Flank Tumors Research Articles

Factors impacting the efficacy of the in-situ vaccine with CpG and OX40 agonist

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

Targeting of Head and Neck Cancer by Radioiodinated CLR1404 in Murine Xenograft Tumor Models with Partial Volume Corrected Theranostic Dosimetry.

Background: Delivery of radiotherapeutic dose to recurrent head and neck cancer (HNC) is primarily limited by locoregional toxicity in conventional radiotherapy. As such, HNC patients stand to benefit from the conformal targeting of primary and remnant disease achievable with radiopharmaceutical therapies. In this study, the authors investigated the tumor targeting capacity of 131I-CLR1404 (iopofosine I-131) in various HNC xenograft mouse models and the impact of partial volume correction (PVC) on theranostic dosimetry based on 124I-CLR1404 (CLR 124) positron emission tomography (PET)/computed tomography (CT) imaging. Methods: Mice bearing flank tumor xenograft models of HNC (six murine cell line and six human patient derived) were intravenously administered 6.5-9.1 MBq of CLR 124 and imaged five times over the course of 6 d using microPET/CT. In vivo tumor uptake of CLR 124 was assessed and PVC for 124I was applied using a novel preclinical phantom. Using subject-specific theranostic dosimetry estimations for iopofosine I-131 based on CLR 124 imaging, a discrete radiation dose escalation study (2, 4, 6, and 8 Gy) was performed to evaluate tumor growth response to iopofosine I-131 relative to a single fraction of external beam radiation therapy (6 Gy). Results: PET imaging demonstrated consistent tumor selective uptake and retention of CLR 124 across all HNC xenograft models. Peak uptake of 4.4% ± 0.8% and 4.2% ± 0.4% was observed in squamous cell carcinoma-22B and UW-13, respectively. PVC application increased uptake measures by 47%-188% and reduced absolute differences between in vivo and ex vivo uptake measurements from 3.3% to 1.0 percent injected activity per gram. Tumor dosimetry averaged over all HNC models was 0.85 ± 0.27 Gy/MBq (1.58 ± 0.46 Gy/MBq with PVC). Therapeutic iopofosine I-131 studies demonstrated a variable, but linear relationship between iopofosine I-131 radiation dose and tumor growth delay (p < 0.05). Conclusions: Iopofosine I-131 demonstrated tumoricidal capacity in preclinical HNC tumor models and the theranostic pairing with CLR 124 presents a promising new treatment approach for personalizing administration of iopofosine I-131.

Abstract 2872: Site specific tumor microenvironments modulate the immune checkpoint inhibitors response in models of CT26 colon carcinoma

Abstract Syngeneic models provide a robust platform to test new and cutting edge immune-oncology therapies. CT26 is a popular syngeneic colon carcinoma model that is known to respond to immune checkpoint inhibitors when engrafted into Balb/c mice as a subcutaneous flank tumor. The subcutaneous flank implant location provides a robust model, that is easy to set up, and allows for continuous measurement of the tumor growth by calipering. However, the location of tumor implant has been shown to impact tumor microenvironment and immune response for a number of cancer cell lines (Zhao 2017, Brand 2021, Oliver 2018). In addition to subcutaneous flank tumors, CT26 can be implanted to generate a lung metastasis or peritoneal metastasis model. We were interested to see if the lung metastasis or peritoneal metastasis CT26 model responded differently to immune checkpoint inhibition compared with the subcutaneous model and whether the different implant locations resulted in changes to the tumor infiltrating lymphocytes. CT26 cells were either injected intravenously to induce a lung metastasis model, intraperitoneally to produce a peritoneal metastasis model, or subcutaneously to generate a solid flank tumor model. Immune checkpoint inhibitor (ICI) therapy began on Day 5 following implant in each model, and tumor progression or survival provided the endpoint for analysis. In addition, tumor infiltrating leukocytes were assessed by flow cytometry to determine differences in the overall tumor microenvironment for each model. The response to both anti-PD-1 and anti-CTLA-4 monotherapies were strongest in the subcutaneous flank model, followed by moderate responses in the lung metastasis model, and no significant response to either ICI in the peritoneal metastasis model. This provides researchers with an opportunity to assess the ability of novel immune-oncology therapies to provoke an immunogenic response in models with identical antigenicity, but different tumor microenvironments based on the tumor location. Citation Format: Fei Zhao, Caitlin Thompson, Nicole Barnes, José Roques, Terrence Coleman, Tyler Rowe, Joseph Kolb, Chassidy Hall, Patrick Fadden. Site specific tumor microenvironments modulate the immune checkpoint inhibitors response in models of CT26 colon carcinoma [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 2872.

Abstract 4156: Site specific tumor response to combination chemotherapy and immune checkpoint inhibitor in the syngeneic Pan02 pancreatic ductal adenocarcinoma model

Abstract Syngeneic tumor models provide a platform to assess novel immune-oncology therapeutics in fully immuno-competent mice. A subcutaneous flank tumor implant provides a convenient and accessible location to monitor the tumor growth and response to therapy. However, the tumor microenvironment, and therefore, the responsiveness to immuno-oncology therapies, is greatly impacted by the location in which the tumor develops (Ho 2021, Oliver 2018). Given the potential of different immuno-suppressive features driven by the specific tumor location, we wanted to compare the tumor infiltrating leukocyte populations in the subcutaneous flank and orthotopic pancreatic Pan02 syngeneic models, as well as the response to standard of care therapies. Pan02 cells were either implanted orthotopically in the pancreas or subcutaneously in the flank and allowed to establish before dosing with either vehicle, oxaliplatin, anti-CTLA-4 or the combination of therapies. Established tumors from untreated orthotopic and flank models were sampled for flow cytometry to assess infiltration by a broad range of immune cell populations. Response to therapy was clearly dependent on tumor location with flank tumors being resistant to both monotherapy and combination therapy, while the orthotopic tumors responded modestly to monotherapy but displayed synergy with the combination therapy. The results suggest that for a given syngeneic tumor cell line with identical mutational load and antigenicity, we can generate unique tumor microenvironments dependent on implant location leading to different outcomes when testing novel immune-oncology therapies. These types of models can help define the context in which novel immune-oncology therapeutics can be expected to drive an effective immune response. Citation Format: Andrew Wong, Patrick Fadden, Cara Clouse, Patrick Wood, Marianna Brown-Augustine, Fei Zhao, Elliot Ser, Joseph Kolb, Chassidy Hall, Elizabeth Rainbolt. Site specific tumor response to combination chemotherapy and immune checkpoint inhibitor in the syngeneic Pan02 pancreatic ductal adenocarcinoma model. [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 4156.

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 &amp;lt;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, &amp;gt;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 1161: Primary and metastatic tumors exhibit systems-level differences in dependence on mitochondrial respiratory function

Abstract The Warburg effect, aerobic glycolysis, is a hallmark feature of cancer cells grown in culture. However, the relative roles of glycolysis and respiratory metabolism in supporting in vivo tumor growth and processes such as tumor dissemination and metastatic growth remain poorly understood, particularly on a systems level. Using a CRISPRi mini-library enriched for mitochondrial ribosomal protein and respiratory chain genes in multiple human lung cancer cell lines we analyzed in vivo metabolic requirements in xenograft tumors grown in distinct anatomic contexts. While knockdown of mitochondrial ribosomal protein and respiratory chain genes (mito-respiratory genes) has little impact on growth in vitro, tumor cells depend heavily on these genes when grown in vivo as either flank or primary orthotopic lung tumor xenografts. In contrast, respiratory function is comparatively dispensable for metastatic tumor growth. RNA-Seq and metabolomics analysis of tumor cells expressing individual sgRNAs against mito-respiratory genes indicate overexpression of glycolytic genes and increased sensitivity of glycolytic inhibition compared to control when grown in vitro, but when grown in vivo as primary tumors these cells downregulate glycolytic mechanisms. Metabolic shifts associated with silencing mito-respiratory genes can be detected in vivo with metabolic imaging. These studies demonstrate that discrete perturbations of mitochondrial respiratory chain function impact in vivo tumor growth in a context-specific manner with differential impacts on primary and metastatic tumors. Citation Format: Neal K. Bennett, Hiroki J. Nakaoka, Danny Laurent, Ross Okimoto, Yoshitaka Sei, Andrew E. Horvai, Trever G. Bivona, Georgios Batsios, Pavithra Viswanath, Johanna ten Hoeve, Thomas G. Graeber, Ken Nakamura, Jean L. Nakamura. Primary and metastatic tumors exhibit systems-level differences in dependence on mitochondrial respiratory function [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 1161.

Abstract 6178: A novel second-generation nano-fluoropyrimidine to treat metastatic colorectal cancer and overcome 5-fluorouracil resistance

Abstract Colorectal cancer (CRC) is the 3rd leading cause of cancer-related mortality. The mortality associated with colon cancer results notably from metastatic disease (mCRC), with a 5-year survival rate for patients with stage IV CRC being &amp;lt; 14%. Despite extensive efforts towards developing personalized therapies, limited success has been achieved and chemotherapeutic regimens that include the fluoropyrimidine (FP) drug 5-fluorouracil (5-FU) are central to patient management. While using 5-FU-based regimens such as FOLFOX and FOLFIRI in combination with biologics confer a survival benefit, their ineffectiveness at promoting long-term survival in a substantial fraction of mCRC patients and associated toxicities limits their use. This underscores the pressing need to develop improved FP drugs. To overcome the limitations of 5-FU that include resistance due to elevated expression of thymidylate synthase (TS) and inefficient anabolic metabolism to FdUMP, the TS-inhibitory/active metabolite, we are developing FP polymers consisting of single-stranded DNA with FdUMP (only active metabolite) as the repeating nucleotide unit. Our therapeutic objective is to initiate a Phase I clinical trial with a new 2nd generation nanoscale FP polymer (CF10) that demonstrates promising anticancer effects in several preclinical models and significantly lower systemic toxicity. We observed that CF10 displayed significantly higher anti-tumor activity than 5-FU in flank and syngeneic tumor models. Additionally, CF10 treatment improved survival (84.5 days vs 32 days; P &amp;lt; 0.0001) relative to 5-FU in an orthotopic HCT-116-luc colorectal cancer mice model that spontaneously metastasized to the liver. Interestingly, a reduction in metastatic tumor burden in the CF10 treatment group led us to hypothesize that CF10 could inhibit the TS-mediated EMT phenotype and pro-metastatic activities in colorectal cancer cells (CRCs). This was supported by increased vimentin, decreased E-cadherin expression, and increased cell migration and invasion in TS-overexpressing CRC cells (“Tet-on” system). Alkaline comet assay, immunofluorescence imaging, and immunoblot identified significantly higher replication stress with CF10 treatment than 5-FU in mouse, rat, and human-established CRC cell lines and primary CRC cells. Strikingly, we also discovered that CF10 attenuates the growth of TS-overexpressing primary colonoids. In conclusion, CF10 induces increased replication stress compared to a similar concentration of conventional drugs. Our findings suggest the significance of elevated TS in CRC metastatic progression and 5-FU resistance and demonstrate that CF10 may be effective at inhibiting CRC metastatic progression. CF10 may be a successful candidate for early-phase clinical trials to treat mCRC. Citation Format: Naresh Sah, Pamela Luna, Chinnadurai Mani, William Gmeiner, Komaraiah Palle. A novel second-generation nano-fluoropyrimidine to treat metastatic colorectal cancer and overcome 5-fluorouracil resistance [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 6178.

Abstract 6284: SUV420H1 as a novel target in HPV-negative head and neck squamous cell carcinoma

Abstract Human Papilloma Virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type in the world and its outcomes are mostly unchanged for the past few decades, especially for HPV-negative HNSCC. The TCGA recently revealed a plethora of genetic alterations in chromatin modifiers in HPV-negative HNSCC, including protein lysine methyltransferases which methylate lysine residues on histone tails and regulate gene expression. One of these enzymes, SUV420H1, is known to trimethylate H4K20 and is recurrently amplified in approximately 6% of HPV-negative HNSCC tumors (TCGA). It has also been found to be significantly overexpressed in multiple HNSCC cell lines compared with normal keratinocytes. This study aims to investigate whether SUV420H1 has oncogenic activity and could be a novel therapeutic target in HPV-negative HNSCC. To evaluate the effect of SUV420H1 depletion on HPV-negative HNSCC cell lines, siRNA-mediated knockdown was performed in four SUV420H1 overexpressing HPV-negative HNSCC cell lines, and MTT assays showed significantly reduced cell viability by approximately 80-90% after 12 days of siRNA treatment. Colony formation assays, cell cycle analysis and apoptosis assays are ongoing, and these assays are planned in SUV420H1 CRISPR KO cell lines. Gene Set Enrichment Analysis in 434 HPV-negative HNSCC tumors (TCGA) showed significant enrichment of immune signatures, EMT, cell cycle and apoptosis pathways. Ongoing experiments aim to investigate the effects of SUV420H1 knockdown on the global histone methylome, and to identify direct downstream targets of SUV420H1 through combined genome-wide mapping of SUV420H1 using CUT&amp;RUN assays and RNA-seq in HPV-negative HNSCC cell lines. Syngeneic and xenograft mouse models are also planned to evaluate the effect of SUV420H1 depletion on flank tumor growth in vivo. These studies may elucidate the oncogenic effects and mechanisms of SUV420H1, and may provide evidence to support SUV420H1 as a potential novel target in the treatment of HPV-negative HNSCC tumors with SUV420H1 amplification or overexpression. Citation Format: Arfa Moshiri, Hui Cheng, Sohyoung Kim, Vassiliki Saloura. SUV420H1 as a novel target in HPV-negative head and neck squamous cell carcinoma [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 6284.

Abstract 1993: Sulfobutylether-β-cyclodextrin hydrogel microspheres delivering TLR 7/8 agonist for transarterial immunoembolization

Abstract Drug-eluting bead transcatheter arterial chemoembolization (DEB-TACE) is a treatment procedure for liver cancer that involves the selective catheterization and subsequent embolization of tumor-feeding arteries with drug-eluting beads (DEBs). DEB-TACE elicits ischemic cell death in the embolized tumor while simultaneously delivering a local, sustained release of chemotherapy. We hypothesize that the application of DEBs loaded with an immunostimulatory adjuvant in the DEB-TACE procedure will promote local antigen presenting cells to utilize the antigens released by dying tumor cells to generate a systemic, adaptive anti-tumor immune response. This approach represents a novel form of transarterial immunoembolization (TIE). 558 is a highly potent, small molecule Toll-like receptor 7/8 agonist that activates both innate and adaptive immune responses to eliminate tumor cells in various preclinical tumor models. Hydrogel microspheres composed of cross-linked sulfobutylether-β-cyclodextrin (SBE-βCD) were investigated as DEBs for 558 in the current study. SBE-βCD hydrogel microspheres (SBE-βCDMS) of 10 - 300 μm diameter were synthesized via suspension polymerization of SBE-βCD and ethylene glycol diglycidyl ether followed by wet sieving. 558 loading was achieved by incubating blank SBE-βCDMS in aqueous solutions of 558. Under non-saturating conditions, SBE-βCDMS absorbed almost the entirety of 558 from loading solutions in 4 h. The dose of 558 loaded in SBE-βCDMS was tuned by altering the initial amount of 558 in solution, up to a maximum loading of 0.28 mg 558/mg dry SBE-βCDMS determined under saturating conditions. The time to 50% release of 558 from loaded SBE-βCDMS was less than 30 min when phosphate buffered saline was used as release media. However, the release of 558 was negligible when deionized water was used as release media. The released drug was as effective as free 558 in stimulating cytokine response from human peripheral blood mononuclear cells in vitro. As a surrogate for TIE, we evaluated plasma and tumor pharmacokinetics upon intratumoral injection of 558-loaded SBE-βCDMS (50 - 100 μm diameter) or free 558 at a dose of 100 μg in C57BL/6 mice bearing B16F10-OVA flank tumors. The gradual release of 558 from loaded SBE-βCDMS prevented an initial spike in plasma concentration that was observed for mice administered with free 558, and maintained constant tumor concentrations for at least 4 h post-injection. High-resolution MALDI mass spectrometry imaging of 15 μm-thick tumor cryosections indicated that 558 was initially concentrated within SBE-βCDMS after intratumoral injection, and extensively released into the surrounding tumor tissue 24 h post-injection. Taken together, these results suggest that 558-loaded SBE-βCDMS are a promising platform for local drug delivery and immune cell stimulation via TIE. Citation Format: Joel Updyke, Shubhmita Bhatnagar, Nitu Bhaskar, Rachel Parise, Swati Nagar, John Schultz, David Ferguson, Tamara Kucaba, Thomas Griffith, Ronald Siegel, Jayanth Panyam. Sulfobutylether-β-cyclodextrin hydrogel microspheres delivering TLR 7/8 agonist for transarterial immunoembolization [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 1993.

Abstract 5088: MDSC-targeted TFF2-MSA synergizes with PD-1 blockade therapy in diffuse-type gastric cancer

Abstract Aims: Recent studies revealed myeloid-derived suppressor cell (MDSC) in the tumor microenvironment as an appealing therapeutic target to sensitize intestinal-type gastric cancer (GC) to PD-1 blockade therapy. However, whether MDSC inhibition can improve survival in the more aggressive diffuse-type GC is unknown. Trefoil factor family 2 (TFF2), a secreted anti-inflammatory peptide, suppresses MDSC expansion partly via CXCR4 receptor. Here, we developed a novel MDSC-targeted peptide TFF2-MSA and investigated whether it can synergize with anti-PD1 to prolong survival in syngeneic diffuse GC mouse models. Methods: Murine serum albumin (MSA) was appended to the murine TFF2 to generate TFF2-MSA peptide with an extended serum half-life. ACKP (Atp4b-Cre; Cdh1-/-; LSL-KrasG12D; Trp53-/-) GC cells developed from a highly malignant diffuse GC mouse model were grafted subcutaneously into HDC-GFP transgenic mice in which the more immunosuppressive histidine decarboxylase (HDC)-expressing myeloid cells were traced with GFP. These recipient mice subsequently received either TFF2-MSA or anti-PD-1 antibody or both. Further, to assess treatment efficacy in inhibiting spontaneous lung metastasis, subcutaneous flank tumors were resected in ACKP cell-grafted mice once tumors reached 500 mm3, and mice then received treatment with TFF2-MSA or anti-PD-1 or the combination. At the endpoint, flow cytometry and histopathological analyses were performed to examine immune profiles and tumor metastasis. In another model, ACKP-luc cells were implanted orthotopically to stomach submucosa, and recipient mice received the same treatments started from 1 week later to assess their efficacy. Results: While either TFF2-MSA or PD-1 antibody showed little benefit as a single agent (TGI 15% and 25% respectively, p&amp;gt;0.05), their combination dramatically suppressed ACKP s.c. tumor growth (TGI 78%, p&amp;lt;0.0001) and markedly prolonged median survival (64 days vs. 32.5 days in control) of host mice in a synergistic manner. The combination therapy efficiently reduced HDC-GFP+ MDSCs (by 67% vs. control, p&amp;lt;0.01), and profoundly increased tumor-infiltrating cytotoxic CD8+ T cells (by 18 fold, p&amp;lt;0.001). Further, while standard chemotherapy (5-fluorouracil and oxaliplatin) showed limited efficacy in this model, addition of chemotherapy to the TFF2-MSA/PD-1 antibody combination further improved treatment efficacy (TGI 91%) and extended median survival to 73 days. In addition, combination of TFF2-MSA/PD-1 antibody significantly reduced 80% lung metastasis (vs. control, p&amp;lt;0.0001), in contrast to minimal inhibition observed with either monotherapy (p&amp;gt;0.05). Finally, in the orthotopic model, the combo regimen eradicated GC in 50% mice compared to 0% in either monotherapy treatment. Conclusion: Targeting MDSCs using TFF2-MSA synergizes with PD-1 blockade therapy in advanced and metastatic syngeneic mouse models of diffuse-type GC. Citation Format: Jin Qian, Sandra Ryeom, Bruce Daugherty, Seth Lederman, Timothy C. Wang. MDSC-targeted TFF2-MSA synergizes with PD-1 blockade therapy in diffuse-type gastric cancer. [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 5088.

Abstract 2715: Utilizing a pH sensitive peptide (pHLIP) for tumor targeted delivery of an immunogenic peptide motif

Abstract Chimeric antigen receptor (CAR) T cell and immune-cell therapies are revolutionizing how patients are treated in the clinic by targeting tumor-specific antigens. Specifically, advances in antigen targeting of CD20 and CD19 with CAR T cell therapy has increased overall survival of this patient population and has influenced to expand T cell repertoire to novel antigen targets. However, limitations to these cell-based therapies are the lack of development against solid tumor antigens, partly due to the paucity of antigen candidates that are sufficiently specific to epithelial cancers. Therefore, technologies that can mark tumor cells for immune recognition could prove beneficial in the treatment of these solid cancers. This project aims to artificially deliver an antigen to tumor cells by harnessing a universal property of tumors: acidic microenvironment. The pH low insertion peptide (pHLIP) is a technology that targets solid tumors based on the low pH produced by the Warburg effect. pHLIP-conjugates have undergone extensive development for drug delivery, with one notable candidate CBX12 entering phase I clinical trials in 2021. Our system uses a pHLIP-conjugated SIINFEKL peptide (derived from ovalbumin) to serve as our model antigen system, with SIINFEKL-targeting T cells (OT1) that can be used as a tool to investigate antigen delivery, recognition, and functionality. We have observed through immunofluorescence that B16 F10 mouse melanoma cells treated in vitro with the 1uM pHLIP-SIINFEKL construct under acidic conditions can recruit OT1 cells and induce a T cell synapse. Preliminary in vivo experiments in C57BL/6-Tg OT1 mice bearing B16 F10 flank tumors showed that treatment with pHLIP-SIINFEKL by intravenous administration led to a suppression of tumor growth compared to controls, with approximately 50% smaller fold change in tumor volume. Cumulatively, these initial results suggest that pHLIP-SIINFEKL can be delivered to tumors to recruit OT1 cells to engage in antigen recognition and lead to efficacy in tumor growth delay in vivo. This technology has the capability to provide a novel approach to decorate solid tumors with engineered antigens for enhanced immune recognition and anti-tumor efficacy. Citation Format: Annali M. Yurkevicz, Yanfeng Liu, Peter M. Glazer. Utilizing a pH sensitive peptide (pHLIP) for tumor targeted delivery of an immunogenic peptide motif [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 2715.

Acss2/HIF-2 signaling facilitates colon cancer growth and metastasis.

The microenvironment of solid tumors is characterized by oxygen and glucose deprivation. Acss2/HIF-2 signaling coordinates essential genetic regulators including acetate-dependent acetyl CoA synthetase 2 (Acss2), Creb binding protein (Cbp), Sirtuin 1 (Sirt1), and Hypoxia Inducible Factor 2α (HIF-2α). We previously shown in mice that exogenous acetate augments growth and metastasis of flank tumors derived from fibrosarcoma-derived HT1080 cells in an Acss2/HIF-2 dependent manner. Colonic epithelial cells are exposed to the highest acetate levels in the body. We reasoned that colon cancer cells, like fibrosarcoma cells, may respond to acetate in a pro-growth manner. In this study, we examine the role of Acss2/HIF-2 signaling in colon cancer. We find that Acss2/HIF-2 signaling is activated by oxygen or glucose deprivation in two human colon cancer-derived cell lines, HCT116 and HT29, and is crucial for colony formation, migration, and invasion in cell culture studies. Flank tumors derived from HCT116 and HT29 cells exhibit augmented growth in mice when supplemented with exogenous acetate in an Acss2/HIF-2 dependent manner. Finally, Acss2 in human colon cancer samples is most frequently localized in the nucleus, consistent with it having a signaling role. Targeted inhibition of Acss2/HIF-2 signaling may have synergistic effects for some colon cancer patients.

Efficient Synthesis of Chlorin e6 and Its Potential Photodynamic Immunotherapy in Mouse Melanoma by the Abscopal Effect.

Photodynamic therapy (PDT) can eradicate not only cancer cells but also stimulate an antitumor immune response. Herein, we describe two efficient synthetic methodologies for the preparation of Chlorin e6 (Ce6) from Spirulina platensis and address the phototoxic effect of Ce6 in vitro along with antitumor activity in vivo. Melanoma B16F10 cells were seeded and phototoxicity was monitored by the MTT assay. The C57BL/6 mice were subcutaneously inoculated on the left and right flank with B16F10 cells. The mice were intravenously injected with Ce6 of 2.5 mg/kg and then exposed to red light (660 nm) on the left flank tumors 3 h after the injection. The immune response was studied by analyzing Interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and Interleukin-2 (IL-2) of the right flank tumors through qPCR. Our results revealed that the tumor was suppressed not only in the left flank but also in the right flank, where no PDT was given. The upregulated gene and protein expression of IFN-γ, TNF-α, and IL-2 revealed antitumor immunity due to Ce6-PDT. The findings of this study suggest an efficient methodology of Ce6 preparation and the efficacy of Ce6-PDT as a promising antitumor immune response.

An optimized IgG-based B7-H3xCD3 bispecific antibody for treatment of gastrointestinal cancers

T cell-based immunotherapy has revolutionized oncological treatment. However, many patients do not respond to treatment, and long-term remissions remain rare, particularly in gastrointestinal cancers like colorectal cancer (CRC). B7-H3 is overexpressed in multiple cancer entities including CRC on both tumor cells and tumor vasculature, the latter facilitating influx of effector cells into the tumor site upon therapeutic targeting. We generated a panel of Tcell-recruiting B7-H3xCD3 bispecific antibodies (bsAbs) and show that targeting a membrane-proximal B7-H3 epitope allows for a 100-fold reduction of CD3 affinity. Invitro, our lead compound CC-3 showed superior tumor cell killing, Tcell activation, proliferation, and memory formation, whereas undesired cytokine release was reduced. Invivo, CC-3 mediated potent antitumor activity in three independent models using immunocompromised mice adoptively transferred with human effector cells with regard to prevention of lung metastasis and flank tumor growth as well as elimination of large established tumors. Thus, fine-tuning of both target and CD3 affinities as well as binding epitopes allowed for the generation of a B7-H3xCD3 bsAbs with promising therapeutic activity. CC-3 is presently undergoing good manufacturing practice (GMP) production to enable evaluation in a clinical "first-in-human" study in CRC.

Cisplatin and Albumin-Based Gold–Cisplatin Nanoparticles Enhance Ablative Radiation Therapy–Induced Antitumor Immunity in Local and Distant Tumor Microenvironment

Ablative radiation therapy (RT) is an important strategy to eliminate primary tumor and can potentially induce the abscopal effect. Human serum albumin nanoparticle (NP) was used for controlled release of cisplatin to decrease cisplatin's systemic toxicity, and gold (Au) was added to increase RT-induced immunogenic cell death and potentiate the abscopal antitumor immunity. The designed albumin-based cisplatin-conjugated AuNPs were administered concurrently with ablative RT. C57BL/6 mice implanted with syngeneic murine Lewis lung carcinoma or murine MB49 tumor models were treated with ablative RT (12 Gy per fraction for 2 fractions, total 24 Gy), cisplatin, or Au-cisplatin NPs. Combining ablative RT with cisplatin or Au-cisplatin NPs both destroyed the primary tumor effectively and elicited immunogenic cell death accompanied by release of danger-associated molecular patterns. This enhanced recruitment of effector tumor-infiltrating immune cells, including natural killer T cells and CD8+ T cells, and elicited an increased percentage of professional antigen-presenting CD11c+ dendritic cells. Transient weight loss, accompanying hepatotoxicity, nephrotoxicity, and hematopoietic suppression, was observed as a systemic adverse event in the cisplatin but not the Au-cisplatin NPs group. Cisplatin and Au-cisplatin NPs both showed equivalent ability to reduce metastatic potential when combined with ablative RT, confirmed by suppressed unirradiated flank tumor growth and decreased metastatic lung tumor burden, which translated to improved survival. Mobilization and abundance of effector tumor-infiltrating immune cells including CD8+ T cells and dendritic cells were observed in the distant lung tumor microenvironment after ablative RT with cisplatin or Au-cisplatin NPs, demonstrating increased antitumor immunotherapeutic activity as an abscopal effect. Compared with cisplatin, the albumin-based Au-cisplatin NPs exhibited equivalent but no superior antitumor immunotherapeutic activity while reducing systemic adverse events and can be safely administered concurrently with ablative RT. Alternative NP formulations may be designed to further improve anticancer outcomes.

Photothermic therapy with cuttlefish ink-based nanoparticles in combination with anti-OX40 mAb achieve remission of triple-negative breast cancer.

Immunostimulatory monoclonal antibodies (IS-mAb) have been proven to enhance the therapeutic effectiveness of various anticancer therapy. In the present investigation, we launched a separate combinational therapy for the treatment of triple-negative breast cancer (TNBC) using cuttlefish ink-based nanoparticles (CINPs) for photothermal therapy (PTT) and anti-OX40 antibody. Our goal was to increase the therapeutic response to the disease. CINPs were characterized by their physicochemical properties, which revealed that they had a hydrodynamic diameter ranging from 128 to 148nm, a negative surface charge, and a high photothermal conversion efficiency under both in vitro and in vivo settings. In TNBC model, we evaluated the therapeutic effectiveness of the following groups: CINP-PTT+anti-OX40 Ab (G1), CINPs-PTT (G2), CINPs+anti-OX40 Ab (G3), anti-OX40 (G4) or PBS (G5). In each case, we assessed the efficacy of these groups against one another. The intratumor administration of all of the substances and therapies was performed. CINP-PTT+anti-OX40 Ab and CINP+anti-OX40 Ab (particularly CINP-PTT+anti-OX40 Ab) induced significant tumor regression in treated (breast) and non-treated (flank) tumor, and completely inhibited lung metastasis, thereby inducing a higher survival rate in mice in comparison to CINP-PTT, anti-OX40 Ab, or PBS. This was the case because in CINPs-treated tumors, particularly those treated with CINPs-PTT, intratumoral injection of CINPs increased the frequency of OX40, CD8 double-positive T cells. CINPs improved the conversion of the macrophage phenotype from M2 to M1 in vitro, which is significant from an immunological point of view. In addition, anti-OX40 Ab combined with CINPs or, more specifically, CINPs-PPT produced a larger frequency of preexisting and newly formed tumor-specific CD8 T cells, as well as an enhanced frequency of CD8 T cells infiltrating non-treated tumors, in comparison to respective monotherapies. When the data were taken into consideration as a whole, it seemed that CINPs-based PTT may effectively enhance the antitumor response effectiveness of anti-OX40 Ab.

Anti-tumor immunity enhancement by photodynamic therapy with talaporfin sodium and anti-programmed death 1 antibody

Photodynamic therapy (PDT) is a relatively non-invasive anti-cancer therapy that employs a photosensitizer with a specific wavelength of light irradiation. PDT induces direct cell killing and enhancement effects on tumor immunity, but its underlying mechanism remains unknown. Here, we perform a basic analysis of the anti-tumor effect of talaporfin sodium (TS)-PDT as well as its synergism with the immune checkpoint inhibitor anti-programmed death 1 (anti-PD-1) antibody. Weestimate the cell death mechanism induced by TS-PDT and the induction of damage-associated molecular patterns (DAMPs) by TS-PDT invitro. We establish a syngeneic mouse model ofbilateral flank tumors and verify the enhancement of the abscopal effect on the non-irradiated side. TS-PDT induced apoptosis, necrosis, and autophagy-associated cell death invitro. TS-PDT induced the release and/or expression of DAMPs invitro. Tumor growth was inhibited in the TS-PDT and anti-PD-1 antibody combination group compared with other single-treatment or non-treatment groups invivo. In summary, TS-PDT induces the release and/or expression of DAMPs, indicating that it activates innate immunity. PD-1 blockage enhances the anti-tumor immunity induced by TS-PDT. Thus, our results demonstrate that the combination of TS-PDT and anti-PD-1 antibody can potentially be used for anti-tumor therapy.

Radiation to all macroscopic sites of tumor permits greater systemic antitumor response to in situ vaccination

BackgroundThe antitumor effects of external beam radiation therapy (EBRT) are mediated, in part, by an immune response. We have reported that a single fraction of 12 Gy EBRT combined with...

Layer-by-layer interleukin-12 nanoparticles drive a safe and effective response in ovarian tumors.

Ovarian cancer is especially deadly, challenging to treat, and has proven refractory to known immunotherapies. Cytokine therapy is an attractive strategy to drive a proinflammatory immune response in immunologically cold tumors such as many high grade ovarian cancers; however, this strategy has been limited in the past due to severe toxicity. We previously demonstrated the use of a layer-by-layer (LbL) nanoparticle (NP) delivery vehicle in subcutaneous flank tumors to reduce the toxicity of interleukin-12 (IL-12) therapy upon intratumoral injection. However, ovarian cancer cannot be treated by local injection as it presents as dispersed metastases. Herein, we demonstrate the use of systemically delivered LbL NPs using a cancer cell membrane-binding outer layer to effectively target and engage the adaptive immune system as a treatment in multiple orthotopic ovarian tumor models, including immunologically cold tumors. IL-12 therapy from systemically delivered LbL NPs shows reduced severe toxicity and maintained anti-tumor efficacy compared to carrier-free IL-12 or layer-free liposomal NPs leading to a 30% complete survival rate.

Targeting Sumoylation with Subasumstat (TAK-981), a Selective Inhibitor of SUMO-Activating Enzyme (SAE), Induces Mitochondrial Dysfunction in Preclinical Models of Non-Hodgkin Lymphoma (NHL)

SUMOylation is a post-translational modification which leads to covalent attachment of small ubiquitin-like modifiers (SUMO) proteins to lysine residues of a target protein. SUMOylation is essential for the regulation of genomic integrity, gene expression and intracellular signaling. Many tumors exhibit deregulation of SUMOylation pathway. Subasumstat, (TAK-981), a novel selective inhibitor of SUMO-activating enzyme (SAEi), has entered early phase II clinical trials. Here we studied SAEi in pre-clinical models of B-cell non-Hodgkin's lymphoma (NHL). TAK-981 was provided by Takeda Development Center Americas, Inc. (Lexington, MA). Diffuse large B-cell lymphoma (DLBCL) cell lines (OCI-LY3, VAL, U-2932, SU-DHL10), and primary mantle cell lymphoma (MCL) cells were assayed for survival, mitochondrial function, and metabolic phenotype. In vivo experiments were performed using DLBCL cell line xenografts and MCL PDX models. Treatment with TAK-981 restricted growth of DLBCL cell lines with an IC50~10 nM. SAEi led to rapid (within 2 h) protein desumoylation in a dose dependent manner, prominently in the ~90 kDa region. TAK-981 induced pronounced double strand DNA breaks in DLBCL cells (comet assay) and cell cycle arrest (propidium iodine). RNA-Seq and ATAC-Seq analysis of TAK-981-treated DLBCL cells revealed upregulation of the DNA damage, NF-κB, and OXPHOS signaling pathways. We noted that in addition to cytoplasmic proteins, mitochondrial proteins were heavily de-SUMOylated upon treatment of NHL cells with TAK-981. In vitro exposure to TAK-981 resulted in mitochondrial membrane depolarization, loss of mitochondrial integrity (by electron microscopy) and a change of mitochondrial dynamics as assessed by expression of mitochondrial proteins (eg, Drp1). Seahorse respirometry analysis revealed dramatic downmodulation of OXPHOS following SAEi. Furthermore, metabolomic profiling of DLBCL cells treated with TAK-981 showed downmodulation of TCA substrates, while glycolysis substrates remained unchanged. The mitochondrial disruption observed following TAK-981 treatment was accompanied by rapid accumulation of reactive oxygen species, a mechanism that has been previously shown to lead to DNA damage in cancer cells. Consistent with cell line data, exposure of primary MCL cells to TAK-981 in stromal conditions (CD40L- or BAFF expressing stroma) induced apoptosis and reduced OXPHOS. Next, we conducted genome-wide CRISPR-Cas9 loss-of-function library screens and identified that loss of genes in the NFκB, TP53, DNA damage and centromere/telomere gene pathways contributed to resistance to SAEi in DLBCL cells. By means of genetic knockout we demonstrated that TP53 and its transcriptional target BAX contributed to TAK-981-induced G2 arrest and apoptosis, further implicating mitochondrial dysfunction in sensitivity to SAEi. For in vivo experiments, we used DLBCL xenografts. OCI-LY3 and U-2932 cells were inoculated subcutaneously in NSG mice. Once tumors reached 100 mm3, mice received 10 twice-weekly IV doses of 7.5 mg/kg TAK-981 (over 5 weeks), leading to resolution of flank tumors and improved survival compared with control. Finally, we used an MCL PDX model where MCL cells were inoculated intravenously. Once cells became detectable in the blood, mice were treated as above. Treatment with TAK-981 resulted in delayed tumor expansion in blood and spleen and extended animal survival. TAK-981-exposed splenocytes exhibited a reduction of maximal respiration by Seahorse analysis. Conclusions. Pharmacologic SAEi with TAK-981 demonstrated pre-clinical activity in NHL models in vitro and in vivo, accompanied by DNA damage, mitochondrial dysfunction, and metabolic reprogramming.