IL-2 DEPENDENT NK-92 CELLS WITH STABLE FC RECEPTOR EXPRESSION
Provided herein are populations of IL2 Dependent haNK® cells, which express a high affinity CD16 but does not express IL-2. These cells maintain stable expression of Fc receptor CD16 while retaining cytotoxicity. In some embodiments, the expression level of CD16 decreases no more than 20% when the cells are activated as compared to expression level of CD16 on the cells before activation. Compositions and kits comprising the cells, and methods of making and using the IL2 Dependent haNK® cells are also provided.
1 . A population of modified NK-92® cells expressing CD16 (SEQ ID NO:1), wherein the modified NK-92® cells do not express IL-2, and wherein the population comprises one or more of the modified NK-92® cells, wherein the expression level of CD16 does not decrease or decreases no more than 20% when the cells are activated as compared to expression level of CD16 on the cells before activation.
2 . The population of modified NK-92® cells of claim 1 , wherein the modified NK-92® cells comprises a nucleic acid of CD16 (SEQ ID NO:2).
3 . The population of modified NK-92® cells of claim 1 , wherein the modified NK-92® cells have antibody-dependent cell-mediated cytotoxicity (ADCC).
4 . The population of modified NK-92® cells of claim 1 , wherein the modified NK-92® cells maintain a steady state of cytotoxicity for at least 5 hours from the initiation of the activation.
5 . The population of modified NK-92® cells of claim 1 , wherein the cells express higher level of CD16 than NK cells from a donor.
6 . The population of modified NK-92® cells of claim 1 , wherein the percentage of cells that are positive for CD16 decreases no more than 20% after the cells are activated as compared to the cells before activation.
7 . (canceled)
8 . The population of modified NK-92® cells of claim 1 ,
wherein the cells are activated by a PHA stimulation, an innate pathway activation via co-incubation with K562 cells, an ADCC activation via co-incubation with Rituxan and DOHH,
wherein the cells are activated by one or more compounds selected from the group consisting of PMA, ionomycin, and LPS, or
wherein the cells are activated by contacting target tumor cells, or
wherein the cells are activated by incubating the cells with an antibody and a target cell, wherein the incubating results in ADCC.
9 - 10 . (canceled)
11 . The population of modified NK-92® cells of claim 8 , wherein the target tumor cells are selected from the group consisting of K562 cells and SKBR-3 cells.
12 . The population of modified NK-92® cells of claim 8 , wherein the CD16 expression decreases no more than 10% as compared to the modified NK-92® cells before the activation.
13 . The population of modified NK-92® cells of claim 8 , wherein the CD16 expression decreases no more than 5% as compared to the modified NK-92® cells before the activation, or
wherein the percentage of cells that are positive for CD16 decreases no more than 10% after the cells are activated by contacting the target cells as compared to the cells before the activation.
14 - 15 . (canceled)
16 . The population of modified NK-92® cells of claim 8 , wherein the antibody is anti-CD20 antibody and the target cell is a DOHH-2 cell, or
antibody is anti-HER2 antibody and the target cell is a SKBR3 cells.
17 . (canceled)
18 . The population of modified NK-92® cells of claim 8 , wherein the effector to target ratio is 1:1 to 1:10.
19 . The population of modified NK-92® cells of claim 1 , wherein the modified NK-92® cells additionally express a chimeric antigen receptor and/or a suicide gene.
20 . The population of modified NK-92® cells of claim 1 , wherein the modified NK-92® cells have direct cytotoxicity of at least 60% when the effector to target ratio is 5:1.
21 . The population of modified NK-92® cells of claim 1 , wherein the modified NK-92® cells have ADCC activity of at least 40%.
22 . A method of producing a population of modified NK-92® cells that are capable of maintaining expression of CD16 during activation, wherein the method comprises introducing CD16 (SEQ ID NO:2), but not IL-2, into NK-92® cells, wherein the expression of CD16 on the activated modified NK-92® cells is no less than 50% of the CD16 expression on the modified NK-92® cells before the activation.
23 . The method of claim 22 , wherein the CD16 is introduced into the NK-92® cells through lentiviral infection.
24 . A kit comprising the population of modified NK-92® cells of claim 1 .
25 . The kit of claim 24 , wherein the kit further comprises an antibody.
26 . A pharmaceutical composition comprising the population of cells of claim 1 and a pharmaceutically acceptable excipient.
27 . A method of treating a subject comprising administering to the subject the pharmaceutical composition of claim 26 .
28 . (canceled)
29 . The population of modified NK-92® cells of claim 19 , wherein the suicide gene is selected from the group consisting of a thymidine kinase (TK) gene, a Cytosine deaminase, cytochrome P450, and iCas9.