The Enhanced Effect Of Novel Bispecific Nucleic Acid Aptamers On The Killing Effect Of NK Cells Against Adenocarcinoma

Objective:A promising strategy in cancer immunotherapy is the employment of a bispecific agent that can bind with both tumor cells and immunocytes for recruitment of lymphocytes to tumor sites and enhancement of anticancer immune reactions.Mucin 1(MUC1)is a tumor marker overexpressed in almost all adenocarcinomas,making it a potentially important therapeutic target.CD 16 is expressed in several types of immunocytes,including NK cells,??-T cells,monocytes,and macrophages.Aptamers are short,single-stranded DNA or RNA(ssDNA or ssRNA)that can form complicated three-dimensional structures and bind to target molecules with high affinity and specificity.In this study,two MUC1 aptamers and two CD 16 aptamers were integrated into one single molecule.The avidity of this newly-designed bivalent bispecific aptamer(BBiApt)to target cells was evaluated and compared with monovalent MUC1 aptamer or CD 16 aptamer.We also studied whether BBiApt could bring CD 16-positive immunocytes to the vicinity of MUC1-positive tumor cells,and strengthen the immune cytotoxicity of CD 16-positive immunocytes against the target MUC1-positive tumor cells.Method:Both the monovalent bispecific aptamer and BBiApt were constructed by overlapping PCR.Large amounts of BBiApt were produced by helper phage culturing.The binding avidity and binding cites of BBiApt to target cells were evaluated by flow cytometry,and compared with monovalent MUC1 or CD 16 aptamers.Trypsin digestion experiment was conducted to confirm if BBiApt bound to the extracellular domains of membrane proteins.Fluorescent microscope was used to evaluate whether BBiApt could tether the MUC1-expressing tumor cells and NK cells together.Immune cytotoxicity of NK cells against the target cells was evaluated by a standard MTS assay.Result:In this study,we constructed the first bispecific aptamer targeting both MUC1 and CD 16.This aptamer consisted of two MUC1 aptamers and two CD 16 aptamers linked together by three 60 nt DNA spacers.Compared with monovalent MUC1 or CD 16 aptamers,BBiApt showed more potent avidities to both MUC1-positive tumor cells and CD 16-positive immunocytes.Competition experiments indicated that BBiApt and monovalent aptamers bound to the same sites on the target cells.Moreover,BBiApt recruited more CD 16-positive immunocytes around MUC1-positive tumor cells and enhanced the immune cytotoxicity against the tumor cells in vitro.Conclusion:BBiApt had higher avidities to both MUC1-positive tumor cells and CD 16-positive immunocytes,tethered the two types of cells together,and significantly enhanced the antitumor cytotoxicity in vitro.The results suggest that,apart from bispecific antibodies,bispecific aptamers may also potentially serve as a novel strategy for targeted enhancement of antitumor immune reactions against MUC1-expressing malignancies.