Innate Antitumor Immune Responses Research Articles

Tumor cell-directed STING agonist antibody-drug conjugates induce type III interferons and anti-tumor innate immune responses

Activating interferon responses with STING agonists (STINGa) is a current cancer immunotherapy strategy, and therapeutic modalities that enable tumor-targeted delivery via systemic administration could be beneficial. Here we demonstrate that tumor cell-directed STING agonist antibody-drug-conjugates (STINGa ADCs) activate STING in tumor cells and myeloid cells and induce anti-tumor innate immune responses in in vitro, in vivo (in female mice), and ex vivo tumor models. We show that the tumor cell-directed STINGa ADCs are internalized into myeloid cells by Fcγ-receptor-I in a tumor antigen-dependent manner. Systemic administration of STINGa ADCs in mice leads to STING activation in tumors, with increased anti-tumor activity and reduced serum cytokine elevations compared to a free STING agonist. Furthermore, STINGa ADCs induce type III interferons, which contribute to the anti-tumor activity by upregulating type I interferon and other key chemokines/cytokines. These findings reveal an important role for type III interferons in the anti-tumor activity elicited by STING agonism and provide rationale for the clinical development of tumor cell-directed STINGa ADCs.

β-Mangostin targets and suppresses glioma via STING activation and tumor-associated microglia polarization

Glioma, a common and highly malignant central nervous system tumor, markedly influences patient prognosis via interactions with glioma-associated macrophages. Previous research has revealed the anticancer potential of β-mangostin, a xanthone derivative obtained from the mangosteen fruit. This research investigated the role of β-mangostin on microglia in the glioma microenvironment and evaluated the efficacy of β-mangostin combined with anti-PD-1 antibody (αPD-1) in glioma-bearing mice. The results showed that, β-mangostin attenuated M2 polarization in BV2 cells and promoted M1-related interleukin (IL)-1β and IL-6 secretion, thereby inhibiting glioma invasion. In addition, β-mangostin improved the anti-glioma effects of αPD-1 and increased CD8+T cell and M1-type microglia infiltration. Mechanistically, β-mangostin bound to the stimulator of interferon genes (STING) protein, which is crucial for the anti-tumor innate immune response, and promoted STING phosphorylation in microglia, both in vivo and in vitro. These results provide insights into its mode of action and supporting further investigation into β-mangostin as a therapeutic agent.

Intravesical local administration of pembrolizumab for treatment of bladder cancer: A novel strategy to minimize toxicity.

e14687 Background: A major hallmark of bladder cancer (BCa) is a higher treatment success rate than prostate cancer following stimulation of innate anti-tumor immune response either by intravesical BCG or to FDA approved intravenous anti-PD1 checkpoint antibody (pembrolizumab) approved for BCG resistant BCa since 2021. However, injected pembrolizumab is characterized by a markedly higher incidence (66%) of serious adverse events than the median duration of complete response of ~40% for non-muscle invasive BCa (NMIBC) and 15-20% for invasive BCa. Since the toxicity (fatigue, Pneumonitis and diarrhea) of injected pembrolizumab is linked to systemic breakdown of T-cell mediated immune surveillance we enquired whether analogous to BCG, pembrolizumab can be administered intravesically to safely induce a localized anti-tumor response in mouse model of BCa. Methods: Six weeks old female mice of B6D2F1 strain (n=10) were ad libitum fed carcinogen, N-butyl-N-4-hydroxybutyl nitrosamine (BBN) in drinking water (0.05% w/v) for 12 weeks. The presence of BBN evoked tumor was ascertained by unenhanced T2 weighted MRI at week 11 prior to single intravesical dosing by 24G transurethral catheter under isoflurane anesthesia of 0.1mL vehicle (sterile water) with or without 0.3mg of Pembrolizumab (Lipella Pharmaceuticals). 6 weeks later, T2 weighted MRI was repeated together with T1 mapping by ICE-MRI prior to organ harvest. Results: BBN feeding evoked multiclonal tumors (red*) of comparable size on T2 weighted MRI at week 11 but repeat MRI 7 weeks after treatment revealed that tumor progression and perivesical invasion noted in vehicle group was arrested in pembrolizumab group. Longest dimension of BBN tumor in vehicle treated mice was ≥ 1mm longer (p<0.05) than in pembrolizumab treated mice. T1 mapping revealed thickened bladder wall missed by T2 MRI. Conclusions: These findings on single intravesical dose of pembrolizumab (10mg/kg) in mice are consistent with human study on multiple intravesical dosing of BCG plus pembrolizumab (1-2mg/kg) in BCG resistant NMIBC patients. Intravesical pembrolizumab mirrors the limited systemic uptake of radiolabelled antibodies together with substantially higher ingress into tumor foci. The tumor selective ingress for BCa immunotherapy without any toxicity will be enhanced with Lipella Pharmaceutical’s liposomal delivery of Pembrolizumab.

Abstract PO2-18-05: Blocking soluble TNF to Improve potency of trastuzumab deruxtecan by increasing internalization and antitumor innate immune response in a resistant HER2-positive breast cancer model

Abstract Background Trastuzumab deruxtecan (T-DXd) administration improves response for patients with HER2-positive metastatic breast cancer (HER2+ BC). Unfortunately, 50% of patients relapse after 2 years. T-DXd resistance mechanisms are being explored. For trastuzumab we have shown that mucin 4 (MUC4) expression is an independent predictor of poor response in HER2+BC patients. MUC4 is upregulated by soluble TNF (sTNF) secreted by the tumor, confers resistance to trastuzumab by hiding its epitope on the HER2, reducing its binding and decreasing anti-tumor phagocytic function. In preclinical models of de novo trastuzumab-resistant tumors, combination of a sTNF blocking agent INB03, (DN), with T-DXd decreases tumor growth compared to T-DXd alone. To disclose the underlying mechanism, we studied whether DN improved internalization of T-DXd in tumor cells and modified the innate immune response to enhance T-DXd antitumor effects in a multiple HER2-targeted therapy-resistant model. Methods Nude mice bearing HER2+MUC4+ JIMT-1 tumor, primary resistant to trastuzumab, pertuzumab and lapatinib, were treated with IgG 5 mg/kg, T-DXd 5 mg/kg (T-DXd 5), 2.5 mg/kg (T-DXd 2.5) or 1.25 mg/kg (T-DXd 1.25), DN 10 mg/kg or the combined therapies. T-DXd and IgG were administered i.v. on days 0, 7 and 14. DN was administered i.p. twice a week for 3 weeks. Tumor growth was monitored. Mitotic figures/field (mean) were analyzed in H&E tumor sections. Tumor-infiltrating macrophages were studied by flow cytometry. IFNƔ was determined in tumor extracts by ELISA. Internalization of T-DXd in JIMT-1 cells was studied in a S1 Incucyte along 18h by Fab-rhodo red labeling. Results T-DXd dose-response curves showed inhibition in tumor growth of 83%, 61% and 37% for T-DXd 5, 2.5 and 1.25 mg/kg treatment respectively compared with IgG-treated tumors. DN alone exhibited no antitumor activity. T-DXd+DN increased the antitumor effect by 10%, 33% and 97% for T-DXd dose of 5, 2.5 and 1.25 mg/kg respectively. Tumor growth inhibition of T-DXd 5 was similar to combination of T-DXd1.25+DN. Addition of DN did not have toxicity. DN increases IFNƔ production in the TME of T-DXd treated tumors vs T-DXd alone and promoted macrophage recruitment to the tumor bed and polarization to the antitumor M1-like phenotype. Histopathological analysis of the tumor showed a significant decrease in proliferation in all the combined and in the T-DXd 5mg/kg (2.8-3.7 mitotic figures/field) vs IgG (6.0 mitotic figures/field). In vitro, JIMT-1 cells treated with DN internalized 40% more T-DXd than its vehicle-treated counterparts. Conclusions Neutralizing sTNF with DN enhances T-DXd effect in a multiple HER2 targeted therapy resistant model of MUC4 expressing HER2+ BC. Combination of DN with T-DXd increases tumor response at all dose levels tested. The largest effect was seen at lower doses (T-DXd 1.25+DN), which mimicked the effect of 4 times the dose of T-DXd alone, suggesting significant synergy with DN as dose of T-DXd decreases. Combination of DN with T-DXd in MUC4 expressing HER2+ BC improves response to T-DXd alone by increasing T-DXd internalization and improving anti-tumor innate immune responses in the TME without increasing toxicity. The results suggest this combination should be investigated in clinical trials in patients who have MUC4 expressing tumors when T-DXd is started or become resistant to T-DXd therapy. Citation Format: Sofia Bruni, Florencia Mauro, Sofia Naveiro, Rosalia Cordo-Russo, Agustina Dupont, Mercogliano María Florencia, Roxana Schillaci. Blocking soluble TNF to Improve potency of trastuzumab deruxtecan by increasing internalization and antitumor innate immune response in a resistant HER2-positive breast cancer model [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-18-05.

Abstract CT023: Prostate cancer in situ autovaccination with the intratumoral viral mimic poly-ICLC: Making a cold tumor hot

Abstract PURPOSE To test the safety of sequential intratumoral plus systemic intramuscular injection of poly-ICLC, and its efficacy in priming antitumor immune responses in patients with prostate cancer (PCa). EXPERIMENTAL PROCEDURES This is an open-label dose-escalating phase 1 neoadjuvant clinical trial of poly-ICLC (NCT03262103) in 12 patients diagnosed with clinically localized PCa with plans to undergo radical prostatectomy (RP). Poly-ICLC was administered intratumorally (Artemis MRI-TRUS-guided) and intramuscularly (e.g., deltoid muscle). Comprehensive transcriptional profiling of tissues, phenotypic and transcriptional analysis of longitudinally collected peripheral blood, and analysis of the prostate tumor microenvironment (TME) were performed before and after poly-ICLC treatment in order to identify innate and adaptive antitumor immune responses within the tumor and in peripheral blood. This trial is the first to use human intratumoral immunotherapy instead of systemic immunomodulation for high-risk PCa patients. RESULTS Poly-ICLC was well tolerated (safe) in all 12 patients. There were no dose-limiting toxicities or TEAE (treatment-emergent adverse events) withdrawals during treatment. Eight of 10 evaluable patients (80%) had undetectable PSA (<0.1 ng/ml) at 1 year of follow-up post-RP. Eight of 12 evaluable patients (66.7%) and 7 of 10 patients (70%) with biopsy Gleason 8-10 had a lower Gleason score in the post-treatment RP specimen. Transcriptome profiling of paired biopsy and RP specimens showed significant upregulation of gene signatures associated with immune cell infiltration and TP53-associated metabolic genes, but downregulation of gene signatures associated with metastasis and DNA replication. Genes upregulated by poly-ICLC were associated with a good prognosis. Treatment with poly-ICLC increased systemic immune responses, as demonstrated by an increase in cytolytic signatures, T- and NK-cell signatures, and NK-cell subsets in the blood. Multiplex immunohistochemistry analysis revealed a significant increase in the densities of CD4- and CD8-T cells and B cells, as well as evidence of tertiary lymphoid structures (TLS) within the TME of post-treatment RP specimens compared to baseline. CONCLUSIONS Poly-ICLC treatment can reliably transform the cold TME of PCa into a hot, immune-enhanced ecosystem. The identified baseline and response biomarkers, tertiary lymphoid structures, potential clinical benefit, and immunologic correlates require validation in larger studies. Citation Format: Sujit S. Nair, Dimple Chakravarty, Sreekumar Balan, Alexander Hakansson, Manuel Duval, Elai Davicioni, Yang Liu, Swati Bhardwaj, Tin Htwe Thin, Monica Garcia-barros, Kenneth Haines, Majd Al Shaarani, Rachel Weil, Marcia Meseck, Parita Ratnani, Monali Fatterpekar, Ivan Jambor, Elena Gonzalez-Gugel, Adam Farkas, Vinayak Wagaskar, Kacie Schlussel, Cristina Pasat-karasik, Kamala Bhatt, Zachary Dovey, Adriana Pedraza, Akriti Gupta, Dara Lundon, Ante Peros, Sneha Parekh, Lily Davenport, Xiangfu Zhang, Raghav Gupta, Macy Robison, Cynthia Knauer, Ethan Ellis, Dmitry Rykunov, Boris Reva, Babu Padanilam, Matthew D. Galsky, Rachel Brody, Mani Menon, Andres Salazar, Nina Bhardwaj, Ashutosh K. Tewari. Prostate cancer in situ autovaccination with the intratumoral viral mimic poly-ICLC: Making a cold tumor hot [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT023.

Abstract 6639: Mechanisms of action of a killed, bacteria-based, multiple immune receptor agonist in development for pulsed anti-tumor immunotherapy

Abstract Activation of immune receptors, such as Toll-like (TLR), NOD-like (NLR) and Stimulator of Interferon Genes (STING) is required for efficient innate and adaptive immunity. Gram-negative bacteria (G-NB) contain multiple TLR, NOD and STING agonists. Potential utility of G-NB for cancer immunotherapy is supported by observations of tumor regression in the setting of infection and Coley’s Toxins. Coley reported that intravenous (i.v.) administration was likely most effective but produced toxicity. The discovery of TLRs and their agonists, particularly the potent/multi-functional TLR4 agonist lipopolysaccharide (LPS)-endotoxin, comprising ~75% of the outer membrane of G-NB, suggests that it may be both a critical active ingredient and responsible for dose-limiting toxicity of i.v. G-NB. We have produced killed, intact bacteria products from non-pathogenic G-NB with ~90% reduction of LPS activity. One product, Decoy10, contained TLR2,4,8,9, NOD2 and STING agonist activity and exhibited reduced i.v. toxicity in mice and rabbits relative to unprocessed cells. Decoy10 and closely related Decoy20 produced single agent activity or combination-mediated durable regressions, with immunological memory, in mice with syngeneic or human breast, colorectal, hepatocellular, pancreatic carcinomas or non-Hodgkin’s lymphoma. Regressions were observed in combination with chemotherapy, a non-steroidal anti-inflammatory drug, anti-PD-1, or rituximab, were associated with induction of 18-26 plasma cytokines/chemokines, activation of innate and adaptive immune pathways in tumors, and were dependent on NK, CD4+ and CD8+ T cells (Newman, Cancer Res 2023;83(7 Suppl):Abstract nr 4165). These and preliminary clinical results demonstrating rapid clearance of Decoy20 with transient induction of >50 plasma cytokines/chemokines (Newman et al., J Immunother Cancer 2023;11(Suppl 2)782-E:A1770) are supportive of a “pulse-prime” mechanism, whereby Decoy bacteria produce transient, but broad innate and adaptive immune activation. We have now analyzed immune activation by Decoy bacteria in vitro using human peripheral blood mononuclear cells (PBMCs). Decoy10 enhanced polarization of CD14+ monocytes to M1 macrophages and inhibited Treg polarization. Decoy10 enhanced monocyte-derived dendritic cell maturation and enhanced Th1, Th2 and Th17 CD4+ polarization, with Th1 at 10-100-fold lower Decoy10 concentrations than Th2 or Th17. Decoy10 also activated NKT and T cells, and enhanced PBMC-mediated tumor cell killing. The results demonstrate that Decoy bacteria stimulate polarization, maturation, or activation of cellular mediators of innate and adaptive anti-tumor immune responses, inhibit an immune-suppressive mechanism, and enhance immune cell killing of tumor cells. The results are consistent with and significantly extend our previously reported in vivo data. Citation Format: Michael J. Newman, Deepak Singh. Mechanisms of action of a killed, bacteria-based, multiple immune receptor agonist in development for pulsed anti-tumor immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6639.

Abstract 5522: Elucidating the role of P-selectin/P-selectin ligand-1 axis in lung cancer brain metastasis

Abstract Lung cancer (LC) stands as a prominent cause of cancer-related mortality. Brain metastasis (BM) is a common occurrence in LC, constituting nearly 50% of all BM cases with the brain emerging as the preferred site for LC metastasis. Unfortunately, current LCBM treatment options yield limited success, as reflected in the median survival of BM patients at 7.8 months. Consequently, there is an imperative need for novel and more efficacious therapeutic approaches targeting LCBM. Evidence suggests that brain microenvironment cells, particularly microglia and tumor-associated macrophages (TAMs), play a pivotal role in facilitating the colonization and growth of LCBM. Previous investigations from our lab have identified the involvement of the P-selectin/P-selectin glycoprotein ligand-1 (SELP/PSGL-1) axis in microglia/TAMs immunophenotypes, promoting pro-tumorigenic activity. This led us to explore the role of the SELP/PSGL-1 axis in the interactions between LCBM cells and the brain microenvironment1. Thus, we conducted Immunostaining on human tissue samples, revealing increased expression of SELP, PSGL-1, PD-1, PD-L1, IBA-1, and CD68 in LCBM tissues. Simultaneously, we employed advanced 3D models to investigate the intricate interactions between cancer cells and microglia/TAMs. Actively inhibiting the SELP/PSGL-1 axis with pharmacological agents led to a significant decrease in migration and invasion when LC cells were co-cultured with microglia and TAMs. Moreover, SELP inhibition prompted a shift in microglia/TAMs state towards an M1-like phenotype, enhancing the ability of CD4/CD8 cells to target cancer cells in our unique 3D LCBM models. This effect was further enhanced when co-treated with a PD-1 inhibitor, demonstrating a potential synergistic impact in targeting both pathways for therapeutic intervention. Our results suggest that combining SELP inhibition with other immunomodulators, including a small molecule PD-L1 inhibitor developed in our lab2, could synergistically enhance both innate and adaptive anti-tumor immune responses. Taken together, this study may enhance our understanding of LCBM immunogenicity and contribute to the development of novel immunotherapeutic approaches that can improve patient outcomes.1.Yeini, E., et al. P-selectin axis plays a key role in microglia immunophenotype and glioblastoma progression. Nat Commun 12, 1912 (2021). 2. Acúrcio, R.C., et al. Therapeutic targeting of PD-1/PD-L1 blockade by novel small-molecule inhibitors recruits cytotoxic T cells into solid tumor microenvironment. J Immunother Cancer 10(2022). Citation Format: Koren Salomon, Eilam Yeini, Neta Frommer, Irisi Kamer, Jair Bar, Iris Barshack, Ronit Satchi-Fainaro. Elucidating the role of P-selectin/P-selectin ligand-1 axis in lung cancer brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5522.

Effects of a humanized CD47 antibody and recombinant SIRPα proteins on triple negative breast carcinoma stem cells.

Signal regulatory protein-α (SIRPα, SHPS-1, CD172a) expressed on myeloid cells transmits inhibitory signals when it engages its counter-receptor CD47 on an adjacent cell. Elevated CD47 expression on some cancer cells thereby serves as an innate immune checkpoint that limits phagocytic clearance of tumor cells by macrophages and antigen presentation to T cells. Antibodies and recombinant SIRPα constructs that block the CD47-SIRPα interaction on macrophages exhibit anti-tumor activities in mouse models and are in ongoing clinical trials for treating several human cancers. Based on prior evidence that engaging SIRPα can also alter CD47 signaling in some nonmalignant cells, we compared direct effects of recombinant SIRPα-Fc and a humanized CD47 antibody that inhibits CD47-SIRPα interaction (CC-90002) on CD47 signaling in cancer stem cells derived from the MDA-MB- 231 triple-negative breast carcinoma cell line. Treatment with SIRPα-Fc significantly increased the formation of mammospheres by breast cancer stem cells as compared to CC-90002 treatment or controls. Furthermore, SIRPα-Fc treatment upregulated mRNA and protein expression of ALDH1 and altered the expression of genes involved in epithelial/mesenchymal transition pathways that are associated with a poor prognosis and enhanced metastatic activity. This indicates that SIRPα-Fc has CD47-mediated agonist activities in breast cancer stem cells affecting proliferation and metastasis pathways that differ from those of CC-90002. This SIRPα-induced CD47 signaling in breast carcinoma cells may limit the efficacy of SIRPα decoy therapeutics intended to stimulate innate antitumor immune responses.

Engineered Bacterial Biomimetic Vesicles Reprogram Tumor-Associated Macrophages and Remodel Tumor Microenvironment to Promote Innate and Adaptive Antitumor Immune Responses.

Tumor-associated macrophages (TAMs) are among the most abundant infiltrating leukocytes in the tumor microenvironment (TME). Reprogramming TAMs from protumor M2 to antitumor M1 phenotype is a promising strategy for remodeling the TME and promoting antitumor immunity; however, the development of an efficient strategy remains challenging. Here, a genetically modified bacterial biomimetic vesicle (BBV) with IFN-γ exposed on the surface in a nanoassembling membrane pore structure was constructed. The engineered IFN-γ BBV featured a nanoscale structure of protein and lipid vesicle, the existence of rich pattern-associated molecular patterns (PAMPs), and the costimulation of introduced IFN-γ molecules. In vitro, IFN-γ BBV reprogrammed M2 macrophages to M1, possibly through NF-κB and JAK-STAT signaling pathways, releasing nitric oxide (NO) and inflammatory cytokines IL-1β, IL-6, and TNF-α and increasing the expression of IL-12 and iNOS. In tumor-bearing mice, IFN-γ BBV demonstrated a targeted enrichment in tumors and successfully reprogrammed TAMs into the M1 phenotype; notably, the response of antigen-specific cytotoxic T lymphocyte (CTL) in TME was promoted while the immunosuppressive myeloid-derived suppressor cell (MDSC) was suppressed. The tumor growth was found to be significantly inhibited in both a TC-1 tumor and a CT26 tumor. It was indicated that the antitumor effects of IFN-γ BBV were macrophage-dependent. Further, the modulation of TME by IFN-γ BBV produced synergistic effects against tumor growth and metastasis with an immune checkpoint inhibitor in an orthotopic 4T1 breast cancer model which was insensitive to anti-PD-1 mAb alone. In conclusion, IFN-γ-modified BBV demonstrated a strong capability of efficiently targeting tumor and tuning a cold tumor hot through reprogramming TAMs, providing a potent approach for tumor immunotherapy.

Abstract A013: Selective HDAC6 inhibition by SP-2-225 enhances the innate anti-tumor immune response after radiation exposure

Abstract Circulating tumor DNA fragments and other antigens resulting from radiation therapy cause activation of the STING pathway and initiate an inflammatory anti-tumor immune response. However, when pro-inflammatory immune cells interact with the tumor microenvironment, they are rapidly converted to their anti-inflammatory, pro-tumor state associated with wound healing. HDAC6 presents an important target due to its role in immune regulatory processes involved in antitumor immune responses. We have shown that SP-2-225, a selective inhibitor of HDAC6, regulates macrophages to recognize, phagocyte, and present antigens to the adaptive immune system. Treatment with SP-2-225 enhances the production of cancer-associated antigens and macrophage antigen cross-presentation to T cells. Through a non-cytotoxic mechanism, SP-2-225 significantly reduced the tumor volume in syngeneic SM1 melanoma and 4T1 breast models. Tumor analysis from treated animals showed a shift in the M1/M2 ratio of the infiltrating macrophages as compared to controls, demonstrating that SP-2-225 caused a pro-inflammatory, antitumor shift in the macrophage equilibrium present in the tumor microenvironment. The putative mechanism was identified through adoptive cell therapy where macrophages from naive animals were harvested and treated ex-vivo with SP-2-225. Reimplantation of these SP-2-225 ex-vivo treated macrophages resulted in reduced tumor volumes when compared to both vehicle and untreated macrophage animal cohorts. We propose that this selective inhibitor of HDAC6 offers potential in combination with radiation therapy for cancer. Citation Format: Scott Grindrod, Satish Noonepalle, Xingtang Li, David Quiceno, Mira Jung, Anatoly Dritschilo, Alejandro Villagra. Selective HDAC6 inhibition by SP-2-225 enhances the innate anti-tumor immune response after radiation exposure [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A013.

Impaired activation of plasmacytoid dendritic cells via toll-like receptor 7/9 and STING is mediated by melanoma-derived immunosuppressive cytokines and metabolic drift.

Plasmacytoid dendritic cells (pDCs) infiltrate a large set of human cancers. Interferon alpha (IFN-α) produced by pDCs induces growth arrest and apoptosis in tumor cells and modulates innate and adaptive immune cells involved in anti-cancer immunity. Moreover, effector molecules exert tumor cell killing. However, the activation state and clinical relevance of pDCs infiltration in cancer is still largely controversial. In Primary Cutaneous Melanoma (PCM), pDCs density decreases over disease progression and collapses in metastatic melanoma (MM). Moreover, the residual circulating pDC compartment is defective in IFN-α production. The activation of tumor-associated pDCs was evaluated by in silico and microscopic analysis. The expression of human myxovirus resistant protein 1 (MxA), as surrogate of IFN-α production, and proximity ligation assay (PLA) to test dsDNA-cGAS activation were performed on human melanoma biopsies. Moreover, IFN-α and CXCL10 production by in vitro stimulated (i.e. with R848, CpG-A, ADU-S100) pDCs exposed to melanoma cell lines supernatants (SN-mel) was tested by intracellular flow cytometry and ELISA. We also performed a bulk RNA-sequencing on SN-mel-exposed pDCs, resting or stimulated with R848. Glycolytic rate assay was performed on SN-mel-exposed pDCs using the Seahorse XFe24 Extracellular Flux Analyzer. Based on a set of microscopic, functional and in silico analyses, we demonstrated that the melanoma milieu directly impairs IFN-α and CXCL10 production by pDCs via TLR-7/9 and cGAS-STING signaling pathways. Melanoma-derived immunosuppressive cytokines and a metabolic drift represent relevant mechanisms enforcing pDC-mediated melanoma escape. These findings propose a new window of intervention for novel immunotherapy approaches to amplify the antitumor innate immune response in cutaneous melanoma (CM).

IMM01 Plus Tislelizumab in Prior Anti-PD-1 Failed Classic Hodgkin Lymphoma: An Open Label, Multicenter, Phase 2 Study (IMM01-04) Evaluating Safety As Well As Preliminary Anti-Tumor Activity

Background: IMM01, a recombinant SIRPa-Fc fusion protein, can activate macrophages to enhance anti-tumor activity by blocking CD47-SIRPa interaction. IMM01 showed unique property of weak human erythrocyte conjugation in preclinical studies, and low incidence of anemia in early clinical trials with no need for a priming dose. IMM01 plus tislelizumab has the potential to augment both innate and adaptive anti-tumor immune responses. Methods: IMM01 at a dose of 2.0mg/kg was administered once a week and tislelizumab (200mg) was administered once every 3 weeks. 2.0mg/kg of IMM01 was determined as RP2D when combo with tislelizumab in patients with advanced solid tumors (e.g. SCLC, NSCLC, HNSCC). The primary objective of this phase 2 study was to evaluate the preliminary anti-tumor activity as well as safety of IMM01 plus tislelizumab in patients with classic Hodgkin lymphoma (cHL) who have failed prior check point inhibitor treatment. A total of 32 patients are planned to be enrolled in this phase 2 study according to pre-plan statistical assumption. Adverse events (AEs) were reported according to CTCAE v5.0. The anti-tumor activity was assessed according to the Lugano 2014 criteria. Results: As of 19 Jul 2023, 20 cHL patients (median age: 34 years, range: 23-73 years) who have failed prior anti-PD-1 treatment were enrolled, 18 patients were relapsed on anti-PD1, and 2 patients were anti-PD1 refractory. 18 (90%) patients received 3 or more prior lines of therapy. All patients had Ann Arbor stage III-IV disease. Among total 14 evaluable patients, 2 patients achieved complete response (CR), 7 patients achieved partial response (PR). The objective response rate (ORR) and disease control rate (DCR) were 64.3% and 100%, respectively. Among the responders, 5 patients failed prior tislelizumab. As of 7 Jul 2023, for all 20 treated patients, treatment-related adverse events (TRAEs) of any grade occurred in 18 (90.0%) patients. The most frequent TRAEs (≥ 20%) were platelet count decreased (50.0%), lymphocyte count decreased (35.0%), anemia (30.0%), white blood count decreased (30.0%), neutrophil count decreased (25.0%) and Gamma-glutamyl transferase increased (20.0%). Most TRAEs were grade 1-2. TRAEs of grade ≥3 occurred in 7 (35%) patients, including lymphocyte count decreased (30.0%), platelet count decreased (10.0%), neutrophil count decreased (5.0%) and white blood count decreased (5.0%). 1 patient had treatment related SAE (platelet count decreased, grade 3) and recovered without bleeding. There was no reported hemolytic anemia or hemolysis in any of the patients. No TRAEs leading to drug discontinuation or death. Conclusions: IMM01-04 showed a robust anti-tumor effectivity with a well-tolerated safety profile in prior anti-PD-1 failed classical Hodgkin Lymphoma patients. The phase 2 study is ongoing.

Small molecule inhibitors for cancer immunotherapy and associated biomarkers - the current status.

Following the success of cancer immunotherapy using large molecules against immune checkpoint inhibitors, the concept of using small molecules to interfere with intracellular negative regulators of anti-tumor immune responses has emerged in recent years. The main targets for small molecule drugs currently include enzymes of negative feedback loops in signaling pathways of immune cells and proteins that promote immunosuppressive signals within the tumor microenvironment. In the adaptive immune system, negative regulators of T cell receptor signaling (MAP4K1, DGKα/ζ, CBL-B, PTPN2, PTPN22, SHP1), co-receptor signaling (CBL-B) and cytokine signaling (PTPN2) have been preclinically validated as promising targets and initial clinical trials with small molecule inhibitors are underway. To enhance innate anti-tumor immune responses, inhibitory immunomodulation of cGAS/STING has been in the focus, and inhibitors of ENPP1 and TREX1 have reached the clinic. In addition, immunosuppressive signals via adenosine can be counteracted by CD39 and CD73 inhibition, while suppression via intratumoral immunosuppressive prostaglandin E can be targeted by EP2/EP4 antagonists. Here, we present the status of the most promising small molecule drug candidates for cancer immunotherapy, all residing relatively early in development, and the potential of relevant biomarkers.

IL-1β+ Tumor Associated Macrophages Induced by Type I Interferon Initiates Radiotherapy-Mediated Abscopal Effect

Both experimental and clinical studies have proved that radiotherapy can lead to not only local control of irradiated tumors, but also tumor regression of non-irradiated area, termed as abscopal effect. Abscopal effect has been attributed to radiotherapy-induced innate and adaptive anti-tumor immune responses. However, the detailed molecular mechanisms and key cellular components are still unclear. So, the purpose of this study is to reveal the key molecular mechanisms and essential cells in inducing abscopal effect. Bilateral MC38 subcutaneous tumor mouse models were established, and primary tumors were subjected to one fraction of 15Gy. CD45+ cells were sorted from both primary and abscopal tumors 3-, 7-, 17- and 24-day post radiotherapy respectively and subjected to single-cell RNA sequencing followed by standard bioinformatic analysis with R studio. To verify the findings, flow cytometry, mIHC and real-time qPCR were carried out to analyze the cellular and molecular components in tumor microenvironments. Cellular depletion experiments and conditional knockout mice were finally employed to confirm key mechanisms that contribute to abscopal effect. The primary and abscopal immune microenvironments showed different repertoires time-dependently. Radiotherapy induced durable type I interferon (IFN-I) responses with dramatic infiltration and activation of CD8+ T cells and tumor associated macrophages (TAMs) in primary tumors. However, in abscopal tumors, we found that TAMs rather than CD8+ T cells were the predominant population activated in early stages (3- and 7-day post radiotherapy), and functional CD8+ T cells enriched until late stages (24-day post radiotherapy). Thorough analysis of scRNA-seq and experimental validation discovered a unique subset of TAMs characterized by high expression of IL-1β emerged as early as 3-day post radiotherapy in both primary and secondary tumor immune microenvironments, termed as IL-1β+ TAMs herein. IL-1β+ TAMs were the main activated component in abscopal tumors in early stage, and strongest respondent to IFN-I pathway. Abscopal effect was significantly attenuated when IFN-I response was abolished in either primary or abscopal tumors or TAMs were depleted from abscopal tumors. Our data identified a subset of immune cells, IL-1β+ TAMs, and IFN-I as the essential cellular and molecular components that contribute to abscopal effect. Mechanically, radiotherapy-induced dramatical IFN-I response in primary tumors lead to enrichment of IL-1β+ TAMs, which initiated the anti-tumor immune response in abscopal tumors. All these findings provided theoretical basis for understanding and improving radiotherapy-induced abscopal effect.

Nanoscale coordination polymer synergizes photodynamic therapy and toll-like receptor activation for enhanced antigen presentation and antitumor immunity

While activating antitumor immunity with toll-like receptor (TLR) agonists provides a promising approach toward cancer immunotherapy, existing TLR agonists, including resiquimod (R848), have shown poor tumor selectivity and ineffective TLR activation in tumors for optimal antitumor effects. We hypothesized that improved delivery of TLR agonists to tumors and their effective combination with tumor antigens could significantly enhance their antitumor efficacy. Here, we report a novel nanoscale coordination polymer, Ce6/R848, for the co-delivery of Ce6 photosensitizer to elicit immunogenic cell death via photodynamic therapy (PDT) and cholesterol-conjugated R848 (Chol-R848) for tumor-selective TLR7/8 activation. Upon light irradiation, Ce6-mediated PDT released tumor antigens while selectively delivered R848 activated TLR7/8 in the tumors to synergistically activate antigen-presenting cells and prime T cells for enhanced innate and adaptive antitumor immune responses. Ce6/R848 achieved a 50% cure rate and 99.4% inhibition of tumor growth in subcutaneous MC38 colorectal tumors with minimal systemic toxicity.

Engineered Bacteria Labeled with Iridium(III) Photosensitizers for Enhanced Photodynamic Immunotherapy of Solid Tumors.

Despite metal-based photosensitizers showing great potential in photodynamic therapy for tumor treatment, the application of the photosensitizers is intrinsically limited by their poor cancer-targeting properties. Herein, we reported a metal-based photosensitizer-bacteria hybrid, Ir-HEcN, via covalent labeling of an iridium(III) photosensitizer to the surface of genetically engineered bacteria. Due to its intrinsic self-propelled motility and hypoxia tropism, Ir-HEcN selectively targets and penetrates deeply into tumor tissues. Importantly, Ir-HEcN is capable of inducing pyroptosis and immunogenic cell death of tumor cells under irradiation, thereby remarkably evoking anti-tumor innate and adaptive immune responses in vivo and leading to the regression of solid tumors via combinational photodynamic therapy and immunotherapy. To the best of our knowledge, Ir-HEcN is the first metal complex decorated bacteria for enhanced photodynamic immunotherapy.

Innate immune response restarts adaptive immune response in tumors.

The imbalance of immune response plays a crucial role in the development of diseases, including glioblastoma. It is essential to comprehend how the innate immune system detects tumors and pathogens. Endosomal and cytoplasmic sensors can identify diverse cancer cell antigens, triggering the production of type I interferon and pro-inflammatory cytokines. This, in turn, stimulates interferon stimulating genes, enhancing the presentation of cancer antigens, and promoting T cell recognition and destruction of cancer cells. While RNA and DNA sensing of tumors and pathogens typically involve different receptors and adapters, their interaction can activate adaptive immune response mechanisms. This review highlights the similarity in RNA and DNA sensing mechanisms in the innate immunity of both tumors and pathogens. The aim is to enhance the anti-tumor innate immune response, identify regions of the tumor that are not responsive to treatment, and explore new targets to improve the response to conventional tumor therapy and immunotherapy.

Breaking Tumor Immunosuppressive Network by Regulating Multiple Nodes with Triadic Drug Delivery Nanoparticles.

Inside the tumor microenvironment, a complicated immunosuppressive network is constituted by tumor cells and suppressive immune cells as its nodes, including myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and regulatory T cells, which have mutual promotion on each other and superimposed inhibition on natural killer (NK) cells and cytotoxic T cells. Breaking the whole balance of this web is critical to tumor immunotherapy since modulation on a single node may be diluted by other factors in the network. To achieve multifaceted regulation on antitumor immunity against triple-negative breast cancer, in this work, a micelle, termed BEM, co-delivering the MDSC inhibitor, entinostat (ENT), and the immune checkpoint inhibitor, BMS-1, was constructed with pH-sensitive amphiphilic poly(β-amino ester) derivatives. Then, BEM and the scavenger receptor A (SR-A) ligand dextran sulfate (DXS) formed a negatively charged nanoparticle (BEN). DXS detached from BEN in the weakly acidic tumor microenvironment and blocked SR-A on TAMs, reprogramming TAMs toward the M1 type. The positively charged BEM with facilitated intratumoral penetration and cellular uptake dissociated in the lysosomes, accompanied by the release of ENT and BMS-1 to suppress MDSCs and block the programmed cell death protein (PD)-1/PD-ligand 1 pathway, respectively. As a result, NK cells and CD8+ T cells in tumors were increased, as were their effector cytokines. The activated innate and adaptive antitumor immune responses suppressed the growth and metastasis of tumors and prolonged survival of 4T1 tumor-bearing mice. BEN provides a reliable approach for improving cancer immunotherapy by destroying the immunosuppression web in tumors via multinode regulation.

The effect of m2 peptide targeted nanoliposomes containing crocin on induction of phenotypic change in tumor macrophages to M1 state

AimsCrocin has immunomodulatory and anticancer effects. In this study, crocin was used to induce the M1 phenotype in mouse tumor macrophages. Main methodsA targeted liposomal formulation with m2 peptide was prepared and characterized to deliver crocin to the M2 macrophages present in the tumor environment. RT-qPCR and IHC were performed for in vitro and in vivo (in C26 colon carcinoma mouse model at a dose of 50 mg/kg) assessment of M1 induction, respectively. Key findingsIn vitro results indicated that liposome modified with m2 peptide was non-toxic to macrophages and had an improved uptake by macrophages compared to the non-targeted formulation and induced M1 phenotype through an IL6-independent pathway. M2 peptide- modified liposome showed considerable tumor accumulation and anti-tumor effects and significantly shifted the phenotype of tumor macrophages towards an anti-tumor M1 phenotype. SignificanceProbably the remarkable anti-tumor responses observed in this study with m2 peptide-targeted liposomal formulations containing crocin were due to the enhanced delivery of crocin to the tumor macrophage and the subsequent initiation of anti-tumor immune responses.

DIPG-05. EVALUATION OF TELOMERE-MODIFYING COMPOUND THIO AS A RADIOSENSITIZER AND IMMUNOMODULATOR TO TREAT DIPG

Abstract Diffuse intrinsic pontine glioma (DIPG) is one of the most aggressive tumors of the central nervous system in children. Radiotherapy remains the only standard treatment but is rarely curative. In recent years, several immunotherapy strategies have emerged as an option to treat DIPG. However, the low mutational burden and rare infiltration of T lymphocytes, render these tumors immunologically “cold” and therefore pose challenges for general immunotherapy. We have previously reported in pediatric brain tumors the preclinical validation of THIO (6-thio-2’-deoxyguanosine), a telomerase substrate precursor analog. Treatment of cancer cells with THIO caused both telomeric and genomic DNA damage and rapid cell death. In vivo treatment delayed tumor growth in mouse models of high-risk medulloblastoma and DIPG. Importantly, THIO crosses the blood-brain barrier and specifically targets tumor cells in an orthotopic mouse model of DIPG. The cGAS-STING pathway is a cellular mechanism that senses cytosolic double stranded DNA, stimulating type I interferons (IFNs) and IFN-stimulated genes, thereby activating the innate and adaptive anti-tumor immune responses. Micronuclei resulting from cell division in the presence of unresolved DNA damage, has been shown to be recognized by the cGAS-STING pathway. THIO was shown to induce anti-tumor immunity through micronuclei formation in colon and lung tumor models. We hypothesized that THIO treatment will activate anti-tumor immunity in DIPG. Our results indicate that THIO treatment sensitized DIPG cells to ionizing radiation (IR) leading to a significant decrease in DIPG cell proliferation. We are currently testing this combination in vivo in an orthotopic mouse model of DIPG. We will present results on the potential of THIO to activate anti-tumor immunity through the activation of Sting pathway in a syngeneic mouse model of DIPG. These preclinical studies will support potential use of THIO in combination with IR to treat children with high-risk pediatric brain tumors.