AU2007202312A1 - IFNAR2 mutants, their production and use - Google Patents
IFNAR2 mutants, their production and use Download PDFInfo
- Publication number
- AU2007202312A1 AU2007202312A1 AU2007202312A AU2007202312A AU2007202312A1 AU 2007202312 A1 AU2007202312 A1 AU 2007202312A1 AU 2007202312 A AU2007202312 A AU 2007202312A AU 2007202312 A AU2007202312 A AU 2007202312A AU 2007202312 A1 AU2007202312 A1 AU 2007202312A1
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- Prior art keywords
- ifn
- ifnar2
- anyone
- mutant polypeptide
- ifnar2 mutant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Description
AUSTRALIA
Patents Act 1990 COMPLETE SPECIFICATION Standard Patent Applicant: YEDA RESEARCH AND DEVELOPMENT CO. LTD.
Invention Title: IFNAR2 MUTANTS, THEIR PRODUCTION AND USE The following statement is a full description of this invention, including the best method for performing it known to us:
I
la IFNAR2 MUTANTS, THEIR PRODUCTION AND USE FIELD OF THE INVENTION The present invention relates to mutant polypeptides of the beta chain of the type I IFN receptor (MIFNAR2) with enhanced affinity for interferon-p as compared to the wild type protein for prolonging the effect of IFNP in vivo.
The entire disclosure in the complete specification of our Australian Patent Application No.
2002366976 is by this cross-reference incorporated into the present specification.
BACKGROUND OF THE INVENTION Interferons are classified either as the leukocyte and fibroblast derived Type I interferons, or as 1i the mitogen induced or "immune" Type 11 interferons (Pestka et al, 1987). Through analysis of sequence identities and common biological activities, type I interferons include interferon alpha (IFN-a), interferon beta (IFN-P) and interferon omega (IFNw), while type II interferon includes interferon gamma (IFN7).
The IFNa, IFNp and IFNw genes are clustered on the short arm 25 of chromosome 9 (Lengyl, 1982). There are at least 25 non-allelic IFNa genes, 6 non-allelic IFNw genes and a single IFNp gene. All are believed to have evolved from a single common ancestral gene. Within species, IFNa genes share at least 80% sequence identity with each other. The IFNP gene shares approximately 50% sequence identity with IFNa; and the IFNw gene shares homology with IFNa (Weissman et al, 1986; Dron et al, 1992). IFNa has a molecular weight range of 17-23 kDa (165-166 amino acids), IFNp, about 23 kDa (166 amino acids) and IFNo, about 24 kDa (172 amino acids).
Type I interferons are pleiotropic cytokines having activity such as host defense against viral and parasitic infections, anti-cancer properties and as immune modulators (Baron et al, 1994; Baron et al, 1991). Type I interferon physiological responses include anti-proliferative activity on normal and transformed cells, stimulation of cytotoxic activity in lymphocytes, natural killer cells and phagocytic cells, modulation of cellular differentiation, stimulation of expression of class I MHC antigens, N \MeIboum\Cass\Pateie\50O-SS9n'S\3416 AU l\Specis\P53416 AU I Spccification 2007 5 21 doc 21/05/07 inhibilion of class II MHC, and modulation of a variety of cell surface receptors. Under normal physiological conditions, IFNa and IFNP (IFNa/p) are secreted constitutively by most human cells at low levels with expression being up-regulated by addition of a variety of inducers, comprising infectious agents (viruses, bacteria, mycoplasma and protozoa), dsRNA, and cytokines (M-CSF, IL-la, IL-2, TNFa). The actions of Type I interferon in vivo can be monitored using the surrogate markers, neopterin, oligoadenylate synthetase, and P2 microglobulin (Alam et al, 1997; Fierlbeck et al, 1996; S;lmon et al, 1996).
Type I interferons (IFNa/p/co) act through a cell surface receptor complex to induce specific biologic effects, such as anti-viral, anti-tumor, and immune modulators. The type I IFN receptor (IFNAR) is a hetero- multimeric receptor complex composed of at least two different polypeptide chains (Colamonici et al, 1992; Colamonici et al, 1993; Platanias et al, 1993). The genes coding for these chains are found on chromosome 21, and their proteins are expressed on the surface of most cells (Tan et al, 1973). The receptor chains were originally designated alpha and beta and have been renamed IFNAR1 for the alpha subunit and IFNAR2 for the beta subunit. In most cells, IFNAR1 (alpha chain, Uze subunit) (Uze et al, 1990) has a molecular weight of 100- 130 kDa, while IFNAR2 (beta chain, 3 L, IFNa/pR) has a molecular weight of 100 kDa.
In certain cell types (monocytic cell lines and normal bone marrow cells) an alternate receptor complex has been identified, where the IFNAR2 subunit 3 s) is expressed as a truncated receptor with a molecular weight of 51 kDa. The IFNAR1 and IFNAR2 3s and PL subunits have been cloned (Novick et al, 1994; Domanski et al, 1995). The IFNAR2 Ps and PL subunits have identical extracellular and transmembrane domains; however, in the cytoplasmic domain they only share identity in the first 15 amino acids. The IFNAR2 subunit alone is able to bind IFNa/p, while the IFNARI subunit is unable to bind IFNa/p. When the human IFNAR1 receptor subunit alone was transfected into murine L-929 fibroblasts, no human IFNas except IFNa8/IFNaB were able to bind to the cells (Uze et al, 1990). The human IFNAR2 subunit, transfected into L cells in the absence of the human IFNAR1 subunit, bind human IFNa, binding with a Kd of approximately 0.45 nM. When human IFNAR2 subunits were transfected in the presence of the human IFNARI subunit, high affinity binding could be shown with a Kd of 0.026-0.114 nM (Novick et al, 1994; Domanski et al, 1995). It is estimated that from 500- 20,000 high affinity and 2,000-100,000 low affinity IFN binding sites exist on most cells. Although the IFNAR1/2 complex (a/Ps or a/L) subunits bind IFNa with high affinity, only the a/PL pair appears to be a functional signaling receptor.
Transfeclion of the IFNAR1 and the IFNAR2 PL subunits into mouse L-929 cells, followed by incubation with IFNa2, induces an anti-viral state, initiates intracellular protein phosphorylation, and causes the activation of intracellular kinases (Jakl and Tyk2) and transcription factors (STAT 1, 2, and 3) (Novick et al, 1994; Domanski et al, 1995). In a corresponding experiment, transfection of the IFNAR2 ps subunit was unable to initiate a similar response. Thus, the IFNAR2 pI subunit is required for r functional activity (anti-viral response) with maximal induction occurring in association with the IFNAR1 subunit.
In addition to membrane bound cell surface IFNAR forms, a soluble IFNAR has been identified in both human urine and serum (Novick et al, 1994; Novick et al, 1995; Novick et al, 1992; Lutfalla et al, 1995). The soluble IFNAR isolated from serum has an apparent molecular weight of 55 kDa on SDS- PAGE, while the soluble IFNAR from urine has an apparent molecular weight of 40-45 kDa (p40). Transcripts for the soluble p40 IFNAR2 are present at the mRNA level and encompass almost the entire extracellular domain of the IFNAR2 subunit with two additional amino acids at the carboxy terminal end. There are five potential glycosylation sites on the soluble IFNAR2 receptor. The soluble p40 IFNAR2 has been shown to bind IFNa2 and IFNp and to inhibit in vitro the anti-viral activity of a mixture of IFNa species ("leukocyte IFN") and individual Type I IFNs (Novick et al, 1995). A recombinant IFNAR2 subunit Ig fusion protein was shown to inhibit the binding of a variety of Type I IFN species (IFNaA, IFNaB, IFNaD, IFNp, IFNa Conl and IFNo) to Daudi cells and a/ps subunit double transfected COS cells.
Type I IFN signaling pathways have been identified (Platanias et al, 1996; Yan et al., 1996; Qureshi et al. 1996; Duncan et al., 1996; Sharf et al, 1995; Yang et al, 1996).
Initial events leading to signaling are thought to occur by the binding of IFNa/P/co to the IFNAR2 subunit, followed by the IFNAR1 subunit associating to form an IFNARI/2 complex (Platanias et al, 1994). The binding of IFNa/P/o to the IFNAR1/2 complex results in the activation of two Janus kinases (Jakl and Tyk2), which are believed to phosphorylate specific tyrosines on the IFNAR1 and IFNAR2 subunits.
Once these subunits are phosphorylated, STAT molecules (STAT 1, 2 and 3) are phosphorylated, which results in dimerization of STAT transcription complexes followed by nuclear localization of the transcription complex and the activation of specific IFN inducible genes.
A randomized, double-blinded, placebo-controlled, two-year multicenter study demonstrated that natural human fibroblast interferon (interferon beta) administered intrathecally (IT) is effective in reducing the exacerbations of exacerbating-remitting multiple sclerosis The mean reduction in exacerbation rate of 34 patients with MS who received interferon beta administered IT was significantly greater during the sludy than that of 35 control patients who received placebo (Jacobs et al. 1987).
It) The pharmacokinetics and pharmacodynamics of Type 1 IFNs have been assessed in humans (Alan et al, 1997; Fierlbeck et al, 1996; Salmon et al, 1996). The clearance of IFNP is fairly rapid with the bioavailability of IFNP lower than expected for most cytokines. Although the pharmacodynamics of IFNp has been assessed in humans, no clear correlation has been established between the bioavailability of IFNp and clinical efficacy. In normal healthy human volunteers, administration of a single intravenous (iv) bolus dose (6 MIU) of recombinant CHO derived IFNp resulted in a rapid distribution phase of 5 minutes and a terminal half-life of about 5 hours (Alam et al, 1997). Following subcutaneous (sc) or intramuscular (im) administration of IFNp, serum levels are flat with only about 15% of the dose systemically available. The pharmacodynamics of IFNp following iv, im or sc administration (as measured by changes in 2 5/-oligoadenylate synthetase 5'-AS) activity in PBMCs) were elevated within the first 24 hours and slowly decreased to baseline levels over the next 4 clays. The magnitude and duration of the biologic effect was the same regardless of the route of administration.
A multiple dose pharmacodynamic study of IFNO has been conducted in human melanoma patients (Fierlbeck et al, 1996) with IFNp being administrated by sc route, three times per week at 3 MIU/dose over a six-month period. The pharmacodynamic markers, 5' -AS synthetase, 32- microglobulin, neopterin, and NK cell activation peaked by the second injection (day 4) and dropped off by 28 days, remaining only slightly elevated out to six months.
Purification and refolding of the extracellular part of human IFNAR2 (IFNAR2-EC) expressed in Escherichia coli and its characterization with respect to its interaction with interferon alpha2 (IFNc2) has been reported (Piehler and Schreiber 199A). The 25 kDa, nonglycosylated IFNAR2-EC was shown to be a stable, fully active protein, which inhibits antiviral activity of IFNa2. The stoichiometry of binding IFNa2 is 1:1, as determined by gel filtration, chemical cross-linking and solid-phase detection. The affinity of this interaction was found to be about 3 nM (Piehler and Schreiber 2001). The rate of complex formation is relatively high compared to other cytokine-receptor interactions. The salt dependence of the association kinetics suggests a limited but significant contribution of electrostatic forces towards the rate of complex formation. The dissociation constant increases with decreasing pH according to the protonation of a base with a pKa of 6.7. The affinity of IFN/ to IFNAR2 is about two-fold higher than that of IFNa2 to IFNAR2 (Piehler and Schreiber 1999B).
Single mutations in the binding site of IFNAR2 allowed mapping of differences in binding of IFa2 and IFN3 (Piehler and Schreiber 1999B). For example, a mutation H-78A was found to stabilize the complex with IFN3 nearly by two fold, while destabilized the complex with IF.2 more than two fold.
A mutation N100A was found to hardly affect the rates for binding IFa2, whereas it decreased the dissociation rate constant for IFN/ by almost four folds.
EP1037658 discloses that the in vivo effect of Type I interferon (IFN) can be prolonged by administering the interferon in the form of a complex with an IFN binding chain of the human 2 0 interferon alpha beta receptor (IFNAR) i.e. IFNAR behaves as a carrier protein for IFN. Such a complex also improves the stability of the IFN and enhances the potency of the IFN. The complex may be a non-covalent complex or one in which the IFN and the IFNAR are bound by a covalent bond or a peptide. EP1037658 also discloses that storing IFN in the form of such a complex improves the storage life of the IFN and pennits storage under milder conditions than would otherwise be possible.
There exists a need for an IFNAR2 with improved affinity towards IFNf3, but not to IFNa2, making IFNAR2 a better and specific carrier for IFN3.
All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.
H:\deborhk\keep\speci\202 3 66976.doc 30/10/06 SUMMARY OF THE INVENTION The invenlion provides an IFNAR2 mutant polypeptide (MIFNAR2) mutated at amino acid residues histidine 78 and asparagine 100, having higher affinity for interferon-P (IFN-3) than the wild type polypeptide, or an analog, functional derivative, fusion protein or salt thereof. The mutations are substitutions of amino acids, preferable conservative amino acids, more preferable alanine, aspartic acid and histidine. The IFNAR2 mutant has about 25, preferably 50 and more preferably 100fold higher affinity than the wild type protein and a preferred of value is about pM.
More particularly the invention provides an IFNAR2 mutant polypeptide fragment comprising the extracellular domain.
In addition the invention provides a DNA encoding the IFNAR2 mutant polypeptide of the invention, a vector comprising said DNA, host cells comprising said vector and methods for producing a polypeptide mutant of the invention by cultivating said host cells and isolating the produced polypeptide mutant.
In another aspect the invention provides the use of an IFNAR2 mutant polypeptide for the manufacture of a medicament for modulating the effects of IFN, preferably IFNp, in vivo.
The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of an IFNAR2 mutant or its extracellular domain fragment, to be administrated alone or co-administrated with IFN, more preferably IFNp, separately or covalently bound. More specifically the invention provides pharmaceutical compositions for augmenting the anti-viral, anti-cancer and immune modulating properties of IFNP and for treatment autoimmune diseases, such as 3( multiple sclerosis, rheumatoid arthritis, myasthenia gravis, diabetes, ulcerative colitis iand lupus.
SFurthermore, the invention provides methods of treatment of autoimmune diseases, viral disease and cancer, comprising administration of an IFNAR2 mutant polypeptide of the invention.
CN, In adldilion the invention provides the use of IFNAR2 mutant polypeptide, preferably co-administered with an IFN antagonist, for inhibition of IFN activity in a disease which is aggravated or caused by IFN.
Cl€ C, 10 The invention also provides the use of the IFNAR2 mutant polypeptide of the O invention in a formulation to prevent IFN oligomerization BRIEF DESCRIPTION OF THE FIGURES Figure (left) depicts a simulation of the concentrations of bound and free IFNP using a constant concentration of IFN (50 pM) and increasing concentrations of with -type -IFNAR2 EC (left) and mutant IFNAR2 EC (right), with-a Kd of 3 nM and pM respectively, calculated in accordance with the law of mass action.
Figure 2 shows the amino acid sequence of the extracellular domain IFNAR2 protein (not including the leader sequence) and the modified amino acid residues (marked with an asterisk).
Figure 3 shows the binding of IFNp and IFNa2 to the IFNAR2 EC H78A/N100A mutant. Association and disassociation of IFNp and IFNa2 to the Wild type IFNAR2 EC (upper panel), to the IFNAR2 EC H78A/N100A mutant (middle panel) and the binding of the wild type and mutant IFNAR2 EC H78A/N100A mutant to IFNp (Lower panel) was measured using reflectometric interference spectroscopy (RifS), 8 with IFNAR2 immobilized to the surface (described in Piehler and Schreiber 2001). Y-axis signal (nanometer) and the X-axis time (seconds).
Figure 4 shows occlusion of IFN/3 by IFNAR2 wild type and mutants. A constant amount of IFN/ (10 pM) was mixed with different concentrations of IFNAR2 (R2) wild type and mutants (single mutants R2NI00A and R2H78A, double mutants R2 H78A/N100A, R2 H78A/N100H and R2 H78A/N100D), and the residual antiviral activity at equilibrium was determined in WISH cells. In the upper box, a plot of the antiviral activity of IFN3 as a function of its concentration in the absence of IFNAR2 is shown (Y-axis survival index). This plot is used as a standard to determine how much of the IFN/ is free (active) in the anti-viral assay.
DETAILED DESCRIPTION OF THE INVENTION In the claims which follow and in the description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.
The invention relates to a mutant of the beta chain of the type I IFN receptor (IFNAR2), mutated at amino acid residues H78 and N100 (see DNA sequence of wild type IFNAR2 in Figure 2, SEQ ID N: 1) having increased affinity to IFN/3, but not to IFNc2 (MIFNAR2). The invention relates also to a drug carrier system to enhance activity of IFN comprising the extracellular domain (EC) of MIFNAR2. The invention relates to MIFNAR2, or an analog, functional derivative, fusion protein, 2 5 fragment thereof or salts thereof.
Carriers are usually administered to prolong the intra-vascular retention time of proteins having molecular weight below 50,000 daltons interferon). Particularly beneficial are such carriers that bind non-covalently and permit constant release of the drug. Using such a carrier is desirable in order to have at any time some portion of the free drug available for curative activity (about and some amount of drug bound to the carrier and protected (about Figure 1 (left panel) depicts a simulation of the concentration of bound and free IFN3 in the presence of different concentrations of IFNAR2 based on the law of mass action and on a Kd of 3 5 3nM (tested by reflectometric interference spectroscopy [RifS]). This H \deborahk\keep\peci\2002 3 66976.doc 30/10/06 simulation shows that in order to achieve 20 of free IFNf (10 pM, which equals about 100 Units), and 80 bound, a very high concentration of IFNAR2 protein such as 12.5 nM (which is equivalent to 300 pg/Kg of non-glycosilated IFNAR2) is needed.
Thus, using an IFNAR2 mutant with 50 fold and higher affinities to IFNB as a carrier (see simulation Figure 1, right panel), would be advantageous since with such a mutant theoretically only about 0.24 nM will be required to get 20 IFN3 free (which is equivalent to 6 pg/Kg).
A mutant of the IFNAR2 with increased affinity to IFN (MIFNAR2) was generated.
To get MIFNAR2 EC, the wild type IFNAR2 EC (Figure 1, SEQIDNOI) was modified in two amino acid residues, residue 78 histidine and residue 100 asparagine (see Figures 2, 3 and 4, SEQ ID sNO: 2, 3, and This mutant IFNAR2 EC proteins turned out to be a better carrier specifically for IFN-0 i.e. has improved affinity for IFN-8 while its affinity towards IFNo2 remains unchanged. The affinity of the mutants for IFNB was found to be 26, 40 and above 50 fold higher than that of the wild type (Table The results obtained show that despite the increased affinity of this mutated soluble receptor (Kd of the H78A/N100A IFNAR2 mutant 30pM versus Kd of WT protein 3nM), enough IFNB remains unbound and therapeutically active, as evidenced by the anti-viral protective activity of VSV challenged WISH cells (Figure The results show also that the levels of IFN occlusion (bound IFN at equilibrium conditions) obtainable with wild type IFNAR2 EC could be accomplished using lower concentrations of IFNAR mutants EC. The best results are obtained with mutants modified in both residues, particularly when both amino acids are mutated to alanine, H78A/N100A IFNAR2, e.g. in order to get 80% ofIFNf bound (8 pM occluded and 2 pM free IFN) about 30 fold less H78A/N100A IFNAR mutant is required over the wild type IFNAR2 protein.
This result show that the double mutated IFNAR2 occludes more effectively IFNf and administration of considerably lower amounts is required to fulfill its carrier activity towards IFNO.
The advantages of using M IFNAR2 EC are that it is possible to administrate lower quantities (thus technically feasible) of the receptor as a carrier (II) because of the stabilizing activity of the mutant it is possible to reduce the amount of IFN/3 9 SUBSTITUTE SHEET (RULE 26) administrated, and consequently to reduce some of the unwanted side effects of inlerleron Ireatment (111) the increase in the activity by the mutant is specific to IFNp, O and (IV) that in some inflammatory disorders, where it may be required to lower the IFN concentrations, it is possible under certain conditions to use this mutant as an effective antagonist specifically towards IFNp, but not IFNa2.
MIFNAR2-EC may be administered alone to stabilize and enhance the activity of endogenous IFNp, this is particularly useful for the treatment of patients having a disease or condition which naturally causes the elevation of native IFN, so that the 1 10 IFN will already be circulating in the body for its intended natural effect of fighting N' such disease or condition. MIFNAR2-EC will act specifically on endogenous IFNp, O but less towards IFNa2. Alternatively, MIFNAR2-EC may be co-administrated together with IFN, preferably IFNP or may be administrated covalently bound to IFNp to modulate the activity of IFN-P. Preferably, MIFNAR2 and IFNp used to generate the complex are recombinant molecules.
The technology required to produce the fusion protein of the mutant EC IFNAR2 and IFN is similar to the technology described for wild type IFNAR/IFN complex production which is described in detail in W09932141, wherein the IFNAR2 mutated in H78 and N100 (MIFNAR2) is used instead of the wild type version.
The implications of using a MIFNAR2/ IFNp non covalently bound complex according to the invention is that lower concentrations of the IFNAR2 EC are required and may be used for a variety of therapeutic indications in which IFN by itself is therapeutically active.
These indications include those in which free IFNs have shown some therapeutic activity, such as anti-viral, anti-cancer and immune modulatory activity. It is expected that the mutant IFNAR2/IFN complex, by virtue of its greater potency, enhanced activity and/or improved pharmacokinetics half-life), will be more efficacious in treating viral, oncologic and autoimmune disorders.
When administered in vivo, the interferon receptor complex enhances the bioavailability, pharmacokinetics, and/or pharmacodynamics of the IFN, thus augmenting the anti-viral, anti-cancer and immune modulating properties of the IFN.
The preferred molecules for use in the complexes of the present invention comprise the amino acid sequence of native IFN-P and MIFNAR2 (SEQ ID NOs: 2, 3, and The native sequence is that of a naturally occurring human IFN-p. Such sequences are known and can be readily found in the literature. Naturally occurring allelic variations are also considered to be native sequences.
The present invention also concerns analogs of the above MIFNAR2 EC.
Such analogs may be ones in which up to about 30, preferably up to 20 and most preferably 10 amino acid residues may be deleted, added or substituted by others in the proteins, except mutations in residues 78 and 100 which results in a decrease in the affinity of MIFNAR2 for IFN-p to the wild type IFNAR2 affinity for IFN-p.
These analogs are prepared by known synthesis and/or by site-directed mutagenesis techniques or any other known technique suitable therefore.
Any such analog preferably has a sequence of amino acids sufficiently duplicative of that of the basic MIFNAR2 such as to have substantially similar activity thereto. Thus, it can be determined whether any given analog has substantially the same activity and/or stability as the protein and complex of the invention by means of routine experimentation, comprising subjecting each such analog to binding and biological activity tests. MIFNAR2 EC analogs may bind IFNp with at least 15 folds and about to 100 fold higher affinity over the wild type protein wherein the affinity towards IFNa2 is not significantly changed. The MIFNAR2 EC analogs my exhibit a Kd of about 30 pM and lower towards IFNp. The binding tests for MIFNAR2 and IFN interaction may involve analytical gel filtration, optical heterogeneous phase detection (such as surface plasmon resonance [SPR], or reflectometric interference spectroscopy [RifS] which resembles the widely used BIACORE technique) and fluorescent spectroscopy (Piehler and Schreiber 1999A Piheler and Schreiber 2001).
Analogs of the complex which can be used in accordance with the present invention, or nucleic acid sequence coding therefore, include a finite set of substantially corresponding sequences as substitution peptides or polynucleotides which can be routinely obtained by one of ordinary skill in the art, without undue experimentation, based on the teachings and guidance presented herein. For a detailed description of protein chemistry and structure, see Schulz et al, Principles of Protein Structure, Springer Verlag, New York (1978); and Creighton, proteins: Structure and Molecular Properties, W.H. Freeman Co, San Francisco (1983), which are hereby incorporated by reference.
For a presentation of nucleotide sequence substitutions, such as codon preferences, see Ausubel et al (1987, 1992), A.1. I-A. 1.24, and Sambrook et al (1987, 1992), 6.3 and 6.4, at Appendices C and D.
Preferred changes for analogs in accordance with the present invention are what are known as "conservative" substitutions. Conservative amino acid substitutions of those in the sequence of the proteins in the invention may include synonymous amino acids within a group, which have sufficient similar physicochemical properties that substitution between members of the group will preserve the biological function of the molecule (Grantham, 1974). It is clear that insertions and deletions of amino acids may also be made in the above-defined sequences without altering their function, particularly if the insertions or deletions only involve a few amino acids, under thirty, and preferably under ten, and do not remove or displace amino acids which are critical to a functional conformation, cysteine residues (Anfinsen, 1973). Analogs produced by such deletions and or insertions come within the purview of the present invention. Preferably, the synonymous amino acid groups are those defined in Table I.
More preferably, the synonymous amino acid groups are those defined in Table II; and most preferably the synonymous amino acid groups are those defined 5 in Table III.
TABLE I Preferred Groups of Synonymous Amino Acids Amino Acid Synonymous Group Ser Ser, Thr, Gly, Asn A rg Arg, Gin, Lys, Giu, His Lett Ilie, Phe, Tyr, Met, Val, Leu Pro Gly, Aia, Thr, Pro JO) Tiir Pro, Ser, Ala, Gly, His, Gin, Thr Ala Gly, Thr, Pro, Ala Val Met, Tyr, Phe, Ile, Leu, Val Gly Ala, Thr, Pro, Ser, Gly Ile Met, Tyr, Phe, Val, Leu, Ile Phe Tip, Met, Tyr, Ilie, Val, Leu, Phe Tyr Tip, Met, Phe, Ile, Vai, Leu, Tyr Cys Ser, Thr, Cys His Giu, Lys, Gin, Thr, Arg, His Gin Giu, Lys, Asn, His, Thr, Arg, Gin Asn Gin, Asp, Ser, Asn Lys Giu, Gin, His, Arg, Lys Asp Glu, Asn, Asp Giu Asp, Lys, Asn, Gin, His, Arg, Glu Met Phe, Ilie, Val, Leu, Met Trp Trp TAI3LE2 More Prefcrred Groups of Synonymous Amino Acids Amino Acid Synonymous Group Ser Ser Arg His, Lys, Arg ILu Leu, Ilie, Phe, Met it)Pro Ala, Pro Thr Thr Ala Pro, Ala Vai Val, Met, Ile Gly Gly Ile Ile, Met, Phe, Val, Leu Phe Met, Tyr, Ile, Leu, Phe Tyr Phe, Tyr Cys Cys, Ser His His, Gin, Arg Gin Glu, Gin, His Asn Asp, Asn Lys Lys, Arg Asp Asp, Asn Glu Giu, Gin Me[ Met, Phe, Ile, Val, Leu Trp Trp CN TABLE3 Most Preferred Groups of Synonymous Amino Acids Amino Acid Synonymous Group Scr Ser Arg Arg Leu Leu, lie, Met S1) Pro Pro Thr Thr Ala Ala Val Val Gly Gly lie lie, Met, Leu Phe Phe Tyr Tyr Cys Cys, Ser His His Gin Gin Asn Asn Lys Lys Asp Asp Glu Glu Met Met, lie, Leu Trp Met Examples of production of amino acid substitutions in proteins which can be used for obtaining analogs of MIFNAR2 or MIFNAR2 EC for use in the present invention include any known method steps, such as presented in U.S. patents RE 33,653; 4,959,314; 4,588,585 and 4,737,462, to Mark et al; 5,116,943 to Koths et al; 4,965,195 1o Namen et al; and 5,017,691 to Lee, et al, and lysine substituted proteins presented in US patent 4,904,584 (Shaw et al).
The term "essentially corresponding to" is intended to comprehend analogs with minor changes to the sequence of the basic MIFNAR2 or MIFNAR2 EC which do not affect the basic characteristics thereof e.g. its specific enhanced binding and affinity to IFN3. The type of changes which are generally considered to fall within the "essentially corresponding to" language are those which would result from conventional mutagenesis techniques of the DNA encoding the complex of the invention, resulting in a few minor modifications, and screening for the desired activity in the manner discussed above.
Preferably, the MIFNAR2 portion of the complex will have a core sequence which is the same as that of the native sequence or biologically active fragment thereof, or a variant thereof which has an amino acid sequence having at least 70% identity to the native amino acid sequence and retains the biological activity thereof. More preferably, such a sequence has at least 85% identity, at least 90% identity, or most preferably at least 95% identity to the native sequence.
With respect to the IFN portion of the complex, the core sequence which may be used is the native sequence, or a biologically active fragment thereof, or a variant thereof which has an amino acid sequence having at least 70% identity thereto, more preferably, at least 85% or at least 90% identity, and most preferably at least identity. Such analogs must retain the biological activity of the native IFN sequence or fragment thereof, or have antagonist activity as discussed herein below.
The term "sequence identity" as used herein means that the sequences are compared as follows. The sequences are aligned using Version 9 of the Genetic Computing Group GAP (global alignment program), using the default (BLOSUM62) matrix (values -4 to +1 with a gap open penalty of -12 (for the first null of a gap) and a gap extension penalty of -4 (per each additional consecutive null in the gap). After alignment, percentage identity is calculated by expressing the number of matches as a percentage of the number of amino acids in the claimed sequence.
Analogs in accordance with the present invention may also be determined in accordance with the following procedure. With respect to either the MIFNAR2 portion of the complex or the IFN portion of the complex, the DNA of the IFNAR and IFN sequence are known to the prior art and is either found in the literature cited in the background section of the present specification or can be readily located by those of ordinary skill in the art. Polypeptides encoded by any nucleic acid, such as DNA or t) RNA, which hybridize to the complement of the native DNA or RNA under highly stringent or moderately stringent conditions, as long as that polypeptide maintains the biological activity of the native sequence or, in the case of IFN, either maintains the biological activity of MIFNAR2 or MIFNAR2 EC or possesses antagonistic activity, are also considered to be within the scope of the present invention.
"stringent conditions" refers to hybridization and subsequent washing conditions, which those of ordinary skill in the art conventionally refer to as "stringent". See Ausubel et al., Current Protocols in Molecular Biology, supra, Interscience, N.Y., and 6.4 (1987, 1992), and Sambrook et al. (Sambrook, J. Fritsch, E. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor 21) Laboratory Press, Cold Spring Harbor, NY).
Without limitation, examples of stringent conditions include washing conditions 12-20C below the calculated Tm of the hybrid under study in, 2 x SSC and SDS for 5 minutes, 2 x SSC and 0.1% SDS for 15 minutes; 0.1 x SSC and 05% SDS at 37"C for 30-60 minutes and then, a 0.1 x SSC and 05% SDS at 68"C for 30-60 minutes. Those of ordinary skill in this art understand that stringency conditions also depend on the length of the DNA sequences, oligonucleotide probes (such as 10-40 bases) or mixed oligonucleotide probes. If mixed probes are used, it is preferable to use letramethyl ammonium chloride (TMAC) instead of SSC. See Ausubel, supra.
"Functional derivatives" as used herein covers derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, they do not destroy the biological activity of the corresponding protein of the complex as described herein and do not confer toxic properties on compositions containing it O or the complex made therefore. Derivatives may have chemical moieties, such as CK", carbohydrate or phosphate residues, provided such a fraction has the same biological activity and remains pharmaceutically acceptable.
C. For example, derivatives may include aliphatic esters of the carboxyl of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives or free amino groups of SII) the amino acid residues formed with acyl moieties alkanoyl or carbocyclic Saroyl groups) or O-acyl derivatives of free hydroxyl group that of seryl or threonyl residues) formed with acyl moieties. Such derivatives may also include for example, polyethylene glycol side-chains, which may mask antigenic sites and extend the residence of the complex or the portions thereof in body fluids.
The term "fused protein" refers to a polypeptide comprising an MIFNAR2, or MIFNAR2 EC or a analog or fragment thereof, fused with another protein, which, has an extended residence time in body fluids. An MIFNAR2 or MIFNAR2 EC may thus be fused to another protein, polypeptide or the like, e.g., an immunoglobulin or a fragment thereof.
A "fragment" according to the present invention may e.g. be a fragment of MIFNAR2 or MIFNAR2 EC. The term fragment refers to any subset of the molecule, that is, a shorter peptide that retains the desired biological activity.
Fragments may readily be prepared by removing amino acids from either end of the MIFNAR2 molecule and testing the resultant fragment for its properties to bind to IFN-P. Proteases can be used for removing one amino acid at a time from either the N-terminal or the C- terminal of a polypeptide are known, and so determining fragments, which retain the desired biological activity, involves only routine experimentation.
As active fragment of an MIFNAR2, analogs and fused proteins thereof, the present invention further covers any fragment or precursors of the polypeptide chain of the protein molecule alone or together with associated molecules or residues linked thereto, sugar or phosphate residues, or aggregates of the protein molecule or the sugar residues by themselves, provided said fragment has subslantially similar activity.
The term "salts" herein refers to both salts of carboxyl groups and to acid addition salts ol amino groups of the complex of the invention or analogs thereof. Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with mineral acids, such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids, such as, for example, acetic acid or oxalic acid. Of course, any such salts must have substantially similar biological activity to the complex of the invention or its analogs.
The lerm "biological activity" as used herein is interpreted as follows. Insofar as the MIFNAR2 is concerned, the important biological activity is its ability to bind to IFNI 2( with increased affinity. Thus, analogs or variants, salts and functional derivatives must be those chosen so as to maintain this interferon-binding ability.
This can be tested by routine binding assay experiments. In addition, fragments of the MIFNAR2, or analogs thereof, can also be used as long as they retain their interferonenhanced binding activity. Fragments may readily be prepared by removing amino acids from either end of the interferon-binding polypeptide and testing the resultant for interferon-binding properties.
Additionally, the polypeptide which has such interferon-binding activity, be it MIFNAR2, MINFAR2 EC, an analog functional derivative, fragment, can also contain additional amino acid residues flanking the interferon-binding polypeptide. As long as the resultant molecule retains the increased interferon-binding ability of the core polypeptide, one can determine whether any such flanking residues affect the basic and novel characteristics of the core peptide, its interferon-binding characteristics, by routine experimentation. The term "consisting essentially of", when referring to a specified sequence, means that additional flanking residues can be present which do not affect the basic and novel characteristic of the specified sequence.
This term does not comprehend substitutions, deletions or additions within the specilied sequence.
While MIFNAR2 or MIFNAR2 EC have been used throughout this description and in the examples, it should be understood that this is merely the preferred example and that the IFNARI subunit, and particularly its extracellular domain, may be used together with MIFNAR2 or MIFNAR2 EC.
With respect to the interferon part of the complex of the present invention, the biological activity which must be maintained in any analog, functional derivative, fusion protein or fragment is the activity of the interferon relied upon for the intended utility. In most instances, this will be the ability to bind to a native cell surface receptor and thereby mediate signal production by the receptor. Thus, any such analog, derivative or fragment should maintain such receptor agonist activity to be useful in the present invention for such a utility. On the other hand, it is sometimes useful to have a molecule with antagonist activity on the receptor so as to prevent the biological activity of native interferon. Such an antagonist can also be used for prolonged beneficial effect by means of the complex of the present invention. For such utilities in which it is desired to eliminate an undesired effect of interferon, analogs which are still bound by the receptor and by the IFNAR portion of the complex but which do not mediate a signal and block signal generation by the native interferon on that receptor (i.e interferon antagonist), may also be considered to be biologically active for the purpose of this invention and to be encompassed by the term interferon when used with respect to the complexes of the present invention. Straightforward assays can determine whether any such analog maintains such receptor agonist activity or has receptor antagonist activity and would, thus, be useful for one of the utilities of the present invention.
The present invention also relates to DNA sequences encoding MIFNAR2 EC e.g.
DNA encoding the amino acid sequences in SEQIDNOs: 2, 3 and 4 or analogs, and fragments thereof, as well as DNA vectors carrying such DNA sequences for expression in suitable prokaryotic or eukaryotic host cells.
The ability to generate large quantities of heterologous proteins using a recombinant protein expression system has led to the development of various therapeutic agents, II) I-PA and EPO (Edington, 1995). The various expression hosts from which recombinant proteins can be generated range from prokaryotic in origin bacteria) (Olins, 1993), through lower eukaryotes yeast) (Ratner, 1989) to higher eukaryotic species insect and mammalian cells (Reuveny, 1993; Reff, 1993). All of these systems rely upon the same principle introducing the DNA sequence of the protein of interest into the chosen cell type (in a transient or stable fashion, as an integrated or episomal element) and using the host transcription, translation and transportation machinery to over-express the introduced DNA sequence as a heterologous protein (Keown, 1990).
Various protocols for the production of recombinant heterologous proteins are described (Ausubel et al., Current Protocols in Molecular Biology, Greene Publications and Wiley Interscience, New York, NY, 1987-1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989).
In addition to the expression of native gene sequences, the ability to manipulate DNA at the nucleotide level has expedited the development of novel engineered sequences which, although based on natural proteins, possess novel activities as a result of the alteration in primary protein structure (Grazia, 1997).
Moreover, chosen sequences of DNA can be physically linked to generate transcripts 31) which develop into novel fusion proteins where once independent proteins are now expressed as one polypeptide unit (Ibanez, 1991). The activity of such fusion proteins can be different, more potent, than either of the individual proteins (Curtis, 1991).
For co-administration of MIFNAR2 EC with IFN, human IFNP may be derived from a production process, which uses the mammalian Chinese hamster ovary cell (CHO) as C( disclosed in EP220574. Type 1 interferons can be expressed in a variety of host cells including bacteria (Utsumi, 1987), insect (Smith, 1983) and human (Christofinis, 198 Also human MIFNAR2 or a fragment thereof may be expressed using the CHO hosi cell. For secretion of MIFNAR2 EC from CHO cells, MIFNAR2 EC DNA sequence may be ligated to the sequence of the human growth hormone signal peptide as described in the patent application W00022146. Alternatively, soluble receptors, such as MIFNAR2 EC, may be expressed successfully in bacterial expression systems (Terlizzese, 1996).
O The invention also relates to a pharmaceutical composition comprising as active ingredient an, MIFNAR2, MIFNAR2 EC, MIFNAR2 EC/IFN complex or analogs, fusion proteins, functional derivatives, fragments thereof or mixtures thereof or salts thereof and a pharmaceutical acceptable carrier, diluent or excipient. An embodiment of the pharmaceutical composition of the invention includes a pharmaceutical composition for enhanced IFN type action, in the treatment of viral diseases, in anticancer therapy, in immune modulation therapy e.g. in autoimmune diseases and other applications of interferons and cytokines related thereto.
The pharmaceutical compositions of the invention are prepared for administration by mixing the an, MIFNAR2, MIFNAR2 EC, MIFNAR2 EC/IFN complex or analogs, fusion proteins, functional derivatives, fragments thereof or mixtures thereof or salts thereof with physiologically acceptable stabilizers and/or excipients, and prepared in dosage form, by lyophilization in dosage vials. The method of administration can be via any of the accepted modes of administration for similar agents and will depend on the condition to be treated, intravenously, intramuscularly, and subcutaneously, by local injection or topical application, or continuously by infusion, etc. The amount of active compound to be administered will depend on the route of adminis ration, the disease to be treated and the condition of the patient.
The invention relates to method for treatment of autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, myasthenia gravis, diabetes, lupus and ulcerative colitis, comprising aministration of a theurapeutically effective amount of an, MIFNAR2, MIFNAR2 EC, MIFNAR2 EC/IFN complex or analogs, fusion proteins, functional derivatives, fragments thereof or mixtures thereof or salts thereof.
The invention relates to method for treatment of a viral disease such as granulomatous discase, condyloma acuminatum, juvenile laryngeal papillomatosis, hepatitis A or chronic infection with hepatitis B and C viruses, comprising aministration of a theurapeutically effective amount of an, MIFNAR2, MIFNAR2 EC, MIFNAR2 EC/IFN complex or analogs, fusion proteins, functional derivatives, fragments thereof or mixtures thereof or salts thereof.
The invention relates to method for treatment of various types of cancer such as such as hairy cell leukemia, Kaposi's sarcoma, multiple myeloma, chronic myelogenous leukemia, non-Hodgkins's lymphoma or melanoma, comprising aministration of a theurapeutically effective amount of an, MIFNAR2, MIFNAR2 EC, MIFNAR2 EC/IFN complex or analogs, fusion proteins, functional derivatives, fragments thereof or mixtures thereof or salts thereof.
In the above methods the MIFNAR2, MIFNAR2 EC, MIFNAR2 EC/IFN complex or analogs, fusion proteins, functional derivatives, fragments thereof or mixtures thereof or salts thereof may be administered together with IFN, preferably IFN-3.
A "therapeutically effective amount" is such that when administered, an, MIFNAR2, MIFNAR2 EC, MIFNAR2 EC/IFN complex or analogs, fusion proteins, functional derivatives, fragments thereof or mixtures thereof or salts thereof results in modulation of the biological activity of IFN-P. The dosage administered, as single or multiple doses, to an individual may vary depending upon a variety of factors, including the route of administration, patient conditions and characteristics (sex, age, body weight, health, size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired. Adjustment and manipulation of established dosage ranges are well within the ability of those skilled in the art, as well as in vitro and in vivo methods of determining the activity of an, MIFNAR2, MIFNAR2 EC, MIFNAR2 EC/IFN complex or analogs, fusion proteins, functional derivatives, Iragments thereof or mixtures thereof or salts thereof.
Local injection, for instance, will require a lower amount of the protein on a body weight basis than will intravenous infusion.
Free IFNP has a tendency to oligomerize. To suppress this tendency, present day formulations of IFN3 have an acidic pH, which may cause some localized irritation when administered. As an, MIFNAR2, MIFNAR2 EC, or analogs, fusion proteins, functional derivatives, fragments thereof or mixtures thereof or salts thereof can serve as a superior stabilizing over the wild type version factor for IFNp and thereby prevent oligomerization, its use in IFNp formulations can serve to stabilize the IFNp and thereby obviate the necessity of acidic formulations. Accordingly, a non-acidic pharmaceutical composition containing an, MIFNAR2, MIFNAR2 EC, or analogs, fusion proteins, functional derivatives, fragments thereof or mixtures thereof or salts thereof, along with other conventional pharmaceutically acceptable excipients, is also a part of the present invention.
The present invention also concerns uses of an, MIFNAR2, MIFNAR2 EC, MIFNAR2 EC/IFN complex or analogs, fusion proteins, functional derivatives, fragments thereof or mixtures thereof or salts thereof for anti-viral, anti-cancer and immune modulation therapy. Specifically, the mutant interferon receptor and mutant interferon receptor and interferon complexes of this invention are useful for anti-viral therapy in such therapeutic indications as chronic granulomatous disease, condyloma acuminatum, juvenile laryngeal papillomatosis, hepatitis A and chronic infection with hepatitis B and C viruses.
In particular, the mutant interferon receptor and mutant interferon receptor and interferon complexes of this invention are useful for anti-cancer therapy in such therapcutic indications as hairy cell leukemia, Kaposi's sarcoma, multiple myeloma, chronic myelogenous leukemia, non-Hodgkins's lymphoma and melanoma.
The mutant interferon receptor and mutant interferon receptor and interferon complexes of this invention are also useful for immune modulation therapy, in auloimmune diseases e.g. multiple sclerosis, rheumatoid arthritis, myasthenia gravis, diabetes, lupus, ulcerative colitis etc.
"An autoimmune disorder" is a disease in which a person's immune system begins to attack his or her own body. The immune system creates antibodies against its own tissues. Virtually every part of the body is susceptible to an autoimmune disorder.
The mutant interferon receptor and mutant interferon receptor and interferon complexes are also useful for treating neurodegenerative diseases, preferably multiple sclerosis.
The invention further relates to a pharmaceutical composition comprising an, MIFNAR2, MIFNAR2 EC, MIFNAR2 EC/IFN complex or analogs, fusion proteins, functional derivatives, fragments thereof or mixtures thereof or salts thereof to a pharmaceutical composition comprising an expression vector, in particular a lentiviral gene therapy vector expressing an, MIFNAR2, MIFNAR2 EC, MIFNAR2 EC/IFN complex or analogs, fusion proteins, fragments thereof.
The terms "treating" as used herein should be understood as preventing, inhibiting, attenuating, ameliorating or reversing any or all symptoms or cause(s) of the disease.
Having now described the invention, it will be more readily understood by reference to the following examples that are provided by way of illustration and are not intended to be limiting of the present invention.
EXAMPLES
Example 1: Protein expression and purification.
IFNAR2-EC (extracellular domain) and IFNa was expressed in E. coli purified by ion exchange and size-exclusion chromatography as described (Piehler Schreiber, 1999A). The levels of expression of IFNAR2-EC mutants were as high as the wild type. Wild type, glycosylated IFNp produced in CHO (disclosed in EP220574).
Protein concentrations were determined from absorbance at 280 nm (Piehler Schreiber, 1999A) with 1:280 18,070 M-1 for IFNa2, 1:280 30,050 M-1 for 1FNp and 1:280 26,500 M-l for IFNAR2-EC (corrected to 1:280 21,100 M-1 for the tryptophan mutants of IFNAR2- EC W102A and W74F). Protein purity was analyzed by SD5-PAGE under non-reducing conditions.
Example 2: Generation ofIFNAR EC mutants.
Site-directed mutagenesis was carried out by PCR with the template pT72CR2 (Piehler and Schreiber 1999) and with 18-21 nucleotide primers containing the mutated codon using high fidelity polymerases pwo (Boehringer Mannheim) and Pfu (Stratagene) as described in detail (Albeck Schreiber, 1999). After phosphorylation and ligation, the mutated plasmids were used to transform E. coli TG1 cells. The sequence of the whole expressed gene containing the mutation was verified by DNA sequencing (Ausubel et al., Current Protocols in Molecular Biology, Greene Publications and Wiley Interscience, New York, NY, 1987-1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989).
Mutants were generated in which two amino acid residues, histidine 78 (H78) and Asparagine 100 (N100), were mutated: A- both to alanine residue (H78A/N100A mutant), 1- to alanine and aspartic acid respectively (H78A/N100D) and C- to alanine and hislidine respectively (H78A/N100H).
Example 3: Thermodynamic and kinetic analysis.
All ihcrmodynamic and kinetic data were obtained from label-free heterogeneous phase detection. The interaction between IFNp2 and IFNAR2-EC was monitored by rcflectometric interference spectroscopy (RIfS) under flow-through conditions as described (Piehler Schreiber, 1999A). This technique is similar to Biacore and is used to accurately measure affinity of binding between two proteins. IFNAR2-EC (wild-type or mutant) was immobilized trough immobilized specific antibodies (as described by Piehler and Schreiber 2001). All measurements with IFNP, IFNa2 and IFNAR2-EC were carried out in 50 mM Hepes with 500 mM NaCI and 0.01 Triton XI00 at pH 7.4. The interaction was measured at 500 mM NaCI in order to eliminate non-specific interactions with the surface, which was observed with IFNp at 150 mM NaCI.
Association and dissociation kinetics were measured by standard injection protocols and corrected by blank runs. Dissociation rate constants were measured at IFN concentration in the range of 1-1000 nM in order to saturate the surface. The total range of dissociation was used for fitting a 1:1 kinetic model (Piehler Schreiber, 2001).
Example 4: Anti viral activity assay.
Anti-viral activity of IFN3 was assayed as the inhibition of the cytophatic effect of vesicular stomatitis virus (VSV) on human WISH cells (Rubinstein et al., 1981).
Example 5: Measurement of IFN binding to mutant IFNAR2 Binding of the IFNf and IFNo2 to the H78A/N100A mutant (example 2) was measured and compared to the wild type EC receptor by RifS (example While the association rate of IFN3 to the H78A/N 00A mutant was found to be similar to that of the wild type (Figure 3) the disassociation rate was found to be significantly lower.
The calculated affinity of IFNf to H78A/N100A mutant is about 30 pM versus the affinity to the WT protein of about 3 nM. In contrast to IFN/3, both the association and disassociation rate of IFNa2 to the H78A/Nl00A mutant, were found to be similar to the rates obtained with the wild type protein (Figure These results show that the affinity of the IFNAR2 mutant mutant was found to be approximately 100 times higher than the wild type towards IFN0 and unchanged towards IFNo2.
Example 6: Relative affinities of interferon towards the mutant IFNAR2.
The binding and affinities of IFNAR EC receptor and mutant receptor EC (example 2) to IFNO and IFNa2 were measured using RIS, with IFNAR2 wild type or mutant immobilized to the surface trough specific antibodies (example After measuring the affinities, the relative affinities were obtained by comparing the Kd of the mutant receptor over the Kd of the wild type receptor (Table 4).
The Kd of binding of interferon to IFNAR2 extracellular domain (EC) was measured by RIfS and was found to be about 3nM (example The Kd of IFNO binding to H78A/N100A (EC) mutant was about 30 pM. The exact measurement of Kd for this mutant was not possible, because binding was to tight to get good data from RIfS.
The Kd of IFN2 to the H78A/N I00A EC mutant was found to be similar to the wild type receptor. The results in Table 4 show the relative affinities of the IFNAR EC mutants compared to the wild type IFNAR2 receptor EC. The mutants were the following: mutated in one amino acid residue, H78A or NI00A, and mutated in two amino acids H78A/N100A, H78A/N100D and H78A/N100H, wherein the amino acid N100 is mutated into alanine, aspartic acid or histidine respectively (example The results demonstrate that the single mutations in IFNAR2 increase the affinity of the complex from 4.6 up to 7.3 fold, while the double mutation causes a synergistic effect, increasing the affinity of the complex by 26 and to above 50-fold. The best mutant in terms of affinity was found to be the double mutant with the N100 modified to alanine, exhibiting over 50 fold increased affinity versus the wild type version.
ifnar2 Wt 1.0 S78A 0.4, 4.61 N I00A 2.01 7.3 H-8 A/ N10 A 0.7 H78 A 0D 1.0 40.0 H78A/N00 OOH 0.9 26.0! Table 4 Example 7: Occlusion of interferon beta by the IFNAR2 mutant.
The capability of IFNAR2 EC wild type and mutants EC to serve as carriers of IFNp was compared. For that purpose antiviral activity of IFNP left (free) in samples comprising a constant concentration of IFNP (10 pM) mixed with varying concentrations of recombinant soluble IFNAR2 EC or IFNAR2 mutants EC (example 6) was monitored. In the antiviral assay, the mixture (IFNAR2/IFN complex) was added to WISH cells (human amniotic cells). These WISH cells were then challenged with vcsicular stomatitis virus (VSV), and the residual (free) anti-viral activity of IFN3 was monitored as the degree of cell survival following 24-hour incubation (example 4).
The free IFNp present in samples having different amount of WT or mutant IFNAR2 EC (R2) concentration was determined from a survival dose curve of antiviral activity as a function of IFNp concentration carried out in the absence of IFNAR2 (Figure 2 upper plot).
The mutants tested were the following: IFNAR2 EC mutated in one amino acid residue, H78A or N100A, and mutated in two amino acids H78A/N100A, H78A/N100D and H78A/N100H wherein the amino acid N100 is mutated into alanine, aspartic acid and histidine respectively example The double mutant of IFNAR2 H78A/N100A (example 2) showed the highest affinity of all the generated mutants (Kd of about 30 pM and lower, see examples 5 and 6).
Figure 4 shows that in the presence of 25 nM of wild type IFNAR2 EC about IFNp is bound to the soluble receptor (occluded), while in the presence of only 0.2 nM of the double mutant EC H78A/N100A 50 of IFNp is bound and using only 0.4nM of H78A/N100A mutant EC 80 of the IFNp is bound. The biological assay demonstrated also, that the same extent of occluded IFN3 (bound of IFNp under equilibrium conditions) and the residual antiviral activity (free IFNP) obtainable with wild type IFNAR2 could be accomplished using about 30 fold lower concentration of the H78A/N100A IFNAR2 mutant EC. The results show also that the double modified mutant yield the best results, particularly the one in which both amino acids were mutated to alanine, H78A/N100A IFNAR2.
This result shows that the double mutated IFNAR2 occlude more effectively IFN3 and therefore administration of considerably lower amounts will be required to accomplish its carrier activity.
REFERENCES
Alani ct al, Pharmaceutical Research 14:546-549 (1997).
Anfinsen, Science 181 :223-230 (1973).
Ausubel et al, Current Protocols in Molecular Bioloqy, Greene Publications and Wiley kIte rscience (New York, 1987-1992).
Baron cl al, Antiviral Res. 24:97-110 (1994).
Baron, et al, J. Am. Med. Assoc.266:1375-1383 (1991) Ii) Christofinis, GJ Journal of General Virology_52:169-171 (1981) Colamonici et al, J. Immunol. 148:2126- 2132 (1992).
Colamonici et al, J. Biol. Chem. 268:10895-10899 (1993).
Curtis, Proc. Nall. Acad. Sci. 88:5809-5813 (1991).
Domanski et al, The Journal of BioloGical Chemistry_270:6 (1995).
Duncan ci al, J. Exp. Med. 184:2043-2048 (1996).
Dron et al, "Interferon a#3 gene structure and regulation" in Interferon: princiDles and Medical ADDlications, Baron et al, Editors, (University of Texas Medical Branch: Galveston, TX, 1992) pp. 33-45 Edingiton, "Biotech Products as Drug Leads" BioTechnoloqy 13:649 (1995) Fierlheck et al, Journal of Interferon and Cvtokine Research 16:777 (1996).
Grantham, Science 185 :X62-X64 (1974).
Grazia Cusi, Mo, Immunotechnoloqy 3:61-69 (1997).
Ibanez, EMBO Journal 10:2105-2110 (1991).
Jacobs L, et al. Arch Neurol 1987 Jun;44(6):589-95.
Keown, Methods in Enzvmolov 185 :527-537 (1990).
Lengyl, P. Ann. Rev. Biochem. 51:251-282 (1982).
Lutfaila et al, EMBO Journal_14:5100-5108 (1995).
Novick et al, FEBS Lett._314:445-448 (1992).
Novick et al, Cell 77:391-400 (1994).
Novick et al, J. Leuk. Bio.57:712-718 (1995).
Pichler and Schreiber J. ol. Biol. 1999A 289, 57-67.
Pichler and Schreiber J. ol. Biol. 1999B 294, 223-237.
Pichler and Schreiber Analytical Biochemistry 289, 173-186.
Platanias ei al, 1993 J. Immunology 150 3382-3388.
Plata"niuset al, 1996 J.Biol. Chem. 271 :23630-3.
Salmon e( al, 1996 Journal of Interferon and Cytokine Research 16 759.
Shari ci al, 1995 J. Biol. Chem. 270 13063-9.
Tan ef al, 1973. J. Exp. Med. 137: 317-330.
Uzcel al, 1990 Cell 60 225-34.
Yang ci al, 1996 J. Biol. Chem. 271 8057-61.
Yan el al. 1996 Mol. Cell Bio. 16 2074-82.
Claims (52)
1. An IFNAR2 mutant polypeptide (MIFNAR2) mutated at amino acid residues histidine 78 and asparagine 100, having higher affinity for interferon-P (IFN-p) than the wild type polypeptide, or an analog, functional derivative, fusion protein or salt thereof.
2. An IFNAR2 mutant polypeptide according to claim 1, wherein the mutations are substitutions.
3. An IFNAR2 mutant polypeptide according to claim 2, wherein the substitutions are non-conservative.
4. An IFNAR2 mutant polypeptide according to anyone of claims 1 to 3, wherein the histidine residue 78 is substituted by alanine. An IFNAR2 mutant polypeptide according to anyone of claims 1 to 4 wherein the asparagine residue 100 is substituted by alanine, aspartic acid or histidine.
6. An IFNAR2 mutant polypeptide according to claims 4 and 5, wherein both residues 78 and 100 are substituted by alanine.
7. An IFNAR2 mutant polypeptide according to claim sequence in SEQ ID NO: 2.
8. An IFNAR2 mutant polypeptide according to claim sequence in SEQ ID NO: 3.
9. An IFNAR2 mutant polypeptide according to claim sequence in SEQ ID NO: 4. An IFNAR2 mutant polypeptide according to anyone wherein its affinity to IFN-0 is about 30 pM. 1 comprising the 1 comprising the 1 comprising the of claims 1 to 9, II. An IFNAR2 mutant polypeptide according to anyone of claims 1 to 9, wherein its affinity to IFN-P is about 25, preferably up to 50 and more preferably up to 100-fold higher than the affinity of the wild type IFNAR2.
12. An IFNAR2 mutant polypeptide according to anyone of claims 1 to 9, the fragment comprises the extracellular domain (MIFNAR2 EC).
13. An IFNAR2 mutant polypeptide according to anyone of claims 1 to 12 being covalently bound to IFN.
14. An IFNAR2 mutant polypeptide according to claim 13, wherein the IFN is IFN-P. 1) 15. An IFNAR2 mutant polypeptide according to anyone of claims 1-14, wherein the IFNAR mutant is PEGylated.
16. A DNA encoding a polypeptide according to anyone of claims 1-14.
17. A DNA according to claim 16, wherein the DNA is fused to a signal peptide sequence.
18. A DNA according to claim 17, wherein the signal peptide sequence is that of the human growth hormone.
19. A vector comprising a DNA according to anyone of claims 16 to 18, capable of expressing the polypeptide encoded by said DNA in a prokaryotic host cell.
20. A vector comprising a DNA according to anyone of claims 16 to 18, capable of expressing the polypeptide encoded by said DNA in a eukaryotic host cell.
21. A prokaryotic host cell comprising the vector according to claim 19.
22. A eukaryotic cell comprising the vector according to claim
23. A method for producing an IFNAR2 mutant polypeptide according to anyone of claims 1 to 14, comprising cultivating a cell according to claim 21 and isolating the IFNAR mutant polypeptide produced.
24. A method for producing an IFNAR2 mutant polypeptide according to anyone of claims 1 to 14, comprising cultivating the cell according to claim 22 and isolating the IFNAR mutant polypeptide produced. The use of the IFNAR2 mutant according to anyone of claims 1 to 15 in the manufacture of a medicament.
26. The use according to claim 25, wherein the medicament further comprises IFN.
27. The use according to claim 26, wherein the IFN is IFN-p. 21. The use according to claim 25, wherein the medicament further comprises an IFN antagonist.
29. The use according to anyone of claims 25 to 28, for modulating the effects of IFN.
30. The use according to claim 29, for enhancing the activities of IFN.
31. The use according to calim 30, for enhancing the anti-cancer activities of IFN.
32. The use according to claim 30, for enhancing the immune modulatory therapeutic properties of IFN.
33. The use accoprding to claim 30, for enhancing the immune modulatory activities of IFN in autoimmune diseases selected from multiple sclerosis, rheumatoid arthritis, myasthenia gravis, diabetes, lupus and ulcerative colitis.
34. The use according to claim 28 or 29, for inhibiting the activity of IFN.
35. A pharmaceutical composition comprising a therapeutically effective amount of the IFNAR2 mutant according to anyone of claims 1 to
36. A pharmaceutical composition comprising a gene therapy expression vector, expressing a therapeutically effective amount of the IFNAR2 mutant according to anyone of claims 1 to
37. A pharmaceutical composition according to claim 35 or 36, further comprising IFN.
38. A pharmaceutical composition according to claim 37, wherein the IFN is IFN-P.
39. A pharmaceutical composition according to claim 35 or 36, further comprising an IFN antagonist. A pharmaceutical composition according to claim 38, wherein the IFNAR2 mutant and IFN-P are covalently bound.
41. A pharmaceutical composition according to claims 35 and 40, wherein the fragment of IFNAR2 mutant comprises the extracellular domain.
42. A pharmaceutical composition according to anyone of claims 35 to 41, for augmenting the anti-viral properties of IFN.
43. A pharmaceutical composition according to claim 42, for the treatment of t1) chronic granulomatous disease, condyloma acuminatum, juvenile laryngeal papillomatosis, hepatitis A or chronic infection with hepatitis B and C viruses.
44. A pharmaceutical composition according to anyone of claims 35 to 41, for augmenting the anti-cancer properties of IFN.
45. A pharmaceutical composition according to claim 44, for the treatment of hairy cell leukemia, Kaposi's sarcoma, multiple myeloma, chronic myelogenous leukemia, non-Hodgkins's lymphoma or melanoma.
46. A pharmaceutical composition according to anyone of claims 35 to 41, for augmenting the immune modulating properties of IFN.
47. A pharmaceutical composition according to claim 46, for treatment of diseases selected from multiple sclerosis, rheumatoid arthritis, myasthenia gravis, diabetes, ulcerative colitis and lupus.
48. A pharmaceutical composition according to claim 39, for the inhibition of immune modulating properties of IFN.
49. A method of treatment of an autoimmune disease comprising aministration of a theurapeutically effective amount of an IFNAR2 mutant polypeptide according to anyone of claims 1 to A method according to claim 49, wherein the autoimmune disease is selected from multiple sclerosis, rheumatoid arthritis, myasthenia gravis, diabetes, lupus and ulcerative colitis.
51. A method of treatment of a viral disease comprising aministration of a theurapeutically effective amount of an IFNAR2 mutant polypeptide according to anyone of claims 1 to
52. A method of treatment according to claim 51, for the treatment of chronic disease, condyloma acuminatum, juvenile laryngeal papillomatosis, hepatitis A or chronic infection with hepatitis B and C viruses.
53. A method of treatment of cancer comprising aministration of a theurapeutically effective amount of an IFNAR2 mutant polypeptide according to anyone of claims 1 to
54. A method according to claim 53, for the treatment of hairy cell leukemia, Kaposi's sarcoma, multiple myeloma, chronic myelogenous leukemia, non- Hodgkins's lymphoma or melanoma. A method of treatment according to anyone of claims 49 to 54, further comprising a therapeutically effective amount of IFN-P
56. A method of treatment of a disease caused or aggravated by IFN-P comprising the inhibition of immune modulating properties of IFN by administration of an IFNAR2 mutant polypeptide according to anyone of claims 1-15.
57. A method of treatment according to claim 56, further comprising the administration of an IFN-P antagonist.
58. The use of the IFNAR2 mutant polypeptide according to anyone of claims 1 to 14 in a formulation to prevent IFN oligomerization.
59. The use according to claim 58, to prevent IFN-P oligomerization.
60. An IFN formulation comprising the IFNAR2 mutant polypeptide according to anyone of claims 1 to 14.
61. A formulation according to claim 60, wherein the formulation comprises IFN-P. 3()
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