Car-expressing nk-92 cells as cell therapeutic agents

Abstract

The present invention relates to an ErbB2-specific NK-92 cell or cell line containing a lentiviral vector encoding a chimeric antigen receptor and preferably two vector integration loci in its cellular genome. The present invention further relates to the use of the ErbB2-specific NK-92 cell or cell line in the prevention and/or treatment of cancer, preferably ErhB2-expressing cancers. The present invention further relates to the use of the ErbB2-specific NK-92 cell or cell line as targeted cell therapeutic agent and/or for adoptive cancer immunotherapy. The present invention further relates to a method for generating an ErbB2-specific NK-92 cell or cell line as well as to a method for identifying au ErbB2-specific NK-92 cell or cell line and to the ErbB2-specific NK-92 cell or cell line obtained or identified by the methods as well as their uses.

1 .- 15 . (canceled) 
     
     
         16 . A method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of ErbB2-specific NK-92 cells, the cells containing a lentiviral vector encoding a chimeric antigen receptor comprising an ErbB2-specific scFv antibody fragment, a hinge region, transmembrane and intracellular domains of CD28, and intracellular domain of CD3 zeta, and wherein said vector is genomically integrated, wherein administration treats the cancer in the subject. 
     
     
         17 . The method of  claim 16 , wherein said vector is genomically integrated (i) in an intergenic region on chromosome 2, and (ii) in the TRAF2 gene on chromosome 9. 
     
     
         18 . The method of  claim 16 , wherein the cell or cell line is characterized in that by PCR analysis of the genomic DNA of said cell or cell line at least one of the following amplification products is obtained:
 PCR with primers of SEQ ID NOs. 1 and 2 yields an amplification product with the nucleotide sequence of SEQ ID NO. 9;   PCR with primers of SEQ ID NOs. 3 and 4 yields an amplification product with the nucleotide sequence of SEQ ID NO. 10;   PCR with primers of SEQ ID NOs. 5 and 6 yields an amplification product with the nucleotide sequence of SEQ ID NO. 11;   PCR with primers of SEQ ID NOs. 7 and 8 yields an amplification product with the nucleotide sequence of SEQ ID NO. 12.   
     
     
         19 . The method of  claim 16 , wherein the cells show reduced or no natural cytotoxicity to ErbB2-negative cells. 
     
     
         20 . The method of  claim 16 , wherein the cells show increased cytotoxicity to ErbB2-expressing tumor cells compared to unmodified NK-92 cells. 
     
     
         21 . The method of  claim 16 , wherein the ErbB2-specific scFv antibody fragment comprises the amino acid sequence of SEQ ID NO. 13 (scFv FRP5). 
     
     
         22 . The method of  claim 16 , wherein the ErbB2-specific scFv antibody fragment is encoded by the nucleotide sequence of SEQ ID NO. 14. 
     
     
         23 . The method of  claim 16 , wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO. 15 (full-length CAR). 
     
     
         24 . The method of  claim 16 , wherein the chimeric antigen receptor is encoded by the nucleotide sequence of SEQ ID NO. 16. 
     
     
         25 . The method of  claim 16 , wherein the cancer is an ErbB2-expressing cancer. 
     
     
         26 . The method of  claim 16 , wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, gastric cancer, prostate cancer, squamous cell carcinoma, head and neck cancer, colon cancer, pancreatic cancer, uterine cancer, renal cell cancer, glioblastoma, medulloblastoma, sarcoma, and lung cancer. 
     
     
         27 . The method of  claim 16 , wherein the method comprises pre-treatment of the cells by irradiation, preferably γ-irradiation. 
     
     
         28 . The method of  claim 16 , wherein 1×10 5  to 1×10 7  cells are administered to the subject. 
     
     
         29 . The method of  claim 16 , wherein the cells are administered intravenously.