Low-dose ionizing γ-radiation elicits the extrusion of neutrophil extracellular traps.

PURPOSE: Cancer patients frequently undergo radiotherapy in their clinical management with unintended irradiation of blood vessels and copiously irrigated organs in which polymorphonuclear leukocytes circulate. Following the observation that such low doses of ionizing radiation are able to induce neutrophils to extrude neutrophil extracellular traps (NETs), we have investigated the mechanisms, consequences and the occurrence of such phenomena in patients undergoing radiotherapy. EXPERIMENTAL DESIGN: NETosis was analyzed in cultures of neutrophils isolated from healthy donors, cancer patients and cancer-bearing mice under confocal microscopy. Cocultures of radiation-induced NETs, immune effector lymphocytes and tumor cells were used to study the effects of irradiation-induced NETs on immune cytotoxicity. Radiation-induced NETs were intravenously injected to mice assessing their effects on metastasis. Circulating NETs in irradiated cancer patients were measured by ELISA methods detecting MPO-DNA complexes and citrullinated H3. RESULTS: Very low γ-radiation doses (0.5-1 Gy) given to neutrophils elicit NET formation in a manner dependent on oxidative stress, NADPH oxidase activity and autocrine interleukin-8. Radiation-induced NETs interfere with NK- and T-cell cytotoxicity. As a consequence, pre-injection of irradiation-induced NETs increases the number of successful metastases in mouse tumor models. Increases in circulating NETs were readily detected in two prospective series of patients following the first fraction of their radiotherapy courses. CONCLUSIONS: NETosis is induced by low-dose ionizing irradiation in a neutrophil-intrinsic fashion and radiation-induced NETs are able to interfere with immune-mediated cytotoxicity. Radiation-induced NETs foster metastasis in mouse models and can be detected in the circulation of patients undergoing conventional radiotherapy treatments.

Cytotoxicity as a form of immunogenic cell death leading to efficient tumor antigen cross-priming.

Antigen cross-priming of CD8+ T cells is a critical process necessary for the effective expansion and activation of CD8+ T cells endowed with the ability to recognize and destroy tumor cells. The cross-presentation of tumor antigens to cross-prime CD8+ T cells is mainly mediated, if not only, by a subset of professional antigen-presenting cells termed type-1 conventional dendritic cells (cDC1). The demise of malignant cells can be immunogenic if it occurs in the context of premortem stress. These ways of dying are termed immunogenic cell death (ICD) and are associated with biochemical features favoring cDC1 for the efficient cross-priming of tumor antigens. Immunosurveillance and the success of immunotherapies heavily rely on the ability of cytotoxic immune cells, primarily CD8+ T cells and NK cells, to detect and eliminate tumor cells through mechanisms collectively known as cytotoxicity. Recent studies have revealed the significance of NK- and CTL-mediated cytotoxicity as a prominent form of immunogenic cell death, resulting in mechanisms that promote and sustain antigen-specific immune responses. This review focuses on the mechanisms underlying the cross-presentation of antigens released during tumor cell killing by cytotoxic immune cells, with an emphasis on the role of cDC1 cells. Indeed, cDC1s are instrumental in the effectiveness of most immunotherapies, underscoring the significance of tumor antigen cross-priming in contexts of immunogenic cell death. The notion of the potent immunogenicity of cell death resulting from NK or cytotoxic T lymphocyte (CTL)-mediated cytotoxicity has far-reaching implications for cancer immunotherapy.

Interleukin-18 in cancer immunology and immunotherapy.

INTRODUCTION: Interleukin-18 (IL-18) is a myeloid leukocyte inflammatory mediator whose main known function is to elicit IFNγ secretion from T and NK cells. AREAS COVERED: This function offers potential in cancer immunotherapy but as a single treatment, preclinical and clinical antitumor activities are modest. IL-18 bioactivity is chiefly downregulated by a decoy soluble receptor named IL18-binding protein (IL-18BP) that is induced by IFNγ as a negative feedback mechanism. Recent advances indicate promising efficacy of IL-18 at armoring CAR-T cells for the treatment of hematological malignancies. Preclinical research has also yielded IL-18 constructs that do not bind IL-18BP but have preserved activity on the receptor and exert markedly increased antitumor effects. Indeed, agents of this kind are undergoing clinical trials. The synergistic effects of IL-18 and IL-12 in combination to induce IFNγ are extremely potent but are toxic if systemically delivered. In mouse models, IL-12 and decoy-resistant variants of IL-18 can be efficaciously used as local treatments for tumors by exploiting mRNA intratumoral co-delivery. Moreover, antitumor T cells can be transiently engineered with mRNAs encoding this combination of cytokines to attain efficacious synergistic effects also upon intratumoral delivery. EXPERT OPINION: IL-18 certainly holds promise for immunotherapy in combination with other agents and for local approaches.

Intratumoral co-injection of NK cells and NKG2A-neutralizing monoclonal antibodies.

NK-cell reactivity against cancer is conceivably suppressed in the tumor microenvironment by the interaction of the inhibitory receptor NKG2A with the non-classical MHC-I molecules HLA-E in humans or Qa-1b in mice. We found that intratumoral delivery of NK cells attains significant therapeutic effects only if co-injected with anti-NKG2A and anti-Qa-1b blocking monoclonal antibodies against solid mouse tumor models. Such therapeutic activity was contingent on endogenous CD8 T cells and type-1 conventional dendritic cells (cDC1). Moreover, the anti-tumor effects were enhanced upon combination with systemic anti-PD-1 mAb treatment and achieved partial abscopal efficacy against distant non-injected tumors. In xenografted mice bearing HLA-E-expressing human cancer cells, intratumoral co-injection of activated allogeneic human NK cells and clinical-grade anti-NKG2A mAb (monalizumab) synergistically achieved therapeutic effects. In conclusion, these studies provide evidence for the clinical potential of intratumoral NK cell-based immunotherapies that exert their anti-tumor efficacy as a result of eliciting endogenous T-cell responses.

Druggable Targets in Cytokine Release Syndromes.

Bispecific T-cell engagers and chimeric antigen receptor T cells share the problem of eliciting acute systemic inflammation episodes known as cytokine release syndrome. Knowledge on the sequential waves of cytokines that can be neutralized with clinically available agents is crucial to prevent or treat this condition without jeopardizing the antitumor therapeutic outcome. See related article by Leclercq-Cohen et al., p. 4449.

CD137 (4-1BB) requires physically associated cIAPs for signal transduction and antitumor effects.

CD137 (4-1BB) is a member of the TNFR family that mediates potent T cell costimulatory signals upon ligation by CD137L or agonist monoclonal antibodies (mAbs). CD137 agonists attain immunotherapeutic antitumor effects in cancer mouse models, and multiple agents of this kind are undergoing clinical trials. We show that cIAP1 and cIAP2 are physically associated with the CD137 signaling complex. Moreover, cIAPs are required for CD137 signaling toward the NF-κB and MAPK pathways and for costimulation of human and mouse T lymphocytes. Functional evidence was substantiated with SMAC mimetics that trigger cIAP degradation and by transfecting cIAP dominant-negative variants. Antitumor effects of agonist anti-CD137 mAbs are critically dependent on the integrity of cIAPs in cancer mouse models, and cIAPs are also required for signaling from CARs encompassing CD137's cytoplasmic tail.

Intratumoral immunotherapy with mRNAs encoding chimeric protein constructs encompassing IL-12, CD137 agonists, and TGF-ß antagonists.

Intratumoral immunotherapy strategies for cancer based on interleukin-12 (IL-12)-encoding cDNA and mRNA are under clinical development in combination with anti-PD-(L)1 monoclonal antibodies. To make the most of these approaches, we have constructed chimeric mRNAs encoding single-chain IL-12 fused to single-chain fragment variable (scFv) antibodies that bind to transforming growth factor ß (TGF-ß) and CD137 (4-1BB). Several neutralizing TGF-ß agents and CD137 agonists are also undergoing early-phase clinical trials. To attain TGF-ß and CD137 binding by the constructions, we used bispecific tandem scFv antibodies (taFvs) derived from the specific 1D11 and 1D8 monoclonal antibodies (mAbs), respectively. Transfection of mRNAs encoding the chimeric constructs achieved functional expression of the proteins able to act on their targets. Upon mRNA intratumoral injections in the transplantable mouse cancer models CT26, MC38, and B16OVA, potent therapeutic effects were observed following repeated injections into the tumors. Efficacy was dependent on the number of CD8+ T cells able to recognize tumor antigens that infiltrated the malignant tissue. Although the abscopal effects on concomitant uninjected lesions were modest, such distant effects on untreated lesions were markedly increased when combined with systemic PD-1 blockade.

Facts and Hopes on Neutralization of Protumor Inflammatory Mediators in Cancer Immunotherapy.

In cancer pathogenesis, soluble mediators are responsible for a type of inflammation that favors the progression of tumors. The mechanisms chiefly involve changes in the cellular composition of the tumor tissue stroma and in the functional modulation of myeloid and lymphoid leukocytes. Active immunosuppression, proangiogenesis, changes in leukocyte traffic, extracellular matrix remodeling, and alterations in tumor-antigen presentation are the main mechanisms linked to the inflammation that fosters tumor growth and metastasis. Soluble inflammatory mediators and their receptors are amenable to various types of inhibitors that can be combined with other immunotherapy approaches. The main proinflammatory targets which can be interfered with at present and which are under preclinical and clinical development are IL1ß, IL6, the CXCR1/2 chemokine axis, TNFα, VEGF, leukemia inhibitory factor, CCL2, IL35, and prostaglandins. In many instances, the corresponding neutralizing agents are already clinically available and can be repurposed as a result of their use in other areas of medicine such as autoimmune diseases and chronic inflammatory conditions.

Dendritic cells, headhunters for anti-tumor CD8+ T cells in triple-negative breast cancer.

How do we identify patients most likely to benefit from immune checkpoint blockade therapies? This month in Med, Wu and colleagues1 identify that CCL19+ mature dendritic cells correlated with responses to anti-PD-(L)1 immunotherapy in triple-negative breast cancer patients, suggesting the use of CCL19 as a biomarker to predict patient outcomes.

Intratumoral neoadjuvant immunotherapy based on the BO-112 viral RNA mimetic.

BO-112 is a poly I:C-based viral mimetic that exerts anti-tumor efficacy when intratumorally delivered in mouse models. Intratumoral BO-112 synergizes in mice with systemic anti-PD-1 mAbs and this combination has attained efficacy in PD1-refractory melanoma patients. We sought to evaluate the anti-tumor efficacy of BO-112 pre-surgically applied in neoadjuvant settings to mouse models. We have observed that repeated intratumoral injections of BO-112 prior to surgical excision of the primary tumor significantly reduced tumor metastasis from orthotopically implanted 4T1-derived tumors and subcutaneous MC38-derived tumors in mice. Such effects were enhanced when combined with systemic anti-PD-1 mAb. The anti-tumor efficacy of this neoadjuvant immunotherapy approach depended on the presence of antigen-specific effector CD8 T cells and cDC1 antigen-presenting cells. Since BO-112 has been successful in phase-two clinical trials for metastatic melanoma, these results provide a strong rationale for translating this pre-surgical strategy into clinical settings, especially in combination with standard-of-care checkpoint inhibitors.

Synergistic effects of combined immunotherapy strategies in a model of multifocal hepatocellular carcinoma.

Immune checkpoint-inhibitor combinations are the best therapeutic option for advanced hepatocellular carcinoma (HCC) patients, but improvements in efficacy are needed to improve response rates. We develop a multifocal HCC model to test immunotherapies by introducing c-myc using hydrodynamic gene transfer along with CRISPR-Cas9-mediated disruption of p53 in mouse hepatocytes. Additionally, induced co-expression of luciferase, EGFP, and the melanosomal antigen gp100 facilitates studies on the underlying immunological mechanisms. We show that treatment of the mice with a combination of anti-CTLA-4 + anti-PD1 mAbs results in partial clearance of the tumor with an improvement in survival. However, the addition of either recombinant IL-2 or an anti-CD137 mAb markedly improves both outcomes in these mice. Combining tumor-specific adoptive T cell therapy to the aCTLA-4/aPD1/rIL2 or aCTLA-4/aPD1/aCD137 regimens enhances efficacy in a synergistic manner. As shown by multiplex tissue immunofluorescence and intravital microscopy, combined immunotherapy treatments enhance T cell infiltration and the intratumoral performance of T lymphocytes.

mRNAs encoding IL-12 and a decoy-resistant variant of IL-18 synergize to engineer T cells for efficacious intratumoral adoptive immunotherapy.

Interleukin-12 (IL-12) gene transfer enhances the therapeutic potency of adoptive T cell therapies. We previously reported that transient engineering of tumor-specific CD8 T cells with IL-12 mRNA enhanced their systemic therapeutic efficacy when delivered intratumorally. Here, we mix T cells engineered with mRNAs to express either single-chain IL-12 (scIL-12) or an IL-18 decoy-resistant variant (DRIL18) that is not functionally hampered by IL-18 binding protein (IL-18BP). These mRNA-engineered T cell mixtures are repeatedly injected into mouse tumors. Pmel-1 T cell receptor (TCR)-transgenic T cells electroporated with scIL-12 or DRIL18 mRNAs exert powerful therapeutic effects in local and distant melanoma lesions. These effects are associated with T cell metabolic fitness, enhanced miR-155 control on immunosuppressive target genes, enhanced expression of various cytokines, and changes in the glycosylation profile of surface proteins, enabling adhesiveness to E-selectin. Efficacy of this intratumoral immunotherapeutic strategy is recapitulated in cultures of tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR) T cells on IL-12 and DRIL18 mRNA electroporation.

CD137 (4-1BB)-Based Cancer Immunotherapy on Its 25th Anniversary.

Twenty-five years ago, we reported that agonist anti-CD137 monoclonal antibodies eradicated transplanted mouse tumors because of enhanced CD8+ T-cell antitumor immunity. Mouse models indicated that anti-CD137 agonist antibodies synergized with various other therapies. In the clinic, the agonist antibody urelumab showed evidence for single-agent activity against melanoma and non-Hodgkin lymphoma but caused severe liver inflammation in a fraction of the patients. CD137's signaling domain is included in approved chimeric antigen receptors conferring persistence and efficacy. A new wave of CD137 agonists targeting tumors, mainly based on bispecific constructs, are in early-phase trials and are showing promising safety and clinical activity. SIGNIFICANCE: CD137 (4-1BB) is a costimulatory receptor of T and natural killer lymphocytes whose activity can be exploited in cancer immunotherapy strategies as discovered 25 years ago. Following initial attempts that met unacceptable toxicity, new waves of constructs acting agonistically on CD137 are being developed in patients, offering signs of clinical and pharmacodynamic activity with tolerable safety profiles.

Intratumoral Gene Transfer of mRNAs Encoding IL12 in Combination with Decoy-Resistant IL18 Improves Local and Systemic Antitumor Immunity.

IL12-based local gene therapy of cancer constitutes an active area of clinical research using plasmids, mRNAs, and viral vectors. To improve antitumor effects, we have experimentally tested the combination of mRNA constructs encoding IL12 and IL18. Moreover, we have used a form of IL18 [decoy-resistant IL18 (DR-18)] which has preserved bioactivity but does not bind to the IL18 binding protein decoy receptor. Both cytokines dramatically synergize to induce IFNγ release from mouse splenocytes, and, if systemically cotransferred to the liver, they mediate lethal toxicity. However, if given intratumorally to B16OVA tumor-bearing mice, the combination attains efficacy against the directly treated tumor and moderate tumor-delaying activity on distant noninjected lesions. Cotreatment was conducive to the presence of more activated CD8+ T cells in the treated and noninjected tumors. In keeping with these findings, the efficacy of treatment was contingent on the integrity of CD8+ T cells and cDC1 dendritic cells in the treated mice. Furthermore, efficacy of IL12 plus DR-18 local mRNA coinjection against distant concomitant tumors could be enhanced upon combination with anti-PD-1 mAb systemic treatment, thus defining a feasible synergistic immunotherapy strategy.

Depletion of Conventional Type-1 Dendritic Cells in Established Tumors Suppresses Immunotherapy Efficacy.

The ability of conventional type-1 dendritic cells (cDC1) to cross-present tumor antigens to CD8+ T cells is critical for the induction of antitumor CTLs. Mice that are constitutively deficient in cDC1 cells have been reported to fail to respond to immunotherapy strategies based on checkpoint inhibitors. However, further work is needed to clarify the precise time during immunotherapy treatment that cDC1 cells are required for the beneficial effect of treatment. Here, we used a refined XCR1-DTR-Venus transgenic mouse model to acutely deplete cDC1 cells and trace their behavior using intravital microscopy. Diphtheria toxin-mediated cDC1 depletion prior to immunotherapy treatment with anti-PD-1 and/or anti-CD137 immunostimulatory mAbs completely ablated antitumor efficacy. The efficacy of adoptive T-cell therapy was also hampered by prior cDC1 depletion. After the onset of immunotherapy treatment, depletion of cDC1s only moderately reduced the therapeutic efficacy of anti-PD-1 and anti-CD137 mAbs. Intravital microscopy of liver-engrafted tumors revealed changes in the intratumoral behavior of cDC1 cells in mice receiving immunotherapy, and treatment with diphtheria toxin to deplete cDC1s impaired tumor T-cell infiltration and function. These results reveal that the functional integrity of the cDC1 compartment is required at the onset of various immunotherapies to successfully treat established tumors. SIGNIFICANCE: These findings reveal the intratumoral behavior of cDC1 dendritic cells in transgenic mouse models and demonstrate that the efficacy of immunotherapy regimens is precluded by elimination of these cells.

Novel strategies exploiting interleukin-12 in cancer immunotherapy.

Interleukin-12 is considered a potent agent to enhance antitumor immune responses. It belongs to a family of heterodimeric cytokines with key roles in the up-regulation and down-regulation of cellular immunity. Since its discovery, recombinant IL-12 was found to exert potent antitumor effects in rodent tumor models and was rapidly tested in the clinic with an unfavorable benefit/toxicity profile. Localized delivery of IL-12 dramatically improves the therapeutic index and this approach is being applied in the clinic based on in-vivo electroporation of naked plasmid DNA encoding IL-12, mRNA formulations, viral vectors and tumor-targeted fusion proteins. Other biotechnology strategies such as IL-12-engineered local adoptive cell therapy and pro-cytokines can also be used to improve results and broaden the therapeutic window. Combination strategies of such localized IL-12-based approaches with checkpoint inhibitors are yielding promising results both preclinically and in the early-phase clinical trials.

Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies.

BACKGROUND: On the basis of efficacy in mouse tumor models, multiple CD137 (4-1BB) agonist agents are being preclinically and clinically developed. The costimulatory molecule CD137 is inducibly expressed as a transmembrane or as a soluble protein (sCD137). Moreover, the CD137 cytoplasmic signaling domain is a key part in approved chimeric antigen receptors (CARs). Reliable pharmacodynamic biomarkers for CD137 ligation and costimulation of T cells will facilitate clinical development of CD137 agonists in the clinic. METHODS: We used human and mouse CD8 T cells undergoing activation to measure CD137 transcription and protein expression levels determining both the membrane-bound and soluble forms. In tumor-bearing mice plasma sCD137 concentrations were monitored on treatment with agonist anti-CD137 monoclonal antibodies (mAbs). Human CD137 knock-in mice were treated with clinical-grade agonist anti-human CD137 mAb (Urelumab). Sequential plasma samples were collected from the first patients intratumorally treated with Urelumab in the INTRUST clinical trial. Anti-mesothelin CD137-encompassing CAR-transduced T cells were stimulated with mesothelin coated microbeads. sCD137 was measured by sandwich ELISA and Luminex. Flow cytometry was used to monitor CD137 surface expression. RESULTS: CD137 costimulation upregulates transcription and protein expression of CD137 itself including sCD137 in human and mouse CD8 T cells. Immunotherapy with anti-CD137 agonist mAb resulted in increased plasma sCD137 in mice bearing syngeneic tumors. sCD137 induction is also observed in human CD137 knock-in mice treated with Urelumab and in mice transiently humanized with T cells undergoing CD137 costimulation inside subcutaneously implanted Matrigel plugs. The CD137 signaling domain-containing CAR T cells readily released sCD137 and acquired CD137 surface expression on antigen recognition. Patients treated intratumorally with low dose Urelumab showed increased plasma concentrations of sCD137. CONCLUSION: sCD137 in plasma and CD137 surface expression can be used as quantitative parameters dynamically reflecting therapeutic costimulatory activity elicited by agonist CD137-targeted agents.