Highly efficient PD-1-targeted CRISPR-Cas9 for tumor-infiltrating lymphocyte-based adoptive T cell therapy
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Adoptive T cell therapy (ACT) with expanded tumor-infiltrating lymphocytes (TIL) can induce durable responses in cancer patients from multiple histologies, with response rates of up to 50%. Antibodies blocking the engagement of the inhibitory receptor programmed cell death protein 1 (PD-1) have been successful across a variety of cancer diagnoses. We hypothesized that these approaches could be combined by using CRISPR-Cas9 gene editing to knock out PD-1 in TILs from metastatic melanoma and head-and-neck, thyroid, and colorectal cancer. Non-viral, non-plasmid-based PD-1 knockout was carried out immediately prior to the traditional 14-day TIL-based ACT rapid-expansion protocol. A median 87.53% reduction in cell surface PD-1 expression was observed post-expansion and confirmed at the genomic level. No off-target editing was detected, and PD-1 knockout had no effect on final fold expansion. Edited cells exhibited few phenotypic differences and matched control functionality. Pre-clinical-scale results were confirmed at a clinical scale by generating a PD-1-deficient TIL product using the good manufacturing practice facilities, equipment, procedures, and starting material used for standard patient treatment. Our results demonstrate that simple, non-viral, non-plasmid-based CRISPR-Cas9 methods can be feasibly adopted into a TIL-based ACT protocol to produce treatment products deficient in molecules such as PD-1, without any evident negative effects.
| Originalsprog | Engelsk |
|---|---|
| Tidsskrift | Molecular Therapy - Oncolytics |
| Vol/bind | 24 |
| Sider (fra-til) | 417-428 |
| Antal sider | 12 |
| DOI | |
| Status | Udgivet - 2022 |
Bibliografisk note
Funding Information:
First and foremost, the authors sincerely thank all the patients who donated the samples used in the study. All funding sources are thanked for their generous support: Lundbeck Foundation (grants R296-2018-991 , R307-2018-3636 ), The Danish Cancer Society (grants R180-A11339 , R149-A10124 ), and Herlev and Gentofte Hospital Research Council (Clinician-Scientist grant to M.D.). All physicians and technicians involved in patient enrollment and sample procurement are heartily thanked for their efforts. Dr. Stine Kiær Larsen, Martin Jørgensen, and Dora Furda Selimovic are thanked for their roles in the GMP clinical-scale production process. Dr. Morten Hansen and Dr. Michael Douglas Crowther are thanked for technical assistance with flow cytometry set-up. Kasper Mølgaard Jensen is thanked for assistance with mycoplasma testing. Camilla Andersen is thanked for IDAA assay-related assistance. Dr. Ignacio Ortea is acknowledged for his work on the LC-MS proteomics analysis. The graphical abstract was created using BioRender.com .
Funding Information:
First and foremost, the authors sincerely thank all the patients who donated the samples used in the study. All funding sources are thanked for their generous support: Lundbeck Foundation (grants R296-2018-991, R307-2018-3636), The Danish Cancer Society (grants R180-A11339, R149-A10124), and Herlev and Gentofte Hospital Research Council (Clinician-Scientist grant to M.D.). All physicians and technicians involved in patient enrollment and sample procurement are heartily thanked for their efforts. Dr. Stine Ki?r Larsen, Martin J?rgensen, and Dora Furda Selimovic are thanked for their roles in the GMP clinical-scale production process. Dr. Morten Hansen and Dr. Michael Douglas Crowther are thanked for technical assistance with flow cytometry set-up. Kasper M?lgaard Jensen is thanked for assistance with mycoplasma testing. Camilla Andersen is thanked for IDAA assay-related assistance. The graphical abstract was created using BioRender.com. Conceptualization: C.A.C. E.P.B. ?M. Data curation: C.A.C. E.P.B. A.H.K. M.D. ?.M. Formal analysis: C.A.C. E.P.B. ?.M. Funding acquisition: CAC, EPB, IMS, MD, ?M. Investigation: C.A.C. E.P.B. ?.M. Methodology: C.A.C. E.P.B. ?.M. Project administration: CAC, EPB, ?M. Resources: E.P.B. A.H.K. I.M.S. M.D. ?.M. Supervision: I.M.S. M.D. ?.M. Validation: C.A.C. E.P.B. A.H.K. ?.M. Visualization: C.A.C. E.P.B. ?.M. Writing ? original draft: C.A.C. E.P.B. ?.M. Writing ? review & editing: All authors. E.P.B. declares that a patent application covering the IDAA method is pending and acts as a scientific advisor to Cobo Technologies. IDAA and ProfileIt are commercialized by Cobo Technologies. M.D. has received honoraria from Roche (past two years) and acts as a consultant for Achilles Therapeutics UK Limited.
Publisher Copyright:
© 2022 The Authors
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