B16F10 Tumor Research Articles

18F-Labeled Amidobenzimidazole Analogue for Visualizing STING Expression in Tumor.

The stimulator of interferon genes (STING) is pivotal in mediating STING-dependent type I interferon production, which is crucial for enhancing tumor rejection. Visualizing STING within the tumor microenvironment is valuable for STING-related treatments, yet the availability of suitable STING imaging probes is limited. In this study, we developed [18F]AlF-ABI, a novel 18F-labeled agent featuring an amidobenzimidazole core structure, for positron emission tomography (PET) imaging of STING in B16F10 and CT26 tumors. [18F]AlF-ABI was synthesized with a decay-corrected radiochemical yield of 38.0 ± 7.9% and radiochemical purity exceeding 97%. The probe exhibited a nanomolar STING binding affinity (KD = 35.6 nM). Upon administration, [18F]AlF-ABI rapidly accumulated at tumor sites, demonstrating significantly higher uptake in B16F10 tumors compared to CT26 tumors, consistent with STING immunofluorescence patterns. Specificity was further validated through in vitro cell experiments and in vivo blocking PET imaging. These findings suggest that [18F]AlF-ABI holds promise as an effective agent for visualizing STING in the tumor microenvironment.

Neutrophils bearing adhesive polymer micropatches as a drug-free cancer immunotherapy.

Tumour-associated neutrophils can exert antitumour effects but can also assume a pro-tumoural phenotype in the immunosuppressive tumour microenvironment. Here we show that neutrophils can be polarized towards the antitumour phenotype by discoidal polymer micrometric 'patches' that adhere to the neutrophils' surfaces without being internalized. Intravenously administered micropatch-loaded neutrophils accumulated in the spleen and in tumour-draining lymph nodes, and activated splenic natural killer cells and T cells, increasing the accumulation of dendritic cells and natural killer cells. In mice bearing subcutaneous B16F10 tumours or orthotopic 4T1 tumours, intravenous injection of the micropatch-loaded neutrophils led to robust systemic immune responses, a reduction in tumour burden and improvements in survival rates. Micropatch-activated neutrophils combined with the checkpoint inhibitor anti-cytotoxic T-lymphocyte-associated protein 4 resulted in strong inhibition of the growth of B16F10 tumours, and in complete tumour regression in one-third of the treated mice. Micropatch-loaded neutrophils could provide a potent, scalable and drug-free approach for neutrophil-based cancer immunotherapy.

Cancer-associated Fibroblast-specific Expression of the Matricellular Protein CCN1 Coordinates Neovascularization and Stroma Deposition in Melanoma Metastasis.

In human patients, the expression of proangiogenic matricellular protein CCN1 in CAFs correlates positively with expression of stroma and angiogenic markers and progressive disease/resistance to checkpoint inhibitor therapy. In an animal model, loss of CCN1 from CAFs impaired metastasis of melanoma cells, neovascularization, and collagen deposition, emphasizing that CAFs coordinate cellular behavior in a tumor microenvironment and that CCN1 may be a novel target.

Benzimidazole-oxindole hybrids as multi-kinase inhibitors targeting melanoma

In the current study, a series of benzimidazole-oxindole conjugates 8a-t were designed and synthesized as type II multi-kinase inhibitors. They exhibited moderate to potent inhibitory activity against BRAFWT up to 99.61 % at 10 µM. Notably, compounds 8e, 8k, 8n and 8s demonstrated the most promising activity, with 99.44 to 99.61 % inhibition. Further evaluation revealed that 8e, 8k, 8n and 8s exhibit moderate to potent inhibitory effects on the kinases BRAFV600E, VEGFR-2, and FGFR-1. Additionally, compounds 8a-t were screened for their cytotoxicity by the NCI, and several compounds showed significant growth inhibition in diverse cancer cell lines. Compound 8e stood out with a GI50 range of 1.23 – 3.38 µM on melanoma cell lines. Encouraged by its efficacy, it was further investigated for its antitumor activity and mechanism of action, using sorafenib as a reference standard. The hybrid compound 8e exhibited potent cellular-level suppression of BRAFWT, VEGFR-2, and FGFR-1 in A375 cell line, surpassing the effects of sorafenib. In vivo studies demonstrate that 8e significantly inhibits the growth of B16F10 tumors in mice, leading to increased survival rates and histopathological tumor regression. Furthermore, 8e reduces angiogenesis markers, mRNA expression levels of VEGFR-2 and FGFR-1, and production of growth factors. It also downregulated Notch1 protein expression and decreased TGF-β1 production. Molecular docking simulations suggest that 8e binds as a promising type II kinase inhibitor in the target kinases interacting with the key regions in their kinase domain.

Reinforced Immunogenic Endoplasmic Reticulum Stress and Oxidative Stress via an Orchestrated Nanophotoinducer to Boost Cancer Photoimmunotherapy.

Cancer progression and treatment-associated cellular stress impairs therapeutic outcome by inducing resistance. Endoplasmic reticulum (ER) stress is responsible for core events. Aberrant activation of stress sensors and their downstream components to disrupt homeostasis have emerged as vital regulators of tumor progression as well as response to cancer therapy. Here, an orchestrated nanophotoinducer (ERsNP) results in specific tumor ER-homing, induces hyperthermia and mounting oxidative stress associated reactive oxygen species (ROS), and provokes intense and lethal ER stress upon near-infrared laser irradiation. The strengthened "dying" of ER stress and ROS subsequently induce apoptosis for both primary and abscopal B16F10 and GL261 tumors, and promote damage-associated molecular patterns to evoke stress-dependent immunogenic cell death effects and release "self-antigens". Thus, there is a cascade to activate maturation of dendritic cells, reprogram myeloid-derived suppressor cells to manipulate immunosuppression, and recruit cytotoxic T lymphocytes and effective antitumor response. The long-term protection against tumor recurrence is realized through cascaded combinatorial preoperative and postoperative photoimmunotherapy including the chemokine (C-C motif) receptor 2 antagonist, ERsNP upon laser irradiation, and an immune checkpoint inhibitor. The results highlight great promise of the orchestrated nanophotoinducer to exert potent immunogenic cell stress and death by reinforcing ER stress and oxidative stress to boost cancer photoimmunotherapy.

Synthesis and preclinical evaluation of a novel molecular probe [18F]AlF-NOTA-PEG2-Asp2-PDL1P for PET imaging of PD-L1 positive tumor

Immunotherapy has brought great benefits to cancer patients, but only some patients benefit from it. Noninvasive, real-time and dynamic monitoring of the effectiveness of immunotherapy through PET imaging may provide assistance for the treatment plan of immunotherapy. In this study, we designed and synthesized a new targeted PD-L1 peptide NOTA-PEG2-Asp2-PDL1P, which was labeled with nuclide 18F to obtain a new imaging agent [18F]AlF-NOTA-PEG2-Asp2-PDL1P. The total radiochemical yield of [18F]AlF-NOTA-PEG2-Asp2-PDL1P was 13.7 % (Uncorrected radiochemical yield, n > 5). [18F]AlF-NOTA-PEG2-Asp2-PDL1P achieved high radiochemical purity (>95 %) with a molar activity more than 51.2 GBq/μmol. [18F]AlF-NOTA-PEG2-Asp2-PDL1P exhibited good hydrophilicity and had good stability both in vivo and in vitro, it can specifically targets B16F10 tumor with PD-L1 expression, and had a relatively high retention in tumor, a relatively fast clearance in vivo and a higher tumor-to-non-target ratio, all of which could make [18F]AlF-NOTA-PEG2-Asp2-PDL1P a potential tracer for PD-L1 prediction before clinical immunotherapy.

Modification of the tumor microenvironment enhances immunity with plasmid gene therapy.

Local intratumor delivery with electroporation of low levels of plasmids encoding molecules, induces an antitumor effect without causing systemic toxicity. However, previous studies have predominately focused on the function of the delivered molecule encoded within the plasmid, and ignored the plasmid vector. In this study, we found vectors pUMVC3 and pVax1 induced upregulation of MHC class I (MHC-I) and PD-L1 on tumor cell surface. These molecules participate in a considerable number of immunoregulatory functions through their interactions with and activating inhibitory immune cell receptors. MHC molecules are well-known for their role in antigen (cross-) presentation, thereby functioning as key players in the communication between immune cells and tumor cells. Increased PD-L1 expression on tumor cells is an important monitor of tumor growth and the effectiveness of immune inhibitor therapy. Results from flow cytometry confirmed increased expression of MHC-I and PDL-1 on B16F10, 4T1, and KPC tumor cell lines. Preliminary animal data from tumor-bearing models, B16F10 melanoma, 4T1 breast cancer and KPC pancreatic cancer mouse models showed that tumor growth was attenuated after pUMVC3 intratumoral electroporation. Our data also documented that pSTAT1 signaling pathway might not be associated with plasmid vectors' function of upregulating MHC-I, PD-L1 on tumor cells.

An Engineered M13 Filamentous Nanoparticle as an Antigen Carrier for a Malignant Melanoma Immunotherapeutic Strategy.

Bacteriophages, prokaryotic viruses, hold great potential in genetic engineering to open up new avenues for vaccine development. Our study aimed to establish engineered M13 bacteriophages expressing MAGE-A1 tumor peptides as a vaccine for melanoma treatment. Through in vivo experiments, we sought to assess their ability to induce robust immune responses. Using phage display technology, we engineered two M13 bacteriophages expressing MAGE-A1 peptides as fusion proteins with either pVIII or pIIII coat proteins. Mice were intraperitoneally vaccinated three times, two weeks apart, using two different engineered bacteriophages; control groups received a wild-type bacteriophage. Serum samples taken seven days after each vaccination were analyzed by ELISA assay, while splenocytes harvested seven days following the second boost were evaluated by ex vivo cytotoxicity assay. Fusion proteins were confirmed by Western blot and nano-LC-MS/MS. The application of bacteriophages was safe, with no adverse effects on mice. Engineered bacteriophages effectively triggered immune responses, leading to increased levels of anti-MAGE-A1 antibodies in proportion to the administered bacteriophage dosage. Anti-MAGE-A1 antibodies also exhibited a binding capability to B16F10 tumor cells in vitro, as opposed to control samples. Splenocytes demonstrated enhanced CTL cytotoxicity against B16F10 cells. We have demonstrated the immunogenic capabilities of engineered M13 bacteriophages, emphasizing their potential for melanoma immunotherapy.

Brachytherapy at the nanoscale with protein functionalized and intrinsically radiolabeled [169Yb]Yb2O3 nanoseeds.

Classical brachytherapy of solid malignant tumors is an invasive procedure which often results in an uneven dose distribution, while requiring surgical removal of sealed radioactive seed sources after a certain period of time. To circumvent these issues, we report the synthesis of intrinsically radiolabeled and gum Arabic glycoprotein functionalized [169Yb]Yb2O3 nanoseeds as a novel nanoscale brachytherapy agent, which could directly be administered via intratumoral injection for tumor therapy. 169Yb (T½ = 32days) was produced by neutron irradiation of enriched (15.2% in 168Yb) Yb2O3 target in a nuclear reactor, radiochemically converted to [169Yb]YbCl3 and used for nanoparticle (NP) synthesis. Intrinsically radiolabeled NP were synthesized by controlled hydrolysis of Yb3+ ions in gum Arabic glycoprotein medium. In vivo SPECT/CT imaging, autoradiography, and biodistribution studies were performed after intratumoral injection of radiolabeled NP in B16F10 tumor bearing C57BL/6 mice. Systematic tumor regression studies and histopathological analyses were performed to demonstrate therapeutic efficacy in the same mice model. The nanoformulation was a clear solution having high colloidal and radiochemical stability. Uniform distribution and retention of the radiolabeled nanoformulation in the tumor mass were observed via SPECT/CT imaging and autoradiography studies. In a tumor regression study, tumor growth was significantly arrested with different doses of radiolabeled NP compared to the control and the best treatment effect was observed with ~ 27.8MBq dose. In histopathological analysis, loss of mitotic cells was apparent in tumor tissue of treated groups, whereas no significant damage in kidney, lungs, and liver tissue morphology was observed. These results hold promise for nanoscale brachytherapy to become a clinically practical treatment modality for unresectable solid cancers.

Systemic Delivery of Paclitaxel by Find-Me Nanoparticles Activates Antitumor Immunity and Eliminates Tumors.

Local delivery of immune-activating agents has shown promise in overcoming an immunosuppressive tumor microenvironment (TME) and stimulating antitumor immune responses in tumors. However, systemic therapy is ultimately needed to treat tumors that are not readily locatable or accessible. To enable systemic delivery of immune-activating agents, we employ poly(lactic-co-glycolide) (PLGA) nanoparticles (NPs) with a track record in systemic application. The surface of PLGA NPs is decorated with adenosine triphosphate (ATP), a damage-associated molecular pattern to recruit antigen-presenting cells (APCs). The ATP-conjugated PLGA NPs (NPpD-ATP) are loaded with paclitaxel (PTX), a chemotherapeutic agent inducing immunogenic cell death to generate tumor antigens in situ. We show that the NPpD-ATP retains ATP activity in hostile TME and provides a stable "find-me" signal to recruit APCs. Therefore, the PTX-loaded NPpD-ATP helps populate antitumor immune cells in TME and attenuate the growth of CT26 and B16F10 tumors better than a mixture of PTX-loaded NPpD and ATP. Combined with anti-PD-1 antibody, PTX-loaded NPpD-ATP achieves complete regression of CT26 tumors followed by antitumor immune memory. This study demonstrates the feasibility of systemic immunotherapy using a PLGA NP formulation that delivers ICD-inducing chemotherapy and an immunostimulatory signal.

Immunomodulatory Function of Pien Tze Huang in T Cell-Mediated Anti-tumor Activity against B16-F10, MC38 and Hep1-6 Tumor Models.

To investigate the anti-tumor effects of Pien Tze Huang (PZH) in mouse models of B16-F10 melanoma, MC38 colorectal cancer, Hep1-6 hepatocellular carcinoma and chemically induced hepatocellular carcinoma model. Various tumor models, including B16-F10, MC38 and Hep1-6 tumor hypodermic inoculation models, B16-F10 and Hep1-6 pulmonary metastasis models, Hep1-6 orthotopic implantation model, and chemically induced hepatocellular carcinoma model, were utilized to evaluate the anti-tumor function of PZH. Tumor growth was assessed by measuring tumor size and weight of solid tumors isolated from C57BL/6 mice. For cell proliferation and death of tumor cells in vitro, as well as T cell activation markers, cytokine production and immune checkpoints analysis, single-cell suspensions were prepared from mouse spleen, lymph nodes, and tumors after PZH treatment. PZH demonstrated significant therapeutic efficacy in inhibiting tumor growth (P<0.01). Treatment with PZH resulted in a reduction in tumor size in subcutaneous MC38 colon adenocarcinoma and B16-F10 melanoma models, and decreased pulmonary metastasis of B16-F10 melanoma and Hep1-6 hepatoma (P<0.01). However, in vitro experiments showed that PZH only had slight impact on the cell proliferation and survival of tumor cells (P>0.05). Nevertheless, PZH exhibited a remarkable ability to enhance T cell activation and the production of interferon gamma, tumor necrosis factor alpha, and interleukin 2 in CD4+ T cells in vitro (P<0.01 or P<0.05). Importantly, PZH substantially inhibited T cell exhaustion and boosted cytokine production by tumor-infiltrating CD8+ T cells (P<0.01 or P<0.05). This study has confirmed a novel immunomodulatory function of PZH in T cell-mediated anti-tumor immunity, indicating that PZH holds promise as a potential therapeutic agent for cancer treatment.

Comparative Study of the Immune Microenvironment in Heterotopic Tumor Models.

The tumor microenvironment (TME) is pivotal in cancer progression and the response to immunotherapy. A "hot" tumor typically contains immune cells that promote anti-tumor immunity, predicting positive prognosis. "Cold" tumors lack immune cells, suggesting a poor outlook across various cancers. Recent research has focused on converting "cold" tumors into "hot" tumors to enhance the success of immunotherapy. A prerequisite for the studies of the TME is an accurate knowledge of the cell populations of the TME. This study aimed to describe the immune TME of lung and colorectal cancer and melanoma, focusing on lymphoid and myeloid cell populations. We induced heterotopic immunocompetent tumors in C57BL/6 mice, using KP and LLC (Lewis lung carcinoma) cells for lung cancer, MC38 cells for colorectal cancer, and B16-F10 cells for melanoma. Immune cell infiltration was analyzed using multicolor flow cytometry in single-cell suspensions after tumor excision. KP cell tumors showed an abundance of neutrophils and eosinophils; however, they contained much less adaptive immune cells, while LLC cell tumors predominated in monocytes, neutrophils, and monocyte-derived dendritic cells. Monocytes and neutrophils, along with a significant T cell infiltration, were prevalent in MC38 tumors. Lastly, B16-F10 tumors were enriched in macrophages, while showing only moderate T cell presence. In conclusion, our data provide a detailed overview of the immune TME of various heterotopic tumors, highlighting the variabilities in the immune cell profiles of different tumor entities. Our data may be a helpful basis when investigating new immunotherapies, and thus, this report serves as a helpful tool for preclinical immunotherapy research design.

Preclinical Ultra-High Dose Rate (FLASH) Proton Radiation Therapy System for Small Animal Studies

BackgroundAnimal studies with ultra-high dose-rate radiation therapy (FLASH, >40Gy/s) preferentially spare normal tissues without sacrificing anti-tumor efficacy compared to conventional dose-rate radiation therapy (CONV). At XXXX, we developed a cyclotron-generated preclinical scattered proton beam with FLASH dose rates. We present the technical details of our FLASH radiation system and preliminary biological results from whole pelvis radiation. MethodsA Scanditronix MC50 compact cyclotron beamline has been modified to produce a 48.7 MeV proton beam at dose rates between 0.1-150 Gy/s. The system produces a 6 cm diameter scattered proton beam (flat to +/-3%) at the target location. Female C57BL/6 mice 5-6 weeks old were used for all experiments. To study normal tissue effects in the distal colon, mice were irradiated using the entrance region of the proton beam to the whole pelvis, 18.5 Gy at different dose rates: control, CONV (0.6–1 Gy/s) and FLASH (50–80 Gy/s). Survival was monitored daily and EdU (5-ethynyl-2´-deoxyuridine) staining was performed at 24-hours and 96-hours post-radiation. Cleaved caspase-3 (C-CASP3) staining was performed 24-hours post-radiation. To study tumor control, allograft B16F10 tumors were implanted in the right flank and received 18 Gy CONV or FLASH proton radiation. Tumor growth and survival were monitored. ResultsAfter 18.5 Gy whole pelvis radiation, survival was 100% in the control group, 0% in the CONV group, and 44% in the FLASH group (p<0.01). EdU staining showed cell proliferation was significantly higher in the FLASH versus CONV group at both 24-hours and 96-hours post-radiation in the distal colon, although both radiation groups showed decreased proliferation compared to controls (P<0.05). Lower C-CASP3 staining was seen in the FLASH versus conventional group post-radiation (p<0.05). Comparable flank tumor control was observed in the CONV and FLASH groups. ConclusionWe present our preclinical FLASH proton radiation system and biological results showing improved survival after whole pelvis radiation, with equivalent tumor control.

Celastrol, which targets IL-2/CD25 binding inhibition, induces T cell-mediated antitumor activity in melanoma

Interleukin-2 (IL-2) induces contrasting immune responses depending on its binding receptor subunit; thus, selective receptor binding is considered a key challenge in cancer therapeutic strategies. In this study, we aimed to investigate the inhibition of IL-2 action and antitumor activity of celastrol (CEL), a compound identified in a screen for IL-2/CD25 binding inhibitors, and to elucidate the underlying role of CEL in immune cells. We found that CEL selectively impairs the binding of IL-2 and CD25 and directly binds to IL-2 but not to CD25. CEL significantly suppressed the proliferation and signaling of IL-2-dependent murine T cells and interfered with IL-2-responsive STAT5 phosphorylation in IL-2 reporter cells and human PBMCs. After confirming the impact of CEL on IL-2, we evaluated its antitumor activity in C57BL/6 mice bearing B16F10 tumors and found that CEL significantly inhibited tumor growth by increasing CD8+ T cells. We also found that CEL did not inhibit tumor growth in T cell-deficient BALB/c nude mice, suggesting that its activity was mediated by the T-cell response. Moreover, combination therapy with low-dose CEL and a TNFR2 antagonist synergistically improved the therapeutic efficacy of the individual monotherapies by increasing the ratio of intratumoral CD8/Treg cells and suppressing Foxp3 expression.These findings suggest that CEL, which inhibits CD25 binding by targeting IL-2, exerts antitumor activity by mediating the T-cell response and could be a promising candidate for combination therapy in cancer immunotherapy against melanoma.

Abstract A005: Synergistically Engaging a b-Selective IL-2 Agonist with PD1/PDL-1 Blockade in a Bifunctional Superkine, MDNA223

Abstract Introduction: Combination treatment with a long-acting IL-2 superkine (MDNA109FEAA-Fc; aka MDNA19) and anti-PD1 or anti-CTLA4 has shown clear evidence of synergy in blunting tumor growth. To further leverage this synergism, we designed MDNA223, a BiSKIT (Bifunctional SuperKine for ImmunoTherapy) comprising MDNA109FEAA with enhanced affinity for IL-2Rb and no binding to IL-2Ra, fused to either human or mouse anti-PD1. We present in vitro and in vivo characterization of MDNA223 and demonstrate its enhanced IL-2R agonism and effective PD1/PDL-1 blockade in multiple “hot” and “cold” syngeneic tumor models. Experimental Procedure: In vitro studies included human PBMCs and cell-based reporter assays. In vivo studies were conducted in CT26, B16F10, E0771 and TRAMP-C1 tumor models by intraperiotonneal (IP) administration. Intratumoral treatment was performed in a CT26 bilateral tumor model with or without a STING agonist. Analysis of tumor infiltrating lymphocytes (TILs) was performed using flow cytometry. Results: In studies with human PBMCs, MDNA223 showed increased p-STAT5 signaling in CD8+ T cells and reduced activity in Tregs compared to rhIL-2, consistent with its enhanced receptor selectivity. MDNA223 and anti-PD1 were similarly potent at PD1/PDL-1 blockade in a cell-based reporter assay. In mice, MDNA223 induced proliferation (based on Ki67 expression) and expansion of peripheral CD8+ T cells for over 7 days. IP treatment with MDNA223 resulted in significantly greater inhibition of CT26, B16F10, E0771 and TRAMP-C1 tumor growth when compared to combination of MDNA19 with anti-PD1 at equimolar dosage. This observation is consistent with the proposed cis-binding mechanism of MDNA223 to both, IL-2R and PD1 on the same effector immune cell. In a bilateral CT26 tumor model, IT treatment with MDNA223 not only inhibited the treated tumor but also exhibited an abscopal effect by slowing growth of the untreated tumor implanted on the opposite flank. TILs analysis of B16F10 melanoma following a single dose of MDNA223 showed increased infiltration of CD8+ T cells over Tregs compared to single agent treatment with MDNA19 or anti-PD1 as well as their combination. Treatment with MDNA223 also resulted in a larger population of functional effector CD8+ T cells expressing low levels of the Tim-3 exhaustion marker and high levels of the cytotoxic Granzyme-B protease. Conclusion: MDNA223 demonstrated superior therapeutic activity over the combination of MDNA19 and anti-PD1 in both immunologically ‘hot’ (CT26) and ‘cold’ (B16F10, E0771 and TRAMP-C1) tumor models. Additional studies with variants of MDNA223 capable of tumor selective targeting and/or conditional activation within the tumor micro-environment are in progress to maximize activity at the tumor site with tunable peripheral stimulation. Citation Format: Aanchal Sharma, Minh D To, Hardeep Kataria, Qian Liu, Rosemina Merchant, Fahar Merchant. Synergistically Engaging a b-Selective IL-2 Agonist with PD1/PDL-1 Blockade in a Bifunctional Superkine, MDNA223 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A005.

Abstract A012: STING agonists drive intrinsic type I interferon responses in monocytes for optimal anti-tumor immunity

Abstract The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is triggered by the sensing of cytosolic DNA, triggering the production of type I interferon (IFN-I) and play a crucial role in host defense against pathogens. This pathway also induces an anti-tumor response when tumor-derived DNA is sensed. Small molecule agonists called STING agonists activate this pathway and have been shown to cause tumor regression in mice. However, it is unclear how immune cells respond to STING agonist-induced IFN-I. In our study, we observed that monocytes were abundantly recruited to tumors during STING agonist therapy, and the lack of CCR2+ Ly6Chi inflammatory monocytes in CCR2-knockout (KO) mice led to impaired responses to STING agonist therapy of B16F10 and MC-38 tumors. Single-cell RNA sequencing (scRNA-Seq) of CD45+ cells from lymph nodes and tumors of Ifnar1 mixed bone marrow chimeric mice in which half of the immune cells lacked IFN-I receptors revealed that inflammatory monocytes upregulated chemokine receptor ccr5 and genes associated with MHC-I restricted antigen presentation in response to IFN-I. Using mice with B16.OVA.Tomato red fluorescent tumors, we demonstrated that IFN-I-responsive monocytes captured fluorescent antigens in tumors and transported them to lymph nodes. Additionally, the administration of CCR5 antagonist, maraviroc, significantly reduced the number of fluorescent antigen-carrying monocytes in the lymph nodes of red fluorescent tumor-bearing mice treated with STING agonists, indicating the involvement of CCR5 in monocyte-mediated antigen transport. Furthermore, NicheNet analysis of scRNA-Seq data showed that IL-18 - IL-18R1 interaction was the top predicted interaction between inflammatory monocytes and CD8+ T cells or NK cells. Blocking IL-18 remarkedly reduced the therapeutic efficacy of STING agonist treatment. Moreover, IFN-I acted on monocytes to upregulate glucocorticoid-induced TNFR-related (GITRL) providing a pro-survival signal to T cells. Deletion of GITRL on CCR2+ monocytes led to impaired tumor control, and the absence of the cognate receptor GITR on T cells led to impaired anti-tumor effector T cell responses in the tumors treated with and tissue-resident memory T cell accumulation in the skins that rejected tumors with STING agonist therapy. Taken together, our data suggests that STING agonists induce IFN-I-responsive monocytes to promote anti-tumor responses through secreting IL-18, potentially through transporting antigens to lymph nodes, and through providing a pro-survival signal for T cell accumulation in the tumors during STING agonist therapy. Citation Format: Tianning Yu, Melanie Girard, Tania H Watts. STING agonists drive intrinsic type I interferon responses in monocytes for optimal anti-tumor immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A012.

Abstract A074: Discovery of WTX-330, a clinical stage conditionally activated IL-12 INDUKINETM therapeutic with potent antitumor activity in murine syngeneic tumor models resistant to checkpoint blockade

Abstract Background: IL-12 is a pleotropic inflammatory cytokine with broad stimulatory effects on immune cells and robust antitumor activity in syngeneic murine tumor models, making it an attractive target for cancer immunotherapy. Unfortunately, systemic administration of recombinant human IL-12 causes severe toxicity in patients. With the goal of improving the therapeutic index for IL-12 therapy, we have engineered an IL-12 prodrug referred to as an INDUKINETM molecule that is dosed systemically but designed to be selectively activated in tumors. Methods: Mice bearing MC38, CT26, B16F10, and EMT-6 syngeneic tumors were treated with surrogate mWTX-330 (a chimeric IL-12 containing INDUKINETM molecule), and tumor growth and body weight were monitored over time. Tumor tissues were harvested at various timepoints and analyzed by flow cytometry, NanoString GeoMx Digital Spatial Profiling (DSP), and high-plex immunofluorescence to characterize the mechanisms associated with response. Results: mWTX-330 is comprised of a half-life extension domain and an inactivation domain tethered to chimeric IL-12 by tumor protease-sensitive linkers. These linkers were chosen for their selective processing by a majority of human tumors in vitro as well as mouse tumors in vitro and in vivo. The linkers have been shown in preclinical studies to remain stable in normal human tissues including serum. Systemic administration of mWTX-330 in mice was well tolerated, resulted in robust antitumor immunity in multiple tumor models, including those resistant to checkpoint blockade, and preferentially activated tumor-infiltrating immune cells versus peripheral cells. Antitumor activity was dependent on in vivo processing of the protease cleavable linkers and required CD8+ T cells for full efficacy, although NK and CD4+ T cells also contributed to tumor growth inhibition. Within the tumor, mWTX-330 increased the frequency of cross-presenting dendritic cells (DCs), activated natural killer (NK) cells, skewed conventional CD4+ T cells toward a T helper 1 (TH1) phenotype, drove regulatory T cells (Treg) fragility, and increased the frequency of polyfunctional CD8+ T cells. mWTX-330 treatment also increased the clonality of tumor-infiltrating T cells by expanding underrepresented T-cell receptor (TCR) clones, drove CD8+ T and NK cells towards increased mitochondrial respiration and fitness, and decreased the frequency of TOX+ exhausted CD8+ T cells within the tumor. Conclusion: Based on the strong biological rational and preclinical studies of surrogate mWTX-330, we created WTX-330, a fully human IL-12 INDUKINETM molecule that is stable in human serum and is reliably and selectively processed by human tumor samples in vitro. A first-in-human clinical trial to evaluate the safety profile, pharmacokinetics, and preliminary antitumor activity of WTX-330 in patients with relapsed/refractory advanced or metastatic solid tumors or non-Hodgkin lymphoma is now underway (NCT05678998). Citation Format: Christopher J Nirschl, Heather R Brodkin, Sameer S Chopra, Connor J Dwyer, Daniel J Hicklin, Randi Isaacs, Nesreen Ismail, Cynthia Seidel-Dugan, Zoe Steuert, Jenna M Sullivan, William M Winston, Andres Salmeron. Discovery of WTX-330, a clinical stage conditionally activated IL-12 INDUKINETM therapeutic with potent antitumor activity in murine syngeneic tumor models resistant to checkpoint blockade [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A074.

Abstract IA011: Engineered IL-12 delivery to the tumor matrix to achieve enhanced efficacy and safety in multiple advanced tumors

Abstract Background. Our goal is to create an effective and novel immunotherapy against multiple solid tumors with minimal toxicity. IL-12 is an “ideal” immune-activator, capable of activating both innate and acquired immune response. We tested more than 20 reported pro-inflammatory cytokines, and found IL-12 shows the most potent anti-tumor activity. Peripheral toxicity limits the use of systemic IL-12 therapy in the clinic. We have hypothesized that the tumor-specific drug delivery system would improve IL-12 immunotherapies. We took original approaches of protein engineering. Methods. We developed technologies to turn the tumor collagen into a drug reservoir after intravenous injections. The recombinant fusion of IL-12 to a collagen binding domain (CBD) derived from the VWF A3 domain allows for targeted IL-12 delivery into the tumor stroma. Here, we evaluated the pharmacology, safety, efficacy and the effects of CBD-IL-12 to the tumor microenvironment. Results. Intravenously-injected CBD proteins preferentially localized to the tumor, but not in other organs in mice. Single intravenous injection of CBD-IL-12 (recombinant fusion protein) eradicated most EMT6 breast cold tumors. CBD-IL-12-treated mice that were tumor-free developed systemic immunological memory, as 12 mice out of 13 rejected rechallenge with the breast tumor cells in their contralateral mammary fat pad. Single intravenous injection of CBD-IL-12 induced 67% complete remission of established B16F10 tumors. CBD fusion to IL-12 reduced the systemic toxicity, such as 50% reduction of hepatotoxicity markers and cytokine storm. CBD-IL-12 treatment increased intratumoral cytokine and immune cell numbers. In short, this tumor collagen-targeting approach improves both safety and efficacy of IL-12. We showed that the CBD can target multiple solid tumor types, because the CBD exploits the abnormality of tumor vasculature-specific pathological structure (e.g. leakiness and exposure of collagen to the bloodstream). So far, we have shown that CBD-IL-12 has significant anti-tumor efficacy in breast, lung, ovarian, liver, colon, prostate, melanoma, head and neck, pancreatic cancers and melanoma, sarcoma, and glioblastoma in syngeneic mouse models. CBD-IL-12 is effective in cold, advanced, spontaneous tumors, low-mutation burden tumors, and prevents metastasis and recurrence. CBD-IL-12 stimulated antigen-presenting cells and T cells. Also, humanized mice treated with humanized CBD-IL-12 show great anti-tumor effects in patient-derived lung and ovarian tumors. Humanized CBD-IL-12 was tested in dogs, and showed great tolerability at the therapeutic dose. Human CBD-IL-12 molecule was successfully manufactured in a clinical-grade. Conclusions. Intravenously injected CBD–IL-12 is effective to multiple difficult, immunologically cold tumors with low mutation burdens. Collagen is an attractive target because abnormally high expression of collagen is a shared nature of tumors, regardless of tumor types and patients. VWF A3 domain naturally exists in our body, minimizing the risk of immunogenicity. Citation Format: Jun Ishihara. Engineered IL-12 delivery to the tumor matrix to achieve enhanced efficacy and safety in multiple advanced tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr IA011.

Comparison of Tumor Binding Across Tumor Types and Cell Lines to Support Free Drug Considerations for Oncology Drug Discovery

Tumor binding is an important parameter to derive unbound tumor concentration to explore pharmacokinetics (PK) and pharmacodynamics (PD) relationships for oncology disease targets. Tumor binding was evaluated using eleven matrices, including various commonly used ex vivo human and mouse xenograft and syngeneic tumors, tumor cell lines and liver as a surrogate tissue. The results showed that tumor binding is highly correlated among the different tumors and tumor cell lines except for the mouse melanoma (B16F10) tumor type. Liver fraction unbound (fu) has a good correlation with B16F10 tumor binding. Liver also demonstrates a two-fold equivalency, on average, with binding of other tumor types when a scaling factor is applied. Predictive models were developed for tumor binding, with correlations established with LogD (acids), predicted muscle fu (neutrals) and measured plasma protein binding (bases) to estimate tumor fu when experimental data are not available. Many approaches can be applied to obtain and estimate tumor binding values. One strategy proposed is to use a surrogate tumor tissue, such as mouse xenograft ovarian cancer (OVCAR3) tumor, as a surrogate for tumor binding (except for B16F10) to provide an early assessment of unbound tumor concentrations for development of PK/PD relationships.

Crosstalk between NFAT and NFκB Signaling Regulates NK Cell Immunosurveillance

One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T Cells), controlling lymphocyte development and activity. The role of NFAT signaling is well defined in T cells, the cytotoxic lymphocytes of the adaptive immunity. However, surprisingly little is known regarding the relevance of this transcription factor family in NK cells as effector cells of the innate immunity. Available data indicate that NFAT activity is dispensable for development of NK cells, whereas effects of the immunosuppressive drugs cyclosporin A and tacrolimus, that inhibit calcineurin and consecutively NFAT, implicate an involvement of the NFAT family in NK cell function. We here employed different genetic mouse models and functional analyses to unravel the role of NFAT1 (NFATc2) and NFAT2 (NFATc1) in NK cell reactivity. In vitro knockout (KO) of NFAT1 (NFATc2) or NFAT2 (NFATc1) enhanced NK cell degranulation and resulted in increased production of granzyme B and perforin upon stimulation of activating receptors like NK1.1 or Nkp46 or upon co-culture with different leukemia and solid tumor cells. In line, cytotoxicity assays revealed increased lysis of YAC-1 and B16F10 tumor cells by both NFAT1- and NFAT2-deficient NK cells as compared to wildtype (WT) controls. The inhibitory effect of NFAT transcription factors on NK cell effector function could also be confirmed in vivo by employing WT and NFAT KO animals in syngeneic B16F10 melanoma, RMA-S lymphoma and LL/2-luc lung carcinoma models, which revealed a significantly reduced tumor burden in NFAT1 and NFAT2 KO mice. Furthermore, we detected higher numbers of tumor infiltrating NK cells in NFAT KO mice, which suggested improved NK cell migration and homing to the tumor side. Comparative analyses with single NFAT as well as NFAT1+NFAT2 double KO and WT animals further confirmed the inhibitory effect of NFAT1 and NFAT2 and pointed to additive effects of NFAT1 and NFAT2 in NK cell tumor immunosurveillance. Proteomics analysis of NK cells from the different NFAT KO strains revealed altered expression of molecules which are regulating chemokine receptor expression as well as a decreased expression of NFκB inhibitory molecules, like NFKBIB (NFKB inhibitor beta) Commd6 (COMM domain containing 6). NFκB signaling was shown to regulate granzyme B and perforin synthesis and expression. Thus, we suggest that NFAT1 and NFAT2 inhibit NFκB signaling in NK cells, and NFAT1 and NFAT2 KO reinforce NFκB induced granzyme B and perforin expression and thereby increase NK cell cytotoxicity. Taken together, our results identify NFAT as a negative regulator of NK cell function. In addition, we provide the first evidence for a direct functional involvement of NFAT1 and NFAT2 in NK cell antitumor reactivity by regulation tumor infiltration and cytotoxicity of NK cell.