Antibodies have long been recognised as potent vectors for carrying diagnostic medical radionuclides, contrast agents and optical probes to diseased tissue for imaging. The area of ImmunoPET combines the use of positron emission tomography (PET) imaging with antibodies to improve the diagnosis, staging and monitoring of diseases. Recent developments in antibody engineering and PET radiochemistry have led to a new wave of experimental ImmunoPET imaging agents that are based on a range of antibody fragments and affibodies. In contrast to full antibodies, engineered affibody proteins and antibody fragments such as minibodies, diabodies, single‐chain variable region fragments (scFvs), and nanobodies are much smaller but retain the essential specificities and affinities of full antibodies in addition to more desirable pharmacokinetics for imaging. Herein, recent key developments in the PET radiolabelling strategies of antibody fragments and related affibody molecules are highlighted, along with the main PET imaging applications of overexpressed antigen‐associated tumours and immune cells.
The use of biologics in positron emission tomography (PET) imaging is an important area of radiopharmaceutical development and new automated methods are required to facilitate their production. We report an...
Non-invasive positron emission tomography (PET) imaging of immune cells is a powerful approach for monitoring the dynamics of immune cells in response to immunotherapy. Despite the clinical success of many immunotherapeutic agents, their clinical efficacy is limited to a subgroup of patients. Conventional imaging, as well as analysis of tissue biopsies and blood samples do not reflect the complex interaction between tumour and immune cells. Consequently, PET probes are being developed to capture the dynamics of such interactions, which may improve patient stratification and treatment evaluation. The clinical efficacy of cancer immunotherapy relies on both the infiltration and function of cytotoxic immune cells at the tumour site. Thus, various immune biomarkers have been investigated as potential targets for PET imaging of immune response. Herein, we provide an overview of the most recent developments in PET imaging of immune response, including the radiosynthesis approaches employed in their development.
Hypoxia is a complex microenvironmental condition known to regulate choline kinase α (CHKA) activity and choline transport through transcription factor hypoxia-inducible factor-1α (HIF-1α) and, therefore, may confound the uptake of choline radiotracer [18F]fluoromethyl-[1,2-2H4]-choline ([18F]-D4-FCH). The aim of this study was to investigate how hypoxia affects the choline radiotracer dynamics. Three underlying mechanisms by which hypoxia could potentially alter the uptake of the choline radiotracer, [18F]-D4-FCH, were investigated: 18F-D4-FCH import, CHKA phosphorylation activity, and the efflux of [18F]-D4-FCH and its phosphorylated product [18F]-D4-FCHP. The effects of hypoxia on [18F]-D4-FCH uptake were studied in CHKA-overexpressing cell lines of prostate cancer, PC-3, and breast cancer MDA-MB-231 cells. The mechanisms of radiotracer efflux were assessed by the cell uptake and immunofluorescence in vitro and examined in vivo (n = 24). The mathematical modelling methodology was further developed to verify the efflux hypothesis using [18F]-D4-FCH dynamic PET scans from non-small cell lung cancer (NSCLC) patients (n = 17). We report a novel finding involving the export of phosphorylated [18F]-D4-FCH and [18F]-D4-FCHP via HIF-1α-responsive efflux transporters, including ABCB4, when the HIF-1α level is augmented. This is supported by a graphical analysis of human data with a compartmental model (M2T6k + k5) that accounts for the efflux. Hypoxia/HIF-1α increases the efflux of phosphorylated radiolabelled choline species, thus supporting the consideration of efflux in the modelling of radiotracer dynamics.
Antibody-drug conjugates (ADCs) are emerging targeted agents against cancer. Current studies of ADCs are performed on monolayer cultures which do not mimic the biophysical property of a tumour. Hence, in vitro models that can better predict the efficacy of ADCs in vivo are needed. In this study, we aim to optimise 3-dimentional cancer spheroid systems, which preserve the features of the tumour structure, to test the efficacy of two ADCs (T-DM1 and T-vcMMAE). Firstly, a set of reproducible spheroid models using epithelial ovarian cancer cell lines were established. Subsequently, phenotypic changes in spheroids were characterised upon ADC treatment. The penetration dynamics of ADCs into 3D tumour structure were also studied. Our data revealed that spheroids are less sensitive to ADCs compared to monolayer cultures. Interestingly, the small molecule component of ADCs- the cytotoxic payload- showed a similar decrease in efficacy in spheroids compared to monolayer cultures. Furthermore, we also gained new insight into ADC penetration dynamics and showed that ADCs can fully penetrate a tumour-like spheroid within 24h. The results suggest that although ADCs, as large molecule biological drugs, are likely to have slower penetration dynamics than small molecule compounds such as their cytotoxic payload, they could have comparable capability to kill cancer cells in 3D structures. This may be explained by the fact that multiple cytotoxic payloads are conjugated with each single antibody, which compensates the penetration deficiency of the large molecules. In conclusion, our work confirms that the tumour 3D structure could limit the therapeutic efficacy of ADCs. Nevertheless, optimising ADC design such as adjusting drug-to-antibody ratios could help to overcome this hurdle.
Messenger RNA (mRNA) has been proposed as a therapeutic agent for various diseases, including cancer. To ensure effective transfection of cancer cells, mRNA needs to be transported with a delivery system that protects its integrity and functionality. In this regard, cationic lipid nanoparticles composed of dioleoylphosphatidylethanolamine (DOPE) and 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) have emerged as common vectors to deliver mRNA. In this project, we aim to use luciferase mRNA as a reporter to synthesise mRNA-loaded cationic lipid nanoparticles, and optimise their mRNA encapsulation and transfection efficiency in ovarian cancer cells. The optimisation process included: 1) adjusting the lipid formulation; 2) adjusting the input mRNA concentration before lipid nanoparticle extrusion; and 3) adjusting the extrusion methods. After optimisation, the encapsulation efficiency was optimised to 62%, thus achieving a relatively high transfection luminescence signal (9.4 times compared to baseline). The lipid nanoparticles also demonstrated stable physical characteristics and high biocompatibility (above 75% cell viability after treatment) within 24 hours. Overall, this project evaluated the synthesis of DOPE/DC-Chol cationic lipid nanoparticles, and optimised their mRNA encapsulation and transfection efficiency in ovarian cancer cell lines. The optimised lipid nanoparticles can be utilised as an ideal system for mRNA delivery, which could be further developed as a potential platform for the immunotherapy in ovarian cancer.
Antibody drug conjugate (ADC) is a novel class of therapeutic agent which recently showed great success in both solid tumour and blood cancer. ADCs deliver potent cytotoxic payloads to tumour via cancer specific monoclonal antibodies. A few ADCs are currently evaluated in clinical trials for high grade serous ovarian cancer (HGSOC). However, most of them target FOLR1-alpha, a conventional HGSOC target which also expresses in vital organs including lungs. Thus, it is desirable to discover more specific antigen targets, and these new targets will be the foundation for the next-generation ADC for HGSOC.We propose that ovarian tumour specificity of an ADC target could contributed by three factors: 1. Lineage (e.g. fallopian tube marker FOLR1); 2. DNA amplification (e.g. ERBB2); 3. Other carcinogenesis process including epigenetic reprogramming. Then, by combining the GTEX, TCGA dataset with our own WES, long-reads RNA-sequencing and Mass spectrometry-based proteomics cohort, an array of differentially expressed genes in HGSOC tumours are identified. Out of over 50 candidates, two most promising targets are chosen for monoclonal antibodies (mAbs) production and subsequent ADC development. First, the affinity and specificity of the mAbs were confirmed by ELISA, flow cytometry and immunofluorescence. The cellular internalisation characteristics of these antibodies are confirmed by a fluorescence-based assay. Then, ADCs for these two targets are produced by conjugating monomethyl auristatin E payload to the mAbs via a cleavable linker. Finally, both ADCs show highly target-specific and sub-nanomolar cytotoxicity in model ovarian cancer cell lines. In conclusion, we identify a panel of HGSOC-specific ADC targets and develop two ADCs candidates for further pre-clinical and clinical investigations. Citation Format: Ruisi Fu, Hantao Lou, Ning Wang, Dongnan Yan, Clara Tresserras-Segura, Haonan Lu, Eric Aboagye. Development of an antibody-drug conjugate panel targeting high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 455.
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