1
CONCLUSION • ADU-1604 binds to a unique epitope on hCTLA4 • ADU-1604 was well tolerated when given weekly for 5 dosages at doses up to 30 mg/kg to cynomolgus monkeys • ADU-1604 enhanced T cell responses in a non-humane primate HBsAg immunization model and induced tumor growth inhibition in humanized NSCLC PDX models • Anti-CTLA-4 therapy enhanced anti-tumor activity of STING agonist ADU-S100 in a syngeneic mouse model • Intratumoral administration of anti-CTLA-4 induced tumor growth inhibition and T cell modulation in a syngeneic mouse model • The recommended starting dose in humans of 0.3 mg/kg ADU-1604 has a 100-fold safety factor based on the HNSTD • ADU-1604 will be clinically evaluated in a phase I study in advanced melanoma patients Assessment of pharmacology and toxicology of anti-CTLA-4 antibody (ADU-1604) in non-human primates and evaluation of local anti-CTLA-4 application Maaike Hendriks 1 , Joost Kreijtz 1 , Paul Vink 1 , David Lutje Hulsik 1 , Imke Lodewijks 1 , Astrid Bertens 1 , Jos van de Crommert 1 , Maurice Habraken 1 , Wout Janssen 1 , Judith Stammen-Vogelzang 1 , Lilian Driessen 1 , Weiwen Deng 2 , Meredith Leong 2 , Victor Lira 2 , Sarah McWhirter 2 , Hans van Eenennaam 1 , Andrea van Elsas 1 1: Aduro Biotech Europe, Oss, The Netherlands; 2: Aduro Biotech, Berkeley, CA, USA EC50; IC50 CD80 / IC50 CD86 0.04 nM; 1.98 nM / 1.49 nM Species cross-reactivity Cross-reacts with cynomolgus monkey CTLA-4 (EC50: 0.24 nM) No cross-reactivity with mouse or rat CTLA-4 ADCC/CDC induction Capable of ADCC / CDC In vitro/vivo Cytokine Release Assay No ADU-1604 induced cytokine production observed in human PBMCs and cynomolgus monkeys Epitope Unique (compared to ipilimumab/tremelimumab) Tissue Cross-reactivity No relevant off-target binding was observed with ADU-1604 on human and cynomolgus monkey tissues ADU-1604 CHARACTERISTICS INTRODUCTION Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a negative regulator of T-cell responses, also known as an immune checkpoint. The immune checkpoint CTLA-4 can be targeted with antibodies that are applied as single agent therapy or in combination with other therapies. The clinical relevance of CTLA-4 blockade was demonstrated by the approval of ipilimumab (YERVOY®) for the treatment of melanoma, both in the adjuvant as well as metastatic settings. Aduro Biotech has generated a humanized hIgG1 CTLA-4 antagonist designated ADU-1604. To evaluate the safety profile of ADU-1604 and to facilitate the selection of a safe clinical starting dose in adult patients with metastatic melanoma, non-clinical safety testing was conducted. The cynomolgus monkey was selected as the relevant non-clinical toxicology species. Here we present the in vitro and in vivo characteristics of the antibody, and an in vivo proof of concept study that illustrates the potential of the combination of CTLA-4 blockade with Aduro’s proprietary STING agonist (ADU-S100), and the results from the single dose PK study and the GLP 4-week repeat-dose safety study. HBsAg vaccine titer in cynomolgus monkey. T cell-dependent antibody responses against HBsAg surface antigen were measured after two intramuscular administrations (day 1 and day 29) of the Engerix-B vaccine with or without double intravenous dosing (day 1 and day 29) of ADU- 1604. A star (*) indicates P<0.05 by non- parametric Kruskal-Wallis testing ADU-1604 PK profile in cynomolgus monkey. Exploratory analysis of ADU-1604 including anti- drug antibodies after intravenous dosing on day 1 at 1 mg/kg (group 2; left graph) and single dose 10 mg/kg (group 3; middle graph) or dosing on day 1 and day 29 of 10 mg/kg (group 5; right graph). ADU-1604 PK PROFILE AND HBsAg ANTIBODY RESPONSE IN NHP Group. Test Article Dose Level b (mg/kg/dose) Dose Volume (mL/kg) Dose Concentration (mg/mL) No. of Animals a Main Study Recovery Study Males Females Males Females 1 Control c 0 5 0 3 3 2 2 2 ADU-1604 3 5 0.6 3 3 2 2 3 ADU-1604 10 5 2 3 3 2 2 4 ADU-1604 30 5 6 3 3 2 2 GLP REPEAT-DOSE SAFETY STUDY IN NON HUMAN PRIMATES a:10mM Histidine,75 mM Arginine, 3% Sorbitol, 0.01% Polysorbate 20, pH 6.0 No. = number, IV = intravenous slow bolus injection, IM = intramuscular injection. a Animals designated for terminal necropsy (3/sex/group) underwent 4 weeks of dosing (5 dosages) and were euthanized Day 30 of the dosing phase. Animals designated for recovery necropsy (2/sex/group) underwent 4 weeks of dosing (5 dosages) followed by 8-week recovery period b Doses given intravenously once weekly on Days 1, 8, 15, 22 and 29 of the dosing phase. c 10mM L-Histidine, 75mM L-Arginine, 3% (w/v) sorbitol, 0.01% (w/v) Polysorbate-20, pH 6.0 PROOF OF CONCEPT: ANTI-CTLA-4 + STING AGONIST • ADU-1604 was well tolerated at 5 weekly dosages (doses up to 30 mg/kg) to cynomolgus monkeys. • Enlarged lymph nodes were noted for one monkey at 10 mg/kg/dose and four monkeys at 30 mg/kg/dose on Day 22 • Changes in hematology parameters were primarily noted at the high dose of 30 mg/kg. These included increases in WBC, lymphocyte, monocyte, basophil and LUC counts on Day 8 of the dosing phase, which trended toward baseline by Day 30 • Administration of ADU-1604 resulted in increases in T-lymphocytes and subsets, B-lymphocytes, and NK cells for most animals in the 30 mg/kg/dose group • Primary target organs identified microscopically were the esophagus (tunica muscularis) and salivary gland at ≥ 10 mg/kg/dose. The increased severity of the mononuclear infiltration and degeneration/regeneration of the esophagus at 30 mg/kg/dose were considered adverse; this finding was tolerated and reversible after an 8-week recovery period • The NOAEL was established as 10 mg/kg. Toxicities at the highest dose tested in cynomolgus monkey, 30 mg/kg, were not severe or life threatening; 30 mg/kg was established as the HNSTD. • The recommended FIH starting dose of 0.3 mg/kg has a 100-fold safety factor (based on the HNSTD) and is expected not to compromise subject safety • The pharmacokinetic properties of ADU-1604 were determined after single and repeated intravenous dosing to cynomolgus monkey • Observed clearance was low (0.029 L/day) and observed volume of distribution was limited (0.03-0.06 L at steady-state) • The observed clearance and distribution volume are typical for a monoclonal antibody. • Half-life was extrapolated on the basis of the linear part of the pharmacokinetic curve and calculated effective half-lives • The plausible range for human half-life was determined as: 13-21 days • The extrapolated PK profile, including half-life and extrapolated clearance, is consistent with the proposed dosing interval of 3 weeks (Q3W) SUMMARY OF SINGLE AND TWO-DOSE IV ADU-1604 IN NHP SUMMARY OF SINGLE AND REPEAT DOSE PK OF IV ADU-1604 IN NHP ADU-1604 ENHANCES T-CELL ACTIVITY IN VITRO Group No. of Animals Day of Dosing Test Article Dose Route 1 3 1 Control a 0 mg/kg IV 2 3 1 ADU-1604 1 mg/kg IV 3 3 1 ADU-1604 10 mg/kg IV 4 3 1 and 29 Engerix-B 10 μg/animal IM 5 3 1 and 29 Engerix-B ADU-1604 10 μg/animal IM 10 mg/kg IV • Exposure (Cmax, AUC) increased proportional to the dose level in the range of 1-10 mg/kg • After the second dose of 10 mg/kg, observed exposure (AUC) was approximately 3-fold lower • Half-life as determined during the linear part of the pharmacokinetic curve was 5-8 days • Half-life and general PK profile supported once-weekly dosing in the GLP repeated dose study • ADU-1604 increased anti-HBsAg titers upon vaccination with Engerix-B, as compared to Engerix-B alone ADU-1604 EPITOPE MAPPING tremelimumab PDB ID: 5GGV ADU-1604 PDB ID: n.a. mapped on 5GGV ipilimumab PDB ID: 5TRU mapped on 5GGV ADU-1604 binds to a unique epitope on hCTLA-4. The epitope was determined by deuterated chemical crosslinking followed by enzymatic digestion and peptide mass fingerprinting using mass spectrometry (CovalX, Zurich, Switzerland). The same experimental procedure was used to determine the binding region of ipilimumab. The epitope residues determined for ipilimumab overlap with the residues identified in the X-ray structure as described by Ramagopal et al (2017) PNAS 114: E4223-4232 (PDB ID: 5TRU). The tremelimumab binding region was not determined here, its graphic representation is based on the epitope as described by Lee et al (2016) Nat Commun 7: 13354-13354 (PDB ID: 5GGV). ADU-1604 INHIBITS TUMOR GROWTH IN NSCLC huPDX MODELS ADU-1604 anti-tumor activity in NSCLC huPDX models. NOD/SCID/IL-2Rγnull (NOG) mice were engrafted with human CD34+ cells. 12 weeks post-engraftment, successful humanization was determined by flow cytometry after which the PDX tumor was implanted. Animals bearing a tumor of 50 - 250 mm3 in volume, were randomized and treated intraperitoneally with 100 μg of one of the respective antibodies, on day 0, 3, 7, 10. Tumor growth was followed for the course of this experiment (single mouse trial design). SINGLE AND TWO-DOSE PK STUDY IN NON HUMAN PRIMATES For the SEB PBMC assay, concentration ranges of ADU-1604 were tested for their potential to enhance SEB-induced IL- 2 production by PBMCs from healthy donors. As shown above, ADU-1604 enhanced IL-2 production and its potency was similar to 10D1* in this assay. CP-675,206* has a reduced potency in this assay. INTRATUMORAL APPLICATION *Reference antibodies: Benchmark antibody 10D1 in patent US20020086014 (referred to as ipilimumab) and antibody CP-675,206 in patent WO2007113648 (referred to as tremelimumab) were generated based on the sequences described. Ipilimumab was purchased for the epitope mapping and the NSCLC huPDX models Combination potential of anti-CTLA-4 plus ADU-S100 in a B16 melanoma tumor model. C57BL/6 mice bearing B16.F10 tumors were treated IT on day 17, 20 and 24 with ADU-S100 (50 μg), or vehicle (n=8). Mice were treated IP on days 20 and 24 with anti- CTLA4 (clone 9D9) or isotype control (100 ug). Results are shown as mean tumor volume ± SEM. * P < 0.05. Intratumoral injection of anti-CTLA-4 delays tumor growth and induces CD8 T cell modulation in CT26 tumor model comparable to IP injection. Balb/c mice were implanted with 2x10 5 CT26 tumor cells. On days 7, 10, 14 and 20 mice were administered 100 μg control IgG or anti-CTLA4 (9D9) IP or IT. Systemic and local administration of Anti-CTLA-4 increases CD8 T cell content in the tumor. CD8+ T/Treg ratio increases in the TME 0 1 0 2 0 3 0 0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 D a y s P o s t Im p la n ta tio n T u m o r V o lu m e (m m 3 ) is o typ e IP a n ti-C T L A -4 IP isotype IT a n ti-C T L A -4 IT D a y s P o s t T u m o r C h a lle n g e T u m o r V o lu m e (m m 3 ) 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 0 5 0 0 1 0 0 0 V e h ic le a n ti-C T L A 4 A D U -S 1 0 0 A D U -S 1 0 0 + a n ti-C T L A 4 C D N IT * + /- a n ti-C T L A 4 # 1702 ADU-1604 PK curve in cynomolgus monkey. Half-life was extrapolated on the basis of the linear part of the pharmacokinetic curve and calculated effective half-lives.
