Conditionally active chimeric antigen receptors for modified t-cells

Abstract

This disclosure relates to a chimeric antigen receptor for binding with a target antigen. The chimeric antigen receptor comprises at least one antigen specific targeting region including a multispecific antibody evolved from a wild-type antibody or a fragment thereof and having at least one of: (a) a decrease in activity in the assay at the normal physiological condition compared to the wild-type antibody or the fragment thereof, and (b) an increase in activity in the assay under the aberrant condition compared to the wild-type antibody or the fragment thereof. A method for using the chimeric antigen receptor and cytotoxic cells for cancer treatment is also provided. A method for producing the chimeric antigen receptor is also provided.

What is claimed is: 
     
         1 . A chimeric antigen receptor for binding with a target antigen, comprising:
 i. at least one antigen specific targeting region evolved from a wild-type protein or a domain thereof and having at least one of: (a) a decrease in activity in an assay at the normal physiological condition compared to the antigen specific targeting region of the wild-type protein or a domain thereof, and (b) an increase in activity in an assay under the aberrant condition compared to the antigen specific targeting region of the wild-type protein or a domain thereof;   ii. a transmembrane domain; and   iii. an intracellular signaling domain.   
     
     
         2 . The chimeric antigen receptor of  claim 1 , wherein the antigen specific targeting region has both (a) a decrease in activity in an assay at the normal physiological condition compared to the antigen specific targeting region of the wild-type protein or a domain thereof, and (b) an increase in activity in an assay under the aberrant condition compared to the antigen specific targeting region of the wild-type protein or a domain thereof. 
     
     
         3 . The chimeric antigen receptor of  claim 1 , further comprising an extracellular spacer domain or at least one co-stimulatory domain. 
     
     
         4 . The chimeric antigen receptor of  claim 3 , wherein the extracellular spacer domain is selected from an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence and combinations thereof. 
     
     
         4 . The chimeric antigen receptor of  claim 3 , wherein the extracellular spacer domain has a first conformation at the aberrant condition for the at least one antigen specific targeting region to bind to the target antigen at a higher binding activity than a binding activity of a second conformation of a second conformation of the extracellular spacer domain at the normal physiological condition. 
     
     
         5 . The chimeric antigen receptor of  claim 3 , wherein the extracellular spacer domain has an enhanced ubiquitylation-resistance level at the aberrant condition than at the normal physiological condition. 
     
     
         6 . The chimeric antigen receptor of  claim 1 , wherein the at least one antigen specific targeting region comprises two antigen specific targeting regions that are connected with a linker. 
     
     
         7 . The chimeric antigen receptor of  claim 6 , wherein the two antigen specific targeting regions each binds with a different target antigen or a different epitope of the same target antigen. 
     
     
         8 . The chimeric antigen receptor of  claim 6 , wherein the linker has a first conformation at the aberrant condition for the at least two antigen specific targeting regions to bind to the target antigen at a higher binding activity than a binding activity of a second conformation of the linker at the normal physiological condition. 
     
     
         9 . The chimeric antigen receptor of  claim 1 , wherein the at least one antigen specific targeting region is selected from an antibody, a fragment of an antibody, a divalent single chain antibody or a diabody, a ligand, a receptor binding domain of a ligand, a receptor, a ligand binding domain of a receptor, and an affibody. 
     
     
         10 . The chimeric antigen receptor of  claim 9 , wherein the antibody is a multiple specific antibody. 
     
     
         11 . The chimeric antigen receptor of  claim 1 , wherein the transmembrane domain is selected from an artificial hydrophobic sequence and transmembrane domains 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, and CD154. 
     
     
         12 . The chimeric antigen receptor of  claim 1 , wherein the co-stimulatory domain is selected from co-stimulatory domains of proteins in the TNFR superfamily, CD28, CD 137, CD 134, DaplO, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS LIGHT, NKG2C, and B7-H3. 
     
     
         13 . The chimeric antigen receptor of  claim 1 , wherein the intracellular signaling domain is selected from cytoplasmic signaling domains of a human CD3 zeta chain, FcyRIII, FcsRI, a cytoplasmic tail of a Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. 
     
     
         14 . The chimeric antigen receptor of  claim 1 , wherein the normal physiological condition is selected from one or more of temperature, pH, osmotic pressure, osmolality, oxidative stress, an electrolyte concentration, a concentration of a small organic molecule, a concentration of inorganic molecule, cell types, and nutrient availability. 
     
     
         15 . The chimeric antigen receptor of  claim 1 , wherein the aberrant condition is a condition selected from conditions present in a tumor microenvironment, a brain extracellular fluid, a stem cell niche, a lymph node, a tonsil, an adenoid, a sinus, and a synovial fluid. 
     
     
         16 . An expression vector, comprising a polynucleotide sequence encoding the chimeric antigen receptor of  claim 1 . 
     
     
         17 . The expression of  claim 16 , wherein the expression vector is selected from lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno associated virus vectors, adenovirus vectors, pox virus vectors, herpes virus vectors, engineered hybrid viruses, and transposon mediated vectors. 
     
     
         18 . A genetically engineered cytotoxic cell, comprising a polynucleotide sequence encoding the chimeric antigen receptor of  claim 1 . 
     
     
         19 . The genetically engineered cytotoxic cell of  claim 17 , wherein the cytotoxic cell is selected from a T cell, natural killer cell, an activated NK cells, a neutrophil, an eosinophil, a basophil, a B-cell, a macrophage and a lymphokine-activated killer cell. 
     
     
         20 . A method for treating a cancer in a subject, comprising the steps of:
 a. introducing an expression vector comprising a polynucleotide sequence encoding the chimeric antigen receptor of  claim 1  into a cytotoxic cell obtained from the subject to produce an genetically engineered cytotoxic cell; and   b. administering the genetically engineered cytotoxic cell to the subject.   
     
     
         21 . 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 the steps of
 generating the at least one antigen specific targeting region from a wild-type protein or a domain thereof that binds specifically with a target antigen, by:
 i. evolving the DNA which encodes the wild-type protein or a domain thereof using one or more evolutionary techniques to create mutant DNAs; 
 ii. expressing the mutant DNAs to obtain mutant polypeptides; 
 iii. subjecting the mutant polypeptides and the wild-type protein or a domain thereof to an assay under a normal physiological condition and to an assay under an aberrant condition; and 
 iv. selecting a conditionally active antigen specific targeting region from the mutant polypeptides expressed in step (iii) which exhibits at least one of: (a) a decrease in activity in the assay at the normal physiological condition compared to the antigen specific targeting region of the wild-type protein or a domain thereof, and (b) an increase in activity in the assay under the aberrant condition compared to the antigen specific targeting region of the wild-type protein or a domain thereof.