ImmunoPET imaging of Trop2 in patients with solid tumours.

Accurately predicting and selecting patients who can benefit from targeted or immunotherapy is crucial for precision therapy. Trophoblast cell surface antigen 2 (Trop2) has been extensively investigated as a pan-cancer biomarker expressed in various tumours and plays a crucial role in tumorigenesis through multiple signalling pathways. Our laboratory successfully developed two 68Ga-labelled nanobody tracers that can rapidly and specifically target Trop2. Of the two tracers, [68Ga]Ga-NOTA-T4, demonstrated excellent pharmacokinetics in preclinical mouse models and a beagle dog. Moreover, [68Ga]Ga-NOTA-T4 immuno-positron emission tomography (immunoPET) allowed noninvasive visualisation of Trop2 heterogeneous and differential expression in preclinical solid tumour models and ten patients with solid tumours. [68Ga]Ga-NOTA-T4 immunoPET could facilitate clinical decision-making through patient stratification and response monitoring during Trop2-targeted therapies.

Preclinical development of novel PD-L1 tracers and first-in-human study of [68Ga]Ga-NOTA-RW102 in patients with lung cancers.

BACKGROUND: The programmed cell death protein-1 (PD-1)/programmed death receptor ligand 1 (PD-L1) axis critically facilitates cancer cells' immune evasion. Antibody therapeutics targeting the PD-1/PD-L1 axis have shown remarkable efficacy in various tumors. Immuno-positron emission tomography (ImmunoPET) imaging of PD-L1 expression may help reshape solid tumors' immunotherapy landscape. METHODS: By immunizing an alpaca with recombinant human PD-L1, three clones of the variable domain of the heavy chain of heavy-chain only antibody (VHH) were screened, and RW102 with high binding affinity was selected for further studies. ABDRW102, a VHH derivative, was further engineered by fusing RW102 with the albumin binder ABD035. Based on the two targeting vectors, four PD-L1-specific tracers ([68Ga]Ga-NOTA-RW102, [68Ga]Ga-NOTA-ABDRW102, [64Cu]Cu-NOTA-ABDRW102, and [89Zr]Zr-DFO-ABDRW102) with different circulation times were developed. The diagnostic efficacies were thoroughly evaluated in preclinical solid tumor models, followed by a first-in-human translational investigation of [68Ga]Ga-NOTA-RW102 in patients with non-small cell lung cancer (NSCLC). RESULTS: While RW102 has a high binding affinity to PD-L1 with an excellent KD value of 15.29 pM, ABDRW102 simultaneously binds to human PD-L1 and human serum albumin with an excellent KD value of 3.71 pM and 3.38 pM, respectively. Radiotracers derived from RW102 and ABDRW102 have different in vivo circulation times. In preclinical studies, [68Ga]Ga-NOTA-RW102 immunoPET imaging allowed same-day annotation of differential PD-L1 expression with specificity, while [64Cu]Cu-NOTA-ABDRW102 and [89Zr]Zr-DFO-ABDRW102 enabled longitudinal visualization of PD-L1. More importantly, a pilot clinical trial shows the safety and diagnostic value of [68Ga]Ga-NOTA-RW102 immunoPET imaging in patients with NSCLCs and its potential to predict immune-related adverse effects following PD-L1-targeted immunotherapies. CONCLUSIONS: We developed and validated a series of PD-L1-targeted tracers. Initial preclinical and clinical evidence indicates that immunoPET imaging with [68Ga]Ga-NOTA-RW102 holds promise in visualizing differential PD-L1 expression, selecting patients for PD-L1-targeted immunotherapies, and monitoring immune-related adverse effects in patients receiving PD-L1-targeted treatments. TRIAL REGISTRATION NUMBER: NCT06165874.

ImmunoPET/CT imaging of clear cell renal cell carcinoma with [18F]RCCB6: a first-in-human study.

PURPOSE: The cluster of differentiation (CD70) is a potential biomarker of clear cell renal cell carcinoma (ccRCC). This study aims to develop CD70-targeted immuno-positron emission tomography/computed tomography (immunoPET/CT) imaging tracers and explore the diagnostic value in preclinical studies and the potential value in detecting metastases in ccRCC patients. METHODS: Four novel CD70-specific single-domain antibodies (sdAbs) were produced and labelled with 18F by the aluminium fluoride restrained complexing agent (AlF-RESCA) method to develop radiotracers. The visualisation properties of the tracers were evaluated in a subcutaneous ccRCC patient-derived xenograft (PDX) model. In a registered prospective clinical trial (NCT06148220), six patients with pathologically confirmed RCC were included and underwent immunoPET/CT examination exploiting one of the developed tracers (i.e., [18F]RCCB6). RESULTS: We engineered four sdAbs (His-tagged RCCB3 and RCCB6, His-tag-free RB3 and RB6) specifically targeting recombinant human CD70 without cross-reactivity to murine CD70. ImmunoPET/CT imaging with [18F]RCCB3 and [18F]RCCB6 demonstrated a high tumour-to-background ratio in a subcutaneous ccRCC PDX model, with the latter showing better diagnostic potential supported by higher tumour uptake and lower bone accumulation. In comparison, [18F]RB6, developed by sequence optimisation, has significantly lower kidney accumulation than that of [18F]RCCB6. In a pilot translational study, [18F]RCCB6 immunoPET/CT displayed ccRCC metastases in multiple patients and demonstrated improved imaging contrast and diagnostic value than 18F-FDG PET/CT in a patient with ccRCC. CONCLUSION: The work successfully developed a series of CD70-targeted immunoPET/CT imaging tracers. Of them, [18F]RCCB6 clearly and specifically identified inoculated ccRCCs in preclinical studies. Clinical translation of [18F]RCCB6 suggests potential for identifying recurrence and/or metastasis in ccRCC patients.

ImmunoPET imaging of Trop2 expression in solid tumors with nanobody tracers.

PURPOSE: The high expression of the transmembrane glycoprotein trophoblast cell-surface antigen 2 (Trop2) was strongly associated with the progression of solid tumors, including pancreatic and gastric cancers. Our study aimed to construct Trop2-specific immuno-positron emission tomography (immunoPET) probes and assess the diagnostic abilities in preclinical pancreatic and gastric cancer models. METHODS: The expression of Trop2 in pancreatic cancer was determined by single-cell sequencing and immunohistochemistry on tissue microarray (TMA). Flow cytometry was used to screen the expression of Trop2 in pancreatic cancer cell lines. Two nanobodies (i.e., RTD98 and RTD01) targeting Trop2 were developed and labeled with gallium-68 (68Ga, T1/2 = 1.1 h) to construct immunoPET imaging probes. The agents were researched in cell-derived pancreatic and patient-derived gastric cancer models expressing varying Trop2. RESULTS: Single-cell sequencing results showed high expression of Trop2 in pancreatic ductal cells as well as acinar cells and immunohistochemical staining of TMA from pancreatic cancers showed significantly higher expression of Trop2 in cancerous than in paracancerous tissues. ImmunoPET utilizing [68Ga]Ga-NOTA-RTD98 could clearly delineate subcutaneous tumors, both in cell-derived pancreatic cancer models and patient-derived gastric cancer models, superior to imaging using [18F]-FDG or a non-specific probe [68Ga]Ga-NOTA-RTD161. Another probe with improved pharmacokinetics targeting Trop2, [68Ga]Ga-NOTA-RTD01, was further prepared and showed advantageous diagnostic capabilities in preclinical pancreatic cancer models. CONCLUSION: In the work, we reported two nanobody tracers targeting human Trop2 which may facilitate better use of Trop2-targeted therapeutics by noninvasively displaying expression dynamics of the target.

Site-Specific Modification of Single Domain Antibodies by Enzyme-Immobilized Magnetic Beads.

Nanobodies as imaging agents and drug conjugates have shown great potential for cancer diagnostics and therapeutics. However, site-specific modification of a nanobody with microbial transglutaminase (mTGase) encounters problems in protein separation and purification. Here, we describe a facile yet reliable strategy of immobilizing mTGase onto magnetic beads for site-specific nanobody modification. The mTGase immobilized on magnetic beads (MB-mTGase) exhibits catalytic activity nearly equivalent to that of the free mTGase, with good reusability and universality. Magnetic separation simplifies the protein purification step and reduces the loss of nanobody bioconjugates more effectively than size exclusion chromatography. Using MB-mTGase, we demonstrate site-specific conjugation of nanobodies with fluorescent dyes and polyethylene glycol molecules, enabling targeted immunofluorescence imaging and improved circulation dynamics and tumor accumulation in vivo. The combined advantages of MB-mTGase method, including high conjugation efficiency, quick purification, less protein loss, and recycling use, are promising for site-specific nanobody functionalization and biomedical applications.

Biodistribution and radiation dosimetry of multiple tracers on total-body positron emission tomography/computed tomography.

Background: [18F]F-FDG, [68Ga]Ga-PSMA-11, and [68Ga]Ga-FAPI-04 have achieved good results in multiple clinical trials and clinical practice, but the imaging of these tracers is limited to traditional short-axis positron emission tomography/computed tomography (PET/CT). Therefore, we aimed to use total-body PET/CT dynamic scanning to describe whole-body biodistribution of these three tracers and to calculate more precise radiation doses. Methods: Total-body PET/CT (uExplorer, United Imaging Healthcare) dynamic scanning was performed on 54 patients, including 30 patients with [18F]F-FDG, 10 patients with [68Ga]Ga-PSMA-11, and 14 patients with [68Ga]Ga-FAPI-04. A 60-minute dynamic scanning of whole body was performed simultaneously after bedside bolus injection of the corresponding tracers. The dynamic sequence of 92 frames was quantitatively analyzed by the Pmod4.0 software. Whole body biodistribution was calculated as time-activity curves (TACs) describing dynamic uptake patterns in the subject's major organs, followed by calculation of tracer kinetics and cumulative organ activity. Finally, combined with the OLINDA/EXM software, effective doses of the different tracers and individual organ doses were calculated. Results: In a systematic TAC analysis of three tracers, we identified distinct biodistribution patterns in major organs. [68Ga]Ga-PSMA-11 showed a trend of rapid increasing and slow decreasing in liver, spleen, muscle, and bone. In the heart, stomach, brain, and lung, tracer decreased rapidly after rapid increasing. Similarly, tracer uptake in the kidney and urinary bladder increased gradually. [68Ga]Ga-FAPI-04 showed a rapid increasing and rapid decreasing trend in brain, lung, liver, spleen, bone, heart, kidney, and stomach. The mean effective dose of [68Ga]Ga-PSMA-11 was 1.47E-02 mSv/MBq, and the mean effective doses of [18F]F-FDG and [68Ga]Ga-FAPI-04 were comparable (2.52E-02 mSv/MBq and 2.23E-02 mSv/MBq). The mean effective dose of [18F]F-FDG was lower than that reported in the literature measured by previous short-axis PET, while both [68Ga]Ga-PSMA-11 and [68Ga]Ga-FAPI-04 had higher value than previously reported value. Conclusions: [18F]F-FDG, [68Ga]Ga-PSMA-11 and [68Ga]Ga-FAPI-04 have good biodistribution in human organs. Real-time high-sensitivity dynamic scanning with total-body PET/CT is a very effective way to accurately calculate biodistribution and effective dose of positron-labeled radiopharmaceuticals.

Improved [68Ga]Ga-PSMA-11 image qualities reconstructed by total variation regularized expectation maximization on total-body PET/CT.

Background: Total variation regularized expectation maximization (TVREM) reconstruction algorithm on the image quality of gallium (68GA) prostate-specific membrane antigen-11 ([68Ga]Ga-PSMA-11) total-body positron emission tomography/computed tomography (PET/CT). Methods: Images of a phantom with small hot sphere inserts and the total-body PET/CT scans of 51 prostate cancer patients undergoing [68Ga]Ga-PSMA-11 were reconstructed using TVREM with 5 different penalization factors between 0.09 and 0.45 and for 20-, 40-, 60-, 120-, and 300-second acquisition, respectively. As a comparison, the same data were also reconstructed using the ordered subset expectation maximization (OSEM) with 3 iterations, 20 subsets, and 300 second acquisition. The contrast recovery coefficients (CRC) and background variability (BV) of the phantom, the tumor-to-background ratios (TBR), the contrast recovery (CR) ratio, the image noise of the liver, and maximum standard uptake value (SUVmax) of the lesions were calculated to evaluate the image quality. The clinical performance of the images was evaluated by 2 radiologists with a 5-point scale (1-poor, 5-excellent). Results: The TVREM reconstructions groups fwith 120 second acquisition and the penalization of 0.27 to 0.45 showed the best performance in terms of CR, TBR, image noise, and the gain of SUVmax compared to that obtained in the OSEM 300 second group. Even the image noise of the TVREM 120 second group with a penalization factor of 0.27 and 0.36 was comparable to the OSEM 300 second group; the lesions' SUVmax increased by 28% whereas the image noise decreased by 5% and 14%, respectively. The TVREM 120 second group with a penalization factor of 0.36 (5.00±0.00) had the highest qualitative score that equaled OSEM and TVREM for the 300 second (P>0.05) group. Conclusions: Our study has shown the potential of the TVREM reconstruction algorithm with optimized penalization factors to achieve comparable [68Ga]Ga-PSMA-11 total-body PET/CT image quality with a shorter acquisition time, compared with the conventional OSEM reconstruction algorithm.

ImmunoPET Imaging of CD47 with VHH-Derived Tracers in Pancreatic Cancers.

Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor with insidious onset, rapid progression, and a very poor prognosis. CD47 is a transmembrane protein associated with the development and poor prognosis of pancreatic cancer. The aim of this study was to evaluate the diagnostic value of novel immunoPET tracers targeting CD47 in preclinical pancreatic cancer models. The association of CD47 expression with pancreatic cancer was analyzed using the Gene Expression Profiling Interactive Analysis platform. Immunohistochemical analysis of tissue microarrays was performed to detect CD47 expression in PDAC. CD47 expression levels on BxPC-3 and AsPC-1 cell membranes were compared using flow cytometry. A VHH (C2)-targeting human CD47 and its albumin-binding derivative (ABDC2) were labeled with 68Ga or 89Zr, respectively. The developed tracers were evaluated by immuno-positron emission tomography (immunoPET) imaging in tumor-bearing nude and CD47-humanized mice. [68Ga]Ga-NOTA-C2 effectively detected tumor lesions in nude mice models and further showed confirmative imaging capacity in CD47-humanized PDAC models. Compared with [68Ga]Ga-NOTA-C2, [89Zr]Zr-DFO-ABDC2 had a significantly prolonged circulation time, increased tumor uptake, and reduced accumulation in the kidneys. Finally, biodistribution and histological staining confirmed the results of the immunoPET imaging studies. In this study, we validated that two novel VHH-derived molecular imaging tracers for immunoPET imaging ([68Ga]Ga-NOTA-C2 and [89Zr]Zr-DFO-ABDC2) can effectively annotate CD47 expression and diagnose PDAC in a target-specific manner. Clinical application of the imaging strategies may help select patients for CD47-targeted therapies and assess the response thereafter.

Claudin18.2-targeted cancer theranostics.

Claudin 18.2 (CLDN18.2) is an emerging target for the treatment of CLDN18.2-expressing cancers such as gastric and pancreatic cancers. Cell and antibody therapies targeting CLDN18.2 are under intensive clinical trials. In this setting, how to efficiently and specifically detect CLDN18.2 expression before and after the therapies is a clinical challenge. In recent years, molecular imaging with radiolabeled antibodies or antibody fragments have shown promise in noninvasively annotating antigen expression across the body. In this Perspective, we will bring together the most recent progress on CLDN18.2-targeted imaging and therapy of solid tumors.

Development and Characterization of Nanobody-Derived CD47 Theranostic Pairs in Solid Tumors.

Overexpression of CD47 is frequently observed in various types of human malignancies, inhibiting myeloid-mediated elimination of tumor cells and affecting the prognosis of cancer patients. By mapping biomarker expression, immuno-positron emission tomography has been increasingly used for patient screening and response monitoring. By immunization alpacas with recombinant human CD47, we prepared a CD47-targeting nanobody C2 and developed [68Ga]Ga-NOTA-C2, followed by an exploration of the diagnostic value in CD47-expressing tumor models including gastric-cancer patient-derived xenograft models. By fusing C2 to an albumin binding domain (ABD), we synthesized ABDC2, which had increased in vivo half-life and improved targeting properties. We further labeled ABDC2 with 68Ga/89Zr/177Lu to develop radionuclide theranostic pairs and evaluated the pharmacokinetics and theranostic efficacies of the agents in cell- and patient-derived models. Both C2 and ABDC2 specifically reacted with human CD47 with a high K D value of 23.50 and 84.57 pM, respectively. [68Ga]Ga-NOTA-C2 was developed with high radiochemical purity (99 >%, n = 4) and visualized CD47 expression in the tumors. In comparison to the rapid renal clearance and short half-life of [68Ga]Ga-NOTA-C2, both [68Ga]Ga-NOTA-ABDC2 and [89Zr]Zr-DFO-ABDC2 showed prolonged circulation and increased tumor uptake, with the highest uptake of [89Zr]Zr-DFO-ABDC2 occurring at 72 h post-injection. Moreover, [177Lu]Lu-DOTA-ABDC2 radioimmunotherapy suppressed the tumor growth but was associated with toxicity, warranting further optimization of the treatment schedules. Taken together, we reported a series of nanobody-derived CD47-targeted agents, of which [68Ga]Ga-NOTA-C2 and [89Zr]Zr-DFO-ABDC2 are readily translatable. Optimization and translation of CD47-targeted theranostic pair may provide new prospects for CD47-targeted management of solid tumors.

Development and Characterization of Novel FAP-Targeted Theranostic Pairs: A Bench-to-Bedside Study.

Fibroblast activation protein (FAP) is among the most popular targets in nuclear medicine imaging and cancer theranostics. Several small-molecule moieties (FAPI-04, FAPI-46, etc.) are used for developing FAP-targeted theranostic agents. Nonetheless, the circulation time of FAP inhibitors is relatively short, resulting in rapid clearance via kidneys, low tumor uptake, and associated unsatisfactory treatment efficacy. To address the existing drawbacks, we engineered 3 peptides named FD1, FD2, and FD3 with different circulation times through solid-phase peptide synthesis. All the 3 reported peptides bind to human and murine FAP with single-digit nanomolar affinity measured by surface plasmon resonance. The diagnostic and therapeutic potential of the agents labeled with 68Ga and 177Lu was assessed in several tumor models exhibiting different levels of FAP expression. While radiolabeled FD1 was rapidly excreted from kidneys, radiolabeled FD2/FD3 have significantly prolonged circulation, increased tumor uptake, and decreased kidney accumulation. Our findings indicated that [68Ga]Ga-DOTA-FD1 positron emission tomography (PET) effectively detected FAP dynamics, whereas [177Lu]Lu-DOTA-FD2 and [177Lu]Lu-DOTA-FD3 exhibited remarkable therapeutic efficacy in FAP-overexpressing tumor models, including pancreatic cancer cell models characterized by abundant stroma. Moreover, a pilot translational investigation demonstrated that [68Ga]Ga-DOTA-FD1 had the capability to identify both primary and metastatic tumors with precision and distinction. In summary, we developed [68Ga]Ga-DOTA-FD1 for same-day PET imaging of FAP dynamics and [177Lu]Lu-DOTA-FD2 and [177Lu]Lu-DOTA-FD3 for effective radioligand therapy of FAP-overexpressing tumors.

Development and comparison of 68Ga/18F/64Cu-labeled nanobody tracers probing Claudin18.2.

Claudin 18.2 (CLDN18.2) is an emerging target for the treatment of gastric cancers. We aim to develop tracers to image the expression of CLDN18.2. A humanized nanobody targeting CLDN18.2 (clone hu19V3) was produced and labeled with 68Ga, 64Cu, and 18F. The tracers were investigated in subcutaneous and metastatic models established using two different mouse types (nude and Balb/c mice) and two different cell lines (CHO-CLDN18.2 and CT26-CLDN18.2). Gastric cancer patient-derived xenograft (PDX) models were further established for validation experiments. Three novel CLDN18.2-targeted tracers (i.e., [68Ga]Ga-NOTA-hu19V3, [64Cu]Cu-NOTA-hu19V3, and [18F]F-hu19V3) were developed with good radiochemical yields and excellent radiochemical purities. [68Ga]Ga-NOTA-hu19V3 immuno-positron emission tomography (immunoPET) rapidly delineated subcutaneous CHO-CLDN18.2 lesions and CT26-CLDN18.2 tumors, as well as showing excellent diagnostic value in PDX models naturally expressing CLDN18.2. While [68Ga]Ga-NOTA-hu19V3 had high kidney accumulation, [64Cu]Cu-NOTA-hu19V3 showed reduced kidney accumulation and improved image contrast at late time points. Moreover, [18F]F-hu19V3 was developed via click chemistry reaction under mild conditions and precisely disseminated CHO-CLDN18.2 lesions in the lungs. Furthermore, region of interest analysis, biodistribution study, and histopathological staining results correlated well with the in vivo imaging results. Taken together, immunoPET imaging with the three tracers can reliably visualize CLDN18.2 expression.

Rare pulmonary metastases of atypical meningioma diagnosed on total-body 18F-FDG PET/CT.

Here we reported a 59-year-old male who had undergone brain surgery three times and the pathological results showed atypical meningioma (2015, WHO grade I; 2018, WHO grade II; 2019, WHO grade II-III), with multiple pulmonary nodules, which arose during follow-up. A total-body 18F-FDG PET/CT showed multiple solid nodules with increased 18F-FDG metabolism (SUVmax = 8.6). The patient underwent a CT-guided lung biopsy and the histopathological study showed positive staining of epithelial membrane antigen (EMA), vimentin (VIM), SSTR2, Ki67 (20%), and negative staining of CK, TTF-1, CD34, SY, PR, P40, respectively. Based on the history and immunohistology results, multiple pulmonary metastases from atypical meningioma were finally diagnosed, since double positive staining of EMA and VIM supported the diagnosis of meningioma and negative staining excluded primary lung cancers. The patient has given up any treatment because of personal reasons. Pulmonary metastasis from meningioma is rare, accurate diagnosis should be based on medical history, imaging characteristics, and histopathological findings.

Single-Domain Antibody Theranostics on the Horizon.

Single-domain antibody (sdAb) is among the most promising vectors for developing molecular imaging tracers. Several sdAb tracers targeting human epidermal growth factor receptor 2 or programmed death ligand 1 have entered clinical practice. However, radiolabeled single-valent sdAbs generally have high kidney retention, limiting their therapeutic applications. Therefore, engineering strategies such as PEGylation or incorporation of renal cleavable linkers can be adapted to improve pharmacokinetics and reduce kidney retention. In this Focus on Molecular Imaging review, we try to summarize the latest developments in sdAb-derived agents and propose potential strategies that can be used to improve the theranostic value of radiolabeled sdAbs.