David Savitsky

Abstract 3204: INCAGN01949: an anti-OX40 agonist antibody with the potential to enhance tumor-specific T-cell responsiveness, while selectively depleting intratumoral regulatory T cells

OX40 (CD134, TNFRSF4) is a T cell co-stimulatory receptor that potentiates T cell receptor (TCR) signaling during CD4+ and CD8+ T cell priming, effector cell differentiation and memory T cell recall responses. In preclinical mouse tumor models, surrogate anti-OX40 agonist antibodies have shown remarkable single agent anti-tumor efficacy, as well as the ability to combine effectively with other immunomodulatory antibodies and immune education strategies, such as therapeutic cancer vaccines. Agonistic antibodies targeting OX40 are predicted to counteract the immunosuppressive tumor microenvironment and promote tumor-specific T cell immunity via two primary mechanisms: 1) binding and activating OX40 signaling in tumor-specific effector and memory T cells, thereby enhancing their responsiveness to tumor-associated antigens, and 2) co-engaging Fcγ receptors expressed by tumor-associated effector cells, and facilitating the selective depletion of intratumoral regulatory T cells. INCAGN01949 is a novel fully human IgG1 monoclonal antibody identified using the Retrocyte Display™ platform that is being developed for the treatment of advanced malignancies. INCAGN01949 recognizes human and cynomolgus monkey OX40 with comparable binding affinity. INCAGN01949 has been optimized to potently mediate receptor forward signaling under conditions of suboptimal TCR stimulation, leading to features like enhanced production of TNFα and IFNγ, and concomitant suppression of IL-10. INCAGN01949 achieves this functionality through OX40 clustering and downstream activation of the NFκB pathway in T cells, which is sustained across a broad range of antibody concentrations. Consistent with mouse preclinical tumor models, OX40 was found to be selectively overexpressed by intratumoral regulatory T cells from a variety of primary human tumor samples. Commensurate with its human IgG1 Fc region, INCAGN01949 can effectively co-engage activating Fcγ receptors on immune effector cells, including natural killer cells and macrophages. Therefore INCAGN01949 has the potential to mediate selective effector cell activity toward intratumoral populations of regulatory T cells. The biophysical and functional attributes of INCAGN01949 make it suited for clinical development, both as a single agent and in combination with other immunomodulatory antibodies or immune education strategies. Citation Format: Ana Maria Gonzalez, Mariana L. Manrique, Ekaterina Breous, David Savitsky, Jeremy Waight, Randi Gombos, Yuqi Liu, Shiwen Lin, Taha Merghoub, Daniel Hirschhorn-Cymerman, Gerd Ritter, Jedd Wolchok, Peggy Scherle, Gregory Hollis, Reid Huber, Marc Van Dijk, Robert Stein, Nicholas S. Wilson. INCAGN01949: an anti-OX40 agonist antibody with the potential to enhance tumor-specific T-cell responsiveness, while selectively depleting intratumoral regulatory T cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3204.

Toxicological and pharmacological assessment of AGEN1884, a novel human IgG1 anti-CTLA-4 antibody

CTLA-4 and CD28 exemplify a co-inhibitory and co-stimulatory signaling axis that dynamically sculpts the interaction of antigen-specific T cells with antigen-presenting cells. Anti-CTLA-4 antibodies enhance tumor-specific immunity through a variety of mechanisms including: blockade of CD80 or CD86 binding to CTLA-4, repressing regulatory T cell function and selective elimination of intratumoral regulatory T cells via an Fcγ receptor-dependent mechanism. AGEN1884 is a novel IgG1 antibody targeting CTLA-4. It potently enhanced antigen-specific T cell responsiveness that could be potentiated in combination with other immunomodulatory antibodies. AGEN1884 was well-tolerated in non-human primates and enhanced vaccine-mediated antigen-specific immunity. AGEN1884 combined effectively with PD-1 blockade to elicit a T cell proliferative response in the periphery. Interestingly, an IgG2 variant of AGEN1884 revealed distinct functional differences that may have implications for optimal dosing regimens in patients. Taken together, the pharmacological properties of AGEN1884 support its clinical investigation as a single therapeutic and combination agent.

Abstract 3220: A novel agonist antibody (INCAGN01876) that targets the costimulatory receptor GITR

Activation of costimulatory receptors of the tumor necrosis factor receptor (TNFR) superfamily in T cells is considered a promising alternative approach to potentiate anti-tumor immunity that may complement strategies focused on the blockade of co-inhibitory pathways such PD-1/PDL1. Glucocorticoid-induced TNFR-related protein (GITR, CD357 or TNFRSF18) is an important T cell costimulatory receptor that can potentiate T cell receptor (TCR) signaling during CD4+ and CD8+ T cell priming, effector cell differentiation and memory T cell recall responses. In humans GITR expression is generally restricted to subsets of T cells responding to TCR stimulation, and is co-expressed with OX40. Like other TNFR family members, GITR co-stimulation can enhance T cell responsiveness to suboptimal TCR signaling by activating the NFκB pathway, leading to enhanced cytokine responses and survival. GITR signaling in T cells may also promote resistance to the immune suppressive effects of regulatory T cells, thereby enhancing T cell responsiveness to weakly immunogenic tumor-associated antigens. INCAGN01876 is a humanized IgG1 monoclonal antibody being developed for the treatment of advanced malignancies. INCAGN01876 potently binds to human and non-human primate GITR but does not cross-react with related TNFR family members. INCAGN01876 has been optimized to mediate receptor forward signaling under suboptimal TCR stimulatory conditions, leading to increased production of TNFα and IFNγ by both CD4+ and CD8+ T cells. INCAGN01876 achieves this functionality by virtue of its ability to facilitate GITR clustering in TCR-stimulated T lymphocytes. In mouse preclinical tumor models, GITR was found to be selectively overexpressed by intratumoral regulatory T cells, a finding that was also observed in primary human tumor samples from diverse tumor types. In mouse models, this feature enabled a surrogate anti-GITR antibody to co-engage activating Fcγ receptors expressed by tumor-associated effector cells, and mediate the selective depletion of intratumoral regulatory T cells. Consistent with this, INCAGN01876 was designed to co-engage activating Fcγ receptors and was shown to efficiently mediate immune effector cell mechanisms, including ADCC and ADCP. Taken together, the biophysical and functional attributes of INCAGN01876 make it ideally suited for clinical development, both as a single agent and in combination with other immunomodulatory agents. Citation Format: Ana Maria Gonzalez, Ekaterina Breous, Mariana L. Manrique, David Savitsky, Jeremy Waight, Randi Gombos, Yuqi Liu, Shiwen Lin, Olivier Leger, Volker Seibert, Takemasa Tsuji, Taha Merghoub, Sadna Budha, Roberta Zappasodi, Gerd Ritter, Jedd Wolchok, Peggy Scherle, Gregory Hollis, Reid Huber, Marc Van Dijk, Robert Stein, Nicholas Wilson. A novel agonist antibody (INCAGN01876) that targets the costimulatory receptor GITR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3220.

New tricks for old targets: Anti-CTLA-4 antibodies re-envisioned for cancer immunotherapy

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) has emerged as an effective target for cancer immunotherapy [1]. As a potent negative regulator of T cell priming and immune cell activation, James Allison and colleagues hypothesized that blockade of CTLA-4 may enhance antitumor T cell activity in cancer patients by “releasing the brakes” on the immune system [1]. The approval of ipilimumab (Yervoy®, Bristol MyersSquibb) for unresectable or metastatic melanoma in 2011 has validated this hypothesis. Nevertheless, restriction of durable antitumor responses to only a subset of patients has motivated us and others to better understand CTLA4 biology and the mechanisms by which anti-CTLA-4 antibodies mediate their antitumor effects [2]. An early view for how anti-CTLA-4 antibodies increased immune responses was through liberation of B7 (CD80/86) from CTLA-4, ultimately permitting costimulation through B7-CD28 and activation of naïve T cells [1]. Antagonism of CTLA-4-mediated lipid raft disruption, trans-endocytosis of, and reverse signaling into B7 have since been described as potential mechanisms of action [3]. In parallel, an evolving understanding of Fc gamma receptor (FcγR) interactions and their importance for the activity of a range of immunotherapeutic antibodies has led to the discovery that antibody Fc-FcγR coengagement is critically important for the antitumor effects of CTLA-4 antibodies [4, 5]. In preclinical mouse models, this Fc-dependent antitumor activity has been correlated with the depletion of intratumoral regulatory T cells (Tregs). However, we found that while treatment of tumor-bearing mice with anti-CTLA-4 antibody (i) results in profound tumor control and (ii) requires FcFcγR co-engagement, intratumoral Treg cell depletion is often incomplete [6]. Therefore, we asked whether the Fcdependent activity of CTLA-4 antibodies is exclusively dependent on Treg cell depletion or whether the story is more nuanced. Using a tumor-free mouse system (staphylococcal enterotoxin B administration), we interrogated the dependence of anti-CTLA-4 Fc-FcγR co-engagement on antigen-specific T cell activity in vivo. Consistent with observations in tumor-bearing mice, we found enhanced antigen-specific T cell responses to anti-CTLA-4 therapy could be controlled by simply modifying the Fc backbone of the antibody or lost following blockade of activating FcγRIV (CD16-2). Remarkably, we observed that responses elicited by anti-CTLA-4 were maintained in the absence of Treg cells. Our results implicated an unappreciated Fc-dependent mechanism employed by anti-CTLA-4 antibodies to enhance T cell responsiveness independent of Treg cell depletion. A central aspect of our study was the concordance in observations between murine and human cells. In particular, we showed that FcγRIIIA (CD16) engagement was required for promoting anti-CTLA-4 activity, again highlighting the importance of activating FcγRs in facilitating antibody function. As an orthogonal approach, we generated several anti-CTLA-4 Fc variants and observed a robust correlation between affinity to FcγRIIIA and functional activity. This improved functional activity was associated with enhanced apical T cell receptor (TCR) signaling events, as determined by ZAP70 phosphorylation. Our data also suggest that this enhanced activity is T cell intrinsic and unlikely to be due to antigen presenting cell (APC) conditioning via reverse signaling into FcγRIIIA. We hypothesized that anti-CTLA-4 FcFcγRIIIA co-engagement helps “bridge” T cells and APCs to improve immune synapse quality and promote more effective T cell priming, an attribute that can be modulated by Fc engineering (Figure 1). This hypothesis aligns well with the model of kinetic segregation in which physical exclusion of phosphatases through tight T cell:APC Editorial

Abstract 4703: INCAGN1949, an anti-OX40 antibody with an optimal agonistic profile and the ability to selectively deplete intratumoral regulatory T cells

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 Fcγ receptors expressed by tumor-associated effector cells, and facilitating the selective elimination of OX40high 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 NFκB 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 OX40high 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). Citation Format: Ana M. Gonzalez, Mariana L. Manrique, Lukasz Swiech, Thomas Horn, Ekaterina Breous, Jeremy Waight, David Savitsky, Yuqi Liu, Shiwen Lin, Christopher Clarke, Taha Merghoub, Daniel Hirschhorn-Cymerman, David Schaer, Gerd Ritter, Jennifer Pulini, Kevin Heller, Peggy Scherle, Gregory Hollis, Reid Huber, Marc van Dijk, Jennifer Buell, Robert Stein, Nicholas Wilson. INCAGN1949, an anti-OX40 antibody with an optimal agonistic profile and the ability to selectively deplete intratumoral regulatory T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4703. doi:10.1158/1538-7445.AM2017-4703

Abstract 3654: AGEN1884, an IgG1 anti-CTLA-4 antibody, combines effectively with PD-1 blockade in primary human T cell assays and in a non-human primate pharmacodynamic (PD) model

Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) play important non-redundant roles in negatively regulating T cell immune responses. Therapeutic blockade of CTLA-4 or PD-1 pathways has been demonstrated to enhance T cell reactivity to tumor-specific antigens, translating to a significant improvement in overall survival. This anti-tumor effect can be further augmented when PD-1 and CTLA-4 antagonist antibodies are co-administered. The therapeutic impact of this combination is exemplified by the approval of this combination for advanced melanoma, as well as clinical benefit of the combination observed in NSCLC, mRCC, and most recently, mUC. AGEN1884, a human IgG1 antibody directed against CTLA-4, potently inhibits CTLA-4 binding to CD80 and CD86, resulting in enhanced T cell responsiveness in vitro, as well as in a vaccination model in non-human primates. A Phase 1 clinical study (NCT02694822) is currently ongoing to evaluate the safety and pharmacokinetic (PK)/pharmacodynamic (PD) relationships in patients with advanced solid tumors. AGEN2034 is a human IgG4 antibody that binds selectively to PD-1 with high affinity and potentiates T cell responsiveness via the blockade of PD-L1 and PD-L2 binding to PD-1. Here we evaluated the pharmacologic effect of combining AGEN1884 with AGEN2034, and other molecules targeting the PD-1/PD-L1 pathway, on primary human T cell immune responses. AGEN1884 combined effectively with AGEN2034, and other antibodies targeting the PD1/PD-L1 pathway, to promote superior T cell immune responses compared to monotherapies. Consistent with these in vitro findings, the co-administration of AGEN1884 with an anti-PD-1 antibody in cynomolgus monkeys (Macaca fascicularis) induced a dynamic PD effect, including a proliferative T cell response in peripheral blood, as compared to animals receiving either antibody alone. Finally, co-administration of an anti-mouse CTLA-4 antibody together with Agenus’ tumor-specific neo-epitope-based vaccine (AutoSynVax™) in mice induced effective amplification of vaccine-driven T cell responses, compared to animals that received the vaccine alone. These data further exemplify the versatility of harnessing antibody-mediated CTLA-4 blockade to influence apical events involved in T cell priming by antigen presenting cells. Taken together, these in vitro and in vivo findings demonstrate that the combination of AGEN1884 with PD-1 pathway blockade or with neo-epitope-based vaccines has the potential to provide therapeutic activity that is superior to that of either checkpoint- or vaccine-based monotherapies. Citation Format: Elise E. Drouin, David Savitsky, Ana M. Gonzalez, Randi Gombos, Dhan Chand, Jeremy Waight, Xia Yang, Mithun Khattar, Benjamin Morin, Mark Findeis, David Schaer, Taha Merghoub, Gerd Ritter, Antoine Tanne, Marc van Dijk, John M. Goldberg, Daniel Levey, John Castle, Jean-Marie Cuillerot, Jennifer S. Buell, Robert Stein, Nicholas S. Wilson. AGEN1884, an IgG1 anti-CTLA-4 antibody, combines effectively with PD-1 blockade in primary human T cell assays and in a non-human primate pharmacodynamic (PD) model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3654. doi:10.1158/1538-7445.AM2017-3654

Abstract 5005: AGEN1884 and AGEN2041: Two functionally distinct anti-CTLA-4 antagonist antibodies

CTLA-4 is an important negative regulator of T cell function. Together with CD28, these two co-receptors exemplify a co-inhibitory/stimulatory system that is critical in the regulation of T cell immune responses. Key to this regulatory system are the shared ligands, CD80 and CD86, whose engagement determines whether T cells receive stimulatory (CD28) or inhibitory (CTLA-4) signals. Pre-clinical and clinical studies have shown that anti-CTLA-4 antibodies can enhance tumor-specific immunity through a combination of mechanisms including: 1) blockade and or displacement of CD80/CD86 binding to CTLA-4, leading to CD28 activation; 2) prevention of the trans-endocytosis of CD80/CD86 from the surface of antigen presenting cells by CTLA-4 expressing regulatory T cells; and 3) the selective depletion of CTLA-4 expressing intratumoral regulatory T cells by an Fcγ receptor-mediated mechanism. AGEN1884 and AGEN2041, two fully human anti-CTLA-4 antibodies identified using the Retrocyte Display™ platform, are being developed for the treatment of advanced malignancies. The antibodies share heavy and light chain complementarity determining regions (CDRs), but differ in their IgG Fc region (AGEN1884, an IgG1, and AGEN2041, an IgG2). AGEN1884 and AGEN2041 selectively bind to human and cynomolgus monkey CTLA-4 with low single digit nM affinity. Further, both antibodies bind CTLA-4 expressed on T cells, and potently block engagement of CD80 and CD86, leading to enhanced T cell responsiveness. In a T cell-dependent antibody response (TDAR) study in cynomolgus monkeys, administration of a vaccine in combination with either AGEN1884 or AGEN2041 augmented the antibody response to the vaccine antigen. This finding demonstrates that both antibodies are functional in non-human primates, and exemplifies their utility in promoting immunity to co-administered antigens in patients, such as therapeutic cancer vaccines. Consistent with this, an anti-mouse CTLA-4 antibody produced potent tumor regressions in combination with a heat-shock protein-based vaccine (a surrogate vaccine resembling Prophage™ heat shock protein-based autologous vaccine). The distinct IgG backbones of AGEN1884 and AGEN2041 enable distinct optimal effector functions, such as the ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP). We anticipate that these differences in effector function may be exploited in certain tumors depending on immune cell composition. Taken together, the biochemical and functional attributes of AGEN1884 and AGEN2041 are ideally suited for clinical development, both as single agents and also in combination with other immune education approaches, such as cancer vaccines and immunomodulatory antibodies or small molecule therapies. Citation Format: Elise E. Drouin, Ana Gonzalez, Hao Tang, David Savitsky, Randi Gombos, Jeremy Waight, Benjamin Duckless, Andrea Schuster, Lili Wang, Shiwen Lin, Cornelia Mundt, Gerd Ritter, Taha Merghoub, Kyle Draleau, Jedd Wolchok, Daniel Levey, Jennifer Buell, Marc van Dijk, John M. Goldberg, Robert Stein, Nicholas S. Wilson. AGEN1884 and AGEN2041: Two functionally distinct anti-CTLA-4 antagonist antibodies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5005.