WO2015000585A1

WO2015000585A1 - Muteins of cytokines of the gamma-chain receptor family conjugated to a non-protein group

Info

Publication number: WO2015000585A1
Application number: PCT/EP2014/001796
Authority: WO
Inventors: Walter Sebald
Original assignee: Walter Sebald
Priority date: 2013-07-02
Filing date: 2014-07-01
Publication date: 2015-01-08

Abstract

The present invention relates to novel mutein conjugates, to compositions comprising said novel mutein conjugates, and to their uses. More specifically, the invention relates to mutein conjugates of cytokines of the common gamma-chain receptor family, to a pharmaceutical composition that includes such a mutein conjugate of a cytokine of the common gamma-chain receptor family as well as to various uses of the mutein conjugates of cytokines of the common gamma-chain receptor family.

Description

MUTEINS OF CYTOKINES OF THE GAMMA-CHAIN RECEPTOR FAMILY

CONJUGATED TO A NON-PROTEIN GROUP

Field of the invention

Background of the invention

Polymer therapeutics encompass polymer-macromolecule conjugates. These polymer therapeutics can accomplish several desirable objectives: a longer in vivo half-life; reduced immunogenicity, toxicity, and clearance rate through the kidneys; successful transportation across a cell membrane; protection against proteolysis; modification of electro-osmotic flow; increased pH and thermal stability; a low volume of distribution and sustained adsorption from the injection site; and improved formulation properties of the protein. These superior properties can increase effective potency, improve response to the drug, increase patient tolerance and reduce side effects, and reduce overall dosage. For example, many biological macromolecules, including proteins and peptides, have proven useful for the treatment of various health problems. In recent years, numerous polymer therapeutics have been developed and some of them have already received market approval. An overview of polymer therapeutics is, for example, presented in Polymer Therapeutics I and II: Polymers as Drugs, Conjugates and Gene Delivery Systems (Advances in Polymer Science; eds. R. Satchi-Fainaro and R. Duncan; Springer Berlin Heidelberg; 2006).

The cytokines of the common gamma-chain receptor family are the following six interleukins:

Interleukin 2 (IL-2), Interleukin 4 (IL-4), Interleukin 7 (IL-7), Interleukin 9 (IL-9), Interleukin 15 (IL- 15), and Interleukin 21 (IL-21). All members of this family signal through receptor complexes that contain the common cytokine receptor gamma-chain subunit. The receptors for IL-4, IL-7, EL-9, and EL- 21 are heterodimeric complexes consisting of the common gamma-chain and a unique cytokine-specific subunit. The receptors for IL-2 and IL-15 are heterotrimeric complexes consisting of either IL-2 R alpha or IL-15 R alpha, IL-2/IL-15 R beta and the common gamma-chain. Signaling pathways activated by this cytokine family include the PI 3-K-Akt pathway, the Ras-MAPK pathway, and the Jak-STAT pathway. Cytokines of the common gamma-chain receptor family are essential for the establishment and maintenance of normal immune system functions. They have both unique and overlapping effects on a number of cell types including T cells, B cells, natural killer cells, mast cells, and myeloid and erythroid progenitors.

IL-2 was initially identified as a T cell growth factor, and has since been shown to play a critical role in maintaining T cell homeostasis and preventing self-reactivity.

IL-4 is one of the major regulatory cytokines determining differentiation and growth of immune cells, which is produced and secreted primarily by Th2-biased CD4+ T cells, mast cells, basophils and eosinophils. IL-4 is a small 4-helix bundle cytokine containing three disulfide bridges and no free cysteine residue. It employs two types of heterodimeric receptors for signalling into a cell. Both receptor types contain an IL-4Ra chain which binds IL-4 with high affinity (KD about 100 pM) and provides most of the IL-4 affinity of the whole receptor complex. The type I receptor employs the common gamma-chain (yc) as second chain and it is expressed predominantly on immune and other hematopoietic cells. The type II receptor contains IL-13Rctl as the second chain and it is found mainly on nonhematopoietic cells. The type II receptor is promiscuous in using not only IL-4 but also IL-13. IL-4 is of high medical interest, since together with its close relative IL-13, another Th2 cytokine, IL-4 plays a dominant role in the development and maintenance of allergic inflammation and asthma

[References 1-4]. Many findings support the view that IL-4 acts mainly as "immunoregulatory" cytokine and EL- 13 largely as an "effector" cytokine. In addition to its crucial role in the immune system, EL-4 functions as survival factor and inhibitor of apoptosis in cancer cells [References 5, 6].

IL-7 is required for T cell development and homeostatic proliferation, mouse B cell development, and the generation of memory T cells.

IL-9 promotes mouse T cell and mast cell growth, regulates B cell immunoglobulin production, enhances mast cell protease expression, and promotes goblet cell hyperplasia and mucus production.

IL-15 is required for the development, survival, and activation of natural killer cells, the homeostasis of natural killer T cells and intraepithelial lymphocytes, and the maintenance of naive and memory CD8⁺ T cells. IL-21 has been shown to be required for Thl7 cell differentiation and the generation of T follicular helper cells. In addition, it regulates B cell activities and the cytotoxicity of CD8⁺ T cells and natural killer cells.

Various muteins and mutein conjugates of cytokines of the common gamma-chain receptor family have been described in the art, e.g. WO 1999/003887 and WO 2001/087925. For instance, IL-4 double mutein (IL-4DM, pitrakinra) with substitutions R121D and Y124D retains binding to the IL-4Ra chain, but binding to the yc and IL-13Ral chains is disrupted [Reference 7]. This IL-4DM has no agonist activity, but competes efficiently with endogenous wild type IL-4 for receptor binding. It is being developed as receptor antagonist (IL-4Ra blocker) and therapeutic for the treatment of eosinophilic asthma and eczema [References 8, 9]. For this application it is relevant (1) that by antagonizing IL-4Ra both IL-4 and IL-13 dependent responses become inhibited, and (2) that IL-4Ra alone has a very high affinity for EL-4 (KD ~ ΙΟΟρΜ), which is only slightly lower than the IL-4 affinity of the heterodimeric type I or II complex existing in the cell membrane. On the other hand, an IL-4 single mutein with the substitution R121E was shown to be a selective agonist stimulating immune cells via interacting with yc but to be inactive in eliciting inflammatory responses due to the loss of interaction with EL-13al

[Reference 10].

Site specific modification of IL-4 has also been used in the past for conjugation with a truncated Pseudomonas exotoxin (PE35) [Reference 11]. For achieving this, a series of EL-4 analogues were prepared in which residues 28, 38, 68, 70, 97, or 105 were individually substituted by cysteine.

Disulfide-mediated conjugation with PE35 yielded IL-4-toxins with improved binding affinity and biological activity compared to an IL-4-PE35 fusion protein. A major concern in these studies was to preserve the function of IL-4, this means the receptor-binding affinity, as much as possible. Another study described the site-specific modification of IL-4DM (and IL-4TM) with polyethylene glycol (PEG) [Reference 12]. Residues 28, 36, 37, 104, 105, or 106 were selected for substitution with cysteine and subsequent conjugation, since all of them were considered to be located outside the binding epitopes of IL-4 for the receptor. Conjugation with maleimide-PEG yielded IL-4 TM analogues with the aimed bioactivity [Reference 13]. In both studies sites selected for chemical modification were outside the binding epitopes of the receptors.

It would, therefore, be advantageous to provide novel, alternative mutein conjugates of cytokines of the common gamma-chain receptor family, which exhibit new and advantageous properties, e.g. reduced immunogenicity, superior pharmacokinetics, inhibiting IL-4 as well as IL-13 signalling, etc., and thus have utility as protein therapeutics and/or as research tools. Description of the invention

The present invention was made in view of the prior art and the needs described above, and, therefore, the object of the present invention is to provide novel mutein conjugates of cytokines of the common gamma-chain receptor family, a pharmaceutical composition that includes such a mutein conjugate of at least one cytokine of the common gamma-chain receptor family, and various uses of the mutein conjugates of cytokines of the common gamma-chain receptor family.

These objects are solved by the subject matter of the attached claims.

These and other aspects of the present invention will become apparent upon reference to the following detailed description and definitions.

The inventors established that mutein conjugates according to the invention behave as antagonists in cellular assays. In particular, the inventors established that mutein conjugates according to the invention were more potent inhibitors than the IL-4DM double mutein.

The inventors further showed that mutein conjugates according to the invention can reduce binding affinity to the common gamma-chain receptor by more than 99%.

The inventors also showed that mutein conjugates according to the invention can reduce binding affinity to IL-13Ral by more than 95%.

The inventors, moreover, found that mutein conjugates according to the invention inhibit IL-4 as well IL-13 signalling.

Furthermore, the inventors selected residues for substitution with cysteine and subsequent conjugation, which are located within the binding epitopes for the receptors, e.g. an IL-4 position within the binding epitopes for the receptors yc and IL-13Ral . In particular, the inventors found that chemical

modification at only one certain position results in a virtually complete inhibition of yc and IL-13Ral binding and a substantial loss of biological activity.

Taken together, the inventors demonstrate that mutein conjugates according to the invention exhibit new properties. These properties are the result of a chemical modification of the mutein at a position which is located within the binding epitopes for the receptors. These surprising and unexpected results allow an alternative therapeutic, preventive and/or curative role to be conceived for the mutein conjugates of the cytokines of the common gamma-chain receptor family according to the invention in the prevention and/or treatment of various conditions or disorders associated with cytokines of the common gamma-chain receptor family.

Accordingly, the present invention is directed to a mutein conjugate, the mutein conjugate comprising a mutein moiety and a non-protein moiety, wherein

the mutein moiety comprises or has an amino acid sequence as set forth in SEQ ED No. 1 to SEQ ED No. 12, or a fragment thereof;

the non-protein moiety comprises a group that is reactive with a side chain sulfhydryl group (SH) of the mutein; and

the mutein moiety is linked to the non-protein moiety through covalent bonding of the side chain sulfhydryl group of the residue corresponding to position 121 of SEQ ED No. 1, corresponding to position 122 of SEQ ED No. 2, corresponding to position 126 of SEQ ED No. 3, corresponding to position 127 of SEQ ED No. 4, corresponding to position 143 of SEQ ED No. 5, corresponding to position 144 of SEQ ED No. 6, corresponding to position 115 of SEQ ED No. 7, corresponding to position 1 16 of SEQ ED No. 8, corresponding to position 108 of SEQ ED No.9, corresponding to position 109 of SEQ ED No. 10, corresponding to position 1 16 of SEQ ED No. 1 1 , or corresponding to position 1 17 of SEQ ED No. 12, wherein the covalent bond is formed via a thioether, sulfur or disulfide bond.

The term "mutein moiety" as used herein refers to a mutant protein or peptide carrying a single or multiple amino acid substitution(s), i.e. exchange of an amino acid for another amino acid, compared to the naturally occurring polypeptide. A mutein moiety of the present invention also refers to the deletion or insertion of one or more amino acids, provided that the overall tertiary structure and bioactivity of the naturally occurring polypeptide is essentially retained in the shortened or extended polypeptide. The mutein moiety of the present invention includes at least one substitution of a native amino acid by a cysteine residue at the indicated or a corresponding position in comparison to the naturally occurring polypeptide.

The term "fragment" as used herein refers to a polypeptide that comprises a portion of the full length sequences of SEQ ED NOs. 1 to 12, generally the receptor binding region thereof. The fragments derived from the mutein moieties according to SEQ ED NOs. 1 to 12 may be any as long as they have substantially equivalent activity to said mutein moieties. For example, the fragments of the mutein moieties according to SEQ ID NOs. 1 to 12 include peptides having a sequence with at least 75 or more, preferably 80 or more, still preferably 90 or more, more preferably 100 or more, still more preferably 110 or more amino acid residues contained in the mutein moieties according to SEQ ID NOs. 1 to 12, preferably wherein said amino acid residues are contiguous. Preferable examples thereof are those having at least 85 %, at least 90 %, at least 93 %, at least 95 %, at least 97 % identity to the corresponding section of the sequence of SEQ ID NOs. 1 to 12, with the proviso that the altered position is retained.

The term "identity" refers to a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100.

The term "non-protein moiety" includes all chemical groups that are suitable for conjugation to a polypeptide, except groups comprising two or more amino acid residues arranged in a linear chain, wherein the individual amino acids in the organic compound are linked by peptide bonds, i.e. an amide bond formed between adjacent amino acid residues.

The terms "protein", "polypeptide", "peptide" as used herein define an organic compound made of two or more amino acid residues arranged in a linear chain, wherein the individual amino acids in the organic compound are linked by peptide bonds, i.e. an amide bond formed between adjacent amino acid residues. By convention, the primary structure of a protein is reported starting from the amino-terminal (N) end to the carboxyl-terminal (C) end.

The term "position" as used herein means the position of an amino acid within an amino acid sequence depicted herein. The term "corresponding" as used herein includes that a position is not only determined by the number of the preceding amino acids. Accordingly, the position of a given amino acid in accordance with the invention which may be substituted may very due to deletion or addition of amino acids elsewhere in the respective cytokine of the common gamma-chain receptor family.

Thus, under a "corresponding position" in accordance with the invention it is preferably to be understood that amino acids may differ in the indicated number but may still have similar neighboring amino acids. Said amino acids which may be exchanged, deleted or added are also comprised by the term "corresponding position".

Specifically, in order to determine whether an amino acid residue of the amino acid sequence of a mutein moiety of the cytokine of the common gamma-chain receptor family different from a mutein moiety of the invention corresponds to a certain position in the amino acid sequence as described in any of SEQ ID NOs. 1 to 12, a skilled artisan can use means and methods well-known in the art, e.g.

alignments, either manually or by using computer programs such as BLAST2.0, which stands for Basic Local Alignment Search Tool or ClustalW or any other suitable program which is suitable to generate sequence alignments.

As used herein, "comprising", "including", "containing", "characterized by", and grammatical equivalents thereof are inclusive or open-ended terms that do not exclude additional, unrecited elements or method steps. "Comprising" is to be interpreted as including the more restrictive terms "consisting of and "consisting essentially of."

As used herein, "consisting of and grammatical equivalents thereof exclude any element, step, or ingredient not specified in the claim.

As used herein, "consisting essentially of and grammatical equivalents thereof limit the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic or characteristics of the claimed invention.

When trade names are used herein, it is intended to independently include the trade name product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product.

In general, unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and are consistent with general textbooks and dictionaries.

Preferably, the mutein conjugate comprises a mutein moiety and a non-protein moiety, wherein

the mutein moiety has an amino acid sequence as set forth in SEQ ID No. 1 to SEQ ID No. 12; the non-protein moiety comprises a group that is reactive with a side chain sulfhydryl group

(SH) of the mutein; and

the mutein moiety is linked to the non-protein moiety through covalent bonding of the side chain sulfhydryl group of the residue at position 121 of SEQ ID No. 1 , at position 122 of SEQ ID No. 2, at position 126 of SEQ ID No. 3, at position 127 of SEQ ID No. 4, at position 143 of SEQ ID No. 5, at position 144 of SEQ ID No. 6, at position 1 15 of SEQ ID No. 7, at position 1 16 of SEQ ED No. 8, at position 108 of SEQ ID No.9, at position 109 of SEQ ED No. 10, at position 1 16 of SEQ ED No. 11 , or at position 117 of SEQ ID No. 12, wherein the covalent bond is formed via a thioether, sulfur or disulfide bond.

The non-protein moiety of the mutein conjugate according to the invention may further comprise a nonprotein polymer. This non-protein polymer may be a water-soluble polymer of synthetic or natural origin.

Preferably, the non-protein polymer is polyethylene glycol (PEG), polypropylene glycol,

polyoxyalklylene, poly(N-vinylpyrrolidone), N-hydroxy-ethyl methacrylamide copolymer, poly(2- ethyl-2-oxazoline), poly(N-acryloylmorpholine), hyaluronic acid, or polysyalic acid.

Further preferred, the non-protein polymer can be a linear or branched polyethylene glycol having a molecular weight (MW) of from 400 to 50000 Da.

In the mutein conjugate according to the invention, the group of the non-protein moiety that is reactive with the side chain sulfhydryl group (SH) of the mutein can be a sulfhydryl-reactive group selected from maleimides, brom- or iodoalkyls, pyridyldisulfides, thiosulfonates, vinylsulfones, and compounds containing a thiol group.

In the mutein conjugate according to the invention, the mutein moiety can be linked to the non-protein moiety via a thioether or disulfide bond.

In the mutein conjugate according to the invention, the non-protein moiety can be a thiol compound selected from glutathione; cysteamine; thioglycolate; MeO-[(CH₂)2-0]_n-(CH₂)2-SH (or MeO-(PEG)„- SH), n being an integer of from 4 to 150; and MeO-(PEG) _n-SH having a molecular weight of about 5000 Da.

In the mutein conjugate according to the invention, the non-protein moiety can also be a maleimide compound selected from N-ethyl-maleimide, N-(2-ethylamine)-maleimide, N-(S)-alanine-maleimide, and TMM(PEG)12.

In the mutein conjugate according to the invention, the mutein moiety has preferably an amino acid sequence as set forth in SEQ ID No. 1 or SEQ ID No. 2.

Preferably, an inventive mutein conjugate binds to the common gamma chain with a K_D of >0,1 mM. Also, an inventive mutein conjugate can inhibit the response of human Jurkat cells to EL-4 with an IC₅₀ of 0,1 nM to 10 nM.

The mutein conjugate according to the invention can also reduce the cell activating ability by 50 % or more, preferably by 95% or more, relative to wildtype IL-4.

Also preferred according to the invention is a mutein conjugate, wherein the mutein has an amino acid sequence as set forth in SEQ ID No. 1 ;

the non-protein moiety is glutathione, cysteamine, thioglycolate, MeO-(PEG)-SH, N-ethyl- maleimide, N-(2-ethylamine)-maleimide, N-(S)-alanine-maleimide, or TMM(PEG)12; and

the mutein moiety is linked to the non-protein moiety through covalent bonding of the side chain functional group of the cysteine residue at position 121 of SEQ ID No. 1 , wherein the covalent bond is formed via a thioether or disulfide bond.

The mutein conjugates according to the invention can, for example, be prepared as mixed disulfides according to Reaction Scheme 1 , or as maleimide or maleimido-compounds according to Reaction Scheme 2, wherein IL-4 is representative of all cytokines of the common gamma-chain receptor family.

Reaction Scheme 2

A variety of compounds containing a thiol group that are reactive with the side chain sulfhydryl group (SH) of the mutein, including thiol-PEGs, are commercially available, including those depicted in Table A below.

Table A

Similarly, a variety of maleimide compounds, including maleimide-PEGs, are commercially available, including those depicted in Table B below. Table B

The therapeutic use of the mutein conjugates according to the invention and also formulations and pharmaceutical compositions containing the same are within the scope of the present invention. The present invention also relates to the use of said inventive mutein conjugates as active ingredients in the preparation or manufacture of a medicament. A pharmaceutical composition according to the present invention comprises one or more mutein conjugate(s) according to the invention and, optionally, at least one carrier substance, excipient and/or adjuvant.

Carrier substances are, for example, cyclodextrins such as hydroxypropyl β-cyclodextrin, micelles or liposomes, excipients and/or adjuvants. Pharmaceutical compositions may additionally comprise, for example, one or more of water, buffers such as, e.g., neutral buffered saline or phosphate buffered saline, ethanol, mineral oil, vegetable oil, dimethylsulfoxide, carbohydrates such as e.g., glucose, mannose, sucrose or dextrans, mannitol, adjuvants, antioxidants, chelating agents such as EDTA or glutathione and/or preservatives. Furthermore, one or more other active ingredients may (but need not) be included in the pharmaceutical compositions provided herein. For instance, the compounds of the invention may advantageously be employed in combination with an antibiotic, anti-fungal, or anti-viral agent, an anti histamine, a non-steroidal anti-inflammatory drug, a disease modifying anti-rheumatic drug, a cytostatic drug, a drug with smooth muscle activity modulatory activity or mixtures of the aforementioned.

Pharmaceutical compositions may be formulated for any appropriate route of administration, including, for example, parenteral administration. The term parenteral as used herein includes subcutaneous, intradermal, intravascular such as, e.g., intravenous, intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and intraperitoneal injection, as well as any similar injection or infusion technique.

Customary excipients include, for example, inert diluents such as, e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate, granulating and disintegrating agents such as, e.g., corn starch or alginic acid, binding agents such as, e.g., starch, gelatin or acacia, and lubricating agents such as, e.g., magnesium stearate or stearic acid.

For the prevention and/or treatment of diseases mediated by the six cytokines of the common gamma- chain receptor family - IL-2, IL-4, BL-7, IL-9, IL-15, and EL-21 - the dose of the biologically active mutein conjugate according to the invention may vary within wide limits and may be adjusted to individual requirements. Active mutein conjugates according to the present invention are generally administered in a therapeutically effective amount. The expression "therapeutically effective amount" denotes a quantity of the mutein conjugate(s) that produce(s) a result that in and of itself helps to ameliorate, heal, or cure the condition or disease mediated by the six cytokines of the common gamma- chain receptor family. Preferred doses range from about 0.1 mg to about 140 mg per kilogram of body weight per day (about 0.5 mg to about 7 g per patient per day). The daily dose may be administered as a single dose or in a plurality of doses. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Dosage unit forms will generally contain between from about 1 mg to about 500 mg of an active ingredient.

It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination (i.e. other drugs being used to treat the patient) and the severity of the particular disease undergoing therapy.

Preferred compounds of the invention will have certain pharmacological properties. Such properties include, but are not limited to reduced immunogenicity and advantageous pharmacokinetic

characteristics, such that the dosage forms can provide therapeutically effective levels of the mutein conjugate in vivo.

The mutein conjugate according to the invention as well as the pharmaceutical composition according to the invention can be used as a medicament, which can be administered to a patient (e.g., a human or an other mammal) parenterally, and will be present within at least one body fluid or tissue of the patient while modulating common gamma-chain receptor activity. Accordingly, the present invention also provides methods for treating patients suffering from a condition or disease mediated by any one of the six cytokines of the common gamma-chain receptor family.

The mutein conjugate of the invention, or the pharmaceutical composition according to the invention can be used in the prevention and/or treatment of a condition or disease mediated by any one of the six cytokines of the common gamma-chain receptor family, wherein the condition or disease is asthma, eczema, cancer, pancreatitis, type I diabetes (IDDM), Graves Disease, inflammatory bowel disease (IBD), Crohn's Disease, ulcerative colitis, irritable bowel syndrome, multiple sclerosis, rheumatoid arthritis, diverticulosis, systemic lupus erythematosus, psoriasis, ankylosing spondylitis, scleroderma, systemic sclerosis, psoriatic arthritis, osteoarthritis, atopic dermatitis, vitiligo, graft vs. host disease (GVHD), cutaneous T cell lymphoma (CTCL), Sjogren's syndrome, glomerulonephritis, IgA nephropathy, transplant rejection, atopic dermatitis, or anti-phospholipid syndrome. As used herein, the term "treatment" encompasses both disease-modifying treatment and symptomatic treatment, either of which may be prophylactic, i.e., before the onset of symptoms, in order to prevent, delay or reduce the severity of symptoms, or therapeutic, i.e., after the onset of symptoms, in order to reduce the severity and/or duration of symptoms.

Preferably, the method of preventing and/or treating any one of the above conditions or diseases mediated by any one of the six cytokines of the common gamma-chain receptor family according to the invention comprises administering to a subject in need thereof at least an effective amount of an inventive mutein conjugate or of a pharmaceutical composition according to the invention. The method of preventing or treating said conditions or disorders may, moreover, be characterized in that the active mutein conjugate or composition is intended to be administered by injection.

The mutein conjugate according to the invention may also be used as a research tool. For instance, the mutein conjugate according to the invention may be used as a diagnostic agent or theranostic, whereby such diagnostic agent may be used for the diagnosis of the diseases and conditions which can be addressed by the mutein conjugates of the present invention for therapeutic purposes as disclosed herein. For instance, for use as a research tool, the mutein conjugate of the invention can be labelled by isotopes, fluorescence or luminescence markers, or any other affinity label. The labelled compounds of the invention are, for example, useful for mapping the location of receptors in vivo, in vitro and in situ (e.g. in tissue sections via autoradiography) and as radiotracers for positron emission tomography (PET) imaging, single photon emission computerized tomography (SPECT) and the like to characterize those receptors in living subjects or other materials, e.g. tissue samples. Such uses and their respective conditions are known to those skilled in the art.

The activity of the mutein conjugate according to the invention can, for example, be determined in appropriate in vitro and/or in vivo assays. For instance, the biological activity of the mutein conjugates according to the present invention may be determined via a cell proliferation assay known to those skilled in the art. For example, human IL-2 activity may be measured in a cell proliferation assay using CTLL-2 mouse cytotoxic T cells as described by Gearing and Bird in Lymphokines and Interferons, A Practical Approach (Clemens, M.J. et al. (eds): IRL Press. 295 (1987)); the ED₅₀ for this effect is typically 0.05 - 0.25 ng/mL. The human IL-4 activity may, for example, be measured in a cell proliferation assay using TF-1 human erythroleukemic cells (Kitamura, T. et al. (1989) J. Cell Physiol. 140:323); the ED₅₀ for this effect is typically 0.05-0.2 ng/mL. Human IL-7 activity can, for example, be measured in a cell proliferation assay using PHA-activated human peripheral blood lymphocytes (PBL) as described by Yokota, T. et al. (Proc. Natl. Acad. Sci. USA 83:5894 (1986)); the ED₅₀ for this effect is typically 0.1-0.5 ng/mL. Human IL-9 activity can, for example, be determined in a cell proliferation assay using M07e human megakaryocyte leukemic cells (Avanzi, G. et al. (1988) Br. J. Haematol. 69:359); the ED₅₀ for this effect is typically 0.1-0.6 ng/mL. Human IL-15 activity may be measured in the same assay as human IL-9 activity, the ED₅₀ for this effect being typically 0.8-4 ng/mL. Human EL- 21 activity can, for example, be determined in a cell proliferation assay using Nl 186 human T cells (Parrish-Novak, J. et al. (2000) Nature 408:57); the ED₅₀ for this effect is typically 4 - 30 ng/mL.

Brief description of the figures

Figure 1. Mass spectrometry of purified IL-4 analogues (mixed disulfides) with site-specific chemical modification.

Figure 2. Mass spectrometry of purified IL-4 analogues (maleimide derivatives) with site-specific chemical modification.

Figure 3. Surface plasmon resonance (SPR) analysis of the interaction of selected IL-4 analogues with yc and IL-13Ral receptor ectodomain. The IL-4 analogues were bound to BL-4Ra ectodomain immobilized on a CM5 biosensor chip (see Figure 4). Then at time point 0 s perfusion with 1 μΜ yc (A, C) or 2 μΜ IL-13Ral (B, D) was started for a duration of 120 s after which the biosensor is again perfused with buffer to measure dissociation started at time 120 s. The sensorgrams recorded during perfusion with alone buffer was subtracted from the sensorgrams obtained with the receptor proteins.

Figure 4. Surface plasmon resonance (SPR) analysis of the interaction of selected IL-4 analogues with yc and IL-13Ral receptor ectodomain. The EL-4 analogues at 20 nM concentrations were bound to IL- 4Ra ectodomain immobilized on a CM5 biosensor chip for duration of 300 s. Then perfusion with 1 μΜ yc, or 2 uM IL-13Ral, or alone buffer was started for a duration of 120 s after which the biosensor is again perfused with buffer to measure dissociation. The sensorgrams recorded during in series perfusion of a surface without receptor protein has been subtracted.

Figure 5. Agonist activity of IL-4 analogues and inhibition of IL-4 or IL-13 dependent activity in HEK Blue cells. HEK Blue cells were incubated with 1 nM IL-4 analogues or with 1 nM EL-4 analogues plus 20 pM IL-4WT or plus 20 pM IL-13 as detailed under METHODS. The induced SEAP activity measured in all samples was related to the SEAP activity induced by alone 20 pM IL-4WT as 100%. Figure 6. Dose-dependent inhibition of IL-4 activity by IL-4 analogues in Jurkat cells. The level of STAT6 pY641 (see Figure S4) was evaluated at 300 pM IL-4WT in the presence of IL-4 analogues at the indicated concentrations. The activity of the IL-4SH mutein is included as reference.

Figure 7. Dose dependent stimulation of SEAP activity by IL-4 and IL-13 in HEK Blue cells. HEK Blue cells were incubated with the indicated concentrations of IL-4 or IL-13 for 30 min before SEAP activity was measured in the supernatant.

Figure 8. Dose dependent stimulation of STAT6 phosphorylation by IL-4 in Jurkat cells. Cells were incubated for 15 min with the indicated concentrations of IL-4. Then cell lysates were submitted to SDS-PAGE and Western blot analysis for the detection of phosphorylated STAT pY641.

Figure 9. Detection of EL-4 dependent STAT6 phosphorylation in Jurkat cells. Jurkat cells were preincubated for 5 min with the indicated concentrations of IL-4 analogues 121 SH and 121 -MA-PEG, and thereafter for another 15 min with the analogue plus 300 pM 11-4. Then cell lysates were submitted to SDS PAGE and Western blot analysis. For a quantitative evaluation the intensity of the band corresponding to STAT6 p641 was normalized to a-tubulin analysed in the same lysate.

The present invention is now further illustrated by the following examples from which further features, embodiments and advantages of the present invention may be taken.

Examples

Materials and Methods

Chemicals. E. coli glutaredoxin (GRX-01) was obtained from IMCO (Stockholm, Sweden), glutathione from Fluka, NADPH and yeast glutathione reductase (GR) from Sigma (St. Louis, MO, USA).

TMM(PEG)12 [(Methyl-PEG12)3-PEG4-Maleimide, MW 2360,75] was purchased from Thermo Scientific (Dreieich, Germany). MeOH-PEG-SH (Mr 5079) was obtained from Iris Biotech (Markt Redwitz, Germany). N-malenoyl-(S)-alanine was from GL Chemtech, Oakville, Canada. N-(2- aminoethyl)maleimide was from Sigma Aldrich.

Preparation of EL-4 Cysteine Muteins as Mixed Disulfides. The cDNA of IL-4 cysteine muteins was generated by recombinant PCR. The IL-4 muteins were expressed in E. coli, extracted from the inclusion bodies and refolded as detailed elsewhere [Reference 14]. Oxidized and reduced thiol compounds were added during refolding to generate specific mixed disulfides. The mixed disulfide with PEG (121-SS-PEG) was produced by adding lmM MeOH-PEG-SH and 0.1 mM dithio-dipyridine to the refolding mixture. IL-4 analogues were purified by SP-Sepharose chromatography and C4 HPLC as described [Reference 14], freeze-dried and stored in aliquots at -20oC until further use.

Enzymatic Reduction of BL-4 Cysteine Muteins with GRX1 from E. coli. The mixed disulfide of IL- 4 mutein and glutathione was reduced enzymatically employing E. coli glutaredoxin-1 [Reference 14]. The final reaction conditions were 100 mM potassium phosphate pH 7, 2 mM EDTA, 3 μΜ glutathione reductase, 0.2 mM NADPH, 0.5 mM glutathione (reduced form), and 50 μΜ glutathionylated IL-4 protein. The redox-reaction was started by adding 0.01 volume of a 300 μΜ glutaredoxin solution prepared according to the manufacturer's recommendation. The progress of the reaction was monitored by the oxidation of NADPH, which can be measured by the decrease in the extinction at 340 nm. After 30 min 0.025 volumes of 4M ammonium acetate pH 5 were added. The reaction mixture was immediately loaded on a 1 ml SOURCE 15S column (GE Healthcare) equilibrated with 25 mM ammonium acetate pH 5. The IL-4 protein was eluted via a salt gradient increasing from 0.5 M to 1.5 M sodium chloride in 60 min at a flow rate of 0.5 ml min-1. The protein-containing fractions from the ion exchange chromatography step (containing about 0.75 M NaCl) were immediately loaded onto a C4 reversed phase HPLC column (Vydac 214TP54, 250 x 4.6 mm) equilibrated with 0.1% TFA for further purification. The IL-4 protein was eluted via gradient increasing from 20% to 80% acetonitrile in 60 min at a flow rate of 0.8 ml min-1. IL-4 protein containing fractions were pooled, freeze-dried and dissolved in water at about 100 μΜ concentration.

Reaction of maleimido-analogues. Reaction of the reduced IL-4 muteins at preparative scale was performed at a 3-fold molar excess of the maleimido compound in PE7 buffer (0.1 M potassium phosphate pH 7.2 mM EDTA) at 20°C for 30 min. The conjugated proteins were first submitted to a SP-Sepharose chromatography at pH 5. The eluted protein was then mixed with 0.1% TFA to a final volume of 5 ml and loaded on a C4 revered phase HPLC column (Vydac 214TP54, 250 x 4.6 mm) equilibrated with 0.1% TFA. The modified protein was purified by applying an acetonitrile gradient running from 20 to 80% acetonitrile in 60 min and using a flow rate of 0.8 ml min-1. Protein containing fractions were pooled and freeze-dried. The conjugated IL-4 protein was dissolved in water at concentrations of about 100 uM, and stored at -20oC until further use.

Electrospray Ionization Mass Spectrometry Analysis (ESI-MS). ESI-MS was performed using an APEX-II FT-ICR (Bruker Daltonic GmbH, Bremen) equipped with a 7.4 T magnet and an Apollo ESI ion source in positive mode. Desalted proteins were dissolved in methanol/water/acetic acid

(49.5/49.5/1) to yield a sample concentration ranging between 1-5 μΜ. The sample was injected using a Hamilton syringe at a speed of 2 μΐ. per minute with a capillary voltage of 360 mV. Detection range was typically set to 300-3000 m/z in initial measurements. The detection range was then optimized to the signal-containing area. An accumulation of 256 scans was combined at a resolution of 256 K.

For evaluation, the mass spectra were deconvolved to the single protonated ion mode using the Bruker Xmas software. The most intense isotope signal was selected for mass determination.

Surface Plasmon Resonance (SPR) Interaction Analysis. Biosensor experiments were carried out employing a BIAcore 2000 system (GE Healthcare, Freiburg, Germany) at 25°C and a flow rate of 10 μΐ, min^"1 as described [Reference 16]. In short, biotinylated ectodomain of IL-4Ra receptor was immobilized on a CM5 biosensor chip loaded with streptavidin. Using the command COINJECT the IL-4Ra was first saturated with IL-4 protein by a 300-s perfusion with a solution of 10 to 20 nM IL-4 or IL-4 analogue. Thereafter a solution of recombinant ectodomain of yc at 1 uM concentration, respectively of IL-13al at 2 μΜ concentration was applied for 120 s.

Cellular assays for IL-4 and IL-13 bioactivity.

HEK-Blue™ IL-4/IL-13 cells (Cat. Code hkb-stat6 ; CAYLA - INVF/OGEN EUROPE, Toulouse) were grown in DMEM medium supplemented with 4.5g/l glucose, 10% fetal bovine serum, 50U/ml penicillin, 50 mg/ml streptomycin, 100 mg/ml normocinTM, 2 mM L-glutamine at 37oC and 5% C02. Cells were maintained in growth medium supplemented 10 μg/ml blasticidin and 100 μg/ml zeocynTM. Induction of SEAP (soluble alkaline phosphatase) by IL-4WT and IL-4 analogues was measured in the growth medium supplemented with heat-inactivated (30 min at 56 oC) fetal bovine serum according to the manufacturer's instructions.

Human acute T-cell leukemia Jurkat cells (ATCC ΤΓΒ-152) were grown at 37oC and 5% C02 in RPMI 1640 medium plus 10% fetal bovine serum. 1 ml of cells (2.5 x 106 / ml) in RPMI plus 1 % heat inactivated fetal bovine serum were plated in a 24-well dish for 4 h. Cells were preincubated for 5 min with the indicated concentration of IL-4 analogue, before they were incubated for another 15 min with IL-4 (300 pM). Cells were harvested at 4oC in a 1.5 ml vial by first centrifuging 2 x 1 min at 1000 rpm and then 10 sec at 5000 rpm. The sedimented cells were washed by resuspending in ice cold PBS (phosphate buffered saline) and centrifuging 2 min at 2500 rpm. Cell lysates were prepared by adding 180 μΐ Triton X-100 lysis buffer (20 mM Tris, pH 7.5,150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10 mM NaF, 1 mM Na3V04, 1 mM phenylmethylsulfonylfluoride, 5 μg / ml aprotinin, and 5 μg / ml leupeptidin) and thoroughly vortexed as described [Reference 17]. After incubation for 15 min on ice the mixture was centrifuged at 4 oC for 15 min at 14 000 rpm. Supernatants were transferred to new vials. After mixing with 3 volumes of 4 x concentrated SDS sample buffer 20 μΐ aliquots were submitted to SDS PAGE on 10% PAA gels and proteins were transferred to a

PVDF(polyvinylidenedifluoride) by western blotting. Polyclonal antibodies against STAT6 pY641 (Cell Signaling, Cat. No. 9361) were applied for the detection of phosphorylated STAT6. After stripping, the membranes were reprobed with monoclonal a-tubulin antibody (Sigma Aldrich, Cat. No. T6074) to verify equal loading. The ECL signals were quantified using the FluorChemQ Imaging System (ProteinSimple, Santaclara, USA). The STAT6 pY641 signals were normalized to the tubulin signal in each sample.

Protein Analysis. Protein concentrations of purified IL-4 proteins were determined by measuring a UV absorption spectrum between 250 and 320 nm. The molar extinction coefficient was calculated from the amino acid sequence using the ProtParam tool and used to determine the protein concentration according to the law of Lambert-Beer (molar extinction coefficient ε278 = 8900 M-l cm-1). SDS- PAGE was performed using a 12% polyacrylamide gel as described previously [Reference 14]. Protein samples for SDS-PAGE analysis were diluted 2- to 5-fold with sample buffer with 50 mM DTT (for analysis under reducing conditions) or without reducing agent (gel under non-reducing conditions to determine disulfide bonding). The gels were stained with Coomassie Blue R-250 for lh and destained overnight. Molecular weight standards phosphorylase b (97 kDa), bovine albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor soybean (20.1 kDa), and a-lactalbumin (14.4 kDa) were obtained from GE Healthcare (Freiburg, Germany).

Example 1

Preparation IL-4 cysteine muteins mutated at the interface with the yc/IL-13al interfaces.

Previous alanine scanning mutational analysis revealed that the main determinant of IL-4 for yc binding is Tyrl24, whereas for IL-13al binding it is Argl21 [Reference 15]. The structural interfaces show that the side chains of these residues are located at the centre of the respective binding epitope. More peripheral determinants are Ilel 1, Glul 10, Glul 14, Lysl 17, Thrl 18, and Glul 22. When expressed in E. coli the mutated proteins were recovered in the insoluble fraction ("inclusion bodies"). They were refolded and purified as described earlier [Reference 14]. As detailed before, the mono-glutathionylated proteins were recovered when a redox couple of oxidized and reduced glutathione was applied during the refolding step.

Example 2

Mixed disulfides of IL-4 cysteine mutein R121C. The mutein conjugates were prepared as mixed disulfides with glutathione according to Reaction Scheme 1 employing a glutathione redox couple during refolding (red/ox = 2/5 mM). Mass

spectrometry analysis (Fig. 1 , Table 1 below) revealed a molecular weight in accordance with the conclusion that the muteins were recovered with all three native disulfide bonds formed, and that the 7^th cysteine was completely conjugated with the thiol compound. The yield of conjugated R121C and of the other cysteine muteins (with the exception of IL-4 II 1C which was not further analysed) was similar to that of IL-4 wild type.

The mixed disulfides of IL-4 121C with cysteamine or thioglycolate could be generated in the similar way. For this, EL-4 analogue conjugated with thio-PEG (121-SS-PEG) was obtained by adding the reduced thiol compound plus an appropriate concentration of dithiodipyridine (see Methods). SDS PAGE analysis (Figure 3) of the PEGylated R121C analogue (121-SS-PEG) showed that only a high- molecular weight analogue is present in the non-reduced sample, which could be converted to a protein with IL-4 like mobility under reducing conditions. In conclusion, these results document that the formation of mixed IL-4 disulfides during protein refolding represents an efficient method to generate site-specific modification.

Example 3

Preparation of IL-4 cysteine mutein R121C with a free thiol group and of analogues modified by maleimide or maleimido-compounds.

Maleimidation was applied as a second highly specific thiol modifying reaction according to Reaction Scheme 2. A variety of maleimide compounds, including maleimide-PEGs, are commercially available, including those depicted in Table B above. Some However, it is a prerequisite that the cysteine mutein contains the engineered cysteine in a non-conjugated form. Previous experiments have shown that isolation of IL-4 cysteine muteins with a fully reduced engineered cysteine can be obtained by employing specific refolding conditions [Reference 14]. Also a selective chemical reduction by for instance DTT of EL-4 mutein mixed disulfides could not be achieved since the structure stabilizing native disulfide bonds were reduced simultaneously. An enzymatic reduction of glutathionylated mutein employing glutaredoxin 1 from E. coli (GRX1) proved to be more specific and allowed preparation of the non-conjugated mutein in good yield. For instance, the IL-4 analogue R121-SS-G became rapidly reduced enzymatically within 5 - 10 min to a species with one free thiol group. Thereafter, additional free thiol groups are generated in the R121C mutein as can be detected by SDS PAGE analysis of the MA-PEGylated species. The purified non-conjugated mutein (121 SH) can be reacted with various maleimide compounds. The selectivity of the reaction is documented with purified analogues modified with N-ethyl-maleimide (NEM; 121-MA-E), N-Maleonyl-(S)-alanine (121 -MA- ALA), or N-(2-Aminoethyl)maleimide (121- MA-EA), which showed after mass spectrometry analysis only one protein species with the expected mass (Figure 2; Table 1 below)

Table 1

Mass spectrometry analysis of mixed disulfides and maleimide derivatives of IL-4 cysteine mutein 121SH

Minor species represent oxidized (+16 Mr) or deaminated (-16 Mr) analogue. Mass spectrometry of PEGylated analogues was not possible, since the molecular weight of the PEG reagents were not homogeneous. However, SDS-PAGE analysis revealed that non-conjugated protein was present at only minor amounts and that the attached PEG was not cleaved off by reduction.

Example 4

Receptor interaction analysis shows that binding of yc and/or IL- 13 Ral can be lost after chemical modification of IL-4. More than a dozen residues of human IL-4 contribute to the interaction with the y_c or EL-13Ral receptor chain. The percentage of buried surface area for IL-4 residues (Table C), which upon complex formation come into contact with y_c or IL-13Ral provide a first estimate of the location of the residues within and their contribution to the receptor binding epitope.

Table C

Surface areas of IL-4WT residues buried upon complex formation with y_c or IL-13Ral

aThe buried surface area (BSA) was calculated from the difference between the accessible surface area (ASA) of respective IL-4 residues in complex with either y_c (PDB entry 3BPL) or IL-13Rccl (PDB entry 3BPN) and their accessible surface area in free IL-4. The BSA was normalized by setting the accessible surface area of the respective residue in free IL-4 as 100%.

SPR-based interaction analysis was performed in order to see if IL-4 conjugates are affected in their binding affinity for yc, IL-13Ral , or EL-4Ra. As detailed under Materials and Methods, the ectodomain of IL-4Ra was immobilized at a sensor chip by the streptavidin/biotin method. The receptor was first saturated by perfusion with the IL-4 analogue and then the ectodomain of yc or EL-13Ral was perfused and their binding recorded. The sensorgrams shown in Figure 4 were generated by subtracting the recording obtained from a control surface without immobilized receptor. IL-4WT and the analogues at 20 nM concentration bind rapidly within the first 100 to 200 sec to immobilized IL-4Ra ectodomain. Then equilibrium binding is achieved where the receptor is virtually saturated with the ligand. When after 300 sec perfusion is continued with buffer alone a slow dissociation of IL-4 proteins from the IL- 4Ra receptor (half-life tl/2 ~ 10 min) is observed. When buffer plus yc or IL-13al ectodomains are perfused an instantaneous rapid binding commences during the association phase and a rapid dissociation when after 120 sec buffer alone is applied again. The differential sensorgrams presented in Figure 6 show only the specific signals generated during the period of yc and IL-13 l association and dissociation.

The mixed disulfides of IL-4 as well as the maleimide analogues retained binding to the IL-4R ectodomain. The initial velocities measuring the diffusion limited phase of IL-4 binding to IL-4Ra were comparable between IL-4WT and the EL-4 analogues (see Fig. 2). Also the saturation levels obtained with IL-4 or IL-4 analogues were similar with the exception of the PEGylated analogues where a slightly higher signal at saturation level was obtained. For the measurement of the kinetic and equilibrium constants of the interaction between 11-4 and IL-4Ra low levels (<50 RU) of the IL-4Ra ectodomain had to be immobilized. Some IL-4 analogues were analysed under this condition and the constants were found to be comparable (Table 2). This suggests that the conformation of the IL-4 protein and its binding affinity for the IL-4Ra receptor is not altered by the R121C mutation itself or by the site specific modification at position 121. Table 2

Interaction of IL-4 mutein conjugates with the IL-4Ra receptor ectodomain. The kinetic rate constants for dissociation (k ) and association (kj of the complex were evaluated from separate SPR experiments performed at low levels (<I00 RU) of immobilized IL-4Ra ectodomain (see [Reference 16]). The initial rate constants (k) representing diffusion limited receptor association and equilibrium binding (RU_eq) were evaluated from experiments as shown, e.g., in Figure 4.

The IL-4 mutein R121C bound to the yc ectodomain at levels comparable to the IL-4 WT (Fig. 3, Table 3). However, the binding of the R121C mutein to the IL-13al receptor was strongly reduced (Figure 3). All analogues of R121 C mutein exhibited a strongly reduced affinity for the yc and IL-13Ral receptors. Remarkably, also the wild type like binding of R121C to yc is severely disrupted by the chemical modification (Table 3, Fig. 3). The faint residual binding of 121 -SS-G and 121 -SS-PEG to yc receptor was reproducibly observed and therefore appears to be significant. However, the binding of all other R121C analogues is below the limit of detection (<1%). The calculated residual binding of the analogues to IL-13Ral showed a larger variability than the binding to yc, since the equilibrium binding of IL-13Ral (2 uM) to IL-4WT was only 30 to 50 RU. A closer inspection of the sensorgrams reveals however, that the SPR curves of the maleimide R121C analogues are virtually super imposable. Table 3

Interaction of IL-4 mutein conjugates with receptor y_c and IL-I3RaI ectodomains. Sensorgrams as shown in Figures 3 and 4 have been evaluated for equilibrium binding of 1 μΜ γ₀ or 2 μΜ IL-13 l ectodomain. The equilibrium binding with IL-4 WT was normalized to account for 100%.

In conclusion, biosensor interaction analysis demonstrates that chemical modification at position 121 of IL-4 can reduce its binding affinity to the receptor yc by more than 99%, and binding affinity to IL- 13al by more than 95%.

Example 5

Chemically modified IL-4 analogues behave as antagonists in cellular assays.

HEK-BlueTM IL-4/EL-13 cells have been engineered to respond to IL-4 or BL-13 by the expression of a secreted alkaline phosphatase (SEAP). These cells express the EL-4Ra and IL-13Ral receptors. The response is very sensitive with EC50 values of 3 pM for DL-4 and 8 pM for IL-13 (Figure 7).

The IL-4 R121C analogues even at 1 nM concentration exhibited a low activity in HEKBlue cells. Compared to 20 pM IL-4WT (Figure 5, Table 4) the activity of the 121-SS-CA analogue amounted to 25%. The PEGylated analogues were virtual inactive similar to the IL-4DM double mutein, which was analysed in parallel. The mixed disulfides 121-SS-G and 121-SSTG showed about 3% residual activity, and the maleimide analogues between 7.4 and 11%. This means that the activity of the analogues in the HEK Blue assay can be estimated to be more than 1000-fold lower than that of IL-4 WT. Table 4

Agonist activity and inhibition ofIL-4 or IL-13 dependent SEAP expression (antagonist activity) ofIL-4 mutein conjugates in HEKBlue cells. All SEAP expression levels were related to that of 20 pM IL-4WT as 100%. For comparison the agonist and antagonist activity of 1 nM IL-4DM double mutein is included in the last line of the Table.

Remarkably, the IL-4 R121C analogues at 1 nM concentration inhibited the IL-4 or the IL-13 dependent response to levels of 11 to 55% of the 20 pM IL-4WT control. Most of the analogues were more potent inhibitors than the IL-4DM double mutein.

During a second biological assay the T-cell leukemia Jurkat cells were employed, which express EL- 4Ra and the yc receptor. They respond to IL-4 by the tyrosine phosphorylation of the STAT6 protein with an EC50 of about 50 pM (Figures 8 and 9). The cytokine EL-13 is not active in Jurkat cells, since no IL-13Ral receptor is present.

The 121SH mutein at 10 nM concentration showed almost the same agonist activity as 300 pM EL-4WT and almost no inhibitory activity. This finding is in accordance with the receptor binding assay where a wild type-like affinity for the yc receptor was observed (Table 3). For the various analogues at 10 nM concentration the agonist activity was found to be 3- to 50-times lower than that of 0.3 nM IL-4WT. No significant activity was found with IL-4DM double mutein (2 nM) and IL-13 (1 nM).

The residual activity measured in the presence of 0.3 nM IL-4WT plus 10 nM analogue was similar to the agonist activity of the analogue (Figure 6; Table 5). The 121 -MA-EA analogue similar as the IL- 4DM had only minimal agonist activity and it attenuated 300 pM IL-4WT activity to very low levels (Figure 6). Half maximal inhibition of 300 pM IL-4WT occurred at 300 pM concentration of analogues 121-SS-G, 121-SS-CA and 121-MA-EA (Figure 6). This suggests that these analogues bind as efficiently to the receptor as normal EL-4WT.

In conclusion, the results indicate that chemical modification at a single IL-4 position (position 121) can generate analogues with low agonist activity and strong antagonist activity comparable to that of the IL- 4DM double mutein. More specifically, the results presented herein demonstrate that a single chemical modification at position 121 of IL-4 can disrupt yc as well as IL-13al binding. The resulting analogues inhibit IL-4 and IL-13 dependent responses in immunological and inflammatory cells. Thus, IL-4 analogues with modification at position 121 have antagonist properties similar to the IL-4DM double mutein. Three types of residues were linked to EL-4 mutein R121 C either via a disulphide bond or via a N-maleimide group: (1) A positively charged residue (ethylamine), (2) a negatively charged residue (glycolate or alanine), and (3) a bulky and polar residue (branched TMS(PEG)12 , Mr 2360.75, and linear MeO-PEG, Mr 5079). All three types of analogues retained less than 1.2 % of the yc binding compared to IL-4WT or the IL-4 R121C muteine. The 1 μΜ concentration of yc ectodomain used during the SPR analysis was below the about 3 uM KD [Reference 15]. Therefore, the level of residual yc binding to the analogues signifies a roughly proportional loss in affinity. This means the affinity is reduced more than 80-fold compared to DL-4WT or the IL-4 R121C muteine. The equally large deficiency in affinity of all analogues suggests that already small geometrical constraints in the binding epitope are responsible for the disruption of yc binding. Charge and size of the attached residue appear to be of secondary importance since the analogue obtained with N-ethyl maleimide exerts a comparably low affinity as the other maleimide analogues.

Table 5

Agonist and antagonist activity of IL-4 mutein conjugates in Jurkat cells. STAT6 phosphorylation (see Figure 9) was determined for the mutein conjugates at 10 nM concentration in the absence and in the presence of 300 pM IL-4WT. The STAT6 phosphorylation in the presence of 300 pM IL-4WT was taken as 100 %. For comparison the agonist/antagonist activities of 2 nM IL-4DM and the agonist activity of 1 nMIL-13 is included in the lower part of the table. IL-4 mutein conjugate Mutein conjugate Mutein conjugate [10 nM]

[10 nM] plus IL-4WT [300 pM]

STAT6 pY641 [% max]

121-SS-G 9 19

121-SS-CA 18 10

121-SS-TG 32 39

121-SS-PEG n.m. n.m

121 SH 82 91

121-MA-NEM 20 27

121 MA-EA 2 3

121 -MA- ALA n.m. n.m.

121 MA-PEG 24 28

300 pM IL-4WT =100

2 nM IL-4DM 1 2

1 nM IL-13 2

Not surprisingly, the binding to the IL-13Ral receptor ectodomain is strongly reduced in all analogues. IL-4 Argl21 is the major determinant for IL-13Ral binding affinity and seems to exert a specific role in the contact, since already the free thiol group at position 121 in the mutein results in a loss of binding affinity for IL-13Ral .

In conclusion, the IL-4 analogues modified at position 121 exhibit a strongly reduced binding affinity not only for the receptor EL-13Ral but also for the common gamma chain yc. The mechanism causing the inactivation of the yc binding epitope appears to be the generation of steric hindrance for the association of the yc receptor rather than the loss or mismatch of positive or negative charge.

As also shown in the present experiments "oxido shuffling" reagents can be used to generate site specific conjugation of thiol compounds with engineered cysteine side chains of IL-4. In addition to conjugates with glutathione, those with cysteamine, thioglycolate, or even PEG could be generated and purified. Importantly, functional analysis and mass spectrometry indicated that the three native disulfide bonds of IL-4 were readily formed in parallel under the applied conditions. The disulfide-conjugated analogues appear to be stable in cell culture medium during the period of the assay, since a pronounced antagonist activity of the purified analogues could be observed.

Example 6

Comparison of IL-4 analogues modified at different positions

A series of glutathionylated IL-4 analogues modified at positions within or close to the receptor contacts was prepared and tested for biological activity and receptor binding. For details of the preparation and the assays see the Materials and Methods section and the Examples above. The biological activities and receptor binding properties of the glutathionylated IL-4 analogues tested are compiled and presented in Table D.

Table D

Biological activities and receptor binding of glutathionylated IL-4 analogues

"The glutathione-modified analogs were obtained from IL-4 cysteine muteins after refolding involving a glutathione redox couple. ^bReceptor binding was evaluated from surface plasmon resonance (SPR) interaction analysis. Biological activity was measured during a HEK Blue receptor gene assay and during a Jurkat STAT Y641 phosphorylation assay.

As can be taken from the results presented in Table D, all IL-4 analogues but IL-4 121 -SS-G showed substantial biological activities. Specifically, only chemical modification at position 121 resulted in a virtually complete inhibition of y_c and IL-13Ral binding and a more than 90% loss of biological activity in HEK Blue and Jurkat cell assays. Particularly noteworthy, chemical modification at position 124 disrupted biological activity and receptor binding only partially, even though Tyrl24 (as Argl21) is located at the center of the binding epitopes and is nearly completely buried upon complex formation. Since the residue at position 121 (Argl21) has been established to be critical for IL-13Ral but not for y_c binding, the finding that the single chemical modification at position 121 disrupted y_c as well as IL- 13Ral interaction is surprising.

References

1. Brandt, E. B. & Sivaprasad, U. (2011) Th2 Cytokines and Atopic Dermatitis, Journal of clinical & cellular immunology. 2.

2. Ingram, J. L. & Kraft, M. (2012) IL-13 in asthma and allergic disease: asthma phenotypes and targeted therapies, The Journal of allergy and clinical immunology. 130, 829-42; quiz 843-4.

3. Kuperman, D. A. & Schleimer, R. P. (2008) Interleukin-4, interleukin-13, signal transducer and activator of transcription factor 6, and allergic asthma, Current

molecular medicine. 8, 384-92.

4. Arron, J. R., Scheerens, H. & Matthews, J. G. (2013) Redefining approaches to asthma: developing targeted biologic therapies, Adv Pharmacol. 66, 1-49.

5. Li, Z., Jiang, J., Wang, Z., Zhang, J., Xiao, M., Wang, C, Lu, Y. & Qin, Z. (2008) Endogenous interleukin-4 promotes tumor development by increasing tumor cell resistance to apoptosis, Cancer research. 68, 8687-94.

6. Di Stefano, A. B., Iovino, F., Lombardo, Y., Eterno, V., Hoger, T., Dieli, F., Stassi, G. & Todaro, M. (2010) Survivin is regulated by interleukin-4 in colon cancer stem cells, Journal of cellular physiology. 225, 555-61.

7. Tony, H. P., Shen, B. J., Reusch, P. & Sebald, W. (1994) Design of human interleukin-4 antagonists inhibiting interleukin-4-dependent and interleukin-13 -dependent responses in T-cells and B-cells with high efficiency, European journal of biochemistry / FEBS. 225, 659-65.

8. Tomkinson, A., Tepper, J., Morton, M., Bowden, A., Stevens, L., Harris, P., Lindell, D., Fitch, N., Gundel, R. & Getz, E. B. (2010) Inhaled vs subcutaneous effects of a dual IL-4/IL-13 antagonist in a monkey model of asthma, Allergy. 65, 69-77.

9. Wenzel, S., Wilbraham, D., Fuller, R., Getz, E. B. & Longphre, M. (2007) Effect of an interleukin-4 variant on late phase asthmatic response to allergen challenge in asthmatic patients: results of two phase 2a studies, Lancet. 370, 1422-31. 10. Shanafelt, A. B., Forte, C. P., Kasper, J. J., Sanchez-Pescador, L., Wetzel, M., Gundel, R. & Greve, J. M. (1998) An immune cell-selective interleukin 4 agonist, Proceedings of the National Academy of Sciences of the United States of America. 95, 9454-8.

1 1. Kreitman, R. J., Puri, R. K., Leland, P., Lee, B. & Pastan, I. (1994) Site-specific conjugation to interleukin 4 containing mutated cysteine residues produces interleukin 4-toxin conjugates with improved binding and activity, Biochemistry. 33, 1 1637-44.

12. Pan, C, Roczniak, S., Greve, J. M., Yung, S. L., Longphre, M., Wong, T. M. & Tomkinson, A. (2008) Modified IL-4 mutein receptor antagonists, United States Patent. US 7,404,957 B2.

13. Pan, C, Roczniak, S., Greve, J. M., Yung, S. L., Longphre, M., Wong, T. M., Tomkinson, A., Boisvert, D., Burmeister Getz, E. & Delaria, K. (2010) Modified IL-4 mutein receptor antagonists, United States Patent. US 7,785,580 B2.

14. Duppatla, V., Gjorgjevikj, M., Schmitz, W., Kottmair, M., Mueller, T. D. & Sebald, W. (2012) Enzymatic deglutathionylation to generate interleukin-4 cysteine muteins with free thiol, Bioconjugate chemistry. 23, 1396-405.

15. Letzelter, F., Wang, Y. & Sebald, W. (1998) The interleukin-4 site-2 epitope determining binding of the common receptor gamma chain, European journal of biochemistry / FEBS. 257, 1 1 -20.

16. Shen, B. J., Hage, T. & Sebald, W. (1996) Global and local determinants for the kinetics of interleukin-4/interleukin-4 receptor alpha chain interaction. A biosensor study employing recombinant interleukin-4-binding protein, European journal of biochemistry / FEBS. 240, 252-61.

17. Drechsler, J., Grotzinger, J. & Hermanns, H. M. (2012) Characterization of the rat oncostatin M receptor complex which resembles the human, but differs from the murine cytokine receptor, PloSone. 7, e43155.

Claims

1. A mutein conjugate, the mutein conjugate comprising a mutein moiety and a non-protein moiety, wherein

the mutein moiety has an amino acid sequence as set forth in SEQ ID No. 1 to SEQ ID No. 12; the non-protein moiety comprises a group that is reactive with a side chain sulfhydryl group (SH) of the mutein; and

the mutein moiety is linked to the non-protein moiety through covalent bonding of the side chain sulfhydryl group of the residue at position 121 of SEQ ID No. 1 , at position 122 of SEQ ID No. 2, at position 126 of SEQ ID No. 3, at position 127 of SEQ ID No. 4, at position 143 of SEQ ID No. 5, at position 144 of SEQ ID No. 6, at position 115 of SEQ ID No. 7, at position 1 16 of SEQ ID No. 8, at position 108 of SEQ ID No.9, at position 109 of SEQ ID No. 10, at position 1 16 of SEQ ID No. 1 1, or at position 117 of SEQ ID No. 12, wherein the covalent bond is formed via a thioether, sulfur or disulfide bond.

2. The mutein conjugate according to claim 1, wherein the non-protein moiety further comprises a nonprotein polymer.

3. The mutein conjugate according to claim 2, wherein the non-protein polymer is a water-soluble polymer of synthetic or natural origin.

4. The mutein conjugate according to claim 2 or 3, wherein the non-protein polymer is polyethylene glycol (PEG), polypropylene glycol, polyoxyalklylene, poly(N-vinylpyrrolidone), N-hydroxy-ethyl methacrylamide copolymer, poly(2-ethyl-2-oxazoline), poly(N-acryloylmorpholine), hyaluronic acid, or polysyalic acid.

5. The mutein conjugate according to any one of claims 2 to 4, wherein the non-protein polymer is a linear or branched polyethylene glycol having a molecular weight (MW) of from 400 to 50000 Da.

6. The mutein conjugate according to any one of claims 1 to 5, wherein the group of the non-protein moiety that is reactive with the side chain sulfhydryl group (SH) of the mutein is a sulfhydryl-reactive group selected from maleimides, brom- or iodoalkyls, pyridyldisulfides, thiosulfonates, vinylsulfones, and compounds containing a thiol group.

7. The mutein conjugate according to any one of claims 1 to 6, wherein the mutein moiety is linked to the non-protein moiety via a thioether or disulfide bond.

8. The mutein conjugate according to any one of claims 1 to 7, wherein the non-protein moiety is a thiol compound selected from glutathione; cysteamine; thioglycolate; MeO-[(CH₂)2-0]n-(CH₂)2-SH, n being an integer of from 4 to 150; and MeO-(PEG_n)-SH having a molecular weight of about 5000 Da.

9. The mutein conjugate according to any one of claims 1 to 7, wherein the non-protein moiety is a maleimide compound selected from N-ethyl-maleimide, N-(2-ethylamine)-maleimide, N-(S)-alanine- maleimide, and TMM(PEG)12.

10. The mutein conjugate according to any one of claims 1 to 9, wherein the mutein moiety has an amino acid sequence as set forth in SEQ ID No. 1 or SEQ ID No. 2.

1 1. The mutein conjugate according to any one of claims 1 to 10, wherein the mutein conjugate binds to the common gamma chain with a KD of >0,1 mM.

12. The mutein conjugate according to any one of claims 1 to 1 1 , wherein the mutein has an amino acid sequence as set forth in SEQ ID No. 1 ;

the non-protein moiety is glutathione, cysteamine, thioglycolate, MeO-(PEG)-SH, N-ethyl- maleimide, N-(2-ethylamine)-maleimide, N-(S)-alanine-maleimide, or TMM(PEG) 12; and

13. A pharmaceutical composition that comprises one or more mutein conjugate(s) according to any one of claims 1 to 12 and, optionally, at least one carrier substance, excipient and/or adjuvant.

14. . The mutein conjugate according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 13, for use as a medicament.

15. The mutein conjugate according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 13, for use in the prevention and/or treatment of asthma, eczema, cancer, pancreatitis, type I diabetes (IDDM), Graves Disease, inflammatory bowel disease (IBD), Crohn's Disease, ulcerative colitis, irritable bowel syndrome, multiple sclerosis, rheumatoid arthritis, diverticulosis, systemic lupus erythematosus, psoriasis, ankylosing spondylitis, scleroderma, systemic sclerosis, psoriatic arthritis, osteoarthritis, atopic dermatitis, vitiligo, graft vs. host disease (GVHD), cutaneous T cell lymphoma (CTCL), Sjogren's syndrome, glomerulonephritis, IgA nephropathy, transplant rejection, atopic dermatitis, anti-phospholipid syndrome.