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Prophylactic TNF blockade uncouples efficacy and toxicity in dual CTLA-4 and PD-1 immunotherapy

Abstract

Combined PD-1 and CTLA-4-targeted immunotherapy with nivolumab and ipilimumab is effective against melanoma, renal cell carcinoma and non-small-cell lung cancer1,2,3. However, this comes at the cost of frequent, serious immune-related adverse events, necessitating a reduction in the recommended dose of ipilimumab that is given to patients4. In mice, co-treatment with surrogate anti-PD-1 and anti-CTLA-4 monoclonal antibodies is effective in transplantable cancer models, but also exacerbates autoimmune colitis. Here we show that treating mice with clinically available TNF inhibitors concomitantly with combined CTLA-4 and PD-1 immunotherapy ameliorates colitis and, in addition, improves anti-tumour efficacy. Notably, TNF is upregulated in the intestine of patients suffering from colitis after dual ipilimumab and nivolumab treatment. We created a model in which Rag2−/−Il2rg−/− mice were adoptively transferred with human peripheral blood mononuclear cells, causing graft-versus-host disease that was further exacerbated by ipilimumab and nivolumab treatment. When human colon cancer cells were xenografted into these mice, prophylactic blockade of human TNF improved colitis and hepatitis in xenografted mice, and moreover, immunotherapeutic control of xenografted tumours was retained. Our results provide clinically feasible strategies to dissociate efficacy and toxicity in the use of combined immune checkpoint blockade for cancer immunotherapy.

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Acknowledgements

We thank M. Fernandez de Sanmamed and I. Etxeberria for scientific discussion, P. Miller for English editing and X. Morales for ultrasound imaging. The figures contain elements from Servier Medical Art (https://smart.servier.com/), licensed under Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/). This work was supported by the International Immuno-Oncology Network (II-ON) from Bristol-Myers Squibb; a Worldwide Cancer Research Grant (15-1146); the Asociación Española Contra el Cancer (AECC) Foundation under grant GCB15152947MELE; the Instituto Carlos III (under grants PI14/01686, PI13/00207 and PI16/00668) co-financed with FEDER funds; and the European Union's Horizon 2020 Program (grant agreement no. 635122 PROCROP). P.B. is supported by a Miguel Servet II (CPII15/00004) contract from Instituto de Salud Carlos III; E.P.-R is supported by the Carmen Lavigne training program of the Asociación Española contra el Cancer and by Consejeria de Salud de la Junta de Andalucía; and A.T. has received financial support through la Caixa Banking Foundation (LCF/BQ/LR18/11640014).

Reviewer information

Nature thanks Frances Balkwill, Kevin Tracey and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

E.P.-R., L.M., P.B. and I.M. designed experiments. E.P.-R., V.B., C.M. and M.C.O. performed the in vivo experiments in Figs. 1, 2 and Extended Data Fig. 1. L.M. performed the experiments in Figs. 3a–f, 4c–g and Extended Data Fig. 8. I.O. performed the in vitro experiments with human PBMCs (Fig. 3g and Extended Data Fig. 7) and in vivo experiments with the anti-IL6 monoclonal antibody (Extended Data Fig. 2). Maite Alvarez performed the ELISA determinations in Extended Data Figs. 3, 4, and flow cytometry experiments in Extended Data Figs. 5, 6. A.T. contributed to the experiment in Extended Data Fig. 5. C.d.A. performed immunohistochemistry analysis and scored intestinal inflammation (Fig. 1e). M.E.R.-R., J.L.P.-G., I.M.-R. and C.L. provided human tissue samples (Fig. 4a, b). Martina Alvarez and V.D.L. performed NanoString analysis (Fig. 4a, b). L.M. and P.B. performed all statistical analyses. E.P.-R., L.M., P.B. and I.M. analysed the data. I.M. wrote the manuscript, assisted by E.P.-R, L.M. and P.B. for the figures. All authors performed a critical revision of the manuscript content and gave their final approval.

Competing interests

I.M. reports advisory roles with Roche-Genentech, Bristol-Myers Squibb, CYTOMX, Incyte, MedImmune, Tusk, F-Star, Genmab, Molecular Partners, Alligator, Bioncotech, MSD, Merck-Serono and Bayer, and research funding from Roche, BMS, Alligator and Bioncotech. P.B. reports advisory roles with Tusk and Moderna, research funding from Sanofi, Moderna and Bavarian Nordic and speaker honoraria from BMS, MSD, Novartis and AstraZeneca. I.M.-R. reports advisory roles with Roche-Genentech, Bristol-Myers Squibb, Incyte, Merck, Amgen, Pierre Fabre, Novartis, and Bioncotech. J.L.P.-G. reports advisory roles with Roche, MSD and BMS, travel support from Roche, BMS and MSD and research funding from Roche, BMS, MSD, Ipsen, Eisai, Incyte and Janssen. E.P.-R. reports speaker honoraria and travel support from BMS, MSD and Novartis. The rest of the authors have no conflict of interest to declare.

Correspondence to Pedro Berraondo or Ignacio Melero.

Extended data figures and tables

Extended Data Fig. 1 Anti-tumour activity of double immune checkpoint blockade on B16-OVA-derived tumours on TNF blockade.

a, Schematic representation of experiments. Experiments were performed as in Fig. 2a, but on mice bearing B16-OVA-derived tumours, treated in different groups. b, Overall survival of treatment groups. The numbers of biologically independent mice are indicated in c. P values were calculated using a two-sided log-rank test. c, Individual follow-up of tumour size, depicting the fraction of mice that completely rejected their tumours. One representative experiment out of two is shown. Source data

Extended Data Fig. 2 Prophylactic IL-6 blockade hinders the anti-tumour activity of the combined anti-PD-1 and anti-CTLA-4 immunotherapy regimen.

a, Schematic representation of the treatments applied to mice that were subcutaneously engrafted with MC38 colon carcinoma cells (the results from these mice are presented in b). b, Individual follow-up of tumour mean diameters, depicting the fraction of mice that completely rejected established tumours. c, Schematic representation of the treatments applied to mice that were subcutaneously engrafted with B16-OVA melanoma cells (the results from these mice are presented in d). d, Individual follow-up of tumour size, as in b. Data are pooled from two independent experiments. Source data

Extended Data Fig. 3 Administration of etanercept causes mice to develop anti-drug antibodies.

An assay to detect anti-drug antibodies was performed on serum that was collected from MC38 tumour-bearing mice 4 or 8 days after the start of treatment with TNF blockade (as in Fig. 2d). a, c, Percentages of normalized absorbance. Percentages reflect the levels of antibody against rat anti-TNF or anti-etanercept, as measured by ELISA on day 4 (a) and day 8 (c). b, d, EC50 values for day 4 (b) and day 8 (d). Data are mean ± s.d. n = 15 biologically independent mice for DSS, n = 13 for DSS + ICB + anti-TNF and n = 9 for DSS + ICB + etanercept. P values were calculated using a one-way ANOVA followed by Dunnett’s test. A sigmoidal dose–response equation was used to determine EC50. The continuous line represents the nonlinear regression curve fit, and the dotted line represents the s.d. Data are pooled from three independent experiments. Source data

Extended Data Fig. 4 Increased concentrations of TNF in the tumour microenvironment after immune checkpoint blockade therapy.

Tumour homogenates were prepared from mice 24 h after they finished the treatment regimen described in Fig. 2d. Levels of TNF in the tumour are shown. Data are mean ± s.d. n = 4 biologically independent mice. P values were calculated using a two-sided t-test. TNF was undetectable in serum samples from the same mice. One representative experiment out of two is shown. Source data

Extended Data Fig. 5 Downregulation of PD-1 expression on tumour-infiltrating antigen-specific CD8+ T cells following the combined anti-PD-1 and anti-CTLA-4 immunotherapy regimen.

Tumours from MC38 and B16-OVA mouse models were collected 24 h after the mice finished the treatment regimen described in Fig. 2d, and cell suspensions were analysed by flow cytometry. a, The percentage of total TCRβ+CD8+ T cells among viable CD45+ cells for MC38 (top) and B16-OVA (bottom) tumours. b, Median fluorescence intensity (MFI) of surface PD-1 for PD-1+ cells previously gated on viable CD45+CD19TCRβ+CD8+ gp70 pentamer+ cells (top) or OVA tetramer+ cells (bottom). c, MFI of surface TIM3 for TIM3+ cells previously gated on viable CD45+CD19TCRβ+CD8+gp70 pentamer+ cells (top) or CD45+CD19TCRβ+CD8+OVA tetramer+ cells (bottom). Data are mean ± s.d. For the MC38 models (top), n = 7 biologically independent mice for DSS and DSS + ICB and n = 6 for the groups treated with anti-TNF or etanercept; for the B16-OVA models (bottom), n = 6 for DSS + ICB+ anti-TNF and n = 10 for the other groups. P values were calculated using a one-way ANOVA followed by Dunnett’s test, and each condition was compared with the DSS group as a control. d, Representative contour plots of PD-1 and TIM3 in the experimental groups described in b for viable CD45+CD19TCRβ+CD8+gp70 pentamer+ cells. Fluorescence minus one (FMO) negative controls were used. Data are representative of two independent experiments. Source data

Extended Data Fig. 6 Expression of T cell exhaustion-related markers on T cells following immune checkpoint blockade treatment with or without TNF blockade.

af, Tumour-specific CD8+ T cells recognizing gp70 (top) or OVA (bottom), in tumour-cell suspensions that were derived one day after completion of the indicated treatments, were analysed by multicolour flow cytometry. Expression of surface CTLA-4 (a), LAG3 (b), BTLA (c), 2B4 (d), CD160 (e) and intracellular Ki67 (f) are shown. Data are MFI (mean ± s.d.) for surface markers (ae) and percentage of positive cells (mean ± s.d.) for Ki67 (f). For the MC38 models (top), n = 3; for the B16-OVA models (bottom), n = 4 for the DSS + ICB group and n = 5 for the other groups. P values were calculated using a one-way ANOVA followed by Dunnett’s test, and each condition was compared with the DSS group as a control. Data are representative of two independent experiments. Source data

Extended Data Fig. 7 Gating strategy and representative contour plots of AICD protection by TNF blockade in human PBMCs.

a, Gating strategy for flow cytometry analysis. b, Representative contour plots showing annexin V-positive cells among CD8+ T lymphocytes after stimulation with anti-CD3 and anti-CD28, with or without TNF blockade with infliximab or etanercept (the experimental groups are those described in Fig. 3g).

Extended Data Fig. 8 The TNF axis is involved in immune-mediated colitis, as exacerbated by immune checkpoint blockade, in immune-deficient mice reconstituted with human PBMCs.

Fresh human PBMCs were injected intraperitoneally on day 0 into immunodeficient Rag2−/−Il2rg−/− mice. On days 0, 4, 7 and 10, mice were injected intraperitoneally with ipilimumab (200 µg) and nivolumab (200 µg), with or without etanercept (40 µg; given subcutaneously). As an antibody control, we used human polyclonal IgG. a, Normalized follow-up of body weight. Data are mean ± s.d. n = 4 biologically independent mice for all groups, except n = 5 for PBMCs + ICB + etanercept. P values were calculated using an extra sum-of-squares F test. b, Ultrasound assessments of intestinal wall thickness. Data are mean ± s.d. n = 4 biologically independent mice for PBMCs + IgG, n = 3 for PBMCs + ICB and n = 5 for PBMCs + ICB + etanercept. P values were calculated using a one-way ANOVA followed by Dunnett’s-test. c, Representative ultrasound images estimating colon wall thickness in the different experimental groups described in b. Red bars indicate intestinal wall thickness. Data are from a single experiment. Source data

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Fig. 1: DSS-induced colitis, exacerbated by anti-PD-1 and anti-CTLA-4, is ameliorated by prophylactic TNF blockade.
Fig. 2: Prophylactic TNF blockade does not hinder—and even enhances—the anti-tumour activity of combined anti-PD-1 and anti-CTLA-4 immunotherapy.
Fig. 3: TNF blockade increases the infiltration of tumour-specific T cells in MC38-derived tumours and decreases AICD in CD8+ T cells from mice and humans.
Fig. 4: TNF is involved in immune checkpoint blockade-induced colitis in patients and in a humanized mouse model.
Extended Data Fig. 1: Anti-tumour activity of double immune checkpoint blockade on B16-OVA-derived tumours on TNF blockade.
Extended Data Fig. 2: Prophylactic IL-6 blockade hinders the anti-tumour activity of the combined anti-PD-1 and anti-CTLA-4 immunotherapy regimen.
Extended Data Fig. 3: Administration of etanercept causes mice to develop anti-drug antibodies.
Extended Data Fig. 4: Increased concentrations of TNF in the tumour microenvironment after immune checkpoint blockade therapy.
Extended Data Fig. 5: Downregulation of PD-1 expression on tumour-infiltrating antigen-specific CD8+ T cells following the combined anti-PD-1 and anti-CTLA-4 immunotherapy regimen.
Extended Data Fig. 6: Expression of T cell exhaustion-related markers on T cells following immune checkpoint blockade treatment with or without TNF blockade.
Extended Data Fig. 7: Gating strategy and representative contour plots of AICD protection by TNF blockade in human PBMCs.
Extended Data Fig. 8: The TNF axis is involved in immune-mediated colitis, as exacerbated by immune checkpoint blockade, in immune-deficient mice reconstituted with human PBMCs.

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