Directed Evolution of Genetically Encoded LYTACs for Cell-Mediated Delivery

Jonathan Lee Yang; Sean A Yamada-Hunter; Louai Labanieh; Elena Sotillo; Joleen S Cheah; David S Roberts; Crystal L Mackall; Alice Y Ting; Carolyn R Bertozzi

doi:10.1101/2023.11.14.567117

Directed Evolution of Genetically Encoded LYTACs for Cell-Mediated Delivery

bioRxiv [Preprint]. 2023 Nov 15:2023.11.14.567117. doi: 10.1101/2023.11.14.567117.

Authors

Jonathan Lee Yang¹, Sean A Yamada-Hunter², Louai Labanieh^{3

2}, Elena Sotillo^{3

2}, Joleen S Cheah⁴, David S Roberts¹, Crystal L Mackall^{2

5

6

7}, Alice Y Ting^{1

4

8}, Carolyn R Bertozzi^{1

9}

Affiliations

¹ Department of Chemistry and Sarafan ChEM-H, Stanford University, Stanford, CA, USA.
² Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.
³ Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
⁴ Departments of Biology, and Genetics Stanford University, Stanford, CA 94305, USA.
⁵ Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA.
⁶ Department of Medicine, Stanford University School of Medicine.
⁷ Parker Institute for Cancer Immunotherapy, San Francisco, CA 94305, USA.
⁸ Chan Zuckerberg Biohub-San Francisco, San Francisco, CA 94158, USA.
⁹ Howard Hughes Medical Institute, Stanford, CA, USA.

Abstract

Lysosome-targeting chimeras (LYTACs) are a promising therapeutic modality to drive the degradation of extracellular proteins. However, early versions of LYTAC contain synthetic glycopeptides that cannot be genetically encoded. Here we present our designs for a fully genetically encodable LYTAC (GELYTAC), making our tool compatible with integration into therapeutic cells for targeted delivery at diseased sites. To achieve this, we replaced the glycopeptide portion of LYTACs with the protein insulin like growth factor 2 (IGF2). After showing initial efficacy with wild type IGF2, we increased the potency of GELYTAC using directed evolution. Subsequently, we demonstrated that our engineered GELYTAC construct not only secretes from HEK293T cells but also from human primary T-cells to drive the uptake of various targets into receiver cells. Immune cells engineered to secrete GELYTAC thus represent a promising avenue for spatially-selective targeted protein degradation.

Keywords: Cell therapy; Chemical biology; Targeted protein degradation; synthetic biology.

Publication types

Preprint

Abstract

Publication types

Grants and funding