Master project 2025-2027, University of Oslo, Department of Biosciences.
Aims, content and approach
Background: Immunotherapy is a growing field of research and offers alternative treatments for solid tumours that are hard to treat. We have designed and validated different Chimeric Antigen Receptor (CAR) molecules from cancer-specific antibodies 1–5. When expressed in patient T cells, CAR can stimulate signalling without binding to a target, which is known as tonicity. This can lead to problems with the manufacturing of high-quality therapeutic products. Different methods have been proposed to go around tonicity; we have previously reported about the use of kinase inhibitors [1]. Recently, different labs have shown that combination of genome editing and targeted mutation in the CAR signalling domain could lead to a less tonic and more sustain CAR T cell product [2-4]. We will include the ITAM mutation in the CD28 co-signalling motif, CD28_1XX and will replace the retroviral transduction by genome editing using CRISPR-Cas9 based knock in/knock out strategy.
Main aim: The candidate will clone CAR molecules from our collection in the CD28z_1XX format, namely: K101CAR [5], HH1CAR [6] and CD19CAR, which are targeted against ovarian cancer, AML and B-cell malignancies, respectively. These mutants will be compared to the existing product in side-by-side assays. We will then move these constructs to a genome editing format, where they will be transduced to primary T cells in the TRAC locus. We will compare their expression in this format with the classical retroviral format. This work will guide our future strategy to clone and express CAR molecules.
Tentative title:
Production and Validation of next generation genome edited 1XX_CAR
Methods
1) In vitro assay Jurkat cell line expressing an NFAT-GFP reporter gene super infected with the CARCD28z_1xx and the original CAR. After transduction and sorting, these cells will be co-cultured with target cancer cell lines, and GFP will be detected (GFP = activation). Live-fluorescent microscopy we will follow the kinetic of the GFP signal using an advanced device for live-cell imaging (IncuCyte).
2) Primary T cell experiments: T cells isolated from 3-4 healthy donors will be used to perform comparative studies. We will confirm their killing capacity (Bioluminescence based assay, live cell microscopy, flow cytometry), rechallenge assays, quantify their cytokine release upon stimulation (Bioplex, ELISA), assess their metabolic status upon stimulation (Seahorse) and perform a cellular profiling (flowcytometry, CyTOF).
3) Genome editing: we will subclone the 1xx_CAR into our genome editing vector and proceed to electroporation. CAR expression will be monitored and genome edited cells will be compared to retrovirally transduced cells in functional assays. If time allows, selected genome edited 1XX_CAR will be tested in vivo.
Profile of the candidate
Immunotherapy is a very competitive field, and the Master student will have to be dedicated and hard working. She/he will learn a lot of techniques related with immunology and immunotherapy, including work with primary T cells and the possibility to test constructs in animals. If the objectives are reached, the results will be included in a scientific publication.
References
1. Rosselle, L., T. Leray, S. Joaquina, B. Caulier, E. McCormack, P. Gelebart, S. Walchli and E.M. Inderberg, STAR Protoc, 2024.
2. Eyquem, J., J. Mansilla-Soto, T. Giavridis, S.J. van der Stegen, M. Hamieh, K.M. Cunanan, A. Odak, M. Gonen and M. Sadelain, Nature, 2017.
3. Shang, K., D. Huang, J. Liu, Z. Yu, W. Bian, J. Chen, Y. Zhao, L. Liu, J. Jiang, Y. Wang, Y. Duan, J. Ge, S. Zhang, C. Zhou, Y. Han, Y. Hu, W. Zheng, J. Sun, H. Huang, S. Pei and J. Sun, Cell Reports Medicine, 2025/06/17.
4. Feucht, J., J. Sun, J. Eyquem, Y.J. Ho, Z. Zhao, J. Leibold, A. Dobrin, A. Cabriolu, M. Hamieh and M. Sadelain, Nat Med, 2019.
5. Casey, N.P., K. Kleinmanns, C. Forcados, P.F. Gelebart, S. Joaquina, M. Lode, E. Benard, F. Kaveh, B. Caulier, C. Helgestad Gjerde, E. Garcia de Jalon, D.J. Warren, K. Lindemann, E. Rokkones, B. Davidson, M.R. Myhre, G. Kvalheim, L. Bjorge, E. McCormack, E.M. Inderberg and S. Walchli, J Immunother Cancer, 2024.
6. Caulier, B., S. Joaquina, P. Gelebart, T.H. Dowling, F. Kaveh, M. Thomas, L. Tandaric, P. Wernhoff, N.U. Katyayini, C. Wogsland, M.E. Gjerstad, Y. Floisand, G. Kvalheim, C. Marr, S. Kobold, J.M. Enserink, B.T. Gjertsen, E. McCormack, E.M. Inderberg and S. Walchli, Cell Rep Med, 2024.