Study On The Construction Of Dual Targeted Nano-immune Cluster Against CD47 And PD-L1 And Its Effect In The Treatment Of Bladder Cancer

BackgroundBladder cancer(BC)is the tenth most common cancer in the world and the most common malignancy of urinary system in China.BC has the high recurrence rate and progression rate,and the prognosis of patient with BC is poor.At present,immunotherapy including inhibitors of PD1/PD-L1 has the promising efficacy in the treatment of bladder cancer.Five PD1/PD-L1 inhibitors have been approved by the US FDA for the treatment of patients with locally advanced or metastatic urothelial carcinoma,but,the objective response rate is only 15%-24.4%,which means only minority of BC patients can benefit from this therapy.The combination with other innate immune checkpoint inhibitors provides an improved strategy to enhance the efficacy of PD1/PD-L1 inhibitors for BC patients.PD-L1 is a signal of "can’t find me" on the surface of tumor cells and an adaptive immune checkpoint,while CD47 is a signal of "don’t eat me" and an innate immune checkpoint.Both PD-L1 and CD47 are highly expressed on the bladder cancer cells.CD47 was expressed on more than 80% bladder cancer cells and higher on bladder cancer stem cells.The sensitivity and specificity of fluorescence-labeled CD47 antibody in the diagnosis of BC in blue light cystoscopy were 82.9% and 90.5%,respectively.Therefore,the combination of anti-CD47 m Abs and anti-PD-L1 m Abs is expected to significantly enhance the efficacy of anti-PD-L1 m Abs,reduce the recurrence and progression of bladder cancer,and improve the prognosis of patients with BC.Previous experimental studies had shown that the combination of anti-PD-L1 m Abs and anti-CD47 m Abs or the fusion proteins targeted to both PD-L1 and CD47 could significantly improve the anti-tumor effect on mouse models of breast and colon cancer compared with free antibodies.However,both of them are single antibodies with lower tumor enrichment and more side effects relatively,which limit the efficacy of the combined immunotherapy.Nano drugs can increase their enrichment in tumors by enhanced penetration and retention effects and achieve active targeting and synergistic anti-tumor effects by loading different antibodies.Hence,multiple different m Abs can be connected into a whole through nanotechnology to achieve targeting and therapeutic effects of different antibodies,which lead to better anti-tumor effect compared with the combined use of different free antibodies.ObjectiveThis study aimed to fabricate a dual targeted nano-immune cluster focusing on blocking innate and adaptive immune suppression,and explore its effect as well as the mechanism in the treatment of bladder cancer.Methods 1.Compare the expression of CD47 and PD-L1 between with SV-HUC-1 cells and 5637,HT-1376,T24 cells by flow cytometry(FCM).Construct a subcutaneous bladder cancer model of C3H/He N mice with MBT2 cells and compare the expression of CD47 and PD-L1 between with MBT2 cells and primary tumor cells from the mice by FCM.2.A dual targeted nano-immune cluster(NCPC)was constructed through anchoring anti-CD47 m Abs and anti-PD-L1 m Abs to the branched polyethyleneimine(PEI)with a molecular weight of 750 k Da.The connection between the antibody and PEI was confirmed by SDS-PAGE and Zeta potential measurement.The particle size distribution and 3D morphology was characterized by dynamic laser scattering(DLS) and atomic force microscope(AFM),respectively.The biocompatibility of PEI and its intermediates was evaluated by CCK-8 kit.3.In vitro: We evaluated the antigen-binding and dissociation ability of NCPC and CPB (blend of anti-CD47 m Abs and anti-PD-L1 m Abs)through immunofluorescence microscope,and the cytotoxic and apoptosis-promoting effects of NCPC and CPB on 5637 cells;We further evaluated their effects on phagocytosis of macrophages and tumor-infiltrating lyphocytes(TIL)activation.4.Extract the RNA from 5637 cells untreated and treated with CPB and NCPC for 24 h for transcriptome sequencing.Bioinformatics analysis was used to explore the possible mechanism of NCPC on bladder cancer cells.5.The morphological and mechanical change of 5637 cells after exposure to NCPC was detected by AFM.The effect of NCPC on mitochondrial membrane potential of 5637 cells was evaluated by FCM with JC-1 probe.Westing Bloting was used to evaluate the expression of capase 3 after 5637 cells were treated with NCPC.6.The distribution of NCPC in tumor-bearing BLAB/c nude mice was evaluated by in vivo imaging system(IVIS)and fluorescence microscope.Based on the subcutaneous bladder cancer model of C3H/He N mice,we evaluate the tumor suppression effect of NCPC in vivo.FCM and FCM based on fluorescent microsphere technology were used to detect distribution of tumor infiltrating immune cells in tumor tissues and cytokine concentration in tumor tissues and serum from mice after different adminstration, respectively.7.The safety of NCPC was evaluated by body weitht changes of tumor-bearing mice,the blood routine examination,liver and kidney function test,and changes in the cell morphology and structure of the main organs tissues from the mice after treatment.Results 1.The expression of CD47 and PD-L1 on the surface of 5637,HT-1376,and T24 bladder cancer cells were higher than that of SV-HUC-1 cells.MBT2 cells had increased CD47 and PD-L1 expression in the tumor microenvironment.5637 and MBT2 cell lines as well as C3H/He N mice were selected as the objects in the follow-up experiments.2.SDS-PAGE showed that the molecular weight of NCPC was significantly increased compared with free m Abs.Zeta potential measurement showed that the Zeta potential of NCPC approached zero,when the Zeta potential of PEI and free m Abs was positive and negative,respectively.Both of them indicatied that the m Abs were anchored to the PEI successfully.DLS showed that the mean particle size distribution of NCPC was about 133 nm.AFM showed that the 3D morphology of NCPC was like the net.CCK-8 experiment indicated that PEI and its intermediates were highly biocompatible at the choiced concentrations in this study.3.In vitro: Fluorescence microscopy showed that the NCPC had intact antigen recognition and binding ability,moreover,dissociation rate with the antigen is slower than that of the free m Abs;NCPC could kill bladder cancer cells by promting phagocytosis of macrophages and activating TIL activity.4.RNA-seq showed that the NCPC caused more differentially expressed genes than CBP, suggesting that NCPC has an unique mechanism on bladder cancer cell.Further GO and KEGG enrichment analysis showed that NCPC might change cells morphological structure by binding to the antigen on the membrane,then activated downstream signaling pathways(including MAPK and IL-17 pathways)and futher affected the development and apoptosis of bladder cancer cells.5.AFM showed that NCPC acted on the membrane of 5637 cells in a network shape, which caused the cell become round and Young’s modulus increased.In addition, NCPC could cause depolarization of mitochondrial membrane potential in 5637 cells and lead to increased expression of caspase 3.6.IVIS showed that NCPC more enrich in the tumor tissues than CPB.In vivo,NCPC can significantly reduce the tumor burden in C3H/He N mice compared with free m Abs and CPB.Detection of tumor infiltrating immune cells and cytokines showed that NCPC can increase the proportion of M1 macrophages,NKT cells,Tc cells and other immune effector cells in the tumor microenvironment,and promote the secretion of anti-tumor cytokine such as TNF-α and IFN-γ,which could significantly enhance its anti-tumor activities by activating innate and adaptive immunity.7.The evaluation of body weight chang,blood routine examination,liver and kidney function test and structure in main organ tissues all indicated NCPC was safe in the administration.ConclusionThis study showed that NCPC simultaneously blocking two kinds of innate and adaptive immune checkpoints was successfully constructed,and it has excellent ability in surpressing bladder cancer in vitro and in vivo through induceing apoptosis,promting phagocytosis of macrophages and activating TIL activity.