Search

Supplementary Figure from Targeting Macrophages with CAR T Cells Delays Solid Tumor Progression and Enhances Antitumor Immunity

Supplementary Table from Targeting Macrophages with CAR T Cells Delays Solid Tumor Progression and Enhances Antitumor Immunity

Tumor-associated macrophages (TAM) are one of the most abundant cell types in many solid tumors and typically exert protumor effects. This has led to an interest in macrophage-depleting agents for cancer therapy, but approaches developed to date have had limited success in clinical trials. Here, we report the development of a strategy for TAM depletion in mouse solid tumor models using chimeric antigen receptor (CAR) T cells targeting the macrophage marker F4/80 (F4.CAR-T). F4.CAR-T cells effectively killed macrophages in vitro and in vivo without toxicity. When injected into mice bearing orthotopic lung tumors, F4.CAR-T cells infiltrated tumor lesions and delayed tumor growth comparably with PD-1 blockade, and significantly extended mouse survival. Antitumor effects were mediated by F4.CAR-T–produced IFNγ, which promoted upregulation of MHC molecules on cancer cells and tumor-infiltrating myeloid cells. Notably, F4.CAR-T promoted expansion of endogenous CD8 T cells specific for tumor-associated antigen and led to immune editing of highly antigenic tumor cell clones. Antitumor impact was also observed in mouse models of ovarian and pancreatic cancer. These studies provide proof of principle to support CAR T-cell targeting of TAMs as a means to enhance antitumor immunity.

Supplementary figure 1. Similar survival results with anti-PD-1 or anti-PD-L1 in the combination with SFV-IL12 for B16-OVA-derived tumors.

Supplementary figure legends

Migratory CD103+ DCs are the main mediators of cross-priming at the tumor-draining LNs.

Systemic sFlt3L and local intratumoral poly-ICLC expand and mature DCs in B16-OVA bearing mice.

CTLs against the Adpgk neoantigen of MC38 are induced by anti-CD137 and anti-PD-1 mAbs in a fraction of WT mice, but not in Batf3-/- mice.

T and NK lymphocytes express CD137 (4-1BB), a costimulatory receptor of the TNFR family whose function is exploitable for cancer immunotherapy. Mitochondria regulate the function and survival of T lymphocytes. Herein, we show that CD137 costimulation provided by agonist mAb and CD137L (4-1BBL) induced mitochondria enlargement that resulted in enhanced mitochondrial mass and transmembrane potential in human and mouse CD8+ T cells. Such mitochondrial changes increased T-cell respiratory capacities and were critically dependent on mitochondrial fusion protein OPA-1 expression. Mass and function of mitochondria in tumor-reactive CD8+ T cells from cancer-bearing mice were invigorated by agonist mAb to CD137, whereas mitochondrial baseline mass and function were depressed in CD137-deficient tumor reactive T cells. Tumor rejection induced by the synergistic combination of adoptive T-cell therapy and agonistic anti-CD137 was critically dependent on OPA-1 expression in transferred CD8+ T cells. Moreover, stimulation of CD137 with CD137 mAb in short-term cultures of human tumor-infiltrating lymphocytes led to mitochondria enlargement and increased transmembrane potential. Collectively, these data point to a critical link between mitochondrial morphology and function and enhanced antitumor effector activity upon CD137 costimulation of T cells. Cancer Immunol Res; 6(7); 798–811. ©2018 AACR.

Supplementary figure 2. Effects of treatment with SFV-IL12 + anti-PD-1 combination on the B16.F10 and 4T1 tumor models.

Supplementary Figure Legends

Virotherapy and checkpoint inhibitors can be combined for the treatment of cancer with complementarity and potential for synergistic effects. We have developed a cytolytic but nonreplicative viral vector system based on Semliki Forest virus that encodes IL12 (SFV-IL12). Following direct intratumoral injection, infected cells release transgenic IL12, die, and elicit an inflammatory response triggered by both abundantly copied viral RNA and IL12. In difficult-to-treat mouse cancer models, such as those derived from MC38 and bilateral B16-OVA, SFV-IL12 synergized with an anti–PD-1 monoclonal antibody (mAb) to induce tumor regression and prolong survival. Similar synergistic effects were attained upon PD-L1 blockade. Combined SFV-IL12 + anti–PD-1 mAb treatment only marginally increased the elicited cytotoxic T-lymphocyte response over SFV-IL12 as a single agent, at least when measured by in vivo killing assays. In contrast, we observed that SFV-IL12 treatment induced expression of PD-L1 on tumor cells in an IFNγ-dependent fashion. PD-L1–mediated adaptive resistance thereby provides a mechanistic explanation of the observed synergistic effects achieved by the SFV-IL12 + anti–PD-1 mAb combination. Cancer Immunol Res; 3(5); 449–54. ©2015 AACR.

Combinations of immunomodulatory anti-CD137 and anti-PD-1 mAbs synergize with sFlt3L and poly-ICLC against grafted B16F10 and B16-OVA melanomas.

Figure S1 shows that CD8 T cells with high mitochondrial transmembrane potential after CD137 stimulation have enlarged mitochondria. Fig S2 shows that CD137 induced mitochondrial changes are not due to enrichment of specific CD8 T cells subsets. Figure S3 shows that mitochondrial enhanced functions induced by CD137 activation are not a consequence of autocrine secretion of cytokines. Fig S4 shows that spare respiratory capacity induced by CD137 activation is dependent on fatty acid oxidation. Figure S5 explores regulation of mitochondrial biogenesis and fusion related genes upon CD137 activation. Fig S6 shows functional effects of OPA knockdown in CD8 T cells. Figure S7 shows the implication of Akt pathway on enhanced mitochondrial function by CD137 activation. Figure S8 shows that CD137KO CD8 T cells have intrinsic mitochondrial defects upon CD28 activation. Figure S9 shows that CD137 activation enhances mitochondrial parameters in tumor infiltrating lymphocytes. Fig S10 shows engrafment abilities and memory markers of OTI cells in B16OVA tumor bearing mice upon OPA silencing and CD137 mAb treatment.

Multiple lines of evidence indicate a critical role of antigen cross-presentation by conventional BATF3-dependent type 1 classical dendritic cells (cDC1) in CD8-mediated antitumor immunity. Flt3L and XCL1, respectively, constitute a key growth/differentiation factor and a potent and specific chemoattractant for cDC1. To exploit their antitumor functions in local immunotherapy, we prepared Semliki Forest Virus (SFV)–based vectors encoding XCL1 and soluble Flt3L (sFlt3L). These vectors readily conferred transgene expression to the tumor cells in culture and when engrafted as subcutaneous mouse tumor models. In syngeneic mice, intratumoral injection of SFV-XCL1-sFlt3L (SFV-XF) delayed progression of MC38- and B16-derived tumors. Therapeutic activity was observed and exerted additive effects in combination with anti–PD-1, anti-CD137, or CTLA-4 immunostimulatory mAbs. Therapeutic effects were abolished by CD8β T-cell depletion and were enhanced by CD4 T-cell depletion, but not by T regulatory cell predepletion with anti-CD25 mAb. Antitumor effects were also abolished in BATF3- and IFNAR-deficient mice. In B16-OVA tumors, SFV-XF increased the number of infiltrating CD8 T cells, including those recognizing OVA. Consistently, following the intratumoral SFV-XF treatment courses, we observed increased BATF3-dependent cDC1 among B16-OVA tumor-infiltrating leukocytes. Such an intratumoral increase was not seen in MC38-derived tumors, but both resident and migratory cDC1 were boosted in SFV-XF–treated MC38 tumor-draining lymph nodes. In conclusion, viral gene transfer of sFlt3L and XCL1 is feasible, safe, and biologically active in mice, exerting antitumor effects that can be potentiated by CD4 T-cell depletion.Significance:These findings demonstrate that transgenic expression of sFLT3L and XCL1 in tumor cells mediates cross-priming of, and elicits potent antitumor activity from, CD8 T lymphocytes, particularly in combination with CD4 T-cell depletion.

Cytokine production profiling of CD4+ T cells in draining lymph nodes from mice intratumorally treated with SFV-XF.

FACS Gating strategies to analyze tumor-infiltrating and lymph node cell suspensions

Suppplementary materials and methods

Expression of immunomodulatory antibody targets on tumor infiltrating lymphocytes following SFV-XF treatment.

The SFV-XF vector exerts maximal antitumor efficacy as compared to SFV vectors encoding each single transgene and is effective against B16F10-derived melanomas, PANC02 pancreatic cancer and CT26 colon cancer.

Administration of SFV-XF into MC38 tumors shows no detectable T-cell infiltration increases.

SFV-XF intratumoral treatment combined with CD4 depletion control the progression of uninjected contralateral tumors.

CD4 Treg depletion or inhibition does not recapitulate the enhancement of efficacy of SFV-XF treatment found with CD4 T-cell depletion.

Tumor-associated macrophages (TAMs) are one of the most abundant cell types in many solid tumors and typically exert protumor effects. This has led to an interest in macrophage-depleting agents for cancer therapy, but approaches developed to date have had limited success in clinical trials. Here, we report the development of a strategy for TAM depletion in mouse solid tumor models using chimeric antigen receptor (CAR) T cells targeting the macrophage marker F4/80 (F4.CAR-T). F4.CAR-T cells effectively killed macrophages in vitro and in vivo without toxicity. When injected into mice bearing orthotopic lung tumors, F4.CAR-T cells infiltrated tumor lesions and delayed tumor growth comparably to PD1 blockade, and significantly extended mouse survival. Anti-tumor effects were mediated by F4.CAR-T-produced IFN-γ, which promoted upregulation of MHC molecules on cancer cells and tumor-infiltrating myeloid cells. Notably, F4.CAR-T promoted expansion of endogenous CD8 T cells specific for tumor-associated antigens and led to immune editing of highly antigenic tumor cell clones. Antitumor impact was also observed in mouse models of ovarian and pancreatic cancer. These studies provide proof-of-principle evidence to support CAR-T targeting of TAMs as a means to enhance antitumor immunity.

Conventional type 1 dendritic cells (cDC1s) are central to antitumor immunity and their presence in the tumor microenvironment associates with improved outcomes in patients with cancer. DNGR-1 (CLEC9A) is a dead cell-sensing receptor highly restricted to cDC1s. DNGR-1 has been involved in both cross-presentation of dead cell-associated antigens and processes of disease tolerance, but its role in antitumor immunity has not been clarified yet.

BackgroundThe immune response to cancer is often conceptualized with the cancer immunity cycle. An essential step in this interpretation is that antigens released by dying tumors are presented by dendritic cells to naive or memory T cells in the tumor-draining lymph nodes. Whether tumor cell death resulting from cytotoxicity, as mediated by T cells or natural killer (NK) lymphocytes, is actually immunogenic currently remains unknown.MethodsIn this study, tumor cells were killed by antigen-specific T-cell receptor (TCR) transgenic CD8 T cells or activated NK cells. Immunogenic cell death was studied analyzing the membrane exposure of calreticulin and the release of high mobility group box 1 (HMGB1) by the dying tumor cells. Furthermore, the potential immunogenicity of the tumor cell debris was evaluated in immunocompetent mice challenged with an unrelated tumor sharing only one tumor-associated antigen and by class I major histocompatibility complex (MHC)-multimer stainings. Mice deficient inBatf3,Ifnar1andSting1were used to study mechanistic requirements.ResultsWe observe in cocultures of tumor cells and effector cytotoxic cells, the presence of markers of immunogenic cell death such as calreticulin exposure and soluble HMGB1 protein. Ovalbumin (OVA)-transfected MC38 colon cancer cells, exogenously pulsed to present the gp100 epitope are killed in culture by mouse gp100-specific TCR transgenic CD8 T cells. Immunization of mice with the resulting destroyed cells induces epitope spreading as observed by detection of OVA-specific T cells by MHC multimer staining and rejection of OVA+EG7 lymphoma cells. Similar results were observed in mice immunized with cell debris generated by NK-cell mediated cytotoxicity. Mice deficient inBatf3-dependent dendritic cells (conventional dendritic cells type 1, cDC1) fail to develop an anti-OVA response when immunized with tumor cells killed by cytotoxic lymphocytes. In line with this, cultured cDC1 dendritic cells uptake and can readily cross-present antigen from cytotoxicity-killed tumor cells to cognate CD8+T lymphocytes.ConclusionThese results support that an ongoing cytotoxic antitumor immune response can lead to immunogenic tumor cell death.

Live 17D is widely used as a prophylactic vaccine strain for yellow fever virus that induces potent neutralizing humoral and cellular immunity against the wild‐type pathogen. 17D replicates and kills mouse and human tumor cell lines but not non‐transformed human cells. Intratumoral injections with viable 17D markedly delay transplanted tumor progression in a CD8 T‐cell‐dependent manner. In mice bearing bilateral tumors in which only one is intratumorally injected, contralateral therapeutic effects are observed consistent with more prominent CD8 T‐cell infiltrates and a treatment‐related reduction of Tregs. Additive efficacy effects were observed upon co‐treatment with intratumoral 17D and systemic anti‐CD137 and anti‐PD‐1 immunostimulatory monoclonal antibodies. Importantly, when mice were preimmunized with 17D, intratumoral 17D treatment achieved better local and distant antitumor immunity. Such beneficial effects of prevaccination are in part explained by the potentiation of CD4 and CD8 T‐cell infiltration in the treated tumor. The repurposed use of a GMP‐grade vaccine to be given via the intratumoral route in prevaccinated patients constitutes a clinically feasible and safe immunotherapy approach., The attenuated vaccine for yellow fever virus exerts antitumor effects when intratumorally injected which are mediated by immune responses and enhanced in pre‐vaccinated mice.

Immune checkpoint blockade has shown anti-cancer efficacy, but requires systemic administration of monoclonal antibodies (mAbs), often leading to adverse effects. To avoid toxicity, mAbs could be expressed locally in tumors. We developed adeno-associated virus (AAV) and Semliki Forest virus (SFV) vectors expressing anti-programmed death ligand 1 (aPDL1) mAb. When injected intratumorally in MC38 tumors, both viral vectors led to similar local mAb expression at 24 h, diminishing quickly in SFV-aPDL1-treated tumors. However, SFV-aPDL1 induced >40% complete regressions and was superior to AAV-aPDL1, as well as to aPDL1 mAb given systemically or locally. SFV-aPDL1 induced abscopal effects and was also efficacious against B16-ovalbumin (OVA). The higher SFV-aPDL1 antitumor activity could be related to local upregulation of interferon-stimulated genes because of SFV RNA replication. This was confirmed by combining local SFV-LacZ administration and systemic aPDL1 mAb, which provided higher antitumor effects than each separated agent. SFV-aPDL1 promoted tumor-specific CD8 T cells infiltration in both tumor models. In MC38, SFV-aPDL1 upregulated co-stimulatory markers (CD137/OX40) in tumor CD8 T cells, and its combination with anti-CD137 mAb showed more pronounced antitumor effects than each single agent. These results indicate that local transient expression of immunomodulatory mAbs using non-propagative RNA vectors inducing type I interferon (IFN-I) responses represents a potent and safe approach for cancer treatment.

Preclinical and clinical evidence indicate that the proimmune effects of radiotherapy can be synergistically augmented with immunostimulatory mAbs to act both on irradiated tumor lesions and on distant, nonirradiated tumor sites. The combination of radiotherapy with immunostimulatory anti-PD1 and anti-CD137 mAbs was conducive to favorable effects on distant nonirradiated tumor lesions as observed in transplanted MC38 (colorectal cancer), B16OVA (melanoma), and 4T1 (breast cancer) models. The therapeutic activity was crucially performed by CD8 T cells, as found in selective depletion experiments. Moreover, the integrities of BATF-3–dependent dendritic cells specialized in crosspresentation/crosspriming of antigens to CD8+ T cells and of the type I IFN system were absolute requirements for the antitumor effects to occur. The irradiation regimen induced immune infiltrate changes in the irradiated and nonirradiated lesions featured by reductions in the total content of effector T cells, Tregs, and myeloid-derived suppressor cells, while effector T cells expressed more intracellular IFNγ in both the irradiated and contralateral tumors. Importantly, 48 hours after irradiation, CD8+ TILs showed brighter expression of CD137 and PD1, thereby displaying more target molecules for the corresponding mAbs. Likewise, PD1 and CD137 were induced on tumor-infiltrating lymphocytes from surgically excised human carcinomas that were irradiated ex vivo. These mechanisms involving crosspriming and CD8 T cells advocate clinical development of immunotherapy combinations with anti-PD1 plus anti-CD137 mAbs that can be synergistically accompanied by radiotherapy strategies, even if the disease is left outside the field of irradiation. Cancer Res; 76(20); 5994–6005. ©2016 AACR.

Introduction Anti–programmed cell death 1 (anti–PD-1) and anti–programmed cell death ligand 1 (PD-L1) antagonist monoclonal antibodies (mAbs) against metastatic non–small cell lung cancer with special efficacy in patients with squamous cell lung cancer are being developed in the clinic. However, robust and reliable experimental models to test immunotherapeutic combinations in squamous lung tumors are still lacking. Methods We generated a transplantable squamous cell carcinoma cell line (UN-SCC680AJ) from a lung tumor induced by chronic N -nitroso-tris-chloroethylurea mutagenesis in A/J mice. Tumor cells expressed cytokeratins, overexpressed p40, and lacked thyroid transcription factor 1, confirming the squamous lineage reported by histological analysis. More than 200 mutations found in its exome suggested potential for antigenicity. Immunocompetent mice subcutaneously implanted with this syngeneic cell line were treated with anti-CD137 and/or anti–PD-1 mAbs and monitored for tumor growth/progression or assessed for intratumoral leukocyte infiltration using immunohistochemical analysis and flow cytometry. Results In syngeneic mice, large 12-day–established tumors derived from the transplantable cell line variant UN-SCC680AJ were amenable to curative treatment with anti–PD-1, anti–PD-L1, or anti-CD137 immunostimulatory mAbs. Single-agent therapies lost curative efficacy when treatment was started beyond day +17, whereas a combination of anti–PD-1 plus anti-CD137 achieved complete rejections. Tumor cells expressed weak baseline PD-L1 on the plasma membrane, but this could be readily induced by interferon-γ. Combined treatment efficacy required CD8 T cells and induced a leukocyte infiltrate in which T lymphocytes co-expressing CD137 and PD-1 were prominent. Conclusions These promising results advocate the use of combined anti–PD-1/PD-L1 plus anti-CD137 mAb immunotherapy for the treatment of squamous non–small cell lung cancer in the clinical setting.

CD137 (4-1BB, TNF-receptor superfamily 9) is a surface glycoprotein of the TNFR family which can be induced on a variety of leukocyte subsets. On T and NK cells, CD137 is expressed following activation and, if ligated by its natural ligand (CD137L), conveys polyubiquitination-mediated signals via TNF receptor associated factor 2 that inhibit apoptosis, while enhancing proliferation and effector functions. CD137 thus behaves as a bona fide inducible costimulatory molecule. These functional properties of CD137 can be exploited in cancer immunotherapy by systemic administration of agonist monoclonal antibodies, which increase anticancer CTLs and enhance NK-cell-mediated antibody-dependent cell-mediated cytotoxicity. Reportedly, anti-CD137 mAb and adoptive T-cell therapy strongly synergize, since (i) CD137 expression can be used to select the T cells endowed with the best activities against the tumor, (ii) costimulation of the lymphocyte cultures to be used in adoptive T-cell therapy can be done with CD137 agonist antibodies or CD137L, and (iii) synergistic effects upon coadministration of T cells and antibodies are readily observed in mouse models. Furthermore, the signaling cytoplasmic tail of CD137 is a key component of anti-CD19 chimeric antigen receptors that are used to redirect T cells against leukemia and lymphoma in the clinic. Ongoing phase II clinical trials with agonist antibodies and the presence of CD137 sequence in these successful chimeric antigen receptors highlight the importance of CD137 in oncoimmunology.

Background: The immune response to cancer is often conceptualized with the Cancer Immunity Cycle. An essential step in this interpretation is that antigens released by dying tumors are presented by dendritic cells to naive or memory T cells in the tumor-draining lymph nodes. Whether tumor cell death resulting from cytotoxicity, as mediated by T cells or NK lymphocytes, is actually immunogenic currently remains unknown. Methods: In this study, tumor cells were killed by antigen-specific T-cell receptor (TCR) transgenic CD8 T cells or activated natural killer cells. Immunogenic cell death was studied analyzing the membrane exposure of calreticulin and the release of high mobility group box 1 (HMGB1) by the dying tumor cells. Furthermore, the potential immunogenicity of the tumor cell debris was evaluated in immunocompetent mice challenged with an unrelated tumor sharing only one tumor-associated antigen and by class I MHC-multimer stainings. Mice deficient in BATF-3, IFNAR and STING were used to study mechanistic requirements. Results: We observe in co-cultures of tumor cells and effector cytotoxic cells, the presence of markers of immunogenic cell death such as calreticulin exposure and soluble HMGB1 protein. OVA-transfected MC38 colon cancer cells, exogenously pulsed to present the gp100 epitope are killed in culture by mouse gp100 specific TCR transgenic CD8 T cells. Immunization of mice with the resulting destroyed cells induces epitope spreading as observed by detection of OVA-specific T cells by MHC multimer staining and rejection of OVA+ EG7 lymphoma cells. Similar results were observed in mice immunized with cell debris generated by NK-cell mediated cytotoxicity. Mice deficient in BATF3-dependent dendritic cells (cDC1) fail to develop an anti-OVA response when immunized with tumor cells killed by cytotoxic lymphocytes. In line with this, cultured cDC1 dendritic cells uptake and can readily cross-present antigen from cytotoxicity-killed tumor cells to cognate CD8+ T lymphocytes. Conclusion: These results support that an ongoing cytotoxic antitumor immune response can lead to immunogenic tumor cell death. Citation Format: Luna Minute, Alvaro Teijeira, Alfonso Rodriguez Sánchez-Paulete, Maria del Carmen Ochoa, Maite Alvarez, Itziar Otano, Iñaki Etxeberria, Elixabet Bolaños, Arantza Azpilikueta, Saray Garasa, Noelia Casares, José Luiz Pérez-Gracia, Maria Esperanza Rodríguez-Ruiz, Pedro Berraondo, Ignacio Melero. Cellular cytotoxicity is a form of immunogenic cell death [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1698.

Multiple lines of evidence indicate a critical role of antigen cross-presentation by conventional BATF3-dependent type 1 classical dendritic cells (cDC1) in CD8-mediated antitumor immunity. Flt3L and XCL1, respectively, constitute a key growth/differentiation factor and a potent and specific chemoattractant for cDC1. To exploit their antitumor functions in local immunotherapy, we prepared Semliki Forest Virus (SFV)-based vectors encoding XCL1 and soluble Flt3L (sFlt3L). These vectors readily conferred transgene expression to the tumor cells in culture and when engrafted as subcutaneous mouse tumor models. In syngeneic mice, intratumoral injection of SFV-XCL1-sFlt3L (SFV-XF) delayed progression of MC38- and B16-derived tumors. Therapeutic activity was observed and exerted additive effects in combination with anti-PD-1, anti-CD137, or CTLA-4 immunostimulatory mAbs. Therapeutic effects were abolished by CD8β T-cell depletion and were enhanced by CD4 T-cell depletion, but not by T regulatory cell predepletion with anti-CD25 mAb. Antitumor effects were also abolished in BATF3- and IFNAR-deficient mice. In B16-OVA tumors, SFV-XF increased the number of infiltrating CD8 T cells, including those recognizing OVA. Consistently, following the intratumoral SFV-XF treatment courses, we observed increased BATF3-dependent cDC1 among B16-OVA tumor-infiltrating leukocytes. Such an intratumoral increase was not seen in MC38-derived tumors, but both resident and migratory cDC1 were boosted in SFV-XF-treated MC38 tumor-draining lymph nodes. In conclusion, viral gene transfer of sFlt3L and XCL1 is feasible, safe, and biologically active in mice, exerting antitumor effects that can be potentiated by CD4 T-cell depletion. SIGNIFICANCE: These findings demonstrate that transgenic expression of sFLT3L and XCL1 in tumor cells mediates cross-priming of, and elicits potent antitumor activity from, CD8 T lymphocytes, particularly in combination with CD4 T-cell depletion. We are in debt to Eneko Elizalde for excellent animal facility care. Critical reading and suggestions by Drs. Sandra Hervas, Carmen Ochoa, Jose L. Perez-Gracia, Ana Rouzaut, and Juan Jose Lasarte are also much appreciated. This work was supported by grants MINECO SAF 2014-52361-R and SAF 2017-83267-C2-1R and Cancer Research Institute (CRI) CLIP Grant 2017 and the Horizon 2020 Programme from the European Comission (PROCROP project, ref. #635122) to I. Melero; FIS (PI14/01442 and PI17/01859 (to C. Smerdou); and Fundacion Echebano fellowship (to M.C. Ballesteros-Briones). Sí

T and NK lymphocytes express CD137 (4-1BB), a costimulatory receptor of the TNFR family whose function is exploitable for cancer immunotherapy. Mitochondria regulate the function and survival of T lymphocytes. Herein, we show that CD137 costimulation provided by agonist mAb and CD137L (4-1BBL) induced mitochondria enlargement that resulted in enhanced mitochondrial mass and transmembrane potential in human and mouse CD8+ T cells. Such mitochondrial changes increased T-cell respiratory capacities and were critically dependent on mitochondrial fusion protein OPA-1 expression. Mass and function of mitochondria in tumor-reactive CD8+ T cells from cancer-bearing mice were invigorated by agonist mAb to CD137, whereas mitochondrial baseline mass and function were depressed in CD137-deficient tumor reactive T cells. Tumor rejection induced by the synergistic combination of adoptive T-cell therapy and agonistic anti-CD137 was critically dependent on OPA-1 expression in transferred CD8+ T cells. Moreover, stimulation of CD137 with CD137 mAb in short-term cultures of human tumor-infiltrating lymphocytes led to mitochondria enlargement and increased transmembrane potential. Collectively, these data point to a critical link between mitochondrial morphology and function and enhanced antitumor effector activity upon CD137 costimulation of T cells. Cancer Immunol Res; 6(7); 798-811. ©2018 AACR. This project was supported by imCORE Network (Roche Genentech), MINECO (SAF2014-52361-R, 2017-83267-C2-1-R; I. Melero), European Commission VII Framework and Horizon 2020 programs (IACT and PROCROP), Fundacion de la Asociaci on Espa nola Contra el C ~ ancer (AECC), and Fundacion BBVA. A. Teijeira receives support from a Juan de la Cierva contract, MINECO. Electron and light microscopy CIMA services as well as Flow Cytometry CIMA facilities (Diego Alignani) and personnel at blood bank of Navarra are acknowledged for their technical support. We are also grateful to Drs. Lasarte and HervasStubbs for scientific discussion and to Dr. Paul Miller for editing the manuscript. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Sí

Dendritic cells (DCs) are the main professional antigen-presenting cells for induction of T-cell adaptive responses. Cancer cells express tumor antigens, including neoantigens generated by nonsynonymous mutations, but are poor for antigen presentation and for providing costimulatory signals for T-cell priming. Mounting evidence suggests that antigen transfer to DCs and their surrogate presentation on major histocompatibility complex class I and II molecules together with costimulatory signals is paramount for induction of viral and cancer immunity. Of the great diversity of DCs, BATF3/IRF8-dependent conventional DCs type 1 (cDC1) excel at cross-presentation of tumor cell-associated antigens. Location of cDC1s in the tumor correlates with improved infiltration by CD8+ T cells and tumor-specific T-cell immunity. Indeed, cDC1s are crucial for antitumor efficacy using checkpoint inhibitors and anti-CD137 agonist monoclonal antibodies in mouse models. Enhancement and exploitation of T-cell cross-priming by cDC1s offer opportunities for improved cancer immunotherapy, including in vivo targeting of tumor antigens to internalizing receptors on cDC1s and strategies to increase their numbers, activation and priming capacity within tumors and tumor-draining lymph nodes.

Retroviral gene transfer of interleukin-12 (IL-12) into T cells markedly enhances antitumor efficacy upon adoptive transfer but has clinically shown unacceptable severe side effects. To overcome the toxicity, we engineered tumor-specific CD8+ T cells to transiently express IL-12. Engineered T cells injected intratumorally, but not intravenously, led to complete rejections not only of the injected lesion but also of distant concomitant tumors. Efficacy was further enhanced by co-injection with agonist anti-CD137 mAb or by transient co-expression of CD137 ligand. This treatment induced epitope spreading of the endogenous CD8+ T cell immune response in a manner dependent on cDC1 dendritic cells. Mouse and human tumor-infiltrating T lymphocyte cultures can be transiently IL-12 engineered to attain marked immunotherapeutic effects.

Adoptive T-cell therapy with TILs, CAR-T cells, and TCR-engineered T-cells is achieving remarkable clinical efficacy. Retroviral gene transfer of IL-12 into the T cell cultures markedly enhances efficacy but has shown unacceptable severe side effects in the clinic. To ensure transient gene transfer, tumor specific CD8 T cells were engineered by electroporation of mRNA encoding single chain IL-12 into OT1 and Pmel-1 TCR transgenic lymphocytes. Engineered T cells were injected intratumorally and achieved complete rejections not only of the injected lesion but also of distant concomitant tumors. Efficacy was further enhanced if a small dose of agonist anti-CD137 mAb was co-injected or if CD137-ligand mRNA was co-electroporated. Importantly, intratumor T-cell administration was more efficacious that the intravenous or subcutaneous routes. Therapeutic effects were dependent on IFNγ, cDC1 dendritic cells and endogenous T cells. Interestingly, treatment induced epitope spreading of the immune response to antigens not recognized by the adoptively transferred lymphocytes. Efficacy was also achieved when mouse TILs cultures were used and human TIL cultures can also be transiently IL-12 mRNA engineered. Intratumor release, transient IL-12 mRNA-engineering and repeated administration jointly constitute a safe, feasible and powerful cancer immunotherapy strategy. Note: This abstract was not presented at the meeting. Citation Format: Inaki Etxeberria, Elixabet Bolaños, Alvaro Teijeira, Arantza Azpilicueta, Jose Ignacio Quetglas, Alfonso Rodriguez Sanchez-Paulete, Itziar Otano, Uxua Mancheño, Sandra Hervas-Stubbs, Susana Inoges, Saray Garasa, Maite Alvarez, Pedro Berraondo, Ignacio Melero. Intratumor adoptive transfer of IL-12 mRNA transiently engineered anti-tumor CD8+ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2331.

Introduction: Yellow Fever attenuated virus vaccine (17D) induces very potent cellular and humoral immunity. Its delivery in situ in the tumor bed may induce an immune response leading to the killing of infected cells. Experimental procedures: 17D virus was propagated in Vero cells and infectious supernatants were harvested. To evaluate in vitro sensitivity of tumors to 17D infection, a panel of human and mouse tumor cell lines were infected with increasing MOIs and viability was quantified. To evaluate the antitumor efficacy of intratumor (i.t.) injection of 17D, 4x106pfu/50μl were injected (4 doses) in MC38- and in B16-OVA-tumor bearing C57BL/6 mice. Systemic antitumor effects of i.t 17D were examined in bilateral MC38 models in which one of the tumors was left uninjected. Depletion experiments were performed to assess the contribution of CD4, CD8, Gr1 and NK cells. To determine the role of conventional type-I dendritic cells (cDC1), identical experiments were performed in BATF3-/- mice. Tumor and lymph node immune infiltrates were examined by multiparametric flow cytometry. Combination with intraperitoneal anti-PD1 or anti-CD137 was performed to improve antitumor efficacy. To evaluate the effects of 17D-preexisting immunity, mice were preimmunized with 17D prior to MC38 bilateral inoculation and were subsequently treated. To assess the individual cell type contribution to the antitumor effects induced upon 17D pre-immunization, CD8, CD4, CD8+CD4 or sera from preimmunized mice were passively transferred 24h prior to first 17D i.t injection to MC38-tumor bearing mice. Results: 17D live attenuated strain is able to induce cell death of mouse and human tumor cell lines. In MC38 or B16-OVA mice models, repeated i.t. injections of 17D clearly delayed tumor progression. Regarding the systemic effect of i.t. 17D, certain contralateral therapeutic efficacy was induced upon i.t injection in bilateral MC38 models. These local and distant effects were dependent on CD8+ and cDC1 cells. 17D i.t injection increased in CD8+ T cell infiltrates and decreased in Tregs in injected tumors, while in contralateral tumors induced an increase of CD8+ and NK cells expressing key immunomodulatory receptors. I.t. 17D with anti-CD137 monoclonal antibody (mAb) induced a strong synergistic effect, while combination with anti-PD-1 mAb showed mild local additive efficacy compared to 17D monotherapy. Both combinatorial treatments delayed contralateral tumor growth. Notably, 17D preimmunization induced an important improvement of local and distant antitumor immunity. Such beneficial effects of preimmunization were transferred along with CD8+ T cells. Conclusions: 17D can be safely injected i.t. in preclinical models, giving rise to immune-mediate antitumor effects that can be combined with other immunotherapy agents. The repurposed use of this GMP-grade vaccine in preimmunized patients represents a promising and feasible immunotherapy approach. Citation Format: M. Angela Aznar, Carmen Molina, Alvaro Teijeira, Arantza Azpilikueta, Saray Garasa, Alfonso R. Sanchez-Paulete, Iñaki Etxeberria, Maite Alvarez, Pedro Berraondo, Ignacio Melero. Repurposing the yellow fever vaccine for intratumoral immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1474.

Immune mechanisms have evolved to cope with local entry of microbes acting in a confined fashion but eventually inducing systemic immune memory. Indeed, in situ delivery of a number of agents into tumors can mimic in the malignant tissue the phenomena that control intracellular infection leading to the killing of infected cells. Vascular endothelium activation and lymphocyte attraction, together with dendritic cell–mediated cross-priming, are the key elements. Intratumoral therapy with pathogen-associated molecular patterns or recombinant viruses is being tested in the clinic. Cell therapies can be also delivered intratumorally, including infusion of autologous dendritic cells and even tumor-reactive T lymphocytes. Intralesional virotherapy with an HSV vector expressing GM-CSF has been recently approved by the Food and Drug Administration for the treatment of unresectable melanoma. Immunomodulatory monoclonal Abs have also been successfully applied intratumorally in animal models. Local delivery means less systemic toxicity while focusing the immune response on the malignancy and the affected draining lymph nodes.

UNLABELLED: Weak and ineffective antitumor cytotoxic T lymphocyte (CTL) responses can be rescued by immunomodulatory mAbs targeting PD-1 or CD137. Using Batf3(-/-) mice, which are defective for cross-presentation of cell-associated antigens, we show that BATF3-dependent dendritic cells (DC) are essential for the response to therapy with anti-CD137 or anti-PD-1 mAbs. Batf3(-/-) mice failed to prime an endogenous CTL-mediated immune response toward tumor-associated antigens, including neoantigens. As a result, the immunomodulatory mAbs could not amplify any therapeutically functional immune response in these mice. Moreover, administration of systemic sFLT3L and local poly-ICLC enhanced DC-mediated cross-priming and synergized with anti-CD137- and anti-PD-1-mediated immunostimulation in tumor therapy against B16-ovalbumin-derived melanomas, whereas this function was lost in Batf3(-/-) mice. These experiments show that cross-priming of tumor antigens by FLT3L- and BATF3-dependent DCs is crucial to the efficacy of immunostimulatory mAbs and represents a very attractive point of intervention to enhance their clinical antitumor effects. SIGNIFICANCE: Immunotherapy with immunostimulatory mAbs is currently achieving durable clinical responses in different types of cancer. We show that cross-priming of tumor antigens by BATF3-dependent DCs is a key limiting factor that can be exploited to enhance the antitumor efficacy of anti-PD-1 and anti-CD137 immunostimulatory mAbs. Work at the I. Melero lab is funded by MICINN (SAF200803294 and SAF2011-22831), Departamento de salud del Gobierno de Navarra, Redes temáticas de investigación cooperativa RETIC (RD06/0020/0065), and the European commission 7th framework program (ENCITE and IACT). Work in the D. Sancho laboratory is funded by the CNIC and grants from the Spanish Ministry of Economy and Competitiveness (SAF-2013-42920R) and the European Research Council (ERC Starting Independent Researcher Grant 2010, ERC-2010-StG 260414). The CNIC is supported by the Spanish Ministry of Economy and Competitiveness and the Pro-CNIC Foundation. I. Melero and D. Sancho are funded by the European Commission (635122-PROCROP H2020). Sí

CD69 is an early activation marker on the surface of T lymphocytes undergoing activation by cognate antigen. We observed intense expression of CD69 on tumor-infiltrating T-lymphocytes that reside in the hypoxic tumor microenvironment and hypothesized that CD69 could be, at least partially, under the control of the transcriptional hypoxia response. In line with this, human and mouse CD3-stimulated lymphocytes cultured under hypoxia (1% O2) showed increased expression of CD69 at the protein and mRNA level. Consistent with these findings, mouse T lymphocytes that had recently undergone hypoxia in vivo, as denoted by pimonidazole staining, were more frequently CD69+ in the tumor and bone marrow hypoxic tissue compartments. We found evidence for HIF-1α involvement both when using T-lymphocytes from inducible HIF-1α−/− mice and when observing tumor-infiltrating T-lymphocytes in mice whose T cells are HIF-1α−/−. Direct pro-transcriptional activity of HIF-1α on a newly identified hypoxia response element (HRE) found in the human CD69 locus was demonstrated by ChIP experiments. These results uncover a connection between the HIF-1α oxygen-sensing pathway and CD69 immunobiology.

Virotherapy and checkpoint inhibitors can be combined for the treatment of cancer with complementarity and potential for synergistic effects. We have developed a cytolytic but nonreplicative viral vector system based on Semliki Forest virus that encodes IL12 (SFV-IL12). Following direct intratumoral injection, infected cells release transgenic IL12, die, and elicit an inflammatory response triggered by both abundantly copied viral RNA and IL12. In difficult-to-treat mouse cancer models, such as those derived from MC38 and bilateral B16-OVA, SFV-IL12 synergized with an anti–PD-1 monoclonal antibody (mAb) to induce tumor regression and prolong survival. Similar synergistic effects were attained upon PD-L1 blockade. Combined SFV-IL12 + anti–PD-1 mAb treatment only marginally increased the elicited cytotoxic T-lymphocyte response over SFV-IL12 as a single agent, at least when measured by in vivo killing assays. In contrast, we observed that SFV-IL12 treatment induced expression of PD-L1 on tumor cells in an IFNγ-dependent fashion. PD-L1–mediated adaptive resistance thereby provides a mechanistic explanation of the observed synergistic effects achieved by the SFV-IL12 + anti–PD-1 mAb combination. Cancer Immunol Res; 3(5); 449–54. ©2015 AACR.

Radiotherapy is considered an efficacious local tool to erradicate or at least control cancer progression. However, recent lines of preclinical and clinical evidence indicate that proimmune effects of radiotherapy can be synergistically augmented with immunostimulatory monoclonal antibodies (mAb) to act both on irradiated tumor lessions and on distant, non-irradiated tumor sites. The combination of radiotherapy with immunostimulatory anti-PD1 and anti-CD137 mAbs was conducive to favourable effects on distant non-irradiated tumor lesions as observed on transplanted MC38 (colorectal cancer), B16OVA (melanoma) and 4T1 (breast cancer) models. Immunotherapy and radiotherapy synergized both when irradiation was given using external beams or provided with brachytherapy. The therapeutic activity was crucially performed by CD8 T cells, as found in selective depletion experiments. The irradiation regimen induced immune infiltrate changes in the irradiated and non-irradiated lesions featured by reductions in the content of effector T cells, Tregs, and myeloid-derived supresor cells (MDSC), while effector T cells were expressing more intracellular IFN gamma in both the irradiated and contralateral tumors. Importantly, 48h following irradiation CD8+ TILs showed brighter expression of CD137 and PD-1 thereby displaying more target molecules for the activity of the corresponding monoclonal antibodies. Likewise, PD-1 and CD137 were induced on tumor infiltrating lymphocytes from surgically excised of human carcinoma lessions that were irradiated ex-vivo. These findings advocate for clinical development of immunotherapy combinations with anti-PD1 plus anti-CD137 mAbs that can be synergistically accompained by radiotherapy strategies on treatable lesions, even if leaving disease outside the irradiation field. Citation Format: MariaE. Rodríguez-Ruiz, Inmaculada Rodriguez, Saray Garasa, Benigno Barbes, Jose Luis Solorzano, Jose Luis Perez Gracia, Sara Labiano, Arantza Azpilikueta, Elixabet Bolanos, Alfonso R. Sanchez-Paulete, M. Angela Aznar, Ana Rouzaut, Maria Jure-Kunkel, Iñaki Etxeberria, Carlos Alfaro, Carmen Oñate, Mariano Ponz, Ignacio Melero. Improving radiotherapy abscopal effects with anti-PD1 and anti-CD137-based immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4012.

Weak and ineffective antitumor cytotoxic T lymphocyte (CTL) responses can be rescued by immunomodulatory monoclonal antibodies (mAbs) targeting PD-1 or CD137. Using Batf3-/- mice, which are defective for cross-presentation of cell-associated antigens, we show that Batf3-dependent dendritic cells (DCs) are essential for the response to therapy with anti-CD137 or anti-PD-1 mAbs. Batf3-/- mice failed to prime an endogenous CTL-mediated immune response toward tumor-associated antigens, including neoantigens. As a result, the immunomodulatory mAbs could not amplify any therapeutically functional immune response in these mice. Moreover, administration of systemic sFlt3L and local poly-ICLC enhanced DC-mediated cross-priming and synergized with anti-CD137- and anti-PD-1-mediated immunostimulation in tumor therapy against B16-OVA-derived melanomas, whereas this function was lost in Batf3-/- mice. These experiments show that cross-priming of tumor antigens by Flt3L- and Batf3-dependent DCs is crucial to the efficacy of immunostimulatory mAbs and represents a very attractive point of intervention to enhance their clinical antitumor effects. Citation Format: Alfonso R. Sánchez-Paulete, Francisco J. Cueto, María Martínez-López, Sara Labiano, Aizea Morales-Kastresana, Maria E. Rodríguez-Ruiz, Maria Jure-Kunkel, Arantza Azpilikueta, José I. Quetglas, David Sancho, Ignacio Melero. Cancer immunotherapy with immunomodulatory anti-CD137 and anti-PD-1 monoclonal antibodies requires Batf3-dependent dendritic cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4908.

Hypoxia is a common feature in solid tumors that has been implicated in immune evasion. Previous studies from our group have shown that hypoxia upregulates the co-stimulatory receptor CD137 on activated T lymphocytes and on vascular endothelial cells. In this study, we show that exposure of mouse and human tumor cell lines to hypoxic conditions (1% O2) promotes CD137 transcription. However, the resulting mRNA is predominantly an alternatively spliced form that encodes for a soluble variant, lacking the transmembrane domain. Accordingly, soluble CD137 (sCD137) is detectable by ELISA in the supernatant of hypoxia-exposed cell lines and in the serum of tumor-bearing mice. sCD137, as secreted by tumor cells, is able to bind to CD137-Ligand (CD137L). Our studies on primed T lymphocytes in co-culture with stable transfectants for CD137L demonstrate that tumor-secreted sCD137 prevents co-stimulation of T lymphocytes. Such an effect results from preventing the interaction of CD137L with the transmembrane forms of CD137 expressed on T lymphocytes undergoing activation. Indeed, silencing CD137 with shRNA renders more immunogenic tumor-cell variants upon inoculation to immunocompetent mice but which readily grafted on immunodeficient or CD8+ T-cell-depleted mice. These mechanisms are interpreted as a molecular strategy deployed by tumors to repress lymphocyte co-stimulation via CD137/CD137L.

CD137 (4-1BB) is a surface protein initially discovered to mark activated T lymphocytes. However, its broader expression pattern also encompasses activated NK cells, B cells and myeloid cells, including mature dendritic cells. In this study, we have immunostained for CD137 on paraffin-embedded lymphoid tissues including tonsils, lymph nodes, ectopic tertiary lymphoid tissue in Hashimoto thyroiditis and cancer. Surprisingly, immunostaining mainly decorated intrafollicular lymphocytes in the tissues analyzed, with only scattered staining in interfollicular areas. Moreover, pathologic lymphoid follicles in follicular lymphoma and tertiary lymphoid tissue associated with non-small cell lung cancer showed a similar pattern of immunostaining. Multispectral fluorescence cytometry demonstrated that CD137 expression was restricted to CD4+ CXCR5+ follicular T helper lymphocytes (TFH cells) in tonsils and lymph nodes. Short-term culture of lymph node cell suspensions in the presence of either an agonistic anti-CD137 monoclonal antibody (mAb) or CD137-ligand stimulated the functional upregulation of TFH cells in 3 out of 6 cases, as indicated by CD40L surface expression and cytokine production. As a consequence, immunostimulatory monoclonal antibodies targeting CD137 (such as urelumab and PF-05082566) should be expected to primarily act on this lymphocyte subset, thus modifying ongoing humoral immune responses in patients with autoimmune disease and cancer.