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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.

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 cytotoxic T lymphocytes. 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 monoclonal antibodies (mAbs) completely ablated anti-tumor 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. This work was supported by Spanish Ministry of Economy and Competitiveness and Spanish Ministry of Research (MINECO SAF2014-52361-R and SAF 2017-83267-C2-1R and PID2020-112892RB-100, PID2020-113174-RA-100 [AEI/FEDER,UE], financed by MCIN/AEI/10.13039/501100011033), Cancer Research Institute under the CRI-CLIP, Asociación Española Contra el Cancer (AECC) Foundation under Grant GCB15152947MELE, Joint Translational Call for Proposals 2015 (JTC 2015) TRANSCAN-2 (code: TRS-2016-00000371), projects PI14/01686, PI13/00207, PI16/00668, PI19/01128, funded by Instituto de Salud Carlos III and co-funded by European Union (ERDF, “A way to make Europe”), European Commission within the Horizon 2020 Programme (PROCROP - 635122), Gobierno de Navarra Proyecto LINTERNA Ref: 0011–1411, Mark Foundation, Fundación BBVA and Fundación Olga Torres. AT is supported by the Ramon y Cajal program from the Spanish Ministry of Science (RYC2019-026406-I financiada por MCIN/AEI /10.13039/501100011033 y por El FSE invierte en tu futuro). Sí

Supplementary figures 1-8 and supplementary table 1

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.

Supplementary figures 1-5 Supplementary methods

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.

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Interleukin-12-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 interleukin-12 (IL-12) and interleukin-18 (IL-18). Moreover, we have used a form of IL-18 (DR-18) which has preserved bioactivity but does not bind to the IL-18BP decoy receptor. Both cytokines dramatically synergize to induce IFNγ release from mouse splenocytes, and, if systemically co-transferred 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 uninjected lesions. Co-treatment was conducive to the presence of more activated CD8+ T cells in the treated and non-injected 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 IL-12 plus DR-18 local mRNA co-injection against distant concomitant tumors could be enhanced upon combination with anti-PD-1 mAb systemic treatment, thus defining a feasible synergistic immunotherapy strategy.

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.

CD137 (4-1BB) is a member of the TNFR family, whose expression is induced on antigen-primed T cells and that mediates potent costimulatory signals upon ligation by CD137L or agonist monoclonal antibodies. 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 polyprotein complex. Moreover, cIAPs are required for CD137 signaling towards the NF-κB and MAPK pathways and for costimulation of human and mouse T lymphocytes. Functional evidence was substantiated with SMAC-mimetics that trigger proteasomal degradation of cIAPs and transfecting dominant-negative variants of cIAPs. Importantly, antitumor effects of agonist anti-CD137 mAbs are critically dependent on the integrity of cIAPs in cancer mouse models. Citation Format: Javier Glez-Vaz, Arantza Azpilikueta, Maria Carmen Ochoa, Irene Olivera, Gabriel Gomis, Asunta Cirella, Carlos Luri-Rey, Maite Álvarez, Sergio Ciordia, Pedro Berraondo, Álvaro Teijeira, Fernando Corrales, Juan M Zapata, Ignacio Melero. CD137 (4-1BB) requires physically associated cIAPs for signal transduction and antitumor effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 614.

BackgroundOn 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.MethodsWe 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.ResultsCD137 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.ConclusionsCD137 in plasma and CD137 surface expression can be used as quantitative parameters dynamically reflecting therapeutic costimulatory activity elicited by agonist CD137-targeted agents.

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.

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 co-stimulation 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 of the membrane-bound and soluble forms. In tumor-bearing mice plasma sCD137 concentrations were monitored upon treatment with agonist anti-CD137 mAbs. Human CD137 knock-in mice were treated with clinical-grade agonist anti-human CD137 (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 ELISAs and Luminex and flow cytometry was used to monitor CD137 surface expression. Results: CD137 co-stimulation 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 co-stimulation inside subcutaneous implanted Matrigel® plugs. CD137 signaling domain-containing CAR T cells readily released sCD137 upon antigen recognition in a manner favored by the CD137 signaling domain. 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 co-stimulatory activity elicited by agonist CD137-targeted agents. Citation Format: Javier Glez-Vaz, Arantza Azpilikueta, Irene Olivera, Assunta Cirella, Álvaro Teijeira, Carmen Ochoa, Maite Álvarez, Iñaki Eguren, Carlos Luri-Rey, Miguel F. Sanmamed, Ignacio Melero. Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 628.