CD40, a member of the tumor necrosis factor receptor (TNFR) superfamily, is essential for activating both innate and adaptive immune systems. CD40 is expressed by antigen-presenting cells (APC) including dendritic cells and B cells. The engagement by its natural ligand (CD40L) on T cells activates APC thus enhancing immune responses. CD40 is also expressed on many cancer cells, in particular B cell lymphomas, and agonist CD40 antibodies can lead to apoptosis and impaired growth of cancer cells due to direct signaling effects. There are several CD40 agonist antibodies in clinical development, which are differentiated by their level of agonist activity, route of administration, and associated side effect profile. We have generated a panel of CD40-specific fully human antibodies derived from human immunoglobulin transgenic mice with the aim to select a clinical candidate with strong agonist activity and a safety profile that allows for systemic dosing. CDX-1140 is a human IgG2 antibody that stimulates CD40 signaling without the requirement for cross-linking or Fc receptor interactions. CDX-1140 is a potent stimulator of human B cells and dendritic cells in vitro, and has potent direct anti-tumor activity against B cell lymphoma cell lines transplanted into immunocompromised mice. To demonstrate the contribution of immune cell activation to the anti-lymphoma activity in vivo, we included human PBMCs in subsequent xenograft studies. Using human B cell lymphoma cell lines Raji and Ramos as tumor models and severe combined immunodeficiency mice (SCID-beige) as hosts, we found that addition of human PBMC alone had a minor impact on tumor growth (median survival 32 days, compared to 26 days with no treatment, p<0.01). Administration of CDX-1140 alone showed partial anti-tumor activity (median survival 39 days, p< 0.001), while the combination of human PBMCs and CDX-1140 provided a significant increase in survival with all mice alive at day 45. These studies provide evidence that CDX-1140 is able to induce in vivo activation and anti-tumor activity of effector cells derived from injected human PBMC, in addition to the direct signaling effects on lymphoma cells. We are investigating anti-lymphoma efficacy of combining CDX-1140 with other immunotherapies including varlilumab, our CD27 agonist antibody, which is currently in clinical development. Together with our prior preclinical studies characterizing this antibody, these results suggest that CDX-1140 is a promising candidate for clinical development in patients with lymphomas. Disclosures He: Celldex Therapeutics, Inc.: Employment. Testa:Celldex Therapeutics, Inc.: Employment. Anna:Celldex Therapeutics, Inc.: Employment. Jeffery:Celldex Therapeutics, Inc.: Employment. Sisson:Celldex Therapeutics, Inc.: Employment. Vitale:Celldex Therapeutics, Inc.: Employment. O'Neill:Celldex Therapeutics, Inc.: Employment. Crocker:Celldex Therapeutics, Inc.: Employment. Widger:Celldex Therapeutics, Inc.: Employment. Goldstein:Celldex Therapeutics, Inc.: Employment. Marsh Jr.:Celldex Therapeutics, Inc.: Employment. Keler:Celldex Therapeutics: Employment, Equity Ownership.

2861 CD27, a lymphoid cell-specific TNFR superfamily member, is constitutively expressed on the majority of T cells, some NK cells and memory B cells. Through interaction with its ligand CD70, CD27 transduces a co-stimulatory signal promoting T cell and NK cell activation and cytotoxicity. In addition, CD27 is also expressed on many lymphoid-originated hematological neoplastic cells, such as chronic lymphocytic leukemia, mantle cell lymphoma, and Waldenstrom macroglobulinemia, thus being a potential direct target for antibody therapy. To generate potential antibodies for clinical development, we immunized human Ig transgenic mice and developed a panel of CD27 specific human mAbs. Clone 1F5 was identified as a lead based on its high affinity to both human and monkey CD27, enhanced co-stimulation of T cells, and ADCC of CD27-expressing lymphoblastic cell lines. Using SCID mice challenged with CD27-expressing human lymphoid cell lines, we demonstrated that 1F5 mediates conventional antibody effector function. Compared to human IgG1 isotype control (huIgG1), 1F5 at doses ranging 33 μg – 500 μg (x 6) significantly delayed the growth of Burkitt's lymphoma Raji even when administration was initiated 1 week after tumor inoculation. Similar anti-tumor activity was observed against other CD27-expressing tumor lines including, Daudi and T-originated acute lymphoblastic leukemia CCRF-CEM. In order to investigate 1F5 in vivo agonistic activities and T cell-mediated tumor eradication, a human CD27 transgenic mouse model (hCD27-Tg) was generated and backcrossed onto C57BL/6 and BALB/c backgrounds. The expression profile and regulation of the human CD27 transgene driven by its own promoter were similar to that observed with endogenous mouse CD27. In addition to enhancing T cell responses when combined with vaccination, 1F5 treatment was highly effective against syngeneic mouse tumors including lymphoma BCL1 (BALB/c) and thymoma EL4-derived E.G7 (C57BL/6). For the BCL1 model, various dose levels of 1F5 mAb were delivered to mice intraperitoneally on days 3, 5, 7, 9 and 11 after i.v. administration of 107 BCL1 cells to huCD27 Tg and control animals. Controls including hCD27-Tg mice treated with saline or isotype control, or WT mice treated with 1F5 all performed consistently, leading to 50% survival approximately 23 days after tumor challenge. Treatment of mice with mAb, 1F5 substantially improved the 50% survival in a dose dependent fashion to >70 days post tumor challenge at the higher dose levels. Based on the promising efficacy data with anti-CD27 mAb 1F5 in immunocompromised and immunocompetent lymphoma models, a clinical grade product, referred to as CDX-1127 was manufactured and tested for safety. To assess the potential for 1F5 to mediate lymphocyte activation, we investigated its ability to induce proliferation and cytokine release from human PBMC or purified T cell cultures. Consistent with the known biology of CD27 we demonstrated the 1F5 mAb does not lead to direct activation of lymphocytes in the absence of additional signals. However, combining 1F5 with suboptimal levels of T cell receptor stimulation using anti-CD3 mAb (OKT3) was shown to enhance proliferation of human T cells. Two studies were performed using cynomolgus macaques. There were no CDX-1127 related mortalities or changes noted in the clinical condition, food appetence, body weights and body temperature, ophthalmic, electrocardiographic and clinical pathology assessments, organ weights and bone marrow assessments. In addition, there were no major differences in the percentage of lymphocyte populations between control and CDX-1127 treated animals at the end of the study demonstrating that the antibody did not significantly deplete normal CD27-expresssing cells. Based on the pre-clinical studies we are planning a Phase 1 clinical trial of CDX-1127 in patients with hematological malignancies and selected solid tumors. The trial is designed with separate arms to independently assess the safety and activity of CDX-1127 in hematologic malignancies, in which the antibody may act through multiple mechanisms, and in solid tumors where it would be fully dependent on indirect immune mechanisms. Disclosures: He: Celldex Therapeutics, Inc.: Employment. Thomas:Celldex Therapeutics, Inc.: Employment. Weidlick:Celldex Therapeutics, Inc.: Employment. Vitale:Celldex Therapeutics, Inc.: Employment. O'Neill:Celldex Therapeutics, Inc.: Employment. Prostak:Celldex Therapeutics, Inc.: Employment. Sundarapandiyan:Celldex Therapeutics, Inc.: Employment. Marsh:Celldex Therapeutics, Inc.: Employment. Yellin:Celldex Therapeutics, Inc.: Employment. Davis:Celldex Therapeutics, Inc.: Employment. Keler:Celldex Therapeutics, Inc.: Employment.

CD27 is a member of TNFR superfamily. It constitutively expresses on the majority of T cell and a subset of NK cells, playing key roles in T cell activation and survival and in NK cell proliferation and cytotoxicity upon interaction with ligand CD70. Some antibodies to mouse CD27 have been reported that display agonistic and anti-tumor activities while other mAbs had less anti-tumor activity and were depleting. We hypothesized that differences in these antibodies may be due to Fc receptor engagement, as has recently been shown for the adjuvant and anti-tumor activities of agonistic mouse CD40 mAbs, which is also member of TNFR superfamily. We have developed and previously described a human anti-human CD27 antibody (1F5) and a human CD27 transgenic mouse model (hCD27-Tg) to explore the therapeutic potential of targeting CD27. In this study, we examined the effect of modifying the constant regions of the 1F5 mAb on its ability to enhance antigen specific T cell responses. With the original 1F5 hIgG1 as template, a panel of 1F5 variants was made including 1F5 mIgG1, 1F5 mIgG2a, 1F5 mIgG1D265A and 1F5 hIgG1N297S using molecular cloning techniques. All of the variants retained equal binding to hCD27 as shown by ELISA and flow cytometry studies. In addition, Biacore analysis confirmed the expected pattern of binding to human and mouse FcαRs. Co-injection of 1F5 or its variants with ovalbumin enhanced antigen-specific CD8 T cell response to different extents, as detected by SIINFEKL-specific IFNα-ELISPOT and ICS. The 1F5 mIgG1 induced the highest number of IFNα-producing CD8+ cells, whereas 1F5 mIgG2a was very weak at enhancing the CD8 T cell response. The hIgG1 version of 1F5 was intermediate in activity. Introduction of the D265A mutation that disrupts FcαRs binding into the mIgG1 eliminated the co-stimulatory function of 1F5. Similarly, the 1F5 hIgG1N297S also showed reduced activity compared to the original 1F5 hIgG1. The isotype-specific effects on our anti-hCD27 mAb are surprisingly consistent with the findings described for the agonistic anti-mCD40 mAbs, and imply that engagement of the inhibitory Fcα receptors (FcαRIIb) is driving the co-stimulatory activity in this model. Interestingly, the 1F5 hIgG1 triggered a significant T cell response, despite the lack of FcαRIIb binding by Biacore analysis. The effect of these variants on anti-tumor activity in hCD27 transgenic mice is currently being investigated. The 1F5 hIgG1 mAb (CDX-1127) is currently undergoing clinical evaluation in a phase 1 trial of patients with advanced cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3519. doi:1538-7445.AM2012-3519