Therapeutic Effects And Mechanisms Of Antibody-IL-2 Conjugated Nano-immunomodulators In Experimental Autoimmune Encephalomyelitis

Background:In recent years,Interleukin-2(IL-2)has garnered significant attention for its role in treating multiple sclerosis(MS)and experimental autoimmune encephalomyelitis(EAE)by balancing regulatory T cells(Treg)and effector T cells(Teff).However,the off-target activation of Teff cells by IL-2 limits its potential clinical application.To overcome this limitation,various engineered IL-2 strategies have emerged,such as IL-2 immune complexes,IL-2 fusion proteins,and de novo-designed IL-2,aimed at enhancing IL-2 selectivity for Treg cells to modulate immune tolerance.Nevertheless,these IL-2 therapies still encounter challenges due to insufficient selectivity for Treg cells and low expansion efficiency,thus affecting their therapeutic efficacy.This study is based on the high expression of T cell immunoglobulin and ITIM domain proteins(TIGIT)in Treg cells,as well as the selective expansion characteristics of Treg cells mediated by the IL-2/JES6-1 complexes(IL2C).Therefore,a rapidly prepared nano-immunomodulators termed a T-IL2C NPs was developed,which specifically recognized Treg cells with high TIGIT expression thanks to the presence of an anti-TIGIT(aTIGIT)and an IL2C being delivered to Treg cells but not to Teff cells with low TIGIT expression.Then,IL2C released IL-2 due to the specific expression of the high-affinity IL-2receptor on Treg cells,thus enabling the active targeting and selective proliferation of Treg cells.Moreover,the aTIGIT of a T-IL2C NPs selectively inhibited the proliferation of Teff cells while leaving the proliferation of Treg cells unaffected.In addition,since the IL-2 receptor on Teff cells had medium-affinity,the IL2C hardly released IL-2 to Teff cells,thus enabling the inhibition of Teff cell proliferation.Purpose:This study developed antibody-IL-2-conjugated nano-immunomodulators,which actively target and selectively expand Treg cells.We assessed the regulatory effects of these nanoparticles on Treg cells at both the material and cellular levels.Additionally,we investigated their therapeutic effects and immunomodulatory mechanisms in a mouse model of EAE induced by myelin oligodendrocyte glycoprotein(35-55),offering a potential new strategy for MS therapyMethods:1.The EAE model was established,and the expression of TIGIT on various T cell subsets(Treg,CD4~+Teff,CD8~+T cells)in mice with EAE at the peak of the disease was assessed by flow cytometry.The Pep-PLG-GEE nano-carrier was synthesized using a one-pot method,which involves grafting glycine ethyl ester(GEE)and the Fc-III-4C peptide onto poly(L-glutamic acid).The high-affinity interaction between the Fc-III-4C peptide in Pep-PLG-GEE and the Fc domain of monoclonal antibodies(aTIGIT,JES6-1)was responsible for the rapid synthesis of nano-immunomodulators(aTIGIT-IL-2/JES6-1 NPs,aTIGIT-JES6-1 NPs,and IgG NPs)in an aqueous environment.The nano-immunomodulators were characterized based on their particle size,zeta potential,and the efficiency of Fc-III-4C peptide binding to monoclonal antibodies.2.In vitro experiments:The binding ability of aTIGIT-JES6-1 NPs to Treg cells was assessed by flow cytometry.The active targeting and targeting shielding of aTIGIT-JES6-1 NPs to Treg cells were examined by confocal laser scanning microscopy(CLSM).The selective activation of Treg cells by aTIGIT-IL-2/JES6-1 NPs was detected by flow cytometry,and the optimal molar ratio of aTIGIT to JES6-1 was screened out.The number of antibodies connected to the surface of each Pep-PLG-GEE nanoparticle was tested by a Nano Sight and then calculated.3.In vivo experiments:The drug metabolism of IgG NPs in Sprague-Dawley rats was detected by microplate reader.The distribution of a T-IL2C NPs in various organs was observed using an ex vivo imaging system.EAE mice were treated according to a predetermined dosing regimen,and the regulatory effects of a T-IL2C NPs on Treg cells and antigen-specific T cell responses were evaluated by flow cytometry,CLSM,and ELISA.The therapeutic efficacy of a T-IL2C NPs in the EAE model was validated.The immunomodulatory mechanisms of a T-IL2C NPs were investigated through flow cytometric analysis of immune cells in the spinal cord and spleen of mice at the peak of EAE disease,as well as changes in cytokines in the serum at different stages of disease onset.The biosafety of a T-IL2C NPs was explored through hematoxylin and eosin(H&E)staining and hepatic and renal function tests of major organs in mice at the end of treatment.Results:1.Flow cytometric analysis at the peak of EAE disease revealed high expression of TIGIT on the surface of splenic Treg cells,with levels approximately 21.0-fold and46.5-fold higher than those on CD4~+Teff cells and CD8~+T cells,respectively.Nano-immunomodulators,such as aTIGIT-IL-2/JES6-1 NPs,aTIGIT-JES6-1 NPs,and IgG NPs,were successfully synthesized.Dynamic light scattering measurements indicated that the diameter of IgG NPs was(121.6±26.7)nm,and transmission electron microscopy images confirmed their uniformly distributed spherical structure.The Fc-III-4C peptide exhibited high-affinity binding to IgG,with minimal free IgG observed after agitation for more than 3 h.IgG NPs remained stable in PBS and high-concentration IgG solution for 48 h.Pep-PLG-GEE could simultaneously bind to two monoclonal antibodies.2.In vitro experiments:aTIGIT-JES6-1 NPs can actively target and bind with Treg cells.aTIGIT-IL-2/JES6-1 NPs can selectively expand Treg cells,and the optimal molar ratio of aTIGIT to JES6-1 was determined to be 20:1 based on the level of pSTAT5 in Treg cells,termed a T-IL-2 NPs.Nano Sight testing revealed that approximately 94 aTIGIT and IL-2/JES6-1 were conjugated to the surface of each Pep-PLG-GEE nanoparticle in a T-IL2C NPs.According to the fitted results of the standard normal distribution curve,the proportion of a T-IL2C NPs containing 2-6 JES6-1 reached 99.08%.3.In vivo experiments:The half-life of IgG NPs is(29.6±1.9)h,primarily metabolized in the liver and kidneys.After three tail vein injections,a T-IL2C NPs significantly increased the frequency of Treg cells,with no significant effect on CD8~+T cells,while significantly increasing the expression of CD25,Foxp3,PD-1,and CTLA-4,and reducing the frequency of antigen-specific Th17 cells and Th1 cells.After five consecutive tail vein injections,a T-IL2C NPs significantly delayed the onset and reduced the severity of EAE,alleviating the infiltration of lymphocytes and demyelination in the spinal cord tissue.Flow cytometric analysis at the peak of disease showed that a T-IL2C NPs significantly increased the frequency of Treg cells in the spleen and spinal cord tissues,while significantly reducing the frequency of inflammatory Th17 cells and Th1 cells.a T-IL2C NPs significantly reduced the number of activated microglia cells,increased the number of M2-type microglia cells,and significantly increased the number of nissl bodies and the expression level of MBP.At different stages of the disease,a T-IL2C NPs significantly increased the expression of anti-inflammatory cytokine IL-10 and effector molecule fibrinogen-like protein 2 in the blood,while significantly inhibiting the secretion of pro-inflammatory cytokines IL-17A and IFN-γ.H&E staining of major organs and blood biochemical analysis in mice at the end of treatment did not show any significant abnormalities.Conclusion:1.In EAE mice,Treg cells exhibit high expression of TIGIT,providing a potential target for Treg cell specific drug delivery.2.In this study,we successfully designed and validated an antibody-IL-2conjugate nano-immunomodulator(a T-IL2C NPs)with active targeting and selective expansion of Treg cells,while inhibiting Teff cell proliferation.The aT-IL2C NPs significantly delayed the onset of EAE and reduced its severity.3.The a T-IL2C NPs demonstrated significant demyelination improvement and neuroprotective effects by reshaping the Th17/Treg cell balance and modulating the inflammatory microenvironment.In conclusion,the antibody-interleukin-2 conjugated nano-immunomodulators prepared in this study offer a promising therapeutic strategy for MS therapy,while also providing new insights into the precise treatment of other autoimmune diseases.