Carbonic anhydrase IX (CAIX), which is overexpressed in tumor cells under hypoxic stress, is a promising target for cancer diagnosis and therapy. To enhance tumor uptake and pharmacokinetics, we designed a series of new bivalent CAIX-targeting probes by integrating a hypoxia-sensitive 2-nitroimidazole moiety into the DPI-4452 scaffold. Among these, the new agent [68Ga]Ga-IPM-N001 demonstrated superior higher tumor uptake and significantly improved tumor-to-background ratios (T/K and T/L > 5.0) in the PET/CT imaging studies using OS-RC-2 tumor-bearing mice. This probe also exhibited rapid clearance from the gallbladder, intestines, and kidneys while maintaining strong and prolonged tumor retention, thereby limiting potential systemic toxicity in the normal tissues. Surface plasmon resonance analysis further demonstrated that the precursor IPM-N001 possesses a comparable or improved CAIX-binding affinity relative to DPI-4452. These findings indicate that this nitroimidazole-containing bivalently targeted agent holds promise as a candidate for the theranostics of clear cell renal cell carcinoma.

To develop a novel molecular scaffold designed for dual targeting via site-specific[99mTc] Tc labeling, we report its radiochemical evaluation and preliminary targeting efficacy in acidic tumor models. The pHLIP-RGD scaffold was synthesized by conjugating pHLIP variant 7 (var7) with cyclo(RGDfK) peptide.Pharmacokinetic assessment of [99mTc] Tc-pHLIP-RGD was performed in MDA-MB-231 xenograft-bearing mice through quantitative biodistribution studies and small-animal single photon emission computed tomography (SPECT) imaging. [99mTc] Tc-pHLIP-RGD demonstrated high stability in mouse serum for at least 4h and exhibited strong binding affinity and specificity both in vitro and in vivo. Biodistribution studies revealed rapid tumor accumulation and prolonged retention, with uptake values of 7.01 ± 1.28, 4.23 ± 0.44, 8.04 ± 0.63, and 9.60 ± 1.26%ID/g at 0.5, 1.0, 2.0, and 4.0h post-injection, respectively. Off-target accumulation was primarily observed in the liver. In blocking studies, the administration of non-radioactive pHLIP-RGD partially reduced tumor uptake of [99mTc] Tc-pHLIP-RGD, with tumor distribution values at 0.5, 1.0, 2.0, and 4.0h of 4.66 ± 0.49, 3.25 ± 0.36, 3.04 ± 1.15, and 3.75 ± 0.57%ID/g, respectively. SPECT imaging findings were consistent with biodistribution data, showing clear visualization of tumors at all time points. Tumor visibility was significantly reduced in the blocking study, with a corresponding increase in liver uptake. The heterodimeric radiotracer [99mTc] Tc-pHLIP-RGD exhibited high radiochemical yield, good stability, and favorable tumor uptake and retention characteristics. These proof-of-concept results suggest the potential of the dual-targeting design strategy for developing diagnostic imaging agents for triple-negative breast cancer (TNBC).

We report the design and synthesis of new conjugates (H2L1-4) consisting of a bifunctional bis(thiosemicarbazone) chelator conjugated to prostate-specific membrane antigen pharmacophores. Radiolabeling H2L1-4 with the 99mTc nitrido core in a one-pot synthesis at 95 °C for 10 min produced the complexes [99mTc][TcN(L1-4)] with high radiochemical purity (RCP > 95%) and excellent stability. In vitro evaluation of the [99mTc]Tc PSMA complexes demonstrated high cell-surface receptor binding and internalization in cancer cells expressing PSMA (PSMA+), which was inhibited by an excess of a PSMA-specific inhibitor. The most lipophilic of the complexes, [99mTc][TcN(L2)], also showed significant nonspecific uptake in the control cell line (PSMA-). SPECT-CT imaging of the four complexes, at 1 and 4 h postinjection and biodistribution studies at 5 h postinjection in BALB/c nude mice with both PSMA+ and PSMA- tumors, demonstrated significant differences in the receptor-specific tumor uptake and clearance pathways. [99mTc][TcN(L3)] and the bivalent [99mTc][TcN(L4)] showed predominantly renal clearance and the highest PSMA+ tumor uptake and retention. Conversely, [99mTc][TcN(L1)] and [99mTc][TcN(L2)] displayed gastrointestinal clearance and lower PSMA+ tumor uptake. The work demonstrates that bis(thiosemicarbazones) are effective bifunctional chelators for the development of 99mTc radiopharmaceuticals targeting PSMA with the potential to be easily modified to target other diseases.

[18F]PSMA-1007 is widely used for prostate cancer imaging, but suffers from nonspecific accumulation in healthy tissues. Our previous work demonstrated that linker manipulation with beta3 (β3)-amino acid effectively reduces salivary gland accumulation and enhances tumor uptake of PSMA-617. Here, we redesigned the scaffold of [18F]PSMA-1007 by incorporating β3-amino acid linker and a DOTA chelator, yielding PSMA-HK9. Preclinical evaluations showed that [68Ga]Ga-PSMA-HK9 demonstrated enhanced binding affinity, significantly reduced uptake in kidneys and salivary glands, and increased tumor uptake compared with the 68Ga-labeled analog of [18F]PSMA-1007. A first-in-human study further characterized the in vivo pharmacokinetics of [68Ga]Ga-PSMA-HK9, revealing a trend toward higher tumor uptake compared with [68Ga]Ga-PSMA-617. Therapeutically, [177Lu]Lu-PSMA-HK9 demonstrated efficient cellular internalization and superior tumor inhibition compared with [177Lu]Lu-PSMA-617, with acceptable safety profiles. These findings indicate the linker manipulation with β3-amino acid as an effective and promising strategy for optimizing the pharmacokinetics and pharmacodynamics performance of PSMA-targeted radioligands.

Preclinical studies have demonstrated that [68Ga]Ga/[177Lu]Lu-DOTA-NI-FAPI-04, a bivalent agent containing an extra hypoxia-sensitive 2-nitroimidazole (NI) group, exhibited favorable tumor binding affinity and improved tumor uptake and retention than [68Ga]Ga/[177Lu]Lu-DOTA-FAPI-04. This study aims to further investigate the value of clinical application for [68Ga]Ga-DOTA-NI-FAPI-04 PET/CT via a direct head-to-head comparison with [68Ga]Ga-DOTA-FAPI-04. A total of 50 patients underwent paired [68Ga]Ga-DOTA-NI-FAPI-04 and [68Ga]Ga-DOTA-FAPI-04 PET/CT within 1 week interval. Among these, four patients underwent serial dynamic [68Ga]Ga-DOTA-NI-FAPI-04 PET scans for dosimetry evaluation, and the others underwent scans at 60min and 120min. Additionally, we calculated the SUVmax differences (ΔSUVmax) by [68Ga]Ga-DOTA-NI-FAPI-04 minus [68Ga]Ga-DOTA-FAPI-04 PET/CT for further analysis. Immunohistochemistry for FAP and hypoxia-inducible factor-1 alpha (HIF-1α) was performed in 22 primary tumors. The effective absorbed dose of [68Ga]Ga-DOTA-NI-FAPI-04 PET/CT was calculated as 1.95E-02 mSv/MBq. Tumor uptake of [68Ga]Ga-DOTA-NI-FAPI-04 showed rapid uptake and steady values (average SUVmax 11.6-13.0 from 3 to 120min). [68Ga]Ga-DOTA-NI-FAPI-04 exhibited significantly higher uptake in tumor lesions compared to [68Ga]Ga-DOTA-FAPI-04 PET/CT, particularly in primary tumors (P < 0.05), nodal metastases (P < 0.001), bone metastases (P < 0.001), and liver metastases (P < 0.05). That of FAP expression was correlated with that of HIF-1α (r = 0.661, P < 0.001), and the expression of HIF-1α showed a positive correlation with ΔSUVmax (r = 0.528, P = 0.011). [68Ga]Ga-DOTA-NI-FAPI-04 showed significantly higher tumor uptake and retention over [68Ga]Ga-DOTA-FAPI-04, with particularly enhanced visualization of hypoxic lesions, suggesting that hypoxia-sensitive moiety may play an important role in detection of tumors. Further study of [177Lu]Lu-DOTA-NI-FAPI-04 in humans is warranted to explore its clinical applications. URL OF REGISTRY: https://clinicaltrials.gov/study/NCT06688305 . ClinicalTrials.gov, NCT06688305, Registered 14 November 2024, retrospectively registered.

Preclinical evaluation of 68Ga-labeled acetazolamide derivatives as radiotracers targeting carbonic anhydrase IX in clear cell renal cell carcinoma.

Benzothiazole diimine rhenium(I) '2+1' complexes: Synthesis, structural characterization and anticancer activity.

Comparative study of 47Sc and 177Lu-labeled albumin binder-conjugated FAPI radiopharmaceuticals.

H3RESCA chelator-enabled [18F]AlF labeling: An optimized temperature-resilient platform for PSMA-targeted PET tracers in prostate cancer.

Mesothelin-targeted alpha therapy in PDAC with [225Ac]Ac-Macropa-PEG6-Amatuximab.

68Ga- and 177Lu-labeled theranostic companion tracers have become a mainstay in the clinical management of SST2 overexpressing neuroendocrine tumors. Despite the excellent radionuclide characteristics of [18F]fluorine for PET imaging, 18F-labeled SST2-targeted tracers remain underrepresented. Novel radiohybrid SST2-tracers with DOTA as a bridging unit were designed, allowing radiolabeling with either 18F or 68Ga. Seven DOTA-TATE derivatives (rhTATE1/2: N-SiFAlin-N, N-Me2-Gly-D-Dap/Lys(trans-DOTA-TATE)-OH and (rhTATE2.1-2.5: H-AA1-AA2-AA3-D-Dap(N-SiFAlin-N, N-Me2-Gly)-D-Lys(trans-DOTA-TATE)-OH) with different linkers and hydrophilic modifiers (AA1-AA3) were synthesized and compared to [18F]SiTATE. Competitive binding studies (IC50) were performed using hSST2-CHO cells and [125I]TOC. Internalization was investigated using AR42J cells. Biodistribution and PET/CT studies were performed using AR42J xenograft bearing CD1 nu/nu mice. SST2 specificity was confirmed in a blocking study (+/- co-injection of octreotide). While the first-generation compounds showed good affinity (IC50: [natGa]rhTATE1: 5.6 ± 1.4 nM, [natGa]rhTATE2: 5.7 ± 0.2 nM) but high lipophilicity (LogDpH=7.4 = -1.03 and - 1.19), the inclusion of hydrophilic modifiers ([natGa]rhTATE2.1-[natGa]rhTATE2.5) improved affinity (IC50: 2.6 to 3.7 nM) and hydrophilicity (LogDpH=7.4 = -2.30 to -2.12). The compounds demonstrated efficient internalization (357% to 841% compared to [125I]TOC), and variable human serum albumin affinity (84.8% to 98.8%). [18F][natGa]rhTATE2.2 showed the highest tumor accumulation (27.9 ± 4.8%iD/g), while [18F][natGa]rhTATE2.5 showed lower tumor uptake (18.6 ± 6.2%iD/g), but substantially lower background accumulation, providing improved tumor-to-organ ratios. This study demonstrates a SST2-targeted radiohybrid concept using bifunctionalized DOTA as a bridging unit. N-terminal modifications with hydrophilic tripeptides improved the pharmacokinetic properties. [18F][natGa]rhTATE2.5 (D-Glu-D-Glu-D-Glu) compares particularly well to [18F]SiTATE regarding background clearance and tumor accumulation, with the additional advantage of radiohybrid radiolabeling.

Despite radiotheranostics being at the forefront of Precision Oncology, most radiopharmaceuticals in use today are based on synthetic ligands and/or synthetic peptides, which are often costly and unavailable in many geographies (Hofman et al., 2020; Hope et al., 2019). India’s medicinal plants are among the richest sources of bioactive phytochemicals, which remain untapped to develop low-cost and potent radiotheranostic agents (Sharma et al., 2010; Newman &amp; Cragg, 2020). This review presents the first Indo Reflective Radiotheranostic Phytotechnology (IRR) and lays down the science to transform some of the Indian plants to act as a source of radiopharmaceuticals. The methodology begins with high-throughput computational screening, including ADME profiling and receptor docking, to identify phytochemicals with strong tumour-binding potential (Daina et al., 2017; Morris et al., 2009). Radiolabeling feasibility is assessed using established clinical radionuclides—such as ⁹⁹mTc, ⁶⁸Ga, ¹³¹I, ¹⁷⁷Lu, and ²²⁵Ac—supported by rational chelation chemistry (Price &amp; Orvig, 2014; Velikyan, 2015). In vitro evaluation examines receptor affinity, internalization, radiosensitization, and cytotoxicity (Smith et al., 2012; Kunnumakkara et al., 2017). In vivo biodistribution and small-animal imaging establish tumour uptake, pharmacokinetics, and safety profiles (Workman et al., 2010; Bolch et al., 2015). This includes curcumin, withaferin A, nimbolide, eugenol, bacosides, and guggulsterones— these plants are anticancer, and these are compatible with radiochemicals (Aggarwal &amp; Sung, 2009; Kakar et al., 2020; Subapriya &amp; Nagini, 2005; Baliga et al., 2013; Deepak et al., 2014; Shishodia et al., 2003). Key challenges include differences in plants, radiochemicals not being stable, there being no or little of some important substances and issues with regulations (Ekor, 2014; Memon &amp; Jensen, 2020). These include radiolabeling, using nanoparticles, using AI to make the ligands, using plants- using plants that are GMP grade, and using integrated dosimetry (Yallapu et al., 2012; Kratochwil et al., 2019). Overall, IORP is meant to improve and make it cheaper to improve the bioavailability of some radiopharmaceuticals to make them usable in precision oncology.

Fibroblast activation protein (FAP), which is abundantly expressed in cancer-associated fibroblasts, is an attractive target for tumour imaging and therapy. [68Ga]Ga-FAPI-46, a gallium-68-labelled FAP inhibitor, has demonstrated favourable pharmacokinetics for PET. This study evaluated the radiolabelling efficiency, stability, and biodistribution of [68Ga]Ga-FAPI-46 in a breast cancer mouse model using MCF-7 cell line, which is known to lack FAP expression. [68Ga]Ga-FAPI-46 was radiolabeled directly from FAPI-46 using 68Ga in acetate buffer and analysed for radiochemical purity (RCP) by radio-TLC. Stability at room temperature was assessed up to 5 h post-radiolabeling. Biodistribution studies were conducted in female BALB/c nude mice with MCF-7 tumours. Organ uptake was determined 1 h after injection and expressed as a percentage of injected dose per gramme of tissue (%ID/g). A comparative study between [68Ga]Ga-FAPI-46 and free 68Ga was also performed. [68Ga]Ga-FAPI-46 consistently achieved high RCP values (>95%, maximum 99.1%) and remained stable for up to 5 h without added radioprotectant. At 1 h, [68Ga]Ga-FAPI-46 showed higher uptake in kidneys (4.15%ID/g), heart (3.59%ID/g), and liver (1.70%ID/g), and low uptake in muscle (0.31%ID/g) and bone (0.57%ID/g). Mean tumour uptake was low (0.92%ID/g), consistent with the FAP-negative status of MCF-7 cells. In contrast, free 68Ga showed a non-specific distribution in blood (29.13%ID/g), lungs (18.50%ID/g), and bone (13.37%ID/g). [68Ga]Ga-FAPI-46 showed efficient radiolabelling with high RCP and short-term stability. Its low accumulation in FAP-negative tumours supports tracer selectivity and provides a baseline for comparative studies in FAP-expressing tumour models.

Prostate-specific membrane antigen (PSMA) is a validated target for prostate cancer imaging and therapy. [68Ga]Ga-HTK03149 is a PSMA inhibitor designed to improve biodistribution by incorporating a 2-aminoadipic acid moiety as an alternative PSMA binding group. We report the first-in-human safety, biodistribution, and dosimetry evaluation of [68Ga]Ga-HTK03149 and explore organ uptake relevant to future theranostic use. Methods: Men with biochemical recurrence of prostate cancer (prostate-specific antigen, >0.4 ng/mL) after definitive local therapy were enrolled. Each participant underwent a 60-min dynamic PET/CT scan (heart-focused for the first 6 min and then serial whole-body passes), followed by static whole-body acquisitions at 1 and 2 h after injection. In 5 participants, intravenous furosemide (40 mg) was given after the 1-h scan. Volumes of interest were drawn manually, and organ absorbed doses were calculated using OLINDA/EXM version 2.2. Results: Ten men (median age, 68 y; range, 61-74 y; median prostate-specific antigen, 3.3 ng/mL; range, 0.42-9.6 ng/mL) completed imaging without adverse events. Vital signs and laboratory parameters showed no clinically relevant changes. [68Ga]Ga-HTK03149 cleared rapidly from blood and soft tissue, with prominent uptake in the lacrimal and salivary glands, liver, spleen, kidneys, and small intestine, as well as urinary excretion. The kidneys received the highest absorbed dose (0.107 ± 0.020 mGy/MBq), followed by the salivary glands (0.067 ± 0.019 mGy/MBq) and lacrimal glands (0.063 ± 0.026 mGy/MBq). The mean effective dose was 0.012 ± 0.005 mSv/MBq. Forced diuresis significantly reduced the urinary bladder wall dose (0.029 ± 0.0097 vs. 0.056 ± 0.0268 mGy/MBq, P = 0.04) but did not affect the kidney dose (P = 0.99). Lesions (n = 16 in 9/10 patients) showed high uptake and improved contrast at 2 h, with an SUVmax of 7.13 (range, 5.44-10.99) at 1 h versus 9.52 (range, 7.07-13.67) at 2 h (P < 0.001) and a tumor-to-background ratio of 15.74 (range, 10.85-21.09) at 1 h versus 20.42 (range, 12.50-27.51) at 2 h (P < 0.001). Conclusion: [68Ga]Ga-HTK03149 is a safe PSMA-targeted PET radiopharmaceutical with biodistribution and radiation dosimetry comparable to those of existing tracers. Its high tumor uptake and improved delayed lesion contrast support further clinical evaluation for prostate cancer imaging and theranostic applications.

Radiopharmaceutical therapy with [177Lu]Lu-PSMA-617 has improved outcomes for patients with advanced prostate cancer (PCa), yet modest survival benefits underscore the need for more effective agents. Here, we describe the development of [177Lu]Lu-ART-101, a next-generation prostate-specific membrane antigen (PSMA)-targeting radioligand, and evaluate its biodistribution, dosimetry, therapeutic efficacy, and toxicity in advanced PSMA-expressing PCa models. Methods: Lipophilic ART-101 was synthesized and radiolabeled with 177Lu. [177Lu]Lu-ART-101 longitudinal biodistribution and dosimetry were assessed through SPECT/CT imaging in human PC3-PIP (PC3 cells engineered to overexpress PSMA) xenograft-bearing mice and compared with those of [177Lu]Lu-PSMA-617. The therapeutic efficacy of [177Lu]Lu-ART-101 was evaluated in the PC3-PIP and LNCaP xenograft-bearing mice, whereas toxicity was assessed in normal ICR mice. Results: [177Lu]Lu-ART-101 demonstrated patterns of cell binding and internalization similar to those of [177Lu]Lu-PSMA-617 in PC3-PIP cells. Internalization was effectively blocked by PSMA-617, indicating the PSMA-specific binding of [177Lu]Lu-ART-101. In vitro competition assays revealed potent nanomolar binding, with unlabeled ART-101 competitively displacing [177Lu]Lu-PSMA-617, with 0.4 ± 0.1 nM of inhibitory concentration of 50%, in PC3-PIP cells. In nude mice bearing PC3-PIP xenografts, [177Lu]Lu-ART-101 displayed higher tumor uptake (peak at 14.3 ± 1.5 %IA/g at 48 h after injection) and prolonged retention (8.1 ± 0.8 and 6.4 ± 1.0 %IA/g at 120 and 168 h, respectively) versus [177Lu]Lu-PSMA-617 (peak at 10.0 ± 2.0 %IA/g at 2 h) followed by a rapid decrease (2.2 ± 1.5 %IA/g at 120 h), leading to a 2-fold higher tumor absorbed dose with [177Lu]Lu-ART-101 (1.2 ± 0.1 vs. 0.6 ± 0.1). Notably, [177Lu]Lu-ART-101 showed a distinctive normal tissue distribution compared with [177Lu]Lu-PSMA-617, with longer blood circulation, negligible salivary gland uptake, and primarily hepatic rather than renal clearance. [177Lu]Lu-ART-101's favorable tumor dosimetry translated to enhanced tumor control and prolonged survival in both the PC3-PIP and LNCaP xenograft models. Safety studies in ICR mice revealed mild transient cytopenia in animals receiving the highest tested injected activity (55.5 MBq), which resolved by day 28, without concomitant renal, hepatic, or histologic abnormalities, including those in the bone marrow and salivary glands. Conclusion: [177Lu]Lu-ART-101 demonstrates significant potential as a novel radiopharmaceutical for advanced PCa, with superior therapeutic efficacy and a favorable safety profile. These findings support our ongoing clinical translation effort in advanced PCa.

Previous studies reported low [18F] fluorodeoxyglucose ([18F]FDG) PET uptake in estrogen receptor-positive breast tumours, potentially missing detection of distant metastases. This study assessed the proportion of estrogen receptor-positive hypometabolic tumours, clinical factors influencing [18F]FDG uptake, and the prognostic impact. Baseline [18F]FDG PET/computed tomography (CT) and [18F]FDG PET/MRI exams of female patients diagnosed with estrogen receptor-positive locally advanced (cT3-4N0 or cT1-4N+), metastatic, or recurrent breast cancer between 2013-2022 were retrospectively collected. Different thresholds of maximum standardised uptake value (SUVmax) and tumour-to-background ratio (TBR; SUVmax tumour/SUVmax background) were applied to determine the proportion of hypometabolic [18F]FDG PET exams. Logistic regression and survival analysis were performed. 119 patients underwent [18F]FDG PET/CT and 31 [18F]FDG PET/MRI. The proportion of hypometabolic tumours for SUVmax thresholds 2.0, 2.5, 3.0, TBR of contralateral breast less than or equal to 1, and TBR of liver less than or equal to 1 was 8.4, 15.1, 21.8, 5.1, and 28.6%, respectively for [18F]FDG PET/CT and 16.1, 19.4, 29.0, 6.9, and 35.5% for [18F]FDG PET/MRI. Clinically tumour status (cT-status), histology type, and tumour grade were associated with the presence of a hypometabolic tumour. No PET-derived variables were associated with recurrence-free survival. A considerable proportion of estrogen receptor-positive breast tumours showed low SUVmax, indicating potential suboptimal staging on [18F]FDG PET. In patients with lower cT-status, lobular histology and low-grade estrogen receptor-positive tumour, [18F]FDG PET may be less reliable as staging procedure. Further research is necessary to determine the optimal metabolic threshold for defining a hypometabolic tumour.

Fibroblast activation protein inhibitor (FAPI) PET/CT has emerged as an investigational alternative which targets cancer-associated fibroblasts, abundant in the ILC tumor stroma. This narrative review synthesizes emerging evidence on radiolabeled FAPI PET/CT in ILC and highlights potential advantages, limitations, and research gaps. A purposive literature search of SCOPUS, PubMed/MEDLINE, and Web of Science up to August 2025 identified 10 clinical publications, including three prospective studies, two retrospective comparisons, and five case reports; mixed-cohort studies were included when ILC-specific findings were extractable. Across these small, heterogeneous early studies, gallium-68 FAPI PET/CT consistently demonstrated higher tumor uptake and greater lesion conspicuity than 18F-FDG PET/CT in several ILC-relevant scenarios, particularly infiltrative soft-tissue metastases, peritoneal and serosal involvement, and sclerotic osseous lesions. Comparative studies also reported identification of additional multifocal or multicentric primary foci and greater nodal involvement, while case reports described enhanced visualization of gastric, orbital, peritoneal, and ovarian metastases, sites often inconspicuous on 18F-FDG PET/CT. Despite these promising findings, available evidence remains limited, predominantly observational, and lacks lesion-level histopathologic confirmation, standardized imaging protocols, and data on management impact or patient outcomes. Overall, early results suggest that gallium-68 FAPI PET/CT may offer improved lesion conspicuity in selected ILC settings; however, these observations remain hypothesis generating. Larger, multicentre prospective studies with standardized acquisition parameters, rigorous lesion validation, and clinically meaningful endpoints are required to establish the diagnostic and therapeutic relevance of FAPI PET/CT and determine whether it has a role in the routine clinical evaluation of ILC.

Delivery strategies exploiting red blood cells (RBCs) have been widely pursued, particularly for the purpose of achieving sustained systemic exposure to small molecule drugs. However, limited efforts have been focused on applying RBC-inspired delivery strategies to biologic therapies. We aimed to evaluate RBC binding as a potential strategy to achieve systemic half-life extension or reduced tissue accessibility of antibodies, including those having intravascular targets or safety liabilities within peripheral tissues. The effects of RBC targeting on systemic pharmacokinetic (PK) properties was evaluated by measuring exposures of bispecific antibodies targeting a murine RBC surface target, the TER119 antigen, with or without point mutations that diminish binding to the neonatal Fc receptor (FcRn). Additionally, the effects of RBC targeting on specific (tumor) and nonspecific (normal) peripheral tissue uptake of a bispecific antibody targeting the TER119 antigen and a tumor cell surface tumor target (HER2) was assessed by noninvasive imaging and gamma counting. HER2 was used solely as a surrogate peripheral antigen to model potential safety-relevant tissue engagement rather than as a target expected to benefit from RBC binding. Results from an imaging study in tumor bearing mice revealed that RBC binding reduced peripheral uptake in both tumor and normal tissues but increased uptake in the spleen, which acts as both a reservoir for viable RBCs and a site for hemolysis of senescent RBCs. Furthermore, PK and biodistribution studies in normal mice indicated that RBC binding significantly increased the antibody half-life by ∼14-fold in the absence of FcRn-binding. These efforts lay an initial foundation for developing a next generation of biologic therapies with improved PK and therapeutic windows by taking advantage of the unique properties of RBCs.

The programmed cell death protein-1/programmed cell death ligand-1 (PD-1/PD-L1) axis plays a central role in tumor immune regulation, with PD-L1 expression serving as a critical biomarker for patient stratification and response prediction. Accurate, noninvasive assessment of PD-L1 expression is, therefore, essential for guiding clinical decision-making. KN035 is an ∼79.6 kDa fusion protein comprising a humanized single-domain antibody linked to an Fc fragment, offering a smaller molecular size than conventional monoclonal antibodies. In this study, KN035 was conjugated with p-SCN-Bn-NOTA and radiolabeled with 64Cu to generate [64Cu]Cu-NOTA-KN035 for PET imaging of PD-L1. The tracer showed high radiochemical purity (>95%) and strong binding specificity in vitro. In vivo PET imaging and biodistribution studies were performed in H1975 (high PD-L1 expression) and A549 (low PD-L1 expression) nonsmall cell lung cancer (NSCLC) xenograft models. Clear tumor visualization was achieved at 4 h postinjection (5.62 ± 0.55%ID/g in H1975; 4.16 ± 0.18%ID/g in A549), with peak uptake at 48 h (12.32 ± 0.66 and 5.72 ± 0.21%ID/g, respectively). Tumor uptake decreased significantly after blocking with excess KN035, confirming the specificity. These results demonstrate the high PD-L1-targeting specificity of [64Cu]Cu-NOTA-KN035, suggesting its great potential as a noninvasive diagnostic tool for immunotherapy-based treatments in the future.

CD105 (endoglin) is a proliferation-associated transmembrane glycoprotein selectively expressed on activated endothelial cells in tumor neovasculature and serves as an attractive biomarker for imaging tumor angiogenesis. Here, we report the development of a novel CD105-targeted PET tracer, 68Ga-DOTA-CDP, based on a high-affinity peptide (KD = 13.5 nM) identified from a combinatorial library. The radiotracer was obtained with high radiochemical purity (>97%), excellent stability in phosphate-buffered saline and fetal bovine serum, and favorable hydrophilicity. In vitro confocal imaging and flow cytometry demonstrated specific binding of CDP to CD105-positive HUVECs with minimal uptake in CD105-negative cells. Micro-PET imaging in multiple tumor-bearing mouse models, including 4T1, A549, H1975, MDA-MB-231, and JIMT-1 xenografts, enabled rapid tumor visualization at early time points following injection. Tracer uptake was significantly higher in CD105-high tumors compared with CD105-low tumors, with the highest accumulation observed in the triple-negative breast cancer model MDA-MB-231. Biodistribution studies revealed predominant renal clearance, low hepatic uptake, and favorable tumor-to-background ratios. Blocking experiments with excess unlabeled peptide markedly reduced tumor uptake, confirming receptor-mediated targeting. Immunohistochemical analysis further validated heterogeneous CD105 expression in tumor neovasculature and demonstrated a positive correlation between CD105 expression levels and PET-derived tumor uptake. Overall, 68Ga-DOTA-CDP shows promise as a peptide-based PET tracer for noninvasive tumor angiogenesis imaging.