Quantitative systems pharmacology model of GITR-mediated T cell dynamics in tumor microenvironment.

T cell interaction in the tumor microenvironment is a key component of immuno-oncology therapy. Glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR) is expressed on immune cells including regulatory T cells (Tregs) and effector T cells (Teffs). Preclinical data suggest that agonism of GITR in combination with Fc-γ receptor-mediated depletion of Tregs results in increased intratumoral Teff:Treg ratio and tumor shrinkage. A novel quantitative systems pharmacology (QSP) model was developed for the murine anti-GITR agonist antibody, DTA-1.mIgG2a, to describe the kinetics of intratumoral Tregs and Teffs in Colon26 and A20 syngeneic mouse tumor models. It adequately captured the time profiles of intratumoral Treg and Teff and serum DTA-1.mIgG2a and soluble GITR concentrations in both mouse models, and described the response differences between the two models. The QSP model provides a quantitative understanding of the trade-off between maximizing Treg depletion versus Teff agonism, and offers insights to optimize drug design and dose regimen.

Rationale for anti-GITR cancer immunotherapy.

Over the past decade, our understanding of cancer immunotherapy has evolved from assessing peripheral responses in the blood to monitoring changes in the tumour microenvironment. Both preclinical and clinical experience has taught us that modulation of the tumour microenvironment has significant implications to generating robust antitumour immunity. Clinical benefit has been well documented to correlate with a tumour microenvironment that contains a dense infiltration of CD8+CD45RO+ T effectors and a high ratio of CD8+ T cells to FoxP3+ regulatory T cells (Tregs). In preclinical tumour models, modulation of the Glucocorticoid induced TNF receptor (GITR)/GITR ligand (GITRL) axis suggests this pathway may provide the desired biological outcome of inhibiting Treg function while activating CD8+ T effector cells. This review will focus on the scientific rationale and considerations for the therapeutic targeting of GITR for cancer immunotherapy and will discuss possible combination strategies to enhance clinical benefit.

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

Fc γ receptor (FcγR) coengagement can facilitate antibody-mediated receptor activation in target cells. In particular, agonistic antibodies that target tumor necrosis factor receptor (TNFR) family members have shown dependence on expression of the inhibitory FcγR, FcγRIIB. It remains unclear if engagement of FcγRIIB also extends to the activities of antibodies targeting immunoregulatory TNFRs expressed by T cells. We have explored the requirement for activating and inhibitory FcγRs for the antitumor effects of antibodies targeting the TNFR glucocorticoid-induced TNFR-related protein (GITR; TNFRSF18; CD357) expressed on activated and regulatory T cells (T reg cells). We found that although FcγRIIB was dispensable for the in vivo efficacy of anti-GITR antibodies, in contrast, activating FcγRs were essential. Surprisingly, the dependence on activating FcγRs extended to an antibody targeting the non-TNFR receptor CTLA-4 (CD152) that acts as a negative regulator of T cell immunity. We define a common mechanism that correlated with tumor efficacy, whereby antibodies that coengaged activating FcγRs expressed by tumor-associated leukocytes facilitated the selective elimination of intratumoral T cell populations, particularly T reg cells. These findings may have broad implications for antibody engineering efforts aimed at enhancing the therapeutic activity of immunomodulatory antibodies.

Differential regulation of the TRAIL death receptors DR4 and DR5 by the signal recognition particle.

TRAIL (TNF-related apoptosis-inducing ligand) death receptors DR4 and DR5 facilitate the selective elimination of malignant cells through the induction of apoptosis. From previous studies the regulation of the DR4 and DR5 cell-death pathways appeared similar; nevertheless in this study we screened a library of small interfering RNA (siRNA) for genes, which when silenced, differentially affect DR4- vs. DR5-mediated apoptosis. These experiments revealed that expression of the signal recognition particle (SRP) complex is essential for apoptosis mediated by DR4, but not DR5. Selective diminution of SRP subunits by RNA interference resulted in a dramatic decrease in cell surface DR4 receptors that correlated with inhibition of DR4-dependent cell death. Conversely, SRP silencing had little influence on cell surface DR5 levels or DR5-mediated apoptosis. Although loss of SRP function in bacteria, yeast and protozoan parasites causes lethality or severe growth defects, we observed no overt phenotypes in the human cancer cells studied--even in stable cell lines with diminished expression of SRP components. The lack of severe phenotype after SRP depletion allowed us to delineate, for the first time, a mechanism for the differential regulation of the TRAIL death receptors DR4 and DR5--implicating the SRP complex as an essential component of the DR4 cell-death pathway.

Synthetic lethal targeting of MYC by activation of the DR5 death receptor pathway.

The genetic concept of synthetic lethality provides a framework for identifying genotype-selective anticancer agents. In this approach, changes in cellular physiology that arise as a consequence of oncogene activation or tumor suppressor gene loss, rather than oncoproteins themselves, are targeted to achieve tumor selectivity. Here we show that agonists of the TRAIL death receptor DR5 potently induce apoptosis in human cells overexpressing the MYC oncogene, both in vitro and as tumor xenografts in vivo. MYC sensitizes cells to DR5 in a p53-independent manner by upregulating DR5 cell surface levels and stimulating autocatalytic processing of procaspase-8. These results identify a novel mechanism by which MYC sensitizes cells to apoptosis and validate DR5 agonists as potential MYC-selective cancer therapeutics.

Activation and suppression of the TRAIL death-receptor pathway in chemotherapy sensitive and resistant follicular lymphoma cells.

Aberrant expression of the apoptosis inhibitor bcl-2 provides a survival advantage throughout oncogenesis and can facilitate chemotherapeutic resistance in a variety of human cancers. Follicular lymphoma (FL) for example, is characterized by the chromosomal translocation t(14;18), which results in bcl-2 overexpression and initiates lymphomagenesis. Although FL cells possess ample amounts of bcl-2, they respond remarkably well to standard first-round chemotherapy. However, the vast majority of patients relapses and becomes progressively resistant to therapy. We obtained cell lines derived from chemosensitive and chemoresistant FL patients, that are characterized by the chromosomal translocation t(14;18) and expression of bcl-2, to investigate how chemotherapeutic drugs can circumvent bcl-2 anti-apoptotic function and to identify alterations in those pathways that may facilitate resistance to DNA damaging drugs. In chemosensitive FL cells, we found that DNA damaging drugs promote apoptosis through p53-dependent upregulation of the TRAIL-DR5 receptor, resulting in activation of caspase-8 and downstream executioner caspases, thereby evading bcl-2 mediated suppression of apoptosis. Examination of drug resistant FL cell lines revealed that at least two defects in this pathway can contribute to chemotherapeutic resistance: 1. p53 gene mutations that disable the transcriptional response to DNA damaging drugs, including expression of the TRAIL-DR5 receptor, and 2. transcriptional repression of the cell-death executioner enzyme caspase-3.

Bag1 proteins regulate growth and survival of ZR-75-1 human breast cancer cells.

Bag1 proteins bind heat shock protein M(r) 70,000 (Hsp 70) family molecular chaperones and regulate diverse pathways involved in cell proliferation, apoptosis, and stress responses. Four isoforms of Bag1 can be produced from a single gene in humans, including a nuclear-targeted long version (Bag1L)and a shorter cytosolic isoform (Bag1). Because overexpression of Bag1and Bag1L has been reported in breast cancers, we explored the effects of Bag1 and Bag1L on the growth of ZR-75-1 human breast cancer cells cultured in vitro and in tumor xenograft models using immunocompromised mice. Cells stably transfected with expression plasmids encoding either Bag1 or Bag1L displayed comparable rates of growth in cultures containing 10% serum, compared with control-transfected ZR-75-1 cells. In contrast, ZR-75-1 cells stably expressing mutants of Bag1 or Bag1L, which lack the COOH-terminal domain (DeltaC) required for heat shock protein M(r) 70,000 binding, displayed retarded growth rates. When cultured without serum, the viability of control-transfected, as well as Bag1DeltaC- and Bag1LDeltaC-expressing, cells declined with time, whereas Bag1- and Bag1L-overexpressing ZR-75-1 cells survived for over a week in culture. Caspase protease activation induced by serum deprivation was also prevented by stable expression of either Bag1 or Bag1L in ZR-75-1 cells. In addition, sensitivity to anchorage dependence was restored partially in ZR-75-1 cells expressing dominant-negative Bag1DeltaC and Bag1LDeltaC. In tumor xenograft studies involving injection of ZR-75-1 cells into mammary fat pads of female nu/nu mice, ZR-75-1 cells expressing Bag1 or Bag1L formed 1.4-1.6-fold larger tumors compared with control-transfected cells, whereas tumors formed by Bag1DeltaC- and Bag1LDeltaC-expressing cells grew very slowly and reached sizes < one-third of tumors generated by Neo-control ZR-75-1 cells. Altogether, these findings demonstrate that Bag1 and Bag1L provoke similar changes in breast cancer cell growth and survival and suggest that interference with Bag1 or Bag1L function might be a useful strategy for opposing breast cancer.