yeast-based compositions and methods for treating or preventing infection by the hepatitis delta vir

专利摘要:
IMMUNOTHERAPY COMPOSITIONS BASED ON YEAST AND USES OF THE SAME. The present invention relates to immunotherapeutic compositions that elicit a specific immune response to the hepatitis delta virus (HDV) when administered to a subject, wherein the compositions comprise (a) a yeast vehicle; and (b) a fusion protein comprising HDV antigens. The present invention also relates to uses of said compositions in the preparation of medicaments to treat HDV infection or to immunize a population of individuals against HDV.
公开号:BR112013032381B1
申请号:R112013032381-7
申请日:2012-06-14
公开日:2021-01-12
发明作者:Thomas H. King；David Apelian
申请人:Globeimmune, Inc.；
IPC主号:

专利说明:

[0001] [0001] This application claims priority benefit, in accordance with 35 USC § 119 (e), of provisional U.S. Patent Application No. 61 / 497,039, filed on June 14, The entire exposition of the northern patent application American Provisional Order No. 61 / 497.039 is hereby incorporated by reference. REFERENCE TO A SEQUENCE LISTING
[0002] [0002] This order contains a Sequence Listing submitted electronically as a text file via EFS-Web. The text file, called "3923-39-PCT_ST25", has a size in bytes of 66 KB and was recorded on June 12, 2012. The information contained in the text file is incorporated by reference in its entirety based on at 37 CFR § 1.52 (e) (5). FIELD OF THE INVENTION
[0003] [0003] The present invention relates generally to immunotherapeutic compositions and methods for the prevention and / or treatment of hepatitis delta virus (HDV) infection. BACKGROUND OF THE INVENTION
[0004] [0004] Hepatitis D is a disease caused by infection with a small circular envelope RNA virus known as the hepatitis delta virus (HDV). HDV was first discovered in 1977 (Rizzetto et al., Gut 1977; 18: 997-1003), and was later shown to be the infectious agent of a new form of hepatitis (Rizzetto et al., J Infect Dis 1980; 141 : 590-602; Rizzetto et al., Proc Natl Acad Sci USA 1980; 77: 6124-8; Wang et al., Nature 1986; 323: 508-14; Mason et al., In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA, eds. Eight Report of the International Committee on Taxonomy of Viruses, London: Elsevier / Academic Press, 2005; 735-8). It has recently been estimated that 15 - 20 million people are infected with HDV, which requires concomitant infection with the hepatitis B virus (HBV) in order to get a life cycle, although this number may be underrepresented due, in part , the lack of systematic screening for HDV infection in individuals infected with HBV (Pascarella and Negro, Liver International 2011,31: 7-21).
[0005] [0005] Hepatitis D viruses are spherical particles that contain a nucleus structure formed by an HDV genomic RNA that forms a complex of about 70 HDAg molecules (both small and large) (Ryu et al., J Virol 1993; 67: 32817). HDV is believed to enter the cell using the same receptor as HBV, using the HBV envelope proteins as its outer shell. The shell consists of approximately 100 copies of HBV surface antigen proteins (small, medium and large HBsAg). Large HDAg and HBsAg are sufficient to form viral particles, which are not infectious, unless HDV RNA is also included, and small HDAg increases the packaging efficiency of the virus (Chen et al., J Virol 1992; 66 : 2853-9; Wang et al., J Virol 1994; 68: 6363-71).
[0006] [0006] Once inside the cell, HDV uses host cell RNA polymerases. Three RNAs accumulate during viral replication processes. The HDV genome is a circular negative single-stranded RNA of about 1,672 - 1,697 nucleotides (Radjef et al., J Virol 2004; 78: 2537-44) and contains a ribozyme domain, which comprises nucleotides 680 - 780, and a suspected HDAg RNA promoter site (Beard et al., J Virol 1996; 70: 4986-95). The antigenome, which contains the open reading frame encoding HDAg and a ribozyme domain (Sharmeen et al., J Virol 1988; 62: 2674-9; Ferre-D'amare et al., Nature 1998; 395: 567-74 ) is the perfect complement of the genome, and its replication occurs through RNA-directed RNA synthesis without any DNA intermediates (Chen et al., Proc Natl Acad Sci USA 1986; 83: 8774-8). The mRNA drives the synthesis of HDAg.
[0007] [0007] There is only one known protein encoded by the HDV genome, and it consists of two forms, a large HDAg (L) of 27 kDa (HDAg-L or L-HDAg) (214 amino acids) and a small HDAg (S) 24 kDa (HDAg-S or S-HDAg) (195 amino acids). Proteins differ by about 19 amino acids at the C-terminus of the large HDAg. The N-terminus of the HDV antigen is responsible for signaling nuclear location, the middle domain of the HDV antigen is responsible for the binding of RNA, and the C-terminus is involved in assembling the virion and inhibiting the assembly of the RNA. HDAg-S is produced in the early stages of viral infection and supports viral replication. HDAg-L is produced later in viral infection, inhibits viral infection and is required for the assembly of viral particles.
[0008] [0008] HDV infection occurs only in individuals who are co-infected with a different virus, the hepatitis B virus (HBV), and, more specifically, only in individuals positive for HBV surface antigen (HBsAg). As discussed above, HDV requires HBsAg from HBV for the formation and transmission of particles and therefore HBV is essential for the assembly and release of HDV virions. There are two main known ways in which HDV infects an individual. In the first, called co-infection, HDV and HBV can simultaneously co-infect an individual as an acute infection, and this type of infection results in about 95% recovery for most people, similar to recovery rates for infection only. acute by HBV. The second type of HDV infection, which is more common, is "superinfection", in which HDV sharply infects an individual who already has chronic HBV infection. In this case, HDV infection progresses to chronic HDV infection in about 80 - 90% of individuals, and it is this chronic HDV infection that is the most serious form of the disease. Consequently, superinfection can be classified as the initial acute HDV superinfection of a chronic HBV carrier or the chronic posterior HDV infection. A third, but controversial, form of potential HDV infection, called latent helper-independent infection, was first reported in 1991 and has been described as occurring during a liver transplant (Ottobrelli et al., Gastroenterology 1991; 101: 1649- 55). In this form of infection, a patient's hepatocytes could be infected only with HDV (for example, during a liver transplant, when HBV transmission is prevented by administering hepatitis B immunoglobulins), but if residual HBV escapes neutralization , or the patient is otherwise exposed to HBV subsequently, HDV infected cells can be "rescued". This form has been demonstrated in animal models, but this infection of human hepatocytes remains controversial.
[0009] [0009] Chronic hepatitis D is currently considered to be the most severe form of viral hepatitis in humans (for a detailed review of the disease and associated HDV, see Grabowski and We-demeyer, 2010, Dig. Disease 28: 133-138 ; or Pascarella and Negro, Liver International 2011, 31: 7-21). Individuals chronically infected with HDV have an accelerated progression to fibrosis, an increased risk of hepatocellular carcinoma and early decompensation in the establishment of cirrhosis. The disease can be asymptomatic or present with non-specific symptoms, and the diagnosis can occur only when complications appear in the cirrhosis stage of the disease. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are persistently elevated in most patients and can be used to monitor the disease. In 5 - 10 years, up to 70 - 80% of patients with chronic hepatitis D may develop cirrhosis (Rizzetto et al., Ann Intern Med 1983; 98: 437-41; Govindarajan et al., Hepatology 1986; 6: 640-4 ) and 15% in 1 - 2 years (Saracco et al., J Hepatol 1987; 5: 274-81). HDV infection can also accelerate the development of hepatocellular carcinoma (HCC).
[0010] [00010] HDV infection is more prevalent in the Mediterranean basin, the Middle East, Central and North Asia, West and Central Africa, the Amazon basin, Venezuela, Colombia and certain Pacific islands, although the virus is present and / or arising in all over the world (for example, Russia, North India, Southern Albania, mainland China and some Pacific islands). HDV infection is transmitted parenterally, most typically through drug use or exposure to blood or blood products. Sexual transmission of HDV is less common, and perinatal transmission of the virus is rare.
[0011] [00011] Despite the mode of HDV infection, there is currently no good option for the treatment or prevention of HDV infection. Antiviral drugs used to treat other viruses that infect hepatocytes (for example, antiviral drugs for HBV or HCV) are not effective against HDV. Immunomodulatory drugs, such as corticosteroids or lemivasol, were not effective (Rizzetto et al., Ann Intern Med 1983; 98: 437-41; Arrigoni et al., Ann Intern Med 1983; 98: 1024), nor the thymus-derived peptides ( Rosina et al., Dig Liver Dis 2002; 34: 285-9; Zavaglia et al., J Clin Gastroenterol 1996; 23: 162-3). This leaves treatment with interferon (for example, pegylated interferon-α; pegIFN-α) as the only treatment currently approved for HDV infection. However, it is known that HDV can interfere with IFN-α signaling in vitro and, in fact, treatment of HDV with pegIFN-α suffers from treatment failures and low response rates. In a prospective trial, only 21% of patients achieved HDV RNA negativity, and only 26% had a biochemical response (Niro et al., Hepatology 2006; 44: 713-20), and similar results were obtained in others trials, in which a sustained response to therapy (cure) remains very low. Consequently, there is a need in the technique for new prophylactic and therapeutic approaches to HDV infection. SUMMARY OF THE INVENTION
[0012] [00012] One embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising HDV antigens. The composition elicits an immune response specific to HDV when administered to a subject.
[0013] [00013] In one aspect of this embodiment of the invention, HDV antigens consist of at least one immunogenic domain of a large HDV antigen (HDAg-L) or a small HDV antigen (HDAg-S), wherein the sequence of Nuclear localization (NLS) was inactivated by replacing or deleting one or more NLS amino acids. For example, an NLS can be inactivated by deleting the entire NLS, although the invention is not limited to this example.
[0014] [00014] In one aspect of this embodiment of the invention, the HDV antigen consists of at least one full-length HDAg-L or HDAg-S, except that the nuclear location sequence (NLS) of HDAg-L or HDAg-S has been inactivated by substitution or deletion of one or more NLS amino acids.
[0015] [00015] In another aspect of this embodiment of the invention, the HDV antigen consists of a fusion of two or more full-length HDAg-L or HDAg-S, except that the nuclear localization sequence (NLS) of each HDAg-L or HDAg-S was inactivated by replacing or deleting one or more NLS amino acids. For example, in one aspect, the NLS is deleted. In one aspect of this modality, each HDAg-L or HDAg-S is of a different HDV genotype.
[0016] [00016] In yet another aspect of this embodiment of the invention, the HDV antigen consists of a fusion of three full-length HDAg-L or HDAg-S, except that the nuclear localization sequence (NLS) of each HDAg-L or HDAg -S was inactivated by replacing or deleting one or more NLS amino acids. In one aspect of this modality, each HDAg-L or HDAg-S is of a different HDV genotype. In an example of this HDV antigen, a full-length HDAg-L genotype 1 sequence represented by SEQ ID NO: 2 (or a corresponding sequence from another HDV strain) with a deletion of positions 66 - 75 (the NLS) is linked to a full-length HDAg-L genotype 2 sequence represented by SEQ ID NO: 5 (or a corresponding sequence from another HDV strain) with a deletion of positions 66 - 75 (the NLS) which is linked to a sequence of HDAg-L genotype 3 of full length represented by SEQ ID NO: 8 (or a corresponding sequence from another HDV strain) with a deletion of positions 66 - 75 (the NLS). The arrangement of these three HDAg’s in the fusion protein can be modified in any order other than the above. In one respect, the three HDAg’s are of different HDV genotypes or subgenotypes in addition to those listed above.
[0017] [00017] In one aspect of this embodiment of the invention, the HDV antigen comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NO: 34, SEQ ID NO: 28, or a sequence of corresponding amino acid from another HDV strain. In one aspect, the HDV antigen comprises an amino acid sequence that is at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to an amino acid sequence selected from SEQ ID NO: 34 , SEQ ID NO: 28, or a corresponding amino acid sequence from another HDV strain. In one aspect, the HDV antigen is selected from SEQ ID NO: 34, SEQ ID NO: 28, or a corresponding sequence from another HDV strain.
[0018] [00018] In one aspect of this embodiment of the invention, the fusion protein has an amino acid sequence selected from SEQ ID NO: 36 or SEQ ID NO: 30 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 36 or SEQ ID NO: 30, respectively.
[0019] [00019] In yet another modality related to the immunotherapeutic composition of the invention, HDV antigens consist of at least one immunogenic domain of a large HDV antigen (HDAg-L) or small HDV antigen (HDAg-S), in that the HDV antigen is smaller than a full-length HDAg-L or HDAg-S protein. In one aspect, the HDV antigen consists of a fusion of at least two or more HDAg-L or HDAg-S proteins, where at least one of the HDAg-L or HDAg-S proteins is smaller than an HDAg-L or Full-length HDAg-S. In one respect, each HDAg-L or HDAg-S protein is of a different HDV genotype. In one aspect, the HDV antigen comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NO: 10, SEQ ID NO: 16, or a corresponding amino acid sequence from another HDV strain . In one aspect, the HDV antigen comprises an amino acid sequence that is at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical, to an amino acid sequence selected from SEQ ID NO : 10, SEQ ID NO: 16, or a corresponding amino acid sequence from another HDV strain. In one aspect, the HDV antigen is selected from SEQ ID NO: 10, SEQ ID NO: 16, or a corresponding sequence from another HDV strain.
[0020] [00020] In one aspect of this embodiment of the invention, the fusion protein is selected from SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 18.
[0021] [00021] In any of the above described embodiments of the invention, in one aspect, HDAg-L has an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or a sequence of corresponding amino acid from another HDV strain. In one aspect, HDAg-S has an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, or a corresponding sequence from another HDV strain. In one aspect, the HDV antigen comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or an amino acid sequence corresponding from another HDV strain.
[0022] [00022] In any of the above described modalities related to an immunotherapeutic composition of the invention, in one aspect, the HDV antigen is expressed by the yeast vehicle. In one aspect, the yeast vehicle is an entire yeast. In one respect, the entire yeast is dead. In one respect, the entire yeast is thermally inactivated. In one aspect, the yeast vehicle is from a yeast genus selected from the group consisting of: Saccharomyces, Candida, Cryptococcus, Hansenula, Kluyveromyces, Pi-chia, Rhodotorula, Schizosaccharomyces and Yarrowia. In one aspect, the yeast vehicle is Saccharomyces. In one aspect, the yeast vehicle is Saccharomyces cerevisiae.
[0023] [00023] In any of the above described embodiments related to an immunotherapeutic composition of the invention, in one aspect, the composition is formulated in a pharmaceutically acceptable excipient suitable for administration to a subject.
[0024] [00024] In any of the above described modalities related to an immunotherapeutic composition of the invention, in one aspect, the composition contains more than 90% yeast protein.
[0025] [00025] Another embodiment of the invention relates to a method for treating hepatitis D virus (HDV) infection or at least one symptom resulting from HDV infection in a subject or improving the survival of a subject who is infected with HDV. The method includes a step of administration to a subject who has been infected with HDV of at least one immunotherapeutic composition as described above or elsewhere here, where administration of the composition to the subject reduces HDV infection or at least one resulting symptom. of HDV infection in a subject. In one aspect, the method also includes administering to the subject one or more additional agents useful for treating or ameliorating a symptom of HDV infection. For example, this agent may include, but is not limited to, an interferon. Interferons include, but are not limited to, interferon-α, including, but not limited to, pegylated interferon-a2a. In one respect, interferon is interferon-λ.
[0026] [00026] In one aspect of this embodiment of the invention, the subject is chronically infected with hepatitis B virus (HBV). In one aspect, the method also includes a step of administering an antiviral compound to the subject to treat HBV infection. Such an antiviral compound may include, but is not limited to: tenofovir, lamivu-dine, adefovir, telbivudine, entecavir and their combinations.
[0027] [00027] Yet another embodiment of the invention relates to a method for eliciting a cell-mediated and antigen-specific immune response against an HDV antigen, comprising administering to a subject at least one immunotherapeutic composition as described above or elsewhere on here.
[0028] [00028] Another embodiment of the invention relates to a method for preventing HDV infection in a subject, comprising administering to a subject who has not been infected with HDV at least one immunotherapeutic composition as described above or elsewhere here. In one aspect of this modality, the subject is chronically infected with hepatitis B virus (HBV). In one aspect, the method also includes administering to the subject an antiviral compound to treat HBV infection.
[0029] [00029] Yet another embodiment of the invention relates to a method for immunizing a population of individuals against HDV, comprising administering to the population of individuals at least one immunotherapeutic composition as described above or elsewhere here. In one aspect of this embodiment of the invention, the population of individuals is chronically infected with HBV.
[0030] [00030] Another embodiment of the invention relates to an immunotherapeutic composition as described above or elsewhere here, for use in the treatment of HDV infection.
[0031] [00031] Yet another embodiment of the invention relates to an immunotherapeutic composition as described above or elsewhere here, for use in preventing HDV infection in a subject. In one aspect, the subject is chronically infected with HBV.
[0032] [00032] Another embodiment of the invention relates to the use of at least one immunotherapeutic composition as described above or elsewhere here in the preparation of a medicament to treat HDV infection.
[0033] [00033] Yet another embodiment of the invention relates to the use of at least one immunotherapeutic composition as described above or elsewhere in the preparation of a medicament to prevent HDV infection. BRIEF DESCRIPTION OF THE DRAWINGS OF THE INVENTION
[0034] [00034] Fig. 1 is a sequence alignment showing the parts of the HDAg used in the construct represented by SEQ ID NO: 16 aligned, to illustrate the homology between the genotypes used in this fusion protein (the "genotype1" sequence in the line 1 are positions 1 - 140 of SEQ ID NO: 16, the sequence of "genotype2" in line 2 is positions 141 - 280 of SEQ ID NO: 16; the sequence of "genotype-po3" in line 3 is positions 281-420 of SEQ ID NO: 16).
[0035] [00035] Fig. 2 is a scanned image showing the expression of the yeast-based immunotherapeutic known as HDV1 (which expresses SEQ ID NO: 30) and HDV2 (which expresses SEQ ID NO: 33), grown in U2 medium and UL2, and compared to a set of NS3-His standards.
[0036] [00036] Fig. 3 is a scanned image showing the expression of the yeast-based immunotherapeutic known as HDV1 (which expresses SEQ ID NO: 30) and HDV2 (which expresses SEQ ID NO: 33), grown in U2 medium and UL2, and compared to a second set of NS3-His standards.
[0037] [00037] Fig. 4 is a scanned image showing the expression of the yeast-based immunotherapeutic known as HDV3 (which expresses SEQ ID NO: 36) grown in U2 and UL2 media.
[0038] [00038] Fig. 5A is a graph showing an ELISpot of interferon-γ (IFNy) from mice C57BL / 6 vaccinated with the HDV1 and HDV3 yeast-based HDV immunotherapeutic compositions using the HDV-P2 peptide (OVAX = yeast from control that expresses irrelevant antigen).
[0039] [00039] Fig. 5B is a graph showing the same ELISpot results as Fig. 5A, with the bottom of the medium subtracted.
[0040] [00040] Fig. 6A is a graph showing an ELISpot of interleukin-2 (IL-2) from C57BL / 6 mice vaccinated with the immunotherapeutic composition for HDV-1 using the HDV-P1 peptide (OVAX = control yeast that expresses irrelevant antigen).
[0041] [00041] Fig. 6B is a graph showing the same ELISpot results as Fig. 6A, with the bottom of the medium subtracted. DETAILED DESCRIPTION OF THE INVENTION
[0042] [00042] This invention relates generally to compositions and methods for the prevention and / or treatment of hepatitis delta virus (HDV) infection. The invention includes a yeast-based immunotherapeutic composition (also called yeast-based HDV immunotherapy) comprising a yeast vehicle and HDV antigen (s) that have been designed to elicit a prophylactic and / or therapeutic immune response against HDV infection in a subject, and the use of these compositions to prevent and / or treat HDV infection. The invention also includes the recombinant nucleic acid molecules used in the yeast-based compositions of the invention, as well as the proteins encoded by them, for use in any immunotherapeutic composition and / or any therapeutic or prophylactic protocol for HDV, including any therapeutic protocol or prophylactic combination of the HDV-specific yeast-based compositions of the invention with any one or more other therapeutic or prophylactic compositions, agents, drugs, compounds and / or protocols for HDV and / or related co-infections.
[0043] [00043] Yeast-based immunotherapeutic compositions are administered as biological agents or pharmaceutically acceptable compositions. Therefore, instead of using yeast as an antigen production system, followed by purification of the yeast antigen, the entire yeast vehicle as described herein must be suitable for, and formulated for, administration to a patient. This contrasts with the use of yeast to produce recombinant proteins for subunit vaccines, in which the proteins, once expressed, are subsequently released from the yeast by rupture and purified from the yeast, so that the final vaccine combined with an adjuvant does not contain any. Detectable yeast DNA and contains a maximum of 1 - 5% yeast protein. The yeast-based immunotherapeutic compositions for HDV of the invention, on the other hand, contain readily detectable yeast DNA and contain substantially more than 5% yeast protein (i.e., more than 5% of the total protein in the vaccine belongs to or is contributed by yeast); generally, yeast-based immunotherapies of the invention contain more than 70%, more than 80%, or generally more than 90% yeast protein.
[0044] [00044] Yeast-based immunotherapeutic compositions are administered to a patient to immunize the patient for therapeutic and / or prophylactic purposes. In one embodiment of the invention, yeast-based compositions are formulated for administration in a pharmaceutically acceptable excipient or formulation. The composition must be formulated, in one aspect, to be suitable for administration to a human subject (for example, the manufacturing conditions must be suitable for use in humans, and any excipients or formulations used to finish the composition and / or prepare it) the dose of the immunotherapy for administration must be suitable for use in humans). In one aspect of the invention, yeast-based immunotherapeutic compositions are formulated for administration by injection into the patient or subject, such as via a parenteral route (for example, by subcutaneous, intraperitoneal, intramuscular or intradermal injection, or another suitable parenteral route).
[0045] [00045] In one embodiment, the yeast expresses the antigen (for example, detectable by a Western blot), and the antigen is not aggregated in the yeast, the antigen does not form inclusion bodies in the yeast and / or does not form virus-like particles (VLPs) or other large antigen particles in yeast. In one embodiment, the antigen is produced as a protein soluble in yeast and / or is not secreted from yeast or not substantial or mainly secreted from yeast. Yeast-based immunotherapies must be promptly phagocyted by dendritic cells of the immune system, and the yeast and antigens readily processed by these dendritic cells, to elicit an effective immune response against HDV. Hepatitis D Virus Antigens, Constructs and Compositions of the Invention
[0046] [00046] One embodiment of the present invention relates to a yeast-based immunotherapeutic composition that can be used to prevent and / or treat HDV infection and / or to alleviate at least one symptom resulting from HDV infection. The composition comprises: (a) a yeast vehicle; and (b) one or more HDV antigens comprising HDV protein (s) and / or its immunogenic domain (s), as described in detail herein. Together with the yeast vehicle, HDV antigens are more typically expressed as recombinant proteins by the yeast vehicle (for example, by an intact yeast or yeast spheroplast, which can optionally be further processed into a yeast cytoplasm , yeast phantom or yeast membrane extract or its fraction), although it is an embodiment of the invention that one or more of these HDV antigens are loaded in a yeast vehicle or otherwise in complex with, attached to, mixed with or administered with a yeast vehicle as described herein to form a composition of the present invention. According to the present invention, reference to a "heterologous" protein or "heterologous" antigen, including a heterologous fusion protein, with respect to a yeast vehicle of the invention, means that the protein or antigen is not a protein or antigen that is naturally expressed by yeast, although a fusion protein that includes heterologous antigen or heterologous protein may also include yeast sequences or proteins or parts thereof that are also naturally expressed by yeast (for example, a pre-pro sequence of alpha factor as described here).
[0047] [00047] One embodiment of the invention relates to HDV antigens usable in an immunotherapeutic composition of the invention and, in one aspect, in a yeast-based immunotherapeutic composition of the invention. As discussed above, there is only one known protein encoded by the HDV genome, and it consists of two forms: a large HDAg of 27 kDa (L) (HDAg-L or L-HDAg) (214 amino acids) and a small HDAg of 24 kDa (S) (HDAg-S or S-HDAg) (195 amino acids). Proteins differ by about 19 amino acids at the C-terminus of the large HDAg.
[0048] [00048] Although at least eight distinct HDV genotypes are currently known (Le Gal et al., Emerg Infect Dis 2006; 12: 1447-50), three genotypes are the most prevalent, known as 1 (or I) 2 (or II), and 3 (or III). The genotype (s) can (s) be determined in an individual by routine methods (for example, restriction fragment length polymorphism (RFLP), analysis of polymerase chain reaction products (PCR), sequencing and / or immunohistochemical staining). Highly conserved domains among HDV genotypes are located around the genetically and antigenically clinically autocatalytic RNA sites and the HDAg RNA-binding domain (Chao et al., Virology 1990; 178: 384-92; Wu et al ., Hepatology 1995; 22: 1656-60).
[0049] [00049] Genotype 1 is currently the most dominant HDV genotype and is found worldwide, but particularly in Europe, North America, Africa and some Asian regions. An example of a genotype 1 HDV genome is represented by Database Accession No. AF104263 or GI: 11022740, also represented here by SEQ ID NO: 1. SEQ ID NO: 1 encodes an HDAg-L represented by SEQ ID NO: 2 (also under Accession No. AAG26087.1 or GI: 11022742) and an HDAg-S represented by SEQ ID NO: 3. Genotype 2 is found in Japan, Taiwan and Russia. An example of a partial genome of HDV genotype 2 is represented by Database Accession No. AJ309880 or GI: 15212076, also represented here by SEQ ID NO: 4. SEQ ID NO: 4 encodes an HDAg-L represented by SEQ ID NO: 5 (also under Accession No. CAC51366.1 or GI: 15212077) and an HDAg-S represented by SEQ ID NO: 6. Genotype 3 is found in South America (for example, Peru, Colombia, Venezuela). An example of a genotype 3 HDV genome is represented by Database Accession No. L22063.1 or GI: 410182, also represented here by SEQ ID NO: 7. SEQ ID NO: 7 encodes an HDAg-L represented by SEQ ID NO: 8 (also under Accession No. P0C6M3.1 or GI: 226737601) and an HDAg-S represented by SEQ ID NO: 9. Genotype 4 is found in Japan and Taiwan, and genotypes 5 - 8 are found in Africa. Several HDAg sequences have been described for these genotypes and can be found in public databases. Multiple genotypes can infect a single patient, although one genotype typically dominates.
[0050] [00050] The nucleic acid and amino acid sequence for many HDV genomes and the HDAg proteins (large and small) encoded by them are known in the art for each of the known genotypes. The sequences described above are exemplary (representative). It should be noted that small variations in the amino acid sequence can occur between different viral isolates of the same protein or domain of the same HDV genotype. However, the HDAg antigen has essentially the same global structure between different strains and genotypes, so that those skilled in the art can readily determine from a given HDAg sequence the corresponding HDAg sequence of a strain / isolate or HDV genotype. different. Consequently, using the guidance presented here and the reference to exemplary HDV sequences, those skilled in the art will readily be able to produce various HDV-based proteins, including fusion proteins, of any strain (isolated) or HDV genotype, for use in the compositions and methods of the present invention, and therefore the invention is not limited to the specific sequences presented herein. Reference to an HDV protein or HDV antigen anywhere in this exposure, or to any functional, structural or immunogenic domain thereof, can therefore be made by reference to a particular sequence from one or more of the sequences presented in this exhibition, or by reference to the same, similar or corresponding sequence of a different HDV isolate (strain), including a different genotype or subgenotype of the reference isolate / strain.
[0051] [00051] Although the use of any of the full-length or near-full length HDV antigens (HDAg-L or HDAg-S) described herein is within the scope of the invention, additional HDV antigens are considered, particularly to optimize or increase the usefulness of HDV antigens as clinical products, including in the context of a yeast-based immunotherapeutic composition. HDV antigens that are useful in the present invention were designed to produce a yeast-based immunotherapy product for HDV that achieves one or more of the following purposes: (1) inclusion of a maximized number of known T cell epitopes (MHC Class I and MHC Class II); (2) maximizing or prioritizing the inclusion of immunogenic domains and, more particularly, T cell epitopes (CD4 + and / or CD8 + epitopes and dominant and / or subdominant epitopes), which are the most conserved among the genotypes and / or subgenotypes HDV, or that can be readily modified in a consensus sequence or included in two or more ways to cover the most important sequence differences between the target genotypes; (3) minimization of the number of unnatural junctions within the HDV antigen sequence in the product; (4) minimizing or eliminating sequences that may interfere with the expression of a protein by yeast (for example, hydrophobic domains); and / or (5) minimizing or eliminating sequences that may be essential for viral function (the nuclear localization sequence), for example, modifications to disable the virus. In one embodiment, HDV antigens can be designed to meet the guidelines of the Recombinant DNA Advisory Committee (RAC) of the National Institutes of Health (NIH).
[0052] [00052] In an embodiment of the invention, an HDV antigen usable in the invention consists of HDAg that are encoded by less than about: 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92 %, 91%, 90%, 85%, 80%, 75%, 70%, or 66.67% of an HDV genome, although multiple copies of the same HDAg that meet this parameter, or two or more different HDAg that meet the parameters, but each coming from a different genotype, subgenotype or HDV strain / isolate, can be provided in the form of a fusion protein in the present invention.
[0053] [00053] In an embodiment of the invention, an HDV antigen usable in the invention consists of HDAg containing a mutation sufficient to deactivate or remove the nuclear localization sequence (NLS; represented, for example, by the published AGA-PPAKRAR sequence (SEQ ID NO: 27) or by EGAPPAKRAR corresponding to positions 66 - 75 of SEQ ID NO: 2, or the corresponding sequence of a different HDV genotype or HDV strain / isolate). This mutation can consist of a deletion or substitution of one, two, three, four, five, six, seven, eight, nine or all ten amino acid residues that make up the NLS. Additional amino acids flanking the NLS can also be deleted or replaced, although this is not typically required, and it is more preferable to retain amino acid residues from natural T cell epitopes in the HDV antigen. Removing the NLS is useful for at least two reasons. First, concerns about the potential biological activity of the HDV antigen used in the invention are avoided (the virus is inactivated) and, second, the inventors found that deletion of this functional site improves the expression of the fusion protein or protein resulting in yeast, while improving the growth rate and eliminating an abnormal agglomeration morphology that the yeast acquired when expressing the HDV antigen containing the NLS sequence.
[0054] [00054] Table 1 shows several published HDV T cell epitopes (positions given with respect to an HDAg-L protein), any one or more of which can be used on an HDV antigen according to the present invention. In one aspect of the invention, an HDV antigen includes at least one, two, three, four, five or more of these epitopes or other T cell epitopes.
[0055] [00055] HDV antigens and fusion proteins of the invention are useful in an immunotherapeutic composition of the invention, including a yeast-based immunotherapeutic composition of the invention. These antigens, fusion proteins and / or the recombinant nucleic acid molecules that encode these proteins can also be used in, in combination with or to produce a non-yeast immuno-therapeutic composition, which may include, without limitation, a DNA vaccine, a protein subunit vaccine, an immunotherapeutic composition based on recombinant virus, a dead or inactivated pathogen vaccine and / or a dendritic cell vaccine. In another embodiment, these fusion proteins can be used in a diagnostic assay for HDV and / or to generate antibodies against HDV.
[0056] [00056] One embodiment of the invention relates to new HDV antigens and fusion proteins and recombinant nucleic acid molecules that encode those antigens and proteins. Several different new HDV antigens are described herein for use in a yeast-based immunotherapeutic composition or other composition (for example, other immunotherapeutic or diagnostic compositions).
[0057] [00057] According to the present invention, the present general use of the term "antigen" refers to: any part of a protein (peptide, partial protein, full length protein), in which the protein is naturally occurring or synthetically derived, to a cellular composition (whole cell, cell lysate or disrupted cells), an organism (whole organism, lysate or disrupted cells) or a carbohydrate, or other molecule or part of it. An antigen can elicit an antigen-specific immune response (for example, a humoral and / or cell-mediated immune response) against the same or similar antigens that are found by an element of the immune system (for example, T cells, antibodies ).
[0058] [00058] An antigen can be as small as a single epitope, a single immunogenic domain or larger and can include multiple epitopes or immunogenic domains. Thus, the size of an antigen can be as small as about 8 - 12 amino acids (ie, a peptide) and as large as: a full-length protein, a multimer, a fusion protein, a chimeric protein, a cell whole, an entire microorganism or any of its parts (for example, whole cell lysates or extracts of microorganisms). In addition, antigens can include carbohydrates, which can be loaded into a yeast vehicle or a composition of the invention. It should be noted that, in some modalities (for example, when the antigen is expressed by the yeast vehicle from a recombinant nucleic acid molecule), the antigen is a protein, fusion protein, chimeric protein or fragment thereof, in instead of an entire cell or microorganism.
[0059] [00059] When the antigen must be expressed in yeast, an antigen has a minimum size capable of being expressed recombinantly in yeast and is typically at least or more than 25 amino acids in length, or at least or more than 26, at least or more than 27, at least or more than 28, at least or more than 29, at least or more than 30, at least or more than 31, at least or more than 32, at least or more than 33, at least or more than 34, at least or more than 35, at least or more than 36, at least or more than 37, at least or more than 38, at least or more than 39, at least or more than 40, at least or more 41, at least or more than 42, at least or more than 43, at least or more than 44, at least or more than 45, at least or more than 46, at least or more than 47, at least or more than 48, at least or more than 49, or at least or more than 50 amino acids in length, or is at least 25 - 50 amino acids in length, at least 30 - 50 amino acids in length length, or at least 35 - 50 amino acids in length, or at least 40 - 50 amino acids in length, or at least 45 - 50 amino acids in length. Smaller proteins can be expressed, and considerably larger proteins can be expressed (for example, hundreds of amino acids in length or even a few thousand amino acids in length). In one aspect, a full-length protein, or its structural or functional domain, or its immunogenic domain, which is devoid of one or more amino acids with the N and / or C termination can be expressed (for example, devoid of about 1 to about 20 amino acids of the N and / or C termination). Fusion proteins and chimeric proteins are also antigens that can be expressed in the invention. A "target antigen" is an antigen that is the specific target of an immunotherapeutic composition of the invention (i.e., an antigen against which it is desired to elicit an immune response). An "HDV antigen" is an antigen derived, designed or produced from one or more HDV proteins, so that targeting the antigen also targets the hepatitis D virus.
[0060] [00060] When referring to the stimulation of an immune response, the term "immunogen" is a subset of the term "antigen" and, consequently, in some cases, can be used interchangeably with the term "antigen". An immunogen, as used herein, describes an antigen that elicits a humoral and / or cell-mediated immune response (that is, it is immunogenic), so that administration of the immunogen to an individual creates a specific immune response to antigen against equal antigens or similar that are found by the individual's immune system. In one embodiment, an immunogen elicits a cell-mediated immune response, including a CD4 + T cell immune response (eg, TH1, TH2 and / or TH17) and / or a CD8 + T cell immune response (eg, a response CTL).
[0061] [00061] An "immunogenic domain" of a given antigen can be any part, fragment or epitope of an antigen (for example, a peptide fragment or subunit or an antibody epitope or other conformational epitope) that contains at least one epitope that act as an immunogen when administered to an animal. Consequently, an immunogenic domain is larger than a single amino acid and is at least large enough to contain at least one epitope that acts as an immunogen. For example, a single protein can contain multiple different immunogenic domains. Immunogenic domains need not be linear sequences within a protein, as in the case of a humoral immune response, when considering conformational domains.
[0062] [00062] A "functional domain" of a given protein is a functional part or unit of the protein that includes a sequence or structure that is directly or indirectly responsible for at least one biological or chemical function associated with, attributed to or performed by the protein. For example, a functional domain can include an active site for enzymatic activity, a ligand binding site, a receptor binding site, a binding site for a molecule or fraction such as calcium, a phosphorylation site or a transactivation domain . Examples of HDV functional domains include, but are not limited to, the nuclear localization sequence (NLS), RNA binding domain, and domains involved in virion assembly and inhibition of RNA assembly.
[0063] [00063] A "structural domain" of a given protein is a part of the protein or an element in the overall structure of the protein that has an identifiable structure (for example, it may be a primary or tertiary structure belonging to and indicative of several proteins within a protein class or family), is self-stabilizing and / or can fold independently of the rest of the protein. A structural domain is often associated with or appears prominently in the biological function of the protein to which it belongs.
[0064] [00064] An epitope is defined here as a single immunogenic site within a given antigen that is sufficient to elicit an immune response when presented to the immune system in the context of appropriate costimulatory signals and / or activated cells of the immune system. In other words, an epitope is the part of an antigen that is actually recognized by components of the immune system and can also be mentioned as an antigenic determinant. Those skilled in the art will recognize that T cell epitopes are different in size and composition with respect to B cell or antibody epitopes, and that epitopes presented through the MHC Class I pathway differ in structural size and attribute of epitopes presented through the MHC pathway. Class II. For example, T cell epitopes presented by MHC Class I molecules are typically between 8 and 11 amino acids in length, whereas epitopes presented by MHC Class II molecules are less length restricted and can have 8 amino acids up to 25 amino acids or more. In addition, T cell epitopes have predicted structural characteristics depending on the specific MHC molecules linked by the epitope. Some T cell epitopes have been identified in HDV strains and are identified in Table 1. Epitopes can be linear sequence epitopes or conformational epitopes (conserved binding regions). Most antibodies recognize conformational epitopes.
[0065] [00065] In any of the HDV antigens described here, including any of the fusion proteins, the following additional modalities can be applied. First, the N-terminal expression sequence and the C-terminal tag included in some of the antigen constructs are optional and, if used, can be selected from several different sequences described here elsewhere to provide resistance to degradation proteasome and / or stabilize or improve expression, stability and / or allow protein identification and / or purification. Alternatively, one or both of the N or C-terminal sequences are completely omitted. In addition, many different promoters suitable for use in yeast are known in the art and are encompassed for use in the expression of HDV antigens according to the present invention. Suitable promoters include, but are not limited to, CUP1 and TEF2. In addition, short intervening link sequences (for example, peptides of 1, 2, 3, 4 or 5, or more, amino acids) can be introduced between parts of the fusion protein for several reasons, including the introduction of enzyme sites from restriction to facilitate cloning and future manipulation of constructs. Finally, as discussed here in detail elsewhere, the sequences described here are exemplary and can be modified as described here in detail elsewhere to replace, add or delete sequences to accommodate preferences for HDV genotype, HDV strain or isolate or consensus sequences and inclusion of preferred T-cell epitopes, including dominant and / or sub-dominant T-cell epitopes. A description A description of several different exemplary HDV antigens usable in the invention is presented below.
[0066] [00066] In an embodiment of the invention, the HDV antigen (s) for use in a composition or method of the invention is a protein or fusion protein comprising HDV antigens, wherein the HDV antigens comprise or consist of all in at least one HDAg-L or HDAg-S protein and / or at least one immunogenic domain thereof. In one aspect, the HDAg-L or HDAg-S protein is either full-length or almost full-length. According to any embodiment of the present invention, reference to a "full length" protein (or a full length functional domain or full length immune domain) includes the full length amino acid sequence of the protein or functional domain or immune domain , as described herein or as otherwise known or described in a publicly available sequence. A protein or domain that is "almost full-length", which is also a type of protein homologue, differs from a full-length protein or domain by adding or deleting or omitting 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the N and / or C terminus of this full-length protein or full-length domain. The generic reference to a protein or domain can include both full-length and near-full length proteins, as well as other counterparts to them.
[0067] [00067] The HDV sequences used to design these constructs and the others described and / or exemplified here are based on isolates or strains of a particular HDV genotype. However, it is a modality of the invention to add or replace any part of an HDV antigen described herein that is based on or derived from a particular genotype, subgenotype or strain / isolate, a corresponding sequence, or even a single or small substitution, insertion or deletion of amino acids that occurs in a corresponding sequence, of any other genotype (s), subgenotype (s) or strain (s) of HDV (s). In one embodiment, an HDV antigen can be produced by replacing an entire sequence (s) of an HDV antigen described here by the corresponding sequence (s) of one or more genotypes, subgenotypes or different HDV strain / isolates. The addition or replacement of a sequence of an HDV genotype or subgenotype with another, for example, allows adaptation of the immunotherapeutic composition to a particular individual or population of individuals (for example, a population of individuals within a given country or region of a country, to target the HDV genotype (s) that is most prevalent in that country or region of the country). Likewise, it is also a modality of the invention to use all or part of a consensus sequence derived from, determined by or published for a given HDV strain, genotype or subtype to make changes to the sequence of a given HDV antigen for corresponding more closely or exactly with the consensus sequence. According to the present invention and as generally understood in the art, a "consensus sequence" is typically a sequence based on the most common nucleotide or amino acid at a particular position in a given sequence after aligning multiple sequences.
[0068] [00068] As a particular example of the aforementioned types of modifications, an HDV antigen can be modified to alter a T cell epitope in a given sequence of an isolate to more closely or exactly match a T cell epitope of an isolate different, or to correspond more closely or exactly to a consensus sequence for the T cell epitope. In fact, according to the invention, HDV antigens can be designed to incorporate consensus sequences from various genotypes and / or subtypes of HDV, or mixtures of sequences of different HDV genotypes and / or subtypes. Alignments of HDV's HDAg’s between exemplary sequences from each of the largest known genotypes can be readily generated using publicly available software, which will inform the generation of consensus sequences, for example. Examples of such modifications are illustrated and exemplified here.
[0069] [00069] An exemplary embodiment of the invention relates to an HDV antigen that has the amino acid sequence represented by SEQ ID NO: 10, which is a truncated HDV genotype 1 HDAg-L (less than the total length). This antigen includes the sequence KLEDLERDL or SEQ ID NO: 20 (positions 5 - 13 of SEQ ID NO: 10) which is an MHC Class I T cell epitope; and the sequence KLE-DENPWL or SEQ ID NO: 19 (positions 22 - 30) which is another MHC Class I T cell epitope (see Table 1 above). Positions 22 - 140 of SEQ ID NO: 10 are regions rich in MHC Class II binding epitopes (see Table 2). This antigen can be produced using the corresponding sequence of any HDV genotype, subgenotype, or strain (that is, the same HDAg-L amino acid positions of a different HDV strain can be used instead of that selected for the SEQ ID NO: 10). For use in the production of a yeast-based immunotherapeutic composition, yeast (for example, Saccharomyces cerevisiae) is manipulated to express the HDV antigen under the control of a suitable promoter. The construction and production of yeast-based immunotherapeutic products is described in more detail below.
[0070] [00070] As generically discussed above and further described in more detail below, the antigen represented by SEQ ID NO: 10, and any other HDV antigen described here, can also be attached to the N termination to add a sequence that provides resistance to degradation proteasome and / or stabilize protein expression, such as the following protein: MADEAP (SEQ ID NO: 11). Additional suitable N-terminal sequences are discussed below. Optionally, this antigen, and any HDV antigen described herein, can be modified to include a C-terminal sequence that can assist in protein stabilization, identification and / or isolation, such as a hexa-histidine sequence, which is usable to identify a protein. As an example, a fusion protein comprising the N-terminal sequence of SEQ ID NO: 11 and a hexa-histidine C termination is represented by SEQ ID NO: 12, where positions 1 - 6 of SEQ ID NO: 12 correspond to SEQ ID NO: 11, positions 7 - 146 of SEQ ID NO: 12 correspond to SEQ ID NO: 10, and positions 147 - 152 of SEQ ID NO: 12 are a hexa-histidine tag.
[0071] [00071] The antigen of SEQ ID NO: 10, which can be replaced by the corresponding sequence of a different HDV genotype, subgenotype or strain, can also be attached to the N-terminus to add a pre-pro alpha factor sequence to stabilize the expression (here represented by SEQ ID NO: 13 or SEQ ID NO: 14, which are both exemplary of alpha factor pre-pro sequences), and, again, can be optionally appended to the C termination to add a hex sequence -histidine, if desired. One such fusion protein is represented here by SEQ ID NO: 15, which can be replaced by the corresponding sequence of a different HDV strain (incorporating SEQ ID NO: 10 and SEQ ID NO: 13, as well as a hexa-histidine tag ). Positions 1 - 89 of SEQ ID NO: 15 correspond to SEQ ID NO: 13, positions 90 - 229 of SEQ ID NO: 15 and SEQ ID NO: 10, and positions 230 - 235 of SEQ ID NO: 15 they are a hexa-histidine tag.
[0072] [00072] Another HDV antigen usable in the invention is represented by the amino acid sequence of SEQ ID NO: 16. In this antigen, a copy of a truncated HDAg (selected to maximize T cell epitopes) from a strain from each of the three different genotypes known as genotypes 1, 2 and 3 is provided as a single fusion protein to produce a more universal construct. . This antigen can be produced using the corresponding sequence any HDV genotype (s) or strain (s), and in addition, any part of the HDAg can be used to create this fusion (ie, the parts of the HDAg's used in the SEQ ID NO: 16 are exemplary, but smaller parts or larger parts, up to the total length of HDAg's could be used). In this construct, a part of an HDAg from an HDV genotype 1 strain ("genotype1" below) corresponds to positions 1 -140 of SEQ ID NO: 16; a part of a HDAg from a genotype 2 strain ("genotype2" below) corresponds to positions 141 - 280 of SEQ ID NO: 16; and a portion of a HDAg from a genotype 3 strain ("ge-notype3" below) corresponds to positions 281 - 420 of SEQ ID NO: 16.
[0073] [00073] Another similar construct that can be produced and related to the sequence represented by SEQ ID NO: 16 is a fusion of three or more full-length HDAg-L proteins, where each HDAg-L protein is from a genotype of Different HDV (for example, genotypes 1, 2, and 3, or different combinations of genotypes), except that, compared to the full-length sequences, each HDAg-L protein was modified to inactivate an NLS region, as described above (for example example, by replacing and / or deleting waste within that site). In an example of this construct, a full-length HDAg-L genotype 1 sequence represented by SEQ ID NO: 2 (or a corresponding sequence from another HDV strain) with a deletion of positions 66 - 75 (the NLS) is linked to a full-length HDAg-L genotype 2 sequence represented by SEQ ID NO: 5 (or a corresponding sequence from another HDV strain) with a deletion of positions 66 - 75 (the NLS) which is linked to a sequence of HDAg- L full-length genotype 3 represented by SEQ ID NO: 8 (or a corresponding sequence from another HDV strain) with a deletion of positions 66 - 75 (the NLS). These sequences can also be fused in a different order, or different or additional HDV genotypes or subgenotypes can be added to the fusion to produce a "universal" immunotherapeutic composition. In addition, several residues from any of the individual genotype sequences can be modified (for example, by substitution of amino acids) to more closely match the consensus sequence for a given genotype, and / or several residues can be modified (for example, by amino acid substitution) to more closely match known consensus T cell epitopes that have been associated with immune responses to HDV.
[0074] [00074] Fig. 1 shows an alignment of the HDAg parts used in the SEQ ID NO: 16 construct, to illustrate the homology between genotypes used in this SEQ ID NO: 16 fusion protein. This alignment also illustrates that it is a straightforward process to identify the corresponding sequence between different HDV genotypes, subgenotypes or strains / isolates, as each of the sequences in SEQ ID NO: 16 is a truncated part of a full-length HDV HDAg-L .
[0075] [00075] As with the HDV antigens discussed elsewhere here, the N and / or C terminations of SEQ ID NO: 16 or a similar fusion protein can be attached to include multiple sequences (for example, SEQ ID NO: 11, 13 or 14). For example, using an N-terminal peptide of SEQ ID NO: 11 and a C-terminal hexa-histidine sequence together with SEQ ID NO: 16, the fusion protein represented by SEQ ID NO: 17 is produced. Positions 1 - 6 of SEQ ID NO: 17 correspond to SEQ ID NO: 11; positions 7 - 426 of SEQ ID NO: 17 correspond to SEQ ID NO: 16, and positions 427 - 432 of SEQ ID NO: 17 correspond to a hexa-histidine tag. Using the N-terminal sequence of SEQ ID NO: 13 together with SEQ ID NO: 16 and appending the C termination as above, the fusion protein represented by SEQ ID NO: 18 is produced, where positions 1 - 89 of SEQ ID NO: 18 correspond to SEQ ID NO: 13, positions 90 -509 of SEQ ID NO: 18 correspond to SEQ ID NO: 16, and positions 510 - 515 of SEQ ID NO: 18 correspond to a hex label -histidine.
[0076] [00076] Additional HDV antigens, including fusion proteins, and compositions comprising HDV antigens are described in Example 1. In one construct, yeast (for example, Saccharomyces cerevisiae) was manipulated to express an HDV antigen under the control of the copper-inducible promoter, CUP1 (an optional promoter may include, but is not limited to, the TEF2 promoter). This HDV antigen comprises a single copy of an HDAg-L of HDV genotype 1. The antigen was constructed using a single copy of the HDAg represented here as SEQ ID NO: 2 as the template for the main structure, although a corresponding sequence from any other HDV strain / isolate, or any other HDV genotype or subgenotype, could be used instead of that included in this construct. In this construct, the HDV antigen represented by SEQ ID NO: 2 was modified to delete the nuclear localization sequence of 10 amino acid residues (ie, EGAPPAKRAR, or positions 66 - 75 of SEQ ID NO: 2) to increase the profile potential safety of the resulting construct and the yeast-based immunotherapy that expresses this antigen, and to potentially increase the expression of that construct in yeast. This HDV isolate (SEQ ID NO: 2) contains several T cell epitopes that are expected to elicit an immune response against HDV. The resulting protein did not contain known transmembrane domains and was free of extremely hydrophobic elements, and therefore further modification to compensate for hydrophobicity was not considered to be an important factor in the design of this HDV antigen. The amino acid sequence of the resulting antigen is represented here by SEQ ID NO: 28, which can be replaced by the corresponding sequence of a different HDV strain, genotype or subgenotype. An N-terminal sequence corresponding to SEQ ID NO: 11 and a C-terminal sequence of a hexa-histidine tag were added to SEQ ID NO: 28 to improve yeast antigen expression (N-terminal sequence) and to facilitate the identification of the antigen (C-terminal sequence). A two amino acid link (Thr-Ser) that introduces a SpeI restriction enzyme site has been inserted between SEQ ID NO: 11 and SEQ ID NO: 28 to facilitate cloning of the construct. The resulting fusion protein has the amino acid sequence represented by SEQ ID NO: 30, where positions 1 - 6 of SEQ ID NO: 30 are the N-terminal peptide of SEQ ID NO: 11; positions 7 - 8 of SEQ ID NO: 30 are the link of two amino acids; positions 9 - 212 of SEQ ID NO: 30 are the HDV antigen of SEQ ID NO: 28; and positions 213 - 218 of SEQ ID NO: 30 are the hexa-histidine tag. SEQ ID NO: 30 is encoded by a nucleic acid sequence represented here by SEQ ID NO: 29, which is optimized for expression in yeast.
[0077] [00077] In a second construct based on the HDV HDAg of SEQ ID NO: 2 and described in Example 1, the NLS sequence was maintained to evaluate the resulting antigen with respect to yeast biology and yeast antigen expression. The amino acid sequence of the resulting antigen is represented here by SEQ ID NO: 31, which can be replaced by the corresponding sequence of a different HDV strain, genotype or subgenotype. An N-terminal sequence corresponding to SEQ ID NO: 11 and a C-terminal sequence of a hexa-histidine tag were added to SEQ ID NO: 31 to improve yeast antigen expression (N-terminal sequence) and to facilitate antigen identification (C-terminal sequence). A two amino acid link (Thr-Ser) was inserted between SEQ ID NO: 11 and SEQ ID NO: 31 to facilitate cloning of the construct. The resulting fusion protein has the amino acid sequence represented by SEQ ID NO: 33, where positions 1 - 6 of SEQ ID NO: 33 are the N-terminal peptide of SEQ ID NO: 11; positions 7 - 8 of SEQ ID NO: 33 are the link of two amino acids; positions 9 - 222 of SEQ ID NO: 33 are the HDV antigen of SEQ ID NO: 31; and positions 223 - 228 of SEQ ID NO: 33 are the hexa-histidine tag. SEQ ID NO: 33 is encoded by a nucleic acid sequence represented here by SEQ ID NO: 32, which is optimized for expression in yeast.
[0078] [00078] In a third construct described in Example 1, yeast (for example, Saccharomyces cerevisiae) was manipulated to express an HDV antigen under the control of the copper-inducible promoter, CUP1. This antigen is considered to be a more "universal" immunotherapy or vaccine and is a single fusion protein comprising modified HDAg-L from two different HDV genotypes, in this case, genotypes 1 and 2. This HDV antigen was constructed using a single copy of HDAg here represented as SEQ ID NO: 2 (genotype 1) as a template of main structure, and a single copy of HDAg here represented as SEQ ID NO: 5 (genotype 2) as a second template of main structure. A corresponding sequence from any other HDV strain / isolate, or any other HDV genotype or subgenotype, could be used instead of one or both of the sequences included in this construct, and similar fusions can be prepared using a third, fourth, fifth or more genotypes, subgenotypes or strains. In that fusion protein, the HDV antigen represented by SEQ ID NO: 2 was modified to delete the 10-residue nuclear localization sequence (i.e., EGAPPAKRAR, or positions 66 - 75 of SEQ ID NO: 2) to increase the profile potential safety potential of the resulting construct and the yeast-based immuno-therapy that expresses this antigen. Likewise, the HDV antigen represented by SEQ ID NO: 5 has been modified to delete the 10-residue nuclear localization sequence (EGAPPAKRAR, or positions 66 - 75 of SEQ ID NO: 5), also to increase the safety profile the resulting construct and the yeast-based immunote-fast that expresses this antigen. This fusion protein contains several T cell epitopes in each of the sequences of the HDV isolate templates (SEQ ID NO: 2 and SEQ ID NO: 5) which are expected to elicit an immune response against HDV. The amino acid sequence of the resulting antigen is here represented by SEQ ID NO: 34, which can be replaced by the corresponding sequence of different HDV strains, genotypes or subgenotypes. In SEQ ID NO: 34, the HDV antigen genotype 1 sequence is represented by positions 1 - 204 of SEQ ID NO: 34, and the sequence of HDV genotype 2 antigen is represented by positions 205 -408 of SEQ ID NO: 34.
[0079] [00079] An N-terminal sequence corresponding to SEQ ID NO: 11 and a C-terminal sequence of a hexa-histidine tag have been added to SEQ ID NO: 34 to improve yeast antigen expression (N-terminal sequence) and to facilitate the identification of the antigen (C-terminal sequence). A two amino acid link (Thr-Ser) was inserted between SEQ ID NO: 11 and SEQ ID NO: 34 to facilitate cloning of the construct. The resulting fusion protein has the amino acid sequence represented by SEQ ID NO: 36, where positions 1 - 6 of SEQ ID NO: 36 are the N-terminal peptide of SEQ ID NO: 11; positions 7 - 8 of SEQ ID NO: 36 are the link of two amino acids; positions 9 - 212 of SEQ ID NO: 36 are the HDV antigen genotype 1 of SEQ ID NO: 34; positions 213 - 416 of SEQ ID NO: 36 are the genotype 2 antigen of SEQ ID NO: 34, and positions 417 -422 of SEQ ID NO: 36 are the hexa-histidine tag. SEQ ID NO: 36 is encoded by a nucleic acid sequence represented here by SEQ ID NO: 35, which is optimized for expression in yeast.
[0080] [00080] The invention also includes homologues of any of the HDV antigens and fusion proteins described above, as well as the use of homologues, variants or mutants of the individual HDV proteins or parts thereof (including any functional and / or immunogenic domains) that are part of those fusion proteins. In one aspect, an HDV antigen usable in the present invention (including any of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 36) comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98% or 99% of the linear sequence of a full-length HDV antigen (HDAg-L or HDAg-S), or of an HDV antigen that has been modified to delete or replace between 1 and 10 amino acids from the NLS domain, or from a part of the HDAg-L or HDAg-S protein that comprises at least one immunogenic domain of HDAg-L or HDAg-S. In one aspect, the HDV antigen (including any of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 36) is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a full-length HDV antigen (HDAg-L or HDAg-S), or an HDV antigen that has been modified to delete or replace between 1 and 10 amino acids in the NLS domain, or to a part of the HDAg-L or HDAg-S protein that comprises at least one immunogenic domain of HDAg-L or HDAg-S. The addition of an N-terminal expression sequence and the C-terminal tag are optional, and can be selected from the many different sequences described here elsewhere to improve expression, stability and / or allow identification and / or purification of the protein , or one or both of the N or C-terminal sequences are completely omitted. In addition, many different promoters suitable for use in yeast are known in the art. In addition, short intervening link sequences (for example, peptides of 1, 2, 3, 4 or 5, or more, amino acids) can be introduced between parts of a fusion protein for several reasons, including the introduction of enzyme sites restrictions to facilitate the cloning and future manipulation of the constructs.
[0081] [00081] In some aspects of the invention, insertions, deletions and / or substitutions of amino acids can be made for one, two, three, four, five, six, seven, eight, nine, ten or more amino acids of an HDV protein of wild type or reference or its immunogenic domain, provided that the resulting HDV protein, when used as an antigen in a yeast HDV immunotherapeutic composition of the invention, elicits an immune response against the wild type or HDV target or protein. reference, which may include an enhanced immune response, a decreased immune response, or a substantially similar immune response. For example, the invention includes the use of HDV agonist antigens, which may include one or more T cell epitopes that have mutated to enhance the T cell response against the HDV agonist, such as by improving the epitope's avidity or affinity by an MHC molecule or by the T cell receptor that recognizes the epitope in the context of MHC presentation. HDV protein agonists can therefore improve the potency or effectiveness of a T cell response against native HDV proteins that infect a host.
[0082] [00082] Recombinant nucleic acid molecules and the proteins encoded by them, including any HDV antigens described herein, as an embodiment of the invention, can be used in yeast-based immunotherapy compositions or, in other embodiments, for any other purpose suitable for HDV antigen (s), including in an in vitro assay, for the production of antibodies, or in another immunotherapy composition, including another vaccine that is not based on the yeast-based immunotherapy described herein (for example, a vaccine viral vector, a dendritic cell vaccine or as a component of a fusion or binding to another immuno-therapeutic fraction). Expression of proteins by yeast is a preferred modality, although other expression systems can be used to produce proteins for applications other than a yeast-based immunotherapeutic composition.
[0083] [00083] Yeast-based Immunotherapy Compositions. In various embodiments of the invention, the invention includes the use of at least one "yeast-based immunotherapeutic composition" (this expression can be used interchangeably with "yeast-based immunotherapy product", "yeast-based immunotherapy composition" yeast "," yeast-based composition "," yeast-based immunotherapy "," yeast-based vaccine "or derivatives of these expressions). An "immunotherapeutic composition" is a composition that elicits an immune response sufficient to obtain at least one therapeutic benefit in a subject. As used herein, yeast-based immunotherapeutic composition refers to a composition that includes a yeast vehicle component and that elicits an immune response sufficient to obtain at least one therapeutic benefit in a subject. More particularly, a yeast-based immunotherapeutic composition is a composition that includes a yeast vehicle component and can elicit or induce an immune response, such as a cellular immune response, including without limitation, a T-cell-mediated cellular immune response. in one aspect, a yeast-based immunotherapeutic composition usable in the invention is capable of inducing an immune response mediated by CD8 + and / or CD4 + T cells and, in one aspect, an immune response mediated by CD8 + and CD4 + T cells. Optionally, a yeast-based immunotherapeutic composition is capable of eliciting a humoral immune response. A yeast-based immunotherapeutic composition usable in the present invention can, for example, elicit an immune response in an individual so that the individual is protected from HDV infection and / or is treated for HDV infection or symptoms resulting from infection by HDV.
[0084] [00084] Yeast-based immunotherapy compositions of the invention can be "prophylactic" or "therapeutic". When used prophylactically, the compositions of the present invention are provided prior to the detection or observation of any identifier or symptom of HDV infection. This composition could be administered at birth, in early childhood or to adults and can be useful, in one modality, to immunize populations of individuals in whom HDV infection is a risk, or where HBV infection is a risk. Prophylactic administration of the immunotherapy compositions serves to prevent subsequent HDV infection, to resolve an infection more quickly or more completely, if HDV infection subsequently arises, and / or to improve symptoms of HDV infection, if infection subsequently arises. When used therapeutically, immunotherapy compositions are provided on or after the onset of HDV infection, for the purpose of ameliorating at least one symptom of the infection and, preferably, for the purpose of eliminating the infection, providing a long-lasting remission infection and / or confer long-term immunity against subsequent infections or virus reactivations.
[0085] [00085] Typically, a yeast-based immunotherapeutic composition includes a yeast vehicle and at least one antigen or its immunogenic domain expressed by, attached to, or mixed with the yeast vehicle, wherein the antigen is heterologous to yeast, and wherein the antigen comprises one or more HDV antigens. In some embodiments, the HDV antigen is provided as a fusion protein. Various HDV fusion proteins suitable for use in the compositions and methods of the invention have been described above. In one aspect of the invention, the fusion protein can include two or more antigens, such as a repeat of the same antigen or two or more HDAg, where each antigen is of a different genotype, subgenotype or strain. In one aspect, the fusion protein can include two or more immunogenic domains of one or more antigens, or two or more epitopes of one or more antigens.
[0086] [00086] In any of the yeast-based immunotherapy compositions used in the present invention, the following aspects related to the yeast vehicle are included in the invention. According to the present invention, a yeast vehicle is any yeast cell (for example, an entire or intact cell) or its derivative (see below) that can be used in conjunction with one or more antigens, their immunogenic domains or their epitopes in a therapeutic composition of the invention, or, in one aspect, the yeast vehicle can be used alone or as an adjuvant. The yeast vehicle may therefore include, but is not limited to, an intact (whole) living yeast microorganism (i.e., a yeast cell with all its components, including a cell wall), a yeast microorganism killed (killed) or inactivated inactivated (whole), or intact (whole) yeast derivatives, including: a yeast esfroplast (ie, a yeast cell devoid of a cell wall), a yeast cytoplasm (ie , a yeast cell devoid of a cell wall and nucleus), a yeast phantom (i.e., a yeast cell devoid of a cell wall, nucleus and cytoplasm), a subcellular yeast membrane extract or its fraction (also called yeast membrane particle and, previously, subcellular yeast particle), any other yeast particle or a yeast cell wall preparation.
[0087] [00087] Yeast spheroplasts are typically produced by enzymatic digestion of the yeast cell wall. This method is described, for example, in Franzusoff et al., 1991, Meth. Enzymol. 194, 662-674., Hereby incorporated by reference in its entirety.
[0088] [00088] Yeast cytoplasts are typically produced by enucleation of yeast cells. This method is described, for example, in Coon, 1978, Natl. Cancer Inst. Monogr. 48, 45-55, hereby incorporated by reference in its entirety.
[0089] [00089] Yeast ghosts are typically produced by re-waterproofing a permeabilized or lysed cell and may, but need not, contain at least some of the cell's organelles. This method is described, for example, in Franzusoff et al., 1983, J. Biol. Chem. 258, 3608-3614 and Bussey et al., 1979, Bio-chim. Biophys. Minutes 553, 185-196, each incorporated herein by reference in its entirety.
[0090] [00090] A yeast membrane particle (subcellular yeast membrane extract or its fraction) refers to a yeast membrane devoid of a natural nucleus or cytoplasm. The particle can be of any size, including sizes ranging from the size of a natural yeast membrane to microparticles produced by sonication or other membrane disruption methods known to those skilled in the art, followed by re-permeabilization. A method for producing subcellular yeast membrane extracts is described, for example, in Franzusoff et al., 1991, Meth. Enzymol. 194, 662-674. Fractions of yeast membrane particles that contain yeast membrane parts can also be used and, when the antigen or other protein was recombinantly expressed by the yeast before the preparation of the yeast membrane particles, the antigen or other protein of interest . Antigens or other proteins of interest can be transported into the membrane, on any surface of the membrane or combinations thereof (that is, the protein can be both inside and outside the membrane and / or across the membrane of the yeast membrane particle) . In one embodiment, the yeast membrane particle is a recombinant yeast membrane particle that can be an intact, broken or broken and re-waterproofed yeast membrane, which includes at least one desired antigen or other protein of interest on the membrane surface or at least partially embedded in the membrane.
[0091] [00091] An example of a yeast cell wall preparation is an isolated yeast cell wall preparation carrying an antigen on its surface or at least partially embedded within the cell wall, so that the yeast cell wall preparation, when administered to an animal, stimulate a desired immune response against a disease target.
[0092] [00092] Any yeast strain can be used to produce a yeast vehicle of the present invention. Yeasts are single-celled microorganisms that belong to one of three classes: As-comycetes, Basidiomycetes and Fungi Imperfecti. One consideration for selecting a type of yeast for use as an immune modulator is the pathogenicity of the yeast. In one embodiment, yeast is a non-pathogenic strain, like Saccharomyces cerevisiae. The selection of a non-pathogenic yeast strain minimizes any adverse effects for the individual to whom the yeast vehicle is administered. However, pathogenic yeast can be used if the yeast's pathogenicity can be neutralized by any means known to those skilled in the art (for example, mutant strains). In accordance with an aspect of the present invention, non-pathogenic yeast strains are used.
[0093] [00093] Genera of yeast strains that can be used in the invention include, but are not limited to, Saccharomyces, Candida, Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schi-zosaccharomyces and Yarrowia. In one aspect, the yeast genera are selected from Saccharomyces, Candida, Hansenula, Pichia or Schizosaccharomyces, and in one aspect, the yeast genera are selected from Saccharomyces, Hansenula and Pichia, and, in one aspect, Saccharomyces are used. . Species of yeast strains that can be used in the invention include, but are not limited to, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida albicans, Candida kefyr, Candida tropicalis, Cryptococcus laurentii, Cryptococcus neoformans, Hansenula anomala, Hansenulavermymy , Kluyveromyces marxianus var. lactis, Pichia pastoris, Rhodotorula rubra, Schizosaccharomyces pom-be and Yarrowia lipolytica. It should be noted that numerous of these species include several subspecies, types, subtypes and others that must be included within the species mentioned above. In one aspect, yeast species used in the invention include S. cerevisiae, C. albicans, H. polymorpha, P. pastoris and S. pombe. S. cerevisiae is useful as it is relatively easy to manipulate and is "Generically Recognized as Safe" or "GRAS" for use as food additives (GRAS, FDA Proposed Rule 62FR18938, April 17, 1997). One embodiment of the present invention is a yeast strain that is capable of replicating plasmids in a particularly high copy number, such as a S. cerevisiae strain cir °. The S. cerevisiae strain is one of those strains that is capable of supporting expression vectors that allow one or more target antigens and / or fusion proteins of antigens and / or other proteins to be expressed at high levels. In addition, any mutant yeast strains can be used in the present invention, including those that exhibit reduced post-translational modifications of the target antigens or other expressed proteins, such as mutations in the enzymes that extend N-linked glycosylation.
[0094] [00094] In most embodiments of the invention, the yeast-based immunotherapy composition includes at least one antigen, its immunogenic domain or its epitope. Antigens considered for use in this invention include any HDV antigen or its immunogenic domain, including HDV protein mutants, variants and agonists or their domains, against which it is desired to elicit an immune response for the purpose of prophylactic or therapeutic immunization of a host against HDV infection. HDV antigens that are usable in various embodiments of the invention have been described in detail above.
[0095] [00095] As discussed above, the compositions of the invention include at least one HDV antigen and / or at least one immunogenic domain of at least one HDV antigen to immunize a subject. In some embodiments, the antigen is a fusion protein, the various examples of which have been described above.
[0096] [00096] Optionally, proteins, including fusion proteins, which are used as a component of the yeast-based immunotherapeutic composition of the invention are produced using constructs that are particularly useful for improving or enhancing expression, or expression stability. , of recombinant antigens in yeast. Typically, the desired antigenic protein (s) or peptide (s) is (are) fused (s) at its amino-terminal end to: (a) a specific synthetic peptide that stabilizes expression of the fusion protein in the yeast vehicle or prevent post-translational modification of the expressed fusion protein (these peptides are described in detail, for example, in U.S. Patent Publication No. 2004-0156858 A1, published on 12 August 2004, hereby incorporated by reference in its entirety); (b) at least a portion of an endogenous yeast protein, including, but not limited to, alpha factor, in which the fusion partner provides better stability of protein expression in yeast and / or prevents post-translational protein modification yeast cells (these proteins are also described in detail, for example, in U.S. patent publication No. 2004-0156858 A1, supra); and / or (c) at least a portion of a yeast protein that causes the fusion protein to be expressed on the surface of the yeast (for example, an Aga protein, described in more detail here). In addition, the present invention optionally includes the use of peptides that are fused to the C-terminus of the antigen-encoding construct, particularly for use in protein selection and identification. Such peptides include, but are not limited to, any synthetic or natural peptide, such as a peptide tag (for example, 6X His) or any other short epitope tag. Peptides attached to the C-terminus of an antigen according to the invention can be used with or without the addition of the N-terminal peptides discussed above.
[0097] [00097] In one embodiment, a synthetic peptide usable in a fusion protein is attached to the N-terminus of the antigen, the peptide consisting of at least two amino acid residues that are heterologous to the antigen, in which the peptide stabilizes the expression of the fusion in the yeast vehicle or prevents post-translational modification of the expressed fusion protein. The synthetic peptide and the N-terminal part of the antigen together form a fusion protein that has the following requirements: (1) the amino acid residue at position one of the fusion protein is a methionine (that is, the first amino acid in the synthetic peptide is a methionine); (2) the amino acid residue at position two of the fusion protein is not a glycine or a proline (that is, the second amino acid in the synthetic peptide is not a glycine or a proline); (3) none of the amino acid residues at positions 2 - 6 of the fusion protein is a methionine (i.e., the amino acids at positions 2 - 6, either part of the synthetic peptide or the protein, if the synthetic peptide is less than 6 amino acids , do not include a methionine); and (4) none of the amino acids at positions 2 - 6 of the fusion protein is a lysine or an arginine (that is, the amino acids at positions 2 - 6, either part of the synthetic peptide or the protein, if the synthetic peptide is less than 5 amino acids, do not include lysine or arginine). The synthetic peptide can be as short as two amino acids, but in one respect it has 2 - 6 amino acids (including 3, 4, 5 amino acids) and can have more than 6 amino acids, in whole numbers, up to about 200 amino acids, 300 amino acids, 400 amino acids, 500 amino acids or more.
[0098] [00098] In one embodiment, a fusion protein comprises an amino acid sequence of M-X2-X3-X4-X5-X6, where M is methionine; where X2 is any amino acid except glycine, proline, lysine or arginine; where X3 is any amino acid except methionine, lysine or arginine; where X4 is any amino acid except methionine, lysine or arginine; where X5 is any amino acid except methionine, lysine or arginine; and where X6 is any amino acid except methionine, lysine or arginine. In one embodiment, residue X6 is proline. An exemplary synthetic sequence that increases the stability of antigen expression in a yeast cell and / or prevents post-translational modification of the protein in yeast includes the sequence M-A-D-E-A-P (SEQ ID NO: 11). Another exemplary synthetic sequence with the same properties is M-V. In addition to the greater stability of the expression product, these fusion partners do not appear to have a negative impact on the immune response against the immunizing antigen in the construct. In addition, synthetic fusion peptides can be designed to provide an epitope that can be recognized by a selection agent, such as an antibody.
[0099] [00099] In one embodiment, the HDV antigen is linked at the N-terminus to a yeast protein, as a pre-pro alpha factor sequence (also called the leading alpha factor signal sequence, whose amino acid sequence is exemplified here by SEQ ID NO: 13 or SEQ ID NO: 14. Other sequences for the yeast alpha factor pre-pro sequence are known in the art and are encompassed for use in the present invention.
[0100] [000100] In one aspect of the invention, the yeast vehicle is manipulated in such a way that the antigen is expressed or supplied by distribution or translocation of a protein product expressed, partially or totally, on the surface of the yeast vehicle (extracellular expression). One method of accomplishing this aspect of the invention is to use a spacer arm to position one or more proteins on the surface of the yeast vehicle. For example, a spacer arm can be used to create a fusion protein from the antigen (s) or other protein of interest with a protein that targets the antigen (s) or other protein from interest to the yeast cell wall. For example, one of those proteins that can be used to target other proteins is a yeast protein (for example, cell wall protein 2 (cwp2), Aga2, Pir4 or Flo1 protein) that allows the antigen (s) ( s) or another protein targets the yeast cell wall, so that the antigen or other protein is located on the surface of the yeast. Proteins other than yeast proteins can be used for the spacer arm; however, for any spacer arm protein, it is more desirable to have the immunogenic response directed against the target antigen, rather than against the spacer arm protein. Thus, if other spacer arm proteins are used, then the spacer arm protein that is used should not generate a large immune response against the spacer arm protein itself, so that the immune response against the antigen (s) ( s) target is overcome. Those skilled in the art should seek a small immune response against the spacer arm protein with respect to the immune response to the target antigen (s). Spacer arms can be constructed to have cleavage sites (for example, protease cleavage sites) that allow the antigen to be promptly removed or processed from the yeast, if desired. Any known method of determining the magnitude of immune responses can be used (for example, antibody production, lytic assays and the like) and are readily known to those skilled in the art.
[0101] [000101] Another method for positioning the target antigen (s) or other proteins to be exposed on the yeast surface is to use signal sequences, such as glycosylphosphatidyl inositol (GPI), to anchor the target to the yeast cell wall. Alternatively, positioning can be performed by attaching signal sequences that target the antigen (s) or other proteins of interest in the secretory pathway by translocation to the endoplasmic reticulum (ER), so that the antigen binds to a protein that is bound to the cell wall (for example, cwp).
[0102] [000102] In one aspect, the spacer arm protein is a yeast protein. The yeast protein can consist of between about two and about 800 amino acids of a yeast protein. In one embodiment, the yeast protein has about 10 to 700 amino acids. In another embodiment, the yeast protein has about 40 to 600 amino acids. Other embodiments of the invention include the yeast protein having at least 250 amino acids, at least 300 amino acids, at least 350 amino acids, at least 400 amino acids, at least 450 amino acids, at least 500 amino acids, at least 550 amino acids, at least 600 amino acids, or at least 650 amino acids. In one embodiment, the yeast protein is at least 450 amino acids in length. Another consideration for optimizing expression on the antigen surface, if desired, is whether the combination of antigen and spacer arm should be expressed as a monomer or as a dimer or trimer, or even more units connected together. This use of monomers, dimers, trimers and the like allows for appropriate spacing or folding of the antigen, so that some, if not all, of the antigen is presented on the surface of the yeast vehicle in a way that makes it more immunogenic.
[0103] [000103] The use of yeast proteins can stabilize the expression of fusion proteins in the yeast vehicle, prevent post-translational modification of the expressed fusion protein and / or direct the fusion protein to a particular compartment in the yeast (for example , to be expressed on the yeast cell surface). For distribution in the yeast secretory pathway, exemplary yeast proteins include, but are not limited to: Aga (including, but not limited to, Agal and / or Aga2); SUC2 (yeast invertase); leading alpha factor signal sequence; CPY; Cwp2p for its location and retention on the cell wall; BUD genes for localization in the yeast cell bud during the initial phase of daughter cell formation; Flo1p; Pir2p; and Pir4p.
[0104] [000104] Other sequences can be used to target, retain and / or stabilize the protein in other parts of the yeast vehicle, for example, in the cytosol or mitochondria or in the endoplasmic reticulum or in the nucleus. Examples of suitable yeast proteins that can be used for any of the above embodiments include, but are not limited to, TK, AF, SEC7 gene products; phospho-nolpyruvate carboxy kinase PCK1, phosphoglycerokinase PGK and triose phosphate isomerase TPI for its repressible expression in glucose and cytosolic location; heat shock proteins SSA1, SSA3, SSA4, SSC1, whose expression is induced and whose proteins are more thermostable by exposing cells to heat treatment; the mitochondrial protein CYC1 for import into mitochondria; ACT1.
[0105] [000105] Methods of producing yeast and expression vehicles, combining and / or associating yeast vehicles with antigens and / or other proteins and / or agents of interest to produce yeast-based immunotherapy compositions are considered by the invention.
[0106] [000106] According to the present invention, the term "yeast-antigen vehicle complex" or "yeast-antigen complex" is used generically to describe any association of a yeast vehicle with an antigen, and can be used interchangeably with "yeast-based immunotherapy composition" when that composition is used to elicit an immune response as described above. This association includes the expression of the antigen by yeast (a recombinant yeast), introduction of an antigen in the yeast, physical attachment of the antigen to the yeast, and mixing the yeast and antigen with each other, as in a buffer or other solution or formulation. These types of complexes are described in detail below.
[0107] [000107] In one embodiment, a yeast cell used to prepare the yeast vehicle is transfected with a heterologous nucleic acid molecule that encodes a protein (for example, the antigen), so that the protein is expressed by the yeast cell . This yeast is also called recombinant yeast or recombinant yeast vehicle. The yeast cell can then be loaded into the dendritic cell as an intact cell, or the yeast cell can be killed or derivatized, as by forming yeast spheroplasts, cytoplasts, ghosts or subcellular particles, which can be followed by loading the derivative into the dendritic cell. Yeast spheroplasts can also be directly transfected with a recombinant nucleic acid molecule (for example, the spheroplast is produced from an entire yeast and then transfected) to produce a recombinant spheroplast that expresses an antigen or other protein.
[0108] [000108] In general, the yeast vehicle and the antigen (s) and / or other agents can be combined by any technique described herein. In one aspect, the yeast vehicle was loaded intracellularly with the antigen (s) and / or agent (s). In another aspect, the antigen (s) and / or agent (s) were covalently or non-covalently attached to the yeast vehicle. In yet another aspect, the yeast vehicle and the antigen (s) and / or agent (s) were combined by mixing. In another aspect, and in one embodiment, the antigen (s) and / or agent (s) are expressed recombinantly by the yeast vehicle or by the yeast cell or yeast spheroplast from which the yeast vehicle was derived .
[0109] [000109] Numerous antigens and / or other proteins to be produced by a yeast vehicle of the present invention are any number of antigens and / or other proteins that can reasonably be produced by a yeast vehicle, and typically range from at least one to at least about 6 or more, including about 2 to about 6 heterologous antigens and / or other proteins.
[0110] [000110] The expression of an antigen or other protein in a yeast vehicle of the present invention is carried out using techniques known to those skilled in the art. Briefly, a nucleic acid molecule that encodes at least one desired antigen or other protein is inserted into an expression vector so that the nucleic acid molecule is operationally linked to a transcription control sequence to be able to effect the constitutive or regulated expression of the nucleic acid molecule when transformed into a host yeast cell. Nucleic acid molecules that encode one or more antigens and / or other proteins can be one or more expression vectors operably linked to one or more expression control sequences. Particularly important expression control sequences are those that control the initiation of transcription, such as promoter and upstream activation sequences. Any suitable yeast promoter can be used in the present invention, and several such promoters are known to those skilled in the art. Promoters for expression in Saccharomyces cerevisiae include, but are not limited to, gene promoters that encode the following yeast proteins: alcohol dehydrogenase I (ADH1) or II (ADH2), CUP1, phosphoglycerate kinase (PGK), triose phosphate isomerase (TPI ), translational stretching factor EF-1 alpha (TEF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also called TDH3, for triose phosphate dehydrogenase), galactokinase (GAL1), galactose-1-uridyl transferase (GAL7) , UDP-galactose epimerase (GAL10), cytochrome c1 (CYC1), Sec7 protein (SEC7) and acid phosphatase (PHO5), including hybrid promoters, such as the ADH2 / GAPDH and CYC1 / GAL10 promoters, and including the ADH2 / GAPDH promoter, which is induced when glucose concentrations in the cell are low (for example, from about 0.1 to about 0.2 percent), as well as the CUP1 promoter and the TEF2 promoter. Likewise, numerous upstream activation sequences (UASs), also called intensifiers, are known. Upstream activation sequences for expression in Saccharomyces cerevisiae include, but are not limited to, the UASs of genes encoding the following proteins: PCK1, TPI, TDH3, CYC1, ADH1, ADH2, SUC2, GAL1, GAL7 and GAL10, as well as other UASs activated by the GAL4 gene product, with UAS ADH2 being used in one respect. As UAS ADH2 is activated by the product of the ADR1 gene, it may be preferable to overexpress the ADR1 gene when a heterologous gene is operationally linked to UAS ADH2. Transcription termination sequences for expression in Saccharomyces cerevisiae include the termination sequences of the α factor genes, GAPDH and CYC1.
[0111] [000111] Transcriptional control sequences to express genes in methyltrophic yeast include the transcriptional control regions of the genes encoding alcohol oxidase and dehydrogenase format.
[0112] [000112] The transfection of a nucleic acid molecule into a yeast cell according to the present invention can be carried out by any method by which a nucleic acid molecule can be introduced into the cell and includes, but is not limited to, diffusion , active transport, bath sonication, electroporation, microinjection, lipofection, adsorption and fusion of protoplasts. Transfected nucleic acid molecules can be integrated into the yeast chromosome or maintained in extrachromosomal vectors using techniques known to those skilled in the art. Examples of yeast vehicles carrying these nucleic acid molecules are presented here in detail. As discussed above, yeast cytoplasm, yeast phantom and yeast membrane particles or cell wall preparations can also be produced recombinantly by transfecting intact yeast microorganisms or yeast spheroplasts as desired nucleic acid molecules, producing there the antigen, and then manipulation of microorganisms or spheroplasts using techniques known to those skilled in the art to produce subcellular yeast membrane cytoplasm, phantom or extract or their fractions containing desired antigens or other proteins.
[0113] [000113] Effective conditions for the production of recombinant yeast vehicles and expression of antigen and / or other protein by the yeast vehicle include an effective medium in which a yeast strain can be grown. An effective medium is typically an aqueous medium comprising sources of assimilable carbohydrate, nitrogen and phosphate, as well as salts, minerals, metals and other appropriate nutrients, such as vitamins and growth factors. The medium can comprise complex nutrients or it can be a defined minimum medium. The yeast strains of the present invention can be grown in various containers, including, but not limited to, bioreactors, Erlenmeyer flasks, test tubes, microtiter plates and Petri dishes. Cultivation is carried out at a temperature, pH and oxygen content appropriate for the yeast strain. These cultivation conditions are within the competence of those skilled in the art (see, for example, Guthrie et al. (Eds.), 1991, Methods in Enzymology, vol. 194, Academic Press, San Diego).
[0114] [000114] In some embodiments of the invention, yeast is grown under neutral pH conditions. As used herein, the generic use of the term "neutral pH" refers to a pH range between about pH 5.5 and about pH 8 and, in one aspect, between about pH 6 and about 8. Those versed in the art they will realize that small fluctuations (for example, tenths or hundredths) can occur when measuring with a pH meter. Thus, the use of neutral pH to grow yeast cells means that yeast cells are grown at neutral pH most of the time that they are in culture. In one embodiment, the yeast is grown in a medium maintained at a pH level of at least 5.5 (that is, the pH of the culture medium is not allowed to fall below pH 5.5). In another aspect, yeast is grown at a pH level maintained at about 6, 6.5, 7, 7.5 or 8. The use of a neutral pH in yeast cultivation promotes several biological effects that are desirable characteristics for use of yeast as a vehicle for immunomodulation. For example, growing the yeast at neutral pH allows good yeast growth without a negative effect on cell generation time (eg delaying doubling time). Yeast can continue to grow at high densities without losing its cell wall flexibility. The use of a neutral pH allows the production of yeast with flexible cell walls and / or yeast that is more sensitive to cell wall digesting enzymes (eg, glucanase) at all harvest densities. This trait is desirable, because a yeast with flexible cell walls can induce different or better immune responses compared to a yeast grown under more acidic conditions, for example, by promoting cytokine secretion by antigen presenting cells that have phagocytosed the yeast. (for example, TH1-type cytokines including, but not limited to, IFN-γ, interleukin-12 (IL-12), and IL-2, as well as pro-inflammatory cytokines, such as IL-6). In addition, greater accessibility to antigens located on the cell wall is permitted by these culture methods. In another aspect, the use of neutral pH for some antigens allows the disulfide-bound antigen to be released by treatment with dithiothreitol (DTT), which is not possible when this antigen-expressing yeast is grown in a lower pH environment (for example, example, pH 5).
[0115] [000115] In one embodiment, control of the amount of glycosylation in the yeast is used to control the expression of antigens by the yeast, particularly on the surface. The amount of yeast glycosylation can affect the immunogenicity and antigenicity of the antigen expressed on the surface, since sugar fractions tend to be voluminous. Thus, the existence of sugar fractions on the yeast surface and its impact on the three-dimensional space around the target antigen (s) must be considered in the modulation of the yeast according to the invention. Any method can be used to reduce the amount of glycosylation in the yeast (or increase it, if desired). For example, a mutant strain of yeast that has been selected to have low glycosylation (for example, mnn1, och1 and mnn9 mutants) can be used, or the glycosylation receptor sequences in the target antigen could be mutated. Alternatively, a yeast with abbreviated glycosylation patterns can be used, for example, Pichia. Yeast can also be treated using methods that reduce or alter glycosylation.
[0116] [000116] In one embodiment of the present invention, as an alternative to the expression of an antigen or other protein recombinantly in the yeast vehicle, a yeast vehicle is loaded intracellularly with the protein or peptide, or with carbohydrates or other molecules that serve as an antigen and / or are useful as immunomodulatory or biological response modifying agents according to the invention. Subsequently, the yeast vehicle, which now contains the antigen and / or other proteins intracellularly, can be administered to an individual or loaded into a vehicle, such as a dendritic cell. Peptides and proteins can be inserted directly into yeast vehicles of the present invention by techniques known to those skilled in the art, such as by diffusion, active transport, liposome fusion, electroporation, phagocytosis, freeze-thaw cycles and bath sonication. Yeast vehicles that can be directly loaded with peptides, proteins, carbohydrates or other molecules include intact yeast, as well as spheroplasts, ghosts or cytoplasts, which can be loaded with antigens and other agents after production. Alternatively, intact yeast can be loaded with the antigen and / or agent and then spheroplasts, ghosts, cytoplasts or subcellular particles can be prepared from it. Any number of antigens and / or other agents can be loaded into a yeast vehicle in this mode, from at least 1, 2, 3, 4 or any whole number up to hundreds or thousands of antigens and / or other agents, as would be provided by the loading of a microorganism or its parts, for example.
[0117] [000117] In another embodiment of the present invention, an antigen and / or other agent is physically attached to the yeast vehicle. The physical attachment of the antigen and / or other agent to the yeast vehicle can be accomplished by any method suitable in the art, including covalent and non-covalent combination methods, which include, but are not limited to, chemical crosslinking of the antigen and / or other agent to the outer surface of the yeast vehicle or biological antigen binding and / or other agent to the outer surface of the yeast vehicle, such as by the use of an antibody or other binding partner. Chemical cross-linking can be achieved, for example, by methods that include glutaraldehyde binding, photo-affinity labeling, treatment with carbodiimides, treatment with chemicals capable of forming disulfide bonds and treatment with other cross-linking chemicals common in the art. Alternatively, a chemical substance can be contacted with the yeast vehicle that alters the yeast membrane lipid bilayer load or cell wall composition, so that the outer surface of the yeast is more likely to fuse or bind to antigens and / or another agent with particular loading characteristics. Targeting agents, such as antibodies, binding peptides, soluble receptors and other ligands, can also be incorporated into an antigen such as a fusion protein or otherwise associated with an antigen to bind the antigen to the yeast vehicle.
[0118] [000118] When the antigen or other protein is expressed in or physically attached to the surface of the yeast, spacer arms can, in one aspect, be carefully selected to optimize the expression of antigen or other protein or the content on the surface. The size of the spacer arm (s) can affect how much of the antigen or other protein is exposed for binding to the yeast surface. Thus, depending on which antigen (s) or other protein (s) is (are) being used, those skilled in the art will select a spacer arm that performs appropriate spacing for the antigen or other protein on the surface of the surface. In one embodiment, the spacer arm is a yeast protein of at least 450 amino acids. Spacer arms were discussed in detail above.
[0119] [000119] In yet another embodiment, the yeast vehicle and the antigen or other protein are linked together by a more passive, non-specific or non-covalent binding mechanism, such as by gently mixing the yeast vehicle and the antigen or other protein each other in a buffer or other suitable formulation (for example, mixture).
[0120] [000120] In one embodiment of the invention, the yeast vehicle and the antigen or other protein are both loaded intracellularly into a vehicle, such as a dendritic cell or macrophage, to form the therapeutic composition or vaccine of the present invention. Alternatively, an antigen or other protein can be loaded into a dendritic cell in the absence of the yeast vehicle.
[0121] [000121] In one embodiment, intact yeast (with or without expression of heterologous antigens or other proteins) can be ground or processed in order to produce yeast cell wall preparations, yeast membrane particles or yeast fragments (ie, not intact) and yeast fragments may, in some embodiments, be supplied with or administered with other compositions that include antigens (for example, DNA vaccines, protein subunit vaccines, killed or inactivated pathogens) to increase responses immune. For example, enzymatic treatment, chemical treatment or physical strength (for example, mechanical shear or sonication) can be used to break up the yeast into parts that are used as an adjuvant.
[0122] [000122] In one embodiment of the invention, yeast vehicles usable in the invention include yeast vehicles that have been killed or inactivated. Yeast death or inactivation can be accomplished by any of several suitable methods known in the art. For example, yeast thermal inactivation is a standard way of inactivating yeast, and those skilled in the art can monitor structural changes to the target antigen, if desired, by standard methods known in the art. Alternatively, yeast inactivation methods such as chemical, electrical, radioactive or UV methods can be used. See, for example, the methodology presented in standard yeast culture textbooks, such as Methods of Enzymology, Vol. 194, Cold Spring Harbor Publishing (1990). Any inactivation strategy used must take the secondary, tertiary or quaternary structure of the target antigen into consideration and preserve that structure to optimize its immunogenicity.
[0123] [000123] Yeast vehicles can be formulated in immunotherapy compositions based on yeast or products of the present invention, including preparations to be administered to a subject directly or first loaded into a vehicle, such as a dendritic cell, using numerous techniques known for those skilled in the art. For example, yeast vehicles can be lyophilized. Formulations comprising yeast vehicles can also be prepared by compacting the yeast into a pie or tablet, as is done for the yeast used in baking or fermentation operations. In addition, yeast vehicles can be mixed with a pharmaceutically acceptable excipient, such as an isotonic buffer that is tolerated by a host or host cell. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution and other physiologically balanced aqueous saline solutions. Non-aqueous vehicles, such as fixed oils, sesame oil, ethyl oleate or triglycerides, can also be used. Other usable formulations include suspensions containing viscosity-increasing agents, such as sodium carboxymethylcellulose, sorbitol, glycerol or dextran. Excipients can also contain small amounts of additives, such as substances that increase isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include time-rosal, m- or o-cresol, formalin and benzyl alcohol. Standard formulations can be injectable liquids or solids that can be dissolved in a suitable liquid such as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient may comprise, for example, dextrose, human serum albumin and / or preservatives to which water or sterile saline can be added prior to administration.
[0124] [000124] In one embodiment of the present invention, the composition may include additional agents, which may also be called biological response modifying compounds, or the ability to produce such agents / modifiers. For example, a yeast vehicle can be transfected with or loaded with at least one antigen and at least one biological response modifying agent / compound, or a composition of the invention can be administered together with at least one response modifying agent / agent. biological. Biological response modifiers include adjuvants and other compounds that can modulate immune responses, which can be called immunomodulatory compounds, as well as compounds that modify the biological activity of another compound or agent, such as a yeast-based immunotherapeutic, this biological activity not being limited to effects on the immune system. Certain immunomodulatory compounds can stimulate a protective immune response, while others can suppress a harmful immune response, and whether an immunomodulator is usable in combination with a yeast-based immunotherapeutic agent may depend, at least in part, on the state of the disease or condition to be treated or prevented and / or the individual to be treated. Certain biological response modifiers preferably increase the cell-mediated immune response, while others preferably increase the humoral immune response (i.e., they can stimulate an immune response where there is an increased level of cell-mediated immunity compared to humoral , or vice versa.). Certain biological response modifiers have one or more properties in common with the biological properties of yeast-based immunotherapeutic agents or augment or complement the biological properties of yeast-based immunotherapeutic agents. There are numerous techniques known to those skilled in the art to measure the stimulation or suppression of immune responses, as well as to differentiate cell-mediated immune responses from humoral immune responses, and to differentiate one type of cell-mediated response from another (for example, a TH 17 response versus a TH1 response).
[0125] [000125] Biological response agents / modifiers usable in the invention may include, but are not limited to, cytokines, chemokines, hormones, lipid derivatives, peptides, proteins, polysaccharides, small molecule drugs, antibodies and their antigen-binding fragments ( including, but not limited to, anti-cytokine antibodies, anti-cytokine receptor antibodies, anti-chemokine antibodies), vitamins, polynucleotides, nucleic acid binding fractions, aptamers and growth modulators. Some suitable agents include, but are not limited to, IL-1 or IL-1 or IL-1R agonists, anti-IL-1 or other IL-1 antagonists; IL-6 or IL-6 or IL-6R agonists, anti-IL-6 or other IL-6 antagonists; IL-12 or IL-12 or IL-12R agonists, anti-IL-12 or other IL-12 antagonists; IL-17 or IL-17 or IL-17R agonists, anti-IL-17 or other IL-17 antagonists; IL-21 or IL-21 or IL-21R agonists, anti-IL-21 or other IL-21 antagonists; IL-22 or agonists of IL-22 or IL-22R, anti-IL-22 or other IL-22 antagonists; IL-23 or agonists of IL-23 or IL-23R, anti-IL-23 or other IL-23 antagonists; IL-25 or IL-25 or IL-25R agonists, anti-IL-25 or other IL-25 antagonists; IL-27 or agonists of IL-27 or IL-27R, anti-IL-27 or other IL-27 antagonists; type I interferon (including IFN-α) or type I interferon agonists or antagonists or its receptor; type II interferon (including IFN-γ) or type II interferon agonists or antagonists or its receptor; anti-CD40, CD40L, anti-CTLA-4 antibody (for example, to release anergic T cells); T cell co-stimulators (for example, anti-CD137, anti-CD28, anti-CD40); alemtuzumab (for example, CamPath®), denileukin diftitox (for example, ONTAK®); anti-CD4; anti-CD25; anti-PD-1, anti-PD-L1, anti-PD-L2; agents that block FOXP3 (for example, to stem the activity / death of CD4 + / CD25 + T regulatory cells); Flt3 ligand, imiquimod (AldaraTM), granulocyte-macrophage colony stimulating factor (GM-CSF); granulocyte colony stimulating factor (G-CSF), sargramostim (Leukine®); hormones, including, without limitation, prolactins and growth hormone; Toll-like receptor (TLR) agonists, including, but not limited to, TLR-2 agonists, TLR-4 agonists, TLR-7 agonists and TLR-9 agonists; TLR antagonists, including, but not limited to, TLR-2 antagonists, TLR-4 antagonists, TLR-7 antagonists and TLR-9 antagonists; anti-inflammatory and immunomodulatory agents, including, but not limited to, COX-2 inhibitors (eg, Celecoxib, NSAIDS), glucocorticoids, statins and thalidomide and their analogues, including IMiD ™ s (which are structural and functional analogs of thalidomide (for example, example, REVLI-MID® (lenalidomide), ACTIMID® (pomalidomide)); pro-inflammatory agents, such as fungal or bacterial components or any pro-inflammatory cytokine or chemokine; immunotherapeutic vaccines, including, but not limited to, vaccines based on viruses, bacterial-based vaccines or antibody-based vaccines, and any other immunomodulators, immunopotentivizers, anti-inflammatory agents and / or pro-inflammatory agents. Any combination of these agents is considered by the invention, and any of these agents combined with or administered in a protocol with (for example, concomitantly, sequentially or in other formats with) a yeast-based immunotherapeutic is a composition encompassed by the invention. Such agents are well known in the art. These agents can be used alone or in combination with other agents described herein.
[0126] [000126] Agents can include agonists and antagonists of a given protein or peptide or its domain. As used herein, an "agonist" is any compound or agent, including, without limitation, small molecules, proteins, peptides, antibodies, nucleic acid binding agents and the like, that binds to a receptor or ligand and produces or triggers a response, which may include agents that mimic the action of a naturally occurring substance that binds to the receptor or ligand. An "antagonist" is any compound or agent, including, without limitation, small molecules, proteins, peptides, antibodies, nucleic acid binding agents and others, that block or inhibit or reduce the action of an agonist.
[0127] [000127] The compositions of the invention may also include or can be administered with (concomitantly, sequentially or intermittently with) any other compounds or compositions that are usable to prevent or treat HDV infection or any compounds that treat or ameliorate any symptom of infection by HDV. These agents include, but are not limited to, small molecule drugs against HDAg, or interferons, such as interferon-a2a or pegylated interferon-a2a (PEGASYS®). In addition, the compositions of the invention can be used in conjunction with other immunotherapeutic compositions, including prophylactic and / or therapeutic immunotherapy. Although no immunotherapy composition has been approved in the U.S. for the treatment of HDV, those compositions may include HDV protein or epitope subunit vaccines, HDV viral vector vaccines, cytokines, and / or other immunomodulatory agents (for example, TLR agonists, immunomodulatory drugs).
[0128] [000128] The invention also includes a kit comprising any of the compositions described herein or any of the individual components of the compositions described herein.
[0129] [000129] Methods for the Administration or Use of Compositions of the Invention
[0130] [000130] The compositions of the invention, which may include any one or more (e.g., combinations of two, three, four, five or more) of the yeast-based immunotherapeutic compositions described herein, HDV antigens, including HDV proteins and fusion proteins, and / or recombinant nucleic acid molecules encoding those HDV proteins or fusion proteins described above, and other compositions comprising those yeast-based compositions, antigens, proteins, fusion proteins or recombinant molecules described herein, can be used in various in vivo and in vitro methods, including, but not limited to, to treat and / or prevent HDV infection and its sequelae, in diagnostic assays for HDV or to produce antibodies against HDV.
[0131] [000131] HDV infection is most typically detected by measurement of HDV RNA, detection of HDV antigen (HDAg) and / or detection of anti-HDV (antibodies against HDV). Detection of HDV RNA can be carried out, for example, by nucleotide hybridization assays (which may include in situ hybridization) or reverse transcriptase-polymerase chain reaction (RT-PCR). RT-PCR is the most sensitive of these assays and detects 10 genomes / mL. Serum HDAg or IgM or anti-HDV IgG are typically detected by enzyme linked immunoassay (ELISA) or radioimmunoassay (RIA), and HDAg can also be detected by immunofluorescence or immunohistochemical staining of liver biopsies. Since HBV infection is essential for HDV infection, the presence of HBV surface antigen (HBsAg) usually precedes HDV detection.
[0132] [000132] Acute HDV co-infection (HDV / HBV) is characterized by high titers of anti-HBc IgM (antibodies against HBV core antigen), which are antibodies that disappear in chronic HBV infection. HDAg is also an early marker of acute HDV infection (both in the co-infection and superinfection environments). Anti-HD antibodies are late markers, but can be used to establish the diagnosis if early markers are no longer present and are an indicator of progression to chronic infection. In chronic HDV infection, HDAg was complex with high titers of anti-HD. At this stage, it is difficult to detect HDAg in the liver, but HDV RNA can typically be detected in serum, and high anti-HD titers are the main biomarker used to diagnose chronic HDV infection.
[0133] [000133] HDV is considered to have been eliminated when both HDV RNA in serum and HDAg in the liver become persistently undetectable (Pascarella et al., Supra), although only when accompanied by or followed by HBsAg clearance ( HBV surface antigen) is that the cure is considered to be definitive. The development of anti-HD antibodies is believed to protect against reinfection by HDV, and the clearance of the virus is typically characterized by the normalization of ALT levels, reduced liver necroinflammation and halted progression of liver fibrosis.
[0134] [000134] One embodiment of the invention relates to a method for treating chronic hepatitis D virus (HDV) infection and / or for preventing, ameliorating or treating at least one symptom of chronic HDV infection, in an individual or population of individuals . The method includes the step of administering to an individual or a population of individuals who are chronically infected with HDV of one or more immunotherapeutic compositions of the invention. In one aspect, the composition is an immunotherapeutic composition comprising one or more HDV antigens as described herein, which may include one or more yeast-based immunotherapeutic compositions. In one aspect, the composition includes a fusion protein or protein comprising HDV antigens as described herein and / or a recombinant nucleic acid molecule that encodes that fusion protein or protein. In one aspect, the individual subject or the population of subjects is additionally treated with at least one other therapeutic compound usable for the treatment of HDV infection, such as a type I interferon (e.g., IFN-α).
[0135] [000135] In this embodiment of the invention, the subject to be treated, with chronic HDV infection, will also, consequently, be infected with the hepatitis B virus (HBV). Therefore, it is an additional aspect of this method of the invention to treat the subject concomitantly or sequentially (before or after) HBV infection. Several agents are known to be useful for preventing and / or treating or ameliorating HBV infection. These agents include, but are not limited to, antiviral compounds, including, but not limited to, nucleotide analog reverse transcriptase inhibitors (nRTIs). In one aspect of the invention, suitable antiviral compounds include, but are not limited to: tenofovir (VIREAD®), lamivudine (EPIVIR®), adefovir (HEPSE-RA®), telbivudine (TYZEKA®), entecavir (BARACLUDE®), and their combinations, and / or interferons, such as interferon-a2a or pegylated interferon-a2a (PEGASYS®) or interferon-λ. For the treatment of HBV, these agents are typically administered over long periods of time (for example, daily or weekly for up to one to five years or more).
[0136] [000136] In addition, other compositions for the treatment of HBV can be combined with the compositions of the invention for the treatment of HDV, such as various prophylactic and / or immunote-rapid compositions for HBV. Prophylactic vaccines for HBV have been commercially available since the early 1980s, and are non-infectious viral subunit vaccines that feature the purified recombinant hepatitis B virus (HBsAg) surface antigen and can be administered from birth. There is currently no approved therapeutic vaccine for HBV.
[0137] [000137] Another embodiment of the invention relates to a method for immunizing an individual or population of individuals against HDV to prevent HDV infection, prevent chronic HDV infection and / or reduce the severity of HDV infection in the individual or population of individuals. The method includes the step of administering to an individual or population of individuals who are not infected with HDV (or believed to be not infected with HDV) a composition of the invention. In one aspect, the composition is an immunotherapeutic composition comprising one or more HDV antigens as described herein, including one or more yeast-based immunotherapeutic compositions. In one aspect, the composition includes a fusion protein comprising HDV antigens as described herein, or the recombinant nucleic acid molecule encoding that fusion protein.
[0138] [000138] In one aspect of this embodiment of the invention, the subject or population to be immunized against HDV infection using a composition of the invention is already chronically infected with HBV. In this aspect of the invention, the subject who is already chronically infected with HBV can be newly diagnosed, diagnosed, but currently untreated for HBV infection, regardless of the time since the diagnosis of HBV, or currently undergoing treatment for chronic HBV infection, and may include subjects who have been on treatment for HBV infection for a long period of time (for example, years). These subjects may also include subjects who have been treated for HBV previously and who are believed to be cured of HBV infection at the present time. An HDV immunotherapeutic composition of the invention can also be used to immunize individuals or populations of individuals who may be at a higher risk of becoming infected with HBV and HDV, regardless of the status of HBV infection (for example, these individuals can be immunized even if HBV negative, or if the individual's HBV status is not known), for example, due to the location in an area of the world where HDV is endemic or highly prevalent. Individuals or populations of individuals who are at higher risk of exposure to HBV infection compared to the general population (for example, due to the location, occupation and / or high-risk practices associated with HBV transmission) can also be immunized against HDV (concomitantly), particularly if these individuals are located in an area where HDV is endemic or prevalent.
[0139] [000139] As used herein, the term "treat" HDV infection, or any of its permutations (for example, "treated for HDV infection" and others), refers generally to the application or administration of a composition of the invention once infection (acute or chronic) has occurred, for the purpose of reducing or eliminating detectable viral titers (for example, reducing viral RNA), reducing at least one symptom resulting from infection in the individual, delaying or preventing the onset and / or severity of symptoms and / or sequelae downstream caused by infection, reduction of injury to organs or physiological systems (eg, cirrhosis) resulting from infection (eg, reduction of abnormal ALT levels, reduction of liver inflammation, reduction of liver fibrosis), prevention and / or reduction in the frequency and incidence of hepatocellular carcinoma (HCC), improvement in the function of the organ or system that has been negatively impacted by the infection (normalization of serum d levels) and ALT, improvement in liver information, improvement in liver fibrosis), improvement of immune responses against infection, improvement of immune responses of long-term memory against infection, reduced reactivation of the HDV virus, better general health of the individual or population of individuals and / or better overall survival of the individual or population of individuals. All of these parameters are compared to the status of the individual or the population of individuals in the absence of the use of the HDV immunotherapeutic compositions of the invention.
[0140] [000140] In one aspect, a purpose of treatment is a viral clearance maintained for at least 6 months after the end of therapy. In one aspect, one purpose of treatment is the loss of detectable HDV RNA in the serum and HDAg in the liver, followed by clearance of HBsAg (HBV surface antigen), the latter of which can be achieved when the corresponding HBV infection is treated concomitant or sequentially with the treatment for HDV described herein, such as by concomitant or sequential administration of antiviral drugs for HBV, or other therapies for HBV, including, but not limited to, interferons, immunotherapeutic agents for HBV or other therapeutic treatments for HBV. In one aspect, one purpose of treatment is the development of antibodies (seroconverting) against HDAg (anti-HD). Seroconversion can be determined by radioimmunoassay, enzyme immunoassay. Additional results from successful treatment of HDV infection include normalization of ALT levels, reduction in liver necroinflammation and halting the progression of liver fibrosis.
[0141] [000141] "Preventing" HDV infection, or any of its permutations (for example, "preventing HDV infection" and others), refers generally to the application or administration of a composition of the invention before an HDV infection has occurred for the purpose of preventing HDV infection, preventing chronic HDV infection (that is, allowing an individual to clear an acute HDV infection without further intervention) or at least reducing the severity and / or duration of the infection, and / or the physical injury caused by chronic infection, including by improving survival, in an individual or population of individuals if the infection occurs later. In one aspect, the present invention can be used to prevent chronic HDV infection, such as allowing an individual who becomes acutely infected with HDV subsequent to administration of a composition of the invention to clear the infection and not become chronically infected. In one aspect, the present invention is used to prevent or reduce the occurrence or severity of HDV infection in an individual chronically infected with HBV, even if the HBV infection has not been cured.
[0142] [000142] The present invention includes the distribution (administration, immunization) of one or more immunotherapeutic compositions of the invention, including a yeast-based immunotherapeutic composition for HDV, to a subject. The administration process can be carried out ex vivo or in vivo, but it is typically carried out in vivo. Ex vivo administration refers to performing part of the regulatory step outside the patient, such as administering a composition of the present invention to a population of cells (dendritic cells) removed from a patient, under conditions where a yeast vehicle, antigen (s) ) and any other agents or compositions are loaded into the cell, and the cells are returned to the patient. The therapeutic composition of the present invention can be returned to a patient, or administered to a patient, by any suitable mode of administration.
[0143] [000143] The administration of a composition can be systemic, mucous and / or proximal to the location of the target site (for example, close to an infection site). Appropriate routes of administration will be clear to those skilled in the art, depending on the type of condition to be prevented or treated, the antigen used and / or the target cell or tissue population. Various acceptable methods of administration include, but are not limited to, intravenous administration, intraperitoneal administration, intramuscular administration, intranodal administration, intracoronary administration, intraarterial administration (for example, in a carotid artery), subcutaneous administration, transdermal delivery, intratracheal administration, administration intraarticular, intraventricular administration, inhalation (for example, aerosol), intracranial, intraspinal, intraocular, aural, intranasal, oral, pulmonary administration, catheter impregnation and direct injection into a tissue. In one aspect, routes of administration include: intravenous, intraperitoneal, subcutaneous, intradermal, intranodal, intramuscular, transdermal, inhaled, intranasal, oral, intraocular, intraarticular, intracranial and intraspinal. Parenteral distribution can include intradermal, intramuscular, intraperitoneal, intrapleural, intrapulmonary, intravenous, subcutaneous routes, via atrial catheter and venous catheter. Aural distribution can include ear drops, intranasal distribution can include nasal drops or intranasal injection, and intraocular distribution can include eye drops. Aerosol delivery (inhalation) can also be performed using standard methods in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA 189: 11277-11281, 1992). Other routes of administration that modulate mucosal immunity may be useful in the treatment of viral infections. These routes include the bronchial, intradermal, intramuscular, intranasal, other inhalations, rectal, subcutaneous, topical, transdermal, vaginal and urethral. In one aspect, an immunotherapeutic composition of the invention is administered subcutaneously.
[0144] [000144] With respect to the yeast-based immunotherapy compositions of the invention, in general, a suitable single dose is a dose that is capable of effectively delivering a yeast vehicle and an antigen (if included) to a given cell type, tissue or region of the patient's body, in an amount effective to elicit an antigen-specific immune response against one or more HDV antigens or epitopes, when administered one or more times over an appropriate period of time. For example, in one embodiment, a single dose of a yeast vehicle of the present invention is from about 1 x 105 to about 5 x 107 yeast cell equivalents per kilogram of body weight of the organism to which the composition is administered. In one aspect, a single dose of a yeast vehicle of the present invention is about 0.1 Y. (1 x 106 cells) at about 100 Y.U. (1 x 109 cells) per dose (ie, per organism), including any intermediate dose, in increments of 0.1 x 106 cells (ie, 1.1 x 106, 1.2 x 106, 1.3 x 106). In one embodiment, doses include doses between 1 Y.U and 40 Y.U., doses between 1 Y.U. and 50 Y.U., doses between 1 Y.U. and 60 Y.U., doses between 1 Y.U. and 70 Y.U., or doses between 1 Y.U. and 80 Y.U. and, in one aspect, between 10 Y.U. and 40 Y.U., 50 Y.U., 60 Y.U., 70 Y.U., or 80 Y.U. In one embodiment, doses are administered at different locations in the individual, but over the same dosing period. For example, a dose of 40 Y.U. can be administered by injection of 10 Y.U. at four different locations in the subject over a dosing period, or a dose of 20 Y.U. can be administered by injection of 5 Y.U. at four different locations in the individual or by injecting doses of 10 Y.U. at two different locations in the individual during the same dosing period. The invention includes administration of a quantity of the yeast-based immunotherapy composition (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14,15, 16 , 17, 18, 19, 20 YU or more) at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different locations in an individual, to form a single dose.
[0145] [000145] "Reinforcers" or "reinforcers" of a therapeutic composition are administered, for example, when the immune response against the antigen has decreased or when necessary to provide an immune response or induce a memory response against a particular antigen or antigens. Reinforcers can be administered about 1, 2, 3, 4, 5, 6, 7 or 8 weeks apart from each other every month, every two months, every four months, annually or several years after the original administration. In one embodiment, an administration schedule is one in which from about 1 x 105 to about 5 x 107 yeast cell equivalents of a composition per kg of body weight are administered at least 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10 or more times over a period of time from weeks to months to years. In one embodiment, doses are administered weekly for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses, followed by monthly doses when necessary to achieve the desired inhibition or elimination of HDV. For example, doses can be administered until the individual reaches seroconversion, until HDV RNA titers are persistently undetectable, until HDAg is not detected in the liver, until HBV HBsAg is not detected and / or until ALT levels normalize. In one embodiment, doses are administered in a 4-week protocol (every 4 weeks or day 1, week 4, week 8, week 12, and so on to enter 2 and 10 doses or more, as determined by the medium ). Additional doses may be administered even after the individual has achieved seroconversion, if desired, although this dosage may not be necessary.
[0146] [000146] Immunotherapeutic compositions for HDV of the invention, including yeast-based immunotherapeutic compositions for HDV, can be administered with one or more additional therapeutic or prophylactic agents. Such therapeutic or prophylactic agents may include agents that are useful for the prevention and / or treatment of HDV and may, in addition, include agents that are useful for the prevention and / or treatment of HBV. In one aspect of the invention, one or more additional therapeutic or prophylactic agents are administered sequentially with the yeast-based immunotherapy composition. In another embodiment, one or more additional therapeutic or prophylactic agents are administered before the yeast-based immunotherapy composition is administered. In another embodiment, one or more additional therapeutic or prophylactic agents are administered after the yeast-based immunotherapy composition for HDV is administered. In one embodiment, one or more additional therapeutic or prophylactic agents are administered in alternating doses with the yeast-based immunotherapy composition, or in a protocol in which the yeast-based HDV composition is administered at prescribed intervals between or with a or more consecutive doses of the additional agents, or vice versa. In one embodiment, the yeast-based HDV immunotherapy composition is administered in one or more doses over a period of time before the administration of the additional agents begins. In other words, the yeast-based immunotherapeutic composition for HDV is administered as a monotherapy over a period of time and then administration of therapeutic or prophylactic agent is added, concomitantly with new doses of yeast-based HDV immunotherapy. or alternatively with yeast-based immunotherapy. Alternatively, the agent can be administered over a period of time before administration of the yeast-based immunotherapy composition for HDV begins.
[0147] [000147] In an embodiment of the invention, an additional therapeutic agent to be used in conjunction with the yeast-based immunotherapeutic composition for yeast-based HDV is an interferon. In one aspect, interferon is interferon-α and, in one aspect, interferon-a2b (administered by subcutaneous injection 3 times a week); or pegylated interferon-a2a (for example, PEGASYS®). As used herein, the term "interferon" refers to a cytokine that is typically produced by cells of the immune system and a wide variety of cells in response to the presence of double-stranded RNA. Interferons assist the immune response by inhibiting viral replication within host cells, activating natural killer cells and macrophages, increasing antigen presentation to lymphocytes and inducing host cell resistance to viral infection. Type I interferons include interferon-α. Type III interferons include interferon-λ. Interferons usable in the methods of the present invention include any type I or type III interferon, including interferon-α, interferon-a2, and, in one aspect, longer-lasting forms of interferon, including, but not limited to, interferons pegylates, interferon fusion proteins (albumin-fused interferon) and controlled release formulations comprising interferon (for example, interferon in microspheres or interferon with polyamino acid nanoparticles). An interferon, PEGASYS®, pegylated interferon-a2a, is a covalent recombinant interferon-a2a conjugate (approximate molecular weight [PM] of 20,000 daltons) with a single branching bis-monomethoxy polyethylene glycol (PEG) chain (approximate MP 40,000 daltons). The PEG fraction is linked in a single site to the interferon-α fraction through a stable amide bond to lysine. Pegylated interferon-a2a has an approximate molecular weight of 60,000 daltons.
[0148] [000148] For the treatment of HDV, interferon is typically administered by intramuscular or subcutaneous injection and is usually administered in high doses over a long period of time. In one embodiment, standard IFN-α is administered at about 9 million units three times a week or 5 million units daily for 12 months, which can be extended if HBsAg is not cleared. Pegylated IFN-α can be administered weekly at a dose between 3 and 10 million units, with 3 million units being preferred in one modality, and higher doses being preferred in other modalities (eg 4 million units, 5 million units, 6 million units, 7 million units, 8 million units, 9 million units or 10 million units). In general, interferon doses are administered on a regular schedule, which can vary from 1, 2, 3, 4, 5 or 6 times a week, to weekly, biweekly, every three weeks, or monthly, and depending on the type of administered interferon, patient tolerance and resolution of infection A typical dose of interferon that is currently available is provided weekly (pegylated IFN-α), and this is a preferred dosing schedule for interferon, according to the present invention.
[0149] [000149] In one aspect of the invention, when a course of treatment with interferon therapy begins, additional doses of the immunotherapeutic composition of the invention are administered over the same period of time, or for at least part of that time, and may continue to be administered once the interferon course has ended. However, the dosing schedule for immunotherapy throughout the period can be, and is expected to typically be, different from that for interferon. For example, the immunotherapeutic composition can be administered on the same days or at least 3 - 4 days after the last (most recent) given dose of interferon (or any suitable number of days after the last dose), and can be administered daily, weekly. , biweekly, monthly, bimonthly or every 3 - 6 months, or at longer intervals, as determined by the doctor. During an initial monotherapy period, the administration of the immunotherapeutic composition, if used, the immunotherapeutic composition is preferably administered weekly between 4 and 12 weeks, followed by monthly administration (regardless of when additional interferon is added to the protocol). In one aspect, the immunotherapeutic composition is administered weekly for four or five weeks, followed by monthly administration thereafter, until the completion of the complete treatment protocol. In one aspect of the invention, the use of an HDV immunotherapeutic composition of the invention is done in an interferon-free protocol (i.e., as monotherapy or in combination with one or more non-interferon agents).
[0150] [000150] In one aspect of the invention, an additional therapeutic agent to be administered in conjunction with the yeast-based immunotherapy composition for yeast-based HDV is an antiviral compound that is effective for the treatment of coexisting HBV infection in the subject. As used herein, the term "antiviral" refers to any compound or drug, typically a small molecule inhibitor or antibody, that targets one or more steps in the virus life cycle, with direct antiviral therapeutic effects. Suitable antiviral compounds include, but are not limited to: tenofovir (VIREAD®), lamivudine (EPIVIR®), adefovir (HEPSERA®), telbivudine (TYZEKA®), entecavir (BARACLUDE®), and combinations thereof.
[0151] [000151] Tenofovir (tenofovir diisoproxyl fumarate or TDF), or phosphonic acid ({[(2R) -1- (6-amino-9H-purin-9-yl) propan-2-yl] oxy} methyl), is a nucleotide analog reverse transcriptase inhibitor (nRTIs). For the treatment of HBV infection, tenofovir is typically administered to adults as a pill taken at a dose of 300 mg (tenofovir diisoproxil fumarate) once daily. The dosage for pediatric patients is based on the patient's body weight (8 mg per kg body weight, up to 300 mg once daily) and can be supplied as a tablet or oral powder.
[0152] [000152] Lamivudine, or 2 ', 3'-dideoxy-3'-thiacitidine, commonly called 3TC, is a potent inhibitor of nucleoside analog reverse transcriptase (nRTI). For the treatment of HBV infection, lamivudine is administered as a pill or oral solution taken at a dose of 100 mg once daily (3.1 - 4.4 mg / kg (1.4 - 2 mg / lb) twice a day for children from 3 months to 12 years old).
[0153] [000153] Adefovir (adefovir dipivoxil), or 9- [2 - [[bis [(pivaloyloxy) methoxy] -phosphinyl] -methoxy] ethyl] adenine, is an orally administered nucleotide analog reverse transcriptase inhibitor (ntRTI) . For the treatment of HBV infection, adefovir is administered as a pill taken at a dose of 10 mg once a day.
[0154] [000154] Telbivudine, or 1- (2-deoxy-β-L-erythro-pentofuranosyl) -5-methylpyrimidine-2,4 (1H, 3H) -dione, is a synthetic thymidine nucleoside analogue (the L isomer of thymidine ). For the treatment of HBV infection, telbivudine is administered as a pill or oral solution taken at a dose of 600 mg once a day.
[0155] [000155] Entecavir, or 2-Amino-9 - [(1S, 3R, 4S) -4-hydroxy-3- (hydroxy-tyl) -2-methylidenocyclopentyl] -6,9-dihydro-3H-purin-6- ona, is a nucleoside analog (guanine analog) that inhibits reverse transcription, DNA replication and virus transcription. For the treatment of HBV infection, entecavir is administered as a pill or oral solution taken at a dose of 0.5 mg once daily (1 mg daily for mutations refractory to lamivudine or resistant to telbivudine).
[0156] [000156] In aspects of the invention, an immunotherapeutic composition and other agents can be administered together (concurrently). As used herein, concomitant use does not necessarily mean that all doses of all compounds are administered on the same day at the same time. Rather, concomitant use means that each of the components of therapy (for example, immunotherapy and interferon therapy) is started at approximately the same period (hours apart up to 1 - 7 days apart or more (weeks or months away) ), but administered as part of the same protocol) and are administered over the same general period of time, it should be noted that each component may have a different dosing schedule (eg interferon, weekly, immunotherapy, monthly). In addition, before or after concomitant administration, any of the immunotherapeutic agents or compositions can be administered without the other agent (s).
[0157] [000157] In the present invention, it is considered that the use of an immunotherapeutic composition of the invention with an interferon, such as IFN-α, allows a shorter course of time for the use of the interferon, or may allow the elimination of the interferon. Dosage requirements for interferon can also be reduced or modified as a result of combining with the immunotherapeutic of the invention to generally improve the patient's tolerance to the drug. In addition, the immunotherapeutic composition of the invention is considered to allow seroconversion or maintained viral responses for patients in whom only interferon therapy is unable to achieve these goals. In other words, more patients will achieve viral negativity or seroconversion when an immunotherapeutic composition of the invention is combined with an interferon than they would achieve viral negativity or seroconversion with the use of interferon alone.
[0158] [000158] In the method of the present invention, therapeutic compositions and compositions can be administered to an animal, including any vertebrate, and particularly to any member of the class Vertebrates, Mammalia, including, without limitation, primates, rodents, cattle and pets . Livestock includes mammals to be consumed or that produce useful products (for example, sheep for wool production). Mammals to be treated or protected include humans, dogs, cats, mice, rats, goats, sheep, cattle, horses and pigs.
[0159] [000159] An "individual" is a vertebrate, like a mammal, including, without limitation, a human being. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, mice and rats. The term "individual" can be used interchangeably with the term "animal", "subject" or "patient". General Techniques Usable in the Invention
[0160] [000160] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry and immunology, which are well known for those skilled in the art. These techniques are explained in more detail in the literature, such as Methods of Enzymology, Vol. 194, Guthrie et al., Eds., Cold Spring Harbor Laboratory Press (1990); Biology and activities of yeasts, Skinner, et al., Eds., Academic Press (1980); Methods in yeast genetics: a laboratory course manual, Rose et al., Cold Spring Harbor Laboratory Press (1990); The Yeast Saccharomyces: Cell Cycle and Cell Biology, Pringle et al., Eds., Cold Spring Harbor Laboratory Press (1997); The Yeast Saccharomyces: Gene Expression, Jones et al., Eds., Cold Spring Harbor Laboratory Press (1993); The Yeast Saccharomyces: Genome Dynamics, Protein Synthesis, and Energetics, Broach et al., Eds., Cold Spring Harbor Laboratory Press (1992); Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001), (together here called "Sambrook"); Current Protocols in Molecular Biology (F.M. Ausubel et al., Eds., 1987, including supplements until 2001); PCR: The Polymerase Chain Reaction, (Mullis et al., Eds., 1994); Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York; Harlow and Lane (1999) Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (collectively called "Harlow and Lane" here), Beaucage et al. eds., Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, Inc., New York, 2000); Casarett and Doull’s Toxicology The Basic Science of Poisons, C. Klaassen, ed., 6th edition (2001), and Vaccines, S. Plotkin and W. Orenstein, eds., 3rd edition (1999). General Settings
[0161] [000161] A "TARMOGEN®" (Globelmmune, Inc., Louisville, Colorado) refers generically to a yeast vehicle that expresses one or more heterologous antigens extracellularly (on its surface), intracellularly (internally or cytosolically) or as much ex-tracellularly, when intracellularly. TARMOGEN® products have been generally described (see, for example, U.S. Patent No. 5,830,463). Certain yeast-based immunotherapy compositions, and methods of their preparation and generic use, are also described in detail, for example, in U.S. Patent No. 5,830,463, U.S. Patent No. 7,083,787, North Patent American No. 7,736,642, Stubbs et al., Nat. Med. 7: 625-629 (2001), Lu et al., Cancer Research 64: 5084-5088 (2004), and in Bernstein et al., Vaccine , January 24, 2008; 26 (4): 509-21, each incorporated herein by reference in its entirety.
[0162] [000162] As used herein, the term "analog" refers to a chemical compound that is structurally similar to another compound, but differs slightly in composition (as in the replacement of an atom by an atom of a different element or in the presence of a particular functional group, or the replacement of a functional group by another functional group). Thus, an analog is a compound that is similar or comparable in function and appearance, but with a different structure or origin in relation to the reference compound.
[0163] [000163] The terms "substituted", "substituted derivative" and "derivative", when used to describe a compound, mean that at least one hydrogen bonded to the unsubstituted compound is replaced by a different atom or chemical fraction.
[0164] [000164] Although a derivative has a physical structure similar to the parent compound, the derivative may have different chemical and / or biological properties than the parent compound. These properties may include, but are not limited to, increased or decreased activity of the parent compound, new activity compared to the parent compound, increased or decreased bioavailability, increased or decreased efficacy, increased or decreased stability in vitro and / or in and / or increased or decreased absorption properties.
[0165] [000165] In general, the term "biologically active" indicates that a compound (including a protein or peptide) has at least one detectable activity that has an effect on the metabolic or other processes of a cell or organism, as measured or observed in live (ie, in a natural physiological environment) or in vitro (ie, under laboratory conditions).
[0166] [000166] According to the present invention, the term "modular" can be used interchangeably with "regular" and refers generally to the upward or downward regulation of a particular activity. As used herein, the term "regulate up" can be used generically to describe any of: provoke, initiate, increase, magnify, reinforce, improve, intensify, amplify, promote or provide in relation to a particular activity. Likewise, the term "downward" can be used generically to describe any of: decrease, reduce, inhibit, improve, shorten, block or prevent in relation to a particular activity.
[0167] [000167] In one embodiment of the present invention, any of the amino acid sequences described herein can be produced with at least one to about 20 additional heterologous amino acids flanking each of the C and / or N-terminal ends of the specified amino acid sequence. The resulting protein or polypeptide can be called "consisting essentially of" the specified amino acid sequence. According to the present invention, heterologous amino acids are a sequence of amino acids that are not naturally found (i.e., not found in nature, in vivo) flanking the specified amino acid sequence, or that are not related to the function of the amino acid sequence specified, or that are not encoded by the nucleotides that flank the naturally occurring nucleic acid sequence that encodes the specified amino acid sequence as it occurs in the gene, if those nucleotides in the naturally occurring sequence are translated using standard codons for the organism from which the given amino acid sequence is derived. Likewise, the term "consisting essentially of", when used with reference to a nucleic acid sequence here, refers to a nucleic acid sequence that encodes a specified amino acid sequence that can be flanked by at least one and up to about 60 additional heterologous nucleotides at each of the 5 'and / or 3' ends of the nucleic acid sequence encoding the specified amino acid sequence. Heterologous nucleotides are not naturally found (that is, they are not found in nature, in vivo) flanking the nucleic acid sequence that encodes the specified amino acid sequence as occurs in the natural gene or encodes a protein that provides any additional function to the protein or alter the function of the protein with the specified amino acid sequence.
[0168] [000168] According to the present invention, the term "selectively binds" refers to the ability of an antibody, antigen binding fragment or binding partner of the present invention to preferentially bind to specified proteins. More specifically, the term "selectively binds" refers to the specific binding of one protein to another (for example, an antibody, its fragment or binding partner to an antigen), where the level of binding, as measured by any standard assay (for example, an immunoassay), is statistically significantly higher than the background assay control. For example, when performing an immunoassay, controls typically include a reaction well / tube that contains only antibody or antigen-binding fragment (that is, in the absence of antigen), where an amount of reactivity (for example, binding well-specific) by the antibody or its antigen-binding fragment in the absence of the antigen is considered to be the bottom. Binding can be measured using a number of methods standardized in the art, including enzyme immunoassays (eg, ELISA, immunoblotting assays and others).
[0169] [000169] Reference to a protein or polypeptide used in the present invention includes full-length proteins, fusion proteins or any fragment, domain, conformational or homologous epitope of those proteins, including functional domains and immunological domains of proteins. More specifically, an isolated protein according to the present invention is a protein (including a polypeptide or peptide) that has been removed from its natural environment (that is, that has been subjected to human manipulation) and can include proteins purified, partially purified proteins, proteins produced recombinantly and proteins produced synthetically, for example. Thus, "isolated" does not reflect the extent to which the protein has been purified. Preferably, an isolated protein of the present invention is produced recombinantly. According to the present invention, the terms "modification" and "mutation" can be used interchangeably, particularly with respect to modifications / mutations in the amino acid sequence of proteins or their parts (or nucleic acid sequences) described herein .
[0170] [000170] As used herein, the term "homologous" is used to refer to a protein or peptide that differs from a naturally occurring protein or peptide (i.e., the "prototype" or "wild-type" protein) by small modifications to the naturally occurring protein or peptide, but maintaining the protein structure and basic collateral chain of the naturally occurring form. These changes include, but are not limited to: changes in one or a few collateral chains of amino acids; changes to one or a few amino acids, including deletions (for example, a truncated version of the protein or peptide) insertions and / or substitutions; changes in the stereochemistry of one or a few atoms; and / or small derivations, including, but not limited to: methylation, glycosylation, phosphorylation, acetylation, mylysylation, prenylation, palmitation, amidation and / or addition of glycosylphosphidyl inositol. A homologue may have increased, decreased or substantially similar properties compared to the naturally occurring protein or peptide. A homolog can include a protein agonist or a protein antagonist. Homologues can be produced using techniques known in the art for protein production including, but not limited to, direct modifications of the isolated naturally occurring protein, direct protein synthesis or modifications of the nucleic acid sequence encoding the protein using, for example, classic or recombinant DNA techniques to perform random or targeted mutagenesis.
[0171] [000171] A homologue of a given protein may comprise, consist essentially of or consist of an amino acid sequence that is at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91% identical, or at least about 92% identical, or at least about 93% identical, or at least about 94% identical, or at least about 95% identical, or at least at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least about 99% identical (or any percentage of identity between 45% and 99%, in whole increments) , to an amino acid sequence of the reference protein. In one embodiment, the homologue comprises, consists essentially of, or consists of, an amino acid sequence that is less than 100% identical, less than about 99% identical, less than about 98% identical, less than about 97% identical, less than about 96% identical, less than about 95% identical, and so on, in 1% increments, unless about 70% identical to the naturally occurring amino acid sequence of the reference protein.
[0172] [000172] A homologue can include proteins or protein domains that are "almost full-length", which means that that homologue differs from the full-length protein, functional domain or immune domain (how that protein, functional domain or immune domain is described herein or otherwise known or described in a publicly available sequence) by adding or deleting 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the N and / or C terminus of that protein full length or full length functional domain or full length immune domain.
[0173] [000173] As used herein, unless otherwise specified, reference to a percentage (%) of identity refers to a homology assessment that is performed using: (1) a BLAST 2.0 Basic BLAST homology search using blastp for amino acid searches and blastn for nucleic acid searches with standard predetermined parameters, where the query string is filtered to regions of low complexity by default (described in Altschul, SF, Madden, TL, Schääffer, AA, Zhang, J., Zhang, Z., Miller, W. & Lipman, DJ (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs." Nucleic Acids Res. 25: 3389-3402, incorporated herein by reference in its entirety); (2) a BLAST 2 alignment (using the parameters described below); (3) and / or PSI-BLAST with the default predetermined parameters (BLAST with position specific iteration). It should be noted that, due to some differences in the standard parameters between BLAST 2.0 Basic BLAST and BLAST 2, two specific strings could be recognized as having significant homology using the BLAST 2 program, whereas a search performed in BLAST 2.0 Basic BLAST using one of the strings as the query string could not identify the second string in the main matches. In addition, PSI-BLAST provides an automated, easy-to-use version of a "profile" search, which is a sensitive way to search for sequence counterparts. The program first performs a search in the BLAST database with gaps. The PSIBLAST program uses information from any significant alignments provided to build a position-specific counting matrix, which replaces the query string for the next search round in the database. Consequently, it must be understood that the percentage of identity can be determined using any of these programs.
[0174] [000174] Two specific sequences can be aligned with each other using the BLAST 2 sequence as described in Tatusova and Madden, (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174: 247-250, hereby incorporated by reference in its entirety. The BLAST 2 sequence alignment is performed in blastp or blastn using the BLAST 2.0 algorithm to perform a BLAST search with Gap (BLAST 2.0) between the two sequences, allowing the introduction of gaps (deletions and insertions) in the resulting alignment. For the sake of clarity here, a BLAST 2 sequence alignment is performed using the following standard predetermined parameters.
[0175] [000175] For blastn, using matrix 0 BLOSUM62: Correspondence reward = 1 Mismatch penalty = -2 Open span penalties (5) and span span (2) Word size (on) filter (on) expected (10) from x_ span drop
[0176] [000176] For blastp, using matrix 0 BLOSUM62: Open span penalties (11) and span span (1) Word size filter (on) (3) expected (10) of x_fall span
[0177] [000177] An isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural environment (ie, that has been subjected to human manipulation), its natural environment being the genome or chromosome in which the acid molecule nucleic acid is found in nature. Thus, "isolated" does not necessarily reflect which nucleic acid molecule was purified, but it does indicate that the molecule does not include an entire genome or an entire chromosome in which the nucleic acid molecule is found in nature. An isolated nucleic acid molecule can include a gene. An isolated nucleic acid molecule that includes a gene is not a fragment of a chromosome that includes that gene, but instead includes the coding region and regulatory regions associated with the gene, but no additional agents that are naturally found on the same chromosome . An isolated nucleic acid molecule can also include a specified nucleic acid sequence flanked (that is, at the 5 'and / or 3' ends of the sequence) by additional nucleic acids that do not normally flank the specified nucleic acid sequence in nature (i.e. that is, heterologous sequences). The isolated nucleic acid molecule can include DNA, RNA (for example, mRNA), or derivatives of DNA or RNA (for example, cDNA). Although the expression "nucleic acid molecule" refers mainly to the physical nucleic acid molecule, and the expression "nucleic acid sequence" refers mainly to the nucleotide sequence in the nucleic acid molecule, the two expressions can be used in different ways. interchangeably, particularly with respect to a nucleic acid molecule, or nucleic acid sequence, that is capable of encoding a protein or domain of a protein.
[0178] [000178] A recombinant nucleic acid molecule is a molecule that can include at least one of any nucleic acid sequence that encodes any one or more proteins described herein operably linked to at least one of any transcription control sequence capable of effectively regulating the expression of the nucleic acid molecule (s) in the cell to be transfected. Although the term "nucleic acid molecule" refers primarily to the physical nucleic acid molecule, and the term "nucleic acid sequence" refers primarily to the nucleotide sequence in the nucleic acid molecule, the two expressions can be used interchangeably , particularly with respect to a nucleic acid molecule, or nucleic acid sequence, that is capable of encoding a protein or domain of a protein. In addition, the term "recombinant molecule" refers primarily to a nucleic acid molecule operably linked to a transcriptional control sequence, but can be used interchangeably with the term "nucleic acid molecule" that is administered to an animal .
[0179] [000179] A recombinant nucleic acid molecule includes a recombinant vector, which is any nucleic acid sequence, typically a heterologous sequence, that is operably linked to the isolated nucleic acid molecule encoding a fusion protein of the present invention, which is capable of to allow recombinant production of the fusion protein, and that is capable of delivering the nucleic acid molecule to a host cell according to the present invention. This vector can contain nucleic acid sequences that are not found naturally adjacent to the isolated nucleic acid molecules to be inserted into the vector. The vector can be RNA or DNA, prokaryotic or eukaryotic and, preferably in the present invention, is a virus or plasmid. Recombinant vectors can be used in cloning, sequencing and / or other forms of manipulation of nucleic acid molecules and can be used in the distribution of these molecules (for example, as in a DNA composition or a viral vector-based composition). Recombinant vectors are preferably used in the expression of nucleic acid molecules and can also be called expression vectors. Preferred recombinant vectors are capable of expression in a transfected host cell.
[0180] [000180] In a recombinant molecule of the present invention, the nucleic acid molecules are operably linked to expression vectors containing regulatory sequences, such as transcription control sequences, translation control sequences, origins of replication and other regulatory sequences that are compatible with the host cell and which control the expression of nucleic acid molecules of the present invention. In particular, recombinant molecules of the present invention include nucleic acid molecules that are operably linked to one or more expression control sequences. The term "operably linked" refers to the binding of the nucleic acid molecule to an expression control sequence so that the molecule is expressed when transfected (i.e., transformed, transduced or transfected) in a host cell.
[0181] [000181] According to the present invention, the term "transfection" is used to refer to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into a cell . The term "transformation" can be used interchangeably with the term "transfection" when that term is used to refer to the introduction of nucleic acid molecules into microbial cells, such as algae, bacteria and yeast. In microbial systems, the term "transformation" is used to describe an inherited change due to the acquisition of exogenous nucleic acids by the microorganism and is essentially synonymous with the term "transfection". Consequently, transfection techniques include, but are not limited to, transformation, chemical treatment of cells, bombardment of particles, electroporation, mi-chrojection, lipofection, adsorption, infection and protoplast fusion.
[0182] [000182] The following experimental results are presented for purposes of illustration and are not intended to limit the scope of the invention. EXAMPLES Example 1
[0183] [000183] The following example describes the production of a yeast-based immunotherapeutic composition for the treatment or prevention of hepatitis D virus (HDV) infection.
[0184] [000184] In this experiment, yeast (for example, Saccharomyces cerevisiae) was manipulated to express an HDV antigen under the control of the copper-inducible promoter, CUP1. The HDV antigen was a single polypeptide of approximately 218 amino acids, with the following sequence elements fused in the frame from the N to C terminus, represented by SEQ ID NO: 30: (1) an N-terminal peptide to confer resistance to degradation pro-teassomic and stabilize expression (positions 1 to 6 of SEQ ID NO: 30); 2) a two amino acid spacer (Thr-Ser) to introduce a SpeI restriction enzyme site (positions 7 to 8 of SEQ ID NO: 30); 3) the amino acid sequence of a large HDV genotype 1 (L) antigen (HDAg-L) that has been modified to delete the nuclear localization sequence (positions 9 to 212 of SEQ ID NO: 30, also represented here by SEQ ID NO: 28); and 4) a hexa-histidine tag (positions 213 to 218 of SEQ ID NO: 30). A nucleic acid sequence encoding the fusion protein of SEQ ID NO: 30 (from codons optimized for expression in yeast) is represented here by SEQ ID NO: 29. SEQ ID NO: 30 (and SEQ ID NO: 28) contains multiple epitopes or domains believed to increase the immunogenicity of the HDV antigen. For example, positions 34 to 42 and positions 51 to 59 of SEQ ID NO: 30 comprise known MHC Class I T cell epitopes, and positions 34 to 49, positions 58 to 73, positions 74 to 87, positions 104 to 119 and positions 120 - 151 comprise known MHC Class II T cell epitopes. A yeast immunotherapy composition that expresses SEQ ID NO: 30 is also referred to here as HDV1.
[0185] [000185] A second product that expresses the same HDV antigen as above, but with the retained NLS, was produced as follows. Yeast (eg, Saccharomyces cerevisiae) was engineered to express the HDV antigen under the control of the copper-inducible promoter, CUP1. The HDV antigen was a single polypeptide of approximately 228 amino acids, with the following sequence elements fused in the frame from the N to the C terminus, represented by SEQ ID NO: 33: (1) an N-terminal peptide to confer resistance to degradation protosomal and stabilize expression (positions 1 to 6 of SEQ ID NO: 33); 2) a two amino acid spacer (Thr-Ser) to introduce a SpeI restriction enzyme site (positions 7 to 8 of SEQ ID NO: 33); 3) the amino acid sequence of a large antigen (L) of HDV genotype 1 (HDAg-L) corresponding to SEQ ID NO: 2 except for the substitution of an alanine in place of glutamine at position 66 of SEQ ID NO: 2 (positions 9 to 222 of SEQ ID NO: 33, also represented here by SEQ ID NO: 31); and 4) a hexa-histidine tag (positions 223 to 228 of SEQ ID NO: 33). A nucleic acid sequence encoding the fusion protein of SEQ ID NO: 33 (from codons optimized for expression in yeast) is represented here by SEQ ID NO: 32. SEQ ID NO: 33 (and SEQ ID NO: 31) contains multiple epitopes or domains believed to increase the immunogenicity of the HDV antigen. For example, positions 34 to 42 and positions 51 to 59 of SEQ ID NO: 33 comprise known MHC Class I T cell epitopes, and positions 34 to 49, positions 58 to 73, positions 74 to 89, positions 82 to 97, positions 114 to 129 and positions 130 to 161 comprise known MHC Class II T cell epitopes. A yeast immunotherapy composition that expresses SEQ ID NO: 33 is also referred to here as HDV2.
[0186] [000186] Briefly, to produce the yeast immunotherapy compositions HDV1 and HDV2, the DNA encoding the HDV antigens described above was optimized in terms of codons for expression in yeast and then digested with EcoRI and NotI and inserted behind the CUP1 promoter (pGI-100) in 2 μm yeast expression vectors. The nucleotide sequence of SEQ ID NO: 29 encodes the fusion protein represented by SEQ ID NO: 30, and the nucleotide sequence of SEQ ID NO: 32 encodes the fusion protein represented by SEQ ID NO: 33. The resulting plasmids were introduced into the yeast Saccharomyces cerevisiae W303a by transfection with lithium acetate / polyethylene glycol, and the primary transfectants were selected on minimal solid plates devoid of uracil (UDM; uridine abandonment medium). The colonies were reapplied in UDM or ULDM (uridine and leucine abandonment medium) and allowed to grow for 3 days at 30 ° C. Liquid cultures devoid of uridine (U2 medium: 20 g / L glucose; 6.7 g / L nitrogen base for yeast containing ammonium sulfate; 0.04 mg / mL each of histidine, leucine, tryptophan and adenine) or devoid of uridine and leucine (UL2 medium: 20 g / L glucose; 6.7 g / L nitrogen base for yeast containing ammonium sulfate; and 0.04 mg / mL each of histidine, tryptophan and adenine) were inoculated plates, and starter cultures were grown for 20 h at 30 ° C, 250 rpm. Primary cultures were used to inoculate final cultures of the same formulation, and growth was continued until a density of 1.1 to 4.0 YU / mL was achieved. Cultures were induced with 400 μΜ of copper sulfate at this starting density of 1 - 4 YU / mL for 3 hours at 30 ° C. The cells from each culture were then harvested, washed with PBS and thermally killed at 56 ° C for 1 hour in PBS.
[0187] [000187] After killing the cultures with heat, the cells were washed three times in PBS, and the total protein was isolated by rupture with glass globules, followed by boiling in SDS lysis buffer. Quantification of total protein was done by a starch / nitrocellulose binding assay, and HDV antigen content was measured by Western blot using a His-tag monoclonal antibody probe, followed by interpolation to a standard protein curve. HCV NS3 labeled His.
[0188] [000188] The results are shown in Figs. 2 and 3. Fig. 2 shows the copper-inducible expression of HDV1 and HDV2: U2 versus UL2 using a set of internal standards (HDV1 and HDV2 loaded at 4 μg of protein / lane), and Fig. 3 shows the expression copper-inducible from HDV1 and HDV2: U2 versus UL2 using a second set of internal standards (HDV1 and HDV2 loaded at 4 μg of protein / lane). These figures show that each of the HDV yeast-based immunotherapy compositions of the invention described above express the HDV protein and can be identified by Western blot. Antigen expression was better using UL2 medium. The calculated antigen expression was ~ 7,171 ng of protein per Y.U. for yeast expressing SEQ ID NO: 30 (HDV1) and ~ 6,600 ng of protein per Y.U. for yeast expressing SEQ ID NO: 33 (HDV2) (Yeast Unit; a Yeast Unit (Y.U.) is 1 x 107 yeast cells or yeast cell equivalents) or 76 pmol of protein per Y.U.). However, it was observed that HDV2 agglomerates and has a much longer doubling time than HDV1. Cell agglomeration and extremely slow growth are undesirable characteristics for candidates for yeast-based immunotherapy and, therefore, HDV1 was selected for further experiments. Example 2
[0189] [000189] The following example describes the production of another yeast-based immunotherapeutic composition for the treatment or prevention of hepatitis D virus (HDV) infection.
[0190] [000190] In this experiment, yeast (for example, Saccharomyces cerevisiae) was manipulated to express an HDV antigen under the control of the copper-inducible promoter, CUP1. The HDV antigen was a single polypeptide of approximately 422 amino acids, with the following sequence elements fused in the frame from the N to the C terminus, represented by SEQ ID NO: 36: (1) an N-terminal peptide to confer resistance to degradation pro-teassomic and stabilize expression (positions 1 to 6 of SEQ ID NO: 36); 2) a two amino acid spacer (Thr-Ser) to introduce a SpeI restriction enzyme site (positions 7 to 8 of SEQ ID NO: 36); 3) the amino acid sequence of a large HDV genotype 1 (L) antigen (HDAg-L) that has been modified to delete the nuclear localization sequence (positions 9 to 212 of SEQ ID NO: 36); 4) the amino acid sequence of a large HDV genotype 2 (L) antigen (HDAg-L) that has been modified to delete the nuclear localization sequence (positions 213 to 416 of SEQ ID NO: 36); and 5) a hexa-histidine tag (positions 417 to 422 of SEQ ID NO: 36). An amino acid sequence representing only the HDV sequences in that fusion protein is SEQ ID NO: 34. A nucleic acid sequence encoding the fusion protein of SEQ ID NO: 36 (from codons optimized for expression in yeast) is represented here by SEQ ID NO: 35. SEQ ID NO: 36 (and SEQ ID NO: 34) contains multiple epitopes or domains believed to increase the immunogenicity of the HDV antigen. For example, within the HDV antigen genotype 1 sequence, positions 34 to 42 and positions 51 to 59 of SEQ ID NO: 36 comprise known MHC Class I T cell epitopes, and positions 34 to 49, positions 58 to 73, positions 74 to 89, positions 82 to 97, positions 114 to 129 and positions 130 to 161 comprise known MHC Class II T cell epitopes. Corresponding sequences can be identified in the sequence of the HDV antigen genotype 2. A yeast immunotherapy composition that expresses SEQ ID NO: 36 is also referred to here as HDV3.
[0191] [000191] Briefly, to produce the yeast immunotherapeutic composition HDV3, the DNA encoding the HDV antigen described above was optimized in terms of codons for expression in yeast and then digested with EcoRI and NotI and inserted behind the promoter CUP1 (pGI-100) in 2 μm yeast expression vectors. The nucleotide sequence of SEQ ID NO: 35 encodes the fusion protein represented by SEQ ID NO: 36. The resulting plasmids were introduced into the yeast Saccharomyces cerevisiae W303a by transfection with lithium acetate / polyethylene glycol, and the primary transfectants were selected on minimal solid plates devoid of uracil (UDM; uridine abandonment medium). The colonies were reapplied in UDM or ULDM (uridine and leucine abandonment medium) and allowed to grow for 3 days at 30 ° C. Liquid cultures devoid of uridine (U2 medium: 20 g / L glucose; 6.7 g / L nitrogen base for yeast containing ammonium sulfate; 0.04 mg / mL each of histidine, leucine, tryptophan and adenine) or devoid of uridine and leucine (UL2 medium: 20 g / L glucose; 6.7 g / L nitrogen base for yeast containing ammonium sulfate; and 0.04 mg / mL each of histidine, tryptophan and adenine) were inoculated plates, and starter cultures were grown for 20 h at 30 ° C, 250 rpm. Primary cultures were used to inoculate final cultures of the same formulation, and growth was continued until a density of 1.1 to 4.0 YU / mL was achieved. Cultures were induced with 400 μΜ of copper sulfate at this starting density of 1 - 4 YU / mL for 3 hours at 30 ° C. The cells from each culture were then harvested, washed with PBS and thermally killed at 56 ° C for 1 hour in PBS.
[0192] [000192] After killing the cultures with heat, the cells were washed three times in PBS, and the total protein was isolated by rupture with glass globules, followed by boiling in SDS lysis buffer. Quantification of total protein was done by a starch / nitrocellulose binding assay, and HDV antigen content was measured by Western blot using a His-tag monoclonal antibody probe, followed by interpolation to a standard protein curve. HCV NS3 labeled His. The results are shown in Fig. 4. Fig. 4 shows the copper-inducible expression of HDV3 in each U2 versus UL2 medium using two different sets of internal standards. The results show that HDV3 expresses high levels of the HDV antigen, and can be identified by Western blot. Antigen expression was better using UL2 medium. The calculated antigen expression was ~ 2.3861 ng of protein per Y.U. for yeast expressing SEQ ID NO: 36 (HDV3). HDV3 was selected for further experimentation. Example 3
[0193] [000193] The following example describes preclinical experiments on mice to demonstrate the immunogenicity of the yeast-based HDV immunotherapy compositions of the invention when administered in vivo.
[0194] [000194] In these experiments, three groups of C57BL / 6 mice were immunized subcutaneously as in Table 2. The mice were immunized with HDV1 (SEQ ID NO: 30, see Example 1) and HDV3 (SEQ ID NO: 36, see Example 2) and with a control yeast composition known as OVAX2010 (that yeast expresses chicken ovalbumin comprising a leading N-terminal alpha factor peptide, expression activated by the CUP1 promoter).
[0195] [000195] The mice were immunized with a total of 5 YU (in two locations at 2.5 YU per injection site) of the indicated yeast-based immunotherapy composition once a week for a total of 3 weeks. Eight days after the third immunization, the mice were sacrificed, and the spleen and lymph nodes were removed, macerated in single cell suspensions and counted. The cells were plated in 96-well U-bottom plates at 200,000 cells / well (106 cells / ml), and HDV-specific peptide antigens were added at 30 μg / ml. After a 4-day incubation in a humidified incubator at 37 ° C / 5% CO2, 150 μL (150,000 cells) were transferred to interleukin-2 / interferon-Y (IL-2 / IFNy) ELISpot plates (R&D) Systems) for 24 hours. The plates were developed according to the manufacturer's instructions, and points were counted using instrumentation and validated point counting software (CTL, Inc.). The HDV peptides used in this assay were: HDV 26-34 (HLA-A2 linker): KLEDLERDL; SEQ ID NO: 20 HDV 43-51 (HLA-A2 linker): KLEDENPWL; SEQ ID NO: 19
[0196] [000196] The results of this experiment are shown in Figs. 5A, 5B, 6A and 6B. Fig. 5A shows that vaccination with HDV1 or HDV3 elicits a HDV-specific IFNy ELISpot response that is specifically developed by the ex vivo addition of a known HDV T cell epitope peptide (P2: HDV 43-51 or SEQ ID NO: 19; p = 0.0008 HDV3 versus OVAX). This result is significant because IFNy is a key component of a functional adaptive immune response; it is produced by effector T cells of CD4 + Th1 and CD8 + cytotoxic T lymphocytes (CTL) in the development of functional immunity. Although there is a remarkable level of bottom ELISpots in wells containing only growth medium, the specificity for the immune response antigen is clear, even after subtracting this bottom from the peptide-treated ELISpot counts (Fig. 5B). Notably, HDV3 proved an ELISpot response 3.5 times greater than HDV1 (Fig. 5B; 42 points for HDV3 versus 12 points for HDV1) and contains an HDV antigen content ~ 3.3 times greater than HDV1 (23,861 ng / YU for HDV3 versus 7,171 ng / YU for HDV1). This finding illustrates that a higher antigen content per yeast cell correlates with a higher frequency of antigen-specific T cells caused by a yeast immunotherapy.
[0197] [000197] The results of the IL-2 ELISpot assay also revealed the induction of an antigen-specific immune response by vaccination with HDV-1. Fig. 6A shows that vaccination with HDV1 elicits an ILISpot ELISpot response that is specifically developed by the ex vivo addition of a different HDV peptide (P1: HDV-26-34 or SEQ ID NO: 20; p = 0.02 HDV1 versus OVAX). This result attests to the quality of the immune response induced by the yeast immunotherapy for HDV by yeast, because IL-2 stimulates the growth, differentiation and survival of cytotoxic T cells specific for the antigen. Although there is a remarkable level of IL-2 ELISpots for samples incubated with growth medium only ("No Estimate" in Fig. 6A), the specificity for the antigen of the immune response is clear even after subtracting this background from the treated ELISpot counts with peptide (Fig. 6B). The number of background corrected IL-2 ELISpots for mice vaccinated with HDV1 is double that of mice vaccinated with OVAX.
[0198] [000198] Taken together, IFNy and IL-2 ELISpot data show that the yeast-based immunotherapeutic compositions for HDV of the invention cause antigen-specific T cells as a result of in vivo administration, producing cytokines that are known markers of CTL induction, and illustrating the usefulness of these compositions for inducing functional anti-HDV responses. Example 4
[0199] [000199] The following example describes a phase 1 clinical trial in healthy volunteers.
[0200] [000200] A phase 1 clinical study with 12-week open label dose escalation is performed using a yeast-based HDV immunotherapy composition described here (for example, the HDV immunotherapy composition described in Examples 1 or 2). The subjects are immunoactive and healthy volunteers, with no previous or current indication or record of HDV infection or HBV infection.
[0201] [000201] Approximately 48 subjects (6 arms, 8 subjects per arm) who meet these criteria receive administration of the yeast-based HDV immunotherapy composition in a sequential dose group escalation protocol using one of two different treatment protocols dosage as follows:
[0202] [000202] Protocol A: Primary Dosage-Booster (4 weekly doses starting on Day 1, followed by 2 monthly doses on Week 4 and Week 8)
[0203] [000203] Arm 1A: 20 Yeast Units (Y.U.) (administered in doses of 10 Y.U. to 2 different sites);
[0204] [000204] Arm 2A: 40 Y.U. (administered in doses of 10 Y.U. at 4 different sites);
[0205] [000205] Arm 3A: 80 Y.U. (administered in doses of 20 Y.U. at 4 different sites)
[0206] [000206] Dosage in 4 weeks (three total doses administered on Day 1, Week 4 and Week 8)
[0207] [000207] Arm 1B: 20 Y.U. (administered in doses of 10 Y.U. at 2 different sites);
[0208] [000208] Arm 2B: 40 Y.U. (administered in doses of 10 Y.U. at 4 different sites);
[0209] [000209] Arm 3B: 80 Y.U. (administered in doses of 20 Y.U. at 4 different sites)
[0210] [000210] All doses are administered subcutaneously, and the dose is divided between two or four sites on the body (at each visit), as indicated above. Safety and immunogenicity are assessed (eg, antigen-specific T cell responses measured by ELISpot and T cell proliferation). Specifically, an ELISpot-based algorithm is developed for categorical responsives. ELISpot assays measuring regulatory T cells (Treg) are also evaluated, and the proliferation of CD4 + T cells in response to HDV antigens is assessed and correlated with the development of anti-Saccharomyces cerevisiae (ASCA) antibodies.
[0211] [000211] The yeast-based HDV immunotherapeutic agent is expected to be well tolerated and to show immunogenicity as measured by one or more of the ELISpot assay, lymph proliferation assay (LPA), stimulation of T cells ex vivo by antigens from HBV and / or ASCA. Example 5
[0212] [000212] The following example describes a phase 1b / 2a clinical trial in subjects chronically infected with either hepatitis D virus, or with hepatitis B virus (therapeutic arm) or monoinfected with hepatitis B virus (prophylactic arm) .
[0213] [000213] A phase 1b / 2a open-label, dose-escalation clinical trial is performed using a yeast-based HDV immunotherapy composition described herein (for example, the HDV immunotherapy composition described in Examples 1 or two). In Arm 1 (therapeutic treatment arm for HDV), the subjects are immunoactive and critically infected with both hepatitis B virus (HBV) and hepatitis D virus (HDV). In Arm 2 (prophylactic arm for HDV), subjects are immunoactive and chronically infected with HBV, with no evidence of HDV co-infection (ie, HBV mono-infection). In each arm, chronic HBV infection is well controlled by antiviral therapy for conventional HBV (for example, tenofovir disoproxil fumarate, or TDF (VIREAD®)) as measured by HBV DNA levels.
[0214] [000214] In stage one of this study, subjects in both arms who meet the relevant criteria receive administration of the yeast-based HDV immunotherapy composition in a sequential dose group escalation protocol using dose ranges of 0, 05 YU at 80 Y.U. Optionally, a single patient group will receive subcutaneous injections of placebo (PBS) on the same schedule as immunotherapy, in addition to continuing antiviral therapy. Conservative interruption rules are employed for ALT outbreaks and signs of decompression.
[0215] [000215] In the second stage of each arm of this trial, subjects are randomized into groups of equal numbers to continue with only the antiviral or antiviral plus the yeast-based immunotherapy protocol for HDV (dose 1 and dose 2) for up to 48 weeks . Patients in Arm 2 can optionally be followed up for a longer period (beyond 48 weeks) to monitor HDV infection rates. In Arm 2, a single patient group will receive subcutaneous injections of placebo (PBS) on the same schedule as immunotherapy for HDV, in addition to continuing antiviral therapy for HBV.
[0216] [000216] In Arm 1, safety, HDV viral kinetics, HDAg seroconversion and HDV specific immunogenicity (for example, antigen-specific T cell responses measured by ELISpot) are evaluated, as well as HBsAg seroconversion to measure effects of treatment for HBV concomitantly. In addition, dose-dependent biochemistry (ALT) is monitored.
[0217] [000217] In Arm 2, safety and specific immunogenicity for HDV (for example, antigen-specific T cell responses measured by ELISpot) are evaluated, as well as HBsAg seroconversion concomitantly, and subjects are routinely monitored for indicators of HDV infection, including detection of HDV RNA, detection of HDV antigen (HDAg) and / or detection of anti-HDV (antibodies against HDV).
[0218] [000218] In Arm 1 (therapeutic), the immunotherapy composition for yeast-based HDV is expected to provide a therapeutic benefit for patients chronically infected with HDV. Immunotherapy is expected to be safe and well tolerated in all doses provided. Patients receiving at least the highest dose of yeast-based HDV immunotherapy will show HDV specific T cell responses emerging from treatment, as determined by ELISPOT, and patients with previous specific T cell responses are expected for baseline HDV show improved HDV-specific T cell responses while on treatment. Patients receiving yeast-based HDV immunotherapy are expected to show reductions in HDV RNA and / or improvement in HDV seroconversion rates compared to the antiviral group and / or compared to the placebo control group, used case. Improvements in ALT normalization are expected in patients receiving yeast-based HDV immunotherapy.
[0219] [000219] In Arm 2 (prophylaxis), the immunotherapy composition for yeast-based HDV is expected to provide protection against co-infection of HBV monoinfected patients with HDV, including evidence of decreased rates of HDV co-infection, reduced severity of symptoms and sequelae associated with HDV infection, increased overall survival and / or increased clearance of HDV infection as an acute disease, compared to the control (placebo group). In addition, patients who receive at least the highest dose of yeast-based HDV immunotherapy are expected to show HDV-specific T cell responses emerging from treatment as determined by ELISPOT.
[0220] [000220] Although several modalities of the present invention have been described in detail, it is clear that modifications and adaptations of these modalities will occur for those skilled in the art. It should be expressly understood, however, that these modifications and adaptations are within the scope of the present invention, as set out in the following claims.

权利要求:
Claims (7)
[0001]
Immunotherapeutic composition, characterized by the fact that it comprises: (a) a yeast vehicle; and (b) a fusion protein comprising an amino acid sequence selected from SEQ ID NO: 36 or SEQ ID NO: 30.
[0002]
Immunotherapeutic composition according to claim 1, characterized by the fact that the fusion protein has an amino acid sequence of SEQ ID NO: 36.
[0003]
Immunotherapeutic composition according to claim 1, characterized by the fact that the fusion protein has an amino acid sequence of SEQ ID NO: 30.
[0004]
Immunotherapeutic composition, according to claim 1, characterized by the fact that the fusion protein is expressed by the yeast vehicle.
[0005]
Immunotherapeutic composition, according to claim 1, characterized by the fact that the yeast vehicle is an entire yeast, thermally inactivated.
[0006]
Immunotherapeutic composition, according to claim 1, characterized by the fact that the yeast vehicle is Saccharomyces cerevisiae.
[0007]
Immunotherapeutic composition according to claim 1, characterized by the fact that the composition is formulated in a pharmacologically acceptable excipient suitable for administration to an individual.

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

FR2486400B1|1980-07-09|1986-05-30|Univablot|MEDICINAL PRODUCTS BASED ON YEAST OR THEIR INSOLUBLE EXTRACTS|

---

NZ199722A|1981-02-25|1985-12-13|Genentech Inc|Dna transfer vector for expression of exogenous polypeptide in yeast;transformed yeast strain|

---

US4775622A|1982-03-08|1988-10-04|Genentech, Inc.|Expression, processing and secretion of heterologous protein by yeast|

---

US5310654A|1985-07-31|1994-05-10|The Board Of Trustees Of The Leland Stanford Junior University|Method for determining virulence of Yersinia|

---

US5234830A|1988-02-03|1993-08-10|Suntory Limited|DNA encoding a KEX2 endoprotease without a C-terminal hydrophobic region|

---

IL95019D0|1989-08-09|1991-06-10|Mycogen Corp|Process for encapsulation of biologicals|

---

US5413914A|1993-07-07|1995-05-09|The Regents Of The University Of Colorado|Yeast assay to identify inhibitors of dibasic amino acid processing endoproteases|

---

US5830463A|1993-07-07|1998-11-03|University Technology Corporation|Yeast-based delivery vehicles|

---

EP1728800A1|1994-10-07|2006-12-06|Loyola University Of Chicago|Papilloma virus-like particles, fusion proteins and process for producing the same|

---

US5858378A|1996-05-02|1999-01-12|Galagen, Inc.|Pharmaceutical composition comprising cryptosporidium parvum oocysts antigen and whole cell candida species antigen|

---

AUPO434196A0|1996-12-24|1997-01-23|Crown In The Right Of The Queensland Department Of Health, The|An improved therapeutic|

---

US6844171B1|1998-07-02|2005-01-18|President And Fellows Of Harvard College|Oligomerization of hepatitis delta antigen|

---

US20020044948A1|2000-03-15|2002-04-18|Samir Khleif|Methods and compositions for co-stimulation of immunological responses to peptide antigens|

---

AU2001219510B2|2000-04-06|2007-06-14|Allertein Therapeutics, Llc|Microbial delivery system|

---

US7083787B2|2000-11-15|2006-08-01|Globeimmune, Inc.|Yeast-dendritic cell vaccines and uses thereof|

---

FR2830019B1|2001-09-24|2005-01-07|Assist Publ Hopitaux De Paris|NUCLEIC ACID MOLECULES OF HDV, THEIR FRAGMENTS AND THEIR APLLICATIONS|

---

CN1622828A|2001-12-18|2005-06-01|基因创新有限公司|Purified hepatitis c virus envelope proteins for diagnostic and therapeutic use|

---

US7439042B2|2002-12-16|2008-10-21|Globeimmune, Inc.|Yeast-based therapeutic for chronic hepatitis C infection|

---

ES2676503T3|2002-12-16|2018-07-20|Globeimmune, Inc.|Yeast-based vaccines as immunotherapy|

---

BRPI0516356A|2004-10-18|2008-09-02|Globeimmune Inc|yeast therapy for chronic hepatitis c infections|

---

JP4320357B2|2005-02-25|2009-08-26|ファイザー・プロダクツ・インク|N protein variant of porcine genital respiratory syndrome virus|

---

WO2007008780A2|2005-07-11|2007-01-18|Globeimmune, Inc.|Compositions and methods for eliciting an immune response to escape mutants of targeted therapies|

---

US20070172503A1|2005-12-13|2007-07-26|Mycologics,Inc.|Compositions and Methods to Elicit Immune Responses Against Pathogenic Organisms Using Yeast Based Vaccines|

---

EP2468296A3|2006-02-02|2013-12-04|Globeimmune, Inc.|Yeast-based vaccine for inducing an immune response|

---

CN101448848B|2006-03-27|2013-12-04|全球免疫股份有限公司|Ras mutation and compositions and methods related thereto|

---

US20080138354A1|2006-07-21|2008-06-12|City Of Hope|Cytomegalovirus vaccine|

---

DK2121013T3|2007-02-02|2015-01-19|Globeimmune Inc|Methods for preparing yeast-based vaccines|

---

US8501167B2|2007-03-19|2013-08-06|Globeimmune, Inc.|Compositions and methods for targeted ablation of mutational escape of targeted therapies for cancer|

---

US8728489B2|2008-09-19|2014-05-20|Globeimmune, Inc.|Immunotherapy for chronic hepatitis C virus infection|

---

WO2010065626A1|2008-12-02|2010-06-10|Globeimmune, Inc.|Genotyping tools, methods and kits|

---

ITTO20080964A1|2008-12-22|2010-06-23|Natimab Therapeutics S R L|ANTI-HCV MONOCLONAL ANTIBODY AS A MEDICATION FOR THERAPEUTIC TREATMENT AND THE PREVENTION OF HCV INFECTIONS|

---

US10383924B2|2009-04-17|2019-08-20|Globeimmune, Inc.|Combination immunotherapy compositions against cancer and methods|

---

SG178254A1|2009-08-07|2012-03-29|Transgene Sa|Composition for treating hbv infection|

---

WO2011032119A1|2009-09-14|2011-03-17|The Regents Of The University Of Colorado|Modulation of yeast-based immunotherapy products and responses|

---

US20130121964A1|2010-03-14|2013-05-16|Globeimmune, Inc.|Pharmacogenomic and Response-Guided Treatment of Infectious Disease Using Yeast-Based Immunotherapy|

---

WO2012019127A2|2010-08-05|2012-02-09|The Regents Of The University Of Colorado|Combination yeast-based immunotherapy and arginine therapy for the treatment of myeloid-derived supressor cell-associated diseases|

---

WO2012083302A2|2010-12-17|2012-06-21|Globeimmune, Inc.|Compositions and methods for the treatment or prevention of human adenovirus-36 infection|

---

US8877205B2|2011-02-12|2014-11-04|Globeimmune, Inc.|Yeast-based therapeutic for chronic hepatitis B infection|

---

ES2627979T3|2011-03-17|2017-08-01|Globeimmune, Inc.|Yeast-Brachyury immunotherapeutic compositions|

---

CA2838950C|2011-06-14|2020-10-27|Globeimmune, Inc.|Yeast-based compositions and methods for the treatment or prevention of hepatitis delta virus infection|

---

EP2744918A4|2011-08-17|2015-06-10|Globeimmune Inc|Yeast-muc1 immunotherapeutic compositions and uses thereof|WO2012083302A2|2010-12-17|2012-06-21|Globeimmune, Inc.|Compositions and methods for the treatment or prevention of human adenovirus-36 infection|

---

US8877205B2|2011-02-12|2014-11-04|Globeimmune, Inc.|Yeast-based therapeutic for chronic hepatitis B infection|

---

ES2627979T3|2011-03-17|2017-08-01|Globeimmune, Inc.|Yeast-Brachyury immunotherapeutic compositions|

---

CA2838950C|2011-06-14|2020-10-27|Globeimmune, Inc.|Yeast-based compositions and methods for the treatment or prevention of hepatitis delta virus infection|

---

EP2744918A4|2011-08-17|2015-06-10|Globeimmune Inc|Yeast-muc1 immunotherapeutic compositions and uses thereof|

---

WO2014003853A1|2012-06-26|2014-01-03|Biodesix, Inc.|Mass-spectral method for selection, and de-selection, of cancer patients for treatment with immune response generating therapies|

---

WO2014186047A1|2013-03-19|2014-11-20|Globeimmune, Inc.|Yeast-based immunotherapy for chordoma|

---

TWI626948B|2013-03-26|2018-06-21|環球免疫公司|Compositions and methods for the treatment or prevention of human immunodeficiency virus infection|

---

WO2015031778A2|2013-08-30|2015-03-05|Globeimmune, Inc.|Compositions and methods for the treatment or prevention of tuberculosis|

---

CN106456677A|2014-04-11|2017-02-22|全球免疫股份有限公司|Yeast-based immunotherapy and type i interferon sensitivity|

---

US10076512B2|2014-05-01|2018-09-18|Eiger Biopharmaceuticals, Inc.|Treatment of hepatitis delta virus infection|

---

EP3226973A4|2014-12-04|2018-05-30|Eiger Biopharmaceuticals, Inc.|Treatment of hepatitis delta virus infection|

---

CN107530383A|2015-01-09|2018-01-02|埃图比克斯公司|Method and composition for research of Ebola vaccine inoculation|

---

ES2844848T3|2015-04-21|2021-07-22|Eiger Biopharmaceuticals Inc|Pharmaceutical compositions comprising Lonafarnib and Ritonavir|

---

KR20180054587A|2015-08-03|2018-05-24|글로브이뮨|Modified yeast-brachyury immunotherapeutic composition|

---

WO2017143253A1|2016-02-19|2017-08-24|Eiger Biopharmaceuticals, Inc.|Treatment of hepatitis delta virus infection with interferon lambda|

---

CN107641661A|2016-12-28|2018-01-30|成都百泰赛维生物科技有限公司|A kind of kit for being used to detect the Hans HDV viruses|

---

CN107653342A|2016-12-28|2018-02-02|成都百泰赛维生物科技有限公司|A kind of method of PCR methods detection the Hans HDV viruses|

法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law|

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

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2018-07-31| B07E| Notice of approval relating to section 229 industrial property law|

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2019-07-02| B06T| Formal requirements before examination|

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2020-08-11| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application according art. 36 industrial patent law|

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2020-11-10| B09A| Decision: intention to grant|

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2021-01-12| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 14/06/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161497039P| true| 2011-06-14|2011-06-14|

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US61/497,039|2011-06-14|

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PCT/US2012/042426|WO2012174220A1|2011-06-14|2012-06-14|Yeast-based compositions and methods for the treatment or prevention of hepatitis delta virus infection|

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