Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Reexamination Certificate
2001-08-24
2003-10-07
Eyler, Yvonne (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
C530S350000, C424S085200, C514S002600, C435S069520
Reexamination Certificate
active
06630576
ABSTRACT:
BACKGROUND OF THE INVENTION
Human interleukin 13 (hIL13) is a 114 amino acid cytokine secreted by activated T cells. Minty et al. (1993) Nature, 362:248-250; and McKenzie et al. (1993) Proc. Natl. Acad. Sci. USA, 90:3735-3739. hIL13 is involved in regulating several different physiological responses. Among these, hIL13 has been shown to downregulate the production of cytokines involved in inflammation. Minty et al., supra; and de Waal Malefyt et al. (1993) J. Immunol., 151:6370-6381. It has also been shown to upregulate expression of major histocompatibility class II molecules and CD23 on monocytes, and to regulate various aspects of B cell function De Waal Malefyt et al. (1993) Res. Immunol. 144:629-633; McKenzie et al., supra; and de Waal Malefyt et al. (1993) J. Immunol., 151:6370-6381. In addition to regulating cells of the immune system, IL-13 has also been shown to act on other cell types. For example, IL13 has been shown to modulate expression of vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells. Sironi et al. (1994) Blood, 84:1913-1921; Bochner et al. (1995) J. Immunol., 154:799-803; and Schnyder et al. (1996) Blood, 87:4286-4295.
Based on its predicted secondary structure, hIL13 has been added to a growing family of growth hormone-like cytokines that all exhibit bundled alpha-helical core topology. Bamborough et al. (1994) Prot. Engin,. 7:1077-1082. Structural analyses indicated that hIL13 is a globular protein comprised mainly of four alpha-helical regions (helices A, B, C, and D) arranged in a “bundled core.” Miyajima et al. (1992) Ann. Rev. Immunol., 10, 295-331.
While dissimilar at the primary amino acid level, hIL13 and human interleukin 4 (hIL4) bind and signal through a shared receptor complex. Zurawski et al. (1993) EMBO J., 12:2663-2670; and Tony et al. (1994) Eur. J. Biochem., 225:659-66. This shared receptor is a heterodimer that includes a first subunit of approximately 140 kDa termed p 140, and a second subunit of approximately 52 kDa termed a′ or IL13Ra1. Idzerda et al. (1990) J. Exp. Med., 173:861-873; Obiri et al. (1995) J. Biol. Chem., 270:8797-8804; Hilton et al. (1996) Proc. Natl. Acad. Sci. USA, 93:497-501; and Miloux et al. (1997) FEBS Letters, 401:163-166. Unlike hIL4, hIL13 does not bind p140 in the absence of a′. Vita et al. (1995) J. Biol. Chem., 270:3512-3517. In addition to the shared receptor, another hIL13 receptor termed the restricted (IL4 independent) receptor exists. In contrast to the shared receptor, the latter receptor binds hIL13 but not hIL4. The restricted receptor is also sometimes called the glioma-associated receptor because it is preferentially expressed at high levels in certain malignant cells, including those in high grade human gliomas. Debinski et al. (1995) Clin. Cancer Res., 1:1253-1258; and Debinski et al. (1996) J. Biol. Chem., 271, 22428-22433. In addition to being associated with malignancies, hIL13 has also been associated with other pathological conditions. Notably, IL13 has been shown to be involved in pathways that regulate airway inflammation, suggesting that this cytokine might play an important role in asthma and perhaps other allergic pathologies. Webb et al., (2000) J. Immunol.165:108-113; and Djukanovic, R. (2000) Clin. Exp. Allergy 30 Suppl 1:46-50.
SUMMARY OF THE INVENTION
The invention relates to the development and characterization of several mutants of hIL13. Using these mutants, three regions of native hIL13 were identified as being required for signaling through the shared receptor. These regions were localized to alpha-helices A, C and D and were generally separated from the regions involved in binding to the restricted receptor. Glutamic acids at positions 13 and 16 in hIL13 alpha-helix A, arginine and serine at positions 66 and 69 in helix C, and arginine at position 109 in helix D were found to be important in inducing biological signaling because these mutations resulted in the loss and/or gain of functional phenomena.
Mutants within the invention include those having one or more of the native amino acids of hIL13 at positions 13, 16, 17, 66, 19, 99, 102, 104, 105, 106, 107, 108, 109, 112, 113, and 114 replaced with a different amino acid. These mutants are expressed herein as hIL13.X
1
PX
2
, where P is a number corresponding to the position of the mutated amino acid in hIL13, X
1
is the letter abbreviation of the amino acid that was replaced, and X
2
is the letter abbreviation of the replacement amino acid. Mutants with multiple mutations are indicated in the same fashion as hIL13X
1
PX
2
.X
3
P
1
X
4
for a double amino acid substitution mutant hIL13X
1
PX
2
.X
3
P
1
X
4
.X
5
P
2
X
6
for a triple amino acid substitution mutant; and hIL13X
1
PX
2
.X
3
P
1
X
4
.X
5
P
2
X
6
.X
7
P
3
X
8
for a quadruple amino acid substitution mutant. For example, hIL13.E13K represents a mutant form of hIL13 that has the glutamic acid residue that naturally occurs at position 13 in native hIL13 replaced with a lysine residue; and hIL13.E13K.S69D represents a mutant form of hIL13 that has the glutamic acid residue that naturally occurs at position 13 in native hIL13 replaced with a lysine residue and the serine residue that naturally occurs at position 69 in native hIL13 replaced with an aspartic acid residue. Representative single amino acid substitution mutants within the invention include hIL13.E13K, hIL13.E13I, hIL13.E13C, hIL13.E13S, hIL13.E13R, hIL13.E13Y, hIL13.E13D, hIL13.E16K, hIL13.E17K, hIL13.R66D, hIL13.S69D, hIL13.D99K, hIL13.L102A, hIL13.L104A, hIL13.K105D, hIL13.K106D, hIL13.L107A, hIL13.F108Y, hIL13.R109D, hIL13.R112D, hIL13.F113D, and hIL13.N114D. The invention also includes double, triple, and quadruple amino acid substitution mutants including: hIL13.E13K.S69D (SEQ ID NO:2); hIL13.E13K.R109D (SEQ ID NO:3); hIL13.E13K.R112D (SEQ ID NO:4); hIL13.E13Y.R66D (SEQ ID NO:5); hIL13.E13Y.S69D (SEQ ID NO:6); hIL13.E13K.R66D.S69D (SEQ ID NO:7); hIL13.E13Y.R66D.S69D (SEQ ID NO:8); and hIL13.E13K.R66D.S69D.R112D (SEQ ID NO:9).
Accordingly, the invention features a purified mutant hIL13 molecule including an amino acid sequence (a) having at least 90% sequence identity to the native hIL13 sequence (SEQ ID NO:1) and (b) differing from the native hIL13 sequence by at least a first amino acid substitution occurring in the A alpha helix and a second amino acid substitution occurring in the D alpha helix.
Also within the invention is a purified mutant hIL13 molecule including an amino acid sequence (a) having at least 90% sequence identity to the native hIL13 sequence (SEQ ID NO:1) and (b) differing from the native hIL13 sequence by at least three amino acid substitutions. In one variation of the foregoing, the amino acid sequence differs from the native hIL13 sequence by at least a first amino acid substitution occurring in the A alpha helix, a second amino acid substitution occurring in the D alpha helix, and a third amino acid substitution occurring in the C alpha helix. In another variation of the foregoing, the amino acid sequence differs from the native hIL13 sequence by at least four amino acid substitutions, e.g., with at least a first amino acid substitution occurring in the A alpha helix, a second amino acid substitution occurring in the D alpha helix, and a third amino acid substitution occurring in the C alpha helix.
The invention further includes a purified mutant hIL13 molecule that includes a polypeptide having or consisting of an amino acid sequence of one of SEQ ID NOs: 2-9.
The purified mutant hIL13 molecule of the invention can further include a pharmaceutically acceptable carrier and/or can be conjugated to an effector molecule such as a cytotoxin (e.g., a Pseudomonas exotoxin such as PE38QQR, PE1E, and PE4E, Diptheria toxin, ricin, abrin, saporin, and pokeweed viral protein), a detectable label, an antibody, a liposome, and a lipid. The effector molecule can also be a radionuclide.
In another aspect, the invention features a purified nucleic acid encoding a polypeptide including or consisting of an amino acid sequence of one of SEQ ID NOs: 2-9.
The invention ad
Akerman & Senterfitt
Andres Janet L.
Eyler Yvonne
Kim Stanley A.
Pennsylvania State Research Foundation
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