CAR-expressing NK-92 cells as cell therapeutic agents
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 ErbB2-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 an 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. An ErbB2-specific NK-92 cell or cell line, 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 in: i) an intergenic region on chromosome 2 and ii) the Tumor necrosis factor Receptor Associated Factor 2 (TRAF2) gene on chromosome 9, wherein the NK-92 cell is NK-92/5.28.z having accession number DSM ACC3244.
2. The NK-92 cell or cell line according to claim 1 , 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.
3. The NK-92 cell or cell line according to claim 1 , showing reduced or no natural cytotoxicity to ErbB2-negative cells which are lysed by unmodified NK-92 cells.
4. The NK-92 cell or cell line according to claim 1 , wherein the ErbB2-specific scFv antibody fragment comprises the amino acid sequence of SEQ ID NO. 13 (scFv FRP5).
5. The NK-92 cell or cell line according to claim 1 , wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO. 15 (full-length CAR).
6. The NK-92 cell or cell line according to claim 1 for use as targeted cell therapeutic agent.
7. The NK-92 cell or cell line according to claim 1 , wherein the cells have been treated by irradiation.
8. The NK-92 cell or cell line according to claim 1 , wherein the cell shows increased cytotoxicity to ErbB2-expressing tumor cells compared to unmodified NK-92 cells.
9. The NK-92 cell or cell line according to claim 1 , wherein the ErbB2-specific scFv antibody fragment is encoded by the nucleotide sequence of SEQ ID NO. 14.
10. The NK-92 cell or cell line according to claim 1 , wherein the chimeric antigen receptor is encoded by the nucleotide sequence of SEQ ID NO. 16.
11. The NK-92 cell or cell line according to claim 7 , wherein the cells have been treated by γ-irradiation.