Construction and Evaluation of Guanylyl Cyclase C–Specific Antibody for Noninvasive Diagnosis and Targeted Therapy of Colorectal Cancer
Zhuona Rong, Hongjin Liu, Xia Teng, Lin Chen, Yanlun Gu, Bingqi Dong, Xiaojiang Duan, Xin Wang, Xiaocong Pang
TL;DR
This study develops a targeted antibody for colorectal cancer that can be used for diagnosis and therapy, showing strong tumor selectivity and effective imaging and treatment outcomes.
Contribution
The development of a GUCY2C-specific monoclonal antibody probe with applications in PET, NIR imaging, and targeted radiotherapy for colorectal cancer.
Findings
PR15-7 antibody showed strong GUCY2C binding affinity and tumor selectivity.
NIR-II probe ICG-PR15-7 enabled precise intraoperative tumor visualization and complete resection.
Therapeutic administration of [177Lu]Lu-DOTA-PR15-7 significantly inhibited tumor growth.
Abstract
Colorectal cancer (CRC) remains the leading cause of cancer mortality worldwide despite therapeutic advances. Guanylyl cyclase C (GUCY2C), an intestinal epithelial receptor, is emerging as a promising diagnostic and therapeutic target for CRC. Thus, we are interested in developing a monoclonal antibody probe targeting GUCY2C for both in vitro and in vivo diagnosis and treatment of CRC. Methods: The GUCY2C-specific monoclonal antibody, PR15-7, was generated by hybridoma fusion. We developed [89Zr]Zr-DFO-PR15-7 for PET imaging, Cy5-PR15-7 for near-infrared fluorescence I (NIR-I) detection, and ICG-PR15-7 for NIR-II–guided surgical navigation. The therapeutic potential was evaluated using [177Lu]Lu-DOTA-PR15-7 for targeted radiotherapy. Biologic properties and antitumor activity of PR15-7 probes were evaluated in vitro and in vivo. Results: PR15-7 showed strong GUCY2C binding affinity and…
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TopicsRadiopharmaceutical Chemistry and Applications · Cell Adhesion Molecules Research · Monoclonal and Polyclonal Antibodies Research
Colorectal cancer (CRC) is the third most prevalent and lethal malignancy worldwide (1), accounting for a substantial proportion of cancer cases and deaths, representing a significant public health burden (2). Despite significant advances in treatment strategies, including surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy, the prognosis for patients with CRC, especially those with advanced or metastatic disease, remains dismal (3). Although surgical resection offers a potential cure for early-stage CRC, the risk of postoperative recurrence and metastasis remains a significant challenge (4). Current postoperative surveillance and management strategies, such as regular imaging and biopsy, have limitations in detecting early recurrence and metastasis, often leading to delays in intervention and suboptimal patient outcomes (5). Therefore, the development of targeted diagnostic and therapeutic approaches is imperative to improve early detection, treatment efficacy, and ultimately patient prognosis.
Guanylyl cyclase C (GUCY2C), a type I transmembrane receptor, has emerged as a promising tumor antigen for targeted therapy because of its restricted expression in normal intestinal epithelia and consistent overexpression in gastrointestinal malignancies, particularly CRC (6–8). Furthermore, the disruption of the tight junctions results in the distribution of GUCY2C across the apical–basolateral membranes of tumor cells (9). This unique expression pattern makes GUCY2C an ideal target for therapeutics. Previous research has introduced PF-07062119, an anti-GUCY2C/anti-CD3ε bispecific antibody, which demonstrated robust antitumor efficacy with manageable toxicity in CRC xenograft tumor models (10). Additionally, CAR-T cells targeting GUCY2C showed potential therapeutic benefit without inducing collateral autoimmunity in patients with metastatic CRC (11–13). These studies demonstrated the feasibility of developing GUCY2C-targeted therapies. However, rapid and effective identification of patients with GUCY2C-positive CRC is crucial to maximize the benefits for this patient population.
Leveraging the high specificity and affinity of monoclonal antibodies (mAbs) and the sensitivity of PET (14–17), we developed an integrated antibody-based PET imaging strategy to noninvasively visualize in vivo expression and distribution of targeted antigen. In this study, we introduced an antibody-based PET approach using ^89^Zr- and Cy5-labeled GUCY2C mAb (PR15-7), enabling simultaneous PET and fluorescence imaging of lesions and assessment of GUCY2C expression. Intraoperative imaging systems, capable of generating real-time and high-sensitivity images, may reduce postoperative recurrence and metastasis (18,19). Building on our previous work with the claudin18.2 mAb labeled with a near-infrared-II (NIR-II) fluorescence (FD1080) for gastric cancer imaging (20), we employed NIR-II technology to achieve deeper tissue penetration and superior contrast relative to that of NIR-I, because of reduced scattering and autofluorescence (21–23). Although indocyanine green (ICG) is clinically approved for tumor margin assessment (24), its lack of targeting specificity limits its utility in CRC. To overcome this, we demonstrated that ICG-PR15-7 enables sensitive identification and resection of tumors in subcutaneous and orthotopic mouse models, improving surgical navigation. Furthermore, to advance clinical translation, we explored radioimmunotherapy and SPECT imaging using ^177^Lu-labeled PR15-7, seamlessly integrating diagnosis and therapy. Together, we present a biocompatible GUCY2C-targeting mAb imaging platform with strong potential for early diagnosis and effective treatment of CRC.
MATERIALS AND METHODS
Details of cell lines, animal models (25), mAb generation, RNA isolation, and real-time quantitative polymerase chain reaction analysis, conjugation and radiolabeling, in vitro saturation binding assay, cellular uptake study, cellular immunofluorescence, flow cytometry and enzyme-linked immunosorbent assay ionization (ELISA), immunoprecipitation and Western blot, fluorescence imaging and surgery in xenograft models, ex vivo incubation and imaging of fresh animal-derived CRC tissues, PET imaging and biodistribution of [^89^Zr]Zr-DFO-PR15-7, biologic safety evaluation of [^177^Lu]Lu-DOTA-15A7, histologic staining, and statistical analysis are provided in the supplemental materials (available at http://jnm.snmjournals.org).
RESULTS
Expression of GUCY2C in Human CRC Cells
To investigate and confirm the viability of GUCY2C as a potential target for CRC, we examined its expression among CRC cell lines (HCT116, LoVo, CACO2, SW1463, and T84). Real-time quantitative polymerase chain reaction and Western blot analysis confirmed messenger RNA and protein expression in CACO2, SW1463, and T84, consistent with Cancer Cell Line Encyclopedia dataset patterns, whereas HCT116 and LoVo showed no expression. This finding was further corroborated by immunofluorescence and flow cytometry results showing high expression of GUCY2C in CRC cell lines with cell membrane localization (Fig. 1). The results confirmed that the expression profile of GUCY2C was well suited for the development of antibody-based therapy.
*Analysis of GUCY2C expression in human CRC cell lines. Messenger RNA (mRNA) levels were assessed using real-time quantitative polymerase chain reaction and Cancer Cell Line Encyclopedia datasets, and protein levels were evaluated via immunoblotting with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as control. Immunofluorescence showed GUCY2C (green) localization in cell lines, with nuclei stained blue (Hoechst); scale bar = 50 μm. Flow cytometry provided histogram of GUCY2C, indicating mean fluorescence intensity (MFI). *P < 0.05; ***P < 0.001; ***P < 0.0001.
Screening of mAbs Specific for GUCY2C
We established an efficient method for generating anti-GUCY2C antibodies (Supplemental Fig. 1). The extracellular domain of GUCY2C was fused to His-tag via a glycine-rich linker, cloned into a pcDNA3.1(+) vector, expressed in 293T cells, with peak protein yield observed on day 5. The recombinant GUCY2C–His protein was purified by nickel affinity chromatography and imidazole elution, showing an apparent molecular weight of 55–85 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) due to glycosylation modifications, despite a theoretic mass of 47.9 kDa. BALB/c mice were immunized with recombinant GUCY2C–His chimeric proteins using a water-soluble adjuvant. After 3 immunizations, serum titers reached 1:32,000, indicating a strong immune response suitable for hybridoma fusion. To enable screening for GUCY2C-specific clones, we constructed HCT116^GUCY2C^-overexpressing cells based on GUCY2C-negative HCT116 as reported previously (12).
After subcloning, we obtained 8 mAbs against GUCY2C (Supplemental Figs. 2 and 3), named PR1-5, PR2-8, PR3-12, PR7-10, PR7-11, PR9-1, PR12-2, and PR15-7, all identified as IgG1 with high purity by ELSIA and SDS-PAGE. Immunologic characterization confirmed specific binding to the extracellular domain of GUCY2C, with half-maximal effective concentration values in the nanomolar to subnanomolar range. Based on its unique CDR profile, PR15-7 was chosen for further development.
Cellular Uptake and Biodistribution of [89Zr]Zr-DFO-PR15-7 In Vitro
[^89^Zr]Zr-DFO-PR15-7 was synthesized with high radiochemical purity and specific activity (>95%, 111–124 MBq/mg) (Supplemental Figs. 4A–4C). In vitro binding assays confirmed highly specific targeting of GUCY2C, with HCT116^GUCY2C^ cells demonstrating markedly strong binding (76.2 ± 7.2% of the total radioactivity) of [^89^Zr]Zr-DFO-PR15-7, which was significantly greater than the minimal binding observed in wild-type HCT116 cells (8.5% ± 1.2%, P < 0.0001). Binding was competitively inhibited by unlabeled PR15-7 (32.5% ± 4.5%, P < 0.0001) (Fig. 2A), confirming specificity. The control probe [^89^Zr]Zr-DFO-IgG showed no specific binding. Saturation binding assays on HCT116^GUCY2C^ cells quantified the binding of [^89^Zr]Zr-DFO-PR15-7 to GUCY2C. The assay revealed saturable and specific binding with a dissociation constant of 2.0 ± 0.5 nM and a maximum binding capacity of 1,653 fmol/mg, corresponding to approximately 1.25 × 10^5^ receptors per cell (Supplemental Fig. 4D).
*[89Zr]Zr-DFO-PR15-7 antibody–based PET/CT in overexpressed GUCY2C xenograft models by competitive blocking. (A) Cellular uptake of [89Zr]Zr-DFO-PR15-7 on HCT116 and HCT116GUCY2C cell lines. (B) Representative and quantitative analysis of PET/CT images in overexpressed GUCY2C xenograft models of 4 different groups from 4 to 168 h: experimental group, HCT116GUCY2C with [89Zr]Zr-DFO-PR15-7; isotype group, HCT116GUCY2C with [89Zr]Zr-DFO-IgG; blocking group, HCT116GUCY2C with [89Zr]Zr-DFO-IgG and unlabeled PR15-7; negative group, HCT116 with [89Zr]Zr-DFO-PR15-7. (C) Hematoxylin and eosin (H&E) staining and GUCY2C immunohistochemistry (IHC) images of HCT116/HCT116GUCY2C tumor tissues. Scale bars = 100 and 500 μm. ***P < 0.0001.
[89Zr]Zr-DFO-PR15-7–Based PET/CT in a GUCY2C-Overexpressing Xenograft Model
Real-time and noninvasive assessment of the in vivo distribution and metabolic properties of both [^89^Zr]Zr-DFO-PR15-7 (experimental group) and a nontargeting control, [^89^Zr]Zr-DFO-IgG (isotype group), was performed using small-animal PET/CT imaging from 4 to 168 h after injection. To reinforce the specific targeting of GUCY2C by [^89^Zr]Zr-DFO-PR15-7, we also performed competition studies with unlabeled PR15-7 (blocking group) and HCT116 tumors with [^89^Zr]Zr-DFO-PR15-7 (negative group). As shown in Figure 2B, maximum-intensity projection images revealed that although [^89^Zr]Zr-DFO-PR15-7 accumulated in the heart and blood pool at an early time point (4 h), which was physiologically expected, PET imaging clearly detected HCT116^GUCY2C^ tumors at 24 h. The uptake pattern in the other 3 control groups was similar to that of the experimental group, except for reduced tumor uptake due to blocking by additional PR15-7, the use of nontargeting [^89^Zr]Zr-DFO-IgG probes, or the lack of GUCY2C expression in HCT116 cells. [^89^Zr]Zr-DFO-PR15-7 showed specific and time-dependent accumulation in GUCY2C-positive tumors, reaching a peak SUV_max_ of 2.36 ± 0.14 at 72 h. In contrast, significantly lower uptake was observed in the isotype group (0.72 ± 0.05, P < 0.001) and the negative control group (0.91 ± 0.08, P < 0.001). Preblocking with unlabeled PR15-7 notably reduced tumor uptake in the blocking group (1.00 ± 0.08, P < 0.001), confirming the specificity of [^89^Zr]Zr-DFO-PR15-7 for GUCY2C (Fig. 2B). To evaluate the potential risks posed by full-length antibody-radiolabeled tracers to central parenchymal organs, we quantified PET data to analyze the dynamic changes of tracer accumulation in major organs at different time points after injection (Supplemental Fig. 5A). Meanwhile, radioactivity in major organs (heart, liver, spleen, lungs, kidneys) decreased over time across all groups, indicating normal clearance and minimal nonspecific retention.
Biodistribution results were consistent with PET imaging, demonstrating progressively increasing tumor-to-blood and tumor-to-muscle ratios from 4 to 168 h, confirming specific targeting of HCT116^GUCY2C^ tumors by [^89^Zr]Zr-DFO-PR15-7 (Supplemental Figs. 5B and 5C). Tumor accumulation in the experimental group (16.52 ± 1.12) was significantly higher than that in the other 3 control groups (7.45 ± 0.22 for the blocking control, 3.81 ± 0.47 for the isotype control, and 4.92 ± 0.25 for the negative control; P < 0.0001). No significant differences in uptake were observed in normal tissues, including the lungs, liver, stomach, intestine, bladder, muscle, heart, blood, or brain (Supplemental Fig. 5D). In terms of both the tumor-to-blood and tumor-to-muscle ratios, [^89^Zr]Zr-DFO-PR15-7 showed a trend toward greater tumor accumulation in the GUCY2C-positive xenograft model when compared with the other 3 control groups (Supplemental Fig. 5E). The high and persistent tumor uptake supports the potential of [^177^Lu]Lu-DOTA-PR15-7 for radioimmunotherapy.
Hematoxylin and eosin and immunohistochemical staining of paraffin-embedded tumor sections (Fig. 2C) revealed high anaplastic cells, accompanied by necrosis-related features in both HCT116 and HCT116^GUCY2C^ xenografts. Immunohistochemistry confirmed strong GUCY2C expression in the HCT116^GUCY2C^ tumors, whereas no positive signals were detected in the HCT116 tumors, supporting the use of these 2 models for evaluating the in vivo targeting specificity of PR15-7–based tracers.
[89Zr]Zr-DFO-PR15-7–Based PET/CT in Endogenous GUCY2C Xenograft Model
In T84 tumor models with endogenous GUCY2C expression, [^89^Zr]Zr-DFO-PR15-7 exhibited higher and sustained tumor uptake compared with the blocking group (Fig. 3A). The SUV_max_ increased from 0.57 ± 0.09 at 4 h to 1.65 ± 0.19 at 168 h. Uptake in the blocking group remained low (from 0.38 ± 0.06 to 0.67 ± 0.14). Notably, there were statistically significant differences in tumor uptake between the 2 groups at every time point, ranging from 4 to 168 h after injection. Uptake in spleen, kidney, lungs, and heart decreased over time, with no significant liver uptake difference between groups (Supplemental Fig. 5F). In vitro, [^89^Zr]Zr-DFO-PR15-7 showed specific binding to T84 cells (60.6 ± 2.7%), which was reduced by unlabeled PR15-7 (44.3 ± 2.3%) (Fig. 3A), confirming target specificity in endogenously expressing models. Hematoxylin and eosin and immunohistochemical staining of the T84 xenograft tissues revealed distinct membrane and cytoplasmic localization of GUCY2C, further confirming that [^89^Zr]Zr-DFO-PR15-7 was a highly promising imaging technique for delineating GUCY2C-positive tumors (Fig. 3B).
*[89Zr]Zr-DFO-PR15-7 antibody–based PET/CT in endogenous GUCY2C xenograft models by competitive blocking. (A) Representative and quantitative analysis of PET/CT images from 4 to 168 h after injection in T84 xenograft models and cellular uptake of [89Zr]Zr-DFO-PR15-7 on T84 cell line. (B) Hematoxylin and eosin (H&E) and GUCY2C immunohistochemistry (IHC) images of T84 tumor tissues. Scale bars = 100 and 500 μm. ***P < 0.001; ***P < 0.0001.
Near-Infrared Fluorescence (NIRF) Imaging of Cy5-PR15-7
Motivated by the PET results, we developed a NIRF probe, Cy5-PR15-7, for real-time imaging (Supplemental Fig. 6A). As shown in Figure 4A, in vivo NIRF imaging showed significantly higher fluorescence intensity in GUCY2C-positive tumors compared with blocking, isotype, and negative control groups (15.15 ± 1.18 vs. 6.76 ± 0.89, 3.38 ± 1.49, and 6.62 ± 1.75 arbitrary units, respectively, at 168 h). Ex vivo fluorescence imaging further confirmed that GUCY2C-positive tumors had a higher uptake of Cy5-PR15-7 compared with other groups, mirroring the in vivo findings. HCT116^GUCY2C^ tumors displayed significantly greater Cy5-PR15-7 uptake in biodistribution studies, whereas Cy5-IgG showed weaker fluorescence signals (Supplemental Figs. 6B and 6C). Flow cytometry and immunofluorescence in vitro further validated specific binding to HCT116^GUCY2C^ cells, which was competitively inhibited by unlabeled PR15-7, with clear membrane localization and minimal background signal in controls (Figs. 4B and 4C).
*NIRF imaging of Cy5-PR15-7 in overexpressed GUCY2C xenograft model by competitive blocking. (A) Representative and quantitative analysis of NIRF images observed from 4 to 168 h after injection in overexpressed GUCY2C xenograft model of 4 different groups. (B and C) Competitive blocking assay of Cy5-PR15-7 evaluated by flow cytometry (B) and immunofluorescence (C) in HCT116 and HCT116GUCY2C cell lines. Cy5-PR15-7 (red), nuclei (Hoechst, blue). Scale bar = 50 μm. ***P < 0.0001.
Surgical Resection of GUCY2C-Positive Tumors Guided by ICG-PR15-7 NIR-II Imaging
To enable real-time intraoperative surgical navigation in CRC, we synthesized an ICG-conjugated NIR-II probe, ICG-PR15-7, along with a nonspecific antibody control ICG-IgG. Conjugation success was confirmed by SDS-PAGE and NIRF detection (Supplemental Figs. 7A and 7B). Subcutaneous and orthotopic xenograft tumor models using HCT116^GUCY2C^ cells were established to evaluate the feasibility of ICG-PR15-7–guided tumor surgery. Tumor-bearing mice were injected intravenously with ICG-PR15-7, followed by tumor excision 72 h after injection, with adjacent tissues included as the surgical margin. The detailed operation videos are shown in the supplemental materials. Tumors as small as 50 mm^3^ are clearly visible during NIR-II fluorescence–guided surgery (Fig. 5A). After surgery, residual tumor tissue in surgical margins, with detection sensitivity down to 0.3 mm in fresh tissue and approximately 200 μm in histologic sections. Hematoxylin and eosin staining and NIR imaging of sections confirmed tumor-specific fluorescence and distinct histology. These results support ICG-PR15-7 as a highly sensitive tool for intraoperative identification and resection of CRC.
*NIR-II imaging-guided surgical excision of GUCY2C-positive tumors using ICG-PR15-7. (A) Representative NIR-II fluorescence images of subcutaneous (S) and orthotopic (O) HCT116GUCY2C tumor resections captured 72 h postintravenous administration of ICG-PR15-7. Hematoxylin and eosin (H&E) staining and fluorescence (Odyssey FL) images of GUCY2C-positive tumors after NIR-II imaging-guided surgery are presented. Scale bar = 2 mm. (B) Fluorescence imaging and quantitative analysis of HCT116 and HCT116GUCY2C tumor tissues incubated with ICG-PR15-7 probes in vitro. *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001.
ICG-PR15-7 NIR-II Imaging of Freshly Resected Tumor Tissue Ex Vivo
To facilitate clinical translational, we evaluated ICG-PR15-7 as a diagnostic probe in fresh surgical tissues across multiple exposure times (200–500 ms). Under optimized ex vivo staining conditions (50-μg/mL probe, 20-min incubation), ICG-PR15-7 consistently exhibited significantly higher positive-to-negative tumor ratios than the isotype control at all exposure times tested (Fig. 5B; Supplemental Fig. 7C), with the most pronounced difference observed at 300 ms (3.56 vs. 1.35, P < 0.0001). These results support the potential of ICG-PR15-7 for targeted intraoperative pathologic detection in CRC.
Radioimmunotherapy and SPECT/CT Imaging of [177Lu]Lu-DOTA-PR15-7
Capitalizing on the strong tumor uptake of PR15-7–based probes, we evaluated the therapeutic efficacy of [^177^Lu]Lu-DOTA-PR15-7 in the HCT116^GUCY2C^ tumor model (Fig. 6; Supplemental Fig. 8). A single injection of [^177^Lu]Lu-DOTA-PR15-7 produced pronounced and dose-dependent tumor suppression. The high-dose group showed the strongest suppression effect, with a tumor volume of 208 ± 55% at day 16, significantly lower than control groups (phosphate-buffered saline, 1,184% ± 379%; unlabeled PR15-7, 1,173% ± 318%; [^177^Lu]Lu^3+^ alone, 1,174% ± 168%; [^177^Lu]Lu-IgG, 993% ± 394%; P < 0.0001). The low-dose group also showed significant inhibition (348% ± 66%, P < 0.001), confirming the potential of [^177^Lu]Lu-DOTA-PR15-7 for target radiotherapy of GUCY2C-positive tumors. All mice showed mild weight loss, attributable to tumor burden in control groups (phosphate-buffered saline and PR15-7 only) and to radiation exposure in the ^177^Lu treatment groups. The reduction was slightly greater in treatment groups without being statistically significant.
*Radioimmunotherapy of [177Lu]Lu-DOTA-PR15-7. Representative photographs of tumor-bearing mice, standardized tumor volume, and body weight over 16 d after a single injection in HCT116GUCY2C mouse model. ***P < 0.001; ***P < 0.0001; ns = not significant. PBS = phosphate-buffered saline.
Hematologic analysis at day 15 revealed reductions in white blood cells, neutrophils, and lymphocytes in all high-dose [^177^Lu]Lu^3+^-treated groups, indicating transient myelosuppression (Fig. 7A). Long-term monitoring showed dose-dependent recovery of these parameters (Supplemental Fig. 9A), further supporting the reversible nature of these effects. Serum biochemistry markers (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, creatinine, urea, and lactate dehydrogenase) remained within reference ranges, suggesting no significant renal, hepatic, and cardiac toxicity (Fig. 7A). Histopathologic examination showed no necrosis or structural damage in major organs (Fig. 7B), though mild hemosiderin deposition and splenic fibrosis were observed. Overall, [^177^Lu]Lu-DOTA-PR15-7 demonstrated an acceptable safety profile in this model.
Safety assessment of [177Lu]Lu-DOTA-PR15-7. (A) Hematologic analysis and serum chemistry at 6 and 15 d after injection in [177Lu]Lu-DOTA-PR15-7 treated HCT116GUCY2C mouse model. (B) H&E staining of the major organs of [177Lu]Lu-DOTA-PR15-7 at 16 d after injection in HCT116GUCY2C mouse model, scale bar = 200 and 400 μm. ALP = alkaline phosphatase; ALT = alanine aminotransferase; AST = aspartate aminotransferase; CREA = creatinine; LDH = lactate dehydrogenase; PBS = phosphate-buffered saline.
SPECT imaging in HCT116^GUCY2C^ and HCT116 tumor models revealed tumor accumulation of [^177^Lu]Lu-DOTA-PR15-7 in GUCY2C-positive tumors that was significantly higher than that with control groups [^177^Lu]Lu-DOTA-IgG, blocking group, and GUCY2C-negative tumors (Supplemental Fig. 9B). Minimal tracer retention was observed in HCT116 tumors. Pharmacokinetic analysis demonstrated favorable in vivo stability with a prolonged half-life of 44.4 ± 1.3 h (Supplemental Fig. 9C). Biodistribution results confirmed the imaging findings (Supplemental Figs. 9D–9G). Dosimetry estimates derived from biodistribution studies in tumor-bearing mice (Supplemental Table 1) informed the selection of [^177^Lu]Lu-DOTA-PR15-7 doses that maximize tumor response while maintaining radiation exposure to healthy tissues within tolerable limits. Human dosimetry estimates indicated the liver received the highest absorbed dose (2.06 mSv/MBq, 70 kg; Supplemental Table 2), consistent with anticipated biodistribution patterns.
DISCUSSION
CRC remains a leading cause of cancer-related mortality globally, necessitating improved therapeutic strategies. GUCY2C has emerged as a promising target because of its frequent expression in metastatic CRC tissues (83% of samples) (12) and its role as an “orphan receptor” after ligand loss (26–30). Current clinical development of GUCY2C-targeting agents, including antibody-based therapies (31), antibody–drug conjugates (32), and CAR-T cell (12) across 12 registered clinical trials, underscores its therapeutic relevance.
Antibody-based PET combines the high targeting specificity of mAbs and the sensitivity of PET imaging, enabling visualization of tumor markers (33), immune cells (34), immune checkpoint molecules (35), and inflammatory sites (36). In this study, we developed [^89^Zr]Zr-DFO-PR15-7 for PET imaging and demonstrated specific visualization of GUCY2C-expressing tumors. Although the tracer showed significantly higher uptake in target-positive tumors compared with controls, we acknowledge the moderate absolute uptake values achieved. Similarly, our development of NIR probes (Cy5-PR15-7 and ICG-PR15-7) enabled real-time tumor delineation, though NIR imaging remains constrained by tissue attenuation. This limited uptake is likely due to the slow penetration and high nonspecific retention (e.g., enhanced permeability and retention effect) associated with the full-length IgG format. To overcome this limitation, we propose engineering smaller, high-affinity antibody formats (e.g., mini-antibodies) for improved tumor penetration and pharmacokinetics, coupled with predosing protocols to minimize Fc-mediated off-target uptake, thereby significantly enhancing target-specific accumulation and imaging contrast.
Radionuclide labeling, although valuable in diagnostic imaging because of its use of ionizing radiation (37,38), is unsuitable for surgical guidance. Capitalizing on the targeting success of [^89^Zr]Zr-DFO-PR15-7, we developed ICG-PR15-7, an NIR-II fluorescent conjugate that demonstrated clear tumor delineation in both subcutaneous and orthotopic models. This approach addresses the critical need for precise intraoperative guidance while overcoming limitations of NIR-I imaging, positioning fluorescence-guided surgery as a promising extension of GUCY2C-targeted theranostics.
The Food and Drug Administration has authorized the use of ^177^Lu for oncology treatments (39). We used [^177^Lu]Lu-DOTA-PR15-7 to investigate its therapeutic efficacy in the treatment of CRC. These findings showed that ^177^Lu-labeled PR15-7 had a significantly greater tumor inhibitory effect than [^177^Lu]Lu^3+^ alone. The observed toxicity profile of [^177^Lu]Lu-DOTA-PR15-7, including hematologic effects, reflects both on-target binding and off-target Fc-mediated uptake—a challenge inherent to radioimmunoconjugates. The modest tumor-to-blood ratios further highlight the impact of prolonged IgG circulation on the background signal. Dosimetry estimates derived from the biodistribution data further indicate radioactive accumulation in the liver and spleen. We have conducted preclinical studies on the diagnostic and therapeutic applications of the PR15-7 antibody, which would complete the screening and validation phase of antibody candidates. Although the murine origin of PR15-7 currently limits clinical application, our comprehensive preclinical evaluation supports advancing to humanized versions for future development.
From a translational perspective, although our T84 model confirms membrane localization of GUCY2C, the notable heterogeneity of its expression patterns in human CRC specimens highlights the necessity of careful patient stratification for future clinical developments. The use of both endogenous and GUCY2C-overexpressing mouse xenograft models enables controlled quantification, so these systems may not fully represent the clinical relevance of patient samples. To enhance the clinical translation, we plan to establish patient-derived organoid and xenograft models for validating humanized or engineered antibodies, thereby enhancing the clinical relevance of our GUCY2C-targeted theranostic platform.
CONCLUSION
This study developed a versatile GUCY2C-targeted theranostic platform by conjugating the specific antibody PR15-7 to ^89^Zr, Cy5, ICG, and ^177^Lu, enabling antibody-based PET, NIR-I and NIR-II imaging, and targeted radiopharmaceutical therapy. After optimization and clinical translation, this probe will be a promising tool for precise tumor localization, surgical navigation, and treatment of GUCY2C-positive cancers.
DISCLOSURE
This work was funded by the Beijing Municipal Natural Science Foundation (L248076, L234038, and JQ24059, China), National Natural Science Foundation of China (82372860 and 22106006), and National High Level Hospital Clinical Research Funding (24QZ011, 2024CX15, and 2025CX25, China). No other potential conflict of interest relevant to this article was reported.
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