Murine Colon Adenocarcinoma Research Articles

Improved antitumor effectiveness of oncolytic HSV-1 viruses engineered with IL-15/IL-15Rα complex combined with oncolytic HSV-1-aPD1 targets colon cancer

Oncolytic virotherapy is emerging as a promising therapeutic avenue for cancer treatment, harnessing both innate and tumor-specific immune responses for targeted tumor elimination. In this study, we present a novel oncolytic virus (oHSV1-IL15B) derived from herpes simplex virus-1 (HSV-1), armed with IL-15/IL-15Rα complex, with a focus on treating colon cancer combined with oncolytic HSV-1 expressing anti-PD-1 antibody (oHSV1-aPD1). Results from our study reveal that recombinant oHSV-1 virus equipped with IL-15/IL-15Rα complex exhibited significant anti-tumor effects in a murine CT26 colon adenocarcinoma model. Notably, oHSV1-IL15B combined with oHSV-1-aPD1 demonstrates superior tumor inhibition and prolonged overall survival compared to oHSV1-mock and monotherapy groups. Further exploration highlights the impact of oHSV1-IL15B, oHSV-1-aPD1 and combined group on antitumor capacity, revealing a substantial increase in CD8+ T and CD4+ T cell proportions of CT26-bearing BALB/c mice and promoting apoptosis in tumor tissue. The study emphasizes the pivotal role of cytotoxic CD8+T cells in oncolytic virotherapy, demonstrating that recombinant oHSV1-IL15B combined with oncolytic HSV-1-aPD1 induces a robust tumor-specific T cell response. RNA sequence analysis highlighted oHSV1-IL15B combined with oHSV1-aPD1 improved tumors immune microenvironment on immune response, antiviral response-related genes and apoptosis-related genes, which contributed to anti-tumor immunotherapy. The findings underscore the promising antitumor activity achieved through the combination of IL-15/IL-15Rα complex and anti-PD-1 antibody with oHSV-1. This research opens avenues for diverse therapeutic strategies, suggesting the potential of synergistically utilizing cytokines and anti-PD-1 antibody with oncolytic viruses to enhance immunotherapy for cancer management.

Abstract 5293: Preclinical development of NI-3201, a PD-L1xCD28 bispecific antibody mediating CD28 costimulation upon PD-L1 blockade

Abstract NI-3201 is a PD-L1xCD28 bispecific antibody generated on the κλ body platform that blocks the PD-L1/PD-1 immune checkpoint pathway and conditionally provides T cell activation “signal 2” when engaging PD-L1+ tumor or immune cells (e.g., antigen-presenting cells). By combining two distinct mechanisms of action into a single molecule, NI-3201 overcomes two major immune suppression mechanisms commonly observed in a variety of solid tumors, namely: [1] suppression of T cell response (induced by PD-L1+ cells engaging PD-1 on T cells) and [2] restricted/limited T cell activation (due to the lack of costimulatory signal associated to the scarcity of CD28 ligands in the TME). Importantly, NI-3201 was designed to require primary T cell stimulation (“signal 1”) to afford its activity: in the absence of TCR engagement, NI-3201 is unable to activate T cells, thus preventing unintended systemic inflammation. The single agent pharmacological activity of NI-3201 was assessed in vitro through a Cytomegalovirus (CMV) recall assay in which CMV+ PBMCs were cocultured with PD-L1+ cancer cell lines ectopically loaded with CMV-specific MHC-I peptides, thus providing “signal 1”. NI-3201 induced the activation and proliferation of CMV-specific T cells which resulted in killing of the target tumor cells. In vivo, the single agent activity of NI-3201 was shown in immunocompetent huCD28-transgenic mice engrafted with huPD-L1-expressing murine colon adenocarcinoma MC38 cells, a model in which NI-3201 induced durable anti-tumor immunological memory response. In humanized mouse models where T cell engagers (TCEs) were ineffective, NI-3201 was able to synergize with TCEs to control tumor growth, even inducing tumor regression in one model. The pharmacokinetics (PK) and tolerability of NI-3201 were investigated in non-human primates (NHP) after single and repeated-dosing. NI-3201 demonstrated favorable PK with close to dose-proportional concentrations being achieved. Upon repeated NI-3201 administration, no hypercytokinemia was observed; only a mild and transient IL-6 release and associated increase in C-reactive protein (CRP) levels were seen, with no clinical signs reported. The favorable profile seen in NHP was supported by data generated in vitro further de-risking the cytokine release syndrome (CRS) potential. Quantitative systems pharmacology (QSP) modelling integrating both in vitro and NHP data predicted a drug concentration required for optimal NI-3201 activity in the TME, and preliminary data suggests optimal patient activity at Q2W-Q3W dosing schedule. The anti-tumor activity as a single agent or as universal combination partner for TCEs, shown in vitro and in vivo, together with the favorable safety profile of NI-3201, support its clinical development planned to start in Q1 2025. Citation Format: Sara Majocchi, Nadia Anceriz, Alizée Viandier, Adeline Lesnier, Laura Cons, Lise Nouveau, Emily A. Pace, Bjorn L. Millard, Bruno Daubeuf, Valéry Moine, Susana Salgado-Pires, José Saro, Limin Shang, Krzysztof Masternak, Walter G. Ferlin. Preclinical development of NI-3201, a PD-L1xCD28 bispecific antibody mediating CD28 costimulation upon PD-L1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5293.

Development of rice bran-derived nanoparticles with excellent anti-cancer activity and their application for peritoneal dissemination

BackgroundRice bran a by-product of the rice milling process is currently underutilized. Recent studies have shown that plant-derived nanoparticles (pdNPs) can be mass-produced at a low cost and exhibit biological and therapeutic activities. Rice bran contains various anti-cancer compounds, including γ-oryzanol and γ-tocotrienol, and rice bran-derived nanoparticles (rbNPs) can be employed as novel therapeutic agents for cancer treatment.ResultsKoshihikari rice bran was suspended in water, and the suspension was centrifuged and filtered through a 0.45-µm-pore size syringe filter. The filtrate was ultracentrifuged, and the precipitates were suspended to obtain rbNPs. The rbNPs were negatively charged exosome-like nanoparticles with an average diameter of approximately 130 nm. The rbNPs exhibited cytotoxic activities against cancer cells but not against normal cells. The cytotoxic activity of rbNPs to murine colon adenocarcinoma colon26 cells was significantly greater than DOXIL® or other pdNPs. The rbNPs induced cell cycle arrest and apoptosis, and reduced the expression of proliferative proteins, including β-catenin and cyclin D1. Intraperitoneal injections of rbNPs into mice bearing peritoneal dissemination of colon26 cells significantly suppressed tumor growth with no significant adverse effects.ConclusionThese results indicated that rbNPs are promising nanoparticles, hold significant potential for anti-cancer applications, and are expected to play a vital role in cancer treatment.

Nanoparticles Synergize Ferroptosis and Cuproptosis to Potentiate Cancer Immunotherapy.

The recent discovery of copper-mediated and mitochondrion-dependent cuproptosis has aroused strong interest in harnessing this novel mechanism of cell death for cancer therapy. Here the design of a core-shell nanoparticle, CuP/Er, for the co-delivery of copper (Cu) and erastin (Er) to cancer cells for synergistic cuproptosis and ferroptosis is reported. The anti-Warburg effect of Er sensitizes tumor cells to Cu-mediated cuproptosis, leading to irreparable mitochondrial damage by depleting glutathione and enhancing lipid peroxidation. CuP/Er induces strong immunogenic cell death, enhances antigen presentation, and upregulates programmed death-ligand 1 expression. Consequently, CuP/Er promotes proliferation and infiltration of T cells, and when combined with immune checkpoint blockade, effectively reinvigorates T cells to mediate the regression of murine colon adenocarcinoma and triple-negative breast cancer and prevent tumor metastasis. This study suggests a unique opportunity to synergize cuproptosis and ferroptosis with combination therapy nanoparticles to elicit strong antitumor effects and potentiate current cancer immunotherapies.

Magnetic Bead-Based Workflow for Sensitive and Streamlined Cell Surface Proteomics.

Cell surface proteins represent an important class of molecules for therapeutic targeting and cellular phenotyping. However, their enrichment and detection via mass spectrometry-based proteomics remains challenging due to low abundance, post-translational modifications, hydrophobic regions, and processing requirements. To improve their identification, we optimized a Cell-Surface Capture (CSC) workflow that incorporates magnetic bead-based processing. Using this approach, we evaluated labeling conditions (biotin tags and catalysts), enrichment specificity (streptavidin beads), missed cleavages (lysis buffers), nonenzymatic deamidation (digestion and deglycosylation buffers), and data acquisition methods (DDA, DIA, and TMT). Our findings support the use of alkoxyamine-PEG4-biotin plus 5-methoxy-anthranilic acid, SDS/urea-based lysis buffers, single-pot solid-phased-enhanced sample-preparation (SP3), and streptavidin magnetic beads for maximal surfaceome coverage. Notably, with semiautomated processing, sample handling was simplified and between ∼600 and 900 cell surface N-glycoproteins were identified from only 25-200 μg of HeLa protein. CSC also revealed significant differences between in vitro monolayer cultures and in vivo tumor xenografts of murine CT26 colon adenocarcinoma samples that may aid in target identification for drug development. Overall, the improved efficiency of the magnetic-based CSC workflow identified both previously reported and novel N-glycosites with less material and high reproducibility that should help advance the field of surfaceomics by providing insight in cellular phenotypes not previously documented.

Synthesis and biological evaluation of 99mTc-tricarbonyl complexes of C-3 carboxy derivatives of fluoroquinolones in infection and tumor animal models

Fluoroquinolones (FQ) are clinically used antibacterial agents. Antibacterial fluoroquinolone derivatives have been shown to exhibit cytotoxic properties in tumor cells. In this work, C3-carboxy fluoroquinolones derivatives were radiolabeled with the diagnostic radionuclide technetium-99 m and were evaluated as infection- and tumor-targeted agents. First, ciprofloxacin and enrofloxacin were converted to 3-hydrazides and then to the respective pyridine hydrazones (L1 and L2). Secondly, 3-carboxamide of enrofloxacin was converted to dipicolylamine (L3), and picolylaminoacetate (L4) tridentate chelators. The respective rhenium-tricarbonyl complexes of L1-L4 were synthesized and characterized by spectroscopic methods and were used as non-radioactive standards to characterize the analogous 99mTc-radiotracers. The 99mTc-tricarbonyl complexes were synthesized and their lipophilicity and stability were evaluated in vitro. The most stable tracers 99mTcL3, 99mTcL4, were incubated with E. coli cells and CT-26 murine colon adenocarcinoma cells to assess their cellular uptake. Also, the 99mTc-tracers were injected in mice, infected with E. coli, as well as in mice, bearing CT-26 tumors.

Carbon Ion and Photon Radiation Therapy Show Enhanced Antitumoral Therapeutic Efficacy With Neoantigen RNA-LPX Vaccines in Preclinical Colon Carcinoma Models

Personalized liposome-formulated mRNA vaccines (RNA-LPX) are a new powerful tool in cancer immunotherapy. In preclinical tumor models, RNA-LPX are known to achieve potent results when combined together with conventional X-ray radiotherapy (XRT). Densely ionizing radiation used in carbon-ion radiotherapy (CIRT) may induce distinct effects in combination with immunotherapy, compared to sparsely ionizing X-rays. Within this study, we investigate the potential of CIRT and isoeffective doses of XRT to mediate tumor growth inhibition and survival in murine colon adenocarcinoma models in conjunction with neoantigen (neoAg)-specific RNA-LPX vaccines, encoding both MHC class I and II-restricted tumor-specific neoantigens. We characterize tumor immune infiltrates and antigen-specific T cell responses by flow cytometry and IFNγ ELISpot respectively. RNA-LPX vaccines significantly potentiate radiotherapy-mediated tumor growth inhibition. CIRT and XRT alone marginally prime neoAg-specific T cell responses in the tumor, but not in the blood or spleen of the mice. The infiltration and cytotoxicity of neoAg-specific T cells is strongly driven by RNA-LPX vaccines and accompanied by reduced expression of the inhibitory markers PD-1 and Tim-3 on these cells. NeoAg RNA-LPX vaccines shows similar overall therapeutic efficacy in combination with both CIRT and XRT, even if the physical radiation dose is lower for carbon ions than X-rays. We hence conclude that the combination of CIRT and neoAg RNA-LPX is a promising strategy for treatment of radioresistant tumors.

Singlet oxygen-generating cell-adhesive glass surfaces for the fundamental investigation of plasma membrane-targeted photodynamic therapy

Recently, plasma membrane-targeted photodynamic therapy has attracted attention as an effective cancer immunotherapeutic strategy. However, the released photosensitizers do not only adhere to the plasma membrane but may also be internalized in the cytosol, in endosomes/lysosomes, hindering investigations of the effects of photosensitizers attached to the plasma membrane. In this study, we developed a cell culture dish with singlet oxygen-generating cell-adhesive glass surfaces that allows investigation of the effects of photosensitizers attached to the plasma membrane. For cell adhesion, poly[N-(3-aminopropyl)methacrylamide] conjugated with hematoporphyrin PA–HpD was immobilized on the glass surfaces. Singlet oxygen was produced from the PA–HpD-immobilized glass surface upon laser irradiation at 635 nm. When murine colon adenocarcinoma 26 (Colon-26) cells were cultured on the PA–HpD-immobilized surface, the cells were swollen and ruptured, leading to effective apoptotic cell death using laser irradiation at 635 nm. In addition, microvesicles of approximately 10 μm in diameter were released from the plasma membrane into the culture medium. These phenomena were due to the oxidation of lipids in the cellular membrane, caused by the plasma membrane-targeted photodynamic therapy. In contrast, these phenomena were not observed on poly[N-(3-aminopropyl)methacrylamide]-immobilized glass surfaces. These results indicate that cell culture dishes with singlet oxygen-generating cell-adhesive glass surfaces can be used to investigate fundamental mechanisms in plasma membrane-targeted photodynamic therapy.

Biodegradable lipophilic polymeric mRNA nanoparticles for ligand-free targeting of splenic dendritic cells for cancer vaccination

Nanoparticle (NP)-based mRNA cancer vaccines hold great promise to realize personalized cancer treatments. To advance this technology requires delivery formulations for efficient intracellular delivery to antigen-presenting cells. We developed a class of bioreducible lipophilic poly(beta-amino ester) nanocarriers with quadpolymer architecture. The platform is agnostic to the mRNA sequence, with one-step self-assembly allowing for delivery of multiple antigen-encoding mRNAs as well as codelivery of nucleic acid-based adjuvants. We examined structure-function relationships for NP-mediated mRNA delivery to dendritic cells (DCs) and identified that a lipid subunit of the polymer structure was critical. Following intravenous administration, the engineered NP design facilitated targeted delivery to the spleen and preferential transfection of DCs without the need for surface functionalization with targeting ligands. Treatment with engineered NPs codelivering antigen-encoding mRNA and toll-like receptor agonist adjuvants led to robust antigen-specific CD8+ T cell responses, resulting in efficient antitumor therapy in invivo models of murine melanoma and colon adenocarcinoma.

Exploring the effect of different linkers on self-assembly, ROS-sensitivity and anticancer efficacy of hydroxyethyl starch-curcumin prodrug

Curcumin is a BCPR inhibitor, antiproliferative agent and apoptotic inducer to cancer cells, which is poorly soluble in water. In order to broaden the application of curcumin, conjugations of quaternary ammonium substituted hydroxyethyl starch (QHES) with curcumin by thioether bond (QHES-2S-CUR), disulfide bond (QHES-SS-CUR), and carbon-carbon bond (QHES-CC-CUR) were implemented, respectively. The effect of above three linkers on the self-assembly behavior of QHES-curcumin prodrugs was evaluated. QHES-2S-CUR and QHES-SS-CUR could facilely self-assemble into spherical micelles in water, while QHES-CC-CUR was lack of conformational stability. In vitro drug release profiles of prodrug micelles were further investigated. Concretely, QHES-2S-CUR presented ROS-sensitive drug release, with 73.57 ± 4.22% of curcumin released under the addition of H2O2 over 24 h. The release of curcumin from QHES-SS-CUR lasted for 54 h due to the slower oxidization of disulfide bonds, and QHES-CC-CUR was insensitive to ROS. In addition, QHES-2S-CUR prodrug micelles demonstrated superior cytotoxicity to murine colonic adenocarcinoma (CT-26) cells compared to QHES-SS-CUR and QHES-CC-CUR, which were internalized by CT-26 cells with high efficiency. These results reveal the potential of QHES-2S-CUR prodrug micelles as tumor microenvironment-responsive anticarcinogen.

Topology of cell-free DNA from tumour cells influences its immunogenicity in macrophages and dendritic cells in vitro

Abstract Activation of DNA-sensing pathways can induce an anti-tumour immune response, but the mechanisms by which tumour-derived cell-free DNA (cfDNA) activates pro-inflammatory signaling is not well-understood. In particular, the topological features of cfDNA that contribute to DNA sensor activation in phagocytes are unknown. As such, we investigated how tumour-derived cfDNA topology influences DNA sensor activation in macrophages and dendritic cells. We treated a murine colon adenocarcinoma cell line (MC38) with chemotherapy (topotecan or staurosporine) for varying durations to induce cfDNA release. We then harvested cfDNA, separated the protected (membrane-bound) and accessible (non-membranous) fractions, and quantified nuclear and mitochondrial cfDNA abundance. The cfDNA fractions were then added to murine RAW 264.7 macrophages or bone marrow-derived dendritic cells (BMDCs), and a panel of inflammatory markers was measured by RT-qPCR. We found that accessible cfDNA from MC38 cells treated with chemotherapy for 48h increased Il6expression in RAW 264.7, but other inflammatory markers were not elevated. Interestingly, both nuclear and mitochondrial cfDNA abundance directly correlated with Il6expression. Surprisingly, cfDNA did not induce pro-inflammatory signalling in BMDCs. These results demonstrate that tumour-derived cfDNA immunogenicity depends on its mechanistic origins, abundance, and the immune cell type to which it is exposed. We are currently examining the impact of other cfDNA biophysical properties on macrophage activation, as well as elucidating the DNA sensing pathways responsible for inducing Il6expression. Our findings will help uncover the role of cfDNA in mediating anti-tumour immunity. E.Z. Malkin is supported by the Frederick Banting and Charles Best Canada Graduate Scholarship (FRN: FBD-175873) through CIHR. S.V. Bratman is supported by the Gattuso-Slaight Personalized Cancer Medicine Fund at the Princess Margaret Cancer Centre and the Dr. Mariano Elia Chair in Head and Neck Cancer Research at University Health Network and the University of Toronto.

Defective N-glycosylation in tumor-infiltrating CD8+ T cells impairs IFN-γ-mediated effector function.

T cell-mediated antitumor immunity is modulated, in part, by N-glycosylation. However, the interplay between N-glycosylation and the loss of effector function in exhausted T cells has not yet been fully investigated. Here, we delineated the impact of N-glycosylation on the exhaustion of tumor-infiltrating lymphocytes in a murine colon adenocarcinoma model, focusing on the IFN-γ-mediated immune response. We found that exhausted CD8+ T cells downregulated the oligosaccharyltransferase complex, which is indispensable for N-glycan transfer. Concordant N-glycosylation deficiency in tumor-infiltrating lymphocytes leads to loss of antitumor immunity. Complementing the oligosaccharyltransferase complex restored IFN-γ production and alleviated CD8+ T cell exhaustion, resulting in reduced tumor growth. Thus, aberrant glycosylation induced in the tumor microenvironment incapacitates effector CD8+ T cells. Our findings provide insights into CD8+ T cell exhaustion by incorporating N-glycosylation to understand the characteristic loss of IFN-γ, opening new opportunities to amend the glycosylation status in cancer immunotherapies.

Abstract 5129: Neutrophil extracellular traps induced by surgical stress regulate cancer metabolism leading to tumor growth

Abstract Introduction: Surgery is a crucial intervention to cure malignancies. However, evidence shows that the surgical insult and its accompanying immune response can enhance tumor growth and metastasis. Neutrophil Extracellular Traps (NETs) released systemically during surgical stress were previously shown to contribute to this process. In fact, to generate increased energy demands required for growth and metastasis, cancer cells in a stressful microenvironment need an adaptive mechanism where metabolic reprogramming to fatty acid (FA) oxidation has been emerging as an important process. Here, we aim to explore the direct effects of surgical NETs in regulating cancer cell metabolism leading to tumor growth and metastasis. Methods: For the indolent tumor model, C57BL/6 mice were injected with a small number of LLC-1 (Lewis Lung Cancer-1) cells subcutaneously (SQ) and a group was subjected to laparotomy for 30 mins, with or without intraperitoneal DNAse (NET inhibitor) injection perioperatively. Tumor growth was then followed for 4 weeks. For the tumor metastasis model, LLC-1 cells were injected SQ and allowed to grow for 3 weeks before resection with or without perioperative DNAse. Tumor metastasis to the lungs was assessed after 3 weeks. In vitro experiments on LLC-1 and MC-38 (murine colon adenocarcinoma) cancer cells were performed using NETs released from neutrophils freshly isolated from mice bone marrow and stimulated with Phorbol myristate acetate (PMA). Results: For the indolent tumor model, injection of cancer cells did not lead to any significant tumor growth after 4 weeks unless mice underwent surgical stress via laparotomy. Treatment with DNAse significantly reversed their growth. For the tumor metastasis model, tumor resection alone led to significant lung metastasis, but DNAse treatment during resection or resection in PAD-4 KO mice (unable to form NETs) led to significantly less metastatic burden. Furthermore, in vitro treatment of cancer cells with NETs lead to significant cell proliferation, relatively more under hypoxic and serum conditions. This was paralleled with a significant increase in fluorescently labeled FA uptake into cancer cells as detected by flow cytometry, as well as a significant upregulation of gene expression of FA transport proteins and oxidation enzymes. Conclusion: Surgical stress can promote tumor growth and metastasis through cancer cell metabolic reprogramming induced by systemically released NETs. We hope that elucidating the molecular mechanism behind this process can help in finding therapeutics to prevent the protumorigenic effects of surgical stress. Citation Format: Tony Haykal, Ruiqi Yang, Celine Tohme, Ronghua Wang, David Geller, Christof Kaltenmeier, Hamza Yazdani, Samer Tohme. Neutrophil extracellular traps induced by surgical stress regulate cancer metabolism leading to tumor growth. [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 5129.

Abstract 3579: Assessment of target-mediated biodistribution of an 89Zr labeled PD-L1/4-1BB bispecific Mabcalin protein

Abstract Background: PRS-344/S095012 is a novel 4-1BB (CD137) and programmed death-ligand 1 (PD-L1) bispecific antibody-Anticalin® fusion protein (MabcalinTM protein) designed to cluster 4-1BB on activated T cells exclusively in the presence of PD-L1 expressing cells. We aimed to study PRS-344/S095012 in vivo biodistribution and pharmacokinetics with 89Zr-positron emission tomography (PET) at a dose with antitumoral activity in mice and evaluate the contribution of each targeting arm. Methods: PRS-344/S095012 lacks cross-reactivity to murine 4-1BB and PD-L1. To explore the PRS-344/S095012 biodistribution in a humanized 4-1BB knock-in mouse model, we synthesized the surrogate 89Zr-Atezo-J10 with the same 4-1BB building block and cross-reactivity to murine PD-L1. Humanized 4-1BB knock-in C57BL/6J (h4-1BB KI B6) and C57BL/6J (B6) mice (n=4-6 per group) were subcutaneously engrafted with murine wildtype MC38 colon adenocarcinoma cells. Tumors were grown to a minimum of ≥50 mm3 (average 163 mm3) before tracer injection. Mice received intravenously 30 µg (2.5 MBq) of 89Zr-PRS-344/S095012 or 89Zr-Atezo-J10 supplemented with PRS-344/S095012 or Atezo-J10 up to 10 mg/kg. Four mice groups were formed to distinguish between bispecific (Atezo-J10 in h4-1BB KI B6), monospecific PD-L1 (Atezo-J10 in B6), monospecific 4-1BB (PRS-344/S095012 in h4-1BB KI B6), and isotype (PRS-344/S095012 in B6) binding up to 4 days post-injection (pi). In addition, a fifth group (5 MBq 89Zr-Atezo-J10) was studied to visualize the bispecific biodistribution up to 7 days pi. At days 1, 2, 4, or 2, 4, 7 pi, mice underwent serial PET imaging to obtain mean and maximum standardized uptake (SUVmean/max) and retro-orbital blood sampling, followed by ex vivo biodistribution. Results: PET imaging showed 89Zr-Atezo-J10 specific tumor accumulation with higher tumor-to-blood ratios of respectively 2.2-, 2.6-, and 2.4-fold (p<0.01) at 4 days pi compared to monospecific binding of PD-L1, 4-1BB, and isotype. The ex vivo biodistribution demonstrated the same trend with respectively 4.2-, 5.5-, and 6.8-fold (p<0.01) increase in tumor-to-blood uptake for 89Zr-Atezo-J10 versus monospecific binding of PD-L1, 4-1BB, and isotype 89Zr-Atezo-J10 spleen uptake was comparable (ns) with monospecific binding of PD-L1 but elevated (p<0.01) compared to 4-1BB or isotype distribution. The uptake in lymph nodes (axillary, cervical, tumor-draining, and mesenteric) did not differ between the groups. Conclusion: 89Zr-Atezo-J10 specific accumulation in PD-L1 expressing tumors is due to both PD-L1 and 4-1BB binding and is higher than with PD-L1 and 4-1BB mono-targeting. This preclinical study supports the clinical evaluation of 89Zr-PRS-344/S095012’s whole-body distribution and the development of tumor-specific 4-1BB targeting bispecifics. Citation Format: Claudia A. van Winkel, Xiaoyu Fan, Danique Giesen, Glenn Gauderat, Lucia Pattarini, Thomas Jaquin, Anissa Barakat, Anne-Marie De La Bigne, Marleen Richter, Nicole Andersen, Julie Legrand, Helene Lelièvre, Elisabeth G. de Vries, Aizea Morales-Kastresana, Marjolijn N. Lub- de Hooge. Assessment of target-mediated biodistribution of an 89Zr labeled PD-L1/4-1BB bispecific Mabcalin protein. [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 3579.

Increased Enzyme Loading in PICsomes via Controlling Membrane Permeability Improves Enzyme Prodrug Cancer Therapy Outcome.

Mesoscopic-sized polyion complex vesicles (PICsomes) with semi-permeable membranes are promising nanoreactors for enzyme prodrug therapy (EPT), mainly due to their ability to accommodate enzymes in their inner cavity. Increased loading efficacy and retained activity of enzymes in PICsomes are crucial for their practical application. Herein, a novel preparation method for enzyme-loaded PICsomes, the stepwise crosslinking (SWCL) method, was developed to achieve both high feed-to-loading enzyme efficiency and high enzymatic activity under in vivo conditions. Cytosine deaminase (CD), which catalyzes the conversion of the 5-fluorocytosine (5-FC) prodrug to cytotoxic 5-fluorouracil (5-FU), was loaded into PICsomes. The SWCL strategy enabled a substantial increase in CD encapsulation efficiency, up to ~44% of the feeding amount. CD-loaded PICsomes (CD@PICsomes) showed prolonged blood circulation to achieve appreciable tumor accumulation via enhanced permeability and retention effect. The combination of CD@PICsomes and 5-FC produced superior antitumor activity in a subcutaneous model of C26 murine colon adenocarcinoma, even at a lower dose than systemic 5-FU treatment, and showed significantly reduced adverse effects. These results reveal the feasibility of PICsome-based EPT as a novel, highly efficient, and safe cancer treatment modality.

Tumor Cell-Intrinsic E3 Ligase TRIM21 Restrains Radiation-Induced Antitumor Immunity by Decreasing Mitochondrial DNA Release from VDAC2 Oligomers

<h3>Purpose/Objective(s)</h3> Radiotherapy has been proved to activate antitumor immunity but how aberrantly expressed genes in cancer regulated this process remains elusive. <h3>Materials/Methods</h3> We identified TRIM21, an E3 ligase gene, by bioinformatics analysis based on gross sequencing and single-cell sequencing data of nasopharyngeal carcinoma (NPC). The impact of TRIM21on radiation-induced antitumor immune response was identified in vitro and in vivo by TRIM21 knockout. We also determined the prognosis value of TRIM21 in NPC tissues with immunohistochemical analysis. <h3>Results</h3> Tumor cell-intrinsic E3 ligase tripartite motif-containing 21(TRIM21) is inversely associated with CD8+ T cell antitumor immunity and radiation response in NPC. Knockout of TRIM21 promoted radiation-induced antigen presentation, chemokine expression, dendritic cells, and CD8+ T cells activation in vitro, and led to enhanced radiation efficacy and immune response in MC38 murine colon adenocarcinoma, and transplanted NPC tumor in humanized mice in vivo. In mechanism, knockout of TRIM21 reversed its degradation of mitochondrial voltage-dependent anion-selective channel protein 2 (VDAC2), which resulted in sufficient VDAC2 oligomerization, mitochondrial DNA release, and thus strengthened activation of STING and type I interferon signaling during radiation. Clinically, high expression of TRIM21 in NPC indicated poor prognosis and tumor relapse after radiation. <h3>Conclusion</h3> Tumor cell-intrinsic TRIM21 suppressed radiation-induced antitumor immunity and predicted poor survival in NPC patients.

Tumor Ablation by Irreversible Electroporation Augments PD-1 Checkpoint Inhibitor Immunotherapy in Colon Adenocarcinoma

INTRODUCTION: Programmed Cell Death Protein 1 (PD-1) checkpoint inhibitors have recently been approved for microsatellite instability high (MSI-H) or mismatch repair deficient (dMMR) colorectal cancer (CRC). Response rates are promising in MSI-H-dMMR CRC but remain dismal for microsatellite-stable (MSS) or mismatch repair proficient (pMMR) CRC. >80% of CRCs are MSS-pMMR. Increasing tumor-infiltrating lymphocytes (TILs) may improve checkpoint inhibitor efficacy in CRC. New data suggest irreversible electroporation (IRE) therapy whereby electric pulses cause tumor necrosis and apoptosis; increase tumor-associated protein uptake by dendritic cells, subsequently boosting neoantigen presentation and TILs. We hypothesized that IRE combined with anti–PD-1 antibody, when compared with IRE or anti–PD-1 antibody alone, would increase survival, cause tumor regression by increasing TILs, and guard against secondary tumor challenge by increasing tumor antigen-specific CD8 T cells. METHODS: Using the MC38 cell line derived from murine colon adenocarcinoma, immunohistochemistry, and anti-CD8 antibodies, we evaluated antitumor immune responses in mice. RESULTS: IRE with anti–PD-1 antibody showed statistically significant survival, tumor regression, and increased TILs compared with IRE or anti–PD-1 antibody alone. Seventeen of 18 mice treated with IRE and anti–PD-1 antibody rejected a secondary tumor challenge. Conversely, IRE with anti–PD-1 antibody failed to protect against tumor rechallenge in mice that underwent CD8 T-cell depletion. Zero of 10 CD8 T-cell–depleted mice survived after secondary tumor challenge (Figure).FigureCONCLUSION: These data suggest IRE and anti–PD-1 antibody work in concert to bolster tumor-specific CD8 T-cell immune responses, serving as a pragmatic strategy for overcoming clinical challenges in checkpoint inhibitor immunotherapy for CRC.

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.

Abstract 3319: Purified poloxamer 188 mitigates radiation-induced lung toxicity

Abstract The dose-limiting factor in radiation therapy (RT) is damage to healthy tissues, which presents as radiation-induced lung toxicity (RILT). Acute RILT, pneumonitis, is an early inflammatory-driven toxicity that develops within weeks to months following RT. Purified poloxamer 188 (PP188) is a triblock copolymer that associates with and protects cell membranes from oxidative insult, minimizing secondary inflammatory damage. This study aimed to investigate the effects of PP188 to mitigate the onset of acute radiation pneumonitis in a rat model of RILT, and to test the effect of PP188 on the efficacy of RT on tumor growth of a syngeneic CT26.WT murine colon carcinoma in Balb/c mice. To evaluate the efficacy of PP188 to mitigate RILT in healthy tissues, Sprague Dawley rats were given 200mg/kg PP188 or vehicle via IV 1hr prior to a single dose of partial volume (6mm) 20Gy X-irradiation to the right lung, and then were followed for 6 weeks. To validate PP188 treatment does not interfere with RT efficacy to reduce tumor volume, Balb/c mice were implanted with CT26.WT murine colon adenocarcinoma in the high axilla space, given 5Gy SARRP irradiation, and tumor volume was monitored. Rats were evaluated for histopathological changes indicative of acute RILT, including leakage of proteins into alveolar space, thickening of alveolar septa, and alteration of bronchial epithelium and capillaries; for presence of inflammatory infiltrate (Hanker-Yates Peroxidase Leukocyte kit); and for immunohistochemical expression of alpha-smooth muscle actin (SMA). Without PP188, significant inflammatory infiltrate was observed in all lungs bilaterally (p&amp;lt;0.001) contributing to significant bronchiole epithelium thickening (p&amp;lt;0.001) and alveolar septa thickening (p&amp;lt;0.001). Within the thickened alveoli there was significant SMA expression (p&amp;lt;0.001) indicative of cells undergoing a myofibroblast transition that was not observed in the PP188+RT animals or controls. Morphological assessment demonstrated that PP188 significantly attenuated pathological changes in the functional subunits of the lung, alveoli, as well as the vasculature including bronchiole epithelium and small capillaries, with no significant differences from control animals. Finally, it was shown that PP188 does not abrogate radiation-induced tumor volume reduction. The unique mechanism of PP188 provides radioprotection to healthy tissue without interfering with tumor control, and has the potential to mitigate radiation-induced toxicities in the majority of cancers treated with RT. Citation Format: Sarah G. Dennis-Little, Wilfred Ngwa, Marikki Laiho, Graham Warren, Michael J. Yost. Purified poloxamer 188 mitigates radiation-induced lung toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3319.

Abstract 1326: Loss of p53 sensitizes tumor cells to immune checkpoint blockade therapy via upregulation of IL-33

Abstract Recent studies suggest p53 contributes to poor survival in the context of immune checkpoint blockade (ICB). Mutations to p53 significantly improve the response rate to PD-1 ICB in lung cancer patients. Tumor-derived IL-33 is required for the anti-tumor immune response and efficacy of ICB. However, the relationship between p53 and IL-33 during ICB is unknown. In this study, we characterize the role of the p53/IL-33 axis in regulating the response of the tumor microenvironment to ICB. We use CRISPR-Cas9 to delete Trp53 from murine MC38 colon adenocarcinoma. Deletion of Trp53 and PD-1 ICB synergistically inhibits tumor growth. Il33 is upregulated in the Trp53-deficient and treated tumor cells, and its expression increases with time and response to treatment. Cd4+ and Cd8+ T cell infiltration increases adjacent to IL-33 expressing tumor cells, while Treg infiltration is decreased. Simultaneous deletion of Il33 in MC38 tumors reverses the efficacy PD-1 ICB. Trp53-deficient MC38 in ST2-/- (IL-33 receptor) mice also show no response to PD-1 ICB. Our findings depict a novel mechanism by which the loss of p53 in tumors treated with ICB unleashes the expression of tumoral IL-33 and induces downstream signaling. p53 mutations may be a double-edged sword for cancer, i.e. the loss of the tumor suppressor initially facilitates tumorigenesis, but eventual buildup of mutations and activation of damage pathways, such as NF-kB, leads to upregulation of danger signals in the tumor. These danger signals, such as IL-33, mediate the anti-tumor effect of ICB. Citation Format: David Shihong Gao, Yang Li, Jason Shoush, Runzi Sun, Binfeng Lu. Loss of p53 sensitizes tumor cells to immune checkpoint blockade therapy via upregulation of IL-33 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1326.