In recent years, quinoline-based fibroblast activation protein (FAP) inhibitors (FAPI) have shown promising results in the diagnosis of cancer and several other diseases, making them the hotspot of much productive research. This review summarizes the literature for the state-of-the-art FAPI-PET imaging for cancer diagnosis compared with fluorodeoxyglucose (FDG)-PET. We also summarize the use of FAPI-PET for therapeutic regimen improvement and fibroblast activation protein (FAP)-targeted molecule modification strategies, as well as preliminary clinical studies regarding FAP-targeted radionuclide therapy. Our qualitative summary of the literature to date can inform future research directions, medical guidelines, and optimal clinical decision-making.

Fibroblast activation protein-based theranostics in cancer research: A state-of-the-art review

Fibroblast activation protein (FAP) has become an attractive target for diagnosis and therapy, and a series of FAP inhibitor (FAPI) based radiotracers have been developed and performed excellent diagnosis outcome in clinical applications. Yet, their fast clearance and insufficient tumor retention have hampered their further clinical applications for cancer treatment. In this study, we developed two albumin binder-conjugated FAPI radiotracers, TEFAPI-06 and TEFAPI-07. They are derived from FAPI-04, and optimized by conjugating two types of well-studied albumin binders, 4-(p-iodophenyl) butyric acid moiety (TEFAPI-06) and truncated Evans blue moiety (TEFAPI-07), to try to overcome the above limitations at the expense of prolonging the blood circulation. Methods: TEFAPI-06 and TEFAPI-07 were synthesized and labeled with 68Ga, 86Y and 177Lu successfully. A series of cell assays were performed to identify the binding affinity and FAP specificity in vitro. PET imaging, SPECT imaging and biodistribution study were performed to evaluate the pharmacokinetics in the pancreatic cancer patient-derived xenografts (PDX) animal models. The cancer treatment efficacy of 177Lu-TEFAPI-06 and 177Lu-TEFAPI-07 have been evaluated and the comparative study with 177Lu-FAPI-04 has also been performed in pancreatic cancer PDX-bearing mice. Results: The binding affinity (Kd) to FAP of 68Ga-TEFAPI-06 and 68Ga-TEFAPI-07 is 10.16 ± 2.56 nM and 7.81 ± 2.28 nM, respectively, which were comparable with that of 68Ga-FAPI-04. Comparative PET imaging of HT-1080-FAP and HT-1080 tumor-bearing mice and blocking study showed the FAP targeting ability in vivo of these two tracers. Compared with 177Lu-FAPI-04, PET imaging, SPECT imaging and biodistribution studies of TEFAPI-06 and TEFAPI-07 have demonstrated their remarkably enhanced tumor accumulation and retention, respectively. Notable tumor growth inhibitions of 177Lu-TEFAPI-06 and 177Lu-TEFAPI-07 have been observed, while the control groups and the group treated by 177Lu-FAPI-04. Conclusion: Two albumin binder-conjugated FAPI radiopharmaceuticals have been developed and evaluated in vitro and in vivo. Notably improved tumor uptake and retention have been observed compared to the original FAPI tracer. Both 177Lu-TEFAPI-06 and 177Lu-TEFAPI-07 showed remarkable growth inhibition to PDX tumors while the side effect is negligible, showing that they are promising for further clinical translational studies.

https://jnm.snmjournals.org/content/jnumed/early/2021/09/30/jnumed.121.262533.full.pdf

Albumin Binder-Conjugated Fibroblast Activation Protein Inhibitor Radiopharmaceuticals for Cancer Therapy.

Rationale: Fibroblast activation protein (FAP) targeted molecular imaging radiotracers have shown promising preclinical and clinical results in tumor diagnosis. However, rapid clearance and inadequate tumor retention of these molecules have hindered them for further clinical translation in cancer therapy. In this study, we aimed to develop a series of albumin binder-truncated Evans blue (EB) modified FAP targeted radiotracers, and optimize the pharmacokinetic (PK) characteristics to overcome the existing limitations in order to apply in the radionuclide therapy of cancer. Methods: A series of compounds with the general structure of EB-FAPI-Bn were synthesized based on a FAP inhibitor (FAPI) variant (FAPI-02) and radiolabeled with 177LuCl3. To verify the binding affinity and FAP targeting specificity of these tracers in vitro, U87MG cell uptake and competition assays were performed. Preclinical PK was evaluated in U87MG tumor-bearing mice using SPECT imaging and biodistribution studies. The lead compound EB-FAPI-B1 was selected and cancer therapeutic efficacy of 177Lu-EB-FAPI-B1 was assessed in U87MG tumor-bearing mice. Results: 177Lu-EB-FAPI-B1, B2, B3, B4 were stable in PBS (pH 7.4) and saline for at least 24 h. EB-FAPI-B1 showed high binding affinity (IC50 = 16.5 nM) to FAP in vitro, which was comparable with that of FAPI-02 (IC50 = 10.9 nM). SPECT imaging and biodistribution studies of 177Lu-EB-FAPI-B1, B2, B3, B4 have proved their prominently improved tumor accumulation and retention at 96 h post-injection, especially for 177Lu-EB-FAPI-B1, high tumor uptake and low background signal make it the optimal compound. Compared to the saline group, noteworthy tumor growth inhibitions of 177Lu-EB-FAPI-B1 have been observed after administration of different dosages. Conclusion: In this study, several EB modified FAPI-02 related radiopharmaceuticals have been synthesized successfully and evaluated. High binding affinity and FAP targeting specificity were identified in vitro and in vivo. Remarkably enhanced tumor uptake and retention of EB-FAPI-B1 were found over the unmodified FAPI-02. 177Lu-EB-FAPI-B1 showed remarkable tumor growth suppression in U87MG tumor model with negligible side effects, indicating that 177Lu-EB-FAPI-B1 is promising for clinical application and transformation.

Evans blue-modified radiolabeled fibroblast activation protein inhibitor as long-acting cancer therapeutics

Tumor-induced osteomalacia in the head and neck region remains a challenging diagnosis to manage. Literature pertaining to management and outcome details remains sparse. We describe two cohorts: cohort 1 included seven patients from a single center in Western India with tumors located in paranasal sinuses (n = 3), intracranial (n = 2) and maxilla (n = 2). The unique features from our series is the management of persistent disease with radiation therapy (n = 2) and peptide receptor radionuclide therapy (PRRT) (n = 1). Cohort two has 163 patients identified from 109 publications for systematic review. Paranasal sinuses, mandible, intracranial disease, maxilla and oral cavity, in descending order, are reportedly common tumor sites. Within this cohort, mean age was 46 ± 14 years at presentation with 44.1% having local symptoms. Duration of symptoms varied from 1 to 240 months. Pre-surgery mean serum phosphorus was 1.4 ± 0.4 mg/dL and median FGF-23 levels were 3.6 (IQR:1.8-6.8) times of normal upper limit of normal. Majority (97.5%) were managed primarily with surgical excision; however, primary radiotherapy (n = 2) and surgery combined with radiotherapy (n = 2) were also reported. Twenty patients had persistent disease while nine patients had recurrence, more commonly noted with intracranial and oral cavity tumors. Surgery was the most common second mode of treatment employed succeeded by radiotherapy. Four patients had metastatic disease. The most common histopathological diagnosis reported is PMT mixed connective tissue, while the newer terminology 'PMT mixed epithelial and connective tissue type' has been described in 15 patients.

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Tumor induced osteomalacia in head and neck region: single center experience and systematic review

Radiopharmaceuticals that target cancer-associated fibroblasts (CAFs) have become an increasingly attractive strategy for cancer theranostics. Recently, a series of fibroblast activation protein inhibitor (FAPI)-based radiopharmaceuticals have been successfully applied to the diagnosis of a variety of cancers and exhibited excellent tumor selectivity. Nevertheless, CAF-targeted radionuclide therapy encounters difficulties in cancer treatment, as the tumor uptake and retention of FAPIs are insufficient. To meet this challenge, we tried to conjugate albumin-binding moiety to FAPI molecule for prolonged circulation that may increase the accumulation and retention of radiopharmaceuticals in tumor. Two fatty acids, lauric acid (C12) and palmitic acid (C16), were conjugated to FAPI-04 to give two albumin-binding FAPI radiopharmaceuticals, denoted as FAPI-C12 and FAPI-C16, respectively. They had been radiolabeled with gallium-68, yttrium-86, and lutecium-177 for stability study, binding affinity assay, PET and SPECT imaging, biodistribution, and radionuclide therapy study to systematically evaluate their potential for CAF-targeted radionuclide therapy. FAPI-C12 and FAPI-C16 showed high binding affinity to FAP with the IC50 of 6.80 ± 0.58 nM and 5.06 ± 0.69 nM, respectively. They were stable in both saline and plasma. The tumor uptake of [68Ga]Ga-FAPI-04 decreased by 56.9% until 30 h after treated with FAPI-C16 before, and the uptakes of [86Y]Y-FAPI-C12 and [86Y]Y-FAPI-C16 in HT-1080-FAP tumor were both much higher than that of HT-1080-Vehicle tumor which identified the high FAP specific of these two radiopharmaceuticals. Both FAPI-C12 and FAPI-C16 showed notably longer circulation and significantly enhanced tumor uptake than those of FAPI-04. [177Lu]Lu-FAPI-C16 had the higher tumor uptake at both 24 h (11.22 ± 1.18%IA/g) and 72 h (6.50 ± 1.19%IA/g) than that of [177Lu]Lu-FAPI-C12 (24 h, 7.54 ± 0.97%IA/g; 72 h, 2.62 ± 0.65%IA/g); both of them were much higher than [177Lu]Lu-FAPI-04 with the value of 1.24 ± 0.54%IA/g at 24 h after injection. Significant tumor volume inhibition of [177Lu]Lu-FAPI-C16 at the high activity of 29.6 MBq was observed, and the median survival was 28 days which was much longer than that of the [177Lu]Lu-FAPI-04 treated group of which the median survival was only 10 days. This proof-of-concept study validates the hypothesis that conjugation of albumin binders may shift the pharmacokinetics and enhance the tumor uptake of FAPI-based radiopharmaceuticals. This could be a general strategy to transform the diagnostic FAP-targeted radiopharmaceuticals into their therapeutic pairs.

Fatty acid-conjugated radiopharmaceuticals for fibroblast activation protein-targeted radiotherapy

Rationale: The low response rate of immunotherapy, such as anti-PD-L1/PD-1 and anti-CTLA4, has limited its application to a wider population of cancer patients. One widely accepted view is that inflammation within the tumor microenvironment is low or ineffective for inducing the sufficient infiltration and/or activation of lymphocytes. Here, a highly tumor-selective anti-PD-L1 (αPD-L1) antibody was developed through PET imaging screening, and it was radiolabeled with Lu-177 for PD-L1-targeted radioimmunotherapy (RIT) and radiation-synergized immunotherapy. Methods: A series of αPD-L1 antibodies were radiolabeled with zirconium-89 for PET imaging to screen the most suitable antibodies for RIT. Mice were divided into an immunotherapy group, a RIT group and a radiation-synergized immunotherapy group to evaluate the therapeutic effect. Alterations in the tumor microenvironment after treatment were assessed using flow cytometry and immunofluorescence microscopy. Results: Radiation-synergistic RIT can achieve a significantly better therapeutic effect than immunotherapy or RIT alone. The dosages of the radiopharmaceuticals and αPD-L1 antibodies were reduced, the infiltration of CD4+ and CD8+ T cells in the tumor microenvironment was increased, and no side effects were observed. This radiation-synergistic RIT strategy successfully showed a strong synergistic effect with αPD-L1 checkpoint blockade therapy, at least in the mouse model. Conclusions: PET imaging of 89Zr-labeled antibodies is an effective method for antibody screening. RIT with a 177Lu-labeled αPD-L1 antibody could successfully upregulate antitumor immunity in the tumor microenvironment and turn "cold" tumors "hot" for immunotherapy.

PET imaging facilitates antibody screening for synergistic radioimmunotherapy with a 177Lu-labeled αPD-L1 antibody.

Objective Ga-DOTATATE PET/CT is currently the most common imaging modality in localizing culprit tumors, which can result in tumor-induced osteomalacia (TIO). Fracture, which is one of the most common consequences of the TIO, can also lead to increased Ga-DOTATATE activity and potentially affect the accuracy of Ga-DOTATATE PET/CT imaging. The aim of this investigation is to evaluate whether the increased Ga-DOTATATE activity at the sites of the fracture will cause interpretation difficulty in the localizing the culprit tumor causing TIO. Method The images of Ga-DOTATATE PET/CT scan from a total of 54 patients who had multiple foci of increased Ga-DOTATATE PET/CT on PET/CT were retrospectively analyzed. Not only was the intensity of the activity on PET but also the appearance of the activity on CT taken into consideration when the interpretation of the images occurred. The results from imaging analysis were compared with the clinical chart record. All patients had tentative clinical diagnosis of TIO. Results The causative tumors in 53 patients were eventually identified. In 1 patient, the causative tumor was not identified. Among the 53 patients with confirmed TIO, 52 tumors were accurately localized. Conclusions Mild activity at the sites of fracture is not a major challenging factor in the interpretation of Ga-DOTATATE PET/CT in the evaluation of TIO when both intensity on PET and morphology on CT were assessed.

Increased Activity Due to Fractures Does Not Significantly Affect the Accuracy of 68Ga-DOTATATE PET/CT in the Detection of Culprit Tumor in the Evaluation of Tumor-Induced Osteomalacia.

Phosphaturic mesenchymal tumors are benign tumors causing majority of tumor-induced osteomalacia These tumors are most commonly located in the lower extremities followed by craniofacial regions However, intracranial lesion is very rare Here we describe Ga-DOTATATE PET/CT findings of a recurrent intracranial phosphaturic mesenchymal tumor in a 68 year old woman

Jie Ding

Papers

Albumin Binder-Conjugated Fibroblast Activation Protein Inhibitor Radiopharmaceuticals for Cancer Therapy.

Fatty acid-conjugated radiopharmaceuticals for fibroblast activation protein-targeted radiotherapy

PET imaging facilitates antibody screening for synergistic radioimmunotherapy with a 177Lu-labeled αPD-L1 antibody.

Increased Activity Due to Fractures Does Not Significantly Affect the Accuracy of 68Ga-DOTATATE PET/CT in the Detection of Culprit Tumor in the Evaluation of Tumor-Induced Osteomalacia.

Recurrent/Residual Intracranial Phosphaturic Mesenchymal Tumor Revealed on 68Ga-DOTATATE PET/CT