U.S. patent application number 13/225889 was filed with the patent office on 2014-04-10 for natural killer cell lines and methods of use.
This patent application is currently assigned to Conkwest, Inc.. The applicant listed for this patent is Hans Klingemann. Invention is credited to Hans Klingemann.
Application Number | 20140099714 13/225889 |
Document ID | / |
Family ID | 44741147 |
Filed Date | 2014-04-10 |
United States Patent
Application |
20140099714 |
Kind Code |
A1 |
Klingemann; Hans |
April 10, 2014 |
NATURAL KILLER CELL LINES AND METHODS OF USE
Abstract
This invention relates to a natural killer cell line termed
NK-92. The invention provides a vector for transfecting a mammalian
cell which includes a nucleic acid sequence encoding a cytokine
that promotes the growth of NK-92. Additionally, the invention
provides an NK-92 cell, or an NK-92 cell modified by transfection
with a vector conferring advantageous properties, which is unable
to proliferate and which preserves effective cytotoxic activity.
The invention further provides a modified NK-92 cell that is
transfected with a vector encoding a cytokine that promotes the
growth of NK-92 cells. The cell secretes the cytokine upon being
cultured under conditions that promote cytokine secretion, and
furthermore secretes the cytokine in vivo upon being introduced
into a mammal. In a significant embodiment, the cytokine is
interleukin 2. The present invention also provides methods of
purging cancer cells from a biological sample, of treating a cancer
ex vivo in a mammal, and of treating a cancer in vivo in a mammal
employing a natural killer cell, such as NK-92 itself, an NK-92
cell which is unable to proliferate and which preserves effective
cytotoxic activity, or natural killer cells transfected with a
vector encoding a cytokine.
Inventors: |
Klingemann; Hans; (Winnetka,
IL) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Klingemann; Hans |
Winnetka |
IL |
US |
|
|
Assignee: |
Conkwest, Inc.
Del Mar
CA
|
Family ID: |
44741147 |
Appl. No.: |
13/225889 |
Filed: |
September 6, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10456237 |
Jun 6, 2003 |
8034332 |
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13225889 |
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10008955 |
Dec 7, 2001 |
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10456237 |
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09403910 |
Oct 27, 1999 |
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PCT/US98/08672 |
Apr 30, 1998 |
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10008955 |
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60045885 |
Apr 30, 1997 |
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Current U.S.
Class: |
435/375 |
Current CPC
Class: |
C12N 5/0093 20130101;
C12N 5/0646 20130101; C07K 14/55 20130101; C12N 2510/00 20130101;
A61K 38/00 20130101; A61K 2035/124 20130101 |
Class at
Publication: |
435/375 |
International
Class: |
C12N 5/0783 20060101
C12N005/0783 |
Claims
30. A modified NK-92 cell, wherein the cell secretes a
cytokine.
31. The cell of claim 30, wherein the cytokine is
interleukin-2.
32. The cell of claim 30, wherein the cell comprises a transfected
polynucleotide encoding interleukin-2.
33. The cell of claim 32, where in the transfected polynucleotide
comprises MFG-hIL or pCEP4-LTRhIL-2 polynucleotide.
34. The cell of claim 33, wherein the cell is ATCC CRL-2408 or ATCC
CRL-2409.
35. A method of making the cell of claim 30, comprising physically
treating a NK-92 cell to secrete a cytokine.
36. The method of claim 35, wherein the physically treating a NK-92
cell is transfecting a polynucleotide encoding interleukin-2 into
the cell.
37. The method of claim 36, wherein the transfected polynucleotide
comprises a MFG-hIL or pCEP4-LTRhIL-2 polynucleotide.
Description
RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 09/403,910, filed on Oct. 27, 1999, which was
based on, and claimed benefit of, U.S. Provisional Application Ser.
No. 60/045,885, filed on Apr. 30, 1997.
FIELD OF THE INVENTION
[0002] This invention relates to natural killer cells and their use
in the treatment of pathologies related to cancer or viral
infections. Specifically, a particular cell line, NK-92, and
modifications thereof, are disclosed. These cells are shown to be
highly effective in the treatment of these pathologies.
BACKGROUND OF THE INVENTION
[0003] Certain cells of the immune system have cytotoxic activity
against particular target cells. Cytotoxic T lymphocytes (CTLs) are
specifically directed to their targets via antigen-derived peptides
bound to MHC class I-specific markers. Natural killer (NK) cells,
however, are not so restricted. NK cells, generally representing
about 10-15% of circulating lymphocytes, bind and kill target
cells, including virus-infected cells and many malignant cells,
nonspecifically with regard to antigen and without prior immune
sensitization (Herberman et al., Science 214:24 (1981)). Killing of
target cells occurs by inducing cell lysis. MHC class restriction
likewise is not involved. In these ways the activity of NK cells
differs from antigen-specific and MHC class-specific T cells, such
as cytotoxic T lymphocytes. Use of NK cells in the immunotherapy of
tumors and malignancies is suggested by these properties, since
many tumors are MHC class I deficient and therefore do not attract
CTL activity. Adhesion molecules may also be involved in the
targeting of NK cells; for example, it is observed that the
Fc.gamma. receptor (CD16) is expressed on NK cells. NK cells are
large granular lymphocytes which lack CD3, and in addition to CD16,
also may express Leu19 (Lanier et al., J. Immunol. 136; 4480
(1986)).
[0004] NK cells are activated when exposed to cytokines such as
interleukin-2 (IL-2), IL-7, IL-12, and interferons (Alderson et
al., J. Exp. Med. 172:577-587 (1990); Robertson et al., J. Exp.
Med. 175:779-788 (1992)). The resulting cells are called lymphokine
activated killer (LAK) cells. The spectrum of target cells is
altered in activated NK cells compared to nonactivated cells,
although the mechanism of killing may be identical or similar
(Philips et al., J. Exp. Med. 164:814-825 (1986)).
[0005] More generally, killing activity in the cells of the immune
system may be induced by treating a population of cells, such as
peripheral blood mononuclear cells (PBMCs), with lymphokines. Such
preparations contain LAK cells. LAK cells may also be generated
from autologous samples of peripheral blood lymphocytes. LAK cells
have antitumor killing activity while having essentially no effect
on normal cells. They appear to purge leukemia (Long et al.,
Transplantation 46:433 (1988); Xhou et al., Proc. Am. Assoc. Cancer
Res. 34:469 (1993; abstract)), lymphoma (Schmidt-Wolf et al., J.
Exp. Med. 174: 139 (1991); Gambacorti-Passerini et al., Br. J.
Haematol. 18:197 (1991)) and neuroblastoma (Ades et al., Clin.
Immunol. Immunopathol. 46:150 (1988)). NK cells, activated NK
cells, and LAK cells are distinguishable by their cell surface
markers and by the identity of the target cells that they kill.
[0006] Activated and expanded (i.e., cultured to proliferate) NK
cells and LAK cells have been used in both ex vivo therapy and in
vivo treatment in patients with advanced cancer. Some success with
ex vivo therapy has been observed in bone marrow related diseases,
such as leukemia, breast cancer and certain types of lymphoma. In
vivo treatment may be directed toward these and other forms of
cancer, including malignant melanoma and kidney cancer (Rosenberg
et al., N. Engl. J. Med. 316:889-897 (1987)). LAK cell treatment
requires that the patient first receive IL-2, followed by
leukophoresis and then an ex vivo incubation and culture of the
harvested autologous blood cells in the presence of IL-2 for a few
days. The LAK cells must be reinfused along with relatively high
doses of IL-2 to complete the therapy. This purging treatment is
expensive and can cause serious side effects. These include fluid
retention, pulmonary edema, drop in blood pressure, and high fever.
In some cases in which these side effects occur, intensive care
unit management is required.
[0007] Purging techniques have been applied in other circumstances
as well. Cytotoxic drugs or monoclonal antibodies combined with
complement, and toxins, may be administered in order to bring about
remission. In such cases bone marrow or blood stem cells, purged to
reduce the number of residual leukemic cells present, have been
infused back into the patient after the drug treatment (Uckun et
al., Blood 79:1094 (1992)). Gene marking studies have shown that
unpurged bone marrow may contribute to relapse in patients presumed
to be in remission (Brenner et al., Lancet 341:85 (1993)). This
suggests that some form of purging of autologous marrow or blood
prior to transplantation is necessary (Klingemann et al., Biol.
Blood Marrow Transplant. 2:68-69 (1996)).
[0008] Recently, preclinical studies have also demonstrated
promising antitumor activity in vivo with a lethally irradiated,
MHC-unrestricted, cytotoxic T-cell leukemic clone (TALL-104)
(Cesano et al., Cancer Immunol. Immunother. 40:139-151 (1995);
Cesano et al., Blood 87:393-403 (1996)). These cells were derived
from leukemia T cell lines obtained from patients having acute T
lymphoblastic leukemias (ALL). They bear the CD3 cell surface
marker, but not the CD56 marker, in distinction to NK cells which
have the converse immunophenotype (CD3.sup.- CD56.sup.+). Adoptive
transfer of these cells was able to eliminate human leukemic cell
lines in xenografted severe combined immunodeficient (SCID) mice
and to induce remissions of spontaneous lymphomas in dogs without
producing T-cell leukemia in the animal models (Cesano et al.
(1995); Cesano et al. (1996); Cesano et al., J. Clin Invest.
94:1076-1084 (1994); Cesano et al., Cancer Res. 563021-3029
(1996)).
[0009] In spite of the advantageous properties of NK cells in
killing tumor cells and virus-infected cells, they remain difficult
to work with and to apply in immunotherapy. It is difficult to
expand NK cells ex vivo that maintain their tumor-targeting,
tumoricidal, and viricidal capabilities in vivo. This remains a
major obstacle to their clinical use in adoptive cell immunotherapy
(Melder et al., Cancer Research 48:3461-3469 (1988); Stephen et
al., Leuk. Lymphoma 377-399 (1992); Rosenberg et al., New Engl. J.
Med. 316:889-897 (1987)). Studies of the mechanisms whereby NK
cells exert their tumoricidal and viricidal effects are also
limited by difficulties in enriching the NK cell fractions without
compromising their biological functions and in obtaining pure NK
cells that are not contaminated by T cells or other immune effector
cells. In an attempt to overcome these problems, many investigators
have turned to the use of established NK-like cell lines to explore
the mechanisms whereby target cells are susceptible to cytotoxic
cells (Hercend et al., Nature 301:158-160 (1983); Yodoi et al., J.
Immunol. 134:1623-1630 (1985); Fernandez et al., Blood 67:925-930
(1986); Robertson et al., Exp. Hematol. 24:406-415 (1996); Gong et
al., Leukemia 8:652-658 (1994)). NK cell lines described in earlier
work carry T lymphocyte-associated surface markers, in spite of the
fact that they were developed from precursor cells depleted of T
cells (Rosenberg, et al. (1987); Hercend, et al., (1983)).
[0010] There thus remains a need for a method of treating a
pathology related to cancer or a viral infection with a natural
killer cell line that maintains viability and therapeutic
effectiveness against a variety of tumor classes. This need
encompasses therapeutic methods in which samples from a mammal are
treated ex vivo with natural killer cells, as well as methods of
treatment of these pathologies with natural killer cells in vivo in
a mammal. There is also a need for a natural killer cell line that
maintains its own propensity for viability and cytolytic activity
by secreting a cytokine which fosters these properties. There also
remains a need for such natural killer cell lines which are
modified to be more effective, convenient, and/or useful in
treatment of cancer and viral infection. It is the objective of
this invention to provide NK cells and methods that address these
needs.
SUMMARY OF THE INVENTION
[0011] The cell line described by Gong et al. (1994), termed NK-92,
proliferates in the presence of IL-2 and has high cytolytic
activity against a variety of cancers. The present invention
employs the NK-92 cell line, as well as modified NK-92 cell lines,
to provide cancer treatment and virus treatment systems. The
invention also provides the vectors that transfect NK-92, as well
as the modified NK-92 cells. For purposes of this invention and
unless indicated otherwise, the term "NK-92" is intended to refer
to the original NK-92 cell lines as well as the modified NK-92 cell
lines disclosed herein.
[0012] One aspect of the invention provides a vector for
transfecting NK-92 cells, wherein the vector includes a nucleic
acid sequence encoding a protein that is either a cytokine which
promotes the growth of the NK-92 cells, a cellular component
responsive to an agent, a cancer cell receptor molecule, or any
combination of these proteins. When transfected with the vector,
the NK-92 cells constitutively express the protein. In an important
embodiment, the protein is the cytokine interleukin 2. In
especially important embodiments of this aspect of the invention,
the vectors are MFG-hIL-2 and pCEP4-LTRhIL-2. In additional
significant embodiments, the protein is a cellular component
responsive to an agent, such that when the vector transfects NK-92
cells and the agent is taken up by the cells, the cells are
inactivated. In still more significant embodiments the agent is
either acyclovir or gancyclovir.
[0013] A further embodiment of the invention provides a cell
population containing NK-92 cells that have been modified by a
physical treatment or by transfection with a vector.
[0014] In significant embodiments of this population, the physical
treatment renders them non-proliferative yet does not significantly
diminish the cytotoxicity of the cells, and in particularly
significant embodiments, the treatment is irradiation. In
additional important embodiments the cells have been transfected by
a vector that encodes a cytokine promoting the growth of the cells.
The cells secrete the cytokine both upon being cultured under
conditions that promote cytokine secretion or in vivo upon being
introduced into a mammal. In particularly important embodiments of
this aspect of the invention, the cytokine is interleukin 2. In
still further important embodiments, the NK-92 cells are the cells
NK-92MI, modified by transfection with the vector MFG-hIL-2
encoding, and the cells NK-92CI modified by transfection with the
vector pCEP4-LTRhIL-2 encoding interleukin-2. The NK-92MI and
NK-92CI cell lines have been in the American Type Culture
Collection under the designations CRL-2408 and CRL-2409,
respectively. In additional important embodiments, the NK-92 cells
are transfected by a vector including a sequence that encodes a
cellular component responsive to an agent such that, when the NK-92
cell so transfected takes up the agent, the cell is inactivated. In
particularly important embodiments thereof, the agent is acyclovir
or gancyclovir. In yet additional embodiments, the cell population
is transfected with a vector encoding a cancer cell receptor
molecule.
[0015] The present invention also provides a method of purging
cells related to a pathology from a biological sample including the
steps of (i) obtaining a biological sample from a mammal that is
suspected of containing cells related to the pathology, and (ii)
contacting the sample with a medium comprising NK-92 or modified
NK-92 natural killer cells, wherein the modified NK-92 cells have
been modified by a physical treatment or by transfection with a
vector. In significant embodiments of this method, the pathology is
a cancer, or is an infection by a pathogenic virus such as human
immunodeficiency virus (HIV), Epstein-Barr virus (EBV),
cytomegalovirus (CMV), or herpes virus. In additional important
embodiments, the modified NK-92 cells have undergone a physical
treatment that renders them non-proliferative, yet which does not
significantly diminish their cytotoxicity, or have been transfected
with a vector, or they have been treated by any combination of
these modifications. In significant embodiments of this method, the
vector encodes a cytokine that promotes the growth of the cells, a
protein that is responsive to an agent, a cancer cell receptor
molecule, or a combination of these coding sequences. In a further
embodiment, the medium also includes a cytokine that promotes the
growth of the cells. The sample, once purged of cancer cells, may
be further treated, including, for example, being returned to the
mammal from which it was obtained. In important embodiments of the
method, the biological sample is blood or bone marrow, the mammal
is a human, and/or the natural killer cell is immobilized on a
support.
[0016] The invention additionally provides a method of treating a
pathology ex vivo in a mammal including the steps of (i) obtaining
a biological sample suspected of containing cells related to the
pathology from the mammal; (ii) contacting the biological sample
with a medium including natural killer cells, either NK-92 cells or
modified NK-92 cells that have been modified by a physical
treatment or by transfection with a vector, thereby selectively
destroying the cells related to the pathology in the sample and
producing a purged sample, and (iii) returning the purged sample to
the mammal. The pathology may be a cancer, such as a leukemia, a
lymphoma, or a multiple myeloma. Alternatively, the pathology may
be infection by a pathogenic virus such as HIV, EBV, CMV, or
herpes. In this method the natural killer cells may be NK-92 itself
or modified NK-92 cells. Examples of such modified NK-92 cells
include those that have been modified by a physical treatment that
renders them non-proliferative yet does not significantly diminish
their cytotoxicity, and modification by transfection with a vector.
The vector encodes a cytokine that promotes the growth of the
cells, or a protein that is responsive to an agent, or a cancer
cell receptor molecule, or the vector may include any combination
of these modifications. In important embodiments of this method,
the biological sample is blood or bone marrow, the mammal is a
human, and/or the natural killer cell is immobilized on a support.
In additional significant embodiments, the medium further includes
a cytokine that promotes the growth of the cells, and/or the cancer
is a leukemia, a lymphoma or a multiple myeloma.
[0017] The present invention further provides a method of treating
a pathology in vivo in a mammal including the step of administering
to the mammal a medium comprising natural killer cells, either
NK-92 cells or NK-92 cells that have been modified by a physical
treatment that renders them non-proliferative yet does not
significantly diminish their cytotoxicity, by treatment that
inhibits expression of HLA antigens on the NK-92 cell surface, or
by transfection with a vector. The vector encodes a cytokine that
promotes the growth of the cells, or a protein that is responsive
to an agent, or a cancer cell receptor molecule, or they have been
treated by any combination of these modifications. In important
embodiments, the pathology is a cancer, such as a leukemia, a
lymphoma, or a multiple myeloma. Alternatively, in important
embodiments the pathology is infection by a pathogenic virus such
as HIV, EBV, CMV, or herpes. Advantageous embodiments of this
method include administering the cells intravenously to a human and
administering a cytokine that promotes the growth of the cells to
the mammal in conjunction with administering the medium comprising
the natural killer cell. The present methods are especially adapted
for the treatment of leukemia, lymphoma or multiple myeloma.
[0018] In yet an additional embodiment of the in vivo method of
treating cancer, the NK-92 is modified by transfection with a
vector comprising an element responsive to an agent such that when
the agent is taken up by the cell, the cell is inactivated.
According to this method, an amount of the agent effective to
inactivate the cell can be administered to a mammal after a time
sufficient for the natural killer cell to treat the cancer has
elapsed or at a time desirable to effectively end the treatment. A
significant aspect of this embodiment is one in which the agent is
acyclovir or gancyclovir. Such transfected cells can, in effect, be
"turned off" as desired by administering the agent.
BRIEF DESCRIPTION OF THE DRAWING
[0019] FIG. 1. Cytotoxic activity of NK-92 against different
leukemic target cell lines tested in a 4 hour .sup.51Cr release
assay. The results represent the mean.+-.the standard deviation
(SD) for three replicate experiments.
[0020] FIG. 2. Cytotoxicity of NK-92 after IL-2 deprivation. NK-92
cells were cultured in enriched alpha medium (Myelocult.TM.,
StemCell Technologies, Vancouver, BC) without IL-2. Cytotoxicity
was measured daily with the .sup.51Cr-release assay against
K562-neo.sup.r or Daudi target cells. The Figure shows results from
one representative experiment at the E:T ratio of 10:1.
[0021] FIG. 3. Effect of various doses of .gamma. radiation on the
cytolytic potential of NK-92 cells. NK-92 cells were irradiated
with a .sup.137Cs source using doses ranging from 200 to 1000 cGy.
To allow for recovery, cells were left in medium containing IL-2
for 24 hours before cytotoxicity was measured in a 4 hour .sup.51Cr
release assay against the target cell line K562.
[0022] FIG. 4. Survival curves of NK-92 cells after
.gamma.-irradiation. NK-92 cells were irradiated with a .gamma. ray
source at doses of 300, 500, 1000, and 3000 cGy. Viability of NK-92
cells was determined by trypan blue staining. The maximal
achievable concentration of the non-irradiated NK-92 cells in
culture was about 1.5.times.10.sup.6/mL. The cells had to be fed to
prevent overgrowth.
[0023] FIG. 5. Effect of .gamma.-irradiation on the in vitro colony
formation of NK-92 cells. NK-92 cells were cultured in agar-based
medium supplemented with recombinant human IL-2 (rhIL-2).
[0024] FIG. 6. Effect of various radiation doses on the cytolytic
potential of NK-92 cells. NK-92 cells were .gamma.-irradiated at
doses of 300, 500, 1000, and 3000 cGy. .sup.51Cr-labeled leukemic
target cells K562 (Panel A) and HL60 (Panel B) as well as two
patient-derived leukemic samples TA27 (Panel C) and BA25 (Panel D)
were tested for susceptibility to cytolysis by irradiated and
non-irradiated (NR) NK-92 cells. The results of 4 hr chromium
release assays are expressed as 30% lytic units/10.sup.8 effector
cells.
[0025] FIG. 7. Selective killing of patient-derived leukemic cells
by NK-92 cells. .sup.51Cr-labeled leukemic target cells derived
from 40 patients [9 acute myeloid leukemia (AML) cases, 11 chronic
myeloid leukemia (CML) cases, 14 B-lineage-acute lymphoblastic
leukemia (ALL) cases and 6 T-ALL cases] and T cell depleted normal
bone marrow cells from 14 normal donors were tested for
susceptibility to cytolysis by NK-92 cells at four different E:T
ratios. The results of a 4 hr chromium release assay are expressed
as 30% lytic units/10.sup.8 effector cells.
[0026] FIG. 8. In vitro (Panels A and B) and in vivo (Panels C and
D) antileukemic efficacy of NK-92 cells against K562 and HL60
leukemias as compared to human LAK cells and other effectors.
.sup.51Cr labeled K562 (Panel A) and HL60 (Panel B) cells were
tested for susceptibility to cytolysis by NK-92 cells in comparison
with various known effector cells [LAK, NK (CD3.sup.- CD56.sup.+),
and T cells (CD3+CD56.sup.-)] at indicated E:T ratios in a 4 hr CRA
assay. Results are means.+-.SD of three separate tests for NK-92
cells, and two tests of different donor-derived effectors for LAK,
CD56.sup.+ and CD3.sup.+ cells. SCID mice were inoculated
subcutaneously with K562 cells (Panel C) or HL60 cells (Panel D)
(5.times.10.sup.6 cells per mouse) alone or in combination with
NK-92, LAK, or NK cells at a 4:1 E:T ratio. As a measure of the
tumor sizes, their surface areas were measured once a week post
inoculation (n=5).
[0027] FIG. 9. Antileukemic effect of NK-92 cells, allogeneic
cytotoxic T lymphocyte (CTL) cells and other effector cells against
a patient-derived acute T lymphoblastic leukemia (T-ALL) determined
in vitro and in vivo. Panel A: In vitro specific killing of T-ALL
(TA27) target cells by NK-92, CTL, and other effector cells, was
determined by a 4 hr .sup.51Cr-release assay using the indicated
E:T ratios. Results are means.+-.SD of two or three separate tests.
Panel B: SCID mice were inoculated subcutaneously with TA27 cells
(5.times.10.sup.6 each mouse) alone or co-inoculated with NK-92,
CTL or other effector cells at a 4:1 E:T ratio. Recombinant human
IL-2 (rhIL-2) was administered to the mice intraperitoneally for
two weeks at the dose of 5.times.10.sup.4 U every other day.
Leukemic tumor areas were measured once a week post inoculation
(n=5).
[0028] FIG. 10. Survival of SCID mice bearing T-ALL (TA27) leukemia
co-inoculated with NK-92 cells as compared with co-inoculation with
allogenic CTL or irradiated TALL-104 cells.
[0029] FIG. 11. Survival of SCID mice bearing T-ALL (TA27) after
treatment with NK-92 cells. Mice received 5.times.10.sup.6 TA27
cells intraperitoneally (I.P.). NK-92 cells (2.times.10.sup.7) were
injected I.P. once, or 5 times (on days 1, 3, 5, 7 and 9), with or
without the addition of rhIL-2 every other day for two weeks.
[0030] FIG. 12. Survival of SCID mice bearing pre-B-ALL (BA31)
after treatment with NK-92 cells. Mice received 5.times.10.sup.6
BA31 cells I.P. NK-92 (2.times.10.sup.7) cells were injected I.P.
for a total of 5 doses, on days 1, 3, 5, 7 and 9. Mice in the
indicated groups received rhIL-2 every other day for two weeks.
[0031] FIG. 13. Survival of SCID mice bearing human AML (MA26)
after treatment with NK-92 cells. Mice received 5.times.10.sup.6
MA26 leukemia cells I.P. NK-92 (2.times.10.sup.7) cells were
injected I.P. on days 1, 3, 5, 7 and 9 for a total of five doses.
Mice in the indicated groups received rhIL-2 every other day for
two weeks.
[0032] FIG. 14. Diagrammatic map of plasmid MFG-hIL-2.
[0033] FIG. 15. Diagrammatic map of plasmid pCEP4-LTR.hIL-2.
[0034] FIG. 16. PCR analysis of NK-92, NK-92MI and NK-92CI for
human IL-2 cDNA. DNA isolated from the parental NK-92 and from the
NK-92MI and NK-92CI transfectants was subjected to PCR analysis
with primers flanking the first exon of the human IL-2 gene. PCR
products were resolved on a 2% agarose gel, stained with ethidium
bromide and viewed on a UV Transilluminator (Panel A). DNA was
transferred to a nylon membrane and analyzed by Southern blot
analysis with a radiolabelled probe for the hIL-2 gene (Panel
B).
[0035] FIG. 17. Northern blot analysis of cytokine expression in
NK-92. NK-92MI and NK-92CI. RNA samples isolated from the parental
and transfected cell lines were separated by agarose gel
electrophoresis blotted to nylon membrane by capillary transfer and
hybridized with probes for human IL-2 in (Panel A) and TNF-.alpha.
(Panel B).
[0036] FIG. 18. Cytotoxicity of NK-92, NK-92MI and NK-92CI against
K562 and Raji target cells. The cytotoxic activities of the IL-2
transfectants were compared to that of the parental cell line. NK
cells were mixed with .sup.51Cr-labeled K562 (Panel A) or Raji
(Panel B) cells at effector:target ratios of 1:1, 5:1, 10:1 and
20:1 for a 4 hour chromium release assay. The cytotoxicities of
NK-92 ( ), NK-92MI (.tangle-solidup.) and NK-92CI (.box-solid.) are
shown.
[0037] FIG. 19. Effect of NK-92 MI and NK-92CI on hematopoietic
progenitors. To assay the effect of the NK-92 cells on normal
hematopoietic progenitors, a clonogenic assay was performed. Normal
PBMCs were incubated with irradiated NK-92MI or NK-92CI at various
NK:PBMC ratios ranging from 1:1 to 1:1000 for 48 hours. The cells
were plated in methylcellulose at concentrations to give 10-100
colonies per dish after 14 days. Clonogenic output of PBMCs
incubated with NK-92MI (white bars) and NK-92CI (gray bars) is
expressed as either total number of colonies or subclassified on
the basis of colony type (BFU-E, CFU-GM and CFU-GEMM).
[0038] FIG. 20. Effect of irradiation on NK-92, NK-92MI and NK-92CI
proliferation and viability. To assess the effect of irradiation on
the parental and transfected NK-92 cells, cells were exposed to 0,
500, 1,000, 1,500, and 2,000 cGy doses of radiation and assayed for
proliferation by a standard .sup.3H-thymidine incorporation assay.
Panel A: Proliferation of NK-92 ( ), NK-92MI (.tangle-solidup.) and
NK-92CI (.box-solid.) is expressed as a percentage of control
(unirradiated cells). Panel B: Cells were exposed to 0, 250, 500,
1,000, and 2,000 cGy of irradiation and assessed by trypan blue
exclusion for viability after 24 (black bars), 48 (gray bars) and
72 hours (white bars).
[0039] FIG. 21. Effect of irradiation on NK-92, NK-92MI and NK-92CI
cytotoxicity. To assess the effect of irradiation on cytotoxicity
of the NK cells, NK-92, NK-92MI and NK-92CI were irradiated at 0,
1,000, and 2,000 cGy and tested after three days for cytotoxicity
against K562 (Panel A) and Raji (Panel B) cells.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The present invention relates to methods of treating a
biological sample or a mammal suspected of having a pathology such
as a cancer or an infection by a virus. Certain natural killer
cells which are cytolytic for the cells affected by the pathology
are employed. The treatment results in significant diminution of
the number, or, in some cases, the elimination, of malignant or
cancerous cells, or virus-infected cells, in the sample or mammal.
The natural killer cells of this invention are designated NK-92
cells and include certain treated or transfected modifications of
NK-92 cells. These cells are highly effective in purging cancer
cells ex vivo and in destroying cancer cells in vivo.
[0041] As used in the present invention, "cytotoxic T lymphocytes"
(CTL) relate to immune cells which kill antigen-specific target
cells. CTL are MHC class I-restricted. As used in the present
invention, lymphokine activated killer (LAK) cells relate to cells
of the immune system that have antitumor killing activity. They are
obtained from a population of cells, such as peripheral blood
mononuclear cells, upon activation by treatment with lymphokines.
LAK cells have essentially no effect on normal cells.
[0042] As used to describe the present invention, "natural killer
(NK) cells" are cells of the immune system that kill target cells
in the absence of a specific antigenic stimulus, and without
restriction according to MHC class. Target cells may be tumor cells
or cells harboring viruses. NK cells are characterized by the
presence of CD56 and the absence of CD3 surface markers. The
present invention is based on an immortal NK cell line, NK-92,
originally obtained from a patient having non-Hodgkin's lymphoma.
As used to describe the present invention, a modified NK-92 cell is
an NK-92 cell which has been further treated to endow it with
properties not found in the parent from which it is derived. Such
treatments include, for example, physical treatments, chemical
and/or biological treatments, and the like. The treatments confer
properties upon the modified NK-92 cells that render them more
advantageous for the purposes of the invention.
[0043] As used to describe the present invention, the terms
"cytotoxic" and "cytolytic", when used to describe the activity of
effector cells such as NK cells, are intended to be synonymous. In
general, cytotoxic activity relates to killing of target cells by
any of a variety of biological, biochemical, or biophysical
mechanisms. Cytolysis refers more specifically to activity in which
the effector lyses the plasma membrane of the target cell, thereby
destroying its physical integrity. This results in the killing of
the target cell. Without wishing to be bound by theory, it is
believed that the cytotoxic effect of NK cells is due to
cytolysis.
[0044] As used to describe the present invention, "target cells"
are the cells that are killed by the cytotoxic activity of the NK
cells of the invention. These include in particular cells that are
malignant or otherwise derived from a cancer, and cells that are
infected by pathogenic viruses such as HIV, EBV, CMV, or
herpes.
[0045] As used to describe the present invention, "purging" relates
to killing of target cells by effector cells such as NK cells ex
vivo. The target cells may be included in a biological sample
obtained from a mammal believed to be suffering from a pathology
related to the presence of the target cell in the sample. The
pathology may be a cancer or malignancy due to tumor cells in the
sample, and may be treated by purging the sample of the tumor cells
and returning the sample to the body of the mammal.
[0046] As used to describe the present invention, "inactivation" of
the NK-92 cells renders them incapable of growth and/or their
normal function, in particular, their cytotoxic activity.
Inactivation may also relate to the death of the NK-92 cells. It is
envisioned that the NK-92 cells may be inactivated after they have
effectively purged an ex vivo sample of cells related to a
pathology in a therapeutic application, or after they have resided
within the body of a mammal a sufficient period of time to
effectively kill many or all target cells residing within the body.
Inactivation may be induced, by way of nonlimiting example, by
administering an inactivating agent to which the NK-92 cells are
sensitive.
[0047] As used herein, a "vector" relates to a nucleic acid which
functions to incorporate a particular nucleic acid segment, such as
a sequence encoding a particular gene, into a cell. In most cases,
the cell does not naturally contain the gene, so that the
particular gene being incorporated is a heterologous gene. A vector
may include additional functional elements that direct and/or
regulate transcription of the inserted gene or fragment. The
regulatory sequence is operably positioned with respect to the
protein-encoding sequence such that, when the vector is introduced
into a suitable host cell and the regulatory sequence exerts its
effect, the protein is expressed. Regulatory sequences may include,
by way of non-limiting example, a promoter, regions upstream or
downstream of the promoter such as enhancers that may regulate the
transcriptional activity of the promoter, and an origin of
replication. A vector may additionally include appropriate
restriction sites, antibiotic resistance or other markers for
selection of vector containing cells, RNA splice junctions, a
transcription termination region, and so forth.
[0048] As used to describe the present invention, "cancer",
"tumor", and "malignancy" all relate equivalently to a hyperplasia
of a tissue or organ. If the tissue is a part of the lymphatic or
immune system, malignant cells may include non-solid tumors of
circulating cells. Malignancies of other tissues or organs may
produce solid tumors. In general, the methods of the present
invention may be used in the treatment of lymphatic cells,
circulating immune cells, and solid tumors.
[0049] As used to describe the present invention, a "pathogenic
virus" is a virus causing disease in a host. The pathogenic virus
infects cells of the host animal and the consequence of such
infection is a deterioration in the health of the host. Pathogenic
viruses envisioned by the present invention include, but are not
limited to, HIV, EBV, CMV, and herpes.
[0050] Natural Killer Cell NK-92.
[0051] The NK-92 cell line has been described by Gong et al.
(1994). It is found to exhibit the CD56.sup.bright, CD2, CD7,
CD11a, CD28, CD45, and CD54 surface markers. It furthermore does
not display the CD1, CD3, CD4, CD5, CD8, CD10, CD14, CD16, CD19,
CD20, CD23, and CD34 markers. Growth of NK-92 cells in culture is
dependent upon the presence of recombinant interleukin 2 (rIL-2),
with a dose as low as 10 IU/mL being sufficient to maintain
proliferation. IL-7 and IL-12 do not support long-term growth, nor
do other cytokines tested, including IL-1.alpha., IL-6, tumor
necrosis factor .alpha., interferon .alpha., and interferon
.gamma.. NK-92 is highly effective in killing certain tumor cells,
such as K562 (erythroleukemia) and Daudi (Burkitt lymphoma) cells,
for it has high cytotoxicity even at a low effector:target (E:T)
ratio of 1:1 (Gong et al. (1994)). In addition, NK-92 cells have
high cytotoxic activity against 8E5 cells, which are infected with
HIV and produce HIV virions. NK-92 cells are deposited with the
American Type Culture Collection, designation CRL-2407.
[0052] NK-92 cells are readily maintained in culture medium, such
as enriched alpha minimum essential medium (MEM; Sigma Chemical
Co., St. Louis, Mo.) supplemented with fetal calf serum (for
example, at 12.5%; Sigma Chemical Co., St. Louis, Mo.), and horse
serum (for example, at 12.5%; Sigma Chemical Co., St. Louis, Mo.).
Initially, 10.sup.-6 M hydrocortisone is required, but in
subsequent passages it is found that hydrocortisone may be omitted.
In addition, IL-2, such as recombinant human IL-2 (500 U/mL;
Chiron, Emeryville, Calif.), is required for long-term growth. When
suspension cultures are maintained in this fashion with semiweekly
changes of medium, the cells exhibit a doubling time of about 24
h.
[0053] NK-92 cells in vitro demonstrate lytic activity against a
broad range of malignant target cells. These include cell lines
derived from circulating target cells such as acute and chronic
lymphoblastic and myelogenous leukemia, lymphoma, myeloma,
melanoma, as well as cells from solid tumors such as prostate
cancer, neuroblastoma, and breast cancer cell lines. This effect is
observed even at very low effector:target ratios. This lysis is
superior to cytotoxicity obtained from normal peripheral blood
mononuclear cells stimulated for four days with IL-2.
[0054] Vector for Transfecting Mammalian Cells to Produce
Cytokine.
[0055] The present invention provides NK-92 cells which have been
modified by transfection with a vector that directs the secretion
of a cytokine, such as IL-2. In order that NK-92 cells maintain
long-term growth and cytolytic function, they generally must be
supplied with IL-2. A vector encoding the gene for human IL-2, and
which also contains a control element directing the synthesis of
the IL-2 gene product is therefore of great utility in the
invention. NK-92 cells bearing such a vector secrete the IL-2
needed for cytolytic activity in a therapeutic setting; thus, IL-2
from an exogenous source is not required. The control element is
one which directs the synthesis of IL-2 as a constitutive product,
i.e., one that is not dependent upon induction. Methods for
constructing and employing vectors are described in general terms
in "Current Protocols in Molecular Biology", Ausubel et al., John
Wiley and Sons, New York (1987, updated quarterly), and "Molecular
Cloning: A Laboratory Manual 2nd Ed.", Sambrook, Fritsch and
Maniatis, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
(1989), which are incorporated herein by reference.
[0056] Modified NK-92 Transfected to Produce Cytokine and Method of
Transfecting.
[0057] Modified NK-92 cells that secrete a cytokine may be prepared
by inserting a vector that directs the synthesis and secretion of
the cytokine into the cells. In important aspects of the invention,
the cytokine is IL-2. Methods of introducing a vector into a
mammalian cell are well known to workers of ordinary skill in
molecular biology and cellular immunology, and are described in
Ausubel et al. (1987, updated quarterly) and Sambrook et al.
(1989). The vectors encoding the cytokine encompass as well control
elements that lead to constitutive synthesis of the cytokine when
incorporated into the NK-92 cells.
[0058] When cultured under appropriate conditions that promote
cytokine secretion the transfected NK-92 cells secrete IL-2 or
other cytokine. Since the vector directs constitutive synthesis of
the cytokine, nutrient cultures in which the NK-92 cells are known
to grow and to exhibit their normal cytolytic function are
sufficient for the transfected cells to secrete the cytokine. For
the same reason, the transfected cells secrete the cytokine in vivo
when they are introduced within the body of a mammal.
[0059] NK-92 cells transfected with a vector that directs secretion
of a cytokine such as IL-2 are useful in the ex vivo treatment of a
biological sample drawn from a mammal which is suspected of
containing malignant cells. By treating the malignant cells with
these modified NK-92 cells, the need for applying exogenous IL-2 or
other cytokine is obviated. These modified NK-92 cells are useful,
for the same reasons, in the in vivo treatment of a mammal
suffering from a malignancy. The modified NK-92 cells exert their
cytolytic effect against the malignant cells when introduced into
the body of the mammal. Examples of such cells in the present
invention are designated NK-92MI and NK-92CI.
[0060] NK-92 Cells that are Cytolytic but not Capable of
Proliferation.
[0061] An additional modified NK-92 cell of the invention is one
that has been treated in such away that it is no longer able to
proliferate, yet whose cytotoxic activity is preserved. One way of
achieving this state is by .gamma. irradiation. Additional forms of
radiation, including, for example, ultraviolet radiation, may be
employed. Suitable sources to use for this purpose include, for
example, a .sup.137Cs source (Cis-US, Bedford, Mass.; Gammacell 40,
Atomic Energy of Canada Ltd., Canada). Additionally, proliferative
activity may be abrogated by treatment with chemical agents which
inhibit DNA synthesis. An example of such an agent is mitomycin
C.
[0062] Vector for transfecting NK-92 with an element responsive to
an inactivating agent. The NK-92 cells may also be modified by
transfection with a vector such that, when the cell takes up a
specific agent, the cell is inactivated. The vector includes a
sequence that encodes a cellular component responsive to the agent,
such that when the vector transfects a cell and the agent is taken
up by the cell, the cell is inactivated. In preferred embodiments,
the agent is acyclovir or gancyclovir. The vector also contains a
control element directing the synthesis of the cellular component
as a constitutive product.
[0063] The NK-92 cell transfected with the vector described in the
preceding paragraph maintains its characteristic growth and
cytolytic activity in the absence of the agent. At a point in time,
for example, when an ex vivo sample has been purged of malignant
cells by the action of the NK-92 cells, or when the NK-92 cells
administered in vivo have effectively exerted their cytolytic
activity within a mammalian body, or when it desired that the
treatment be stopped for any reason, the agent may be administered.
The agent interacts with the cellular component sensitive to the
agent encoded in the vector. The interaction of the agent with the
cellular component induces the inactivation of the NK-92 cells.
Inactivation may range from loss of characteristic cytolytic
function to death of the cells.
[0064] This property of the modified NK-92 cells is significant
because the parent NK-92 cells are derived from a tumor cell line
that may continue propagating in a sample reintroduced into a
mammal after ex vivo therapy, or in vivo when so administered. It
is therefore important to ablate the cells after they have carried
out their therapeutic function. Rendering the cells sensitive to an
agent, such as acyclovir or gancyclovir, is an advantageous way of
achieving this objective.
[0065] Vector for Transfecting NK-92 with an Altered HLA Cell
Surface Molecule.
[0066] The HLA cell surface protein, involved in presenting
antigens to other cells of the immune system, includes a
non-immunospecific subunit, the protein .beta..sub.2-microglobulin.
If this protein is altered or mutated, the HLA protein loses its
affinity for the T-cell receptor to which it ordinarily binds. The
.beta..sub.2-microglobulin gene in NK-92 cells of the invention may
be mutated by site specific mutagenesis in order to transform its
properties in this way. The result is an NK-92 cell which no longer
has a high affinity for T-cell receptors. As a result, the NK-92
cell modified in this way remains within the host organism for a
longer period of time, rather than being eliminated by the action
of the host's cellular immune response.
[0067] Vector for Transfecting NK-92 with a Gene Encoding a Cancer
Cell Receptor Molecule.
[0068] The NK-92 cells may also be modified by transfection with a
vector such that the cells constitutively express a receptor for a
cancer cell. Cancer cells express cell surface molecules that are
idiosyncratic for the origin of the cancer, and frequently are also
idiosyncratic for the individual host. The CTL population in such
diseased patients may have been activated by exposure to the cells
of the growing cancer. Such activated CTL express cell surface
proteins that are specific for, or target, the cells of the cancer.
These CTL may be isolated, the gene for the targeting receptor
identified, isolated, and transfected into the NK-92 cells of the
invention. This confers on the NK-92 cells the capability of
likewise specifically targeting the cancer cells present in the
individual host. This has the effect of enhancing the specificity
of the cytotoxic activity of the NK-92 cells toward the cancer
cells of that individual. The corresponding process wold be carried
out for each host suffering from cancer, taking advantage of the
idiosyncratic specificity of the CTL targeting moiety in each
case.
[0069] Methods of Treating.
[0070] The natural killer cells of the invention are employed in
methods of treating biological samples in order to purge them of
cells from a cancer, a malignancy, or a tumor, or cells infected by
a pathogenic virus. The NK cells include by way of nonlimiting
example, NK-92, and modified NK-92 cells, such as NK-92MI and
NK-92CI, as well as other modified NK-92 cells envisioned within
the scope of this invention. The NK-92MI and NK-92CI cells are
modified by transfection with vectors that result in the secretion
of IL-2. In addition, any of the NK-92, NK-92MI, and NK-92CI cells
may be treated such that they maintain the cytolytic activity of
the untreated cells but cannot proliferate. The NK cells so treated
may also be equivalent cell lines which have the properties such as
cytotoxicity and NK-specific cell surface markers described herein.
Malignancies of the immune system, the lymphatic system, and the
hematopoietic system may be treated by the methods of the
invention. In addition, formed tumors and solid tumors may also be
treated. Infections by pathogenic viruses, such as HIV, EBV, CMV,
and herpes may also be treated.
[0071] Treating a Biological Sample.
[0072] In vitro biological samples may be treated experimentally or
therapeutically in order to eliminate malignant cells, or
virus-infected cells, in an effective manner. The sample may be
drawn from a mammal and maintained in vitro in an appropriate
culture medium. Such media are well known to workers of skill in
cell biology, cellular immunology, and oncology. Media and cell
culture techniques are presented in general terms in, for example,
Freshney, R. I., "Culture of Animal Cells, 3rd Ed.", Wiley-Liss,
New York (1994), and in Martin, B. M., "Tissue Culture Techniques,
An Introduction", Birkhauser, Boston, Mass. (1994), which are
incorporated herein by reference. The biological sample is
established in culture in vitro, and contacted with a medium that
includes the natural killer cells of the present invention. The
cytolytic activity of the NK cells effectively eliminates the
malignant cells or the virus-infected cells from the sample. The
prevalence and depletion of the target cells may be traced by any
of a number of methods well known to those of skill in the fields
of cell biology and cellular immunology. These include indirect
immunofluorescence microscopy to assay for intact tumor cells or
virus-bearing cells, fluorescent-activated cell sorting, chromium
release assays, and the like.
[0073] Treating a Cancer or Virus Infection Ex Vivo: Purging.
[0074] The present invention additionally encompasses the ex vivo
treatment of a biological sample suspected of containing cancer
cells or virus-infected cells by contacting the sample with the NK
cells of the invention. The biological sample is drawn from the
body of a mammal, such as a human, and may be blood, bone marrow
cells, or similar tissues or cells from an organ afflicted with a
cancer. Methods for obtaining such samples are well known to
workers in the fields of cellular immunology, oncology, and
surgery. They include sampling blood in well known ways, or
obtaining biopsies from the bone marrow or other tissue or organ.
The cancer cells or virus-infected cells contained in the sample
are effectively eliminated due to the cytotoxic activity of the
NK-92 cells. The sample may then be returned to the body of the
mammal from which it was obtained.
[0075] The NK-92 cells used to treat the sample may be freely
suspended in the medium. It is generally preferred that the purged
sample, prior to being returned to the body of the mammal from
which it was obtained, be rid of NK-92 cells that may continue
growing, since they arose originally from a proliferating lymphoma.
The invention envisions several ways of accomplishing this
objective. In one embodiment, the NK cells, prior to use, are
irradiated with .gamma. rays or with ultraviolet light to the
extent that they maintain their cytolytic activity but are not
capable of growth. In an additional embodiment, the NK cells are
permanently immobilized on a macroscopic solid support. The support
with the NK cells attached may then be physically separated from
the cells of the biological sample, for example by centrifugation,
or filtration with a column which permits the unbound cells of the
sample to pass through, or like technique. Suitable solid supports
include particles of polyacrylamide, agarose, cellulose,
Sepharose.TM. (Pharmacia, Piscataway, N.J.), celite, and the like,
and may be supplied with groups such as an aldehyde,
carbonyldiimidazole, broamoacetyl, epichlorhydrin, and the like,
which are activated for reaction with cell surface groups. The
activated groups on the support react with groups such as amino or
carboxyl groups, for example, on the cell surface, thereby
immobilizing the cells on the support.
[0076] In yet a further embodiment, the NK cells may be modified
with a vector directing the synthesis of a cellular component
sensitive to an agent, such that when the agent is administered to
the ex vivo sample, the NK-92 cells are inactivated. Examples of
such agents include acyclovir or gancyclovir, by way of nonlimiting
example. Functionally equivalent vectors, directing the synthesis
of alternative cellular components sensitive to different agents,
are also envisioned within the scope of this embodiment.
[0077] The NK cells to be used in the methods of the invention may
require a cytokine such as IL-2 to maintain their functional
effectiveness as cytolytic cells. The cytokine may simply be added
to the ex vivo preparation. Alternatively, if desired, a modified
NK-92 cell bearing a vector directing the constitutive synthesis of
the cytokine may be employed. In this way the necessity of
furnishing exogenous cytokine is avoided.
[0078] Treating a Cancer or Virus Infection In Vivo: Administering
NK-92.
[0079] A further method of the invention is directed toward
treatment of a cancer or a virus infection in vivo in a mammal
using NK-92 cells. The cells are administered in a variety of ways.
By way of nonlimiting example, the cells may be delivered
intravenously, or into a body cavity adjacent to the location of a
solid tumor, such as the intraperitoneal cavity, or injected
directly within or adjacent to a solid tumor. Intravenous
administration, for example, is advantageous in the treatment of
leukemias, lymphomas, and comparable malignancies of the lymphatic
system, as well as in the treatment of viral infections.
[0080] As has been described in detail in the preceding section, it
is desirable to employ methods that eliminate or ablate the NK-92
cells after they have effectively lysed (or otherwise destroyed)
the target cells. Certain methods described above may be employed
for this purpose, namely, use of irradiated NK-92 cells, and use of
NK-92 cells harboring a vector directing the synthesis of a
cellular component sensitive to an agent, such that when the agent
is administered, the NK-92 cells are inactivated, and equivalent
methods. When the cells produce such a component sensitive to the
specific agent, administration of the agent to the mammal is
effective to inactivate the NK-92 cells within the mammal.
[0081] The NK-92 cells may be administered in conjunction with a
cytokine such as IL-2 in order to maintain the functional
effectiveness of the cells as cytotoxic effectors. As used to
describe the invention, the term "in conjunction" indicates that
the cytokine may be administered shortly prior to administration of
the NK-92 cells, or it may be given simultaneously with the cells,
or shortly after the cells have been administered. The cytokine may
also be given at two such times, or at all three times with respect
to the time of administering the NK-92 cells. Alternatively, NK-92
cells harboring a vector directing the constitutive synthesis of
the cytokine may be employed in the in vivo method of treating a
cancer. This effectively eliminates the need to furnish exogenous
cytokine.
[0082] The following examples are included to illustrate the
invention and not to limit the invention. All publications or
references cited in the present specification are hereby
incorporated by reference. All deposits referred to in the present
specification are in the process of being submitted to ATCC.
EXAMPLES
Example 1
NK-92 Cells
[0083] NK-92 cells (Gong et al. (1994)) were derived from cells
obtained from a patient suffering from non-Hodgkin's lymphoma. PBMC
from the patient were cultured in enriched alpha MEM supplemented
with fetal calf serum (12.5%) and horse serum (12.5%) plus
10.sup.-6 M hydrocortisone and 1000 U/mL of recombinant human IL-2
(rhIL-2). Cells were cultured at 37.degree. C. in humidified air
containing 5% CO.sub.2. Subcultures were made after 4 weeks, and
propagated indefinitely with twice-weekly changes in medium. In
these later stages the hydrocortisone could be omitted without any
effect on cell growth. This culture has been designated NK-92 and
has been deposited with the American Type Culture Collection (ATCC;
Rockville, Md.) under designation CLR-2407.
[0084] The cells have the morphology of large granular lymphocytes.
The cells bear the CD56.sup.bright, CD2, CD7, CD11a, CD28, CD45,
and CD54 surface markers. In contrast, they do not display the CD1,
CD3, CD4, CD5, CD8, CD10, CD14, CD16, CD19, CD20, CD23, and CD34
markers. Growth of NK-92 cells in culture is dependent upon the
presence of recombinant interleukin 2 (IL-2), with a dose as low as
10 IU/mL being sufficient to maintain proliferation. IL-7 and IL-12
do not support long-term growth, nor do other cytokines tested,
IL-1.alpha., IL-6, tumor necrosis factor .alpha., interferon
.alpha., and interferon .gamma.
Example 2
Cytotoxic Activity of NK-92 against Different Leukemic Cell
Lines
[0085] The cytotoxic activity of NK-92 against K562, Daudi, TF-1,
AML-193, and SR-91 cells was determined (Gong et al. (1994)). K562
(erythroleukemia) and Daudi (Burkitt) lymphoma cell lines were
obtained from ATCC. They were maintained in continuous suspension
culture in RPMI 1640 medium supplemented with 10% fetal calf serum
(FCS). TF-1 is a myelomonocytic cell line (Kitamura et al., J. Cell
Physiol. 140:323-334 (1989)) that requires the presence of medium
containing 2 ng/mL of human GM-CSF. AML-193 is a myeloid cell line
that is maintained in the presence of 10% 5637-conditioned medium
(Lange et al., Blood 70:192-199 (1987)). Both TF-1 and AML-193
cells were obtained from Dr. D. Hogge, Terry Fox Laboratory,
University of British Columbia, Vancouver, BC. SR-91 is a cell line
with features of early progenitor cells established by Gong et al.
(1994) from a patient with acute lymphoblastic leukemia (ALL)
(Klingemann et al., Leuk. Lymphoma, 12, 463-470 (1994). It is
resistant to both NK and activated-NK (A-NK) cell cytotoxicity.
SR-91 is also maintained in RPMI 1640/10% FCS. This cell line can
be rendered sensitive to killing by NK-92 by treatment with
cytokine. Naki et al., "Induction of sensitivity to the NK-mediated
cytotoxicity by TNF-.alpha. treatment: Possible role of ICAH-3 and
CD44," Leukemia, in press.
[0086] The cytotoxic activity of NK-92 (effector) against these
target cells was assessed by means of a .sup.51Cr release assay
(Gong et al. (1994)) using the procedure described by Klingemann et
al. (Cancer Immunol. Immunother. 33:395-397 (1991)). The percentage
of specific cytotoxicity in triplicate specimens was calculated
as:
% 51 Cr release = ( average experimental cpm - average spontaneous
cpm ) ( average maximum cpm - average spontaneous cpm ) .times.
100. ##EQU00001##
FIG. 1 presents the results of this determination. It is seen that
NK-92 cells kill K562 and Daudi cells with high efficiency. Even at
the low E:T ratio of 1:1, 83% of K562 cells and 76% of Daudi cells
were killed by NK-92 cells. Susceptibility to killing by NK-92
cells was lower for TF-1 cells (23% at E:T=1:1) and for AML-193
cells (6% at E:T=1:1). SR-91 cells appear to be resistant to the
cytotoxic effect of the NK-92 cells. Without wishing to be bound by
theory, it is believed that SR-91 cells lack adhesion molecules
necessary to mediate initial binding with NK-92 cells.
Example 3
Cytotoxicity of NK-92 Against Leukemia, Lymphoma, and Myeloma
Target Cell Lines
[0087] K562 (Ph-chromosome positive [Ph.sup.+] erythroleukemia),
HL60 (promyelocytic), U937 (myelomonocytic), KG1a (variant subline
of the AML cell line KG1), DHL-10 (B-cell lymphoma), Daudi
(Burkitt's lymphoma), Raji (B-cell lymphoma), Jurkat (T-cell
lymphoma), U266 (IgE myeloma), NCI H929 (IgA myeloma), and RPMI
8226 (myeloma, light chain secreting) cell lines were obtained from
ATCC. The lymphoma-derived cell lines Ly3 (B-lineage, diffuse large
cell), Ly8 (immunoblastic), and Ly13.2 (T-lineage, diffuse large
cell) were provided by Dr. H. Messner, Toronto, Ontario. Their
characteristics have been described (Chang et al., Leuk. Lymphoma
19:165 (1995)). All lines were maintained in RPM1 1640 medium
supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM
nonessential amino acids, 50 U/mL penicillin, 25 mM HEPES (StemCell
Technologies), and 5% heat-inactive FCS(RPMI/5% FCS) at 37.degree.
C. in a humidified atmosphere of 5% CO.sub.2 in air.
[0088] Cell lysis was determined by a 4-hour .sup.51Cr release
assay using various E:T ratios. To allow for comparison, PBMCs from
normal donors were activated with IL-2 (500 U/mL) for 4 days and
.sup.51Cr release measured against the same target cells
concurrently. The mean of two separate experiments is
presented.
[0089] Results of NK-92-mediated cytotoxicity (.sup.51Cr release
assay) against various leukemia, lymphoma, and myeloma target cell
lines are summarized in Table 1. For comparison, lysis of the same
tumor target cells was also tested in the same experiment with
PBMCs obtained from normal donors. Those cells had been activated
by IL-2 (500 U/mL) for 4 days prior to testing. Results show that
NK-92 cells very effectively lyse all target cells tested. High
cytotoxicity is observed even at the low E:T ratio of 1:1. The
cytotoxicity achieved with these cells is significantly higher than
that observed with normal (allogeneic) PBMCs activated under
optimal conditions with IL-2 for all the target cells except RPMI
8226 and U266.
TABLE-US-00001 TABLE 1 Cytotoxic activity of NK-92 cells against
various leukemia, lymphoma, and myeloma cell lines Target 50:1 20:1
10:1 5:1 1:1 HL-60 NK-92 97 90 77 46 40 PBMCs + IL-2 31 26 17 2 0
K562 NK-92 68 68 64 59 50 PBMCs + IL-2 63 73 67 51 19 KG1a NK-92 90
91 80 67 39 PBMCs + IL-2 15 11 12 6 0 U937 NK-92 99 98 96 91 85
PBMCs + IL-2 57 43 23 13 2 DHL-10 NK-92 95 95 92 94 80 PBMCs + IL-2
60 40 24 19 5 Daudi NK-92 94 87 71 48 39 PBMCs + IL-2 65 57 29 16 6
Jurkat NK-92 100 100 98 93 80 PBMCs + IL-2 67 50 36 27 4 Ly 3 NK-92
63 59 53 42 28 PBMCs + IL-2 47 35 18 6 095 Ly 8 NK-92 95 104 102 88
42 PBMCs + IL-2 67 65 62 59 44 Ly 13.2 NK-92 104 105 100 97 67
PBMCs + IL-2 61 63 52 4 13 Raji NK-92 81 75 74 70 54 PBMCs + IL-2
32 67 57 35 13 NCI H929 NK-92 94 89 89 86 51 PBMCs + IL-2 75 58 39
24 5 RPMI NK-92 82 72 70 72 41 8224 PBMCs + IL-2 95 83 81 67 25
U266 NK-92 84 77 85 81 53 PBMCs + IL-2 84 74 73 56 21
Example 4
Effect of Deprivation of IL-2 on Cytotoxic Activity of NK-92
[0090] To test how long NK-92 cells would maintain their cytolytic
activity without IL-2 present in the culture medium. NK-92 cells
were deprived of IL-2 and .sup.51Cr-release was measured in 24-hour
intervals. Results, summarized in FIG. 2, suggest that the cells
maintain full cytotoxic activity for at least 48 hours. Thereafter,
the activity drops precipitously to negligible levels. Thus, for
short-term purging, IL-2 does not have to be present in the
cultures to achieve suitable effect.
Example 5
Co-Culture of K562-neo.sup.r Cells with PBMC's and NK-92
[0091] The transfection of the K562 cells with the
neomycin-resistance (neo.sup.r) gene has been described (Wong et
al., Bone Marrow Transplant 18:63 (1966)). Briefly,
5.times.10.sup.7 K562 cells were suspended in 0.8 mL RPMI 1640/5%
FCS and incubated on ice for 10 minutes with 30 .mu.g of the
pMC1-Neoplasmid (provided by Dr. K. Humphries, Terry Fox
Laboratory, Vancouver, BC). The cells were then exposed too single
voltage pulse (125 .mu.F/0.4 kV) at room temperature, allowed to
remain in buffer for 10 minutes, transferred into 25-cm.sup.2
tissue culture flasks (Falcon, Lincoln Park, N.J.), and incubated
at 37.degree. C. in a humidified atmosphere of 5% CO.sub.2 in air
for 2 days. Transfected cells, were selected in 0.8% Iscove's
methylcellulose medium (StemCell Technologies) supplemented with
30% FCS, 10.sup.-4 M 2-mercaptoethanol, and 2 mM glutamine,
containing 0.8 mg/mL G418 (neomycin) (Gibco-BRL, Grand Island,
N.Y.). Neo.sup.r clones of K562 cells were identified after 2
weeks, plucked, and maintained in RPMI/10% FCS containing 0.8 mg/mL
neomycin. K562-neo.sup.r cells cultured for 2 days showed a
neo.sup.r clonogenic cell doubling time of 36-42 hours.
[0092] Normal PBMCs (10.sup.4/mL) were spiked with 10%
K562-neo.sup.r cells, and NK-92 cells were added to yield different
effector:target (E:T) ratios of NK-92:K562-neo.sup.r cells. (Wong
et al. (1996)). Briefly, PBMCs were suspended in enriched alpha
medium (Myelocult.TM.) as described above. This medium has been
shown to provide optimal conditions for supporting both IL-2
activation of PBMCs and hematopoietic progenitor cell function
(Klingemann et al., Exp. Hematol. 21:1263 (1993)). The final
concentration of PBMCs in 35-mm tissue culture dishes (Corning,
East Brunswick, N.J.) was 1.times.10.sup.6/mL, and the proportion
of input K562-neo.sup.r cells was kept at 10% for all experiments.
Various numbers of irradiated (1000 cGy) NK-92 cells (see Examples
7 and 8) were added, resulting in various E:T ratios as specified
in Table 2. These mixtures were cultured in an atmosphere of 5%
CO.sub.2 in air for 4 or 48 hours at 37.degree. C. with and without
IL-2 (500 units/mL).
[0093] After the culture, cells were washed in RPMI/5% FCS,
10.sup.3 cells were to suspended in 0.8% Iscove's methylcellulose
containing 0.8 mg/mL neomycin, and 1.1-mL volumes were plated in
3-mm petri dishes. After 7 days at 37.degree. C. in a humidified
atmosphere of 5% CO.sub.2 in air, colonies were counted. The number
of neo.sup.r colonies provided a measure for the number of
surviving clonogenic K562-neo.sup.r cells present in the cell
suspension originally plated. Percent survival values for
co-cultures containing various numbers of NK-92 cells were
determined by comparing the number of clonogenic K562-neo.sup.r
cells present in test co-cultures with the number of those present
in control co-cultures (no NK-92 cells added) and harvesting after
the same period of incubation. At an input number of 10.sup.3
K562-neo.sup.r cells prior to purging, the absolute number of
clonogenic K562-neo.sup.r cells after 4 hours with no NK-92 cells
present was 6400.+-.820 cells and after 48 hours 28,300.+-.2100
cells. The mean.+-.SEM of four to eight experiments is
reported.
[0094] When only PBMCs were plated in the neomycin-containing
methylcellulose medium, no colonies were ever observed. To
quantitate the purging capacity of NK-92 cells, PBMCs were spiked
with 10% K562-neo.sup.r cells and cultured for 4 or 48 hours in
medium in the presence or absence of IL-2. Results, summarized in
Table 2, show that NK-92 cells used at E:T ratios of 10:1 and 5:1
eliminated the K562-neo.sup.r cells from PBMCs, and that very low
survival was observed at E:T of 1:1. The presence of IL-2 during
the purging did not result in any increase in the number of K562
cells purged compared to no IL-2 (Table 2).
TABLE-US-00002 TABLE 2 Purging effect of NK-92 cells % Survival
(-IL-2) % Survival (+IL-2) NK-92:K562-neo' of K562-neo' of
K562-neo' E:T Ratio 4 hrs. 48 hrs. 4 hrs. 48 hrs. 10:1 0 0 0 0 5:1
0 0 0 0 1:1 10.5 .+-. 2.1 15.4 .+-. 7.2 6.5 .+-. 3 15.2 .+-. 5.9
0.1:1.sup. 56 .+-. 14.1 68.5 .+-. 19.5 54.4 .+-. 13 69.6 .+-.
16.8
Example 6
Effect of NK-92 Cells on Hematopoietic Progenitor Cells
[0095] The effect of NK-92 cells on PBMCs was determined (Cashman
et al., Blood 75:96 (1990)). Briefly, normal PBMCs were co-cultured
with irradiated (1000 cGy) NK-92 cells (see Examples 7 and 8) for 2
days. Cells were then plated in replicate 1.1-mL aliquots of
methylcellulose-containing media at densities adjusted to give
approximately 10-100 large colonies of erythroid cells (from
burst-forming units-erythroid [BFU-E]), granulocytes and
macrophages (from colony-forming units-granulocyte/macrophage
[CFU-GM]), and combinations of all of these (from CFU
granulocyte/erythroid/macrophage/megakaryocyte [CFL-GEMM]).
Colonies were counted under an inverted microscope 2 weeks
later.
[0096] The number and growth kinetics of clonogenic hematopoietic
cells were quantified at a 1:1 ratio of NK-92:PBMC after 2 days of
co-culture with irradiated (1000 cGy) NK-92 cells. The cells were
plated in standard methylcellulose and counted 2 weeks later.
Results obtained from three different normal donors are presented
as percentage of normal controls in Table 3. No growth inhibitory
effect on hematopoietic progenitors by NK-92 cells was noted.
TABLE-US-00003 TABLE 3 Effect of NK-92 cells on colony formation of
normal hematopoietic progenitor cells. Experiment Number CFU-GEMM
BFU-E CFU-C 1 100 46 94 2 200 98 64 3 33 104 103
Example 7
.gamma.-Irradiation of NK-92 Cells
[0097] NK-92 cells were irradiated in T25 flasks (Corning, Newark,
N.J.) with the dose indicated using a cesium source (Cis-US,
Bedford, Mass.). A dose range of 200-2000 cGy was tested. After
irradiation, the cells were washed twice in RPMI, resuspended in
medium, and cultured for 72 hours at 37.degree. C. in the presence
of 500 IU/mL IL-2. Cytotoxicity (.sup.51Cr-release assay) was
performed with these cells as described above in Example 2. Prior
to performing the .sup.51Cr-release assay, the cells were left for
24 hours in medium supplemented with IL-2 to allow for recovery.
Proliferation was assessed by means of a .sup.3H-thymidine
incorporation assay. Prior to adding .sup.3H-thymidine (0.5
.mu.Ci/cell), NK-92 cells were resuspended in thymidine-free RPMI.
Uptake of .sup.3H-thymidine was measured in a liquid scintillation
counter 4 hours later. (Klingemann et al., Leuk. Lymphoma 12:463
(1994)). The counts per minute (cpm) from three different
experiments are presented.
[0098] Clinical use of this cell line to purge cancerous cells
requires that NK-92 cells not undergo significant growth and
proliferation. This was achieved by irradiating the cells.
Proliferation, as measured by .sup.3H incorporation, was
effectively reduced at a dose of 1000 cGy (Table 4). The
cytotoxicity of NK-92 cells after administration of various
radiation doses is presented in FIG. 3. At doses up to 1000 cGy an
essentially undiminished cytolytic response was maintained.
TABLE-US-00004 TABLE 4 Effect of irradiation on the proliferation
of NK-92 cells Experiment Radiation dose (cGy) Number 0 500 1000
1500 2000 1 5766 3071 406 125 114 2 4236 2411 1216 1192 562 3 3994
2046 824 689 748
Example 8
Radiation Susceptibility of NK-92 Cells
[0099] NK-92 cells were irradiated by a .gamma. ray source
(Gammacell 40, Atomic Energy of Canada. Ltd., Canada). A dose range
of 100-3000 cGy was tested. After irradiation, the cells were
washed and resuspended in culture medium with rhIL-2. Colony
assays, viability and cytotoxic activity of the irradiated NK-92
cells were performed using standard techniques (Yan et al.,
Leukemia, 7:131-139 (1993)). To quantify clonogenic NK-92 cells,
NK-92 cells (500 cells per mL culture medium) were cultured in a
0.3% agar-based medium supplemented with 12.5% FCS, 12.5% horse
serum, 2 mM 4-glutamine, 100 .mu.g/mL penicillin 50 .mu.g/mL
streptomycin, 10.sup.-5 M mercaptoethanol, and 500 U/mL rhIL-2 at
37.degree. C. for 14 days. An additional aliquot of 500 U/mL rhIL-2
was added at day 7 during the culture. Triplicate cultures were
performed for each data point.
[0100] The viability of NK-92 cells, determined by trypan blue
staining, and the recovery of the ability of the NK-92 cells to
generate colonies after exposure to various radiation doses is
shown in FIGS. 4 and 5, respectively. The NK-92 cells maintained
substantial survival for 3 or 4 days after exposure to high doses
of radiation (1000-3000 cGy). However, in vitro clonogenic NK-92
cells were significantly depleted after low doses of radiation and
totally eliminated by doses above 300 cGy. FIG. 6 shows the
cytotoxicity of NK-92 cells to K562, HL60 and 2 patient-derived
leukemic samples after exposure to the different doses of
radiation. Doses of 300, 500, and 1000 cGy allow for substantial
cytolysis against leukemic cell lines and primary leukemias 1-2
days after radiation.
[0101] These experiments suggest that NK-92 cells, irradiated to an
extent that renders them nonclonogenic, retain their cytolytic
activity against a wide spectrum of target cells. They therefore
may be used ex vivo in the purging of tumor cells as well as in the
treatment of various cancers in vivo.
Example 9
Cytolysis of Human Primary Leukemic Cells by NK-92
[0102] a. Patient-Derived Leukemic Samples.
[0103] Samples were obtained, with informed consent, during routine
diagnostic blood studies or bone marrow (BM) aspirates from
patients with newly diagnosed or relapsed leukemias. 9 acute
myeloid leukemia (AML) cases, 11 chronic myeloid leukemia (CML)
cases (6 chronic phase, 1 accelerated phase and 4 blast crisis), 14
B-lineage-acute lymphoblastic leukemia (ALL) cases (13 pre-B-ALLs
and 1 B-ALL) and 6 T-ALL cases, were studied (see Table 5).
Blast-enriched mononuclear cells were isolated by Ficoll Hypaque
(Pharmacia, Piscataway, N.J.) density gradient separation and
washed in RPMI 1640 medium.
[0104] b. Effector Cells.
[0105] NK-92 cells were cultured and maintained in .alpha.-MEM
medium supplemented with 12.5% FCS, 12.5% horse serum and rhIL-2
(500 U/mL Chiron, Emeryville, Calif.). TALL-104 cells (a
MHC-unrestricted human cytotoxic T cell clone, generously provided
by Drs. D. Santoli and A. Cesano, The Wistar Institute,
Philadelphia) were maintained in Iscove's modified Dulbecco's
medium supplemented with 10% FCS and rhIL-2 (100 U/mL) (Cesano et
al., Blood, 87:393-403 (1996)). Another human NK cell clone. YT,
was maintained in RPM, 1640 medium with 10% FCS and rhIL-2 (100
U/mL) (Yodoi et al., J. Immunol., 134:1623-1630 (1985)).
[0106] c. Cytotoxicity Assays.
[0107] The cytotoxic activity of non-irradiated NK-92 and
responding T cells against leukemic targets was measured in a
standard 4-hour chromium release assay (CRA). Some of the samples
were also measured in an 18-hour CRA. A fixed number of
.sup.51Cr-labeled target cells (5.times.10.sup.3/well) was tested
for susceptibility to 4 effector cell concentrations. .sup.51Cr
release of target cells alone (spontaneous release, determined by
placing target cells in 5% Triton) was always <25% of maximal
.sup.51Cr release. CRA data were expressed as specific lysis (%) at
a given effector:target (E:T) ratio or were converted to lytic
units (LU) defined as the number of effectors resulting in 30%
lysis of target cells (Cesano et al., Cancer Immunol. Immunother.,
40:139-151 (1995)). The degree of sensitivity of patient-derived
leukemic cell targets to each effector was defined as insensitive
(-/+: <10/10-19% lysis), sensitive (++/+++/++++:
20-29/30-39/40-49% lysis) and highly sensitive (+++++/++++++:
50-59/>60% lysis) at an E:T ratio of 9:1.
[0108] d. Results: Cytolysis of Human Primary Leukemic Cells by
NK-92 Cells.
[0109] The sensitivity of patient-derived leukemic cells to the
cytotoxic effect of NK-92 cells is summarized in column 4 of Table
5. Of the 40 patient-derived leukemic samples shown in Table 5, 26
(65%) were sensitive or highly sensitive to NK-92 mediated in vitro
cytotoxicity. Six of the samples that were insensitive to the NK-92
cells in the standard 4 hr CRA (sole or first entries), became
sensitive after 18 hours incubation (second entries, enclosed in
parentheses). Leukemia blasts derived from 6 out of 9 (67%) AML, 6
of 6 (100%) T-ALL and 6 of 14 (43%) B-lineage-ALL were either
sensitive or highly sensitive to the NK-92 mediated lysis. 7 of 8
acute leukemia samples which demonstrated high sensitivity to the
cytotoxic effect of NK-92 cells were derived from relapsed patients
and 1 was from a newly diagnosed patient. Out of 11 CML samples, 8
(73%) were sensitive (5 in chronic phase) or highly sensitive (2 in
blast crisis; 1 in accelerated phase) to the NK-92 mediated
cytolysis (Table 5).
[0110] In comparison, the last two columns in Table 5 present
results obtained with cell lines known in the field to have
cytolytic activity against tumor cells, namely, TALL-104 cells and
YT cells. Only 16 out of the 37 leukemic samples tested (43%) were
sensitive (4 AMLs, 5 B-lineage ALLs and 3 CMLs) or highly sensitive
(1 AML, 1 B-lineage-ALL and 2 CMLs) to the MHC unrestricted
cytotoxic T cell clone TALL-104 mediated cytolysis. Leukemias
sensitive to the TALL-104 cells were not consistently sensitive to
NK-92 cells, and cells that were lysed by NK-92 cells were not
always lysed by TALL-104 cells. In addition, the cytolytic activity
of TALL-104 cells was usually detected only after 18 hours of
incubation (second entries, enclosed in parentheses). Only four of
16 (25%) of the target samples that were lysed at 18 hours were
also lysed in the standard 4 hr CRA. The remaining 12 (75%)
responded only after the 18 hour incubation, with the response
being generally lower than that observed with the NK-92 cells of
the invention. Without wishing to be bound by theory, these
observations may be due to the possibility that 1) different target
structures are recognized by TALL-104 vs NK-92 cells, or 2) a
different pathway may be involved in the NK-92 and in the TALL-104
cell mediated cytolysis.
[0111] The majority of leukemic samples treated with YT cells, the
other NK-like clone tested, were found to be resistant, with the
exception of 2 samples (a CML in blast crisis and a T-ALL) (see
Table 5).
[0112] In conclusion, the NK-92 cells of the invention are
surprisingly and significantly more effective in lysing
patient-derived tumor cells, and exert their effect in a shorter
time, than do the cells from two cytolytic cell lines an known in
the field.
TABLE-US-00005 TABLE 5 Cytotoxicity of NK-92, T-ALL104 and YT Clone
to Patient-Derived Leukemic Cells.sup.a Disease Blast (%) in
Cytotoxic Sensitivity Patient Status Sample NK-92 TALL-104 YT AML 1
M4.quadrature. Relapse PB (66%) ++++++ +++++ - 2 (M1) Relapse PB
(50%) +++++ - - 3 (M3) Relapse PB (50%) +++ (++++) + (++++) - (-) 4
(M4) Refractory PB (90%) ++ (++) - (+) - (-) 5 (M2) New BM (90%)
+++ (+++) + (+++) ND 6 (M4) New BM (97%) - - - 7 (M4) New PB (39%)
- (-) - (++) - (-) 8 (M3) New PB (55%) - (++) - (+++) + (-) 9 (M3)
New BM (32%) - - - T-ALL 1 Relapse BM (98%) ++++++ - - 2 Relapse PB
(85%) ++++++ - (-) +++ (+++) 3 Relapse PB (77%) ++++++ - (+) - (-)
4 Relapse PB (60%) +++++ - (-) + (-) 5 New BM (40%) +++ - - 6 New
BM (66%) +++ - - B-Lineage-All 1 .cndot. Relapse BM (78%) +++++
++++ - 2 New BM (30%) ++++ ND ND 3 Relapse BM (75%) +++ (++++) +
(++++) ++ (++) 4 New BM (97%) ++ (+++) + (+++) - (-) 5 Relapse BM
(60%) + (+) - (+) - (-) 6 Relapse BM (80%) - ND ND 7 Relapse PB
(80%) - - (-) - 8 New BM (68%) - - - 9 New BM (33%) - - (+) - 10
Relapse BM (87%) - - (++) - 11 Relapse BM (75%) - (+++) - (++++) -
12 New BM (30%) - - ND 13 New PB 90%) - (+++) - (+++) ND 14 New BM
(81%) - - ND CML 1 BC PB (45%) ++++++ +++++ +++ 2 AC PB (22%)
++++++ ++ - 3 BC PB (93%) +++++ + - 4 CP PB (15%) D ++++ + - 5 CP
PB (8%) D ++ (++++) ND ND 6 CP BM (12%) D + (+++) + (+) ND 7 CP BM
(10%) D + (+++) + (++++) ND 8 BC PB (60%) + - - 9 BC BM (48%) + -
(-) - 10 CP PB (21%) D + (++) - (++++) - (-) 11 CP PB (11%) D - -
(+++++) - (-) Notes and Abbreviations. .sup.aColumns show results
of chromium release assays at E:T = 9:1 after 4 h without
parentheses, and (results after 18 h enclosed in parentheses); New:
newly diagnosed; ND: none done; o: FAB classification; D: blast and
promyelocyte; BM: bone marrow; PB: peripheral blood; I: B-ALL; BC:
blast crisis; AC: accelerated phase; CP: chronic phase.
Example 10
Cytotoxicity of NK-92 Towards Human Leukemic Cell Lines
[0113] The following human leukemic cell lines were cultured at
37.degree. C. in 5% CO.sub.2 in RPMI 1640 medium supplemented with
10% heat-inactivated fetal calf serum (FCS). L-glutamine and
antibiotics: K562 (Chronic myeloid leukemia in blast crisis), HL60
(acute promyelocytic leukemia), KG1 (erythroleukemia), NALM6 (acute
pre-B lymphoblastic leukemia), Raji (Burkitt's lymphoma), CEM/S
(acute T lymphoblastic leukemic cell line sensitive to methotrexate
(MTX), a commonly used antitumor drug) as well as CEM/T
(methotrexate-resistant subline of CEM/S) (Mini, E. et al., Cancer
Res. 45:325-330 (1985)).
[0114] NK-92 cells were highly cytotoxic to all the 8 leukemic cell
lines tested in a 4 hr standard CRA (Table 6). The MTX-sensitive
T-ALL cell line CEM/S as well as its MTX-transport resistant
subline CEM/T displayed a similar sensitivity to the NK-92 cells.
This suggests that tumors that are not responsive to MTX treatment
could be treated by administering NK-92 cells of the instant
invention. Table 6 surprisingly shows highly effective cytolytic
activity for NK-92 against all the target cells tested. The results
obtained at the low E:T ratio of 1:1 are especially noteworthy.
[0115] In contrast, the cytolytic cell lines TALL-104 and YT have
little or virtually no cytolytic activity against many of these
target cells under these conditions; when active, their activity is
generally lower than that for NK-92 at E:T of 1:1. TALL-104 was
cytotoxic to K562, NALM6 and HL60 cells, however, Raji cells
exhibited only 22.2% lysis at 9:1 E:T ratio and KG1 cells, CEM/S as
well as CEM/T were resistant. The YT clone did not exhibit
significant cytotoxic activity. Activity was found only against
K562 cells and Raji cells, which showed a 32% and 25% lysis at 9:1
E:T ratio, respectively.
[0116] As shown in Table 6, NK-92 cells of the present invention
have a significantly wider range of action and higher activities
than the known cytolytic cell lines TALL-104 and YT. These
activities are higher than any previously reported values in the
field of tumor cytotherapy.
TABLE-US-00006 TABLE 6 Specific Lysis of Human Leukemia Cell Lines
by Natural Killer Cell ClonesNK-92, TALL-104, and YT. Specific
Lysis (%) NK92 TALL-104 YT Effector:Target Ratio Target 9:1 3:1 1:1
9:1 3:1 1:1 9:1 3:1 1:1 K562 94.1 91.2 82.1 88.5 85.2 72.5 34.2
28.2 18.4 HL60 87.9 75.3 79.6 43.0 16.0 6.9 2.1 1.1 1.5 KG1 64.6
53.8 43.7 2.7 0.5 0 0.1 0 0 NALM6 72.6 56.8 52.4 67.8 55.6 33.3 1.0
0.5 0 Raji 86.0 75.4 70.0 22.2 10.2 0.3 25.1 18.0 14.2 TALL-104
57.3 53.2 44.1 -- -- -- 3.2 1.4 0.9 CEM/S 56.6 48.8 34.7 2.7 1.6
0.9 0.9 0.4 0.3 CEM/T 57.5 42.1 39.1 1.5 0.6 0.3 1.2 0.1 0.2
Example 11
Effect of NK-92 Cells on Normal Human Bone Marrow Hematopoietic
Cells
[0117] Heparinized bone marrow collected from normal donors was
separated by Ficoll Hypaque density gradient isolation to produce
the mononuclear cells. Enrichment of hematopoietic cells and
depletion of T cells was achieved by soybean lectin agglutination
(SLA) of mature marrow elements and removal of residual T cells by
resetting with sheep red blood cells (Reisner et al., Lancet,
2:327-31 (1981)).
[0118] Hematopoietic cell enriched fractions of normal bone marrows
from 14 normal donors were tested by standard CRA to determine
their susceptibility to lysis by NK-92 cells. All of the normal
bone marrow samples were insensitive to NK-92 mediated cytolysis
(FIG. 7).
Example 12
In Vivo Leukemogenesis of NK-92 Cells in SCID Mice
[0119] a. Experimental Animals.
[0120] Severe combined immunodeficient (SCID) mice (CB17 scid/scid
and pfp/Rag-2) (6 to 8 weeks old; Taconic Farms, Germantown, N.Y.)
were maintained in microisolator cages under sterile conditions
with a specific pathogen-free environment. To determine the
potential of NK-92 cells to induce leukemia in vivo,
2.times.10.sup.7 viable NK-92 cells in 0.3 mL phosphate buffered
saline (PBS) were administrated by either intraperitoneal (I.P.) or
intravenous (I.V.) route every other day for 5 injections in each
animal. For subcutaneous (S.C.) inoculations, 2.times.10.sup.7
NK-92 cells were injected in the right flank of SCID mouse, as
described previously (Yan et al., Blood, 88:3137-3146 (1996)).
Thereafter, all the experimental animals were administered rhIL-2,
5.times.10.sup.4 U every other day for 2 weeks by S.C. injection.
Survival of the animals was followed for at least 6 months after
inoculation.
[0121] b. Tissue Analysis.
[0122] From each group, 2 SCID mice were sacrificed at the end of
observation and tissues from peripheral blood, bone marrow, spleen,
liver, kidney, lung, and brain were collected for histopathological
and/or fluorescence activated cell sorting (FACS) analysis. Tissue
sections from sacrificed SCID mice were fixed in 10% neutral
buffered formalin, dehydrated and embedded in paraffin, sectioned
and stained according to standard histological techniques.
[0123] Viable cells recovered from various tissues were stained by
fluorescein isothiocyanate-conjugated (FITC) or
phycoerythrin-conjugated (PE) Mab, as described (Yan et al.,
(1996)). A FAGS scan flow cytometer (Becton Dickinson) was used for
analysis. Monoclonal antibodies (Mabs) directed against the
respective human cell surface antigens were used for determination
of their presence: CD2, CD3, CD5, CD7, HLA-DR, CD45, CD56 (Becton
Dickinson). A fluorescein isothiocyanate (FITC)-conjugated rat
anti-mouse Mab mCD45 (Boehringer-Mannheim, Indianapolis, Ind.) was
used for Characterization of murine leukocyte common antigen.
[0124] c. Leukemogenesis.
[0125] CB-17 scid/scid mice as well as pfp-Rag-2 mice were
inoculated with NK-62 cells by I.V. (n=3, for each group), S.C.
(n=2, each group) and I.P. (CB-17: n=8; pfp-Rag-2: n=3) injection.
Survival of the animals was followed at least 6 months after
inoculation. At the end of the six month period, all animals
appeared healthy; there was no hepatosplenomegaly, lymphadenopathy
or leukemic nodular growth, which would have indicated leukemia
development. Leukemic cellular infiltration was not detected in the
different tissues of the sacrificed animals by histopathology. No
cells of human origin were detectable in the tissues by FAGS
analysis.
Example 13
Comparison of Antileukemic Effect of NK-92 Cells with LAK, NK and T
Cells Against Human Leukemic Cell Lines
[0126] To isolate the NK cell populations, a Ceprate.RTM. cell
separation system based on avidin-biotin immunoaffinity (CellPro,
Bothel, Wash.) was used to purify a CD56+ cell fraction from
cultured LAK cells. Briefly, the harvested cells were washed and
resuspended in PBS with 1% bovine serum albumin (BSA). To each
1-2.times.10.sup.8 cells/mL, 40 .mu.L primary monoclonal antibody
(mouse anti-human CD56) was added and the cells were incubated at
4.degree. C. for 25 minutes. After incubation, the cells were
washed and resuspended to a concentration of 1.times.10.sup.8
cells/mL in PBS with 1% BSA. Then, to each one mL cell suspension,
20 .mu.L biotin labeled rat antimouse IgG1 antibody was added and
the cells were incubated again at 4.degree. C. for 25 minutes.
After incubation, the cells were washed and resuspended at a
concentration of 1.times.10.sup.8 cells per mL in PBS with 5% BSA
and slowly passed through the avidin column. The CD56+ cells were
captured; other cells, including the T cell fraction, were
eliminated from the column. After washing the column, the adherent
cells were then disassociated from the column by agitation and
elimination. After separation, the NK cell-enriched populations
contained >85% CD56+CD3- NK cells. The majority of the other
cells in the fraction (>95%) were CD3+CD56- T cells.
[0127] To generate leukemia-reactive allocytotoxic T lymphocytes
(CTLs), peripheral blood mononuclear cells (PBMC) isolated from
normal donors were cultured with irradiated leukemic stimulating
target cells and irradiated autologous PBMC as feeder cells.
Cultures were started in 60 well plates at 1000 responder cells per
well in RPMI 1640 medium containing 15% human serum and rhIL-2 100
U/mL at 37.degree. C., 5% CO.sub.2. The ratios of stimulator cells
and feeder cells to responder cells were 5:1 and 10:1,
respectively. After 10-12 days culture, CTLs were harvested from
growth-positive wells and specific lysis toward leukemic target
cells and K562 cells was quantitated by .sup.51Cr-release assay.
The CTLs were continuously cultured and fed with stimulator and
feeder cells in flasks. After 2-3 weeks culture, the monoclonal
antibody OKT3 (Ortho Biotech, Raritan, N.J.) was added to the
culture for rapid expansion of the CTL lines.
[0128] The antileukemic effects of NK-92 cells, human LAK cells, NK
cells (CD56+ CD3-; CD56+ in FIG. 8), and T cells (CD3+CD56-; CD3+
in FIG. 8) were assessed by measuring in vitro cytolytic activity
in standard CRA (FIG. 8, Panels A and B), and by measuring
inhibition of leukemic cell xenograft growth in vivo (FIG. 8,
Panels C and D) when the effector cells and targets were
co-inoculated subcutaneously into SCID mice. In order to evaluate
the inhibition of growth in vivo, the area of the subcutaneous
growths of leukemic nodules as a measure of their size was
determined once a week after inoculation, and survival of the
animals was also followed. NK-92 cells displayed the highest in
vitro cytotoxicity against K562 (FIG. 8, Panel A) and HL60 (FIG. 8,
Panel B) of the cells tested, with a mean specific lysis of 89% and
78%, respectively. This was superior to the killing mediated by
human LAK (52% and 11%, respectively), NK (72% and 28%,
respectively) and T cells (12% and 1.2%, respectively).
[0129] Correspondingly, the NK-92 cells demonstrated more effective
in vivo inhibition of the growth of K562 (FIG. 8, Panel C) and HL60
(FIG. 8, Panel D) leukemic cells xenografts than did the human LAK
and NK cells. The results shown in FIG. 8 indicate that the NK-92
cells of the present invention have cytolytic activity in vitro and
tumor-inhibiting activity in vivo that is superior to those
activities manifested by the known preparations of cytolytic cells
normally present in humans. These activities are therefore
unexpected by a worker in the field of tumor cytotherapy.
Example 14
Comparison of Antileukemic Effect of NK-92 Cells with Allogeneic
Leukemic-Reactive CTL Cells
[0130] To examine the in vivo effects of NK-92 cells and other
effector cells on the growth of human leukemia xenografts,
5.times.10.sup.6 leukemic target cells alone or mixed with
2.times.10.sup.7 NK-92 or other effector cells (E:T ratio=4:1) were
injected S.C. into SC ID mice. The TALL-104 effector cells were
irradiated with 3000 cGy before inoculation to prevent
leukemogenesis in SCID mice. RhIL-2 was administered to the mice,
as in Example 13. The Log-rank test and Wilcoxon test were used for
the comparison of the survival of leukemia bearing SCID mice.
[0131] The antileukemia effect of NK-92 cells was evaluated using
allogeneic leukemia-reactive CTL cells (derived from a patient with
T-ALL (TA27)). Both NK-92 and CTL cells activated by exposure to
TA27 displayed a significantly higher specific cytolysis (70% and
58% at 9:1 E:T ratio, respectively) than the other effectors (LAK
cells: 22%; NK cells (designated CD56+ in FIG. 9): 38%; TALL-104:
8%; and T cells (CD3+ in FIG. 9): 1.5% specific lysis) against the
TA27 leukemic cells (FIG. 9, Panel A). Correspondingly, the
subcutaneous growth of TA27 leukemic cells was inhibited by
co-injection of either NK-92 cells or anti-TA27-CTL cells (FIG. 9,
Panel B). The survival of those animals which were co-inoculated
with TA27 leukemic cells plus NK-92 or with anti-TA27-CTL cells was
significantly prolonged beyond that of the animals bearing TA27
leukemia alone (NK-92 cells: p=0.001; TA27-CTL cells: p=0.002; see
FIG. 10). In contrast the TALL-104 cells did not show significant
in vitro killing against TA27 leukemic cells by CRA (FIG. 9, Panel
A). However, moderate inhibition of the leukemic tumor growth in
vivo (FIG. 9, Panel B), coupled with a statistically insignificant
(p>0.05) increase in survival, was observed in the animals
co-inoculated with TA27 leukemic cells and irradiated TALL-104
cells (FIG. 10).
Example 15
Antileukemia Effect of NK-92 Cells in Human Leukemia Xenograft SCID
Mice Model
[0132] For study of the in vivo tumoricidal capacity of NK-92
cells, leukemic cells derived from a T-ALL patient (TA27), an AML
patient (MA26), and a pre-B-ALL patient (BA31) were adoptively
grown and expanded in SCID mice by S.C. inoculation. Leukemic cells
recovered from the leukemic nodules in the mice (first passage)
were used in these experiments. The SCID mice in each group were
inoculated I.P. with 5.times.10.sup.6 leukemic cells from the first
passage in 0.2 mL PBS, and 24 hours later 2.times.10.sup.7 NK-92
cells in 0.4 mL PBS were administered by I.P. injection. The
animals received either 1 dose or a series of 5 doses of NK-92
cells which were administered on days 1, 3, 5, 7, and 9, with and
without rhIL-2, as indicated in the Figures.
[0133] All the human leukemias grew aggressively in SCID mice.
Leukemic cells derived from a patient (TA27) with T-ALL and a
patient (MA26) with AML M4 leukemia were highly sensitive in vitro
to the NK-92 cells (73% and 66% specific killing at 9:1 E:T ratio
determined by CRA, respectively), whereas cells from a patient with
pre-B-ALL (BA31) were insensitive to the NK-92 cells (4% specific
killing at 9:1 E:T ratio assessed by CRA). FIG. 11 shows that the
survival of mice bearing TA27 leukemia was significantly prolonged
by the administration of NK-92 cells. The median survival time
(MST) of the animals with no treatment or rhIL-2 alone was 72 days
(n=5) and 63 days (n=5) (p>0.05), respectively. All these
animals died of leukemia. Treatment with NK-92 cells (alone or with
rhIL-2) increased the MST to 102 days (n=6) and 114 days (n=6),
respectively, for the 1 dose injection schedule (2.times.10.sup.7
NK-92 cells, day 1). The MST increased to 160 days (n=6) and 129
days (n=6), respectively, with 5 doses NK-92 with or without rhIL-2
injection (FIG. 11). Three animals that received 5 doses of NK-92
cell injections with or without rhIL-2 administration survived
without any signs of leukemia development 6 months after
inoculation. There was no significant difference in survival
between the mice receiving treatments with or without rhIL-2
administration, whether in the group receiving 1 dose of NK-92
cells (p=0.75), or the in the group receiving 5 doses (p=0.45).
Compared to the group receiving 1 dose of NK-92 cells, with or
without rhIL-2 treatment, survival was significantly extended in
animals that received 5 doses of NK-92 cells without rhIL-2
treatment (p=0.009 and p=0.009, respectively).
[0134] In SCID mice inoculated with human pre-B-ALL (BA31)
leukemia, with or without rhIL-2 treatment, the MST were 63 days
(n=5) and 64 days (n=5), respectively (see FIG. 12). For the
animals that received 5 doses of 2.times.10.sup.7 NK-92 cells, with
or without rhIL-2 administration, the MST was increased to 79 days
(n=5) and 76 days (n=5), respectively. These survival times were
not significantly different from those for the animals that were
not treated by NK-92 cells (p>0.05).
[0135] In animals bearing human AML (MA26), MST was 97 days (n=6)
(see FIG. 13). The MST was extended to 173 days among the animals
that received 5 doses of 2.times.10.sup.7 NK-92 cells (p<0.01)
(n=6). Three of the 6 animals that received NK-92 cells remained
alive 6 months after leukemia inoculation. Two of these appeared
healthy without any signs of leukemia development. One mouse had an
enlarged abdomen indicating residual leukemia. The 6 animals that
received NK-92 cells plus rhIL-2 treatment were all alive 6 months
after leukemia inoculation without any signs suggestive of leukemia
development.
[0136] The results presented in FIGS. 11-13 show that in vivo
treatment of leukemic tumors can result in enhanced longevity of
the subject mice. The extent of the prolongation of life, and of
the improvement in the health of the animals, is dependent on the
particular leukemic tumor involved, and ranges from modest or
insignificant (FIG. 12) to very dramatic (FIG. 13). Based on these
results, it is concluded that treatment of tumors in vivo by
administering NK-92 cells, depending on the tumor in question, can
be surprisingly effective.
Example 16
Preparation of Modified NK-92 Cell Lines Secreting IL-2
[0137] In order to generate NK-92 cells that constitutively secrete
IL-2, two plasmids encoding human IL-2 were employed.
[0138] a. Methods.
[0139] DNA Clones: The MG-hIL-2 vector (FIG. 7) was generously
provided by Dr. Craig Jordan (formerly of Somatix Corp., Alameda,
Calif.). The pCEP4-LTR-hIL-2 vector (FIG. 8) was created by
excising the Hin DIII-Bam HI fragment from the MFG-hIL-2 vector,
containing the 5' LTR and hIL-2 gene, and inserting it into the
complementary sites of the pCEP4 episomal vector backbone
(InVitrogen, Carlsbad, Calif.).
[0140] Particle-Mediated Gene Transfer: NK cells were transduced by
particle-mediated gene transfer using the Biolistic PDS-1000/He
Particle Delivery System (BioRad Laboratories, Hercules, Calif.).
Cells were transduced according to the manufacturer's instructions.
Briefly, 1.0 or 1.6 .mu.m gold particles were coated with 5 .mu.g
of DNA using calcium chloride spermidine, and ethanol. NK-92 cells
were prepared for bombardment by adherence to poly-L-lysine (Sigma,
St. Louis, Mo.) coated 35 mm tissue culture plates. Cells were
bombarded in an evacuated chamber (vacuum of 20 inches mercury) and
DNA-coated particles were accelerated by a 1,100 psi helium pulse.
Cells were returned to IL-2 supplemented Myelocult media
immediately following bombardment and allowed to recover for 24
hours prior to transfer to IL-2-free media. Media was changed
periodically. Cells were selected for IL-2-independent growth.
Preliminary experiments showed heat transfer efficiencies of 5-15%
were obtained under the conditions used.
[0141] PCR and Southern Blot Analysis: The transfection of the
NK-92 cells was confirmed by polymerase chain reaction (PCR)
analysis of DNA isolated from both the parental and transfected
NK-92 cell lines for the presence of genomic and cDNA forms of the
human IL-2 gene. DNA was isolated using DNAzol (Gibco Life
Technologies Inc., Burlington, ON). Briefly, cells were lysed in
DNAzol and DNA was precipitated with ethanol at room temperature.
DNA pellets were collected, washed in 95% ethanol and briefly air
dried. DNA was resuspended in 8 mM NaOH at 62.degree. C. and the
solution was neutralized with HEPES buffer. DNA was quantitated by
absorbance at 260 nm. Primers flanking exon 1 of the human IL-2
gene (forward: 5'-CAA CTC CTG TCT TGC ATT GC-3' and reverse: 5'-GCA
TCC TGG TGA GTT TGG G-3', Gibco Lift Technologies Inc., Burlington,
ON) were used to amplify the DNA (30 cycles, 1 min 95.degree. C., 2
min 50.degree. C. and 2 min 72.degree. C.). PCR products were
resolved on a 2% agarose gel. For Southern blot analysis, DNA was
transferred to Hybond+nylon membrane (Amersham Life Sciences,
Arlington Heights, Ill.) by capillary transfer in 10.times.SSC
(1.5M NaCl, 1.5M NaCitrate) and fixed by UV cross-linking
(StrataLinker Stratagene, La Jolla, Calif.). The blot was
hybridized with a .sup.32P radiolabeled human IL-2 probe for 8-12
hours, washed and visualized by autoradiography at -70.degree. C.
with Kodak X-Omat XAR film.
[0142] Northern Blot Analysis: Cytokine and chemokine gene
expression was analyzed by Northern blot analysis. RNA was
extracted from parental and transfected NK-92 cell lines using
Trizol reagent (Gibco Life Technologies Inc., Burlington, ON)
according to the manufacturer's instructions. Briefly, cells were
lysed in Trizol and the lysate extracted with chloroform. The
aqueous phase was then precipitated with isopropanol. The RNA
pellet was collected, briefly air-dried and then resuspended in
DEPC-treated water (diethyl-pyrocarbonate; Sigma Chemical Co., St.
Louis, Mo.). RNA was quantitated by determining OD.sub.260 nm.
Fifteen micrograms of RNA was resolved on a 1% formaldehyde agarose
gel in 1% MOPS (3-[N-Morpholino]propanesulfonic acid, Sigma, St.
Louis, Mo.) and blotted as described previously for Southern blot
analysis. The blot was hybridize with .sup.32P radiolabeled probes
for human IL-2 and TNF-.alpha..
[0143] DNA probes for Northern and Southern blot analysis were
radiolabeled by random primer extension. DNA probes for human IL-2
and TNF-.alpha. were purified by digestion with appropriate
restriction endonucleases and agarose electrophoresis. The DNA was
excised from the gel and purified by centrifugation through a
Spin-X tube filter (Corning Costar, Cambridge, Mass.),
phenol:chloroform extraction and ethanol precipitation. DNA probe
was labeled with .alpha.-.sup.32P-dCTP (Sp. Ac. 3000 Ci/mmol; ICN,
Montreal, PQ).
[0144] Cytokine Determination: IL-2 production by NK-92 was
determined by ELISA. Aliquots of 1.times.10.sup.6 of the parental
or transfected NK-92 cells were cultured in 8 ml of IL-2 free
Myelocult media for 1, 2, and 3 days. Supernatants were collected
from at -20.degree. C. until all samples were collected. Samples
were thawed and assayed for IL-2 levels by ELISA according to the
manufacturers' instructions (Quantikine; R&D Systems,
Minneapolis, Minn.). The ELISA is a horseradish
peroxidase/tetramethylbenzidine based colorimetric assay and the
ELISA microtiter plates were read at 450 nm (with a 540 nm
correction) in a microplate reader (Moidel EK309, Bio-Tek
Instruments Inc., Winooski, Vt.).
[0145] Irradiation of NK-92 Cells: To determine the sensitivity of
both parental and transfected NK-92 cells to irradiation, cells
were irradiated using a Cis BioInternational 437c cesium source
(Cis-US, Bedford, Mass.). Cells were collected, washed and
resuspended in medium and irradiated in 15 or 50 ml conical
centrifuge tubes (Becton Dickinson, Franklin Lakes, N.J.).
Following irradiation, cells were washed and resuspended in
Myelocult with (for parental NK-92) or without (for transfected
cells) IL-2. Cells were cultured for 24, 48 and 72 hours and
assayed for viability by trypan blue exclusion, for proliferation
by .sup.3H thymidine incorporation and for cytotoxicity by
.sup.51Cr-release assay (as described above).
[0146] b. Plasmid MFG-hIL-2.
[0147] For NK cells transfected with the MFG-hIL-2 vector, 85-95%
of cells died after 4-7 days following transfer to unsupplemented
media. A small number of cells, however, remained viable. These
were assumed to be cells that had been successfully transfected.
However, even with these cells, no viable cells were detectable
after two to three weeks. This was expected as the MFG-hIL-2 vector
construct did not contain the genetic elements required for
replication and maintenance in eukaryotic cells such as a mammalian
origin of replication. Therefore, as the transfected cells were
maintained in culture and began to replicate, the vector construct
would have been lost from cells and the cells would have reverted
to their IL-2-dependent phenotype. These cultures were nevertheless
propagated for several weeks. Surprisingly, a small number of
viable cells appeared in the cultures after approximately 4-5 weeks
following initial transfer of the cells to IL-2-free media. These
cells were capable of IL-2-independent growth upon subculturing to
fresh media and appeared to be stably transfected, maintaining
their IL-2 independent phenotype during prolonged culturing. Since
the vector was unable to replicate, the appearance of stably
transfected cells suggests that the vector had integrated into the
genome of a transfected cell. Since this would be a very rare
event, these transfected cells probably arose from one or a very
small number of cells. IL-2-ihdependent NK-92 cells arising from
transfection with the MFG-hIL-2 were denoted as NK-92MI.
[0148] c. Plasmid pCEP4-LTR.hIL-2.
[0149] Initial observations for cells transfected with the episomal
vector pCEP4-LTR.hIL-2 were identical to those seen with NK-92MI.
The majority of the transfected cells died within 4-7 days
following transfer to IL-2-free Myelocult media. However, unlike
the NK-92MI cells, the remainder of the cells did not lose their
IL-2-independent phenotype or vitality and die after the initial
2-3 week period. Instead, the cells that were initially
IL-2-independent were immediately capable of long-term
IL-2-independent growth and survival. This was expected since the
pCEP-LTR.hIL-2 vector contains elements that enable it to be
maintained in eukaryotic cells as an autonomously replicating
genetic element. Therefore, any cell that was initially transfected
should maintain its IL-2-independent phenotype for an indefinite
length of time. Although cells harboring episomal vectors are not
stably transferred by strict definition, these cells are under
constant selection pressure in IL-2-free media in favor of cells
maintaining the vector. Therefore, these cells are capable of
long-term culturing. IL-2-independent NK-92 cells arising from
transfection with the pCEP4-LTR.hIL-2 are denoted as NK-92CI.
[0150] To confirm that NK-92MI and NK-92CI have in fact been
transfected with hIL-2 gene, PCR analysis was performed on the
parental and transfected cell lines. Primers flanking exon 1 of the
hIL-2 gene, which has 88 base pairs (bp), were used to amplify DNA
isolated from NK-92, NK-92MI and NK-92CI to assay for the presence
of the genomic and cDNA forms. Agarose gel electrophoresis of the
PCR products from the parental line revealed a single 263 bp
fragment corresponding to the size expected for the DNA fragment
amplified from the genomic IL-2 gene (FIG. 16, Panel A). However,
analysis of both the NK-92MI and NK-92CI products revealed two
bands, the 263 bp fragment corresponding to the genomic hIL-2 gene
as well as a 175 bp fragment resulting from the amplification of
the hIL-2 cDNA. To confirm the identity of these DNA fragments,
Southern blot analysis with a radiolabeled probe specific for hIL-2
probe was performed. As seen in FIG. 16, Panel 13, both the 263 bp
genomic fragment and the 175 bp cDNA fragment hybridized with the
probe. These data indicate that both NK-92MI and NK-92CI had been
successfully transferred and contain the cDNA for hIL-2.
[0151] d. Analysis of Gene Expression.
[0152] To analyze expression of specific cytokines in the parental
and transfected cell lines, they were analyzed by Northern blot
analysis. RNA isolated form the NK-92, NK-92MI, and NK-92CI cells
was separated by electrophoresis, transferred to a nylon membrane
and hybridized with probes for the cytokines hIL-2 and hTNF-.alpha.
(see FIG. 17). Northern blot analysis of IL-2 in these cells
revealed that IL-2 RNA was not detectable in the parental cell line
(FIG. 17, Panel A, Lane 1). However, hIL-2 was found in RNA from
both the NK-92MI and NK-92CI (Lanes 2 and 3, respectively). Two
mRNA transcripts were seen in NK-92MI, a major RNA species of
approximately 1.9 kDa and a less intense transcript at 2.4 kDa. In
NK-92CI, a hIL-2 mRNA transcript of approximately 1.4 kDa was
detected. As well, a very faint band was seen at 2.5 kDa. These
data confirm that the transfected cells expressed IL-2 while the
parental NK-92 cells did not. The significance of the multiple
hIL-2 mRNA transcripts in the two transfectants is not clear,
although it is possibly a consequence of the different vector
constructs. Furthermore, in the case of NK-92MI, the integration of
the hIL-2 gene into the genomic DNA may also have affected the RNA
size.
[0153] TNF-.alpha. expression in the NK cells was also examined
using this technique (FIG. 17, Panel B). It is seen that all three
lines expressed the gene for this cytokine. A TNF-.alpha. probe
hybridized to a 1.6 kDa band in RNA isolated from NK-92, NK-92MI
and NK-92CI (FIG. 17, Panel B). These results indicate that
although transfection of NK-92 cells with the IL-2 gene resulted in
expression of the IL-2 in the transfectants, this did not influence
the expression of another cytokine.
[0154] e. Secretion of hIL-2.
[0155] After confirming expression of the IL-2 gene by Northern
blot analysis, cells were assayed for production and secretion of
hIL-2 by ELISA. Aliquots of 10.sup.6 NK-92, NK-92MI and NK-92CI
cells were plated in 8 mL aliquots and cultured in Myelocult in the
absence of IL-2. Supernatants were collected after 24, 48 and 72
hours for IL-2 analysis by ELISA. Background levels of IL-2 were
detected in the supernatant of NK-92 cells at all three time points
(2-3 pg/mL). Elevated IL-2 levels were detected in both NK-92MI and
NK-92CI supernatants (Table 7). NK-92MI produced much higher levels
of IL-2 in comparison to NK-92CI, with levels ranging from
60.times. higher after 24 hours (9.3 pg/mL vs 549.3 pg/mL) to about
80.times. higher after 48 hours (15.7 pg/mL vs 1,260.3 pg/mL) and
72 hours (27.2 pg/L vs 2,248.3 pg/mL).
TABLE-US-00007 TABLE 7 Synthesis of Human IL-2 by NK-92, NK-92MI,
and NK-92CI IL-2 (pg/ml) in Experiment #1 #2 #3 Ave .+-. S.D. NK-92
Day 1 0 7 1 2.7 .+-. 3.8 Day 2 0 4 1 1.7 .+-. 2.1 Day 3 0 3 3 2.0
.+-. 1.7 NK- Day 1 517 568 545 549.3 .+-. 34.7 92MI Day 2 977 1462
1342 1260.3 .+-. 252.6 Day 3 1872 2610 2263 1148.3 .+-. 369.2 NK-
Day 1 7 13 8 9.3 .+-. 3.2 92CI Day 2 14 16 17 15.7 .+-. 1.5 Day 3
52 18 13 27.7 .+-. 21.2
[0156] f. Comparison of Cell Surface Antigens in NK-92, NK-92MI and
NK-92CI.
[0157] To compare the IL-2-independent transfectants with the
parental cells, NK-92MI and NK-92CI were analyzed for CD2, CD3,
CD4, CD8, CD10, CE16, CD28, CD56, ICAM-1, ICAM-2, ICAM-3 and LFA-1
expression by fluorescent activated cell sorting (FACS) analysis.
The transfected cells revealed a pattern of expression identical to
tat seen on the untransfected parental cell line with the exception
of the IL-2 receptor. FACS analysis of CD25 (the IL-2 receptor
.alpha.-chain) on NK-92 cells indicated that the receptor was
expressed on the surface of NK-92 cells and that its expression is
down-regulated in response to IL-2. This confirmed similar findings
obtained in earlier work (Gong et al., 1994). Therefore, NK-92
cells in unsupplemented media had relatively high levels of CD25 on
their surface while cells in media supplemented with as low as 100
U/mL had low levels of CD25 cell surface expression.
[0158] CD25 expression in the high IL-2-producing transfectant
NK-92MI was decreased both in unsupplemented media and in media
supplemented with 100 U/mL or 1000 U/mL of IL-2. These results are
consistent with those seen with the parental cells. Since the
levels of endogenously produced IL-2 in NK-92MI were high,
down-regulation of IL-2 receptor levels is expected even in the
absence of exogenously administered IL-2.
[0159] Culture of NK-92CI in media supplemented with 100 U/mL and
1000 U/mL IL-2 resulted in CD25 upregulation and increased cell
surface expression. However, the results for NK-92CI in
unsupplemented media are not as clear. Two distinct populations
appear, a population expressing very low CD25 levels, similar to
NK-92MI, and a population expressing high levels, similar to the
NK-92 parental cells. This suggests that NK-92CI consists of a
polyclonal population consisting of high and low IL-2 expressing
cells rather than a uniform population of cells expressing an
intermediate to low level of IL-2. Therefore, when cultured in
IL-2-free media, the cells expressing high levels of IL-2 would
have low surface levels of CD25 while low IL-2 expressing cells
would have high CD25 levels on their surface.
Example 17
Cytotoxicity of NK-92 Transfected to Produce IL-2
[0160] To evaluate the cytotoxicity of these transfected cells, a
standard 4 hour .sup.51Cr-release assay was performed to compare
the toxicity of the parental cells to NK-92MI and NK-92CI to the
standard test target cells K562 and Raji. The cytotoxicity of
NK-92MI and NK-92CI was comparable to that seen with the parent
cells (FIG. 18). The transfected cell lines show cytotoxic
activities against K562 and Raji that are very similar to that of
the parental cells. Cytotoxicity of NK-92 against K562 ranged from
82 to 67% while NK-92MI and NK-92CI had cytotoxicity ranges of 77
to 62% and 82 to 62%, respectively. For Raji cells, NK-92 had
cytotoxicity of 81 to 47%. NK-92M| had cytotoxicity of 75 to 65%
and NK-92CI had cytotoxicity of 82 to 52%.
Example 18
Effect of Transfected NK-92 Cells on Hematopoietic Progenitor
Cells
[0161] One potential clinical application of the NK-92. NK-92MI and
NK-92CI cells is as an ex vivo purging agent for autologous grafts.
In order for the NK cells to be suitable for such a purpose, they
must be able to purge the malignant cells without killing the
hematopoietic progenitor cells in the graft or influencing their
hematopoietic potential. In order to assay this, a colony-forming
cell assay (CFC) was performed where the clonogenic output of PBMCs
was examined following a 48 hour incubation with NK-92MI and
NK-92CI at various E:T ratios. NK-92 was previously shown to have
minimal effect on hematopoietic stem cells (Example 6). In this
example. NK-92M| and NK-92CI also show little or no effect on
clonogenic output. The number of total colonies following
incubation with either NK-92MI or NK-92CI was very similar to
control, although a slight decrease was seen with the highest
effector:PBMC ratio of 1:1 (FIG. 19). Total clonogenic output from
both NK-92MI and NK-92CI was approximately 80% of control under
this condition. However, no consistent trend was seen in terms of
clonogenic output with respect to the ratio of NK:PBMCs. In terms
of specific colony types, there were no detectable differences in
the number of output BFU-E colonies, which are the most numerous.
Some effect was seen with both the CFU-GM and CFU-GEMM colonies.
However, the absolute numbers of these colonies are very low,
making any conclusions difficult since small variations in the
number of colonies has a large effect on the calculation of
clonogenic output. An influence on CFU-GM and CFU-GEMM is seen at
higher ratios, but no consistent correlation between ratio and
output was noted.
Example 19
Irradiation of the Transfected NK-92 Cells
[0162] To establish an effective irradiation dose to inhibit
proliferation and maintain cytotoxicity, NK-92MI and NK-92CI cells
were irradiated at 500, 1,000, 1,500 and 2,000 cGy and assayed for
proliferation by the .sup.3H thymidine incorporation assay (see
Examples 7 and 8). Both NK-92MI and NK-92CI were more sensitive to
irradiation than the parental NK-92 cell. Proliferation of NK-92MI
and NK-92CI was found to be more strongly suppressed than NK-92 at
all radiation doses tested (FIG. 20, Panel A). For NK-92MI and
NK-9201, proliferation was completely suppressed by a radiation
dose between 500 and 1,000 cGy. The level of thymidine
incorporation reached a plateau at approximately 20% of
unirradiated control cells for NK-92CI and 10% for NK-92MI. For
determination of viability, NK-92, NK-92MI and NK-92CI cells were
irradiated at 250, 500, 1,000 and 2,000 cGy and trypan blue
exclusion was determined 24, 48 and 72 hours following irradiation.
It was found that greater percentages of both NK-92MI and NK-92CI
were found to be killed by irradiation as compared to the parental
cells at equivalent doses (FIG. 20, Panel B). Viability of NK-92
was higher than that of both transfectants at all dose rates
tested.
[0163] The cytotoxicity of these cells following irradiation is
shown in FIG. 21. Cells irradiated at 0, 1,000 and 2,000 cGy were
tested after three days for cytotoxicity against K562 and Raji
cells at effector:target ratios of 20:1, 10:1, 5:1 and 1:1.
Cytotoxicity of NK-92 cells three days following irradiation at
1,000 cGy was determined to be approximately 10-30% K562 (FIG. 21,
Panel A) and 30-50% and for Raji (FIG. 21, Panel B). Irradiation at
2,000 cGy resulted in cytotoxicity of 1-5% against K562 and 3-13%
against Raji. In contrast, NK-92MI had only 0-5% and 0-1% cytotoxic
activity against K562 and 0-1% and 0% against Raji three days after
irradiation doses of 1,000 and 2,000 cGy, respectively. NK-92CI had
only 1-4% cytotoxicity to K562 and 2-7% to Raji three days after
irradiation at 1000 cGy and 0% to K562 and 0-2% after irradiation
with 2000 cGy.
[0164] In the data reported here, IL-2 transfectants are seen to be
more sensitive to irradiation than the parental strain.
Proliferation and cytotoxicity of both NK-92MI and NK-92CI cells
were suppressed at a lower radiation level than for the parental
strains, and radiation-induced lethality was much greater in the
IL-2-independent modified cells in NK-92 at equivalent radiation
doses. The high IL-2-producing NK-92MI is more sensitive to
radiation than the low IL-2 producing NK-92CI variant. As a result
of the increased radiation sensitivity, a reduced level of
irradiation would be sufficient to adequately control proliferation
while minimizing lethality to the cells and inhibition of
cytotoxicity. In routine experiments, the worker of ordinary skill
would be able to repeat experiments such as those described in this
example. By using lower radiation doses, in the range between 0 and
1000 cGy optimal doses can be determined that inhibit proliferation
while maintaining viability and cytolytic activity in NK-92MI and
NK-92CI.
Example 20
Transfection of NK-92 with a Gene for Thymidine Kinase
[0165] NK-92 cells are to be transfected with a vector bearing a
gene for thymidine kinase (TK). The resulting TK-modified NK-92
cells are thereby rendered susceptible to the toxic effects of the
guanosine analogs, gancyclovir, and acyclovir.
[0166] A vector suitable for transfecting a mammalian cell is to be
constructed, such as a retroviral vector harboring a herpes simplex
virus (HSV) TK gene, under the control of the HSV TK promoter, and
containing its own polyA addition site. Transfection is to be
carried out by a method known to those skilled in cell biology and
mammalian molecular biology, such as by electroporation (Bio-Rad
Gene Pulser.TM.), or by lipofection (Feigner et al., Proc. Natl.
Acad. Sci. USA 84:7413 (1987)). The transfected NK-92 cells so
produced are susceptible to inactivation by administering
gancyclovir or acyclovir.
Example 21
Mutation of NK-92 HLA Cell Surface Protein
[0167] NK-92 cells are to be obtained from the cell line described
by Gong et al. (1994). The chromosome bearing the
.beta..sub.2-microglobulin gene is to be isolated, and the DNA
contained within this chromosome is to be purified away from
histones and other DNA-bound proteins. The gene fragment bearing
.beta..sub.2-microglobulin is to be excised with restriction
nucleases, and site specific mutagenesis is to be conducted via an
oligonucleotide cassette harboring the mutated nucleotide sequence.
These procedures employ techniques commonly known in recombinant
DNA technology, as set forth, for example, in "Current Protocols in
Molecular Biology", Ausubel et al., John Wiley and Sons, New York
1987 (updated quarterly), and "Molecular Cloning: A Laboratory
Manual", 2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 1989, incorporated
herein by reference. The mutated .beta..sub.2-microglobulin is to
be reincorporated into the cellular DNA, and reintroduced into the
NK-92 cells. This preparation of cells will then express cell
surface HLA molecules incorporating mutated
.beta..sub.2-microglobulin moieties, and will have lost the ability
to bind T-cell receptors.
Example 22
NK-92 Cells Expressing Receptors for a Cancer Cell
[0168] The CTL of a patient suffering from a cancer are to be
harvested by differential centrifugation on a density gradient. The
CTL are to be immunoaffinity purified to contain predominantly the
CTL targeting a receptor on the cancer cell from the patient. The
DNA of the CTL population obtained is to be isolated, and the genes
for the WIC class I receptor in the cancer-targeted CTL isolated by
restriction nuclease cleavage. The genes so purified are to be
amplified using the polymerase chain reaction, and the resulting
amplified genes incorporated into a vector suitable for the
constitutive expression of the genes in NK-92 cells. The vectors
are to be transfected into NK-92 cells, and the modified NK-92
cells so obtained are to be selected using, for example, an
antibiotic resistance marker incorporated into the vector. The
cells so selected are to be cultured to increase their number. They
may then be employed to target specifically the cancer cells in the
patient, and treat the cancer occurring in the patient either ex
vivo or in vivo.
Example 23
Use of NK-92 Cells to Kill HIV-Infected Cells
[0169] 8E5 is a cell line harboring HIV that produces HIV virions.
8E5L is a corresponding cell line infected with HIV which does not
produce virions. In a cytotoxic activity experiment in which the
chromium release assay was used to evaluate activity, the results
presented in Table 8 were obtained. In these experiments. A3.01
cells are an uninfected control cell line.
TABLE-US-00008 TABLE 8 Cytotoxic Activity of NK-92 Cells on
HIV-Infected Cells. Target E:T Ratio % Cytotoxicity A3.01 50:1 43
20:1 51 5:1 44 1:1 44 8E5L 50:1 43 20:1 37 5:1 44 1:1 40 8E5 50:1
76 20:1 69 5:1 77 1:1 65
It is seen from Table 8 that 8E5 cells which produce HIV particles
elicit a higher cytotoxic activity than do 8E5L cells, which do not
produce HIV particles, and higher than control cells. Without
wishing to be bound by theory, it is believed that the anti-viral
effect of NK-92 cells is due to factors such as a direct cytotoxic
effect, as well as inhibition through MIP-1.alpha., which is
produced by NK-92 cells in high concentrations (Bluman et al., J.
Clin. Investig. 97, 2722 (1996)). The results indicate that NK-92
cells effectively lyse HIV-producing cells in vitro.
Sequence CWU 1
1
2120DNAArtificial Sequenceprimer oligonucleotide based on human
sequence 1caactcctgt cttgcattgc 20219DNAArtificial Sequencereverse
primer oligonucleotide based on human sequence 2gcatcctggt
gagtttggg 19
* * * * *