Intratumoral regulatory T (Treg) cell abundance associates with diminished antitumor immunity and poor prognosis in human cancers. Recent work demonstrates that CD25, the high-affinity receptor subunit for interleukin (IL)-2, is a selective target for Treg depletion in mouse and human malignancies; however, anti-human CD25 antibodies have failed to deliver clinical responses against solid tumors due to bystander IL-2 receptor signaling blockade on effector T cells, which limits their antitumor activity. Here we demonstrate potent single-agent activity of anti-CD25 antibodies optimized to deplete Treg cells, while preserving IL-2-STAT5 signaling on effector T cells and show synergy with immune checkpoint blockade in vivo. Pre-clinical evaluation of an anti-human CD25 (RG6292) antibody with equivalent features demonstrates, in both nonhuman primates and humanized mouse models, efficient Treg cell depletion with no overt immune-related toxicities. Our data support the clinical development of RG6292 and evaluation of new combination therapies incorporating non-IL-2-blocking anti-CD25 antibodies in clinical studies.
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
The datasets generated during and/or analyzed during this current study have been deposited or are available from the corresponding author on reasonable request. Crystal structure coordinates and structure factors have been deposited with the PDB under accession code 6YIO. Source data are provided with this paper.
Codes used for the analysis of flow cytometry data in Fig. 4 can be obtained from the corresponding author upon request.
Plitas, G. & Rudensky, A. Y. Regulatory T cells: differentiation and function. Cancer Immunol. Res. 4, 721–725 (2016).
Elpek, K. G., Lacelle, C., Singh, N. P., Yolcu, E. S. & Shirwan, H. CD4+ CD25+ T regulatory cells dominate multiple immune evasion mechanisms in early but not late phases of tumor development in a B cell lymphoma model. J. Immunol. 178, 6840–6848 (2007).
Golgher, D., Jones, E., Powrie, F., Elliott, T. & Gallimore, A. Depletion of CD25+ regulatory cells uncovers immune responses to shared murine tumor rejection antigens. Eur. J. Immunol. 32, 3267–3275 (2002).
Jones, E. et al. Depletion of CD25+ regulatory cells results in suppression of melanoma growth and induction of autoreactivity in mice. Cancer Immun. 2, 1 (2002).
Onizuka, S. et al. Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor-α) monoclonal antibody. Cancer Res. 59, 3128–3133 (1999).
Quezada, S. A., Peggs, K. S., Curran, M. A. & Allison, J. P. CTLA4 blockade and GM-CSF combination immunotherapy alters the intratumor balance of effector and regulatory T cells. J. Clin. Invest. https://doi.org/10.1172/JCI27745 (2006).
Mihm, M. C. et al. Immunologic and clinical effects of antibody blockade of cytotoxic T lymphocyte-associated antigen 4 in previously vaccinated cancer patients. Proc. Natl Acad. Sci. USA https://doi.org/10.1073/pnas.0712237105 (2008).
Simpson, T. R. et al. Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma. J. Exp. Med. https://doi.org/10.1084/jem.20130579 (2013).
Wilson, N. S. et al. Activating Fc γ receptors contribute to the antitumor activities of immunoregulatory receptor-targeting antibodies. J. Exp. Med. 210, 1685–1693 (2013).
Selby, M. et al. Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells. Cancer Immunol. Res. 1, 32–43 (2013).
Sharma, A. et al. Anti-CTLA-4 immunotherapy does not deplete Foxp3 þ regulatory T cells (Tregs) in human cancers. Clin. Cancer Res. 25, 1233–1238 (2019).
Vargas, F. A. et al. Fc effector function contributes to the activity of human anti-CTLA-4 antibodies. Cancer Cell 33, 649–663 (2018).
Waight, J. D. et al. Selective FcγR Co-engagement on APCs modulates the activity of therapeutic antibodies targeting T cell antigens. Cancer Cell 33, 1033–1047 (2018).
Ha, D. et al. Differential control of human Treg and effector T cells in tumor immunity by Fc-engineered anti-CTLA-4 antibody. Proc. Natl Acad. Sci. USA 116, 609–618 (2019).
Chevrier, S. et al. An immune atlas of clear cell renal cell carcinoma. Cell 169, 736–749.e18 (2017).
Azizi, E. et al. Single-cell map of diverse immune phenotypes in the breast tumor microenvironment resource single-cell map of diverse immune phenotypes in the breast tumor microenvironment. Cell https://doi.org/10.1016/j.cell.2018.05.060 (2018).
Vargas, F. A. et al. Fc-optimized anti-CD25 depletes tumor-infiltrating regulatory T cells and synergizes with PD-1 blockade to eradicate established tumors. Immunity 46, 577–586 (2017).
Kapic, E., Becic, F. & Kusturica, J. Basiliximab, mechanism of action and pharmacological properties. Med. Arh. 58, 373–376 (2004).
Baldassari, L. E. & Rose, J. W. Daclizumab: development, clinical trials, and practical aspects of use in multiple sclerosis. Neurotherapeutics 14, 842–858 (2017).
Moreau, J.‐L. et al. Monoclonal antibodies identify three epitope clusters on the mouse p55 subunit of the interleukin 2 receptor: relationship to the interleukin 2‐binding site. Eur. J. Immunol. 17, 929–935 (1987).
Spolski, R., Li, P. & Leonard, W. J. Biology and regulation of IL-2: from molecular mechanisms to human therapy. Nat. Rev. Immunol. 18, 648–659 (2018).
Arenas-Ramirez, N. & Woytschak, J. Interleukin-2: biology, design and application. Trends Immunol. 36, 763–777 (2015).
Liao, W., Lin, J. X. & Leonard, W. J. Interleukin-2 at the crossroads of effector responses, tolerance, and immunotherapy. Immunity 38, 13–25 (2013).
Scheffold, A., Murphy, K. M. & Höfer, T. Competition for cytokines: T reg cells take all. Nat. Immunol. 8, 1285–1287 (2007).
Kohm, A. P. & Miller, S. D. Response to comment on “Cutting edge: anti-CD25 monoclonal antibody injection results in the functional inactivation, not depletion, of CD4+ CD25+ T regulatory cells”. J. Immunol. 177, 2037–2038 (2006).
Malek, T. R. & Castro, I. Interleukin-2 receptor signaling: at the interface between tolerance and immunity. Immunity 33, 153–165 (2010).
Van Elsas, A. et al. Elucidating the autoimmune and antitumor effector mechanisms of a treatment based on cytotoxic T lymphocyte antigen-4 blockade in combination with a B16 melanoma vaccine: comparison of prophylaxis and therapy. J. Exp. Med. https://doi.org/10.1084/jem.194.4.481 (2001).
Hashimoto, M. et al. CD8 T cell exhaustion in chronic infection and cancer: opportunities for interventions. Annu. Rev. Med. https://doi.org/10.1146/annurev-med-012017-043208 (2018).
Blank, C. U. et al. Defining ‘T cell exhaustion’. Nat. Rev. Immunol. https://doi.org/10.1038/s41577-019-0221-9 (2019).
Bulliard, Y. et al. OX40 engagement depletes intratumoral Tregs via activating FcγRs, leading to antitumor efficacy. Immunol. Cell Biol. https://doi.org/10.1038/icb.2014.26 (2014).
Wolchok, J. D. et al. Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma. J. Exp. Med. https://doi.org/10.1084/jem.20130579 (2013).
Furness, A. J. S., Vargas, F. A., Peggs, K. S. & Quezada, S. A. Impact of tumour microenvironment and Fc receptors on the activity of immunomodulatory antibodies. Trends Immunol. https://doi.org/10.1016/j.it.2014.05.002 (2014).
Das, R. et al. Combination therapy with anti-CTLA-4 and anti-PD-1 leads to distinct immunologic changes in vivo. J. Immunol. https://doi.org/10.4049/jimmunol.1401686 (2015).
National Cancer Institute. First-in-human study of monoclonal antibody BMS-986218 by itself and in combination with nivolumab in patients with advanced solid tumors. Clinical Trials https://www.cancer.gov/about-cancer/treatment/clinical-trials/search/v?id=NCI-2017-00920&r=1 (2019).
Du, J. et al. Structural basis for the blockage of IL-2 signaling by therapeutic antibody basiliximab. J. Immunol. https://doi.org/10.4049/jimmunol.0903178 (2009).
Kim, J. M., Rasmussen, J. P. & Rudensky, A. Y. Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nat. Immunol. https://doi.org/10.1038/ni1428 (2007).
Bacac, M. et al. CD20-TCB with obinutuzumab pretreatment as next-generation treatment of hematologic malignancies. Clin. Cancer Res. https://doi.org/10.1158/1078-0432.CCR-18-0455 (2018).
Goubier, A. et al. Fc-optimized anti-CD25 for tumour specific cell depletion. World patent WO2018167104 (2018).
Roy, G. et al. A novel bicistronic gene design couples stable cell line selection with a fucose switch in a designer CHO host to produce native and afucosylated glycoform antibodies. MAbs https://doi.org/10.1080/19420862.2018.1433975 (2018).
Schlothauer, T. et al. Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions. Protein Eng. Des. Sel. https://doi.org/10.1093/protein/gzw040 (2016).
Wolfgang, Kabsch XDS. Acta Crystallogr. Sect. D D66, 125–132 (2010).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. Sect. D https://doi.org/10.1107/S0907444910045749 (2011).
Bricogne, G. et al. BUSTER version 2.9.5. (United Kingdom Glob. Phasing Ltd., Cambridge, 2011).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of coot. Acta Crystallogr. Sect. D D66, 486–501 (2010).
Parker, R. & Fahl, S. P100 Exploring dissociated human tissues as an alternative to fresh tissue for multiple downstream applications. In Poster session presented at the Society for Immunotherapy of Cancer (SITC 2018) 33rd Annual Meeting (2018).
Becht, E. et al. Dimensionality reduction for visualizing single-cell data using UMAP. Nat. Biotechnol. 37, 38–44 (2019).
Van Gassen, S. et al. FlowSOM: using self-organizing maps for visualization and interpretation of cytometry data. Cytom. Part A 87, 636–645 (2015).
Nowicka, M. et al. CyTOF workflow: differential discovery in high-throughput high-dimensional cytometry datasets. F1000Research 6, 748 (2017).
Lun, A. T. L., Richard, A. C. & Marioni, J. C. Testing for differential abundance in mass cytometry data. Nat. Methods 14, 707–709 (2017).
Wang, X., Rickert, M. & Garcia, K. C. Structure of the quaternary complex of interleukin-2 with its α, β and γc receptors. Science https://doi.org/10.1126/science.1117893 (2005).
S.A.Q. is funded by a Cancer Research UK (CRUK) Senior Cancer Research Fellowship (C36463/A22246) and a CRUK Biotherapeutic Program Grant (C36463/A20764). K.S.P. receives funding from the NIH-RBTRU for Stem Cells and Immunotherapies (167097), of which he is the Scientific Director. This work was undertaken at University College London with support from the CRUK-UCL Centre (C416/A18088), the Cancer Immunotherapy Accelerator Award (CITA-CRUK) (C33499/A20265) and CRUK funding schemes for National Institute for Health Research Biomedical Research Centres and Experimental Cancer Medicine Centres. The authors thank T. Singer for the guidance provided on the nonclinical safety assessment of RG6292.
Two patent applications, WO/2018/167104 and US20190284287, with relevance to this work have been filed by Cancer Research Technology Limited and Tusk Therapeutics, and we declare our relationship with this patent. I.S. F.A.V., S.A.Q., K.S.P., A.G., J.S. and P.M. are named inventors on this patent. F.A.V., S.A.Q., I.S. and K.S.P. receive royalties related to this patent. S.A.Q. is an advisor to TUSK/Roche. M.A., R.F., J.E., C.M., J.S., B.J., L.L., H.K., J.B., S.B., C.B., E.M.-B. and R.S. are employees of Roche, which plans clinical development of the drug. M.A., R.F., J.E., C.M., J.S., B.J., H.K., J.B., S.B., C.B., E.M.-B. and R.S. have shares in the companies to which the patent belongs.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Anti-CD25NIB promotes single dose, single agent activity against established tumors.
(a) Balb/C mice were injected with 500,000 CT26 tumor cells. Treatment was started on day 6 post-tumor inoculation. (b) Mean tumor volume of the tumor-bearing mice in (A), n=10 mice/group. Data are presented as mean values ±SEM. Source data
C57BL6 mice were injected with 500,000 MC38 tumor cells. Once tumors were palpable, on day 7, mice were injected IP with αPC61/αCD25NIB/αCD25NIB + αIL2 (200μg). Tumors and LN were harvested on day 15 post-tumor inoculation and processed as described in materials and methods section. (a) Graph showing % FoxP3+ cells of total CD4+ cells. (b) Absolute number of Tregs shown as number of Tregs/g of tumor. p-value=0.0003 between No Tx and αCD25NIB group. ****=p-value <0.0001 (c) Ratio of effector T cells over Tregs. For CD8/Treg ratio, P-value between No tx versus αCD25NIB=0.0008, and between No tx and αCD25NIB + αIL2 =0.0045. For CD4 Eff/Treg ratio, p-value between no tx and /αCD25PC61 = 0.0056, between No tx and αCD25NIB=0.0002, between no tx and αCD25NIB + αIL2=0.0001. For NK/Treg ratio, p-value=0.0001 for no tx versus αCD25PC61, 0.0010 for no tx versus αCD25NIB and 0.0004 for No tx versus αCD25NIB + αIL2. (d) Representative FACS plots showing Granzyme B expression versus Ki67 expression in CD8, CD4 effectors and NK cells. (e) Graph showing percentage of Granzyme B+ cells in different effector subsets. (f) Graph showing the Mean Fluorescence Intensity of Granzyme of the effector cells plotted in (e). For CD8 cells, p-value between No tx group and αCD25NIB =0.0001. For CD4 Eff, p-values between No tx versus αCD25NIB =0.0007, for αCD25NIB versus αCD25PC61=0.0009, and between αCD25NIB and αCD25NIB + αIL2 group= 0.0002. For NK cells, p-value between No tx group and αCD25NIB group=0.0164 and between αCD25NIB and αCD25NIB + αIL2 group=0.0280. Quantification plots: mean ± SEM, 1-way ANOVA, Tukey’s multiple comparisons test (ns=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Source data
Extended Data Fig. 3 Binding of anti- CD25NIB, RG6292 and Daclizumab to mouse, human and cynomolgus CD25 positive cells. ADCC and ADCP capacity of RG6292.
For binding experiments with RG6292 and Daclizumab, SU-DHL1 tumor cells (human CD25+, (a)) and HSC-F cells (cynomolgus CD25+, (b)) were used. To quantify binding of anti-CD25NIB, splenocytes were isolated of spleens resected from female C57BL6-Foxp3tm1Flv/J mice (c) Cells were incubated with indicated serial dilutions of the test antibody detected then by fluorescently labeled 2nd antibody against human and mouse Fcγ, respectively. Living mouse Treg cells (Aqua−, mRFP+ singlets) and tumor cells (Aqua−, singlets), respectively, were gated and the mean fluorescence intensity of the secondary antibody was plotted. EC50 values were calculated by as described in the data analysis section in materials and methods. Shown are technical duplicates of one representative experiment out of several independent ones conducted (n>2). (d) RG6292 (and the fully fucosylated version RG6292 (FF)) depleted via ADCC in-vitro differentiated Treg cells using purified, IL-2 activated NK cells. Shown are technical duplicates of one representative experiment out of several independent ones conducted (n>2). (e) RG6292 and RG6292 (FF) mediated ADCP of in-vitro differentiated Treg cells when co-cultured with MCSF differentiated macrophages. Flow cytometric analysis was performed to determine percentage of phagocytosis. Shown are technical duplicates of one representative experiment out of several independent ones conducted (n>2). (f) Schematics of binder selection. Source data
Extended Data Fig. 4 Anti-human-CD25NIB (RG6292) depletes Treg and drives T cell activation in tumor-bearing humanized mice.
Stem cell humanized female NOG mice bearing an established s.c. BxPC-3 tumor were injected i.p. with vehicle, RG6292 [4 mg/kg] or Ipilimumab [10 mg/kg]. After 72 hrs, splenocytes, blood lymphocytes and tumor infiltrating lymphocytes were isolated and evaluated for counts of activated CD8+ T cells (huCD45+, huCD3+, huCD8+ huCTLA-4+) and Tregs (huCD45+, huCD3+, huCD4+, huFoxP3+) as well as for markers of recent T cell activation. (a) Ipilimumab as well as RG6292 decreased the intratumoral Treg counts. An increase of intratumoral activated CD8+ T cell count was only evident after administration of RG6292. Normalized counts were plotted for the respective treatment groups. Each symbol represents one animal (n=5 mice), CD8 and Treg cells are connected for the same animals (b) Intratumoral CD8+ T cells after RG6292 treatment were highly activated and had increased levels of HLA-DR, PD-1 and CTLA-4 (MFI as well as % of positive cells). Each symbol represents one animal (n=5 mice). The box and whiskers plots show minima and maxima and the median. Statistical analysis of RG6292 and Ipilimumab treated groups against vehicle group is indicated. Data was analyzed using 2-way ANOVA, Dunnet’s multiple comparisons test (ns=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) (p-value between RG6292 and Ipilimumab was 0.0001 for CTLA4 MFI on CD8 T cells and 0.0008 for HLA-DR on CD8 T cells. (c) Representative FACS plots showing CD25 expression versus FoxP3 expression in CD4+ T cells and PD-1 expression versus CTLA-4 expression in CD8+ T cells for vehicle, RG6292 and Ipilimumab treated animals. Source data
CD25 shown as surface colored in yellow with residues contributing to the interface highlighted in salmon. Fab light and heavy chain are colored in cyan and blue. Residues from the heavy chain CDR1, CDR2 and CDR3 contributing to the interface are labeled and the surface colored in dark pink, magenta and light pink, respectively. Light chain residues of CDR1 to 3 contributing to the interface are labeled and shown in yellow, lime green and green, respectively.
About this article
Cite this article
Solomon, I., Amann, M., Goubier, A. et al. CD25-Treg-depleting antibodies preserving IL-2 signaling on effector T cells enhance effector activation and antitumor immunity. Nat Cancer (2020). https://doi.org/10.1038/s43018-020-00133-0