Abstract 3643: INCAGN1876, a unique GITR agonist antibody that facilitates GITR oligomerization
Ana María GonzálezMariana ManriqueLukasz SwiechThomas HornJeremy D. WaightYuqi LiuS.J. LinDennis UnderwoodEkaterina BreousOlivier LegerVolker SeibertTaha MerghoubRoberta ZappasodiGerd RitterDavid SchaerKevin N. HellerKimberli J. BrillPeggy ScherleGregory HollisReid HuberMarc Van DijkJennifer S. BuellRobert B. SteinNicholas S. Wilson
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Abstract 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.AM2017-3643Keywords:
CD137
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Cancer-immunotherapy targeting programmed cell death 1 (PD-1) activates tumor-specific T cells and provides clinical benefits in various cancers. However, the molecular basis of PD-1 function is still enigmatic. Especially, it is unclear which signaling pathway PD-1 primarily targets. Besides, the capacity of PD-1 to inhibit the T cell receptor (TCR)-dependent activation of T cells in the presence of co-stimulation is also controversial. Here we used co-culture systems of T cells and antigen-presenting cells with targeted deletion and overexpression of co-receptors and ligands and examined the inhibitory potency of PD-1 against T cell activation upon TCR stimulation with CD28 and ICOS co-stimulation. As an unambiguous criterion of T cell activation, we used the acquisition of cytokine production capacity, which represents one of the most important functions of T cells. PD-1 inhibited functional T cell activation upon TCR stimulation in the absence as well as in the presence of CD28 co-stimulation, indicating that PD-1 can directly inhibit TCR signal. Notably, CD28 co-stimulation rather attenuated the efficiency of PD-1 in inhibiting TCR-dependent functional T cell activation. In addition, PD-1 inhibited TCR-dependent functional T cell activation with ICOS co-stimulation as efficiently as that with CD28 co-stimulation. Furthermore, we found that the maintenance of antigen-induced TFH cells that required ICOS co-stimulation was persistently restrained by PD-1 in vivo. These findings indicate that PD-1 primarily targets TCR signal in the inhibition of functional T cell activation. Thus, PD-1 functions as the rheostat of T cell activation rather than an inhibitor of a specific stimulatory co-receptor.
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T cell activation requires two signals: a signal from the TCR and a co-stimulatory signal provided by antigen-presenting cells (APC). In addition to CD28, multiple molecules on the T cell have been described to deliver co-stimulatory signals. Here, we investigated whether there exist quantitative or qualitative differences in the co-stimulatory capacity between CD28 and other molecules. Anti-CD28 monoclonal antibody (mAb) and mAb against CD5, CD9, CD2, CD44 or CD11a all induced activation of naive T cells in the absence of APC when co-immobilized with a submitogenic dose of anti-CD3 mAb. [ 3 H]Thymidine incorporation determined 2 days after co-stimulation was all comparable. In contrast to progressive T cell proliferation induced by CD28 co-stimulation, co-stimulation by other T cell molecules led to a decrease in viable cell recovery along with the induction of apoptosis of once activated T cells. This was associated with a striking difference in IL-2 production; CD28 co-stimulation induced progressively increasing IL-2 production, whereas co-stimulation by other molecules produced limited amounts of IL-2. Addition of recombinant IL-2 to the latter cultures corrected the induction of apoptosis, resulting in levels of cellular proliferation comparable to those observed for CD28 co-stimulation. These results indicate that a fundamental difference exists in the nature of co-stimulation between CD28 and other molecules, which can be evaluated by the levels of IL-2 production, but not simply by [ 3 H]thymidine incorporation.
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T cell activation requires two signals: a signal from the TCR and a co-stimulatory signal provided by antigen-presenting cells (APC). In addition to CD28, multiple molecules on the T cell have been described to deliver co-stimulatory signals. Here, we investigated whether there exist quantitative or qualitative differences in the co-stimulatory capacity between CD28 and other molecules. Anti-CD28 monoclonal antibody (mAb) and mAb against CD5, CD9, CD2, CD44 or CD11a all induced activation of naive T cells in the absence of APC when co-immobilized with a submitogenic dose of anti-CD3 mAb. [ 3 H]Thymidine incorporation determined 2 days after co-stimulation was all comparable. In contrast to progressive T cell proliferation induced by CD28 co-stimulation, co-stimulation by other T cell molecules led to a decrease in viable cell recovery along with the induction of apoptosis of once activated T cells. This was associated with a striking difference in IL-2 production; CD28 co-stimulation induced progressively increasing IL-2 production, whereas co-stimulation by other molecules produced limited amounts of IL-2. Addition of recombinant IL-2 to the latter cultures corrected the induction of apoptosis, resulting in levels of cellular proliferation comparable to those observed for CD28 co-stimulation. These results indicate that a fundamental difference exists in the nature of co-stimulation between CD28 and other molecules, which can be evaluated by the levels of IL-2 production, but not simply by [ 3 H]thymidine incorporation.
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Our present study provides evidence that the 4-1BB signal is critical to CD28 co-stimulation in maintaining T cell activation when CD28 has been down-regulated because of repeated stimulation. The 4-1BB signal synergized with CD28 co-stimulation by lowering the threshold of anti-CD28 required to sustain proliferation and IL-2 production. The 4-1BB signal also modulated CD28-mediated cytokine profiles by markedly enhancing Th1 but suppressing Th2-type cytokine production. The 4-1BB signal generated Th1-type cells, as identified by intracellular IFN-γ production. IFN-γ induction was detected preferentially in 4-1BB-expressing cells, but not in those expressing CD30. 4-1BB and CD30 were induced in both CD4+ and CD8+ cells, but the location of the two molecules was mutually exclusive in each T cell subset. Our study suggests that the 4-1BB signal regulates CD28 co-stimulation in the targeted subset cells to favor Th1 development and maintain long-term cell growth.
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T cells that play role in cell-mediated immunity must be activated in order to generate immune responses. The two signals are required for T cell activation. The first of these signals is provided by the antigen. The second signal is provided by the co-stimulatory molecules. For many years it was believed that T cells do not require co-stimulation for activation. However, in vitro and in vivo studies have shown that T cells need the co-stimulatory molecules for activation and expansion. Co-stimulation for T-cell activation and growth is provided by molecules of CD28 and the tumor necrosis factor (TNF) family members. Four co-stimulatory molecules belonging to the CD28 family have been identified. These family members are CD28, inducible co-stimulator (ICOS), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, CD152) and programmed cell death protein 1 (PD-1). Although CD28 and ICOS showed positive effects, CTLA- 4 and PD-1 molecule have a negative effect on T cell activation. In this review, we discussed co-stimulatory molecules belonging to the CD28 family, the ligands of these molecules, and the interactions of co-stimulatory molecules with ligands.
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T cells that play role in cell-mediated immunity must be activated in order to generate immune responses. The two signals are required for T cell activation. The first of these signals is provided by the antigen. The second signal is provided by the co-stimulatory molecules. For many years it was believed that T cells do not require co-stimulation for activation. However, in vitro and in vivo studies have shown that T cells need the co-stimulatory molecules for activation and expansion. Co-stimulation for T-cell activation and growth is provided by molecules of CD28 and the tumor necrosis factor (TNF) family members. Four co-stimulatory molecules belonging to the CD28 family have been identified. These family members are CD28, inducible co-stimulator (ICOS), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, CD152) and programmed cell death protein 1 (PD-1). Although CD28 and ICOS showed positive effects, CTLA- 4 and PD-1 molecule have a negative effect on T cell activation. In this review, we discussed co-stimulatory molecules belonging to the CD28 family, the ligands of these molecules, and the interactions of co-stimulatory molecules with ligands.
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