U.S. patent application number 11/097993 was filed with the patent office on 2007-05-03 for methods of reducing aggregation of il-1ra.
Invention is credited to Bruce Kerwin, Andrei Raibekas.
Application Number | 20070098684 11/097993 |
Document ID | / |
Family ID | 34964860 |
Filed Date | 2007-05-03 |
United States Patent
Application |
20070098684 |
Kind Code |
A9 |
Raibekas; Andrei ; et
al. |
May 3, 2007 |
Methods of reducing aggregation of IL-1ra
Abstract
Methods of reducing aggregation of an aggregating IL-1ra
comprising incubating IL-1ra with at least one accessory molecule
are provided. Kits comprising IL-1ra and at least one accessory
molecule are also provided. Pharmaceutical compositions comprising
IL-1ra and at least one accessory molecule are also provided.
Inventors: |
Raibekas; Andrei; (Thousand
Oaks, CA) ; Kerwin; Bruce; (Thousand Oaks,
CA) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20050271618 A1 |
December 8, 2005 |
|
|
Family ID: |
34964860 |
Appl. No.: |
11/097993 |
Filed: |
April 1, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60558879 |
Apr 2, 2004 |
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60559161 |
Apr 2, 2004 |
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60601216 |
Aug 12, 2004 |
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60601229 |
Aug 12, 2004 |
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Current U.S.
Class: |
424/85.2 ;
514/1.1; 514/53; 514/9.7 |
Current CPC
Class: |
A61K 47/26 20130101;
A61K 33/42 20130101; A61K 31/7012 20130101; A61K 47/02 20130101;
A61K 38/20 20130101; A61P 19/02 20180101; A61P 1/00 20180101; A61P
29/00 20180101; C07K 14/54 20130101; C07K 14/545 20130101; A61K
47/12 20130101; Y02A 50/30 20180101; A61K 9/0019 20130101; A61P
1/04 20180101; A61P 13/12 20180101; A61P 35/02 20180101; A61K
31/7012 20130101; A61K 2300/00 20130101; A61K 33/42 20130101; A61K
2300/00 20130101; A61K 38/20 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/085.2 ;
514/012; 514/053 |
International
Class: |
A61K 38/20 20060101
A61K038/20; A61K 31/7012 20060101 A61K031/7012 |
Claims
1. A method of reducing aggregation of an aggregating interleukin-1
receptor antagonist (IL-1ra) comprising incubating IL-1ra with at
least one accessory molecule at a concentration sufficient to
reduce aggregation of the IL-1ra or reduce the rate of aggregation
of the IL-1ra, wherein at least one of the at least one accessory
molecule is selected from a sugar and a multiple-charge anion.
2. The method of claim 1, wherein at least one accessory molecule
is a multiple-charge anion.
3. The method of claim 2, wherein said multiple-charge anion is 1
to 20 mM pyrophosphate.
4. The method of claim 2, wherein said multiple-charge anion is 1
to 20 mM citrate.
5. The method of claim 1, wherein at least one accessory molecule
is a sugar.
6. The method of claim 5, wherein said sugar is glycerol, sorbitol,
or sucrose.
7. The method of claim 5, wherein said sugar is at a concentration
of from 1 to 3 percent.
8. The method of claim 1, wherein at least one accessory molecule
is selected from a lysine-reactive accessory molecule and an
arginine-reactive accessory molecule.
9. The method of claim 1, wherein at least one accessory molecule
is selected from
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid
(NBD-X), methyl acetyl phosphate (MAP), and citraconic
anhydride.
10. A method of preparing an interleukin-1 receptor antagonist
(IL-1 ra) drug formulation comprising incubating an aggregating
IL-1ra with at least one accessory molecule at a concentration
sufficient to reduce aggregation of the IL-1ra or reduce the rate
of aggregation of the IL-1ra, wherein at least one of the at least
one accessory molecule is selected from a sugar and a
multiple-charge anion, wherein aggregation is reduced.
11. The method of claim 10, wherein at least one accessory molecule
is a multiple-charge anion.
12. The method of claim 11, wherein said multiple-charge anion is 1
to 20 mM pyrophosphate.
13. The method of claim 11, wherein said multiple-charge anion is 1
to 20 mM citrate.
14. The method of claim 10, wherein at least one accessory molecule
is a sugar.
15. The method of claim 14, wherein said sugar is glycerol,
sorbitol, or sucrose.
16. The method of claim 14, wherein said sugar is at a
concentration of from 1 to 3 percent.
17. The method of claim 10, wherein at least one accessory molecule
is selected from a lysine-reactive accessory molecule and an
arginine-reactive accessory molecule.
18. The method of claim 10, wherein at least one accessory molecule
is selected from
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid
(NBD-X), methyl acetyl phosphate (MAP), and citraconic
anhydride.
19. A method of treating a patient comprising administering to the
patient a composition comprising (i) a therapeutically effective
amount of an aggregating interleukin-1 receptor antagonist (IL-1ra)
and (ii) at least one accessory molecule at a concentration
sufficient to reduce aggregation of the IL-1ra or reduce the rate
of aggregation of the IL-1ra, wherein at least one of the at least
one accessory molecule is selected from a sugar and a
multiple-charge anion.
20. The method of claim 19, wherein at least one accessory molecule
is a multiple-charge anion.
21. The method of claim 20, wherein said multiple-charge anion is 1
to 20 mM pyrophosphate.
22. The method of claim 20, wherein said multiple-charge anion is 1
to 20 mM citrate.
23. The method of claim 19, wherein at least one accessory molecule
is a sugar.
24. The method of claim 23, wherein said sugar is glycerol,
sorbitol, or sucrose.
25. The method of claim 23, wherein said sugar is at a
concentration of from 1 to 3 percent.
26. The method of claim 19, wherein at least one accessory molecule
is selected from a lysine-reactive accessory molecule and an
arginine-reactive accessory molecule.
27. The method of claim 19, wherein at least one accessory molecule
is selected from
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid
(NBD-X), methyl acetyl phosphate (MAP), and citraconic
anhydride.
28. A method of treating a patient having rheumatoid arthritis
comprising administering to the patient a composition comprising
(i) a therapeutically effective amount of an aggregating
interleukin-1 receptor antagonist (IL-1ra) and (ii) at least one
accessory molecule at a concentration sufficient to reduce
aggregation of the IL-1ra or reduce the rate of aggregation of the
IL-1ra, wherein at least one of the at least one accessory molecule
is selected from a sugar and a multiple-charge anion.
29. The method of claim 28, wherein at least one accessory molecule
is a multiple-charge anion.
30. The method of claim 29, wherein said multiple-charge anion is 1
to 20 mM pyrophosphate.
31. The method of claim 29, wherein said multiple-charge anion is 1
to 20 mM citrate.
32. The method of claim 28, wherein at least one accessory molecule
is a sugar.
33. The method of claim 32, wherein said sugar is glycerol,
sorbitol, or sucrose.
34. The method of claim 32, wherein said sugar is at a
concentration of from 1 to 3 percent.
35. The method of claim 28, wherein at least one accessory molecule
is selected from a lysine-reactive accessory molecule and an
arginine-reactive accessory molecule.
36. The method of claim 28, wherein at least one accessory molecule
is selected from
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid
(NBD-X), methyl acetyl phosphate (MAP), and citraconic
anhydride.
37. A method of treating a patient having osteoarthritis comprising
administering to the patient a composition comprising (i) a
therapeutically effective amount of an aggregating interleukin-1
receptor antagonist (IL-1ra) and (ii) at least one accessory
molecule at a concentration sufficient to reduce aggregation of the
IL-1ra or reduce the rate of aggregation of the IL-1ra, wherein at
least one of the at least one accessory molecule is selected from a
sugar and a multiple-charge anion.
38. The method of claim 37, wherein at least one accessory molecule
is a multiple-charge anion.
39. The method of claim 38, wherein said multiple-charge anion is 1
to 20 mM pyrophosphate.
40. The method of claim 38, wherein said multiple-charge anion is 1
to 20 mM citrate.
41. The method of claim 37, wherein at least one accessory molecule
is a sugar.
42. The method of claim 41, wherein said sugar is glycerol,
sorbitol, or sucrose.
43. The method of claim 41 wherein said sugar is at a concentration
of from 1 to 3 percent.
44. The method of claim 37, wherein at least one accessory molecule
is selected from a lysine-reactive accessory molecule and an
arginine-reactive accessory molecule.
45. The method of claim 37, wherein at least one accessory molecule
is selected from
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid
(NBD-X), methyl acetyl phosphate (MAP), and citraconic
anhydride.
46. A pharmaceutical composition comprising an aggregating
interleukin-1 receptor antagonist (IL-1ra) and at least one
accessory molecule at a concentration sufficient to reduce
aggregation of the IL-1ra or reduce the rate of aggregation of the
IL-1ra, wherein at least one of the at least one accessory molecule
is selected from a sugar and a multiple-charge anion, and wherein
aggregation is reduced.
47. The pharmaceutical composition of claim 46, wherein at least
one accessory molecule is a multiple-charge anion.
48. The pharmaceutical composition of claim 47, wherein said
multiple-charge anion is -1 to 20 mM pyrophosphate.
49. The pharmaceutical composition of claim 47, wherein said
multiple-charge anion is 1 to 20 mM citrate.
50. The pharmaceutical composition of claim 46, wherein at least
one accessory molecule is a sugar.
51. The pharmaceutical composition of claim 50, wherein said sugar
is glycerol, sorbitol, or sucrose.
52. The pharmaceutical composition of claim 50, wherein said sugar
is at a concentration of from 1 to 3 percent.
53. The pharmaceutical composition of claim 46, wherein at least
one accessory molecule is selected from a lysine-reactive accessory
molecule and an arginine-reactive accessory molecule.
54. The pharmaceutical composition of claim 46, wherein at least
one accessory molecule is selected from
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid
(NBD-X), methyl acetyl phosphate (MAP), and citraconic anhydride.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/558,879, filed Apr. 2, 2004, U.S. Provisional
Application No. 60/559,161, filed Apr. 2, 2004, U.S. Provisional
Application No. 60/601,216, filed Aug. 12, 2004, and U.S.
Provisional Application No. 60/601,229, filed Aug. 12, 2004. U.S.
Provisional Application Nos. 60/558,879, 60/559,161, 60/601,216,
and 60/601,229 are incorporated by reference herein for any
purpose.
FIELD
[0002] The present invention relates to methods of reducing
aggregation of an aggregating interleukin-1 receptor antagonist
(IL-1ra). The present invention also relates to methods of
improving drug formulations comprising reducing aggregation of
IL-1ra. The present invention also relates to methods of treating
diseases using IL-1ra whose aggregation has been reduced. Finally,
the present invention relates to compositions and kits comprising
an IL-1ra whose aggregation has been reduced.
BACKGROUND
[0003] Interleukin-1 alpha (IL-1.alpha.), interleukin-1 beta
(IL-1.beta.), and interleukin-1 receptor antagonist (IL-1ra) each
binds to the type 1 IL-1 receptor (IL-1-RI), which is found on the
surface of certain cell types. IL-1.alpha. and IL-1.beta. have
physiological effects on a number of different target cells,
including certain cells that are involved in the inflammatory and
immune responses. IL-1ra, in contrast, binds to IL-1RI, but does
not elicit comparable downstream biological responses. Rather,
IL-1ra competitively inhibits IL-1.alpha.and IL-1.beta. binding to
IL-1RI. Anakinra, an E. coli-produced version of IL-1ra, is
marketed for treatment of rheumatoid arthritis.
SUMMARY
[0004] In certain embodiments, a method of reducing aggregation of
an aggregating interleukin-1 receptor antagonist (IL-1ra) is
provided. In certain embodiments, the method comprises incubating
IL-1ra with at least one accessory molecule.
[0005] In certain embodiments, a method of preparing an
interleukin-1 receptor antagonist (IL-1ra) drug formulation is
provided. In certain embodiments, the method comprises incubating
the aggregating IL-1ra with at least one accessory molecule. In
certain embodiments, aggregation is reduced.
[0006] In certain embodiments, a method of treating a patient is
provided. In certain embodiments, a method of treating a patient
having arthritis is provided. In certain embodiments, a method of
treating a patient having rheumatoid arthritis is provided. In
certain embodiments, a method of treating a patient having
osteoarthritis is provided. In certain embodiments, a method of
treating a patient having at least one of Crohn's disease,
ulcerative colitis, glomerulonephritis, or leukemia is provided. In
certain embodiments, a method of treating a patient having an
adverse effect of IL-1 is provided. In certain embodiments, the
method of treating a patient comprises administering to the patient
a composition comprising (i) a therapeutically effective amount of
an aggregating interleukin-1 receptor antagonist (IL-1ra) and (ii)
at least one accessory molecule.
[0007] In certain embodiments, kits are provided. In certain
embodiments, a kit comprises an aggregating interleukin-1 receptor
antagonist (IL-1ra) and at least one accessory molecule.
[0008] In certain embodiments, pharmaceutical compositions are
provided. In certain embodiments, pharmaceutical compositions
comprise an aggregating interleukin-1 receptor antagonist (IL-1ra)
and at least one accessory molecule.
[0009] In certain embodiments, at least one accessory molecule is
at a concentration that reduces aggregation of an aggregating
IL-1ra. In certain embodiments, at least one accessory molecule is
at a concentration that reduces the rate of aggregation of an
aggregating IL-1ra. In certain embodiments, at least one accessory
molecule is selected from a sugar and a multiple-charge anion. In
certain embodiments, at least one accessory molecule is a
multiple-charge anion. In certain embodiments, at least one
accessory molecule is 1 to 20 mM pyrophosphate. In certain
embodiments, at least one accessory molecule is 1 to 20 mM citrate.
In certain embodiments, at least one accessory molecule is at least
one sugar. In certain embodiments, at least one of said sugars is
glycerol, sorbitol, or sucrose. In certain embodiments, at least
one of such sugars is at a concentration of from 1 to 3
percent.
[0010] In certain embodiments, at least one accessory molecule is
selected from a lysine-reactive accessory molecule and an
arginine-reactive accessory molecule. In certain embodiments, at
least one accessory molecule is selected from
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid
(NBD-X), methyl acetyl phosphate (MAP), and citraconic
anhydride.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1 shows the IL-1ra wild-type protein aggregation
profile over time in either PSE (10 mM phosphate, pH 6.5, 140 mM
NaCl, 0.5 mM EDTA) or CSE (10 mM citrate, pH 6.5, 140 mM NaCl, 0.5
mM EDTA) discussed in Example 2.
[0012] FIG. 2 shows the reverse-phase high performance liquid
chromatography (RP-HPLC) of IL-1ra wild-type protein derivatized
with NBD-X discussed in Example 3. The upper panel shows the
absorbance of NBD-X labeled IL-1ra at 215 nm. The lower panel shows
the fluorescent emission at 535 nm following excitation at 480
nm.
[0013] FIG. 3 shows the rates of aggregation for IL-1ra wild-type
protein in the presence of increasing concentrations of phosphate,
pyrophosphate, and citrate anions discussed in Example 4.
[0014] FIG. 4 shows the aggregation rates for IL-1ra wild-type
protein in the presence of increasing concentrations of glycerol,
sorbitol, or sucrose discussed in Example 5.
[0015] FIG. 5 shows the nucleotide sequence (SEQ ID NO: 1) and
amino acid sequence (SEQ ID NO: 2) of precursor human IL-1ra, which
includes a secretory leader sequence.
[0016] FIG. 6 shows the amino acid sequence of human IL-1ra lacking
the secretory leader sequence (SEQ ID NO: 3). The dot (.cndot.)
indicates the lysine at position 93. The plus (+) indicates the
arginine at position 97. The locations of tryptophan-16 (.DELTA.)
and tyrosine-34 (.largecircle.) are also indicated.
[0017] FIG. 7 shows an exemplary x-ray crystal structure of
IL-1ra.
[0018] FIG. 8 shows a portion of the interface between the two
subunits of the asymmetric IL-1ra dimer in the x-ray crystal
structure described for FIG. 1.
[0019] FIG. 9 shows the urea-induced equilibrium unfolding of
IL-1ra detected by circular dichroism discussed in Example 6.
[0020] FIG. 10 shows the urea-induced equilibrium unfolding of
IL-1ra detected by intrinsic fluorescence discussed in Example
6.
[0021] FIG. 11 shows the guanidinium hydrochloride-induced
unfolding of IL-1ra by circular dichroism discussed in Example
6.
[0022] FIG. 12 shows the guanidinium hydrochloride-induced
unfolding of IL-1ra by intrinsic fluorescence discussed in Example
6.
[0023] FIG. 13 shows the reverse-phase HPLC of IL-1ra affinity
labeled with methyl acetyl phosphate (MAP) in the presence of 10 mM
citrate, pH 6.5, discussed in Example 7.
[0024] FIG. 14 shows the reverse-phase HPLC of IL-1ra affinity
labeled with methyl acetyl phosphate (MAP) in the presence of 10 mM
phosphate, pH 6.5, discussed in Example 7.
[0025] FIG. 15 shows the overlay of the reverse-phase HPLC profiles
of IL-1ra affinity labeled with methyl acetyl phosphate for 4.5
hours (270 minutes) in the presence of 10 mM citrate, pH 6.5, 10 mM
phosphate, pH 6.5, or 10 mM pyrophosphate, pH 6.4, discussed in
Example 7. Four weakly resolved peaks are labeled as 1, 2, 3, and
4.
[0026] FIG. 16 shows the rate of derivatization of IL-1ra with
methyl acetyl phosphate in the presence of 10 mM citrate, pH 6.5,
discussed in Example 7. FIG. 16A shows a reproduction of the
chromatograms from FIG. 13. FIG. 16B shows the results of
deconvoluting each chromatogram from FIG. 16A. FIG. 16C shows a
plot of the integration of each of the deconvoluted peaks from FIG.
16B versus time.
[0027] FIG. 17 shows a comparison of the rates of derivatization of
IL-1ra with MAP in the presence of various buffers at various pH,
discussed in Example 7.
[0028] FIG. 18 shows the overlay of the reverse-phase HPLC profiles
of IL-1ra affinity-labeled with methyl acetyl phosphate for 5.5
hours in the presence of 10 mM citrate or 10 mM phosphate at
various pH levels, discussed in Example 7.
[0029] FIG. 19 shows the IL-1ra wild-type protein aggregation
profile over time in various concentrations of phosphate in a
buffer with 100 mM NaCl, pH 6.5, discussed in Example 8.
[0030] FIG. 20 shows citrate binding to IL-1ra as a function of
increasing concentration of citrate, as discussed in Example 9.
[0031] FIG. 21 shows competition for citrate binding to IL-1ra by
increasing concentrations of pyrophosphate or phosphate, as
discussed in Example 10.
[0032] FIG. 22 shows a plot of the K.sup.d.sub.Lapp for citrate
binding to IL-1ra as a function of pyrophosphate concentration in
the solution, as discussed in Example 10. The K.sup.d.sub.X for
pyrophosphate binding to the anion-binding site of IL-1ra was
calculated to be 2.994 mM.
[0033] FIG. 23 shows the nucleotide sequence (SEQ ID NO: 4) and
amino acid sequence (SEQ ID NO: 5) of an exemplary IL-1ra.
[0034] FIG. 24 shows the amino acid sequence of an exemplary
IL-1ra, referred to as icIL-1ra (SEQ ID NO: 6). SEQ ID NO: 6 has
the same sequence as SEQ ID NO: 3, but with an additional 7 amino
acids at the N-terminus.
DETAILED DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS
[0035] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All references cited in this application are
expressly incorporated by reference herein for any purpose.
[0036] In this application, the use of the singular includes the
plural unless specifically stated otherwise. In this application,
the use of "or" means "and/or" unless stated otherwise.
Furthermore, the use of the term "including", as well as other
forms, such as "includes" and "included", is not limiting. Also,
terms such as "element" or "component" encompass both elements and
components comprising one unit and elements and components that
comprise more than one subunit unless specifically stated
otherwise.
[0037] In various embodiments, standard techniques may be used for
recombinant DNA, oligonucleotide synthesis, tissue culture,
transformation and transfection. In various embodiments, enzymatic
reactions and purification techniques may be performed according to
manufacturer's specifications or as commonly accomplished in the
art or as described herein. In various embodiments, techniques and
procedures may be generally performed according to conventional
methods known in the art and as described in various general and
more specific references that are cited and discussed throughout
the present specification and/or that are known to one skilled in
the art. See e.g., Sambrook et al. Molecular Cloning: A Laboratory
Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989). Unless specific definitions are provided, the
nomenclatures utilized in connection with, and the laboratory
procedures and techniques of, analytical chemistry, synthetic
organic chemistry, and medicinal and pharmaceutical chemistry
described herein are those known and commonly used in the art. In
various embodiments, standard techniques may be used for chemical
syntheses, chemical analyses, pharmaceutical preparation,
formulation, delivery, and treatment of patients. In certain
embodiments, where an amino acid is "replaced" with another amino
acid, that replacement may be done recombinantly, e.g., by mutating
the codon in the polynucleotide that encodes the amino acid to the
codon that encodes another amino acid. In certain embodiments,
mutating the codon may be done by any method known in the art.
Definitions
[0038] As utilized in accordance with the present disclosure, the
following terms, unless otherwise indicated, shall be understood to
have the following meanings:
[0039] The term "isolated polynucleotide" shall mean a
polynucleotide of genomic, cDNA, or synthetic origin, or some
combination thereof, which (1) is not associated with at least a
portion of a polynucleotide in which it is found in nature, or (2)
is linked to a polynucleotide to which it is not linked in nature,
or (3) does not occur in nature.
[0040] The term "operably linked" refers to components that are in
a relationship permitting them to function in their intended
manner. For example, a control sequence "operably linked" to a
coding sequence is ligated in such a way that expression of the
coding sequence is achieved under conditions compatible with the
operation of the control sequences.
[0041] The term "control sequence" refers to polynucleotide
sequences which may effect the expression and processing of coding
sequences to which they are ligated. The nature of such control
sequences may differ depending upon the host organism. According to
certain embodiments, control sequences for prokaryotes may include
promoters, ribosomal binding sites, and transcription termination
sequences. According to certain embodiments, control sequences for
eukaryotes may include promoters and transcription termination
sequence. In certain embodiments, "control sequences" can include
leader sequences and/or fusion partner sequences.
[0042] The term "polynucleotide" means a polymeric form of
nucleotides having naturally occurring and/or modified
ribonucleotides and/or deoxyribonucleotides which are linked
together by naturally occurring and/or non-naturally occurring
linkages. In certain embodiments, a polynucleotide is least 10
bases in length. The term includes single and double stranded forms
of DNA/RNA, and DNA/RNA hybrid, or modified forms thereof.
[0043] The term "oligonucleotide" includes polymers having
naturally occurring and/or modified ribonucleotides and/or
deoxyribonucleotides, which are linked together by naturally
occurring and/or non-naturally occurring linkages. Oligonucleotides
are a subset of polynucleotides and generally comprise about 200
bases or fewer. In certain embodiments, oligonucleotides are about
10 to about 60 bases in length. In certain embodiments,
oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 to
40 bases in length. Oligonucleotides may be single stranded or
double stranded. Oligonucleotides may be sense or antisense
oligonucleotides.
[0044] The term "naturally occurring nucleotides" includes
deoxyribonucleotides and ribonucleotides. The term "modified
nucleotides" includes nucleotides with modified or substituted
sugar groups and/or modified or substituted nucleotide base groups
and the like. The terms "oligonucleotide linkage" and
"polynucleotide linkage" include linkages such as phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the
like. See, e.g., LaPlanche et al. Nucl. Acids Res. 14:9081 (1986);
Stec et al. J. Am. Chem. Soc. 106:6077 (1984); Stein et al. Nucl.
Acids Res. 16:3209 (1988); Zon et al. Anti-Cancer Drug Design 6:539
(1991); Zon et al. Oligonucleotides and Analogues: A Practical
Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press,
Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;
Uhlmann and Peyman Chemical Reviews 90:543 (1990). In certain
embodiments, an oligonucleotide or polynucleotide may include a
label.
[0045] The term "naturally occurring" as applied to an object
refers to an object that can be found in nature. For example, a
polypeptide or polynucleotide sequence that is present in an
organism (including viruses) that can be isolated from a source in
nature and which has not been modified by man in the laboratory or
otherwise is naturally occurring.
[0046] The term "isolated protein" means a protein made by
synthetic means or a protein encoded by genomic DNA, cDNA, RNA, or
other polynucleotide, which (1) is free, of at least some proteins
with which it would normally be found; or (2) is essentially free
of other proteins from the same source, e.g., from the same
species; or (3) is expressed by a cell from a different species; or
(4) does not occur in nature. In the polypeptide notation used
herein, the left-hand direction is the amino-terminal direction and
the right-hand direction is the carboxy-terminal direction, in
accordance with standard usage and convention, unless specifically
indicated otherwise.
[0047] Similarly, unless specifically indicated otherwise, the
left-hand end of single-stranded polynucleotide sequences is the 5'
end; the left-hand direction of double-stranded polynucleotide
sequences is referred to as the 5' direction. The direction of 5'
to 3' addition to nascent RNA transcripts is referred to as the
transcription direction; sequence regions on the DNA strand that
are 5' to the 5' end of the RNA transcript are referred to as
"upstream sequences" and are "upstream of the coding region";
sequence regions on the DNA strand that are 3' to the 3' end of the
RNA transcript are referred to as "downstream sequences" and are
"downstream of the coding region".
[0048] As used herein, the terms "label" or "labeled" refer to the
presence of a detectable moiety. A detectable moiety may be
incorporated during synthesis of a polynucleotide or polypeptide or
may be attached, either covalently or non-covalently, after
synthesis. Labeling may be, e.g., incorporation of a radiolabeled
amino acid, attachment of biotin moieties that can be detected with
labeled avidin (e.g., streptavidin containing a fluorescent moiety
or enzymatic activity that can be detected by optical or
colorimetric methods). In certain embodiments, the label or
detectable moiety can be therapeutic. Various methods of labeling
polypeptides and/or polynucleotides are known in the art. Examples
of labels for polypeptides and/or polynucleotides include, but are
not limited to: radioisotopes or radionuclides (e.g., .sup.3H,
.sup.14C, .sup.15N, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In,
.sup.125I, .sup.131I), fluorescent labels (e.g., FITC, rhodamine,
lanthanide phosphors), enzymatic labels (e.g., horseradish
peroxidase, .beta.-galactosidase, luciferase, alkaline
phosphatase), chemiluminescent labels, biotinyl groups,
predetermined polypeptide epitopes recognized by a secondary
reporter (e.g., leucine zipper pair sequences, binding sites for
secondary antibodies, metal binding domains, epitope tags). In
certain embodiments, labels are attached by spacer arms of various
lengths to reduce the potential for steric hindrance.
[0049] As used herein, the twenty conventional amino acids and
their abbreviations follow conventional usage. See Immunology--A
Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer
Associates, Sunderland, Mass. (1991)). Stereoisomers (e.g., D-amino
acids) of the twenty conventional amino acids, unnatural amino
acids, such as .alpha.-, .alpha.-disubstituted amino acids, N-alkyl
amino acids, lactic acid, and other unconventional amino acids, may
also be suitable components for polypeptides of the present
invention. Non-limiting exemplary unconventional amino acids
include 4-hydroxyproline, .gamma.-carboxyglutamate,
.epsilon.-N,N,N-trimethyllysine, .epsilon.-N-acetyllysine,
O-phosphoserine, N-acetylserine, N-formylmethionine,
3-methylhistidine, 5-hydroxylysine, .sigma.-N-methylarginine, and
other similar amino acids and imino acids.
[0050] A skilled artisan will be able to identify suitable variants
of a polypeptide with well-known techniques. In certain
embodiments, one skilled in the art may identify suitable regions
of a polypeptide that may be changed without destroying activity by
targeting regions not believed to be important for activity. In
certain embodiments, one can identify residues and portions of a
polypeptide that are conserved among similar polypeptides. In
certain embodiments, even areas that may be important for
biological activity or for structure may be amenable to
conservative amino acid substitutions without destroying the
biological activity or without adversely affecting the polypeptide
structure.
[0051] Additionally, in certain embodiments, one skilled in the art
can review structure-function studies and identify residues in
similar polypeptides that are important for activity or structure.
In view of such a comparison, one can predict the importance of
amino acid residues in a protein that correspond to amino acid
residues which are important for activity or structure in similar
proteins. In certain embodiments, one skilled in the art may opt
for chemically similar amino acid substitutions for such predicted
important amino acid residues.
[0052] In certain embodiments, one skilled in the art can analyze
the three-dimensional structure and amino acid sequence in relation
to known structures in similar polypeptides. Moreover, in certain
embodiments, one skilled in the art may generate test variants
containing a single amino acid substitution at each desired amino
acid residue. In certain embodiments, the variants can then be
screened using activity assays known to those skilled in the art.
Such variants could be used to gather information about suitable
variants. For example, in certain embodiments, if one discovered
that a change to a particular amino acid residue resulted in
destroyed, undesirably reduced, or unsuitable activity, variants
with such a change may be avoided. In other words, based on
information gathered from such routine experiments, one skilled in
the art can readily determine the amino acids where further
substitutions should be avoided either alone or in combination with
other mutations.
[0053] In certain embodiments, deletions, insertions, and/or
substitutions (individually or collectively referred to as
"variant(s)") are made within the amino acid sequence of a IL-1ra
wild-type protein. As used herein, "IL-1ra wild-type protein"
refers to a protein having the amino acid sequence of SEQ ID NO: 3,
optionally having an additional methionine residue at its
N-terminus, such that the N-terminal sequence is MRPSGR . . . . The
therapeutic protein having the generic name "anakinra" falls within
this definition of IL-1ra wild-type protein. Anakinra has the
sequence of SEQ ID NO: 5, which is identical to SEQ ID NO: 3, but
with an N-terminal methionine. In certain embodiments, alterations
to the IL-1ra wild-type protein, such as chemical or enzymatic
modification, are made after translation or synthesis of the
protein. Such altered IL-1ra proteins are individually or
collectively referred to as "derivative(s)". The term IL-1ra
encompasses IL-1ra wild-type proteins, as well as naturally
occurring and non-naturally occurring IL-1ra variants and
derivatives that have antagonist activity for the IL-1ra
receptor.
[0054] In certain embodiments, an "amino acid that does not have a
positive charge" is selected from alanine, cysteine, aspartic acid,
glutamic acid, phenylalanine, glycine, histidine, isoleucine,
leucine, methionine, asparagine, proline, glutamine, serine,
threonine, valine, tryptophan, and tyrosine. Amino acids that do
not have a positive charge also include, but are not limited to,
unconventional amino acids that do not have a positive charge. One
skilled in the art can determine whether or not a particular amino
acid variant has a positive charge when incorporated into a
polypeptide.
[0055] In certain embodiments, an "amino acid that does not have a
charge" is selected from alanine, cysteine, phenylalanine, glycine,
histidine, isoleucine, leucine, methionine, asparagine, proline,
glutamine, serine, threonine, valine, tryptophan, and tyrosine.
Amino acids that do not have a charge also include, but are not
limited to, unconventional amino acids that do not have a charge.
One skilled in the art can determine whether or not a particular
unconventional amino acid variant has a charge when incorporated
into a polypeptide.
[0056] In certain embodiments, a "polar amino acid that does not
have a charge" is selected from cysteine, glycine, glutamine,
asparagine, serine, threonine, and tyrosine. Polar amino acids that
do not have a charge also include, but are not limited to,
unconventional amino acids that are polar but do not have a charge.
One skilled in the art can determine whether or not a particular
unconventional amino acid variant is polar and whether it has a
charge when incorporated into a polypeptide.
[0057] In certain embodiments, a "non-aromatic amino acid" is
selected from alanine, arginine, cysteine, aspartic acid, glutamic
acid, glycine, histidine, isoleucine, leucine, lysine, methionine,
asparagine, proline, glutamine, serine, threonine, and valine.
Non-aromatic amino acids also include, but are not limited to,
unconventional amino acids that are not aromatic. One skilled in
the art can determine whether or not a particular unconventional
amino acid variant is aromatic when incorporated into a
polypeptide.
[0058] The term "cation-pi interaction" refers to a non-covalent
interaction between a cationic amino acid and an aromatic amino
acid. In certain embodiments, the cationic amino acid may be
lysine. In certain embodiments, the cationic amino acid may be
arginine. In certain embodiments, the aromatic amino acid may be
phenylalanine. In certain embodiments, the aromatic amino acid may
be tyrosine. In certain embodiments, the aromatic amino acid may be
tryptophan. The cation-pi interaction may be within a single
polypeptide (i.e., intramolecular) or the cation-pi interaction
maybe between two or more polypeptides (i.e., intermolecular).
[0059] In certain embodiments, certain amino acid residues of
IL-1ra are involved in a cation-pi interaction. FIG. 1 shows an
x-ray crystal structure of IL-1ra. The crystal structure was
prepared using a 1 ILR.pdb file and Vector NTI 3D Molecular Viewer
(InforMax). IL-1ra crystallized as an asymmetric dimer. See, e.g.,
Vigers et al., J. Biol. Chem., 269: 12874-12879 (1994). FIG. 2
shows a portion of the crystal structure of FIG. 1. In that crystal
structure, lysine-93 on one IL-1ra subunit appears to be involved
in a cation-pi interaction with tryptophan-16 on the other IL-1ra
subunit. Similarly, arginine-97 on one IL-1ra subunit appears to be
involved in a cation-pi interaction with tyrosine-34 on the other
IL-1ra subunit.
[0060] The term "polypeptide fragment" as used herein refers to a
polypeptide that has an amino-terminal and/or a carboxy-terminal
deletion. In certain embodiments, fragments are at least 5 to 201
amino acids long. It will be appreciated that in certain
embodiments, fragments are at least 5, 6, 8, 10, 14, 20, 50, 70,
80, 90, 100, 110, 125, 150, 170, 175, 176, 177, 180, 185, 190, or
200 amino acids long.
[0061] The term "biological sample", as used herein, includes, but
is not limited to, any quantity of a substance from a living thing
or formerly living thing. Such living things include, but are not
limited to, humans, mice, monkeys, rats, rabbits, and other
animals. Such substances include, but are not limited to, blood,
serum, urine, cells, organs, tissues, bone, bone marrow, lymph
nodes, and skin.
[0062] As used herein, "substantially pure" means an object
macromolecular species is the predominant macromolecular species
present (i.e., on a molar basis it is more abundant than any other
individual macromolecular species in the composition). In certain
embodiments, a substantially purified fraction is a composition
wherein the object macromolecular species comprises at least about
50 percent (on a weight basis) of all macromolecular species
present. In certain embodiments, in a substantially pure
composition, the object macromolecular species will comprise more
than about 80%, 85%, 90%, 95%, or 99% by weight of all
macromolecular species present in the composition. In certain
embodiments, the object macromolecular species is purified to
essentially homogeneity (i.e., contaminant species cannot be
detected in the composition by conventional detection methods). In
certain embodiments, the composition consists essentially of a
single macromolecular species.
[0063] The term patient includes human and animal subjects.
[0064] Interleukin-1 receptor antagonist (IL-1ra) is a human
protein that acts as an inhibitor of interleukin-1 activity and is
a member of the IL-1 family, which also includes IL-1.alpha. and
IL-1.beta.. A non-exclusive, non-limiting, non-exhaustive list of
IL-1 receptor antagonists includes Kineret.RTM. (anakinra) (e.g., a
protein having the amino acid sequence of SEQ ID NO: 5), IL-1ra
wild-type protein (including, but not limited to, a protein having
the sequence of SEQ ID NO: 3 or a protein having the sequence of
SEQ ID NO: 5), intracellular IL-1ra (icIL-1ra) (including, but not
limited to, a protein having the sequence of SEQ ID NO: 6), IL-1ra
.beta. (see, e.g., PCT Publication No. WO 99/36541), IL-1ra
variants, and IL-1ra derivatives. Certain IL-1ra receptor
antagonists, including IL-1ra and variants and derivatives thereof,
as well as methods of making and using them, are described, e.g.,
in U.S. Pat. No. 5,075,222; U.S. Pat. No. 6,599,873 B1; U.S. Pat.
No. 5,863,769; U.S. Pat. No. 5,858,355; U.S. Pat. No. 5,739,282;
U.S. Pat. No. 5,922,573; U.S. Pat. No. 6,054,559; WO 91/08285; WO
91/17184; WO 91/17249; AU 9173636; WO 92/16221; WO 93/21946; WO
94/06457; WO 94/21275; FR 2706772; WO 94/21235; DE 4219626, WO
94/20517; WO 96/22793; WO 96/12022; WO 97/28828; WO 99/36541; WO
99/51744. An IL-1 receptor antagonist may be glycosylated or
non-glycosylated.
[0065] Exemplary IL-1ras include, but are not limited to, a
polypeptide comprising an amino acid sequence as set forth in SEQ
ID NO: 2 and fragments, variants, and derivatives of such a
polypeptide that have an antagonist activity for the interleukin-1
receptor; a polypeptide comprising an amino acid sequence as set
forth in SEQ ID NO: 3 and fragments, variants, and derivatives of
such a polypeptide that have an antagonist activity for the
interleukin-1 receptor; a polypeptide comprising an amino acid
sequence as set forth in SEQ ID NO: 5 and fragments, variants, and
derivatives of such a polypeptide that have an antagonist activity
for the interleukin-1 receptor; and a polypeptide comprising an
amino acid sequence as set forth in SEQ ID NO: 6 and fragments,
variants, and derivatives of such a polypeptide that have an
antagonist activity for the interleukin-1 receptor.
[0066] In certain embodiments, the term IL-1ra includes, but is not
limited to, IL-1ra variants that have antagonist activity for the
interleukin-1 receptor. In certain embodiments, IL-1ra variants are
naturally occurring. In certain embodiments, IL-1ra variants are
artificially constructed. Exemplary IL-1ra variants include, but
are not limited to, amino acid sequences having one or more amino
acid substitutions, deletions, and/or additions as compared to SEQ
ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6. In certain
embodiments, IL-1ra variants comprise an amino acid sequence that
is 95% identical to the amino acid sequence of SEQ ID NO: 3. In
certain embodiments, IL-1ra variants comprise an amino acid
sequence that is 90% identical to the amino acid sequence of SEQ ID
NO: 3. In certain embodiments, IL-1ra variants comprise an amino
acid sequence that is 85% identical to the amino acid sequence of
SEQ ID NO: 3. In certain embodiments, IL-1ra variants comprise an
amino acid sequence that is 75% identical to the amino acid
sequence of SEQ ID NO: 3.
[0067] In certain embodiments, the term IL-1ra includes, but is not
limited to, IL-1ra fragments that have antagonist activity for the
interleukin-1 receptor. In certain embodiments, IL-1ra fragments
are naturally occurring. In certain embodiments, IL-1ra fragments
are artificially constructed. Exemplary IL-1ra fragments include,
but are not limited to, fragments of the sequence set forth in SEQ
ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6. IL-1ra
fragments are a subset of IL-1ra variants.
[0068] In certain embodiments, the term IL-1ra includes, but is not
limited to, IL-1ra derivatives that have antagonist activity for
the interleukin-1 receptor. In certain embodiments, IL-1ra
derivatives are naturally occurring. In certain embodiments, IL-1ra
derivatives are artificially constructed. Exemplary IL-1ra
derivatives include, but are not limited to, chemically or
enzymatically modified forms of the sequence set forth in SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6. Exemplary
IL-1ra derivatives also include, but are not limited to, chemically
or enzymatically modified forms of variants of the sequence set
forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO:
6.
[0069] In certain embodiments, the term IL-1ra includes, but is not
limited to, an IL-1ra having a secretory leader sequence. In
certain embodiments, IL-1ra having a secretory leader sequence is
referred to as "precursor IL-1ra." An exemplary precursor IL-1ra
amino acid sequence is set forth in SEQ ID NO: 2. The term
"precursor IL-1ra" includes fragments, variants, and derivatives of
SEQ ID NO: 2 that are capable of being secreted and processed into
a form having antagonist activity for the interleukin-1
receptor.
[0070] The term IL-1ra includes both aggregating IL-1ra and IL-1ra
having reduced aggregation. Aggregating IL-1ra proteins have a
lysine at position 93 and an arginine at position 97, but not all
IL-1ra proteins with a lysine at position 93 and an arginine at
position 97 are aggregating IL-1ras. "Aggregating IL-1ra" includes
IL-1ra wild-type, variant, and derivative proteins that aggregate
at 39.degree. C. according to the following assay. A solution of
100 mg/ml of the subject IL-1ra is incubated at 39.degree. C. in 10
mM phosphate, 140 mM NaCl, 0.5 mM EDTA, pH 6.5 (PSE). As a
reference, a solution of 100 mg/ml of an IL-1ra wild-type protein
having the sequence of SEQ ID NO: 5, is incubated in PSE at
39.degree. C. The optical density of each solution is measured at
405 nm after 2 hours of incubation at 39.degree. C. If the subject
IL-1ra has an optical density after 2 hours of incubation that is
at least 60% of the optical density of the IL-1ra wild-type protein
after 2 hours of incubation, then the subject 1 L-1 ra is an
aggregating 1 L-1 ra.
[0071] "IL-1ra having reduced aggregation" includes IL-1ra variant
and derivative proteins that have an optical density that is less
than 60% of the optical density of the IL-1ra wild-type protein in
the assay described above.
[0072] In certain embodiments, the term "an IL-1ra that has
antagonist activity for the interleukin-1 receptor" refers to an
IL-1ra wild-type, variant, or derivative protein that is at least
50% as active as an IL-1ra wild-type protein having the amino acid
sequence of SEQ ID NO: 5, in the IL-1ra signaling complex formation
assay described in Example 3. "At least 50% as active" is
determined by comparing the EC50 of the subject IL-1ra to the EC50
of IL-1ra wild-type protein.
[0073] The term "Arginine-97" refers to the amino acid residue at
the 97th position in SEQ ID NO: 3 or the amino acid position in an
IL-1ra that corresponds to the amino acid residue at the 97th
position in SEQ ID NO: 3. For example, the amino acid that
corresponds to arginine-97 of SEQ ID NO: 3 is the arginine at the
98th position of SEQ ID NO: 5. That arginine is still referred to
as arginine-97 of IL-1ra. In certain embodiments, Arginine-97 is
referred to as "R97," wherein the R is the single-letter code for
arginine and 97 refers to its position in SEQ ID NO: 3. In certain
embodiments, if R97 is replaced with another amino acid, the
mutation may be referred to as R97X, wherein X is the single-letter
code for the replacement amino acid. Thus, as a non-limiting
example, if R97 is replaced by alanine, the mutation may be
referred to as R97A.
[0074] The term "Lysine-93" refers to the amino acid residue at the
93rd position in SEQ ID NO: 3 or the amino acid position in an
IL-1ra that corresponds to the amino acid residue at the 93rd
position in SEQ ID NO: 3. For example, the amino acid that
corresponds to lysine-93 of SEQ ID NO: 3 is the lysine at the 94th
position of SEQ ID NO: 5. That lysine is still referred to as
lysine-93 of IL-1ra. In certain embodiments, Lysine-93 is referred
to as "K93," wherein the K is the single-letter code for lysine and
93 refers to its position in SEQ ID NO: 3. In certain embodiments,
if K93 is replaced with another amino acid, the mutation may be
referred to as K93X, wherein X is the single-letter code for the
replacement amino acid. Thus, as a non-limiting example, if K93 is
replaced by alanine, the mutation may be referred to as K93A.
[0075] The term "Tryptophan-16" refers to the amino acid residue at
the 16th position in SEQ ID NO: 3 or the amino acid position in an
IL-1ra that corresponds to the amino acid residue at the 16th
position in SEQ ID NO: 3. For example, the amino acid that
corresponds to tryptophan-16 of SEQ ID NO: 3 is the tryptophan at
the 17th position of SEQ ID NO: 5. That tryptophan is still
referred to as tryptophan-16 of IL-1ra. In certain embodiments,
Tryptophan-16 is referred to as "W16," wherein the W is the
single-letter code for tryptophan and 16 refers to its position in
SEQ ID NO: 3. In certain embodiments, if W16 is replaced with
another amino acid, the mutation may be referred to as W16X,
wherein X is the single-letter code for the replacement amino acid.
Thus, as a non-limiting example, if W16 is replaced by alanine, the
mutation may be referred to as W16A.
[0076] The term "Tyrosine-34" refers to the amino acid residue at
the 34th position in SEQ ID NO: 3 or the amino acid position in an
IL-1ra that corresponds to the amino acid residue at the 34th
position in SEQ ID NO: 3. For example, the amino acid that
corresponds to tyrosine-34 of SEQ ID NO: 3 is the tyrosine at the
35th position of SEQ ID NO: 5. That tyrosine is still referred to
as tyrosine-35 of IL-1ra. In certain embodiments, Tyrosine-34 is
referred to as "Y34," wherein the Y is the single-letter code for
tyrosine and 34 refers to its position in SEQ ID NO: 3. In certain
embodiments, if Y34 is replaced with another amino acid, the
mutation may be referred to as Y34X, wherein X is the single-letter
code for the replacement amino acid. Thus, as a non-limiting
example, if Y34 is replaced by alanine, the mutation may be
referred to as Y34A.
[0077] In certain embodiments, "reduced aggregation" is defined as
(1) aggregation of a polypeptide under condition A that is reduced
relative to aggregation of the polypeptide under condition B;
and/or (2) aggregation of a polypeptide variant under condition A
that is reduced relative to aggregation of the wild-type
polypeptide under the same condition A; and/or (3) aggregation of a
polypeptide variant under condition A that is reduced relative to
aggregation of a different polypeptide variant under the same
condition A. As a non-limiting example, relative aggregation may be
determined for case (1) as follows. The optical density at 405 nm
of a polypeptide under condition A is measured at various times.
The optical density at 405 nm of the polypeptide under condition B
is then measured at the same various times. The aggregation curve
for the object polypeptide under each condition is plotted with
optical density on the y-axis and time on the x-axis. If the
polypeptide under condition A has a lower optical density at time t
than the polypeptide under condition B at the same time t, then the
polypeptide under condition A is said to have reduced aggregation
relative to the polypeptide under condition B.
[0078] Examples of different conditions include, but are not
limited to, differences in buffer composition, differences in
temperature, differences in polypeptide concentration, the presence
and absence of accessory molecules, differences in accessory
molecule concentration, etc.
[0079] The term "accessory molecule" refers to a molecule that
reduces aggregation of one or more polypeptides. In certain
embodiments, an accessory molecule reduces aggregation of one or
more polypeptides nonspecifically. In certain embodiments, an
accessory molecule reduces aggregation by interacting with one or
more amino acids of the polypeptide. In certain embodiments, an
accessory molecule interacts covalently with one or more amino
acids of the polypeptide and is referred to as a "covalent
accessory molecule." In certain embodiments, an accessory molecule
interacts non-covalently with one or more amino acids of the
polypeptide and is referred to as a "non-covalent accessory
molecule."
[0080] In certain embodiments, the reduction in aggregation of a
polypeptide is related to the concentration of the accessory
molecule. In certain embodiments, an accessory molecule may
substantially eliminate aggregation of a polypeptide. In certain
embodiments, an accessory molecule reduces aggregation of a
polypeptide by at least 10%. In certain embodiments, an accessory
molecule reduces aggregation of a polypeptide by at least 20%. In
certain embodiments, an accessory molecule reduces aggregation of a
polypeptide by at least 30%. In certain embodiments, an accessory
molecule reduces aggregation of a polypeptide by at least 40%. In
certain embodiments, an accessory molecule reduces aggregation of a
polypeptide by at least 50%. In certain embodiments, an accessory
molecule reduces aggregation of a polypeptide by at least 60%. In
certain embodiments, an accessory molecule reduces aggregation of a
polypeptide by at least 70%. In certain embodiments, an accessory
molecule reduces aggregation of a polypeptide by at least 75%. In
certain embodiments, an accessory molecule reduces aggregation of a
polypeptide by at least 80%. In certain embodiments, an accessory
molecule reduces aggregation of a polypeptide by at least 85%. In
certain embodiments, an accessory molecule reduces aggregation of a
polypeptide by at least 90%. In certain embodiments, an accessory
molecule reduces aggregation of a polypeptide by at least 95%.
[0081] In certain embodiments, an accessory molecule reduces the
rate of aggregation of a polypeptide. In certain embodiments, the
reduction in the rate of aggregation of a polypeptide is dependant
on the concentration of the accessory molecule. In certain
embodiments, an accessory molecule substantially eliminates
aggregation of a polypeptide. In certain embodiments, an accessory
molecule reduces the rate of aggregation of a polypeptide by at
least 10%. In certain embodiments, an accessory molecule reduces
the rate of aggregation of a polypeptide by at least 20%. In
certain embodiments, an accessory molecule reduces the rate of
aggregation of a polypeptide by at least 30%. In certain
embodiments, an accessory molecule reduces the rate of aggregation
of a polypeptide by at least 40%. In certain embodiments, an
accessory molecule reduces the rate of aggregation of a polypeptide
by at least 50%. In certain embodiments, an accessory molecule
reduces the rate of aggregation of a polypeptide by at least 60%.
In certain embodiments, an accessory molecule reduces the rate of
aggregation of a polypeptide by at least 70%. In certain
embodiments, an accessory molecule reduces the rate of aggregation
of a polypeptide by at least 75%. In certain embodiments, an
accessory molecule reduces the rate of aggregation of a polypeptide
by at least 80%. In certain embodiments, an accessory molecule
reduces the rate of aggregation of a polypeptide by at least 85%.
In certain embodiments, an accessory molecule reduces the rate of
aggregation of a polypeptide by at least 90%. In certain
embodiments, an accessory molecule reduces the rate of aggregation
of a polypeptide by at least 95%.
[0082] In certain embodiments, an accessory molecule interacts
covalently or non-covalently with a polypeptide at one or more
amino acid residues. In certain embodiments, an accessory molecule
interacts with one or more specific amino acid residues. In certain
embodiments, an accessory molecule does not substantially reduce
the activity of the polypeptide. In certain embodiments, an
accessory molecule does not reduce the activity of the polypeptide
by more than 10%. In certain embodiments, accessory molecules to
not reduce the activity of the polypeptide by more than 20%. In
certain embodiments, an accessory molecule does not reduce the
activity of the polypeptide by more than 30%. In certain
embodiments, an accessory molecule does not reduce the activity of
the polypeptide by more than 50%. In certain embodiments, an
accessory molecule does not reduce the activity of the polypeptide
by more than 75%.
[0083] In certain embodiments, an accessory molecule removes a
charge present at an amino acid residue. In certain embodiments, an
accessory molecule removes a charge by covalently modifying the
amino acid residue. In certain embodiments, an accessory molecule
removes a charge by non-covalently interacting with the amino acid
residue, thereby "masking" the charge.
[0084] Exemplary covalent accessory molecules include, but are not
limited to, 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic
acid (NBD-X), methyl acetyl phosphate (MAP), and citraconic
anhydride.
[0085] Exemplary non-covalent accessory molecules include, but are
not limited to, sugars, single-charge anions, and multiple-charge
anions. Exemplary sugars that may be accessory molecules include,
but are not limited to, glycerol, sucrose, mannitol, and sorbitol.
Exemplary single-charge anions that may be accessory molecules
include, but are not limited to, phosphate and chloride. Exemplary
multiple-charge anions that may be accessory molecules include, but
are not limited to, pyrophosphate and citrate.
[0086] The term "arginine-reactive accessory molecule" refers to an
accessory molecule that specifically interacts with arginine
residues. In certain embodiments, an arginine-reactive accessory
molecule interacts solely with arginine. In certain embodiments, an
arginine-reactive accessory molecule interacts with arginine in
addition to other amino acids. In certain embodiments, an
arginine-reactive accessory molecule interacts covalently with
arginine. In certain embodiments, an arginine-reactive accessory
molecule interacts non-covalently with arginine. In certain
embodiments, an arginine-reactive accessory molecule does not
substantially reduce the activity of the polypeptide that contains
the arginine.
[0087] The term "lysine-reactive accessory molecule" refers to an
accessory molecule that specifically interacts with lysine
residues. In certain embodiments, an lysine-reactive accessory
molecule interacts solely with lysine. In certain embodiments, an
lysine-reactive accessory molecule interacts with lysine in
addition to other amino acids. In certain embodiments, an
lysine-reactive accessory molecule interacts covalently with
lysine. In certain embodiments, an lysine-reactive accessory
molecule interacts non-covalently with lysine. In certain
embodiments, an lysine-reactive accessory molecule does not
substantially reduce the activity of the polypeptide that contains
the lysine.
[0088] The term "multiple-charge anions" refers to molecules that
comprise more than one negative charge at pH 6.5 and 25.degree. C.
In certain embodiments, multiple-charge anions have on average more
than one, but less than two, negative charges at pH 6.5 and
25.degree. C. In certain embodiments, multiple-charge anions have
on average two or more negative charges at pH 6.5 and 25.degree. C.
In certain embodiments, multiple-charge anions have on average
between two and four negative charges at pH 6.5 and 25.degree. C.
In various embodiments, one skilled in the art can determine
whether a particular anion is a multiple-charge anion at pH 6.5 and
25.degree. C., e.g., from the published pKa values for the anion.
The term "single-charge anion" refers to an anion that has on
average one, or less than one, negative charge at pH 6.5 and
25.degree. C. In various embodiments, one skilled in the art can
determine whether a particular anion is a single-charge anion at pH
6.5 and 25.degree. C., e.g., from the published pKa values for the
anion.
[0089] The term "sugar" refers to a carbohydrate. Exemplary sugars
include, but are not limited to, monosaccharides, disaccharides,
and trisaccharides. Non-limiting exemplary sugars include, but are
not limited to, glycerol, sucrose, mannitol, and sorbitol.
[0090] A disease or medical condition is considered to be an
"interleukin-1 mediated disease" if the spontaneous or experimental
disease or medical condition is associated with elevated levels of
IL-1 in bodily fluids or tissue and/or if cells or tissues taken
from the body produce elevated levels of IL-1 in culture. In
certain embodiments, such interleukin-1 mediated diseases are also
recognized by one or more of the following two conditions: (1)
pathological findings associated with the disease or medical
condition can be mimicked experimentally in animals by
administration of IL-1 or upregulation of expression of IL-1;
and/or (2) a pathology induced in experimental animal models of the
disease or medical condition can be inhibited or abolished by
treatment with agents that inhibit the action of IL-1. In certain
embodiments, one or more of the above conditions are met in an
IL-1-mediated disease. In certain embodiments, all of the
conditions are met in an IL-1-mediated disease.
[0091] Acute and chronic interleukin-1 (IL-1) mediated diseases
include, but are not limited to, acute pancreatitis; amyotrophic
lateral sclerosis (ALS, or Lou Gehrig's disease); Alzheimer's
disease; cachexia/anorexia, including, but not limited to,
AIDS-induced cachexia; asthma and other pulmonary diseases;
atherosclerosis; autoimmune vasculitis; chronic fatigue syndrome;
Clostridium associated illnesses, including, but not limited to,
Clostridium-associated diarrhea; coronary conditions and
indications, including, but not limited to, congestive heart
failure, coronary restenosis, myocardial infarction, myocardial
dysfunction (e.g., related to sepsis), and coronary artery bypass
graft; cancer, including, but not limited to, leukemias, including,
but not limited to, multiple myeloma leukemia and myelogenous
(e.g., AML and CML), and tumor metastasis; diabetes (including, but
not limited to, insulin-dependent diabetes); endometriosis; fever;
fibromyalgia; glomerulonephritis; graft versus host disease and/or
transplant rejection; hemohorragic shock; hyperalgesia;
inflammatory bowel disease; inflammatory conditions of a joint,
including, but not limited to, osteoarthritis, psoriatic arthritis,
and rheumatoid arthritis; inflammatory eye disease, including, but
not limited to, those associated with, for example, corneal
transplant; ischemia, including, but not limited to, cerebral
ischemia (including, but not limited to, brain injury as a result
of, e.g., trauma, epilepsy, hemorrhage or stroke, each of which may
lead to neurodegeneration); Kawasaki's disease; learning
impairment; lung diseases (including, but not limited to, acute
respiratory distress syndrome, or ARDS); multiple sclerosis;
myopathies (e.g., muscle protein metabolism, including, but not
limited to, muscle protein metabolism in sepsis); neurotoxicity
(including, but not limited to, such condition induced by HIV);
osteoporosis; pain, including, but not limited to, cancer-related
pain; Parkinson's disease; periodontal disease; pre-term labor;
psoriasis; reperfusion injury; septic shock; side effects from
radiation therapy; temporal mandibular joint disease; sleep
disturbance; uveitis; and inflammatory conditions resulting from,
e.g., strain, sprain, cartilage damage, trauma, orthopedic surgery,
infection, or other disease processes.
DETAILED DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS
[0092] Methods of reducing aggregation of an aggregating IL-1ra are
provided. In certain embodiments, the aggregating IL-1ra whose
aggregation is to be reduced comprises the amino acid sequence in
SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6. In
certain embodiments, the aggregating IL-1ra whose aggregation is to
be reduced comprises a fragment, variant, or derivative of a
polypeptide having the amino acid sequence shown in SEQ ID NO: 2,
SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6, where that fragment,
variant, or derivative has an antagonist activity for the
interleukin-1 receptor.
[0093] Aggregation of IL-1ra may result from one or more cation-pi
interactions between surface residues of two IL-1ra polypeptides.
For example, as shown in FIG. 2, lysine-93 of one IL-1 polypeptide
may form a cation-pi interaction with tryptophan-16 of a second
IL-1 polypeptide. Similarly, arginine-97 of one polypeptide may
form a cation-pi interaction with tyrosine-34 of a second
polypeptide. Those interactions may cause two IL-1 ra polypeptides
to bind to one another. Furthermore, because that binding is
asymmetric, meaning that binding does not occur between the same
face on both polypeptides, each polypeptide may be able to bind to
two IL-1ra polypeptides simultaneously. In fact, if additional
asymmetric binding contacts are possible between IL-1ra
polypeptides, an IL-1ra polypeptide may be able to bind to more
than two IL-1ra polypeptides simultaneously. If each IL-1ra
polypeptide is capable of binding to at least two other IL-1ra
polypeptides, e.g., through the asymmetric cation-pi interaction
shown in FIG. 8, then those interactions may lead to aggregation of
IL-1ra in solution.
[0094] In certain embodiments, aggregation of an aggregating IL-1ra
may be reduced by reducing the positive charge at lysine-93, at
arginine-97, or at both lysine-93 and arginine-97. In certain
embodiments, if the positive charge at one or both of those
positions is sufficiently reduced, the cation-pi interaction may
not form, or may be weakened such that it is no longer stable
enough to cause aggregation of IL-1ra. In certain embodiments,
aggregation of an aggregating IL-1ra may be reduced by other
mechanisms. The method is not limited by the mechanism of the
reduction in aggregation. Any of the described methods of reducing
aggregation may occur by the exemplary proposed mechanism discussed
above or by any other mechanism that achieves the result described.
Molecules that are capable of reducing aggregation of an
aggregating IL-1ra are collectively referred to as "accessory
molecules," regardless of their mechanism of reducing aggregation
and regardless of whether they act covalently or
non-covalently.
[0095] In certain embodiments, an accessory molecule may reduced
aggregation of an aggregating IL-1ra by reducing the positive
charge at one or both of lysine-93 or arginine-97. In various
embodiments, an accessory molecule may reduce the positive charge
at lysine-93, at arginine-97, or at both lysine-93 and arginine-97
covalently or non-covalently. An accessory molecule postulated to
reduce the positive charge at one or both of those amino acids may
also reduce aggregation through other mechanisms, or may act by
other mechanisms entirely.
[0096] In certain embodiments, incubation of an aggregating IL-1ra
with single-charge anionic or multiple-charge anionic molecules may
reduce aggregation of IL-1ra. In certain embodiments, incubation of
an IL-1ra having reduced aggregation with single-charge anionic or
multiple-charge anionic molecules may further reduce aggregation of
IL-1ra. In certain embodiments, that reduction in aggregation may
result from the single-charge anionic or multiple-charge anionic
molecule interacting with the positive charge at lysine-93, at
arginine-97, or at both lysine-93 and arginine-97. As discussed in
the work described in Example 2 and shown in FIG. 1, incubating an
aggregating IL-1ra in CSE, which contains 10 mM citrate, reduced
aggregation (as measured by the optical density at 405 nm) over
time more than incubating the aggregating IL-1ra in PSE, which
contains 10 mM phosphate. In that experiment, aggregation in CSE
reached an OD.sub.405 of about 0.55, while aggregation in PSE
reached an OD.sub.405 of about 1.2. Thus, incubation in CSE reduced
aggregation by more than 50% relative to aggregation in PSE in that
experiment.
[0097] Similarly, in the work described in Example 2, the rate of
aggregation of aggregating IL-1ra in CSE was lower than the rate of
aggregation of aggregating IL-1ra in PSE. The rate of aggregation
in PSE in FIG. 1 is represented by the slope of the line marked as
K.sub.agg. A similar line may be drawn for CSE in FIG. 1, and that
line would have a shallower slope, indicating a decreased rate of
aggregation in CSE relative to PSE.
[0098] In certain embodiments, citrate may be more effective at
reducing aggregation because it has a greater negative charge than
phosphate at pH 6.5. In certain embodiments, certain amounts of
negative charge may be more effective than others at reducing
aggregation. Furthermore, in certain embodiments, certain
configurations of negative charge, e.g., the distance between
negative charges on an accessory molecule and how "fixed" those
negative charges are in space, may also affect the effectiveness of
accessory molecules. Thus, in certain embodiments, the
effectiveness of various accessory molecules may be affected by the
amount of negative charge, but the amount of negative charge may
not always be determinative. In certain embodiments, a
multiple-charge anion may be more effective at reducing aggregation
than a single-charge anion. In various embodiments, one skilled in
the art can select accessory molecules and determine which is
appropriate for the specific application contemplated. For example,
certain accessory molecules may be more or less effective at
certain temperatures. In various embodiments, one skilled in the
art can select an accessory molecule that is effective at the
particular temperature at which the aggregating IL-1ra will be
incubated or stored. In certain embodiments, an accessory molecule
is selected that is effective at reducing aggregation between about
20.degree. C. and 45.degree. C. In certain embodiments, an
accessory molecule is selected that is effective at reducing
aggregation between about 25.degree. C. and 45.degree. C. In
certain embodiments, an accessory molecule is selected that is
effective at reducing aggregation between about 30.degree. C. and
45.degree. C. In certain embodiments, an accessory molecule is
selected that is effective at reducing aggregation between about
35.degree. C. and 45.degree. C.
[0099] In certain embodiments, altering the concentration of an
accessory molecule may affect the reduction in aggregation or the
reduction in the rate of aggregation of an aggregating IL-1ra. The
work discussed in Example 4 and shown in FIG. 3 considered the rate
of aggregation of an aggregating IL-1ra incubated in various
concentrations of phosphate, citrate, or pyrophosphate. In that
experiment, citrate and pyrophosphate showed a similar correlation
between a reduction in the rate of aggregation and the
concentration of the accessory molecule. Both citrate and
pyrophosphate showed an almost 90% reduction in the rate of
aggregation at 10 mM accessory molecule. That reduction increased
for both citrate and pyrophosphate at 20 mM, and then leveled off
up to over 100 mM. In contrast, phosphate showed only about a 20%
reduction in the rate of aggregation at 10 mM, and the reduction
didn't level off until about 80 mM. The results of the experiment
discussed in Example 4 suggest that pyrophosphate and citrate are
substantially equally effective at reducing the rate of aggregation
of an aggregating IL-1ra at 29.degree. C. Both citrate and
pyrophosphate have a greater negative charge than phosphate at pH
6.5, suggesting that, in certain embodiments, the amount of
negative charge may affect the efficiency of certain accessory
molecules.
[0100] In certain embodiments, a sugar may be an accessory
molecule. Exemplary sugars that may be accessory molecules include,
but are not limited to, sucrose, glycerol, mannitol, and sorbitol.
Example 5 describes an exemplary experiment in which increasing
concentrations of sucrose, glycerol, or sorbitol reduced the rate
of aggregation of an aggregating IL-1ra (see FIG. 4). At 3%, each
of the three sugars reduced the rate of aggregation to less than 5
aggregation units (a.u.; 1 a.u. is equal to an increase of 1
milli-optical density unit at 405 nm per minute using a volume of
200 .mu.l and a SpectroMax.TM. plate-reading spectrophotometer),
compared to a rate of aggregation of greater than 20 a.u. at 0%
sugar.
[0101] In various embodiments, one skilled in the art can determine
the appropriate concentration of a non-covalent accessory molecule
for a particular application by using, e.g., the methods of Example
4 or Example 5. In certain embodiments, an accessory molecule may
be present at a concentration of 1 to 100 mM. In certain
embodiments, an accessory molecule may be present at a
concentration of 1 to 50 mM. In certain embodiments, an accessory
molecule may be present at a concentration of 1 to 20 mM. In
certain embodiments, an accessory molecule may be present at a
concentration of 10 mM.
[0102] In certain embodiments, an accessory molecule may be present
at a concentration of between 0 and 10%. In certain embodiments, an
accessory molecule may be present at a concentration of between 0
and 5%. In certain embodiments, an accessory molecule may be
present at a concentration of 1%, 2%, or 3%.
[0103] In certain embodiments, the K.sub.d of a non-covalent
accessory molecule for IL-1ra is less than 10 mM. In certain
embodiments, the K.sub.d of a non-covalent accessory molecule for
IL-1ra is less than 7 mM. In certain embodiments, the K.sub.d of a
non-covalent accessory molecule for IL-1ra is less than 5 mM. In
certain embodiments, the K.sub.d of a non-covalent accessory
molecule for IL-1ra is less than 4 mM. In certain embodiments, the
K.sub.d of a non-covalent accessory molecule for IL-1ra is less
than 3 mM. In certain embodiments, the K.sub.d of a non-covalent
accessory molecule for IL-1ra is between 0.1 mM and 5 mM. In
certain embodiments, the K.sub.d of a non-covalent accessory
molecule for IL-1ra is between 1 mM and 5 mM. In certain
embodiments, the K.sub.d of a non-covalent accessory molecule for
IL-1ra is between 0.1 mM and 4 mM. In certain embodiments, the
K.sub.d of a non-covalent accessory molecule for IL-1ra is between
1 mM and 4 mM. In certain embodiments, the K.sub.d of a
non-covalent accessory molecule for IL-1ra is between 2 mM and 4
mM.
[0104] In certain embodiments, an accessory molecule may reduce
aggregation by covalently modifying an aggregating IL-1ra. In
certain embodiments, an accessory molecule may further reduce
aggregation by covalently modifying an IL-1ra having reduced
aggregation. In certain embodiments, the covalent modification
removes a positive charge at lysine-93, at arginine-97, or at both
lysine-93 and arginine-97. In certain embodiments, a covalent
accessory molecule may reduce aggregation by another mechanism,
e.g., by sterically inhibiting formation of one or more cation-pi
interactions or other interactions between aggregating IL-1ra
polypeptides.
[0105] Non-limiting exemplary accessory molecules that may
covalently modify an IL-1ra include, but are not limited to, NBD-X,
MAP, and citraconic anhydride. In certain embodiments, NBD-X forms
a derivative with primary amines. In the work discussed in Example
3 and shown in FIG. 2, incubation of IL-1ra wild-type protein with
NBD-X, SE resulted in derivatization of the amino-terminal amine
group of the polypeptide and of lysine-93. In certain embodiments,
derivatization with NBD-X may remove the positive charge at
lysine-93 and may result in reduced aggregation of derivatized IL-1
ra. In certain embodiments, MAP acetylates lysine residues and the
N-terminal amino groups of polypeptides. In the work discussed in
Example 7 and shown in FIGS. 13-15, incubation of IL-1ra with MAP
at various times of incubation, resulted in derivatization of the
N-terminal amino group of IL-1ra; derivatization of the N-terminal
amino group and lysine-6 of IL-1ra; derivatization of the
N-terminal amino group, lysine-6, and lysine-93 of IL-1ra; or
derivatization of the N-terminal amino group, lysine-6, lysine-93,
and lysine-96 of IL-1ra. In certain embodiments, derivatization
with MAP may remove the positive charge at lysine-93 and may result
in reduced aggregation of derivatized IL-1ra.
[0106] In certain embodiments, other amine-reactive molecules may
reduce aggregation of an aggregating IL-1ra by removing one or more
positive charges or through another mechanism. In certain
embodiments, IL-1 ra that has been derivatized with a covalent
accessory molecules is at least 90% as active as an IL-1ra
wild-type protein that has not been similarly derivatized. In
certain embodiments, IL-1ra that has been derivatized with a
covalent accessory molecules is at least 80% as active as an IL-1ra
wild-type protein that has not been similarly derivatized. In
certain embodiments, IL-1ra that has been derivatized with a
covalent accessory molecules is at least 75% as active as an IL-1ra
wild-type protein that has not been similarly derivatized. In
certain embodiments, IL-1ra that has been derivatized with a
covalent accessory molecules is at least 50% as active as an IL-1ra
wild-type protein that has not been similarly derivatized.
[0107] In certain embodiments, kits comprising IL-1ra and at least
one accessory molecule are provided. Kits may optionally include
instructions for combining the IL-1ra and the at least one
accessory molecule, if the components are provided separately. In
certain embodiments, kits include instructions for using the IL-1ra
and the at least one accessory molecule. In certain embodiments,
the kits may comprise at least one covalent accessory molecule
and/or at least one non-covalent accessory molecule. When the kit
comprises at least one covalent accessory molecule, the kit may
further comprise instructions for removing any remaining unreacted
accessory molecule from the derivatized IL-1ra following incubation
of IL-0.1ra with the covalent accessory molecule. In certain
embodiments, kits contain IL-1ra that has already been derivatized
with at least one covalent accessory molecule. Similarly, in
certain embodiments, a kit may contain an IL-1ra that is already in
a composition with at least one non-covalent accessory
molecule.
[0108] In certain embodiments, pharmaceutical compositions are
provided comprising a therapeutically effective amount of IL-1ra
together with a pharmaceutically acceptable diluent, carrier,
solubilizer, emulsifier, preservative and/or adjuvant.
[0109] In certain embodiments, acceptable formulation materials are
nontoxic to recipients at the dosages and concentrations
employed.
[0110] In certain embodiments, a pharmaceutical composition may
comprise formulation materials for modifying, maintaining and/or
preserving, for example, the pH, osmolarity, viscosity, clarity,
color, isotonicity, odor, sterility, stability, rate of dissolution
or release, adsorption, and/or penetration of the composition.
Exemplary formulation materials include, but are not limited to,
amino acids (such as glycine, glutamine, asparagine, arginine, and
lysine); antimicrobials; antioxidants (such as ascorbic acid,
sodium sulfite, and sodium hydrogen-sulfite); buffers (such as
borate, bicarbonate, Tris-HCl, citrates, phosphates, and other
organic acids); bulking agents (such as mannitol and glycine);
chelating agents (such as ethylenediamine tetraacetic acid (EDTA));
complexing agents (such as caffeine, polyvinylpyrrolidone,
beta-cyclodextrin, and hydroxypropyl-beta-cyclodextrin); fillers;
monosaccharides; disaccharides; and other carbohydrates (such as
glucose, mannose, and dextrins); proteins (such as serum albumin,
gelatin, and 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, and hydrogen peroxide); solvents (such as glycerin,
propylene glycol, and polyethylene glycol); sugar alcohols (such as
mannitol and sorbitol); suspending agents; surfactants or wetting
agents (such as pluronics, PEG, sorbitan esters, polysorbates such
as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin,
cholesterol, tyloxapal); stability enhancing agents (such as
sucrose and sorbitol); tonicity enhancing agents (such as alkali
metal halides, such as sodium and potassium chloride, mannitol,
sorbitol); delivery vehicles; diluents; excipients and
pharmaceutical adjuvants. (See, e.g., Remington's Pharmaceutical
Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company
(1990).
[0111] In certain embodiments, an IL-1ra and/or one or more
additional therapeutic agent are linked to a half-life extending
vehicle. Exemplary vehicles include, but are not limited to, the Fc
domain, polyethylene glycol, and dextran. Certain exemplary
vehicles are described, e.g., in U.S. application Ser. No.
09/428,082 and published PCT Application No. WO 99/25044.
[0112] In certain embodiments, an optimal pharmaceutical
composition will be determined by one skilled in the art depending
upon, for example, the intended route of administration, delivery
format, and desired dosage. See, for example, Remington's
Pharmaceutical Sciences, supra. In certain embodiments, such
compositions may influence the physical state, stability, rate of
in vivo release, and rate of in vivo clearance of the IL-1ra.
[0113] In certain embodiments, the primary vehicle or carrier in a
pharmaceutical composition may be either aqueous or non-aqueous in
nature. For example, in certain embodiments, a suitable vehicle or
carrier may be water for injection, physiological saline solution
or artificial cerebrospinal fluid, possibly supplemented with other
materials common in compositions for parenteral administration. In
certain embodiments, neutral buffered saline or saline mixed with
serum albumin are further exemplary vehicles. In certain
embodiments, pharmaceutical compositions comprise Tris buffer of
about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may
further include sorbitol or a suitable substitute therefor. In
certain embodiments, a composition comprising an IL-1ra, with or
without at least one accessory molecule and/or one or more
additional therapeutic agents, may be prepared for storage by
mixing the selected composition having the desired degree of purity
with optional formulation agents (Remington's Pharmaceutical
Sciences, supra) in the form of a lyophilized cake or an aqueous
solution. Further, in certain embodiments, a composition comprising
an IL-1ra, with or without at least one accessory molecule and/or
one or more additional therapeutic agents, may be formulated as a
lyophilizate using appropriate excipients such as sucrose.
[0114] In certain embodiments, the pharmaceutical compositions of
the invention can be selected for parenteral delivery. In certain
embodiments, the compositions may be selected for inhalation or for
delivery through the digestive tract, such as orally. The
preparation of such pharmaceutically acceptable compositions is
within the skill of the art.
[0115] In certain embodiments, when parenteral administration is
contemplated, a therapeutic composition may be in the form of a
pyrogen-free, parenterally acceptable aqueous solution comprising
the desired IL-1ra, with or without at least one accessory molecule
and/or one or more additional therapeutic agents, in a
pharmaceutically acceptable vehicle. In certain embodiments, a
vehicle for parenteral injection is sterile distilled water in
which the IL-1ra, with or without at least one accessory molecule
and/or one or more additional therapeutic agents, is formulated as
a sterile, isotonic solution, properly preserved. In certain
embodiments, the preparation can involve the formulation of the
desired molecule with an agent, such as injectable microspheres,
bio-erodible particles, polymeric compounds (such as polylactic
acid or polyglycolic acid), beads or liposomes, that may provide
for the controlled or sustained release of the product which may
then be delivered via a depot injection. In certain embodiments,
hyaluronic acid may also be used, and may have the effect of
promoting sustained duration in the circulation. In certain
embodiments, implantable drug delivery devices may be used to
introduce the desired molecule.
[0116] In certain embodiments, a pharmaceutical composition may be
formulated for inhalation. In certain embodiments, an IL-1ra, with
or without at least one accessory molecule and/or one or more
additional therapeutic agents, may be formulated as a dry powder
for inhalation. In certain embodiments, an inhalation solution
comprising an IL-1ra, with or without at least one accessory
molecule and/or one or more additional therapeutic agents, may be
formulated with a propellant for aerosol delivery. In certain
embodiments, solutions may be nebulized. Pulmonary administration
is further described in PCT application no. PCT/US94/001875, which
describes pulmonary delivery of chemically modified proteins.
[0117] In certain embodiments, it is contemplated that formulations
may be administered orally. In certain embodiments, an IL-1ra, with
or without at least one accessory molecule and/or one or more
additional therapeutic agents, that is administered in this fashion
may be formulated with or without those carriers customarily used
in the compounding of solid dosage forms such as tablets and
capsules. In certain embodiments, a capsule may be designed to
release the active portion of the formulation at the point in the
gastrointestinal tract when bioavailability is maximized and
pre-systemic degradation is minimized. In certain embodiments, at
least one additional agent can be included to facilitate absorption
of the IL-1ra and/or any accessory molecules and/or any additional
therapeutic agents. In certain embodiments, diluents, flavorings,
low melting point waxes, vegetable oils, lubricants, suspending
agents, tablet disintegrating agents, and binders may also be
employed.
[0118] In certain embodiments, a pharmaceutical composition may
involve an effective quantity of IL-1ra, with or without at least
one accessory molecule and/or one or more additional therapeutic
agents, in a mixture with non-toxic excipients which are suitable
for the manufacture of tablets. In certain embodiments, by
dissolving the tablets in sterile water, or another appropriate
vehicle, solutions may be prepared in unit-dose form. In certain
embodiments, suitable excipients include, but are not limited to,
inert diluents, such as calcium carbonate, sodium carbonate or
bicarbonate, lactose, or calcium phosphate; or binding agents, such
as starch, gelatin, or acacia; or lubricating agents such as
magnesium stearate, stearic acid, or talc.
[0119] Additional pharmaceutical compositions will be evident to
those skilled in the art, including formulations involving IL-1ra,
with or without at least one accessory molecule and/or one or more
additional therapeutic agents, in sustained- or controlled-delivery
formulations. In certain embodiments, techniques for formulating a
variety of other sustained- or controlled-delivery products, such
as liposome carriers, bio-erodible microparticles or porous beads
and depot injections, are also known to those skilled in the art.
See for example, PCT Application No. PCT/US93/00829 which describes
the controlled release of porous polymeric microparticles for the
delivery of pharmaceutical compositions. In certain embodiments,
sustained-release preparations may include semipermeable polymer
matrices in the form of shaped articles, e.g. films, or
microcapsules. Sustained release matrices may include polyesters,
hydrogels, polylactides (see, e.g., U.S. Pat. No. 3,773,919 and EP
058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate
(Sidman et al., Biopolymers, 22:547-556 (1983)), poly
(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater.
Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)),
ethylene vinyl acetate (Langer et al., supra) or
poly-D(-)-3-hydroxybutyric acid (EP 133,988). In certain
embodiments, sustained release compositions may include liposomes,
which can be prepared by any of several methods known in the art.
See e.g., Eppstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692
(1985); EP 036,676; EP 088,046 and EP 143,949.
[0120] The pharmaceutical composition to be used for in vivo
administration typically is sterile. In certain embodiments, this
may be accomplished by filtration through sterile filtration
membranes. In certain embodiments, where the composition is
lyophilized, sterilization using this method may be conducted
either prior to or following lyophilization and reconstitution. In
certain embodiments, the composition for parenteral administration
may be stored in lyophilized form or in a solution. In certain
embodiments, parenteral compositions generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0121] In certain embodiments, after the pharmaceutical composition
has been formulated, it may be stored in sterile vials as a
solution, suspension, gel, emulsion, solid, or as a dehydrated or
lyophilized powder. In certain embodiments, such formulations may
be stored either in a ready-to-use form or in a form (e.g.,
lyophilized) that is reconstituted prior to administration.
[0122] In certain embodiments, kits are provided for producing a
single-dose administration unit. In certain embodiments, the kits
may each contain both a first container having a dried protein and
a second container having an aqueous formulation. In certain
embodiments, kits containing single and multi-chambered pre-filled
syringes (e.g., liquid syringes and lyosyringes) are included.
[0123] In certain embodiments, the effective amount of a
pharmaceutical composition comprising an IL-1ra, with or without at
least one accessory molecule and/or one or more additional
therapeutic agents, to be employed therapeutically will depend, for
example, upon the therapeutic context and objectives. One skilled
in the art will appreciate that the appropriate dosage levels for
treatment, according to certain embodiments, will thus vary
depending, in part, upon the molecule delivered, the indication for
which the IL-1ra, with or without at least one accessory molecule
and/or one or more additional therapeutic agents, 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. In certain embodiments, the clinician may
titer the dosage and modify the route of administration to obtain
the optimal therapeutic effect. In certain embodiments, a typical
dosage may range from about 0.1 .mu.g/kg to up to about 100 mg/kg
or more, depending on the factors mentioned above. In certain
embodiments, the dosage may range from 0.1 .mu.g/kg up to about 100
mg/kg; or 1 .mu.g/kg up to about 100 mg/kg; or 5 .mu.g/kg up to
about 100 mg/kg.
[0124] In certain embodiments, the frequency of dosing will take
into account the pharmacokinetic parameters of the IL-1ra and/or
any accessory molecules and/or any additional therapeutic agents in
the formulation used. In certain embodiments, a clinician will
administer the composition until a dosage is reached that achieves
the desired effect. In certain embodiments, 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. In certain embodiments, appropriate dosages may be
ascertained through use of appropriate dose-response data.
[0125] In certain embodiments, 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.
[0126] In certain embodiments, the composition may be administered
locally via implantation of a membrane, sponge or another
appropriate material onto which the desired molecule has been
absorbed or encapsulated. In certain embodiments, where an
implantation device is used, the device may be implanted into any
suitable tissue or organ, and delivery of the desired molecule may
be via diffusion, timed-release bolus, or continuous
administration.
[0127] In certain embodiments, it may be desirable to use a
pharmaceutical composition comprising an IL-1ra, with or without at
least one accessory molecule and/or one or more additional
therapeutic agents, in an ex vivo manner. In such instances, cells,
tissues and/or organs that have been removed from the patient are
exposed to a pharmaceutical composition comprising an IL-1ra, with
or without at least one accessory molecule and/or one or more
additional therapeutic agents, after which the cells, tissues
and/or organs are subsequently implanted back into the patient.
[0128] In certain embodiments, an IL-1ra having reduced aggregation
and/or any accessory molecules and/or any additional therapeutic
agents can be delivered by implanting certain cells that have been
genetically engineered, using methods known in the art, to express
and secrete the polypeptides. In certain embodiments, such cells
may be animal or human cells, and may be autologous, heterologous,
or xenogeneic. In certain embodiments, the cells may be
immortalized. In certain embodiments, in order to decrease the
chance of an immunological response, the cells may be encapsulated
to avoid infiltration of surrounding tissues. In certain
embodiments, the encapsulation materials are typically
biocompatible, semi-permeable polymeric enclosures or membranes
that allow the release of the protein product(s) but prevent the
destruction of the cells by the patient's immune system or by other
detrimental factors from the surrounding tissues.
EXAMPLES
[0129] The following examples, including the experiments conducted
and results achieved are provided for illustrative purposes only
and are not to be construed as limiting the present invention.
Example 1
Production of IL-1ra Wild-Type Protein
[0130] Human recombinant interleukin-1 receptor antagonist (IL-1ra)
having the sequence of SEQ ID NO: 5, can be prepared at Amgen
manufacturing facilities according to the method discussed in
European Patent No. EP 0 502 956 B1, in which the cell recovery
step is optional. Alternatively, anakinra, which has the amino acid
sequence of SEQ ID NO: 5, may be obtained from Amgen Inc., Thousand
Oaks, Calif. Purified human IL-1 ra having the amino acid sequence
of SEQ ID NO: 5 was used in the examples described herein.
Example 2
IL-1ra Aggregation
[0131] IL-1ra aggregation in phosphate buffer and citrate buffer at
39.degree. C. was determined as follows. Ten ml of an IL-1ra stock
solution (200-220 mg/ml protein in 10 mM sodium citrate, 140 mM
NaCl, 0.5 mM EDTA, pH 6.5 (CSE)) was dialyzed overnight at
4.degree. C. against either 2.times.2L of 10 mM phosphate, 140 mM
NaCl, 0.5 mM EDTA, pH 6.5 (PSE) or 2.times.2L of CSE. The dialyzed
solution was filtered through a 0.2 .mu.m filter and the IL-1ra
protein concentration was adjusted to 140 mg/ml by diluting with
the appropriate buffer.
[0132] Aggregation of IL-1ra in each buffer was measured using a
96-well glass plate (Zissner) and a temperature-controlled plate
reading spectrophotometer, SpectraMax Plus (Molecular Devices). The
sample size per well was 180 .mu.l.mu.l. The plates were incubated
in the spectrophotometer at 39.degree. C. and the optical density
measured at 405 nm every 1 minute. FIG. 1 shows the optical density
at 405 nm plotted as a function of time for this experiment. The
rate of aggregation was determined as the slope of the initial
linear region of the saturation curve using the SoftMax Pro program
(Molecular Devices). The rate of aggregation of IL-1ra incubated in
CSE is reduced relative to the rate of aggregation of IL-1ra
incubated in PSE. Furthermore, the extent of aggregation of IL-1ra
incubated in CSE is reduced relative to the extent of aggregation
of IL-1ra incubated in PSE.
Example 3
NBD-X Labeling of IL-1ra
[0133] Surface-exposed amino groups were identified by labeling
IL-1ra with NBD-X, SE (succinimidyl
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4yl)amino)hexanoate, Molecular
Probes). NBD-X becomes fluorescent upon derivitization with amino
groups. Twenty .mu.l.mu.l of 0.2 mM IL-1ra in PSE was diluted with
480 .mu.l.mu.l PSE. Eight .mu.l of 50 .mu.g/.mu.l.mu.l NBD-X, SE
solution in dimethyl formamide was added to the IL-1ra solution in
2 .mu.l.mu.l increments at room temperature. The reaction was
incubated for one hour at room temperature, then stopped by the
addition of 50 .mu.l.mu.l of a 1.5 M hydroxylamine solution, pH
8.5. The reaction mixture was passed through a desalting gel
filtration column that was pre-equilibrated with PSE. The protein
peak was collected and further analyzed by RP-HPLC and LC-MS/MS as
follows.
[0134] Reverse-phase HPLC was conducted using a Phenomenex
Jupiter.TM. 5.mu., C4 column (300 .ANG., 250.times.4.6 mm) and an
HP1100 HPLC system equipped with on-line absorbance and
fluorescence detectors. The absorbance was measured at 215 nm to
detect the presence of the polypeptide. Fluorescent emission was
measured at 535 nm following excitation at 480 nm to detect the
presence of the amine-derivatized NBD-X. The elution was performed
with a linear 30-45% gradient of methanol in 0.1% trifluoroacetic
acid (TFA) and water (v/v/v). FIG. 2 shows the absorbance of NBD-X
labeled IL-1ra at 215 nm and the fluorescent emission of NBD-X
labeled IL-1ra at 535 nm following excitation at 480 nm for this
experiment. The identity of the fluorescing peaks was determined as
follows.
[0135] Two major fluorescing peaks were identified, an
early-eluting peak at 16.75 minutes and a later-eluting peak at
17.5 minutes. See FIG. 2, lower panel. The peaks were collected
manually and digested with Lys-C endoproteinase as follows. Each
peak was collected from five separate HPLC runs. All of the
fractions primarily containing the 16.75 peak were pooled together.
All of the fractions primarily containing the 17.5 minute peak were
pooled together. The two pooled samples were then dried with
centrifugation using a SpeedVac concentrator. Each of the pooled
samples was then dissolved in 490 .mu.l.mu.l of digestion buffer,
which contained 10 mM Tris, 0.8 M guinidinium hydrochloride, pH
8.0. Ten .mu.l of a Lys-C proteinase solution, prepared by
dissolving 5 .mu.g Lys-C proteinase (sequencing grade, Roche
Diagnostics) in 50 .mu.l digestion buffer, was then added to each
pooled sample. The samples were digested overnight at 37.degree. C.
Following digestion, the peptides generated were subjected to
LC-MS/MS using a Finnegan LCQ Deca equipped with a Beckman System
Gold HPLC according to the standard manufacturer's protocol.
[0136] For the early-eluting peak (16.75 min), digestion yielded a
unique peptide of mass 1107.8 m/z, corresponding to an
amino-terminal peptide of IL-1ra with an added mass of 276 amu,
which suggests the presence of an NBD derivative. MS/MS analysis of
the peptide produced fragments consistent with the amino acid
sequence MRPSGRK plus an extra mass of 276 amu on the methionine
residue, suggesting that the amino-terminal methionine of IL-1ra
was derivatized with NBD-X.
[0137] Digestion of the later-eluting peak (17.5 min) yielded a
unique peptide of mass 1145.9 m/z, which suggested an IL-1ra
peptide corresponding to residues 72-96 comprising an NBD
derivative. MS/MS analysis of the peptide produced fragments
consistent with the amino acid sequence corresponding to residues
72-96 with an NBD-derivatized lysine at position 93.
[0138] According to the foregoing results, the lysine at position
93 of IL-1 ra is capable of derivatization with NBD-X under the
conditions used in this experiment. These results also suggest that
the lysine at position 93 is exposed and is a solvent-accessible
residue in the intact IL-1ra protein.
Example 4
Rate of Aggregation of IL-1ra in the Presence of Phosphate,
citrate, or Pyrophosphate
[0139] The rate of aggregation of IL-1ra was determined in several
different buffer compositions. Ten ml of an IL-1ra stock solution
(220 mg/ml in CSE) was dialyzed against 2.times.4L of 140 mM NaCl
using Pierce Snakeskin dialysis tubing (3.5 kDa cut-off) at
4.degree. C. Aliquots of the dialyzed IL-1ra solution were brought
to various concentrations of citrate, phosphate, or pyrophosphate
by addition of 0.45M citrate, 0.5M phosphate, or 0.45M
pyrophosphate stock solutions (all pH 6.5) and an appropriate
volume of 140 mM NaCl to result in a final concentration of IL-1ra
in each sample of 140 mg/ml. The final concentration of citrate,
phosphate, or pyrophosphate in each sample ranged from 1 to 125
mM.
[0140] Aggregation of IL-1ra in each sample was monitored for 4
hours at 29.degree. C. by measuring the light scattering at 405 nm
at one minute intervals as described in Example 2 above. The rate
of aggregation was determined as the slope of the linear region of
the saturation curve calculated using the SoftMax Pro program
(Molecular Devices). FIG. 3 shows that in this experiment, the rate
of aggregation of IL-1ra decreased with increasing concentrations
of citrate, phosphate, or pyrophosphate. Notably, the rate of
aggregation of IL-1ra in citrate or pyrophosphate buffer decreased
more rapidly than the rate of aggregation in phosphate buffer.
[0141] The estimated pKa's of the three ionizable hydrogens on
phosphate are 2.148, 7.198, and 12.35 at pH 6.5 and 39.degree. C.
The estimated pKa's of the three ionizable hydrogens on citrate are
3.128, 4.761, and 6.396 at pH 6.5 and 39.degree. C. The estimated
pKa's of the four ionizable hydrogens on pyrophosphate are 0.83,
2.26, 6.72, and 9.46 at pH 6.5 and 39.degree. C. See, e.g.,
Goldberg et al., Thermodynamic quantities for the ionization
reactions of buffers. J. Phys. Chem. Ref. Data, 31(2): 231-370
(2002). Accordingly, phosphate is predicted to have predominantly
one negative charge at pH 6.5 and 39.degree. C., while citrate is
predicted to have three negative charges at pH 6.5 and 39.degree.
C. and pyrophosphate is predicted to have between two and three
negative charges at pH 6.5 and 39.degree. C., as estimated
according to the literature-reported pKas discussed above.
[0142] Thus, these results suggest that multiple-charge anions may
reduce the rate of aggregation of IL-1ra more effectively than
single-charge anions at a particular pH. Consistent with that
conclusion, NaCl, which is a single-charge anion, was found to have
a similar aggregation rate profile as phosphate (data not
shown).
Example 5
Rate of Aggregation of IL-1 ra in the Presence of Sucrose,
Glycerol, or Sorbitol
[0143] The rate of aggregation of IL-1ra was determined in the
presence of several different sugars. Ten ml of an IL-1ra stock
solution (220 mg/ml in CSE) was dialyzed against 2.times.4 L of 140
mM NaCl using Pierce. Snakeskin dialysis tubing (3.5 kDa cut-off)
at 4.degree. C. Aliquots of the dialyzed IL-1 ra solution were
brought to various percent concentrations of sucrose, glycerol, or
sorbitol by addition of 25% sucrose, 25% glycerol, or 25% sorbitol
stock solutions and an appropriate volume of 140 mM NaCl to result
in a final concentration of IL-1ra in each sample of 140 mg/ml. The
final concentration of sucrose, glycerol, or sorbitol in each
sample was 0%, 1%, 2%, or 3%. The rate of aggregation was
determined by measuring the optical density of each IL-1ra solution
at 39.degree. C. using the method described in Example 2. FIG. 4
shows that in this experiment, increasing concentrations of sugar
resulted in decreasing rates of aggregation of IL-1ra. At 0% sugar,
the rate of aggregation of IL-1ra in this experiment was about 22
aggregation units (a.u., measured as the increase in milli-optical
density at 405 nm per minute). At 3% sucrose, glycerol, or
sorbitol, the rate of aggregation of IL-1ra was reduced to between
0 and 5 a.u.
Example 6
Urea- and Guanidinium Hydrochloride-Induced Unfolding of IL-1ra
[0144] Equilibrium urea-induced unfolding of IL-1ra was performed
as follows. For far-UV circular dichroism studies, 1-1.5 ml samples
of 0.86 mg/ml IL-1ra and various concentrations of urea (between
about 0 and 10M) were wrapped in foil to protect them from light
and incubated overnight (>12 hours) at room temperature. The
circular dichroism at 230 nm was then determined for each sample at
23.degree. C. FIG. 9 shows the circular dichroism signal at 230 nm
for IL-1ra incubated in various concentrations of urea for this
experiment. The data were plotted and the unfolding curve was
fitted to a 3-state model, native state <-> intermediate
state <-> unfolded state. In this model, ml, which is the
slope of the first transition, and C.sub.m1, which is the midpoint
of the first transition were constrained. Those parameters were
constrained because far-UV CD cannot distinguish between the native
and the intermediate states. The constrained values for m1 and
C.sub.m1 were obtained by averaging the values from several
fluorescence measurements, which were able to distinguish the
native state from the intermediate state. As shown in FIG. 9,
IL-1ra underwent a global unfolding transition between about 4 and
6 M urea in this experiment.
[0145] For fluorescence studies, 1-1.5 ml samples of 0.086 mg/ml
IL-1ra and various concentrations of urea (between about 0 and 10M)
were wrapped in foil to protect them from light and incubated
overnight (>12 hours) at room temperature. FIG. 10 shows the
intrinsic fluorescence at 353 nm of IL-1ra incubated in various
concentrations of urea. The data were plotted and the unfolding
curve was fitted to the 3-state model discussed above. No
constraints were applied in this experiment. As shown in FIG. 10,
the fluorescence experiment suggests the presence of an unfolding
transition between 0 and about 1.5 M urea. Since that transition is
not observed in the far-UV CD experiment, it may reflect
accumulation of an intermediate state with a native-like secondary
structure. That intermediate state may involve more local changes
to the structure, e.g., destabilization of surface loops, while the
hydrophobic core of IL-1ra may remain substantially
unperturbed.
[0146] Equilibrium guanidinium hydrochloride (GuHCl)-induced
unfolding was performed as follows. For far-UV circular dichroism
studies, 1-1.5 ml samples of 0.86 mg/ml IL-1ra and various
concentrations of GuHCl (between about 0 and 5M) were wrapped in
foil to protect them from light and incubated overnight (>12
hours) at room temperature. The circular dichroism at 230 nm was
then determined for each sample at 23.degree. C. FIG. 11 shows the
circular dichroism signal at 230 nm for IL-1ra incubated in various
concentrations of GuHCl. The data were plotted and the unfolding
curve was fitted to the 3-state model, native state <->
intermediate state <-> unfolded state. In this model, m1,
C.sub.m1, "ns", which is the slope of the native state baseline,
and "is", which is the slope of the intermediate state baseline,
were constrained. The constrained values of m1 and C.sub.m1 for
GuHCl-induced unfolding were determined substantially as described
above for urea-induced unfolding. "Ns" and "is" were constrained to
be greater than zero. Those parameters were constrained because
far-UV CD cannot distinguish between the native and the
intermediate states. As shown in FIG. 11, IL-1ra underwent a global
unfolding transition between about 1 and 2 M GuHCl in this
experiment.
[0147] For fluorescence studies, 1-1.5 ml samples of 0.086 mg/ml
IL-1 ra and various concentrations of GuHCl (between about 0 and
5M) were wrapped in foil to protect them from light and incubated
overnight (>12 hours) at room temperature. FIG. 12 shows the
intrinsic fluorescence at 353 nm of IL-1ra incubated in various
concentrations of urea. The data were plotted and the unfolding
curve was fitted to the 3-state model discussed above. No
constraints were applied in this experiment. FIG. 12 shows the
intrinsic fluorescence at 353 nm of IL-1ra incubated in various
concentrations of GuHCl. The data were plotted and the unfolding
curve was fitted to the 3-state model discussed above. No
constraints were applied in this experiment. As shown in FIG. 12,
the fluorescence data suggests the presence of the an unfolding
transition between about 0 and 0.6 M GuHCl. Since that transition
is not observed in the far-UV CD experiment, it may reflect
accumulation of an intermediate state with a native-like secondary
structure. That intermediate state may involve more local changes
to the structure and/or involve destabilization of surface loops,
while the hydrophobic core of IL-1ra may remain substantially
unperturbed.
[0148] The difference between the fluorescence data for
urea-induced unfolding and GuHCl-induced unfolding may result from
differential effects of the two denaturants on the intrinsic
fluorescence of IL-1ra.
[0149] Furthermore, the difference between the CD data and the
fluorescence data for each denaturant may be due to the ability of
tryptophan intrinsic fluorescence to detect local changes in
protein tertiary structure, while CD detects changes in protein
secondary structure.
Example 7
Derivatization of IL-1ra with Methyl Acetyl Phosphate (MAP)
[0150] In certain embodiments, methyl acetyl phosphate (MAP)
acetylates lysine residues and the N-terminal amines of
polypeptides. MAP was prepared as described in Kluger et al. (1980)
J. Org. Chem., 45: 2723. The identity of the product was confirmed
by NMR and mass spectrometry.
[0151] IL-1ra was derivatized with MAP in the presence of citrate
or phosphate as follows. For the derivitization in the presence of
citrate, 20 .mu.l of MAP (10 mM stock in water) and 20 .mu.l IL-1ra
(10 mM stock in CSE) were added to 160 .mu.l of 10 mM citrate, pH
6.5 in an HPLC vial. The reaction was incubated at 37.degree. C.
and 25 .mu.l aliquots were taken using an HPLC autosampler at 0,
45, 90, 135, 180, 225, and 270 minutes. Each aliquot was analyzed
by reverse-phase HPLC using an Agilent 110 HPLC equipped with a
Jupiter 5u C4 300A reverse-phase HPLC column (4.6.times.250 mm,
Phenomenex, Torrance, Calif.), an on-line UV detector and a
temperature-controlled 100-well sample tray set at 37.degree. C.
The flow rate was set at 1 ml/min and the column was maintained at
a temperature of 50.degree. C. The column was pre-equilibrated for
15 min (flow rate 1 ml/min) with 65% buffer A (0.1% trifluoroacetic
acid (TFA) in water) and 35% buffer B (90% acetonitrile, 0.1% TFA
in water). After loading, the analytes were eluted using a gradient
ramping from 35% to 50% buffer B (90% acetonitrile, 0.1% TFA in
water) over 25 minutes.
[0152] For derivatization in the presence of phosphate, 20 .mu.l of
MAP (10 mM stock in water) and 20 .mu.l IL-1ra (10 mM stock in CSE)
were added to 160 .mu.l of 10 mM phosphate, pH 6.5 in an HPLC vial.
The reaction was incubated at 37.degree. C. and 25 .mu.l aliquots
were taken using an HPLC autosampler at 0, 45, 90, 135, 180, 225,
and 270 minutes. Each aliquot was analyzed by reverse-phase HPLC as
described above for derivatization in the presence of citrate.
[0153] The results of the derivatization in the presence of citrate
are shown in FIG. 13. The results of the derivatization in the
presence of phosphate are shown in FIG. 14. Both HPLC profiles
reveal four new, weakly-resolved IL-1ra peaks resulting from
derivatization with MAP in this experiment.
[0154] IL-1ra was also derivatized with MAP in the presence of 10
mM pyrophosphate, pH 6.4. Twenty .mu.l of MAP (10 mM stock in
water) and 20 .mu.l IL-1ra (10 mM stock in CSE) were added to 160
.mu.l of 10 mM pyrophosphate, pH 6.4 in an HPLC vial. The reaction
was incubated at 37.degree. C. and 25 .mu.l aliquots were taken
using an HPLC autosampler at 0, 45, 90, 135, 180, 225, and 270
minutes. Each aliquot was analyzed by reverse-phase HPLC as
described above for derivatization in the presence of citrate. FIG.
15 shows an overlay of the results of reverse-phase HPLC of IL-1ra
derivatization with MAP for 4.5 hours (270 minutes) in the presence
of citrate, in the presence of phosphate, and in the presence of
pyrophosphate. Four weakly resolved peaks were identified in this
experiment and are indicated on FIG. 15.
[0155] Each of the peaks from the 270 minute time point for
derivitazation in the presence of citrate, shown in FIG. 15, was
further analyzed in order to determine the identity of each, as
follows. The four major peaks were collected from the HPLC run
discussed above. The samples were concentrated on a SpeedVac to
5-10 .mu.l final volume. Ninety .mu.l of digest buffer (50 mM tris,
0.8 M guanidinium-HCl, pH 8.0) was then added to each sample,
followed by 10 .mu.l of endoproteinase lys-C (1 .mu.g in 10 .mu.l
digest buffer). The samples were incubated at 37.degree. C. for 16
hours. The resulting peptides were analyzed by LC-MS/MS using an
Agilent 110 HPLC equipped with an on-line MS detection with a
Finnegan ion trap. The HPLC column used was a Jupiter 5u C18 100A
reverse-phase HPLC column (2.times.150 mm, Phenomenex, Torrance,
Calif.). The flow rate was set at 0.2 ml per minute and the column
was maintained at a temperature of 50.degree. C. The column was
pre-equilibrated for 22 minutes with 98% buffer A ((0.1% TFA in
water) and 2% buffer B (90% acetonitrile, 0.1% TFA in water). After
loading, analytes were eluted from the HPLC column using a gradient
ramping from 2% to 45% buffer B over 50 minutes. Ten percent of the
eluent from the HPLC column was subjected to to MS analysis. The
presence of an additional acetyl group on a peptide resulted in an
increase of 42 m/z on the MS profile relative to unmodified
peptide. The identity of the detected peptides and the specific
amino acid residues that were modified was verified by MS/MS.
[0156] The IL-1ra polypeptide of peak 1 appeared to be derivatized
at its N-terminal amine only. The IL-1ra polypeptide of peak 2
appeared to be derivatized at its N-terminal amine and at lysine-6.
The IL-1ra polypeptide of peak 3 appeared to be derivatized at its
N-terminal amine, at lysine-6, and at lysine-93. Finally, the
IL-1ra polypeptide of peak 4 appeared to be derivatized at its
N-terminal amine, at lysine-6, at lysine-93, and at lysine-96.
[0157] The level of derivatization at all positions except for the
N-terminal amine appeared to be greater in the presence of
phosphate than in the presence of citrate or pyrophosphate. These
results suggest that citrate and/or pyrophosphate may protect the
positively-charged lysine groups in IL-1 ra from derivatization
with MAP better than phosphate. These results may suggest that
citrate and/or pyrophosphate has a stronger affinity for the
positively-charged lysine residues than does phosphate.
[0158] The rate of derivatization of IL-1ra with MAP was determined
using the data shown in FIG. 13 (and reproduced in FIG. 16A) for
IL-1ra derivatization in the presence of citrate. In particular,
each chromatogram from FIG. 16A was deconvoluted using PeakFit.TM.
(Systat Software Inc.). The area under the each of the deconvoluted
peaks (peaks 1-4) at each time point (0, 45, 90, 135, 180, 225, and
270 minutes) was then integrated. An exemplary deconvoluted profile
for one time point is shown in FIG. 16B. Deconvoluted peaks 1-4 are
labeled on FIG. 16B and the integrated area under each one is shown
below. Peak 1 had an integrated area of 160.28590. Peak 2 had an
integrated area of 190.41964. Peak 3 had an integrated area of
181.76548. Peak 4 had an integrated area of 178.18844. The
integrated area of deconvoluted peak 4 at time points 0, 45, 90,
135, 180 minutes was then plotted against the incubation time of
the reaction that produced that peak. That plot is shown in FIG.
16C. The resulting line has a slope of 68.931 hour.sup.-1.
[0159] The rate of derivatization of IL-1ra with MAP was then
calculated for various experiments in the presence of various
buffers and at various pHs, according to the methods discussed
above. The various rates were then plotted on a bar chart, shown in
FIG. 17. Specifically, the rate of derivatization of IL-1ra with
MAP was determined in the presence of 10 mM phosphate, pH 6.3; 10
mM citrate, pH 6.3; 10 mM pyrophosphate, pH 6.3; 10 mM phosphate,
pH 6.5; 20 mM phosphate, pH 6.5; 10 mM citrate, pH 6.5; 20 mM
citrate, pH 6.5; 10 mM pyrophosphate, pH 6.5; 20 mM pyrophosphate,
pH 6.5; 10 mM phosphate, pH 6.7; 10 mM citrate, pH 6.7; and 10 mM
pyrophosphate, pH 6.7.
[0160] At each pH, the rate of derivatization of IL-1ra with MAP
was slower in citrate or pyrophosphate buffer than it was in
phosphate buffer. See FIG. 17. Moreover, the rate of derivatization
in the presence of 20 mM citrate or pyrophosphate was slower than
the rate of derivatization in the presence of 10 mM citrate or
pyrophosphate. These results suggest that citrate and/or
pyrophosphate may protect the lysine residues of IL-1ra from
derivatization with MAP more effectively than does phosphate. Thus,
the rate of derivatization of IL-1ra with MAP is slower in the
presence of citrate buffer or pyrophosphate buffer than it is in
the presence of phosphate buffer.
[0161] Finally, IL-1ra was also derivatized with MAP in the
presence of 10 mM citrate at pH 6.3, pH 6.5, or pH 6.7; or in the
presence of 10 mM phosphate, pH 6.3, pH 6.5, or pH 6.7, for 5.5
hours, according to the methods discussed above. FIG. 18 shows an
overlay of the results of reverse-phase HPLC of IL-1ra
derivatization with MAP for 5.5 hours in the presence of each of
those buffers. That figure shows that IL-1ra is derivatized to a
greater extent in the presence of phosphate than in the presence of
citrate at all pH levels tested. Those results suggest that citrate
may protect the lysine residues of IL-1ra from derivatization with
MAP more effectively than does phosphate.
[0162] The data shown in FIG. 18 also suggest that IL-1ra is
derivatized to a greater extent as the pH is increased.
Example 8
IL-1ra Aggregation in the Presence of Various Concentrations of
Phosphate
[0163] Ten ml of IL-1ra stock (220 mg/ml in CSE) was dialyzed at
4.degree. C. for 48 hours against a total of 8 L (2 L with three
buffer changes of 2 L each) 10 mM phosphate, 100 mM NaCl, pH 6.5.
The IL-1ra concentration was adjusted to 167 mg/ml with 10 mM
phosphate, 100 mM NaCl, pH 6.5. A 1 M stock of phosphate was
prepared by dissolving an appropriate amount of sodium phosphate
into the 10 mM phosphate, pH 6.5, 100 mM NaCl. Twenty mM, 60 mM,
110 mM, 210 mM, 310 mM, 410 mM, 510 mM, 610 mM, 710 mM, 810 mM, 910
mM, and 1 M working stock solutions of phosphate, pH 6.5 were made
from the 1 M stock by diluting with 10 mM phosphate, pH 6.5, 100 mM
NaCl.
[0164] Aggregation of IL-1ra in various concentrations of phosphate
was measured using a 96-well glass plate (Zissner) and a
temperature-controlled plate reading spectrophotometer, SpectraMax
Plus (Molecular Devices). One hundred and eighty microliters of the
167 mg/ml ll-1ra stock solution was added to each well. Twenty
.mu.l of a phosphate working stock solution was then added to each
well (one working stock per well). The final concentration of
phosphate in the wells was 0 mM, 2 mM, 6 mM, 11 mM, 21 mM, 31 mM,
41 mM, 51 mM, 61 mM, 71 mM, 81 mM, 91 mM, 100 mM. The plate was
incubated in the spectrophotometer at 39.degree. C. and the optical
density was measured at 405 nm every 1 minute. FIG. 19 shows the
optical density at 405 nm plotted as a function of time for each
concentration of phosphate for this experiment. The data in FIG. 19
suggests that the extent of aggregation of IL-1ra decreases with
increasing concentration of phosphate.
Example 9
Measurement of Citrate Binding to IL-1ra
[0165] The number of citrate ion binding sites and the K.sub.d of
citrate binding to IL-1ra were determined as follows. Solutions of
1 ml of 1 mM IL-1 ra were prepared in buffers having various
concentrations of citrate, pH 6.5 by diluting IL-1ra stock (220
mg/ml in CSE) in appropriate buffers. Specifically, 1 ml of 1 mM
solutions of IL-1ra in each of 5 mM, 10 mM, and 20 mM citrate, pH
6.5 were prepared. Each solution also contained 70 mM NaCl. Four
hundred microliters of each solution was placed in a
Microsep.TM.-10 spin column (Pall Corporation; one solution per
column). The columns were spun at 18.degree. C. for 35 minutes at
4000.times.g. After spinning, the IL-1ra remains in the retentate,
bound to some of the citrate, while some of the buffer containing
unbound citrate passes through the filter as filtrate. After
spinning, about half of the solution had passed through the filter
as filtrate.
[0166] The amount of citrate in each of the retentate and filtrate
was determined as follows. Twenty .mu.l of filtrate or retentate
was loaded onto a reverse phase HPLC column (Supelcosil.TM. LC-18
column, 15 cm.times.4.6 mm, Sigma-Aldrich cat. no. 58985). The flow
rate was 1 ml per minute with 0.1 M phosphoric acid as running
buffer (isocratic elution). The elution was carried out at room
temperature. The amount of citric acid eluted from the column was
measured at 215 nm. The concentration of citrate in the sample
being assayed (the retentate or filtrate) was then calculated from
that measurement (the system was calibrated with 0-10 mM citrate
solution standards, pH 6.5).
[0167] FIG. 20 shows a plot of the concentration of citrate bound
to IL-1ra (determined as the concentration of citrate in the
retentate minus the concentration of citrate in the filtrate)
versus the concentration of total citrate in the solution. The
maximum concentration of citrate bound, B.sub.max, was extrapolated
from that data to be 1.8218 mM. The data from FIG. 20 was used to
create a Scatchard plot in order to determine the K.sub.d for
citrate binding to IL-1ra. The K.sub.d was calculated as 3.846 mM
for this experiment (data not shown). Furthermore, the number of
binding sites for citrate on IL-1ra was determined from the
x-intercept on the Scatchard plot. The number of binding sites was
0.94 for this experiment (data not shown). These data suggest that
there is one citrate binding site in IL-1ra.
Example 10
Competition for the IL-1ra Anion Binding Site
[0168] Competition for the citrate binding site on IL-1ra by
pyrophosphate was determined as follows. Four hundred microliters
of a solution containing 1 mM IL-1ra, 10 mM citrate, pH 6.5, 70 mM
NaCl, and a test concentration of pyrophosphate was placed in a
Microsep.TM.-10 spin columns (Pall Corporation) and spun at
18.degree. C. for 35 minutes at 4000.times.g. The concentrations of
pyrophosphate tested were 0 mM, 5 mM, 10 mM, and 20 mM. After
spinning, about half of the solution passed through the filter as
filtrate, while the remaining solution remained above the filter as
retentate. The IL-1ra remains in the retentate, bound to some of
the citrate and/or pyrophosphate, while some of the buffer
containing unbound citrate and pyrophosphate will pass through the
filter as filtrate. The concentration of citrate in both the
retentate and the filtrate was determined as discussed above in
Example 9. Competition for the citrate binding site on IL-1ra by
phosphate was determined as described above for pyrophosphate.
[0169] FIG. 21 shows a plot of the concentration of citrate bound
by IL-1ra versus the ratio of the total concentration of competing
anion (pyrophosphate or phosphate) to the total concentration of
citrate in the solution. The data suggest that pyrophosphate is
more effective at competing with citrate for the citrate binding
site on IL-1ra than is phosphate. Because both pyrophosphate and
phosphate appear to be capable of competing for the IL-1ra binding
site (i.e., the site is not specific for citrate), we refer to the
citrate binding on IL-1ra site as the anion binding site.
[0170] From the data shown in FIG. 21, the K.sub.d for
pyrophosphate binding to the anion binding site on IL-1ra was
determined using the following calculations. See, e.g., Stinson and
Holbrook, Biochem. J. (1973) 131: 719-728. The competition between
citrate and pyrophosphate binding can be expressed as two separate
equilibria: ##STR1## Where P=protein, which is IL-1ra in this case;
L=ligand, which is citrate in this case; and X=competing anion,
which is pyrophosphate in this case. K.sup.d.sub.L is the K.sub.d
for ligand binding to protein and is equal to the concentration of
P (free protein) times the concentration of L (free ligand),
divided by the concentration of PL (protein bound to ligand).
K.sup.d.sub.X is the K.sub.d for competitive anion binding to
protein and is equal to the concentration of P (free protein) times
the concentration of X (free competing anion), divided by
concentration of PX (protein bound to competing anion).
[0171] The apparent, or observed K.sub.d of citrate
(K.sup.d.sub.Lapp) can be expressed as follows: K L d .times. app =
( L free ) .function. [ ( P free ) + ( PX ) ] ( PL ) ##EQU1## Where
L.sub.free is the concentration of free ligand (citrate),
P.sub.free is the concentration of free protein (IL-1ra), PX is the
concentration of protein bound to competitive anion
(pyrophosphate), and PL is the concentration of protein bound to
ligand. By replacing the (PX) term with
(X.sub.free)(P.sub.free)/K.sup.d.sub.X, which is derived from the
equilibrium equation shown above, the expression for
K.sup.d.sub.Lapp can be reduced to:
K.sup.d.sub.Lapp=K.sup.d.sub.L[1+(X.sub.free/K.sup.d.sub.X)] Thus,
if K.sup.d.sub.Lapp is plotted versus X.sub.free, the y-intercept
will be equal to K.sup.d.sub.L and the slope will be equal to
K.sup.d.sub.L/K.sup.d.sub.X. Rearranging, K.sup.d.sub.X will be
equal to the y-intercept divided by the slope.
[0172] K.sup.d.sub.Lapp for citrate binding is calculated at each
concentration of competing anion as the concentration of free
protein (IL-1ra) times the concentration of free ligand (citrate),
divided by the concentration of bound ligand (citrate bound to
IL-1ra). The concentration of free protein is calculated to be the
total concentration of protein in the sample minus the
concentration of bound protein (which is equal to the concentration
of bound ligand). X.sub.free, or the concentration of free
competing anion, is equal to the total concentration of competing
anion minus the change in the concentration of bound ligand
(citrate bound to IL-1ra) as the total concentration of competing
anion is increased (i.e., as citrate is replaced at the IL-1ra
anion binding sites by the competing anion). In other words,
X.sub.free=X.sub.total-.DELTA.X.sub.bound as X.sub.total is
increased.
[0173] FIG. 22 shows a plot of K.sup.d.sub.Lapp for citrate versus
the amount of free pyrophosphate, as calculated using the data from
FIG. 20 and the calculations discussed above. The K.sup.d.sub.X for
pyrophosphate was calculated as 2.994 mM for this experiment. As
stated above, the y-intercept should be equal to K.sup.d.sub.L,
which is the K.sub.d of citrate. In this experiment, the
K.sup.d.sub.L was 3.894 mM, which agrees well with the previous
experimental determination of the K.sub.d for citrate binding to
IL-1ra, 3.846 mM, discussed in Example 9.
[0174] A similar plot (data not shown) was created for the
phosphate data shown in FIG. 21. In this experiment, the
K.sup.d.sub.X for phosphate was calculated to be 12.641 mM. The
K.sup.d.sub.L for citrate, as determined from the y-intercept of
the plot for this experiment (data not shown), was 4.996 mM.
[0175] These data suggest that citrate and pyrophosphate have lower
K.sub.ds for the anion binding site of IL-1ra. Furthermore,
pyrophosphate may have a slightly lower K.sub.d than citrate for
than anion binding site. The lower K.sub.ds seen in this experiment
correlate with the greater ability of pyrophosphate and citrate to
reduce aggregation of IL-1ra, as shown in Examples 2 and 4.
Sequence CWU 1
1
6 1 600 DNA Homo sapiens 1 gaattccggg ctgcagtcac agaatggaaa
tctgcagagg cctccgcagt cacctaatca 60 ctctcctcct cttcctgttc
cattcagaga cgatctgccg accctctggg agaaaatcca 120 gcaagatgca
agccttcaga atctgggatg ttaaccagaa gaccttctat ctgaggaaca 180
accaactagt tgctggatac ttgcaaggac caaatgtcaa tttagaagaa aagatagatg
240 tggtacccat tgagcctcat gctctgttct tgggaatcca tggagggaag
atgtgcctgt 300 cctgtgtcaa gtctggtgat gagaccagac tccagctgga
ggcagttaac atcactgacc 360 tgagcgagaa cagaaagcag gacaagcgct
tcgccttcat ccgctcagac agtggcccca 420 ccaccagttt tgagtctgcc
gcctgccccg gttggttcct ctgcacagcg atggaagctg 480 accagcccgt
cagcctcacc aatatgcctg acgaaggcgt catggtcacc aaattctact 540
tccaggagga cgagtagtac tgcccaggcc tgctgttcca ttcttgcatg gcaaggactg
600 2 177 PRT Homo sapiens 2 Met Glu Ile Cys Arg Gly Leu Arg Ser
His Leu Ile Thr Leu Leu Leu 1 5 10 15 Phe Leu Phe His Ser Glu Thr
Ile Cys Arg Pro Ser Gly Arg Lys Ser 20 25 30 Ser Lys Met Gln Ala
Phe Arg Ile Trp Asp Val Asn Gln Lys Thr Phe 35 40 45 Tyr Leu Arg
Asn Asn Gln Leu Val Ala Gly Tyr Leu Gln Gly Pro Asn 50 55 60 Val
Asn Leu Glu Glu Lys Ile Asp Val Val Pro Ile Glu Pro His Ala 65 70
75 80 Leu Phe Leu Gly Ile His Gly Gly Lys Met Cys Leu Ser Cys Val
Lys 85 90 95 Ser Gly Asp Glu Thr Arg Leu Gln Leu Glu Ala Val Asn
Ile Thr Asp 100 105 110 Leu Ser Glu Asn Arg Lys Gln Asp Lys Arg Phe
Ala Phe Ile Arg Ser 115 120 125 Asp Ser Gly Pro Thr Thr Ser Phe Glu
Ser Ala Ala Cys Pro Gly Trp 130 135 140 Phe Leu Cys Thr Ala Met Glu
Ala Asp Gln Pro Val Ser Leu Thr Asn 145 150 155 160 Met Pro Asp Glu
Gly Val Met Val Thr Lys Phe Tyr Phe Gln Glu Asp 165 170 175 Glu 3
152 PRT Homo sapiens 3 Arg Pro Ser Gly Arg Lys Ser Ser Lys Met Gln
Ala Phe Arg Ile Trp 1 5 10 15 Asp Val Asn Gln Lys Thr Phe Tyr Leu
Arg Asn Asn Gln Leu Val Ala 20 25 30 Gly Tyr Leu Gln Gly Pro Asn
Val Asn Leu Glu Glu Lys Ile Asp Val 35 40 45 Val Pro Ile Glu Pro
His Ala Leu Phe Leu Gly Ile His Gly Gly Lys 50 55 60 Met Cys Leu
Ser Cys Val Lys Ser Gly Asp Glu Thr Arg Leu Gln Leu 65 70 75 80 Glu
Ala Val Asn Ile Thr Asp Leu Ser Glu Asn Arg Lys Gln Asp Lys 85 90
95 Arg Phe Ala Phe Ile Arg Ser Asp Ser Gly Pro Thr Thr Ser Phe Glu
100 105 110 Ser Ala Ala Cys Pro Gly Trp Phe Leu Cys Thr Ala Met Glu
Ala Asp 115 120 125 Gln Pro Val Ser Leu Thr Asn Met Pro Asp Glu Gly
Val Met Val Thr 130 135 140 Lys Phe Tyr Phe Gln Glu Asp Glu 145 150
4 475 DNA Artificial coding sequence for anakinra 4 catatgcgac
cgtccggccg taagagctcc aaaatgcagg ctttccgtat ctgggacgtt 60
aaccagaaaa ccttctacct gcgcaacaac cagctggttg ctggctacct gcagggtccg
120 aacgttaacc tggaagaaaa aatcgacgtt gtaccgatcg aaccgcacgc
tctgttcctg 180 ggtatccacg gtggtaaaat gtgcctgagc tgcgtgaaat
ctggtgacga aactcgtctg 240 cagctggaag cagttaacat cactgacctg
agcgaaaacc gcaaacagga caaacgtttc 300 gcattcatcc gctctgacag
cggcccgacc accagcttcg aatctgctgc ttgcccgggt 360 tggttcctgt
gcactgctat ggaagctgac cagccggtaa gcctgaccaa catgccggac 420
gaaggcgtga tggtaaccaa attctacttc caggaagacg aataatggga agctt 475 5
153 PRT Artificial anakinra sequence 5 Met Arg Pro Ser Gly Arg Lys
Ser Ser Lys Met Gln Ala Phe Arg Ile 1 5 10 15 Trp Asp Val Asn Gln
Lys Thr Phe Tyr Leu Arg Asn Asn Gln Leu Val 20 25 30 Ala Gly Tyr
Leu Gln Gly Pro Asn Val Asn Leu Glu Glu Lys Ile Asp 35 40 45 Val
Val Pro Ile Glu Pro His Ala Leu Phe Leu Gly Ile His Gly Gly 50 55
60 Lys Met Cys Leu Ser Cys Val Lys Ser Gly Asp Glu Thr Arg Leu Gln
65 70 75 80 Leu Glu Ala Val Asn Ile Thr Asp Leu Ser Glu Asn Arg Lys
Gln Asp 85 90 95 Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser Gly Pro
Thr Thr Ser Phe 100 105 110 Glu Ser Ala Ala Cys Pro Gly Trp Phe Leu
Cys Thr Ala Met Glu Ala 115 120 125 Asp Gln Pro Val Ser Leu Thr Asn
Met Pro Asp Glu Gly Val Met Val 130 135 140 Thr Lys Phe Tyr Phe Gln
Glu Asp Glu 145 150 6 159 PRT Artificial icIL-1ra sequence 6 Met
Ala Leu Glu Thr Ile Cys Arg Pro Ser Gly Arg Lys Ser Ser Lys 1 5 10
15 Met Gln Ala Phe Arg Ile Trp Asp Val Asn Gln Lys Thr Phe Tyr Leu
20 25 30 Arg Asn Asn Gln Leu Val Ala Gly Tyr Leu Gln Gly Pro Asn
Val Asn 35 40 45 Leu Glu Glu Lys Ile Asp Val Val Pro Ile Glu Pro
His Ala Leu Phe 50 55 60 Leu Gly Ile His Gly Gly Lys Met Cys Leu
Ser Cys Val Lys Ser Gly 65 70 75 80 Asp Glu Thr Arg Leu Gln Leu Glu
Ala Val Asn Ile Thr Asp Leu Ser 85 90 95 Glu Asn Arg Lys Gln Asp
Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser 100 105 110 Gly Pro Thr Thr
Ser Phe Glu Ser Ala Ala Cys Pro Gly Trp Phe Leu 115 120 125 Cys Thr
Ala Met Glu Ala Asp Gln Pro Val Ser Leu Thr Asn Met Pro 130 135 140
Asp Glu Gly Val Met Val Thr Lys Phe Tyr Phe Gln Glu Asp Glu 145 150
155
* * * * *