| Background and Purpose In recent years,tumor immunotherapy has gradually become the focus of malignant tumor treatment.With the deepening of research,immunotherapy has provided a new breakthrough for cancer treatment.Immunotherapy is a method of treating disease by regulating the body’s own immune system.Immunotherapy means a method of treating diseases by managing the native immune system of the body.As a relatively novel therapeutic strategy,immunotherapy has received increasing attention due to its low potential side effects and high specificity.For example,cancer immunotherapy has made great progress in recent decades,especially for the therapies of recurrent and metastatic cancer.There are a variety of immunotherapeutic strategies for different diseases,such as vaccine-based therapies.CD47 and 5T4 antigens,new targets after pd-1,have become two star molecules in tumor immunotherapy with the deepening of research on them.CD47 and 5T4 have expressed in some abnormal cells in the body,especially in tumor cells,the common tumors such as breast cancer,pancreatic cancer,colorectal cancer,prostate cancer,ovarian cancer,research has shown that these two kinds of antigens in the body after the spread of the tumor metastasis and prognosis is closely related to,as for CD47 and respectively 5T4 antigen specific antibody preparation of success,they in early clinical trials have been encouraging results have been achieved.Double specific antigen has been half a century of development,because of its preparation and purification of the rapid development of technology and equipment,double specificity antibody development is growing by leaps and bounds in recent years,several pairs of specific antibody appeared,especially in the treatment of tumor showed huge application prospect,CD47-5T4 double specificity antibodies possess the characteristics of two kinds of single resistance,signal conduction between cells,mediated phagocytosis of tumor cell to gobble up,accelerate the apoptosis of many aspects,such as all showed an incomparable advantages of double antibody specificity,This study focused on the preparation of bispecific antibody to cd47-5t4 and its toxicity to single administration in rats,and analyzed the research value and clinical and market application prospect of bispecific antibody.Methods1.Preparation and identification of cd47-5t4 bifunctional antibody The same amount of CD47 a antibody and 5T4 b antibody(50mg each)were taken and ultrafiltration was used to replace the buffer into PBS,and the concentration was adjusted to 10mg/ml,p H7.0~7.4.TCEP 10 mg was weighed and dissolved in 1ml PBS solution.10 mg dichloroacetone was weighed and dissolved with 1ml DMA.TCEP(190 l)of 20 times the amount of substance was added to the solution of CD47 a antibody and 5T4 b antibody respectively,and reduced for 3 hours at 37°C,and then left at room temperature overnight.The reduced CD47 a antibody and 5T4 b antibody solution were mixed and placed at room temperature for 1.5h.Cd47-5t4 bispecific antibody was prepared by adding 30 times the amount of dichloroacetone-dma solution(250 l)to the mixture.2.Toxicity test of cd47-5t4 rats by single intravenous injection Thirty healthy SD rats were taken,half male and half female.According to gender and weight,the stratified random method was used to divide the solvent control(PBS)group,CD47-5T4 low(5 mg / kg)and high(20 mg / kg)two dose groups,each group containing 5 animals / sex.Animals in each group were injected with test substance or vehicle by tail vein injection,and the administration volume was 10 ml / kg.Observe the movement immediately after the administration Response of things.Starting on the first day after the administration,the death or dying of the animals and the clinical symptoms were observed once a day for 21 consecutive days.Rats in each group were weighed on the day of administration and on days 1,2,5,8,12,15,19,and 21 after administration;on D1-D2,D4-D6,D11-D13,D18-D20 The food consumption of each cage animal was measured.2 / sex animals and 3 / sex animals in each group were examined clinically(hematology,hemagglutination,and blood biochemistry)and pathology(general anatomy and organ weight)at D3 and D22,respectively.Results1.Preparation and identification of cd47-5t4 bifunctional antibody Ultrafiltration was used to replace the buffer solution into PBS with the equivalent CD47 a antibody and 5T4 b antibody(50mg each),and the concentration was adjusted to 10mg/ml,p H7.0~7.4.TCEP 10 mg was weighed and dissolved in 1ml PBS solution.10 mg dichloroacetone was weighed and dissolved with 1ml DMA.Cd47-5t4 bispecific antibody was obtained by reduction and coupling after equal mixing,and its molecular weight was still 150 KDa under reduced and non-reduced conditions after analysis by sds-page.It indicates that the conjugation has been successful,that is,the preparation of cd47-5t4 bispecific antibody has been successful.2.Toxicity test of cd47-5t4 rats by single intravenous injection General symptom observation: no abnormal response associated with bisspecific antibody administration was observed in 5 mg/kg and 20 mg/kg cd47-5t4 animals on the day of administration and during the 21-day symptom observation period after administration.Body weight and food intake: cd47-5t4 the average body weight and food intake of 5mg/kg and 20 mg/kg animals were comparable to that of the solvent control group.Clinical laboratory: in the tests of D3 and D22,compared with the solvent control,there were no changes in blood routine indexes and blood coagulation indexes of cd47-5t4 5 mg/kg and 20 mg/kg animals.D22 showed a slight increase in AST in 5 mg/kg and 20 mg/kg cd47-5t4 animals,but no changes were observed in liver gross anatomy and organ weight,suggesting that this bispecific antibody drug might cause liver functional changes.Pathology: cd47-5t4 5 mg/kg and 20 mg/kg bispecific antibodies did not show any general changes or organ weight changes associated with administration.Conclusions1.Preparation and identification of cd47-5t4 bifunctional antibody The same amount of CD47 a antibody and 5T4 b antibody(50mg each)were added to PBS,and the concentration was adjusted to 10mg/ml,p H7.0~7.4.Take TCEP 10mg;Dichloroacetone 10mg;TCEP(190 l)was added to the solution of CD47 a antibody and 5T4 b antibody respectively,and was reduced for 3 hours at 37°C,and left overnight at room temperature.The reduced CD47 a antibody and 5T4 b antibody solution were mixed and placed at room temperature for 1.5h.Cd47-5t4 bispecific antibody was prepared by adding 30 times the amount of dichloroacetone-dma solution to the mixture.Bispecific antibody has been successfully prepared by electrophoresis and chromatography.2.Toxicity test of cd47-5t4 rats by single intravenous injection Under the present experimental conditions,SD rats were given a single intravenous injection of cd47-5t4 5 and 20 mg/kg,and 5 and 20 mg/kg showed a slight increase in AST.No changes were observed in the clinical symptoms,body weight,food intake,clinical examination(hematology,hemagglutinology,blood biochemistry)and pathology(gross anatomy and organ weight)of 20 mg/kg.The maximum tolerated dose(MTD)is greater than 20 mg/kg. |
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