U.S. patent application number 13/145537 was filed with the patent office on 2011-11-10 for compositions and methods of treating inflammatory and autoimmune diseases.
This patent application is currently assigned to AMGEN INC.. Invention is credited to Marc A. Gavin, Li Li.
Application Number | 20110274650 13/145537 |
Document ID | / |
Family ID | 42356180 |
Filed Date | 2011-11-10 |
United States Patent
Application |
20110274650 |
Kind Code |
A1 |
Gavin; Marc A. ; et
al. |
November 10, 2011 |
COMPOSITIONS AND METHODS OF TREATING INFLAMMATORY AND AUTOIMMUNE
DISEASES
Abstract
Described herein are immunosuppressive molecules including
immunosuppressive variants of IL-2, and use of such molecules to
treat inflammatory and autoimmune disorders.
Inventors: |
Gavin; Marc A.; (Vashon,
WA) ; Li; Li; (Sammamish, WA) |
Assignee: |
AMGEN INC.
Thousand Oaks
CA
|
Family ID: |
42356180 |
Appl. No.: |
13/145537 |
Filed: |
January 20, 2010 |
PCT Filed: |
January 20, 2010 |
PCT NO: |
PCT/US10/21519 |
371 Date: |
July 20, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61146111 |
Jan 21, 2009 |
|
|
|
Current U.S.
Class: |
424/85.2 ;
435/375; 530/351 |
Current CPC
Class: |
A61P 19/02 20180101;
A61P 3/10 20180101; A61P 29/00 20180101; A61P 37/08 20180101; C07K
14/55 20130101; A61P 11/06 20180101; A61P 43/00 20180101; A61K
38/00 20130101; A61P 37/06 20180101 |
Class at
Publication: |
424/85.2 ;
435/375; 530/351 |
International
Class: |
A61K 38/20 20060101
A61K038/20; A61P 11/06 20060101 A61P011/06; A61P 3/10 20060101
A61P003/10; C07K 14/55 20060101 C07K014/55; A61P 37/08 20060101
A61P037/08; A61P 37/06 20060101 A61P037/06; C12N 5/0783 20100101
C12N005/0783; A61P 29/00 20060101 A61P029/00; A61P 19/02 20060101
A61P019/02 |
Claims
1. A method of treating an inflammatory disorder in a subject, said
method comprising administering to a subject in need thereof a
therapeutically effective amount of an IL-2 variant, wherein said
IL-2 variant (a) comprises a sequence of amino acids at least 80%
identical to SEQ ID NO:1; (b) stimulates STAT5 phosphorylation in
FOXP3-positive regulatory T cells; and (c) has a reduced ability
compared to the polypeptide set forth as SEQ ID NO:1 to induce
phosphorylation of STAT5 in FOXP3-negative T cells.
2. The method of claim 1, wherein the inflammatory disorder is
selected from the group consisting of asthma, diabetes, arthritis,
allergy, organ graft rejection and graft-versus-host disease.
3. The method of claim 1, wherein said IL-2 variant comprises a
sequence of amino acids at least 90% identical to SEQ ID NO:1.
4. The method of claim 1, wherein said IL-2 variant comprises a
sequence of amino acids at least 95% identical to SEQ ID NO:1.
5. The method of claim 1, wherein the IL-2 variant has a higher
affinity for IL-2R.alpha. than does the polypeptide set forth as
SEQ ID NO:1;
6. The method of claim 1, wherein the IL-2 variant promotes
FOXP3-positive regulatory T cell growth or survival in vitro.
7. The method of claim 1, wherein the IL-2 variant comprises a
mutation in the polypeptide sequence set forth in SEQ ID NO:1 at a
position selected from the group consisting of amino acid 30, amino
acid 31, amino acid 35, amino acid 69, and amino acid 74.
8. The method of claim 7, wherein the mutation at position 30 is
N30S.
9. The method of claim 7, wherein the mutation at position 31 is
Y31H.
10. The method of claim 7, wherein the mutation at position 35 is
K35R.
11. The method of claim 7, wherein the mutation at position 69 is
V69A.
12. The method of claim 7, wherein the mutation at position 74 is
Q74P.
13. The method of claim 1, wherein the IL-2 variant induces STAT5
phosphorylation in ex vivo FOXP3-positive T cells comprising a
functional IL-2 receptor complex but has a reduced ability to
induce phosphorylation of STAT5.
14. The method of claim 13, wherein the IL-2 variant comprises a
mutation in the polypeptide sequence set forth in SEQ ID NO:1 at
position 88.
15. The method of claim 14, wherein the mutation at position 88 is
N88D.
16. The method of claim 1, wherein the IL-2 variant is conjugated
to a chemical or polypeptide that extends the serum half-life of
said IL-2 variant in vivo.
17. A method of promoting FOXP3-postive regulatory T cell growth or
survival, said method comprising contacting a FOXP3-positive
regulatory T cell with an IL-2 variant, wherein said IL-2 variant
(a) comprises a sequence of amino acids at least 80% identical to
SEQ ID NO:1; (b) stimulates STAT5 phosphorylation in said
FOXP3-positive regulatory T cells; and (c) has a reduced ability
compared to the polypeptide set forth as SEQ ID NO:1 to induce
phosphorylation of STAT5 in FOXP3-negative T cells.
18. The method of claim 16, wherein the FOXP3-positive regulatory T
cell is contacted in vitro.
19. The method of claim 18, wherein the IL-2 variant is conjugated
to a chemical or polypeptide that extends the serum half-life of
said IL-2 variant in vivo.
20. Use of an IL-2 variant in the preparation of a medicament for
the treatment of an inflammatory disease, wherein said IL-2 variant
(a) comprises a sequence of amino acids at least 80% identical to
SEQ ID NO:1; (b) stimulates STAT5 phosphorylation in FOXP3-positive
regulatory T cells; and (c) has a reduced ability compared to the
polypeptide set forth as SEQ ID NO:1 to induce phosphorylation of
STAT5 in FOXP3-negative T cells.
21. The method of claim 20, wherein the IL-2 variant is conjugated
to a chemical or polypeptide that extends the serum half-life of
said IL-2 variant in vivo.
Description
[0001] This application claims priority to U.S. Provisional Patent
Application No. 61/146,111, filed Jan. 21, 2009, which is
incorporated herein by reference in its entirety.
BACKGROUND
[0002] IL-2 binds three transmembrane receptor subunits:
IL-2R.beta. and IL-2R.gamma. which together activate intracellular
signaling events upon IL-2 binding, and CD25 (IL-2R.alpha.) which
serves to present IL-2 to the other 2 receptor subunits. The
signals delivered by IL-2R.beta..gamma. include those of the
PI3-kinase, Ras-MAP-kinase, and STAT5 pathways.
[0003] T cells require expression of CD25 to respond to the low
concentrations of IL-2 that typically exist in tissues. T cells
that express CD25 include both CD4.sup.+ FOXP3.sup.+ regulatory T
cells (T-reg cells)--which are essential for suppressing autoimmune
inflammation--and FOXP3.sup.- T cells that have been activated to
express CD25. FOXP3.sup.-CD25.sup.- T effector cells (T-eff) may be
either CD4.sup.+ or CD8.sup.+ cells, both of which can be
pro-inflammatory and may contribute to autoimmunity, organ graft
rejection or graft-versus-host disease. IL-2-stimulated STAT5
signaling is crucial for normal T-reg cell growth and survival and
for high FOXP3 expression.
[0004] Because of the low affinity IL-2 possesses for each of the
three IL-2R chains, a further reduction in affinity for IL-2R.beta.
and/or IL-2R.gamma. could be offset by an increased affinity for
CD25. Mutational variants of IL-2 have been generated that exhibit
up to 170-fold higher affinity for CD25 (US Patent Application
Publication No. 2005/0142106; Rao et al., Biochemistry 44,
10696-701 (2005)). These variants were reported to associate for
several days with cell surface CD25 and to chronically promote
growth of an IL-2-dependent cell line. The authors report that the
mutants stimulate persistent T cell growth and, thus, may be useful
in methods of viral immunotherapy and in treating cancer or other
hyperproliferative disorders. High doses of IL-2 (Proleukin) are
administered to cancer patients to induce anti-tumor immunity, a
treatment that is often associated with undesirable toxicity. U.S.
Pat. No. 6,955,807 describes IL-2 variants that are said to have
reduced toxicity. The patent attributes the toxicity to
IL-2-induced stimulation of natural killer (NK) cells, which only
express IL-2R.beta. and IL-2R.gamma.. The IL-2 variants described
therein were said to have reduced toxicity because they selectively
activate CD25.sup.+ T cells over NK cells. Again the IL-2 variants
were said to be useful in therapeutic methods wherein it is
beneficial to generally stimulate the immune system, e.g., the
treatment of cancer or infectious diseases.
SUMMARY
[0005] Provided herein are immunosuppressive mutational variants of
IL-2 that preferentially promote the growth/survival of FOXP3.sup.+
regulatory T cells (T-reg cells) over the growth/survival of
potentially proinflammatory FOXP3.sup.-CD25.sup.+ T cells. By
increasing the ratio of T-reg to other T cells and/or by increasing
FOXP3 expression in T-reg without activating FOXP3.sup.-CD25.sup.+
T cells, these variants should suppress undesirable
inflammation.
[0006] The unique properties of these IL-2 variants stem from two
sets of mutations. One set of mutations results in a reduced
affinity for the signaling chains of the IL-2 receptor
(IL-2R.beta./CD122 and/or IL-2R.gamma./CD132) and/or a reduced
capacity to induce a signaling event from one or both subunits of
the IL-2 receptor. The second set of mutations confers higher
affinity for CD25 (IL-2R.alpha.) and may include mutations
described by Rao et al. (US Patent Application Publication No.
2005/0142106).
[0007] As described herein, certain IL-2 variants induce signaling
events that preferentially induce survival, proliferation,
activation and/or function of T-reg cells. In certain embodiments,
the IL-2 variant retains the capacity to stimulate, in T-reg cells,
STAT5 phosphorylation and/or phosphorylation of one or more of
signaling molecules downstream of the IL-2R, e.g., p38, ERK, SYK
and LCK. In other embodiments, the IL-2 variant retains the
capacity to stimulate, in T-reg cells, transcription or protein
expression of genes or proteins, such as FOXP3 or IL-10, that are
important for T-reg cell survival, proliferation, activation and/or
function. In other embodiments, the IL-2 variant exhibits a reduced
capacity to stimulate endocytosis of IL-2/IL-2R complexes on the
surface of CD25.sup.+ T cells. In other embodiments, the IL-2
variant demonstrates inefficient, reduced, or absence of
stimulation of PI3-kinase signaling, such as inefficient, reduced
or absent phosphorylation of AKT and/or mTOR (mammalian target of
rapamycin). In yet other embodiments, the IL-2 variant retains the
ability of wt IL-2 to stimulate STAT5 phosphorylation and/or
phosphorylation of one or more of signaling molecules downstream of
the IL-2R in T-reg cells, yet demonstrates inefficient, reduced, or
absent phosphorylation of STAT5, AKT and/or mTOR or other signaling
molecules downstream of the IL-2R in FOXP3.sup.-CD4.sup.+ or
CD8.sup.+ T cells or NK cells. In other embodiments, the IL-2
variant is inefficient or incapable of stimulating survival,
growth, activation and/or function of FOXP3.sup.-CD4.sup.+ or
CD8.sup.+ T cells or NK cells.
[0008] Provided are methods of treating an inflammatory or
autoimmune disorder. The methods comprise administering a
therapeutically effective amount of one or more immunosuppressive
IL-2 variants to a subject.
[0009] Further provided are methods of promoting proliferation,
survival, growth, or activation of regulatory T cells. The methods
comprising contacting a FOXP3-positive (FOXP3.sup.+) T cell with an
immunosuppressive IL-2 variant.
[0010] Also provided is the use of an immunosuppressive IL-2
variant in the preparation of a medicament for the treatment of an
inflammatory or autoimmune disorder.
[0011] Also provided are compositions of IL-2 variants conjugated
to additional protein sequences or other chemical that prolong the
stability and/or half-life of the therapeutic in vivo.
BRIEF EXPLANATION OF THE DRAWINGS
[0012] FIG. 1. Sequences of IL-2 variants. Sequences that vary from
germline human IL-2 are not shaded in gray.
[0013] FIG. 2A-2F. FIG. 2A An example of flow cytometric data and
gating strategy. Representative data is shown in FIG. 1A where 9.5%
of CD4.sup.+ cells are CD25.sup.+FOXP3.sup.+, 9.9% of CD4.sup.+
cells are CD25.sup.+FOXP3.sup.- and 6.7% of CD8.sup.+ cells are
CD25.sup.+FOXP3.sup.-. FOXP3.sup.+CD8.sup.+ T cells are typically
very infrequent. FIG. 2B. Relative number of
CD8.sup.+CD25.sup.+FOXP3.sup.- T cells. FIG. 2C. Relative number of
CD4.sup.+CD25.sup.+FOXP3.sup.- T cells. FIG. 2D. Relative number of
CD4.sup.+CD25.sup.+FOXP3.sup.+ T cells. FIG. 2E. Ratio of
FOXP3.sup.+/FOXP3.sup.- cells among CD25.sup.+CD4.sup.+ T cells.
FIG. 2F. IL-2-mediated FOXP3 upregulation in FOXP3.sup.+CD4.sup.+ T
cells.
[0014] FIG. 3. IL-2 muteins stimulate phospho-STAT5 in T-reg but
are inefficient at stimulating signals in other T cells. T cells
were stimulated with IL-2 as described in Example 3. Phospho-STAT5
was measured by flow cytometry and phospho-AKT was measured by
ELISA (MesoScale Discovery). Abbreviations are as follows: T-reg,
FOXP3.sup.+CD4.sup.+ T cells; CD4 T-eff,
CD4.sup.+CD25.sup.+FOXP3.sup.- "effector" T cells; CD8 T-eff,
CD8.sup.+CD25.sup.+FOXP3.sup.- "effector" T cells.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0015] FOXP3.sup.+ regulatory T cells (T-reg cells) are essential
for maintaining normal immune homeostasis and immune tolerance to
self tissues, as well as for suppressing undesirable inflammation.
T-reg cells exert their suppressor and regulatory functions through
multiple mechanisms which are likely to be regulated by temporal
and environmental factors. Current immunosuppressive therapeutics
generally target individual proinflammatory pathways and as such
often exhibit partial efficacy or are applicable to specific
diseases. An alternative immunosuppressive modality might involve
the elevation of the numbers and activation state of natural
suppressor cells to better enable them to deliver appropriate
suppressor molecules/activities at sites of inflammation.
[0016] Described herein are therapeutic agents that selectively
promote T-reg cell proliferation, survival, activation and/or
function. By "selectively promote," it is meant the therapeutic
agent promotes the activity in T-reg cells but has limited or lacks
the ability to promote the activity in non-regulatory T cells.
Further described herein are assays to screen for agents that
selectively promote T-reg cell proliferation, survival, activation
and/or function. Agents that may be screened include, but are not
limited to, small molecules, peptides, polypeptides, proteins
including antibodies, e.g., monoclonal, humanized, human,
monovalent, bivalent, and multivalent antibodies.
[0017] In certain embodiments, the agent is an IL-2 variant. In
particular, the IL-2 variant promotes these activities of T-reg
cell growth/survival but have a reduced ability, as compared to
wild-type IL-2, to promote non-regulatory T-cell
(FOXP3.sup.-CD25.sup.+) and NK cell proliferation, survival,
activation and/or function. In certain embodiments, such IL-2
variants function through a combination of elevated affinity for
the IL-2R subunit IL-2R.alpha. (CD25) and a reduced affinity for
the signaling subunits IL-2R.beta. and/or IL-2R.gamma.. Whereas
IL-2 and variants thereof have been used in the art as
immunostimulatory agents, e.g., in methods of treating cancer or
infectious diseases, the IL-2 variants described herein are
particularly useful as immunosuppressive agents, e.g., in methods
of treating inflammatory disorders.
[0018] IL-2 variants comprise a sequence of amino acids at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 91%, at least 92%, at least 93% at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% identical to
wild-type IL-2. IL-2 variants further include a sequence of amino
acids at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 91%, at least 92%, at least 93% at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% identical to a functional fragment of wild-type IL-2. As used
herein, "wild-type IL-2" shall mean the polypeptide having the
following amino acid sequence:
TABLE-US-00001 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMP
KKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVL
ELKGSETTFMCEYADETATIVEFLNRWITFXQSIISTLT
[0019] wherein X is C, S, A or V (SEQ ID NO:1).
[0020] Variants may contain one or more substitutions, deletions,
or insertions within the wild-type IL-2 amino acid sequence.
Residues are designated herein by the one letter amino acid code
followed by the IL-2 amino acid position, e.g., K35 is the lysine
residue at position 35 of SEQ ID NO:1. Substitutions are designated
herein by the one letter amino acid code followed by the IL-2 amino
acid position followed by the substituting one letter amino acid
code, e.g., K35A is a substitution of the lysine residue at
position 35 of SEQ ID NO:1 with an alanine residue.
[0021] In one aspect, the invention provides immunosuppressive IL-2
variants that have a higher affinity for IL-2R.alpha. than
wild-type IL-2. U.S. Published Patent Application No. 2005/0142106
(incorporated herein by reference in its entirety) describes IL-2
variants that have higher affinity for IL-2R.alpha. than does
wild-type IL-2 and methods of making and screening for such
variants. Preferred IL-2 variants contain one or more mutations in
positions of the IL-2 sequence that either contact IL-2R.alpha. or
alter the orientation of other positions contacting IL-2R.alpha.,
resulting in higher affinity for IL-2R.alpha.. The mutations may be
in or near areas known to be in close proximity to IL-2R.alpha.
based on published crystal structures (Xinquan Wang, Mathias
Rickert, K. Christopher Garcia. Science 310:1159 2005). IL-2
residues believed to contact IL-2R.alpha. include K35, R38, F42,
K43, F44, Y45, E61, E62, K64, P65, E68, V69, L72, and Y107.
[0022] IL-2 variants having greater affinity for IL-2R.alpha. can
include a change in N29, N30, Y31, K35, T37, K48, E68, V69, N71,
Q74, S75, or K76. Preferred variants include those having one or
more of the following mutations: N29S, N30S, N30D, Y31H, Y31S,
K35R, T37A, K48E, V69A, N71R, and Q74P.
[0023] Immunosuppressive IL-2 variants also include variants that
demonstrate altered signaling through certain pathways activated by
wild-type IL-2 via the IL-2R and result in preferential
proliferation/survival/activation of T-reg. Molecules known to be
phosphorylated upon activation of the IL-2R include STAT5, p38,
ERK, SYK, LCK, AKT and mTOR. Compared to wild-type IL-2, the
immunosuppressive IL-2 variant can possess a reduced PI3K signaling
ability in FOXP3.sup.- T cells, which can be measured by a
reduction in the phosphorylation of AKT and/or mTOR as compared to
wild-type IL-2. Such variants may include mutations in positions
that either contact IL-2R.beta. or IL-2R.gamma. or alter the
orientation of other positions contacting IL-2R.beta. or
IL-2R.gamma.. IL-2 residues believed to contact IL-2R.beta. include
L12, Q13, H16, L19, D20, M23, R81, D84, S87, N88, V91, I92, and
E95. IL-2 residues believed to contact IL-2R.gamma. include Q11,
L18, Q22, E110, N119, T123, Q126, S127, I129, S130, and T133. In
certain embodiments, the IL-2 variant comprises a mutation at E15,
H16, Q22, D84, N88, or E95. Examples of such mutations include
E15Q, H16N, Q22E, D84N, N88D, and E95Q.
[0024] In certain embodiments, the IL-2 variant comprises a
combination of mutations. Examples of IL-2 variants having a
combination of mutations are provided in FIG. 1 and include haWT
(SEQ ID NO:2), haD, (SEQ ID NO:3), haD.1 (SEQ ID NO:4), haD.2 (SEQ
ID NO:5), haD.4 (SEQ ID NO:6), haD.5 (SEQ ID NO:7), haD.6 (SEQ ID
NO:8), haD.8 (SEQ ID NO:9), and haD.11 (SEQ ID NO:10)d. In
preferred embodiments, the IL-2 variant stimulates STAT5
phosphorylation in FOXP3-positive regulatory T cells but has
reduced ability to induce STAT5 and AKT phosphorylation in
FOXP3-negative T cells as compared to wild-type IL-2. Preferred
variants having such properties include haD, haD.1, haD.2, haD.4,
haD.5, haD.6, and haD.8.
[0025] The IL-2 variants may further comprise one or more mutations
as compared to the wild-type IL-2 sequence that do not have an
effect on the affinity for IL-2R.beta. or IL-2R.gamma., provided
the IL-2 variant promotes the preferential proliferation, survival,
activation or function of FOXP3.sup.+ T-reg over that of other T
cells that do not express FOXP3. In preferred embodiments, such
mutations are conservative mutations.
[0026] The IL-2 variant may comprise one or more compounds to
increase the serum-half-life of the IL-2 variant when administered
to a patient. Such half-life extending molecules include water
soluble polymers (e.g., polyethylene glycol (PEG)), low- and
high-density lipoproteins, antibody Fc (monomer or dimer),
transthyretin (TTR), and TGF-.beta. latency associated peptide
(LAP). Also contemplated are IL-2 variants comprising a combination
of serum half-life extending molecules, such as PEGylated TTR (US
Pat. Appl. Publ. No. 2003/0195154).
[0027] Methods of Making an Immunosuppressive IL-2 Variant
[0028] The immunosuppressive IL-2 variants can be produced using
any suitable method known in the art, including those described in
U.S. Pat. No. 6,955,807 for producing immunostimulatory IL-2
variants (incorporated herein by reference). Such methods include
constructing a DNA sequence encoding the IL-2 variant and
expressing those sequences in a suitably transformed host. This
method will produce the recombinant variant of this invention.
However, the variants may also be produced by chemical synthesis or
a combination of chemical synthesis and recombinant DNA technology.
Batch-wise production or perfusion production methods are known in
the art. See Freshey, R. I. (ed), "Animal Cell Culture: A Practical
Approach," 2nd ed., 1992, IRL Press. Oxford, England; Mather, J. P.
"Laboratory Scaleup of Cell Cultures (0.5-50 liters)," Methods Cell
Biolog 57: 219-527 (1998); Hu, W. S., and Aunins, J. G.,
"Large-scale Mammalian Cell Culture," Curr Opin Biotechnol 8:
148-153 (1997); Konstantinov, K. B., Tsai, Y., Moles, D.,
Matanguihan, R., "Control of long-term perfusion Chinese hamster
ovary cell culture by glucose auxostat," Biotechnol Prog 12:100-109
(1996).
[0029] In one embodiment of a recombinant method for producing a
variant, a DNA sequence is constructed by isolating or synthesizing
a DNA sequence encoding the wild type IL-2 and then changing one or
more codons by site-specific mutagenesis. This technique is well
known. See, e.g., Mark et. al., "Site-specific Mutagenesis Of The
Human Fibroblast Interferon Gene", Proc. Natl. Acad. Sci. USA 81,
pp. 5662-66 (1984); and U.S. Pat. No. 4,588,585, incorporated
herein by reference.
[0030] Another method of constructing a DNA sequence encoding the
IL-2 variant would be chemical synthesis. This for example includes
direct synthesis of a peptide by chemical means of the protein
sequence encoding for an IL-2 variant exhibiting the properties
described herein. This method may incorporate both natural and
unnatural amino acids at positions that affect the interactions of
IL-2 with the IL2R.alpha., IL-2R.beta., or IL-2R.gamma..
Alternatively, a gene which encodes the desired IL-2 variant may be
synthesized by chemical means using an oligonucleotide synthesizer.
Such oligonucleotides are designed based on the amino acid sequence
of the desired IL-2 variant, and preferably selecting those codons
that are favored in the host cell in which the recombinant variant
will be produced. In this regard, it is well recognized that the
genetic code is degenerate--that an amino acid may be coded for by
more than one codon. For example, Phe (F) is coded for by two
codons, TTC or TTT, Tyr (Y) is coded for by TAC or TAT and his (H)
is coded for by CAC or CAT. Trp (W) is coded for by a single codon,
TGG. Accordingly, it will be appreciated that for a given DNA
sequence encoding a particular IL-2 variant, there will be many DNA
degenerate sequences that will code for that IL-2 variant.
[0031] The DNA sequence encoding the IL-2 variant, whether prepared
by site directed mutagenesis, chemical synthesis or other methods,
may or may not also include DNA sequences that encode a signal
sequence. Such signal sequence, if present, should be one
recognized by the cell chosen for expression of the IL-2 variant.
It may be prokaryotic, eukaryotic or a combination of the two. It
may also be the signal sequence of native IL-2. The inclusion of a
signal sequence depends on whether it is desired to secrete the
IL-2 variant from the recombinant cells in which it is made. If the
chosen cells are prokaryotic, it generally is preferred that the
DNA sequence not encode a signal sequence. If the chosen cells are
eukaryotic, it generally is preferred that a signal sequence be
encoded and most preferably that the wild-type IL-2 signal sequence
be used.
[0032] Standard methods may be applied to synthesize a gene
encoding an IL-2 variant. For example, the complete amino acid
sequence may be used to construct a back-translated gene. A DNA
oligomer containing a nucleotide sequence coding for an IL-2
variant may be synthesized. For example, several small
oligonucleotides coding for portions of the desired polypeptide may
be synthesized and then ligated. The individual oligonucleotides
typically contain 5' or 3' overhangs for complementary
assembly.
[0033] Once assembled (by synthesis, site-directed mutagenesis or
another method), the DNA sequences encoding an IL-2 variant will be
inserted into an expression vector and operatively linked to an
expression control sequence appropriate for expression of the IL-2
variant in the desired transformed host. Proper assembly may be
confirmed by nucleotide sequencing, restriction mapping, and
expression of a biologically active polypeptide in a suitable host.
As is well known in the art, in order to obtain high expression
levels of a transfected gene in a host, the gene must be
operatively linked to transcriptional and translational expression
control sequences that are functional in the chosen expression
host. The choice of expression control sequence and expression
vector will depend upon the choice of host. A wide variety of
expression host/vector combinations may be employed.
[0034] Any suitable host may be used to produce the IL-2 variant,
including bacteria, fungi (including yeasts), plant, insect,
mammal, or other appropriate animal cells or cell lines, as well as
transgenic animals or plants. More particularly, these hosts may
include well known eukaryotic and prokaryotic hosts, such as
strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi,
yeast, insect cells such as Spodoptera frugiperda (Sf9), animal
cells such as Chinese hamster ovary (CHO) and mouse cells such as
NS/O, African green monkey cells such as COS 1, COS 7, BSC 1, BSC
40, and BNT 10, and human cells, as well as plant cells in tissue
culture. For animal cell expression, CHO cells and COS 7 cells in
cultures and particularly the CHO cell line CHO (DHFR-) or the HKB
line are preferred.
[0035] It should of course be understood that not all vectors and
expression control sequences will function equally well to express
the DNA sequences described herein. Neither will all hosts function
equally well with the same expression system. However, one of skill
in the art may make a selection among these vectors, expression
control sequences and hosts without undue experimentation. For
example, in selecting a vector, the host must be considered because
the vector must replicate in it. The vectors copy number, the
ability to control that copy number, and the expression of any
other proteins encoded by the vector, such as antibiotic markers,
should also be considered. For example, preferred vectors for use
in this invention include those that allow the DNA encoding the
IL-2 variants to be amplified in copy number. Such amplifiable
vectors are well known in the art. They include, for example,
vectors able to be amplified by DHFR amplification (see, e.g.,
Kaufman, U.S. Pat. No. 4,470,461, Kaufman and Sharp, "Construction
Of A Modular Dihydrafolate Reductase cDNA Gene: Analysis Of Signals
Utilized For Efficient Expression", Mol. Cell. Biol., 2, pp.
1304-19 (1982)) or glutamine synthetase ("GS") amplification (see,
e.g., U.S. Pat. No. 5,122,464 and European published application
338,841).
[0036] The IL-2 variants may be glycosylated or unglycosylated
depending on the host organism used to produce the variant. If
bacteria are chosen as the host, then the IL-2 variant produced
will be unglycosylated. Eukaryotic cells, on the other hand, will
glycosylate the IL-2 variant, although perhaps not in the same way
as native IL-2 is glycosylated. The IL-2 variant produced by the
transformed host can be purified according to any suitable method.
Various methods are known for purifying IL-2. See, e.g Current
Protocols in Protein Science, Vol 2. Eds: John E. Coligan, Ben M.
Dunn, Hidde L. Ploehg, David W. Speicher, Paul T. Wingfield, Unit
6.5 (Copyright 1997, John Wiley and Sons, Inc).
[0037] The biological activity of the IL-2 variants can be assayed
by any suitable method known in the art. Such assays include those
described in the Examples below.
[0038] Indications
[0039] Diseases, disorders, or conditions may be amenable to
treatment with or may be prevented by administration of a
T-reg-selective IL-2 variant to a subject. Such diseases,
disorders, and conditions include, but are not limited to,
inflammation, autoimmune disease, paraneoplastic autroimmune
diseases, cartilage inflammation, fibrotic disease and/or bone
degradation, arthritis, rheumatoid arthritis, juvenile arthritis,
juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoid
arthritis, polyarticular juvenile rheumatoid arthritis, systemic
onset juvenile rheumatoid arthritis, juvenile ankylosing
spondylitis, juvenile enteropathic arthritis, juvenile reactive
arthritis, juvenile Reter's Syndrome, SEA Syndrome (Seronegativity,
Enthesopathy, Arthropathy Syndrome), juvenile dermatomyositis,
juvenile psoriatic arthritis, juvenile scleroderma, juvenile
systemic lupus erythematosus, juvenile vasculitis, pauciarticular
rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic
onset rheumatoid arthritis, ankylosing spondylitis, enteropathic
arthritis, reactive arthritis, Reter's Syndrome, SEA Syndrome
(Seronegativity, Enthesopathy, Arthropathy Syndrome),
dermatomyositis, psoriatic arthritis, scleroderma, systemic lupus
erythematosus, vasculitis, myolitis, polymyolitis, dermatomyolitis,
osteoarthritis, polyarteritis nodossa, Wegener's granulomatosis,
arteritis, ploymyalgia rheumatica, sarcoidosis, scleroderma,
sclerosis, primary biliary sclerosis, sclerosing cholangitis,
Sjogren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis,
inverse psoriasis, pustular psoriasis, erythrodermic psoriasis,
dermatitis, atopic dermatitis, atherosclerosis, lupus, Still's
disease, Systemic Lupus Erythematosus (SLE), myasthenia gravis,
inflammatory bowel disease (IBD), Crohn's disease, ulcerative
colitis, celiac disease, multiple schlerosis (MS), asthma, COPD,
Guillain-Barre disease, Type I diabetes mellitus, thyroiditis
(e.g., Graves' disease), Addison's disease, Raynaud's phenomenon,
autoimmune hepatitis, GVHD, transplantation rejection, and the
like. In specific embodiments, pharmaceutical compositions
comprising a therapeutically effective amount of a T-reg-selective
IL-2 variant are provided.
[0040] The term "treatment" encompasses alleviation or prevention
of at least one symptom or other aspect of a disorder, or reduction
of disease severity, and the like. A T-reg-selective IL-2 variant
need not effect a complete cure, or eradicate every symptom or
manifestation of a disease, to constitute a viable therapeutic
agent. As is recognized in the pertinent field, drugs employed as
therapeutic agents may reduce the severity of a given disease
state, but need not abolish every manifestation of the disease to
be regarded as useful therapeutic agents. Similarly, a
prophylactically administered treatment need not be completely
effective in preventing the onset of a condition in order to
constitute a viable prophylactic agent. Simply reducing the impact
of a disease (for example, by reducing the number or severity of
its symptoms, or by increasing the effectiveness of another
treatment, or by producing another beneficial effect), or reducing
the likelihood that the disease will occur or worsen in a subject,
is sufficient. One embodiment of the invention is directed to a
method comprising administering to a patient A T-reg-selective IL-2
variant in an amount and for a time sufficient to induce a
sustained improvement over baseline of an indicator that reflects
the severity of the particular disorder.
[0041] Pharmaceutical Compositions
[0042] In some embodiments, the invention provides pharmaceutical
compositions comprising a therapeutically effective amount of one
or a plurality of T-reg-selective IL-2 variants of the invention
together with a pharmaceutically acceptable diluent, carrier,
solubilizer, emulsifier, preservative, and/or adjuvant. In
addition, the invention provides methods of treating a patient by
administering such pharmaceutical composition. The term "patient"
includes human and animal subjects.
[0043] In certain embodiments, acceptable formulation materials
preferably are nontoxic to recipients at the dosages and
concentrations employed. In certain embodiments, the pharmaceutical
composition may contain formulation materials for modifying,
maintaining or preserving, for example, the pH, osmolality,
viscosity, clarity, color, isotonicity, odor, sterility, stability,
rate of dissolution or release, adsorption or penetration of the
composition. In such embodiments, suitable formulation materials
include, but are not limited to, amino acids (such as glycine,
glutamine, asparagine, arginine or lysine); antimicrobials;
antioxidants (such as ascorbic acid, sodium sulfite or sodium
hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,
citrates, phosphates or other organic acids); bulking agents (such
as mannitol or glycine); chelating agents (such as ethylenediamine
tetraacetic acid (EDTA)); complexing agents (such as caffeine,
polyvinylpyrrolidone, beta-cyclodextrin or
hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;
disaccharides; and other carbohydrates (such as glucose, sucrose,
mannose or dextrins); proteins (such as serum albumin, gelatin or
immunoglobulins); coloring, flavoring and diluting agents;
emulsifying agents; hydrophilic polymers (such as
polyvinylpyrrolidone); low molecular weight polypeptides;
salt-forming counterions (such as sodium); preservatives (such as
benzalkonium chloride, benzoic acid, salicylic acid, thimerosal,
phenethyl alcohol, methylparaben, propylparaben, chlorhexidine,
sorbic acid or hydrogen peroxide); solvents (such as glycerin,
propylene glycol or polyethylene glycol); sugar alcohols (such as
mannitol or sorbitol); suspending agents; surfactants or wetting
agents (such as pluronics, PEG, sorbitan esters, polysorbates such
as polysorbate 20, polysorbate, triton, tromethamine, lecithin,
cholesterol, tyloxapal); stability enhancing agents (such as
sucrose or sorbitol); tonicity enhancing agents (such as alkali
metal halides, preferably sodium or potassium chloride, mannitol
sorbitol); delivery vehicles; diluents; excipients and/or
pharmaceutical adjuvants. See, REMINGTON'S PHARMACEUTICAL SCIENCES,
18'' Edition, (A. R. Genrmo, ed.), 1990, Mack Publishing
Company.
[0044] The therapeutically effective amount of T-reg-selective IL-2
variant-containing pharmaceutical composition to be employed will
depend, for example, upon the therapeutic context and objectives.
One skilled in the art will appreciate that the appropriate dosage
levels for treatment will vary depending, in part, upon the
molecule delivered, the indication for which the T-reg-selective
IL-2 variant is being used, the route of administration, and the
size (body weight, body surface or organ size) and/or condition
(the age and general health) of the patient.
[0045] In certain embodiments, the clinician may titer the dosage
and modify the route of administration to obtain the optimal
therapeutic effect. A typical dosage may range from about 0.1
.mu.g/kg to up to about 30 mg/kg or more, depending on the factors
mentioned above. In specific embodiments, the dosage may range from
0.1 .mu.g/kg up to about 30 mg/kg, optionally from 1 .mu.g/kg up to
about 30 mg/kg or from 10 .mu.g/kg up to about 5 mg/kg.
[0046] Dosing frequency will depend upon the pharmacokinetic
parameters of the particular T-reg-selective IL-2 variant in the
formulation used. Typically, a clinician administers the
composition until a dosage is reached that achieves the desired
effect. The composition may therefore be administered as a single
dose, or as two or more doses (which may or may not contain the
same amount of the desired molecule) over time, or as a continuous
infusion via an implantation device or catheter. Further refinement
of the appropriate dosage is routinely made by those of ordinary
skill in the art and is within the ambit of tasks routinely
performed by them.
[0047] The route of administration of the pharmaceutical
composition is in accord with known methods, e.g., orally, through
injection by intravenous, intraperitoneal, intracerebral
(intra-parenchymal), intracerebroventricular, intramuscular,
intra-ocular, intraarterial, intraportal, or intralesional routes;
by sustained release systems or by implantation devices. In certain
embodiments, the compositions may be administered by bolus
injection or continuously by infusion, or by implantation
device.
[0048] Combination Therapies
[0049] In further embodiments, T-reg-selective IL-2 variant is
administered in combination with other agents useful for treating
the condition with which the patient is afflicted. Examples of such
agents include both proteinaceous and non-proteinaceous drugs. When
multiple therapeutics are co-administered, dosages may be adjusted
accordingly, as is recognized in the pertinent art.
"Co-administration" and combination therapy are not limited to
simultaneous administration, but also include treatment regimens in
which a T-reg-selective IL-2 variant is administered at least once
during a course of treatment that involves administering at least
one other therapeutic agent to the patient.
[0050] In certain embodiments, a T-reg-selective IL-2 variant is
administered in combination with an inhibitor of the PI3-K/AKT/mTOR
pathway, e.g., rapamycin (rapamune, sirolimus). Inhibitors of this
pathway in combination with IL-2 favor T-reg enrichment.
[0051] The invention having been described, the following examples
are offered by way of illustration, and not limitation.
EXAMPLES
Example 1
Panel of IL-2 Mutants
[0052] To examine the potential for generating IL-2 variants with
reduced capacity to stimulate FOXP3.sup.-CD25.sup.+ "effector" T
cells (T-eff) but not T-reg, a series of IL-2 mutants was generated
in which amino acids predicted to interact with the IL-2R.beta.
and/or IL-2R.gamma. chain were altered. These variants also
contained a set of previously described mutations that conferred
high affinity for CD25 (variant "2-4" in Rao et al., Biochemistry
44, 10696-701 (2005)). This series of variants is shown in FIG. 1.
Variant haWT contained only the mutations that contributed to the
high affinity for CD25. Variants haD, haD.1, haD.2, etc, also
contained mutations predicted to alter interactions with
IL-2R.beta. and/or IL-2R.gamma.. In all assays, variant haD.11 was
not capable of inducing any signal or altering any cellular
phenotype and, as such, served as a control for CD25 binding
without IL-2R signaling. All the IL-2 variants contained the C125S
mutation for improved manufacturability and terminated with FLAG
and HIS-tag sequences (DYKDDDDKGSSHHHHHH) (SEQ ID NO:11)
[0053] Several assays were used to assess the ability of the IL-2
variants to induce signaling events and T cell growth. These
included assays to detect: [0054] 1. Growth and survival of T cell
subsets and measurement of FOXP3 expression. [0055] 2. Cell
signaling (e.g. detection of phosphorylated STAT5 and AKT using
flow cytometric and ELISA-based methods).
Example 2
Enrichment of FOXP3.sup.+ Cells and Retention of FOXP3 Upregulation
During Long Term T Cell Culture
[0056] Total PBMC were activated in 24-well plates at
4.times.10.sup.6 cells per well with 100 ng/ml anti-CD3 (OKT3). On
day 3 of culture, cells were washed 3 times and rested in fresh
media for 3 days. Cells were then washed and seeded in 96 well
flat-bottom plates with IL-2 variants at either 10 nM or 100 pM.
Three days later cells were counted and analyzed by flow cytometry.
(FIG. 2A)
[0057] As expected, CD8.sup.+CD25.sup.+ T cells were especially
responsive to WT IL-2 and variant haWT, however, all variants that
contained mutated IL-2R.beta. and/or .gamma. contact residues were
very inefficient at promoting accumulation of activated
CD8.sup.+CD25.sup.+ T cells (FIG. 2B). A similar trend was observed
for CD4.sup.+CD25.sup.+FOXP3.sup.- T cells (FIG. 2C). In contrast,
the growth/survival of FOXP3.sup.+CD4.sup.+ T cells was stimulated
by several IL-2 variants to a degree similar to that of WT IL-2
(FIG. 2D). As a result, the ratio of FOXP3.sup.+ to FOXP3.sup.- T
cells among CD4.sup.+ CD25.sup.+ T cells was increased by several
IL-2 variants with IL-2R.beta..gamma.-contact residue mutations
(FIG. 2E). Furthermore, the mutations did not impair
IL-2-stimulated FOXP3 upregulation in T-reg (FIG. 2F).
Example 3
Mutations that Reduce Signaling in FOXP3.sup.-T Cells but Stimulate
STAT5 Signaling in T-Reg
[0058] The IL-2 variants were screened for their ability to
stimulate AKT and STAT5 phosphorylation in T cell subsets. Several
IL-2 variants were as potent, or nearly as potent, as wt IL-2 at
stimulating STAT5 in FOXP3.sup.+ T cells 10 min after stimulation.
Three hours after washing IL-2 from the media, some IL-2 variants
(haD, haD.1, haD.2, haD.4, haD.6, and haD.8) continued to stimulate
sustained STAT5 signaling at levels higher than that seen with wt
IL-2. In contrast, for FOXP3.sup.-T cells, STAT5 and AKT responses
to the haD variants after 10 min stimulation were not nearly as
high as those stimulated by wt IL-2 or haWT. After 3 hrs, weak
STAT5 and AKT signals similar to those seen with wt IL-2 were
observed in T-eff, however, at this late timepoint wt IL-2
signaling had diminished greatly. In FOXP3.sup.+ T cells, AKT
signaling is not normally stimulated by IL-2 (Zeiser R, et al, 2008
Blood 111:453) thus the phospho-AKT signal observed in total T cell
lysates can be attributed to T-eff.
[0059] Methods: Previously activated (with anti-CD3 for 2-3 days)
and rested (in fresh culture medium for 2-5 days) T cells were
exposed to 1 nM wt or mutant IL-2 for 10 min at 37.degree. C. Cells
were then stained (10 min timepoint) or washed and cultured for an
additional 3 hrs (3 hr timepoint). To measure phospho-AKT by ELISA,
a 50 .mu.l culture was stopped by adding an equal volume of
2.times. lysis buffer and lysates were measured for phospho-AKT
with multiplex ELISA plates according to the manufacturer's
protocols (MesoScale Discovery, Gaithersburg, Md.). To measure
phospho-STAT5 by flow cytometry, a 50 .mu.l culture was stopped by
adding 1 ml of FOXP3 Fix/Perm Buffer (BioLegend, San Diego,
Calif.), incubation at 25.degree. C. for 20 min, and staining for
cell surface markers, FOXP3 and phospho-STAT5 according the
BioLegend FOXP3 staining protocol.
Sequence CWU 1
1
111133PRTHomo sapiensMISC_FEATURE(125)..(125)Xaa is Cysteine,
Serine, Alanine or Valine 1Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr
Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu
Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu
Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His
Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu
Asn Leu Ala Gln Ser Lys Asn Phe His Leu65 70 75 80Arg Pro Arg Asp
Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser
Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr
Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Xaa Gln Ser Ile 115 120
125Ile Ser Thr Leu Thr 1302133PRTArtificialIL-2 Variant 2Ala Pro
Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu
Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Ser Asn His Lys 20 25
30Asn Pro Arg Leu Ala Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Glu
35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu
Lys 50 55 60Pro Leu Glu Glu Ala Leu Arg Leu Ala Pro Ser Lys Asn Phe
His Leu65 70 75 80Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile
Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr
Ala Asp Glu Thr Ala 100 105 110Thr Ile Val Glu Phe Leu Asn Arg Trp
Ile Thr Phe Ser Gln Ser Ile 115 120 125Ile Ser Thr Leu Thr
1303133PRTArtificialIL-2 Variant 3Ala Pro Thr Ser Ser Ser Thr Lys
Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met
Ile Leu Asn Gly Ile Ser Asn His Lys 20 25 30Asn Pro Arg Leu Ala Arg
Met Leu Thr Phe Lys Phe Tyr Met Pro Glu 35 40 45Lys Ala Thr Glu Leu
Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu
Ala Leu Arg Leu Ala Pro Ser Lys Asn Phe His Leu65 70 75 80Arg Pro
Arg Asp Leu Ile Ser Asp Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys
Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105
110Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile
115 120 125Ile Ser Thr Leu Thr 1304133PRTArtificialIL-2 Variant
4Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Gln His1 5
10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Ser Asn His
Lys 20 25 30Asn Pro Arg Leu Ala Arg Met Leu Thr Phe Lys Phe Tyr Met
Pro Glu 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu
Glu Leu Lys 50 55 60Pro Leu Glu Glu Ala Leu Arg Leu Ala Pro Ser Lys
Asn Phe His Leu65 70 75 80Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn
Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys
Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr Ile Val Glu Phe Leu Asn
Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125Ile Ser Thr Leu Thr
1305133PRTArtificialIL-2 Variant 5Ala Pro Thr Ser Ser Ser Thr Lys
Lys Thr Gln Leu Gln Leu Glu Asn1 5 10 15Leu Leu Leu Asp Leu Gln Met
Ile Leu Asn Gly Ile Ser Asn His Lys 20 25 30Asn Pro Arg Leu Ala Arg
Met Leu Thr Phe Lys Phe Tyr Met Pro Glu 35 40 45Lys Ala Thr Glu Leu
Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu
Ala Leu Arg Leu Ala Pro Ser Lys Asn Phe His Leu65 70 75 80Arg Pro
Arg Asp Leu Ile Ser Asp Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys
Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105
110Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile
115 120 125Ile Ser Thr Leu Thr 1306133PRTArtificialIL-2 Variant
6Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5
10 15Leu Leu Leu Asp Leu Glu Met Ile Leu Asn Gly Ile Ser Asn His
Lys 20 25 30Asn Pro Arg Leu Ala Arg Met Leu Thr Phe Lys Phe Tyr Met
Pro Glu 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu
Glu Leu Lys 50 55 60Pro Leu Glu Glu Ala Leu Arg Leu Ala Pro Ser Lys
Asn Phe His Leu65 70 75 80Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn
Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys
Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr Ile Val Glu Phe Leu Asn
Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125Ile Ser Thr Leu Thr
1307133PRTArtificialIL-2 Variant 7Ala Pro Thr Ser Ser Ser Thr Lys
Lys Thr Gln Leu Gln Leu Gln Asn1 5 10 15Leu Leu Leu Asp Leu Gln Met
Ile Leu Asn Gly Ile Ser Asn His Lys 20 25 30Asn Pro Arg Leu Ala Arg
Met Leu Thr Phe Lys Phe Tyr Met Pro Glu 35 40 45Lys Ala Thr Glu Leu
Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu
Ala Leu Arg Leu Ala Pro Ser Lys Asn Phe His Leu65 70 75 80Arg Pro
Arg Asp Leu Ile Ser Asp Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys
Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105
110Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile
115 120 125Ile Ser Thr Leu Thr 1308133PRTArtificialIL-2 Variant
8Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5
10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Ser Asn His
Lys 20 25 30Asn Pro Arg Leu Ala Arg Met Leu Thr Phe Lys Phe Tyr Met
Pro Glu 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu
Glu Leu Lys 50 55 60Pro Leu Glu Glu Ala Leu Arg Leu Ala Pro Ser Lys
Asn Phe His Leu65 70 75 80Arg Pro Arg Asn Leu Ile Ser Asp Ile Asn
Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys
Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr Ile Val Glu Phe Leu Asn
Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125Ile Ser Thr Leu Thr
1309133PRTArtificialIL-2 Variant 9Ala Pro Thr Ser Ser Ser Thr Lys
Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met
Ile Leu Asn Gly Ile Ser Asn His Lys 20 25 30Asn Pro Arg Leu Ala Arg
Met Leu Thr Phe Lys Phe Tyr Met Pro Glu 35 40 45Lys Ala Thr Glu Leu
Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu
Ala Leu Arg Leu Ala Pro Ser Lys Asn Phe His Leu65 70 75 80Arg Pro
Arg Asp Leu Ile Ser Asp Ile Asn Val Ile Val Leu Gln Leu 85 90 95Lys
Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105
110Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile
115 120 125Ile Ser Thr Leu Thr 13010133PRTArtificialIL-2 Variant
10Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1
5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Ser Asn His
Lys 20 25 30Asn Pro Arg Leu Ala Arg Met Leu Thr Phe Lys Phe Tyr Met
Pro Glu 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu
Glu Leu Lys 50 55 60Pro Leu Glu Glu Ala Leu Arg Leu Ala Pro Ser Lys
Asn Phe His Leu65 70 75 80Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn
Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys
Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr Ile Val Glu Phe Leu Asn
Arg Trp Ile Thr Phe Ser Glu Ser Ile 115 120 125Ile Ser Thr Leu Thr
1301117PRTArtificialFLAG-His 11Asp Tyr Lys Asp Asp Asp Asp Lys Gly
Ser Ser His His His His His1 5 10 15His
* * * * *