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Metastatic, hormone therapy-resistant breast cancers expressing estrogen receptor (ER+) account for most breast cancer deaths. A lack of tumor-infiltrating lymphocytes (TILs) and low tumor mutation burden render most ER+ breast cancers immunologically “cold” and unresponsive to immune checkpoint inhibitors (ICI). Radiation therapy (RT) can augment tumor immune susceptibility, but RT may also increase immunosuppressive cell recruitment to the tumor. Recent studies demonstrate that ER inhibition may antagonize the trafficking and activation of myeloid derived suppressive cells (MDSCs) in tumors. We hypothesized that combining RT and a selective ER degrader (SERD), fulvestrant, would cooperate to relieve immunosuppression, increase tumor immune susceptibility, and facilitate response to ICI in a syngeneic murine model of ER+ breast cancer. Using a cell line (TC11) developed in the Schuler laboratory, cells were transplanted to caudal mammary fat pads of female FVB/N mice. When tumors reached ~200mm3, mice were treated with combinations of vehicle, fulvestrant (250mg/kg sc, weekly), 8Gy RT on 3 consecutive days (8Gy x 3, days -1, 0, 1), and/or anti-PDL1 (200µg, IP, at days 3, 6, and 9). Age-matched controls that received identical treatments were included. Tumors from these mice were collected at day 28, and cytokines in tumor lysates were evaluated using a multiplex immunoassay. In a similar experiment, tumors were collected at day 10 for qPCR and flow cytometry analysis. Therapeutic intervention demonstrated that fulvestrant with RT slowed tumor growth compared to either treatment alone. 8Gy RT elicited a type-1 interferon response in TC11 tumors as measured by upregulated expression of Mx1, Pdl1, Oas2, Oas3, Trex1, and IfnB mRNAs peaking at 3-5 days post RT and persisting to day 10. Cytokine analysis from tumor lysate revealed significantly increased IP-10 in tumors from mice treated with both RT and fulvestrant compared to controls, and similar trends were seen for IL6, VEGF, and MCSF. With the addition of RT and fulvestrant to anti-PDL1, IL2, IL3, IL6, IL7, IL17, and IP-10 were significantly increased compared to anti-PDL1 alone. At day 28 RT and fulvestrant together primed a greater response to anti-PDL1. Combined RT and fulvestrant may reduce immunosuppression compared to RT alone and render this immunologically cold tumor model more responsive to anti-PDL1 blockade. RT increases tumor cell immune susceptibility by activation of a type I IFN response and combining fulvestrant with RT does not affect this IFN1 response. These results demonstrate a cooperative interaction between RT and fulvestrant in favorably modulating the TME and response to ICIs in an immunologically cold, hormone-therapy resistant, murine ER+ breast cancer model. Further experiments are needed to explore the mechanisms of these immune effects. Citation Format: Erin J. Nystuen, Amber M. Bates, Kathleen A. O'Leary, Sarah E. Emma, Elizabeth G. Sumiec, Linda A. Schuler, Zachary S. Morris. Fulvestrant and radiation modify the tumor immune microenvironment in ER+ metastatic breast cancer and cooperate to enhance response to anti-PDL1 checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 676.

We have observed in preclinical studies that the delivery of low dose targeted radionuclide therapy (TRT) therapy to sites of primary and metastatic cancer can improve the anti-tumor immune response to immune checkpoint inhibition (ICI) with anti-CTLA4 or anti-PDL1. NM600, an alkylphosphocholine that can be radiolabeled with 90Y, is taken up and retained in most cancer cells following intravenous injection. Bempegaldesleukin (BEMPEG) is a first in class, CD122-preferential IL2 pathway agonist that can selectively stimulate an immune response. We hypothesized that combining low dose TRT with BEMPEG would increase immune activation to enhance the response to ICIs in immunologically “cold” murine cancer models. C57Bl/6 female mice were subcutaneously engrafted in the flank with the murine head and neck squamous cell carcinoma (HNSCC) cell line, MOC2. In vivo dosimetry performed using the Monte Carlo based RAPID platform following serial 86Y-NM600 PET/CT imaging demonstrated that 100 µCi of 90Y-NM600 would deliver ~8 Gy to the MOC2 tumor. Mice bearing MOC2 tumors (mean volume ~100 mm3) received combinations of 90Y-NM600 (100 µCi, day 1 IV), BEMPEG (16 µg, days 6, 15, and 24 IV), and anti-CTLA4 (200 µg, days 4, 7, and 10 IP) using a 2x2x2 study design. Tumor growth and survival were monitored. Blood was collected weekly and analyzed on the Abaxis VetScan HM5 to evaluate for toxicity. In a parallel 2x2x2 survival study, an orthotopic model generated by engrafting MOC2 tumors in the cheek was used, and mice were treated with combinations of BEMPEG, 90Y-NM600, and anti-PDL1 (200 µg, days 4, 7, and 10, IP). Mice were euthanized when >25% weight loss was observed. Similar experiments using 90Y-NM600 and BEMPEG were performed in additional syngeneic mouse cancer models including SCC7 (HNSCC), 4T1 (breast), LLC (lung), and Panc02 (pancreatic). In the immunologically “cold” MOC2 HNSCC model, 62.5% of mice treated with 90Y-NM600 (TRT), BEMPEG, and anti-CTLA4; and 44.4% of mice treated with 90Y-NM600, BEMPEG, and anti-PDL1 experienced complete tumor response with no observable primary or metastatic disease at day 60. In mice treated with 90Y-NM600 and BEMPEG, tumor regression followed by escape was seen without ICI. By day 60, no mice receiving single or dual treatment combinations exhibited a complete tumor response. Comprehensive whole blood analysis did not show any major hematologic toxicities. This treatment was explored in mice bearing SCC7, 4T1, Panc02, and LLC tumors, and similar trends were seen. Combination of 90Y-NM600, BEMPEG, and ICI displays robust anti-tumor activity that prevents metastatic disease progression and prolongs survival in spontaneously metastatic, immunologically “cold” tumor models. Clinical studies are warranted to test the safety and efficacy of this promising combined modality treatment regimen. Citation Format: Elizabeth G. Sumiec, Amber M. Bates, Reinier Hernandez, Joseph J. Grudzinski, Ian R. Marsh, Sarah E. Emma, Erin J. Nystuen, Justin C. Jagodinsky, Alexander A. Pieper, Gustavo A. Sosa, Bryan P. Bednarz, Ravi B. Patel, Jamey Weichert, Zachary S. Morris. In vivo synergy of 90Y-NM600 and Bempegaldesleukin improves anti-tumor efficacy of immune checkpoint inhibitors in syngeneic murine cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 679.

The purpose of this study was to evaluate mechanisms of cooperative therapeutic effects of bempegaldesleukin (BEMPEG; NKTR-214) and 90Y-NM600 in head and neck squamous cell carcinoma (HNSCC). BEMPEG is a first in class, CD122-preferential interleukin-2 (IL2) pathway agonist tailored to stimulate antitumor immunity through promoting activation and proliferation of CD8+ T and NK cells. We have observed that targeted radionuclide therapy (TRT) delivered at low doses to sites of metastatic cancer can enhance immune susceptibility in immunologically “cold” tumors such as the MOC2 syngeneic mouse HNSCC model. NM600 is an alkylphosphocholine analog that is selectively sequestered and retained by most human and murine cancer cells. We hypothesized that combining BEMPEG and 90Y-NM600 would cooperate to increase immunosusceptibility and immune cell tumor infiltration and create an overall more favorable immune microenvironment in MOC2 HNSCC tumors. MOC2 tumors were engrafted in the flank of C57BL/6 female mice. Once tumors reached ~100 mm³, mice were randomized into one of eight groups for a survival study utilizing varying combinations of BEMPEG (16 µg, days 6, 15, 24 IV), 90Y-NM600 (100 µCi, day 1 IV), and anti-CTLA4 (200 µg, days 4, 7, 10 IP). In vivo dosimetry was performed prior to day 1 using the Monte Carlo based RAPID platform. Serial 86Y-NM600 PET/CT imaging indicated the dose delivered to the tumor was ~8 Gy. Cohorts of mice in a parallel study were treated with PBS (control), BEMPEG, 90Y-NM600, or BEMPEG and 90Y-NM600. Flow cytometry, qPCR analysis, multiplex cytokine analyses were used to evaluate tumors collected at day 14. In this immunologically “cold” murine HNSCC model, a complete tumor response was observed in 62.5% of mice treated with the triple combination therapy of BEMPEG, 90Y-NM600, and anti-CTLA4. Compared to single therapy groups, mice treated with BEMPEG and 90Y-NM600 had increased CD8+ T cell tumor infiltrate, and 90Y-NM600 induced increased expression of the IL2βγ receptor, CD122, on the surface of CD8 T cells. Tumors from mice treated with BEMPEG and 90Y-NM600 had increased expression of genes associated with tumor cell immune susceptibility (Pdl1, Mhc1, Fas), a type 1 interferon response (Mx1, Trex1, Oas2, Oas3), tumor immune cell recruitment (MIP1α , Tnfα, Mac1, Csf1, Cxcl11) activation of cytotoxic T lymphocytes (Ifny, Cxcl10), and production of immune stimulatory cytokines (IFNy, IL3, IL4, IL5, IL17, RANTES, TNFα, MIP1β, MIP1α, CXCL10, CXCL9). These results suggest a synergistic interaction between BEMPEG and 90Y-NM600 that improves the immune microenvironment in this difficult to treat murine model of HNSCC, which may thereby enhance the response to immune checkpoint blockade. Further studies are warranted to examine the therapeutic implications of this combination in patients. Citation Format: Sarah E. Emma, Amber M. Bates, Reinier Hernandez, Joseph J. Grudzinski, Ian R. Marsh, Justin C. Jagodinsky, Bryan P. Bednarz, Alexander A. Pieper, Elizabeth G. Sumiec, Erin J. Nystuen, Gustavo A. Sosa, Ravi B. Patel, Jamey Weichert, Zachary S. Morris. Mechanisms of cooperative response to bempegaldesleukin (BEMPEG) and 90Y-NM600 targeted radionuclide therapy in the treatment of a syngeneic murine model of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 508.

BackgroundSome patients with cancer treated with anticancer monoclonal antibodies (mAbs) develop antidrug antibodies (ADAs) that recognize and bind the therapeutic antibody. This response may neutralize the therapeutic mAb, interfere with mAb effector function or cause toxicities. We investigated the potential influence of ADA to modify the tumor-binding capability of a tumor-reactive ‘immunocytokine’ (IC), namely, a fusion protein (hu14.18-IL2) consisting of a humanized, tumor-reactive, anti-GD2 mAb genetically linked to interleukin 2. We characterize the role of treatment delivery of IC (intravenous vs intratumoral) on the impact of ADA on therapeutic outcome following IC treatments in an established antimelanoma (MEL) regimen involving radiotherapy (RT) +IC.MethodsC57BL/6 mice were injected with human IgG or the hu14.18-IL2 IC to develop a mouse anti-human antibody (MAHA) response (MAHA+). In vitro assays were performed to assess ADA binding to IC using sera from MAHA+ and MAHA− mice. In vivo experiments assessed the levels of IC bound to tumor in MAHA+ and MAHA− mice, and the influence of IC route of delivery on its ability to bind to B78 (GD2+) MEL tumors.ResultsMAHA is inducible in C57BL/6 mice. In vitro assays show that MAHA is capable of inhibiting the binding of IC to GD2 antigen on B78 cells, resulting in impaired ADCC mediated by IC. When B78-bearing mice are injected intravenously with IC, less IC binds to B78-MEL tumors in MAHA+ mice than in MAHA− mice. In contrast, when IC is injected intratumorally in tumor-bearing mice, the presence of MAHA does not detectibly impact IC binding to the tumor. Combination therapy with RT+IT-IC showed improved tumor regression compared with RT alone in MAHA+ mice. If given intratumorally, IC could be safely readministered in tumor-bearing MAHA+ mice, while intravenous injections of IC in MAHA+ mice caused severe toxicity. Histamine levels were elevated in MAHA+ mice compared with MAHA− mice after reintroduction of IC.ConclusionsIntratumoral injection may be a means of overcoming ADA neutralization of therapeutic activity of tumor-reactive mAbs or ICs and may reduce systemic toxicity, which could have significant translational relevance.

We have recently demonstrated the effectiveness of combining external beam radiation therapy (RT), intratumoral immunocytokine (IT-IC), and anti-CTLA4 (αCTLA4) in eliminating large (500mm3) B78 melanomas (MEL), prolonging survival and invoking T cell immune memory in mice. This highly translational therapy is currently being investigated in a phase I clinical trial. Despite the success of this regimen against large single subcutaneous MEL tumors, many mice with large tumors and intravenously (IV) injected metastatic MEL, receiving this same regimen, still die from metastatic disease. A recent report has shown that fusokine GIFT4, a recombinant chimera of GMCSF and IL4 programs B cells (GIFT4BC) to acquire a unique phenotype with augmented capacity for antigen presentation and anti-MEL activity. Herein, we report the preliminary results of studies combining RT+IC+αCTLA4 with intratumoral (IT) GIFT4BCs therapy. We hypothesized that GIFT4BCs enhance the anti-tumor and anti-metastatic efficacy of RT+IC+αCTLA4. C57BL/6 mice were engrafted with 2 × 106 B78 MEL intradermally in the flank. At a tumor volume of ~250mm3, mice were intravenously injected with 3.5 × 105 B16 MEL (a metastatic parental line of B78 MEL) to induce metastases. Mice were treated with 12 Gy RT on day 1 (D1), 1 × 107 IT- GIFT4BC on D4, IC (IT, 50ug) on D6-D10, and αCTLA4 (ip, 200ug) on D3, 6, and 9 (designated RT+IC+αCTLA4+GIFT4BC). Control groups received no treatment, GIFT4BCs alone, or RT+IC+αCTLA4. Tumor growth and survival were monitored. At the time of euthanasia for tumor burden or illness, necropsy was performed to quantify metastases. A pre-determined cohort of mice from each treatment group were euthanized on D12. From these, tumor-draining lymph nodes were fixed for histology and tumors were harvested for qPCR, histology, and flow cytometry. Preliminary results indicate that the addition of GIFT4BCs to RT+IC+αCTLA4 improves survival. Lungs from treated mice showed reduced metastatic burden in RT+IC+αCTLA4+GIFT4BC mice compared to controls. GIFT4BCs were identified in the tumor-draining lymph nodes using confocal microscopy in RT+IC+αCTLA4+GIFT4BC mice. qPCR analysis of tumor samples revealed an upregulation of interferon gamma (Ifny) in RT+IC+αCTLA4+GIFT4BC mice compared to control groups. Flow cytometry analysis show increased CD45+ tumor infiltrating lymphocytes in RT+IC+αCTLA4+GIFT4BC compared to RT+IC+αCTLA4 groups. Combination of RT+IC+αCTLA4+GIFT4BC displays anti-MEL activity that reduces metastatic burden and prolongs survival in murine MEL models. These preliminary findings support published hypotheses which postulate that GIFT4BC-mediated mechanisms may augment the T cell response through B cell antigen presentation and chemotactic cytokine production in vivo. Future studies will elucidate the mechanisms of anti-tumor efficacy of RT+IC+αCTLA4+GIFT4BC. Citation Format: Elizabeth G. Sumiec, Claire C. Baniel, Amber M. Bates, Sarah Emma, Erin Nystuen, Alexander L. Rakhmilevich, Amy K. Erbe, Pradyut Paul, Jacques Galipeau, Paul M. Sondel, Zachary S. Morris. Augmentation of anti-melanoma response by combining radiation-based in situ vaccination with GIFT4 B cell therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 902.

Metastatic, hormone therapy-resistant breast cancers expressing estrogen receptor (ER+) account for the majority of breast cancer deaths. ER+ breast cancers are immunologically “cold” with low tumor mutation burden and few tumor-infiltrating lymphocytes (TILs). Consequently, application of immune checkpoint inhibitors (ICIs) to ER+ breast cancers has not proven effective. Radiation therapy (RT) can increase immune susceptibility of “cold” tumors, but can also increase recruitment of immunosuppressive myeloid-derived suppressor cells (MDSCs) to the radiated tumor microenvironment (TME). Recent studies demonstrate that ER inhibition may antagonize the trafficking and activation of MDSCs in tumors. We hypothesized that combining RT and a selective ER degrader (SERD), fulvestrant, would cooperate to relieve immunosuppression, increase tumor immune susceptibility, and facilitate response to ICIs in a syngeneic murine model of ER+ breast cancer. Using a murine breast cancer model of ER+ breast cancer developed (TC11), we examined effects in vitro and in vivo. In vitro, TC11 cells were treated with 8Gy RT and/or fulvestrant (1uM) to evaluate cell autonomous effects. In vivo, 25,000 TC11 cells were transplanted to caudal mammary fat pads of female FVB/N mice. When tumors reached ~200mm3, mice were randomized and treated. We initially evaluated the time course of immune responses to 8Gy RT in the TC11 TME, euthanizing mice every 5 days up to 20 days after RT. We subsequently treated mice with vehicle, fulvestrant (250mg/kg sc, weekly), 8Gy RT, and fulvestrant+8Gy RT. Flow cytometry was performed on day 5 and day 10 after RT to examine the innate and adaptive immune cell infiltrates, respectively. A therapeutic intervention study was performed using combinations of fulvestrant, 8Gy RT, and/or anti-PDL1 (200µg, ip, at days 3, 6, and 9 after RT). 8Gy RT elicited a type-1 interferon response in TC11 cells in vitro and in vivo; as measured by upregulated expression of Mx1, Pdl1, Oas2, Oas3, Trex1, and IfnB mRNAs peaking at 3-5 days post RT and persisting to day 10 in TC11 tumors. RT increased MDSCs (Ly6C+Ly6G+) in the TC11 TME compared to vehicle or fulvestrant alone in tumors taken at day 5, but MDSCs were reduced in tumors treated with fulvestrant and RT. Together, fulvestrant and RT increased TILs (CD45+) and CD8+ T cells at day 10 compared to vehicle control and single treatments. Preliminary data indicate RT and fulvestrant together primed a greater response to anti-PDL1 ICI compared to single or dual combinations of these treatments. These results demonstrate a cooperative interaction between RT and fulvestrant in favorably modulating the TME and response to ICIs in an immunologically cold, hormone-therapy resistant, murine ER+ breast cancer model. Further study is warranted to determine whether this combination may elicit a more effective in situ vaccine effect compared to RT alone. Citation Format: Amber M. Bates, Kathleen A. O'Leary, Sarah Emma, Erin Nystuen, Elizabeth G. Sumiec, Linda A. Schuler, Zachary S. Morris. Enhancing immunogenicity in immunologically cold ER+ breast cancer using estrogen receptor blockade and radiation therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2255.

In preclinical studies, we have demonstrated that delivering low dose radiation therapy to all sites of metastatic cancer using targeted radionuclide therapy (TRT) can improve the response to immune checkpoint blockade. NM600 is a tumor-targeting alkylphosphocholine radiolabeled with 90Y. Following intravenous administration, NM600 is selectively taken up and retained in most murine and human cancer cells. Bempegaldesleukin (NKTR-214) is a first in class, CD122-preferential interleukin-2 (IL2) pathway agonist being studied for its ability to influence the IL2 pathway and selectively stimulate an immune response. The primary objective of this study was to test the hypothesis that NKTR-214 and 90Y-NM600 would increase the response to immune checkpoint blockade in the immunologically “cold” MOC2 syngeneic mouse model of head and neck squamous cell carcinoma (HNSCC). C57BL/6 female mice were engrafted with MOC2, a murine HNSCC cell line, in the right flank. When mean tumor volume reached ~100mm3, mice were randomized into eight treatment groups using a 2 × 2 × 2 study design for combinations of NKTR-214, 90Y-NM600, and anti-CTLA4. 100 µCi 90Y-NM600 was administered intravenously (IV, treatment day 1). Prior in vivo dosimetry performed using the Monte Carlo based RAPID platform following serial 86Y-NM600 PET/CT imaging demonstrated that this activity delivered ~8Gy to the MOC2 tumor. 200 µg anti-CTLA4 was delivered by intraperitoneal injection on days 4, 7, and 10. 16 µg NKTR-214 was given IV on days 6, 15, and 24. Tumor growth was monitored. In a parallel study, cohorts of mice were treated with PBS (control), NKTR-214, 90Y-NM600, or NKTR-214 + 90Y-NM600, and tumors were collected at day 14 for flow cytometry analysis. In the spontaneously metastatic, immunologically “cold” MOC2 HNSCC tumor model, 62.5% of mice treated with the combination of 90Y-NM600, NKTR-214, and anti-CTLA4 experienced complete tumor response, and these mice showed no observable primary or metastatic disease 60 days after treatment initiation. No mice receiving single or dual therapy combinations exhibited complete tumor response (p = Citation Format: Gustavo A. Sosa, Amber M. Bates, Ravi Patel, Reinier Hernandez, Joseph J. Grudzinski, Ian Marsh, Bryan Bednarz, Alexander Pieper, Erin Nystuen, Sarah Emma, Elizabeth G. Sumiec, Jamey P. Weichert, Zachary S. Morris. In vivo efficacy of bempegaldesleukin, immune checkpoint inhibition, and targeted radionuclide therapy in immunocompetent murine model of head and neck cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 903.