The transmembrane cell adhesion receptor α_Vβ₃ integrin (α_Vβ₃) has been identified as an important molecular target for cancer imaging and therapy. We have developed a tetrameric cyclic RGD (Arg-Gly-Asp) peptide-based radiotracer ⁶⁴Cu-cyclam-RAFT-c(-RGDfK-)₄, which successfully captured α_Vβ₃-positive tumors and angiogenesis by positron emission tomography. Here, we subsequently evaluated its therapeutic potential and side effects using an established α_Vβ₃-positive tumor mouse model. Mice with subcutaneous U87MG-glioblastoma xenografts received single administrations of 37 and 74 MBq of ⁶⁴Cu-cyclam-RAFT-c(-RGDfK-)₄ (37 MBq/nmole), peptide control or vehicle solution and underwent tumor growth evaluation. Side effects were assessed in tumor-bearing and tumor-free mice in terms of body weight, routine hematology, and hepatorenal functions. Biodistribution of ⁶⁴Cu-cyclam-RAFT-c(-RGDfK-)₄ with ascending peptide doses (0.25−10 nmole) and with the therapeutic dose of 2 nmole were determined at 3 h and at various time points (2 min−24 h) post-injection, respectively, based on which radiation-absorbed doses were estimated. The results revealed that ⁶⁴Cu-cyclam-RAFT-c(-RGDfK-)₄ dose-dependently slowed down the tumor growth. The mean tumor doses were 1.28 and 1.81 Gy from 37 and 74 MBq of ⁶⁴Cu-cyclam-RAFT-c(-RGDfK-)₄, respectively. Peptide dose study showed that the tumor uptake of ⁶⁴Cu-cyclam-RAFT-c(-RGDfK-)₄ dose-dependently decreased at doses {greater than or equal to}1 nmole, indicating a saturation of α_Vβ₃ with the administered therapeutic doses (1 and 2 nmole). Combined analysis of the data from tumor-bearing and tumor-free mice revealed no significant toxicity caused by 37−74 MBq of ⁶⁴Cu-cyclam-RAFT-c(-RGDfK-)₄. Our study demonstrates the therapeutic efficacy and safety of ⁶⁴Cu-cyclam-RAFT-c(-RGDfK-)₄ for α_Vβ₃-targeted radionuclide therapy. ⁶⁴Cu-cyclam-RAFT-c(-RGDfK-)₄ would be a promising theranostic drug for cancer imaging and therapy.

Peptide-based radiopharmaceuticals targeting integrin α5β1 show promise for precise tumor diagnosis and treatment. However, current peptide-based radioligands that target α5β1 demonstrate inadequate in vivo performance owing to limited tumor retention. The use of PEGylation to enhance the tumor retention of radiopharmaceuticals by prolonging blood circulation time poses a risk of increased blood toxicity. Therefore, a PEGylation strategy that boosts tumor retention while minimizing blood circulation time is urgently needed. Here, we developed a PEGylation-enabled peptide multidisplay platform (PEGibody) for PR_b, an α5β1 targeting peptide. PEGibody generation involved PEGylation and self-assembly. [⁶⁴Cu]QM-2303 PEGibodies displayed spherical nanoparticles ranging from 100 to 200 nm in diameter. Compared with non-PEGylated radioligands, [⁶⁴Cu]QM-2303 demonstrated enhanced tumor retention time due to increased binding affinity and stability. Importantly, the biodistribution analysis confirmed rapid clearance of [⁶⁴Cu]QM-2303 from the bloodstream. Administration of a single dose of [¹⁷⁷Lu]QM-2303 led to robust antitumor efficacy. Furthermore, [⁶⁴Cu]/[¹⁷⁷Lu]QM-2303 exhibited low hematological and organ toxicity in both healthy and tumor-bearing mice. Therefore, this study presents a PEGibody-based radiotheranostic approach that enhances tumor retention time and provides long-lasting antitumor effects without prolonging blood circulation lifetime. The PEGibody-based radiopharmaceutical [⁶⁴Cu]/[¹⁷⁷Lu]QM-2303 shows great potential for positron emission tomography imaging-guided targeted radionuclide therapy for α5β1-overexpressing tumors.

Purpose: Ovarian cancer peritoneal metastases (OCPMs) are a pathophysiologically heterogeneous group of tumors that are rarely curable. α_Vβ₃ Integrin (α_Vβ₃) is overexpressed on tumoral neovessels and frequently on ovarian cancer cells. Here, using two clinically relevant α_Vβ₃-positiveOCPM mouse models, we studied the theranostic potential of an α_Vβ₃-specific radiopeptide, ⁶⁴Cu-cyclam-RAFT-c(-RGDfK-)₄ (⁶⁴Cu-RaftRGD), and its intra- and intertumoral distribution in relation to the tumor microenvironment. Experimental Design:α_Vβ₃-Expressing peritoneal and subcutaneous models of ovarian carcinoma (IGR-OV1 and NIH:OVCAR-3) were established in nude mice. ⁶⁴Cu-RaftRGD was administered either intravenously or intraperitoneally. We performed intratumoral distribution (ITD) studies, PET/CT imaging and quantification, biodistribution assay and radiation dosimetry, and therapeutic efficacy and toxicity studies. Results: Intraperitoneal administration was an efficient route for targeting ⁶⁴Cu-RaftRGD to OCPMs with excellent tumor penetration. Using the fluorescent surrogate Cy5.5-RaftRGD in our unique high-resolution multifluorescence analysis, we found that the ITD of ⁶⁴Cu-RaftRGD was spatially distinct from but complementary to that of hypoxia. ⁶⁴Cu-RaftRGD-based PET enabled clear visualization of multiple OCPM deposits and ascites; biodistribution analysis demonstrated an inverse correlation between tumor uptake and tumor size (1.2-17.2 mm). ⁶⁴Cu-RaftRGD at a radiotherapeutic dose (148 MBq/0.357 nmol) showed antitumor activities by inhibiting tumor cell proliferation and inducing apoptosis, and negligible toxicity. Conclusions: Collectively, these results demonstrate the all-in-one potential of ⁶⁴Cu-RaftRGD for imaging-guided radiotherapy of OCPM by targeting both tumoral neovessels and cancerous cells. Based on the ITD finding, we propose that pairing α_Vβ₃- and hypoxia-targeted radiotherapies could improve therapeutic efficacy by overcoming the heterogeneity of ITD encountered with single-agent treatments.

The present study proposed a new concept for targeted radionuclide therapy (TRT) to improve the intratumoral distribution of radioactivity using two different radiopharmaceuticals. We examined the efficacy of a combination of a tetrameric cyclic Arg-Gly-Asp (cRGD) peptide-based radiopharmaceutical, 64Cu-cyclam-RAFT-c(-RGDfK-)4 (64Cu-RaftRGD, an αVβ3 integrin [αVβ3] tracer), and 64Cu-diacetyl-bis (N4-methylthiosemicarbazone) (64Cu-ATSM, a supposed tracer for hypoxic metabolism) in a small animal tumor model. Mice with subcutaneous αVβ3-positive U87MG glioblastoma xenografts were used. The intratumoral distribution of a near-infrared dye, Cy5.5-labeled RAFT-c(-RGDfK-)4 (Cy5.5-RaftRGD), 64Cu-RaftRGD, and 64Cu-ATSM was visualized by fluorescence imaging and autoradiography of the co-injected Cy5.5-RaftRGD with 64Cu-RaftRGD or 64Cu-ATSM at 3 h postinjection. Mice were treated with a single intravenous dose of the vehicle solution (control), 18.5 or 37 MBq of 64Cu-RaftRGD or 64Cu-ATSM, or a combination (18.5 MBq of each agent). The tumor volume, tumor cell proliferation, body weight, survival, and tumor and organ uptake of radiopharmaceuticals were assessed. It was shown that Cy5.5-RaftRGD colocalized with 64Cu-RaftRGD and could be used as a surrogate for the radioactive agent. The intratumoral distribution of Cy5.5-RaftRGD and 64Cu-ATSM was discordant and nearly complementary, indicating a more uniform distribution of radioactivity achievable with the combined use of 64Cu-RaftRGD and 64Cu-ATSM. Neither 64Cu-RaftRGD nor 64Cu-ATSM showed significant effects on tumor growth at 18.5 MBq. The combination of both (18.5 MBq each) showed sustained inhibitory effects against tumor growth and tumor cell proliferation and prolonged the survival of the mice, compared to that by either single agent at 37 MBq. Interestingly, the uptake of the combination by the tumor was higher than that of 64Cu-RaftRGD alone, but lower than that of 64Cu-ATSM alone. The kidneys showed the highest uptake of 64Cu-RaftRGD, whereas the liver exhibited the highest uptake of 64Cu-ATSM. No obvious adverse effects were observed in all treated mice. The combination of 64Cu-RaftRGD and 64Cu-ATSM achieved an improved antitumor effect owing to the more uniform intratumoral distribution of radioactivity. Thus, combining different radiopharmaceuticals to improve the intratumoral distribution would be a promising concept for more effective and safer TRT.

Radiometal-labeled peptide-based radiopharmaceuticals (RLPB-radiopharmaceuticals) are promising for cancer imaging and targeted radiotherapy; however, their effectiveness is often compromised by the high retention of nonspecific radioactivity in the kidneys due to renal excretion pathways. Current strategies to address this issue have limitations, highlighting the need for innovative approaches to improve targeting specificity and therapeutic efficacy. We aimed to evaluate the applicability of the Gly-Phe-Lys (GFK) tripeptide, a renal brush border (RBB) enzyme-cleavable linkage, to reduce renal radioactivity in RLPB-radiopharmaceuticals using the integrin-targeting radiopeptide [⁶⁴Cu]Cu-cyclam-RAFT-c(-RGDfK-)₄ ([⁶⁴Cu]Cu-cyclam-RaftRGD). We designed and synthesized the model compound [⁶⁴Cu]Cu-cyclam-GFK(benzoyl [Bz]), its predictive metabolites, and GFK-incorporated [⁶⁴Cu]Cu-cyclam-RaftRGD derivatives [⁶⁴Cu]Cu-cyclam-GFK-RaftRGD and [⁶⁴Cu]Cu-cyclam-GFK(beta-alanine [βA])₃-RaftRGD. In vitro studies showed that dual radiometabolites, namely, [⁶⁴Cu]Cu-cyclam-G and [⁶⁴Cu]Cu-cyclam-GF, were simultaneously released from [⁶⁴Cu]Cu-cyclam-GFK(Bz) by different RBB enzymes, whereas both RaftRGD derivatives released only [⁶⁴Cu]Cu-cyclam-GF. When injected into mice, [⁶⁴Cu]Cu-cyclam-GFK(Bz) and the two RaftRGD derivatives led to the urinary excretion of [⁶⁴Cu]Cu-cyclam-G and [⁶⁴Cu]Cu-cyclam-GF, respectively. PET imaging and biodistribution studies showed the increased rates of reduction in renal radioactivity levels for the two RaftRGD derivatives compared to the parental [⁶⁴Cu]Cu-cyclam-RaftRGD (e.g., PET: 1 to 24 h postinjection, 73.0 ± 2.3 and 75.6 ± 1.8 vs 43.0 ± 4.5%, p < 0.0001; biodistribution: 3 to 24 h, 61.1 and 74.4 vs 22.8%). Taken together, these results indicate that the designed renal cleavage occurred in vivo. We also noted the steric interference of the RaftRGD moiety on enzyme access, the spacer effect of the trimeric βA sequence (reduced steric hindrance), and the altered radiopharmacokinetics (e.g., initially increased renal accumulation) of the RaftRGD compounds upon linker incorporation. These findings provide important insights into the chemical design of RLPB-radiopharmaceuticals with reduced renal retention based on the RBB strategy.

PH (low) insertion peptides (pHLIPs) have been developed for cancer imaging and therapy targeting the acidic extracellular microenvironment. However, the characteristics of intratumoral distribution (ITD) of pHLIPs are not yet fully understood. This study aimed to reveal the details of the ITD of pHLIPs and their spatial relationship with other tumor features of concern. The fluorescent dye-labeled pHLIPs were intravenously administered to subcutaneous xenograft mouse models of U87MG and IGR-OV1 expressing α_Vβ₃ integrins (using large necrotic tumors). The α_Vβ₃ integrin-targeting Cy5.5-RAFT-c(-RGDfK-)₄ was used as a reference. In vivo and ex vivo fluorescence imaging, whole-tumor section imaging, fluorescence microscopy, and multiplexed fluorescence colocalization analysis were performed. The ITD of fluorescent dye-labeled pHLIPs was heterogeneous, having a high degree of colocalization with necrosis. A direct one-to-one comparison of highly magnified images revealed the cellular localization of pHLIP in pyknotic, karyorrhexis, and karyolytic necrotic cells. pHLIP and hypoxia were spatially contiguous but not overlapping cellularly. The hypoxic region was found between the ITDs of pHLIP and the cRGD peptide and the Ki-67 proliferative activity remained detectable in the pHLIP-accumulated regions. The results provide a better understanding of the characteristics of ITD of pHLIPs, leading to new insights into the theranostic applications of pHLIPs.