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metabolic remodeling during tumor treatment, as a result of environmental stressors, which may co nf ound interpretation of the image signal output [ 204].
6.6 Adoptively transferred T-cell imaging
Extensive clinical interest in CAR T-cell therapy, especially in hematological cancers, has been noted in recent years [205]. Tumor-specific T lymphocytes, usually from the patient, are collected and expanded in vitro, subsequently transduced to express a CAR targeting the tumor antigen of interest for the patient’s malignancy, before being reintroduced back into the patient. An important component contributing to the success of CAR T-cell therapy is the localization of the T-cells to the tumor target. Furthermore, identifying their functional status in real-time is also likely useful. Longitudinal nonin­vasive imaging is therefore crucial for monitoring the dynamics of these processes [206].
Multiple T-cell labeling strategies have been developed for this purpose. Passive
uptake of labeled lipophilic complexes has been explored for T-cell labeling. Initial stud­ies utilized ex vivo labeling with 111In-oxine of CD4 + T-cells to monitor infiltrating lymphocytes in Hodgkin’s lymphoma [207] and melanoma patients [208, 209] using SPECT/CT. Pittet et al. confirmed the requirement of antigen-specific binding for infil­tration of adoptively transferred T-cells using SPECT/CT imaging of 111In-oxine labeled T-cells combined with intravital microscopy [210]. Currently, a clinical trial is underway to monitor the dynamics of ex vivo 111In-oxine-labeled autologous CD8 + T-cell tumor infiltration in nonsmall cell lung cancer patients receiving neo-adjuvant PD-L1 (NCT03853187). T-cell populations can be radiation-sensitive, although recent studies suggest that certain T-cell subsets (e.g., CD4, Treg) are more radioresistant than others [178]. Stanton et al. found that 111In-oxine labeled adoptively labeled T-cells did not significantly impact their viability or function [211]. Further studies should con­tinue to monitor the effect of radioisotopes upon T-cell function.
PET agents have also been explored to directly label T-cells [32], including using 64Cu conjugated to lipophilic complexes such as tropolonate, polyethylenimine, and pyruvaldehyde-bis(N of these agents within cells, and subsequent accumulation of the effluxed agents in the liver, limit their use for clinical purposes. 89Zr-oxine labeling has been alternatively pur­sued, with better labeling yield compared with 111In-oxine, and 89Zr-oxine labeled γδ T-cells were able to be followed for over a week in mouse models of breast cancer [212]. Given concern for the sequestration of free 89Zr to the bone marrow, Bansal et al. explored the use of 89Zr-desferrioxamine-NCS (89Zr-DBN) for cell labeling, showing stable labeling to the cell surface of human mesenchymal stem cells for up to seven days. Other direct T-cell labeling approaches include labeling with iron oxide nanoparticles
[213], gold nanoparticles complexed to 64Cu, and PFC nanoparticles for 19F-MRI [214, 215], although none have been translated for human use to date. T-cell labeling
4
-methylthiosemicarbazone). However, the short residence time
451Image-guided cancer immunotherapy
452 Thomas S.C. Ng and Miles A. Miller
Данная книга находится в списке для перевода на русский язык сайта https://meduniver.com/
with an optimized highly derivatized cross-linked iron oxide nanoparticle was efficient, nontoxic, and allowed high-resolution visualization of heterogeneous tumor accumula­tion of T-cells. PFC labeling of T-cells offers high sensitivity given low background sig­nal in tissue and quantification capabilities using 19F-MRI but may suffer from dilution of signal upon cell division and PFC persistence in dead cells.
With the need for ex vivo transduction before reintroduction to patients, CAR T-cells
may be well-suited for reporter gene labeling. Gene reporters for several imaging modal­ities have been developed. Moroz et al. performed a comparative analysis of multiple report gene systems for nuclear imaging of T-cells [216], including a human deoxycytidine kinase double mutant (hdCKDM), the human sodium-iodide symporter (hNIS), human norepinephrine transporter (hNET), and herpes simplex virus type 1 thy­midine kinase (hsvTK) reporter gene systems. They found the hNET/meta-18F­fluorobenzylguanidine (18F-MFBG) reporter system to be the most sensitive, detecting 35-40 10
3
Tcells without significant toxicity. Krebs et al. transduced DOTA antibody
reporter 1 (DAbr1) with GFP in CD3/CD28 activated human T-cells and 1928z CAR T cells. This moiety is expressed on the cell surface and binds irreversibly to lanthanoid (S)-2-(4-acrylamidobenzyl)-DOTA (AABD) [217]. They showed the feasibility of this system for PET and SPECT imaging using 86Y and 177Lu AABD and demonstrated the highest T-cell to background contrast in animal models within 16 h postinjection. Somatostatin receptor 2 (SSTR2) is the target for the FDA-approved agents 68Ga­DOTATATE and DOTATOC. While used clinically to image neurorendocrine tumors, among other malignancies, it is not significantly expressed in normal tissues. Vedvyas et al. thus expressed SSTR2 in CAR T-cells for monitoring by 68Ga­DOTATOC [218]. In this system, peak T-cell tumor accumulation lagged tumor cell burden by several days in CAR-T cell therapy responders, using a mouse model of met­astatic anaplastic thyroid cancer. Results suggested enduring cognate antigen-mediated signals, causing continued CAR T cell expansion before eventual exhaustion and con­traction of the T-cell population. This pattern was not observed in nonresponders. In a similar vein, Minn et al. transduced anti-CD19 CAR T-cells with the prostate-specific membrane antigen (PSMA). In a mouse model of acute lymphoblastic leukemia, PET/CT imaging of CD19 dynamics showed a discrepancy between CAR T-cells levels in periph­eral blood compared to uptake levels within the tumor, highlighting the need for T-cell infiltration monitoring by imaging [219]. A reporter gene system that has no background expression and minimal immunogenicity are ideal. Sellmyer et al. adapted the expression of the Escherichia coli dihydrofolate reductase enzyme (eDHFR) together with fluorine-18 labeled small-molecule antibiotic trimethoprim (18F-TMP) as a novel CAR T-cell reporter system [220]. GD2+ CAR T-cells engineered with eDHFR, yellow fluorescent protein (YFP), and Renilla luciferase (rLuc) showed preferential targeting for GD2+ tumors in mice and showed gradual migration from the spleen to the tumor over 2 weeks. Given its small size, eDHFR (18kDa, compared to 46 kDa for HSV-tk) is potentially less
immunogenic than the other tested systems while maintaining high specificity in targeting.
To date, only one T-cell reporter gene system has been tested in patients. Keu et al. expressed the HSV1-tk reporter gene in IL13Ra2-CAR T-cells. Using 9-[4-[18F] fluoro-3-(hydroxymethyl)butyl]guanine (18F-FHBG), cell trafficking was monitored by PET/CT in patients with recurrent glioma [221, 222] (Fig. 9). Significantly increased imaging signal was noted in tumors injected with T-cells, suggestive of their localization.
453Image-guided cancer immunotherapy
Fig. 9 CAR T-cell imaging in clinical trials. 18F-FHBG PET imaging was performed in a 60-year-old male with multifocal left-hemispheric glioma. T-cells were injected into the medial left frontal lobe tumor (yellow arrows). (A) Tumor size was delineated with T panels). PET images were fused with MRI images (bottom left panels), and 3D volumes of interest were outlined in red. (B) MRI and MR-PET images 1 week after T-cell infusions. 18F-FHBG activity was addi­tionally assessed in a noninjected tumor focus (red arrows) before (C) and after (D) T-cell infusions. These findings show both local and region trafficking of injected labeled CAR T-cells. Further, images show nonspecific uptake pre-T-cell injection which needs to be accounted for during analysis.
(Adapted from Keu KV, Witney TH, Yaghoubi S, Rosenberg J, Kurien A, Magnusson R, et al. Reporter gene imaging of targeted T cell immunotherapy in recurrent glioma. Sci Transl Med. 2017;9(373):eaag2196.)
-weighted (T1W) contrast-enhanced MRI (top left
1
454 Thomas S.C. Ng and Miles A. Miller
Данная книга находится в списке для перевода на русский язык сайта https://meduniver.com/
However, the slightly increased signal was also found in untreated tumors, underscoring the need to account for nonspecific uptake.
Understanding the functional status of CAR T-cells localized at the tumor site may offer insights into mechanisms affecting their efficacy and resistance. To this end, reporter gene technology may be tailored to be expressed under T-cell activation pro­moters. In a proof of concept study, P onomarev et al. monitored T-cell receptor (TCR) dependent, nuclear factor of activated T cells (NFAT) mediated activation of T cells by optical fluorescence imaging (OFI) and positron emission tomography (PET), enabling moni toring of T-cell activation in vivo [223].Toimprovethespec­ificity of the T-cell activation signal, Uchibori et al. transduced CD-19 targeting CAR T-cells with an inducible reporter (CAR-T/iReporter) that is strongly induced only in the presence of CD19 positive cells. Imaging applicability of this system was demon­strated in vivo with a coexpressed lucifer ase reporter, showing targeted induction only at CD19 + tumor sites [224].
7. Imaging to guide bispecific T-cell engager therapy (BiTE)
Bispecific T-cell engager (BiTE) therapy is another promising immunotherapy modality explored for clinical use [225]. BiTe is a recombinant bispecific protein­containing single-chain variable fragments (scFv) targeting a tumor-associated antigen and a T-cell activating molecule, thus supporting the coengagement of tumor cells with activated T-cells. Imaging can play an important role in evaluating BiTE therapy by (1) identifying patients with appropriate tumor burden expressing the tumor antigen of interest, and (2) monitoring appropriate T-cell recruitment and response.
In a phase I study, Moek et al. evaluated the use of
directed to carcinoembryonic antigen (CEA) on tumor cells and CD3 on T-cells
89
[226].
Zr- AMG 211 was imaged in patients with gastric adenocarcinoma over 2 days, showing uptake in lymphoid tissues (spleen and bone marrow) commensurate with its CD3 targeting capabilities as well as heterogeneous tumor uptake. Imaging of other BiTE constructs is being actively explored [227]. Successful translation of BiTe therapies to the clinic may also benefit from patient stratification offered by imaging. For example, PSMA-targeting PET tracers may identify potentially responsive prostate cancer patients (NCT04221542, NCT03792841), while immunoPET agents may inform BiTE treat­ment for hematologic malignancies (NCT00274742, NCT01741792).
89
Zr-labeled BiTE AMG 211
8. Image-guided interventional immunotherapies
Conventional delivery of chemotherapy is via intravenous injection or oral inges­tion. However, these modes of delivery have several limitations. Systemic delivery can result in dose-limiting toxicities precluding adequate dosing and poor tumor drug
penetration. This can hamper efficacy and can potentially result in drug resistance. Local­ized drug delivery can overcome some of these barriers, and imaging can guide these modes of tumor drug delivery. Transarterial delivery of chemo- (TACE) and radio­embolization (TARE) therapies directed at hepatocellular carcinoma lesions exemplify the concept [228]. For these therapies, 99mTc-MAA agents are used to identifying appropriate hepatic arterial selection for subsequent therapy delivery and ensure minimal off-target delivery to the lungs and adjacent bowel. Interventional devices and imaging techniques used for these treatments can similarly be applied for the guided delivery of immunotherapies. For example, preclinical studies have explored both intraarterial and intravenous delivery of adoptive cell therapy. Using fluorescently labeled OT1 T-cells and immunofluorescence, Visioni et al. demonstrated improved infiltration of activated T-cells into tumors via intraarterial infusion as compared with intravenous injection. The latter resulted in increased lung retention of T-cells [229].
Recent preclinical studies have demonstrated the improved efficacy of concentrated delivery of immunostimulatory agents into the local tumor site, both for a sustained local­ized and systemic response. In 2015, the US FDA approved the first oncolytic virus, talimogene laherparepvec (TVEC), as immunotherapy for the treatment of metastatic melanoma lesions that cannot be surgically resected. There are many clinical trials ongoing for intratumoral injections of oncolytic viruses (NCT02977156), immune checkpoint inhibitors (NCT03707808, NCT02812524, NCT03892525), immunostimulatory com­pounds such as polymers (NCT03993678), cytokines (NCT04362722), or mRNA (NCT03788083) and CAR T cells (NCT01837602).
Multimodal interventional imaging techniques can be harnessed for the tumoral delivery of immunotherapy agents. Noninvasive imaging probes described in earlier sec­tions can be used to identify the target tumors and intratumoral targets for precise delivery of immunotherapeutic agents. These can be combined with novel deployment devices to ensure adequate drug injection. Sheth et al. performed a case study review of 85 cases with patients receiving intratumoral guided delivery of immunotherapies in advanced solid organ malignancies [230]. No adverse events were noted with the technical component of the procedures, with major adverse events due to immune-related systemic toxic effects occurring in 2% of investigational agents and 4% of standard-of-care procedures (4%). These results suggest that intratumoral delivery of immunotherapeutics is feasible.
Interventional oncology (IO) is a subspecialty within interventional radiology. Several techniques actively in use in IO can stimulate the immune system (e.g., thermal ablation, TACE, TARE, high-intensity focused ultrasound) and can be synergistic with concurrently administered immunotherapy to sustain an immune response
[231]. For example, anti-CTLA-4 can be combined with TACE to unmask tumor-
associated antigen-specific responses [232], which has been associated with improved patient survival. Efforts are underway to understand t he evolving tumor response of
455Image-guided cancer immunotherapy
456 Thomas S.C. Ng and Miles A. Miller
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IO therapies. Imaging probes described above are well-poised to monitor these pro­cesses in vivo.
9. Summary and future directions
As reviewed in this chapter, many promising clinical imaging approaches are being
explored to monitor cancer immunotherapies. These companion imaging diagnostics promise to give more precise readouts of immune system activation in vivo and comple­ment the current mainstay of anatomic clinical imaging. These strategies must be robustly evaluated in concert with the therapies that they seek to monitor. Rigorous clinical trials providing evidence-based motivation for using the imaging modalities described above will facilitate their widespread adoption. Concurrently it should be recognized that these imaging techniques can also improve or fine-tune the indications for immunotherapies based on precise patient stratification and early treatment response monitoring. Further­more, imaging data are increasingly being analyzed in the context of the broader clinical and laboratory information available. Integration of genomic information from tissue samples [233, 234], biomarkers from liquid biopsies [235, 236], for example, will max­imize the utility of the combined data to tailor individual patient care. Identification and realization of such opportunities will require close collaboration between imaging scien­tists, physicians, cancer biology researchers, and clinical oncologists.
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