A Single Domain Antibody Targeting The PD-1 Ligand Binding Interface

PD-1/PD-L1 is the star target of current anti-cancer drug research and development.As of March 2018,there are already five drugs for this pathway in the world that have been successfully marketed and have achieved good results.The antibody drug targeting this pathway blocks the binding of PD-1 to its ligand,renders the immune checkpoint dysfunctional,and eliminates the negative regulatory effect of PD-1protein on the antigen presenting signal,so that the T-cell can be normally activated and proliferated.The delivery of the presentation signal keeps T cells asleep and avoids excessive immune responses.However,this pathway was used by tumor cells overexpressed by PD-L1,but it caused immune escape of tumor cells.In view of the characteristics of this pathway,a high-affinity nanobody was designed for the ligand interface region of PD-1 and PD-L1 binding,and the T cell was restored by blocking the binding of PD-1 extracellular domain to PD-L1 the killing effect on tumor cells.Methods:1.Panning target high-affinity antibodies by phage display technologyBy means of bioinformatics,the binding of PD-1 to PD-L1 was simulated to determine the sequence in which the extracellular domain is responsible for binding to the ligand.This segment of the sequence is the ligand binding interface,and this sequence is artificially synthesized using nucleosidase.After the plate was coated,multiple rounds of ELISA elution from the commercial human single domain antibody phage display library resulted in high affinity single domain antibodies to the sequence.After its DNA sequence was obtained by sequencing,an amino acid sequence was obtained by translation.2.Eukaryotic expression system's construction of the single domain antibody(1)Optimization of single-domain antibody amino acid sequences:Amino acids that may produce mutations are modified by comparison with other homologous human single-domain antibodies.(2)Synthesis and vector construction of eukaryotic expression genesThe optimized antibody sequence was optimized using Pichia pastoris for codon usage.MF-?signal peptide was added to the front end of the gene,6 His and terminator were added to the end of the gene,and the restriction site XhoI was added at both ends.Synthesize this gene with XbaI.Amplified by PCR.After double digestion with pGAPz?-A empty vector,it was ligated with T4 ligase and transformed into E.coli JM109.(3)Construction and screening of Pichia pastoris X-33 high expressing strainTherecombinantplasmidwasextracted,linearizedand electroporated into the host Pichia pastoris X-33.Positive transformants were obtained by colony PCR and high resistance screening,and high expression strains were obtained by expression identification.(4)Purification of Expression Products from Eukaryotic Expression Systems.Take the fermentation supernatant and remove most of the impurities by ammonium sulfate precipitation and re-dissolve it with the CM column equilibration solution.Purified by molecular sieve and nickel column.(5)Identification of glycosylation:The purified product was glycosylated using endoglycosidase(Endo H).3.Prokaryotic expression system's construction of the single domain antibody(1)Synthesis and Vector Construction of Prokaryotic Expression Target GenesAfter the optimized sequence was added,His and terminator were added to the end,and two restriction sites Nde I and Hind III contained in the pET21b vector were added at both ends to synthesize and PCR amplification,and the enzyme was ligated to pET21b by double digestion test.On the other hand,heat shock was converted to E.coli DH5?.(2)Construction of E.coli BL21(DE)high efficient expression strains:The recombinant plasmids were extracted and transformed into E.coli BL21(DE)in the same way.High expression screening was performed using colony PCR and expression identification methods.(3)Purification of soluble expression productsThe engineered bacteria were used to induce soluble expression in the form of low-temperature culture.The expression pattern was confirmed by the same method,and the disrupted bacterial supernatant was purified by CM column and nickel column.4.Activity identificationThe ELISA method was used to determine the affinity and the obtained purified product was co-incubated with human PD-1 protein,PD-L1 protein-overexpressing tumor cells were tested by it.FITC labeled His tag antibody was used as a secondary antibody,and fluorescence was detected by flow cytometry.Results:1.Phage screening results17 strains of positive strains were successfully obtained.The sequence of the 16 positive bacteria was highly homologous and the protein sequences of the 16 V-regions encoded by the positive strains were identical.A high affinity single domain antibody targeting the PD-1ligand binding interface was successfully screened.2.Results of construction of eukaryotic expression systemThe optimized antibody was named Nb:PD-1.The autocrine Pichia pastoris X-33 highly expressed strain of Nb:PD-1 was successfully obtained.The activity of glycosylation enzyme of the engineered bacteria was insufficient.The CM column and nickel column were used successfully to obtain high-purity expression products,and internal glycosylation was confirmed by endoglycosidase.3.Results of construction of prokaryotic expression systemSuccessfully obtained Nb:PD-1 E.coli BL21(DE)high expressing strain.The soluble expression was successfully constructed,and successfully obtained high-purity expression products.4.Activity identification resultThe indirect ELISA method measured the eukaryotic expression of Nb:PD-1 with an affinity of 10~6 mol/L,and the prokaryotic expression of Nb:PD-1 with an affinity of 10~5 mol/L.The obtained antibodies'activity was identified by flow cytometry.Conclusion:A single domain antibody targeting the PD-1 ligand binding interface was successfully screened and the amino acid sequence of the antibody was optimized.Two eukaryotic and prokaryotic expression systems were successfully constructed,and purified higher expression products were successfully obtained.In this study,a single domain antibody Nb:PD-1 targeting the PD-1 ligand binding interface was successfully prepared.