Drug – bio-affecting and body treating compositions – Lymphokine – Interleukin
Reexamination Certificate
2000-10-05
2003-06-10
Eyler, Yvonne (Department: 1646)
Drug, bio-affecting and body treating compositions
Lymphokine
Interleukin
C424S001410, C424S185100, C424S192100, C424S197110, C530S351000
Reexamination Certificate
active
06576232
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 histo-compatibility 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 p140, and a second subunit of approximately 52 kDa termed &agr;′ or IL13R&agr;1. 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 &agr;′. 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 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, 69, 99, 102, 104, 105, 106, 107, 108, 109, 112, 113, and 114 replaced with a different amino acid. These mutants are expressed herein as hIL13X
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. 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. Representative 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.
Also within the invention are compositions including a mutant hIL13 having an amino acid sequence having at least 90% sequence identity to native hIL13 (SEQ ID NO:1). Such mutants can have a mutation in a domain corresponding to the A, C, or D alpha-helices of native hIL13. Exemplary mutants include those with a polypeptide having an amino acid sequence of one of SEQ ID NOs: 2-23.
Mutants of hIL13 within the invention can be those that specifically bind the shared IL4/IL13 receptor but not the restricted (IL4-independent) receptor; those that specifically bind the restricted (IL4-independent) receptor but not the shared IL4/IL13 receptor; or those that bind both receptors.
Some hIL13 mutants of the invention specifically bind to an hIL13 receptor associated with a cell in a manner that induces a measurable change in the cell's physiology. This change can be of greater or less magnitude than a change in the cell's physiology that would be induced by specifically binding the IL13 receptor with native hIL13.
Compositions within the invention can include both an hIL13 mutant and a pharmaceutically acceptable carrier.
Mutants of hIL13 within the invention can be conjugated to an effector molecule such as a cytotoxin (e.g., Pseudomonas exotoxin, PE38QQR, PE1E, PE4E, Diptheria toxin, ricin, abrin, saporin, and pokeweed viral protein), a detectable label (e.g., radionuclide), an antibody, a liposome, or a lipid.
In another aspect the invention includes a purified nucleic acid encoding a mutant hIL13. Also within the invention is an antibody that specifically binds a mutant hIL13 molecule, but not a native hIL13 molecule. And in another aspect, the invention features a method of delivering a mutant hIL13 to a cell. The method can include the steps of: providing a mutant hIL13 and a cell; and contacting the cell with the mutant hIL13.
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Commonly understood definitions of molecular biology terms can be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994.
As used herein, the phrase “native hIL13” means the mature form of human interleukin 13, the amino acid sequence of which is shown herein as SEQ ID NO:1.
The phrase “hIL13 mutant” or a “mutant hIL13 molecule” means an hIL13 in which one or more of the amino acids differ from the corresponding amino acids in the native hIL13. Thus, for example, where a native hIL13 has a glutamic acid at position 13, a mutant hIL13 can have an amino acid other than glutamic acid at position 13 (e.g., glutamic acid is substituted with lysine). It will appreciated that mutant IL13 molecules of this invention include mutant IL13 molecules of other mammalian species (e.g., rat, murine, porcine, ovine, goats, non-human primates, bovine, canus, and the like) and this invention contemplates the use of mutant IL13 in veterinary as well as human me
Debinski Waldemar
Thompson Jeffrey P.
Akerman & Senterfitt
Andres Janet L.
Eyler Yvonne
Kim Stanley A.
The Penn State Research Foundation
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