Note: Descriptions are shown in the official language in which they were submitted.
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NATURAL KILLER CELL LINES AND METHODS OF USE
RELATED APPLICATION
This application is based on, and claims benefit of, United States Provisional
Application Serial Number 60/97,163, filed on April 30, 1997.
FIELD OF THE INVENTION
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
n the treatment of these pathologies.
BACKGROUND OF THE INVENTION
Certain cells of the immune system have cytotoxic activity against particular
target
?o 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-IS%
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
~5 sensitization (Herberman et al., Science 214:24 (1981)). Killing of target
cells occurs by
inducing cell lysis. MHC class restriction likewise ~s 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
~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
Fcy receptor
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(CD16) is expressed on NK cells. NI< cells are large granular lymphocytes
which lack
CD3, and in addition to CD16, also may express Leul9 (Lanier et al., J.
Immunol. 136;
4480 ( 1986)).
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.,
.1. 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., .1. Exp. Med. 164:814-825 ( 1986)).
m 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
is 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-Passerine et al., Br. J. Haematol. 18:197 ( I
991 )) 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
2o the identity of the target cells that they kill.
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
z5 directed toward these and other forms of cancer, including malignant
melanoma a.nd 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
3o complete the therapy. This purging treatment is expensive and can cause
serious side
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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.
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
m 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)).
Recently, preclinical studies have also demonstrated promising antitumor
activity in
vivo with a lethally irradiated, MHC-unrestricted, cytotoxic T-cell leukemic
clone (TALL
15 104) {Cesano et al., Cancer Immunol. Immunother. 40:139-1 S 1 ( 1995);
Cesano et al.,
Blood 87:393-403 ( 1996)). These cells were derived from leukemia T cell lines
obtained
from patients having acute T iymphoblastic 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- CD56'-). Adoptive transfer of these cells was
able to
?n 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.
56:3021-3029 (1996)).
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);
3c:~ Stephen et al., Leuk. Lymphoma 377-399 ( 1992); Rosenberg et al., New
Engl. J. Med.
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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. Lmmunol. 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
n~ 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)).
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
is 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
2o 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.
25 SUMMARY OF THE INVENTION
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
3o cancer treatment and virus treatment systems. The invention also provides
the vectors that
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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.
One aspect of the invention provides a vector for transfecting NK-92 cells,
wherein
a 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
m 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.
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.
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
2~ 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-~2MI and NK-92CI cell lines have been in the American
Type
Culture Collection under the designations and
respectively. In additional important embodiments, the NK-92 cells are
transfected by a
zo vector including a sequence that encodes a cellular component responsive to
an agent such
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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.
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 patholog~;~. 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
~i~ 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,
~ 5 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.
2o 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.
The invention additionally provides a method of treating a pathology ex vivo
in a
za mammal including the steps of (i) obtaining a biological sample suspected
of containing
cells related to the pathology from the mamma(; (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
:n purged sample, and (iii) returning the purged sample to the mammal. The
pathology may
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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 receptov molecule, or the vector may include any combination
of these
modifications. In important embodiments of this method, the biological sample
is blood or
~t~ bone marrow, the mammal is a human, and/or the natural killer cell is
immobilized on a
support 1n 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.
The present invention further provides a method of treating a pathology in
vivo in a
~s mammal including the step of administering to the mamma( 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-proiiferative 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
2o 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
_'S 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.
In yet an additional embodiment of the in vivo method of treating cancer, the
NK-
zo 92 is modified by transfection with a vector comprising an element
responsive to an agent
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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
a 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
to Figure 1. Cytotoxic activity of NK-92 against different leukemic target
cell lines
tested in a 4 hour ''Cr release assay. The results represent the mean t the
standard
deviation (SD) for three replicate experiments.
Figure 2. Cytotoxicity of NK-92 after IL-2 deprivation. NK-92 cells were
cultured
in enriched alpha medium (MyelocultT"', StemCell Technologies. Vancouver, BC)
without
I a IL-2. Cvtotoxicity was measured daily with the 5'Cr-release assay against
K562-neo' or
Daudi target cells. The Figure shows results from one representative
experiment at the E:T
ratio of 10:1.
Figure 3. Effect of various doses of y radiation on the cytolytic potential of
NK-92
cells. NK-92 cells were irradiated with a '3'Cs 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 ''Cr release assay against the
target cell line
K562.
Figure 4. Survival curves of NK-92 cells after y-irradiation. NK-92 cells were
irradiated with a y ray source at doses of 300, 500, 1000, and 3000 cGy.
Viability of NK-
Z> 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 x 10~/mL. The cells
had to be fed
to prevent overgrowth.
Figure 5. Effect of y-irradiation on the in vitro colony formation of NK-92
cells.
NK-92 cells were cultured in agar-based medium supplemented with recombinant
human
3u IL-2 (rhIL-2).
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Figure 6. Effect of various radiation doses on the cytolytic potentiaE of NK-
92
cells. NK-92 cells were y-irradiated at doses of 300, 500, 1000, and 3000 cGy.
5'Cr-
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-J2 cells.
The results
of 4 hr chromium release assays are expressed as 30% lytic units/10~ effector
cells.
Figure 7. Selective killing of patient-derived leukemic cells by NK-92 cells.
5'Cr-
labeled leukemic target cells derived from 40 patients (9 acute myeloid
leukemia (AML)
cases, 1 1 chronic myeloid leukemia (CML) cases, 14 B-lineage-acute
lymphoblastic
m 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~ effector cells AT AN E:T RATIO OF ... .
Figure 8. 1n 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. 5'Cr 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~CD56+), and T cells (CD3'CD56-)] at indicated E:T
ratios in
a 4 hr CRA assay. Results are means t SD of three separate tests for NK-92
cells, and two
2ci tests of different donor-derived effectors for LAK, CD56' and CD3' cells.
SCID mice were
inoculated subcutaneousiy with K562 cells (Panel C) or HL60 cells (Panel D)
(5x106 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).
2> Figure 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 s'Cr-release assay using the indicated E:T ratios. Results are means ~ SD
of two or three
3« separate tests. Panel B: SCID mice were inoculated subcutaneously with TA27
cells
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(5x10'' 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 Sx 10' U every other day.
Leukemic tumor
areas were measured once a week post inoculation (n=S).
Figure 10. Survival of SCID mice bearing T-ALL (TA27) leukemia co-inoculated
with NK-92 cells as campared with co-inoculation with allogenic CTL or
irradiated TALL-
104 cells.
Figure 1 1. Survival of SCID mice bearing T-ALL (TA27) after treatment with
NK-92 cells. Mice received 5x10'' TA27 cells intraperitoneally (LP.). NK-92
cells (2x10')
n~ were injected I.P. once, or S times (on days 1, 3, S, 7 and 9), with or
without the addition
of rhIL-2 every other day for two weeks
Figure 12. Survival of SLID mice bearing pre-B-ALL (BA31 ) after treatment
with
NK-92 cells. Mice received 5x10'' BA31 cells l.P. NK-92 (2x10') cells were
injected I.P.
for a total of S doses, on days 1, 3, S, 7 and 9. Mice in the indicated groups
received rhlL-
i s 2 every other day for two weeks.
Figure 13. Survival of SCID mice bearing human AML (MA26) after treatment
with NK-92 cells. Mice received 5x106 MA26 leukemia cells l.P. NK-92 (2x10')
cells
were injected I.P. on days 1, 3, S, 7 and 9 for a total of five doses. Mice in
the indicated
groups received rhlL-2 every other day for two weeks.
zo Figure 14. Diagrammatic map of plasmid MFG-hIL-2.
Figure 15. Diagrammatic map of piasmid pCEP4-LTR.hIL-2.
Figure 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
~5 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).
Figure 17. Northern blot analysis of cytokine expression in NK-92, NK-92MI and
~c~ NK-92CI. RNA samples isolated from the parental and transfected cell lines
were
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separated by agarose gel electrophoresis blotted to nylon membrane by
capillary transfer
and hybridized with probes for human IL-2 (Panel A) and TNF-cx (Panel B).
Figure 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 5'Cr-labeled KSf>2 (Panel A) or
Raji (Panel
B) cells at effector:target ratios of l:l, 5:1, 10:1 and 20:1 for a 4 hour
chromium release
assay. The cytotoxicities of NK-92 (~), NK-92M1 (~) and NK-92CI {~) are shown.
Figure 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
~u assay was performed. Normal PBMCs were incubated with irradiated NK-92M1 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-
92C1
(gray bars) is expressed as either total number of colonies or subclassified
on the basis of
I ~ colony type {BFU-E, CFU-GM and CFU-GEMM).
Figure 20. Effect of irradiation on NK-92, NK-92M1 and NK-92CI proliferation
and viability. To assess the effect of irradiation on the parental and
cransfected 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 'H-thymidine incorporation assay.
Panel A'
?o Proliferation of NK-92 (~), NK-92MI (~) and NK-92CI (~) is expressed as a
percentage of
control (unirradiated cells). Pane) 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).
Figure 21. Effect of irradiation on NK-92, NK-92MI and NK-92CI cytotoxicity.
To
25 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 KS62 (Panel A) and Raji (Panel B) cells.
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DETAILED DESCRIPTION OF THE INVENTION
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.
a 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
to effective in purging cancer cells ex vivo and in destroying cancer cells in
vivo.
As used in the present invention, "cytotoxic T lymphocytes" (CTL) relate to
immune cells which kill antigen-specific target cells. CTL are MHC :;lass 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
la of cells, such as peripheral blood mononuclear cells, upon activation by
treatment with
lymphokines. LAK cells have essentially no effect on normal cells.
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
?r~ 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
2a 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.
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
3o synonymous. In general, cytotoxic activity relates to killing of target
cells by any of a
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variety of biological, biochemical, or biophysical mechanisms. Cytolysis
refers more
specifically to activity it 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.
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.
As used to describe the present invention, "purging" relates to killing of
target cells
by effector cells such as NK cells ex viva. 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
I5 the tumor cells and returning the sample to the body of the mammal.
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 thev
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.
As used herein, a "vector" relates to a nucleic acid which functions to
incorporate a
2s 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 ~s operably positioned with respect to the protein-encoding sequence
such that,
3o when the vector is introduced into a suitable host cell and the regulatory
sequence exerts
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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.
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 ~s a
part of the
lymphatic or immune system, malignant cells may include non-solid tumors of
circulating
lc~ 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.
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
~ 5 consequence of such infection is a deterioration in the health of the
host. Pathogenic
viruses envisioned by the present invention include, but are not limitr:d to.
HIV, EBV,
CMV, and herpes.
Natural Killer Cell NK-92
2o The NK-92 cell line has been described by Gong et al. ( 1994). It is found
to
exhibit the CD56""s'", CD2, CD7, CDlla, CD28, CD45, and CD54 surface markers.
It
furthermore does not display the CD1, CD3, CD4, CDS, CDB, 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
~5 sufficient to maintain proliferation. IL-7 and IL-12 do not support long-
term growth, nor
do other cytokines tested, including IL-la, IL-6, tumor necrosis factor a,
interferon a,, and
interferon y. 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 iow effectoraarget (E:T) ratio of 1:1 (Gong et a1. ( 1994)). In addition, NK-
92 cells have
~o high cytotoxic activity against 8E5 cells, which are infected with HIV and
produce HIV
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virions. NK-92 cells are deposited with the American Type Culture Collection,
designation
NK-92 cells are readily maintained in culture medium, such as enriched alpha
minimum essential medium (MEM; Sigma Chemical Co.. St. Louis, MO) supplemented
a 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-'' 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, CA), is required for long-term growth. When
suspension
n cultures are maintained in this fashion with semiweekly changes of medium,
the cells
exhibit a doubling time of about 24 h.
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 a.nd myelogenous leukemia, lymphoma, myeloma, melanoma,
as
~ s 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.
2o Vector for transfectin<T mammalian cells to produce cytokine
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
2a control element directin~> 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, 1L-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
3c~ are described in general terms in "Current Protocols in Molecular
Biulogy", Ausubel et al.,
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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, NY ( 1989), which are incorporated herein by
reference.
s Modified NK-92 transfected to produce cytokine, and method of transfectin~
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
a immunology, and are described in Ausubel et al. ( 1987, updated quarterly)
and Sambrook
et al. ( I 989). 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.
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
s 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.
NK-92 cells transfected with a vector that directs secretion of a cytokine
such as
3~~ 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
2~ cytolytic effect against tire 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.
NK-92 cells that are~tolytic but not capable of proliferation
An additional modified NK-92 cell of the invention is one that has been
treated in
a such a way that it is no longer able to proliferate, yet whose cytotoxic
activity is preserved.
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One way of achieving this state is by y irradiation. Additional forms of
radiation,
including, for example, ultraviolet radiation, may be employed. Suitable
sources to use for
this purpose include, for example, a "'Cs source (Cis-US, Bedford, MA;
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 mitomy;.in C.
Vector for transfectin~ NK-92 with an element responsive to an inactivatin~l
went
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
inactmated. 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.
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
2o 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.
2p 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
3o advantageous way of achieving this objective.
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Vector for transfecting NK-92 with an altered HLA cell surface molecule.
The HLA cell surface protein, involved in presenting antigens to other cells
of the
immune system, includes a non-immunospecific subunit, the protein (3,-
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 ~3~-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
nr immune response.
Vector for transfecting NK-92 with a gene encoding a cancer cell receptor
molecule.
The NK-~)2 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
la 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
3o 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
<5 CTL targeting moiety in each case.
Methods of treating
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,
~o or cells infected by a pathogenic virus. The NK cells include by way of
nonlimiting
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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 H1V, EBV, CMV, and herpes
may also
be treated.
Treating a biological sample. In vitro biological samples may be treated
experimentally or therapeutically in order to eliminate malignant cells, or
virus-infected
oa cells, in an effective manner. The sample may be drawn from a mammal and
maintained
an intro 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
~c~ Introduction", Birkhauser, Boston, MA ( 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 anu
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.
Treating a cancer or virus infection ex vivo: pur ink. The present invention
~c~ additionally encompasses the ex vivo treatment of a biological sample
suspected of
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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
bodv of the mammal from which it was obtained.
m 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-~2 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
~ a irradiated with y 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
2n the sample to pass through, or like technique. Suitable solid supports
include particles of
polyacrylamide, agarose, cellulose, SepharoseT"' (Pharmacia, Piscataway, NJ),
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
2> carboxyl groups, for example, on the cell surface, thereby immobilizing the
cells on the
support.
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
~o agents include acyclovir or gancyclovir, by way of nonlimiting example.
Functionally
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equivalent vectors, directing the synthesis of alternative cellular components
sensitive to
different agents, are also envisioned within the scope of this embodiment.
The NK cells to he 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.
Treating a cancer or virus infection in vivo: administering NK-92. A further
m method of the invention is directed toward treatment of a cancer or a virus
infection in
v~vo 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
o 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.
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
'o 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.
~s 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
:u cytokine may also be given at two such times, or at all three times with
respect to the time
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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.
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.
m EXAMPLES
Example 1. NK-92 Cells. 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%)
n and horse serum (12.5%) plus 10-6 M hydrocortisone and 1000 U/mL of
recombinant
human IL-2 (rhIL-2). Cells were cultured at 37°C in humidified air
containing 5% CO,.
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
2u with the American Type Culture Collection (ATCC: Rockville, MD) under
designation
The cells have toe morphology of large granular lymphocytes. The cells bear
the
CD56b"~n~, CD2, CD7, CD1 la, CD28, CD45, and CD54 surface markers. In
contrast, they
do not display the CD 1, CD3, CD4, CDS, CDB, CD 10, CD 14, CD 16, CD 19, CD20,
CD23,
25 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-la., IL-6. tumor necrosis factor a., interferon oc, and
interferon y.
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Example 2. Cytotoxic Activity of NK-92 against Different Lenkemic
Cell Lines. The cytotoxic activity of NK-92 against K562, Daudi, TF-l, 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 m RPMI 1640 medium supplemented with 10°o
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 (Large et al., Blood 70:192-199 (1987)). Both TF-1 and AML-
193
to 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 parent 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.
la 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-
a treatment:
Possible role of ICAH-3 and CD44," Leukemia, in press.
The cytotoxic activity of NK-92 (effector) against these target cells was
assessed by
means of a "Cr release assay (Gong et al. (1994)) using the procedure
described by
'_c> Klingemann et al. (Cancer Immunol. Immunother. 33:395-397 (19911). The
percentage of
specific cytotoxicity in triplicate specimens was calculated as:
% 5'Cr release = ~averare experimental cpm - average ~ontaneous~m)
(average maximum cpm - average spontaneous cpm) x 100
Figure 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
KS62 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°io at E:T = I:1) and for AML-193
cells (6% at E:T=l:l).
SR-91 cells appear to be resistant to the cytotoxic effect of the NK-92 cells.
Without
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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 C.'ell Lines. K562 (Ph-chromosome positive [Ph-']
erythroleukemia),
HL60 (promyelocytic), U937 (myelomonocytic), KGl a (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.
m 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,
15 25 mM HEPES (StemCell Technologies), and 5% heat-inactive FCS (RPMI/5% FCS)
at
37° C. in a humidified atmosphere of 5% CO~ in air.
Cell lysis was determined by a 4-hour 5'Cr 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 s'Cr release measured against the same target cells
concurrently. The
2o mean of two separate experiments is presented.
Results of NK-92-mediated cytotoxicity (5'('.r release assay) against various
leukemia, lymphoma, and myeloma target cell lines are summarized in Table I.
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
(S00
's 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 fo~ all the
target cells
except RPMI 8226 and L1266.
'~fl
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Table 1. Cytotoxic activity of NK-92 cells against various leukemia, lymphoma
and
myeloma cell lines
Target 5C):l 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
m K562 NK-92 68 68 64 59 50
PBMCs + IL-2 63 73 67 51 19
KGla NK-92 90 91 80 67 39
PBMCs + IL-2 15 11 12 E> 0
n
U937 NK-92 99 98 96 91 85
PBMCs + IL-2 57 43 23 13 2
DHL-I0 NK-92 95 95 92 94 80
'- PBMCs + TL-2 00 40 24 19 5
Daudi NK-92 94 87 71 48 39
PBMCs + IL-2 65 57 29 16 6
2aJurkat 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 0
3c~
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 0l 63 52 4 13
Raji NK-92 81 75 74 70 54
PBMCs + IL-2 32 67 57 35 13
aoNCI H929 NK-92 ~ 94 89 89 86 51
PBMCs + IL-2 75 58 39 24 5
RPMI 8224 NK-92 82 72 70 72 41
PBMCs + IL-2 95 83 81 67 25
4p
U266 NK-92 84 77 85 81 53
PBMCs + IL-2 84 74 73 56 21
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Example 4. Effect of Deprivation of IL-2 on Cytotoxic Activity of NK-92.
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 "Cr-release was
measured
in 24-hour intervals. Results, summarized in Figure 2, suggest that tire 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 a suitable effect.
Example 5. Co-culture of K562-neo' Cells with PBMC'S and NK-92.
m The transfection of the h562 cells with the neomycin-resistance (neo') gene
has been
described (along et al., Bone Marrov~ Transplan~ 18:63 ( 1966)). Briefly, 5 x
10' K562
cells were suspended in 0.8 mL RPMI 1640/5% FCS and incubated on ice for 10
minutes
with 30 pg of the pMCI-Neo plasmid (provided by Dr. K. Humphries, Terry Fox
Laboratory, Vancouver, BC). The cells were then exposed to a single voltage
pulse ( 125
~ i pF/0.4kV) at room temperature, allowed to remain in buffer for 10 minutes,
transferred into
25-cm- tissue culture flasks {Falcon, Lincoln Park, NJ), and incubated at
37°C in a
humidified atmosphere of 5% CO, in air for 2 days. Transfected cells were
selected in
0.8% Iscove's methylcellulose medium (StemCefl Technologies) supplemented with
30%
FCS, 10-a M 2-mercapto°thanol, and 2 mM glutamine, containing 0.8
mg/mL 6418
20 (neomycin) (Gibco-BRL, Grand Island, NY). Neo' clones of K562 cells were
identified
after 2 weeks, plucked, and maintained in RPMI/10% FCS containing 0.8 mg/mL
neomycin. K562-neo~ cells cultured for 2 days showed a neo' clonogenic cell
doubling
time of 36-42 hours.
Normal PBMCs ( 1 Oa/mL) were spiked with I 0% K562-neo' cells, and NIt-92
cells
25 were added to yield different effector:target (E:T) ratios of NK-92:K562-
neo' cells. (along
et al. (1996)). Briefly, PBMCs were suspended in enriched alpha medium
(MyelocultT"')
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., Exh. Hematol. 21:1263 (1993)). The final concentration of
PBMCs in
au 35-mm tissue culture dishes (Corning, East Brunswick, NJ) was 1 x 106/mL,
and the
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proportion of input K562-neo' 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= in air for 4 or 48 hours at 37"C with and without IL-2 (500
units/mL).
After the culture, cells were washed in RPMI/5% FCS, 10' cells were suspended
in
0.8% Iscove's methylcellulose containing 0.8 mg/mL neomycin, and I.l-mL
volumes were
plated in 3-mm petri dishes. After 7 days at 37"C in a humidified atmosphere
of 5% CO,
in air, colonies were counted. The number of neo' colonies provided a measure
for the
number of surviving clonogenic K562-neo' cells present in the cell suspension
originally
lu plated. Percent survival values for co-cultures containing various numbers
of NK-92 cells
were determined by comparing the number of clonogenic K562-neo' 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~
K562-neo'
cells prior to purging, the absolute number of clonogenic K562-neo' cells
after 4 hours with
~ S no NK-92 cells present was 6400 ~ 820 cells and after 48 hours 28,300
X2100 cells. The
mean ~ SEM of four to eight experiments is reported.
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' 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' cells
from
PBMCs, and that very low survival was observed at E:T of I :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).
,;
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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 hours 48 hours 4 hours 98 hours
10:1 0 0 0 0
5:1 0 0 0 0
W l:l i0.5 ~ 2.1 15.4 ~ 7.2 6.5 ~ 3 15.2 ~ 5.9
0.1:1 56 ~ 14.1 68.5 ~ 19.5 54.4 ~ 13 69.6 ~ 16.8
Example G. Effect of NK-92 Cells on Hematonoietic Proeenitor Cells.
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 l.l-mL
aliquots of
2c~ methylcelfulose-containing media at densities adjusted to give
approx..imately 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
[CFU-GEMM]}. Coloni~a were counted under an inverted microscope 2 weeks later.
The number and growth kinetics of clonogenic hematopoietic cells were
quantified
at a 1: I 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
o cells was noted.
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Table 3 Effect of NK-92 cells on colony formation of normal hematopoietic
progenitor
cells.
Experiment
Number CFU-GEMM BFIJ-E CFLI-C
1 100 46 94
2 200 98 64
l0 3 33 104 103
Example 7. y-Irradiation of NK-92 ('ells. NK-92 cells were irradiated in T25
~ ~ flasks (Corning, Newark, N.1) with the dose indicated using a cesium
source (Cis-US,
Bedford, MA). A dose range of 200-2000 cCiy was tested. After irradiation, the
cells were
washed twice in RPMI, resuspended in medium, and cultured for 72 hours at
37°C in the
presence of 500 IU/mL IL-2. Cytotoxicity (S~Cr-release assay) was performed
with these
cells as described above in Example 2. Prior to performing the 5'Cr-release
assay, the cells
2t~ were left for 24 hours in medium supplemented with IL-2 to allow for
recovery.
Proliferation was assessed by means of a ;H-thymidine incorporation assay.
Prior to
adding ~H-thymidine (0.5 pCi/cell), NK-92 cells were resuspended in thymidine-
free RPMI.
Uptake of ;H-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.
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 'H incorporation, was effectively reduced at a
dose of 1000
cGy (Table 4). The cytotoxicity of NK-92 cells after administration of various
radiation
zo doses is presented in Figure 3. At doses up to 1000 cGy an essentiaiiy
undiminished
cytolytic response was maintained.
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Table 4. Effect of irradiation on the proliferation of NK-92 cells
Experiment Radiation dose (cGv)
number 0 500 1000 1 S00 2000
1 5766 3071 406 125 114
2 4236 2411 1216 1192 S62
3 3994 2046 824 689 748
l0
Example 8. Radiation susceptibility of NK-92 cells. NK-92 cells were
irradiated by a y ray source (Gammacell 40, Atomic Energy of Canada. Ltd.,
Canada). A
I a dose range of 100-3000 cGy was tested. After irradiation, the cells wire
washed and
resuspended in culture rr~dium with rhIL-2 Colony assays, viability and
cytotoxic activity
of the irradiated NK-92 cells were performed using standard techniques (Yap et
al.,
Leukemia, 7:131-139 (1993)). To duantify clonogenic NK-92 cells, NK-92 cells
(500 cells
per mL culture medium) were cultured in a 0.3% agar-based medium supplemented
with
2c~ 12.5% FCS, 12.5% horse serum, 2 mM L-glutamine, 100 pg/mL penicillin 50
~rg/mL
streptomycin, 10'' M mercaptoethanol, and 500 U/mL rhIL-2 at 37°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.
The viability of NK-92 cells, determined by trypan blue staining, and the
recovery
25 of the ability of the NK-92 cells to generate colonies after exposure to
various radiation
doses is shown in Figures 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 depleteu after low
doses of
radiation and totally eliminated by doses above 300 cGy. Figure 6 shows the
cytotoxicity
;o of NK-92 cells to K562, HL60 and 2 patient-derived leukernic samples after
exposure to
the different doses of radiation. Doses of 300, 500, and 1000 cGy allow for
substantial
cytolysis against leukemic cell fines and primary leukemias 1-2 days after
radiation.
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.
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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. Cvtolysis of humanprim~ry (eukemic cells by NK-92.
a. Patient-derived leukemic samples. 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, 1 l chronic myeloid leukemia (CML) cases (6 chronic phase. 1
accelerated phase and
4 blast crisis), 14 B-lineage-acute lymphoblastic leukemia (ALL) cas~.s (13
pre-B-ALLs
and 1 B-ALL) and 6 T-ALL cases, were studied (see Table S). Blast-enriched
mononuclear cells were isolated by Ficoll Hypaque (Pharmacia, Piscataway, NJ)
density
gradient separation and washed in RPMI 1640 medium.
b. Effector cells. NK-92 cells were cultured and maintained iii a,-MEM medium
supplemented with 12.5% FCS, 12.5% horse serum and rhlL-2 (500 U/mL Chiron,
p Emeryville. CA). 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 RPMI 1640 medium with 10% FCS and rhIL-2 ( 100
U/mL)
?o (Yodoi et al., J. Immunol., 134:1623-1630 (1985)).
c. Cytotoxicity assays. 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 5'Cr-labeled target cells (Sx103/well) was tested for
Susceptibility to 4
25 effector cell concentrations. 5'Cr release of target cells alone
(spontaneous release,
determined by placing target cells in 5% Triton) was always <25% of maximal
s'Cr
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)).
:o The degree of sensitivity of patient-derived leukemic cell targets to each
effector was
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defined as insensitive (-!+: <10/10-19% lysis), sensitive (++/+++/++-++: 20-
29/30-39/40-
49% lysis) and highly sensitive (+++++/+++++-t-: 50-59/>60% iysis) at an E:T
ratio of 9:1.
d. Results: ('ytolysis of human~rimary leukemic cells by NK-92 cells. The
sensitivity of patient-derived leukemic cells to the cytotoxic effect of NK-92
cells is
a 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
to (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 t
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
is mediated cytolysis (Table 5).
In comparison, the last two columns in Table 5 present result: obtained with
cell
lines known in the field to have cytolytic activity against tumor cells,
namely, TALL-104
cells and YT cells. Onlv 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~~ 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
zs target samples that were iysed at 18 hours were also Iysed 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 inve~~tion.
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) x different
pathway
~o may be involved in the NK-92 and in the TALL-104 cell mediated cytolysis.
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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).
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 known in the field.
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Table 5. Cytotoxicity
of NK-92,
T-ALL104 and
YT Clone to
Patient-Derived
Leukemic
Cel Is''
C:ytotoxic Sertsitivih
Patients Uiseasc Blast(~~) NK-92 TALL-I04 YT
Status in Sample
ANIL
1 (M4)~ Relapse f'B (GG~o) +++-+-~- t. _
2. (M 1 ) Relapse I'B (50%) , ,.-.y _ _
~
3. (M3) Relapse I'B (50~0) --~-- (++~+), (++++) -
(-)
~ ~. (M2) New BM (28':01 -+-~ (+-;-.-). (+++) NU
()
4. (M4) Rel~actoryPB (90~0) n--,- (++)- (~ ) -
(-)
G. (M4) New 13M (97%) - - _
7 (M4) Nev F'B (39r~) - (-) - (+-+) -
(
8. (M3) Ncw 1'B (550) - (++) - (+++) ~
(_)
1 9. (M3) Nun- BM (32:~) - _ _
~
T_,ILI.
1. Relapse BM (98%) +-r+;+-, -
2. Relapse PB (85~0) +t+-+-~-r _ (_) -+T
(+-.--)
3. Relapse PB (77~0) ~+.~+-~+ _ (, ) _
(_)
204. Relapse I'B (60io) +++-+t - (-) +
(-)
New BM (40%) ~-~-~ _ _
6. New BM (GG%) +-~, _ _
I3_LIIILGfr('-All
1. Relap;c BM (78%) ~++++ +-++~ _
t. New BM (300) +t+~ NU NI)
3. Relansc BM (75'..) .-+-F (++++). (.-++,-) ++
(++)
4 Near BM (97'!0) + (+--) + (t++)
Relapse BM (G(1.o) r (+) - (+) _
(-)
6. Relapse BM (80ro) - NU NU
3(17. Relapse PB (80io) _ - (_) _
8. New- BM (G8%0) - - _
9. New $M (33'0) _ _ (+) _
10. Relapse BM (870) _ _ t~ ;) _
1 1. Relapse BM (75 o) - (-~-++) - (+++~-T) _ (_)
3512. New BM (30io) - - ND
13. New: PB (90al - (-t+) _ (+++) NU
14- New BM (81io) - - ND
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'Ruble 5. (continued)
C.'A fl.
I. BC PR (450) ++-r+~. +++~-, -t+.
2. AL: Pfi (22"-~) ,-+,--.-:-,++ _
> ~. F3C: Pf3 (93~) ~__-~-. t _
d. CP PCB (IS%)1) ~, ~- _
5. CP I'f3 (8,o)I) +, (.,~..:+)NI) NL>
6- CP BM (12o)D (+++) + (t) N17
7. Cf' LM (10.=o)U t (-~-~-~)(++~-+) NI)
1 (I 8. BC PLi (60"r) _
9. HC BM (48-0) + _ (_) _
10. CP Pti (21~b)f) ~- (-,-r) _ (-,-,--~+) _
(_)
11. C'f' 1'I3 (11~)f) - . (,,+~..) _ (_)
I
Notes and Abbreviations. a) (:olumns show results of chromium rclvasc assays
at C.'R c):I after 4 h without parentheses,
and (results utter 18 h enclosed in parentheses); New: ncwfy diagnosed; Nf):
none done; o: FAI3 classilication; ():blast
2ll and promyelocyte: IBM: bone marrow: YB: peripheral blood: I:f3-AI.I.: R(::
blast crisis; A(:: accelerated phase: CP:
chronic phase.
Example 10. Cytotoxicity of NK-92 towards human leukemic cell lines.
3a The following human leukemic cell lines were cultured at 37°C in 5'o
CO, 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). KGl {erythroleukemia), NALM6 (acute pre-B lymphoblastic leukemia),
Raji
(Burkitt's lymphoma), CEM/S (acute T lymphoblastic leukemic cell line
sensitive to
3o 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)).
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
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effective cytolytic actimty for NK-92 against all the target cells tested. The
results
obtained at the low E:T ratio of l:l are especially noteworthy.
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 KGl 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.
IO 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 6. Specific Lysis of Human Leukemia Cell Lines by Nahvral Killer Cell
ClonesNK-92, TALL-104, and YT.
Snecitic t_vsis (°rol
NK92 TAI.(.-104 YT
Fy.ffcctor:Ta~et
Ratio
2(1 'I'ar~et 9:1 3:l I:1 9:1 3:1 1:1 9:1 3:1 1:1
K562 94.1 91.2 82.188.5 85.2 72.5 34.228.2 18.4
F3L(,0 87.9 75.3 79.643.0 16.0 6.9 2.1 1.1 l.p
27 KG 1 64.6 53.8 43.72.7 0.5 0 0.1 0 0
NALM6 72.6 56.8 52.467.8 55.6 33.3 I.0 0.5 0
Raji 86.0 75.4 70.022.2 10.2 0.3 25.118.0 14.2
30
TALL-10-1 57.3 53.2 44.1- - - 3.2 1.4 0.9
CEM/S 56.6 48.R 34.72.7 I.(. 0.9 0.9 0.4 0.3
3O CEMi'1' 57.5 42.1 39.11.5 (1.6 0.3 1.2 0.1 0.2
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Example 11. Effect of NK-92 cells on normal human bone marrow
hematonoietic cells. 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 ( l 981 )).
Hematopoietic cell enriched fractions of normal bone marrows from 14
normal donors were tested by standard CRA to determine their susceptibility to
lysis
m by NK-92 cells. All of the normal bone marrow samples were insensitive to NK-
92
mediated cytolysis (Figure 7).
Example 12. In vivo leukemoeenesis of NK-92 cells in SCID mice.
a. Experimental animals. Severe combined immunodeficient (SCID) mice
I S (CB 17 scid/scid and pfp/Rag-2) (6 to 8 weeks old; Taconic Farms,
Germantown,
NY) 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, 2x 10' viable NK-92 cells in 0.3 mL phosphate buffered
saline
(PBS) were administrated by either intraperitoneal (I.P.) or intravenous (LV.)
route
2tf every other day for 5 injections in each animal. For subcutaneous (S.C.)
inoculations,
2x 10' NK-92 cells were injected in the right flank of SCID mouse, as
described
previously (Yap et al., Blood, 88:3137-3146 (1996)). Thereafter, all the
experimental
animals were administered rhIL-2, SxlOa U every other day for 2 weeks by S.C.
injection. Survival of the animals was followed for at least 6 months after
25 inoculation.
b. Tissue analysis. From each group, 2 SCID mice were sacrificed at the end
of observation anc! tissues from peripheral blood, bone marrow, spleen, liver,
kidney,
lung, and brain were collected for h~stopathological and/or fluorescence
activated cell
sorting (FACS) analysis. Tissue sections from sacrificed SLID mice were fixed
in
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10% neutral buffered formalin, dehydrated and embedded in paraffin, sectioned
and
stained according to standard histological techniques.
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 t7ow 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, CDS, CD7, HLA-DR, CD45, CD56 (Becton Dickinson). A fluorescein
isothiocyanate (FI'fC)-conjugated rat anti-mouse Mab mCD45 (Boehringer-
m Mannheim, Indianapolis, IN) was used for characterization of murine
leukocyte
common anti<~en.
c. Leukemo~enesis. CB-17 scid/scid mice as well as pfp-Rag-2 mice were
inoculated with NK-92 cells by I.V. (n = 3, for each group), S.C. (n = 2, each
~~roup)
and I.P. (CB-17: n = 8; pfp-Rag-2: n = 3) injection. Survival of the animals
was
15 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
2o the tissues by FAGS analysis.
Exnmnle 13. Comparison of antileukemic effect of NK-92 cells with
LAK, NK and T cells against human leukemic cell lines. To isolate the NK cel)
populations, a CeprateR cell separation system based on avidin-biotin
immunoaffinity
25 (CellPro, Bothel, WA) 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 1-2x10k cells/mL, 40pL primary monoclonal
antibody (mouse anti-human CD56) was added and the cells were incubated at
4°C
for 25 minutes. After incubation, the cells were washed and resuspended to a
3o concentration of lxl0~ cells/mL in PBS with 1% BSA. Then, to each one mL
cell
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suspension, 20 pL biotin labeled rat antimouse IgGI antibody was added and the
cells were incubated again at 4°C for 2S minutes. After incubation, the
cells were
washed and resuspended at a concentration of 1x10 cells per mL in PBS with S%
BSA and slowly passed through the avidin column. The CDSi,+ 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 >8S% CDS6+CD3- NK cells. The majority of the other cells in the
fraction (>9S%) were CD3+CDS6- T cells.
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 1 S% human serum and rhIL-2 100 U/mL at
37°C,
~5 S% CO,. The ratios of stimulator cells and feeder cells to responder cells
were S: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 KS62 cells
was
quantitated by 5'Cr-release assay. The CTLs were continuously cultured and fed
with
stimulator and feeder cells in flasks. After 2-3 weeks culture, the monoclonal
2O antibody OKT3 (Ortho Biotech, Raritan, N.J.) was added to the culture for
rapid
expansion of the CTL lines.
The antileukemic effects of NK-92 cells, human LAK cells, NK cells (CD56+
CD3-: CD56+ in Figure 8), and T cells (CD3+CDS6-; CD3+ in Figure 8) were
assessed by measuring in vitro cytolytic activity in standard Cf.A (Figure 8,
Panels
2s A and B), and by measuring inhibition of leukemic cell xenograft growth in
vivo
(Figure 8, Panels C and D) when the effeclor cells and targets were co-
inoculated
subcutaneously into SC1D mice. In order to evaluate the inhibition of growth
in vivo,
the area of the subcutaneous ~>rowths of leukemic nodules as a measure of
their size
was determined once a week after inoculation, and survival of the animals was
also
z« followed. NK-92 cells displayed the highest in vitro cytotoxicity against
K562
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(Figure 8, Panel A) and HL60 (Figure 8, Panel B) of the cells tested, with a
mean
specific lysis of R9% 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).
Correspondingly, the NK-92 cells demonstrated more effective in vivo
inhibition of the growth of K562 (Figure 8, Panel C) and HL60 (Figure 8, Panel
D)
leukemic cells xenografts than did the human LAK and NK cells. The results
shown
in Figure 8 indicate that the NK-92 cells of the present invention have
cytolytic
activity m vitro and tumor-inhibiting activity in vivo that is superior to
those
activities manifested by the known preparations of cytolytic ce:ls normally
present in
humans. These activities are therefore unexpected by a worker in the field of
tumor
cytotherapy.
Example 14. Comnlrison of :~ntileukemic effect of NK-92 cells with
is allo~eneic leukernic-reactive CTL cells. To examine the in vivo effects of
NK-92
cells and other effector cells on the growth of human leukemia xenografts,
Sx106
leukemic target cells alone or mixed with 2x10' NK-92 or other effector cells
(E:T
ratio=4:1) were injected S.C. into SCID mice. The TALL-104 effector cells were
irradiated with 3000 cGy before inoculation to prevent leukemogenesis in SCID
2« 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
SLID mice.
The antileukemia effect of NK-92 cells was evaluated using ailogeneic
leukemia-reactive CTL cells (derived from a patient with T-ALL (TA27)). Both
zs 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 Figure 9): 38%; TALL-
104: 8°io; and T cells (CD3+ in Figure 9): 1.5% specific lysis) against
the TA27
leukemic cells (Figure 9, Panel A). Correspondingly, the subcutaneous growth
of
3o TA27 leukemic cells was inhibited by co-injection of either NK-92 cells or
anti-
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TA27-CTL cells (Figure 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 Figure l 0). In contrast
the
s TALL-104 cells did not show significant in vitro killing against TA27
leukemic cells
by CRA (Figure 9, Panel A). However, moderate inhibition of the leukemic tumor
growth in vivo (Figure 9, Panel B), coupled with a statistically insignificant
(p>0.05)
increase in surviv:-~I, was observed in the animals co-inoculated with TA27
leukemic
cells and irradiated TALL-104 cells (Figure 10).
Exzmnle IS. Antileukemia effect of NK-92 cells in human leukemia
xenograft SCID mice model. 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
~s 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 LP. with 5x10'' leukemic cells from the first passage in
0.2
mL PBS, and 24 hours later 2x10' 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
2« cells which were administered on days 1, 3. S. 7, and 9, with and without
rhIL-2, as
indicated in the Figures.
Ali 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
2p 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). Figure 1 1 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
3o rhIL-2 alone was 72 days (n=5) and 63 days (n=5) (p>0.05), respectively.
All these
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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 (2x10' 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
a injection (Figure 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-~°? cells
(p=0.75), or the
Its 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).
In SCID mice inoculated with human pre-B-ALL (BA31 ) leukemia, with or
U without rhIL-2 treatment, the MST were 63 days (n=5) and 64 days (n=5),
respectively (see Figure 12). For the animals that received 5 doses of 2x10'
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).
2o In animals bearing human AML (MA26), MST was 97 days (n=6) (see
Figure 13). The MST was extended to 173 days among the animals that received 5
doses of 2x10' NK-92 cells (p<0.01)(n=6). Three of the 6 anir.~als 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
?5 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.
The results presented in Figures 11-13 show that in viva treatment of
leukemic tumors can result in enhanced longevity of the subject mice. The
extent of
zo the prolongation of life, and of the improvement in the health of the
animals, is
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dependent on the particular leukemic tumor involved, and ranges from modest or
insignificant (Figure 12) to very dramatic (Figure 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. In order to generate NK-92 cells that constitutively secrete IL-2, two
plasmids encoding human IL-2 were employed.
a. Methods. DNA Clones: The MG-hIL-2 vector (Figure 7) was generously
to provided by Dr. Craig Jordan (formerly of Somatix Corp., Alameda, CA). The
pCEP4-LTR-hIL-2 vector (Figure 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, CA).
t5 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, CA). Cells were transduced according to the
manufacturer's instructions. Briefly, 1.0 or 1.6 ~tm gold particles were
coated with 5
~tg of DNA using calcium chloride spermidine, and ethanol. i\K-92 cells were
2o prepared for bombardment by adherence to poly-L-lysine (Sigma, St. Louis,
MO)
coated 35mm 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
2~ 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.
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
3o the parental and transfected NK-92 cell lines for the presence of genomic
and cDNA
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forms of the hunu.n IL-2 gene. DNA was isolated using DNAzoI (Gibco Life
Technologies Inc., Burlington. ON). Briefly, cells were lysed in DNAzoI 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°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 TG(: 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°C, 2 min 50°C and 2
min 72°C).
to 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, IL) by capillary transfer in 10 X SSC (1.SM NaCI, 1.5M NaCitrate) and
fixed by LIV cross-linking (StrataLinker Stratagene, La Jolla, CA). The blot
was
hybridized with a 'yP radiolabeled human IL-2 probe for 8-12 hours, washed and
15 visualized by autoradiography at -70°C with Kodak X-Omat XAR film.
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 Trizoi reagent (Gibco Life Technologies
Inc.,
Burlington, ON) according to the manufacturer's instructions. Briefly, cells
were
20 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 ODZbon~,.
Fifteen micrograms of RNA was resolved on a 1 % formaldehyde agarose gel in I
2~ MOPS (3-[N-Morpholino]propanesulfonic acid. Sigma, St. Louis, MO) and
blotted as
described previously for Southern blot analysis. The blot was hybridize with
32P
radiolabeled probes for human IL-2 and TNF-a..
DNA probes for Northern and Southern blot analysis were radiolabeled by
random primer extension. DNA probes for human IL-2 and TNF-a, were purified by
3o digestion with appropriate restriction endonucleases and agarose
electrophoresis.
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The DNA was excised from the gel and purified by centrifugation through a Spin-
X
tube filter (Corning Costar, Cambridge, MA), phenol:chloroform extraction and
ethanol precipitation. DNA probe was labeled with a,-''P-dCTP (Sp. Ac. 3000
Ci/mmol; ICN, Montreal, PQ).
Cytokine Determination: IL-2 production by NK-92 cell lines was
determined by ELiSA. Aliquots of IxlO'' of the parental or transfected NK-92
cells
were cultured in 8 ml of IL-2 free Myelocult media for I, 2, and 3 days.
Supernatants were collected from at -20°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, MN). The ELISA is a
horseradish perox~dase/tetramethylbenzidine based colorimetric assay and the
ELISA
microriter plates were read at 450 nm (with a 540 nm correction} in a
microplate
reader (Model EK309, Bio-Tek Instruments Inc., Winooski, VT).
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, MA). Cells were
collected,
washed and resuspended in medium and irradiated in 15 or 50 ml conical
centrifuge
tubes (Becton Dickinson, Franklin Lakes, NJ). Following irradiation, cells
were
washed and resuspended in Myelocult with (for parental NK-92) or without (for
2« transfected cells) IL-2. Cells were cultured for 24, 48 and 72 hours and
assayed for
viability by trypan blue exclusion, for proliferation by ;H thymidine
incorporation
and for cytotoxicity by s'Cr-release assay (as described above).
b. Plasmid MFG-hIL-2. 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
3O replication Therefore, as the transfected cells were maintained in culture
and began
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WO 98/49268 PCT/US98/08672
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 stabiy 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-independent NK-92 cells arising from transfection with the MFG-hIL-
2
were denoted as NK-92MI.
c. Plasmid pCEP4-LTR.hIL-2. Initial observations for cells transfected with
the episomal vector pCEP4-LTR.hIL-2 were identical to those seen with NK-92MI.
ns 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
2o 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
25 pressure in IL-2-free media in favor of cells maintaining the vector.
Therefore, these
cells are capable ~~~ long-term culturing. IL-2-independent NK-92 cells
arising from
transfection with the pCEP4-LTR.hIL-2 are denoted as NK-92CI.
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 I of the hIL-2 gene, which has 88 base pairs (bp), were
used
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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 by fragment
corresponding to
the size expected for the DNA fragment amplified from the genomic IL-2 gene
(Figure 16, Panel .A). However, analysis of both the NK-92MI and NK-92CI
products revealed two bands, the 263 by fragment corresponding to the genomic
hIL-
2 gene as well as a 175 by 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 Figure
16,
W Panel B, both the 263 by genomic fragment and the i 75 by cDNA fragment
hybridized with the probe. These data indicate that both NK-92M1 and NK-92C1
had been successfully transferred and contain the cDNA for hIL-2.
d. Analysis of Gene Expression. To analyze expression of specific cytokines
in the parental and transfected cell lines, they were analyzed by Northern
blot
~s analysis. RNA isolated form the NK-92. NK-92MI, and NK-92C1 cells was
separated
by electrophoresis. transferred to a nylon membrane and hybridized with probes
for
the cytokines hIL-2 and hTNF-cx (see Figure 17). Northern blot analysis of IL-
2 in
these cells reveale,I that IL-2 RNA was not detectable in the parental cell
line
(Figure 17, Panel A, Lane 1 ). However, hIL-2 was found in RNA from both the
2o 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
25 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.
TNF-a expression in the NK cells was also examined using this technique
3n (Figure 17, Panel B). It is seen that all three lines expressed the gene
for this
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CA 02289915 1999-10-29
WO 98/49268 PCT/US98108672
cytokine. A TNF-oc probe hybridized to a 1.6 kDa band in RNA isolated from NK-
92, NK-92MI and NK-92CI (Figure 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
p cytokine.
e. Secretion of h1L-2. 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'' NK-92. NK-92MI and NK-92C1 cells were plated in 8 mL
aliquots and cultured in Myelocult in the absence of IL-2. Supernatants were
n 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 60x higher after 24 hours (9.3
~ 5 pg/mL vs 549.3 pg/mL) to about 80x 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).
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CA 02289915 1999-10-29
WO 98/49268 PCT/US98/08672
N
00 "' h ~ N N ~ N
Ov
V7 M N .. M ~O M ..-.
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W h h O M M ~, h h
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rt
~nN
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~ r- M
..~
.~ ~Y~h
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C h ~ M ~ ~ M ~O00
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""'' ~ tnp~ ~
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49
CA 02289915 1999-10-29
WO 98/49268 PCT/US98/08672
f. Comparisonof cell surface antigens in NK-92. NK-92MI and NK-92CI. To
compare the IL-2-independent transfectants With the parental cells, NK-92MI
and NK-92CI
were analyzed for CD2_ CD3, CD4, CDB, CD10, CE16, CD28, CD56, ICAM-1, ICAM-2,
ICAM-3 and LFA-I expression by fluorescent activated cell sorting (FAGS)
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 oc-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
m 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.
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
5 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.
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
2O 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 polycional 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.
25 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.
3o To evaluate the cytotoxicity of these transfected cells, a standard 4 hour
s'Cr-release assay
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was performed to compare the toxicity of the parental cells to NK-92MI and NK-
92C1 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 (Figure 18). The transfected
cell lines
show cytotoxic activities against K562 and Raji that are very similar to that
of the parental
cells. Cytotoxicity of I~rK-92 against K562 ranged from 82 to 67% while NK-
92MI and
NK-92C1 had cytotoxicity ranges of 77 to 62% and 82 to 62%, respectively. For
Raji
cells, NK-92 had cytotoxicity of 81 to 47°~0, NK-92MI had cytotoxicity
of 75 to 65% and
NK-92CI had cytotoxicity of 82 to 52%.
to Example 18. Effect of Tr~nsfected NK-92 Cells on Hematonoietic
Progenitor Celts. 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
I a 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-92C1 at various E:T ratios. NK-92 was previously sh: wn to have
minimal
effect on hematopoietic stem cells (Example 6). In this example, NK-92MI and
NK-92CI
also show little or no effect on clonogenic output. The number of total
colonies following
Zo incubation with either NK-92MI or NK-92C1 was very similar to control,
although a slight
decrease was seen with the highest effector:PBMC ratio of 1:1 (Figure 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 term 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, whic,~ 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
?o higher ratios, but no consistent correlation between ratio and output was
noted.
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Example 19. Irradiation of the Tr~nsfected NK-92 Celts. 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 ar;d assayed
for
proliferation by the 'H thymidine incorporation assay (see Examples 7 and 8).
Both NK-
s 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 (Figure 20, Panel A). For NK-92MI and NK-
92CI,
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
1c! unirradiated control cells for NK-92CI and 10% for NK-92MI. For
determination of
viability, NK-92, NK-92MI and NK-92C1 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
~ ~ (Figure 20, Panel B). Viability of NK-92 was higher than that of both
transfectants at all
dose rates tested.
The cytotoxicity of these cells following irradiation is shown in Figure 21.
Cells
irradiated at 0, 1,000 and 2,000 eGy 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
?o NK-92 cells three days following irradiation at 1,000 cGy was determined to
be
approximately 10-30% K562 {Figure 21, Panel A) and 30-50% and for Raji (Figure
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°io against Raji three days after
irradiation doses of 1,000 and
2, 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°io
after irradiation with
2000 cGy.
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
3u NK-92CI cells were suppressed at a lower radiation level than for the
parental strains, and
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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
s 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.
IO Example 20. Transfection of NK-92 with a gene for thvmidine kinase.
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 analot~~s, gancyclovir, and acyclovir.
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 PulserT"' ), or by
lipofection (Felgner et
al., Proc. Natl. Acad. Sci. CJSA 84:7413 ( I 987)). The transfected NK-92
cells so produced
2o are susceptible to inactivation by administering gancyclovir or acyclovir.
Example 21. Mutation of NK-92 HLA cell surface protein. NK-92
cells are to be obtained from the cell line described by Gong et al. ( 1994).
The
chromosome bearing the (3~-microglobulin gene is to be isolated, and the DNA
contained
within this chromosome is to be purified away from histones and other DNA-
bound
2s proteins. The gene fragment bearing ~3~-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
"Curr,~nt
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,
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Fritsch and Maniatis, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY,
1989,
incorporated herein by r°ference. The mutated (3=-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 (3=-
microglobulin
s moieties, and will have. lost the ability to bind T-cell receptors.
Example 22. NK-92 Cells Expressing Receptors for a Cancer Cell.
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
Ic> of the CTL population obtained is to be isolated, and the genes for the
MHC 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
l5 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. LJse of NK-92 Cells to Kill HIV-Infected Cells. 8E5 is
2n a cell line harboring HIV that produces HIV virions. 8ESL 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.
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Table 8. Cytotoxic Activity of NK-92 Cells on HIV-Infected Cells
Ta__ rget E:T Ratio % Cytotoxicity
A3.01 50:1 43
20: I 5 I
5:1 44
1.1 44
8E5L 50:1 43
20:1 3 7
S:1 44
I:1 40
8E5 50.1 76
20: I 6<)
5:1 77
l:l 65
l~
It is seen from Table 8 that 8E5 cells which produce HIV particles elicit a
higher cytotoxic
activity than do 8ESL cells, which do not produce FIIV 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
2o MIP-la, 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
eff°ctively lyse HIV-
producing cells in vitro.
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