cdx-1140, a novel agonist cd40 antibody with potent anti ...cdx-1140 (µ g/ml) human b cells 21.4.1...

• 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.

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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 ActivityLi-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.

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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

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CDX-1140 Agonist Activity Is Fc-independent

CDX-1140 Induces Cytokines Production

NFkB activation

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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.

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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 ActivityRamos

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Ramos cells 0.5x106 were s.c. inoculated on day 0; N=6. CDX-1140 0.3 mg i.p. on day 1, 6, 13.

Raji cells 1.0x106 were s.c. inoculated on day 0; N=5. CDX-1140 0.3 mg i.p. on day 1, 6, 13.

Raji cells 0.5x106 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

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CDX-1140 in Combination with PBMC

Immunotherapy. 2015 August ; 7(8): 899–911. Tianshu Zhang, Richard N Pierson III Agnes M Azimzadeh

Ramos cells 1.0x106 with or without 3x106 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

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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.

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PBMC donor 1

Raji cells 1.0x106 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 0.5x106 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.

cdx-1140, a novel agonist cd40 antibody with potent anti ...cdx-1140 (µ g/ml) human b cells 21.4.1...

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