Cell-mediated Antitumor Immunity Research Articles

Abstract 5481: Combination therapy with anti-PD-1 and YH29407, a novel IDO1 inhibitor, enhances T cell-mediated antitumor immunity in MC38 tumor-bearing mice

Abstract Background: The IDO is an enzyme responsible for catabolizing tryptophan (Trp) to kynurenine (Kyn). The kynurenine exert important immunosuppressive functions by activating Treg cells and myeloid-derived suppressor cells. YH29407 is a novel IDO1 inhibitor, improved the pharmacodynamics compared to previous IDO inhibitors. Methods: To evaluate antitumor effects and immune profiles of YH29407, YH29407 was dosed at 50 or 100 mg/kg twice daily (B.I.D.) alone or in combination with anti-PD-1 (10 mg/kg, B.I.W., i.p.) and BMS-986205 was dosed at 125 mg/kg once daily (Q.D.) alone or in combination with anti-PD-1 in a MC38 syngeneic tumor model. The antitumor effects during 11 days of administration of YH29407 alone or combinations and BMS-986205 alone or combination in the MC38 model were measured. Measurements were carried out up to day 50 for survival testing. After 3 days of treatment for each condition, immune cell profiles were evaluated by flow cytometry. Results: The YH29407 at dose of 100 mg/kg B.I.D combination treatment group showed the best effects on tumor size reduction. The group constituting >TGI:70% contained 100% (15/15) of the combination treatment group, whereas 15% (2/13) of competitor BMS-986205 combination group were contained. In addition, in the combination treatment group, most tumors showed an aspect of decrease including complete responses of 5 mice. Moreover, there was a complete response in YH29407 alone group but, not observed in BMS-986205 group. According to flow cytometry analysis, the combination treatment group showed higher CD3+ total T cells compared to the vehicle group (*p<0.05). Also, the effector T cells were respectively increased in the combination treatment group than BMS-986205 alone group. Significantly, helper T cells were increased in the combination treatment group against vehicle group (***p<0.001) and BMS-986205 combination group (*p<0.05). In addition, total macrophages were increased in the combination treatment group than BMS-986205 alone group (*p<0.05). On the other hand, M-MDSC were significantly decreased in the combination treatment group than BMS-986205 combination group (*p<0.05). We evaluated the tumor-infiltrating immune cells by immunohistochemistry, infiltrated CD3+ T cells were increased in the tumor of the combination treatment group than the vehicle group and BMS-986205 combination group (both *p<0.05). Likewise, tumor infiltrated CD8+ T cells were increased in the combination treatment group than vehicle group (**p<0.01) and BMS-986205 combination group (*p<0.05). However, dose-response effect or synergistic effect on immune cell profiles between YH29407 50 mg/kg alone or combination and YH29407 100 mg/kg alone or combination were not observed. Conclusion: Taken together, we suggest that YH29407 is the best combination partner with immune checkpoint inhibitors for solid tumor. Citation Format: Kyoung-Ho Pyo, Dong Kwon Kim, Se-Woong Oh, Gyu-Jin Lee, Ho-Woong Kang, Jae Hwan Kim, Youngseon Byeon, Young Seob Kim, Chun-Bong Synn, Wongeun Lee, Su Hwan Lee, Seul Lee, Seung Min Yang, Soyeon Lee, Yunjoo Joo, Eun Ji Lee, Sun Min Lim, Byoung Chul Cho. Combination therapy with anti-PD-1 and YH29407, a novel IDO1 inhibitor, enhances T cell-mediated antitumor immunity in MC38 tumor-bearing mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5481.

Abstract CT243: Phase 1 study of TAK-676 + pembrolizumab following radiation therapy in patients with advanced non-small-cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), or squamous-cell carcinoma of the head and neck (SCCHN)

Abstract Background: Radiation therapy has immune-modulating effects resulting from apoptosis of tumor cells and DNA damage. The downstream generation of cytosolic DNA activates the cyclic GMP-AMP Synthase (cGAS)-STimulator of INterferon Gene (STING) signaling axis in both tumor and nearby immune cells, leading to increased induction of type I interferon (IFN-I) and other immune stimulating molecules. TAK-676 is a novel synthetic STING agonist being investigated (+/- pembrolizumab) in an ongoing first-in-human phase 1 study (NCT04420884). TAK-676 is a potent modulator of the innate immune system and leads to downstream activation of the adaptive immune system to produce antitumor responses in preclinical studies. In contrast to intratumorally injected STING agonists, TAK-676 is optimally designed for reduced serum degradation and enhanced permeability, allowing systemic IV delivery and access to tumor sites and lymphatics. The addition of TAK-676 following radiation therapy may enhance the immune response by increasing the STING-mediated IFN-I release and further stimulate T cell-mediated antitumor immunity, particularly in combination with anti-PD-1/PD-L1 therapies. Impaired IFN signaling has been linked to checkpoint inhibitor (CPI) resistance. Preclinical studies show that addition of a STING agonist may reverse the mechanisms of resistance in tumors with prior exposure to CPIs. This phase 1 trial was designed to investigate the safety and preliminary antitumor activity of TAK-676 + pembrolizumab following radiation therapy (NCT04879849). Methods: Patients aged ≥18 years with advanced NSCLC, TNBC, or SCCHN who have progressed on CPIs with ≥2 lesions, one of which can be targeted with radiation, are being enrolled. Patients receive 8 Gy x 3 fractions of image-guided radiation therapy followed by (after a minimum of 40 hours) IV pembrolizumab 200 mg on day 1 plus escalating doses of IV TAK-676 on days 1, 8, and 15 of a 21-day cycle. TAK-676 dose escalation is guided by the Bayesian Optimal Interval design. Patients receive TAK-676 + pembrolizumab until disease progression, intolerance, or withdrawal of consent. Once pharmacologically active dose levels of TAK-676 have been observed, paired biopsies will be collected in patients with a safely accessible lesion outside the radiation field at screening and between days 15 and 21 of cycle 1. The primary objective is to determine the safety and tolerability of TAK-676 + pembrolizumab following radiation therapy; secondary objectives are to determine the recommended phase 2 dose of TAK-676 + pembrolizumab following radiation therapy, and to assess preliminary antitumor activity both locally (in the radiation field) and systemically (non-radiated lesions). As of January 2022, nearly 10% of the planned patients have been enrolled. Citation Format: Benjamin Cooper, Steven J. Chmura, Jason J. Luke, Stephen L. Shiao, Reva K. Basho, Wade T. Iams, David B. Page, Cong Li, Richard C. Gregory, Michael H. Shaw, Kristin H. Horn, John P. Gibbs, Vicky A. Appleman, Allison J. Berger, Adnan O. Abu-Yousif, Neil B. Lineberry, Kate F. Stumpo, Aymen Elfiky, Naamit K. Gerber. Phase 1 study of TAK-676 + pembrolizumab following radiation therapy in patients with advanced non-small-cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), or squamous-cell carcinoma of the head and neck (SCCHN) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT243.

Abstract SY19-04: Next-generation macrophage and T cell activation reporters to define functional immune states in solid tumors

Abstract Although chronic activation is considered a major driver of T cell dysfunction in the tumor microenvironment (TME), there is a virtual absence of methodologies to discern how T cells spatiotemporally progress through activation states into that dysfunction. Furthermore, T cells and other immune populations in the TME communicate extensively with tumor-associated macrophages (TAMs) which are typically simply described as ‘suppressive’, but whose functions are certainly more complex. To address these critical knowledge gaps, we designed two types of novel genetic marking systems to visualize, track and manipulate (i) T cell subsets with distinct activation histories and (ii) specific subsets of macrophages, modulating primarily those in the TME, to study and define these co-evolving immune states. We generated a novel T cell activation reporter mouse, which is a transcriptional reporter of CD69 expressing the fluorescent protein TFP. By recording transcriptional activity of a locus where the protein expression is under tight, activation-dependent post-transcriptional control, we generated a reporting system to read out contemporary as well as historic T cell triggering. While cell surface CD69 reliably indicates current activation, resting TFP (Cd69 mRNA) expression is downregulated with successive TCR triggering and hence an inverse marker of chronic stimulation. Using multiple tumor models in vivo, tumor slice cultures ex vivo and scRNA-Seq, we thus defined and tracked over time distinct functional states of intratumoral T cells based on their activation histories. These data revealed key insights including the delineation of quiescent versus activated states within progenitor, effector and exhausted T cells and the trajectory of inter-conversion that leads to the emergence of these states in the TME. Probing the inflection points in this differentiation trajectory reveals a particularly interesting subset of CD69+TFP+ cells, representing intratumorally activated effector CD8 T cells prior to their dysfunctional adaptation to chronic stimulation. Dissecting their phenotype led to the discovery of a previously unexplored subset of intratumoral T cells of functional significance. Indeed, these cells were enriched specifically in the context of T cell-mediated tumor control in mice and their phenotypic markers correlated with improved outcomes and survival in human patients. To complement this system, we generated a second genetic strain of mice that allowed specific depletion of TAMs. The prevailing view of M1 vs. M2 TAMs as ostensibly anti-tumor vs. pro-tumor, although useful, is almost certainly over-simplistic and has lacked further delineation, limited by the quality of available tools to specifically manipulate TAMs in situ. Remarkably, till now, a method to specifically deplete TAMs, without global depletion of all macrophages (e.g CSF1R blockade, Clodronate) did not exist. We alleviated this problem by using this novel mouse strain, in which conditional (lox-Stop-lox flanked) YFP and DTR genes are knocked into the endogenous CD206 locus. Since, within macrophages, CD206 is largely expressed in tumors, an intercross of this allele with a Csf1r-Cre (to specify monocyte-derived lineage) provides a 2-allele gating system to limit and robustly penetrate reporter and depletion function to TAMs. In multiple TAM-rich tumor models, this revealed that TAMs assumed context-dependent divergent roles in anti-tumor immunity. On the one hand, their removal led to a mitigation of T cell exhaustion, consistent with the popular notion that they are immunosuppressive and in need of global elimination. However, our data also reveals that macrophage depletion not only fails to alleviate the tumor burden but also prevents successful T cell-mediated anti-tumor immunity. We linked the latter effect to an obligate role for TAMs in the recruitment and maintenance of cDC1s and CD8 T cells in the TME, respectively. By live imaging of TAMs in vivo using the reporter and subsequent spatially resolved scRNA-Seq, the key TAM subsets and interactional networks involved in these processes were established. Importantly, in responsive tumor models, TAM depletion subverted T cell-mediated tumor control, demonstrating their importance in organizing anti-tumor adaptive immunity. We thus combine the use of novel engineered mice in vivo, real time imaging and spatial transcriptomics with functional studies to shed new light on immune dysfunction in the TME. Defining the ontology of functional immune states in the TME provides a clear target space to maneuver cancer immunotherapies. Herein, we offer prime examples of dissecting the spatiotemporal biology of CD8 T cells and TAMs in anti-tumor immunity to define such interdependent states. These findings and the newly established models therein pave the way to deepen as well as broaden our understanding of the complex immune landscape in solid tumors. Citation Format: Arja Ray, Kenneth Hu, Bushra Samad, Grace Hernandez, Matthew Krummel. Next-generation macrophage and T cell activation reporters to define functional immune states in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY19-04.

TAK-676 in combination with pembrolizumab after radiation therapy in patients (pts) with advanced non–small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), or squamous-cell carcinoma of the head and neck (SCCHN): Phase 1 study design.

TPS2698 Background: The cyclic GMP-AMP Synthase (cGAS)–STimulator of INterferon Genes (STING) pathway is an important modulator of the innate immune system via induction of type I interferon (IFN-I). Cytosolic DNA generated as a result of tumor cell death following radiation therapy has been demonstrated to activate the cGAS-STING signaling axis resulting in antitumor immunogenicity. TAK-676 is a novel, synthetic STING agonist and it has been shown in preclinical studies to potently modulate the innate immune system and subsequently activate the adaptive immune system to produce antitumor responses. TAK-676 is designed for prolonged half-life in serum and enhanced tissue permeability compared with other STING agonists designed for intratumoral injection, allowing for systemic IV delivery with access to tumor sites and lymphatic tissue. IFN signaling impairment has been linked to checkpoint inhibitor (CPI) resistance in tumors. Treatment with TAK-676 after radiation therapy has the potential to stimulate T cell-mediated antitumor immunity via STING-mediated IFN-I release, particularly when used with anti-PD-1/PD-L1 therapies. Preclinical data support the addition of STING agonists to reverse resistance in tumors with prior exposure to CPIs. TAK-676 is being investigated (+/- pembrolizumab) in an ongoing first-in-human phase 1 study (NCT04420884). Here, we describe another phase 1 trial to investigate the safety and preliminary antitumor activity of TAK-676 plus pembrolizumab following radiation therapy in pts with advanced or metastatic NSCLC, TNBC, or SCCHN (NCT04879849). Methods: Adult pts with progressive disease (PD) following CPI treatment and who have ≥2 lesions, 1 of which can be targeted with radiation, are being enrolled. Pts receive 8 Gy x 3 fractions of image-guided radiation followed (after ≥40 hours) by IV pembrolizumab 200 mg on day 1 plus escalating doses of IV TAK-676 on days 1, 8, and 15 of a 21-day cycle. TAK-676 dose escalation is guided by the Bayesian optimal interval design. Pts receive TAK-676 plus pembrolizumab until PD, intolerance to treatment, or withdrawal. Pts enrolled at TAK-676 dose levels shown to have pharmacodynamic activity, and who have a safely accessible lesion outside the radiation field, will have paired biopsies collected at screening and between days 15 and 21 of cycle 1. The primary objective is to determine the safety and tolerability of TAK-676 plus pembrolizumab following radiation therapy; secondary objectives are to establish the recommended phase 2 dose of TAK-676 plus pembrolizumab following radiation therapy, and to assess preliminary antitumor activity both locally (within the radiation field) and systemically (non-radiated lesions). As of February 2022, we have enrolled ̃10% of the planned pts. Clinical trial information: NCT04879849.

Reversing insufficient photothermal therapy-induced tumor relapse and metastasis by regulating cancer-associated fibroblasts

Insufficient tumor accumulation and distribution of photosensitizers as well as low antitumor immunity severely restrict the therapeutic efficacy of photothermal therapy (PTT). Cancer-associated fibroblasts (CAFs) play a key role in tumor extracellular matrix (ECM) remodeling and immune evasion. Reshaping tumor microenvironment via CAF regulation might provide a potential approach for complete tumor elimination in combination with PTT. Here, tumor cell-derived microparticles co-delivering calcipotriol and Indocyanine green (Cal/ICG@MPs) are developed to modulate CAFs for improved PTT efficacy. Cal/ICG@MPs efficiently target tumor tissues and regulate CAFs to reduce tumor ECM, resulting in enhanced tumor accumulation and penetration of ICG to generate strong PTT efficacy and activate CD8+ T cell-mediated antitumor immunity. In addition, Cal/ICG@MPs-triggered CAF regulation enhances tumor infiltration of CD8+ T cells and ameliorates CAF-induced antigen-mediated activation-induced cell death of tumor-specific CD8+ T cells in response to PTT, eliciting long-term antitumor immune memory to inhibit tumor recurrence and metastasis. Our results support Cal/ICG@MPs as a promising drug to improve PTT efficacy in cancer treatment.

Fueling Cancer Vaccines to Improve T Cell-Mediated Antitumor Immunity.

Cancer vaccines offer the potential to enhance T cell-mediated antitumor immunity by expanding and increasing the function of tumor-specific T cells and shaping the recall response against recurring tumors. While the use of cancer vaccines is not a new immunotherapeutic approach, the cancer vaccine field continues to evolve as new antigen types emerge and vaccine formulations and delivery strategies are developed. As monotherapies, cancer vaccines have not been very efficacious in part due to pre-existing peripheral- and tumor-mediated tolerance mechanisms that limit T cell function. Over the years, various agents including Toll-like receptor agonists, cytokines, and checkpoint inhibitors have been employed as vaccine adjuvants and immune modulators to increase antigen-mediated activation, expansion, memory formation, and T effector cell function. A renewed interest in this approach has emerged as better neoepitope discovery tools are being developed and our understanding of what constitutes an effective cancer vaccine is improved. In the coming years, cancer vaccines will likely be vital to enhance the response to current immunotherapies. In this review, we discuss the various types of therapeutic cancer vaccines, including types of antigens and approaches used to enhance cancer vaccine responses such as TLR agonists, recombinant interleukin-2 and interleukin-2 derivatives, and checkpoint inhibitors.

Tumoral Expression of NAC1 Restrains Antitumor Immunity through the LDHA-mediated Immune Evasion

Abstract T cell-mediated antitumor immunity has an important role in cancer prevention and treatment; however, immunosuppressive tumor microenvironment (TME) constitutes a major contributor to immune evasion that weakens antitumor immunity. We report here that tumoral expression of nucleus accumbens-associated protein-1 (NAC1), a nuclear factor of the broad complex, tramtrack, bric-a-brac/poxvirus and zinc finger (BTB/POZ) gene family, negatively impacts the CD8+ T cell - mediated antitumor immunity via through LDHA-mediated suppressive tumor microenvironment. We show that NAC1 could positively regulate the expression of lactate dehydrogenase A (LDHA) at transcriptional level, and LDHA-mediated lactic acid accumulation only inhibited cytokine production but also induced the exhaustion and apoptosis of CD8+ cytotoxic T lymphocytes (CTLs), impairing their cell-killing ability. We further show that in the immunocompetent and immunodeficient mice bearing melanoma, tumors with NAC1 depletion grew significantly slower and had elevated infiltration of tumor antigen specific CTLs following adoptive cell transfer, as compared with the control groups. These findings imply that tumor expression of NAC1 contributes substantially to immune evasion through its regulatory role in LDHA expression and lactic production. Thus, therapeutic targeting of NAC1 warrants further exploration as a potential strategy to reinforce cancer immunotherapy such as adoptive cell transfer of CTLs.

Targeting Metabolic Reprogramming of T-Cells for Enhanced Anti-Tumor Response.

Cancer immunotherapy is an effective treatment option against cancer. One of the approaches of cancer immunotherapy is the modification of T cell-based anti-tumor immune responses. T-cells, a type of adaptive immune response cells responsible for cell-mediated immunity, have long been recognized as key regulators of immune-mediated anti-tumor immunity. T-cell activities have been reported to be suppressed or enhanced by changes in cell metabolism. Moreover, metabolic reprogramming during activation of T cells is required for the development of distinct differentiation profiles of these cells, which may allow the development of long-term cell-mediated anti-tumor immunity. However, T cells have been shown to undergo metabolic exhaustion in tumor microenvironment (TME) as it poses several obstacles to their function. Applications of several mechanistic solutions to improve the efficacy of T cell-based therapies including chimeric antigen receptor (CAR) T cell therapy are yet to be determined. Modifying the metabolic properties of these cells and employing them in cancer immunotherapy is a potential strategy for improving their anti-tumor activity and therapeutic efficacy. To give an insight, in this review paper, we endeavoured to cover metabolic reprogramming in cancer and T cells, signalling mechanisms involved in immuno-metabolic regulation, the effects of the TME on T cell metabolic fitness, and targeting metabolic reprogramming of T cells for an enhanced anti-tumor response.

Albumin and interferon-β fusion protein serves as an effective vaccine adjuvant to enhance antigen-specific CD8+ T cell-mediated antitumor immunity

BackgroundType I interferons (IFN) promote dendritic cells maturation and subsequently enhance generation of antigen-specific CD8 +T cell for the control of tumor. Using type I interferons as an adjuvant to...

Ephrin receptor A10 monoclonal antibodies and the derived chimeric antigen receptor T cells exert an antitumor response in mouse models of triple-negative breast cancer

Expression of the receptor tyrosine kinase ephrin receptor A10 (EphA10), which is undetectable in most normal tissues except for the male testis, has been shown to correlate with tumor progression and poor prognosis in several malignancies, including triple-negative breast cancer (TNBC). Therefore, EphA10 could be a potential therapeutic target, likely with minimal adverse effects. However, no effective clinical drugs against EphA10 are currently available. Here, we report high expression levels of EphA10 in tumor regions of breast, lung, and ovarian cancers as well as in immunosuppressive myeloid cells in the tumor microenvironment. Furthermore, we developed anti-EphA10 monoclonal antibodies (mAbs) that specifically recognize cell surface EphA10, but not other EphA family isoforms, and target tumor regions precisely in vivo with no apparent accumulation in other organs. In syngeneic TNBC mouse models, we found that anti-EphA10 mAb clone #4 enhanced tumor regression, therapeutic response rate, and T cell–mediated antitumor immunity. Notably, the chimeric antigen receptor T cells derived from clone #4 significantly inhibited TNBC cell viability in vitro and tumor growth in vivo. Together, our findings suggest that targeting EphA10 via EphA10 mAbs and EphA10-specific chimeric antigen receptor–T cell therapy may represent a promising strategy for patients with EphA10-positive tumors.

Targeting KDM4C enhances CD8+ T cell mediated antitumor immunity by activating chemokine CXCL10 transcription in lung cancer

BackgroundAlthough immune checkpoint blockade (ICB) has been proven to achieve a persistent therapeutic response in various tumor types, only 20%–40% of patients benefit from this treatment. Radiotherapy (RT) can enhance...

Targeting transforming growth factor-β2 by antisense oligodeoxynucleotide accelerates T cell-mediated tumor rejection in a humanized mouse model of triple-negative breast cancer.

Transforming growth factor-beta (TGF-β) pathway mediates suppression of antitumor immunity and is associated with poor prognosis in triple-negative breast cancer (TNBC). In this study, we generated a humanized animal model by transplanting human peripheral blood mononuclear cells into immunodeficient mice followed by inoculation of MDA-MB-231 cells and subsequently analyzed the role of TGF-β2 in the interaction between human T cells and human tumor cells. Following reconstitution of the human immune system, inhibition of TGF-β signaling by TGF-β2 antisense oligodeoxynucleotide (TASO) resulted in accelerated tumor growth inhibition. TGF-β2 inhibition also resulted in downregulation of peripheral Foxp3 + regulatory T cells (Treg), whereas no effect was seen in the expression of CD8 + cytotoxic T cells. Analysis of the TASO-treated mice serum revealed elevated levels of human IFN-γ and reduced levels of human IL-10 and TGF-β2. Moreover, TGF-β2 inhibition resulted in increased CD8 + T cell infiltration, whereas the reduced infiltration of Tregs into the tumor partly resulted from decreased expression of CCL22. Decreased intratumoral Tregs facilitated the activation of cytotoxic T cells, associated with increased granzyme B expression. These results indicate that TASO potentiated T cell-mediated antitumor immunity, and it is proposed that TGF-β2 may be a promising target in the immunotherapeutic strategy of TNBC.

Hepatic interferon regulatory factor 8 expression suppresses hepatocellular carcinoma progression and enhances the response to anti-programmed cell death protein-1 therapy.

Therapeutic blockade of the programmed cell death protein-1 (PD-1) immune checkpoint pathways has resulted in significant reactivation of T cell-mediated antitumor immunity and is a promising clinical anticancer treatment modality in several tumor types, but the durable response rate remains relatively low (15%-20%) in most patients with HCC for unknown reasons. Evidence reveals that the interferon signaling pathway plays a critical role in modulating the efficacy and sensitivity of anti-PD-1 therapy against multiple tumor types, but the mechanisms are unclear. Using Kaplan-Meier survival analysis based on HCC databases, we found that deceased expression of interferon regulatory factor (IRF) 8 in HCC, among all the nine IRF members that regulate interferon signals, was associated with poor prognosis of patients with HCC. Moreover, gene set enrichment analysis identified the interferon-gamma and PD-1 signaling signatures as the top suppressed pathways in patients with IRF8-low HCC. Contrarily, overexpression of IRF8 in HCC cells significantly enhanced antitumor effects in immune-competent mice, modulating infiltration of tumor-associated macrophages (TAMs) and T cell exhaustion in tumor microenvironment. We further demonstrated that IRF8 regulated recruitment of TAMs by inhibiting the expression of chemokine (C-C motif) ligand 20 (CCL20). Mechanically, IRF8-mediated repression of c-fos transcription resulted in decreased expression of CCL20, rather than directly bound to CCL20 promoter region. Importantly, adeno-associated virus 8-mediated hepatic IRF8 rescue significantly suppressed HCC progression and enhanced the response to anti-PD-1 therapy. This work identified IRF8 as an important prognostic biomarker in patients with HCC that predicted the response and sensitivity to anti-PD-1 therapy and uncovered it as a therapeutic target for enhancing the efficacy of immune therapy.

Naringenin potentiates anti-tumor immunity against oral cancer by inducing lymph node CD169-positive macrophage activation and cytotoxic T cell infiltration

The CD169+ macrophages in lymph nodes are implicated in cytotoxic T lymphocyte (CTL) activation and are associated with improved prognosis in several malignancies. Here, we investigated the significance of CD169+ macrophages in oral squamous cell carcinoma (OSCC). Further, we tested the anti-tumor effects of naringenin, which has been previously shown to activate CD169+ macrophages, in a murine OSCC model. Immunohistochemical analysis for CD169 and CD8 was performed on lymph node and primary tumor specimens from 89 patients with OSCC. We also evaluated the effects of naringenin on two murine OSCC models. Increased CD169+ macrophage counts in the regional lymph nodes correlated with favorable prognosis and CD8+ cell counts within tumor sites. Additionally, naringenin suppressed tumor growth in two murine OSCC models. The mRNA levels of CD169, interleukin (IL)-12, and C-X-C motif chemokine ligand 10 (CXCL10) in lymph nodes and CTL infiltration in tumors significantly increased following naringenin administration in tumor-bearing mice. These results suggest that CD169+ macrophages in lymph nodes are involved in T cell-mediated anti-tumor immunity and could be a prognostic marker for patients with OSCC. Moreover, naringenin is a new potential agent for CD169+ macrophage activation in OSCC treatment.

Combination of ultrasound-based mechanical disruption of tumor with immune checkpoint blockade modifies tumor microenvironment and augments systemic antitumor immunity

BackgroundDespite multimodal adjuvant management with radiotherapy, chemotherapy and hormonal therapies, most surgically resected primary breast cancers relapse or metastasize. A potential solution to late and distant recurrence is to augment...

Abstract P053: Ablation of CCR1 relieves immunosuppression in pancreatic cancer

Abstract Pancreatic Cancer is one of the deadliest malignancies, with 5-year survival rate of 10%. The tumor microenvironment of pancreatic ductal adenocarcinoma (PDA) includes abundant fibroblasts and infiltrating immune cells, the latter largely immunosuppressive. Mono-immunotherapy or combination immunotherapy approaches has been ineffective in pancreatic cancer, pointing to the need for additional avenues to target in pancreatic cancer microenvironment. We previously showed that targeting regulatory T cell (Treg), a prevalent T cell population in pancreatic cancer, failed to relieve immunosuppression and led to accelerated tumor progression. We discovered that Treg depletion reprogrammed tumor associated fibroblasts and increased immunosuppressive myeloid cell recruitment, an effect that was partially mediated by CCLs/CCR1signaling. We found that tumor educated macrophages express the highest levels of Ccr1 compared to non-activated (M0), pro-inflammatory (M1), or anti-inflammatory (M2) bone marrow derived macrophage subsets. Thus, we sought to investigate the functional role of CCR1 in pancreatic cancer. By single cell RNA sequencing, we found CCR1 to be mainly expressed by tumor associated macrophages (TAMs) and neutrophils (or granulocytes) in both human and mouse PDA. We then orthotopically transplanted syngeneic mouse pancreatic cancer cells in CCR1 knockout hosts and observed reduced tumor growth which was rescued by CD8 T cell depletion. Histological analysis showed elevated Granzyme B expression in infiltrating T cells, as well as an increase in apoptotic cells in tumors implanted in Ccr1−/− mice. Through cytometry by time of flight (CyTOF) and co-immunofluorescence we also discovered that TAMs in tumors implanted in Ccr1−/− mice expressed less Arginase 1 and CD206 -both markers of immunosuppressive macrophages- compared to TAMs in wild type tumors. Thus, our data is consistent with the notion that tumor associated macrophages lacking CCR1 expression are less immunosuppressive, consequently allowing increased CD8 T cell-mediated anti-tumor immunity. We are currently exploring combination approaches targeting CCR1 in pancreatic cancer. Citation Format: Yaqing Zhang, Kristee L. Brown, Wei Yan, Zeribe C. Nwosu, Eileen K. Carpenter, Katelyn L. Donahue, Ashley Velez-Delgado, Sion Yang, Marina Pasca di Magliano. Ablation of CCR1 relieves immunosuppression in pancreatic cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P053.

CpG Oligodeoxynucleotides for Anticancer Monotherapy from Preclinical Stages to Clinical Trials.

CpG oligodeoxynucleotides (CpG ODNs), the artificial versions of unmethylated CpG motifs that were originally discovered in bacterial DNA, are demonstrated not only as potent immunoadjuvants but also as anticancer agents by triggering toll-like receptor 9 (TLR9) activation in immune cells. TLR9 activation triggered by CpG ODN has been shown to activate plasmacytoid dendritic cells (pDCs) and cytotoxic T lymphocytes (CTLs), enhancing T cell-mediated antitumor immunity. However, the extent of antitumor immunity carried by TLR agonists has not been optimized individually or in combinations with cancer vaccines, resulting in a decreased preference for TLR agonists as adjuvants in clinical trials. Although various combination therapies involving CpG ODNs have been applied in clinical trials, none of the CpG ODN-based drugs have been approved by the FDA, owing to the short half-life of CpG ODNs in serum that leads to low activation of natural killer cells (NK cells) and CTLs, along with increases of pro-inflammatory cytokine productions. This review summarized the current innovation on CpG ODNs that are under clinical investigation and explored the future direction for CpG ODN-based nanomedicine as an anticancer monotherapy.

Theranostic near-infrared-IIb emitting nanoprobes for promoting immunogenic radiotherapy and abscopal effects against cancer metastasis

Radiotherapy is an important therapeutic strategy for cancer treatment through direct damage to cancer cells and augmentation of antitumor immune responses. However, the efficacy of radiotherapy is limited by hypoxia-mediated radioresistance and immunosuppression in tumor microenvironment. Here, we construct a stabilized theranostic nanoprobe based on quantum dots emitting in the near-infrared IIb (NIR-IIb, 1,500–1,700 nm) window modified by catalase, arginine–glycine–aspartate peptides and poly(ethylene glycol). We demonstrate that the nanoprobes effectively aggregate in the tumor site to locate the tumor region, thereby realizing precision radiotherapy with few side-effects. In addition, nanoprobes relieve intratumoral hypoxia and reduce the tumor infiltration of immunosuppressive cells. Moreover, the nanoprobes promote the immunogenic cell death of cancer cells to trigger the activation of dendritic cells and enhance T cell-mediated antitumor immunity to inhibit tumor metastasis. Collectively, the nanoprobe-mediated immunogenic radiotherapy can boost the abscopal effect to inhibit tumor metastasis and prolong survival.

Abstract PO-129: Targeting CCR1 reprograms tumor associated macrophages in pancreatic cancer

Abstract The tumor microenvironment of pancreatic ductal adenocarcinoma (PDA) includes abundant fibroblasts and infiltrating immune cells, the latter largely immunosuppressive. We previously showed that targeting regulatory T cell (Treg), a prevalent T cell population in pancreatic cancer, failed to relieve immunosuppression and led to accelerated tumor progression. We discovered that Treg depletion reprogrammed tumor associated fibroblasts and increased immunosuppressive myeloid cell recruitment, an effect that was partially mediated by CCLs/CCR1signaling. Thus, we sought to investigate the potential therapeutic effect of targeting CCR1 in pancreatic cancer. By single cell RNA sequencing, we found CCR1 to be mainly expressed by tumor associated macrophages (TAMs) and neutrophils (or granulocytes) in both human and mouse PDA. We then orthotopically transplanted syngeneic mouse pancreatic cancer cells in CCR1 knockout hosts, and observed reduced tumor growth which was rescued by CD8 T cell depletion. Histological analysis showed elevated Granzyme B expression in infiltrating T cells, as well as an increase in cleaved caspase 3 positive cancer cells in tumors implanted in Ccr1−/− mice. Through cytometry by time of flight (CyTOF) and co-immunofluorescence we also discovered that TAMs in tumors implanted in Ccr1−/− mice expressed less Arginase 1 and CD206 -both markers of immunosuppressive macrophages- compared to TAMs in wild type tumors. Thus, our data is consistent with the notion that tumor associated macrophages lacking CCR1 expression are less immunosuppressive, consequently allowing increased CD8 T cell-mediated anti-tumor immunity. We are currently exploring combination approaches targeting CCR1 in pancreatic cancer. Citation Format: Yaqing Zhang, Kristee L. Brown, Wei Yan, Zeribe C. Nwosu, Eileen K. Carpenter, Katelyn L. Donahue, Ashley Velez-Delgado, Sion Yang, Marina Pasca di Magliano. Targeting CCR1 reprograms tumor associated macrophages in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-129.

Pharmacologic Inhibition of SUMO-Activating Enzyme Potentiates Interferon Response and T Cell-Mediated Anti-Tumor Immunity in Chronic Lymphocytic Leukemia (CLL) and Lymphoma Models

Introduction: CLL is characterized by deficient immunity which clinically manifests as increased predisposition towards malignancies and infectious complications. T-cells from patients with CLL exhibit a skewed repertoire with predominance of Tregs as well as impaired immune synapse formation and cytotoxic function. Small ubiquitin-like modifier (SUMO) family proteins regulate a variety of cellular processes, including nuclear trafficking, gene transcription and cell cycle progression, via post-translational modification of target proteins. Sumoylation regulates NFκB signaling, IFN response and NFAT activation, processes indispensable in immune cell activation. Despite this, the role of sumoylation in T cell biology in context of cancer is not known. TAK-981 is a small molecule inhibitor of the SUMO-activating enzyme (SAE) that forms a covalent adduct with an activated SUMO protein, thereby preventing its transfer to the SUMO-conjugating enzyme (Ubc9). Here, we investigated the immunomodulatory effects of TAK-981 in CLL.Methods: T cells from patients with CLL were purified using Dynabeads. For polarization assays, FACS-sorted naïve CD4+ T cells were cultured for 7 days in control or differentiation media. For gene expression profiling (GEP; Clariom S), RNA was harvested after 3 and 24 hours of TCR engagement from FACS-sorted naïve CD4+ T cells. For in vivo immunization experiments, CD4+KJ1-26+ cells were inoculated IV into BALB/cJ mice. Mice received 100 µg IV ovalbumin ± R848 followed by TAK-981 7.5 mg/kg or vehicle control IV twice weekly for 10 days prior to spleen collection. Both recipient and transplanted splenocytes were analyzed. For analysis of tumor-infiltrating lymphocytes (TILs), BALB/c mice were injected with 1x10 6 A20 lymphoma cells and treated as above. TAK-981 was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA).Results: T cells from patients with CLL demonstrated high baseline protein sumoylation that slightly increased following TCR engagement (αCD3/CD28). Treatment with TAK-981 significantly downregulated SUMO1 and SUMO2/3-modified protein levels yet did not disrupt early TCR signaling as evidenced by sustained ZAP70, p65/NFκB and NFAT activation detected by immunoblotting, immunocytochemistry and GEP. Treatment with TAK-981 resulted in dose-dependent upregulation of the early activation marker CD69 in CD4 + T cells following 72 and 96 hours of TCR stimulation vs. control. Meanwhile, expression of CD25, HLA-DR and CD40L was delayed in the presence of TAK-981. Interestingly, CD38, an IFN response target, was induced two-fold in TAK-981-treated cells after 24 hours and persisted at high levels at subsequent timepoints. T cell proliferation was reduced in the presence of high (1 μM) but not low/intermediate concentrations of TAK-981, accompanied by reduced S phase entry and decreased synthesis of IL-2. However, T cells did not undergo apoptosis under those conditions.Targeting SAE in either control or Th1/Treg polarizing conditions facilitated an increase in IFNγ and loss of FoxP3 expression (accompanied by decreased IL-2/STAT5), suggesting a shift towards Th1 and away from Treg phenotype, respectively. GEP (Reactome, GSEA) confirmed a dramatically upregulated IFN response in TAK-981-treated CD4 + naïve T cells. Furthermore, targeting SAE enhanced degranulation (CD107a), IFNγ and perforin secretion in cytotoxic CD8+ T cells and potentiated T cell cytotoxicity in allogeneic assays with lymphoma cells (OCI-LY3, U2932) as targets.Consistent with our in vitro data, OVA-stimulated transplanted transgenic KJ1-26+ splenocytes, as well as total CD4+ T cells from recipient mice treated with TAK-981 in vivo exhibited a significant reduction in expression of FoxP3 and an increased production of IFNγ (Figure 1). In the A20 syngeneic model, treatment with TAK-981 similarly downregulated FoxP3 expression in CD4+ TILs and induced IFNγ secretion in CD8+ TILs.Conclusion. Using a combination of in vitro and in vivo experiments, we demonstrate that pharmacologic targeting of sumoylation with TAK-981 does not impair proximal TCR signaling in T cells obtained from patients with CLL, but leads to rebalancing toward healthy immune T cell subsets via induction of IFN response and downmodulation of Tregs. These data provide a strong rationale for continued investigation of TAK-981 in CLL and lymphoid malignancies. [Display omitted] DisclosuresSiddiqi: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; TG Therapeutics: Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncternal: Research Funding; Janssen: Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Danilov: SecuraBio: Research Funding; Bayer Oncology: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Takeda Oncology: Research Funding; TG Therapeutics: Consultancy, Research Funding; Rigel Pharm: Honoraria; Abbvie: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Gilead Sciences: Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding.