HEPATOLOGY Vol. 34, No. 4, Pt. 2, 2001 AASLD ABSTRACTS 4 4 7 A

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~;VEEK 48 RESISTANCE SURVEILLANCE IN CHRONIC HEPATITS B PA- TIENTS ENROLLED IN A PHASE II I CLINICAL STUDY OF ADEFOVIR DIPIVOX1L Christopher E Westland, Hulling Yang, William E Delaney Iv, Craig S Gibbs, Michael D Miller, Richard Fallis, John Fry, Carol L Brosgart, Hamid Namini, Shelly Xiong, Gilead Sciences, Foster City, CA

Background: 515 chronic hepatitis B patients (HBeAg+) were enrolled in a random- ized, double-blind, placebo-controlled Phase Ill clinical study (GS-98-437) to eval- uate the safety and efficacy of adefovir dipivoxil (ADV) for the treatment of chronic hepatitis B. Patients received ADV 10 nag, 30 mg or placebo once daily for 48 weeks in a 1:1:1 ratio. To date, no HBV mutations associated with resistance to adefovir have been identified in vitro or in patients treated with ADV in Phase II trials. Aim: To monitor for the emergence of potential adefovir resistance mutations in all pa- tients in the first year of this Phase III clinical study of ADV. Methods: A prospective, bi[inded genotypic analysis was performed on serum HBV isolates from all patient samples with detectable HBV DNA (~" 400 copies/mL, Roche PCR) at baseline, week 48, or (for patients terminated before wk 48) the last available on-therapy visit. The HBV polymerase gene (amino acids 349 to 692, Ref. GenBauk X02763) was se- quenced for all HBV isolates that could be amplified by PCR. The baseline and post-treatment sequences were compared for each patient to identify any mutations that emerged. Mutations observed at conserved sites of HBV polymerase were fur- ther characterized in vitro in a cell culture model of drug susceptibility. Results: Paired baseline and post-treatment sequences were obtained for 370 (72%) patients. For the remaining patients, 117 had undetectable HBV DNA at week 48, 2 patients never received study medication, a PCR product could not be generated from 12 samples, unambiguous sequence could not be obtained from 5 PCR products, and 9 patients had unmatched baseline and post-treatment sequences (> 2.6% sequence divergence) after resequencing verification. 76 of 370 (21%) patients with paired baseline and post-treatment sequences developed a total of 135 amino acid substi- tntions. Most changes (93%) were observed at natural polymorphic sites of HBV

olymerase and are considered unlikely to be associated with resistance. The most equently observed amino acid substitution (482D) occurred in 6 patients and

reflected a conversion to the more dominant amino acid observed in 70 published GenBank HBV sequences. There were 6 novel substitutions (R366K, $467A, H481 L, V562A, E566K, K589R) observed once each at conserved sites in HBV polymerase. In vitro cell culture assays showed that HBV mutants carrying either the R366K, Sq67A, V562A, E566K, or K589R mutations remained fully susceptible to adefovir v~;Lth 1C~0 values increased by less than 1.5-fold compared to the wild-type, suggest- ing that these mutations were not associated with adefovir resistance. In vitro anal- ysis for the H481L mutation is ongoing. Conclusions: Following 1 year of ADV or placebo therapy the majority of substitutions observed were at natural polymorphic sites in HBV polymerase and the rare substitutions that were seen at conserved sites were not associated with decreased susceptibility to adefovir in vitro. This blinded genotypic analysis suggests the frequency of resistance after 1 year ADV therapy is very low. Future unbfinded analyses will allow correlation of the emergence of specific mutations with treatment assignment and clinical response.

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HEPATITIS B VIRUS GENOTYPES AND RESPONSE TO INTERFERON ALPHA TREATMENT IN CHRONIC HEPATITIS B. Jinlin Hou, galf Schill- ing, University College London, London United Kingdom; Harry L Janssen, Erasmus University Hospital, Rotterdam Netherlands; J Chen, D Liu, Nanfang Hospital, Guangzhou People'S Rep Of China; Rudolf Heijtink, Erasmus Uni- versity Hospital, Rotterdam Netherlands; K Luo, Nanfang Hospital, Guang- zhou People'S Rep Of China; George KLan, Queen Mary Hospital, Hong Kong People'S Rep Of China; Erwin Sablon, Innogenetics, Ghent Belgium; Roger Williams, University College London, London United Kingdom; Solko W Schalm, Erasmus University Hospital, Rotterdam Netherlands; Nikolai V Naoumov, University College London, London United Kingdom

The impact of different HBV genotypes on the response to interferon-alpha (IFN-c~) treat- ment is not known. This is of increasing importance because of the re-emerging use of IFN-~x or pegylated interferon in combination with other antiviral agents. We investigated the relationship between the outcome of IFN-a treatment with the molecular characteris- tics of the HBV genome, in particular: i) HBV genotype; ii) the genotype ability to develop mutations, which down-regulate the expression of HBeAg (in the core promoter and in the precore region) and iii) the variability in the HBV core gene. The study includes a homo- geneous group of 148 patients with HBeAg (+) chronic hepatitis B (103 from Europe and 45 from China). Treatment response was defined as sustained loss ofHBeAg. Methods:HBV genotypes were determined by InnoLipa assay (Innogenetics, Belgium). The HBV core promoter and precore region were analyzed by innoLipa and DNA sequencing, both at baseline and at the end of treatment. The entire HBV Core gene was also sequenced at both time points. Results:In European patients the most common genotypes were A 46/103 (45%) and D 35/103 (34%), while Chinese patients were infected with either genotype C 29/45 (64%) or B 16/45 (36%). The response to IFN-a treatment was higher in genotype A vs. D and in genotype C vs. B. At baseline, core promoter mutations (CPM) were more frequent in treatment responders vs. non-responders (p=0.005). The prevalence of these mutations further increased at the end of treatment in the responder group (p=0,01), but not in non-responders. In contrast, the precore stop mutation (PCM) was equally distrib- uted between responders and non-responders at baseline and there was no significant change in the prevalence of this mutation at the end of treatment. The development of CPM occurred more frequently in patients infected with HBV genotype A (48%) vs. D (12%) and in genotype C (42%) vs B (21%). In contrast, the PCM was significantly more often in genotype D (43%) vs. A (7%) and in genotype B (65%) vs. C (33%). The treatment response was associated with a lower variation in the HBV core gene: the number of amino acid (aa) changes per patient were 2.4 + 2.0 in responders vs. 4.1 z 2.4 in non-responders (p =0.007). Genotype A had the lowest core gene variation: 1.7 +- 1.9 an/patient, compared to D 4.0 +- 2.3 (p=0.0004); B 3.7---2.1 (p=0.007) or C 3.3+2.5 (p=0.002). Failure to respond to IFN-a was not associated with the selection on HBV strains containing new core gene mutations at the end of treatment. Conclusion: The response to IFN-a treatment differs belween HBV genotypes, depending on the molecular characteristics of the core promoter and core gene variability. The higher response to treatment, observed in HBV genotype A, is associated with its greater tendency to develop core promoter mutations and with less variations in the nucleoeapsid protein.

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COMBINATION OF HEPBZYME TM, AN ANTI-ItBV NUCLEASE RESIS- TANT RIBOZYME, WITH LAMIVUDINE OR INTERFERON IMPROVES INHIBITION OF HBV REPLICATION IN CELL CULTURE. David V Mor- rissey, Stacey L Overly, David A Johnson, Victor Mokler, Pamela A Pavco, Lawrence M Blatt, Ribozyme Pharmaceuticals, Inc, Boulder, CO

Hepatitis B virus (HBV) is the prototypic member of the Hepadnaviridae family and is a double-stranded circular DNA virus that utilizes a reverse transcriptase step to complete its lifecycle. Infection with HBV is responsible for >350 million cases of chronic Hepatitis worldwide and 1.2 million deaths each year. To, explore the use of ribozymes as novel therapeutic agents for the treatment of chronic Hepatitis B, nuclease-resistant ribozymes containing nucleotide modifications such as 2'-O-methyl and 2'-C-allyl nucleotides, a T-inverted abasic cap and four phosphorothioate linkages were synthesized. As previously reported these ribozymes target highly conserved regions of HBV RNA and ex]hibit marked antiviral activity. These synthetic ribozymes have the potential to ,:leave several of the major HBV RNA transcripts and may also block the HBV lifecycle by cleavage of the pregenomic RNA. One of these ribozymes (site 273: HepBzyme) has been shown in a HBV transgenic mouse to significantly reduce viremia compared to saline-treated animals and was as effective as treatment with lamivudine (P<0.01). The therapeutic use of HepBzyme either alone or in combination with current therapies (lamivudine or type 1 IFN) may lead to improved HBV treatment modalities. To assess the potential of combination therapy, HepG2 cells transfected with a replication competent HBV cDNA, were treated with HepBzyme, Infergen® (Amgen, Thousand Oaks Ca), Lami- vudine (Epivir®: GlaxoSmithKline, Research Triangle Park NC) either alone or in combination. Initial results indicated that combination treatment with either HepBzyme plus lnfergen or combination of HepBzyme plus lamivudine results in additive down regulation of HBsAg expression (P<0.001). Addi- tional studies in lamivudine resistant cell lines are being carried out and will be reported. These preliminary results suggest the potential for combination ther- apy, of HepBzyme plus currently available therapies for the treatment of chronic Hepatitis B.

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PREVENTION OF SPONTANEOUS DEVELOPMENT OF HEPATOCELLU- LAR CARCINOMA (HCC) IN C3H/HEN MICE BY IMMUNIZATION W I T H FUSIONS OF DENDRITIC AND HCC CELLS. Masaki Irie, Sadamu Homma, Hiroki Takahashi, Mikio Zeniya, Div of Gastroenterology and Hepatology, Dep of Int Med, Tokyo Japan; Tsuneya Ohno, Institute of DNA Medicine, Tokyo Japan; Gotaro Toda, Div of Gastroenterology and Hepatology, Dep of Int Med, Tokyo Japan

Patients with HBV- and/or HCV-associated chronic hepatitis or liver cirrhosis are at high risk of HCC. Furthermore the recurrence rate of HCC after surgical resection, ablation or embolization therapy is also high, partly because of multicemric development of HCC. Thus prevention of HCC development is a crucial problem in these patients. In the last few years, advance in cancer immunology have led to an intense interest in cancer immunotherapy utilizing dendritic cells loaded with tumor associated antigens as an adjuvant. In a previous report, we showed that immunization with dendritic cells (DCs) fused transplantable HCC cells (BNL cells), not DCs or irradiated BNL cells, inhibited the growth of BNL cells tranplanted. In the present study, using C3H/HeN mice of which about 80% have HCC at 16 months of age, we tried to prevent the spontaneous development of HCC by immunization with DCs fused with HCC cells. Methods. A new HCC cell line, designated as MIH-2, was established from HCC which has grow spontaneously in C3H/HeN mice. Bone marrow-derived DCs were prepared according to inaba et at (J Exp Med 176:1693,1992). DCs and MIH-2 cells were mixed at a ratio of 2:l and treated with 50% polyeth- yleneglycol to be fused. Fusion efficiency was estimated by flow cytometric analysis. Fusion cells (FC) were suspended in phosphate-buffered saline (PBS) and administered subcutaneously to 13-month old C3H mice four times at intervals of 6 days between each injection at the dose of 0.5 x 10 s (low dose), L5 x 105 (middle dose) or 4.5 x 103 (high dose) cells/mouse. The mice were sacrificed at 16 months of age. HCC in the liver was counted and the size of each HCC was measured. In another series of experiments, the middle dose of FC was injected 8 times at intervals of 2 to 3 days between each injection. The control mice received the same volume of PBS instead of FC suspension. Each group consisted of 7 - 9 mice. For assay of cytotoxicity of splenocytes to MIH-2 cells, 6-week old mice were treated with high dose of FC twice at one week interval and sacrificed one week after the last treatment. The splenocyts were cultured with FC for 4 days and then incubated with 51Cr-labelled MIH-2 cells at effector : target ratio from 20:1 to 80:1. Statistical analyses were performed by chi square test and Student's t test. Results. Flow cytometric analysis showed that about 50% of DCs were fused with MIH-2 cells. The incidence of HCC was 11.1% (1/9) at 13 months of age when immunization was started. In the untreated mice, the incidence rose to 77.8% (7/9) at 16 months of age. In the mice treated with low, middle and high dose of FC, the incidence was 44.4% (4/9), 44.4% (4/9) and 11.1% (1/9), respectively. The incidence in the mice treated with high dose of FC was significantly lower than in untreated mice (P<0.01) and identical with the base line incidence. The increase in the frequency of immunization with middle dose of FC did not reduce the incidence significantly as compared with that in the untreated mice (77.8 vs 33.3%, P>0.05). The mean size of HCC did not differ significantly among the groups of mice. Per cent cytolysis by the splenocytes of FC-treated mouse, 36+/-2 ;(mean+/-SD:n=3), was significantly higher than 5.5+/-1.5 (n=3), that by the sprenocytes from the untreated mouse (P<0.0001). Conclusion. This is the first demonstration that the immunization with DCs fused with tumor cells was effective in the prevention of spontaneous HCC development. Immunization with FC is promising in the prevention of recurrence of HCC not only after surgical treatment of HCC, but a/so ablation or embolization therapy for HCC, when HCC cells obtained by biopsy were used as the fuision partner.