Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/29—Hepatitis virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/29—Hepatitis virus
  • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2730/00—Reverse transcribing DNA viruses
  • C12N2730/00011—Details
  • C12N2730/10011—Hepadnaviridae
  • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
  • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• the present invention relates to an immunotherapeutic for the treatment of chronic Hepatitis B virus (“HBV”) infection and disease.
• HBV Hepatitis B virus
• HBV infection results primarily from cellular immune responses to infected hepatocytes.
• HBV infection The mechanisms responsible for chronic HBV infection are unclear.
• Hepatitis B e-antigen (“HBeAg” or "HBe”, pre-core polypeptide) is associated with active viral replication and is indicative of increased infectiousness and severity.
• HBsAg Hepatitis B surface antigen
• HBsAg loss with the appearance of neutralizing anti-HBs antibodies is indicative of clearance of infection.
• Spontaneous seroconversion to anti-HBs is extremely rare. While approximately 10 % of the patients per year experience spontaneous seroconversion from HBeAg to anti-HBe, seroconversion from HBs to anti-HBs occurs in only about 1 % of the patients per year.
• Newer antiviral agents such as lamivudine, can reduce viral loads, but lead to anti-HBs seroconversion in only a few patients. Further, they must be used long-term — discontinuation leads to the reappearance of the virus, making the requirement for lifetime treatment a possibility ⁇ and resistant mutants can emerge.
• Immunotherapies are also available. Some immunotherapies are based upon the administration of antibody. Anti-HBs antibody (“HBIG”), when used alone, can have therapeutic activity, as evidenced in liver transplantation for HBV infection. Protein Design Labs is pursuing the use of a humanized anti-HBs antibody for Ig infusion therapy.
• HBIG Anti-HBs antibody
• Antigen-based immunotherapy utilizes yeast-expressed HBsAg with an oil/water emulsion with RIBI® and QS-21 (SmithKline Beecham). It is currently in Phase 2 trial for chronic infection. Another utilizes PreSl + PreS2 + S/alum (Medeva). Still another, currently in an 150 subject immunotherapy trial, uses PreS2+S/alum (Institut Pasteur). Another antigen-based immunotherapy utilizes core-derived peptide for the purpose of inducing a cytotoxic T lymphocyte response (Cytel).
• the present invention relates to a method for treating chronic HBV infection comprising the administration of a composition comprising recombinant PreS2+S protein of HBsAg and a metabolizable oil adjuvant and, optionally, an anti-viral agent or compound.
• the present invention relates to a kit for immunotherapy comprising PreS2 + S protein of HBsAg, a metabolizable oil adjuvant, and, optionally, an anti-viral agent or compound.
• Figure 1 depicts the HBV genome with restriction sites of interest and the adaptors for preparing the expression plasmid.
• Figure 2 schematically depicts the cloning strategy.
• Figures 3a-b, 4a-b, and 5 a-b depict the HBe/Anti-HBe(a) and serum
• ALT/HBV DNA results for three patients, the second exhibiting a transient flare.
• the present invention is directed to an antigen-based immunotherapy for treating chronic HBV infection.
• treating includes the amelioration and/or elimination of chronic HBV infection, as well as the stimulation of an immune response thereto, preferably an anti-HBsAg or anti-HBe response.
• Human HBV is a member of the hepadnavirus family of liver-tropic D ⁇ A viruses that infect avian and mammalian species.
• the outer membrane-associated antigen — HBsAg ⁇ is responsible for eliciting a neutralizing protective antibody response.
• HBsAg is composed of three related proteins which share common amino acid sequences ⁇ S, preS2 + S, and PreSl + PreS2 + S.
• Antigen-containing compositions to be used in the present invention comprise PreS2 + S antigen of HBV and a metabolizable oil adjuvant.
• the PreS2 + S antigen is recombinantly produced in Chinese Hamster Ovary cells.
• the metabolizable oil adjuvant is the adjuvant MF59, described below.
• the foregoing compositions are administered to a subject.
• an anti -viral agent is administered before, at the same time as, or subsequent to the administration of the antigen-containing compositions.
• Administration of the antiviral agent before the administration of the antigen-containing compositions can range from one month to 24 months prior.
• an effective amount of the foregoing compositions is administered to a subject presenting with chronic HBV infection.
• the term "effective amount,” refers to the amount required to achieve an intended purpose for treatment without undesirable side effects, such as toxicity, irritation, or allergic response. Although individual needs may vary, the determination of optimal ranges for effective amounts of formulations is within the skill of the art.
• the dosage required to provide an effective amount of a formulation will vary depending on several factors, including the age, health, physical condition, weight, type and extent of the disease or disorder of the recipient, frequency of treatment, the nature of concurrent therapy, if required, and the nature and scope of the desired effect(s) (Nies et al., Chapter 3 In: Goodman & Gilman 's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al, eds., McGraw-Hill, New York, NY, 1996). A dosage in the range of about 5 to 100 ⁇ g is contemplated.
• compositions may be required.
• the time between administrations depends upon the number of administrations to be given. For example, if two administrations are given, the first can occur at zero months and the second can occur at one, two, or six months; if four administrations are given, they can occur at 0, 1, 2, and 6 months, respectively.
• the administrations can occur at monthly intervals.
• the time between multiple administrations can be readily determined by one skilled in the art. In one embodiment, eight administrations are given.
• the term "administering” includes, but is not limited to, transdermal, parenteral, subcutaneous, intra-muscular, oral, and topical delivery. A common requirement for any route of administration is efficient and easy delivery.
• the compositions are administered intra-muscularly.
• the intended purpose of the methods of the disclosed invention is the amelioration of chronic HBV infection. Amelioration can be determined by, for example, a decrease in HBV DNA in blood, as measured using nucleic acid assays; a decrease in serum alanine aminotransferase (ALT) as measured by routine tests; or a transient flare in transaminases, as measured by routine tests.
• the treatment according to the invention will result in the appearance of anti-HBe, with the concurrent decrease in/disappearance of HBe or, more preferably, the appearance of anti-HBs, with the concurrent decrease in/disappearance of HBsAg, all of which can be measured by routine immunoassays.
• normal ALT levels are defined as about 6 to 34 IU/L for females and about 6 to 43 IU/L for males.
• the term "metabolizable oil adjuvant” refers to adjuvants that comprise an oil that is non-toxic to and metabolizable by the subject to which it is administered, preferably one of about 6 to about 30 carbon atoms including, but not limited to, alkanes, alkenes, alkynes, and their corresponding acids and alcohols, the ethers and esters thereof, and mixtures thereof.
• the oil can be any vegetable oil, fish oil, animal oil or synthetically prepared oil which can be metabolized by the body of the host animal to which the adjuvant will be administered and which is not toxic to the subject.
• the host animal is typically a mammal, and preferably a human. Mineral oil and similar toxic petroleum distillate oils are expressly excluded from this invention.
• Exemplary sources for vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, exemplify the nut oils. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the grain group, corn oil is the most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale and the like can also be used. The technology for obtaining vegetable oils is well developed and well known.
• compositions of these and other similar oils can be found in, for example, the Merck Index, and source materials on foods, nutrition and food technology.
• the 6-10 carbon fatty acid esters of glycerol and 1,2-propanediol can be prepared by hydrolysis, separation and esterification of the appropriate materials starting from the nut and seed oils. These products are commercially available under the name NEOBEE® from PVO International, Inc., Chemical Specialties Division, 416 Division Street, Boongon, NJ, and others.
• Oils from any animal source can also be employed in the adjuvants and immunogenic compositions of this invention.
• Animal oils and fats are usually solids at physiological temperatures due to the fact that they exist as triglycerides and have a higher degree of saturation than oils from fish or vegetables.
• fatty acids are obtainable from animal fats by partial or complete triglyceride saponification which provides the free fatty acids.
• Fats and oils from mammalian milk are metabolizable and can therefore be used in the practice of this invention.
• the procedures for separation, purification, saponification and other means necessary for obtaining pure oils from animal sources are well known in the art.
• cod liver oil cod liver oil
• shark liver oils and whale oil such as spermaceti exemplify several of the fish oils which can be used herein.
• a number of branched chain oils are synthesized biochemically in 5-carbon isoprene units and are generally referred to as terpenoids.
• Shark liver oil contains a branched, unsaturated terpenoid known as squalene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene which is particularly preferred herein.
• Squalane the saturated analog to squalene
• Fish oils, including squalene and squalane are readily available from commercial sources or can be obtained by methods known in the art.
• oil component of these adjuvants and immunogenic compositions will be present in an amount from about 0.5% to about 20% by volume but preferably no more than about 15%, especially in an amount of about 1% to about 12%. It is most preferred to use from about 1% to about 4% oil.
• Metabolizable oil adjuvants as contemplated herein are described in EP 0 399 843 BI, incorporated by reference.
• a preferred adjuvant is MF59, described therein and in Ott et al., "MF59, Design and Evaluation of a Sage and Potent Adjuvant for Human Vaccines," Chapter 10, pp. 277-296, Vaccine Design: The Subunit and Adjuvant Approach, Michael F. Powell and Mark J. Newman, eds., Plenum Press, New York, 1995, incorporated herein by reference.
• MF59 is formulated as follows: 4.3 % w/v squalene, 0.5% w/v Tween 80® , 0.5% Span 85, and 400 ⁇ g/ml MTP-PE (N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-[l,2-dipalmitoyl- sn-glycero-3-3(hydroxyphosphoryloxy)]ethylamide). MF59's mechanism of action appears to be the development of a strong CD4 + T cell response.
• the adjuvant can also comprise emulsifying and/or immunostimulatory agents.
• emulsifying and suspending agents are generally used in the pharmaceutical sciences. These include naturally derived materials such as gums from trees, vegetable protein, sugar-based polymers such as alginates and cellulose, and the like.
• Certain oxypolymers or polymers having a hydroxide or other hydrophilic substituent on the carbon backbone have surfactant activity, for example, povidone, polyvinyl alcohol, and glycol ether-based mono- and poly-functional compounds.
• Long chain fatty-acid-derived compounds form a third substantial group of emulsifying and suspending agents which could be used in this invention. Any of the foregoing surfactants are useful so long as they are non-toxic.
• immunostimulatory agents include, for example, synthetic adjuvants that increase host immunity such as levamisole and isoprinosine.
• Levamisole is the levo isomer of tetramisole and potentiates humoral and cellular immunity through a T cell-dependent mechanism.
• Isoprinosine a complex containing inosine, the purine precursor of adenosine and guanosine, promotes T cell mitogenesis.
• Tuftsin a 4 amino acid peptide (Thr-Lys-Pro-Arg) homologous to a sequence in the immunoglobulin (Ig) heavy chain, primarily stimulates macrophages.
• the term "subject” refers to an animal, typically a mammal, preferably a human. As used herein, the term “about” means ⁇ about 10%.
• chronic HBV infection generally refers to an HBV infection that has persisted beyond the acute stage. Chronic infection can result from chronic disease acquired either in childhood or as an adult. Chronic infection as a result of disease can generally be identified by the presence of HBsAg with abnormal levels of ALT, with or without detectable levels of HBeAg. Normal ALT levels generally range from 10 to 32 U/L (9-24 U/L for women, with the normal range for infants being twice that of adults).
• Chronic infection also includes, however, asymptomatic carriers that have normal ALTs; patients initially treated with anti-virals, such as interferon, to reduce virus below detectable levels; and post-liver transplant patients who are on either maintenance anti-viral therapy or receiving Ig infusions.
• anti-virals such as interferon
• post-liver transplant patients who are on either maintenance anti-viral therapy or receiving Ig infusions.
• the persistence of HBsAg for greater than 6 months following an acute infection is the hallmark of chronic infection.
• a defective antiviral T cell response has also been documented in patients with chronic infection (Boni et al., J. Clin. Invest., 102(5):968-975, September, 1998).
• anti-viral agent or “anti-viral compound” includes, without limitation, interferon and nucleoside analogues, including lamivudine.
• Lamivudine has been used in reducing liver damage caused by chronic HBV and has been orally administered at doses of 25 mg - 300 mg per patient per day for periods of up to a year.
• the appropriate dosage of the anti-viral agent will depend upon, inter alia, the agent and the patient.
• a vaccine comprising recombinant HBV PreS2+S antigen (10 ⁇ g) and the metabolizable oil-in-water adjuvant MF59 has been shown to enhance immunogenicity in adults (Poland et al., 37th ICAAC, p. 216, September 28-October
• PreS2+S/MF59 vaccine were over 100 times greater than those of a control vaccine containing aluminum as the adjuvant (Poland et al., supra). It was predicted by the present inventor that similar compositions can be used as immunotherapeutic agents for treating chronic HBV infection.
• Example 1 A composition comprising recombinant PreS2+S in combination with the oil- in-water adjuvant MF59 was prepared. Recombinant PreS2+S was prepared as described below. All media and supplements were supplied by Hyclone.
• the recombinant plasmid pKSVAdPreS-Bglll was constructed by the insertion of the S + PreS2 gene using two adaptors ( Figures 1-2).
• the cloning vector used in the construction of the recombinant expression system was pKSV-10 from Pharmacia.
• the pKSV-10 vector is7.2 Kb long and the cloning site is Bglll.
• Viral DNA was isolated as described in Valenzuela et al., Nature, 280:815-819, August,30, 1979, incorporated herein by reference.
• An EcoRI/Bglll fragment of about 1880 bps from the viral genomic DNA was partially digested with Hhal to isolate a 1230 bps EcoRI/Hhal fragment.
• Adaptors were designed to be ligated with EcoRI/Hhal fragments to create Bglll/Bglll fragments for cloning into pKSV-10.
• pKSV-10/BglII was then ligated with Pre S Bglll/Bglll fragments to yield the DNA plasmid vector pKSVAdPreS-Bglll. This was followed by transformation of competent cells HB101. Transformants were screened and maxi-preparations were made.
• Cloning vector pSV7d/AML-dhfr was constructed by the insertion of the dhfr sequence into pSV7d (Stibbe et al., Virology, 123:436-442, 1982, incorporated herein by reference).
• DG44 (dhgr-) CHO cells (Urlaub et al., PNAS USA, 77:4216-4220, 1980, obtained at approximately passage number 100 in September, 1985 from Dr. Leslie Rail of Chiron) were co-transfected with pKSVAdPres-Bglll and pAML/dhfr. Thereafter, 48 individual clones were grown in 24 well microwell plates.
• HEP 30 cells were expanded to prepare Master/Working Cell Banks, designated as HBV B30 cells. Cells from the HBV B30 Master Cell bank were cloned and expanded in
• MTX methotrexate
• MF59 was prepared as a sterile oil-in-water emulsion as described in Ott et al., supra, incorporated herein by reference. Ten ⁇ g of PreS2 + S in a 0.25 ml volume antigen was combined with 0.25 ml MF59 for a final dose volume of 0.5 ml.
• the vaccine components are listed in Tables 1 and 2 below.
• Sorbitan Trioleate (Span 85TM) 1.18 mg Sodium Citrate, dihydrate, USP 2.15 mg
• Citric Acid monohydrate, USP 0.04 mg
• Example 1 Five hundred microliters of the composition described in Example 1 were administered intra-muscularly into the deltoid of 13 human patients presenting with chronic HBV as determined by the presence of HBsAg for longer than six months. Eight patients were HBe positive. All 13 patients had elevated serum ALT (greater than 1.2 x ULN i.e., upper limits of normal), and had no evidence of decompensated liver disease. Administrations were performed at 0, 1, 2, and 6 months. Efficacy was determined by measuring decreases in HBV DNA, serum ALT levels, and decreases in HBeAg levels, as well as the appearance of anti-HBe antibodies one month after the second administration. HBV DNA was measured using the bDNA method
• HBeAg For measuring HBeAg, 100 ⁇ of serum was combined with 100 ⁇ of human anti-HBe coupled to latex magnetic particles (600 g/ml), and incubated for 18 minutes at 37 °C. One hundred ⁇ l of human anti-HBe coupled to DMAE (dimethyl acridinium ester, 300 30 million relative light units, "RLU", per ml) was added and incubated for 18 minutes at 37 °C. The particles were washed three times with buffer and then chemiluminscence measured.
• DMAE dimethyl acridinium ester, 300 30 million relative light units, "RLU", per ml
• the buffer used had the following composition: 50 mM TRIS, pH 8.0; 500 mM potassium chloride; 0.09% sodium azide; 1 mM EDTA; 0.05% TWEEN 20; and 1.75% BSA, sulfhydryl modified and was filtered using a MILLIPAK-60 0.22 ⁇ m filter unit.
• HBsAg For measuring HBsAg, 100 ⁇ l of serum was diluted 1/100 in the buffer described above and combined with 100 ⁇ l of mouse anti-HBs (specifically binding to the anti-"a" neutralizing epitope of HBsAg) coupled to paramagnetic particles (for antibody concentrations of 280 ⁇ g/ml and 150 ⁇ g/ml, respectively) and 50 ⁇ l DMAE coupled to mouse anti-HBs (anti-"a", at 75.0 ng/ml) diluted in the buffer described above, and incubated for 18 minutes at 37 °C. The particles were washed three times with buffer and then chemiluminscence measured.
• HBeAg SOD fusion protein 50 ng/ml
• human anti-HBe coupled to latex magnetic particles 600 ⁇ g/ml
• DMAE 300 x 10 E 6 RLU/ ml
• the buffer used had the following composition: 100 mM borate buffer, pH 8.9; 0.09% sodium azide; 5 mM EDTA; 0.05% TWEEN 20; and 0.2% gelatin (fish) and was filtered using a MILLIPAK-60 0.22 ⁇ m filter unit.
• Anti-HBs was measured using standard enzyme immunoassays (Abbott).
• Example 1 Five hundred microliters of the composition described in Example 1, and 100 mg of lamivudine, are administered to a subject at time zero. Another 500 ⁇ l of the composition described in Example 1 and another 100 mg of lamivudine are administered at one month. Efficacy is determined by measuring anti-HBs titers as well as assaying for HBs presence one month after the second administration. If detectable HBs is still present, a third administration is performed. This process is repeated until no more HBs is detected.
• One hundred mg of lamivudine is administered to a subject starting at time zero and administered on a continuous once daily dose schedule.
• Five hundred ⁇ l of the composition described in Example 1 are administered to a subject intra-muscularly at 1, 2, 3, and 7 month.
• Efficacy is determined by measuring HBeAg and anti-HBe titers as well as assaying for HBV DNA one month after the administration of the composition of Example 1. If detectable HBe is still present, a further administration of the composition of Example 1 is performed. If detectable HBe is present one month after this further administration, an additional administration is performed. This process is repeated until no more HBe is detected.
• Example 1 Five hundred ⁇ l of the composition described in Example 1 are administered to a subject at time zero. One hundred mg of lamivudine are administered to a subject at one month. Efficacy is determined by measuring anti-HBs titers as well as assaying for HBs and HBe presence one month after the administration of the composition of Example 1. If detectable HBs or HBe is still present, a second administration of the composition of Example 1 is performed. If detectable HBs or HBe is still present, a third administration is performed. This process is repeated until no more HBs is detected.
• Example 7 One hundred mg of lamivudine are administered daily for 6 months. Five hundred ⁇ l of the composition described in Example 1 are administered to a subject at months 7, 8, 9, and 13. If detectable HBs or HBe is present, additional administrations can be performed. Example 7
• Chiron ® HBV/MF59 Antigen: Recombinant HBV PreS2+S (20 ⁇ g/ dose) produced and purified from CHO cells.
• Adjuvant MF59, a microfluidized oil-in- water emulsion.
• the antigen and MF59 were provided in separate vials to be mixed at the time of injection. Each dose was given intramuscularly in a total volume of 0.5 mL.
• the schedule consisted of four doses administered on a 0-, 1-, 2-, and 6-month schedule (Table 6).
• Lamivudine (Epivir ® , Glaxo Wellcome): Patients must have been treated for at least 1 month prior to initiation of Chiron ® HBV/MF59 therapy. Lamivudine was continued for 7 months thereafter (i.e., one month after the fourth injection) at the dose level prior to Chiron ® HBV/MF59 treatment (100 mg to 300 mg per day). Measures of efficacy: At the conclusion of the study, the proportion of patients with each of the following parameters was/will be determined: loss of or reduction in HBsAg, loss of HBeAg, and anti-HBs or anti-HBe seroconversion.
• HBV/MF59 will be summarized during and following the Chiron ® HBV/MF59 treatment period.
• available efficacy data were limited to selected timepoints and a full analysis has not been performed.
• a composition comprising recombinant PreS2+S antigens combined with MF59, an oil-in-water adjuvant, e.g. Chiron ® HBV/MF59 (Table 2) was given to patients with chronic HBV infection receiving lamivudine.
• Subjects received four injections intramuscularly of Chiron ® HBV/MF59 on a 0-, 1-, 2-, 6-month schedule. Patients continued treatment with lamivudine throughout the 6 month period of Chiron ®
• HBV/MF59 injections and continued to receive lamivudine for one additional month after the fourth dose, after which lamivudine was discontinued.
• Eligible subjects included patients with chronic HBV infection (defined by a history of positive HBsAg in serum for at least 6 months and positive HBsAg on the screening examination) with compensated liver disease who were receiving lamivudine or were candidates for initiating therapy with lamivudine at screening.
• Subjects on lamivudine at the time of screening received lamivudine treatment for at least 1 month prior to the time of administration of the first Chiron ® HBV/MF59 dose. These subjects continued lamivudine at their pre-study dose (between 100 to 300 mg per day).
• Subjects who were not receiving lamivudine treatment at the time of screening were treated for at least one month prior to initiating Chiron ® HBV/MF59. These subjects received lamivudine at the approved dose of 100 mg per day. All patients with any evidence of poorly compensated or advanced liver disease based upon history, physical examination, clinical laboratory evaluation, or previous liver biopsy were to be excluded from participation.
• HBV infection both qualitative and quantitative measures: HBsAg, HBeAg, anti-HBs, and anti-HBe. Additional assays included: liver function tests (LFTs): ALT, AST, alkaline phosphatase, bilirubin (total and direct), prothrombin time, total protein and albumin; complete blood count (CBC); and a urinalysis for protein, blood, and casts.
• LFTs liver function tests
• ALT, AST alkaline phosphatase
• bilirubin total and direct
• prothrombin time total protein and albumin
• CBC complete blood count
• a urinalysis for protein, blood, and casts.
• Quantitative HBV DNA level was measured by polym erase chain reaction (PCR).
• Subjects were excluded if they had evidence of a lamivudine-resistant mutant as determined either prior to screening or at the time of screening (based upon elevated HBV DNA levels). Additional clinical laboratory tests at the screening visit included: anti-HCV antibody, anti-HIV antibody, and hepatitis delta antibody. Subjects who were positive for any of these were excluded from participation.
• Subjects who met all of the inclusion criteria and none of the exclusion criteria returned to receive their first intramuscular injection. They were observed for 30 minutes following each injection for evidence of immediate local and systemic reactions. They completed diary cards to describe local (e.g., pain, warmth, erythema, induration at the injection site) and systemic (e.g., fatigue, malaise, fever, chills, myalgia, arthralgia, nausea, headache, rash) reactions for 7 days post-injection. Diary cards were maintained by the subject to record medical problems and medications taken during the 7-day period. Subjects were contacted by telephone 7 days following each injection.
• local e.g., pain, warmth, erythema, induration at the injection site
• systemic e.g., fatigue, malaise, fever, chills, myalgia, arthralgia, nausea, headache, rash
• a subject experienced any unusual, severe, or serious adverse event that may be therapy-related, they were instructed to be seen immediately in the clinic. Subjects were seen 14 days prior to the second, third, and fourth injections (i.e., at 1-month, 2- months, and 6-months, respectively) in order to have clinical laboratory tests (LFTs, urinalysis, creatinine) checked prior to receiving the next injection.
• LFTs clinical laboratory tests
• Subject Characteristics and Demography The mean age of subjects was 42 years of age (range: 27 to 63 years). Out of the 24 subjects, 20 (83%) were male.
• Chiron ® HBV/MF59 Twenty-two patients have been followed for a minimum of two months post discontinuation of lamivudine. Among these 22 patients data were collected up to Month 10.5 in five patients, while two patients had completed the study entirely (Month 12).
• Interferon Alfa (intron-a) 201, 203, 204, 208, 401, 402, 403, 404, 501
• Interferon (CIFN (consensus 202 interferon))
• Hepatitis B Vaccine (CV-1899) 101,207 Clinical laboratory evaluation: Historical information on pre-study serum ALT levels were collected as extensively as possible. The period and number of available data varied for each subject. Many subjects had widely fluctuating levels of ALT over variable time periods including substantial elevations of ALT levels pre- study prior to receiving lamivudine. Among the 24 subjects, nine had documented evidence of pre-study hepatitis flares with peak serum ALT levels over 700 IU/L. The median peak ALT level among these nine patients was 1035 IU/L with a range of 782 IU/L to 31 16 IU/L.
• Serum ALT levels were either normal or minimally elevated ( ⁇ 2 times upper limit of normal) in most subjects both at the time of screening and during the first seven months of study participation. This would be expected since, during this period, all subjects were receiving lamivudine.
• the serum ALT levels were persistently abnormal, with the maximum recorded level on- study while receiving lamivudine being 112 IU/L and 119 IU/L respectively. Both patients had HBV DNA detectable by bDNA assay while receiving lamivudine, and patient 601 showed a marked increase in the level of HBV DNA while receiving lamivudine, suggesting the possibility of the development of lamivudine-resistant mutant.
• Table 10 shows that maximum ALT level after lamivudine discontinuation for the 22 of the 24 patients for whom data were available up to the most recent visit.
• maximum ALT values were less than 50 IU/L.
• Six patients had maximum ALT values between 50 IU/L to 100 IU/L, 5 patients had maximum values between 101 IU/L to 200 IU/L, and a single patient (502) had a peak ALT value >200 IU/L, with a peak level of 1870 IU/L.
• Patient 502 exhibited an early virologic relapse upon the discontinuance of lamivudine that was resolved by 10.5 months.
• One month after discontinuing lamivudine (Month 8), the HBV DNA level had increased to 511 Meq/mL and the ALT level increased to 63 IU/L, indicating an early virologic relapse was taking place.
• Two months after discontinuing the lamivudine treatments (month 9), the ALT level of patient 502 peaked at 1870 IU/L, however the HBV DNA level had decreased to 118 mEq/ml. After month 9, the ALT levels of patient 502 decreased steadily to 46 IU/L at month 10.5.
• HBV DNA evaluation Data on quantitative HBV DNA levels are available for a limited number of time points. Three subjects (402, 504, and 601) had significant increases (>100-fold) in HBV DNA while on lamivudine. The likely reason for these increases is the emergence of HBV escape mutants with resistance to lamivudine. HBV genotyping of these samples will be performed. Early information on viral relapse following discontinuation of lamivudine were available in 16 patients who had HBV DNA levels measured at Month 8 or 9.
• HBeAg and anti-HBe evaluation Limited data on quantitative changes in HBeAg and anti-HBe levels were evaluated. HBeAg and anti-HBe titers were measured by an in-house EIA. The lower limit of detection of the in-house HBeAg assay is comparable to a commercial EIA (Abbott).
• EIA EIA
• 4 subjects 102, 404, 505, and 602 had quantitative HBeAg below the level of detection after the final injection.
• Two of these four patients (102 and 505) have also had significant (>4-fold) increases in anti-HBe titer (Table 12).
• Table 12 The foregoing examples are meant to illustrate the invention and not to limit it in any way. Those skilled in the art will recognize modifications within the spirit and scope of the invention as exemplified in the appended claims.

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