Modulating T cell homeostatic mechanisms with checkpoint blockade can efficiently promote endogenous anti-tumor T cell responses1,2,3,4,5,6,7,8,9,10,11. However, many patients still do not benefit from checkpoint blockade12, highlighting the need for targeting of alternative immune pathways13. Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) is an attractive target for immunotherapy, owing to its capacity to promote effector T cell (Teff) functions14,15 and hamper regulatory T cell (Treg) suppression16,17,18,19,20. On the basis of the potent preclinical anti-tumor activity of agonist anti-GITR antibodies, reported by us and others16,21,22, we initiated the first in-human phase 1 trial of GITR agonism with the anti-GITR antibody TRX518 (NCT01239134). Here, we report the safety profile and immune effects of TRX518 monotherapy in patients with advanced cancer and provide mechanistic preclinical evidence to rationally combine GITR agonism with checkpoint blockade in future clinical trials. We demonstrate that TRX518 reduces circulating and intratumoral Treg cells to similar extents, providing an easily assessable biomarker of anti-GITR activity. Despite Treg reductions and increased Teff:Treg ratios, substantial clinical responses were not seen. Similarly, in mice with advanced tumors, GITR agonism was not sufficient to activate cytolytic T cells due to persistent exhaustion. We demonstrate that T cell reinvigoration with PD-1 blockade can overcome resistance of advanced tumors to anti-GITR monotherapy. These findings led us to start investigating TRX518 with PD-1 pathway blockade in patients with advanced refractory tumors (NCT02628574).
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The TCR sequencing data that support the findings are available on the immuneACCESS Analyzer portal, https://clients.adaptivebiotech.com/pub/zappasodi-2019-natmed. All other relevant data are available upon reasonable request.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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We thank the Immune Monitoring, Flow Cytometry and Molecular Cytology Core Facilities at Memorial Sloan Kettering Cancer Center for technical assistance. This research was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA008748, NIH/NCI R01 CA056821, the Swim Across America, Ludwig Institute for Cancer Research, Ludwig Center for Cancer Immunotherapy at Memorial Sloan Kettering, Cancer Research Institute, Parker Institute for Cancer Immunotherapy and Breast Cancer Research Foundation. R.Z. was supported by the Parker Institute for Cancer Immunotherapy scholar award.
a, Patients with the indicated tumor types enrolled in the seven highest dose cohorts, for whom pre- and post-therapy PBMC samples were analyzed by flow cytometry. Asterisks indicate patients for whom pre- and post-therapy tumor biopsies were also available for immunofluorescent staining. b, Mean ± s.e.m. absolute numbers per ml of blood of the indicated cell subsets (CD3−CD56+ NK cells, CD3+CD8+ T cells, CD3+CD4+Foxp3− Teff, CD3+CD4+Foxp3+ Treg cells, GITR+ Treg cells, CD45RA-Foxp3hiCD4+ effector Treg (eTreg) cells and CD45RA+Foxp3lowCD4+ naive Treg (nTreg) cells) at baseline and at the indicated time points after treatment with TRX518. c, Fold changes relative to baseline of numbers per ml of blood of the same cell subsets as in b. For one patient in cohort 3, lymphocyte counts for the baseline sample used for FACS analysis are not available and it is not possible to calculate fold changes in cell numbers relative to baseline. Data are mean ± s.e.m. in patients grouped by dose cohort for each time point. d, Mean ± s.e.m. fold change in CD3+CD4+Foxp3+ Treg cells (percentage of CD3+CD4+) at the indicated time points after TRX518 relative to baseline in patients with the most prevalent tumor types grouped by dose cohort as reported in a. Treg cells were gated after exclusion of doublets, CD19+, CD14+ and dead cells. Wk, week; hrs, hours; Ca, cancer.
a, HEK293 cells transfected with human GITR (HEK293-GITR) were incubated with TRX518 (blue) or the isotype control (ctrl, gray) (10 µg ml−1) and then stained with an AF647-conjugated anti-human Ig alone or together with the flow cytometry (FC) anti-human GITR monoclonal antibody (mAb) eBioAITR (black). In parallel, HER293-GITR were stained with the eBioAITR alone (red). Plot shows results of one representative out of two independent tests. b, Example in 1 of the 37 patients tested (Extended Data Fig. 1a) of GITR staining on circulating Treg cells at baseline and at the indicated time points after TRX518 treatment from a pancreatic cancer patient receiving the highest dose of TRX518 (cohort 9). c, Mean ± s.e.m. fold changes in circulating GITR+CD4+Foxp3− effector T cells (Teff) (percentage of CD3+CD4+Foxp3−) and GITR+CD8+ T cells (percentage of CD3+CD8+) at the indicated time points after TRX518 relative to baseline in patients grouped by dose cohort as detailed in Extended Data Fig. 1a. Cell subsets were gated after exclusion of doublets, CD19+, CD14+ and dead cells. d, Flow cytometry analysis of GITR expression in circulating CD4+ and CD8+ T cell subsets at baseline in patients enrolled in cohort 8 and 9 (naive, CCR7+CD45RA+; CM, central memory, CCR7+CD45RA-; EM, effector memory, CCR7−CD45RA−; TEMRA, terminally differentiated, CCR7−CD45RA+; Teff, CD4+Foxp3−). Representative FACS plots show GITR expression in total circulating CD8+ and CD4+ T cells. Mean ± s.e.m. (n = 10), two-sided paired t-test, **P < 0.01 (Treg cells versus CD8+ naïve, P = 0.0024; Treg cells versus CD8+ CM, P = 0.0042; Treg cells versus CD8+ EM, P = 0.0038; Treg cells versus CD8+ TEMRA, P = 0.0033; Treg cells versus Teff naive, P = 0.0029; Treg cells versus Teff CM, P = 0.0050; Treg cells versus Teff EM, P = 0.0044; Treg cells versus Teff TEMRA, P = 0.0030.
Extended Data Fig. 3 Changes in frequency of GITR+CD4+ T cell subsets and their expression of cytokine receptors after TRX518.
a,b, Frequency of GITR+ Teff (blue) and GITR+ Treg cells (red) (percentages of Teff or Treg cells respectively) (top) and expression of IL7 receptor (CD127) and IL-2 receptor alpha (CD25) on these cell subsets (bottom) in peripheral blood at baseline and after treatment with TRX518 at the indicated time points in cohort 5 (a) and cohort 7 (b) patients. CD127 and CD25 expression in GITR-negative Foxp3−CD4+ T cells (GITRneg Teff, black) is also reported as control. Mean ± s.d. values of PBMC samples tested in duplicate at each time point.
a, Representative gating strategy of Foxp3+ Treg cells (Foxp3+CD4+), GITR+ Treg cells (GITR+Foxp3+CD4+), Foxp3lowGITR+ Treg cells and Foxp3hiGITR+ Treg cells. Cell subsets were gated after exclusion of doublets, CD19+, CD14+ and dead cells. b, Fold changes in total Foxp3+GITR+ Treg cells (as in Fig. 1b), Foxp3low and Foxp3hi GITR+ Treg cells (percentage of Treg cells) at the indicated time points after treatment with TRX518 relative to baseline. c, Frequency of Foxp3low and Foxp3hi cells among GITR+ Treg cells at baseline and after treatment with TRX518 at the indicated time points. b,c, Mean ± s.d. values in one representative patient per dose cohort tested in duplicate.
a, Quantification of Foxp3+CD4+ Treg cells, CD8+ T cells and CD4+Foxp3− Teff in seven-day mixed leukocyte reaction cultures with PBMCs from three different donors treated with 10 µg ml−1 soluble TRX518 or isotype control. Each data point is the mean of two to three technical replicates; paired data are isotype- and TRX518-treated samples from the same donor (two-sided ratio paired t-test; Treg cells absolute number, P = 0.0037; Treg cells percentage of live cells, P = 0.0364; Treg cells percentage of CD4+, P = 0.0200; n.s., not significant). b–d, Standard proliferation/suppression assays with anti-CD3/anti-CD28-stimulated CFSE-labeled autologous CD8+ T cells cultured alone or with CD4+CD25hi Treg cells (1:1 ratio), or CD4+CD25− Teff as control, immunomagnetically purified from donor-derived PBMCs, in the presence of 10 µg ml−1 plate-bound TRX518 or the isotype control. b, Frequency of proliferating activated CFSElow (top) and CD25+ (bottom) CD8+ T cells in each condition after 72 h incubation in one of two independent experiments with different donors are shown (mean ± s.d.; CD8 alone, n = 3; CD8:Teff, n = 3; CD8:Treg cells, n = 2). c, Proportion of CD4+ T cells (Teff or Treg cells) expressing Foxp3 and/or Tbet (left), and proportion of dead Treg cells expressing Foxp3 and/or Tbet (right) in the indicated T cell co-cultures from two biologically independent donor-derived PBMC samples after 72 h incubation (mean ± s.d., n = 3). d, Frequency of dead cells in proliferating (CellTraceViolet (CTV)low) and non-proliferating (CTVhi) Teff and Treg cells in the same cultures as in c. e, Proportion of cells stained with a caspase 3/7 fluorogenic substrate (casp) and/or 7AAD viability dye in monocultures of immunomagnetically purified CD4+CD25hi Treg cells, CD8+ and CD4+CD25− Teff activated with anti-CD3/-CD28 in the presence of 10 µg ml−1 plate-bound TRX518, isotype control (IgG), or a trimeric recombinant human GITRL (rhGITRL) after 16 and 32-hour incubation (mean ± s.d., n = 3 replicates). f, Schematic of the sequence of events leading to Treg loss after GITR stimulation. g, Expression of the anti-apoptotic gene BCL2L1 (BCL-XL) in eTreg cells sorted from two melanoma patients at baseline and at the indicated time points after treatment (mean ± s.d., n = 3). FACS plot depicts the gating strategy to sort viable eTreg cells on the basis of CD25 and CD45RA expression in CD4+ T cells. b–e,g, Two-sided unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n.a., not assessable.
Extended Data Fig. 6 Representative Treg modulations in peripheral blood and tumor after treatment with TRX518.
a, Flow cytometry plots of Foxp3, CD25 and CD45RA expression in circulating CD14−CD19−dead−CD3+CD4+ T cells from a patient with melanoma (0102-0003, left) and a patient with lung cancer (0002-0004, right) in cohort 7 (2 mg kg−1 TRX518) at baseline (Pre-Tx) and after treatment (Post-Tx) at the same time point as tumor biopsies were collected, as representative examples of patients showing Treg decreases or increases after TRX518 as shown in Fig. 2a. Gates showing frequencies of total Foxp3+ Treg cells and Foxp3hiCD45RA− eTreg cells are depicted. b, Immunofluorescent staining of Foxp3 (pink), CD4 (green) and DAPI (blue) on tumor tissue sections from the same patients and at the same time points shown in a, as representative examples of results shown in Fig. 2b for eight patients (scale bar, 50 µm; 40× original magnification; inset, 60× original magnification). White arrows indicate Foxp3+CD4+ Treg cells.
Extended Data Fig. 7 GITR stimulation cannot overcome T cell exhaustion in the setting of an immunosuppressive advanced tumor microenvironment.
a, B16F10-melanoma-bearing mice were treated with a single administration of the anti-GITR antibody DTA-1 (αGITR) or the matched isotype IgG control at day 4 or day 7 after tumor injection and 6 d later flow cytometry analyses of tumor infiltrating lymphocytes (TILs) were performed. Data are mean ± s.e.m. from one representative experiment out of those averaged in Fig. 3b (two-sided unpaired t-test; αGITR, n = 4; IgG, n = 5). b, B16F10-melanoma-bearing Foxp3-diphtheria-toxin-receptor transgenic mice were treated with diphtheria toxin (DT) to deplete Treg cells 4 d or 7 d after tumor implantation as indicated in the schema. A separate group of mice was injected with PBS as control. Mean ± s.e.m. averaged shortest and longest tumor diameters (n = 5; two-way analysis of variance with Bonferroni correction; P < 0.0001) and frequency of circulating Foxp3+ Treg cells at the indicated time points after treatment (n = 5; unpaired t-test) from one representative of two independent experiments. c, Frequencies, ratios and phenotype of CD8+ TILs and intra-tumor Foxp3+ Treg cells (TM-Treg) from B16F10-melanoma-bearing untreated mice at day 4 or day 7 after tumor implantation. Data are mean ± s.e.m. of five mice per group from one representative of two to four independent experiments depending on the parameter (two-sided unpaired t-test). d, Flow cytometry analysis of granzyme B (GzmB) and PD-1 expression in CD8+ TILs and PD-1 on GzmB+CD8+ TILs from B16F10 tumors harvested 6 d after optimal (αGITR day 4) or suboptimal (αGITR day 7) anti-GITR therapy or control treatments (IgG). Data are mean ± s.e.m. of four or five mice in αGITR- and IgG-treated groups respectively from one representative of two independent experiments (unpaired t-test). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n.s., not significant.
CD8+ TILs pooled from five mice per group 6 d after αGITR day 4, αGITR day 7, αPD-1 day 7, αGITR + αPD-1 day 7 or isotype control treatments as in Fig. 4c were processed for TCR β-chain CDR3 sequencing analysis (ImmunoSEQ, Adaptive Biotechnology). a,b, Overview of TCR β-chain CDR3 clonality results (a) and frequencies of the indicated top rearrangements (b, productive frequency by amino acid sequences) in each treatment condition. Values for clonality range from 0 (polyclonal samples) to 1 (monoclonal or oligoclonal samples). Productive clonality is calculated by normalizing productive entropy using the total number of unique productive rearrangements and subtracting the result from 1. Accordingly, the higher is the frequency of top rearrangements within a sample the higher is the clonality.
Representative immunofluorescent staining of PD-1 (green), GzmB (red), CD8 (white) and DAPI (blue) on tumor tissue sections from patient 0002–0009 (cohort 8, colon cancer) before (Pre-Tx) and after (Post-Tx) TRX518 treatment (scale bar, 20 µm; 40× original magnification). White arrows indicate PD-1+GzmB+CD8+ T cells (top). Box and whiskers (min to max and median) plots showing frequencies of PD-1-expressing CD8+ TILs and PD-1-expressing GzmB+CD8+ TILs and absolute numbers of PD-1+CD8+ and PD-1+GzmB+CD8+ TILs normalized relative to tumor area (µm2) (Pre-Tx, n = 10 optical fields; Post-Tx, n = 3 optical fields) (bottom).
Extended Data Fig. 10 Model of effects of anti-GITR therapy in combination with PD-1 blockade in the setting of advanced tumors.
Advanced tumors are generally characterized by abundant Treg infiltration and dysfunctional CD8+ T cells (1). Administration of anti-GITR in this setting can reduce intra-tumor Treg cells promoting increases in CD8+ T cell:Treg ratios (2–3). This however may be insufficient to cause effective anti-tumor T cell responses because T cells remain dysfunctional (3). Changes in tumor-infiltrating T cell quality is also key to ensure induction of anti-tumor immunity. This may be achieved by concurrent treatment with PD-1 blockade (4) that, leading to T cell reinvigoration/rejuvenation (5), allows for optimal anti-tumor T cell activation in the absence of Treg cells (6).
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Nature Reviews Clinical Oncology (2019)