Anti-robo1 car-t cell, and preparation and application thereof

Abstract

Provided is a method for modifying a chimeric antigen receptor-modified T cell (CAR-T cell). The method comprises expressing an ScFv-CD8-4-1 BB-CD3ζ molecule in a T cell. The CAR-T cell prepared using the method can specifically recognize and bind to a tumor cell with elevated expression of a ROBO1 protein, and can be used to prevent and treat a corresponding tumor-related disease

1 - 9 . (canceled) 
     
     
         10 : A method of making an anti-ROBO1 CAR-T cell, comprising:
 (1) cloning a gene encoding an anti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein into a lentiviral expression vector;   (2) packaging and preparing a lentivirus by expressing a lentiviral envelop plasmid and the lentiviral expression vector of step (1) in a 293 T cell;   (3) isolating and expanding human peripheral blood T lymphocytes and infecting the T lymphocytes with the lentivirus of step (2) to express the ScFv-CD8-4-1 BB-CD3ζ fusion protein in the T lymphocytes; wherein the ScFv portion of the fusion protein is expressed on a surface of the CAR-T cell and the 4-1 BB-CD3ζ portion of the fusion protein is expressed inside the CAR-T cell.   
     
     
         11 : The anti-ROBO1 CAR-T cell of  claim 10 , wherein the amino acid sequence of ScFv in the anti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein is SEQ ID NO:5; and the amino acid sequence of CD8 in the anti-ROBO1-ScFv-CD8-4-1 BB-CD3ζ fusion protein is SEQ ID NO:1. 
     
     
         12 : The anti-ROBO1 CAR-T cell of  claim 10 , wherein the amino acid sequence of 4-1BB in the anti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein is SEQ ID NO:2; wherein the 4-1 BB in the anti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein can be replaced by CD28 that has the amino acid sequence of SEQ ID NO:3. 
     
     
         13 : The anti-ROBO1-ScFv CAR-T cell of  claim 10 , wherein the amino acid sequence of CD3ζ in the anti-ROBO1-ScFv-CD8-4-1 BB-CD3ζ fusion protein is SEQ ID NO:4. 
     
     
         14 : The anti-ROBO1 CAR-T cell of  claim 10 , wherein the amino acid sequence of the anti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein is SEQ ID NO:6. 
     
     
         15 : The anti-ROBO1-ScFv CAR-T cell of  claim 10 , wherein the T cell is derived from human periphery blood T lymphocytes. 
     
     
         16 : A method of treating tumor by administering an anti-ROBO1 CAR-T cell expressing an anti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein; wherein the anti-ROBO1 portion of the fusion protein is expressed on a surface of the CAR-T cell and the 4-1 BB-CD3ζ portion of the fusion protein is expressed inside the CAR-T cell. 
     
     
         17 : The method of  claim 16 , wherein the amino acid sequence of ScFv in the anti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein is SEQ ID NO:5; and the amino acid sequence of CD8 in the anti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein is SEQ ID NO:1. 
     
     
         18 : The method of  claim 16 , wherein the amino acid sequence of 4-1BB in the anti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein is SEQ ID NO:2; wherein the 4-1 BB in the anti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein can be replaced by CD28 that has the amino acid sequence of SEQ ID NO:3. 
     
     
         19 : The method of  claim 16 , wherein the amino acid sequence of CD34 in the anti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein is SEQ ID NO:4. 
     
     
         20 : The method of  claim 16 , wherein the amino acid sequence of the anti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein is SEQ ID NO:6. 
     
     
         21 : The method of  claim 16 , wherein the tumor is characterized by high expression level of ROBO1.