U.S. patent application number 09/953323 was filed with the patent office on 2002-08-22 for modulation of il-2- and il-15-mediated t cell responses.
Invention is credited to Li, Xian Chang, Strom, Terry B..
Application Number | 20020114781 09/953323 |
Document ID | / |
Family ID | 22872402 |
Filed Date | 2002-08-22 |
United States Patent
Application |
20020114781 |
Kind Code |
A1 |
Strom, Terry B. ; et
al. |
August 22, 2002 |
Modulation of IL-2- and IL-15-mediated T cell responses
Abstract
The present invention is based, in part, on expression studies
of IL-2 and IL-15 receptor subunits by cycling T cells in vivo. In
one embodiment, the invention generally features novel combinations
of IL-2 and IL-15 antagonists and methods of suppressing the immune
response by administering these antagonists. In each case,
suppression is achieved by administration of a first agent that
targets an IL-15 molecule or an IL-15 receptor (IL-15R) and a
second agent that targets an IL-2 molecule or an IL-2 receptor
(IL-2R). More generally, the invention features novel combinations
of agents that, when administered to a patient (or to a transplant
ex vivo), reduce the number of antigen-reactive T cells. For
example, the invention features compositions (e.g.,
pharamaceutically acceptable compositions) that include two or more
agents, each of which promote T cell death. Alternatively, the
composition can contain at least one agent that promotes T cell
death and at least one agent that inhibits T cell proliferation.
The agent that promotes T cell death can promote AICD (activation
induced cell death), passive cell death, ADCC (antibody dependent
cell-mediated cytotoxicity) or CDC (complement directed
cytotoxicity).
Inventors: |
Strom, Terry B.; (Brookline,
MA) ; Li, Xian Chang; (Newton, MA) |
Correspondence
Address: |
LEE CREWS, PH.D.
Fish & Richardson P.C.
225 Franklin Street
Boston
MA
02110-2804
US
|
Family ID: |
22872402 |
Appl. No.: |
09/953323 |
Filed: |
September 14, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60232251 |
Sep 14, 2000 |
|
|
|
Current U.S.
Class: |
424/85.2 ;
424/145.1 |
Current CPC
Class: |
A61P 17/00 20180101;
A61P 43/00 20180101; A61P 21/00 20180101; A61P 35/00 20180101; A61P
17/06 20180101; A61P 29/00 20180101; A61P 21/04 20180101; A61P 5/30
20180101; A61P 3/10 20180101; A61K 38/2013 20130101; A61P 37/02
20180101; C07K 14/5443 20130101; A61P 19/04 20180101; A61K 38/2013
20130101; A61K 38/2086 20130101; A61K 2300/00 20130101; A61P 19/02
20180101; A61P 1/00 20180101; A61K 2300/00 20130101; A61P 31/18
20180101; A61P 19/08 20180101; A61K 38/2086 20130101; C07K 2319/30
20130101; A61P 25/00 20180101; A61P 37/06 20180101; C07K 14/55
20130101; C07K 2319/00 20130101; A61P 5/14 20180101 |
Class at
Publication: |
424/85.2 ;
424/145.1 |
International
Class: |
A61K 039/395; A61K
038/20 |
Goverment Interests
[0001] The work described herein was supported in part by a grant
from the National Institutes of Health. The U.S. government may,
therefore, have certain rights in the invention.
Claims
What is claimed is:
1. A therapeutic composition comprising a first agent that targets
an interleukin-15 receptor (IL-15R) and a second agent that targets
an interleukin-2 receptor (IL-2R).
2. The therapeutic composition of claim 1, wherein the first agent
comprises a substantially pure mutant IL-15 polypeptide that binds
a subunit of an IL-15R.
3. The therapeutic composition of claim 2, wherein the subunit is
an IL-15R.alpha. subunit.
4. The therapeutic composition of claim 3, wherein the mutant IL-15
polypeptide has a mutation at position 156 of SEQ ID NO: 2.
5. The therapeutic composition of claim 4, wherein the mutant IL-15
polypeptide also has a mutation at position 149 of SEQ ID NO:
2.
6. The therapeutic composition of claim 4, wherein the mutation at
position 156 of SEQ ID NO: 2 is a substitution of aspartate for
glutamine.
7. The therapeutic composition of claim 5, wherein the mutation at
position 149 of SEQ ID NO: 2 is a substitution of aspartate for
glutamine.
8. The therapeutic composition of claim 5 wherein the mutant IL-15
polypeptide has a substitution of aspartate for glutamine at
positions 149 and 156 of SEQ ID NO: 2.
9. The therapeutic composition of claim 2, wherein the first agent
further comprises a moiety that leads to the elimination of
IL-15R-bearing cells.
10. The therapeutic composition of claim 9, wherein the moiety that
lyses IL-15R-bearing cells is an Fc region of an IgG molecule.
11. The therapeutic composition of claim 1, wherein the first agent
comprises a substantially pure anti-IL15R antibody.
12. The therapeutic composition of claim 1, wherein the second
agent comprises an antibody that specifically binds IL-2 or an
IL-2R.
13. A method of suppressing an immune response in a patient, the
method comprising administering to the patient a therapeutic
composition comprising a first agent that targets an IL-15R and a
second agent that targets an IL-2R.
14. The method of claim 13, wherein the patient has an immune
disease, particularly autoimmune disease or is at risk of
developing an immune disease, particularly autoimmune disease.
15. The method of claim 14, wherein the autoimmune disease is a
rheumatic disease selected from the group consisting of systemic
lupus erythematosus, Sjogren's syndrome, scleroderma, mixed
connective tissue disease, dermatomyositis, polymyositis, Reiter's
syndrome, and Behcet's disease.
16. The method of claim 14, wherein the autoimmune disease is
rheumatoid arthritis.
17. The method of claim 14, wherein the autoimmune disease is type
I diabetes.
18. The method of claim 14, wherein the autoimmune disease is an
autoimmune disease of the thyroid selected from the group
consisting of Hashimoto's thyroiditis and Graves' Disease.
19. The method of claim 14, wherein the autoimmune disease is an
autoimmune disease of the central nervous system selected from the
group consisting of multiple sclerosis, myasthenia gravis, and
encephalomyelitis.
20. The method of claim 14, wherein the autoimmune disease is a
variety of phemphigus selected from the group consisting of
phemphigus vulgaris, phemphigus vegetans, phemphigus foliaceus,
Senear-Usher syndrome, and Brazilian phemphigus.
21. The method of claim 14, wherein the autoimmune disease is
psoriasis.
22. The method of claim 14, wherein the autoimmune disease is
inflammatory bowel disease.
23. The method of claim 13, wherein the patient has acquired immune
deficiency syndrome (AIDS).
24. The method of claim 13, wherein the patient has received a
transplant of a biological organ, tissue, or cell.
25. The method of claim 13, wherein the patient has a graft versus
host disease.
26. A method of eliminating a cell that expresses a receptor for
IL-15, the method comprising exposing the cell to the therapeutic
composition comprising a first agent that targets an IL-15R and a
second agent that targets an IL-2R.
27. The method of claim 26, wherein the cell is a cell of the
immune system.
28. The cell of claim 26, wherein the cell is a malignant cell.
29. A method of diagnosing a patient as having a disease or
condition that can be treated with the therapeutic composition of
claim 1, the method comprising determining whether a biological
sample obtained from the patient contains a cell that is bound by a
polypeptide comprising IL-15 and an antigenic tag, the occurrence
of binding indicating that the cell can be bound by an agent that
targets an IL-15R in vivo and thereby inhibited from proliferating
in response to wild-type IL-15 in vivo.
30. A pharmaceutically acceptable composition comprising two or
more agents, each of which promote T cell death.
31. The pharmaceutical composition of claim 30, further comprising
an agent that inhibits T cell proliferation.
32. The pharmaceutical composition of claim 31, wherein the
composition comprises a lytic IL-2/Fc molecule, a mutant IL-15
molecule that antagonizes and IL-15 receptor, and rapamycin.
33. A pharmaceutically acceptable composition comprising at least
one agent that promotes T cell death and at least one agent that
inhibits T cell proliferation.
34. The pharmaceutical composition of claim 32, wherein the T cell
death is AICD (activation induced cell death), passive cell death,
ADCC (antibody dependent cell-mediated cytotoxicity) or CDC
(complement directed cytotoxicity).
Description
This application claims the benefit of U.S. patent application Ser.
No. 60/232,251, filed Sep. 14, 2000.
TECHNICAL FIELD
[0002] This invention relates to immunology, transplant rejection,
and diseases associated with the immune system.
BACKGROUND
[0003] Two of the interleukins, IL-2 and IL-15, are functionally
redundant in stimulating T cell proliferation in vitro. However,
their role in primary immune activation and immune homeostasis in
vivo is much less clear. In vivo, IL-2 and IL-15 may have distinct
functions and regulate distinct aspects of T cell activation. For
example, IL-2 may prime activated T cells for apoptosis (Lenardo,
Nature 353:858-861, 1991), while IL-15 may support cell survival
(Dooms et al., J. Immunol. 161:2141-2150, 1998; Bulfone et al.
Nature Medicine 3:1124-1128, 1997). IL-15 also appears to drive the
proliferation of memory type CD8.sup.+ T cells in vivo while IL-2
limits their continued expansion (Ku et al., Science 288:675-678,
2000). In addition, the phenotype of IL-2 deficient mice is
lymphoproliferative and autoimmune (Horak et al., Immunol. Rev.
148:35-44, 1995), whereas IL-15 deficient mice are somewhat
lymphopenic and unable to mount a primary response to viral
challenge (Kennedy et al., J. Exp. Med. 191:771-780, 2000; Lodolce
et al., Immunity 2:669-676, 1998). The molecular basis for this
striking dichotomy remains enigmatic.
[0004] The functional receptors for IL-2 and IL-15 consist of a
private .alpha. chain, which defines the binding specificity for
IL-2 or IL-15, and shared IL-2 receptor .beta. and .gamma. chains.
The .gamma. chain is also a critical signaling component of the
IL-4, IL-7, and IL-9 receptors (Sugamura et al., Ann. Rev. Immunol.
14:179-205, 1996). In the lymphoid compartment, these receptor
subunits can be expressed individually or in various combinations
resulting in the formation of receptors with different affinities
and/or with distinct signaling capabilities (Sugamura et al.,
supra). For example, the .beta. chain can associate with either the
a chain or the y chain to form dimeric structures, or with both the
.alpha. and .gamma. chains to form trimeric structures. Similarly,
the .gamma. chain can interact with the .beta. chain and, through
the .beta. chain, with the .alpha. chain of either the IL-2
receptor or the IL-15 receptor. The IL- 15 receptor .alpha. chain
alone, in contrast to the IL- 2 receptor .alpha. chain, can bind to
IL-15 with a remarkably high affinity (Giri et al., EMBO J.
14:3654-3663, 1995). However, similar to IL-2 receptor .alpha.
chain, this interaction is not believed to trigger signaling
events. Thus, trimerization of .alpha., .beta., and .gamma. chain
subunits is essential for the functional integrity of high affinity
receptors for both IL-2 and IL-15.
[0005] In vitro studies have shown that activated T cells can
express both IL-2 receptor .alpha. chain and IL-15 receptor .alpha.
chain (Chae et al., J. Immunol. 157:2813-2819, 1996) and the .beta.
and .gamma. chains are constitutively expressed by activated T
cells (Ishii et al., Int. Immunol. 6:1273-1277, 1994). Furthermore,
both IL-2 and IL-15 are readily detected during immune activation
in vivo (Li et al., J. Immunol. 161:890-896, 1998). Thus, it is
unclear how activated T cells distinguish between IL-2, IL-15, and
other .gamma. chain dependent cytokines in vivo.
SUMMARY
[0006] The present invention is based, in part, on expression
studies of IL-2 and IL-15 receptor subunits by cycling T cells in
vivo. Surprisingly, these subunits direct activated T cell
responses to IL-2 or IL-15 in a selective manner and, thereby,
regulate the T cell response in vivo. In other words (and contrary
to the conventional wisdom that IL-2 and IL-15 are redundant), IL-2
and IL-15 perform different roles in controlling T cell
proliferation in vivo. In particular, IL-15 is critical for
initiating T cell division, whereas IL-2 controls T cell expansion
via down-regulation of .gamma.c expression. Accordingly, in one
embodiment, the invention generally features novel combinations of
IL-2 and IL-15 antagonists and methods of suppressing the immune
response by administering these antagonists. In each case,
suppression is achieved by administration of a first agent that
antagonizes an IL-15 molecule or an IL-15 receptor (IL-15R) and a
second agent that antagonizes an IL-2 molecule or an IL-2 receptor
(IL-2R). In alternative embodiments, the compositions of the
invention can include (in place of, or in addition to, the agents
described above), agents that inhibit the expression of the nucleic
acids (e.g., DNA or RNA) that encode an interleukin (e.g., IL-2 or
IL-15) or an interleukin receptor (e.g., an IL-2 or an IL-15
receptor).
[0007] More generally, the invention features novel combinations of
agents that, when administered to a patient, reduce the number of
antigen-reactive T cells. For example, the invention features
compositions (e.g., pharamaceutically acceptable compositions) that
include two or more agents, each of which promote T cell death.
Alternatively, the composition can contain at least one agent that
promotes T cell death and at least one agent that inhibits T cell
proliferation. The agent that promotes T cell death can promote
AICD (activation induced cell death), passive cell death, ADCC
(antibody dependent cell-mediated cytotoxicity) or CDC (complement
directed cytotoxicity).
[0008] Agents that promote AICD include IL-2 and related molecules
(e.g., IL-2/Fc or other molecules that function as agonists of IL-2
or the IL-2 receptor (e.g., an antibody that specifically binds to
the IL-2 receptor and mimics the binding of the receptor's natural
ligand)). Another agent that promotes AICD is the Fas Ligand
(FasL). Agents that promote passive cell death include agents that
antagonize IL-15(by targeting, e.g., binding to, and thereby
inhibiting the activity of, IL-15, an IL-15 receptor, or a
component of the intracellular signaling pathway that is activated
once a receptor is bound) or any other factor required for T cell
survival (e.g., IL-4, IL-7, OX-4 ligand, IFN-.beta., 4-1 BB, or
IGF-I). In alternative embodiments, the compositions of the
invention can include (in place of, or in addition to, one or more
of the agents described above), agents that inhibit the expression
of the nucleic acids (e.g., DNA or RNA) that encode an interleukin
(e.g., IL-2 or IL-15) or an interleukin receptor (e.g., an IL-2 or
an IL-15 receptor).
[0009] One can promote ADCC or CDC by exposing a T cell to an agent
that binds to the T cell surface and contains an Fc portion that
activates ADCC or CDC. More specifically, agents that promote ADCC
or CDC include fusion proteins that contain an interleukin (e.g.,
IL-2 or a mutant IL-15) and an Fc region (e.g., IL-2/Fc) as well as
antibodies or other Fc-containing proteins that bind to an
interleukin receptor (e.g., an IL-2 or an IL-15 receptor).
[0010] As stated above, the compositions of the invention can
include not only an agent that promotes T cell death, but also an
agent that inhibits T cell proliferation. Agents that inhibit T
cell proliferation include rapamycin, mycophenolate mofetil (MMF),
azathioprine, and any of the other agents known and used in the art
to prevent cellular proliferation (including chemotherapeutic
agents). The use of an agent that inhibits T cell proliferation is
particularly useful in combination with agents that promote AICD
and also stimulate T cell proliferation (such as IL-2/Fc). For
example, the invention features a pharmaceutically acceptable
composition that includes IL-2/Fc (which, for example, promotes
AICD and cellular lysis via ADCC or CDC), an IL-15 antagonist
(which, for example, promotes passive cell death by antagonizing
IL-15, a factor required for T cell survival), and rapamycin (which
inhibits T cell proliferation). Compositions containing other
combinations of agents are described below.
[0011] Notably, when two or more agents are employed, they need not
be physically separate from one another. While an agent can be a
single entity that has primarily one functional activity (e.g., an
antibody that targets IL-2 or IL-15 by specifically binding IL-2 or
IL-15), it can also be a single entity that has at least two
functional activities (e.g., IL-2/Fc, mIL-15/Fc, or an anti-IL-2 or
anti-IL-15 antibody; in these molecules, the interleukin mediates
AICD and the Fc portion of the molecule mediates CDC and ADCC).
Thus, a composition that includes (1) an agent that induces AICD,
(2) an agent that induces CDC, and (3) an agent that inhibits
cellular proliferation may include only two active ingredients
(e.g., (1) an IL-2/Fc molecule, which induces AICD and CDC, and (2)
rapamycin, which inhibits cellular proliferation).
[0012] The compositions described herein are useful in treating
patients who would benefit from immune suppression (e.g., a patient
who has received, or is scheduled to receive, a transplant; a
patient who has an immune disease, particularly an autoimmune
disease; a patient who has cancer (e.g., a cancer of the immune
system), or a patient suffering from graft versus host disease
(GVHD)). GVHD is characterized by a response of donor leukocytes
against antigens in the recipient. This response is particularly
problematic in bone marrow transplants, but also occurs in whole
organ transplants; donor leukocytes resident in transplanted organs
are always co-transplanted.
[0013] Although the compositions of the invention can contain more
than one agent, the methods of the invention are not limited to
those in which the agents are administered simultaneously. For
example, a patient could receive a composition containing an IL-15
antagonist or an IL-1 SR antagonist before receiving a composition
containing an IL-2 antagonist or an IL-2R antagonist. Similarly, a
patient could receive a composition containing rapamycin before
receiving a composition containing an IL-2 agonist. Moreover, the
compositions of the invention (applied simultaneously or
sequentially) can be used to treat an organ or cellular graft
before it is implanted in a patient. The agents of the invention,
and methods for their use, are described further below.
[0014] Many of the agents used in the context of the present
invention have advantageous therapeutic characteristics. For
example, agents that target an IL-15R can be fusion proteins that
include a mutant IL-15(mIL-15) polypeptide. These agents are
unlikely to be immunogenic because the mutant IL-15 portion of the
fusion protein can differ from wild type IL-15 by only a few
substituted residues. In addition, since miL-15 polypeptides can
bind the IL-15R.alpha. with high affinity, they can compete
effectively with wild type IL-15 for the receptor. Further, agents
of the invention can activate components of the host immune system,
such as complement and phagocytes, which ultimately mediate
elimination of (or depletion of) cells bearing the receptor (e.g.,
an IL-2 receptor) to which the agent binds. For example, agents of
the invention can mediate lysis or phagocytosis of targeted cells.
As the alpha subunit of the IL-15 receptor (IL-15R.alpha.) is
expressed by activated or malignant immune cells, but not by
resting immune cells, agents of the invention can be used to
specifically target those cells that have been activated (e.g.,
antigen-activated T cells) or that have become malignant. Thus,
although T cells represent a preferred target for the agents of the
invention, the compositions of the invention can also be used to
target other cells involved in the pathogenesis of immunological
disorders, such as other cells of the immune system or
hyperproliferating cells of tissues.
[0015] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. All
publications, patent applications, patents and other references
mentioned herein are incorporated by reference in their
entirety.
[0016] Other features and advantages of the invention will be
apparent from the drawings, the detailed description, and claims.
Although materials and methods similar or equivalent to those
described herein can be used in the practice or testing of the
invention, the preferred materials and methods are described
below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a representation of a wild-type IL-15 nucleic acid
sequence (SEQ ID NO: 1) and the predicted amino acid sequence (SEQ
ID NO: 2).
[0018] FIG. 2 is a representation of a mutant IL-15 nucleic acid
sequence (SEQ ID NO: 3) and the predicted amino acid sequence (SEQ
ID NO: 4). The wild-type codon encoding glutamine at position 149,
CAG, and the wild-type codon encoding glutamine at position 156,
CAA, have both been changed to GAC, which encodes aspartate. (These
positions (149 and 156) correspond to positions 101 and 108,
respectively, in the mature IL-15 polypeptide, which lacks a
48-amino acid signal sequence).
[0019] FIG. 3A is a series of plots depicting expression of IL-2
receptor .alpha., .beta. and .gamma.c chains by dividing T cells in
a host spleen 3 days after intravenous (i.v.) injection of
CFSE-labeled cells. Representative data of six experiments are
shown.
[0020] FIG. 3B is a series of plots depicting expression of IL-2
receptor .alpha., .beta. and .gamma. chains by dividing T cells in
vitro. CFSE-labeled cells lymphocytes were stimulated with
anti-CD3(2 .mu.g/ml) in vitro for three days. Cell division and the
expression of IL-2 receptor subunits were analyzed by
fluorescence-activated cell sorting (FACS).
[0021] FIG. 3C is a plot depicting expression of L-selectin by
dividing T cells in vivo. Cells were harvested from lymph nodes of
host mice three days after intravenous injection of CFSE-labeled
cells and stained with PE-anti-CD62mAb. The quadrant was set based
on cells stained with isotype control mAb.
[0022] FIG. 3D is a series of plots depicting differential
expression of IL-2 receptor .alpha. chain between in vivo dividing
T cells. CFSE-labeled cells were stimulated in vivo for three days
and cells in the second cell division were sorted. The sorted cells
were re-stimulated in vitro with anti-CD3 and anti-CD28 for three
days. Cells division and expression of the IL-2 receptor .alpha.
chain were analyzed by FACS.
[0023] FIG. 4A is a series of plots depicting expression of IL-15
receptor a chain by dividing T cells in vivo three days after
intravenous injection of CFSE-labeled cells. Cells were stained
with an IL-15-FLAG fusion protein, followed by staining with
biotinylated anti-FLAG mAb and PE-streptavidin. Cell staining in
the absence of IL-15-FLAG was included as a control.
[0024] FIG. 4B is a bar graph depicting the different response of
in vivo dividing T cells to IL-2 and IL-15 in vitro. CFSE-labeled
lymphocytes were stimulated in vivo for three days and cell
division was analyzed by examining the CFSE profile. T cells in the
second cell division were sorted and cells (1.times.10.sup.4) were
cultured in vitro with IL-2 or IL-15 for two days. Cellular
proliferation was determined by .sup.3H-TdR uptake. The results are
presented as the mean CPM.+-.SD of triplicate assays.
[0025] FIG. 4C is a pair of graphs depicting the effect of
anti-CD25 treatment on T cell division in vivo. Host mice were
given anti-CD25 mAb intraperiotoneally (i.p.) at 1 mg/day for three
days immediately before intravenous injection of CFSE-labeled
cells. Mice treated with isotype control mAb (rat IgG1) were
included as a control.
[0026] FIG. 5A is a series of plots depicting intracellular IL-2
staining of in vivo dividing T cells. CFSE-labeled cells were
stimulated in vivo for three days. Cells harvested from the host
spleen were stimulated in vitro with PMA and ionomycin for four
hours in the presence of GolgiStop.TM.. Cells were then fixed,
permeablized, and stained for IL-2 production. Cells stained with
isotype control mAb were included as a control.
[0027] FIG. 5B is a pair of graphs depicting expression of .gamma.c
by CD4.sup.+ T cells from IL-2 deficient mice. Spleen cells from
IL-2 deficient mice and wild type control mice were stained with
PE-anti-mouse CD4 mAb and FITC-anti-mouse IL-2 receptor .gamma.c
mAb. The expression of .gamma.c by CD4.sup.+ T cells was analyzed
by FACS.
[0028] FIG. 5C is a series of plots depicting the effect of
anti-CD25 treatment on .gamma.c expression by dividing T cells in
vivo. Host mice were given anti-CD25 mAb at 1 mg/day (i.p.) for
three days immediately before intravenous injection of CFSE-labeled
cells. Expression of IL-2 receptor .beta. and .gamma. chains on
dividing T cells in vivo was determined on day three (i.e., three
days after injection of CFSE-labeled cells). Mice treated with an
isotype control mAb (rat IgG1) were included as a control.
[0029] FIG. 5D is a series of plots depicting apoptotic cell death
of in vivo dividing T cells. CFSE-labeled cells were stimulated in
vivo for three days. Cells were harvested from the host spleen and
stained with PE-annexin V. Cell division and apoptic cell death
were analyzed by FACS.
[0030] FIG. 5E is a series of plots depicting intracellular Bcl-2
expression by dividing T cells in vivo. CFSE-labeled cells were
stimulated in vivo for three days. Cells harvested from the host
spleen were stained with PE-anti-mouse Bcl-2 mAb or an isotype
control mAb. Cell division and expression of Bcl-2 were analyzed by
FACS.
[0031] FIG. 6 is a bar graph depicting the results of an experiment
in which cell death (assessed by release of the isotope .sup.51Cr
from CTLL-2 cells; see the x axis) was assessed following treatment
with various agents. NP40 is a detergent; IL-2/Fc is a fusion
protein that contains IL-2 and the Fc region of an IgG molecule
(this molecule is lytic); C' is rat complement; IL-2/FC-/- is a
non-lytic IL-2-containing fusion protein; and mIg is a murine
immunoglobulin. This study supports the conclusion that cytolytic
IL-2/Fc lyses IL-2R bearing CTLL-2 cells, but non-lytic IL-2/Fc
does not.
[0032] FIG. 7 is a series of histographs. The fluorescence
intensity of FcRI on CHO (Chinese hamster ovary) cells was measured
after the cells were exposed to phosphate buffered saline (PBS;
upper left), a murine immunoglobulin (mIgG2a; upper right), a
non-lytic IL-2/Fc molecule (IL-2/Fc-/-; lower left), and a lytic
IL-2 containing fusion protein (IL-2 /Fc; lower right). In each
histograph, cell number is plotted against the fluorescence
intensity of FcRI/CHO. This study supports the conclusion that
cytolytic IL-2/Fc binds to FcRI, but nonlytic IL-2/Fc does not.
[0033] FIG. 8 is a series of eight plots depicting the
proliferative response of CD4.sup.+ (left-hand side) and CD8.sup.+
(right-hand side) T cells in vivo. The cells were labeled with CFSE
and stimulated in vivo for three days with a lytic molecule
(IL-2/Fc), a cell proliferation agent (rapamycin (Rap)), or the two
agents combined. As a negative control, one group of animals was
not treated. IL-2/FC was analyzed by their CFSE profile. This study
supports the conclusion that rapamycin inhibits IL-2 proliferative
signaling.
[0034] FIG. 9 is a series of four plots obtained from an experiment
in which CFSE-labeled lymphocytes were stimulated in vivo for three
days. The expression of an IL-2R .alpha. chain on dividing T cells
was assessed by FACS in animals that received no treatment (upper
left), rapamycin alone (Rap; upper right), IL-2/Fc alone (lower
left), or a combination of rapamycin and IL-2/Fc (Rap+IL-2/Fc;
lower right). This study supports the conclusion that treatment
with rapamycin and IL-2/Fc promotes expression of the .alpha.
subunit of the IL-2R during early T cell proliferation in vivo.
[0035] FIG. 10 is a pair of plots obtained when CFSE-labeled
lymphocytes were stimulated in vivo for three days and analyzed by
FACS. The expression of Annexin V on dividing T cells (CD4.sup.+)
was assessed in animals that received no treatment (left hand
panel) or rapamycin and IL-2/Fc (Rap+IL-2/Fc; right hand panel).
This study supports the conclusion that rapamycin and IL-2/Fc
treatment promotes apoptosis of CD4+ cells during early T cell
proliferation in vivo.
[0036] FIG. 11 is a Table showing the results of experiments that
examined islet allograft survival in autoimmune non-obese diabetic
(NOD) mice. The grafts were assessed in terms of primary allograft
function (the percentages shown in this column represent the
percentage of mice in which the allograft functioned (function was
assessed by monitoring blood glucose levels)) and the mean survival
time (MST) of the functioning grafts. n=the number of animals
tested. The treatments are indicated under the heading "Treatment"
(see also the legend that accompanies the Table and the description
below). The results presented here support the conclusion that
treatment with a combination of rapamycin, IL-2/Fc and mIL-15/Fc
results in long-term survival of islet allografts.
[0037] FIG. 12 is a Table showing the results of experiments that
examined the survival of skin allografts in NOD mice. The grafts
were assessed in terms of the mean survival time (MST) of
functioning grafts. n=the number of animals tested. The treatments
are indicated under the heading "Treatment" (see also the legend
that accompanies the Table and the description below). The results
presented here support the conclusion that treatment with a
combination of rapamycin, IL-2/Fc and mIL-15/Fc results in
long-term survival of skin allografts.
[0038] FIG. 13 is a line graph that plots the % of animals that
remained diabetes free over time following treatment with a lytic
IL-2/Fc molecule, a murine immunoglobulin (mIg), and a non-lytic
IL-2/Fc molecule. Cytolytic IL-2/Fc blocked autoimmunity, but lytic
IL-2/Fc did not.
DETAILED DESCRIPTION
[0039] An effective immune response begins when an antigen or
mitogen triggers the activation of T cells. In the process of T
cell activation, numerous cellular changes occur, which include the
expression of cytokines and cytokine receptors. One of the
cytokines involved in the immune response is interleukin-15(IL-15),
which is a T cell growth factor that stimulates the proliferation
and differentiation of B cells, T cells, natural killer (NK) cells,
and lymphocyte-activated killer (LAK) cells in vitro. In vivo, the
proliferation of these cell types enhances the immune response.
Another cytokine involved in the immune response, and described
herein, is IL-2.
[0040] The compositions of the present invention include agents
that target IL-15, or its receptor, and IL-2, or its receptor, and
methods in which those compositions are used to suppress an immune
response (e.g., a humoral or cellular immune response). Patients
benefit from suppression of the immune response in a number of
circumstances, for example, in the event of organ transplantation
or immune disease, particularly autoimmune disease, or Graft Versus
Host Disease. In other circumstances, for example when select
immune cells have become malignant or autoaggressive, it is
beneficial to actively destroy them.
[0041] The present invention is based on the discovery of novel
ways to inhibit the immune response. Inhibition can be achieved by
administering of a combination of agents, one of which targets
IL-15 or an IL-15R and one of which targets IL-2 or an IL-2R (modes
of administration, including ex vivo treatment of grafts, are known
in the art and described further below). More generally, one can
reduce the number of antigen-reactive T cells by activating
signaling pathways that lead to the death of activated T cells (by,
e.g., AICD);
[0042] depriving cells of factors that are required for their
survival (cells that die following such deprivation are said to die
by passive cell death); or targeting activated cells for lysis by
components of the immune system (cells that die in this way are
said to die by ADCC or CDC). Accordingly, the compositions of the
invention include agents that achieve one or more of these ends
(i.e., that promote T cell death via a recognized cell death
pathway (e.g., AICD, passive cell death, ADCC, or CDC)). In
addition to containing one or more agents that promote T cell
death, the compositions of the invention can include one or more
agents that inhibit T cell proliferation (as occurs, e.g., in
response to an antigen). For example, the invention features a
composition (e.g., a pharmaceutically acceptable composition or one
formulated for application to an organ or cell culture) that
includes IL-2/Fc (which, for example, promotes AICD and cellular
lysis via ADCC or CDC), mIL-15/Fc (which antagonizes IL-15(and
thereby promotes passive cell death) and promotes cellular lysis
via ACDD or CDC), and rapamycin (which inhibits T cell
proliferation).
[0043] The term "agent" is meant to encompass essentially any type
of molecule that can be used as a therapeutic agent. Proteins, such
as antibodies, fusion proteins, and soluble ligands, any of which
may either be identical to a wild-type protein or contain a
mutation (i.e., a deletion, addition, or substitution of one or
more amino acid residues), and the nucleic acid molecules that
encode them (or that are "antisense" to them; e.g., an
oligonucleotide that is antisense to the nucleic acids that encode
IL-2, IL-15, or a component (e.g., a subunit) of their receptors),
are all "agents." The agents of the invention can either be
administered systemically, locally, or by way of cell-based
therapies (i.e., an agent of the invention can be administered to a
patient by administering a cell that expresses that agent to the
patient). The cell can be a cell administered to the patient solely
for the purpose of expressing the therapeutic agent. The cell can
also be a cell of a cellular, tissue, or organ transplant. For
example, transplanted cells (e.g., islet cells) or cells within
tissues or organs (e.g., cells within a patch of skin or a liver,
kidney, or heart) can be treated with an agent or transduced with a
nucleic acid molecule that encodes an agent ex vivo (e.g., prior to
transplantation). In this way, the transplanted cell produces its
own immunosuppressive agents. For example, a cell with a desirable
phenotype (e.g., an insulin producing cell) can be modified to
include a gene producing one or more of the immunosuppresive
factors of the invention. The transplanted cell, tissue, or organ
can be treated either prior to or subsequent to transplantation.
Methods of administering agents to patients (or to cells or organs
in culture) are known and routinely used by those of ordinary skill
in the art and are discussed further below.
[0044] Agents that Target IL-15 or an IL-15R
[0045] The compositions of the invention can include one or more
agents that target IL-15 or an IL-15 receptor. As noted above, a
single agent can have multiple functional domains. Agents that
target IL-15 or an IL-15R include agents that bind to (or otherwise
interact with) IL-15, an IL-15R, or the nucleic acids that encode
them as well as agents that bind to and subsequently destroy
IL-15R-bearing cells, such as activated T cells. Thus, agents
useful in achieving immune suppression can contain two functional
moieties: a targeting moiety that targets the agent to an
IL-15R-bearing cell and a target-cell depleting (e.g., lytic)
moiety that leads to the elimination of the IL-15R-bearing cell. In
one embodiment, the targeting moiety binds an IL-15R without
effectively transducing a signal through that receptor. For
example, the targeting moiety can include a mutant IL-15
polypeptide, and the target-cell depleting moiety can include the
Fc region of an immunoglobulin molecule. The Fc region can be
derived from an IgG, such as human IgG1, IgG2, IgG3, IgG4, or
analogous mammalian IgGs or from an IgM, such as human IgM or
analogous mammalian IgMs. In a preferred embodiment, the Fc region
includes the hinge, CH2 and CH3 domains of human IgG1 or murine
IgG2a. Although the invention is not limited to agents that work by
any particular mechanism, it is believed that the Fc region
mediates complement and phagocyte-driven elimination of
IL-15R-bearing cells.
[0046] Mutant IL-15 polypeptides that bind the IL-15 receptor
complex with an affinity similar to wild-type IL-15, but fail to
fully activate signal transduction, have been produced. These
mutant polypeptides compete effectively with wild-type IL-15
polypeptides and can inhibit one or more of the events that
normally occur in response to IL-15 signaling, such as cellular
proliferation. The "wild-type IL-15 polypeptide" referred to herein
is a polypeptide that is identical to a naturally occurring IL-15
(e.g., a wild-type IL-15 polypeptide is shown in FIG. 1). In
contrast, a "mutant IL-15 polypeptide" is a polypeptide that has at
least one mutation relative to wild-type IL-15 and that inhibits at
least one of the in vivo or in vitro activities that are usually
promoted by wild-type IL-15.
[0047] A mutant IL-15 polypeptide that can be used according to the
present invention will generally block at least 40%, more
preferably at least 70%, and most preferably at least 90% of one or
more of the activities of the wild-type IL-15 molecule. The ability
of a mutant IL-15 polypeptide to block wild-type IL-15 activity can
be assessed by numerous assays, including the BAF-BO3 cell
proliferation assay described herein (in which the cells were
transfected with a construct encoding IL-2R.beta.). Further, mutant
polypeptides of the invention can be defined according the
particular percent identity they exhibit with wild-type IL-15. When
examining the percent identity between two polypeptides, the length
of the sequences compared will generally be at least 16 amino
acids, preferably at least 20 amino acids, more preferably at least
25 amino acids, and most preferably at least 35 amino acids. The
term "identity," as used in reference to polypeptide or DNA
sequences, refers to the identity between subunits (amino acid
residues of proteins or nucleotides of DNA molecules) within the
two polypeptide or DNA sequences being compared. When a subunit
position in both of the molecules is occupied by the same monomeric
subunit (i.e., the same amino acid residue or nucleotide), then the
molecules are identical at that position. The similarity between
two amino acid sequences or two nucleotide sequences is a direct
function of the number of identical positions. Thus, a polypeptide
that is 50% identical to a reference polypeptide that is 100 amino
acids long can be a 50 amino acid polypeptide that is completely
identical to a 50 amino acid long portion of the reference
polypeptide. It might also be a 100 amino acid long polypeptide
that is 50% identical to the reference polypeptide over its entire
length. Of course, many other polypeptides will meet the same
criteria. Identity is typically and most conveniently measured
using sequence analysis software, such as the Sequence Analysis
Software Package of the Genetics Computer Group at the University
of Wisconsin Biotechnology Center (1710 University Avenue, Madison,
Wis. 53705), with the default parameters thereof.
[0048] A mutant IL-15 polypeptide of the invention can be at least
65%, preferably at least 80%, more preferably at least 90%, and
most preferably at least 95% (e.g., 96%, 97%, 98% or 99%) identical
to wild-type IL-15. The mutation can consist of a change in the
number or content of amino acid residues. For example, the mutant
IL-15 can have a greater or a lesser number of amino acid residues
than wild-type IL-15. Alternatively, or in addition, the mutant
polypeptide can contain a substitution of one or more amino acid
residues that are present in the wild-type IL-15. The mutant IL-15
polypeptide can differ from wild-type IL15 by the addition,
deletion, or substitution of a single amino acid residue, for
example, an addition, deletion or substitution of the residue at
position 156. Similarly, the mutant polypeptide can differ from
wild-type by an addition, deletion, or substitution of two amino
acid residues, for example, the residues at positions 156 and 149.
For example, the mutant IL-15 polypeptide can differ from wild-type
IL-15 by the substitution of aspartate for glutamine at residues
156 and 149 (as shown in FIG. 2). Mutant polypeptides useful as
targeting agents, like wild-type IL-15, recognize and bind a
component of the IL-15R. In one embodiment, the mutation of IL-15
is in the carboxy-terminal domain of the cytokine, which is
believed to bind IL-2R.gamma. (the IL-2 receptor subunit).
Alternatively, or in addition, mutant IL-15 polypeptides can
include one or more mutations within IL-2R.beta. (the IL-2 receptor
.beta. subunit) binding domain.
[0049] In the event a mutant IL-15 polypeptide contains a
substitution of one amino acid residue for another, the
substitution can be, but is not necessarily, a conservative
substitution, which includes a substitution within the following
groups: glycine, alanine; valine, isoleucine, leucine; aspartic
acid, glutamic acid; asparagine, glutamine; serine, threonine;
lysine, arginine; and phenylalanine, tyrosine.
[0050] Instead of using, or in addition to using, an IL-15
targeting polypeptide (e.g., a mutant IL-15 polypeptide), the
therapeutic agent can be an antibody. For example, IL-15 can be
targeted (i.e., specifically bound) with an antibody. Similarly,
the IL-15R can be targeted with antibodies that bind a component of
the IL-15R (e.g., the IL-15R.alpha. subunit). The methods by which
antibodies, including humanized antibodies, can be generated
against a component of the IL-15R are well known in the art. The
antibodies preferably should be able to activate complement and
phagocytosis, for example, human IgG3 and IgG1 (preferably the
latter) subclasses, or murine IgG2a subclass.
[0051] The methods of the invention can also be carried out with
compositions that contain: (a) two or more agents, each of which
promote T cell death or (b) at least one agent that promotes T cell
death and at least one agent that inhibits T cell proliferation.
The agent that promotes T cell death can do so by promoting passive
cell death, which occurs when a T cell is deprived of a factor
required for its survival. IL-15 is one such agent (others are
described below). Thus, agents that interfere with the ability of
IL-15 to serve as a survival factor (e.g., an antibody that
specifically binds to IL-15 or the IL-15 receptor) can be included
in the compositions of the invention (e.g., a composition can
include an agent that promotes AICD, an agent that promotes passive
cell death (e.g., an anti-IL-15 antibody), and, optionally, an
agent that inhibits T cell proliferation.
[0052] As described above, agents useful in achieving immune
suppression can contain two functional moieties: a targeting moiety
that targets the agent to an IL-15R-bearing cell (such as the
mutant IL-15 molecule just described) and a target-cell depleting
moiety that, for example, lyses or otherwise leads to the
elimination of, the IL-15R-bearing cell. Thus, the agent can be a
chimeric polypeptide that includes a mutant IL-15 polypeptide and a
heterologous polypeptide such as the Fc region of the IgG and IgM
subclasses of antibodies. The Fc region may include a mutation that
inhibits complement fixation and Fc receptor binding, or it may be
target-cell depleting (i.e., able to destroy cells by binding
complement or by another mechanism, such as antibody-dependent
complement lysis).
[0053] The Fc region can be isolated from a naturally occurring
source, recombinantly produced, or synthesized (just as any
polypeptide featured in the present invention can be). For example,
an Fc region that is homologous to the IgG C-terminal domain can be
produced by digestion of IgG with papain. IgG Fc has a molecular
weight of approximately 50 kDa. The polypeptides of the invention
can include the entire Fc region, or a smaller portion that retains
the ability to lyse cells. In addition, full-length or fragmented
Fc regions can be variants of the wild-type molecule. That is, they
can contain mutations that may or may not affect the function of
the polypeptide.
[0054] Reference is made herein to agents that "target" an
interleukin or an interleukin receptor. Targeting occurs when an
agent directly or indirectly binds to, or otherwise interacts with,
an interleukin or an interleukin receptor in a way that affects the
activity of the interleukin or the interleukin receptor. Activity
can be assessed by those of ordinary skill in the art and with
routine laboratory methods. For example, one can assess the
strength of signal transduction or another downstream biological
event that occurs, or would normally occur, following receptor
binding. The activity generated by an agent that targets an
interleukin or an interleukin receptor can be, but is not
necessarily, different from the activity generated when a naturally
occurring interleukin binds a naturally occurring interleukin
receptor. For example, an agent that targets an IL-2 receptor falls
within the scope of the invention even if that agent generates
substantially the same activity that would occur had the receptor
been bound by naturally occurring IL-2. When an agent generates
activity that is substantially the same as, or greater than, the
activity generated by a naturally occurring ligand, the agent can
be described as a receptor agonist (the agent and the natural
ligand being examined under the same conditions). When an agent
generates activity that is less than the activity generated by a
naturally occurring ligand, the agent can be described as an
antagonist of the receptor (if the agent's primary interaction is
with the receptor; e.g., mIL-15) or of the interleukin (if the
agent's primary interaction is with the interleukin; e.g., an
anti-IL-15 antibody). Here again, levels of activity are assessed
by testing the agent and the naturally occurring receptor (or
ligand) under the same conditions.
[0055] The Fc region that can be part of the agents of the
invention can be "target-cell depleting" or "non-target-cell
depleting." A non-target-cell depleting Fc region typically lacks a
high affinity Fc receptor binding site and a C'1q binding site. The
high affinity Fc receptor binding site of murine IgG Fc includes
the Leu residue at position 235 of IgG Fc. Thus, the murine Fc
receptor binding site can be destroyed by mutating or deleting Leu
235. For example, substitution of Glu for Leu 235 inhibits the
ability of the Fc region to bind the high affinity Fc receptor. The
murine C'1q binding site can be functionally destroyed by mutating
or deleting the Glu 318, Lys 320, and Lys 322 residues of IgG. For
example, substitution of Ala residues for Glu 318, Lys 320, and Lys
322 renders IgG1 Fc unable to direct antibody-dependent complement
lysis. In contrast, a target-cell depleting IgG Fc region has a
high affinity Fc receptor binding site and a C'1q binding site. The
high affinity Fc receptor binding site includes the Leu residue at
position 235 of IgG Fc, and the C'1q binding site includes the Glu
318, Lys 320, and Lys 322 residues of IgG1. Target-cell depleting
IgG Fc has wild-type residues or conservative amino acid
substitutions at these sites. Target-cell depleting IgG Fc can
target cells for antibody dependent cellular cytotoxicity or
complement directed cytolysis (CDC). Appropriate mutations for
human IgG are also known (see, e.g., Morrison et al., The
Immunologist 2:119-124, 1994; and Brekke et al., The Immunologist
2:125, 1994).
[0056] Agents that target the IL-15R can mutant IL-15 polypeptides,
optionally fused to an antigenic tag (e.g., a FLAG sequence). FLAG
sequences are recognized by biotinylated, highly specific,
anti-FLAG antibodies, as described herein (see also Blanar et al.,
Science 256:1014, 1992; LeClair et al., Proc. Natl. Acad. Sci. USA
89:8145, 1992).
[0057] In addition, soluble IL-15R .alpha. chain can be used as
antagonist. While the IL-15 receptor complex consists of .alpha.
.beta. .gamma. subunits, the .alpha. chain alone displays a high
affinity for IL-15. Thus, soluble IL-15R .alpha. chain will bind
IL-15 and prevent IL-15 from binding to a cell surface-bound IL-15R
complex. Thus, a soluble IL-15R .alpha. chain can act as a
receptor-specific antagonist.
[0058] Construction of soluble IL-15R .alpha. chain involves
cloning the extracellular fragment of the IL-15R .alpha. chain from
receptor-positive cells, such as activated T cells or receptor
expressing cell lines, and, optionally, fusing it to a molecular
tag sequence. The tag sequence can be, for example, FLAG, GST, or
Histidine. This genetic construct in an expression vector can be
transfected into expressing cell lines. The tagged soluble IL-15R
.alpha. chain produced by expressing cell lines will be purified
using mAbs specific for the Tag sequence. Furthermore, an IL-15R
extracellular domain can be linked (e.g., fused by way of a peptide
bond) to an immunoglobulin Fc domain (e.g. hinge, CH2 and CH3
domains of Immunoglogulin G), preferably of an IgG or IgM subtype.
Such a fusion protein could be expressed in a suitable cell type,
many of which are known to those of ordinary skill in the art.
[0059] Agents that Target IL-2 or an IL-2Receptor
[0060] To inhibit an immune response, the agents that target
IL-15R-bearing cells, described above, can be administered with an
agent that targets IL-2 or an IL-2R. An agent that is administered
"with" another may be, but is not necessarily, administered at the
same time or in the same manner (while this comment is stated in
the context of a discussion of IL-2-related agents, it is
applicable for any of the agents or molecules combined in the
compositions of the invention). For example, an agent that targets
an IL-15R may be administered before or after an agent that targets
an IL-2R. Similarly, an agent that targets IL-15 or an IL15R can be
administered ex vivo (to treat, for example, a cell, tissue, or
organ that is slated for transplantation) while an agent that
targets IL-2 or an IL-2R can be administered systemically (e.g.,
intravenously) to a patient (e.g. a patient who has received a
transplant that was treated ex vivo with an agent that targets
IL-15). Similarly, one can administer an agent that promotes AICD
at a different time or in a different manner than an agent that
inhibits cellular proliferation. Thus, in the methods of the
invention, any of the agents or types of molecules that are
combined in the compositions of the invention can be administered
separately.
[0061] To inhibit an IL-2R, one can administer any agent that binds
to and antagonizes IL-2 or an IL-2R. Agents that target IL-2 or an
IL-2R include agents that bind to IL-2 or an IL-2R as well as
agents that bind to and subsequently destroy IL-2R-bearing cells,
such as activated T cells. As described above in the context of
IL-15 targeting, agents useful in achieving immune suppression can
contain a moiety that targets the agent to an IL-2R-bearing cell
and a target-cell depleting (e.g., lytic) moiety that leads to the
elimination of the IL-2R-bearing cell. For example, the targeting
moiety can bind an IL-2R without effectively transducing a signal
through that receptor. In the event an Fc region is included, that
region can be derived from the same immunoglobulin molecules
described above.
[0062] Targeting agents, such as an IL-2/Fc agent (e.g., see Zheng
et al., J. Immunol. 163:4041-4048, 1999) can be administered with
an agent that prevents IL-2-mediated IL-2R signaling, such as
rapamycin. Agents that inhibit cellular proliferation are well
known to those of ordinary skill in the art (and are discussed
further below).
[0063] Instead of using, or in addition to using, an IL-2R
targeting polypeptide (e.g., an IL-2 polypeptide), the therapeutic
agent used in combination with an IL-15 antagonist can be an
anti-IL-2 or an anti-IL-2R antibody (e.g., a humanized antibody)
that antagonizes IL-2 or the IL-2R, respectively.
[0064] As explained above, the methods of the invention (e.g.,
methods of inhibiting an immune response (e.g., a cellular immune
response), methods of inhibiting transplant rejection, and methods
of treating cancer) can also be carried out with compositions
(e.g., pharmaceutically acceptable compositions) that contain: (a)
two or more agents, each of which promote T cell death or (b) at
least one agent that promotes T cell death and at least one agent
that inhibits T cell proliferation. The agent that promotes T cell
death can do so by promoting AICD (activation induced cell death),
and such agents include IL-2 and molecules that function as IL-2
agonists. For example, IL-2/Fc, mutants of IL-2 that retain the
ability to bind and transduce a signal through the IL-2 receptor,
and antibodies that specifically bind and agonize the IL-2 receptor
(e.g., an antibody that specifically binds the .alpha. subunit of
the IL-2 receptor) can be included in the compositions of the
invention. Other agents that promote AICD include Fas Ligand
(FasL), which stimulates T cell death by activating the Fas signal
transduction cascade on activated T cells, and biologically active
mutants thereof.
[0065] Agents that Promote Passive Cell Death
[0066] Passive T cell death occurs when a T cell is deprived of an
agent that is required for its survival. In addition to IL-15,
factors including IL-4, IL-7, OX-40 ligand, IFN.beta., 4-1BB and
IGF-I are essential (i.e., T cells die in the absence of each of
these factors; see, e.g., Tu et al., J. Immunol. 165:1331-1336,
2000; Tsuda et al., J. Immunol. Meth. 236:37-51, 2000; Bertolino et
al., Int. Immunol. 11: 1225-1238, 1999; Takahashi et al., J.
Immunol. 162:5037-5040, 1999; Pilling et al., Eur. J. Immunol.
29:1041-1050, 1999; Chu et al., J. Immunol. 162:1896-1903, 1999;
and Weinberg et al., Semin. Immunol. 10:471-480, 1998). One can
deprive T cells of one or more of these factors (IL-15, IL-4, IL-7,
etc.) by, for example, exposing the cells, in vivo or in culture,
to agents that selectively bind to one or more of the factors or
otherwise prevent them from interacting with the T cell as they
normally would (the result of the deprivation being passive cell
death).
[0067] Agents that Promote ADCC or CDC
[0068] ADCC and CDC can be provoked by agents that bind to the T
cell surface and that contain an Fc portion of an immunoglobulin
molecule that activates ADCC or CDC. Examples of such agents
include antibodies that bind to cell surface structures that are
expressed on activated immune cells (e.g., cell surface receptors
such as CD154, the IL-2 receptor, and the IL-15 receptor). In
addition, one can use a ligand/Fc chimeric fusion protein, which
binds to receptor proteins on the surface of activated cells (e.g.,
an IL-2/Fc or a mIL-15/Fc). Given these examples, other suitable
agents will be apparent to those of ordinary skill in the art.
[0069] Agents that Inhibit Cellular Proliferation
[0070] Agents that inhibit cellular proliferation include rapamycin
(Sirolimus), mycophenolate mofetil (MMF), azathioprine, and any
other of the agents that are known to be useful for the treatment
of hyperproliferative disorders (such as cancer).
Well-characterized chemotherapeutics include agents that inhibit
nucleic acid metabolism (such as purine and pyrimidine biosynthesis
inhibitors, RNA synthesis inhibitors, and DNA binding, DNA
modifying, or intercalating agents). These agents are especially
useful when the composition used to, for example, inhibit an immune
response, also contains an agent such as IL-2/Fc, which not only
promotes AICD but also stimulates T cell proliferation.
[0071] Agents that inhibit cellular proliferation also include
folic acid antimetabolites such as methotrexate (MTX) and
pyrimethamine; purine antimetabolites (such as 6-mercaptopurine
(6-MP) and azathioprine) and pyrimidine antagonists such as
cytarabine (ara-C), 5-azacytidine, and 5-fluorouracil (these
categories were mentioned above); alkylating and other DNA-linking
agents (e.g., cyclophosphamide (CPA); mitomycin C, and Doxorubicin
(Adriamycin)); vinca alkaloids (e.g., vincristine); and calcineurin
inhibitors (e.g., Cyclosporin A, FK506, and Brequinar).
[0072] Other agents that can be used to inhibit cellular
proliferation include agents that interfere directly with proteins
involved in cell cycle regulation (such as anti-CDKs (Cell Division
Kinase) or anti-cyclins) or proteins that affect cell proliferation
check points (all proliferating cells have check points at
different stages of the cell cycle that prevent them from entering
the next stage of the cell division cycle (CDC) before they have
concluded the previous step). Pathways that feed into check point
controls include DNA-, RNA- and protein-synthesis inhibitors (e.g.,
S6 kinase and PI-3-kinase inhibitors). Cytokinesis inhibitors can
also be used.
[0073] Procedures for Screening Agents that Inhibit the Immune
Response
[0074] In addition to testing a candidate agent (e.g., a mutant
IL-15 or IL-2 polypeptide) in the in vitro assays described in the
examples below, one can use any of the following in vivo assays to
test which particular combinations of the agents described herein
most effectively bring about immune suppression. For example, one
can test one or more of the agents that target the IL-15R in
combination with one or more of the agents that antagonize IL-2 or
its receptor. These in vivo assays represent only some of the
routine ways in which one of ordinary skill in the art could
further test the efficacy of agents of the invention. They were
selected for inclusion here because of their relevance to the
variety of clinical conditions amenable to treatment with agents
that target IL-2, IL-15, and their receptors. For example, the
assays are relevant to organ transplantation, immune disease,
particularly autoimmune disease, graft versus host disease and
cancers of the immune system (e.g. cancers that arise when T cells
become malignant).
[0075] Transplantation Paradigms
[0076] To determine whether a combination of agents of the
invention achieves immune suppression, the combination can be
administered (either directly, by gene-based therapy, or by
cell-based therapy) in the context of well-established
transplantation paradigms.
[0077] Agents of the invention, nucleic acid molecules encoding
them (or that hybridize with and thereby inhibit them), can be
systemically or locally administered by standard means to any
conventional laboratory animal, such as a rat, mouse, rabbit,
guinea pig, or dog, before an allogeneic or xenogeneic skin graft,
organ transplant, or cell implantation is performed on the animal.
Strains of mice such as C57 B1-10, B10.BR, and B10.AKM (Jackson
Laboratory, Bar Harbor, Me.), which have the same genetic
background but are mismatched for the H-2 locus, are well suited
for assessing various organ grafts.
[0078] Heart Transplantation
[0079] A method for performing cardiac grafts by anastomosis of the
donor heart to the great vessels in the abdomen of the host was
first published by Ono et al. (J. Thorac. Cardiovasc. Surg. 57:225,
1969; see also Corry et al., Transplantation 16:343, 1973). By way
of this surgical procedure, the aorta of a donor heart is
anastomosed to the abdominal aorta of the host, and the pulmonary
artery of the donor heart is anastomosed to the adjacent vena cava
using standard microvascular techniques. Once the heart is grafted
in place and warmed to 37.degree. C. with Ringer's lactate
solution, normal sinus rhythm will resume. Function of the
transplanted heart can be assessed frequently by palpation of
ventricular contractions through the abdominal wall. Rejection is
defined as the cessation of myocardial contractions. Agents of the
invention (e.g., a combination of mutant IL-15/Fc and an antibody
that binds to and inhibits IL-2 or IL-2R, or a combination of a
mutant IL-15/FC, IL-2/Fc, and rapamycin) would be considered
effective in reducing organ rejection if hosts that received these
agents experienced a longer period of engraftment of the donor
heart than did untreated hosts.
[0080] Skin Grafting
[0081] The effectiveness of various combinations of the agents of
the invention can also be assessed following a skin graft. To
perform skin grafts on a rodent, a donor animal is anesthetized and
the full thickness skin is removed from a part of the tail. The
recipient animal is also anesthetized, and a graft bed is prepared
by removing a patch of skin from the shaved flank. Generally, the
patch is approximately 0.5.times.0.5 cm. The skin from the donor is
shaped to fit the graft bed, positioned, covered with gauze, and
bandaged. The grafts can be inspected daily beginning on the sixth
post-operative day, and are considered rejected when more than half
of the transplanted epithelium appears to be non-viable. Agents of
the invention (e.g., a combination of mutant IL-15/Fc and an
antibody that binds to and inhibits IL-2 or IL-2R, or a combination
of a mutant IL- 15/FC, IL-2/Fc, and rapamycin) would be considered
effective in reducing skin graft rejection if hosts that received
these agents experienced a longer period of engraftment of the
donor skin than did untreated hosts.
[0082] A typical example of a skin grafting experiment, the results
of which demonstrate the usefulness of a composition containing
IL-2/Fc, mIL-15/Fc and rapamycin, is described in the Examples
(below) and summarized in FIG. 12.
[0083] Islet Allograft Model
[0084] DBA/2 J islet cell allografts can be transplanted into
rodents, such as 6-8 week-old B6 AFI mice rendered diabetic by a
single intraperitoneal injection of streptozotocin (225 mg/kg;
Sigma Chemical Co., St. Louis, Mo.). As a control, syngeneic islet
cell grafts can be transplanted into diabetic mice. Islet cell
transplantation can be performed by following published protocols
(for example, see Gotoh et al., Transplantation 42:387, 1986).
Briefly, donor pancreata are perfused in situ with type IV
collagenase (2 mg/ml; Worthington Biochemical Corp., Freehold,
N.J.). After a 40-minute digestion period at 37.degree. C., the
islets are isolated on a discontinuous Ficoll gradient.
Subsequently, 300-400 islets are transplanted under the renal
capsule of each recipient. Allograft function can be followed by
serial blood glucose measurements (Accu-Check III.TM.; Boehringer,
Mannheim, Germany). Primary graft function is defined as a blood
glucose level under 11.1 mmol/l on day 3 post-transplantation, and
graft rejection is defined as a rise in blood glucose exceeding
16.5 mmol/l (on each of at least 2 successive days) following a
period of primary graft function.
[0085] Models of Autoimmune Disease
[0086] Models of autoimmune disease provide another means to assess
combinations of the agents of the invention in vivo. These models
are well known to those of ordinary skill in the art and can be
used to determine whether a given combination of agents, which
includes, for example, an agent that targets an IL-15R, would be
therapeutically useful in treating a specific autoimmune disease
when delivered either directly, via genetic therapy, or via
cell-based therapies.
[0087] Autoimmune diseases that have been modeled in animals
include rheumatic diseases, such as rheumatoid arthritis and
systemic lupus erythematosus (SLE), type I diabetes, and autoimmune
diseases of the thyroid, gut, and central nervous system. For
example, animal models of SLE include MRL mice, BXSB mice, and NZB
mice and their F.sub.1 hybrids. These animals can be crossed in
order to study particular aspects of the rheumatic disease process;
progeny of the NZB strain develop severe lupus glomerulonephritis
when crossed with NZW mice (Bielschowsky et al., Proc. Univ. Otago
Med. Sch. 37:9, 1959; see also Fundamental Immunology, Paul, Ed.,
Raven Press, New York, N.Y., 1989). Similarly, a shift to lethal
nephritis is seen in the progeny of NBZ X SWR matings (Data et al.,
Nature 263:412, 1976). The histological appearance of renal lesions
in SNF.sub.1 mice has been well characterized (Eastcott et al., J.
Immunol. 131:2232, 1983; see also Fundamental Immunology, supra).
Therefore, the general health of the animal as well as the
histological appearance of renal tissue can be used to determine
whether the administration of agents that target an IL-15R and,
e.g., target the IL-2R, can effectively suppress the immune
response in an animal model of SLE.
[0088] One of the MRL strains of mice that develops SLE,
MRL-lpr/lpr, also develops a form of arthritis that resembles
rheumatoid arthritis in humans (Theofilopoulos et al., Adv.
Immunol. 37:269, 1985). Alternatively, an experimental arthritis
can be induced in rodents by injecting rat type II collagen (2
mg/ml) mixed 1:1 in Freund's complete adjuvant (100 .mu.l total)
into the base of the tail. Arthritis develops 2-3 weeks after
immunization. The ability of nucleic acid molecules encoding agents
of the invention (e.g., agents that target the IL-15R and agents
that target the IL-2R or that bind to and inactivate
antigen-activated T cells) to suppress an immune response can be
assessed by targeting the nucleic acid molecules to T lymphocytes.
One way to target T lymphocytes is as follows. Spleen cell
suspensions are prepared 2-3 days after the onset of arthritis and
incubated with collagen (100 .mu.g/ml) for 48 hours to induce
proliferation of collagen-activated T cells. During this time, the
cells are transduced with a vector encoding the polypeptide agent
of interest. As a control, parallel cultures are grown but not
transduced or, transduced with an "empty" vector.
[0089] The cells are then injected intraperiotoneally
(5.times.10.sup.7 cells/animal). The effectiveness of the treatment
is assessed by following the disease symptoms during the subsequent
2 weeks, as described by Chernajovsky et al. (Gene Therapy
2:731-735, 1995). Lesser symptoms, compared to control, indicate
that the combined agents of the invention, and the nucleic acid
molecules that encode them, function as immunosuppressants and are
therefore useful in the treatment of immune disease, particularly
autoimmune disease.
[0090] The ability of various combinations of agents to suppress
the immune response in the case of Type I diabetes can be tested in
the BB rat strain, which was developed from a commercial colony of
Wistar rats at the Bio-Breeding Laboratories in Ottawa. These rats
spontaneously develop autoantibodies against islet cells and
insulin, just as occurs with human Type I diabetes. Alternatively,
NOD (non-obese diabetic) mice can be used as a model system. A
typical example of an experiment in which blood sugar levels are
restored in NOD mice following transplantation of allogeneic donor
islets is described below and summarized in FIG. 11. The animals
were treated with a combination of IL-2/Fc, IL-15/Fc, and
rapamycin. The result was long-term engraftment.
[0091] Autoimmune diseases of the thyroid have been modeled in the
chicken. Obese strain (OS) chickens consistently develop
spontaneous autoimmune thyroiditis resembling Hashimoto's disease
(Cole et al., Science 160:1357, 1968). Approximately 15% of these
birds produce autoantibodies to parietal cells of the stomach, just
as in the human counterpart of autoimmune thyroiditis. The
manifestations of the disease in OS chickens, which could be
monitored in the course of any treatment regime, include body size,
fat deposit, serum lipids, cold sensitivity, and infertility.
[0092] Models of autoimmune disease in the central nervous system
(CNS) can also be experimentally induced. An inflammation of the
CNS, which leads to paralysis, can be induced by a single injection
of brain or spinal cord tissue with adjuvant in many different
laboratory animals, including rodents and primates. This model,
referred to as experimental allergic encephalomyelitis (EAE) is T
cell mediated. Similarly, experimentally induced myasthenia gravis
can be produced by a single injection of acetylcholine receptor
with adjuvants (Lennon et al., Ann. N.Y. Acad. Sci. 274:283,
1976).
[0093] Autoimmune diseases of the gut can be modeled in IL-2 or
IL-10 "knock out" mice, or in mice that receive enemas containing
bovine serum albumin.
[0094] Nucleic Acid Molecules That Encode Agents of the
Invention
[0095] Polypeptide agents of the invention, including those that
are fusion proteins (e.g., the mutant IL-15/Fc and IL-2/Fc
molecules) can not only be obtained by expression of a nucleic acid
molecule in a suitable eukaryotic or prokaryotic expression system
in vitro and subsequent purification of the polypeptide agent, but
can also be administered to a patient by way of a suitable gene
therapeutic expression vector encoding a nucleic acid molecule.
Further more a nucleic acid can be introduced into a cell of a
graft prior to transplantation of the graft. Thus, nucleic acid
molecules encoding the agents described above are within the scope
of the invention. Just as polypeptides of the invention can be
described in terms of their identity with wild-type polypeptides,
the nucleic acid molecules encoding them will necessarily have a
certain identity with those that encode the corresponding wild-type
polypeptides. For example, the nucleic acid molecule encoding a
mutant IL-15 polypeptide can be at least 65%, preferably at least
75%, more preferably at least 85%, and most preferably at least 95%
(e.g., 96%, 97%, 98%, or 99% ) identical to the nucleic acid
encoding wild-type IL-15. For nucleic acids, the length of the
sequences compared will generally be at least 50 nucleotides,
preferably at least 60 nucleotides, more preferably at least 75
nucleotides, and most preferably 110 nucleotides.
[0096] The nucleic acid molecules that encode agents of the
invention can contain naturally occurring sequences, or sequences
that differ from those that occur naturally, but, due to the
degeneracy of the genetic code, encode the same polypeptide. These
nucleic acid molecules can consist of RNA or DNA (for example,
genomic DNA, cDNA, or synthetic DNA, such as that produced by
phosphoramidite-based synthesis), or combinations or modifications
of the nucleotides within these types of nucleic acids. In
addition, the nucleic acid molecules can be double-stranded or
single-stranded (i.e., either a sense or an antisense strand).
[0097] The nucleic acid molecules of the invention are referred to
as "isolated" because they are separated from either the 5' or the
3' coding sequence with which they are immediately contiguous in
the naturally occurring genome of an organism. Thus, the nucleic
acid molecules are not limited to sequences that encode
polypeptides; some or all of the non-coding sequences that lie
upstream or downstream from a coding sequence can also be included.
Those of ordinary skill in the art of molecular biology are
familiar with routine procedures for isolating nucleic acid
molecules. They can, for example, be generated by treatment of
genomic DNA with restriction endonucleases, or by performance of
the polymerase chain reaction (PCR). In the event the nucleic acid
molecule is a ribonucleic acid (RNA), molecules can be produced by
in vitro transcription.
[0098] The isolated nucleic acid molecules of the invention can
include fragments not found as such in the natural state. Thus, the
invention encompasses recombinant molecules, such as those in which
a nucleic acid sequence (for example, a sequence encoding a mutant
IL-15) is incorporated into a vector (for example, a plasmid or
viral vector) or into the genome of a heterologous cell (or the
genome of a homologous cell, at a position other than the natural
chromosomal location).
[0099] As described above, agents of the invention can be fusion
proteins. In addition to, or in place of, the heterologous
polypeptides described above, a nucleic acid molecule encoding an
agent of the invention can contain sequences encoding a "marker" or
"reporter." Examples of marker or reporter genes include
.beta.-lactamase, chloramphenicol acetyltransferase (CAT),
adenosine deaminase (ADA), aminoglycoside phosphotransferase
(neo.sup.r, G418.sup.r), dihydrofolate reductase (DHFR),
hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ
(encoding .beta.-galactosidase), and xanthine guanine
phosphoribosyltransferase (XGPRT). As with many of the standard
procedures associated with the practice of the invention, one of
ordinary skill in the art will be aware of additional useful
reagents, for example, of additional sequences that can serve the
function of a marker or reporter.
[0100] The nucleic acid molecules of the invention can be obtained
by introducing a mutation into an agent of the invention (e.g., an
IL-15 molecule or an IL-2 molecule) obtained from any biological
cell, such as the cell of a mammal, or produced by routine cloning
methods. Thus, the nucleic acids of the invention can be those of a
mouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, monkey,
baboon, dog, or cat. Preferably, the nucleic acid molecules will be
those of a human.
[0101] The nucleic acid molecules described above can be contained
within a vector that is capable of directing their expression in,
for example, a cell that has been transduced with the vector.
Accordingly, in addition to polypeptide agents, expression vectors
containing a nucleic acid molecule encoding those agents and cells
transfected with those vectors are among the preferred
embodiments.
[0102] Vectors suitable for use in the present invention include
T7-based vectors for use in bacteria (see, e.g., Rosenberg et al.,
Gene 56:125, 1987), the pMSXND expression vector for use in
mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988),
yeast expression systems, such as Pichia pastoris (for example the
PICZ family of expression vectors from Invitrogen, Carlsbad,
Calif.) and baculovirus-derived vectors (for example the expression
vector pBacPAK9 from Clontech, Palo Alto, Calif.) for use in insect
cells. The nucleic acid inserts, which encode the polypeptide of
interest in such vectors, can be operably linked to a promoter,
which is selected based on, for example, the cell type in which
expression is sought. For example, a T7 promoter can be used in
bacteria, a polyhedrin promoter can be used in insect cells, and a
cytomegalovirus or metallothionein promoter can be used in
mammalian cells. Also, in the case of higher eukaryotes,
tissue-specific and cell type-specific promoters are widely
available. These promoters are so named for their ability to direct
expression of a nucleic acid molecule in a given tissue or cell
type within the body. One of ordinary skill in the art is well
aware of numerous promoters and other regulatory elements that can
be used to direct expression of nucleic acids.
[0103] In addition to sequences that facilitate transcription of
the inserted nucleic acid molecule, vectors can contain origins of
replication, and other genes that encode a selectable marker. For
example, the neomycin-resistance (neo.sup.r) gene imparts G418
resistance to cells in which it is expressed, and thus permits
phenotypic selection of the transfected cells. Other feasible
selectable marker genes allowing for phenotypic selection of cells
include various fluorescent proteins, e.g. green fluorescent
protein (GFP) and variants thereof. Those of skill in the art can
readily determine whether a given regulatory element or selectable
marker is suitable for use in a particular experimental
context.
[0104] Viral vectors that can be used in the invention include, for
example, retroviral, adenoviral, and adeno-associated vectors,
herpes virus, simian virus 40 (SV40), and bovine papilloma virus
vectors (see, e.g., Gluzman (Ed.), Eukaryotic Viral Vectors, CSH
Laboratory Press, Cold Spring Harbor, N.Y.).
[0105] Prokaryotic or eukaryotic cells that contain a nucleic acid
molecule that encodes an agent of the invention and express the
protein encoded in that nucleic acid molecule in vitro are also
features of the invention. A cell of the invention is a transfected
cell, i.e., a cell into which a nucleic acid molecule, for example
a nucleic acid molecule encoding a mutant IL-15 polypeptide, has
been introduced by means of recombinant DNA techniques. The progeny
of such a cell are also considered within the scope of the
invention. The precise components of the expression system are not
critical. For example, a mutant IL-15 polypeptide can be produced
in a prokaryotic host, such as the bacterium E. coli, or in a
eukaryotic host, such as an insect cell (for example, Sf21 cells),
or mammalian cells (e.g., COS cells, CHO cells, 293 cells, NIH 3T3
cells, or HeLa cells). These cells are available from many sources,
including the American Type Culture Collection (Manassas, Va.). In
selecting an expression system, it matters only that the components
are compatible with one another. One of ordinary skill in the art
is able to make such a determination. Furthermore, if guidance is
required in selecting an expression system, one can consult Ausubel
et al. (Current Protocols in Molecular Biology, John Wiley and
Sons, New York, N.Y., 1993) and Pouwels et al. (Cloning Vectors: A
Laboratory Manual, 1985 Suppl. 1987).
[0106] Eukaryotic cells that contain a nucleic acid molecule that
encodes the agent of the invention and express the protein encoded
in such nucleic acid molecule in vivo are also features of the
invention.
[0107] Furthermore, eukaryotic cells of the invention can be cells
that are part of a cellular transplant, a tissue or organ
transplant. Such transplants can comprise either primary cells
taken from a donor organism or cells that were cultured, modified
and/or selected in vitro before transplantation to a recipient
organism (e.g., eurkaryotic cells lines, including stem cells or
progenitor cells). Since, after transplantation into a recipient
organism, cellular proliferation may occur, the progeny of such a
cell are also considered within the scope of the invention. A cell,
being part of a cellular, tissue or organ transplant, can be
transfected with a nucleic acid encoding a mutant Il -15
polypeptide and subsequently be transplanted into the recipient
organism, where expression of the mutant IL-15 polypeptide occurs.
Furthermore, such a cell can contain one or more additional nucleic
acid constructs allowing for application of selection procedures,
e.g. of specific cell lineages or cell types prior to
transplantation into a recipient organism.
[0108] The expressed polypeptides can be purified from the
expression system using routine biochemical procedures, and can be
used as diagnostic tools or as therapeutic agents, as described
below.
[0109] Agents that Target an IL-15R are Useful in Making
Diagnoses
[0110] Agents that target an IL-15R can be used to determine
whether a patient has a disease (e.g., an immune disease,
particularly autoimmune disease) that is amenable to treatment with
a combination of the agents described herein. The diagnostic method
can be carried out, for example, by obtaining a sample of tissue
from a patient suspected of having an immune disease, particularly
autoimmune disease or a cancer that is manifest as malignant immune
cells and exposing that tissue to an antigenically-tagged
polypeptide that targets an IL-15R. The sample may be any
biological sample, such as a blood, urine, serum, or plasma sample.
In addition, the sample may be a tissue sample (e.g.., biopsy
tissue), or an effusion obtained from a joint (e.g., synovial
fluid), from the abdominal cavity (e.g., ascites fluid), from the
chest (e.g., pleural fluid), or from the central nervous system
(e.g., cerebral spinal fluid). The sample may also consist of
cultured cells that were originally obtained from a patient (e.g.,
peripheral blood mononuclear cells). The sample can be obtained
from a mammal, such as a human patient. If the sample contains
cells that are bound by the agent to which they are exposed, it is
highly likely that they would be bound by that agent (e.g. an agent
that targets an IL-15R) in vivo and could thereby be inhibited from
proliferating or destroyed in vivo. The presenting symptoms of
candidate patients for such testing and the relevant tissues to be
sampled given a particular set of symptoms are well known to one of
ordinary skill in the art.
[0111] Patients Amenable to Treatment
[0112] The compositions of the invention are useful in inhibiting T
cells that are involved, or would be involved, in an immune
response (e.g., a cellular immune response) to an antigen; in
inhibiting other cells involved in the pathogenesis of
immunological disorders (e.g., monocytes, macrophages, and other
antigen presenting cells such as dendritinc cells, NK cells, and
granulocytes); and in destroying hyperproliferating cells (as seen,
for example, in tissues involved in immunological disorders such as
synovial fibroblasts (which are affected in rheumatoid arthritis)
keratinocytes (which are affected in psoriasis), or dermal
fibroblases (which are affected in systemic lupus erythematosis).
Given these examples, other cell types that can usefully be
targeted will be apparent to those of ordinary skill in the art.
Hyperproliferative cells may also be cancerous cells (e.g.,
malignant T cells).
[0113] Thus, the compositions of the invention can be used to treat
patients who are suffering from an immune disease, particularly
autoimmune disease, including but not limited to the following: (1)
a rheumatic disease such as rheumatoid arthritis, systemic lupus
erythematosus, Sjogren's syndrome, scleroderma, mixed connective
tissue disease, dermatomyositis, polymyositis, Reiter's syndrome or
Behcet's disease (2) type I or type II diabetes (3) an autoimmune
disease of the thyroid, such as Hashimoto's thyroiditis or Graves'
Disease (4) an autoimmune disease of the central nervous system,
such as multiple sclerosis, myasthenia gravis, or encephalomyelitis
(5) a variety of phemphigus, such as phemphigus vulgaris,
phemphigus vegetans, phemphigus foliaceus, Senear-Usher syndrome,
or Brazilian phemphigus, (6) diseases of the skin such as psoriasis
or neurodermitis, and (7) inflammatory bowel disease (e.g.,
ulcerative colitis or Crohn's Disease). Combinations of the agents
of the invention can also be used to treat acquired immune
deficiency syndrome (AIDS). Similarly, methods by which these
agents are administered can be used to treat a patient who has
received a transplant of synthetic or biological material, or a
combination of both. Such transplants can be organ, tissue or cell
transplants, or synthetic grafts seeded with cells, for example,
synthetic vascular grafts seeded with vascular cells. In addition,
patients suffering from GVHD or patients who have received a
vascular injury would benefit from this method.
[0114] Because the invention encompasses administration of a
target-cell depleting form of an agent that targets the IL-15R (or
an IL-2 receptor, or a combination of IL-15 or the IL-15R and IL-2
or the IL-2R), it is possible to selectively kill autoreactive or
"transplant destructive" immune cells without massive destruction
of normal T cells. Accordingly, the invention features a method of
killing cells that express the IL-15R in vivo, which includes
activated or autoreactive or "transplant destructive" immune cells
or malignant cells. These methods can be carried out by
administering to a patient a combination of agents that includes an
agent that targets the IL-15R and that activates the complement
system, lyses cells by the ADCC mechanism, or otherwise kills cells
expressing the wild-type IL-15 receptor complex. This method can be
used to treat patients who have IL-15R .sup.+ leukemia, lymphoma,
or other IL-15R.sub.+ malignant diseases, such as colon cancer.
[0115] Formulations for Use and Routes of Administration
[0116] The methods of the present invention and the therapeutic
compositions used to carry them out contain "substantially pure"
agents. For example, in the event the agent is a polypeptide, the
polypeptide is at least 60% by weight (dry weight) the polypeptide
of interest, e.g., a polypeptide that binds and destroys
IL-15R-bearing cells. Preferably, the agents (e.g., the
polypeptides) are at least 75%, more preferably at least 90%, and
most preferably at least 99%, by weight, the agent of interest.
Purity can be measured by any appropriate standard method, e.g.,
column chromatography, polyacrylamide gel electrophoresis, or HPLC
analysis.
[0117] Although agents useful in the methods of the present
invention can be obtained from naturally occurring sources, they
can also be synthesized or otherwise manufactured (e.g., agents
that bind and destroy IL-15R-bearing cells can be produced by
expression of a recombinant nucleic acid molecule). Polypeptides
that are derived from eukaryotic organisms or synthesized in E.
coli, or other prokaryotes, and polypeptides that are chemically
synthesized will be substantially free from their naturally
associated components.
[0118] In the event the polypeptide is a chimera, it can be encoded
by a hybrid nucleic acid molecule containing one sequence that
encodes all or part of the agent (e.g., a sequence encoding a
mutant IL-15 polypeptide and sequence encoding an Fc region of
IgG). Agents of the invention (e.g., polypeptides) can be fused to
a hexa-histidine tag to facilitate purification of bacterially
expressed protein, or to a hemagglutinin tag to facilitate
purification of protein expressed in eukaryotic cells.
[0119] The techniques that are required to make the agents of the
invention are routine in the art, and can be performed without
resort to undue experimentation by one of ordinary skill in the
art. For example, a mutation that consists of a substitution of one
or more of the amino acid residues in IL-15 can be created using
the PCR-assisted mutagenesis technique described herein for the
creation of the mutant IL-15 polypeptide in which glutamine
residues at positions 149 and 156 were changed to aspartic acid
residues. Mutations that consist of deletions or additions of amino
acid residues (to an IL-15 polypeptide or to any of the other
useful polypeptides described herein, e.g., polypeptides that
inhibit costimulation or that bind activated T cells) can also be
made with standard recombinant techniques. In therapeutic
applications, agents of the invention can be administered with a
physiologically acceptable carrier, such as physiological saline.
The therapeutic compositions of the invention can also contain a
carrier or excipient, many of which are known to one of ordinary
skill in the art. Excipients that can be used include buffers
(e.g., citrate buffer, phosphate buffer, acetate buffer, and
bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid,
phospholipids, proteins (e.g., serum albumin), EDTA, sodium
chloride, liposomes, mannitol, sorbitol, and glycerol. The agents
of the invention can be formulated in various ways, according to
the corresponding route of administration. For example, liquid
solutions can be made for ingestion or injection; gels or powders
can be made for ingestion, inhalation, or topical application.
Methods for making such formulations are well known and can be
found in, for example, "Remington's Pharmaceutical Sciences."
[0120] Routes of administration are also well known to skilled
pharmacologists and physicians and include intraperitoneal,
intramuscular, subcutaneous, and intravenous administration.
Additional routes include intracranial (e.g., intracisternal or
intraventricular), intraorbital, opthalmic, intracapsular,
intraspinal, intraperitoneal, transmucosal, topical, subcutaneous,
and oral administration. It is expected that the intravenous or
intra-arterial routes will be preferred for the administration of
agents that target an IL-15 receptor. The subcutaneous route may
also be used frequently as the subcutaneous tissue provides a
stable environment for polypeptides, from which they can be slowly
released.
[0121] In case of cell-based therapies (gene therapies), the
cells/tissues/organs could either be transfected by incubation,
infusion or perfusion prior to transplantation with a nucleic acid
composition, such that the therapeutic protein is expressed and
subsequently released by the transplanted cells/tissues/organs
within the recipient organism. As well, the cells/tissues/organs
could undergo a pretreatment by perfusion or simple incubation with
the therapeutic protein prior to transplantation in order to
eliminate transplant-associated immune cells adherent to the donor
cells/tissues/organs (although this is only a side aspect, which
will probably not be of any clinical relevance). In the case of
cell transplants, the cells may be administered either by an
implantation procedure or with a catheter-mediated injection
procedure through the blood vessel wall. In some cases, the cells
may be administered by release into the vasculature, from which the
subsequently are distributed by the blood stream and/or migrate
into the surrounding tissue (this is done in islet cells
transplantation, where the islet cells are released into the portal
vein and subsequently migrate into liver tissue).
[0122] It is well known in the medical arts that dosages for any
one patient depend on many factors, including the general health,
sex, weight, body surface area, and age of the patient, as well as
the particular compound to be administered, the time and route of
administration, and other drugs being administered concurrently.
Dosages for the polypeptide of the invention will vary, but can,
when administered intravenously, be given in doses on the order of
magnitude of 1 microgram to 10 mg/kg body weight or on the order of
magnitude of 0.01 mg/l to 100 mg/l of blood volume. A dosage can be
administered one or more times per day, if necessary, and treatment
can be continued for prolonged periods of time. Determining the
correct dosage for a given application is well within the abilities
of one of ordinary skill in the art.
EXAMPLES
[0123] Reagents
[0124] The following reagents were used in the studies described
herein: recombinant human IL-2 was obtained from Hoffman-La Roche
(Nutley, N.J.); rapamycin was obtained from Wyeth-Ayerst
(Princeton, N.J.); cyclosporine-A (CsA) was obtained from Sandoz
(East Hanover, N.J.); RPMI-1640 and fetal calf serum (FCS) were
obtained from BioWittaker (Walkersville, Md.); penicillin,
streptomycin, G418, and strepavidin-RED670 were obtained from
Gibco-BRL (Gaithersburg, Md.); dexamethasone, PHA, lysozyme,
Nonidet P-40, NaCl, HEPES, and PMSF were obtained from Sigma (St.
Louis, Mo.); Ficoll-Hypaque was obtained from Pharmacia Biotech
(Uppsala, Sweden); recombinant human IL-15 and anti-human IL-15 Ab
were obtained from PeproTech (Rocky Hill, N.J.); anti-FLAG Ab and
anti-FLAG-affinity beads were obtained from International
Biotechnologies, Inc. (Kodak, New Haven, Conn.); pRcCMV was
obtained from InVitrogen Corporation (San Diego, Calif.); genistein
was obtained from ICN Biomedicals (Irvine, Calif.); disuccinimidyl
suberate (DSS) was obtained from Pierce (Rockford, Ill.);
restriction endonucleases were obtained from New England Biolabs
(Beverly, Mass.); [.sup.3H]TdR was obtained from New England
Nuclear (Boston, Mass.); and fluorescent dye conjugated antibodies
CD25-PE.sup.3, CD14-PE, CD16-PE, CD122-PE, CD4-FITC, CD8-FITC,
IgG1-PE or IgG1-FITC were obtained from Beckton/Dickinson (San
Jose, Calif.). FLAG peptide was synthesized in the Peptide
Synthesis Facility at Harvard Medical School.
[0125] Production of FLAG-HMK-IL-15 Fusion Protein
[0126] To study the cellular pattern of human IL-15 receptor
expression, a plasmid that could be used to express an IL-15 fusion
protein was constructed. The plasmid encodes an IL-15 polypeptide
having an N-terminus covalently bound to the 18 amino acid
FLAG-HMK-sequence (FLAG-HMK-IL-15). FLAG sequences are recognized
by biotinylated, highly specific anti-FLAG antibodies (Blanar et
al., Science 256:1014, 1992); LeClair et al., Proc. Natl. Acad.
Sci. USA 89:8145,1992) while HMK (Heart Muscle Kinase recognition
site) sequences allow introduction of radioactive label [.sup.32P]
into the molecule (Blanar et al., supra, LeClair et al.,
supra).
[0127] For the construction of the plasmid FLAG-HMK-IL-15, a 322 bp
cDNA fragment encoding mature IL-15 protein was amplified by PCR
utilizing synthetic oligonucleotides [sense
5'-GGAATTCAACTGGGTGAATGTAATA-3' (SEQ ID NO: 5; EcoRI site
(underlined) plus bases 145-162); antisense
[0128] 5'-CGGGATCCTCAAGAAGTGTTGATGAA-3' (SEQ ID NO: 5; BamHI site
[underlined] plus bases 472-489)]. The template DNA was obtained
from PHA-activated human PBMCs. The PCR product was purified,
digested with EcoRI and BamHI, and cloned into the pAR(DRI)59/60
plasmid digested with EcoRi-BamHI as described (Blanar et al.,
Science 256:1014, 1992; LeClair et al., Proc. Natl. Acad. Sci. USA
89:8145, 1992). The backbone of the pAR(DRI)59/60 plasmid contains
in frame sequences encoding the FLAG and HMK recognition peptide
sequences (Blanar et al, Science 256:1014, 1992; LeClair et al.,
Proc. Natl. Acad. Sci. USA 89:8145, 1992).
[0129] Expression and Purification of FLAG-HMK-IL-15 Fusion
Protein
[0130] The IL-15-related fusion construct, FLAG-HMK-IL-15, was
expressed in BL-21 strain E. coli and affinity purified with
anti-FLAG coated beads as described (Blanar et al., Science
256:1014, 1992; LeClair et al., Proc. Natl. Acad. Sci. USA 89:8145,
1992). The fusion protein was eluted from affinity columns after
extensive washing with 0.1 M glycine (pH 3.0). The eluate
containing FLAG-HMK-IL-15 was dialyzed against a buffer containing
50 mM Tris (pH 7.4) and 0.1 M NaCl for 18 hours at 4.degree. C.,
filtered through a 0.2 .mu.m membrane, and stored at -20.degree.
C.
[0131] FLAG-HMK-IL-15 binds the IL-15R.alpha. subunit
[0132] The purified FLAG-HMK-IL-15 fusion protein was tested to
determine whether it interacts with cell surface IL-15 receptors.
As described above, [.sup.32P]-FLAG-HMK-IL-15 was added to cultures
of PBMCs that were activated by a mitogen, PHA. In order to
permanently bind interactive proteins to one another, the chemical
cross-linker disuccinimidyl suberate (DSS) was added. The cells
were washed, lysed, centrifuged, and detergent-soluble proteins
were separated by SDS-PAGE. Autoradiography of SDS-PAGE separated
proteins revealed a single 75-80 kDa band corresponding to the
combined molecular weight of FLAG-HMK-IL-15 (15 kDa) and the human
IL-15R.alpha. subunit (60-65 kDa). The identity of this band as the
IL-15R.alpha. subunit was confirmed by cross-linking experiments
conducted in the presence of a molar excess of hIL-15. Under these
conditions, we failed to detect the radio labeled 15 kDa band.
Thus, the conformation of [.sup.32P]-FLAG-HMK-IL-15 fusion proteins
allows site specific binding to the 60-65 kDa IL-15R.alpha. subunit
expressed on the surface of mitogen-activated PBMCs.
[0133] FLAG-HMK-IL-15 is a Biologically Active Growth Factor that
Requires Expression of IL-2R.beta.
[0134] In the next series of experiments, the FLAG-HMK-IL-15 fusion
protein was tested to determine whether it could function as a
biologically active growth factor. PHA-activated human PBMCs
proliferate in response to either FLAG-HMK-IL-15 or human
recombinant IL-2, as detected via the [.sup.3H]-TdR incorporation
assay. A FLAG peptide lacking the IL-15 sequence does not stimulate
cell proliferation. As does IL-2, the FLAG-HMK-IL-15 fusion protein
stimulates proliferation of IL-2R.gamma..sup.+BAF-BO3 cell
transfectants that express the IL-2 R.beta. subunit. The
FLAG-HMK-IL-15 fusion protein does not, however, stimulate the
proliferation of parental BAF-BO3 cells that were transfected with
a vector lacking IL-2R.beta. chain sequences. Thus, FLAG-HMK-IL-15
is a biologically active growth factor that requires expression of
IL-2R.beta. chains upon target cells in order to stimulate cellular
proliferation.
[0135] Mitogen-activated, but not Resting, PBMCs Express the
IL-15R.alpha. Subunit
[0136] The FLAG-HMK-IL-15 fusion protein, biotinylated anti-FLAG
antibody, and streptavidin-RED670 were employed to detect
expression of IL-15 binding sites on human PBMCs by
cytofluorometric analysis. The PBMCs tested were either freshly
isolated or PHA-activated. These cells were washed and incubated
with either medium alone or FLAG-HMK-IL-15 followed by anti-FLAG
biotinylated Ab and streptavidin-RED670. The stained cells were
analyzed by flow cytometry. PBMCs that were activated with PHA
expressed IL-5 R.alpha. proteins but resting PBMCs did not. In
keeping with the result of the cross-linking experiments described
above, binding of FLAG-HMK-IL-15 to PHA activated PBMCs is blocked
by a molar excess of rIL-15, thereby demonstrating the specificity
of FLAG-HMK-IL-15 binding for IL-15 binding sites. Both activated
CD4.sup.+ and CD8.sup.+ cells express IL-15 .alpha. chains.
Activation induced IL-15R.alpha. chains were also detected on
CD14.sup.+ (monocyte/macrophage) cells and CD 16.sup.+ (natural
killer) cells.
[0137] IL-2R.alpha. and IL-2R.beta. Subunits Are Not Required for
IL-15 Binding
[0138] FACS analysis of PHA-activated PBMCs stained with
FLAG-HMK-IL-15 proteins and anti-CD25 Mab, against the IL-2R.alpha.
subunit, reveals cell populations expressing both IL-15R.alpha. and
IL-2R.alpha. subunits, as well as cell populations that express
either subunit, but not both. There are IL-2R.alpha..sup.+ cells
that do not bind FLAG-K-IL-15. Almost all PBMCs that were
stimulated with PHA for only one day express either IL-15R.alpha.
or IL-2R.beta. chains, but not both proteins. In contrast, 3 days
following PHA stimulation, a far larger population of
IL-15R.alpha..sup.+, IL-2R.beta..sup.+ cells (double positive) and
a far smaller population of IL-15R.alpha..sup.+, IL-2R.beta..sup.+
cells (single positive) were noted. Interestingly, there are
IL-2R.beta..sup.+ cells that fail to bind IL-15. Therefore,
expression of IL-2R.beta. chains is not sufficient for IL-15
binding.
[0139] Taken together, these data indicate that IL-15 can bind
IL-15R.alpha..sup.+, IL-2R.alpha..sup.-, and IL-2R.beta..sup.-
cells. A similar conclusion was reached through experimentation
that probed the interaction of IL-15 with IL-2R.alpha..sup.-,
.beta..sup.- cells transfected with IL-15R.alpha. subunit (Anderson
et al., J. Biol. Chem. 270:29862, 1995; Giri et al., EMBO J.
14:3654, 1995). In addition to the requirement for IL-15R.alpha.
subunit expression, the IL-2R.beta. and IL-2R.gamma. subunits are
required to render cells sensitive to IL-15 triggered growth.
[0140] In summary, the experiments presented above have
demonstrated that: (i) IL-15R.alpha. subunits are rapidly expressed
by activated macrophages, T cells, and NK cells, and (ii) induction
of the IL-15R.alpha. subunit is blocked by dexamethasone but not by
CsA or rapamycin. In addition, the experiments have confirmed that
the IL-15R.alpha. subunit is necessary and sufficient for IL-15
binding and that the FLAG-HMK-IL-15 fusion protein is an extremely
useful tool for studying IL-15 receptors.
[0141] The IL-2R.beta. Subunit is Critical for both IL-2 and IL-15
Signal Transduction
[0142] Decreasing the viability of activated T cells and thereby
depleting activated T cells provides a way to decrease the
production of lymphokines and mitogens that contribute to
accelerated atherosclerosis, allograft rejection, certain leukemias
and other immune-mediated pathologies. In addition, blocking the
signal transduction pathway activated by IL-15 also provides a way
to decrease the production of lymphokines and mitogens that
contribute to accelerated atherosclerosis, allograft rejection,
certain leukemias and other immune-mediated pathologies. When
activated, T cells proliferate and express receptors on their cell
surface for interleukins. In addition, activated T cells release at
least 3 lymphokines: gamma interferon, B cell differentiation
factor II, and IL-3. These lymphokines can produce various
undesirable events, such as allograft rejection. In contrast,
resting T cells and long-term memory T cells do not express
lymphokine receptors. This difference in receptor expression
provides a means to target activated immune cells without
interfering with resting cells.
[0143] Molecules designed to recognize some subunit of the IL-15R
will recognize activated monocytes/macrophages as well as activated
T cells and can be used to selectively inhibit or destroy these
cells. Derivatives of IL-15 that bind to an IL-15R subunit but that
lack IL-15 activity, either because they block the binding and/or
uptake of bona fide IL-15, are useful in the method of the
invention. The mutant IL-15 molecule described below provides a
working example of such a derivative.
[0144] A Mutant IL-15 Polypeptide that Targets an IL-15R
[0145] Genetic Construction of mutant IL-15
[0146] The human IL-15 protein bearing a double mutation (Q149 D;
Q156 D) was designed to target the putative sites critical for
binding to the IL-2R.gamma. subunit. The polar, but uncharged
glutamine residues at positions 149 and 156 were mutated into
acidic residues of aspartic acid utilizing PCR-assisted
mutagenesis. A cDNA encoding the double mutant of IL-15 was
amplified by PCR utilizing a synthetic sense oligonucleotide
[5'-GGAATTCAACTGGGTGAATGTAATA-3' (SEQ ID NO: ______ ); EcoRI site
(underlined hexamer) plus bases 145-162] and a synthetic antisense
oligonucleotide
(5'-CGGGATCCTCAAGAAGTGTTGATGAACATGTCGACAAT-ATGTACAAAACTGT-
CCAAAAAT-3'(SEQ ID NO: ______); BamHI site (underlined hexamer)
plus bases 438-489; mutated bases are singly underlined]. The
template was a plasmid containing cDNA that encodes human
FLAG-HMK-IL-15. The amplified fragment was digested with
EcoRI/BamHI and cloned into the pAR(DRI)59/60 plasmid digested with
EcoRI/BamRI as described (LeClair et al., Proc. Natl. Acad. Sci.
USA 89:8145, 1989). The presence of a mutation at residue 156 was
confirmed by digestion with SalI; the mutation introduces a new
SalI restriction site. In addition, mutations were verified by DNA
sequencing, according to standard techniques. The FLAG-HMK-IL-15
(Q149D; Q156D) double mutant protein was produced, purified, and
verified by sequencing as described above for the FLAG-HMK-IL-15
wild-type protein.
[0147] Using this same strategy, mutants that contain a single
amino acid substitution, either at position 149 or at position 156
were prepared. As described above, these positions (149 and 156)
correspond to positions 101 and 108, respectively, in the mature
IL-15 polypeptide, which lacks a 48-amino acid signal sequence.
[0148] Similarly, this strategy can be used to incorporate any
other amino acid in place of the glutamine residues at positions
149 or 156 or to introduce amino acid substitutions at positions
other than 149 and/or 156.
[0149] Proliferation of BAF-BO3 Cells in the Presence of IL-15
Related Proteins
[0150] The double mutant IL-15 polypeptide may inhibit BAF-BO3
proliferation in a dose dependent manner: addition of 30 .mu.m
(approximately 50 .mu.g/ml) of the double mutant IL-15 inhibited
proliferation more completely than did addition of 20 .mu.L of the
same concentration of the double mutant IL-15.
[0151] Proliferation of PHA-Stimulated Human PBMCs
[0152] The ability of the FLAG-HMK-IL-15 double mutant polypeptide
to bind PHA activated human PBMCs was demonstrated as follows.
PHA-activated PBMCs were washed and incubated with medium alone, or
with the FLAG-HMK-IL-15 double mutant. The cells were then
incubated with an anti-FLAG biotinylated antibody and stained with
streptavidin conjugated to RED670. The stained cells were analyzed
by flow cytometry.
[0153] FACS Analysis of Leukemic Cell Lines Stained with Wild-Type
FLAG-HMK-IL-15
[0154] In a series of experiments similar to those above, the
ability of the wild-type FLAG-HMK-IL-15 polypeptide to bind
leukemia cells was shown. The cells treated were obtained from the
leukemic cell lines MOLT-14, YT, HuT-102, and from cell lines
currently being established at Beth Israel Hospital (Boston,
Mass.), and named 2A and 2B. The cultured cells were washed and
incubated with either medium alone or with medium containing the
FLAG-HMK-IL-15 wild-type polypeptide. The cells were then incubated
with the biotinylated anti-FLAG antibody and stained with
RED670-conjugated streptavidin. The stained cells were analyzed by
flow cytometry.
[0155] Genetic Construction of Additional Mutant IL-15 Chimeric
Polypeptides
[0156] In addition to the FLAG-HMK-IL-15 chimera, which provides
the mutant IL-15 with an antigenic tag, numerous other polypeptides
can be linked to any mutant of IL-15 or IL-2. For example, mutant
IL-15 or IL-2 can be linked to the Fc fragment of the IgG subclass
of antibodies according to the following method.
[0157] Genetic Construction of Mutant IL-15/Fc
[0158] cDNA for Fc.gamma.2a can be generated from mRNA extracted
from an IgG2a secreting hybridoma using standard techniques with
reverse transcriptase (MMLV-RT; Gibco-BRL, Grand Island, N.Y.) and
a synthetic oligo-dT (12-18) oligonucleotide (Gibco BRL). The
mutant IL-15 cDNA can be amplified from a plasmid template by PCR
using IL-15 specific synthetic oligonucleotides.
[0159] The 5' oligonucleotide is designed to insert a unique NotI
restriction site 40 nucleotides 5' to the translational start
codon, while the 3' oligonucleotide eliminates the termination
codon and modifies the C-terminal Ser residue codon usage from AGC
to TCG to accommodate the creation of a unique BamHI site at the
mutant IL-15/Fc junction. Synthetic oligonucleotides used for the
amplification of the Fc.gamma.2a domain cDNA change the first codon
of the hinge from Glu to Asp in order to create a unique BamHI site
spanning the first codon of the hinge and introduce a unique XbaI
site 3' to the termination codon.
[0160] The Fc fragment can be modified so that it is
non-target-cell depleting, i.e., not able to activate the
complement system. To make the non-target-cell depleting mutant
IL-15 construct (mIL-15/Fc), oligonucleotide site directed
mutagenesis is used to replace the C'1q binding motif Glu318,
Lys320, Lys322 with Ala residues. Similarly, Leu235 is replaced
with Glu to inactivate the Fc.gamma.R I binding site. Ligation of
cytokine and Fc.DELTA. components in the correct translational
reading frame at the unique BamHI site yields a 1,236 basepair open
reading frame encoding a single 411 amino acid polypeptide
(including the 18 amino acid IL-15 signal peptide) with a total of
13 cysteine residues. The mature secreted homodimeric IL-15/Fc is
predicted to have a total of up to eight intramolecular and three
inter-heavy chain disulfide linkages and a molecular weight of
approximately 85 kDa, exclusive of glycosylation.
[0161] Expression and Purification of mIL-15 Receptor Fc Fusion
Proteins
[0162] Proper genetic construction of mIL-15/Fc can be confirmed by
DNA sequence analysis following cloning of the fusion gene as a
NotI-XbaI cassette into the eukaryotic expression plasmid pRc/CMV
(In vitrogen, San Diego, Calif.). This plasmid carries a CMV
promoter/enhancer, a bovine growth hormone polyadenylation signal
and a neomycin resistance gene for selection with G418. Plasmids
carrying the mIL-15/Fc fusion gene is transfected into Chinese
hamster ovary cells (CHO-K1, available from the American Type
Culture Collection) by electroporation (1.5 kV/3 .mu.F/0.4 cm/PBS)
and selected in serum-free Ultra-CHO media (BioWhittaker Inc.,
Walkerville, Md.) containing 1.5 mg/ml of G418 (Geneticin, Gibco
BRL). After subcloning, clones that produce high levels of the
fusion protein are selected by screening supernatants from IL-15 by
ELISA (PharMingen, San Diego, Calif.). mIL-15/Fc fusion proteins
are purified from culture supernatants by protein A sepharose
affinity chromatography followed by dialysis against PBS and 0.22
.mu.m filter sterilization. Purified proteins can be stored at
-20.degree. C. before use.
[0163] Western blot analysis following SDS-PAGE under reducing
(with DTT) and nonreducing (without DTT) conditions can be
performed using monoclonal or polyclonal anti-mIL-15 or anti
Fc.gamma. primary antibodies to evaluate the size and isotype
specificity of the fusion proteins. The functional activity of
mutant IL-15/Fc can be assessed by a standard T cell proliferation
assay, as described above. The following mAbs were obtained from
PharMingen (San Diego, Calif.): PE-anti-mouse CD25 (IL-2R .alpha.
chain, IgG1, PC61), rat anti-mouse CD122 (IL-2R .beta. chain,
IgG2b, TM-b1), rat anti-mouse CD132 (IL-2R .gamma.c, IgG2b, TUGm2),
hamster anti-mouse CD3 (IgG, 145-2C11), hamster anti-mouse CD28
(IgG, 37.51), PE-anti-mouse CD62 (IgG2a, MEL14), PE conjugated
hamster anti-mouse Bcl-2 (IgG, 3F11), PE conjugated anti-mouse IL-2
(IgG2b, JES6-5H4), PE-annexin V, biotinylated anti-rat IgG2b,
PE-streptoavidin, PE-CyChrome, and PE conjugated isotype control
mAbs. A biotinylated mouse anti-FLAG mAb and a rat IgG1 control mAb
were obtained from Sigma Chemical Co. (St Louis, Mo.). A B-cell
hybridoma secreting rat anti-mouse CD25 mAb (TIB 222, IgG1) was
obtained from the American Type Culture Collection (ATCC; Manassas,
Va.). Cells were grown in serum free UltraCulture medium
(BioWhittaker, Walkerville, Md.) and the mAb in the culture
supernatant was purified with a protein G column.
[0164] Expression Studies of IL-2 and IL-15 in vivo.
[0165] Recombinant human IL-2 and IL-15 were purchased from R &
D System (Minneapolis, Minn.). IL-15-FLAG and IL-15 mutant/Fc
fusion proteins were constructed, expressed, and tested as
previously reported (Chae et al., J. Immunol. 157:2813-2819, 1996;
Kim et al., J. Immunol. 161:5742-5748, 1998). Rat anti-mouse
.gamma.c mAbs (4G3/3E12, IgG2b) were used as previously reported
(Li et al. J. Immunol. 164:1193-1199, 2000).
[0166] Lymphocytes were labeled with fluorochrome
5-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular
Probes, Inc., Portland, Ore.) as follows. Spleens and peripheral
lymph nodes were harvested from donor mice and single cell
suspensions were prepared in Hanks balanced salt solution (HBSS).
Red blood cells were lysed by hypotonic shock. Cells were
resuspended in HBSS at 1.times.10.sup.7/ml and labeled with CFSE as
described by Wells et al. (J. Clin. Invest. 100:3173-3183,
1997).
[0167] To activate CFSE-labeled T cells in vivo, DBA/2 mice were
irradiated (1000 rad) with a Gammacell Exactor (Kanata, Ontario,
Canada). Each mouse then received 4 to 6.times.10.sup.7
CFSE-labeled cells in 0.5 ml HBSS via the tail vein. Three days
later, the host mice were sacrificed and spleens and peripheral
lymph nodes were harvested separately. Single cell suspensions were
prepared for cell surface staining and FACS analysis.
[0168] In some experiments, irradiated host mice were treated with
anti-CD25 mAb or anti-.gamma.c mAbs (i.p. at 1 mg/day for 3 days
starting at i.v. injection of CFSE-labeled cells). Cell division in
vivo was determined on the third day following injection of
CFSE-labeled cells. Treatment with IL-15 mutant /Fc fusion protein
consisted of 1.5 .mu.g i.p. daily, for three days, starting at i.v.
injection of labeled cells.
[0169] CFSE-labeled cells activated in vivo in irradiated
allogeneic hosts were stained for the expression of IL-2 and IL-15
receptor subunits. To detect IL-2 receptor .alpha. chain
expression, cells (2.times.10.sup.6) were stained with
PE-anti-mouse CD25 mAb on ice for 30 minutes, washed, and
resuspended in 1 ml PBS containing 0.5% BSA. To detect IL-2R
.beta., and .gamma.c expression, cells were incubated with a rat
anti-mouse .beta. chain (IgG2b) or .gamma.c mAb (IgG2b) on ice for
30 minutes, followed by incubation with a biotinylated anti-rat
IgG2b. Cells were washed and further stained with PE-streptoavidin
for 20 minutes. Cells were washed and resuspended in PBS-0.5% BSA
for analysis. To detect IL-15R .alpha. chain expression, cells were
incubated with an IL-15-FLAG fusion protein that binds to the
.alpha. chain (Chae et al., J. Immunol. 157:2813-2819, 1996) and
then stained with biotinylated mouse anti-FLAG mAb. The cells were
then washed and stained with PE-streptoavidin. Isotype matched
control mAbs were included in each experiment as a control. All
samples were analyzed using FACSort with CellQuest.TM. software
(Becton Dickinson, Mountain View, Calif.). Data were collected and
analyzed by gating onto CFSE.sup.+ cells. All dividing CFSE.sup.+
cells were T cells, as defined by the expression of CD3. At least
100,000 events were collected for each sample.
[0170] Apoptosis of dividing T cells in vivo was analyzed as
follows. CFSE-labeled lymphocytes were stimulated in vivo in
irradiated allogeneic hosts as described above. Cells were
harvested from the host spleen or peripheral lymph nodes three days
later and stained with PE conjugated annexin V on ice for 15
minutes in labeling buffer. Cell division was identified based on
the cells' CFSE profile, and apoptotic cell death in each distinct
cell division was analyzed by annexin V staining.
[0171] Cells were also stained for intracellular IL-2 and Bcl-2
cytokine expression. CFSE-labeled cells that had been activated in
vivo for three days were harvested from the host spleen and lymph
nodes. Cells were restimulated in vitro with PMA (50 ng/ml) and
ionomycin (500 ng/ml) for four hours and GolgiStop.TM. (PharMingen)
was added for the last two hours of culture. Cells were fixed and
permeablized with Cytofix/Cytoperm (PharMingen) at 4.degree. C. for
10 minutes, and then stained with PE-conjugated anti-mouse IL-2
mAb, isotype matched control mAb was included as a control. For
Bcl-2 staining, cells were fixed and permeablized with
Cytofix/Cytoperm for 10 minutes and stained with PE-conjugated
anti-Bcl-2 mAb or isotype control Ab for 30 minutes. Cells were
washed and analyzed by FACS.
[0172] Cell sorting and in vitro re-stimulation was carried out as
follows. CFSE-labeled cells were prepared from irradiated
allogeneic hosts three days after i.v. injection of labeled cells.
Cell proliferation in vivo was identified through analysis of their
CFSE profiles. The second cell divisions were selected, gated, and
sorted with FACS Vantage.TM. sorter (Becton Dickinson) at 2000
events/second. The sorted cells were resuspended in RPMI 1640
medium supplemented with 10% FCS and 1% penicillin and streptomycin
at 5.times.10.sup.5/ml and plated on anti-CD3 (2 .mu.g/ml) coated
plates along with anti-CD28 mAb (1 .mu.g/ml). Three days later,
cells were harvested and stained with PE-conjugated anti-mouse CD25
and isotype control Ab. Cell proliferation and IL-2 receptor
.alpha. chain expression were analyzed by FACS.
[0173] Cell sorting and in vitro proliferation assays were carried
out as follows. CFSE-labeled cells were prepared from irradiated
allogeneic hosts three days after intravenous injection of labeled
cells, and cell proliferation in vivo was identified by analysis of
the cells' CFSE profile. The second cell division was selected,
gated, and sorted with FACS Vantage.TM.. Cells
(1.times.10.sup.4/ml) were resuspended in RPMI 1640 medium with 10%
FCS and 1% penicillin and streptomycin, and stimulated with IL-2
(40 .mu.ml to 500 .mu./ml) or IL-15 (5 ng/ml) for 48 hours. Cells
were pulsed with 1 mCi 3H-TdR (Amersham, Boston, Mass.) for 16
hours and .sup.3H-TdR uptake was determined by scintillation
counting (Beckman Instrument, Columbia, Md.).
[0174] The reagents and techniques described above provided the
basis for several findings. First, CFSE-labeled B6AF1 (H-2b/d.k)
allogeneic lymphocytes, in contrast to syngeneic controls (Li et
al., Nature Medicine 5:1298-1302, 1999), proliferated vigorously in
irradiated DBA/2 (H-2d) hosts. Approximately 20% of the
CFSE-labeled T cells recovered from the host spleen entered the
cell cycle within three days of adoptive transfer, and seven to
eight discrete rounds of cell division were clearly identified
(FIG. 3A). Surprisingly, the IL-2 receptor .alpha. chain, which is
required for high affinity IL-2 receptor signaling, could not be
detected during the first 5 divisions, i.e., this receptor subunit
is expressed only after five cell divisions. In contrast, .beta.
subunits of the IL-2 receptor were expressed constitutively by all
dividing T cells, and their level of expression was increased
progressively as cells continued to divide. The pattern of .gamma.c
expression in vivo differed strikingly from that of the .alpha.
chain and the .beta. chain (FIG. 3A). Undivided T cells (0
division) expressed very low levels of .gamma. chain (<10% ).
Following entry into the cell cycle, .gamma. chain was highly
expressed by dividing T cells, and the levels of expression
continued to increase after each consecutive cell division. After
five cell divisions, however, .gamma. chain expression was
drastically down regulated, nearly reaching the basal level after
the sixth cell division (FIG. 3A).
[0175] The differential expression of IL-2 receptor subunits is not
due to selective accumulation of a subset of activated T cells in
the host spleen, as CFSE-labeled cells harvested from peripheral
lymph nodes displayed a remarkably similar pattern of expression
for the three subunits of the IL-2 receptor.
[0176] Second, stimulation of CFSE-labeled T cells in vitro
resulted in a uniform expression of all three subunits of the IL-2
receptor (FIG. 3B). This suggests that regulation of IL-2 receptor
expression in vivo is distinct from that in vitro. The IL-2
receptor .alpha. chain is known to be sensitive to proteolytic
cleavage in vivo in a manner that is similar to the selectins
(Hemar et al., J. Cell. Biol. 129:55-64, 1995). Staining for
L-selectin expression by dividing T cells in vivo showed that
L-selectin was expressed at high levels during the first five cell
divisions (FIG. 3C), suggesting that the failure to detect IL-2
receptor .alpha. chain expression during the first five cell
divisions is not due to rapid proteolytic cleavage. To determine
whether T cells in the first five cell divisions are capable of
expressing the IL-2 receptor .alpha. chain, T cells at the second
cell division, which did not express IL2 receptor .alpha. chain,
were sorted and stimulated in vitro with immobilized anti-CD3 and
soluble anti-CD28 for three days. These sorted T cells continued to
divide upon in vitro restimulation, and all dividing T cells
expressed the IL-2 receptor .alpha. chain. Clearly, expression of
IL-2 receptor .alpha. chain in vivo and in vitro is differentially
regulated.
[0177] As the receptor for IL-15 also uses the IL-2 receptor .beta.
and .gamma. chains as critical signaling components (Tagaya et al.,
Immunity 4:329-336, 1996), which are highly expressed during the
first five cell divisions, we asked whether cells express an IL-15
receptor a chain that renders them responsive to IL-15 during
initial cell divisions. Application of an IL-15-FLAG fusion protein
as a primary staining reagent (Chae et al., J. Immunol.
157:2813-2819, 1996), demonstrated that the IL-15 receptor .alpha.
chain is clearly detectable, albeit at low levels, on dividing T
cells regardless the number of cell divisions (FIG. 4A). The
.alpha. chain for IL-2 receptor was not detected on all dividing T
cells in vivo. Thus, selective expression of the .alpha. chain for
IL-15 receptor, but not for IL-2 receptor, along with the
expression of shared .beta. and .gamma. chains during the first
five cell divisions, suggests that initial cell division in vivo is
likely IL-15- but not IL-2-dependent.
[0178] To test this hypothesis, IL-2 production was assessed in
dividing T cells in vivo. Intracellular IL-2 staining revealed that
IL-2 was highly expressed only by cells that have divided more than
five times. Treatment of host mice with saturating doses of
cytolytic anti-CD25 mAb failed to inhibit the first five cell
divisions (relative to control Ab treated mice), and dividing cells
in the first and fifth divisions were remarkably similar in
anti-CD25 treated mice and in control mice. Furthermore, T cells at
the second cell division in vivo were sorted and cultured in vitro
in the presence of IL-2 or IL-15, and cell proliferation was
analyzed by .sup.3H-TdR uptake. IL-2, provided in doses as high as
500 u/ml in culture, failed to support T cell proliferation. In
contrast, IL-15 stimulated vigorous cell proliferation (FIG. 4B).
See Li et al. Nature Medicine 7:114-118, 2001
[0179] The pattern of IL-2 expression in vivo is closely associated
with upregulation of the IL-2 receptor .alpha. and .beta. chains,
and with markedly decreased expression of the common .gamma. chain
(FIG. 5A). This suggests that IL-2 regulates .gamma. chain
expression in vivo. To test this possibility, .gamma. chain
expression was examined in T cells from IL-2 deficient mice and
wild type control mice. CD4.sup.+ T cells from IL-2 deficient mice
expressed very high levels of .gamma. chain on the cell surface as
compared to wild type controls (FIG. 5B). Treatment of host mice
with anti-CD25 inhibited .gamma. chain down regulation on dividing
T cells in vivo, but this treatment had no effect on IL-2 receptor
.beta. chain expression (FIG. 5C).
[0180] The .gamma. chain is a critical signaling element for all
known T cell growth factors and .gamma. chain signals are essential
for cell survival, which is accomplished at least in part via
sustained expression of Bcl-2 family anti-apoptotic proteins
(Nakajima et al., J. Exp. Med. 185:189-195, 1997). To determine
whether decreased .gamma. chain expression after five cell
divisions in vivo regulates clonal expansion, CFSE-labeled cells
were stained with PE-annexin V after recovery from the hosts and
apoptotic cell death of dividing T cells was analyzed in vivo.
Precipitous cell death occurred after four cell divisions.
Undivided cells (0 division) had <10% annexin V positive cells.
After the sixth cell division, however, .about.40% of the cells
were annexin V positive. This type of cell death is not Fas
dependent, as T cells from Fas mutant MRL-lpr mice had similar
pattern of apoptotic cell death in vivo (Li et al., J. Immunol.
163:2500-2507, 1999). Staining for Bcl-2 expression showed that the
mean channel fluorescence intensity of Bcl-2 staining was markedly
decreased after four cell divisions (FIG. 5E). Thus, the signaling
events upon .gamma. chain down-regulation may fail to support
sustained Bcl-2 expression and cells become susceptible to
apoptotic cell death (Nakajima et al., J. Exp. Med. 185:189-195,
1997).
[0181] These results suggest that blocking IL-2 or IL-15 signaling
will have different effects on T cell expansion in vivo. To explore
this possibility further, CFSE-labeled lymphocytes from IL-2
deficient mice (H-2b) were injected into irradiated DBA/2 hosts
(H-2d), cell division was analyzed in vivo three days later and
compared with that in wild type control mice. T cells from IL-2
deficient mice continued to divide and expand in vivo. About 30% of
CFSE-labeled cells entered the cell cycle, and the majority of the
cells divided more than five times, compared to control.
[0182] Treating host mice with an IL-15 mutant /Fc, which acts as
an IL-15 receptor specific antagonist (Kim et al., J. Immunol.
161:5742-5748, 1998), markedly reduced the proliferation frequency
of CFSE-labeled T cells, and an overwhelming majority of
CFSE-labeled cells failed to enter the cell cycle in the treated
mice. Furthermore, treatment of host mice with blocking mAbs
against the common y chain, a shared signaling component of IL-2
and IL-15 receptors, also markedly inhibited T cell division in
vivo. Thus, IL-2 and IL-15 regulate distinct aspects of T cell
expansion in vivo, and administration of antagonists for these
interleukins can suppress the immune response, as discussed
above.
[0183] These results also demonstrate that .gamma. chain
downregulation requires T cell activation and cell cycle
progression as well as IL-2 signaling. Clearly, .gamma. chain
downregulation in vivo is closely associated with IL-2 production
and high affinity IL-2 receptor expression. In the absence of IL-2,
.gamma. chain is expressed at extremely high levels and blockade of
IL-2 receptor inhibits .gamma. chain downregulation in vivo on
cycling T cells. Thus, these studies provide novel evidence that
IL-2 and IL-15 regulate distinct aspects of primary T cell
activation in vivo. Contrary to traditional beliefs and conclusions
based on in vitro studies, IL-15 is a critical growth factor in
initiating T cell division in vivo and IL-2's unique role in vivo
is to control the magnitude of clonal expansion by regulating y
chain expression on cycling T cells.
[0184] These results support the clinical applications described
above. Attempts to boost IT cell response with exogenous IL-2 in
tumor immunity and AIDS may promote premature T cell death and
therapies to block IL-2 in tolerance induction and autoimmunity may
induce unwanted T cell expansion. Furthermore, staged and combined
targeting of IL-15 and IL-2 represent an important way to block T
cell activation in T cell dependent cytopathic conditions.
[0185] Lytic IL-2/Fc lyses IL-2R-bearing cells and binds to
FcRI
[0186] Cells of a T cell line (CTLL-2 cells; 10.sup.6) were labeled
with 100 mCi .sup.51Cr and incubated with a lytic form of IL-2/Fc
and rat low-toxic complement (C'), a non-lytic form of IL-2/Fc and
C', murine immunoglobulin and C' (a negative control) or C' alone
(a negative control at 0.5 .mu.g/ml). Another group of the same
cells was treated with a detergent (1% NP40)(a positive control).
Cell lysis was measured by .sup.51Cr release. The degree of lysis
observed in the presence of the detergent represents 100% lysis.
Specific lysis following treatment as described above was
calculated according to the formula: % specific
lysis=[(experimental cpm-background cpm)/(total release
cpm-background cpm).times.100% ]. The results, which are shown in
FIG. 6, support the conclusion that cytolytic IL-2/Fc lyses
IL-2R-bearing CTLL-2 cells, but non-lytic IL-2/Fc does not.
[0187] To assess the ability of lytic and nonlytic IL-2/Fc to bind
Fc receptors on FcRI-transfected CHO cells (murine FCRI, FcRII, and
IL-2R-negative), FcRI transfectants were pre-incubated with PBS,
mIgG2a, lytic IL-2/Fc, or nonlytic IL-2/Fc. After washing,
fluorescent-conjugated goat anti-mouse Fc was used to stain the
cells for FACS analysis. As shown in FIG. 7, cytolytic IL-2/Fc can
bind FcRI, but lytic IL-2/Fc cannot.
[0188] Lytic IL-2/Fc is effective in preventing diabetes in an
adoptive transfer model
[0189] Two different variants of IL-2/Fc were created. The first
contained an Fc terminus derived from murine IgG2a that will
mediate CDC and CDCC (IL-2/Fc), and the second was a point mutated
Fc portion that would not activate CDC or CDCC (IL-2/Fc-/-).
Whereas IL-2/Fc mediates CDC in IL-2R bearing cells (such as those
of the murine CTLL-2 T cell line) and binds to FcRI, IL-2/Fc-/-
does not (FIGS. 6 and 7). Moreover, the lytic IL-2/Fc molecule will
prevent the development of diabetes in an NOD adoptive transfer
model, but a non-lytic form of the molecule (IL-2/Fc-/-) is
uneffective (FIG. 13). In the animal model, monodispersed spleen
cells were depleted of erythrocytes by treatment with ACK Lysing
Buffer (BioWhittaker Inc., Walkersville, Md.). Eight to 12 week-old
irradiated (700-rad) NOD male recipients, which were non-diabetic,
were then injected with 2.times.10.sup.7 splenic leukocytes from
acutely diabetic female NOD mice (hyperglycemia<two weeks).
Blood glucose levels (BGL) were tested weekly, and diabetes was
diagnosed when the BGL was greater than 16.5 mmol/L on any single
measurement or greater than 13.8 mmol/L on 3 consecutive days. As
shown in FIG. 13, most of the animals remained diabetes free even
after the treatment was discontinued.
[0190] As noted above, the assays and animal models presented
herein can be used to test various combinations of the agents of
the invention for therapeutic efficacy.
[0191] Rapamycin inhibits T cell proliferation in an in vivo graft
versus host model, but does not inhibit apoptosis of activated T
cells
[0192] In an in vivo graft versus host model (as described above),
the fate of host reactive T cells in the presence of IL-2/Fc and
rapamycin was analyzed. As shown in FIG. 8, rapamycin will inhibit
the proliferation of CD4.sup.+ and CD8.sup.+ T cells, even in the
presence of IL-2/Fc, but will allow the expression of a functional
IL-2R. As evidenced by Annexin V staining, antigen activated host
reactive T cells will undergo apoptosis in the presence of IL-2/Fc
and rapamycin (FIG. 10).
[0193] The compositions of the invention permit long-term survival
of islet and skin allografts
[0194] To prevent rejection of either allogeneic islets
transplanted into acutely diabetic NOD recipients or rejection of
skin grafts transplanted onto NOD mice, a combination of agents
promoting T cell death and inhibiting T cell proliferation was
used. As shown in FIGS. 11 and 12, a combination of lytic IL-2/Fc,
mutIL-15/Fc and rapamycin proved effective in preventing graft
rejection (and more effective than other treatment regimens
tested). Graft survival and graft function persisted throughout the
observation period shown, and most grafts survived after
discontinuation of the therapy. This dramatic effect is believed to
be due to the combination of agents used (agents that promote T
cell death as well as inhibit T cell proliferation).
[0195] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
* * * * *