Smart Medical Imaging

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DTP P11 - An Indirect Labelling Approach to Track the Therapeutic CAR T-cells with Dual PET and Fluorescence Imaging

First Supervisor: Dr Ran Yan
Second Supervisor: Dr John Maher

Cell-based therapies have shown great promise in cancer treatment. One fundamental challenge in the successful development and clinical application of cellular therapeutics is the need to better understand the in vivo behaviour of adoptively infused cell products. This interdisciplinary project aims to develop a generic indirect labelling approach to track the persistence and proliferation of the c-myc tag-containing CAR T-cells with dual PET and fluorescence imaging. A dual reporter bioconjugation reagent, 124I-Green, labelled anti-c-myc 9E10 antibody, its scFv or F(ab)2 fragments will be systematically evaluated for this purpose. This dual PET and fluorescence imaging approach would enable detecting the in vivo behaviour of the c-myc tag-containing CAR T-cells across cellular to macroscopic scales. PET would provide real-time images of the living CAR T-cells and quantitatively measure their migration and proliferation in the whole-body. Meanwhile, the high-resolution ex vivo fluorescence imaging will unveil precisely the distribution of these CAR T-cells in the target and non-target tissues.

Background and hypothesis
The chimeric antigen receptor (CAR) technology is increasingly being harnessed to generate a novel T-cell based adoptive immunotherapeutic approaches for cancer and other disease types. One fundamental challenge in the successful development and clinical application of cell based therapeutics is the need to better understand the in vivo behaviour of adoptively infused cell products.
Recently, Maher engineered a second-generation CAR named A20-28z that re-targets T-cells against v6 integrin expressing tumours of diverse origin. Myc epitope tags were engineered into the hinge/spacer of this CAR which can be recognised by the 9E10 antibody (Fig. 1A). Given its small size and linear nature, the myc epitope tag can easily be engineered into any CAR design. Moreover, commercially available anti-c-myc 9E10 antibody, its single-chain variable fragment (scFv), and F(ab)2 fragments have nanomolar binding affinity for the myc epitope. Consequently, the in vivo persistence and proliferation of the myc epitope tagged CAR T-cells could be detected by the anti-c-myc 9E10 antibody, its scFv, or F(ab)2 fragments labelled with a contrast reagent. Ran has developed an iodine-124/fluorescein-based dual-modality labelling reagent, 124I-Green. It constitutes a generic tool for one-step installation of a PET and a fluorescent reporter to any antibody or antibody fragment. When coupled with a cancer targeting antibody, the 124I-Green dual-labelled A5B7 was successfully applied to undertake cancer PET imaging and fluorescence-guided surgery (Fig. 1B).
We envisage that a generic indirect labelling approach to track the persistence and proliferation of myc tagged CAR T-cells with dual PET and fluorescence imaging would be developed using the 124I-Green labelled anti-c-myc 9E10 antibody, its scFv, or F(ab)2 fragments. PET will provide real-time images of the living CAR T-cells and quantitatively measure their migration and proliferation in the whole-body. Meanwhile, the high-resolution ex vivo fluorescence imaging will unveil precisely the distribution of these CAR T-cells in the target and non-target tissues.


Figure1 A) the dimeric v6 integrin-specific CAR(A20-28z) containing myc epitope tags on T-cell surface; B) 124I-Green dual-labelled A5B7 antibody to enable cancer PET imaging and fluorescence-guided surgery


Road map
Stage 1: 124I-Green anti-c-myc 9E10 antibody/ fragments labelling and characterisation. (1-8 month)
1. Anti-c-myc 9E10 antibody, its scFv, and F(ab)2 bioconjugation with 124I-Green and different amount of its non-radioactive reference to install 1 to 4 fluorophores per biomolecule;
2. Dual labelled antibody/ fragments fluorescence emission spectrum measurement, HPLC analysis, and EC50 determination.
The dual labelled antibody/ fragments with similar HPLC retention time and EC50 to the corresponding native antibody/ fragments and emitting stronger fluorescence will be selected for in vitro evaluation. 
Stage 2: 124I-Green anti-c-myc antibody/ fragments in vitro evaluation (9-18 month)
1. The CAR(A20-28z) and control T-cells without the c-myc tag (5×104, 5×105, 5×106) incubation with the 124I-Green antibody/ fragments with and without addition of the corresponding native antibody/ fragments;
2. Evaluation of the in vitro anti-tumour activity of CAR(A20-28z) T-cell prior and post 124I-Green anti-c-myc antibody/ fragments incubation with variousv6 integrin expressing cell lines (Panc0403, CFPAC-1, BxPC3), and comparison with the negative control C20-28z CAR T-cells withoutv6 binding activity.
The 124I-Green antibody/ fragments having specific uptake by the CAR(A20-28z) T-cells in a cell number-dependent manner and with retention of anti-tumour activity will be selected for in vivo evaluation. 
Stage 3: PET/CT imaging and biodistribution of 124I-Green anti-c-myc antibody/ fragments (19-26 month)
1. Sequential PET/CT scanning on the subcutaneous Panc0403 xenograft bearing SCID Beige mice following the intravenous administration of the 124I-Green-anti-c-myc antibody at 24 h, 2, 3, and 5 days, 124I-Green-scFv at 1, 12, 24 h, and 2 days or 124I-Green-F(ab)2 at 6, 24 h, 2, and 3 days post injection, respectively;
2. biodistribution study at above time points to quantify the distribution of the 124I-Green anti-c-myc antibody/ fragments in this animal model.
The time points that the 124I-Green anti-c-myc antibody/ fragments are largely cleared from the blood pool, tumour xenograft, and major thoraco-abdominal organs will be selected for PET imaging of inoculated CAR(A20-28z) T-cells.
Stage 4 In vivo tracking of CAR(A20-28z) T-cells by 124I-Green anti-c-myc antibody/ fragments. (27-36 months)
1. Intra-tumoural injection of CAR(A20-28z) T-cells (1×106, 10×106, and 20×106) followed by the intravenous injection of 124I-Green anti-c-myc antibody/ fragments to the Panc0403 xenograft bearing SCID Beige mice for PET imaging at the time points determined in stage 3, respectively;
2. Additional PET/CT imaging on day 7, and day 14 of the CAR(A20-28z) T-cell inoculation with the corresponding 124I-Green anti-c-myc antibody/ fragments;
3. Control PET imaging experiments using the c-myc tag free CAR T-cells;
4. Biodistribution study after the final PET imaging;
5. Preparing tumours for sequential ex vivo autoradiography and fluorescence imaging.
The 124I-Green anti-c-myc antibody/ fragments met the following criteria will be deemed successful: i) can detect the smallest number of CAR(A20-28z) T-cells with PET imaging;
ii) can monitor the persistence of the CAR(A20-28z) T-cells over time;
iii) have negligible tumour radioactivity retention in animals that received c-myc tag free CAR T-cells;
iv) distribution patterns of radioactivity and fluorescence in the same tumour tissue section were largely identical.
Stage 5: PhD thesis writing up. Conference presentation. Preparation of manuscript(s). (37-42 month)

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