CHIMERIC ANTIGEN RECEPTORS (CARs) HAVING MUTATIONS IN THE FC SPACER REGION AND METHODS FOR THEIR USE

Abstract

Adoptive immunotherapy using T cells genetically redirected via expression of chimeric antigen receptors (CARs) is a promising approach for cancer treatment. However, this immunotherapy is dependent in part on the optimal molecular design of the CAR, which involves an extracellular ligand-binding domain connected to an intracellular signaling domain by spacer and/or transmembrane sequences.

What is claimed is: 
     
         1 . A recombinant chimeric antigen receptor (CAR) having impaired binding to an Fc receptor (FcR) comprising:
 an antigen recognition domain;   a spacer domain derived from a modified immunoglobulin Fc region having one or more mutations in its CH2 region resulting in impaired binding to an FcR; and   an intracellular signaling domain.   
     
     
         2 . The method of  claim 1 , wherein the antigen recognition domain is an scFv. 
     
     
         3 . The method of  claim 1 , wherein the antigen recognition domain targets a cancer associated antigen selected from the group consisting of 5T4, 8H9, αvβ6 integrin, alphafetoprotein (AFP), B7-H6, CA-125 carbonic anhydrase 9 (CA9), CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD52, CD123, CD171, carcionoembryonic antigen (CEA), EGFrvIII, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbB1/EGFR, ErbB2/HER2/neu/EGFR2, ErbB3, ErbB4, epithelial tumor antigen (ETA), FBP, fetal acetylcholine receptor (AchR), folate receptor-α, G250/CAIX, ganglioside 2 (GD2), ganglioside 3 (GD3), HLA-A1, HLA-A2, high molecular weight melanoma-associated antigen (HMW-MAA), IL-13 receptor α2, KDR, k-light chain, Lewis Y (LeY), L1 cell adhesion molecule, melanoma-associated antigen (MAGE-A1), mesothelin, Murine CMV infected cella, mucin-1 (MUC1). mucin-16 (MUC16), natural killer group 2 member D (NKG2D) ligands, nerve cell adhesion molecule (NCAM), NY-ESO-1, Oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor-tyrosine kinase-like orphan receptor 1 (ROR1), TAA targeted by mAb IgE, tumor-associated glycoprotein-72 (TAG-72), tyrosinase, and vascular endothelial growth factor (VEGF) receptors. 
     
     
         4 . The method of  claim 1 , wherein the modified immunoglobulin Fc region is a modified IgG1, IgG2, IgG3, or IgG4 Fc region. 
     
     
         5 . The method of  claim 1 , wherein the one or more mutations of the modified immunoglobulin Fc region comprise one or more amino acid substitutions selected from an S228P amino acid substitution, an L235E amino acid substitution, an N297Q amino acid substitution, or a combination thereof. 
     
     
         6 . The method of  claim 1 , wherein the one or more mutations of the modified immunoglobulin Fc region comprise one or more deletions. 
     
     
         7 . The method of  claim 1 , further comprising a transmembrane domain. 
     
     
         8 . The method of  claim 1 , wherein the intracellular signaling domain is a T cell receptor (TCR) zeta chain signaling domain. 
     
     
         9 . The method of  claim 8 , further comprising one or more costimulatory intracellular signaling domain derived from CD28, inducible costimulatory (ICOS), OX40, CD27, DAP10, 4-1BB, p56lck, or 2B4. 
     
     
         10 . The method of  claim 1 , wherein the CAR is encoded by a nucleic acid sequence which that is inserted within a viral vector. 
     
     
         11 . A population of human immune cells transduced by a viral vector comprising an expression cassette that includes a CAR gene, the gene comprising a nucleotide sequence that encodes:
 an antigen recognition domain;   a spacer domain derived from a modified immunoglobulin Fc region having one or more mutations in its CH2 region resulting in impaired binding to an FcR; and   an intracellular signaling domain;   wherein the population of human immune cells expresses the CAR gene.   
     
     
         12 . The method of  claim 11 , wherein the antigen recognition domain targets a cancer associated antigen selected from the group consisting of 5T4, 8H9, αvβ6 integrin, alphafetoprotein (AFP), B7-H6, CA-125 carbonic anhydrase 9 (CA9), CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD52, CD123, CD171, carcionoembryonic antigen (CEA), EGFrvIII, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbB1/EGFR, ErbB2/HER2/neu/EGFR2, ErbB3, ErbB4, epithelial tumor antigen (ETA), FBP, fetal acetylcholine receptor (AchR), folate receptor-α, G250/CAIX, ganglioside 2 (GD2), ganglioside 3 (GD3), HLA-A1, HLA-A2, high molecular weight melanoma-associated antigen (HMW-MAA), IL-13 receptor α2, KDR, k-light chain, Lewis Y (LeY), L1 cell adhesion molecule, melanoma-associated antigen (MAGE-A1), mesothelin, Murine CMV infected cella, mucin-1 (MUC1). mucin-16 (MUC16), natural killer group 2 member D (NKG2D) ligands, nerve cell adhesion molecule (NCAM), NY-ESO-1, Oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor-tyrosine kinase-like orphan receptor 1 (ROR1), TAA targeted by mAb IgE, tumor-associated glycoprotein-72 (TAG-72), tyrosinase, and vascular endothelial growth factor (VEGF) receptors. 
     
     
         13 . The method of  claim 11 , wherein the modified immunoglobulin Fc region is a modified IgG1, IgG2, IgG3, or IgG4 Fc region. 
     
     
         14 . The method of  claim 11 , wherein the one or more mutations of the modified immunoglobulin Fc region comprise one or more amino acid substitutions selected from an S228P amino acid substitution, an L235E amino acid substitution, an N297Q amino acid substitution, or a combination thereof. 
     
     
         15 . The method of  claim 11 , wherein the one or more mutations of the modified immunoglobulin Fc region comprise one or more deletions. 
     
     
         16 . The method of  claim 11 , wherein the intracellular signaling domain is a T cell receptor (TCR) zeta chain signaling domain. 
     
     
         17 . The method of  claim 16 , further comprising one or more costimulatory intracellular signaling domain derived from CD28, inducible costimulatory (ICOS), OX40, CD27, DAP10, 4-1BB, p56lck, or 2B4. 
     
     
         18 . A method of treating a cancer in a subject comprising administering a population of human immune cells transduced with a CAR gene to the subject, wherein the CAR gene comprises a nucleotide sequence that encodes:
 an antigen recognition domain that targets a cancer associated antigen specific to the cancer;   a spacer domain derived from a modified immunoglobulin Fc region having one or more mutations in its CH2 region resulting in impaired binding to an FcR; and   an intracellular signaling domain.   
     
     
         19 . The method of  claim 18 , wherein the impaired binding to the FcR results in improved persistence of the human immune cells as compared to human immune cells transduced with a CAR gene comprising a nucleotide sequence that encodes a spacer domain derived from an unmodified immunoglobulin Fc region. 
     
     
         20 . The method of  claim 18 , further comprising administering the population of human immune cells transduced with the CAR gene in combination with one or more anti-cancer therapy selected from stem cell transplantation, radiation therapy, surgical resection, chemotherapeutics, immunotherapeutics, targeted therapeutics or a combination thereof. 
     
     
         21 . A recombinant chimeric antigen receptor (CAR) having impaired binding to an Fc receptor (FcR) comprising:
 an antigen recognition domain comprising an scFv;   a spacer domain derived from a modified immunoglobulin Fc region having one or more mutations in its CH2 region resulting in impaired binding to an FcR, wherein the one or more mutations are selected from an S228P amino acid substitution, an L235E amino acid substitution, an N297Q amino acid substitution, or a combination thereof; and   an intracellular signaling domain.