U.S. patent application number 11/938123 was filed with the patent office on 2009-05-14 for expanded nk cells.
This patent application is currently assigned to Avaris AB. Invention is credited to Evren Alici, Sirac Dilber.
Application Number | 20090123442 11/938123 |
Document ID | / |
Family ID | 40623914 |
Filed Date | 2009-05-14 |
United States Patent
Application |
20090123442 |
Kind Code |
A1 |
Dilber; Sirac ; et
al. |
May 14, 2009 |
EXPANDED NK CELLS
Abstract
The present invention relates to expanded NK cells. The NK cells
have been expanded ex vivo, are activated and have a cytotoxic
phenotype. The cytotoxicity against malignant cells is markedly
increased compared to non-expanded NK cells. The invention also
relates to a method of treatment.
Inventors: |
Dilber; Sirac; (Huddinge,
SE) ; Alici; Evren; (Bandhagen, SE) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
425 MARKET STREET
SAN FRANCISCO
CA
94105-2482
US
|
Assignee: |
Avaris AB
Stockholm
SE
|
Family ID: |
40623914 |
Appl. No.: |
11/938123 |
Filed: |
November 9, 2007 |
Current U.S.
Class: |
424/93.71 ;
435/372 |
Current CPC
Class: |
A61K 2039/585 20130101;
C12N 2501/33 20130101; A61K 2039/572 20130101; A61K 35/12 20130101;
A61K 35/17 20130101; A61K 2039/804 20180801; A61P 35/00 20180101;
C12N 2500/44 20130101; C12N 2500/84 20130101; C12N 2501/23
20130101; A61K 2039/5158 20130101; C12N 2500/90 20130101; C12N
2501/515 20130101; C12N 5/0646 20130101; C12N 2501/2302 20130101;
A61K 39/0011 20130101; C12N 2500/32 20130101; A61K 2035/124
20130101 |
Class at
Publication: |
424/93.71 ;
435/372 |
International
Class: |
A61K 35/12 20060101
A61K035/12; C12N 5/08 20060101 C12N005/08; A61P 35/00 20060101
A61P035/00 |
Claims
1. Natural killer (NK) cells that are ex vivo expanded and said
expanded NK cells are active and have a CD56.sup.+CD3.sup.-
phenotype, said phenotype being cytotoxic against autologous tumor
cells.
2. The NK cells according to claim 1, wherein said NK cells have an
upregulated expression of at least one activating receptor.
3. The NK cells according to claim 2, wherein said activating
receptor is upregulated by at least about 50%.
4. The NK cells according to claim 2, wherein said at least one
upregulated activating receptor is a natural cytotoxic receptor
(NCR).
5. The NK cells according to claim 2, wherein said at least one
activating receptor is selected from the group consisting of 2B4,
CD8, CD16, CD 27, CD226, NKG2D, NKp30, NKp44 and NKp46.
6. The NK cells according to claim 1, wherein said NK cells have at
least about 100% increased cytotoxicity compared to freshly
isolated non-expanded NK cells.
7. The NK-cells according to claim 1, wherein said NK cells have
been expanded for at least about 5 days.
8. The NK-cells according to claim 1, wherein said NK cells have
been expanded for at least about 10 days.
9. The NK cells according to claim 1, wherein said NK cells are
cytotoxic against tumor cells, said tumor cells are cells from
haematological tumors.
10. The NK cells according to claim 1, wherein said NK cells are
cytotoxic against autologous tumor cells, said tumor cells being
selected from the group consisting of cells from multiple myeloma,
leukaemia and lymphoma.
11. The NK cells according to claim 1, wherein said NK cells are
cytotoxic against autologous tumor cells, said tumor cells being
cells from multiple myeloma.
12. The NK cells according to claim 1, wherein said NK cells is
cytotoxic against autologous tumor cells, said tumor cells being
cells from solid tumors.
13. The NK cells according to claim 1, wherein said NK cells are
cytotoxic against autologous tumor cells, said tumor cells being
selected from the group consisting of cells from hepatocellular
tumors, gastrointestinal tumors, ovarian tumors, renal tumors, lung
tumors and pancreatic tumors.
14. The NK cells according to claim 1, wherein said NK cells
exhibit higher degranulation activity compared to non-expanded and
activated NK cells as determined by CD107a expression.
15. A composition comprising NK cells according to claim 1.
16. Method of curative or prophylactic treatment, wherein NK cells
according to claim 1 are administered to a patient with a malignant
disease following allogeneic stem cell transplantation, or a
patient undergoing autologous stem cell transplantation for cancer,
or a patient with haematological malignancies, or a patient with
solid tumors, in a pharmaceutically effective dose.
17. The method according to claim 16, wherein the NK cells are
administered to a patient to prevent recurrence of a malignant
disease.
18. The method according to claim 16, wherein the NK cells are ex
vivo expanded for at least about 5 days prior to administration to
the patient.
19. The method according to claim 16, wherein the NK cells are
expanded at least about 100 fold prior to administration to the
patient.
20. Method of curative or prophylactic treatment, wherein the
composition according to claim 15 is administered to a patient with
a malignant disease following allogeneic stem cell transplantation,
or a patient undergoing autologous stem cell transplantation for
cancer, or a patient with haematological malignancies, or a patient
with solid tumors, in a pharmaceutically effective dose.
21. The method according to claim 20, wherein the composition is
administered to a patient to prevent recurrence of a malignant
disease.
22. The method according to claim 20, wherein the NK cells are ex
vivo expanded for at least about 5 days prior to administration to
the patient.
23. The method according to claim 20, wherein the NK cells are
expanded at least about 100 fold prior to administration to the
patient.
Description
TECHNICAL FIELD
[0001] The present invention relates to expanded natural killer
(NK) cells being cytotoxic against malignant cells and a method of
treatment of malignant disease.
BACKGROUND ART
[0002] NK cells are cytotoxic lymphocytes that lyse certain tumor
and virus infected cells without any prior stimulation or
immunization. NK cells are also potent producers of various
cytokines, e.g. IFN-.gamma., TNF-.alpha., GM-CSF and IL-3.
Therefore, NK cells are also believed to function as regulatory
cells in the immune system, influencing other cells and
responses.
[0003] In humans, NK cells are broadly defined as
CD56.sup.+CD3.sup.- lymphocytes. The cytotoxic activity of NK cells
is tightly controlled by a balance between the activating and
inhibitory signals from receptors on the cell surface. A main group
of receptors that inhibits NK cell activation are the inhibitory
killer immunoglobulin-like receptors (KIRs). Upon recognition of
self MHC class I molecules on the target cells, these receptors
deliver an inhibitory signal that stops the activating signalling
cascade, keeping cells with normal MHC class I expression from NK
cell lysis. Activating receptors include the natural cytotoxicity
receptors (NCR) and NKG2D that push the balance towards cytolytic
action through engagement with different ligands on the target cell
surface. Thus, NK cell recognition of target cells is tightly
regulated by processes involving the integration of signals
delivered from multiple activating and inhibitory receptors.
[0004] Several strategies have been used to enhance NK cell
responses to tumors. Cytokines are used in the treatment of some
human cancers and NK cell differentiation and activation is
affected by cytokines such as interleukins (e.g. IL-2, IL-12.
IL-15, IL-18 and IL-21). The effect of IL-2 administration on
activation and expansion of NK cells in cancer patients have been
assessed in several trials with mixed outcomes depending on type of
tumor and the conditions used for IL-2 administration. One example
of cellular therapy is the NK cell-mediated killing of leukaemia
cells which is based on NK cell alloreactivity.
[0005] Multiple myeloma (MM) is a plasma cell neoplasm
characterized by the clonal proliferation of plasma cells in the
bone marrow (BM). The malignant cells are associated with the
synthesis of monoclonal immunoglobulin and a high incidence of
osteolytic bone lesions. The disease accounts for about 2% of all
cancer deaths and nearly 20% of deaths caused by hematological
malignancies. Although allogeneic stem cell transplantation
occasionally cures these patients and drugs like thalidomide,
lenalidomide and bortezomib have improved outcome, high-dose
chemotherapy followed by autologous stem cell transplantation
(ASCT) still appears to be the best treatment for patients up to
65-70 years of age. However the great majority of patients with MM
are incurable due to the persistence of minimal residual disease.
Thus, novel methods for complementing or improving current
treatments are needed.
[0006] In order to use NK cells in adoptive immunotherapeutic
strategies, the availability of functionally active NK cells on a
clinical scale is crucial. Clinical trials that have been performed
using autologous NK cells were hampered by the fact that the cell
dose was inadaptable to the demands of clinical trials. Therefore,
the development of protocols for large-scale generation of NK cells
is important to evaluate the potential of NK cell-based therapeutic
protocols. Examples of reports that deal with ways of expanding and
culturing human NK-cells are U.S. Ser. No. 10/242,788 and WO
2006/052534.
[0007] Human ex vivo expanded NK cells would be favorable
candidates for immunotherapeutic approaches against malignant
disease if they could target tumor cells.
Aims of the Invention
[0008] One aim of the invention is to provide expanded and active
CD56.sup.+CD3.sup.- NK cells.
[0009] Another aim of the invention is to provide expanded
CD56.sup.+CD3.sup.- NK cells that are cytotoxic against cells of
malignant disease.
[0010] Yet another aim is to provide a method of treatment of a
malignant disease.
SUMMARY OF THE INVENTION
[0011] The present invention relates to ex vivo expanded natural
killer (NK cells) that can be used for immunotherapy of tumor
cells, the NK cells showing no significant cytotoxicity against
normal cells. The NK cells according to the invention are ex vivo
expanded and active cells having the phenotype CD56.sup.+CD3.sup.-.
This phenotype is specifically toxic against tumor cells.
Upregulation of at least one activating receptor contributes to the
cytotoxic activity of the expanded NK cells. In one embodiment the
invention relates to a composition comprising the NK cells
according to the invention.
[0012] In another embodiment the present invention relates to a
method of treatment of a malignant disease wherein NK cells
according to the invention or a composition according to the
invention are/is administered to a patient with a malignant
disease, or a patient undergoing autologous stem cell
transplantation for cancer, or a patient with a malignant disease
following allogeneic stem cell transplantation, or a patient with
haematological malignancies, or a patient with solid tumors, in a
pharmaceutically effective dose.
DISCLOSURE OF THE INVENTION
[0013] Before the present invention is described, it is to be
understood that the terminology employed herein is used for the
purpose of describing particular embodiments only and is not
intended to be limiting, since the scope of the present invention
will be limited only by the appended claims and equivalents
thereof.
[0014] It must be noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
[0015] Also, the term "about" is used to indicate a deviation of
+/-2% of the given value, preferably +/-5%, and most preferably
+/-10% of the numeric values, where applicable.
[0016] Given their strong cytolytic activity and the potential for
auto-reactivity, natural killer (NK) cell activity is tightly
regulated. In order to kill cells with a missing or abnormal MHC
class I expression the NK cells need to be activated. NK cells must
receive an activating signal which can come in a variety of forms,
the most important of which are cytokines, Fc receptors, activating
and inhibitory receptors. In the content of the present invention
the term "activated NK cells" refers to NK cells that have received
an activating signal, thus being capable of killing cells with
deficiencies in MHC class I expression.
[0017] In the content of the present invention the term "NK cell
expansion" relates to the culturing of NK cells that go through a
series of cell division and thus expand in number of cells present
in the culture. The term "expanded NK cells" relates to NK cells
obtained through NK cell expansion. More specifically, in one
embodiment the term "expanded NK cells" relates to a polyclonal
group of chronically activated CD56.sup.+CD3.sup.- cells, expanded
in a specific cGMP grade environment and cGMP grade medium.
[0018] In the content of the present invention the term
"upregulation of receptor" relates to the increase in receptor
density per cell.
[0019] In the content of the present invention cells that have a
"cytotoxic phenotype" relates to cells that are toxic, i.e. they
induce the death of other cells such as, but not limited to, tumor
cells, infected cells or cells that are otherwise damaged or
dysfunctional. Cytotoxic cells of the present invention are mainly
toxic to tumor cells. The cytotoxicity of NK cells towards other
cells can easily be measured, for example, by traditional cell
counting before and after exposure to the NK cells according to the
invention. Such methods are well known to a person skilled in the
art. Examples of suitable methods are, but not limited to,
fluorescent cell counting assay, immunofluorescent cell counting
assay, cell viability assay, and flow cytometry-based cytotoxicity
assay.
[0020] In the context of the present invention the term "effector
cell" relates to a cell that performs a specific function in
response to a stimulus such as cells in the immune system. In one
embodiment effector cells are a type of lymphocytes that are
actively engaged in secreting antibodies. Non-limiting examples of
effector cells are NK cells, T cells and NK-like T cells
[0021] Ex vivo expansion and reinfusion of natural killer (NK)
cells to patients afflicted with a malignant disease offers a new
potentially interesting therapeutic approach to combat such a
disease. A prerequisite for this is the possibility to expand and
use NK cells that meet the demands of clinical trials. The present
inventors have surprisingly shown that ex vivo expanded NK cells
can be used for immunotherapy of tumor cells that often express
varying levels of MHC class I molecules, the NK cells showing no
significant cytotoxicity against normal cells.
[0022] Accordingly, the present invention relates to ex vivo
expanded and activated natural killer cells having the phenotype
CD56.sup.+CD3.sup.-. The NK cells according to the invention are
expanded, active and have a phenotype cytotoxic against tumor
cells, preferably autologous tumor cells. The NK cells can be
obtained from any conventional source and are preferably derived
from peripheral blood, bone marrow, cord blood, cell lines or
cytokine stimulated peripheral blood. NK cells can, for example; be
expanded from a sample of peripheral blood mononuclear cells
(PBMCs). PBMCs are a mixture of monocytes and lymphocytes; blood
leucocytes from which granulocytes have been separated and removed.
The culture conditions are outlined below in "Experimentals".
According to the present invention the NK cells should be expanded
for at least about 5 days, preferably not less than about 10 days,
more preferably not less than about 15 days and most preferably not
less than about 20 days. Already after 5 days the NK cells can show
anti-tumor activity.
[0023] Furthermore, in one embodiment of the invention the NK cells
according to the invention should be expanded at least about 100
fold, preferably at least about 200 fold, more preferably at least
about 400 fold, more preferably at least about 600 fold, more
preferably at least about 1000 fold and even more preferably at
least about 1500 fold.
[0024] The activating pathway of NK cells also includes a series of
different receptors. Activating receptors do not directly signal
through their cytoplasmic tail, but instead associate
non-covalently with other molecules containing ITAMs
(immunoreceptor tyrosine-based activation motifs), that serve as
the signal transducing proteins. Thus, according to one embodiment
of the invention the ex vivo expanded and activated NK cells have
an upregulated expression of at least one activating receptor.
Non-limiting examples of activating receptors are: CD16
(Fc.gamma.RIII), CD25 (IL-2R.alpha.), CD27, CD28, CD69, CD94, CD122
(IL-2R.beta.), CD161, CD226 (DNAM-1), 2B4 (CD244), CD314 (NKG2D),
KIR2S, KIR3S, NCRs (natural cytotoxicity receptors) such as NKp30,
NKp44, and NKp46, CD85H (ILT-1) and IFN-.alpha./.beta.R. Preferably
said at least one activating receptor is selected from the group
consisting of 2B4, CD8, CD16, CD 27, CD226, NKG2D, NKp30, NKp44 and
NKp46. In one embodiment of the present invention the at least one
activating receptor is 2B4 or CD226.
[0025] The NK cells according to the present invention exhibit
higher degranulation activity compared to non-expanded NK cells.
The degranulation activity can be estimated through the
determination of the CD107a expression. CD107a surface expression
correlates closely with degranulation and release of cytotoxic
granules. Degranulation as measured by CD107a expression correlates
to cytotoxic activity of an effector cell, such as an NK cell. The
method of determining degranulation activity through the
determination of CD107a expression is well known to a person
skilled in the art. See, for example, Alter G, Malenfant J M,
Altfeld M. CD107a as a functional marker for the identification of
natural killer cell activity. J Immunol Methods. 2004;
294:15-22.
[0026] The change in receptor expression can be calculated by mean
fluorescence intensity (MFI) ratios:
MFI.sub.dayX/MFI.sub.day0
where x is the number of days of expansion of the NK cell.
[0027] When the MFI for day X samples is higher than for day 0, the
MFI ratio will be higher than 1, which indicates the relative
extent of upregulation in that receptor. Thus, a MFI ratio of e.g.
1.5 would mean a 50% of upregulation of a specific receptor. The
calculation of MFI ratios is well known to persons skilled in the
art.
[0028] According to one embodiment of the present invention the at
least one activating receptor is upregulated by at least about 50%,
preferably at least about 75, more preferably at least about 100%
and most preferably at least about 200%.
[0029] According to another embodiment of the invention the NK
cells have a down-regulated expression of at least one receptor,
such as an inhibitory receptor or a chemokine receptor (e.g. CCR7).
Non-limiting examples of inhibiting receptors are inhibitory killer
immunoglobulin-like receptors (KIRs). Further non-limiting examples
of inhibiting receptors are GL183, KIR2DL1, Lir-1, NKB1, and NKG2A.
It is possible that none inhibitory receptor can be downregulated
in some patients, one inhibitory receptor downregulated in some
patients and more than one in some patients. Thus, in one
embodiment of the invention the NK cells according to the invention
can have an upregulated expression of at least one activating
receptor and a down-regulated expression of at lest one receptor.
In another embodiment the NK cells according to the invention can
have an upregulated expression of at least one activating receptor
and a downregulated expression of at lest one inhibitory
receptor.
[0030] According to another embodiment of the present invention the
expanded and activated NK cells have at least about 100% increased
cytotoxicity compared to non-ex vivo expanded NK cells. Preferably
at least about 150%, more preferably at least about 200% and most
preferably at least about 400% increased cytotoxicity compared to
non-expanded NK cells. Non expanded NK cells relates to freshly
isolated NK cells and short-term activated NK cells. In the context
of this invention the expression "short term activated" means NK
cells that have been expanded over night only.
[0031] The increased cytotoxicity of the expanded and activated NK
cells can also be an effect mediated by an upregulation of a
combination of receptors. Thus, in yet another embodiment the
present invention relates to expanded and activated NK cells having
an upregulated expression of more than one receptor. Thus,
recognition of the target cell by NK cells can involve a
combination of receptors that synergistically deliver activating
signals.
[0032] Another embodiment of the present invention relates to a
composition comprising the expanded and active NK cells as
described above. In addition to the NK cells, a composition
according to the invention may also contain any suitable
physiological buffer, such as, but not limited to, phosphate
buffered saline. Suitable physiological buffers are well known to
the skilled person. Other substances, such as, but not limited to
cytokines such as IL-2 and its derivatives, immunomodulatory drugs
and proteosome inhibitors, that increase the effect of the
administered cells can also be added to the composition or
administered separately. In one embodiment the composition also
contains lower levels of other cells. Non-expanded PBMCs usually
contain a mixture of different cells, for example, NK cells
(CD56.sup.+CD3.sup.-), NK-like T cells (CD56.sup.+CD3.sup.+) and T
cells (CD56.sup.-CD3.sup.+). After expansion NK cells are the
dominating cell type in the culture but also other cell types can
be present in the expanded culture.
[0033] The NK cells according to the invention can also be
administered or used in combination with other cancer therapies,
such as, but not limited to, surgery, radiation and cytotoxic
drugs.
[0034] Several experimental studies have shown that NK cells can
eliminate cancer cells. The results of the study underlying the
present invention suggest that ex vivo expanded and activated cells
have a promising anti-tumor potential compared to resting NK cells.
Thus, the present invention also relates to NK cells being
cytotoxic against tumor cells, said tumor cells preferably being
autologous tumor cells. Autologous tumor cells refer to cells that
are reimplated in the same individual as they came from, i.e. the
donor and the recipient are the same person. Allogeneic tumor
cells, on the other hand, refers to cells taken from different
individuals of the same species, i.e. genetically non-identical
individuals. Two or more individuals are said to be allogeneic to
one another when the genes at one or more loci are not
identical.
[0035] The NK cells according to the present invention are
cytotoxic against tumor cells, said tumor cells being selected
from, but not limited to, the group consisting of haematological
tumors or malignancies and solid tumors or malignancies.
Haematological tumors or malignancies include types of cancer that
affects the blood, the bone marrow and the lymph nodes. In
particular the NK cell according to the invention is cytotoxic
against cells of lymphoma (e.g. Hodgkin's disease and non-Hodgkin's
lymphoma), multiple myeloma (MM), and leukaemia (e.g. acute
lympoblastic leukemia, acute myelogeneous leukaemia, chronic
myelogeneous leukaemia, chronic lymphocytic leukaemia, hairy cell
leukaemia). Examples of solid tumors are, but not limited to,
hepatocellular tumors, gastrointestinal tumors (such as colon
tumors), ovarian tumors, renal tumors, lung tumors and pancreatic
tumors. In one particular embodiment of the present invention the
NK cells are cytotoxic against multiple myeloma cells.
[0036] Since large numbers of activated NK cells now can be
produced and used in the setting of adoptive immunotherapy, the
present invention also relates to a method of treatment. In
leukemia patients autologous ex vivo expanded and activated NK
cells might be helpful for treatment of minimal residual disease
(MRD) after autologous stem cell transplantation. According to the
invention it would be advantageous to administer autologous ex vivo
cultured NK cells, either prophylactically or therapeutically, to
patients undergoing autologous hematopoietic stem cell
transplantation for diseases such as multiple myeloma which have in
general a poor prognosis with high incidence of progressive disease
post transplant. Ex vivo expanded NK cells of donor origin can be
used for the treatment of recurrent malignant disease following
allogeneic stem cell transplantation. Autologous NK cells can be
administered, either prophylactically or therapeutically, to
patients undergoing autologous stem cell transplantation for
cancer. Other ways of using autologous NK cells is for ex vivo
purging of malignant cells in the harvest, for treatment of
patients with hematological malignancies, and as a cellular therapy
for solid tumors.
[0037] Another embodiment of the invention is therefore a method of
curative or prophylactic treatment, wherein expanded, activated and
cytotoxic NK cells with the phenotype CD56.sup.+CD3.sup.-, as
described above, are administered to patients with a malignant
disease, or to a patient with a malignant disease following
allogeneic stem cell transplantation, or patients undergoing
autologous stem cell transplantation for cancer, or patients with
hematological malignancies, or patients with solid tumors, in a
pharmaceutically effective dose. A patient can also be treated with
the inventive method in order to prevent recurrence of a malignant
disease.
[0038] In another embodiment of the inventive method a composition
comprising the NK cells is administered to patients with a
malignant disease or patients undergoing autologous stem cell
transplantation for cancer, or to a patient with a malignant
disease following allogeneic stem cell transplantation or patients
with hematological malignancies, or patients with solid tumors, in
a pharmaceutically effective dose. The composition according to the
invention can also be administered to a patient in order to prevent
recurrence of a malignant disease.
[0039] Examples of malignancies/tumors that can be treated with the
inventive method are outlined above.
[0040] In the inventive method a sample (e.g from peripheral blood,
bone marrow, or cord) is taken from a patent afflicted with a
malignant disease. In one embodiment the patient has been treated
with conventional cancer therapies but the treatment has been
unsuccessful or the malignancy has recurred. The method can also be
a prophylactic treatment, for example, to prevent recurrence of a
malignant disease. Before culturing the cells (e.g. PBMCs), they
are purified and separated according to methods well known for the
skilled person. The cells are thereafter ex vivo expanded as
outlined above and in the "Examples". Subsequently, the expanded
cells are administered to the patient. The patient is thereafter
carefully followed-up in order to determine if the patient has
responded well to the treatment and to determine if the treatment
is to be repeated. Samples can, for example, be taken from blood,
bone marrow and or urine for follow-up of the treatment at regular
time intervals.
[0041] In the inventive method the NK cells are preferably are ex
vivo expanded for at least about 5 days, preferably not less than
about 10 days, more preferably not less than about 15 days and most
preferably not less than about 20 days before administration to the
patient.
[0042] In another embodiment the NK cells have been expanded at
least about 100 fold, preferably at least about 200 fold, and more
preferably at least about 400 fold, preferably at least about 600
fold, more preferably at least about 1000 fold and even more
preferably at least about 1500 fold compared to day 0 of expansion,
before administration to a patient.
[0043] The inventive method can be performed once or repeated
several times. In one embodiment the NK cells according to the
invention are administered to the patient about 1-10 times,
preferably about 1-7 times, more preferably about 1-5 times and
most preferably about 1-3 times. The administration route can be
any suitable way of administration well known to the skilled person
for example, but not limited to; intravenous, intraperitoneal and
intratumoral administration. The dosage can be the same in all
administrations or for example high in the first administration(s)
and than lower in subsequent administrations.
[0044] Administration, alone or in combination with the NK cells of
the invention, of subcutaneous IL-2 or its derivatives as well as
immunomodulatory drugs such as, but not limited to, thalidomide or
proteosome inhibitors such as, but not limited to, bortezomib, may
further increase the effect of administered cells.
[0045] The features of the ex vivo expanded and activated NK cells
to be used in the method of treatment are as described above for
the NK cells according to the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0046] The invention is further described in the description,
examples and claims with reference to the attached figures in
which:
[0047] FIG. 1 shows expansion dynamics of blood and bone marrow
samples obtained from seven multiple myeloma patients and cultured
for 20 days under identical conditions. Bulk cells in culture
expanded to a total of 511 fold (A). Initial OKT-3 treatment lead
to an increased percentage of T cells in the culture during the
first five days of culture followed by a decrease after withdrawal
of OKT-3 (B). The subsequent increase continued until finally NK
cells dominated in the culture (C). Results are shown as means
+SD.
[0048] FIG. 2 shows changes in the receptor expression patterns of
NK cells following expansion and activation. Representative data
(Patient 5) showing comparative phenotyping of day 0 (grey) and day
20 (white) cells (A). Each patient's MFI ratios () of day 20 to day
0 as well as the medians (-) of seven patients were plotted for
each receptor (B). The dashed line shows MFI ratios=1 which
indicates unaltered receptor expression during the expansion
period. Values above the line (MFI ratio>1) indicate
upregulation and below (MFI ratio<1) denote downregulation.
[0049] FIG. 3 shows the results from the cell mediated cytotoxicity
assays. NK cell cytotoxicity against K562 cell line was measured by
the standard chromium release assay and flow cytometry based
cytotoxicity assay (A). The flow cytometry based cytotoxicity assay
demonstrates an increased cytotoxicity of expanded NK cells (day
20) against autologous MM cells compared to freshly isolated NK
cells (B). Expansion and activation of the cells did not affect the
cytotoxicity against autologous normal BM cells (B). Representative
data (Patient 5) is shown to demonstrate the analysis procedure
(C).
[0050] FIG. 4 shows blocking of activating receptors on NK cells.
The results are shown as mean 522 inhibition+SD (N=3).
[0051] FIG. 5 shows CD107a mAb based degranulation assay against
primary MM cells. Representative data (Patient 5) demonstrating the
expression of CD107a on NK cells after contact with the NK
sensitive K562 cell line and autologous MM cells (A). Comparison of
CD107a expression on day 0, 5 and 20 NK cells upon contact with 519
autologous MM cells (B). The results are shown as percent CD107a
cells +SD (N=3).
MODE(S) FOR CARRYING OUT THE INVENTION
[0052] The invention will now be further described in the
experiments outlined below wherein efficient ex vivo NK cell
expansion from PBMCs (peripheral blood mononuclear cells) of
Multiple Myeloma (MM)+patients using clinical grade components is
demonstrated. Furthermore, the ability of these NK cells to kill
autologous tumor cells is shown. These data suggest the possibility
of using autologous ex vivo expanded NK cells for immunotherapy of
MM.
EXPERIMENTAL
Patients and Acquisition of Patient Material
[0053] Peripheral blood and bone marrow (BM) samples from seven
newly diagnosed patients at different stages of MM were included in
the study. The patients were followed at the Department of
Hematology, Karolinska University Hospital Huddinge, Stockholm,
Sweden. The study was approved by the local research ethics
committee. Informed consents were obtained from all patients.
Patients' characteristics at the time of blood and BM sampling for
the study are given in Table 1.
TABLE-US-00001 TABLE1 Patient characteristics at the time of blood
and bone marrow sampling for the study. MM Stage Patient* Age
Gender (Durie &Salmon) MM Type 1 41 Male IIIB IgG-.lamda. 2 80
Male IB IgG-.lamda. 3 80 Male IIIB Light chain-.lamda. 4 75 Female
IIA IgG-.lamda. 5 66 Female IIB IgG-.lamda. 6 53 Female IA
IgG-.kappa. 7 64 Male IIB Light chain-.kappa. *All the patients
included in this study were diagnosed in the first half of 2006 and
had not yet started any treatment regimen at sampling time.
[0054] PBMCs as well as BM mononuclear cells (BMMCs) were isolated
by gradient centrifugation, using Lymphoprep (Axis-Shield, Oslo,
Norway). PBMCs and BMMCs were washed twice with phosphate-buffered
saline (PBS) (Gibco, Grand Island, N.Y., USA), and cell viability
was assessed by Trypan blue exclusion. To avoid inter-experimental
variability, PBMCs and BMMCs were directly frozen in human serum
albumin (Baxter) containing 6% DMSO (Wak-chemie medical, Germany)
for subsequent phenotyping and cytotoxicity experiments.
Example 1
Ex Vivo Expansion of NK Cells from PBMCs
[0055] Material & Methods: The culture conditions for the
expansion of cytotoxic cells have previously been optimized on
PBMCs from healthy individuals (Carlens et al., Hum. Immunol. 2001;
62:1092-1098). Briefly, PBMCs were initially thawed and cultured in
T25 flasks (TPP, Trasadingen, Switzerland) at a concentration of
0.5.times.10.sup.6 cells/ml in CellGro SCGM serum-free medium
(CellGenix, Freiburg, Germany) with the addition of 5% human serum
(Biowhittaker-Cambrex, Md., USA) and 500 U/ml rhlL-2
(Proleukin.RTM., Chiron Corporation, Emeryville, Calif., USA). For
the first 5 days, the medium was further supplemented with anti-CD3
antibody (Orthoclone OKT-3, Ortho Biotech Inc., Raritan, N.J., USA)
to a final concentration of 10 ng/ml. On day 5 of culture, the
OKT-3-containing medium was washed out, and fresh medium with IL-2
(500 U/ml) and 5% human serum was added. The cultures were then
replenished with fresh medium every other day throughout the
culture period. Total cell numbers were assessed by staining cells
with Trypan blue dye on days 0, 5-6, 9-10, 14-15, and 20 of
culture. Absolute cell counts were calculated by multiplying the
total number of cells with the percentage of these subsets
determined by flow cytometry. To prevent contact inhibition of cell
growth, the cells were transferred to bigger flasks when necessary.
The final products were evaluated for purity, viability and
phenotype.
[0056] Results: In order to study whether it is possible to expand
NK cells from MM patients using GMP grade components, cultures of
PBMCs from seven patients with MM were established. At the start of
the culture (day 0), the mean percentage of NK cells
(CD56.sup.+CD3.sup.-) was 11% (range: 7-17%) whereas T cells
constituted 57% (range: 36-81%). NK cell expansion approached
log-linearity after an initial non-proliferative phase of about
five days. By day 20, the total cell population had expanded on
average 511-fold (range: 123-1545) (FIG. 1A) and, of these, NK
cells had expanded on average 1625-fold (range: 502-2658) (FIG.
1B). Due to the relatively greater expansion of NK cells compared
to the other cell types, NK cells dominated the culture towards the
end of the culture period, reaching on average 66% of the cells by
day 20 (FIG. 1C). The percentage of NK-like T cells
(CD56.sup.+CD3.sup.+) did not change significantly during the
culture period (day 0: 14%, day 20: 18%), while the percentage of T
cells (CD56.sup.-CD3.sup.+) declined following withdrawal of OKT3
at day 5, decreasing to an average of 14%. These results show that
NK cells from MM patients can be expanded efficiently ex vivo by
using the present 20 day culture approach.
Example 2
Flow Cytometry Based Phenotyping of NK Cells and NK Ligands on
Multiple Myeloma (MM) Cells
[0057] Material & Methods: The cell phenotype and expansion
dynamics of subpopulations were analyzed by flow cytometry on days
0, 5-6, 9-10, 14-15 and 20 of culture using standard procedures
with fluorochrome conjugated mAbs against the following surface
antigens CD3, CD14, CD38, CD56 and CD138.
[0058] Day 0 and day 20 cells from all patients were subjected to a
more detailed immunophenotypic analysis. To avoid inter-acquisition
variability, all frozen samples were simultaneously thawed for a
detailed phenotypic characterization of CD56.sup.+CD3.sup.- (NK)
cell subset by flow cytometry. This panel included fluorochrome
conjugated mAbs against the following surface antigens: CD2
(RPA-2.10), CD3 (UCHT-1), CD4 (SK3), CD7 (M-T701), CD8 (HIT8a),
CD14 (MOP9), CD16 (3G8), CD19 (HIB19), CD25 (M-A251), CD27
(M-T271), CD38 (HIT2), CD56 (B159), CD57 (NK-1), CD161 (DX12),
CD183 (3D12), CD184 (12G5), CD195 (2D7/CCR5), CD197 (1C6/CXCR3),
CD226 (DX11), NKB1 (DX9), LFA-1 (HI111), CD62L (DREG56), CD69
(FN50) and CD138 (MI15) purchased from BD Biosciences, San Jose,
Calif., USA; CD48 (MEM102) from Biosource AB, Stockholm, Sweden;
CD158B1/B2,j (GL183), CD244 (2B4) (C1.7), NKG2D (ON71), NKp30
(Z25), NKp44 (Z231), NKp46 (BAB281), LIR-1 (HP-F1), Valpha24 (C15),
Vbeta11 (C21) from Beckman Coulter Inc., Fullerton Calif., USA;
NKG2A (131411), NKG2C (134591), KIR2DL1 (143211), KIR2DL3 (180701)
from R&D Systems, Minneapolis, Minn., USA.
[0059] All antibody stainings for flow cytometry were done
according to the following protocol. Fc receptors were blocked by
incubation with 1 .mu.g human IgG per 105 cells for 15 minutes on
ice. The cells were then washed once with PBS and incubated with
appropriate amounts of antibody at 4.degree. C. for 30 minutes
followed by another wash with PBS. For both panels, LIVE/DEAD
Fixable Red Dead Cell Stain (Invitrogen, Carlsbad, Calif., USA) was
used for dead cell exclusion according to the manufacturer's
instructions. Briefly, 1 .mu.l of dye was applied to 1.times.106
cells resuspended in 1 ml of PBS and incubated on ice for 30
minutes. The labeled cells were then washed with PBS and fixed in
4% PFA prior to data acquisition. Cells were analyzed by nine-color
flow cytometry (CyAn.TM. ADP LX, Dako A/S, Glostrup, Denmark)
calibrated with CompBeads.TM. and appropriate isotype controls (BD
Biosciences). The acquired data were analyzed with Dako Cytomation
Summit software versions 4.2 and 4.3 (Dako A/S) and FlowJo software
version 7.2 for PC (Tree Star Inc., Ashland, Oreg., USA) setting
appropriate SSC/FSC gates around the lymphocyte population and
using LIVE/DEAD Fixable Red Dead Cell Stain negative cells. From
the lymphocyte gate, NK cells were gated as the CD56.sup.+CD3.sup.-
population. NK-like T cells and T cells were gated as CD3+CD56+ and
CD3+CD56- populations respectively. MM cells were gated as
CD38+CD138+. In each sample a minimum of 105 cells was
analyzed.
[0060] For each cell surface receptor analyzed, mean fluorescence
intensity (MFI) values were calculated for day 0 and day 20
samples. To estimate the change in receptor expression, MFI ratios
were calculated (MFlday20/MFlday0) for each receptor. When the MFI
for day 20 samples was higher than for day 0, the MFI ratio was
higher than 1, which indicated the relative extent of up regulation
in that receptor. Likewise, an MFI ratio below 1 was interpreted as
down regulation in the expression of that receptor.
[0061] Results: To characterize the final expansion product with
respect to the starting material, a detailed flow cytometric
analysis was undertaken (representative data of one patient is
shown in FIG. 2A). For each receptor analyzed, MFI values were
calculated in samples from day 0 and day 20 cultures and used their
ratio (MFlday20/MFlday0) as an indicator of the change. FIG. 2B
illustrates the MFI ratios of all patients for all receptors
analyzed as well as the median values. Briefly, in the final
product, the inventors observed a significantly upregulated
expression of the following activating receptors: 2B4, CD8, CD16,
CD27, CD226, NKG2C, NKG2D, NKp30, NKp44 and NKp46. The inhibitory
receptors KIR2DL3 and LIR-1 were also upregulated. The chemokine
receptor CCR7 was significantly down-regulated whereas CXCR3 was
markedly upregulated. No significant changes in the expression
levels of CD2, CD7, CD57, CD62L, CD69, CD161, LFA-1, GL183,
KIR2DL1, NKB1, NKG2A, CCR5 or CXCR4 were observed.
Example 3
Evaluation of Cell-Mediated Cytotoxicity
[0062] Material & Methods: The cytotoxic capacity of NK cells
before and after expansion was evaluated in vitro with a standard 4
hour 51 Cr-release assay against NK-sensitive K562 cells. Because
the 51 Crrelease assay is not suitable for primary MM cells 16-18,
a flow cytometry based cell mediated cytotoxicity assay was
used.
[0063] For the 51 Cr-release assay, K562 target cells were labelled
with 100 .mu.Ci of 51Cr for one hour at 37.degree. C., washed twice
with PBS, and resuspended in 1 ml of RPMI medium. A total of
3.times.104 target cells in 100 .mu.l RPMI medium was placed in
triplicates into V-bottom 96-well plates and incubated for 4 hours
with 100 .mu.l of effector cells at appropriate concentrations to
obtain effector:target ratios from 1:3 to 10:1. Aliquots of
supernatants were then counted using a Packard Cobra Auto-Gamma
5000 Series Counting System (Meriden, Conn., USA). The percentage
of specific 51Cr release was calculated according to the formula:
percent specific release=[(experimental release-spontaneous
release)/(maximum release-spontaneous release)].times.100.
[0064] For the flow cytometry based assay, autologous MM cells or
K562 controls were labelled with TFL4 reagent of the
CytoToxiLux-PLUS kit (Oncolmmunin Inc., Gaithersburg, Md., USA)
according to the manufacturer's instructions. 5.times.104 target
cells were placed in tubes together with different amounts of
effector cells to obtain effector:target ratios from 1:3 to 10:1 in
a final volume of 300 .mu.l RPMI medium and incubated at 37.degree.
C. for 4 hours. The cells were then washed once with PBS. Following
Fc receptor blockade with IgG (1 .mu.g/105 cells) on ice for 20
minutes to avoid antibody-dependent cellular cytotoxicity, the
cells were incubated with appropriate amounts of fluorochrome
conjugated mAbs against CD3, CD34, CD38, CD56 and CD138 at
4.degree. C. for 30 minutes. After washing with PBS, the cells were
resuspended in 500 .mu.l of PBS containing 5 .mu.g
7-aminoactinomycin D (7-AD; Invitrogen, Carlsbad, Calif., USA) and
incubated in the dark for an additional 15 minutes at 4.degree. C.
before data acquisition by nine-color flow cytometry. Cytotoxicity
was assessed according to the following formula: percent killing
=[(experimental death-spontaneous death)/(maximum death-spontaneous
death)].times.100.
[0065] To compare cytotoxicity of short term activated and expanded
cells, day 5 cells from three patients were also tested for
cytotoxicity against K-562 cell line and primary autologous myeloma
cells.
[0066] For blocking experiments, effector cells were preincubated
at 4.degree. C. for 30 minutes with 10 .mu.g/ml of isotype controls
or the mAbs against the following receptors: 2B4 (C1.7)19, CD226
(DX11)20, NKG2C (134522), NKG2D (1D11)21, NKp30 (Z25)22, NKp44
(Z231)22, NKp46 (BAB281)22, and CD27 (1A4)23.
[0067] Results: The inventors next investigated the cytotoxic
activity of ex vivo expanded NK cells against a standard NK target
cell line, K562. Cytotoxicity against K562, measured by a flow
cytometry-based cytotoxicity assay and confirmed by a standard
4-hour chromium release assay, was markedly increased by day 20 NK
cells when compared to day 0 and day 5 cells (FIG. 3A). At a 10:1
effector to target cell ratio, 62% of the K562 targets were killed
by the day 20 NK cells whereas day 0 and day 5 cells killed only 8%
and 29% of K562 targets, respectively, at a similar effector to
target ratio.
[0068] In an in vivo immunotherapy approach, the present NK cells
would only be useful if they are able to target autologous MM
cells. The inventors thus assessed cytotoxicity against autologous
MM cells by a flow cytometry based assay. At day 20, marked
cytotoxic activity of NK cells against autologous MM cells was
observed whereas both day 0 and day 5 cells showed no or only low
levels of cytotoxicity (FIG. 3B). At a 10:1 effector:target ratio,
61% of autologous MM cells were killed by day 20 cells
(representative data of one patient is shown in FIG. 3c). Notably,
no significant cytotoxicity against non-MM (CD138-) cells was
observed. Ex vivo expanded NK cells show increased cytotoxicity
against autologous MM cells.
[0069] To determine the relative contributions of different
activating receptors on autologous MM cytotoxicity, effector cells
were preincubated with blocking antibodies against several
individual activation receptors, or their combinations, and then
co-incubated with autologous MM cells. Cytotoxicity was partially
inhibited by blocking 2B4 (60% inhibition), CD226 (DNAM-1; 53%),
NKG2C (48%), NKG2D (49%), CD27 (50%), NKp30 (57%), NKp44 (55%), and
NKp46 (59%) (FIG. 4). This indicates that several activating
receptors may contribute to MM cytotoxicity in line with current
knowledge of receptor synergy for induction of cytotoxicity. Thus,
the cytotoxicity against autologous MM involves target cell
interaction with activating NK cell receptors.
Example 4
Analysis of NK Cell Degranulation
[0070] Material & Methods: NK cells were coincubated with
target cells at a ratio of 1:1 in a final volume of 200 .mu.l in
round-bottomed 96-well plates at 37.degree. C. and 5% CO.sub.2 for
6 h. Fluorochrome-conjugated anti-CD107a mAb or the corresponding
IgG1 isotype control was added at the initiation of the assay.
After 1 h of coincubation, Monensin (GolgiStop, Becton Dickinson)
was added at a 1:100 dilution. Surface staining was done by
incubating cells with anti-CD3 and anti-CD56 mAbs for 30 mins on
ice. The cells were then washed, resuspended in PBS and immediately
analyzed by flow cytometry.
[0071] Results: In order to better pinpoint the active population
within the final expansion product showing cytotoxicity against
autologous MM cells, the inventors shifted focus from MM cell lysis
to effector cell activation by analyzing the surface expression of
CD107a on different subpopulations upon contact with MM cells.
CD107a expression correlates closely with degranulation and release
of cytotoxic granules. Approximately 30% of Day 20 NK cells
expressed CD107a on the cell surface on contact with K562 cell
line. Similar degranulation was observed against autologous MM
cells (representative data of one patient is shown in FIG. 5A).
Analysis of expanded cells showed that NK cells were the main
degranulating population following challenge with autologous MM
cells (FIG. 5B). Thus, autologous MM cells trigger degranulation of
ex vivo expanded NK cells.
Discussion (Example 1-4)
[0072] Although previous reports suggest that cytokine activation
of NK cells may lead to a better recognition of MM cells, MM cells
are considered to be resistant to lysis by resting and short term
activated autologous NK cells. Similar to the immune system defects
mentioned above, this resistance has been explained by NK cell
dysfunctions in MM patients including impaired NK cytotoxicity and
increased levels of soluble IL-2 receptors as well as decreased
expression of a number of activating receptors compared to healthy
controls. The results indicate that expansion and/or long term
activation of NK cells may reverse this potential dysfunction,
since it was paralleled by induction of surface expression of
CD107a and cytotoxicity of autologous MM. Based on assessment of
induction of degranulation measured by expression of CD107a, it was
concluded that expanded NK cells are the major effector compartment
exerting autologous anti-MM activity under the present experimental
conditions. Within the course of the present studies the inventors
also phenotyped the expanded NK cells. This allowed a comparison of
day 0 and day 20 NK cells. Since a balance of activating and
inhibitory signals regulates NK cell function, optimal NK cell
effector function is expected to occur in situations where the
expression of activating NK cell receptors is adequate and not
suppressed by inhibitory signals. Reduced 2B4 expression in NK
cells from MM patients has been suggested to play a role in the
immune escape mechanism of MM expressing its ligand CD4841, however
it cannot be excluded that it might be a consequence of
interactions between NK and MM cells. The upregulation of 2B4 after
ex vivo expansion is likely one factor contributing to the
cytotoxicity observed against autologous MM cells. Furthermore,
NCRs and NKG2D, which presumably take part in the recognition of MM
cells by NK cells, are significantly upregulated, suggesting
possible pathways for autologous MM cell killing. The upregulation
of CD226 which is a tumor surveillance receptor on NK cells, and a
potent inducer of cytotoxicity against many tumor cell lines of
hematopoetic and non-hematopoetic origin, could also contribute to
the increase in cytotoxicity. Furthermore, it has previously been
shown that CD27 NK cells are tightly regulated by inhibitory
receptors whereas CD27 high NK cell subset is more cytotoxic. The
upregulation of CD27 during expansion may also contribute to
elevated levels of cytotoxicity.
[0073] Recently published results of clinical trials testing NK
cell based immunotherapy involve infusion of resting and short-term
IL-2 activated NK cells to patients with malignancies. These trials
have shown that adoptively transferred NK cells are well tolerated.
The present results shows that the present ex vivo expanded
autologous NK cells have a promising anti-MM potential compared to
resting or short-term activated NK cells. The high levels of NK
cell expansion using cGMP quality components is of particular
importance in relation to reaching an appropriate infusion dose in
settings of immunotherapy.
[0074] A concern for the use of activated NK cells, especially in
the allogeneic settings, is that they could cause a tissue damaging
reaction. The present data shows that the recognition of autologous
MM cells by ex vivo expanded NK cells involve a certain degree of
specificity. The inventors demonstrated that day 20 NK cells lysed
MM cells but spared non-MM cells from the same patient.
[0075] Although particular embodiments have been disclosed herein
in detail, this has been done by way of example for purposes of
illustration only, and is not intended to be limiting with respect
to the scope of the appended claims that follow. In particular, it
is contemplated by the inventor that various substitutions,
alterations, and modifications may be made to the invention without
departing from the spirit and scope of the invention as defined by
the claims.
* * * * *