B16F10 Tumor-bearing Mice Research Articles

Combination of PD-1 Checkpoint Blockade and Botulinum Toxin Type A1 Improves Antitumor Responses in Mouse Tumor Models of Melanoma and Colon Carcinoma

ABSTRACT Background Tumor innervation has been shown to be utilized by some solid cancers to support tumor initiation, growth, progression, and metastasis, as well as confer resistance to immune checkpoint blockade through suppression of antitumor immunologic responses. Since botulinum neurotoxin type A1 (BoNT/A1) blocks neuronal cholinergic signaling, its potential use as an anticancer drug in combination with anti-PD-1 therapy was investigated in four different syngeneic mouse tumor models. Methods Mice implanted with breast (4T1), lung (LLC1), colon (MC38), and melanoma (B16-F10) tumors were administered a single intratumoral injection of 15 U/kg BoNT/A1, repeated intraperitoneal injections of 5 mg/kg anti-PD-1 (RMP1-14), or both. Results Compared to the single-agent treatments, anti-PD-1 and BoNT/A1 combination treatment elicited significant reduction in tumor growth among B16-F10 and MC38 tumor-bearing mice. The combination treatment also lowered serum exosome levels in these mice compared to the placebo control group. In the B16-F10 syngeneic mouse tumor model, anti-PD-1 + BoNT/A1 combination treatment lowered the proportion of MDSCs, negated the increased proportion of Treg cells, and elicited a higher number of tumor-infiltrating CD4+ and CD8+ T lymphocytes into the tumor microenvironment compared to anti-PD-1 treatment alone. Conclusion Our findings demonstrate the synergistic antitumor effects of BoNT/A1 and PD-1 checkpoint blockade in mouse tumor models of melanoma and colon carcinoma. These findings provide some evidence on the potential application of BoNT/A1 as an anticancer drug in combination with immune checkpoint blockade and should be further explored.

Preparation and Bioevaluation of 18F-Labeled Small-Molecular Radiotracers via Sulfur(VI) Fluoride Exchange Chemistry for Imaging of Programmed Cell Death Protein Ligand 1 Expression in Tumors.

Nowadays, one of the most effective methods of tumor immunotherapy is blocking programmed cell death protein 1/programmed cell death protein ligand 1 (PD-1/PD-L1) immune checkpoints. However, there is still a significant challenge in selecting patients to benefit from immune checkpoint therapies. Positron emission tomography (PET), a noninvasive molecular imaging technique, offers a new approach to accurately detect PD-L1 expression and allows for a better prediction of response to PD-1/PD-L1 target immunotherapy. Here, we designed and synthesized a novel group of aryl fluorosulfate-containing small-molecule compounds (LGSu-1, LGSu-2, LGSu-3, and LGSu-4) based on the phenoxymethyl-biphenyl scaffold. After screening by the time-resolved fluorescence resonance energy transfer (TR-FRET) assay, the most potent compound LGSu-1 (half maximal inhibitory concentration (IC50): 15.53 nM) and the low-affinity compound LGSu-2 (IC50: 189.70 nM) as a control were selected for 18F-radiolabeling by sulfur(VI) fluoride exchange chemistry (SuFEx) to use for PET imaging. [18F]LGSu-1 and [18F]LGSu-2 were prepared by a one-step radiofluorination reaction in over 85% radioconversion and nearly 30% radiochemical yield. In B16-F10 melanoma cell assays, [18F]LGSu-1 (5.00 ± 0.06%AD) showed higher cellular uptake than [18F]LGSu-2 (2.55 ± 0.04%AD), in which cell uptake could be significantly blocked by the nonradioactivity LGSu-1. In vivo experiments, micro-PET imaging of B16-F10 tumor-bearing mice and radiographic autoradiography of tumor sections showed that [18F]LGSu-1 was more effectively accumulated in the tumor due to the higher binding affinity with PD-L1. The above experimental results confirmed the potential of the small-molecule probe LGSu-1 as a targeting PD-L1 imaging tracer in tumor tissues.

The effectiveness of calcium electroporation combined with gene electrotransfer of a plasmid encoding IL-12 is tumor type-dependent.

In calcium electroporation (CaEP), electroporation enables the cellular uptake of supraphysiological concentrations of Ca2+, causing the induction of cell death. The effectiveness of CaEP has already been evaluated in clinical trials; however, confirmatory preclinical studies are still needed to further elucidate its effectiveness and underlying mechanisms. Here, we tested and compared its efficiency on two different tumor models to electrochemotherapy (ECT) and in combination with gene electrotransfer (GET) of a plasmid encoding interleukin-12 (IL-12). We hypothesized that IL-12 potentiates the antitumor effect of local ablative therapies as CaEP and ECT. The effect of CaEP was tested in vitro as well as in vivo in murine melanoma B16-F10 and murine mammary carcinoma 4T1 in comparison to ECT with bleomycin. Specifically, the treatment efficacy of CaEP with increasing calcium concentrations alone or in combination with IL-12 GET in different treatment protocols was investigated. We closely examined the tumor microenvironment by immunofluorescence staining of immune cells, as well as blood vessels and proliferating cells. In vitro, CaEP and ECT with bleomycin reduced cell viability in a dose-dependent manner. We observed no differences in sensitivity between the two cell lines. A dose-dependent response was also observed in vivo; however, the efficacy was better in 4T1 tumors than in B16-F10 tumors. In 4T1 tumors, CaEP with 250 mM Ca resulted in more than 30 days of growth delay, which was comparable to ECT with bleomycin. In contrast, adjuvant peritumoral application of IL-12 GET after CaEP prolonged the survival of B16-F10, but not 4T1-bearing mice. Moreover, CaEP with peritumoral IL-12 GET modified tumor immune cell populations and tumor vasculature. Mice bearing 4T1 tumors responded better to CaEP in vivo than mice bearing B16-F10 tumors, even though a similar response was observed in vitro. Namely, one of the most important factors might be involvement of the immune system. This was confirmed by the combination of CaEP or ECT with IL-12 GET, which further enhanced antitumor effectiveness. However, the potentiation of CaEP effectiveness was also highly dependent on tumor type; it was more pronounced in poorly immunogenic B16-F10 tumors compared to moderately immunogenic 4T1 tumors.

MBD2 facilitates tumor metastasis by mitigating DDB2 expression

Despite past extensive studies, the pathoetiologies underlying tumor metastasis remain poorly understood, which renders its treatment largely unsuccessful. The methyl-CpG-binding domain 2 (MBD2), a “reader” to interpret DNA methylome-encoded information, has been noted to be involved in the development of certain types of tumors, while its exact impact on tumor metastasis remains elusive. Herein we demonstrated that patients with LUAD metastasis were highly correlated with enhanced MBD2 expression. Therefore, knockdown of MBD2 significantly attenuated the migration and invasion of LUAD cells (A549 and H1975 cell lines) coupled with attenuated epithelial–mesenchymal transition (EMT). Moreover, similar results were observed in other types of tumor cells (B16F10). Mechanistically, MBD2 selectively bound to the methylated CpG DNA within the DDB2 promoter, by which MBD2 repressed DDB2 expression to promote tumor metastasis. As a result, administration of MBD2 siRNA-loaded liposomes remarkably suppressed EMT along with attenuated tumor metastasis in the B16F10 tumor-bearing mice. Collectively, our study indicates that MBD2 could be a promising prognostic marker for tumor metastasis, while administration of MBD2 siRNA-loaded liposomes could be a viable therapeutic approach against tumor metastasis in clinical settings.

Abstract 1839: CLN-617 is a first-in-class fusion protein that retains IL-2 and IL-12 in the injected tumor and potently triggers systemic anti-tumor immunity

Abstract Despite clinical evidence of anti-tumor activity, cytokine therapies have been hampered by a narrow therapeutic window and limited efficacy. Two cytokines of interest are IL-2 and IL-12, which synergize to proliferate and activate T cells and natural killer cells. However, the only approved human IL-2 product (Proleukin) is rarely used in the clinic due to systemic toxicities, and no IL-12 product has been approved to date due to severe dose-limiting toxicities. Here, we describe CLN-617, a first-in-class therapeutic designed for intratumoral (IT) injection and retention, that co-delivers IL-2 and IL-12 in a safe and effective manner. CLN-617 is a single-chain fusion protein comprised of human IL-2, leukocyte-associated immunoglobulin-like receptor 2 (LAIR2), human serum albumin (HSA), and human IL-12. LAIR2 and HSA functionally retain CLN-617 in the injected tumor by binding collagen and increasing molecular weight, respectively. A murine surrogate of CLN-617, mCLN-617, was delivered either IV or IT in B16F10 tumor-bearing mice. The concentration of mCLN-617 as measured in serum two hours after IT administration was <5% that of the concentration following IV administration, demonstrating robust retention of mCLN-617 in the tumor. Following mCLN-617 treatment, durable and complete responses with no significant body weight loss were observed in checkpoint-refractory tumor models, including B16F10, CT26, and MC38. mCLN-617 triggered a robust systemic anti-tumor immune response based on three lines of evidence. First, in mice implanted with two MC38 tumors, 70% of injected tumors and 40% of non-injected tumors were eradicated. When combined with IV-administered anti-PD1 antibody, >90% of both injected and non-injected tumors showed a complete response, while anti-PD1 alone exhibited no anti-tumor activity. Second, in mice implanted with an MC38 tumor in the flank and with luciferase-expressing MC38 cells delivered to the liver via intra-splenic injection, the injected flank tumor exhibited ~90% growth inhibition with either mCLN-617 monotherapy or anti-PD1 co-administration. Growth of established metastatic tumors, as measured by luciferase signal, was inhibited by mCLN-617 monotherapy, and metastatic tumor burden significantly regressed in combination with anti-PD1, while anti-PD1 alone exhibited no anti-tumor activity in the metastases. Third, in mice bearing two MC38 flank tumors, tumor-specific CD8+ T cells expanded in both the injected and non-injected tumors and in peripheral blood. We conclude that IT-administered mCLN-617 is efficiently retained in the injected tumor, thereby preventing systemic toxicity of the two cytokines, yet can activate and remodel systemic immunosurveillance for clearance of non-injected distal lesions. A phase I trial of CLN-617 in patients with advanced solid tumors is expected to commence in 2023. Citation Format: Naveen K. Mehta, Kavya Rakhra, Kristan Meetze, K. Dane Wittrup, Jennifer S. Michaelson, Patrick A. Baeuerle. CLN-617 is a first-in-class fusion protein that retains IL-2 and IL-12 in the injected tumor and potently triggers systemic anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1839.

Abstract 2282: Immune modulation of the T cell repertoire in mice following immunotherapy and chemotherapy combination treatments

Abstract Background: Immune checkpoint blockade therapeutics have revolutionized cancer therapy. However, most patients are either unresponsive or develop acquired resistance. Understanding patient-specific temporal T cell repertoires in the context of immune interventions will contribute to a better understanding of potential improvement strategies for better T cell anti-tumor immunity. Current methods to understand the T cell repertoire changes over time are limited due to the limited amount of material and prohibitive cost. In this study, we designed and performed longitudinal TCR sequencing in mice treated with multiple therapies. Methods: C57BL/6 mice were used in each treatment group, with blood drawn overtime before, during and after treatment. Mice were treated with a combination of immunotherapeutic agents and OVA peptide immunization, 100µl blood was drawn at multiple pre-, on- and post-treatment time points followed subsequently by TCR sequencing. Cyclophosphamide (CTX) is known to cause lymphodepletion followed by homeostatic proliferation. Thus, we also treated B16-F10 tumor bearing mice with CTX, and performed TCR sequencing on blood draws pre-, on-, and post-treatment to track TCR clone diversity and clonality over time. Results: We find that our method is sensitive enough to detect effects of immunotherapies on TCR diversity in small sample quantities. Using our method, we discovered that certain immunotherapeutic agents, most evidently CD40, have a significant but transient effect on the diversity of the T cell repertoire. We observed an initial increase in repertoire clonality, seven days post CD40 treatment followed by contraction. We show how we can identify antigen specific T cells and follow reacting T cell clones. In addition, we find that CTX treatment is associated with increased clonality in tumor bearing mice particularly in the week following treatment reflecting changes in the TCR repertoire. Conclusion: We have developed a method by which we can follow the TCR repertoire in mice treated with immunotherapy or chemotherapy. This enabled us to isolate and study the effects of different immune modulatory drugs of the endogenous T cell repertoire overtime. Citation Format: Yuval Elhanati, Mariam M. George, Hyejin Choi, Rachana Maniyar, Linda Hamadane, Neeman Mohibullah, Benjamin D. Greenbaum, Taha Merghoub. Immune modulation of the T cell repertoire in mice following immunotherapy and chemotherapy combination treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2282.

Glycol-Split Heparin-Linked Prodrug Nanoparticles Target the Mitochondrion Apparatus for Cancer Metastasis Treatment.

The progression and metastasis of solid tumors rely strongly on neovascularization. However, angiogenesis inhibitors alone cannot meet the needs of tumor therapy. This study prepared a new drug conjugate (PTX-GSHP-CYS-ES2, PGCE) by combining polysaccharides (heparin without anticoagulant activity, GSHP), chemotherapeutic drugs (paclitaxel, PTX), and antiangiogenic drugs (ES2). Furthermore, a tumor-targeted prodrug nanoparticle delivery system is established. The nanoparticles appear to accumulate in the mitochondrial of tumor cells and achieve ES2 and PTX release under high glutathione and acidic environment. It has been confirmed that PGCE inhibited the expression of multiple metastasis-related proteins by targeting the tumor cell mitochondrial apparatus and disrupting their structure. Furthermore, PGCE nanoparticles inhibit migration, invasion, and angiogenesis in B16F10 tumor-bearing mice and suppress tumor growth and metastasis in vitro. Further in vitro and in vivo experiments show that PGCE has strong antitumor growth and metastatic effects and exhibits efficient anti-angiogenesis properties. This multi-targeted nanoparticle system potentially enhances the antitumor and anti-metastatic effects of combination chemotherapy and antiangiogenic drugs.

Development of [18F]F-5-OMe-Tryptophans through Photoredox Radiofluorination: A New Method to Access Tryptophan-Based PET Agents.

Although various radiolabeled tryptophan analogs have been developed to monitor tryptophan metabolism using positron emission tomography (PET) for various human diseases including melanoma and other cancers, their application can be limited due to the complicated synthesis process. In this study, we demonstrated that photoredox radiofluorination represents a simple method to access novel tryptophan-based PET agents. In brief, 4-F-5-OMe-tryptophans (l/d-T13) and 6-F-5-OMe-tryptophans (l/d-T18) were easily synthesized. The 18F-labeled analogs were produced by photoredox radiofluorination with radiochemical yields ranging from 2.6 ± 0.5% to 32.4 ± 4.1% (3 ≤ n ≤ 5, enantiomeric excess ≥ 99.0%) and over 98.0% radiochemical purity. Small animal imaging showed that l-[18F]T13 achieved 9.58 ± 0.26%ID/g tumor uptake and good contrast in B16F10 tumor-bearing mice (n = 3). Clearly, l-[18F]T13 exhibited prominent tumor uptake, warranting future evaluations of its potential usage in precise immunotherapy monitoring.

Charge reversal yolk-shell liposome co-loaded JQ1 and doxorubicin with high drug loading and optimal ratio for synergistically enhanced tumor chemo-immunotherapy via blockade PD-L1 pathway

Antitumor immunotherapy has become a powerful therapeutic modality to identify and kill various malignant tumors by harnessing the immune system. However, it is hampered by the immunosuppressive microenvironment and poor immunogenicity in malignant tumors. Herein, in order to achieve multi-loading of drugs with different pharmacokinetic properties and targets, a charge reversal yolk-shell liposome co-loaded with JQ1 and doxorubicin (DOX) into the poly (D,L-lactic-co-glycolic acid) (PLGA) yolk and the lumen of the liposome respectively was engineered to increase hydrophobic drug loading capacity and stability under physiological conditions and further enhance tumor chemotherapy via blockade programmed death ligand 1 (PD-L1) pathway. This nanoplatform could release less JQ1 compared to traditional liposomes to avoid drug leakage under physiological conditions due to the protection of liposomes on JQ1 loaded PLGA nanoparticles while the release of JQ1 increased in an acidic environment. In the tumor microenvironment, released DOX promoted immunogenic cell death (ICD), and JQ1 blocked the PD-L1 pathway to strengthen chemo-immunotherapy. The in vivo antitumor results demonstrated the collaborative treatment of DOX and JQ1 in B16-F10 tumor-bearing mice models with minimized systemic toxicity. Furthermore, the orchestrated yolk-shell nanoparticle system could enhance the ICD effect, caspase 3 activation, and cytotoxic T lymphocyte infiltration while inhibiting PD-L1 expression, provoking a strong antitumor effect, whereas yolk-shell liposomes encapsulating only JQ1 or DOX showed modest tumor therapeutic effects. Hence, the cooperative yolk-shell liposome strategy provides a potential candidate for enhancement of hydrophobic drug loading and stability, showing potential for clinic application and synergistic cancer chemo-immunotherapy.

Engineered Norovirus-Derived Nanoparticles as a Plug-and-Play Cancer Vaccine Platform.

In recent years, virus-derived self-assembled protein nanoparticles (NPs) have emerged as attractive antigen delivery platforms for developing both preventive and therapeutic vaccines. In this study, we exploited the genetically engineered Norovirus S domain (Nov-S) with SpyCatcher003 fused to the C-terminus to develop a robust, modular, and versatile NP-based carrier platform (Nov-S-Catcher003). The NPs can be conveniently armed in a plug-and-play pattern with SpyTag003-linked antigens. Nov-S-Catcher003 was efficiently expressed in Escherichia coli and self-assembled into highly uniform NPs with a purified protein yield of 97.8 mg/L. The NPs presented high stability at different maintained temperatures and after undergoing differing numbers of freeze-thaw cycles. Tumor vaccine candidates were easily obtained by modifying Nov-S-Catcher003 NPs with SpyTag003-linked tumor antigens. Nov-S-Catcher003-antigen NPs significantly promoted the maturation of bone marrow-derived dendritic cells in vitro and were capable of efficiently migrating to lymph nodes in vivo. In TC-1 and B16F10 tumor-bearing mice, the subcutaneous immunization of NPs elicited robust tumor-specific T-cell immunity, reshaped the tumor microenvironment, and inhibited tumor growth. In the TC-1 model, the NPs even completely abolished established tumors. In conclusion, the Nov-S-Catcher003 system is a promising delivery platform for facilitating the development of NP-based cancer vaccines.

An injectable, in situ forming and NIR-responsive hydrogel persistently reshaping tumor microenvironment for efficient melanoma therapy

BackgroundMelanoma is a highly aggressive form of skin cancer with increasing incidence and mortality rates. Chemotherapy, the primary treatment for melanoma, is limited by hypoxia-induced drug resistance and suppressed immune response at the tumor site. Modulating the tumor microenvironment (TME) to alleviate hypoxia and enhance immune response has shown promise in improving chemotherapy outcomes.MethodsIn this study, a novel injectable and in situ forming hydrogel named MD@SA was developed using manganese dioxide (MnO2) nanosheets pre-loaded with the chemotherapy drug doxorubicin (DOX) and mixed with sodium alginate (SA). The sustainable drug delivery, oxygen generation ability, and photothermal property of MD@SA hydrogel were characterized. The therapeutic efficacy of hydrogel was studied in B16F10 in vitro and B16F10 tumor-bearing mice in vivo. The immune effects on macrophages were analyzed by flow cytometry, real-time quantitative reverse transcription PCR, and immunofluorescence analyses.ResultsThe MD@SA hydrogel catalyzed the tumoral hydrogen peroxide (H2O2) into oxygen, reducing the hypoxic TME, down-regulating hypoxia-inducible factor-1 alpha (HIF-1α) and drug efflux pump P-glycoprotein (P-gp). The improved TME conditions enhanced the uptake of DOX by melanoma cells, enhancing its efficacy and facilitating the release of tumor antigens. Upon NIR irradiation, the photothermal effect of the hydrogel induced tumor apoptosis to expose more tumor antigens, thus re-educating the M2 type macrophage into the M1 phenotype. Consequently, the MD@SA hydrogel proposes an ability to constantly reverse the hypoxic and immune-inhibited TME, which eventually restrains cancer proliferation.ConclusionThe injectable and in situ forming MD@SA hydrogel represents a promising strategy for reshaping the TME in melanoma treatment. By elevating oxygen levels and activating the immune response, this hydrogel offers a synergistic approach for TME regulation nanomedicine.

Lactate oxidase/vSIRPα conjugates efficiently consume tumor-produced lactates and locally produce tumor-necrotic H2O2 to suppress tumor growth

Aggressive tumor formation often leads to excessive anaerobic glycolysis and massive production and accumulation of lactate in the tumor microenvironment (TME). To significantly curb lactate accumulation in TME, in this study, lactate oxidase (LOX) was used as a potential therapeutic enzyme and signal regulatory protein α variant (vSIRPα) as a tumor cell targeting ligand. SpyCatcher protein and SpyTag peptide were genetically fused to LOX and vSIRPα, respectively, to form SC-LOX and ST-vSIRPα and tumor-targeting LOX/vSIRPα conjugates were constructed via a SpyCatcher/SpyTag protein ligation system. LOX/vSIRPα conjugates selectively bound to the CD47-overexpressing mouse melanoma B16-F10 cells and effectively consumed lactate produced by the B16-F10 cells, generating adequate amounts of hydrogen peroxide (H2O2), which induces drastic necrotic tumor cell death. Local treatments of B16-F10 tumor-bearing mice with LOX/vSIRPα conjugates significantly suppressed B16-F10 tumor growth in vivo without any severe side effects. Tumor-targeting vSIRPα may allow longer retention of LOX in tumor sites, effectively consuming surrounding lactate in TME and locally generating adequate amounts of cytotoxic H2O2 to suppress tumor growth. The approach restraining the local lactate concentration and H2O2 in TME using LOX and vSIRPα could offer new opportunities for developing enzyme/targeting ligand conjugate-based therapeutic tools for tumor treatment.

High-Dose Ascorbate in Combination with Anti-PD1 Checkpoint Inhibition as Treatment Option for Malignant Melanoma.

Ascorbate acts as a prooxidant when administered parenterally at high supraphysiological doses, which results in the generation of hydrogen peroxide in dependence on oxygen. Most cancer cells are susceptible to the emerging reactive oxygen species (ROS). Accordingly, we evaluated high-dose ascorbate for the treatment of the B16F10 melanoma model. To investigate the effects of ascorbate on the B16F10 cell line in vitro, viability, cellular impedance, and ROS production were analyzed. In vivo, C57BL/6NCrl mice were subcutaneously injected into the right flank with B16F10 cells and tumor-bearing mice were treated intraperitoneally with ascorbate (3 g/kg bodyweight), immunotherapy (anti-programmed cell death protein 1 (PD1) antibody J43; 2 mg/kg bodyweight), or both treatments combined. The efficacy and toxicity were analyzed by measuring the respective tumor sizes and mouse weights accompanied by histological analysis of the protein levels of proliferating cell nuclear antigen (Pcna), glucose transporter 1 (Glut-1), and CD3. Treatment of B16F10 melanoma-carrying mice with high-dose ascorbate yielded plasma levels in the pharmacologically effective range, and ascorbate showed efficacy as a monotherapy and when combined with PD1 inhibition. Our data suggest the applicability of ascorbate as an additional therapeutic agent that can be safely combined with immunotherapy and has the potential to potentiate anti-PD1-based immune checkpoint blockades.

Peptide-Based [68Ga]Ga Labeled PET Tracer for Tumor Imaging by Targeting Tumor-Associated Macrophages.

Tumor-associated macrophages (TAMs) are known to promote cancer development and metastasis. In this study, a TAMs-targeting peptide named M2pep was selected to investigate the feasibility of [68Ga]Ga-labeled M2pep as a noninvasive probe in targeted TAMs imaging. The peptide M2pep was conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and radiolabeled with 68Ga. The cellular uptake and binding assay were assessed in M2 macrophages and in the B16F10 cell line. Micro-PET imaging and a biodistribution study were performed on B16F10 tumor-bearing mice. High radiochemical purity [68Ga]Ga-DOTA-M2pep (>95%) was prepared and was stabilized in saline and bovine serum at 37 °C for 2 h. In vitro studies demonstrated high uptake of [68Ga]Ga-DOTA-M2pep in M2 macrophages, which was effectively blocked by the "cold" M2pep (free peptide). The micro-PET imaging and biodistribution study revealed that [68Ga]Ga-DOTA-M2pep reached the tumor site rapidly and showed high accumulation in the tumor at 1 h post-injection. In addition, the probe was rapidly cleared from the blood and mainly excreted via the kidneys, resulting in a high tumor/background ratio. Preclinical studies have shown that [68Ga]Ga-DOTA-M2pep specifically targets TAMs and might be a promising molecular probe for the noninvasive visualization of TAMs expression.

H3TPAN-Triazole-Bn-NH2: Tripicolinate Clicked-Bifunctional Chelate for [225Ac]/[111In] Theranostics.

A new, high-denticity, bifunctional ligand─H3TPAN-triazole-Bn-NH2─has been synthesized and studied in complexation with [225Ac]Ac3+ and [111In]In3+ for radiopharmaceutical applications. The bifunctional chelator is readily synthesized, using a high-yielding four-step prep, which is highly adaptable and allows for straightforward incorporation of different covalent linkers using CuI-catalyzed alkyne-azide cycloaddition (click) chemistry. Nuclear magnetic resonance (NMR) studies of H3TPAN-triazole-Bn-NH2 with La3+ and In3+ metal ions show the formation of a single, asymmetric complex with each ion in solution, corroborated by density functional theory (DFT) calculations. Radiolabeling studies with [225Ac]Ac3+ and [111In]In3+ showed highly effective complexation, achieving quantitative radiochemical conversions at low ligand concentrations (<10-6 M) under mild conditions (RT, 10 min), which is further accompanied by high stability in human serum. The bioconjugate─H3TPAN-triazole-Bn-Aoc-Pip-Nle-CycMSHhex─was prepared for targeting of MC1R-positive tumors, and the corresponding 111In-radiolabeled tracer was studied in vivo. SPECT/CT and biodistribution studies in C57BL/6J mice bearing B16-F10 tumors were performed, with the radiotracer showing good in vivo stability; tumor uptake was achieved. This work highlights a new promising and versatile bifunctional chelator, easily prepared and encouraging for 225Ac/111In theranostics.

Localized ablative immunotherapy drives de novo CD8+ T-cell responses to poorly immunogenic tumors

BackgroundLocalized ablative immunotherapies hold great promise in stimulating antitumor immunity to treat metastatic and poorly immunogenic tumors. Tumor ablation is well known to release tumor antigens and danger-associated molecular patterns...

147 (PB027) - Combination Therapy with Poly-gamma-glutamic Acid and Anti-PD-1 Results in Synergistic Therapeutic Effects against Cancers

Background: Combinatorial therapeutic strategies based on innate immunity have the potential to enhance the treatment efficacy of cancer therapies. We have been demonstrated that poly-γ-glutamic acid (γ-PGA) has a potent innate immune modulating activities including natural killer cell activation and interferon secretion through Toll-like receptor 4 and may be a useful mean for immunotherapeutic as reported previously. The programmed death protein 1 (PD-1) plays a critical part in downregulating immune responses and limiting immunogenic response in T cells. Here, we report the anti-cancer effects of γ-PGA combined with PD-1 blockade in murine colon adenocarcinoma and murine melanoma. Material and methods: It was performed to evaluate the in vivo combinatorial anti-cancer effects by oral administration of γ-PGA and intraperitoneal injection of anti-PD-1 in C57BL/6N mice transplanted with MC38 cell, a colorectal cancer cell. And the another study was carried out to examine the synergetic therapeutic efficacy by γ-PGA and anti-PD-1 against B16F10 melanoma tumor model in mice. Results: Combination therapy with γ-PGA and anti-PD-1 produced significantly longer tumor growth delay than other treatment and almost completely inhibited melanoma tumor growth, and also the improved longterm survival rate was shown compared to γ-PGA or anti-PD-1 alone. Similar experiment was performed in melanoma tumor and the result shows that co-treatment with γ-PGA and anti-PD-1 significantly prolonged survival rate compared with anti-PD-1 alone in MC38 tumor model and dramatically reduced MC38 tumor growth but had no synergistic effect. The results showed that the survival rate of the combinatorial treatment was superior to anti-PD-1 alone. Conclusions: Based on the resistance mechanism of PD-1 blockade with low response rates in many patients, γ-PGA treatment having an innate immune modulating activity combined with anti-PD-1 can induce stronger anti-tumor immunity and destroy the target cancer cells. In this study, oral administration of γ-PGA with intraperitoneal injection of anti-PD-1 dramatically inhibited tumor growth and enhanced the survival rate in MC38 and B16F10 tumor-bearing mice. The study demonstrates that the tumor microenvironment was controlled by administration of γ-PGA, thus results in exertion of immune checkpoint inhibitor. These data provide a strong preclinical evidence for a novel combinatorial therapeutic strategy that can be assessed in prospective clinical trials. No conflict of interest.

H4picoopa─Robust Chelate for 225Ac/111In Theranostics.

The nuclear decay characteristics of 225Ac (Eα = 5-8 MeV, linear energy transfer (LET) = ∼100 keV/μm, t1/2 = 9.92 days) are well recognized as advantageous for the treatment of primary and metastatic tumors; however, suitable chelation systems are required, which can accommodate this radiometal. Since 225Ac does not possess any suitable low-energy, high abundance γ-ray emissions for nuclear imaging, there is a clear need for the development of other companion radionuclides with similar coordination characteristics and comparable half-lives, which can be applied in diagnostics. H4picoopa was designed and executed as a high-denticity ligand for chelation of [225Ac]Ac3+, and the complexation characteristics have been explored through nuclear magnetic resonance (NMR) spectroscopy, solution thermodynamic stability studies, and radiolabeling. The ligand shows highly favorable complexation with La3+ (pM = 17.6), Lu3+ (pM = 21.3), and In3+ (pM = 31.2) and demonstrates effective radiolabeling of both [225Ac]Ac3+ and [111In]In3+ ions achieving quantitative radiochemical conversions (RCCs) under mild conditions (RT, 10 min), accompanied by high serum stability (>97% radiochemical purity (RCP) over 6 days). A bifunctional analogue of H4picoopa was synthesized and conjugated to the Pip-Nle-CycMSHhex peptide for targeting of MC1R positive melanoma tumors. In vivo single-photon emission computed tomography (SPECT) and biodistribution studies of the 111In-radiolabeled bioconjugate in mice bearing B16-F10 tumors showed good radiotracer stability, although improved tumor targeting could not be achieved for imaging purposes. This work highlights H4picoopa as a very promising platform for application of [225Ac]Ac3+ and [111In]In3+ as a theranostic pair and allows great versatility for the incorporation of other directing vectors. The logical synthetic approach reported here for bifunctional H4picoopa, involving an azide-functionalized covalent linker and CuI-catalyzed alkyne-azide cycloaddition, allows for ease of optimization of bioconjugate pharmacokinetics and will be valuable for further radiopharmaceutical applications moving forward.

Suppression of high mobility group box 1 in B16F10 tumor does not inhibit the induction of neoantigen-specific T cells.

Accumulated clinical data of immune checkpoint blockades have suggested the importance of neoantigens in cancer immunity. Tumor antigens are released from dead cancer cells together with cellular components, such as damage-associated molecular patterns (DAMPs), into the tumor microenvironment. We recently reported that high mobility group box 1 (HMGB1), a representative DAMP molecule, showed a negative impact on anti-tumor immunity. However, a positive role of HMGB1 in the initiation of innate and subsequent adaptive immunity has also been demonstrated; thus, the effects of HMGB1 on anti-tumor immunity have not been well understood. In this study, we identified nine immunogenic neoantigen epitopes of B16F10 murine melanoma cells and subsequently investigated the effects of suppression of HMGB1 on the induction of neoantigen-specific immunity using HMGB1-knockout tumors. Neoantigen-reactive T cells were expanded in B16F10 tumor-bearing mice, and T cell receptor repertoire analysis suggested that neoantigen-reactive T cells were oligo-clonally increased in B16F10 tumor bearers. An increase of neoantigen-reactive T cells and oligoclonal expansion of the T cells were similarly detected in HMGB1-knockout tumor-bearing mice. The induction of neoantigen-specific immunity under the suppression of HMGB1 in the tumor microenvironment shown in this study supports further development of combination therapy of HMGB1 suppression with neoantigen-targeted cancer immunotherapies, including immune checkpoint blockade therapy.

Exercise Promotes Pro-Apoptotic Ceramide Signaling in a Mouse Melanoma Model.

Simple SummaryExercise has been shown to improve the efficacy of chemotherapy against several tumor models using mice through modulating tumor vascular perfusion, immune function, circulating growth factors, hypoxia, and metabolism in tumor cells and their surrounding microenvironment. However, little is known about the effect of exercise on tumor-cell-intrinsic death mechanisms, such as apoptosis. Ceramide is a bioactive lipid that can promote cell death. The strategy of increasing intracellular ceramide has potential as an anticancer treatment for melanoma with dysregulated ceramide metabolism, but there is not yet a clinically relevant method to do so. We found that moderate aerobic exercise increases pro-apoptotic ceramide in melanoma in mice, and activates p53 signaling, promoting tumor cell apoptosis. This finding suggests that exercise may be most effective as an adjuvant therapy to sensitize cancer cells to anticancer treatments in tumors that exhibit downregulated ceramide generation to evade cell death.Ceramides are essential sphingolipids that mediate cell death and survival. Low ceramide content in melanoma is one mechanism of drug resistance. Thus, increasing the ceramide content in tumor cells is likely to increase their sensitivity to cytotoxic therapy. Aerobic exercise has been shown to modulate ceramide metabolism in healthy tissue, but the relationship between exercise and ceramide in tumors has not been evaluated. Here, we demonstrate that aerobic exercise causes tumor cell apoptosis and accumulation of pro-apoptotic ceramides in B16F10 but not BP melanoma models using mice. B16F10 tumor-bearing mice were treated with two weeks of moderate treadmill exercise, or were control, unexercised mice. A reverse-phase protein array was used to identify canonical p53 apoptotic signaling as a key pathway upregulated by exercise, and we demonstrate increased apoptosis in tumors from exercised mice. Consistent with this finding, pro-apoptotic C16-ceramide, and the ceramide generating enzyme ceramide synthase 6 (CerS6), were higher in B16F10 tumors from exercised mice, while pro-survival sphingosine kinase 1 (Sphk1) was lower. These data suggest that exercise contributes to B16F10 tumor cell death, possibly by modulating ceramide metabolism toward a pro-apoptotic ceramide/sphingosine-1-phosphate balance. However, these results are not consistent in BP tumors, demonstrating that exercise can have different effects on tumors of different patient or mouse origin with the same diagnosis. This work indicates that exercise might be most effective as a therapeutic adjuvant with therapies that kill tumor cells in a ceramide-dependent manner.