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Simple Summary Radiation therapy and immune checkpoint inhibitors (ICIs) have been demonstrated to cooperatively activate adaptive anti-tumor immunity with curative potential in preclinical models of melanoma. Receptor-targeted radionuclide therapy can be systemically injected to selectively deliver ionizing radiation to tumor sites throughout the body, potentially rendering all tumor sites more susceptible to anti-tumor immune response. In this study, we demonstrated the feasibility of delivering alpha-particle radiation to murine melanoma tumors using a 212Pb radiolabeled peptide [212Pb]VMT01 that targets the melanocortin 1 receptor (MC1R). Our data showed anti-tumor cooperation between [212Pb]VMT01 and ICIs in melanoma, mediated by induction of tumor-specific immunity. The immunogenicity of [212Pb]VMT01 in melanoma was also evidenced by enhanced tumor infiltrating lymphocytes and tumor vaccination assays. Abstract Radiotherapy can facilitate the immune recognition of immunologically “cold” tumors and enhance the efficacy of anti-PD-1 and anti-CTLA-4 immune checkpoint inhibitors (ICIs) in melanoma. Systemic administration of receptor-targeted radionuclide therapy has the potential to selectively deliver radionuclides to multiple tumors throughout the body in metastatic settings. By triggering immunologic cell death and increasing the immune susceptibility of surviving tumor cells in these locations, targeted radionuclide therapies may overcome resistance to ICIs and render immunologically “cold” tumors throughout the body responsive to ICIs and immunologically “hot”. Here, we show the anti-tumor cooperation of targeted α-particle radionuclide therapy (α-TRT) and ICIs in preclinical models of melanoma. Melanocortin 1 receptor (MC1R)-targeted radiopeptide [212Pb]VMT01 was employed to deliver α-radiation to melanoma tumors in mice. A single injection of 4.1 MBq [212Pb]VMT01 significantly slowed the tumor growth of B16-F10 melanoma and the combination of [212Pb]VMT01 and ICIs induced a cooperative anti-tumor effect leading to 43% complete tumor response with no sign of malignancy on autopsy. Animals with complete response developed anti-tumor immunity to reject further tumor inoculations. This therapeutic cooperation was completely abolished in RAG1 KO mice, which are deficient in T-cell maturation. In addition, the anti-tumor cooperation was compromised when fractionated [212Pb]VMT01 was used in the combination. We also demonstrated that [212Pb]VMT01 induced immunogenic cell death in tumor vaccination assays and in vitro exposure to [212Pb]VMT01 sensitized immunotolerant melanoma to ICIs treatment in vivo. Enhanced tumor infiltrating CD3+, CD4+, CD8+ lymphocytes were observed following injection of 1.4 MBq [212Pb]VMT01. Overall, we demonstrated anti-tumor cooperation between α-TRT and ICIs in melanoma that is mediated by tumor specific immunity.

The Toll-like receptor 8 (TLR8) agonist VTX-2337 (motolimod) is an anti-cancer immunotherapeutic agent that is believed to augment natural killer (NK) and dendritic cell (DC) activity. The goal of this work is to examine the role of TLR8 expression/activity in head and neck squamous cell carcinoma (HNSCC) to facilitate the prediction of responders to VTX-2337-based therapy. The prognostic role of TLR8 expression in HNSCC patients was assessed by TCGA and tissue microarray analyses. The anti-tumor effect of VTX-2337 was determined in SCCVII/C3H, mEERL/C57Bl/6 and TUBO-human EGFR/BALB/c syngeneic mouse models. The effect of combined VTX-2337 and cetuximab treatment on tumor growth, survival and immune cell recruitment was assessed. TLR8 expression was associated with CD8+ T cell infiltration and favorable survival outcomes. VTX-2337 delayed tumor growth in all 3 syngeneic mouse models and significantly increased the survival of cetuximab-treated mice. The anti-tumor effects of VTX-2337+ cetuximab were accompanied by increased splenic lymphoid DCs and IFNγ+ CD4+ and tumor-specific CD8+ T cells. Depletion of CD4+ T cells, CD8+ T cells and NK cells were all able to abolish the anti-tumor effect of VTX-2337+ cetuximab. Altogether, VTX-2337 remains promising as an adjuvant for cetuximab-based therapy however patients with high TLR8 expression may be more likely to derive benefit from this drug combination compared to patients with low TLR8 expression.

BackgroundCMP-001 is a novel Toll-like receptor-9 agonist that consists of an unmethylated CpG-A motif-rich G10 oligodeoxynucleotide (ODN) encapsulated in virus-like particles.In situvaccination of CMP-001 is believed to activate local tumor-associated plasmacytoid dendritic cells (pDCs) leading to type I interferon secretion and tumor antigen presentation to T cells and systemic antitumor T cell responses. This study is designed to investigate if CMP-001 would enhance head and neck squamous cell carcinoma (HNSCC) tumor response to anti-programmed cell death protein-1 (anti-PD-1) therapy in a human papilloma virus-positive (HPV+) tumor mouse model.MethodsImmune cell activation in response to CMP-001±anti-Qβ was performed using co-cultures of peripheral blood mononuclear cells and HPV+/HPV- HNSCC cells and then analyzed by flow cytometry.In situvaccination with CMP-001 alone and in combination with anti-PD-1 was investigated in C57BL/6 mice-bearing mEERL HNSCC tumors and analyzed for anti-Qβ development, antitumor response, survival and immune cell recruitment. The role of antitumor immune response due to CMP-001+anti-PD-1 treatment was investigated by the depletion of natural killer (NK), CD4+T, and CD8+T cells.ResultsResults showed that the activity of CMP-001 on immune cell (pDCs, monocytes, CD4+/CD8+ T cells and NK cells) activation depends on the presence of anti-Qβ. A 2-week ‘priming’ period after subcutaneous administration of CMP-001 was required for robust anti-Qβ development in mice.In situvaccination of CMP-001 was superior to unencapsulated G10 CpG-A ODN at suppressing both injected and uninjected (distant) tumors.In situvaccination of CMP-001 in combination with anti-PD-1 therapy induced durable tumor regression at injected and distant tumors and significantly prolonged mouse survival compared with anti-PD-1 therapy alone. The antitumor effect of CMP-001+anti-PD-1 was accompanied by increased interferon gamma (IFNγ)+CD4+/CD8+T cells compared with control-treated mice. The therapeutic and abscopal effect of CMP-001+ anti-PD-1 therapy was completely abrogated by CD8+T cell depletion.ConclusionsThese results demonstrate thatin situvaccination with CMP-001 can induce both local and abscopal antitumor immune responses. Additionally, the antitumor efficacy of CMP-001 combined with α-PD-1 therapy warrants further study as a novel immunotherapeutic strategy for the treatment of HNSCC.

The Measures for Management of Soil Environment in Agricultural Land (Trial, Nov. 01, 2017, China) recently came into effect and highlighted the proper management of contaminated croplands to lower risks of exceedances of contaminants, especially toxic trace metals in agricultural produce. We aimed to develop local soil criteria for lead (Pb) in Hezhang county of southwestern China by the inverse use of reliable models linking Pb contamination levels between soils and vegetables. Dilute nitric acid (0.43 M) extraction, a new ISO standard (ISO-17586:2016) for extracting the geochemically reactive Pb fraction (PbNA), and calcium chloride (0.01 M) extraction (ISO-14255: 1998) for estimating the plant-available Pb (PbCC) were performed in fifty historically polluted and newly Pb-spiked soils with differing soil types, properties (pH 4.1–8.0), and total soil Pb levels (PbT, 20–6153 mg kg−1). Greenhouse experiments for Brassica pekinensis L., and in-situ soil porewater measurement for Pb were conducted to investigate the mechanism of Pb uptake, and to establish reliable Pb soil-plant relationships. The results indicated that about 83% of the variation for Pb concentrations in vegetable (PbCL, 0.009–1.06 mg kg−1) was contributable to free Pb2+ activity in soil porewater, which was mainly influenced by pH and dissolved organic matter. PbCL was satisfactorily predicted using PbNA and key soil properties (adj. R2 0.852). Soil Pb criteria for PbT and PbNA are then derived based on food standard. The full implementation of criteria derived for PbNA (i.e., 27–127 mg kg−1, soil pH 5.5–8.0) can avoid the exceedance of Pb in 95% of cabbage samples in this study, 95% of cabbage cultivars by model extrapolation, and one widely cultivated root vegetable, radish, in the study region. We provide a successful case study that has effectively tackled the challenge for the complexity of the soil management in contaminated croplands.

High concentrations of trace metal(loid)s in cigarette tobacco are likely the result of soil contamination and seriously threaten the health of smokers and non-smokers. This study aims to improve soil protection guidelines by carefully examining metal(loid)s uptake by tobacco leaves and human exposure to metal(loid)s through the soil–tobacco–smoker pathway. Soil and tobacco tests and health risk assessment were performed. Soil results indicated that the exchangeable fractions, greatly influenced by soil pH in historically contaminated (HC) agricultural soils were small but the geochemically reactive fractions were very large, representing a great potential of phytoavailability. Greenhouse experiments for tobacco (Nicotiana tabacum L.) grown on both HC and artificially metal-spiked (AS) soils indicated that tobacco leaves accumulated high concentrations of Zn, followed by Pb, Cu, Cd, Cr, Ni and As. Exposure model indicates that consuming the contaminated tobacco can result in an unacceptable inhalation intake of Zn (138 mg kg−1 year−1), As (0.64), Cd (4.9), Pb (5.9) and Cu (8.9) for women in particular. Plant-available fractions of As, Cd, Pb, Zn, Cu and Cr in soils explained 85–100% of the variation for tobacco metal(loid)s. Reliable soil-tobacco transfer models were derived and validated with field data. In the present study, contaminated soils are unsafe for growing tobacco identified by the inverse use of empirical models based on regulatory limits estimated for tobacco metal(loid)s. This work provides a basis in a regulatory context of tobacco metal(loid)s exposure.

Historically metal-polluted agricultural soils are an important source of human exposure to toxic metals such as arsenic (As), cadmium (Cd), and lead (Pb) through food chains, especially in remote rural areas like Hezhang county, China. This paper aimed to investigate the processes by which a staple food crop, maize (Zea mays L.), became contaminated, identify the dominant exposure pathway and dietary intake risks, identify soils that were unsafe for growing maize, and share findings with farmers and decision-makers. A greenhouse experiment was performed using 20 historically metal-polluted soils with different soil types, properties, and contamination levels. Metal(loid) concentrations in maize grains, soil porewater (in situ), and DTPA (0.005 M) extracts of rooting-zone soils were measured to investigate the factors that influence the availability and the uptake of metal(loid)s. An investigation was performed to identify the main exposure pathway. Regression models describing metal(loid)s transfer from soils to maize grains were inversely used to derive bioavailability-based soil criteria. Soil results indicated that 28% of total Cd and 17% of total Pb in soils were potentially plant-available whereas the labile fractions in soil porewater were negligible due to high soil pH (> 7.5). For As in soils, approximately 2% was the non-specifically adsorbed fraction. Grain metal(loid)s (i.e., As, Cd, and Pb) levels were satisfactorily predicted using porewater concentrations and key properties such as pH and dissolved organic carbon contents (adj. R2 0.814 - 0.887). The bioaccumulation of As in maize was very limited. However, the level of Cd in 73% and Pb in 25% of grain samples exceeded Chinese food quality standard, thereby threatening food safety. Metal(loid)s transfer through maize → animal → human pathway would result in a substantial dietary intake of metals, e.g., 79.7 μg Pb day−1 and 217.9 μg Cd day−1. A more implementable soil criterion for DTPA-extractable Cd (0.6 mg kg−1) was derived allowing identification of soils that were unsafe for growing maize. Hezhang residents experience dietary intake risks of Pb and Cd. Excessive Cd and Pb in maize are the result of soil pollution. Soil with DTPA-extractable Cd concentration

Immunotherapy involving immune checkpoint inhibitors (i.e. anti-PD1) has shown promise for the treatment of head and neck squamous cell carcinoma (HNSCC). However, only a small subset of HNSCC patients respond to these therapies. Therefore, the identification of alternative immunotherapeutic strategies is necessary to improve HNSCC patient outcomes. CMP-001 is a novel toll-like receptor-9 agonist that consists of an unmethylated CpG motif-rich G10 oligonucleotide encapsulated in virus-like particles. In situ vaccination of CMP-001 is believed to activate tumor-associated plasmacytoid dendritic cells (pDCs) leading to tumor antigen presentation to T cells and anti-tumor T cell responses. This study is designed to investigate if CMP-001 would enhance HNSCC tumor response to anti-PD1 therapy in a human papilloma virus-positive (HPV+) tumor mouse model. C57BL/6 mice were subcutaneously inoculated with murine HPV+ mEERL HNSCC cells on both left and right flanks of each mouse. CMP-001 (100 µg/mouse) was administered intratumorally (i.t.) 3 times over the course of 1 week into the left tumor as a single agent and in combination with anti-PD1 (50 µg/mouse, intraperitoneally (i.p.)) 3 times per week for 2 weeks. Succinate buffer i.t. in combination with human IgG i.p. was administered as controls. The role of anti-tumor immune response due to CMP-001+anti-PD1 treatment was investigated by the depletion of NK cells, CD4+ and CD8+ T cells. Tumor growth, survival and immune cell recruitment was monitored and analyzed in response to drug treatment. Results showed that in situ vaccination of CMP-001 alone and in combination with anti-PD1 therapy induced tumor regression in the injected tumors and the regression of CMP-001+anti-PD1-injected tumors was durable. Additionally, CMP-001+anti-PD1 induced tumor regression at the distant sites which was not observed in the other treatment groups. CMP-001+anti-PD1 also significantly prolonged mouse survival compared with other groups. The anti-tumor effect of CMP-001+anti-PD1 was accompanied by increased DCs, macrophages, IFNγ+ CD4+/CD8+ T cells, HPV+ CD8+ T cells and activated NK cells in tumors and lymph nodes near the injected site compared with control. The therapeutic and abscopal effect of CMP-001+anti-PD1 therapy was partially abrogated by NK cell depletion and completely abrogated by CD8+ T cell depletion. These results demonstrate the synergistic anti-tumor efficacy of CMP-001 combined with α-PD1 therapy and warrants further study of this combination as a novel immunotherapeutic strategy for the treatment of HNSCC. Citation Format: Yinwen Cheng, Caitlin D. Lemke-Miltner, Carlos H. Chan, Aliasger Salem, George Weiner, Andrean Simons. In situ immunization with the TLR9-agonist CMP-001 enhances anti-PD1 therapy in head and neck tumors [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 6641.

Reliable models describing Pb transfer from soils to food crops are useful in the improvement of soil protection guidelines. This study provides mechanistic insights from in-situ soil solution measurement on the Pb uptake in the root tissues (RF) of radish, grown in 25 representative Pb-contaminated agricultural soils. Lead speciation and regression analysis indicate that >88.6% of the variation in RF Pb is attributable to free Pb2+ activity (aPb2+) in the soil solution, which is predominantly controlled by pH and DOC. Higher DOC would increase the total dissolved Pb (CSol-Pb) in the soil solution but reduce the bioavailability of Pb to radish. CSol-Pb performs poorly in predicting RF Pb unless pH and DOC are included. However, 0.01M CaCl2 extractable Pb (CCC-Pb) alone can satisfactorily predict RF Pb, attributable to the fact that CCC-Pb is consistent with aPb2+. CCC-Pb can be predicted using CSol-Pb and pH. Total soil Pb (CT-Pb), or 0.43M HNO3 extractable Pb (CNA-Pb) has a strong, non-linear correlation with CSol-Pb or CCC-Pb and it is therefore not surprising that CT-Pb or CNA-Pb, together with pH and CEC, can also satisfactorily predict RF Pb. Derived models are effective in identification of soils where RF Pb exceeds the food quality standard (FQS). Soil Pb criteria based on CT-Pb, CNA-Pb and CCC-Pb are derived by inverse use of empirical models. The derived Pb criterion (target value) based on CCC-Pb is 0.02mgkg-1 and the stricter criterion (safe value) is 0.01mgkg-1, which allows a 5% probability for RF Pb to exceed FQS. Safe values based on CT-Pb and CNA-Pb ranged from 26 to 1036mgkg-1 and 9 to 745mgkg-1, respectively.

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