Conditionally active chimeric antigen receptors for modified t-cells

Abstract

This disclosure relates to a chimeric antigen receptor for binding with a tumor specific target antigen. The chimeric antigen receptor comprises at least one antigen specific targeting region evolved from a parent protein or a fragment thereof and having a decrease in activity in the assay at the normal physiological condition compared to the activity in the assay under the aberrant condition. A method for producing the chimeric antigen receptor is also provided.

What is claimed is: 
     
         1 . A method for producing a chimeric antigen receptor comprising at least one antigen specific targeting region, a transmembrane domain and an intracellular signaling domain, said method comprising generating the at least one antigen specific targeting region from a parent or wild-type protein or a domain thereof that binds specifically with a tumor specific target antigen on the surface of a cancer cell, by steps of:
 i) evolving DNA which encodes the parent or wild-type protein or the domain thereof using one or more non-stochastic evolutionary techniques to create mutant DNAs;   ii) expressing the mutant DNAs to obtain mutant polypeptides;   iii) subjecting the mutant polypeptides to a target antigen binding assay under a normal physiological pH of 7.4 and to a target antigen binding assay under an aberrant tumor microenvironment pH of from 6.0 to 7.0;   iv) selecting the at least one antigen specific targeting region from the mutant polypeptides expressed in step ii) which exhibits a decrease in binding activity in the assay at the normal physiological pH of 7.4 compared to the binding activity in the assay under the aberrant tumor microenvironment pH of from 6.0 to 7.0;   v) ligating the polynucleotide sequences encoding the antigen specific targeting region selected in step iv), the transmembrane domain and the intracellular signaling domain to form a polynucleotide; and   vi) expressing the polynucleotide formed in step v) to obtain the chimeric antigen receptor.   
     
     
         2 . The method of  claim 1 , further comprising inserting at least one of an extracellular spacer domain or a co-stimulatory domain into the chimeric antigen receptor. 
     
     
         3 . The method of  claim 1 , wherein the antigen specific targeting region is an antibody or antibody fragment. 
     
     
         4 . The method of  claim 3 , wherein the antibody or antibody fragment is a single chain antibody, a diabody, an Fab fragment, and Fab′ fragment, an (Fab)2 fragment or an Fv fragment. 
     
     
         5 . The method of  claim 1 , wherein the transmembrane domain is an artificial hydrophobic sequence or transmembrane domain of a Type I transmembrane protein, an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154. 
     
     
         6 . The method of  claim 1 , wherein the non-stochastic evolutionary technique is site saturation mutagenesis. 
     
     
         7 . An isolated expression vector, comprising a polynucleotide sequence encoding the chimeric antigen receptor produced by the method of  claim 1 . 
     
     
         8 . The isolated expression vector of  claim 7 , wherein the expression vector is selected from lentivirus vectors, adenovirus vectors, pos virus vectors, herpes virus vectors, engineered hybrid vectors, and transposon mediated vectors. 
     
     
         9 . A genetically engineered cytotoxic cell, comprising a polynucleotide sequence encoding the chimeric antigen receptor produced by the method of  claim 1 . 
     
     
         10 . The genetically engineered cytotoxic cell of  claim 9 , wherein the cytotoxic cell is a T-cell. 
     
     
         11 . The genetically engineered cytotoxic cell of  claim 10 , wherein the T-cell is selected from a naïve T-cell, a central memory T-cell and an effector memory T-cell. 
     
     
         12 . The genetically engineered cytotoxic cell of  claim 9 , wherein the cytotoxic cell is selected from a natural killer cell, an activated NK cell, a neutrophil, an eosinophil, a basophil, a B-cell macrophage and a lymphokine-activated killer cell.