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 . (canceled) 
     
     
         2 . The genetically engineered cytotoxic cell of  claim 18 , wherein the antigen specific targeting region of the chimeric antigen receptor also has at least one of (a) a decrease in binding activity to the cancer cell surface antigen in the assay at the normal physiological pH compared to the binding activity to the cancer cell surface antigen of an antigen specific targeting region of the parent protein or the domain thereof in the assay at the normal physiological pH, and (b) an increase in binding activity in the assay under the aberrant pH compared to the binding activity to the cancer cell surface antigen of the antigen specific targeting region of the parent protein or the domain thereof in the assay at the aberrant pH. 
     
     
         3 . The genetically engineered cytotoxic cell of  claim 18 , wherein the chimeric antigen receptor further comprises an extracellular spacer domain or at least one co-stimulatory domain. 
     
     
         4 . The genetically engineered cytotoxic cell 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 . (canceled) 
     
     
         5 . The genetically engineered cytotoxic cell of  claim 3 , wherein the extracellular spacer domain of the chimeric antigen receptor has an enhanced ubiquitylation-resistance level at the aberrant pH as compared to the ubiquitylation-resistance at the normal physiological pH. 
     
     
         6 . The genetically engineered cytotoxic cell of  claim 18 , wherein the at least one antigen specific targeting region of the chimeric antigen receptor comprises two antigen specific targeting regions that are connected by a linker. 
     
     
         7 . The genetically engineered cytotoxic cell of  claim 6 , wherein the two antigen specific targeting regions each bind with a different target antigen or a different epitope of the same target antigen. 
     
     
         8 . The genetically engineered cytotoxic cell of  claim 6 , wherein the linker has a first conformation at the aberrant pH for the at least two antigen specific targeting regions to bind to the target antigen with a higher binding activity than a binding activity of a second conformation of the linker at the normal physiological pH. 
     
     
         9 . The genetically engineered cytotoxic cell of  claim 18 , wherein the at least one antigen specific targeting region of the chimeric antigen receptor 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 genetically engineered cytotoxic cell of  claim 9 , wherein the antibody is a multi-specific antibody. 
     
     
         11 . The genetically engineered cytotoxic cell of  claim 18 , wherein the transmembrane domain of the chimeric antigen receptor 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 genetically engineered cytotoxic cell of  claim 18 , wherein the co-stimulatory domain of the chimeric antigen receptor is selected from co-stimulatory domains of proteins in the TNFR superfamily, CD28, CD137, CD134, Dap1O, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS LIGHT, NKG2C, and B7-H3. 
     
     
         13 . The genetically engineered cytotoxic cell of  claim 18 , wherein the intracellular signaling domain of the chimeric antigen receptor is selected from cytoplasmic signaling domains of a human CD3 zeta chain, FcγRIII, 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 . (canceled) 
     
     
         15 . The genetically engineered cytotoxic cell of  claim 18 , wherein the aberrant pH is a pH present in one of 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 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . A genetically engineered cytotoxic cell, comprising a polynucleotide sequence encoding a chimeric antigen receptor for binding with a target antigen that is a cancer cell surface antigen, said chimeric antigen receptor comprising:
 i. at least one antigen specific targeting region evolved from a parent protein or a domain thereof and having a decrease in binding activity to the cancer cell surface antigen in an assay at a normal physiological pH compared to a same binding activity of the antigen specific targeting region to the cancer cell surface antigen in an assay at an aberrant pH that differs from the normal physiological pH;   ii. a transmembrane domain; and   iii. an intracellular signaling domain.   
     
     
         19 . The genetically engineered cytotoxic cell of  claim 18 , 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 a chimeric antigen receptor for binding with a target antigen that is a cancer cell surface antigen, said chimeric antigen receptor comprising:   i. at least one antigen specific targeting region evolved from a parent protein or a domain thereof and having a decrease in binding activity to the cancer cell surface antigen in an assay at a normal physiological pH compared to a same binding activity of the antigen specific targeting region to the cancer cell surface antigen in an assay at an aberrant pH that differs from the normal physiological pH;   ii. a transmembrane domain; and   iii. an intracellular signaling domain, into a cytotoxic cell obtained from the subject to produce a genetically engineered cytotoxic cell; and   b. administering the genetically engineered cytotoxic cell to the subject.   
     
     
         21 . (canceled) 
     
     
         22 . The genetically engineered cytotoxic cell 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 cancer cell surface antigen at a higher binding activity than a binding activity to the cancer cell surface antigen of a second conformation of a second conformation of the extracellular spacer domain at the normal physiological condition. 
     
     
         23 . The genetically engineered cytotoxic cell of  claim 18 , wherein the cancer cell surface antigen is a tyrosine kinase growth factor receptor. 
     
     
         24 . The genetically engineered cytotoxic cell of  claim 18 , wherein the normal physiological pH is a pH in a range of 7.2 to 7.6. 
     
     
         25 . The genetically engineered cytotoxic cell of  claim 18 , where in the aberrant pH is a lower pH than the normal physiological pH. 
     
     
         26 . The method of  claim 20 , wherein the cancer cell surface antigen is a tyrosine kinase growth factor receptor. 
     
     
         27 . The method of  claim 20 , wherein the normal physiological pH is a pH in a range of 7.2 to 7.6. 
     
     
         28 . The method of  claim 20 , where in the aberrant pH is a lower pH than the normal physiological pH.