OA16530A - Yeast-based therapeutic for chronic hepatitis B infection. - Google Patents

Yeast-based therapeutic for chronic hepatitis B infection. Download PDF

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Publication number
OA16530A
OA16530A OA1201300338 OA16530A OA 16530 A OA16530 A OA 16530A OA 1201300338 OA1201300338 OA 1201300338 OA 16530 A OA16530 A OA 16530A
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OAPI
Prior art keywords
seq
positions
hbv
antigen
amino acid
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OA1201300338
Inventor
David Apelian
Thomas H. King
Zhimin Guo
Claire Coeshott
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Globeimmune, Inc.
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Publication of OA16530A publication Critical patent/OA16530A/en

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Abstract

Disclosed are yeast-based immunotherapeutic compositions, hepatitis B virus (HBV) antigens, and fusion proteins for the treatment and/or prevention of HBV infection and symptoms thereof, as well as methods of using the yeastbased immunotherapeutic compositions, HBV antigens, and fusion proteins for the prophylactic and/or therapeutic treatment of HBV and/or symptoms thereof.

Description

Compositions and Methods for the Treatment or Prévention of Hepatitis B Virus Infection
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) from each of U.S. Provisionai Application No. 61/442,204, filed February 12, 2011, U.S. Provisional Application No. 61/496,945, filed June 14, 2011, and U.S. Provisionai Application No. 61/507,361, filed July 13, 2011. The entire disclosure of each of U.S. Provisionai Application No. 61/442,204, U.S. Provisionai Application No. 61/496,945, and U.S. Provisionai Application No. 61/507,361 is incorporated herein by reference.
REFERENCE TO A SEQUENCE LISTING
[0002] This application contains a Sequence Listing submitted électron icall y as a text file by EFS-Web. The text file, named 3923-32-PCT_ST25, has a size in bytes of 476 KB, and was recorded on February 7, 2012. The information contained in the text file is incorporated herein by reference in its entirety pursuant to 37 CFR § 1.52(e)(5).
FIELD OF THE INVENTION
[0003] The présent invention generally relates to immunotherapeutic compositions and methods for preventing and/or treating hepatitis B virus (HBV) infection.
BACKGROUND OF THE INVENTION
[0004] Hepatitis B virus (HBV) is a member of the hepadnavirus family and is a causative agent of acute and chronic hepatitis worldwide. HBV épidémies hâve been prévalent in Asia and Africa, and HBV infection is endemic in China (Williams, R. (2006), Global challenges in liver disease, Hepatology (Baltimore, Md,) 44 (3): 521-526). More than 2 billion people hâve been infected with the virus, and it is estimated that there are
350 million chronically HBV-infected individuals worldwide (“Hepatitis B”, World
Health Organization, 2009; “FAQ About Hepatitis B”, Stanford School of Medicine. 2008-07-10). Routes of infection are through blood and bodily fluid contact, including blood transfusions and IV drug use, sexual transmission, bites and lésions, and vertical transmission (e.g., childbirth).
[0005] HBV is found as one of four major serotypes (adr, adw, ayr, ayw) that are determined based on antigenic epitopes within its envelope proteins. There are eight different génotypes (A-H) based on the nucléotide sequence variations in the genome.
Génotype différences impact disease severity, disease course and likelihood of complications, response to treatment and possibly response to vaccination (Kramvis et al., (2005), Vaccine 23 (19): 2409-2423; Magnius and Norder, (1995), Intervirology 38 (1-2):
24-34).
[0006] The clinical incubation period for HBV is usually 2-3 months; approximately two thirds of those acutely infected are asymptomatic or hâve mild, subclinical symptoms. The remaining one third of acutely infected individuals may expérience jaundice, inflammation of the liver, vomiting, aches and/or mild fever, but the disease is eventually resolved in most adults and rarely leads to liver failure. Indeed, approximately 95% of adults recover completely from HBV infection and do not become chronically infected. However, approximately 90% of infants and 25%-50% of children aged 1-5 years will remain chronically infected with HBV (Centers for Disease Control and Prévention as of September 2010). Approximately 25% of those who become chronically infected during childhood and 15% of those who become chronically infected after childhood die prematurely from cirrhosis or hepatocellular carcinoma, and the majority of chronically infected individuals remain asymptomatic until onset of cirrhosis or end-stage liver disease (CDC as of September 2010). 1 million deaths per year worldwide (about 2000-4000 deaths per year in the U.S.) resuit from chronic HBV infection. Chronically infected individuals hâve elevated sérum alanine aminotransferase (ALT) levels (a marker of liver damage), liver inflammation and/or fibrosis upon liver biopsy. For those patients who develop cirrhosis, the 5 year survival rate is about 50%.
[0007] HBV infection and its treatment are typically monitored by the détection of viral antigens and/or antibodies against the antigens. Upon infection with HBV, the first détectable antigen is the hepatitis B surface antigen (HBsAg), followed by the hepatitis B “e” antigen (HBeAg). Clearance of the virus is indicated by the appearance of IgG antibodies in the sérum against HBsAg and/or against the core antigen (HBeAg), also known as séroconversion. Numerous studies indicate that viral réplication, the level of viremia and progression to the chronic state in HBV-infected individuals are influenced directly and indirectly by HBV-specific cellular immunity mediated by CD4+ helper (Th) and CD8+ cytotoxic T lymphocytes (CTLs). Patients progressing to chronic disease tend to hâve absent, weaker, or narrowly focused HBV-specific T cell responses as compared to patients who clear acute infection. See, e.g., Chisari, 1997, J Clin Invest 99: 14721477; Maini et al., 1999, Gastroenterology 117:1386-1396; Rehermann et al., 2005, Nat Rev Immunol 2005; 5:215-229; Thimme et al., 2001, J Virol 75: 3984-3987; Urbani et al., 2002, J Virol 76: 12423-12434; Wieland and Chisari, 2005, J Virol 79: 9369-9380;
Webster et al., 2000, Hepatology 32:1117-1124; Penna et al., 1996, J Clin Invest 98:11851194; Sprengers et al., 2006, J Hepatol 2006; 45: 182-189.
[0008] Vaccines for the prévention of HBV hâve been commercially available since the early 1980’s. Current commercial vaccines are non-infectious, subunit viral vaccines providing purified recombinant hepatitis B virus surface antigen (HBsAg), and can be administered beginning at birth. The vaccines hâve been effective at reducing the incidence of infection in countries where the vaccine is routinely administered. While a few immunotherapeutics are in development, including various HBV protein or epitope vaccines and cytokines, there are currently no approved immunotherapeutics for the treatment of active HBV infection in the United States.
[0009] Current standard of care (SOC) therapy for HBV infection includes primarily antiviral drugs, such as tenofovir (VIREAD®), lamivudine (EPIVIR®), adefovir (HEPSERA®), telbivudine (TYZEKA®) and entecavir (BARACLUDE®), as well as interferon-a2a and pegylated interferon-a2a (PEGASYS®). These drugs, and particularly the antiviral drugs, are typically administered for long periods of time (e.g., daily or weekly for one to five years or longer), and although they slow or stop viral réplication, they typically do not provide a complété “cure” or éradication of the virus. Interferonbased approaches are toxic and hâve modest remission rates. The antiviral thérapies inhibit viral réplication and are better tolerated than interferon, but as mentioned above, these drugs typically do not provide a complété viral cure, and in some cases long term rémission rates are not achieved. Moreover, in some cases, development of drug résistance ensues. For example, lamivudine is a potent oral antiviral that inhibits HBV reverse transcriptase (Pol). As lamivudine is well tolerated, and because it is now a generic drug, lamivudine is an option for HBV antiviral therapy in developing countries. However, a 20% annual viral résistance rate from point mutations in the Pol sequence limits the utility of lamivudine for HBV. Moreover, response to current anti-viral and interferon treatment is differently effective among HBV génotypes (Cao, World Journal of Gastroenterology 2009;15(46):5761-9) and in some patients, because the hepatitis B virus DNA can persist in the body even after infection clears, réactivation of the virus can occur over time.
[0010] Accordîngly, while standard of care (SOC) therapy provides the best currently approved treatment for patients suffering from chronic HBV, the length of time for therapy and the significant adverse effects of the regimens can lead to noncompliance, dose réduction, and treatment discontinuation, combined with viral escape, réactivation of
the virus, and patients who still fail to respond or sustain response to therapy. Therefore, there remains a need in the art for improved therapeutic treatments for HBV infection.
SUMMARY OF THE INVENTION
[0011] One embodiment of the invention relates to an immunotherapeutic composition for the treatment and/or prévention of hepatitis B virus (HBV) infection and/or a symptom of HBV infection. The immunotherapeutic composition comprises: (a) a yeast vehicle; and (b) one or more HBV antigens. In one aspect, the HBV antigens are provided as one or more fusion proteins, although single protein HBV antigens may also be provided. The HBV antigens consist of: (i) an HBV surface antigen comprising at least 10 one immunogenic domain of a full-length HBV large (L), medium (M) and/or small (S) surface antigen; (ii) an HBV polymerase antigen comprising at least one immunogenic domain of a full-length HBV polymerase or domain thereof (e.g., a reverse transcriptase (RT) domain); (iii) an HBV core antigen or HBV e-antigen comprising at least one immunogenic domain of a full-length HBV core protein and/or a full-length HBV e15 antigen, respectively; and/or (iv) an HBV X antigen comprising at least one immunogenic domain of a full-length HBV X antigen. The composition elicits an HBV-specific immune response against one or more HBV antigens in the composition and/or against one or more antigens in a hepatitis B virus that has infected, or may infect, an individual.
[0012] In any of the embodiments of the invention described herein, including any 20 embodiment related to an immunotherapeutic composition, HBV antigen, fusion protein or use of such composition, HBV antigen or fusion protein, in one aspect, the amino acid sequence of the HBV large surface antigen (L) can include, but is not limited to, an amino acid sequence represented by SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO: 11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ DI NO:27 or SEQ ID NO:31, or a 25 corresponding sequence from another HBV strain/isolate. The amino acid sequence of HBV polymerase can include, but is not limited to, an amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26 or SEQ ID NO:30, a domain of these sequences, such as the reverse transcriptase (RT) domain, or a corresponding sequence from another HBV 30 strain/isolate. The amino acid sequence of HBV precore protein, which includes both HBV core protein sequence and HBV e-antigen sequence, can include, but is not limited to, an amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO: 17, SEQ ID NO:21, SEQ ID NO:25, or SEQ ID NO:29, or a corresponding sequence from another HBV strain/isolate. The amino acid sequence of an
HBV X antigen can include, but is not limited to, an amino acid sequence represented by
SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO:20, SEQ ID
NO:24, SEQ ID NO:28, or SEQ ID NO:32, or a corresponding sequence from another
HBV strain/isolate.
[0013] In any of the embodiments of the invention described herein, including any embodiment related to an immunotherapeutic composition, HBV antigen, fusion protein or use of such composition, HBV antigen or fusion protein, in one aspect, an amino acid of an HBV surface antigen useful as an HBV antigen or in a fusion protein or an immunotherapeutic composition of the invention can include, but is not limited to, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO: 11, positions 21-47 of SEQ ID NO: 11, positions 176400 of SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, positions 9-407 of SEQ ID NO:34, positions 6-257 of SEQ ID NO:36, positions 6-257 of SEQ ID NO:41, positions 92-343 of SEQ ID NO:92, positions 90-488 of SEQ ID NO:93, SEQ ID NO:97, positions 90-338 of SEQ ID NO: 101, positions 7-254 of SEQ ID NO:102, positions 1-249 of SEQ ID NO:107, positions 1-249 of SEQ ID NO:108, positions 1-249 of SEQ ID NO:109, positions 1-249 of SEQ ID NO:110, positions 1-399 of SEQ ID NO:112, positions 1-399 of SEQ ID NO:114, or positions 1399 of SEQ ID NO:116, positions 1-399 of SEQ ID NO:118, positions 1-399 of SEQ ID NO: 120, positions 1-399 of SEQ ID NO:122, positions 1-399 of SEQ ID NO:124, positions 1-399 of SEQ ID NO:126, positions 231-629 of SEQ ID NO:128, positions 63461 of SEQ ID NO: 130, positions 289-687 of SEQ ID NO: 132, positions 289-687 of SEQ ID NO: 134, or a corresponding sequence from a different HBV strain.
[0014] In any of the embodiments of the invention described herein, including any embodiment related to an immunotherapeutic composition, HBV antigen, fusion protein or use of such composition, HBV antigen or fusion protein, in one aspect, an amino acid of an HBV polymerase antigen useful as an HBV antigen or in a fusion protein or an immunotherapeutic composition of the invention can include, but is not limited to, positions 383-602 of SEQ ID NO:2, positions 381-600 of SEQ ID NO:6, positions 381600 of SEQ ID NO: 10, positions 453 to 680 of SEQ ID NO: 10, positions 370-589 of SEQ ID NO:14, positions 380-599 of SEQ ID NO: 18, positions 381-600 of SEQ ID NO:22, positions 380-599 of SEQ ID NO:26, positions 381-600 of SEQ ID NO:30, positions 260 to 604 of SEQ ID NO:36, positions 7-351 of SEQ ID NO:38, positions 7-351 of SEQ ID NO:40, 260 to 604 of SEQ ID NO:41, positions 346 to 690 of SEQ ID NO:92, positions 90-434 of SEQ ID NO:94, SEQ ID NO:98, positions 339 to 566 of SEQ ID NQ:101, positions 255 to 482 of SEQ ID NO:102, positions 250-477 of SEQ ID NO: 107, positions 250-477 of SEQ ID NO: 108, positions 250-477 of SEQ ID NO: 109, positions 250-477 of SEQ ID NO: 110, positions 582 to 809 of SEQ ID NO: 120, positions 582 to 809 of SEQ ID NO: 124, positions 642 to 869 of SEQ ID NO: 126, positions 1 to 228 of SEQ ID NO:128, positions 1 to 228 of SEQ ID NO:132, positions 61 to 288 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain.
[0015] In any of the embodiments of the invention described herein, including any embodiment related to an immunotherapeutic composition, HBV antigen, fusion protein or use of such composition, HBV antigen or fusion protein, in one aspect, an amino acid of an HBV core antigen useful as an HBV antigen or in a fusion protein or an immunotherapeutic composition of the invention can include, but is not limited to, positions 31-212 of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212 of SEQ ID NO:9, positions 37 to 188 of SEQ ID NO:9, positions 31-212 of SEQ ID NO:13, positions 31-212 of SEQ ID NO:17, positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO:29, positions 408-589 of SEQ ID NO:34, positions 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ ID NO:39, positions 605-786 of SEQ ID NO:41, positions 691872 of SEQ ID NO:92, positions 90-271 of SEQ ID NO:95, SEQ ID NO:99, positions 567 to 718 of SEQ ID NO:101, positions 483 to 634 of SEQ ID NO;102, positions 2-183 of SEQ ID NO:105, positions 184-395 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions 579-761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338520 of SEQ ID NO:106, positions 478-629 of SEQ ID NO:107, positions 478-629 of SEQ ID NO: 108, positions 478-629 of SEQ ID NO: 109, positions 478-629 of SEQ ID NO: 110, positions 400-581 of SEQ ID NO:112, positions 400-581 of SEQ ID NO:114, positions 400-581 of SEQ ID NO:116, positions 400-581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:120, positions 400 to 581 of SEQ ID NO:122, positions 400 to 581 of SEQ ID NO:124, positions 400 to 581 of SEQ ID NO:126, positions 630 to 811 of SEQ ID NO: 128, positions 462 to 643 of SEQ ID NO: 130, positions 688 to 869 of SEQ ID NO:132, positions 688 to 869 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain.
[0016] In any of the embodiments of the invention described herein, including any embodiment related to an immunotherapeutic composition, HBV antigen, fusion protein or use of such composition, HBV antigen or fusion protein, in one aspect, an amino acid of an HBV X antigen useful as an HBV antigen or in a fusion protein or an immunotherapeutic composition of the invention can include, but is not limited to, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, positions 2 to 154 of SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, positions 52-68 followed by positions 84-126 of SEQ ID NO:4, positions 52-68 followed by positions 84126 of SEQ ID NO:8, positions 52-68 followed by positions 84-126 of SEQ ID NO: 12, positions 52-68 followed by positions 84-126 of SEQ ID NO:16, positions 52-68 followed by positions 84-126 of SEQ ID NO:20, positions 52-68 followed by positions 84-126 of SEQ ED NO:24, positions 52-68 followed by positions 84-126 of SEQ ID NO:28, positions 52-68 followed by positions 84-126 of SEQ ID NO:32, positions 787 to 939 of SEQ ID NO:36, positions 7-159 of SEQ ID NO:39, positions 873-1025 of SEQ ID NO:92, positions 90-242 of SEQ ID NO:96, SEQ ID NO: 100, positions 719-778 of SEQ ID NO:101, positions 635-694 of SEQ ID NO:102, positions 184-337 of SEQ ID NQ:106, positions 521-674 of SEQ ID NO:106, positions 630-689 of SEQ ID NO:107, positions 630-689 of SEQ ID NO: 108, positions 630-689 of SEQ ID NO:109, positions 630-689 of SEQ ID NO: 110, positions 582-641 of SEQ ID NO: 122, positions 810-869 of SEQ ID ΝΟ.Ί24, positions 582-641 of SEQ ID NO:126, positions 1-60 of SEQ ID NO:130, positions 229 to 288 of SEQ ID NO: 132, positions 1 to 60 of SEQ ID NO: 134, or a corresponding sequence from a different HBV strain.
[0017] In one embodiment, the présent invention includes an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of: (i) an HBV X antigen comprising at least one immunogenic domain of a full-Iength HBV X antigen; (ii) an HBV surface antigen comprising at least one immunogenic domain of a full-length HBV large surface antigen (L), and; (iii) an HBV core antigen comprising at least one immunogenic domain of a fulllength HBV core protein. In one aspect of this embodiment, the immunotherapeutic composition comprises: (a) a yeast vehicle; and (b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of: (i) an HBV X antigen having an amino acid sequence that is at least 80% identical to positions 52 to 126 of a full-length HBV X antigen; (ii) an HBV surface antigen having an amino acid sequence that is at least 95% identical to an amino acid sequence of a full-length HBV large surface antigen (L), and; (iii) an HBV core antigen having an amino acid sequence that is at least 95% identical to an amino acid sequence of a full-length HBV core protein. The composition elicits an HBV-specific immune response.
[0018] In one aspect of this embodiment of the invention, the amino acid sequence of HBV X antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 1-60 of SEQ ID NO:130, positions 630-689 of SEQ ID NO:110, positions 582-641 of SEQ ID NO:122, positions 630-689 of SEQ ID NO:107, positions 630-689 of SEQ ID NO: 108, positions 630-689 of SEQ ID NO: 109, positions 52-68 followed by positions 84-126 of SEQ ID NO:4, positions 52-68 followed by positions 84-126 of SEQ ID NO:8, positions 52-68 followed by positions 84-126 of SEQ ID NO:12, positions 52-68 followed by positions 84-126 of SEQ ID NO:16, positions 5268 followed by positions 84-126 of SEQ ID NO:20, positions 52-68 followed by positions 84-126 of SEQ ID NO:24, positions 52-68 followed by positions 84-126 of SEQ ID NO:28, positions 52-68 followed by positions 84-126 of SEQ ID NO:32, SEQ ID NO: 100, positions 719-778 of SEQ ID NO:101, positions 635-694 of SEQ ID NO:102, positions 810-869 of SEQ ID NO: 124, positions 582-641 of SEQ ID NO:126, positions 229 to 288 of SEQ ID NO: 132, positions 1 to 60 of SEQ ID NO: 134, or a corresponding sequence from a different HBV strain. In one aspect, the amino acid sequence of HBV X antigen is selected from: positions 1-60 of SEQ ID NO:130, positions 630-689 of SEQ ID NO:110, positions 582-641 of SEQ ID NO:122, positions 630-689 of SEQ ID NO:109, positions 630-689 of SEQ ID NO:108, positions 630-689 of SEQ ID NO:107, SEQ ID NO:100, or a corresponding sequence from a different HBV strain.
[0019] In one aspect of this embodiment of the invention, the amino acid sequence of the HBV surface antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 63-461 of SEQ ID NO:130, positions 1-399 of SEQ ID NO: 118, positions 1-399 of SEQ ID NO: 122, positions 9-407 of SEQ ID NO:34, positions 1-399 of SEQ ID NO:112, positions 1-399 of SEQ ID NO:114, positions 1-399 of SEQ ID NO:116, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO;19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, positions 90-488 of SEQ ID NO:93, positions 1-399 of SEQ ID NO: 120, positions 1-399 of SEQ ID NO: 124, positions 1-399 of SEQ ID NO:126, positions 231-629 of SEQ ID NO:128, positions 289-687 of SEQ ID NO:132, positions 289-687 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain. In one aspect, the amino acid sequence of the HBV surface antigen is selected from: positions 63-461 of SEQ ID NO:130, positions 1-399 of SEQ ID NO:118, positions 1-399 of SEQ ID NO:122, positions 9-407 of SEQ ID NO:34, positions
1-399 of SEQ ID NO: 112, positions 1-399 of SEQ ID NO: 114, positions 1-399 of SEQ ID
NO:116, or a corresponding sequence from a different HBV strain.
[0020] In one aspect of this embodiment of the invention, the amino acid sequence of the HBV core antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 462 to 643 of SEQ ID NQ:130, positions 400-581 of SEQ ID NO: 118, positions 400 to 581 of SEQ ID NO:122, positions 408-589 of SEQ ID NO:34, positions 400-581 of SEQ ID NO:112, positions 400-581 of SEQ ID NO:114, positions 400-581 of SEQ ID NO:116, positions 31-212 of SEQ ID NO:1, positions 31212 of SEQ ID NO:5, positions 31-212 of SEQ ID NO:9, positions 31-212 of SEQ ID NO:13, positions 31-212 of SEQ ID NO:17, positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO;29, positions 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ ID NO:39, positions 605-786 of SEQ ID NO:41, positions 691-872 of SEQ ID NO:92, positions 90-271 of SEQ ID NO:95, positions 2-183 of SEQ ID NO:105, positions 184395 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions 579-761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO: 106, 338-520 of SEQ ID NO:106, positions 400 to 581 of SEQ ID NO:120, positions 400 to 581 of SEQ ID NO:124, positions 400 to 581 of SEQ ID NO:126, positions 630 to 811 of SEQ ID NO:128, positions 688 to 869 of SEQ ID NO:132, positions 688 to 869 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain. In one aspect, the amino acid sequence of the HBV core antigen is selected from: positions 462 to 643 of SEQ ID NO: 130, positions 400-581 of SEQ ID NO: 118, positions 400 to 581 of SEQ ID NO:122, positions 408-589 of SEQ ID NO:34, positions 400-581 of SEQ ID NO:116, positions 400-581 of SEQ ID NO:112, positions 400-581 of SEQ ID NO:114, or a corresponding sequence from a different HBV strain.
[0021] In one aspect of this embodiment of the invention, the HBV antigens are arranged in the following order, from N- to C-terminus, in the fusion protein: HBV X antigen, HBV surface antigen, HBV core antigen. In one aspect of this embodiment of the invention, the HBV antigens are arranged in the following order, from N- to C-terminus, in the fusion protein: HBV surface antigen, HBV core antigen, HBV X antigen.
[0022] In one aspect of this embodiment of the invention, the fusion protein comprises an amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical,
to an amino acid sequence selected from SEQ ID NO:130, SEQ ID NO: 122, or SEQ ID NO: 150.
[0023] Yet another embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a whole, heat-inactivated yeast from Saccharomyces 5 cerevisiae; and (b) an HBV fusion protein expressed by the yeast, wherein the fusion protein comprises SEQ ID NO: 130.
[0024] Another embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a whole, heat-inactivated yeast from Saccharomyces cerevisiae', and (b) an HBV fusion protein expressed by the yeast, wherein the fusion 10 protein comprises SEQ ID NO:150.
[0025] Yet another embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a whole, heat-inactivated yeast from Saccharomyces cerevisiae-, and (b) an HBV fusion protein expressed by the yeast, wherein the fusion protein comprises SEQ ID NO: 122. In one aspect, the fusion protein is a single polypeptide with the following sequences fused in frame from N- to C-terminus: (1) an amino acid sequence of SEQ ID NO:37; (2) a two amino acid linker peptide of threonineserine; (3) an amino acid sequence of SEQ ID NO: 122; and (4) a hexahistidine peptide.
[0026] In another embodiment of the invention, the immunotherapeutic composition includes: (a) a yeast vehicle; and (b) a fusion protein comprising HBV antigens consisting 20 of: (i) at least one immunogenic domain of HBV large surface antigen (L) and (ii) at least one immunogenic domain of HBV core protein or HBV e-antigen. The composition elicits an HBV-specific immune response, such as an immune response against HBV large surface antigen (L) and/or HBV core protein or HBV e-antigen.
[0027] In one embodiment, the présent invention includes an immunotherapeutic 25 composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising HBV antigens consisting of: (i) an HBV surface antigen having an amino acid sequence that is at least 95% identical to an amino acid sequence of a full-length HBV large surface antigen (L), and; (ii) an HBV core antigen having an amino acid sequence that is at least 95% identical to an amino acid sequence of a full-length HBV core protein. The 30 composition elicits an HBV-specific immune response. In one aspect of this embodiment, the HBV antigens consist of an amino acid sequence comprising at least 95% of a fulllength HBV large surface antigen (L) fused to an amino acid sequence comprising at least 95% of a full-length HBV core protein or HBV e-antigen. In one aspect of this embodiment, the HBV antigens consist of an amino acid sequence comprising at least
95% of a full-length HBV large surface antigen (L) fused to the N-terminus of an amino acid sequence comprising at least 95% of a full-length HBV core protein. In one aspect, the HBV antigens consist of: amino acids 2 to 400 of HBV large surface antigen (L); and amino acids 31 to 212 of the HBV precore protein comprising HBV core protein and a portion of HBV e-antigen.
[0028] In one aspect of this embodiment of the invention, the amino acid sequence of the HBV surface antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 1-399 of SEQ ID NO: 118, positions 9-407 of SEQ ID NO:34, positions 1-399 of SEQ ID NO:116, positions 1-399 of SEQ ID NO:112, positions 1-399 of SEQ ID NO: 114, SEQ ID NO:3 or positions 2-400 of SEQ ID NO:3, SEQ ID NO:7 or positions 2-400 of SEQ ID NO:7, SEQ ID NO: 11 or positions 2-400 of SEQ ID NO:11, SEQ ID NO:15 or positions 2-389 of SEQ ID NO:15, SEQ ID NO:19 or positions 2-399 of SEQ ID NO:19, SEQ ID NO:23 or positions 2-400 of SEQ ID NO:23, SEQ ID NO:27 or positions 2-399 of SEQ ID NO:27, SEQ ID NO:31 or positions 2-400 of SEQ ID NO:31, positions 90-488 of SEQ ID NO:93, positions 1-399 of SEQ ID NO:120, positions 1-399 of SEQ ID NO:122, positions 1-399 of SEQ ID NO:124, positions 1-399 of SEQ ID NO:126, positions 231-629 of SEQ ID NO:128, positions 63461 of SEQ ID NO: 130, positions 289-687 of SEQ ID NO:132, positions 289-687 of SEQ ID NO: 134, or a corresponding sequence from a different HBV strain. In one aspect, the amino acid sequence of the HBV surface antigen is selected from: positions 1-399 of SEQ ID NO:118, positions 9-407 of SEQ ID NO:34, positions 1-399 of SEQ ID NO:112, positions 1-399 of SEQ ID NO: 114, positions 1-399 of SEQ ID NO:116, or a corresponding sequence from a different HBV strain.
[0029] In one aspect of this embodiment of the invention, the amino acid sequence of the HBV core antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 400-581 of SEQ ID NO: 118, positions 408-589 of SEQ ID NO:34, positions 400-581 of SEQ ID NO:116, positions 400-581 of SEQ ID NO:112, positions 400-581 of SEQ ID NO:114, positions 31-212 of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212 of SEQ ID NO:9, positions 31-212 of SEQ ID NO: 13, positions 31-212 of SEQ ID NO:17, positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO:29, positions 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ ID
NO:39, positions 605-786 of SEQ ID NO:41, positions 691-872 of SEQ ID NO:92, positions 90-271 of SEQ ID NO:95, positions 2-183 of SEQ ID NO:105, positions 184395 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions 579-761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338-520 of SEQ ID NO:106, positions 400 to 581 of SEQ ID NO: 120, positions 400 to 581 of SEQ ID NO: 122, positions 400 to 581 of SEQ ID NO:124, positions 400 to 581 of SEQ ID NO:126, positions 630 to 811 of SEQ ID NO:128, positions 462 to 643 of SEQ ID NO:130, positions 688 to 869 of SEQ ID NO:132, positions 688 to 869 of SEQ ID ΝΟ.Ί34, or a correspondîng sequence from a different HBV strain. In one aspect, the amino acid sequence of the HBV core antigen is selected from: positions 400-581 of SEQ ID NO: 118, positions 408-589 of SEQ ID NO:34, positions 400-581 of SEQ ID NO: 116, positions 400-581 of SEQ ID NO: 112, positions 400-581 of SEQ ID NO: 114, or a correspondîng sequence from a different HBV strain.
[0030] In one aspect of this embodiment of the invention, the HBV antigens consist of amino acids 9 to 589 of SEQ ID NO:34, or a correspondîng sequence from a different HBV strain. In one aspect, the HBV antigens consist of an amino acid sequence that is at least 95% identicai, or at least 96% identicai, or at least 97% identicai, or at least 98% identicai, or at least 99% identicai, or is identicai, to an amino acid sequence selected from: SEQ ID NO:118, SEQ ID NO: 116, positions 9-589 of SEQ ID NO:34, SEQ ID NO:112, SEQ ID NO:114, or a correspondîng sequence for a different HBV strain. In one aspect, the HBV antigens consist of a full-length or near fuil-length HBV large surface antigen (L) and a full-length or near full-length HBV core protein.
[0031] In one aspect of this embodiment of the invention, any of the fusion proteins can include an N-terminal amino acid sequence (appended to the N-terminus of the fusion protein) of SEQ ID NO:37. In another aspect, any of the fusion proteins can include an Nterminal amino acid sequence selected from SEQ ID NO:89 or SEQ ID NO:90. In one aspect, the fusion protein comprises an amino acid sequence of SEQ ID NO: 151.
[0032] Yet another embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a whole, heat-inactivated yeast from Saccharomyces cerevisiae; and (b) an HBV fusion protein expressed by the yeast, wherein the fusion protein comprises SEQ ID NO:118.
[0033] Another embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a whole, heat-inactivated yeast from Saccharomyces
cerevisiae; and (b) an HBV fusion protein expressed by the yeast, wherein the fusion protein comprises SEQ ID NO: 151.
[0034] Yet another embodiment of the invention relates to an îmmunotherapeutic composition comprising: (a) a whole, heat-inactivated yeast from Saccharomyces 5 cerevisiae; and (b) an HBV fusion protein expressed by the yeast, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:34.
[0035] In another embodiment, the présent invention includes an îmmunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising HBV antigens. The HBV antigens consist of: (i) an HBV surface antigen consisting of at least 10 one immunogenic domain of full-length HBV large (L), medium (M) or small (S) surface antigen; (ii) an HBV polymerase antigen consisting of at least one immunogenic domain of full-length HBV polymerase or of the reverse transcriptase (RT) domain of HBV polymerase; (iii) an HBV core antigen consisting of at least one immunogenic domain of full-length HBV core protein or of full-length HBV e-antigen; and (iv) an HBV X antigen 15 consisting of at least one immunogenic domain of full-length HBV X antigen. The composition elicits an HBV-specific immune response. In one aspect of this embodiment, the HBV surface antigen comprises at least one immunogenic domain of hépatocyte receptor région of Pre-S 1 of the HBV large surface antigen (L) and at least one immunogenic domain of HBV small surface antigen (S).
[0036] In one aspect of this embodiment, the HBV antigens consist of: at least 95% of the full-length hépatocyte receptor of Pre-Sl of the HBV large surface antigen (L), at least 95% of the full-length HBV small surface antigen (S), at least 95% of the reverse transcriptase domain of HBV polymerase, at least 95% of the full-length HBV core protein or HBV e-antigen, and at least 95% of the full-length X antigen. In one aspect, the 25 HBV antigens consist of: an HBV large surface antigen (L) comprising at least 95% of amino acids 120 to 368 of HBV large surface antigen (L); an RT domain of HBV polymerase comprising at least 95% of amino acids 453 to 680 of the RT domain of HBV polymerase; an HBV core protein comprising at least 95% of amino acids 37 to 188 of HBV core protein; and an HBV X antigen comprising at least 80% of amino acids 52 to 30 127 of HBV X antigen. In one aspect, the HBV antigens consist of: amino acids 21 to 47 of HBV large surface antigen (L) comprising the hépatocyte receptor domain of Pre-Sl; amino acids 176 to 400 of HBV large surface antigen (L) comprising HBV small surface antigen (S); amino acids 247 to 691 of HBV polymerase comprising the reverse transcriptase domain; amino acids 31 to 212 of HBV precore protein comprising HBV core protein and a portion of HBV e-antigen; and amino acids 2 to 154 of HBV X antigen. In one aspect, the HBV antigens consist of: an amino acid sequence at least 95% identical to amino acids 120 to 368 of HBV large surface antigen (L); an amino acid sequence at least 95% identical to amino acids 453 to 680 of the RT domain of HBV polymerase; an amino acid sequence at least 95% identical to amino acids 37 to 188 of HBV core protein; and an amino acid sequence at least 80% identical to amino acids 52 to 127 of HBV X antigen. In one aspect, the HBV antigens hâve been modified to incorporate one or more T cell epitopes set forth in Table 5 and represented herein by SEQ ID NOs:42 to 88 or SEQ ID NOs: 135-140. In one aspect, the HBV large surface antigen (L) comprises an amino acid sequence of SEQ ID NO:97 or a sequence that is 95% identical to SEQ ID NO:97. In one aspect, the RT domain of an HBV polymerase comprises an amino acid sequence of SEQ ID NO:98 or a sequence that is 95% identical to SEQ ID NO:98. In one aspect, the HBV core protein comprises an amino acid sequence of SEQ ID NO:99 or a sequence that is 95% identical to SEQ ID NO:99. In one aspect, the HBV X antigen comprises an amino acid sequence of SEQ ID NO:100 or a sequence that is 95% identical to SEQ ID NO: 100.
[0037] In one aspect of this embodiment of the invention, the amino acid sequence of the HBV surface antigen is at least 95% identical to an amino acid sequence of a fulllength HBV large surface antigen (L). In one aspect, the amino acid sequence of the HBV surface antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 1-399 of SEQ ID NO: 124, positions 1-399 of SEQ ID NO:126, positions 289-687 of SEQ ID NO:132, positions 289-687 of SEQ ID NO.134, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, positions 9-407 of SEQ ID NO:34, positions 90488 of SEQ ID NO:93, positions 1-399 of SEQ ID NO:112, positions 1-399 of SEQ ID NO:114, positions 1-399 of SEQ ID NO:116, positions 1-399 of SEQ ID NO:118, positions 1-399 of SEQ ID NO: 120, positions 1-399 of SEQ ID NO: 122, positions 231629 of SEQ ID NO:128, positions 63-461 of SEQ ID NO:130, or a corresponding sequence from a different HBV strain.
[0038] In one aspect of this embodiment, the amino acid sequence of the HBV surface antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: SEQ ID NO:97, positions 1-249 of SEQ ID NQ:107, positions 116530
249 of SEQ ID NO: 108, positions 1-249 of SEQ ID NO: 109, positions 1-249 of SEQ ID
NO:110, positions 21-47 of SEQ ID NO:11, positions 176-400 of SEQ ID NO:11, positions 6-257 of SEQ ID NO:36, positions 6-257 of SEQ ID NO:41, positions 92-343 of
SEQ ID NO:92, positions 90-338 of SEQ ID NO: 101, positions 7-254 of SEQ ID NO: 102, or a corresponding sequence from a different HBV strain.
[0039] In one aspect of this embodiment, the HBV polymerase antigen consists of at least one immunogenic domain of the RT domain of HBV polymerase. In one aspect, the amino acid sequence of the HBV polymerase antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: SEQ ID NO:98, positions 582 to 809 of SEQ ID NO:124, positions 642 to 869 of SEQ ID NO:126, positions 1 to 228 of SEQ ID NO:132, positions 61 to 288 of SEQ ID NO:134, positions 250-477 of SEQ ID NO: 107, positions 250-477 of SEQ ID NO: 108, positions 250-477 of SEQ ID NO: 109, positions 250-477 of SEQ ID NO:110, positions 383-602 of SEQ ID NO:2, positions 381600 of SEQ ID NO:6, positions 381-600 of SEQ ID NO: 10, positions 453 to 680 of SEQ ID NO:10, positions 370-589 of SEQ ID NO:14, positions 380-599 of SEQ ID NO:18, positions 381-600 of SEQ ID NO:22, positions 380-599 of SEQ ID NO;26, positions 381600 of SEQ ID NO:30, positions 260 to 604 of SEQ ID NO:36, positions 7-351 of SEQ ID NO:38, positions 7-351 of SEQ ID NO:40,260 to 604 of SEQ ID NO:41, positions 346 to 690 of SEQ ID NO:92, positions 90-434 of SEQ ID NO:94, positions 339 to 566 of SEQ ID NO:101, positions 255 to 482 of SEQ ID NO:102, positions 582 to 809 of SEQ ID NO:120, positions 1 to 228 of SEQ ID NO: 128, or a corresponding sequence from a different HBV strain.
[0040] In one aspect of this embodiment, the amino acid sequence of the HBV core antigen is at least 95% identical to an amino acid sequence of a full-length HBV core protein. In one aspect, the amino acid sequence of the HBV core antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 400 to 581 of SEQ ID NO:124, positions 400 to 581 of SEQ ID NO:126, positions 688 to 869 of SEQ ID NO: 132, positions 688 to 869 of SEQ ID NO: 134, positions 408-589 of SEQ ID NO:34, positions 400-581 of SEQ ID NO:112, positions 400-581 of SEQ ID NO:114, positions 400-581 of SEQ ID NO: 116, positions 400-581 of SEQ ID NO: 118, positions 31-212 of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212 of SEQ ID NO:9, positions 31-212 of SEQ ID NO: 13, positions 31-212 of SEQ ID NO: 17, positions
31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ
ID NO:29, positions 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ ID NO:39, positions 605-786 of SEQ ID NO:41, positions 691872 of SEQ ID NO:92, positions 90-271 of SEQ ID NO:95, positions 2-183 of SEQ ID
NO:105, positions 184-395 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions 579-761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338-520 of SEQ ID NO:106, positions 400 to 581 of SEQ ID NO:120, positions 400 to 581 of SEQ ID NO:122, positions 630 to 811 of SEQ ID NO:128, positions 462 to 643 of SEQ ID NO:130, or a corresponding sequence from a different HBV strain.
[0041] In one aspect of this embodiment, the amino acid sequence of the HBV core antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions SEQ ID NO:99, 37 to 188 of SEQ ID NO:9, positions 567 to 718 of SEQ ID NO:101, positions 483 to 634 of SEQ ID NO: 102, positions 478-629 of SEQ
ID NO:107, positions 478-629 of SEQ ID NO:108, positions 478-629 of SEQ ID NO:109, positions 478-629 of SEQ ID NO: 110, or a corresponding sequence from a different HBV strain.
[0042] In one aspect of this embodiment, the HBV X antigen consists of an amino acid sequence that is at least 95% identical to a full-Iength HBV X antigen. In one aspect, 20 the HBV X antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO: 12, positions 2 to 154 of SEQ ID NO:12, SEQ ID NO.16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, positions 787 to 939 of SEQ ID NO;36, positions 7-159 of SEQ ID 25 NO:39, positions 873-1025 of SEQ ID NO:92, positions 90-242 of SEQ ID NO:96, positions 184-337 of SEQ ID NO:106, positions 521-674 of SEQ ID NO: 106, or a corresponding sequence from a different HBV strain.
[0043] In one aspect, the HBV X antigen consists of an amino acid sequence that is at least 80% identical to positions 52 to 126 of a full-Iength HBV X antigen. In one aspect, 30 the amino acid sequence of HBV X antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: SEQ ID NO:100, positions 810-869 of SEQ ID NO:124, positions 582-641 of SEQ ID NO:126, positions 229 to 288 of SEQ ID NO:132, positions 1 to 60 of SEQ ID NO:134, positions 630-689 of SEQ ID NO:107,
positions 630-689 of SEQ ID ΝΟ.Ί08, positions 630-689 of SEQ ID NO:109, positions 630-689 of SEQ ID NO.110, positions 52-68 followed by positions 84-126 of SEQ ID NO:4, positions 52-68 followed by positions 84-126 of SEQ ID NO:8, positions 52-68 followed by positions 84-126 of SEQ ID NO;12, positions 52-68 followed by positions 5 84-126 of SEQ ID NO:16, positions 52-68 followed by positions 84-126 of SEQ ID
NO:20, positions 52-68 followed by positions 84-126 of SEQ ID NO:24, positions 52-68 followed by positions 84-126 of SEQ ID NO:28, positions 52-68 followed by positions
84-126 of SEQ ID NO:32, positions 719-778 of SEQ ID NO:101, positions 635-694 of SEQ ID NO: 102, positions 582-641 of SEQ ID NO:122, positions 1-60 of SEQ ID
ΝΟ.Ί30, or a corresponding sequence from a different HBV strain.
[0044] In one aspect of this embodiment, the HBV antigens hâve an amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 6 to 939 of SEQ ID NO:36, positions 92 to 1025 of 15 SEQ ID NO:92, positions 90 to 778 of SEQ ID NO: 101, positions 7 to 694 of SEQ ID
NO: 102, or a corresponding sequence from a different HBV strain.
[0045] In one aspect of this embodiment, the fusion protein comprises an amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid 20 sequence selected from: SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO; 109, SEQ ID
NO:110, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:132 or SEQ ID NO: 134.
[0046] Any of the fusion proteins may, in one aspect, comprise an N-terminal sequence selected from SEQ ID NO:37, SEQ ID NO:89, or SEQ ID NO:90.
[0047] In one aspect of this embodiment, the fusion protein comprises an amino acid 25 sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: SEQ ID NO:36, SEQ ID NO:92, SEQ ID NO:101, or SEQ ID ΝΟ.Ί02.
[0048] Another embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of: (i) an HBV surface antigen consisting of at least one immunogenic domain of hépatocyte receptor région of Pre-S 1 of the HBV large surface antigen (L) and at least one immunogenic domain of HBV small surface antigen (S); (d) an HBV polymerase antigen consisting of at least one immunogenic domain of
reverse transcriptase domain of HBV polymerase; and (iii) an HBV core antigen consisting of at least one immunogenic domain of HBV core protein. The composition elicits an HBV-specific immune response. In one aspect, the HBV antigens consist of at least 95% of full-length hépatocyte receptor of Pre-Sl of HBV large surface antigen (L), at 5 least 95% of full-length HBV small surface antigen, at least 95% of full-length reverse transcriptase domain of HBV polymerase, and at least 95% of full-length HBV core protein. In one aspect, the HBV antigens consist of at least 95% of full-length HBV large surface antigen (L), at least 95% of full-length reverse transcriptase domain of HBV polymerase, and at least 95% of full-length HBV core protein.
[0049J In one aspect of this embodiment, the amino acid sequence of the HBV surface antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 1-399 of SEQ ID NO: 120, positions 231-629 of SEQ ID NO:128, positions 1-399 of SEQ ID NO:112, positions 1-399 of SEQ ID NO:114, positions 1-399 of SEQ ID NO:116, positions 1-399 of SEQ ID NO:118, positions 6-257 of SEQ ID NO:41, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, positions 21-47 of SEQ ID NO:11, positions 176-400 of SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, positions 9-407 of SEQ ID NO:34, positions 6257 of SEQ ID NO:36, positions 92-343 of SEQ ID NO;92, positions 90-488 of SEQ ID
NO:93, SEQ ID NO:97, positions 90-338 of SEQ ID NO: 101, positions 7-254 of SEQ ID
NO:102, positions 1-249 of SEQ ID NO:107, positions 1-249 of SEQ ID NO:108, positions 1-249 of SEQ ID NO:109, positions 1-249 of SEQ ID NO:110, positions 1-399 of SEQ ID NO:122, positions 1-399 of SEQ ID NO:124, positions 1-399 of SEQ ID NO:126, positions 63-461 of SEQ ID NO: 130, positions 289-687 of SEQ ID NO: 132, positions 289-687 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain.
[0050] In one aspect of this embodiment of the invention, the amino acid sequence of the HBV polymerase antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an 30 amino acid sequence selected from: positions 582 to 809 of SEQ ID NO: 120, positions 1 to 228 of SEQ ID NO: 128, positions 250-477 of SEQ ID NO: 107, positions 250-477 of SEQ ID NO:108, positions 250-477 of SEQ ID NO:109, positions 250-477 of SEQ ID NO: 110,260 to 604 of SEQ ID NO:41, positions 383-602 of SEQ ID NO:2, positions 381600 of SEQ ID NO:6, positions 381-600 of SEQ ID NO: 10, positions 453 to 680 of SEQ
ID NO-,10, positions 370-589 of SEQ ID NO:14, positions 380-599 of SEQ ID NO:18, positions 381-600 of SEQ ID NO:22, positions 380-599 of SEQ ID NO:26, positions 381600 of SEQ ID NO:30, positions 260 to 604 of SEQ ID NO:36, positions 7-351 of SEQ ID NO:38, positions 7-351 of SEQ ID NO:40, positions 346 to 690 of SEQ ID NO:92, positions 90-434 of SEQ ID NO:94, SEQ ID NO;98, positions 339 to 566 of SEQ ID NQ:101, positions 255 to 482 of SEQ ID NO:102, positions 582 to 809 of SEQ ID NO:124, positions 642 to 869 of SEQ ID NO:126, positions 1 to 228 of SEQ ID NO:132, positions 61 to 288 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain.
[0051] In one aspect of this embodiment of the invention, the amino acid sequence of the HBV core antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 400 to 581 of SEQ ID NO:120, positions 630 to 811 of SEQ ID NO:128, positions 400-581 of SEQ ID NO:112, positions 400-581 of SEQ ID NO:114, positions 400-581 of SEQ ID NO:116, positions 400-581 of SEQ ID NO:118, positions 605-786 of SEQ ID NO:41, positions 31-212 of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212 of SEQ ID NO:9, positions 37 to 188 of SEQ ID NO:9, positions 31-212 of SEQ ID NO:13, positions 31-212 of SEQ ID NO:17, positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO:29, positions 408-589 of SEQ ID NO:34, positions 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ ID NO:39, positions 691872 of SEQ ID NO:92, positions 90-271 of SEQ ID NO:95, SEQ ID NO:99, positions 567 to 718 of SEQ ID NO: 101, positions 483 to 634 of SEQ ID NO: 102, positions 2-183 of SEQ ID NO:105, positions 184-395 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions 579-761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338520 of SEQ ID NO: 106, positions 478-629 of SEQ ID NO:107, positions 478-629 of SEQ ID NO:108, positions 478-629 of SEQ ID NO:109, positions 478-629 of SEQ ID NO:110, positions 400 to 581 of SEQ ID NO: 122, positions 400 to 581 of SEQ ID NO: 124, positions 400 to 581 of SEQ ID NO: 126, positions 462 to 643 of SEQ ID NO:130, positions 688 to 869 of SEQ ID NO:132, positions 688 to 869 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain.
[0052] In one aspect of this embodiment of the invention, the fusion protein has an amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an
amino acid sequence selected from: SEQ ID NO: 120, SEQ ID NO:128, positions 6-786 of SEQ ID NO:41, or SEQ ID NO:41, or a corresponding sequence from a different HBV strain.
[0053] Another embodiment of the invention relates to an immunotherapeutic 5 composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of: (i) an HBV polymerase antigen consisting of at least one immunogenic domain of the reverse transcriptase (RT) domain of HBV polymerase; and (ii) an HBV core antigen consisting of at least one immunogenic domain of HBV core protein. The composition elicits an HBV-specific immune response. In one 10 aspect of this embodiment of the invention, the HBV antigens consist of: an amino acid sequence that is at least 95% identical to full-length RT domain of HBV polymerase and an amino acid sequence that is at least 95% identical to full-length HBV core protein.
[0054] In one aspect of this embodiment of the invention, the amino acid sequence of the HBV polymerase antigen is at least 95% identical, or at least 96% identical, or at least 15 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 7-351 of SEQ ID NO:38, positions 383-602 of SEQ ID NO:2, positions 381-600 of SEQ ID NO:6, positions 381-600 of SEQ ID NO:10, positions 453 to 680 of SEQ ID NO:10, positions 370-589 of SEQ ID NO:14, positions 380-599 of SEQ ID NO:18, positions 381-600 of SEQ ID NO:22, positions 38020 599 of SEQ ID NO:26, positions 381-600 of SEQ ID NO:30, positions 260 to 604 of SEQ
ID NO:36, positions 7-351 of SEQ ID NO:40, 260 to 604 of SEQ ID NO:41, positions 346 to 690 of SEQ ID NO:92, positions 90-434 of SEQ ID NO:94, SEQ ID NO:98, positions 339 to 566 of SEQ ID NO:101, positions 255 to 482 of SEQ ID NO:102, positions 250477 of SEQ ID NQ:107, positions 250-477 of SEQ ID NO;108, positions 250-477 of SEQ 25 ID NO:109, positions 250-477 of SEQ ID NO:110, positions 582 to 809 of SEQ ID
NO.120, positions 582 to 809 of SEQ ID NO: 124, positions 642 to 869 of SEQ ID NO:126, positions 1 to 228 of SEQ ID NO:128, positions 1 to 228 of SEQ ID NO:132, positions 61 to 288 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain.
[0055] In one aspect of this embodiment of the invention, the amino acid sequence of the HBV core antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 352-533 of SEQ ID NO:38, positions 31-212 of
SEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212 of SEQ ID NO:9,
positions 37 to 188 of SEQ ÏD N0:9, positions 31-212 of SEQ ID N0:13, positions 31212 of SEQ ID NO:17, positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO:29, positions 408-589 of SEQ ID NO:34, positions 605 to 786 of SEQ ID NO:36, positions 160-341 of SEQ ID NO:39, positions
605-786 of SEQ ID NO:41, positions 691-872 of SEQ ID NO:92, positions 90-271 of
SEQ ID NO:95, SEQ ID NO:99, positions 567 to 718 of SEQ ID NO:101, positions 483 to 634 of SEQ ID NO:102, positions 2-183 of SEQ ID NO:105, positions 184-395 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions 579-761 of SEQ ID NO:1Û5, positions 2-183 of SEQ ID NO: 106, 338-520 of SEQ ID NO: 106, positions 478-629 of 10 SEQ ID NO:107, positions 478-629 of SEQ ID NO:108, positions 478-629 of SEQ ID
NO:109, positions 478-629 of SEQ ID NO:110, positions 400-581 of SEQ ID NO:112, positions 400-581 of SEQ ID NO:114, positions 400-581 of SEQ ID NO:116, positions 400-581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:120, positions 400 to 581 of SEQ ID NO:122, positions 400 to 581 of SEQ ID NO:124, positions 400 to 581 of 15 SEQ ID NO:126, positions 630 to 811 of SEQ ID ΝΟ.Ί28, positions 462 to 643 of SEQ
ID NO:130, positions 688 to 869 of SEQ ID NO:132, positions 688 to 869 of SEQ ID NO: 134, or a corresponding sequence from a different HBV strain.
[0056] In one aspect of this embodiment of the invention, the fusion protein has an amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 20 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence of SEQ ID NO:38, or a corresponding sequence from a different HBV strain.
[0057] Yet another embodiment of the invention relates to an immunotherapeutic composition comprising; (a) a yeast vehicle; and (b) a fusion protein comprising HBV 25 antigens, wherein the HBV antigens consist of: (i) an HBV X antigen consisting of at least one immunogenic domain of HBV X antigen; and (ii) an HBV core antigen consisting of at least one immunogenic domain of HBV core protein. The composition elicits an HBVspecific immune response. In one aspect of this embodiment, the HBV antigens consist of: an amino acid sequence that is at least 95% identical to full-length HBV X antigen and 30 an amino acid sequence that is at least 95% identical to full-length HBV core protein.
[0058] In one aspect of this embodiment of the invention, the amino acid sequence of the HBV core antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 160-341 of SEQ ID NO:39, positions 31-212 of
Φ 22
SEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212 of SEQ ID NO:9, positions 37 to 188 of SEQ ID NO:9, positions 31-212 of SEQ ID NO:13, positions 31212 of SEQ ID NO:17, positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO:29, positions 408-589 of SEQ ID NO:34, positions 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38, positions 605-786 of SEQ ID NO:41, positions 691-872 of SEQ ID NO:92, positions 90-271 of
SEQ ID NO:95, SEQ ID NO:99, positions 567 to 718 of SEQ ID NO:101, positions 483 to 634 of SEQ ID NO:102, positions 2-183 of SEQ ID NO:105, positions 184-395 of SEQ ID
NO:105, positions 396-578 of SEQ ID NO:105, positions 579-761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO.106, 338-520 of SEQ ID NO:106, positions 478-629 of SEQ ID NO: 107, positions 478-629 of SEQ ID NO: 108, positions 478-629 of SEQ ID NO:109, positions 478-629 of SEQ ID NO:110, positions 400-581 of SEQ ID NO:112, positions 400-581 of SEQ ID NO:114, positions 400-581 of SEQ ID NO:116, positions 400-581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:120, positions 400 to
581 of SEQ ID NO:122, positions 400 to 581 of SEQ ID NO-.124, positions 400 to 581 of
SEQ ID NO:126, positions 630 to 811 of SEQ ID NO;128, positions 462 to 643 of SEQ
ID NO: 130, positions 688 to 869 of SEQ ID NO:132, positions 688 to 869 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain.
[0059] In one aspect of this embodiment of the invention, the amino acid sequence of the HBV X antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 7-159 of SEQ ID NO:39, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, positions 2 to 154 of SEQ ID NO:12, SEQ ID NO:16, SEQ ID
NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, positions 52-68 followed by positions 84-126 of SEQ ID NO:4, positions 52-68 followed by positions 84-126 of SEQ ID NO:8, positions 52-68 followed by positions 84-126 of SEQ ID NO:12, positions 52-68 followed by positions 84-126 of SEQ ID NO:16, positions 52-68 followed by positions 84-126 of SEQ ID NO;20, positions 52-68 followed by positions 84-126 of SEQ ID NO:24, positions 52-68 followed by positions 84-126 of SEQ ID NO:28, positions 52-68 followed by positions 84-126 of SEQ ID NO:32, positions 787 to 939 of SEQ ID NO:36, positions 873-1025 of SEQ ID NO:92, positions 90-242 of SEQ ID NO:96, SEQ ID
NO:100, positions 719-778 of SEQ ID NO:101, positions 635-694 of SEQ ID NO:102, positions 184-337 of SEQ ID NO:106, positions 521-674 of SEQ ID NO:106, positions
630-689 of SEQ ID NO: 107, positions 630-689 of SEQ ID NO: 108, positions 630-689 of
SEQ ID NO:109, positions 630-689 of SEQ ID NO:110, positions 582-641 of SEQ ID
NO:122, positions 810-869 of SEQ ID NO:124, positions 582-641 of SEQ ID NO:126, positions 1-60 of SEQ ID NO: 130, positions 229 to 288 of SEQ ID NO: 132, positions 1 to of SEQ ID NO: 134, or a corresponding sequence from a different HBV strain.
[0060] In one aspect of this embodiment of the invention, the fusion protein has the amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence to SEQ ID NO:39, or a corresponding sequence from a different HBV strain.
[0061] Another embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising an HBV surface antigen consisting of at least one immunogenic domain of an HBV large surface antigen (L), wherein the composition elicits an HBV-specific immune response. In one aspect of this embodiment, the HBV surface antigen consists of at least 95% of full-length
HBV large surface antigen (L). In one aspect, the amino acid sequence of the HBV surface antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 90-488 of SEQ ID NO:93, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, positions 21-47 of SEQ ID NO:11, positions 176-400 of SEQ ID
NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, positions 9-407 of SEQ ID NO:34, positions 6-257 of SEQ ID NO:36, positions 6-257 of SEQ ID NO:41, positions 92-343 of SEQ ID NO:92, positions 90-488 of SEQ ID NO:93, SEQ ID NO:97, positions 90-338 of SEQ ID NO: 101, positions 7-254 of SEQ ID NO:102, positions 1-249 of SEQ ID NO: 107, positions 1-249 of SEQ ID NO: 108, positions 1-249 of SEQ ID NO:109, positions 1-249 of SEQ ID NO:110, positions 1-399 of SEQ ID NO:112, positions 1-399 of SEQ ID NO:114, positions 1-399 of SEQ ID NO:116, positions 1-399 of SEQ ID NO: 118, positions 1-399 of SEQ ID NO: 120, positions 1-399 of SEQ ID NO: 122, positions 1-399 of SEQ ID NO:124, positions 1-399 of SEQ ID NO: 126, positions 231-629 of SEQ ID NO: 128, positions 63-461 of SEQ ID NO: 130, positions 289-687 of SEQ ID NO: 132, positions 289-687 of SEQ ID NO: 134, or a corresponding sequence from a different HBV strain. In one aspect, the fusion protein has the amino acid sequence that is at least 95% identical, or at least 96% identical, or at least
97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an
amino acid sequence of SEQ ID NO:93, or a corresponding sequence from a different HBV strain.
[0062] Yet another embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising an HBV 5 polymerase antigen consisting of at least one immunogenic domain of a reverse transcriptase domain of HBV polymerase, wherein the composition elicits an HBVspecific immune response. In one aspect of this embodiment of the invention, the HBV polymerase antigen consists of at least 95% of full-length reverse transcriptase domain of HBV polymerase. In one aspect, the amino acid sequence of the HBV polymerase antigen 10 is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 7-351 of SEQ ID NO:40, positions 90-434 of SEQ ID NO:94, positions 383-602 of SEQ ID NO:2, positions 381-600 of SEQ ID NO:6, positions 381-600 of SEQ ID NO:10, positions 453 to 680 of SEQ ID NO:10, positions 370-589 of SEQ ID NO:14, 15 positions 380-599 of SEQ ID NO: 18, positions 381-600 of SEQ ID NO:22, positions 380599 of SEQ ID NO:26, positions 381-600 of SEQ ID NO:30, positions 260 to 604 of SEQ ID NO:36, positions 7-351 of SEQ ID NO:38, 260 to 604 of SEQ ID NO:41, positions 346 to 690 of SEQ ID NO:92, SEQ ID NO:98, positions 339 to 566 of SEQ ID NO: 101, positions 255 to 482 of SEQ ID NO: 102, positions 250-477 of SEQ ID NO: 107, positions 20 250-477 of SEQ ID NO: 108, positions 250-477 of SEQ ID NO:109, positions 250-477 of
SEQ ID NO: 110, positions 582 to 809 of SEQ ID NO: 120, positions 582 to 809 of SEQ
ID NO:124, positions 642 to 869 of SEQ ID NO:126, positions 1 to 228 of SEQ ID NO: 128, positions 1 to 228 of SEQ ID NO:132, positions 61 to 288 of SEQ ID NO: 134, or a corresponding sequence from a different HBV strain. In one aspect, the fusion protein 25 has the amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence of SEQ ID NO:40 or SEQ ID NO:94, or a corresponding sequence from a different HBV strain.
[0063] Another embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising an HBV core antigen consisting of at least one immunogenic domain of an HBV core protein, wherein the composition elicits an HBV-specific immune response. In one aspect of this embodiment of the invention, the HBV antigens consist of at least 95% of full-length HBV core protein. In one aspect, the amino acid sequence of the HBV core antigen is at least
95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 90-271 of SEQ ID NO:95, positions 31-212 of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212 of SEQ ID NO:9, positions 37 to 188 of SEQ ID NO:9, positions 31-212 of SEQ ID NO:13, positions 31-212 of SEQ ID NO:17, positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO:29, positions 408-589 of SEQ ID NO:34, positions 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ ID NO:39, positions 605786 of SEQ ID NO:41, positions 691-872 of SEQ ID NO:92, SEQ ID NO:99, positions 567 to 718 of SEQ ID NO:101, positions 483 to 634 of SEQ ID NO:102, positions 2-183 of SEQ ID NO:105, positions 184-395 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions 579-761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338520 of SEQ ID NO:106, positions 478-629 of SEQ ID NO:107, positions 478-629 of SEQ ID NO:108, positions 478-629 of SEQ ID NO:109, positions 478-629 of SEQ ID NO:110, positions 400-581 of SEQ ID NO:112, positions 400-581 of SEQ ID NO:114, positions 400-581 of SEQ ID NO: 116, positions 400-581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:120, positions 400 to 581 of SEQ ID NO:122, positions 400 to 581 of SEQ ID NO:124, positions 400 to 581 of SEQ ID NO:126, positions 630 to 811 of SEQ ID NO:128, positions 462 to 643 of SEQ ID NO: 130, positions 688 to 869 of SEQ ID NO:132, positions 688 to 869 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain. In one aspect, the protein has the amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence of SEQ ID NO:95, or a corresponding sequence from a different HBV strain.
[0064] Yet another embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising an HBV X antigen consisting of at least one immunogenic domain of a full-length HBV X antigen, wherein the composition elicits an HBV-specific immune response. In one aspect, the HBV antigen consists of at least 95% of full-length HBV X antigen. In one aspect, the amino acid sequence of the HBV X antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 90-242 of SEQ ID NO:96, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, positions 2 to 154 of SEQ ID NO:12, SEQ ID NO: 16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, positions 5216530 followed by positions 84-126 of SEQ ID NO:4, positions 52-68 followed by positions 84-126 of SEQ ID NO:8, positions 52-68 followed by positions 84-126 of SEQ ID NO:12, positions 52-68 followed by positions 84-126 of SEQ ID NO: 16, positions 52-68 followed by positions 84-126 of SEQ ID NO:20, positions 52-68 followed by positions 84-126 of SEQ ID NO:24, positions 52-68 followed by positions 84-126 of SEQ ID NO:28, positions 52-68 followed by positions 84-126 of SEQ ID NO:32, positions 787 to 939 of SEQ ID NO:36, positions 7-159 of SEQ ID NO:39, positions 873-1025 of SEQ ID NO:92, SEQ ID NOrlOO, positions 719-778 of SEQ ID NO: 101, positions 635-694 of SEQ ID NO:102, positions 184-337 of SEQ ID NO:106, positions 521-674 of SEQ ID NO:106, positions 630-689 of SEQ ID NO:107, positions 630-689 of SEQ ID NO:108, positions 630-689 of SEQ ID NO: 109, positions 630-689 of SEQ ÏD NO: 110, positions 582-641 of SEQ ID NO:122, positions 810-869 of SEQ ID NO:124, positions 582-641 of SEQ ID NO:126, positions 1-60 of SEQ ID NO:130, positions 229 to 288 of SEQ ID NO:132, positions 1 to 60 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain. In one aspect, the protein has the amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence of SEQ ID NO:96, or a corresponding sequence from a different HBV strain.
[0065] Another embodiment of the invention relates to an Îmmunotherapeutic composition comprising any two, three or four of the îmmunotherapeutic compositions described above, or elsewhere herein and in particular, any two, three, or four of the îmmunotherapeutic compositions described above that relate to single HBV proteins.
[0066] Yet another embodiment of the invention relates to an îmmunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of at least one immunogenic domain of two, three or four HBV surface antigen proteins, wherein each of the HBV surface antigen proteins is from a different HBV génotype. The composition elicits an HBV-specific immune response.
[0067] Yet another embodiment of the invention relates to an îmmunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of at least one immunogenic domain of two, three or four HBV polymerase proteins, wherein each of the HBV polymerase proteins is from a different HBV génotype. The composition elicits an HBV-specific immune response.
[0068] Yet another embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of at least one immunogenic domain of two, three or four HBV X antigens, wherein each of the HBV X antigens is from a different
HBV génotype. The composition elicits an HBV-specific immune response.
[0069] Yet another embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of at least one immunogenic domain of two, three or four HBV core proteins, wherein each of the HBV core proteins is from a different HBV génotype. The composition elicits an HBV-specific immune response. In one aspect, each of the HBV core proteins consists of at least 95% of a full-length HBV core protein. In one aspect, each of the HBV core proteins consists of amino acids 31 to 212 of HBV core protein. In one aspect, the HBV génotypes include génotype C, and in one aspect, the HBV génotypes include génotype D, and in one aspect, the HBV génotypes include génotype A, and in one aspect, the HBV génotypes include génotype B. In one aspect, each of the HBV core proteins consists of amino acids 37 to 188 of HBV core protein. In one aspect, the fusion protein comprises four HBV core proteins from génotype A, génotype B, génotype C and génotype D.
[0070] In one aspect of this embodiment of the invention, the amino acid sequence of any one or more of the HBV core antigens is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 90-271 of SEQ ID NO:95, positions 31-212 of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212 of SEQ ID NO:9, positions 37 to 188 of SEQ ID NO:9, positions 31-212 of SEQ ID NO:13, positions 31-212 of SEQ ID NO: 17, positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO;29, positions 408-589 of SEQ ID NO:34, positions 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ ID NO:39, positions 605-786 of SEQ ID NO:41, positions 691872 of SEQ ID NO:92, SEQ ID NO:99, positions 567 to 718 of SEQ ID NO: 101, positions 483 to 634 of SEQ ID NO:102, positions 2-183 of SEQ ID NO:105, positions 184-395 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions 579-761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338-520 of SEQ ID NO:106, positions 478-629 of SEQ ID NO: 107, positions 478-629 of SEQ ID NO: 108, positions 478-629 of SEQ ID NO:109, positions 478-629 of SEQ ID NO: 110, positions 400-581 of
SEQ ID NO:112, positions 400-581 of SEQ ID NO:114, positions 400-581 of SEQ ID
NO:116, positions 400-581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:120, positions 400 to 581 of SEQ ID NO:122, positions 400 to 581 of SEQ ID NO: 124, positions 400 to 581 of SEQ ID NO:126, positions 630 to 811 of SEQ ID NO:128, positions 462 to 643 of SEQ ID NO: 130, positions 688 to 869 of SEQ ID NO: 132, positions 688 to 869 of SEQ ID NO: 134, or a corresponding sequence from a different
HBV strain. In one aspect, the HBV antigens have an amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence of SEQ ID NO: 105, or a corresponding sequence from a different HBV strain.
[0071] Yet another embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising at least two HBV Core proteins and at least two HBV X antigens, where each of the HBV Core proteins is from a different HBV génotype and where each of the HBV X antigens is from a different HBV génotype. The composition elicits an HBV-specific immune response. In one aspect, the HBV génotypes include génotype C; in one aspect, the HBV génotypes include génotype D; in one aspect, the HBV génotypes include génotype A; and in one aspect, the HBV génotypes include génotype B. In one aspect, each of the HBV core proteins consists of at least 95% of a full-length HBV Core protein. In one aspect, each of the HBV core proteins comprises amino acids 31 to 212 of HBV Core protein. In one aspect, each of the HBV core proteins comprises amino acids 37 to 188 of HBV Core protein. In one aspect, each of the HBV X antigens comprises at least 95% of a full-length of HBV X antigen. In one aspect, each of the HBV X antigens comprises amino acids 52 to 127 of HBV X antigen.
[0072] In one aspect, the amino acid sequence of the HBV core antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 90-271 of SEQ ID NO:95, positions 31-212 of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212 of SEQ ID NO:9, positions 37 to 188 of SEQ ID NO:9, positions 31-212 of SEQ ID NO:13, positions 31-212 of SEQ ID NO:17, positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO:29, positions 408-589 of SEQ ID NO:34, positions 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ ID NO:39, positions 605786 of SEQ ID NO:41, positions 691-872 of SEQ ID NO:92, SEQ ID NO:99, positions
567 to 718 of SEQ ID NO:101, positions 483 to 634 of SEQ ID NO:102, positions 2-183 of SEQ ID NO: 105, positions 184-395 of SEQ ID NO:105, positions 396-578 of SEQ ID
NO:105, positions 579-761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338520 of SEQ ID NO: 106, positions 478-629 of SEQ ID NO: 107, positions 478-629 of SEQ
ID NO:108, positions 478-629 of SEQ ID NO:109, positions 478-629 of SEQ ID NO:110, positions 400-581 of SEQ ID NO:112, positions 400-581 of SEQ ID NO:114, positions 400-581 of SEQ ID NO:116, positions 400-581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO: 120, positions 400 to 581 of SEQ ID NO: 122, positions 400 to 581 of SEQ ID NO: 124, positions 400 to 581 of SEQ ID NO: 126, positions 630 to 811 of SEQ ID
NO:128, positions 462 to 643 of SEQ ID NÛ:130, positions 688 to 869 of SEQ ID
NO: 132, positions 688 to 869 of SEQ ID NO: 134, or a corresponding sequence from a different HBV strain.
[0073] In one aspect, the amino acid sequence of the HBV X antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 90242 of SEQ ID NO:96, SEQ ID NO;4, SEQ ID NO:8, SEQ ID NO:12, positions 2 to 154 of SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ
ID NO:32, positions 52-68 followed by positions 84-126 of SEQ ID NO:4, positions 52-68 followed by positions 84-126 of SEQ ID NO:8, positions 52-68 followed by positions 8420 126 of SEQ ID NO:12, positions 52-68 followed by positions 84-126 of SEQ ID NO:16, positions 52-68 followed by positions 84-126 of SEQ ID NO:20, positions 52-68 followed by positions 84-126 of SEQ ID NO:24, positions 52-68 followed by positions 84-126 of SEQ ID NO:28, positions 52-68 followed by positions 84-126 of SEQ ID NO:32, positions 787 to 939 of SEQ ID NO:36, positions 7-159 of SEQ ID NO:39, positions 87325 1025 of SEQ ID NO:92, SEQ ID NO: 100, positions 719-778 of SEQ ID NO: 101, positions 635-694 of SEQ ID NO:102, positions 184-337 of SEQ ID NO:106, positions 521-674 of SEQ ID NO:106, positions 630-689 of SEQ ID NO:107, positions 630-689 of SEQ ID NO: 108, positions 630-689 of SEQ ID NO: 109, positions 630-689 of SEQ ID NO:110, positions 582-641 of SEQ ID NO:122, positions 810-869 of SEQ ID NO:124, positions 582-641 of SEQ ID NO:126, positions 1-60 of SEQ ID NO:130, positions 229 to
288 of SEQ ID NO:132, positions 1 to 60 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain.
[0074] In one aspect of this embodiment of the invention, the fusion protein has an amino acid sequence that is at least 95% identical, or at least 96% identical, or at least
97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence of SEQ ID NO: 106, or a corresponding sequence from a different HBV strain.
[0075] In any of the embodiments described herein, including above and below, 5 related to a fusion protein, HBV antigens, or immunotherapeutic composition comprising such a fusion protein or HBV antigens, in one further embodiment, the fusion protein can be appended at its N-terminus to add an additional sequence. In one aspect, the Nterminal sequence is selected from an amino acid sequence that is 95% identical to SEQ ID NO:37, an amino acid sequence that is 95% identical to SEQ ID NO:89, or an amino 10 acid sequence that is 95% identical to SEQ ID NO:90. In one aspect, the N-terminal sequence is selected from SEQ ID NO:37, positions 1 to 5 of SEQ ID NO:37, SEQ ID NO:89, or SEQ ID NO:90, or a corresponding sequence from a different HBV strain.
[0076] In one aspect of any of the embodiments of the invention described above or elsewhere herein, the fusion protein is expressed by the yeast vehicle. In another aspect of 15 any of the embodiments of the invention described above or elsewhere herein, the yeast vehicle is a whole yeast. The whole yeast, in one aspect is killed. In one aspect, the whole yeast is heat-inactivated.
[0077] In one aspect of any of any of the embodiments of the invention described above or elsewhere herein, the yeast vehicle can be from a yeast genus selected from: 20 Saccharomyces, Candida, Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula,
Schizosaccharomyces and Yarrowia. In one aspect, the yeast vehicle is from Saccharomyces. In one aspect, the yeast vehicle is from Saccharomyces cerevisiae.
[0078] In one aspect of any of the embodiments of the invention described above or elsewhere herein, the composition is formulated for administration to a subject or patient.
In one aspect, the composition is formulated for administration by injection of a subject or patient (e.g., by a parentéral route, such as subcutaneous or intraperitoneal or intramuscular injection). In one aspect, the composition is formulated in a pharmaceutically acceptable excipient that is suitable for administration to a human. In one aspect, the composition contains greater than 90% yeast protein. In one aspect, the 30 composition contains greater than 90% yeast protein and is formulated for administration to a patient.
[0079] In one aspect of any of the embodiments of the invention described above or elsewhere herein, the fusion protein is not aggregated in the yeast. In one aspect, the fusion protein does not form inclusion bodies in the yeast. In one aspect, the fusion protein does not form VLPs or other large antigen particles in the yeast. In one aspect, the fusion protein does form VLPs or other large antigen particles in the yeast.
[0080] In one aspect of any embodiment of the invention described above or elsewhere herein, in one aspect, the HBV sequences are from HBV génotype A. In another aspect, the HBV sequences are from HBV génotype B. In another aspect, the HBV sequences are from HBV génotype C. In another aspect, the HBV sequences are from HBV génotype D. In another aspect, the HBV sequences are from HBV génotype E. In another aspect, the HBV sequences are from HBV génotype F. In another aspect, the HBV sequences are from HBV génotype G. In another aspect, the HBV sequences are from HBV génotype H. In one aspect, the HBV sequences are from a combination of any of the above-referenced HBV génotypes or of any known HBV génotypes or subgenotypes.
[0081] Another embodiment of the invention relates to any of the fusion proteins described above as part of an immunotherapeutic composition of the invention, or elsewhere herein. In one aspect of this embodiment, a fusion protein comprises HBV antigens, the HBV antigens selected from, but not limited to: (a) HBV antigens consisting of: HBV large surface antigen (L), HBV core protein and HBV X antigen; (b) HBV antigens consisting of: HBV large surface antigen (L) and HBV core protein; (c) HBV antigens consisting of: hépatocyte receptor of Pre-Sl of the HBV large surface antigen (L), HBV small surface antigen (S), the reverse transcriptase domain of HBV polymerase, HBV core protein or HBV e-antîgen, and HBV X antigen; (d) HBV antigens consisting of: HBV large surface antigen (L), the reverse transcriptase domain of HBV polymerase, HBV core protein or HBV e-antigen, and HBV X antigen; (e) HBV antigens consisting of: HBV large surface antigen (L), the reverse transcriptase domain of HBV polymerase, and HBV core protein; (f) HBV antigens consisting of: HBV polymerase (RT domain) and HBV core protein; (g) HBV antigens consisting of: HBV X antigen and HBV core protein;
(h) HBV antigens consisting of: hépatocyte receptor of Pre-Sl of the HBV large surface antigen (L), HBV small surface antigen (S), the reverse transcriptase domain of HBV polymerase, and HBV core protein or HBV e-antigen; (i) HBV antigens consisting of HBV large surface antigen (L); (j) HBV antigens consisting of HBV core antigen; (k) HBV antigens consisting of: HBV polymerase including the reverse transcriptase domain; (1) HBV antigens consisting of HBV X antigen; (m) HBV antigens consisting of between two and four HBV surface antigens, HBV polymerase antigens, HBV core antigens, or HBV X antigens, where each of the between two and four HBV antigens is from a different HBV génotype; and (n) HBV antigens consisting of two HBV core antigens and two HBV X antigens, wherein each of the two HBV core antigens and each of the two
HBV X antigens are from a different HBV génotype. Aspects of the invention related to each of the HBV antigens, including a variety of sequences useful in these antigens, hâve been described above.
[0082] In one aspect of this embodiment of the invention, the fusion protein comprises an amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: SEQ ID NO: 130, SEQ ID NO: 150, SEQ ID NO:118, SEQ ID NO:151, SEQ ID NO:34, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID N0:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, and SEQ ID NO:110.
[0083] Another embodiment of the invention relates to a recombinant nucleic acid molécule encoding any of the fusion proteins described herein. In one aspect, the recombinant nucleic acid molécule comprises a nucleic acid sequence selected from, but not limited to: SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:91, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO: 121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, or SEQ ID NO:133.
[0084] Yet another embodiment of the invention relates to an isolated cell transfected with any of the recombinant nucleic acid molécules described herein. In one aspect, the cell is a yeast cell.
[0085] Another embodiment of the invention relates to a composition comprising any of the fusion proteins described herein. Yet another embodiment of the invention relates to a composition comprising any of the recombinant nucleic acid molécules described herein. Another embodiment of the invention relates to a composition comprising any of the isolated cells described herein.
[0086] Yet another embodiment of the invention relates to a method to treat hepatitis B virus (HBV) infection or at least one symptom resulting from HBV infection in a subject, comprising administering to a subject that is infected with HBV at least one of any of the immunotherapeutic compositions, încluding any HBV antigen, fusion protein, or yeast-based immunotherapeutic composition, described herein. The administration of the composition to the subject reduces HBV infection or at least one symptom resulting from HBV infection in a subject.
[0087] Yet another embodiment of the invention relates to a method to elicit an antigen-specifïc, ceil-mediated immune response against an HBV antigen, comprising administering to a subject any one or more of the compositions, încluding any HBV antigen, fusion protein, or yeast-based immunotherapeutic composition, described herein.
[0088] Yet another embodiment of the invention relates to a method to prevent HBV infection in a subject, comprising administering to a subject that has not been infected with HBV, any one or more of the compositions, încluding any HBV antigen, fusion protein, or yeast-based immunotherapeutic composition, described herein.
[0089] Another embodiment of the invention relates to a method to immunize a population of individuals against HBV, comprising administering to the population of indivîduals any one or more of the compositions, încluding any HBV antigen, fusion protein, or yeast-based immunotherapeutic composition, described herein.
[0090] Another embodiment of the invention relates to any one or more of the compositions, încluding any HBV antigen, fusion protein, or yeast-based immunotherapeutic composition, described herein, for use to treat HBV infection or a symptom thereof.
[0091] Another embodiment of the invention relates to any one or more of the compositions, încluding any HBV antigen, fusion protein, or yeast-based immunotherapeutic composition, described herein, for use to prevent HBV infection or a symptom thereof.
[0092] Yet another embodiment of the invention relates to the use of any one or more of the compositions, încluding any HBV antigen, fusion protein, or yeast-based immunotherapeutic composition, described herein in the préparation of a médicament to treat HBV infection or a symptom thereof.
[0093] Yet another embodiment of the invention relates to the use of any one or more of the compositions, încluding any HBV antigen, fusion protein, or yeast-based immunotherapeutic composition, described herein in the préparation of a médicament to prevent HBV infection or a symptom thereof.
[0094] In one aspect of any of the embodiments related to methods or uses of the invention described above or elsewhere herein, the method can include administration of
at least two, three, four or more of the compositions, including any HBV antigen, fusion protein, or yeast-based immunotherapeutic composition, described herein. In one aspect, additional compositions or compounds useful for the prévention or treatment of HBV infection can be administered (e.g., anti-viral compounds, interferons, other 5 immunotherapeutic compositions, or combinations thereof). In one aspect, the various compositions or compounds are administered concurrently to an individual. In one aspect, the various compositions or compounds are administered sequentially to an individual. In one aspect, each of the various compositions is administered by injection to a different site on the individual. In one aspect, a single dose of a yeast-based HBV immunotherapeutic 10 composition of the invention is between 40 Y.U. total and 80 Y.U. total, administered in equal parts at two, three or four different sites on an individual, per dose.
[0095] In one aspect of any of the embodiments related to methods or uses of the invention described above or elsewhere herein, administration of the composition to the subject causes séroconversion in the subject or improves séroconversion rates in a 15 population of subjects. In one aspect, administration of the composition to the subject reduces sérum HBsAg or results in loss of sérum HBsAg in the subject or improves rates of loss of sérum HBsAg in a population of subjects. In one aspect, administration of the composition to the subject reduces sérum HBeAg or results in loss of sérum HBeAg in the subject or improves rates of loss of sérum HBeAg in a population of subjects. In one 20 aspect, administration of the composition to the subject reduces HBV viral load in the subject or improves rates in réduction of HBV viral load in a population of subjects. In one aspect, administration of the composition to the subject results in undetectable HBV DNA in infected cells in the subject or results in higher rates of HBV DNA negativity in a population of subjects. In one aspect, administration of the composition to the subject 25 reduces liver damage or improves liver function in the subject or reduces the rate of liver damage or increases the rate of improved liver function in a population of subjects. In one aspect, administration of the composition to the subject improves ALT normalization in the subject or in a population of subjects.
[0096] In any of the embodiments related to an HBV antigen, fusion protein, immunotherapeutic composition, or any method of use of the HBV antigen, fusion protein or immunotherapeutic composition described herein, in one aspect, the composition further comprises, or is used in conjunction with, at least one biological response modifier.
In one aspect, the composition further comprises, or is used in conjunction with, one or more additional compounds useful for treating or ameliorating a symptom of HBV
infection. In one aspect, the composition further comprises, or is used in conjunction with, at least one anti-viral compound. In one aspect, the anti-viral is a nucléotide analogue reverse transcriptase inhibitor. An anti-viral compound can include, but is not limited to, tenofovir, lamivudine, adefovir, telbivudine, entecavîr, and combinations thereof. In one aspect, the anti-viral compound is tenofovir. In one aspect, the anti-viral compound is entecavir. In one aspect, the composition further comprises, or is used in conjunction with, at least one interferon. In one aspect, the interferon is interferon-α. In one aspect, the interferon is pegylated interferon-a2a. In one aspect, the interferon is interferon-λ.
BRIEF DESCRIPTION OF THE DRAWINGS
[0097] Fig. 1 is a schematic drawing showing the hepatitis B virus genome arrangement.
[0098] Fig. 2 is a schematic drawing showing the basic structure of a recombinant nucleic acid molécule encoding an HBV surface antigen/core fusion protein useful in a yeast-based immunotherapeutic composition of the invention.
[0099] Fig. 3 is a schematic drawing showing the basic structure of a recombinant nucleic acid molécule encoding an HBV surface antigen/polymerase/core/X fusion protein useful in a yeast-based immunotherapeutic composition of the invention.
[00100] Fig. 4 is a schematic drawing showing the basic structure of a recombinant nucleic acid molécule encoding an HBV polymerase/core fusion protein useful in a yeast20 based immunotherapeutic composition of the invention.
[00101] Fig. 5 is a schematic drawing showing the basic structure of a recombinant nucleic acid molécule encoding an HBV X/core fusion protein useful in a yeast-based immunotherapeutic composition of the invention.
[00102] Fig. 6 is a schematic drawing showing the basic structure of a recombinant 25 nucleic acid molécule encoding an HBV polymerase fusion protein useful in a yeast-based immunotherapeutic composition of the invention.
[00103] Fig. 7 is a schematic drawing showing the basic structure of a recombinant nucleic acid molécule encoding an HBV surface antigen/polymerase/core fusion protein useful in a yeast-based immunotherapeutic composition of the invention.
[00104] Fig. 8 is a schematic drawing showing the basic structure of a recombinant nucleic acid molécule encoding an HBV surface antigen/core/polymerase fusion protein useful in a yeast-based immunotherapeutic composition of the invention.
[00105] Fig. 9 is a schematic drawing showing the basic structure of a recombinant nucleic acid molécule encoding an HBV surface antigen/core/X fusion protein useful in a yeast-based immunotherapeutic composition of the invention.
[00106] Fig. 10 is a schematic drawing showing the basic structure of a recombinant nucleic acid molécule encoding an HBV surface antigen/core/polymerase/X fusion protein useful in a yeast-based immunotherapeutic composition of the invention.
[00107] Fig. 11 is a schematic drawing showing the basic structure of a recombinant nucleic acid molécule encoding an HBV surface antigen/core/X/polymerase fusion protein useful in a yeast-based immunotherapeutic composition of the invention.
[00108] Fig. 12 is a schematic drawing showing the basic structure of a recombinant nucleic acid molécule encoding an HBV polymerase/surface antigen/core fusion protein useful in a yeast-based immunotherapeutic composition of the invention.
[00109] Fig. 13 is a schematic drawing showing the basic structure of a recombinant nucleic acid molécule encoding an HBV X/surface antigen/core fusion protein useful in a yeast-based immunotherapeutic composition of the invention.
[00110] Fig. 14 is a schematic drawing showing the basic structure of a recombinant nucleic acid molécule encoding an HBV polymerase/X/surface antigen/core fusion protein useful in a yeast-based immunotherapeutic composition of the invention.
[00111] Fig. 15 is a schematic drawing showing the basic structure of a recombinant nucleic acid molécule encoding an HBV X/polymerase/surface antigen/core fusion protein useful in a yeast-based immunotherapeutic composition of the invention.
[00112] Fig. 16 is a digital image of a Western blot showing expression of several yeast-based immunotherapeutic compositions expressing an HBV Surface antigen/Core fusion protein (heat-killed, whole yeast).
[00113] Fig. 17 is a digital image of a Western blot showing expression of several yeast-based immunotherapeutic compositions expressing an HBV Surface antigen/Core fusion protein (live, whole yeast).
[00114] Fig. 18 is a digital image of a Western blot showing expression of several yeast-based immunotherapeutic compositions expressing an HBV surface antigen/polymerase/core/X fusion protein.
[00115] Fig. 19 is a digital image of a Western blot showing expression of several yeast-based immunotherapeutic compositions expressing an HBV surface antigen/polymerase/core/X fusion protein.
[00116] Fig. 20 is a digital image of a Western blot showing expression of several yeast-based immunotherapeutic compositions expressing HBV antigens comprising surface-core fusion proteins (Sc) or surface-polymerase-core-X fusion proteins (Sp).
[00117] Fig. 21 is a digital image of a Western blot showing expression of HBV antigens from several yeast-based HBV immunotherapeutic compositions cultured in UL2 medium.
[00118] Fig. 22 is a bar graph showing the average expression of HBV antigens from several yeast-based HBV immunotherapeutic compositions cultured in UL2 medium or U2 medium (error bars are Standard Déviation).
[00119] Fig, 23 is a graph showing the prolifération of splenic CD4+ T cells from mice immunized with a yeast-based immunotherapeutic product expressing an HBV SurfaceCore antigen (SCORE) to an S/Core antigen mix or to a MHC Class II SAg mimetope peptide (error bars are Standard Déviation).
[00120] Fig. 24 is a graph showing the prolifération of lymph node T cells from mice 15 immunized with a yeast-based immunotherapeutic product expressing an HBV SurfaceCore antigen (SCORE) to an S/Core antigen mix or to a MHC Class II SAg mimetope peptide (error bars are Standard Déviation).
[00121] Fig. 25 is a graph showing the interferon-γ (IFN-γ) ELISpot response of lymph node T cells from mice immunized with a yeast-based immunotherapeutic product 20 expressing an HBV Surface-Core antigen (SCORE) to an S/Core antigen mix or to a MHC Class II SAg mimetope peptide.
[00122] Fig. 26 is a graph showing the prolifération of splenic CD4+ T cells from mice immunized with a yeast-based immunotherapeutic product expressing an HBV SurfacePol-Core-X antigen (denoted a-Spex) to an S/Core antigen mix or to a MHC Class II SAg 25 mimetope peptide (error bars are Standard Déviation).
[00123] Fig. 27 is a graph showing IL-Ιβ production in splénocytes from mice immunized with: (a) a yeast-based immunotherapeutic product expressing an HBV Surface-Pol-E/Core-X antigen (denoted Sp), left columns; or (b) a yeast-based immunotherapeutic product expressing an HBV Surface-Core antigen (denoted Sc) (error 30 bars are Standard Déviation).
[00124] Fig. 28 is a graph showing IL-12p70 production in splénocytes from mice immunized with: (a) a yeast-based immunotherapeutic product expressing an HBV Surface-Pol-Core-X antigen (denoted Sp), left columns; or (b) a yeast-based immunotherapeutic product expressing an HBV Surface-Core antigen (denoted Sc) (error bars are Standard Déviation).
[00125] Figs. 29A and 29B are graphs showing interferon-γ (IFN-γ) production in splénocytes from mice immunized with: (Fig. 29A) a yeast-based immunotherapeutic product expressing an HBV Surface-Core antigen (denoted Sc) or (Fig. 29B) a yeast-based immunotherapeutic product expressing an HBV Surface-Pol-Core-X antigen (denoted Sp) (error bars are Standard Déviation).
[00126] Figs. 30A-D are graphs showing IL-1 β (Fig. 30A), IL-6 (Fig. 30B), IL-13 (Fig. 30C), and IL-12p70 (Fig. 30D) production in splénocytes from mice immunized with: (a) a yeast-based immunotherapeutic product expressing an HBV Surface-Pol-Core-X antigen (denoted Sp), left columns; or (b) a yeast-based immunotherapeutic product expressing an HBV Surface-Core antigen (denoted Sc).
[00127] Fig. 31 is a bar graph showing that mice immunized with GI-13002 or GI13002 + anti-CD40 antibody, but not YVEC, elicited comparable protection from challenge with EL4 tumors expressing the target HBV antigen (error bars are Standard Déviation).
[00128] Fig. 32 is a bar graph showing the results of an IFN-γ ELISpot assay comparing T cell responses of mice immunized with GI-13008 (SCORE-C) and GI-13013 (SPEXv2) as compared to YVEC using a variety of HBV peptides and antigens (error bars are Standard Déviation).
[00129] Fig. 33 is a bar graph showing IFN-γ ELISpot responses to stimulation with GI-13002 from a human subject pre- and post-immunization, and post-boost, with a prophylactic HBV vaccine.
[00130] Fig. 34 is a bar graph showing HBV antigen-specific IFN-γ ELISpot responses from lymph node cells isolated from HLA-A2 transgenic mice immunized with GI-13009 (SCORE-D) or GI-13020 (X-SCORE) as compared to mice immunized with a yeast control (YVEC) (error bars are Standard Déviation).
[00131] Fig. 35 is a bar graph showing HBV antigen-specific IFN-γ ELISpot responses from spleen cells isolated from HLA-A2 transgenic mice immunized with GI-13009 (SCORE-D) as compared to mice immunized with a yeast control (YVEC) (error bars are Standard Error).
[00132] Fig. 36 is a bar graph showing HBV antigen-specific IFN-γ ELISpot responses from lymph node cells isolated from C57BL/6 mice immunized with GI-13009 (SCORE16530
D) or GI-13020 (X-SCORE) as compared to mice immunized with a yeast control (YVEC) or Naïve mice (error bars are Standard Déviation).
[00133] Fig. 37 is a line graph showing HBV antigen-specific CD8+ T cell responses to an MHC Class I-restricted HBV peptide in C57BL/6 mice immunized with GI-13009 (SCORE-D) or GI-13020 (X-SCORE) as compared to mice immunized with a yeast control (YVEC) or a yeast-based immunotherapeutic expressing ovalbumin (OVAX).
[00134] Fig. 38 is a bar graph showing HBV antigen-specific CD4+ T cell responses to an MHC Class II-restricted HBV peptide in C57BL/6 mice immunized with GI-13009 (SCORE-D) or GI-13020 (X-SCORE) as compared to mice immunized with a yeast control (YVEC) or a yeast-based immunotherapeutic expressing ovalbumin (OVAX).
DETAILED DESCRIPTION OFTHE INVENTION
[00135] This invention generally relates to compositions and methods for preventing and/or treating hepatitis B virus (HBV) infection. The invention includes a yeast-based immunotherapeutic composition (also referred to as “yeast-based HBV immunotherapy”) comprising a yeast vehicle and HBV antigen(s) that hâve been designed to elicit a prophylactic and/or therapeutic immune response against HBV infection in an individual, and the use of such compositions to prevent and/or treat HBV infection and related symptoms thereof. The invention also includes the recombinant nucleic acid molécules used in the yeast-based compositions of the invention, as well as the proteins and fusion proteins encoded thereby, for use in any immunotherapeutic composition and/or any therapeutic or prophylactic protocol for HBV infection, including any therapeutic or prophylactic protocol that combines the HBV-specific yeast-based compositions of the invention with any one or more other therapeutic or prophylactic compositions, agents, drugs, compounds, and/or protocole for HBV infection.
[00136] The yeast-based, HBV-specific immunotherapeutic compositions are unique among various types of immunotherapy, in that these compositions of the invention induce innate immune responses, as well as adaptive immune responses that specifically target HBV, including CD4-dependent TH17 and TH1 T cell responses and antigen-specific CD8+ T cell responses. The breadth of the immune response elicited by HBV-specific yeast-based immunotherapy is well-suited to target HBV. First, HBV is believed to évadé the innate immune response early in infection by “hiding” from the innate response and thereby not inducing it, rather than by directly counteracting innate immunity (Wieland and Chisari, 2005, J. Virol. 15:9369-9380; Wieland, et al., 2004, PNAS USA 101:66696674). Accordingly, it can be expected that HBV will be sensitive to innate immune responses if they are activated by another mechanism, i.e., the yeast-based immunotherapeutic compositions of the invention. Second, HBV produces high-level antigen expression in infected host cells that is expected to be visible to the adaptive immune response (Guidotti, et al., 1999, Science 284:825-829; Thimme et al., 2003, J. Virol. 77:68-76), and clearance of acute infection has been associated with robust CD4+ and CD8+ T cell responses (Maini et al., 1999, Gastroenterol. 117:1386-1396; Rehermann et al., 1995, J. Exp. Med. 181:1047-1058; Thimme et al., 2003, J. Virol. 77:68-76; Wieland and Chisari, 2005, J. Virol. 15:9369-9380). Therefore, yeast-based HBV immunotherapy, by activating the adaptive immune response, is expected to effectively target HBV-infected cells for destruction and/or is expected to effectively enhance viral clearance. Moreover, the immune response generated by yeast-based immunotherapy is believed to be interferon-independent and interferon-dependent (Tamburini et al., 2012, J. Immunother. 35(1): 14-22); accordingly, the ability, or lack thereof, of an individual to respond to interferon-based therapy, which is one standard of care treatment for HBV, is not believed to directly impact the ability of the subject to respond to yeast-based immunotherapy of the invention. In addition, the yeast-based HBV immunotherapy compositions described herein are designed to target immunogenic and conserved régions of HBV, multiple CTL epitopes, and include régions of HBV that may be targeted for escape (allowing for modifications of the compositions as needed to target such escape mutations), making it a highly adaptable therapy for HBV that optimizes the opportunity for effective immune responses against this virus.
[00137] In addition, and without being bound by theory, yeast-based immunotherapy for HBV is believed to induce an immune response that is not only directed specifically against the target antigen carried by the yeast-based immunotherapeutic product, but that also evolves to be directed against other immunological epitopes on the virus (i.e., other than those carried by the yeast-antigen composition). In other words, a primary cellular immune response to the antigen(s) and/or epitope(s) contained in the yeast-based immunotherapeutic can lead to secondary cellular immune responses to antigen(s) and/or epitope(s) that are present in the infected cells in the treated subject but that are not present in the yeast-based immunotherapeutic, thereby leading to the évolution of complex and unpredictable immune response profiles that are unique to each treated subject. These secondary immune responses are spécifie to the molecular profile of the HBV infection in each subject treated, and the yeast-based immunotherapeutic may drive these downstream effects in a unique manner when compared to other treatment modalities, including other
îmmunotherapy platforms. This phenomenon may also be generally referred to as “epitope spreading” and represents an advantage of using yeast-based HBV îmmunotherapy, because induction of an immune response against a particular HBV antigen or even against a particular HBV génotype (e.g., by providing that antigen in the 5 context of the yeast îmmunotherapeutic), is expected to resuit in the cascading targeting of the immune system against a variety of additional HBV antigens, which may resuit in effective immune responses against antigens from different HBV génotypes or strains than those represented in the yeast-based îmmunotherapeutic composition.
[00138] As discussed above, patients who become chronically infected with HBV tend 10 to hâve weaker (or absent) and more narrow HBV-specific, T cell-mediated immunity.
Accordingly, the yeast-based HBV îmmunotherapy compositions of the invention address the need for therapeutic compositions to treat patients who are actively infected with HBV, including chronically infected patients, and further provide an additional vaccine for the prévention of HBV infection that may hâve advantages with respect to the production of 15 durable memory immune responses. Indeed, the yeast-based HBV îmmunotherapy compositions of the invention are expected to promote durable memory T cell responses against HBV, which can prevent infection, as well as provide long term benefits that can protect a chronically infected patient from viral réactivation. Yeast-based HBV îmmunotherapy compositions as monotherapy or in combination with other therapeutic 20 approaches for the treatment of HBV (e.g., in combination with anti-viral compounds) are expected to increase the percentage of chronically infected patients who achieve clearance of HBsAg and HBeAg, who achieve complété séroconversion, and/or who achieve sustained viral clearance for at least 6 months after the completion of therapy.
[00139] Accordingly, yeast-based HBV îmmunotherapy can be combined with anti25 viral drugs and/or interferon therapy, and/or with other thérapies for HBV, in order to reduce the viral load in an individual to a level that can be more effectively handled by the immune system. HBV viral titers are typically very high (as many as 1011 hépatocytes may be infected) and thus may overwhelm an individual’s ability to mount an effective CTL response; accordingly, réduction of viral load using anti-viral drugs in combination 30 with induction of HBV-specific CTL activity using yeast-based îmmunotherapy is expected to be bénéficiai to the infected individual. In addition, réduction of viral load through the use of anti-viral drugs may also reduce négative effects, if any, of immune activation in the context of a high number of infected hépatocytes being targeted for destruction. Yeast-based HBV îmmunotherapy is also expected to play a rôle in reducing and/or eliminating compartments of latent viral infection. For example, there are many tissues that have been shown to be HBV-positive by PCR, and that are considered potential sanctuaries for re-activation of HBV. HBV DNA can intégrale into the host genome, which provides for a quiescent persistence of HBV, and cccDNA is a supercoiled, dormant form of the HBV genome that also contributes to quiescence. Without being bound by theory, the inventors believe that yeast-based HBV immunotherapy described herein will play a rôle in eliminating ail of these types of HBV “sanctuaries” that likely contribute to the low disease-free cure rate observed with the current anti-viral approaches. [00140] In another scénario, use of a yeast-based HBV immunotherapeutic of the invention, alone or in combination with an anti-viral or other HBV therapeutic, if sufficient to achieve complété clearance of HBsAg, but not sufficient to achieve anti-HB production, may be followed by, or further combined with, existing prophylactic subunit vaccines to achieve complété séroconversion. Alternatively, any of the fusion proteins described herein may also be used as subunit vaccines to achieve complété séroconversion, or to protect a subject from HBV infection, alone or in combination with a yeast-based HBV immunotherapeutic of the invention. Finally, the immunotherapeutic composition of the invention is weli-suited for modification and/or combination with additional immunotherapeutic compositions, including any described herein, to treat escape mutations of HBV that are elicited by treatment with anti-viral drugs.
[00141] Yeast-based immunotherapeutic compositions are administered as biologics or pharmaceutically acceptable compositions. Accordingly, rather than using yeast as an antigen production system followed b y purification of the antigen from the yeast, the entire yeast vehicle as described herein must be suitable for, and formulated for, administration to a patient. In contrast, existing commercial HBV vaccines as well as many in development, comprise recombinant HBV proteins (e.g., HBsAg proteins) that are produced in Saccharomyces cerevisiae, but are subsequently released from the yeast by disruption and purified from the yeast so that the final vaccine, combined with an adjuvant (e.g., aluminum hydroxyphosphate sulfate or aluminum hydroxide), eontains no détectable yeast DNA and eontains no more than 1-5% yeast protein. The HBV yeastbased immunotherapeutic compositions of the invention, on the other hand, contain readily détectable yeast DNA and contain substantially more than 5% yeast protein; generally, yeast-based immunotherapeutics of the invention contain more than 70%, more than 80%, or generally more than 90% yeast protein.
[00142] Yeast-based immunotherapeutic compositions are administered to a patient in order to immunize the patient for therapeutic and/or prophylactic purposes. In one embodiment of the invention, the yeast-based compositions are formulated for administration in a pharmaceutically acceptable excipient or formulation. The composition should be formulated, in one aspect, to be suitable for administration to a human subject (e.g., the manufacturing conditions should be suitable for use in humans, and any excipients or formulations used to finish the composition and/or préparé the dose of the immunotherapeutic for administration should be suitable for use in humans). In one aspect of the invention, yeast-based immunotherapeutic compositions are formulated for 10 administration by injection of the patient or subject, such as by a parentéral route (e.g., by subcutaneous, intraperitoneal, intramuscular or intradermal injection, or another suitable parentéral route).
[00143] In one embodiment, the yeast express the antigen (e.g., détectable by a Western blot), and the antigen is not aggregated in the yeast, the antigen does not form 15 inclusion bodies in the yeast, and/or does not form very large particles (VLPs) or other large antigen particles in the yeast. In one embodiment, the antigen is produced as a soluble protein in the yeast, and/or is not secreted from the yeast or is not substantially or primarily secreted from the yeast. In another embodiment, without being bound by theory, the présent inventors believe that particular combinations and perhaps, arrangements, of 20 antigens in an HBV fusion protein including surface antigen and core antigen, described in detail herein, may form VLPs or aggregate to some extent within the yeast expressing the antigens. As a resuit, the antigen expressed by the yeast has immunogenic properties which appear to be related to its overall structure and form, as a separate characteristic from the immunogenic properties of the immune epitopes (e.g., T cell epitopes) carried 25 within the antigen. When the yeast expressing such fusion proteins are provided in a yeast-based HBV immunotherapeutic of the invention, the immunotherapeutic composition dérivés properties that activate the innate immune system not only from the yeast vehicle as discussed above (as with ail yeast-based immunotherapeutics described herein), but also in part from the fusion protein antigen structure (e.g., the surface-core 30 fusion protein as expressed in the yeast also has adjuvant-like properties); in addition, the immunotherapeutic composition dérivés properties that activate the adaptive immune system in an antigen-specific manner from the fusion protein (via provision of various T cell epitopes), as with ail of the yeast-based immunotherapeutics described herein. This spécifie combination of properties appears to be unique to yeast-based
immunotherapeutics expressing particular surface-core fusion proteins from HBV described herein. However, in ail of the embodiments of the invention described herein, the yeast-based immunotherapeutics should be readily phagocytosed by dendritic cells of the immune System, and the yeast and antigens readily processed by such dendritic cells, 5 in order to elicit an effective immune response against HBV.
Compositions of the Invention
[00144] One embodiment of the présent invention relates to a yeast-based immunotherapy composition which can be used to prevent and/or treat HBV infection and/or to alleviate at least one symptom resulting from the HBV infection. The 10 composition comprises: (a) a yeast vehicle; and (b) one or more antigens comprising HBV protein(s) and/or immunogenic domain(s) thereof. In conjunction with the yeast vehicle, the HBV proteins are most typically expressed as recombinant proteins by the yeast vehicle (e.g., by an intact yeast or yeast spheroplast, which can optionally be further processed to a yeast cytoplast, yeast ghost, or yeast membrane extract or fraction thereof), 15 although it is an embodiment of the invention that one or more such HBV proteins are loaded into a yeast vehicle or otherwise complexed with, attached to, mixed with or administered with a yeast vehicle as described herein to form a composition of the présent invention. According to the présent invention, reference to a heterologous protein or heterologous antigen, încluding a heterologous fusion protein, in connection with a yeast 20 vehicle of the invention, means that the protein or antigen is not a protein or antigen that is naturally expressed by the yeast, although a fusion protein that includes heterologous antigen or heterologous protein may also include yeast sequences or proteins or portions thereof that are also naturally expressed by yeast (e.g., an alpha factor prepro sequence as described herein).
[00145] One embodiment of the invention relates to various HBV fusion proteins. In one aspect, such HBV fusion proteins are useful in a yeast-based immunotherapeutic composition of the invention. Such fusion proteins, and/or the recombinant nucleic acid molécules encoding such proteins, can also be used in, in combination with, or to produce, a non-yeast-based immunotherapeutic composition, which may include, without limitation, a DNA vaccine, a protein subunit vaccine, a recombinant viral-based immunotherapeutic composition, a killed or inactivated pathogen vaccine, and/or a dendritic cell vaccine. In another embodiment, such fusion proteins can be used in a diagnostic assay for HBV and/or to generate antibodies against HBV. Described herein are exemplary HBV fusion proteins providing selected portions of HBV antigens, încluding, for example, selected
portions of and/or modified polymerase; selected portions of and/or modified surface antigen; selected portions of and/or modified core (including at least portions of or most of e-antigen); selected portions of and/or modified X antigen; as well as selected portions of and/or arrangements of any one, two, three or ail four of the antigens (surface antigen, core, X and polymerase), such as, but not limited to, selected portions and/or arrangements of surface antigen and core (including at least portions of or most of e-antigen); selected portions and/or arrangements of surface antigen, core (including at least portions of or most of e-antigen), polymerase and X antigen; selected portions and/or arrangements of surface antigen, core (including at least portions of or most of e-antigen), and polymerase;
and selected portions and/or arrangements of surface antigen, core (including at least portions of or most of e-antigen), and X antigen.
[00146] In one embodiment, HBV antigens, including immunogenic domains of fulllength proteins, as described herein, are fused to host proteins that are overexpressed in HBV infected, but not in non-infected, host cells. In one embodiment, HBV antigens, 15 including immunogenic domains of fuil-length proteins, as described herein, are fused to protein R2, a host factor required for HBV réplication, which in one embodiment, is expressed in hépatocytes. R2 is a protein component of ribonucleotide reductase (RNR), and is critical for the HBV life-cycle (see, e.g., Cohen et al., 2010, Hepatol. 51(5):15381546). Other embodiments of the invention will be apparent in view of the disclosure 20 provided herein.
[00147] Hepatitis B Virus, Genes, and Proteins. Hepatitis B virus (HBV) is a member of the Hepadnaviridae (hepadnavirus) family of viruses and causes transient and chronic infections of the liver in humans and the great apes. The hepadnaviruses that infect mammals hâve similar DNA sequences and genome organization, and are grouped in the 25 genus Orthohepadnavirus. The hepatitis B virus particle has an outer envelope containing lipid and surface antigen particles known as HBsAg. A nucleocapsid core containing core protein (HBcAg) surrounds the viral DNA and a DNA polymerase with reverse transcriptase activity. As reviewed in Seeger and Mason, 2000, Microbiol. Mol. Biol. Rev. 64(1):51-68, HBV has a 3.2 kb partially double-stranded relaxed-circular DNA (rcDNA) 30 genome that is converted into a covalently closed circular double-stranded DNA (cccDNA) molécule upon delivery of the viral genome to the nucléus of an infected hépatocyte. The host cell RNA polymerase II transcribes four viral RNAs from the cccDNA template which are transported to the host cell cytoplasm. The viral RNAs include mRNAs that are transcribed to produce the viral core and envelope structural
proteins and the precore, polymerase and X nonstructural viral proteins. The RNA that is translated to produce core and polymerase also serves as the pregenomic RNA (pgRNA) which is the template for reverse transcription. pgRNA and the polymerase are encapsulated by the core protein, producing the viral nucleocapsid where the pgRNA is 5 reverse transcribed into rcDNA. These rcDNA-containing nucleocapsids are then enclosed by envelope proteins and secreted from the host cell as mature virions or shuttled to the nucléus to amplify the viral cccDNA.
[00148] The structural and non-structural proteins produced by the HBV genome are shown in Table 1. The partially double-stranded HBV genome contains four genes known 10 as C, X, Pt and 5 (see also Fig. 1).
Table 1. HBV genes and gene products
Gene Protein Function(s)
C core protein (HBcAg) Forms viral capsid surrounding viral pgRNA and polymerase
e antigen (HBeAg) Function unknown; may be HBV-specific immune suppressive factor for adaptive immune response
P polymerase Polymerase for viral DNA réplication Domain 1: terminal protein (TP) domain packages pgRNA and primes minus strand DNA Domain 2: reverse transcriptase (RT) domain, RNase H; dégradés pgRNA
S S HBsAg (surface antigen; small) Envelope protein and forms surface antigen particles; may suppress Immune function
M HBsAg (surface antigen; middle = Pre-S2 + S) Envelope protein and forms surface antigen particles together with S; may suppress immune function
L HBsAg (surface antigen; large = Pre-S1 + pre-S2 + S) Envelope protein and forms surface antigen particles together with S; pre-SI domain provides ligand for core particles during assembly of viral envelope; hépatocyte receptor; may suppress immune function
X X antigen (HBx) Transcriptional transactivation; régulation of DNA repair pathways; élévation of cytosolic calcium levels; modulation of protein dégradation pathways; modulation of cell cycle progression and cell prolifération pathways In host cell; stimulation of HBV réplication
[00149] Gene C encodes two closely related antigens: a 21-kDa protein called “core 15 protein” or “core antigen” (HBcAg) which forms the virai capsid, and a 17-kDa protein called e-antigen (HBeAg) that forms dimers but that does not assemble into capsid. Fulllength core protein is an approximately 183 amino acid protein, comprising ail but the Nterminal 10 amino acids of e-antigen and comprising approximately 34 additional amino acids at the C-terminus that are proteolytically cleaved in the production of e-antigen. In 20 other words, core protein and e-antigen hâve 149 amino acid residues in common (this section sometimes being referred to as the hepatitis core antigen), but differ at the N16530
terminal and C-terminal régions. Precore protein is a precursor protein comprising an amino acid sequence that includes sequence from both core and e-antigen, from which eantigen is produced via proteolytic processing. Intracellular HBeAg includes precore residues -29 to -1 (the residue numbering in this particular description is provided with the 5 first amino acid residue of core protein within the precore protein being denoted as position 1”), which contains a signal sequence that directs the protein to the endoplasmic réticulum at which point amino acids -29 to -11 are cleaved; another proteolytic cleavage between amino acids 149 and 150 removes the C-terminai portion of precore (which is présent in full-length core protein), and the remaining HBeAg (consisting of amino acids 10 10 to -1 of precore plus amino acids 1-149 of HBeAg or core) is then secreted as e-antigen (Standing et al., 1988, PNAS USA 85: 8405-8409; Ou et al., 1986, PNAS USA 83:15781582; Bruss and Gerlich, 1988, Virology 163:268-275; Takahashi et al., 1983, J. lmmunol. 130:2903-2907). HBeAg consisting of the entire precore région has also been found in human sera (Takahashi et al., 1992, J. lmmunol. 147:3156-3160). As mentioned, HBeAg 15 (core) forms dimers that assemble into the viral capsid and contain the polymerase and viral DNA or pgRNA. The fonction of HBeAg (e-antigen) is unknown, but it is not required for HBV réplication or infection, and it is thought to be an immune suppressive factor that protects HBV against attack by the immune systern (Milich et al., 1990, PNAS USA 87:6599-6603; Che et al., 2004, PNAS USA 101:14913-14918; Wieland and Chisari, 20 2005, J. Virol. 79:9369-9380). For clarity, in the HBV sequences described herein (e.g., see Table 3), the sequence for precore from représentative HBV génotypes is provided, and the positions of core protein and e-antigen are denoted within the precore sequence, with the first amino acid of precore designated as position 1.
[00150] Gene P encodes the HBV DNA polymerase (Pol), which consists of two major 25 domains linked by a spacer. The N-terminal domain of the polymerase (also referred to as “terminal protein” or TP) is involved in the packaging of pgRNA and in the priming of non-sense strand DNA. The C-terminal domain is a reverse transcriptase (RT) that has RNase H (RH) activity.
[00151] Gene S has multiple start codons and encodes three envelope proteins (also referred to herein generally as “surface protein” or “surface antigen”) denoted S, M and L, which are ail components of the infectious viral particles, also known as Dane particles. S, by itself, and together with M and L, also form surface antigen particles (HBsAg) which can be secreted from infected cells in large quantities (Seeger and Mason, 2000, Microbiol.
Mol. Biol. Rev. 64(1):51-68; Beck, (2007), Hepatitis B virus réplication, World Journal
of Gastroenterology : WJG 13(1):48-64). The codons for M and L are located approximately 165 (M) and 489 (L) nucléotides, respectively, upstream of the initiation codon for S. S or “small” surface antigen is the smallest and most abundant of the surface antigens. Antibodies produced against this antigen represent séroconversion in infected 5 individuals. M or “mîddle” surface antigen has an extra protein domain, as compared to S, known as pre-S2, and the protein domain that is unique to L or “large” surface antigen is known as pre-Sl (L therefore also contains pre-S2 and the additional sequence belonging to M and S). Pre-Sl contains the viral hépatocyte receptor domain (hépatocyte receptor binding site), which is located approximately between amino acid positions 21 and 47 of 10 Pre-Sl. Epitopes in pre-Sl can elicit virus-neutralizing antibodies. In addition, the pre-Sl domain provides the ligand for core particles during the assembly of the viral envelope. Surface antigen particles (HBsAg) may also suppress immune élimination of infected cells by functioning as a high-dose toleragen (Reignat et al., 2002, J. Exp. Med. 195:1089-1101; Webster et al., 2004, J. Virol. 78:5707-5719).
[00152] Gene X encodes X antigen (HBx) (which may also be referred to as “X protein”) which is involved in transcriptional transactivation, régulation of DNA repair pathways, élévation of cytosolic calcium levels, modulation of protein dégradation pathways, and modulation of cell cycle progression and cell prolifération pathways in the host cell (Gearhart et al., 2010, J. Virol.), which enhances stimulation of HBV réplication.
HBx is also associated with the development of liver cancer (Kim et al., Nature 1991, 351:317-320; Terradillos et al., Oncogene 1997,14:395-404).
[00153] HBV is found as one of four major serotypes (adr, adw, ayr, ayw) that are determined based on antigenic epitopes within its envelope proteins. There are eight different HBV génotypes (A-H) based on the nucléotide sequence variations in the 25 genome. The geographical distribution of the génotypes is shown in Table 2 (Kramvis et al., 2005, Vaccine 23(19):2409-2423; Magnius and Norder, 1995, Intervirology 38(12):24-34; Sakamoto et al., 2006, J. Gen. Virol. 87:1873-1882; Lim et al., 2006, Int. J. Med. Sci. 3:14-20).
Table 2
HBV génotype Prévalent Geographical Distribution
HBV/A Americas, Europe, Africa, Southeast Asia
HBV/B Asia (China, Japan, Southeast Asia), United States
HBV/C Asia (China, Japan, Southeast Asia), United States
HBV/D United States, Mediterranean, Mlddle East and India
HBV/E Sub-Saharan and West Africa
HBV/F Central and South America
HBV/G France, Germany, United States
H B V/H Central America, United States (California)
[00154] The nucleic acid and amino acid sequence for HBV genes and the proteins encoded thereby are known in the art for each of the known génotypes. Table 3 provides 5 reference to sequence identifiers for exemplary (représentative) amino acid sequences of ail of the HBV structural and non-structural proteins in each of the eight known génotypes of HBV, and further indicates the position of certain structural domains. It is noted that small variations may occur in the amino acid sequence between different viral isolâtes of the same protein or domain from the same HBV génotype. However, as discussed above, 10 strains and serotypes of HBV and génotypes of HBV display high amino acid identity even between serotypes and génotypes (e.g., see Table 4). Therefore, using the guidance provided herein and the reference to the exemplary HBV sequences, one of skill in the art will readily be able to produce a variety of HBV-based proteins, including fusion proteins, from any HBV strain (isolate), serotype, or génotype, for use in the compositions and 15 methods of the présent invention, and as such, the invention is not limited to the spécifie sequences disclosed herein. Reference to an HBV protein or HBV antigen anywhere in this disclosure, or to any functional, structural, or immunogenic domain thereof, can accordingly be made by reference to a particular sequence from one or more of the sequences presented in this disclosure, or by reference to the same, similar or 20 corresponding sequence from a different HBV isolate (strain).
Table 3
Organlsm, Génotype, Gene Protein Sequence Identifier (Database Accession No.)
HBV, Génotype A, C Precore SEQ ID NO:1 (Accession No. AAX83988.1)
• Core (HBcAg) ♦Positions 30/31-212 of SEQ ID NO:1
• e-antigen (HBeAg) ♦Positions 20-178 of SEQ ID NO:1
HBV, Génotype A, P Polymerase SEQ ID NO:2 (Accession No. BAI81985)
• reverse transcriptase ♦Positions 383-602 of SEQ ID NO:2
HBV, Génotype A, S Surface HBsAg (L) SEQ ID NO:3 (Accession No. BAD91280.1)
Surface HBsAg (M) ♦Positions 120-400 of SEQ ID NO:3
Surface HBsAg (S) ♦Positions 175-400 of SEQ ID NO:3
HBV, Génotype A, X X (HBx) SEQ ID NO:4 (Accession No. ΑΑΚ971Θ9.1)
HBV, Génotype B, C Precore SEQ ID NO:5 (Accession No. BAD90067)
• Core (HBcAg) ♦Positions 30/31-212 of SEQ ID NO:5
• e-antigen (HBeAg) ♦Positions 20-178 of SEQ ID NO:5
HBV, Génotype B, P Polymerase SEQ ID NO:6 (Accession No. BAD90068.1)
• reverse transcriptase ♦Positions 381-600 of SEQ ID NO:6
HBV, Génotype B, S Surface HBsAg (L) SEQ ID NO:7 (Accession No. BAJ06634.1)
Surface HBsAg (M) ♦Positions 120-400 of SEQ ID NO:7
Surface HBsAg (S) ♦Positions 175-400 of SEQ ID NO:7
HBV, Génotype B, X X (HBx) SEQ ID NO:8 (Accession No. BAD90066.1)
HBV, Génotype C, C Precore SEQ ID NO:9 (Accession No. YP 355335)
• Core (HBcAg) ♦Positions 30/31-212 of SEQ ID NO:9
• e-antigen (HBeAg) ♦Positions 20-178 of SEQ ID NO:9
HBV, Génotype C, P Polymerase SEQIDNQ:10 (Accession No. ACH57822)
• reverse transcriptase ♦Positions 381-600 of SEQ ID NO:10
HBV, Génotype C, S Surface HBsAg (L) SEQIDNO:11 (Accession No. BAJ06646.1)
Surface HBsAg (M) ♦Positions 120-400 ofSEQ ID NO:11
Surface HBsAg (S) ♦Positions 175-400 of SEQ ID NO:11
HBV, Génotype C,X X (HBx) SEQIDNO:12 (Accession No. BAJ06639.1)
:·· ·;γ Vv' · ν;·τ ·.·,·· ng- -··'
HBV, Génotype D, C Precore SEQ IDNO:13 (Accession No. ADF29260.1)
• Core (HBcAg) ♦Positions 30/31-212 of SEQ ID NO:13
Organlsm, Génotype, Gene Protein Sequence Identifier (Database Accession No.)
• e-antigen (HBeAg) *Positions 20-178 of SEQ ID NO:13
HBV, Génotype D, P Polymerase SEQ ID NO:14 (Accession No. ADD12642.1)
• reverse transcriptase ♦Positions 370-589 of SEQ ID NO:14
HBV, Génotype D, S Surface HBsAg (L) SEQIDNO:15 (Accession No. ACP20363.1)
Surface HBsAg (M) ♦Positions 109-389 of SEQ ID NO: 15
Surface HBsAg (S) ♦Positions 164-389 of SEQ ID NO:15
HBV, Génotype D,X X (HBx) SEQ ID NO:16 (Accession No. BAF47226.1)
HBV, Génotype E, C Precore SEQ IDNO:17 (Accession No. ACU25047.1)
• Core (HBeAg) ♦Positions 30/31-212 of SEQ ID NO:17
• e-antigen (HBeAg) ♦Positions 20-178 of SEQ ID NO:17
HBV, Génotype E, P Polymerase SEQ ID NO118 (Accession No. ACO89764.1)
• reverse transcriptase ♦Positions 380-599 of SEQ ID NO:18
HBV, Génotype E, S Surface HBsAg (L) SEQIDNO:19 (Accession No. BAD91274.1)
Surface HBsAg (M) ♦Positions 119-399 of SEQ ID NO:19
Surface HBsAg (S) ♦Positions 174-399 of SEQ ID NO:19
HBV, Génotype E, X X (HBx) SEQ ID NO:20 (Accession No. ACU24870.1)
HBV, Génotype F, C Precore SEQ ID NO:21 (Accession No. BAB17946.1)
• Core (HBeAg) ♦Positions 30/31-212 of SEQ ID NO:21
• e-antigen (HBeAg) ♦Positions 20-178 of SEQ ID NO:21
HBV, Génotype F, P Polymerase SEQ ID NO:22 (Accession No. ACD03788.2)
• reverse transcriptase ♦Positions 381-600 of SEQ ID NO:22
HBV, Génotype F, S Surface HBsAg (L) SEQ ID NO;23 (Accession No. BAD98933.1)
Surface HBsAg (M) ♦Positions 120-400 of SEQ ID NO:23
Surface HBsAg (S) ♦Positions 175-400 of SEQ ID NO:23
HBV, Génotype F, X X (HBx) SEQ ID NO:24 (Accession No. AAM09054.1)
· 1 ' * · . ’ frt
HBV, Génotype G, C Precore SEQ ID NQ:25 (Accession No. ADD62622.1)
• Core (HBeAg) ♦Positions 14-194 of SEQ ID NO:25
• e-antigen (HBeAg) ♦Positions 4-161 of SEQ ID NO:25
HBV, Génotype G, P Polymerase SEQ ID NO:26 (Accession No. ADD62619.1)
Organism, Génotype, Gene Protein Sequence Identifier (Database Accession No.)
• reverse transcriptase ♦Positions 380-599 of SEQ ID NO:26
HBV, Génotype G, S Surface (HBsAg) (L) SEQ ID NO:27 (Accession No. ADD62620.1)
Surface HBsAg (M) ♦Positions 119-399 ofSEQ ID NO:27
Surface HBsAg (S) ♦Positions 174-399 of SEQ ID NO:27
HBV, Génotype G, X X (HBx) SEQ ID NO:28 (Accession No. BAB82400.1)
HBV, Génotype H, C Precore SEQ ID NO:29 (Accession No. BAD91265.1)
• Core (HBcAg) ♦Positions 30/31-212 of SEQ ID NO:29
• e-antigen (HBeAg) ♦Positions 20-178 of SËQ ID NO:29
HBV, Génotype H, P Polymerase SEQ ID NO:30 (Accession No. BAF49208.1)
* reverse transcriptase ♦Positions 381-600 of SEQ ID NO:30
HBV, Génotype H, S Surface HBsAg (L) SEQ ID NO:31 (Accession No. BAE20065.1)
Surface HBsAg (M) ♦Positions 120-400 of SEQ ID NO:31
Surface HBsAg (S) ♦Positions 175-400 of SEQ ID NO:31
HBV, Génotype H, X X (HBx) SEQ ID NO:32 (Accession No. BAF49206.1)
♦Position numbering is approxlmate and may Include additional amino acids flanking either side of the Indicated position
[00155] Hepatitis B Virus Antigens and Constructs. One embodiment of the invention relates to novel HBV antigens and fusion proteins and recombinant nucleic acid molécules encoding these antigens and proteins. Described herein are several different novel HBV antigens for use in a yeast-based immunotherapeutic composition or other composition (e.g., other immunotherapeutic or diagnostic composition) that provide one or multiple (two, three, four, five, six, seven, eight, nine or more) antigens and/or 10 immunogenic domains from one or more proteins, ail contained within the same fusion protein and encoded by the same recombinant nucleic acid construct (recombinant nucleic acid molécule). The antigens used in the compositions of the invention include at least one HBV protein or immunogenic domain thereof for immunizing an animal (prophylactically or therapeutically). The composition can include one, two, three, four, a 15 few, several or a plurality of HBV antigens, including one, two, three, four, five, six, seven, eight, nine, ten, or more immunogenic domains of one, two, three, four or more HBV proteins. In some embodiments, the antigen is a fusion protein. In one aspect of the
invention, fusion protein can include two or more proteine. In one aspect, the fusion protein can include two or more immunogenic domains and/or two or more epitopes of one or more proteins. An immunotherapeutic composition containing such antigens may provide antigen-specific immunization in a broad range of patients. For example, an 5 antigen or fusion protein encompassed by the invention can include at least a portion of, or the full-length of, any one or more HBV proteins selected from: HBV surface protein (also called surface antigen or envelope protein or HBsAg), including the large (L), middle (M) and/or small (S) forms of surface protein and/or the pre-Sl and/or pre-S2 domains thereof; HBV precore protein; HBV core protein (also called core antigen or HBeAg);
HBV e-antigen (also called HBeAg); HBV polymerase (including one or both domains of the polymerase, called the RT domain and the TP domain); HBV X antigen (also called X, X antigen, or HBx); and/or any one or more immunogenic domains of any one or more of these HBV proteins. In one embodiment, an antigen useful in an immunotherapeutic composition of the invention is from a single HBV protein (full-length, near full-length, or portion thereof comprising at least, one, two, three, four or more immunogenic domains of a full-length protein). In one embodiment of the invention, an immunotherapeutic composition includes one, two, three, four, five or more individual yeast vehicles, each expressing or containing a different HBV antigen(s).
[00156] Combinations of HBV antigens useful in the présent invention include, but are 20 not limited to (in any order within the fusion protein):
(1) surface protein (L, M and/or S and/or any one or combination of functional and/or immunological domains thereof, including, but not limited to pre-Sl and/or pre-S2 and/or the hépatocyte receptor domain of pre-Sl) in combination with any one or more of: (a) precore/core/e (precore, core, e-antigen, and/or any one or combination of functional and/or immunological domains thereof); (b) polymerase (full-length, RT domain, TP domain and/or any one or combination of functional and/or immunological domains thereof); and/or (c) X antigen (or any one or combination of functional and/or immunological domains thereof);
(2) precore/core/e (precore, core, e-antigen, and/or any one or combination of functional and/or immunological domains thereof) in combination with any one or more of: (a) surface protein (L, M and/or S and/or any one or combination of functional and/or immunological domains thereof, including, but not limited to preSl and/or pre-S2 and/or the hépatocyte receptor domain of pre-Sl); (b) polymerase (full-length, RT domain, TP domain and/or any one or combination of functional
and/or immunological domains thereof); and/or (c) X antigen (or any one or combination of functional and/or immunological domains thereof);
(3) polymerase (full-length, RT domain, TP domain and/or any one or combination of functional and/or immunological domains thereof) in combination with any one or more of: (a) surface protein (L, M and/or S and/or any one or combination of functional and/or immunological domains thereof, including, but not limited to preS1 and/or pre-S2 and/or the hépatocyte receptor domain of pre-Sl); (b) precore/core/e (precore, core, e-antigen, and/or any one or combination of functional and/or immunological domains thereof); and/or (c) X antigen (or any 10 one or combination of functional and/or immunological domains thereof); or (4) X antigen (or any one or combination of functional and/or immunological domains thereof) in combination with any one or more of: (a) surface protein (L, M and/or S and/or any one or combination of functional and/or immunological domains thereof, including, but not limited to pre-Sl and/or pre-S2 and/or the hépatocyte receptor domain of pre-Sl); (b) polymerase (full-length, RT domain, TP domain and/or any one or combination of functional and/or immunological domains thereof); and/or (c) precore/core/e (precore, core, e-antigen, and/or any one or combination of functional and/or immunological domains thereof).
[00157] Recombinant nucleic acid molécules and the proteins encoded thereby, 20 including fusion proteins, as one embodiment of the invention, may be used in yeast-based immunotherapy compositions, or for any other suitable purpose for HBV antîgen(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 yeastbased immunotherapy described herein. Expression of the proteins by yeast is one 25 preferred embodiment, although other expression Systems may be used to produce the proteins for applications other than a yeast-based immunotherapy composition.
[00158] According to the présent invention, the general use herein of the term antigen refers: to any portion of a protein (peptide, partial protein, full-length protein), wherein the protein is naturally occurring or synthetically derived, to a cellular 30 composition (whole cell, cell lysate or disrupted cells), to an organism (whole organism, lysate or disrupted cells) or to a carbohydrate, or other molécule, or a portion thereof. An antigen may elicit an antigen-specific immune response (e.g., a humoral and/or a cellmediated immune response) against the same or similar antigens that are encountered by an element of the immune System (e.g., T cells, antibodies).
[00159( An antigen can be as small as a single epitope, a single immunogenic domain or larger, and can include multiple epitopes or immunogenic domains. As such, the size of an antigen can be as small as about 8-12 amino acids (i.e., a peptide) and as large as: a full length protein, a multimer, a fusion protein, a chimeric protein, a whole cell, a whole microorganism, or any portions thereof (e.g., lysâtes of whole cells or extracts of microorganisms). In addition, antigens can include carbohydrates, which can be loaded into a yeast vehicle or into a composition of the invention. It will be appreciated that in some embodiments (e.g., when the antigen is expressed by the yeast vehicle from a recombinant nucleic acid molécule), the antigen is a protein, fusion protein, chimeric 10 protein, or fragment thereof, rather than an entire cell or microorganism.
[00160] When the antigen is to be expressed in yeast, an antigen is of a minimum size capable of being expressed recombinantly in yeast, and is typically at least or greater than 25 amino acids in length, or at least or greater than 26, at least or greater than 27, at least or greater than 28, at least or greater than 29, at least or greater than 30, at least or greater 15 than 31, at least or greater than 32, at least or greater than 33, at least or greater than 34, at least or greater than 35, at least or greater than 36, at least or greater than 37, at least or greater than 38, at least or greater than 39, at least or greater than 40, at least or greater than 41, at least or greater than 42, at least or greater than 43, at least or greater than 44, at least or greater than 45, at least or greater than 46, at least or greater than 47, at least or 20 greater than 48, at least or greater than 49, or at least or greater than 50 amino acids in length, or is at least 25-50 amino acids in length, at least 30-50 amino acids in 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 may be expressed, and considerably larger proteins (e.g., hundreds of amino acids in length or even a few thousand amino acids in 25 length) may be expressed. In one aspect, a full-length protein, or a structural or functional domain thereof, or an immunogenic domain thereof, that is lacking one or more amino acids from the N- and/or the C-terminus may be expressed (e.g., lacking between about 1 and about 20 amino acids from the N- and/or the C-terminus). Fusion proteins and chimeric proteins are also antigens that may be expressed in the invention. A “target 30 antigen” is an antigen that is speeifically targeted by an immunotherapeutic composition of the invention (i.e., an antigen against which elicitation of an immune response is desired). An “HBV antigen” is an antigen derived, designed, or produced from one or more HBV proteins such that targeting the antigen also targets the hepatitîs B virus.
[00161] When referring to stimulation of an immune response, the term “immunogen” is a subset of the term “antigen”, and therefore, in some instances, can be used interchangeably with the term antigen. An immunogen, as used herein, describes an antigen which elicits a humoral and/or cell-mediated immune response (i.e., is 5 immunogenic), such that administration of the immunogen to an individual mounts an antigen-specific immune response against the same or similar antigens that are encountered by the immune system of the individual. In one embodiment, an immunogen elicits a cell-mediated immune response, including a CD4+ T cell response (e.g., ΊΉ1, ΤΉ2 and/or TH17) and/or a CD8+ T cell response (e.g., a CTL response).
[00162] An “immunogenic domain” of a given antigen can be any portion, fragment or epitope of an antigen (e.g., a peptide fragment or subunit or an antibody epitope or other conformational epitope) that contains at least one epitope that acts as an immunogen when administered to an animal. Therefore, an immunogenic domain is larger than a single amino acid and is at least of a size sufficient to contain at least one epitope that can act as an immunogen. For example, a single protein can contain multiple different immunogenic domains. Immunogenic domains need not be linear sequences within a protein, such as in the case of a humoral immune response, where conformational domains are contemplated.
[00163] A “functîonal domain” of a given protein is a portion or functîonal unit of the protein that includes sequence or structure that is directly or indirectly responsible for at 20 least one biological or chemical function associated with, ascribed to, or performed by the protein. For example, a functîonal domain can include an active site for enzymatîc activity, a ligand binding site, a receptor binding site, a binding site for a molécule or moiety such as calcium, a phosphorylation site, or a transactivation domain. Examples of HBV functîonal domains include, but are not limited to, the viral hépatocyte receptor 25 domain in pre-Sl, or the reverse transcriptase domain or RNase H domain of polymerase.
[00164] A “structural domain” of a given protein is a portion of the protein or an element in the protein’s overall structure that has an identifiable structure (e.g., it may be a primary or tertiary structure belonging to and indicative of several proteins within a class or family of proteins), is self-stabilizïng and/or may fold independently of the rest of the 30 protein. A structural domain is frequently associated with or features prominently in the biological function of the protein to which it belongs.
[00165] An epitope is defined herein as a single immunogenic site within a given antigen that is sufficient to elicit an immune response when provided to the immune system in the context of appropriate costimulatory signais 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 may also be referred to as an antigenic déterminant. Those of skill in the art will recognize that T cell epitopes are different in size and composition from B cell or antibody epitopes, and that epitopes presented through the Class I MHC pathway differ in size and structural attributes from epitopes presented through the Class II MHC pathway. For example, T cell epitopes presented by Class I MHC molécules are typically between 8 and 11 amino acids in length, whereas epitopes presented by Class II MHC molécules are less restricted in length and may be from 8 amino acids up to 25 amino acids or longer. In addition, T cell epitopes 10 hâve predicted structural characteristics depending on the spécifie MHC molécules bound by the epitope. Multiple different T cell epitopes hâve been identified in various HBV strains and for many human HLA types, several of which are identified in Table 5. In addition, epitopes for certain murine MHC haplotypes hâve been newly discovered herein and are also presented in Table 5 or in the Examples. Epitopes can be linear sequence 15 epitopes or conformational epitopes (conserved binding régions). Most antibodies recognize conformational epitopes.
[00166] One exemplary embodiment of the invention relates to a fusion protein comprising an HBV antigen that is a multi-protein HBV antigen, and in this example, a fusion comprised of HBV large (L) surface antigen, including ail of the hydrophobie 20 transmembrane domains, and core antigen (HBcAg), described in detail below. Surface antigen and core are abundantly expressed in infected cells, are required for viral réplication, and contain multiple CD4+ and CD8+ T cell epitopes. In addition, these antigens, particularly surface antigen, contain known mutation sites that can be induced by anti-viral therapy; these régions can therefore be modified, as needed, to provide 25 additional immunotherapy compositions to target the “escape” mutations. An additional advantage of targeting these proteins, and particularly both proteins in a single immunotherapeutic composition, is the high degree of conservation at the amino acid level among different HBV génotypes. Both the core and surface (L) proteins are highly conserved between HBV génotypes A and C or between A and H, for example (see Table 30 4), which are génotypes prévalent in the Americas and Asia (Table 2). The core protein displays a 95% amino acid identity between génotypes A and C and between génotypes A and H. The large (L) surface protein is also highly conserved among the different HBV génotypes; a 90% amino acid identity exists between génotypes A and C, and 82% amino acid identity exists between génotypes A and H.
Table 4
Comparison Core Surface (L) X Polymerase
HBV Génotype Avs. HBV Génotype C 95 90 89 90
HBV Génotype A vs. HBV Génotype H 95 82 79 82
[00167] Therefore, one immunotherapeutic composition designed using one HBV génotype can be expected to induce an effective immune response against a highly similar HBV génotype, either through direct targeting of conserved epitopes or through epitope spreading as a resuit of initially targeting epitopes that are conserved between génotypes. Alternatively, because of the ease of producing the yeast-based immunotherapy compositions of the invention, it is straightforward to modify a sequence to encode a protein, domain, or epitope from a different génotype, or to include in the same construct different T cell epitopes or entire domains and/or proteins from two or more different HBV génotypes, in order to increase the wide applicability of the immunotherapy. Examples of such HBV antigens are described in detail and exemplified below. While one immunotherapeutic composition of the présent invention was designed to target two HBV antigens, surface and core protein, in a single product, this approach can readily be expanded to incorporate the protein sequences of other essential, conserved, and immunogenic HBV viral proteins to resuit in even broader cellular immune responses. Such additional fusion proteins and immunotherapeutic compositions are described and exemplified herein.
[00168] In one embodiment of the invention, the HBV antigen(s) for use in a composition or method of the invention are selected from HBV antigens that hâve been designed to optimize or enhance their usefulness as clinical products, including in the context of a yeast-based immunotherapeutic composition. Such HBV antigens hâve been designed to produce an HBV yeast-based immunotherapeutic product that achieves one or more of the foilowing goals: (1) compliance with the guidelines of the Recombinant DNA Advisory Committee (RAC) of the National Institutes of Health (NIH), wherein no more than two thirds (2/3) of the genome of an infectious agent may be used in a recombinant therapeutic or vaccine; (2) inclusion of a maximized number of known T cell epitopes associated with immune responses to acute/self-limiting HBV infections and/or chronic HBV infections (with prioritization in one aspect based on the acute/self-limiting epitope répertoire, as discussed below); (3) 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), that are the most conserved among HBV génotypes and/or sub-genotypes, or that can be readily modified to a consensus sequence or included in two or more forms to cover the most important sequence différences among 5 target génotypes; and/or (4) minimizing the number of non-natural junctions within the sequence of the HBV antigen in the product.
[00169] Accordingly, the invention includes, in some embodiments, modification of HBV antigens from their naturally occurring or wild-type sequences in a given strain to meet one or more of criteria described above, as well as to include design éléments and/or 10 antigen design criteria described elsewhere herein. Such criteria and antigen design guidance is applicable to yeast-based immunotherapeutics comprising HBV antigens that are individual HBV proteins or domains, as well as HBV antigens that include combinations of HBV proteins or domains and particularly, muiti-protein antigens/fusion t
proteins (e.g., HBV antigens from two or more different HBV proteins and/or domains 15 thereof, such as combinations of antigens from HBV surface protein, polymerase, core, eantigen, and/or X antigen). It will be appreciated that as the complexity of the HBV antigen increases, the utilization of more of these criteria are implemented in the construction of the antigen.
[00170] Therefore, in one embodiment of the invention, an HBV antigen useful in the 20 présent invention as a protein or fusion protein to be expressed by a yeast includes HBV sequences encode d b y nucleotîde sequences representing less than two thirds (2/3) of the HBV genome (i.e., the antigens are encoded by nucleic acid sequences that in total make up less than two thirds (2/3) of the HBV genome or meet the requirements of RAC for recombinant therapeutics and prophylactics). In one aspect, this embodiment can be 25 achieved by selecting HBV antigens for expression in a yeast-based îmmunotherapeutic that meet the RAC requirements in their full-length or near-full-length form (e.g., X antigen is small and when used alone would meet the RAC requirements). In another aspect, this embodiment is achieved by modifying the structure of the protein(s) and/or domain(s) to be included in the HBV antigen, such as by délétion of sequence to truncate 30 proteins or remove internai sequences from proteins, by including only selected functional, structural or immunogenic domains of a protein, or by choosing to eliminate the inclusion of a particular protein in the antigen construct altogether. In addition, HBV yeast-based immunotherapeutics may, in one embodiment, be produced as individual antigen constructs, and then used in combination in a manner that does not contravene any restrictions related to the viral genome.
[00171] In another embodiment of the invention, as discussed above, the inclusion of T cell epitopes in an HBV antigen construct (protein or fusion protein) is maximized, for example, if the HBV antigen included in the immunotherapeutic has been modified to meet another design considération, such as the RAC requirement discussed above. In this embodiment, HBV antigens useful in a yeast-based immunotherapeutic are modified with the goal of maximizing the number of immunogenic domains, and in one aspect, the number of T cell epitopes, that are retained in the HBV antigen. In one aspect, the inclusion of T cell epitopes in an HBV antigen is prioritized as follows:
Epitopes identified in immune responses to both acute/self-limiting HBV infections and chronic HBV infections > Epitopes identified in immune responses to acute/self-limiting HBV infections > Epitopes identified in immune responses to chronic HBV infections.
In this embodiment, without being bound by theory, the inventors believe that immune responses from individuals who had acute or self-limiting HBV infections may be more productive in eliminating the viral infection than the immune responses from individuals who hâve chronic HBV infections. Therefore, the inclusion of T cell epitopes that appear to be associated with clearance of virus in these acute or self-limiting infections (whether dominant or sub-dominant) is prioritized as being more lîkely to elicit a bénéficiai immune response in an immunized individual. In addition, and again without being bound by theory, the inventors believe that the génération of an immune response against one or more HBV target antigens using yeast-based immunotherapy will resuit in an immune response in the immunized individual against not only the epitopes included in the yeastbased immunotherapeutic, but also against other HBV epitopes présent in the individual. This phenomenon, referred to as “epitope spreading” allows for the design of HBV antigens that are focused on epitopes that appear to be most relevant to therapeutic benefit, and the mechanism of action of a yeast-based immunotherapeutic product then allows the immune System to expand the immune response to cover additional target epitopes, thereby enhancing a therapeutically productive or bénéficiai immune response against HBV.
[00172] Accordingly, an HBV antigen in one embodiment comprises one or more CTL epitopes (e.g., epitopes that are recognized by a T cell receptor of a cytotoxic T lymphocyte (CTL), when presented in the context of an appropriate Class I MHC
molécule). In one aspect, the HBV antigen comprises one or more CD4+ T cell epitopes (e.g., epitopes that are recognized by a T cell receptor of a CD4+ T cell, in the context of an appropriate Class II MHC molécule). In one aspect, the HBV antigen comprises one or more CTL epitopes and one or more CD4+ T cell epitopes. In one aspect, an HBV antigen 5 useful in an immunotherapeutic composition of the invention comprises one or more of the exemplary HBV CTL epitopes described in Table 5. One of skill in the art will readily be able to identify the position of the corresponding sequence for each epitope in Table 5 in a given HBV sequence of any génotype, sub-genotype, or strain/isolate, given the guidance provided below, even though some amino acids may differ from those in Table 5.
Examples of such différences are illustrated in Table 5. The invention is not limited to antigens comprising these epitopes as others will be known in the art and are contemplated for use in the invention. In one embodiment, the epitope can be modified to correspond to the sequence of the epitope within a given génotype, sub-genotype or strain/isolate of HBV, since there may be one or more amino acid différences at these epitopes among 15 génotypes, sub-genotypes or stain/isolates.
Table 5
Epitope Sequence Identifier HBV Antigen HLA Preference
FLLTRILTI1·23 SEQ ID NO:42 Surface (e.g. positions 20-28 of S; e.g. corresponding to positions 194-202 of SEQ ID NO:11, positions 201-209 of SEQ ID NO:34, or positions 51-59 of SEQ ID NO:36) A*0201
GLSPTVWLSV5 SEQ ID NO.43 Surface (e.g. positions 185-194 of S; e.g. corresponding to positions 359-368 of SEQ ID NO:11, positions 366-375 of SEQ ID NO:34, or positions 216-225** ofSEQ ID NO:36) A*0201
FLPSDFFPSI2'3,4 SEQ ID NO:44 Core (e.g. positions 47-56 of Precore; e.g. corresponding to positions 47-56 of SEQ ID NO:9, positions 424-433 of SEQ ID NO:34, or positions 621-630 of SEQ ID NO:36) A*0201
FLLSLGIHL1 SEQ ID NO:45 Polymerase (e.g. positions 575-583 of Pol; e.g. corresponding to positions 573-581 of SEQ ID NO:10, or positions 486-494 of SEQ ID NO:36) A*0201
WLSLLVPFV1,3,9 SEQ ID NO:46 Surface (e.g. positions 172-180 of S; e.g. corresponding to positions 346-354 of SEQ ID NO:11, positions 353-3611 of SEQ ID NO:34, or positions 203-211 of SEQ ID NO:36) A*0201
Epitope Sequence Identifier HBV Antigen HLA Preference
KYTSFPWLL SEQ ID NO:47 Polymerase (e.g. positions 756-764 of Pol; e.g. corresponding to positions 756-764 of SEQIDNO:10) A*2402
YVNVNMGLK4 SEQ ID NO:48 Core (e.g. positions 117-125 of Precore; e.g. corresponding to positions 117125 of SEQ ID NO:9, positions 494502 of SEQ ID NO:34, or positions 691-699 of SEQ ID NO:36) A*1101
EYLVSFGVW SEQ ID NO:49 Core (e.g. positions 146-154 of Precore; e.g. corresponding to positions 146154 of SEQ ID NO:9, positions 523531 of SEQ ID NO:34, or positions 720-72Θ of SEQ ID NO:36) A*2402
GLSRYVARL3 SEQ ID NO:50 Polymerase (e.g. positions 455-463 of Pol; e.g. corresponding to positions 453-461* of SEQ ID NO:10, or positions 366374 of SEQ ID NO:36) A*0201
CLFKDWEEL® SEQ ID N0:51 X (e.g. positions 115-123 of X; e.g. corresponding to positions 115-123* of SEQ ID NO:12, or positions 900908* of SEQ ID NO:36) A*02
PLGFFPDH® SEQ ID NO:52 Surface (e.g. positions 21-28 of Pre-S1 ; e.g. corresponding to positions 21-28 of SEQ ID NO:11, positions 28-35 of SEQ ID NO:34;, or positions 6-13 of SEQ ID NO:36) A*11
IPIPSSWAF8 SEQ ID NO:53 Surface (e.g. positions 150-158 of S; e.g. corresponding to positions 324-332 of SEQ ID NO:11, positions 331-339 of SEQ ID NO:34, or positions 181-189 of SEQ ID NO:36) B*07
LPSDFFPSV5 SEQ ID NO:54 Core (e.g. positions 48-56 of Precore; e.g. corresponding to positions 48-561 of SEQ ID NO:9, positions 425-433* of SEQ ID NO:34, or positions 619-630* Of SEQ ID NO:36) B*51
MQWNSTALHQALQDP5 SEQ ID NO:55 Surface (e.g. positions 1-15 of pre-S2; e.g. ***corresponding to positions 120-134 of SEQ ID NO:3) A*3
LLDPRVRGL8 SEQ ID NO:56 Surface (e.g. positions 12-20 of pre-S2; e.g. ***corresponding to positions 131-139 of SEQ ID NO:3) A*2
SILSKTGDPV8 SEQ ID NO:57 Surface (e.g. positions 44-53 of a pre-S2; e.g. ***corresponding to positions 163-172 of SEQ ID NO:3) A*2
Epitope Sequence Identifier HBV Antigen HLA Preference
VLQAGFFLL5 SEQ ID NO:58 Surface (e.g. positions 14-22 of S; e.g. ***corresponding to positions 188-196 of SEQ ID N0:3) A*2
FLLTRILTI8 SEQ ID NO:59 Surface (e.g. positions 20-28 of S; e.g. ***corresponding to positions 194-202 of SEQ ID NO:3) A*2
FLGGTPVCL5 SEQ ID NO:60 Surface (e.g. positions 41-49 of S; e.g. ***corresponding to positions 215-223 of SEQ ID NO:3) A*2
LLCLIFLLV® SEQ ID NO;61 Surface (e.g. positions 88-96 of S; e.g. ***corresponding to positions 262-270 of SEQ ID NO:3) A*2
LVLLDYQGML® SEQ ID NO:62 Surface (e.g. positions 95-104 of S; e.g. ***corresponding to positions 269-278 of SEQ ID NO:3) A*2
LLDYQGMLPV5 SEQ ID NO:63 Surface (e.g. positions 97-106 of S; e.g. ***corresponding to positions 271-280 of SEQ ID NO:3) A*2
SIVSPFIPLL5 SEQ ID NO:64 Surface (e.g. positions 207-216 of S; e.g. ***corresponding to positions 381-390 of SEQ ID NO:3) A*2
ILSPFLPLL5 SEQ ID NO:65 Surface (e.g. positions 208-216 of S; e.g. ***corresponding to positions 382-390 of SEQ ID NO:3) A*2
TPARVTGGVF5 SEQ ID NQ:66 Polymerase (e.g. positions 367-376 of Pol; e.g. ***corresponding to positions 367-376 of SEQ ID NO:2) B*7
LWDFSQFSR5 SEQ ID NO:67 Polymerase (e.g. positions 390-399 of Pol; e.g. ***corresponding to positions 390-399 of SEQ ID NO:2) A*3
SAICSWRR9 SEQ ID NO:68 Polymerase (e.g. positions 533-541 of Pol; e.g. ***correspondîng to positions 533-541 of SEQ ID NO:2) A*3
YMDDWLGA5 SEQ ID NO:69 Polymerase (e.g. positions 551-559 of Pol; e.g. ***corresponding to positions 551-559 of SEQ ID NO:2) A*2
ALMPLYACI5 SEQ ID NO:70 Polymerase (e.g. positions 655-663 of Pol; e.g. ***corresponding to positions 655-663 of SEQ ID NO:2) A*2
QAFTFSPTYK9 SEQ ID NO:71 Polymerase (e.g. positions 667-676 of Pol; e.g. ***corresponding to positions 667-676 of SEQ ID NO:2) A*3
Epitope Sequence Identifier HBV Antigen HLA Preference
ATVELLSFLPSDFFPSV® SEQ ID NO:72 Core (e.g. positions 40-56 of Precore; e.g. ***corresponding to positions 40-56 of SEQ ID NO;1) A*2
LPSDFFPSV9 SEQ ID NO:73 Core (e.g. positions 48-56 of Precore; e.g. ***corresponding to positions 48-56 of SEQ ID NO:1) B*51
CLTFGRETV9 SEQ ID NO:74 Core (e.g. positions 136-144 of Precore; e.g. ***correspondîng to positions 136-144 of SEQ ID NO;1) A*2
VLEYLVSFGV5 SEQ ID NO:75 Core (e.g. positions 144-153 of Precore; e.g. ***corresponding to positions 144-153 of SEQ ID NO:1) A*2
ILSTLPETTV9 SEQ ID NO:76 Core (e.g. positions 168-177 of Precore; e.g. ***corresponding to positions 168-177 of SEQ 1DNO:1) A*2
STLPETTWRR® SEQ ID NO:77 Core (e.g. positions 170-180 of Precore; e.g. ***corresponding to positions 170-180 of SEQ ID NO:1) A*3
HLSLRGLFV® SEQ ID NO:78 X (e.g. positions 52-60 of X; e.g. ***corresponding to positions 52-60 of SEQ ID NO:4) A*2
VLHKRTLGL8 SEQ IDNO:79 X (e.g. positions 92-100 of X; e.g. ***corresponding to positions 92-100 of SEQ ID NO:4) A*2
GLSAMSTTDL8 SEQ ID NO:80 X (e.g. positions 99-108 of X; e.g. ***corresponding to positions 99-108 of SEQ ID NO:4) A*2
VLGGCRHKL8 SEQ ID NO:81 X (e.g. positions 133-141 ofX; e.g. ***corresponding to positions of 133141 SEQ ID NO:4) A*2
NVSIWTHK5 SEQ ID NO:82 Polymerase (e.g. positions 49-57 of Pol; e.g. ***corresponding to positions 49-57 of SEQ ID NO:2) A*3
KVGNFTGLY8 SEQ ID NO:83 Polymerase (e.g. positions 57-65 of Pol; e.g. ***corresponding to positions 57-65 of SEQ ID NO:2) A*3
GLYSSTVPV5 SEQ ID NO:84 Polymerase (e.g. positions 63-71 of Pol; e.g. ***corresponding to positions 63-71 of SEQ ID NO;2) A*2
TLWKAGILYK8 SEQ ID NO:85 Polymerase (e.g. positions 152-161 of Pol; e.g. ***corresponding to positions of SEQ ID NO:2) A*3
Epitope Sequence Identifier HBV Antigen HLA Preference
KYTSFPWLL? SEQ ID NO:86 Polymerase (e.g. positions 756-764 of Pol; e.g. •corresponding to positions 758-766 of SEQ ID NO:2) A*24
ILRGTSFVYV1 2 * * 5 * 7 * 9 SEQ ID NO:87 Polymerase (e.g. positions 773-782 of Pol; e.g. ***corresponding to positions 773-782 of SEQ ID NO:2) A*2
SLYADSPSV® SEQ ID NO:88 Polymerase (e.g. positions 816-824 of Pol; e.g. ***corresponding to positions 816-824 of SEQ ID NO:2) A*2
KLHLYSHPI6 SEQ ID NO:135 Polymerase (e.g., positions 502-510 of Pol; e.g., ***corresponding to positions 502-510 of SEQ ID NO:2) A*2
LLVPFVQWFV6'7 SEQIDNO:136 Surface (e.g., positions 349-358 of S; e.g., •corresponding to positions 349-358 of SEQ ID NO;3) A*2
HLYSHPIIL® SEQ ID NO:137 Polymerase (e.g., positions 504-512 of Pol; e.g., •corresponding to positions 504-512 of SEQ ID NO:2) A*2
WSPQAQGIL® SEQ ID NO:138 Surface (e.g., positions 77-84 of S; e.g., •corresponding to positions 77-84 of SEQ ID NO:3) H-2Db
VLLDYQGM10 SEQ ID NO:139 Surface (e.g., positions 270-277 of S; e.g., •corresponding to positions 270-277 of SEQ ID NO:3) H-2Kb
ASVRFSWL10 SEQ IDNO:140 Surface (e.g., positions 340-347 of S; e.g., •corresponding to positions 340-347 of SEQ ID NO:3) H-2Kb
**Substitutlon of an Ala for Val at position 9 of SEQ ID NO:43; at position 225 in SEQ ID NO:36. Substitution of Gln-Ala for Leu-Val at positions 5 and 6 of SEQ ID NO:46; at positions 357 and 358 in SEQ ID NO:34.
Substitution of Pro for Ser at position 3 of SEQ ID NO:50; at position 455 in SEQ ID NO:10.
Substitution of Val for Leu at position 2 of SEQ ID NO:51 ; at position 116 in SEQ ID NO:12 and position 901 In SEQ ID NO:36.
Substitution of Ile for Val at position 9 of SEQ ID NO:54; at position 56 in SEQ ID NO:9, position 433 of SEQ ID N0:34, and position 630 of SEQ ID NO:36.
***One or more amino acid différences between the epitope sequence and the actual sequence of the corresponding larger protein or domain may exist due to génotype, sub-genotype or strain différences, although position of the epitope within the larger protein or domain can readlly be determined.
1Zhang et al., Journal of Hepatology 50:1163-1173 (2009) 2Lopes étal., J. Clin. Invest. 118:1835-1845 (2008) ’Boettler et al., J Virol 80(7):3532-3540 (2006) 4Peng et al., Mol. Immunol. 45:963-970 (2008) 5Desmond 2008; vyww.allelefrequencies.net or Desmond et al., Antiviral Ther. 13:16-175 (2008) ®Webster et al., 2004, J. Virol. 78(11)5707-5719 7Vitiello, 1997, Eur. J. Immunol. 27(3): 671-678 eSette et al., 1994, J. Immunol. 153(12): 5586-5592 9Murine H-2Db epitope, not previously reported 10Murine H-2Kb epitope, not previously reported
[00173] In one embodiment of the invention, useful HBV antigens can include in one or more yeast-based immunotherapeutic compositions an antigen comprising one or more T cell epitopes that has been described as or determined to be a “dominant” epitope (i.e., a T cell epitope that contributes to the development of a T cell response against the whole 5 protein, and/or that is among the relatively small number of T cell epitopes within the large group of possible epitopes that most likely or most readily elicit CD4+ and CD8+ T cell responses, also referred to as an “immunodominant epitope”). In another embodiment, HBV antigens useful in the invention can include in the same or a different or additional yeast-based compositions, an HBV antigen comprising one or more T cell epitopes that 10 has been described as or determined to be a “subdominant” epitope (i.e., a T cell epitope that is immunogenic, but to a lesser extent than an immunodominant epitope; the immune response generated by a sub-dominant epitope may be suppressed or outcompeted by the immune response to an immunodominant epitope). For an example of this effect with CTL responses to HBV T cell epitopes in mice, see Schirmbeck R., et al. J. immunology 15 168: 6253-6262, 2010; or Sette et al. J Immunology 166:1389-1397, 2001. In one aspect of the invention, different compositions comprising immunodominant or sub-dominant epitopes could be administered at the same site in an individual, or in one embodiment, at different sites in an individual (i.e., the composition comprising dominant epitopes being administered to one site and the composition comprising sub-dominant epitopes being 20 administered to a different site). In some cases, a sub-dominant epitope may elicit a more therapeutically bénéficiai immune response than a dominant epitope. Therefore, if administered to separate sites, it may decrease the chance that an immune response to a dominant epitope would suppress or outcompete an immune response to a sub-dominant epitope, thereby maximizing the immune response as a whole and maximizing the 25 protective or therapeutic benefit in an individual. This approach of providing different antigens in different compositions administered to different sites in the individual can also be utilized even if ail epitopes are dominant or sub-dominant. Immunodominant epitopes and sub-dominant epitopes hâve been recognized to play a rôle in HBV infection and immune responses (see, e.g., Sette et al., 2001, supra, and Schirmbeck et al., 2002, supra).
[00174] In one embodiment of the invention, an HBV antigen useful in a yeast-based immunotherapeutic maximizes the inclusion of immunogenic domains, and particularly, T cell epitopes, that are conserved among génotypes and/or sub-genotypes, and/or includes immunogenic domains from several different génotypes and/or sub-genotypes and/or includes immunogenic domains that can readily be modified to produce multiple yeast16530 based immunotherapeutic products that differ in some minor respects, but are tailored to treat different individuals or populations of individuals based on the HBV genotype(s) or sub-genotype(s) that infect such individuals or populations of individuals. For example, the HBV antigen can be produced based on a génotype or sub-genotype that is most prévalent among individuals or populations of individuals to be protected or treated, and the HBV antigen includes the most conserved immunogenic domains from those génotypes. Alternatively or in addition, immunogenic domains can be modified to correspond to a consensus sequence for that domain or epitope, or more than one version of the epitope can be included in the construct.
[00175] In any embodiment of the invention related to the design of an HBV antigen for a yeast-based immunotherapeutic composition, in one aspect, artificial junctions between segments of a fusion protein comprising HBV antigens is minimized (i.e., the inclusion of non-natural sequences is limited or minimized to the extent possible). Without being bound by theory, it is believed that natural évolution has resulted in: i) contiguous sequences in the virus that most likely to be expressed well in another cell, such as a yeast; and ii) an immunoproteasome in antigen presenting cells that can properly digest and présent those sequences to the immune system. The yeast-based immunotherapeutic product of the invention allows the host immune system to process and présent target antigens; accordingly, a fusion protein with many unnatural junctions may be less useful in a yeast-based immunotherapeutic as compared to one that retains more of the natural HBV protein sequences.
[00176] In any of the HBV antigens described herein, including any of the fusion proteins, the following additional embodiments can apply. First, the N-terminal expression sequence and the C-terminal tag included in some of the fusion proteins are optional, and if used, may be selected from several different sequences described elsewhere herein to improve expression, stability, and/or allow for identification and/or purification of the protein. Alternatively, one or both of the N- or C-terminal sequences are omitted altogether. In addition, many different promoters suitable for use in yeast are known in the art and are encompassed for use to express HBV antigens according to the présent invention. Furthermore, short intervening linker sequences (e.g., 1, 2, 3,4, or 5, or larger, amino acid peptides) may be introduced between portions of the fusion protein for a variety of reasons, including the introduction of restriction enzyme sites to facilitate cloning and future manipulation of the constructs. Finally, as discussed in detail elsewhere herein, the sequences described herein are exemplary, and may be modified as described in detail elsewhere herein to substitute, add, or delete sequences in order to accommodate preferences for HBV génotype, HBV subgenotype, HBV strain or isolate, or consensus sequences and inclusion of preferred T cell epitopes, including dominant and/or subdominant T cell epitopes. A description of several different exemplary HBV antigens useful în the invention is provided below.
[00177] In any of the embodiments of the invention described herein, including any embodiment related to an immunotherapeutic composition, HBV antigen, fusion protein or use of such composition, HBV antigen or fusion protein, in one aspect, an amino acid of an HBV surface antigen useful as an HBV antigen or in a fusion protein or an immunotherapeutic composition of the invention can include, but is not limited to, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, positions 21-47 of SEQ ID NO:11, positions 176400 of SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, positions 9-407 of SEQ ID NO:34, positions 6-257 of SEQ ID NO:36, positions 6-257 of SEQ ID NO:41, positions 92-343 of SEQ ID NO:92, positions 90-488 of SEQ ID NO:93, SEQ ID NO:97, positions 90-338 of SEQ ID NO: 101, positions 7-254 of SEQ ID NO:102, positions 1-249 of SEQ ID NO:107, positions 1-249 of SEQ ID NO:108, positions 1-249 of SEQ ID NO:109, positions 1-249 of SEQ ID NO:110, positions 1-399 of SEQ ID NO:112, positions 1-399 of SEQ ID NO:114, or positions 1399 of SEQ ID NO: 116, positions 1-399 of SEQ ID NO: 118, positions 1-399 of SEQ ID NO:120, positions 1-399 of SEQ ID NO:122, positions 1-399 of SEQ ID NO:124, positions 1-399 of SEQ ID NO:126, positions 231-629 of SEQ ID NO: 128, positions 63461 of SEQ ID NO: 130, positions 289-687 of SEQ ID NO: 132, positions 289-687 of SEQ ID NO: 134, or a corresponding sequence from a different HBV strain.
[00178] In any of the embodiments of the invention described herein, including any embodiment related to an immunotherapeutic composition, HBV antigen, fusion protein or use of such composition, HBV antigen or fusion protein, in one aspect, an amino acid of an HBV polymerase antigen useful as an HBV antigen or in a fusion protein or an immunotherapeutic composition of the invention can include, but is not limited to, positions 383-602 of SEQ ID NO:2, positions 381-600 of SEQ ID NO:6, positions 381600 of SEQ ID NO: 10, positions 453 to 680 of SEQ ID NO: 10, positions 370-589 of SEQ ID NO:14, positions 380-599 of SEQ ID NO:18, positions 381-600 of SEQ ID NO:22, positions 380-599 of SEQ ID NO:26, positions 381-600 of SEQ ID NO:30, positions 260 to 604 of SEQ ID NO:36, positions 7-351 of SEQ ID NO:38, positions 7-351 of SEQ ID NO:40, 260 to 604 of SEQ ID NO:41, positions 346 to 690 of SEQ ID NO:92, positions
90-434 of SEQ ID NO:94, SEQ ID NO:98, positions 339 to 566 of SEQ ID NO:101, positions 255 to 482 of SEQ ID NO: 102, positions 250-477 of SEQ ID NO: 107, positions
250-477 of SEQ ID NO: 108, positions 250-477 of SEQ ID NO: 109, positions 250-477 of
SEQ ID NO:110, positions 582 to 809 of SEQ ID NO:120, positions 582 to 809 of SEQ
ID NO:124, positions 642 to 869 of SEQ ID NO:126, positions 1 to 228 of SEQ ID
NO:128, positions 1 to 228 of SEQ ID NO:132, positions 61 to 288 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain.
[00179] In any of the embodiments of the invention described herein, including any embodiment related to an immunotherapeutic composition, HBV antigen, fusion protein or 10 use of such composition, HBV antigen or fusion protein, in one aspect, an amino acid of an HBV core antigen useful as an HBV antigen or in a fusion protein or an immunotherapeutic composition of the invention can include, but is not limited to, positions 31-212 of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212 of
SEQ ID NO:9, positions 37 to 188 of SEQ ID NO:9, positions 31-212 of SEQ ID NO: 13, 15 positions 31-212 of SEQ ID NO:17, positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO:29, positions 408-589 of SEQ ID NO:34, positions 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ ID NO:39, positions 605-786 of SEQ ID NO:41, positions 691872 of SEQ ID NO:92, positions 90-271 of SEQ ID NO:95, SEQ ID NO:99, positions 567 20 to 718 of SEQ ID NO: 101, positions 483 to 634 of SEQ ID NO: 102, positions 2-183 of
SEQ ID NO: 105, positions 184-395 of SEQ ID NO: 105, positions 396-578 of SEQ ID NO:105, positions 579-761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338520 of SEQ ID NO: 106, positions 478-629 of SEQ ID NO: 107, positions 478-629 of SEQ ID NO:108, positions 478-629 of SEQ ID NO:109, positions 478-629 of SEQ ID NO:110, 25 positions 400-581 of SEQ ID NO:112, positions 400-581 of SEQ ID NO:114, positions
400-581 of SEQ ID NO:116, positions 400-581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:120, positions 400 to 581 of SEQ ID NO:122, positions 400 to 581 of SEQ
ID NO: 124, positions 400 to 581 of SEQ ID NO: 126, positions 630 to 811 of SEQ ID
NO: 128, positions 462 to 643 of SEQ ID NO:130, positions 688 to 869 of SEQ ID 30 NO:132, positions 688 to 869 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain.
[00180] In any of the embodiments of the invention described herein, including any embodiment related to an immunotherapeutic composition, HBV antigen, fusion protein or use of such composition, HBV antigen or fusion protein, in one aspect, an amino acid of
an HBV X antigen useful as an HBV antigen or in a fusion protein or an immunotherapeutic composition of the invention can include, but is not limited to, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, positions 2 to 154 of SEQ ID NO:12, SEQ ID NO: 16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, positions 52-68 followed by positions 84-126 of SEQ ID NO:4, positions 52-68 followed by positions 84126 of SEQ ID NO:8, positions 52-68 followed by positions 84-126 of SEQ ID N0:12, positions 52-68 followed by positions 84-126 of SEQ ID N0:16, positions 52-68 followed by positions 84-126 of SEQ ID NO:20, positions 52-68 followed by positions 84-126 of SEQ ID NO:24, positions 52-68 followed by positions 84-126 of SEQ ID NO:28, positions 52-68 followed by positions 84-126 of SEQ ID NO:32, positions 787 to 939 of SEQ ID NO:36, positions 7-159 of SEQ ID NO:39, positions 873-1025 of SEQ ID NO:92, positions 90-242 of SEQ ID NO:96, SEQ ID NO:100, positions 719-778 of SEQ ID NO:101, positions 635-694 of SEQ ID NO:102, positions 184-337 of SEQ ID NO:106, positions 521-674 of SEQ ID NO: 106, positions 630-689 of SEQ ID NO; 107, positions
630-689 of SEQ ID NO:108, positions 630-689 of SEQ ID NO:109, positions 630-689 of
SEQ ID NO:110, positions 582-641 of SEQ ID NO:122, positions 810-869 of SEQ ID NO: 124, positions 582-641 of SEQ ID NO: 126, positions 1-60 of SEQ ID NO: 130, positions 229 to 288 of SEQ ID NO:132, positions 1 to 60 of SEQ ID NO: 134, or a corresponding sequence from a different HBV strain.
[00181] HBV Antigens Comprising Surface Antigen and Core Protein. In one embodiment of the invention, the HBV antigen(s) for use in a composition or method of the invention is a fusion protein comprising HBV antigens, wherein the HBV antigens comprise or consist of HBV large (L) surface antigen or at least one immunogenic domain thereof and HBV core protein (HBeAg) or at least one immunogenic domain thereof. In 25 one aspect, the HBV large (L) surface antigen and/or the HBV core protein is full-length or near full-length. According to any embodiment of the présent invention, reference to a “full-length” protein (or a full-length functional domain or full-length immunological domain) includes the full-length amino acid sequence of the protein or functional domain or immunological domain, as described herein or as otherwise known or described in a 30 publicly available sequence. A protein or domain that is “near full-length”, which is also a type of homologue of a protein, differs from a full-length protein or domain, by the addition or délétion or omission of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the Nand/or C-terminus of such a full-length protein or full-length domain. General reference
to a protein or domain can include both full-length and near full-length proteins, as well as other homologues thereof.
[00182] In one aspect, the HBV large (L) surface antigen or the HBV core protein comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% 5 of the linear sequence of a full-length HBV large (L) surface antigen or HBV core protein, respectively, or of the linear sequence of a portion of HBV large surface antigen that comprises the hépatocyte receptor binding portion of pre-Sl and ail or a portion of HBV small (S) surface antigen, of the linear amino acid sequences represented by SEQ ID NO:97 (optimized HBV surface antigen, described below), SEQ ID NO:99 (optimized 10 core protein, described below), or a corresponding sequence from another HBV strain, as applicable. A variety of other sequences for suitable HBV surface antigens and HBV core antigens useful in the invention are described herein. In one aspect, the HBV large (L) surface antigen or lhe HBV core protein is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a full-length HBV large (L) surface antigen or 15 HBV core protein, respectively, or to another HBV surface antigen or HBV core antigen described herein, including the amino acid sequence represented by SEQ ID NO;97 (optimized HBV surface antigen, described below), SEQ ID NO:99 (optimized core protein, described below), or a corresponding sequence from another HBV strain, as applicable.
[00183] Such a fusion protein is schematically represented in Fig. 2. One example of a composition comprising such a fusion protein is described in Example 1. In this embodiment, yeast (e.g,, Saccharomyces cerevisiae) were engineered to express various HBV surface-core fusion proteins as shown in Fig. 2 under the control of the copperinducible promoter, CUP1, or the TEF2 promoter. In each case, the HBV fusion protein 25 was a single polypeptide with the following sequence éléments fused in frame from N- to
C-terminus, represented by SEQ ID NO:34: (1) an N-terminal peptide to impart résistance to proteasomal dégradation and stabilize expression (e.g., positions 1 to 6 of SEQ ID NO:34); 2) a two amino acid spacer to introduce a Spel restriction enzyme site; 3) the amino acid sequence of a near full-length (minus position 1) HBV génotype C large (L) 30 surface antigen (e.g., positions 9 to 407 of SEQ ID NO:34 or positions 2-400 of SEQ ID
NO:11 (which differs from SEQ ID NO:34 at positions 350-351 of SEQ ID NO:11, where a Leu-Val sequence in SEQ ID NO: 11 îs replaced with a Gln-Ala sequence at positions 357-358 of SEQ ID NO:34)); 4) the amino acid sequence of an HBV core antigen (e.g., positions 31-212 of SEQ ID NO:9 or positions 408 to 589 of SEQ ID NO:34); and 5) a hexahistidine tag (e.g., positions 590-595 of SEQ ID NO:34). Positions 28-54 of SEQ ID NO:34 comprise the hépatocyte receptor portion of large (L) surface protein. SEQ ID NO:34 contains multiple epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. For example, positions 209-220, positions 389-397, positions 360-367, and positions 499-506, with respect to SEQ ID NO:34, comprise known MHC Class I binding and/or CTL epitopes. Positions 305-328 of SEQ ID NO:34 comprise an antibody epitope. A nucleic acid sequence encoding the fusion protein of SEQ ID NO:34 (codon optimized for yeast expression) is represented herein by SEQ ID NO:33. A yeast-based immunotherapy composition expressing this fusion protein is also referred to herein as GI-13002,
[00184] The amino acid segments used in any of the fusion proteins described herein can be modifîed by the use of additional amino acids flanking either end of any domain; the descriptions provided herein are exemplary. For example, a fusion protein according to this embodiment can include 1) the amino acid sequence of a near full-length (minus position 1) HBV génotype C large (L) surface antigen (e.g., positions 2-400 of SEQ ID NO: 11 or positions 9 to 407 of SEQ ID NO:34); and 2) the amino acid sequence of an HBV core antigen (e.g., positions 31-212 of SEQ ID NO:9 or positions 408 to 589 of SEQ ID NO:34), and utilize no N- or C-terminal sequences, or utilize different N- or C-terminal sequences and/or linkers or no linkers between HBV sequences. In one embodiment, instead of the N-terminal peptide represented by positions 1-6 of SEQ DI NO:34, an Nterminal peptide represented by SEQ ID NO:89 or SEQ ID NO:90 is utilized, followed by the remainder of the fusion protein, including or not including the hexahistidine Cterminal tag. The fusion protein may also include one, two, three, four, five, six, or more linker (spacer) amino acids between HBV proteins or domains. The same alternate embodiments apply to any fusion protein or HBV antigen construct used in the invention as described herein.
[00185] The HBV sequences used to design this fusion protein and many of the others described and/or exemplified herein are based on isolâtes of a particular HBV génotype (e.g., génotype A, B, C, or D). However, it is an embodiment of the invention to add to or substitute into any portion of an HBV antigen described herein that is based on or derived from one particular génotype, sub-genotype, or strain, a corresponding sequence, or even a single or small amino acid substitution, insertion or délétion that occurs in a corresponding sequence, from any other HBV genotype(s), sub-genotype(s), or strain(s). In one embodiment, an HBV antigen can be produced by substituting an entire sequence(s) of an
HBV antigen described herein with the corresponding sequence(s) from one or more different HBV génotypes, sub-genotypes or strain/isolates. Adding to or substituting a sequence from one HBV génotype or sub-genotype for another, for example, allows for the customization of the immunotherapeutic composition for a particular individual or population of individuals (e.g., a population of individuals within a given country or région of a country, in order to target the HBV genotype(s) that is most prévalent in that country or région of the country). Similarly, it is also an embodiment of the invention to use ail or a portion of a consensus sequence derived from, determined from, or published for, a given HBV strain, génotype or subtype to make changes in the sequence of a given HBV antigen to more closely or exactly correspond to the consensus sequence. According to the présent invention and as generally understood in the art, a “consensus sequence” is typically a sequence based on the most common nucléotide or amino acid at a particular position of a given sequence after multiple sequences are aligned.
[00186] As a particular example of the above-mentioned types of modifications, an HBV antigen can be modified to change a T cell epitope in a given sequence from one isolate to correspond more closely or exactly with a T cell epitope from a different isolate, or to correspond more closely or exactly with a consensus sequence for the T cell epitope. Such T cell epitopes can include dominant epitopes and/or sub-dominant epitopes. Indeed, according to the invention, HBV antigens can be designed that incorporate consensus sequences from a variety of HBV génotypes and/or subtypes, or mixtures of sequences from different HBV génotypes and/or subtypes. Alignments of major HBV proteins across exemplary sequences from each of the major known génotypes can be readily generated using publicly available software, which will inform the génération of consensus sequences, for example. Furthermore, consensus sequences for many HBV proteins have been published. Since there is a high degree of conservation at the amino acid level among different HBV génotypes, sub-genotypes and strains, it is straightforward to use the corresponding portions of HBV proteins from génotypes, sub-genotypes or strains other than those exemplified herein to create HBV antigens having a similar or the same overall structure as those described herein. Examples of such modifications are illustrated and exemplified herein.
[00187] By way of example, there can be minor différences among sequences of the same protein even within the same serotype and génotype (i.e., due to strain or isolate variations), although such différences in sequence identity will typically be less than 20% across the full length of the sequences being compared (i.e., the sequences will be at least
80% identical), and more typically, the sequences will be at least 85% identical, 90% identical, 91% identical, 92% identical, 93% identical, 94% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical, or 100% identical, over the full length of the compared sequences. For ex ample, in the fusion protein described above (SEQ ID NO:34), the sequence for the large (L) surface antigen used in the fusion (positions 9-407 of SEQ ID NO:34) is from an HBV génotype C isolate, and is about 99% identical to positions 2-400 of SEQ ID NO:11, which is also from large (L) surface antigen from an HBV génotype C isolate (i.e., there are two different amino acids, at positions 350-351 of SEQ ID NO: 11 (Gln-Ala) as compared to positions 357-358 of SEQ ID NO:34 (Leu-Val). However, either sequence is suitable for use in a fusion protein described herein, as are sequences from other HBV strains. Accordingly, in one embodiment, the sequences utilized in any of the HBV antigens described herein, including any of the fusion proteins described herein, can include the corresponding sequences from one or more different HBV génotypes, sub-genotypes, or strains.
[00188] The above-described utilization of consensus sequences and individual HBV génotypes has been applied to various HBV antigene described herein. For example, consensus sequence design has been applied to the fusion protein described above with reference to SEQ ID NO:34, which contains HBV surface proteins and HBV core proteins. Example 7 describes additional fusion proteins that are similar in design to the fusion protein represented by SEQ ID NO:34, but that are based on a consensus sequence for HBV génotypes A, B, C and D, respectively. A fusion protein comprising HBV surface and core proteins that is based on a consensus sequence for HBV génotype A, which is also schematically illustrated in Fig. 2, is a single polypeptide with the foilowing sequence éléments fused in frame from N- to C-tenninus, represented by SEQ ID NO: 112 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO: 112, but may be added to this sequence as in the construct described in Example 7): (1) optionally, an N-termînal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37, which may be substituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids linker sequences of one, two, three or more amino acids, such as the two amino acid linker of Thr-Ser; (3) the amino acid sequence of a near full-Iength (minus position 1) a consensus sequence for HBV génotype A large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO: 112; (4) the amino acid sequence of a consensus sequence for HBV génotype A core antigen represented by positions 400 to 581 of SEQ ID NO: 112; and (5) optionally, a hexahistidine tag. A nucleic acid sequence encoding the fusion protein of SEQ ID NO: 112 (codon optimized for yeast expression) is represented herein by SEQ ID NO:111. A yeast-based immunotherapy composition expressing this fusion protein is also referred to herein as GI-13006.
[00189] Example 7 also describes a fusion protein that is similar in design to the fusion protein represented by SEQ ID NO:34, but that is based on a consensus sequence for HBV génotype B. This fusion protein, which is also schematically illustrated in Fig. 2, is a single polypeptide with the following sequence éléments fused in frame from N- to Cterminus, represented by SEQ ID NO;114 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO:114, but may be added to this sequence as in the construct described in Example 7): (1) optionally, an N-terminai peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37, which may be substituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids linker sequences of one, two, three or more amino acids, such as the two amino acid linker of Thr-Ser; (3) the amino acid sequence of a near full-length (minus position 1) a consensus sequence for HBV génotype B large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO:114; (4) the amino acid sequence of a consensus sequence for HBV génotype B core antigen represented by positions 400 to 581 of SEQ ID NO: 114; and (5) optionally, a hexahistidine tag. A nucleic acid sequence encoding the fusion protein of SEQ ID NO: 114 (codon optimized for yeast expression) is represented herein by SEQ ID NO: 113. A yeast-based immunotherapy composition expressing this fusion protein is also referred to herein as GI-13007.
[00190] Example 7 describes a fusion protein that is similar in design to the fusion protein represented by SEQ ID NO:34, but that is based on a consensus sequence for HBV génotype C. This fusion protein, which is also schematically illustrated in Fig. 2, is a single polypeptide with the following sequence éléments fused in frame from N- to Cterminus, represented by SEQ ID NO: 116 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO: 116, but may be added to this sequence as in the construct described in Example 7): (1) optionally, an N-terminal
peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37, which may be substituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminal peptide suitable for use with a yeast-based îmmunotherapeutic as 5 described herein; (2) optionally, a linker peptide of from one to three or more amino acids linker sequences of one, two, three or more amino acids, such as the two amino acid linker of Thr-Ser; (3) the amino acid sequence of a near full-length (minus position 1) a consensus sequence for HBV génotype C large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO: 116; (4) the amino acid sequence of a consensus 10 sequence for HBV génotype C core antigen represented by positions 400 to 581 of SEQ ID NO:116; and (5) optionally, a hexahistidine tag. A nucleic acid sequence encoding the fusion protein of SEQ ID NO:116 (codon optimized for yeast expression) is represented herein by SEQ ID NO: 115. A yeast-based îmmunotherapy composition expressing this fusion protein is also referred to herein as GI-13008.
[00191] Example 7 describes a fusion protein that is similar in design to the fusion protein represented by SEQ ID NO:34, but that is based on a consensus sequence for HBV génotype D. This fusion protein, which is also schematically illustrated in Fig. 2, is a single polypeptide with the following sequence éléments fused in frame from N- to Cterminus, represented by SEQ ID NO:118 (optional sequences that are not HBV sequences 20 are not included in the base sequence of SEQ ID NO:118, but may be added to this sequence as in the construct described in Example 7): (1) optionally, an N-terminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37, which may be substituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or 25 another N-terminal peptide suitable for use with a yeast-based îmmunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids linker sequences of one, two, three or more amino acids, such as the two amino acid linker of Thr-Ser; (3) the amino acid sequence of a near full-length (minus position 1) a consensus sequence for HBV génotype D large (L) surface antigen represented by 30 positions 1 to 399 of SEQ ID NO:118; (4) the amino acid sequence of a consensus sequence for HBV génotype D core antigen represented by positions 400 to 581 of SEQ ID NO: 118; and (5) optionally, a hexahistidine tag. The amino acid sequence of a complété fusion protein described in Example 7 comprising SEQ ID NO: 118 and including the N- and C-terminal peptides and lînkers is represented herein by SEQ ID
NO:151. A nucleic acid sequence encoding the fusion protein of SEQ ID N0:118 or SEQ ID NO: 151 (codon optimized for yeast expression) is represented herein by SEQ ID NO:117. A yeast-based immunotherapy composition expressing this fusion protein is also referred to herein as GI-13009.
[00192] HBVAntigens Comprising Surface Antigen, Core Protein, Polymerase and X
Antigen. In one embodiment of the invention, the HBV antigen(s) for use in a composition or method of the invention is a fusion protein comprising HBV antigens, wherein the HBV antigens comprise or consist of: the HBV surface antigen (large (L), medium (M) or small (S)) or at least one structural, functional or immunogenic domain 10 thereof), HBV polymerase or at least one structural, functional or immunogenic domain thereof, the HBV core protein (HBcAg) or HBV e-antigen (HBeAg) or at least one structural, functional or immunogenic domain thereof, and the HBV X antigen (HBx) or at least one structural, functional or immunogenic domain thereof. In one aspect, any one or more of the HBV surface antigen, HBV polymerase, HBV core protein, HBV e-antigen, 15 HBV X antigen, or domain thereof, is full-Iength or near full-Iength. In one aspect, any one or more of the HBV surface antigen, HBV polymerase, HBV core protein, HBV eantigen, HBV X antigen, or domain thereof comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of a full-Iength HBV surface antigen, HBV polymerase, HBV core protein, HBV e-antigen, HBV X antigen, or 20 domain thereof, respectively, or of the linear amino acid sequences represented by SEQ ID
NO:97 (optimized HBV surface antigen, described below), SEQ ID NO:98 (optimized HBV polymerase, described below), SEQ ID NO:99 (optimized core protein, described below), SEQ ID NO: 100 (optimized X antigen, described below), or a corresponding sequence from another HBV strain, as applicable. In one aspect, any one or more of the 25 HBV surface antigen, HBV polymerase, HBV core protein, HBV e-antigen, HBV X antigen, or domain thereof is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a full-Iength HBV surface antigen, HBV polymerase, HBV core protein, HBV e-antigen, HBV X antigen, or domain thereof, respectively, or to the amino acid sequences represented by SEQ ID NO:97 (optimized HBV surface antigen, 30 described below), SEQ ID NO:98 (optimized HBV polymerase, described below), SEQ ID
NO:99 (optimized core protein, described below), or SEQ ID NO:100 (optimized X antigen, described below), or a corresponding sequence from another HBV strain, as applicable. A variety of suitable and exemplary sequences for HBV surface antigens, HBV polymerase antigens, HBV core antigens, and HBV X antigens are described herein.
[00193] In one embodiment of the invention, the HBV antigen(s) for use in a composition or method of the invention is a fusion protein comprising HBV antigens, wherein the HBV antigens comprise or consîst of: the hépatocyte receptor portion of PreS1 of the HBV large (L) surface antigen or at least one immunogenic domain thereof, an 5 HBV small (S) surface antigen (HBsAg) or at least one immunogenic domain thereof, the reverse transcriptase (RT) domain of HBV polymerase or at least one immunogenic domain thereof, the HBV core protein (HBcAg) or at least one immunogenic domain thereof, and the HBV X antigen (HBx) or at least one immunogenic domain thereof. In one aspect, any one or more of the hépatocyte receptor portion of Pre-Sl of the HBV large 10 (L) surface antigen, the HBV small (S) surface antigen, the RT domain of HBV polymerase, the HBV core protein, X antigen, or domain thereof, is full-length or near full-length. In one aspect, any one or more of the hépatocyte receptor portion of Pre-Sl of the HBV large (L) surface antigen, the HBV small (S) surface antigen, the RT domain of HBV polymerase, the HBV core protein, X antigen, or domain thereof, comprises at least 15 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of a full-length Pre-Sl of the HBV large (L) surface antigen, the HBV small (S) surface antigen, the RT domain of HBV polymerase, the HBV core protein, X antigen, or domain thereof, respectively. In one aspect, any one or more of the hépatocyte receptor portion of Pre-Sl of the HBV large (L) surface antigen, the HBV small (S) surface antigen, 20 the RT domain of HBV polymerase, the HBV core protein, X antigen, or domain thereof is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a full-length hépatocyte receptor portion of Pre-Sl of the HBV large (L) surface antigen, the HBV small (S) surface antigen, the RT domain of HBV polymerase, the HBV core protein, X antigen, or domain thereof, respectively.
[00194] Such a fusion protein is schematically represented in Fig. 3. An example of a composition comprising this fusion protein is described in Example 2. In this embodiment, yeast (e.g., Saccharomyces cerevisiaé) were engineered to express various HBV fusion proteins as schematically shown in Fig. 3 under the control of the copper-inducible promoter, CUP1, or the TEF2 promoter. In one case, the fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:36: (1) an N-terminal peptide to impart résistance to proteasomal dégradation and stabilize expression (e.g., positions 1 to 5 of SEQ ID
NO:36); 2) the amino acid sequence of an HBV génotype C hépatocyte receptor domain of the pre-Sl portion of HBV large (L) surface protein (unique to L) (e.g., positions 21-47 of
SEQ ID NO:11 or positions 6 to 32 of SEQ ID NO:36); 3) the amino acid sequence of a full-length HBV génotype C small (S) surface antigen (e.g., positions 176 to 400 of SEQ ID NO: 11 or positions 33 to 257 of SEQ ID NO:36); 4) a two amino acid spacer/linker to facilitate cloning and manipulation of the sequences (e.g., positions 258 and 259 of SEQ ID NO:36); 5) the amino acid sequence of a portion of the HBV génotype C polymerase including the reverse transcriptase domain (e.g., positions 247 to 691 of SEQ ID NO: 10 or positions 260 to 604 of SEQ ID NO:36); 6) an HBV génotype C core protein (e.g., positions 31-212 of SEQ ID NO:9 or positions 605 to 786 of SEQ ID NO:36); 7) the amino acid sequence of an HBV génotype C X antigen (e.g., positions 2 to 154 of SEQ ID NO: 12 or positions 787 to 939 of SEQ ID NO:36); and 8) a hexahistidine tag (e.g., positions 940 to 945 of SEQ ID NO:36). A nucleic acid sequence encoding the fusion protein of SEQ ID NO:36 (codon optimized for yeast expression) is represented herein by SEQ ID NO:35. A yeast-based immunotherapy composition expressing this fusion protein is referred to herein as GI-13005.
[00195] In one altemate example of this embodiment, a fusion protein according to the embodiment described above or that below can include 1) the amino acid sequence of an HBV génotype C hépatocyte receptor domain of the pre-Sl portion of HBV large (L) surface protein (unique to L) (e.g., positions 21-47 of SEQ ID NO:11 or positions 6 to 32 of SEQ ID NO:36); 2) the amino acid sequence of a full-length HBV génotype C small (S) surface antigen (e.g., positions 176 to 400 of SEQ ID NO: 11 or positions 33 to 257 of SEQ ID NO:36); 3) the amino acid sequence of a portion of the HBV génotype C polymerase including the reverse transcriptase domain (e.g., positions 247 to 691 of SEQ ID NO: 10 or positions 260 to 604 of SEQ ID NO:36); 4) an HBV génotype C core protein (e.g., positions 31-212 of SEQ ID NO:9 or positions 605 to 786 of SEQ ID NO:36); and 5) the amino acid sequence of an HBV génotype C X antigen (e.g., positions 2 to 154 of SEQ ID NO:12 or positions 787 to 939 of SEQ ID NO:36), and utilize no N- or C-terminal sequences, or utilize different N- or C-terminal sequences, and/or use linkers or no linkers between HBV sequences.
[00196] In one embodiment, instead of the N-terminal peptide represented by positions
1-5 of SEQ DI NO:36, an N-terminal peptide represented by SEQ ID NO:89 or SEQ ID
NO:9Û is utilized (or a homologue thereof), followed by the remainder of the fusion protein as described. Ex ample 2 describes such a fusion protein, which is also illustrated by the schematic depiction of the construct in Fig. 3. In this embodiment, yeast (e.g.,
Saccharomyces cerevisiae) were again engineered to express various HBV fusion proteins
as schematically shown in Fig. 3 under the control of the copper-indu cible promoter, CUP1, or the TEF2 promoter. In this second case, the fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:92: (1) an N-terminal peptide to impart résistance to proteasomal dégradation and stabilize or enhance expression (SEQ ID NO:89, positions 1 to 89 of SEQ ID NO:92); 2) a two amino acid spacer/linker (Thr-Ser) to facilitate cloning and manipulation of the sequences (positions 90 to 91 of SEQ ID NO:92); 3) the amino acid sequence of an HBV génotype C hépatocyte receptor domain of the pre-Sl portion of HBV large (L) surface protein (unique to L) (e.g., positions 21-47 of SEQ ID NO:11 or 10 positions 92 to 118 of SEQ ID NO:92); 4) the amino acid sequence of a full-length HBV génotype C small (S) surface antigen (e.g., positions 176 to 400 of SEQ ID NO: 11 or positions 119 to 343 of SEQ ID NO:92); 5) a two amino acid spacer/linker (Leu-Glu) to facilitate cloning and manipulation of the sequences (e.g., positions 344 to 345 of SEQ ID NO:92); 6) the amino acid sequence of a portion of the HBV génotype C polymerase 15 including the reverse transcriptase domain (e.g., positions 247 to 691 of SEQ ID NO:10 or positions 346 to 690 of SEQ ID NO:92); 7) an HBV génotype C core protein (e.g., positions 31-212 of SEQ ID NO:9 or positions 691 to 872 of SEQ ID NO:92); 8) the amino acid sequence of an HBV génotype C X antigen (e.g., positions 2 to 154 of SEQ ID NO:12 or positions 873 to 1025 of SEQ ID NO:92); and 9) a hexahistidine tag (e.g., 20 positions 1026 to 1031 of SEQ ID NO;92). A nucleic acid sequence encoding the fusion protein of SEQ ID NO:92 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO:91. A yeast-based immunotherapy composition expressing this fusion protein is referred to herein as GI-13004.
[00197] SEQ ID NO:36 and SEQ ID NO:92 contain multiple epitopes or domains that 25 are believed to enhance the immunogenicity of the fusion protein, including several described above for SEQ ID NO:34. In addition, the reverse transcriptase domain used in this fusion protein contains several amino acid positions that are known to become mutated as a drug-resistance response to treatment with various anti-viral drugs, and therefore, any one or more of these may be mutated in this fusion protein in order to 30 provide a therapeutic or prophylactic immunotherapeutic that targets spécifie drug résistance (escape) mutations. These amino acid positions are, with respect to SEQ ID NO:36, at amino acid position: 432 (Val, known to mutate to a Leu after lamivudine therapy); position 439 (Leu, known to mutate to a Met after lamivudine therapy); position 453 (Ala, known to mutate to a Thr after tenofovir therapy); position 463 (Met, known to
mutate to an Ile or Val after lamivudine therapy); and position 495 (Asn, known to mutate to Thr after adefovir therapy). These amino acid positions are, with respect to SEQ ID
NO:92, at amino acid position: 518 (Val, known to mutate to a Leu after lamivudine therapy); position 525 (Leu, known to mutate to a Met after lamivudine therapy); position
539 (Ala, known to mutate to a Thr after tenofovir therapy); position 549 (Met, known to mutate to an Ile or Val after lamivudine therapy); and position 581 (Asn, known to mutate to Thr after adefovir therapy). Additional drug résistance mutations that are identifîed or that hâve been identified can be added, as desired, to create additional immunotherapeutics targeting such mutations, using the guidance provided herein.
[00198] In one embodiment of the invention, the valine at position 901 in SEQ ID
NO:36 or the valine at position 987 of SEQ ID NO:92 (or the valine at position 116 of
SEQ ID NO: 12 or in any X antigen or domain thereof containing this corresponding position) is substituted with a leucine, to create the T cell epitope identified as SEQ ID NO:51 (see Table 5).
[00199] As discussed above, the invention includes the modification of HBV antigens from their naturally occurring or wild-type sequences for inclusion in a yeast-based immunotherapeutic that improve the clinical utility or meet required criteria for therapeutics or prophylactics related to infectious agents. By way of example, the following discussion and Examples 5-8 describe the design and construction of yeast20 based immunotherapeutics that takes into considération one or more criteria of RAC requirements, maximization of immunogenic domains associated with the most bénéficiai immune responses, maximization of conserved T cell epitopes, utilization of consensus sequences for a particular HBV génotype, and/or minimization of artificial junctions within the HBV antigen. For example, the following yeast-based immunotherapeutic composition exemplifies an HBV fusion protein meeting the requirements of the goals specified above, and comprising portions of each of the HBV major proteins: HBV surface antigen, polymerase, core and X antigen. To design this fusion protein, individual HBV antigens within the fusion were optimized or modified to reduce the size of the segments in the protein (e.g., to ensure that the protein represented less than 2/3 of the
HBV genome), as well as to maximize the inclusion of T cell epitopes that hâve been associated with an immune response in acute/self-limiting HBV infection and/or chronic
HBV infection, to maximize conserved epitopes, and to minimize non-natural sequences.
One of skill in the art using this guidance can produce altemate optimized HBV proteins for use in an HBV antigen of the invention.
[00200] As described in more detail in Example 5, to construct an HBV surface antigen segment, a full-length large (L) surface antigen protein from HBV génotype C was reduced in size by troncation of the N- and C-terminal sequences, while maximizing the inclusion of known MHC T cell epitopes, using the prioritization for inclusion of T cell 5 epitopes associated with acute/self-limiting infections. The resulting surface antigen segment is represented by SEQ ID NO:97.
[00201] To construct the segment of the fusion protein comprising HBV polymerase (see Example 5), substantial portions of a full-length polymerase from HBV génotype C were eliminated by focusing on inclusion of the active site domain (from the RT domain), 10 which is the most conserved région of the protein among HBV génotypes and isolâtes, and which includes several sites where drug résistance mutations have been known to occur.
The HBV polymerase segment was designed to maximize known T cell epitopes, using the prioritization strategy discussed above, and to modify one of the T cell epitopes to correspond exactiy to a known T cell epitope that differed by a single amino acid. The 15 resulting HBV polymerase antigen segment îs represented by SEQ ID NO:98.
[00202] To construct the segment of the fusion protein comprising HBV Core antigen (see Example 5), a full-length Core protein from HBV génotype C was modified to reduce the size of the protein while maximizing the number of T cell epitopes by inclusion and by modification of sequence to created perfect matches to certain known T cell epitopes. In 20 addition, sequence was removed that contained exceptionally positively charged Cterminus which may be toxic to yeast by compétitive interférence with natural yeast RNA binding proteins which often are arginine rich (positively charged). The resulting HBV Core antigen segment is represented by SEQ ID NO:99.
[00203] To construct the segment of the fusion protein comprising HBV X antigen (see 25 Example 5), a full-length X antigen from HBV génotype C was truncated to reduce the size of the protein, while maximizing the rétention of most of the known T cell epitopes. Single amino acid changes were also introduced to correspond to the published T cell epitope sequences, and sequence flanking the T cell epitopes at the ends of the segment was retained to facilitate efficient processing and présentation of the correct epitopes by an 30 antigen presenting cell. The resulting HBV X antigen segment is represented by SEQ ID
NQ:100.
[00204] Finally, as described in Example 5, a complété fusion protein was constructed by linking the four HBV segments described above to form a single protein optimized for clinical use. Two different exemplary fusion proteins were created, each with a different
N-terminal peptide added to enhance and/or stabilize expression of the fusion protein in yeast. As described previously herein with respect to ail of the other proteins used in a yeast-based immunotherapeutic compositions described herein, the N-terminal peptide can be replaced with a different synthetic or natural N-terminal peptide or with a homologue 5 thereof, or the N-terminal peptide can be omitted altogether and a méthionine included at position one. In addition, linker sequences of one, two, three or more amino acids may be added between segments of the fusion protein, if desired. For example, a two amino acid linker sequence such as Thr-Ser may be inserted between the N-terminal peptide and the first HBV antigen in the fusion protein, and/or between two HBV antigens in the fusion 10 protein. Also, while these constructs were designed using HBV proteins from génotype C as the backbone, any other HBV génotype, sub-genotype, or HBV proteins from different strains or isolâtes can be used to design the protein segments. In one aspect, consensus sequences from a given HBV génotype can be used to design or form the protein segments, as described in additional fusion proteins below. Finally, if one or more segments are 15 excluded from the fusion protein as described herein, then the sequence from the remaining segments can be expanded in length, if desired, to include additional T cell epitopes and/or flanking régions of the remaining proteins.
[00205] Example 5 describes an HBV fusion protein, which is also illustrated by the schematic depiction of the construct in Fig. 3, that is a single polypeptide with the 20 following sequence éléments fused in frame from N- to C-terminus, represented by SEQ
ID NO: 101: (1) an N-terminal peptide that is an alpha factor prepro sequence, to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:89 (positions 1-89 of SEQ ID NO:101); (2) an optîmized portion of an HBV large (L) surface antigen represented by SEQ ID NO:97 (positions 90 to 338 of SEQ ID NO:101, 25 e.g., corresponding to positions 120 to 368 of SEQ ID NO: 11 plus optimization of epitopes); (3) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase represented by SEQ ID NO:98 (positions 339 to 566 of SEQ ID NO: 101, e.g., corresponding to positions 453 to 680 of SEQ ID NO: 10 plus optimization of epitopes);
(4) an optimized portion of HBV Core protein represented by SEQ ID NO:99 (positions
567 to 718 of SEQ ID NO:101 e.g., corresponding to positions 37 to 188 of SEQ ID NO:9 plus optimization of epitopes); (5) an optimized portion of HBV X antigen represented by
SEQ ID NO: 100 (positions 719 to 778 of SEQ ID NO: 101, e.g., corresponding to positions 52 to 127 of SEQ ID NO: 12 plus optimization of epitopes); and (6) a hexahistidine tag (e.g., positions 779 to 784 of SEQ ID NO: 101). In one embodiment, the lmker sequence of threonine (Thr or T)-serine (Ser or S) is used between the N-terminal peptide of SEQ ID NO:89 and the first HBV protein (optimized portion of HBV large surface antigen), thereby extending the total length of SEQ ID NO: 101 by two amino acids. [00206] Example 5 also describes a fusion protein, which is also illustrated by the schematic depiction of the construct in Fig. 3, that is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO: 102: (1) an N-terminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37 (positions 1-6 of SEQ ID NO:102); (2) an optimized portion of an HBV large (L) surface antigen represented by positions 2 to 248 of SEQ ID NO:97 (positions 7 to 254 of SEQ ID NO: 102, e.g., corresponding to positions 120 to 368 of SEQ ID NO: 11 plus optimization of epitopes); (3) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase represented by SEQ ID NO:98 (positions 255 to 482 of SEQ ID NO: 102, e.g., corresponding to positions 453 to 680 of SEQ ID NO:10 plus optimization of epitopes); (4) an optimized portion of HBV Core protein represented by SEQ ID NO:99 (positions 483 to 634 of SEQ ID NO:102, e.g., corresponding to positions 37 to 188 of SEQ ID NO:9 plus optimization of epitopes); (5) an optimized portion of HBV X antigen represented by SEQ ID NO:100 (positions 635 to 694 of SEQ ID NO:102, e.g., corresponding to positions 52 to 127 of SEQ ID NO: 12 plus optimization of epitopes); and (6) a hexahistidine tag (e.g., positions 695 to 700 of SEQ ID NO: 102). In one embodiment, the linker sequence of threonine (Thr or T)-serine (Ser or S) is used between the N-terminal peptide of SEQ ID NO:37 and the first HBV protein (optimized portion of HBV large surface antigen), thereby extending the total length of SEQ ID NO: 102 by two amino acids. In one embodiment, an optimized portion of an HBV large (L) surface antigen used in the fusion protein described above is represented by positions 1 to 248 of SEQ ID NO;97 (thereby extending the total length of SEQ ID NO: 102 by one amino acid). In one embodiment both the T-S linker and positions 1-248 of SEQ ID NO:97 are used in SEQ ID NO: 102.
[00207] As dîscussed above, the invention includes the modification of HBV antigens from their naturally occurring or wild-type sequences for inclusion in a yeast-based immunotherapeutic that improve the clinical utility or meet required criteria for therapeutics or prophylactics related to infectious agents, utilizing consensus sequences from a given HBV génotype to design or form the protein segments. By way of example, additional HBV antigens for use in a yeast-based immunotherapeutic of the invention were designed to illustrate this type of modification. As in the design of the HBV fusion proteins represented described above, to produce these additional fusion proteins, individual HBV antigens within the fusion were optimized or modified to reduce the size of the segments in the protein (e.g., to ensure that the protein represented less than 2/3 of the HBV genome), as well as to maximize the inclusion of T cell epitopes that have been associated with an immune response in acute/self-limiting HBV infection and/or chronic HBV infection, to maximize conserved epitopes, to minimize non-natural sequences, and also to utilize consensus sequences for each of génotype A-D that were built from multiple sources of HBV sequences (e.g., Yu and Yuan et al, 2010, for S, Core and X, where consensus sequences were generated from 322 HBV sequences, or for Pol (RT), from the Stanford Universily HIV Drug Résistance Database, HBVseq and HBV Site Release Notes). In desîgning the following four exemplary fusion proteins comprising HBV antigens, the consensus sequence for the given HBV génotype was used unless using the consensus sequence altered one of the known acute self-limiting T cells epitopes or one of the known polymerase escape mutation sites, in which case, these positions followed the published sequence for these epitopes or mutation sites. Additional antigens could be constructed based solely on consensus sequences or using other published epitopes as they become known.
[00208] Example 7 describes a fusion protein that is similar in design to the fusion protein represented by SEQ ID NO:101 or SEQ ID NO:102 (illustrated schematically by Fig. 3), but that is based on a consensus sequence for HBV génotype A. This fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO: 107 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO:107, but may be added to this sequence as in the construct described in Example 7): (1) optionally, an N-terminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37, which may be substituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids linker sequences of one, two, three or more amino acids, such as the two amino acid linker of Thr-Ser; (3) an optimized portion of an HBV large (L) surface antigen represented by positions 1 to 249 of SEQ ID NO: 107, which is a consensus sequence for HBV génotype A utilizing the design strategy discussed above; (4) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase represented by positions 250 to 477 of SEQ ID NO: 107, which is a consensus sequence for HBV génotype A utilizing the design strategy discussed above; (5) an optimized portion of HBV Core protein represented by positions 478 to 629 of SEQ ID NO: 107, which is a consensus sequence for HBV génotype A utilizing the design strategy discussed above; (6) an optimized portion of HBV X antigen represented by positions 630 to 689 of SEQ ID NO: 107, which is a consensus sequence for HBV génotype A utilizing the design strategy discussed above; and (7) optionally, a hexahistidine tag. A yeast-based immunotherapy composition expressing this fusion protein is also referred to herein as GI-13010.
[00209] Example 7 also describes a fusion protein that is similar in design to the fusion protein represented by SEQ ID NO:101 or SEQ ID NO:102 (illustrated schematically by Fig. 3), but that is based on a consensus sequence for HBV génotype B. This fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:108 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO: 108, but may be added to this sequence as in the construct described in Example 7): (1) optionally, an N-terminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37, which may be substituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids linker sequences of one, two, three or more amino acids, such as the two amino acid linker of Thr-Ser; (3) an optimized portion of an HBV large (L) surface antigen represented by positions 1 to 249 of SEQ ID NO: 108, which is a consensus sequence for HBV génotype B utilizing the design strategy discussed above; (4) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase represented by positions 250 to 477 of SEQ ID NO:108, which is a consensus sequence for HBV génotype B utilizing the design strategy discussed above; (5) an optimized portion of HBV Core protein represented by positions 478 to 629 of SEQ ID NO: 108, which is a consensus sequence for HBV génotype B utilizing the design strategy discussed above; (6) an optimized portion of HBV X antigen represented by positions 630 to 689 of SEQ ID NO:108, which is a consensus sequence for HBV génotype B utilizing the design strategy discussed above; and (7) optionally, a hexahistidine tag. A yeast-based immunotherapy composition expressing this fusion protein is also referred to herein as GI-13011.
[00210] Example 7 also describes a fusion protein that is similar in design to the fusion protein represented by SEQ ID NO: 101 or SEQ ID NO: 102 (illustrated schematically by Fig. 3), but that is based on a consensus sequence for HBV génotype C. This fusion protein is a single polypeptide with the foilowing sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO: 109 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO:109, but may be added to this sequence as in the construct described in Example 7): (1) optionally, an N-terminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37, which may be substituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids linker sequences of one, two, three or more amino acids, such as the two amino acid linker of Thr-Ser; (3) an optimized portion of an HBV large (L) surface antigen represented by positions 1 to 249 of SEQ ID NO: 109, which is a consensus sequence for HBV génotype C utilizing the design strategy discussed above; (4) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase represented by positions 250 to 477 of SEQ ID NO: 109, which is a consensus sequence for HBV génotype C utilizing the design strategy discussed above; (5) an optimized portion of HBV Core protein represented by positions 478 to 629 of SEQ ID NO: 109, which is a consensus sequence for HBV génotype C utilizing the design strategy discussed above; (6) an optimized portion of HBV X antigen represented by positions 630 to 689 of SEQ ID NO: 109, which is a consensus sequence for HBV génotype C utilizing the design strategy discussed above; and (7) optionally, a hexahistidine tag. A yeast-based immunotherapy composition expressing this fusion protein is also referred to herein as GI-13012.
[00211] Example 7 also describes a fusion protein that is similar in design to the fusion protein represented by SEQ ID NO:101 or SEQ ID NO:102 (illustrated schematically by Fig. 3), but that is based on a consensus sequence for HBV génotype D. This fusion protein is a single polypeptide with the foilowing sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO: 110 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO: 110, but may be added to this sequence as in the construct described in Example 7): (1) optionally, an N-terminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37, which may be
substituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids linker sequences of one, two, three or more amino acids, such as the two amino acid linker 5 of Thr-Ser; (3) an optimized portion of an HBV large (L) surface antigen represented by positions 1 to 249 of SEQ ID NO:110, which is a consensus sequence for HBV génotype D utilizing the design strategy discussed above; (4) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase represented by positions 250 to 477 of SEQ ID NO: 110, which is a consensus sequence for HBV génotype D utilizing the design 10 strategy discussed above; (5) an optimized portion of HBV Core protein represented by positions 478 to 629 of SEQ ID NO: 110, which is a consensus sequence for HBV génotype D utilizing the design strategy discussed above; (6) an optimized portion of HBV X antigen represented by positions 630 to 689 of SEQ ID NO: 110, which is a consensus sequence for HBV génotype D utilizing the design strategy discussed above; and (7) 15 optionally, a hexahistidine tag. A yeast-based immunotherapy composition expressing this fusion protein which comprises an N-terminal sequence represented by SEQ ID NO:37 is referred to herein as GI-13013. A yeast-based immunotherapy composition expressing this fusion protein which comprises an N-terminal sequence represented by SEQ ID NO:89 is referred to herein as GI-13014.
[00212] As discussed above, it is one embodiment of the invention to change the order of HBV protein segments within a fusion protein described herein. Accordingly, although the constructs utilizing four HBV proteins as described above are provided in the order of a surface antigen fused to a polymerase antigen fused to a Core antigen fused to an X antigen, the invention is not limited to this particular order of proteins within the construct, 25 and indeed, other arrangements of fusion segments may be used and in some aspects, may improve the resulting immunotherapeutic compositions. For example, rearrangement of segments within a fusion protein may improve or modify expression of the HBV antigen in yeast, or may improve or modify the immunogenicity or other functional attribute of the HBV antigen. In one aspect of this embodiment, the invention contemplâtes beginning 30 with one HBV antigen that expresses well in yeast and/or provides positive functional data (e.g., is immunogenic), and adding additional HBV proteins or domains to that HBV antigen in order to expand the potential antigens or epitopes that are contained within the HBV antigen. Example 8 provides an example of additional arrangements of the four HBV proteins described above.
[00213] Example 8 describes a fusion protein that contains sequences from HBV surface antigen, core protein, polymerase and X antigen, where the sequences were derived from segments of the fusion proteins represented by SEQ ID NO: 110 and SEQ ID NO: 118, and where the fusion protein utilizes a different order of fusion segments as compared to SEQ ID NO: 110. This antigen is based on a consensus sequence for HBV génotype D; however, it would be straightforward to produce a fusion protein having a similar overall structure using the corresponding fusion segments from the fusion proteins represented by SEQ ID ΝΟ.Ί07 or SEQ ID NO.112 (génotype A), SEQ ID ΝΟ.Ί08 or SEQ ID NO:114 (génotype B), SEQ ID NO:109 or SEQ ID NO:116 (génotype C), or using the corresponding sequences from a different HBV génotype, sub-genotype, consensus sequence or strain. In this example, yeast (e.g., Saccharomyces cerevisiae) were engineered to express this fusion protein under the control of the copper-inducible promoter, CUP1, and the resulting yeast-HBV îmmunotherapy composition can be referred to herein as GI-13017, schematically illustrated in Fig. 10. The fusion protein represented by SEQ ID NO: 124 comprises, in order, surface antigen, core, polymerase and X antigen sequences, as a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:124 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO: 124, but may be added to this sequence as in the construct described in Example 8): (1) optionally, an N-terminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37 (in the construct described in Example 8), which may be substituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminal peptide suitable for use with a yeast-based îmmunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids, such as the two amino acid linker of Thr-Ser (in the construct described in Example 8); (3) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV génotype D large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO:124 (corresponding to positions 1 to 399 of SEQ ID NO: 118); 4) the amino acid sequence of a consensus sequence for HBV génotype D core antigen represented by positions 400 to 581 of SEQ ID NO:124 (corresponding to positions 400 to 581 of SEQ ID NO:118); (5) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase using a consensus sequence for HBV génotype D, represented by positions 582 to 809 of SEQ ID NO: 124 (corresponding to positions to 250 to 477 of SEQ ID NO:110); (6) an optimized portion of HBV X antigen using a consensus sequence for HBV génotype D, represented by positions 810 to 869 of SEQ ID NO:124 (corresponding to positions 630 to 689 of SEQ ID NO:110); and (7) optionally, a hexahistidine tag (in the construct described in Example 8). SEQ ID NO: 124 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein of SEQ ID NO:124 (codonoptimized for expression in yeast) is represented herein by SEQ ID NO: 123.
[00214] Example 8 also describes another fusion protein that contains sequences from HBV surface antigen, core protein, X antigen, and polymerase, where the sequences were derived from segments of the fusion proteins represented by SEQ ID NO: 110 and SEQ ID NO: 118, but where the fusion protein utilizes a different order of fusion segments as compared to SEQ ID NO: 110. This antigen is also based on a consensus sequence for HBV génotype D; however, it would be straightforward to produce a fusion protein having a similar overall structure using the corresponding fusion segments from the fusion proteine represented by SEQ ID NO:107 or SEQ ID NO:112 (génotype A), SEQ ID NO:108 or SEQ ID NO:114 (génotype B), SEQ ID NO:109 or SEQ ID NO:116 (génotype C), or using the corresponding sequences from a different HBV génotype, sub-genotype, consensus sequence or strain. In this example, yeast (e.g., Saccharomyces cerevisiaé) were engîneered to express this fusion protein under the control of the copper-inducible promoter, CUP1, and the resulting yeast-HBV immunotherapy composition can be referred to herein as GI-13018, schematically illustrated in Fig. 11. The fusion protein represented by SEQ ID NO:126 comprises, in order, surface antigen, core, X antigen, and polymerase sequences, as a single polypeptide with the following sequence éléments fused in frame from N- to C-termînus, represented by SEQ ID NO: 126 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO: 126, but may be added to this sequence as in the construct described in Example 8): (1) optionally, an N-terminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37 (in the construct described in Example 8), which may be substituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids, such as the two amino acid linker of Thr-Ser (in the construct described in Example 8); (3) the amino acid sequence of a near full-Iength (minus position 1) consensus sequence for HBV génotype D large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO: 126 (corresponding to positions 1 to 399 of SEQ ID NO:118); 4) the amino acid sequence of a consensus sequence for HBV génotype D core antigen represented by positions 400 to 581 of SEQ ID NO:126 (corresponding to positions 400 to 581 of SEQ ID NO: 118); (5) an optimized portion of HBV X antigen using a consensus sequence for HBV génotype D, represented by positions 582 to 641 of SEQ ID NO:126 (corresponding to positions 630 to 689 of SEQ ID NO: 110); (5) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase using a consensus sequence for HBV génotype D, represented by positions 642 to 869 of SEQ ID NO: 126 (corresponding to positions to 250 to 477 of SEQ ID NQ:110); and (7) optionally, a hexahistidine tag (in the construct described in Example 8). SEQ ID NO: 126 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein of SEQ ID ΝΟ.Ί26 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO: 125.
[00215] Example 8 describes another fusion protein that contains sequences from HBV polymerase, X antigen, surface antigen, core protein, where the sequences were derived from segments of the fusion proteins represented by SEQ ID NO:110 and SEQ ID NO:118, but where the fusion protein utilizes a different order of fusion segments as compared to SEQ ID NO:110. This antigen is based on a consensus sequence for HBV génotype D; however, it would be straightforward to produce a fusion protein having a similar overall structure using the corresponding fusion segments from the fusion proteins represented by SEQ ID NO:107 or SEQ ID NO:112 (génotype A), SEQ ID NO:108 or SEQ ID NO:114 (génotype B), SEQ ID NO: 109 or SEQ ID NO: 116 (génotype C), or using the corresponding sequences from a different HBV génotype, sub-genotype, consensus sequence or strain. In this example, yeast (e.g., Saccharomyces cerevisiaé) were engineered to express this fusion protein under the control of the copper-inducible promoter, CÜP1, and the resulting yeast-HBV immunotherapy composition can be referred to herein as GI-13021, schematically illustrated in Fig. 14. The fusion protein represented by SEQ ID NO:132 comprises, in order, polymerase, X antigen, surface antigen, and core, as a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO: 132 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO: 132, but may be added to this sequence as in the construct described in Example 8): (1) optionally, an Nterminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37 (in the construct described in Example 8), which may be substituted by an N-terminal peptide
represented by SEQ ID NO:89, SEQ ID NO:90, or another N-termînal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids, such as the two amino acid linker of Thr-Ser (in the construct described in Example 8); (3) an optimîzed portion of the reverse 5 transcriptase (RT) domain of HBV polymerase using a consensus sequence for HBV génotype D, represented by positions 1 to 228 of SEQ ID NO: 132 (corresponding to positions to 250 to 477 of SEQ ID NO:110); (4) an optimized portion of HBV X antigen using a consensus sequence for HBV génotype D, represented by positions 229 to 288 of SEQ ID NO: 132 (corresponding to positions 630 to 689 of SEQ ID NO: 110); (5) the 10 amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV génotype D large (L) surface antigen represented by positions 289 to 687 of SEQ ID NO:132 (corresponding to positions 1 to 399 of SEQ ID NO:118); (6) the amino acid sequence of a consensus sequence for HBV génotype D core antigen represented by positions 688 to 869 of SEQ ID NO:132 (corresponding to positions 400 to 581 of SEQ ID 15 NO:118); and (7) optionally, a hexahistidine tag (in the construct described in Example 8).
SEQ ID NO:132 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein of SEQ ID NO:132 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO:131.
[00216] Example 8 also describes a fusion protein that contains sequences from HBV 20 X antigen, polymerase, surface antigen, and core protein, where the sequences were derived from segments of the fusion proteine represented by SEQ ID NO:110 and SEQ ID NO: 118, but where the fusion protein utilizes a different order of fusion segments as compared to SEQ ID NO:110. This antigen is based on a consensus sequence for HBV génotype D; however, it would be straightforward to produce a fusion protein having a 25 similar overall structure using the corresponding fusion segments from the fusion pro te ins represented by SEQ ID NO:107 or SEQ ID NO:112 (génotype A), SEQ ID NO:108 or SEQ ID NO.114 (génotype B), SEQ ID NO:109 or SEQ ID NO:116 (génotype C), or using the corresponding sequences from a different HBV génotype, sub-genotype, consensus sequence or strain. In this example, yeast (e.g., Saccharomyces cerevisiae) 30 were engineered to express this fusion protein under the control of the copper-inducible promoter, CUP1, and the resulting yeast-HBV immunotherapy composition can be referred to herein as GI-13022, schematically illustrated in Fig. 15. The fusion protein represented by SEQ ID NO:134 comprises, in order, X antigen, polymerase, surface antigen, and core protein, as a single polypeptide with the foliowing sequence éléments
fused in frame from N- to C-terminus, represented by SEQ ID NO: 134 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO:134, but may be added to this sequence as in the construct described in Example 8): (1) optionally, an N-terminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ
ID NO:37 (in the construct described in Example 8), which may be substituted by an Nterminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids, such as the two 10 amino acid linker of Thr-Ser (in the construct described in Example 8); (3) an optimized portion of HBV X antigen using a consensus sequence for HBV génotype D, represented by positions 1 to 60 of SEQ ID NO: 134 (corresponding to positions 630 to 689 of SEQ ID NO:110); (4) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase using a consensus sequence for HBV génotype D, represented by positions 61 15 to 288 of SEQ ID NO: 134 (corresponding to positions to 250 to 477 of SEQ ID NO: 110);
(5) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV génotype D large (L) surface antigen represented by positions 289 to 687 of SEQ
ID NO: 134 (corresponding to positions 1 to 399 of SEQ ID NO: 118); (6) the amino acid sequence of a consensus sequence for HBV génotype D core antigen represented by 20 positions 688 to 869 of SEQ ID NO: 134 (corresponding to positions 400 to 581 of SEQ ID
NO:118); and (7) optionally, a hexahistidine tag (in the construct described in Example 8). SEQ ID NO: 134 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein of SEQ ID NO: 134 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO: 133.
[00217] HBV Antigens Comprising Surface Antigen, Core Protein and X Antigen. In one embodiment of the invention, the HBV antigen(s) for use in a composition or method of the invention is a fusion protein comprising HBV antigens, wherein the HBV antigens comprise or consist of: the HBV surface antigen (large (L), medium (M) or small (S)) or at least one structural, functional or immunogenic domain thereof), the HBV core protein 30 (HBcAg) or HBV e-antigen (HBeAg) or at least one structural, functional or immunogenic domain thereof, and the HBV X antigen (HBx) or at least one structural, functional or immunogenic domain thereof. In one aspect, any one or more of the HBV surface antigen, HBV core protein, HBV e-antigen, HBV X antigen, or domain thereof, is full-length or near full-length. In one aspect, any one or more of the HBV surface antigen, HBV core protein, HBV e-antigen, HBV X antigen, or domain thereof comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of a full-length HBV surface antigen, HBV core protein, HBV e-antigen, HBV X antigen, or domain thereof, respectively, or of the amino acid sequences represented by SEQ ID NO:97 (optimized HBV surface antigen), SEQ ID NO:99 (optimized core protein), SEQ ID NO: 100 (optimized X antigen), or a corresponding sequence from another HBV strain, as applicable. In one aspect, any one or more of the HBV surface antigen, HBV core protein, HBV e-antigen, HBV X antigen, or domain thereof is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a full-length HBV surface antigen, HBV core protein, HBV e-antigen, HBV X antigen, or domain thereof, respectively, or to the amino acid sequences represented by SEQ ID NO:97 (optimized HBV surface antigen), SEQ ID NO:99 (optimized core protein), SEQ ID NO: 100 (optimized X antigen), or a corresponding sequence from another HBV strain, as applicable. A variety of suitable and exemplary sequences for additional HBV surface antigens, HBV core antigens, and HBV X antigens useful in this construct are described herein.
[00218] Example 8 describes a fusion protein that contains sequences from HBV surface antigen, core protein, and X antigen, where the sequences were derived from segments of the fusion proteins represented by SEQ ID NO: 110 and SEQ ID NO: 118. This antigen is based on a consensus sequence for HBV génotype D; however, it would be straightforward to produce a fusion protein having a similar overail structure using the corresponding fusion segments from the fusion proteins represented by SEQ ID NO: 107 or SEQ ID NO:112 (génotype A), SEQ ID NO:108 or SEQ ID NO:114 (génotype B), SEQ ID NO: 109 or SEQ ID NO: 116 (génotype C), or using the corresponding sequences from a different HBV génotype, sub-genotype, consensus sequence or strain. In this example, yeast (e.g., Saccharomyces cerevisiae) were engineered to express this fusion protein under the control of the copper-inducible promoter, CUP1, and the resulting yeast-HBV immunotherapy composition can be referred to herein as GI-13016, schematically illustrated in Fig. 9. The fusion protein represented by SEQ ID NO: 122 comprises, in order, surface antigen, core, and X antigen sequences, as a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO: 122 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO:122, but may be added to this sequence as in the construct described in Example 8): (1) optionally, an N-terminal peptide that is a synthetic N16530
terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37 (in the construct described in Example 8), which may be substituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminal peptide suitable for use with a yeast-based 5 immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids, such as the two amino acid linker of Thr-Ser (in the construct described in Example 8); (3) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV génotype D large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO: 122 (corresponding to positions 1 to 399 of SEQ ID 10 NO:118); 4) the amino acid sequence of a consensus sequence for HBV génotype D core antigen represented by positions 400 to 581 of SEQ ID NO: 122 (corresponding to positions 400 to 581 of SEQ ID NO: 118); (5) an optimized portion of HBV X antigen using a consensus sequence for HBV génotype D, represented by positions 582 to 641 of SEQ ID NO:122 (corresponding to positions 630 to 689 of SEQ ID NO:110); and (6) 15 optionally, a hexahistidine tag (in the construct described in Example 8). SEQ ID NO: 122 eontains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encodîng the fusion protein of SEQ ID NO: 122 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO: 121.
[00219] Example 8 also describes a fusion protein that eontains sequences from HBV 20 surface antigen, core protein, and X antigen, where, as in the fusion protein comprising
SEQ ID NO:122, the sequences were derived from segments of the fusion proteins represented by SEQ ID NO: 110 and SEQ ID NO: 118. This fusion protein differs from the fusion protein comprising SEQ ID NO: 122, however, in the arrangement of the fusion segments within the fusion protein. This antigen is based on a consensus sequence for 25 HBV génotype D; however, it would be straightforward to produce a fusion protein having a similar overall structure using the corresponding fusion segments from the fusion proteins represented by SEQ ID NO: 107 or SEQ ID NO: 112 (génotype A), SEQ ID NO:108 or SEQ ID NO:114 (génotype B), SEQ ID NO:109 or SEQ ID NO:116 (génotype C), or using the corresponding sequences from a different HBV génotype, sub-genotype, 30 consensus sequence or strain. In this example, yeast (e.g., Saccharomyces cerevisiae) were engineered to express this fusion protein under the control of the copper-inducible promoter, CUP1, and the resulting yeast-HBV immunotherapy composition can be referred lo herein as GI-13020, schematically illustrated in Fig. 13. The fusion protein represented by SEQ ID NO: 130 comprises, in order, X antigen, surface antigen, and core
antigen sequences, as a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:130 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO: 130, with the exception of the Leu-Glu linker between the X antigen segment and the surface antigen segment in the construct exemplified here, but may be added to this sequence as in the construct described in Example 8): (1) optionally, an N-terminal peptide that is a synthetic
N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37 (in the construct described in Example 8), which may be substituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ 10 ID NO:90, or another N-terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids, such as the two amino acid linker of Thr-Ser (in the construct described in Example 8); (3) an optimized portion of HBV X antigen using a consensus sequence for HBV génotype D, represented by positions 1 to 60 of SEQ ID NO: 130 15 (corresponding to positions 630 to 689 of SEQ ID NO: 110); (4) optionally, a linker peptide of from one to three or more amino acids, such as the two amino acid linker of Leu-Glu (in the construct described in Example 8), represented by positions 61 to 62 of
SEQ ID NO: 130; (5) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV génotype D large (L) surface antigen represented by 20 positions 63 to 461 of SEQ ID NO: 130 (corresponding to positions 1 to 399 of SEQ ID
NO: 118); (6) the amino acid sequence of a consensus sequence for HBV génotype D core antigen represented by positions 462 to 643 of SEQ ID NO: 130 (corresponding to positions 400 to 581 of SEQ ID NO:118); and (7) optionally, a hexahistidine tag (in the construct described in Example 8). SEQ ID NO: 130 contains multiple T cell epitopes 25 (human and murine), which can be found in Table 5. The amino acid sequence of the complété fusion protein described in Example 8 comprising SEQ ID NO: 130 and including the N- and C-terminal peptides and ail linkers is represented herein by SEQ ID
NO: 150. A nucleic acid sequence encoding the fusion protein of SEQ ID NO: 130 or SEQ
ID NO: 150 (codon-optimized for expression in yeast) is represented herein by SEQ ID 30 NO:129.
[00220] HBV Antigens Comprising Surface Antigen, Core Protein and Polymerase. In one embodiment of the invention, the HBV antigen(s) for use in a composition or method of the invention is a fusion protein comprising HBV antigens, wherein the HBV antigens comprise or consist of: the HBV surface antigen (large (L), medium (M) or small (S)) or at least one structural, functional or immunogenic domain thereof), the HBV core protein (HBcAg) or HBV e-antigen (HBeAg) or at least one structural, functional or immunogenic domain thereof, and the HBV polymerase or at least one structural, functional or immunogenic domain thereof (e.g., the reverse transcriptase (RT) domain). In one aspect, any one or more of the HBV surface antigen, HBV core protein, HBV eantigen, HBV polymerase, or domain thereof, is full-length or near full-length. In one aspect, any one or more of the HBV surface antigen, HBV core protein, HBV e-antigen, HBV polymerase, or domain thereof comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of a full-length HBV surface antigen, HBV core protein, HBV e-antigen, HBV polymerase, or domain thereof, respectively, or of the amino acid sequences represented by SEQ ID NO:97 (optimized HBV surface antigen), SEQ ID NO:99 (optimized core protein), SEQ ID NO:98 (optimized polymerase), or a corresponding sequence from another HBV strain, as applicable. In one aspect, any one or more of the HBV surface antigen, HBV core protein, HBV e-antigen, HBV polymerase, or domain thereof is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a full-length HBV surface antigen, HBV core protein, HBV e-antigen, HBV polymerase, or domain thereof, respectively, or to the amino acid sequences represented by SEQ ID NO:97 (optimized HBV surface antigen), SEQ ID NO:99 (optimized core protein), SEQ ID NO:98 (optimized polymerase), or a corresponding sequence from another HBV strain, as applicable. A variety of suitable and exemplary sequences for HBV surface antigens, HBV polymerase antigens, and HBV core antigens are described herein.
[00221] One example of such a fusion protein is schematically represented in Fig. 7. An example of a composition comprising this fusion protein is described in Example 3. In this embodiment, yeast (e.g., Saccharomyces cerevisiae) are engineered to express various HBV surface-polymerase-core fusion proteins under the control of the copper-inducible promoter, CUP1, or the TEF2 promoter. In each case, the fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:41: (1) an N-terminal peptide to impart résistance to proteasomal dégradation and stabiiize expression (e.g., positions 1 to 5 of SEQ ID NO:41); 2) an amino acid sequence of the amino HBV hépatocyte receptor domain of the pre-Sl portion of HBV large (L) surface protein (unique to L) (e.g., positions 21-47 of SEQ ID NO: 11 or positions 6 to 32 of SEQ ID NO:41); 3) the amino acid sequence of an HBV small (S) surface protein (e.g., positions 176 to 400 of SEQ ID NO:11 or positions
to 257 of SEQ ID NO:41); 4) a two amino acid spacer/Iinker to facilitate cloning and manipulation of the sequences (e.g., positions 258 and 259 of SEQ ID N0:41); 5) the amino acid sequence of an HBV polymerase comprising the reverse transcriptase domain (e.g., positions 247 to 691 of SEQ ID NO: 10 or positions 260 to 604 of SEQ ID NO:41);
6) the amino acid sequence of an HBV core protein (e.g., positions 31-212 of SEQ ID
NO:9 or positions 605 to 786 of SEQ ID NO:41); and 7) a hexahistidine tag (e.g., positions 787 to 792 of SEQ ID NO:41). The sequence also contains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. In addition, in one embodiment, the sequence of this construct can be modifîed to introduce one or more or ail of the following anti-viral résistance mutations: rtM2041, rtL180M, rtM204V, rtV173L, rtN236T, rtA194T (positions given with respect to the full-length amino acid sequence for HBV polymerase). In one embodiment, six different immunotherapy compositions are created, each one containing one of these mutations. In other embodiments, ail or some of the mutations are included in a single fusion protein. In one embodiment, this construct also contains one or more anti-viral résistance mutations in the surface antigen. The amino acid segments used in any of the fusion proteins described herein can be modifîed by the use of additional amino acids flanking either end of any domain; the examples provided herein are exemplary. For example, a fusion protein according to this embodiment can include 1) an amino acid sequence of the amino HBV hépatocyte receptor domain of the pre-Sl portion of HBV large (L) surface protein (unique to L) (e.g., positions 21-47 of SEQ ID NO:11 or positions 6 to 32 of SEQ ID NO:41); 2) the amino acid sequence of an HBV small (S) surface protein (e.g., positions 176 to 400 of SEQ ID NO: 11 or positions 33 to 257 of SEQ ID NO:41); 3) the amino acid sequence of an HBV polymerase comprising the reverse transcriptase domain (e.g., positions 247 to 691 of SEQ
ID NO: 10 or positions 260 to 604 of SEQ ID NO:41); and 4) the amino acid sequence of an HBV core protein (e.g., positions 31-212 of SEQ ID NO:9 or positions 605 to 786 of SEQ ID NO:41), and utilize no N- or C-terminal sequences, or utilîze different N- or Cterminal sequences, and/or use lînkers or no linkers between HBV sequences. In one embodiment, instead of the N-terminal peptide represented by positions 1-5 of SEQ ID
NO:41, an N-terminal peptide represented by SEQ ID NO:89 or SEQ ID NO:90 is utilized, followed by the remainder of the fusion protein as described.
[00222] Another example of such a fusion protein is described in Example 8. Example exemplifies a fusion protein that contains sequences from HBV surface antigen, core protein, and polymerase where the sequences were derived from segments of the fusion
proteins represented by SEQ ID NO:110 and SEQ ID NO:118. This antigen is based on a consensus sequence for HBV génotype D; however, it would be straightforward to produce a fusion protein having a similar overall structure using the correspondîng fusion segments from the fusion proteins represented by SEQ ID NO:107 or SEQ ID NO:112 5 (génotype A), SEQ ID NO:108 or SEQ ID N0:114 (génotype B), SEQ ID NO:109 or SEQ
ID NO: 116 (génotype C), or using the correspondîng sequences from a different HBV génotype, sub-genotype, consensus sequence or strain. In this example, yeast (e.g., Saccharomyces cerevisiae) were engineered to express this fusion protein under the control of the copper-inducible promoter, CUP1, and the resulting yeast-HBV immunotherapy composition can be referred to herein as GI-13015, schematically illustrated in Fig. 8. The fusion protein represented by SEQ ID NO: 120 comprises, in order, surface antigen, core protein, and polymerase sequences, as a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO: 120 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO: 120, but may be added to this sequence as in the construct described in Example 8): (1) optionally, an N-terminal peptide that is a synthetic N-termînal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37 (in the construct described in Example 8), which may be substituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ
ID NO:90, or another N-terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids, such as the two amino acid linker of Thr-Ser (in the construct described in Example 8); (3) the amino acid sequence of a near full-length (minus position
1) consensus sequence for HBV génotype D large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO:120 (correspondîng to positions 1 to 399 of SEQ ID
NO: 118); (4) the amino acid sequence of a consensus sequence for HBV génotype D core antigen represented by positions 400 to 581 of SEQ ID NO:120 (correspondîng to positions 400 to 581 of SEQ ID NO: 118); (5) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase using a consensus sequence for HBV 30 génotype D, represented by positions 582 to 809 of SEQ ID NO: 120 (correspondîng to positions to 250 to 477 of SEQ ID NO: 110); and (6) optionally, a hexahistidine tag (in the construct described in Example 8). SEQ ID NO: 120 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding
the fusion protein of SEQ ID NO: 120 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO: 119.
[002231 Yet another example of such a fusion protein is described in Example 8. Example 8 exemplifies a fusion protein that contains sequences from HBV polymerase, 5 surface antigen, and core protein, where the sequences were derived from segments of the fusion proteins represented by SEQ ID NO:110 and SEQ ID NO:118. This fusion protein differs from the fusion protein comprising SEQ ID NO: 120 in the anangement of the fusion segments within the fusion protein, This antigen is based on a consensus sequence for HBV génotype D; however, it would be straightforward to produce a fusion protein 10 having a similar overall structure using the corresponding fusion segments from the fusion proteins represented by SEQ ID NO: 107 or SEQ ID NO: 112 (génotype A), SEQ ID NO:108 or SEQ ID NO:114 (génotype B), SEQ ID NO.109 or SEQ ID NO:116 (génotype C), or using the corresponding sequences from a different HBV génotype, sub-genotype, consensus sequence or strain. In this example, yeast (e.g., Saccharomyces cerevisiaé) 15 were engineered to express this fusion protein under the control of the copper-inducible promoter, CUP1, and the resulting yeast-HBV immunotherapy composition can be referred to herein as GI-13019, schematically illustrated in Fig. 12. The fusion protein represented by SEQ ID NO: 128 comprises, in order, polymerase, surface antigen, and core sequences, as a single polypeptide with the following sequence éléments fused in frame 20 from N- to C-terminus, represented by SEQ ID NO:128 (optional sequences that are not
HBV sequences are not included in the base sequence of SEQ ID NO: 128, with the exception of the Leu-Glu linker between the polymerase segment and the surface antigen segment in the construct exemplified here, but may be added to this sequence as in the construct described in Example 8): (1) optionally, an N-terminal peptide that is a synthetic 25 N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37 (in the construct described in Example 8), which may be substituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ ID NO;90, or another N-terminai peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to 30 three or more amino acids, such as the two amino acid linker of Thr-Ser (in the construct described in Example 8); (3) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase using a consensus sequence for HBV génotype D, represented by positions 1 to 228 of SEQ ID NO: 128 (corresponding to positions to 250 to 477 of SEQ ID NQ:110); (4) optionally, a linker peptide of from one to three or more amino acids,
101 such as the two amino acid linker of Leu-Glu (in the construct described in Example 8), represented by positions 229 to 230 of SEQ ID NO: 128; (5) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV génotype D large (L) surface antigen represented by positions 231 to 629 of SEQ ID NO: 128 (corresponding to positions 1 to 399 of SEQ ID NO: 118); (6) the amino acid sequence of a consensus sequence for HBV génotype D core antigen represented by positions 630 to 811 of SEQ ID NO:128 (corresponding to positions 400 to 581 of SEQ ID NO:118); and (7) optionally, a hexahistidine tag (in the construct described in Example 8). SEQ ID NO: 128 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein of SEQ ID NO: 128 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO: 127.
[00224] HBVAntigens Comprising Polymerase and Core Protein. In one embodiment of the invention, the HBV antigen(s) for use in a composition or method of the invention is a fusion protein comprising HBV antigens, wherein the HBV antigens comprise or consist of HBV polymerase (the RT domain) or at least one immunogenic domain thereof and an HBV core protein (HBcAg) or at least one immunogenic domain thereof. In one aspect, one or both of the RT domain of HBV polymerase or the HBV core protein is full-length or near full-length. In one aspect, one or both of the RT domain of HBV polymerase or the HBV core protein or a domain thereof comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of a full-length the RT domain of HBV polymerase or the HBV core protein or a domain thereof, respectively, or to the amino acid sequences represented by SEQ ID NO:98 (optimized HBV polymerase), SEQ ID NO:99 (optimized core protein), or a corresponding sequence from another HBV strain, as applicable. In one aspect, one or both of the RT domain of HBV polymerase or the HBV core protein or a domain thereof is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a full-length RT domain of HBV polymerase or the HBV core protein or a domain thereof, respectively, or to the amino acid sequences represented by SEQ ID NO:98 (optimized HBV polymerase), SEQ ID NO;99 (optimized core protein), or a corresponding sequence from another HBV strain, as applicable. A variety of suitable and exemplary sequences for HBV polymerase antigens and HBV core antigens are described herein.
[00225] One example of this antigen is schematically represented in Fig. 4. One example of a composition comprising this fusion protein is described in Example 3. In this embodiment, yeast (e.g., Saccharomyces cerevisiae) are engineered to express various
102
HBV polymerase-core fusion proteins as shown schematically in Fig. 4 under the control of the copper-inducible promoter, CUP1, or the TEF2 promoter. In each case, the fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:38: (1) an N-terminal peptide to impart résistance to proteasomal dégradation and stabilize expression (e.g., SEQ ID NO:37 or positions 1 to 6 of SEQ ID NO:38); 2) the amino acid sequence of a portion of the HBV génotype C polymerase including the reverse transcriptase domain (e.g., positions 347 to 691 of SEQ ID NO:10 or positions 7 to 351 of SEQ ID NO:38); 3) an HBV génotype C core protein (e.g., positions 31 to 212 of SEQ ID NO:9 or positions 352 to 533 of SEQ ID NO:38); and 4) a hexahistidine tag (e.g., positions 534 to 539 of SEQ ID NO:38). The sequence also contains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. The amino acid segments used in any of the fusion proteins described herein can be modified by the use of additional amino acids flanking either end of any domain; the examples provided herein are exemplary. For example, a fusion protein according to this embodiment can include 1) the amino acid sequence of a portion of the HBV génotype C polymerase including the reverse transcriptase domain (e.g., positions 347 to 691 of SEQ ID NO: 10 or positions 7 to 351 of SEQ ID NO:38); and 2) an HBV génotype C core protein (e.g., positions 31 to 212 of SEQ ID NO:9 or positions 352 to 533 of SEQ ID NO:38), and utilize no N- or C-terminal sequences, or utilize different N- or C-terminal sequences, and/or use linkers or no linkers between HBV sequences. In one embodiment, instead of the N-terminal peptide represented by SEQ ID NO:37, an N-terminal peptide represented by SEQ ID NO:89 or SEQ ID NO:90 is utilized, followed by the remainder of the fusion protein.
[00226] HBVAntigens Comprising X Antigen and Core Protein. In one embodiment of the invention, the HBV antigen(s) for use in a composition or method of the invention is a fusion protein comprising HBV antigens, wherein the HBV antigens comprise or consist of HBV X antigen or at least one immunogenic domain thereof and HBV core protein (HBcAg) or at least one immunogenic domain thereof. In one aspect, one or both of the HBV X antigen or the HBV core protein is full-length or near full-length. In one aspect, one or both of the HBV X antigen or the HBV core protein or a domain thereof comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of a full-length HBV X antigen or HBV core protein or domain thereof, respectively, or to the amino acid sequences represented by SEQ ID NO:99 (optimized core protein), SEQ ID NO;100 (optimized X antigen), or a corresponding sequence from
103 another HBV strain, as applicable. In one aspect, one or both of the HBV X antigen or the HBV core protein or a domain thereof is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a full-length HBV X antigen or HBV core protein or domain thereof, respectively, or to the amino acid sequences represented by
SEQ ID NO:99 (optimized core protein), SEQ ID NO: 100 (optimized X antigen), or a corresponding sequence from another HBV strain, as applicable. A variety of suitable and exemplary sequences for HBV core antigens and HBV X antigens are described herein.
[00227] This fusion protein is schematically represented in Fig. 5. An example of a composition comprising this fusion protein is described in Example 3. In this embodiment, 10 yeast (e.g., Saccharomyces cerevisiae) are engineered to express various HBV X-core fusion proteins as shown schematically in Fig. 5 under the control of the copper-inducible promoter, CUP1, or the TEF2 promoter. In each case, the fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:39 (1) an N-terminal peptide to impart résistance to 15 proteasomal dégradation and stabilize expression (e.g. SEQ ID NO:37 or positions 1 to 6 of SEQ ID NO:39); 2) the amino acid sequence of a near full-length (minus position 1) HBV génotype C X antigen (e.g,, positions 2 to 154 of SEQ ID NO: 12 or positions 7 to 159 of SEQ ID NO:39); 3) an HBV génotype C core protein (e.g., positions 31 to 212 of SEQ ID NO:9 or positions 160 to 341 of SEQ ID NO:39); and 4) a hexahistidine tag (e.g., 20 positions 342 to 347 of SEQ ID NO:39). The sequence also contains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. The amino acid segments used in any of the fusion proteins described herein can be modified by the use of additional amino acids flanking either end of any domain; the examples provided herein are exemplary. For example, a fusion protein according to this embodiment can include 1) 25 the amino acid sequence of a near full-length (minus position 1) HBV génotype C X antigen (e.g., positions 2 to 154 of SEQ ID NO: 12 or positions 7 to 159 of SEQ ID NO:39); and 2) an HBV génotype C core protein (e.g., positions 31 to 212 of SEQ ID NO:9 or positions 160 to 341 of SEQ ID NO:39), and utilize no N- or C-terminal sequences, or utilize different N- or C-terminal sequences, and/or use linkers or no linkers 30 between HBV sequences. In one embodiment, instead of the N-terminal peptide represented by SEQ ID NO:37, an N-terminal peptide represented by SEQ ID NO:89 or SEQ ID NO:90 is utilized, followed by the remainder of the fusion protein as described.
[00228] HBV Antigens Comprising Single HBV Proteins. In one embodiment of the invention, an HBV antigen is comprised of a single HBV protein (e.g., one HBV protein
selected from surface, core, e-antigen, polymerase, or X antigen) or one or more domains (structural, functional, and/or immunological) from a single HBV protein. This embodiment of the invention is particularly useful for creating a yeast-based immunotherapeutic composition that can be used, for example, in combination with one or 5 more other yeast-based immunotherapeutic compositions for the treatment or prophylaxis of HBV, or in sequence with one or more other yeast-based immunotherapeutic compositions for the treatment or prophylaxis of HBV, or to follow a prophylactic approach with a therapeutic approach if the patient becomes infected. For example, the yeast-based immunotherapeutic composition including an HBV surface antigen of this 10 embodiment can be combined with a second yeast-based immunotherapeutic composition including a different HBV protein/antigen, such as an HBV X antigen (described below), and further, with additional “single HBV protein” yeast-based immunotherapeutics, as desired (e.g., a yeast-based immunotherapeutic composition including an HBV Precore, Core or e-antigen and/or a yeast-based immunotherapeutic composition including an HBV 15 polymerase antigen or domain thereof). These “single HBV protein yeast immunotherapeutics” can be used in combination or sequence with each other and/or in combination or sequence with other multi-HBV protein yeast-based immunotherapeutics, such as those described in the Examples or elsewhere herein. Alternatively, or in addition, a “single HBV protein yeast immunotherapeutic” such as this HBV surface antigen yeast20 based immunotherapeutic can be produced using the HBV sequence for any given génotype or sub-genotype, and additional HBV surface antigen yeast-based immunotherapeutics can be produced using the HBV sequences for any one or more additional génotype or sub-genotype. This strategy effectively créâtes a “spice rack” of different HBV antigens and génotypes and/or sub-genotypes to each of which is provided 25 in the context of a yeast-based immunotherapeutic of the invention, or in a strategy that includes at least one yeast-based immunotherapeutic of the invention. Accordingly, any combination of one, two, three, four, five, six, seven, eight, nine, ten or more of these yeast-based immunotherapeutics can be selected for use to treat a particular patient or population of patients who are infected with HBV, illustratïng the flexibility of the présent 30 invention to be customized or tailored to meet the needs of a particular patient, population of patients, démographie, or other patient grouping.
[00229] In one embodiment of the invention, the HBV antigen(s) for use in a composition or method of the invention is an HBV antigen comprising or consisting of: (a) an HBV surface antigen protein and/or one or more domains (structural, functional or
immunogenic) thereof, which can include the hépatocyte receptor portion of Pre-Sl of the HBV large (L) surface antigen, the HBV large (L) surface antigen, the HBV middle (M) surface antigen, the HBV small (S) surface antigen (HBsAg), or any domain or combination thereof; (b) an HBV polymerase antigen, which can include one or more 5 domains (structural, functional, or immunogenic) of HBV polymerase, such as the reverse transcriptase (RT) domain (a functional domain) of HBV polymerase; (c) an HBV precore antigen, an HBV core antigen and/or HBV e-antigen, or one or more domains thereof (structural, functional or immunogenic), which can include one or more domains or portions of HBV Precore containing sequences from both HBV core and HBV e-antigen, 10 or one or the other of these proteins; or (d) an HBV X antigen, which can include one or more domains (structural, functional or immunogenic) of HBV X antigen. In one aspect, any one or more of these proteins or domains is full-length or near full-length. In one aspect, one or more of these proteins or domains comprise or consist of 1, 2, 3,4,5,6, 7, 8, 9, or 10 or more immunogenic domains. In one aspect, any one or more of these proteins 15 or domains comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% of the linear sequence of the corresponding full-length sequence or a domain thereof. In one aspect, any one or more of these proteins or domains is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding full-length sequence or a domain thereof. A variety of suitable and exemplary sequences 20 for HBV surface antigens, HBV polymerase antigens, HBV core antigens, and HBV X antigens are described herein.
[00230] An example of a composition comprising a surface antigen protein is described in Example 5. In this embodiment, yeast (e.g., Saccharomyces cerevisiae) are engineered to express HBV surface proteins under the control of a suitable promoter, such 25 as the copper-inducible promoter, CUP1, or the TEF2 promoter. The protein is a single polypeptide comprising HBV near-full-length HBV large (L) surface antigen (to accommodate the presence of an N-terminal sequence selected to enhance or stabilize expression of the antigen), represented by SEQ ID NO:93: (1) an N-terminal peptide of SEQ ID NO:89 (positions 1-89 of SEQ ID NO:93); 2) the amino acid sequence of a near 30 full-length (minus position 1) HBV génotype C large (L) surface antigen (e.g., positions 2400 of SEQ ID NO:11 or positions 90 to 488 of SEQ ID NO:93); and 3) a hexahîstidine tag (e.g., positions 489 to 494 of SEQ ID NO:93). Alternatively, the N-terminal peptide can be replaced with SEQ ID NO:37 or a homologue thereof or another N-terminal peptide described herein. In one embodiment, this construct also contains one or more
anti-viral résistance mutations in the surface antigen. While this example utilizes large (L) surface antigen as an HBV antigen that may maximize the exposure of immunogenic epitopes generated by the immune System, small portions of surface antigen, încluding any domains or combinations of domains of surface antigen, can be produced using the 5 guidance provided herein. In addition, while the exemplary immunotherapeutic is shown using a génotype C sequence, sequences from other génotypes, sub-genotypes, and/or strains or isolâtes of HBV can be used instead.
[00231] An example of a composition comprising an HBV polymerase antigen is described in Example 3 and also in Example 5. The HBV antigen described in Example 5 10 îs schematically represented in Fig. 6. In this embodiment, yeast (e.g., Saccharomyces cerevisiae) are engineered to express various HBV polymerase proteins under the control of the copper-inducible promoter, CUP1, or the TEF2 promoter. In each case, the fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:40 (1) an N-terminal peptide to impart 15 résistance to proteasomal dégradation and stabilize expression (SEQ ID NO:37, or positions 1 to 6 of SEQ ID NO:40; 2) the amino acid sequence of a portion of the HBV génotype C polymerase încluding the reverse transcriptase domain (e.g., positions 347 to 691 of SEQ ID NO:10 or positions 7 to 351 of SEQ ID NO:40); and 3) a hexahistidine tag (e.g., positions 352 to 357 of SEQ ID NO:40). The sequence also contains epitopes or 20 domains that are believed to enhance the immunogenicity of the fusion protein. In addition, in one embodiment, the sequence of this construct can be modified to introduce one or more or ail of the following anti-viral résistance mutations: rtM2041, rtL180M, rtM204V, rtV173L, rtN236T, rtA194T (positions given with respect to the full-length amino acid sequence for HBV polymerase). In one embodiment, six different 25 immunotherapy compositions are created, each one containing one of these mutations. In other embodiments, ail or some of the mutations are included in a single fusion protein. The amino acid segments used in any of the fusion proteins described herein can be modified by the use of additional amino acids flanking either end of any domain; the examples provided herein are exemplary. For example, a fusion protein according to this 30 embodiment can include the amino acid sequence of a portion of the HBV génotype C polymerase încluding the reverse transcriptase domain (e.g., positions 347 to 691 of SEQ ID NO: 10 or positions 7 to 351 of SEQ ID NO:40), and utilîze no N- or C-terminal sequences, or utilize different N- or C-terminal sequences, and/or use linkers or no linkers between HBV sequences. In one embodiment, instead of the N-terminal peptide
represented by SEQ ID NO:37, an N-terminal peptide represented by SEQ ID NO:89 or SEQ ID NO:90 is utilized, followed by the remainder of the fusion protein as described.
[00232] In the embodiment shown in Example 5, yeast (e.g., Saccharomyces cerevisiae) are engineered to express HBV polymerase proteins under the control of a 5 suitable promoter, such as the copper-inducible promoter, CUP1, or the TEF2 promoter.
The protein is a single polypeptide comprising HBV reverse transcriptase (RT) domain of polymerase (Pol), represented by SEQ ID NO:94: (1) an N-terminal peptide of SEQ ID NO:89 (positions 1-89 of SEQ ID NO:94); 2) the amino acid sequence of a portion of the HBV génotype C polymerase including the reverse transcriptase domain (e.g., positions 10 347 to 691 of SEQ ID NO: 10 or positions 90 to 434 of SEQ ID NO:94); and 3) a hexahistidine tag (e.g., positions 435 to 440 of SEQ ID NO:94). The sequence also contains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. In addition, in one embodiment, the sequence of this construct can be modified to introduce one or more or ail of the following anti-viral résistance mutations: 15 rtM2041, rtL180M, rtM204V, rtV173L, rtN236T, rtA194T (positions given with respect to the full-length amino acid sequence for HBV polymerase). Alternatively, the N-terminal peptide can be replaced with SEQ ID NO:37 or a homologue thereof or another Nterminal peptide described herein.
[00233] An example of a composition comprising an HBV Precore, Core or e-antigen 20 is described in Example 5. Yeast (e.g., Saccharomyces cerevisiae) are engineered to express HBV Core proteins under the control of a suitable promoter, such as the copperinducible promoter, CUP1, or the TEF2 promoter. The protein is a single polypeptide comprising near full-length HBV Core protein, represented by SEQ ID NO:95: (1) an Nterminal peptide of SEQ ID NO:89 (positions 1-89 of SEQ ID NO:95); 2) the amino acid 25 sequence of a portion of the HBV génotype C Core protein (e.g., positions 31 to 212 of
SEQ ID NO:9 or positions 90 to 271 of SEQ ID NO:95); and 3) a hexahistidine tag (e.g., positions 272 to 277 of SEQ ID NO:95). The sequence also contains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. Alternatively, the N-terminal peptide can be replaced with SEQ ID NO:37 or a homologue thereof or 30 another N-terminal peptide described herein.
[00234] An example of a yeast-based îmmunotherapeutic composition comprising an HBV X antigen is described in Example 5. Yeast (e.g., Saccharomyces cerevisiae) are engineered to express HBV X antigens under the control of a suitable promoter, such as the copper-inducible promoter, CUP1, or the TEF2 promoter. The protein is a single
108 polypeptide comprising near full-Iength HBV X antigen, represented by SEQ ID NO:96: (1) an N-terminal peptide of SEQ ID NO:89 (positions 1-89 of SEQ ID NO:96); 2) the amino acid sequence of a portion of the HBV génotype C X antigen (e.g., positions 2 to 154 of SEQ ID NO: 12 or positions 90 to 242 of SEQ ID NO:96); and 3) a hexahistidine tag (e.g., positions 243 to 248 of SEQ ID NO:96). The sequence also contains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. Alternatively, the N-terminal peptide can be replaced with SEQ ID NO:37 or a homologue thereof or another N-terminal peptide described herein.
[00235] HBV Antigens Comprising HBV Proteins from Two or More Génotypes. Another embodiment of the invention relates to HBV antigens for use in an immunotherapeutic composition of the invention that maximizes the targeting of HBV génotypes and/or sub-genotypes in order to provide compositions with the potential to treat a large number of individuals or populations of individuals using one composition. Such compositions are generally more efficient to produce (i.e., have a production advantage by including multiple antigens and/or a consensus approach to targeting génotypes) and are more efficient to utilize in a wide variety of clinical settings (e.g., one composition may serve many different types of patient populations in many different geographical settings). As discussed above, to produce such HBV antigens, conserved antigens and/or conserved domains (among HBV génotypes) can be selected, and the antigens can be designed to maximize the inclusion of conserved immunological domains.
[00236] In one aspect of this embodiment, an HBV antigen is provided that includes in a single yeast-based immunotherapeutic a single HBV protein or domain thereof (e.g., surface, polymerase, core/e or X) that is repeated two, three, four, five or more times within the antigen construct, each time using a sequence from a different HBV génotype or subgenotype. In this aspect, multiple dominant or prévalent génotypes can be targeted in one yeast-based immunotherapeutic, increasing clinical and manufacturing efficacy. These antigens can be modified, if desired, to maximize the inclusion of consensus sequences, including consensus T cell epitopes within the antigens, which may otherwise contain subtle différences due to sub-genotype, strain or isolate différences.
[00237] Accordingly, in one embodiment of the invention, the HBV antigen(s) for use in a composition or method of the invention is an HBV antigen comprising or consisting of two or more repeated HBV antigens of the same protein or domain, but of different
HBV génotypes (e.g., two or more HBV Core or e-antigens, which can include one or more domains (structural, functional or immunogenic) of HBV Core or e-antigen, wherein
the antigens include the same or similar antigen from each of HBV génotype C and HBV génotype D, to form a Core-Core fusion where each Core protein is a different génotype). In one aspect, the HBV protein used in such constructs is full-length or near full-length protein or domain. In one aspect, the HBV antigen comprises or consists of 1, 2,3,4, 5,6, 5 7, 8, 9, or 10 or more immunogenic domains. In one aspect, any one or more of these proteins or domains comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of the corresponding full-length sequence. In one aspect, any one or more of these proteins or domains is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of the 10 corresponding full-length sequence.
[00238] Such an antigen is exemplified in Example 6. In this embodiment, yeast (e.g., Saccharomyces cerevisiae) are engineered to express an HBV fusion protein under the control of a suitable promoter, such as the copper-inducible promoter, CUP1, or the TEF2 promoter. The protein is a single polypeptide comprising four Core antigens, each one 15 from a different génotype (HBV génotypes A, B, C and D), represented by SEQ ID
NO:105: 1) an N-terminal méthionine at position 1 of SEQ ID NO:105; 2) the amino acid sequence of a near full-length Core protein from HBV génotype A (e.g., positions 31 to 212 of SEQ ID NO:1 or positions 2 to 183 of SEQ ID NO: 105); 3) the amino acid sequence of a near full-length Core protein from HBV génotype B (e.g., positions 30 to 20 212 of SEQ ID NO:5 or positions 184 to 395 of SEQ ID NO: 105); 4) the amino acid sequence of a near full-length Core protein from HBV génotype C (e.g., positions 30 to 212 of SEQ ID NO:9 or positions 396 to 578 of SEQ ID NO: 105); 5) the amino acid sequence of a near full-length Core protein from HBV génotype D (e.g., positions 30 to 212 of SEQ ID NO:13 or positions 579 to 761 of SEQ ID NO: 105); and 5) a hexahistidine 25 tag (e.g., positions 762 to 767 of SEQ ID NO: 105). The sequence also contains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. The Nterminal méthionine at position 1 can be substituted with SEQ ID NO:37 or a homologue thereof, or with an alpha prepro sequence of SEQ ID NO:89 or SEQ ID NO:90, or a homologue thereof, or any other suitable N-terminal sequence if desired. In addition, 30 linker sequences can be inserted between HBV proteins to facilitate cloning and manipulation of the construct, if desired. This is an exemplary construct, as any other combination of HBV génotypes and/or sub-genotypes can be substituted into this design as desired to construct a single antigen yeast-based HBV immunotherapeutic product with broad clinical applicability and efficient design for manufacturing. The amino acid
sequence of SEQ ID NO: 105 also contains several known T cell epitopes, and certain epitopes hâve been modified to correspond to the published sequence for the given epitope (see Table 5).
[00239] In another aspect of this embodiment, more than one protein or domain from a single HBV génotype is included in an HBV antigen useful in the invention, which may be selected to maximize the most conserved protein sequences encoded by the HBV genome or to maximize the inclusion of therapeutically or prophylactically useful immunogenic domains within the antigen, These antigens are then repeated within the same fusion protein, but using the same or similar sequences from a different HBV génotype or 10 subgenotype. Ln this aspect, multiple dominant or prévalent génotypes can also be targeted in one yeast-based immunotherapeutic, again increasing clinical and manufacturing efficacy. These antigens can also be modified, if desired, to maximize the inclusion of consensus T cell epitopes within the antigens, which may otherwise contain subtle différences due to sub-genotype, strain or isolate différences.
[00240] Accordingly, in one embodiment of the invention, the HBV antigen(s) for use in a composition or method of the invention is an HBV antigen comprising or consisting of at least two different HBV proteins or domains thereof, each of which is repeated two or more times, but wherein the repeated sequences are from different HBV génotypes (e.g., two or more HBV Core and two or more X antigens, or domains thereof, wherein the 20 antigens include the same or similar antigen from each of HBV génotype C and HBV génotype D, to form a Core-X-Core-X fusion (or any other order of segments within the fusion) where each Core protein is a different génotype and each X antigen is a different génotype). In one aspect, the HBV protein used in such constructs is full-length or near full-length protein or domain. In one aspect, the HBV antigen comprises or consiste of 1,
2, 3,4,5, 6, 7, 8, 9, or 10 or more immunogenic domains. In one aspect, any one or more of these proteins or domains comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of the corresponding full-length sequence. In one aspect, any one or more of these proteins or domains is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to the sequence of the 30 corresponding full-length sequence.
[00241] Such an antigen is exemplified in Example 6. In this embodiment, yeast (e.g., Saccharomyces cerevisiaè) are engineered to express an HBV fusion protein under the control of a suitable promoter, such as the copper-inducible promoter, CUP1, or the TEF2 promoter. The protein is a single polypeptide comprising two Core antigens and two X
111 antigens, each one of the pair from a different génotype (HBV génotypes A and C), represented by SEQ ID NO: 106: 1) an N-terminal méthionine at position 1 of SEQ ID NO:106; 2) the amino acid sequence of a near full-length Core protein from HBV génotype A (e.g., positions 31 to 212 of SEQ ID NO:1 or positions 2 to 183 of SEQ ID NO: 106); 3) the amino acid sequence of a full-length X antigen from HBV génotype A (e.g., positions SEQ ID NO:4 or positions 184 to 337 of SEQ ID NO: 106); 4) the amino acid sequence of a near full-length Core protein from HBV génotype C (e.g., positions 30 to 212 of SEQ ID NO:9 or positions 338 to 520 of SEQ ID NO: 106); 5) the amino acid sequence of a full-length X antigen from HBV génotype C (e.g., SEQ ID NO:8 or positions 521 to 674 of SEQ ID NO: 106); and 5) a hexahistidine tag (e.g., positions 675 to 680 of SEQ ID NO: 106). The sequence also contains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. The N-terminal méthionine at position 1 can be substituted with SEQ ID NO:37 or a homologue thereof, or with an alpha prepro sequence of SEQ ID NO:89 or SEQ ID NO:90, or a homologue thereof. The amino acid sequence of SEQ ID NO: 106 also contains several known T cell epitopes, and certain epitopes hâve been modified to correspond to the published sequence for the given epitope (see Table 5).
[00242] Additional Embodiments Regarding HBV Antigens. In some aspects of the invention, amino acid insertions, délétions, and/or substitutions can be made for one, two, three, four, five, six, seven, eight, nine, ten, or more amino acids of a wild-type or reference HBV protein, provided that the resulting HBV protein, when used as an antigen in a yeast-HBV immunotherapeutic composition of the invention, elicits an immune response against the target or wild-type or reference HBV protein, which may include an enhanced immune response, a diminished immune response, or a substantially similar immune response. For example, the invention includes the use of HBV agonist antigens, which may include one or more T cell epitopes that hâve been mutated to enhance the T cell response against the HBV agonist, such as by improving the avidity or affinity of the epitope for an MHC molécule or for the T cell receptor that recognizes the epitope in the context of MHC présentation. HBV protein agonists may therefore improve the potency or efficiency of a T cell response against native HBV proteins that infect a host.
[00243] Referring to any of the above-described HBV antigens, including the fusion proteins that hâve amino acid sequences including or represented by SEQ ID NO:34, SEQ
ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID
NO:92, SEQ ID NQ:101, SEQ ID NQ:102, SEQ ID NQ:107, SEQ ID NO:108, SEQ ID
NO:109, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:150 or SEQ ID NO: 151, it is an aspect of the invention to use one or more of the HBV antigens from individual HBV proteins within the fusion protein (e.g., from HBV surface antigen, HBV polymerase, HBV core/e-antigen, and/or HBV X antigen) to construct “single protein” antigens (e.g., antigens from only one of these HBV proteins), or to construct fusion proteins using only two or three of the HBV protein segments, if applicable to the given reference fusion protein. It is also an aspect of the invention to change the order of HBV 10 protein segments within the fusion protein. As another altemate design, HBV génotypes and/or consensus sequences can be combined, where two, three, four or more génotypes and/or consensus sequences are used to construct the fusion protein.
[00244] The invention also includes homologues of any of the above-described fusion proteins, as well as the use of homologues, variants, or mutants of the individual HBV 15 proteins or portions thereof (including any functional and/or immunogenic domains) that are part of such fusion proteine or otherwise described herein. In one aspect, the invention includes the use of fusion proteins or individual (single) HBV proteins or HBV antigens, having amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one 20 of the fusion proteins or individual HBV proteins or HBV antigens, respectively, described herein, including any of the HBV proteins, HBV antigens and fusion proteins referenced by a spécifie sequence identifier herein, over the full length of the fusion protein, or with respect to a defined segment in ±e fusion protein or a defined protein or domain thereof (immunogenic domain or functional domain (i.e., a domain with at least 25 one biological activity)) that forms part of the fusion protein. Many CTL epitopes (epitopes that are recognized by cytotoxic T lymphocytes from patients infected with HBV) and escape mutations (mutations that arise in an HBV protein due to sélective pressure from an anti-viral drug) are known in the art, and this information can also be used to make substitutions or create variants or homologues of the HBV antigens 30 described herein in order to provide a spécifie sequence in the HBV antigen of the invention.
[00245] Yeast-Based Immunotherapy Compositions. In various embodiments of the invention, the invention includes the use of at least one “yeast-based immunotherapeutic composition” (which phrase may be used interchangeably with “yeast-based
113 immunotherapy product”, “yeast-based immunotherapy composition”, “yeast-based composition”, “yeast-based immunotherapeutic”, “yeast-based vaccine”, or dérivatives of these phrases). An “immunotherapeutic composition” is a composition that elicits an immune response sufficient to achieve 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 achieve 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 useful in the invention is capable of inducing a CD8+ and/or a CD4+ T cell-mediated immune response and in one aspect, a CD8+ and a CD4+ T cell-mediated immune response. Optionally, a yeast-based immunotherapeutic composition is capable of eliciting a humoral immune response. A yeast-based immunotherapeutic composition useful in the présent invention can, for example, elicit an immune response in an individual such that the individual is protected from HBV infection and/or is treated for HBV infection or for symptoms resulting from HBV infection.
[00246] Yeast-based immunotherapy compositions of the invention may be either prophylactic or therapeutic. When provided prophylactically, the compositions of the présent invention are provided in advance of any symptom of HBV infection. Such a composition could be administered at birth, in early childhood, or to adults. The prophylactic administration of the immunotherapy compositions serves to prevent subséquent HBV infection, to résolve an infection more quickly or more completely if HBV infection subsequently ensues, and/or to ameliorate the symptoms of HBV infection if infection subsequently ensues. When provided therapeutically, the immunotherapy compositions are provided at or after the onset of HBV infection, with the goal of ameliorating at least one symptom of the infection and preferably, with a goal of eliminating the infection, providing a long lasting rémission of infection, and/or providing long term immunity against subséquent infections or réactivations of the virus. In one aspect, a goal of treatment is loss of détectable HBV viral load or réduction of HBV viral load (e.g., below détectable levels by PCR or <2000 IU/ml). In one aspect, a goal of treatment is sustained viral clearance for at least 6 months after the completion of therapy. In one aspect, a goal of treatment is the loss of détectable sérum HBeAg and/or HBsAg proteins. In one aspect, a goal of treatment is the development of antibodies against the
114 hepatitis B surface antigen (anti-HBs) and/or antibodies against HBeAg. In one aspect, the goal of treatment is séroconversion, which may be defined by: (a) 10 or more sample ratio units (SRU) as determined by radioimmunoassay; (b) a positive resuit as determined by enzyme immunoassay; or (c) détection of an antibody concentration of >10 mlU/ml (10 SRU is comparable to 10 mlU/mL of antibody). In one aspect, a goal of treatment is normalization of sérum alanine aminotransferase (ALT) levels, improvement in liver inflammation and/or improvement in liver fibrosis.
[00247] Typically, a yeast-based immunotherapy composition includes a yeast vehicle and at least one antigen or immunogenic domain thereof expressed by, attached to, or mixed with the yeast vehicle, wherein the antigen is heterologous to the yeast, and wherein the antigen comprises one or more HBV antigens or immunogenic domains thereof. In some embodiments, the antigen or immunogenic domain thereof is provided as a fusion protein. Several HBV fusion proteins suitable for use in the compositions and methods of the invention hâve been described above. In one aspect of the invention, fusion protein can include two or more antigens. 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.
[00248] In any of the yeast-based immunotherapy compositions used in the présent invention, the following aspects related to the yeast vehicle are included in the invention. According to the présent invention, a yeast vehicle is any yeast cell (e.g., a whole or intact cell) or a dérivative thereof (see below) that can be used in conjunction with one or more antigens, immunogenic domains thereof or epitopes thereof 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 can therefore include, but is not limited to, a live intact (whole) yeast microorganism (i.e., a yeast cell having ail its components including a cell wall), a killed (dead) or inaclivated intact yeast microorganism, or dérivatives of intact/whole yeast including: a yeast spheroplast (i.e., a yeast cell lacking a cell wall), a yeast cytoplast (i.e., a yeast cell lacking a cell wall and nucléus), a yeast ghost (i.e., a yeast cell lacking a cell wall, nucléus and cytoplasm), a subcellular yeast membrane extract or fraction thereof (also referred to as a yeast membrane particle and previously as a subcellular yeast particle), any other yeast particle, or a yeast cell wall préparation.
[00249] Yeast spheroplasts are typically produced by enzymatic digestion of the yeast cell wall. Such a method is described, for example, in Franzusoff et al., 1991, Meth. Enzymol. 194,662-674., incorporated herein by reference in its entîrety.
[00250] Yeast cytoplasts are typically produced by énucléation of yeast cells. Such a method is described, for example, in Coon, 1978, Natl. Cancer Inst. Monogr. 48, 45-55 incorporated herein by reference in its entirety.
[00251] Yeast ghosts are typically produced by resealing a permeabilized or lysed cell 5 and can, but need not, contain at least some of the organelles of that cell. Such a method is described, for example, in Franzusoff et al., 1983, J. Biol. Chem. 258, 3608-3614 and Bussey et al., 1979, Biochim, Biophys. Acta 553, 185-196, each of which is incorporated herein by reference in its entirety.
[00252] A yeast membrane particle (subcellular yeast membrane extract or fraction 10 thereof) refers to a yeast membrane that lacks a natural nucléus 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 dîsruption methods known to those skilled in the art, followed by resealing. A method for producing subcellular yeast membrane extracts is described, for example, in Franzusoff et al., 1991, Meth. Enzymol.
194, 662-674. One may also use fractions of yeast membrane particles that contain yeast membrane portions and, when the antigen or other protein was expressed recombinantly by the yeast prior to préparation of the yeast membrane particles, the antigen or other protein of interest. Antigens or other proteins of interest can be carrîed inside the membrane, on either surface of the membrane, or combinations thereof (i.e., the protein 20 can be both inside and outside the membrane and/or spanning the membrane of the yeast membrane particle). In one embodiment, a yeast membrane particle is a recombinant yeast membrane particle that can be an intact, disrupted, or disrupted and resealed yeast membrane that includes at least one desired antigen or other protein of interest on the surface of the membrane or at least partially embedded within the membrane.
[00253] An example of a yeast cell wall préparation is a préparation of isolated yeast cell walls carrying an antigen on its surface or at least partially embedded within the cell wall such that the yeast cell wall préparation, when administered to an animal, stimulâtes a desired immune response against a disease target.
[00254] Any yeast strain can be used to produce a yeast vehicle of the présent invention. Yeast are unicellular microorganisms that belong to one of three classes:
Ascomycètes, Basidiomycetes and Fungi Imperfecti. One considération for the sélection of a type of yeast for use as an immune modulator is the pathogenicity of the yeast. In one embodiment, the yeast is a non-pathogenic strain such as Saccharomyces cerevisiae. The sélection of a non-pathogenic yeast strain minimizes any adverse effects to the indivîdual
116 to whom the yeast vehicle is administered. However, pathogenic yeast may be used if the pathogenicity of the yeast can be negated by any means known to one of skill in the art (e.g., mutant strains).
[00255] Généra of yeast strains that may be used in the invention include but are not limited to Saccharomyces, Candida, Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia. In one aspect, yeast généra are selected from Saccharomyces, Candida, Hansenula, Pichia or Schizosaccharomyces, and in one aspect, yeast généra are selected from Saccharomyces, Hansenula, and Pichia, and in one aspect, Saccharomyces is used. Species of yeast strains that may 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, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianus var. lac lis, Pichia pastoris, Rhodotorula rubra, Schizosaccharomyces pombe, and Yarrowia lipolytica. It is to be appreciated that a number of these species include a variety of subspecies, types, subtypes, etc. that are intended to be included within the aforementioned species. 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 being Generally Recognized As Safe or GRAS for use as food additives (GRAS, FDA proposed Rule 62FR18938, April 17, 1997). One embodiment of the présent invention is a yeast strain that is capable of replicating plasmids to a particularly high copy number, such as a S. cerevisiae cir° strain. The 5. cerevisiae strain is one such strain that is capable of supporting expression vectors that allow one or more target antigen(s) and/or antigen fusion protein(s) and/or other proteins to be expressed at high levels. In addition, any mutant yeast strains can be used in the présent invention, including those that exhibit reduced post-translational modifications of expressed target antigens or other proteins, such as mutations in the enzymes that extend N-linked glycosylation.
[00256] In most embodiments of the invention, the yeast-based immunotherapy composition includes at least one antigen, immunogenic domain thereof, or epitope thereof.
The antigens contemplated for use in this invention include any HBV antigen or immunogenic domain thereof, including mutants, variants and agonists of HBV proteins or domains thereof, against which it is desired to elicit an immune response for the purpose of prophylactically or therapeutically immunizing a host against HBV infection. HBV
117 antigens that are useful in various embodiments of the invention have been described in detail above.
[00257] Optionally, proteins, including fusion proteins, which are used as a component of the yeast-based immunotherapeutic composition of the invention are produced using 5 constructs that are particularly useful for improving or enhancing the expression, or the stability of expression, of recombinant antigens in yeast. Typically, the desired antigenic protein(s) or peptide(s) are fused at their amino-terminal end to: (a) a spécifie synthetic peptide that stabilizes the expression of the fusion protein in the yeast vehicle or prevents posttranslational modification of the expressed fusion protein (such peptides are described 10 in detail, for example, in U.S. Patent Publication No. 2004-0156858 Al, published August
12, 2004, incorporated herein by reference in its entirety); (b) at least a portion of an endogenous yeast protein, including but not limited to alpha factor, wherein either fusion partner provides improved stability of expression of the protein in the yeast and/or a prevents post-translational modification of the proteins by the yeast cells (such proteins 15 are also described in detail, for example, in U.S. Patent Publication No. 2004-0156858 Al, 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 (e.g., an Aga protein, described in more detail herein). In addition, the présent invention optionally includes the use of peptides that are fused to the C-terminus of the antigen-encoding construct, particularly for use in the sélection and 20 identification of the protein. Such peptides include, but are not limited to, any synthetic or natural peptide, such as a peptide tag (e.g., hexahistidine) 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.
[00258] In one embodiment, a synthetic peptide useful in a fusion protein is linked to 25 the N-terminus of the antigen, the peptide consisting of at least two amino acid resîdues that are heterologous to the antigen, wherein the peptide stabilizes the expression of the fusion protein in the yeast vehicle or prevents posttranslational modification of the expressed fusion protein. The synthetic peptide and N-terminai portion of the antigen together form a fusion protein that has the following requirements: (1) the amino acid 30 residue at position one of the fusion protein is a méthionine (i.e., the first amino acid in the synthetic peptide is a méthionine); (2) the amino acid residue at position two of the fusion protein is not a glycine or a proline (i.e., the second amino acid in the synthetic peptide is not a glycine or a proline); (3) none of the amino acid resîdues at positions 2-6 of the fusion protein is a méthionine (i.e., the amino acids at positions 2-6, whether part of the
118 synthetic peptide or the protein, if the synthetic peptide is shorter than 6 amino acids, do not include a méthionine); and (4) none of the amino acids at positions 2-6 of the fusion protein is a lysine or an arginine (i.e., the amino acids at positions 2-6, whether part of the synthetic peptide or the protein, if the synthetic peptide is shorter than 5 amino acids, do not include a lysine or an arginine). The synthetic peptide can be as short as two amino acids, but in one aspect, is 2-6 amino acids (including 3, 4, 5 amino acids), and can be longer than 6 amino acids, in whole integers, up to about 200 amino acids, 300 amino acids, 400 amino acids, 500 amino acids, or more.
[00259] In one embodiment, a fusion protein comprises an amino acid sequence of MX2-X3-X4-X5-X6, wherein M is méthionine; wherein X2 is any amino acid except glycine, proline, lysine or arginine; wherein X3 is any amino acid except méthionine, lysine or arginine; wherein X4 is any amino acid except méthionine, lysine or arginine; wherein X5 is any amino acid except méthionine, lysine or arginine; and wherein X6 is any amino acid except méthionine, lysine or arginine. In one embodiment, the X6 residue is a proline. An exemplary synthetic sequence that enhances the stability of expression of an antigen in a yeast cell and/or prevents post-translational modification of the protein in the yeast includes the sequence M-A-D-E-A-P (SEQ ID NO:37). Another exemplary synthetic sequence with the same properties is M-V. In addition to the enhanced stability of the expression product, these fusion partners do not appear to negatively impact the immune response against the immunizing antigen in the construct. In addition, the synthetic fusion peptides can be designed to provide an epitope that can be recognized by a sélection agent, such as an antibody.
[00260] In one embodiment, the HBV antigen is linked at the N-terminus to a yeast protein, such as an alpha factor prepro sequence (also referred to as the alpha factor signal leader sequence, the amino acid sequence of which is exemplified herein by SEQ ID NO:89 or SEQ ID NO:90. Other sequences for yeast alpha factor prepro sequence are known in the art and are encompassed for use in the présent invention.
[00261] In one aspect of the invention, the yeast vehicle is manipulated such that the antigen is expressed or provided by delivery or translocation of an expressed protein product, partially or wholly, on the surface of the yeast vehicle (extracellular expression). One method for accomplishing this aspect of the invention is to use a spacer arm for positioning one or more protein(s) on the surface of the yeast vehicle. For example, one can use a spacer arm to create a fusion protein of the antigen(s) or other protein of Înterest with a protein that targets the antigen(s) or other protein of interest to the yeast cell wall.
119
For example, one such protein that can be used to target other proteins is a yeast protein (e.g., cell wall protein 2 (cwp2), Aga2, Pir4 or Flol protein) that enables the antigen(s) or other protein to be targeted to the yeast cell wall such that the antigen or other protein is located on the surface of the yeast. Proteins other than yeast proteins may be used for the spacer arm; however, for any spacer arm protein, it is most désirable to hâve the immunogenic response be directed against the target antigen rather than the spacer arm protein. As such, if other proteins are used for the spacer arm, then the spacer arm protein that is used should not generate such a large immune response to the spacer arm protein itself such that the immune response to the target antigen(s) is overwhelmed. One of skill in the art should aim for a small immune response to the spacer arm protein relative to the immune response for the target antigen(s). Spacer arms can be constructed to hâve cleavage sites (e.g., protease cleavage sites) that allow the antigen to be readily removed or processed away from the yeast, if desired. Any known method of determining the magnitude of immune responses can be used (e.g., antibody production, lytic assays, etc.) and are readily known to one of skill in the art.
[00262] 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. Alternatîvely, positioning can be accomplished by appending signal sequences that target the antigen(s) or other proteins of interest into the secretory pathway via translocation into the endoplasmic réticulum (ER) such that the antigen binds to a protein which is bound to the cell wall (e.g., cwp).
[00263] 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 is about 10 to 700 amino acids. In another embodiment, the yeast protein is about 40 to 600 amino acids. Other embodiments of the invention include the yeast protein being 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 considération for optimizing antigen surface expression, if that is desired, is whether the antigen and spacer arm combination should be expressed as a monomer or as dimer or as a trimer, or even more units connected together. This use of monomers, dimers, trimers, etc. allows for appropriate spacing or folding of the antigen such that some part, if not ail, of the
120 antigen is displayed on the surface of the yeast vehicle in a manner that makes it more immunogenic.
[00264] Use of yeast proteins can stabilize the expression of fusion proteins in the yeast vehicle, prevents posttranslational modification of the expressed fusion protein, and/or targets the fusion protein to a particular compartment in the yeast (e.g., to be expressed on the yeast cell surface). For delivery into the yeast secretory pathway, exemplary yeast proteins to use include, but are not limited to: Aga (including, but not limited to, Agal and/or Aga2); SUC2 (yeast invertase); alpha factor signal leader sequence; CPY; Cwp2p for its localization and rétention in the cell wall; BUD genes for localization at the yeast cell bud during the initial phase of daughter cell formation; Flolp; Pir2p; and Pir4p.
[00265] Other sequences can be used to target, retain and/or stabilize the protein to other parts of the yeast vehicle, for example, in the cytosol or the mitochondria or the endoplasmic réticulum or the nucléus. Examples of suitable yeast protein that can be used for any of the embodiments above include, but are not limited to, TK, AF, SEC7; phosphoenolpyruvate carboxykinase PCK1, phosphoglycerokinase PGK and triose phosphate isomerase TPI gene products for their repressible expression in glucose and cytosolic localization; the heat shock proteins SSA1, SSA3, SSA4, SSCI, whose expression is induced and whose proteins are more thermostable upon exposure of cells to heat treatment; the mitochondrial protein CYC1 for import into mitochondria; ACT1.
[00266] Methods of producing yeast vehicles and expressing, combining and/or associating yeast vehicles with antigens and/or other proteins and/or agents of interest to produce yeast-based immunotherapy compositions are contemplated by the invention.
[00267] According to the présent invention, the term yeast vehicle-antigen complex or yeast-antigen complex is used generically to de scribe any association of a yeast vehicle with an antigen, and can be used interchangeably with “yeast-based immunotherapy composition” when such composition is used to elicît an immune response as described above. Such association includes expression of the antigen by the yeast (a recombinant yeast), introduction of an antigen into a yeast, physical attachment of the antigen to the yeast, and mixing of the yeast and antigen together, such as in a buffer or other solution or formulation. These types of complexes are described in detail below.
[00268] In one embodiment, a yeast cell used to préparé the yeast vehicle is transfected with a heterologous nucleic acid molécule encoding a protein (e.g., the antigen) such that the protein is expressed by the yeast cell. Such a yeast is also referred to herein as a
recombinant yeast or a 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 it can be derivatized such as by formation of yeast spheroplasts, cytoplasts, ghosts, or subcellular particles, any of which is followed by loading of the dérivative into the dendritic cell. Yeast 5 spheroplasts can also be directly transfected with a recombinant nucleic acid molécule (e.g., the spheroplast is produced from a whole yeast, and then transfected) in order to produce a recombinant spheroplast that expresses an antigen or other protein.
[00269] In general, the yeast vehicle and antigen(s) and/or other agents can be associated by any technique described herein. In one aspect, the yeast vehicle was loaded 10 intracellularly with the antigen(s) and/or agent(s). In another aspect, the antigen(s) and/or agent(s) was 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 associated by mixing. In another aspect, and in one embodiment, the antigen(s) and/or agent(s) is expressed recombinantly by the yeast vehicle or by the yeast cell or yeast spheroplast from which the 15 yeast vehicle was derived.
[00270] A number of antigens and/or other proteins to be produced by a yeast vehicle of the présent invention is any number of antigens and/or other proteins that can be reasonably produced by a yeast vehicle, and typically ranges from at least one to at least about 6 or more, including from about 2 to about 6 heterologous antigens and or other 20 proteins.
[00271] Expression of an antigen or other protein in a yeast vehicle of the présent invention is accomplished using techniques known to those skilled in the art. Briefly, a nucleic acid molécule encoding at least one desired antigen or other protein is inserted into an expression vector in such a manner that the nucleic acid molécule is operatively linked 25 to a transcription control sequence in order to be capable of effectîng either constitutive or regulated expression of the nucleic acid molécule when transformed into a host yeast cell. Nucleic acid molécules encoding one or more antigens and/or other proteins can be on one or more expression vectors operatively linked to one or more expression control sequences. Partîcularly important expression control sequences are those which control transcription 30 initiation, such as promoter and upstream activation sequences. Any suitable yeast promoter can be used in the présent invention and a variety of such promoters are known to those skilled in the art. Promoters for expression in Saccharomyces cerevisiae include, but are not limited to, promoters of genes encoding the foilowing yeast proteins: alcohol dehydrogenase I (ADH1) or II (ADH2), CUP1, phosphoglycerate kinase (PGK), triose
122 phosphate isomerase (TPI), translational élongation factor EF-1 alpha (TEF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also referred to as TDH3, for triose phosphate dehydrogenase), galactokinase (GAL1), gai actose-1 -phosphate uridyltransferase (GAL7), UDP-galactose epimerase (GAL10), cytochrome cl (CYC1), Sec7 protein (SEC7) and acid phosphatase (PHO5), including hybrid promoters such as ADH2/GAPDH and CYC1/GAL10 promoters, and including the ADH2/GAPDH promoter, which is induced when glucose concentrations in the cell are low (e.g.t about 0.1 to about 0.2 percent), as well as the CUP1 promoter and the TEF2 promoter. Likewise, a number of upstream activation sequences (UASs), also referred to as enhancers, 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 the ADH2 UAS being used in one aspect. Since the ADH2 UAS is activated by the ADR1 gene product, it may be préférable to overexpress the ADR1 gene when a heterologous gene is operatively linked to the ADH2 UAS. Transcription termination sequences for expression in Saccharomyces cerevisiae include the termination sequences of the α-factor, GAPDH, and CYC1 genes.
[00272] Transcription control sequences to express genes in methyltrophic yeast include the transcription control régions of the genes encoding alcohol oxidase and formate dehydrogenase.
[00273] Transfection of a nucleic acid molécule into a yeast cell according to the présent invention can be accomplished by any method by which a nucleic acid molécule can be introduced into the cell and includes, but is not limited to, diffusion, active transport, bath sonication, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. Transfected nucleic acid molécules can be integrated into a yeast chromosome or maintained on extrachromosomal vectors using techniques known to those skilled in the art. Examples of yeast vehicles carrying such nucleic acid molécules are disclosed in detail herein. As discussed above, yeast cytoplast, yeast ghost, and yeast membrane particles or cell wall préparations can also be produced recombinantly by transfecting intact yeast microorganisms or yeast spheroplasts with desired nucleic acid molécules, producing the antigen therein, and then further manipulating the microorganisms or spheroplasts using techniques known to those skilled in the art to produce cytoplast, ghost or subcellular yeast membrane extract or fractions thereof containing desired antigens or other proteins.
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[00274] Effective conditions for the production of recombinant yeast vehicles and expression of the antigen and/or other protein by the yeast vehicle include an effective medium in which a yeast strain can be cultured. An effective medium is typically an aqueous medium comprising assimilable carbohydrate, nitrogen and phosphate sources, as well as appropriate salts, minerais, metals and other nutrients, such as vitamine and growth factors. The medium may comprise complex nutrients or may be a defined minimal medium. Yeast strains of the présent invention can be cultured in a variety of containers, including, but not limited to, bioreactors, Erlenmeyer flasks, test tubes, microtiter dishes, and Pétri plates. Culturing is carried out at a température, pH and oxygen content appropriate for the yeast strain. Such culturing conditions are well within the expertise of one of ordinary skill in the art (see, for example, Guthrie et al, (eds.), 1991, Methods in Enzymology, vol. 194, Academie Press, San Diego).
[00275] In some embodiments of the invention, yeast are grown under neutral pH conditions. As used herein, the general 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. One of skill the art will apprecîate that minor fluctuations (e.g., tenths or hundredths) can occur when measuring with a pH meter. As such, the use of neutral pH to grow yeast cells means that the yeast cells are grown in neutral pH for the majority of the time that they are in culture. In one embodiment, yeast are grown in a medium maintained at a pH level of at least 5.5 (i.e., the pH of the culture medium is not allowed to drop below pH 5.5). In another aspect, yeast are grown at a pH level maintained at about 6, 6.5, 7, 7.5 or 8. The use of a neutral pH in culturing yeast promûtes several biological effects that are désirable characteristics for using the yeast as vehicles for immunomodulation. For example, culturing the yeast in neutral pH allows for good growth of the yeast without négative effect on the cell génération time (e.g., slowing of doubling time). The yeast can continue to grow to high densities without losing their cell wall pliability. The use of a neutral pH allows for the production of yeast with pliable cell walls and/or yeast that are more sensitive to cell wall digesting enzymes (e.g., glucanase) at ail harvest densities. This trait is désirable because yeast with flexible cell walls can induce different or improved immune responses as compared to yeast grown under more acidic conditions, e.g., by promoting the sécrétion of cytokines by antigen presenting cells that hâve phagocytosed the yeast (e.g., THl-type cytokines including, but not limited to, IFN-γ, interleukin-12 (IL12), and IL-2, as well as proinflammatory cytokines such as IL-6). In addition, greater accessibility to the antigens located in the cell wall is afforded by such culture methods.
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In another aspect, the use of neutral pH for some antigens allows for release of the disulfîde bonded antigen by treatment with dithiothreitol (DTT) that is not possible when such an antigen-expressing yeast is cultured in media at lower pH (e.g., pH 5).
[00276] In one embodiment, control of the amount of yeast glycosylation is used to 5 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 moieties tend to be bulky. As such, the existence of sugar moieties on the surface of yeast and its impact on the three-dimensional space around the target antigen(s) should be considered in the modulation of yeast according to 10 the invention. Any method can be used to reduce the amount of glycosylation of the yeast (or increase it, if desired). For example, one could use a yeast mutant strain that has been selected to hâve low glycosylation (e.g., mnnl, ochl and mnn9 mutants), or one could elimînate by mutation the glycosylation acceptor sequences on the target antigen. Alternatively, one could use a yeast with abbreviated glycosylation patterns, e.g., Pichia. 15 One can also treat the yeast using methods that reduce or alter the glycosylation.
[00277] In one embodiment of the présent invention, as an alternative to 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 molécules that serve as an antigen and/or are useful as immunomoduiatory agents or biological response 20 modifiers 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 carrier such as a dendritic cell. Peptides and proteins can be inserted directly into yeast vehicles of the présent invention by techniques known to those skilled in the art, such as by diffusion, active transport, liposome fusion, electroporation, phagocytosis, 25 freeze-thaw cycles and bath sonication. Yeast vehicles that can be directly loaded with peptides, proteins, carbohydrates, or other molécules 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 30 therefrom. Any number of antigens and/or other agents can be loaded into a yeast vehicle in this embodiment, from at least 1, 2, 3, 4 or any whole integer up to hundreds or thousands of antigens and/or other agents, such as would be provided by the loading of a microorganism or portions thereof, for example.
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[00278] In another embodiment of the présent invention, an antigen and/or other agent is physically attached to the yeast vehicle. Physical attachment of the antigen and/or other agent to the yeast vehicle can be accomplished by any method suitable in the art, încluding covalent and non-covalent association methods which include, but are not limited to, chemically crosslinking the antigen and/or other agent to the outer surface of the yeast vehicle or biologically linking the antigen and/or other agent to the outer surface of the yeast vehicle, such as by using an antibody or other binding partner. Chemical crosslinking can be achieved, for example, by methods încluding glutaraldehyde linkage, photoaffinity labeling, treatment with carbodiimides, treatment with chemicals capable of linking di-sulfide bonds, and treatment with other cross-linking chemicals standard in the art. Altematîvely, a chemical can be contacted with the yeast vehicle that alters the charge of the lipid bilayer of yeast membrane or the composition of the cell wall so that the outer surface of the yeast is more likely to fuse or bind to antigens and/or other agent having particular charge characteristics. Targeting agents such as antibodies, binding peptides, soluble receptors, and other ligands may also be incorporated into an antigen as a fusion protein or otherwise associated with an antigen for binding of the antigen to the yeast vehicle.
[00279] When the antigen or other protein is expressed on or physically attached to the surface of the yeast, spacer arms may, in one aspect, be carefully selected to optimize antigen or other protein expression or 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 on the surface of the yeast. Thus, depending on which antigen(s) or other protein(s) are being used, one of skill in the art will select a spacer arm that effectuâtes appropriate spacing for the antigen or other protein on the yeast surface. In one embodiment, the spacer arm is a yeast protein of at least 450 amino acids. Spacer arms hâve been discussed in detail above.
[00280] In yet another embodiment, the yeast vehicle and the antigen or other protein are associated with each other by a more passive, non-specifîc or non-covalent binding mechanism, such as by gently mixing the yeast vehicle and the antigen or other protein together in a buffer or other suitable formulation (e.g., admixture).
[00281] In one embodiment of the invention, the yeast vehicle and the antigen or other protein are both loaded intracellularly into a carrier such as a dendritic cell or macrophage to form the therapeutic composition or vaccine of the présent invention. Alternatively, an antigen or other protein can be loaded into a dendritic cell in the absence of the yeast vehicle.
[00282] In one embodiment, intact yeast (with or without expression of heterologous antigens or other proteins) can be ground up or processed in a manner to produce yeast cell wall préparations, yeast membrane particles or yeast fragments (i.e., not intact) and the yeast fragments can, in some embodiments, be provided with or administered with 5 other compositions that include antigens (e.g., DNA vaccines, protein subunit vaccines, killed or inactivated pathogens) to enhance immune responses. For example, enzymatic treatment, chemical treatment or physicaï force (e.g., mechanical shearing or sonication) can be used to break up the yeast into parts that are used as an adjuvant.
[00283] In one embodiment of the invention, yeast vehicles useful in the invention 10 include yeast vehicles that have been killed or inactivated. Killing or inactivating of yeast can be accomplished by any of a variety of suitable methods known in the art. For example, heat inactivation of yeast is a standard way of inactivating yeast, and one of skill in the art can monitor the structural changes of the target antigen, if desired, by standard methods known in the art. Alternatively, other methods of inactivating the yeast can be 15 used, such as chemical, electrical, radioactive or UV methods. See, for example, the methodology disclosed in standard yeast culturing textbooks such as Methods of Enzymology, Vol. 194, Cold Spring Harbor Publishing (1990). Any of the inactivation strategies used should take the secondary, tertiary or quatemary structure of the target antigen into considération and preserve such structure as to optimize its immunogenicity.
[00284] Yeast vehicles can be formulated into yeast-based immunotherapy compositions or products of the présent invention, including préparations to be administered to a subject directly or first loaded into a carrier such as a dendritic cell, using a number of techniques known to those skilled in the art. For example, yeast vehicles can be dried by lyophilization. Formulations comprising yeast vehicles can also 25 be prepared by packing yeast in a cake or a tablet, such as is done for yeast used in baking or brewing operations. In addition, yeast vehicles can be mixed with a pharmaceutically acceptable excipient, such as an isotonie 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 aqueous physiologically balanced sait solutions. Nonaqueous 30 vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycérides may also be used. Other useful formulations include suspensions containing viscosity-enhancing agents, such as sodium carboxymethylcellulose, sorbitol, glycerol or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and
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Tris buffer, while examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise, for example, dextrose, human sérum albumin, and/or preservatives to which stérile water or saline can be added prior to administration.
[00285] In one embodiment of the present invention, a composition can include additional agents, which may also be referred to as biological response modifier 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 agent/biological response modifier compound, or a composition of the invention can be administered in conjunction with at least one agent/biological response modifier. Biological response modifiers include adjuvants and other compounds that can modulate immune responses, which may be referred to as immunomodulatory compounds, as well as compounds that modify the biological activity of another compound or agent, such as a yeast-based immunotherapeutic, such biological activity not being limited to immune System effects. Certain immunomodulatory compounds can stimulate a protective immune response whereas others can suppress a harmful immune response, and whether an immunomodulatory is useful in combination with a given yeast-based immunotherapeutic may dépend, at least in part, on the disease state or condition to be treated or prevented, and/or on the individual who is to be treated. Certain biological response modifiers preferentially enhance a cell-mediated immune response whereas others preferentially enhance a humoral immune response (i.e., can stimulate an immune response in which there is an increased level of cell-mediated compared to humoral immunity, or vice versa.). Certain biological response modifiers hâve one or more properties in common with the biological properties of yeast-based immunotherapeutics or enhance or complément the biological properties of yeast-based immunotherapeutics. There are a number of techniques known to those skilled in the art to measure 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 (e.g., a TH17 response versus a TH1 response).
[00286] Agents/biological response modifiers useful in the invention may include, but are not limited to, cytokines, chemokines, hormones, lipidic dérivatives, peptides, proteins, polysaccharides, small molécule drugs, antibodies and antigen binding fragments thereof
128 (including, but not limited to, anti-cytokine antibodies, anti-cytokine receptor antibodies, anti-chemokine antibodies), vitamins, polynucleotides, nucleic acid binding moieties, aptamers, and growth modulators. Some suitable agents include, but are not limited to, IL-1 or agonists of IL-1 or of IL-1R, anti-IL-1 or other IL-1 antagonists; IL-6 or agonists of IL-6 or of IL-6R, anti-IL-6 or other IL-6 antagonists; IL-12 or agonists of IL-12 or of IL-12R, anti-IL-12 or other IL-12 antagonists; IL-17 or agonists of IL-17 or of IL-17R, anti-IL-17 or other IL-17 antagonists; IL-21 or agonists of IL-21 or of IL-21R, anti-IL-21 or other IL-21 antagonists; IL-22 or agonists of IL-22 or of IL-22R, anti-IL-22 or other IL22 antagonists; IL-23 or agonists of IL-23 or of IL-23R, anti-IL-23 or other IL-23 antagonists; 1L-25 or agonists of IL-25 or of IL-25R, anti-IL-25 or other IL-25 antagonists; IL-27 or agonists of IL-27 or of IL-27R, anti-IL-27 or other IL-27 antagonists; type I interferon (including IFN-α) or agonists or antagonists of type I interferon or a receptor thereof; type II interferon (including IFN-γ) or agonists or antagonists of type II interferon or a receptor thereof; anti-CD40 antibody, CD40L, antiCTLA-4 antibody (e.g., to release anergie T cells); T cell co-stimulators (e.g., anti-CD137, antî-CD28, anti-CD40); alemtuzumab (e.g., CamPath®), denileukin diftitox (e.g., ONTAK®); anti-CD4; anti-CD25; anti-PD-1, anti-PD-Ll, anti-PD-L2; agents that block FOXP3 (e.g., to abrogate the activity/kill CD4+/CD25+ T regulatory cells); Flt3 ligand, Îmiquimod (Aldara™), granulocyte-macrophage colony stimulating factor (GM-CSF); granulocyte-colony stimulating factor (G-CSF), sargramostim (Leukine®); hormones including without limitation prolactin 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 agents and immunomodulators, including but not limited to, COX-2 inhibitors (e.g., Celecoxib, NSAIDS), glucocorticoïde, statins, and thalidomide and analogues thereof including IMiD™s (which are structural and functional analogues of thalidomide (e.g., REVLIMID® (lenalîdomide), ACTIMID® (pomalidomide)); proinflammatory agents, such as fungal or bacterial components or any proinflammatory cytokine or chemokine; îmmunotherapeutic vaccines including, but not limited to, virus-based vaccines, bacteria-based vaccines, or antibody-based vaccines; and any other immunomodulators, immunopotentiators, antiinflammatory agents, and/or pro-inflammatory agents. Any combination of such agents is contemplated by the invention, and any of such agents combined with or administered in a protocol with (e.g., concurrently, 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 may be used alone or in combination with other agents described herein.
[00287] Agents can include agonists and antagonists of a given protein or peptide or 5 domain thereof. As used herein, an “agonist” is any compound or agent, including without limitation small molécules, proteins, peptides, antibodies, nucleic acid binding agents, etc., 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 10 molécules, proteins, peptides, antibodies, nucleic acid binding agents, etc., that blocks or inhibits or reduces the action of an agonist.
[00288] Compositions of the invention can further include or can be administered with (concurrently, sequentialiy, or intermittently with) any other compounds or compositions that are useful for preventing or treating HBV infection or any compounds that treat or 15 ameliorate any symptom of HBV infection. A variety of agents are known to be useful for preventing and/or treating or ameliorating HBV infection. Such agents include, but are not limited to, anti-viral compounds, including, but not limited to, nucléotide analogue reverse transcriptase inhibitor (nRTIs). In one aspect of the invention, suitable anti-viral compounds include, but are not limited to: tenofovir (VIREAD®), lamivudine (EPIVIR®), 20 adefovir (HEPSERA®), telbivudine (TYZEKA®), entecavir (BARACLUDE®), and combinations thereof, and/or interferons, such as interferon-a2a or pegylated interferonct2a (PEGASYS®) or interferon-λ. These agents are typically administered for long periods of time (e.g., daily or weekly for up to one to five years or longer). In addition, compositions of the invention can be used together with other immunotherapeutic 25 compositions, including prophylactic and/or therapeutic immunotherapy. For example, prophylactic vaccines for HBV hâve been commercially available since the early 1980’s. These commercial vaccines are non-infectious, subunit viral vaccines providing purified recombinant hepatitis B virus surface antigen (HBsAg), and can be administered beginning at birth. While no therapeutic immunotherapeutic compositions hâve been 30 approved in the U.S. for the treatment of HBV, such compositions can include HBV protein or epitope subunit vaccines, HBV viral vector vaccines, cytokines, and/or other immunomodulatory agents (e.g., TLR agonists, immunomodulatory drugs).
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[00289] The invention also includes a kit comprising any of the compositions described herein, or any of the individual components of the compositions described herein.
Methods for Administration or Use of Compositions of the Invention
[00290] Compositions of the invention, which can include any one or more (e.g., combinations of two, three, four, five, or more) yeast-based immunotherapeutic compositions described herein, HBV antigens including HBV proteins and fusion proteins, and/or recombinant nucleic acid molécules encoding such HBV proteins or fusion proteins described above, and other compositions comprising such yeast-based compositions, 10 antigens, proteins, fusion proteins, or recombinant molécules described herein, can be used in a variety of in vivo and in vitro methods, including, but not limited to, to treat and/or prevent HBV infection and its sequelae, in diagnostic assays for HBV, or to produce antibodies against HBV.
[00291] One embodiment of the invention relates to a method to treat chronic hepatitis 15 B virus (HBV) infection, and/or to prevent, ameliorate or treat at least one symptom of chronic HBV 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 HBV one or more immunotherapeutic compositions of the invention. In one aspect, the composition is an immunotherapeutic composition 20 comprising one or more HBV antigens as described herein, which can include a yeastbased immunotherapeutic composition. In one aspect, the composition includes a protein or fusion protein comprising HBV antigens as described herein, and/or recombinant nucleic acid molécule encoding such protein or fusion protein. In one embodiment, the individual or population of individuals has chronic HBV infection. In one aspect, the 25 individual or population of individuals is additionally treated with at least one other therapeutic compound useful for the treatment of HBV infection. Such therapeutic compounds include, but are not limited to, direct-acting antiviral drugs (e.g., those described above or elsewhere herein) and/or interferons and/or other immunotherapeutic or immunomodulatory agents. In one aspect, such therapeutic compounds include host30 targeted therapeutics (e.g., cyclophilin inhibitors which can interfère with viral réplication, or re-entry inhibitors that can interfère with the viral life cycle (re-infection)).
[00292] “Standard Of Care” or “SOC” generally refers to the current approved standard of care for the treatment of a spécifie disease. In chronic HBV infection, SOC may be one of several different approved therapeutic protocole, and include, but may not
131 be limited to, interferon therapy and/or anti-viral therapy. Currently approved anti-viral drugs for the treatment of HBV infection include tenofovir (VIREAD®), lamivudîne (EPIVIR®), adefovir (HEPSERA®), telbivudine (ΤΎΖΕΚΑ®) and entecavir (BARACLUDE®). The anti-viral drugs prescribed most often for chronic HBV infection currently are tenofovir and entecavir. Interferon useful for the treatment of chronic HBV infection includes a type I interferon such as interferon-α, including, but not limited to interferon-a2 or pegylated Înterferon-a2 (e.g., PEGASYS®). In one embodiment, the interferon is a type III interferon, including without limitation, interferon-λΐ, interferon-X2, and/or interferon-X3. The immunotherapeutic composition of the invention can be administered prior to, concurrently with, intermittently with, and/or after one or more antiviral(s) and/or interferon and/or other immunotherapeutic or immunomodulatory agents. The other therapeutic compounds may also be administered prior to or after treatment with the immunotherapeutic compositions of the invention.
[00293] HBV infection is typically diagnosed in an individual by détection of HBsAg (hepatitis B virus surface antigen) and/or HBeAg (e-antigen) in the blood of the infected individual. The détection of HBeAg in the sérum reflects active viral réplication, and clinical outcome of infection can be correlated with e-antigen status, although long-term remission (or cure) is better predicted using HBsAg séroconversion when using current thérapies (see below). Détection of IgM core antibody may also be used to detect acute HBV infection during the first 6-12 months of infection. Persistence of HBsAg in the blood for more than 6 months typically identifies chronic HBV infection. In addition, chronic HBV infection can be diagnosed by identîfying HBV DNA (>2000 IU/ml), which can be combined with détection or identification of elevated sérum alanine aminotransferase (ALT) and/or aspartate aminotrasferase (AST) levels (e.g., more than twice the upper limit of normal).
[00294] Recovery from the viral infection (complété response, or the endpoint for a treatment of HBV) is determined by HBeAg/HBsAg séroconversion, which is loss of HBeAg and HBsAg, respectively, and the development of antibodies against the hepatitis B surface antigen (anti-HBs) and/or antibodies against HBeAg. Clinical studies hâve defined séroconversion, or a protective antibody (anti-HBs) level as: (a) 10 or more sample ratio units (SRU) as determined by radioimmunoassay; (b) a positive resuit as determined by enzyme immunoassay; or (c) détection of an antibody concentration of >10 mlU/ml (10 SRU is comparable to 10 mlU/mL of antibody). Séroconversion can take years to develop in a chronically infected patient under current standard of care treatment
132 (i.e., anti-viral drugs or interferon). Patients can also be monitored for loss or marked réduction of viral DNA (below détectable levels by PCR or <2000 IU/ml), normalization of sérum alanine aminotransferase (ALT) levels, and improvement in liver inflammation and fibrosis. “ALT” is a well-validated measure of hepatîc injury and serves as a surrogate for hepatic inflammation. In prior large hepatitis trials, réductions and/or normalization of ALT levels (ALT normalization) hâve been shown to correlate with împroved liver function and reduced liver fibrosis as determined by serial biopsy.
[00295] Another embodiment of the invention relates to a method to immunize an individual or population of individuals against HBV in order to prevent HBV infection, prevent chronic HBV infection, and/or reduce the severity of HBV infection in the individual or population of individuals. The method includes the step of administering to an individual or population of individuals that is not infected with HBV (or believed not to be infected with HBV), a composition of the invention. In one aspect, the composition is an immunotherapeutic composition comprising one or more HBV antigens as described herein, including one or more yeast-based immunotherapeutic compositions. In one aspect, the composition includes a fusion protein comprising HBV antigens as described herein, or recombinant nucleic acid molécule encoding such fusion protein.
[00296] As used herein, the phrase “treat” HBV infection, or any permutation thereof (e.g., “treated for HBV infection”, etc.) generally refers to applying or administering a composition of the invention once the infection (acute or chronic) has occurred, with the goal of réduction or élimination of détectable viral titer (e.g., réduction of viral DNA (below détectable levels by PCR or <2000 IU/ml)), reaching séroconversion (development of antibodies against HBsAg and/or HBeAg and concurrent loss or réduction of these proteins from the sérum), réduction in at least one symptom resulting from the infection in the individual, delaying or preventing the onset and/or severity of symptoms and/or downstream sequelae caused b y the infection, réduction of organ or physiological System damage (e.g., cirrhosis) resulting from the infection (e.g., réduction of abnormal ALT levels, réduction of liver inflammation, réduction of liver fibrosis), prévention and/or réduction in the frequency and incidence of hepatocellular carcinoma (HCC), improvement in organ or system function that was negatively impacted by the infection (normalization of sérum ALT levels, improvement in liver inflammation, improvement in liver fibrosis), improvement of immune responses against the infection, improvement of long term memory immune responses against the infection, reduced réactivation of HBV virus, and/or improved general health of the individual or population of individuals.
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[00297] In one aspect, a goal of treatment is sustained viral clearance for at least 6 months after the completion of therapy. In one aspect, a goal of treatment is the loss of détectable sérum HBeAg and/or HBsAg proteins. In one aspect, a goal of treatment is the development of antibodies against the hepatitis B surface antigen (anti-HBs) and/or antibodies against HBeAg. In one aspect, the goal of treatment is séroconversion, which may be defined by: (a) 10 or more sample ratio units (SRU) as determined by radioimmunoassay; (b) a positive resuit as determined by enzyme immunoassay; or (c) détection of an antibody concentration of >10 mlU/ml (10 SRU is comparable to 10 mlU/mL of antibody).
[00298] To “prevent” HBV infection, or any permutation thereof (e.g., “prévention of HBV infection”, etc.), generally refers to applying or administering a composition of the invention before an infection with HBV has occurred, with the goal of preventing infection by HBV, preventing chronic infection by HBV (i.e., enabling an individual to clear an acute HBV infection without further intervention), or, should the infection later occur, at least reducing the severity, and/or length of infection and/or the physiological damage caused by the chronic infection, including preventing or reducing the severity or incidence of at least one symptom resulting from the infection in the individual, and/or delaying or preventing the onset and/or severity of symptoms and/or downstream sequelae caused by the infection, in an individual or population of individuals. In one aspect, the présent invention can be used to prevent chronic HBV infection, such as by enabling an individual who becomes acutely infected with HBV subséquent to administration of a composition of the invention to clear the infection and not become chronically infected.
[00299] The présent invention includes the delivery (administration, immunization) of one or more immunotherapeutic compositions of the invention, including a yeast-based immunotherapy composition, to a subject. The administration process can be performed ex vivo or in vivo, but is typically performed in vivo. Ex vivo administration refers to performing part of the regulatory step outside of the patient, such as administering a composition of the présent invention to a population of cells (dendritic cells) removed from a patient under conditions such that a yeast vehicle, antigen(s) and any other agents or compositions are loaded into the cell, and retuming the cells to the patient. The therapeutic composition of the présent invention can be returned to a patient, or adminîstered to a patient, by any suitable mode of administration.
[00300] Administration of a composition can be systemic, mucosal and/or proximal to the location of the target site (e.g., near a site of infection). Suitable routes of
134 administration will be apparent to those of skill in the art, depending on the type of condition to be prevented or treated, the antigen used, and/or the target cell population or tissue. Various acceptable methods of administration include, but are not limited to, intravenous administration, intraperitoneal administration, intramuscular administration, intranodal administration, intracoronary administration, intraarterial administration (e.g., into a carotid artery), subcutaneous administration, transdermal delivery, intratracheal administration, intraarticular administration, intraventricular administration, inhalation (e.g., aérosol), intracranial, intraspinal, intraocular, aurai, intranasal, oral, pulmonary administration, imprégnation of a cathéter, 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. Parentéral delivery can include intradermal, intramuscular, intraperitoneal, intrapleural, intrapulmonary, intravenous, subcutaneous, atrial cathéter and vénal cathéter routes. Aurai delivery can include ear drops, intranasal delivery can include nose drops or intranasal injection, and intraocular delivery can include eye drops. Aérosol (inhalation) delivery can also be performed using methods standard 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. Such routes include bronchial, intradermal, intramuscular, intranasal, other inhalatory, rectal, subcutaneous, topical, transdermal, vaginal and uréthral routes. In one aspect, an îmmunotherapeutic composition of the invention is administered subcutaneously.
[00301] 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 providing a yeast vehicle and an antigen (if included) to a given cell type, tissue, or région of the patient body in an amount effective to elicit an antigen-specific immune response against one or more HBV antigens or epitopes, when administered one or more times over a suitable time period. For example, in one embodiment, a single dose of a yeast vehicle of the présent invention is from about 1 x 105 to about 5 x 107 yeast cell équivalents per kilogram body weight of the organism being administered the composition. In one aspect, a single dose of a yeast vehicle of the présent invention is from about 0.1 Y.U. (1 x 106 cells) to about 100 Y.U. (1 x 109 cells) per dose (i.e., per organism), including any intérim dose, in incréments of 0.1 x 106 cells (i.e., 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
135 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, the doses are administered at different sites on the individual but during the same dosing period. For example, a 40 Y.U. dose may be administered via by injecting 10 Y.U. doses to four different sites on the individual during one dosing period, or a 20 Y.U. dose may be administered by injecting 5 Y.U. doses to four different sites on the individual, or by injecting 10 Y.U. doses to two different sites on the individual, during the same dosing period. The invention includes administration of an amount 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 Y.U. or more) at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different sites on an individual to form a single dose.
[00302] Boosters or boosts of a therapeutic composition are administered, for example, when the immune response against the antigen has waned or as needed to provide an immune response or induce a memory response against a particular antigen or antigen(s). Boosters can be administered from about 1, 2, 3,4,5,6,7, or 8 weeks apart, to monthly, to bimonthly, to quarterly, to annually, to several years after the original administration. In one embodiment, an administration schedule is one in which from about 1 x 10 to about 5x10 yeast cell équivalents of a composition per kg body weight of the organism is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times over a time period of from weeks, to months, to years. In one embodiment, the doses are administered weekly for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses, followed by monthly doses as needed to achieve the desired inhibition or élimination of the HBV virus. For example, the doses can be administered until the individual achieves séroconversion, until HBV DNA titers fall below 2000 IU/ml, and/or until ALT levels normalize. In one embodiment, the doses are administered in a 4-weekly protocol (every 4 weeks, or on day 1, week 4, week 8, week 12, etc., for between 2 and 10 doses or longer as determined by the clinician). Additional doses can be administered even after the individual achieves séroconversion, if desired, although such dosing may not be necessary.
[00303] With respect to administration of yeast-based immunotherapeutic compositions described herein, a single composition can be administered to an individual or population of individuals or combination of such compositions can be administered.
For example, the invention provides several “single protein” compositions or compositions directed against a particular génotype, as well as multi-protein compositions and compositions that target multiple génotypes, or sub-genotypes. Accordingly, two or more
compositions can be selected in a “spice rack” approach to most effectively prevent or treat HBV infection in a given individual or population of individuals.
[00304] In one aspect of the invention, one or more additional therapeutic agents are administered sequentially with the yeast-based immunotherapy composition. In another 5 embodiment, one or more additional therapeutic agents are administered before the yeastbased immunotherapy composition is administered. In another embodiment, one or more additional therapeutic agents are administered after the yeast-based immunotherapy composition is administered. In one embodiment, one or more additional therapeutic agents are administered in alternating doses with the yeast-based immunotherapy 10 composition, or in a protocol in which the yeast-based composition is administered at prescribed intervals in between or with one or more consecutive doses of the additional agents, or vice versa. In one embodiment, the yeast-based immunotherapy composition is administered in one or more doses over a period of time prior to commencing the administration of the additional agents. In other words, the yeast-based 15 immunotherapeutic composition is administered as a monotherapy for a period of time, and then the agent administration is added, either concurrently with new doses of yeastbased immunotherapy, or in an alternating fashion with yeast-based immunotherapy. Alternatively, the agent may be administered for a period of time prior to begînning administration of the yeast-based immunotherapy composition. In one aspect, the yeast is 20 engineered to express or carry the agent, or a different yeast is engineered or produced to express or carry the agent.
[00305] In one aspect of the invention, when a treatment course of interferon or antiviral compound therapy begins, additional doses of the immunotherapeutic composition are administered over the same period of time, or for at least a portion of that time, and may continue to be administered once the course of interferon or anti-viral compound has ended. However, the dosing schedule for the immunotherapy over the entire period may be, and is expected to typically be, different than that for the interferon or the anti-viral compound. For example, the immunotherapeutic composition may be administered on the same days or at least 3-4 days after the last given (most recent) dose of interferon or anti30 viral (or any suitable number of days after the last dose), and may be administered daily, weekly, biweekly, monthly, bimonthly, or every 3-6 months, or at longer intervals as determined by the physician. During an initial period of monotherapy administration of the immunotherapeutic composition, if utilized, the immunotherapeutic composition is preferably administered weekly for between 4 and 12 weeks, followed by monthly
137 administration (regardless of when the additional interferon or anti-viral therapy is added into the protocol). In one aspect, the immunotherapeutic composition is administered weekly for four or five weeks, followed by monthly administration thereafter, until conclusion of the complété treatment protocol.
[00306] In aspects of the invention, an immunotherapeutic composition and other agents can be administered together (concurrently). As used herein, concurrent use does not necessarily mean that ail doses of ail compounds are administered on the same day at the same time. Rather, concurrent use means that each of the therapy components (e.g., immunotherapy and interferon therapy, or immunotherapy and anti-viral therapy) are started at approximately the same period (within hours, or up to 1-7 days of each other) and are administered over the same general period of time, noting that each component may hâve a different dosing schedule (e.g., interferon weekly, immunotherapy monthly, anti-viral daily or weekly). In addition, before or after the concurrent administration period, any one of the agents or immunotherapeutic compositions can be administered without the other agent(s).
[00307] It is contemplated by the présent invention that the use of an immunotherapeutic composition of the invention with an anti-viral such as tenofovir or entecavir will enable a shorter time course for the use of the anti-viral drug. Similar results are expected when combining an immunotherapeutic of the invention with interferon. Dosing requirements for the anti-viral or interferon may also be reduced or modified as a resuit of combination with the immunotherapeutic of the invention to generally improve the tolérance of the patient for the drug. In addition, it is contemplated that the immunotherapeutic composition of the invention will enable séroconversion or sustained viral responses for patients in whom anti-viral therapy alone fails to achieve these endpoints. In other words, more patients will achieve séroconversion when an immunotherapeutic composition of the invention is combined with an anti-viral or interferon than will achieve séroconversion by using anti-virals or interferon alone. Under current SOC for HBV infection, anti-virals may be administered for 6 months to one year, two years, three years, four years, five years, or longer (e.g., indefinitely). By combining such therapy with an immunotherapeutic composition of the invention, the time for the administration of the anti-viral may be reduced by several months or years. It is contemplated that use of the immunotherapeutic compositions of the présent invention, as a monotherapy or in combination with anti-viral and/or immunomodulatory approaches will be effective to achieve loss of HBsAg and/or HBeAg; HBeAg séroconversion,
138
HBsAg séroconversion, or complété séroconversion; and in many mdividuals, sustained viral clearance for at least 6 months after the completion of therapy. In some patients, immunotherapy according to the présent invention, when used as a monotherapy or in combination with anti-viral and/or immunomodulatory approaches, may achieve loss of HBsAg and/or HBeAg, but not achieve séroconversion (development of anti-HBs or antiHBeAg). In this scénario, it is an embodiment of the invention to additionally use, alone or in combination with the yeast-based immunotherapy of the invention and/or anti-virals or other immunomodulatory agents, an agent such as the current prophylactic recombinant HBV subunit vaccine, in order to achieve complété response in the patient.
[00308] As used herein, the term “anti-viral” refers to any compound or drug, typically a small-molecule inhibitor or antibody, which targets one or more steps in the virus life cycle with direct anti-viral therapeutic effects. In one embodiment of the invention, the anti-viral compound or drug to be administered in the same therapeutic protocol with an immunotherapeutic composition of the invention is selected from tenofovir (VIREAD®), lamivudine (EPIVIR®), adefovir (HEPSERA®), telbivudine (TYZEKA®) and entecavir (BARACLUDE®), or any analog or dérivative thereof, or any composition comprising or containing such compound, drug, analog or dérivative.
[00309] Tenofovir (tenofovir disoproxil fumarate or TDF), or ({[(2R)-l-(6-amino-9Hpurin-9-yl)propan-2-yl]oxy}methyl)phosphonic acid, is a nucléotide analogue 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 disproxil fumarate) once daily. Dosage for pédiatrie patients is based on body weight of the patient (8 mg per kg body weight, up to 300 mg once daily) and may be provided as tablet or oral powder.
[00310] Lamivudine, or 2',3'-dideoxy-3'-thiacytidine, commonly called 3TC, is a potent nucleoside analog reverse transcriptase inhibitor (nRTI). For the treatment of HBV infection, lamivudine is administered as a pill or oral solution taken at a dose of lOOmg once a day (1.4-2mg/lb. twice a day for children 3 months to 12 years old).
[00311] Adefovir (adefovir dipivoxil), or 9-[2-[[bis[(pivaloyloxy)methoxy]phosphinyl]-methoxy]ethyl]adenine, is an orally-administered nucléotide 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 daily.
[00312] Telbivudine, or l-(2-deoxy-p-L-erythro-pentofuranosyl)-5-methylpyrimidine2,4(lH,3H)-dione, is a synthetic thymidine nucleoside analogue (the L-isomer of
139 thymidine). For the treatment of HBV infection, telbivudine is administered as a pill or oral solution taken at a dose of 600 mg once daily.
[00313] Entecavir, or 2-Amino-9-[(lS,3R,4S)-4-hydroxy-3-(hydroxymethyl)-2methylidenecyclopentyl]-6,9-dihydro-3H-purin-6-one, is a nucleoside analog (guanine analogue) that inhibits reverse transcription, DNA réplication and transcription of the virus. 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 lamivudine-refractory or telbivudine résistance mutations).
[00314] In one embodiment of the invention, the interferon to be administered in a therapeutic protocol with an immunotherapeutic composition of the invention is an interferon, and in one aspect, interferon-α, and in one aspect, interferon-a2b (administered by subcutaneous injection 3 times per week); or pegylated interferon-a2a (e.g. PEGASYS®). As used herein, the term “interferon” refers to a cytokine that is typically produced by cells of the immune system and by a wide variety of cells in response to the presence of double-stranded RNA. Interferons assist the immune response by inhibiting viral réplication within host cells, activating natural killer cells and macrophages, increasing antigen présentation to lymphocytes, and inducing the résistance of host cells to viral infection. Type I interferons include interferon-α. Type III interferons include interferon-λ. Interferons useful in the methods of the présent 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, pegylated interferons, interferon fusion proteins (interferon fused to albumin), and controlled-release formulations comprising interferon (e.g., interferon in microspheres or interferon with polyaminoacid nanoparticles). One interferon, PEGASYS®, pegylated interferon-a2a, is a covalent conjugate of recombinant interferon-a2a (approximate molecular weight [MW] 20,000 daltons) with a single branched bis-monomethoxy polyethylene glycol (PEG) chain (approximate MW 40,000 daltons). The PEG moiety is linked at a single site to the interferon-α moiety via a stable amide bond to lysine. Pegylated interferon-a2a has an approximate molecular weight of 60,000 daltons.
[00315] Interferon is typically administered by intramuscular or subcutaneous injection, and can be administered in a dose of between 3 and 10 million units, with 3 million units being preferred in one embodiment. Doses of interferon 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, A typical dose of interferon that is currently available is
140 provided weekly, and that is a preferred dosing schedule for interferon, according to the présent invention. For the treatment of HBV, pegylated interferon-a2a is currently administered subcutaneously once a week at a dose of 180 mg (1.0 ml viral or 0.5 ml prefilled syringe), for a total of 48 weeks. The dose amount and timing can be varied according to the préférences and recommendations of the physician, as well as according to the recommendations for the particular interferon being used, and it is within the abilities of those of skill in the art to détermine the proper dose. It is contemplated that by using interferon therapy together with an immunotherapeutic composition of the invention, the dose strength and/or number of doses of interferon (length of time on interferon and/or intervals between doses of interferon) can be reduced.
[00316] In the method of the présent invention, compositions and therapeutic compositions can be administered to animal, including any vertebrate, and particularly to any member of the Vertebrate class, Mammalia, including, without limitation, primates, rodents, livestock and domestic pets. Livestock include mammals to be consumed or that produce useful products (e.g., sheep for wool production). Mammals to treat or protect include humans, dogs, cats, mice, rats, goats, sheep, cattle, horses and pigs.
[00317] An “individual” is a vertebrate, such as a rnammal, including without limitation a human. Mammals include, but are not limited to, farm animais, sport animais, pets, primates, mice and rats. The term “individual” can be used interchangeably with the term “animal”, “subject” or “patient”.
General Techniques Useful in the Invention
[00318] The practice of the présent invention will employ, uniess otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as, Methods of Enzymologv. Vol. 194, Guthrie et al., eds., Cold Spring Harbor Laboratory Press (1990); Biology and activities of yeasts, Skinner, et al., eds., Academie 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 édition (Sambrook et al., 1989) and Molecular
141
Cloning: A Laboratory Manual, third édition (Sambrook and Russel, 2001), (jointly referred to herein as “Sambrook”); Current Protocols in Molecular Biology (F.M. Ausubel et aL, eds., 1987, including suppléments through 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) Usine Antibodies: A Laboratory ManuaL Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (jointly referred to herein as “Harlow and Lane”), Beaucage et al. eds., Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, Inc., New York, 2000); Casarett and Douil’s Toxicoloev The Basic Science of Poisons. C. Klaassen, ed., 6th édition (2001), and Vaccines, S. Plotkin and W. Orenstein, eds., 3rd édition (1999). General Définitions
[00319] A “TARMOGEN®” (Globelmmune, Inc., Louisville, Colorado) generally refers to a yeast vehicle expressing one or more heterologous antigens extracellularly (on its surface), intracellularly (internally or cytosolically) or both extracellularly and intracellularly. TARMOGEN® products hâve been generally described (see, e.g., U.S. Patent No. 5,830,463). Certain yeast-based immunotherapy compositions, and methods of making and generally using the same, are also described in detail, for example, in U.S. Patent No. 5,830,463, U.S. Patent No. 7,083,787, U.S. Patent 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 2008 Jan 24;26(4):509-21, each of which is incorporated herein by reference in its entirety.
[00320] 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 one atom by an atom of a different element or in the presence of a particular functional group, or the replacement of one functional group by another functional group). Thus, an analog is a compound that is similar or comparable in function and appearance, but has a different structure or origin with respect to the reference compound.
[00321] The terms substituted, substituted dérivative and dérivative, when used to describe a compound, means that at least one hydrogen bound to the unsubstituted compound is replaced with a different atom or a chemical moiety.
[00322] Although a dérivative has a similar physical structure to the parent compound, the dérivative may hâve different chemical and/or biological properties than the parent compound. Such properties can include, but are not limited to, increased or decreased
142 activity of the parent compound, new activity as compared to the parent compound, enhanced or decreased bioavailability, enhanced or decreased efficacy, enhanced or decreased stability in vitro and/or in vivo, and/or enhanced or decreased absorption properties.
[00323] In general, the term biologically active indicates that a compound (încluding a protein or peptide) has at least one détectable activity that has an effect on the metabolic or other processes of a cell or organism, as measured or observed in vivo (Le., in a natural physiological environment) or in vitro (i.e., under laboratory conditions).
[00324] According to the présent invention, the term “modulate” can be used interchangeably with “regulate” and refers generally to upregulation or downregulatîon of a particular activity. As used herein, the term “upregulate” can be used generally to describe any of: elicitation, initiation, increasing, augmenting, boosting, improving, enhancing, amplifying, promoting, or providing, with respect to a particular activity. Similarly, the term “downregulate” can be used generally to describe any of: decreasing, reducing, inhibiting, ameliorating, diminishing, lessening, blocking, or preventing, with respect to a particular activity.
[00325] In one embodiment of the présent invention, any of the amino acid sequences described herein can be produced with from at least one, and up 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 referred to as consisting essentially of the specified amino acid sequence. According to the présent invention, the heterologous amino acids are a sequence of amino acids that are not naturally found (Âe., not found in nature, in vivo) flanking the specified amino acid sequence, or that are not related to the function of the specified amino acid sequence, or that would not be encoded by the nucléotides that flank the naturally occurring nucleic acid sequence encoding the specified amino acid sequence as it occurs in the gene, if such nucléotides in the naturally occurring sequence were translated using standard codon usage for the organism from which the given amino acid sequence is derived. Similarly, the phrase consisting essentially of, when used with reference to a nucleic acid sequence herein, refers to a nucleic acid sequence encoding a specified amino acid sequence that can be flanked by from at least one, and up to as many as about 60, additional heterologous nucléotides at each of the 5' and/or the 3' end of the nucleic acid sequence encoding the specified amino acid sequence. The heterologous nucléotides are not naturally found (i.e., not found in nature, in vivo) flanking the nucleic acid sequence encoding the specified
143 amino acid sequence as it occurs in the natural gene or do not encode a protein that imparts any additional function to the protein or changes the function of the protein having the specified amino acid sequence.
[00326] According to the présent invention, the phrase selectively binds to refers to the ability of an antibody, antigen-binding fragment or binding partner of the présent invention to preferentially bind to specified proteins. More specifically, the phrase selectively binds refers to the spécifie binding of one protein to another (e.g., an antibody, fragment thereof, or binding partner to an antigen), wherein the level of binding, as measured by any standard assay (e.g., an immunoassay), is statistically significantly higher than the background control for the assay. For example, when performing an immunoassay, controls typically include a reaction well/tube that contain antibody or antigen binding fragment alone (i.e., in the absence of antigen), wherein an amount of reactivity (e.g., non-specific binding to the well) by the antibody or antigen-binding fragment thereof in the absence of the antigen is considered to be background. Binding can be measured using a variety of methods standard in the art including enzyme immunoassays (e.g., ELISA, immunoblot assays, etc.).
[00327] Reference to a protein or polypeptide used in the présent invention includes full-iength proteins, fusion proteins, or any fragment, domain, conformational epîtope, or homologue of such proteins, including functional domains and îmmunological domains of proteins. More specifically, an isolated protein, according to the présent invention, is a protein (including a polypeptide or peptide) that has been removed from its natural milieu (/.e., that has been subject to human manipulation) and can include purified proteins, partially purified proteins, recombinantly produced proteins, and synthetically produced proteins, for example. As such, isolated does not reflect the extent to which the protein has been purified. Preferably, an isolated protein of the présent invention is produced recombinantly. According to the présent invention, the terms modification and mutation can be used interchangeably, particularly with regard to the modifications/mutations to the amino acid sequence of proteins or portions thereof (or nucleic acid sequences) described herein.
[00328] As used herein, the term homologue is used to refer to a protein or peptide which differs from a naturally occurring protein or peptide (i.e., the prototype or wildtype protein) by minor modifications to the naturally occurring protein or peptide, but which maintains the basic protein and side chain structure of the naturally occurring form.
Such changes include, but are not limited to: changes in one or a few amino acid side
144 chains; changes one or a few amino acids, including délétions (e.g., a truncated version of the protein or peptide) insertions and/or substitutions; changes in stereochemistry of one or a few atoms; and/or minor derivatizations, including but not limited to: méthylation, glycosylation, phosphorylation, acétylation, myristoylation, prénylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol. A homologue can hâve enhanced, decreased, or substantially similar properties as compared to the naturally occurring protein or peptide. A homologue can include an agonist of a protein or an antagonist of a protein. Homologues can be produced using techniques known in the art for the production of proteins including, but not limited to, direct modifications to the isolated, naturally occurring protein, direct protein synthesis, or modifications to the nucleic acid sequence encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.
[00329] A homologue of a given protein may comprise, consist essentially of, or consîst 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 about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least about 99% identical (or any percent identity between 45% and 99%, in whole integer incréments), to the 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 incréments of 1%, to less than about 70% identical to the naturally occurring amino acid sequence of the reference protein.
[00330] A homologue may include proteins or domains of proteins that are “near fulllength”, which means that such a homologue differs from the full-length protein, functional domain or immunologicai domain (as such protein, functional domain or immunological domain is described herein or otherwise known or described in a pubiicly available sequence) by the addition of or délétion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the N- and/or the C-terminus of such full-length protein or full-length functional domain or full-length immunological domain.
145
[00331] As used herein, unless otherwise specified, reference to a percent (%) identity refers to an évaluation of homology which 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 default parameters, wherein the query sequence is filtered for low complexity régions by default (described in Altschul, S.F., Madden, T.L., Schiiaffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J. (1997) Gapped BLAST and PSIBLAST: a new génération 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 standard default parameters (Position-Specific Iterated BLAST. It is noted that due to some différences in the standard parameters between BLAST 2.0 Basic BLAST and BLAST 2, two spécifie sequences might 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 sequences as the query sequence may not identify the second sequence in the top matches. In addition, PSI-BLAST provides an automated, easy-to-use version of a profile search, which is a sensitive way to look for sequence homologues. The program first performs a gapped BLAST database search. The PSI-BLAST program uses the information from any significant alignments returned to construct a position-specific score matrix, which replaces the query sequence for the next round of database searching. Therefore, it is to be understood that percent identity can be determined by using any one of these programs.
[00332] Two spécifie sequences can be aligned to one another using BLAST 2 sequence as described in Tatusova and Madden, (1999), Blast 2 sequences - a new tool for comparing protein and nucléotide sequences, FEMS Microbiol Lett. 174:247-250, incorporated herein by reference in its entirety. BLAST 2 sequence alignment is performed in blastp or blastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search (BLAST 2.0) between the two sequences allowing for the introduction of gaps (délétions and insertions) in the resulting alignment. For purposes of clarity herein, a BLAST 2 sequence alignment is performed using the standard default parameters as follows.
For blastn, using 0 BLOSUM62 matrix:
Reward for match = 1
Penalty for mismatch = -2
Open gap (5) and extension gap (2) penalties gap x_dropoff (50) expect (10) word size (11) filter (on)
146
For blastp, using 0 BLOSUM62 matrix:
Open gap (11) and extension gap (1) penalties gap x_dropoff (50) expect (10) word size (3) filter (on).
[00333] An isolated nucleic acid molécule is a nucleic acid molécule that has been removed from its naturel milieu (i.e., that has been subject to human manipulation), its natural milieu being the genome or chromosome in which the nucleic acid molécule is found in nature. As such, isolated does not necessarily reflect the extent to which the nucleic acid molécule has been purified, but indicates that the molécule does not include an entire genome or an entire chromosome in which the nucleic acid molécule is found in nature. An isolated nucleic acid molécule can include a gene. An isolated nucleic acid molécule that includes a gene is not a fragment of a chromosome that includes such gene, but rather includes the coding région and regulatory régions associated with the gene, but no additional genes that are naturally found on the same chromosome. An isolated nucleic acid molécule can also include a specified nucleic acid sequence flanked by (i.e., at the 5' and/or the 3' end of the sequence) additional nucleic acids that do not normally flank the specified nucleic acid sequence in nature (i.e., heterologous sequences). Isolated nucleic acid molécule can include DNA, RNA (e.g., mRNA), or dérivatives of either DNA or RNA (e.g., cDNA). Although the phrase nucleic acid molécule primarily refers to the physical nucleic acid molécule and the phrase nucleic acid sequence primarily refers to the sequence of nucléotides on the nucleic acid molécule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molécule, or a nucleic acid sequence, being capable of encoding a protein or domain of a protein.
[00334] A recombinant nucleic acid molécule is a molécule that can include at least one of any nucleic acid sequence encoding any one or more proteins described herein operatively linked to at least one of any transcription control sequence capable of effectively regulating expression of the nucleic acid molecule(s) in the cell to be transfected. Although the phrase nucleic acid molécule primarily refers to the physical nucleic acid molécule and the phrase nucleic acid sequence primarily refers to the sequence of nucléotides on the nucleic acid molécule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molécule, or a nucleic acid sequence, being capable of encoding a protein. In addition, the phrase recombinant molécule primarily refers to a nucleic acid molécule operatively linked to a transcription
control sequence, but can be used interchangeably with the phrase nucleic acid molécule which is administered to an animal.
[00335] A recombinant nucleic acid molécule includes a recombinant vector, which is any nucleic acid sequence, typically a heterologous sequence, which is operatively linked 5 to the isolated nucleic acid molécule encoding a fusion protein of the présent invention, which is capable of enabling recombinant production of the fusion protein, and which is capable of delivering the nucleic acid molécule into a host cell according to the présent invention. Such a vector can contain nucleic acid sequences that are not naturally found adjacent to the isolated nucleic acid molécules to be inserted into the vector. The vector 10 can be either RNA or DNA, either prokaryotic or eukaryotic, and preferably in the présent invention, is a virus or a plasmid. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating of nucleic acid molécules, and can be used in delivery of such molécules (e.g., as in a DNA composition or a viral vector-based composition). Recombinant vectors are preferably used in the expression of nucleic acid 15 molécules, and can also be referred to as expression vectors. Preferred recombinant vectors are capable of being expressed in a transfected host cell.
[00336] In a recombinant molécule of the présent invention, nucleic acid molécules are operatively linked to expression vectors containing regulatory sequences such as transcription control sequences, translation control sequences, origins of réplication, and 20 other regulatory sequences that are compatible with the host cell and that control the expression of nucleic acid molécules of the présent invention. In particular, recombinant molécules of the présent invention include nucleic acid molécules that are operatively linked to one or more expression control sequences. The phrase operatively linked refers to linking a nucleic acid molécule to an expression control sequence in a manner 25 such that the molécule is expressed when transfected (i.e., transformed, transduced or transfected) into a host cell.
[00337] According to the présent invention, the term “transfection” is used to refer to any method by which an exogenous nucleic acid molécule (i.e., a recombinant nucleic acid molécule) can be inserted into a cell. The term transformation can be used 30 interchangeably with the term transfection when such term is used to refer to the introduction of nucleic acid molécules 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. Therefore, transfection techniques
148 include, but are not limited to, transformation, chemical treatment of cells, particle bombardment, electroporation, microinjection, lipofection, adsorption, infection and protoplast fusion.
[00338] The following experimental results are provided for purposes of illustration and are not intended to limit the scope of the invention.
EXAMPLES
Example 1
[00339] The following example describes the production of a yeast-based immunotherapeutic composition for the treatment or prévention of hepatitis B virus (HBV) infection.
[00340] In this experiment, yeast (e.g., Saccharomyces cerevisiae) were engineered to express various HBV surface-core fusion proteins, each having the basic structure shown in Fig. 2, under the control of the copper-inducible promoter, CUP1, or the TEF2 promoter. In each case, the HBV fusion protein was a single polypeptide of approximately 595 amino acids, with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:34 (1) an N-terminal peptide to impart résistance to proteasomal dégradation and stabilize expression (positions 1 to 6 of SEQ ID NO:34); 2) a two amino acid spacer (Thr-Ser) to introduce a Spel restriction enzyme site; 3) the amino acid sequence of a near full-length (minus position 1) HBV génotype C large (L) surface antigen (e.g., positions 9 to 407 of SEQ ID NO:34, corresponding to positions 2-400 of SEQ ID NO:11, which dîffers from SEQ ID NO:34 at positions 350-351 of SEQ ID NO:11, where a Leu-Val sequence in SEQ ID NO:11 is replaced with a Gln-Ala sequence at positions 357-358 of SEQ ID NO:34); 4) the amino acid sequence of an HBV core antigen (e.g., positions 408 to 589 of SEQ ID NO:34 or positions 31-212 of SEQ ID NO:9); and 5) a hexahistidine tag (positions 590-595 of SEQ ID NO:34). A nucleic acid sequence encoding the fusion protein of SEQ ID NO:34 (codon optimized for yeast expression) is represented herein by SEQ ID NO:33. Positions 28-54 of SEQ ID NO:34 comprise the hépatocyte receptor portion of large (L) surface protein. SEQ ID NO:34 contains multiple epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. For example, at positions 209-220, positions 389-397, positions 360367, and positions 499-506, with respect to SEQ ID NO:34, comprise known MHC Class I binding and/or CTL epitopes. Positions 305-328 of SEQ ID NO:34 comprise an antibody epitope. This fusion protein and corresponding yeast-based immunotherapeutic
149 comprising this protein can be generally referred to herein as Score”, “MADEAP-Score”, “Μ-Score”, or “GI-13002”.
[00341] Briefly, DNA encoding nearly full length large surface antigen (L) fused to full length core antigen was codon optimized for expression in yeast, and then digested 5 with EcoRI and Notl and inserted behind the CUP1 promoter (pGI-100), or the TEF2 promoter (pTK57-l), in yeast 2 um expression vectors. The fusion protein encoded by these constructs is represented herein by SEQ ID NO:34 (encoded by nucléotide sequence SEQ ID NO:33) and has an expected approximate molecular weight of 66 kDa. The resulting plasmids were introduced into Saccharomyces cerevisiae W303a yeast by 10 Lithium acetate/polyethylene glycol transfection, and primary transfectants were selected on solid minimal plates lacking uracil (UDM; uridine dropout medium). Colonies were re-streaked onto UDM or ULDM (uridine and leucine dropout medium) and allowed to grow for 3 days at 30°C. Liquid cultures lacking uridine (U2 medium: 20g/L glucose; 6.7 g/L of yeast nitrogen base containing ammonium sulfate; 0.04 mg/mL each of histidine, 15 leucine, tryptophan, and adenine) or lacking uridine and leucine (UL2 medium: 20g/L glucose; 6.7 g/L of yeast nitrogen base containing ammonium sulfate; and 0.04 mg/mL each of his, tryptophan, and adenine) were inoculated from plates and starter cultures were grown for 20h at 30°C, 250 rpm. pH buffered media containing 4.2g/L of Bis-Tris (BTU2; BT-UL2)was also inoculated to evaluate growth of the yeast under neutral pH 20 conditions. Primary cultures were used to inoculate final cultures of the same formulation and growth was contînued until a density or 1.1 to 4.0 YU/mL was reached.
[00342] For TEF2 strains (constitutive expression), cells were harvested, washed and heat killed at 56°C for lh in PBS. Live cells were also processed for comparison. For CUP1 strains (inducible expression), expression was induced in the same medium with 0.5 25 mM copper sulfate for 5h at 30°C, 250 rpm. Cells were harvested, washed and heat killed at 56°C for lh in PBS. Live cells were also processed for comparison.
[00343] After heat kill of TEF2 and CUP1 cultures, cells were washed three times in PBS. Total protein expression was measured by a TCA precipitation/nitrocellulose binding assay and antigen expression was measured by western blot using an anti-his tag 30 monoclonal antibody. The antigen was quantified by interpolation from a standard curve of recombinant, hexa-histidine tagged NS3 protein that was processed on the same western blot. Results are shown in Fig. 16 (heat-killed) and Fig. 17 (live yeast). These figures show that the yeast-based immunotherapy composition of the invention expresses the HBV surface-core fusion protein well using both promoters, and can be identified by
150
Western blot in both heat-killed and live yeast cells. The calculated antigen expression by this yeast-based immunotherapeutic was ~5000 ng protein per Y.U. (Yeast Unit; One
Yeast Unit (Y.U.) is 1 x 107 yeast cells or yeast cell équivalents) or 76 pmol protein per
Y.U.
Example 2
[00344] The following example describes the production of another yeast-based immunotherapeutic composition for the treatment or prévention of hepatitis B virus (HBV) infection.
[00345] Yeast (e.g., Saccharomyces cerevisiaë) were engineered to express various 10 HBV fusion proteins, each having the structure schematically shown in Fig. 3, under the control of the copper-inducible promoter, CUP1, or the TEF2 promoter. In each case, the fusion protein was a single polypeptide of approximately 945 amino acids, with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:36: (1) an N-terminal peptide to impart résistance to proteasomal dégradation and 15 stabilize expression (positions 1 to 5 of SEQ ID NO:36); 2) the amino acid sequence of an
HBV génotype C hépatocyte receptor domain of the pre-Sl portion of HBV large (L) surface protein (unique to L) (e.g., positions 21-47 of SEQ ID NO: 11 or positions 6 to 32 of SEQ ID NO:36); 3) the amino acid sequence of a full-length HBV génotype C small (S) surface antigen (e.g., positions 176 to 400 of SEQ ID NO: 11 or positions 33 to 257 of 20 SEQ ID NO:36); 4) a two amino acid spacer/linker (Leu-Glu) to facilitate cloning and manipulation of the sequences (positions 258 and 259 of SEQ ID NO:36); 5) the amino acid sequence of a portion of the HBV génotype C polymerase including the reverse transcriptase domain (e.g., positions 247 to 691 of SEQ ID NO: 10 or positions 260 to 604 of SEQ ID NO:36); 6) an HBV génotype C core protein (e.g., positions 31-212 of SEQ ID 25 NO:9 or positions 605 to 786 of SEQ ID NO:36); 7) the amino acid sequence of an HBV génotype C X antigen (e.g., positions 2 to 154 of SEQ ID NO: 12 or positions 787 to 939 of SEQ ID NO:36); and 8) a hexahistidine tag (positions 940 to 945 of SEQ ID NO:36). This fusion protein and correspondîng yeast-based immunotherapeutic comprising this protein can be generally referred to herein as “MADEAP-Spex”, “M-Spex”, or “GI30 13005”.
[00346] A nucleic acid sequence encoding the fusion protein of SEQ ID NO:36 (codon optimized for yeast expression) is represented herein by SEQ ID NO:35. SEQ ID NO:36 has an expected approximate molecular weight of 106-107 kDa. SEQ ID NO:36 contains multiple epitopes or domains that are believed to enhance the immunogenicity of the
151 fusion protein, including several described above for SEQ ID NO:34. In addition, the reverse transcriptase domain used in this fusion protein contains several amino acid positions that are known to become mutated as a drug-resistance response to treatment with anti-viral drugs, and therefore, may be mutated in this fusion protein in order to 5 provide a therapeutic or prophylactic immunotherapeutic that targets spécifie drug résistance (escape) mutations. These amino acid positions are, with respect to SEQ ID NO:36, at amino acid position: 432 (Val, known to mutate to a Leu after lamivudine therapy); position 439 (Leu, known to mutate to a Met after lamivudine therapy); position 453 (Ala, known to mutate to a Thr after tenofovir therapy); position 463 (Met, known to 10 mutate to an Ile or Val after lamivudine therapy); and position 495 (Asn, known to mutate to Thr after adefovir therapy).
[00347] To create a second yeast-based immunotherapeutic utilizing a different Nterminal peptide in the antigen, yeast (e.g., Saccharomyces cerevisiaë) were engineered to express various HBV fusion proteins, also having the basic structure schematically shown 15 in Fig. 3, under the control of the copper-inducible promoter, CUP1, or the TEF2 promoter. In this second case, an alpha factor prepro sequence (represented by SEQ ID
NO:89) was used in place of the synthetic N-terminal peptide described above in the fusion represented by SEQ ID NO:36. Briefly, the new fusion protein was a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, 20 represented by SEQ ID NO:92: (1) an N-terminal peptide to impart résistance to proteasomal dégradation and stabilize or enhance expression (SEQ ID NO:89, positions 1 to 89 of SEQ ID NO:92); 2) a two amino acid spacer/linker (Thr-Ser) to facilitate cloning and manipulation of the sequences (positions 90 to 91 of SEQ ID NO:92); 3) the amino acid sequence of an HBV génotype C hépatocyte receptor domain of the pre-Sl portion of 25 HBV large (L) surface protein (unique to L) (e.g., positions 21-47 of SEQ ID NO:11 or positions 92 to 118 of SEQ ID NO:92); 4) the amino acid sequence of a full-length HBV génotype C small (S) surface antigen (e.g., positions 176 to 400 of SEQ ID NO:11 or positions 119 to 343 of SEQ ID NO:92); 5) a two amino acid spacer/linker (Leu-Glu) to facilitate cloning and manipulation of the sequences (e.g., positions 344 to 345 of SEQ ID 30 NO:92); 6) the amino acid sequence of a portion of the HBV génotype C polymerase including the reverse transcriptase domain (e.g,, positions 247 to 691 of SEQ ID NO:10 or positions 346 to 690 of SEQ ID NO:92); 7) an HBV génotype C core protein (e.g., positions 31-212 of SEQ ID NO:9 or positions 691 to 872 of SEQ ID NO:92); 8) the amino acid sequence of an HBV génotype C X antigen (e.g., positions 2 to 154 of SEQ ID
152
NO: 12 or positions 873 to 1025 of SEQ ID NO:92); and 9) a hexahistidine tag (e.g., positions 1026 to 1031 of SEQ ID NO:92). This fusion protein and corresponding yeastbased immunotherapeutic comprising this protein can be generally referred to herein as “alpha-Spex”, a-Spex”, or GI-13004”.
[00348] A nucleic acid sequence encoding the fusion protein of SEQ ID NO:92 (codon optimized for yeast expression) is represented herein by SEQ ID NO:91. SEQ ID NO:92 has an expected approximate molecular weight of 123 kDa. SEQ ID NO:92 contains multiple epitopes or domains that are believed to enhance the immunogenicity of the fusion protein, including several described above for SEQ ID NO:34 and SEQ ID NO:36. In addition, the reverse transcriptase domain used in this fusion protein contains several amino acid positions that are known to become mutated as a drug-resistance response to treatment with anti-viral drugs, and therefore, may be mutated in this fusion protein in order to provide a therapeutic or prophylactic immunotherapeutic that targets spécifie drug résistance (escape) mutations. These amino acid positions are, with respect to SEQ ID NO:92, at amino acid position: 518 (Val, known to mutate to a Leu after lamivudine therapy); position 525 (Leu, known to mutate to a Met after lamivudine therapy); position 539 (Ala, known to mutate to a Thr after tenofovir therapy); position 549 (Met, known to mutate to an Ile or Val after lamivudine therapy); and position 581 (Asn, known to mutate to Thr after adefovir therapy).
[00349] To create these immunotherapeutic compositions comprising the amino acid sequences represented by SEQ ID NO:36 and SEQ ID NO:92, DNA encoding the abovedescribed conserved régions of surface antigen (hépatocyte receptor région of pre-Sl or large surface antigen, and full-length small surface antigen) and the reverse transcriptase région of polymerase were fused to full length core and full length X antigen. The DNA was codon-optimized for expression in yeast and then digested with EcoRI and Notl and inserted behind the CUP1 promoter (pGI-100) or the TEF2 promoter (pTK57-l) in yeast 2 um expression vectors. The resulting plasmids were introduced into Saccharomyces cerevisiae W3O3a yeast by Lithium acetate/polyethylene glycol transfection, and primary transfectants were selected on solid minimal plates lacking Uracil (UDM; uridine dropout medium). Colonies were re-streaked onto UDM or ULDM (uridine and leucine dropout medium) and allowed to grow for 3 days at 30°C.
[00350] Liquid cultures lacking uridine (U2) or lacking uridine and leucine (UL2) were inoculated from plates and starter cultures were grown for 20h at 30°C, 250 rpm. pH buffered Media containing 4.2g/L of Bis-Tris (BT-U2; BT-UL2) were also inoculated to
153 evaluate growth of the yeast under neutral pH conditions (data not shown). Primary cultures were used to inoculate final cultures of the same formulation and growth was continued until a density or 1.1 to 4.0 YU/mL was reached. For TEF2 strains (constitutive expression), cells were harvested, washed and heat kiiled at 56°C for lh in PBS. For CUP1 strains (inducible expression), expression was induced in the same medium with 0.5 mM copper sulfate for 5h at 30°C, 250 rpm. Cells were harvested, washed and heat kiiled at 56°C for lh in PBS. Live cells were also processed for comparison (data not shown).
[00351] After heat kill of TEF2 and CUP1 cultures, cells were washed three times in PBS. Total protein expression was measured by a TCA precipitation/nitrocellulose binding assay and antigen expression was measured by western blot using an anti-his tag monoclonal antibody. The antigen was quantified by interpolation from a standard curve of recombinant, hexa-histidine tagged NS3 protein that was processed on the same western blot.
[00352] For the yeast-based immunotherapeutic expressing the fusion protein represented by SEQ ID NO:36 (GI-13005), results are shown in Fig. 18. Fig. 18 shows that the yeast-based immunotherapy composition of the invention expresses the fusion protein well using both promoters, and can be identified by Western blot in heat-killed yeast cells (expression was also achieved in live yeast cells, data not shown). The calculated antigen expression by this yeast-based immunotherapeutic was -1200 ng protein per Y.U. or 11 pmol protein per Y.U., for growth in UL2.
[00353] For the yeast-based immunotherapeutic expressing the fusion protein represented by SEQ ID NO:92 (GI-13004), results are shown in Fig. 19. Fig. 19 shows expression of this yeast-based immunotherapy composition under the control of the CUP1 promoter (identified in Fig. 19 as Alpha-SPEX) as compared to a yeast-based immunotherapeutic that expresses an unrelated antigen (Control Yeast) and to the yeastbased immunotherapeutic composition expressing an HBV fusion protein represented by SEQ ID NO:36 (SPEX). Fig. 19 shows that the yeast-based immunotherapeutics expresses the relevant fusion proteins well, and can be identified by Western blot in heatkilled yeast cells. The calculated antigen expression by this yeast-based immunotherapeutic (Alpha-SPEX) was -5000 ng protein per Y.U. or 41 pmol protein per Y.U. for growth in UL2.
Example 3
[00354] The following example describes the production of additional yeast-based immunotherapeutic composition for the treatment or prévention of hepatitis B virus (HBV) infection.
[00355] In this experiment, yeast (e.g., Saccharomyces cerevisiaé) are engineered to express various HBV polymerase-core fusion proteins, as shown schematically in Fig. 4, under the control of the copper-inducible promoter, CUP1, or the TEF2 promoter. In each case, the fusion protein is a single polypeptide of approximately 527 amino acids, with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:38: (1) an N-terminal peptide to impart résistance to proteasomal dégradation and 10 stabilize expression (SEQ ID NO:37; positions 1 to 6 of SEQ ID NO:38); 2) the amino acid sequence of a portion of the HBV génotype C polymerase including the reverse transcriptase domain (e.g., positions 347 to 691 of SEQ ID NO:10 or positions 7 to 351 of SEQ ID NO:38); 3) an HBV génotype C core protein (e.g., positions 31 to 212 of SEQ ID NO:9 or positions 352 to 533 of SEQ ID NO:38); and 4) a hexahistîdine tag (e.g., 15 positions 534 to 539 of SEQ ID NO:38). SEQ ID NO:38 has a predicted molecular weight of approximately 58 kDa. The sequence also eontains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. In additional constructs, the N-terminal peptide of SEQ ID NO:37 is replaced with a different synthetic N-terminal peptide represented by a homologue of SEQ ID NO:37 that meets the same basic 20 structural requirements of SEQ ID NO:37 as described in detail in the spécification, or the N-terminal peptide of SEQ ID NO:37 is replaced with the N-terminal peptide of SEQ ID NO:89 or SEQ ID NO:90, and in another construct, the N-terminal peptide is omitted and a méthionine is included at position one.
[00356] In another experiment, yeast (e.g., Saccharomyces cerevisiaé) are engineered to express various HBV X-core fusion proteins as shown schematically in Fig. 5 under the control of the copper-inducible promoter, CUP1, or the TEF2 promoter. In each case, the fusion protein is a single polypeptide of approximately 337 amino acids with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:39 (1) an N-terminal peptide to impart résistance to proteasomal dégradation and stabilize 30 expression (SEQ ID NO:37; positions 1 to 6 of SEQ ID NO:39); 2) the amino acid sequence of a near full-length (minus position 1) HBV génotype C X antigen (e.g., positions 2 to 154 of SEQ ID NO:12 or positions 7 to 159 of SEQ ID NO:39); 3) an HBV génotype C core protein (e.g., positions 31 to 212 of SEQ ID NO:9 or positions 160 to 341 of SEQ ID NO:39); and 4) a hexahistîdine tag (positions 342 to 347 of SEQ ID NO:39).
155
SEQ ID NO:39 has a predicted approximate molecular weight of 37 kDa. The sequence also contains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. In additional constructs, the N-terminal peptide of SEQ ID NO:37 is replaced with a different synthetic N-terminal peptide represented by a homologue of SEQ ID NO:37 that meets the same basic structural requirements of SEQ ID NO:37 as described in detail in the spécification, or the N-terminal peptide of SEQ ID NO:37 is replaced with the N-terminal peptide of SEQ ID NO:89 or SEQ ID NO:90, and in another construct, the N-terminal peptide is omitted and a méthionine is included at position one. [00357] In another experiment, yeast (e.g., Saccharomyces cerevisiae) are engineered to express various HBV polymerase proteins as shown schematically in Fig. 6 under the control of the copper-inducible promoter, CUP1, or the TEF2 promoter. In each case, the fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:40 (1) an N-terminal peptide to impart résistance to proteasomal dégradation and stabilize expression (SEQ ID NO:37, or positions 1 to 6 of SEQ ID NO:40; 2) the amino acid sequence of a portion of the HBV génotype C polymerase including the reverse transcriptase domain (e.g., positions 347 to 691 of SEQ ID NO: 10 or positions 7 to 351 of SEQ ID NO:40); and 3) a hexahistidine tag (positions 352 to 357 of SEQ ID NO:40). The sequence also contains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. In addition, in one embodiment, the sequence of this construct can be modifîed to introduce one or more or ail of the following anti-viral résistance mutations: rtM2041, rtL180M, rtM204V, rtV173L, rtN236T, rtA194T (positions given with respect to the full-length amino acid sequence for HBV polymerase). In one embodiment, six different immunotherapy compositions are created, each one containing one of these mutations. In other embodiments, ail or some of the mutations are included in a single fusion protein. In additional constructs, the Nterminal peptide of SEQ ID NO:37 is replaced with a different synthetic N-terminal peptide represented b y a homologue of SEQ ID NO: 37 that meets the same basic structural requirements of SEQ ID NO:37 as described in detail in the spécification, or the N-terminal peptide of SEQ ID NO:37 is replaced with the N-terminal peptide of SEQ ID NO:89 or SEQ ID NO:90, and in another construct, the N-terminal peptide is omitted and a méthionine îs included at position one.
[00358] In another experiment, yeast (e.g., Saccharomyces cerevisiae) are engineered to express various HBV polymerase-surface-core fusion proteins as shown schematically in Fig. 7 under the control of the copper-inducible promoter, CUP1, or the TEF2 promoter.
156
In each case, the fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:41: (1) an Nterminal peptide to impart résistance to proteasomal dégradation and stabilize expression (e.g., positions 1 to 5 of SEQ ID NO:41); 2) an amino acid sequence of the amino HBV hépatocyte receptor domain of the pre-Sl portion of HBV large (L) surface protein (unique to L) (e.g., positions 21-47 of SEQ ID NO: 11 or positions 6 to 32 of SEQ ID NO:41); 3) the amino acid sequence of an HBV small (S) surface protein (e.g., positions 176 to 400 of SEQ ID NO:11 or positions 33 to 257 of SEQ ID NO:41); 4) a two amino acid spacer/linker to facilitate cloning and manipulation of the sequences (e.g., positions 258 and 259 of SEQ ID NO:41); 5) the amino acid sequence of an HBV polymerase comprising the reverse transcriptase domain (e.g., positions 247 to 691 of SEQ ID NO:10 or positions 260 to 604 of SEQ ID NO:41); 6) the amino acid sequence of an HBV core protein (e.g., positions 31-212 of SEQ ID NO:9 or positions 605 to 786 of SEQ ID NO:41); and 7) a hexahistidine tag (e.g., positions 787 to 792 of SEQ ID NO:41). The sequence also contains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. In addition, in one embodiment, the sequence of this construct can be modified to introduce one or more or ail of the following anti-viral résistance mutations: rtM2041, rtL180M, rtM204V, rtV173L, rtN236T, rtA194T (positions given with respect to the full-length amino acid sequence for HBV polymerase). In one embodiment, six different immunotherapy compositions are created, each one containing one of these mutations. In other embodiments, ail or some of the mutations are included in a single fusion protein. In one embodiment, this construct also contains one or more anti-viral résistance mutations in the surface antigen. In additional constructs, the Nterminal peptide represented by positions 1 to 5 of SEQ ID NO:41 is replaced with a different synthetic N-terminal peptide represented by a homologue of positions 1 to 5 of SEQ ID NO:41 that meets the same basic structural requirements of positions 1 to 5 of SEQ ID NO:41 (or of SEQ ID NO:37) as described in detail in the spécification, or the Nterminal peptide of positions 1 to 5 of SEQ ID NO:41 is replaced with the N-terminal peptide of SEQ ID NO:89 or SEQ ID NO:90, and in another construct, the N-terminal peptide is omitted and a méthionine is included at position one.
[00359] To produce any of the above-described fusion proteins and yeast-based immunotherapy compositions expressing such proteins, briefly, DNA encoding the fusion protein is codon optimized for expression in yeast and then digested with EcoRI and Notl and inserted behind the CUP1 promoter (pGI-100) or the TEF2 promoter (pTK57-l) in
157 yeast 2 um expression vectors. The resulting plasmids are introduced into Saccharomyces cerevisiae W3O3a yeast by Lithium acetate/polyethylene glycol transfection, and primary transfectants are selected on solid minimal plates lacking Uracil (UDM; uridine dropout medium). Colonies are re-streaked onto UDM or ULDM (uridine and leucine dropout medium) and allowed to grow for 3 days at 30°C.
[00360] Liquid cultures lacking uridine (U2) or lacking uridine and leucine (UL2) are inoculated from plates and starter cultures were grown for 20h at 30°C, 250 rpm. pH buffered Media containing 4.2g/L of Bis-Tris (BT-U2; BT-UL2) can also be inoculated to evaluate growth of the yeast under neutral pH conditions. Primary cultures are used to inoculate final cultures of the same formulation and growth is continued until a density or 1.1 to 4.0 YU/mL is reached. For TEF2 strains (constitutive expression), cells are harvested, washed and heat killed at 56°C for lh in PBS. For CUP1 strains (inducible expression), expression is induced in the same medium with 0.5 mM copper sulfate for 5h at 30°C, 250 rpm. Cells are harvested, washed and heat killed at 56°C for lh in PBS. Live cells are also processed for comparison.
[00361] After heat kill of TEF2 and CUP1 cultures, cells are washed three times in PBS. Total protein expression is measured by a TCA precipîtation/nitrocellulose binding assay and protein expression is measured by western blot using an anti-his tag monoclonal antibody. Fusion protein is quantified by interpolation from a standard curve of recombinant, hexa-histidine tagged NS3 protein that was processed on the same western blot.
Example 4
[00362] The following example describes the production of additional yeast-based îmmunotherapeutic compositions for the treatment or prévention of hepatitis B virus (HBV) infection.
[00363] This example describes the production of four different yeast-based îmmunotherapeutic compositions, each one designed to express one HBV protein. These “single HBV protein yeast immunotherapeutics” can be used in combination or in sequence with each other and/or in combination or in sequence with other yeast-based immunotherapeutics,, such as those described in any of Examples 1-3 and 5-8, including multi-HBV protein yeast-based immunotherapeutics described herein. In addition, a “single HBV protein yeast îmmunotherapeutic”, such as those described in this example, can be produced using the HBV sequence for any given génotype or sub-genotype, and additional HBV surface antigen yeast-based immunotherapeutics can be produced using
158 the HBV sequences for any one or more additional génotypes or sub-genotypes, in order to provide a “spice rack” of different HBV antigens and génotypes and/or subgenotypes, each of which is provided in the context of a yeast-based immunotherapeutic of the invention, or in an immunization/administration strategy that includes at least one yeastbased immunotherapeutic of the invention.
[00364] In this example, the following four yeast-based immunotherapeutic products are produced:
[00365] HBVSurface Antigen. Saccharomyces cerevisiae are engineered to express an HBV surface protein under the control of the copper-inducible promoter, CUP1, or the TEF2 promoter. In each case, the fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:93: 1) an N-terminal peptide of SEQ ID NO:89 (positions 1-89 of SEQ ID NO:93); 2) the amino acid sequence of a near full-length (minus position 1) HBV génotype C large (L) surface antigen (e.g., positions 2-400 of SEQ ID NO: 11 or positions 90 to 488 of SEQ ID NO:93); and 3) a hexahistidine tag (e.g., positions 489 to 494 of SEQ ID NO:93). Altematively, the N-terminal peptide can be replaced with SEQ ID NO:37 or a homologue thereof or another N-terminal peptide described herein.
[00366] HBV Polymerase Antigen. Saccharomyces cerevisiae are engineered to express the following HBV Polymerase protein under the control of the copper-inducible promoter, CUP1, or the TEF2 promoter. In each case, the fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:94: 1) an N-terminal peptide of SEQ ID NO:89 (positions 189 of SEQ ID NO:94); 2) the amino acid sequence of a portion of the HBV génotype C polymerase încluding the reverse transcriptase domain (e.g., positions 347 to 691 of SEQ ID NO:10 or positions 90 to 434 of SEQ ID NO;94); and 3) a hexahistidine tag (e.g., positions 435 to 440 of SEQ ID NO:94). Altematively, the N-terminal peptide can be replaced with SEQ ID NO:37 or a homologue thereof or another N-terminal peptide described herein.
[00367] HBV Core Antigen. Saccharomyces cerevisiae are engineered to express the following HBV Core protein under the control of the copper-inducible promoter, CUP1, or the TEF2 promoter. In each case, the fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ
ID NO:95: 1) an N-terminal peptide of SEQ ID NO:89 (positions 1-89 of SEQ ID
NO:95); 2) the amino acid sequence of a portion of the HBV génotype C Core protein
159 (e.g., positions 31 to 212 of SEQ ID NO:9 or positions 90 to 271 of SEQ ID NO:95); and
3) a hexahistidîne tag (e.g., positions 272 to 277 of SEQ ID NO:95). Alternatively, the Nterminal peptide can be replaced with SEQ ID NO:37 or a homologue thereof or another
N-terminal peptide described herein.
[00368] HBV X Antigen. Saccharomyces cerevisiae are engineered to express the following HBV X antigen under the control of the copper-inducible promoter, CUP1, or the TEF2 promoter. In each case, the fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:96: 1) an N-terminal peptide of SEQ ID NO:89 (positions 1-89 of SEQ ID NO:96); 2) the amino acid sequence of a portion of the HBV génotype C X antigen (e.g., positions 2 to 154 of SEQ ID NO: 12 or positions 90 to 242 of SEQ ID NO:96); and 3) a hexahistidîne tag (e.g., positions 243 to 248 of SEQ ID NO:96). Alternatively, the Nterminal peptide can be replaced with SEQ ID NO:37 or a homologue thereof or another N-terminal peptide described herein.
[00369] To create these immunotherapeutic compositions, briefly, DNA encoding the fusion protein is codon optimized for expression in yeast and then digested with EcoRI and Notl and inserted behind the CUPI promoter (pGI-100) or the TEF2 promoter (pTK57-l) in yeast 2 um expression vectors. The resulting plasmids are întroduced into Saccharomyces cerevisiae W303a yeast by Lithium acetate/polyethylene glycol transfection, and primary transfectants are selected on solid minimal plates lacking uracil (UDM; uridine dropout medium). Colonies are re-streaked onto UDM or ULDM (uridine and leucine dropout medium) and allowed to grow for 3 days at 30°C.
[00370] Liquid cultures lacking uridine (U2) or lacking uridine and leucine (UL2) are inoculated from plates and starter cultures were grown for 20h at 30°C, 250 rpm. pH buffered Media containing 4.2g/L of Bis-Tris (BT-U2; BT-UL2) may also be inoculated to evaluate growth of the yeast under neutral pH conditions. Primary cultures are used to inoculate final cultures of the same formulation and growth is continued until a density or 1.1 to 4.0 YU/mL is reached. For TEF2 strains (constitutive expression), cells are harvested, washed and heat killed at 56°C for lh in PBS. For CUPI strains (inducible expression), expression is induced in the same medium with 0.5 mM copper sulfate for 5h at 30°C, 250 rpm. Cells are harvested, washed and heat killed at 56°C for lh in PBS. Live cells are also processed for comparison.
[00371] After heat kill of TEF2 and CUPI cultures, cells are washed three times in PBS. Total protein expression is measured by a TCA precipitation/nitrocellulose binding
160 assay and protein expression is measured by western blot using an anti-his tag monoclonal antibody. Fusion protein is quantified by interpolation from a standard curve of recombinant, hexa-histidine tagged NS3 protein that was processed on the same western blot.
Example 5
[00372] The following example describes the production of several different yeastbased immunotherapeutic compositions for the treatment or prévention of hepatitis B virus (HBV) infection.
[00373] This example describes the production of yeast-based immunotherapeutics 10 expressing proteins that hâve been designed to achieve one or more of the following goals:
(1) produce a multi-antigen HBV construct that comprises less than about 690 amino acids (corresponding to less than two thirds of the HBV genome), in order to produce a yeastbased immunotherapeutic clinical product that is compilant with the guidelînes of the Recombinant DNA Advisory Committee (RAC), if necessary; (2) produce a multi-antigen 15 HBV construct containing a maximized number of known T cell epitopes associated with immune responses to acute/self-limiting HBV infections and/or chronic HBV infections;
(3) produce a multi-antigen HBV construct containing T cell epitopes that are most conserved among génotypes; and/or (4) produce a multi-antigen HBV construct modified to correspond more closely to one or more consensus sequences, consensus epitopes, 20 and/or epitope(s) from particular génotypes. The modifications demonstrated in this example can be applied individually or together to any other yeast-based immunotherapeutic described or contemplated herein.
[00374] In one experiment, a yeast-based immunotherapeutic composition that comprises a yeast expressing a fusion protein meeting the requirements of the goals 25 specified above, and comprising portions of each of the HBV major proteins: HBV surface antigen, polymerase, core and X antigen, was designed. To design this fusion protein, individual HBV antigens within the fusion were reduced in size (as compared to full-length), and the fusion segments were individually modified to maximize the inclusion of known T cell epitopes corresponding to those identified in Table 5. Inclusion 30 of T cell epitopes in this fusion protein was prioritized as follows:
Epitopes identified in immune responses to both acute/self-limiting HBV infections and chronic HBV infections > Epitopes identified in immune responses to acute/self-limiting HBV infections > Epitopes identified in immune responses to chronic HBV infections
161
[00375] Artificial junctions were also minimized in the design of each segment of this fusion protein because, without being bound by theory, it is believed that natural évolution has resulted in: i) contiguous sequences in the virus that express well; and ii) an immunoproteasome in antigen presenting cells that can properly digest and présent those sequences to the immune System. Accordingly, a fusion protein with many unnatural junctions may be less useful in a yeast-based immunotherapeutic as compared to one that retains more of the natural HBV protein sequences.
[00376] To construct a segment comprising HBV surface antigen for use in a fusion protein, a full-length large (L) surface antigen protein from HBV génotype C was reduced in size by troncation of the N- and C-terminal sequences (positions 1 to 119 and positions 369 to 400 of large antigen were removed, as compared to a full-length L surface antigen protein, such as that represented by SEQ ID NO: 11). The remaining portion was selected, in part, to maximize the inclusion of known MHC Class IT cell epitopes corresponding to those identified in Table 5, using the prioritization for inclusion of T cell epitopes described above. The resulting surface antigen segment is represented by SEQ ID NO:97.
[00377] To construct the segment comprising HBV polymerase for use in a fusion protein, substantial portions of a full-length polymerase from HBV génotype C, which is a very large protein of about 842 amino acids, were eliminated by focusing on inclusion of the active site domain (from the RT domain), which is the most conserved région of the protein among HBV génotypes and isolâtes. The RT domain also includes several sites where drug résistance mutations hâve been known to occur; thus, this portion of the construct can be further modified in other versions, as needed, to target escape mutations of targeted therapy. In fusion proteins including fewer HBV proteins, the size of the polymerase segment can be expanded, if desired. The selected portion of the HBV polymerase was included to maximize known T cell epitopes, using the prioritization strategy discussed above. Sequence of full-length polymerase that was therefore eliminated included sequence outside of the RT domain, and sequences within the RT domain that contained no known T cell epitopes, or that included two epitopes identified in less than 17% or 5%, respectively, of génotype A patients where these epitopes were identified (see Desmond et al., 2008 and Table 5). Ail but one of the remaining T cell epitopes in the HBV polymerase génotype C segment were perfect matches to the published epitopes from the génotype A analysis, and the one epîtope with a single amino acid mismatch was modified to correspond to the published epîtope. The resulting HBV polymerase antigen segment is represented by SEQ ID NO:98.
162
[00378] To construct the segment comprising HBV core antigen for use in a fusion protein, a full-length Core protein (e.g., similar to positions 31-212 of SEQ ID NO:9) from HBV génotype C was modified as follows: i) a single amino acid within a T cell epitope of the génotype C-derived protein was modified to create a perfect match to a known T cell epitope described in Table 5; ii) seven amino acids of the N-terminus, which did not contain a T cell epitope, were removed, preserving some flanking amino acids N-terminal to the first known T cell epitope in the protein; and iii) the 24 C terminal amino acids of Core were removed, which does not delete known epitopes, but which does remove an exceptionally posîtively charged C-terminus. A positively charged C-terminus is a good candidate for removal from an antigen to be expressed in yeast, as such sequences may, in some constructs, be toxic to yeast by compétitive interférence with natural yeast RNA binding proteins which often are arginine rich (positively charged). Accordingly, removal of this portion of Core is acceptable. The resulting HBV Core antigen segment is represented by SEQ ID NO:99.
[00379] To construct a segment comprising HBV X antigen for use in a fusion protein, a full-length X antigen from HBV génotype C (e.g., similar to SEQ ID NO: 12) was truncated at the N- and C-terminus to produce a segment of X antigen that includes most of the known T cell epitopes from Table 5, which are clustered in the X antigen. Two of the epitopes were modified by single amino acid changes to correspond to the published T cell epitope sequences, and sequence flanking the T cell epitopes at the ends of the segment was retained to facilitate efficient processing and présentation of the correct epitopes by an antigen presenting cell. The resulting HBV X antigen segment is represented by SEQ ID NO: 100.
[00380] To construct a complété fusion protein containing ail four HBV protein segments, the four HBV segments described above were linked (surface-pol-core-X) to form a single protein that optimizes the inclusion of T cell epitopes spanning ail proteins encoded by the HBV genome, and that is expected to meet criteria for viral proteins for anticipated clinical use.
[00381] Two different fusion proteins were ultimately created, each with a different Nterminal peptide added to enhance and/or stabilize expression of the fusion protein in yeast.
In addition, a hexahistidine peptide was included at the C-terminus to aid with the identification of the protein. As for ail of the other proteins used in the yeast-based immunotherapeutic compositions described herein, in additional constructs, the N-terminal peptide of SEQ ID NO:37 or SEQ ID NO:89 utilized in this example can be replaced with
163 a different synthetic N-terminal peptide (e.g., a homologue of SEQ ID NO:37 that meets the same basic structural requirements of SEQ ID NO:37 as described in detail in the spécification), or with a homologue of the N-terminal peptide of SEQ ID NO;89 or SEQ ID NO:90, and in another construct, the N-terminal peptide is omitted and a méthionine is included at position one. In addition, linker sequences of one, two, three or more amino acids may be added between segments of the fusion protein, if desired. Also, while these constructs were designed using HBV proteins from génotype C as the backbone, any other HBV génotype, sub-genotype, or HBV proteins from different strains or isolâtes can be used to design these protein segments, as exemplified in Example 7. Finally, if one or more segments are excluded from the fusion protein as described herein, then the sequence from the remaining segments can be expanded to include additional T cell epitopes and flanking régions of the proteins (e.g., see Example 8).
[00382] To produce yeast-based immunotherapeutic compositions comprising a fusion protein constructed of the HBV segments described above, yeast (e.g., Saccharomyces cerevisiae) are engineered to express various HBV surface-polymerase-core-X fusion proteins, optimized as discussed above, under the control of the copper-inducible promoter, CUP1, or the TEF2 promoter.
[00383] In one construct, the fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:101: (1) an N-terminal peptide that is an alpha factor prepro sequence, to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:89 (positions 1-89 of SEQ ID NO: 101); (2) an optimized portion of an HBV large (L) surface antigen represented by SEQ ID NO:97 (positions 90 to 338 of SEQ ID NO:101); (3) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase represented by SEQ ID NO:98 (positions 339 to 566 of SEQ ID NO:101); (4) an optimized portion of HBV Core protein represented by SEQ ID NO:99 (positions 567 to 718 of SEQ ID NO: 101); (5) an optimized portion of HBV X antigen represented by SEQ ID NO:100 (positions 719 to 778 of SEQ ID NO: 101); and (6) a hexahistidine tag (e.g., positions 779 to 784 of SEQ ID NO:101).
[00384] In a second construct, the fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ
ID NO;102: (1) an N-terminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ
ID NO:37 (positions 1-6 of SEQ ID NO: 102); (2) an optimized portion of an HBV large
164 (L) surface antigen represented by positions 2 to 248 of SEQ ID NO:97 (positions 7 to 254 of SEQ ID NO: 102); (3) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase represented by SEQ ID NO:98 (positions 255 to 482 of SEQ ID NO: 102); (4) an optimized portion of HBV Core protein represented by SEQ ID NO:99 (positions 483 to 634 of SEQ ID NO: 102); (5) an optimized portion of HBV X antigen represented by SEQ ID NO: 100 (positions 635 to 694 of SEQ ID NO:102); and (6) a hexahistidine tag (e.g., positions 695 to 700 of SEQ ID NO: 102).
[00385] Yeast-based immunotherapy compositions expressing these fusion proteins are produced using the same protocol described in detail in Example 1-4.
Example 6
[00386] The following example describes the production of additional yeast-based HBV immunotherapeutic compositions that maximize the targeting of HBV génotypes and/or sub-genotypes in conjunction with conserved antigen and/or epitope inclusion within a single composition, in order to provide single compositions with the potential to treat a large number of individuals or populations of individuals.
[00387] To préparé a construct comprising multiple different génotypes within the same yeast-based immunotherapeutic, yeast (e.g., Saccharomyces cerevisiae) are engineered to express an HBV fusion protein under the control of a suitable promoter, such as the copper-inducible promoter, CUP1, or the TEF2 promoter. The protein is a single polypeptide comprising four Core antigens, each one from a different génotype (HBV génotypes A, B, C and D), represented by SEQ ID NO: 105: 1) an N-terminal méthionine at position 1 of SEQ ID NO: 105; 2) the amino acid sequence of a near fullIength Core protein from HBV génotype A (e.g., positions 31 to 212 of SEQ ID NO:1 or positions 2 to 183 of SEQ ID NO: 105); 3) the amino acid sequence of a near full-Iength Core protein from HBV génotype B (e.g., positions 30 to 212 of SEQ ID NO:5 or positions 184 to 395 of SEQ ID NO: 105); 4) the amino acid sequence of a near full-Iength Core protein from HBV génotype C (e.g., positions 30 to 212 of SEQ ID NO:9 or positions 396 to 578 of SEQ ID NO: 105); 5) the amino acid sequence of a near full-Iength Core protein from HBV génotype D (e.g., positions 30 to 212 of SEQ ID NO: 13 or positions 579 to 761 of SEQ ID NO: 105); and 5) a hexahistidine tag (e.g., positions 762 to 767 of SEQ ID NO: 105). The sequence also contains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. The N-terminal méthionine at position 1 can be substituted with SEQ ID NO:37 or a homologue thereof, or with an alpha prepro sequence of SEQ ID NO:89 or SEQ ID NO:90, or a homologue thereof, or
165 any other suitable N-terminal sequence if desired. In addition, linker sequences can be inserted between HBV proteins to facilitate cloning and manipulation of the construct, if desired. This is an exemplary construct, as any other combination of HBV génotypes and/or subgenotypes can be substituted into this design as desired to construct a single antigen yeast-based HBV immunotherapeutic product with broad clinical applicability and efficient design for manufacturing. The amino acid sequence of SEQ ID NO:105 also contains several known T cell epitopes, and certain epitopes hâve been modified to correspond to the published sequence for the given epitope, which can be identified by comparison of the sequence to the epitopes shown in Table 5, for example.
[00388] To préparé a construct comprising more than one HBV antigen and more than one génotype within the same yeast-based immunotherapeutic, yeast (e.g., Saccharomyces cerevisiae) are engineered to express an HBV fusion protein under the control of a suitable promoter, such as the copper-inducible promoter, CUP1, or the TEF2 promoter. The protein is a single polypeptide comprising two Core antigens and two X antigens, each one of the pair from a different génotype (HBV génotypes A and C), represented by SEQ ID NO:106: 1) an N-terminal méthionine at position 1 of SEQ ID NO:106; 2) the amino acid sequence of a near full-length Core protein from HBV génotype A (e.g., positions 31 to 212 of SEQ ID NO:1 or positions 2 to 183 of SEQ ID NO:106); 3) the amino acid sequence of a full-length X antigen from HBV génotype A (e.g., positions SEQ ID NO:4 or positions 184 to 337 of SEQ ID NO:106); 4) the amino acid sequence of a near fulllength Core protein from HBV génotype C (e.g., positions 30 to 212 of SEQ ID NO:9 or positions 338 to 520 of SEQ ID NO: 106); 5) the amino acid sequence of a full-length X antigen from HBV génotype C (e.g., SEQ ID NO:8 or positions 521 to 674 of SEQ ID NO:106); and 5) a hexahistidine tag (e.g., positions 675 to 680 of SEQ ID ΝΟ.Ί06). The sequence also contains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. The N-terminal méthionine at position 1 can be substituted with SEQ ID NO:37 or a homologue thereof, or with an alpha prepro sequence of SEQ ID NO:89 or SEQ ID NO:90, or a homologue thereof. The amino acid sequence of SEQ ID NO: 106 also contains several known T cell epitopes, and certain epitopes hâve been modified to correspond to the published sequence for the given epitope, which can be identified by comparison of the sequence to the epitopes shown in Table 5, for example.
[00389] Yeast-based immunotherapy compositions expressing these fusion proteins are produced using the same protocol described in detail in Example 1-4.
Example 7
166
[00390] The following example describes the production of additional yeast-based HBV immunotherapeutic compositions that utilize consensus sequences for HBV génotypes, further maximizing the targeting of HBV génotypes and/or sub-genotypes in conjunction with conserved antigen and/or epitope inclusion, in order to provide compositions with the potential to treat a large number of individuals or populations of individuals using one composition.
[00391] To design several constructs that include HBV segments from each of surface protein, core, polymerase, and X antigen, the fusion protein structure described in Example 5 for SEQ ID NO:101 and SEQ ID NO:102 (and therefore the subparts of these fusion proteins represented by SEQ ID NO:97 (Surface antigen), SEQ ID NO:98 (Polymerase), SEQ ID NO:99 (Core antigen), and SEQ ID NO: 100 (X antigen)) was used as a template. With reference to consensus sequences for each of HBV génotype A, B, C and D that were built from multiple sources of HBV sequences (e.g., Yu and Yuan et al, 2010, for S, Core and X, where consensus sequences were generated from 322 HBV sequences, or for Pol (RT), from the Stanford University HIV Drug Résistance Database, HBVseq and HBV Site Release Notes), sequences in the template structure were replaced with consensus sequences corresponding to the same positions, unless using the consensus sequence altered one of the known acute self-limiting T cells epitopes or one of the known polymerase escape mutation sites, in which case, these positions followed the published sequence for these epitopes or mutation sites. Additional antigens could be constructed based solely on consensus sequences or using other published epitopes as they become known.
[00392] A first construct based on a consensus sequence for HBV génotype A was designed as follows. Using SEQ ID NO;97, SEQ ID NO;98, SEQ ID NO:99 and SEQ ID NO: 100, which were designed to reduce the size of the fusion segments (as compared to full-length), to maximize the inclusion of known T cell epitopes corresponding to those identified in Table 5 (priority as discussed above), and to minimize artificial junctions, new fusion segments were created based on a consensus sequence for HBV génotype A. The new surface antigen segment is represented by positions 1-249 of SEQ ID NO:107. The new polymerase (RT) segment îs represented by positions 250-477 of SEQ ID NO: 107. The new Core segment is represented by positions 478-629 of SEQ ID NO: 107. The new X antigen segment is represented by positions 630-689 of SEQ ID NO: 107. This complété fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, wherein the HBV sequences are represented by
167
SEQ ID NO: 107 (non-HBV sequences denoted as “optional” were not included in the base sequence of SEQ ID NO: 107, but were actually added to the fusion protein described in this example): (1) an optional N-terminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) an optimized portion of an HBV large (L) surface antigen represented by positions 1 to 249 of SEQ ID NO:107, which is a consensus sequence for HBV génotype A utilizing the design strategy dîscussed above; (4) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase represented by positions 250 to 477 of SEQ ID NO: 107, which is a consensus sequence for HBV génotype A utilizing the design strategy dîscussed above; (5) an optimized portion of HBV Core protein represented by positions 478 to 629 of SEQ ID NO: 107, which is a consensus sequence for HBV génotype A utilizing the design strategy dîscussed above; (6) an optimized portion of HBV X antigen represented by positions 630 to 689 of SEQ ID NO: 107, which is a consensus sequence for HBV génotype A utilizing the design strategy dîscussed above; and (7) an optional hexahistidine tag (six histidine residues following position 689 of SEQ ID NO: 107). A yeast-based immunotherapy composition expressing this complété fusion protein is also referred to herein as GI-13010. The fusion protein and corresponding yeast-based immunotherapeutic can also be referred to herein as “SPEXv2-A” or “Spex-A”.
[00393] A second construct based on a consensus sequence for HBV génotype B was designed as follows. Using SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99 and SEQ ID NO: 100, which were designed to reduce the size of the fusion segments (as compared to full-length), to maximize the inclusion of known T cell epitopes corresponding to those identified in Table 5 (priority as dîscussed above), and to minimize artificial junctions, new fusion segments were created based on a consensus sequence for HBV génotype B. The new surface antigen segment is represented by positions 1-249 of SEQ ID NO: 108. The new polymerase (RT) segment is represented by positions 250-477 of SEQ ID NO: 108. The new Core segment is represented by positions 478-629 of SEQ ID NO: 108. The new X antigen segment is represented by positions 630-689 of SEQ ID NO: 108. This fusion protein is a single polypeptide with the following sequence éléments fiised in frame from N- to C-terminus, represented by SEQ ID NO: 108 (non-HBV sequences denoted as “optional” were not included in the base sequence of SEQ ID NO: 108, but were actually added to the fusion protein described in this example): (1) an optional N-terminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal
168 dégradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) an optimized portion of an HBV large (L) surface antigen represented by positions 1 to 249 of SEQ ID NO: 108, which is a consensus sequence for HBV génotype B utilizing the design strategy discussed above; (4) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase represented by positions 250 to 477 of SEQ ID NO: 108, which is a consensus sequence for HBV génotype B utilizing the design strategy discussed above; (5) an optimized portion of HBV Core protein represented by positions 478 to 629 of SEQ ID NO: 108, which is a consensus sequence for HBV génotype B utilizing the design strategy discussed above; (6) an optimized portion of HBV X antigen represented by positions 630 to 689 of SEQ ID NO: 108, which is a consensus sequence for HBV génotype B utilizing the design strategy discussed above; and (7) an optional hexahistidine tag. A yeast-based immunotherapy composition expressing this complété fusion protein is also referred to herein as GI-13011. The fusion protein and correspondîng yeast-based immunotherapeutic can also be referred to herein as “SPEXv2-B” or “Spex-B”.
[00394] A third construct based on a consensus sequence for HBV génotype C was designed as follows. Using SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99 and SEQ ID NO: 100, which were designed to reduce the size of the fusion segments (as compared to full-length), to maximize the inclusion of known T cell epitopes correspondîng to those identified in Table 5 (priority as discussed above), and to minimize artificial junctions, new fusion segments were created based on a consensus sequence for HBV génotype C. The new surface antigen segment is represented by positions 1-249 of SEQ ID NO: 109. The new polymerase (RT) segment is represented by positions 250-477 of SEQ ID NO:109. The new Core segment is represented by positions 478-629 of SEQ ID NO:109. The new X antigen segment îs represented by positions 630-689 of SEQ ID NO: 109. This fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO: 109 (non-HBV sequences denoted as “optional” were not included in the base sequence of SEQ ID NO: 109, but were actually added to the fusion protein described in this example): (1) an optional N-terminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) an optimized portion of an HBV large (L) surface antigen represented by positions 1 to 249 of SEQ ID NO: 109, which is a consensus sequence for HBV génotype C utilizing the design strategy discussed above; (4) an optimized portion of
169 the reverse transcriptase (RT) domain of HBV polymerase represented by positions 250 to 477 of SEQ ID NO: 109, which is a consensus sequence for HBV génotype C utilizing the design strategy discussed above; (5) an optimized portion of HBV Core protein represented by positions 478 to 629 of SEQ ID NO: 109, which is a consensus sequence for HBV génotype C utilizing the design strategy discussed above; (6) an optimized portion of HBV X antigen represented by positions 630 to 689 of SEQ ID NO: 109, which is a consensus sequence for HBV génotype C utilizing the design strategy discussed above; and (7) an optional hexahistidine tag. A yeast-based immunotherapy composition expressing this complété fusion protein is also referred to herein as GI-13012. The fusion protein and corresponding yeast-based immunotherapeutic can also be referred to herein as “SPEXv2-C” or “Spex-C”.
[00395] A fourth construct based on a consensus sequence for HBV génotype D was designed as follows. Using SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99 and SEQ ID NO: 100, which were designed to reduce the size of the fusion segments (as compared to full-length), to maximize the inclusion of known T cell epitopes corresponding to those identified in Table 5 (priority as discussed above), and to minimize artificial junctions, new fusion segments were created based on a consensus sequence for HBV génotype D. The new surface antigen segment is represented by positions 1-249 of SEQ ID NO: 110. The new polymerase (RT) segment is represented by positions 250-477 of SEQ ID NO:110. The new Core segment is represented by positions 478-629 of SEQ ID NO:110. The new X antigen segment is represented by positions 630-689 of SEQ ID NO: 110. This fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO: 110 (non-HBV sequences denoted as “optional” were not included in the base sequence of SEQ ID NO:110, but were actually added to the fusion protein described in this example): (1) an optional N-terminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) an optimized portion of an HBV large (L) surface antigen represented by positions 1 to 249 of SEQ ID NO: 110, which is a consensus sequence for HBV génotype D utilizing the design strategy discussed above; (4) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase represented by positions 250 to 477 of SEQ ID NO: 110, which is a consensus sequence for HBV génotype D utilizing the design strategy discussed above; (5) an optimized portion of HBV Core protein represented by positions 478 to 629 of SEQ ID NO: 110, which is a consensus sequence
170 for HBV génotype D utilizing the design strategy discussed above; (6) an optimized portion of HBV X antigen represented by positions 630 to 689 of SEQ ID NO: 110, which is a consensus sequence for HBV génotype D utilizing the design strategy discussed above; and (7) an optîonal hexahistidine tag. A yeast-based immunotherapy composition expressing this complété fusion protein is also referred to herein as GI-13013. A yeastbased immunotherapy composition expressing a similar fusion protein (containing SEQ ID NCkllO), except that the N-terminal peptide of SEQ ID NO:37 is substituted with the alpha factor sequence of SEQ ID NO:89, is referred to herein as GI-13014. The fusion protein and corresponding yeast-based immunotherapeutic can also be referred to herein as “SPEXv2-D”, “Spex-D”, or “M-SPEXv2-D” (for GI-13013) or “a-SPEXv2-D” for (GI13014).
[00396] Additional HBV fusion proteins for use in a yeast-based immunotherapeutic were designed using the application of consensus sequences for four HBV génotypes to demonstrate how alterations similar to those made in the fusion proteins described above (SEQ ID NOs: 107-110) can be made in a different HBV fusion protein, such as that described by SEQ ID NO:34, which contains HBV Surface proteins and HBV Core proteins. To design these additional HBV antigens and corresponding yeast-based immunotherapy compositions, the fusion protein structure described above for SEQ ID NO:34 (and therefore the subparts of these fusion proteins (Surface antigen and Core) was used as a template. As above for the constructs described above, consensus sequences for each of HBV génotype A, B, C and D were built from multiple sources of HBV sequences (e.g., Yu and Yuan et al, 2010, for S and Core), and sequences in the template structure were replaced with consensus sequences corresponding to the same positions, unless using the consensus sequence altered one of the known acute self-limiting T cells epitopes or one of the known polymerase escape mutation sites, in which case, these positions followed the published sequence for these epitopes or mutation sites.
[00397] A first construct based on a consensus sequence for HBV génotype A was designed as follows. Using SEQ ID NO:34 as a template, a new fusion protein was created based on a consensus sequence for HBV génotype A, represented here by SEQ ID NO: 112. This fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO: 112 (non-HBV sequences denoted as “optional” are not included in the base sequence of SEQ ID NO: 112, but were actually added to the fusion protein described in this example): (1) an optional Nterminal peptide that is a synthetic N-terminal peptide designed to impart résistance to
171 proteasomal dégradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) a consensus sequence for HBV génotype A large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO:112; 4) the amino acid sequence of a consensus sequence for HBV génotype A core antigen represented by positions 400 to 581 of SEQ ID NO:112; and (5) an optional hexahistidine tag. A nucleic acid sequence encoding the fusion protein comprising SEQ ID NO: 112 (codon optimized for yeast expression) is represented herein by SEQ ID NO: 111. A yeast-based immunotherapy composition expressing this fusion protein is also referred to herein as GI13006. The fusion protein and corresponding yeast-based immunotherapeutic can also be referred to herein as “Score-A”.
[00398] A second construct based on a consensus sequence for HBV génotype B was designed as follows. Using SEQ ID NO:34 as a template, a new fusion protein was created based on a consensus sequence for HBV génotype B, represented here by SEQ ID NO: 114. This fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:114 (non-HBV sequences denoted as “optional” are not included in the base sequence of SEQ ID NO: 114, but were actually added to the fusion protein described in this example): (1) an optional Nterminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) a consensus sequence for HBV génotype B large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO:114; 4) the amino acid sequence of a consensus sequence for HBV génotype B core antigen represented by positions 400 to 581 of SEQ ID NO: 114; and (5) an optional hexahistidine tag. A nucleic acid sequence encoding the fusion protein comprising SEQ ID NO: 114 (codon optimized for yeast expression) is represented herein by SEQ ID NO:113. A yeast-based immunotherapy composition expressing this fusion protein is also referred to herein as GI13007. The fusion protein and corresponding yeast-based immunotherapeutic can also be referred to herein as “Score-B”.
[00399] A third construct based on a consensus sequence for HBV génotype C was designed as follows. Using SEQ ID NO:34 as a template, a new fusion protein was created based on a consensus sequence for HBV génotype C, represented here by SEQ ID
NO: 116. This fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO: 116 (non-HBV sequences denoted as “optional” are not included in the base sequence of SEQ ID NO.116,
172 but were actually added to the fusion protein described in this example): (1) an optional Nterminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) a consensus sequence for HBV génotype C large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO: 116; 4) the amino acid sequence of a consensus sequence for HBV génotype C core antigen represented by positions 400 to 581 of SEQ ID NO:116; and (5) an optional hexahistidine tag. A nucleic acid sequence encoding the fusion protein comprising SEQ ID NO: 116 (codon optimized for yeast expression) is represented herein by SEQ ID NO: 115. A yeast-based îmmunotherapy composition expressing this fusion protein is also referred to herein as GI13008. The fusion protein and corresponding yeast-based îmmunotherapeutic can also be referred to herein as “Score-C”.
[00400] A fourth construct based on a consensus sequence for HBV génotype D was designed as foliows. Using SEQ ID NO:34 as a template, a new fusion protein was created based on a consensus sequence for HBV génotype D, represented here by SEQ ID NO: 118. This fusion protein is a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, represented by SEQ ID NO:118 (non-HBV sequences denoted as “optional” are not included in the base sequence of SEQ ID NO:118, but were actually added to the fusion protein described in this example): (1) an optional Nterminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) a consensus sequence for HBV génotype D large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO:118; 4) the amino acid sequence of a consensus sequence for HBV génotype D core antigen represented by positions 400 to 581 of SEQ ID NO:118; and (5) an optional hexahistidine tag. The amino acid sequence of the complété fusion protein comprising SEQ ID NO:118 and the N- and C-terminal peptides and linker peptide is represented herein by SEQ ID NO:151. A nucleic acid sequence encoding the fusion protein comprising SEQ ID NO: 118 or SEQ ID NO:151 (codon optimized for yeast expression) is represented herein by SEQ ID NO:117. A yeast-based îmmunotherapy composition expressing this fusion protein is also referred to herein as GI-13009. The fusion proteins and corresponding yeast-based îmmunotherapeutic can also be referred to herein as “Score-D”.
[00401] The yeast-based îmmunotherapy compositions of GI-13010 (comprising SEQ ID NO: 107), GI-13011 (comprising SEQ ID NO: 108), GI-13012 (comprising SEQ ID
173
NO: 109), GI-13013 (comprising SEQ ID NO: 110), GI-13006 (comprising SEQ ID NO: 112), GI-13007 (comprising SEQ ID NO: 114), GI-13008 (comprising SEQ ID NO: 116) and GI-13009 (comprising SEQ ID NO: 118) were produced as described for other compositions above. Briefly, DNA encoding the fusion protein was codon optimized for expression in yeast and then inserted behind the CUP1 promoter (pGl-100) in yeast 2 um expression vectors. The resulting plasmids were introduced into Saccharomyces cerevisiae W303a yeast by Lithium acetate/polyethylene glycol transfection. Yeast transformants of each plasmid were isolated on solid minimal plates lacking uracil (UDM; uridine dropout medium). Colonies were re-streaked onto ULDM (uridine and leucine dropout medium) and allowed to grow for 3 days at 30°C. Liquid starter cultures lacking uridine and leucine (UL2; formulation provided in Example 1) were inoculated from plates and starter cultures were grown for 18h at 30°C, 250 rpm. Primary cultures were used to inoculate final cultures of UL2 and growth continued until a density of 2 YU/mL was reached. Cultures were induced with 0.5 mM copper sulfate for 3h and then cells were washed in PBS, heat-killed at 56°C for lh, and washed three times in PBS. Total protein content was measured by a TCA precipitation/nitrocellulose binding assay and HBV antigen expression was measured by western blot using an antî-his tag monoclonal antibody.
[00402] The results are shown in Fig. 20. The lanes in the blot shown in Fig. 20 contain protein from the following yeast-based immunotherapeutics: Lane 1 (vl.0; Score) = GI-13002 (expressing SEQ ID NO:34); Lane 2 (v2.0; ScA) = GI-13006 (expressing SEQ ID NO: 112); Lane 3 (v2.0; ScB) = GI-13007 (expressing SEQ ID NO: 114); Lane 4 (v2.0; ScC) = GI-13008 (expressing SEQ ID NO:116); Lane 5 (v2.0; ScD) = GI-13009 (expressing SEQ ID NO:118); Lane 6 (vl.0; Sp) = GI-13005 (expressing SEQ ID NO:36); Lane 7 (vl.0; a-Sp) = GI-13004 (expressing SEQ ID NO:92); Lane 8 (v2.0; SpA) = GI13010 (expressing SEQ ID NO: 107); Lane 9 (v2.0; SpB) = GI-13011 (expressing SEQ ID NO:108); Lane 10 (v2.0; SpC) = GI-13012 (expressing SEQ ID NO:109); Lane 11 (v2.0; SpD) = GI-13013 (expressing SEQ ID NO: 110).
[00403] The results show that each of the HBV antigens comprising the combination of surface antigen and core (“Score” antigens), i.e., GI-13002 (Score), GI-13006 (ScA;
Score-A), GI-13007 (ScB; Score-B), GI-13008 (ScC; Score-C), and GI-13009 (ScD;
Score-D) expressed robustly in yeast. Typical Score v2.0 expression levels in these and similar experiments were in the range of approximately 90 to 140 pmol/YU (i.e., 5940 ng/YU to 9240 ng/YU). Expression levels of the HBV antigens comprising ail four HBV
174 proteins (surface, polymerase, core and X, or “Spex”) was variable. Specifically, expression of the antigens from GI-13010 (SpA; Spex-A), Gl-13011 (SpB; Spex-B), GI13012 (SpC; Spex-D) and Gl-13013 (SpD; Spex-D) was substantially lower than expression of the “Score” antigens, as well as the antigens from GI-13005 (Sp; Spex) and GI-13004 (a-Sp; a-Spex). Expression of the antigen in Gl-13012 (SpC; Spex-C) was barely détectable. Taken together, these results indicate that as a group, HBV antigens comprising surface antigen and core express very well in yeast, whereas HBV antigens comprising ail of surface antigen, polymerase, core and X hâve variable expression in yeast, and generally express less well than the “Score” antigens.
Example 8
[00404] The foliowing example describes the production of additional yeast-based HBV immunotherapeutic compositions that utilize consensus sequences for HBV génotypes, and additionally demonstrate the use of alternate configurations/arrangements of HBV protein segments within a fusion protein in order to modify or improve the expression of an HBV antigen in yeast and/or improve or modify the immunogenicity or other functional attribute of the HBV antigen.
[00405] In this example, new fusion proteins were designed that append X antigen and/or polymerase antigens to the N- or C-terminus of the combination of surface antigen fused to core. These constructs were designed in part based on the rationale that because the fusion proteins arranged in the configuration generally referred to herein as “Score” (e.g., SEQ ID NO:34, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116 and SEQ ID NO:118) express very well in yeast, it may be advantageous to utilize this base configuration (i.e., surface antigen fused to core protein) to produce HBV antigens comprising additional HBV protein components. Such strategies may improve expression of multi-protein antigens and/or improve or modify the functionality of such antigens in the context of immunotherapy. For example, without being bound by theory, the inventors proposed that the expression of an HBV antigen using three or ail four HBV proteins could be improved by constructing the fusion protein using surface-core (in order) as a base, and then appending the other antigens to this construct.
[00406] Accordingly, to exemplify this embodiment of the invention, eight new fusion proteins were designed and constructed, and yeast-based immunotherapy products expressing these proteins were produced. In each case, the fusion protein used a surfacecore fusion protein as a base that was derived from segments of the fusion protein represented by SEQ ID NO:118, which is a surface-core fusion protein described in
175
Example 7 utilizing a consensus sequence for HBV génotype D and optimized to maximize the use of conserved immunological epitopes. AU possible arrangements of a polymerase segment and/or an X antigen segment were appended to this base configuration, utilizing segments derived from the fusion protein represented by SEQ ID NO: 110, which is a multi-protein HBV fusion protein described in Example 7 that was constructed to reduce the size of the protein segments, maximize the use of conserved immunological epitopes, and utilize a consensus sequence for HBV génotype D. While these eight resulting antigens are based on a consensus sequence for HBV génotype D, it would be straightforward to produce a fusion protein having a similar overall structure using the corresponding fusion segments from the fusion proteins represented by SEQ ID NO: 107 and/or SEQ ID NO: 112 (génotype A), SEQ ID NO: 108 and/or SEQ ID NO: 114 (génotype B), SEQ ID NO: 109 and/or SEQ ID NO: 116 (génotype C), or using the corresponding sequences from a different HBV génotype, sub-genotype, consensus sequence or strain.
[00407] To produce the first composition, yeast (e.g., Saccharomyces cerevisiae) were engineered to express a new HBV fusion protein, schematically illustrated in Fig. 8, under the control of the copper-inducible promoter, CUP1. The resulting yeast-HBV immunotherapy composition can be referred to herein as GI-13015. This fusion protein, also referred to herein as “Score-Pol” and represented by SEQ ID NO: 120, comprises, in order, surface antigen, core protein, and polymerase sequences, as a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus (non-HBV sequences denoted as “optional” were not included in the base sequence of SEQ ID NO: 120, but were actually added to the fusion protein described in this example): (1) an optional N-terminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV génotype D large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO: 120 (corresponding to positions 1 to 399 of SEQ ID NO: 118); (4) the amino acid sequence of a consensus sequence for HBV génotype D core antigen represented by positions 400 to 581 of SEQ ID NO:120 (corresponding to positions 400 to 581 of SEQ ID NO.118); (5) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase using a consensus sequence for HBV génotype D, represented by positions 582 to 809 of SEQ ID NO: 120 (corresponding to positions to 250 to 477 of SEQ ID NO: 110); and (6) an optional hexahistidine tag.
176
SEQ ID NO: 120 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein of SEQ ID NO: 120 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO:119.
[00408] To produce the second composition, yeast (e.g., Saccharomyces cerevisiae) were engineered to express a new HBV fusion protein, schematically illustrated in Fig. 9, under the control of the copper-inducible promoter, CUP1. The resulting yeast-HBV immunotherapy composition can be referred to herein as GI-13016. This fusion protein, also referred to herein as “Score-X” and represented by SEQ ID NO:122, comprises, in order, surface antigen, core, and X antigen sequences, as a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus (non-HBV sequences denoted as “optional” were not included in the base sequence of SEQ ID NO:122, but were actually added to the fusion protein described in this example): (1) an optional Nterminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV génotype D large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO: 122 (corresponding to positions 1 to 399 of SEQ ID NO:118); 4) the amino acid sequence of a consensus sequence for HBV génotype D core antigen represented by positions 400 to 581 of SEQ ID NO: 122 (corresponding to positions 400 to 581 of SEQ ID NO:118); (5) an optimized portion of HBV X antigen using a consensus sequence for HBV génotype D, represented by positions 582 to 641 of SEQ ID NO:122 (corresponding to positions 630 to 689 of SEQ ID NO: 110); and (6) an optional hexahistidine tag. SEQ ID NO: 122 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein comprising SEQ ID NO: 122 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO:121.
[00409] To produce the third composition, yeast (e.g., Saccharomyces cerevisiae) were engineered to express a new HBV fusion protein, schematically illustrated in Fig. 10, under the control of the copper-inducible promoter, CUP1. The resulting yeast-HBV immunotherapy composition can be referred to herein as GI-13017. This fusion protein, also referred to herein as “Score-Pol-X” and represented by SEQ ID NO: 124 comprises, in order, surface antigen, core, polymerase and X antigen sequences, as a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus (non-HBV sequences denoted as “optional” were not included in the base sequence of SEQ ID
177
NO: 124, but were actually added to the fusion protein described in this example): (1) an optional N-terminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV génotype D large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO: 124 (corresponding to positions 1 to 399 of SEQ ID NO:118); 4) the amino acid sequence of a consensus sequence for HBV génotype D core antigen represented by positions 400 to 581 of SEQ ID NO: 124 (corresponding to positions 400 to 581 of SEQ ID NO: 118); (5) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase using a consensus sequence for HBV génotype D, represented by positions 582 to 809 of SEQ ID NO:124 (corresponding to positions to 250 to 477 of SEQ ID NO: 110); (6) an optimized portion of HBV X antigen using a consensus sequence for HBV génotype D, represented by positions 810 to 869 of SEQ ID NO:124 (corresponding to positions 630 to 689 of SEQ ID NO:110); and (7) an optional hexahistidine tag. SEQ ID NO: 124 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein comprising SEQ ID NO:124 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO: 123.
[00410] To produce the fourth composition, yeast (e.g., Saccharomyces cerevisiae) were engineered to express a new HBV fusion protein, schematically illustrated in Fig. 11, under the control of the copper-inducîble promoter, CUP1. The resulting yeast-HBV immunotherapy composition can be referred to herein as GI-13018. This fusion protein, also referred to herein as “Score-X-Pol” and represented by SEQ ID NO:126 comprises, in order, surface antigen, core, X antigen, and polymerase sequences, as a single polypeptide with the foilowing sequence éléments fused in frame from N- to C-terminus (non-HBV sequences denoted as “optional” were not included in the base sequence of SEQ ID NO:126, but were actually added to the fusion protein described in this example): (1) an optional N-terminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV génotype D large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO:126 (corresponding to positions 1 to 399 of SEQ ID NO:118); 4) the amino acid sequence of a consensus sequence for HBV génotype D core antigen represented by positions 400 to 581 of SEQ ID NO: 126
178 (corresponding to positions 400 to 581 of SEQ ID NO: 118); (5) an optimized portion of HBV X antigen using a consensus sequence for HBV génotype D, represented by positions 582 to 641 of SEQ ID NO:126 (corresponding to positions 630 to 689 of SEQ ID NO: 110); (5) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase using a consensus sequence for HBV génotype D, represented by positions 642 to 869 of SEQ ID NO:126 (corresponding to positions to 250 to 477 of SEQ ID NO:110); and (7) an optional hexahistidine tag. SEQ ID NO:126 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein comprising SEQ ID NO: 126 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO: 125.
[00411] To produce the fifth composition, yeast (e.g., Saccharomyces cerevisiae) were engineered to express a new HBV fusion protein, schematically illustrated in Fig. 12, under the control of the copper-inducible promoter, CUP1. The resulting yeast-HBV immunotherapy composition can be referred to herein as GI-13019. This fusion protein, also referred to herein as “Pol-Score” and represented by SEQ ID NO: 128 comprises, in order, polymerase, surface antigen, and core sequences, as a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus (non-HBV sequences denoted as “optional” were not included in the base sequence of SEQ ID NO: 128, with the exception of the Leu-Glu linker between the polymerase segment and the surface antigen segment in the construct exemplified here, but were actually added to the fusion protein described in this example): (1) an optional N-terminal peptide that is a synthetic Nterminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase using a consensus sequence for HBV génotype D, represented by positions 1 to 228 of SEQ ID NO:120 (corresponding to positions to 250 to 477 of SEQ ID NO:110); (4) a linker peptide (optional) of Leu-Glu, represented by positions 229 to 230 of SEQ ID NO: 128; (5) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV génotype D large (L) surface antigen represented by positions 231 to 629 of SEQ ID NO:128 (corresponding to positions 1 to 399 of SEQ ID NO:118); (6) the amino acid sequence of a consensus sequence for HBV génotype D core antigen represented by positions 630 to 811 of SEQ ID NO:128 (corresponding to positions 400 to 581 of SEQ ID NO:118); and (7) an optional hexahistidine tag. SEQ ID NO:128 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence
179 encoding the fusion protein comprising SEQ ID NO: 128 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO:127.
[00412] To produce the sixth composition, yeast (e.g., Saccharomyces cerevisiae) were engineered to express a new HBV fusion protein, schematically illustrated in Fig. 13, under the control of the copper-inducible promoter, CUP1. The resulting yeast-HBV immunotherapy composition can be referred to herein as GI-13020. This fusion protein, also referred to herein as “X-Score” and represented by SEQ ID NO:130 comprises, in order, X antigen, surface antigen, and core sequences, as a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus, (non-HBV sequences denoted as “optional” were not included in the base sequence of SEQ ID NO: 130, with the exception of the Leu-Glu linker between the X segment and the surface antigen segment in the construct exemplified here, but were actually added to the fusion protein described in this example): (1) an optional N-terminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) an optimized portion of HBV X antigen using a consensus sequence for HBV génotype D, represented by positions 1 to 60 of SEQ ID NO: 130 (corresponding to positions 630 to 689 of SEQ ID NO: 110); (4) a linker peptide (optional) of Leu-Glu, represented by positions 61 to 62 of SEQ ID NO: 130; (5) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV génotype D large (L) surface antigen represented by positions 63 to 461 of SEQ ID NO: 130 (corresponding to positions 1 to 399 of SEQ ID NO: 118); (6) the amino acid sequence of a consensus sequence for HBV génotype D core antigen represented by positions 462 to 643 of SEQ ID NO:130 (corresponding to positions 400 to 581 of SEQ ID NO: 118); and (7) an optional hexahistidine tag. SEQ ID NO:130 contains multiple T cell epitopes (human and murine), which can be found in Table 5. The amino acid sequence of the complété fusion protein comprising SEQ ID NO: 130 and the N- and C-terminal peptides and linkers is represented herein by SEQ ID NO: 150. A nucleic acid sequence encoding the fusion protein comprising SEQ ID NO: 130 or SEQ ID NO:150 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO: 129.
[00413] To produce the seventh composition, yeast (e.g., Saccharomyces cerevisiae) were engineered to express a new HBV fusion protein, schematically illustrated in Fig. 14, under the control of the copper-inducible promoter, CUP1. The resulting yeast-HBV immunotherapy composition can be referred to herein as GI-13021. This fusion protein,
180 also referred to herein as “Pol-X-Score” and represented by SEQ ID NO: 132 comprises, in order, polymerase, X antigen, surface antigen, and core, as a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus (non-HBV sequences denoted as “optional” were not included in the base sequence of SEQ ID NO: 132, but were actually added to the fusion protein described in this example): (1) an optional Nterminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase using a consensus sequence for HBV génotype D, represented by positions 1 to 228 of SEQ ID NO: 132 (corresponding to positions to 250 to 477 of SEQ ID NO: 110); (4) an optimized portion of HBV X antigen using a consensus sequence for HBV génotype D, represented by positions 229 to 288 of SEQ ID NO:132 (corresponding to positions 630 to 689 of SEQ ID NO: 110); (5) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV génotype D large (L) surface antigen represented by positions 289 to 687 of SEQ ID NO:132 (corresponding to positions 1 to 399 of SEQ ID NO: 118); (6) the amino acid sequence of a consensus sequence for HBV génotype D core antigen represented by positions 688 to 869 of SEQ ID NO:132 (corresponding to positions 400 to 581 of SEQ ID NO: 118); and (7) an optional hexahistidine tag. SEQ ID NO: 132 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein comprising SEQ ID NO: 132 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO:131.
[00414] To produce the eighth composition, yeast (e.g., Saccharomyces cerevisiae) were engineered to express a new HBV fusion protein, schematically illustrated in Fig. 15, under the control of the copper-inducible promoter, CVP1. The resulting yeast-HBV immunotherapy composition can be referred to herein as GI-13022. This fusion protein, also referred to herein as “X-Pol-Score” and represented by SEQ ID NO:134 comprises, in order, X antigen, polymerase, surface antigen, and core protein, as a single polypeptide with the following sequence éléments fused in frame from N- to C-terminus (non-HBV sequences denoted as “optional” were not included in the base sequence of SEQ ID NO:134, but were actually added to the fusion protein described in this example): (1) an optional N-terminal peptide that is a synthetic N-terminal peptide designed to impart résistance to proteasomal dégradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) an optimized portion of HBV X
181 antigen using a consensus sequence for HBV génotype D, represented by positions 1 to 60 of SEQ ID NO:134 (corresponding to positions 630 to 689 of SEQ ID NO:110); (4) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase using a consensus sequence for HBV génotype D, represented by positions 61 to 288 of SEQ ID NO:134 (corresponding to positions to 250 to 477 of SEQ ID NO:110); (5) the amino acid sequence of a near full-Iength (minus position 1) consensus sequence for HBV génotype D large (L) surface antigen represented by positions 289 to 687 of SEQ ID NO: 134 (corresponding to positions 1 to 399 of SEQ ID NO;118); (6) the amino acid sequence of a consensus sequence for HBV génotype D core antigen represented by positions 688 to 869 of SEQ ID NO:134 (corresponding to positions 400 to 581 of SEQ ID NO:118); and (7) an optional hexahistidine tag. SEQ ID NO.134 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein comprising SEQ ID NO: 134 (codon-optîmized for expression in yeast) is represented herein by SEQ ID NO: 133.
[00415] To produce each of the yeast-based immunotherapy compositions described above, yeast transformants of each plasmid were isolated on solid minimal plates lacking uracil (UDM; uridine dropout medium). Colonies were re-streaked onto ULDM and UDM plates and allowed to grow for 3 days at 30°C. Liquid starter cultures lacking uridine and leucine (UL2) or lacking uridine (U2) were inoculated from plates and starter cultures were grown for 18h at 30°C, 250 rpm. Primary cultures were used to inoculate intermediate cultures of U2 or UL2 and growth was continued until a density of approximately 2 YU/mL was reached. Intermediate cultures were used to inoculate final cultures to a density of 0.05 YU/mL and these were incubated until the cell density reached 1-3 YU/mL. Final cultures were then induced with 0.5 mM copper sulfate for 3h and cells were washed in PBS, heat killed at 56°C for lh, and washed three times in PBS. Total protein content was measured with a TCA precipitation/nitrocellulose binding assay and HBV antigen expression was measured by Western blot using an anti-his tag monoclonal antibody. Lysâtes from two yeast immunotherapeutic compositions described in Example 7 as GI-13008 (SEQ ID NO: 116; “Score-C”) or GI-13009 (SEQ ID NO: 118; “Score-D”) were used as a basis of comparison to a yeast expressing the base surface-core antigen product.
[00416] Fig. 21 is a blot showing the expression of ail eight constructs in yeast cultured in UL2 medium (1 pg of protein loaded) as compared to expression of the construct in the yeast immunotherapeutic described in Example 7 as GI-13009 (SEQ ID
182
NO: 118; “Score-D”). Referring to Fig. 21, lanes 1 and 2 contain molecular weight markers, and lanes 4-6 contain recombinant hexahistidine tagged NS3 protein that was processed on the same blot in order to quantify antigen by interpolation from a standard curve generated from these lanes. Lanes 7-14 contain lysâtes from each yeast-based immunotherapeutic denoted by number (e.g., GI-13015) grown in UL2 medium, and lane 15 contains the lysate from the GI-13009 comparison. Additional western blots from yeast cultured in U2 medium, as well as additional blots evaluating different amounts of protein loading on the gel are not shown here, but overall, the results indicated that ail eight antigens were expressed to détectable levels in at least one growth medium.
[00417] The overall expression results are summarized in Fig. 22 as a bar graph for those cultures that had détectable expression of target antigen in U2 or UL2 medium as compared to expression of antigens in GI-13008 (Score-C) and GI-13009 (Score-D). Referring to Fig. 22, the HBV antigens are denoted below each bar using the reference to antigen arrangement in the fusion protein as described for each construct above, along with the medium used to culture the corresponding yeast that expressed the antigen (i.e., “Pol-Score-U2” refers to the HBV antigen that is a polymerase-surface-core fusion protein, represented by SEQ ID NO: 128 and expressed by GI-13019 in U2 medium). The results indicated that expression of the antigen denoted “X-Score” (expressed by GI-13020; SEQ ID NO: 130) was particularîy robust, at ~122 pmol/YU, which was approximately 79-80% of the expression level obtained for Score-C (GI-13008) or Score-D (GI-13009) on a molar basis (either medium). Expression of the antigens expressed by GI-13015 (ScorePol; SEQ ID NO:120), GI-13016 (Score-X; SEQ ID NO:122), GI-13017 (Score-Pol-X; SEQ ID NO:124) and GI-13018 (Score-X-Pol; SEQ ID NO:126) in U2 medium was below the level of quantification in this experiment, although each of these antigens were expressed when the same yeast-based immunotherapeutic was grown in UL2 medium (see Fig. 22). In general, antigen configurations containing the polymerase reverse transcriptase (RT) domain accumulated to lower levels than those containing only S-core with or without the addition of X antigen. Taking the data shown in this and prior Examples as a whole, the antigen configurations of surface-core (“Score” or “SCORE”; ali similar constructs) and X-surface-core (“X-Score” or “X-SCORE”; GI-13020) were the highest expressing antigen configurations among ail yeast-based HBV immunotherapeutics tested.
Example 9
183
[00418] The following example describes preclinical experiments in mice to demonstrate the safety, immunogenicity, and in vivo efficacy of yeast-based HBV immunotherapy compositions of the invention,
[00419] To evaluate the yeast-based HBV immunotherapy compositions in preclinical studies, a variety of in vitro and in vivo assays that detect induction of antigen-specific lymphocytes by yeast-based HBV immunotherapy compositions of the invention were employed, including lymphocyte prolifération, cell-mediated cytotoxicity, cytokine sécrétion, and protection from tumor challenge (e.g., killing of tumors engineered to express HBV proteins in vivo).
[00420] To support these studies, yeast-based HBV immunotherapy compositions described in Examples 1 and 2 were used initially, with additional studies performed using yeast-based HBV immunotherapy compositions described in 7 and 8 or elsewhere herein. However, these studies can be readily applied to any yeast-based HBV immunotherapy composition of the invention, and the results provided herein can be extrapolated to other HBV compositions comprising the same antigen base or similar antigen constructs. The results of these initial experiments are described below.
[00421] As a general protocol that can be adapted for any yeast-based HBV immunotherapy composition, mice (e.g., female BALB/c and/or C57BL/6 mice) are injected with a suitable amount of a yeast-based HBV immunotherapy composition, e.g., 4-5 YU (administered subcutaneously in 2-2.5 YU injections at 2 different injection sites). Optionally, an injection of anti-CD40 antibody is administered the day following the yeast compositions. Mice are immunized weekly or biweekly, for 1, 2, or 3 doses, and a final booster dose is optionally administered 3-4 weeks after the last weekly or biweekly dose. Mice are sacrificed 7-9 days after the final injection. Spleen cell suspensions, and/or lymph node suspensions, pooled from each group, are prepared and subjected to in vitro stimulation (IVS) conditions utilizing HBV-specîfic stimuli in the form of HBV peptides and/or HBV antigens, which may include yeast expressing HBV antigens. Control cultures are stimulated with non-HBV peptides, which can include an ovalbumin peptide, or a non-relevant viral peptide (e.g., a peptide from HIV). Standard assays are employed to evaluate immune responses induced by administration of yeast-based HBV immunotherapy compositions and include lymphocyte prolifération as assessed by Hthymidîne incorporation, cell-mediated cytotoxicity assays (CTL assays) employing 51Crlabeled target cells (or other targets labeled for overnight CTL), quantification of cytokine sécrétion by cytokine assay or ELISPOT (e.g., IFN-γ, IL-12, TNF-α, IL-6, and/or IL-2,
184 etc.), and protection from tumor challenge (e.g.} in vivo challenge with tumor cells recombinantly engineered to express HBV antigens).
[00422] Yeast-based HBV immunotherapy compositions are expected to be immunogenic as demonstrated by their ability to elicit HBV antigen-specific T cell responses as measured by the assays described above.
[00423] In initial experiments, two of the yeast-based HBV immunotherapy products described in Examples 1 and 2 were tested in lymphocyte prolifération assays (LPA) to détermine whether immunization with these products elicits antigen-specific CD4+ T cell prolifération. More specifically, the yeast-based immunotherapy product (GI-13002) expressing a fusion protein represented by SEQ ID NO:34 under the control of the CUP1 promoter, also known as “SCORE” and more specifically described in Example 1 above, and the yeast-based immunotherapy product (GI-13004) expressing a fusion protein represented by SEQ ID NO:92 under the control of the CUP1 promoter and also known as “a-SPEX” and more specifically described in Example 2, were each used to immunize mice and evaluate CD4+ T cells spécifie for the surface and/or Core antigens that are targeted in both products using lymphocyte prolifération assays (LPAs).
[00424] Female BALB/c mice were immunized three times weekly with 5 YU of “SCORE” or a-SPEX subcutaneously at 2 different sites on the mouse (2.5 YU/flank). Control mice were vaccinated with empty vector yeast (denoted “YVEC”) or nothing (denoted “Naïve”). One week after the third immunization, mice were humanely sacrificed and spleens and periaortal and inguinal draining lymph nodes (LNs) were removed and processed to single cell suspensions. LN cells from the two types of nodes were pooled and stimulated in vitro (IVS) with a mixture of recombinant core and surface antigen (S/Core mix) or a class II restricted mimetope peptide (GYHGSSLY, SEQ ID NO:103, denoted “Class II SAg mimetope peptide”), previously reported to elicit prolifération of T cells from SAg-immunized BALB/c mice (Rajadhyaksha et al (1995). PNAS 92: 1575-1579).
[00425] Spleen cells were subjected to CD4+ T cell enrichment by Magnetic Activated Cell Sorting (MACS) and incubated with the same antigens as described for LN. After 4 days incubation, IVS cultures were pulsed with tritiated (3H) thymidine for 18h, and cellular DNA was harvested on glass fiber microfilters. The level of încorporated 3Hthymidine was measured by scintillation counting. Replicate LN cultures from SCOREimmunized mice were assayed in parallel. Interferon gamma (IFN-γ) production by ELISpot was used as an additional means to assess T cell activation.
185
[00426] As shown in Fig. 23, Fig. 24 and Fig. 26, CD4 T cells from SCORE- or aSPEX-immunized mice proliferated in response to the recombinant S- and Core antigen mixture. Splenic T cells from SCORE-immunized mice (Fig. 23) showed >5 fold higher level of prolifération than T cells from YVEC-immunized (empty vector control) or Naive mice, indicating that the effect is spécifie for the Surface-Core fusion protein (i.e., antigenspecific T cell response). T cells from SCORE-immunized mice incubated with the HBV mimetope peptide also proliferated to higher levels than peptide-pulsed YVEC or Naive controls, providing further évidence of the antigen-specificity of the yeast-based immunotherapeutic product response. These effects are also dépendent upon the amount of antigen added to IVS, with optimal activity occurring at 3 pg/ml (recombinant antigen) or 30 pg/mL (peptide).
[00427] As shown in Fig. 24, LN cells from SCORE-immunized mice also proliferated in response to IVS with these same antigens, although the différence in prolifération between SCORE vs. Naive or YVEC-immunized animais was smaller than for isolated splenic CD4+ T cells.
[00428] The ELISpot data (Fig. 25) indicate that LN préparations from SCOREimmunized mice re-stimulated with S+C mix contain > 10-fold more IFN-γ secreting cells than LNs from Naive animais. IVS with HBV peptide (SEQ ID NO: 103) also elicited an IFN-γ response. Specifically, the SCORE LN preps contained > 3.5-fold more IFN-γproducing cells than Naive LN preps (Fig. 25). These data collectively show that SCORE (yeast-based immunotherapy expressing the fusion protein comprising surface antigen and core) elicits HBV antigen-specific T cell responses in both spleen and LN, and that these responses can be amplified by IVS with purified antigens in a dose-dependent fashion.
[00429] Similar analyses with a-SPEX (Fig. 26) showed that this yeast-based HBV immunotherapeutic product also elicits T cell proliférative responses. a-SPEX elicited about a 30% increase as compared to YVEC in IVS performed with the recombinant antigen mixture. Overall, the responses observed with a-SPEX were lower than those observed with SCORE. The différence in magnitude of the response may reflect the fact that antigen expression in a-SPEX is less than half that of SCORE on a molar basis. Alternatively, without being bound by theory, these results may indicate that the configuration of the antigens expressed by the yeast influence expression level, processing efficiency through the endosome/proteasome, or other parameters of the immune response. The prolifération of T cells from a-SPEX mice using the 100 pg/mL peptide was at least 2 -fold greater than the prolifération in YVEC vaccinated mice (Fig. 26, right three columns).
186
Example 10
[00430] The following example describes the immunological évaluation of two yeastbased HBV immunotherapeutics of the invention using cytokine profiles.
[00431] One way to characterize the cellular immune response elicited as a resuit of immunization with yeast-based HBV immunotherapeutics of the invention is to evaluate the cytokine profiles produced upon ex vivo stimulation of spleen préparations from the immunized animais.
[00432] In these experiments, female C57B1/6 mice were immunized with GI-13002 (“SCORE”, a yeast-based immunotherapeutic expressing the HBV surface-core fusion protein represented by SEQ ID NO:34, Example 1) and GI-13005 (“M-SPEX”, a yeastbased immunotherapeutic expressing the HBV surface-pol-core-X fusion protein represented by SEQ ID NO:36 under the control of the CUP1 promoter, Example 2), YVEC (empty vector control yeast), or nothing (Naïve) as follows: 2 YU of yeast-based immunotherapeutic or control yeast were injected subcutaneously at 2 different sites on the animal on days 0, 7, & 28. Anti-CD40 antibody was administered by intraperitoneal (IP) injection on day 1 to provide additional activation of dendritic cells (DCs) beyond the level of activation provided by yeast-based therapeutic. The anti-CD40 antibody treatment is optional, but the use of the antibody can boost the level of antigen-specific CD8+ T cells when attempting to detect these cells by direct pentamer staining (such data not shown in this experiment). Nine days after the last immunization, spleens were removed and processed into single cell suspensions. The cells were put into in vitro stimulation (IVS) cultures for 48h with a mixture of 2 HBV peptides pools (denoted “P” in Fig. 27 and Fig. 28 and “HBVP” in Figs. 29A and 29B), or with mitomycin C-treated naive syngeneic splénocytes pulsed with the 2 peptides (denoted “PPS” in Fig. 27 and Fig. 28 and “HPPS” in Figs. 29A and 29B). The peptides are H-2Kb- restricted and hâve following sequences: ILSPFLPLL (SEQ ID NO:65, see Table 5) and MGLKFRQL (SEQ ID NO:104). The cultures were subjected to replicate Luminex analysis of IL1 β, IL-12, and IFN-γ.
[00433] These cytokines were evaluated because they are associated with the types of immune responses that are believed to be associated with a productive or effective immune response against HBV. IL-Ιβ is a pro-inflammatory cytokine produced by antigen presenting cells, and is a cytokine known to be induced by immunization with yeast-based immunotherapy compositions. IL-12 is also produced by antigen presenting cells and promûtes CD8+ cytotoxic T lymphocyte (CTL) activity. IFN-γ is produced by
187
CD8+ cytotoxic T lymphocytes in the development of the adaptive immune response and also promoted Thl CD4+ T cell différentiation.
[00434] The results, shown in Fig. 27 (IL-Ιβ), Fig. 28 (IL-12), Fig. 29A (IFN-y; SCORE-immunized), and Fig. 29B (IFN-γ; M-SPEX-immunized) show that ail three 5 cytokines are produced by splénocytes from Score-immunized mice (denoted “Sc” in Fig.
27, Fig. 28 and Fig. 29A) in response to direct IVS with peptide pool alone, and that the response is greater for SCORE-immunized than for YVEC (denoted “Y” in Fig. 27, Fig.
and Figs. 29A and 29B) or Naïve (denoted “N” in Fig. 27, Fig. 28 and Figs. 29A and 29B) mice, demonstrating that immunization with SCORE elicits an antigen-specific 10 immune response resulting in production of these three cytokines. IVS with peptidepulsed syngeneic splénocytes also elicited an antigen spécifie response although of lower magnitude. Splénocytes from M-SPEX-vaccinated mice (denoted “Sp” in Fig. 27, Fig. 28 and Fig. 29B) produced an overall lower level of the cytokines than those from SCOREvaccinated mice. Nevertheless, the amount of IL12p70 produced in response to M-SPEX 15 is higher than the amount produced by YVEC or Naïve, indicating an antigen-specific immune response induced by this yeast-based immunotherapeutic composition. It is expected that a-SPEX (GI-13004; Example 2), which expressed higher levels of antigen and induced a CD4+ proliférative response in the assays described in Example 9, will elicit higher levels of cytokine production.
[00435] Additional cytokine assays were performed using female BALB/c mice immunized with one of the same two yeast-based immunotherapeutic products. In these experiments, female BALB/c mice were immunized with SCORE (GI-13002; denoted “Sc” in Figs. 30A-30D), M-SPEX (GI-13005; denoted “Sp” in Figs. 30A-30D), YVEC (denoted “Y” in Figs. 3OA-3OD), or nothing (Naïve, denoted “N” in Figs. 30A-30D) as 25 follows: 2 YU of yeast product were administered at 2 sites on days 0,11, 39, 46, 60, and
67. As in the experiment above, anti-CD40 antibody was administered i.p. Nine days after the last immunization (day 76) spleens were removed, processed into single cell suspensions, and subjected to IVS for 48h with a mixture of recombinant HBV Surface and Core proteins (denoted “HBV Sag+Core Ag” in Figs. 30A-30D). Supernatants were 30 collected and evaluated by Luminex for production of ΙΕΙβ, IL-6, IL-13, and IL12p70.
IL-6 is a pro-inflammatory cytokine produced by antigen presenting cells and T cells and is believed to be an important cytokine in the mechanism of action of yeast-based immunotherapeutic products. IL-13 is also a pro-inflammatory cytokine produced by T cells and is closely related to IL-4 and promotion of a Th2 CD4+ immune response.
188
[00436] The results, shown in Fig. 30A (IL-1 β), Fig. 30B (IL-6), Fig. 30C (IL-13) and Fig. 30D (IL-12) show that splénocytes from SCORE-immunized mice produced IL-Ιβ, IL-6, IL12p70, and IL-13 in response to the surface and core antigen mix and that the magnitude of the response was higher than for splénocytes from YVEC-immunized or Naïve mice. This antigen specificity is consistent with results obtained for LPA in BALB/c mice (see Example 9) and for cytokine release assays in C57B1/6 mice (see above).
[00437] Splénocytes from M-SPEX immunized mice produced antigen-specific signais for IL-1 β (Fig. 30A) but not for the other cytokines. As with the findings in C57B1/6, this apparent différence in potency between SCORE and M-SPEX may be explained by the lower antigen content of the latter. It is expected that a-SPEX (expressing a fusion protein represented by SEQ ID NO:92, described in Example 2), which expresses higher levels of antigen, will induce improved antigen-specific cytokine production, and in addition, IVS assays featuring the additional antigens expressed by this product or others that incorporate other HBV antigens (HBV X and Polymerase antigens) are expected to reveal additional immunogenicity.
Example 11
[00438] The following example describes immunogenicity testing in vivo of a yeastbased immunotherapeutic composition for HBV.
[00439] In this experiment, the yeast-based immunotherapy product (GI-13002) expressing a fusion protein represented by SEQ ID NO:34 under the control of the CUP1 promoter, also known as “SCORE” and more specifically described in Example 1 was used in an adoptive transfer method in which T cells from SCORE-immunized mice were transferred to récipient Severe Combined Immune Déficient (SCID) mice prior to tumor implantation in the SCID mice.
[00440] Briefly, female C57BL/6 mice (âge 4-6 weeks) were subcutaneously immunized with GI-13002 (SCORE), YVEC (yeast containing empty vector), or nothing (naïve) at 2 sites (2.5 YU flank, 2.5 YU scruff) on days 0, 7 and 14. One cohort of SCORE-immunized mice was additionally injected intraperitoneally (i.p.) with 50 pg of anti-CD40 antibody one day after each îmmunization. On day 24, mice were sacrificed and total splénocytes were prepared and counted. Twenty-five million splénocytes in 200 pL PBS were injected i.p. into naive récipient 4-6 week old female SCID mice. Twenty four hours post-transfer, the récipients were challenged subcutaneously (s.c.) in the
189 nbcage area with 300,000 SCORE-antigen expressing EL4 tumor cells (denoted “EL-4Score”), or tumor cells expressing irrelevant ovalbumin antigen. Tumor growth was monitored by digital caliper measurement at 1 to 2 day intervals starting at day 10 post tumor challenge.
[00441] The results at 10 days post tumor challenge, shown in Fig. 31, demonstrated that splénocytes from mice immun ized with GI-13002 (SCORE) or GI-13002 + anti-CD40 antibody, but not from YVEC or naive mice, elicited comparable protection from challenge with EL4 tumors expressing the SCORE antigen (Fig. 31, first and second bars from left). The number of mice with tumors 10 days post challenge are indicated above each bar in Fig. 31. T cells from GI-13002-immunized mice had no effect on the growth of EL4 tumors expressing an unrelated antigen (not shown). Splénocytes from YVECimmunized mice (Fig. 31, middle bar) did not affect tumor growth, as the size and number of tumors in this group were comparable to those of mice receiving no splénocytes (Fig. 31, far right bar) or those mice receiving splénocytes from naïve mice (Fig. 31, second bar from right). These results indicate that immunization with a yeast-based immunotherapeutic composition expressing a surface antigen-core fusion protein generates an antigen-specific immune response that protects SCID mice from tumor challenge. Co-administration of the dendritic cell (DC)-activating anti-CD40 antibody did not influence the extent of protection.
Example 12
[00442] The following example describes the immunogenicity testing of two yeastbased immunotherapy compositions for HBV using interferon-γ (IFN-γ) ELISpot assays.
[00443] This experiment was designed to evaluate two optimized yeast-based immunotherapy compositions described in Example 7 for the ability to induce HBV antigen-specific T cells in mice immunized with these compositions. The experiment also tested whether novel HBV peptide sequences designed with computational algorithms and sequences obtained from the published literature can be used to re-stimulate T cell responses that were generated by these immunotherapy compositions.
[00444] In this experiment, the yeast-based immunotherapy composition described in
Example 7 as GI-13008 (“Score-C”, comprising SEQ ID NO:116) and the yeast-based immunotherapy composition described in Example 7 as GI-13013 (“Spex-D”, comprising
SEQ ID NO: 110) were evaluated for immunogenicity. Peptide sequences used in this experiment are shown in Table 7. The sequences denoted ZGP-5 and ZGP-7 are from the published literature whereas the remaining peptides were identified computationally with
BIMAS or SYFPEITHI prédictive algorithme. The préfixés “Db” or “Kb” refer to the haplotype of C57BL/6 mice: H-2Db and H2-Kb, respectively.
Table 7
Sequence MHC HBV
Peptide name Amino acid sequence Identifier Class Antigen
Db9-84 WSPQAQGIL SEQIDNO:138 I Sag
Db9-94 TVPANPPPA SEQ ID NO: 141 I Sag
Db9-283 GMLPVCPLL SEQ ID NO:142 I Sag
Db9-499 MGLKIRQLL SEQ ID NO:143 I Core
Kb8-249 ICPGYRWM SEQ ID NO:144 I Sag
Kb8-262 IIFLFILL SEQ ID NO:145 I Sag
Kb8-277 VLLDYQGM SEQ ID NQ:139 I Sag
Kb8-347 ASVRFSWL SEQ ID NO:140 I Sag
Kb8-360 FVQWFVGL SEQ ID NO:146 I Sag
Kb8-396 LLPIFFCL SEQ ID NO:147 I Sag
ZGP-5 VSFGVWIRTPPAYRPPNAPIL SEQ ID NO:148 II Core
ZGP-7 ILSPFLPL SEQIDNO:149 I Sag
[00445] Female C57BL/6 mice (âge 4-6 weeks) were subcutaneously immunized with
GI-13008 (Score-C), GI-13013 (Spex-D), YVEC (empty vector yeast control), or nothing (naïve) at 2 sites (2.5 YU flank, 2.5 YU scruff) on days 0, 7 and 14. On day 20, mice were sacrificed and total splénocytes were prepared, depleted of red blood cells, counted, and incubated at 200,000 cells/well for four days in complété RPMI containing 5% fêtai calf 10 sérum plus the peptide stimulants listed in Table 7 (10 μΜ for Db and Kb peptides; 30 pg/mL for ZGP peptides) or a mixture of recombinant HBV SAg and Core antigen (3 pg/mL total). Concanavalin A was added as a positive control stimulant.
[00446] The results (Fig. 32) show that immunization of C57BL/6 mice with GI-13008 (Score-C) elicits IFNy ELISpot responses directed against HBV surface (S) and core 15 antigens with particular specificity for the following peptides: Db9-84, Kb8-277 and/or
Kb8-347, ZGP-5, and ZGP-7. These peptides elicited IFNy responses greater than those from wells containing medium alone, or from wells containing splénocytes from GI-13013 (Spex-D)-immunized, YVEC-immunized, or Naïve mice. Recombinant S+Core antigen mixture also elicited an IFNy response, although the YVEC control cells in that particular 20 stimulant group produced background signal which precluded the évaluation of an antigen-specific contribution for the S+Core antigen mix. These data indicate that GI13008 (Score-C), which expresses a surface-core fusion protein, elicits HBV-antigen spécifie immune responses that can be re-stimulated with selected peptides ex vivo, and that these responses are more readily détectable than those elicited by GI-13013 (Spex-D).
Example 13
[00447] The following example describes an experiment in which a yeast-based îmmunotherapy composition for HBV was tested for the ability to stimulate IFNy production from peripheral blood mononuclear cells (PBMCs) from a subject vaccinated with a commercial HBV prophylactic vaccine.
[00448] In this experiment, the yeast-based îmmunotherapy product known as GI13002 (“Score”, comprising SEQ ID NO:34, Example 1) was tested for its ability to stimulate IFNy production from PBMCs isolated from a subject who was vaccinated with commercial HBV prophylactic vaccine (ENGERIX-B®, GlaxoSmithKline), which is a prophylactic vaccine containing a recombinant purified hepatitis B virus surface antigen 10 (HBsAg) adsorbed on an aluminum-based adjuvant.
[00449] Briefly, blood was collected and PBMCs were isolated from a healthy HBVnaive human subject expressing the HLA-A*0201 allele. The PBMCs were frozen for later analysis. The subject was then vaccinated with ENGERIX-B® (injection 1), blood was collected at days 12 and 29 post-injection, and PBMCs were isolated and frozen. The 15 subject was vaccinated a second time with ENGERIX-B® (injection 2, boost) and blood was collected on days 10, 21, and 32 post-boost, PBMCs were isolated and frozen for each time point.
[00450] After the sériés of PBMC samples was acquired and frozen, the cells from ail time points were thawed, washed, and incubated with the empty vector yeast control 20 (YVEC) or with GI-13002 at a 5:1 yeast:PBMC ratio for 3 days in a 37°C/5% CO2 incubator. The cells were then transferred to an IFNy ELISpot plate, incubated for 18h, and processed to develop ELISpots according to standardized procedures.
[00451] As shown in Fig. 33 (columns dénoté time periods pre- and post-priming immunization or post-boost), a substantial ELISpot response was observed for GI-1300225 treated PBMCs that was higher than that of YVEC-treated PBMCs at the day 21 postboost time-point (GI-13002 ELISpots minus YVEC ELISpots ~ 230 spots per one million PBMCs). The level of YVEC-subtracted Score ELISpots was above the number observed for other time-points and 2.8 fold above the signal obtained for the pre-vaccination sample. The only substantial structural différence between the yeast-based compositions of GI30 13002 and YVEC is the presence of the surface-core fusion protein (the HBV antigen) within the vector carried by GI-13002 (i.e., YVEC has an “empty” vector). Therefore, the resuit indicates that GI-13002 elicited antigen-specific stimulation of T cells in the PBMCs of the subject. Because the ENGERIX-B® vaccine contains recombinant surface
192 antigen, but not core antigen, and because the subject was négative for HBV virus (had not been infected with HBV), this resuit also indicates that the IFNy production observed was derived from HBsAg-specific (surface antigen-specific), rather than core antigen-specific,
T cells.
Example 14
[00452] The following example describes the évaluation of yeast-based immunotherapy compositions for HBV in vivo in murine immunization models.
[00453] In this experiment, the yeast-based immunotherapy product known as GI13009 (“SCORE-D”, comprising SEQ ID NO:118, Example 7), and the yeast-based immunotherapy product known as GI-13020 (“X-SCORE”, comprising SEQ ID NO: 130, Example 8) were administered to C57BL/6 mice, BALB/c mice and HLA-A2 transgenic mice (B6.Cg-Tg(HLA-A/H2-D)2Enge/J; The Jackson Laboratory, provided under a license from the University of Virginia Patent Foundation). The HLA-A2 transgenic mice used in these experiments express an interspecies hybrid class I MHC gene, AAD, which contains the alpha-1 and alpha-2 domains of the human HLA-A2.1 gene and the alpha-3 transmembrane and cytoplasmic domains of the mouse H-2Dd gene, under the direction of the human HLA-A2.1 promoter. The chimeric HLA-A2.1/H2-Dd MHC Class I molécule médiates efficient positive sélection of mouse T cells to provide a more complété T cell répertoire capable of recognizing peptides presented by HLA-A2.1 Class I molécules. The peptide epitopes presented and recognized by mouse T cells in the context of the HLAA2.1/H2-Dd class I molécule are the same as those presented in HLA-A2.1* humans. Accordingly, this transgenic strain enables the modeling of human T cell immune responses to HLA-A2 presented antigens.
[00454] The goal of these experiments was to evaluate the breadth and magnitude of HBV antigen-specific immune responses that are generated by immunization with the yeast-based HBV immunotherapeutics in mice with varied MHC alleles, including one expressing a human MHC (HLA) molécule. Immunogenicity testing was done postimmunization by ex vivo stimulation of spleen or lymph node cells with relevant HBV antigens, followed by assessment of T cell responses by: IFN-y/IL-2 dual color ELISpot, lymphocyte prolifération assay (LPA), Luminex multi-cytokine analysis, and/or intracellular cytokine staining (ICCS). ICCS was used to détermine the contribution of CD4+ and CD8+ T cells to the antigen-specific production of IFN-γ and TNF-a.
[00455] In each of the experiments described below, mice were vaccinated subcutaneously with yeast-based HBV immunotherapeutics or yeast-based control (described below) according to the same regimen: injection at 2 sites (flank, scruff) with 2.5 YU of the yeast composition per site, once per week for 3 weeks. Controls included 5 YVEC (control yeast containing an empty vector, i.e., no antigen), OVAX2010 (a control yeast immunotherapy composition that expresses the non-HBV antigen, ovalbumin), and the combination of YVEC with soluble recombinant antigens (ovalbumin or HBV antigens) and anti-CD40 antibody. The spécifie experiments and treatment cohorts are shown in Tables 8 and 9 below. Mice were euthanized 8 days (HLA-A2 transgenic, 10 Experiment 1) or 14 days (C57BL/6 and BALB/c, Experiment 2) after the third immunization, and spleen and inguinal lymph nodes were dissected and incubated with various antigenic stimuli (HBV class I and class II MHC-restricted peptides, recombinant proteins, and HBV-antigen expressing tumor cell lines) for 5 days, as indicated below. For Luminex analysis, culture supernatants were harvested and evaluated for the 15 production of 10 different cytokines (Thl and Th2 type) at 48h after antigen addition. For
ELISpot assays, cells were incubated on IFN-γ antibody-coated plates for the last 24h of the 5 day in vitro stimulation (IVS), followed by standardized spot détection and counting. For LP As, cells were pulsed with 3H-thymidine for the last 18h of the 5 day IVS, and the amount of isotope incorporated into newly synthesized DNA was then measured by 20 scintillation counting. For ICCS, after a full 7 day stimulation, were subjected to Ficoll gradient centrifugation to eliminate dead cells, and 1 million viable cells per well (96 well U-bottom plates) were then incubated for 5 hours with the same antigenic stimuli at a range of concentrations (titration), and then permeablized, and subjected to staining with fluorochrome coupled-antibodies recognizing intracellular IFN-γ and TNF-α plus cell 25 surface markers CD4 and CD8. The percentage of CD4+ or CD8+ T cells expressing the cytokines was determined by flow cytometry.
[00456] Table 8 describes the experimental cohorts and protocol for Experiment 1. In this experiment, HLA-A2 cohorts of mice were immunized using the protocol described above, and the mice were euthanized for immune analysis 8 days after the third 30 immunization. Group A (“YVEC”) received the yeast YVEC control according to the immunization schedule described above; Group B (“SCORE-D (GI-13009-LJL2)”) received Gl-13009 grown in UL2 medium (see Example 7) according to the immunization schedule described above; and Group C (“X-SCORE (GI-13020-U2)”) received GI-13020
194 grown in U2 medium (see Example 8) according to the immunization schedule described above.
Table 8
Group HLA-A2 Mice (#, treatment)
A 3, YVEC
B 3, SCORE-D/GI-13009-UL2
C 3, X-SCORE/GI-13020-U2
[004571 IFN-γ ELISpot assay results from the lymph node cells harvested from mice in Experiment 1 are shown in Fig. 34. This figure shows the results of restimulation of lymph node cells from the immunized mice with various HBV peptides as compared to a medium control (note that the peptide denoted “TKO20” is a peptide from X antigen (X52-60) that is contained within the immunotherapeutic X-SCORE, but is not présent in SCORE-D). The results indicated that lymph node cells from both SCORE-D (GI-13009)immunized and X-SCORE (GI-13020)-immunized mice possess T cells that produce IFNγ in response to in vitro stimulation with a mixture of 3 pg/ml each of recombinant HBV surface and core antigens (Fig. 34; denoted “S&C3”). This ELISpot response was greater than that observed for YVEC-immunized mice (yeast controls) treated with the same stimulant, indicating that the HBV surface and/or core antigens within the yeast-based immunotherapeutic compositions (SCORE-D and X-SCORE) are required for the induction of the IFN-γ response. Furthermore, the results indicate that the restimulation using HBV antigen in the IVS results in efficient IFN-γ production, since wells containing medium alone showed a much lower ELISpot response.
[00458] Fig. 34 also shows that a selected HLA-A2-restricted epitope from HBV core (TKP16; Core:115-124 VLEYLVSFGV; SEQ ID NO:75) known in the field to be important in patients with acute HBV exposure and clearance, elicits a response in XSCORE-immunized mice that is greater than that observed for media only wells. Further refinement of the peptide concentration and incubation times for the ELISpot assay is expected to increase the magnitude and reduce the variability in the observed response for these antigens.
[00459] Fig. 35 shows the IFN-γ ELISpot assay results from the spleen cells harvested from SCORE-D-immunized mice in Experiment 1. The HBV core peptide denoted “Corel 1-27” (ATVELLSFLPSDFFPSV (SEQ ID NO:72)) is contained within the antigen
expressed by SCORE-D, whereas the HBV X peptide denoted “X92-100” is not contained within the antigen expressed by SCORE-D and is therefore a control peptide in this experiment. As shown in Fig. 35, spleen cells from the SCORE-D immunized HLA-A2 transgenic mice produced an IFN-γ ELISpot response upon in vitro stimulation with the 5 known HLA-A2 restricted HBV core epitope, denoted “Core 11-27”. This response was greater than that observed from spleen cells that were stimulated in vitro with an irrelevant peptide (denoted “X92-100”), medium alone, or for any IVS treatment wells for splénocytes from YVEC-immunized mice.
[00460] Therefore, the initial results from Experiment 1 show that both SCORE-D and 10 X-SCORE elicit HBV antigen-specific T cell responses in HLA-A2 transgenic mice immunized with these yeast-based immunotherapy compositions.
[00461] Table 9 describes the experimental cohorts and protocol for Experiment 2. In this experiment, C57BL/6 and BALB/c cohorts of mice were immunized using the protocol described above, and the mice were euthanized for immune analysis two weeks 15 after the third immunization. Group A (“Naïve”) received no treatment; Group B (“YVEC”) received the yeast YVEC control according to the immunization schedule described above; Group C (“X-SCORE (GI-13020-U2)”) received GI-13020 grown in U2 medium (see Example 8) according to the immunization schedule described above; Group D (“SCORE-D (GI-13009-UL2)”) received GI-13009 grown in UL2 medium (see 20 Example 7) according to the immunization schedule described above; and Group E (“OVAX2010”) received the yeast control expressing ovalbumin according to the immunization schedule described above.
Table 9
Group C57BL/6 Mice (#, treatment) BALB/c Mice (#, treatment)
A Θ, Naïve 8, Naïve
B 8, YVEC 8, YVEC
C 8, X-SCORE (GI-13020-U2) 8, X-SCORE (GI-13020-U2)
D 8, SCORE-D (GI-13009-UL2) 8, SCORE-D (GI-13009-UL2)
E 8, OVAX2010 7, OVAX2010
[00462] Fig. 36 shows the results of the ELISpot assays for lymph node cells isolated from C57BL/6 mice immunized as indicated in Experiment 2 (Table 9). These results
196 demonstrated that both X-SCORE and SCORE-D elicited IFN-γ responses in wild type C57BL/6 mice. Lymph node cells from X-SCORE-immunized mice stimulated in vitro with purified, Pichia pastoris-Qxpiesscd surface and core antigens (denoted yS&C; IVS with 1:1 mix of PicAm-expressed surface and core antigens, 3 gg/mL each) produced a meaningful IFN-γ immune response. The same lymph node cell préparations from both X-SCORE- and SCORE-D-immunized mice, when stimulated with E. co/z-expressed surface and core antigens (denoted cS&C; IVS with 1:1 mix of E. coli expressed surface and core antigens, 3 gg/mL each), produced higher overall ELISpot responses than those observed for the PicZiza-expressed recombinant antigens. The column labeled “no stim” in Fig. 36 dénotés IVS conditions where cRPMI medium alone was provided (no antigen). In general, immunization with X-SCORE elicited a greater effect than SCORE-D, and both HBV yeast-based immunotherapy compositions elicited a greater response than that observed for YVEC-immunized (yeast control) or Naive mice. These data indicate that both SCORE-D and X-SCORE produce HBV-antigen spécifie immune responses that are détectable in ex vivo lymph node cell préparations from C57BL/6 mice.
[00463] Fig. 37 shows the intracellular cytokine staining (ICCS) assay results for C57BL/6 mice conducted in Experiment 2 (Table 9). The results showed that immunization of C57BL/6 mice with either X-SCORE or SCORE-D elicits IFN-γproducing CD8+ T cells that are spécifie for the MHC Class I, H-2Kb-restricted peptide from surface antigen, denoted “VWL” (VWLSVIWM; SEQ ID NO: 152). This effect was dépendent upon the concentration of peptide added to cells during the 5 hour incubation of the ICCS procedure; greater concentrations of peptide resulted in an increasing différence in the level of IFN-γ producing CD8+ T cells for HBV yeast-based immunotherapeutics (SCORE-D and X-SCORE) versus irrelevant the yeast controls (Ovax and Yvec), with maximal séparation occurring at 10 gg/mL of peptide.
[00464] The ICCS assays of Experiment 2 also showed that immunization of C57BL/6 mice with X-SCORE and SCORE-D elicited IFN-y-producing CD4+ T cells spécifie for the MHC Class Il-restricted HBV peptide from core protein denoted “ZGP-5” (VSFGVWIRTPPAYRPPNAPIL; SEQ ID NO: 148), with 0.5 gg/mL of peptide added during the 5 hour incubation period of the ICCS procedure (Fig. 38).
[00465] Taken together with the ELISpot results described above, these data indicate that both SCORE-D and X-SCORE yeast-based immunotherapeutic compositions elicit HBV antigen-specific, effector CD4+ and CD8+ T cells that are detected by ex vivo stimulation of lymph node and spleen cells with recombinant HBV antigens and HBV
197 peptides. The T cell responses occur in both wild type C57BL/6 mice (H2-Kb) and in
HLA-A2 transgenic mice, indicating the potential of these vaccines to elicit immune responses in the context of diverse major histocompatibility types.
[00466] Based on the results described in this Example above for HLA-A2 mice and C57BL/6 mice, as well as the results of the experiments described in Examples 9, 10, 11 and 12, it is expected that results with BALB/c mice immunized with either SCORE-D or X-SCORE will also demonstrate that the yeast-based HBV immunotherapeutic compositions elicit HBV antigen-specific, effector CD4+ and CD8+ T cells in these mice. Indeed, initial results from the BALB/c cohorts were positive for CD8+ T cell responses (data not shown). It is further expected that lymphocyte prolifération assays and Luminex cytokine release analyses will show that both SCORE-D and X-SCORE induce immune responses specifically targeted to the HBV antigen sequences présent in the yeast immunotherapeutics, and that these responses will be observed in ail three mouse strains (HLA-A2 transgenic, C57B176 and BALB/c).
Example 15
[00467] The following example describes an experiment in which yeast-based immunotherapy compositions for HBV are evaluated for the ability to stimulate IFNy production from PBMCs isolated from donors of varied HBV antigen exposure.
[00468] In this experiment, the yeast-based immunotherapy product known as GI13009 (“Score-D”, comprising SEQ ID NO: 118, Example 7), and the yeast-based immunotherapy product known as GI-13020 (“X-Score”, comprising SEQ ID NO: 130, Example 8) are tested for their ability to stimulate IFNy production from PBMCs isolated from donors of varied HBV antigen exposure. In this experiment, one group of donors has previously been vaccinated with ENGERIX-B® (GlaxoSmithKline) or with RECOMBIVAX HB® (Merck & Co., Inc.), one group of donors is naïve to HBV antigen (“normal”), and one group of donors is a chronic HBV patient (a subject chronically infected with HBV). ENGERIX-B® is a prophylactic recombinant subunit vaccine containing a recombinant purified hepatitis B virus surface antigen (HBsAg) produced in yeast cells, purified and then adsorbed on an aluminum-based adjuvant. RECOMBIVAX HB® is a prophylactic recombinant subunit vaccine derived from HBV surface antigen (HBsAg) produced in yeast cells and purified to contain less than 1% yeast protein. Ail donors express the HLA-A*0201 allele.
198
[00469] The donor PBMCs are incubated in 6-well flat-bottomed tissue culture plates (107 PBMCs per well) for 3h in a 5% CO2 incubator in complété RPMI medium containing 10% fêtai bovine sérum. Non-adherent cells are removed and discarded and the adhèrent cells are treated with recombinant human interleukin-4 (IL-4) and recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) (20 and 50 ng/mL, respectively) for 5 days to generate immature dendritic cells (iDCs). The iDCs are then incubated with ant-CD40 antibody (1 pg/ml), YVEC (yeast control comprising an empty vector), or the yeast-based products GI-13020 or GI-13009, for 48h in a 5% CO2 incubator at 37°C, to generate mature DCs. For anti-CD40 antibody-treated DCs, cells are additionally pulsed with HLA-A*0201-restricted HBV peptides using standard methods. AU DC groups are PBS-washed and then removed from plates with a cell harvester in PBS. Cells are irradiated (30 Gy) and used to stimulate the autologous donor PBMCs at a DC:PBMCs ratio of 1:10. Stimulation is conducted for 7 days (round 1) of which the last 4 days are conducted in medium containing recombinant human IL-2. The stimulated PBMCs are then subjected to Ficoll gradient centrifugation, and the isolated viable cells subjected to a second round of IVS with yeast-pulsed or peptide-pulsed DCs prepared as described above. The stimulated PBMCs are then incubated with HBV peptide(s) or controls in the presence of 20 U/mL rhIL-2 in 96 well plates coated with antibody spécifie for IFN-γ, and ELISpot détection is conducted using standard manufacturer procedures. It is expected that PBMCs stimulated with autologous SCORE-D- or X-SCORE-fed DCs, or with HBV peptide-pulsed DCs, will respond to exogenous HBV peptides to a greater degree than PBMCs stimulated with YVEC-fed or unpulsed DCs, and that this effect will be more pronounced for HBV ENGERIX® or RECOMBIVAX HB® vaccine récipients than for donors who are naive to HBV antigen exposure.
Example 16
[00470] The following example describes preclinical experiments using human PBMCs to demonstrate the immunogenicity of yeast-based HBV immunotherapy compositions of the invention in humans.
[00471] Specifically, these experiments are designed to détermine whether HBV surface antigen-specifîc and/or HBV core antigen-specifîc CD8+ T cells can be detected in the peripheral blood mononuclear cells (PBMCs) of HBV carriers following 2 rounds of in vitro stimulation (IVS) with yeast-based HBV immunotherapy compositions containing
HBV surface antigen and HBV core.
[00472] PBMCs are obtained from human donors confirmed to be positive for HBV (based on sérum HBsAg status). Total DNA is isolated from 0.5 mL whole blood and typed for HLA in order to identify the correct HBV pentamer for testing (see table below).
Table 10
HI A type Pentamer Peptid e Sequence Antigen
A*0201 FLLTRILTI (SEQ ID NO:42) Surface
A*0201 GLSPTVWLSV (SEQ ID NO:43) Surface
A*0201 FLPSDFFPSI (SEQ ID NO:44) Core
A*1101 YVNVNMGLK (SEQ ID NO:48) Core
A*2402 EYLVSFGVW (SEQ ID NO:49) Core
[00473] Dendritic cells (DCs) are prepared from the PBMCs isolated from the donors described above by culturing PBMCs for 5 days in the presence of GM-CSF and IL-4. The DCs are subsequently incubated with yeast-based HBV immunotherapy compositions (e.g., those described in any of Examples 1-8 or elsewhere herein) or control yeast (e.g., 10 “YVEC”, which is Saccharomyces cerevisiae yeast that is transformed with an empty vector, or vector that does not contain an antigen-encoding insert), at a ratio of 1:1 (yeast:DCs). Control DC cultures also include DCs incubated with HBV peptides, control peptides (non-HBV peptides), or nothing.
[00474] After 48-hours in co-culture, the DCs are used as antigen presenting cells 15 (APCs) for stimulation of autologous T cells (i.e., T cells from the donors). Each cycle of stimulation, designated as 1VS (in vitro stimulation), consists of 3 days culture in the absence of IL-2, followed by 4 additional days in the presence of recombinant IL-2 (20 U/mi). At the end of IVS 2, T cells are stained with a control tetramer or pentamer or a tetramer or pentamer spécifie for an HBV peptide epitope identified above. The 20 percentage of CD8+ T cells that stain positive with the tetramer or pentamer is quantified by flow cytometry.
[00475] It is expected that stimulation of donor T cells from HBV-positive donors with a yeast-HBV immunotherapeutic of the invention increases the percentage of tetramer/pentamer-positive CD8+ T cells in at least some or a majority of the donors, as 25 compared to controls, indicating that human T cells from HBV-infected individuals have the capacity to recognize HBV proteins carried by the yeast-based immunotherapy as immunogens.
[00476] Additional expérimente similar to those above are run using donor PBMCs from normal (non-HBV infected) individuals. It is expected that stimulation of donor T cells from normal donors with a yeast-HBV immunotherapeutic of the invention increases
200 the percentage of tetramer/pentamer-positive CD8 T cells in at least some or a majority of the donors, as compared to controls, indicating that human T cells from non-infected individuals also hâve the capacity to recognize HBV proteins carried by the yeast-based immunotherapy as immunogens.
[00477] In an additional experiment, HBV-specific T cells from three of the donors from the experiments described above are expanded in vitro using DCs incubated with HBV yeast-based immunotherapeutics (e.g., those described in any of Examples 1-8 or elsewhere herein) for 2 cycles of IVS (as described above). A third IVS is carried out with DCs matured in presence of CD40L and pulsed with the HBV peptide(s). At day 5, CD8+ T cells are isolated and used in an overnight cytotoxic T lymphocyte (CTL) assay against tumor cell targets expressing HBV antigens, at various effectorrtarget (ET) ratios. The percentage of CD8+ T cells that stain positive with a control tetramer/pentamer versus an HBV-specific tetramer/pentamer is measured.
[00478] It is expected that T cells from some or ail of the donors will be capable of generating CD8+ CTLs that can kill targets expressing HBV antigens. These data will demonstrate that yeast-HBV immunotherapeutic compositions can generate HBV-specific CTLs that are capable of killing an HBV antigen-expressing tumor cell.
Example 17
[00479] The following example describes a phase 1 clinical trial in healthy volunteers. [00480] A 12-week, open-label dose escalation phase 1 clinical study is performed using a yeast-based HBV immunotherapy composition described herein as GI-13009 (“SCORE-D”, comprising SEQ ID NO: 118, Example 7), or alternatively, the yeast-based HBV immunotherapy composition described herein as GI-13020 (“X-SCORE”, comprising SEQ ID NO: 130, Example 8) is used. Other yeast-based HBV immunotherapy compositions described herein (e.g., any of those described in Examples 1-8) can be utilized in a similar phase 1 clinical trial. The yeast-based HBV immunotherapy product for the phase 1 clinical trial is selected from pre-clinical studies (e.g., those described in any one of Examples 9-17) on the basis of considérations including strongest net immune response profile (e.g., amplitude of response for T cell epitopes that are most prédictive of positive outcome, and/or breadth of immune response across the range of epitopes).
201
[00481] Subjects are immune active and healthy volunteers with no prior or current indication or record of HBV infection.
[00482] Approximately 48 subjects (6 arms, 8 subjects per arm) meeting these criteria are administered the yeast-based HBV immunotherapy composition in a sequential dose cohort escalation protocol utilizing one of two different dosing protocols as follows: Protocol A: Prime-Boost Dosing (4 weekly doses starting at Day 1, followed by 2 monthly doses at Week 4 & Week 8)
Arm IA: 20 Y.U. (administered in 10 Y.U. doses to 2 different sites);
Arm 2A: 40 Y.U. (administered in 10 Y.U. doses to 4 different sites);
Arm 3A: 80 Y.U. (administered in 20 Y.U. doses to 4 different sites)
4-Weely Dosing (three total doses administered at Day 1, Week 4 and Week 8) Arm IB: 20 Y.U. (administered in 10 Y.U. doses to 2 different sites);
Arm 2B: 40 Y.U. (administered in 10 Y.U. doses to 4 different sites);
Arm 3B: 80 Y.U. (administered in 20 Y.U. doses to 4 different sites)
[00483] Ail doses are administered subcutaneously and the dose is divided among two or four sites on the body (every visit) as indicated above. Safety and immunogenicity (e.g., antigen-specific T cell responses measured by ELISpot and T cell prolifération) are assessed. Specifically, an ELlSpot-based algorithm is developed for categorical responders. ELISpot assays measuring regulatory T cells (Treg) are also assessed and CD4+ T cell prolifération in response to HBV antigens is assessed and correlated with the development of anti-SaccAaromyces cerevisiae antibodies (ASCA).
[00484] It is expected that the yeast-based HBV immunotherapeutic will be welltolerated and show immunogenicity as measured by one or more of ELISpot assay, lymphocyte prolifération assay (LPA), ex vivo T cell stimulation by HBV antigens, and/or ASCA.
Example 18
[00485] The following example describes a phase lb/2a clinical trial in subjects chronically infected with hepatitis B virus.
[00486] Due to a tendency of HBV infected patients to expérience destabilizing exacerbations of hepatitis as part of the natural history of the disease, yeast-based HBV immunotherapy is initiated after some period of partial or complété virologie control using anti-viral-based therapy, with a primary efficacy goal of improvîng séroconversion rates. In this first consolidation approach, yeast-based HBV immunotherapy is used in patients
202 after they achieve HBV DNA negativity by PCR to détermine whether séroconversion rates can be improved in combination with continued anti-viral therapy.
[00487] An open-label dose escalation phase lb/2a clinical trial is run using a yeastbased HBV immunotherapy composition described herein as GI-13009 (“SCORE-D”, comprising SEQ ID NO:118, Example 7), or alternatively, the yeast-based HBV immunotherapy composition described herein as GI-13020 (“X-SCORE”, comprising SEQ LD NO: 130, Example 8) is used. Other yeast-based HBV immunotherapy compositions described herein (e.g., any of those described in Examples 1-8) can be utilized in a similar phase 1 clinical trial. Subjects are immune active and chronically infected with hepatitis B virus (HBV) that is well controlled by anti-viral therapy (i.e., tenofovir disoproxil fumarate, or TDF (VIREAD®)) as measured by HBV DNA levels. Subjects are négative for HBV DNA (below détectable levels by PCR or <2000 lU/ml), but to qualify for this study, subjects must be HBeAg positive and have no evidence of cirrhosis or decompensation.
[00488] In stage one of this study, approximately 40 subjects (-5 subjects per arm) meeting these criteria are administered the yeast-based HBV immunotherapy composition in a sequential dose cohort escalation protocol utilizing dose ranges from 0.05 Y.U. to 80 Y.U. (e.g., 0.05 Y.U., 10 Y.U., 20 Y.U., and 40-80 Y.U.). In one protocol, 5 weekly doses will be administered subcutaneously (weekly dosing for 4 weeks), followed by 2-4 monthly doses also administered subcutaneously, with continued anti-viral therapy during treatment with the yeast-based HBV immunotherapy (prime-boost protocol). In a second protocol, a 4-weekly dosing protocol is followed, where subjects receive a total of three doses administered on day 1, week 4 and week 8, using the same escalating dose strategy as set forth above. Optionally, in one study, a single patient cohort (5-6 patients) will receive subcutaneous injections of placebo (PBS) on the same schedule as the immunotherapy plus continued anti-viral therapy. Conservative stopping rules are in place for ALT flares and signs of décompression.
[00489] In the second stage of this trial, subjects (n=60) are randomized 30 per arm to continue on anti-viral (TDF) alone or anti-viral plus the yeast-based HBV immunotherapeutic protocol (dose 1 and dose 2) for up to 48 weeks.
[00490] Safety, HBV antigen kinetics, HBeAg and HBsAg séroconversion, and immunogenicity (e.g., antigen-specific T cell responses measured by ELISpot) are assessed. In addition, dose-dependent biochemical (ALT) and viral load is monitored. Specifically, measurement of sérum HBsAg décliné during treatment between the 316530
203 treatment arms at weeks 12, 24, 48 is measured, and HBsAg-loss/seroconversion is measured at week 48.
[00491] An increase in rates of HBsAg loss and/or séroconversion to >20% at 48 weeks in subjects receiving the yeast-based immunotherapy and TDF, as compared to 5 subjects receiving TDF alone, is considered a clinically meaningful advancement. The yeast-based HBV immunotherapy composition is expected to provide a therapeutic benefit to chronically infected HBV patients. The immunotherapy is expected to be safe and welltolerated at ail doses delivered. Patients receiving at least the highest dose of yeast-based HBV immunotherapy are expected to show treatment-emergent, HBV-specific T cell 10 responses as determined by ELISPOT, and patients with prior baseline HBV-specific T cell responses are expected to show improved HBV-specific T cell responses while on treatment. Patients receiving yeast-based HBV immunotherapy are expected to show improvement in séroconversion rates as compared to the anti-viral group and/or as compared to the placebo controlled group, if utilized. Improvements in ALT 15 normalization are expected in patients receiving yeast-based HBV immunotherapy.
[00492] In an alternate trial, HBeAg négative patients meeting the other criteria (immune active, chronically HBV infected, well-controlled on anti-virals, with no signs of decompensation) are treated in a similar dose escalation trial as described above (or at the maximum tolerated dose or best dose identified in the trial described above). Patients are 20 monitored for safety, immunogenicity, and HBsAg séroconversion.
Example 19
[00493] The following example describes a phase lb/2a clinical trial in subjects chronically infected with hepatitis B virus.
[00494] An open-label dose escalation phase lb/2a clinical trial is run using a yeast25 based HBV immunotherapy composition described herein as GI-13009 (“SCORE-D”, comprising SEQ ID NO:118, Example 7), or alternatively, the yeast-based HBV immunotherapy composition described herein as GI-13020 (“X-SCORE”, comprising SEQ ID NO:130, Example 8) is used. Other yeast-based HBV immunotherapy compositions described herein (e.g., any of those described in Examples 1-8) can be 30 utilized in a similar phase lb/2a clinical trial. Subjects are immune active and chronically infected with hepatitis B virus (HBV) that has been controlled by anti-viral therapy (e.g. tenofovir (VIREAD®)) for at least 3 months. Subjects are not required to hâve completely cleared the virus to enroll in the study, i.e., patients may be positive or négative for HBV DNA (negativity determined as below détectable levels by PCR or <2000 IU/ml); however,
204 to qualify for this study, subjects hâve no evidence of cîrrhosis or decompensation.
Patients may be HBeAg-positive, although HBeAg-negative patients can be included in the study.
[00495] 30-40 subjects (6-10 patients per cohort) meeting these criteria are administered the yeast-based HBV immunotherapy composition in a sequential dose cohort escalation protocol utilizing dose ranges from 0.05 Y.U. to 40 Y.U. (e.g., 0.05 Y.U., 0.5 Y.U., 4 Y.U., 40 Y.U.), or utilizing dose ranges from 0.05 Y.U. to 80 Y.U. (e.g., 0.05 Y.U., 10 Y.U., 20 Y.U., 40/80 Y.U.). In one protocol, 5 weekly doses will be administered subcutaneously (weekly dosing for 4 weeks), followed by 2-4 monthly doses also administered subcutaneously, with continued anti-viral therapy during treatment with the yeast-based HBV immunotherapy (prime-boost protocol). In a second protocol, a 4weekly dosing protocol is followed, where subjects receive a total of three doses administered on day 1, week 4 and week 8, using the same escalating dose strategy as set forth above. In one study, a single patient cohort (5-6 patients) will receive subcutaneous injections of placebo (PBS) on the same schedule as the immunotherapy plus continued anti-viral therapy. Conservative stopping rules are in place for ALT flares and signs of décompression.
[00496] Safety, HBeAg and HBsAg séroconversion, viral control (e.g., development of viral negativity or trend toward viral negativity), and immunogenicity (e.g., antigenspecific T cell responses measured by ELISpot) are assessed. In addition, dose-dependent biochemical (ALT) and viral load is monitored,
[00497] >llogl0 réduction in HB-SAg by 24 weeks or >llogl0 réduction in HB-eAg by 12 weeks are considered to be endpoints for phase 2a. For HBV séroconversion, an SAg séroconversion of 10% by 24 weeks, and 15% by 48 weeks, and/or an eAg séroconversion rate of 25% by 24 weeks or 50% by 48 weeks are success criteria.
[00498] The yeast-based HBV immunotherapy composition is expected to provide a therapeutic benefit to chronîcally infected HBV patients. The immunotherapy is expected to be safe and well-tolerated at ail doses delivered. Patients receiving at least the highest dose of yeast-based HBV immunotherapy are expected to show treatment-emergent, HBV-specific T cell responses as determined by ELISPOT and patients with prior baseline HBV-specific T cell responses show improved HBV-specific T cell responses while on treatment. Patients receiving yeast-based HBV immunotherapy will show improvement in séroconversion rates as compared to available comparative data for the given anti-viral and/or as compared to the placebo controlled group. Patients receiving yeast-based HBV
205 immunotherapy will show împrovement in viral loss (e.g., viral negativity as measured by
PCR). Improvements in ALT normalization are expected in patients receiving yeast-based
HBV immunotherapy.
Example 20
[00499] The following example describes a phase 2 clinical trial in subjects chronically infected with hepatitis B virus.
[00500] A randomized phase 2 clinical trial in patients chronically infected with HBV treats treatment-naïve, HBeAg-positive (and possibly HBeAg-negative) subjects with ALT>2x ULN and viral loads > 1 million copies. The subjects (~60 subjects per arm adjusted based on phase 1 study signal) must hâve at least 6 months of prior anti-viral therapy, and hâve viral negativity for 2 consecutive visits at least one month apart. Subjects are randomized into two arms. Arm 1 patients receive 24-48 weeks of yeastbased HBV immunotherapy (e.g., yeast-based HBV immunotherapy composition described herein as GI-13009 (“SCORE-D”, comprising SEQ ID NO:118, Example 7), or alternatively, the yeast-based HBV immunotherapy composition described herein as GI13020 (“X-SCORE”, comprising SEQ ID NO: 130, Example 8). Ail patients receiving immunotherapy continue anti-viral therapy (e.g., tenofovir (VIREAD®)). Arm 2 patients receive a placebo (PBS control injection) with continued anti-viral therapy. The primary endpoint is séroconversion and viral negativity. Additional yeast-based HBV immunotherapy compositions described herein (e.g., any of those described in Examples 1-8) can also be utilized in a phase 2 trial with similar design.
[00501] Patients who achieve séroconversion receive 6-12 month consolidation therapy on either yeast-immunotherapy and antivirals (Arm 1) or antivirals alone (Arm 2), followed by a 6 month treatment holiday. The number of patients remaining in remission after completion of the 6 month holiday represent the secondary endpoint of the study. Additional endpoints include safety, immunogenicity and ALT normalization, as discussed in the Examples describing human clinical trials above.
[00502] The yeast-based HBV immunotherapy composition is expected to provide a therapeutic benefit to chronically infected HBV patients. The immunotherapy is expected to be safe and well-tolerated. Patients receiving yeast-based HBV immunotherapy are expected to show treatment-emergent, HBV-specific T cell responses as determined by ELISPOT and patients with prior baseline HBV-specific T cell responses show improved HBV-specific T cell responses while on treatment. Patients receiving yeast-based HBV immunotherapy are expected to show an improvement in séroconversion rates as
206 compared to the placebo controlled group. Patients receiving yeast-based HBV immunotherapy are expected to show an improvement in viral loss (e.g., viral negativity as measured by PCR). Improvements in ALT normalization are expected in patients receiving yeast-based HBV immunotherapy.
[00503] While various embodiments of the présent invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the présent invention, as set forth in the following claims.

Claims (75)

  1. Wnat is claimed is:
    1. An immunotherapeutic composition comprising:
    a) a yeast vehicle; and
    b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of:
    i) an HBV X antigen having an amino acid sequence that is at least 80% identical to positions 52 to 126 of a full-length HBV X antigen;
    ii) an HBV surface antigen having an amino acid sequence that is at least 95% identical to an amino acid sequence of a full-length HBV large surface antigen (L), and;
    iii) an HBV core antigen having an amino acid sequence that is at least 95% identical to an amino acid sequence of a full-length HBV core protein;
    wherein the composition elicits an HBV-specific immune response.
  2. 2. The immunotherapeutic composition of Claim 1, wherein the amino acid sequence of HBV X antigen is at least 95% identical to an amino acid sequence selected from: positions 1-60 of SEQ ID NO:130, positions 630-689 of SEQ ID NO:110, positions 582-641 of SEQ ID NO: 122, positions 630-689 of SEQ ID NO: 107, positions 630-689 of SEQ ID NO: 108, positions 630-689 of SEQ ID NO: 109, positions 52-68 followed by positions 84-126 of SEQ ID NO:4, positions 52-68 followed by positions 84-126 of SEQ ID NO:8, positions 52-68 followed by positions 84-126 of SEQ ID NO: 12, positions 52-68 followed by positions 84-126 of SEQ ID NO:16, positions 52-68 followed by positions 84-126 of SEQ ID NO:20, positions 52-68 followed by positions 84-126 of SEQ ID NO:24, positions 52-68 followed by positions 84-126 of SEQ ID NO:28, positions 52-68 followed by positions 84-126 of SEQ ID NO:32, SEQ ID NO: 100, positions 719-778 of SEQ ID NO: 101, positions 635-694 of SEQ ID NO:102, positions 810-869 of SEQ ID NO:124, positions 582-641 of SEQ ID NO:126, positions 229 to 288 of SEQ ID NO:132, positions 1 to 60 of SEQ ID NO: 134, or a corresponding sequence from a different HBV strain.
  3. 3. The immunotherapeutic composition of Claim 1, wherein the amino acid sequence of HBV X antigen is selected from: positions 1-60 of SEQ ID NO: 130, positions 630-689 of SEQ ID NO: 110, positions 582-641 of SEQ ID NO: 122, positions 630-689 of SEQ ID NO: 109, positions 630-689 of SEQ ID NO: 108, positions 630-689 of
    208
    SEQ ID NO: 107, SEQ ID NO: 100, or a correspondîng sequence from a different HBV strain.
  4. 4. The immunotherapeutic composition of any one of Claims 1 to 3, wherein the amino acid sequence of the HBV surface antigen is at least 95% identicai to an amino acid sequence selected from: positions 63-461 of SEQ ID NO:130, positions 1-399 of SEQ ID NO:118, positions 1-399 of SEQ ID NO:122, positions 9-407 of SEQ ID NO:34, positions 1-399 of SEQ ID NO:112, positions 1-399 of SEQ ID NO:114, positions 1-399 of SEQ ID NO: 116, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, positions 90-488 of SEQ ID NO:93, positions 1-399 of SEQ ID NO: 120, positions 1-399 of SEQ ID NO:124, positions 1-399 of SEQ ID NO:126, positions 231-629 of SEQ ID NO:128, positions 289-687 of SEQ ID NO:132, positions 289-687 of SEQ ID NO:134, or a correspondîng sequence from a different HBV strain.
  5. 5. The immunotherapeutic composition of any one of Claims 1 to 4, wherein the amino acid sequence of the HBV surface antigen is selected from: positions 63-461 of SEQ ID NO:130, positions 1-399 of SEQ ID NO:118, positions 1-399 of SEQ ID NO:122, positions 9-407 of SEQ ID NO:34, positions 1-399 of SEQ ID NO: 112, positions 1-399 of SEQ ID NO:114, positions 1-399 of SEQ ID NO:116, or a correspondîng sequence from a different HBV strain.
  6. 6. The immunotherapeutic composition of any one of Claims 1 to 5, wherein the amino acid sequence of the HBV core antigen is at least 95% identicai to an amino acid sequence selected from: positions 462 to 643 of SEQ ID NO: 130, positions 400-581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:122, positions 408-589 of SEQ ID NO:34, positions 400-581 of SEQ ID NO:112, positions 400-581 of SEQ ID NO:114, positions 400-581 of SEQ ID NO:116, positions 31-212 of SEQ ID NO:1, positions 31212 of SEQ ID NO:5, positions 31-212 of SEQ ID NO:9, positions 31-212 of SEQ ID NO:13, positions 31-212 of SEQ ID NO:17, positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO:29, positions 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ ID NO:39, positions 605-786 of SEQ ID NO:41, positions 691-872 of SEQ ID NO:92, positions 90-271 of SEQ ID NO:95, positions 2-183 of SEQ ID NO:105, positions 184395 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions 579-761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338-520 of SEQ ID NO:106, positions 400 to 581 of SEQ ID NO:120, positions 400 to 581 of SEQ ID NO:124, positions 400 to
    209
    581 of SEQ ID NO:126, positions 630 to 811 of SEQ ID NO:128, positions 688 to 869 of
    SEQ ID NO: 132, positions 688 to 869 of SEQ ID NO: 134, or a corresponding sequence from a different HBV strain.
  7. 7. The immunotherapeutic composition of any one of Claims 1 to 6, wherein the amino acid sequence of the HBV core antigen is selected from: positions 462 to 643 of SEQ ID NO:130, positions 400-581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:122, positions 408-589 of SEQ ID NO:34, positions 400-581 of SEQ ID NO:116, positions 400-581 of SEQ ID NO:112, positions 400-581 of SEQ ID NO:114, or a corresponding sequence from a different HBV strain.
  8. 8. The immunotherapeutic composition of any one of Claims 1 to 7, wherein the fusion protein comprises an N-terminal amino acid sequence of SEQ ID NO:37.
  9. 9. The immunotherapeutic composition of any one of Claims 1 to 8, wherein the HBV antigens are arranged in the following order, from N- to C-terminus, in the fusion protein: HBV X antigen, HBV surface antigen, HBV core antigen.
  10. 10. The immunotherapeutic composition of any one of Claims 1 to 8, wherein the fusion protein comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NO: 130, SEQ ID NO: 150 or SEQ ID NO: 122.
  11. 11. The immunotherapeutic composition of any one of Claims 1 to 8, wherein the fusion protein comprises an amino acid sequence selected from SEQ ID NO: 130, SEQ ID NO: 150 or SEQ ID NO: 122.
  12. 12. An immunotherapeutic composition comprising:
    a) a yeast vehicle; and
    b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of:
    i) an HBV surface antigen having an amino acid sequence that is at least 95% identical to an amino acid sequence of a full-Iength HBV large surface antigen (L), and;
    îi) an HBV core antigen having an amino acid sequence that is at least 95% identical to an amino acid sequence of a full-Iength HBV core protein;
    wherein the composition elicits an HBV-specific immune response.
  13. 13. The immunotherapeutic composition of Claim 12, wherein the HBV antigens consist of an amino acid sequence comprising at least 95% of a full-Iength HBV
    210 large surface antigen (L) fused to the N-terminus of an amino acid sequence comprising at least 95% of a full-length HBV core protein.
  14. 14. The îmmunotherapeutic composition of Claim 12 or Claim 13, wherein the amino acid sequence of the HBV surface antigen is at least 95% identical to an amino acid sequence selected from: positions 1-399 of SEQ ID NO;118, positions 9-407 of SEQ ID NO:34, positions 1-399 of SEQ ID NO:116, positions 1-399 of SEQ ID NO:112, positions 1-399 of SEQ ID NO:114, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, positions 90-488 of SEQ ID NO:93, positions 1-399 of SEQ ID NO:120, positions 1-399 of SEQ ID NO:122, positions 1-399 of SEQ ID NO:124, positions 1-399 of SEQ ID NO:126, positions 231629 of SEQ ID NO:128, positions 63-461 of SEQ ID NO: 130, positions 289-687 of SEQ ID NO: 132, positions 289-687 of SEQ ID NO: 134, or a corresponding sequence from a different HBV strain.
  15. 15. The îmmunotherapeutic composition of any one of Claims 12 to 14, wherein the amino acid sequence of the HBV surface antigen is selected from: positions 1-399 of SEQ ID NO:118, positions 9-407 of SEQ ID NO:34, positions 1-399 of SEQ ID NO:112, positions 1-399 of SEQ ID NO:114, positions 1-399 of SEQ ID NO:116, or a corresponding sequence from a different HBV strain.
  16. 16. The îmmunotherapeutic composition of any one of Claims 12 to 15, wherein the amino acid sequence of the HBV core antigen is at least 95% identical to an amino acid sequence selected from: positions 400-581 of SEQ ID NO:118, positions 408589 of SEQ ID NO:34, positions 400-581 of SEQ ID NO: 116, positions 400-581 of SEQ ID NO: 112, positions 400-581 of SEQ ID NO: 114, positions 31-212 of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212 of SEQ ID NO:9, positions 31-212 of SEQ ID NO: 13, positions 31-212 of SEQ ID NO: 17, positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO:29, positions 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ ID NO:39, positions 605-786 of SEQ ID NO:41, positions 691-872 of SEQ ID NO:92, positions 90-271 of SEQ ID NO:95, positions 2-183 of SEQ ID NO: 105, positions 184395 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions 579-761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338-520 of SEQ ID NO:106, positions 400 to 581 of SEQ ID NO:120, positions 400 to 581 of SEQ ID NO:122, positions 400 to 581 of SEQ ID NO: 124, positions 400 to 581 of SEQ ID NO: 126, positions 630 to 811 of SEQ ID NO:128, positions 462 to 643 of SEQ ID NO:130, positions 688 to 869 of SEQ
    211
    ID NO: 132, positions 688 to 869 of SEQ ID NO: 134, or a corresponding sequence from a different HBV strain.
  17. 17. The immunotherapeutic composition of any one of Claims 12 to 15, wherein the amino acid sequence of the HBV core antigen is selected from: positions 400581 of SEQ ID NO:118, positions 408-589 of SEQ ID NO:34, positions 400-581 of SEQ ID NO: 116, positions 400-581 of SEQ ID NO: 112, positions 400-581 of SEQ ID NO: 114, or a corresponding sequence from a different HBV strain.
  18. 18. The immunotherapeutic composition of any one of Claims 12 to 17, wherein the HBV antigens consist of an amino acid sequence that is at least 95% identical to an amino acid sequence selected from: SEQ ID NO: 118, SEQ ID NO: 116, positions 9589 of SEQ ID NO:34, SEQ ID NO:112, SEQ ID NO:114, or a corresponding sequence for a different HBV strain.
  19. 19. The composition of any one of Claims 12 to 18, wherein the fusion protein comprises an N-terminal amino acid sequence of SEQ ID NO:37.
  20. 20. The immunotherapeutic composition of any one of Claims 12 to 18, wherein the fusion protein comprises an amino acid sequence of SEQ ID NO: 151 or SEQ ID NO:34.
  21. 21. An immunotherapeutic composition comprising:
    a) a yeast vehicle; and
    b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of:
    i) an HBV surface antigen consisting of at least one immunogenic domain of full-length HBV large (L), medium (M) or small (S) surface antigen;
    ii) an HBV polymerase antigen consisting of at least one immunogenic domain of full-length HBV polymerase or of the reverse transcriptase (RT) domain of HBV polymerase;
    iii) an HBV core antigen consisting of at least one immunogenic domain of full-length HBV core protein or of full-length HBV e-antigen; and iv) an HBV X antigen consisting of at least one immunogenic domain of full-length HBV X antigen;
    wherein the composition elicits an HBV-specific immune response.
    212
  22. 22. The immunotherapeutic composition of Claim 21, wherein the HBV antigens consist of the amino acid sequence that is at least 95% identical to an amino acid sequence selected from: SEQ ID NO: 134, SEQ ID NO: 132, positions 6 to 939 of SEQ ID NO:36, positions 92 to 1025 of SEQ ID NO:92, positions 90 to 778 of SEQ ID NO: 101, positions 7 to 694 of SEQ ID NO: 102, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:124, SEQ ID NO:126, or a corresponding sequence from a different HBV strain.
  23. 23. An immunotherapeutic composition comprising:
    a) a yeast vehicle; and
    b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of:
    i) an HBV surface antigen consisting of at least one immunogenic domain of hépatocyte receptor région of Pre-Sl of the HBV large surface antigen (L) and at least one immunogenic domain of HBV small surface antigen (S);
    ii) an HBV polymerase antigen consisting of at least one immunogenic domain of reverse transcriptase domain of HBV polymerase; and iii) an HBV core antigen consisting of at least one immunogenic domain of HBV core protein.
    wherein the composition elicits an HBV-specific immune response.
  24. 24. The immunotherapeutic composition of Claim 23, wherein the HBV antigens consist of an amino acid sequence that is at least 95% identical to an amino acid sequence selected from: SEQ ID NO:128, SEQ ID NO:120, positions 6-786 of SEQ ID NO:41, or a corresponding sequence from a different HBV strain.
  25. 25. An immunotherapeutic composition comprising:
    a) a yeast vehicle; and
    b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of:
    i) an HBV polymerase antigen consisting of at least one immunogenic domain of the reverse transcriptase (RT) domain of HBV polymerase; and ii) an HBV core antigen consisting of at least one immunogenic domain of HBV core protein;
    213 wherein the composition elicits an HBV-specific immune response.
  26. 26. The immunotherapeutic composition of Claim 25, wherein the fusion protein has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:38 or a corresponding sequence from a different HBV strain.
  27. 27. An immunotherapeutic composition comprising:
    a) a yeast vehicle; and
    b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of:
    i) an HBV X antigen consisting of at least one immunogenic domain of HBV X antigen; and ii) an HBV core antigen consisting of at least one immunogenic domain of HBV core protein;
    wherein the composition elicits an HBV-specific immune response.
  28. 28. The immunotherapeutic composition of Claim 27, wherein the fusion protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:39 or a corresponding sequence from a different HBV strain.
  29. 29. An immunotherapeutic composition comprising:
    a) a yeast vehicle; and
    b) a fusion protein comprising an HBV antigen selected from the group consisting of:
    i) HBV surface antigen consisting of at least one immunogenic domain of an HBV large surface antigen (L);
    ii) an HBV polymerase antigen consisting of at least one immunogenic domain of a reverse transcriptase domain of HBV polymerase;
    iii) an HBV core antigen consisting of at least one immunogenic domain of an HBV core protein; and iv) an HBV X antigen consisting of at least one immunogenic domain of a full-length HBV X antigen;
    wherein the composition elicits an HBV-specific immune response.
  30. 30. The immunotherapeutic composition of Claim 29, wherein the fusion protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:40, SEQ ID NO:95, SEQ ID NO:96, or a corresponding sequence from a different HBV strain.
    214
  31. 31. The immunotherapeutic composition of Claim 29, comprising any two, three or four of the immunotherapeutic compositions of (i), (ii), (iii) or (iv).
  32. 32. An immunotherapeutic composition comprising:
    a) a yeast vehicle; and
    b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of at least one immunogenic domain of two, three or four HBV antigens selected from HBV surface antigens, HBV polymerase antigens, HBV core antigens or HBV X antigens, wherein each of the HBV antigens is from a different HBV génotype;
    wherein the composition elicits an HBV-specific immune response.
  33. 33. The immunotherapeutic composition of Claim 32, wherein the HBV antigens consist of four HBV core antigens, and wherein the fusion protein has an amino acid sequence that is at least 95% identical to SEQ ID NO: 105, or a corresponding sequence from a different HBV strain.
  34. 34. An immunotherapeutic composition comprising:
    a) a yeast vehicle; and
    b) a fusion protein comprising at least two HBV core proteins and at least two HBV X antigens, where each of the HBV core proteins is from a different HBV génotype and where each of the HBV X antigens is from a different HBV génotype;
    wherein the composition elicits an HBV-specific immune response.
  35. 35. The immunotherapeutic composition of Claim 34, wherein the fusion protein has an amino acid sequence that is at least 95% identical to SEQ ID NO: 106, or a corresponding sequence from a different HBV strain.
  36. 36. The immunotherapeutic composition of any one of Claims 1 to 35, wherein the fusion protein is expressed by the yeast vehicle.
  37. 37. The immunotherapeutic composition of any one of Claims 1 to 36, wherein the yeast vehicle is a whole yeast.
  38. 38. The immunotherapeutic composition of Claim 37, wherein the whole yeast is killed.
  39. 39. The immunotherapeutic composition of Claim 37, wherein the whole yeast is heat-inactivated.
  40. 40. The immunotherapeutic composition of any one of Claims 1 to 39, wherein the yeast vehicle is from Saccharomyces cerevisiae.
    215
  41. 41. The immunotherapeutic composition of any one of Claims 1 to 40, wherein the composition contains greater than 90% yeast protein and is formulated for administration by injection of a patient.
  42. 42. An immunotherapeutic composition comprising:
    a) a whole, heat-inactivated yeast from Saccharomyces cerevisiae; and
    b) an HBV fusion protein expressed by the yeast, wherein the fusion protein comprises SEQ ID NO: 130.
  43. 43. An immunotherapeutic composition comprising:
    a) a whole, heat-inactivated yeast from Saccharomyces cerevisiae; and
    b) an HBV fusion protein expressed by the yeast, wherein the fusion protein comprises SEQ ID NO:150.
  44. 44. An immunotherapeutic composition comprising:
    a) a whole, heat-inactivated yeast from Saccharomyces cerevisiae; and
    b) an HBV fusion protein expressed by the yeast, wherein the fusion protein comprises SEQ ID NO:118.
  45. 45. An immunotherapeutic composition comprising;
    a) a whole, heat-inactivated yeast from Saccharomyces cerevisiae; and
    b) an HBV fusion protein expressed by the yeast, wherein the fusion protein comprises SEQ ID NO: 151.
  46. 46. An immunotherapeutic composition comprising:
    a) a whole, heat-inactivated yeast from Saccharomyces cerevisiae; and
    b) an HBV fusion protein expressed b y the yeast, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:34.
  47. 47. A fusion protein comprising HBV antigens, wherein the fusion pTOtein comprises an amino acid sequence that is at least 95% identical to an amino acid sequence of: SEQ ID NQ:130, SEQ ID NO:150, SEQ ID NO:118, SEQ ID NO:151, SEQ ID NO:34, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO: 109, and SEQ ID NO: 110.
    216
  48. 48. A recombinant nucleic acid molécule encoding the fusion protein of Claim 47.
  49. 49. The recombinant nucleic acid molécule of Claim 48, wherein the recombinant nucleic acid molécule comprises a nucleic acid sequence selected from: SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:91, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO: 127, SEQ ID NO:129, SEQ ID NO:131, or SEQ ID NO:133.
  50. 50. An isolated cell transfected with the recombinant nucleic acid molécule of Claim 48 or Claim 49.
  51. 51. The isolated cell of Claim 50, wherein the cell is a yeast cell.
  52. 52. A composition comprising the fusion protein of Claim 47.
  53. 53. A composition comprising the recombinant nucleic acid molécule of Claim 48 or Claim 49.
  54. 54. A composition comprising the isolated cell of Claim 50 or Claim 51.
  55. 55. Use of a yeast vehicle and a fusion protein according to any one of daims 1 - 41 or a whole, heat-inactivated yeast and an HBV fusion protein according to any one of daims 42 - 46 or the fusion protein of claim 47 or the recombinant nucleic acid molécule of claim 48 or daim 49 or the isolated cell of claim 50 or claim 51 in the manufacture of at least one composition for treating hepatitis B virus (HBV) infection or at least one symptom resulting from HBV infection in a subject, by administration to a subject that has been infected with HBV of said at least one composition, wherein administration of the composition to the subject reduces HBV infection or at least one symptom resulting from HBV infection in the subject.
  56. 56. The use of Claim 55, wherein administration of the composition to the subject results in séroconversion in the subject.
  57. 57. The use of Claim 55, wherein administration of the composition to the subject reduces HBV viral load in the subject.
  58. 58. The use of Claim 55, wherein administration of the composition to the subject reduces liver damage in the subject.
  59. 59. The use of Claim 55, wherein said administration further comprises administration to the subject of one or more additional compounds useful for treating or ameliorating a symptom of HBV infection.
  60. 60. The use of Claim 59, wherein the compound is an anti-viral compound.
    217
  61. 61. The use of Claim 60, wherein the anti-viral compound is a nucléotide analogue reverse transcriptase inhibitor.
  62. 62. The use of Claim 60, wherein the anti-viral compound is selected from the group consisting of: tenofovir, lamivudine, adefovir, telbivudine, entecavir, and combinations thereof.
  63. 63. The use of Claim 60, wherein the anti-viral compound is tenofovir.
  64. 64. The use of Claim 59, wherein the compound is an interferon.
  65. 65. Use of a yeast vehicle and a fusion protein according to any one of claims 1
    - 41 or a whole, heat-inactivated yeast and an HBV fusion protein according to any one of claims 42 - 46 or the fusion protein of claim 47 or the recombinant nucleic acid molécule of claim 48 or claim 49 or the isolated cell of claim 50 or claim 51 in the manufacture of at least one composition for eliciting an antigen-specific, cell-mediated immune response against an HBV antigen, by administration to a subject of said at least one composition.
  66. 66. Use of a yeast vehicle and a fusion protein according to any one of claims 1
    - 41 or a whole, heat-inactivated yeast and an HBV fusion protein according to any one of claims 42 - 46 or the fusion protein of claim 47 or the recombinant nucleic acid molécule of claim 48 or claim 49 or the isolated cell of claim 50 or claim 51 in the manufacture of at least one composition for preventing HBV infection in a subject, by administration to a subject that has not been infected with HBV, of said at least one composition,
  67. 67. Use of a yeast vehicle and a fusion protein according to any one of claims 1
    - 41 or a whole, heat-inactivated yeast and an HBV fusion protein according to any one of claims 42 - 46 or the fusion protein of claim 47 or the recombinant nucleic acid molécule of claim 48 or claim 49 or the isolated cell of claim 50 or claim 51 in the manufacture of at least one composition for immunizing a population of individuals against HBV, by administration to the population of individuals of said at least one composition.
  68. 68. The use of any one of Claims 55 to 67, in which said administration comprises administration of at least two compositions according to any one of Claims 1 to 46 or 52 to 54.
  69. 69. The use of Claim 68, wherein the compositions are for administration concurrently to an individual.
  70. 70. The use of Claim 68, wherein the compositions are for administration sequentially to an individual.
  71. 71. The use of Claim 68, wherein each of the compositions are for administration by injection to a different site on the individual.
  72. 72. The composition of any one of Claims 1 to 46 or 52 to 54, for use to treat HBV infection or a symptom thereof.
  73. 73. The composition of any one of Claims 1 to 46 or 52 to 54, for use to prevent HBV infection or a symptom thereof.
    5
  74. 74. Use of the composition of any one of Claims 1 to 46 or 52 to 54 in the préparation of a médicament to treat HBV infection.
  75. 75. Use of the composition of any one of Claims 1 to 46 or 52 to 54 in the préparation of a médicament to prevent HBV infection.
OA1201300338 2011-02-12 2012-02-09 Yeast-based therapeutic for chronic hepatitis B infection. OA16530A (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US61/442,204 2011-02-12
US61/496,945 2011-06-14
US61/507,361 2011-07-13

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Publication Number Publication Date
OA16530A true OA16530A (en) 2015-10-22

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