Suppression of cytokine release syndrome in chimeric antigen receptor cell therapy

Abstract

Disclosed herein are methods of gene editing, or endogenous suppression, of cytokines/chemokines/transcription factors secreted from chimeric antigen receptor (CAR)-bearing immune effector cell such as CAR-T cells for the mitigation of cytokine release syndrome and/or CAR-T associated neuropathy. These methods involve insertion of the CAR into a locus of a cytokine gene, blocking its expression. Also disclosed herein are (CAR)-bearing immune effector cells with CARs inserted into a locus of a cytokine gene, and methods of treatment of diseases with immunotherapy with a reduced incidence of cytokine release syndrome and/or CAR-T associated neuropathy.

1 . A chimeric antigen receptor (CAR)-bearing immune effector cell that is deficient in a cytokine or in a chemokine or in a transcription factor that is involved in cytokine release syndrome. 
     
     
         2 . The cell as recited in  claim 1 , wherein the cytokine or chemokine or transcription factor deficiency is effected by deletion or suppression of a gene encoding the cytokine or chemokine or transcription factor, or by suppression of a gene transcript of the cytokine or chemokine or transcription factor gene. 
     
     
         3 . The cell as recited in  claim 2 , wherein the deletion or suppression of the gene encoding the cytokine or chemokine or transcription factor is effected by inserting the CAR into a locus of the cytokine or chemokine or transcription factor gene, or wherein the suppression of the gene transcript of the cytokine or chemokine or transcription factor is effected by transfection of one or more types of small interfering RNAs (siRNA) or by transduction of one or more types of short hairpin RNAs (shRNA). 
     
     
         4 . The cell as recited in  claim 3 , wherein the CAR is part of a construct that also includes a selectable marker chosen from a green fluorescence (GFP) gene, a YFP gene, a tCD34 gene, or a tEGFR gene. 
     
     
         5 .- 6 . (canceled) 
     
     
         7 . The cell as recited in  claim 1 , wherein deletion or suppression is effected using CRISPR, Cas9-CRISPR, Transcription Activator-like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). 
     
     
         8 . (canceled) 
     
     
         9 . The cell as recited in  claim 7 , wherein the Cas9 is delivered into the cell as mRNA or protein, and/or wherein a guide RNA (gRNA) targeting the gene to be deleted or suppressed is delivered contemporaneously with the Cas9. 
     
     
         10 .- 15 . (canceled) 
     
     
         16 . The cell of  claim 2 , wherein:
 the cell expresses at least one CAR that is inserted into a locus of the gene encoding the cytokine or chemokine or transcription factor;   the gene encoding the cytokine or chemokine or transcription factor is deleted or suppressed by a method chosen from Transcription Activator-like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), and Clustered Regularly Interspaces Short Palindromic Repeats (CRISPR) editing;   the cytokine or chemokine or transcription factor is suppressed by expression of an scFv with an endoplasmic reticulum (ER) binding tether to bind the cytokine or chemokine in the ER and prevent secretion;   the cytokine or chemokine or transcription factor gene transcript is suppressed by transfection of small interfering RNAs (siRNAs);   the cytokine or chemokine or transcription factor gene transcript is suppressed by transduction of short hairpin RNAs (shRNAs); or   the gene that is deleted or suppressed is a T cell surface receptor that is integrated into CAR-T cell signaling.   
     
     
         17 . The cell as recited in  claim 1 , wherein the cell is chosen from a chimeric antigen receptor T cell (CAR-T), a CAR-bearing iNKT cell (iNKT-CAR), and a CAR-bearing natural killer (NK) cell (NK-CAR), or a CAR-bearing macrophage. 
     
     
         18 . (canceled) 
     
     
         19 . The cell as recited in  claim 17 , wherein the cell is a dual or tandem CAR-T or iNKT-CAR or CAR-bearing macrophage. 
     
     
         20 .- 23 . (canceled) 
     
     
         24 . The cell as recited in  claim 1 , wherein the cytokine or chemokine or transcription factor contributes to the development of cytokine release syndrome and is selected from among those recited in Table 10. 
     
     
         25 . (canceled) 
     
     
         26 . The cell as recited in  claim 24 , wherein the cytokine or chemokine or transcription factor is produced by T cells that activate or localize myeloid cells, or is a T cell surface receptor gene that activates myeloid or CAR-T cells. 
     
     
         27 .- 28 . (canceled) 
     
     
         29 . The cell as recited in  claim 1 , wherein the cytokine or chemokine or transcription factor drives T cell/CAR-T cell differentiation, or wherein the cytokine or chemokine is a transcription factor that drives T cell/CAT-T cell differentiation. 
     
     
         30 . (canceled) 
     
     
         31 . The cell as recited in  claim 1 , wherein the cytokine or chemokine or transcription factor is chosen from MCP1 (CCL2), MCP-2, GM-CSF, G-CSF, M-CSF, Il-4, and IFNγ. 
     
     
         32 . (canceled) 
     
     
         33 . The cell as recited in  claim 31 , wherein the cell is a GM-CSF deficient CAR-T cell or a GM-CSF deficient iNKT-CAR cell. 
     
     
         34 .- 36 . (canceled) 
     
     
         37 . The cell as recited in  claim 1 , wherein the chimeric antigen receptor(s) specifically binds at least one antigen expressed on a malignant cell, a malignant T cell, a malignant B cell, a malignant mesothelial cell, or a malignant plasma cell. 
     
     
         38 . The cell as recited in  claim 37 , wherein the at least one antigen expressed on a malignant cell is chosen from BCMA, CS1, CD38, CD138, CD19, CD33, CD123, CD371, CD117, CD135, Tim-3, CD5, CD7, CD2, CD4, CD3, CD79A, CD79B, APRIL, CD56, and CD1a; or
 wherein the at least one antigen expressed on a malignant T cell is chosen from CD2, CD3ε, CD4, CD5, CD7, TCRA, and TCRβ; or   wherein the at least one antigen expressed on a malignant B cell is chosen from CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD38, and CD45; or   wherein the at least one antigen expressed on a malignant mesothelial cell is mesothelin; or   wherein the at least one antigen expressed on a malignant plasma cell is chosen from BCMA, CS1, CD38, and CD19.   
     
     
         39 .- 47 . (canceled) 
     
     
         48 . The cell as recited in  claim 37 , wherein the chimeric antigen receptor expresses the extracellular portion of the APRIL protein, the ligand for BCMA and TACI, effectively co-targeting both BCMA and TACI. 
     
     
         49 . (canceled) 
     
     
         50 . The cell as recited in  claim 1 , wherein endogenous T cell receptor mediated signaling is negligible in the cell. 
     
     
         51 . The cell as recited in  claim 50 , wherein the cell does not induce alloreactivity, graft-versus-host disease, or fratricide. 
     
     
         52 . (canceled) 
     
     
         53 . A method of treatment of cancer in a patient, which has a reduced incidence of cytokine release syndrome and/or CAR-T associated neuropathy, comprising the administration of cells as recited in  claim 1 . 
     
     
         54 . The method as recited in  claim 53 , wherein the cancer is a hematologic malignancy chosen from a T-cell malignancy, multiple myeloma, or acute myeloid leukemia (AML), or a solid tumor chosen from cervical cancer, pancreatic cancer, ovarian cancer, mesothelioma, or lung cancer. 
     
     
         55 . (canceled) 
     
     
         56 . The method as recited in  claim 54 , wherein the T cell malignancy is T-cell acute lymphoblastic leukemia (T-ALL), or non-Hodgkin's lymphoma. 
     
     
         57 .- 61 . (canceled) 
     
     
         61 . A method of prevention or reduction of cytokine release syndrome or CAR-T associated neuropathy in a patient being treated for cancer and receiving chimeric antigen receptor T cell (CAR-T), CAR-bearing iNKT cell (iNKT-CAR), CAR-bearing natural killer (NK) cell (NK-CAR), or CAR-bearing macrophage (CAR-macrophage) immunotherapy, comprising the administration of cells as recited in  claim 1  as the immunotherapy. 
     
     
         62 . (canceled) 
     
     
         63 . The method as recited in  claim 62 , wherein the cancer is a hematologic malignancy chosen from T-cell malignancy, multiple myeloma, or AML, or a solid tumor chosen from cervical cancer, pancreatic cancer, ovarian cancer, mesothelioma, or lung cancer. 
     
     
         64 . (canceled) 
     
     
         65 . The method as recited in  claim 64 , wherein the T cell malignancy is T-cell acute lymphoblastic leukemia (T-ALL) or non-Hodgkin's lymphoma. 
     
     
         66 .- 73 . (canceled) 
     
     
         74 . A method of making a CAR-T (immune effector) cell that does not cause or contribute to CRS or CAR-T-associated neuropathy (CAN) comprising deleting or suppressing a cytokine or chemokine or transcription factor gene, wherein deleting or suppressing the cytokine or chemokine or transcription factor gene does not reduce CAR-T cell-mediated killing. 
     
     
         75 . (canceled) 
     
     
         76 . The method as recited in  claim 74 , wherein the deletion or suppression is effected by inserting the CAR into a locus of the cytokine or chemokine or transcription factor gene, by suppression of the cytokine or chemokine or transcription factor gene transcript by transduction of one or more types of short hairpin RNAs (shRNA), or by transducing a construct encoding a protein expression blocker (PEBL). 
     
     
         77 . The method as recited in  claim 74 , wherein the CAR is part of a construct that also includes a selectable marker chosen from a green fluorescence (GFP) gene, a YFP gene, a tCD34 gene, or a tEGFR gene. 
     
     
         78 . (canceled) 
     
     
         79 . The method of  claim 74 , wherein the insertion of a CAR with a selectable marker into the cytokine or chemokine or transcription factor gene allows a single-step purification of TCR-negative cells or CAR+ cytokine negative cells. 
     
     
         80 . (canceled) 
     
     
         81 . The method as recited in  claim 74 , wherein the deletion or suppression is effected using Transcription Activator-like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), or Cas9-CRISPR. 
     
     
         82 .- 83 . (canceled) 
     
     
         84 . The method as recited in  claim 81 , wherein the Cas9 is delivered into the cell as mRNA or protein and/or wherein a guide RNA (gRNA) targeting the gene to be deleted or suppressed is delivered contemporaneously with the Cas9. 
     
     
         85 .- 90 . (canceled) 
     
     
         91 . The method of  claim 76 , wherein the construct encodes an antibody-derived single-chain variable fragment specific for the cytokine, chemokine or transcription factor gene. 
     
     
         92 .- 94 . (canceled)