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 evolved from a wild-type protein or a domain thereof and having 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. A method for generating the chimeric antigen receptor and cytotoxic cells that express the chimeric antigen receptor are also provided.

1 . A chimeric antigen receptor for binding with a target antigen, comprising:
 i. at least one antigen specific targeting region evolved from a parent protein or a domain thereof and having at least one of: (a) a decrease in activity in an assay at normal physiological condition compared to an antigen specific targeting region of the parent protein or the domain thereof, and (b) an increase in activity in an assay under an aberrant condition that deviates from a normal range of a physiological condition, compared to the antigen specific targeting region of the parent protein or the domain thereof;   ii. a transmembrane domain; and   iii. an intracellular signaling domain.   
     
     
         2 - 4 . (canceled) 
     
     
         5 . The chimeric antigen receptor of  claim 1 , configured such that a protein containing the chimeric antigen receptor has an increase in expression level compared to the parent protein or the domain thereof, as measured in a same expression host. 
     
     
         6 . The chimeric antigen receptor of  claim 1 , further comprising an extracellular spacer domain or at least one co-stimulatory domain. 
     
     
         7 . The chimeric antigen receptor of  claim 1 , wherein the at least one antigen specific targeting region comprises two antigen specific targeting regions and each antigen specific targeting region binds with a different target antigen or a different epitope of the same target antigen. 
     
     
         8 . The chimeric antigen receptor of  claim 1 , wherein the at least one antigen specific targeting region is selected from an antibody, a ligand, a receptor binding domain of a ligand, a receptor, a ligand binding domain of a receptor, and an affibody. 
     
     
         9 . The chimeric antigen receptor of  claim 8 , wherein the chimeric antigen receptor is an antibody selected from a full length antibody, a single-chain antibody, an Fab fragment, an Fab′ fragment, an (Fab′)2 fragment, an Fv fragment, and a divalent single chain antibody or a diabody. 
     
     
         10 . (canceled) 
     
     
         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 . (canceled) 
     
     
         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 target antigen is located on a surface of cancer cells. 
     
     
         15 . The chimeric antigen receptor of  claim 1 , wherein the target antigen is selected from 4-1BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD 152, CD 19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DRS, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, and vimentin. 
     
     
         16 . The chimeric antigen receptor of  claim 1 , wherein the normal physiological condition is selected from one or more of normal physiological temperature, pH, osmotic pressure, osmolality, oxidative stress, and electrolyte concentration. 
     
     
         17 . (canceled) 
     
     
         18 . An expression vector, comprising a polynucleotide sequence encoding the chimeric antigen receptor of  claim 1 . 
     
     
         19 . The expression vector of  claim 18 , where 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. 
     
     
         20 . A genetically engineered cytotoxic cell, comprising a polynucleotide sequence encoding the chimeric antigen receptor of  claim 1 . 
     
     
         21 . The genetically engineered cytotoxic cell of  claim 20 , wherein the cytotoxic cell is a T cell. 
     
     
         22 . The genetically engineered cytotoxic cell of  claim 21 , wherein the T cell is selected from a naive T cell, a central memory T cell, and an effector memory T cell. 
     
     
         23 . The genetically engineered cytotoxic cell of  claim 20 , wherein the cytotoxic cell is selected from a natural killer cell, an activated NK cells, a neutrophil, an eosinophil, a basophil, a B-cell, a macrophage and a lymphokine-activated killer cell. 
     
     
         24 - 25 . (canceled) 
     
     
         26 . A pharmaceutical composition, comprising:
 a. one or more of the chimeric antigen receptors of  claim 1 , the expression vector of  claim 18 , and the genetically engineered cytotoxic cell of  claim 20 ; and   b. a pharmaceutically acceptable excipient.   
     
     
         27 . 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 steps of
 i. generating the at least one antigen specific targeting region from a parent protein or a domain thereof that binds specifically with a target antigen, by:   ii. evolving DNA which encodes the parent protein or the domain thereof using one or more evolutionary techniques to create mutant DNAs;   iii. expressing the mutant DNAs to obtain mutant polypeptides;   iv. subjecting the mutant polypeptides and the parent protein or the domain thereof to at least one of an assay under a normal physiological condition and an assay under an aberrant condition that deviates from a normal range of a physiological condition; and   v. 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 an antigen specific targeting region of the parent protein or the domain thereof, and (b) an increase in activity in the assay under the aberrant condition compared to the antigen specific targeting region of the parent protein or the domain thereof.   
     
     
         28 - 31 . (canceled) 
     
     
         32 . The method of  claim 27 , wherein the normal physiological condition is selected from one or more of temperature, pH, osmotic pressure, osmolality, oxidative stress and electrolyte concentration. 
     
     
         33 - 34 . (canceled) 
     
     
         35 . 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 genetically engineered cytotoxic cell; and   b. administering the genetically engineered cytotoxic cell to the subject.   
     
     
         36 - 44 . (canceled)