Tumor Active Immunotherapy By Multipotent Artificial Antigen-presenting Cells Based On PLGA Microparticle

Artificial antigen-presenting cells（aAPCs）play a vital role in facilitating T cells proliferation and tumor immunotherapy.There are two classes of aAPCs according to the carrier materials.The first one is cellular aAPCs that co-expressing the peptide-loaded major histocompotability complex（pMHC）and co-stimulatory molecules on the surface of xenogeneic or allogeneic cells such as murine fibroblasts（3T3）,human erythroleukemia cells（K562）and drosophila cells,by transfection of multiple recombinant genes.The second one is cell-free aAPCs that have the acellular scaffolds such as magnetic beads,polystyrene microspheres,exosome and liposome.Recombinant pMHC multimers and co-stimulatory molecules were co-coupled onto their surface.The in vivo observational studies reported by us and other researchers have demonstrated that the static and solid acellular aAPCs have the potent ability to activate and proliferate tumor antigen specific T cells in an antigen-specific manner with the infitring prospects for clinical uses.However,these previous cell-free aAPCs reported only present two or three kinds of surface molecules in design;these works do not elucidate the in vivo mechanisms,spatial and temporal distribution and fates of aAPCs after administration;furthermore,their scaffold materials,such as magnetic beads and polystyrene microspheres etc.,are not biodegradable and are not suitable for clinical application.Based on the previous works,this study aims to thoroughly optimize and improve the cell-free aAPCs system in design,and investigate the precise mechanisms by which aAPCs achieve their therapeutic effects in vivo.（1）The biodegradable,biocompatible and non-toxic poly（lactic-co-glycolic acid）microparticles（PLGA-MPs）were used as scaffold for the putative use in human.（2）Encapsulating the mixture of IL-2,IL-15,CCL21,blockade of both CTLA-4 and PD-1 inside the PLGA-MPs to recruit and activate T cells by activating multiple stimulatory signal pathways and blocking the inhibitory signal pathways,in a paracrine delivery manner.（3）Co-coupling pMHC mutilmers carrying two kinds of tumor antigen epitopes,anti-CD28,anti-4-1BB,anti-CD2 and CD47 molecules onto the surface of PLGA-MPs to provide two antigen-specific signals,co-stimulatory signals,adhesion molecules,and anti-phagocytosis molecules for the activation of antigen-specific T cells by surface presentation.The biomimetic,multivalent and multipotent artificial antigen-presenting cells（MaAPCs）will be generated by using the biodegradable PLGA-MPs and 11 kinds of immune molecules,and administered into melanoma-bearing mice followed by the investigation of therapeutic outcome,precise mechanism,tissue distribution,contacts with immune cells,overall innune function,side effects and organ toxicity.This approach will provide a novel strategy for the antigen-specific active immunotherapy of cancers or persistent infections.Objective:To generate the multipotent aAPCs carrying 11 kinds of immune molecules,investigate their capability to expand antigen-specific T cells both in vitro and in vivo,and elucidate the therapeutic outcomes to inhibit tumor growth and the precise in vivo mechanisms.Methods and Results:1.Fabrication,characterization and function detection of surface modified PLGA microparticles:PLGA-MPs encapsulating BSA with a diameter of 4.6μm were prepared using a double-emulsion solvent evaporation method and followed by surface modification in EDC/NHS chemistry method.The shape,size distribution and zeta potential of PLGA-MPs were characterized by scanning electron microscopy,dynamic light scattering technique and PALS zeta instrument,respectively.BCA protein quantification and flow cytometry were used to determine the capability of PLGA-MPs to couple and encapsulate protein.The results showed that the MPs displayed a spherical shape with smooth surface under the SEM,and in a diameter of4.6±1.9μm;BSA encapsulation efficiency was 91.0±4.1%and the encapsulated protein can release constantly;the surface modified PLGA-MPs with the zeta potential of 31.3±5.58 mV showed strong capability to couple protein.The maximum adsorption of BSA was up to 73μg/5×10⁶ MPs.These results indicate the well characterization and protein encapsulation and absorption capability of the in-house PLGA-MPs.2.Generation and characterization of MaAPCs.The MaAPCs were developed by co-coupling pMHC multimers（H-2K^b/TRP2-Ig and H-2D^b/gp100-Ig）,anti-CD28,anti-4-1BB,anti-CD2 and CD47-Fc onto the PLGA-MPs and encapsulating the mixture of IL-2,IL-15,CCL21,anti-CTLA-4 and anti-PD-1 inside.ELISA assay revealed that each kind of soluble molecules can be encapsulated into MaAPCs and can release from MaAPCs in a constant manner,and without the interference on each other.All the 6 kinds of surface molecules have been immobilized onto the MaAPCs as determined by flow cytometry and confocal microscope.These results indicate that MaAPCs carrying 11 kinds of immune molecules have been successfully generated in house,with the correct phenotype and paracrine release.3.MaAPCs expand antigen-specific CTL cells in vitro.Blank-MPs,MPs co-coupled with 6 signals on the surface(MPs⁶⁺),MPs encapsulated with 5 signals inside(MPs₅₊)and MaAPCs carrying 11 signals were prepared and co-cultured with spleen lymphocytes from C57BL/6 mice for 7 days,respectively.Then the cells were stained by H-2K^b/TRP2-Ig dimer or H-2D^b/gp100-Ig dimer followed by flow cytometry to detect the frequencies of TRP2₁₈₀–₁₈₈88 or gp100_25-33-specific CD8⁺T cells in the splenic lymphocytes.As compared with the Blank-MPs group,TRP2_180-18880-188 antigen-specific CD8⁺T cells were successfully expanded around 268.4 times by MaAPCs,124.2 times by MPs⁶⁺,15.5 times by MPs₅₊during 7 days under the current co-culture conditions without the presence of IL-2.And gp100_25-335-33 antigen-specific CD8⁺T cells were successfully expanded around 333.1 times by MaAPCs,166.9 times by MPs⁶⁺,16.6 times by MPs₅₊during 7 days when compared with Blank-MPs group under the same condition.4.MaAPCs expand antigen-specific CTL cells and inhibit tumor growth in vivo.aAPCs carrying different molecules were prepared,such as MP²⁺（MPs co-coupled with H-2K^b/TRP2-Ig and H-2D^b/gp100-Ig）,MP⁵⁺(MP²⁺co-coupled with anti-CD2,anti-4-1BB and anti-CD28),MP⁶⁺(MP⁵⁺co-coupled with CD47-Fc),MP₂₊⁶⁺(MP⁶⁺encapsulated with IL-2 and IL-15),MP3⁶+⁺(MP₂₊⁶⁺encapsulated with CCL21)and MaAPCs（MP3⁶+⁺encapsulated with anti-CTLA-4 and anti-PD-1）.These aAPCs were administered,respectively,into the C57BL/6 mice via tail vein on days 7,9 and 11after B16 cells challenge.The tumor growth was monitored at 3-day interval.On day28,peripheral blood,spleens and tumor tissues were harvested from each mouse and processed to single cell suspensions.H-2K^b/TRP2-Ig dimer or H-2D^b/gp100-Ig dimer staining and flow cytometry were performed to detect the frequencies of TRP2₁₈₀–₁₈₈or gp100_25-33-specific CD8⁺T cells in these cell suspensions.As compared with the control aAPCs,MaAPCs displayed much stronger capability to expand antigen specific T cells in vivo,increase the infiltration of TRP2₁₈₀–₁₈₈88 and gp100_25-33-specific CD8⁺T cells in tumor tissue,thus inhibit the tumor growth and prolong the survival of tumor-bearing mice.5.The in vivo mechanism by which MaAPCs inhibit tumor growth.Blank-MPs,MP²⁺,MP⁵⁺,MP⁶⁺,MP₂₊⁶⁺,MP3⁶+⁺and MaAPCs were injected,respectively,into the C57BL/6 mice via tail vein on days 7,9 and 11 after B16 cells challenge.On day 14,spleen cells were prepared from each mouse and were used in several experiments to elucidate the anti-tumor immune responses initiated by MaAPCs.When compared with these control aAPCs,MaAPCs treatment much more effectively expanded the antigen-specific CTLs,increased the proportion of activated and memory cells in the antigen-specific CTLs population,enhanced the degranulation of CD8⁺T cells and TRP2₁₈₀–₁₈₈88 or gp100_25-33-specific CTLs,and more effectively facilitated the cytotoxicity of CTLs on tumor cells in an antigen-specific manner.Meanwhile,the MaAPCs treatment more effectively inhibited Treg proliferation and the apoptosis of CD8⁺T cells and TRP2₁₈₀–₁₈₈88 or gp100_25-33-specific CTLs,enhanced the activation and proliferation of CD8+T cells and TRP2₁₈₀–₁₈₈88 or gp100_25-33-specific CTLs ex vivo,more effectively promoted inflammatory cytokines secretion and reduced the inhibitory cytokines production.6.In vivo tracking and tissue distribution of MaAPCs and contacts with immune cells in vivo:Indocyanine green（ICG）-encapsulated MaAPCs,H-2K^b-aAPCs（non-targeting MaAPCs without pMHC dimers）,CD4^7-aAPCs（MaAPCs without CD47-Fc）and Blank-MPs were injected,respectively,into the C57BL/6 mice via tail vein on day 11 after B16 cells challenge.Then whole-body near-infrared imaging at various time points and ex vivo imaging of excised organs were carried out for each mouse to determine the spatial and temporal location of MaAPCs.PE-labeled MaAPCs were injected into the melanoma-bearing mice through tail vein on day 11and spleens were dissected after 4 hours to prepare the frozen section.FITC-labeled CD4,CD8,CD11b,F4/80 and CD19 mAbs staining were performed to display the co-localizations of MaAPCs with various immune cells under laser scanning confocal microscope.As shown,MaAPCs can rapidly distribute into various organs and tissues after tail vein injection with a retention time of over 36 hours in vivo,but much more MaAPCs accumulated in lymphoid tissues such as spleen and lymph nodes as compared with H-2K^b-aAPCs,CD^47-aAPCs and Blank-MPs.In the spleen sections,MaAPCs displayed lots of contacts with CD⁸⁺T cells,but few co-localizations with CD⁴⁺T cells,DCs,macrophages and B cells.7.MaAPCs do not lead to the impairment of host overall immune function and visible organ toxicity.MaAPCs,Blank-MPs and PBS were injected,respectively,into the C57BL/6 mice via tail vein on days 7,9 and 11 after B16 cells challenge.On day14,the frequencies or function of many immune cells in spleen cells were detected.On days 14,21 and 28,the blood routine test,biochemical analysese for the function of liver and kidney,and HE staining for main organs were carried out.As shown,the MaAPCs treatment increased the proportions of CD3⁺T cells and CD8⁺T cells in the spleen lymphocyte population,elevated the numbers of white blood cells and lymphocytes and the frequency of lymphocytes in the peripheral blood,but did not change the nummers or frequencies of other immune cells in spleen and peripheral blood.The MaAPCs treatment enhanced the alloreactivity of host T cells in response to allogenic spleen cells and the cytotoxicity of host T cells against non-melanoma tumor cells（S180 and SP2/0）to some extent,but did not inhibit the NK cells and their cytotoxicity against tumor cells,and also did not change the level of melanoma cell-specific IgG antibodies in host serum.The treatment did not impair the function of liver and kidney and the parameters of blood routine test,and also did not induce visible organ toxicity for spleen,kidney,liver,heart and lung,at several time points.Conclusion:The biodegradable multipotent artificial antigen-presenting cells delivering 11kinds of immune molecules by surface presentation and paracrine release have been successfully generated.The MaAPCs can powerfully expand tumor antigen-specific CTL cells both in vitro and in vivo,bystimulating multiple signal pathways.After injecting into tumor-bearing mice,the MaAPCs can directly contact antigen-specific CTL cells in vivo and greatly facilitate the T cells activation,proliferation and cytotoxicity;increase the frequency of memory T cells;inhibit the proliferation of regulatory T cells and the apoptosis of antigen-specific CTL cells;promote inflammatory cytokines secretion and reduce inhibitory cytokines production.Underlying these mechanisms,MaAPCs treatment induced powerful active immune responses to tumor and strong effects on tumor inhibition,without the impairment of overall immune function and visible organ toxicity.This study demonstrates a novel strategy and immune agent for the antigen-specific active immunotherapy of tumors.