Shaun O'Brien

Potentiating Endogenous Antitumor Immunity to Prostate Cancer through Combination Immunotherapy with CTLA4 Blockade and GM-CSF

CTL-associated antigen 4 (CTLA4) is a costimulatory molecule expressed on activated T cells that delivers an inhibitory signal to these T cells. CTLA4 blockade with antibody treatment has been shown to augment antitumor immunity in animal models and is being developed as a treatment for cancer patients. As has been seen in preclinical models, combining CTLA4 blockade and granulocyte macrophage colony-stimulating factor (GM-CSF)-based immunotherapies can enhance the antitumor efficacy of this approach. We therefore examined whether CTLA4 blockade could be combined with GM-CSF administration. We treated 24 patients with metastatic, castration-resistant prostate cancer in a phase I trial where sequential cohorts were treated with increasing doses of ipilimumab, a fully human anti-CTLA4 antibody. Study subjects also received s.c. injections of GM-CSF at a fixed dose. Of the six patients treated at the highest dose level, three had confirmed PSA declines of >50%, including one patient that had a partial response in visceral metastases. Expansion of activated, circulating CD25(+) CD69(+) CD8(+) T cells occurred more frequently at higher doses of treatment and was greater in magnitude than was seen in patients who received the same doses of either ipilimumab or GM-CSF alone. By screening sera with protein arrays, we showed that our treatment can induce antibody responses to NY-ESO-1. These results show that this combination immunotherapy can induce the expansion not only of activated effector CD8 T cells in vivo but also of T cells that are specific for known tumor-associated antigens from the endogenous immune repertoire.

CTLA4 blockade expands FoxP3+ regulatory and activated effector CD4+ T cells in a dose-dependent fashion

Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) delivers inhibitory signals to activated T cells. CTLA4 is constitutively expressed on regulatory CD4(+) T cells (Tregs), but its role in these cells remains unclear. CTLA4 blockade has been shown to induce antitumor immunity. In this study, we examined the effects of anti-CTLA4 antibody on the endogenous CD4(+) T cells in cancer patients. We show that CTLA4 blockade induces an increase not only in the number of activated effector CD4(+) T cells, but also in the number of CD4(+) FoxP3(+) Tregs. Although the effects were dose-dependent, CD4(+) FoxP3(+) regulatory T cells could be expanded at lower antibody doses. In contrast, expansion of effector T cells was seen only at the highest dose level studied. Moreover, these expanded CD4(+) FoxP3(+) regulatory T cells are induced to proliferate with treatment and possess suppressor function. Our results demonstrate that treatment with anti-CTLA4 antibody does not deplete human CD4(+) FoxP3(+) Tregs in vivo, but rather may mediate its effects through the activation of effector T cells. Our results also suggest that CTLA4 may inhibit Treg proliferation similar to its role on effector T cells. This study is registered at http://www.clinicaltrials.gov/ct2/show/NCT00064129, registry number NCT00064129.

Pericyte Requirement for Anti-Leak Action of Angiopoietin-1 and Vascular Remodeling in Sustained Inflammation

Blood vessel leakiness is an early, transient event in acute inflammation but can also persist as vessels undergo remodeling in sustained inflammation. Angiopoietin/Tie2 signaling can reduce the leakiness through changes in endothelial cells. The role of pericytes in this action has been unknown. We used the selective PDGF-B-blocking oligonucleotide aptamer AX102 to determine whether disruption of pericyte-endothelial crosstalk alters vascular leakiness or remodeling in the airways of mice under four different conditions: i) baseline, ii) acute inflammation induced by bradykinin, iii) sustained inflammation after 7-day infection by the respiratory pathogen Mycoplasma pulmonis, or iv) leakage after bradykinin challenge in the presence of vascular stabilization by the angiopoietin-1 (Ang1) mimic COMP-Ang1 for 7 days. AX102 reduced pericyte coverage but did not alter the leakage of microspheres from tracheal blood vessels at baseline or after bradykinin; however, AX102 exaggerated leakage at 7 days after M. pulmonis infection and increased vascular remodeling and disease severity at 14 days. AX102 also abolished the antileakage effect of COMP-Ang1 at 7 days. Together, these findings show that pericyte contributions to endothelial stability have greater dependence on PDGF-B during the development of sustained inflammation, when pericyte dynamics accompany vascular remodeling, than under baseline conditions or in acute inflammation. The findings also show that the antileakage action of Ang1 requires PDGF-dependent actions of pericytes in maintaining endothelial stability.

Angiopoietin/Tie2 Signaling Transforms Capillaries into Venules Primed for Leukocyte Trafficking in Airway Inflammation

Vascular endothelial growth factor (VEGF) is a key angiogenic factor in tumors, but less is known about what drives vascular remodeling in inflammation, where plasma leakage and leukocyte influx are prominent features. In chronic airway inflammation in mice infected by the bacterium Mycoplasma pulmonis (M. pulmonis), the segment of the microvasculature that supports leukocyte adhesion and migration expands through remodeling of capillaries into vessels with features of venules. Here, we report that the angiopoietin/Tie2 pathway is an essential driving force for capillary remodeling into venules in M. pulmonis-infected mouse airways. Similar to M. pulmonis infection, systemic overexpression of angiopoietin-1 (Ang1) resulted in remodeling of airway capillaries into venular-like vessels that expressed venous markers like P-selectin, ICAM-1, and EphB4 and were sites of leukocyte adhesion during lipopolysaccharide-induced acute inflammation. Ang1 and Ang2 protein increased in M. pulmonis-infected mouse airways but came from different cellular sources: Ang1 was expressed in infiltrating neutrophils and Ang2 in endothelial cells. Indeed, systemic administration of soluble Tie2 inhibited capillary remodeling, induction of venous markers, and leukocyte influx in M. pulmonis-infected mouse airways. Together, these findings suggest that blockade of the Ang/Tie2 pathway may represent a therapeutic approach in airway inflammation.

Abstract 1641: Dual antibody blockade of TIM3 and PD1 on NYESO1 redirected human T cells leads to augmented control of lung cancer tumors

Background: Immunotherapy using checkpoint blockade has demonstrated impressive, durable responses in select patients with solid malignancies. This is especially true for the blockade of programmed death 1 (PD1). Currently, efforts are focused on understanding non-response. One hypothesis is that multiple inhibitory receptors (IRs) are upregulated on hypofunctional tumor-infiltrating lymphocytes (TILs) and necessitates blocking multiple IRs to increase the response rate. We describe data from a unique xenograft model of human lung cancer where tumor-reactive human T cells become hypofunctional and upregulate multiple IRs including PD1 and TIM3 (T cell immunoglobulin and mucin-domain containing-3). Combining a single IV dose of tumor-reactive T cells with blockade of both PD1 and TIM3 led to greater control of tumor growth than combining it with blockade of either alone. Materials/Methods: The A549 human lung cancer cell line was transduced to stably express the cancer-testis antigen, NYESO1, expressed in the contact of HLA-A2 (A549-A2-ESO). 5x106 A549-A2-ESO cells were injected subcutaneously into the flanks NSG mice. Activated T cells from healthy donors were lentivirally transduced with a TCR targeting NYESO1 (Ly95). After two weeks when tumors were established and measured ~150mm3, the mice were randomly assigned to one of the following treatments: 1) untreated, 2) non-transduced (NTD) T cells plus anti-PD1 and anti-TIM3 Ab, 3) Ly95 T cells, 4) Ly95 T cells + anti-PD1 Ab, 5) Ly95 T cells + anti-TIM3 Ab, 6) Ly95 T cells + anti-PD1 and anti-TIM3 Ab. T cells were injected IV at a single dose of 10x106/mouse. Abs were injected intraperitonealy (IP) at 10mg/kg every 5 days from the time of T cell injection. Tumor volumes were measured serially. At the end, the mice were sacrificed and the tumors were harvested, digested, and processed into single cell suspension. Flow cytometry was performed to look at degree of TIL infiltration and expression of surface markers. Ficoll gradient was used to isolate the T cells to conduct functional analyses. Results/Conclusion: By twenty days post T cell injection, the tumors in the Ly95 T cell group (3) were ~30% smaller than that of the untreated (1) and the NTD T cell + Ab group (2). Anti-PD1 Ab augmented Ly95 T cell control of tumor (4) (further decrease in size by ~30%) (4). Anti-TIM3 Ab had no effect on Ly95 T cell tumor control (5). Anti-PD1 plus anti-TIM3 Abs had the greatest augmentation on Ly95 T cell control of tumor size (6) (greater than 50% reduction in tumor size compared Ly95 T cells alone) The greatest TIL infiltration and ex-vivo TIL function were seen in tumors that received Ly95 T cell + anti-PD1 and anti-TIM3 Abs. Flow cytometry analysis revealed dynamic, adaptive upregulation of one IR when another IR was blocked. This explained why augmentation of Ly95 T cell control of tumor was greatest with combinatorial Ab blockade. Citation Format: Edmund Moon, Soyeon Kim, Shaun O9Brien, Naomi Saint Jean, Raluca Verona, Linda Snyder, Yangbing Zhao, Steven Albelda. Dual antibody blockade of TIM3 and PD1 on NYESO1 redirected human T cells leads to augmented control of lung cancer tumors [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 1641. doi:10.1158/1538-7445.AM2017-1641