Construction Of Bi-specific Aptamer And Preliminary Antitumor Activity Study

Aptamer is single-strand oligonucleotide,which binds to target with its unique spatial structure.The bispecific aptamer consist of two single aptamers interacting or bridging to form a bifunctional aptamer with dual aptamers.Single or bispecific aptamers have high affinity with target molecules.Due to its high specificity and low immunogenicity,it has potential application incancer therapy.Bispecific aptamers has the advantages of bi-targeting and bi-specificity,which can stimulate or regulate the immune reaction between effector cells and tumour cells.This paper focuses on the proof-of-concept study of bispecificaptamers similar to the bi-specific antibodies,the focus in the biotech drugs R&D,by constructing bispecific aptamers targeting tumor cells and immune cells to study its pharmacodynamics in mediating tumor targeting by immune cells.By using exponentially enriched ligand system evolution(SELEX)technique,candidate aptamers with specificity and affinity were obtained by using human phosphatidylinositol proteoglycan(Glypican-1,GPC1)and NKG2 D molecules on the surface of human NK cells as targets,respectively.Then two candidate aptamers are truncated and linked with Poly A or other random oligonucleotide sequences.Finally,a double specific aptamer N-G-Dual Apt was constructed and its pharmacodynamics was preliminary investigatedin vitro.Part Ⅰ: Optimization of SELEX Screening and NKG2 D,GPC1 aptamers selectionUsing the optimized SELEX screening technique,the candidate aptamers of GPC1 and NKG2 D were obtained after 10 rounds of screeningby library 1 and library 2,respectively.Affinity of aptamers were evaluated using enzyme linked immunosorbent assay(ELISA).The Kd of candidate aptamers G-#35 targeting GPC1 is 186±27n M,and the Kd of the NKG2 D candidate aptamer is 126 ±17n M.Specificity of the two candidates were determined using flow cytometry in PANC-1 cell line with high expression of GPC1 and human NK cells with high expression of NKG2 D.Part Ⅱ: Characteristics of GPC1 and NKG2 D truncated aptamersSeries of GPC1 and NKG2 D truncated aptamers were designed and affinity and specificity were evaluated.The affinity constants of the truncated aptamer were determined by ELISA experiment.The affinity constant ofcandidate GPC1 truncted aptamer(-30-G-30)and NKG2 D truncted aptamer(-20-N-15)was 198±19 n M and 174 ±18 n M,respectively.Flow cytometry experiments further demonstrated that aptamer #-30-G-30 had specificity to PANC-1 cell line(75%),and aptamer #-20-N-15 had specificity to NK cells(41%)with 293 T cell as control.Part Ⅲ: Construction and Identification of Bispecific Aptamer N-G-Dual AptThe bispecific aptamer was constructed using candidate-30-N-30 aptamers and-20-N-15 aptamers with different combinations,different lengths of Poly A or random oligonucleotides as linkers.Flow cytometry experiments were performed to verify the binding specificity of different N-G-Dual Aptwith PANC-1 cell line and human NK cell with 293 T as control.Results showed that N-G-Dual Apt kept the similar specificity to cells asindividual aptamaer controls.The affinity of bispecific aptamer N-G-Dual Apt was determined by ELISA with Kd 204±6n M and 235 ±12n M,respectively.Part Ⅳ: Study on Pharmacodynamics of Bispecific Aptamer N-G-Dua Apt in vitroTumor cell growth inhibition of N-G-Dual Apt was determined in vitro by co-culture of PANC-1and NK cells in addition of N-G-Dual Apt and individual control.NK cells activation status evaluations were also performed by investigate the expression levels of NKG2 D,Perforin and Granzyme B genes using real-time quantitative PCR(q PCR).Significcant tumor cells apoptosis were observed and the m RNA expression of NKG2 D,Perforin and Granzyme B were up-regulated,which showed the role of N-G-Dual Apt in the cytotoxic killing of PNAC-1 by NK cells.In summary,we optimized the SELEX technique route and successfully screened aptamers targeting GPC1 and NKG2 D.Bispecific aptamer construction strategy was determined and preliminary tumor killing efficacy of the N-G-Dual Apt was evaluated.The proof-of-concept study here provied potential immunotheray role of bispecific aptamers.