Anticancer Immune Response Research Articles

Abstract 5830: Presence of androgens improves efficacy of PD1 blockade

Abstract Immune therapies have revolutionized the treatment of cancer, however, until now only a minority of patients derive long-term benefit. Notably, despite widely reported significant differences between the immune system of males and females, there continues to exist a knowledge gap regarding sex disparities in anti-cancer immune responses. Recent meta-analyses indicate that immune checkpoint blockade (ICB) treatment has higher efficacy in male patients compared to females, regardless of the cancer type. To investigate the mechanisms underlying these sex-specific differences we utilized the syngeneic colorectal cancer model MC38 because it mirrors the clinical situation yielding responding and non-responding mice. Therefore, we inoculated tumor cells subcutaneously (s.c.) into male and female mice, and treated them with either IgG2a control Ab or anti-PD1 Ab. When analyzing the tumor response to anti-PD1 treatment we observed significant sex-biased growth kinetics upon ICB favoring males. To asses a potential role of androgens in impacting observed sex differences to ICB response in vivo, we performed surgical androgen deprivation therapy (orchiectomy) in male mice, as well as sham-surgery in male and female mice as control. After a recovery period, we proceeded with s.c. cell injection and ICB treatment as previously described. Sham male mice showed a very high response rate to anti-PD1 (92%) compared to sham females (58%), but more importantly, testosterone suppression by surgical androgen deprivation therapy led to a lower response rate of castrated mice (71%). To further confirm whether the presence of testosterone was beneficial for ICB response, we implanted s.c. sham or testosterone pumps into male and female mice and proceeded as before. Males with sham pumps showed a very high response rate to anti-PD1 (90%) compared to females with sham pumps (50%), while testosterone supplementation in females led to enhanced response rate (85%). Overall, this demonstrated that regardless of biological sex, presence of testosterone is sufficient to positively impact ICB therapy outcome. Furthermore, immunophenotyping analysis by flow cytometry showed that females supplemented with testosterone and treated with anti-PD1 had significantly increased intratumoral stem-like CD8+TCF1+PD1+ T cells compared to sham females. Additionally, these findings also correlated with enhanced intratumoral terminal differentiated effector CD8+TCF1-PD1+ T cells in both sham males and females supplemented with testosterone, compared to sham females. In summary, androgens can modulate anti-cancer immune responses by contributing to a more sustained anti-tumor CD8+ T cell response and consequently better responses upon treatment with anti-PD1. These findings are in concordance with male cancer patients responding better to ICB and warrant therapy escalation in female patients. Citation Format: María Elena Vargas-Delgado, Lara Meier, Jonas Waizenegger, Julia Oberbauer, Nikolaus Berenbrok, Janik Engelmann, Victoria Gensch, Franziska Heilmann, Jochim Reinert, Kristoffer Riecken, Boris Fehse, Hannelore Lotter, Dorothee Schwinge, Christoph Schramm, Hans-Willi Mittruecker, Isabel Ben-Batalla, Sonja Loges. Presence of androgens improves efficacy of PD1 blockade [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 5830.

Abstract 6706: Co-culture of circulating tumor cells (CTCs)-derived 3D organoids and autologous cytotoxic CD8+ T cells: A new functional precision oncology platform

Abstract Greater than 10 million patients succumb to cancer each year. Cancer metastasis is responsible for more than 90% of all cancer-associated deaths due to treatment resistance and increased tumor burden. As a seed for metastases, circulating tumor cells (CTCs) present a new dimension and horizon for clinical doctors in diagnosis, prognosis prediction, treatment monitoring, disease mechanism, and drug development. CTCs could gradually replace tissue biopsies which are painful and may be difficult to obtain depending on tumor location. CTC isolation is feasible after minimally invasive liquid biopsy and provides the basis for a multitude of ex vivo and in vivo studies including establishment of CTCs-derived 2D and 3D cultures. Organoids are miniscule models of tissues that grow in a 3D semisolid extracellular matrix medium with specific growth factors supplied. CTCs-derived 3D organoids play a vital role in precision oncology because they can preserve tumor heterogeneity, imitate the tumor microenvironment (TME), mimic cancer hypoxia in the TME, and maintain cancer and metastasis phenotypes. Cytotoxic CD8+ T cells are the most powerful effectors in the anticancer immune response. We hypothesized that co-culture of CTCs-derived 3D organoids and autologous cytotoxic CD8+ T cells could maximize patient-relevance of laboratory assessment of cancer-treatment immune-system interactions to facilitate precision oncology practice. TellBio’s novel TellDx CTC technology allows for isolation of viable and intact CTCs in liquid biopsies, regardless of cancer type. Unlabeled CTCs were cultured in growth factor reduced Matrigel with organoid culture WENRAS medium. Magnetic beads labeled white blood cells (WBCs) were cultured with T cell culture medium - WBCs and magnetic beads usually separate in three days - cytotoxic CD8+ T cells were isolated with the EasySep™ Human CD8+ T Cell Isolation Kit. CTCs-derived 3D organoids and autologous cytotoxic CD8+ T cells were co-cultured with or without different drug treatments - cytotoxicity was measured with CellTiterGlo® 3D Cell Viability Assay and imaging, and further mechanistic studies were feasible. Our co-culture platform enables us to utilize a patient's peripheral blood or pleural effusions to create a patient-specific, in vivo-like TME and immune microenvironment to model and assess ex vivo responses to investigational and FDA-cleared cancer therapies, and potentially provide oncologists with insights to improve clinical outcomes. Citation Format: Lanlan Zhou, Leiqing Zhang, Lindsey Carlsen, Kelsey E. Huntington, Vida Tajiknia, Andrew George, Arielle De La Cruz, Arunasalam Navaraj, Praveen Srinivasan, Maximilian Schwermann, Benedito A. Carneiro, Wafik S. El-Deiry. Co-culture of circulating tumor cells (CTCs)-derived 3D organoids and autologous cytotoxic CD8+ T cells: A new functional precision oncology platform [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 6706.

Abstract NG14: Dissecting immune microenvironment of T-cell acute lymphoblastic leukemia

Abstract NG14: Dissecting immune microenvironment of T-cell acute lymphoblastic leukemia

Abstract 1800: An oral triple inhibitor of PI3Kδ/γ and DNA-PK elicits anticancer immunity and potentiates therapeutic efficacy of immune checkpoint blockade

Abstract Background: PI3Kδ and PI3Kγ are mainly expressed in leukocytes, indicating that they are involved in immune modulation of the tumor microenvironment (TME). Additionally, DNA-dependent protein kinase (DNA-PK) plays a pivotal role as a mediator in repairing DNA damage in cancer cells. Here, we developed a triple inhibitor of PI3Kδ/γ and DNA-PK, BR101801, to promote antitumor immune responses and thereby enhance therapeutic efficacy of the PD-1 immune checkpoint inhibitor. Methods: CT26 and MC38 colon tumor-bearing immunocompetent mice were treated with BR101801 (50 mg/kg, PO) daily and/or anti-PD-1 (8 mg/kg, IP) twice a week. The tumors were comprehensively analyzed using flow cytometry, multiplex tissue imaging, and NanoString profiling methods. Results: Oral administration of BR101801 suppressed tumor growth without significant toxicities. The optimal schedule for BR101801 was determined by activation of T cell immunity in the TME. BR101801 monotherapy significantly increased CD8+ cytotoxic T cells and decreased CD4+CD25+Foxp3+ regulatory T cells, while no changes were observed in tumor-associated macrophages. Moreover, BR101801 increased AH-1+ tumor-specific CD8+ T cells within BR101801-treated CT26 tumors. BR101801 induced extensive immune remodeling of the TME by altering immune-related genes. Notably, gene signatures related to DNA damage and inflammatory responses were more enriched in BR101801-treated tumors. Furthermore, the combination immunotherapy of BR101801 and anti-PD-1 antibody strongly suppressed tumor growth and improved antitumor immunity within the TME, leading to complete tumor regression. Conclusion: Our study demonstrates that BR101801, an oral triple inhibitor of PI3Kδ/γ and DNA-PK, effectively elicited anticancer immune responses within the TME and potently inhibited tumor progression in combination with immune checkpoint blockade. Citation Format: Seung Joon Lee, Won Suk Lee, Eun-Jin Go, Hannah Yang, Hong Ro Kim, Bong-Seog Kim, Chan Kim, Hong Jae Chon. An oral triple inhibitor of PI3Kδ/γ and DNA-PK elicits anticancer immunity and potentiates therapeutic efficacy of immune checkpoint blockade [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 1800.

Abstract 2011: Polycondensate-epitope vaccine augments anti-cancer T lymphocyte responses and induces long-term immune memory

Abstract Background: Personalized cancer vaccines are promising treatments to induce or augment antitumor responses. Despite the observed benefits in early-phase clinical studies, the clinical efficacy of cancer vaccines remains modest. Here, we aim to develop a vaccine delivery strategy to enhance the antitumor efficacy of cancer vaccines. Method: We developed polycondensate-epitope vaccines (PEVs) for co-delivering of major histocompatibility complex (MHC) I and II-restricted peptides (OVA257-264 and OVA323-339) to elicit antigen-specific CD4+ and CD8+ T cells for long-term antitumor immunity. PEVs were prepared by polymerizing amino-functionalized MHC I and MHC II-restricted peptides with a TLR1/2 agonist, Pam3CSK4, using a responsive linker. We characterized the physical properties (size, MW) and responsiveness of the prepared PEVs. The immunogenicity was evaluated in mice via flow cytometry and ELISpot assay. We finally assessed the antitumor efficacy in both prophylactic and therapeutic tumor models. Results: The prepared PEVs have an average size of 20 nm to 50 nm, >95% incorporating efficiency, and a remarkably high loading capacity of antigens and adjuvants (57.18~ 61.3 wt/wt %). PEVs consisting of both MHC I and MHC II-restricted OVA peptides induced robust antigen-specific CD4+ and CD8+ T cell responses and long-term memory immune response against tumors. The vaccine-induced CD8+ and CD4+ T cells exhibited high proliferative capacity and polyfunctionality upon antigen restimulation ex vivo. In a prophylactic model, mice immunized by PEVs completely rejected the first and the second challenge of YUMM1.7-OVA tumor cells with 100% rejection rate. In a therapeutic model, treatment with PEVs loaded OVA257-264 and OVA323-339 peptides induced marked tumor regression in mice bearing established YUMM1.7-OVA tumors showing enhanced anti-tumor efficacy as compared to PEVs loaded with CD8 epitope alone. Conclusion: Our results demonstrate that PEV is a promising vaccine delivery strategy for eliciting highly effective and durable anti-cancer immune responses. Citation Format: Xiaomeng Hu, Li Tang. Polycondensate-epitope vaccine augments anti-cancer T lymphocyte responses and induces long-term immune memory [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 2011.

Abstract 6376: Discovery and preclinical characterization of dual antagonist antibodies targeting both LILRB1 and LILRB2 that enhance innate and adaptive anti-cancer immune responses

Abstract Background: One cause for the failure of checkpoint inhibitors is the immunosuppressive nature of the tumor microenvironment. LILRB1 (ILT2) and LILRB2 (ILT4) are ITIM-containing inhibitory receptors that recognize HLA Class 1 and nonclassical ligands (e.g., HLA-A, HLA-G, etc.). LILRB1 is expressed on myeloid cells and subsets of B, NK, and T cells, while LILRB2 expression is mostly restricted to myeloid cells. Interaction of LILRB1 and LILRB2 receptors with HLA ligands promotes an inhibitory milieu that prevents T cells from attacking cancer cells. The distinct pattern of expression and function of these lymphoid and myeloid checkpoints suggests complementary targeting approaches for cancer immunotherapy. Dual blockade of LILRB1 and LILRB2 receptors by a single antibody that restores both innate and adaptive immune responses is a promising strategy to enhance efficacy of checkpoint inhibitors. Methods: Dual LILRB1 and LILRB2 targeting antibodies were cloned from B cells derived from rabbits immunized with human LILRB2 recombinant protein, and subsequently humanized. Antibodies were evaluated for binding to human LILRB1 and LILRB2 proteins. Dual targeting antibodies were evaluated in a panel of functional and phenotypic assays. Selected antibodies were further tested for efficacy in a humanized NSG-SGM3 tumor model. Results: Dual antibodies were selected based on binding to recombinant human LILRB1 and LILRB2 protein, as well as blocking of HLA-G binding. These antibodies demonstrated binding to cells expressing LILRB1 and LILRB2, with no appreciable binding to other family members. Lead antibodies demonstrated activity in functional cell-based assays modeling LILRB1- or LILRB2-mediated immunosuppression. Dual antibodies also enhanced IFN-γ production by LPS-stimulated human PBMC. Selected clones restored T-cell function from M2c macrophage-mediated suppression in coculture with CD8+ T cell, and enhanced the tumoricidal activity of NK cells. Importantly, the lead antibody demonstrated in vivo efficacy with significant tumor growth inhibition and tumor regression in an SK-MEL-5 tumor model in humanized NSG-SGM3 mice. Conclusions: We have identified dual antagonist antibodies targeting both LILRB1 and LILRB2 antibodies that restore both innate and adaptive immune responses. Additionally, dual antibodies restored CD8+ T cell activation from macrophage-mediated suppression and enhanced NK cell cytotoxic activity. These data provide a strong rationale for further development of dual antibodies as an anti-cancer immunotherapy. Citation Format: Meghan Zuck, Myriam Bouchlaka, Huyen Dinh, Kevin Green, Francisco Zapata, Ramya Chandrasekaran, Tatyana Pisarenko, Lauren Loh, Gajendra S. Naika, Meilyn Sylvestre, Jacob Heit, Raymond Fox, Darbie Whitman, Tom Graddis, Kamal D. Puri, Peter Probst. Discovery and preclinical characterization of dual antagonist antibodies targeting both LILRB1 and LILRB2 that enhance innate and adaptive anti-cancer immune responses [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 6376.

Abstract 828: DNA origami nanostructures as a targeted payload delivery system

Abstract DNA origami is a platform nanotechnology allows for the generation of nanostructures with precisely defined geometric shape that may be easily functionalized with a variety of payloads for therapeutic delivery. These payloads include nucleic acids (miRNA, siRNA, gene sequences), associated Cas protein molecules, therapeutic small molecules drugs, functional peptides (adjuvants or cell penetrating peptides), and incorporated targeting and/or therapeutic antibodies. Hence, DNA origami is a highly promising vehicle to deliver targeted therapeutic payloads to cancer cells or other diseases. Previous studies by our laboratory and others revealed DNA origami mediated drug delivery efficiently delivered daunorubicin or doxorubicin to cancer cells while outperforming free drug in both solid and liquid tumor model systems in vitro and in vivo. Our recent findings showed significant survival advantage by doxorubicin-loaded DNA origami over delivery of free doxorubicin in an aggressive, acute monocytic leukemia model. We also showed antibody-directed drug delivery by DNA origami may be directly targeted to monocytic leukemia cells expressing the target surface antigen, and superior efficacy in vitro against acute myeloid leukemia cells when loaded with daunorubicin relative to free drug at low concentrations. Thus, antibody targeted drug loaded DNA origami represent a promising novel precision medicine approach. Additionally, recent findings by our laboratory in vivo determined that DNA origami nanostructures alone administered at high dose (12.0mg/kg) distribute well, are non-toxic, and illicit mild immunogenicity, making them an attractive candidate for further development towards therapeutic applications. We recently developed a DNA origami structure functionalized at a high density with adjuvant (CpG) and antigenic peptides (OVA) to stimulate a directed antigen-specific immune response. We have shown this DNA origami vaccine platform displays significant improvement in efficacy by an OVA-pulsed antigen-killing assay in vivo relative to alum and CFA standard adjuvant formulations. Thus, our DNA origami vaccine platform represents a promising novel approach to optimize an anti-cancer immune response, a finding also shown by other laboratories.Collectively, DNA Nanobot technology offers 1) a promising a cancer drug delivery system that can deliver a high number of therapeutic drugs (100s of drug molecules per targeted delivery system) utilizing existing ADC conjugation methods; 2) An optimal vaccine delivery method to enhance efficacy; and 3) cell specific targeted delivery of therapeutic nucleic acids and genes to modify oncogene and/or tumor suppressor gene expression. Citation Format: Patrick Halley, Niksa Roki, Nicholas Vantangoli, Tom Zupancic, Jeff Spitzner, Meixiao Long, Karilyn Karilyn Larkin, John Byrd, Carlos Castro, Chris R. Lucas. DNA origami nanostructures as a targeted payload delivery system [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 828.

Abstract 4430: Sacituzumab-Interferon beta mutein fusion protein, ABN202 (anti Trop2-interferon beta mutein), is a potent therapeutics for Trop-2-positive urothelial cancer

Abstract Background: Trop2, trophoblast cell-surface antigen 2, is a type 1 transmembrane glycoprotein overexpressed in many solid tumors such as breast, lung, pancreatic, gastric, colon, urothelial cancer. Overexpression of Trop2 is associated with aggressive disease progression and poor prognosis. Recently, Trop2 targeting antibody-drug conjugate such as sacituzumab-govitecan (Trodelvy®) was approved for the treatment of triple-negative breast cancer and urothelial cancer (UC) patients. Interferon-beta (IFN-beta), a pleiotropic cytokine, can induce direct/in-direct tumor killing and activate anti-tumor immune response in tumor microenvironments. However, it is challenging to develop IFN-beta as an anti-cancer agent due to its low biophysical properties, short half-life, and the systemic toxicity. Previously, we developed IFN-beta mutein (ABN102), which showed improved stability and biophysical properties compared to wild-type IFN-beta. We hypothesis to reduce any toxicity of ABN102 by fusing with tumor targeting antibody. In this study, we generated antibody cytokine fusion protein (ABN202), anti Trop2-interferon-beta mutein, and evaluated the efficacy of ABN202 against Trop2-positive UC cell line models. Method & Result: We aimed to determine the direct anti-cancer efficacy of ABN202 using Trop2-positive UC cell lines and the indirect immune cell mediated effect of ABN202 using Trop2-positive UC cell lines co-cultured with human peripheral blood mononuclear cell (PBMC). The in vivo efficacy of ABN202 was further confirmed in Trop2 positive tumor mouse model. Our results revealed that ABN202 directly inhibited proliferation and activates the apoptosis-related signaling mechanisms via Type 1 IFN signaling, showing better efficacy than either anti Trop2 antibody (sacituzumab) or ABN102 alone. ABN202 induces the activation of PBMC subsets and enhanced immune cell-mediated cancer cell killing when UC cell lines were co-cultured with PBMC. The anti-cancer efficacy of ABN202 were confirmed in Trop2-positive tumor mouse model. Conclusion: We evaluated the direct and indirect anti-cancer efficacy of ABN202 (anti Trop2-interferon-beta mutein) in Trop2-positive urothelial cancer. Our results support that ABN202 is a potent drug candidate in Trop2-positive urothelial cancer by directly inhibiting tumor growth and activating anti-cancer immune responses through type 1 IFN signaling. Citation Format: Jiyang Lee, Myeung-Ryun Seo, Heegeon Park, YeongMun Kim, Saehyung Lee, Na Young Kim, Chan Gyu Lee, Hae Min Jeong, Jun Young Choi, Young kee Shin. Sacituzumab-Interferon beta mutein fusion protein, ABN202 (anti Trop2-interferon beta mutein), is a potent therapeutics for Trop-2-positive urothelial 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 4430.

Abstract 1263: Hypoxia induces down-regulation of stimulator of interferon genes (STING) that is synergized with oncometabolites

Abstract The cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway, a component of the innate immune system, functions to detect the presence of cytosolic DNA, and STING signaling is necessary for the anti-cancer immune response. However, STING expression is suppressed or lost in the majority of cancers, which correlates with poor survival in cancer patients. Hypoxia is a key feature of solid tumors that adversely affects prognosis by altering gene expression, creating an immunosuppressive tumor microenvironment, promoting genome instability, and hampering therapy responses. In this study, we found that that prolonged moderate hypoxia induces downregulation of STING expression in multiple cancer cell lines, causing decreased STING pathway activation after treatment with a STING agonist, or with DNA damaging chemotherapy. Mechanically, we found that hypoxia suppressed histone 3 lysine 4 (H3K4) methylation at the STING promoter based on chromatin immunoprecipitation (ChIP) assays in H1975 cells, a non-small cell lung cancer cell line, and knockdown of the H3K4 demethylases, KDM5A or KDM1A, prior to hypoxic exposure prevented hypoxia-induced down-regulation of STING expression. Moreover, decreased STING expression in hypoxic cells was reversed by treatment with the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine. Next, we found that prolonged hypoxia increased both D-2-hydroxyglutarate (D-2-HG) and L-2-HG levels in H1975 cells, and that 2-HG treatment, by itself, decreased STING expression under normoxic conditions in H1975 cells. These findings are consistent with analysis of TCGA data showing that STING expression at the mRNA level is reduced in malignancies with mutations in isocitrate dehydrogenase 1 (IDH1) which causes elevated D-2-HG. Furthermore, we found that hypoxia increased Branched Chain Amino Acid Transaminase 1 (BCAT1) expression, and overexpression of BCAT1 accelerated STING downregulation in hypoxic condition. Functionally, expression of interferon (IFN)-stimulated genes (ISGs), CCL5 and interferon-β, was low in hypoxic H1975 cells compared to normoxic cells after treatment with poly(dA:dT), a small molecule STING agonist, and doxorubicin. Immunogenic cell death (ICD) was also reduced in hypoxic H1975 cells. In summary, the results suggest that hypoxia suppresses STING expression through epigenetic regulation, leading to STING silencing, and that this process synergizes with oncometabolites. The decreased STING caused by hypoxia further causes reduction of innate immune responses in cancer cells. Our study provides new understanding of the underlying mechanisms for STING silencing, which will help not only to study cancer immunity but also to develop novel therapeutic strategies. Citation Format: Yuhong Lu, Annali Yurkevicz, Yanfeng Liu, Jonathan Dow, Peter M. Glazer. Hypoxia induces down-regulation of stimulator of interferon genes (STING) that is synergized with oncometabolites [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 1263.

Rigid Shell Decorated Nanodevice with Fe/H2O2 Supply and Glutathione Depletion Capabilities for Potentiated Ferroptosis and Synergized Immunotherapy

AbstractThe overexpressed glutathione peroxidase4 (GPX4) and insufficient H2O2 in tumor cells weaken ferroptosis therapy and the elicited anticancer immune response. Herein, a rigid metal‐polyphenol shell decorated nanodevice ssPPELap@Fe‐TA is constructed to successfully overcome the drawbacks of ferroptosis therapy. The ssPPELap@Fe‐TA consists of a rigid Fe‐TA network‐based shell and disulfide‐containing polyphosphoester (ssPPE) core with β‐lapachone loading. The rigid Fe‐TA network‐based shell of ssPPELap@Fe‐TA enables its efficient internalization by tumor cell and then disintegrates in the acidic endosome/lysosome to initiate Fe3+/Fe2+ conversion‐driven ferroptosis. The ssPPE core will deplete glutathione (GSH) via the disulfide‐thiol exchange reaction to inactivate GPX4, and also trigger the release of β‐lapachone to significantly increase intracellular H2O2 and then promote Fe3+‐mediated Fenton reaction, eventually achieving strong inhibition of tumor progression. Moreover, ssPPELap@Fe‐TA elicites a robust systemic antitumor immune response by promoting dendritic cells (DCs) maturation and T cell infiltration, and synergizes with anti‐PD‐L1 antibody (a‐PD‐L1) to strikingly suppress 4T1 tumor growth and lung metastasis.

MP04-14 T CELL AND B CELL RECEPTOR SEQUENCING REVEALS ANTI-CANCER IMMUNE RESPONSE ASSOCIATED WITH IMMUNE-RELATED ADVERSE EVENTS IN ADVANCED RENAL CELL CARCINOMA PATIENTS TREATED WITH IMMUNE CHECKPOINT INHIBITORS

You have accessJournal of UrologyCME1 Apr 2023MP04-14 T CELL AND B CELL RECEPTOR SEQUENCING REVEALS ANTI-CANCER IMMUNE RESPONSE ASSOCIATED WITH IMMUNE-RELATED ADVERSE EVENTS IN ADVANCED RENAL CELL CARCINOMA PATIENTS TREATED WITH IMMUNE CHECKPOINT INHIBITORS Taigo Kato, Kazuma Kiyotani, Yu Ishizuya, Yoshiyuki Yamamoto, Koji Hatano, Atsunari Kawashima, and Norio Nonomura Taigo KatoTaigo Kato More articles by this author , Kazuma KiyotaniKazuma Kiyotani More articles by this author , Yu IshizuyaYu Ishizuya More articles by this author , Yoshiyuki YamamotoYoshiyuki Yamamoto More articles by this author , Koji HatanoKoji Hatano More articles by this author , Atsunari KawashimaAtsunari Kawashima More articles by this author , and Norio NonomuraNorio Nonomura More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003215.14AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Recently, several data are available on the relationship between immune-related adverse events (irAEs) and clinical efficacy in patients with immune checkpoint inhibitors (ICIs). In this study, we aimed to elucidate T cell receptor (TCR) and B cell receptor (BCR) repertoire in peripheral blood mononuclear cells (PBMCs) before and after ICI treatment, and at the onset of irAE for understanding of irAE-induced anti-cancer immune responses. METHODS: We collected metastatic tumor tissues and peripheral blood samples from 56 patients with advanced renal cell carcinoma before and 1 month after anti-programmed cell death protein 1 (PD-1) treatment initiation. Furthermore, we applied a next-generation sequencing approach to characterize TCR and BCR repertoires using tumor tissues and PBMCs. RESULTS: The number of irAEs was significantly higher in responder for PD-1 treatment (p<0.0001). TCR repertoire analysis revealed that diversity index (DI) for TCR alpha and beta in irAE present patients were significantly decreased at 1 month after treatment, indicating expansion of certain T cell clones even in PBMCs after the treatment (Figure 1A). We also examined morisita index to measure T cell similarity between pre- and post- treatment than irAE absent patients, indicating a larger change in the T cell clones, and inducing newly expanded T cells in post- treatment PBMC samples (Figure 1B). Importantly, irAE present patients had significantly higher numbers of peripheral T cell clones shared with metastatic tumor-infiltrating T cells than those in irAE absent patients at 1 month after treatment (p=0.038). We also confirmed that DI for BCR IgM and IgG in irAE present patients were significantly decreased at 1 month after the treatment, indicating expansion of certain B cell clones (Figure 1C). CONCLUSIONS: Our findings revealed that a certain number of irAE-induced T cell and B cell clones can circulate systemically and attack tumor cells synergistically in distant regions, leading to durable response in the patients with irAEs. Source of Funding: This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (C), grant number 18K09133 and 21K09343 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e38 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Taigo Kato More articles by this author Kazuma Kiyotani More articles by this author Yu Ishizuya More articles by this author Yoshiyuki Yamamoto More articles by this author Koji Hatano More articles by this author Atsunari Kawashima More articles by this author Norio Nonomura More articles by this author Expand All Advertisement PDF downloadLoading ...

Immune checkpoint blockade induces gut microbiota translocation that augments extraintestinal antitumor immunity.

Gut microbiota, specifically gut bacteria, are critical for effective immune checkpoint blockade therapy (ICT) for cancer. The mechanisms by which gut microbiota augment extraintestinal anticancer immune responses, however, are largely unknown. Here, we find that ICT induces the translocation of specific endogenous gut bacteria into secondary lymphoid organs and subcutaneous melanoma tumors. Mechanistically, ICT induces lymph node remodeling and dendritic cell (DC) activation, which facilitates the translocation of a selective subset of gut bacteria to extraintestinal tissues to promote optimal antitumor T cell responses in both the tumor-draining lymph nodes (TDLNs) and the primary tumor. Antibiotic treatment results in decreased gut microbiota translocation into mesenteric lymph nodes (MLNs) and TDLNs, diminished DC and effector CD8+ T cell responses, and attenuated responses to ICT. Our findings illuminate a key mechanism by which gut microbiota promote extraintestinal anticancer immunity.

The Blockade of Mitogen-Activated Protein Kinase 14 Activation by Marine Natural Product Crassolide Triggers ICD in Tumor Cells and Stimulates Anti-Tumor Immunity

Immunogenic cell death (ICD) refers to a type of cell death that stimulates immune responses. It is characterized by the surface exposure of damage-associated molecular patterns (DAMPs), which can facilitate the uptake of antigens by dendritic cells (DCs) and stimulate DC activation, resulting in T cell immunity. The activation of immune responses through ICD has been proposed as a promising approach for cancer immunotherapy. The marine natural product crassolide, a cembranolide isolated from the Formosan soft coral Lobophytum michaelae, has been shown to have cytotoxic effects on cancer cells. In this study, we investigated the effects of crassolide on the induction of ICD, the expression of immune checkpoint molecules and cell adhesion molecules, as well as tumor growth in a murine 4T1 mammary carcinoma model. Immunofluorescence staining for DAMP ectolocalization, Western blotting for protein expression and Z'-LYTE kinase assay for kinase activity were performed. The results showed that crassolide significantly increased ICD and slightly decreased the expression level of CD24 on the surface of murine mammary carcinoma cells. An orthotopic tumor engraftment of 4T1 carcinoma cells indicated that crassolide-treated tumor cell lysates stimulate anti-tumor immunity against tumor growth. Crassolide was also found to be a blocker of mitogen-activated protein kinase 14 activation. This study highlights the immunotherapeutic effects of crassolide on the activation of anticancer immune responses and suggests the potential clinical use of crassolide as a novel treatment for breast cancer.

Older but Stronger: Development of Platinum-Based Antitumor Agents and Research Advances in Tumor Immunity

Platinum (Pt) drugs have developed rapidly in clinical applications because of their broad and highly effective antitumor effects. In recent years, with the rapid development of immunotherapy, Pt-based antitumor agents have gained new challenges and opportunities. Since the discovery of their pharmacological effects in immunotherapy and tumor microenvironment regulation, research into Pt drugs has progressed to multi-ligand and multi-functional Pt precursors and their own shortcomings have been further highlighted. With the development of antitumor immunotherapy and the rise of combination therapy, the development of Pt-based drugs has started to move in the direction of multi-targeting, nanocarrier modification, immunotherapy and photodynamic therapy. In this paper, we first overview the recent applications of Pt-based drugs in antitumor inorganic chemistry, with a focus on summarizing the application of Pt-based drugs and their precursors in the anticancer immune response. The paper also provides a reasonable outlook on the future development of Pt-based drugs from the chemical and immunological perspectives, relying on the existing content and problems of Pt-based drug development. On the basis of the gathered information, joint multidisciplinary programs on implementing comprehensive immune analyses for the future development of novel anticancer metal compounds should be initiated.

Pathologic Response to Neoadjuvant Sequential Chemoradiation Therapy in Locally Advanced Breast Cancer: Preliminary, Translational Results from the French Neo-APBI-01 Trial

Radiation therapy (RT), a novel approach to boost the anticancer immune response, has been progressively evaluated in the neoadjuvant setting in breast cancer (BC). We aimed to evaluate immunity-related indicators of response to neoadjuvant chemoradiation therapy (NACRT) in BC for better treatment personalization. We analyzed data of the first 42 patients included in the randomized phase 2 Neo-APBI-01 trial comparing standard neoadjuvant chemotherapy (NACT) and NACRT regimen in locally advanced triple-negative (TN) and luminal B (LB) subtype BC. Clinicopathological parameters, blood counts and the derived parameters, total tumor-infiltrating lymphocytes (TILs) and their subpopulation, as well as TP53 mutation status, were assessed as predictors of response. Twenty-one patients were equally assigned to each group. The pathologic complete response (pCR) was 33% and 38% in the NACT and NACRT groups, respectively, with a dose-response effect. Only one LB tumor reached pCR after NACRT. Numerous parameters associated with response were identified, which differed according to the assigned treatment. In the NACRT group, baseline hemoglobin of ≥13 g/dL and body mass index of <26 were strongly associated with pCR. Higher baseline neutrophils-to-lymphocytes ratio, total TILs, and T-effector cell counts were favorable for pCR. This preliminary analysis identified LB and low-TIL tumors as poor responders to the NACRT protocol, which delivered RT after several cycles of chemotherapy. These findings will allow for amending the selection of patients for the trial and help better design future trials of NACRT in BC.

Pan-cancer genomic analysis shows hemizygous PTEN loss tumors are associated with immune evasion and poor outcome

In tumors, somatic mutations of the PTEN suppressor gene are associated with advanced disease, chemotherapy resistance, and poor survival. PTEN loss of function may occur by inactivating mutation, by deletion, either affecting one copy (hemizygous loss) leading to reduced gene expression or loss of both copies (homozygous) with expression absent. Various murine models have shown that minor reductions in PTEN protein levels strongly influence tumorigenesis. Most PTEN biomarker assays dichotomize PTEN (i.e. presence vs. absence) ignoring the role of one copy loss. We performed a PTEN copy number analysis of 9793 TCGA cases from 30 different tumor types. There were 419 (4.28%) homozygous and 2484 (25.37%) hemizygous PTEN losses. Hemizygous deletions led to reduced PTEN gene expression, accompanied by increased levels of instability and aneuploidy across tumor genomes. Outcome analysis of the pan-cancer cohort showed that losing one copy of PTEN reduced survival to comparable levels as complete loss, and was associated with transcriptomic changes controlling immune response and the tumor microenvironment. Immune cell abundances were significantly altered for PTEN loss, with changes in head and neck, cervix, stomach, prostate, brain, and colon more evident in hemizygous loss tumors. These data suggest that reduced expression of PTEN in tumors with hemizygous loss leads to tumor progression and influences anticancer immune response pathways.

Therapeutic efficacy of plant-produced Nivolumab in transgenic C57BL/6-hPD-1 mouse implanted with MC38 colon cancer

The therapeutic blockade of inhibitory PD-1 signaling has emerged as an effective approach for cancer immunotherapy. Nivolumab (Opdivo®), a monoclonal antibody (mAb) targeting the PD-1 immune checkpoint, is approved for treatment of several cancer indications. It functions by blocking the PD-1-mediated T-cell inhibition thus reinstating anticancer immune responses. Tremendous advances in plant biotechnology offer an alternative and economical strategy to produce therapeutic mAbs for immune-based therapies. In this study, recombinant anti-PD-1 Nivolumab was produced in Nicotiana benthamiana and the plant-produced anti-PD-1 mAb was exploited for cancer treatment in syngeneic mice model C57BL/6 mice that were used to test the antitumor efficacy of plant produced Nivolumab, along with commercial Opdivo®. C57BL/6 syngeneic mice treated with plant produced anti-PD-1 mAb exhibited reduction in the growth of established MC38 tumors. The plant produced Nivolumab treatment showed 82.9% antitumor effect in decreasing the tumor volume along with 50% tumor-free mice, whereas Opdivo® showed 90.26% reduction in volume without any tumor-free mice. Finally, plant-derived anti-PD-1 therapy was also well tolerated in tumor-bearing mice that correlated with no significant body weight changes. Overall, our plant-produced Nivolumab elicits significant inhibition of tumor growth in vivo and provides a proof-of-concept for the production of immunotherapy targeting PD-1.

Modification with Conventional Surfactants to Improve a Lipid-Based Ionic-Liquid-Associated Transcutaneous Anticancer Vaccine

Transcutaneous vaccination is one of the successful, affordable, and patient-friendly advanced immunization approaches because of the presence of multiple immune-responsive cell types in the skin. However, in the absence of a preferable facilitator, the skin's outer layer is a strong impediment to delivering biologically active foreign particles. Lipid-based biocompatible ionic-liquid-mediated nanodrug carriers represent an expedient and distinct strategy to permit transdermal drug delivery; with acceptable surfactants, the performance of drug formulations might be further enhanced. For this purpose, we formulated a lipid-based nanovaccine using a conventional (cationic/anionic/nonionic) surfactant loaded with an antigenic protein and immunomodulator in its core to promote drug delivery by penetrating the skin and boosting drug delivery and immunogenic cell activity. In a follow-up investigation, a freeze-dry emulsification process was used to prepare the nanovaccine, and its transdermal delivery, pharmacokinetic parameters, and ability to activate autoimmune cells in the tumor microenvironment were studied in a tumor-budding C57BL/6N mouse model. These analyses were performed using ELISA, nuclei and HE staining, flow cytometry, and other biological techniques. The immunomodulator-containing nanovaccine significantly (p < 0.001) increased transdermal drug delivery and anticancer immune responses (IgG, IgG1, IgG2, CD8+, CD207+, and CD103+ expression) without causing cellular or biological toxicity. Using a nanovaccination approach, it is possible to create a more targeted and efficient delivery system for cancer antigens, thereby stimulating a stronger immune response compared with conventional aqueous formulations. This might lead to more effective therapeutic and preventative outcomes for patients with cancer.

Type I interferon signaling in malignant blasts contributes to treatment efficacy in AML patients

While type I interferon (IFN) is best known for its key role against viral infection, accumulating preclinical and clinical data indicate that robust type I IFN production in the tumor microenvironment promotes cancer immunosurveillance and contributes to the efficacy of various antineoplastic agents, notably immunogenic cell death inducers. Here, we report that malignant blasts from patients with acute myeloid leukemia (AML) release type I IFN via a Toll-like receptor 3 (TLR3)-dependent mechanism that is not driven by treatment. While in these patients the ability of type I IFN to stimulate anticancer immune responses was abolished by immunosuppressive mechanisms elicited by malignant blasts, type I IFN turned out to exert direct cytostatic, cytotoxic and chemosensitizing activity in primary AML blasts, leukemic stem cells from AML patients and AML xenograft models. Finally, a genetic signature of type I IFN signaling was found to have independent prognostic value on relapse-free survival and overall survival in a cohort of 132 AML patients. These findings delineate a clinically relevant, therapeutically actionable and prognostically informative mechanism through which type I IFN mediates beneficial effects in patients with AML.

Repurposing fluphenazine to suppress melanoma brain, lung and bone metastasis by inducing G0/G1 cell cycle arrest and apoptosis and disrupting autophagic flux

Brain metastasis is the main cause of treatment failure and melanoma-related death. Inadequate concentrations of therapeutic drugs in the brain due to the blood-brain barrier (BBB) pose a major challenge in the treatment of brain metastasis. Antipsychotics can cross the BBB to reach the brain. Fluphenazine (FPZ) inhibits the survival of melanoma cells in vitro. However, its efficacy in suppressing the metastasis of melanoma, especially brain metastasis, remains unknown. Therefore, we explored whether fluphenazine (FPZ) can be repurposed for treating melanoma metastasis. A subcutaneous tumor model, and experimental metastasis models that simulate the outgrowth of melanoma cells in the brain, lung, and bone were established to verify the inhibitory effect of FPZ on melanoma cells. FPZ showed potential inhibitory effects against melanoma both in vivo and in vitro. It induced G0/G1 phase arrest and-mitochondrion-mediated intrinsic apoptosis, and inhibited autophagic flux in melanoma cells in vitro. In vivo, subcutaneous tumor, brain, lung, and bone models of metastatic melanoma were established. Intraperitoneal injection of FPZ (8mg/kg) significantly inhibited melanoma growth in the subcutaneous and experimental metastasis models. In a lung metastasis model, FPZ reduced the proportion of M2 macrophages and increased the proportion of CD8+ T cells and NK cells in vivo, thereby promoting an anticancer immune response. The findings of this study indicate that FPZ is a potential drug candidate for treating metastatic melanoma.