US20080247990A1

US20080247990A1 - Genetically modified human natural killer cell lines

Info

Publication number: US20080247990A1
Application number: US12/106,665
Authority: US
Inventors: Kerry S. Campbell
Original assignee: Fox Chase Cancer Center
Current assignee: Fox Chase Cancer Center
Priority date: 2004-07-10
Filing date: 2008-04-21
Publication date: 2008-10-09
Also published as: DE602005016683D1; AU2005277831B2; AU2005277831A1; ES2678094T3; US9150636B2; EP3406631B1; ES2925912T3; WO2006023148A3; US8313943B2; CA2572938C; US7618817B2; US20100086996A1; EP2921500A1; CA3052445A1; EP2801583B1; EP2921500B1; US9181322B2; US20230372545A1; EP2112162A1; US20130040386A1

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.

Description

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent Application 60/586,581, filed Jul. 10, 2004, entitled A GENETICALLY MODIFIED HUMAN NATURAL KILLER (NK) CELL LINE, the entirety of which is herein incorporated by reference; U.S. Non-Provisional patent application Ser. No. 11/178,258, filed Jul. 8, 2005, entitled GENETICALLY MODIFIED HUMAN NATURAL KILLER CELL LINES; U.S. Provisional Patent Application 60/913,072, filed Apr. 20, 2007, entitled A GENETICALLY MODIFIED NK CELL LINE EXPRESSING THE HIGH AFFINITY FORM OF THE FC RECEPTOR, CD16; and U.S. Provisional Patent Application 60/991,285, filed Nov. 30, 2007, entitled BLOCKING NI CELL INHIBITORY SELF RECOGNITION PROMOTES ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY (ADCC).

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with grants from the National Institutes of Health: NIH R01 CA083859 (NCI; 2000-2009), entitled “Negative signaling by killer cell Ig-like receptors” and NIH R01 CA100226 (NCI; 2004-2009), entitled “Mechanisms of NK cell activation by the KIR2DL4 receptor.” The government may have certain rights in the invention.

FIELD OF THE INVENTION

Natural killer (NK) cell lines that are genetically engineered to express a cell surface receptor protein that participates in antibody-dependent cellular cytotoxicity (ADCC) responses are disclosed. More specifically, NK-92 cells modified to express at least one of the following are disclosed: an Fc cell surface receptor protein such as CD16; one or more of the CD16-associated accessory signaling proteins such as FcεRI-γ or TCR-ζ; a cytokine such as IL-2 or IL-15; a chemokine; or one or more inhibitory killer cell immunoglobulin-like receptors (KIR).

BACKGROUND

NK-92 is an NIL-like cell line that was initially isolated from the blood of a subject suffering from a large granular lymphoma and subsequently propagated in cell culture. The NK-92 cell line has been described in Gong et al. (1994) and Klingemann (2002). NK-92 cells have a CD3−/CD56+ phenotype that is characteristic of NK cells. They express most of the known NK cell-activating receptors except CD 16 and they lack the major MHC class I-recognizing NK cell inhibitory receptors, IIR, which engage with MHC Class 1 molecules on self cells to block NK cell activation. NK-92 cells do express NKG2A/CD94 and ILT2/LIR1 inhibitory receptors at low levels. Furthermore, NK-92 is a clonal cell line that, unlike the polyclonal NK cells isolated from blood, expresses these receptors in a consistent manner with respect to both type and cell surface concentration.
NK-92 cells are not immunogenic and do not elicit an immune rejection response when administered therapeutically to a human subject. Indeed NK-92 cells are well tolerated in humans with no known detrimental effects on normal tissues. While NK-92 cells have been engineered to express novel proteins by means of transduction using retroviral vectors (Campbell et al, 2004; Kikuchi-Maki et al., 2003; Klingemann, 2002; Yusa and Campbell, 2003; Yusa et al., 2002; Yusa et al., 2004), such engineering has proved difficult as evidenced by lack of previous reports that describe engineering NK-92 cells to express an Fc receptor or a KIR. More particularly, despite the clear potential benefits that could be anticipated from an NK-92 cell line modified to express at least one of CD16 or KIRs, such genetic modification had not been achieved in fact until the present disclosure.
A number of antibodies, most notably Rituximab (MabThera®; Hoffmann-LaRoche, Ltd; Basel, Switzerland) and Herceptin® (Genentech, Inc.; South San Francisco, Calif.), have shown significant therapeutic value as highly selective and effective anti-tumor agents. Although these antibodies can bind to specific antigens on the tumor cells, their anti-tumor activity depends at least in part on the subsequent binding of NK cells to the Fc (constant) portion of the antibody through the CD16 Fc receptor with consequent destruction of the tumor cell via an antibody dependent cellular cytotoxicity (ADCC) mechanism. However, unmodified NK-92 cells do not express CD16 and therefore are ineffective in killing target cells via ADCC. Although NK-92 cells are widely used as a model system for the study of NK cell activation, action and inhibition, the lack of CD16 expression precludes the use of NK-92 cells for the evaluation of efficacy of antibodies as therapeutic agents and the use of NK-92 cells as a therapeutic agent that is co-administered with an antibody. In an embodiment, the modified NK-92 cells disclosed herein address this limitation by causing NK-92 cells to express CD16. Additionally, many tumor cells and virus-infected cells down modulate expression of MHC class 1 molecules and thereby become targets for NK cell attack due to a lack of tolerizing negative signals from inhibitory KIR. In an embodiment, the modified NK-92 cells disclosed herein either lack these inhibitory KIRs to promote the ADCC responses by lacking inhibitory self-recognition or have been co-transduced to express these inhibitory KIR to improve tolerance toward normal MHC class I-expressing cells of the body if administered as a therapeutic agent.
Additional utility and benefit of the disclosed modified NK-92 cells will become apparent in the following descriptions.

SUMMARY

In an embodiment, a modified NK-92 cell comprising an NK-92 cell modified to express a CD16 receptor on a surface of the cell is disclosed. In examples, the modified cell is further modified to co-express at least one of an associated accessory signaling polypeptide, a cytokine, an inhibitory killer cell immunoglobulin (KIR), or fragments thereof.
In another embodiment, a modified NK-92 cell comprising an NK-92 cell modified to express a KIR on a surface of the cell is disclosed. In examples, the KIR is at least one of KIR2DL1, KIR2DL2, or KIR3DL1.
In yet another embodiment, a method for in vitro assessment of the efficacy of an antibody to induce target cell death is disclosed. The method compress the steps of: 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.
In another embodiment, an in vitro method for detecting cytotoxic and apoptosis-inducing activity is disclosed. The method of detecting comprises the steps of: exposing a target cell in the presence of anti-target cell antibodies to an NK-92 cell modified to express a CD16 receptor; and monitoring the target cell for cytotoxic or apoptotic activity. In an example, the NK-92 cell is further modified to co-express at least one of an associated accessory signaling polypeptide, a cytokine, an inhibitory killer cell immunoglobulin (KIR), or fragments thereof.
In another embodiment, a method of assaying the efficacy of an antibody to treat at least one of a tumor, an infection or a lesion is disclosed. The method comprises the steps of: 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.
In another embodiment, a method of treating a subject who has a tumor, infection or other lesion is disclosed. The method comprises the steps of: 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 CD16 receptor or a IR. In an example, the NK-92 cell is further modified to co-express at least one of an associated accessory signaling polypeptide, a cytokine, an inhibitory killer cell immunoglobulin (KR), or fragments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects, features and advantages of the present invention will be more readily appreciated upon reference to the following disclosure when considered in conjunction with the accompanying drawings.

FIG. 1 is a graph showing a Florescence Activated Cell Sorter (FACS) analysis of surface expression of CD16 transduced with the native form (GFP-CD16-F176.NK-92) and high affinity variant (GFP-CD16-F176V.NK-92). Cells were stained with mouse anti-human CD16 monoclonal antibody (CLBFc) supernatant and phycoerythrin-conjugated anti-mouse Kappa light chain antibody. Samples were analyzed with a FACScan analyzer and data were processed with FlowJo software. Surface expression of both the native form and the variant is nearly equivalent on transduced NK-92 cells.

FIG. 2 shows a dose response of Herceptin-induced ADCC of SKOV3 cells by unmodified NK-92 cells (NK-92), or NK-92 cells modified to express either the native form (GFP-CD16-F176.NK-92) or the high affinity variant (GFP-CD16-F176V.NK-92) of CD16. The cells were stimulated with IL-2 two days prior to the assay. The cytotoxicity assay was performed by ⁵¹Cr-release at 37° C. for three hours.

FIG. 3 shows flow cytometer scatter diagrams of NK-92 cells transduced with CD16 cDNA (F176V) using the pBMN-IRES-EGFP vector after staining with no primary antibody (FIG. 3A) and with both primary (anti-CD16) and secondary anti-mouse IgG antibody (FIG. 3B). EGFP expression is assessed on the x-axis, and surface CD16 expression is assessed on the y-axis.

FIG. 4 shows flow cytometer scatter diagrams showing the expression of CD16 by NK-92 cells transduced with CD16-F176V cDNA alone (expressed in pBMN-No-GFP vector) (FIG. 4A), and the increase in expression of CD 16 by NK-92 cells transduced with CD16-F176V cDNA in combination with FcεRI-γ cDNA (γ; in pBMN-IRES-EGFP vector) (FIG. 4B) or TCR-(cDNA (4; FIG. 4C). The x-axis shows EGFP expression, which directly correlates with the expression of FcεRI-γ or TCR 4 expressed from pBMN-IRES-EGFP, and the y-axis denotes surface staining for CD16 expression.

FIG. 5 is a graph showing redirected cytotoxicity of FcγRII/III⁺ P815 target cells by CD16-F176V.NK-92 cells induced by anti-CD16 antibody (3G8; +), but not antibodies toward CD56 (B159; Δ) or KIR (DX9; □), which correlated with cytotoxicity level in the absence of monoclonal antibody (No mAb; o). Cells were assayed using ⁵¹Cr release from P815 target cells at the indicated effector to target ratios.

FIG. 6 is a graph showing redirected cytotoxicity of FcγRII/III⁺ THP-1 target cells by CD16-F176V.NK-92 cells (filled symbols) induced by anti-CD16 antibody (3G8; squares), but not anti-NKR-P1 antibody (B199; triangles). Redirected cytotoxicity was not induced by anti-CD16 antibody in NK-92 cells transduced instead with mouse IgM cDNA (open symbols).

FIG. 7 is a graph showing redirected cytotoxicity of SKOV-3 target cells by CD16-F176V.NK-92 cells (Δ), but not mouse IgM-transduced NK-92 (□), induced by bi-specific 2B1 antibody (▴, ▪). 2B1 contains F(ab) domains recognizing both Her2/neu antigen on SKOV-3 cells and CD16 on GFP-CD16-F176V.NK-92 cells or mouse IgM heavy chain with GFP.

FIG. 8 is a graph showing redirected cytotoxicity of the following cells against P815 target cells in combination with the indicated concentration of 2B1 chimeric bi-specific monoclonal antibody:

No GFP-CD16-F176V.NK-92 cells (o);

GFP-CD16-F176V/FcεRI-γ.NK-92-cells (); and

GFP-CD16-F176 V/TCR-ζ.NK-92 cells (+).

FIG. 9. Panel A shows IUR expression on NK-92.26.5 cells. Flow cytometry was used to determine IR expression on NK-92.26.5 cells. The Abs HP3E4 (thin black histogram) binding KIR2DL1, KIR2DS1, and KIR2DS4; DX9 (gray-shaded histogram) binding KIR3DL1; GL183 (thick gray histogram) binding KIR2DL2, KIR2DL3, and KIR2DS2; and 5.133 (thick black histogram) binding KIR3DL1, KIR3DL2, and KIR2DS4 are used. Thin gray histogram is isotype control stain. Panel B shows that KIR3DL1 is a functional inhibitory receptor on the NK-92.26.5 cell line. NK-92.26.5 cells were incubated with ⁵¹Cr-labeled Fc R-expressing P815 cells (redirected cytotoxicity assay) at various E:T ratios with no Ab (open circles), anti-CD56 (B159) (filled square), or anti-KIR3DL1 (DX9) (filled circle) Abs at 1 ug/ml each. ⁵¹Cr release was measured 4 h later.

FIG. 10 is a graph showing a FACS analysis of NK-92.26.5 cells modified to express CD16. NK-92.26.5 cells were transduced with CD16 cDNA encoding either the native form (F176) or the high affinity variant (F176V). Cells were stained with anti-CD16 antibodies to compare expression. Anti- CD16 antibodies are CLB-Fc (solid grey histogram) and 3G8 (black line). Reactivity was detected with an R-phycoerythrin conjugated anti-mouse kappa antibody (thin black line- secondary antibody alone) (A). Unmodified NK-92.26.5 cells (B) GFP-CD16-F176.NK-92.26.5 cells, GFP-CD16-F176V.NK-92.26.5 cells.

FIG. 11 is a graph showing that ADCC is induced to different levels by NK-92.26.5 cells transduced with the native form (F176) or the high affinity variant (F176V). Original KIR3DL1-expressing NK-92.26.5 cells (KIR3DL1.NK-92.26.5) (open bars), GFP-CD16-F176.NK-92.26.5 cells (grey bars) or GFP-CD16-F176V.NK-92.26.5 cells (black bars) were incubated with (A) ⁵¹Cr labeled 721.221 B-cells expressing CD120 and HLA-B51 (the KIR3DL1 ligand; 721.221-B*5101), or (B) ⁵¹Cr labeled SKOV-3, c-erbB2 expressing ovarian cancer cells, at a 10:1 ratio, in the presence of the indicated concentrations of rituximab (anti-CD20 antibody) or trastuzumab (anti-c-erbB2), respectively. ⁵¹Cr-release was measured 4 hours later. Results shown are mean ±SD.

FIG. 12 is a graph showing that KIR3DL1 blocking antibody induces cytotoxicity against 721.221-B-15101 cells by NK-92.26.5 cells. (A) Unmodified NK-92.26.5 cells were incubated at an E:T ratio of 5:1 with ⁵¹Cr labeled 721.221-B*5101 (HLA-Bw4 transfected B-cells) in the presence of various concentrations of anti-NKR-P1 (B159; open square) or anti-KIR3DL1 (DX9; black circle) antibodies. The anti-KIR3DL1 antibody blocked interaction of the receptor with the HLA-B*5101 MHC class I molecule on the target cells, thereby reversing KIR-mediated inhibition and allowing target cell lysis. (B) Cytotoxicity was measured at different E:T ratios with no antibody (grey triangle), or in the presence of B159 (open square, binding CD56), DX9 (filled circle, binding KIR3DL1) or GL183 (open circle, binding KIR2DL2, KIR2DL3, and KIR2DS2), all at 1 μg/ml. ⁵¹Cr-release was measured 4 hours later. Results shown are mean ±.

FIG. 13 is a graph showing that ADCC is augmented by blocking the inhibitory self-recognition receptor KIR3DL1. (A, C, E) NK-92.26.5 cells were transduced with either the native form (CD16-F176.NK-92.26.5) or (B, D, F) the high affinity variant (CD16-F176V.NK-92.26.5) and incubated with ⁵¹Cr labeled 721.221-B*5101 cells, at a 5:1 E:T ratio and with (A, B) various concentrations of DX9 and with (filled square) or without (open square) 10 ng/ml rituximab, or with (C, D) various concentrations of rituximab and with (filled circle) or without (open circle) 0.1 μg/ml DX9. (E, F) Cells were incubated with no antibody (open bars), DX9 alone (grey bars, 0.1 μg/ml), rituximab (striped bars, 10 ng/ml) or with rituximab+DX9 (black bars) antibodies. ⁵¹Cr-release was measured 4 hour later. Results shown are mean ±SD of a representative of at least three independent experiments.

DETAILED DESCRIPTION OF THE INVENTION

While the modified NK-92 cells disclosed herein are susceptible of embodiment in many different forms, there are shown in the drawings, and will be described herein in detail, specific embodiments and examples thereof, with the understanding that the present disclosure is to be considered as an exemplification and is not intended to be limiting in any way.

DEFINITIONS

In order to properly understand the disclosure made herein, certain terms used in the disclosure are described in the following paragraphs. This description of the following terms in no way disclaims the ordinary and accustomed meanings of these terms.
“Antibody” (Ab) refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site (Fab region) that specifically binds an antigen. Ab as used herein includes single Abs directed against an antigenic epitope, antigen-specific compositions with poly-epitope specificity, single chain anti-epitope variants, and fragments of Abs.
A “monoclonal antibody” is obtained from a population of substantially homogeneous Abs, in which all Abs have common binding specificity for a desired antigen, i.e., the individual Abs comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies can be produced by any method known in the art or obtained commercially.
A “bi-specific Ab” is a monoclonal Ab, preferably human or humanized, that has binding specificities for at least two different antigens.
“Antibody dependent cellular cytotoxicity” (ADCC) is an anti-tumor mechanism that is dependent upon interactions between Fc domains of an Ab and Fc receptors expressed by effector cells, especially NK cells.
“CD16”, also known as “FcγRIII-A,” refers to one type of Fc receptor. The polynucleotide sequence encoding the native form of CD16 is shown in SEQ ID NO:3 and the polypeptide sequence encoding the native form is shown in SEQ ID NO: 1. The native form is designated herein as “F176”.
“CD16.NK-92” or “CD16.NK-92.26.5” refers generally to NK-92 or NK-92.26.5 cells, respectively, modified to express CD16 receptors without specific reference to the native form or any variant of CD16.
“CD16 chimeric polypeptide” or “CD16 fusion polypeptide” comprises CD16 fused to a non-CD16 polypeptide.
“CD16 polypeptide variant” includes a CD16 polypeptide having at least one of the following: (1) at least about 70% amino acid sequence identity with a full-length native CD16 sequence, (2) a CD16 sequence lacking a signal peptide, (3) an extracellular domain of a CD16, with or without a signal peptide, or (4) any other fragment of a full-length CD16 sequence. For example, CD16 polypeptide variants include those wherein one or more amino acid residues are substituted within the sequence or added or deleted at the N- or C-terminus of the full-length native amino acid sequence. An example of a CD16 polypeptide (high affinity) variant is shown in SEQ ID NO:2. The high affinity variant is designated herein as “F176V.”
“CD16 variant polynucleotide” or “CD16 variant polynucleotide sequence” includes a polynucleotide molecule which encodes a CD16 polypeptide that (1) has at least about 70% polynucleotide sequence identity with a polynucleotide acid sequence encoding a full-length native CD16, (2) a full-length native CD16 lacking the signal peptide, (3) an extracellular domain of a CD16, with or without the signal peptide, or (4) any other fragment of a full-length CD16.
“Homologous polynucleotide sequence” or “homologous amino acid sequence” refer to sequences characterized by a homology at the polynucleotide level or amino acid level, respectively.
“Host cell” and “recombinant host cell” are used interchangeably and refer to a particular subject cell and to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term.
“NK-92.26.5” refers to a variant of the original NK-92 cell line that was treated with 5-aza-2′-deoxycytidine, as described (Binmyamin et al., 2008). The treatment induced expression of several members of the killer cell immunoglobulin-like receptor (KIR) family, including KIR3DL1. NK-92.26.5 subclone was isolated and retains expression of KIR3DL1, even after long-term culture.
A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of CD16 without altering CD16 function in the methods and compositions disclosed herein, whereas an “essential” amino acid residue is required for activity.
“Percent (%) amino acid sequence identity” is defined as the percentage of amino acid residues that are identical with amino acid residues in a CD16 sequence in a candidate sequence when the two sequences are aligned.
“Percent (%) polynucleotide sequence identity” with respect to CD16-encoding polynucleotide sequences is defined as the percentage of polynucleotides in the CD16 polynucleotide sequence of interest that are identical with the polynucleotides in a candidate sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

Unmodified NK-92 Cells

The unmodified NK-92 cell line was deposited to the general depository of the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, Va. 20110-2209) on Sep. 3, 1998 and was assigned Deposit Number CRL-2407. The unmodified NK-92 cell line was transferred to the patent depository of ATCC on Apr. 11, 2005, and was assigned Deposit Number PTA-6670.

Modified NK-92 Cells

As set forth in greater detail below, NK-92 cells modified to express at least one of the following are disclosed: CD16; a cytokine such as IL-2 or IL-1 5; a chemokine; at least one KIR; an accessory signaling protein such as FcεRI-γ or TCR-4; or a variant thereof. Optionally, the modified NK-92 cells are NK-92MI cells modified by transfection with the vector MFG-hIL2 encoding interleukin-2 or NK-92CI cells modified by transfection with vector pCEP4-LTRhIL-2 encoding interleukin-2. The NK-92MI and NK-92CI cell lines were deposited with ATCC on Sep. 3, 1998 and were assigned Accession Nos. CRL-2408 and CRL-2409, respectively. As used herein, reference to NK-92 cells includes the substitution of NK-92MI or NK-92CI cells for unmodified NK-92 cells.
In an embodiment, NK-92 cells that are modified to express an Fc receptor protein such as CD16 either with (GFP-CD16.NK-92) or without (no-GFP-CD16.NK-92) co-expression of enhanced green fluorescent protein (EGFP), respectively, which is used as an endogenous surrogate marker for expression of the gene product of interest as co-expressed from a bicistronic (IRES-containing) expression vector, are disclosed. In alternate examples, other fluorescent proteins, such as yellow, red, cyan, etc., can be substituted for EGFP in a bicistronic vector without departing from the scope of this disclosure. Optionally, more than one fluorescent protein is used, as long as the proteins are of different colors so that their expression can mark co-expression of multiple gene products in the same transduced cell. Alternatively, an expression vector that lacks the IRES-fluorescent protein sequences can be used to express or co-express a protein that can be detected on the cell surface using a fluorescent antibody in conjunction with a fluorescence activated cell sorter (FACS). In an example, CD16 is a low affinity form having a phenylalanine (Phe) at position 176 (referred to herein as “F176”) (SwissProt database entry P08637; SEQ ID NO:1). In another example, the CD16 is a high affinity variant having a valine (Val) substituted at position 176 (referred to herein as “F176V”) (VAR_—003960; SEQ ID NO:2). The complete sequences are shown in Tables 1 and 2. The polynucleotide encoding SEQ ID NO:1 is presented in Table 3 (SEQ ID NO:3). As shown in FIG. 1, Fluorescent Activated Cell Sorter (FACS) analysis of surface expression of the high and low affinities of CD16 is substantially equivalent on modified NK-92 cells. Cells were stained with mouse anti-tumor CD16 monoclonal Ab (CLB Fc) supernatant and phycoerythrin-conjugated anti-mouse kappa light chain secondary antibody. Samples were analyzed on a FACScan analyzer and data were processed with FlowJo software. Florescence intensity of bound antibody is in log scale on the x-axis.
The NK-92 cell line modified to express the high affinity variant of CD16 without GFP (F176V; SEQ ID NO:2) was deposited with ATCC on Sep. 9, 2005, and was assigned Patent Deposit Designation No. PTA-6967. The NK-92 cell line modified to express the native form of CD16 (F176; SEQ ID NO:1) plus GFP and the NK-92 cell line modified to express the high affinity variant of CD16 (F176V; SEQ ID NO:2) plus GFP were deposited with ATCC on Dec. 13, 2007. The NK-92 cell line transduced to co-express GFP and the native form of CD16 (GFP-CD16-F176.NK-92) was assigned ATCC Patent Deposit Designation No. PTA-8837. The NK-92 cell line transduced to co-express GFP and the high affinity variant of CD16 (GFP-CD16-F176V.NK-92) was assigned ATCC Patent Deposit Designation No. PTA-8836.
In another embodiment, NI-92 cells are modified to express at least one inhibitory killer cell immunoglobulin-like receptor (KIR) (KIR.NK-92 cells). The IUR expressed on the modified NK-92 cell is a member of the KIR family that has long cytoplasmic domains, including KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR3DL1, KIR3DL2, and KIR3DL3. In an example, CD16 and KIR are co-expressed on the NK-92 cells. The modified cells are optionally co-expressed with one of the fluorescent proteins described above in the absence of CD16.
Such modifications may be made by any mechanism known to those of skill in the art. In an example, KIR cDNA constructs are ligated into the bicistronic retroviral expression vector, pBMN-IRES-EGFP, to produce recombinant retrovirus for generation of NK-92 cells with stably integrated cDNA. In an example, A 1.35-kb cDNA fragment encoding human KIR3DL1 (NKAT3) (obtained from M. Colonna, Washington University, St. Louis, Mo.) is subcloned using BamHI and NotI restriction sites. The packaging cell line, Phoenix-Amphotropic, is transfected with the pBMN-IRES-EGFP vector containing the KIR gene using Lipofectamine Plus reagent (Life Technologies). Supernatants of these transfected cells grown in serum-free Opti-MEM medium (Life Technologies) for 2 days are then co-cultured with NK-92 cells for 8 h in the presence of Lipofectamine Plus reagent, and then complete α-MEM medium containing IL-2 is added for 3 days before sorting by FACS. The transduced NK cells are then sorted for expression of EGFP or KIR3DL1 (KIR3DL1-specific PE-conjugated DX9 mAb; BD PharMingen, San Diego, Calif.).
In another embodiment, CD16.NK-92 or KIR.NK-92 cells are further modified to express a cytokine. The choice of host cell dictates the preferred technique for introducing the polynucleotide of interest. Introduction of polynucleotides into NK-92 cells may also be done with ex vivo techniques that use an in vitro method of transfection, as well as other established genetic techniques, such as through the use of lentivirus or adenovirus.

Generation of Modified NK-92 Cells

In an embodiment, the native form (F176; SEQ ID NO:1) or high affinity variant (F176V; SEQ ID NO:2) cDNA for CD16 is transduced in unmodified NK-92 cells. In another embodiment, the NK-92 cells are transduced with cDNA for at least one KIR. In still another embodiment, the NK-92 cells are modified to co-express CD16 and at least one KIR. Any vector and packaging cell line may be used to modify the NK-92 cells, including the pBMN-IRES-EGFP vector and the Phoenix-Amphotropic packaging cell line (see e.g., Example 1 below), as well as those now-known and later-developed. By way of example, these alternative methods include, but are not limited to the p-JET vector in conjunction with FLYA13 packaging cells (Gerstmayer et al., 1999), the plasmid-based cat retroviral transduction system, and DFG-hIL-2-neo/CRIP (Nagashima et al., 1998).
Likewise, the disclosure is not limited to transduction. In alternate examples, the cells are transfected with a mammalian expression vector containing the CD16 gene. For example, a wide variety of mammalian expression vectors in conjunction with electroporation, lipofection, nucleofection, and “gene gun”-mediated introduction of the vector into the packaging cells may also be used.
In another embodiment, NK-92 cells are further modified to express a chemokine or cytokine such as IL-2 or IL-15, such as where the NK-92 cell is transduced with the IL-2 gene by retroviral transduction (Nagashima et al., 1998).
In alternate examples and as described in greater detail below, the NK-92 cells are further modified to express at least one accessory signaling protein, including for example FcεRI-γ or TCR-4, such as by sequential transduction with CD16 and/or KIR.

TABLE 1

Polypeptide sequence for SEQ ID NO:1 (CD16, Native Form; F176)

Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala
1 5 10 15

Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro
20 25 30

Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln
35 40 45

Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu
50 55 60

Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr
65 70 75 80

Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu
85 90 95

Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln
100 105 110

Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys
115 120 125

His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn
130 135 140

Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro
145 150 155 160

Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe
165 170 175

Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln
180 185 190

Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln
195 200 205

Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly
210 215 220

Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp
225 230 235 240

Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys
245 250

TABLE 2

Polypeptide sequence for SEQ ID NO:2 (CD16, High Affinity
Variant; F176V)

Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala
1 5 10 15

Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro
20 25 30

Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln
35 40 45

Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu
50 55 60

Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr
65 70 75 80

Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu
85 90 95

Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln
100 105 110

Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys
115 120 125

His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn
130 135 140

Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro
145 150 155 160

Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val
165 170 175

Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln
180 185 190

Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln
195 200 205

Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly
210 215 220

Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp
225 230 235 240

Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys
245 250

TABLE 3

Polynucleotide sequence (mRNA) for SEQ ID NO:3
(CD16, Native Form; F176)

atgtggcagc tgctcctccc aactgctctg ctacttctag tttcagctgg catgcggact	60

gaagatctcc caaaggctgt ggtgttcctg gagcctcaat ggtacagggt gctcgagaag	120

gacagtgtga ctctgaagtg ccagggagcc tactcccctg aggacaattc cacacagtgg	180

tttcacaatg agagcctcat ctcaagccag gcctcgagct acttcattga cgctgccaca	240

gtcgacgaca gtggagagta caggtgccag acaaacctct ccaccctcag tgacccggtg	300

cagctagaag tccatatcgg ctggctgttg ctccaggccc ctcggtgggt gttcaaggag	360

gaagacccta ttcacctgag gtgtcacagc tggaagaaca ctgctctgca taaggtcaca	420

tatttacaga atggcaaagg caggaagtat tttcatcata attctgactt ctacattcca	480

aaagccacac tcaaagacag cggctcctac ttctgcaggg ggctttttgg gagtaaaaat	540

gtgtcttcag agactgtgaa catcaccatc actcaaggtt tggcagtgtc aaccatctca	600

tcattctttc cacctgggta ccaagtctct ttctgcttgg tgatggtact cctttttgca	660

gtggacacag gactatattt ctctgtgaag acaaacattc gaagctcaac aagagactgg	720

aaggaccata aatttaaatg gagaaaggac cctcaagaca aatga	765

CD16 Polynucleotide Sequences

In examples, a CD16 variant polynucleotide has at least about 70% polynucleotide sequence identity with the polynucleotide sequence encoding a full-length, native CD16 (F176; SEQ ID NO:3). In examples, a CD16 variant polynucleotide encodes full-length native CD16 lacking the signal peptide, an extracellular domain of CD16 with or without the signal sequence, or any other fragment of a full-length CD16, or a chimeric receptor encompassing at least partial sequence of CD16 fused to an amino acid sequence from another protein. In other examples, an epitope tag peptide, such as FLAG, myc, polyhistidine, or V5, is added to the amino terminal domain of the mature polypeptide to assist in cell surface detection by using anti-epitope tag peptide monoclonal or polyclonal antibodies.
In examples, CD16 variant polynucleotides are about 150 to about 900 polynucleotides in length, although CD16 variants having more than 900 polynucleotides are within the scope of the disclosure.
Homologous polynucleotide sequences encode polypeptide sequences coding for variants of CD16. Homologous polynucleotide sequences also include naturally occurring allelic variations related to SEQ ID NO:3. Transduction of an NI-92 cell with any polynucleotide encoding a polypeptide having the amino acid sequence shown in either SEQ ID NO: 1, SEQ ID NO:2, a naturally occurring variant thereof, or a sequence that is at least 70% identical to SEQ ID NO:1 or SEQ ID NO:2 is within the scope of the disclosure. In examples, homologous polynucleotide sequences encode conservative amino acid substitutions in SEQ ID NO:1 or SEQ ID NO:2. In an example, NK-92 cells are transduced using a degenerate homologous CD16 polynucleotide sequence that differs from the polynucleotide sequence shown in SEQ ID NO:3, but encodes a protein with the same amino acid sequence as that encoded by the polynucleotide sequence shown in SEQ ID NO:3.
In other examples, cDNA sequences having polymorphisms that change the CD16 amino acid sequences are used to modify NK-92 cells, such as, for example, the allelic variations among individuals that exhibit genetic polymorphisms in CD16 genes. In other examples, CD16 genes from other species that have a polynucleotide sequence that differs from the sequence of SEQ ID NO:3 are used to modify NK-92 cells.
In examples, the variant polypeptides are made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter, 1986; Zoller and Smith, 1987), cassette mutagenesis, restriction selection mutagenesis (Wells et al., 1985) or other known techniques can be performed on the cloned DNA to produce CD16 variants(Ausubel, 2002; Sambrook and Russell, 2001).
In an example, SEQ ID NO:3 is mutated to incur alterations in the amino acid sequence encoding for CD16 without altering the function of CD16. For example, polynucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in SEQ ID NO: 1 or SEQ ID NO:2.
Useful conservative substitutions are shown in Table 4. Conservative substitutions in SEQ ID NO:1 or SEQ ID NO:2, whereby an amino acid of one class is replaced with another amino acid of the same class, fall within the scope of the disclosed CD16.NK-92 cells as long as the substitution does not materially alter the activity of the modified NK-92 cell. Non-conservative substitutions that affect (1) the structure of the polypeptide backbone, such as a β-sheet or α-helical conformation, (2) the charge, (3) the hydrophobicity, or (4) the bulk of the side chain of the target site can modify CD16 polypeptide function or immunological identity. Non-conservative substitutions entail exchanging a member of one of these classes for another class. Substitutions may be introduced into conservative substitution sites or more preferably into non-conserved sites.

TABLE 4

Preferred substitutions

Original		Preferred
residue	Exemplary substitutions	substitutions

Ala (A)	Val, Leu, Ile	Val

Arg (R)	Lys, Gln, Asn	Lys

Asn (N)	Gln, His, Lys, Arg	Gln

Asp (D)	Glu	Glu

Cys (C)	Ser	Ser

Gln (Q)	Asn	Asn

Glu (E)	Asp	Asp

Gly (G)	Pro, Ala	Ala

His (H)	Asn, Gln, Lys, Arg	Arg

Ile (I)	Leu, Val, Met, Ala, Phe,	Leu
	Norleucine

Leu (L)	Norleucine, Ile, Val, Met,	Ile
	Ala, Phe

Lys (K)	Arg, Gln, Asn	Arg

Met (M)	Leu, Phe, Ile	Leu

Phe (F)	Leu, Val, Ile, Ala, Tyr	Leu

Pro (P)	Ala	Ala

Ser (S)	Thr	Thr

Thr (T)	Ser	Ser

Trp (W)	Tyr, Phe	Tyr

Tyr (Y)	Trp, Phe, Thr, Ser	Phe

Val (V)	Ile, Leu, Met, Phe, Ala,	Leu
	Norleucine

CD16 Polypeptide Variants

In examples, CD16 polypeptide variants have at least about 70% amino acid sequence identity with a full-length native sequence CD16 sequence and are at least about 50 amino acids to more than about 300 amino acids in length, although variants having more than 300 amino acids are within the scope of the disclosure.
A non-CD16 polypeptide is not substantially homologous to CD16 (SEQ ID NO:1 or SEQ ID NO:2). A CD16 fusion polypeptide includes any portion of CD16 or an entire CD16 fused with a non-CD16 polypeptide. Fusion polypeptides are created using recombinant methods. In an example, a polynucleotide encoding CD16 (e.g., SEQ ID NO:3) is fused in-frame with a non-CD 16 encoding polynucleotide such as TCR-ζ or FcεRIγ to the CD16 C-terminus or internally in order to replace up to about 30% of the CD16 cytoplasmic domain, thereby enhancing ADCC responsiveness. In other examples, chimeric proteins, such as domains from other lymphocyte activating receptors, including but not limited to Ig-α, Ig-β, CD3-ε, CD3-γ, CD3-δ, DAP-12, and DAP-10, replace a portion of the CD16 cytoplasmic domain. In still another example, fusion genes are synthesized by conventional techniques, including automated DNA synthesizers and PCR amplification using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (Ausubel, 2002). Many vectors are commercially available that facilitate sub-cloning CD 16 in-frame to a fusion moiety.
In other embodiments, fusion polypeptide variants of other proteins, such as KIR, FcεRI-γ, TCR-ζ, or naturally occurring minor polymorphic variants, are formed as described above.

ADCC Assay

Examples of methods of measuring cytoxicity using modified NK-92 cells, such as CD16.NK-92, are presented below, but any method of measuring cytotoxicity is within the spirit and scope of the disclosure.
In an example, CD16.NK-92 cells are used in ADCC assays as a pure “effector” cell population having defined and consistent characteristics to determine the efficacy of antibodies being developed as potential therapeutic agents. In examples, NK-92 cells that are modified to express intermediate levels of CD16 expression relative to that expressed by the native form are used to generate dose-response curves and as “ladder” type calibrators for CD16 activity. In other examples, NIK-92 cells transduced with either the native form (CD16-F176.NIK-92) or the high affinity variant (CD16-F176V.NK-92) are used. In still other examples and as described in greater detail below, the NK-92 cells are modified to co-express at least one of the following: a cytokine such as IL-2 (CD16/IL-2.NK-92); a chemokine; at least one KIR (CD16/KIR.NK-92); an accessory signaling protein such as FcεRI-γ (CD16/FcεRI-γ.NK-92) or TCR-ζ (CD16/TCR-ζ.NK-92); or a variant thereof.
In an example, redirected cytotoxicity is tested using a chimeric bi-specific antibody, such as 2B1 (Clark et al., 1997; Weiner et al., 1995a; Weiner et al., 1995b), which expresses two F(ab) regions, one which binds the CD16 receptor on the CD16.NK-92 cell and another that binds the Her2/neu antigen on an appropriate target cell line, such as SKOV-3. In still another example, monoclonal antibodies that specifically bind antigens that are uniquely expressed on the target cells are used to directly test ADCC. In this format, the F(ab) portion of the antibody binds to the corresponding antigenic epitope on the target cell while the CD16 receptor on the CD16.NK-92 cells bind to the Fc portion of the antibody. The resulting cross-link between the antigen on the target cell and the CD16 receptor results in lysis of the target cell via the ADCC pathway.
In an example, the method of performing the ADCC assay comprises: loading target cells with an indicator material such as ⁵¹Cr or a Europium chelate; treating the indicator-loaded target cells with the antibody to be evaluated; exposing the target cells to CD16.NK-92 effector cells; and measuring the quantity of the indicator in the assay supernatant concentration by any suitable method known to those skilled in the art, such as for example gamma or scintillation counting (⁵¹Cr), fluorescence intensity, or lifetime determination (Europium chelate). In an example, cytotoxicity is estimated by measuring the quantity of label released into the culture supernatants using the formula:
% Specific Lysis=100×[Experimental Release (mean cpm)−Spontaneous Release (mean cpm)]/[Total Counts (mean cpm)- Spontaneous Release (mean cpm)],
Where experimental release is defined as mean counts per minute (cpm) released by target cells in the presence of effector cells and/or antibody and the spontaneous release is defined as the mean cpm released by target cells alone. Such measurements may be made by any method known to those skilled in the art.
FIG. 2 shows a dose response curve of Herceptin-induced ADCC of SKOV-3 target cells by CD16-F176.NK-92 cells and CD16-F176V.NK-92 cells. These data show that the CD16-F176V.NK-92 cells offer improved ADCC responsiveness in vitro.
Optionally, the method further comprises the step of measuring at least one of the following: cytokine/chemokine production, including for example interferon-γ, tumor necrosis factor (TNF)-α, granulocyte-macrophage colony stimulating factor (GM-CSF), MIP-1α, MIP-1β, Rantes, and IL-8; apoptosis, such as by using fluorescent peptides to measure caspase activation, analyzing mitochondrial integrity, or labeling with fluorphore-conjugated Annexin V; measuring the expression of cell surface activation markers such as CD69, CD95L, and CD25; accumulation of cytolytic granule components, such as CD107A or CD107B, on the NK-92 cell surface as a measure of granule exocytosis by FACS; or activation of NK-92 cell transcription factors such as NF-AT or NF-κB. In these examples, measurements are made using standard methods such as enzyme-linked immunosorbent assays (ELISA), ELISPOT, or intracellular staining with fluorosphore-tagged anticytokine/antichemokine antibodies. The above-mentioned measures of NK cell activation are examples only and other methods of measuring activation are within the scope of this disclosure.
Optionally, the method further comprises at least one of the steps of decreasing IL-2 concentration in the culture medium or assaying the supernatant four days after passing the cells into fresh IL-2-containing medium in order to reduce baseline cytolytic capacity of NK-92 cells.
Optionally, the functionality of CD16 introduced into CD16.NI-92 cells, CD16/FcεRI-γ.NI-92 cells, or CD16/TCR-ζ.NK-92 cells is determined using either the ADCC assay or the redirected cytotoxicity assay (described below).
Optionally, the method of performing the ADCC assay further comprises the step of blocking known activating receptors on NK-92 cells in order to reduce baseline killing of target cells by NIL-92 cells. Such methods and agents are well-known in the art (see for example Pende et al., 1999; Pessino et al., 1998; Vitale et al., 1998). 1 in an example, masking antibodies are used (Pessino et al., 1998).
Unmodified NK-92 cells, which do not express CD16, serve as a unique and valuable control in the ADCC assay because they permit differentiation between ADCC-mediated cytotoxicity and other cytolytic effects that NK-92 cells exert on the target cells. These control conditions are used to assess baseline cytotoxicity by NK-92 cells toward the target cell and are substantially not influenced by addition of antibodies if the effect of the antibody is dependent upon triggering an ADCC response by the NK-92 cells.
In an example, the target cells in the ADCC assay express an antigen to which the antibody being evaluated binds and that has low susceptibility to lysis by the unmodified NK-92 cell line. Target cells suitable for use in the cytotoxicity assays include cells transduced/transfected to express the specific antigen the ovarian carcinoma line SKOV-3 (e.g., ATCC Deposit HTB-77) (Tam et al, 1999); U373MG and T98G (e.g., ATCC Deposit CRL-1690) (Komatsu and Kajiwara, 1998); AML-193 (myeloid; e.g., ATCC Deposit CRL-9589) and SR-91 (lymphoid progenitor) (Gong et al., 1994); and ALL1 and REH (B-cell acute lymphocytic leukemia) (Reid et al, 2002). In other examples, target cells such as the FcγRII/III⁺ murine mastocytoma cell line P815 (e.g., ATCC Deposit No. TIB-64) and the FcγRII/III⁺ myelocytic leukemia line THP-1 (e.g., ATCC Deposit No. TIB-202) that have limited (between 5 and 30%) susceptibility to lysis by unmodified NK-92 cells are used in order to ensure sufficient assay dynamic range for the detection of significant effects through CD16. In still other examples, other cell types with limited cytolytic potential that express or are engineered to express specific cell surface markers of interest are employed as targets.
In an example, NK-92 cells modified to co-express inhibitory KIR are used to establish the impact of co-engagement of this inhibitory receptor with MHC class I ligand on the target cells. The interaction of KIR with MHC class I ligand transduces an inhibitory signal that diminishes NK cell activation toward the target cell. As such, the method further comprises the step of testing the degree to which the antibody overcomes KIR inhibition to trigger an ADCC or redirected cytotoxicity response (described below).
In an example, at least one of FcεRI-γ (Genbank Accession No. M33195; SEQ ID NO:4, Table 5) (polynucleotide) and SEQ ID NO:5, Table 6 (polypeptide)) or TCR-ζ (Genbank Accession NO. J04132; SEQ ID NO:6, Table 7 (polynucleotide) and SEQ ID NO:7, Table 8 (polypeptide) are co-introduced into NK-92 cells by sequential transduction with CD16. Preferably a no-GFP vector is used to transduce the NK-92 cell with CD16 cDNA. The sequential transduction comprises the steps of: transducing the parental NK-92 cells with the CD16 vector; immunostaining the transduced cells with a fluorescently labeled anti-CD16 antibody; sorting the cells for CD16 expression; transducing the CD16.NM-92 cells with a vector containing cDNA for both the accessory protein and EGFP; and sorting the doubly transduced cells on the basis of EGFP expression. The signaling activity initiated by FcεRI-γ or TCR-ζ results in transduced NK-92 cells that exhibit higher levels of surface CD16 expression and enhanced cytotoxicity and cytokine release activities than do CD16.NK-92 cells (FIG. 8).

Redirected Cytotoxicity Assay

The modified NK-92 cells are also used in redirected cytotoxicity assays using the method steps disclosed above with respect to ADCC assays. In examples such as those related to the evaluation of bi- or poly-functional antibodies or in the study of activation mechanisms and other characteristics of NK-92 cells with monoclonal antibodies to engage NK-92 cell surface components, the ADCC assay described above is restructured as a “redirected cytotoxicity” assay. For example, a bi-functional antibody having one domain that specifically binds to an antigen of interest on the target cells and a second domain that specifically binds to CD16 on CD16.NK-92 cells are evaluated in the manner described above. In this example, the bi-functional antibody cross-links the antigen on the target cell to CD16 on the NK-92-CD16 cell and triggers a redirected cytotoxicity response. The redirected cytotoxicity assay can be used for research purposes, for example by treating a target cell that expresses another Fc receptor with an antibody that is directed against CD16. Exposing the anti-CD16-labeled target cells to CD16.NK-92 cells results in the cross-linking of CD16 at the target cell interface with consequent triggering of redirected cytotoxicity. Differentiation between the ADCC and redirected cytotoxicity formats is based upon whether the CD16.NK-92 effector cell CD16 receptor binds to the Fc portion of an antibody bound to an antigen on the target cell surface (ADCC) or whether CD16 is aggregated upon binding by the F(ab) portion of an anti-CD16 antibody, which is simultaneously engaged via its Fc domain to the Fc receptor on the target cell surface (redirected cytotoxicity). As both binding arrangements can trigger similar cytotoxicity responses in the target cell, the choice between the ADCC and redirected formats is, for example, a matter of the target cells and the characteristics of the Ab or Ab construct to be evaluated.
In order to ensure maximum assay dynamic range, target cells that are minimally susceptible to lysis by the parental NK-92 cell line are selected for use in the assay. In examples, the target cell is selected to exhibit NK-92 mediated lysis of between about 0% and about 30%. Target cell lines such as SKOV-3 exhibit minimal (about 5% to about 30%) susceptibility to lysis by NK-92 cells and they constitutively express certain cell surface antigens that are of particular interest as targets for therapeutic antibodies. Optionally, the cells are transduced or transfected to express other antigens of interest and utility.
Optionally, the redirected cytotoxicity and ADCC assays described above are used to identify antibodies and antibody constructs that are useful as therapeutic agents for the treatment of cancers and infections.

TABLE 5

FcεRI-γ polynucleotide sequence (SEQ ID NO:4)

cagaacggcc gatctccagc ccaagatgat tccagcagtg gtcttgctct tactcctttt	60

ggttgaacaa gcagcggccc tgggagagcc tcagctctgc tatatcctgg atgccatcct	120

gtttctgtat ggaattgtcc tcaccctcct ctactgtcga ctgaagatcc aagtgcgaaa	180

ggcagctata accagctatg agaaatcaga tggtgtttac acgggcctga gcaccaggaa	240

ccaggagact tacgagactc tgaagcatga gaaaccacca cagtagcttt agaatagatg	300

cggtcatatt cttctttggc ttctggttct tccagccctc atggttggca tcacatatgc	360

ctgcatgcca ttaacaccag ctggccctac ccctataatg atcctgtgtc ctaaattaat	420

atacaccagt ggttcctcct ccctgttaaa gactaatgct cagatgctgt ttacggatat	480

ttatattcta gtctcactct cttgtcccac ccttcttctc ttccccattc ccaactccag	540

ctaaaatatg ggaagggaga acccccaata aaactgccat ggactggact c	591

TABLE 6

FcεRI-γ polypeptide sequence (SEQ ID NO:5)

Met Ile Pro Ala Val Val Leu Leu Leu Leu Leu Leu Val Glu Gln Ala
1 5 10 15

Ala Ala Leu Gly Glu Pro Gln Leu Cys Tyr Ile Leu Asp Ala Ile Leu
20 25 30

Phe Leu Tyr Gly Ile Val Leu Thr Leu Leu Tyr Cys Arg Leu Lys Ile
35 40 45

Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val
50 55 60

Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys
65 70 75 80

His Glu Lys Pro Pro Gln
85

TABLE 7

TCR-ζ polynucleotide sequence (SEQ ID NO:6)

cttttctcct aaccgtcccg gccaccgctg cctcagcctc tgcctcccag cctctttctg	60

agggaaagga caagatgaag tggaaggcgc ttttcaccgc ggccatcctg caggcacagt	120

tgccgattac agaggcacag agctttggcc tgctggatcc caaactctgc tacctgctgg	180

atggaatcct cttcatctat ggtgtcattc tcactgcctt gttcctgaga gtgaagttca	240

gcaggagcgc agagcccccc gcgtaccagc agggccagaa ccagctctat aacgagctca	300

atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg gaccctgaga	360

tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag	420

ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg aggggcaagg	480

ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac gacgcccttc	540

acatgcaggc cctgccccct cgctaacagc caggggattt caccactcaa aggccagacc	600

tgcagacgcc cagattatga gacacaggat gaagcattta caacccggtt cactcttctc	660

agccactgaa gtattcccct ttatgtacag gatgctttgg ttatatttag ctccaaacct	720

tcacacacag actgttgtcc ctgcactctt taagggagtg tactcccagg gcttacggcc	780

ctgccttggg ccctctggtt tgccggtggt gcaggtagac ctgtctcctg gcggttcctc	840

gttctccctg ggaggcgggc gcactgcctc tcacagctga gttgttgagt ctgttttgta	900

aagtccccag agaaagcgca gatgctagca catgccctaa tgtctgtatc actctgtgtc	960

tgagtggctt cactcctgct gtaaatttgg cttctgttgt caccttcacc tcctttcaag	1020

gtaactgtac tgggccatgt tgtgcctccc tggtgagagg gccgggcaga ggggcagatg	1080

gaaaggagcc taggccaggt gcaaccaggg agctgcaggg gcatgggaag gtgggcgggc	1140

aggggagggt cagccagggc ctgcgagggc agcgggagcc tccctgcctc aggcctctgt	1200

gccgcaccat tgaactgtac catgtgctac aggggccaga agatgaacag actgaccttg	1260

atgagctgtg cacaaagtgg cataaaaaac agtgtggtta cacagtgtga ataaagtgct	1320

gcggagcaag aggaggccgt tgattcactt cacgctttca gcgaatgaca aaatcatctt	1380

tgtgaaggcc tcgcaggaag acgcaacaca tgggacctat aactgcccag cggacagtgg	1440

caggacagga aaaacccgtc aatgtactag gg	1472

TABLE 8

TCR-ζ polypeptide sequence (SEQ ID NO:7)

Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu
1 5 10 15

Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys
20 25 30

Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala
35 40 45

Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Glu Pro Pro Ala Tyr
50 55 60

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
65 70 75 80

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
85 90 95

Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
100 105 110

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
115 120 125

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
130 135 140

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
145 150 155 160

Pro Pro Arg

Antibodies (Abs)

The disclosed method of performing the ADCC and redirected cytotoxicity assays use Abs and antibody fragments, such as Fab or (Fab′)₂, that bind specifically to their epitopes. Both ADCC and redirected cytotoxicity assays require that the Ab contains an Fc domain with the capacity to bind to the Fc receptor on either the NK-92 cell or on the target cell.

Humanized and Human Abs

The CD16.NK-92 cells are used for ADCC assay with antibodies that encompass Fc regions. Inclusion of humanized Fc regions would improve recognition by the human CD16 incorporated in the cell line.
A humanized antibody has one or more amino acid residues introduced from a non-human source. In an example, non-human amino acid residues are taken from an “import” variable domain. Humanization is accomplished by substituting rodent complementary determining regions (CDRs) or CDR sequences in the Fab domains for the corresponding sequences of a human antibody (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al., 1988). Such “humanized” Abs are chimeric Abs, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized Abs are typically human Abs in which some CDR residues and possibly some Fc residues are substituted by residues from analogous sites in Abs generated in rodents or other non-human species. In some instances, corresponding non-human residues replace F_vframework residues of the human Ig. In examples, humanized Abs comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which most, if not all, of the CDR regions correspond to those of a non-human Ig and most if not all of the framework regions are those of a human Ig consensus sequence. The humanized antibody optionally also comprises at least a portion of an Ig constant region (F_c), typically that of a human Ig (Jones et al., 1986; Presta, 1992; Riechmaim et al., 1988).

Pharmaceutical Compositions

In an embodiment, a pharmaceutical composition comprising modified NK-92 cells and a pharmaceutically acceptable carrier is disclosed. For example, the NK-92 cell is modified to express at least one of the following: CD16; a cytokine such as IL-2 or IL-15; a chemokine; at least one KIR; an accessory signaling protein such as FcεRI-γ or TCR-ζ; or a variant thereof. Optionally, the composition further comprises an agent that enhances function such as cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. In an example, the composition further comprises polypeptides, Abs, and derivatives, fragments, analogs and homologs thereof. The pharmaceutically acceptable carrier comprises any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration (Gennaro, 2000). Examples of such carriers include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Other examples include liposomes and non-aqueous vehicles such as fixed oils. Optionally, the composition further comprises supplementary active compounds
In an example, where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the epitope is used. For example, peptide molecules are designed that bind a preferred epitope based on the variable-region sequences of a useful antibody. Such peptides are synthesized chemically or produced by recombinant DNA technology (Marasco et al., 1993). Formulations optionally contain more than one active compound for a particular treatment, preferably those with activities that do not adversely affect each other.
The formulations to be used for in vivo administration to a subject are highly preferred to be sterile. This is readily accomplished by filtration through sterile filtration membranes or any of a number of techniques.
In an example, the active ingredients comprising the pharmaceutical composition are entrapped in microcapsules prepared by coacervation techniques or by interfacial polymerization; for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethaciylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
In another example, sustained-release preparations of the pharmaceutical compositions described above are prepared, such as semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (Boswell and Scribner, 1973), copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as injectable microspheres composed of lactic acid-glycolic acid copolymer, and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
In another example, the pharmaceutical compositions described above are prepared as a sterile injectable solution comprising the modified NK-92 cells and a sterile vehicle that contains a basic dispersion medium. Optionally, sterile powders are used for the preparation of sterile injectable solutions.

Screening Assays

In another embodiment, ADCC or redirected cytotoxicity assays using the modified NK-92 cells are used to screen purified antibody preparations or hybridoma supernatants for the presence of ADCC-inducing monoclonal antibodies. The ADCC and redirected cytotoxicity assays, such as the redirected cytotoxicity assay described in Example 6 below, are used to screen clones to identify those sub-clones that secrete potentially useful antibodies of the IgG isotypes. Optionally, these same assays are subsequently used to support the evaluation, characterization and further development of these antibodies. In another embodiment an assay for confirming the uniformity of batches of production grade therapeutic antibodies for ADCC capacity is disclosed. The CD16.NK-92 cells provide a consistent biological assay that allows comparative measurements. In another embodiment, an assay that compares the capacity of ADCC or redirected cytotoxicity of a variety of therapeutic antibodies with respect to CD16 polymorphism is disclosed. Since the high affinity variant (F176V) can be triggered to imitate ADCC with a lower dose of a standard anti-tumor antibody as compared to the native form (F176), distinct populations of CD16.NK-92 cells bearing each receptor can be separately tested for capacities to induce ADCC or redirected cytotoxicity. In this way ADCC or redirected cytotoxicity triggering capacity of an antibody can be assayed in vitro toward each of the human polymorphic CD16 variants.
In an embodiment, a method of measuring capacity of ADCC or redirected cytoxicity by human NK cells is disclosed. The method comprises the steps of: introducing a tumor bearing the antigen detected by the antibody to a subject, such an animal model; allowing the antigen to establish residence to an appropriate level; adding an amount of antibody; adding an amount of CD16.NK-92 cells; and measuring the effects of the antibody on the tumor thereafter. In examples, the animal model is the RAG-deficient/common Y chain-deficient mice (lacking T, B, and NK cells) or SCID mice (lacking T and B cells) available from The Jackson Laboratory, Bar Harbor, Me. The suppression of the native immune system in such immuno-compromised animals facilitates differentiation between responses induced by the treatment and normal immune responses. Measuring includes, for example, survival time of the subject or measuring the size, growth, metabolism, etc. of the tumor on subsequent days, weeks, or months and comparing the results to those in mice that did not receive either Ab or CD16. NK-92 cells, or which received unmodified NK-92 cells. Measurements of tumor mass and growth characteristics are made using standard methods known to those skilled in the art, including excising and weighing the tumor, NMR, labeling or prelabeling the tumor, or injecting the tumor with a contrast agent.

Cellular Immunotherapeutic Use of Modified NIK-92 Cells; Method of Treating a Subject

In an embodiment, a method of treating a subject who has a tumor, infection, or other lesion is disclosed. The method comprises the steps of: administering to the subject antibodies that specifically bind to the tumor, infection, or lesion; administering to the subject NK-92 cells modified to express at least one of CD16, a cytokine, a chemokine, a KIR, or an accessory signaling protein; and monitoring at least one of expression of IFN-γ or cytokines, reduction in the tumor, infection, or lesion size, metabolism, or growth to indicate a therapeutic response to the method of treatment. In examples, the antibody is the anti-Her2/neu antibody, Herceptin, and the treatment of B cell leukemias with the anti-CD20 antibody, Rituximab. In examples, the step of administering is carried out by infusing either intravenously or intraperitoneally at least one of the antibody or the NK-92.CD16 cells. In other examples, the step of administering is carried out by injecting at least one of the antibody or the modified NK-92 cells directly into the solid tumor or other focal lesions or into the areas surrounding the solid tumors or other focal lesions. Optionally, a split-dose regimen is used, such as when IL-2 is not being co-administered, in order to maintain a high level of active, transduced NK-92 cells in the subject. In another example, the method comprises the steps of administering the antibody by infusion and administering the transduced cells by direct injection. The efficacy of the treatment is measured by lesion reduction/clearance, cytokine profile or other physiological parameters that allow one to establish impacts on the tumor or lesion.
In examples, the subject is a human, a bovine, a swine, a rabbit, an alpaca, a horse, a canine, a feline, a ferret, a rat, a mouse, a fowl or a buffalo. Optionally, the method further comprises the step of infusing the subject with a pharmacologically effective dose of IL-2 or IL-15 for at least one of immediately prior to or subsequent to administering the modified NK-92 cells and tumor-specific antibody. Preferably, the infusion is continued for a period of time afterwards. In an example, the period of time is one to a plurality of days, such as a week.
Alternatively, the CD16-NK-92 cells are prepared from cells of the NK-92MI or NK-92CI cell lines (described above) that have been engineered to constitutively express IL-2. Concurrent treatment with IL-2 increases the survival of the administered NK-92 cells.
Optionally, the method of treatment further comprises the step of irradiating NK-92 cells prior to administering the modified NI-92 cells to the subject at doses that suppress proliferation of the modified NI-92 cells while substantially maintaining cytotoxicity and cell survival. In an example, NK-92 cells are irradiated at doses of between about 250 and 1000 Grays.
Optionally, the method of cellular immunotherapeutic use of the modified NK-92 cells comprises the steps of: large-scale expanding the modified NI-92 cells under conditions of good manufacturing practices (GMP) in the presence of 500 U/ml IL-2 and 5% heat inactivated fresh frozen plasma; treating the modified NK-92 cells with 250-1000 Grays of gamma irradiation to prevent further cell division; and injecting the subject with the irradiated modified NK-92 cells and an anti-tumor antibody, such as Herceptin, Rituximab, etc. The modified NK-92 cells and the antibody may be injected simultaneously. Optionally, the method further comprises the step of administering Interleukin-2 or Interleukin-15 to the subject in order to promote survival of the modified NK-92 cells in the patient. Another aspect of development in this area is directed toward the creation of chimeric antibodies that incorporate two or more antigen-binding [F(ab)] domains having differing specificities. A chimeric “bi-specific antibody” can, by way of example, incorporate one F(ab) binding domain that specifically binds to a cell surface marker that is uniquely or characteristically expressed on the target tumor or infected cells and a second F(ab) domain that specifically engage CD16 on CD16.NK-92 effector cells. Such chimeric antibodies are exemplified by the monoclonal antibody 2B1 (Clark et al., 1997; Weiner et al., 1995a; Weiner et al., 1995b) which incorporates one F adb domain that specifically binds to the ErbB2 (HER2/neu) antigen and a second F_(ab)domain that specifically binds to CD16. Cells of the ErbB2⁺ ovarian cancer line SKOV-3 are only slightly susceptible to cytotoxicity by NK cells or NK-92-CD16 cells. However, SKOV-3 cells become highly susceptible to NK-92-CD16 cell-mediated cytotoxicity in the presence of 2B1 antibody which ligates the ErbB2 antigen on a SKOV-3 cell to the CD16 activating receptor on a NK-92-CD16 cell. (See FIG. 7). Optionally, the NK-92.CD16 cells are used therapeutically in combination with 2B1 or related chimeric Abs.
In another embodiment, an NK-92 cell line transduced to express CD16, KIR2DL1, and KIR2DL2/3, is used to treat a subject (CD16/KIR2DL1/KIR2DL2/3.NK-92 cells). Such a modified cell line offers high potency ADCC responses and is tolerant toward HLA-C-expressing normal cells when injected into essentially all human subjects or tested in vitro using MHC class I-expressing target cells. The method comprises the steps of: administering to the subject antibodies that specifically bind to the tumor, infection, or lesion; and administering to the subject a therapeutic composition comprising the CD16/KIRDL1/KIR2DL2/3.NK-92 cells. Preferably, the in vivo method further comprises the step of irradiating the CD16/KIR2DL1/KIR2DL2/3.NK-92 cells prior to administering them to the patient. Optionally, the method further comprises the step of administering a cytokine to the patient either with or subsequent to administering the CD16/KIR2DL1/KIR2DL2/3.NK-92 cells. In an example, the NK-92 cells are further modified to express a cytokine or an accessory signaling protein. Optionally, the method further comprises the step of monitoring at least one of expression of IFN-γ or cytokines, reduction in the tumor, infection, or lesion size, metabolism, or growth to indicate a therapeutic response to the method of treatment.

EXAMPLES

The following examples are for illustrative purposes only and should not be interpreted as limitations of the claimed invention. There are a variety of alternative techniques and procedures available to those of skill in the art which would similarly permit one to successfully employ the use of the modified NK-92 cells disclosed herein.

Example 1

Preparation of CD16 Recombinant Retrovirus

CD16 cDNA encoding the native form of CD16 (F176) was constructed by first cloning the cDNA into pBMN-IRES-EGFP using BamHI and NotI restriction enzymes. Residue 176 was modified to encode phenylalanine (F) using standard polymerase chain reaction (PCR) methods in combination with the oligonucleotides: 5′-CTT CTG CAG GGG GCT TTT TGG GAG TAA AAA TGT GTC T and 5′-AGA CAC ATT TTT ACT CCC AAA AAG CCC CCT GCA GAA G, as well as primers overlapping to the pBMN-IRES-EGFP vector 5′-GCA TCG CAG CTT GGA TAC AC and 5′-GGC GGA ATT TAC GT AGC G.
The Phoenix-Amphotropic retroviral packaging cell line was transfected with the pBMN-IRES-EGFP vector containing the CD16 gene and retrovirus-containing supernatants of these transfected cells were co-cultured with NK-92 cells. Transduced NK-92 cells expressing CD16 on their surface and co-expressing EGFP in the cytoplasm were separated from the residual non-transduced NK-92 cells using a fluorescence activated cell sorter (FACS).
The recombinant vector was mixed with 10 μL of PLUS™ Reagent (Invitrogen; Carlsbad, Calif.); diluted to 100 μL with pre-warmed, serum-free Opti-MEM® (Invitrogen; MEM, minimum essential media); further diluted by the addition of 8 SL Lipofectamine (Invitrogen) in 100 mL pre-warmed serum-free Opti-MEM®; and incubated at room temperature for 15 minutes. This mixture was then brought to a total volume of 1 mL by the addition of pre-warmed serum-free Opti-MEM®. Phoenix-Amphotropic packaging cells (obtained from G. Nolan, Stanford University, Stanford, Calif.; (Kinsella and Nolan, 1996)) were grown to 70-80% confluence in a 6-well plate and washed with 6 mL of pre-warmed serum-free Opti-MEM® medium (Invitrogen). After removal of the medium, 1 mL of the solution of recombinant vector in Lipofectamine™ PLUS™ Reagent was added to each well, and the cells were incubated for at least three hours at 37° C. under a 7% CO₂/balance air atmosphere. Four mL of pre-warmed RPMI medium containing 10% fetal bovine serum (FBS) was added to each well, and the cells incubated overnight at 37° C., under a 7% CO₂/balance air atmosphere. The media was then removed; the cells washed with 6 mL pre-warmed serum-free Opti-MEM®; 2 mL serum-free Opti-MEM® added; and the cells incubated at 37° C., under a 7% CO₂/balance air atmosphere for an additional 48 hours.
The virus-containing supernatant was collected into a 15 mL plastic centrifuge tube; centrifuged at 1300 rpm for 5 minutes to remove cells and cell fragments; and the supernatant transferred to another 15 mL plastic centrifuge tube. Immediately before use, 20 μL of PLUS™ Reagent was added to the virus suspension; the mixture incubated at room temperature for 15 minutes; 8 μL Lipofectamine™ added to the mixture; and the mixture incubated for an additional 15 minutes at room temperature.

Example 2

Retroviral Transduction of CD16 into NK-92 Cells

NK-92 cells cultured in α-MEM (Sigma; St. Louis, Mo.) supplemented with 12.5% FBS, 12.5% fetal horse serum (FHS) and 500 IU rhIL-2/mL (Chiron; Emeryville, Calif.) were collected by centrifugation at 1300 rpm for 5 minutes, and the cell pellet was re-suspended in 10 mL serum-free Opti-MEM® medium. An aliquot of cell suspension containing 5×10⁴cells was sedimented at 1300 rpm for 5 minutes; the cell pellet re-suspended in 2 mL of the retrovirus suspension described in Example 1, and the cells plated into 12-well culture plates. The plates were centrifuged at 1800 rpm for 30 minutes and incubated at 37° C. under an atmosphere of 7% CO₂/balance air for 3 hours. This cycle of centrifugation and incubation was then repeated a second time. The cells were diluted with 8 mL of α-MEM, transferred to a T-25 flask, and incubated at 37° C. under a 7% CO₂/balance air until the cells were confluent. The transduced cells were collected, re-suspended in serum-free Opti-MEM(V medium, and sorted on the basis of their level of EGFP expression using a fluorescence activated cell sorter (FACS), EGFP being co-expressed with, and a surrogate marker for, CD16. Cell-surface expression of CD16 was confirmed by immuno-staining the transduced cells with an anti-CD16 antibody. The transduced cells, which are designated as CD16.NK-92 cells, were passed with fresh IL-2 every 4 days and assayed for cell-surface expression of CD16 before use.
FIG. 3 shows flow cytometer scatter diagrams of NK-92 cells transduced with CD16 cDNA using the pBMN-IRES-EGFP vector after staining with secondary phycoerythrin (PE)-conjugated anti-mouse IgG antibody alone (FIG. 3A) or anti-CD16 antibody (3G8 (Fleit et al, 1982; Perussia and Trinchieri, 1984); mouse IgG)+PE-anti-mouse IgG (FIG. 3B) and analysis using a FACS (Becton Dickinson; Franklin Lakes, N.J.) flow cytometer. EGFP expression is assessed on the x-axis and surface CD16 expression is expressed on the y-axis. The data shown in FIG. 3 confirm that the CD16-F176.NK-92 cell line expresses CD16 on the cell surface when stained with a monoclonal anti-CD16 antibody.

Example 3

.NK-92 CELLS CO-EXPRESSING CD16 and an Accessory Signaling Protein FcεRI-γ or TCR-ζ

Recombinant retroviruses incorporating inserted genes for the expression of FcεRI-γ (SEQ ID NO:5) or TCR-4 (SEQ ID NO:7) were prepared by using standard methods to ligate the corresponding cDNA into the pBMN-IRES-EGFP vector and transfecting this construct into the Phoenix-Amphotropic packaging cell line in the presence of Lipofectamine™ Plus as described in Example 1. The resulting FcεRI-γ or TCR-ζ recombinant retroviruses were used to transduce NK-92 cells as described in Example 2 with the following further modifications.
NK-92 cells transduced with retrovirus incorporating pBMN vector constructs were collected, re-suspended in serum-free Opti-MEM® medium and sorted on the basis of their level of the co-expressed EGFP using a FACS. CD16 cDNA was ligated into a version of the pBMN vector lacking the IRES and EGFP sequences, called pBMN-NoGFP, for co-transfection purposes in combination with another cDNA ligated into pBMN-IRES-EGFP (Yusa et al., 2002). The FcεRI-γ or TCR-ζ transduced NK-92 cells (FcεRI-γ.NK-92 or TCR-ζ.NK-92, respectively) were secondarily co-transduced with CD16-pBMN-NoGFP using the same retroviral transduction method as described in Example 2. The co-transduced cells (CD16/FcεRI-γ.NK-92 or CD16/TCR-4.NK-92) were suspended in α-MEM, transferred to a T-25 flask, and grown to confluency at 37° C. under a 7% CO₂/balance air. After reaching confluency, the co-transduced cells were immuno-stained with an anti-CD16 antibody and sorted by FACS for cell-surface expression of CD16. The selected cells were sub-cultured with fresh IL-2 every four days and assayed for cell-surface expression of CD16 before use.
In this Example, only one of the two vectors contained the gene for EGFP in order to facilitate the determination of the levels at which the other (co-expressed) protein could be directly detected on the cell surface. In this example, the accessory protein (FcεRI-γ or TCR-ζ) was co-expressed with EGFP. Thus EGFP fluorescence is a surrogate indicator for the level of expression of the second protein. An anti-CD16 antibody conjugated to a fluorophore having an emission spectrum different from that of EGFP was employed to determine the level of surface expression of CD16.
FIG. 4 shows flow cytometer scatter diagrams showing the expression of CD16 by NK-92 cells transduced with CD16 alone (CD16.NI-92; FIG. 4A) and the increase in CD16 expression when NK-92 cells are transduced with CD16 cDNA in combination with FcεRI-γ cDNA (CD16/FcεRI-γ.NK-92; FIG. 4B); or TCR-ζ cDNA (CD16/TCR-ζ.NK-92; FIG. 4C). These data show that when CD16 is co-expressed with FcεRI-γ or TCR-ζ in the NK-92 cell line, the cell-surface expression of CD16 is increased over that obtained when NK-92 cells are transduced with CD16 alone.

Example 4

Redirected Cytotoxicity Assays

Effector cells (NM-92, CD16.NK-92, CD16/TCR-4.NK-92, and CD16/FcεRI-γ.NK-92) were washed by suspension in α-MEM without IL-2 and sedimented at 1300 rpm for 5 minutes. The cell pellet was suspended in α-MEM, cells counted, and aliquots prepared at cell concentrations of 1×10⁵/mL (effector to target cell ratio (E:T)=1:1), 5×10⁵/mL (E:T=5:1), 1×10⁶/mL (E:T=10:1), 2×10⁶/mL (E:T=20:1) or as appropriate to the determination being performed. The transduced NK-92 cells used in these assays were generally selected for maximal CD16 expression as previously described by FACS either through direct labeling with fluorophore-conjugated anti-CD16 antibody or through detection of coordinate levels of EGFP expression.
The type of target cell used in the redirected cytotoxicity assay was selected on the basis of the requirements of the particular determination being performed. Raji cells (e.g., ATCC Deposit No. CCL-86), which are known to be moderately susceptible (about 50% lysis under these conditions) to lysis by NK-92 cells, were used for most purposes, including verification of the cytotoxicity of the effector cells.
Approximately 2×10⁶of the selected target cells were washed by suspension in RPMI medium and sedimentation at 1300 rpm for 5 minutes. After removal of the supernatant, 20 μL of FBS and 100 μCi of Na[⁵¹Cr]chromate was added and the cells incubated at 37° C. for 60-90 minutes with mixing every 30 minutes. The labeled target cells were washed three times by suspension in 10 mL of RPMI medium and sedimentation at 1500 rpm for 5 minutes. The final cell pellet was re-suspended in α-MEM and diluted to a concentration of 1×10⁵/mL. Target cells for use in redirected cytotoxicity assays were further incubated with the appropriate antibody at a final concentration of 0.01-5 μg/mL for 10-15 minutes at room temperature.
One-hundred μL of the selected type of target cells and 100 μL of the effector cells at cell concentrations of 1×10⁵/ml (E:T=1:1), 5×10⁵/ml (E:T=5:1), 1×10⁶/ml (E:T=10:1), 2×10⁶/ml (E:T=20:1) (and optionally tip to an E:T=100:1) were added to each well of a 96 well V-bottom plate in order to evaluate a spectrum from low to nearly complete cytotoxicity. Three to six replicate wells were prepared at each E:T ratio. At least 6 wells were allocated to each of a spontaneous lysis control (effector cells replaced with 100 μL of α-MEM) and total release control (effector cells replaced with 100 μL of 2% t-Octylphenoxypolyethoxyethanol (Triton X-100®) detergent in α-MEM). An additional six or more wells were allocated to the use of unmodified NIL-92 effector cells that do not express CD16 as a procedural control and internal standard. The plate was then centrifuged at 500 rpm for 3 minutes and incubated for 4 hours at 37° C. in an atmosphere of 7% CO₂/balance air. At the end of the incubation period, the plate was centrifuged at 1500 rpm for 8 minutes, and 100 μL of the supernatant was collected from each well for counting in a γ counter to measure of ⁵¹Cr release. The percentage of specific target cell lysis was calculated as described above.

Example 5

CD16-Mediated Cell Lysis

A monoclonal antibody specific for CD16 was used in redirected cytotoxicity assays in those cases where a target cell line that expressed a Fc receptor other than CD16 was available. In particular, the FcγR+ mouse mastocytoma cell line P815 and human FcγR⁺ myelocytic cell line THP-1 were used as targets in combination with the anti-CD16 monoclonal antibody (mAb) 3G8 (Fleit et al., 1982; Perussia and Trinchieri, 1984) to evaluate CD16.NK-92, CD16/FcεRI-γ.NK-92, and CD16/TCR-4.NK-92 cells (FIGS. 5 and 6).
Alternatively, redirected cytotoxicity assays were performed using target cells that express a unique antigen, but which do not express a Fc receptor, in conjunction with a bi-specific antibody construct (FIGS. 7 and 8). The evaluation of CD16.NK-92, CD16/FcεRI-γ.NK-92, or CD16/TCR-4.NK-92 cells in this assay was carried out using SKOV-3 as target cells and the chimeric antibody 2B1 as the cross-linking agent. The 2B1 chimeric bi-specific antibody has one binding domain that is specific for HER2/neu and a second binding domain that is specific for CD16 (Clark et al., 1997; Weiner et al., 1995a; Weiner et al., 1995b) (FIG. 7). FIG. 8 illustrates the cytotoxicity of CD16.NM-92, CD16/FcεRI-γ.NK-92, or CD16/TCR-4.NK-92 cells against P815 target cells in a redirected cytotoxicity assay using 2B1 chimeric antibody after performing a ⁵¹Cr-release assay for four hours. FIG. 8 shows that at less than saturating antibody concentrations, cytotoxicity is a function of both antibody concentration and the level of expression of CD16 on the NK-92 effector cells.
These assays can also employ NK-92 cells modified to express a variant of one of the proteins the cells are modified to express. These variants provide a broad dynamic range of assay sensitivities. Although this Example is described with reference to monoclonal antibodies and bi-specific antibody constructs, polyclonal antibodies and other types of antibody constructs having the appropriate characteristics are within the scope of this disclosure. FIG. 4 demonstrates that introduction of FcεRI-γ or TCR-ζ into NK-92 cells increases expression of CD16 on the surface of the NK-92 cell. The increased CD16 expression results in redirected cytotoxicity at a lower does of antibody, thus making the cells more sensitive to mediate ADCC.

Example 6

Screening and Evaluation of Therapeutic Antibodies

The selected target cells were labeled with Na[⁵¹Cr]chromate as described in Example 4 and adjusted to a concentration of 1×10⁵cells/mL before use. A 100 μL aliquot of labeled target cells was then transferred to each well of the requisite number to achieve a desired E:T ratio of 96-well plates. The immunoglobulin concentrations in the hybridoma supernatants to be screened were adjusted to a convenient nominal concentration of 1 μg/mL. At least 100 μL aliquots of each hybridoma supernatant was added to each of three target cell containing wells; incubated for 15 minutes at room temperature; washed with α-MEM; and re-suspended in 100 μL of α-MEM. The effector cell concentration was adjusted to achieve the desired E:T ratio in the assay which was between about 1:1 and 1:20. The assay was initiated by adding 100 μL of effector cells to each well. The plates were then centrifuged at 500 RPM for 3 minutes and incubated for 4 hours at 37° C. in an atmosphere of 7% CO₂/balance air. At the end of the incubation period, the plate was centrifuged at 1500 rpm for 8 minutes and 100 μL of the supernatant was collected from each well for counting in a y counter as a measure of ⁵¹[Cr] release due to cytotoxicity. The percentage of specific lysis was calculated as described in Example 4. At least six wells were allocated to each of a spontaneous lysis control (effector cells replaced with 100 μL of α-MEM) and a total release control (effector cells replaced with 100 μL of 2% Triton X-100 detergent in α-MEM) on each plate. An additional six wells in each set of plates was allocated to each of a “no antibody” control (target cells not treated with antibody) and an NK-92 cell control (unmodified NK-92 cells). Specific lysis was reported as the average of three replicate wells after correction for the appropriate controls.
Efficacy is likewise assessed by the measurement of surrogate indicators such as cytokine release by the NK-92, CD16 cells, the up-regulation of NK cell activation markers such as CD25, CD69, CD107, and/or CD95L, activation of transcription factors, such as NF-AT or NF-κB within the NK-92 cells, or the activation of caspases or other markers of apoptosis in the target cells.
In most cases, relatively small numbers (often only one) of antibody constructs were prepared. In such cases, screening was not necessary, and the construct was more conveniently evaluated using a direct assay, such as the one described in Example 5. Similarly, the relatively few potentially useful antibodies detected during screening were subsequently characterized in more detail using assays such as described in Example 5. As shown in FIG. 8, efficacies of antibodies at varied concentrations can be tested for inducing cytotoxicity. Furthermore, comparative testing of cytotoxicity potential between antibodies on NK-92 cells bearing the native form (F176) and those bearing the higher affinity variant (F176V) of CD16 was used to assess therapeutic efficacies of individual antibodies in the context of both of these known human alleles of CD16. FIGS. 2, 10, and 12 show comparisons of the native form and the high affinity variant. Such comparisons circumvented the need for the user to identify specific donors that are homozygous for each of the two alleles for such assays.

Example 7

CD16 Mediated Cytokine Production

The production of cytokines by NIC-92, CD16.NK-92, CD16/FcεRI-γ.NK-92, and CD16/TCR-ζ.NK-92 cells in response to CD16 mediated stimulation was determined by the redirected cytotoxicity assays described in Examples 4 and 5.
Effector cells (NK-92, CD16.NK-92, CD16/FcεRI-γ.NK-92, and CD16/TCR-ζ.NK-92 cells) were washed by suspension in α-MEM (without IL-2) and sedimentation at 1300 rpm for 5 minutes. The cell pellet was suspended in α-MEM, the cells counted, and aliquots prepared at cell concentrations of 1×10⁵/mL (E:T=1:1), 5×10⁵/mL (E:T=5:1), 1×10⁶/mL (E:T=10:1), 2×10⁶/mL (E:T=20:1), or as appropriate to establish an effective dynamic range of cytotoxicity in the assay being performed.
Targets cells exhibiting low level basal killing by NK-92 cells used in these assays were selected to allow for a further measurable increase in cytotoxicity due to the engagement of CD16 in the ADCC or redirected cytotoxicity assay. If a target cell line expressing the desired antigen that also exhibits low basal killing by NK-92 cannot be identified, target cells known to exhibit low level killing, such as SKOV-3 and other target cells listed above, can be transfected to express the desired antigen for use in an ADCC or redirected cytotoxicity assay.
One hundred μL of varying concentrations of effector cells were combined with a constant concentration of antibody treated target cells (not labeled with ⁵¹[Cr]) in wells of a 96-well V-bottom plate. Three to six replicate wells were prepared at each E:T ratio to be evaluated. At least 6 wells each were allocated as controls for non-CD 16 specific effector cell activation in which the target cells were replaced with 100 mL of α-MEM (spontaneous release) or with 100 μL of 2% Triton X-100 (total release). Additional controls using target cells that were not antibody treated and target or effector cells that were treated with F(ab′)₂fragments to suppress non-CD16 specific effector cell activation were also included. The plate was centrifuged at 500 rpm for 3 minutes and incubated for 4 hours at 37° C. in an atmosphere of 7% CO₂/balance air. At the end of the incubation period, the plate was centrifuged at 1500 rpm for 8 minutes, and aliquots of the supernatant were collected from each well to quantify cytokine concentrations, using commercially available cytokine ELISA kits (e.g., BD Phramingen; San Diego, Calif.). Effector cell cytokine production was generally determined to track effector cell cytotoxicity and could therefore be taken as an alternative indicator of effector cell activation.

Example 8

NK-92 Cell Stimulation By IL-2

Certain cytokines, particularly IL-2, IL-12, IL-15 and IL-18, are known to promote the growth, survival, cytotoxicity and cytokine-releasing activities of NK, NK-92, CD16.NK-92, CD16/FcεRI-γ.NK-92, and CD16/TCR-ζ.NK-92 cells, and other NK-92 variant cells both in vitro and in vivo. The cells transduced in Examples 2 and 3 were proliferated and exhibited stable levels of CD 16 expression, cytotoxicity and cytokine response for several months without the need for antibiotic selection when sub-cultured with fresh IL-2-containing medium every 4 days. When these same cells were passed without the addition of IL-2, they exhibited cytotoxicity and cytokine production levels that declined with time through the 4-day culture period and returned to higher levels on the first day after fresh IL-2 addition. Furthermore, cells maintained in the absence of IL-2 specifically lysed a narrower range of cell types than did cells maintained in the presence of IL-2. This behavior of transduced NK-92 cells and derivatives closely reflects that of unmodified primary NK cells. For these reasons, cells are assayed and transduced at consistent intervals after passage with defined concentrations of IL-2. Similarly, it is desirable to co-administer IL-2 when these cells are being used for in vivo therapeutic purposes. It is important that NK-92 cells are consistently assayed on the same day every time after IL-2 stimulation in order to assure that the NK-92 cells are at a similar level of activation.
Optionally, the NK-92MI and NK-92CI cell lines described above may be substituted for the NK-92 cells in these examples, wherein these modified cells are transduced in the same manners described for the NK-92 cell line in Examples 2 and 3.

Example 9

High Affinity (F176V) CD16 Variant Mediates ADCC at Lower Antibody Doses than the Native Form (F176)

NK-92 cells were transduced to express either the native form or the high affinity variant of CD16 as described above in Example 1. Target cells (SKOV-3; about 2 million) were pre-labeled with radioactive chromium (Na₂ ⁵, CrO₄; 100 μCi/million target cells for 1 hi at 37° C. in 500 μl fetal bovine serum) and washed before assembling the assay in 200 ul/well of a 96-well U-bottom culture plate. A total of 10,000 target cells were introduced into each well with 100,000 NK-92 cells (for 10:1 effector to target ratio)±antibody (the Her2/neu tumor antigen-specific IgG antibody, Herceptin), in α-MEM medium lacking IL-2. All assays were performed in triplicate. The plate was centrifuged briefly to pellet the effector and target cells prior to a three hour incubation at 37° C. (7% CO₂). The plate was then centrifuged again and 100 ul of culture supernatant was removed and counted on a gamma radiation counter or to quantify the release of ⁵¹Cr from lysed target cells in counts per minute (cpm). Some wells did not receive antibody (background cytotoxicity), some received untransduced parent NK-92 cells, some did not receive NK-92 cells (spontaneous release measurement), and others received 1% final concentration of Triton X-100 detergent (to measure total release). Percent cytotoxicity was calculated as described above.
Both variants are expressed at nearly equivalent levels on the surface of the transduced NK-92 cells when stained with the anti-CD16 monoclonal antibody CLB Fc and analyzed on a fluorescence activated cell sorter (FACS; data were analyzed using FlowJo software; see FIG. 1). CD16.NK-92 cells were tested for their capacities to elicit ADCC toward two distinct target cell lines. While both the CD16-F176.NK-92 form and the CD16-F176V.NK-92 variant demonstrate strong ADCC responses, cells expressing the F176V variant elicit an ADCC response at a substantially lower concentration (by about one log) of anti-tumor antibody and at a higher intensity than cells expressing the native form, F176 (see FIG. 2). These results demonstrate the biological benefit of the higher affinity variant and suggest that the F176V variant of CD16 is more effective in eliciting an ADCC response than the F176 form in an in vitro assay system.

Example 10

Blocking NK Cell Inhibitory Self Recognition Promotes ADCC

This example was carried out in order to use NK-92 cells and NK-92.26.5 cells (described below) and target cell models to study the impact of blocking self inhibitory receptor interactions with antibodies to increase NK-92 mediated ADCC.

Cells

NK-92 and NK-92.26.5 (a subclone generated to express novel genes such as KIRs by treatment with 5-aza-2′-deoxycytidine) were maintained in α-MEM medium (Life Technologies, Rockville, Md.) containing 10% fetal bovine serum (FBS, HyClone Laboratories, Logan, Utah), 10% horse serum (Life Technologies), 2 mM L-glutamate (Life Technologies), 100 μg/ml penicillin (Life Technologies), 100 μg/ml streptomycin (Life Technologies), 1 mM sodium pyruvate (Life Technologies), 100 μM 2-ME (Fisher, Pittsburgh, Pa.), 2 mM folic acid (Sigma-Aldrich, St. Louis, Mo.), 20 mM myoinositol (Sigma-Aldrich), and supplemented with 2% culture supernatant of J558L cells transfected with the human IL-2 gene. Cells were passed with fresh IL-2 every 4 days. NK-92.26.5 cells were generated by, cloned by, and obtained from Dr. Charles Lutz (university of Kentucky; Binyamin et al., 2008) as a representative example of the functional capacity of an NK-92 cell line expressing KIR. To generate NK-92.26.5, the unmodified NK-92 cell line (CRL-2407) was treated with 5-aza-2′-deoxycytidine to promote expression of KIRs. In response to this treatment, individual NK-92 clones permanently expressed a variety of the available KIR genes. In this example, the NK-92.26.5 subclone was selected for the capacity to express KIR3DL1.
Lymphoblast transfectant cell lines 721.221-B*5101 (referred to herein as B51) and 721.221-Cw4 were maintained in RPMI containing 10% FBS (HyClone Laboratories), 2 mM L-glutamate (Life Technologies), 100 μg/ml penicillin (Life Technologies), 100 μg/ml streptomycin (Life Technologies), 1 mM sodium pyruvate (Life Technologies), 1 mM HEPES buffer and 50 μM 2-ME (Fisher). SKOV3 ovarian carcinoma cells were maintained in DMEM containing 10% FBS (HyClone Laboratories), 2 mM L-glutamate (Life Technologies), 100 μg/ml penicillin (Life Technologies), 100 μg/ml streptomycin (Life Technologies) and 50 μM 2-ME (Fisher).

Generation of CD16 Expressing NK-92 Cells

CD16 cDNA (F176V polymorphic variant) was ligated into the bicistronic retroviral expression vector pBMN-IRES-EGFP to produce recombinant retrovirus for transduction of NK cell lines with stably integrated cDNA. The oligos 5′CTT CTG CAG GGG GCT TTT TGG GAG TAA AAA TGT GTC T and 5′ AGA CAC ATT TTT ACT CCC AAA AAG CCC CCT GCA GAA G were used to generate the CD-16 F176 variant as well as primers overlapping to the pBMN-IRES-EGFP vector 5′ GCA TCG CAG CTT GGA TAC AC and 5′ GGC GGA ATT TAC GT AGC G and digestion with the BamHI and NotI restriction sites. The integrity of all constructs was confirmed by sequencing.
Transduction of the CD16 construct was carried out using the packaging cell line, Phoenix-Amphotrophic, which was transfected with the pBMN-IRES-EGFP vector containing the CD16 gene using Lipofectamine Plus reagent (Life Technologies). Supernatants of these transfected cells grown in serum-free Opti-MEM medium (Life Technologies) for 2 days were co-cultured with NI-92 or NK-92.26.5 cell lines for 8 h in the presence of Lipofectamine Plus reagent. Complete α-MEM medium containing IL-2 was added for 3 days. At that time, 5-10% of the infected NK-92 or NK-92.26.5 cells efficiently expressing EGFP and CD16 were sorted on a FACS Vantage flow cytometer.

Antibodies

KIR and NK cell receptor-directed antibodies used in this example were: DX9, (binds to KIR3DL1, produced from a hybridoma), GL183 (binds to KIR2DL2, KIR2DL3 and KIR2DS2, Immunotech); and 5.133 (binds to IIR3DL1, KIR3DL2 and KIR2DS4, produced from a hybridoma). B159 (binds CD56, produced from a hybridoma) was used as control antibody in the ADCC assay. CD16 antibodies CLB-Fc and 3G8 (BD Pharmingen) were used to detect CD16 expression of the two polymorphic variants at residue 176 (i.e., F176V or F176). Herceptin (anti-Her2/neu), Rituximab (anti CD20), and trastuzumab (anti c-erbB2) antibodies were used to direct NK-92 and NK-92.26.5 ADCC.

ADCC Assays

ADCC studies were performed as described above. Target cells were labeled with Na₂ ⁵¹CrO₄(100 μCi/10⁶targets; PerkinElmer Life Sciences) for 1 h at 37° C. in 500 ul FBS. The ⁵¹Cr-labeled target cells were washed twice and resuspended at the desired concentration in RPMI-1640. Ten thousand cells were added to individual wells of 96-well flat-bottomed plates (Costar, Cambridge, Mass.) containing NK-92 or NK-92.26.5 (effector) cells at indicated E:T ratios and/or indicated concentrations antibodies in supplemented RPMI 1640. Each well contained a total volume of 200 μl, and all assays were performed in triplicate. The plates were centrifuged at 300×g for 3 min, incubated for 4 h in a 5% (v/v) CO₂incubator at 37° C., and then centrifuged again at 300×g for 3 min. One hundred microliters of supernatant were removed from each well for counting on a Packard Instruments Cobra Quantum, Series 5002 (PerkinElmer Life Sciences). Cytotoxicity was estimated by measuring the quantity of label released into culture supernatants using the formula disclosed above.

Flow Cytometry

The expression levels of CD16 and KIRs on NK-92 and NK-92.26.5 were determined by flow cytometry with techniques known to those of skill in the art. Briefly, 1×10⁶cells were incubated with the relevant antibody for 30 min at 4° C. The cells were washed before the addition of fluorochrome-conjugated goat anti-mouse kappa antibody (SouthernBiotech). The degree of fluorescence was determined using a FACScan flow cytometer (Becton-Dickinson, San Jose, Calif.) and was analyzed using FlowJo software (Tree Star, Inc.).

KIR Expression on Effector Cells

To determine the impact of KIR blockade we first used the NK-92 subclone NK-92.26.5. These cells have been described elsewhere and here we confirmed by RT-PCR that different KIR mRNA, including KIR3DL1 mRNA, are expressed (Table 9) and by flow cytometry that KIRs, including KIR3DL1, are expressed on the surface (FIG. 9A). Antibodies that have been reported to functionally block KIR recognition were used for the flow cytometry analysis, as shown in FIG. 9B. These antibodies are HP3E4, DX9, GL183 and 5.133. Of these antibodies, only DX9 binds solely to one inhibitory receptor, i.e., KIR3DL1. To study the inhibitory function of KIR3DL1 in NK-92.26.5 cells, a redirected cytotoxicity assay was performed (FIG. 9B). Mouse Fe receptor-positive P815 target cells were incubated with NK-92.26.5 cells at varying effector: target ratios and with either DX9 antibody (murine IgG1) or anti-CD56 antibody (murine IgG1; binds to NK-92 cells but does not mediate signaling). Inhibition of P815 lysis by NK-92.26.5 cells was observed when DX9 antibody was added, indicating that KIR3DL1 inhibitory signaling was triggered by engagement with DX9 antibody at the target cell interface via Fc receptors on the P815 cells. These results indicate that KIR3DL1 is functional on NK-92.26.5 cells, as it delivers a dominant negative signal to those cells.

CD16 Expression on the Effector Cells

To determine CD16 expression levels on the cells, flow cytometry assays were performed using two different anti-CD16 antibodies, CLB-Fc and 3G8 (FIG. 10). Using the CLB-Fc antibody, it was confirmed that CD16 expression was at a comparable level in cell lines transduced with F176 or F176 variant forms of the receptor. Fluorescence intensity with 3G8 was higher with CD16-F176V.NK-92 cells. Accordingly, the difference between the two variants was more pronounced when staining with the 2B1 antibody (a bispecific antibody that binds monovalently to both CD16 (3G8 derived) and c-erbB2 (data not shown). The flow cytometry analysis showed that 2B1 binds to cells that express the high affinity variant but not the native form of CD16.

ADCC

To confirm that the CD16.NK-92.26.5 cells mediate ADCC, the CD20 positive 721.22 1-B*5101 (which will be referred to as B51) or c-erbB2 positive SKOV-3 target cells were used in the presence of serial dilution of the antigen-reactive mAbs rituximab or trastuzumab, respectively. ADCC was mediated only toward target cells that expressed the specific antigen (see FIGS. 11A, B). CD16-F176V.NK-92.26.5 cells mediated higher maximal cytotoxicity and were more sensitive to lower concentrations of antibody compared to corresponding CD16-F176.NK-92.26.5 cells. ADCC was not mediated by CD16 negative NK-92.26.5 cells or by antibody that does not bind to CD20-expressing target cells (e.g., trastuzumab to B51 or rituximab to SKOV-3).

Increasing NK Cell Cytotoxicity by KIR Blockade

To determine if cytotoxicity of NK-92.26.5 against relevant target cells was increased by blocking inhibitory self-recognition, cytotoxicity assays using B51 cells as targets were performed. 721.221-B*5101 B-cells were transfected solely to express HLA-B*5101, containing the HLA-Bw4 epitope for the engagement of HLA-B51 by KIR3DL1 receptor. Blockade of the KIR3DL1 interaction by the DX9 antibody promoted cytotoxicity in a concentration-dependent manner (FIG. 12A). Attempted blockade of the other inhibitory receptor KIR2DL2 and KIR2DL3 by GL183 blocking antibody failed to induce cytotoxicity against the B51 target cells (FIG. 12B). Since these mechanisms are CD16 independent, results are shown for the NK-92.26.5 cells but were comparable with the CD16-F176V.NK-92.26.5 cells and the CD16-F176.NK-92.26.5 cells (not shown).

Blocking Inhibitory Self-Recognition Improves ADCC

The impact of combining the NK cell activating mechanisms of CD16 engagement and blockade of inhibitory self-recognition was then assessed. Effector cells (CD16-F176V.NK-92.26.5 or CD16-F176.NK-92.26.5) were incubated with B51 targets at varied E:T ratios (5:1 E:T ratio is shown) using different concentrations of DX9 and in the presence or absence of rituximab (10 ng/ml, see FIG. 13A, 13B). The data suggest that at any given DX9 concentration, adding rituximab improved NM cell-mediated target cell lysis. At this low rituximab concentration, the amplitude of the effect was higher with the higher affinity CD16 variant (F176V). Next, the effector and target cells were incubated in different concentrations of rituximab and in the presence or absence of DX9 (0.1 ug/ml, FIG. 13C, 13D). As shown, combining DX9 with any given rituximab concentration yielded higher NK cell-mediated target cell lysis than with rituximab alone. Cytotoxicity was compared using the different combinations of antibodies employed in these experiments at various E:T ratios (FIG. 13E, 13F). A combination of rituximab plus DX9 yielded the highest NK cell-mediated target cell lysis, when either CD16-F176V.NK-92.26.5 or CD16-F176.NK-92.26.5 were the effector cells (FIGS. 13E and 13F, respectively). KIR3DL1 blockade alone efficiently promoted B51 cell killing. In this cell line model system, KIR3DL1 is the only receptor that contributes to inhibitory self recognition. In the CD16-F176V.NK-92.26.5 effector cell setting, rituximab alone mediated a significant level of killing and target cell lysis was only modestly promoted when rituximab and DX9 were used together (FIG. 13E). In the CD16-F176.NK-92.26.5 effector cell setting, the level of cytotoxicity significantly increased when combining DX9 and rituximab compared to rituximab alone (FIG. 13F) and was equivalent to the level of cytotoxicity achieved by the CD16-F176V.NK-92.26.5 cells when tested with rituximab alone. These data demonstrate that blockade of inhibitory self-recognition can be applied to increase the degree of ADCC by human NK cells.
This example demonstrates the strong negative impact on ADCC response by engagement of inhibitory KIR with MUC class I molecules on target cells. MHC class I molecules are commonly down regulated from the surface of tumors and virus infected cells to avoid detection by cytolytic T cells, but thereby become targets for NIL cells. It is important to note that the unmodified NK-92 cell line lacks expression of inhibitory KIRs, and therefore, ADCC function by NK-92 would not be down-regulated by expression of MHC class I on target cells in either in vitro or in vivo uses, thereby reinforcing their value for both direct experimental analysis of ADCC in vitro and therapeutic ADCC responsiveness in vivo. On the other hand, IUR engagement with MHC class I on normal cells is the major mechanism by which NIL cells are tolerant to normal cells in the body. Therefore, the introduction of inhibitory KIR in CD16-expressing NK-92 cells is useful for in vitro analyses of antibody efficacies or in certain clinical situations in which tolerance toward normal cells is required. Each KIR gene product recognizes a distinct subset of the numerous polymorphic alleles of MHC class I (HLA-A, -B, and -C) that exist in the human population, but the introduction of KIR2DL1 in combination with KIR2DL2 (or KIR2DL3) offers universal tolerance toward all alleles of HLA-C molecules, all of which contain either lysine or asparagine at position 80 that are detected by KIR2DL1 or KIR2DL2/KIR2DL3, respectively (Vilches and Parham, 2002).
From the foregoing, it will be observed that numerous variations and modifications can be effected without departing from the spirit and scope of the novel concept of the invention. It is to be understood that no limitation with respect to the specific methods and apparatus illustrated herein is intended or should be inferred. It is, of course, intended to cover by the appended claims all such modifications as fall within the scope of the claims.

Claims

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.