Development Of A New Approach For High Speed Screening Aptamer And Preparation Of Anti-CD33 Aptamer- Drug Conjugates(ADCs)

Objective:Present study was separated to two different parts. In the first part, we used the Classical Protein-SELEX method to compare with our unique Smart Cell-SELEX approach. The aim was to obtain high binding affinity of anti-CD33 aptamers and to shorten screening time by our Smart Cell-SELEX approach. In addition, we further studied change in binding affinity and stability of aptamer after modification of its aptamer structure. In part two, we mainly focused on the construction of Aptamers-Drug Conjugates (ADCs). In particular, we predominantly tested the different linkers conjugated with anticancer drug Doxorubicin (Dox), and then compared the anticancer effects and release of drug from the Dox-SPDP~?and Dox-SPDP~? complexes.Methods:Part one:Two different screening approaches were used to current study for selection of anti-CD33 aptamers. Here, the Protein Based-SELEX was used as standard screening approach (8-16 rounds), while our developed Smart Cell-SELEX approach (2 rounds) is a new screening approach for challenging classical SELEX methods. In this new approach, the CD33~+ transfected CD33~+-HEK-293T (Positive cells) and HEK-293T cells (Negative cells) were used to optimize the classical approach. The binding affinity of selected aptamers was determined by CD33 positive HL60 cells, NB4 cells and CD33 negative Jurkat cells. In addition, western blot and immunofluorescence is used to detect the expression of CD33 protein and location of CD33 in different cell lines; the dot-blot hybridization assay and flow cytometry are applied to measure the binding affinity of selected aptamers. Calculation of Gibbs free energy and simulation of the three-dimensional structure of the CD33 protein are established by theoretical calculation the computer Docking methods.Part two:The ADCs is constructed by chemical methods. We used the N-succinimidyl-3-(2-pyridyldithiol) propionate, SPDP, (as linker) to conjugate the aptamers and Dox. Before the construction of ADC, we tested the the Dox-SPDP could affect the Dox itself activity or not. Thus, SPDP was chemically conjugated with amido bond or carbon-nitrogen double bond of Dox and then determined by HPLC, LC-MS and ~1HNMR. As a result, we successfully constructed the Dox-SPDP complexs, the anticancer effect was determined by MTT and flow cytometry.Results:Part one:Based on the classical protein-SELEX method, we obtained the two specific CD33 binding aptamers (e.g., A88 and 97), and the screening process was spent more than 3 months for getting its aptamers. Differently from the classical approaches, we developed the new approach (called as Smart Approach) which based on the classical Cell-SELEX have significantly decreased the screening time from 3 months to 1 day and increased the binding affinity and stability of aptamers (e.g., S30)Part two:HPLC,~1HNMR and LC-MS results show that two different conjugates of Dox-SPDP~? and Dox-SPDP~? were successfully generated. However, results of MTT and flow cytometry shown that the anticancer effect of Dox-SPDP~? conjugate was remarkably decreased when compared with Dox alone, while the conjugation of SPDP~? with Dox (Dox-SPDP~?) moderated inhibited Dox itself activity.Conclusions:Part one:Compared with the Classical screening approach, our smart Cell-SELEX screening approach could absolutely shorten the screening time from 3 months to 1 day. In addition, our new smart Cell-SELEX approach is also able to remove most of the non-specific binding aptamers from the negative selection process and significantly increase the rate of CD33 binding affinity aptamers. Moreover, we also confirmed the binding affinity of aptamer is related to its special hairpin and loop structure, while the stability of aptamers is dependence on its the length of sequences.Part two:We have successfully constructed Dox-SPDP~? (amido bonds) and Dox-SPDP~? (carbon-nitrogen double bonds) two conjugates, and we found that the Dox-SPDP~? (but not Dox-SPDP~?) has maintained the anticancer effect, suggesting the amino group on Dox is supposed to be the functional group.