ArticleMarch 23, 2026

Peptide-Based ROR1-Targeting PET Ligands for Melanoma Tumor Imaging: Design and Preclinical Evaluation
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Donglan Huang
Donglan Huang
Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, P.R. China
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Xingru Long
Xingru Long
Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430022, P.R. China
Hubei Key Laboratory of Molecular Imaging, Wuhan, Hubei 430022, P.R. China
Key Laboratory of Biological Targeted Therapy, The Ministry of Education, Wuhan, Hubei 430022, P.R. China
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Li Zhong
Li Zhong
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, P.R. China
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Yajing Wang
Yajing Wang
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, P.R. China
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Xuan Di
Xuan Di
Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, P.R. China
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Zihan Wang
Zihan Wang
Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, P.R. China
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Shuhan Zhou
Shuhan Zhou
Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, P.R. China
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Xiaoyu Du
Xiaoyu Du
Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, P.R. China
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Yang Zhang*
Yang Zhang
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, P.R. China
*Email: [email protected]
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Hai Qian*
Hai Qian
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, P.R. China
Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical University, Nanjing 210009, P.R. China
*Email: [email protected], [email protected]; Phone: +86-25-86185286; Fax: +86-25-83271050.
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https://orcid.org/0000-0002-3827-0992
Dawei Jiang*
Dawei Jiang
Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430022, P.R. China
Hubei Key Laboratory of Molecular Imaging, Wuhan, Hubei 430022, P.R. China
Key Laboratory of Biological Targeted Therapy, The Ministry of Education, Wuhan, Hubei 430022, P.R. China
*Email: [email protected]
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https://orcid.org/0000-0002-4072-0075
Hualong Fu*
Hualong Fu
Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, P.R. China
*Email: [email protected]
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https://orcid.org/0000-0002-7197-8087

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Journal of Medicinal Chemistry

Cite this: J. Med. Chem. 2026, 69, 7, 8628–8639

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https://doi.org/10.1021/acs.jmedchem.6c00570

Published March 23, 2026

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Abstract

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Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is overexpressed in multiple cancers while remaining largely absent in adult tissues, which makes it an attractive target for both tumor diagnosis and therapy. To enable noninvasive imaging of ROR1, four peptide-based PET ligands [⁶⁸Ga]1–4 were rationally designed and evaluated for melanoma imaging. In vitro assays confirmed reasonable ROR1 binding affinity (K_D = 481.0 and 44.9 nM, respectively) and specific cellular uptake of [⁶⁸Ga]2 and [⁶⁸Ga]3, which are functionalized with serum albumin-binding groups. Notably, microPET/CT imaging and biodistribution studies in B16F10, A375, and SK-MEL-28 tumor-bearing mice demonstrated that [⁶⁸Ga]2 achieved the most favorable imaging performance, characterized by high tumor accumulation (up to 9.18% ID/g), sustained retention, and a relatively lower nonspecific background signal. These findings highlight [⁶⁸Ga]2 as a promising candidate for ROR1-targeting PET imaging and underscore the potential of peptide-based ROR1 PET probes for tumor imaging and therapy guidance.

This publication is licensed for personal use by The American Chemical Society.

Subjects

Introduction

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Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a transmembrane receptor protein that belongs to the receptor tyrosine kinase (RTK) superfamily, specifically the ROR subfamily alongside ROR2. (1,2) Initially classified as an orphan receptor due to its unidentified ligand, ROR1 is now recognized as a receptor for noncanonical Wnt signaling molecules, primarily Wnt5a. (3−5) While ROR1 exhibits broad expression and plays pivotal roles during embryonic development, (6) its expression is typically low or absent in most adult postnatal tissues, except in specific cell types such as endocrine glands and immature B lymphocytes. (7) In contrast, ROR1 is aberrantly overexpressed in many malignancies, including chronic lymphocytic leukemia (CLL) and a wide range of solid tumors, such as melanoma, lung cancer, and triple-negative breast cancer (TNBC). (5,7−10) Furthermore, ROR1 contributes to oncogenesis by activating multiple Wnt5a-mediated signaling pathways, which are critical for cell proliferation, survival, migration, and metastasis. (5,10−14) Therefore, ROR1 has emerged as a promising therapeutic target in oncology, which is underscored by its tumor-specific overexpression and critical role in promoting cancer cell survival and metastasis.

Therapeutic strategies targeting ROR1 are rapidly developing and encompass diverse modalities, including antibody-based immunotherapies, bispecific T-cell engagers (BiTEs), (15−17) peptide-drug conjugates (PDCs), (18) and small molecule inhibitors, (19−24) with several candidates currently under clinical studies. (5,25) Among these, the antibody-based approaches, such as monoclonal antibodies (mAbs), (26,27) antibody-drug conjugates (ADCs), (28,29) and chimeric antigen receptor-modified T (CAR-T) cells, (30,31) have demonstrated notable progress. For example, the anti-ROR1 mAb Zilovertamab is undergoing clinical trials for CLL, breast cancer, and mantle cell lymphoma (MCL), (27,32−36) while the ADC VSL-101 (37) and CAR-T cell PRGN-3007 (38) have also advanced to clinical evaluation. Despite their high affinity and specificity, antibody-based therapies face limitations, including inadequate solid tumor penetration due to large molecular size and binding site barriers, (39) immunoreactivity risks, and high production costs. (40) These challenges highlight the need for alternative strategies, where peptide-based approaches offer distinct advantages. (40−42) First, the compact size of the peptides potentially enables deeper tumor infiltration and improved tissue distribution while retaining high target affinity and selectivity. Second, their rapid pharmacokinetics promote fast clearance from bloodstream and nontarget tissues, minimizing off-target effects. In addition, peptides are cost-effective and easily synthesized, facilitating the design of novel targeted therapies such as PDCs and radionuclide drug conjugates (RDCs). (18,40−45) Furthermore, peptide structures can be feasibly engineered to improve stability, bioavailability, and therapeutic efficacy. (40−42) Particularly, PDCs have recently emerged as a promising next-generation treatment modality, achieving significant success in cancer therapy. (40−42,45) Of note, two PDCs, Lutathera ([¹⁷⁷Lu]DOTA-TATE) (46,47) and Pluvicto ([¹⁷⁷Lu]PSMA-617), (48) which also qualify as RDCs, were approved by the U.S. Food and Drug Administration (FDA) for the treatment of somatostatin receptor-positive gastroenteropancreatic neuroendocrine tumors (GEP-NETs) and metastatic castration-resistant prostate cancer, respectively.

Positron emission tomography (PET) imaging plays a pivotal and expanding role in oncology, establishing a paradigm for noninvasive target characterization and molecular-level tumor profiling, together with the development of advanced PET ligands. (49,50) Furthermore, the emergence of innovative radiolabeling strategies, such as water-compatible S_N2 ¹⁸F-fluorination, (51,52) has facilitated the development of excellent PET ligands, thereby driving the progress of PET imaging. Given the crucial therapeutic relevance of ROR1 in oncology, the creation of ROR1-targeted PET probes holds significant potential to 1) enable real-time, dynamic visualization of ROR1 expression in tumors; 2) assess and monitor therapeutic responses to anti-ROR1 therapies; 3) guide patient stratification and treatment optimization; and 4) accelerate the clinical translation of novel ROR1-targeted drugs. Despite these opportunities, few ROR1-specific PET tracers have been reported to date. Recently, three ⁶⁸Ga-labeled ROR1 PET ligands, [⁶⁸Ga]DOTA-PEG4-PR3, [⁶⁸Ga]DOTA-PEG4-PR7, and [⁶⁸Ga]DOTA-KGGG-1036, were initially developed. [⁶⁸Ga]DOTA-PEG4-PR3 and [⁶⁸Ga]DOTA-PEG4-PR7 were demonstrated to have fast accumulation in ROR1-positive NCI-H1975 tumor-bearing mice with specific binding, while no such tumor uptake was observed for [⁶⁸Ga]DOTA-KGGG-1036. (53) Subsequent efforts focused on the development of PR7 peptides, leading to both linear ([⁶⁸Ga]Ga-DP1 and [¹⁸F]AlF-NP1) and cyclic ([⁶⁸Ga]Ga-DP2, [⁶⁸Ga]Ga-DP3 and [¹⁸F]AlF-NP2) peptide derivatives. (54) Among these, [⁶⁸Ga]Ga-DP1 and [¹⁸F]AlF-NP1 displayed favorable performance in visualizing various tumor models such as MC38, B16F10, A549, and HepG2 tumors and exhibited specific binding in ROR1-positive MC38 tumors, despite their short tumor retention. With continued efforts, we aimed to develop efficient PET ligands for ROR1 imaging, with the goal of validating ROR1 as a viable target for tumor monitoring. Recent advances include the downsizing of the bispecific antibody scFv R11 (PDB: 6BA5) (55) into smaller ROR1-binding peptide mimetics with moderate to high affinity (1.21–713 nM). (18) These peptides provided valuable targeting entities for the ROR1 PET tracer development. In this study, leveraging a high-affinity peptide PR3 (sequence: INSGPGYSTYYGDF, K_D = 1.21 nM), (18) we designed three novel PET ligands, namely [⁶⁸Ga]PR3 ([⁶⁸Ga]1), [⁶⁸Ga]PR3-ABCF3 ([⁶⁸Ga]2), and [⁶⁸Ga]PR3-FA16 ([⁶⁸Ga]3), and evaluated their utility as targeted tracers in ROR1-positive melanoma-bearing mice. Furthermore, [⁶⁸Ga]MCP-14 ([⁶⁸Ga]4) was also developed from a ROR1-binding macrocyclic peptide, which is a binder to extracellular cysteine-rich domain (CRD) of human ROR1 with high affinity (IC₅₀ = 2.2 nM). (56) The radioligands incorporate a poly(ethylene glycol) (PEG) linear polymer with eight ethylene oxide units (PEG-8, for [⁶⁸Ga]1, Figure 1A) or a lysine moiety (for [⁶⁸Ga]2–4, Scheme 1) as a spacer to reduce steric hindrance between the bulky peptide-binding moiety and the chelator, thus minimizing steric interference during radiolabeling and preserving binding affinity. Additionally, [⁶⁸Ga]2 and [⁶⁸Ga]3 are functionalized with serum albumin-binding groups of 4-(4-(trifluoromethyl)phenyl)butyric acid (ABCF3, where 4-(4-(trifluoromethyl)phenyl)butyric acid is denoted as “CF3” due to its unique trifluoromethyl moiety) and a 16-carbon long-chain fatty acid (palmitic acid, C16), respectively, to enhance serum half-life.

Figure 1. Ligand design and primary evaluations. (A) Schematic diagram and chemical structure of [⁶⁸Ga]1. (B) MicroPET/CT images (left panel) and biodistribution results (right panel) in B16F10 tumor-bearing mice (n = 2–4) after intravenous injection of [⁶⁸Ga]1. BL, bladder; K, kidneys; T, tumor.

Scheme 1. Chemical Structures of [⁶⁸Ga]2–4

Results and Discussion

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Ligand Design

The structure of a peptide-based radiopharmaceutical typically comprises a peptide as the tumor receptor-targeting entity and a radionuclide-chelating moiety for radiolabeling, which are systematically combined with a linker group (Figure 1A). Accordingly, three ROR1-targeting peptides, including PR2, PR3, and PR7, bind to the extracellular Kringle (Kr) domain of ROR1 with significantly high affinity (K_D < 3 nM). (18) Among these, PR7 (K_D = 1.57 nM) has been used to create efficient PDCs to deliver cytotoxic payloads to ROR1-positive tumors, (18) indicating the feasibility of using these peptides as warheads in ROR1-targeting drugs. Inspired by this, an ROR1-targeting PET ligand ([⁶⁸Ga]1, Figure 1A) was initially established based on PR3, which has the highest affinity (K_D = 1.21 nM) and smallest molecular weight (M.W. = 1539.6), to verify its utility in developing a peptide-based ROR1 PET ligand. In addition, PEG polymers have been approved for human use by FDA and are widely used to enhance the pharmacokinetics of therapeutic peptides due to its unique advantages in increasing blood-residence time, as well as reducing proteolytic degradation and immunogenicity. (41,57−59) As a result, the peptide PR3 was modified with a PEG-8 chain and subsequently coupled with the bifunctional chelator (for radiolabeling of both ⁶⁸Ga and ¹⁷⁷Lu) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and radiosynthesized via ⁶⁸Ga³⁺-DOTA chelation to obtain [⁶⁸Ga]1. Subsequently, an initial evaluation of [⁶⁸Ga]1 was carried out using tumor PET imaging in B16F10 melanoma-bearing mice. As shown in Figure 1B, [⁶⁸Ga]1 quickly accumulated in the tumor at 30 min post-injection (1.7 ± 0.2% of the injected dose per gram of tissue, % ID/g, n = 3) with a desirable tumor-to-muscle (T/M) ratio of 5. Notably, its tumor uptake was significantly higher than that of the previously reported ROR1-targeted PET ligands [⁶⁸Ga]Ga-DP1 (∼0.35% ID/g) and [¹⁸F]AlF-NP1 (∼0.60% ID/g). This improvement can likely be attributed to the optimized linker design of [⁶⁸Ga]1, specifically the incorporation of the PEG-8 chain, which prolongs blood circulation time and thereby enhances tumor delivery. (54) However, the tumor uptake was rapidly decreased to 0.3 ± 0.07% ID/g over 120 min, in accordance with the performance of the developed ROR1-targeting PET ligands such as [⁶⁸Ga]DOTA-PEG4-PR3, [⁶⁸Ga]DOTA-PEG4-PR7, [⁶⁸Ga]Ga-DP1 and [¹⁸F]AlF-NP1. (53,54) In addition, the highest radioactivity uptake of [⁶⁸Ga]1 was observed in the kidneys with a value of 6.1 ± 1.0% ID/g at 30 min, which decreased to 2.6 ± 0.2% ID/g at 120 min post-injection. These results indicated the capability of [⁶⁸Ga]1 in the detection of ROR1-positive tumors with a high T/M ratio yet with low tumor retention. The fast pharmacokinetics of [⁶⁸Ga]1 may be caused by its short blood circulation time in vivo. To address this issue, two albumin-binding ROR1 PET ligands were constructed by conjugating ABCF3 and C16 to the peptide PR3 (Scheme 1), denoted as [⁶⁸Ga]PR3-ABCF3 ([⁶⁸Ga]2) and [⁶⁸Ga]PR3-C16 ([⁶⁸Ga]3), respectively, which were expected to have prolonged circulation that may increase the accumulation and retention of radioligands in the tumor. The noncovalent complexation with albumin could also protect the radioligand against protease hydrolysis. (40,60) Specifically, compared with linear peptides, cyclic peptides have the advantage of superior structural rigidity, which leads to enhanced biochemical stability and resistance to proteolytic degradation. In addition, cyclization may also improve membrane permeability, and binding affinity and specificity for their targets. (61) In this context, [⁶⁸Ga]MCP-14 ([⁶⁸Ga]4, Scheme 1) that possessed a ROR1-targeting cyclic peptide was designed and evaluated for ROR1 detection.

Binding Potency

To evaluate the binding affinities of our newly developed peptide-DOTA conjugates with ROR1 protein, biolayer interferometry (BLI) analysis was employed to measure the dissociation constants (K_Ds). As shown in Figure 2, PR3-C16-DOTA exhibited the most potent binding affinity (K_D = 44.9 nM, Figure 2C) among the tested peptides, which was driven by both a high association rate (K_on = 2.5 × 10⁵ M^–1 s^–1) and a low dissociation rate (K_off = 1.1 × 10^–2 s^–1). These results indicated stable complex formation between PR3-C16-DOTA and ROR1. PR3-ABCF3-DOTA and MCP-14-DOTA demonstrated submicromolar affinities (K_D = 481.0 and 414.6 nM, respectively, Figure 2B,D) with slow dissociation rates; however, the low association rates resulted in weaker overall binding strength compared to PR3-C16-DOTA. In contrast, PR3-DOTA exhibited the weakest binding (K_D = 193.0 μM, Figure 2A), primarily due to a much slower association rate and a relatively faster dissociation. Overall, PR3-DOTA exhibited a sharp decline in binding potency compared to that of PR3. This reduction is likely attributable to steric hindrance introduced by the linker and DOTA chelator, which may obstruct the PR3-ROR1 interaction interface and restrict spatial accessibility at the binding site. Conversely, the incorporation of ABCF3 and C16 moieties enhances binding affinity relative to PR3-DOTA, potentially by augmenting hydrophobic interactions or optimizing the peptide orientation. MCP-14-DOTA, which was designed from a cyclic peptide, displayed a relatively higher association rate (K_on = 8.0 × 10³ M^–1 s^–1) than PR3-DOTA, consistent with its more stable binding. These data demonstrate that the structural modification of peptide ligands significantly impacts their affinity toward ROR1. In particular, the conjugation of serum albumin-binding groups ABCF3 and C16 markedly improves the binding affinity toward ROR1, highlighting their potential utility in peptide modification.

Figure 2. BLI binding curves of the interaction of newly developed ligands with the ROR1 protein. A–D correspond to PR3-DOTA, PR3-ABCF3-DOTA, PR3-C16-DOTA, and MCP-14-DOTA, respectively.

Radiolabeling and In Vitro Stability

Comprehensive synthetic procedures for the peptide PR3 conjugates (PR3-DOTA, PR3-ABCF3-DOTA, PR3-C16-DOTA, and MCP-14-DOTA) are provided in Schemes S1–4 of Supporting Information (SI). Structural confirmation and purity assessment of these compounds were performed using liquid chromatography–mass spectrometry (LC-MS) and high-performance liquid chromatography (HPLC), respectively. All four peptide conjugates were determined to have high purity of higher than 96% (Figure S1 in the SI). The radiolabeling routes of [⁶⁸Ga]1–4 were illustrated in Scheme S5 in SI. Briefly, peptide-DOTA precursors (50 μg) were reacted with ⁶⁸GaCl₃ in an acidic sodium acetate buffer (pH = ∼4.0) at 95 ([⁶⁸Ga]1–3) or 90 °C ([⁶⁸Ga]4) for 20 min. The resulting ⁶⁸Ga-labeled radioligands were subsequently purified via solid-phase extraction, yielding high radiochemical yields (RCYs > 60%, Table 1) and high radiochemical purity (RCP > 92%, Table 1, Figure S2 in SI). As summarized in Table 1, [⁶⁸Ga]1–4 displayed high stability in saline upon incubation for 2 h at 37 °C, with ≥ 90% of the parent radioligands remaining. However, reduced stability was observed for [⁶⁸Ga]1–3 in mouse plasma (<90%), which likely attributes to protease hydrolysis of the linear PR3 peptide. Meanwhile, [⁶⁸Ga]4, a derivative based on a cyclic peptide scaffold, exhibited poor stability in mouse plasma, with only 7% of the parent radioligand remaining. Furthermore, the log D_7.4 values were determined to range from −2.84 ± 0.28 to −0.93 ± 0.15 (Table 1), indicating the polar and hydrophilic characteristics of these radioligands.

Table 1. Comparison of RCYs, RCP, In Vitro Stability, and Lipophilicity of [⁶⁸Ga]1–4

Radioligand	RCY	RCPa	Stability in salineb	Stability in mouse plasmab	Log D_7.4
[⁶⁸Ga]1	94% (n = 3)	93%	90% (2 h)	81% (2.5 h)	–2.84 ± 0.28
[⁶⁸Ga]2	85% (n = 3)	95%	95% (2 h)	73% (2.5 h)	–2.76 ± 0.15
[⁶⁸Ga]3	70% (n = 3)	96%	96% (2 h)	88% (2 h)	–1.93 ± 0.08
[⁶⁸Ga]4	61% (n = 2)	96%	95% (2 h)	7% (2 h)	–0.93 ± 0.15

^{^a}

Determined upon the purification with a C18 cartridge.

^{^b}

In vitro stability was measured after incubation for 2 or 2.5 h, as indicated in the parentheses.

Cellular Uptake and Self-Blocking Assays

To evaluate the cell permeability and binding specificity of [⁶⁸Ga]1–4, cellular uptake and self-blocking assays were performed using three melanoma cell lines, including highly metastatic murine melanoma B16F10 and two human malignant melanoma lines, A375 and SK-MEL-28. As shown in Figure 3A, the cellular uptake of [⁶⁸Ga]1 remained low in all three cell lines (<4% at 2 h), consistent with its weak affinity for ROR1. In contrast, [⁶⁸Ga]2 and [⁶⁸Ga]3, which incorporate serum albumin-binding groups, exhibited markedly enhanced uptake in B16F10 cells, reaching maximum uptake values of 54.4% at 1 h and 33.1% at 2 h, respectively. Both radioligands showed negligible uptake in A375 cells (<0.5%), likely reflecting the low expression of ROR1 in this cell line. (10,62) A notable difference was observed in SK-MEL-28 cells, where [⁶⁸Ga]3 demonstrated significantly higher uptake (46.5% at 2 h) than that of [⁶⁸Ga]2 (<0.5%), which may be attributed to its stronger binding affinity for ROR1. Uptake of [⁶⁸Ga]4 in B16F10 cells displayed a rapid initial increase (10.6% at 0.5 h), followed by a gradual decline (8.5% at 2 h), possibly due to the lower stability of the MCP14-DOTA–ROR1 complex (K_off = 3.3 × 10^–3 s^–1, Figure 2D) compared with other tracers.

Figure 3. Cell uptake assay (A) and self-blocking assay (B) of [⁶⁸Ga]1–4 in melanoma cell lines B16F10, A375, and SK-MEL-28; n.d., not determined. Asterisks indicate statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001 vs control. Data were analyzed using a two-tailed unpaired Student’s t-test.

Self-blocking experiments were conducted to assess the specific binding of [⁶⁸Ga]1–3 for ROR1 (Figure 3B). [⁶⁸Ga]1 only exhibit significant inhibition in SK-MEL-28 cell line (p < 0.05), yet with a low uptake level. [⁶⁸Ga]2 showed substantial specific binding in B16F10 cells, with 78% inhibition under self-blocking conditions (p < 0.001), but lower inhibition in A375 and SK-MEL-28 cells (36% and 45%, respectively). In contrast, [⁶⁸Ga]3 displayed remarkable inhibition in both B16F10 and SK-MEL-28 cells, with rates of 97% (p < 0.001) and 75% (p < 0.001), respectively, while a more modest inhibition was observed in A375 cells (46%, p < 0.01). In summary, [⁶⁸Ga]1 failed to exhibit sufficient specific ROR1 binding in the tested cells, consistent with its low affinity and cellular uptake. [⁶⁸Ga]2 showed high specificity in B16F10 cells but limited binding in SK-MEL-28 cells due to its low uptake. Notably, [⁶⁸Ga]3, with its superior affinity for ROR1, demonstrated strong specific binding in both the B16F10 and SK-MEL-28 cells. The incorporation of a C16 moiety may further enhance the membrane permeability of [⁶⁸Ga]3. (63)

MicroPET/CT Imaging of [⁶⁸Ga]1–4 in B16F10 Tumor-Bearing Mice

Motivated by the favorable cellular uptake results, [⁶⁸Ga]1–4 was subsequently evaluated in PET imaging studies using B16F10 tumor-bearing mice. Compared with that of [⁶⁸Ga]1 (Figure 1B), the incorporation of serum albumin-binding moieties in [⁶⁸Ga]2 and [⁶⁸Ga]3 markedly enhanced their tumor uptake. Both radioligands showed rapid tumor uptake within 0.5 h postinjection (Figure 4A,C), achieving maximum standardized uptake values (SUV_max) of 1.72 and 1.82, respectively, which increased by ∼5-fold relative to [⁶⁸Ga]1. At this initial time point, both agents displayed a high T/M ratio of 6. Furthermore, [⁶⁸Ga]2 and [⁶⁸Ga]3 demonstrated prolonged tumor retention, with uptake values of 1.66 and 1.99, respectively, at 2 h postinjection. These values represent an ∼28-fold increase compared to [⁶⁸Ga]1, while consistent T/M ratios of 6 were maintained. Notably, in an independent PET study (Table S6 and Figure S6 in the SI), high levels of tumor radioactivity were still observed for [⁶⁸Ga]2 and [⁶⁸Ga]3 at 4 h post-injection with SUV_max of 0.99 and 2.77, respectively, indicating prolonged residence time within the tumor. These findings establish a solid foundation for the subsequent development of ROR1-targeted therapeutic radiopharmaceuticals. The enhanced tumor uptake and retention are primarily attributed to the extended blood circulation and improved membrane permeability conferred by the serum albumin-binding groups, which promote radioligand extravasation into the tumor. However, PET imaging also revealed notable nonspecific uptake in the heart (Figure 4A,C), particularly for [⁶⁸Ga]3, which displayed a higher cardiac retention and slower clearance. In contrast, [⁶⁸Ga]4 exhibited negligible tumor uptake with predominant hepatic accumulation (Figure S3 in the SI), likely due to its poor stability (Table 1), thereby precluding its further development as an ROR1-targeted PET ligand.

Figure 4. (A, C) MicroPET/CT images of B16F10 tumor-bearing mice following the injection of [⁶⁸Ga]2 (A) and [⁶⁸Ga]3 (C), respectively. Self-blocking studies were performed by coinjecting the corresponding nonradioactive competitors, PR3-ABCF3-DOTA (0.25 mg/mouse) for [⁶⁸Ga]2 and PR3-C16-DOTA (0.33 mg/mouse) for [⁶⁸Ga]3. (B,D) The radioactivity uptake values of [⁶⁸Ga]2 (B) and [⁶⁸Ga]3 (D) are expressed as SUV_max in tumor and muscle. BL, bladder (red arrow); H, heart (black arrow); T, tumor (white arrow).

Self-blocking studies were conducted by coinjection of corresponding peptide-DOTA conjugates to evaluate the binding specificity of [⁶⁸Ga]2 and [⁶⁸Ga]3 toward tumor ROR1. For [⁶⁸Ga]2, the coinjection with PR3-ABCF3-DOTA significantly suppressed tumor radioactivity uptake (Figure 4A and Figure S4 in the SI), showing 25%, 35%, and 41% inhibition at 0.5, 1, and 2 h postinjection, respectively (Figure 4B). Conversely, no such inhibition was observed in the muscle, confirming its target-specific binding of [⁶⁸Ga]2 to ROR1 in tumors. Despite high ROR1 affinity, [⁶⁸Ga]3 exhibited reduced blocking efficacy, with only 23% inhibition observed at 2 h postinjection (Figure 4D). This attenuation may result from its excessively high serum albumin binding, which impedes the access of the competing ligand PR3-C16-DOTA to tumor ROR1 sites. Similar to the case for [⁶⁸Ga]2, no appreciable inhibition was detected in the muscle for [⁶⁸Ga]3 (Figure 4D). In summary, both [⁶⁸Ga]2 and [⁶⁸Ga]3 demonstrated enhanced tumor uptake and retention in B16F10 tumor-bearing mice, and notably, [⁶⁸Ga]2 exhibited superior specificity for tumor ROR1. Moreover, the incorporation of serum albumin-binding moieties (ABCF3 and C16, Scheme 1) conferred favorable pharmacokinetic properties, including elevated accumulation and prolonged retention in tumors. Nevertheless, this strategy also elevated background signals due to increased blood pool radioactivity, which consequently compromises the tumor-to-background contrast. Thus, the rational design of albumin-binding radioligands requires careful optimization to balance improved pharmacokinetics with other physicochemical properties.

Ex Vivo Biodistribution Studies of [⁶⁸Ga]2 and [⁶⁸Ga]3 in B16F10 Tumor-Bearing Mice

Whole-body ex vivo biodistribution studies were conducted in B16F10 tumor-bearing mice following intravenous (i.v.) administration of [⁶⁸Ga]2 and [⁶⁸Ga]3 at 1 and 2 h postinjection. The results were summarized in Figure 5 and Tables S1 and S2 in the SI. Both radioligands showed high tumor uptake, with values of 7.61 ± 0.96 for [⁶⁸Ga]2 and 8.06 ± 1.20% ID/g for [⁶⁸Ga]3 at 1 h. Furthermore, they exhibited excellent tumor retention over time. After 2 h postinjection, [⁶⁸Ga]2 maintained a high uptake of 6.73 ± 0.52% ID/g, while the uptake of [⁶⁸Ga]3 increased to 8.97 ± 1.78% ID/g. In contrast, muscle uptake remained low for both radioligands (<2.5% ID/g), resulting in favorable T/M ratios exceeding 3.5 (Tables S1 and S2 in the SI). As expected, the presence of albumin-binding moieties led to significant radioactivity retention in the blood pool. At 1 h postinjection, blood uptake reached 20.48 ± 2.72% ID/g for [⁶⁸Ga]2 and 44.26 ± 3.16% ID/g for [⁶⁸Ga]3, demonstrating slow clearance kinetics with 14.03 ± 1.84% and 37.99 ± 3.46% ID/g remaining at 2 h, respectively. Furthermore, [⁶⁸Ga]2 exhibited a blood half-life of 2.47 h in ICR mice (Figure S7 and Table S7, SI). This observed reversible kinetics likely contributes to its improved pharmacodynamic outcomes. Notably, [⁶⁸Ga]3 displayed elevated uptake in several nontarget organs at 1 h postinjection, including the heart (10.45 ± 1.45% ID/g), liver (12.11 ± 1.54% ID/g), lung (20.67 ± 4.64% ID/g), and kidneys (11.52 ± 0.93% ID/g), with subsequently slow washout from these tissues (Table S1 in SI). In comparison, [⁶⁸Ga]2 exhibited a more favorable pharmacokinetic profile, with lower nontarget accumulation (e.g., < 10% ID/g in lung and kidneys) and more rapid clearance, which contributed to reduced background signal in PET imaging. Taken together with the PET imaging results, these biodistribution findings indicate that [⁶⁸Ga]2 is a more suitable radioligand for noninvasive PET detection of ROR1-positive tumors by virtue of its high tumor uptake and retention combined with relatively lower blood uptake and nonspecific binding.

Figure 5. *Ex vivo* biodistribution of [⁶⁸Ga]2 (A) and [⁶⁸Ga]3 (B) in B16F10 tumor-bearing mice at 1 and 2 h postinjection. Data are expressed as the percentage of injected dose per gram of tissue (% ID/g), with the exception of the stomach and small intestine, for which uptake is presented as %ID per organ (% ID/organ).

MicroPET/CT Imaging of [⁶⁸Ga]1–4 and Ex Vivo Biodistribution Studies of [⁶⁸Ga]2 in A375 Tumor-Bearing Mice

To expand their application, microPET/CT imaging studies of [⁶⁸Ga]1–4 were performed in A375 tumor-bearing mice. Different from B16F10, A375 is a highly aggressive human melanoma cell line that lacks melanin production. As shown in Figure 6A–C, all radioligands rapidly accumulated in tumors within 0.5 h postinjection, with SUV_max values ranging from 0.35 to 1.11 (Figure 6C and Table S4 in SI). Based on PET images (Figure 6A,B), [⁶⁸Ga]1–4 displayed distinct tumor-to-background ratios (TBRs), with [⁶⁸Ga]1 and [⁶⁸Ga]2 exhibiting higher TBRs than [⁶⁸Ga]3 and [⁶⁸Ga]4. Among them, [⁶⁸Ga]1 showed the lowest tumor uptake at all time points and rapid washout kinetics (Figure 6A,C and Table S4 in the SI), consistent with its performance in B16F10 tumor-bearing mice (Figure 1B) that can likely be attributed to its poor binding affinity for ROR1. In contrast, [⁶⁸Ga]2 achieved the highest tumor accumulation at early time points (0.5 and 1 h), with SUV_max values of 1.11 ± 0.13 and 1.03 ± 0.12, respectively, although this was lower than its uptake in the B16F10 model (Figure 4A,B). However, coinjection with PR3-ABCF3-DOTA (0.25 mg per mouse) in a self-blocking study did not inhibit tumor uptake (Table S4 in SI), which likely results from the low ROR1 expression level in this cell line. (10,62) Meanwhile, [⁶⁸Ga]3 and [⁶⁸Ga]4 exhibited moderate tumor uptake with SUV_max values ranging from 0.51 to 0.83 (Figure 6C and Table S4 in the SI), but their potential for ROR1-targeted PET imaging was limited by high nonspecific accumulation in the heart and liver. Furthermore, ex vivo biodistribution results for [⁶⁸Ga]2 in A375 tumor-bearing mice (Figure 6D and Table S2 in the SI) confirmed its high tumor uptake (9.18 ± 0.99% ID/g at 1 h) and prolonged retention (8.79 ± 1.35% ID/g at 2 h). As expected, high radioactivity levels were observed in the blood (>25% ID/g) and lungs (>15% ID/g), comparable to the results in B16F10 model, which contributes to increased background signals in PET imaging. In summary, [⁶⁸Ga]2 exhibited the most favorable tumor uptake profile in the A375 model; however, its specific binding to ROR1 could not be conclusively verified under these conditions. This limitation may reflect the relatively lower ROR1 expression in A375 cells and suggests that a higher dose of competitor may be required in blocking studies to fully assess ROR1-mediated binding.

Figure 6. MicroPET/CT imaging and *ex vivo* biodistribution studies in A375 tumor-bearing mice. (A, B) MicroPET/CT images of A375 tumor-bearing mice following the injection of [⁶⁸Ga]1–4. Representative images acquired at selected time points postinjection. [⁶⁸Ga]1 (A) and [⁶⁸Ga]2 (B), which achieved high T/M ratios, were shown in three time points of 0.5, 1, and 2 h, while [⁶⁸Ga]3 and [⁶⁸Ga]4 (B) that had lower T/M ratios were displayed at the 2 h time point only. (C) Quantitative analysis of tumor uptake for [⁶⁸Ga]1–4 expressed as SUV_max. (D) *Ex vivo* biodistribution of [⁶⁸Ga]2 in A375 tumor-bearing mice at 1 and 2 h postinjection. Data are expressed as % ID/g, with the exception of the stomach and small intestine, for which uptake is presented as % ID/organ. BL, bladder (red arrow); H, heart (purple arrow); L, liver (yellow arrow); and T, tumor (white arrow).

MicroPET/CT Imaging of [⁶⁸Ga]1–3 and Ex Vivo Biodistribution Studies of [⁶⁸Ga]2 in SK-MEL-28 Tumor-Bearing Mice

Building on previous results, [⁶⁸Ga]1–3 were further evaluated by microPET/CT imaging and ex vivo biodistribution studies in SK-MEL-28 tumor-bearing mice. The SK-MEL-28 cell line is a human-derived malignant melanoma model commonly used for studying cutaneous melanoma. As shown in Figure 7A and B, [⁶⁸Ga]1 exhibited fast tumor accumulation within 0.5 h postinjection, followed by a rapid washout, consistent with its pharmacokinetic behavior observed in the B16F10 and A375 models. However, its tumor uptake in the SK-MEL-28 model was comparatively lower with a maximum SUV_max of 0.18 ± 0.03 at 0.5 h (Figure 7C and Table S5 in the SI). Similarly, [⁶⁸Ga]2 accumulated rapidly in tumors, reaching a peak SUV_max of 1.11 ± 0.17 at 0.5 h, followed by a gradual decline to 0.72 ± 0.15 at 2 h, mirroring the kinetic profile observed in the A375 model. Notably, [⁶⁸Ga]3 showed the highest tumor uptake, with SUV_max values >1.31 and a gradual increase over time, peaking at 1.51 ± 0.01 at 2 h. This sustained accumulation aligns with its high cellular uptake in the SK-MEL-28 cell line (Figure 3A). However, high background signals of [⁶⁸Ga]3 resulting from nonspecific uptake in the heart, lung, and liver (Figure 7B and Figure S5 in the SI) reduced the overall TBR, indicating that later imaging time points may be required to achieve optimal contrast. Ex vivo biodistribution of [⁶⁸Ga]2 in the SK-MEL-28 model confirmed its tumor kinetics, showing an initial uptake of 2.32 ± 0.36% ID/g at 1 h, which decreased to 1.80 ± 0.27% ID/g by 2 h (Table S2 in the SI). These values are lower than those observed in the B16F10 and A375 models. Meanwhile, reduced radioactivity levels were observed in the blood (11.57 ± 2.90% ID/g), lung (6.02 ± 1.09% ID/g), and kidneys (4.64 ± 0.88% ID/g, Figure 7D and Table S2 in the SI). Collectively, these results suggest the capability of [⁶⁸Ga]2 for detecting tumor ROR1 via PET imaging. Furthermore, [⁶⁸Ga]3 shows significant potential for this application, provided that delayed imaging time points are employed to allow clearance of the nonspecific background signal.

Figure 7. MicroPET/CT imaging and *ex vivo* biodistribution studies in SK-MEL-28 tumor-bearing mice. (A,B) MicroPET/CT images of SK-MEL-28 tumor-bearing mice following the injection of [⁶⁸Ga]1–3. Representative images acquired at selected time points postinjection. [⁶⁸Ga]1 (A) and [⁶⁸Ga]2 (B), which achieved high T/M ratios, were shown in three time points of 0.5, 1, and 2 h, while [⁶⁸Ga]3 (B) that had a lower T/M ratio was displayed at the 2 h time point only. (C) Quantitative analysis of tumor uptake for [⁶⁸Ga]1–3 expressed as SUV_max. (D) *Ex vivo* biodistribution of [⁶⁸Ga]2 in SK-MEL-28 tumor-bearing mice at 1 and 2 h postinjection. Data are expressed as % ID/g, with the exception of the stomach and small intestine, for which uptake is presented as % ID/organ. BL, bladder (red arrow); H, heart (purple arrow); L, liver (yellow arrow); T, tumor (white arrow).

Characterization of ROR1 Expression in Melanoma Models

To evaluate the histopathological features and ROR1 expression of the tumor models, hematoxylin and eosin (HE) and immunohistochemical (IHC) staining was performed on B16F10, A375, and SK-MEL-28 sections. As shown in Figure 8, HE staining revealed typical nested growth patterns of the three tumor models, albeit with distinct morphological profiles. Quantitative analysis of the IHC results revealed a differential ROR1 expression profile across the cell lines. Notably, SK-MEL-28 demonstrated the highest expression level, with a histochemistry score (H-score) of 185.1 ± 21.6, followed by those of B16F10 (167.5 ± 15.7) and A375 (124.6 ± 28.1). In addition, the relatively small standard deviations observed in the H-score distribution indicate a homogeneous expression pattern within the tumor parenchyma. These findings establish a critical foundation for in vivo imaging and ex vivo biodistribution studies of the ROR1-targeted PET ligands.

Figure 8. Histopathological and immunohistochemical (IHC) characterization of B16F10, A375, and SK-MEL-28 melanoma tumors. Representative hematoxylin and eosin (HE)-stained sections (top panels) and corresponding IHC staining (middle panels) of paraffin-embedded tumor tissues. The lower panels show the quantitative analysis of protein expression levels based on histochemistry scores (H-scores). Data are expressed as mean ± SD (n = 10 fields per section). Scale bars: 150 μm.

Conclusion

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In this study, we presented a rational design and comprehensive evaluation of four ⁶⁸Ga-labeled peptide-based PET probes, [⁶⁸Ga]1–4, targeting ROR1 for melanoma imaging. These radioligands were constructed by conjugating high-affinity ROR1-binding peptides (PR3 for [⁶⁸Ga]1–3 and MCP-14 for [⁶⁸Ga]4) to a DOTA chelator through a PEG or lysine linker. [⁶⁸Ga]1, a PEGylated PR3 derivative, showed rapid tumor accumulation but fast clearance and poor retention, limiting its diagnostic performance. To overcome this, serum albumin-binding groups (ABCF3 and C16) were conjugated to generate [⁶⁸Ga]2 and [⁶⁸Ga]3, respectively. These modifications enhanced ROR1 binding affinity and cellular uptake in vitro while increasing tumor accumulation and retention in vivo. Notably, [⁶⁸Ga]2 exhibited a favorable balance between tumor targeting and blood uptake, displaying high tumor uptake (7–9% ID/g) with lower nonspecific accumulation. In contrast, though it exhibited the highest tumor uptake, [⁶⁸Ga]3 showed substantial background accumulation due to its excessive albumin binding, which impeded its application in PET imaging of ROR1. Future structural refinements could involve modulating the fatty acid chain length (approximately C14–C18) to optimize the balance between albumin association and solubility. Such an approach offers a versatile platform for tailoring pharmacokinetic profiles based on target circulatory retention and desired clearance kinetics. This outcome highlights the importance of fine-tuning albumin-binding affinity to balance circulation time and background signal. Additionally, [⁶⁸Ga]4 derived from a cyclic peptide exhibited poor stability in plasma, limiting its utility in PET imaging. Overall, this work validates the feasibility of peptide-based ROR1 PET imaging and underscores the utility of albumin-binding strategies to improve tumor targeting and retention. [⁶⁸Ga]2 emerges as a promising candidate for further development, offering a balanced profile of high specificity, favorable kinetics, and potential clinical application. These findings provide a framework for the rational design of new ROR1-targeted imaging and therapeutic ligands.

Experimental Section

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General Information

Chemical reagents and organic solvents were purchased from commercial suppliers and used without further purification. Radio-HPLC was performed using a Shimadzu SCL-20AVP system equipped with an SPD-20A UV detector at 254 nm and a Bioscan Flow Count 3200 NaI/PMT γ-radiation scintillation detector. The ⁶⁸Ge/⁶⁸Ga generator was purchased from Chengdu New Radiomedicine Technology Co., Ltd. (Sichuan, China). The purity of the DOTA-conjugated peptides (PR3-DOTA, PR3-ABCF3-DOTA, PR3-C16-DOTA, and MCP-14-DOTA) was analyzed by HPLC and verified to be >96% (Figure S1 in the SI). B16F10 and SK-MEL-28 cell lines were kindly provided by the Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, and the A375 cell line was a generous gift from the Institute of Radiation Medicine, Fudan University. B16F10 and SK-MEL-28 cell lines were maintained in RPMI-1640 medium, while A375 cells were cultured in DMEM medium, both supplemented with 10% fetal bovine serum and 1% (w/v) penicillin/streptomycin at 37 °C under 5% CO₂. Female C57BL/6 and BALB/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animals were reared in a pathogen-free, climate-controlled (26 °C) environment with enrichment to support natural behaviors and had continuous access to balanced feed and clean water. All animal experiments were approved by the Institutional Animal Care and Use Committee of Beijing Normal University and were performed in accordance with the relevant guidelines and regulations (BNUCC-EAW-2023–0912–01).

Chemistry and Radiochemistry

The DOTA-conjugated peptides (PR3-DOTA, PR3-ABCF3-DOTA, PR3-C16-DOTA, and MCP-14-DOTA) were synthesized by using standard Fmoc solid-phase synthesis. Detailed information on synthesis was provided in the SI. In radiosynthesis, freshly eluted ⁶⁸Ga³⁺ (in 0.1 M HCl, 2.5 mL) from the ⁶⁸Ge/⁶⁸Ga generator was added to a solution of DOTA-conjugated peptide (50 μg) in NaOAc buffer (1 M, 325 μL). The mixture (pH 4.0) was heated to 90 or 95 °C for 20 min. Purification was performed by solid-phase extraction using preactivated Sep-Pak Plus C18 cartridges (Waters). Radiochemical purity (RCP) was determined with radio-HPLC, and the HPLC profiles were summarized in Figure S2 in the SI.

Binding Kinetics Assays

The binding affinity of the peptide-DOTA conjugates for the ROR1 protein was determined by BLI using an Octet Red96 instrument (Sartorius). Streptavidin (SA) biosensors were prehydrated in phosphate-buffered saline (PBS) for 10 min in a black 96-well plate (Thermo Fisher). ROR1 protein (catalog no. 13968-HCCH1-B, Sino Biological) was diluted to 4.17 μg/mL in PBS and immobilized onto the sensors for 15 min. Subsequently, the sensors were transferred to PBS for 3 min to dissociate weakly and nonspecifically bound proteins. Peptide ligands, prepared at serial concentrations in PBS, were associated with the immobilized ROR1 for 180 s and allowed to dissociate for 180 s at 25 °C. The association rate (K_on, M^–1 s^–1) and dissociation (K_off, s^–1) rate constants were acquired from Octet Red96 Analysis Studio Software. The K_D values were calculated as K_off/K_on.

In Vitro Stability

The in vitro stability of the ⁶⁸Ga-labeled radioligands was determined in both saline and mouse serum. The radioligand was incubated in saline and mouse serum at 37 °C for 2 or 2.5 h. For the stability in saline, the solution was applied to radio-HPLC for analyzing the RCP of the radioligand after incubation. In mouse serum, 200 μL of acetonitrile was added to the incubation solution for protein precipitation, and the supernatant was collected by centrifugation. The RCP of the radioligands was determined by radio-HPLC.

Partition Coefficient

The lipophilicity of [⁶⁸Ga]1–4 was determined by measuring the partition coefficient between 1-octanol and PBS (pH 7.4). Briefly, a pure radioligand (∼3.7 MBq) was added to a test tube containing 3 mL of 1-octanol and 3 mL of PBS (pH 7.4). The mixture was vortexed for 3 min and then centrifuged at 1000 rpm for an additional 5 min. Radioactivity of aliquots from the octanol (500 μL) and aqueous phases (100 μL), respectively, was measured by an automatic γ-counter (WALLAC/Wizard 1470, PerkinElmer, USA). The measurement was performed in triplicate, and the results were used to calculate the log D_7.4 (octanol/buffer). Samples from the aqueous phase were redistributed until consistent distribution coefficient values were obtained, and the entire experiment was repeated three times.

Cellular Uptake and Inhibition Assays

The melanoma cells (B16F10, A375, and SK-MEL-28) were seeded in 24-well plates at a density of 2 × 10⁵ cells per well in 1 mL of fresh growth medium and incubated for 24 h at 37 °C under 5% CO₂. The medium was then removed, and the cells were washed twice with basic medium (500 μL per wash). The cells were divided into control and blocking groups (n = 3–6). To investigate the total uptake of [⁶⁸Ga]1–4 by cells over time, the cells were incubated with radioligand solutions (0.185 MBq in 400 μL of basic medium per well) for 0.5, 1, and 2 h. For blocking studies, the radioligands were coincubated with 20 μg of corresponding unlabeled DOTA-conjugated peptides per well for 1 h. All cells were maintained in 37 °C. After incubation, the cells were washed with cold PBS buffer (2 × 0.5 mL/well) and lysed with NaOH solution (1 M, 300 μL/well) for 10 min at room temperature. The lysates were collected, and the radioactivity was measured using an automatic γ-counter. Cellular uptake was expressed as the percentage of injected activity (%IA/10⁶ cells), calculated as the ratio of radioactivity in the cells to the total radioactivity added per well.

Tumor Model Establishment

To generate a subcutaneous melanoma model, B16F10 cells (1 × 10⁶ in 100 μL of PBS) were subcutaneously injected into the left armpit of female C57BL/6 mice (5–7 weeks old). In parallel, A375 cells (4 × 10⁶) and SK-MEL-28 cells (5 × 10⁶), each in 100 μL of PBS, were incubated into the left armpit of separate groups of female BALB/c mice (4–5 weeks old). Tumor dimensions were measured using a vernier caliper, and tumor volume was estimated using the formula: V = 0.5 × L × W², wherein L and W represent the length and width of the tumor, respectively.

MicroPET/CT Imaging

Animal studies were initiated when tumor volumes reached 200–400 mm³. Tumor-bearing mice (2–3 per group) were intravenously administered with [⁶⁸Ga]1–4 (∼3.7–5.5 MBq, 0.1–0.3 mL, formulated in 8% ethanol saline) via the tail vein. For blocking studies, a separate group of mice received a coinjection of the radiotracer along with the corresponding unlabeled DOTA-conjugated peptide, PR3-ABCF3-DOTA (0.25 mg/mouse) for [⁶⁸Ga]2 and PR3-C16-DOTA (0.33 mg/mouse) for [⁶⁸Ga]3. At 0.5, 1, and 2 h post-injection, the mice were anesthetized with isoflurane and underwent PET/CT scans. PET images were reconstructed by using a 3D ordered-subset expectation maximization (OSEM) algorithm. The maximum standardized uptake value (SUV_max) in tumor and muscle regions was quantified using PMOD software (PMOD Technologies, Switzerland).

Ex Vivo Biodistribution Studies

Melanoma-bearing mice (n = 3–4) were intravenously administered with a solution of each ⁶⁸Ga-labeled radioligand (∼2.2–3.0 MBq, 0.1 mL, formulated in 8% ethanol saline) via the tail vein. The mice were sacrificed at 1 or 2 h post-injection, and the tissues of interest were dissected and weighed. Radioactivity in each sample was measured by an automatic γ-counter. Uptake was expressed as the percentage of injected dose per gram of tissue (% ID/g) or per organ (% ID/organ), calculated as the ratio of the tissue radioactivity counts to the total injected dose. Data are presented as the mean ± SD.

IHC and HE Staining

Paraffin-embedded tumor sections (B16F10, A375, and SK-MEL-28) were deparaffinized in xylene and rehydrated through a graded ethanol series. For IHC, antigen retrieval was performed in citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched with 3% H₂O₂, and nonspecific binding was blocked with 3% BSA. Sections were incubated with anti-ROR1 monoclonal antibody (Proteintech, Cat No. 66923–1-Ig, Proteintech) at 4 °C overnight, followed by a peroxidase-conjugated secondary antibody and visualization with 3,3′-diaminobenzidine (DAB). Nuclei were counterstained with hematoxylin. For HE staining, sections were stained with hematoxylin for 5 min, differentiated in 1% HCl, rinsed, and then stained with eosin for 5 min. After dehydration and clearing in xylene, the mounted sections were observed and photographed by using a Nikon Eclipse Ci upright microscope equipped with a Nikon Fi3 imaging system (Tokyo, Japan).

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.6c00570.

Synthetic routes and procedures of peptide-DOTA conjugates (PR3-DOTA, PR3-ABCF3-DOTA, PR3-C16-DOTA, and MCP-14-DOTA), purity of the peptide-DOTA conjugates with HPLC chromatograms, radiosynthetic routes of [⁶⁸Ga]1–4, HPLC profiles of radioligands, in vitro stability, ex vivo biodistribution data of [⁶⁸Ga]1–3 in tumor-bearing mice, ex vivo biodistribution data of [⁶⁸Ga]2 in ICR mice, tumor and muscle SUV_max from PET imaging, PET images of radioligands in PET imaging studies, time-blood radioactivity curve of [⁶⁸Ga]2, estimated absorbed radiation dosimetry for [⁶⁸Ga]2 in human, and MS spectra of the peptide-DOTA conjugates (PDF)
Molecular formula strings data (CSV)

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Author Information

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Corresponding Authors
- Yang Zhang - Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, P.R. China; Email: [email protected]
- Hai Qian - Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, P.R. China; Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical University, Nanjing 210009, P.R. China; https://orcid.org/0000-0002-3827-0992; Email: [email protected] [email protected]
- Dawei Jiang - Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430022, P.R. China; Hubei Key Laboratory of Molecular Imaging, Wuhan, Hubei 430022, P.R. China; Key Laboratory of Biological Targeted Therapy, The Ministry of Education, Wuhan, Hubei 430022, P.R. China; https://orcid.org/0000-0002-4072-0075; Email: [email protected]
- Hualong Fu - Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, P.R. China; https://orcid.org/0000-0002-7197-8087; Email: [email protected]
Authors
- Donglan Huang - Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, P.R. China
- Xingru Long - Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430022, P.R. China; Hubei Key Laboratory of Molecular Imaging, Wuhan, Hubei 430022, P.R. China; Key Laboratory of Biological Targeted Therapy, The Ministry of Education, Wuhan, Hubei 430022, P.R. China
- Li Zhong - Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, P.R. China
- Yajing Wang - Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, P.R. China
- Xuan Di - Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, P.R. China
- Zihan Wang - Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, P.R. China
- Shuhan Zhou - Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, P.R. China
- Xiaoyu Du - Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, P.R. China
Author Contributions
D.H. and X.L. contributed equally.
Notes
The authors declare no competing financial interest.

Acknowledgments

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This research was supported by the National Natural Science Foundation of China (No. 22306014) and the Fundamental Research Funds for the Central Universities (No. 2233300007).

Abbreviations

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ADC	antibody-drug conjugate
BiTE	bispecific T-cell engager
BLI	biolayer interferometry
CAR-T	chimeric-antigen receptor-modified T
CLL	chronic lymphocytic leukemia
CRD	cysteine-rich domain
DAB	3,3′-diaminobenzidine
DOTA	1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
FDA	Food and Drug Administration
GEP-NET	gastroenteropancreatic neuroendocrine tumor
HE	hematoxylin and eosin
HPLC	high-performance liquid chromatography
H-score	histochemistry score
IHC	immunohistochemical
Kr	Kringle
LC-MS	liquid chromatography–mass spectrometry
mAb	monoclonal antibodies
MCL	mantle cell lymphoma
OSEM	ordered-subset expectation maximization
PBS	phosphate-buffered saline
PDC	peptide-drug conjugate
PEG	polyethylene glycol
PET	positron emission tomography
RCP	radiochemical purity
RCY	radiochemical yield
RDC	radionuclide drug conjugate
ROR1	receptor tyrosine kinase-like orphan receptor 1
RTK	receptor tyrosine kinase
SUV_max	maximum standardized uptake value
TBR	tumor-to-background ratio
T/M	tumor-to-muscle
TNBC	triple-negative breast cancer

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Abstract
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Figure 1
Figure 1. Ligand design and primary evaluations. (A) Schematic diagram and chemical structure of [⁶⁸Ga]1. (B) MicroPET/CT images (left panel) and biodistribution results (right panel) in B16F10 tumor-bearing mice (n = 2–4) after intravenous injection of [⁶⁸Ga]1. BL, bladder; K, kidneys; T, tumor.
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Scheme 1
Scheme 1. Chemical Structures of [⁶⁸Ga]2–4
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Figure 2
Figure 2. BLI binding curves of the interaction of newly developed ligands with the ROR1 protein. A–D correspond to PR3-DOTA, PR3-ABCF3-DOTA, PR3-C16-DOTA, and MCP-14-DOTA, respectively.
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Figure 3
Figure 3. Cell uptake assay (A) and self-blocking assay (B) of [⁶⁸Ga]1–4 in melanoma cell lines B16F10, A375, and SK-MEL-28; n.d., not determined. Asterisks indicate statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001 vs control. Data were analyzed using a two-tailed unpaired Student’s t-test.
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Figure 4
Figure 4. (A, C) MicroPET/CT images of B16F10 tumor-bearing mice following the injection of [⁶⁸Ga]2 (A) and [⁶⁸Ga]3 (C), respectively. Self-blocking studies were performed by coinjecting the corresponding nonradioactive competitors, PR3-ABCF3-DOTA (0.25 mg/mouse) for [⁶⁸Ga]2 and PR3-C16-DOTA (0.33 mg/mouse) for [⁶⁸Ga]3. (B,D) The radioactivity uptake values of [⁶⁸Ga]2 (B) and [⁶⁸Ga]3 (D) are expressed as SUV_max in tumor and muscle. BL, bladder (red arrow); H, heart (black arrow); T, tumor (white arrow).
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Figure 5
Figure 5. Ex vivo biodistribution of [⁶⁸Ga]2 (A) and [⁶⁸Ga]3 (B) in B16F10 tumor-bearing mice at 1 and 2 h postinjection. Data are expressed as the percentage of injected dose per gram of tissue (% ID/g), with the exception of the stomach and small intestine, for which uptake is presented as %ID per organ (% ID/organ).
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Figure 6
Figure 6. MicroPET/CT imaging and ex vivo biodistribution studies in A375 tumor-bearing mice. (A, B) MicroPET/CT images of A375 tumor-bearing mice following the injection of [⁶⁸Ga]1–4. Representative images acquired at selected time points postinjection. [⁶⁸Ga]1 (A) and [⁶⁸Ga]2 (B), which achieved high T/M ratios, were shown in three time points of 0.5, 1, and 2 h, while [⁶⁸Ga]3 and [⁶⁸Ga]4 (B) that had lower T/M ratios were displayed at the 2 h time point only. (C) Quantitative analysis of tumor uptake for [⁶⁸Ga]1–4 expressed as SUV_max. (D) Ex vivo biodistribution of [⁶⁸Ga]2 in A375 tumor-bearing mice at 1 and 2 h postinjection. Data are expressed as % ID/g, with the exception of the stomach and small intestine, for which uptake is presented as % ID/organ. BL, bladder (red arrow); H, heart (purple arrow); L, liver (yellow arrow); and T, tumor (white arrow).
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Figure 7
Figure 7. MicroPET/CT imaging and ex vivo biodistribution studies in SK-MEL-28 tumor-bearing mice. (A,B) MicroPET/CT images of SK-MEL-28 tumor-bearing mice following the injection of [⁶⁸Ga]1–3. Representative images acquired at selected time points postinjection. [⁶⁸Ga]1 (A) and [⁶⁸Ga]2 (B), which achieved high T/M ratios, were shown in three time points of 0.5, 1, and 2 h, while [⁶⁸Ga]3 (B) that had a lower T/M ratio was displayed at the 2 h time point only. (C) Quantitative analysis of tumor uptake for [⁶⁸Ga]1–3 expressed as SUV_max. (D) Ex vivo biodistribution of [⁶⁸Ga]2 in SK-MEL-28 tumor-bearing mice at 1 and 2 h postinjection. Data are expressed as % ID/g, with the exception of the stomach and small intestine, for which uptake is presented as % ID/organ. BL, bladder (red arrow); H, heart (purple arrow); L, liver (yellow arrow); T, tumor (white arrow).
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Figure 8
Figure 8. Histopathological and immunohistochemical (IHC) characterization of B16F10, A375, and SK-MEL-28 melanoma tumors. Representative hematoxylin and eosin (HE)-stained sections (top panels) and corresponding IHC staining (middle panels) of paraffin-embedded tumor tissues. The lower panels show the quantitative analysis of protein expression levels based on histochemistry scores (H-scores). Data are expressed as mean ± SD (n = 10 fields per section). Scale bars: 150 μm.
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Supporting Information
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.6c00570.
- Synthetic routes and procedures of peptide-DOTA conjugates (PR3-DOTA, PR3-ABCF3-DOTA, PR3-C16-DOTA, and MCP-14-DOTA), purity of the peptide-DOTA conjugates with HPLC chromatograms, radiosynthetic routes of [⁶⁸Ga]1–4, HPLC profiles of radioligands, in vitro stability, ex vivo biodistribution data of [⁶⁸Ga]1–3 in tumor-bearing mice, ex vivo biodistribution data of [⁶⁸Ga]2 in ICR mice, tumor and muscle SUV_max from PET imaging, PET images of radioligands in PET imaging studies, time-blood radioactivity curve of [⁶⁸Ga]2, estimated absorbed radiation dosimetry for [⁶⁸Ga]2 in human, and MS spectra of the peptide-DOTA conjugates (PDF)
- Molecular formula strings data (CSV)
- jm6c00570_si_001.pdf (4.52 MB)
- jm6c00570_si_002.csv (1.6 kb)
Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Peptide-Based ROR1-Targeting PET Ligands for Melanoma Tumor Imaging: Design and Preclinical EvaluationClick to copy article linkArticle link copied!

Journal of Medicinal Chemistry

Abstract

Introduction

Figure 1

Scheme 1

Results and Discussion

Ligand Design

Binding Potency

Figure 2

Radiolabeling and In Vitro Stability

Cellular Uptake and Self-Blocking Assays

Figure 3

MicroPET/CT Imaging of [68Ga]1–4 in B16F10 Tumor-Bearing Mice

Figure 4

Ex Vivo Biodistribution Studies of [68Ga]2 and [68Ga]3 in B16F10 Tumor-Bearing Mice

Figure 5

MicroPET/CT Imaging of [68Ga]1–4 and Ex Vivo Biodistribution Studies of [68Ga]2 in A375 Tumor-Bearing Mice

Figure 6

MicroPET/CT Imaging of [68Ga]1–3 and Ex Vivo Biodistribution Studies of [68Ga]2 in SK-MEL-28 Tumor-Bearing Mice

Figure 7

Characterization of ROR1 Expression in Melanoma Models

Figure 8

Conclusion

Experimental Section

General Information

Chemistry and Radiochemistry

Binding Kinetics Assays

In Vitro Stability

Partition Coefficient

Cellular Uptake and Inhibition Assays

Tumor Model Establishment

MicroPET/CT Imaging

Ex Vivo Biodistribution Studies

IHC and HE Staining

Supporting Information

Terms & Conditions

Author Information

Acknowledgments

Abbreviations

References

Cited By

Journal of Medicinal Chemistry

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Abstract

Figure 1

Scheme 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

References

Supporting Information

Supporting Information

Terms & Conditions

Peptide-Based ROR1-Targeting PET Ligands for Melanoma Tumor Imaging: Design and Preclinical Evaluation
Click to copy article linkArticle link copied!

MicroPET/CT Imaging of [⁶⁸Ga]1–4 in B16F10 Tumor-Bearing Mice

Ex Vivo Biodistribution Studies of [⁶⁸Ga]2 and [⁶⁸Ga]3 in B16F10 Tumor-Bearing Mice

MicroPET/CT Imaging of [⁶⁸Ga]1–4 and Ex Vivo Biodistribution Studies of [⁶⁸Ga]2 in A375 Tumor-Bearing Mice

MicroPET/CT Imaging of [⁶⁸Ga]1–3 and Ex Vivo Biodistribution Studies of [⁶⁸Ga]2 in SK-MEL-28 Tumor-Bearing Mice