for the enrichment of UPs and its anti-tumor effect.1)Preparation and identification of α-Al₂O₃-NH2-Vx3: α-Al₂O₃ nanoparticle was first reacted with Triethoxysilane at room temperature,then the modified α-Al₂O₃ was mixed with Glutaraldehyde,All products were tested by Fourier infrared spectrometer;The modified α-Al₂O₃ was reacted with Vx3 at 4°C overnight,fluorescence distribution on the surface of the α-Al₂O₃ was detected by fluorescence microscope after reaction;The amount of Vx3 modified on α-Al₂O₃ was evaluated by collecting and calculating the amount of Vx3 in the supernatant,to examine the stability of α-Al₂O₃-NH2-Vx3,it was dispersed in PBS for different times and Green fluorescence was detected by fluorescence microscope;2)α-Al₂O₃-NH2-Vx3 used for the enrichment of UPs（α-Al₂O₃-NH2-Vx3-UPs）and it’s identification: α-Al₂O₃-NH2-Vx3 was added to cell lysate and stirred at 4°C overnight,UPs enriched were texted by western blot;By calculating concentration difference,the amount of UPs enriched by α-Al₂O₃-NH2-Vx3 was got;To examine the stability of α-Al₂O₃-NH2-Vx3-UPs,it was dispersed in PBS for different times and western blot was used;Transmission electron microscopy was used to detect the surface change of α-Al₂O₃ after conjugation with proteins;3)Immune response induced by α-Al₂O₃-NH2-Vx3-UPs and it’s anti-tumor effort: BABL/c mice were injected with α-Al₂O₃-NH2-Vx3-UPs,α-Al₂O₃/Vx3-UPs,α-Al₂O₃-NH2-Vx3 or α-Al₂O₃/cell lysate,the splenocytes were co-cultured with inactivated tumor cells and released IFN-γ was detected by ELISA;Tumor burden mice was vaccinated withα-Al₂O₃-NH2-Vx3-UPs,α-Al₂O₃/Vx3-UPs,α-Al₂O₃-NH2-Vx3 or α-Al₂O₃/cell lysate,anti-tumor effort was evaluated by tumor size and mice survive.3.Study on the mechanism of anti-tumor immune response induced by α-Al₂O₃-Vx3-UPs.1)α-Al₂O₃-Vx3-UPs phagocytosised by DC2.4 cells and it’s intracellular localization: DC2.4 cells were incubated with α-Al₂O₃-Vx3-UPs for different times,flow cytometry was used to detect the percentage of GFP+ cells;Cell were fixed and treated with Triton X-100,stained with anti-LC3 antibody and images were captured by confocal microscope;2)Antigen from α-Al₂O₃-Vx3-UPs（OVA+）presented by BMDCs: BMDCs was induced by mouse bone marrow plus with cytokines;BMDCs were incubated with α-Al₂O₃-Vx3-UPs（OVA+）for 6 hours,stained with anti-H2Kb-SIINFEKL antibody and flow cytometry was used to detect the percentage of H2Kb-SIINFEKL+ BMDCs;3)T cell proliferation induced by α-Al₂O₃-Vx3-UPs（OVA+）loaded BMDCs: BMDCs were treated with 3-MA for 12 hours,then α-Al₂O₃-Vx3-UPs（OVA+）were added,6 hours later CFSE labeled OT-1 splenocytes were added;Co-culture for 72 hours flow cytometry was used to detect the proliferation of CD8+ T cells.Results 1.Vx3 conjugated to 4-Hydroxybenzoic acid modified α-Al₂O₃（α-Al₂O₃-COOH-Vx3）for the enrichment of UPs and its anti-tumor effect.1)Preparation and identification of α-Al₂O₃-COOH-Vx3: After the reaction between a-Al₂O₃ and 4-Hydroxybenzoic acid –OH on the surface of α-Al₂O₃ was almost replaced by –COOH,Vx3 could be conjugated through-COOH;The reduced –OH could be blocked by 1-butanol,after which α-Al₂O₃-COOH could not adsorb any protein;α-Al₂O₃-COOH-Vx3 presented green fluorescence under the fluorescent microscope,1 mg of α-Al₂O₃ could be conjugated with 50 μg Vx3 protein,and it could be stable at 4 °C for at least 2 weeks;2)α-Al₂O₃-COOH-Vx3 used for the enrichment of UPs（α-Al₂O₃-COOH-Vx3-UPs）and it’s identification: α-Al₂O₃-COOH-Vx3 had the ability to enrich UPs from cell lysate,TEM showed that the clean surface of the single-crystalline α-Al₂O₃ nanoparticles were coated with an amorphous layer after conjugation with UPs;1 mg of α-Al₂O₃-COOH-Vx3 could enrich 150 μg UPs,and it could be stable at 4 °C for at least 2 weeks.3)Immune response induced by α-Al₂O₃-COOH-Vx3-UPs and it’s anti-tumor effort: When co-cultured with inactivated 4T1 tumor cells,IFN-γ released by 1*106 splenocytes from α-Al₂O₃-COOH-Vx3-UPs vaccinated mice was 700 pg/ml;The tumor size was significantly reduced and the survival period was prolonged after the treatment of α-Al₂O₃-COOH-Vx3-UPs,H&E staining showed that there was no difference regarding the features of liver and kidney after the vaccine of α-Al₂O₃-COOH-Vx3-UPs.2.Vx3 conjugated to Triethoxysilane modified α-Al₂O₃（α-Al₂O₃-NH2-Vx3）for the enrichment of UPs and its anti-tumor effect.1)Preparation and identification of α-Al₂O₃-NH2-Vx3: FT-IR results showed that,for α-Al₂O₃,a broad absorption peak centered at about 3448 cm-1 was observed which representing hydrogenbonded OH stretching vibration;After the reaction between a-Al₂O₃ and APTES this absorption peak was significantly reduced and a new absorption band was observed at 2932 cm-1 and could be ascribed to aliphatic γ（CH2）groups;After modified with glutaraldehyde,a small absorption band centered at 1720 cm-1 and a stronger bands centered at 2860-2960 cm-1,referred to as γ（C=O） groups and aliphatic ν（CH2）groups,were found;α-Al₂O₃-NH2-Vx3 presented green fluorescence under the fluorescent microscope,1 mg of α-Al₂O₃ could be conjugated with 75 μg Vx3 protein,and it could be stable at 4 ° C for at least 3 weeks;2)α-Al₂O₃-NH2-Vx3 used for the enrichment of UPs（α-Al₂O₃-NH2-Vx3-UPs）and it’s identification: α-Al₂O₃-NH2-Vx3 had the ability to enrich UPs from cell lysate,TEM showed that the clean surface of the single-crystalline α-Al₂O₃ nanoparticles were coated with an amorphous layer after conjugation with UPs;1 mg of α-Al₂O₃-NH2-Vx3 could enrich 160 μg UPs,and it could be stable at 4°C for at least 3 weeks.3)Immune response induced by α-Al₂O₃-NH2-Vx3-UPs and it’s anti-tumor effort: When cocultured with inactivated 4T1 tumor cells,IFN-γ released by 1*106 splenocytes from α-Al₂O₃-NH2-Vx3-UPs,α-Al₂O₃/Vx3-UPs,α-Al₂O₃-NH2-Vx3 of α-Al₂O₃/cell lysate vaccinated mice were 1240,768,129 and 464 pg/ml;The tumor size was significantly reduced and the survival period was prolonged after the treatment of α-Al₂O₃-Vx3-UPs.3.Study on the mechanism of anti-tumor immune response induced by α-Al₂O₃-Vx3-UPs.1)α-Al₂O₃-Vx3-UPs phagocytosised by DC2.4 cells and it’s intracellular localization: The percentage of GFP+ DC2.4 cells incubated with α-Al₂O₃-Vx3-UPs was significantly higher than that of α-Al₂O₃/Vx3-UPs at different time points;α-Al₂O₃-Vx3-UPs,but not α-Al₂O₃/Vx3-UPs,were co-localized with the autophagosome marker,LC3,after phagocytized by DCs;2)Antigen from α-Al₂O₃-Vx3-UPs（OVA+）presented by BMDCs: CD11 c expressed on the surface of BMDCs was more than 80%;BMDCs loaded with α-Al₂O₃-Vx3-UPs（OVA+）yielded a higher level of H2Kb-SIINFEKL complexes than the BMDCs loaded with α-Al₂O₃/Vx3-UPs（OVA+）;3)T cell proliferation induced by α-Al₂O₃-Vx3-UPs（OVA+）loaded BMDCs: When loaded onto BMDCs,the covalently linked α-Al₂O₃-Vx3-UPs were superior in activating CD8+ T cells as compared to α-Al₂O₃/Vx3-UPs,and this ability could be inhibited by 3-MA.Conclusions 1.Vx3 was successfully conjugated to 4-Hydroxybenzoic acid modified α-Al₂O₃;α-Al₂O₃-COOHVx3 could enrich UPs from cell lysate;the α-Al₂O₃-COOH-Vx3-UPs could induce tumor specific immune response and had anti-tumor effort;2.Vx3 was successfully conjugated to Triethoxysilane modified α-Al₂O₃;α-Al₂O₃-NH2-Vx3 could enrich UPs from cell lysate;the α-Al₂O₃-NH2-Vx3-UPs had anti-tumor effort;Compared with α-Al₂O₃-COOH-Vx3,α-Al₂O₃-NH2-Vx3 could enriched more UPs and α-Al₂O₃-NH2-Vx3-UPs was more stable;Compared with α-Al₂O₃/Vx3-UPs,α-Al₂O₃-NH2-Vx3 or α-Al₂O₃/cell lysate the anti-tumor effort of α-Al₂O₃-NH2-Vx3-UPs was better.3.Compared with UPs absorbed by α-Al₂O₃,DCs could phagocytose more α-Al₂O₃-Vx3-UPs;The functional autophagy pathway is required for the efficient cross-presentation of α-Al₂O₃-Vx3-UPs.