Granzyme B PET Imaging in Response to In Situ Vaccine Therapy Combined with αPD1 in a Murine Colon Cancer Model

Immune checkpoint inhibitors (ICIs) block checkpoint receptors that tumours use for immune evasion, allowing immune cells to target and destroy cancer cells. Despite rapid advancements in immunotherapy, durable response rates to ICIs remains low. To address this, combination clinical trials are underway assessing whether adjuvants can enhance responsiveness by increasing tumour immunogenicity. CpG-oligodeoxynucleotides (CpG-ODN) are synthetic DNA fragments containing an unmethylated cysteine-guanosine motif that stimulate the innate and adaptive immune systems by engaging Toll-like receptor 9 (TLR9) present on the plasmacytoid dendritic cells (pDCs) and B cells. Here, we have assessed the ability of AlF-mNOTA-GZP, a peptide tracer targeting granzyme B, to serve as a PET imaging biomarker in response to CpG-ODN 1585 in situ vaccine therapy delivered intratumourally (IT) or intraperitoneally (IP) either as monotherapy or in combination with αPD1. [18F]AlF-mNOTA-GZP was able to differentiate treatment responders from non-responders based on tumour uptake. Furthermore, [18F]AlF-mNOTA-GZP showed positive associations with changes in tumour-associated lymphocytes expressing GZB, namely GZB+ CD8+ T cells, and decreases in suppressive F4/80+ cells. [18F]AlF-mNOTA-GZP tumour uptake was mediated by GZB expressing CD8+ cells and successfully stratifies therapy responders from non-responders, potentially acting as a non-invasive biomarker for ICIs and combination therapy evaluation in a clinical setting.

PET Imaging Using [18F]olaparib in Mouse Models of Malignant Glioma: Considerations for Molecular Imaging and Radionuclide Therapy Targeting PARP

PurposeRadiopharmaceuticals targeting poly(ADP-ribose) polymerase (PARP) have emerged as promising agents for cancer diagnosis and therapy. PARP enzymes are expressed in both cancerous and normal tissue. Hence, the injected mass, molar activity and potential pharmacological effects are important considerations for the use of radiolabelled PARP inhibitors for diagnostic and radionuclide therapeutic applications. Here, we performed a systematic evaluation by varying the molar activity of [18F]olaparib and the injected mass of [TotalF]olaparib to investigate the effects on tumour and normal tissue uptake in two subcutaneous human glioblastoma xenograft models.Methods[18F]Olaparib uptake was evaluated in the human glioblastoma models: in vitro on U251MG and U87MG cell lines, and in vivo on tumour xenograft-bearing mice, after administration of [TotalF]olaparib (varying injected mass: 0.04-8.0 µg, and molar activity: 1-320 GBq/μmol).ResultsSelective uptake of [18F]olaparib was demonstrated in both models. Tumour uptake was found to be dependent on the injected mass of [TotalF]olaparib (µg), but not the molar activity per sé. An injected mass of 1 μg resulted in the highest tumour uptake (up to 6.9 ± 1.3%ID/g), independent of the molar activity. In comparison, both the lower and higher injected masses of [TotalF]olaparib resulted in lower relative tumour uptake (%ID/g; P<0.05). Ex vivo analysis of U87MG xenograft sections showed that the heterogeneity in [18F]olaparib intratumoural uptake correlated with PARP1 expression. Substantial upregulation of PARP1-3 expression was observed after administration of [TotalF]olaparib (>0.5 µg).ConclusionOur findings show that the injected mass of [TotalF]olaparib has significant effects on tumour uptake. Moderate injected masses of PARP inhibitor-derived radiopharmaceuticals may lead to improved relative tumour uptake and tumour-to-background ratio for cancer diagnosis and radionuclide therapy.

Dose predictions for [177Lu]Lu-DOTA-panitumumab F(ab′)2 in NRG mice with HNSCC patient-derived tumour xenografts based on [64Cu]Cu-DOTA-panitumumab F(ab′)2 – implications for a PET theranostic strategy

Background Epidermal growth factor receptors (EGFR) are overexpressed on many head and neck squamous cell carcinoma (HNSCC). Radioimmunotherapy (RIT) with F(ab')2 of the anti-EGFR monoclonal antibody panitumumab labeled with the β-particle emitter, 177Lu may be a promising treatment for HNSCC. Our aim was to assess the feasibility of a theranostic strategy that combines positron emission tomography (PET) with [64Cu]Cu-DOTA-panitumumab F(ab')2 to image HNSCC and predict the radiation equivalent doses to the tumour and normal organs from RIT with [177Lu]Lu-DOTA-panitumumab F(ab')2. Results Panitumumab F(ab')2 were conjugated to DOTA and complexed to 64Cu or 177Lu in high radiochemical purity (95.6 ± 2.1% and 96.7 ± 3.5%, respectively) and exhibited high affinity EGFR binding (Kd = 2.9 ± 0.7 × 10− 9 mol/L). Biodistribution (BOD) studies at 6, 24 or 48 h post-injection (p.i.) of [64Cu]Cu-DOTA-panitumumab F(ab')2 (5.5–14.0 MBq; 50 μg) or [177Lu]Lu-DOTA-panitumumab F(ab')2 (6.5 MBq; 50 μg) in NRG mice with s.c. HNSCC patient-derived xenografts (PDX) overall showed no significant differences in tumour uptake but modest differences in normal organ uptake were noted at certain time points. Tumours were imaged by microPET/CT with [64Cu]Cu-DOTA-panitumumab F(ab')2 or microSPECT/CT with [177Lu]Lu-DOTA-panitumumab F(ab')2 but not with irrelevant [177Lu]Lu-DOTA-trastuzumab F(ab')2. Tumour uptake at 24 h p.i. of [64Cu]Cu-DOTA-panitumumab F(ab')2 [14.9 ± 1.1% injected dose/gram (%ID/g) and [177Lu]Lu-DOTA-panitumumab F(ab')2 (18.0 ± 0.4%ID/g) were significantly higher (P < 0.05) than [177Lu]Lu-DOTA-trastuzumab F(ab')2 (2.6 ± 0.5%ID/g), demonstrating EGFR-mediated tumour uptake. There were no significant differences in the radiation equivalent doses in the tumour and most normal organs estimated for [177Lu]Lu-DOTA-panitumumab F(ab')2 based on the BOD of [64Cu]Cu-DOTA-panitumumab F(ab')2 compared to those estimated directly from the BOD of [177Lu]Lu-DOTA-panitumumab F(ab')2 except for the liver and whole body which were modestly underestimated by [64Cu]Cu-DOTA-panitumumab F(ab')2. Region-of-interest (ROI) analysis of microPET/CT images provided dose estimates for the tumour and liver that were not significantly different for the two radioimmunoconjugates. Human doses from administration of [177Lu]Lu-DOTA-panitumumab F(ab')2 predicted that a 2 cm diameter HNSCC tumour in a patient would receive 1.1–1.5 mSv/MBq and the whole body dose would be 0.15–0.22 mSv/MBq. Conclusion A PET theranostic strategy combining [64Cu]Cu-DOTA-panitumumab F(ab')2 to image HNSCC tumours and predict the equivalent radiation doses in the tumour and normal organs from RIT with [177Lu]Lu-DOTA-panitumumab F(ab')2 is feasible. RIT with [177Lu]Lu-DOTA-panitumumab F(ab')2 may be a promising approach to treatment of HNSCC due to frequent overexpression of EGFR.

[99mTc]Tc-Galacto-RGD2 integrin αvβ3-targeted imaging as a surrogate for molecular phenotyping in lung cancer: real-world data

Background Epidermal growth factor receptor tyrosine kinase inhibitors (TKIs) are beneficial in patients with lung cancer. We explored the clinical value of [99mTc]Tc-Galacto-RGD2 single-photon emission computed tomography (SPECT/CT) in patients with lung cancer, integrin αvβ3 expression, and neovascularization in lung cancer subtypes was also addressed. Methods A total of 185 patients with lung cancer and 25 patients with benign lung diseases were enrolled in this prospective study from January 2013 to December 2016. All patients underwent [99mTc]Tc-Galacto-RGD2 imaging. The region of interest was drawn around each primary lesion, and tumour uptake of [99mTc]Tc-Galacto-RGD2 was expressed as the tumour/normal tissue ratio(T/N). The diagnostic efficacy was evaluated by receiver operating characteristic curve analysis. Tumour specimens were obtained from 66 patients with malignant diseases and 7 with benign disease. Tumour expression levels of αvβ3, CD31, Ki-67, and CXCR4 were further analysed for the evaluation of biological behaviours. Results The lung cancer patients included 22 cases of small cell lung cancer (SCLC), 48 squamous cell carcinoma (LSC), 97 adenocarcinoma (LAC), and 18 other types of lung cancer. The sensitivity, specificity, and accuracy of [99mTc]Tc-Galacto-RGD2 SPECT/CT using a cut-off value of T/N ratio at 2.5 were 91.89%, 48.0%, and 86.67%, respectively. Integrin αvβ3 expression was higher in non-SCLC compared with SCLC, while LSC showed denser neovascularization and higher integrin αvβ3 expression. Integrin αvβ3 expression levels were significantly higher in advanced (III, IV) than early stages (I, II). However, there was no significant correlation between tumour uptake and αvβ3 expression. Conclusions [99mTc]Tc-Galacto-RGD2 SPECT/CT has high sensitivity but limited specificity for detecting primary lung cancer, integrin expression in the tumour vessel and tumour cell membrane contributes to the tumour uptake.

Development and preclinical evaluation of [68Ga]Ga-Alb-FAPtp-01, a novel tumour-associated fibroblast activation protein tracer for PET imaging of xenograft glioma

Purpose Dynamic changes in tumour-associated fibroblast activation protein (FAP) expression in tumours of different stages may be helpful for prognostic evaluation and treatment response monitoring, making this protein a promising surveillance biomarker for timely diagnosis of malignant tumours and effective planning of patient care. Thus, novel FAP-PET imaging tracers were developed and evaluated for the diagnosis of xenograft glioma tumours. Methods To prospectively verify the prognostic value of the developed FAP tracers, [68Ga]Ga-FAPtp and [68Ga]Ga-Alb-FAPtp-01, measurements of FAP expression and cell uptake and specific binding assays were conducted in U87MG glioma cells. Dynamic/static PET/CT scans were acquired for tumour targeting studies in vivo and in comparison with the reference tracer [68Ga]Ga-FAPI-04 to evaluate diagnostic efficacy. Tumour autoradiography and immunohistochemistry images were acquired to confirm the tracer distribution within the tumour to determine whether it was in accordance with the pathologic data. Results Both [68Ga]Ga-FAPI-04 and [68Ga]Ga-FAPtp demonstrated marked tumour uptake and comparable pharmaceutical profiles in 1 h dynamic PET scans, and [68Ga]Ga-FAPtp had marginally favourable tumour uptake and less kidney and liver uptake. However, both tracers demonstrated rapid clearance from tumours. Thus, the optimized rationally designed FAP-targeting PET tracer [68Ga]Ga-Alb-FAPtp-01, with albumin binding capability, demonstrated prominent longitudinal tumour uptake in tumour xenografts and displayed significant tumour-to-background contrast over time. The tracer uptake values and T/M ratio were 1.775 ± 0.179 SUV and T/M = 5.9, 1.533 ± 0.222 SUV and T/M = 6.7, and 1.425 ± 0.204 SUV and T/M = 9.5, respectively, at 1 h, 2 h and 3 h. Major organs, such as the heart (0.504 ± 0.125% ID/g), muscle (0.156 ± 0.043% ID/g) and brain (0.119 ± 0.039% ID/g), all displayed comparatively low levels of tracer uptake. Conclusion Its improved tumour uptake and pharmacokinetics suggest that the [68Ga]Ga-Alb-FAPtp-01 tracer can noninvasively detect FAP activation in vivo, permitting a precise definition of its roles in tumours of different stages and yielding insights regarding novel FAP-targeted radiotherapeutic strategies at the molecular level.

99mTc-Galacto-RGD2 Integrin αvβ3 Targeted Imaging as a Surrogate for Molecular Phenotyping in Lung Cancer: Prospective Study in the Real World

Purpose: Epidermal growth factor receptor tyrosine kinase inhibitors (TKIs) are beneficial in patients with lung cancer. We explored the clinical value of 99mTc-Galacto-RGD2 single-photon emission computed tomography (SPECT/CT) in patients with lung cancer, Integrin αvβ3 expression, and neovascularization in lung cancer subtypes was also addressed. Methods: A total of 185 patients with lung cancer and 25 patients with benign lung diseases were enrolled in this prostective study from January 2013 to December 2016. All patients underwent 99mTc-Galacto-RGD2 imaging. The region of interest was drawn around each primary lesion, and tumour uptake of 99mTc-Galacto-RGD2 was measured as the tumour/normal tissue ratio（T/N）. The diagnostic efficacy was evaluated by receiver operating characteristic curve analysis. Tumour tissues were obtained from 66 patients with malignant diseases and seven with benign disease. Tumour expression levels of αvβ3, CD31, Ki-67, and CXCR4 were analysed to determine their value for phenotyping and metastasis potential evaluation. Results: The lung cancer patients included 22 cases of small cell lung cancer (SCLC), 48 squamous cell carcinoma (LSC), 97 adenocarcinoma (LAC), and 18 other types of lung cancer. The sensitivity, specificity, and accuracy of 99mTc-Galacto-RGD2 SPECT/CT using a cut-off value of 2.5 were 91.89 %, 48.0 %, and 86.67 %, respectively. Integrin αvβ3 expression was higher in non-SCLC compared with SCLC, while LSC showed denser neovascularization and higher integrin αvβ3 expression. Integrin αvβ3 expression levels were significantly higher in advanced (Ⅲ, Ⅳ) than early stages (Ⅰ, Ⅱ). However, there was no significant correlation between tumour uptake and αvβ3 expression. Conclusion: 99mTc-Galacto-RGD2 SPECT/CT has high sensitivity but limited specificity for detecting primary lung cancer. RGD imaging may help evaluate the biological behaviour and phenotyping, and thus aid management in lung cancer.

Quantification of [18F]afatinib using PET/CT in NSCLC patients, a feasibility study

Introduction: Only a subgroup of non-small cell lung cancer (NSCLC) patients benefit from treatment using epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) such as afatinib. Tumour uptake of [18F]afatinib using positron emission tomography (PET) may identify those patients that respond to afatinib therapy. Therefore, the aim of this study was to find the optimal tracer kinetic model for quantification of [18F]afatinib uptake in NSCLC tumours.Methods: [18F]afatinib PET scans were performed in 10 NSCLC patients. The first patient was scanned for the purpose of dosimetry. Subsequent patients underwent a 20 minutes dynamic [15O]H2O PET-scan (370 MBq) followed by a dynamic [18F]afatinib PET-scan (342 ± 24 MBq) of 60 or 90 minutes. Using the Akaike information criterion (AIC), three pharmacokinetic plasma input models were evaluated with both metabolite corrected plasma sampler input and image-derived (IDIF) input functions in combination with discrete blood samples. Correlation analysis of arterial on-line sampling versus IDIF was performed. In addition, perfusion dependency and simplified measures were assessed.Results: Ten patients were included. The injected dose of [18F]afatinib was 341 ± 37 MBq. 15 tumours could be identified in the field of view of the scanner. Based on AIC, tumour kinetics were best described using an irreversible two-tissue compartment model and a metabolite corrected arterial plasma input function (Akaike 37%). Correlation of plasma-based input functions with metabolite-corrected IDIF was very good (r2=0.93). The preferred simplified uptake parameter was the tumour-to-blood ratio over the 60 to 90 minutes time interval (TBR60-90). Tumour uptake of [18F]afatinib was independent of perfusion.Conclusion: The preferred pharmacokinetic model for quantifying [18F]afatinib uptake in NSCLC tumours was the 2T3K_vb model. TBR60-90 showed excellent correlation with this model and is the best candidate simplified method.

One-Pot Radiosynthesis and Biological Evaluation of a Caspase-3 Selective 5-[123,125I]iodo-1,2,3-triazole derived Isatin SPECT Tracer

Induction of apoptosis is often necessary for successful cancer therapy, and the non-invasive monitoring of apoptosis post-therapy could assist in clinical decision making. Isatins are a class of compounds that target activated caspase-3 during apoptosis. Here we report the synthesis of the 5-iodo-1,2,3-triazole (FITI) analog of the PET tracer [18F]ICMT11 as a candidate tracer for imaging of apoptosis with SPECT, as well as PET. Labelling with radioiodine (123,125I) was achieved in 55 ± 12% radiochemical yield through a chelator-accelerated one-pot cycloaddition reaction mediated by copper(I) catalysis. The caspase-3 binding affinity and selectivity of FITI compares favourably to that of [18F]ICMT11 (Ki = 6.1 ± 0.9 nM and 12.4 ± 4.7 nM, respectively). In biodistribution studies, etoposide-induced cell death in a SW1222 xenograft model resulted in a 2-fold increase in tumour uptake of the tracer. However, the tumour uptake was too low to allow in vivo imaging of apoptosis with SPECT.