Tumor-draining Lymph Nodes Research Articles

Abstract 5243: CAR B cells to accelerate antigen presentation in cancer

Abstract Engineered B cells hold promise for multiple therapeutic applications. Walking Fish Therapeutics is engineering B cells to produce replacement proteins for deficiency diseases, antibodies of defined specificity and effector function, and Chimeric Antigen Receptor (CAR) B cells with and without therapeutic payloads for use in the oncology setting. B cells are professional antigen presenting cells (APCs) with the ability to present antigen to CD4+ T cells. Recent studies have found the presence of tumor infiltrating B cells are associated with favorable outcomes across multiple cancer types. Adoptive transfer of B cells has demonstrated antitumor activity in a syngeneic mouse model. B cells from OVA-vaccinated transgenic luciferase mice were adoptively transferred to mice bearing EG7.OVA-tumors. We observed B cells migrating to the tumor and tumor draining lymph node (TDLN), locations where B cells can engage with antigen. Further, we observed a strong anti-tumor response in mice receiving B cells from OVA-vaccinated donors. Based on these results, we hypothesized that B cells engineered to target tumor specific antigens may engage and activate the immune system leading to an enhanced antitumor immune response. We generated CAR B cells expressing synthetic BCRs with tumor-antigen specific scFvs fused to designated B cell signaling motifs. Expression of different CAR constructs were confirmed in both human and mouse B cells. Upon antigen engagement CAR B cells release pro-inflammatory cytokines. Importantly, we demonstrate in vitro that antigen-specific CAR B cells can recognize cognate cell surface antigen, leading to uptake, processing, and presentation on MHCII as detected by activation of peptide-specific CD4+ T cells. When administered in vivo by IV administration, we observed a significant increase in mouse CAR B cells at the tumor site compared to unengineered B cells. We are currently investigating additional CAR formats that may lead to enhanced anti-tumor activity. Citation Format: Mary Ellen Molloy, Jessica Balero, Ana Contreras-Sandoval, Mark Selby, Hangil Park, Srinivas Kothakota, Kathleen Boyle, Rosa Romano, Smrithi Rajendiran, Moises Hernandez, Ariel Pios, Rusty Williams. CAR B cells to accelerate antigen presentation in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5243.

Abstract 6751: In situ vaccination with Flt3l gene modified CD103+ type 1 conventional dendritic cells synergizes with anti-PD-1 checkpoint blockade in murine models of non-small cell lung cancer

Abstract A major hurdle in treatment of Non-Small Cell Lung Cancer (NSCLC) with anti-PD-1 immune checkpoint blockade (ICB) therapy is a lack of response (primary resistance) and relapse after an initial response (acquired resistance). Recent studies reveal that responses to PD-1/PD-L1 blockade are associated with high tumor mutational burden (TMB), increased CD8+ T cell infiltration and high baseline PD-L1 expression within the tumor microenvironment (TME), while impaired tumor antigen presentation and the immunosuppressive TME have been associated with resistance to ICB. One approach to overcome anti-PD-1 resistance is to intratumorally vaccinate NSCLC tumors with gene modified conventional dendritic cells (cDC), specifically the type I conventional DC (cDC1) lineage. Recent studies have established that generation of an anti-tumor immune response driven by CD8+ T cells requires the cross presentation of tumor associated antigens and that cDC1s are the primary cross presenting APC subtype in vivo, which can license CD8+ T cells to initiate an adaptive anti-tumor immune response. In addition, previous studies have shown that intratumoral administration of the FMS-like tyrosine kinase 3 ligand (FLT3L) protein can expand endogenous CD103+ cDC1s in the TME and augment anti-tumor immune responses to ICB therapy. Here, we engineered murine CD103+ cDC1s to constitutively secrete soluble FLT3L (FLT3L_cDC1) and performed in situ vaccination studies on anti-PD1 resistant murine models of NSCLC with LKB1-deficiency and elevated TMB that better represents human disease. In situ vaccination with FLT3L_cDC1 enhances anti-tumor efficacy compared to non-modified cDC1 vaccination and synergizes with anti-PD-1 ICB to inhibit tumor growth. FLT3L_cDC1 + anti-PD-1 combination therapy induces significant activation and expansion T cells and cDC1s within the TME. Furthermore, combination therapy significantly increases DC progenitor numbers within the tumor draining lymph node, including DC progenitors that are committed to the cDC1 lineage. Our data suggests in situ vaccination with FLT3L_cDC1 may represent a promising strategy to potentiate the efficacy of ICB and improve outcomes for patients with primary resistance PD-1/PD-L1 monotherapy. Citation Format: Jensen W. Abascal, Raymond J. Lim, Ramin Salehi-Rad, Zhe Jing, Michael S. Oh, William P. Crosson, Bitta P. Kahangi, Edgar Perez Reyes, Camelia Dumitras, Diana Reyimjan, Jessie Zhu, Linh M. Tran, Manash Paul, Kostyantyn Krysan, Bin Liu, Steven M. Dubinett. In situ vaccination with Flt3l gene modified CD103+ type 1 conventional dendritic cells synergizes with anti-PD-1 checkpoint blockade in murine models of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6751.

Abstract 4053: Targeting oxygen consumption with metformin and phenformin have differential effects on immune cells in the tumor microenvironment

Abstract T cell immune checkpoint blockade (ICB) has shown remarkable promise in melanoma and other cancers. However, most patients do not show clinical benefit. This is because tumors can activate multiple checkpoints and immunosuppressive pathways to evade anti-tumor immune responses. Inhibition of these immune suppressive mechanisms and immune checkpoints or repolarizing the tumor microenvironment (TME) to become more accessible to the immune system may be necessary for maximal therapeutic efficacy of immunotherapies. There is increasing evidence that tumor hypoxia can attenuate the antitumor immune response by promoting immune suppression and inhibiting direct killing by cytotoxic immune cells. We propose that blocking tumor oxygen consumption using drugs that target the mitochondrial complex I (phenformin and metformin) will enhance the efficacy of immunotherapies such as ICB in preclinical models such as B16 melanoma. In this study, we examined how low oxygen levels (hypoxia) influence T cell effector function such as cytotoxic activity and cytokine production in vitro. We co-cultured B16 melanoma cells in the presence of tumor specific T cells (Pmels) and incubated at 37ºC in either normoxia (~21% O2) or hypoxia (3% O2) and assessed killing and intracellular cytokine production 24 - 48 hours later. We show that the ability of tumor antigen-specific T cells to kill tumor cells or make effector cytokine such as IFNγ and TNFα is significantly reduced in hypoxia settings. We also examined the effects of hypoxia targeting drugs (metformin and phenformin) on T cell priming and activation. When T cells were activated in the presence of each drug in vitro, there was enhanced expression of T cell activation markers (CD25, Granzyme B) as well as an increase in their differentiation into central memory T cells. We next determined whether these drugs could alter the TME in vivo by treating C56BL/6J mice bearing established B16 tumors with phenformin and metformin and then examine the effects on the immune system in the tumor, tumor draining lymph nodes and spleen by flow cytometry. We found that these three drugs have differential effects on both the innate and adaptive the immune system in the tumors and periphery. Finally, we tested whether these drugs could delay B16 melanoma growth in vivo as a monotherapy or in combination with anti-PD1. We found that while both drugs can significantly delay tumor growth as monotherapies, only metformin showed improved anti-tumor efficacy when combined with anti-PD-1 in vivo. These studies show that while both drugs target tumor oxygen consumption (via inhibition of complex I), and can delay B16 tumor growth in vivo, they have differential effects on the TME and will need further considerations when combining with immune based therapies such as ICBs. Citation Format: Sadna Budhu, Anais Assouvie, Mamadou Bah, Svena Verma, Inna Serganova, Mayuresh Mane, Juan Zurita, Jason Koutcher, Vladimir Ponomarev, Jedd D. Wolchok, Taha Merghoub. Targeting oxygen consumption with metformin and phenformin have differential effects on immune cells in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4053.

Abstract 2788: Immunological targeting of integrin β1high/β3high melanoma

Abstract We previously reported two integrin β4 (ITGB4)-targeted immunologic strategies, e.g., ITGB4 protein-pulsed dendritic cells (ITGB4-DC) for vaccination and adoptive transfer of anti-CD3/anti-ITGB4 bispecific antibody (ITGB4 BiAb)-armed tumor draining lymph node (TDLN) T cells. Both immunotherapies significantly demonstrated efficacy in inhibiting local tumor growth and lung metastases. We found these therapeutic efficacies were closely associated with the T cell as well as humoral anti-integrin β4high cancer stem cells (CSCs) in breast (4T1) and head and neck squamous cell carcinoma (SCC7) tumor models. Recently, we further found that integrin subunits β1 (ITGB1) and β3 (ITGB3) are expressed at significantly high level in melanoma cells. In culture cell lines, integrin β1 and β3 are highly expressed on approximately 98% and 95% of the two mouse melanoma cell lines F10 and D5. We confirmed the expression of these two integrin subunits in newly harvested fresh melanoma tumors in vivo, which is very similar to the findings in cell lines. We then assessed ITGB1 and/or ITGB3-pulsed dendritic cell (DC) vaccines using F10 mouse model and demonstrated that ITGB1-DC or ITGB3-DC vaccine both exhibited significant antitumor efficacy inhibiting tumor growth, and ITGB1-DC + ITGB3-DC showed an additive/synergistic anti-tumor effect. Notably, we found that ITGB1 and ITGB3 loaded on separate DCs demonstrated more potent antitumor efficacy than loaded on the same DCs as vaccine. In addition, anti-PD-L1 significantly augmented the antitumor efficacy of ITGB1-DC/ITGB3-DC vaccine to inhibit tumor growth. Considering the crucial role of CSCs in tumor initiation, progression, metastasis, and recurrence, we further evaluated the expression of ITGB1 and ITGB3 on F10 and D5 ALDHhigh CSCs. We detected significantly higher expression of ITGB1 on ALDHhigh F10 CSCs (83.3%) and ALDHhigh D5 CSCs (87.1%) vs. ALDHlow F10 non-CSCs (0.2%) and ALDHlow D5 non-CSCs (2.0%), suggesting that ITGB1 and ITGB3-targeted vaccine-conferred anti-melanoma effects may have significantly involved targeting of ITGB1high/ITGB3high melanoma CSCs. Furthermore, recent studies suggested that bone marrow (BM)-DCs expressing dysfunctional integrin β2 (ITGB2) exhibited more potent antigen presentation function than WT BM-DCs. We found that ITGB2 highly (63.3%) expressed on the surface of BM-DC, but not on F10 or D5 tumor cells. On-going experiments include the investigation of experimental evidence to verify that ITGB1 and ITGB3-targeted vaccines have induced targeting of ITGB1high/ITGB3high melanoma CSCs; and both the cellular and humoral mechanisms underlining the anti- ITGB1high/ITGB3high melanoma CSC immunity. At the same time, generation and use of ITGB2 deficient DCs for the vaccine preparation to improve the efficacy of DC vaccine targeting integrin ITGB1high/ITGB3high melanoma and melanoma CSCs warranty further investigation. Citation Format: Feng Wang, Ying Xin, James J. Moon, Alfred E. Chang, Max Wicha, Qiao Li. Immunological targeting of integrin β1high/β3high melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2788.

Abstract 6734: Preclinical impact of pretreatment with CRS mitigation agents on pharmacodynamic response to TAK-500, a systemically delivered CCR2-targeted STING agonist iADC

Abstract Background: Stimulator of interferon genes (STING) agonists elicit potent innate immune enhancement and mobilize robust adaptive immune responses. Given the potential risk of immunotoxicity from associated cytokine release due to STING agonist treatment, there is a need to identify mitigation strategies that minimize immune-related toxicity while offering a therapeutic window for STING-mediated anti-tumor effects. This strategy is particularly relevant for myeloid-targeted immunotherapies, as cytokine release syndrome (CRS) can be myeloid cell-mediated. We utilized preclinical models to evaluate the pharmacological impact of pretreatment with agents routinely used for management of CRS in advance of delivering TAK-500, a novel, systemic, immune-cell directed STING agonist antibody drug conjugate (iADC) that selectively targets CCR2+ myeloid cells. Methods: Dexamethasone and anti-IL-6R antibodies were tested at clinically relevant doses 1 hour prior to treatment with TAK-500 or mTAK-500, a murine surrogate, to study immunomodulatory and anti-tumor effects in preclinical models. Induction of innate and adaptive immune responses and cytokine release was evaluated in human, ex vivo, and in tumor-bearing mouse models to characterize potential impact of premedication strategies on drug pharmacology and anti-tumor efficacy. Results: TAK-500 triggered dose-dependent monocyte activation and cytokine secretion ex vivo. Monocyte activation was minimally impacted by dexamethasone or anti-IL-6R pretreatment. Suppression of IL-6 and IFN-ƴ and increase in IL-10 were observed following dexamethasone pretreatment, whereas anti-IL-6R maintained a more proinflammatory milieu. In syngeneic mouse models, pretreatment with dexamethasone and anti-mouse IL-6R had minimal impact on mTAK-500 anti-tumor efficacy. Key pharmacodynamic effects, including innate immune cell activation in periphery and tumor-draining lymph node and intratumoral CD8+ T cell accumulation, were minimally impacted by either pretreatment agent. Serum cytokine analyses demonstrated suppression of IL-6, IFN-ƴ and increases in IL-10 with dexamethasone, but not with anti-IL-6R, similar to ex vivo findings. Conclusion: Premedication of TAK-500 and mTAK-500 with dexamethasone or anti-IL-6R antibodies does not substantially impact STING-mediated innate immune activation, mobilization of adaptive immune responses or anti-tumor efficacy, despite altered cytokine profiles compared to (m)TAK-500 without premedication. Clinically, these premedication strategies have been effective in mitigating immunotherapy-associated CRS with T cell therapeutics, and here resulted in maintained anti-tumor activity when used in combination with TAK-500, suggesting a viable potential strategy for mitigation of STING-induced immunotoxicities in patients. Citation Format: Emily Rosentrater, Michelle L. Ganno, Tiquella Hatten, Henry Mack, Arjun Reddy Bollampalli, Dong Mei Zhang, Alex Parent, Jeffrey Raizer, Richard C. Gregory, Neil Lineberry, Vicky Appleman. Preclinical impact of pretreatment with CRS mitigation agents on pharmacodynamic response to TAK-500, a systemically delivered CCR2-targeted STING agonist iADC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6734.

Abstract 1425: Epigenetic modulation synergizes with a novel TCR β chain directed antibody-fusion molecule to suppress checkpoint-refractory tumors irrespective of MHC I status

Abstract Background: Epigenetic silencing of MHC-I, and poor T cell infiltration and function in the tumor microenvironment (TME) are prevalent in solid malignancies and associate with lack of response to immune checkpoint blockade (ICB). Novel combination therapies may overcome these barriers, thereby providing clinical benefit across solid tumors. STAR0602 is a first-in-class bifunctional therapeutic molecule, that simultaneously engages a nonclonal mode of T cell receptor (TCR) activation with costimulation to promote activation and expansion of αβ T cell subsets expressing distinct variable β (Vβ) TCR chains. Here, we examined the tumor-suppressive ability and mode of action of a murine surrogate of STAR0602 (mSTAR1302) in combination with the class I HDAC inhibitor Entinostat, an epigenetic modulator that enhances tumor immune cell recognition, in multiple tumor models, including ICB-refractory. Methods: mSTAR1302 and/or Entinostat were administered to mice bearing ICB-responsive breast EMT6 wild-type (wt) and ICB-refractory EMT6 β2-microglobulin (B2m)-deleted and CT26 (Kras G12Dmut colon) tumors. Anti-tumor efficacy, survival, and protective memory were monitored. Immune cell depletion studies were conducted. Comprehensive proteomic and immune profiling of tumor, tumor-draining lymph node (tdLN), and spleen cell populations was conducted. Antigen-specific IFNg T cell responses were evaluated by ELISpot. Results: Combination therapy demonstrated significant enhancement in tumor suppression and prolonged survival in CT26 (MHC-I+), EMT6 wt (MHC-I+) and EMT6 β2m-deleted (MHC-Inull) tumors relative to single agent therapy, as well as enhanced tumor-specific protective memory responses. Tumor suppression was independent of CD4+ T cell immunity and associated with sustained activation and expansion of Vβ13 CD8+ T cells. Conclusions: Ongoing mechanistic studies indicate combination therapy to elicit complimentary mechanisms of tumor suppression beyond CD8+ T cells. Ongoing proteomic and single-cell transcriptomic investigation of MHC-I competent and null tumors will provide a deeper insight into the mode of action of this combination therapy. Collectively, these data support the use of STAR0602 in combination with Entinostat for the treatment of solid malignancies, including for patients harboring ICB-refractory tumors devoid of MHC class I. Citation Format: Katherine E. Lothstein, Asma S. Khelifa, Masaya Miyamoto, Christine M. Minnar, Lisa Poppe, Nicholas Roller, Andrew Bayliffe, Ke Liu, Jacques Moisan, Madan Katragadda, Zhen Su, Jeffrey Schlom, Sofia R. Gameiro. Epigenetic modulation synergizes with a novel TCR β chain directed antibody-fusion molecule to suppress checkpoint-refractory tumors irrespective of MHC I status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1425.

Abstract 2809: Bifidobacterium species BB_003 identified through metatranscriptomic analysis with experimentally validated antitumor actions

Abstract Introduction & Objectives: A host's microbiome influences both gene expression and phenotype. Whole transcriptome sequencing (WTS) provides information about the microbiome in addition to gene expression. This research probes the correlation between microbial abundance, host gene transcription, and therapeutic efficacy, pinpointing a beneficial microbial strain and its mechanism of action. Materials & Methods: Bladder tissue biopsies from NMIBC patients underwent WTS and subsequent bioinformatic analysis. After in silico evaluation, in vitro assays including WST, quantitative PCR, and immune cell profiling were performed on mouse splenocytes. These assays were followed by in vivo assessment in both subcutaneous and orthotopic murine tumor models. Results: Among 59 biopsies, non-recurrence correlated with heightened presence of Lactococcus species LC_001 and Bifidobacterium species BB_003. Notably, BB_003 was associated with an extended recurrence-free survival (RFS). Gene set enrichment analysis (GSEA) implicated BB_003 in mediating a pro-inflammatory cascade, accentuating macrophages and interferons. In vitro WST assays showed the antitumor potential of BB_003_β, characterized by upregulated pro-inflammatory gene transcription. This strain increased effector CD8 T cells and M2 macrophages. In vivo, direct live BB_003_β introduction into subcutaneous tumors curtailed tumor proliferation. An increase in effector CD8 T cell populations within tumors and elevated cDC1 and M1 macrophage counts in tumor-draining lymph nodes were observed. Intravesical administration of live bacteria markedly decreased bladder weight, amplifying interferon alpha and beta gene expressions compared to controls. Conclusions: According to this study, BB_003_β inhibits tumor growth and is involved in immune response. Our study confirmed the antitumor properties of BB_003_β and suggested that cytokine and immune cell stimulation play a key role in its mechanism of action. Augmented insights warrant further exploration, particularly in synergy with intravesical BCG treatment. Citation Format: Xuan-Mei Piao, Kyungchan Min, Young Joon Byun, Chuang-Ming Zheng, Seon-Kyu Kim, Seong-Hwan Park, Sungmin Moon, Kyeong Kim, Ho Won Kang, Won Tae Kim, Seok Joong Yun, Hansoo Park. Bifidobacterium species BB_003 identified through metatranscriptomic analysis with experimentally validated antitumor actions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2809.

Abstract 1159: Machine learning integrating spatial omics uncovers humoral immunity patterns in intratumoral tertiary lymphoid structures in pancreatic cancer pathologic responders

Abstract Background: In pancreatic ductal adenocarcinoma (PDAC), rare long-term survivors correlate with high intratumoral tertiary lymphoid structure (TLS) density. This finding prompted our investigation of clinically viable strategies to induce TLS in patients with immune-excluded tumors. We previously reported the induction of intratumoral TLS following administration of a neoadjuvant GM-CSF-secreting allogeneic vaccine (GVAX) to PDAC patients (NCT00727441). However, no clinical benefit was observed, likely owing to immune tolerance mechanisms governing the PDAC tumor microenvironment (TME). We previously observed upregulation of both the PD-1 and 4-1BB pathways with GVAX, and thus in a subsequent neoadjuvant trial combined GVAX with PD-1 blockade and 4-1BB agonism (NCT02451982) which was associated with pathologic responses. We hypothesized this combination strategy induced TLS of higher maturity and anti-tumor activity compared to GVAX alone. Methods: To explore how this therapeutic strategy affected TLS morphology and intercellular crosstalk, we leveraged the Visium spatial transcriptomics platform and a 35-marker customized TLS panel for imaging mass cytometry. We generated cellular and molecular maps of the TME after neoadjuvant treatment in 26 PDAC patients (GVAX n=19, GVAX+aPD1 n=2, GVAX+aPD1+a41BB n=5). To compare TLS maturation in parallel with secondary lymphoid organ-mediated tumor immunity, we also profiled tumor-adjacent lymph nodes. We applied unsupervised learning with non-negative matrix factorization (NMF) and trained AI-enabled image classification models to characterize cellular states within tissue structures of interest. Results: We identified spatial gene expression NMF patterns in PDAC TLS spanning across distinct morphologies and neoadjuvant treatment arms. Intratumoral TLS after GVAX were found to propagate activated B cells expressing immunoglobulins that infiltrated into malignant cellular niches. TLS NMF patterns were also associated with autoimmune disease signatures, such as diabetes, in a subset of patients. We scored TLS using tumor-draining lymph nodes as a reference and found increased maturation of TLS after PD-1 blockade, while addition of 4-1BB agonism significantly boosted the cytotoxic NK/T cell compartment compared to GVAX alone. Conclusions: We present machine learning approaches for spatial multi-omics analysis to characterize the TLS-enriched TME. We mined genome-wide spatial TLS gene expression patterns elucidating the spatial dynamics of humoral immunity of rare immunotherapy pathologic responders in PDAC. Altogether our findings shed light on the plasticity of TLS in neoadjuvant immunotherapy and suggest future immunotherapy approaches should target both humoral and cytotoxic NK/T cell compartments to augment responses in solid tumors. Citation Format: Dimitrios N. Sidiropoulos, Sarah M. Shin, Alexander Girgis, Daniel H. Shu, Janelle Montagne, Atul Deshpande, Jeanette A. Johnson, Lucie Dequiedt, Victoria Jacobs, Aleksandra Ogurtsova, Guanglan Mo, Xuan Yuan, Genevieve Stein-O’Brien, Mark Yarchoan, Qingfeng Zhu, Ashley Kiemen, Elizabeth M. Jaffee, Lei Zheng, Won Jin Ho, Robert Anders, Elana J. Fertig, Luciane T. Kagohara. Machine learning integrating spatial omics uncovers humoral immunity patterns in intratumoral tertiary lymphoid structures in pancreatic cancer pathologic responders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1159.

Abstract 4082: Anchored IL-12 synergizes with an epigenetic modulator to promote immune remodeling and overcome anti-PD1-refractory murine tumors

Abstract Background Most patients with solid malignancies harbor innate or acquired resistance to immune checkpoint blockade (ICB), prompting the need for novel therapeutic strategies. Interleukin-12 (IL-12) is a promising cytokine for cancer therapy due to its ability to bridge innate and adaptive immunity. However, a narrow therapeutic index limits the use of systemic IL-12 therapy. Here, we investigated the tumor-suppressive effects and mode of action of intra-tumorally delivered murine Interleukin-12 anchored to aluminum hydroxide (referred as mANK-101) in combination with the class I HDAC inhibitor entinostat, in various ICB-refractory murine tumor models, including CT26 (colorectal) and MOC-1 (HPV16neg). We hypothesized that combining Entinostat with an anchored form of IL-12 could overcome systemic toxicity while maintaining anti-tumor activity. Methods Entinostat and intra-tumoral mANK-101 were administered to mice bearing well-established αPD-1-refractory CT26 (colorectal) and MOC-1 (HPV16neg) tumors. Antitumor activity, survival, and protective memory upon tumor rechallenge were evaluated. Comprehensive proteomic and immune cell analysis was performed in MOC-1 tumors, tumor-draining lymph node (tdLN), and periphery. Tumor-specific T cell responses were examined. Results We demonstrate that intra-tumoral mANK-101 synergizes with entinostat to suppress multiple αPD1-refractory tumors, resulting in significant tumor eradication (62-88%), survival benefit (P < 0.0001), and protective memory, including CT26 (colon, Kras G12Dmut) and MOC-1 (oral, HPV16neg). Analysis of MOC-1 tumor-bearing mice demonstrated these effects to be associated with peripheric activation of CD8+ and NK lymphocytes, augmented polyfunctional IFNγ+/TNFα+-producing CD8+ T cells, CD8+ T cell effector memory, and tumor-specific T cell responses. Significant decrease in CD4+ Tregs and increased CD8/Treg ratio were also observed. Ongoing functional studies, proteomic and immune cell analysis at the tumor site, tdLN, and periphery, including single cell transcriptomics and epigenetic studies, will allow for a deeper understanding of the synergistic effect of mANK-101 with the epigenetic modulator Entinostat. Conclusions Collectively, these findings form a rationale for the clinical combination of intralesional delivery of ANK-101 with entinostat for patients with ICB-refractory malignancies, including colorectal and HPV16neg head and neck cancers. Citation Format: Asma S. Khelifa, Katherine L. Lothstein, Masaya Miyamoto, Rob Tighe, Sailaja Battula, Howard L. Kaufman, Jeffrey Schlom, Sofia R. Gameiro. Anchored IL-12 synergizes with an epigenetic modulator to promote immune remodeling and overcome anti-PD1-refractory murine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4082.

Abstract 6686: The mechanism of gut microbiome-mediated immune activation on the clinical efficacy of PD-1 blocking treatment in solid tumors

Abstract Gut microbiota is associated with antitumor efficacy of immune checkpoint blockade therapies, yet the mechanism is not largely understood. Here, we show that a novel bacterial strain of Ruminococcaceae isolated from the feces of patients who respond to PD-1 (programmed cell death 1) blockade efficiently primes/activates tumor-specific CD8+ T cells through effective stimulation/maturation of CD103+ dendritic cells (DCs) during migration from the gut to the tumor microenvironment (TME). The administration of this bacteria strain with PD-1 blockade significantly inhibited tumor growth, even when adding them to fecal transplantation of non-responder patients in animal models. Mechanistically, the bacterial strain promoted the IRF8-driven differentiation of CD103+ DCs by stimulating multiple Toll-like receptors and the accumulation of these DCs in tumors and tumor-draining lymph nodes. These CD103+ DCs prolonged engagement with cognate antigen-specific CD8+ T cells, which induced the activation of CD8+ T cells specific for a wide variety of tumor antigens and fostered their PD-1 expression through enhanced T-cell activation signals with NFATc1 nuclear translocation, thereby leading to abundant PD-1+CD8+ T cells, a key effector cell population in PD-1 blockade, with a broader T-cell receptor repertoire in the TME. Our findings with novel single bacterium isolated from feces of responder patients demonstrate the mode of action by gut microbiota to augment antitumor immunity. Citation Format: Nina Yi-Tzu Lin, Shota Fukuoka, Daisuke Motooka, Yuko Shigeno, Takuma Irie, Dieter M. Tourlousse, Kazutoshi Murotomi, Yuji Sekiguchi, Hideyuki Tamaki, Mariko Shindo, Takahiro Tsuji, Hiroaki Wake, Koichi Goto, Toshihiko Doi, Kohei Shitara, Keisuke Watanabe, Yuka Maeda, Shota Nakamura, Yoshimi Benno, Hiroyoshi Nishikawa, Shohei Koyama. The mechanism of gut microbiome-mediated immune activation on the clinical efficacy of PD-1 blocking treatment in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6686.

Abstract 4065: Entinostat synergizes with the IL-15 superagonist N-803 and anti-PD-1 to engage cDC1 and stem-like TCF-1+ CD8+ T and NK cells to overcome colorectal tumors refractory to anti-PD-1 therapy

Abstract Background: Immune checkpoint blockade (ICB) has limited efficacy in colorectal carcinoma (CRC), where ~85% of patients harbor microsatellite stability (MSS). A minor subset of CRC patients (~15%) harbor microsatellite instability (MSI) and demonstrate improved responses to ICB ranging from 30-50%. Combination therapy has the potential to increase the response rate. Entinostat, a class I HDAC inhibitor, has demonstrated potential to enhance ICB efficacy, such as αPD-1 and immunostimulatory cytokines. N-803, a heterodimeric IL-15 superagonist that enhances proliferation and activation of CD8+ and NK cells has demonstrated to enhance the clinical benefit of Nivolumab in non-small cell lung cancer patients that were relapsed or refractory to αPD-1 monotherapy. Here, we investigated the antitumor efficacy and mode of action of combination therapy comprising αPD1, Entinostat, and N-803 in αPD1-refractory CT26 and Jak1-deleted MC38 CRC tumor models. Methods: Entinostat, N-803, and αPD1 alone or in combination were administered to mice bearing established CT26 or Jak1-deleted MC38 tumors. Antitumor efficacy, survival, and protective memory upon tumor rechallenge were monitored. Comprehensive flow cytometric profiling of the tumor microenvironment (TME), tumor-draining lymph node (tdLN), and spleen cell populations was conducted. CT26 tumor and tdLN immunomes were examined by single-cell transcriptomics. Cytokines and chemokines were examined in the TME and sera. Antigen-specific IFNg T cell responses were evaluated by ELISpot. Results: We demonstrated that combination therapy with Entinostat, N-803 and αPD1 elicits potent suppression of CT26 and Jak1-deleted MC38 tumors, with median overall survival increasing 33% to not reached. These effects were associated with increased activation, infiltration, cytokine production, and cytolytic function of tumor CD8+ T and NK cells, and increased sera IFNg and TNFα. Tumor resolution and protective memory were associated with development of p15e-specific T cell responses. Increased migration of CD8+ T cells and cross-presenting dendritic cells (cDC1) to the tdLN, as well as significant enrichment of cross-presentation genes in tumor cDC1s was observed. Transcriptomic and phenotypic analyses demonstrated increased stem-like TCF-1+ CD8+ T cell accumulation in the tdLNs and TME of mice treated with triple combination. Conclusions: Combination therapy with Entinostat plus N-803 and αPD1 synergized to promote a sustained antitumor response and prolonged survival. We hypothesize the antitumor effects were due to collaborative efforts of CD8+ T and NK cells, and cDC1 cells in tdLN and TME. These results provide a rationale for the clinical testing of αPD1, Entinostat and N-803 combination for patients with CRC, including those with acquired resistance to ICB. Citation Format: Christine M. Minnar, Masaya Miyamoto, Asma S. Khelifa, Grace Lui, Katherine L. Lothstein, Paul L. Chariou, Thomas J. Meyer, Alexei Lobanov, Margaret Cam, Jeffrey Schlom, Sofia R. Gameiro. Entinostat synergizes with the IL-15 superagonist N-803 and anti-PD-1 to engage cDC1 and stem-like TCF-1+ CD8+ T and NK cells to overcome colorectal tumors refractory to anti-PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4065.

Abstract 4210: Stromal remodeling danger signals regulate “second-touch” antigenic encounters and promote CD8+ T cell effector differentiation in the TME

Abstract Tumor immune infiltration (“hot” tumor microenvironment, TME) constitutes a powerful predictor of outcomes and immunotherapy efficacy, however underlying mechanisms remain elusive. Recent evidence shows that TME-repriming of incoming CD8+ T cells is required for full effector functionality. Reminiscent of the “second-touch” hypothesis proposed by Klaus Ley for CD4+ differentiation, tumor-draining lymph node (TDLN) priming (“first-touch”) generates antigen-specific stem-like TCF1+CD8+ T cells. These stem-like CD8+ T cells then migrate to the tumor periphery where “second-touch” antigenic encounters with non-migratory CCR7neg dendritic cells (DC) (importantly, the cross-presenting cDC1 subset) leads to effector CD8+ T cell differentiation and tumor entry. These second-touch encounters occur within specialized stromal niches that are rich in antigen-presenting cells (APC). We have hypothesized that productive APC:CD8+ interactions require stroma-derived signals that emulate provisional matrix remodeling in embryonic development and/or adult wound healing. Thus, stroma remodeling by the expanding tumor translates into a conserved “danger signal” that licenses APC to activate CD8+ T cell responses. We have focused on a prototypical pathway of provisional matrix remodeling, the regulated proteolysis of the matrix proteoglycan versican (VCAN) to generate a bioactive fragment endowed with immunostimulatory activities, versikine. Stromal VCAN proteolysis at the site releasing versikine is associated with T-cell infiltration across human solid and liquid tumors. To investigate the mechanisms by which versikine regulates first- and second-touch antigenic encounters, we dissected the immune microenvironment of versikine-replete tumors of immunocompetent models at single-cell resolution. Non-migratory Ccr7neg cDC1 in versikine-replete tumors overexpress MHC-I and MHC-II antigen presentation machinery (Tap1, Ciita), maturation (Irf7, ISGs, Tlr3) and co-stimulation markers (Cd86, 4-1BBL), while Ccr7pos DC (mregDC) markedly overexpress Cd40 compared to control mregDC. In parallel, Cd4+ Tfh-like cells (overexpressing Cd40lg, Icos and Maf) accumulate in versikine-replete tumors, mirroring the accumulation of CD4+ T-cells in the stroma of human tumors displaying intense VCAN proteolysis. Consequently, versikine-replete tumors demonstrate expansion of late effector CD8+ T cells (Gzmb+Prf1+). Versikine reverses anti-PD-1 refractoriness in the “cold” model, Lewis Lung Carcinoma (LLC). In “warm” tumor models, versikine with anti-PD-L1 suffices to eradicate tumors and establish immune memory with resistance to tumor rechallenge. Our presentation will focus on the mechanistic dissection of stromal signals regulating antigen presentation across models as well as approaches for translation into novel vaccine platforms. Citation Format: Athanasios Papadas, Duncan Hong, Alexander Cicala, Alicia Gibbons, Daniel J. Lagal, Yun Huang, Yaling Dou, Fotis Asimakopoulos. Stromal remodeling danger signals regulate “second-touch” antigenic encounters and promote CD8+ T cell effector differentiation in the TME [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4210.

Local Enrichment with Convergence of Enriched T-Cell Clones Are Hallmarks of Effective Peptide Vaccination against B16 Melanoma.

Some peptide anticancer vaccines elicit a strong T-cell memory response but fail to suppress tumor growth. To gain insight into tumor resistance, we compared two peptide vaccines, p20 and p30, against B16 melanoma, with both exhibiting good in vitro T-cell responses but different tumor suppression abilities. We compared activation markers and repertoires of T-lymphocytes from tumor-draining (dLN) and non-draining (ndLN) lymph nodes for the two peptide vaccines. We showed that the p30 vaccine had better tumor control as opposed to p20. p20 vaccine induced better in vitro T-cell responsiveness but failed to suppress tumor growth. Efficient antitumor vaccination is associated with a higher clonality of cytotoxic T-cells (CTLs) in dLNs compared with ndLNs and the convergence of most of the enriched clones. With the inefficient p20 vaccine, the most expanded and converged were clones of the bystander T-cells without an LN preference. Here, we show that the clonality and convergence of the T-cell response are the hallmarks of efficient antitumor vaccination. The high individual and methodological dependencies of these parameters can be avoided by comparing dLNs and ndLNs.

Abstract 2: T cells selected from lymph node acquisition for adoptive cell therapyin NSCLC

Abstract Background: A primary limitation of PD-1 inhibitors in non-small cell lung cancer (NSCLC) arises from their inability to act on 'cold' tumors without tumor-reactive T cells, necessitating alternative approaches. Adoptive cell therapy (ACT) using (CAR)-engineered cells, strives to enhance antitumor immunity but faces several challenges such as identifying safe antigens, tumor heterogeneity, antigen escape, cell trafficking, and T cell persistence. To address these issues, we explored a new source of T cells from the benign tumor draining lymph nodes (tdLNs) of NSCLC patients. Our initial results have shown that tdLNs is a reservoir for of tumor-relevant 'stem-like' T cells. We posit that employing these pluripotent T cells for ACT could achieve significant tumor rejection in NSCLC. Methods: Resected tumors, tdLN, non-draining (ndLN), and blood from NSCLC patients, as well as a syngeneic murine lung cancer model (344SQ), underwent analysis. T cells were profiled using flow cytometry, cytotoxicity and proliferation assays. TCR and single cell (sc)RNA sequencing assessed clonal expansion, diversity, and transcriptional profiles of tumor-relevant T cells. T cells were then transduced with an ICAM-1 targeting CAR, in vivo efficacy was evaluated in an A549 murine lung cancer model. Results: T cell subsets with stem-cell memory characteristics, as indicated by PD-1+, TCF1hi, CXCR5+, and CD8+ expression, which were not significantly found in the tumor or PB. These T cells exhibited progenitor-like transcriptional signatures, enhanced SELL and TCF-1 expression, fewer exhaustion markers, and superior in vitro proliferation compared to TILs from both a murine lung cancer model and patient-derived tissues. scRNA sequencing, coupled with TCR "tumor matching" (TM) techniques, exposed a rich clonal diversity of tumor-relevant clones within tdLNs, which showcased a broader transcriptional memory profile and distinct CD4+ and CD8+ phenotypes. Upon analyzing the top 100 expanded (n>3) TM clones, 47 featured the presence of tdLN-derived T cells, covering progenitor, stem cell-like, and central memory clusters. T cell subsets were then transduced with a CAR targeting ICAM-1—a cell surface protein frequently overexpressed in NSCLC tumors. Manufacturing protocol yielded high transduction efficiency and T cell expansion within two weeks in 6/6 patients, consistent with PB-derived CAR T cells and on par with the optimal dosing requirements of an ICAM-1 CAR Phase I trial (NCT04420754). tdLN-CAR T cells showed potent antitumor efficacy compared to the control in an aggressive NSCLC murine model (Median survival 103d vs 66d; respectively; p=0.006). Conclusions: This represents the first reported use of T cells from tdLN for genetically engineered ACT. The data indicate that modifying antigen-experienced, stem-like T cells from tdLN with CAR is a promising and efficient method in a NSCLC murine model, warranting further research. Citation Format: Tatiana Delgado Cruz, Yanping Yang, Rachel Honigsberg, Geoffrey Markowitz, Nasser K Altorki, Vivek Mittal, Moonsoo M. Jin, Jonathan Villena-Vargas. T cells selected from lymph node acquisition for adoptive cell therapyin NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2.

Abstract 5936: A novel class of orally bioavailable small molecules induces potent and sustained tumor growth inhibition and T cell infiltration in various solid tumor mouse models

Abstract The introduction of targeted immunomodulating agents has transformed cancer treatment over the last decade by demonstrating unprecedented efficacy in patients who respond. However, limited clinical response rates as well as adverse events underline the need to identify additional novel modalities in cancer immunotherapy. Here we report on the discovery of a novel class of low molecular weight compounds for oral application that selectively enhance tumor-reactive T cell cytotoxicity. Hit-to-lead development of compound hit series A was performed based on medicinal chemistry to investigate structure-activity-relations. A-306, a representative of the A compound class, induced increased T cell proliferation, elevated levels of the activation markers CD25 and CD69 as well as enhanced secretion of IFN-γ, IL-2 and TNF-α upon anti-CD3/CD28 stimulation. A-306 treatment without T cell receptor ligation had no effect on T cell activation, suggesting selective activation potential only in the context of antigen encounter. Furthermore, A-306 strengthened T-cell mediated killing of M21 melanoma cells and enhanced T cell function in response to viral antigens by showing a Th1 signature. Medicinal chemistry efforts resulted in front runner compounds A-481 and A-687 that show a similar profile and potencies down to nanomolar level. In a murine B16-SIY melanoma and EO771 breast cancer model oral single-agent administration with front runner compounds was well tolerated, showed good bioavailability in lymphoid organs and plasma and resulted in significant tumor growth inhibition beyond the end of treatment (D21). T cells from tumor draining lymph nodes of long-term surviving mice in both cancer models showed a distinct activation pattern indicative of anti-tumor immunity. Furthermore, oral application of A-481 in combination with intra peritoneal administration of anti-PD1 antibodies eradicated B16-SIY melanoma cells in a synergistic manner. Taken together, we report on the identification of a novel class of small molecules possessing high potential for selective anti-tumor activation of the immune system upon oral administration. Front runner compounds A-481 and A-687, which significantly inhibit tumor growth in a B16-SIY melanoma and EO771 breast cancer mouse model, lead to prolonged survival beyond the end of treatment and display a good safety profile. The final candidate compound will undergo testing in toxicity studies and enter a first-in-human trial in selected solid tumors in 2025. Citation Format: Bianca Gapp, Gunther Zischinsky, Maria Urban, Nora Birnbacher, Alice Schwarzböck, Sabine Grünaug, Tommaso Gastaldi, Anton Stütz, Peter Nussbaumer, Stefan Stranner, Julia Buchberger, Andreas Birbach, Daniela Schögl, Mario Kuttke, Alexander Dohnal, Romana Gugenberger. A novel class of orally bioavailable small molecules induces potent and sustained tumor growth inhibition and T cell infiltration in various solid tumor mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5936.

Abstract 709: Enhancing prostate cancer treatment: Synergistic effects of mechano-thermal focused ultrasound and radiation therapy

Abstract Current treatments for prostate cancer primarily rely on radiation and hormone deprivation therapy, yet they often lack effective immunotherapeutic effects. Furthermore, long-term hormone ablation has side effects, such as, impotency and osteoporosis. Addressing this gap, we introduce an innovative dual-frequency focused ultrasound (FUS) platform for non-ablative focal therapy, which induces protein unfolding, ER stress, and increases heat-shock protein expression along with translocation of endoplasmic reticular chaperone proteins (e.g., calreticulin) on the tumor cell surface, thereby sensitizing them for phagocytosis by dendritic cells for efficient antigen presentation and cross priming. We hypothesized that FUS-mediated acoustic immune priming can be combined with ablative therapies, such as, hormone ablation and/or radiation therapy (RT) for induction of tumor-specific adaptive immune response and better tumor control than monotherapy in murine prostate cancer models. Utilizing murine prostate cancer cell lines, MyC-CaP and RM1, we explored the cellular responses to androgen deprivation, observing distinct sensitivities. Twenty-four hours after FUS treatment tumor cells experienced mechano-thermal stress response with 15-fold increase in Hspa1a expression. Transmission Electron Microscopy studies showed formation of myelin figures, nuclear membrane deformation, and lipid droplet formation. In vivo, the combination of FUS (220w/cm2 intensity for 3 sec at focal point) and RT (8-10 Gy daily x two fractions) markedly delayed tumor growth and achieved complete cure in 14-17% of animals. Through multicolor flow cytometry, we detected significant alterations in the immune microenvironment, including increased CD8+ T cell infiltration and reduced tumor-associated macrophages in the tumor bed. However, we also observed a compensatory rise in pro-tumor immune cells such as PMN-MDSCs and Tregs with significant suppression of eosinophils (p<0.05). Interestingly, there was suppression of splenic PMN-MDSCs, Mo-MDSCs, and tumor draining lymph node macrophages, suggesting systemic immune modulation in peripheral lymphoid organs of FUS+RT-treated tumor-bearing animals. Our findings reveal that the combination therapy of FUS and RT improves tumor control in murine prostate cancer and significantly modulates the systemic and tumor immune microenvironment. This approach offers new avenues for immunotherapy, potentially transforming the treatment landscape for prostate cancer, typically resistant to conventional immunotherapeutic strategies. Citation Format: Soumya Chatterjee, Saurabh Singh, Stephen Barry, Chandan Guha. Enhancing prostate cancer treatment: Synergistic effects of mechano-thermal focused ultrasound and radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 709.

Abstract 5572: Microneedle-guided lymphatic delivery of a natural immune modulator toxin for enhanced cancer immunotherapy

Abstract Cancer immunotherapy has demonstrated significant potential as a cancer treatment by enhancing the immune system's ability to recognize and eliminate cancer cells. However, its efficacy can be limited by factors such as tumor heterogeneity, immunosuppressive tumor microenvironments, and systemic toxicities. Recent advances in drug delivery systems have facilitated the development of more targeted and personalized cancer therapies. Microneedles have emerged as a promising platform for non-invasive and highly localized drug delivery, directly administering drugs, vaccines, and other therapeutic agents to the skin. In this study, we designed dissolving microneedles (dMN) based on a biocompatible amphiphilic tri-block copolymer, which enables the self-assembly of nano-micelles containing hydrophobic drugs when applied to the skin. We used the dMN technology to formulate SKKU-06, a hydrophobic natural immune modulator toxin derived from fungi, which exhibits anti-cancer and immunomodulatory properties in melanoma (SKKU-06@dMN). After intratumoral application of SSKU-06@dMN to skin tumors, the drug-loaded nano-micelles can migrate to tumor-draining lymph nodes (TDLN). The dMN-guided delivery of SKKU-06 to skin tumors and TDLN induced immunogenic cell death and stimulated the activation and maturation of antigen-presenting cells (APCs), promoting the development of humoral and cellular anti-tumor immunity. Furthermore, the immunomodulatory effects of SSKU-06@dMN were enhanced when combined with anti-PD-1 treatment, impacting the tumor microenvironment through increased intratumoral CD8+ T cell infiltration and reduced Treg populations. This resulted in efficient growth inhibition of established skin cancer and metastatic cancer, as well as prolonged survival. The dMN-guided lymphatic delivery of SKKU-06 demonstrates the potential for treating metastatic solid tumors and improving cancer immunotherapy efficacy by modulating the tumor microenvironment. Citation Format: Su Yeon Lim, Byung Deok Kim, PIN LIU, Ju hwa Shin, Ji Hoon Jeong. Microneedle-guided lymphatic delivery of a natural immune modulator toxin for enhanced cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5572.

Abstract 3606: Tumor-specific CD8+ Tc9 cells activate host CD4+ T cells to control antigen-lost tumors

Abstract Cancer immunotherapies relying on targeted destruction of cancer cells by potent antitumor T cells have achieved unprecedented success in recent years. As a main form of cancer immunotherapies, adoptive T cell therapy (ACT) has shown a durable response in certain cancer patients. However, this response is often short-lived and tumor relapse occurs due to the outgrowth of antigen-lost-variant (ALV) tumors and poor antitumor immune response. Here, We reported that adoptively transfer of murine tumor-specific CD8+ Tc9 but not Tc1/CTL cells achieved long-term control of tumor growth in vivo. Here, we demonstrated that murine tumor-specific Tc9 cells not only killed antigen-expressing primary tumors but also controlled the outgrowth of antigen-lost relapsed tumors by recruiting and activating host effector CD4+ T cells that recognized relapsed tumors. Tc9 cells secreted IL-24 and recruited CCR7-expressing conventional type-2 dendritic cells (cDC2) into tumor-draining lymph nodes to prime host CD4+ T cell response against relapsed tumors. Depleting host CD4+ T cells or deficiency in CCR7 expression impaired Tc9 cell ability to control the outgrowth of relapsed tumor. We also observed that intratumoral IL24 expression was positively correlated with gene signatures of cDC2 and CD4+ T cells in human cancers and expression of IL24 and cDC2 and CD4+ T cell gene signatures were associated with patient’s overall survival. Thus, this study uncovers a novel mechanism underlying activation of tumor-specific CD4+ T cells in vivo. Citation Format: Liuling Xiao, Qing Yi. Tumor-specific CD8+ Tc9 cells activate host CD4+ T cells to control antigen-lost tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3606.

Abstract 6592: Deciphering the effects of age on T cell immunity and immunotherapy

Abstract Aging induces profound alterations in host biology and correlates with unfavorable clinical outcomes in age-related diseases such as cancer. While the pivotal role of the immune system in regulating tumor progression is well-acknowledged, the precise mechanisms through which aging influences anti-tumor T cell immunity and responsiveness to immune checkpoint blockade (ICB) remain elusive. In this study, we employed syngeneic murine tumor models (B16, MC38, and E0771) in young (2-4 months) and aged (20-24 months) cohorts to elucidate the early impacts of aging on tumor development and T cell responses. Our findings demonstrate that aged mice exhibit accelerated tumor growth across various models. In-depth analysis of tumor-infiltrating lymphocytes (TILs) uncovered distinct alterations in the CD8+ T cell population, characterized by decreased activation status and diminished tumor specificity in aged mice. Correspondingly, aged mice exhibited resistance to anti-PD-1 immunotherapy. To unravel the underlying environmental factors contributing to compromised anti-tumor T cell responses and ICB resistance, we characterized the immune and metabolic landscapes of the aged tumor-draining lymph nodes and tumors. Furthermore, we established a genetically engineered mouse model of lung adenocarcinoma in both young and aged mice, unveiling novel functions and mechanisms associated with age-related T cells actively impeding effective tumor control. In the context of increasing average life expectancy, comprehending the intricate interplay between age-related T cell dysfunction and cancer assumes paramount importance. Such insights hold the key to the development of innovative therapeutic strategies tailored for the growing population of older patients. Citation Format: SeongJun Han, Peter Georgiev, Jaime Schneider, Amy Huang, Zhanel Nugmanova, Thao H. Nguyen, Conghui Yao, Sheila Johnson, Shakchhi Joshi, Ishita Dhiman, Jefte M. Drijvers, Megan Fung, Jared H. Rowe, Brian C. Miller, Gordon J. Freeman, Alison E. Ringel, Marcia C. Haigis, Arlene H. Sharpe. Deciphering the effects of age on T cell immunity and immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6592.

Phototruncation cell tracking with near-infrared photoimmunotherapy using heptamethine cyanine dye to visualise migratory dynamics of immune cells

Noninvasive invivo cell tracking is valuable in understanding the mechanisms that enhance anti-cancer immunity. We have recently developed a new method called phototruncation-assisted cell tracking (PACT), that uses photoconvertible cell tracking technology to detect invivo cell migration. This method has the advantages of not requiring genetic engineering of cells and employing tissue-penetrant near-infrared light. We applied PACT to monitor the migration of immune cells between a tumour and its tumour-draining lymph node (TDLN) after near-infrared photoimmunotherapy (NIR-PIT). PACT showed a significant increase in the migration of dendritic cells (DCs) and macrophages from the tumour to the TDLN immediately after NIR-PIT. This migration by NIR-PIT was abrogated by inhibiting the sphingosine-1-phosphate pathway or Gαi signaling. These results were corroborated by intranodal immune cell profiles at two days post-treatment; NIR-PIT significantly induced DC maturation and increased and activated the CD8+ T cell population in the TDLN. Furthermore, PACT revealed that NIR-PIT significantly enhanced the migration of CD8+ T cells from the TDLN to the tumour four days post-treatment, which was consistent with the immunohistochemical assessment of tumour-infiltrating lymphocytes and tumour regression. Immune cells dramatically migrated between the tumour and TDLN following NIR-PIT, indicating its potential as an immune-stimulating therapy. Also, PACT is potentially applicable to a wide range of immunological research. This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Centre for Cancer Research (grant number: ZIA BC011513 and ZIA BC011506).