NZ620689B2 - Hcv immunotherapy - Google Patents

Abstract

Discloses use of Interleukin-7 (IL-7), for the manufacture of a medicament for the treatment of hepatitis C in a patient infected with hepatitis C virus, wherein the patient has been treated with an antiviral agent or a combination of antiviral agents, so as to reduce viral load, before administration with IL-7, wherein the antiviral agent or combination of antiviral agents reduces the viral load to less than 5 Log10 IU/mL. on with IL-7, wherein the antiviral agent or combination of antiviral agents reduces the viral load to less than 5 Log10 IU/mL.

Description

HCV immunotherapy The present invention s to the field of hepatitis C treatment. More particularly it es a new therapy against hepatitis C, using interieukin—7 (IL-7).

Background of the ion: Hepatitis C is the major cause of chronic liver disease and its complications including liver fibrosis and sis, liver failure and hepatocellular carcinoma.

Hepatitis C virus (HCV) is a major public health problem worldwide. The World Health Organization (WHO) tes that up to 170 million individuals worldwide (3% of the world population) are infected with hepatitis C virus (HCV), more than 130 million of those individuals are chronically infected and at risk of developing liver cirrhosis and liver cancer. Around four million people become infected with HCV each year (World Health Organization. Viral cancers: Hepatitis C. oniine www.who.int; WHO 2010).

Today’s standard-of—care (SOC) for eradication of HCV from the liver consist of Pegylated type I interferon (PegiFN) and synthetic nucleoside ribavirin (RBV) therapy (Fried MW at al; N Eng! J Med. 2002; 347(13):975-82; EASL Clinical Practice Guideline: ment of tis C virus infection, J Hepatol. 2011; 55:245—264). However, this standard therapy has limited and unpredictable efficacy, an extensive toxicity profile frequently leading to treatment discontinuation and is very expensive. Less than half of the cally HCV—infected individuals of genotype 1 and 4 respond to erm ent (48 weeks) of standard therapy N/RBV) (Testino G et al; Hepatogastroenferoiogy 2011; 58(106):536—8).

Interferon (lFN) is a very active ral cytokine but it is lymphopenic, with a poor clinical tolerance. 80 while IFN exhibits antiviral activity, it also blocks the production and maintenance of long term protective central memory T cells. This translates to a high frequency of relapses in chronic HCV-infected patients treated with PegiFN/RBV. In addition, compared to a control group, extended ent with erferon in patients with advanced chronic hepatitis C is associated with excess overall ity (Di Bisceglie AM et al; Hepatology 2011; 53(4):1100-8).

New antiviral compounds have been developed that target inhibition of different steps of the HCV life cycle. Several new antiviral drugs (small molecule inhibitors of viral ation also referred to as direct—acting antivirals (DAAs), including protease inhibitors and polymerase inhibitors) for hepatitis C, are currently in an advanced stages of development. Telaprevir and Boceprevir have reached the market (Ghany et al, Hepathology, 2011, 54(4):1433—1444). These new antiviral agents have been tested in monotherapy or in multidrug therapy, with or without standard of care (PeglFN/RBV).

However, direct—acting antiviral monotherapy generally results in development of drug resistance which considerably reduces its effectiveness and leads to treatment failure.

Drug resistance is a significant tion to the use of DAAs. For example, Telaprevir (an N83/4 protease inhibitor) monotherapy induces a viral load decrease of close to 99% within two days of therapy initiation but frequently, even though treatment continuation, there is a rebound in viral load within ten days (Kieffer TL et al; Hepatology; 2007 Sep; 46(3):631-9) due to emergence of drug resistance (Rong L et al; Sci Trans] Med; 2010 May 5; 2(30):30r332). Chronic infection is maintained by an elevated rate of mutation and a rapid turn-over of hepatitis C viruses, mostly in the liver. This high ility and diversity of the hepatitis C virus causes resistance to one or multiple classes of DAAs.

Consequently, most treatments fail because of replication of variants resistant to antiviral agents. On the other hand, direct—acting antiviral drugs in mono- or ation therapy have shown their potential to increase the response rate and/or shorten the ent duration, but they only work as an add on therapy together with PegIFN/RBV (McHutchison JG et al; N Engl J Med. 2009; 360(18):1827-38). The efficacy of these combined ies has been demonstrated only for genotype-1 infection. Furthermore, they induce more side effects and increase the cost of treatment. Finally, their efficacy s uncertain in terms of potential drug resistance issues.

Several immune—modulating agents (among which are onal antibodies, cytokines such as the new interferon lambda, vaccines, and TLR ts) capable of stimulating a general and specific immune response against HCV are also in development.

A number of scientific groups are currently g to develop both T cell and dy based vaccines to t and also to treat HCV infection, but no vaccine exists so far.

Furthermore, it may not be possible to develop a vaccine that s all HCV genotypes because of the high degree of genetic diversity exhibited by the virus.

IL-7, a cytokine that is critical for T cell pment and homeostasis, has ted interesting antiviral activity in preclinical models of chronic LCMV (Mice infected with LCMV clone-13 having persistent high-level viremia), but this activity only develops at very high doses of lL-7 which are not appropriate for testing in patients (Pellegrini M et al; Cell 2011; 144(4):601-‘|3).

WO 2013017653 3 Despite the fact that the various therapies to control the virus have been improved over the past decade, limitations still remain, among which are treatment duration; treatment efficacy in curing chronic HCV; ent tolerability, excessive cost and inadequate access. Not all fected patients benefit from antiviral treatment. None of the treatments proposed so far are able to offer a broad response rate over a very short term (weeks) — along with a ged effect ing protection from relapses. Today, non- responder HOV-infected patients have limited treatment options. An improved therapy is thus needed to treat HCV infection and HCV—related diseases and deaths.

Summary of the invention: The invention proposes lL-7 therapy to stimulate an efficient immune response against a HCV virus, in ation with a short antiviral ent that decreases the circulating HCV virus concentration.

The invention provides lL—7, for use in treating hepatitis C in a patient infected with hepatitis C virus, in combination or subsequently, with an antiviral agent or a combination of antiviral agents.

Preferably the antiviral agent or combination of antiviral agents is in a therapeutically effective amount that reduces HCV viral load to less than 5 Logm IU/mL, preferably less than 4 L091o IU/mL, more preferably less than 3 Logic lU/mL.

In a preferred embodiment, lL—7 is used in a t who has been treated with an antiviral agent or a ation of antiviral agents, so as to reduce viral load, before administration with |L~7. it is thus provided a new therapeutic regimen for treating or inhibiting Hepatitis C infection in a human subject in need thereof, comprising: - administering an antiviral treatment to decrease hepatitis C viral load and — administering interleukin-7 pharmaceutical composition to restore immune functions and provide a durable cure after tinuation of therapy.

Preferabiy the antiviral agent or combination of antiviral agents reduces the viral load to less than 5 Logm IU/mL, preferably less than 4 Log1o lU/mL, more ably less than 3 Logw lU/mL, before administration with lL-7. 4 PCT/EPZO12/065125 The antiviral agent is advantageously selected from the group consisting of an interferon, a protease inhibitor, a polymerase inhibitor, an inhibitor of virus entry, 3 helicase inhibitor, and ribavirin, or combinations thereof.

In other words, the invention s to the use of Interleukin-7 (lL—7), for the manufacture of a medicament for ng hepatitis C in a patient infected with hepatitis C virus, in combination or subsequently, with an antiviral agent or a combination of antiviral agents.

It is described a method for treating hepatitis C in a patient infected with hepatitis C virus (HCV), which method comprises administering the patient with a therapeutically effective amount of an antiviral agent or of a ation of antiviral agents so as to reduce HCV viral load, while administering the patient with a therapeutically effective amount of |L-7 so as to stimulate an ent immune se against the residual virus.

In a particular embodiment, the treatment with the antiviral agent or the combination of antiviral agents is started simultaneously with the treatment with lL-7, and maintained during at least part of the treatment with lL~7, preferably during 6 to 12 weeks.

In another embodiment, the treatment with the antiviral agent or the combination of antiviral agents is started before the treatment with lL—7, and maintained during at least part of the ent with lL-7, preferably during 6 to 12 weeks. ln another embodiment, the treatment with the antiviral agent or the combination of ral agents is started one week after the treatment with lL—7, and maintained during at least part of the treatment with lL-7, ably during 6 to 12 weeks. This regimen may be useful especially when the antiviral agent is an interferon. Indeed, as Interferon is lymphopenic, starting lL-7 therapy one week before can help the immune system respond efficiently.

The invention makes it possible to induce a broad and stable antiviral immune response targeting many viral quasi species, ng viral escape by mutation, and ting HCV relapse after treatment completion or discontinuation.

WO 17653 5 The invention allows to broaden the oire of the ic immune response in the patients (i.e. the diversity of the TCR oire is broadened). This results in preventing relapses.

A rapid and effective antiviral response develops, and viral clearance is obtained.

In on, sustained protection is achieved and supported by production of long term central memory T cells specific to the host (patient) virus.

Legends to the Figures: Figure 1 is a graph showing the evolution of HCV viral load as determined by quantification of HCV RNA over time (days) in 12 patients subjected to 52 weeks of rd peglFN+RBV (ribavirin) therapy (initiated 9 weeks (median) before lL-7 therapy to confirm lack of response to standard y), to which a short cycle of lL-7 (CYT107) was added (1 Oug/kg, once a week, for 4 weeks starting at Day 0).

Patients who cleared the HCV virus decreased their HCV viral load by 2 Logm lU/mL (mean) n screening and D0 and had a viral load lower than 5 Log1o lU/mL before lL-7 therapy.

Figure 2 is a graph showing the evolution of T cell diversity in 12 patients ted to 52 weeks standard peglFN+RBV (ribavirin) therapy ated 9 weeks (median) before lL-7 therapy to confirm lack of response to standard therapy), to which a short cycle of lL-7 (CYT107) was added (mug/kg, once a week, for 4 weeks starting at Day 0). /12 patients were divpenic, implying that they exhibited moderate to severe reduction of immune diversity, before lL-7 therapy. After lL~7 therapy, normal T cell diversity was restored in all patients and remained stable at least until D56.

Detailed description of the invention: It is herein described a method for treating hepatitis C in a patient infected with a HCV virus, which method comprises administering interleukin-7 (lL-7) as an add—on y in said patient.

Surprisingly, by testing various associations in various patient populations, the inventors have found that while lL-7 therapy seems inactive in chronic HCV infection and unable to WO 20131017653 6 clear the virus in patients with commonly observed high viral loads (i.e. HCV RNA greater than 5 Log“, lUlmL, generaly between 5 to 7 Logw lU/mL), if an antiviral agent is used to decrease the viral load to te or low levels (i.e. HCV RNA lower than 5 Logo lU/mL, preferably lower than 4 Logw lU/mL) then a short additive lL—7 therapy can (1) develop an sting antiviral activity and quickly clear the virus in most patients, (2) enlarge T cell count, diversity and onality, and, (3) induce an efficient and stable immune response, avoiding HCV relapse after treatment discontinuation or completion. The additive lL-7 y can also t liver tis C—associated fibrosis and minimize risk of cirrhosis.

This was well demonstrated in chronic HCV patients, previously identified as non- responders to standard therapy (PeglFN/RBV), who showed a moderate decrease in their viral load after re-introduction of standard therapy and cleared the virus with the addition of lL—7 therapy when the viral load dropped below 4 Logo IU/mL.

Hepatitis C is a viral hepatitis resulting from an ion by a Hepatitis C virus (HCV). Any HCV strain or genotype (1, 2, 3, 4, 5, 6) is contemplated herein. Preferably the patient is infected with HCV genotype 1 or 4. in the context of the invention, the term "treating" or "treatment", as used herein, means curing, reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more ms of such disorder or condition. The term “curing" preferably means that viral clearance is observed.

By “reducing viral load” is meant reducing the quantity of circulating HCV virus that can be measured, 6.9. by quantitative . Viral load is expressed in Logm lU/mL.

According to the invention, the term "patient" or "patient in need thereof" is intended for a human or non-human mammal infected or likely to be ed with HCV. The patient may be a male or a female, of any age, including children or teenagers. The patient may be asymptomatic, or may show early or advanced signs of hepatitis. in a ular embodiment, the patient shows a high HCV viral load when he begins treatment with the antiviral agent. A “high HCV viral load” means lly greater than 2 Logm lU/mL, still preferably greater than 3 Logm lU/mL, more preferably greater than 4 Logm lU/mL, still more preferably greater than 5 Logm lU/mL In another embodiment, any patient, regardless of his/her HCV viral load, may benefit from the treatment of the invention.

Antivira! agents: The HCV viral load is reduced to below about 5 Logm lU/mL, preferably to below about 4 Logw lU/mL, more ably to below about 3 Log10 IU/mL during a first phase of treatment with an antiviral agent or a combination of antiviral agents.

In a ular embodiment, the antiviral agent may include eron, ribavirin, inhibitors of the HCV protease, inhibitors of HCV polymerase ding nucleoside, nucleotide and non—nucleoside polymerase inhibitors), HCV virus entry inhibitors, helicase inhibitors and a combination thereof. Interferon (lFN) includes, but is not limited to, pegylated or not: lFN alpha comprising an IFN alpha variant such as lFN alpha-2a or lFN alpha—2b, lFN lambda or lFN omega, especially interferon alpha—2a, and even preferably pegylated Interferon alpha-2a, combined or not with ribavirin. Pegylated Interferon alpha-2a combined with ribavirin is currently the standard treatment. Combinations of interferon, associated or not with ribavirin, with inhibitors of the HCV protease or inhibitors of HCV polymerase, are also contemplated. Alternatively, combinations of direct-acting antivirals (DAAs), preferably at least one inhibitor of the HCV protease and at least one inhibitor of HCV rase, may be used as antiviral agents.

Generally speaking, the antiviral treatment may comprise any of the below mentioned drugs, especially interferon, ribavirin, inhibitors of the HCV protease, inhibitors of HCV polymerase (including side, nucleotide and non—nucleoside rase inhibitors), entry inhibitors, helicase inhibitors, and other anti—hepatitis C agents, or combinations thereof: (1) Interferon and/or ribavirin; (2) Substrate-based N83 protease inhibitors (WO 98/22496); (3) Non—substrate—based inhibitors such as 2,4,6-trihydroxynitro—benzamide derivatives (Sudo K. et al., mical and sical ch Communications, 238:643-647 (1997); Sudo K., et al. Antiviral Chemistry and Chemotherapy, 9:186 (1998)), including 82 and RD3-4078, the former substituted on the amide with a 14 carbon chain and the latter processing a para-phenoxyphenyl group; (4) Thiazolidine derivatives, which show relevant inhibition in a reverse—phase HPLC assay with an NS3/4A fusion protein and NS5A/SB substrate (Sudo K. et al., Antiviral Research, 32: 9-18 (1996)), especially nd RD6250, possessing a fused cinnamoyl moiety substituted with a long alkyl chain, RD4 6205 and R04 6193; (5) Thiazolidines and benzanilides, identified in Kakiuchi N. et al. J. FEBS Letters 421, 217—220; and Takeshita N. et al. Analytical Biochemistry, 247: 242-246 ; (6) A phenanthrenequinone, which possesses activity against protease in a GE and diography assay and is ed from the fermentation culture broth of Streptomyces sp., Sch 68631 (Chu M. et al., Tetrahedron Letters, 37: 7229-7232 (1996)), and Sch 351633, isolated from the fungus Penicillium griscofuluum, which demonstrates activity in a scintillation proximity assay; (7) Selective N83 inhibitors based on the macromolecule elgin c, isolated from leech (Qasim M. A. et al., Biochemistry, 36: 1598—1607 (1997)); (8) Helicase inhibitors (U.S. Pat. No. 5,633,358); (9) Polymerase inhibitors, such as nucleotide analogues, gliotoxin ri E. et al., Journal of Virology, 73:1649-1654 (1999)), and the natural product cerulenin nn V. et al., Virology, 249: 108-118 (1998)); (10) Antisense phosphorothioate oligodeoxynucleotides ) complementary to sequence hes in the 5' non- coding region (NCR) of the virus, or nucleotides 326—348 comprising the 3‘ end of the NOR and nucleotides 371—388 located in the core coding region of the HCV RNA; (11) Inhibitors of IRES—dependent translation; (12) Nuclease—resistant ribozymes; and (13) Miscellaneous compounds ing 1—amino-alky|oyclohexanes (US. Pat. No. 6,034,134 to Gold et al.), alkyl lipids (U.S. Pat. No. 5,922,757 to Chojkier et al.), vitamin E and other antioxidants (U.S. Pat. No. 5,922,757 to Chojkier et al.), squalene, amantadine, bile acids (US. Pat. No. 5,846,964 to Ozeki et al.), N—(phosphonoacetyl)~L-aspartic acid, (US. Pat.

No. 5,830,905 to Diana et al.), benzenedicarboxamides (U.S. Pat. No. 5,633,388 to Diana et al.), polyadenylic acid tives (US. Pat. No. 5,496,546 to Wang et al.), 2',3' dideoxyinosine (US. Pat. No. 5,026,687 to Yarchoan et al.), and benzimidazoles (U.S.

Pat. No. 5,891,874 to Colacino et al.).

More recently, other anti-viral drugs, also named direct—acting rals (DAAs), have been developed, mainly depending on rase and protease enzymes as targets, and which may be used as antiviral agents as well: (1) se inhibitors such as telaprevir (VX-950) which is a specific omimetic inhibitor of NSS/NS4a protease (Reesink HW Gastroenterology 2006, 131:997-1002) and boceprevir (SCHSO3034) (Sarrazin C Gastroenterology 2007, 132:1270-1278). Other protease inhibitors of interest include evir, vaniprevir. (2) Polymerase inhibitors of 2' and 3' substituted ribonucleoside analogues such as Valopicitabine, a prodrug of the nucleoside analogue 2—C-methylcytidine (NM283) (Pierra C J med chem. 2006, 49:6614—6620), and non nucleoside RNA-dependent RNA polymerase inhibitors, such as benzimidazole derivatives JTK-109 and JTK—003 (Tomei L.

J Virology 2004, 78(2):938—946). cleoside polymerase inhibitors include vir, filibuvir.

Nucleoside or nucleotide polymerase inhibitors e , PSI-7977. lmmune tors capable of inducing an anti-viral response have been developed as well, including the Toll-like receptor agonists such as isatoribine (TLR7) (Horsmans Y, Hepatology 2005, -731), resiquimod (TLR7 and 8) (Pockros PJ, Hepatology 2007, 47:174-182), and CPG10101 (TLR9) (McHutchison JG, Hepatology 2007,4621341—1349).

The antiviral agent preferably is a direct-acting antiviral (DAA) or interferon or ribavirin, used either alone, together or in combination with other antiviral agents. Telaprevir and boceprevir are preferred protease inhibitors useful in the present invention.

Preferred combinations include (i) interferon and ribavirin, (ii) interferon, ribavirin and DAA(s), (iii) interferon and DAA(s), (iv) ribavirin and DAA(s).

Interferons (lFNs) are a well known family of cytokines secreted by a large variety of eukaryotic cells upon re to various mitogens. The interferons have been classified by their chemical and biological teristics into four groups: lFN—alpha (leukocytes), lFN-beta (fibroblasts), mma (lymphocytes), and lFN—lambda. lFN-alpha and beta are known as Type I interferons; mma is known as Type II or immune interferon and mbda is known as Type III interferon. Type | lFNs and Type Ill lFNs exhibit strikingly similar biological activities. Type lll lFNs (lambda eron ) or interleukin-28129), display lFN-like activities, although they exert their action through a receptor complex distinct from the type | lFNs. The lFNs exhibit anti—viral, immunoregulatory, and antiproliferative activities. In the present invention, the interferon to use preferably is interferon—alpha. l suitable interferon-alphas include, but are not limited to, recombinant IFN oc-Zb such as lNTRON A interferon ble from Schering Corporation, Kenilworth, N.J., recombinant lFNa—Za such as ROFERON® interferon available from Hoffmann—La Roche, , N.J., recombinant lFN—a 20 such as Berofor® alpha 2 interferon available from Boehringer Ingelheim Pharmaceutical, lnc., Ridgefield, Conn. lFN- or n1, a d blend of natural alfa interferons such as SUMlFERON® available from Sumitomo, Japan or as WELLFERON® lFN— or n1 (INS) available from the Glaxo-Wellcome Ltd., London, Great Britain, or a consensus alpha interferon such as those described in US. Pat. Nos. 4,897,471 and 4,695,623 (especially Examples 7, 8 or 9 thereof) and the specific product available from Amgen, Inc., Newbury Park, Calif, or lFN— on n3, a mixture of natural alfa interferons made by Interferon Sciences and ble from the Purdue Frederick 00., Norwalk, Conn., as ALFERON® or recombinant eron alpha available from Frauenhoffer Institute, Germany or that is available from Green Cross, South Korea.

Using IFN a -2b or lFN a —2a is preferredJn a most preferred ment, the interferon is in PEGylated form. A PEGylated interferon is a hylene glycol modified conjugate of eron.

Polyethylene-glycol—interferon alfa—Za conjugate is preferred (see EP 809 996), such as PEGASYS®. ted interferon lambda may also be used (as developed by Bristol Myers Squibb for instance).

Furthermore, interferon may be fused or conjugated to a protein such as albumin. For instance, albumin eron alfa-b (alb-lFN) (Albuferon®) is a polypeptide molecule that combines the therapeutic activity of interferon alpha with the long half-life of human serum albumin. in still a preferred embodiment, interferon is used no more than six weeks, especially interferon is used no more than three weeks after the |L-7 treatment.

Indeed, in the present invention, the antiviral agent is preferably a direct—acting antiviral (DAA) agent targeting the HCV viral genotype of the patient such as a protease inhibitor or a polymerase tor, and preferably a combination thereof.

Intefleukin 7: Within the t of the present invention, “IL-7” designates a mammalian (e.g., human, simian, bovine, equine, feline or canine) IL-7 polypeptide. More preferably, the IL-7 polypeptide is a human lL-7 polypeptide.

Preferred human IL-7 polypeptides of this invention se an amino acid ce as bed in EP 314 415 or in W02004/018681 A2, as well as any natural ts and homologs thereof. The sequence of human iL-7 is also available on gene banks. The typical wild-type protein comprises 152 amino acids and, optionally, an additional N— terminal methionine residue. ts thereof include, more preferably, natural allelic variants resulting from natural polymorphism, including SNPs, splicing variants, etc.

The |L-7 polypeptide used in the present invention is preferably a recombinant lL—7. The term “recombinant”, as used , means that the polypeptide is obtained or derived from a recombinant expression system, i.e., from a culture of host cells (e.g., microbial or insect or plant or mammalian) or from transgenic plants or animals engineered to contain wo 2013/017653 a nucleic acid le encoding an lL-7 polypeptide. “Microbial” refers to recombinant ns made in bacterial sion systems. “Mammalian” refers to recombinant glycoproteins made in mammalian expression systems. All of these host cells should preferably express either lly or after transgenesis an appropriate glycosyltransferase and/or sialyltransferase gene. lL-7 polypeptide can also be glycosylated through the use of appropriate in vitro or in vivo glycosyltransferase and/or sialyltransferase les, or by grafting oligosaccharide structures. CHO cells are preferred .

A specific example of a human lL-7 polypeptide is a polypeptide of SEQ ID NO: 1 comprising the disulfide bridges Cys2-Cy592; CysB4—Cy8129 and Cys47~Cysi41, as described in EP 1 527 179.

Also, lL-7 polypeptides of the present invention may comprise the sequence of a mature lL-7 polypeptide, or further comprise additional amino acid es, such as a secretion e for instance. Preferred examples of such secretion peptides include, without tion, a signal peptide selected from the group ting of the EPO signal peptide, SEAP signal peptide, IgGkappa signal peptide, Lactotransferin/vitronectin signal peptide, VlP/vitronectin signal peptide and cytostatin bis signal peptide.

In a red embodiment, lL—7 is in hyperglycosylated form, as described in W02007/010401.

Within the t of the present invention, the term “hyperglycosylated lL—7" designates an lL-7 ptide having at least three glycosylated amino acid residues, an average isoelectric point inferior to 6.5 and an average molecular weight superior to 27 KDa as determined by SDS gel electrophoresis.

The structure and number of oligosaccharide units attached to a particular glycosylation site in the hyperglycosylated lL-7 polypeptide can be variable. These may be, for instance, N-acetyi glucosamine, N—acetyl galactosamine, mannose, ose, glucose, fucose, xylose, glucuronic acid, iduronic acid and/or sialic acids.

More preferably, hyperglycosylated lL—7 polypeptides comprise N-Iinked andlor O—linked carbohydrate chain(s) selected from: a) a mammalian type sugar chain, preferably of the type expressed by CHO cells; WO 20131017653 12 b) a sugar chain comprising a complex N-carbohydrate chain (e.g., a triantenary or biantenary structure), more preferably containing high mannose and acetylglucosamine molecules and high al sialic acid residues; 0) a sugar chain comprising an O—carbohydrate chain without and preferably with a terminal sialic acid residue; d) a sugar chain sialylated by alphaZ,6—sia|yltransferase or alpha2,3- sialyltransferase ; and/0r e) a sialylated sugar chain displaying between 3 to 30 sialyI—N— galactosamine, preferably 7 to 23.

Particularly preferred carbohydrate chain(s) se a triantenary or biantenary structure with partial or complete terminal sialylation. Further preferred carbohydrate chains comprise enary structures and tri or bi—sialylation, and/or a diantenary structure with disialylation.

The lycosylated interleukin—7 polypeptide of interest advantageously has an e isoelectric point inferior to 6,5 and an average apparent molecular weight superior to 27 kDa, between 28 KDa and 65 KDa (theroretical for a 7N + 10 glycosylation), preferably between 28 KDa and 35 KDa (as shown for 3 3N + 10 glycosylation ), by gel electrophoresis (confirmed by Western blot) which is translated to kDa by mass spectrometry analysis.

A sylation site" designates any amino acid residue or region in a polypeptide which is subject to glycosylation, i.e., the attachment of a carbohydrate structure. Such sites are typically N-glycosylation sites (i.e., any amino acid residue or region in a polypeptide which allows the attachment of a carbohydrate structure through N-Iinkage) and/or 0— glycosylation sites (i.e., any amino acid residue or region in a polypeptide which allows the attachment of a carbohydrate structure through O—linkage). Consensus sequences for ylation sites are known per se in the art. As an illustration, a consensus N— glycosylation site typically has the following structure: Asn—X-SerfThr, where X is any amino acid except Proline. Such ylation sites may be either naturally t within an lL-7 polypeptide sequence and/or artificially added or created within said sequence.

A preferred lL—7 composition useful in the t invention comprises at least 80 % human IL—7 recombinant polypeptides having at least three glycosylated amino acid residues, an average isoelectric point inferior to 6.5 and an average molecular weight superior to 27 KDa as determined by SDS gel electrophoresis, and comprising the disulfide bridges Cys2—Cy392; Cys34—Cys129 and Cys47—Cys141.

The lL-7 polypeptides preferably are N—glycosylated on at least three distinct amino acid residues.

In another preferred embodiment, lL-7 is fused to another protein entity. Examples of lL—7 fusion proteins are described in W02005/063820. For instance it is in the form of an lL-7 fusion protein such as (1) an lL—7 functionally attached to a Fc n of an lgG heavy chain, typically through a e hinge region, and the lgG moiety is preferably a human lgG1 or lgG4 as described in W02007/010401, (2) a fusion protein as described in US patents.7,323,549 and 7,589,179, and US patent ation 20090010875 lL-7 , (3) an functionally ated to a human serum albumin (“HSA”) or a portion of a HSA, as a fusion protein, as described in W02007/010401, or (4) an lL-7 functionnally associated to Human Growth Facteor (HGF) or a portion thereof, as a fusion protein. lL-7 variants are encompassed, that show substantial amino acid sequence identity to wild—type mature mammalian lL-7s and substantially equivalent biological activity, e.g., in standard bioassays or assays of lL—7 receptor binding affinity. For example, lL-7 refers to an amino acid sequence of a inant or non-recombinant polypeptide having an amino acid sequence of: i) a native or naturally-occurring allelic variant of an lL-7 polypeptide, ii) a biologically active fragment of an lL—7 polypeptide, iii) a biologically active polypeptide analog of an lL—7 polypeptide, or iv) a biologically active variant of an lL-7 polypeptide.

A "variant" of an IL—7 n is defined as an amino acid sequence that is altered by one or more amino acids. The variant can have "conservative" changes, wherein a tuted amino acid has similar structural or al ties, e.g., replacement of e with isoleucine. More rarely, a variant can have "nonconservative" changes, e.g., replacement of a glycine with a tryptophan. Similar minor variations can also include amino acid deletions or ions, or both. Guidance in determining which and how many amino acid residues may be tuted, inserted or deleted without abolishing biological activity can be found using er ms well known in the art, for example software for molecular modeling or for producing alignments. The variant lL—7 proteins included within the invention e IL—7 proteins that retain lL-7 ty. lL—7 polypeptides which also include additions, substitutions or deletions are also included within the invention as long as the proteins retain substantially equivalent biological lL—7 activity. For example, truncations of |L-7 which retain comparable biological activity as the full length form of the lL-7 protein are included within the invention. The activity of the |L-7 protein can be measured using in vitro ar proliferation . The activity of lL-7 variants of the invention maintain biological activity of at least 30%, at least 40%, 50%, 60%, 70%, preferably at least 80%, 90% or even 99% as compared to wild type lL-7. , 95% t lL—7 proteins also include polypeptides that have at least about 70%, 75%, 80% , 85%, 90%, 95% more sequence identity with wild— type lL-7. To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the ce of a first amino acid or nucleic acid ce for optimal alignment with a second amino acid or nucleic acid sequence). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent homology = # of identical positions/total # of positions.timesx100). The determination of percent homology between two sequences can be accomplished using a mathematical thm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264—68, modified as in Karlin and ul (1993) Proc. Natl. Acad. Sci. USA 90:5873— 77. Such an algorithm is incorporated into the NBLAST and XBLAST programs of ul, et al. (1990) J. Mol. Biol. 215:403—10. BLAST tide searches can be performed with the NBLAST program, score=100, wordlength=12. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Research 25(17):3389—3402. When utilizing BLAST and Gapped BLAST ms, the default ters of the respective programs (e.g., XBLAST and NBLAST) can be used.

Regimen: According to the invention, IL—7 is to be administered preferably once or twice a week, preferably during a period of two to six weeks, preferably four weeks, which s an lL- 7 treatment cycle. Such cycle can be repeated at least once.

In a preferred embodiment, |L-7 is administered once a week during four weeks.

WO 17653 15 In a preferred embodiment, the treatment with the antiviral agent or the combination of antiviral agents is maintained during at least part, of the treatment with IL-7, ably the treatment with the antiviral agent or combination of antiviral agents is not interrupted.

Most preferably, lL-7 is to be administered in combination with the antiviral agent or combination of antiviral agents. IL—7 can then be administered separately, simultaneously or tially with the antiviral agent or combination of antiviral agents.

In a particular embodiment, IL-7 is stered simultaneously with the antiviral agent or combination of antiviral agents.

In a preferred protocol, the patient is to be stered with lL-7 before the antiviral agent or the ation of antiviral agents, preferably one week before.

In another preferred protocol, the patient is to be stered with IL-7 from the tion of therapy at the same time as the antiviral agent or the combination of antiviral , preferably starting between DO and D10, most preferably starting between D3 and D7.

In another preferred protocol, the patient is to be administered with an antiviral agent or a combination of antiviral agents during a first phase, that is preferably of at least one week duration, so as to reduce the viral load, followed by a second phase of preferably 2 to 6 weeks of lL—7, preferably combined with an antiviral agent or a combination of antiviral agents.

The administration of lL—7 may be followed by a third phase lasting at least 1 to 3 weeks, or may be extended beyond 4 or 6 weeks or more of treatment with an antiviral agent or a combination of antiviral agents. Preferably this third phase lasts 1 to 9 weeks.

Altogether the patient is advantageously administered with the antiviral agent or combination of antiviral agents for a period of 6 to 12 weeks.

The antiviral agent or combination of ral agents is preferably the same during all treatment phases. However it can be changed if desired.

In a preferred ment, the protocol involves a inary but quick decrease of the patient viral load, followed by the addition of a short term lL—7 therapy, while the above antiviral treatments are maintained over this period and for a few weeks afterwards. 16 PCTZEP2012/065125 When the treatments are stopped, the patient’s immune system can efficiently and stably l itself the HCV virus.

The amount of antiviral agent such as eron may be from 2 to 10 million IU per week on a weekly, twice or three times a week, or daily basis. In a preferred embodiment, the eron-alpha administered is interferon—alpha-Zb and the amount of eron is stered 3 million lU twice or three times a week. in a particular embodiment, the interferon—alpha administered is a pegylated interferon alpha-2b and the amount of interferon administered is from 0.5 to 2.0 micrograms/kg body weight, per week on a weekly, twice or three times a week, or daily basis. Alternatively, the interferon stered is a pegylated interferon alpha-23 and the amount of interferon administered is from 20 to 250 micrograms/kilogram body weight per week on a weekly, twice or three times a week, or daily basis.

Other antiviral agents such as ribavirin may be administered from about 400 to about 1600 mg per day, preferably about 600 to about 1200 mglday or about 800 to about 1200 mg day and most preferably about 1000 to about 1200 mg/kg a day based on the patient’s weight.

Other antiviral agents such as telaprevir may be administered from about 750 mg three times a day (preferably 7—9 hours apart).

Other antiviral agents such as boceprevir may be administered from about 800mg three times a day (7—9 hours apart).

Preferably, the effective amount of interleukin-7 to be administered is comprised between about 3 to 30 ug/kg, preferably between about 5 to 20 rig/kg, and is preferably about 10pg/kg body weight, more preferably 20ug/kg body weight. Preferably it is administered on a weekly basis, preferably for 2 to 6 weeks.

If desired, lL-7 can be stered twice a week. in preferred embodiments, lL-7 can be administered once a week, during a cyclic period of two to four weeks. The cycle could be repeated at least once. lL-7 and the antiviral agent may be administered aneously, either tely or within the same formulation. Preferably, they are administered simultaneously, and both therapies may be initiated at the same time or lL~7 may be initiated one week before we 2013I017653 17 antiviral agent. More preferably, they are administered separately, according to different schedules. The antiviral agent dose is preferably administered during the same period of time that the patient receives doses of lL—7.

Pharmaceutical compositions: The pharmaceutical compositions comprising lL—7 may be suitable for oral, , or parenteral routes, more particularly by intravenous, subcutaneous, ermal, intra- arterial, intra~peritonea| or intra—muscular, as well as intranasal route. The parenteral route, especially subcutaneous, is preferred. For ce, the active ingredient is associated with a pharmaceutically acceptable carrier, excipient or diluent which may be selected from neutral to slightly acidic, isotonic, buffered saline, solutions or suspensions and more preferably from sucrose, trehalose, and amino acid. The pharmaceutically compatible carrier is preferably contained in an appropriate buffer to form an isotonic solution. An appropriate buffer has preferably a pH range comprised between 4.5 to 7.5, ably 5.0 to 7.0, even more preferably of about 5.5 and is preferably an organic salt selected from a sodium citrate buffer or an ammonium acetate buffer. The pharmaceutical composition may be in the form of a suspension, solution, gel, powder, solid, etc. The composition is preferably a liquid form.

The composition may comprise stabilizing , such as sugar, amino acids, proteins, surfactants, etc. The composition may comprise any saline on, including phosphates, de, etc.

A ular pharmaceutical composition according to the invention comprises, in addition to the active drug nce, a protein and/or a surfactant. This presence of a protein, or any other high molecular weight le of natural origin, reduces tion of lL-7 to the host immune system and therefore avoids secondary effects. More preferably, the protein is non genic in the subject, such as any protein of human origin. A most preferred example of protein is human serum albumin. The surfactant may be selected from known surfactants such as Polysorbate products, preferably Tween20® or Tween80®. A specific composition of this invention comprises human serum albumin (preferably 2 to 5 mg/ml) or polysorbate (Tween 20 or 80 ally 0.005%» or any other substance such as a tensioactive substance or amino acid (e.g., arginine,, glutamate, or a mixture of arginine and ate) or sugar (e.g., sucrose, trehalose, sorbitol), capable of WO 2013017653 18 preventing lL-7 immunogenicity due to protein aggregation and/or local persistence of the drug product at injection site after administration of the composition. in a ular embodiment, the administration route is the oral route. In comparison to other polypeptide hormones, oral route is indeed acceptable for lL-7, especially in hyperglycosylated form, because of the exceptional stability of this n. The compositions can then be in a solid form, such as a tablet or a powder or a capsule, or in a form of a liquid, such as a syrup or an emulsion, prepared in an appropriate pharmaceutically acceptable carrier. Preferably the carrier itself is stable in the gastro- intestinal tract and in the atory system and exhibits an acceptable plasma half—life.

Gastric acid—resistant capsules, such as gastric acid-resistant capsules ning a micro-emulsion or liposome formulation of |L-7 polypeptide, are advantageous.

Additional active ingredients, such as immuno-stimulating agents, preferably ed from a hematopoietic cell growth factor, a cytokine, an antigenic molecule (or antigen) and an adjuvant, may be used for combined, te or sequential use.

Therapeutic indication: The invention allows a dramatic reduction in the HCV viral load.

Viral clearance and alleviation of the symptoms may be observed within 1 week to 6 months, preferably within 1 week to 3 months after ent.

The invention makes it possible to inhibit the progress of the disease, and to obtain a substantially te nce of the virus. in other words HCV RNA becomes undetectable in the patient.

The invention is particularly useful for preventing or ng any deleterious ion resulting from the HCV infection, in particular any onset of liver fibrosis or cirrhosis or hepatocarcinoma.

The protocol of the ion is of particular interest in a patient who has not responded to a prior treatment. These patients include non-responder patients (also called partial responders or slow responders) or null-responder patients. in particular, non—responder patients (also called partial responders or slow responders) are patients for whom HCV RNA has decreased by 2 logs t week 12 but does not become undetectable by week 24, after initiation of a treatment, especially a prior treatment with interferon alone, or a combination of ribavirin and eron, which is currently the standard treatment. These patients are unlikely to e SVR (sustained viral response) even when retreated with standard therapy. Null-responders are patients for whom HCV RNA has not decreased by at least 1 log (a factor of 10) after 4 weeks of treatment, or by 2 logs after 12 weeks of treatment. These patients are extremely ly to achieve SVR even when retreated with standard therapy.

Absence of viral response to previous treatments is defined as null-response or absence of early viral response (EVR), defined by a decrease of HCV RNA loads lower than 2 logs after 12 weeks as measured by a tative RT PCR test, compared to baseline levels measured by a similar que. Or, absence of end of ent response defined by detectable HCV RNA at the end of treatment.

The protocol of the invention may be also advantageous for treating a nai've patient, Le. a patient who has never been treated for an HCV infection, more particularly a patient who has never been treated with ribavirin or any interferon.

Patients with tis C who have been d for the infection, especially with ribavirin or any interferon, may also be good candidates for the combination therapy of the invention.

These include patients with hepatitis C who have relapsed after initial response to previous ents.

Patients who show viral break-through can also benefit from the treatment of the invention. A viral break—through occurs when a t achieves a response under therapy (especially therapy with interferon) but then loses the response despite the continuous therapy.

Patients having acute or chronic hepatitis C infections are encompassed, including relapsers, non-responders and null-responders.

In a particular ment, the patient has been genotyped for single-nucleotide rphism in the |L28b gene locus that encodes encoding interferon-lambda-3 (see Thomson et al, Gastroenterology. 2010, 139(1):120~9, and international patent application W02011/013019). A CC pe at SNP r512979860 is indicative of a patient responsive to a SOC treatment, ally pegylated interferon-alpha (PEG—IFN-alpha) plus ribavirin (RBV) treatment. A CT or TT pe is indicative of a sponder or null—responder. In a preferred embodiment, a patient with a CT or TT genotype at SNP rs12979860 can advantageously benefit from the treatment of the present ion.

The protocol is useful against the high variability and diversity of hepatitis C viruses, ng emergence of resistance to treatment, benefiting more patients, and ing a faster, efficient and more sustained response.

The protocol of the invention may further be useful in a patient co-infected with HCV and another virus, such as HIV, HBV, HPV, HSV, or CMV.

Especially this method may be useful to HIV/HCV co-infected patients who present with low CD4 T cell counts (<4OO CD4/uL) among which some cannot be treated due to their very low CD4 T cell counts (<250 CD4/pL), which is not compatible with interferon treatment.

In this case the same treatment n may be applied after a preparation cycle of about 2 to 4 weeks of IL—7 or any other lL—7 agonist to restore adequate CD4 T cell counts before applying the protocol bed herein.

The protocol of the invention could further be adapted to the HCV/HBV co-infected patients who present with a detectable viral load of HBV. In this case a preliminary reduction of the HBV viral load could be obtained by a 3 to 4 month pretreatment with a direct anti HBV antiviral, such as entecavir or tenofovir.

The figures and examples illustrate the invention without limiting its scope. wo 2013/017653 EXAMPLES Example 1: Evaluation in hepatitis C Liver disease of lL-7 in a Phase lilla Study Methods: A Phase We study was designed to evaluate the safety and individual benefits of weekly doses of Interleukin—7 in adult ts infected by Genotype 1 or 4 Virus of Hepatitis C and resistant to current “Standard-Of—Care” (SOC) with Peg—interferon and rin after 12 weeks of this standard bi—therapy.

Absence of viral response to current Standard-Of—Care with pegylated interferon-alpha + ribavirin, identified as absence of early viral response (EVR), is defined as a decrease of HCV RNA loads lower than 2 logs, as measured by a quantitative PCR test after 12 weeks of standard therapy, compared to baseline levels measured by a similar technique.

Or, e of end of treatment se defined by detectable HCV RNA at the end of ent (24 weeks or 48 weeks).

In this open-label, dose-escalating study, (3, 10 and 20 pg/kg/week) CYT107 binant human glycosylated lL—7) was administered by subcutaneous route for 4 weeks (D0 to D21) as an add on to 52 weeks SOC therapy initiated 9 weeks (median) before CYT107 to confirm lack of se to SOC. 6 Patients were included at each dose level and 6 more if at least 2 Patients had a HCV RNA drop > 2 logs.

Results: There were no serious Adverse Events or clinically relevant abnormalities in biological parameters d to CYT107 treatment.

At D56, CYT107 (10ug/kg/wk) induced n values): - a T cell increase +341 CD4/ul(+168%) and +209 CD8/pl (+179%) more than correcting the initial pre-CYT107—SOC induced lymphopenia uL CD4). — a broadening of TCR repertoire diversity (+25%) in the 4 patients with low diversity at D0 (45%). - an increased number of CD3 expressing the 014/87 receptors (+ 73%) These increases in T cell counts, diversity and homing were associated with an accelerated rate of HCV viral decrease and clearance at week 12 in 5/12 patients. wo 2013;017653 22 Afterwards, HCV RNA remained undetectable (median current follow up: 11 months).

Responding patients had a moderate viral load (<4.52 log/mL) at CYT107 tion. , As shown on the Figure 1, the 7 patients unable to decrease their viral load during Standard—of—Care oduction did not clear the virus with lL-7 add on therapy (10 ug/kg, once a week, for 4 weeks starting at day 0), while the 5 patients dropping their viral loads below 5 Log1o lU/mL under Standard bi-therapy, cieared the virus with the same lL-7 treatment (given at day 0).

Figure 2 shows that, after |L~7 therapy, normal T cell ity was restored in all patients and remained stable at least until D56.

Conclusions: In chronic HCV patients defined as non—responders to standard bi—therapy with PEGinterferon and ribavirin, |L~7 treatment was safe and expanded both CD4 and CD8 T cells, an effect known to provide an efficient and stable immune response. lL-7 also contributed to an increase of T cell homing in lymphoid , and normalization of the diversity of the TCR repertoire. This effect was systematically associated with viral nce in patients dropping their viral loads below 5 Logo IU/mL under the standard bi- therapy.

Claims (1)

1. CLAIMS Use of Interleukin-7 (IL-7), for the manufacture of a medicament for the treatment of hepatitis C in a patient infected with hepatitis C virus, wherein the patient has been treated with an antiviral agent or a combination of antiviral agents, so as to reduce viral load, before administration with lL-7, wherein the antiviral agent or ation 10 of antiviral agents reduces the viral load to less than 5 Log10 lU/mL. The use according to claim 1, wherein the patient has a viral load less than 4 Log10 lU/mL, before administration of lL-7. 15 The use according to claim 1, wherein the patient has a viral load less than 3 Log10 lU/mL, before administration of lL-7. The use according to any one of claims 1 to 3, wherein the antiviral agent is selected from the group ting of a protease inhibitor such as Telaprevir or Boceprevir, a 20 polymerase inhibitor, an inhibitor of virus entry, and a helicase inhibitor, or combinations thereof, optionally in combination with interferon and/or ribavirin. The use according to any one of claims 1 to 3, wherein the ral agent is interferon, either alone or in ation with another antiviral agent. The use according to claim 5, wherein the interferon is interferon alpha or consensus eron or interferon lambda. The use according to claim 5, wherein the interferon is lFNalpha—Za or lFNalpha—Zb. The use according to claim 5, n the other antiviral agent is ribavirin. The use according to any one of claims 5 to 8, wherein the interferon is in PEGylated form. 10. The use according to any one of claims 1 to 9, wherein the treatment with the antiviral agent or the combination of antiviral agents is started before the treatment with IL-7, and maintained during at least part of the treatment with IL-7. 5 11. The use according to claim 10, wherein the treatment with the antiviral agent or the combination of antiviral agents is for 6 to 12 weeks. 12. The use according to any one of claims 1 to 11, wherein IL-7 is to be administered once a week, thereby defining an IL-7 treatment cycle, that is optionally repeated at 10 least once. 13. The use according to claim 12, wherein the IL-7 is stered during a period of two to six weeks. 15 14. The use according to claim 12, wherein the IL-7 is administered during a period of four weeks. 15. The use ing to any one of claims 1 to 11, wherein the patient is to be administered with an antiviral agent or a ation of antiviral agents during a first 20 phase of one week, so as to reduce the viral load, followed by a second phase of four weeks of IL-7 combined with an antiviral agent or a combination of antiviral agents. 16. The use according to claim 15, n the administration of IL-7 is to be followed by a third phase of 1 to 9 weeks of treatment with an antiviral agent or a combination of antiviral agents. 17. The use according to claim 15 or claim 16, n the antiviral agent or combination of antiviral agents are the same during all treatment phases. 18. The use according to any one of claims 1 to 17, wherein the patient has chronic 30 hepatitis C genotype 1 to 6 ion. 19. The use according to claim 18, wherein the patient has chronic hepatitis C genotype 1 to 4. 35 20. The use according to claim 18, wherein the patient has chronic hepatitis C genotype 21. The use ing to any one of claims 1 to 20, for obtaining HCV viral clearance, preventing or delaying onset of liver fibrosis and sis, and/or preventing relapse of the HCV infection. 5 22. The use according to any one of claims 1 to 21, wherein the IL-7 is in the form of a fusion protein. 23. The use according to claim 22, wherein IL-7 is in fusion with Fc nt of an immunoglobulin. 24. The use according to any one of claims 1 to 22, wherein the IL-7 is a wild-type human IL-7 or a variant thereof. 25. The use according to claim 24, wherein the IL-7 is in hyperglycosylated form. 26. The use according to claim 1, substantially as herein described with reference to any one of the accompanying examples and/or figures thereof.