Construction Of CD20 And PD-L1 Dual Targeting Nano-Immune-System And Mechanism Of Synergistic Effect Of Anti-Lymphoma

Rituximab was approved by the FDA for medical treatment in 1997,and it is the first IgG1 monoclonal antibody approved for clinical non-Hodgkin’s lymphoma(NHL)treatment.Although Rituximab has made some effect in the early treatment of tumor,researchers found that anti-CD20 antibodies having some defects in the clinical treatment,such as short half-life in vivo,the moderate effect,and drug resistance.In addition,due to the presence of immunological checkpoints,tumor cells are prone to immune escape.Therefore,based on the antibody mechanism and tumor microenvironment,the construction of multifunctional antibodies to improve its efficacy is the clinical antibody targeted therapy to solve the problem.Recently,with the development of nanotechnology and materials science,the nano-carrier technology that is based on the delivery system provides a perfect solution.In this study,we selected branching cationic polymer polyethyleneimine(PEI,molecular weight 750 kDa),its side-based NH2 connecting with MAL-PEG-SCM molecules which can react with the antibody.A large number of anti-CD20 and PD-L1 antibodies are attached to the molecules.Then,CD20 and PD-L1 double-targeted nanometer antibody clusters are constructed(abbreviated as PEI-RTX-AZL).The results of Dynamic Laser Light Scattering,Transmission electron microscopy and Marvin Potentiometer demonstrate the successful synthesis of antibody clusters.The antibody has a coupling efficiency of 63%,and the PEI-RTX-AZL has an ellipsoidal topology.Analysis of the binding capacity and dissociation capacity of antibody clusters to Daudi cells showed that the PEI-RTX-AZL compared to Rituximab have similar binding capacity,but the dissociation ability was significantly reduced.That is to say,antibody clusters can be more long time in the target cell surface,and it’s sustained duration more durable,Which lay the foundation for further enhancing the efficacy of antibodies.Next,in the study of antitumor activity in vitro,we found that antibody clusters had a more pronounced antibody-dependent cell-mediated cytotoxicity(ADCC)and programmed cell death(PCD)in addition to proliferation inhibition effects to Daudi cells.In this study,the mechanism of anti-tumor effect of antibody clusters was further explored.Compared with free parental antibodies,the process of antibody clusters to induce Daudi cells apoptosis can be summarized in the following two aspects: On one hand,the Fab fragment of the anti-CD20 antibody specifically targets CD20-positive Daudi cells,and the Fc segment of the antibody binds to FcgR of NK cells and other effector cells.Then,the effector cells attack the target cells to make it crack.In addition,anti-PD-L1 antibody was delivered to the surface of tumor cells.The anti-tumor activity of CTL cells was restored by anti-PD-L1 antibody blocking PD-1 / PD-L1 signaling pathway.Two antibodies play synergistic anti-tumor effect.On the other hand,under the target of Rituximab antibody,750 kDa long-chain PEI binds to CD20 molecules,making the antigen cross-linked or antigen-limited,inducing the lysosome of target cells rupture or distribution of dispersion,and then lysosomal Cathepsin B is realeased to the cytoplasm.Cathepsin B acts on the mitochondria,resulting in increased permeability of the mitochondrial membrane,leading to the release of the apoptotic molecule(cytochrome C)into the cytoplasm.The cytochrome C activates the Caspase pathway and induces cells apoptosis cascade.The anti-tumor effect of antibody clusters was also studied.In order to more intuitively observe the antitumor effect of antibody clusters at animal level,we constructed a subcutaneous solid tumor model of Balb/c nude mice.We observed and analyzed the antitumor effect of antibody clusters PEI-RTX-AZL and monoclonal antibody Rituximab.Studies have shown that antibody clusters than monoclonal antibody has a stronger anti-tumor effect that reduces tumor burden.In conclusion,the antibody cluster specifically targets tumor cells by anti-CD20 antibody,and blocks PD-1/PD-L1 signaling pathway by anti-PD-L1 antibody,and then restores CTL cell anti-tumor activity.Two antibodies play a synergistic anti-tumor effect,and further enhance the proliferation inhibition,ADCC and PCD,and thus give full scope to the effect.In this work,we provide method guidance and theoretical basis for the future design of targeted therapeutic drugs for clinical use by systematic carrier design,antibody conjugation,in vitro/vivo antitumor mechanism research.