• CD40 is a promising and powerful target for immunotherapy, but requires an appropriate balance between antitumor immune activation and harmful side effects of immune stimulation. • CDX-1140 represents a novel CD40 agonist antibody with unique profile: • Potent agonist that functions in the format of human IgG2 isotype and independent of Fc receptors interaction • Strong synergy with sCD40L for enhanced activity • Activation of human B cells, DC and monocytes in vitro • CDX-1140 has potent anti-lymphoma efficacy in xenograft models • Direct anti-tumor activity against CD40 + lymphoma • Enhanced anti-tumor activity when combined with human immune cells • Activation of T cells, B cells, myeloid cells in co-cultures of PBMC with lymphoma cells • Synergy with anti-CD27 mAb varlilumab • Based on these data and a pilot study with non-human primates that confirmed a good safety profile, CDX-1140 is progressing towards clinical trials. • A Phase 1 study with CDX-1140 in advanced cancer patients, including lymphoma patients, is planned to initiate in 2017. • Following dose escalation of CDX-1140, combinations will be explored with immunotherapy and conventional therapies. # 1848 CD40 Introduction CDX-1140 Anti-lymphoma Activity in Xenograph Models Summary and Next Steps Development and Characterization of CDX-1140 CDX-1140, A Novel Agonist CD40 Antibody with Potent Anti-lymphoma Activity Li-Zhen He, James Testa, Anna Wasiuk, Jeffrey Weidlick, Crystal Sisson, Laura A. Vitale, Thomas O’Neill, Andrea Crocker, Jenifer Widger, Joel Goldstein, Henry C. Marsh, Jr.*, Tibor Keler Celldex Therapeutics, Inc., Hampton, NJ 08827 and *Needham, MA 02494 Role of CD40/CD40L in T Cell Activation CD40 Antibody for Lymphoma Therapy • CD40 is a cell surface glycoprotein in the TNF receptor (TNFR) family. • CD40 is expressed constitutively and upregulated upon activation on antigen-presenting cells (APCs), including B cells, dendritic cells (DCs) and macrophages. • CD40 interacted with its ligand (CD40L, aka CD154), which is rapidly induced on T cells following TCR activation, plays essential roles in the modulation of adaptive immunity. • Humoral responses: • B cell proliferation, • immunoglobulin (Ig) production • isotype switching • memory B-cell generation • T-cell-mediated licensing of APCs for antigen-presenting function: • increase surface expression of MHC molecules • upregulate costimulatory molecules to provide “second signals” • release pro-inflammatory cytokines and chemokines to facilitate the polarization of Th1-type immune responses and cytotoxic T lymphocyte priming • Enhancement of macrophage effector functions, such as phagocytosis • CD40 is expressed in a variety of malignances, especially B cell lymphoma, being a target for antibody therapy. • Stimulating CD40 signaling on B cell lymphoma can inhibit proliferation and promote apoptosis. • Ligation of CD40 on APCs can substitute for stimulation normally provided by helper T cells via CD40L to activate and promote antitumor T-cell responses. • Triggering CD40 on myeloid cells, especially macrophages, can activate them with the potential to control malignancies in a T cell-independent manner. • Targeting CD40 on B cell lymphoma with a depleting mAb can induce killing by ADCC, CMC and ADCP. • CD40 mAb in combination can sensitize other anti-cancer agents, including vaccine, cytotoxic drugs, etc. • Due to the broad expression profile of CD40 and the potency of this signaling pathway, toxicity is a critical challenging in exploiting CD40 target as antitumor therapeutics. We set out to develop a panel of human CD40 mAbs with different levels of agonist activity. A lead candidate, named as CDX-1140, for systemic use is identified based on unique properties in agonist activity, anti-lymphoma activity in xenograft models and safety profile in non-human primate. Here we focus on the anti-lymphoma efficacy both in single agent and in combination with anti-CD27 mAb or with human PBMC. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 10 20 30 40 Tumor volume (cm3) Days post tumor inoculation Raji Lymphoma Growth Curves CDX-1140 hIgG2 CDX-1140 CDX-1140+PBMC PBMC hIgG2 ctrl ** ** Days post tumor inoculation Percent survival 0 20 40 60 80 0 20 40 60 80 100 ** * ** hPBMC donor 2 0 20 40 60 80 100 0 5000 10000 15000 0 50 100 150 HLA-DR in Monocytes MFI huIgG2 CDX-1140 PBMC Ramos + - + - - + + - + - + + - + + + huIgG2 CDX-1140 PBMC Ramos + - + - - + + - + - + + - + + + huIgG2 CDX-1140 PBMC Ramos + - + - - + + - + - + + - + + + MFI MFI HLA-DR in B Cells HLA-DR in T Cells Potential Mechanisms of Action of Agonistic CD40 mAb on Various Immune Effectors Robert H. Vonderheide, and Martin J. Glennie Clin Cancer Res 2013;19:1035-1043 ©2013 by American Association for Cancer Research CDX-1140 Binding to CD40 by ELISA and Flow Cytometry 0 50 100 150 200 250 0 20 40 60 80 100 120 140 160 10 1 0.1 Fold of change CDX-1140 huIgG2 CDX-1140 (µg/ml) IL12p40 Secreted by DCs TNFα Secreted by Monocytes 0 100 200 300 400 500 600 700 10 5 2.5 1.25 TNFα (pg/ml) CDX-1140 huIgG2 CDX-1140 (µg/ml) CDX-1140 Agonist Activity Is Fc-independent CDX-1140 Induces Cytokines Production NFkB activation 0 20000 40000 60000 80000 100000 120000 140000 160000 180000 0.001 0.01 0.1 1 10 Mean CPS Concentration (nM) CDX1140 Ab CDX1140 Fab2 huIgG2 huIgG2 Fab2 B cell proliferation 0 2 4 6 8 10 12 14 16 18 20 0.01 0.1 1 10 % Proliferation Concentration (µg/ml) CDX1140 Ab CDX1140 FAb2 huIgG2 Ab huIgG2 FAb2 0 0.5 1 1.5 2 2.5 3 0.001 0.01 0.1 1 10 OD 450 CD40 RANK TNFR2 LTBR HVEM 0 500 1000 1500 2000 0.001 0.01 0.1 1 10 CDX-1140 (µg/ml) Human B cells 21.4.1 FI CDX-114 huIgG2 CDX-1140 MFI Recombinant proteins CDX-1140 (µg/ml) The TNFR antigens were coated on a plate at 2 µg/ml. CDX- 1140 were added and the binding to each antigen was detected with an HRP labeled goat anti-human IgG Ab. Human whole blood was incubated with FITC-CDX-1140. B cells were identified by anti-CD20 mAb. CD40-transfected NFκB-luciferase reporter cells were incubated with CDX-1140 or controls for 6 hours, and then luciferase expression was measured. CD19 + cells were isolated, labeled with CFSE and incubated for 6 days with CDX-1140 or controls. Cells were cultured for 6 days with CDX-1140. Supernatants were harvested and tested for TNFα production by ELISA. (-) B cells = B cell depleted PBMC (Miltenyi CD19 kit) (-) monos = Monocyte depleted PBMC (Miltenyi CD14 kit) DCs were generated by culturing adherent PBMC for 7 days in the presence of 10ng/ml IL4 and 100ng/ml GM-CSF (N=6), and then incubated in the presence of CDX-1140 for 48 hours. The supernatant was analyzed for IL12p40 by ELISA. 0 4000 8000 12000 16000 20000 0.0001 0.001 0.01 0.1 1 MFI Raji Cells huIgG2 CDX1140 0 2000 4000 6000 8000 0.0001 0.001 0.01 0.1 1 MFI Ramos Cells huIgG2 CDX1140 CDX-1140 (µg/ml) CDX-1140 (µg/ml) Lymphoma cells were incubated with CDX-1140 and probed with goat anti-human IgG Fc-specific PE antibody. CDX-1140 Direct Anti-lymphoma Activity Ramos 0 15 30 45 60 75 90 0 20 40 60 80 100 Days post tumor inoculation Percent survival CDX-1140 saline Raji 0 10 20 30 40 50 60 70 0 20 40 60 80 100 Days post tumor inoculation ** *** Ramos cells 0.5x10 6 were s.c. inoculated on day 0; N=6. CDX-1140 0.3 mg i.p. on day 1, 6, 13. Raji cells 1.0x10 6 were s.c. inoculated on day 0; N=5. CDX-1140 0.3 mg i.p. on day 1, 6, 13. Raji cells 0.5x10 6 were s.c. inoculated on day 0; N=6. CDX-1140 or hIgG2 0.3 mg was injected i.p. on day 1, 8, 15. Synergy of CDX-1140 & Varlilumab 0 30 60 90 120 0 20 40 60 80 100 Percent survival Ramos ** ** CDX-1140 Varli CDX-1140+Varli ** CDX-1140 Varli CDX-1140+Varli No treatment ** * 0 15 30 45 60 75 90 0 20 40 60 80 100 Days post tumor inoculation Percent survival * * Raji CDX-1140 in Combination with PBMC Immunotherapy. 2015 August ; 7(8): 899–911. Tianshu Zhang, Richard N Pierson III Agnes M Azimzadeh Ramos cells 1.0x10 6 with or without 3x10 6 PBMC were s.c. inoculated on day 0; N=6. CDX-1140 0.3 mg was injected i.p. on day 1, 8 and 15. PBMC were co-cultured with Ramos cells at a ratio of 5:1 in a 96-well U- bottom plate for 48 hrs in the presence of 0.1 µg/ml CDX-1140 or huIgG2. Cells were stained with antibodies for CD3, CD20 and HLA-DR. • A panel of anti-CD40 monoclonal antibodies (mAbs) were generated by immunization of human Ig transgenic mice (H2L2 strain of Harbour® transgenic mice) with recombinant and cell surface expressed human CD40. • VL and VH sequences were obtained from hybridoma and expressed as huIgG1 and huIgG2a by transient transfection. • CDX-1140 (human IgG2) was identified as a lead candidate after in vitro and in vivo characterization and non-human primate pilot study. CDX-1140 Synergizes with sCD40L 0 5000 10000 15000 20000 25000 0.0001 0.01 1 100 CD95 expression (MFI) Concentration (µg/mL) CDX-1140 huIgG2 CDX-1140 + sCD40L huIgG2 + sCD40L sCD40L alone Ramos cells were incubated overnight with CDX-1140 plus or minus 0.1 µg/ml sCD40L. The cells were then stained with anti-CD95-PE antibody and analyzed by flow cytometry. Percent survival 0 20 40 60 80 100 0 20 40 60 80 100 *** ** *** CDX-1140 CDX-1140+PBMC PBMC No treatment *** ** PBMC donor 1 Raji cells 1.0x10 6 were s.c. inoculated on day 0; N=6. CDX-1140 0.3 mg i.p. on day 2, 7, 12; varlilumab 0.1 mg i.p. on day 5, 12, 19; and the combo. Ramos cells 0.5x10 6 were s.c. inoculated on day 0; N=5. CDX-1140 or/and varlilumab 0.1 mg was injected i.p. on day 5, 12, 19.