Society for Immunotherapy of Cancer (SITC) recommendations on intratumoral immunotherapy clinical trials (IICT): from premalignant to metastatic disease.

BACKGROUND: Intratumorally delivered immunotherapies have the potential to favorably alter the local tumor microenvironment and may stimulate systemic host immunity, offering an alternative or adjunct to other local and systemic treatments. Despite their potential, these therapies have had limited success in late-phase trials for advanced cancer resulting in few formal approvals. The Society for Immunotherapy of Cancer (SITC) convened a panel of experts to determine how to design clinical trials with the greatest chance of demonstrating the benefits of intratumoral immunotherapy for patients with cancers across all stages of pathogenesis. METHODS: An Intratumoral Immunotherapy Clinical Trials Expert Panel composed of international key stakeholders from academia and industry was assembled. A multiple choice/free response survey was distributed to the panel, and the results of this survey were discussed during a half-day consensus meeting. Key discussion points are summarized in the following manuscript. RESULTS: The panel determined unique clinical trial designs tailored to different stages of cancer development-from premalignant to unresectable/metastatic-that can maximize the chance of capturing the effect of intratumoral immunotherapies. Design elements discussed included study type, patient stratification and exclusion criteria, indications of randomization, study arm determination, endpoints, biological sample collection, and response assessment with biomarkers and imaging. Populations to prioritize for the study of intratumoral immunotherapy, including stage, type of cancer and line of treatment, were also discussed along with common barriers to the development of these local treatments. CONCLUSIONS: The SITC Intratumoral Immunotherapy Clinical Trials Expert Panel has identified key considerations for the design and implementation of studies that have the greatest potential to capture the effect of intratumorally delivered immunotherapies. With more effective and standardized trial designs, the potential of intratumoral immunotherapy can be realized and lead to regulatory approvals that will extend the benefit of these local treatments to the patients who need them the most.

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.

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.

Safety, Pharmacokinetics, Pharmacodynamics, and Antitumor Activity from a Phase I Study of Simlukafusp Alfa (FAP-IL2v) in Advanced/Metastatic Solid Tumors.

PURPOSE: Simlukafusp alfa (FAP-IL2v), a tumor-targeted immunocytokine, comprising an interleukin-2 variant moiety with abolished CD25 binding fused to human immunoglobulin G1, is directed against fibroblast activation protein-α. This phase I, open-label, multicenter, dose-escalation and extension study (NCT02627274) evaluated the safety, pharmacokinetics, pharmacodynamics, and antitumor activity of FAP-IL2v in patients with advanced/metastatic solid tumors. METHODS: Participants received FAP-IL2v intravenously once weekly. Dose escalation started at 5 mg; flat dosing (≤25 mg) and intra-participant up-titration regimens (15/20 mg, 20/25 mg, 20/20/35 mg, 20/35/35 mg) were evaluated. Primary objectives were dose-limiting toxicities (DLT), maximum tolerated dose (MTD), recommended expansion dose, and pharmacokinetics. RESULTS: Sixty-one participants were enrolled. DLTs included fatigue (flat dose 20 mg: n = 1), asthenia (25 mg: n = 1), drug-induced liver injury (up-titration regimen 20/25 mg: n = 1), transaminase increase (20/25 mg: n = 1), and pneumonia (20/35/35 mg: n = 1). Up-titration regimen 15/20 mg was the MTD and was selected as the recommended expansion dose. Increases in peripheral blood absolute immune cell counts were seen for all tested doses (natural killer cells, 13-fold; CD4+ T cells [including Tregs], 2-fold; CD8+ T cells, 3.5-fold), but without any percentage change in Tregs. Clinical activity was observed from 5 mg (objective response rate, 5.1% [n = 3]; disease control rate, 27.1% [n = 16]). Responses were durable (n = 3; 2.8 [censored], 6.3, and 43.4 months). CONCLUSIONS: FAP-IL2v had a manageable safety profile and showed initial signs of antitumor activity in advanced/metastatic solid tumors.

The liver as a cytokine factory working on mRNA blueprints for cancer immunotherapy.

mRNA has the potential to encode both vaccines and immunomodulatory proteins for cancer immunotherapy. In this issue, Beck et al. report on lipopolyplexed mRNAs encoding albumin-stabilized interleukin-2 to transduce liver cells. These mRNAs attain antitumor efficacy on subcutaneous mouse tumors even if malignant cells lack major histocompatibility complex class I (MHC class I) expression.

Whole exome sequencing and machine learning germline analysis of individuals presenting with extreme phenotypes of high and low risk of developing tobacco-associated lung adenocarcinoma.

BACKGROUND: Tobacco is the main risk factor for developing lung cancer. Yet, while some heavy smokers develop lung cancer at a young age, other heavy smokers never develop it, even at an advanced age, suggesting a remarkable variability in the individual susceptibility to the carcinogenic effects of tobacco. We characterized the germline profile of subjects presenting these extreme phenotypes with Whole Exome Sequencing (WES) and Machine Learning (ML). METHODS: We sequenced germline DNA from heavy smokers who either developed lung adenocarcinoma at an early age (extreme cases) or who did not develop lung cancer at an advanced age (extreme controls), selected from databases including over 6600 subjects. We selected individual coding genetic variants and variant-rich genes showing a significantly different distribution between extreme cases and controls. We validated the results from our discovery cohort, in which we analysed by WES extreme cases and controls presenting similar phenotypes. We developed ML models using both cohorts. FINDINGS: Mean age for extreme cases and controls was 50.7 and 79.1 years respectively, and mean tobacco consumption was 34.6 and 62.3 pack-years. We validated 16 individual variants and 33 variant-rich genes. The gene harbouring the most validated variants was HLA-A in extreme controls (4 variants in the discovery cohort, p = 3.46E-07; and 4 in the validation cohort, p = 1.67E-06). We trained ML models using as input the 16 individual variants in the discovery cohort and tested them on the validation cohort, obtaining an accuracy of 76.5% and an AUC-ROC of 83.6%. Functions of validated genes included candidate oncogenes, tumour-suppressors, DNA repair, HLA-mediated antigen presentation and regulation of proliferation, apoptosis, inflammation and immune response. INTERPRETATION: Individuals presenting extreme phenotypes of high and low risk of developing tobacco-associated lung adenocarcinoma show different germline profiles. Our strategy may allow the identification of high-risk subjects and the development of new therapeutic approaches. FUNDING: See a detailed list of funding bodies in the Acknowledgements section at the end of the manuscript.

Final results of urelumab, an anti-CD137 agonist monoclonal antibody, in combination with cetuximab or nivolumab in patients with advanced solid tumors.

BACKGROUND: Resistance to immune checkpoint inhibitors and targeted treatments for cancer is common; thus, novel immunotherapy agents are needed. Urelumab is a monoclonal antibody agonist that binds to CD137 receptors expressed on T cells. Here, we report two studies that evaluated urelumab in combination with cetuximab or nivolumab in patients with select, advanced solid tumors. METHODS: CA186-018: Patients with metastatic colorectal cancer or metastatic squamous cell carcinoma of the head and neck (SCCHN) were treated in a dose-evaluation phase with urelumab 0.1 mg/kg (urelumab-0.1) every 3 weeks (Q3W)+cetuximab 250 mg/m2 (cetuximab-250) weekly; and in a dose-expansion phase with urelumab 8 mg flat dose (urelumab-8) Q3W+cetuximab-250 weekly. CA186-107: The dose-escalation phase included patients with previously treated advanced solid tumors (or treated or treatment-naive melanoma); patients received urelumab 3 mg flat dose (urelumab-3) or urelumab-8 every 4 weeks+nivolumab 3 mg/kg (nivolumab-3) or 240 mg (nivolumab-240) every 2 weeks. In the expansion phase, patients with melanoma, non-small cell lung cancer, or SCCHN were treated with urelumab-8+nivolumab-240. Primary endpoints were safety and tolerability, and the secondary endpoint included efficacy assessments. RESULTS: CA186-018: 66 patients received study treatment. The most frequent treatment-related adverse events (TRAEs) were fatigue (75%; n=3) with urelumab-0.1+cetuximab-250 and dermatitis (45%; n=28) with urelumab-8+cetuximab-250. Three patients (5%) discontinued due to TRAE(s) (with urelumab-8+cetuximab-250). One patient with SCCHN had a partial response (objective response rate (ORR) 5%, with urelumab-8+cetuximab-250).CA186-107: 134 patients received study treatment. Fatigue was the most common TRAE (32%; n=2 with urelumab-3+nivolumab-3; n=1 with urelumab-8+nivolumab-3; n=40 with urelumab-8+nivolumab-240). Nine patients (7%) discontinued due to TRAE(s) (n=1 with urelumab-3+nivolumab-3; n=8 with urelumab-8+nivolumab-240). Patients with melanoma naive to anti-PD-1 therapy exhibited the highest ORR (49%; n=21 with urelumab-8+nivolumab-240). Intratumoral gene expression in immune-related pathways (CD3, CD8, CXCL9, GZMB) increased on treatment with urelumab+nivolumab. CONCLUSIONS: Although the addition of urelumab at these doses was tolerable, preliminary response rates did not indicate an evident additive benefit. Nevertheless, the positive pharmacodynamics effects observed with urelumab and the high response rate in treatment-naive patients with melanoma warrant further investigation of other anti-CD137 agonist agents for treatment of cancer. TRIAL REGISTRATION NUMBERS: NCT02110082; NCT02253992.

Obinutuzumab Pretreatment as a Novel Approach to Mitigate Formation of Anti-Drug Antibodies Against Cergutuzumab Amunaleukin in Patients with Solid Tumors.

PURPOSE: The immunocytokine cergutuzumab amunaleukin (CEA-IL2v) showed manageable safety and favorable pharmacodynamics in phase I/Ib trials in patients with advanced/metastatic carcinoembryonic antigen-positive (CEA+) solid tumors, but this was accompanied by a high incidence of anti-drug antibodies (ADA). We examined B-cell depletion with obinutuzumab as a potential mitigation strategy. EXPERIMENTAL DESIGN: Preclinical data comparing B-cell depletion with rituximab versus obinutuzumab are summarized. Substudies of phase I/Ib trials investigated the effect of obinutuzumab pretreatment on ADA development, safety, pharmacodynamics, and antitumor activity of CEA-IL2v ± atezolizumab in patients with advanced/metastatic or unresectable CEA+ solid tumors who had progressed on standard of care. RESULTS: Preclinical data showed superior B-cell depletion with obinutuzumab versus rituximab. In clinical studies, patients received CEA-IL2v monotherapy with (n = 16) or without (n = 6) obinutuzumab pretreatment (monotherapy study), or CEA-IL2v + atezolizumab + obinutuzumab pretreatment (n = 5; combination study). In the monotherapy study, after four cycles (every 2 weeks treatment), 0/15 evaluable patients administered obinutuzumab pretreatment had ADAs versus 4/6 patients without obinutuzumab. Obinutuzumab pretreatment with CEA-IL2v monotherapy showed no new safety signals and pharmacodynamic data suggested minimal impact on T cells and natural killer cells. Conversely, increased liver toxicity was observed in the combination study (CEA-IL2v + atezolizumab + obinutuzumab pretreatment). CONCLUSIONS: These preliminary findings suggest that obinutuzumab pretreatment before CEA-IL2v administration in patients with CEA+ solid tumors may be a feasible and potent ADA mitigation strategy, with an acceptable safety profile, supporting broader investigation of obinutuzumab pretreatment for ADA mitigation in other settings.

Sequential immunotherapy and targeted therapy for metastatic BRAF V600 mutated melanoma: 4-year survival and biomarkers evaluation from the phase II SECOMBIT trial.

No prospective data were available prior to 2021 to inform selection between combination BRAF and MEK inhibition versus dual blockade of programmed cell death protein-1 (PD-1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) as first-line treatment options for BRAFV600-mutant melanoma. SECOMBIT (NCT02631447) was a randomized, three-arm, noncomparative phase II trial in which patients were randomized to one of two sequences with immunotherapy or targeted therapy first, with a third arm in which an 8-week induction course of targeted therapy followed by a planned switch to immunotherapy was the first treatment. BRAF/MEK inhibitors were encorafenib plus binimetinib and checkpoint inhibitors ipilimumab plus nivolumab. Primary outcome of overall survival was previously reported, demonstrating improved survival with immunotherapy administered until progression and followed by BRAF/MEK inhibition. Here we report 4-year survival outcomes, confirming long-term benefit with first-line immunotherapy. We also describe preliminary results of predefined biomarkers analyses that identify a trend toward improved 4-year overall survival and total progression-free survival in patients with loss-of-function mutations affecting JAK or low baseline levels of serum interferon gamma (IFNy). These long-term survival outcomes confirm immunotherapy as the preferred first-line treatment approach for most patients with BRAFV600-mutant metastatic melanoma, and the biomarker analyses are hypothesis-generating for future investigations of predictors of durable benefit with dual checkpoint blockade and targeted therapy.

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.

The significance of the omentum in locoregional immunotherapy for peritoneal carcinomatosis.

Our recent research has unveiled the potential of locoregional immunotherapy. Cytokine-armored viral vectors, such as modified vaccinia virus Ankara vector encoding single-chain interleukin-12 (MVA.scIL-12), can target the omentum and elicit a robust tumor-specific immune response, all the while minimizing toxicity.

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.

Intraperitoneal administration of a modified vaccinia virus Ankara confers single-chain interleukin-12 expression to the omentum and achieves immune-mediated efficacy against peritoneal carcinomatosis.

BACKGROUND: Peritoneal carcinomatosis is an advanced stage of cancer in which the disease has spread to the peritoneal cavity. In order to restore antitumor immunity subverted by tumor cells in this location, we evaluated intraperitoneal administrations of modified vaccinia virus Ankara (MVA) engineered to express single-chain interleukin 12 (scIL-12) to increase antitumor immune responses. METHODS: MVA encoding scIL-12 (MVA.scIL-12) was evaluated against peritoneal carcinomatosis models based on intraperitoneal engraftment of tumor cells. CD8-mediated immune responses, elucidated antitumor efficacy, and safety were evaluated following intravenous, intratumoral, or intraperitoneal administration of the viral vector. The immune response was measured by ELISpot (enzyme-linked immunosorbent spot), RNA sequencing, flow cytometry, intravital microscopy, and depletion of lymphocyte subsets with monoclonal antibodies. Safety was assessed by body-weight follow-up and blood testing. Tissue tropism on intravenous or intraperitoneal administration was assessed by bioluminescence analysis using a reporter MVA encoding luciferase. RESULTS: Intraperitoneal or locoregional administration, but not other routes of administration, resulted in a potent immune response characterized by increased levels of tumor-specific CD8+ T lymphocytes with the ability to produce both interferon-γ and tumor necrosis factor-α. The antitumor immune response was detectable not only in the peritoneal cavity but also systemically. As a result of intraperitoneal treatment, a single administration of MVA.scIL-12 encoding scIL-12 completely eradicated MC38 tumors implanted in the peritoneal cavity and also protected cured mice from subsequent subcutaneous rechallenges. Bioluminescence imaging using an MVA encoding luciferase revealed that intraperitoneal administration targets transgene to the omentum. The omentum is considered a key tissue in immune protection of the peritoneal cavity. The safety profile of intraperitoneal administration was also better than that following intravenous administration since no weight loss or hematological toxicity was observed when the vector was locally delivered into the peritoneal cavity. CONCLUSION: Intraperitoneal administration of MVA vectors encoding scIL-12 targets the omentum, which is the tissue where peritoneal carcinomatosis usually begins. MVA.scIL-12 induces a potent tumor-specific immune response that often leads to the eradication of experimental tumors disseminated to the peritoneal cavity.

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.

Intratumoral injection of IL-12-encoding mRNA targeted to CSFR1 and PD-L1 exerts potent anti-tumor effects without substantial systemic exposure.

IL-12 is a potent cytokine for cancer immunotherapy. However, its systemic delivery as a recombinant protein has shown unacceptable toxicity in the clinic. Currently, the intratumoral injection of IL-12-encoding mRNA or DNA to avoid such side effects is being evaluated in clinical trials. In this study, we aimed to improve this strategy by further favoring IL-12 tethering to the tumor. We generated in vitro transcribed mRNAs encoding murine single-chain IL-12 fused to diabodies binding to CSF1R and/or PD-L1. These targeted molecules are expressed in the tumor microenvironment, especially on myeloid cells. The binding capacity of chimeric constructs and the bioactivity of IL-12 were demonstrated in vitro and in vivo. Doses as low as 0.5 µg IL-12-encoding mRNA achieved potent antitumor effects in subcutaneously injected B16-OVA and MC38 tumors. Treatment delivery was associated with increases in IL-12p70 and IFN-γ levels in circulation. Fusion of IL-12 to the diabodies exerted comparable efficacy against bilateral tumor models. However, it achieved tethering to myeloid cells infiltrating the tumor, resulting in nearly undetectable systemic levels of IL-12 and IFN-γ. Overall, tethering IL-12 to intratumoral myeloid cells in the mRNA-transferred tumors achieves similar efficacy while reducing the dangerous systemic bioavailability of IL-12.

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.

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.

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.