Screening,Identification And Anti-tumor Effects Of Anti-PD-1 And Anti-CTLA-4 Nanobodies

Objective Cancer is the main cause threatening to human life and health,and the increasing number of new cancer cases every year has caused a huge economic burden to our society.Both new happened and death cancer cases in China rank number one in the whole world,posing a serious threat to people’s health and lives.Cancer therapy include surgery,radiotherapy,chemotherapy and immunotherapy.Among them,immune checkpoint blocking therapy is an effective therapeutic method for advanced cancer patients.In this research,natural camel-derived nanobody phage library was used to screen nanobodies targeting human PD-1 and CTLA-4 immune checkpoints,respectively.Recombinant vector was constructed then expressed in prokaryotic expression system,after that,the recombinant nanobodies against PD-1 and CTLA-4 were obtained after purification.The anti-PD-1 and anti-CTLA-4recombinant nanobodies were identified,and their affinity and specificity were analyzed.The antitumor activity of recombinant anti-PD-1-Fc and anti-CTLA-4-Fc nanobodies was studied in vitro and in vivo,in order to obtain therapeutic nanobodies targeting immune checkpoints with independent intellectual property rights.Methods1.Based on the natural camel-derived nanobody phage library constructed by our research group in the early stage(the library capacity reached 15.1×1014 CFU/m L after assisted by phage rescue),the biotinylated PD-1 antigen and biotinylated CTLA-4 antigen were used to screen nanobodies targeting the immune checkpoint PD-1 and CTLA-4 by biotinylated streptavidin liquid phase screening assay,respectively.Monoclonal phage bacteria were identified by Phage-ELISA after the third and fourth round of screening,and the positive phage bacterial fluid was sequenced.NCBI database was used to analyze the all the tested gene sequences.Finally,the gene sequences of anti-PD-1 and anti-CTLA-4 nanobody were obtained.2.The obtained anti-PD-1 and anti-CTLA-4 nanobody gene sequences were connected to human Ig G1 Fc fragment to construct recombinant PCZN1-anti-PD-1-Fc and PCZN1-anti-CTLA-4-Fc vectors,which carried His tags for the later protein purification.The recombinant vector was transferred into escherichia coli expression system for prokaryotic expression.The protein expression was induced by IPTG and the prokaryotic expression conditions were optimized.The recombinant nanobodies were purified by Ni column affinity.Western Blot was used to preliminarily identify the purified anti-PD-1-Fc and anti-CTLA-4nanobody.3.The specificity and affinity of recombinant anti-PD-1-Fc and anti-CTLA-4-Fc nanobodies were detected by ELISA,western blot,SPR and cell immunofluorescence.Human PBMC were stimulated by SEB and added with recombinant nanobodies,the biological activity of them was detected through IL-2 release level.4.Colorectal cancer cell lines with stable luciferase expression were constructed to study the antitumor effect of nanobodies in vitro.The co-culture model of human PBMC and colorectal cancer cells in vitro was built,different effector and target ratios(5:1,10:1,20:1,40:1)were set according to the ratio of PBMC(effector cells)to colorectal cancer cells(target cells).PBMC group,PBMC with different concentrations of recombinant nanobody group,PBMC with corresponding monoclonal antibody group and PBMC with unrelated nanobody group were set up in the experiment.The cytotoxicity of colorectal cancer cells was measured by lucifase gene report assay.5.NOD-SCID mice were subcutaneously injected with a mixture of human PBMC and colorectal cancer cells to construct a xenograft mouse tumor model.Untreated group,PBMC group,PBMC with different concentrations of recombinant nanobody group,PBMC with monoclonal antibody group and PBMC with unrelated nanobody group were set in the experiment.After constructing tumor models,corresponding drugs was given.Tumor volume was measured every 3 days to observe the trend of tumor volume change.On the 9th,18 th and 27 th day of the experiment,bioluminescent imaging was performed to accurately detect the tumor load in vivo.On day 28,the mice were sacrificed to exfoliate the tumors and weighed.The antitumor effects of recombinant anti-PD-1-Fc and anti-CTLA-4-Fc nanobodies in vivo were verified by the above methods.Results1.Phages targeting PD-1 and CTLA-4 were effectively enriched after 4 rounds of liquid eluting screening.12 PD-1 and 7 CTLA-4 positive clones were identified by Phage-ELISA and sent for gene sequencing.Two anti-PD-1 nano-antibody sequences(C11 and C12)and two anti-CTLA-4 nano-antibody sequences(E5 and D11)were obtained,which were consistent with the common characteristics of camel-derived nanobody sequences.After amino acid sequence analysis,anti-PD-1 C12 and anti-CTLA-4 D11 were selected for follow-up study as they possessed stronger solubility and better CDR3 structure.2.The screened anti-PD-1 and anti-CTLA-4 nanobody sequences were conjugated to the Fc segment of human Ig G1,respectively,and then to construct recombinant PCZN1-anti-PD-1-Fc and PCZN1-anti-CTLA-4-Fc vectors.The recombinant vector was transferred into Escherichia coli,and the expression conditions were optimized.The anti-PD-1-Fc nanobody and anti-CTLA-4 nanobody were successfully expressed by IPTG induction.SDS-PAGE analysis showed that the two target proteins existed in the precipitate and were dissolved in PBS after inclusion body modification.The target protein solution was purified by Ni column affinity and identified by Western Blot.The recombinant anti-PD-1-Fc nanobody and recombinant anti-CTLA-4-Fc nanobody with the purity of 90% were obtained.In this expression system,1mg protein could be obtained from 1L bacteria,and 0.33 mg protein could be obtained after purification,with a purification rate of about 33%.3.Preliminary ELISA analysis showed that low concentration of recombinant anti-PD-1-Fc nanobody could bind to PD-1 antigen.SPR assay was further performed to accurately detect its affinity.The affnity is 6.63 n M,which could compete with PD-L1 and PD-1 binding,and the IC50 was 3.015 m M.Western Blot analysis and cell immunofluorescence showed that anti-PD-1-Fc nanobody could specifically recognize PD-1protein on activated T cells and bind to PD-1 receptor on activated T cells.The affinity of recombinant anti-CTLA-4 nanobody is 3.88 n M tested by SPR assay,which could specifically recognize the CTLA-4 protein on activated T cells and bind to the CTLA-4 receptor on activated T cells.Human PBMC was stimulated by SEB,and IL-2 release in anti-PD-1-Fc nanobody or CTLA-4-Fc nanobody was 10 times higher than that of the control group without nanobody(P≤0.01).4.In the co-culture system of PBMC and human colorectal cancer cells,the cytotoxicity of PBMC to tumor cells was related to the effect target ratio,drug concentration and interaction time.The cytotoxicity rate of tumor cells in anti-PD-1-Fc nanobody group was significantly higher than that in PBMC only group and unrelated nanobody group(P<0.05)with different effect target ratio(5:1,10:1,20:1,40:1).When the effector and target ratio was40:1,the cytotoxcity rate in high concentration anti-PD-1-Fc nanobody group(750μg/m L)reached 75%.Similarly,the cytotoxicity rate of tumor cells in anti-CTLA-4-Fc nanobody group was significantly higher than that of control group under different effect target ratio(P<0.05).When the effector and target ratio was 40:1,the cytotoxicity rate in high concentration anti-CTLA4-Fc nano body group(800μg/m L)could reach 74%.5.The NOD-SCID xenograft mouse tumor model was successfully constructed,corresponding antibody drugs were given,bioluminescent imaging was performed regularly,and tumor volume was measured.On day 18,the fluorescence intensity of tumor in PBMC group,recombinant nanobody(anti-PD-1-Fc nanobody group and anti-CTLA-4-Fc nanobody group)and monoclonal antibody group was significantly lower than that in untreated group(P<0.05).On day 27,the fluorescence intensity of medium-dose and high-dose anti-PD-1-Fc nanobody groups(3mg/kg,5mg/kg),medium-dose and high-dose anti-CTLA-4-Fc nanobody groups(2mg/kg,3mg/kg)and monoclonal antibody groups was significantly lower than that of PBMC group(P<0.05).There was no significant difference in fluorescence intensity between control nanobody group and PBMC group.The tumor volume was consistent with the change of tumor fluorescence intensity.Compared with the untreated group,the tumor volume of other groups was significantly decreased(P<0.01).The tumor volume in the medium-dose and high-dose groups of recombinant nanobody and monoclonal antibody treatment group was significantly smaller than that in the PBMC group(P<0.01).The high dose group showed the most obvious inhibitory effect on tumor growth.At the end of the experiment,the tumor tissue was stripped and weighed,which was consistent with the size of the tumor.The tumor quality of the PBMC group and the drug treatment group was significantly lower than that of the untreated group(P<0.01),and the tumor quality of the medium-dose and high-dose recombinant nanobody groups was significantly lower than that of the PBMC group(P<0.01).Conclusion1.The anti-PD-1 nanobody and anti-CTLA-4 nanobody were succesffully Screening,respectively.2.The recombinant anti-PD-1-Fc and anti-CTLA-4-Fc nanobodies with high affinity and specificity were successfully produced.3.The recombinant anti-PD-1-Fc and anti-CTLA-4-Fc nobodies can improve the cytotoxicity of immune cells to tumor cells,and delay the growth of in tumor bearing xenograft mice.