Basic Research On The Application Of Aptamers In Clinical Hematology System

Blood in the human circulatory system is composed of plasma and blood cells,and mainly undertakes the functions of oxygen transport,defense and immunity,hemostasis,thermoregulation,and homeostasis in human life activities.Blood cells are the key to endow the blood with various functions,mainly including red blood cells,white blood cells（immune cells）,and platelets,which are derived from the multipotent hematopoietic stem cells of the bone marrow.Among them,T cell is an important component of lymphocytes,which are mainly responsible for the cellular immunity of the human body and play a key role in resisting disease infection and tumor monitoring.With the development of cancer treatment technology,CAR-T cell therapy,which uses T cells as therapeutic agents,has achieved great success in the clinical treatment of hematologic tumors,but it faces many challenges as well.As the first step of CAR-T cell therapy,T cell isolation from peripheral blood is the basis of CAR-T cell preparation and production,which is directly related to the success or failure of future treatment.However,the current clinical magnetic separation technology based on immunoaffinity binding（antigen-antibody）has limited separation efficiency and purity,and high production cost.Moreover,the surface of T cells isolated by positive selection carries antibodies and magnetic beads contamination（exogenous substances）,which will bring potential safet y problems after reinfusion into patients.Therefore,the development of traceless T cell separation strategy with low cost,high efficiency and purity is crucial to improve the efficacy and safety of CAR-T cell therapy.Additionally,the blood of some special populations（cancer patients and pregnant women）contains circulating rare cells that are extremely rare but hold certain clinical value,such as circulating tumor cells and circulating fetal cells.Among them,fetal nucleated red blood cells（f NRBCs）in maternal blood are immature red blood cells,which are mainly distributed at the stage of terminal erythroid differentiation,including proerythroblast,basophilic erythroblast,polychromatic erythroblast,and orthochromatic erythroblast.Importantly,f NRBCs hold complete fetal genetic information and without confined placental mosaicism,which are recognized as the most ideal targets for non-invasive prenatal diagnosis.With the development of single-cell whole genome sequencing technology,a new generation of non-invasive prenatal diagnosis based on circulating fetal cells is expected to realiz e comprehensive screening and diagnosis of fetal genetic diseases.However,the rarity of f NRBCs in maternal blood is the main factor restricting its clinical translation.Hence,developing superior recognition ligands and establishing efficient separation technologies will further promote the application of f NRBCs in non-invasive prenatal diagnosis.Aptamer,as an emerging recognition ligand,not only has affinity and specificity comparable to antibody but also holds unique advantages,such as low immunogenicity,excellent stability,batch chemical synthesis,low production cost,easy to realize flexible and precise chemical modification,and so on.At present,aptamers have been widely used in many fields such as molecular imaging,disease diagnosis,and t argeted therapy.Meanwhile,aptamers have also emerged in cell isolation applications,becoming a novel type of cell separation and release tool after antibodies.According to the above research background,we successfully identified aptamers against f NRBCs and T cells through a modified cell-SELEX procedure with clinical blood samples and then applied them to non-invasive prenatal diagnosis and T cell traceless separation.The detailed research results are as follows:（1）Establishing the cell-SELEX procedure based on clinical blood samples and discovering aptamer LXD-11 that specifically binds to NRBCs.Then,by optimizing the sequence of aptamer,the obtained LXD-11b not only retains the same target binding specificity as the original sequence but also obtains superior binding affinity.Target identification showed that LXD-11b specifically targeted the transferrin receptor CD71 on the membrane surface of f NRBCs.After identifying the differences in binding sites among LXD-11b,TRF,CD71 antibody（Clone:M-A712）and CD71,we demonstrated the binding sites of LXD-11b on CD71,and the potential competitive mechanism between LXD-11b and TRF by molecular docking simulation approach.（2）Considering the excellent binding affinity and specificity between aptamer and NRBCs,we use LXD-11b as a recognition ligand to capture f NRBCs from maternal blood and realize the screening and diagnosis of fetal genetic diseases.After confirming the recognition and binding ability of LXD-11b to NRBCs in CBMCs,we established the magnetic separation technology system of NRBCs based on aptamer.In subsequent application of NRBCs isolation,LXD-11b displays comparable capture efficiency（>94.0%）with commercialized CD71 antibody and holds a higher purity of enriched cell products（90.83%）.Importantly,the NRBCs captured by LXD-11b could be effectively and non-invasively released under the competition of the complementary sequence（RA1）,which will be more conducive to downstream single-cell whole genome amplification and high-throughput sequencing analysis.Furthermore,LXD-11b could effectively enrich f NRBCs from maternal blood,and FISH（X/Y probes）results showed that NRBCs are derived from the fetus.Finally,we used the enriched f NRBCs for genetic analysis,including fetal sex i dentification and screening for chromosomal abnormalities.Statistical analysis showed that the detection sensitivity and specificity were 91.67%and 83.33%,respectively.Meanwhile,the false positive rate and false negative rate reached 16.67%and 8.33%as well,respectively.Therefore,according to the current clinical sample results,we demonstrate d the potential and feasibility of utilizing aptamer LXD-11b to isolate f NRBCs from maternal blood for non-invasive prenatal diagnosis.（3）In order to obtain aptamers that specifically recognize and bind to various immune cells in peripheral blood,negative and positive selection were conducted with RBCs and WBCs respectively for 14 rounds.After high-throughput sequencing and flow cytometry analysis,we identified aptamer XD-9 and XD-10 that bind to lymphocyte subsets.Meanwhile,we also discovered aptamer XD-2 and XD-5 that simultaneously bind to monocytes and granulocytes.Importantly,each aptamer maintains stable binding ability and specificity in differen t peripheral blood samples and holds superior affinity with target cells as well.（4）Given that CD62L⁺T cells(T_N,T_SCM,and T_CM)hold unique advantages in expansion,survival,and persistence in vivo,which will exhibit better efficacy in CAR-T cell immunotherapy.We first confirmed that XD-10 binding to the T cell subset of lymphocytes through flow cytometry analysis.Meanwhile,target identification revealed that XD-10 specifically targeted CD62L,known as L-selectin.By optimizing the sequence of aptamer,XD-10a still retains its original binding ability,specificity,and affinity.Moreover,XD-10a and CD62L antibody（Clone:DREG-56）might share the same binding site owing to the existence of competitive interaction.The molecular docking simulation results further proved the interaction between XD-10a and CD62L protein,and the competitive mechanism between XD-10a and CD62L antibody from molecular insights.Subsequently,we used XD-10a as a recognition ligand to isolate CD62L⁺T cells and found that XD-10a exhibited significantly superior capture efficiency and comparable cell purity and viability to commercial CD62L antibody.Further analysis of the cell phenotypes by flow cytometry found that the ratio of T_SCMholds unique advantage in XD-10a enriched cell products.Importantly,the CD62L⁺T cells captured by XD-10a could be effectively and tracelessly released under the competition of complementary sequence（RA 6）,and it hardly affected the cell purity,viability,and cell subpopulation,which will conduce to ensure the safety of immunotherapy in the future.（5）In view of the remarkable efficacy of specific CD4⁺T cell and CD8⁺T cell subsets in CAR-T cell immunotherapy,and could effectively reduce the incidence of cytokine release syndrome and neurotoxicity syndrome.We first determined that XD-9binding to the CD8⁺T cell subset of lymphocytes through flow cytometry analysis.Then,Jurkat 76（CD8ab⁺）cells were used to carry out target identification,and final results showed that the binding target of XD-9 was CD8 protein.By optimizing the sequence of aptamer,XD-9a obtains stronger binding ability and affinity,while retaining the same specificity and binding site as aptamer XD-9.Subsequently,we confirmed that XD-9a and CD8 antibody（Clone:HIT8a）might target different binding sites of CD8 proteins based on competition assay.Similarly,we used XD-9a as a recognition ligand to isolate CD8⁺T cells from peripheral blood and found that XD-9a holds the same performance as commercial CD8 antibody.Importantly,the CD8⁺T cells captured by XD-9a could be tracelessly released（>60.58%）under the competition of complementary sequence（RA8）,and it hardly affected the cell purity,viability,cell subpopulation and cell proportion,which could avoid potential negative effects of immunotherapy in the future.