Non-invasive early detection of acute transplant rejection via nanosensors of granzyme B activity

Abstract

The early detection of the onset of transplant rejection is critical for the long-term survival of patients. The diagnostic gold standard for detecting transplant rejection involves a core biopsy, which is invasive, has limited predictive power and carries a morbidity risk. Here, we show that nanoparticles conjugated with a peptide substrate specific for the serine protease granzyme B, which is produced by recipient T cells during the onset of acute cellular rejection, can serve as a non-invasive biomarker of early rejection. When administered systemically in mouse models of skin graft rejection, these nanosensors preferentially accumulate in allograft tissue, where they are cleaved by granzyme B, releasing a fluorescent reporter that filters into the recipient’s urine. Urinalysis then discriminates the onset of rejection with high sensitivity and specificity before features of rejection are apparent in grafted tissues. Moreover, in mice treated with subtherapeutic levels of immunosuppressive drugs, the reporter signals in urine can be detected before graft failure. This method may enable routine monitoring of allograft status without the need for biopsies.

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Fig. 1: Granzyme B activity nanosensors detect onset of acute allograft rejection by amplifying detection signals into urine.
Fig. 2: Activity nanosensors detect proteolytic cleavage by GzmB.
Fig. 3: Sensing GzmB activity during alloreactive T cell killing.
Fig. 4: Granzyme B activity during ACR triggers a urine pharmacokinetic switch.
Fig. 5: Urinary prediction of ACR upon administration of GzmB activity nanosensors.
Fig. 6: Urinary prediction of allograft rejection under subtherapeutic immunosuppression.

Data availability

All data supporting the findings of this study are available within the manuscript and its Supplementary Information. Raw data are available from the corresponding authors.

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Acknowledgements

This work was funded by an NIH Director’s New Innovator Award DP2HD091793 awarded to G.A.K. and National Institutes of Health U01 AI132904 awarded to A.B.A. Q.D.M. is supported by the NSF Graduate Research Fellowships Program (Grant No. DGE-1650044). D.V.M. is supported by National Institutes of Health F30 award number DK109665. B.A.H is supported by the National Institutes of Health GT BioMAT Training Grant under Award Number 5T32EB006343. G.A.K. holds a Career Award at the Scientific Interface from the Burroughs Wellcome Fund. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Q.D.M., D.V.M., A.B.A. and G.A.K. conceived of the idea, designed experiments, and interpreted results. Q.D.M., D.V.M., J.A.K., C.M.S., O.M.D. and B.A.H. carried out the experiments. Q.D.M., D.V.M., B.A.H., A.B.A. and G.A.K. wrote the manuscript.

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Correspondence to Andrew B. Adams or Gabriel A. Kwong.

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Q.D.M., D.V.M., A.B.A. and G.A.K. are listed as inventors on a patent application pertaining to the results of the paper. G.A.K. is co-founder of and serves as consultant to Glympse Bio, which is developing products related to the research described in this paper. This study could affect his personal financial status. The terms of this arrangement have been reviewed and approved by Georgia Tech in accordance with its conflict of interest policies.

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Mac, Q.D., Mathews, D.V., Kahla, J.A. et al. Non-invasive early detection of acute transplant rejection via nanosensors of granzyme B activity. Nat Biomed Eng 3, 281–291 (2019). https://doi.org/10.1038/s41551-019-0358-7

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