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PURPOSE Limited prospective data are available on sequential immunotherapy and BRAF/MEK inhibition for BRAFV600-mutant metastatic melanoma. METHODS SECOMBIT is a randomized, three-arm, noncomparative phase II trial (ClinicalTrials.gov identifier: NCT02631447 ). Patients with untreated, metastatic BRAFV600-mutant melanoma from 37 sites in nine countries were randomly assigned to arm A (encorafenib [450 mg orally once daily] plus binimetinib [45 mg orally twice daily] until progressive disease [PD] -> ipilimumab plus nivolumab [ipilimumab 3 mg/kg once every 3 weeks and nivolumab 1 mg/kg once every 3 weeks × four cycles -> nivolumab 3 mg/kg every 2 weeks]), arm B [ipilimumab plus nivolumab until PD -> encorafenib plus binimetinib], or arm C (encorafenib plus binimetinib for 8 weeks -> ipilimumab plus nivolumab until PD -> encorafenib plus binimetinib). The primary end point was overall survival (OS) at 2 years. Secondary end points included total progression-free survival, 3-year OS, best overall response rate, duration of response, and biomarkers in the intent-to-treat population. Safety was analyzed throughout sequential treatment in all participants who received at least one dose of study medication. RESULTS A total of 209 patients were randomly assigned (69 in arm A, 71 in arm B, and 69 in arm C). At a median follow-up of 32.2 (interquartile range, 27.9-41.6) months, median OS was not reached in any arm and more than 30 patients were alive in all arms. Assuming a null hypothesis of median OS of ≤ 15 months, the OS end point was met for all arms. The 2-year and 3-year OS rates were 65% (95% CI, 54 to 76) and 54% (95% CI, 41 to 67) in arm A, 73% (95% CI, 62 to 84) and 62% (95% CI, 48 to 76) in arm B, and 69% (95% CI, 59 to 80) and 60% (95% CI, 58 to 72) in arm C. No new safety signals emerged. CONCLUSION Sequential immunotherapy and targeted therapy provide clinically meaningful survival benefits for patients with BRAFV600-mutant melanoma.

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.

Interleukin-8 (CXCL8) produced in the tumor microenvironment correlates with poor response to checkpoint inhibitors and is known to chemoattract and activate immunosuppressive myeloid leukocytes. In human cancer, IL8 mRNA levels correlate with IL1B and TNF transcripts. Both cytokines induced IL-8 functional expression from a broad variety of human cancer cell lines, primary colon carcinoma organoids, and fresh human tumor explants. Although IL8 is absent from the mouse genome, a similar murine axis in which TNFα and IL-1β upregulate CXCL1 and CXCL2 in tumor cells was revealed. Furthermore, intratumoral injection of TNFα and IL-1β induced IL-8 release from human malignant cells xenografted in immunodeficient mice. In all these cases, the clinically used TNFα blockers infliximab and etanercept or the IL-1β inhibitor anakinra was able to interfere with this pathogenic cytokine loop. Finally, in paired plasma samples of patients with cancer undergoing TNFα blockade with infliximab in a clinical trial, reductions of circulating IL-8 were substantiated.Significance:IL-8 attracts immunosuppressive protumor myeloid cells to the tumor microenvironment, and IL-8 levels correlate with poor response to checkpoint inhibitors. TNFα and IL-1β are identified as major inducers of IL-8 expression on malignant cells across cancer types and models in a manner that is druggable with clinically available neutralizing agents.This article is highlighted in the In This Issue feature, p. 2007

This first-in-human study evaluated RO7122290, a bispecific fusion protein carrying a split trimeric 4-1BB (CD137) ligand and a fibroblast activation protein α (FAP) binding site that costimulates T cells for improved tumor cell killing in FAP-expressing tumors. Patients with advanced or metastatic solid tumors received escalating weekly intravenous doses of RO7122290 as a single agent ( n = 65) or in combination with a 1200-milligram fixed dose of the anti–programmed death-ligand 1 (anti–PD-L1) antibody atezolizumab given every 3 weeks ( n = 50), across a tested RO7122290 dose range of 5 to 2000 milligrams and 45 to 2000 milligrams, respectively. Three dose-limiting toxicities were reported, two at different RO7122290 single-agent doses (grade 3 febrile neutropenia and grade 3 cytokine release syndrome) and one for the combination (grade 3 pneumonitis). No maximum tolerated dose was identified. The pharmacokinetic profile of RO7122290 suggested nonlinearity in elimination. The observed changes in peripheral and tissue pharmacodynamic (PD) biomarkers were consistent with the postulated mechanism of action. Treatment-induced PD changes included an increase in proliferating and activated T cells in peripheral blood both in the single-agent and combination arms. Increased infiltration of intratumoral CD8 + and Ki67 + CD8 + T cells was observed for both treatment regimens, accompanied by the up-regulation of T cell activation genes and gene signatures. Eleven patients experienced a complete or partial response, six of whom were confirmed to be immune checkpoint inhibitor naive. These results support further evaluation of RO7122290 in combination with atezolizumab or other immune-oncology agents for the treatment of solid tumors.

Supplementary Figure from A Therapeutically Actionable Protumoral Axis of Cytokines Involving IL-8, TNFα, and IL-1β

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.

Interleukin-8 (CXCL8) produced in the tumor microenvironment correlates with poor response to checkpoint inhibitors and is known to chemoattract and activate immunosuppressive myeloid leukocytes. In human cancer, IL8 mRNA levels correlate with IL1B and TNF transcripts. Both cytokines induced IL-8 functional expression from a broad variety of human cancer cell lines, primary colon carcinoma organoids, and fresh human tumor explants. Although IL8 is absent from the mouse genome, a similar murine axis in which TNFα and IL-1β upregulate CXCL1 and CXCL2 in tumor cells was revealed. Furthermore, intratumoral injection of TNFα and IL-1β induced IL-8 release from human malignant cells xenografted in immunodeficient mice. In all these cases, the clinically used TNFα blockers infliximab and etanercept or the IL-1β inhibitor anakinra was able to interfere with this pathogenic cytokine loop. Finally, in paired plasma samples of patients with cancer undergoing TNFα blockade with infliximab in a clinical trial, reductions of circulating IL-8 were substantiated.Significance:IL-8 attracts immunosuppressive protumor myeloid cells to the tumor microenvironment, and IL-8 levels correlate with poor response to checkpoint inhibitors. TNFα and IL-1β are identified as major inducers of IL-8 expression on malignant cells across cancer types and models in a manner that is druggable with clinically available neutralizing agents.This article is highlighted in the In This Issue feature, p. 2007

Supplementary Figure Legens

Experimental treatments (A) and tumor size follow-up until sacrifice (B) of tumors used to prepare cell suspensions for figures 3, 5 and supplementary figure 4. (C) Shows individual tumor lesion weights used to calculate lymphocytes/gram densities.

FACS analyses on cell suspensions (n=4) derived from non-irradiated MC38-derived tumors

Table with Details of Patient Characteristics

Checkpoint inhibitors are being added to standard-of-care chemotherapy in multiple clinical trials. Success has been reported in non–small and small cell lung carcinomas and urothelial, head and neck, gastric, and esophageal cancers, and promising results are already available in triple-negative breast and pancreatic malignancies. The potential mechanisms of synergy include immunogenic tumor cell death, antiangiogenesis, selective depletion of myeloid immunosuppressive cells, and lymphopenia, which reduces regulatory T cells and makes room for proliferation of effector T cells. However, chemotherapy regimens have not been optimized for such combinations, perhaps explaining some recent clinical trial disappointments. Approaches to make the most of chemoimmunotherapy include neoadjuvant and adjuvant schemes.Significance: Immunotherapy of cancer based on PD-1/PD-L1 blockade has prompted a revolution in cancer clinical management. Evidence in phase III clinical trials already supports combinations of immunotherapy with standard-of-care chemotherapy for a number of malignant diseases. This review focuses on such evidence and provides an overview of the potential synergistic mechanisms of action and the opportunities to optimize chemoimmunotherapy regimens.

Myeloid cell content (density per gram) in cell suspension from non-irradiated tumors as those in figure 5. Macrophages are defined as CD45+ CD11b+ F4/80+; Dendritic cells (DC) as CD45+CD11b+CD11c+; myeloid-derived suppressor cells (MDSC) as CD45+CD11b+Ly6C+ or CD45+CD11b+Ly6G+ to estimate monocytic (M-MDSC) and granulocytic (G-MDSC) subtypes.

Radiotherapy can be synergistically combined with immunotherapy in mouse models, extending its efficacious effects outside of the irradiated field (abscopal effects). We previously reported that a regimen encompassing local radiotherapy in combination with anti-CD137 plus anti–PD-1 mAbs achieves potent abscopal effects against syngeneic transplanted murine tumors up to a certain tumor size. Knowing that TGFβ expression or activation increases in irradiated tissues, we tested whether TGFβ blockade may further enhance abscopal effects in conjunction with the anti–PD-1 plus anti-CD137 mAb combination. Indeed, TGFβ blockade with 1D11, a TGFβ-neutralizing mAb, markedly enhanced abscopal effects and overall treatment efficacy against subcutaneous tumors of either 4T1 breast cancer cells or large MC38 colorectal tumors. Increases in CD8 T cells infiltrating the nonirradiated lesion were documented upon combined treatment, which intensely expressed Granzyme-B as an indicator of cytotoxic effector capability. Interestingly, tumor tissue but not healthy tissue irradiation results in the presence of higher concentrations of TGFβ in the nonirradiated contralateral tumor that showed smad2/3 phosphorylation increases in infiltrating CD8 T cells. In conclusion, radiotherapy-induced TGFβ hampers abscopal efficacy even upon combination with a potent immunotherapy regimen. Therefore, TGFβ blockade in combination with radioimmunotherapy results in greater efficacy.

Methods for quantitative immunofluorescence and IFN gamma treatment of PDXs

Supplementary Tables S1-5

Assessment of spontaneous lung metastases of 4T1 tumors in the indicated treatment groups corresponding to mice treated as in figure 2A. Metastases were detected and quantitated -number of lesions in the surface of the lung counted upon inspection under a magnification lens (left) and by real time RT-PCR for tumor specific gp70 RNA (right)-.

Figures S1-14 and Legends

Specific mechanisms by which tumor-infiltrating lymphocytes (TIL) become dysfunctional remain poorly understood. Here, we employed a two-pronged approach using single-cell mass cytometry and tissue imaging technologies to dissect TILs from 25 patients with resectable and 35 patients with advanced non–small cell lung cancer (NSCLC). We identified a burned-out CD8+ TIL subset (Ebo) that specifically accumulated within the tumor microenvironment (TME) but not in adjacent nontumoral tissues. Ebo showed the highest expression of proliferation and activation markers but produced the lowest amount of IFNγ and were the most apoptotic CD8+ TIL subset. Using a humanized patient-derived tumor xenograft model, we demonstrated that Ebo expansion occurred within the TME in a PD-1/B7-H1 pathway-dependent manner. Ebo abundance in baseline tumor tissues was associated with resistance to anti-PD therapy in patients with NSCLC. Our study identifies a dysfunctional TIL subset, with distinct features from previously described exhausted T cells, and implies strategies to overcome immunotherapy resistance.Significance:We identified a highly proliferative, overactivated, and apoptotic dysfunctional CD8+ tumor-infiltrating subpopulation that is functionally distinct from previously described exhausted T cells. This population is expanded in lung cancer tissues in a PD-1/B7-H1-dependent manner, and its abundance is associated with resistance to cancer immunotherapy, thus becoming a potential tissue biomarker.This article is highlighted in the In This Issue feature, p. 1601

Supplementary information regarding chemoimmunotehrapy clinical trials other than phase III or ongoing.

Supplementary Figures S1-S8

PDF file - 946K, Anti-CTLA-4 mAb treatment fails to extend survival of c-myc OVA tg+

PDF file - 953K, TILs recognize unknown antigens in JMJ cell lines.

PDF file - 129K

Purpose: Immunostimulatory monoclonal antibodies (ISmAb) that unleash antitumor immune responses are showing efficacy in cancer clinical trials. Anti-B7-H1 (PD-L1) monoclonal antibodies (mAb) block a critical inhibitory pathway in T cells, whereas anti-CD137 and OX40 mAbs provide T-cell costimulation. A combination of these ISmAbs (anti-CD137 + anti-OX40 + anti-B7-H1) was tested using a transgenic mouse model of multifocal and rapidly progressing hepatocellular carcinoma, in which c-myc drives transformation and cytosolic ovalbumin (OVA) is expressed in tumor cells as a model antigen.Experimental Design: Flow-cytometry and immunohistochemistry were used to quantify tumor-infiltrating lymphocytes (TIL) elicited by treatment and assess their activation status and cytolytic potential. Tolerance induction and its prevention/reversal by treatment with the combination of ISmAbs were revealed by in vivo killing assays.Results: The triple combination of ISmAbs extended survival of mice bearing hepatocellular carcinomas in a CD8-dependent fashion and synergized with adoptive T-cell therapy using activated OVA-specific TCR-transgenic OT-1 and OT-2 lymphocytes. Mice undergoing therapy showed clear increases in tumor infiltration by activated and blastic CD8+ and CD4+ T lymphocytes containing perforin/granzyme B and expressing the ISmAb-targeted receptors on their surface. The triple combination of ISmAbs did not result in enhanced OVA-specific cytotoxic T lymphocyte (CTL) activity but other antigens expressed by cell lines derived from such hepatocellular carcinomas were recognized by endogenous TILs. Adoptively transferred OVA-specific OT-1 lymphocytes into tumor-bearing mice were rendered tolerant, unless given the triple mAb therapy.Conclusion: Extension of survival and dense T-cell infiltrates emphasize the translational potential of combinational immunotherapy strategies for hepatocellular carcinoma. Clin Cancer Res; 19(22); 6151–62. ©2013 AACR.

Supplementary Figure 4. Comparison of IL-8 serum concentrations with the serum concentration of IL-6 and c-reactive protein (CRP) in available samples from 14 melanoma and 19 RCC patients from Figure 2A. (**, p

single dose radiotherapy and hypofractionated doses increase the level of CD137 and PD-1 expression on the surface of tumor-infiltrating T cells.

Supplementary Figure 1B. Time course of the serum concentrations of IL-8 (lower panels) and tumor volume (upper panels) in Rag2-/-IL2Rγc-/- mice xenografted with the indicated cell lines. Individual mice are represented and at the time point indicated by the arrow the tumor was surgically removed. Serum IL-8 concentrations are depicted at 2h and 24h after surgery.

Supplementary Figure 2. Serum IL-8 levels before (day -1) and after (day 5-7) disease-reduction surgery of 7 patients: 1 hepatocellular carcinoma (HCC); 2 colorectal carcinoma (CRC); 1 pancreatic cancer (PC); 3 renal cell carcinoma (RCC).

PDF file - 2976K, HCC cell lines derived from c-myc OVA tg+ mice express OVA and functional antigen presenting molecules.

Radiotherapy reduced the density and absolute numbers of CD4+, CD8+ and CD25+FOXP3+ T cells and Myeloid-derived suppressor cells both in the irradiated and non-irradiated lesions.

Modulation of PD-L1, IDO-1 and B7-H4 levels by cytokines in lung adenocarcinoma cells.

Supplementary figure 3. Number of MDSC in peripheral blood of patients using osmotic lysis of erythrocytes instead of samples from ficoll-gradient centrifugation. Experiments as those shown in figure 3B representing the percentage of positively stained cells analyzed by FACS merely following osmotic lysis of erythrocytes instead of Ficoll gradient enrichment.

PDF file - 1620K, Combo3 treatment induces infiltrates of blastic T lymphocytes and increases the CD8/Treg ratio.

Supplementary video 2. IL-8 induces NET extrusion of GrMDSCs to entrap or adhere HT29 tumor cells. GrMDSCs nuclei were pre-stained with DRAQ5, (cyan), HT29 cells with PKH26 cell membrane dye (Red seen intracellular vesicles) and treated or not with IL-8. The non-cell permeant DNA probe (SYTOXgreen/green) is present in the culture media and only stains DNA when it is extruded extracelullarly. Image on the left shows the complete field of view with several GrMDSCs and an inset of an individual GrMDSC interacting with tumor cells but not extruding NETs since no IL-8 was added to the medium. Image on the left shows the complete microscopy field with several GrMDSCs and an inset with three GrMDSC contacting with HT29 cells and entrapping or adhering to them by formation of the NETs induced upon addition of IL-8. Video is a maximum intensity projection of a Z stack, (DiC is shown for contrast). Video is 300x accelerated.

Statistical comparisons for figures 1, 2, 4 and supplementary figure 1.

Supplementary video 1. IL-8 induces NET extrusion of GrMDSCs using time-lapse in vivo confocal fluorescence microsocopy. GrMDSCs nuclei were pre-stained with DRAQ5 (Red) and treated with IL-8. The non-cell permeant green DNA probe (SYTOXgreen) is present in the culture media and only stains DNA when is extruded extracelullarly. Video represent over five hours a maximum intensity projection of a Zstack. Video is 600x accelerated

4T1-derived spontaneous lung metastases