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1 15 29 43 57 69 IL-2 Secretion OX40 is a T cell co-stimulatory receptor that can enhance the magnitude and durability of T cell immune responses. Anti-OX40 agonist antibodies have shown significant single agent tumoricidal activity in preclinical models, and can combine effectively with other immunomodulatory antibodies, targeted therapies and vaccines. OX40 agonists are able to counteract the immunosuppressive tumor microenvironment and promote tumor-specific cellular immunity via at least two distinct mechanisms: 1) promoting OX40 forward signaling in tumor-specific T cells; and 2) co-engaging Fc receptors expressed by tumor-associated effector cells, and facilitating the selective elimination of OX40 high intratumoral regulatory T cells. INCAGN1949, an anti-OX40 human IgG1 antibody, was selected based on its ability to optimally enhance T cell responsiveness under conditions of suboptimal T cell receptor stimulation. INCAGN1949 was shown to mediate effective apical OX40 clustering that is translated into effective downstream activation of the NFkB pathway. Notably, INCAGN1949 was shown to maintain a sigmoidal dose response curve across a broad range of antibody concentrations. This suggests a wide therapeutic window and may be advantageous for dosing considerations. By contrast, evaluation of reference OX40 antibodies indicated an inverted U-shaped dose response curve, leading to impaired T cell responses at high concentrations. INCAGN1949 was selected for clinical development based on its optimal agonist profile, further reinforced by its ability to combine with other co-inhibitory and co-stimulatory antibodies to augment T cell responsiveness. Prior to human testing, the pharmacology and tolerability of INCAGN1949 was evaluated in non-human primates (NHPs). Pharmacokinetic (PK) and pharmacodynamic (PD) parameters were evaluated including longitudinal measurements of serum cytokines, immune cell populations, activation state and T cell-mediated immune responses to reporter vaccine antigens. INCAGN1949 exhibited a linear PK profile and was well tolerated at all doses tested, with no maximum tolerated dose established. Co-administration of INCAGN1949 and vaccines in NHPs showed an immune-based PD signature across a broad exposure range. These studies were in line with in vitro findings and support a wide PD range for INCAGN1949 in patients. An important secondary mechanism of INCAGN1949 is the ability of its IgG1 Fc region to mediate selective depletion of OX40 high intratumoral regulatory T cells. Immunohistochemistry and flow cytometry analyses support the validity of this regulatory T cell depletion mechanism in a range of tumors. The functional in vitro and in vivo attributes of INCAGN1949 make it suitable for clinical development. It is currently under evaluation in a Phase 1/2 study in subjects with advanced or metastatic tumors (NCT02923349). INCAGN1949, an Anti-OX40 Antibody With an Optimal Agonistic Profile and the Ability to Selectively Deplete Intratumoral Regulatory T Cells Author Disclosures Ana Gonzalez, Mariana Manrique, Ekaterina Breous, David Savitsky, Jeremy Waight, Lukas Swiech, Thomas Horn, Christopher Clarke, Yuqi Liu, Shiwen Lin, Jennifer Buell, Robert Stein, Marc van Dijk, Nicholas S. Wilson: Agenus Inc.: Employment and Stock Ownership. Jennifer Pulini, Kevin Heller, Reid Huber, Peggy Scherle, Gregory Hollis: Incyte Corporation: Employment and Stock Ownership. Taha Merghoub, Daniel Hirschhorn-Cymerman, Gerd Ritter, David Schaer: None. Acknowledgments The authors would like to thank Joseph Connolly and Zhenyu Li for their help characterizing and producing INCAGN1949. Layout and printing support was provided by Evidence Scientific Solutions, Philadelphia, PA, funded by Incyte Corporation. Abstract 4703 Ana Gonzalez, 1 Mariana Manrique, 1 Lukasz Swiech, 1 Thomas Horn, 1 Ekaterina Breous, 1,2 Jeremy Waight, 1 David Savitsky, 1 Yuqi Liu, 1 Shiwen Lin, 1 Christopher Clarke, 1 Taha Merghoub, 3 Daniel Hirschhorn-Cymerman, 3 David Schaer, 3 Gerd Ritter, 4 Jennifer Pulini, 5 Kevin Heller, 5 Peggy Scherle, 5 Gregory Hollis, 5 Reid Huber, 5 Marc van Dijk, 1,2 Jennifer Buell, 1 Robert Stein, 1 and Nicholas S. Wilson 1 Presented at the American Association for Cancer Research Annual Meeting 2017 Washington, DC, USA • April 1–5, 2017 Mechanism 2: Intratumoral Depletion of Treg Cells • INCAGN1949 shows an optimal agonist profile across a broad dose range, which is mediated by its ability to engage and cluster OX40 on T cells • INCAGN1949 cooperates with other immunomodulatory antibodies (both agonist and antagonist) to enhance T cell responsiveness • Primary human tumors contain populations of FOXP3-expressing OX40 high regulatory T cells, with the potential to be selectively depleted by the IgG1 Fc region of INCAGN1949 • INCAGN1949 has a linear PK profile in non-human primates and was well tolerated • INCAGN1949 co-administered with reporter vaccines in non-human primates promotes enhanced T cell–mediated B cell immune responses • INCAGN1949 is an effective agonist of the OX40 pathway and has confirmed immunomodulatory activity in vivo • INCAGN1949 is currently in Phase 1 clinical development (NCT02923349) Summary Scan code to download a copy of this poster or visit: http://bit.ly/2nHuMlq Mechanism 1: OX40 Forward Signaling in Activated T Cells References 1. Croft M. Nat Rev Immunol. 2003;3:609–620. 2. Bulliard Y, et al. Immunol Cell Biol. 2013;92:475–480. 3. Croft M. Annu Rev Immunol. 2010;28:57–78. 4. Voo KS, et al. J Immunol. 2013;191:3641–3650. 5. Piconese S, et al. J Exp Med. 2008;205:825–839. 6. Weinberg A. Cancer Res. 2013;73:7189–7198. INCAGN1949 Demonstrates Increased Activation of Primary T Cells INCAGN1949 Cooperates With Other Immunomodulatory Antibodies to Enhance T Cell Function INCAGN1949 Mediates Effective OX40 Clustering T Cell–Dependent Antibody Response (TDAR) in Cynomolgus Monkeys After Co-administration of Vaccines and INCAGN1949 OX40 Is Selectively Expressed by Intratumoral Treg Cells in a Range of Primary Tumors OX40-mCherry stably transfected Jurkat cells were co-cultured with plate-bound isotype control (A) or INCAGN1949 antibody (B). Arrows point to OX40 clustering. Confocal stack images are shown. A. IL-2 secretion by human primary T cells in the presence of superantigen (Assay #1) and INCAGN1949 or isotype control (representative of n = 8 healthy donors). B. Intracellular cytokine (IFNγ and TNFα) readout using flow cytometry post-anti-CD3 antibody stimulation (Assay #2) and plate bound INCAGN1949 or isotype control (representative of n = 10 healthy donors). INCAGN1949 Is Well Tolerated In Vivo and Demonstrates a Linear PK Profile in Cynomolgus Monkeys Mechanism 2: Anti-OX40 antibodies mediate the selective depletion of intratumoral regulatory T cells (Treg cell), thereby promoting anti-tumor activity (modified from ref. 2). IL-2 secretion by human primary T cells in the presence of superantigen (A–C) or anti-CD3 (D), and INCAGN1949 in combination with (A) anti-PD-1, (B) anti-PD-L1, (C) anti-CTLA-4, or (D) anti-GITR. B A A B B IL-2 secretion by human primary T cells in the presence of superantigen and INCAGN1949 as compared to other anti-OX40 antibody variants (Ab1-Ab10) or isotype control (* significant P<0.05, Mann-Whitney test). A A A B C D A A. Th1 cytokines (IFNγ, GM-CSF, IL-2), Th2 cytokines (IL-6, IL-10), Th17 cytokines (IL-17) in plasma of cynomolgus monkeys administered INCAGN1949 (0.5, 5, 20, and 100 mg/kg) in 5 weekly doses at day 22 post-inoculation (n = 4 monkeys per group). B. Eight-week PK study of cynomolgus monkeys administered 5 weekly doses of INCAGN1949 at 0.5, 5, 20, and 100 mg/kg (n = 6–10 monkeys per group). From 3 different studies, plasma half-lives ranging from 8 to 15 days by either single or repeated administrations were calculated. A. Representative histogram of OX40 expression from tumor-associated T effector cells or Treg cells (non-small cell lung carcinoma [NSCLC]). B. Tabulated summary of OX40 expression by associated T effector cells (CD4 + T cells) and Treg cells. C. Immunohistochemistry (IHC) of NSCLC sample stained for FoxP3 + Treg cells or an isotype control. D. Intensity of OX40 or FoxP3 expression by IHC in a number of positive primary tumors (tumor tissue or tumor stroma tissue; n = 10 per tissue type). Activity of Anti-OX40 Antibody Variants Flow Cytometry IHC OX40 or FoxP3 Expression OX40 Expression INCAGN1949 Enhances Primary T Cell Function Across a Broad Range of Concentrations 10,000 0 IL-2 (pg/mL) 8000 4000 2000 6000 * Isotype Confocal Stacks 3D Top Bottom Top Bottom Bottom Top Top Bottom Bottom Isotype INCAGN1949 Assay #1 IL-2 IFNγ TNFα Assay #2 Antibody concentration (µg/mL) 0.01 3000 0 IL-2 (ng/mL) 2000 1000 0.1 1 10 100 1000 Antibody concentration (µg/mL) 0.001 15 0 %CD8 + IFNγ T cells 10 5 0.01 0.1 1 10 100 5 Antibody concentration (µg/mL) 0.001 15 0 %CD4 + TNFα T cells 10 0.01 0.1 1 10 100 INCAGN1949 Isotype B C D Anti-PD-1 0 Donor 1 Donor 2 20 40 60 10 30 50 IL-2 in Supernatant (Fold Change) Anti-PD-L1 Anti-CTLA-4 Anti-GITR 0 Donor 1 Donor 3 50 100 150 Donor 2 Donor 4 Immunomodulatory antibody INCAGN1949 Combination Serum Cytokine Concentration Indication Samples (n) Teff Cells Treg Cells NSCLC 5 +/− ++++ Colorectal 4 +/− ++++ Endometrial 3 +/− ++++ Renal 3 — +++ Breast 2 — ++ Ovarian 2 — + Liver 2 +/− ++++ Gallbladder 1 ++ + Pancreatic 1 +/− + Gastric 1 ++ ++++ Head and neck 1 ++ ++++ NSCLC Breast Carcinoma Gastric Carcinoma Controls 0.5 mg/kg 5 mg/kg 20 mg/kg 100 mg/kg KLH-Specific B Cell Response (IgM) Controls INCAGN1949 TT-Specific T Cell Response Days post-treatment 0 0 2500 SFU/10 6 total PBMCs 2000 1500 1 8 15 29 36 1000 43 57 66 500 TT-Specific B Cell Response (IgM) 0 500,000 0 µg/mL 400,000 300,000 1 15 29 200,000 43 57 69 100,000 Days post-treatment 0 2000 0 µg/mL 1500 1000 500 0 3000 0 µg/mL 1 15 29 1000 43 57 69 2000 Controls INCAGN1949 B PK Profile of INCAGN1949 10 7 10 1 INCAGN1949 concentration (µg/mL) 10 6 10 5 8 14 20 28 Days 10 4 10 3 10 2 0 2 4 6 10 12 16 18 22 24 26 0.5 mg/kg 5 mg/kg 20 mg/kg 100 mg/kg Intensity of Expression 10 0 Number of tumor samples 8 4 6 2 10 0 8 4 6 2 10 0 8 4 6 2 Tumor tissue Tumor stroma OX40 FoxP3 Tumor tissue Tumor stroma NSCLC (FoxP3) FoxP3 Isotype C D 1 Agenus Inc., Lexington, MA; 2 Agenus Switzerland Inc., Basel, Switzerland; 3 Memorial Sloan Kettering Cancer Center, New York, NY; 4 The Ludwig Institute for Cancer Research, New York, NY; 5 Incyte Corporation, Wilmington, DE Mechanism 1: Anti-OX40 antibodies mediate receptor forward signaling in the context of T cell antigen receptor (TCR) activation, enhance effector T cell activation, cytokine production, and survival, as well as promote memory T cell differentiation and reactivation (modified from ref. 1). Activation CD28 OX40 co-stimulation Memory generation Clonal expansion Effector Contraction Reactivation OX40 co-stimulation Naïve Effector Memory NK CELL Treg cell INCAGN1949 FcRs Treg cell MACROPHAGE INCAGN1949 FcRs NK cell Treg cell Neutrophil Macrophage TUMOR Treg cell depletion and effector T-cell co-stimulation ADCC ADCP KLH-Specific B Cell Response (IgG) T effector cells Treg cells OX40 INCAGN1949 Isotype INCAGN1949 Isotype Cynomolgus monkeys were vaccinated against tetanus toxoid (TT) or keyhole limpet hemocyanin (KLH) and administered 5 mg/kg INCAGN1949 or vehicle at day 1 and day 29; a vaccination booster was also given at day 29 (n = 6 monkeys per group). (A) TT-specific IFNγ T cell responses; (B) TT-specific IgM antibody responses; (C) KLH-specific IgM antibody responses; and (D) KLH-specific IgG antibody responses. IL-17 IL-10 0 1000 0 750 40 24 500 20 250 2 6 pg/mL pg/mL 0 1000 0 pg/mL 750 40 24 500 20 250 2 6 IFNγ IL-6 0 1000 0 400 24 800 200 600 2 6 GM-CSF 0 1000 0 750 40 24 500 20 250 2 6 IL-2 0 1000 0 750 40 24 500 20 250 2 6 0 1000 0 400 24 200 600 2 6 800 Hours Hours Donor 1 Donor 2 0 20 40 60 10 30 50 Donor 1 Donor 2 0 20 40 60 10 30 50
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