CD25-Treg-depleting antibodies preserving IL-2 signaling on effector T cells enhance effector activation and antitumor immunity

Abstract

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.

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Fig. 1: Characterization of non-IL-2 blocking anti-mouse CD25 mAbs.
Fig. 2: Monotherapy anti-CD25NIB drives rejection of established tumors in different mouse models.
Fig. 3: Anti-CD25NIB and anti-CD25PC61 promote equivalent Treg depletion but different Teff cell activation in vivo.
Fig. 4: Anti-CD25NIB as a substrate for combination immunotherapy.
Fig. 5: An anti-human CD25NIB antibody promotes effective Treg depletion in patient-derived tumor samples in vitro.
Fig. 6: Anti-human-CD25NIB (RG6292) depletes Treg cells and drives T-cell activation in tumor-bearing humanized mice and cynomolgus monkeys.

Data availability

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.

Code availability

Codes used for the analysis of flow cytometry data in Fig. 4 can be obtained from the corresponding author upon request.

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Acknowledgements

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.

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Contributions

S.A.Q. and K.S.P. conceived the project. I.S., M.A., E. M.-B., B.J., J.B., A.G., H.K., K.S.P. and S.A.Q. designed the experiments, analyzed the data and wrote the manuscript. I.S., A.G., J.Salimu., P.M., M.A.B., J.S., R.F., L.L. and C.M. performed the experiments. D.Z., C.Q., J.E., M.W.S., D.F.D., J.R.C. and A.U.A. contributed experimentally. J.Y.H., E.G., C.B. and S.B. contributed to analysis of the data. R.S., H.K., F.A.V., A.S. and T.M. contributed intellectually. A. Georgiou provided technical support.

Corresponding authors

Correspondence to Maria Amann or Karl S. Peggs or Sergio A. Quezada.

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Competing interests

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.

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Extended data

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

Extended Data Fig. 2 αCD25NIB depletes Tregs and drives effector immune responses.

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

Extended Data Fig. 5 Open-book representation of the interaction site between CD25 and Fab RG6292.

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.

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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

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