OX40 is a potent immune-stimulating target in late-stage cancer patients

Abstract

OX40 is a potent costimulatory receptor that can potentiate T-cell receptor signaling on the surface of T lymphocytes, leading to their activation by a specifically recognized antigen. In particular, OX40 engagement by ligands present on dendritic cells dramatically increases the proliferation, effector function, and survival of T cells. Preclinical studies have shown that OX40 agonists increase antitumor immunity and improve tumor-free survival. In this study, we performed a phase I clinical trial using a mouse monoclonal antibody (mAb) that agonizes human OX40 signaling in patients with advanced cancer. Patients treated with one course of the anti-OX40 mAb showed an acceptable toxicity profile and regression of at least one metastatic lesion in 12 of 30 patients. Mechanistically, this treatment increased T and B cell responses to reporter antigen immunizations, led to preferential upregulation of OX40 on CD4(+) FoxP3(+) regulatory T cells in tumor-infiltrating lymphocytes, and increased the antitumor reactivity of T and B cells in patients with melanoma. Our findings clinically validate OX40 as a potent immune-stimulating target for treatment in patients with cancer, providing a generalizable tool to favorably influence the antitumor properties of circulating T cells, B cells, and intratumoral regulatory T cells.

Trial registration: ClinicalTrials.gov NCT01644968.

©2013 AACR.

Figures

Figure 1. Clinical observations

(A) Waterfall plot showing percent change in the sum of the diameters of tumor target lesions above or below baseline using RECIST criteria. The numbers on the graph indicate the cohort in which the patients were enrolled. Patients in cohort 1 received one cycle of 0.1mg/kg, cohort 2 0.4mg/kg and cohort 3 2mg/kg of anti-OX40. Patients 10, 18 and 23 did not have follow-up scans due to clinical progression and are PD. (B-C) Regression of a pulmonary nodule in a patient (Cohort 1) with metastatic melanoma, and progression of other nodules. Panel B, imaging before anti-OX40. Panel C, 5 months after anti-OX40 administration. (D-E) Shrinkage of a lymph node in a melanoma patient (cohort 3). Imaging in panel E was obtained 28 days after anti-OX40.

Figure 2. Changes in Ki-67 expression within CD4+ and CD8+ T cell subsets examined over time after anti-OX40

PBMC were analyzed using a multi-color flow cytometry. (A) Cells gated on CD3+, CD95+, CD4+ analyzed for FoxP3 and Ki-67. (B) Cells gated on CD3+ CD95+, CD8+ T cells analyzed for CD28 and Ki-67. (C-F) Average-fold increase in Ki-67 expression for the four lymphocyte sub-types analyzed. The fold increase was calculated by using Ki-67 percentages on various days following anti-OX40 and dividing it by the percentage of Ki-67+ cells on Day 0 (baseline). Statistical analyses were performed as described in material and methods. * p=0.001, ** p=0.013, *** p=0.004, **** p=0.007.

Figure 3. OX40 expression on Treg in TIL and PBL and the percentage of follicular T helper cells in PBL after anti-OX40

PBL and TIL gated on CD3+ CD4+ were analyzed for the expression of OX40 and HLADR on FoxP3+ Treg and CD4+/FoxP3- T cells. Percentage of OX40 positive Treg (B) and conventional CD4+ T cells (C) in PBL and TIL in 3 patients on the OX40 trial and 2 patients with untreated OC, not associated with this trial (open bars PBL, filled bars TIL). (E) PBL gated on CD3+ CCR7+ CD45RA- CD4+ T cells, central memory T cells (TCM) were assessed for expression of the follicular T helper cell marker CXCR5 at different times before and after anti-OX40. (B) Comparison of the mean percent of TCM that express CXCR5 in 6 anti-OX40 treated patients randomly selected from all three cohorts (black circles) and 6 normal donors (black triangles).

Figure 3. OX40 expression on Treg in TIL and PBL and the percentage of follicular T helper cells in PBL after anti-OX40

PBL and TIL gated on CD3+ CD4+ were analyzed for the expression of OX40 and HLADR on FoxP3+ Treg and CD4+/FoxP3- T cells. Percentage of OX40 positive Treg (B) and conventional CD4+ T cells (C) in PBL and TIL in 3 patients on the OX40 trial and 2 patients with untreated OC, not associated with this trial (open bars PBL, filled bars TIL). (E) PBL gated on CD3+ CCR7+ CD45RA- CD4+ T cells, central memory T cells (TCM) were assessed for expression of the follicular T helper cell marker CXCR5 at different times before and after anti-OX40. (B) Comparison of the mean percent of TCM that express CXCR5 in 6 anti-OX40 treated patients randomly selected from all three cohorts (black circles) and 6 normal donors (black triangles).

Figure 4. Anti-OX40 increases HLA-DR and CD38 expression on cycling CD8+ T cells

(A) PBL gated on CD3+ CD8+ T cells and analyzed for Ki-67 from a patient in cohort 2, and CD3+ CD8+ Ki-67+ were assessed for expression of the activation markers HLA-DR and CD38 at different times after anti-OX40 administration. (B) Comparison of the mean percent of cycling CD3+ CD8+ that co-express HLA-DR and CD38 T cells in 11 anti-OX40 treated patients randomly selected from all three cohorts (black circles) and 9 normal donors (black triangles).

Figure 5. Enhancement, by anti-OX40, of the immune response to reporter antigens

(A) Anti-tetanus, (C) anti-KLH antibody titer fold increase as measured by ELISA on Day 15 and 43 (15 days after vaccination). Fold-increase in antibody titers were calculated by dividing post-anti-OX40 titers by pre-anti-OX40 titers. (B) Tetanus-specific T cell proliferation was measured on Day 43 for Arm A and Day 15 for Arm B, respectively 15 days after vaccination. Data are presented as fold changes compared to the proliferation prior to OX40 administration. * p

Figure 6. Anti-OX40 infusion increases tumor-specific immune response

(A-B) PBMC from two patients with melanoma, before and after anti-OX40, were co-cultured with either autologous, HLA-mis-matched melanoma cell lines or flu. IFN-γ in the supernatant was measured by ELISA. Anti-tumor-specific antibodies were measured by Western blot (C) serum from a patient with melanoma was used to probe lysates (15μg of protein/lane) from a melanoma cell line (FEMX) or a human embryonic kidney cell line (HEK 293) at different times after anti-OX40.