PRECLINICAL DEVELOPMENT OF YEAST-BASED IMMUNOTHERAPY FOR CHRONIC INFECTION A. Haller, T. King, Y. Lu, C. Kemmler, D. Bellgrau, G. Gordon, D. Apelian, A. Franzusoff, T. Rodell, and R. Duke GlobeImmune Inc., Aurora, Colorado 80010

Therapy of A20-NS3 tumors Introduction 3500 Figure 6. GI-5005 eliminates Tarmogen Technology 3200 3000 5/5 established tumors Tarmogens (targeted molecular immunogens) are whole, heat-killed recombinant Saccharomyces Naïve-B6 vs. EL4-NS3 Tarmogen Heat inactivated, recombinant Saccharomyces cerevisiae 2500 5/5 2400 cerevisiae yyeasteast engineeredengineered toto eexpressxpress oonene oror moremore ttargetarget pproteinrotein aantigens,ntigens, aandnd aactivatectivate bbothoth aann Pro-B6 vs. EL4-NS3 BALB/c mice were injected with A20-NS3 tumor transfected with the NS3-Core fusion gene (GI-5005) 2000 1600 cells. One week later, the mice were treated with Yeast innate immune response via Toll-Like Receptors (TLRs), as well as an adaptive, antigen-specifi c * 3/5 animals had 3/5 expression 1500 three weekly doses of GI-5005. plasmid immune response. GI-5005 was engineered to express a (HCV) fusion protein no detectable tumor. 1000 The remaining 800 2/5 animals had comprised of large segments of NS3 protease and Core protein sequences. These proteins were 500 2/5* Mean tumor volume (mm^3) Mean tumor volume (mm^3) tumors that did not 0 GI-5005 GI-4014 chosen as targets for immunotherapy because they are essential for virus replication, contain 0 express NS3 Immunotherapy multiple epitopes that are recognized by both CD4+ and CD8+ T cells in acute and chronic 0 7 14 21 Killer T Days after tumor challenge cell (CTL) infections, and are highly conserved among the different HCV genotypes. GI-5005, by expressing multiple antigens, was designed to induce a broad cellular immune response, which is thought to Treatment IVS IL-2 IL-12 GM-CSF IFN-γ TNF-α Phagosome be necessary to achieve a sustained viral response and HCV clearance in patients. PBS (Naïve) Con A + - + ++ + Saccharomyces cerevisiae yeast Proteasome Figure 3. Mice immunized with GI-5005 reject tumors expressing LPS - + - - ++ Tarmogens Targeted HCV antigens Cell Lysis GI-5005 - + - - ++ Toll-like rVV-NS3 - - - - - receptors C57BL/6 mice (5/group) challenged with EL 4-NS3 cells one week after immunization with GI-5005. Dendritic Cell GI-5005 Con A + - + ++ + Endosome Killing of P815-rVV-NS3 Killing of P815-rVV-Core LPS - + + - +++ Uptake of Tarmogens 80 MHC class II Cancer or 60 receptor-peptide virus-infected GI-5005 + + +++ +++ ++++ by dendritic cells results MHC class I complex cell receptor-peptide 60 rVV-NS3 - - ++ - 6000 ++ in the activation of killer complex 40 GI-5005 (5 YU) T cells against HCV Helper T cell 40 GI-5005 (0.1 YU) 5000 infected cells 20 GI-5003 (5 YU) 4000 Table 1. Cytokine profi les from immunized mice 20 % specifi c lysis % specifi % specifi c lysis % specifi GI-5003 (0.1 YU) 3000 BALB/c splenocyte culture supernatants were harvested after in vitro sstimulationtimulation andand analyzedanalyzed 0 0 GI-4014 (5 YU)

40:1 20:1 10:1 5:1 40:1 20:1 10:1 5:1 CPM – Day 5 2000 for cytokine production E : T ratio E : T ratio Abstract 1000 T cells from Naïve mice

0 T cells from protected mice Background: Evidence suggests that control of hepatitis C infection in humans requires Immunized w/ GI-5005 (~1400 ng/mL) Figure 1. T cells from GI-5005 immunized mice kill virus-infected Nil rVV-Gag rVV-NS3 rVV-Core effective T cell-mediated immunity. Previous studies have demonstrated that recombinant, cells expressing HCV NS3 or Core 800 heat-inactivated Saccharomyces cerevisiae yyeasteast (Tarmogens(TarmogensTM) are phagocytosed by and directly activate dendritic cells that present disease-associated proteins contained within the Tarmogen 51Cr release assay was performed to demonstrate antigen-specific killing of P815 tumor cells 600 Figure 4. Antigen-specifi c TH cells in protected mice + + to CD4 and CD8 T cells. Tarmogens are capable of mediating both therapeutic as well as infected with virus expressing NS3 (A) or Core (B) proteins using T cells from 400 BALB/c splenocytes isolated from animals that were protected from tumor challenge were pg/mL prophylactic antigen-specifi c anti-tumor effects (Lu et al. 2004 Can Res 64, 5084). In this study, a BALB/c mice immunized with GI-5005, GI-5003 (produces mutant NS3); GI-4014 which is evaluated in a standard lymphocyte proliferation assay after in vitro sstimulationtimulation withwith vacciniavaccinia 200 IL-6 (IVS w/ GI-5005) Tarmogen that produces an HCV NS3-Core fusion protein (GI-5005) was evaluated for its ability a Tarmogen expressing a non-HCV protein. expressing NS3, Core, HIV-Gag or nothing. 0 GM-CSF (IVS w/ rVV-NS3) to induce protective and therapeutic immunity in mice. 10 YU 1 YU 0.1 YU Methods: C57BL/6 and BALB/c mice were injected subcutaneously with GI-5005. Killing of EL4-rVV-NS3 Killing of EL4-rVV-Core Immunogenicity was determined using assays that measure antigen-specifi c lymphocyte Figure 7. Dose-dependent cytokine production in response to GI-5005 proliferation, cytokine secretion, and cytotoxicity. A surrogate model for hepatitis C infection 50% 35% 30% 100% IVS with GI-5005 100% Pro-infl ammatory cytokine secreting cells induced with different doses of GI-5005. Spleen 40% Immune T cells-IVS employing HCV antigen-expressing syngeneic tumor cells implanted in mice was used to assess 25% 6 80% IVS with YEX 80% with B16-N53 cells (10 spleen cells/well) from BALB/c mice that received three weekly injections of 0.1, 1 or both preventative immunity and therapeutic effi cacy. 30% 20% Naïve Immune T cells 6 60% 60% 10 YU of GI-5005 were stimulated in vitro with GI-5005 (2 x 10 yeast cells/well) or rVV-NS3 20% 15% IVS with rW-N53 1 Injection 6 Results: Immunization with GI-5005 induced dose-dependent NS3 and Core antigen-specifi c 10% (100 x 10 pfu/well). Cell-free supernatants for cytokine analysis were collected at 72 h (IVS w/ 40% 40% Naïve T cells-IVS % specifi c lysis % specifi 10% c lysis % specifi 2 Injections cytotoxic T cell and helper T cell activity associated with secretion of IL-2, IFN-γ, GM-CSF and 5% with B16-N53 GI-5005) or 120 h (IVS w/ rVV-N53) after IVS.

20% c lysis % specifi 20% 0% 0% 3 Injections c lysis % specifi TNF-α. Protective immunity was demonstrated in mice that were immunized prior to tumor Naïve T cells-IVS 1:1 1:2 1:4 1:8 1:1 1:2 1:4 1:8 0% 0% with rW-N53 Effector cell dilution Effector cell dilution challenge with NS3-expressing tumor cells. Therapeutic effi cacy was demonstrated in mice that 40:1 20:1 10:1 5:1 20:1 10:1 5:1 25:1 E : T ratio E : T ratio were immunized seven days after implantation of NS3-expressing tumor cells. No signifi cant Conclusions systemic adverse effects have been observed upon repeated administration of Tarmogens in mice, Figure 2. Dose-dependent induction of CTLs with GI-5005 • GI-5005 induces dose-dependent NS3 and Core-specifi c CTL and T immune responses in mice rats, rabbit and macaques. Figure 5. GI-5005 overcomes antigen ignorance H C57BL/6 mice received one, two or three weekly subcutaneous doses of GI-5005. Isolated T • GI-5005 boosts pre-existing immune responses Conclusion: GI-5005 was found to elicit both protective and therapeutic cytotoxic T cell and cells were stimulated for fi ve days with GI-5005. A 51Cr release assay was carried out to evaluate (A) T cells derived from non-immunized, tumor challenged mice (B) T cells from • GI-5005 induces proinfl ammatory cytokines in mice helper T cell mediated responses specifi c for HCV antigens. A Phase 1 study is being initiated to CTL activity using EL4 tumor cells infected with vaccinia viruses expressing NS3 (A) or Core GI-5005 immunized, tumor challenged mice. EL4-NS3 were used as target cells in a standard • GI-5005 induces therapeutic and protective immunity against HCV antigen-expressing tumors test GI-5005 in humans chronically infected with hepatitis C virus. (B) proteins as target cells. A naïve control group was included. 51Cr release assay. Naïve animals were included as control. YEX, yeast vector alone. • A Phase 1 study of GI-5005 in patients with chronic HCV infection is being initiated