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
1999-07-09
2001-11-06
Mertz, Prema (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.
C530S351000, C530S402000, C930S141000, C424S085200
Reexamination Certificate
active
06313272
ABSTRACT:
BACKGROUND
1. Field of the Invention
The invention is generally related to the fields of pharmacology and immunology. More specifically, the invention is directed to novel variants of the cytokine family, and in particular human Interleukin 4 (IL-4).
2. Description of Related Art
Interleukin-4 is a 15 kDa glycoprotein secreted by activated T cells, (Howard et al.,
J. Exp. Med.
155:914 (1982)), mast cells (Brown et al.,
Cell
50:809(1987)) and basophils (Seder et al.,
Proc. Natl. Acad. Sci. USA
88:2835(1991)) which regulates a wide spectrum of cellular functions in hematopoietic and nonhematopoetic cells. The sequence of IL-4 is disclosed in U.S. Pat. No. 5,017,691. Interleukin 4 (IL-4) is a pleiotropic cytokine, having activities on cells of the immune system, endothelium, and those of fibroblastic nature. Reported in vitro effects of IL-4 administration include proliferation of T and B cells, immunoglobulin class switching in B cells, stimulation of production of cellular surface adhesion molecules in endothelial cells, and stimulation of IL-6 release. In T cells, IL-4 stimulates T cell proliferation after preactivation with mitogens and down regulates IFN-&ggr; production. In monocytes, IL-4 induces class II MHC molecule expression, release of lipopolysaccharide-induced tPA, and CD23 expression. In Endothelial cells (EC), IL-4 induces expression of VCAM-1 and IL-6 release. IL-4 decreases ICAM-1 expression. Maher, D W, et al., Human Interleukin-4: An Immunomodulator with Potential Therapeutic Applications,
Progress in Growth Factor Research,
3:43-56 (1991).
Because of its ability to stimulate proliferation of T cells activated by exposure to IL-2, IL-4 therapy has been pursued. For instance, IL-4 has demonstrated anti-neoplastic activity in animal models of renal carcinomas, and has induced tumor regression in mice (Bosco, M., et al., Low Doses of IL-4 Injected Perilymphatically in Tumor-bearing Mice Inhibit the Growth of Poorly and Apparently Nonimmunogenic Tumors and Induce a Tumor Specific Immune Memory,
J. Immunol.
145:3136-43 (1990)). However, its toxicity limits the dosage in humans (Margolin, K., et al. Phase II Studies of Human Recombinant Interleukin-4 in Advanced Renal Cancer and Malignant Melanoma,
J. Immunotherapy
15:147-153 (1994).
The general structure and function of IL-4 and related monomeric ligands containing 4 antiparallel a-helical domains (A-D) is now known. The three-dimensional structure of IL-4 has been solved (Powers et al.,
Science
256:1673) (1992). The protein contains 4 left hand a-helices and two b-sheets.
The IL-4 receptor consists of at least two chains. The first IL-4R chain, IL-4R&agr;, shares significant homology to the b chain of the IL-2R and other members of the growth factor receptor superfamily (Ldzerda et al.
J. Exp. Med.
171:861(1990)). A second IL-4R chain has been identified, the chain of the IL-2R, also known as the ‘common chain’, &ggr;
c
(Russell et al.,
Science
262:1877(1993). The two binding sites are likely involved in a sequential, two-binding event that results in a ternary 1:1:1 complex. The region of IL-4 likely responsible for binding with IL-4R&agr; is thought to be located on either or both of helices A and C, while the region interactive with g
c
is thought to be located on helix D. Present theory holds that the first binding event involves the ligand contacting IL-4R&agr;, the primary binding component. No cellular signaling activity is associated with this event. The second binding event occurs when the IL-4/IL4R&agr; complex recruits a second chain, gc. After this second binding event, signaling occurs and cellular activity is established. Antagonism of wild-type IL-4 is found when binding interactions mediated by the second region (necessary for cellular activity) are diminished or eliminated, while retaining binding to IL-4R&agr;. Agonism occurs when a candidate ligand constructively interacts, via the first and second regions, to both receptor components.
Antagonists of IL-4 have been reported in the literature. Mutants of IL-4 that function as antagonists include the IL-4 antagonist mutein IL-4/Y124D (Kruse, N., Tony, H. P., Sebald, W., Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid replacement,
Embo J.
11:3237-44, 1992) and a double mutein IL-4[R121D/Y124D] (Tony, H., et al., Design of Human Interleukin-4 Antagonists in Inhibiting Interleukin-4-dependent and Interleukin-13-dependent responses in T-cells and B-cells with high efficiency,
Eur. J. Biochem.
225:659-664 (1994)). The single mutein is a substitution of tyrosine by aspartic acid at position 124 in the D-helix. The double mutein is a substitution of Arginine by Aspartic Acid at position 121, and of tyrosine by aspartic acid at position 124 in the D-helix. Variations in this section of the D helix positively correlate with changes in interactions at the second binding region.
Mutant variants of IL-4 demonstrating agonism or antagonism of wild-type IL-4 may be useful for treating conditions associated with one of the pleiotropic effects of IL-4. For instance, antagonists of IL-4 would be useful in treating conditions exacerbated by IL-4 production such as asthma, allergy, or other inflammatory response-related conditions. Agonists of IL-4 may be useful for treating conditions wherein the presence of IL-4 is associated with the amelioration or attenuation of a disease, for example, an autoimmune disease such as Rheumatoid Arthritis, Multiple Sclerosis, Insulin-dependent Diabetes Mellitus, etc. These autoimmune diseases are characterized by a polarization in production of the T helper cell populations, types 1 and 2 (Th1, Th2). Naive CD4+ T cells differentiate into Th1 or Th2 subsets, depending on the cytokine present during stimulation. An IL-4 agonist would ideally shift production to the T-helper cell desired, i.e., towards Th2, thereby having a therapeutic effect.
PCT/US93/03613 discloses an IL-4 variant having a Phe-Leu or Tyr-Leu sequence in a alpha-helical domain and a negatively-charged amino acid within two amino acids immediately upstream or downstream from the Phe-Leu or Tyr-Leu sequence, the variant having an increased affinity for the IL-4 receptor by virtue of a neutral amino acid substituted for the negatively-charged amino acid. It also discloses that the specific substitution of Trp-Leu or Phe-Leu within an a-helix of IL-4 within 2-residues of a negatively charged residue results in improved affinity. The variant is an IL-4 fusion protein (with diphtheria toxin).
SUMMARY OF THE INVENTION
The invention is directed to a recombinant human IL-4 mutein numbered in accordance with wild-type IL-4 wherein the mutein comprises at least one amino acid substitution in the binding surface of either the A- or C- alpha helices of the wild-type IL-4 whereby the mutein binds to the IL-4R&agr; receptor with at least greater affinity than native IL-4. The substitution is preferably selected from the group of positions consisting of, in the A-helix, positions 13 and 16, and in the C-helix, positions 81 and 89. A most preferred embodiment is the recombinant human IL-4 mutein wherein the substitution at position 13 is Thr to Asp. Pharmaceutical compositions, amino acid and polynucleotide sequences encoding the muteins, transformed host cells, antibodies to the muteins, and methods of treatment are also described.
The invention is also directed to an assay for determining the ability of a mutein to bind to a receptor, comprising the steps of: first introducing into a FlashPlate” coated with streptavidin the binding portion of a receptor chain, the binding portion of a receptor chain having a peptide tag capable of being bound by streptavidin; secondly introducing into the FlashPlate a radiolabelled native ligand having an affinity for the binding portion of a receptor chain; thirdly introducing into the FlashPlate a mutein ligand having an affinity for the binding portion of a receptor chain; the measuring of the amount of signal given off by the FlashPlate after allowing for equilib
Greve Jeffrey M.
Roczniak Steven
Shanafelt Armen B.
Bayer Corporation
Mahoney John W.
Mertz Prema
Prasad Sarada C
Shaw Melissa A.
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