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 bivalent monovalent antibody evolved from a wild-type antibody or a fragment thereof and having at least one of: (a) a decrease CAB-scFv Affinity ELISA 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.

1 - 21 . (canceled) 
     
     
         22 . 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 a decrease in activity in an assay at a normal physiological condition compared to a same activity in an assay under an aberrant condition compared to the antigen specific targeting region evolved from the parent protein or the domain thereof, 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;   ii. a transmembrane domain; and   iii. an intracellular signaling domain.   
     
     
         23 . The chimeric antigen receptor of  claim 22 , wherein the antigen specific targeting region has a decrease in a binding affinity to the target antigen at the normal physiological condition compared to the antigen specific targeting region of the parent protein or the domain thereof. 
     
     
         24 - 25 . (canceled) 
     
     
         26 . The chimeric antigen receptor of  claim 22 , further comprising an extracellular spacer domain or at least one co-stimulatory domain. 
     
     
         27 . (canceled) 
     
     
         28 . The chimeric antigen receptor of  claim 22 , 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, an affibody, and combinations thereof. 
     
     
         29 . The chimeric antigen receptor of  claim 28 , wherein the at least one antigen specific targeting region 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. 
     
     
         30 . The chimeric antigen receptor of  claim 26 , 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. 
     
     
         31 . The chimeric antigen receptor of  claim 22 , 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, CD154, and combinations thereof. 
     
     
         32 . The chimeric antigen receptor of  claim 22 , wherein the co-stimulatory domain is selected from co-stimulatory domains of proteins in the TNFR superfamily, CD28, CD137, CD134, DaplO, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS LIGHT, NKG2C, B7-H3, and combinations thereof. 
     
     
         33 . The chimeric antigen receptor of  claim 22 , 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, CD66d, and combinations thereof. 
     
     
         34 . (canceled) 
     
     
         36 . The chimeric antigen receptor of  claim 22 , wherein the normal physiological condition is selected from a temperature, pH, osmotic pressure, osmolality, oxidative stress, electrolyte concentration, a concentration of glucose, lactic acid, pyruvate, nutrient components, metabolites, oxygen, carbonate, phosphate, or carbon dioxide, cell types, and nutrient availability. 
     
     
         37 . (canceled) 
     
     
         38 . An expression vector, comprising a polynucleotide sequence encoding the chimeric antigen receptor of  claim 22 . 
     
     
         39 . The expression vector of  claim 38 , 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. 
     
     
         40 . A genetically engineered cytotoxic cell, comprising a polynucleotide sequence encoding the chimeric antigen receptor of  claim 22 . 
     
     
         41 . The genetically engineered cytotoxic cell of  claim 40 , wherein the cytotoxic cell is a T cell. 
     
     
         42 . (canceled) 
     
     
         43 . The genetically engineered cytotoxic cell of  claim 40 , 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. 
     
     
         44 - 46 . (canceled) 
     
     
         47 . A chimeric antigen receptor for binding with a target antigen, comprising:
 i. at least one antigen specific targeting region binding with the target antigen;   ii. one component selected from:
 (a) another antigen specific targeting region connected to the at least one antigen specific targeting region by a conditional linker having a first conformation at an aberrant condition for the at least two antigen specific targeting regions to bind to the target antigen at a higher binding activity than in a second conformation of the conditional linker at a normal physiological condition, 
 (b) an extracellular spacer domain having 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 second conformation of the extracellular spacer domain at the normal physiological condition, and 
 (c) an extracellular spacer domain having an enhanced ubiquitylation-resistance at the aberrant condition as compared to the ubiquitylation-resistance of the extracellular spacer domain at the normal physiological condition; 
   iii. a transmembrane domain; and   iv. an intracellular signaling domain.   
     
     
         48 . The chimeric antigen receptor of  claim 47 , wherein the at least one antigen specific targeting region has a decrease in activity in an assay at the normal physiological condition compared to a same activity in an assay under the aberrant condition. 
     
     
         49 - 52 . (canceled) 
     
     
         53 . 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 22  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.   
     
     
         54 . The method of  claim 53 , 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. 
     
     
         55 . The method of  claim 53 , wherein the cytotoxic cell is a T cell. 
     
     
         56 - 61 . (canceled)