NKTR-214 immunotherapy synergizes with radiotherapy to stimulate systemic CD8+ T cell responses capable of curing multi-focal cancer

Background High-dose radiotherapy (RT) is known to be immunogenic, but is rarely capable of driving clinically relevant abscopal antitumor immunity as monotherapy. RT is known to increase antigen presentation, type I/II interferon responses, and immune cell trafficking to irradiated tumors. Bempegaldesleukin (NKTR-214) is a CD122-preferential interleukin 2 (IL-2) pathway agonist that has been shown to increase tumor-infiltrating lymphocytes, T cell clonality, and increase PD-1 expression. NKTR-214 has increased drug half-life, decreased toxicity, and increased CD8+ T cell and natural killer cell stimulation compared with IL-2.Methods Animals bearing bilateral subcutaneous MCA-205 fibrosarcoma or CT26 colorectal tumors were treated with NKTR-214, RT, or combination therapy, and tumor growth of irradiated and abscopal lesions was assessed. Focal RT was delivered using a small animal radiation research platform. Peripheral and tumor-infiltrating immune phenotype and functional analyses were performed by flow cytometry. RNA expression profiling from both irradiated and abscopal lesions was performed using microarray.Results We demonstrate synergy between RT of a single tumor and NKTR-214 systemic therapy resulting in dramatically increased cure rates of mice bearing bilateral tumors compared with RT or NKTR-214 therapy alone. Combination therapy resulted in increased magnitude and effector function of tumor-specific CD8+ T cell responses and increased trafficking of these T cells to both irradiated and distant, unirradiated, tumors.Conclusions Given the increasing role of hypofractionated and stereotactic body RT as standard of care treatments in the management of locally advanced and metastatic cancer, these data have important implications for future clinical trial development. The combination of RT and NKTR-214 therapy potently stimulates systemic antitumor immunity and should be evaluated for the treatment of patients with locally advanced and metastatic solid tumors

Galectin-3 inhibition with belapectin combined with anti-OX40 therapy reprograms the tumor microenvironment to favor anti-tumor immunity.

Treatment with an agonist anti-OX40 antibody (aOX40) boosts anti-tumor immunity by providing costimulation and driving effector T cell responses. However, tumor-induced immune suppression contributes significantly to poor response rates to aOX40 therapy, thus combining aOX40 with other agents that relieve tumor-mediated immune suppression may significantly improve outcomes. Once such target is galectin-3 (Gal-3), which drives tumor-induced immunosuppression by increasing macrophage infiltration and M2 polarization, restricting TCR signaling, and inducing T cell apoptosis. A wide-variety of tumors also upregulate Gal-3, which is associated with poor prognosis. Tumor-bearing (MCA-205 sarcoma, 4T1 mammary carcinoma, TRAMP-C1 prostate adenocarcinoma) mice were treated with a Gal-3 inhibitor (belapectin; GR-MD-02), aOX40, or combination therapy and the extent of tumor growth was determined. The phenotype and function of tumor-infiltrating lymphocytes was determined by flow cytometry, multiplex cytokine assay, and multiplex immunohistochemistry. Gal-3 inhibition synergized with aOX40 to promote tumor regression and increase survival. Specifically, aOX40/belapectin therapy significantly improved survival of tumor-bearing mice through a CD8+ T cell-dependent mechanism. Combination aOX40/belapectin therapy enhanced CD8+ T cell density within the tumor and reduced the frequency and proliferation of regulatory Foxp3+CD4+ T cells. Further, aOX40/belapectin therapy significantly reduced monocytic MDSC (M-MDSCs) and MHC-IIhi macrophage populations, both of which displayed reduced arginase 1 and increased iNOS. Combination aOX40/belapectin therapy alleviated M-MDSC-specific functional suppression compared to M-MDSCs isolated from untreated tumors. Our data suggests that Gal-3 inhibition plus aOX40 therapy reduces M-MDSC-meditated immune suppression thereby increasing CD8+ T cell recruitment leading to increased tumor regression and survival

Galectin-3 inhibition with belapectin combined with anti-OX40 therapy reprograms the tumor microenvironment to favor anti-tumor immunity

Treatment with an agonist anti-OX40 antibody (aOX40) boosts anti-tumor immunity by providing costimulation and driving effector T cell responses. However, tumor-induced immune suppression contributes significantly to poor response rates to aOX40 therapy, thus combining aOX40 with other agents that relieve tumor-mediated immune suppression may significantly improve outcomes. Once such target is galectin-3 (Gal-3), which drives tumor-induced immunosuppression by increasing macrophage infiltration and M2 polarization, restricting TCR signaling, and inducing T cell apoptosis. A wide-variety of tumors also upregulate Gal-3, which is associated with poor prognosis. Tumor-bearing (MCA-205 sarcoma, 4T1 mammary carcinoma, TRAMP-C1 prostate adenocarcinoma) mice were treated with a Gal-3 inhibitor (belapectin; GR-MD-02), aOX40, or combination therapy and the extent of tumor growth was determined. The phenotype and function of tumor-infiltrating lymphocytes was determined by flow cytometry, multiplex cytokine assay, and multiplex immunohistochemistry. Gal-3 inhibition synergized with aOX40 to promote tumor regression and increase survival. Specifically, aOX40/belapectin therapy significantly improved survival of tumor-bearing mice through a CD8+ T cell-dependent mechanism. Combination aOX40/belapectin therapy enhanced CD8+ T cell density within the tumor and reduced the frequency and proliferation of regulatory Foxp3+CD4+ T cells. Further, aOX40/belapectin therapy significantly reduced monocytic MDSC (M-MDSCs) and MHC-IIhi macrophage populations, both of which displayed reduced arginase 1 and increased iNOS. Combination aOX40/belapectin therapy alleviated M-MDSC-specific functional suppression compared to M-MDSCs isolated from untreated tumors. Our data suggests that Gal-3 inhibition plus aOX40 therapy reduces M-MDSC-meditated immune suppression thereby increasing CD8+ T cell recruitment leading to increased tumor regression and survival

NKTR-214 immunotherapy synergizes with radiotherapy to stimulate systemic CD8

BACKGROUND: High-dose radiotherapy (RT) is known to be immunogenic, but is rarely capable of driving clinically relevant abscopal antitumor immunity as monotherapy. RT is known to increase antigen presentation, type I/II interferon responses, and immune cell trafficking to irradiated tumors. Bempegaldesleukin (NKTR-214) is a CD122-preferential interleukin 2 (IL-2) pathway agonist that has been shown to increase tumor-infiltrating lymphocytes, T cell clonality, and increase PD-1 expression. NKTR-214 has increased drug half-life, decreased toxicity, and increased CD8 METHODS: Animals bearing bilateral subcutaneous MCA-205 fibrosarcoma or CT26 colorectal tumors were treated with NKTR-214, RT, or combination therapy, and tumor growth of irradiated and abscopal lesions was assessed. Focal RT was delivered using a small animal radiation research platform. Peripheral and tumor-infiltrating immune phenotype and functional analyses were performed by flow cytometry. RNA expression profiling from both irradiated and abscopal lesions was performed using microarray. RESULTS: We demonstrate synergy between RT of a single tumor and NKTR-214 systemic therapy resulting in dramatically increased cure rates of mice bearing bilateral tumors compared with RT or NKTR-214 therapy alone. Combination therapy resulted in increased magnitude and effector function of tumor-specific CD8 CONCLUSIONS: Given the increasing role of hypofractionated and stereotactic body RT as standard of care treatments in the management of locally advanced and metastatic cancer, these data have important implications for future clinical trial development. The combination of RT and NKTR-214 therapy potently stimulates systemic antitumor immunity and should be evaluated for the treatment of patients with locally advanced and metastatic solid tumors

Tumor radiation results in a myeloid contraction.

<p>a) i) Flow cytometry of fresh whole peripheral blood from naïve and 4T1 tumor-bearing BALB/c mice, showing CD11b⁺SSC^hi myeloid populations. ii) Gr1 and IA (MHC class II) staining on gated CD11b⁺SSC^hi myeloid populations from naïve and 4T1 tumor-bearing mice. b) i) Mean and standard error of leg diameter of BALB/c mice bearing 4T1 tumors left untreated (NT) or treated beginning on day 14 with 3 daily doses of 20 Gy focal radiation (RT). Myeloid cells/µl peripheral blood of ii) mice left untreated and iii) mice treated beginning on day 14 with 3 daily doses of 20 Gy focal radiation. iv) fitted curves from graphs ii) and iii) plotted without measurements. c) i) Clonogenic assays of lung metastases present at euthanasia in BALB/c mice bearing 4T1 tumors left untreated (empty circles – NT) or treated beginning on day 14 with 3 daily doses of 20 Gy focal radiation (filled circles – RT). ii) Myeloid cells/µl peripheral blood of naïve mice (Naïve) and mice day 17 following injection of 4T1 i.v. (i.v.) or s.c. (s.c.) iii) Total spleen cellularity in naïve mice (NT) and mice day 24 following injection of 4T1 i.v. (i.v.) or s.c. (s.c.) d) i) Myeloid cells/µl peripheral blood of C57BL/6 mice bearing Panc02 tumor harvested at different time points. ii) day 24 leg diameter and iii) myeloid cells/µl peripheral blood of C57BL/6 mice bearing Panc02 tumor left untreated (NT) or treated beginning on day 14 with 3 daily doses of 20 Gy focal radiation (RT). In each graph, each symbol represents one mouse. NS = Not significant; * = p<0.05; ** = p<0.01; *** = p<0.005; **** = p<0.001. Data represents multiple replicate experiments.</p

The splenic response to tumor radiation.

<p>a) Freshly excised spleens from d24 4T1 tumor-bearing BALB/c mice left untreated (NT) or treated beginning on day 14 with 3 daily doses of 20 Gy focal radiation (RT). Boxes shown are 3 cm wide. b) i) Total spleen cellularity in naïve mice (No tumor) and mice day 24 following injection of 4T1 left untreated (Tumor NT) or treated beginning on day 14 with 3 daily doses of 20 Gy focal radiation to the tumor (Tumor RT) or to the uninvolved opposite limb (Tumor Leg RT). ii) The number of CD11b⁺ cells per spleen in mice from the experiment shown in i). c) The number of CD11b⁺ cells in the spleen plotted against the number of CD11b⁺ cells/µl peripheral blood at harvest for each tumor and treatment group.</p

Myeloid subpopulations responding to tumor radiation.

<p>a) Flow cytometry of fresh whole peripheral blood from i) naïve mice or mice bearing 4T1 tumors ii) left untreated or iii) irradiated, showing Ly6C and Ly6G within gated CD11b⁺Gr1^hi myeloid populations. iv) Histograms of Ly6C expression in gated CD11b⁺Gr1^hiLy6G⁺ cells, including negative control staining (FMO) and showing the percentage Ly6C⁻ in mice irradiated in the tumor (Tumor RT) or the opposite limb (Tumor Leg RT). v) The percentage of CD11b⁺Gr1^hiLy6G⁺ cells that are Ly6C⁻ from naïve mice (No tumor) and mice day 24 following injection of 4T1 left untreated (Tumor NT) or treated beginning on day 14 with 3 daily doses of 20Gy focal radiation to the tumor (Tumor RT) or to the uninvolved opposite limb (Tumor Leg RT). Each symbol represents one mouse. b) Cytospins of sorted CD11b⁺Gr1^hi cells from untreated mice that are i) Ly6C⁺Ly6G⁻, ii) Ly6C⁺Ly6G⁺, or iii) Ly6C⁻Ly6G⁺. Each cytospin is shown next to the sort purity plot with increased magnification on the inset box. c) Wright-Giemsa stain of d24 blood smears from i) naïve mice (No tumor) and mice day 24 following injection of 4T1 ii) left untreated (Tumor NT) or iii) treated beginning on day 14 with 3 daily doses of 20 Gy focal radiation to the tumor (Tumor RT). The inset box is rotated and shown at increased magnification. NS = Not significant; * = p<0.05; ** = p<0.01; *** = p<0.005; **** = p<0.001. Data represents multiple replicated experiments; each subfigure includes data from a different replicate experiment.</p

Tumor cytokine and growth factor levels following radiation therapy.

<p>a) Bead assay for i) GM-CSF, ii) IL-1α and iii) IL-1β in homogenates of 4T1 tumors at day 24 following injection of 4T1 left untreated (NT) or treated beginning on day 14 with 3 daily doses of 20 Gy focal radiation to the tumor (RT) or to the uninvolved opposite limb (RT Opp). b) d24 following tumor challenge graphs show i) leg diameter ii) tumor weight and iii) splenocytes per spleen/tumor weight for mice left untreated (NT) or treated beginning on day 14 with 3 daily doses of 20 Gy focal radiation (RT). In each graph, each symbol represents one mouse. NS = Not significant; * = p<0.05; ** = p<0.01; *** = p<0.005; **** = p<0.001. Data is representative of three replicate experiments.</p