Construction Of Humanized Semi-synthetic Nanobody Library And Production Of Anti-CD47 Nanobody

Nanobodies are derived from heavy chain antibodies with natural deletion of light chains in camels and chondrofishes such as sharks and cloned from their variable region VHH.As the smallest functional fragment to bind antigen.nanobody has simpler structure and higher stability than complete traditional antibody,and its molecular weight is only 1/10 of it,so it has strong ability to penetrate tissues and is easy for genetic engineering manipulation.therefore,it has a broad application in biotechnology,medical diagnosis and treatment fields.As a member of the immunoglobulin(Ig)superfamily,CD47 molecule represents the signal of "don't eat me" in the tumor immune recognition process,that is,the binding between CD47 molecules on the surface of tumor cells and the protein ligand SIRPa on macrophages can inhibit the phagocytosis of the latter and hence evade from its immune surveillance,while blocking this inhibition can reawaken the silent macrophages.Therefore,CD47 molecules are also endowed with the title of immune checkpoints and has become another potential target for tumor immunotherapy,and the targeted drugs that can block CD47-SIRP? pathway are supposed to become another hot spot in the field of tumor immunotherapy after PD-1 and PD-L1.First,based on the phage display technology of nanobody,a new method for the construction of humanized semi-synthetic nanobody library was discussed in this thesis in which the VHH-vector DNA preparation was carried out without the need of enzyme digestion and was completed in only one PCR reaction.Then,CD47 antigen molecule expressed by eukaryotic expression system was used as the target,and after four rounds of biological panning of VHH library,positive clones of phage antibody were obtained and used for sequence and affinity analysis,and then VHH fragments were expressed by E.coli expression system,and also carried out in Pichia pastoris preliminarily.Finally,the immune blocking activity of VHH was detected by flow cytometry.In the first part,the construction method of humanized semi-synthetic nanobody library was discussed.In this chapter,the reported cAbBCII10 nanobody sequence was used as the basic framework.and the relevant amino acid residues outside the FR2 region were replaced with humanized counterpart.Then.the random sequences of CD3 region was introduced by long primer PCR method into this framework,and the efficiency and feasibilety of constructing nanobody library in this way were confirmed.In the study,it is found that this method can complete the construction of humanized semisynthetie VHH antibody library through one step of PCR reaction,and the products can be used for transformation after gel recovery and ligation,which reduces the work intensity of gene splicing and the loss caused by too many recovery steps,and each batch of electrotransfer can reach a diversity of 1.21×108,and hence improves the work efficiency.The research in this chapter provides a new idea and technical route for the construction of nanobody library.In the second part,the related antigens needed in the biological panning process and subsequent testing stage were prepared by using the eukaryotic expression system.In this chapter,high purity aseptic and endotoxin-free plasmids containing CD47Fc,SIRP ?-Fc and CD47his genes for eukaryotic expression were used to transfect 293Fv suspension cell line,and the eukaryotic expression of the three antigens were successfully carried out.After purification through affinity chromatography,the yield of each of the antigens reached the milligram level,and was ready for subsequent bio-panning and detection procedures.In the third part,the bio-panning,monoclonal identification and affinity determination against CD47 antigen were carried out.First,in this chapter,four rounds of bio-panning process using VHH nanobody library against coated solid-phase CD47 antigen was carried out,and the enrichment effect was obvious.and reached the plateau stage in the third round.Then,the monoclonal clones were selected for testing,and the positive clones were sequenced.Five VHH nanobody sequences targeting CD47 extracellular domain antigens were obtained after sequence analysis.Among which,four sequences were highly similar,and the difference shows only in a few individual amino acid residues of the framework region,and the fifth sequence was quite different from the other four.The affinity test showed that the Kd values of these two VHH classes were 1.5×1010 mol-1 and 7.5×109 mol-1 respectively.The results of this chapter is the foundation for the next stage expression of VHH nanobodies.In the fourth part,the expression,purification and immune activity of VHH nanoantibodies were discussed.In this chapter.the positive phage clones were first transformed into prokaryotic expression vectors of the secretory form by mutating PCR.and the soluble secretory expression of VHH nanobodies was performed and the products were analyzed.During the course of the study,the type of culture medium,culture temperature and the distribution space of secretory expressed VHH were tested.It was found that most of the VHHs were expressed in soluble form.and the spatial distribution of VHH was different between these two VHH classes.Osmotic shock method for periplasmic extraction or extraction from supernatant after ultrasonic disruption or high-pressure homogenization is a better strategy.In addition,this chapter Pichia pastoris expression vector was also used for a preliminary attempt to express VHH secretorily,and VHH antibody molecules be secreted into the culture medium under the guidance of signal peptide,with relatively good yield and little background of miscellaneous proteins in the culture medium,indicating the advantages of yeast expression system.Finally,the immune blocking activities focusing CD47-SIRP ?pathway were tested on these two VHH nanobody classes expressed by E.coli system,the result showed that one of the VHH sequences with relatively good immune blocking activity.The study of this chapter provides a new practical and feasible route and method for the soluble secretory expression of VHH nanobody in E.coli.To sum up.this paper explores a convenient and efficient method for the construction of humanized semi-synthetic VHH nanobody library,bypassing the traditional use of animals,and the built library had a good diversity in capacity,and the capacity of each electrotransfer batch can reach 108 level.,Five VHH nanobody sequences against CD47 antigen were finally obtained through four rounds of bio-panning,and belonged to high affinity VHH clones after affinity analysis.Then the prokaryotic secretory expression of VHH fragments was carried out by using E.coli expression system,and the culture condition and purification strategy were discussed,and a preliminary expression attempt was made in Pichia pastoris.Finally,the immune blocking activity of VHH was detected by flow cytometry,and a VHH nanobody with blocking activity was identified,which is a better candidate for further application and development.