Tigilanol tiglate is an oncolytic small molecule that induces immunogenic cell death and enhances the response of both target and non-injected tumors to immune checkpoint blockade.

BACKGROUND: Tigilanol tiglate (TT) is a protein kinase C (PKC)/C1 domain activator currently being developed as an intralesional agent for the treatment of various (sub)cutaneous malignancies. Previous work has shown that intratumoral (I.T.) injection of TT causes vascular disruption with concomitant tumor ablation in several preclinical models of cancer, in addition to various (sub)cutaneous tumors presenting in the veterinary clinic. TT has completed Phase I dose escalation trials, with some patients showing signs of abscopal effects. However, the exact molecular details underpinning its mechanism of action (MoA), together with its immunotherapeutic potential in oncology remain unclear. METHODS: A combination of microscopy, luciferase assays, immunofluorescence, immunoblotting, subcellular fractionation, intracellular ATP assays, phagocytosis assays and mixed lymphocyte reactions were used to probe the MoA of TT in vitro. In vivo studies with TT used MM649 xenograft, CT-26 and immune checkpoint inhibitor refractory B16-F10-OVA tumor bearing mice, the latter with or without anti-programmed cell death 1 (PD-1)/anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) mAb treatment. The effect of TT at injected and non-injected tumors was also assessed. RESULTS: Here, we show that TT induces the death of endothelial and cancer cells at therapeutically relevant concentrations via a caspase/gasdermin E-dependent pyroptopic pathway. At therapeutic doses, our data demonstrate that TT acts as a lipotoxin, binding to and promoting mitochondrial/endoplasmic reticulum (ER) dysfunction (leading to unfolded protein responsemt/ER upregulation) with subsequent ATP depletion, organelle swelling, caspase activation, gasdermin E cleavage and induction of terminal necrosis. Consistent with binding to ER membranes, we found that TT treatment promoted activation of the integrated stress response together with the release/externalization of damage-associated molecular patterns (HMGB1, ATP, calreticulin) from cancer cells in vitro and in vivo, characteristics indicative of immunogenic cell death (ICD). Confirmation of ICD in vivo was obtained through vaccination and rechallenge experiments using CT-26 colon carcinoma tumor bearing mice. Furthermore, TT also reduced tumor volume, induced immune cell infiltration, as well as improved survival in B16-F10-OVA tumor bearing mice when combined with immune checkpoint blockade. CONCLUSIONS: These data demonstrate that TT is an oncolytic small molecule with multiple targets and confirms that cell death induced by this compound has the potential to augment antitumor responses to immunotherapy.

Respiratory complex I regulates dendritic cell maturation in explant model of human tumor immune microenvironment.

BACKGROUND: Combining cytotoxic chemotherapy or novel anticancer drugs with T-cell modulators holds great promise in treating advanced cancers. However, the response varies depending on the tumor immune microenvironment (TIME). Therefore, there is a clear need for pharmacologically tractable models of the TIME to dissect its influence on mono- and combination treatment response at the individual level. METHODS: Here we establish a patient-derived explant culture (PDEC) model of breast cancer, which retains the immune contexture of the primary tumor, recapitulating cytokine profiles and CD8+T cell cytotoxic activity. RESULTS: We explored the immunomodulatory action of a synthetic lethal BCL2 inhibitor venetoclax+metformin drug combination ex vivo, discovering metformin cannot overcome the lymphocyte-depleting action of venetoclax. Instead, metformin promotes dendritic cell maturation through inhibition of mitochondrial complex I, increasing their capacity to co-stimulate CD4+T cells and thus facilitating antitumor immunity. CONCLUSIONS: Our results establish PDECs as a feasible model to identify immunomodulatory functions of anticancer drugs in the context of patient-specific TIME.

Tetracyclines enhance antitumor T-cell immunity via the Zap70 signaling pathway.

BACKGROUND: Cancer immunotherapy including immune checkpoint inhibitors is only effective for a limited population of patients with cancer. Therefore, the development of novel cancer immunotherapy is anticipated. In preliminary studies, we demonstrated that tetracyclines enhanced T-cell responses. Therefore, we herein investigated the efficacy of tetracyclines on antitumor T-cell responses by human peripheral T cells, murine models, and the lung tumor tissues of patients with non-small cell lung cancer (NSCLC), with a focus on signaling pathways in T cells. METHODS: The cytotoxicity of peripheral and lung tumor-infiltrated human T cells against tumor cells was assessed by using bispecific T-cell engager (BiTE) technology (BiTE-assay system). The effects of tetracyclines on T cells in the peripheral blood of healthy donors and the tumor tissues of patients with NSCLC were examined using the BiTE-assay system in comparison with anti-programmed cell death-1 (PD-1) antibody, nivolumab. T-cell signaling molecules were analyzed by flow cytometry, ELISA, and qRT-PCR. To investigate the in vivo antitumor effects of tetracyclines, tetracyclines were administered orally to BALB/c mice engrafted with murine tumor cell lines, either in the presence or absence of anti-mouse CD8 inhibitors. RESULTS: The results obtained revealed that tetracyclines enhanced antitumor T-cell cytotoxicity with the upregulation of granzyme B and increased secretion of interferon-γ in human peripheral T cells and the lung tumor tissues of patients with NSCLC. The analysis of T-cell signaling showed that CD69 in both CD4+ and CD8+ T cells was upregulated by minocycline. Downstream of T-cell receptor signaling, Zap70 phosphorylation and Nur77 were also upregulated by minocycline in the early phase after T-cell activation. These changes were not observed in T cells treated with anti-PD-1 antibodies under the same conditions. The administration of tetracyclines exhibited antitumor efficacy with the upregulation of CD69 and increases in tumor antigen-specific T cells in murine tumor models. These changes were canceled by the administration of anti-mouse CD8 inhibitors. CONCLUSIONS: In conclusion, tetracyclines enhanced antitumor T-cell immunity via Zap70 signaling. These results will contribute to the development of novel cancer immunotherapy.

Targeting PARG induces tumor cell growth inhibition and antitumor immune response by reducing phosphorylated STAT3 in ovarian cancer.

BACKGROUND: Ovarian cancer is the most lethal gynecological malignancy, with limited treatment options after failure of standard therapies. Despite the potential of poly(ADP-ribose) polymerase inhibitors in treating DNA damage response (DDR)-deficient ovarian cancer, the development of resistance and immunosuppression limit their efficacy, necessitating alternative therapeutic strategies. Inhibitors of poly(ADP-ribose) glycohydrolase (PARG) represent a novel class of inhibitors that are currently being assessed in preclinical and clinical studies for cancer treatment. METHODS: By using a PARG small-molecule inhibitor, COH34, and a cell-penetrating antibody targeting the PARG's catalytic domain, we investigated the effects of PARG inhibition on signal transducer and activator of transcription 3 (STAT3) in OVCAR8, PEO1, and Brca1-null ID8 ovarian cancer cell lines, as well as in immune cells. We examined PARG inhibition-induced effects on STAT3 phosphorylation, nuclear localization, target gene expression, and antitumor immune responses in vitro, in patient-derived tumor organoids, and in an immunocompetent Brca1-null ID8 ovarian mouse tumor model that mirrors DDR-deficient human high-grade serous ovarian cancer. We also tested the effects of overexpressing a constitutively activated STAT3 mutant on COH34-induced tumor cell growth inhibition. RESULTS: Our findings show that PARG inhibition downregulates STAT3 activity through dephosphorylation in ovarian cancer cells. Importantly, overexpression of a constitutively activated STAT3 mutant in tumor cells attenuates PARG inhibitor-induced growth inhibition. Additionally, PARG inhibition reduces STAT3 phosphorylation in immune cells, leading to the activation of antitumor immune responses, shown in immune cells cocultured with ovarian cancer patient tumor-derived organoids and in immune-competent mice-bearing mouse ovarian tumors. CONCLUSIONS: We have identified a novel antitumor mechanism underlying PARG inhibition beyond its primary antitumor effects through blocking DDR in ovarian cancer. Furthermore, targeting PARG activates antitumor immune responses, thereby potentially increasing response rates to immunotherapy in patients with ovarian cancer.

Dual molecule targeting HDAC6 leads to intratumoral CD4+ cytotoxic lymphocytes recruitment through MHC-II upregulation on lung cancer cells.

BACKGROUND: Despite the current therapeutic treatments including surgery, chemotherapy, radiotherapy and more recently immunotherapy, the mortality rate of lung cancer stays high. Regarding lung cancer, epigenetic modifications altering cell cycle, angiogenesis and programmed cancer cell death are therapeutic targets to combine with immunotherapy to improve treatment success. In a recent study, we uncovered that a molecule called QAPHA ((E)-3-(5-((2-cyanoquinolin-4-yl)(methyl)amino)-2-methoxyphenyl)-N-hydroxyacrylamide) has a dual function as both a tubulin polymerization and HDAC inhibitors. Here, we investigate the impact of this novel dual inhibitor on the immune response to lung cancer. METHODS: To elucidate the mechanism of action of QAPHA, we conducted a chemical proteomics analysis. Using an in vivo mouse model of lung cancer (TC-1 tumor cells), we assessed the effects of QAPHA on tumor regression. Tumor infiltrating immune cells were characterized by flow cytometry. RESULTS: In this study, we first showed that QAPHA effectively inhibited histone deacetylase 6, leading to upregulation of HSP90, cytochrome C and caspases, as revealed by proteomic analysis. We confirmed that QAPHA induces immunogenic cell death (ICD) by expressing calreticulin at cell surface in vitro and demonstrated its efficacy as a vaccine in vivo. Remarkably, even at a low concentration (0.5 mg/kg), QAPHA achieved complete tumor regression in approximately 60% of mice treated intratumorally, establishing a long-lasting anticancer immune response. Additionally, QAPHA treatment promoted the infiltration of M1-polarized macrophages in treated mice, indicating the induction of a pro-inflammatory environment within the tumor. Very interestingly, our findings also revealed that QAPHA upregulated major histocompatibility complex class II (MHC-II) expression on TC-1 tumor cells both in vitro and in vivo, facilitating the recruitment of cytotoxic CD4+T cells (CD4+CTL) expressing CD4+, NKG2D+, CRTAM+, and Perforin+. Finally, we showed that tumor regression strongly correlates to MHC-II expression level on tumor cell and CD4+ CTL infiltrate. CONCLUSION: Collectively, our findings shed light on the discovery of a new multitarget inhibitor able to induce ICD and MHC-II upregulation in TC-1 tumor cell. These two processes participate in enhancing a specific CD4+ cytotoxic T cell-mediated antitumor response in vivo in our model of lung cancer. This breakthrough suggests the potential of QAPHA as a promising agent for cancer treatment.

Efficacy of LCMV-based cancer immunotherapies is unleashed by intratumoral injections of polyI:C.

BACKGROUND: Lymphocytic choriomeningitis virus (LCMV) belongs to the Arenavirus family known for inducing strong cytotoxic T-cell responses in both mice and humans. LCMV has been engineered for the development of cancer immunotherapies, currently undergoing evaluation in phase I/II clinical trials. Initial findings have demonstrated safety and an exceptional ability to activate and expand tumor-specific T lymphocytes. Combination strategies to maximize the antitumor effectiveness of LCMV-based immunotherapies are being explored. METHODS: We assessed the antitumor therapeutic effects of intratumoral administration of polyinosinic:polycytidylic acid (poly(I:C)) and systemic vaccination using an LCMV-vector expressing non-oncogenic versions of the E6 and E7 antigens of human papillomavirus 16 (artLCMV-E7E6) in a bilateral model engrafting TC-1/A9 cells. This cell line, derived from the parental TC-1, exhibits low MHC class I expression and is highly immune-resistant. The mechanisms underlying the combination's efficacy were investigated through bulk RNA-seq, flow cytometry analyses of the tumor microenvironment, selective depletions using antibodies and clodronate liposomes, Batf3 deficient mice, and in vivo bioluminescence experiments. Finally, we assessed the antitumor effectiveness of the combination of artLCMV-E7E6 with BO-112, a GMP-grade poly(I:C) formulated in polyethyleneimine, currently under evaluation in clinical trials. RESULTS: Intratumoral injection of poly(I:C) enhanced the antitumor efficacy of artLCMV-E7E6 in both injected and non-injected tumor lesions. The combined treatment resulted in a significant delay in tumor growth and often complete eradication of several tumor lesions, leading to significantly improved survival compared with monotherapies. While intratumoral administration of poly(I:C) did not impact LCMV vector biodistribution or transgene expression, it significantly modified leucocyte infiltrates within the tumor microenvironment and amplified systemic efficacy through proinflammatory cytokines/chemokines such as CCL3, CCL5, CXCL10, TNF, IFNα, and IL12p70. Upregulation of MHC on tumor cells and a reconfiguration of the gene expression programs related to tumor vasculature, leucocyte migration, and the activation profile of tumor-infiltrating CD8+ T lymphocytes were observed. Indeed, the antitumor effect relied on the functions of CD8+ T lymphocytes and macrophages. The synergistic efficacy of the combination was further confirmed when BO-112 was included. CONCLUSION: Intratumoral injection of poly(I:C) sensitizes MHClow tumors to the antitumor effects of artLCMV-E7E6, resulting in a potent therapeutic synergy.

Network-based screening identifies sitagliptin as an antitumor drug targeting dendritic cells.

BACKGROUND: Dendritic cell (DC)-mediated antigen presentation is essential for the priming and activation of tumor-specific T cells. However, few drugs that specifically manipulate DC functions are available. The identification of drugs targeting DC holds great promise for cancer immunotherapy. METHODS: We observed that type 1 conventional DCs (cDC1s) initiated a distinct transcriptional program during antigen presentation. We used a network-based approach to screen for cDC1-targeting therapeutics. The antitumor potency and underlying mechanisms of the candidate drug were investigated in vitro and in vivo. RESULTS: Sitagliptin, an oral gliptin widely used for type 2 diabetes, was identified as a drug that targets DCs. In mouse models, sitagliptin inhibited tumor growth by enhancing cDC1-mediated antigen presentation, leading to better T-cell activation. Mechanistically, inhibition of dipeptidyl peptidase 4 (DPP4) by sitagliptin prevented the truncation and degradation of chemokines/cytokines that are important for DC activation. Sitagliptin enhanced cancer immunotherapy by facilitating the priming of antigen-specific T cells by DCs. In humans, the use of sitagliptin correlated with a lower risk of tumor recurrence in patients with colorectal cancer undergoing curative surgery. CONCLUSIONS: Our findings indicate that sitagliptin-mediated DPP4 inhibition promotes antitumor immune response by augmenting cDC1 functions. These data suggest that sitagliptin can be repurposed as an antitumor drug targeting DC, which provides a potential strategy for cancer immunotherapy.

BTN1A1 is a novel immune checkpoint mutually exclusive to PD-L1.

BACKGROUND: While Programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) blockade is a potent antitumor treatment strategy, it is effective in only limited subsets of patients with cancer, emphasizing the need for the identification of additional immune checkpoints. Butyrophilin 1A1 (BTN1A1) has been reported to exhibit potential immunoregulatory activity, but its ability to function as an immune checkpoint remains to be systematically assessed, and the mechanisms underlying such activity have yet to be characterized. METHODS: BTN1A1 expression was evaluated in primary tumor tissue samples, and its ability to suppress T-cell activation and T cell-dependent tumor clearance was examined. The relationship between BTN1A1 and PD-L1 expression was further characterized, followed by the development of a BTN1A1-specific antibody that was administered to tumor-bearing mice to test the amenability of this target to immune checkpoint inhibition. RESULTS: BTN1A1 was confirmed to suppress T-cell activation in vitro and in vivo. Robust BTN1A1 expression was detected in a range of solid tumor tissue samples, and BTN1A1 expression was mutually exclusive with that of PD-L1 as a consequence of its inhibition of Janus-activated kinase/signal transducer and activator of transcription signaling-induced PD-L1 upregulation. Antibody-mediated BTN1A1 blockade suppressed tumor growth and enhanced immune cell infiltration in syngeneic tumor-bearing mice. CONCLUSION: Together, these results confirm that the potential of BTN1A1 is a bona fide immune checkpoint and a viable immunotherapeutic target for the treatment of individuals with anti-PD-1/PD-L1 refractory or resistant disease, opening new avenues to improving survival outcomes for patients with a range of cancers.

Salmonella-mediated methionine deprivation drives immune activation and enhances immune checkpoint blockade therapy in melanoma.

BACKGROUND: Although immune checkpoint inhibitor (ICI)-based therapy is advantageous for patients with advanced melanoma, resistance and relapse are frequent. Thus, it is crucial to identify effective drug combinations and develop new therapies for the treatment of melanoma. SGN1, a genetically modified Salmonella typhimurium species that causes the targeted deprivation of methionine in tumor tissues, is currently under investigation in clinical trials. However, the inhibitory effect of SGN1 on melanoma and the benefits of SGN1 in combination with ICIs remain largely unexplored. Therefore, this study aims to investigate the antitumor potential of SGN1, and its ability to enhance the efficacy of antibody-based programmed cell death-ligand 1 (PD-L1) inhibitors in the treatment of murine melanoma. METHODS: The antitumor activity of SGN1 and the effect of SGN1 on the efficacy of PD-L1 inhibitors was studied through murine melanoma models. Further, The Cancer Genome Atlas-melanoma cohort was clustered using ConsensusClusterPlus based on the methionine deprivation-related genes, and immune characterization was performed using xCell, Microenvironment Cell Populations-counter, Estimation of Stromal and Immune cells in MAlignant Tumor tissues using Expression data, and immunophenoscore (IPS) analyses. The messenger RNA data on programmed death-1 (PD-1) immunotherapy response were obtained from the Gene Expression Omnibus database. Gene Set Enrichment Analysis of methionine deprivation-up gene set was performed to determine the differences between pretreatment responders and non-responders. RESULTS: This study showed that both, the intratumoral and the intravenous administration of SGN1 in subcutaneous B16-F10 melanomas, suppress tumor growth, which was associated with an activated CD8+T-cell response in the tumor microenvironment. Combination therapy of SGN1 with systemic anti-PD-L1 therapy resulted in better antitumor activity than the individual monotherapies, respectively, and the high therapeutic efficacy of the combination was associated with an increase in the systemic level of tumor-specific CD8+ T cells. Two clusters consisting of methionine deprivation-related genes were identified. Patients in cluster 2 had higher expression of methionine_deprivation_up genes, better clinical outcomes, and higher immune infiltration levels compared with patients in cluster 1. Western blot, IPS analysis, and immunotherapy cohort study revealed that methionine deficiency may show a better response to ICI therapy CONCLUSIONS：: This study reports Salmonella-based SGN1 as a potent anticancer agent against melanoma, and lays the groundwork for the potential synergistic effect of ICIs and SGN1 brought about by improving the immune microenvironment in melanomas.

Targeting the aryl hydrocarbon receptor (AhR) with BAY 2416964: a selective small molecule inhibitor for cancer immunotherapy.

BACKGROUND: The metabolism of tryptophan to kynurenines (KYN) by indoleamine-2,3-dioxygenase or tryptophan-2,3-dioxygenase is a key pathway of constitutive and adaptive tumor immune resistance. The immunosuppressive effects of KYN in the tumor microenvironment are predominantly mediated by the aryl hydrocarbon receptor (AhR), a cytosolic transcription factor that broadly suppresses immune cell function. Inhibition of AhR thus offers an antitumor therapy opportunity via restoration of immune system functions. METHODS: The expression of AhR was evaluated in tissue microarrays of head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). A structure class of inhibitors that block AhR activation by exogenous and endogenous ligands was identified, and further optimized, using a cellular screening cascade. The antagonistic properties of the selected AhR inhibitor candidate BAY 2416964 were determined using transactivation assays. Nuclear translocation, target engagement and the effect of BAY 2416964 on agonist-induced AhR activation were assessed in human and mouse cancer cells. The immunostimulatory properties on gene and cytokine expression were examined in human immune cell subsets. The in vivo efficacy of BAY 2416964 was tested in the syngeneic ovalbumin-expressing B16F10 melanoma model in mice. Coculture of human H1299 NSCLC cells, primary peripheral blood mononuclear cells and fibroblasts mimicking the human stromal-tumor microenvironment was used to assess the effects of AhR inhibition on human immune cells. Furthermore, tumor spheroids cocultured with tumor antigen-specific MART-1 T cells were used to study the antigen-specific cytotoxic T cell responses. The data were analyzed statistically using linear models. RESULTS: AhR expression was observed in tumor cells and tumor-infiltrating immune cells in HNSCC, NSCLC and CRC. BAY 2416964 potently and selectively inhibited AhR activation induced by either exogenous or endogenous AhR ligands. In vitro, BAY 2416964 restored immune cell function in human and mouse cells, and furthermore enhanced antigen-specific cytotoxic T cell responses and killing of tumor spheroids. In vivo, oral application with BAY 2416964 was well tolerated, induced a proinflammatory tumor microenvironment, and demonstrated antitumor efficacy in a syngeneic cancer model in mice. CONCLUSIONS: These findings identify AhR inhibition as a novel therapeutic approach to overcome immune resistance in various types of cancers.

Local and distant response to intratumoral immunotherapy assessed by immunoPET in mice.

BACKGROUND: Despite the promising efficacy of immune checkpoint blockers (ICB), tumor resistance and immune-related adverse events hinder their success in cancer treatment. To address these challenges, intratumoral delivery of immunotherapies has emerged as a potential solution, aiming to mitigate side effects through reduced systemic exposure while increasing effectiveness by enhancing local bioavailability. However, a comprehensive understanding of the local and systemic distribution of ICBs following intratumoral administration, as well as their impact on distant tumors, remains crucial for optimizing their therapeutic potential.To comprehensively investigate the distribution patterns following the intratumoral and intravenous administration of radiolabeled anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and to assess its corresponding efficacy in both injected and non-injected tumors, we conducted an immunoPET imaging study. METHODS: CT26 and MC38 syngeneic colorectal tumor cells were implanted subcutaneously on both flanks of Balb/c and C57Bl/6 mice, respectively. Hamster anti-mouse CTLA-4 antibody (9H10) labeled with zirconium-89 ([89Zr]9H10) was intratumorally or intravenously administered. Whole-body distribution of the antibody was monitored by immunoPET imaging (n=12 CT26 Balb/c mice, n=10 MC38 C57Bl/6 mice). Tumorous responses to injected doses (1-10 mg/kg) were correlated with specific uptake of [89Zr]9H10 (n=24). Impacts on the tumor microenvironment were assessed by immunofluorescence and flow cytometry. RESULTS: Half of the dose was cleared into the blood 1 hour after intratumoral administration. Despite this, 7 days post-injection, 6-8% of the dose remained in the intratumoral-injected tumors. CT26 tumors with prolonged ICB exposure demonstrated complete responses. Seven days post-injection, the contralateral non-injected tumor uptake of the ICB was comparable to the one achieved through intravenous administration (7.5±1.7% ID.cm-3 and 7.6±2.1% ID.cm-3, respectively) at the same dose in the CT26 model. This observation was confirmed in the MC38 model. Consistent intratumoral pharmacodynamic effects were observed in both intratumoral and intravenous treatment groups, as evidenced by a notable increase in CD8+T cells within the CT26 tumors following treatment. CONCLUSIONS: ImmunoPET-derived pharmacokinetics supports intratumoral injection of ICBs to decrease systemic exposure while maintaining efficacy compared with intravenous. Intratumoral-ICBs lead to high local drug exposure while maintaining significant therapeutic exposure in non-injected tumors. This immunoPET approach is applicable for clinical practice to support evidence-based drug development.

SGN-B7H4V, an investigational vedotin ADC directed to the immune checkpoint ligand B7-H4, shows promising activity in preclinical models.

BACKGROUND: SGN-B7H4V is a novel investigational vedotin antibody-drug conjugate (ADC) comprising a B7-H4-directed human monoclonal antibody conjugated to the cytotoxic payload monomethyl auristatin E (MMAE) via a protease-cleavable maleimidocaproyl valine citrulline (mc-vc) linker. This vedotin linker-payload system has been clinically validated in multiple Food and Drug Administration approved agents including brentuximab vedotin, enfortumab vedotin, and tisotumab vedotin. B7-H4 is an immune checkpoint ligand with elevated expression on a variety of solid tumors, including breast, ovarian, and endometrial tumors, and limited normal tissue expression. SGN-B7H4V is designed to induce direct cytotoxicity against target cells by binding to B7-H4 on the surface of target cells and releasing the cytotoxic payload MMAE upon internalization of the B7-H4/ADC complex. METHODS: B7-H4 expression was characterized by immunohistochemistry across multiple solid tumor types. The ability of SGN-B7H4V to kill B7-H4-expressing tumor cells in vitro and in vivo in a variety of xenograft tumor models was also evaluated. Finally, the antitumor activity of SGN-B7H4V as monotherapy and in combination with an anti-programmed cell death-1 (PD-1) agent was evaluated using an immunocompetent murine B7-H4-expressing Renca tumor model. RESULTS: Immunohistochemistry confirmed B7-H4 expression across multiple solid tumors, with the highest prevalence in breast, endometrial, and ovarian tumors. In vitro, SGN-B7H4V killed B7-H4-expressing tumor cells by MMAE-mediated direct cytotoxicity and antibody-mediated effector functions including antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. In vivo, SGN-B7H4V demonstrated strong antitumor activity in multiple xenograft models of breast and ovarian cancer, including xenograft tumors with heterogeneous B7-H4 expression, consistent with the ability of vedotin ADCs to elicit a bystander effect. In an immunocompetent murine B7-H4-expressing tumor model, SGN-B7H4V drove robust antitumor activity as a monotherapy that was enhanced when combined with an anti-PD-1 agent. CONCLUSION: The immune checkpoint ligand B7-H4 is a promising molecular target expressed by multiple solid tumors. SGN-B7H4V demonstrates robust antitumor activity in preclinical models through multiple potential mechanisms. Altogether, these preclinical data support the evaluation of SGN-B7H4V as a monotherapy in the ongoing phase 1 study of SGN-B7H4V in advanced solid tumors (NCT05194072) and potential future clinical combinations with immunotherapies.

Antitumor activity of the investigational B7-H3 antibody-drug conjugate, vobramitamab duocarmazine, in preclinical models of neuroblastoma.

INTRODUCTION: B7-H3 is a potential target for pediatric cancers, including neuroblastoma (NB). Vobramitamab duocarmazine (also referred to as MGC018 and herein referred to as vobra duo) is an investigational duocarmycin-based antibody-drug conjugate (ADC) directed against the B7-H3 antigen. It is composed of an anti-B7-H3 humanized IgG1/kappa monoclonal antibody chemically conjugated through a cleavable valine-citrulline linker to a duocarmycin-hydroxybenzamide azaindole (vc-seco-DUBA). Vobra duo has shown preliminary clinical activity in B7-H3-expressing tumors. METHODS: B7-H3 expression was evaluated by flow-cytometry in a panel of human NB cell lines. Cytotoxicity was evaluated in monolayer and in multicellular tumor spheroid (MCTS) models by the water-soluble tetrazolium salt,MTS, proliferation assay and Cell Titer Glo 3D cell viability assay, respectively. Apoptotic cell death was investigated by annexin V staining. Orthotopic, pseudometastatic, and resected mouse NB models were developed to mimic disease conditions related to primary tumor growth, metastases, and circulating tumor cells with minimal residual disease, respectively. RESULTS: All human NB cell lines expressed cell surface B7-H3 in a unimodal fashion. Vobra duo was cytotoxic in a dose-dependent and time-dependent manner against all cell lines (IC50 range 5.1-53.9 ng/mL) and NB MCTS (IC50 range 17.8-364 ng/mL). Vobra duo was inactive against a murine NB cell line (NX-S2) that did not express human B7-H3; however, NX-S2 cells were killed in the presence of vobra duo when co-cultured with human B7-H3-expressing cells, demonstrating bystander activity. In orthotopic and pseudometastatic mouse models, weekly intravenous treatments with 1 mg/kg vobra duo for 3 weeks delayed tumor growth compared with animals treated with an irrelevant (anti-CD20) duocarmycin-ADC. Vobra duo treatment for 4 weeks further increased survival in both orthotopic and resected NB models. Vobra duo compared favorably to TOpotecan-TEMozolomide (TOTEM), the standard-of-care therapy for NB relapsed disease, with tumor relapse delayed or arrested by two or three repeated 4-week vobra duo treatments, respectively. Further increased survival was observed in mice treated with vobra duo in combination with TOTEM. Vobra duo treatment was not associated with body weight loss, hematological toxicity, or clinical chemistry abnormalities. CONCLUSION: Vobra duo exerts relevant antitumor activity in preclinical B7-H3-expressing NB models and represents a potential candidate for clinical translation.

Polymorphonuclear myeloid-derived suppressor cells and phosphatidylinositol-3 kinase gamma are critical to tobacco-mimicking oral carcinogenesis in mice.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a devastating disease most often associated with tobacco consumption that induces a field of mutations from which a tumor arises. Identification of ways to prevent the emergence of cancer in high-risk patients is an ultimate goal for combatting all types of cancer, including OSCC. METHODS: Our study employs a mouse model of tongue carcinogenesis induced by tobacco carcinogen mimetic, 4-nitroquinoline 1-oxide (4NQO), to establish tongue dysplasia and OSCC. We use conventional histology, immunohistochemistry, multispectral imaging, mass cytometry, novel cell lines, pharmaceutical inhibition of PI3Kγ, T-cell suppression assays and mouse transplant models in our functional experimentation. RESULTS: In our study, we identify Ly6G+ granulocytes as the most abundant immune cell type in a model of tongue carcinogenesis induced by tobacco carcinogen mimetic 4NQO. Targeting Ly6G+ granulocytes with a pharmacologic inhibitor of PI3Kγ, an isoform of PI3K exclusively expressed by myeloid cells, resulted in reduced tongue dysplasia severity, and reduced rates of OSCC. Importantly, we performed functional assays with the Ly6G+ granulocytes induced in cell line models of 4NQO carcinogenesis to demonstrate that these granulocytes have increased polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) activity against T-cell proliferation and these PMN-MDSCs play a functional role in promoting tumor formation by inhibiting tumor regression in a PI3Kγ-dependent manner. CONCLUSIONS: Overall, our data suggest that recruitment of PMN-MDSCs to sites of dysplasia is critical to immune suppression of CD8 T cells, thereby permitting malignancy, and PI3Kγ inhibitors are one mechanism to reduce PMN-MDSC recruitment, immunosuppression and tumorigenesis in OSCC.

ATM inhibition augments type I interferon response and antitumor T-cell immunity when combined with radiation therapy in murine tumor models.

BACKGROUND: Radiation therapy (RT) elicits DNA double-strand breaks, resulting in tumor cytotoxicity and a type I interferon (IFN) response via stimulator of interferon genes (STING) activation. We investigated whether combining RT with an ataxia-telangiectasia mutated inhibitor promoted these effects and amplified tumor immunity. METHODS: Mice-bearing syngeneic flank tumors (MOC2 head and neck squamous cell carcinoma or B78 melanoma) were treated with tumor-directed RT and oral administration of AZD0156. Specific immune cell depletion, type 1 interferon receptor 1 knock-out mice (IFNAR1-KO), and STING-deficient tumor cells were used to investigate tumor-immune crosstalk following RT and AZD0156 treatment. RESULTS: Combining RT and AZD0156 reduced tumor growth compared with RT or AZD0156 alone in mice bearing MOC2 or B78 tumors. Low-dose AZD0156 (1-100 nM) alone did not affect tumor cell proliferation but suppressed tumor cell clonogenicity in combination with RT. Low-dose AZD0156 with RT synergistically increased IFN-ß, major histocompatibility complex (MHC)-I, and programmed death-ligand 1 (PD-L1) expression in tumor cells. In contrast to wild-type mice, IFNAR1-KO mice showed reduced CD8+T cell tumor infiltration and poor survival following RT+AZD0156 treatment. CD8+T cell depletion reduced antitumor response during RT+AZD0156 treatment. STING-deficient MOC2 (MOC2-STING+/-) or B78 (B78-STING-/-) tumors eliminated the effects of RT+AZD0156 on the expression of IFN-ß, MHC-I, and PD-L1, and reduced CD8+T cell infiltration and migration. Additional anti-PD-L1 therapy promoted antitumor response by elevation of tumor-MHC-I and lymphocyte activation. CONCLUSIONS: Combined radiation and AZD0156 increase STING-dependent antitumor response. Tumor-derived cell-autonomous IFN-ß amplification drives both MHC-I and PD-L1 induction at the tumor cell surface, which is required by anti-PD-L1 therapy to promote antitumor immune response following RT and AZD0156 combination therapy.

CLN-978, a novel half-life extended CD19/CD3/HSA-specific T cell-engaging antibody construct with potent activity against B-cell malignancies with low CD19 expression.

BACKGROUND: Despite significant progress in the development of T cell-engaging therapies for various B-cell malignancies, a high medical need remains for the refractory disease setting, often characterized by suboptimal target levels. METHODS: To address this issue, we have developed a 65-kDa multispecific antibody construct, CLN-978, with affinities tuned to optimize the killing of low-CD19 expressing tumor cells. CLN-978 bound to CD19 on B cells with picomolar affinity, and to CD3ε on T cells with nanomolar affinity. A serum albumin binding domain was incorporated to extend serum half-life. In this setting, we biophysically characterize and report the activities of CLN-978 in cell co-culture assays, multiple mouse models and non-human primates. RESULTS: Human T cells redirected by CLN-978 could eliminate target cells expressing less than 300 copies of CD19 on their surface. The half-life extension and high affinity for CD19 led to significant antitumor activity in murine lymphoma models at very low doses of CLN-978. In primates, we observed a long serum half-life, deep and sustained depletion of normal B cells, and remarkable tolerability, in particular, reduced cytokine release when CLN-978 was administered subcutaneously. CONCLUSIONS: CLN-978 warrants further exploration. An ongoing clinical phase 1 trial is investigating safety, pharmacokinetics, pharmacodynamics, and the initial therapeutic potential of subcutaneously administered CLN-978 in patients with non-Hodgkin's lymphoma.

Treating solid tumors with TCR-based chimeric antigen receptor targeting extra domain B-containing fibronectin.

BACKGROUND: The suppression of chimeric antigen receptor (CAR) T cells by the tumor microenvironment (TME) is a crucial obstacle in the T-cell-based treatment of solid tumors. Extra domain B (EDB)-fibronectin is an oncofetal antigen expressed on the endothelium layer of the neovasculature and cancer cells. Though recognized as a T cell therapy target, engineered CAR T cells thus far have failed to demonstrate satisfactory in vivo efficacy. In this study, we report that targeting EDB-fibronectin by redirected TCR-CAR T cells (rTCR-CAR) bypasses the suppressive TME for solid tumor treatment and sufficiently suppressed tumor growth.We generated EDB-targeting CAR by fusing single-chain variable fragment to CD3ε, resulting in rTCR-CAR. Human primary T cells and Jurkat cells were used to study the EDB-targeting T cells. Differences to the traditional second-generation CAR T cell in signaling, immune synapse formation, and T cell exhaustion were characterized. Cytotoxicity of the rTCR-CAR T cells was tested in vitro, and therapeutic efficacies were demonstrated using xenograft models. METHODS: RESULTS: In the xenograft models, the rTCR-CAR T cells demonstrated in vivo efficacies superior to that based on traditional CAR design. A significant reduction in tumor vessel density was observed alongside tumor growth inhibition, extending even to tumor models established with EDB-negative cancer cells. The rTCR-CAR bound to immobilized EDB, and the binding led to immune synapse structures superior to that formed by second-generation CARs. By a mechanism similar to that for the conventional TCR complex, EDB-fibronectin activated the rTCR-CAR, resulting in rTCR-CAR T cells with low basal activation levels and increased in vivo expansion. CONCLUSION: Our study has demonstrated the potential of rTCR-CAR T cells targeting the EDB-fibronectin as an anticancer therapeutic. Engineered to possess antiangiogenic and cytotoxic activities, the rTCR-CAR T cells showed therapeutic efficacies not impacted by the suppressive TMEs. These combined characteristics of a single therapeutic agent point to its potential to achieve sustained control of solid tumors.

Cyclosporine as Therapy for Traumatic Brain Injury.

Drug development in traumatic brain injury (TBI) has been impeded by the complexity and heterogeneity of the disease pathology, as well as limited understanding of the secondary injury cascade that follows the initial trauma. As a result, patients with TBI have an unmet need for effective pharmacological therapies. One promising drug candidate is cyclosporine, a polypeptide traditionally used to achieve immunosuppression in transplant recipients. Cyclosporine inhibits mitochondrial permeability transition, thereby reducing secondary brain injury, and has shown neuroprotective effects in multiple preclinical models of TBI. Moreover, the cyclosporine formulation NeuroSTAT® displayed positive effects on injury biomarker levels in patients with severe TBI enrolled in the Phase Ib/IIa Copenhagen Head Injury Ciclosporin trial (NCT01825044). Future research on neuroprotective compounds such as cyclosporine should take advantage of recent advances in fluid-based biomarkers and neuroimaging to select patients with similar disease pathologies for clinical trials. This would increase statistical power and allow for more accurate assessment of long-term outcomes.

Preclinical proof of concept for VivoVec, a lentiviral-based platform for in vivo CAR T-cell engineering.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformational outcomes in the treatment of B-cell malignancies, but their widespread use is hindered by technical and logistical challenges associated with ex vivo cell manufacturing. To overcome these challenges, we developed VivoVec, a lentiviral vector-based platform for in vivo engineering of T cells. UB-VV100, a VivoVec clinical candidate for the treatment of B-cell malignancies, displays an anti-CD3 single-chain variable fragment (scFv) on the surface and delivers a genetic payload that encodes a second-generation CD19-targeted CAR along with a rapamycin-activated cytokine receptor (RACR) system designed to overcome the need for lymphodepleting chemotherapy in supporting successful CAR T-cell expansion and persistence. In the presence of exogenous rapamycin, non-transduced immune cells are suppressed, while the RACR system in transduced cells converts rapamycin binding to an interleukin (IL)-2/IL-15 signal to promote proliferation. METHODS: UB-VV100 was administered to peripheral blood mononuclear cells (PBMCs) from healthy donors and from patients with B-cell malignancy without additional stimulation. Cultures were assessed for CAR T-cell transduction and function. Biodistribution was evaluated in CD34-humanized mice and in canines. In vivo efficacy was evaluated against normal B cells in CD34-humanized mice and against systemic tumor xenografts in PBMC-humanized mice. RESULTS: In vitro, administration of UB-VV100 resulted in dose-dependent and anti-CD3 scFv-dependent T-cell activation and CAR T-cell transduction. The resulting CAR T cells exhibited selective expansion in rapamycin and antigen-dependent activity against malignant B-cell targets. In humanized mouse and canine studies, UB-VV100 demonstrated a favorable biodistribution profile, with transduction events limited to the immune compartment after intranodal or intraperitoneal administration. Administration of UB-VV100 to humanized mice engrafted with B-cell tumors resulted in CAR T-cell transduction, expansion, and elimination of systemic malignancy. CONCLUSIONS: These findings demonstrate that UB-VV100 generates functional CAR T cells in vivo, which could expand patient access to CAR T technology in both hematological and solid tumors without the need for ex vivo cell manufacturing.

Pharmacological tumor PDL1 depletion with chlorambucil treats ovarian cancer and melanoma: improves antitumor immunity and renders anti-PDL1-resistant tumors anti-PDL1-sensitive through NK cell effects.

BACKGROUND: Tumor intracellular programmed cell death ligand-1 (PDL1) mediates pathologic signals that regulate clinical treatment responses distinctly from surface-expressed PDL1 targeted by αPDL1 immune checkpoint blockade antibodies. METHODS: We performed a drug screen for tumor cell PDL1 depleting drugs that identified Food and Drug Administration (FDA)-approved chlorambucil and also 9-[2-(phosphonomethoxy)ethyl] guanine. We used in vitro and in vivo assays to evaluate treatment and signaling effects of pharmacological tumor PDL1 depletion focused on chlorambucil as FDA approved, alone or plus αPDL1. RESULTS: PDL1-expressing mouse and human ovarian cancer lines and mouse melanoma were more sensitive to chlorambucil-mediated proliferation inhibition in vitro versus corresponding genetically PDL1-depleted lines. Orthotopic peritoneal PDL1-expressing ID8agg ovarian cancer and subcutaneous B16 melanoma tumors were more chlorambucil-sensitive in vivo versus corresponding genetically PDL1-depleted tumors. Chlorambucil enhanced αPDL1 efficacy in tumors otherwise αPDL1-refractory, and improved antitumor immunity and treatment efficacy in a natural killer cell-dependent manner alone and plus αPDL1. Chlorambucil-mediated PDL1 depletion was relatively tumor-cell selective in vivo, and treatment efficacy was preserved in PDL1KO hosts, demonstrating tumor PDL1-specific treatment effects. Chlorambucil induced PDL1-dependent immunogenic tumor cell death which could help explain immune contributions. Chlorambucil-mediated PDL1 reduction mechanisms were tumor cell-type-specific and involved transcriptional or post-translational mechanisms, including promoting PDL1 ubiquitination through the GSK3ß/ß-TRCP pathway. Chlorambucil-mediated tumor cell PDL1 depletion also phenocopied genetic PDL1 depletion in reducing tumor cell mTORC1 activation and tumor initiating cell content, and in augmenting autophagy, suggesting additional treatment potential. CONCLUSIONS: Pharmacological tumor PDL1 depletion with chlorambucil targets tumor-intrinsic PDL1 signaling that mediates treatment resistance, especially in αPDL1-resistant tumors, generates PDL1-dependent tumor immunogenicity and inhibits tumor growth in immune-dependent and independent manners. It could improve treatment efficacy of selected agents in otherwise treatment-refractory, including αPDL1-refractory cancers, and is rapidly clinically translatable.