Development of a high-affinity anti-ROR1 variable region for broad anti-cancer immunotherapy

  • Mol Ther. 2025 Dec 2:S1525-0016(25)00959-1. doi: 10.1016/j.ymthe.2025.11.021.
Joshua K M Wong  1 ,  Pui Yeng Lam  1 ,  Elaina Coleborn  1 ,  Jiya Jose  1 ,  Louisa Alim  1 ,  Cui Tu  1 ,  Magdalena Antczak  2 ,  Bettina Dietmair  3 ,  Arghavan Golbaz Hagh  4 ,  Lucia Noronha  5 ,  Seth W Cheetham  3 ,  John Hooper  6 ,  Paul A Beavis  7 ,  Delphine Merino  8 ,  Jean Berthelet  8 ,  Lauren G Aoude  1 ,  Amy E McCart-Reed  9 ,  Sunil Lakhani  9 ,  Peter T Simpson  10 ,  Gustavo R Rossi  1 ,  Andrew J Brooks  11 ,  Martina L Jones  4 ,  Fiona Simpson  1 ,  Fernando Souza-Fonseca-Guimaraes  12
Affiliations
  • 1. Frazer Institute, The University of Queensland, Woolloongabba, QLD 4102, Australia.
  • 2. Queensland Cyber Infrastructure Foundation Ltd (QCIF) Bioinformatics, Brisbane, QLD 4072, Australia.
  • 3. Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia, QLD 4067, Australia; BASE Facility, University of Queensland, St Lucia, QLD 4067, Australia.
  • 4. Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia, QLD 4067, Australia.
  • 5. Laboratório de Patologia Experimental, Curitiba, Queensland 80215-901, Brazil.
  • 6. Mater Research Institute, The University of Queensland, Brisbane, QLD 4102, Australia.
  • 7. Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, VIC 3000, Australia; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC 3010, Australia.
  • 8. Olivia Newton-John Cancer Research Institute, Heidelberg, VIC 3084, Australia.
  • 9. UQ Centre for Clinical Research, Faculty of Health, Medicine and Behavioural Sciences, The University of Queensland, Herston, QLD 4029, Australia.
  • 10. UQ Centre for Clinical Research, Faculty of Health, Medicine and Behavioural Sciences, The University of Queensland, Herston, QLD 4029, Australia; School of Biomedical Sciences, Faculty of Health, Medicine and Behavioural Sciences, The University of Queensland, Saint Lucia, QLD 4067, Australia.
  • 11. Frazer Institute, The University of Queensland, Woolloongabba, QLD 4102, Australia; School of Science & Technology, University of New England, Armidale NSW 2351, Australia.
  • 12. Frazer Institute, The University of Queensland, Woolloongabba, QLD 4102, Australia. Electronic address: [email protected].
PMID: 41338184 DOI: 10.1016/j.ymthe.2025.11.021
Abstract

Receptor tyrosine kinase-like Orphan Receptor 1 (ROR1) is an emerging target in Cancer Immunotherapy, recognized for its consistent and elevated expression across several epithelial tumors, including triple-negative breast Cancer (TNBC). TNBC is an aggressive and difficult-to-treat Cancer, with limited effective therapeutic options currently available. Therapeutic approaches centered on targeting ROR1 have therefore become increasingly popular, with ROR1 chimeric antigen receptor (CAR) T cells currently in clinical trials to treat TNBC patients. While ROR1-targeting therapies have shown promising preclinical results, single arm treatment has often shown low efficacy as well as off-target toxicity. Natural killer (NK) cell-based immunotherapies, such as antibody-dependent cell cytotoxicity-inducing monoclonal antibodies and CAR NK cells, have also been shown to induce Cancer cell cytotoxicity; however, with less toxicity compared with CAR T cells. Here, we developed and characterized a phage-derived single-chain fragment variable (scFv) against a highly specific ROR1 region and generated scFv-derived chimeric monoclonal antibodies and anti-ROR1-CAR NK cells, which show anti-cancer efficacy against TNBC cells. Additionally, we found TGF-β inhibition using either small-molecule inhibitors or CRISPR-Cas9-edited NK cells could further enhance ROR1-targeting therapy persistence and efficacy in controlling TNBC tumor growth.

Keywords
ADCC; CAR NK cells; CRISPR/Cas9; Frizzled-Kringle domain; TGFBR2 knockout; antibody drug conjugate; natural killer cells; phage display; scFv; triple negative breast cancer.
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