Genetically modified human natural killer cell lines

Abstract

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.