CZ20002287A3 - IFNAR2/IFN complex - Google Patents

Abstract

The effect of in vivo interferon type I (IFN) may be prolonged by administering interferon in the form of a complex with a binding strand Human interferon α / b (IFNAR) receptor IFN. Such the complex also improves IFN stability and enhances IFN activity. The complex may be a non-covalent complex or complex wherein IFN and IFNAR are bound by a covalent bond or peptide. When bound by a peptide bond in the form fusion protein, IFN can be separated from IFNAR by peptide linker. Such a fusion protein may be recombinant DNA technology. Storage of IFN in the form of such a complex improves the storage quality of IFN and allows storage under more benign conditions than any other possible.

Description

Qblast techniques

The present invention provides a type I interferon complex comprising a polypeptide sequence of the extracellular region of the human interferon α / β receptor and an interferon type I (IFNα, 3β and IFNα). Such a complex improves stability, enhances efficacy and prolongs the pharmacokinetics of anti-viral, immunomodulatory and anti-free IFN activity in vivo. The complex is a fusion protein or a covalent complex or a non-covalent complex comprising the polypeptide sequence of the entire extracellular region of IFNAR2 or any interferon binding portion thereof that forms a complex with type I interferon (IFN ?, IFN ?, IFN?) Or any biologically active portion thereof.

BACKGROUND OF THE INVENTION

Interferons are classified as either type I interferons derived from leukocytes and fibroblasts or as induced mitogens or "type II immune interferons" (Pestka et al., Interferons and their actions, Ann. Rev. Biochem. 56: 727777 (1987)). Analysis of sequence identities and common biological activities revealed that type I interferons include interferon alpha (IFNα), interferon beta (ΙΕΝβ) and interferon omega (IFNco), while type II interferon includes interferon gamma (IFNγ). The IFNα, IFNβ, and IFNα genes are found on the short arm of chromosome 9 (Lengyl, P., "Biochemistry of Interferons and Their Actions, Ann. Rev. Biochem. 51: 251-282 (1982)). There are at least 25 non-allelic IFNα genes, 6 non-allelic IFNα genes, and one IFNβ gene. Experts believe that they all evolved from one common parent gene. Among the species ·· tt ·· ·· (University of pp. 33-45.)).

IFNα genes share at least 80% sequence identity. IFNβ genes share approximately 50% sequence identity with IFNα, and the IFNα gene shares 70% homology with IFNα (Weissmann et al., "Prog. Nucleic. Acid Res. 33. 251-302 (1986), Dron et al.," Interferon α. / β gene structure and regulation in Interferon: Principles and Medical Applications, Baron et al., Editors,

Texas Medical Branch: Galveston, TX (1992)

The molecular weight of IFNα is in the range of 17,000 to 23,000 (165 to

166 amino acids), the molecular weight of IFN [beta] is about 23,000 (166 amino acids), and the molecular weight of IFN [alpha] is about 24,000 (172 amino acids).

Type I interferons are pleiotrophic cytokines that exhibit activity in the host defense system against viral and parasitic infections, as cancer cytokines and as immune modulators (Baron et al., "The interferons: A biological system with therapeutic potential in viral infections, Antiviral") Res. 24: 97-110 (1994), Baron et al., "The Interferons. Mechanisms of Action and Clinical Applications, J. Am. Med. Assoc. 266: 1375-1383 (1991)).

Physiological responses of type I interferon proliferative activity on normal and cells, stimulation of cytotoxic activity of natural K cell and phagocytes, cellular modulation. differentiation, stimulation of expression of MHC class I antigens, inhibition of MHC class II include antitransformed lymphocytes, and modulation of various cell surface receptors. Under normal physiological conditions, IFNα and IFNβ (IFNα / IFNβ) are secreted constitutively in a small amount in most human cells, and expression is enhanced by the addition of various inducing agents, including infectious agents (viruses, bacteria, mycoplasma and protozoa), double stranded RNA and cytokines (M-CSF, IL-2α, IL-2, TNFα). In vivo action of type I interferon can be monitored using surrogate markers, neopterin, 2 ', 5'-oligoadenylate synthetase and β-2-microgiobulin (Alam et al., "Comperative Pharmacokinetics and Pharmacodynamics of Two Recombinant Human Interferon Beta-1 (β)" -1α) products administered intramuscularly in healthy male and female volunteers, Parmaceutical Research 14: 546-549 (1997), Fierlbeck et al., "Harmacodynamics of Recombinant IFNP During Ion-Term Treatment of Malignant Melanoma, Journal of Interferon and Cytokine Research 16 : 777 (1996), Salmon et al., "Pharmacokinetics and pharmacodynamics of recombinant human IFNPin healthy male volunteers, Journal of Interferon and Cytokine Research 16: 759 (1996)".

Type I interferons (IFNα / β / ω act through the surface receptor complex to induce specific biological effects such as antiviral, antitumor and immune modulatory activity. The IFN type I receptor (IFNAR) is a heteromultimeric receptor complex consisting of at least from two different polypeptide chains (Colamonici et al., "Multichain structure of the interferon alpha receptor on hematopoietic cells, J. Immunol. 148: 2126-2132 (1992), Colamonici. et al.," Identification of a novel subunit of the Type I interferon receptor localized to human chromosome 21, J. BIOL Chem. 268: 10895-10899 (1993) The genes for these chains are located on chromosome 21 and their proteins are expressed on the surface of most cells (Tan et al., "The line of genes for human interferon-induced antiviral protein and indophenol oxidase-B traits to chromosome G-21, J. Exp. Med. 137: 317-330 (1973). The receptor chains were originally designated as ko alpha and beta, due to their ability to be recognized by the monoclonal antibodies IFNaR3 and IFNaRp1. They were then renamed IFNAR1 for the alpha subunit and IFNR2 for the beta subunit. In most cells, the IFNAR1 molecule (alpha chain, Uze subunit) (Uze et al., "Genetic transfer of a functional human interferon alpha receptor into the mouse cell: cloning and expression of its cDNA, Cell 60: 225-234 (1990)) shows a molecular weight of 100 to 130,000, while a molecular weight of 100 to 130,000

IFNAR2 (beta chain, B _L , IFNα / pR) has a molecular weight of 100,000. In certain cell types (monocyte and normal bone marrow cells) another receptor complex has been identified where IFNAR2 (βε) is expressed as a truncated receptor with a molecular The IFNAR1 and IFNAR2 subunits Ps and βΣ were cloned (Novick et al., "The human interferon α / b receptor: Characterization and molecular cloning, Cell 77: 391-400 (1994), Domanski et al.," Cloningt and Expression of a Long Form of the β subunit of Interferon and β Receptor That is required for Signaling, J. Exp. Med. 184: 2043-2048 (1995)). The IFNAR2 ps and βΣ subunits have identical extracellular and transmembrane domains, but only share the identity of the first 15 amino acids in the cytoplasmic region. The IFNAR2 subunit itself is capable of binding IFNαγ / β, while the IFNAR1 subunit is capable of binding IFNαγ / β. When the human IFNAR1 receptor subunit alone was transfected into murine L-929 fibroblasts, no human IFNαs other than IFNα8 / IFNαB were able to bind to cells (Uze et al., Genetic transfer of a functional human interferon alpha receptor into the mouse cells cloning and expression of its cDNA, Cell 60: 225-234 (1990)). The human IFNAR2 subunit transfected into L cells in the absence of the human IFNAR1 subunit binds human IFNα2. The approximate Kd of binding is 0.45 nM. When human IFNAR2 subunits were transfected in the presence of human IFNAR1 subunit, the high potency binding showed a Kd value of 0.0260.114 nM (Novick et al., "The human interferon α / b receptor: Characterization and molecular cloning, Cell 77: 391-400 (1994), Domanski et al., "Cloning and Expression of the Long Form of the β Subunit of the Interferon and β Receptor. 1995)).

It is estimated that on most cells, there are high affinity binding sites of 500 to 20,000 and low affinity for IFNs with a high affinity of 500 to 20,000. · Φφ

Although complex IFNAR1 / 2 subunits (α / ββ or (α / βΣ) bind with high affinity, only a few (α / βΣ) appear to be a functional signaling receptor.

Transfection of IFNAR1 and IFNAR2 βΣ subunits into mouse L-929 cells, followed by incubation with IFNα2 induces anti-viral stage, initiates intracellular protein phosphorylation and causes intracellular kinase activation (Jak1 and Tyk2) and transcription factors (STATE 1, 2, and 3) (Novick et al., "The Human Interferon α / B Receptor: Characterization and Molecular Cloning, Cell 77: 391-400 (1994), Domanski et al.," Cloning and Expression of the Long Form of the β Subunit of Interferon and β Receptor That is Required for Signaling, J. Exp. Med. 184: 2043-2048 (1995)). In a corresponding transfection experiment, the IFNAR2 βε subunit is unable to initiate a similar response. Thus, the IFNAR2 βΣ subunit is required for functional activity (anti-viral response) with maximal induction that occurs in association with the IFNAR1 subunit.

In addition to membrane bound cell surface forms of IFNAR, soluble IFNAR has been identified in both serum and urine (Novick et al., "The human interferon a / b receptor: Characterization and molecular cloning, Cell 77: 391-400 (1994), Novick et. al., "Soluble and Membrane Novick et al.," The human interferon a / b receptor: Characterization and molecular cloning, Cell 77: 391-400 (1994), non-anchored forms of the human IFN-α / β receptor, J. Leuk Bio 57: 712-718 (1995),

Novick et al., Soluble interferon-alpha receptor molecules are present in body fluids FEBS Lett. 314: 445-448 (1992), Lutfalla et al., "Mutant US cells are complemented by an interferon-α / β receptor subunit generated by an alternative Processing of a new member of the cytokine receptor gene cluster, EMBO Journal 14: 5100- 5108 (1995)). Soluble IFNAR isolated from serum has an apparent molecular weight of 55,000 in SDS-PAGE, whereas soluble IFNAR form derived from β

♦ ··· ·· • ♦ 1 • · «• · ·· at the C-terminus. For five potentially urine, it has an apparent molecular weight of 40-45,000 (p40).

Soluble p40 IFNAR2 transcripts are present at the mRNA level and contain nearly the entire extracellular domain of the subunit

IFNAR2 with two new amino acids soluble IFNAR2 receptor exists glycosylated sites. The soluble form of p40 IFNAR2 has been shown to bind IFNα2 and IFNβ and inhibit the in vitro anti-viral activity of a mixture of IFNα species (leukocyte IFN) and individual IFN type I (Novick et al., "Soluble and membrane Novick et al." interferon α / β receptor: Characterization and molecular cloning, Cell 77: 391-400 (1994), non-anchored forms of the human IFN-α / β receptor, J. Leuk. Bio. 57: 712-718 (1995)) .

Furthermore, the recombinant IFNAR2 Ig fusion protein subunit has been shown to inhibit the binding of various types of IFN type I (IFNaA, IFNaB, IFNaD, IFNP, IFNa Con1 and FNco) to Daudi cells and COS cells were transfected with the α / βε subunit.

IFN type I signaling pathways have been identified (Platanias et al., "Differences in interferon and β signaling, J. Biol. Chem. 271: 23630-23633 (1996), Yan et al.," Molecular characterization of the alpha interferon receptor 1 subunit (IFNαR1) domain required for TYK2 binding and signal transduction, Mol. Bio., 16: 2074-2082 (1996), Yang et al., "Direct association of STAT3 with the IFNAR-1 chain of human Type I interferon receptor, J. Biol. Chem. 271: 8057-8061 (1996), Qureshi et al., "Function of Stat2 protein in transcriptional activation by alpha interferon, Mol. Cell. Bio. 16: 288-293 (1996), Sharf et al., "Functional domain analysis of interferon consensus sequence binding protein (ICSBP) and its association with interferon regulators factors, J. Biol. Chem. 270: 13063-13069 (1995)). It is thought that the initiating features leading to signaling are manifested by the binding of IFNα / β / ω to the IFNAR2 subunit followed by the association of the IFNAR1 subunit to form the IFNAR1 / 2 complex (Platanias et. Al., "Tyrosine phosphorylation of the α and β subunits of the Type I interferon receptor J." Biol. Chem., 269: 17761-17764 (1994). Binding of IFNcc / β / ω to the IFNAR1 / 2 complex results in the activation of two Janus kinases (Jak1 and Tyk2), known to phosphorylate specific tyrosines on the IFNAR1 and IFNAR2 subunits. After these subunits are phosphorylated, the STATE molecules (STATE 1, 2, and 3) also phosphorylate, resulting in dimerization of STATE transcription complexes, followed by nuclear localization of the transcription complex and activation of specific inducible IFN genes.

The pharmacokinetics and pharmacodynamics of type I IFN in humans have been evaluated (Alam et al., "Comperative Pharmacokinetics and Pharmacodynamics of Two Recombinant Human Interferon Beta-1 (ΙΕΝβ-la) Products Combining Intramuscularly in Healthy Male and Female Volunteers, Parmaceutical Research 14: 546- 549 (1997), Fierlbeck et al., "Harmacodynamics of recombinant duringβ during long-term treatment of malignant melanoma, Journal of Interferon and Cytokine Research 16: 777 (1996), Salmon et al.," Pharmacokinetics and pharmacodynamics of recombinant human ΙΓΝβίη healthy male volunteers, Journal of Interferon and Cytokine Research 16: 759 (1996)). Removal of ΙΡΝβ is quite rapid if the bioavailability of IFNP is lower than expected for most cytokinases. Although odynamΕΝβ pharmacodynamics were estimated in humans, there was no clear correlation between biologβ bioavailability and clinical efficacy. In normal healthy human volunteers, administration of a single intravenous (iv) bolus dose (6 MIU) of recombinant Oβ-derived CHO results in a rapid distribution phase of 5 minutes and a half-life of 5 hours (Alam et al., Comperative pharmacokinetics and Pharmacodynamics of two). recombinant human interferon beta1 (β-1α) products administered intramuscularly in healthy male and female volunteers, Parmaceutical Research 14: 546549 (1997)). After subcutaneous (sc) or intramuscular administration 9 9 9 9 9 9

9 · 9 9 · 9 · * 9 9 • 9

ΙΡΝβ, only about 15% of the dose available in systemic administration is contained in serum. The pharmacodynamics of odynamΕ ,β following iv, im or sc administration (measured by changes in 2 ', 5'-oligoadenylated synthetase (2', 5'AS) in PBMC) were evaluated within the first 24 hours. Over the next four days they slowly dropped to the background value. The strength and duration of the biological effect are the same with respect to the mode of administration.

After injecting IM with a single dose of 6 MIU of recombinant ΙΡΝβ, pharmacokinetics (PK) and pharmacodynamics (PD) ΙΡΝβ prepared by two different companies (REBIF®-Serono and AVONEX®-Biogen) were tested (Salmon et al., "Pharmacokinetics and pharmacodynamics of recombinant human β-healthy human volunteers, Journal of Interferon and Cytokine Research 16: 759 (1996)). Serum ΙΡΝβ concentration and ΙΡΝβ spare marker neoptorin marker were monitored over time. Both preparations ΙΓΝβ show similar PK profiles with maximums

Serum ΙΡΝβ was reached after approximately 12 to 15 hours, although REBIF® showed a lower maximum value. The amount of IF? Remains to be evaluated for REBIF® and AVONEX® for at least the first 36 hours after im injection. After 48 hours, the value dropped slightly below the baseline. The amount of neopterin shows a very similar profile for REBIF® and AVONEX®, with the maximum amount of neopterin reached approximately 44 to 50 hours after injection. The remainder was evaluated up to 72 hours after injection and then gradually decreased to baseline over 144 hours.

A multi-dose pharmacodynamic study of ΙΕΝβ was conducted in human melanoma patients (Fierlbeck et al., "Harmacodynamics of recombinant duringβ during long-term treatment of malignant melanoma, Journal of Interferon and Cytokine Research 16: 777 (1996)"), applied by sc, three times a week at 3MIU in a single dose for six months. Maximal pharmacodynamic markers, 2 ', 5'-AS, synthetases, 32-microglobulin, neopterin, and NK cell activation were achieved by a second injection (Day 4) and decreased within 28 days, remaining after six days. months slightly increased.

«Φ φ φ φ φ φ · · · · · · · ·

Elimination of type I interferon in humans is fast. Preparations interferon acting for a long time time will be make a big difference in clinical practice. SUMMARY OF THE INVENTION It was found that the complex interferon type i include e

a soluble form of IFNAR complexed with type I interferons (IFN), exhibits improved stability, increased efficacy, and prolonged pharmacokinetics in vivo as compared to free IFN for anti-viral, anti-cancer, and immune modulating activity.

The present invention provides a type I interferon (IFN) complex that comprises the polypeptide sequence of the extracellular region of the human interferon α / β receptor subunit (IFNAR), which exhibits improved stability, enhanced efficacy and / or prolonged pharmacokinetics in vivo compared to free IFN in anti-viral , against cancer and immune modulating activity. It is preferred that the complex be the extracellular region of IFNAR2 subunit with any type I interferon or IFNAR1 subunit with IFNα.

The complex is a fusion protein or a covalent complex or a non-covalent complex comprising the polypeptide sequence of the entire extracellular region of IFNAR, preferably IFNAR2 or any interferon binding subfraction thereof that complexes with IFNα or β or IFNco, or any biologically active subfraction thereof.

IFNARs include any known extracellular IFNAR receptors as defined above, as well as any active fragments thereof. IFNAR may form a fusion with another protein, for example, an immunoglubulin, such as IgG. The term IFN, IFNa, ΙΝΝβ and IFNco make up one of more than 20 types of i:

♦ «Type I interferon that has been established to date, or any other type I interferon that will be identified in the future.

In one embodiment, the complex comprises IFNα. or IFNβ covalently linked to IFNAR2 via a chemical bond.

Another embodiment of the invention comprises a complex composed of IFNα or IFNβ which forms a non-covalent complex with IFNAR2. Another embodiment of the invention comprises a composition comprising type I IFN and IFNAR2 in any ratio. Type I IFN formulations with an excess of IFNAR2 as defined above are also included in the definition of the term "complex of the invention." The two components may also be administered separately, forming a complex in vivo. In another embodiment of the invention, the complex is a mixture of IFNAR2 and IFN obtained by simultaneous and sequential administration of IFNα or IFNβ and soluble IFNAR2. Further, IFNAR can be administered without co-administration of IFN such that it can form a complex in vivo with endogenous IFN circulation, enhancing the effects of endogenous IFN.

In certain embodiments, the complex comprises IFNα or IFNβ or IFNα, fused to IFNAR2, as a recombinant fusion protein, wherein portions of IFN and IFNAR2 are fused via a flexible peptide linker molecule. This peptide linker is cleavable or not cleavable in vivo.

The invention further provides DNA encoding such fusion proteins, vectors comprising such DNA, host cells transformed with such vectors in the manner described to express the fusion proteins. The invention further provides methods for producing such fusion proteins, culturing said host cells and isolating the fusion proteins that they express.

Further, the invention provides methods of using the complexes of the invention to prolong the effect of IFN in vivo, which can be used to treat any disease or condition treatable by the application of IFN.

Further, the invention describes the use of IFNAR as a stabilizer in IFN formulations. Free IFNβ tends to form oligomers. This is prevented by complexing with IFNAR, especially IFNAR2. The recombinant IFNβ formulation must have an acidic pH that may cause irritation upon administration. If IFNAR is used as a stabilizer, it is possible to form non-acidic compositions.

The invention describes the IFNAR / IFN complex and the technology required to produce the complex. Based on the results, IFN [beta] was chosen as the least restrictive example.

Fusion protein.

The C-terminus of IFNAR2, or any interferon binding sequence thereof, was fused to the N-terminus of IFNP or its biologically active fragments as necessary to bridge the shortest distance between the two molecules. Reverse constructs can also be prepared wherein the C-terminus of IFNβ or fragments thereof is fused to the N-terminus of IFNAR2 or sequences thereof.

From IFNAR2 / IFNβ molecule models, the distance between the tethered C-terminal extracellular region of IFNAR2 and the N-terminal region of IFNP in an active complex model of approximately 80 angstroms was estimated. For example, a repeat of the Gly-Gly-Gly-Gly-Gly-Ser (GGGGS) flexible peptide linker (SEQ ID NO: 1) can be used to design an IFNAR2 / IFNP complex that allows the active complex to be maintained. Alternatively, the linker may be flexible and may form a target for proteolytic cleavage by serum, membrane binding, and / or cellular proteases. An example of a serum protease cleavage site prepared by a complex IFNAR2 / IFNβ fusion may be a Factor Xa cleavage site. Factor Xa cleaves prothrombin at two positions Arg273 and Arg322 and shows the recognized tetrapetide signal Ile-Glu-Glu-Arg (SEQ.

ID NO: 2) (Nagai et al., Nature 309: 810 (1984)). Factor Xa itself was formed in the intrinsic and extrinsic pathways that include tissue factor, which endothelial cells, macrophages and

various activators, release vascular neutrophils.

Factor containing areas a

Xa can act on the IFNAR2 / IFN fusion protein recognized by the Factor Xa sequence in the linker to release the IFNAR2 / IFN complex such that the complex can function as a non-covalent complex.

Alternatively, the complex IFNAR2 / IFNβ fusion may have a cleavage site suitable for a cell membrane protease (e.g., hepsin). Hepsin is a membrane-bound serine protease zymogen of 51,000 molecular weight, which is strongly expressed in liver tissue, but is also found in the kidney, pancreas, lungs, thyroid, pituitary, and testes. One sequence known to be cleaved by hepsin is the Factor VII bound Arg152-Ilel53 peptide. Hepsin is involved in the formation of thrombin in tumor cells (Kazam et al., J. Biol. Chem. 270: 1 (1995)).

Hepsin can act on the SIFNAR2 / IFN fusion protein containing the recognized hepsin sequence in the linker region and releases the SIFNAR2 / IFN complex so that the complex can function in a non-covalent manner.

Alternatively, the SIFNAR2 / IFN fusion complex may comprise an intracellular protease cleavage site. Necrotizing and dying cells release various proteases. These are caspases (proteases similar to interleukin 1-beta converting enzyme), metallo-proteinases, lysosomal proteases (e.g. cathepsin B) and elastase. Elastase is released by granulocytes during the disease stage (e.g., during sepsis) and exhibits broad specificity in terms of amino acid cleavage sequence. They are related to trypsin (Ertel et al.

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Sheet. Surg. 129: 1 (1994); Szilagyi et al., Biochim. Biophys. Acta 1251: 1 (1995)).

Intracellular proteases can act on the SIFNAR2 / IFN fusion protein containing the recognized intracellular protease sequence in the linker region and release the sIFNAR2 / IFN complex so that the complex can function as a non-covalent complex.

In fact, several examples of fusion protein constructs have been prepared in which the C-terminus of SIFNAR2 (P40-ESEFS) is linked to the N-terminus of ΙΕΝβ (MSY) via a flexible linker. Examples of peptide linkers are: ESEFS (GGGGS) "MSY, where n = 5 (SEQ ID NO: 3), 4 (SEQ ID NO: 4), 3 (SEQ ID NO: 5, 2 (SEQ ID NO: 6)) or (SEQ ID NO: 7), ESEFS (hCG-CTP) MSY, wherein hCG-CTP - SSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 8), ESEFS (EFM) _n MSY, wherein n = 5 (SEQ ID NO: 9), 4 (SEQ ID NO: 10) or 3 (SEQ ID NO: 11), ESEFS (EFGAGLVLGGQFM) _n MSY, wherein n = 1 (SEQ ID NO: 12) or 2 (SEQ ID NO: 13) and any other suitable linker , which overlaps the distance between the SIFNAR2 and ΙΕΝβ binding site in a complex model and which does not form an immunogenic epitope between portions of interferon and receptors, it is preferred that these linkers comprise up to 30 amino acids.

Covalent complex

One example of the formation of chemical cross-linked molecules is site-specific modification of IFNAR2 by reacting a biodegradable linker, such as polyethylene glycol (PEG), with cysteines present or introduced into IFNAR2 using amino acid substitutions such as Ser210 to Cys or Asn89 to Cys (site-directed mutagenesis) . The following constructs can be designed as follows: IFNAR (S210C) -PEGn-β (Cysl7), where n = 2,000, 5,000 or 10,000, or IFNAR (N89C) -PEGn- ΙΕΝβ ^ 3ΐ7).

The formation of covalent disulfide bonds between the Cys of two different moieties is also within the scope of the invention.

Non-covalent complex · ♦>>> * »>>>>>>>>>» »» »

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Human IFNAR2 forms a complex with IFN3 under conditions that maximize the formation of the active complex. In order to produce a maximum active complex, an optimal in vitro ratio must be achieved

2.5 ng IFNAR2 per 1 International Unit (IU) ΙΕΝβ as described in the examples. At present, the optimal ratio of IFNAR2 to ΙΤΝβ has been established, which produces maximum active complex activity activity in vivo, although the optimal ratio appears to depend on koncentraciΕΝβ concentration. For example, the optimal ratio of IFNAR2: ΙΕΝβ to increase anti-tumor activity of ΙΕΝβ at 2x10 ⁴ IU / mouse / day is 2.5 ng IFNAR2 per pg ΙΕΝβ, while at 5 x 10 ⁴ IU / mouse / day ΙΕΝβ is optimum 0 , 3 ng IFNAR2 per pg ΙΕΝβ. The same ratio is used to maximize the formation of the active complex in vitro, resulting in prolonged IFN pharmacokinetics in vivo.

Preferably, recombinant IFNAR2 and ΙΕΝβ molecules are used to prepare the complex. In in vitro anti-viral assays, the interferon / receptor complex showed increased activity compared to ΙΕΝβ alone. A constant concentration of ΙΕΝβ was mixed with different concentrations of recombinant SIFNAR2 and this mixture (IFNAR2 / IFN complex) was added to WISH cells (human amniotic cells). These WISH cells were then infected with vesicular stomatitis virus (VSV) and the antiviral activity of IFN was monitored as a number of surviving cells for 48 hours incubation. In each experiment, the addition of IFNAR2 to a constant amount of ΙΕΝβ resulted in an increase in the number of cells that survived VSV infection at the optimal IFNAR2 to IFN ratio, depending on the dose size. These results showed that the IFNAR2-IFNβ complex showed increased activity compared to free ΙΕΝβ in the antiviral assay. The practical implication of this is that the ΙΕΝΑΚ2 / ΙΕΝβ complex has higher efficacy and increased activity compared to free IFN for various therapeutic »» »4

44 indications in which IFN alone is active. These indications include those in which free IFN exhibits therapeutic activity, such as anti-viral, i:

4 * 4 * 4 4 44 # 4 4 ♦ 4 4 ♦ 4 4 »» <4 4 modulating activity of higher efficacy anti-cancer and immune The IFNAR2 / IFN complex is expected to increase activity and / or improve pharmacokinetics (i.e. half-life) will be more effective in the treatment of viral, oncogenic and immune disorders.

When administered in vivo, the interferon receptor complex increases the bioavailability, pharmacokinetics and / or pharmacodynamics of IFN, thereby enhancing the antiviral, anti-cancer, and immune modulating properties of IFN.

Increased complex mediated IFN bioavailability can be achieved either by forming a non-covalent IFN / IFNAR complex, by co-applying free IFN with IFNAR, sequentially applying IFN and IFNAR components, through application of the covalent IFN / IFNAR complex, or by applying the IFNAR / IFN fusion protein.

In another embodiment of the invention, increasing bioavailability can also be achieved by applying the IFNAR component alone without adding IFN. IFNAR will form a "complex in vivo with endogenous IFN" and thus increase the bioavailability, pharmacokinetics and / or pharmacodynamics of endogenous IFN. This is particularly useful in the treatment of patients suffering from a disease or condition that naturally induces native IFN by circulating IFN and naturally counteracting the disease and disorder. The added IFNAR will enhance the effects of native IFN.

Preferred molecules suitable for use in the complexes of the invention exhibit the sequence of native IFN and IFNAR. A natural sequence is one that contains naturally occurring human IFN and IFNAR. Such sequences are known and described in the literature. It is also contemplated that naturally occurring allelic variations are natural sequences.

The invention also provides analogs of the above-described complex

IFNAR2 / IFN according to the invention, which are kept substantially «0» 00 0 · 0 0 0 0 0 0 0 0 0

0 · 0 · 0

0 0 0 0 0 0 00 00 · 0 0 ·

0 '0 0 0 0 ♦ »0« 0 0 0

0 to 0 IFNAR2 and IFN have the same biological activity of a complex having essentially the sequences of native IFNAR2 and IFN. Such analogs may be those that contain deletion, addition and substitution of up to about 30 amino acid residues in portions of the IFNAR and / or IFN complex such that modification of this species does not substantially alter the biological activity of the chimeric protein analog with respect to the complex itself. The various analogs may differ from each other and from the base complex molecules (essentially only naturally occurring IFNAR2 and IFN sequences) at the site of the linker peptide that joins both parts to form the complex. As mentioned above, such a linker forms amino acids and serves to separate parts in the complex. For such a linker, care must be taken to select its sequence (and each such analog must be biologically tested by suitable standard assays) to avoid, for example, the complex folding, which may cause inactivation or increase the activity or activity of the immunogenic complex. resulting in the treatment of the patient to elicit antibodies against the analog. Such an analog is then ineffective at least as a medication with a medium or long duration of action. With respect to the above-described analogs of the complex of the invention, these analogs are those wherein one or more and up to about 30 amino acid residues of the basic complex of the invention are replaced by different amino acid residues or are deleted or one or more to about 30 amino acid residues are added to the original. sequence of the complex of the invention without substantially altering the activity of the resulting product compared to the basic complex of the invention. These analogs are prepared by known synthesis and / or site-directed mutagenesis or by any other method suitable for this purpose.

Any such analog preferably has an amino acid sequence that is essentially a duplicate of the sequence of the IFNAR2 / IFN core complex, which has substantially ♦ φ φ φ φ · · · · φ · · φ φ φ · Stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou stejnou. By basic experiments it is possible to determine whether any analog exhibits substantially the same activity and / or ability as the basic complex of the invention. Such experiments include subjecting the analog to testing for biological activity and stability, as set forth in Examples 2-7 below.

Complex analogs that can be used in accordance with the invention, or the nucleotide sequences encoding them, include a finite set of substantially corresponding sequences, such as peptide or polynucleotide substitutions, which one of ordinary skill in the art can obtain routinely without carrying out experiments. Protein chemistry and structure is described in Schilz et al., Principles of Protein Structure, Springer-Verlag, New York (1978) and Creighton, Τ. E., Proteins: Structure and Molecular Properties, W.H. Freeman and Co ,, San. Francisco (1983). Substitution of the nucleotide sequence present is described in Ausubel et al., Current Protocols in Molecular Biology, Greene Publications and Wiley Interscience (New York, 1987-1992), para. I-A. 1-24 and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 nd Ed., Cold Spring Harbor Press (Cold Spring Harbor, NY, 1989, paragraphs 6.3 and 6.4 in Appendix C and D.)

Preferred changes for analogs in accordance with the invention are those known as conservative substitutions. Conservative amino acid substitutions in a complex that exhibits substantially naturally occurring IFNAR2 and IFN sequences may include synonymous amino acids in a group having substantially similar physicochemical properties such that substitution between members of the group will maintain the biological function of the molecule (Grantham, Science 185: X62-X64 (1974)). Obviously, amino acid insertions and deletions can also be performed in the above sequences without altering their function, particularly when the insertion or deletion comprises only a few amino acids, for example less than 30. Preferably less than 10 amino acids are not deleted or deleted. do not replace • ♦ · »• ·»

4 9

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9

amino acids that are critical in functional alignment. These are, for example, cysteine residues (Anfinsen, Scinece 181: 223230 (1973)). Analogs which arise from such deletions and / or insertions are in accordance with the invention.

Synonymous amino acid groups are those defined in Table I. Preferably, synonymous amino acid groups are those defined in Table II, and most preferably, synonymous amino acid groups are defined in Table 1.

III.

Table I

Preferred groups of synonymous amino acids

9-9 9 9-9 • 9 ♦

9 9 • 9 · ·

9 «99 ·» 9 9

9.9 · 9

9 9 9

99 * 9

9 «9 9 9

9 9 9

99

Aminoacid Synonymous group Ser Ser, Thr, Gly, Asn Arg Arg, Gln, Lys, Glu, His Leu Ile, Phe, Tyr, Met, Val, Leu For Gly, Ala, Thr, Pro Thr Pro, Ser, Ala, Gly, His, Gln, Thr Ala Gly, Thr, Pro, Ala Wall Met, Tyr, Phe, Ile, Leu, Val Gly Ala, Thr, Pro, Ser, Gly Ile Met, Tyr, Phe, Val, Leu, Ile Phe Trp, Met, Tyr, Ile, Val, Leu, Phe Tyr Trp, Met, Ph, Ile, Val, Leu, Tyr Cys Ser, Thr, Cys His Glu, Lys, Gln, Thr, Arg, His Gin Glu, Lys, Asn, His, Thr, Arg, Gln Asn Gln, Asp, Ser, Asn Lys Glu, Gln, His, Arg, Lys Asp Glu, Asn, Asp Glu Asp, Lys, Asn, Gln, His, Arg, Glu Met Phe, Ile, Val, Leu, Met Trp Trp

Table II

More preferred groups of synonymous amino acids 4 4 4 »491

4 4 4 1 44 »995

4 9 4 4 4 4 9 4 4 t · · ··· · 4 4 4 4 4 4 4

44 44 44 »44

Aminoacid Synonymous group Ser Ser Arg His, Lys, Arg Leu Leu, Ile, Phe, Met For Ala, Pro Thr Thr Ala Pro, Ala Wall 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, Gln, Arg Gin Glu, Gln, His Asn Asp, Asn Lys Lys, Arg Asp Asp, Asn Glu Glu, Gin Met Met, Phe, Ile, Val, Leu Trp Trp 1

• 5 · ·

44

44 49

4 4 4 4 9 5

94 9 4 4 9 • 4 5

9 44 44 44

Table III

Most preferred groups of synonymous amino acids

Aminoacid Synonymous group Ser Ser Arg Arg Leu Leu, Ile, Met For For Thr Thr Ala Ala Wall Wall Gly Gly Ile Ile, Met, Leu Phe Phe Tyr Tyr Cys Cys, Ser His His Gin Gin Asn Asn Lys Lys Asp Glu Glu Glu Met Met, Ile, Leu Trp Met .1

·· »« «·

4 • 4 4

4 4 * 4 44 »4» 4 «4

44 ♦ 4 44 • 4 4 4 • 4 4

4 4 4

44

Examples of the production of amino acid substitutions in proteins that can be used to obtain analogs of the IFNAR2 / IFN complex suitable for use in the invention include any known method as described in US Patents RE 33,653; 4,959,314;

4,588,585 and 4,737,462 (Mark et al.,); 5,116,943 (Koths et al.); Nos. 4,965,195 (Namen et al.) And 5,017,691 (Lee et al.) And the lysine-substituted proteins described in U.S. Patent 4,904,584 (Shaw et al.).

In another preferred embodiment of the invention, any analog of the complex suitable for use in the invention has an amino acid sequence substantially corresponding to the sequence of the above described complex of the invention. The term &quot; essentially corresponds to analogs with minor changes in the base complex sequence that does not affect its essential characteristics, particularly its ability to inhibit the proliferation of cancer cells or support bone marrow transplantation. The type of changes that are generally considered to fall within the group "essentially corresponding are those resulting from conventional DNA mutagenesis methods encoding the complex by minor modifications, modifications, and testing required by the method described above.

A portion of the IFNAR2 complex will have a nuclear sequence that is the same as the native sequence, or a biologically active fragment thereof, or a variant thereof, having an amino acid sequence at least 70% identical to the native amino acid sequence and retaining its biological activity. It is preferred that such a sequence show at least 85% identity, at least 90% identity. Most preferably, the sequence is at least 95% identical to the natural sequence.

With respect to the portion of the IFN complex, the core sequence that can be used is a natural sequence or a biologically active fragment or variant thereof, wherein the amino acid sequence has at least 70% identity to it, leading to several activities of the invention and testing for several minor · 4 «

4 • 4 4 · 44 44 4 4 4 4 4> * 44 4 4 44 4 4 44 4 · ♦ ·· 44 94 44 44 preferably at least 85% or at least 90% match and most preferably at least 95% match. Such analogs retain the biological activity of the native IFN sequence or fragment thereof, or exhibit antagonist activity, as discussed below.

The term "sequence identity" means that the sequences are compared as described below. Sequences are arranged using Genetic Computing Group's GAP version 9 using an implicit (BLOSUM62) matrix (-4 to +11) with a gap open penalty of -12 (for the first zero of the gap) and a gap extension penalty of -4 (for each consecutive zero in the gap). After alignment, the percent identity is calculated by expressing the correct pairing number as a percentage of the number of amino acids in the claimed sequence.

Analogs of the invention can also be determined by the following procedure. With respect to part of the IFNAR complex or part of the IFN DNA of the natural sequence, it is known in the art and is described in the literature. Polypeptides encoded by any nucleic acid, such as DNA or RNA, that hybridize to the complement of native DNA or RNA under highly or moderately stringent conditions when the polypeptide retains the biological activity of the natural sequence or, in the case of IFN, retains biological activity or exhibits antagonist activity for the content of the invention.

Stringent conditions are a function of the temperature at which hybridization occurs, the molarity of the monovalent cations, and the percentage of formamide that is contained in the hybridization solution.

To determine the degree of stringency of the conditions in a given set of conditions, the equation described by Meinkoth et al., Anal. Biochem. 138: 267-284 (1984) suitable for determining the stability of 100% identity hybrids expressed as half the denaturation temperature of a Tm DNA-DNA hybrid:

Tm = 81.5 ° C + 16.6 (logM) + 0.41 (% GC) - 0.61 (% form.) 500 / L, where M is monovalent cation molarate,% GC is ·· * · 4 % 44% G and C nucleotides in DNA,% form. is the percentage of formamide in the hybridization solution and L is the length of the hybrid in pairs. For each one degree by which the Tm temperature is reduced from the calculated hybrid value with 100% identity, the amount of incorrect pairs allows it to increase by 1%. Thus, the T m used for any given hybridization experiment at specified salt and formamide concentrations is 10 ° C below the T m calculated for the 100% hybrid according to the Meinkothot equation.

Hybridization occurs even if there are up to approximately 10% mismatch.

The term "high stringency conditions" means those conditions in which up to about 15% sequence divergence is tolerated.

The term "moderate conditions" means those conditions in which up to about 20% sequence divergence is tolerated.

Examples of high stringency conditions (12-15 ° C below the calculated Tm hybrid) and medium stringency conditions (15-20 ° C below the calculated Tm hybrid) use a 2X SSC (standard salt ion citrate) wash solution and 0.5% SDS at a suitable temperature. below the calculated Tm of the hybrid. The limited stringency of the conditions is primarily due to the wash conditions, especially when those hybridization conditions that allow less stable hybrids that are formed with stable hybrids are used. Washing conditions at high stringency then remove less stable hybrids. Common hybridization conditions used with high stringent to medium stringent wash conditions as described above are hybridization in solution of 6 x SSC (or 6 x SSPE), 5 x Denhardt's reagent,

0.5% SDS, 100 ng / ml denatured fragmented salmon sperm DNA at about 20-25 ° C below Tm. If a probe mixture is used, it is preferable to use tetramethylammonium chloride (TMAC) instead of SSC (Ausubel et al., Current Protocols in Molecular Biology, Greene Publications and Wiley Interscience (New York, 1987-1992), paragraph A.I.I-A. 1-24).

»4 φ 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4

· Φ Φ Φ • · Φ 4 • ♦ 4 Φ

4 φ Φ

Φ · ΦΦ

The term "functional derivatives" means derivatives that can be prepared from functional groups that occur as side chain residues or N- or C-terminal groups in a manner known in the art and are subject to the invention as long as they remain pharmaceutically acceptable. That is, they do not disrupt the biological activity of the corresponding complex protein as described above and do not confer toxic properties to the compositions containing them or to the complexes prepared therefrom. The derivatives may have chemical moieties such as carbohydrate or phosphate residues. Such fractions have the same biological activity and remain pharmaceutically acceptable.

Derivatives may, for example, include aliphatic carboxylic acid esters of carboxyl groups, carboxylic amides by reaction with ammonia or with primary or secondary amines, N-acyl derivatives or free amino groups of amino acids of a carbocyclic aroyl group) or O-acyl derivatives of free hydroxyl groups formed with acyl moieties carbocyclic aroyl groups) or O-acyl derivatives of a free hydroxyl group (e.g., hydroxyl groups of seryl and 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 appearance of the complex or a portion thereof in body fluids.

The term "derivatives" includes only those derivatives that do not substitute one amino acid for another of the twenty naturally occurring natural amino acids.

The term "salts" means salts of carboxyl groups and acid addition salts of amino groups of the complex of the invention or analogs thereof. Salts of the carboxyl group can be formed in a manner well known in boron and include inorganic salts such as sodium, calcium, ammonium, ferric or zinc salts and the like and salts with organic bases such as formed with amines such as triethanolamine, arginine or lysine, · ly ly ly ly ly ly ly ly ly ly ly ly ly ly ly ly ly ly ly ly ly ly ly Piperidine, procaine and the like. Acid addition salts include, for example, salts of mineral acids such as hydrochloric acid or sulfuric acid and salts of organic acids such as acetic acid or oxalic acid. Of course, any such salts must have substantially the same biological activity with the complex of the invention or analogues thereof.

The term "biological activity" means the following: For a portion of the IFNAR2 complex, biological activity is important for its ability to bind to type I interferon. Thus, analogs or variants, salts and functional derivatives must be selected to retain the ability to bind interferon. This can be tested by routine binding assays. In addition, IFNAR2 fragments or analogs thereof may also be used as long as they retain their interferon binding activity. Fragments can be readily prepared from any end of the polypeptide to test the resulting interferon binding properties. Proteases that remove an amino acid from the N- or C-terminus of a polypeptide are known, and thus the determination of fragments that retain the ability to bind interferon involves only routine experiments.

In addition, a polypeptide that exhibits interferon binding activity that is IFNAR2, SIFFAR2, an analog or variant, salt, functional derivative or fragment thereof may also contain other amino acid residues flanking the interferon binding polypeptide. If the resulting molecule retains the ability to bind interferon to the nuclear polypeptide, it can be determined using routine experiments whether any such flanking residues affect the basic and characteristics of the nuclear peptide. That. means interferon binding characteristics. A term comprising a specified sequence means that additional flanking residues may be present that do not affect the essential or novel characteristics of the specified sequence.

by removing the interferon-binding amino acids and re-establishing their essence; · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · <· <<<<<<· · · · · · · ·

The term does not include substitutions, deletions or additions in the specified sequence.

While IFNAR2 or SIFNAR2 is used in the present specification and examples, it is to be understood that this is a preferred example and that the IFNAR1 subunit, and in particular its extracellular region, can be replaced at any time by IFNAR2. IFNAR1 can only be used in conjunction with the interferons to which it binds. IFNAR1 is known to bind to IFNα. Any complex using IFNAR1 must consist of interferon species, and the IFNα species to which IFNAR1 binds are preferred.

With respect to the interferon portion of the complex of the invention, the biological activity that any analog or variant, salt, functional derivative, or fragment must retain is the activity of the interferon that is of utility. In many examples, this is the ability to bind to the natural cell surface receptor and thereby mediate signal production by the receptor. Any such analog, derivative or fragment could retain such receptor agonist activity to be used in the invention. On the other hand, it is sometimes advantageous if there is a molecule with an antagonistic activity towards the receptors so that it prevents the biological activity of the natural interferon. Such an antagonist may also be used where it is desired to prolong the positive effect by the methods of the invention. For such uses where it is necessary to eliminate the adverse effect of interferon, analogues that bind to the receptor and portions of the IFNAR complex but which do not mediate the signal and blocking signal by the natural interferon to said receptor, it may also be considered to be biologically active for the purposes of invention. Direct assays can determine whether such any analog retains such receptor agonist activity or whether it exhibits receptor agonist activity and will therefore be useful in the present invention.

The invention further provides DNA sequences encoding the above complex of the invention and analogs thereof, as well as DNA vectors of sequences, proteins carrying such DNA sequences suitable for expression in suitable eukaryotic or prokaryotic host cells. The ability to generate large amounts of heterologous proteins using a recombinant protein expression system has led to the development of various therapeutic agents, such as t-PA and EPO (Edington, S. M., "Biotech Products as Drug Leads, BioTechnology 13: 649 (1995)"). The various expression hosts from which recombinant proteins can be made are initially prokaryotes (e.g., bacteria) (Olins, PO, "Recent Advances in Heterologous Gene Expression in Escherichia coli, Current Opinion in Biotechnology 4: 520-525 (1993)", lower eukaryotes (e.g., yeast) (Ratner, M., "Protein

Expressio in Yeast, Bio / Technology 7: 1129-1133 (1989)) and higher eukaryotes (e.g., insect and mammalian cells) (Reuveny, S., "Production of Recombinant Proteins in High Density Insect Cell Cultures, Biotechnology and Bioengeneering 42: 235"). -239 (1993), Reff, M. "High-level Production of Recombinant Immunoglobulins in Mammalian Cells, Current. Opinion in Biotechnology 4: 573-576 (1993)). All of these systems are based on the same principle. That is, introducing a DNA protein sequence into a cell of a selected type (temporarily or stably, integrating into the chromosome, or remaining as an episomal element) using host transcription, translation, and a transport mechanism to overexpress the introduced DNA sequence as a heterologous protein. in Keown, WA, &quot; Methods for Introducing DNA into Mammalian Cells, Methods in Enzymology 185: 527-537 (1990)].

In addition to the expression of native gene sequences the ability to manipulate DNA at the nucleotide level was expected to develop new ones which, although based on natural, exhibit new activities as a result of a change in the primary protein structure (Grazia Cusi, M., "Harlequin

Granulocyte-Colony Stimulating Factor Interleukin 6 Molecules with Bifunctional and Anatgonistic Activities, Immunotechnology 3: 61-69 (1997)).

Selected DNA sequences can be physically coupled to form transcripts to develop new fusion proteins, where once independent proteins are now expressed as a single polypeptide unit (Ibanez, CF, "Chimeric Molecules with Multiple Neurotrophic Activities"). and BDNF, "EMBO Journal 10: 2105-2110 (1991)"). The activity of such fusion proteins may vary. For example, fusion proteins may be more potent than single proteins (Curtis, M., Enhanced Hematopoietic Activity of a Human Granulocyte / Macrophage Colony-Stimulating Factor-Interleukin 3 Fusion Protein, Proc. Natl. Acad. Sci., 88: 5809-). 5813 (1991)).

Human IFNP was derived from the production process used in the mammalian Chinese Hamster Ovary (CHO) cell. Type 1 interferons can be expressed in a variety of host cells, including bacteria (Utsumi, J., "Characterization of E. coli-derived Recombinant Human Interferon-beta as Compared with Fibroblast Human Interferon Beta, Journal of Biochemistry 101: 1199-1208 (1987) ), insects (Smith, GE, "Production of Human Beta Interferon in Insect Cells Infected with Baculovirus Expression Vector, Molecular and Cellular Biology 3: 2156-2165 (1983)) and Human (Christofinis, GJ," Interferon Production by Human Lymphoblastoid Human sIFNAR2 is also expressed using a CHO host cell, otherwise soluble receptors, such as SIFNAR2, can also be successfully expressed in a bacterial expression system. (Terlizzese, M., "In vitro comparison of inhibiting ability of soluble TNF receptor p75 (TBPII) vs. Soluble TNF receptor p55 (TBPI) versus TNF-α and TNF-β, Journ al of Interferon and Cytokine Research 16: 1047-1053 (1996)). DNA for each gene was introduced

Into the CHO cell genome using a transfection procedure. The result is recombination and integration of the expression vector. Cells that express said protein were then isolated and cultured. The protein was isolated and purified using standard industrial methods well known in the art.

The invention also encompasses a pharmaceutical composition comprising as an active agent an IFNAR2 / IFN complex or an analogue thereof, or a mixture thereof, or a salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient. An embodiment of the pharmaceutical composition of the invention comprises a pharmaceutical composition suitable for enhancing the efficacy of IFN in the treatment of viral disease, in the treatment of cancer, in immunomodulatory therapy and in other applications of interferons and cytokines.

The pharmaceutical compositions of the invention are prepared by applying a mixture of the complex or analogs thereof with physiologically acceptable carriers and / or stabilizers and / or excipients. They are prepared in dosage form, such as by lyophilization into ampoules. The mode of administration is carried out in any acceptable mode as similar agents are administered and will depend on the conditions to be treated. Administration takes place, for example, intravenously, intramuscularly, subcutaneously, by topical injection or by topical administration or without interruption of infusions, etc. The amount of active agent that can be administered depends on the route of administration, the disease being treated and the condition of the patient. For example, a lower amount of protein relative to body weight is sufficient for local injection compared to intravenous infusion.

Free IFNβ tends to oligomerize. In order to suppress this tendency, the IFNβ formulations produced today have an acidic pH which can cause local irritation when applied. When IFNAR serves as an IFNP stabilizing factor, thereby preventing oligomerization, its use in IFNP formation may serve to stabilize ΙΕΝβ while avoiding the necessity of acidity of the formulations. The non-acid pharmaceutical formulation contains ΙΕΝβ and IFNAR and together with sΕΝβ and IFNAR. other conventional pharmaceutically acceptable excipients form part of the invention.

The invention encompasses the use of the complex of the invention or analogs or mixtures thereof suitable for viral disease therapy, cancer therapy and immuno-modulatory therapy. The interferon receptor-interferon complexes of the invention may be used in anti-viral therapy, in therapeutic indications such as chronic granulomatous disease, acute condyloma, juvenile larynx papillomatosis, hepatitis A and chronic hepatitis B and C infection.

The interferon receptor-interferon complexes of the invention may be used in the treatment of cancer in therapeutic indications such as hair cell leukemia, Kaposi's syndrome, multiple myeloma, chronic myelogenous leukemia, lymphoma and melanoma other than Hodkins.

The interferon receptor-interferon complexes of the invention can also be used in immune modulation therapy, such as multiple sclerosis, rheumatoid arthritis, myasthenia gravis, diabetes, AIDS, psoriasis, etc.

The invention also encompasses the complex or analogs thereof or mixtures thereof suitable for use in the preparation of medicaments suitable for the treatment of the above-described diseases or for use in the above indications.

Overview of the drawings

Figure 1 is a graph depicting the anti-viral activity of SIFNAR2 or IFNAR2Ig (containing the extracellular domain of hIFNAR2 fused to human IgG1 lock, CH2 and CH3 regions) in the presence of ΙΕΝβ.

Figure 2 is a graph showing antiviral activity

SIFNAR2 and ΙΕΝβ at a sub-optimal dose of IFΕΝβ. The concomitant antiviral activity of ΙΕΝβ and sIFNAR2 following a one hour pre-incubation was released at the intermediate dose of ΙΕΝβ.

··· · ·· · 99 · · · · ·

9 ··· · 9 · 9 · · · 9 9

Figure 4 is a graph depicting the anti-viral activity of SIFNAR2 and a sub-effective dose of IFNP (0.95 IU / ml) as a function of pre-incubation time. WISH cells were exposed to IFNP (sub-effective dose of 0.95 IU / ml) and SIFNAR2, followed by pre-incubation at the indicated time. Synergistic antiviral activity was observed only after four pre-incubations. Antiviral activity was measured by MTT conversion 48 hours after VSV change. At a sub-therapeutic amount of IFNβ, formation of the IFNAR2 / IFN complex results in potentiation of antiviral activity.

Figure 4 is a graph depicting increased antiviral activity of human IFNβ following pre-incubation with SIFNAR2.

Figure 5 is a graph showing that the increased antiviral activity of human IFNβ associated with sIFNAR is specific for IFNAR2, but not for other proteins.

Figure 6 is a graph depicting the pharmacokinetic comparison of human IFNβ and IFNβ / IFNAR2 complex in mice as determined by ELISA.

Figure 7 is a graph depicting the pharmacokinetic comparison of human IFNβ and IFNβ / IFNAR2 complex in mice as determined in a bioassay.

Figure 8 is a graph depicting the pharmacokinetic comparison of human IFNα and IFNα / IFNAR2 complex in mice as determined by ELISA.

Figure 9 is a graph depicting the pharmacokinetic comparison of human IFNα and IFNα / IFNAR2 complex in mice as determined in a bioassay.

Figure 10 shows the amino acid sequence of the IFNAR2 / IFNβ fusion protein n = 2 (SEQ ID NO: 14). Linker (GGGGS) ₂ (residues 240-249 of SEQ ID NO: 14) is underlined.

Figure 11 is a gel showing restriction enzyme analysis of pCMV-IFNAR2 / IFNP, 10% PAG, lanes 3-7: enzyme digestion

BamHI / XhoI, lane 2: Smal / Xhol digestion, lane 1:

Marker 9: 9 * 94 marker: digestion of pBR322 DNA-MspI, lane # 2 pCMV-IFNAR2 / IFNP, OGS, lane # 3: 1GS, lane # 4: 2GS, lane # 5: 3GS, lane # 2 6: 4GS, lane # 7: 5GS, lane # 8: 'X174 RFIDNA HaelII cleavage marker.

Figure 12 is a restriction endonuclease map of the expression vector IFNAR2 / IFNβ.

Figure 13 shows Western blot analysis of the IFNAR2 / IFNP fusion protein. Lane 1: IFNAR2 / IFNβ construct that does not contain a linker, Lane 2: IFNAR2 / IFNβ construct that contains a Gly ₄ Ser linker (SEQ ID NO: 1), Lane 3: IFNAR2 / IFNβ construct that contains two Gly ₄ Ser linkers ( Lane 4: IFNAR2 / IFNβ construct containing three Gly ₄ Ser linkers (SEQ ID NO: 1), IFNAR2 / IFNP construct containing four Gly ₄ Ser linkers (SEQ ID NO: 1), an IFNAR2 / IFNβ construct that contains five Gly ₄ Ser linkers (SEQ ID NO: 1).

Figure 14 is a graph depicting the anti-viral activity of internally fused molecules expressed in CHO cell supernatants and normalized to IFNβ standard activity.

Figures 15A-B are graphs showing the pharmacokinetics of IFNβ administered after intravenous injection of IFNAR2. IFNβ was injected either alone or as an IFNAR complex or immediately after separate intravenous injection of SIFNAR2. The serum half-life was determined at post-injection ELISA specific for IFNβ (Figure 15) and biological activity in the WISH antiviral assay (Figure 15B).

Figure 16 is a graph depicting the protective effect as a percentage of the cytotoxicity of various doses of the "universal IFN (human IFNaA / D) complex that complexes with SIFNAR2 compared to the application of universal IFN alone or control.

«4

Figure 17 is a graph depicting serum IFN3 concentration as a function of time after single dose intravenous injection of either Interfusion GS5 or hIFNβ alone.

DETAILED DESCRIPTION OF THE INVENTION

Materials and methods

WISH antiviral bioassay and complex preparation:

The WISH assay was developed based on the protocol described in Novick et al., Soluble interferon-alpha receptor molecules present in body fluids FEBS Lett. 314: 445-448.

Material:

• WISH cells (ATCC CCL 25) • Vesicular stomatitis virus stock solution (ATCC V-520-001522), stored at -70 ° C • IFNβ, human recombinant, InterPharm Laboratories LTD 90 x 10 ⁶ IU / ml, specific activities: 264.5 x 10 ^with IU / mg.

Soluble human IFNAR2, Stock concentration in PBS is 373 pg / ml.

• WISH growth medium (high glucose MEM + salts)

Earles + 10% FBS and 1.0% L-glutamine +

Penicillin / streptomycin (100 U / ml, 100 pg / ml)).

• WISH Assay Medium (high glucose MEM + salts)

Earles + 5% FBS and 1.0% L-glutamine + penicillin / streptomycin (100 U / ml, 100 μς / ηΐ), MTT at 5 mg / ml PBS, stored at -70 ° C).

Methods ♦ · ♦ · · 94 94 94 94 94 94 94 94 94 94 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 • 94 4 4 4 4 14 • Dilution of recombinant human IFNβ to 19 IU / ml (4x concentrated predetermined dose of EC50) in WISH assay medium.

• Dilution starts at a concentration of 90 μς [/ ιη1, preparing 11-fold dilutions of human recombinant sIFNAR2 in Eppendorf tubes in WISH assay medium. Twelve tubes contain only WISH media.

• Add 25 μΐ IFNP to each flat-bottomed well on the 96-well plate (to check the effect of IFNAR2 in the absence of IFNP, add WISH-appropriate medium alone to the 3 x 12 well section. 25 μΐ).

• Add 2 5 μΐ of each sIFNAR.2 dilution to 12 rows of 96-well plates in three copies (or test media only to row 12).

Incubate IFNP with sIFNAR2 for 1 to 4 hours at 37 ° C in an incubator and then add WISH cells.

• Trypsin / EDTA solution is added to the logarithmic growth phase of WISH cells, washed with WISH assay medium, and a final suspension of 0.8 χ 10 ⁶ cells / ml is prepared.

• Add 50 μΐ of WISH cell suspension (4 x 10 ⁴ cells per well) to each well. The final concentrations of IFN [beta] and IFNAR2 are those exposed to the cells. This means that the final IFNP concentration is 4.75 IU / ml (1x) and the final concentration of SIFNAR2 in row one is 22.5 μg / ml.

• After incubation for 24 hours in a humidified 5% CO 2 incubator, 50 µΐ 1:10 dilution (in a suitable medium) was added to all wells outside the wells containing no virus (only the same volume of test medium was added). for the WISH assay) of the VSV stock solution (the dose was predetermined to allow 100% lysis of WISH cells within 48 hours).

After incubation for an additional 48 hours, 25 μΐ of MTT was added to all wells. Plates were then incubated for an additional two hours in an incubator.

The well contents were removed by rotating the plate and 200 µΐ of 100% ethanol was added to the wells.

After one hour, plates were read at 595 nm using Soft max Pro software and Spectramax (Molecular Device) spectrophotometric system.

All sequencing reactions were performed using a ThermoSequenase ™ radiolabeled terminator cycle sequencing kit

Cleveland, OH). All sequencing reactions were performed with CastAway ™ Precast kit gels (Amersham Life Scince, manufacturer's recommended protocols) for sequencing (Stratagene, LaJolla, CA) containing 6% polyacrylamide and 7 M urea. T. Sequence gel autoradiographs were read manually, using Genetics Comuter Sequence Analysis Software Package and a UNIX computer for DNA sequence analysis.

Example 1:

To determine the anti-viral activity of human IFNAR2 / IFNP complex and human IFNAR2Ig-IFNP complex, IFNβ at a fixed concentration (4.75 IU / ml) was pre-incubated for 3 hours at 37 ° C with human SIFNAR2 (recombinant p40 ) or with different concentrations of human sIFNAR2Ig (0.25 to 30,000 ng / ml) and then tested in the WISH-VSV cytopaticity assay. No antivirus protection was detected in the absence of IFN (data not shown).

The anti-viral activity of Type I interferons (used at predetermined concentrations of EC50) in the presence of SIFNAR2 at approximately 30 ng / ml, together with IFNP, showed optimal

’.Ι ί

• Λ agonist activity. However, this activity is not shown by the substance alone. Anti-viral activity was measured by MTT conversion 48 hours after the VSV dose.

If IFN is present at the expected EC50 concentration, protection is recorded (shown in Figure 1, absorbance equals 0.45. If no protection is present, the absorbance value is 0.0; equals 1.8)). When IFNAR2 or IFNAR2Ig were titrated at different concentrations, there was approximately a 4-fold increase in IFNP activity up to an IFNAR and IFNAR2 Ig concentration of 32 ng / ml (also described in Example 2 and Figure 2). Concentrations of IFNAR or IFNAR2Ig greater than 32 ng / ml reduce IFNP activity as expected. This causes competition between sIFNAR with membrane IFNAR of ΙΕΝβ. This experiment supports efficacy and increases ΙΕΙβ- activity in the IFNAR2 / IFN complex.

Example 2:

The effect of changes in ΙΕ změnβ concentration on sIFNAR2 / IFN complex activity was tested at various concentrations of různýchΕΝβ except ED50. As can be seen in Figure 2, at množstvíΕΝβ 4.75 IU / ml, pre-incubation of IFNAR2 for one hour increases aktivituβ activity approximately twice at a maximum concentration of approximately 32 ng / ml. At each higher concentration of ΙΕΝβ, it was not possible to detect an increase in the anti-viral activity of ΙΕΝβ, since the activity had already reached its maximum value. The results support the theory that the IFNAR2 / ΙΕΝβ complex exhibits increased IFN activity.

Example 3:

Estimating the anti-viral activity of the sub-effective concentration

ΙΓΝβ (0.95 IU / ml) at different times of pre-incubation with different concentrations of IFNAR2 complex kinetics were tested

IFNAR2 / ΙΕΝβ. As shown in Figure 3, four hours

pre-incubations are necessary to increase the anti-viral activity of IFNβ at this sub-effective dose. Addition of IFNAR2 to a complex amount of IFNP which is not itself active results in a substantial increase in the anti-viral activity of IFN. This experiment supports the theory of increased activity of IFNAR2 / IFNβ complex IFN.

Example 4:

Bioactivities of IFNP were determined to decrease rapidly after in vitro reconstitution at 37 ° C (saline pH 7.4). This is at least in part due to the formation of oligomeric IFNβ structures that exhibit limited activity. To test whether IFNAR2 increases IFNP stability at physiological pH, an experiment was performed in which IFNβ alone was incubated at various concentrations (in RPMI 1640 incubation medium, Gibco) or in the same incubation medium in the presence of IFNAR2 at a constant ratio of IFNAR2 to IFNβ ( 2.5 ng / IU).

IFNP (at a concentration of 500 IU / ml) was pre-incubated with an equal volume of either SIFNAR2 (1.25 μς / ml) or RPMI only for 3 hours at 37 ° C. Titration of both ΙΓΝβ solutions was performed in a WISH assay medium on a 96-well plate before WISH cells were added. VSV was added after 24 hours of incubation as determined by MTT conversion.

As can be seen in Figure 4, IFNP alone exhibits an ED50 of approximately 104 IU / ml, while pre-incubation of IFNP with soluble IFNAR2 results in a substantial increase in ED50 to 7 IU / ml. The high ED50 of IFNβ alone may be due to oligomerization of IFNβ in solution. The above results show enhanced stability of IFNβ when complexed with IFNAR2.

Example 5:

· · · · · «« «« · · · · · · · · · · ·

In order to estimate whether the increased aktivitaβ activity in the presence of IFNAR2 is due to specific protection with sIFNAR, IFNP activity was evaluated by complexing with IFNAR2 or other unrelated proteins at the same concentration (human IgG, bovine serum albumin (BSA)).

As shown in Figure 5, IFNβ (500 IU / ml) was preincubated with an equal volume of either the indicated proteins (1.25 µg / ml) or only in RPMI incubation medium for 4 hours at 37 ° C. Titration of these IFNP solutions in WISH assay medium was performed prior to the addition of WISH cells on a 96-well plate. Freshly prepared IFNβ was also included to determine the effect of pre-incubation on IFNβ activity. VSV was added after 24 hours and after a further 24 hours incubation CPE was estimated as determined by MTT conversion.

Pre-incubation of IFNβ with non-specific proteins (i.e., BSA or human IgG) at a concentration of 2.5 ng / IU IFNβ does not protect IFNβ activity upon reconstitution. The activity of the IFNAR2 / IFNβ complex in this assay is similar to that of freshly added IFNβ. This result supports the finding that SIFNAR2 stabilizes IFNβ activity.

Example 6:

The IFNAR2 / IFNβ complex exhibits a markedly prolonged pharmacokinetic profile of IFNβ in mice as determined by ELISA and biological analysis (Figures 6 and 7).

B6D2F1 mice received a single intravenous injection of either human IFNβ (2.5 χ 10 ⁶ IU / kg) or the same concentration of IFNβ pre-incubated for 1 hour at 4 ° C with human IFNAR2 (2.5 ng / IU IFN) ). Sera were obtained 0.05 to 48 hours after retro-orbital tissue administration and IFNβ concentration and anti-viral IFNβ activity was determined by ELISA (orbase # 6) or WISH bioassay (Figure 7).

ΦΦ ΦΦ «« «

Φ · · · ♦ · · · · · · φ

Serum concentrations that fell below the assay sensitivity (7.55 IU / ml) in the ELISA were not plotted.

Not only did the complex exhibit a higher pharmacokinetic profile, but also by the WISH anti-viral assay, it was found that the level of biological activity of IFNP in mice was significantly increased throughout the test. This suggests increased stability and increased half-life of the IFNAR2 / IFNβ complex in plasma compared to the plasma

ΙΓΝβ.

*

Example 7:

The IFNAR2 / IFNβ complex exhibits a markedly prolonged pharmacokinetic profile of IFN [alpha] 2a in mice, as determined by ELISA and bioassays (FIGS. 8 and 9).

B6D2F1 mice received a single intravenous injection of either human IFNα (1.25 x 10 ⁵ IU / kg) or the same concentration of IFNα pre-incubated for 1 hour at 4 ° C with human IFNAR2 (14.9 ng / IU IFN) . Sera were collected at the indicated time from retro-orbital tissue and concentration

IFNa2a and anti-viral activity were estimated by ELISA (Figure 8) or WISH bioassay (Figure 8).

9) IFNα was determined by an ELISA specific for human IFNα. Serum concentrations below the sensitivity of the assay (30 IU / ml) in the ELISA were not plotted.

Not only did the complex exhibit a higher pharmacokinetic profile, but also by the WISH anti-viral assay, it was found that the level of biological activity of IFNα in mice was significantly increased throughout the test. This suggests increased stability and prolonged plasma half-life of IFNAR2 / IFN compared to IFNα alone.

Example 8: Design of IFNAR2 / IFN Fusion Proteins

Μ 44 * 9 9 9 9 9 • • • • 4 4 4 4 4 4

4 4 9 9 9 4 4 4 4 9 • 44 44 49 99 49

The constructs were prepared by fusing the C-terminus of the extracellular region of IFNAR2 with the N-terminus of mature IFN using the following peptide linkers, where G and S represent the amino acids glycine and serine.

IFNAR2extracellular (linker) mature IFNJ3 ... ESEFS (GGGGS) _n MSY., Where n = 0 (SEQ ID NO: 5), 1 (SEQ ID NO: 7), 2 (SEQ ID NO: 6), 3 ( SEQ ID NO: 5), 4 (SEQ ID NO: 4), and 5 (SEQ ID NO: 3).

The entire amino acid sequence wherein n = 2 of the IFNAR2 / IFNP fusion is shown in Figure 10 (SEQ ID NO: 14).

The pSVEIF expression vector, which contained a gene expressing human recombinant IFN, was used as a template for PCR. Synthetic primers were designed such that only the coding region of the mature human IFN protein (MSY ...) could be amplified from the template. The 5 'primer included the SmaI cleavage site sequences, the last seven amino acids of IFNAR2 (GQESEFS) (residues 344-349 of SEQ ID NO: 14), and the (GGGGS) i (SEQ ID NO: 1) linker. BamHI and XhoI restriction sites were also introduced into the 5 'primers to allow cloning of other cassettes. The 3 'primer contained the AvrII restriction site immediately after the stop codon of the TGA hIFNP. The PCR contained approximately 1 gram of templar DNA, 1 g of each PCR primer, 0.2 mM of each dATP, dCTP, dGTP and dTTP, 1x TermoPol reaction buffer (10 mM KCl, 20 mM Tris-HCl, (pH 8.8 at temperature) 25 ° C), 10 mM (NH4) 2SO4, 2 mM MgSO4, 0.1% Triton X-100: New England Biolabs, Beverly, MA) and 3 mM MgSO4 (5 mM final MgSO4) in a reaction volume of 100 μΐ . After an initial incubation of VENTR® at 99.9 ° C for 30 seconds, 2 units of VENTR® DNA polymerase were added to the reaction. The PCR comprises 20 cycles: a) at 99.9 ° C for 30 seconds, b) at 65 ° C to 55 ° C for 30 seconds, the temperature decreases by 0.5 ° C for each cycle and c) at temperature 75 ° C for 40 seconds. An additional 15 cycles were performed with the above profile while maintaining the hybridization temperature

4 4 ·

4 * 4 4

4 · 4

4 4 4 4

44

43 * 4 4 4 4

4 4 4

4 at 55 ° C. PCR reactions were purified using the DNA Wizard ™ PCR Preps purification system (Promega, Madison, WI). After digestion of the PCR products with AvrII, the reaction contents were purified on low melting point agarose gels. The gel-derived fragment containing the mature GSιΙΕΝβ protein sequence with the 1 GS linker was ligated into the restriction enzyme digested expression vector pCMVp40 (SmaI + AvrII). This vector contains a gene encoding a soluble form of human recombinant IFNAR2. The contents of the ligation reactions were used to transform competent cells of E. coli XLBlue using standard methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 nd Ed., Cold Spring Harbor Press (Cold Spring Harbor, NY)). , 1989, paragraphs 6.3 and 6.4 in Appendix C and D.)). The correct alignment of the construct, which is called ρΟΜν-ΙΕΝΑΡ2 / ΙΕΝΆβ GS, was confirmed by restriction endonuclease digestion and sequencing of the "PCR-generated interfusion" region. Subsequent constructs were constructed using oligonucleotide cassettes each containing a BamHI overhang, a suitable linker (GGGGS) n (SEQ ID NO: 1, where n = 1), and an XhoI overhang. 0 GS cartridges include Smal and Xhol overhang. After creating the vector ρΟΜν-ΙΕΝΑΒ2 / ΙΕΝΑβ 1 GS, this vector was digested with the restriction enzymes BamHI and XhoI and the appropriate cassettes were ligated into the vector. In order to construct the 0 GS construct, the vector was digested with the restriction enzymes SmaI and XhoI to ligate the cassette.

A total of six vectors were generated, ρΟΜν-ΙΕΝΑΚ2 / ΙΕΝβ n (GS), where n represents 0, 1, 2, 3, 4 or 5 GGGGS linker units (SEQ ID NO: 1). The results of the restriction enzyme digestion are shown in Figure 11. Sequencing primers were designed such that the entire cassette was sequenced for each construct. Large-scale plasmid DNA isolation was prepared for each confirmed construct using a commercially available kit and following manufacturer's recommended protocols (Qiagen, Chatsworth, CA).

·· «♦ 43 ·

9 • · * 4 4 4 φ »• 4 4 4 444« • 4444 449 «

94 »» 9 9 4 4 9 9 9 • 99 9 9 99 9 9 9 9 »9 ·· 99 44 * 4 49 44

Figure 12 is a map of plasmid pCMV-IFNAR2 / IFNAP expression plasmid infusion. Transcription of the IFNAR2 / IFNβ fusion protein is driven by the human early CMV promoter. For the 3 'processing of IFNAR2 / IFN3 transcripts, the human growth hormone polyadenylation signal sequence was used.

List of vectors:

number name Exp.vektor Linker GGGGS (SEQ ID NO: 2) 1 pCMV-IFNAR2-IFNβ, OGS PCMV.PA4 ON 2 pCMV-IFNAR2-IFN3, 1GS PCMV.PA4 One 3 pCMV-IFNAR2-IFNβ, 2GS PCMV.PA4 Two 4 pCMV-IFNAR2-IFNβ, 3GS PCMV.PA4 Three 5 pCMV-IFNAR2-IFNβ, 4GS PCMV.PA5 Four 6 pCMV-IFNAR2-IFNβ, 5GS PCMV.PA4 Five

Sequence data was obtained from regions generated by the IFNAR2 / IFNP 1GS PCR fusion. These data indicate that the sequence was predicted in advance. Sequence data was obtained for the peptide linker region of other constructs. The sequence was also predetermined.

Northern blot analysis was performed for cells transfected with each construct. In all lanes containing IFNAR2 / IFNP n GS samples, a band of approximately 1.4 kb appeared for both IFNAR2 probes and IFNβ probe. For the IFNβ probe, an additional 0.9 kb band was observed. . The band of said size corresponds to alternative splice transcripts comprising at least 6 amino acids of IFNAR2, (n) a GS peptide linker, and a sequence encoding the mature hIFN protein. This was confirmed by sequencing of the cDNA prepared from total isolated RNA from the transiently transfected CHO cells described in Example 9.

> 4 »· 4 • 4

44

44 4 4 «>» »► 4 4 I

44

Example 9:

Temporary transfection.

CHO-DUKX cells are clonal mutants of Chinese hamster ovary cells that lack dihydrophylated reductase activity (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216Biol. 2: 93-96 (1982)).

4220 (1980); Graff et al. Mol. Cell

These cells were maintained in aMEM culture medium with ribunicleosides and deoxyribonucleosides supplemented with 10% fetal bovine serum (FBS) and 1% L-glutamine (complete medium). Temporary transfection was performed using Lipofectamine PLUS ™ reagent (GibcoBRL Life Technologies, Gaithersburg, MD) and according to the manufacturer's protocol. Approximately 24 hours prior to transfection, cells were plated on a 100 mm diameter plate at a density of 2 x 10 ⁶ cells per plate. For transfection, 4 µg of double-stranded vector plasmid DNA (pCMV-IFNAR2 / IFN n GS, where n = 0, 1, 2, 3, 4 or 5) was used. Serum-free medium recommended by the manufacturer was used to dilute DNA and PLUS reagent. After incubation at 37 ° C for 48 hours in complete medium, cell supernatants were collected for ELISA (IFNAR2, ΙΕΝβ) and Western analysis.

RNA extraction and northern analysis

Total cell RNA was extracted from the temporarily transfected CHO-DUKX cells (Chomczynski et al., Anal. Biochem. 162: 156-159 (1987)). 10 g of total RNA in each lane was sized by agarose gels containing formaldehyde as denaturing agent. Samples were loaded in two sets. RNA was transferred to a GeneScreen Plus nylon membrane (DuPont / NEW Medical Products, Boston, MA) by capillary transfer using 10 x SSC (1.5 M sodium chloride, 0.15 M sodium citrate). The immobilized RNA was hybridized with ³² P-labeled hIFNAR2 and ΙιΙΕΝβ PCR fragments. The hybridization was performed in a modified solution according to Church et al., Proc. Nati. Acad. Sci. USA 81:

♦ ·

Φ Φ 4 ► · · • Φ Φ

1991-1995 (1984). The buffer contained 0.25 sodný sodium phosphate pH 7.2, 0.25 M sodium chloride, 7% sodium dodecyl sulfate (SDS), 1 mM ethylylenediaminetetraacetic acid, and 100 gg / ml tRNA from E. coli. The ^32- labeled probes were created using commercially available kits ("High Mannheim, Indianapolis, IN)

Boehringer procedures.

Separated radioactivity by Sephadex G-50 column chromatography.

P se

Prime and removed as described herein. After hybridization, the blots were washed. The strictest conditions used are 0.2 x SSC,

0.1% SDS at autoradiography temperature.

'C. The blots were exposed

Expression of interfusion proteins

The supernatants from the interfusion constructs temporarily transfected into CHO cells were analyzed for IFNAR2 and IFNβ expression levels using IFNAR2 specific ELISA and IFNP

ELISA (Toray). The results of this analysis are shown in Table IV.

Table IV: Interfusion constructs temporarily transfected into CHO cells

Identifi kace sample hIFNp * U / ml hIFNR2 + ng / ml hIFNP # μς / πιΐ hIFNp ## pmol hIFNAR2f pmol IFN / IFNA R 0GS 29 175 1094 0.146 6, 571 31,802 0.207 1GS 24 906 994 0.125 5,602 28,890 0, 194 2GS 13 597 600 0,068 3,063 17,442 0, 175 3GS 14 998 718 0,075 3,378 20,857 0, 162 4GS 9 998 535 0.050 2,251 15,538 0, 145 5GS 9 597 540 0,048 2,161 15,698 0.138 IFNAR2 Don't find out lo se 1176 34,200 Cultivator Don't know 0

· 99 99 99

9 9 9 9 9 • 9 99 9 9

100 999 9 «

9 9 9 9 9 9 9

9 9

9 9

9 9 9

9 9 9

99 µl medium * determined using TORAY + kit determined using hIFNAR2 ELISA # based on specific activity 2.0 x 105 U / mg (2 χ 10 ⁸ / mg) ## based on molecular weight 22,200 (average hIFNP molecular weight determined by MALDI-TOF analysis ♦ based on 34,400 molecular weight (MALDI-TOF average molecular weight).

As the length of the linker increases, the amount of detectable IFNAR2 and IFNβ can be observed to decrease. At this time, it is not possible to determine whether this is due to a decreasing amount of an intrusion protein expressed as the length of the linker increases, or if the length of the linker increases with IFNβ binding to the IFNAR2 binding site and therefore not fully detectable by ELISA. It is known that the IFNβ assay detects only non-complex IFNβ.

Western blot analysis of IFNAR2 / IFNβ fusion protein

To determine that the diffusion proteins were expressed at an appropriate molecular weight and that free IFNβ was not expressed, supernatants from transfected cells were analyzed by western blotting using anti-IFNβ antibodies to detect interfusion. The results of this analysis are shown in Figure 13.

15 μΐ of CHO cell supernatants were temporarily transfected with IFNAR2 / IFNP (lane 1 to 6) or IFNAR2 (lane 7), culture medium (lane 8) or IFNP (lane 9) under SDS-PAGE under non-reducing conditions. This was followed by electrical transfer to the PVDF membrane. The membrane was tested ·· 44 · 44 • 4 4 · 4

44 »4 4 fl> 4 44 ► 4 4 <

44 • 4 44 • 4 4 4

I «4 4»

44 with a rabbit anti-IFNβ probe and developed with alkaline phosphatase linked to goat anti-rabbit antibodies using a WesternStar luminescence detection kit.

No free IFNβ was detected in supernatants of any free constructs. Each construct expressed a protein that corresponded to an inert fusion construct at a suitable molecular weight. The molecular weight was determined by western blotting.

Example 10:

Anti-viral activity of diffusion molecules

Each culture supernatant containing interfusion was tested for anti-viral activity by a cytopathicity assay in which WISH cells (human amniotic cells) were exposed to VSV following either IFNP (control) or interfusion. The results are shown in Figure 14.

CHO cell supernatants containing expressed recombinant proteins (as detected by ELISA and Western blotting) or CHO cell culture medium alone were added in duplicate to the top row of flat bottom 96-well plates suitable for 75 µΙ tissue culture. depression. 50 μ zbývající of WISH cell assay medium was added to the remaining wells of the plate. Each sample was diluted in 3-fold increments by removing 25 μΐ from the wells containing the supernatants (top row) and transferring to a secondary row containing 50 μΐ m WISH assay medium. In wells containing positive controls, the supernatant in the top row was replaced with WISH assay medium containing 1000 IU / ml human IFNβ, which was serially diluted 3-fold along the plate length. 50 μΐ of WISH cell suspension (0.6 x 10 ⁶ cells / ml in WISH assay medium) was then added to each well so that

MM ·· · »t t t * * 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 • • • • 9 9 9

99 89 99 98 98 the top row concentration containing REBIF® is 500 IU / ml and the initial dilution of the CHO cell supernatant is 1: 2. After 24 hours incubation in 5% CO 2 at 37 ° C, 50 μΐ of WISH assay medium containing vesicular stomatitis virus (1:10) was added to each well (except those designated as uninfected control wells). After 48 hours of culture, WISH cell viability was determined by MTT conversion.

Anti-viral activity mediated by interfusion molecules was determined by normalizing the supernatant dilution factor necessary to achieve the EC50 to the EC50 determined for the human ΙΕΝβ purified standard. As the linker segment grows, so does the anti-viral activity of the interfusion constructs.

Example 11:

This example is a pharmacokinetic study of the human IFNβ / sIFNAR2 complex in mice after intravenous administration. Preformed complex components and separately injected complex components are compared. 36 mice of strain D2F1 (6-8 weeks old) (20 g each) were separated into four groups:

Group 1 contains nine mice injected intravenously with a single 200 μΐ solution, containing 50,000 IU / ml human ΙΕΝβ (final dose 10,000 IU / mouse).

Group 2 (nine mice) received a 200 μΐ solution containing 50,000 IU / ml human ΙΓΝβ and 125 mg / ml SIFNAR2 (ratio of

2.5 ng / IU).

Group 3 (nine mice) received (1) 200 μΐ of a solution containing 125 mg / ml then (2) 200 ml of a solution containing 50,000 IU / ml human ΙΓΝβ) (2.5 ng / IU).

Group 4 (nine mice) received (1) 200 μΐ of a solution containing 625 mg / ml and then (2) 200 ml of a solution containing

000 IU / ml human β (10 ng / IU ratio).

• 4 ·

4 4

9 9

4

4 4

44

Blood samples were collected at the specified time (volume of one sample is approximately 200 μΐ) by disrupting the retroorbital venous tissue through the capillary. Three mice from each group were sampled at 0.05, 2 and 12 hours post-dose. In three mice from each group, blood samples were collected at 0.54 and 24 hours after dosing and in three mice in each group, blood samples were collected at 1, 8 and 48 hours after dosing. Blood samples were allowed to clot for one hour at room temperature and microcentrifuged. Sera were stored at -70 ° C until all samples were collected. Sera were tested for the presence of human IFNβ by an IFNβ-specific ELISA using the Toray kit for human IFNβ ELISA (TBF, Inc.) and bioactivity using the WISH antiviral assay.

The results of the IFNP-specific ELISA are shown in Figure 15A and the results of the WISH antivirus test are shown in Figure 15B.

It can be seen that the half-life of IFNP injected as an IFNAR2 complex is similar to the half-life injected with IFNP following a separate injection of IFNAR. These results are consistent with the in vivo formation of the IFNβ / IFNAR2 complex with a prolonged half-life.

Example 12:

C57BL / 6 mice were treated with a complex of universal IFN (human IFNaA / D) and SIFNAR2. The protective effect with respect to cytotoxicity was measured and compared with the application of different doses of universal IFN alone or with the control. The first group received 5 x 10 ³ IU universal IFN complexed with 5 ng / IU sIFNAR. The second group received 5 x 10 ³ IU universal IFN. The third group received 5 x 10 ⁴ IU of universal IFN and the fourth group received PBS / 2% NMS. For each of these mice, NK activity was measured as the cytotoxicity of the sessile cells against NK target YAC-1 cells. The results are · r r r r r r r r r r r r «« «« «« «« «« «« «« «« « The NK activity is significantly higher in mice treated with the universal IFN / sIFNAR2 complex compared to mice treated with the universal IFN only.

Example 13:

As noted above, the pharmacokinetic study showed a dramatic increase in the half-life of type I IFN in serum when administered as a complex with SIFNR2, which is a soluble form of IFN receptor subunits. In vitro results indicate that physical association with IFNAR2 results in stabilization of normally labile IFN. To determine whether the enhanced PK profile and the stabilizing effect of IFNAR2 causes IFN-mediated potentiation and prolongation of efficacy in vivo, a model was developed in which mice with severe combined immune deficiency (scid / scid) were administered a lethal dose of Daudi human B cell susceptible lymphoma cell line. to IFN (Ghetie et al., Cancer Research 51: 5876 (1991), Ghetie et al., Blood 80: 2315 (1990)). In these mice, paralysis occurred between 14 and 20 days after tumor cell injection in conjunction with histological evidence of scattered lymphoma. It should be noted that the survival of these mice can be prolonged in a dose-dependent manner by daily subcutaneous administration of human IFNβ. This model is used to evaluate IFNAR2 as an agent that enhances the biological activity associated with IFN type 1 in vivo.

To determine the relationship between mean paralysis time and IFNP dose in Daudi scid model, five groups of 5 BALB / cByJSmn-scid / scid mice, 4 to 5 weeks old, female sex were injected subcutaneously with 200 μΐ human IFNP per mouse and day every day , from day 0 to day 30. The standard dose of Daudi cells obtained from the frozen stock suspension is 5 × 10 ⁶ cells per mouse in PBS. It is injected subcutaneously into the back of the neck on day 0. Groups of mice received the following amounts of IFN.

Group 1: 135 x 10 ⁴ IU / mouse (675 x 10 ⁴ IU / ml)

Group 2: 45 x 10 ⁴ IU / mouse (225 x 10 ⁴ IU / ml)

Group 3: 15 x 10 ⁴ IU / mouse (75 x 10 ⁴ IU / ml)

Group 4: 5 x 10 ⁴ IU / mouse (25 x 10 ⁴ IU / ml)

Group 5: PBS with 2% NMS.

The paralysis time as individual and mean values are shown in Table V.

Table no

Days of paralysis Mean (± SD) Group 1 26, 31, 35, 37, 40 33.8 (5.4) Group 2 20, 23, 23, 23, 26 23.0 (2.1) Group 3 20, 20, 22, 23, 23 21.4 (1.3) Group 4 17, 18, 18, 18, 18 17.8 (0.5) Group 5 14, 14, 15, 15, 15 14.6 (0.6)

It can be seen that the mean paralysis time in the Daudi / scid xenograft model can be prolonged by daily administration of human IFNβ by subcutaneous injection. This method is dose dependent.

Example 14:

To determine whether the antitumor effect of IFNP can be enhanced by complexing with IFNAR2 at a ratio of 2.5 ng / IU, seven groups of five mice were treated according to the same protocol described in Example 13 except that the test materials were applied to each group as follows: :

IFNβ / mouse only IFN3 plus sIFNAR2 / mouse Group 1 2x 10 ² IU Group 4 2x 10 ² IU plus 0.5 pg Group 2 2x 10 ³ IU Group 5 2x 10 ³ IU plus 5.0 pg Group 3 2x 10 ⁴ IU Group 6 2x 10 ⁴ IU plus 50.0

• · · · · · · · · · · · · · · · · ·

μυ Group 7 was administered to Daudi cells, the treatment is only diluted

The paralysis time is given as individual and mean values in the following table:

Table VI:

Days of paralysis Mean (± SD) Group 1 16, 16, 17, 18, 19 17.2 (1.3) Group 2 17, 18, 18, 19, 19 18.2 (0.8) Group 3 17, 17, 17, 18, 18 17.6 (0.9) Group 4 17, 17, 18, 18, 19 17.8 (0.8) Group 5 17, 18, 19, 20 and 20 18.8 (1.3) Group 6 21, 22, 22, 23, 26 22.8 (1.9) * Group 7 16, 16, 17, 17, 17 16.6 (0.6)

* Substantially different (p value less than or equal to 0.05) compared to the same non-complex IFNP concentration, pairwise group comparison was performed by ANOVA.

It can be seen that the anti-tumor activity of low dose IFNβ 2 χ 10 ⁴ IU / mouse / day and the Daudi / scid xenograft model significantly enhanced by the formation of a complex with IFNAR2.

In another experiment (not shown) the effect of injection frequency on mean paralysis time was studied. It has been determined that substantially increased anti-tumor activity in the Daudi / scid xenograft model can be obtained by treatment with IFNβ / IFNAR G2 complex when injected only once per week. This is compared to the effect of free IFNβ which is administered once a day. Further, in another experiment (not shown) the optimal ratio of IFNAR to IFNβ was tested. It has been found that the optimal ratio of IFNAR2 to IFNβ in enhancing anti-tumor activity at one? ί · ί i. ** ,, j ;; IFNβ concentration (2x10 ⁴ / IU / mouse / day) was 2.5 ng IFNAR2 per pg IFNβ.

In the second experiment, it was found that the optimal ratio of IFNAR2 to IFNβ for enhanced anti-tumor activity at a concentration of 5β 5 x 10 ⁴ IU / mouse / day was 0.3 ng IFNAR2 per pg ΙΓΝβ. These two experiments indicate that the optimal ratio depends on the ΙΕΝβ concentration, and it appears that the higher the IFNP concentration, the lower the ratio is required.

In another experiment using the same model, it was determined that application of IFNAR2 alone did not increase the survival rate of mice.

Example 15:

This example is a pharmacokinetic study that was conducted to determine the half-life of the 5GS diffusion molecule in the serum of mice after a single dose by intravenous injection. 21 mice, female, strain B6D2F1 (6-8 weeks) (20 g each) were divided into three groups as follows:

Group 1: contains nine mice injected intravenously as a single dose of 200 μΐ of a solution containing 100,000 Ιϋ / ml 5GS interfusion (final dose is 20,000 IU / mouse or 5 x 10 ⁵ IU / kg).

Group 2: Nine mice received 200 μΐ of a solution containing 100,000 IU / ml human ΙΕΝβ.

Group 3: contains three injected mice that serve as a negative control.

Assuming that the blood volume is approximately 2 ml / mouse the theoretical Cmax and Tmax are 10,000 IU / ml for groups 1 and 2. The blood of three mice from each of groups 1 and 2 was tested at 0.05, 2 and 12 hours after application. Blood from three mice from each of Groups 1 and 2 was tested at 0.5, 4 and 24 hours after administration, and blood from three mice from each of Groups 1 and 2 was tested.

1, 8 and 48 hours after application. Serum was tested for the presence of bioactive human IFNP using the WISH assay.

The results are shown in Figure 17. While IFNβ is removed almost immediately, the diffusion molecule persists in serum long after injection. This suggests that the fusion protein has the desired stabilizing effect.

Claims (22)

PATENT CLAIMS A molecule comprising a type I interferon (IFN) complex and a human α / β (IFNAR) receptor subunit capable of binding to an IFN type I complex, wherein said type I IFNAR binds to said IFNAR by covalent bond or peptide bond wherein the type I IFN has a sequence substantially comprising the sequence (a) natural IFN type I, (b) a fragment of the substance described in (a) that exhibits biological activity of type I IFN; (c) a variant of the substance described in (a) or (b) which has at least 70% sequence identity to the substance described in (a) or (b) and which exhibits biological activity of type I IFN; (d) variants of the substance described in (a) or (b) which are encoded by a DNA sequence that hybridizes to the complement of the natural DNA sequence encoding the substances described in (a) or (b) under moderately stringent conditions and exhibits Type I IFN biological activity; (e) a salt or functional derivative of the substance described in paragraph a), b), c) or d) which exhibits biological activity of type I IFN and wherein said IFNAR has a sequence substantially comprising (f) the polypeptide chain of natural human IFNAR; (g) a fragment of the substance described in paragraph (f) that exhibits biological activity of IFNAR; (h) variants of the substance described in paragraph (f) or (g) which show at least 70% sequence identity to the substance described in paragraph (a) or (b) and which exhibit the biological activity of IFNAR; · 4 4 • 4 4 (i) a variant of the substance described in (f) or (g) that encodes a DNA sequence that hybridizes to the complement of the natural DNA sequence encoding the substance described in (f) or (g) under moderately stringent conditions and that exhibits IFNAR biological activity; (j) a salt or functional derivative of a substance described in paragraph f), g), h) or i) which exhibits biological activity of IFNAR. The molecule of claim 1, wherein said type I IFN binds to said IFNAR by a covalent bond. The molecule of claim 1, wherein said type I IFN binds to said IFNAR by peptide bond. The molecule of claim 3, wherein said type I IFN is linked to said IFNAR by a peptide linker. The molecule of claim 4, wherein said peptide linker is (GGGGS) n, where η = 1 to 5. The molecule of claim 1, wherein the type I IFN is IFNα, IFNβ or IFNα. The molecule of claim 6, wherein said type I IFN is ΙΤΝβ. The molecule of claim 1, wherein said IFNAR is the beta subunit of the human α / β interferon receptor (IFNAR2). 9. A DNA encoding a fusion protein which is a molecule according to claim 3. 10. A host cell characterized in that it has been transformed with the vector carrying the DNA of claim 9 in a manner that allows expression of said fusion protein. 11. A method of preparation comprising the host cells of claim 10 of the fusion protein. fusion that involves protein, culture and recovery of the expressed 12. Method of improvement storage time Type I interferon confesses č u j i c i s e by This includes storage of that interferon ve form of the complex according to of claim 1 or complex, where indicated IFN se binds on IFNAR non-covalent bond. 13. Pharmaceutical composition containing pharmaceutically an acceptable type I interferon carrier (IFN) and a human interferon α / β receptor subunit (IFNAR) capable of binding to an IFN type I complex, wherein said type I IFN has a sequence substantially comprising a sequence (a) natural type I IFN (b) a fragment of the substance described in (a) that exhibits biological activity of type I IFN; (c) a variant of the substance described in (a) or (b) which has at least 70% sequence identity to the substance described in (a) or (b) and which exhibits biological activity of type I IFN; (d) variants of the substance described in (a) or (b) which are encoded by a DNA sequence which hybridizes to the complement of the natural DNA sequence encoding the substance described in (a) or (b) under moderately stringent conditions and which exhibits biological activity of type I or t * 9 8 8 • «0 · (e) a salt or functional derivative of a substance described in (a), (b), (c) or (d), which exhibits biological activity of type I IFN and wherein said IFNAR has a sequence substantially comprising the sequence (f) the polypeptide chain of natural human IFNAR; (g) a fragment of the substance described in paragraph (f) that exhibits biological activity of IFNAR; (h) variants of the substance described in paragraph (f) or (g) that have at least 70% sequence identity to the substance described in paragraph (f) or (g) and which exhibit biological activity of IFNAR, (i) a variant of the substance described in (f) or (g) that encodes a DNA sequence that hybridizes to the complement of the natural DNA sequence encoding the substance described in (f) or (g) under moderately stringent conditions and that exhibits IFNAR biological activity; (j) a salt or functional derivative of a substance described in (f), (g), (h) or (i) which exhibits the biological activity of IFNAR. 14) Use of a human interferon α / β receptor (IFNAR) in the manufacture of a medicament for potentiating the biological effects of type I interferon (IFN), wherein said IFNAR has a sequence substantially comprising the sequence (a) natural type I IFN, (b) a fragment of the substance described in (a) that exhibits biological activity of type I IFN; (c) a variant of the substance described in (a) or (b) which has at least 70% sequence identity to the substance described in (a) or (b) and which exhibits the biological activity of IFNAR; (d) variants of the substance described in (a) or (b) which are encoded by a DNA sequence that hybridizes to the complement of the natural DNA sequence encoding the substance described in (a) or (b) after 4 9 4 4. • 4 • 9 • 9 • 4 4 9 9 • 444 949 49 94 of moderate conditions and exhibiting IFNAR biological activity; or (e) a salt or functional derivative of a substance described in (a), (b), (c) or (d) which exhibits the biological activity of IFNAR. Use of a type I interferon (IFN) complex and a human α / β (IFNAR) receptor subunit capable of binding to an IFN type I complex, in the manufacture of a medicament for prolonging the effect of type I IFN in vivo, wherein said type I IFN has a sequence essentially comprising the sequence (a) natural type I IFN (b) a fragment of the substance described in (a) that exhibits biological activity of type I IFN; (c) a variant of the substance described in (a) or (b) that exhibits at least 70% sequence identity to the substance described in (a) or (b) and which exhibits biological activity of type I IFN, (d) variants of the substance described in (a) or (b) which are encoded by a DNA sequence that hybridizes to the complement of the natural DNA sequence encoding the substance described in (a) or (b) under moderately stringent conditions and that exhibits type I IFN biological activity; (e) a salt or functional derivative of a substance described in (a), (b), (c) or (d), which exhibits biological activity of type I IFN and wherein said IFNAR has a sequence substantially comprising the sequence (f) the polypeptide chain of natural human IFNAR; (g) a fragment of the substance described in paragraph (f) that exhibits biological activity of IFNAR; (h) variants of the substance described in (f) or (g), which show at least 70% sequence identity to the substance described * 9 • 9 9 4 • 9 9 9 9 • 4 9 4 44 99 in paragraph (f) or (g) and which exhibits IFNAR biological activity, (i) a variant of the substance described in (f) or (g) that encodes a DNA sequence that hybridizes to a complement of the natural DNA sequence encoding the substance described in (f) or (g) under moderate conditions and that exhibits IFNAR biological activity; (j) a salt or functional derivative of a substance described in paragraph f), g), h) or i) which exhibits biological activity of IFNAR. 16. A molecule according to claim 1 which is used as a medicament. The use of claim 15, wherein said complex comprises a non-covalent complex of said type I IFN and said IFNAR. The use of claim 16, wherein said type I IFN and said IFNAR are administered separately and said complex is formed in vivo. The use of claim 15, wherein said complex comprises a complex wherein said type I IFN binds to said IFNAR by a covalent bond. The use of claim 15, wherein said complex comprises a fusion protein, wherein said type I IFN binds to said IFNAR by peptide bond. The use of claim 20, wherein said type 1 IFN is linked to said IFNAR by a peptide linker. The use of claim 15, wherein said type I IFN is IFN α, IFNβ or IFNα. The use of claim 22, wherein said type I IFN is ΙΤΝβ. The use of claim 15, wherein said IFNAR is the beta subunit of the human interferon α / β receptor (IFNAR2).