Development Of New NKG2A Monolithic Column Biochromatography Based On Cell-free Synthesis Technique For Membrane Receptors

Natural killer(NK)cells are innate lymphoid cells with natural cytotoxicity and immunomodulatory functions.The activation of NK cells is regulated by the balance of multiple activating and inhibitory receptors expressed on their surfaces.Human natural killer cell family 2 member A(NKG2A)is an inhibitory receptor(immune checkpoint)specifically expressed on NK cells,which mediates NK cells to suppress killing signals by recognizing Human Leukocyte Antigen-E(HLA-E).Blocking NKG2A with the antibody drug Monalizumab is currently the main means of immunotherapy against NKG2A.Due to the complexity and particularity of its transmembrane structure,the research and development of NKG2A small molecule inhibitors are extremely challenging.The key scientific question is how to simulate the natural conformation of NKG2A receptor in vitro,and achieve efficient screening of targeted NKG2A small molecule inhibitors and accurate evaluation of its activity.In this subject,a new method for NKG2A monolithic column biochromatography based on cell-free synthesis technology was established by combining the respective advantages of cell-free protein synthesis(CFPS)and monolithic column technology.It solved the technical problems of single,stable and controllable high expression of NKG2A on the chromatographic stationary phase,while controlling its orientation,and was used for the efficient screening of NKG2A small molecule inhibitors and comprehensive evaluation of their activity.The establishment of this method system has scientific significance for the in-depth understanding of the biological function of immune checkpoints and the development of new small molecule inhibitors,and also provides new ideas and technologies for the precise screening of membrane receptor-targeted drugs.This subject mainly carries out research from the following four aspects.1.The screening of NK cell activators based on CMC analysis systemCell Membrane Chromatography(CMC)is a classical biochromatographic analysis method for the interaction between drugs and membrane receptors.It is widely used in the rapid screening of membrane receptor binding active components of traditional Chinese medicine and the systematic study of drug-target affinity.In this chapter,a comprehensive 2D NK-92MI/CMC/C18/TOFMS analysis system was established to screen potential NK cell activators from Astragali Radix.Firstly,the applicability of the NK-92MI/CMC system was examined.The peak time of the positive drug(Istradefylline,the inhibitor of A2AR receptors)on the NK-92MI/CMC column is about 20 min,while the retention time on the control column is 5 min;the negative drug(Ebselen)has no retention on the two columns.It is proved that the established screening system had good applicability in identifying active ingredients that binded to membrane receptors.Secondly,totally 9 candidate compounds were screened from Astragali Radix by using this system,and verified by using standard solution.Finally,two high abundance compounds(Isoastragaloside I and Astragaloside IV)with strong affinity on NK-92MI/CMC column were selected to further verify the immune activation effect.The viability of NK cells was evaluated by flow cytometry(FCM)and lactate dehydrogenase(LDH).The experimental results showed that isoastragaloside I and astragaloside IV could increase the cytotoxicity of NK-92MI cells in a dose-dependent manner within a certain concentration range.In conclusion,the established comprehensive 2D NK-92MI/CMC system is an effective technique for screening the immunomodulatory active components of traditional Chinese medicine.This study also provides a new idea for screening NK cell activators from other complex traditional Chinese medicine systems.2.Synthesis and methodological investigation of NKG2A monolithic column biochromatography stationary phase based on cell-free synthesis of membrane receptorsAccording to the previous section,although CMC provides a feasible method for rapid screening of membrane receptor-binding drugs,it still has limitations in specificity and accuracy,and cannot achieve the study of specific immune checkpoint NKG2A-interacting compounds.Therefore,how to achieve a single,stable and controllable high expression of NKG2A receptor on a chromatographic stationary phase,so as to simulate its natural conformation in vitro,are very challenging key technical problems in the study of analysis methods of drug-membrane receptor interaction.In this chapter,a novel NKG2A-liposome-monolithic column chromatography stationary phase was constructed and its methodology was investigated.Firstly,NKG2A-liposome complexes were prepared by CFPS technology.The preparation of NKG2A-liposome complexes were investigated by western blot and fluorescence microscope.The results showed that NKG2A receptors could be successfully synthesized by CFPS technology,and the NKG2A receptors were successfully inserted into liposomes.At the same time,the content of NKG2A on the complexes was 20.158±0.386 ng determined by enzyme linked immunosorbent assay(ELISA).Subsequently,through chemical modification,the NKG2A-liposome complexes were immobilized on the monolithic column.The NKG2A-liposome-monolithic columns were characterized by fluorescence microscopy,scanning electron microscopy,and infrared spectroscopy.The results demonstrated that the NKG2A-liposome complexes were successfully immobilized within the monolithic column.Finally,the effectiveness of NKG2A-liposome-monolithic columns was examined.The retention time of positive drug(Monalizumab)on NKG2A-liposome-monolithic column was about 8.5 min,while the retention time on blank liposome-monolithic column was 3.2 min.The retention time of negative drug(Plerixafor)on both columns was the same,which was about 3.2 min and consistent with the result of monalizumab on the negative column.The results proved that the NKG2A receptors on the NKG2A-liposome-monolithic column were active,and the effectiveness of columns met the requirements.Based on the above methodological investigations,it is proved that the preparation of this novel NKG2A-liposome-monolithic column chromatography stationary phase is successful,laying a foundation for the subsequent screening of targeted NKG2A small molecule inhibitors.At the same time,it also provides a feasible idea for the preparation of immune checkpoint chromatography stationary phase.3.The screening and affinity identification of small molecule inhibitors of NKG2AIn this chapter,the NKG2A-liposome-monolithic column was applied to screen potential NKG2A small molecule inhibitors from flavonoids.Firstly,the NKG2A-liposome-monolithic column was loaded into the"heart-cutting"2D biochromatographic analysis system,collected the retained components according to the chromatographic retention behavior,and identified them by UPLC-QTOF/MS.The results showed that the main active ingredients are baicalin and wogonoside.Secondly,standard solutions of baicalin and wogonoside were used to rule out non-specific interference.The results showed that the retention times of baicalin and wogonoside on NKG2A-liposome-monolithic column were about 8 min and 9 min,respectively;while the retention times of these two compounds on blank liposome-monolithic column were about3 min,demonstrating that baicalin and wogonoside could interact with NKG2A receptors on the monolithic column.Finally,frontier affinity chromatography(FAC)was used to determine the affinity of drug-target.The results showed that the K_D values of baicalin and wogonoside were 35.5μm and 23.6μm respectively.The above experimental results show that NKG2A-liposome-monolithic columns were successfully applied for the efficient screening of potential targeted NKG2A small molecule inhibitors,and the accurate evaluation of their affinity.At the same time,this method also provides an effective and practical way for the screening of small molecule inhibitors of immune checkpoint.After that,the immune activating effects of baicalin and wogonoside were further investigated.Firstly,NK-92MI cells were selected as the experimental object and the expression of CD107a on the surface of NK-92MI cells was detected by FCM.The results showed that baicalin and wogonin could up-regulate the expression of CD107a molecule on the surface of NK-92MI cells.With the increase of drug concentration,the expression of CD107a also increased.At the same time,real-time quantitative polynucleotide chain reaction(q PCR)was used to detect the expression of cytokines(TNF-α,IFN-γ,GZMB)in NK-92MI cells from the gene level.The results showed that both baicalin and wogonoside could upregulate the expressions of GZMB,TNF-αand IFN-γ.When the concentration reached 5μmol/L,the expression of IFN-γwas significantly increased compared with the control group(baicalin,p<0.0001;wogonoside,p<0.001).Subsquently,we selected K562 cells as target cells,and detected the killing effect of NK-92MI cells on K562 cells by FCM and LDH methods.The results showed that baicalin and wogonoside enhanced the cytotoxicity of NK-92MI cells in a dose-dependent manner.The results demonstrated that baicalin and wogonoside could activate NK-92MI cells and enhance the killing effect of NK-92MI cells on K562 cells.In summary,this subject established a new method for NKG2A monolithic column biochromatography analysis based on the cell-free synthesis of membrane receptors,which achieved efficient synthesis of NKG2A receptors in vitro.At the same time,the application of monolithic column technology has realized the rapid batch preparation of biochromatography,and has been successfully applied to the high-throughput screening and affinity evaluation of NKG2A small molecule inhibitors.Several active monomers of traditional Chinese medicine such as baicalin and wogonoside were obtained through the verification of NK cell activating effect and killing effect.The establishment of this method not only provides a new idea and method for the in vitro simulation of the natural conformation of membrane receptor and the analysis of drug membrane receptor interaction,but also provides an efficient screening strategy for the development of small molecule inhibitors of immune checkpoints.