Nanobodies-engineered Exosomes And NK Cells For Targeting Tumor Therapy

Exosomes are a class of bilayer membrane nanovesicles of endocytic origin secreted by all cells.As a new type of drug delivery vesicle,exosomes have advantages such as good tissue penetration,high biocompatibility,and low immunogenicity.However,the lack of modification of tumor targeting molecules on the surface of native exosomes greatly limits the efficiency of exosomal drug delivery towards tumors.The development of an efficient,convenient,low-cost exosomal drug delivery system that can target tumor cells is the prerequisite for its clinical application.Natural killer cells（NK cells）are a class of cytotoxic lymphocytes that play vital roles in the innate immune system.NK cells are also widely used in adoptive immunotherapy for tumor due to their unique anti-tumor advantages.Their applications also face with the obstacles of the lack of tumor targeting molecules modifications.In order to endow NK cells with tumor targeting,a series of chimeric antigen receptors were induced onto NK cells（CAR-NK）.It has been reported that CAR technologies designed for CAR-T are also the most commonly used approaches for constructing CAR-NK cells,but NK cells exhibit low sensitive to virus infection,and the use of adenovirus vectors have potential safety risks.Nanobodies are known the smallest antibody at present,which possess the advantages of good stability,high specificity,strong tissue penetration and low immunogenicity.They are a new generation of antibody drugs.Thus,the exosomes and NK cells with nanobodies modification will promote the development and clinincal application of tumor targeted drug delivery and adoptive cell immunotherapy.In this study,we developed a new strategy to render nanobodies onto surface of exosomes and NK cells,and evaluated their tumor targeting ability and tumor therapy effect after modification.The main research contents and results are as follows:（1）Nanobodies were modified onto exosomes that naturally carry tumor inhibitor to achieve targeted drug delivery therapy for tumors via metabolic glycoengineering combined with bioorthogonal reaction strategy.HEK293T cells stably expressed the tumor inhibitor miR-204-5p,established by our group,was selected as a tool cell.These cells were labeled with azide groups after the precursor 1,3,4-O-acetyl-N-azidoacetylmannosamine（Ac₄Man NAz）was metabolized by these cells.They could secrete exosomes loaded with miR-204-5p and modified with azide groups.Moreover,DBCO-modified nanobody 7D12（DBCO-7D12）was synthesized by chemically enzymatic method.Subsequently,azide-modified exosomes were bioorthogonally reacted with DBCO-7D12 to obtain the nanobodies decorated exosomes with drug encapsulation.The exosomal characterization uncovered the properties of these exosomes were not significant differences after nanobody modification.The tumor targeting analyses revealed that the exosomes modified by 7D12 have good targeting abilities for EGFR⁺tumors,and can deliver more miR-204-5p into tumor cells,resulting in excellent antitumor treatment effect.The tumor growth inhibition reached 80%.（2）“Off the shelf”strategy prepared a ready-to-use drug-loaded exosomes with nanobodies modification,which exhibited anti-phagocytosis and tumor-targeting abilities.The recombinant lentivirus plasmid expressed fusion protein was constructed through fusing 7D12and human CD47 gene fragments,and was induced into the HEK293T cells expressed stably the tumor inhibitor miR-204-5p via lentivirus technology.The resultant cells secreted exosomes loading miR-204-5p and decorated with 7D12 and CD47 simultaneously.Both cell and mouse level experiments have shown that these engineered exosomes improve anti-phagocytic effect（70%）through the interaction between the CD47 on tumor cell and SIRPαof macrophage,thereby extending their blood half life.Preliminary mechanism studies indicated that these engineered exosomes can also promote the phagocytosis of tumor cells by macrophages through targeting M2-like tumor-associated macrophages（M2-TAM）and reprogramming M2-TAM to M1.In addition,the engineered exosomes exhibited superior EGFR⁺tumor targeting properties,and could efficiently deliver the tumor inhibitor miR-204-5p into tumor cells.They exert the induction of tumor cells apoptosis and the inhibition of tumor cell proliferation and metastasis,generating excellent antitumor therapeutic effect.（3）Nanobodies-engineered CAR-NK cells mimics were constructed using metabolic glycoengineering combined with bioorthogonal reaction strategy for tumor adoptive immunotherapy.The NK92-MI cell line as a usual clinic“off-the-shelf”product was selected as the tool cell in this case,and was modified with azide groups using medium containing Ac₄Man NAz.The DBCO-7D12 could be further modified to the surface of NK cells through bioorthogonal reaction,generating 7D12-modified NK92-MI cells.The affinity of 7D12-decorated NK92-MI cell line was significantly increased to EGFR⁺tumor cells,thus activating NK cells to release cytokines（Gzms-B,IFNγ,IL2,and perforin）to enhance the tumor killing effects.In vivo animal experiments have shown that compared to unmodified NK92-MI cells,7D12 engineered NK92-MI cells have a 4-fold increase in penetration and a 6-fold increase in tumor inhibition rate in solid tumors.In addition,the same evaluation was conducted in human primary NK cells,further confirming the promotion of 7D12 modification on the anti-tumor effect of NK cells.