Characterization of a novel differentiated anti-CTLA-4 antibody (ADU-1604) in vitro and in vivoMaaike Hendriks1, Joost Kreijtz1, Paul Vink1, David Lutje Hulsik1, Imke Lodewijks1, Astrid Bertens1, Jos van de Crommert1, Maurice Habraken1, Wout Janssen1, Judith Stammen-

Vogelzang1, Lilian Driessen1, Weiwen Deng2, Laura Hix Glickman2, Meredith Leong2, Sarah McWhirter2, Thomas W Dubensky Jr2, Hans van Eenennaam1, Andrea van Elsas1

1: Aduro Biotech Europe, Oss, The Netherlands; 2: Aduro Biotech, Berkeley, CA, USA

CONCLUSION• We generated and functionally characterized a potent humanized hIgG1 CTLA-4 antagonist, designated ADU-1604• ADU-1604 binds to a unique epitope on hCTLA-4 and in vitro and in vivo characterization illustrate that it is at least comparable to 10D1/ipilimumab• ADU-1604 enhanced T cell responses in a non-human primate HBsAg immunization model and induced tumor growth inhibition in a humanized NSCLC PDX model• Anti-CTLA-4 therapy enhanced anti-tumor activity of STING Agonist ADU-S100 and LADD in syngeneic mouse models• ADU-1604 is currently being evaluated in nonclinical safety studies• ADU-1604 is intended to be developed for treatment of cancer in combination with other Aduro immunotherapy assets

Binding of ADU-1604 to hCTLA-4 stablyexpressed on CHO-K1 cells (CHO-K1.hCTLA4).Binding was assessed in a cell-based ELISAformat.

ADU-1604 binds to human CD3+ T cells thatwere activated with anti-CD3/anti-CD28 beads.Data shown represent an average obtained usingthree individual donors.

ADU-1604 blocks the binding of CD80 and CD86 to hCTLA-4 on the CHO-K1.hCTLA4 cell line.Blocking was assessed in a cell-based ELISA format.

ADU-1604 is capable of inducing ADCC through NK-mediated cell lysis (NK cells as effector (E) cells and CDC throughhuman complement mediated cell lysis on CHO-K1.hCTLA4 target (T) cells. 10D1 was used as a reference.

tremelimumabPDB ID: 5GGV

ADU-1604 PDB ID: n.a.

mapped on 5GGV

ipilimumabPDB ID: 5TRU

mapped on 5GGV

To study the functional effect of ADU-1604, CD3+ T-cells were stimulated for two days with PHA-M (mucoproteinvariant of PHA), and subsequently co-cultured for 3 days with irradiated Raji cells that express CD80 and CD86(Raji:TC ratio 1:10). The immunomodulatory effect of ADU-1604, with IL-2 production as a readout, was tested usingT-cells isolated from healthy donors. As shown above both ADU-1604 enhances IL-2 production comparable to 10D1.

For the SEB PBMC assay, concentration ranges of ADU-1604 were tested for their potential to enhance SEB-inducedIL-2 production by PBMCs from healthy donors. As shown above, ADU-1604 enhanced IL-2 production and itspotency was similar to 10D1 in this assay. CP-675,206 has a reduced potency in this assay.

MATERIALS AND METHODSAduro’s proprietary B-select platform was used for the generation of an anti-CTLA-4 antibody.To this end, mice were immunized with a cDNA construct encoding human CTLA-4 (hCTLA-4).hCTLA-4 antibody-producing B cells were isolated and immortalized and secreted antibodieswere purified, characterized and stratified for binding, blocking, functionality and bindingregion to select a lead antibody. The humanization of the lead antibody resulted in the clinicalcandidate: ADU-1604. This antibody has been fully characterized, the results of which aresummarized here. 10D1 (ipilimumab)* and CP-675,206 (tremelimumab)* were taken along asreferences in the experiments.

Antibody Sequence No prohibitive post-translational modifications

EC50; IC50 CD80 / IC50 CD86 0.04 nM; 1.98 nM / 1.49 nM

Species cross-reactivityCross-reacts with cynomolgus monkey CTLA-4 (EC50: 0.24 nM)No cross-reactivity with mouse CTLA-4

ADCC/CDC induction Capable of ADCC / CDC

In vitro Cytokine Release Assay No ADU-1604 induced cytokine production observed in human PBMCs

Epitope Unique (compared to ipilimumab/tremelimumab)

Tissue Crossreactivity No non-specific staining detected of human, cynomolgus and rhesus tissues

ADU-1604 EPITOPE MAPPING

SUMMARY OF ADU-1604 IN VITRO CHARACTERIZATION

PROOF OF CONCEPT: ANTI-CTLA-4 + STING AGONIST

ENHANCEMENT OF IL-2 PRODUCTION BY ADU-1604 IN A T CELL/RAJI ASSAY

hCTLA-4 BINDING AND CD80/CD86 BLOCKING BY ADU-1604

Combination potential of anti-CTLA-4 plus ADU-S100 in a B16 melanoma tumor model.C57BL/6 mice were inoculated with 1×105 B16.F10 tumor cells on the right flank at day 0.Flank tumors were treated IT on day 10 with ADU-S100 (5 µg), or HBSS vehicle control (n =8). Mice were treated IP on days 10, 14 and 17 with anti-mouse CTLA-4 (clone 9D9, 200µg), or control IgG (clone MPC-11, 200 µg). Results are shown as mean tumor volume ±SEM. * P < 0.5, ** P < 0.01.

Anti-CTLA-4 enhances ADU-S100 mediated anti-tumor response in a rechallenge model.C57BL/6 mice (n=8) were implanted with 1×105 B16.F10 tumor cells SC. On days 6, 9, 13,and 20 mice were administered 200 µg control IgG or anti-CTLA-4 (9D9) IP. On day 9, micewere administered 5 µg ADU-S100 IT. On day 16, flank tumors were surgically resected.2*105 B16 cells were implanted IV on D23. Animals were monitored for survival post IVtumor rechallenge.

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 thePDX tumor was implanted. Animals bearing a tumor of 50 - 250 mm3 in volume, wererandomized 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 (singlemouse trial design).

ADU-1604 ENHANCES T-CELL ACTIVITY UPON SEB STIMULATION

ADCC AND CDC EFFECTOR FUNCTION OF ADU-1604

ADU-1604 ENHANCES HBsAg ANTIBODY RESPONSE IN CYNOMOLGUS MONKEYSPROOF OF CONCEPT: ANTI-CTLA-4 + LADD

ADU-1604 INHIBITS TUMOR GROWTH IN NSCLC huPDX MODEL

ADU-1604 binds to a unique epitope on hCTLA-4. The epitope was determined by deuterated chemicalcrosslinking followed by enzymatic digestion and peptide mass fingerprinting using mass spectrometry(CovalX, Zurich, Switzerland). The same experimental procedure was used to determine the bindingregion of ipilimumab. The epitope residues determined for ipilimumab overlap with the residuesidentified 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 basedon the epitope as described by Lee et al (2016) Nat Commun 7: 13354-13354 (PDB ID: 5GGV).

Anti-CTLA-4 Improves Anti-Tumor Efficacy of LADD in 4T1 flank syngeneic mouse model.Balb/c mice (n=8) were implanted with 1x105 4T1 tumor cells SC. On days 7 and 14, micewere administered 100 µg control Ig or anti-CTLA4 (9D9) IP. On day 10, mice wereadministered 1x106 CFU LADD expressing AH1 IV. Animals were monitored daily and tumorswere measured twice weekly. Graphs show growth of tumors in individual animals.

INTRODUCTIONCytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a negative regulator of T-cellresponses, also known as an immune checkpoint. Aduro Biotech has generated a humanizedhIgG1 CTLA-4 antagonist designated ADU-1604. Here we present the in vitro and in vivocharacterization of the antibody and in vivo proof of concept studies that illustrate thepotential of the combination of CTLA-4 blockade with two of Aduro’s proprietary platforms:STING agonists (ADU-S100) and LADD immunotherapy. The cyclic dinucleotide ADU-S100, toactivate all human STING proteins is currently tested in early clinical studies (NCT02675439,NCT03172936). In preclinical models, intratumoral injection of ADU-S100 induced a localinnate response (eg. IFN-β production), and a systemic adaptive immune response byactivation of tumor-resident Dendritic Cells and subsequent priming of tumor specific CD8+ Tcells, leading to rejection of local and distant tumors. LADD (Live Attenuated, Double DeletedListeria monocytogenes) engineered to express tumor antigens to induce innate and adaptiveimmunity to cancer is in clinical development as an immunotherapy platform.

ADU-1604 enhances HBsAg vaccine-induced antibody titers in Cynomolgus Monkeys. To assess T cell modulationby ADU-1604 in vivo Cynomolgus Monkeys were administered ADU-1604 in combination with Hepatitis B surfaceantigen (HBsAg) vaccine on day 1 and day 29. Depicted here are the mean antibody responses for animals thatreceived HBsAg only (solid black line; control group; n=3) or in combination with ADU-1604 (solid red line; n=2).ADU-1604 enhanced antibody responses to the vaccine antigen.

*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 model