Nicholas Wilson

Activating Fc γ receptors contribute to the antitumor activities of immunoregulatory receptor-targeting antibodies

Antibodies that coengage activating FcγRs expressed by tumor-associated leukocytes facilitate the selective elimination of intratumoral T cells.

Harnessing co-stimulatory TNF receptors for cancer immunotherapy: Current approaches and future opportunities

Co-stimulatory tumor necrosis factor receptors (TNFRs) can sculpt the responsiveness of T cells recognizing tumor-associated antigens. For this reason, agonist antibodies targeting CD137, CD357, CD134 and CD27 have received considerable attention for their therapeutic utility in enhancing anti-tumor immune responses, particularly in combination with other immuno-modulatory antibodies targeting co-inhibitory pathways in T cells. The design of therapeutic antibodies that optimally engage and activate co-stimulatory TNFRs presents an important challenge of how to promote effective anti-tumor immunity while avoiding serious immune-related adverse events. Here we review our current understanding of the expression, signaling and structural features of CD137, CD357, CD134 and CD27, and how this may inform the design of pharmacologically active immuno-modulatory antibodies targeting these receptors. This includes the integration of our emerging knowledge of the role of Fcγ receptors (FcγRs) in facilitating antibody-mediated receptor clustering and forward signaling, as well as promoting immune effector cell-mediated activities. Finally, we bring our current preclinical and clinical knowledge of co-stimulatory TNFR antibodies into the context of opportunities for next generation molecules with improved pharmacologic properties.

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.

Gene Expression Ratios Lead to Accurate and Translatable Predictors of DR5 Agonism across Multiple Tumor Lineages

Death Receptor 5 (DR5) agonists demonstrate anti-tumor activity in preclinical models but have yet to demonstrate robust clinical responses. A key limitation may be the lack of patient selection strategies to identify those most likely to respond to treatment. To overcome this limitation, we screened a DR5 agonist Nanobody across >600 cell lines representing 21 tumor lineages and assessed molecular features associated with response. High expression of DR5 and Casp8 were significantly associated with sensitivity, but their expression thresholds were difficult to translate due to low dynamic ranges. To address the translational challenge of establishing thresholds of gene expression, we developed a classifier based on ratios of genes that predicted response across lineages. The ratio classifier outperformed the DR5+Casp8 classifier, as well as standard approaches for feature selection and classification using genes, instead of ratios. This classifier was independently validated using 11 primary patient-derived pancreatic xenograft models showing perfect predictions as well as a striking linearity between prediction probability and anti-tumor response. A network analysis of the genes in the ratio classifier captured important biological relationships mediating drug response, specifically identifying key positive and negative regulators of DR5 mediated apoptosis, including DR5, CASP8, BID, cFLIP, XIAP and PEA15. Importantly, the ratio classifier shows translatability across gene expression platforms (from Affymetrix microarrays to RNA-seq) and across model systems (in vitro to in vivo). Our approach of using gene expression ratios presents a robust and novel method for constructing translatable biomarkers of compound response, which can also probe the underlying biology of treatment response.

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 3643: INCAGN1876, a unique GITR agonist antibody that facilitates GITR oligomerization

Glucocorticoid-induced TNFR family related protein (GITR, CD357 or TNFRSF18) is a member of the tumor necrosis factor receptor superfamily (TNFRSF). Like other T cell co-stimulatory TNFR family members, GITR utilizes multiple oligomerization states to regulate the initiation of downstream signaling during T cell activation by antigen presenting cells (APCs). The formation of receptor superclusters, comprised of two or more trimeric molecules, has been defined for multiple TNFRs as a means of regulating downstream signal amplification. For co-stimulatory TNFRs, like GITR, CD137 and OX40, signaling outcomes in T cells are primarily mediated via the NFκB pathway that promotes cell survival and effector cell activities in response to suboptimal T cell receptor (TCR) stimulation. It has been hypothesized that the manipulation of the oligomeric states of co-stimulatory TNFRs using antibodies may have therapeutic utility in enhancing the activity of tumor-reactive T cells, either as single agents or in combination with other immunomodulatory or immune education strategies. Here we describe a structure-based analysis of two functionally distinct classes of anti-human GITR antibodies that stabilize unique conformational states of the receptor. INCAGN1876, a human IgG1 monoclonal anti-GITR antibody, was found to engage a conformational epitope located within a β-turn of the extracellular domain of GITR. This antibody binding site modified the equilibrium of GITR monomer, dimer and trimers to promote receptor oligomerization, resulting in downstream NFκB signaling. Notably, this mode of INCAGN1876 receptor engagement enabled it to effectively activate the GITR pathway in recently primed T cells. By contrast, a second reference anti-GITR antibody required concomitant TCR co-engagement in order to modulate the GITR pathway. High content confocal analysis was used to evaluate the kinetics of GITR clustering by both classes of anti-GITR antibody, confirming our T cell functional analysis. The ability of INCAGN1876 to engage and effectively activate GITR on recently primed T cells may enable them to overcome suppressive features of the tumor microenvironment. Notably, INCAGN1876 was shown to promote T cell co-stimulation both as a single agent and in combination with other antibodies targeting PD-1, CTLA-4 and OX40. Finally, we compared the pharmacologic activity of INCAGN1876 to Fc variants of this antibody with diminished binding to the inhibitory Fcγ receptor (FcγR), CD32B. The superiority of an IgG1 antibody in these assays was consistent with the potential to achieve optimal GITR clustering by FcγRs, while maintaining the potential for FcγR-mediated effector cell activity directed toward intratumoral GITRhigh regulatory T cells. INCAGN1876 is currently under evaluation in Phase 1/2 studies in subjects with advanced metastatic solid tumors (NCT02697591). Citation Format: Ana M. Gonzalez, Mariana L. Manrique, Lukasz Swiech, Thomas Horn, Jeremy Waight, Yuqi Liu, Shiwen Lin, Dennis Underwood, Ekaterina Breous, Olivier Leger, Volker Seibert, Taha Merghoub, Roberta Zappasodi, Gerd Ritter, David Schaer, Kevin N. Heller, Kimberli Brill, Peggy Scherle, Gregory Hollis, Reid Huber, Marc van Dijk, Jennifer Buell, Robert Stein, Nicholas S. Wilson. INCAGN1876, a unique GITR agonist antibody that facilitates GITR oligomerization [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 3643. doi:10.1158/1538-7445.AM2017-3643

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 A028: Pre-clinical development of a first-in-class fully synthetic heat shock protein-based personalized cancer vaccine

Tumors accumulate spontaneous somatic mutations which can give rise to neo-epitopes (neoantigens) recognizable as non-self by T-cells. Advances in next-generation sequencing and bioinformatics make it possible to analyze individual patients9 tumor genomes and predict immunogenic mutations, which can be synthesized as short peptides, RNA or DNA for use in personalized cancer vaccines. However, these approaches need optimization for safe and effective induction of anti-tumor T-cell responses. We addressed this challenge by complexing synthetic long peptides comprising predicted tumor neo-epitopes to recombinant Hsc70, creating personalized vaccine candidates, AutoSynVax™ (ASV™), administered with QS-21 Stimulon® adjuvant. The same platform comprising Hsc70 plus synthetic long viral peptides was previously validated in a Phase-2 clinical trial, demonstrating effective T-cell responses and reduced viral load in HSV-2+ subjects. ASV immunization is intended to facilitate antigen processing and presentation of neo-epitopes to T-cells, resulting in robust immune responses. The innate immune modulating properties of the Hsc70 protein as well as that of QS-21 Stimulon adjuvant are also under investigation at the vaccine injection site, in draining lymph node, and in cultures of antigen presenting cell populations exposed to the two agents. Finally, ASV is being explored in combination with various agonistic and antagonistic immune modulating monoclonal antibodies with the aim of increasing the duration of immunological memory and durability of tumor control. With an acceptable safety profile associated with Hsc70 and QS-21 Stimulon exposure in humans and demonstrated preclinical anti-tumor activity of ASV, we are poised to begin a first-in-human clinical trial by the first half of 2017. Citation Format: Mithun Khattar, Antoine Tanne, Benjamin Morin, Nicholas Wilson, Mohamed Uduman, Justin Zelin, Robert Stein, Mark Exley, John Castle, Daniel L. Levey. Pre-clinical development of a first-in-class fully synthetic heat shock protein-based personalized cancer vaccine [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A028.