HLA CLASS I-DEFICIENT NK-92 CELLS WITH DECREASED IMMUNOGENICITY
Described herein are modified NK-92 cells comprising a genetic alteration to decrease beta-2-microglobulin (B2M) expression in NK-92 cells to reduce the levels of HLA class I expression; methods of generating such cells; and methods of treating a subject, e.g., that has cancer, with the B2M-modified NK-92 cells.
1 . A beta-2-microglobulin-modified (B2M-modified) NK-92 cell comprising a beta-2 microglobulin-targeted genetic modification to inhibit expression of beta-2 microglobulin.
2 . The B2M-modified NK-92 cell of claim 1 , wherein the cell is produced by knocking down or knocking out beta-2 microglobulin in an NK-92 cell.
3 . The B2M-modified NK-92 cells of claim 2 , comprising an interfering RNA that targets B2M and inhibits its expression.
4 .- 5 . (canceled)
6 . The B2M-modified NK-92 cell of claim 2 , wherein the cell is modified to express a single chain trimer comprising an HLA-E binding peptide, B2M, and HLA-E heavy chain.
7 . The B2M-modified NK-92 cell of claim 6 , wherein the single chain trimer comprises a B2M (β2 microglobulin) signal peptide, a Cw*0304 leader peptide, a mature B2M polypeptide and a mature HLA-E polypeptide.
8 . The B2M-modified NK-92 cell of claim 7 , wherein the Cw*0304 leader peptide is linked to the mature B2M polypeptide by a flexible linker and/or the mature B2M polypeptide is linked to the mature HLA-E polypeptide by a flexible linker.
9 . (canceled)
10 . The B2M-modified NK-92 cell of claim 6 , wherein the HLA-E heavy chain comprises a mature HLA-EG amino acid sequence.
11 . The B2M-modified NK-92 cell of claim 6 , wherein the single chain trimer comprises the amino acid sequence of SEQ ID NO:18.
12 . The B2M-modified NK-92 cell of claim 1 , wherein the B2M-modified NK cell expresses at least one Fc receptor or at least one chimeric antigen receptor (CAR); or at least one Fc receptor and at least one CAR on the cell surface.
13 . The B2M-modified NK-92 cell of claim 12 , wherein the at least one Fc receptor is a human CD16 or a human CD16 polypeptide having a valine at a position corresponding to position 158 of the mature form of the CD16 polypeptide.
14 .- 17 . (canceled)
18 . The B2M-modified NK-92 cell of claim 1 , wherein the cell is further modified to expresses a cytokine.
19 .- 20 . (canceled)
21 . A composition comprising a plurality of cells of claim 1 .
22 . (canceled)
23 . A modified NK-92 cell line comprising a plurality of modified NK-92 cells of claim 1 .
24 . The cell line of claim 23 , wherein the cells undergo less than 10 population doublings.
25 . (canceled)
26 . A method of treating cancer in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the cell line of claim 23 , thereby treating the cancer.
27 .- 28 . (canceled)
29 . A method for producing an NK-92 cell that expresses decreased levels of beta-2 microglobulin relative to a control NK-92 cell, the method comprising genetically modifying the NK-92 cell to inhibit beta-2 microglobulin expression.
30 . The method of claim 29 , wherein the step of genetically modifying beta-2 microglobulin expression comprises modifying the beta-2 microglobulin gene with a zinc finger nuclease (ZFN), a Tale-effector domain nuclease (TALEN), or a CRIPSR/Cas system to eliminate or reduce expression of the beta-2 microglobulin gene; or comprises contacting a NK-92 cell to be modified with an interfering RNA targeting beta-2 microglobulin.
31 . The method of claim 30 , wherein the step of genetically modifying beta-2 microglobulin expression comprises modifying the beta-2 microglobulin gene with a CRIPSR/Cas system to eliminate or reduce expression of the beta-2 microglobulin gene.
32 .- 34 . (canceled)
35 . The method of claim 31 , wherein genetically modifying the beta-2 microglobulin gene expression comprises:
i) introducing a clustered regularly interspaced short palindromic repeat-associated (Cas) protein into the NK-92 cell and
ii) introducing one or more ribonucleic acids in the NK-92 cell to be modified, wherein the ribonucleic acids direct the Cas protein to hybridize to a target motif of the beta-2 microglobulin sequence, and wherein the target motif is cleaved.
36 .- 38 . (canceled)
39 . The method of claim 35 , wherein the target motif is in the first exon of beta 2 microglobulin gene.
40 .- 41 . (canceled)