TW201321016A - Immunotherapy composition and regimen for treating hepatitis C virus infection - Google Patents

Abstract

The present invention relates to the field of immunotherapy and more specifically to a composition comprising non-structural (NS) HCV antigens or expression vector(s) thereof for use for treating a hepatitis C virus (HCV) infection. The present invention as also relates to a therapeutic regimen comprising the step of administrating to a subject such an immunotherapy composition as well as a kit of parts comprising a plurality of containers and instructions for carrying out said therapeutic regimen. The immunotherapy composition, kit and therapeutic regimen can be used in combination with antiviral therapy (e.g. interferon therapy and/or ribavirin).

Description

Immunotherapy composition and treatment for treating hepatitis C virus infection (2) Field of invention

The present invention relates to the field of immunotherapy, and more particularly to the use of a composition comprising a non-structural (NS) HCV antigen or one of its expression vectors for the treatment of hepatitis C virus (HCV) infection. The invention also relates to a course of treatment comprising the step of administering to said subject an immunotherapeutic composition and a kit of reagents comprising a plurality of containers and instructions for performing the course of treatment. The immunotherapeutic compositions, kits, and treatment regimens can be combined with antiviral therapies such as interferon therapy and/or ribavirin.

Background of the invention

HCV belongs to the family Flaviviridae and is a enveloped virus having a positive single-stranded ribonucleic acid (RNA) genome consisting of approximately 9,600 nucleotides and its tissue system common to all HCV strains and isolates. (Lippencott-Raven Press, published in 1996 and edited by Fields, "Virology", Third Edition, pp. 945-51 or later). The genomic RNA is translated into a single polyprotein composed of 3010 to 3030 amino acids according to the genotype, which is subjected to proteolytic cleavage by viruses and cellular proteases to produce functional polypeptides, respectively. Protein (core protein and envelope glycoprotein E1 and E2); a small embedded membrane protein p7 that appears to act as an ion channel; and six non-structural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B, the intracellular processes of their vector viral life cycle. In more detail, NS3 (the length of 631 amino acids and the position in the HCV-1 polyprotein precursor from 1027 to 1657) comprises two distinct domains, each having active serine protease activity and An N-terminal domain involved in the maturation of viral polyproteins, and a C-terminal domain that contains helicase activity and plays a role in viral genome replication. NS4 (315 amino acids in length and located in the HCV-1 polyprotein precursor from 1658 to 1972) was cleaved by NS3-related proteases in NS4A and NS4B polypeptides. They are all hydrophobic and are found to be associated with the membrane. The NS4A polypeptide (located from 1658 to 1711) is a cofactor for the NS3 protease, and the function of NS4B (located from 1712 to 1972) is unknown. NS5 (1039 amino acids and located in the HCV-1 polyprotein precursor from 1973 to 3011) is cleaved by NS3-related proteases in NS5A and NS5B polypeptides, and both NS5A and NS5B polypeptides have been found to be localized. A replication complex associated with the ER membrane. NS5B (from position 2421 to 3011) acts as an RNA-dependent RNA polymerase presumably allowing the synthesis of a negative-stranded RNA intermediate and a positive-stranded progeny copy. NS5A (from position 1973 to 2420) is a phosphoprotein and may be involved in the regulation of RNA-dependent RNA polymerase. In addition, it may be involved in HCV antiviral resistance.

The World Health Organization estimates that in 2011, approximately 200 million people worldwide were chronically infected with HCV, and the number of new infections per year was between three and four million. HCV infection is associated with a high proportion of chronic, and an estimated 75% of seropositive individuals have circulating virus (viremia) and chronic liver disease. 20% of chronically infected patients may have serious complications, and the risk of liver cancer is 1 to 4% per year. This makes hepatitis C the leading cause of liver transplantation in the United States and Europe.

Antiviral therapy (SOC stands for "standard care") for patients with chronic HCV infection in most countries, combined with pegylated interferon alpha (PEG-IFN-alpha) and ribavirin (Neyts) In the 2006 issue of "Antiviral Res." 71, pp. 363-371, B). However, the therapy is expensive and lengthy (24 weeks or 48 weeks depending on the infectious HCV genotype), leading to early termination of treatment with significant side effects in 10% of cases, and for a significant number of patients (Insufficient for those with dysfunctional cirrhosis, autoimmune disease, history of depression, and pregnancy). In addition, responding to SOC and achieving a sustained viral response (SVR; no HCV RNA detected 6 months after the end of SOC), only 40 to 50% of patients infected with the first genotype, but Only 70 to 80% of patients infected with the second and third genotypes. During SOC treatment, the kinetics of viral elimination is two-stage, with one of the first few weeks of rapid reduction in viral load, which reflects the viral clearance in the blood; followed by a progressive phase, which reflects Clearance of infected liver cells. Viral reduction is a predictor of SOC response in patients. The treatment of non-reactive and non-reactive patients is usually terminated. The virus reduction of non-reactive and non-reactive patients after 12 weeks of SOC treatment cannot reach at least 1 log ₁₀ and less than 2 log ₁₀ IU/ Miller.

Recent research efforts to improve the efficacy of SOC have focused on other compounds that calibrate the proteins encoded by the virus. Several NS3/4A, NS5A and NS5B inhibitors are currently in Phase II clinical development, and the most advanced are even in Phase III. Anti-NS3/4A telaprevir/tin U.S. Icivio or Invivek (Vertex) and Boceprevir/Victrelis (Merck) have recently received US food and drug management. The FDA and the European Commission have approved the use of pegylated interferon alpha and ribavirin for the treatment of the first genotype of chronic hepatitis C virus, and are now available in the United States and Europe. When used in combination with pegylated interferon alpha and ribavirin, these compounds are increased despite a sustained viral response (SVR) (eg, up to 25 to 30% in patients infected with the first genotype) It also has significant side effects and may promote the mutation of the calibrated HCV protein to promote the production of drug-resistant HCV variants.

One might expect immunotherapy to be complementary to antiviral therapy, with the goal of providing specific protective immunity in chronic HCV infection. Several candidate programs based on recombinant proteins, synthetic peptides, pyrogenic phenotypic yeast, DNA and viral vectors have emerged (Torresi et al., 2011, J. Hepatol, No. 54, pp. 1273-1285). ). In this regard, WO 2008/113606 describes a method of treatment that relies on the attenuated non-replicating vaccinia virus Ankara strain (MVA) encoding HCV NS3, NS4 and NS5B antigens (referred to as TG4040). The three repeated administrations and the respective administrations were separated by 3 to 10 days. In particular, one of the three injections, including one week apart, is used to prime one of the HCV-specific IFN-g-type T cells. The fourth injection after 4 or 6 months enhances the T cell response. In animal models, this potent and specific T cell-like response elicited by the vaccine lasts for 6 months (Fournillier et al., 2007, Vaccine, 25th, pp. 7339-7353) . In addition, TG4040 confers partial protection in HCV agent challenge analysis based on Listeria monocytogenes expressing HCV NS3 protein.

Currently, the TG4040 has completed Phase I trials in France. The dose escalation portion of the study involved 15 patients who had not been treated for chronic infection with genotype 1 HCV. Such patients received three subcutaneous injection once a week TG4040 action (accelerator operation program based WO2008 / 113606 described) and the dosage form-based increments to ¹⁰⁸ plaques forming units from 10 ⁶ plaques, and Patients treated with the highest dose also received a fourth TG4040 injection in the sixth month. The viral load of 8 of 15 patients decreased by 0.5 to 1.2 log ₁₀ IU/ml, and the largest viral reduction occurred in 2 patients who showed the most significant HCV-specific T cell response; No immune response to NS3 and NS4B virus antigens was detected in these patients with little or no change in load (Honnet et al. Oral report of the 60th AASLD Annual Meeting on November 3, 2009; Habersetzer et al. In the 2011 issue of "Gastroentelology" No. 141, pp. 890-899, B).

Another method recently studied involves a yeast immunotherapy composition encoding an HCV antigen that is suitable for once-weekly and monthly repetitive administration with interferon and ribavirin (WO 2010) /033841). Various treatment settings are disclosed, and the administration of HCV NS3 and the expression core yeast is before, at the same time as, or after SOC treatment.

Due to the chronic and persistent nature of HCV infection, its high incidence, the continued spread of HCV, and the significant incidence of related diseases, it has been expected that HCV will continue to be a serious global health threat for many years to come. Therefore, there is a need to develop more effective, less toxic and shorter-term methods of combining immunotherapeutic methods with antiviral therapies to improve the treatment of HCV infection or HCV-related diseases or disorders and especially chronic HCV infections.

The object of the invention is to provide an optimized therapeutic regimen for administering an immunotherapeutic composition that is actually used in combination with one or more antiviral compounds. It is speculated that this optimal treatment regimen allows the initial action to trigger or stimulate an effective immunity before the initiation of antiviral therapy, and to maintain the immunity caused by the initial action during the duration of the antiviral therapy.

This technical problem is solved by providing an embodiment as defined in the scope of the patent application.

Other and further aspects, features and advantages of the present invention will become apparent in the following description in conjunction with the present invention. These embodiments are provided for the purpose of disclosure.

Summary of invention

One embodiment of the invention relates to an immunotherapeutic composition for the treatment of HCV infection, and in particular chronic HCV infection. The immunotherapeutic composition is administered to a desired body according to a repeated administration mode comprising 4 to 8 administrations varying from 3 to 10 days apart and finally 4 to 5 weeks apart from each other. Up to 15 times of administration. In one aspect of this embodiment, the immunotherapeutic composition comprises a therapeutically effective amount of an active ingredient selected from the group consisting of: (a) polyprotein NS3 of hepatitis C virus/ NS4 and hepatitis C virus peptides NS5B; (b) a performance vector comprising a nucleotide sequence encoding a polyprotein NS3/NS4 of hepatitis C virus and a nucleotide sequence of a polypeptide NS5B encoding a hepatitis virus; or (c) comprising a coding type C One of the nucleotide sequences of one of the polyproteins NS3/NS4 of the hepatitis virus expresses a vector and one of the expression vectors comprising one of the nucleotide sequences of the polypeptide NS5B encoding the hepatitis virus. In one aspect, the mode of administration comprises 4 to 8 administrations ranging from 3 to 10 days apart. Optionally and in a preferred aspect, the administration comprises 4 to 8 administrations varying from 3 to 10 days apart, followed by 4 to 15 administrations varying from 3 to 5 weeks apart.

In another aspect of the above embodiments, the immunotherapeutic composition can be used in combination with antiviral therapy, and in particular with pegylated interferon alpha 2 (PEGylated IFNa2) in combination with ribavirin.

Another embodiment of the invention is directed to a therapeutic procedure comprising the steps of administering the immunotherapeutic composition and ultimately in combination with an antiviral therapy.

A further aspect of the invention includes a kit for use in the treatment of HCV infection and preferably chronic HCV infection, wherein the kit comprises a plurality of containers and instructions for administering the immunotherapeutic composition to one body or performing the treatment regimen .

In yet another aspect of the invention, there is a method of achieving a complete early viral response in a population of individuals chronically infected with HCV compared to cEVR in a population of individuals chronically infected with HCV and treated only with antiviral therapy (cEVR) The proportion of individuals increased by at least 5%.

In yet another aspect, the invention also allows for a safety aspect when used in combination with an antiviral therapy, such as by providing a method The dose or course of shortening antiviral therapy and/or reducing at least one antiviral compound in an individual population of chronically infected HCV treated according to the present invention is compared to an individual population of chronically infected HCV treated with antiviral therapy alone In this case, the proportion of antiviral agent-related adverse events in the HCV patient population treated in accordance with the present invention is reduced (e.g., by at least 5%).

Detailed description of the invention

The present invention relates to an immunotherapy composition comprising a therapeutically effective amount of an active ingredient selected from the group consisting of: - a polyprotein NS3/NS4 of hepatitis C virus and a C Hepatitis virus polypeptide NS5B; - a expression vector comprising a nucleotide sequence encoding a polyprotein NS3/NS4 of hepatitis C virus and a nucleotide sequence of a polypeptide NS5B encoding hepatitis virus; - comprising hepatitis C encoding An expression vector for a nucleotide sequence of one of the polyproteins NS3/NS4 of the virus and an expression vector comprising one of the nucleotide sequences of the polypeptide NS5B encoding the hepatitis virus; and the use as an agent for treating an HCV infection, wherein the agent The mode of administration includes 4 to 8 administrations of the immunotherapeutic composition ranging from 3 to 10 days apart, and finally 4 to 15 of the immunotherapeutic composition ranging from 3 to 5 weeks apart from each other. Sub-dosing effect.

definition

As used in the entire application, "a (a)" and "an", etc. Words are used in the meaning of "at least one of", "at least the first one", "one or more" or "an"

The word "and/or" used in this document includes the meaning of "and", "or" and "all or any combination of the elements to which the word is connected."

The term "about" or "approximately" as used herein means within 10%, preferably within 8%, and more preferably within 5% of a given value or range.

The terms "amino acid", "residue" and "amino acid residue" are used synonymously, and encompass both natural amino acids and amino acid analogs (eg, non-natural, synthetic, and modified amino acids, And includes D or L optical isomers).

The terms "polypeptide", "peptide" and "protein" refer to a polymer of amino acid residues comprising at least 9 or more amino acids bonded via peptide bonds. The polymer may be linear, branched or cyclic, and may comprise naturally occurring amino acids and/or amino acid analogs, with intervening discontinuities in the presence of non-amino acids. In terms of a general expression, if the amino acid polymer has more than 50 amino acid residues, it is preferably referred to as a polypeptide or a protein; if it has a length of 50 amino acids or less, it is called "peptide".

The term "polyprotein" as used herein refers to the fusion of two or more polypeptides/peptides in a single polypeptide chain. Preferably, the fusion is carried out genetically, i.e. by merging the nucleotide sequences encoding each of the polypeptides/peptides in the same reading frame. "Fussing in the same reading frame" means that the expression of the fused coding sequence produces a single protein without any translation terminator between each of the fused polypeptides/peptides. The melt Cooperating may be direct (ie, without any additional amino acid residues in between) or via a linker (eg, a peptide of 3 to 30 amino acids in length consisting of repeating amino acid residues) These amino acid residues such as glycine, serine, threonine, aspartic acid, alanine and/or valine acid).

In the context of the present invention, the terms "nucleic acid", "nucleic acid molecule", "polynucleotide" and "nucleotide sequence" are used interchangeably and are used to define polydeoxyribonucleotides (DNA) (eg, cDNA, genomic DNA, plastid, vector, viral genomic, isolated DNA, probe, primer, and any mixture thereof) or polyribonucleotide (RNA) (eg, mRNA, antisense RNA) Or a polymer of any length consisting of a mixture of polyribose-polydeoxyribonucleotides. They include single or double stranded, linear or cyclic, natural or synthetic polynucleotides. Furthermore, a polynucleotide may comprise a non-naturally occurring nucleotide, and may be interrupted with the presence of a non-nucleotide component.

When used to define a product, a composition, and a method, as used herein, "comprises" and "comprising", such as "comprises" and "comprises", "has" (and Any form such as "have" and "has", "including" (and including any form such as "includes" and "include"). Or the words "including" (and any form such as "contains" and "contains" are open-ended and do not exclude other elements or method steps that are not listed. Thus, a polypeptide "comprises" an amino acid sequence when the amino acid sequence may be part of the final amino acid sequence of the polypeptide. The polypeptide may have up to hundreds of additional amino acid residues. "Substantially composed of" means other groups that will have any substantial meaning Points or steps are excluded. Thus, a composition consisting essentially of the recited components will not exclude minor contaminants and pharmaceutically acceptable carriers. When the amino acid sequence is such that only some additional amino acid residues are present, the polypeptide is "substantially composed of the amino acid sequence". "Consisting of" means that the excluded elements are not only trace elements or steps of other components. For example, when the polypeptide does not contain any amino acid other than the listed amino acid sequence, then the polypeptide is "consisting of the amino acid sequence."

The term "immunotherapy composition" as used herein, refers to a formulation comprising at least one therapeutic agent described herein (eg, a expression vector encoding a NS3/NS4 polyprotein and an NS5B polypeptide) and optionally One or more pharmaceutically acceptable carriers (e.g., carriers, diluents, adjuvants, etc.) can elicit or stimulate an immune response when administered to a subject in accordance with the mode of administration described herein. The elicited or stimulated immune response can be congenital or specific, hormonal or cellular, and can be assessed by various measurement methods including, but not limited to, antibody production and/or interleukin (eg, IFN). -g) activation of production and/or cytotoxic T cells, B lymphocytes, T lymphocytes, antigen presenting cells, helper T cells, dendritic cells, NK cells, and the like.

"HCV" as used herein refers to "hepatitis C virus" which is a member of the Flaviviridae family (also referred to as non-A, non-B hepatitis) that causes hepatitis C.

The term "NS polypeptide" refers to a non-structural (NS) polypeptide derived from any HCV genotype, subtype or isolate currently identified. The term covers an open reading of cDNA or RNA fragments derived from HCV viruses. A native NS polypeptide encoded by a code frame and encompasses an NS derivative and any fragment thereof (eg, an immunogenic peptide).

For purposes of illustration, "NS5B polypeptide" refers to a native NS5B polypeptide encoded by any HCV genotype, subtype or isolate (eg, about 2421 amino acids in the HCV-1 polyprotein precursor to about 3011 amino acids) and any derivatives or fragments thereof. "NS3/NS4 polyprotein" means any HCV genotype, subtype or isolate encoded by or derived from any HCV genotype, subtype or isolate (about 1027 amines in the HCV-1 polyprotein precursor) A fusion between an NS3 polypeptide of a basal acid to about 1972 amino acid and an NS4 polypeptide (such as encompassing both NS4A and NS4B) and any derivative or fragment thereof. For the sake of clarity, the amino acid extensions associated with HCV polypeptides referred to herein are given for their position in the HCV-1 polyprotein precursor (eg, Choo et al., 1991) "Proc. Natl. Acad. Sci. USA", 88th, pp. 2451-2455, or as described in GenBank accession number M62321. However, unless explicitly indicated by the context, when referring to an NS polypeptide or a peptide thereof with reference to an HCV-1 polyprotein precursor, it refers to an NS polypeptide of HCV-1 or a peptide thereof; any other HCV gene An NS polypeptide of the type, subtype or isolate, or a peptide thereof; or a modified NS polypeptide or peptide thereof.

The term "natural" as used herein, when used in reference to an NS polypeptide or polyprotein or to a NS-encoded nucleotide sequence, means that one amino acid sequence can be found, isolated, obtained from a natural source or A nucleotide sequence that differs from an artificially modified or mutated producer (i.e., a mutant or derivative) artificially produced in the laboratory. Such natural sources include infections or Biological samples (such as blood, plasma, serum, semen, saliva, tissue sections, biopsy specimens, etc.) collected by one body exposed to HCV; cultured cells; tissue culture; and recombinant materials. Recombinant materials include, but are not limited to, HCV isolates (such as those available from the registry), HCV genomes, genomic RNA or cDNA libraries, vectors containing HCV genomes or fragments thereof.

The term "derivative" or "mutant" as used herein, refers to a polypeptide/nucleotide sequence that exhibits one or more modifications to a natural counterpart. For the purposes of this description, "NS derivative" refers to an NS polypeptide that has been artificially altered or artificially altered in the laboratory. Any modification can be envisaged, including substitution, insertion and/or deletion of one or more nucleotide/amino acid residues; non-native alignment (eg, fusion with non-HCV polypeptides/peptides) and Any combination of these possibilities. When several mutations are envisaged, they may involve adjacent residues and/or non-adjacent residues. By mutation of some embodiments may be those known to the art skilled generation, such as site-directed mutagenesis mutation (e.g., using the Leizu in France (Les Ullis) of Amersham (Amersham)'s Sculptor ^TM tubes induced mutation system), PCR Mutagenesis, DNA rearrangement techniques, and by chemical synthesis techniques (eg, production of a synthetic nucleic acid molecule).

The term "identity" as used herein refers to the exact correspondence of an amino acid relative to an amino acid or nucleotide relative to a nucleotide between two polypeptide or nucleotide sequences. The percent identity between the two sequences is the number of identical positions shared by the sequences, and will be taken into account for the number of gaps to be included in the optimal alignment and the length of each gap. Desirable in craftsmanship Various computer programs and mathematical algorithms are used to determine the percent identity between amino acid sequences, such as, for example, the basic local alignment search tool (Blast) program or the "protein sequence and structure atlas" available at NCBI (Atlas of ALIGN in Protein Sequence and Structure)" (Dayhoffed, 1981, Supplement No. 3, pp. 482-489). Programs for determining homology between nucleotide sequences can also be obtained in a specialized database (eg Genbank, Wisconsin Sequence Analysis Package, BESTFIT, FASTA, and GAP). Program). For purposes of illustration, as used herein, "at least 80% of sequence identity" means 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

The term "expression carrier" as used herein, refers to a vector, preferably a nucleic acid molecule or a viral particle, and which comprises a HCV nucleotide sequence of interest which is capable of being delivered and propagated in a host cell or in vivo. And / or represent the required elements. The term encompasses extra-chromosomal vectors (eg, multiple sets of plastids) and embedded vectors (eg, nucleic acid molecules that are designed to be inserted into the host cell's genome and that produce additional sets of nucleic acid molecules when the host cell replicates). For the purposes of the present invention, such vectors may be derived from naturally occurring sources of genes, either synthetic or artificial, or some combination of natural and artificial genetic elements.

As used herein, the term "isolated" refers to the removal of a protein, polypeptide, peptide, polynucleotide, vector, etc. from its natural environment (ie, at least one component that is naturally associated with it or naturally occurring). ). For example, when a nucleotide sequence is separated from a sequence that is normally associated with it in nature (eg, When dissociated from a gene, the nucleotide sequence is separated; however, it can be associated with a heterologous sequence.

The term "host cell" as used herein shall be understood broadly and without any limitation as to the composition of the tissue, organ or cell to be isolated. The cells can be a unique type of cell or a group of different types of cells, such as cultured cell lines, primary cells, and proliferating cells. In the context of the present invention, the term "host cell" includes prokaryotic cells; lower eukaryotic cells such as yeast; and other eukaryotic cells such as insect cells, plants and mammalian (eg human or non-human) cells. And a cell which produces an NS polypeptide vector and an expression vector for use in the immunotherapy composition of the invention. The term also encompasses cells that are or have become recipients of the vectors described herein and progeny of such cells.

The term "individual" or "patient" is used interchangeably and generally refers to a vertebrate, particularly a member of a mammalian species, and in particular any product and method of the invention or any product of the invention. Methods may be beneficial to livestock, farm animals, competing animals, and primates, including humans, such as individuals diagnosed with HCV infection or at risk of infection, which are therefore susceptible to HCV infection or with HCV An infection associated with a disease or condition or a person at risk. In a preferred embodiment, the host biological system chronically infects the HCV virus or, optionally, a human patient infected with one of the HCV virus and another virus, such as human immunodeficiency virus (HIV).

As used herein, the term "treatment" (and any form of the word "treating" such as "treatment", "treat"), Means prevention (eg, for the prevention of a body at risk of HCV infection) and/or treatment (eg, for curing or controlling viral replication or disease progression in a subject diagnosed with HCV infection). Therapeutic effect requires the administration of a therapeutic agent, such as the immunotherapeutic compositions described herein, to one body, either externally or orally, and the composition is ultimately used in combination with antiviral therapy, particularly for the treatment of chronic HCV infection and includes Pegylated IFNα and/or ribavirin and/or protease inhibitors and/or polymerase inhibitors, and the like.

The term "administering" as used herein (and any form of administration of a drug such as "administering") refers to the delivery of a therapeutic agent, such as an immunotherapeutic composition described herein, to a body.

The term "dosing mode" as used herein refers to a series of administrations administered in one body in the manner described herein.

HCV sequence

Some HCV sequences suitable for use in the present invention include such sequences which are readily available to researchers in the field, including, but not limited to, HCV sequences as described in Genbank and PubMed. Detailed phylogenetic analysis classifies HCV isolates into six major genotypes (1 to 6) and is subdivided into several subtypes (a, b, c, etc...) in each genotype (Simmons et al. In the 2005 issue of Hepatology, No. 42 (pp. 962-973). HCV sequences derived from any genotype and subtype can be used in the embodiments described herein.

Exemplary HCV types 1a include, but are not limited to, HCV-1 (Choo et al., 1991, "Proc. Natl. Acad. Sci. USA", 88th, pp. 2451-2455), -J1 (Okamoto) Et al. in the 1992 issue "Nucleic Acids Res." No. 20, pp. 6410-6410, B), -H (Inchauspé et al., 1991, "Proc. Natl. Acad. Sci. USA", No. 88, No. 10292-10296) And the isolates described in WO2004/048402. Exemplary Type 1b genotypes of HCV include, but are not limited to, HCV-JA (Kato et al., 1990, "Proc. Natl. Acad. Sci. USA", 87th, pp. 9524-9528) and BK (Takamizawa et al.) In the 1991 issue of J. Virol, No. 65, pp. 1105-1113, B). Exemplary Type 1c genotypes of HCV include, but are not limited to, HCV-G9 (Okamoto et al., J. Gen. Virol., 1994, vol. 45, pp. 629-635). Exemplary second genotype HCV includes, but is not limited to, HCV-J6 (type 2a genotype; Okamoto et al., 1991, J. Gen. Virol., 72, pp. 2697-2704), HCV- J8 (2b genotype; Okamoto et al., 1992, Virology, 188, pp. 331-341) and HCV-BEBE1 (2c genotype; Nako et al., 1996, J. Gen. Virol.”, pp. 701-704, pp. 701). Exemplary third genotype HCV includes, but is not limited to, HCV-NZL1 (type 3a genotype; Sakamoto et al., 1994, J. Gen. Virol., 75th, pp. 1761-1768) and HCV- Tr (3b genotype; Chayama et al., 1994, J. Gen. Virol., 75th, pp. 3623-3628). Exemplary fourth genotype HCV sequences include, but are not limited to, HCV-ED43 genotype 4a; Chamberlain et al., 1997, J. Gen. Virol., 78, pp. 1341-1347, et al. An exemplary fifth genotype HCV sequence is HCV-EUH1480 (5a genotype; Chamberlain et al., 1997, "Biochem. Biophys. Res. Commun.", 236, pp. 44-49). An illustration from the sixth genotype The HCV sequences include, but are not limited to, HCV-EUHK2 (type 6a genotype; Adams et al., 1997, "Biochem. Biophys. Res. Commun.", No. 234, pp. 393-396).

The invention is not intended to be limited to such exemplary HCV viruses. It is true that the nucleotide and amino acid sequences of any or all of the HCV NS sequences used in the present invention may differ depending on the HCV isolate, subtype and genotype, and this natural gene Differences and non-naturally modified effects as described below are encompassed within the scope of the invention.

NS polymorphism

The immunotherapeutic composition used in the present invention comprises at least a non-structural (NS) NS3, NS4 and NS5 BHCV polypeptide, and particularly a NS3 and NS4 HCV polypeptide (i.e., NS3/NS4 polyprotein) in a fusion configuration or for expression. The expression vector of NS3, NS4 (such as a NS3/NS4 polyprotein) and NS5B polypeptide are preferred. The invention preferably excludes the use of an HCV core polypeptide or an expression vector encoding an HCV core polypeptide.

In the context of the present invention, each of the HCV NS3, NS4 and NS5B polypeptides comprised or encoded by the compositions of the present invention may be derived individually from any of the currently recognized HCV genotypes, subtypes or isolates. A strain, such as described herein or any fragment thereof (eg, an immunogenic fragment).

The invention also encompasses a modified NS derivative which is directly or indirectly involved in the modification of one or more amino acid residues (adjacent or non-adjacent) of the enzyme activity exhibited by a native NS polypeptide. The purpose is to destroy the activity of the enzyme. For example, a modification in an enzyme catalytic site can allow for the reduction or removal of related enzyme activity. Can be identified by routine methods The reduction or removal of the modified enzyme activity can be readily determined by appropriate analysis of the amino acid residues of such functional properties and in appropriate assays by methods known to those skilled in the art. In this respect, catalytic residues involving NS3 mediator protease and helicase activity have been identified, and protease deficient NS3 derivatives have been described in the art (e.g., Bartenshlager et al., 1993, J. Virol) "P. 67, pp. 3835-3844, and Tomei et al., 1993, "J. Virol." 67th, pp. 4017-4026, B) and helicase-deficient NS3 derivatives (eg Kim et al.) In the 1997 issue of "J. Virol.", No. 71, p. 9400, et al., and Lin and Kim, 1999, J. Virol., 73, pp. 8798-8807, et al. Polymerase-deficient NS5B derivatives can also be obtained (e.g., Lohmann et al., 1997, J. Virol., 71, pp. 8416-8428).

Other suitable NS derivatives may exhibit a modified cell presentation (eg, by the addition of an appropriate message peptide to the cell membrane anchoring of the transmembrane peptide) and/or after modification with respect to the native NS counterpart Translation processing.

The resulting NS derivative desirably retains immunogenic properties, particularly in the same or optionally higher range as the native NS counterpart, retaining the ability to stimulate a cellular mediating immune response. Therefore, it is best to avoid the modification of the epitopes rich in B, CTL and/or T _H , so as not to impair the immunogenic properties.

In one embodiment, at least two of the NS3, NS4 and NS5B polypeptides comprised or encoded by the composition used in the present invention may be derived from different HCV genotypes, subtypes or isolates. This configuration can thus provide protection against a wider range of HCV genotypes; or by using a region The HCV genotype specific to a domain or a particular patient population, such that the composition or treatment regimen is applicable to that particular geographic region. In this respect, the 1a, 1b, 2 and 3 genotypes are most common in North America, Europe and Asia; the 4th genotype is more common in North Africa and Central Africa; the 5th genotype has so far been mostly found in South Africa; The sixth genotype isolate was mainly found in Vietnam and Hong Kong. For example, the use of one NS3/NS4 polyprotein from the first genotype HCV and one NS5B polypeptide from the third genotype, or vice versa, may allow for the design of one of the compositions suitable for the treatment of individuals in Asia, North America and Europe.

In another embodiment, the NS3, NS4 (eg, NS3/NS4 polyprotein) and NS5B polypeptides contained or encoded by the composition used in the present invention are derived from the same HCV genotype, subtype or isolate. . Suitably, all of these are derived from the first genotype HCV, preferably from the first b genotype, and particularly from the source of the HCV-JA isolate.

Particularly advantageous is an NS3/NS4 polyprotein comprising an amino acid sequence which is at least 80% identical to the amino acid sequence shown in Sequence Identification Number: 1, advantageously at least 85% identical. Preferably, it has at least 90% identity, more preferably at least 95% consistency, and even more preferably 98% consistency. More preferably, it is a NS3/NS4 polyprotein comprising the amino acid sequence shown in Sequence Identification Number: 1.

The NS5B polypeptide optionally or in combination comprises an amino acid sequence which is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 2, advantageously at least 85% identical, preferably It has at least 90% consistency, better at least 95% consistency, and even better 98% consistency. More preferably, it is an NS5B polypeptide comprising sequence recognition No.: The amino acid sequence shown in 2.

NS polypeptides (such as NS3/NS4 polyproteins and NS5B polypeptides) can be produced recombinantly using the expression vectors (or infectious viral particles) described herein. The method generally comprises (a) introducing a expression vector into a suitable host cell to produce a transfected or infected host cell; (b) cultivating the transfected tube in a test tube under conditions suitable for growth of the host cell Or infected host cells; (c) recovering the cell culture; and (d) selectively purifying the NS polypeptide and the polyprotein from the recovered cells and/or culture supernatant.

NS polypeptide expression vector suitable for recombinant production of action include but are not limited for use in prokaryotic host cells such as bacteria (e.g., E. coli (E.coli), Bacillus subtilis (Bacillus subtilis) or Listeria (Listeria)) in the performance Phage, plastid or adhesive plastid vector; vector for expression in yeast (such as Saccharomyces cerevisiae , Saccharomyces pombe , Pichia pastoris ); a baculovirus vector expressed in an insect cell system (such as Sf 9 cells); a viral and plastid vector for expression in a plant cell system (such as Ti plastid, Cauliflower mosaic virus CaMV, Tobacco mosaic virus TMV); A viral and plastid vector for expression in higher eukaryotic cells or organisms. Such vectors are commonly commercially available (eg, from Invitrogen, Stratagene, Amersham Biosciences, Promega, etc.); or Available from depository facilities such as the American Type Culture Collection (ATCC and Rockville, MD); or a large number of publications, sequences, components, and production methods that enable professionals to apply . A performance vector can be introduced into a host cell by a variety of means including, but not limited to, microinjection, transfection of calcium phosphate, transfection of DEAE-polydextrose, liposome infection Action/liposome fusion, gene gun, transduction, electroporation, viral infection, etc. Host cells can be cultured in conventional fermentation bioreactors, flasks, and culture dishes. The culture can be carried out at a temperature, a pH and an oxygen content suitable for a particular host cell. There is no attempt to detail various expression systems, host cells and methods known for the recombinant production of polypeptides.

NS nucleotide sequence

Nucleotide sequences encoding NS3, NS4 and NS5B can be generated from any source using sequence data available in the art and sequence information provided herein. A preferred embodiment relates to the use of a nucleotide sequence encoding one of the NS3/NS4 polyproteins and a nucleotide sequence encoding one of the NS5B polypeptides. Individually isolated from HCV-containing cells, cDNA and genomic libraries, viral genomes, or any of the known techniques known to include such nucleotide sequences, by conventional molecular biology or PCR techniques The nucleotide sequences. Alternatively, they can also be produced by chemical synthesis in an automated process (eg, assembled from overlapping synthetic oligonucleotides or synthetic genes). Modification can be produced by some means known to those skilled in the art, such as chemical synthesis, site-directed mutagenesis, PCR mutagenesis, DNA rearrangement techniques, and the like.

Nucleic acid modification is also envisioned for increasing the level of performance in a particular host cell or individual. When more than one codon can be used to encode a particular amino acid, the mode of use of the codon is indeed observed. Non-random, and codon usage between different hosts is significantly different. Since the nucleotide sequences encompassed by the present invention are mostly derived from viral sources (HCV), their codon usage patterns may not be suitable for efficient expression in host cells such as bacteria, lower or higher eukaryotic cells. In general, one or more codons that are more commonly used to encode the same amino acid in the host cell/individual of interest can be used to replace one of the codons that are not commonly used in the host cell/individual of interest. Codon optimization is performed by a variety of "natural" (eg, HCV) codons. It is not necessary to replace all of the natural codons corresponding to the codons that are not commonly used, because even in the case of partial substitutions, the performance can be increased. In addition, the optimized codon usage can be deviated rather than strictly followed to introduce a restriction site in the resulting nucleic acid molecule.

In addition, the effect of expression in a host cell or an individual can be enhanced by additional modification of the nucleotide sequence, the purpose of which is to prevent clustering of rare, non-optimal codons and/or Negative sequence elements that are expected to negatively affect performance levels (eg, AT-rich or GC-rich sequence extensions; unstable forward or reverse repeats; RNA secondary structures; and/or internal confined regulatory elements such as At least a portion of the internal TATA box, chi address, ribosome entry address, and/or splice donor/acceptor address is inhibited or modified.

In a preferred embodiment, the nucleotide sequence contained in the nucleotide sequence encoding the NS3/NS4 polyprotein is at least 80% identical to the nucleotide sequence shown in SEQ ID NO: 3, suitable The ground has a consistency of at least 85%, advantageously at least 90% consistency, preferably at least 95% Consistency, better with 98% consistency, even better with 100% consistency. Optionally or in combination, the nucleotide sequence comprising the nucleotide sequence encoding the NS5B polypeptide is at least 80% identical to the nucleotide sequence shown in SEQ ID NO: 4, suitably having at least 85 The % consistency, advantageously has at least 90% identity, preferably at least 95% consistency, better 98% consistency, and even better 100% consistency.

Performance carrier

A preferred aspect of the invention relates to an immunotherapeutic composition comprising at least one or both expression vectors encoding the NS3, NS4 and NS5B polypeptides (e.g., NS3/NS4 polyprotein and NS5B polypeptide) described herein.

In the context of the present invention, the term "carrier" should be understood broadly to include both plastid and viral vectors.

"Plastid vector" as used herein refers to a replicable DNA construct for use in various expression systems, the "transport" vector acting in prokaryotic and/or eukaryotic cells, and the "transfer" vector. Transfer to a polynucleotide in a viral vector. Typically, the plastid vector contains a selection marker gene, and the host cell carrying the plastid vector is allowed to be screened or discarded in the presence of a corresponding screening drug. A variety of positive and negative selection marker genes are known in the art. For example, an antibiotic resistance gene can be used as a positive selection marker gene, allowing a host cell to be screened in the presence of the corresponding antibiotic; and a suicide gene can be used as a negative selection marker gene, allowing When the corresponding prodrug is added, the host cell expressing the suicide gene is killed.

Representative examples of suitable plastid carriers include, but are not limited to, pBR Type plastids (eg pBR322), pUC (Gibco), pBluescript (Stratagene), pREP4, pCEP4 (Invitrogen), pCI (Promega) )), pVAX (Invitrogen) and pgWiz (Genetherapy System).

The invention also encompasses carriers (e.g., plastid DNA), such as liposomes, lipid complexes or nanoparticles, that are complexed with a lipid or polymer to form a particulate structure.

The term "viral vector" as used herein, refers to a nucleic acid vector comprising at least one element of a viral genome, and which may be packaged in a viral particle or packaged into a viral particle. The terms "virus", "virion", "virion particle" and "viral vector particle" are used interchangeably and refer to a nucleic acid vector that is transduced to a suitable condition based on the production of infectious viral particles. Viral particles formed when appropriate host cells or cell lines. In the context of the present invention, the term "viral vector" is to be understood broadly to include a nucleic acid vector (such as a DNA viral vector) and the viral particles produced thereby. The term "infectious" refers to the ability of a viral vector to infect and enter a host cell or individual. The viral vector may be competent for replication, or may be genetically ineffective to become a replication defective or a replication impaired. As used herein, the term "replication competent" encompasses selective replication and conditional replication that are engineered to have better or selective replication in a particular host cell, such as an infected cell. Viral vector.

Viral vectors suitable for use in the present invention may be from a variety of different viruses (eg, retrovirus, adenovirus, adeno-associated virus (AAV), pox disease Toxic, herpes virus, measles virus, foam virus, alphavirus, follicular stomatitis virus, etc.). As mentioned above, the term "viral vector" encompasses vector DNA, genomic DNA, and viral particles produced thereby.

In one embodiment, the expression vector contained in the composition used in the present invention is a poxvirus vector. It can be obtained from members of any poxvirus family, such as orthopoxvirus, parapoxvirus, fowlpox (such as canarypox ALVAC and fowlpox), sheeppox virus, rabbitpox virus, porcine poxvirus, mollusc poxvirus and Yatapox virus. Preferably, the poxvirus is an orthopoxvirus and particularly a vaccinia virus, particularly a vaccinia virus (VV) selected from the group consisting of VV Western (Western) retained strain, Copenhagen (Gorebel) strain (Goebel) Et al., 1990, "Virol.", No. 179, pp. 247-266; Johnson et al., 1993, "Virol." 196, pp. 381-401, B), Wyeth and In particular, the modified Ankara (MVA) strain (Antoine et al., 1998, "Virol.", No. 244, pp. 365-396), the latter being preferred, especially MVA 575 (ECCAC V00120707) and MVA BN (ECCAC V00083008). The genome sequences of many members of the poxvirus family are described in the literature and specialized databases (eg, gene bank accession numbers NC_006998, M35027, U94848, and NC_005309NC_001132, respectively, corresponding to Western retained strains, Copenhagen strains, MVA, and The canarypox sequence), and the general conditions for constructing recombinant poxviruses are well known in the art.

The nucleotide sequence encoding the NS3/NS4 polyprotein and the NS5B polypeptide can be inserted into the same vector or a separate vector and inserted into the poxvirus genome. A position is preferably inserted into an unnecessary locus. When the same representation carrier is inserted, the likes may be located in synonymous or antisense orientations of each other. In the VV Copenhagen vaccinia vector, it is particularly suitable for insertion of the thymidine kinase gene; in the MVA vector, it is particularly suitable for insertion of deletions I to VI, and preferably for insertion of deletion II or III.

Another viral vector suitable for use in the present invention is an adenoviral vector. It can be derived from a variety of human or animal adenoviruses (eg, dogs, sheep, apes, etc.). Any serotype can be used. Desirably, the adenoviral vector is defective in replication and is derived from human Ad, in particular from human Ad with a rare serotype, or from chimpanzee Ad. Representative examples of human adenoviruses include Ad2Ad5 and Ad6 of subgenus C; Ad11, Ad34 and Ad35 of subgenus B; and Ad19, Ad24, Ad48 and Ad49 of subgenus D. Representative examples of chimpanzee Ad include, but are not limited to, AdCh3 (Peruzzi et al., 2009 issue "Vaccine" 27th, pp. 1293-1300), AdCh63 (Dudareva et al., 2009 issue "Vaccine" issue 27 Any of those described in the art of the art (see, for example, WO 03/000283; WO 03/046124; WO2005/071093; WO2009/073103; WO 2009/073104; WO2009/105084; WO2009/136977 and WO 2010/086189). A replication-deficient adenoviral vector can be obtained as described in the art, such as by deleting at least a region or portion of an adenoviral genome that is essential for viral replication, particularly by deleting the E1 region (eg, a reference gene bank) Accession number M73260 and Chroboczek et al., 1992, "Virol." 186, pp. 280-285, et al., Type 5 human adenovirus sequence extending from position 459 to 3510) are preferred. The invention also has A carrier with additional deletion/modification in the adenoviral gene, especially in the non-essential E3 region or other necessary deletions/metamorphisms in the E2 and E4 regions, such as WO94/28152 and Lusky et al. In the 1998 issue of J. Virol, No. 72, p. 2022, B.).

Other viral vectors suitable for use in the context of the present invention are the measles virus genus available from the Paramyxoviridae family, with measles virus being the first choice. Various attenuated strains are available in the art (Brandler et al., 2008, CIMID, No. 31, pp. 271-291; Bingh et al., 1999, J. Virol., para. 73(6) Pages 4823-4828, B), such as but not limited to Edmonston A and B strains (Griffin et al., 2001, "Field's in Virology", pp. 1401-1441), Schwarz (Schwartz) strain (Schwarz A in the 1962 issue "Am J Dis Child" 103, page 216), S-191 or C-47 strain (Zhang et al. in the 2009 issue of "J Med Virol" 81st (8) No. 1477-1483 (in Chinese). Among them, it is particularly suitable for insertion between the P and M genes.

The nucleotide sequence contained in the composition used in the present invention, as in the present invention, is suitable for expressing one of the encoded NS polypeptides (such as NS3/NS4 polyprotein and NS5B polypeptide) in a host cell or an individual. It is meant that each of the nucleotide sequences is under the control of an appropriate regulatory sequence. The term "regulatory element" as used herein, refers to any element that permits, facilitates, or modulates the expression of a nucleotide sequence in a given host cell or individual, including the nucleic acid or Proliferation, replication, transcription, splicing, translation, stabilization, and/or delivery of derivatives (ie, mRNA).

Those skilled in the art will appreciate that the choice of regulatory sequences and, in particular, promoters may depend on factors such as the vector itself, the host cell, the desired level of performance, and the like. In the context of the present invention, it can continue to direct the expression of a nucleic acid molecule in a variety of host cell types or against a particular host cell (eg, a liver-specific regulatory sequence), or in response to a particular event or exogenous factor (eg, temperature) , nutrient additives, hormones, etc.) or according to the stage of the virus cycle (such as late or early). Promoters that are inhibited in response to specific events or exogenous factors during the production step can also be used to avoid the potential toxicity of the expressed polypeptide.

Promoters suitable for sustained expression in mammalian cells include, but are not limited to, the cell giant virus (CMV) early promoter (Boshart et al., 1985, "Cell", 41, pp. 521-530), RSV. Promoter, adenovirus major late promoter, phosphoglycerin kinase (PGK) promoter (Adra et al., 1987, "Gene" 60, pp. 65-74), herpes simplex virus (HSV)-1 Thymidine kinase (TK) promoter and T7 polymerase promoter. The vaccinia virus promoter is particularly useful for expression in poxvirus vectors. Representative examples include, but are not limited to, vaccinia 7.5K, H5R, 11K7.5 (Erbs et al. in the 2008 issue of "Cancer Gene Ther." 15th, pp. 18-28), TK, p28, p11 and K1L Promoters, as well as synthetic promoters such as Chakrabarti et al. (in the 1997 issue of "Biotechniques", No. 23, pp. 1094-1097), Hammond et al. (in 1997, J. Virological Methods, No. 66, pp. 135). - Page 138, B) and Kumar and Boyle (in the 1990 issue of Virology, pp. 151, pp. 151-158), and early/late chimeric promoters. Suitable for measles media Promoters for performance, including but not limited to any promoter that directs the expression of measles transcriptional units (Brandler and Tangy, 2008, "CIMID", No. 31, pp. 271-291). Promoters with liver specificity include, but are not limited to, the promoter of Hmg-CoA reductase (Luskey, 1987, "Mol. Cell. Biol", No. 7, pp. 1881-1893); Type 1 sterol regulatory element (SRE-1; Smith et al., 1990, J. Biol. Chem., 265, pp. 2306); albumin (Pinkert et al., 1987, "Genes Dev" "Phase 1 (pp. 268-276)); phosphoenolpyruvate carboxykinase (PEPCK) (Eisenberger et al., 1992, "Mol. Cell Biol.", No. 12, pp. 1396-1403) ; alpha-1 antitrypsin (Ciliberto et al., 1985, "Cell", 41, pp. 531-540); human transferrin (Mendelzon et al., 1990 issue "Nucleia Acids Res." 18th Issues 5717-5721, B); and FIX genes (US Patent No. 5,814,716).

The skilled artisan will appreciate that the regulatory elements that control the expression of the NS (NS3/NS4 and NS5B) nucleotide sequences may further comprise appropriate initiation, regulation and/or termination of transcription for the host cell or individual (eg, Multiple adenine transcriptional termination sequences), mRNA delivery (eg, nuclear localization signal sequences), manipulation (eg, splicing signals), and stability (eg, intron and non-coding 5' and 3' sequences), translation ( Additional elements such as the starting methionine, the triplet leader sequence, the IRES ribosome binding site, the Shine-Dalgarno sequence, and the purification step (eg, a label).

In a preferred embodiment, the composition used in the present invention comprises An MVA expression vector, and in the deletion III of its genome, preferably inserts the following in the same orientation: (a) a nucleotide sequence encoding a NS3/NS4 polyprotein, which is placed under the control of the vaccinia pH5R promoter And (b) a nucleotide sequence encoding a NS5B polypeptide under the control of a vaccinia p7.5K promoter. Preferably, the nucleotide sequence encoding the NS3/NS4 polyprotein comprises the nucleotide sequence shown in SEQ ID NO: 3, and the nucleotide sequence encoding the NS5B polypeptide comprises the sequence identification number: 4 Nucleotide sequence.

If desired, the compositions used in the present invention may further comprise one or more transgenic genes, such as one that is of interest to be expressed in a host cell or individual with a nucleotide sequence encoding NS (NS3/NS4 and NS5B). A gene whose purpose is to ameliorate the therapeutic or protective activity of an HCV infection or any disease or condition caused by or associated with HCV infection. Suitable transgenic genes include, but are not limited to, immunomodulators such as interleukins and any other antigen derived from an organism (e.g., HIV, M. tuberculosis, etc.) that may be associated with infection. If a transgenic gene is used, which can be expressed by a expression vector encoding a NS3/NS4 polyprotein and/or an NS5B polypeptide, or by a separate vector used in combination, the independent vector can be identical to the vector expressing NS3/NS4 or NS5B or different.

According to another preferred embodiment, the expression vector contained in the composition used in the present invention is in the form of infectious virus particles. The viral particles are typically produced by a method, and the steps thereof comprise: (a) introducing a viral expression vector of the invention into a suitable host cell; (b) cultivating the host cell under suitable conditions to permit production of the Infectious viral particles; (c) recovering the resulting viral particles from the culture of the cells; and (d) selectively The recovered virus particles were purified.

Numerous host cells can be used to produce the expression vectors used in the present invention. Host cells can be cultured in conventional bioreactors, rocking bioreactors, microcarriers, roller bottles, flasks or petri dishes in the form of adherent cells or in suspension in the presence or absence of vehicle. The culture is carried out at a temperature, a pH value and an oxygen content suitable for a specific cell using a batch, a feed batch, a continuous system, a hollow fiber or the like.

For example, a poxvirus vector can be produced in avian cells such as chicken embryonic fibroblasts (CEF) and duck cell strains prepared from chicken embryos obtained from fertilized eggs (see, for example, WO03/076601, WO2007/077256, WO2009/004016, WO2010/130756 and US2011-008872). Avian cells are typically cultured at a temperature between 30 ° C and 37 ° C in a suitable culture system for cell growth. The cultured cells are then infected with a poxvirus expression vector and cultured for 1 to 14 days under conditions that promote viral amplification (transcription of viral genomes, production of viral proteins, and assembly into infectious viral particles). In particular, the medium and temperature conditions used during the infection step may be the same or different than the medium and temperature used during the cell growth and/or virus amplification steps.

A replication-deficient adenoviral vector can be produced in a cell strain that provides a trans-deficient function. For example, suitable cell lines for complementing the deleted E1 type adenoviral vector include 293 cells (Graham et al., 1997, J. Gen. Virol., 36, pp. 59-72) and HER. -96 and PER-C6 cells (eg, Fallaux et al., 1998, "Human Gene Ther.", No. 9, pp. 1909-1917; B. WO97/ 00326) or any derivative of such cell lines.

Several media are available in the art, such as Dulbecco's modified Eagle's medium (Invitrogen's DMEM) or Ig's basal medium (British company's BME), etc. Optionally, serum (such as fetal bovine serum (FCS)) and/or amino acids (such as L-glutamine) are added. The cells can also be cultured in a medium free of animal products. Many have been mentioned in the medium free of animal products, and wherein a portion of the configuration made commercially available, such as for example 293 SFM II; 293-F cells, the SFM modified; 293-H cells, the SFM modified; 293fectin ^(TM) Transfection Reagent; the CD 293 AGT ^TM; CD 293 medium; FreeStyle ^TM 293 system performance; FreeStyle ^TM 293 medium; FreeStyle ^TM 293-F cells, SFM modified; VP-SFM; VP-SFM AGT TM; PER.C6® cells for use of adenovirus performance medium (AEM) growth medium; CD 293 AGT ^TM; CD 293 medium; SFM modified; EPISERF® medium; OptiPro ^TM SFM (all from Invitrogen Jieke achieved).

The resulting vector can be recovered from the culture supernatant and/or from the host cell. The cells can be lysed while allowing the producer cells to release the viral particles. Cell membranes can be disrupted by a variety of techniques well known in the art including, but not limited to, freezing/thawing, low osmotic lysis, detergent media lysis, ultrasonic treatment, and microfluidization.

The recovered carrier can be purified if necessary. Various steps can be performed, regardless of the order of the steps, such as the clarification step allowing removal of cellular debris under appropriate conditions (eg, by depth filtration), and the concentration step allows for concentration of the carrier in the crude product (eg, by microfiltration or Ultrafiltration The nuclease treatment step allows degradation of the DNA of the cell (eg, by using benzonase); and/or one or more chromatographic steps (eg, ion exchange, gel filtration, affinity, hydroxyapatite) Stones and especially ion exchange are preferred). For the purpose of illustration, purification of poxvirus particles can be carried out as described in WO2007/147528 and WO2010/130753; such as WO96/27677, WO98/00524, WO98/22588, WO98/26048, WO00/40702, EP1016700 The adenoviral vector was purified as described in WO 00/50573. The conditions and techniques employed depend on factors such as net charge, molecular weight, hydrophobicity, hydrophilicity, and the like, as will be apparent to those skilled in the art. In addition, the degree of purification will depend on the intended use.

Generally, a therapeutic agent that is included or encoded by an immunotherapeutic composition of the invention is administered in a "therapeutically effective amount" which is an amount sufficient to produce a therapeutic benefit, whether administered alone or with one or A variety of antiviral compounds are administered in combination. The therapeutically effective amount may vary depending on various parameters, in particular the type of therapeutic agent (polypeptide, plastid or viral vector); mode of administration; disease state; age and weight of the individual; nature and extent of the symptoms; concurrent treatment type (eg interference) And/or ribavirin and/or anti-protease therapy); and/or frequency and dose of treatment. Further fine calculations are routinely performed by the medical practitioner to determine the appropriate therapeutic dose based on the relevant circumstances.

In accordance with general guidelines, a suitable therapeutically effective amount of a composition comprising a viral vector will vary from about 10 ⁵ to about 10 ¹³ vp (virion particles), iu (infectious units) or pfu (plaque forming units), Depending on the carrier used and the quantitative technique. For example, for administration to an individual dose of vaccinia virus-based composition of the formula comprise from about 5x10 ⁵ to about ¹⁰⁹ plaques forming units or MVA pox virus, advantageously from about 5x10 ⁶ th plaques forming units to about 10 ⁸ lysis plaque forming units, preferably about 5x10 ⁶ th plaques forming units to about 5x10 ⁷ plaque forming units dissolved; and more preferably an amount of about 10 ⁷ plaques forming units. Preferred dosages of the adenoviral vector composition comprise from about 10 ⁵ to about 10 ¹² vp, and especially at a dose of about ⁵ x 10 ⁸ , about 10 ⁹ , about 5 x ^{10 9} , about 10 ¹⁰ , about 5 x ^{10 10} vp or about 10 ¹¹ vp For the first choice. Techniques that can be used to assess the amount of vp, iu, and pfu in a single dose are those employed in the art. Routine potency analysis techniques using appropriate cells (such as BHK-21 or CEF) and appropriate coloring (eg using neutral red; Talavera in the 1992 issue "Methods Mol. Biol." No. 8 Pp. 235-248, B) or immunostaining (eg, using anti-MVA antibodies; Caroll et al., 1997, Virology, 238, pp. 198-211), determination of vaccinia present in a sample Viral vector. The value of adenoviral particles (vp) is usually determined by measuring A260 absorbance or HPLC, the iu titer is determined by quantitative DBP immunofluorescence, and the lysing is determined by counting the number of plaques after allowing the infection of the permissive cells. Spot forming unit. According to FDA guidelines, the vp/iu ratio is preferably less than 50.

The composition of the composition based on the carrier plastid may be between 1 microgram and 25 milligrams, advantageously between 10 micrograms and 20 milligrams, and between 100 micrograms and 2 milligrams, And especially with a dose between 200 micrograms and 1 milligram is preferred. Techniques for assessing the amount of plastid carrier in a sample are routine in the art (e.g., by spectrometry). A protein composition may be administered at a dose of between 10 nanograms and 20 milligrams, more preferably from about 0.1 microgram to about 2 milligrams of immunogenic polypeptide per kilogram of body weight.

The immunotherapeutic compositions used in the present invention are suitable for a variety of administration modes, including parenteral and mucosal routes. Preferably, parenteral administration and especially intradermal, intraepithelial, intranasal, subcutaneous, subcutaneous or intramuscular routes, and especially subcutaneous routes are preferred.

In addition, the administration route, the therapeutic agent, and the therapeutically effective amount of each administration of the immunotherapy composition may be different from each other. For illustrative purposes, an MVA carrier immunotherapy composition can be administered 4 to 8 times apart from each other for 3 to 10 days, and can be separated from each other by an adenoviral vector or plastid carrier immunotherapy composition. 4 to 15 administrations in 5 weeks. It is also conceivable to adopt a combination of different administration routes, such as combined subcutaneous and intramuscular administration.

However, it is preferred to carry out all administrations of the immunotherapy composition by the same route of administration, and the administration route is preferably selected from the group consisting of intradermal, intramuscular and subcutaneous, and the latter is preferred. Subcutaneous administration is particularly useful for compositions comprising viral expression vectors such as MVA, while intramuscular administration is particularly useful for compositions comprising a plastid expression vector.

Administration can be carried out using conventional needles or devices suitable for administration routes, and optionally using one or more compounds or devices that promote or enhance the delivery of the active agent (polypeptide or expression vector). Suitable compounds include, but are not limited to, polyvalent cationic polymers (such as chitosan, polymethacrylate, PEI, etc.) and cationic lipids (such as DC-Chol/DOPE, available from Promega®). The transfectam lipofectin obtained by the company; and suitable devices include, but are not limited to, those that permit electroporation and pressure-mediated delivery. Cast by electroporation The drug action is particularly useful for intramuscular delivery of plastid carrier compositions.

Preferably, the immunotherapeutic composition used in the present invention is administered to the individual according to a mode of administration comprising 4 to 8 times of administration for about one week apart from each other (herein referred to as "weekly" "The administration of the drug" followed by 4 to 15 times of administration for about one month apart (herein referred to as "monthly administration"). "About one week" means 6, 7 or 8 days, and "about one month" means a period of 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 days. In the context of the present invention, the time between secondary administrations may vary within the defined range.

A particularly good mode of administration consists of six weekly dosings followed by five to twelve monthly dosings; more preferably six once a week, followed by six to ten A monthly dosing effect; and even better, six weekly dosings, followed by seven monthly dosings.

Immunotherapy composition

In one embodiment, the immunotherapeutic composition used in the present invention further comprises a pharmaceutically acceptable carrier in addition to a therapeutically effective amount of the active agent.

The term "pharmaceutically acceptable carrier" is intended to include any and all carriers, solvents, diluents, excipients, adjuvants, dispersion media, coatings, which are compatible with the administration of mammals and especially humans. Antibacterial and antifungal agents and delayed absorbents.

The compositions used in the present invention are desirably suitably buffered to be suitable for physiological or weakly alkaline pH values (e.g., from about pH 7 to about pH 9). For human or animal use. Suitable buffers include, but are not limited to, phosphate buffers (such as PBS), bicarbonate buffers, and/or Tris buffers.

The composition used in the present invention may further comprise a diluent suitable for use in one of humans or animals. It is preferably isotonic, low or weakly high and has a relatively low ionic strength. Representative examples include, but are not limited to, sterile water, physiological saline (eg, sodium chloride), Ringer's solution, dextrose, trehalose or sucrose solutions, Hank's solution, and other aqueous physiologically balanced salts. Solutions (see, for example, Lippincott, Williams & Wilkins, Inc., published by A. Gennaro), Remington: Science and Practice of Pharmacy (Remington: The Science and Practice of Pharmacy) "The latest version of the book B)).

The pharmaceutically acceptable carrier can also be stored and stored in a frozen (eg, -70 ° C, -20 ° C), refrigerated (eg, 4 ° C) or ambient temperature for a long period of time (ie, at least 6 months and preferably at least 2 years) The stability of the composition is preserved regardless of the form of the composition (eg, frozen, liquid or solid). A solid (e.g., dry powder or freeze-dried) composition can be obtained by a method involving vacuum drying and freeze drying. For illustrative purposes, sterile phosphate buffered saline (PBS) and saline are suitable for preserving plastid carriers, while buffered formulations comprising sodium chloride and sugar are suitable for preserving viral vectors, such as for glands. Viral vector of 10 mM Tris pH 8.5 with 1 M sucrose, 150 mM sodium chloride, 1 mM magnesium chloride and 54 mg/liter Tween 80, and 5% sucrose (weight/volume) for MVA carrier , 10 mM sodium glutamate and 50 mM sodium chloride Tris 10 mM pH 8.

A pharmaceutically acceptable additional excipient can be used to provide the desired Pharmacological or pharmacodynamic properties, including, for example, the permeability, viscosity, clarity, color, sterility, stability, rate of dissolution of the formulation; delayed release or absorption into the individual; facilitation of delivery through the blood barrier or Penetrate into a specific organ (such as the liver).

Furthermore, the immunotherapeutic compositions used in the present invention may comprise one or more adjuvants. An adjuvant refers to a component of an immunogenic property of an amplifiable immunogen, such as an HCV NS polypeptide, whether the immunogenic property is specific or non-specific, hormonal or cellular. The adjuvant is ideally suited for enhancing immunity, including immunity mediated by a T cell such as a TR receptor such as TLR-7, TLR-8 and TLR-9. Representative examples of suitable adjuvants include, but are not limited to, alum, mineral oil emulsions such as Freund Complete and Incomplete Adjuvant (IFA), Lipopolysaccharides or Derivatives (Plenum Press, New York, USA) Published by Ribi et al., 1986, "Immunology and Immunopharmacology of Bacterial Endotoxins", pp. 407-419), saponin such as QS21 (Sumino et al. 1998) Journal "J. Virol." No. 72, p. 4931, B; WO 98/56415), imidazole-quinoline compounds such as Imiquimod (Suader, 2000 issue "J. Am Acad Dermatol." Issue 43 (page B6), S-27609 (Smorlesi, 2005, "Gene Ther.", No. 12, pp. 1324-1332) and related compounds such as cytosine phosphate as described in WO2007/147529 Avian nucleoside oligodeoxynucleotides such as CpG (Chu et al., 1997, J. Exp. Med., 186, pp. 1623-1631; Tritel, 2003, "J. Immunol." 171, pp. 2538-2547, B) and cationic peptides such as IC-31 (Kritsch et al., 2005, "J. Chromatogr Anal. Technol Biomed Life Sci", No. 822, pp. 263-270).

Combined with other treatments

In a preferred embodiment, the immunotherapeutic composition is used in combination with other treatment modalities intended to treat a subject infected with HCV.

The term "combination" and variants thereof, such as "combined use," refers to administration of two or more entities administered in the same individual, one of which is an immunotherapeutic composition as described herein. The two or more entities used in combination may be administered for different periods via different routes, dosages, and according to different schedules.

The immunotherapy composition used in the present invention is preferably used in combination with one or more antiviral compounds. An "antiviral compound" can be defined as a chemical compound or a biological molecule that inhibits at least one of the steps of the HCV life cycle, either directly (eg, by inhibiting a viral enzyme) or indirectly (eg, by mimicking a viral receptor such as Nucleosides and nucleotide analogues or inhibiting viral replication or entry into a desired cellular protein), or blocking viral infections (such as blocking antibodies), are intended to reduce the number of infected individuals in a temporary or sustained manner. The amount of HCV.

Representative examples of antiviral compounds are selected from the group consisting of interferon, ribavirin, NS3/4A protease inhibitors, NS5A inhibitors, NS5B polymerase inhibitors, nucleoside analogs, nucleotides Analogs, immunomodulators such as interleukins and chemokines and host cell enzyme inhibitors such as cyclic avidin inhibitors or any of their functional analogs (such as Tiribavirin ^(TM ) from Valeant). Preferred antiviral compounds for use in combination with the immunotherapeutic compositions described herein are interferons and/or ribavirin, and optionally an NS3/4A protease inhibitor.

Antiviral therapy is usually provided to individuals for 24 to 48 weeks depending on the type of HCV genotype being infected, the type of antiviral therapy (dimer or triple therapy), and individual tolerance (eg, for the first genotype) 48 weeks and for the 2nd and 3rd genotypes for 24 weeks). Antiviral therapy preferably includes providing interferon with ribavirin for 48 weeks (biotherapy). When antiviral therapy is further involved in NS3/4A protease inhibitors (triple therapy) such as Merck's boceprevir (Victrelis) or Vertex's Trapivir (telaprevir) (Incivek) or Incivo) The duration of antiviral therapy can be shortened to 24 weeks. Optionally, the antiviral therapy can be one that does not contain IFN, and one or more antiviral compounds are provided in the absence of any interferon (such as NS3/4A protease and/or polymerase inhibitor). Antiviral therapy can also include the provision of one or more nucleoside/nucleotide analogs in the absence of any interferon or ribavirin.

Although any interferon may be provided under the circumstances of the present invention, the interferon is advantageously an interferon-α (IFNα), preferably a pegylated interferon-α, and more preferably a polyethylene. Alcoholized interferon-α2a (such as PEGASYS ^R ) or a pegylated interferon-α2b (such as PEGINTRON ^R ). Interferon lambda is also contemplated, and is preferably pegylated interferon lambda. The dosage, route and duration of administration may vary depending on the type of interferon, the manufacturer, and the physician may adopt an appropriate manner suitable for each individual. Pegylated interferon alpha is usually provided by the intramuscular or subcutaneous route, in the case of pegylated interferon-α2a, the dose is about 180 micrograms per week; and in the case of pegylated interferon-α2b The dosage is about 1.5 micrograms per kilogram per week.

Rebavirin (eg COPEGUS ^R ) is a synthetic nucleoside analogue with the chemical name 1-β-D-nucleylfuranyl-1-H-1,2,4-triazole-3-carboxamide . Ribavirin in the form of an oral lozenge is commercially available. Depending on the individual body weight (5 200 mg tablets for individuals under 75 kg and 6 200 mg tablets for individuals over 75 kg) or other factors such as tolerance, preferably about 1000 to about 1200 per day. A dose of milligrams is provided orally in the form of ribavirin (eg 5 or 6 200 mg tablets).

Viral protease inhibitors include, but are not limited to NS / 4A protease inhibitor telaprevir (telaprevir ^TM) (Vertex (Vertex) Corporation), boceprivir (boceprevir ^TM) (Merck (Merck) Corporation), TMC435 (Tibotec ), BI201335 (Boehringer Ingelheim), ABT-450 (Abbott), BMS-650032 (Bristol-Myers Squibb) and RG7227 (Roche) Danoprevir (danoprevir). For illustrative purposes, respectively, preferably from about 2400 to 2250 mg daily dose, orally provide boceprivir (boceprevir ^TM) and telaprevir (telaprevir ^TM).

Inhibition of NS5B polymerase disrupts HCV RNA synthesis and thus prevents the production of genomic HCV RNA required for the production of new virions. Polymerase inhibitors can be divided into two broad categories based on their mechanism of action and chemical structure: nucleoside analogs bind to the active site of the polymerase in a competitive manner, rather than non-competitive inhibitors of nucleoside analogs and with NS5B Multiple discrete addresses are combined. NS5B inhibitors in development include, but are not limited to, RG7128 (Roche, Roche) (mericitabine)), GS-9190 (Gilead), PSI-7977 and PSI-938 (Pharmasset) and VX-222 (Vertex).

The viral protein NS5A, which is involved in viral replication and viral assembly, is also the subject of a new antiviral agent such as BMS-790052 (Bristol-Myers Squibb).

In addition to the virus target, the host involved in viral replication Cellular proteins are also the target of drug developers. In particular, it involves a number of cellular actions such as protein folding and migration of cyclophilin (peptidyl- amidoxime cis-trans isomerase), which is also required for HCV replication. DEB-025 (Aristarii, Novartis) and SCY-635 (Scynexis) are currently hosted cyclic avidin inhibitors in clinical development.

In the context of the present invention, the immunotherapeutic compositions used in the present invention can be administered prior to or simultaneously with or after the provision of antiviral therapy. The present invention encompasses completely separate administration of immunotherapeutic compositions with antiviral therapies (e.g., antiviral therapy is provided after the immunotherapeutic composition and does not begin after the immunotherapeutic treatment is completed) or partially overlapping administration. In a preferred embodiment, the administration of the immunotherapeutic composition used in the present invention begins prior to providing the antiviral therapy, such as at least 4 weeks prior to providing one or more antiviral compounds, desirably at least 8 A week ago, advantageously at least 10 weeks ago and preferably at least 12 weeks ago. In this regard, a particularly suitable mode of administration is the administration of six immunological compositions once a week and then once a month, and about 12 after the first administration of the immunotherapeutic composition. Antiviral therapy is available during the week.

Additional administration of the immunotherapeutic composition can be carried out during antiviral therapy (eg 24 or 48 weeks) or a portion of the period (ie less than 24 or 48 weeks) and can be performed after the end of antiviral therapy . More preferably, the immunotherapeutic composition used in the present invention is administered once a month for a period of 18 to 24 weeks, and especially for about 21 weeks, followed by initiation of antiviral therapy (eg, interferon and Rebavirin and the ultimate NS3/4A inhibitor).

In the context of the present invention, an interferon and immunotherapeutic composition may be provided to the individual on the same day (eg, pegylated IFNα2 once a week may coincide with the administration of the composition described herein), or interfere with The administration of the prime injection and the administration of the immunotherapeutic composition can be separated by one day or several days (2, 3, 4, 5 or 6 days).

An even more preferred mode of administration comprises 13 administrations of the immunotherapeutic compositions used in the present invention, preferably at 0, 1, 2, 3, 4, 5, 9, 13, 17, 21 At 25, 29 and 33 weeks, and at the 12th week, interferon and ribavirin were started.

Even more preferably, the MVA vector system encoding immunotherapy composition contained in the NS3 / NS4 polyprotein and NS5B polypeptide, and by the subcutaneous route comprising from about 5x10 ⁶ to about 1x10 ⁸ th plaques forming units to the name of the administered dose In the individual, the interferon is a pegylated IFNα2 provided by an intramuscular or subcutaneous route, and the antiviral compound is provided by arbavirin orally. The MVA vector immunotherapy composition and more preferably comprises from about 10 ⁷ plaques forming units, and incorporated in its genome encoding the NS3 / NS4 fusion protein of one of the nucleotide sequences, and the NS3 / NS4 fusion protein comprising The amino acid sequence is at least 95% identical to the amino acid sequence shown in Sequence Identification Number: 1; and a nucleotide sequence encoding one of the NS5B polypeptides is included in the vector, and the NS5B polypeptide comprises The amino acid sequence is at least 95% identical to the amino acid sequence shown in Sequence Identification Number: 2; the interferon is pegylated interferon alpha 2a, which is administered in an amount of about 180 micrograms per week. The individual is provided once a week by the subcutaneous route; and the ribavirin is provided by oral administration in an amount of 1000 to 1200 mg per day.

In another embodiment, the immunotherapeutic composition can be used in combination with an antiviral therapy that does not contain IFN, that is, in combination with any one or more antiviral compounds as described above, such as with ribavirin and/or Or a combination of a viral protease inhibitor and/or a polymerase inhibitor and/or a nucleoside/nucleotide analog. In this regard, the ability of immunotherapeutic compositions to elicit innate and adaptive immunity complements the mechanism of action of such antiviral compounds, ie, if antiviral escape mutants are present, the efficacy of immunotherapeutic compositions is not expected to be The effect, because of point mutations, does not interfere with the overall immunity provided by the immunotherapeutic composition. This approach can contribute to the reduction of IFN-related side effects.

In another embodiment, the immunotherapeutic composition can be used in combination with an antiviral therapy without ribavirin, that is, one or more antiviral compounds as described above in addition to ribavirin or any analog thereof. It is used in combination with IFN and/or viral protease inhibitors and/or polymerase inhibitors and/or nucleoside/nucleotide analogs.

Treatment course

Another aspect of the invention relates to a therapeutic procedure, the steps comprising administering the invention to a subject in accordance with the mode of administration described herein The immunotherapeutic composition used. Preferably, the therapeutic treatment is provided in combination with the antiviral therapy described herein, and in particular in the art, currently used to treat HCV infection and particularly chronic HCV infection (eg, PEG-IFNα and/or Rebavirin and ultimately) An antiviral therapy for NS3/4A inhibitors).

In the context of the present invention, the therapeutic regimen is performed using an immunotherapeutic composition or in a manner described herein to provide an anti-HCV compared to the immunotherapeutic composition not being used (eg, compared to providing only antiviral therapy) The therapeutic benefit of infection.

The immunotherapeutic compositions or treatment regimens described herein can be reduced or alleviated in association with HCV infection, as compared to the expected symptoms, disease progression, and survival of the immunotherapeutic compositions or treatment regimens described herein. One or more symptoms (such as liver inflammation, liver cell lysis, liver fibrosis, cirrhosis, and/or liver cancer), reducing the extent of the disease, stabilizing the disease (ie, not worsening), preventing the spread of the disease, delaying or slowing the progression of the disease The efficacy of treatment may be confirmed by worsening, improving or slowing the disease condition, reducing the toxicity associated with SOC, eliminating the virus (whether temporary or persistent), and prolonging the survival period.

In more detail, the immunotherapeutic compositions or therapeutic regimens described herein in combination with antiviral therapy can be achieved, for example, by increasing the proportion of patients who achieve viral reduction or elimination at a particular time point, or by shortening the target. Helping to control HCV infection during the period of antiviral therapy required, and/or by reducing the amount of antiviral therapy that achieves this goal and/or by reducing the frequency of antiviral therapy, and the like.

As generally indicated, it can be experienced by a physician or other skill The health care provider typically measures any relevant markers that reflect HCV infection, and facilitates assessment of therapeutic benefit, such as by quantitative analysis of HCV nucleic acids and/or viral antigens in biological samples taken from one body at different time points. For example, the level of the markers measured in a group of individuals treated in accordance with the present invention may be compared to individuals who have not used the immunotherapeutic composition or treatment at the same time point and at the selected time point. The level of the same marker measured in the comparison is compared to assess the therapeutic benefit. A particularly appropriate marker for assessing therapeutic benefit is the repeated measurement of viral load in the blood at different time points and conversion of these measurements to conventional efficacy measures such as rapid viral response, early viral response, complete early viral response, representation The endpoint of the therapeutic response associated with clearance (ie, cure) or clearance and/or sustained viral response.

Quantitative analysis of HCV ribonucleic acid (RNA) in a given sample (eg, blood or serum sample) collected from a body, typically using quantitative RT-PCR analysis or by any other method recognized in the art, The "viral load" was measured. Although the lower limit of quantitation (LLOQ) for determining the inability to quantify HCV-RNA may vary from assay to analysis, it is desirably less than 29 IU/mL, and preferably less than or equal to 15 IU/mL. A particularly suitable RT-PCR assay is Roche's TaqMan R assay (as described in the accompanying examples) which provides an LLOQ of 15 IU/ml and its determination of HCV. The lower limit of detection for undetectable RNA (LLOD) is less than 10 IU/mL.

In general terms, a common practice is to measure a body at baseline (i.e., prior to the initiation of the course of treatment of the invention or prior to the use of the immunotherapeutic composition) and during the course of treatment or use of the immunotherapeutic composition. The viral load. In embodiments in which the immunotherapeutic composition used and the therapeutic course of treatment performed in combination with antiviral therapy can be performed at different time points (eg, prior to providing antiviral therapy and in antiviral therapy, 2, 4, or 5, Viral load was estimated after 12, 24, 36, and 48 weeks and at the 12th and 24th weeks after the end of antiviral therapy. For example, a reduction in HCV-RNA between two time points of at least 0.5 log ₁₀ IU/ml can be a predictor of therapeutic benefit.

In more detail, the therapeutic benefit is determined if the reduction in the level of serum HCV RNA quantified after a specific time point has reached a significant extent (eg, clearance of HCV RNA) by at least 5% and preferably by at least 10%. .

The objective of the immunotherapy composition or the course of treatment being performed is advantageously to increase the proportion of individuals achieving superviral response (UVR) by at least 5% compared to antiviral therapy that provides only 2 weeks of antiviral therapy. Better increase by at least 10%.

Compared to the antiviral therapy that only provides 4 weeks, the immunotherapeutic composition used or the goal of the treatment course being performed is ideally after 12 weeks of antiviral therapy, enabling rapid viral response (RVR) Or even better, the proportion of individuals who maintain long-term RVR increases by at least 5%, and preferably by at least 10%.

Compared to antiviral therapy that provides only 12 weeks, the immunotherapeutic composition used or the goal of the treatment course is preferably to achieve an early virological response (EVR) or even better to achieve a completely early stage disease. The proportion of individuals who are toxic (cEVR) increases by at least 5%, and preferably by at least 10%.

More preferably, the proportion of individuals achieving sustained viral response (SVR) is increased by at least 5% compared to the goal of providing only 24 or 48 weeks of antiviral therapy, the immunotherapeutic composition used or the course of treatment being performed. And preferably increase by at least 10%.

"at least 5%" means any value between 5% or greater and between 6 and 100, and at least 10% means any value between 10% or greater and between 11 and 100, and especially At least at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34% or at least 35% Preferred.

The term "superviral response" or "UVR" as used herein refers to the inability to detect HCV RNA after 2 weeks of antiviral therapy.

The term "rapid viral response" or "RVR" as used herein refers to the inability to detect HCV RNA after 4 weeks of antiviral therapy.

The term "long-term rapid viral response" or "eRVR" as used herein refers to the inability to detect HCV RNA after 4 and 12 weeks of antiviral therapy.

The term "early viral response" or "EVR" as used herein refers to a reduction in HCV RNA of greater than or equal to 2 log ₁₀ IU/ml after 12 weeks of antiviral therapy.

The term "complete early viral response" or "cEVR" is defined herein as the inability to detect HCV RNA 12 weeks after the completion of 12 weeks of antiviral therapy.

"End of therapeutic response" or "ETR" is defined herein as the inability to detect HCV RNA after completion of antiviral therapy.

The term "sustained viral response" or "SVR" as used herein refers to the inability to detect HCV RNA 12 or 24 weeks after the completion of antiviral therapy.

Preferably, the viral load is quantified at different time points using RT-PCR analysis with a LLOD of less than 10 IU/ml, or any other method approved in the art and having comparable sensitivities.

Optionally or in combination, by measuring the level of liver enzyme activity (such as alanine transaminase (ALT) and/or aspartate transaminase (AST)) in a body of blood or serum The therapeutic benefit was assessed by assessing the level of fibrosis in the liver (eg, by Fibroscan ^R ). For example, normalization of these enzymes or a decrease in elasticity and/or fibrosis stage may indicate a better liver state.

In an advantageous aspect, the immunotherapeutic composition used and the target of the treatment regimen used are shorter periods of antiviral therapy and/or reduced compared to the individual population of chronically infected HCV treated with antiviral therapy alone. A dose or course of treatment of at least one of the antiviral compounds, resulting in an antiviral agent associated with a population of HCV patients treated in accordance with the present invention The proportion of adverse events is reduced (eg, reduced by at least 5%).

In another aspect, the immunotherapeutic composition used and the goal of the course of treatment being performed are to elicit or stimulate the immune system of the individual being treated, thereby making it higher than the baseline state or higher than providing only antiviral therapy (eg, PEG- The expected state of IFNα and ribavirin).

The elicited or stimulated immune response can be non-specific (congenital) and/or specific (anti-HCV and ultimately anti-vector response), hormonal and/or cellular, and especially to elicit or stimulate CD8+ T cells Congenital or specific T cell responses by CD4+ T cells or both CD8+ and CD4+ T cells are preferred. The specific anti-HCV immune response is preferably directed against one or more epitopes present in any of the NS3, NS4 and NS5B polypeptides. Multiple repetitive administrations of immunotherapeutic compositions prior to providing antiviral therapy are expected to elicit an effective innate and/or specific immune response in a treated individual, possibly at the time of administration during antiviral therapy Maintain the immune response.

The ability of the above immunotherapeutic compositions or therapeutic regimens to elicit or stimulate an immune response in a subject being treated can be assessed by a variety of standard assays in the art (a general description of the techniques for assessing the initiation and activation of an immune response) See, for example, the National Institute of Health and the 1992 and 1994 editions of the Immunology Experimental Manual (Current) published by John Wiley & Sons Publishing Company, Coligan et al. Protocols in Immunology) "B) (). The blood test collected at various endpoints can be quantified by measuring the interleukin profile generated by activated cells and those derived from CD4+ and CD8+ T cells (eg, by enzyme-linked immunospot assay (ELIspot) on PBMC. In the case of IL-10 or IFNg-type cells)); by measuring the activation state of immune cells (such as T cell proliferation analysis by conventional [ ³ H] thymidine absorption method); Cellular immunity is assessed by antigen-specific T lymphocytes in individuals (eg, peptide-specific lysis in cytotoxicity assays). The hormonal response can be determined by antibody binding and/or competition assays (see, for example, Cold Spring Harbor Press, published in 1989 and by Harlow, "Antibodies").

In another aspect, the invention also relates to a method of increasing the proportion of individuals achieving complete early viral response (cEVR) in a population of chronically infected HCV by at least 5% and preferably at least 10%, as compared to chronic infection For HCV and cEVR in an individual population treated with only antiviral therapies such as interferon and ribavirin, the method comprises administering an immunotherapeutic composition to the individual population or performing the therapeutic treatment described herein, thereby Initiating or stimulating an immune response mediated by a T cell against one or more epitopes present in the HCV NS3, NS4 and/or NS5B polypeptide.

In another preferred embodiment, the immunotherapeutic compositions and therapeutic treatments used in the present invention are used to treat chronically infected individuals.

The term "chronic HCV infection" refers to an HCV infection that persists in a body for more than 6 months. Although the natural course of chronic HCV infection/chronic hepatitis varies from individual to individual, chronic HCV infection usually leads to liver-related diseases (liver inflammation, liver fibrosis, and ultimately liver cancer).

Preferably, the genotype or subtype of HCV chronically infected by the individual is preferred It is the same as any HCV from which the NS polypeptide contained or encoded by the immunotherapy composition used in the present invention is derived, and particularly the infectious HCV (1a or 1b) of the first genotype. Optionally, the genotype of the HCV infected by the individual is different from the HCV from which the NS therapeutic agent is derived (eg, infectious HCV of the third genotype). Preferably, the individual has not received any interferon therapy for the conventional use of HCV prior to using the immunotherapeutic composition or performing the therapeutic procedure.

In another preferred embodiment, the subject to be treated is a subject who has not received treatment at baseline, and in particular, individuals who have not received conventional IFN therapy prior to using the immunotherapeutic composition or performing the therapeutic procedures described herein .

"Not treated" refers to the treatment of any individual or patient to be treated according to the methods described herein that has not previously been approved for the treatment of any antiviral compound for HCV infection. Therefore, a subject who has not received IFN treatment is a treatment that has not previously been approved for the treatment of any IFNα compound for HCV infection.

The "untreated" subject to be treated preferably has a high viral load at baseline, i.e., prior to the use of the immunotherapeutic composition or the treatment regimen described herein.

In the context of the present invention, "high viral load at baseline" means HCV RNA levels above 400,000 IU/ml and preferably above 600,000 IU/ml.

Optionally, the individual to be treated can be a person who has not previously responded to a treatment for HCV (such as PEG-IFNa and ribavirin) or a partial responder.

In another preferred embodiment, the system to be treated is non-cirrhotic. (i.e., the fibrotic state as determined by routine probing techniques such as liver fibrosis scanner or biopsy method is less than 4 (F0, F1, F2 or F3)).

In another aspect, the invention provides a kit of parts for treating an HCV infection and preferably a chronic HCV infection, wherein the kit comprises a plurality of containers and administering an immunotherapeutic composition to one body or according to the teachings herein Ways to carry out treatment instructions.

Preferably, the kit comprises a) an immunotherapeutic composition comprising a vaccinia MVA vector as defined above; b) an antiviral compound such as interferon and ribavirin; and c) for administration (a) And the instructions in each of (b).

The present invention has been described by way of illustration, and it should be understood that In view of the above teachings, it is apparent that numerous modifications and variations of the present invention are possible. Therefore, it is to be understood that the invention may be practiced otherwise than as specifically described herein within the scope of the appended claims.

The disclosures of all patents, publications, and database items cited above are hereby incorporated by reference in their entirety as references, as if each patent, publication, and database project was incorporated individually and individually. This case is considered as reference material.

Figure 1 is a schematic representation of a randomized, open phase II study conducted in an untreated patient with a first genotype of chronic hepatitis C. The arm A system was treated with SOC consisting of pegylated interferon alpha-2a (Pcgasys ^® ) and ribavirin (Copcgus ^® ). Patients with arm C received multiple TG4040 subcutaneous injections based on two different dosing kinetics (on a weekly and monthly basis) in combination with SOC.

Figure 2 is a schematic representation of the viral response rate (percentage of patients with undetectable HCV RNA) measured in control arm A (SOC treatment) and arm C (SOC + TG4040) patients.

Figure 3 is a graphical representation showing the slope of the viral load decline during the 12 weeks after SOC administration in control arm A (SOC treatment) and arm C (TG4040 + SOC) patients.

Instance

Case 1: TG4040 Phase I Study without Standard Care (SOC)

TG 4040 is described in WO 2004/111082 and WO 2008/113606, the entireties of each of which are expressly incorporated herein by reference. TG4040 is a modified vaccinia virus Ankara (MVA) vector containing two expression combinations of deletion III implanted in the same orientation in the MVA backbone. The first combination is expressed under the ph5r promoter. A fusion between NS3 and the NS4 protein, while the second combination exhibits NS5B under the p7.5 promoter. The NS3, NS4 and NS5B polypeptides are derived from the 1b genotype HCV-JA strain (Kato et al., 1990, "Proc. Natl. Acad. Sci. USA", 87th, pp. 9524-9528). TG4040 was stored at -80 °C with 5% sucrose, 10 mM sodium glutamate and 50 mM sodium chloride and 10 mM Tris-HCl buffered at pH 8. The TG4040 is supplied in individual clear glass vials. Each vial is intended for single use (ie, for one injection of a patient).

This example describes the results of a dose escalation Phase I study with TG4040 (TG4040.01) in patients with non-cirrhotic, untreated chronically infected hepatitis C virus (HCV class 1a or 1b genotype). . Three TG4040 doses of 10 ⁶ , 10 ⁷ and 10 ⁸ plaque forming units were administered subcutaneously (SC) according to generations 1, 2 and 3. Further Study 10 ⁸ units of the dosage form plaques in three generations of extending (4, 5 and 6). The 40 patients included in the analysis received three subcutaneous TG4040 injections once a week and received only 10 ⁸ plaque forming units after 2, 4 or 6 months of the first injection. Injection effect. In more detail, Generation 1 (C1): three patients who received three injections of 10 ⁶ plaque forming units on Day 1, Day 8, and Day 15; - Generation 2 (C2): at Three patients who received three injections of 10 ⁷ plaque forming units on day 1, day 8, and day 15; - generation 3 (C3): received three times on day 1, day 8, and day 10 Injection of ⁸ plaque forming units and 9 patients receiving an additional injection in the 6th month; - Generation 4 (C4): Receiving 3 ⁸ on the 1st, 8th and 15th day Injection of plaque forming units and seven patients who received an additional injection in the sixth month. It is worth noting that these patients have advanced liver disease and high levels of alanine transaminase ALT (between 2 and 5 times the upper limit of normal [ULN] 2 months prior to inclusion in the study) And fibrosis stage is higher than or equal to F2; -Gene 5 (C5): received three injections of 10 ⁸ plaque forming units on Day 1, Day 8 and Day 15 and received in the second month Nine patients who were given an injection; Generation 6 (C6): received three injections of 10 ⁸ plaque forming units on Day 1, Day 8, and Day 15 and received once in the fourth month. Nine patients with injections.

The average age of forty patients was 39.9 years. More male patients (57.5%) were included in the study. The 1a and 1b genotypes were evenly distributed in each dose group.

Virological, biological, and immunological markers were assessed at baseline (before the first TG4040 injection) and at different time points. Viral load (HCV-RNA quantification) was assessed by quantitative RT-PCR (using Roche's TaqMan® analysis); evaluation by ELISpot IFN-g assay MVA and HCV-specific cellular immune responses; and simultaneous evaluation of anti-MVA hormones by ELISA (for total antibodies) and by bioassay based on BHK-21 cell infection (for neutralizing antibodies) Sexual reaction.

result

The TG4040 is shown to be safe and well tolerated. There are no serious adverse events (SAE) associated with the TG4040. The incidence of drug-related injection site induration was more common in the highest dose group (10 ⁸ plaque forming units), with 29 events occurring in 18 of 34 patients (53%); dose group and low dose (10 ⁶ plaques forming units or ¹⁰⁷ units forming plaques) only six patients in two (33%) 2 events. The second most commonly reported adverse drug reaction (ADR) was inflamed at the injection site, and 16 of 40 patients (40%) reported 33 events; followed by erythema at the injection site, 13 of 40 patients The name (33%) notified 20 incidents.

Reduced viral load

The baseline mean viral load for Generation 1 was 5.02 log ₁₀ IU/mL, Generation 2 was 4.97 log ₁₀ IU/mL, and Generation 3 was 5.60 log ₁₀ IU/mL. When considering a viral reduction of better than 0.5 log ₁₀ IU/ml, a higher vaccine efficacy was observed in 3 low- and medium-dose injections per week, as 100% of patients (6/6) The reduction in the apparent viral load is better than the cut point at least once. Conversely, the three highest dose injections per week resulted in a reduction of better than 0.5 log ₁₀ IU/mL observed in only 21% of patients (7 out of 34 patients). The reason for these results may be a higher anti-MVA neutralizing antibody response when administered at the highest dose. It is worth noting that more than half of the 13 patients showed significant reductions in viral at least once, and NADIR was observed on day 37, which is 3 weeks after the last TG4040 initial injection. In generations 3 to 6, the addition of the highest dose did not show an effect on the reduction in viral load.

Results of enzyme-linked immunospot (ELISpot) analysis

Peripheral blood mononuclear cells in patients after stimulation with three peptide pools of NS3 and NS4b antigens derived from TG4040, two peptide pools from NS5a not associated with TG4040, and CEF peptide pools of the positive control group Enzyme-linked immunospot IFN-γ analysis was performed on (PBMC). In Generations 1 to 3, these results show:

- At baseline, 3 out of 15 patients were detected for TG4040 The reaction of the antigen and the antigen not related to TG4040.

- An increase in the response to TG4040-derived antigen was observed in 6 patients after the initial TG4040 injection, but not for the NS5a antigen not associated with TG4040, with peaks at D22 and above the cut point.

Detailed analysis of each patient showed a significant response observed in generations 1 and 2, and in some of these patients, the reduction in viral load was accompanied by an event with a TG4040-specific immune response. Notably, patients with generation 3 who did not have a viral load change due to TG4040 showed only a very weak and sporadic TG4040-related immune response. In addition, TG4040 elicited the most significant immune response (IFN-γ effect) in these chronically infected patients who had not been treated, and their occurrence was accompanied by the largest viral reduction. However, at this level it cannot be concluded that the highest dose does not result in an HCV-specific immune response, and the method for further optimization of generations 4, 5 and 6 is allowed to be 71% (5 out of 7 patients). A significant response was measured in 89% of patients (8 out of 9 patients) and 56% (5 out of 9 patients). After the injection, the intensity of HCV-specific cellular responses was maintained in Generation 5 (in the second month), and 78% of patients in Generation 6 (added in the fourth month) There was a further increase in 7 of the 9 patients, and there was no effect in the generation 4 (additional 6th month).

In summary, the TG4040.01 study showed a good safety profile and promising antiviral efficacy associated with immunogenicity. Based on the efficacy and fewer injection site reactions, selecting ¹⁰⁷ plaques in a unit dosage form for further research.

Case 2: Phase II study of TG4040 in combination with standard care (SOC)

This example describes a randomized, open phase II study conducted in a non-cirrhotic patient who has not received treatment for a first genotype of chronic hepatitis C. Patients were stratified by age (greater than 50 years vs. 50 years or less) and by baseline viral load (greater than 400,000 IU/mL vs. 400,000 IU/mL). The study included multiple TG4040 subcutaneous injections based on two different dosing kinetics (once a week and once a month) and combined use of pegylated interferon alpha-2a (Pegasys ^® ) Standard care consisting of Copegus ^® is used together. The aim of this study was to compare the efficacy and safety of TG4040 with SOC compared to SOC alone. A schematic illustration of the study is shown in Figure 1.

The idea behind the study was to stimulate anti-HCV immunity by multiple repetitive injections of TG4040 (six once-weekly dosing and one monthly dosing) prior to initiation of SOC. And also after the last vaccination (about 3 weeks), prevented the rebound in viral load observed in the Phase I study of TG4040.01. The TG4040 administration was performed on a monthly basis after the SOC was activated, so that the immunity of TG4040 was maintained in the treated patients, while allowing the SOC-mediated viral clearance.

Materials and Methods

10 ⁷ plaque forming units of TG4040 were injected subcutaneously (as described in Example 1 above).

Copegus ^® is a nucleoside analog with antiviral activity. The chemical name of Rebavirin is 1-ß-D-nuclearfuryl-1 H -1,2,4-triazole-3-carboxamide. It can be obtained in the form of an oval or film-coated tablet for oral administration. Each tablet contains 200 mg of RBV and the following non-active ingredients: pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, corn starch, and magnesium stearate. The daily dose of oral RBV is 1000 to 1200 mg/day by weight.

Pegylated interferon α-2a (Pegasys ^®) based recombinant interferon α-2a with one single branched bis (monomethoxy) polyethylene glycol (PEG) chains covalent complex. The dose of Peg-IFN alpha 2a administered subcutaneously per week was 180 micrograms and was provided as a prefilled single-use syringe (180 micrograms per 0.5 milliliter).

Research design

A total of 90 untreated chronic HCV genotype 1 patients were included and stratified by age and viral load. After a random assignment of 1:2, 31 patients were enrolled in control arm A (PegIFN/RBV); 59 patients were enrolled in arm C, and 12 weeks before the start of PegIFN/RBV therapy, Received 10 to 13 TG4040 (10 ⁷ plaque forming units) injection.

In the C arm, according to the following process stage, for patients administered a dose of 10 ⁷ units plaques formed TG4040: six weeks a total of six subcutaneous injection once a week effect, followed by four to seven per Subcutaneous injection once every 4 weeks. The injection is performed alternately on the left and right thighs or arms. The SOC was started and provided for 48 weeks after the first 12 weeks of TG4040 (corresponding to 7 TG4040 injections). Overall, patients with arm C received 33 weeks of TG4040 and 48 weeks of SOC. After 12 weeks from the start of the SOC, patients who continue to receive TG4040 plus SOC if they reach cEVR or fail to achieve it. At any time, it is proposed to continue to administer TG4040 every 4 weeks for up to 48 weeks for patients who cannot tolerate and discontinue SOC. It should be noted that in order to follow the safety correction, the TG4040 injection was stopped during the trial; the patient received an average of 12 injections.

In the arm A of the control group, the patient received the SOC for 48 weeks, and the SOC was administered subcutaneously with a dose of 180 μg of pegylated interferon α-2a (Pegasys ^® ) once a week and orally administered 1000 to 1200. A mg/day by weight dose of Copegus ^® lozenge. After 12 weeks of SOC, patients who achieve EVR (defined as a reduction in viral load (VL) greater than or equal to 2 log ₁₀ IU/mL) will continue for 36 weeks of SOC without experiencing EVR failure. The patient will stop taking SOC treatment. At any time, it is proposed to start TG4040 once a week for a patient who cannot tolerate and stop SOC for a total of 6 weeks, followed by subcutaneous injection every 4 weeks and up to 48 weeks (to Based on the arm C period).

All patients were followed up for up to 24 weeks after treatment or after discontinuation of all study medications.

method

Tracking items included safety over time and baseline, immune parameter assessment, and HCV-RNA monitoring.

By assessing adverse events (AE) and serious adverse events (SAE) Safety was assessed by the overall incidence and for laboratory evaluations of each arm and for the study as a whole.

Viral assessment was performed by quantitative analysis of serum hepatitis C virus (HCV) ribonucleic acid (RNA) on serum samples using Roche's TaqMan® analysis. The detection limit is 10 IU/ml serum. Estimate viral load at different endpoints, especially after 2 weeks of SOC (evaluation of the percentage of patients achieving hyperviral response (UVR)), 4 weeks after SOC (evaluation of disease with rapid viral response (RVR)) Percentage of illness), after 12 weeks of SOC (to assess the percentage of patients with early virologic response (EVR) and complete EVR (cEVR)), and for 24 weeks at SOC (the week when the suspension rule may be used) After, at the end of SOC treatment (to assess the percentage of patients who achieved treatment response (ETR)), 12 and 24 weeks after the end of SOC treatment (evaluation of continuous viral response at 12 and 24 weeks after SOC treatment (SVR12) Percentage of patients with SVR24). In addition, assess the slope of viral clearance at different stages (eg between baseline and week 1 of SOC, between baseline and week 4, and at week 4 and week 12) between).

The kinetics of the initiation and duration of the immune response were assessed at different time points.

In arm A: at the time of screening, at baseline, at week 2, week 12, week 24 of SOC therapy, and 24 weeks after the end of SOC (ie, at week 72).

In arm C: at screening, at baseline, and at week 9 (ie, 4 weeks after TG4040 injection once every six weeks), at 12 weeks (before the start of the SOC), 14th week (that is, 2 weeks after the start of the SOC), 24th week (that is, 12 weeks of SOC), and the 36th week (that is, SOC 24 weeks) and in the 84th week (24 weeks after the end of the SOC).

Enzyme-linked immunospot (ELISpot) IFN- is used in the test tube by using the antigen derived from the vaccine and the HCV reagent not related to the vaccine (the fifteen peptide overlapping 11 amino acids will be used) for PBMC re-stimulation. γ explores cellular immune parameters with HCV specificity. Other immune and inflammatory parameters can be analyzed to further obtain details of the immune pathway involved in the mechanism of action of TG4040, such as by analyzing soluble mediators including interleukins in serum, analyzing anti-MVA antibodies, and analyzing cells with MVA specificity. immune response. The total anti-MVA antibody (plasma IgG) and/or the neutralizing ability of these antibodies were evaluated by ELISA and by inhibiting the infection of BHK21 cell line in the test tube by MVA-GFP, respectively. At each time point, 5 ml of blood was drawn for this purpose. The cell-mediated reaction against MVA was measured by enzyme-linked immunospot (ELISpot) IFN-γ. The soluble medium in the serum was evaluated by ELISA/Raminex technique. For immunological evaluation, a total of 45 ml of blood was drawn: 5 ml for measuring the hormonal immune response and for the soluble medium, and 40 ml for measuring the cellular immune response.

Statistical Analysis

The study was an independent measure of the cEVR rate that can be assessed in patients. Based on the assumption of a three-stage design a result, the number of samples to determine: - cEVR nothing to the reaction rate (r) in terms of hypothesis H ₀ is less than 40% based r; - antagonistic effect hypothesis H _A line r is greater than 60%; - the probability of a false positive result is equal to α = 2.5%, the probability of a false negative result is equal to β = 10%; - the last evaluable patient set (53 in arm C), if at 25 or more If cEVR is observed in fewer patients, the null hypothesis is accepted; if cEVR is observed in at least 29 patients, the null hypothesis is rejected; based on the literature, 40% is chosen as the threshold.

result

The included patients were assigned according to Table 1.

Initial data showed that of the 59 patients who received at least one TG4040 injection in arm C, 27 showed a reduction in viral load at one or more time points of more than 0.5 log ₁₀ IU/mL, and a reduction The range is from 0.50 to over 2 log ₁₀ IU/ml, which is consistent with the results of Phase I test. Interestingly, this antiviral effect lasted for several weeks in responding patients.

After the initiation of SOC in arm C, a significant contrast and unexpectedly, a high proportion of patients were administered in 10 TG4040 doses (six per week prior to SOC initiation with 1 per dose) Serum HCV RNA undetectability (or cEVR) (less than 10 IU/mL) was achieved after a monthly dosing and 3 monthly dosings during the 12-week SOC period. Under the SOC, patients with arm C also began to show a sharp decrease in their viral load at an early stage.

Preliminary data includes the achievement of the primary endpoint (see Figure 2 and Figure 3)

A positive effect of TG4040 pre-vaccination on viral inhibition was observed as early as one week after SOC initiation: the slope of viral load decline in arm C was significantly greater, resulting in a decrease of 1.4 log ₁₀ IU/mL , compared to 0.9 log ₁₀ IU/mL in arm A. After 1 week and 2 weeks of SOC administration, the proportion of patients who had reduced more than 1 log ₁₀ IU/mL in arm C were 56% and 67%, respectively, compared to 35% and 58% in arm A ( Reaction in the second week).

After administration of SOC in arms A and C for 4 weeks or 5 weeks, respectively, the proportion of patients who had reached HCV RNA undetectability (RVR response) in arm C was 23.6% (13 out of 55 patients) Name), relative to 6.5% in arm A (2 out of 31 patients).

Of the 31 and 59 patients enrolled in Arm A and Arm C, 30 and 53 patients were eligible for cEVR assessment. After 12 weeks of SOC administration, the proportion of patients who had reached cEVR in arm C was 64% (34 out of 53 patients), compared to 30% in arm A. Between arm A and arm C The cEVR ratio difference is statistically different (p=0.003 for chi-square test).

When considering patients who have started SOC treatment (ITT population), the proportion of patients who have reached undetectableness in arm C after 7 weeks of SOC is 76.4% (42 out of 55 patients). 67.7% compared to arm A (21 out of 31 patients). The advantage of pre-vaccination of arm C was also observed during ETR assessment, compared to 64% of arm A (20 out of 31 patients), arm C was 69% (38 out of 55 patients) ).

In summary, the data confirms the efficacy of the exemplified therapeutic regimen that combines multiple repetitive TG4040 administrations with SOC and provides TG4040 prior to SOC initiation. The primary objective of the study was to achieve both cEVR and a good balance in terms of IL-28B polymorphism distribution (about 70% non-C-C per arm) in both the evaluable population of arm C and the ITT population.

Although the effect exhibited by viral load on TG4040 alone was significant but limited (1st case), Phase II studies in the second case showed that when TG4040 was used with PEG-IFN and ribavirin therapy Shows powerful synergies. There was no indication that this mode of administration caused a deterioration in liver enzyme levels, and fluctuations in grade 1 and reversibility of these enzymes were observed during TG4040 pre-vaccination. Two safety events were observed in two patients in arm C, possibly due to the worsening of the IFN effect when TG4040 was added to the composition.

These results demonstrate the benefits of TG4040 as an active immunotherapy or cocktail with antiviral compounds and especially antiviral agents that do not contain IFN.

<110> Chuan Sijian Company

<120> Immunotherapy composition and treatment for hepatitis C virus infection (2)

<130> 361604D31579

<150> EP 11306248.3

<151> September 29, 2011

<150> EP 11306433.1

<151> November 4, 2011

<150> EP 11306441.4

<151> November 07, 2011

<160> 4

<170> Patent Application Software Version 3.5

<210> 1

<211> 947

<212> PRT

<213> Hepatitis C virus

<400> 1

<210> 2

<211> 592

<212> PRT

<213> Hepatitis C virus

<400> 2

<210> 3

<211> 2844

<212> DNA

<213> Hepatitis C virus

<400> 3

<210> 4

<211> 1779

<212> DNA

<213> Hepatitis C virus

<400> 4

Claims (42)

An immunotherapeutic composition comprising a therapeutically effective amount of an active ingredient selected from the group consisting of: - a polyprotein NS3/NS4 of hepatitis C virus and a hepatitis C virus polypeptide NS5B - a performance vector comprising a nucleotide sequence encoding a polyprotein NS3/NS4 of hepatitis C virus and a nucleotide sequence of a polypeptide NS5B encoding hepatitis virus; - comprising a polyprotein NS3 encoding hepatitis C virus An expression vector of one of the nucleotide sequences of NS4 and a expression vector comprising one of the nucleotide sequences of the polypeptide NS5B encoding hepatitis virus; as a medicament for treating an HCV infection, wherein the administration mode of the agent includes Four to eight administrations of the immunotherapeutic composition are varied from 3 to 10 days apart, and finally 4 to 15 administrations of the immunotherapeutic composition ranging from 3 to 5 weeks apart. An immunotherapy composition as used in claim 1, wherein the NS3/NS4 polyprotein and the NS5B polypeptide or the nucleotide sequence encoding the NS3/NS4 polyprotein and the NS5B polypeptide are derived from the same HCV genotype Preferably, it is derived from the first genotype, and more preferably from the first genotype. An immunotherapy composition as used in claim 2, wherein the amino acid sequence contained in the NS3/NS4 polyprotein is at least 80% identical to the amino acid sequence shown in the sequence identification number: . An immunotherapy composition as used in claim 2, wherein the NS5B polypeptide comprises an amino acid sequence and sequence identification number: 2 The identity of the amino acid sequences shown is at least 80%. An immunotherapy composition as used in claim 3, wherein the nucleotide sequence encoding the NS3/NS4 polyprotein comprises a nucleotide sequence identical to the nucleotide sequence shown in SEQ ID NO: 3 Sexuality is at least 80%. An immunotherapy composition as used in claim 4, wherein the nucleotide sequence contained in the nucleotide sequence encoding the NS5B polypeptide is at least in conformity with the nucleotide sequence shown in SEQ ID NO: 4 80%. An immunotherapy composition for use according to any one of claims 1 to 6, wherein the expression vector is a plastid vector or a viral vector. An immunotherapy composition for use in claim 7 wherein the viral vector is a poxvirus vector, preferably selected from the group consisting of Western preserved strains, Copenhagen strains, Wyeth strains, and modified strains. A vaccinia virus vector of the group consisting of the Ankara (MVA) strain. An immunotherapy composition as used in claim 8 wherein the immunotherapeutic composition comprises an MVA expression vector and is inserted in the same orientation in its genome and preferably in deletion III: (a) encoding NS3/ a nucleotide sequence of one of the NS4 polyproteins under the control of the vaccinia pH5R promoter; and (b) a nucleotide sequence encoding one of the NS5B polypeptides under the control of the vaccinia p7.5K promoter. An immunotherapy composition for use according to any one of claims 1 to 9, wherein the immunotherapeutic composition further comprises a pharmaceutically acceptable Accepted carrier. An immunotherapeutic composition for use as claimed in any one of claims 1 to 10, which is suitable for parenteral administration, and in particular intradermal, subcutaneous, subcutaneous or intramuscular routes. The use of a patent application in any range of 8 to 11 immunotherapy composition, wherein the dosage for administration to the individual lines of the composition comprising vaccinia virus containing about 5x10 ⁶ th plaques forming units (PFU) to about 10 ⁸ plaques forming units (PFU) of MVA vector or a poxvirus, preferably about 5x10 ⁶ th plaques forming units (PFU) to about 5x10 ⁷ plaque forming units th solution (PFU); and more preferably an amount of about 10 ⁷ plaque forming units (pfu). An immunotherapy composition for use according to any one of claims 1 to 12, wherein the mode of administration comprises 4 to 8 administrations of the immunotherapeutic composition separated from each other for about one week, and then separated from each other It takes about 4 to 15 administrations a month. An immunotherapy composition as used in claim 13 of the patent application, wherein the mode of administration comprises six administrations once a week, followed by five to twelve monthly administrations; more preferably six per Once-a-week administration, followed by six to eleven monthly dosings; and even better six six-weekly dosing, followed by seven monthly dosings. An immunotherapy composition for use according to any one of claims 1 to 14, wherein the immunotherapy composition is used in combination with an antiviral therapy. An immunotherapeutic composition for use in claim 15 wherein the antiviral therapy comprises providing one or more antiviral compounds, the disease resistance The toxic compound is selected from the group consisting of interferon, ribavirin, NS3/4A protease inhibitor, NS5A inhibitor, NS5B polymerase inhibitor, nucleoside analog, nucleotide analog, immunomodulator and host cell. A group of enzyme inhibitors or any functional analog thereof. An immunotherapeutic composition for use in claim 16 wherein the one or more antiviral compounds are interferon and/or ribavirin and optionally an NS3/4A protease inhibitor. An immunotherapy composition for use in claim 17 wherein the interferon is interferon-α (IFNα), preferably pegylated interferon-α and more preferably pegylated interferon-α2a Or pegylated interferon-α2b or interferon λ. An immunotherapy composition as used in claim 18, wherein the pegylated interferon-α is provided by an intramuscular or subcutaneous route, and the amount of pegylated interferon-α2a is about 180 μg per week. The amount of pegylated interferon-α2b is about 1.5 micrograms per kilogram per week. An immunotherapeutic composition for use in claim 18, wherein the ribavirin is provided orally and the dosage is from about 1000 to about 1200 mg per day. An immunotherapeutic composition for use in claim 16 or 17, wherein the NS3/4A protease inhibitor is provided orally and the dosage is about 2250 to 2400 mg per day. An immunotherapy composition for use according to any one of claims 17 to 21, wherein the interferon is simultaneously provided to the individual for 24 to 48 weeks, and preferably for 48 weeks. week. An immunotherapy composition for use according to any one of claims 15 to 17 and 20 to 22, wherein the antiviral therapy system does not contain an interferon. An immunotherapeutic composition for use according to any one of claims 15 to 23, wherein the immunotherapeutic composition is administered at least 4 weeks prior to providing antiviral therapy, advantageously at least 8 weeks ago, ideally At least 10 weeks ago and preferably at least 12 weeks ago. An immunotherapeutic composition for use according to claim 24, wherein the mode of administration comprises administering six times a week prior to providing antiviral therapy and then performing a monthly immunotherapy composition administration, antiviral The therapy is provided about 12 weeks after the first immunotherapy composition is administered. An immunotherapy composition for use according to any one of claims 15 to 25, wherein the immunotherapy composition is administered periodically for a period of 18 to 24 weeks after the initiation of the antiviral therapy, and Preferably it is about 21 weeks. An immunotherapy composition for use according to any one of claims 24 to 26, wherein the mode of administration comprises 13 administrations of the immunotherapeutic composition, preferably at 0, 1, 2, 3, 4 , 5, 9, 13, 17, 21, 25, 29, and 33 weeks, and interferon and ribavirin were provided during the 12th week. Use according to any one of the 15 to 27 range as patent immunotherapy composition, wherein the composition comprises immunotherapy encoding NS3 / NS4 and NS5B proteins Multi one polypeptide MVA vector and to train comprising from about 5x10 ⁶ to about 1 x 10 doses of ⁸ plaque forming units (pfu) administered to the individual by subcutaneous route; the interferon is a pegylated IFNα2 provided by intramuscular or subcutaneous route; and the antiviral compound is administered orally Way to provide Rebaillin. An immunotherapy composition for use in claim 28, wherein the immunotherapy composition comprises about 107 plaque forming units (pfu) of MVA vector, and one of the NS3/NS4 fusion proteins is included in the genome thereof. a nucleotide sequence, wherein the amino acid sequence contained in the NS3/NS4 fusion protein is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 1, and comprises a nucleoside encoding one of the NS5B polypeptides. An acid sequence, wherein the amino acid sequence contained in the NS5B polypeptide is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 2; the interferon is pegylated interferon α2a, The individual is provided once a week by an amount of about 180 micrograms per week and by subcutaneous route; and the ribavirin is provided orally in an amount of from 1000 to 1200 mg per day. A therapeutic procedure comprising the step of administering to a subject an immunotherapeutic composition for use as claimed in any one of claims 1 to 29. The therapeutic treatment of claim 30, wherein the treatment is performed in combination with an antiviral therapy as defined in any one of claims 15 to 29 of the patent application. An immunotherapy composition as used in any one of claims 15 to 29 or a therapeutic treatment as claimed in claim 30 or 31, wherein a fast virus is achieved compared to an antiviral therapy providing only 4 weeks The proportion of individuals in response (RVR) is increased by at least 5%, and preferably by at least 10%. An immunotherapy composition as used in any one of claims 15 to 29 or a therapeutic treatment as claimed in claim 30 or 31, wherein an early virus is achieved compared to an antiviral therapy providing only 12 weeks The proportion of individuals who are learning to respond (EVR) or fully early viral response (cEVR) is increased by at least 5%, and preferably by at least 10%. An immunotherapy composition as used in any one of claims 15 to 29 or a therapeutic treatment as claimed in claim 30 or 31, wherein compared to an antiviral therapy providing only 24 or 48 weeks The proportion of individuals with a sustained viral response (SVR) is increased by at least 5%, and preferably by at least 10%. An immunotherapy composition for use in any one of claims 1 to 29 and 32 to 34, or a treatment course according to any one of claims 30 to 34, for initiating or stimulating an individual The immune system is superior to the baseline state or superior to the use of only the intended state of the antiviral therapy. An immunotherapeutic composition for use in any one of claims 1 to 29 and 32 to 35 or a therapeutic treatment according to any one of claims 30 to 35 for treating a chronically infected individual Use. For example, the immunotherapy composition used in claim 36 of the patent application or the treatment course of claim 36 of the patent application, wherein the system chronically infects the first genotype HCV. An immunotherapy composition as used in claim 36 or 37 or a therapeutic treatment as claimed in claim 36 or 37 for use in a body which has not been approved prior to initiating the mode of administration Any conventional interferon alpha compound treatment for the treatment of HCV infection. The immunotherapy composition as used in any one of claims 36 to 38, or the therapeutic treatment according to any one of claims 36 to 38, wherein the individual has a high viral titer at baseline. A kit for treating HCV infection and preferably chronic HCV infection, wherein the kit comprises a plurality of containers and is used for administering one body as claimed in claims 1 to 29 and items 32 to 39 An immunotherapy composition for use or a method according to the treatment course of any one of claims 30 to 39. A kit of claim 40, comprising: a) an immunotherapeutic composition as defined in any one of claims 1 to 29 and 32 to 39; b) an antiviral compound such as Interferon and ribavirin; and c) instructions for administering each of (a) and (b). A method that achieves a proportion of individuals achieving complete early viral response (cEVR) in a population of individuals chronically infected with hepatitis C virus (HCV) compared to cEVR in a population of individuals chronically infected with HCV and treated with only antiviral therapy. Increasing at least 5% and preferably by at least 10%, the method comprising administering to the individual population an immunotherapeutic composition as used in any one of claims 1 to 29 and 32 to 39, or as claimed The course of treatment of any one of items 30 to 39, which elicits or stimulates a T cell-mediated immune response against one or more epitopes present in the HCV NS3, NS4 and/or NS5B polypeptide.