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

John Blazeck1, Christos S Karamitros1, Kyle Ford1

  • 1Department of Chemical Engineering, University of Texas at Austin (UT Austin), Austin, Texas, USA.

Nature Catalysis
|December 5, 2022
PubMed

Insights

Researchers engineered a human enzyme to effectively degrade kynurenine (KYN), a tumor metabolite that suppresses the immune system. This enhanced enzyme demonstrated significant anti-tumor effects in mice, offering a promising cancer therapy.

Area of Science:

  • Biochemistry
  • Enzyme Engineering
  • Cancer Immunology

Background:

  • The tryptophan metabolite kynurenine (KYN) promotes tumor immunosuppression.
  • Bacterial kynureninases (KYNases) can reduce tumor immunosuppression but pose immunogenicity risks.
  • The human KYNase (HsKYNase) has insufficient activity for therapeutic use.

Purpose of the Study:

  • To engineer a human KYNase with enhanced activity and therapeutic potential.
  • To overcome limitations of bacterial KYNases for cancer immunotherapy.

Main Methods:

  • Directed evolution and protein engineering of human KYNase (HsKYNase).
  • Fitness selections and exploration of evolutionary trajectories (>10^9 variants).
  • Introduction of bacterial KYNase-conserved amino acid substitutions.
  • Pre-steady-state kinetic analysis and structural/dynamic characterization.

Main Results:

  • Engineered HsKYNase variant with 27-fold higher activity initially.
  • Introduction of specific substitutions enabled rapid evolution to ~500-fold improved activity.
  • Achieved reversed substrate specificity and potent anti-tumor effects in mouse models.
  • Identified a switch in the rate-determining step linked to structural and dynamic changes.

Conclusions:

  • Rational protein engineering can overcome evolutionary barriers to create highly active enzymes.
  • The evolved HsKYNase demonstrates significant therapeutic potential for cancer immunotherapy.
  • This approach offers a safer alternative to bacterial KYNases for clinical applications.

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