Directed evolution-based discovery of ligands for in vivo restimulation of chimeric antigen receptor T cells

Tomasz M Grzywa¹, Alexandra Neeser^#^{1

2}, Ranjani Ramasubramanian^#^{1

2}, Anna Romanov^{3

4}, Ryan Tannir^{1

5}, Naveen K Mehta³, Benjamin Cossette³, Duncan M Morgan^{3

6}, Beatriz Goncalves^{1

7}, Ina Sukaj³, Elisa Bergaggio⁸, Stephan Kadauke⁹, Regina M Myers^{10

11}, Luca Paruzzo^{12

13

14}, Guido Ghilardi^{12

13

14}, Austin Cozzone¹, Stephen J Schuster^{13

14}, Noelle Frey^{13

14}, Libin Zhang³, Parisa Yousefpour³, Wuhbet Abraham³, Heikyung Suh³, Marco Ruella^{12

13

14}, Stephan A Grupp^{9

10

11}, Roberto Chiarle^{8

15}, K Dane Wittrup^{3

4

6}, Leyuan Ma^{16

17

18

19

20}, Darrell J Irvine^{21

22

23

24

25

26}

Affiliations

¹ The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
² Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA, USA.
³ David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA.
⁴ Department of Biological Engineering, MIT, Cambridge, MA, USA.
⁵ College of Arts and Sciences, University of Pennsylvania, Philadelphia, PA, USA.
⁶ Department of Chemical Engineering, MIT, Cambridge, MA, USA.
⁷ Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁸ Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA.
⁹ Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
¹⁰ Division of Oncology, Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
¹¹ Cellular Therapy and Transplant Section and Cancer Immunotherapy Program, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
¹² Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, PA, USA.
¹³ Division of Hematology/Oncology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.
¹⁴ Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA.
¹⁵ Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy.
¹⁶ The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA. Leyuan.ma@pennmedicine.upenn.edu.
¹⁷ David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA. Leyuan.ma@pennmedicine.upenn.edu.
¹⁸ Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Leyuan.ma@pennmedicine.upenn.edu.
¹⁹ Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, PA, USA. Leyuan.ma@pennmedicine.upenn.edu.
²⁰ Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA. Leyuan.ma@pennmedicine.upenn.edu.
²¹ David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA. djirvine@scripps.edu.
²² Department of Biological Engineering, MIT, Cambridge, MA, USA. djirvine@scripps.edu.
²³ Department of Materials Science and Engineering, MIT, Cambridge, MA, USA. djirvine@scripps.edu.
²⁴ Ragon Institute of Massachusetts General Hospital, Cambridge, MA, USA. djirvine@scripps.edu.
²⁵ Howard Hughes Medical Institute, Chevy Chase, MD, USA. djirvine@scripps.edu.
²⁶ Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA. djirvine@scripps.edu.

^# Contributed equally.

PMID: 40855124
DOI: 10.1038/s41551-025-01470-0

Directed evolution-based discovery of ligands for in vivo restimulation of chimeric antigen receptor T cells

Tomasz M Grzywa et al. Nat Biomed Eng. 2025.

. 2025 Aug 25.

doi: 10.1038/s41551-025-01470-0. Online ahead of print.

Authors

Affiliations

¹ The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
² Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA, USA.
³ David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA.
⁴ Department of Biological Engineering, MIT, Cambridge, MA, USA.
⁵ College of Arts and Sciences, University of Pennsylvania, Philadelphia, PA, USA.
⁶ Department of Chemical Engineering, MIT, Cambridge, MA, USA.
⁷ Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁸ Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA.
⁹ Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
¹⁰ Division of Oncology, Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
¹¹ Cellular Therapy and Transplant Section and Cancer Immunotherapy Program, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
¹² Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, PA, USA.
¹³ Division of Hematology/Oncology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.
¹⁴ Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA.
¹⁵ Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy.
¹⁶ The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA. Leyuan.ma@pennmedicine.upenn.edu.
¹⁷ David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA. Leyuan.ma@pennmedicine.upenn.edu.
¹⁸ Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Leyuan.ma@pennmedicine.upenn.edu.
¹⁹ Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, PA, USA. Leyuan.ma@pennmedicine.upenn.edu.
²⁰ Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA. Leyuan.ma@pennmedicine.upenn.edu.
²¹ David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA. djirvine@scripps.edu.
²² Department of Biological Engineering, MIT, Cambridge, MA, USA. djirvine@scripps.edu.
²³ Department of Materials Science and Engineering, MIT, Cambridge, MA, USA. djirvine@scripps.edu.
²⁴ Ragon Institute of Massachusetts General Hospital, Cambridge, MA, USA. djirvine@scripps.edu.
²⁵ Howard Hughes Medical Institute, Chevy Chase, MD, USA. djirvine@scripps.edu.
²⁶ Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA. djirvine@scripps.edu.

^# Contributed equally.

PMID: 40855124
DOI: 10.1038/s41551-025-01470-0

Abstract

Chimeric antigen receptor (CAR) T cell therapy targeting CD19 elicits remarkable clinical efficacy in B cell malignancies, but many patients relapse owing to failed expansion and/or progressive loss of CAR-T cells. We recently reported a strategy to potently restimulate CAR-T cells in vivo, enhancing their functionality by administration of a vaccine-like stimulus comprised of surrogate peptide ligands for a CAR linked to a lymph node-targeting amphiphilic PEG-lipid (amph-vax). Here we demonstrate a general strategy to discover and optimize peptide mimotopes enabling amph-vax generation for any CAR. We use yeast surface display to identify peptide binders to FMC63 (the scFv used in clinical CD19 CARs), which are then subsequently affinity matured by directed evolution. CAR-T vaccines using these optimized mimotopes triggered marked expansion and memory development of CD19 CAR-T cells in both syngeneic and humanized mouse models of B-acute lymphoblastic leukaemia/lymphoma, and enhanced control of disease progression compared with CD19 CAR-T-only-treated mice. This approach enables amph-vax boosting to be applied to any clinically relevant CAR-T cell product.

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Conflict of interest statement

Competing interests: L.M. and D.J.I. are inventors on patents filed related to the amphiphile-mimotope vaccine technology (WO2021221782A1, WO2021221783A1), which has been licensed to Elicio Therapeutics. D.J.I. is a consultant and holds equity in Elicio Therapeutics. G.G. served as a scientific consultant for viTToria Biotherapeutics. M.R. holds patents related to CD19 CAR-T cells, served as a consultant for NanoString, Bristol Myers Squibb, GlaxoSmithKline, Scaylite, Bayer and AbClon, and receives research funding from AbClon, NanoString, Oxford NanoImaging, viTToria Biotherapeutics, CURIOX and Beckman Coulter. M.R. is the scientific founder of viTToria Biotherapeutics. The remaining authors declare no competing interests.

Update of

Directed evolution-based discovery of ligands for in vivo restimulation of CAR-T cells.
Grzywa T, Mehta N, Cossette B, Romanov A, Paruzzo L, Ramasubramanian R, Cozzone A, Morgan D, Sukaj I, Bergaggio E, Tannir R, Kadauke S, Myers R, Yousefpour P, Ghilardi G, Schuster S, Neeser A, Frey N, Goncalves B, Zhang L, Abraham W, Suh H, Ruella M, Grupp S, Chiarle R, Wittrup KD, Ma L, Irvine DJ. Grzywa T, et al. bioRxiv [Preprint]. 2024 Oct 1:2024.04.16.589780. doi: 10.1101/2024.04.16.589780. bioRxiv. 2024. Update in: Nat Biomed Eng. 2025 Aug 25. doi: 10.1038/s41551-025-01470-0. PMID: 38659938 Free PMC article. Updated. Preprint.

References

1. Cappell, K. M. & Kochenderfer, J. N. Long-term outcomes following CAR T cell therapy: what we know so far. Nat. Rev. Clin. Oncol. 20, 359–371 (2023). - PubMed - PMC
1. Irvine, D. J., Maus, M. V., Mooney, D. J. & Wong, W. W. The future of engineered immune cell therapies. Science 378, 853–858 (2022). - PubMed - PMC
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Directed evolution-based discovery of ligands for in vivo restimulation of chimeric antigen receptor T cells

Affiliations

Directed evolution-based discovery of ligands for in vivo restimulation of chimeric antigen receptor T cells

Authors

Affiliations

Abstract

Conflict of interest statement

Update of

References

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