Background:
Immunotherapy is a growing field of research and offers alternatives to traditional cancer treatments which are not always effective as is the case for solid tumours. We have designed and validated different Chimeric Antigen Receptor (CAR) molecules from cancer-specific antibodies 1–5. In order to facilitate their path to the clinic, their safety profile should be evaluated. A main concern resides in the fact that they are all of murine origin, which can trigger an anti-mouse response once injected in a human being. An accepted method to avoid this effect is to render the mouse molecule “more human”, using a process called “humanization”, by replacing exposed amino acids with ones more frequently occurring in human sequences with the intention to not affect the specificity of the molecule.
Main aim:
The candidate will isolate a CAR molecules (K101CAR) targeted against an ovarian cancer epitope. A selection of the most functionally related to the original murine CAR will be performed using different immunological tools. in addition, he/she will study specific constructs for their ability to not only equal the original murine construct but also to enhance it. Indeed, it was recently reported that humanization might improve CAR fitness6. If the progression is satisfying, the selected CAR constructs will even be tested in animal models.
Methods:
1) In vitro assay using model T-cell line, Jurkat expressing an NFAT-GFP reporter gene together with the selected humanized K101CAR construct. After retroviral transduction and sorting, Jurkat-OSCAR will be co-incubated with ovarian cancer cell lines, the reporter gene will be activated and GFP will be produced. Thus, by fluorescent microscopy we will follow the kinetic of the GFP signal using an advanced device for live-cell imaging (IncuCyte). This will help us to perform a first selection of the hK101CAR: only the construct showing similar kinetics to the mK101CAR will be kept.
2) Primary T cell experiments: hK101CAR will be transduced into T cells isolated from 3-4 healthy donors and a series of assays will be run to validate their potency and verify that their specificity is maintained. We will confirm their killing capacity (Bioluminescence based assay, live cell microscopy, flowcytometry), quantify their cytokine release upon stimulation (Bioplex, ELISA), assess their metabolic status upon stimulation (Seahorse)7 and perform a cellular profiling (flowcytometry, CyTOF). If time allows, we will also test their activity against complex 3D structure (spheroids)8 and prepare large amounts of material for in vivo assay.
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:
- K?ksal, H. et al. Combinatorial CAR design improves target restriction. J Biol Chem 296, 100116 (2021).
- K?ksal, H. et al. Preclinical development of CD37CAR T-cell therapy for treatment of B-cell lymphoma. Blood Adv 3, 1230–1243 (2019).
- Mensali, N. et al. ALPL-1 is a target for chimeric antigen receptor therapy in osteosarcoma. Nat Commun 14, 3375 (2023).
- Casey, N. P. et al. Efficient CAR T cell targeting of the CA125 domain of MUC16. In preparation.
- Forcados, C. et al. Targeting carbohydrates in adenocarcinomas: STn-CAR T cells. In preparation.
- Landoni, E. et al. Modifications to the Framework Regions Eliminate Chimeric Antigen Receptor Tonic Signaling. Cancer Immunol Res (2021) doi:10.1158/2326-6066.CIR-20-0451.
- Joaquina, S., Forcados, C., Caulier, B., Inderberg, E. M. & W?lchli, S. Determination of CAR T cell metabolism in an optimized protocol. Frontiers in Bioengineering and Biotechnology 11, (2023).
- Dillard, P. et al. Colorectal cysts as a validating tool for CAR therapy. BMC Biotechnol 20, 30 (2020).