T-cell Mediated Antitumor Immunity Research Articles

Interleukin-34-orchestrated tumor-associated macrophage reprogramming is required for tumor immune escape driven by p53 inactivation

As the most frequent genetic alteration in cancer, more than half of human cancers have p53 mutations that cause transcriptional inactivation. However, how p53 modulates the immune landscape to create a niche for immune escape remains elusive. We found that cancer stem cells (CSCs) established an interleukin-34 (IL-34)-orchestrated niche to promote tumorigenesis in p53-inactivated liver cancer. Mechanistically, we discovered that Il34 is a gene transcriptionally repressed by p53, and p53 loss resulted in IL-34 secretion by CSCs. IL-34 induced CD36-mediated elevations in fatty acid oxidative metabolism to drive M2-like polarization of foam-like tumor-associated macrophages (TAMs). These IL-34-orchestrated TAMs suppressed CD8+ Tcell-mediated antitumor immunity to promote immune escape. Blockade of the IL-34-CD36 axis elicited antitumor immunity and synergized with anti-PD-1 immunotherapy, leading to a complete response. Our findings reveal the underlying mechanism of p53 modulation of the tumor immune microenvironment and provide a potential target for immunotherapy of cancer with p53 inactivation.

ZG16 enhances the maturation of dendritic cells via induction of CD40 and contributes to the antitumor immunity in pancreatic cancer.

Dendritic cells (DCs) are critical mediators of antigen priming and T-cell activation. Zymogen granule protein 16 (ZG16) is demonstrated as an anti-oncogene in T-cell mediated antitumor immunity, but its effect on DCs is largely unknown. Herein, we wonder whether ZG16 affects the activation of DCs in pancreatic cancer. Firstly, the increased ZG16 expression was observed during the maturation of DCs derived from mouse bone marrow or human peripheral blood. Then, overexpression of ZG16 or exogenous introduction of recombinant ZG16 protein induced the expression of MHC II, CD86, CD84, and CCR7 on the surface of DCs, thereby facilitating the secretion of proinflammatory mediators IL-1β, IL-6, TNF-α, and IL-12/p70, supporting the promoting effect of ZG16 on DC maturation. By establishing the subcutaneous and orthotopic mouse models of pancreatic cancer, we confirmed that intraperitoneal injection of recombinant ZG16 protein (Re-mZG16) could induce tumor regression by stimulating DC maturation and enhancing antitumor responses of CD4 + , CD8 + , PD-1 + , and Ctla4+ cells. Besides, Re-mZG16 in combination with gemcitabine showed a synergistic effect in the treatment of pancreatic cancer. Mechanistically, we demonstrated that ZG16 inhibited the ubiquitination and degradation of CD40, which depended on the lectin domain of ZG16. In conclusion, this study provided a novel insight into the role of ZG16-CD40 axis in DC-based immunotherapy for pancreatic cancer.

Hypoxia-driven protease legumain promotes immunosuppression in glioblastoma

Glioblastoma (GBM) is a hypoxic and "immune-cold" tumor containing rich stromal signaling molecules and cell populations, such as proteases and immunosuppressive tumor-associated macrophages (TAMs). Here, we seek to profile and characterize the potential proteases that may contribute to GBM immunosuppression. Legumain (LGMN) emerges as the key protease that is highly enriched in TAMs and transcriptionally upregulated by hypoxia-inducible factor 1-alpha (HIF1α). Functionally, the increased LGMN promotes TAM immunosuppressive polarization via activating the GSK-3β-STAT3 signaling pathway. Inhibition of macrophage HIF1α and LGMN reduces TAM immunosuppressive polarization, impairs tumor progression, enhances CD8+ Tcell-mediated anti-tumor immunity, and synergizes with anti-PD1 therapy in GBM mouse models. Thus, LGMN is a key molecular switch connecting two GBM hallmarks of hypoxia and immunosuppression, providing an actionable therapeutic intervention for this deadly disease.

Mechanotransduction in response to ECM stiffening impairs cGAS immune signaling in tumor cells

The tumor microenvironment (TME) plays decisive roles in disabling Tcell-mediated antitumor immunity, butthe immunoregulatory functions of its biophysical properties remain elusive. Extracellular matrix (ECM) stiffening is a hallmark of solid tumors. Here, we report that the stiffened ECM contributes to the immunosuppression in TME via activating the Rho-associated coiled-coil-containing protein kinase (ROCK)-myosin IIA-filamentous actin (F-actin) mechanosignaling pathway in tumor cells to promote the generation of TRIM14-scavenging nonmuscle myosin heavy chain IIA (NMHC-IIA)-F-actin stress fibers, thus accelerating the autophagic degradation of cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) to deprive tumor cyclic GMP-AMP (cGAMP) and further attenuating tumor immunogenicity. Pharmacological inhibition of myosin IIA effector molecules with blebbistatin (BLEB) or the RhoA upstream regulator of this pathway with simvastatin (SIM) restored tumor-intrinsic cGAS-mediated cGAMP production and enhanced antitumor immunity. Our work identifies that ECM stiffness is an important biophysical cue to regulate tumor immunogenicity via the ROCK-myosin IIA-F-actin axis and that inhibiting this mechanosignaling pathway could boost immunotherapeutic efficacy for effective solid tumor treatment.

Polyclonal lymphoid expansion drives paraneoplastic autoimmunity in neuroblastoma

Neuroblastoma is a lethal childhood solid tumor of developing peripheral nerves. Two percent of children with neuroblastoma develop opsoclonus myoclonus ataxia syndrome (OMAS), a paraneoplastic disease characterized by cerebellar and brainstem-directed autoimmunity but typically with outstanding cancer-related outcomes. We compared tumor transcriptomes and tumor-infiltrating T and B cell repertoires from 38 OMAS subjects with neuroblastoma to 26 non-OMAS-associated neuroblastomas. We found greater B and Tcell infiltration in OMAS-associated tumors compared to controls and showed that both were polyclonal expansions. Tertiary lymphoid structures (TLSs) were enriched in OMAS-associated tumors. We identified significant enrichment of the major histocompatibility complex (MHC) class II allele HLA-DOB∗01:01 in OMAS patients. OMAS severity scores were associated with the expression of several candidate autoimmune genes. We propose a model in which polyclonal auto-reactive B lymphocytes act as antigen-presenting cells and drive TLS formation, thereby supporting both sustained polyclonal Tcell-mediated anti-tumor immunity and paraneoplastic OMAS neuropathology.

Abstract B038: Toll like receptor 4 (TLR4) agonism by lipopolysaccharide (LPS) in pancreatic cancer model stimulates anti-tumor immunity in CD4+T cell dependent fashion

Abstract Introduction: Pancreatic cancer, one of the most lethal malignancies, has a 5-year overall survival of approximately 10%. It is an immunologically cold tumor and is poorly responsive to immunotherapy. Globally, researchers are in quest of immunomodulatory modalities to improve the therapeutic response. lipopolysaccharide (LPS), a known toll like receptor 4 (TLR4) agonist is known to activate immune response. Here, we evaluate the effect of LPS mediated TLR4 agonism on the anti-tumor immune machinery of pancreatic cancer. Methods: We implanted KPC (Kras LSL-G12D; Trp53 LSL-R172H/+; Pdx1Cre/+) pancreatic cancer cells in C57BL/6 mice subcutaneously. The animals were treated intraperitoneally with LPS (1mg/kg) or saline (control) at day 10 and 17. Tumor samples were isolated at endpoint and were analyzed using flow cytometry and real time polymerase chain reaction (RT-PCR) for infiltration of immunocytes and expression of immune-associated genes respectively. Functional response of CD4+ T-cells to tumor antigen re-challenge was assessed by measuring interferon-γ (IFN-γ) release in-vitro. In another experiment, LPS or saline treatment was administered along with depletion of CD4+ or CD8+ T-cells employing monoclonal antibodies. CD4+ T-cells extracted from spleens of tumor bearing control or LPS treated mice were subjected to Calcein AM based cytotoxicity assay. Cytotoxicity assay was also performed by blocking major histo-compatibility II (MHCII) on KPC cells. Results: LPS administration in tumor bearing mice significantly reduced the tumor burden as compared to control group. Flow cytometry on tumor samples revealed increased infiltration of CD4+ T-cells in LPS group whereas CD8+ T-cell repertoire was unaffected. Tumor samples from the LPS group showed higher expression of genes for anti-tumor cytokines such as IFN-γ, granzymes and perforin. Depletion of CD4+ T cells, but not CD8+ T cells attenuated the effect of LPS on tumor growth. When the splenic CD4+ T-cells from both groups were pulsed with KPC cell lysate, LPS treated mice derived CD4+ T-cells demonstrated a greater ability to secrete IFN-γ. Splenic CD4+ T-cells from LPS treated mice showed significantly higher cytotoxicity against KPC cells as compared to control CD4+ T-cells. However, blocking MHCII molecules on KPC cells reduced the cytotoxicity in CD4+ T-cells derived from LPS treated mice. This suggests that CD4 cytotoxicity ensues in an MHCII dependent manner. Conclusions: LPS treatment reduces tumor burden in the subcutaneous tumor model of pancreatic cancer by upregulating the infiltration of CD4+ T-cells and translating them to a cytotoxic phenotype. It also stimulates the upregulation of anti-tumor immunity related cytokines in the tumor microenvironment. We have also found that the cytotoxic CD4+ T cells terminate the pancreatic cancer cells in an MHCII dependent manner. Therefore, our findings provide a novel insight into how “TLR4 agonism can stimulate CD4+ T-cell mediated antitumor immunity”, which can be harnessed as a potential source for pancreatic cancer immunotherapy. Citation Format: Utpreksha Vaish, Anthony R. Ferrantella, Tejeshwar Jain, Prateek Sharma, Srikanth Iyer, Vikas Dudeja. Toll like receptor 4 (TLR4) agonism by lipopolysaccharide (LPS) in pancreatic cancer model stimulates anti-tumor immunity in CD4+T cell dependent fashion [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B038.

A Phase 1 Study of TAK-676, a Novel STING Agonist, Plus 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)

<h3>Purpose/Objective(s)</h3> Radiation therapy-induced cell death produces cytosolic DNA that activates the cyclic GMP-AMP synthase (cGAS)–STimulator of INterferon Genes (STING) pathway, crucial for the induction of Type I interferons (IFN-I). Checkpoint inhibitor (CPI) resistance mechanisms have been linked to impaired IFN signaling. Preclinical data have shown STING agonists to reverse CPI resistance in tumors with prior exposure, particularly when used with anti-PD-1/PD-L1 therapies. TAK-676 (a synthetic STING agonist) potently modulates the innate immune system, leading to cytokine release, adaptive immune activation, and antitumor responses in preclinical studies (Appleman et al., AACR 2022). TAK-676 is being investigated as a single agent, and in combination with pembrolizumab, for advanced solid tumors, in a first-in-human phase 1 study (NCT04420884). TAK-676 is optimally designed for intravenous (IV) delivery, with a prolonged half-life in serum and enhanced tissue permeability, allowing access to tumor sites and lymphatic tissue. Following radiation therapy, TAK-676 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. Here, we present a second phase 1 study to investigate the safety and preliminary antitumor activity of TAK-676 in combination with pembrolizumab following radiation therapy, in patients with advanced or metastatic NSCLC, TNBC, or SCCHN who have progressed on CPIs (NCT04879849). <h3>Materials/Methods</h3> Patients aged ≥18 years who progressed on CPIs and have ≥2 lesions, of which one is targetable with radiation, are being enrolled. Patients receive 8 Gy x 3 followed by (after a minimum of 40 hours) escalating doses of IV TAK-676 on days 1, 8 and 15 of a 21-day cycle, and 200 mg of IV pembrolizumab on day 1 of each cycle until disease progression, intolerance, or withdrawal of consent. Dose escalation of TAK-676 will be guided by the Bayesian Optimal Interval design. At screening and between days 15–21 of cycle 1, patients with a safely accessible lesion outside of the radiation field will have paired biopsies collected once the pharmacologically active dose levels of TAK-676 have been observed. The primary objective is to determine the safety and tolerability of TAK-676 in combination with pembrolizumab following radiation therapy. Secondary objectives are to determine the recommended phase 2 dose of TAK-676 in combination with pembrolizumab following radiation therapy, and to assess the local (within the radiation field) and systemic (non-radiated lesions) preliminary antitumor activity. As of February 2022, ∼10% of the planned patients have been enrolled. <h3>Results</h3> TBD <h3>Conclusion</h3> TBD

Tannins in Terminalia bellirica inhibits hepatocellular carcinoma growth via re-educating tumor-associated macrophages and restoring CD8+T cell function

Tumor-associated macrophages (TAMs) are the major immunosuppressive components infiltrating the tumor microenvironment (TME). Targeting TAMs has emerged as a promising strategy to remodel immunosuppressive TME and enhance T-cell mediated anti-tumor immunity for cancer therapy. In this study, we investigate the effect and mechanism of total tannin fraction of Terminalia bellirica (Gaertn.) Roxb. (TB-TF) against hepatocellular carcinoma (HCC) using established Hepa1–6 orthotopic mouse model and murine bone marrow derived macrophage polarization model. Here we showed that TB-TF significantly inhibited orthotopic tumor growth and promoted the polarization of M2-TAMs toward the anti-tumor M1 phenotype in vivo. Further studies showed that TB-TF reversed tumor-conditioned medium induced M2 polarization of macrophages as indicated by increased expression of TNF-α, IL-1β, and iNOS, and decreased expression of Arg-1, thereby re-educating macrophages co-cultured with tumor-conditioned medium into M1 phenotype. In addition, we found that TB-TF also promoted T cell infiltration mediated by chemokines such as CCL5 and CXCL10, and restored the cytotoxic function of CD8+T cells as evidenced by upregulated expression of Granzyme B, Perforin, and IFN-γ. Our data suggest TB-TF as a promising anti-cancer agent, mediates its anti-tumor effects via remodeling the tumor immunosuppressive microenvironment, indicating its potential in the immunotherapy for hepatocellular carcinoma.

Notch signaling is required for generation of conventional type 1 dendritic cells from human induced pluripotent stem cells

Abstract Compelling evidence shows a critical role of conventional type 1 dendritic cells (cDC1) in T-cell mediated antitumor immunity; however, obtaining large numbers of cDC1 is difficult. Theoretically, this limitation can be overcome by using induced pluripotent stem cells (iPSC) that could provide an unlimited source of autologous cDC1. However, generation of cDC1 from human iPSC remains elusive. Here, we hypothesized that Notch signaling would facilitate generation of cDC1 from human iPSC. We found that human iPSC gave rise to CD141+XCR1+CLEC9A+HLA-DR+ cells on OP9 feeder cells expressing the Notch ligand delta-like 1 (OP9-DL1) while the majority of iPSC-derived cells differentiated on OP9 cells were monocytic cells. Single-cell RNA sequencing analyses confirmed the presence of cDC1 as well as identified the heterogeneity of iPSC-derived CD1c+ cells; cDC2A, cDC2B, CD5+cDC2, and DC3. Inhibition of Notch signaling during co-culture of iPSC-derived CD34+ hematopoietic progenitor cells with OP9-DL1 cells abrogated generation of cDC1, and made expression of CD5, CLEC4A, CLEC10A and CD163 in CD1c+ DC similar to those differentiated on OP9 cells. Notch-activated human iPSC-derived XCR1+CLEC9A+HLA-DR+CD11c+ cells exhibited similar gene expression profile with peripheral blood cDC1, and phagocytotic, T-cell proliferative, and cytokine producing functions. Our study demonstrates that Notch signaling is required for the differentiation of cDC1-like cells, and facilitates generation of various cDC subsets from human iPSC. These findings provide insights into the future development of personalized treatment with unlimited numbers of autologous cDC1 from human iPSC. This work was supported by National Cancer Institute (NCI) grant P30CA016056 involving the use of Roswell Park’s Flow and Image Cytometry, Bioinformatics, and Genomic Shared Resources. This work was supported by the Melanoma Research Alliance and the Sarcoma Foundation of America (F. Ito), Uehara Memorial Foundation (T. Oba), and National Cancer Institute (NCI) grant, U24CA232979 (M. Long and S. Liu), K08CA197966 and R01CA255240-01A1 (F. Ito).

Targeting CD73 with AB680 (Quemliclustat), a Novel and Potent Small-Molecule CD73 Inhibitor, Restores Immune Functionality and Facilitates Antitumor Immunity.

T cells play a critical role in the control of cancer. The development of immune checkpoint blockers (ICB) aimed at enhancing antitumor T-cell responses has revolutionized cancer treatment. However, durable clinical benefit is observed in only a subset of patients, prompting research efforts to focus on strategies that target multiple inhibitory signals within the tumor microenvironment (TME) to limit tumor evasion and improve patient outcomes. Adenosine has emerged as a potent immune suppressant within the TME, and CD73 is the major enzyme responsible for its extracellular production. CD73 can be co-opted within the TME to impair T-cell-mediated antitumor immunity and promote tumor growth. To target this pathway and block the formation of adenosine, we designed a novel, selective, and potent class of small-molecule inhibitors of CD73, including AB680 (quemliclustat), which is currently being tested in patients with cancer. AB680 effectively restored T-cell proliferation, cytokine secretion, and cytotoxicity that were dampened by the formation of immunosuppressive adenosine by CD73. Furthermore, in an allogeneic mixed lymphocyte reaction where CD73-derived adenosine had a dominant suppressive effect in the presence of PD-1 blockade, AB680 restored T-cell activation and function. Finally, in a preclinical mouse model of melanoma, AB680 inhibited CD73 in the TME and increased the antitumor activity of PD-1 blockade. Collectively, these data provide a rationale for the inhibition of CD73 with AB680 in combination with ICB, such as anti-PD-1, to improve cancer patient outcomes.

Microbial metabolite as icebreaker for immunotherapy

Immunotherapy has limited success in triple-negative breast cancer (TNBC). In this issue of Cell Metabolism, Wang etal. found that microbial metabolite TMAO boosts CD8+ Tcell-mediated antitumor immunity by inducing pyroptosis in tumor cells, enhancing the efficacy of immunotherapy in TNBC (Wang etal., 2022).

The microbial metabolite trimethylamine N-oxide promotes antitumor immunity in triple-negative breast cancer

Immunotherapy has achieved limited success in patients with triple-negative breast cancer (TNBC), an aggressive disease with a poor prognosis. Commensal microbiota have been proven to colonize the mammary gland, but whether and how they modulate the tumor microenvironment remains elusive. We performed a multiomics analysis of a cohort of patients with TNBC (n= 360) and found genera under Clostridiales, and the related metabolite trimethylamine N-oxide (TMAO) was more abundant in tumors with an activated immune microenvironment. Patients with higher plasma TMAO achieved better responses to immunotherapy. Mechanistically, TMAO induced pyroptosis in tumor cells by activating the endoplasmic reticulum stress kinase PERK and thus enhanced CD8+ Tcell-mediated antitumor immunity in TNBC invivo. Collectively, our findings offer new insights into microbiota-metabolite-immune crosstalk and indicate that microbial metabolites, such as TMAO or its precursor choline, may represent a novel therapeutic strategy to promote the efficacy of immunotherapy in TNBC.

Targeting PIM1-Mediated Metabolism in Myeloid Suppressor Cells to Treat Cancer

There is a strong correlation between myeloid-derived suppressor cells (MDSC) and resistance to immune checkpoint blockade (ICB), but the detailed mechanisms underlying this correlation are largely unknown. Using single-cell RNA sequencing analysis in a bilateral tumor model, we found that immunosuppressive myeloid cells with characteristics of fatty acid oxidative metabolism dominate the immune-cell landscape in ICB-resistant subjects. In addition, we uncovered a previously underappreciated role of a serine/threonine kinase, PIM1, in regulating lipid oxidative metabolism via PPARγ-mediated activities. Enforced PPARγ expression sufficiently rescued metabolic and functional defects of Pim1 -/- MDSCs. Consistent with this, pharmacologic inhibition of PIM kinase by AZD1208 treatment significantly disrupted the myeloid cell-mediated immunosuppressive microenvironment and unleashed CD8+ T-cell-mediated antitumor immunity, which enhanced PD-L1 blockade in preclinical cancer models. PIM kinase inhibition also sensitized nonresponders to PD-L1 blockade by selectively targeting suppressive myeloid cells. Overall, we have identified PIM1 as a metabolic modulator in MDSCs that is associated with ICB resistance and can be therapeutically targeted to overcome ICB resistance.

A Phase Ib Trial of Personalized Neoantigen Therapy Plus Anti-PD-1 in Patients with Advanced Melanoma, Non-small Cell Lung Cancer, or Bladder Cancer

Neoantigens arise from mutations in cancer cells and are important targets of Tcell-mediated anti-tumor immunity. Here, we report the first open-label, phase Ib clinical trial of a personalized neoantigen-based vaccine, NEO-PV-01, in combination with PD-1 blockade in patients with advanced melanoma, non-small cell lung cancer, or bladder cancer. This analysis of 82 patients demonstrated that the regimen was safe, with no treatment-related serious adverse events observed. De novo neoantigen-specific CD4+ and CD8+ Tcell responses were observed post-vaccination in all of the patients. The vaccine-induced T cells had a cytotoxic phenotype and were capable of trafficking to the tumor and mediating cell killing. In addition, epitope spread to neoantigens not included in the vaccine was detected post-vaccination. These data support the safety and immunogenicity of this regimen in patients with advanced solid tumors (Clinicaltrials.gov: NCT02897765).

Abstract A15: Rewiring the tumor microenvironment with an oncolytic virus to enhance and support tumor-infiltrating lymphocytes for ovarian cancer immunotherapy

Abstract The natural occurrence of immune cells in the tumor microenvironment of ovarian cancer (OC) facilitates the ex vivo generation and expansion of tumor-infiltrating lymphocytes (TIL) for infusion in the context of adoptive T-cell transfer. Yet, the lack of tumor-reactive TILs and the suppressive microenvironment have hindered the translation of TIL therapy into meaningful clinical responses in OC patients. Therefore, generating a proinflammatory tumor microenvironment with oncolytic adenoviruses, in order to induce a steady and potent antitumor activity, may prove an effective add-on to TIL therapy for OC. Besides their well-established efficacy in OC, oncolytic adenoviruses genetically modified to carry the interleukin-2 (IL-2) and tumor necrosis factor alpha (TNFa) transgenes may further support and promote T-cell mediated antitumor immunity. In this study, we assessed the capability of an oncolytic virus coding for IL-2 and TNFa (Ad5/3-E2F-D24-hIL-2-IRES-TNFa; TILT-123) to enhance the reactivity of TILs towards OC in a suppressive microenvironmental context. Fresh tumor tissue from regional metastatic or primary sites was obtained from stage III-IV OC patients. Part of the samples were diced in small fragments for the generation and rapid expansion of TILs under clinically relevant in vitro conditions. Phenotypic characterization of TILs was performed by flow cytometry after rapid expansion. Following TIL production and characterization, TILs were co-cultured with T cell-depleted autologous OC single-cell suspensions in the presence or absence of oncolytic virus. TIL reactivity was determined by quantifying Interferon (IFN)-gamma in culture supernatants through ELISA. The remainder of the tumor sample was dissociated overnight and cultured in the presence or absence of oncolytic virus. Supernatants were harvested at different days post-infection for detection of relevant cytokines. Following a 14-day rapid expansion period, the final TIL product was composed mostly of CD8+ T cells (56%), CD4+ T cells (27%) and a small fraction of CD3+CD56+ NK-like T cells (2%). When TILs were cocultured with T-cell depleted OC single-cell suspensions, treatment with oncolytic virus coding for IL-2 and TNFa caused a significant increase in the release of IFNg by TILs as compared to conditions where no virus or the backbone oncolytic virus (without cytokine transgenes) was added (p&amp;lt;0.05). Microenvironmental changes by the cytokine-encoding oncolytic adenovirus were evidenced by increased release of proinflammatory cytokines such as, TNFa, IL-2 and IFNg in 7-day cell cultures of ex vivo single-cell suspensions derived from OC patients. In summary, tailoring the tumor microenvironment with IL-2 and TNFa using oncolytic viruses sharpens the TIL reactivity in an immune-suppressive OC microenvironmental context. Hence, this approach may enable effective responses in OC patients treated with TIL therapy. This evidence supports further clinical validation. Citation Format: João Manuel Santos, Camilla Hëinio, Victor Cervera-Carrascon, Mikko Siurala, Dafne Quixabeira, Tanja De Gruijl, Heini Lassus, Anna Kanerva, Akseli Hemminki. Rewiring the tumor microenvironment with an oncolytic virus to enhance and support tumor-infiltrating lymphocytes for ovarian cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A15.

Selective and Potent CDK8/19 Inhibitors Enhance NK-Cell Activity and Promote Tumor Surveillance.

Natural killer (NK) cells play a pivotal role in controlling cancer. Multiple extracellular receptors and internal signaling nodes tightly regulate NK activation. Cyclin-dependent kinases of the mediator complex (CDK8 and CDK19) were described as a signaling intermediates in NK cells. Here, we report for the first time the development and use of CDK8/19 inhibitors to suppress phosphorylation of STAT1S727 in NK cells and to augment the production of the cytolytic molecules perforin and granzyme B (GZMB). Functionally, this resulted in enhanced NK-cell-mediated lysis of primary leukemia cells. Treatment with the CDK8/19 inhibitor BI-1347 increased the response rate and survival of mice bearing melanoma and breast cancer xenografts. In addition, CDK8/19 inhibition augmented the antitumoral activity of anti-PD-1 antibody and SMAC mimetic therapy, both agents that promote T-cell-mediated antitumor immunity. Treatment with the SMAC mimetic compound BI-8382 resulted in an increased number of NK cells infiltrating EMT6 tumors. Combination of the CDK8/19 inhibitor BI-1347, which augments the amount of degranulation enzymes, with the SMAC mimetic BI-8382 resulted in increased survival of mice carrying the EMT6 breast cancer model. The observed survival benefit was dependent on an intermittent treatment schedule of BI-1347, suggesting the importance of circumventing a hyporesponsive state of NK cells. These results suggest that CDK8/19 inhibitors can be combined with modulators of the adaptive immune system to inhibit the growth of solid tumors, independent of their activity on cancer cells, but rather through promoting NK-cell function.

Potency of CD8+ T-cell mediated antitumor immunity from intratumoral immunotherapy with STING agonist, ADU-S100, in an esophageal adenocarcinoma model.

408 Background: Esophageal adenocarcinoma (EAC) is a deadly disease with poor prognosis due to limited treatment options. STING is a transmembrane protein that activates the transcription of type I IFN genes, resulting in the stimulation of APCs and enhanced CD8+ T-cell infiltration. Recently, STING agonists have demonstrated durable anticancer activity in solid tumors when used alone and in combination with either chemotherapy, radiation or immunotherapy. In this study we evaluated the efficacy and immunomodulatory effects of STING agonist +/- radiation in an established EAC model. Methods: Esophagojejunostomy was performed on rats to induce reflux leading to the development EAC. At 32 weeks post operatively, rats received either STING (ADU-S100) or placebo (PBS), +/- 16Gy radiation. Drug efficacy was evaluated by pre- and post- treatment MRI, serial biopsies, histology and RT-PCR. Additionally, immunofluorescence was performed using CD8 and PD-L1 antibodies. Results: A comparison of MRIs in the study groups between 32 and 40 weeks demonstrated a mean increase in percentage tumor volume of 76.7 % and 152.4% in the P and P+R arms and a decrease of 30.1 % and 50.8% in the S and S+R arms, respectively (ANOVA test p &lt; 0.0001) Overall, the S+R group demonstrated the best results with maximum mean volume reduction with all cases responding. Downstream gene expression, pre, on, and post- treatment demonstrated significant upregulation of IFNβ, TNFα, IL-6 and CCL-2 in the treatment groups compared with placebo. On and post treatment, radiation alone, ADU-S100 alone and ADU-S100 + radiation groups demonstrated enhanced PD-LI expression, induced by higher densities of IFNγ producing CD8+ T-cells (p &lt; 0.01). Conclusions: ADU-S100 +/- radiation exhibits potent anti-tumor efficacy and a promising immunomodulatory profile in a de novo EAC model providing the rationale for clinical testing, likely concurrently in combination with immune checkpoint inhibitors.

Phosphoinositide 3-Kinase Signaling Can Modulate MHC Class I and II Expression

Molecular events activating the PI3K pathway are frequently detected in human tumors and the activation of PI3K signaling alters numerous cellular processes including tumor cell proliferation, survival, and motility. More recent studies have highlighted the impact of PI3K signaling on the cellular response to interferons and other immunologic processes relevant to antitumor immunity. Given the ability of IFNγ to regulate antigen processing and presentation and the pivotal role of MHC class I (MHCI) and II (MHCII) expression in T-cell-mediated antitumor immunity, we sought to determine the impact of PI3K signaling on MHCI and MHCII induction by IFNγ. We found that the induction of cell surface MHCI and MHCII molecules by IFNγ is enhanced by the clinical grade PI3K inhibitors dactolisib and pictilisib. We also found that PI3K inhibition increases STAT1 protein levels following IFNγ treatment and increases accessibility at genomic STAT1-binding motifs. Conversely, we found that pharmacologic activation of PI3K signaling can repress the induction of MHCI and MHCII molecules by IFNγ, and likewise, the loss of PTEN attenuates the induction of MHCI, MHCII, and STAT1 by IFNγ. Consistent with these in vitro studies, we found that within human head and neck squamous cell carcinomas, intratumoral regions with high phospho-AKT IHC staining had reduced MHCI IHC staining. IMPLICATIONS: Collectively, these findings demonstrate that MHC expression can be modulated by PI3K signaling and suggest that activation of PI3K signaling may promote immune escape via effects on antigen presentation.

5PD - The role of EGFR inhibitor (EGFRi) in immune cell infiltration and CD8+ T-cell activation in EGFR mutant lung cancer

BackgroundOncogenic MAP kinase pathway activating mutations have been shown to drive immune suppression in melanoma and colorectal cancers. In this study, we explored whether oncogenic EGFR mutations play an analogous role in non-small cell lung cancer (NSCLC), and whether EGFRi can relieve tumor-associated immune suppression. MethodsLung cancer cell lines expressing wild-type or mutated EGFR were treated with EGFRi, followed by collection of RNA and cell supernatants. In parallel, tumor biopsies from NSCLC patients receiving a personalized peptide vaccine with or without concurrent EGFRi treatment were obtained. RNAseq-based transcriptome profiles of tumor cell lines and patient tumor biopsies were compared to assess common gene signatures driven by EGFRi. Gene expression changes were confirmed at the protein level using Western blot, flow cytometry, and cytokine/chemokine Luminex. The impact of EGFRi on T-cell migration and tumor cell recognition by antigen-specific CD8+ T cells was also assessed. ResultsIn addition to downregulating genes associated with cell proliferation, apoptosis and survival, EGFRi increased the transcription of genes associated with TNFa and TRAIL signaling, and antigen presentation. HLA class I protein upregulation was confirmed and correlated with increased recognition of tumor cells by cytotoxic T cells. Several chemokines and cytokines were up- or down-regulated following EGFRi treatment, and Luminex analysis confirmed changes to 10 of them. Migration assays demonstrated that chemotaxis of T cells towards EGFR-mutant cell supernatants increased in an EGFRi dose-dependent manner. Transcriptome profiling of tumor biopsies revealed similar gene expression changes in on-EGFRi treatment tumor samples. Furthermore, increased tumor immune cell infiltration observed in EGFRi-treated patient tumors was consistent with the upregulation of EGFRi signature chemokines at the tumor site. ConclusionsThese results provide evidence that EGFRi has the capacity to facilitate modulation of the tumor microenvironment to favor immune cell infiltration and promote T-cell mediated antitumor immunity. Legal entity responsible for the studyThe University of Texas MD Anderson Cancer Center. FundingTianjin HengJia Biotechnology Development Co., Ltd. DisclosureF. Li: Shareholder/Stockholder/Stock options: Tianjin HengJia Biotechnology Development Co., Ltd.. G. Lizee: Advisory/Consultancy: HengJia Neoantigen Biotechnology (Tianjin) Co., Ltd.. P. Hwu: Advisory/Consultancy: Dragonfly Therapeutics; Advisory/Consultancy: GlaxoSmithKline; Advisory/Consultancy, Shareholder/Stockholder/Stock options: Immatics; Advisory / Consultancy: Sanofi; Research grant/Funding (institution): Genentech. L. Deng: Full/Part-time employment: Tianjin HengJia Biotechnology Development Co., Ltd.. Q. Zou: Full/Part-time employment: Tianjin HengJia Biotechnology Development Co., Ltd.. Y. Wang: Full/Part-time employment: Tianjin HengJia Biotechnology Development Co., Ltd.. R. Ataullakhanov: Full/Part-time employment: Bostongene. Llc. Bagaev: Full/Part-time employment: Bostongene. Llc. N. Kotlov: Full/Part-time employment: Bostongene. Llc. V. Svekolkin: Full/Part-time employment: Bostongene. Llc. N. Miheecheva: Full/Part-time employment: Bostongene. Llc. F. Frenkel: Full/Part-time employment: Bostongene. Llc. All other authors have declared no conflicts of interest.

Targeting Tumors with IL-10 Prevents Dendritic Cell-Mediated CD8+ T Cell Apoptosis

Increasing evidence demonstrates that interleukin-10 (IL-10), known as an immunosuppressive cytokine, induces antitumor effects depending on CD8+ Tcells. However, it remains elusive how immunosuppressive effects of IL-10 contribute to CD8+ Tcell-mediated antitumor immunity. We generated Cetuximab-based IL-10 fusion protein (CmAb-(IL10)2) to prolong its half-life and allow tumor-targeted delivery of IL-10. Our results demonstrated potent antitumor effects of CmAb-(IL10)2 with reduced toxicity. Moreover, we revealed a mechanism of CmAb-(IL10)2 preventing dendritic cell (DC)-mediated CD8+ tumor-infiltrating lymphocyte apoptosis through regulating IFN-γ production. When combined with immune checkpoint blockade, CmAb-(IL10)2 significantly improves antitumor effects in mice with advanced tumors. Our findings reveal a DC-regulating role of IL-10 to potentiate CD8+ Tcell-mediated antitumor immunity and provide a potential strategy to improve cancer immunotherapy.