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Metabolic modulation of tumours with engineered bacteria for immunotherapy

Abstract

The availability of l-arginine in tumours is a key determinant of an efficient anti-tumour T cell response1,2,3,4. Consequently, increases of typically low l-arginine concentrations within the tumour may greatly potentiate the anti-tumour responses of immune checkpoint inhibitors, such as programmed death-ligand 1 (PD-L1)-blocking antibodies5. However, currently no means are available to locally increase intratumoural l-arginine levels. Here we used a synthetic biology approach to develop an engineered probiotic Escherichia coli Nissle 1917 strain that colonizes tumours and continuously converts ammonia, a metabolic waste product that accumulates in tumours6, to l-arginine. Colonization of tumours with these bacteria increased intratumoural l-arginine concentrations, increased the number of tumour-infiltrating T cells and had marked synergistic effects with PD-L1 blocking antibodies in the clearance of tumours. The anti-tumour effect of these bacteria was mediated by l-arginine and was dependent on T cells. These results show that engineered microbial therapies enable metabolic modulation of the tumour microenvironment leading to enhanced efficacy of immunotherapies.

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Fig. 1: l-Arginine synergizes with PD-L1 blockade to promote MC38 tumour rejection.
Fig. 2: Metabolically engineered bacteria produce l-arginine and colonize tumours.
Fig. 3: l-Arg bacteria synergize with PD-L1 blockade to promote MC38 tumour rejection.
Fig. 4: l-Arg bacteria enhance anti-tumour immunity and can be administered systemically.

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD027167Source data are provided with this paper.

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Acknowledgements

R.G. is supported in part by Synlogic, the European Research Council (803150) and Swiss Cancer Research (KFS-4593-08-2018). F.S. is supported by the Helmut Horten Foundation.

Author information

Affiliations

Authors

Contributions

R.G. and J.M.L. conceived the project. F.P.C., C.B., G.A., M.P., S.G., J.N. and W.J. performed experiments with mouse tumour models. N.L. and M.J.J. generated bacterial strains. J.-P.T. analysed tissue sections. A.S. and K.A.W. contributed to discussions. R.G., F.S. and J.M.L. supervised the work. D.S.L., J.M.L. and R.G. wrote the manuscript.

Corresponding author

Correspondence to Roger Geiger.

Ethics declarations

Competing interests

R.G. and J.L. are inventors on a patent application related to l-Arg bacteria. R.G. received research funding from Synlogic. N.L., A.S., M.J, D.S.L, K.A.W. and J.M.L. are or were employees and stockholders of Synlogic.

Additional information

Peer review information Nature thanks Jeff Hasty 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 Intratumoral injections of an L-arginine solution does not affect the growth of MC38 tumors.

(a) MC38 tumors were established in C57BL/6 WT mice. Ten days later, 50 μl of a saturated L-arginine solution was injected into tumors. This corresponds to 0.25 mg L-arginine/g body weight and is the maximal volume that tumors can take up. Where indicated, mice were treated with 200µg anti-PD-L1 antibodies or isotype control antibodies by i.p injection. Mice received a total of four injections (bi-weekly). MC38 tumor growth curves. Values represent mean tumor volume ± s.e.m. Number of mice are indicated in the graph. Two experiments. (b) Same experiment as in (a) but survival curves are shown. (c) Same experiment as in (a) but growth curves of individual mice are shown. (d) Example of the gating strategy to quantify CD4 and CD8 T cells used in Figs. 3b, c, f, g

Source data.

Extended Data Fig. 2 EcN colonization does not affect the growth of MC38 tumors.

MC38 tumors were established in C57BL/6 WT mice. Mice were treated with 5 x 106 CFU EcN (i.t.) or with i.t. injections of PBS, bi-weekly (four treatments in total). MC38 tumor growth curves. Values represent mean tumor volume ± s.e.m. Number of mice are indicated in the graph. Two experiments

Source data.

Extended Data Fig. 3 The anti-tumor effect of L-Arg bacteria is T cell-dependent.

(a) MC38 tumors were established in C57BL/6 WT mice and in CD3e-/- mice and treated via intratumoral injection with 5 x 106 CFUs of L-Arg bacteria or EcN. Tumors were harvested and homogenized after 24 h, and bacterial abundance was measured by CFU assay. n = 2 (b) MC38 tumors were established in CD3e-/- mice. Tumors of the control group were treated with 5 x 106 CFUEcN (i.t.) twice a week. A second group was treated with 5 x 106 CFU L-Arg bacteria (i.t.) twice a week. A third group received anti-PD-L1 antibodies i.p. and 5 x 106 CFU EcN i.t. (EcN + α-PD-L1) and a fourth group received anti-PD-L1 antibodies i.p. and 5 x 106 L-Arg bacteria i.t (L-Arg-bac. + α-PD-L1). Tumor growth curve. Values represent mean tumor volume ± s.e.m. n = 5 for each group. (c) Survival curves of mice

Source data.

Extended Data Fig. 4 Effect of bacterial treatments on mouse health.

(a) C57BL/6 WT mice with established MC38 tumors were treated with four i.t. injections of 5 x 106 EcN or L-Arg bacteria, or with PBS. The weight of mice was followed over time. Bars represent the SEM, throughout. (b) C57BL/6 WT mice with established MC38 tumors were treated with a single i.v. injection of 5 x 107 EcN or L-Arg bacteria, or with PBS. The weight of mice was followed over time. The number of mice is indicated in the graph. Two experiments (a, b)

Source data.

Extended Data Fig. 5 Bacterial treatments and PD-L1 blockade have no effect on B16 tumor growth.

C57BL/6 WT mice with established B16.OVA tumors were treated four times with i.t. injections of 5 x 106 EcN or L-Arg bacteria and i.p. injections of anti-PD-L1 antibodies. Tumor growth curves. Values represent mean tumor volume ± s.e.m., n = 5

Source data.

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Canale, F.P., Basso, C., Antonini, G. et al. Metabolic modulation of tumours with engineered bacteria for immunotherapy. Nature 598, 662–666 (2021). https://doi.org/10.1038/s41586-021-04003-2

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