Antitumor Immune Responses In Mice Research Articles

Single-cell RNA-sequencing uncovers compound kushen injection synergistically improves the efficacy of chemotherapy by modulating the tumor environment of breast cancer.

Due to lack of enough specific targets and the immunosuppressive tumor microenvironment (TME) of triple-negative breast cancer (TNBC), TNBC patients often cannot benefit from a single treatment option. This study aims to explore the regulatory effects of Compound kushen injection (CKI) plus chemotherapy on the TME of TNBC from a single cell level. A mouse TNBC model in BALB/c mice was established to evaluate the antitumor efficacy and toxicity of CKI combined with chemotherapy. Flow cytometry was used to observe the influence of CKI on the lymphocyte populations in the tumor bearing mice. Both bulk RNA sequencing (RNA-seq) and single-cell RNA-seq (scRNA-seq) were applied to portray the modulation of CKI combined with chemotherapy on the TME of TNBC mice. CKI significantly enhanced the anticancer activity of chemotherapy in vivo with no obvious side effects. Flow cytometry results revealed a significantly higher activation of CD8+ T lymphocytes in the spleens and tumors of the mice with combination therapy. Bulk RNA-seq indicated that CKI could promote the cytotoxic immune cell infiltrating into tumor tissues. Meanwhile, scRNA-seq further revealed that CKI combined with chemotherapy could enhance the percentage of tumor-infiltrating CD8+ T cells, inhibit tumor-promoting signaling pathways, and promote T cell activation and positive regulation of immune response. In addition, CKI showed obvious anticancer activity against MDA-MB-231 breast tumor cells in vitro. The combination of CKI and chemotherapy might provide a higher efficiency and lower toxicity strategy than a single chemotherapy drug for TNBC. CKI potentiates the anti-TNBC effects of chemotherapy by activating anti-tumor immune response in mice.

Tumor Ablation by Irreversible Electroporation Augments PD-1 Checkpoint Inhibitor Immunotherapy in Colon Adenocarcinoma

INTRODUCTION: Programmed Cell Death Protein 1 (PD-1) checkpoint inhibitors have recently been approved for microsatellite instability high (MSI-H) or mismatch repair deficient (dMMR) colorectal cancer (CRC). Response rates are promising in MSI-H-dMMR CRC but remain dismal for microsatellite-stable (MSS) or mismatch repair proficient (pMMR) CRC. >80% of CRCs are MSS-pMMR. Increasing tumor-infiltrating lymphocytes (TILs) may improve checkpoint inhibitor efficacy in CRC. New data suggest irreversible electroporation (IRE) therapy whereby electric pulses cause tumor necrosis and apoptosis; increase tumor-associated protein uptake by dendritic cells, subsequently boosting neoantigen presentation and TILs. We hypothesized that IRE combined with anti–PD-1 antibody, when compared with IRE or anti–PD-1 antibody alone, would increase survival, cause tumor regression by increasing TILs, and guard against secondary tumor challenge by increasing tumor antigen-specific CD8 T cells. METHODS: Using the MC38 cell line derived from murine colon adenocarcinoma, immunohistochemistry, and anti-CD8 antibodies, we evaluated antitumor immune responses in mice. RESULTS: IRE with anti–PD-1 antibody showed statistically significant survival, tumor regression, and increased TILs compared with IRE or anti–PD-1 antibody alone. Seventeen of 18 mice treated with IRE and anti–PD-1 antibody rejected a secondary tumor challenge. Conversely, IRE with anti–PD-1 antibody failed to protect against tumor rechallenge in mice that underwent CD8 T-cell depletion. Zero of 10 CD8 T-cell–depleted mice survived after secondary tumor challenge (Figure).FigureCONCLUSION: These data suggest IRE and anti–PD-1 antibody work in concert to bolster tumor-specific CD8 T-cell immune responses, serving as a pragmatic strategy for overcoming clinical challenges in checkpoint inhibitor immunotherapy for CRC.

Icaritin and intratumoral injection of CpG treatment synergistically promote T cell infiltration and antitumor immune response in mice

The development of combination therapy that can modulate the tumor immunosuppressive microenvironment is highly desirable for cancer immunotherapy. Icaritin (ICT), a hydrolytic product of icariin from genus Epimedium, has been used as an anti-cancer immunoregulatory agent for many types of cancers. Herein, we design a novel therapeutic strategy for mice melanoma that combines systemic administration of icaritin with intratumoral injection of unmethylated cytosine-guanine oligodeoxynucleotide (CpG). Icaritin induces tumor cell apoptosis and increases tumor immunogenicity. The combination of icaritin with CpG synergistically suppresses tumor growth and significantly prolonged survival time of B16F10 melanoma bearing mice. importantly, the anti-tumor effects of this combination strategy are associated with the reversing of immunosuppressive microenvironment through increased recruitment of functional DCs and tumor-associated macrophages (TAM) in tumors, leading to the infiltration of cytotoxic CD8+ T cells expressing elevated levels of IFN-γ and TNF-α. Furthermore, the combination of icaritin with CpG augments the anti-tumor immune response to anti-PD-1/CTLA-4 immune checkpoint blockade treatment. These results support the combination of icaritin with CpG as a novel strategy to elicit effective T cell-mediated antitumor immune response.

Abstract 5194: BALB/c mice with humanized VISTA: A novel GEMM for preclinical evaluation of therapeutic antibodies against VISTA

Abstract VISTA, also known as PD1H, is an immune-checkpoint receptor mainly expressed in immune cells, including macrophages, conventional dendritic cells, monocytes and circulating neutrophils. As a negative regulator of T cell activation, VISTA inhibits T cell proliferation and attenuates the production of cytokines. Studies have shown that VISTA expression is increased in infiltrating immune cells found in the tumor microenvironment and that blocking pathways that lead to VISTA activation can enhance antitumor immune responses in mice. Based on this, antibodies targeting VISTA are promising leads for cancer therapy. Preclinical efficacy evaluation of antibodies again human VISTA requires the expression of human VISTA in animal models, since difference in extracellular domains may affect antibody recognition while the overall homology between the two proteins is only 77%. To meet this need, we established a humanized mouse model in which the extracellular domain of mVISTA is replaced with that of hVISTA while the trans-membrane and cytoplasmic domains remain intact. In order to confirm the activity of the chimeric protein in our mice, we evaluated the anti-tumor effect of proven VISTA antibodies in homozygous hVISTA mice engrafted with CT26 tumor subcutaneously. Consistent with previous studies, the growth of CT26 tumor was inhibited after VISTA antibody treatment, indicating the humanized VISTA mice are suitable for efficacy evaluation of human VISTA antibodies. Moreover, we evaluated the anti-tumor effect of a combination of PD-L1 antibody and VISTA antibody in these mice. Interestingly, the combined use of PD-L1 antibody and VISTA antibody led to significantly enhanced tumor regression than either antibody. Taken together, BALB/c-hVISTA mouse is a novel non-human animal model appropriate for preclinical efficacy study of human VISTA antibodies. Citation Format: Weiwei Yu, Honghong Bao, Shuai Li, Chao Ju, Hongyan Sun, Hao Qi, Cunxiang Ju, Jing Zhao, Xiang Gao. BALB/c mice with humanized VISTA: A novel GEMM for preclinical evaluation of therapeutic antibodies against VISTA [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 5194.

CD146 bound to LCK promotes T cell receptor signaling and antitumor immune responses in mice.

Initiation of T cell receptor (TCR) signaling involves the activation of the tyrosine kinase LCK; however, it is currently unclear how LCK is recruited and activated. Here, we have identified the membrane protein CD146 as an essential member of the TCR network for LCK activation. CD146 deficiency in T cells substantially impaired thymocyte development and peripheral activation, both of which depend on TCR signaling. CD146 was found to directly interact with the SH3 domain of coreceptor-free LCK via its cytoplasmic domain. Interestingly, we found CD146 to be present in both monomeric and dimeric forms in T cells, with the dimerized form increasing after TCR ligation. Increased dimerized CD146 recruited LCK and promoted LCK autophosphorylation. In tumor models, CD146 deficiency dramatically impaired the antitumor response of T cells. Together, our data reveal an LCK activation mechanism for TCR initiation. We also underscore a rational intervention based on CD146 for tumor immunotherapy.

Toll-Like Receptor-4 Antagonist Enhances the Repair of Ultraviolet Radiation-Induced DNA Damage and Augments Anti-Tumor Immune Responses in Mice.

Simple SummaryUltraviolet B (UVB) radiation is largely responsible for the development of skin cancer. When UVB-induced DNA damage in cells is not repaired, it can lead to the initiation of non-melanoma skin cancers. Xeroderma pigmentosum (XP) disease is caused by a defect in the repair of damaged DNA. Toll-like receptor-4 (TLR4) and NLR family pyrin domain containing 3 (NLRP3) belong to the family of innate immune receptors and are highly expressed in skin tumors. In this study, we determined the mechanism through which TLR4 inhibitor TAK-242 regulates inflammation and prevents skin cancer.Ultraviolet (UV) irradiation of the skin is related to the development of skin cancer. UVB also causes DNA damage in the form of cyclobutane pyrimidine dimers (CPDs), which can result in stable mutations. Toll-like receptor 4 (TLR4), a component of innate immunity, plays a key role in cancer. Previous studies from our laboratory have observed that TLR4 deficiency resulted in the repair of UVB-induced DNA damage, inhibition of UVB-induced immune suppression, and carcinogenesis. In this study, we determined the efficacy of TLR4 antagonist TAK-242 in regulation of UVB-induced DNA damage, inflammation, and tumor development. Our results indicate that TAK-242 treatment increased the expression of xeroderma pigmentosum group A (XPA) mRNA, resulting in the repair of UVB-induced CPDs in skin of SKH-1 mice. Treatment with TAK-242 also inhibited the activation of NLR family pyrin domain containing 3 (NLRP3) in UVB-exposed skin of SKH-1 mice. Cutaneous carcinogenesis was significantly reduced in mice treated with TAK-242 in comparison to vehicle-treated mice. The proinflammatory cytokines IL-1β, IL-6, and TNF-α were also found to be significantly greater in vehicle-treated mice than TAK-242-treated mice. Finally, treatment with TAK-242 augmented anti-tumor immune responses in mice. Our data provide further evidence that activation of the TLR4 pathway promotes the development of UV-induced non-melanoma skin cancer mediated at least in part on its negative effects on DNA damage. Moreover, treatment with the TLR4 inhibitor TAK-242 may be effective for prevention of skin cancer.

A small-molecule SUMOylation inhibitor activates antitumor immune responses and potentiates immune therapies in preclinical models.

SUMOylation, the covalent conjugation of small ubiquitin-like modifier (SUMO) proteins to protein substrates, has been reported to suppress type I interferon (IFN1) responses. TAK-981, a selective small-molecule inhibitor of SUMOylation, pharmacologically reactivates IFN1 signaling and immune responses against cancers. In vivo treatment of wild-type mice with TAK-981 up-regulated IFN1 gene expression in blood cells and splenocytes. Ex vivo treatment of mouse and human dendritic cells promoted their IFN1-dependent activation, and vaccination studies in mice demonstrated stimulation of antigen cross-presentation and T cell priming in vivo. TAK-981 also directly stimulated T cell activation, driving enhanced T cell sensitivity and response to antigen ex vivo. Consistent with these observations, TAK-981 inhibited growth of syngeneic A20 and MC38 tumors in mice, dependent upon IFN1 signaling and CD8+ T cells, and associated with increased intratumoral T and natural killer cell number and activation. Combination of TAK-981 with anti-PD1 or anti-CTLA4 antibodies improved the survival of mice bearing syngeneic CT26 and MC38 tumors. In conclusion, TAK-981 is a first-in-class SUMOylation inhibitor that promotes antitumor immune responses through activation of IFN1 signaling. TAK-981 is currently being studied in phase 1 clinical trials (NCT03648372, NCT04074330, NCT04776018, and NCT04381650) for the treatment of patients with solid tumors and lymphomas.

Recombinant Mycobacterium smegmatis delivering a fusion protein of human macrophage migration inhibitory factor (MIF) and IL-7 exerts an anticancer effect by inducing an immune response against MIF in a tumor-bearing mouse model

BackgroundMacrophage migration inhibitory factor (MIF) is a pleotropic inflammatory cytokine that is overexpressed in a number of cancer types including most types of human cancer. Inhibition of MIF signaling can...

Tumor-Derived Exosomes Enriched by miRNA-124 Promote Anti-tumor Immune Response in CT-26 Tumor-Bearing Mice.

Exosomes have been introduced as a new alternative delivery system for the transmission of small molecules. Tumor-derived exosomes (TEXs) not only contain tumor-associated antigens to stimulate antitumor immune responses but also act as natural carriers of microRNAs. The aim of the current study was to evaluate the efficacy of miR-124-3p-enriched TEX (TEXomiR) as cell-free vaccine in the induction of antitumor immune responses in a mouse model of colorectal cancer. Briefly, the exosomes were isolated from cultured CT-26 cell line, and modified calcium chloride method was used to deliver miR-124-3p mimic into the exosomes. We used a CT-26-induced BALB/c mouse model of colorectal cancer and analyzed the effect of TEXomiR on survival, tumor size, spleen and tumor-infiltrated lymphocytes, and splenocyte proliferation. Furthermore, intra-tumor regulatory T cells, cytotoxic activity of the splenocytes, and cytokine secretion was also evaluated to describe the anti-tumor immune response. When the tumor size reached 100 mm3, the mice were injected with TEXomiR, TEX, and/or phosphate-buffered saline (PBS) subcutaneously three times with 3-day interval, and then tumor size was monitored every 2 days. The in vitro results indicated that TEXs could efficiently deliver functional miR-124-3p mimic. The in vivo evaluation in tumor-bearing mice showed that treatment with TEXomiR can elicit a stronger anti-tumor immune response than unloaded TEX and PBS. Significant tumor growth inhibition and increased median survival time was achieved in tumor-bearing mice treated with TEXomiR. A significant decrease in CD4/CD8 and Treg/CD8 ratio in tumor tissue was demonstrated. Moreover, increased cytotoxicity and proliferation of splenocytes in the TEXomiR group compared to the TEX and PBS groups were identified. Taken together, our data demonstrated that tumor-derived exosomes efficiently deliver miR-124-3p mimic, and TEXomiR promotes anti-tumor immune responses.

Abstract PO072: Nitric oxide tumor ablation stimulates an anti-tumor immune response in mice

Abstract Introduction: Metastases are responsible for a major portion of the morbidity and mortality of cancer, accounting for approximately 90% of all cancer-related deaths. In situ destruction of the tumor mass has been reported to provide the immune system with an antigen source for the induction of antitumor immunity, which can destroy distant metastases. One of the proposed mechanisms involves the induction of antigen presenting cells that may result in the stimulation of adaptive immunity. Our research group is developing an innovative gaseous Nitric Oxide (gNO) based tumor ablation method. NO is a short-lived free radical which, at high doses, possesses anticancer properties. Moreover, NO has been proven to activate innate and adaptive responses of the immune system against tumors. Previous in vivo results showed that all gNO-treated colon tumor-bearing (CT26 model) mice (n=6) were resistant to a secondary CT26 cell inoculation. In the current study, high dose gNO has been used to destroy breast and lung solid tumors in mice. The immune response stimulated following this treatment was tested. Materials and Methods: Breast and lung tumor-bearing mice (4T1 and LLC1 models, respectively) were treated with 50,000 ppm gNO intratumorally. A metastasis model was induced in all gas-treated tumor bearing mice up to 14 days post treatment by challenging the mice with a second cancer cell inoculation (challenge assay). Naïve mice, inoculated with the same cancer cells, served as an internal control. Results: All gNO-treated lung tumor-bearing mice (n=2) were resistant to a second LLC1 lung cancer cell inoculation as compared to 100% tumor take in the control group (n=3, p = 0.03). In gNO-treated breast tumor-bearing mice (n=3), using the highly aggressive 4T1 model that mimics human stage 4 breast cancer, an inhibition in a secondary 4T1 tumor take was observed as compared to the tumor take in the control group (n=3). All naïve mice that were inoculated with 4T1 cells developed a tumor after 5 days, while no gNO-treated 4T1 tumor-bearing mice developed a 4T1 challenge tumor at this time point (p =0.01). At 7 days after 4T1 challenge cell inoculation 1 of 3 mice developed a challenge tumor (p =0.08). Conclusion: The proposed project represents development of a novel treatment for cancer. Our innovative gNO based treatment may serve to treat lethal solid tumors locally and their distant metastases systemically via the stimulation of an anti-tumor immune response. Citation Format: Hila Confino, Shay Yarkoni, Matan Goldshtein, Elya Dekel, Omer Lerner, Shani Puyesky, Steve Lisi, Rinat Kalaora, Pam Golden, Amir Avniel, Prof. Ido Wolf. Nitric oxide tumor ablation stimulates an anti-tumor immune response in mice [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO072.

Activity of tumor-associated macrophage depletion by CSF1R blockade is highly dependent on the tumor model and timing of treatment

Tumor-associated macrophages (TAMs) are abundant in solid tumors where they exhibit immunosuppressive and pro-tumorigenic functions. Inhibition of TAM proliferation and survival through CSF1R blockade has been widely explored as a cancer immunotherapy. To further define mechanisms regulating CSF1R-targeted therapies, we systematically evaluated the effect of anti-CSF1R treatment on tumor growth and tumor microenvironment (TME) inflammation across multiple murine models. Despite substantial macrophage depletion, anti-CSF1R had minimal effects on the anti-tumor immune response in mice bearing established tumors. In contrast, anti-CSF1R treatment concurrent with tumor implantation resulted in more robust tumor growth inhibition and evidence of enhanced anti-tumor immunity. Our findings suggest only minor contributions of CSF1R-dependent TAMs to the inflammatory state of the TME in established tumors, that immune landscape heterogeneity across different tumor models can influence anti-CSF1R activity, and that alternative treatment schedules and/or TAM depletion strategies may be needed to maximize the clinical benefit of this approach.

A Hepatitis B Virus-Derived Peptide Exerts an Anticancer Effect via TNF/iNOS-producing Dendritic Cells in Tumor-Bearing Mouse Model.

Simple SummaryRecently, it has been reported that a tumor necrosis factor (TNF)/inducible nitric oxide synthase (iNOS)-producing dendritic cell (Tip-DC) may play a pivotal role in the anticancer immune response by activating CD8+ T cells in tumor microenvironments. The development of a new immunotherapeutic agent that can enhance the oncolytic effect of Tip-DC has gained increasing attention in the cancer research field. In this study, we introduce a hepatitis B virus-derived peptide, Poly6, which elicited a strong anticancer immune response via enhanced Tip-DC activity. Our findings suggest that Poly6 could be a novel potential adjuvant/co-treatment partner in anticancer immunotherapy approaches.Recently, we reported a 6-mer hepatitis B virus (HBV)-derived peptide, Poly6, that exerts antiviral effects against human immunodeficiency virus type 1 (HIV-1). Here, we explored the immunotherapeutic potential of Poly6 via its administration into dendritic cells (DCs) in a mouse model. Our data revealed that Poly6 treatment led to enhanced production of tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase (iNOS)-producing DCs (Tip-DCs) in a type 1 interferon (IFN-I)-dependent manner via the induction of mitochondrial stress. Poly6 treatment in mice implanted with MC38 cells, a murine colon adenocarcinoma line, led to attenuated tumor formation, primarily due to direct cell death induced by Tip-DC mediated nitric oxide (NO) production and indirect killing by Tip-DC mediated cluster of differentiation 8 (CD8) cytotoxic T lymphocyte (CTL) activation via CD40 activation. Moreover, Poly6 treatment demonstrated an enhanced anticancer effect with one of the checkpoint inhibitors, the anti PD-L1 antibody. In conclusion, our data reveal that Poly6 treatment elicits an antitumor immune response in mice, possibly through NO-mediated oncolytic activity via Tip-DC activation and Tip-DC mediated CTL activation. This suggests that Poly6 represents a potential adjuvant for cancer immunotherapy by enhancing the anticancer effects of immune checkpoint inhibitors.

Polymer-lipid hybrid nanovesicle-enabled combination of immunogenic chemotherapy and RNAi-mediated PD-L1 knockdown elicits antitumor immunity against melanoma

Immunotherapy has revolutionized cancer treatment; however, only a limited portion of patients show responses to currently available immunotherapy regimens. Here, we demonstrate that RNA interference (RNAi) combined with immunogenic chemotherapy can elicit potent antitumor immunity against melanoma. Specially, we developed cationic polymer-lipid hybrid nanovesicles (P/LNVs) as a new delivery system for doxorubicin and small interfering RNA (siRNA) with extensive cytotoxicity and gene silencing efficiency towards B16 cells. The deployment of doxorubicin-loaded P/LNVs augmented the expression and presentation of endogenous tumor antigens directly in situ by inducing the immunogenic cell death of B16 cells through poly(ADP-ribose) polymerase 1-dependent (PARP1) apoptosis pathway; thereby, eliciting remarkable antitumor immune responses in mice. Leveraging dying B16 cells as a vaccination strategy in combination with RNAi-based programmed cell death ligand 1 (PD-L1) knockdown showed efficacy in both prophylactic and metastasis melanoma settings. Strikingly, PD-L1 blockade synergized with a sub-therapeutic dose of doxorubicin triggered robust therapeutic antitumor T-cell responses and eradicated pre-established tumors in 30% of mice bearing B16 melanoma. Our findings indicated that this combination treatment provided a new powerful immunotherapy modality, characterized by markedly increased infiltration of effector CD8+ T cells and effective alleviation of the immunosuppressive microenvironment in tumors. P/LNVs is a versatile and highly scalable carrier that can enable a broad combination of nanomedicine and RNAi, providing new therapeutic strategies for advanced cancers.

Abstract 4522: Potent induction of a tumor-specific immune response by a cyclic dinucleotide STING agonist

Abstract Introduction: Stimulator of interferon genes (STING) is activated by cyclic dinucleotides that are generated by cyclic GMP-AMP synthase in the presence of cytosolic DNA, resulting in a type 1 interferon response and the secretion of pro-inflammatory cytokines. BI-STING mimics the natural ligand of STING and hijacks the STING signaling pathway to trigger a potent anti-tumor immune response. Here we report the pre-clinical characterization of BI-STING in human cells lines in vitro, in human ex vivo systems and in vivo mouse models. Methods: Activity and specificity of BI-STING were tested in vitro in the human monocytic leukemia cell line THP1. Target engagement was confirmed using ex vivo studies in human whole blood and in human precision cut colorectal cancer slices. For in vivo studies, BI-STING was administered intratumorally (i.tu.) in a range of subcutaneous, syngeneic murine tumor models. The modulation of cytokine secretion over time was recorded and the induction of a tumor-specific immune response demonstrated. Results: Using THP1 cells, activation of down-stream signaling events by BI-STING was seen specifically in STING wild type, but not STING knock out cells. Ex vivo treatment of human whole blood and human precision cut colorectal cancer slices with BI-STING resulted in the significant induction of cytokine secretion (including IFNβ) and in an array of transcriptional changes associated with the activation of STING signaling. Treatment of tumor-bearing mice with a single dose of BI-STING i.tu. led to a transient increase of cytokine levels in tumor and plasma. In all tested models, i.tu. administration of BI-STING resulted in dose-dependent local tumor control. Importantly, animals whose primary tumor was cured by BI-STING treatment did not develop tumors when re-challenged with the same tumor cell line and an abscopal delay in tumor growth was seen for non-injected (or anenestic) lesions in mice carrying bilateral tumors. Abscopal tumor control was further improved when combined with anti-PD-1. Finally, ELISpot analysis resulted in a significantly increased number of immunospots in splenocytes from BI-STING treated animals as compared to vehicle control mice, confirming the induction of a tumor-specific immune response. Conclusions: BI-STING is a potent and specific inducer of the STING signaling pathway. In addition to local tumor control, i.tu. treatment with BI-STING results in a systemic anti-tumor immune response in mice, which was enhanced upon anti-PD-1 combination. A clinical trial to evaluate i.tu. administration of BI-STING alone and in combination with anti-PD-1 in patients with advanced solid tumors is ongoing. Citation Format: Gabriela Gremel, Maria A. Impagnatiello, Sebastian Carotta, Otmar Schaaf, Paolo M. Chetta, Thorsten Oost, Thomas Zichner, Marco Hofmann, Sophia Blake, Tom Bretschneider, Martin Fleck, Achim Grube, Herbert Nar, Georg Rast, Esther Schmidt, Ute Klinkhardt, Kirsten Arndt-Schmitz, Thorsten Laux, Vittoria Zinzalla, Jonathon Sedgwick, Norbert Kraut. Potent induction of a tumor-specific immune response by a cyclic dinucleotide STING agonist [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4522.

A therapeutic HPV16 E7 vaccine in combination with active anti-FGF-2 immunization synergistically elicits robust antitumor immunity in mice

FGF-2 accumulates in many tumor tissues and is closely related to the development of tumor angiogenesis and the immunosuppressive microenvironment. This study aimed to investigate whether active immunization against FGF-2 could modify antitumor immunity and enhance the efficacy of an HPV16 E7-specific therapeutic vaccine. Combined immunization targeting both FGF-2 and E7 significantly suppressed tumor growth, which was accompanied by significantly increased levels of IFN-γ-expressing splenocytes and effector CD8 T cells and decreased levels of immunosuppressive cells such as regulatory T cells (Tregs) and myeloid-derived suppressor cells(MDSCs) in both the spleen and tumor; in addition, the levels of FGF-2 and neovascularization in tumors were decreased in the mice receiving the combined immunization, and tumor cell apoptosis was promoted. The combination of an HPV16 E7-specific vaccine and active immunization against FGF-2 significantly enhances antitumor immune responses in mice with TC-1 tumors, indicating a promising strategy for tumor immunotherapy.

Virus-Like Particles Presenting the FGF-2 Protein or Identified Antigenic Peptides Promoted Antitumor Immune Responses in Mice.

BackgroundFibroblast growth factor (FGF)-2 is overexpressed in various tumor tissues. It affects tumor cell proliferation, invasion and survival, promotes tumor angiogenesis and is tightly involved in the development of systemic and local immunosuppressive tumor mechanisms.PurposeThis study aimed to develop an effective vaccine against FGF-2 and to investigate the effects of anti-FGF-2 immunization on tumor growth and antitumor immune responses.MethodsA set of thirteen synthesized overlapping peptides covering all possible linear B-cell epitopes of murine FGF-2 and a recombinant FGF-2 protein were conjugated to virus-like particles (VLPs) of recombinant hepatitis B core antigen (HBcAg). The VLPs were immunized through a preventive or therapeutic strategy in a TC-1 or 4T1 grafted tumor model.ResultsImmunization with FGF-2 peptides or full-length protein-coupled VLPs produced FGF-2-specific antibodies with a high titer. Peptide 12, which is located in the heparin-binding site of FGF-2, or protein-conjugated VLPs presented the most significant effects on the suppression of TC-1 tumor growth. The levels of IFN-γ-expressing splenocytes and serum IFN-γ were significantly elevated; further, the immune effector cells CD8+ IFN-γ+ cytotoxic T lymphocytes (CTLs) and CD4+ IFN-γ+ Th1 cells were significantly increased, whereas the immunosuppressive cells CD4+ CD25+ FOXP3+ Treg cells and Gr-1+ CD11b+ myeloid-derived suppressor cells (MDSCs) were decreased in the immunized mice. In addition, VLP immunization significantly suppressed tumor vascularization and promoted tumor cell apoptosis. In mice bearing 4T1 breast tumor, preventive immunization with FGF-2-conjugated VLPs suppressed tumor growth and lung metastasis, and increased effector cell responses.ConclusionActive immunization against FGF-2 is a new possible strategy for tumor immunotherapy.

Vaccination with CD47 deficient tumor cells elicits an antitumor immune response in mice

Cancer cells are poorly immunogenic and have a wide range of mutations, which makes them unsuitable for use in vaccination treatment. Here, we show that elimination of CD47, a ligand for the myeloid cell inhibitory receptor SIRPα, from tumor cells by genetic deletion or antibody blocking, significantly improves the effectiveness of the immune response to tumour cells. In both solid and hematopoietic mouse tumor models, vaccination with tumor cells or tumor antigen-expressing cells, that lack CD47 or were pre-coated with anti-CD47 antibodies, achieved an antitumor immune response. The efficacy of this approach was synergistically enhanced when used in combination with anti-PD-1 antibodies. The induction of antitumor responses depends on SIRPα+CD11c+ DCs, which exhibit rapid expansion following introduction of CD47-deficient tumor cells. Our results indicate that CD47-deficient whole tumor cells can induce antitumor responses.

Arf1-mediated lipid metabolism sustains cancer cells and its ablation induces anti-tumor immune responses in mice

Cancer stem cells (CSCs) may be responsible for treatment resistance, tumor metastasis, and disease recurrence. Here we demonstrate that the Arf1-mediated lipid metabolism sustains cells enriched with CSCs and its ablation induces anti-tumor immune responses in mice. Notably, Arf1 ablation in cancer cells induces mitochondrial defects, endoplasmic-reticulum stress, and the release of damage-associated molecular patterns (DAMPs), which recruit and activate dendritic cells (DCs) at tumor sites. The activated immune system finally elicits antitumor immune surveillance by stimulating T-cell infiltration and activation. Furthermore, TCGA data analysis shows an inverse correlation between Arf1 expression and T-cell infiltration and activation along with patient survival in various human cancers. Our results reveal that Arf1-pathway knockdown not only kills CSCs but also elicits a tumor-specific immune response that converts dying CSCs into a therapeutic vaccine, leading to durable benefits.

Heat-killed Mycobacterium paragordonae therapy exerts an anti-cancer immune response via enhanced immune cell mediated oncolytic activity in xenograft mice model.

A therapeutic strategy capable of skewing toward a Th1-type immune response is crucial for cancer treatment. Recently, we reported Mycobacterium paragordonae (Mpg) as a potential live vaccine for mycobacterium infections. In this study, we explored the immunotherapeutic potential of heat-killed Mpg (HK-Mpg) in a mouse tumor xenograft model and elucidated its underlying antitumor mechanisms. MC38 cells derived from murine colon adenocarcinoma were implanted by subcutaneously injecting mice. The anticancer effects of HK-Mpg therapy were compared with HK-M. bovis BCG, an effective adjuvant for cancer immunotherapy. HK-Mpg treatment enhanced tumor reduction and mouse survival. Furthermore, HK-Mpg treatment synergistically enhanced the anticancer therapeutic effect of cisplatin. In addition, HK-Mpg enhanced inflammatory cytokine production and recruitment of immune cell into tumor-infiltrating sites and splenocytes in vaccinated mice. Our mechanistic study demonstrates that HK-Mpg therapy elicits a strong antitumor immune response in mice, mainly through natural killer cell-mediated oncolytic activity via the activation of dendritic cells (DCs) and by enhancing inflammatory cytokines production such as IL-12 from DC. Hence, HK-Mpg can be a potential immunotherapy adjuvant, enhancing the effect of cancer chemotherapy.

Abstract 3252: TAK-981: A first in class SUMO inhibitor in Phase 1 trials that promotes dendritic cell activation, antigen-presentation, and T cell priming

Abstract SUMOylation is a reversible post-translational modification that regulates protein function by covalent attachment of the small ubiquitin-like modifier (SUMO) protein to protein substrates. TAK-981 is a novel, selective small molecule inhibitor of the SUMOylation enzymatic cascade, which has demonstrated induction of potent anti-tumor immunity in preclinical models. In this study, we evaluated changes in immune cell composition and states after TAK-981 treatment in preclinical models. In both mouse bone-marrow and human peripheral blood mononuclear cell derived dendritic cells (DCs), TAK-981 treatment ex vivo induced markers of activation and maturation including CD40, CD80 and CD86, as well as increased secretion of inflammatory cytokines like IP-10, MCP1, MIP-1α, MIP1β, IFNα and IFNβ. These effects were reversed by an interferon alpha receptor (IFNAR) blocking antibody, demonstrating dependence of TAK-981 induced pharmacodynamic effects on Type I interferon signaling in DCs. In vivo, a single sub-cutaneous injection of TAK-981 in naïve Balb/c mice at the brachial lymph nodes induced activation of DCs, measured as significant increases in CD40 and CD86 expression. Concomitantly, an increase in expression of the early lymphocyte activation marker CD69 was observed on T cells and NK cells, demonstrating pleiotropic effects of TAK-981 on immune cells. Increased expression of CD69 was also observed in purified human T and NK cells treated with TAK-981 ex vivo. To investigate if enhanced activation of DCs by TAK-981 leads to improved cross-presentation of exogenous antigens, we challenged naïve C57BL/6 mice with chicken-ovalbumin (OVA) protein alone or in combination with TAK-981. Interestingly, an increase in the frequency of Kb-SIINFEKL tetramer positive CD8 T cells was observed in the draining lymph nodes overnight, and in the spleen on day 14, after treatment with TAK-981+OVA, suggesting that TAK-981 promotes cross-presentation of SIINFEKL, the class-I MHC epitope of OVA, to cognate antigen-specific CD8 T cells. We also observed an increase in the frequency of CD8α+ DCs loaded with the peptide ‘SIINFEKL’ on H-2Kb (class-I MHC of C57BL/6 mice), providing direct evidence for enhanced antigen-cross presentation. Similar findings were observed with TAK-981 administered either sub-cutaneously or via the therapeutic route of intra-venous injection in the above OVA immunization model. These results suggest a mechanism by which TAK-981 may promote anti-tumor immune responses in mice via enhanced cross-presentation of exogenous antigens released by dying tumor cells, leading to priming and activation of antigen reactive cytotoxic T cells. Currently, TAK-981 is being evaluated in Phase 1 clinical trials in adult patients with metastatic solid tumors or lymphomas (ClinicalTrials.gov Identifier: NCT03648372). Citation Format: Mithun Khattar, Keli Song, Stephen Grossman, Kristina Xega, Xingyue He, Neeraja Idamakanti, Dennis Huszar. TAK-981: A first in class SUMO inhibitor in Phase 1 trials that promotes dendritic cell activation, antigen-presentation, and T cell priming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3252.