Chimaeric antigen receptor (CAR) therapy is entering the mainstream for the treatment of CD19+ cancers. As is does we learn more about resistance to therapy and the role, risks and management of toxicity. In solid tumour CAR therapy research the route to the clinic is less smooth with a wealth of challenges facing translating this, potentially hugely valuable, therapeutic option for patients. As we strive to understand our successes, and navigate the challenges, having a clear understanding of how adoptively transferred CAR-T-cells behave in vivo and in human trials is invaluable. Harnessing reporter gene imaging to enable detection and tracking of small numbers of CAR-T-cells after adoptive transfer is one way by which we can accomplish this. The compatibility of certain reporter gene systems with tracers available routinely in the clinic makes this approach highly useful for future appraisal of CAR-T-cell success in humans.
This review covers the research to date in reporter gene imaging of gene-modified T-cells.
Chimaeric antigen receptors (CARs) are membrane-spanning fusion molecules in which a targeting moiety, usually a single chain antibody fragment, is coupled via hinge and transmembrane elements to an activating endodomain. CARs can be transduced into human T-cells, via retroviral or lentiviral vectors, with the potential for co-expression of other genes. When expressed in T-cells CARs redirect their specificity against a designated native ‘antigen’, obviating the need for either HLA expression or antigen processing. Immunotherapy using CAR-engineered T-cells is acquiring an increasing niche in the experimental therapy of malignant disease . Recent spectacular results have been achieved in the treatment of CD19+ haematologic malignancies [2–5]. Progress in solid tumours remains less evident [6–10]. Targeting solid tumours requires appropriate antigen selection, optimal CAR design, successful manufacture and administration of CAR-T-cells, CAR-T-cell homing in the patient, T-cell survival and proliferation in immunosuppressive tumour microenvironments and the development of immunological memory to persistently reject the tumour, to name but a few challenges. Toxicity of CAR-T-cell therapy is also a concern as clinical translation progresses [11–13]. Some toxicity may be desirable however, with ‘cytokine storms’ being associated with outcomes, at least in patients with CD19+ acute lymphoid leukaemia (ALL) [4,14].
To optimize efficacy and to predict toxicity of CAR-based immunotherapy, it is desirable to develop systems that allow real-time monitoring of the trafficking, tissue distribution and persistence of these cells in vivo.
Passive labelling of T-cells with 111In and imaging using single photon emission computed tomography (SPECT)-computed tomography (CT) is clinically useful , it is an approach that has been used in the detection of occult infection in patients for years . Imaging is, unfortunately, limited to a few days after T-cell transfer, owing to radioisotope decay [17–19], allowing only transient study of CAR-T-cell behaviour after adoptive transfer .
A more durable imaging approach may be achieved through the co-expression of imaging reporter genes with CARs in the viral vectors used for T-cell transduction. This can be done using internal ribosome entry sites (IRES) or viral T2A systems [20,21]. An ideal reporter would allow sensitive detection of cells through highly specific uptake of radiotracers, would not be immunogenic (removing the risk of elimination of the transduced CAR-T-cells) and would not result in detriment to efficacy of CAR-T-cell function when exposed to radiotracers.
Bioluminescence reporter imaging provides a sensitive approach to image T-cells in animal models [22–25]. It is not amenable to clinical translation as the imaging equipment and substrates have not been developed for human use. Nor will they be as the penetrance of the luminescence is not sufficient to allow detection of T-cells in deep tissue.
A third approach entails the co-expression of SPECT or positron emission tomography (PET) reporter genes that promote the specific uptake of a tracer (Table 1). Pre-eminent among such reporter genes is herpes simplex virus thymidine kinase (HSV-tk), or derivatives that have been mutated to improve function and genetic stability . These have been used both pre-clinically [25,27,28] and clinically , but are disadvantaged by their immunogenicity . Use of a human reporter gene ameliorates this concern. A number of different approaches have been studied including the human noradrenaline transporter gene (hNET), the human sodium iodide symporter (hNIS), the human deoxycytidine kinase double mutant (hdCKDM), the truncated and mutated human mitochondrial thymidine kinase 2 (hΔTK2DM), the somatostatin receptor and the dopamine receptor.
|Reporter .||SPECT tracers .||PET tracers .|
|Reporter .||SPECT tracers .||PET tracers .|
The hNET is a high affinity transporter for catecholamines found in the sympathetic nervous system. Uptake of therapeutic levels of metaiodobenzylguanidine (MIBG) in neural-crest tumours is mediated via the hNET . Genetic modification of cells to express the hNET enables selective uptake of the clinically available imaging tracers for non-invasive imaging 123I-MIBG for SPECT and 124I-MIBG/18F-MFBG for PET [32–34]. Genetic modification of T-cells with hNET enables in vivo dynamic imaging with sensitivity to detect cell numbers as low as 1×105 cells [34,36–38].
Use of hNIS as a T-cell imaging reporter brings several potential advantages. First, it promotes receptor-mediated uptake of the inexpensive, low toxicity and clinically useful SPECT tracer, technetium-99m pertechnetate (99mTcO4−) (Figure 1) as well as the PET tracer 124I. Second, the ectopic expression of NIS is generally well tolerated by host cells. Third, the human (h)NIS transgene is currently being evaluated in clinical studies using oncolytic viruses  (e.g. NCT00450814; NCT01503177), bringing important clinical experience with this reporter. Fourth, hNIS is only functional in viable cells . Ectopically expressed hNIS has been used to monitor the trafficking of leucocytes in vivo in pre-clinical studies [41,42]. T-cells can tolerate both hNIS expression and radiolabelling with large quantities of 99mTcO4− (25 MBq per million cells), without alteration in function or toxicity to these cells and accumulation of injected 99mTcO4−in vivo correlates with the presence of viable hNIS+ T-cells at that site .
SPECT-CT imaging of hNIS expressing CAR-T-cells
The hdCKDM and hΔTK2DM are pyrimidine-specific PET reporter genes used for imaging with 18F-FEAU, 124I-FIAU and 2′-deoxy-2′-18F-fluoro-5-methyl-1-β-L-arabinofuranosyluracil (all substrates of HSV-tk) [35,43]. They are similar in function to HSV-tk with two crucial differences. Firstly they are of human origin and likely not to be immunogenic. Secondly the mutation removes the ability of transduced cells to phosphorylate acycloguanosine-based antivirals such as ganciclovir. This is an advantage in some settings as HSV-tk transduced cells will be efficiently cleared by ganciclovir (beneficial in eliminating cells in the event of toxicity of graft compared with host disease) a drug which is useful for treating viral infections especially in immune compromised hosts, limiting antiviral options for these patients. Cells expressing hdCKDM are resistant to ganciclovir but have acquired sensitivity to pyrimidine-based pro-drugs such as cytarabine and gemcitabine, enabling removal of the transduced cells in the event of severe toxicity.
The somatostatin receptor has relatively broad normal tissue distribution in humans . Identification of receptor sub types has, however, enabled development of the somatostatin receptor subtype 2 (hSSTr2) as an imaging reporter gene. The clinically available PET tracer 68Ga-DOTATOC is specifically taken up by hSSTr2 transduced cells and concentrates tracer with minimal background . Similarly the dopamine type 2-receptor (D2R) can be retrovirally expressed in target cells for functional in vivo PET imaging [46,47]. To date the utility of D2R in adoptively transferred T-cells has not been reported.
These human molecules address the requirement for a non-immunogenic reporter. Most tracers studied have not been found to impact negatively on T-cell function in in vitro assays [34,42] Sensitivity of detection of CAR-T-cells is a more challenging metric to define. Different tracers offer different kinetics of uptake  and levels of transgene expression will vary with viral transduction of T-cells. Moroz et al.  have attempted to address this with a series of experiments looking at in vivo PET and SPECT imaging of subcutaneous injections of T-cells transduced with HSV-tk, hNET, hNIS and hdCKDM. There work has shown that, for the hNET, PET reporter systems have at least 10X the sensitivity of SPECT. For all the PET reporter/tracer combinations studied hNET/18F-MFBG at 4 h performed best with a sensitivity to detect <1×105 subcutaneous transduced T-cells. If we can define thresholds of sensitivity after intravenous injection in vivo this could lead to relative quantification of CAR-T-cell numbers in any given region of interest enabling evaluation of T-cell expansion. This has the potential to inform the kinetics of CAR-T-cell responses and, perhaps, enable modelling of toxicity with the potential for symptomatic intervention in the event of a patient developing a cytokine storm.
Translational research into genetically modified cell therapies for cancer is a fertile field. There have been marked successes in CD19+ B-cell malignancies, but within these successful trials some patients have not responded well. In solid tumour oncology CAR-T-cell therapy has not yet had its break through. There are a number of challenging obstacles to success. Imaging of CAR-T-cells in humans after adoptive transfer would enable detailed study of the behaviour of these cells in real time. This could inform study of failed treatment as well as help aid scheduling of intervention in the event of toxicity. In animal models the same imaging techniques can add understanding to CAR-T-cell behaviour in model systems. An ideal reporter system is human, easily transduced into CAR-T-cells, efficient with minimal background activity and compatible with cheap clinically available tracers. More than one reporter fulfilling these requirements exists and further study should lead to the adoption of these approaches in human clinical trials.
This work was supported by the Medical Research Council and Prostate Cancer UK.
chimaeric antigen receptor
dopamine type 2-receptor
human deoxycytidine kinase double mutant
human noradrenaline transporter gene
human sodium iodide symporter
human somatostatin receptor subtype 2
herpes simplex virus thymidine kinase
truncated and mutated human mitochondrial thymidine kinase 2
positron emission tomography
single photon emission computed tomography
Chimeric Antigen Receptor Therapy in Haematology and Oncology: Current Successes and Challenges: Held at Charles Darwin House, London, U.K., 19–20 October 2015