U.S. patent application number 12/106665 was filed with the patent office on 2008-10-09 for genetically modified human natural killer cell lines.
This patent application is currently assigned to Fox Chase Cancer Center. Invention is credited to Kerry S. Campbell.
Application Number | 20080247990 12/106665 |
Document ID | / |
Family ID | 35968029 |
Filed Date | 2008-10-09 |
United States Patent
Application |
20080247990 |
Kind Code |
A1 |
Campbell; Kerry S. |
October 9, 2008 |
GENETICALLY MODIFIED HUMAN NATURAL KILLER CELL LINES
Abstract
The invention provides a natural killer cell, NK-92, modified to
express a CD16 receptor or an inhibitory killer cell
immunoglobulin-like receptor (KIR) on a surface of the cell. In
examples, the NK-92 cell is further modified to co-express an
associated accessory signaling protein such as
Fc.epsilon.RI-.gamma. or TCR-.zeta., 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.
Inventors: |
Campbell; Kerry S.;
(Wyncote, PA) |
Correspondence
Address: |
COHEN & GRIGSBY, P.C.
11 STANWIX STREET, 15TH FLOOR
PITTSBURGH
PA
15222
US
|
Assignee: |
Fox Chase Cancer Center
Philadelphia
PA
|
Family ID: |
35968029 |
Appl. No.: |
12/106665 |
Filed: |
April 21, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11178258 |
Jul 8, 2005 |
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12106665 |
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60913072 |
Apr 20, 2007 |
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60991285 |
Nov 30, 2007 |
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Current U.S.
Class: |
424/85.2 ;
424/133.1; 424/155.1; 424/172.1; 424/174.1; 435/325; 435/7.21 |
Current CPC
Class: |
A61P 31/00 20180101;
A61K 49/00 20130101; A61K 39/39558 20130101; C12N 2503/02 20130101;
C12N 5/0646 20130101; A61K 35/17 20130101; C12N 2510/00 20130101;
C07K 2317/31 20130101; C07K 16/283 20130101; A61P 35/00 20180101;
G01N 33/5011 20130101; C07K 2317/24 20130101; C12N 2501/23
20130101; C12N 2503/00 20130101; A61K 2035/124 20130101; G01N
33/5047 20130101; A61K 39/39533 20130101; C07K 14/70535 20130101;
C07K 16/28 20130101; C07K 16/32 20130101; C07K 2317/732
20130101 |
Class at
Publication: |
424/85.2 ;
435/325; 435/7.21; 424/172.1; 424/133.1; 424/174.1; 424/155.1 |
International
Class: |
A61K 38/20 20060101
A61K038/20; C12N 5/06 20060101 C12N005/06; G01N 33/566 20060101
G01N033/566; A61K 39/395 20060101 A61K039/395 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] 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.
Foreign Application Data
Date |
Code |
Application Number |
Jul 8, 2005 |
US |
PCT/US05/24229 |
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 Fc.epsilon.RI-.gamma. (SEQ ID NO: 5) or
TCR-.zeta. (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
Fc.epsilon.RI-.gamma. (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 Fc.epsilon.RI-.gamma. (SEQ ID NO:5)
or TCR-.zeta. (SEQ ID NO:7).
42. The method of claim 33 wherein the step of monitoring comprises
measuring at least one of IFN-.gamma. 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
Fc.epsilon.RI-.gamma. (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-.gamma. 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.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] 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).
FIELD OF THE INVENTION
[0003] 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
Fc.epsilon.RI-.gamma. or TCR-.zeta.; a cytokine such as IL-2 or
IL-15; a chemokine; or one or more inhibitory killer cell
immunoglobulin-like receptors (KIR).
BACKGROUND
[0004] 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.
[0005] 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.
[0006] A number of antibodies, most notably Rituximab
(MabThera.RTM.; Hoffmann-LaRoche, Ltd; Basel, Switzerland) and
Herceptin.RTM. (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.
[0007] Additional utility and benefit of the disclosed modified
NK-92 cells will become apparent in the following descriptions.
SUMMARY
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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
[0014] 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.
[0015] 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.
[0016] 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 .sup.51Cr-release at 37.degree.
C. for three hours.
[0017] 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.
[0018] 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 Fc.epsilon.RI-.gamma. cDNA (.gamma.; 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 Fc.epsilon.RI-.gamma. or TCR 4 expressed from
pBMN-IRES-EGFP, and the y-axis denotes surface staining for CD16
expression.
[0019] FIG. 5 is a graph showing redirected cytotoxicity of
Fc.gamma.RII/III.sup.+ P815 target cells by CD16-F176V.NK-92 cells
induced by anti-CD16 antibody (3G8; +), but not antibodies toward
CD56 (B159; .DELTA.) or KIR (DX9; .quadrature.), which correlated
with cytotoxicity level in the absence of monoclonal antibody (No
mAb; o). Cells were assayed using .sup.51Cr release from P815
target cells at the indicated effector to target ratios.
[0020] FIG. 6 is a graph showing redirected cytotoxicity of
Fc.gamma.RII/III.sup.+ 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).
[0021] FIG. 7 is a graph showing redirected cytotoxicity of SKOV-3
target cells by CD16-F176V.NK-92 cells (.DELTA.), but not mouse
IgM-transduced NK-92 (.quadrature.), induced by bi-specific 2B1
antibody (.tangle-solidup., .box-solid.). 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.
[0022] 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:
[0023] No GFP-CD16-F176V.NK-92 cells (o);
[0024] GFP-CD16-F176V/Fc.epsilon.RI-.gamma..NK-92-cells ( );
and
[0025] GFP-CD16-F176 V/TCR-.zeta..NK-92 cells (+).
[0026] 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
.sup.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. .sup.51Cr release was measured
4 h later.
[0027] 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.
[0028] 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) .sup.51Cr labeled 721.221 B-cells expressing CD120 and HLA-B51
(the KIR3DL1 ligand; 721.221-B*5101), or (B) .sup.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.
.sup.51Cr-release was measured 4 hours later. Results shown are
mean .+-.SD.
[0029] 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 .sup.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 .mu.g/ml. .sup.51Cr-release was measured 4 hours later.
Results shown are mean .+-..
[0030] 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 .sup.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
.mu.g/ml DX9. (E, F) Cells were incubated with no antibody (open
bars), DX9 alone (grey bars, 0.1 .mu.g/ml), rituximab (striped
bars, 10 ng/ml) or with rituximab+DX9 (black bars) antibodies.
.sup.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
[0031] 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
[0032] 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.
[0033] "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.
[0034] 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.
[0035] A "bi-specific Ab" is a monoclonal Ab, preferably human or
humanized, that has binding specificities for at least two
different antigens.
[0036] "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.
[0037] "CD16", also known as "Fc.gamma.RIII-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".
[0038] "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.
[0039] "CD16 chimeric polypeptide" or "CD16 fusion polypeptide"
comprises CD16 fused to a non-CD16 polypeptide.
[0040] "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."
[0041] "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.
[0042] "Homologous polynucleotide sequence" or "homologous amino
acid sequence" refer to sequences characterized by a homology at
the polynucleotide level or amino acid level, respectively.
[0043] "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.
[0044] "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.
[0045] 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.
[0046] "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.
[0047] "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
[0048] 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
[0049] 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 Fc.epsilon.RI-.gamma. 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.
[0050] 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.sub.--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.
[0051] 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.
[0052] 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.
[0053] 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 .alpha.-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.).
[0054] 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
[0055] 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).
[0056] 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.
[0057] 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).
[0058] 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
Fc.epsilon.RI-.gamma. or TCR-4, such as by sequential transduction
with CD16 and/or KIR.
TABLE-US-00001 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-US-00002 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-US-00003 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
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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).
[0064] 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.
[0065] 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 .beta.-sheet or .alpha.-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-US-00004 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
[0066] 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.
[0067] 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-.zeta. or Fc.epsilon.RI.gamma. 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-.alpha., Ig-.beta., CD3-.epsilon., CD3-.gamma.,
CD3-.delta., 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.
[0068] In other embodiments, fusion polypeptide variants of other
proteins, such as KIR, Fc.epsilon.RI-.gamma., TCR-.zeta., or
naturally occurring minor polymorphic variants, are formed as
described above.
ADCC Assay
[0069] 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.
[0070] 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 Fc.epsilon.RI-.gamma.
(CD16/Fc.epsilon.RI-.gamma..NK-92) or TCR-.zeta.
(CD16/TCR-.zeta..NK-92); or a variant thereof.
[0071] 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.
[0072] In an example, the method of performing the ADCC assay
comprises: loading target cells with an indicator material such as
.sup.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 (.sup.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.times.[Experimental Release (mean
cpm)-Spontaneous Release (mean cpm)]/[Total Counts (mean cpm)-
Spontaneous Release (mean cpm)],
[0073] 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.
[0074] 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.
[0075] Optionally, the method further comprises the step of
measuring at least one of the following: cytokine/chemokine
production, including for example interferon-.gamma., tumor
necrosis factor (TNF)-.alpha., granulocyte-macrophage colony
stimulating factor (GM-CSF), MIP-1.alpha., MIP-1.beta., 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-.kappa.B. 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.
[0076] 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.
[0077] Optionally, the functionality of CD16 introduced into
CD16.NI-92 cells, CD16/Fc.epsilon.RI-.gamma..NI-92 cells, or
CD16/TCR-.zeta..NK-92 cells is determined using either the ADCC
assay or the redirected cytotoxicity assay (described below).
[0078] 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).
[0079] 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.
[0080] 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 Fc.gamma.RII/III.sup.+
murine mastocytoma cell line P815 (e.g., ATCC Deposit No. TIB-64)
and the Fc.gamma.RII/III.sup.+ 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.
[0081] 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).
[0082] In an example, at least one of Fc.epsilon.RI-.gamma.
(Genbank Accession No. M33195; SEQ ID NO:4, Table 5)
(polynucleotide) and SEQ ID NO:5, Table 6 (polypeptide)) or
TCR-.zeta. (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 Fc.epsilon.RI-.gamma. or TCR-.zeta. 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
[0083] 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.
[0084] 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.
[0085] 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-US-00005 TABLE 5 Fc.epsilon.RI-.gamma. 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-US-00006 TABLE 6 Fc.epsilon.RI-.gamma. 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-US-00007 TABLE 7 TCR-.zeta. 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-US-00008 TABLE 8 TCR-.zeta. 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)
[0086] The disclosed method of performing the ADCC and redirected
cytotoxicity assays use Abs and antibody fragments, such as Fab or
(Fab').sub.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
[0087] 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.
[0088] 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.sub.v framework 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.sub.c),
typically that of a human Ig (Jones et al., 1986; Presta, 1992;
Riechmaim et al., 1988).
Pharmaceutical Compositions
[0089] 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 Fc.epsilon.RI-.gamma. or TCR-.zeta.; 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
[0090] 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.
[0091] 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.
[0092] 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.
[0093] 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.
[0094] 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
[0095] 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.
[0096] 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
[0097] 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-.gamma. 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.
[0098] 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.
[0099] 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.
[0100] 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.
[0101] 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.sub.(ab) domain that specifically binds to CD16. Cells of
the ErbB2.sup.+ 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.
[0102] 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-.gamma. or
cytokines, reduction in the tumor, infection, or lesion size,
metabolism, or growth to indicate a therapeutic response to the
method of treatment.
EXAMPLES
[0103] 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
[0104] 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.
[0105] 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).
[0106] The recombinant vector was mixed with 10 .mu.L of PLUS.TM.
Reagent (Invitrogen; Carlsbad, Calif.); diluted to 100 .mu.L with
pre-warmed, serum-free Opti-MEM.RTM. (Invitrogen; MEM, minimum
essential media); further diluted by the addition of 8 SL
Lipofectamine (Invitrogen) in 100 mL pre-warmed serum-free
Opti-MEM.RTM.; 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.RTM..
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.RTM. medium (Invitrogen).
After removal of the medium, 1 mL of the solution of recombinant
vector in Lipofectamine.TM. PLUS.TM. Reagent was added to each
well, and the cells were incubated for at least three hours at
37.degree. C. under a 7% CO.sub.2/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.degree. C., under a 7% CO.sub.2/balance air atmosphere. The
media was then removed; the cells washed with 6 mL pre-warmed
serum-free Opti-MEM.RTM.; 2 mL serum-free Opti-MEM.RTM. added; and
the cells incubated at 37.degree. C., under a 7% CO.sub.2/balance
air atmosphere for an additional 48 hours.
[0107] 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
.mu.L of PLUS.TM. Reagent was added to the virus suspension; the
mixture incubated at room temperature for 15 minutes; 8 .mu.L
Lipofectamine.TM. 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
[0108] NK-92 cells cultured in .alpha.-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.RTM. medium. An aliquot
of cell suspension containing 5.times.10.sup.4 cells 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.degree. C. under an
atmosphere of 7% CO.sub.2/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 .alpha.-MEM, transferred to a T-25
flask, and incubated at 37.degree. C. under a 7% CO.sub.2/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.
[0109] 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
Fc.epsilon.RI-.gamma. or TCR-.zeta.
[0110] Recombinant retroviruses incorporating inserted genes for
the expression of Fc.epsilon.RI-.gamma. (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.TM. Plus as described in Example 1.
The resulting Fc.epsilon.RI-.gamma. or TCR-.zeta. recombinant
retroviruses were used to transduce NK-92 cells as described in
Example 2 with the following further modifications.
[0111] NK-92 cells transduced with retrovirus incorporating pBMN
vector constructs were collected, re-suspended in serum-free
Opti-MEM.RTM. 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
Fc.epsilon.RI-.gamma. or TCR-.zeta. transduced NK-92 cells
(Fc.epsilon.RI-.gamma..NK-92 or TCR-.zeta..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/Fc.epsilon.RI-.gamma..NK-92 or
CD16/TCR-4.NK-92) were suspended in .alpha.-MEM, transferred to a
T-25 flask, and grown to confluency at 37.degree. C. under a 7%
CO.sub.2/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.
[0112] 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 (Fc.epsilon.RI-.gamma. or TCR-.zeta.) 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.
[0113] 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
Fc.epsilon.RI-.gamma. cDNA (CD16/Fc.epsilon.RI-.gamma..NK-92; FIG.
4B); or TCR-.zeta. cDNA (CD16/TCR-.zeta..NK-92; FIG. 4C). These
data show that when CD16 is co-expressed with Fc.epsilon.RI-.gamma.
or TCR-.zeta. 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
[0114] Effector cells (NM-92, CD16.NK-92, CD16/TCR-4.NK-92, and
CD16/Fc.epsilon.RI-.gamma..NK-92) were washed by suspension in
.alpha.-MEM without IL-2 and sedimented at 1300 rpm for 5 minutes.
The cell pellet was suspended in .alpha.-MEM, cells counted, and
aliquots prepared at cell concentrations of 1.times.10.sup.5/mL
(effector to target cell ratio (E:T)=1:1), 5.times.10.sup.5/mL
(E:T=5:1), 1.times.10.sup.6/mL (E:T=10:1), 2.times.10.sup.6/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.
[0115] 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.
[0116] Approximately 2.times.10.sup.6 of 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 .mu.L of
FBS and 100 .mu.Ci of Na[.sup.51Cr]chromate was added and the cells
incubated at 37.degree. 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
.alpha.-MEM and diluted to a concentration of 1.times.10.sup.5/mL.
Target cells for use in redirected cytotoxicity assays were further
incubated with the appropriate antibody at a final concentration of
0.01-5 .mu.g/mL for 10-15 minutes at room temperature.
[0117] One-hundred .mu.L of the selected type of target cells and
100 .mu.L of the effector cells at cell concentrations of
1.times.10.sup.5/ml (E:T=1:1), 5.times.10.sup.5/ml (E:T=5:1),
1.times.10.sup.6/ml (E:T=10:1), 2.times.10.sup.6/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
.mu.L of .alpha.-MEM) and total release control (effector cells
replaced with 100 .mu.L of 2% t-Octylphenoxypolyethoxyethanol
(Triton X-100.RTM.) detergent in .alpha.-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.degree. C. in an atmosphere of 7%
CO.sub.2/balance air. At the end of the incubation period, the
plate was centrifuged at 1500 rpm for 8 minutes, and 100 .mu.L of
the supernatant was collected from each well for counting in a
.gamma. counter to measure of .sup.51Cr release. The percentage of
specific target cell lysis was calculated as described above.
Example 5
CD16-Mediated Cell Lysis
[0118] 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 Fc.gamma.R+ mouse mastocytoma cell line P815 and
human Fc.gamma.R.sup.+ 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/Fc.epsilon.RI-.gamma..NK-92, and CD16/TCR-4.NK-92
cells (FIGS. 5 and 6).
[0119] 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/Fc.epsilon.RI-.gamma..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/Fc.epsilon.RI-.gamma..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 .sup.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.
[0120] 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
Fc.epsilon.RI-.gamma. or TCR-.zeta. 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
[0121] The selected target cells were labeled with
Na[.sup.51Cr]chromate as described in Example 4 and adjusted to a
concentration of 1.times.10.sup.5 cells/mL before use. A 100 .mu.L
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 .mu.g/mL. At least 100 .mu.L aliquots of each
hybridoma supernatant was added to each of three target cell
containing wells; incubated for 15 minutes at room temperature;
washed with .alpha.-MEM; and re-suspended in 100 .mu.L of
.alpha.-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 .mu.L of
effector cells to each well. The plates were then centrifuged at
500 RPM for 3 minutes and incubated for 4 hours at 37.degree. C. in
an atmosphere of 7% CO.sub.2/balance air. At the end of the
incubation period, the plate was centrifuged at 1500 rpm for 8
minutes and 100 .mu.L of the supernatant was collected from each
well for counting in a y counter as a measure of .sup.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 .mu.L of .alpha.-MEM) and a total release control
(effector cells replaced with 100 .mu.L of 2% Triton X-100
detergent in .alpha.-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.
[0122] 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-.kappa.B within the NK-92 cells, or
the activation of caspases or other markers of apoptosis in the
target cells.
[0123] 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
[0124] The production of cytokines by NIC-92, CD16.NK-92,
CD16/Fc.epsilon.RI-.gamma..NK-92, and CD16/TCR-.zeta..NK-92 cells
in response to CD16 mediated stimulation was determined by the
redirected cytotoxicity assays described in Examples 4 and 5.
[0125] Effector cells (NK-92, CD16.NK-92,
CD16/Fc.epsilon.RI-.gamma..NK-92, and CD16/TCR-.zeta..NK-92 cells)
were washed by suspension in .alpha.-MEM (without IL-2) and
sedimentation at 1300 rpm for 5 minutes. The cell pellet was
suspended in .alpha.-MEM, the cells counted, and aliquots prepared
at cell concentrations of 1.times.10.sup.5/mL (E:T=1:1),
5.times.10.sup.5/mL (E:T=5:1), 1.times.10.sup.6/mL (E:T=10:1),
2.times.10.sup.6/mL (E:T=20:1), or as appropriate to establish an
effective dynamic range of cytotoxicity in the assay being
performed.
[0126] 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.
[0127] One hundred .mu.L of varying concentrations of effector
cells were combined with a constant concentration of antibody
treated target cells (not labeled with .sup.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
.alpha.-MEM (spontaneous release) or with 100 .mu.L 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').sub.2 fragments 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.degree. C. in an atmosphere of 7% CO.sub.2/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
[0128] 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/Fc.epsilon.RI-.gamma..NK-92, and CD16/TCR-.zeta..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.
[0129] 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)
[0130] 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 (Na.sub.2.sup.5, CrO.sub.4; 100
.mu.Ci/million target cells for 1 hi at 37.degree. C. in 500 .mu.l
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 .alpha.-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.degree. C. (7%
CO.sub.2). 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 .sup.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.
[0131] 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
[0132] 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
[0133] 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 .alpha.-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 .mu.g/ml penicillin (Life Technologies),
100 .mu.g/ml streptomycin (Life Technologies), 1 mM sodium pyruvate
(Life Technologies), 100 .mu.M 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.
[0134] 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 .mu.g/ml penicillin (Life Technologies), 100
.mu.g/ml streptomycin (Life Technologies), 1 mM sodium pyruvate
(Life Technologies), 1 mM HEPES buffer and 50 .mu.M 2-ME (Fisher).
SKOV3 ovarian carcinoma cells were maintained in DMEM containing
10% FBS (HyClone Laboratories), 2 mM L-glutamate (Life
Technologies), 100 .mu.g/ml penicillin (Life Technologies), 100
.mu.g/ml streptomycin (Life Technologies) and 50 .mu.M 2-ME
(Fisher).
Generation of CD16 Expressing NK-92 Cells
[0135] 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.
[0136] 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 .alpha.-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
[0137] 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
[0138] ADCC studies were performed as described above. Target cells
were labeled with Na.sub.2.sup.51CrO.sub.4 (100 .mu.Ci/10.sup.6
targets; PerkinElmer Life Sciences) for 1 h at 37.degree. C. in 500
ul FBS. The .sup.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 .mu.l, and all assays were
performed in triplicate. The plates were centrifuged at 300.times.g
for 3 min, incubated for 4 h in a 5% (v/v) CO.sub.2 incubator at
37.degree. C., and then centrifuged again at 300.times.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
[0139] 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.times.10.sup.6 cells were
incubated with the relevant antibody for 30 min at 4.degree. 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
[0140] 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
[0141] 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
[0142] 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
[0143] 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
[0144] 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.
[0145] 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).
[0146] 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.
Sequence CWU 1
1
71254PRTHomo sapiens 1Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu
Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala
Val Val Phe Leu Glu Pro 20 25 30Gln Thr Tyr Arg Val Leu Glu Lys Asp
Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn
Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala
Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly
Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val
Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro
Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120
125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn
130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr
Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe
Cys Arg Gly Leu Phe 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr
Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile
Ser Ser Phe Phe Pro Pro Gly Tyr Gln 195 200 205Val Ser Phe Cys Leu
Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220Leu Thr Phe
Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp225 230 235
240Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 245
2502254PRTHomo sapiens 2Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu
Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala
Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp
Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn
Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala
Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly
Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val
Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro
Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120
125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn
130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr
Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe
Cys Arg Gly Leu Val 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr
Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile
Ser Ser Phe Phe Pro Pro Gly Tyr Gln 195 200 205Val Ser Phe Cys Leu
Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220Leu Tyr Phe
Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp225 230 235
240Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 245
2503765DNAHomo sapiens 3atgtggcagc tgctcctccc aactgctctg ctacttctag
tttcagctgg catgcggact 60gaagatctcc caaaggctgt ggtgttcctg gagcctcaat
ggtacagggt gctcgagaag 120gacagtgtga ctctgaagtg ccagggagcc
tactcccctg aggacaattc cacacagtgg 180tttcacaatg agagcctcat
ctcaagccag gcctcgagct acttcattga cgctgccaca 240gtcgacgaca
gtggagagta caggtgccag acaaacctct ccaccctcag tgacccggtg
300cagctagaag tccatatcgg ctggctgttg ctccaggccc ctcggtgggt
gttcaaggag 360gaagacccta ttcacctgag gtgtcacagc tggaagaaca
ctgctctgca taaggtcaca 420tatttacaga atggcaaagg caggaagtat
tttcatcata attctgactt ctacattcca 480aaagccacac tcaaagacag
cggctcctac ttctgcaggg ggctttttgg gagtaaaaat 540gtgtcttcag
agactgtgaa catcaccatc actcaaggtt tggcagtgtc aaccatctca
600tcattctttc cacctgggta ccaagtctct ttctgcttgg tgatggtact
cctttttgca 660gtggacacag gactatattt ctctgtgaag acaaacattc
gaagctcaac aagagactgg 720aaggaccata aatttaaatg gagaaaggac
cctcaagaca aatga 7654591DNAHomo sapiens 4cagaacggcc gatctccagc
ccaagatgat tccagcagtg gtcttgctct tactcctttt 60ggttgaacaa gcagcggccc
tgggagagcc tcagctctgc tatatcctgg atgccatcct 120gtttctgtat
ggaattgtcc tcaccctcct ctactgtcga ctgaagatcc aagtgcgaaa
180ggcagctata accagctatg agaaatcaga tggtgtttac acgggcctga
gcaccaggaa 240ccaggagact tacgagactc tgaagcatga gaaaccacca
cagtagcttt agaatagatg 300cggtcatatt cttctttggc ttctggttct
tccagccctc atggttggca tcacatatgc 360ctgcatgcca ttaacaccag
ctggccctac ccctataatg atcctgtgtc ctaaattaat 420atacaccagt
ggttcctcct ccctgttaaa gactaatgct cagatgctgt ttacggatat
480ttatattcta gtctcactct cttgtcccac ccttcttctc ttccccattc
ccaactccag 540ctaaaatatg ggaagggaga acccccaata aaactgccat
ggactggact c 591586PRTHomo sapiens 5Met Ile Pro Ala Val Val Leu Leu
Leu Leu Leu Leu Val Glu Gln Ala1 5 10 15Ala Ala Leu Gly Glu Pro Gln
Leu Cys Tyr Ile Leu Asp Ala Ile Leu 20 25 30Phe Leu Tyr Gly Ile Val
Leu Thr Leu Leu Tyr Cys Arg Leu Lys Ile 35 40 45Gln Val Arg Lys Ala
Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val 50 55 60Tyr Thr Gly Leu
Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys65 70 75 80His Glu
Lys Pro Pro Gln 8561472DNAHomo sapiens 6cttttctcct aaccgtcccg
gccaccgctg cctcagcctc tgcctcccag cctctttctg 60agggaaagga caagatgaag
tggaaggcgc ttttcaccgc ggccatcctg caggcacagt 120tgccgattac
agaggcacag agctttggcc tgctggatcc caaactctgc tacctgctgg
180atggaatcct cttcatctat ggtgtcattc tcactgcctt gttcctgaga
gtgaagttca 240gcaggagcgc agagcccccc gcgtaccagc agggccagaa
ccagctctat aacgagctca 300atctaggacg aagagaggag tacgatgttt
tggacaagag acgtggccgg gaccctgaga 360tggggggaaa gccgagaagg
aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag 420ataagatggc
ggaggcctac agtgagattg ggatgaaagg cgagcgccgg aggggcaagg
480ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac
gacgcccttc 540acatgcaggc cctgccccct cgctaacagc caggggattt
caccactcaa aggccagacc 600tgcagacgcc cagattatga gacacaggat
gaagcattta caacccggtt cactcttctc 660agccactgaa gtattcccct
ttatgtacag gatgctttgg ttatatttag ctccaaacct 720tcacacacag
actgttgtcc ctgcactctt taagggagtg tactcccagg gcttacggcc
780ctgccttggg ccctctggtt tgccggtggt gcaggtagac ctgtctcctg
gcggttcctc 840gttctccctg ggaggcgggc gcactgcctc tcacagctga
gttgttgagt ctgttttgta 900aagtccccag agaaagcgca gatgctagca
catgccctaa tgtctgtatc actctgtgtc 960tgagtggctt cactcctgct
gtaaatttgg cttctgttgt caccttcacc tcctttcaag 1020gtaactgtac
tgggccatgt tgtgcctccc tggtgagagg gccgggcaga ggggcagatg
1080gaaaggagcc taggccaggt gcaaccaggg agctgcaggg gcatgggaag
gtgggcgggc 1140aggggagggt cagccagggc ctgcgagggc agcgggagcc
tccctgcctc aggcctctgt 1200gccgcaccat tgaactgtac catgtgctac
aggggccaga agatgaacag actgaccttg 1260atgagctgtg cacaaagtgg
cataaaaaac agtgtggtta cacagtgtga ataaagtgct 1320gcggagcaag
aggaggccgt tgattcactt cacgctttca gcgaatgaca aaatcatctt
1380tgtgaaggcc tcgcaggaag acgcaacaca tgggacctat aactgcccag
cggacagtgg 1440caggacagga aaaacccgtc aatgtactag gg 14727163PRTHomo
sapiens 7Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala
Gln Leu1 5 10 15Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro
Lys Leu Cys 20 25 30Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val
Ile Leu Thr Ala 35 40 45Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala
Glu Pro Pro Ala Tyr 50 55 60Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu
Leu Asn Leu Gly Arg Arg65 70 75 80Glu Glu Tyr Asp Val Leu Asp Lys
Arg Arg Gly Arg Asp Pro Glu Met 85 90 95Gly Gly Lys Pro Arg Arg Lys
Asn Pro Gln Glu Gly Leu Tyr Asn Glu 100 105 110Leu Gln Lys Asp Lys
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 115 120 125Gly Glu Arg
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 130 135 140Ser
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu145 150
155 160Pro Pro Arg
* * * * *