Construction Of PDX Model And Study Of Nano-antibody Immunotoxin In Mice

Colorectal cancer has a serious impact on human health and quality of life,and its treatment methods and drug development continue to bring forth new ideas.Conventional combination of anticancer drugs and chemotherapy has limited curative effect on patients,and it can greatly damage the normal immune system while inhibiting cancer cells.Therefore,it is imperative to develop new therapies that are more effective and can alleviate patients'pain.Targeted immunotherapy using nano-antibodies fused with toxins combined with anticancer embryonic antigen?CEA?has attracted wide attention due to its remarkable efficacy.In this paper,two kinds of linkers?rigid linker-R and flexible linker-V?were inserted into11C12-PE38 to construct fusion immunotoxin 11C12-R/V-PE38 and 11C12-PE38 without linker insertion.Fusion immunotoxins with high affinity were selected by optimizing expression and subsequent analysis and detection.The anti-cancer effect of fusion nano-immunotoxin 11C12-R-PE38was studied at cell level and in PDX model of nude mice transplanted tumors.Firstly,two kinds oflinker were inserted between the nano-antibody of the fusion antibody immunotoxin expression plasmid and the immunotoxin PE38 by means of genetic and molecular engineering,and the expression plasmid pET30a-11C12-R/V-PE38 was constructed.Then,the fusion antibody immunotoxin PE38 was optimized by prokaryotic expression system of Escherichia coli BL21 at the optimum induction temperature of 37?and concentration of1mmol/L.The protein concentrations of 11C12-PE38,11C12-R-PE38 and 11C12-V-PE38were 1.6 mg/mL,1.71 mg/mL and 1.56 mg/mL respectively by His-tag affinity purification.The affinity of PE38 protein with linker-free and inserting linker-R/linker-V was determined by Biacore technique.The results showed that the 11C12-PE38KD constant was1.00×10^-77 mol/L,the 11C12-R-PE38 K_D constant was 2.07×10^-88 mol/L,and the11C12-V-PE38K_D constant was 1.40×10^-7 mol/L.The smaller the equilibrium dissociation constant,the less the dissociation,the higher the affinity.Therefore,according to the measured results,the affinity of 11C12-R-PE38 inserted into rigid linker is the best.Subsequently,11C12-R-PE38 with high affinity was prepared by enlarging the culture scale to evaluate the inhibitory effect of 11C12-R-PE38 on human colon cancer SW480,LoVo and SW403 cells.The results showed that 11C12-R-PE38 could inhibit LoVo,SW403and SW480 cancer cells with inhibition rates of 89%,57%and 31%,respectively.The inhibition rate of 11C12-R-PE38 on LoVo was the highest.Finally,based on the results of cell experiments,a nude mouse transplanted tumor?PDX?model derived from patient's tumor tissue was constructed,and the anti-cancer effect of fusion antibody immunotoxin PE38 was verified by in vivo experiments in mice.The volume of tumors in mice was measured within 10 days.The results showed that the tumors in the low dose group?2?g/kg?and the high dose group?10?g/kg?increased faster than those in the blank control group within 7 days.On the 10th day,the tumor volume of mice in the low dose group,the high dose group and the blank control group were 125 mm³,119 mm³and 199 mm³respectively.Although the tumor volume of mice in the administration group increased within 7-10 days,the growth rate of tumor tissue in the model mice was slowed down,which indicated that the constructed mice containing linker R had a tendency of growth.Fusion immunotoxin has a significant inhibitory effect on colon cancer tissues.