Search

Histotripsy is an emerging noninvasive, nonionizing and nonthermal focal cancer therapy that is highly precise and can create a treatment zone of virtually any size and shape. Current histotripsy systems rely on ultrasound imaging to target lesions. However, deep or isoechoic targets obstructed by bowel gas or bone can often not be treated safely using ultrasound imaging alone. This work presents an alternative x-ray C-arm based targeting approach and a fully automated robotic targeting system.The approach uses conventional cone beam CT (CBCT) images to localize the target lesion and 2D fluoroscopy to determine the 3D position and orientation of the histotripsy transducer relative to the C-arm. The proposed pose estimation uses a digital model and deep learning-based feature segmentation to estimate the transducer focal point relative to the CBCT coordinate system. Additionally, the integrated robotic arm was calibrated to the C-arm by estimating the transducer pose for four preprogrammed transducer orientations and positions. The calibrated system can then automatically position the transducer such that the focal point aligns with any target selected in a CBCT image.The accuracy of the proposed targeting approach was evaluated in phantom studies, where the selected target location was compared to the center of the spherical ablation zones in post-treatment CBCTs. The mean and standard deviation of the Euclidean distance was 1:4 _ 0:5 mm. The mean absolute error of the predicted treatment radius was 0:5 _ 0:5 mm.CBCT-based histotripsy targeting enables accurate and fully automated treatment without ultrasound guidance.The proposed approach could considerably decrease operator dependency and enable treatment of tumors not visible under ultrasound.

Background The in-situ vaccine using CpG oligodeoxynucleotide combined with OX40 agonist antibody (CpG + OX40) has been shown to be an effective therapy activating an anti-tumor T cell response in certain settings. The roles of tumor volume, tumor model, and the addition of checkpoint blockade in the efficacy of CpG + OX40 in-situ vaccination remains unknown. Methods Mice bearing flank tumors (B78 melanoma or A20 lymphoma) were treated with combinations of CpG, OX40, and anti-CTLA-4. Tumor growth and survival were monitored. In vivo T cell depletion, tumor cell phenotype, and tumor infiltrating lymphocyte (TIL) studies were performed. Tumor cell sensitivity to CpG and macrophages were evaluated in vitro. Results As tumor volumes increased in the B78 (one-tumor) and A20 (one-tumor or two-tumor) models, the anti-tumor efficacy of the in-situ vaccine decreased. In vitro, CpG had a direct effect on A20 proliferation and phenotype and an indirect effect on B78 proliferation via macrophage activation. As A20 tumors progressed in vivo, tumor cell phenotype changed, and T cells became more involved in the local CpG + OX40 mediated anti-tumor response. In mice with larger tumors that were poorly responsive to CpG + OX40, the addition of anti-CTLA-4 enhanced the anti-tumor efficacy in the A20 but not B78 models. Conclusions Increased tumor volume negatively impacts the anti-tumor capability of CpG + OX40 in-situ vaccine. The addition of checkpoint blockade augmented the efficacy of CpG + OX40 in the A20 but not B78 model. These results highlight the importance of considering multiple preclinical model conditions when assessing the efficacy of cancer immunotherapy regimens and their translation to clinical testing.

BackgroundThe antitumor effects of external beam radiation therapy (EBRT) are mediated, in part, by an immune response. We have reported that a single fraction of 12 Gy EBRT combined with intratumoral anti-GD2 hu14.18-IL2 immunocytokine (IC) generates an effective in situ vaccine (ISV) against GD2-positive murine tumors. This ISV is effective in eradicating single tumors with sustained immune memory; however, it does not generate an adequate abscopal response against macroscopic distant tumors. Given the immune-stimulatory capacity of radiation therapy (RT), we hypothesized that delivering RT toallsites of disease would augment systemic antitumor responses to ISV.MethodsWe used a syngeneic B78 murine melanoma model consisting of a ‘primary’ flank tumor and a contralateral smaller ‘secondary’ flank tumor, treated with 12 Gy EBRT and intratumoral IC immunotherapy to the primary and additional EBRT to the secondary tumor. As a means of delivering RT to all sites of disease, both known and occult, we also used a novel alkylphosphocholine analog, NM600, conjugated to90Y as a targeted radionuclide therapy (TRT). Tumor growth, overall survival, and cause of death were measured. Flow cytometry was used to evaluate immune population changes in both tumors.ResultsAbscopal effects of local ISV were amplified by delivering as little as 2–6 Gy of EBRT to the secondary tumor. When the primary tumor ISV regimen was delivered in mice receiving 12 Gy EBRT to the secondary tumor, we observed improved overall survival and more disease-free mice with immune memory compared with either ISV or 12 Gy EBRT alone. Similarly, TRT combined with ISV resulted in improved overall survival and a trend towards reduced tumor growth rates when compared with either treatment alone. Using flow cytometry, we identified an influx of CD8+T cells with a less exhausted phenotype in both the ISV-targeted primary and the distant secondary tumor following the combination of secondary tumor EBRT or TRT with primary tumor ISV.ConclusionsWe report a novel use for low-dose RT, not as a direct antitumor modality but as an immunomodulator capable of driving and expanding antitumor immunity against metastatic tumor sites following ISV.

IntroductionCombining CpG oligodeoxynucleotides with anti-OX40 agonist antibody (CpG+OX40) is able to generate an effective in situ vaccine in some tumor models, including the A20 lymphoma model. Immunologically “cold” tumors, which are typically less responsive to immunotherapy, are characterized by few tumor infiltrating lymphocytes (TILs), low mutation burden, and limited neoantigen expression. Radiation therapy (RT) can change the tumor microenvironment (TME) of an immunologically “cold” tumor. This study investigated the effect of combining RT with the in situ vaccine CpG+OX40 in immunologically “cold” tumor models.MethodsMice bearing flank tumors (A20 lymphoma, B78 melanoma or 4T1 breast cancer) were treated with combinations of local RT, CpG, and/or OX40, and response to treatment was monitored. Flow cytometry and quantitative polymerase chain reaction (qPCR) experiments were conducted to study differences in the TME, secondary lymphoid organs, and immune activation after treatment.ResultsAn in situ vaccine regimen of CpG+OX40, which was effective in the A20 model, did not significantly improve tumor response or survival in the “cold” B78 and 4T1 models, as tested here. In both models, treatment with RT prior to CpG+OX40 enabled a local response to this in situ vaccine, significantly improving the anti-tumor response and survival compared to RT alone or CpG+OX40 alone. RT increased OX40 expression on tumor infiltrating CD4+ non-regulatory T cells. RT+CpG+OX40 increased the ratio of tumor-infiltrating effector T cells to T regulatory cells and significantly increased CD4+ and CD8+ T cell activation in the tumor draining lymph node (TDLN) and spleen.ConclusionRT significantly improves the local anti-tumor effect of the in situ vaccine CpG+OX40 in immunologically “cold”, solid, murine tumor models where RT or CpG+OX40 alone fail to stimulate tumor regression.

BackgroundWe, and others, have previously shown that the in-situ vaccine of hypomethylated CG-enriched oligodeoxynucleotide (CpG) with agonist anti-OX40 antibody (OX40) is effective at curing mice in the A20 lymphoma model [1–4]. In separate preclinical models where CpG+OX40 fails to cause tumor regression, radiation therapy (RT) prior to the in-situ vaccine enhances the anti-tumor effect of CpG+OX40 [4]. We investigated the immune response, and specifically the activity of T cells, following treatment with RT+CpG+OX40 in the B78 melanoma model where CpG+OX40 typically fails to cause tumor regression.MethodsC57BL/6 mice were inoculated with 2x106 B78 melanoma cells on the right flank and allowed to grow until the average tumor size was ~150mm3. In two independent experiments, mice were randomized (n=4–5 per group per experiment) and treated with one of the following: 1) PBS, 2) CpG+OX40, 3) RT, 4) RT+CpG+OX40. 12 Gy external beam RT was dosed to the flank tumor on day 0 and intratumoral CpG (50µg)+OX40 (20 µg) were given on days 5, 7, and 9 after RT. Spleens and tumor draining lymph nodes (TDLNs) were harvested on day 12. T cell activation and proliferation were assessed via flow cytometry.ResultsCompared to all other groups in the study, mice treated with RT+CpG+OX40 demonstrated significantly elevated levels of CD4+ and CD8+ T cell activation in the TDLNs, as measured by interferon gamma expression. Similar trends of CD4+ and CD8+ T cell activation were measured in the spleens. Splenic CD8+ T cells from RT+CpG+OX40 treated mice demonstrated significantly elevated levels of proliferation over PBS and RT, as measured by Ki67.ConclusionsIn B78 melanoma, a weakly immunologic tumor model, combining RT with the in-situ vaccine CpG+OX40 enhances the activity of T cells, evidenced by significantly increased CD4+ and CD8+ T cell activation in the TDLN and spleen and elevated CD8+ T cell proliferation in the spleen.ReferencesHouot, R. and Levy, R. T-cell modulation combined with intratumoral CpG cures lymphoma in a mouse model without the need for chemotherapy. Blood, 2009. 113(15):3546–52.Marabelle, A., et al. Depleting tumor-specific Tregs at a single site eradicates disseminated tumors. J Clin Invest, 2013. 123(6):2447–63.Sagiv-Barfi, I., et al. Eradication of spontaneous malignancy by local immunotherapy. Sci Transl Med, 2018. 10(426).Zangl, LM. Et al. External Beam Radiotherapy Required for Tumor Regression When Using CpG-Oligodeoxynucleotide and Anti-OX40 in an Immunologically Cold Tumor Model. Red Journal. 2019. 105:S88.

Introduction Combining CpG oligodeoxynucleotides with anti-OX40 agonist antibody (CpG+OX40) is able to generate an effective in situ vaccine in some tumor models, including the A20 lymphoma model. Immunologically “cold” tumors, which are typically less responsive to immunotherapy, are characterized by few tumor infiltrating lymphocytes (TILs), low mutation burden, and limited neoantigen expression. Radiation therapy (RT) can change the tumor microenvironment (TME) of an immunologically “cold” tumor. This study investigated the effect of combining RT with the in situ vaccine CpG+OX40 in immunologically “cold” tumor models. Methods Mice bearing flank tumors (A20 lymphoma, B78 melanoma or 4T1 breast cancer) were treated with combinations of local RT, CpG, and/or OX40, and response to treatment was monitored. Flow cytometry and quantitative polymerase chain reaction (qPCR) experiments were conducted to study differences in the TME, secondary lymphoid organs, and immune activation after treatment. Results An in situ vaccine regimen of CpG+OX40, which was effective in the A20 model, did not significantly improve tumor response or survival in the “cold” B78 and 4T1 models, as tested here. In both models, treatment with RT prior to CpG+OX40 enabled a local response to this in situ vaccine, significantly improving the anti-tumor response and survival compared to RT alone or CpG+OX40 alone. RT increased OX40 expression on tumor infiltrating CD4+ non-regulatory T cells. RT+CpG+OX40 increased the ratio of tumor-infiltrating effector T cells to T regulatory cells and significantly increased CD4+ and CD8+ T cell activation in the tumor draining lymph node (TDLN) and spleen. Conclusion RT significantly improves the local anti-tumor effect of the in situ vaccine CpG+OX40 in immunologically “cold”, solid, murine tumor models where RT or CpG+OX40 alone fail to stimulate tumor regression.

BackgroundCurrent clinical trials are using radiation therapy (RT) to enhance an antitumor response elicited by high-dose interleukin (IL)-2 therapy or immune checkpoint blockade (ICB). Bempegaldesleukin (BEMPEG) is an investigational CD122-preferential IL-2 pathway agonist with prolonged in vivo half-life and preferential intratumoral expansion of T effector cells over T regulatory cells. BEMPEG has shown encouraging safety and efficacy in clinical trials when used in combination with PD-1 checkpoint blockade. In this study, we investigated the antitumor effect of local RT combined with BEMPEG in multiple immunologically ‘cold’ tumor models. Additionally, we asked if ICB could further enhance the local and distant antitumor effect of RT+BEMPEG in the setting of advanced solid tumors or metastatic disease.MethodsMice bearing flank tumors (B78 melanoma, 4T1 breast cancer, or MOC2 head and neck squamous cell carcinoma) were treated with combinations of RT and immunotherapy (including BEMPEG, high-dose IL-2, anti(α)-CTLA-4, and α-PD-L1). Mice bearing B78 flank tumors were injected intravenously with B16 melanoma cells to mimic metastatic disease and were subsequently treated with RT and/or immunotherapy. Tumor growth and survival were monitored. Peripheral T cells and tumor-infiltrating lymphocytes were assessed via flow cytometry.ResultsA cooperative antitumor effect was observed in all models when RT was combined with BEMPEG, and RT increased IL-2 receptor expression on peripheral T cells. This cooperative interaction was associated with increased IL-2 receptor expression on peripheral T cells following RT. In the B78 melanoma model, RT+BEMPEG resulted in complete tumor regression in the majority of mice with a single ~400 mm3tumor. This antitumor response was T-cell dependent and supported by long-lasting immune memory. Adding ICB to RT+BEMPEG strengthened the antitumor response and cured the majority of mice with a single ~1000 mm3B78 tumor. In models with disseminated metastasis (B78 primary with B16 metastasis, 4T1, and MOC2), the triple combination of RT, BEMPEG, and ICB significantly improved primary tumor response and survival.ConclusionThe combination of local RT, BEMPEG, and ICB cured mice with advanced, immunologically cold tumors and distant metastasis in a T cell-dependent manner, suggesting this triple combination warrants clinical testing.

In a syngeneic murine melanoma (MEL) model, we recently reported an in situ vaccination response to combined radiation (RT) and intra-tumoral (IT) injection of anti-GD2 hu14. 18-IL2 immunocytokine (IC). This combined treatment resulted in 71% complete and durable regression of 5-week tumors, a tumor-specific memory T cell response, and augmented response to systemic anti-CTLA-4 antibody checkpoint blockade. While the ability of radiation to diversify anti-tumor T cell response has been reported, we hypothesize that mice rendered disease-free (DF) by a RT-based ISV might also exhibit a heightened B cell response. C57BL/6 mice were engrafted with 2 × 106 GD2+ B78 MEL and treated at a target tumor size of ~200 mm3 with 12 Gy RT, IT-IC on day (D)6-D10, and anti-CTLA-4 on D3, 6, and 9. Serum was collected via facial vein before tumor injection, before treatment, during treatment, after becoming DF, and following rejection of subcutaneous 2 × 106 B78 MEL re-challenge on D90. Flow cytometry demonstrated the presence of tumor-specific IgG in sera from mice rendered DF and rejecting re-challenge with B78 MEL at D90 after starting treatment. Consistent with an adaptive endogenous anti-tumor humoral memory response, these anti-tumor antibodies bound to B78 cells and parental B16 cells (GD2-), but not to the unrelated syngeneic Panc02 or Panc02 GD2+ cell lines. We evaluated the kinetics of this response and observed that tumor-specific IgG was consistently detected by D22 after initiation of treatment, corresponding to a time of rapid tumor regression. The amount of tumor-specific antibody binding to tumor cells (as measured by flow MFI) did not correlate with host animal prognosis. Incubation of B16 MEL cells in DF serum, vs. naïve serum, prior to IV injection, did not delay engraftment of B16 metastases and showed similar overall survival rates. B cell depletion using anti-CD20 or anti-CD19 and anti-B220 did not impact the efficacy of ISV treatment. Thus, treatment with RT + IC + anti-CTLA-4 results in adaptive anti-tumor humoral memory response. This endogenous tumor-specific antibody response does not appear to have therapeutic efficacy but may serve as a biomarker for an anti-tumor T cell response.

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.

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.

Brain metastases develop in many patients with advanced metastatic cancers, and this challenging clinical scenario demands improved treatment approaches. We previously observed that an in situ vaccination (ISV) regimen combining local radiation (RT) and intratumoral (IT) injection of the radiated tumor site with interleukin-2 (IL2) resulted in enhanced response to immune checkpoint inhibition (ICI: αCTLA4 Ab) in immunologically cold murine models of melanoma. This ISV treatment resulted in complete response (CR) at both the ISV-targeted tumor and at distant extracranial tumor sites in most mice. However, in a model of melanoma brain metastases, this same ISV provided only a modest improvement in survival with all mice succumbing to progressive tumor burden in the brain (Sriramaneni et al., 2020 AACR submitted abstract). Bempegaldesleukin (NKTR-214, Nektar Therapeutics) is a PEGylated derivative of IL2 that can be administered systemically with minimal toxicity, has a longer half-life (~20 hours), and preferentially activates cytotoxic (CD8+) T cells and NK cells over regulatory T-cells within the tumor microenvironment. We hypothesized that these qualities of NKTR-214 could enhance immune response and efficacy of ISV against brain melanoma metastases, and therefore we tested in combination with local RT targeting an extracranial tumor site. Immunologically cold B78 melanoma tumor cells were implanted into the right flank and the right striatum of the brain in syngeneic C57BL/6 mice. Right flank tumors (150-200 mm3) were irradiated (12 Gy) on treatment day 1. NKTR-214 was administered intravenously on treatment days 6, 15, 24. NKTR-214 combined with RT significantly extended survival in these mice, compared to RT or NKTR-214 alone (p=0.008). Twenty percent of these mice treated with right flank RT + NKTR-214 exhibited durable CR at all tumor sites, whereas no CR was observed in mice treated with RT or NKTR-214 alone. Using this same regimen, we treated mice bearing a right flank B78 melanoma tumor (targeted by local RT) and non-radiated B78 melanoma tumors on the left flank and in the brain. In these mice, we compared the immune response in the brain and left flank tumors following treatment with right flank RT + systemic NKTR-214. Analysis of these tumors by microscopy at treatment day 15 demonstrated significantly increased CD8+ T cell infiltrate and an increased ratio of CD8+ T cells: FOXP3+ regulatory T cells in both the left flank and brain tumors compared to untreated control mice. In contrast to our prior observations with RT + IT-IL2, the combination of RT + NKTR-214 triggered a comparable immune response in both the brain and left flank tumors with respect to the degree of CD8+ T cell infiltration and the ratio of CD8+ effector: FOXP3+ regulatory T cells. These results suggest that a combination of RT + NKTR-214 may elicit an ISV effect that is capable of conferring enhanced anti-tumor immune response against brain metastases, and could potentially abrogate the need for direct brain radiation or surgery. Citation Format: Paul A. Clark, Raghava N. Sriramaneni, Alexander Pieper, Amber M. Bates, Bryce R. Anderson, Wonjong Jin, Justin C. Jagodinsky, Alexander L. Rakhmilevich, Zachary S. Morris. Bempegaldesleukin (NKTR-214), a CD122 preferential IL-2 pathway agonist, augments the in situ vaccine response to radiation of an extracranial tumor in a murine melanoma model, conferring response at non-radiated tumor sites in the brain [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 4440.