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Bypassing evolutionary dead ends and switching the rate-limiting step of a human immunotherapeutic enzyme

Nature Catalysis volume 5, pages 952–967 (2022)Cite this article

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Abstract

The Trp metabolite l-kynurenine (KYN) accumulates in numerous solid tumours and mediates potent immunosuppression. Bacterial kynureninases (KYNases), which preferentially degrade KYN, can relieve immunosuppression in multiple cancer models, but immunogenicity concerns preclude their clinical use, while the human enzyme (HsKYNase) has very low activity for KYN and shows no therapeutic effect. Using fitness selections, we evolved a HsKYNase variant with 28-fold higher activity, beyond which exploration of >30 evolutionary trajectories involving the interrogation of >109 variants led to no further improvements. The introduction of two amino acid substitutions conserved in bacterial KYNases reduced enzyme fitness but potentiated rapid evolution of variants with ~500-fold improved activity and reversed substrate specificity, resulting in an enzyme capable of mediating strong anti-tumour effects in mice. Pre-steady-state kinetics revealed a switch in the rate-determining step attributable to changes in both enzyme structure and conformational dynamics. Apart from its clinical significance, our work highlights how rationally designed substitutions can potentiate trajectories that overcome barriers in protein evolution.

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Fig. 1: Directed evolution of HsKYNase and development of a KYN specialist variant.
Fig. 2: Pre-steady-state kinetics analysis.
Fig. 3: Crystal structure of HsKYNase_66 and its comparison to HsKYNase.
Fig. 4: HDX-MS of HsKYNase_66 and HsKYNase in the reaction with KYN.
Fig. 5: Anti-tumour activity of an engineered human KYNase enzyme.

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Data availability

The datasets generated and/or analysed during the current study are attached in the Supplementary Information and additional data are available from the corresponding authors upon reasonable request. The HsKYNase_66 structure has been deposited in the PDB with the accession code 7S3V. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Institutes of Health (1 RO1 CA189623), Cancer Prevention and Research Institute of Texas (grant DP150061) and Ikena Oncology (all to G.G. and E.S.), funding from the University of Texas System Proteomics network (to S.D.), grants R01GM104896 and R01GM125882 from the National Institutes of Health (both to Y.J.Z.) and postdoctoral fellowships from the American Cancer Society (grant 128252-PF-15-143-01-CDD to J.B. and grant 123506-PF-13-354-01-CDD to N.M.).

Author information

Author notes

  1. John Blazeck

    Present address: School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, USA

  2. Christos S. Karamitros

    Present address: Aeglea BioTherapeutics, Austin, TX, USA

  3. Norah Ashoura

    Present address: Elevate Bio, Waltham, MA, USA

  4. Silvia Coma

    Present address: Verastem Oncology, Needham, MA, USA

  5. These authors contributed equally: John Blazeck, Christos S. Karamitros.

Authors and Affiliations

  1. Department of Chemical Engineering, University of Texas at Austin, Austin, TX, USA

    John Blazeck, Christos S. Karamitros, Kyle Ford, Catrina Somody, Ahlam Qerqez, Nicholas Marshall, Anirudh Sivakumar, Wei-Cheng Lu, Bing Tan, Candice Lamb, Yuri Tanno, Menna Y. Siddiqui & George Georgiou

  2. Trajan Scientific Americas, Boston, MA, USA

    Kyle Murray

  3. Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX, USA

    Kyle Murray & Sheena D’Arcy

  4. Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA

    Nathaniel T. Burkholder, Norah Ashoura, Yan Jessie Zhang, Kenneth A. Johnson, Everett Stone & George Georgiou

  5. Ikena Oncology, Cambridge, MA, USA

    Silvia Coma, Xiaoyan M. Zhang, Karen McGovern & Mark Manfredi

  6. Department of Molecular Chemistry and Engineering, Kyoto Institute of Technology, Kyoto, Japan

    Yoichi Kumada

  7. Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA

    Yan Jessie Zhang, Everett Stone & George Georgiou

  8. Department of Oncology, University of Texas Dell Medical School, Austin, TX, USA

    Everett Stone & George Georgiou

  9. Department of Biomedical Engineering, University of Texas at Austin, Austin, TX, USA

    George Georgiou

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Contributions

J.B., C.S.K., K.F., A.Q., C.S., N.M., A.S., B.T., W.-C.L., M.Y.S. and N.A. designed and performed the directed evolution experiments and KYNase enzyme characterizations. C.S.K. and K.A.J. designed and performed the pre-steady-state kinetics experiments. N.T.B. and Y.J.Z. designed and performed the crystallization experiments. C.S.K., K. Murray and S.D. designed and performed the HDX experiments. Y.K., C.L., Y.T., C.S.K., J.B. and C.S. expressed and prepared enzymes for the in vivo and stopped-flow kinetics studies. S.C., M.M., X.M.Z. and K. McGovern designed and performed the in vivo experiments. J.B., C.S.K., G.G., E.S., K.A.J., S.D. and Y.J.Z. interpreted the data. C.S.K., G.G. and J.B. wrote the manuscript.

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Correspondence to John Blazeck, Christos S. Karamitros or George Georgiou.

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Competing interests

J.B., C.S.K., N.M., W.-C.L., G.G. and E.S. are inventors on intellectual property related to this work, including the active patent US9975959B2 and the pending patents US20190350975A1 and US20210207110A1, which are assigned to the Board of Regents of the University of Texas System. G.G., E.S., X.M.Z., K. McGovern, S.C. and M.M. have equity interest in Ikena Oncology, a company pursuing the commercial development of this technology. J.B., C.S.K., C.L. and E.S. have consulted for Ikena Oncology (previously Kyn Therapeutics). The remaining authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–20 and Tables 1–5.

Reporting Summary

Supplementary Table 6

Sequences of the oligonucleotides used in this study.

Source data

Source Data Fig. 5

Statistical source data.

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Blazeck, J., Karamitros, C.S., Ford, K. et al. Bypassing evolutionary dead ends and switching the rate-limiting step of a human immunotherapeutic enzyme. Nat Catal 5, 952–967 (2022). https://doi.org/10.1038/s41929-022-00856-6

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