Cancer immunotherapy via targeted TGF-β signalling blockade in TH cells

Abstract

Cancer arises from malignant cells that exist in dynamic multilevel interactions with the host tissue. Cancer therapies aiming to directly kill cancer cells, including oncogene-targeted therapy and immune-checkpoint therapy that revives tumour-reactive cytotoxic T lymphocytes, are effective in some patients1,2, but acquired resistance frequently develops3,4. An alternative therapeutic strategy aims to rectify the host tissue pathology, including abnormalities in the vasculature that foster cancer progression5,6; however, neutralization of proangiogenic factors such as vascular endothelial growth factor A (VEGFA) has had limited clinical benefits7,8. Here, following the finding that transforming growth factor-β (TGF-β) suppresses T helper 2 (TH2)-cell-mediated cancer immunity9, we show that blocking TGF-β signalling in CD4+ T cells remodels the tumour microenvironment and restrains cancer progression. In a mouse model of breast cancer resistant to immune-checkpoint or anti-VEGF therapies10,11, inducible genetic deletion of the TGF-β receptor II (TGFBR2) in CD4+ T cells suppressed tumour growth. For pharmacological blockade, we engineered a bispecific receptor decoy by attaching the TGF-β-neutralizing TGFBR2 extracellular domain to ibalizumab, a non-immunosuppressive CD4 antibody12,13, and named it CD4 TGF-β Trap (4T-Trap). Compared with a non-targeted TGF-β-Trap, 4T-Trap selectively inhibited TH cell TGF-β signalling in tumour-draining lymph nodes, causing reorganization of tumour vasculature and cancer cell death, a process dependent on the TH2 cytokine interleukin-4 (IL-4). Notably, the 4T-Trap-induced tumour tissue hypoxia led to increased VEGFA expression. VEGF inhibition enhanced the starvation-triggered cancer cell death and amplified the antitumour effect of 4T-Trap. Thus, targeted TGF-β signalling blockade in helper T cells elicits an effective tissue-level cancer defence response that can provide a basis for therapies directed towards the cancer environment.

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Fig. 1: Inducible ablation of TGFBR2 in CD4+ T cells inhibits tumour growth.
Fig. 2: 4T-Trap effectively represses TGF-β signalling in lymph node CD4+ T cells.
Fig. 3: 4T-Trap reprograms the tumour vasculature causing cancer cell hypoxia and cancer cell death.
Fig. 4: 4T-Trap synergizes with VEGF-Trap to induce cancer cell death and suppress tumour growth.

Data availability

Data generated in this study are included within the paper (and its Supplementary Information files) or are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank members of the M.O.L. laboratory for helpful discussions and S. Gong for help with the recombineering technique and BAC DNA purification. This work was supported by a Howard Hughes Medical Institute Faculty Scholar Award (M.O.L.), an award from Mr William H. and Mrs Alice Goodwin and the Commonwealth Foundation for Cancer Research and the Center for Experimental Therapeutics at Memorial Sloan Kettering Cancer Center (M.O.L.) and a Cancer Center Support Grant (P30 CA08748). S.L., C.C. and X.Z. are Cancer Research Institute Irvington Fellows supported by the Cancer Research Institute. M.H.D. and B.G.N. are recipients of F31 CA210332 and F30 AI29273-03 awards from National Institutes of Health. E.G.S. is a recipient of a Fellowship from the Alan and Sandra Gerry Metastasis and Tumour Ecosystems Center of Memorial Sloan Kettering Cancer Center.

Author information

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Authors

Contributions

M.O.L. conceptualized the project and supervised all aspects of this study. S.L. drove the antibody and protein engineering parts of the study, including biochemical characterization and efficacy evaluation, analysis and interpretation of data and writing the manuscript. M.L. initiated the project, established immunoflurescence staining protocols and performed experiments on proliferation of cancer cells, angiogenesis in tumour tissues and inducible TGFBR2 deletion in PyMT mice, interpreted data and wrote the manuscript. H.X. and N.-K.V.C. assisted S.L. for the Biacore assays and interpretation of data. K.J.C., S.G., P.L., B.G.N., W.S., X.Z., C.C., M.H.D. and E.G.S. assisted with mouse colony management and performed experiments.

Corresponding author

Correspondence to Ming O. Li.

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Competing interests

Memorial Sloan Kettering Cancer Center has filed a patent application with the U.S. Patent and Trademark Office directed toward methods and compositions for targeting TGF-β signaling in CD4+ helper T cell for cancer immunotherapy.

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Peer review information Nature thanks Eduard Batlle and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Inducible ablation of TGFBR2 in CD4+ T cells causes enhanced T helper cell responses and increased cancer cell death.

a, Representative immunofluorescence images of CD31 (white), Ki67 (red) and E-Cadherin (green) in mammary tumour tissues from PyMT mice containing unpalpable, 5 × 5-mm or 9 × 9-mm tumours. Isolated CD31+ staining in the tumour parenchyma (yellow arrows) is indicated. b, TGFBR2 expression on CD4+ T cells and CD8+ T cells from the tumour-draining lymph nodes of Tgfbr2fl/fl PyMT and Cd4-creERT2;Tgfbr2fl/fl PyMT mice treated with tamoxifen. c, Representative flow cytometry plots and statistical analyses of IL-4 and IFNγ expression in CD4+FOXP3- T cells from the tumour-draining lymph nodes of Tgfbr2fl/fl PyMT and Cd4-creERT2;Tgfbr2fl/fl PyMT mice treated with tamoxifen. d, Representative immunofluorescence images of E-Cadherin (green), Ki67 (red) and cleaved Caspase 3 (CC3, blue) in mammary tumour tissues from Tgfbr2fl/fl PyMT and Cd4-creERT2;Tgfbr2fl/fl PyMT mice treated with tamoxifen. The percentage of Ki67+E-Cadherin+ cells over total E-Cadherin+ epithelial cells was calculated from 0.02 mm2 regions (n = 9). The percentage of CC3+ areas over total E-Cadherin+ areas was calculated from 0.02 mm2 regions (n = 10). All statistical data are shown as mean ± s.e.m. Two-tailed unpaired t-test (c, d). Data are pooled biological replicates (c) or representative of three independent experiments (a, b, d). Source data

Extended Data Fig. 2 Inducible ablation of TGFBR2 in CD4+ T cells promotes tumour vessel reorganization, hypoxia and cancer cell death.

a, Representative immunofluorescence images of fibrinogen (Fg, white), CD31 (red), cleaved Caspase 3 (CC3, cyan) and E-Cadherin (green) in mammary tumour tissues from Tgfbr2fl/fl PyMT and Cd4-creERT2;Tgfbr2fl/fl PyMT mice treated with tamoxifen. Extravascular (EV) Fg deposition events (magenta arrows) were calculated from 1 mm2 regions (n = 9 for each group). Isolated CD31+ staining (yellow arrows) was counted from 1 mm2 regions (n = 9 for each group). b, Representative immunofluorescence images of NG2+ pericytes (white), CD31+ endothelial cells (red), GP38+ fibroblasts (blue) and E-Cadherin (green) in mammary tumour tissues from Tgfbr2fl/fl PyMT and Cd4-creERT2;Tgfbr2fl/fl PyMT mice treated with tamoxifen. NG2-unbound (magenta arrows) or GP38-unbound (yellow arrows) isolated CD31+ staining was counted from 1 mm2 regions (n = 9 for each group). c, Representative immunofluorescence images of collagen IV (Col IV, white), CD31 (red), fibronectin (FN, cyan) and E-cadherin (green) in mammary tumour tissues from Tgfbr2fl/fl PyMT and Cd4-creERT2;Tgfbr2fl/fl PyMT mice treated with tamoxifen. The average continuous lengths of Col IV and FN were measured in 1 mm2 regions (n = 9 for each group). d, Representative immunofluorescence images of a hypoxia probe (HPP, white), CD31 (red), CC3 (cyan) and E-Cadherin (green) in mammary tumour tissues from Tgfbr2fl/fl PyMT and Cd4-creERT2;Tgfbr2fl/fl PyMT mice treated with tamoxifen. The percentage of HPP+E-Cadherin+ areas over E-Cadherin+ epithelial areas was calculated from 1 mm2 regions (n = 9 for each group). The shortest distance of HPP+ regions (magenta dashed lines) or CC3+ regions (yellow dashed lines) to CD31+ endothelial cells was measured in tumour tissues from Cd4-creERT2;Tgfbr2fl/fl PyMT mice treated with tamoxifen (n = 9). All statistical data are shown as mean ± s.e.m. Two-tailed unpaired t-test (ad) or paired t-test (d). Data are representative of three independent experiments (ad). Source data

Extended Data Fig. 3 Biochemical properties of 4T-Trap and control antibodies.

a, Schematic representation of ibalizumab Fab and TGFBR2 ECD fusion proteins in a murine IgG1 framework. The star indicates a D265A substitution in the CH2 domain, and the semi-circle and moon shapes indicate knob-into-hole (KIH) modifications in the CH3 domain to enable heavy chain heterodimerization. The grey or colored parts indicate mouse or human sequences, respectively. b, c, Yield and aggregation percentage of ibalizumab Fab and TGFBR2 ECD fusion proteins produced in a FreeStyle HEK293-F cell transient expression system. FreeStyle HEK293-F cells transfected with plasmids encoding the indicated fusion antibodies were cultured for 4 days, and the supernatant was collected. Protein G affinity purification and size exclusion chromatography were used to purify these antibodies. d, Molecular weights of anti-CD4, mGO53, 4T-Trap and TGF-β-Trap antibodies detected by Coomassie blue staining of samples run in an SDS–PAGE gel under non-reduced or reduced conditions. Molecular size markers (kDa) are shown on the left. HC, heavy chain; LC, light chain. e, Size exclusion chromatography analyses of mGO53, TGF-β-Trap, anti-CD4 and 4T-Trap antibodies. f, Schematic representation of human CD4 structure and purity examination of recombinant soluble CD4 (sCD4) by SDS–PAGE followed Coomassie blue staining. g, The binding affinities of 4T-Trap and anti-CD4 to human CD4 as well as 4T-Trap and anti-TGF-β (1D11 clone) to human TGF-β1 were determined by surface plasmon resonance. h, Binding of 4T-Trap to human CD4 ectopically expressed on HEK293 cells. Cells were incubated with serial dilutions of 4T-Trap and anti-CD4 antibodies followed by a fluorophore-conjugated anti-mouse IgG secondary antibody. Samples were analysed by flow cytometry. The measured mean fluorescence intensity (MFI) was quantified. i, TGF-β signalling inhibitory functions of 4T-Trap and anti-TGF-β. HEK293 cells transfected with a TGF-β/SMAD firefly luciferase reporter plasmid and a pRL-TK Renilla luciferase reporter plasmid were incubated with the indicated antibodies for 30 min and treated with 10 ng/mL recombinant human TGF-β1 for 12 h before subject to the luciferase assay. RU, relative unit of normalized Firefly luciferase activity to Renilla luciferase activity. Data are representative of three independent experiments (bf, h, i). Source data

Extended Data Fig. 4 Generation and validation of human CD4 transgenic mice.

a, Recombineering a bacterial artificial chromosome (BAC) DNA containing the human CD4 locus with the proximal enhancer (PE) element replaced by its murine equivalent. The shuttle plasmid contains the mouse Cd4 PE flanked by two homologous arms of the human CD4 gene (250 bps), the E. coli. RecA gene to mediate homologous recombination, the SacB gene to mediate negative selection on sucrose, an Ampicillin resistance locus to mediate positive selection and a conditional R6Kγ replication origin. b, Flow cytometry analyses of human CD4 expression on leukocyte populations from wild-type or human CD4 transgenic mice. CD4+ T cells (CD45+TCRβ+CD4+), CD8+ T cells (CD45+TCRβ+CD8+), NK cells (CD45+TCRγ-TCRβ-NKp46+NK1.1+) were isolated from lymph nodes. B cells (CD45+MHCII+Ly6C-B220+), XCR1+ dendritic cells (DCs) (CD45+Lin-F4/80-Ly6C-CD11c+MHCII+XCR1+), CD11b+ DCs (CD45+Lin-F4/80-Ly6C-CD11c+MHCII+CD11b+), Monocytes (CD45+Lin-F4/80+Ly6C+CD11b+) and Macrophages (CD45+Lin-F4/80+CD11b-Ly6C-) were isolated from spleens. Data are representative of three independent experiments (b).

Extended Data Fig. 5 Pharmacokinetics, pharmacodynamics and efficacy study of 4T-Trap and control antibodies.

a, Schematic representation of biotinylated 4T-Trap and control antibodies. b, Mice were administered with a single dose of 150 μg 4T-Trap, anti-CD4, TGF-β-Trap or mGO53 by intravenous injection. Antibody serum levels at different time points were measured by ELISA. c, Mice were administered with a single dose of 50 μg, 100 μg, 150 μg or 450 μg 4T-Trap by intravenous injection. Antibody serum levels were measured by ELISA. d, Mice were administered with a single dose of 150 μg 4T-Trap, anti-CD4, TGF-β-Trap or mGO53 by intravenous injection. TGF-β1 serum levels were measured by ELISA. e, Representative immunofluorescence images of E-Cadherin (green) and phosphorylated Smad2 (pSmad2, magenta) in mammary tumour tissues from mice treated with the indicated antibodies at 12 h post injection. f, Mice were administered with a single dose of 50 μg, 100 μg, 150 μg or 450 μg 4T-Trap by intravenous injection. Percentage of human CD4 molecule occupancy was measured by flow cytometry. g, Immunoblotting analyses of TGF-β-induced SMAD2/3 phosphorylation in mouse CD4+ T cells isolated from human CD4 transgenic mice with different levels of 4T-Trap human CD4 (hCD4) target occupancy (TO). Numbers under lanes indicate SMAD2/3 or pSMAD2/3 band intensity. h, Tumour measurements from CD4 PyMT mice treated with 4T-Trap (n = 4) or combination of anti-CD4 with TGF-β-Trap (n = 5). Two-tailed unpaired t-test (h). Data are pooled biological replicates (h) or representative of three independent experiments (bg). Source data

Extended Data Fig. 6 4T-Trap promotes the generation of a reorganized, nonporous and mature tumour vasculature.

a, Representative immunofluorescence images of fibrinogen (Fg, white), CD31 (red) and E-Cadherin (green) in mammary tumour tissues from CD4 PyMT mice treated with 4T-Trap, anti-CD4, TGF-β-Trap or mGO53 antibodies. Isolated CD31+ staining (yellow arrows) was counted from 1 mm2 regions (n = 13 for each group). Extravascular (EV) Fg deposition events (magenta arrows) were calculated from 1 mm2 regions (n = 13 for each group). b, Representative immunofluorescence images of sulfo-NHS-biotin (white), CD31 (red) and E-Cadherin (green) in mammary tumour tissues from CD4 PyMT mice treated with 4T-Trap, anti-CD4, TGF-β-Trap or mGO53 antibodies. The percentage of sulfo-NHS-biotin areas over E-Cadherin+ epithelial regions was calculated from 1 mm2 regions (n = 15 for each group). Cancer cell-associated sulfo-NHS-biotin deposition events (magenta arrows) in highly perfused regions were calculated from 1 mm2 regions (n = 10 for each group). c, Representative immunofluorescence images of NG2+ pericytes (white), CD31+ endothelial cells (red), GP38+ fibroblasts (cyan) and E-Cadherin (green) in mammary tumour tissues from CD4 PyMT mice treated with 4T-Trap, anti-CD4, TGF-β-Trap or mGO53 antibodies. NG2-unbound (magenta arrows) or GP38-unbound (yellow arrows) isolated CD31+ staining was counted from 1 mm2 regions (n = 13 for each group). d, Representative immunofluorescence images of collagen IV (Col IV, white), CD31 (red), fibronectin (FN, cyan) and E-Cadherin (green) in mammary tumour tissues from CD4 PyMT mice treated with 4T-Trap, anti-CD4, TGF-β-Trap or mGO53 antibodies. The average continuous lengths of Col IV and FN were measured in 1 mm2 regions (n = 13 for each group). All statistical data are shown as mean ± s.e.m. ****P < 0.0001; NS: not significant; one-way ANOVA with post hoc Bonferroni t-test (ad). Data are representative of three independent experiments (ad). Source data

Extended Data Fig. 7 4T-Trap repression of tumour growth and VEGF-Trap construction.

a, A schematic representation of treatment with 4T-Trap and control antibodies. CD4 PyMT mice bearing 9 x 9-mm tumours were administered with 100 μg antibodies by intravenous injection twice a week for 4 weeks. b, Singular tumour measurements from CD4 PyMT mice treated with 4T-Trap, anti-CD4, TGF-β-Trap or mGO53 (n = 7, 6, 7 and 5). c, Representative immunofluorescence images of a hypoxia probe (HPP, white), CD31 (red), cleaved Caspase 3 (CC3, blue) and E-Cadherin (green) in mammary tumour tissues from mice treated with 4T-Trap at the indicated time points. d, Schematic representation of human VEGFR1, VEGFR2 and VEGF-Trap as well as purity examination of recombinant VEGF-Trap by SDS–PAGE followed by Coomassie blue staining. e, VEGF signalling inhibitory function of VEGF-Trap. HEK293 cells transfected with a VEGF/NFAT firefly luciferase reporter plasmid, together with a VEGFR2 expression plasmid and a pRL-TK Renilla luciferase reporter plasmid, were incubated with different concentrations of VEGF-Trap for 30 min followed by 10 ng/mL recombinant human VEGF165 for 12 h before subject to the luciferase assay. RU, relative unit of normalized Firefly luciferase activity to Renilla luciferase activity. ***P < 0.001; NS, not significant; one-way ANOVA with post hoc Bonferroni t-test (b). Data are pooled biological replicates (b) or representative of three independent experiments (ce). Source data

Extended Data Fig. 8 4T-Trap induces T helper cell activation, differentiation and tumour infiltration.

a, Representative flow cytometry plots and statistical analyses of CD62L and CD44 expression in conventional CD4+FOXP3- T cells from the tumour-draining lymph nodes of CD4 PyMT mice treated with 4T-Trap, anti-CD4, TGF-β-Trap or mGO53 antibodies (n = 3 for each group). b, Quantitative RT–PCR analyses of Smad7 and Rgs16 mRNA expression in effector/memory CD4+ T cells from the tumour-draining lymph nodes of CD4 PyMT mice treated with the indicated antibodies. c, Representative flow cytometry plots and statistical analyses of IFNγ and ΙL-4 expression in conventional CD4+FOXP3- T cells from the tumour-draining lymph nodes of CD4 PyMT mice treated with 4T-Trap, anti-CD4, TGF-β-Trap or mGO53 antibodies (n = 3 for each group). d, Representative flow cytometry plots and statistical analyses of TCRβ, NK1.1, CD4, CD8 and FOXP3 expression in tumour-infiltrating leukocytes from CD4 PyMT mice treated with 4T-Trap, anti-CD4, TGF-β-Trap or mGO53 antibodies (n = 3 for each group). All statistical data are shown as mean ± s.e.m. **P < 0.01; ***P < 0.001; ****P < 0.0001; NS: not significant; one-way ANOVA with post hoc Bonferroni t-test (ad). Source data

Extended Data Fig. 9 4T-Trap-triggered anti-tumour immunity is dependent on IL-4.

ab, Tumour measurements from CD4 PyMT mice treated with mGO53 or 4T-Trap in the absence or presence of an IL-4 neutralizing antibody (αIL-4) or an IFNγ neutralizing antibody (αIFN-γ) (n = 5 for each group). c, Representative immunofluorescence images of a hypoxia probe (HPP, white), CD31 (red), cleaved Caspase 3 (CC3, blue) and E-Cadherin (green) in mammary tumour tissues from CD4 PyMT mice treated with mGO53 or 4T-Trap in the absence or presence of αIL-4 or αIFNγ. d, Schematic representation of treatment with 4T-Trap and control antibodies. CD4 mice inoculated subcutaneously with MC38 cancer cells, were treated with 4T-Trap or control antibodies including TGF-β-Trap, anti-CD4 and mGO53 (100 μg/dose), for a total of 5 doses. e, Tumour measurements from CD4 mice bearing MC38 cancer cells treated with 4T-Trap, TGF-β-Trap, anti-CD4, mGO53 and combination of anti-CD4 with TGF-β-Trap (n = 5 for each group). f, Tumour measurements from CD4 mice bearing MC38 cancer cells treated with mGO53, αIL-4, 4T-Trap and combination of αIL-4 with 4T-Trap (n = 5 for each group). g, Tumour measurements from CD4 mice bearing MC38 cancer cells treated with mGO53, αIFNγ, 4T-Trap and combination of αIFΝ-γ with 4T-Trap (n = 5 for each group). *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001; NS: not significant; two-way ANOVA with post hoc Bonferroni t-test (a, b, eg). Data are pooled biological replicates (a, b, eg) or representative of three independent experiments (c). Source data

Extended Data Fig. 10 Cancer therapy landscape.

Cancer therapies are grouped into four categories in terms of targets and targeting strategies. Cancer cell-directed therapies aim to directly destruct cancer cells, which include conventional ‘cancer cell therapy’ with targeting approaches such as chemotherapy to eliminate mitotic cancer cells, and ‘cancer cell immunotherapy’ to engage immune effectors such as cytotoxic T lymphocytes (CTLs), killer innate lymphocytes (ILCs) and killer innate-like T cells (ILTCs) to eradicate cancer cells. The cancer immunosurveillance function of CTLs can be revived by immune checkpoint inhibitors such as PD-1 antibodies (anti-PD-1). Cancer environment-directed therapies aspire to rectify the host tissue pathology that fosters tumour growth. A cancer environment hallmark is angiogenesis characterized by a leaky and immature blood vasculature. Conventional ‘cancer environment therapy’ includes anti-angiogenics such as VEGF antibodies (anti-VEGF) that diminish vasculature abundance. Blockade of TGF-β signalling in helper T (TH) cells with 4T-Trap results in enhanced TH2 cell differentiation that promotes vasculature remodelling and tumour tissue healing with cancer cell hypoxia and cancer cell death instigated in avascular regions. 4T-Trap defines a novel modality of ‘cancer environment immunotherapy’.

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Li, S., Liu, M., Do, M.H. et al. Cancer immunotherapy via targeted TGF-β signalling blockade in TH cells. Nature 587, 121–125 (2020). https://doi.org/10.1038/s41586-020-2850-3

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