The Primary Research On Assembly Of Polyvalent Aptamers Based CAR Analogues And Their Application In Activating T Cells And Targeting Lung Cancer Cells & The Screening And Identification Of Aptamers Against Inactivated H1N1 Virus

Part one:The primary research on assembly of polyvalent aptamers based CAR analogues and their application in activating T cells and targeting lung cancer cellsA number of immunotherapies,including monoclonal antibody,immune adjuvant,tumor vaccine and CAR-T?Chimeric Antigen Receptor T-Cell?immunotherapy,have been widely regarded as a potentially game-changing new tool in treatment of cancer.Amongst which,CAR-T cell therapy ranked the most promising one.CAR-T is a personalized therapy that recognizes antigens characteristic of the patient's cancer type and also a prolonged treatment that engineeried T cells will multiply,recognize and attack tumor cells in vivo.However,the safty issue of modifying and expanding T cells and other challenges still exist.Aptamer has been known as antibody analogue and been widely applied in field of oncology in recent years.In this study,we intend to build up a polyvalent aptamer structure,a CAR analogue,which can activate T cells and target tumor cells simultaneously.The CAR-like polyvalent aptamers were built with different aptamers against immunological molecules and tumor antigens,so as to develop a one-size-fits-all design of aptamer drugs to solve the safty issues of cell immunotherapy.By using the muring CD28 molecule aptamer?CD28Apt7?to activate T cell for supplying the costimulatory signal and the muring CTLA-4?cytotoxic T-lymphocyte-associated protein 4?aptamer?Del 60?for immune checkpoint blockade.As well as a stable nucleic acid three way-junction scaffold,we assembled the CAR analogue of CD28 aptamer dimer and CTLA-4 aptamer tetramer based on the principle of complementary base pairing.The folic acid labeled 3WJ chain used for epithelial tumor cell targeting.The structure was named X polymers for short.Our results showed that the CD28 Apt7 and Del 60 could recognize the recombinant muring CD28 molecules and CTLA-4 molecules respectively by EMSA?Electrophoretic Mobility Shift Assay?and SPR?Surface Plasmon Resonance?assays,and the X polymers could recognize both the recombinant mCD28 and the mCTLA-4molecules.In addition,the X polymers exhibited T cell expanding effect by CFSE cell proliferation assay.ELISA assay showed that the X polymers could also reverse the IL-2 secreting inhibitory effect of exogenous B7.1 molecules on T cells.Confocol imaging and flowcytometry assay confirmed that the X polymers could target both T cells and H460 cells.CCK-8 assay showed that the exogenous X polymers could activate the proliferation of T cells and help the attack of T cells to H460 cells in the coculture of tumorous H460 cells and T lymphocytes.In conclusion,we demonstrated for the first time that the multivalent aptamers–activated T cells could fulfill the function of CAR-T as a chemical drug.Our research provide a new idea for the immunotherapy of tumors.Part two:The screening and identification of aptamers against inactivated H1N1 virusWe evolved H1N1 aptamers using inactivated HIN1 influenza A virus particles as positive selection target and inactivated B virus particles as counter selection target by the SELEX?systematic evolution of ligands by exponential enrichment?procedure.The highly enriched 6^th pool was amplified to dsDNAs and cloned into T vectors for sequencing.Nine sequences were synthesized for further analysis for their recognition to influenza virus,amongst which A8 and A20 showed high selectivity to A type virus of H1N1 and H3N2 in contrast to no recognition to B virus and parainfluenza virus type1-3.The truncation study based on predicted secondary structure and binding assay showed that A8 and A20 aptamers as well as their truncated versions showed selectively high affinity to inactive H1N1 virus and H3N2 virus with the K_d in the low nanomolar range,with A8S8 and A20S ranked highest binding?Kd,A8S8=4.746±2.86/Kd,A20S=5.559±4.141 nM?.We then developed a sandwich ELONA?enzyme-linked oligonucleotide assay?method using A8S8 as capture aptamer and A20S as detection aptamer with a H1N1 detection limit of 0.3ng/?L.In conclusion,we developed the whole virus particle SELEX procedure,evolved H1N1 virus specific aptamer,and thus established the foundation for a safe,fast,low-cost and high sensitive H1N1 detection method.