GENETICALLY MODIFIED HUMAN NATURAL KILLER CELL LINES
The invention provides a natural killer cell, NK-92, modified to express a CD16 receptor or an inhibitory killer cell immunoglobulin-like receptor (KIR) on a surface of the cell. In examples, the NK-92 cell is further modified to co-express an associated accessory signaling protein such as FcεRI-γ or TCR-ζ, chemokines, or cytokines such as interleukin-2 (IL-2) or interleukin-15 (IL-15). Additional methods are disclosed for various assays, assessments, and therapeutic treatments with the modified NK-92 cells.
1 . A modified NK-92 cell comprising an NK-92 cell modified to express a CD16 receptor on a surface of the cell.
2 . The modified cell of claim 1 wherein the CD16 receptor comprises a native form of CD16.
3 . The modified cell of claim 1 wherein the CD16 receptor comprises a variant of a native form of CD16.
4 . The modified cell of claim 1 wherein a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity with SEQ ID NO: 1 is introduced into said cell.
5 . The modified cell of claim 1 wherein the cell is further modified to concurrently express at least one of an associated accessory signaling polypeptide, a cytokine, an inhibitory killer cell immunoglobulin-like receptor (KIR), or a fragment thereof.
6 . The cell of claim 5 wherein the cytokine comprises interleukin-2 or interleukin-15.
7 . The modified cell of claim 5 wherein said KIR is at least one of KIR2DL1, KIR2DL2, or KIR3DL1.
8 . The modified cell of claim 5 wherein said accessory polypeptide is at least one of FcεRI-γ (SEQ ID NO: 5) or TCR-ζ (SEQ ID NO: 7).
9 . The modified cell of claim 1 wherein said modified cell is available from American Type Culture Collection (ATCC) as Deposit No. PTA-6670.
10 . A modified NK-92 cell comprising an NK-92 cell modified to express an inhibitory killer cell immunoglobulin-like receptor (KIR).
11 . The modified cell of claim 10 wherein said KIR is at least one of KIR2DL1, KIR2DL2, or KIR3DL1.
12 . The modified cell of claim 10 wherein said NK-92 cell is available from ATCC as Deposit No. CRL-2407.
13 . A method for in vitro assessment of the efficacy of an antibody to induce cell death, the method comprising: exposing a target cell to the antibody; exposing the target cell to an effector cell comprising an NK-92 cell modified to express at least one of a CD16 receptor or a KIR receptor; and monitoring the target cell for cytotoxicity or apoptosis.
14 . The method of claim 13 wherein said modified NK-92 cell comprises an NK-92 cell having a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO:2 introduced therein.
15 . The method of claim 13 wherein about 5% to about 30% of the target cells lyse or are induced to enter apoptosis in the presence NK-92 cells in the absence of the antibody.
16 . The method of claim 13 wherein an effector:target ratio is between about 0.5:1 and about 100:1.
17 . The method of claim 13 wherein the target cell is one selected from the group consisting of SKOV-3, P815, THP-1, U373MG, T98G, A ML193, SR91, ALL1, and REH or any other target cell exhibiting low baseline cytotoxicity by NK-92 cells.
18 . The method of claim 13 wherein the target cell is a modified cell that has increased expression of the antigen to which the antibody binds.
19 . The method of claim 13 wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, or a chimeric antibody.
20 . The method of claim 13 wherein the antibody is a chimeric antibody comprising at least two dissimilar antigen binding domains.
21 . The method of claim 20 wherein at least one antigen binding domain is adapted to bind to the Fc receptor.
22 . The method of claim 13 wherein the antibody is a hybridoma supernatant.
23 . The method of claim 13 further comprising the step of exposing the target cell to a plurality of unmodified NK-92 cells.
24 . The method of claim 13 wherein said CD16 receptor is the native form (SEQ ID NO:1).
25 . The method of claim 13 wherein said CD 16 receptor is a variant of a native form (SEQ ID NO:2).
26 . The method of claim 13 wherein the NM-92 cell is filter modified to express at least one of an associated accessory signaling polypeptide, a cytokine, or a fragment thereof.
27 . The method of claim 26 wherein the associated accessory signaling polypeptide comprises at least one of FcεRI-γ (SEQ ID NO:5) or TCR-4. (SEQ ID NO:7).
28 . The method of claim 13 further comprising the step of exposing the cells to a cytokine.
29 . The method of claim 28 wherein the cytokine comprises interleukin-2 or interleukin-15.
30 . A method for detecting cytotoxic and apoptosis-inducing activity, comprising: exposing a target cell in the presence of antibodies to an NK-92 cell modified to express a CD16 receptor; and monitoring the target cell for cytotoxic or apoptopic activity.
31 . The method of claim 30 further comprising applying a blocking agent to suppress at least one activating receptor on the modified NK-92 cell.
32 . The method of claim 34 wherein the blocking agent comprises at least one of a polypeptide, an antibody, or fragment thereof that binds specifically to at least one activating receptors.
33 . A method of assaying the efficacy of an antibody to treat at least one of a tumor, an infection or a lesion, comprising: administering the antibody to a subject; administering a plurality of modified NK-92 cells to the subject, the modified NK-92 cells comprising at least one of an NK-92 cell having a polynucleotide having at least 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO:2 introduced therein; and monitoring the tumor, infection or lesion, wherein the efficacy of the antibody correlates with suppression of the tumor, infection or lesion in the subject.
34 . The method of claim 33 wherein the NK-92 cell is further modified to express a KIR.
35 . The method of claim 34 wherein the KIR is at least one of KIR2DL1, KIR2DL2, or KIR3DL1.
36 . The method of claim 33 wherein the antibody is a chimeric antibody.
37 . The method of claim 33 wherein the NK-92 is further modified to express at least one of a cytokine or an associated accessory signaling polypeptide.
38 . The method of claim 37 wherein the cytokine is interleukin-2 or interleukin-15.
39 . The method of claim 33 further comprising the step of administering to the subject an exogenous cytokine.
40 . The method of claim 39 wherein the cytokine is IL-2 or IL-15.
41 . The method of claim 37 wherein the associated accessory signaling polypeptide comprises FcεRI-γ (SEQ ID NO:5) or TCR-ζ (SEQ ID NO:7).
42 . The method of claim 33 wherein the step of monitoring comprises measuring at least one of IFN-γ or cytokine expressed by said cells.
43 . The method of claim 36 , wherein the subject is one selected from the group consisting of humans, bovines, swine, rabbits, alpacas, horses, canines, felines, ferrets, rats, mice, fowl and buffalo.
44 . A method of treating a subject, the subject having a tumor, infection or other lesion, the method comprising: administering to the subject at least one antibody that binds to the tumor, infection or other lesion; and administering to the subject NK-92 cells modified to express at least one of a CD 16 receptor or a KIR.
45 . The method of claim 44 wherein said modified NK-92 cell comprises an NK-92 cell having a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO:2 introduced therein.
46 . The method of claim 45 wherein the polynucleotide sequence is SEQ ID NO:3.
47 . The method of claim 44 wherein the at least one antibody comprises monoclonal or polyclonal antibodies.
48 . The method of claim 44 wherein the at least one antibody comprises a chimeric antibody.
49 . The method of claim 48 wherein at least one antigen binding domain of the chimeric antibody is adapted to bind to the CD16 receptor.
50 . The method of claim 44 wherein the NK-92 is further modified to express at least one of a cytokine or an associated accessory signaling polypeptide.
51 . The method of claim 50 wherein the cytokine is interleukin-2 or interleukin-15.
52 . The method of claim 44 further comprising the step of administering to the subject an exogenous cytokine.
53 . The method of claim 52 wherein the cytokine is IL-2 or IL-15.
54 . The method of claim 50 wherein the associated accessory signaling polypeptide comprises at least one of FcεRI-γ (SEQ ID NO:5) or TCR-4. (SEQ ID NO:7).
55 . The method of claim 44 further comprising the step of determining a therapeutic response.
56 . The method of claim 44 further comprising the step of determining IFN-γ or cytokine expression levels.
57 . The method of claim 44 wherein the subject is one selected from the group consisting of humans, bovines, swine, rabbits, alpacas, horses, canines, felines, ferrets, rats, mice, fowl and buffalo.