Preclinical Evaluation of a Novel Carbonic Anhydrase IX-Targeting 68Ga/177Lu Theranostic Pair for Clear Cell Renal Cell Carcinoma

Preclinical Evaluation of a Novel Carbonic Anhydrase IX-Targeting 68Ga/177Lu Theranostic Pair for Clear Cell Renal Cell Carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Preclinical Evaluation of a Novel Carbonic Anhydrase IX-Targeting 68Ga/177Lu Theranostic Pair for Clear Cell Renal Cell Carcinoma Xinchun Yan, Meixi Liu, Guoyang Zheng, Xiaoyuan Li, Chao Ren, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6954806/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background Carbonic anhydrase IX (CAIX) is a hypoxia-regulated enzyme overexpressed in clear cell renal cell carcinoma (ccRCC) but minimally in normal tissues, making it a promising theranostic target. Current imaging modalities for ccRCC face limitations in accuracy and nephrotoxicity, while systemic therapies are constrained by resistance and variable efficacy. This study evaluates a novel CAIX-targeting theranostic pair, 68 Ga/ 177 Lu-NYM080, for imaging and therapy in preclinical ccRCC models. Results The in vivo and ex vivo biodistribution of 68 Ga/ 177 Lu-NYM080 was evaluated in CAIX-positive OS-RC-2 xenograft bearing models, and the therapeutic efficacy of 177 Lu -NYM080 (74 MBq single dose vs. 74 MBq×2 doses) was also investigated. NYM080 exhibited high CAIX affinity (Kd = 9.69 ± 0.46 nM). 68 Ga-NYM080 showed rapid tumor uptake (peak 10.05%ID/g at 30 min) and specificity (blocking reduced uptake by 55%). Tumor-to-muscle ratios reached 6.5 at 1h. 177 Lu-NYM080 demonstrated prolonged tumor retention (8.79%ID/g at 168 h) and a trend toward enhanced therapeutic efficacy with increased dosing: single dose of 74 MBq delayed tumor doubling to 13.0 days (vs. control, P = 0.064), while 74 MBq×2 doses extended it to 19.5 days (P = 0.0054 vs. control). Minimal off-target uptake and no acute toxicity were observed. Conclusion 68 Ga/ 177 Lu-NYM080 is a promising theranostic pair for ccRCC, combining high tumor specificity, prolonged tumor retention, and favorable therapeutic efficacy. carbonic anhydrase IX clear cell renal cell carcinoma theranostics 68Ga/177Lu radionuclide therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Carbonic anhydrase IX(CAIX) is a membrane-bound enzyme implicated in tumor acidosis, highly expressed in hypoxic tumors and minimally in normal tissues, with the exception of select regions in the gastrointestinal tract.[ 1 – 3 ] As one of the twelve catalytically active carbonic anhydrases, CAIX is regulated by hypoxia-inducible factor 1-alpha (HIF-1α) under hypoxic conditions[ 4 ]. In clear cell renal cell carcinoma (ccRCC), CAIX expression is further intensified due to dysregulation of the von Hippel-Lindau (VHL) tumor suppressor, resulting in sustained HIF-1α activity[ 5 , 6 ]. This overexpression is linked to tumor aggressiveness, metastasis, and poor clinical outcomes, which makes CAIX a valuable target for theranostic applications. Clear cell renal cell carcinoma is the most common subtype of renal cell carcinoma[ 7 ].While diagnosis and staging of ccRCC primarily rely on contrast-enhanced CT (ceCT), its accuracy in differentiating small renal masses remains limited[ 8 ], and its nephrotoxicity makes it unsuitable for patients with renal impairment. Although percutaneous biopsy is recommended before initiating ablative or systemic therapies[ 9 ], it is constrained by limited sampling sites and complications. In terms of therapy, For ccRCC patients presenting with distant metastases at diagnosis, the five-year survival rate is only 13%[ 10 ]. The treatment of advanced or metastatic ccRCC relies on tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs) according to the NCCN Guideline for Kidney Cancer[ 11 ]. However, approximately 30–60% patients have poor response to these therapies or develop secondary acquired resistance, which limits the overall efficacy[ 12 , 13 ]. Furthermore, many patients exhibit poor tolerance to TKIs or require dose reductions, underscoring the need for novel therapeutic approaches. There is an urgent need for new therapies for patients with advanced metastatic ccRCC. In our previous work, we reported 68 Ga-NY104 (also referred to as 68 Ga-NYM005), a small-molecule tracer targeting CAIX, which has recently shown encouraging clinical results in the diagnosis of ccRCC[ 14 , 15 ]. However, NYM005 was not suitable for lutetium labeling due to NOTA chelation. Therefore, we developed NYM080, a novel CAIX-targeted molecular based on acetazolamide core (Fig. S1 ). Compared to NY104, NYM080 links acetazolamide to chelator DOTA through an aliphatic linker and a short peptide to minimize steric interference with CAIX binding. These properties make NYM080 a potential candidate for optimized tumor imaging and therapeutic applications. This preclinical investigation aims to characterize the biodistribution, pharmacokinetics, and therapeutic efficacy of 68 Ga/ 177 LuNYM080 in CAIX-expressing clear cell renal cell carcinoma (ccRCC) models. Methods Detailed experimental procedures are provided in the Supplementary Methods. Radiosynthesis and Quality Control The CAIX-targeting molecule NYM080 precursor was radiolabeled with 68 Ga or 177 Lu based on standardized procedures, using a 68 Ge/ 68 Ga generator (Eckert & Ziegler, Germany) and 177 LuCl 3 (ITG GmbH, Germany), respectively. Radiochemical purity and stability were confirmed by radio-HPLC (Agilent 1260 Infinity II, USA) and Radio-iTLC (Bioscan AR-2000, USA). Binding Affinity and Specificity Binding affinity (Kd) of NYM080 for CAIX was quantified using a Biacore 8K SPR system (Cytiva, USA) with biotinylated CAIX (ACROBiosystems, China) immobilized on a Neutravidin-coated CM5 sensor chip. Cellular and Animal Models Human CAIX-positive OS-RC-2 renal carcinoma cells (ATCC, USA) and CAIX-negative PC3 prostate carcinoma cells (ATCC) were cultured in RPMI-1640 medium (Gibco, USA). Xenografts were established in NCG mice (Vital River, China) by subcutaneous injection of OS-RC-2 cells (1×10⁷ cells/mouse) mixed with Matrigel (Corning, USA). Blood pharmacokinetics of 68 Ga-NYM080 were evaluated in ICR mice following intravenous administration of 3.7 MBq. Blood samples were collected via the lateral tail vein at 5, 15, 30, 60, 90, 120, 180, and 240 min post-injection, and radioactivity was measured using a gamma counter (PerkinElmer 2470, USA). Pharmacokinetic parameters were calculated using DAS software (version 2.1.1, DAS, China). Imaging and Biodistribution PET/CT imaging was performed using a Super Nova® system (Pingseng Healthcare, China) at 1–4 h post-injection of 68 Ga-NYM080 (2.96 MBq). Blocking studies used unlabeled NYM005 (400 µg). Ex vivo biodistribution was analyzed with a gamma counter (PerkinElmer 2470, USA). Therapeutic Efficacy Tumor-bearing mice were allocated into three groups, with six mice per group. Group 1 received single injection of saline as control, Group 2 received a single intravenous injection of 74 MBq of 177 Lu-NYM080 and Group 3 received 148 MBq of 177 Lu-NYM080 in two doses of 74 MBq each, administered 4 days apart. Mice were weighed and tumor volumes measured on the day of the first injection, with subsequent measurements taken twice every week thereafter. Data Analysis Imaging quantification (tumor uptake, %ID/g; tumor-to-muscle ratios) was conducted using PMOD software (version 4.3, PMOD Technologies, Switzerland). All values are presented as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism (version 9.0, GraphPad Software, USA). Differences in tumor growth between treatment groups were assessed by one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. Normality and homogeneity of variances were confirmed using Shapiro-Wilk and Levene’s tests, respectively. A two-tailed P < 0.05 was considered statistically significant. Results Radiochemical purity (RCP) exceeded 96% for both ⁶⁸Ga-NYM080 and ¹⁷⁷Lu-NYM080 (Table S1 ). ⁶⁸Ga-NYM080 had a molar activity of 4.32 MBq/nmol for PET biodistribution, while molar activity of ¹⁷⁷Lu-NYM080 was 20.91 MBq/nmol for therapeutic administration. The molar activities of other experiments are shown in Table S1 . The RCP of ⁶⁸Ga-NYM080 remained > 94% for 4 hours in human serum (96.67% at 1 h, 96.11% at 2 h, 94.08% at 4 h; n = 3), while ¹⁷⁷Lu-NYM080 demonstrated > 91% stability over 72 hours (95.39% at 24 h, 92.13% at 48 h, 91.02% at 72 h; n = 3), with similar retention times (7.13–7.15 min for ⁶⁸Ga and 7.00-7.02 min for ¹⁷⁷Lu), detailed data in Table S2 and Fig. S2 . The binding affinity (kD) of NYM080 for CAIX was assessed in three independent experiments, yielding a mean kD of 9.69 ± 0.46 nM, with a coefficient of variation (CV) of 5.8% (Fig. S3 ). Biodistribution and Blood Pharmacokinetics of Ga-NYM080 in Xenograft Mouse Models The in vivo biodistribution profile of 68 Ga-NYM080 showed remarkable uptake in kidney, which decreased over time (Fig. 1 and Table 1 ). The ex vivo biodistribution study revealed uptake (≥ 6%ID/g) in the kidneys, stomach, intestine and lung (Fig. 2 ). Blood pharmacokinetics of 68 Ga-NYM080 were also evaluated, over 75% of the injected activity was rapidly cleared within the first 90 min (Fig. S4 ), and detailed pharmacokinetic data are presented in Supplementary Table S3 - 4 . The blood clearance curve for 68 Ga-NYM080 followed a biexponential pattern, with a half-life of 3.49 h. The maximum blood concentration of NYM080 was 3.12 µg/kg at 5 min after injection. Table 1 In vivo Biodistribution of ⁶⁸Ga-NYM080 in OS-RC-2 Tumor Model %ID/g-mean Organ 5 min 10 min 15 min 20 min 25 min 30 min 60 min 60 min blocking 60 min PC-3 120 min Brain 0.98 ± 0.12 0.90 ± 0.12 0.87 ± 0.15 0.80 ± 0.11 0.81 ± 0.13 0.76 ± 0.11 0.62 ± 0.08 0.49 ± 0.41 0.67 ± 0.37 0.5 ± 0.09 Heart 4.29 ± 0.27 4.23 ± 0.37 4.18 ± 0.47 4.08 ± 0.51 4.01 ± 0.47 3.94 ± 0.45 2.81 ± 0.4 2.27 ± 1.83 2.63 ± 0.01 2.05 ± 0.3 Lung 4.33 ± 0.54 4.32 ± 0.53 4.49 ± 0.54 4.49 ± 0.45 4.50 ± 0.59 4.56 ± 0.65 3.18 ± 0.74 1.96 ± 1.07 3.41 ± 0.59 2.77 ± 0.47 Liver 5.6 ± 0.13 5.53 ± 0.13 5.33 ± 0.22 5.28 ± 0.20 5.19 ± 0.24 5.15 ± 0.28 4.33 ± 0.08 4.01 ± 1.31 3.53 ± 0.01 4.05 ± 0.21 Kidney 14.37 ± 3.22 12.74 ± 2.70 11.78 ± 2.46 11.19 ± 2.31 10.97 ± 2.50 10.75 ± 2.59 8.76 ± 1.81 13.97 ± 20.27 13.44 ± 1.42 7.34 ± 1.5 Stomach 3.42 ± 1.22 3.28 ± 1.17 3.18 ± 1.19 3.19 ± 1.16 3.14 ± 1.16 3.02 ± 1.18 2.12 ± 0.62 2.06 ± 0.84 8.19 ± 6.19 1.96 ± 0.75 Intestine 3.2 ± 0.57 2.94 ± 0.64 2.82 ± 0.58 2.76 ± 0.53 2.71 ± 0.51 2.69 ± 0.53 2.51 ± 0.54 1.41 ± 0.81 3.12 ± 1.22 2.05 ± 0.39 Muscle 1.6 ± 0.41 1.45 ± 0.28 1.33 ± 0.22 1.36 ± 0.23 1.30 ± 0.19 1.23 ± 0.15 0.82 ± 0.09 0.52 ± 0.19 0.93 ± 0.25 0.69 ± 0.07 Tumor 3.5 ± 1.61 3.90 ± 1.29 4.28 ± 0.88 4.63 ± 0.53 4.89 ± 0.21 5.06 ± 0.14 4.77 ± 0.44 2.45 ± 1.00 0.88 ± 0.08 3.87 ± 0.66 %ID/g-max Organ 5 min 10 min 15 min 20 min 25 min 30 min 60 min 60 min blocking 60 min PC-3 120 min Brain 1.93 ± 0.48 4.00 ± 1.91 1.77 ± 0.49 1.65 ± 0.48 1.67 ± 0.41 1.67 ± 0.4 1.23 ± 0.35 0.92 ± 0.59 0.99 ± 0.21 1.09 ± 0.29 Heart 5.66 ± 0.35 5.88 ± 0.63 5.41 ± 0.54 5.34 ± 0.78 5.54 ± 0.82 5.52 ± 0.91 4.18 ± 0.65 3.15 ± 2.17 3.84 ± 0.01 3.13 ± 0.48 Lung 5.16 ± 0.8 5.13 ± 0.59 5.44 ± 0.70 5.36 ± 0.83 5.23 ± 0.59 5.7 ± 0.95 3.94 ± 0.83 2.39 ± 1.14 4.32 ± 0.61 3.51 ± 0.51 Liver 6.67 ± 0.39 6.53 ± 0.21 6.48 ± 0.62 6.17 ± 0.22 6.19 ± 0.31 6.26 ± 0.39 5.41 ± 0.25 4.77 ± 1.57 4.52 ± 0.09 5.22 ± 0.39 Kidney 17.76 ± 3.73 15.38 ± 2.96 14.41 ± 2.56 13.37 ± 2.19 13.42 ± 2.23 13.31 ± 2.67 10.72 ± 2.02 21.06 ± 33.15 14.58 ± 0.19 9.18 ± 1.77 Stomach 4.85 ± 1.19 4.77 ± 1.05 4.61 ± 1.08 4.59 ± 1.28 4.75 ± 1.33 4.4 ± 1.16 3.64 ± 0.81 3.24 ± 0.9 5.44 ± 2.69 3.26 ± 1.28 Intestine 4.93 ± 0.96 4.79 ± 1.13 4.55 ± 0.58 4.48 ± 0.78 4.30 ± 0.59 4.54 ± 0.53 4.27 ± 1.14 2.38 ± 1.59 7.92 ± 5.54 3.29 ± 0.74 Muscle 2.44 ± 0.46 2.29 ± 0.28 2.20 ± 0.37 2.28 ± 0.19 2.00 ± 0.34 2.11 ± 0.15 1.43 ± 0.12 0.98 ± 0.42 1.68 ± 0.31 1.21 ± 0.25 Tumor 6.87 ± 2.7 7.79 ± 2.18 8.68 ± 1.05 9.21 ± 0.79 9.60 ± 0.24 10.05 ± 0.67 9.33 ± 1.44 4.65 ± 1.64 3.52 ± 0.11 7.91 ± 1.2 Selective and High Tumor Uptake of 68 Ga-NYM080 in ccRCC Xenografts In OS-RC-2 xenograft model, tumor uptake was evident as early as 5 min post-injection, peaking at 10.05%ID/g around 30 min. Blocking studies with cold NYM005 resulted in a significant reduction in tumor uptake, confirming the specificity of 68Ga-NYM080 for CAIX (Fig. 1 ). Notably, the tumor-to-muscle ratio (T/M) gradually increased after injection, peaked at 1 h (T/M = 6.5), and then remained relatively stable thereafter, indicating effective tumor targeting and clearance from non-target tissues. In the ex vivo biodistribution study, tumor accumulation of 68 Ga-NYM080 reached 11.20%ID/g at 1 h, and decreased to 8.41%ID/g at 4 h (detailed data in Supplementary Table S5). Distinct Tumor Accumulation of 177 Lu-NYM080 in ccRCC Xenografts The uptake of ¹⁷⁷Lu-NYM080 exhibited a distinct, time-dependent increase in CAIX-positive OS-RC-2 cells, rising from 2.72% at 4 h to 12.18% at 72 h, while CAIX-negative PC3 cells showed minimal uptake of 1.09% at 72 h (Fig. 3 ), demonstrating the excellent targeting capability of 177 Lu-NYM080 for CAIX-overexpressing cell models. The SPECT/CT imaging from 6 to 168 h post-injection shows significant accumulation and retention of the radiotracer in OS-RC-2 xenograft tumor (Fig. 4 ). Mild retention was observed the kidneys, which gradually decreases from 6 to 48 h. Renal uptake is no longer detectable after 96 h, while tumor retention reaches peak at 168 h, with a %ID/g of 8.79. The ex vivo biodistribution profiles of 177 Lu-NYM080 demonstrated high initial uptake in the kidneys, liver, and tumor, with rapid renal clearance (Fig. 2 ). 177 Lu-NYM080 exhibited sustained and prolonged tumor retention, with uptake decreasing gradually by 168 h (detailed data in Supplementary Table S5), which underscore its potential for therapeutic application. Therapeutic Efficacy of 177 Lu-NYM080 in Xenograft Animal Models In the treatment study, two groups of OS-RC-2 mice were injected with single dose 177 Lu-NYM080 of 74MBq (G2) or 148Mbq (G3) (twice 74 MBq doses, 4 days apart) and were well tolerated. Both doses resulted in a delay of tumor growth compared to the control group receiving normal saline (G1). In the 74 MBq single-dose group (G2), the median tumor doubling time was extended to 13.0 days (interquartile range, 9.0–20.0 days) (P = 0.064 vs. control). In the 148 MBq single-dose group (G3), the median doubling time was further prolonged to 19.5 days (interquartile range, 12.8–23.0 days) (P = 0.0054 vs. control; P = 0.4373 vs. 74 MBq group). While no statistical significance was observed in tumor doubling time between G2 and G3 (P = 0.4373), a dose-dependent tumor suppression trend in tumor growth suppression was evident, with G3 demonstrating more pronounced anti-tumor efficacy compared to G2 (Fig. 5 ). Furthermore, histopathological evaluations of major organs (liver, kidney, heart, lung, and spleen) revealed no significant abnormalities or toxicity compared to controls, no signs of acute nephrotoxicity were observed at the doses studied. These results underscore the therapeutic efficacy of 177Lu-NYM080 and highlight the potential of fractionated dosing to enhance tumor control in ccRCC models. Discussion CAIX is highly expressed in multiple cancers, including ccRCC, pancreatic ductal adenocarcinoma (PDAC), and breast cancer[ 3 , 16 – 18 ]. For an extended period, however, clinical trials targeting CAIX with antibodies and inhibitors have not yielded satisfactory outcomes[ 19 , 20 ]. Among the few promising CAIX inhibitors, clinical studies have either failed to demonstrate objective responses or shown only mild responses, without significant improvement in survival. While the CAIX inhibitor SLC-0111 demonstrated a favorable safety profile in a phase I trial involving solid tumor patients, it failed to elicit objective tumor responses[ 21 ]. Similarly, a phase III adjuvant trial evaluating G250, a monoclonal antibody targeting CAIX, observed no improvement in survival compared with placebo controls in high-risk ccRCC patients[ 22 ]. Therefore, there is a clear need for more effective strategy for CAIX treatment. Radionuclide therapy targeting CAIX has been introduced a promising approach for ccRCC. Studies have demonstrated significant anti-tumor efficacy with the use of the monoclonal antibody cG250, radiolabeled with the β-emitting radionuclide ¹⁷⁷Lu in preclinical evaluation[ 23 , 24 ]. Additionally, Phase I/II clinical trials with 177 Lu-cG250 involving metastatic ccRCC patients also showed that majority of patients achieved disease stabilization[ 25 , 26 ]. Moreover, hG250, a humanized anti-CAIX antibody labeled with either 225 Ac or 177 Lu, both demonstrated excellent tolerability and significant survival prolongation in ccRCC xenograft models[ 27 , 28 ]. These findings underscore the potential of CAIX-targeting radionuclide therapy for ccRCC. Small-molecule peptide are generally non-immunogenic, clear rapidly from circulation and allow use of short-lived radionuclides and imaging at early time points[ 29 , 30 ]. Here, we developed NYM080, a new-generation CAIX-targeting small-molecule which combines acetazolamide with the chelator DOTA through a hybrid linker comprising an aliphatic spacer and a short peptide (Fig. S1 ). The chelator DOTA enables radiolabeling with both ⁶⁸Ga and ¹⁷⁷Lu, while the aliphatic component reduces steric and electronic interference from both the peptide and DOTA chelator, preserving the nanomolar-level affinity of acetazolamide to CAIX. In general, NYM080 exhibits comparable performance to its predecessor (NYM005) in terms of CAIX-binding affinity, biodistribution profile, pharmacokinetics, and target specificity. In the present study, NYM080 showed favorable affinity o NYM080 of 9.69 nM for CAIX, similar to that of NY104 (5.75 nM) [ 15 ]. 68 Ga-NYM080 exhibited rapid blood clearance and accumulated quickly in tumor. 68 Ga-NYM080 exhibits distinct tumor uptake in CAIX-positive OS-RC-2 xenograft models, with a peak uptake of 10%ID/g at 30 min, higher than many previous CAIX-targeted molecule (≤ 5%ID/g)[ 31 – 35 ]. The distinct reduction in tumor uptake after blocking with cold NYM005 confirmed the specificity of 68 Ga-NYM080 for CAIX (Fig. 2 ). Remarkably, T/M reached 6.56 at 1h post-injection then remained stable, highlighting the potential of 68 Ga-NYM080 PET/CT in detecting ccRCC lesions. These findings underscore the promise of 68 Ga-NYM080 as an excellent imaging agent for ccRCC. The most noteworthy feature of 177 Lu-NYM080 is its favorable prolonged tumor accumulation. High uptake of 177 Lu-NYM080 was observed in the CAIX-positive OS-RC-2 cells, while minimal uptake was detected in the CAIX-negative PC3 cells. In xenograft model, 177 Lu-NYM080 showed distinct tumor retention, reaching 8.7%ID/g at 168 h, while the uptake in normal organs decreased overtime (Fig. 4 ), which is comparable to that of 177 Lu-cG250 in the SK-RC-52 model (<10%ID/g in tumors)[ 36 ]. The imaging pattern of 177 Lu-NYM080, characterized by increasing tumor uptake and decreasing background activity, suggests its sustained target engagement and reduced off-target toxicity. The “sink-in” phenomenon observed in SPECT imaging is likely attributed to the high binding affinity and prolonged retention of NYM080 in CAIX-overexpressing tumor tissue, leading to sustained target engagement. Additionally, rapid clearance from non-target tissues such as kidneys and liver may further enhance the visual contrast, creating the apparent “sink-in” imaging effect over time. Similar observations were reported for other therapeutic radioligands such as ¹⁷⁷Lu-PSMA-617, where prolonged tumor retention correlated with improved therapeutic outcomes in prostate cancer[ 37 , 38 ]. Furthermore, 177 Lu-NYM080 exhibited a dose-dependent anti-tumor trend in ccRCC model, with the best tumor suppression observed in the group receiving twice 74MBq injections, suggesting the potential of increasing the administered dose to enhance therapeutic efficacy. For other radioligand therapies such as 177 Lu-DOTATATE in neuroendocrine tumors, dose escalation is a well-established approach to maximize therapeutic effect leading to a more sustained tumor reduction over time while maintaining an acceptable toxicity profile[ 39 ], which also aligns with clinical multiple dosing schedules. The biodistribution profiles vary among different 177 Lu-labeled agents, precluding the establishment of a universal dose range. Preclinical studies show that 111 MBq of 177 Lu‑PSMA‑617 improved survival (53.8% vs 30.7%) over 37 MBq in bone metastasis models[ 40 ], and dose escalation up to 3 × 60 MBq was also safely achieved with 177 Lu‑NeoB[ 41 ]. Given there is no well-established dosing strategy for CAIX-targeted 177 Lu therapies, 74 MBq and 2×74 MBq were optimized to balance treatment efficacy with safety considerations. ¹⁷⁷Lu-NYM080 demonstrated a favorable safety profile. The ex vivo biodistribution of 177 Lu-NYM080 showed minimal accumulation in kidneys and intestines (<1%ID/g at 48h; <0.1%ID/g at 168h), and there was no evidence of organ damage in normal tissues upon histopathological examination in both G2 and G3. This low toxicity profile supports its clinical viability, particularly for repeated dosing regimens. Recently, PSMA PET/CT has been reported as a useful tool in ccRCC lesion detection, with pooled detection rate of 83% in primary or metastatic ccRCC[ 42 ]. Compared to conventional CT for detection of metastatic disease, Rhee et al. reported 68 Ga-PSMA-HBED-CC PET/CT had significantly higher sensitivity (92% vs. 69%) and positive predictive value (97% vs. 80%) [ 43 ]. 18 F-DCFPyL PET/CT also demonstrated superior sensitivity to conventional CT or MRI (95 versus 79%)[ 44 ]. Besides, there is an ongoing phase I/II trial designed to assess the tolerability and effectiveness of 177 Lu-PSMA-1 in individuals with PSMA-positive metastatic ccRCC[ 45 ]. While prostate-specific membrane antigen (PSMA) has been explored as a theranostic target in ccRCC, its clinical utility is limited by its rapid washout due to expression on neovascular endothelial cells, leading to reduced tumor retention over time. In addition, PSMA is expressed on the neovasculature of many other solid malignancies, benign conditions and various RCC subtypes[ 46 – 50 ]. In contrast, CAIX demonstrates prolonged retention as it is stably over-expressed on the cell membrane of ccRCC. Combined with its high specificity for ccRCC, CAIX stands as a promising theranostic target. Currently, there are very few peptide-based CAIX-targeting small molecule 68 Ga/ 177 Lu theranostic pair[ 51 ], and NYM080 offers a novel potential strategy for ccRCC imaging and therapy. The favorable tumor retention and dose-dependent antitumor efficacy demonstrated by NYM080 provide compelling evidence for the translational potential of the CAIX target. These findings strongly support the continued development of CAIX-targeted therapies in the future. Another novel CAIX-targeted theranostic molecule has recently been developed, and a corresponding Phase I clinical study is currently underway to evaluate its theranostic capabilities in metastatic ccRCC patients (ClinicalTrials.gov identifier: NCT06649682). This study has certain limitations. Firstly, due to the proof-of-concept nature of this study, human participant data and radiation dosimetry evaluations were not included, limiting the translation of these findings to clinical settings. Secondly, the pharmacokinetic properties and biodistribution observed in animal models may differ from those in humans, potentially affecting the predictability of therapeutic efficacy and toxicity profiles. Thirdly, while the remarkable tumor uptake and therapeutic efficacy highlight the value of NYM080 as a CAIX-targeted theranostic agent, its non-relatively high initial gastrointestinal and renal uptake necessitates further molecular optimization Furthermore, the short follow-up period did not allow for assessment of the long-term toxicity and side effects of the radiolabeled ligand, long-term toxicity safety profile needs to be assessed in future studies. Conclusion This is preclinical study demonstrated distinct tumor uptake of both 68 Ga-NYM080 and 177 Lu-NYM080. Furthermore, 177 Lu-NYM080 showed marked anti-tumor effect in ccRCC xenograft models, highlighting its therapeutic potential for ccRCC patients. Abbreviations CAIX: Carbonic anhydrase IX ccRCC: Clear cell renal cell carcinoma VHL: von Hippel-Lindau HIF-1α: Hypoxia-inducible factor 1-alpha TKI: Tyrosine kinase inhibitor ICI: Immune checkpoint inhibitor SUV: Standardized uptake value %ID/g: Percentage injected dose per gram SD: Standard deviation T/M: Tumor-to-muscle ratio T/H: Tumor-to-heart ratio DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid NOTA: 1,4,7-triazacyclononane-1,4,7-triacetic acid ANOVA: Analysis of variance PDAC: Pancreatic ductal adenocarcinoma PSMA: Prostate-specific membrane antigen NCCN: National Comprehensive Cancer Network Declarations Ethics approval and consent to participate All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Peking Union Medical College Hospital. All procedures involving animals were performed following the National Research Council’s Guide for the Care and Use of Medical Laboratory Animals (Ministry of Health, China). All efforts were made to minimize animal suffering. Consent for publication Not applicable. Availability of data and material The data will be shared on reasonable request to the corresponding author. Funding This work was supported by the National Natural Science Foundation of China (Grant No. U24A20758); Beijing Natural Science Foundation (Grant No. L242062); National Key R&D Program of China (Grant No. 2024YFC2419400); Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (Grant No. CIFMS-2024-I2M-ZD-001, CIFMS-2023-I2M-2-002, CIFMS-2021-I2M-1-002, CIFMS-2021-I2M-1-003, CIFMS-2021-I2M-1-025); National High Level Hospital Clinical Research Funding (Grant No. 2022-PUMCH-D-001, 2022-PUMCH-D-002). Authors' contributions XY: conceptualization; data curation and analysis; drafting original manuscript. WZ, ML and LH: writing guidance and revision. CR, YL, ZH, and HZ: technical support; data validation. GZ and XL: clinical consultation. All authors read and approved the final manuscript. Acknowledgements We acknowledge the National Natural Science Foundation of China (Grant No. U24A20758); Beijing Natural Science Foundation (Grant No. L242062); National Key R&D Program of China (Grant No. 2024YFC2419400); Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (Grant No. CIFMS-2024-I2M-ZD-001, CIFMS-2023-I2M-2-002, CIFMS-2021-I2M-1-002, CIFMS-2021-I2M-1-003, CIFMS-2021-I2M-1-025); National High Level Hospital Clinical Research Funding (Grant No. 2022-PUMCH-D-001, 2022-PUMCH-D-002) for supporting our work. Competing Interests The authors have no relevant financial or non-financial interests to disclose. 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Phase 1 Radioimmunotherapy Study with Lutetium 177–labeled Anti-Carbonic Anhydrase IX Monoclonal Antibody Girentuximab in Patients with Advanced Renal Cell Carcinoma. European Urology. 2013;64:478–85. Muselaers CHJ, Boers-Sonderen MJ, van Oostenbrugge TJ, Boerman OC, Desar IME, Stillebroer AB, et al. Phase 2 Study of Lutetium 177–Labeled Anti–Carbonic Anhydrase IX Monoclonal Antibody Girentuximab in Patients with Advanced Renal Cell Carcinoma. European Urology. 2016;69:767–70. Kleinendorst SC, Oosterwijk E, Molkenboer-Kuenen J, Frielink C, Franssen GM, Boreel DF, et al. Towards effective CAIX-targeted radionuclide and checkpoint inhibition combination therapy for advanced clear cell renal cell carcinoma. Theranostics. 2024;14:3693–707. Merkx RIJ, Rijpkema M, Franssen GM, Kip A, Smeets B, Morgenstern A, et al. Carbonic Anhydrase IX-Targeted α-Radionuclide Therapy with 225Ac Inhibits Tumor Growth in a Renal Cell Carcinoma Model. Pharmaceuticals. 2022;15:570. Fendler WP, Calais J, Eiber M, Flavell RR, Mishoe A, Feng FY, et al. Assessment of 68 Ga-PSMA-11 PET Accuracy in Localizing Recurrent Prostate Cancer. JAMA Oncol. 2019;5:856. Skoura E, Michopoulou S, Mohmaduvesh M, Panagiotidis E, Al Harbi M, Toumpanakis C, et al. The Impact of 68 Ga-DOTATATE PET/CT Imaging on Management of Patients with Neuroendocrine Tumors: Experience from a National Referral Center in the United Kingdom. J Nucl Med. 2016;57:34–40. Lau J, Zhang Z, Jenni S, Kuo H-T, Liu Z, Vullo D, et al. PET Imaging of Carbonic Anhydrase IX Expression of HT-29 Tumor Xenograft Mice with 68 Ga-Labeled Benzenesulfonamides. Mol Pharmaceutics. 2016;13:1137–46. Pan J, Lau J, Mesak F, Hundal N, Pourghiasian M, Liu Z, et al. Synthesis and evaluation of 18 F-labeled carbonic anhydrase IX inhibitors for imaging with positron emission tomography. Journal of Enzyme Inhibition and Medicinal Chemistry. 2014;29:249–55. Akurathi V, Dubois L, Celen S, Lieuwes NG, Chitneni SK, Cleynhens BJ, et al. Development and biological evaluation of 99mTc-sulfonamide derivatives for in vivo visualization of CA IX as surrogate tumor hypoxia markers. European Journal of Medicinal Chemistry. 2014;71:374–84. Akurathi V, Dubois L, Lieuwes NG, Chitneni SK, Cleynhens BJ, Vullo D, et al. Synthesis and biological evaluation of a 99mTc-labelled sulfonamide conjugate for in vivo visualization of carbonic anhydrase IX expression in tumor hypoxia. Nuclear Medicine and Biology. 2010;37:557–64. Lau J, Pan J, Zhang Z, Hundal-Jabal N, Liu Z, Bénard F, et al. Synthesis and evaluation of 18F-labeled tertiary benzenesulfonamides for imaging carbonic anhydrase IX expression in tumours with positron emission tomography. Bioorganic & Medicinal Chemistry Letters. 2014;24:3064–8. Basaco T, Pektor S, Bermudez JM, Meneses N, Heller M, Galván JA, et al. Evaluation of Radiolabeled Girentuximab In Vitro and In Vivo. Pharmaceuticals. 2018;11:132. Violet J, Jackson P, Ferdinandus J, Sandhu S, Akhurst T, Iravani A, et al. Dosimetry of 177Lu-PSMA-617 in Metastatic Castration-Resistant Prostate Cancer: Correlations Between Pretherapeutic Imaging and Whole-Body Tumor Dosimetry with Treatment Outcomes. Journal of Nuclear Medicine. 2019;60:517–23. Fendler WP, Stuparu AD, Evans-Axelsson S, Lückerath K, Wei L, Kim W, et al. Establishing 177 Lu-PSMA-617 Radioligand Therapy in a Syngeneic Model of Murine Prostate Cancer. J Nucl Med. 2017;58:1786–92. Sundlöv A, Gleisner KS, Tennvall J, Ljungberg M, Warfvinge CF, Holgersson K, et al. Phase II trial demonstrates the efficacy and safety of individualized, dosimetry-based 177Lu-DOTATATE treatment of NET patients. Eur J Nucl Med Mol Imaging. 2022;49:3830–40. Peng C-L, Chen C-T, Tang I-C. Exploring the Therapeutic Potential of 177Lu-PSMA-617 in a Mouse Model of Prostate Cancer Bone Metastases. IJMS. 2025;26:5970. Verhoeven M, Haeck J, de Blois E, Orlandi F, Barbato D, Tedesco M, et al. The Balance Between the Therapeutic Efficacy and Safety of [177Lu]Lu-NeoB in a Preclinical Prostate Cancer Model. Mol Imaging Biol. 2024;26:114–23. Sadaghiani MS, Baskaran S, Gorin MA, Rowe SP, Provost J-C, Teslenko I, et al. Utility of PSMA PET/CT in Staging and Restaging of Renal Cell Carcinoma: A Systematic Review and Metaanalysis. J Nucl Med. 2024;65:1007–12. Rhee H, Blazak J, Tham CM, Ng KL, Shepherd B, Lawson M, et al. Pilot study: use of gallium-68 PSMA PET for detection of metastatic lesions in patients with renal tumour. EJNMMI Res. 2016;6:76. Rowe SP, Gorin MA, Hammers HJ, Som Javadi M, Hawasli H, Szabo Z, et al. Imaging of metastatic clear cell renal cell carcinoma with PSMA-targeted 18F-DCFPyL PET/CT. Ann Nucl Med. 2015;29:877–82. Kryza D, Vinceneux A, Bidaux A-S, Garin G, Tatu D, Cropet C, et al. A multicentric, single arm, open-label, phase I/II study evaluating PSMA targeted radionuclide therapy in adult patients with metastatic clear cell renal cancer (PRadR). BMC Cancer. 2024;24:163. Pyka T, Weirich G, Einspieler I, Maurer T, Theisen J, Hatzichristodoulou G, et al. 68 Ga-PSMA-HBED-CC PET for Differential Diagnosis of Suggestive Lung Lesions in Patients with Prostate Cancer. J Nucl Med. 2016;57:367–71. Ardies PJ, Gykiere P, Goethals L, De Mey J, De Geeter F, Everaert H. PSMA Uptake in Mediastinal Sarcoidosis. Clin Nucl Med. 2017;42:303–5. Wang H, Wang S, Song W, Pan Y, Yu H, Si T, et al. Expression of Prostate-Specific Membrane Antigen in Lung Cancer Cells and Tumor Neovasculature Endothelial Cells and Its Clinical Significance. PLoS ONE. 2015;10:e0125924. Hangaard L, Jochumsen MR, Vendelbo MH, Bouchelouche K. Metastases From Colorectal Cancer Avid on 68Ga-PSMA PET/CT. Clin Nucl Med. 2017;42:532–3. Silver DA, Pellicer I, Fair WR, Heston WD, Cordon-Cardo C. Prostate-specific membrane antigen expression in normal and malignant human tissues. Clin Cancer Res. 1997;3:81–5. Massière F, Wiedemann N, Borrego I, Hoehne A, Osterkamp F, Paschke M, et al. Preclinical Characterization of DPI-4452: A 68 Ga/ 177 Lu Theranostic Ligand for Carbonic Anhydrase IX. J Nucl Med. 2024;65:761–7. Supplementary Files SupplementaryFigures.docx SupplementaryMethods.docx SupplementaryTables.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 05 Sep, 2025 Reviewers invited by journal 04 Sep, 2025 Editor assigned by journal 04 Sep, 2025 First submitted to journal 03 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6954806","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":510437822,"identity":"03becdb3-9385-4226-9094-9dc3e650f733","order_by":0,"name":"Xinchun Yan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYFADCeYDBz78IE0LW+LBmT2kaeExPszBRoRCg+NnDD8X7jgczT+758NhBh4GeX6xA/i1SPbkGEvPPHM4d8adsxsOF1gwGM6cnYBfC78Ej4E0b9vh3A0SuRsOz+BhSDC4TUALG9ALvyFach4c5mEjQgvQFjOoLTkMxGmR7Ekrs+Y9k54740aaATCQJQj7xeD44c23eXdY5/bPSH784cMPG3l+aQJaGBg4DBgYG+A8CULKQYD9AbKWUTAKRsEoGAWYAAAnyUXZS84BZAAAAABJRU5ErkJggg==","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":true,"prefix":"","firstName":"Xinchun","middleName":"","lastName":"Yan","suffix":""},{"id":510437823,"identity":"59de1c16-917e-4bbb-99b1-a65778858764","order_by":1,"name":"Meixi Liu","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"prefix":"","firstName":"Meixi","middleName":"","lastName":"Liu","suffix":""},{"id":510437824,"identity":"6573c947-550e-4886-b1ef-099b4d3a5349","order_by":2,"name":"Guoyang Zheng","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"prefix":"","firstName":"Guoyang","middleName":"","lastName":"Zheng","suffix":""},{"id":510437825,"identity":"33468b4b-6763-48d3-a538-00c8452b9128","order_by":3,"name":"Xiaoyuan Li","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyuan","middleName":"","lastName":"Li","suffix":""},{"id":510437826,"identity":"44611e88-b0cc-44b1-8a22-d0ea0cb538b0","order_by":4,"name":"Chao Ren","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Ren","suffix":""},{"id":510437827,"identity":"9ea1faf8-df72-4a2b-9c2e-0da984006c77","order_by":5,"name":"Yu Liu","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Liu","suffix":""},{"id":510437828,"identity":"94560dba-8b1d-44b1-b723-98b39752d750","order_by":6,"name":"Zhenghai Huang","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhenghai","middleName":"","lastName":"Huang","suffix":""},{"id":510437829,"identity":"a859d0d6-fa60-4438-9001-a560777f8fa2","order_by":7,"name":"Haiqiong Zhang","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"prefix":"","firstName":"Haiqiong","middleName":"","lastName":"Zhang","suffix":""},{"id":510437830,"identity":"2982b6fa-8329-4da8-bfd7-a54d7ee799d4","order_by":8,"name":"Wenjia Zhu","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wenjia","middleName":"","lastName":"Zhu","suffix":""},{"id":510437831,"identity":"e1b17c1d-c3da-434e-aae1-b1aeee5eef29","order_by":9,"name":"Li Huo","email":"","orcid":"https://orcid.org/0000-0003-1216-083X","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Huo","suffix":""}],"badges":[],"createdAt":"2025-06-23 08:59:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6954806/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6954806/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91085382,"identity":"483e6a8d-6839-4dc6-b3e3-df3aaf244407","added_by":"auto","created_at":"2025-09-11 12:21:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6352346,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo biodistribution of \u003csup\u003e68\u003c/sup\u003eGa-NYM080 in the CAIX-positive OS-RC-2 xenograft model (n=4), and tumor uptake was markedly reduced following receptor blockade with cold NYM005 (a-c). PC-3 xenograft models (n=3) exhibited minimal radiotracer uptake (a, c). Detailed biodistribution data are presented in (d). Data are presented as %ID/g max (mean ± SD) across multiple organs and tumor.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6954806/v1/353479088b7eb5f399fd8352.png"},{"id":91085384,"identity":"40950dda-bc8f-4fee-b6d7-9e4c6addb700","added_by":"auto","created_at":"2025-09-11 12:21:32","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":170677,"visible":true,"origin":"","legend":"\u003cp\u003eEx vivo biodistribution of (A) \u003csup\u003e68\u003c/sup\u003eGa-NYM080 in CAIX-positive OS-RC-2 xenografts at 1 and 4 h post-injection and (B) \u003csup\u003e177\u003c/sup\u003eLu-NYM080 at 4, 48, and 168 h post-injection (n=3 for each agent). Data are presented as %ID/g max (mean ± SD) across multiple organs and tumor.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6954806/v1/ccd008a240b54536d33b9279.jpg"},{"id":91087445,"identity":"2cca1f80-8b13-4926-b905-c94ed1d14e99","added_by":"auto","created_at":"2025-09-11 12:37:32","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":40474,"visible":true,"origin":"","legend":"\u003cp\u003eUptake of \u003csup\u003e177\u003c/sup\u003eLu-NYM080 in OS-RC-2 and PC3 cells.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6954806/v1/c29a7ba33e139d8bbd7d0378.jpg"},{"id":91085396,"identity":"36262bc1-96f3-4a82-ad79-08364c49277c","added_by":"auto","created_at":"2025-09-11 12:21:32","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":164482,"visible":true,"origin":"","legend":"\u003cp\u003eTumor growth inhibition with \u003csup\u003e177\u003c/sup\u003eLu-NYM080 (n=6 for each group, G1-G3).\u003cu\u003e \u003c/u\u003eTumor growth delayed in mice treated with normal saline, 74MBq or 148Mbq (two injection of 74MBq, 4 days apart) of \u003csup\u003e177\u003c/sup\u003eLu-NYM080, with G2 showing most pronounced suppression compared to control (a-b). The median tumor doubling time was extended to 13.0 days (interquartile range, 9.0–20.0 days) in G2 (P=0.064 vs. G1) and further prolonged to 19.5 days (interquartile range, 12.8–23.0 days) in G3 (P=0.0054 vs. G1; P=0.4373 vs. G2) (c-d). Blue arrows indicate time of the first and second injection.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6954806/v1/a7589f69c9fdf8a9429869d4.jpg"},{"id":91085397,"identity":"c235bfa4-5c78-4c3c-9dab-29a2a3900a80","added_by":"auto","created_at":"2025-09-11 12:21:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":676948,"visible":true,"origin":"","legend":"\u003cp\u003eSPECT imaging demonstrates significant tumor accumulation of ¹⁷⁷Lu-NYM080 in OS-RC-2 xenograft model, with minimal uptake in normal tissues (a). Tumor uptake of ¹⁷⁷Lu-NYM080 progressively increased over time, peaking at 168 h post-injection (b).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6954806/v1/852007aa2fdd05975f04afe2.png"},{"id":91089325,"identity":"65373877-aecb-4869-a281-4bde0b95dab2","added_by":"auto","created_at":"2025-09-11 12:53:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8545041,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6954806/v1/ebafda00-19ef-4c63-aa5c-18fddbad14d8.pdf"},{"id":91086683,"identity":"ac3743a5-65b2-4872-a6a4-3b53edd7869f","added_by":"auto","created_at":"2025-09-11 12:29:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1436567,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-6954806/v1/fae35a1ae73676a42378a0f8.docx"},{"id":91085380,"identity":"ea7a95a8-f17b-4683-b61d-466a0caf017d","added_by":"auto","created_at":"2025-09-11 12:21:32","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":23937,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMethods.docx","url":"https://assets-eu.researchsquare.com/files/rs-6954806/v1/d07d31be0b2ff3dbe263756e.docx"},{"id":91086672,"identity":"4728ed3e-2e2f-4b36-b341-9eb84bebcc6f","added_by":"auto","created_at":"2025-09-11 12:29:32","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":32566,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6954806/v1/086a3233b6b5030bfd708fb7.docx"}],"financialInterests":"","formattedTitle":"Preclinical Evaluation of a Novel Carbonic Anhydrase IX-Targeting 68Ga/177Lu Theranostic Pair for Clear Cell Renal Cell Carcinoma","fulltext":[{"header":"Background","content":"\u003cp\u003eCarbonic anhydrase IX(CAIX) is a membrane-bound enzyme implicated in tumor acidosis, highly expressed in hypoxic tumors and minimally in normal tissues, with the exception of select regions in the gastrointestinal tract.[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] As one of the twelve catalytically active carbonic anhydrases, CAIX is regulated by hypoxia-inducible factor 1-alpha (HIF-1α) under hypoxic conditions[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In clear cell renal cell carcinoma (ccRCC), CAIX expression is further intensified due to dysregulation of the von Hippel-Lindau (VHL) tumor suppressor, resulting in sustained HIF-1α activity[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This overexpression is linked to tumor aggressiveness, metastasis, and poor clinical outcomes, which makes CAIX a valuable target for theranostic applications.\u003c/p\u003e\u003cp\u003eClear cell renal cell carcinoma is the most common subtype of renal cell carcinoma[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].While diagnosis and staging of ccRCC primarily rely on contrast-enhanced CT (ceCT), its accuracy in differentiating small renal masses remains limited[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and its nephrotoxicity makes it unsuitable for patients with renal impairment. Although percutaneous biopsy is recommended before initiating ablative or systemic therapies[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], it is constrained by limited sampling sites and complications. In terms of therapy, For ccRCC patients presenting with distant metastases at diagnosis, the five-year survival rate is only 13%[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The treatment of advanced or metastatic ccRCC relies on tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs) according to the NCCN Guideline for Kidney Cancer[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, approximately 30\u0026ndash;60% patients have poor response to these therapies or develop secondary acquired resistance, which limits the overall efficacy[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, many patients exhibit poor tolerance to TKIs or require dose reductions, underscoring the need for novel therapeutic approaches. There is an urgent need for new therapies for patients with advanced metastatic ccRCC.\u003c/p\u003e\u003cp\u003eIn our previous work, we reported \u003csup\u003e68\u003c/sup\u003eGa-NY104 (also referred to as \u003csup\u003e68\u003c/sup\u003eGa-NYM005), a small-molecule tracer targeting CAIX, which has recently shown encouraging clinical results in the diagnosis of ccRCC[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, NYM005 was not suitable for lutetium labeling due to NOTA chelation. Therefore, we developed NYM080, a novel CAIX-targeted molecular based on acetazolamide core (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Compared to NY104, NYM080 links acetazolamide to chelator DOTA through an aliphatic linker and a short peptide to minimize steric interference with CAIX binding. These properties make NYM080 a potential candidate for optimized tumor imaging and therapeutic applications.\u003c/p\u003e\u003cp\u003eThis preclinical investigation aims to characterize the biodistribution, pharmacokinetics, and therapeutic efficacy of \u003csup\u003e68\u003c/sup\u003eGa/\u003csup\u003e177\u003c/sup\u003eLuNYM080 in CAIX-expressing clear cell renal cell carcinoma (ccRCC) models.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eDetailed experimental procedures are provided in the Supplementary Methods.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eRadiosynthesis and Quality Control\u003c/h2\u003e\u003cp\u003eThe CAIX-targeting molecule NYM080 precursor was radiolabeled with \u003csup\u003e68\u003c/sup\u003eGa or \u003csup\u003e177\u003c/sup\u003eLu based on standardized procedures, using a \u003csup\u003e68\u003c/sup\u003eGe/\u003csup\u003e68\u003c/sup\u003eGa generator (Eckert \u0026amp; Ziegler, Germany) and \u003csup\u003e177\u003c/sup\u003eLuCl\u003csub\u003e3\u003c/sub\u003e (ITG GmbH, Germany), respectively. Radiochemical purity and stability were confirmed by radio-HPLC (Agilent 1260 Infinity II, USA) and Radio-iTLC (Bioscan AR-2000, USA).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBinding Affinity and Specificity\u003c/h3\u003e\n\u003cp\u003eBinding affinity (Kd) of NYM080 for CAIX was quantified using a Biacore 8K SPR system (Cytiva, USA) with biotinylated CAIX (ACROBiosystems, China) immobilized on a Neutravidin-coated CM5 sensor chip.\u003c/p\u003e\n\u003ch3\u003eCellular and Animal Models\u003c/h3\u003e\n\u003cp\u003eHuman CAIX-positive OS-RC-2 renal carcinoma cells (ATCC, USA) and CAIX-negative PC3 prostate carcinoma cells (ATCC) were cultured in RPMI-1640 medium (Gibco, USA). Xenografts were established in NCG mice (Vital River, China) by subcutaneous injection of OS-RC-2 cells (1\u0026times;10⁷ cells/mouse) mixed with Matrigel (Corning, USA).\u003c/p\u003e\u003cp\u003eBlood pharmacokinetics of \u003csup\u003e68\u003c/sup\u003eGa-NYM080 were evaluated in ICR mice following intravenous administration of 3.7 MBq. Blood samples were collected via the lateral tail vein at 5, 15, 30, 60, 90, 120, 180, and 240 min post-injection, and radioactivity was measured using a gamma counter (PerkinElmer 2470, USA). Pharmacokinetic parameters were calculated using DAS software (version 2.1.1, DAS, China).\u003c/p\u003e\n\u003ch3\u003eImaging and Biodistribution\u003c/h3\u003e\n\u003cp\u003ePET/CT imaging was performed using a Super Nova\u0026reg; system (Pingseng Healthcare, China) at 1\u0026ndash;4 h post-injection of \u003csup\u003e68\u003c/sup\u003eGa-NYM080 (2.96 MBq). Blocking studies used unlabeled NYM005 (400 \u0026micro;g). Ex vivo biodistribution was analyzed with a gamma counter (PerkinElmer 2470, USA).\u003c/p\u003e\n\u003ch3\u003eTherapeutic Efficacy\u003c/h3\u003e\n\u003cp\u003eTumor-bearing mice were allocated into three groups, with six mice per group. Group 1 received single injection of saline as control, Group 2 received a single intravenous injection of 74 MBq of \u003csup\u003e177\u003c/sup\u003eLu-NYM080 and Group 3 received 148 MBq of \u003csup\u003e177\u003c/sup\u003eLu-NYM080 in two doses of 74 MBq each, administered 4 days apart. Mice were weighed and tumor volumes measured on the day of the first injection, with subsequent measurements taken twice every week thereafter.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eData Analysis\u003c/h2\u003e\u003cp\u003eImaging quantification (tumor uptake, %ID/g; tumor-to-muscle ratios) was conducted using PMOD software (version 4.3, PMOD Technologies, Switzerland). All values are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analysis was performed using GraphPad Prism (version 9.0, GraphPad Software, USA). Differences in tumor growth between treatment groups were assessed by one-way ANOVA followed by Tukey\u0026rsquo;s post-hoc test for multiple comparisons. Normality and homogeneity of variances were confirmed using Shapiro-Wilk and Levene\u0026rsquo;s tests, respectively. A two-tailed P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eRadiochemical purity (RCP) exceeded 96% for both ⁶⁸Ga-NYM080 and \u0026sup1;⁷⁷Lu-NYM080 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). ⁶⁸Ga-NYM080 had a molar activity of 4.32 MBq/nmol for PET biodistribution, while molar activity of \u0026sup1;⁷⁷Lu-NYM080 was 20.91 MBq/nmol for therapeutic administration. The molar activities of other experiments are shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The RCP of ⁶⁸Ga-NYM080 remained\u0026thinsp;\u0026gt;\u0026thinsp;94% for 4 hours in human serum (96.67% at 1 h, 96.11% at 2 h, 94.08% at 4 h; n\u0026thinsp;=\u0026thinsp;3), while \u0026sup1;⁷⁷Lu-NYM080 demonstrated\u0026thinsp;\u0026gt;\u0026thinsp;91% stability over 72 hours (95.39% at 24 h, 92.13% at 48 h, 91.02% at 72 h; n\u0026thinsp;=\u0026thinsp;3), with similar retention times (7.13\u0026ndash;7.15 min for ⁶⁸Ga and 7.00-7.02 min for \u0026sup1;⁷⁷Lu), detailed data in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e and Fig.\u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. The binding affinity (kD) of NYM080 for CAIX was assessed in three independent experiments, yielding a mean kD of 9.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 nM, with a coefficient of variation (CV) of 5.8% (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eBiodistribution and Blood Pharmacokinetics of Ga-NYM080 in Xenograft Mouse Models\u003c/h3\u003e\n\u003cp\u003eThe in vivo biodistribution profile of \u003csup\u003e68\u003c/sup\u003eGa-NYM080 showed remarkable uptake in kidney, which decreased over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The ex vivo biodistribution study revealed uptake (\u0026ge;\u0026thinsp;6%ID/g) in the kidneys, stomach, intestine and lung (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Blood pharmacokinetics of \u003csup\u003e68\u003c/sup\u003eGa-NYM080 were also evaluated, over 75% of the injected activity was rapidly cleared within the first 90 min (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e), and detailed pharmacokinetic data are presented in Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e-\u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The blood clearance curve for \u003csup\u003e68\u003c/sup\u003eGa-NYM080 followed a biexponential pattern, with a half-life of 3.49 h. The maximum blood concentration of NYM080 was 3.12 \u0026micro;g/kg at 5 min after injection.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eIn vivo Biodistribution of ⁶⁸Ga-NYM080 in OS-RC-2 Tumor Model\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"11\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"10\" nameend=\"c11\" namest=\"c2\"\u003e\u003cp\u003e%ID/g-mean\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOrgan\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5 min\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10 min\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15 min\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20 min\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e25 min\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e30 min\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e60 min\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e60 min\u003c/p\u003e\u003cp\u003eblocking\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003e60 min\u003c/p\u003e\u003cp\u003ePC-3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003e120 min\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" 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colname=\"c2\"\u003e\u003cp\u003e3.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" 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colname=\"c1\"\u003e\u003cp\u003eMuscle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTumor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" 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colname=\"c11\"\u003e\u003cp\u003e3.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"10\" nameend=\"c11\" namest=\"c2\"\u003e\u003cp\u003e\u003cb\u003e%ID/g-max\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eOrgan\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e5 min\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e10 min\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e15 min\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e20 min\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e25 min\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e30 min\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e60 min\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e60 min\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eblocking\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e60 min\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePC-3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e\u003cb\u003e120 min\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBrain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" 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colname=\"c5\"\u003e\u003cp\u003e5.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e3.15\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e3.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e3.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLung\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e4.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e3.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLiver\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e4.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e4.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e5.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKidney\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e17.76\u0026thinsp;\u0026plusmn;\u0026thinsp;3.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15.38\u0026thinsp;\u0026plusmn;\u0026thinsp;2.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14.41\u0026thinsp;\u0026plusmn;\u0026thinsp;2.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13.37\u0026thinsp;\u0026plusmn;\u0026thinsp;2.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13.42\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e13.31\u0026thinsp;\u0026plusmn;\u0026thinsp;2.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e10.72\u0026thinsp;\u0026plusmn;\u0026thinsp;2.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e21.06\u0026thinsp;\u0026plusmn;\u0026thinsp;33.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e14.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e9.18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.77\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStomach\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e3.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e5.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e3.26\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIntestine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.79\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e7.92\u0026thinsp;\u0026plusmn;\u0026thinsp;5.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e3.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMuscle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTumor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.79\u0026thinsp;\u0026plusmn;\u0026thinsp;2.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e9.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e4.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e3.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e7.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eSelective and High Tumor Uptake of \u003csup\u003e68\u003c/sup\u003eGa-NYM080 in ccRCC Xenografts\u003c/h2\u003e\u003cp\u003eIn OS-RC-2 xenograft model, tumor uptake was evident as early as 5 min post-injection, peaking at 10.05%ID/g around 30 min. Blocking studies with cold NYM005 resulted in a significant reduction in tumor uptake, confirming the specificity of 68Ga-NYM080 for CAIX (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Notably, the tumor-to-muscle ratio (T/M) gradually increased after injection, peaked at 1 h (T/M\u0026thinsp;=\u0026thinsp;6.5), and then remained relatively stable thereafter, indicating effective tumor targeting and clearance from non-target tissues. In the ex vivo biodistribution study, tumor accumulation of \u003csup\u003e68\u003c/sup\u003eGa-NYM080 reached 11.20%ID/g at 1 h, and decreased to 8.41%ID/g at 4 h (detailed data in Supplementary Table S5).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eDistinct Tumor Accumulation of \u003csup\u003e177\u003c/sup\u003eLu-NYM080 in ccRCC Xenografts\u003c/h2\u003e\u003cp\u003eThe uptake of \u0026sup1;⁷⁷Lu-NYM080 exhibited a distinct, time-dependent increase in CAIX-positive OS-RC-2 cells, rising from 2.72% at 4 h to 12.18% at 72 h, while CAIX-negative PC3 cells showed minimal uptake of 1.09% at 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), demonstrating the excellent targeting capability of \u003csup\u003e177\u003c/sup\u003eLu-NYM080 for CAIX-overexpressing cell models.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe SPECT/CT imaging from 6 to 168 h post-injection shows significant accumulation and retention of the radiotracer in OS-RC-2 xenograft tumor (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Mild retention was observed the kidneys, which gradually decreases from 6 to 48 h. Renal uptake is no longer detectable after 96 h, while tumor retention reaches peak at 168 h, with a %ID/g of 8.79.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe ex vivo biodistribution profiles of \u003csup\u003e177\u003c/sup\u003eLu-NYM080 demonstrated high initial uptake in the kidneys, liver, and tumor, with rapid renal clearance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). \u003csup\u003e177\u003c/sup\u003eLu-NYM080 exhibited sustained and prolonged tumor retention, with uptake decreasing gradually by 168 h (detailed data in Supplementary Table S5), which underscore its potential for therapeutic application.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eTherapeutic Efficacy of \u003csup\u003e177\u003c/sup\u003eLu-NYM080 in Xenograft Animal Models\u003c/h2\u003e\u003cp\u003eIn the treatment study, two groups of OS-RC-2 mice were injected with single dose \u003csup\u003e177\u003c/sup\u003eLu-NYM080 of 74MBq (G2) or 148Mbq (G3) (twice 74 MBq doses, 4 days apart) and were well tolerated.\u003c/p\u003e\u003cp\u003eBoth doses resulted in a delay of tumor growth compared to the control group receiving normal saline (G1). In the 74 MBq single-dose group (G2), the median tumor doubling time was extended to 13.0 days (interquartile range, 9.0\u0026ndash;20.0 days) (P\u0026thinsp;=\u0026thinsp;0.064 vs. control). In the 148 MBq single-dose group (G3), the median doubling time was further prolonged to 19.5 days (interquartile range, 12.8\u0026ndash;23.0 days) (P\u0026thinsp;=\u0026thinsp;0.0054 vs. control; P\u0026thinsp;=\u0026thinsp;0.4373 vs. 74 MBq group).\u003c/p\u003e\u003cp\u003eWhile no statistical significance was observed in tumor doubling time between G2 and G3 (P\u0026thinsp;=\u0026thinsp;0.4373), a dose-dependent tumor suppression trend in tumor growth suppression was evident, with G3 demonstrating more pronounced anti-tumor efficacy compared to G2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Furthermore, histopathological evaluations of major organs (liver, kidney, heart, lung, and spleen) revealed no significant abnormalities or toxicity compared to controls, no signs of acute nephrotoxicity were observed at the doses studied. These results underscore the therapeutic efficacy of 177Lu-NYM080 and highlight the potential of fractionated dosing to enhance tumor control in ccRCC models.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCAIX is highly expressed in multiple cancers, including ccRCC, pancreatic ductal adenocarcinoma (PDAC), and breast cancer[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. For an extended period, however, clinical trials targeting CAIX with antibodies and inhibitors have not yielded satisfactory outcomes[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Among the few promising CAIX inhibitors, clinical studies have either failed to demonstrate objective responses or shown only mild responses, without significant improvement in survival. While the CAIX inhibitor SLC-0111 demonstrated a favorable safety profile in a phase I trial involving solid tumor patients, it failed to elicit objective tumor responses[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Similarly, a phase III adjuvant trial evaluating G250, a monoclonal antibody targeting CAIX, observed no improvement in survival compared with placebo controls in high-risk ccRCC patients[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTherefore, there is a clear need for more effective strategy for CAIX treatment. Radionuclide therapy targeting CAIX has been introduced a promising approach for ccRCC. Studies have demonstrated significant anti-tumor efficacy with the use of the monoclonal antibody cG250, radiolabeled with the β-emitting radionuclide \u0026sup1;⁷⁷Lu in preclinical evaluation[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Additionally, Phase I/II clinical trials with \u003csup\u003e177\u003c/sup\u003eLu-cG250 involving metastatic ccRCC patients also showed that majority of patients achieved disease stabilization[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Moreover, hG250, a humanized anti-CAIX antibody labeled with either \u003csup\u003e225\u003c/sup\u003eAc or \u003csup\u003e177\u003c/sup\u003eLu, both demonstrated excellent tolerability and significant survival prolongation in ccRCC xenograft models[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These findings underscore the potential of CAIX-targeting radionuclide therapy for ccRCC. Small-molecule peptide are generally non-immunogenic, clear rapidly from circulation and allow use of short-lived radionuclides and imaging at early time points[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHere, we developed NYM080, a new-generation CAIX-targeting small-molecule which combines acetazolamide with the chelator DOTA through a hybrid linker comprising an aliphatic spacer and a short peptide (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The chelator DOTA enables radiolabeling with both ⁶⁸Ga and \u0026sup1;⁷⁷Lu, while the aliphatic component reduces steric and electronic interference from both the peptide and DOTA chelator, preserving the nanomolar-level affinity of acetazolamide to CAIX.\u003c/p\u003e\u003cp\u003eIn general, NYM080 exhibits comparable performance to its predecessor (NYM005) in terms of CAIX-binding affinity, biodistribution profile, pharmacokinetics, and target specificity. In the present study, NYM080 showed favorable affinity o NYM080 of 9.69 nM for CAIX, similar to that of NY104 (5.75 nM) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. \u003csup\u003e68\u003c/sup\u003eGa-NYM080 exhibited rapid blood clearance and accumulated quickly in tumor. \u003csup\u003e68\u003c/sup\u003eGa-NYM080 exhibits distinct tumor uptake in CAIX-positive OS-RC-2 xenograft models, with a peak uptake of 10%ID/g at 30 min, higher than many previous CAIX-targeted molecule (\u0026le;\u0026thinsp;5%ID/g)[\u003cspan additionalcitationids=\"CR32 CR33 CR34\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The distinct reduction in tumor uptake after blocking with cold NYM005 confirmed the specificity of \u003csup\u003e68\u003c/sup\u003eGa-NYM080 for CAIX (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Remarkably, T/M reached 6.56 at 1h post-injection then remained stable, highlighting the potential of \u003csup\u003e68\u003c/sup\u003eGa-NYM080 PET/CT in detecting ccRCC lesions. These findings underscore the promise of \u003csup\u003e68\u003c/sup\u003eGa-NYM080 as an excellent imaging agent for ccRCC.\u003c/p\u003e\u003cp\u003eThe most noteworthy feature of \u003csup\u003e177\u003c/sup\u003eLu-NYM080 is its favorable prolonged tumor accumulation. High uptake of \u003csup\u003e177\u003c/sup\u003eLu-NYM080 was observed in the CAIX-positive OS-RC-2 cells, while minimal uptake was detected in the CAIX-negative PC3 cells. In xenograft model, \u003csup\u003e177\u003c/sup\u003eLu-NYM080 showed distinct tumor retention, reaching 8.7%ID/g at 168 h, while the uptake in normal organs decreased overtime (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), which is comparable to that of \u003csup\u003e177\u003c/sup\u003eLu-cG250 in the SK-RC-52 model (\u0026lt;10%ID/g in tumors)[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The imaging pattern of \u003csup\u003e177\u003c/sup\u003eLu-NYM080, characterized by increasing tumor uptake and decreasing background activity, suggests its sustained target engagement and reduced off-target toxicity. The \u0026ldquo;sink-in\u0026rdquo; phenomenon observed in SPECT imaging is likely attributed to the high binding affinity and prolonged retention of NYM080 in CAIX-overexpressing tumor tissue, leading to sustained target engagement. Additionally, rapid clearance from non-target tissues such as kidneys and liver may further enhance the visual contrast, creating the apparent \u0026ldquo;sink-in\u0026rdquo; imaging effect over time. Similar observations were reported for other therapeutic radioligands such as \u0026sup1;⁷⁷Lu-PSMA-617, where prolonged tumor retention correlated with improved therapeutic outcomes in prostate cancer[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Furthermore, \u003csup\u003e177\u003c/sup\u003eLu-NYM080 exhibited a dose-dependent anti-tumor trend in ccRCC model, with the best tumor suppression observed in the group receiving twice 74MBq injections, suggesting the potential of increasing the administered dose to enhance therapeutic efficacy. For other radioligand therapies such as \u003csup\u003e177\u003c/sup\u003eLu-DOTATATE in neuroendocrine tumors, dose escalation is a well-established approach to maximize therapeutic effect leading to a more sustained tumor reduction over time while maintaining an acceptable toxicity profile[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], which also aligns with clinical multiple dosing schedules. The biodistribution profiles vary among different \u003csup\u003e177\u003c/sup\u003eLu-labeled agents, precluding the establishment of a universal dose range. Preclinical studies show that 111 MBq of \u003csup\u003e177\u003c/sup\u003eLu‑PSMA‑617 improved survival (53.8% vs 30.7%) over 37 MBq in bone metastasis models[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and dose escalation up to 3 \u0026times; 60 MBq was also safely achieved with \u003csup\u003e177\u003c/sup\u003eLu‑NeoB[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Given there is no well-established dosing strategy for CAIX-targeted \u003csup\u003e177\u003c/sup\u003eLu therapies, 74 MBq and 2\u0026times;74 MBq were optimized to balance treatment efficacy with safety considerations.\u003c/p\u003e\u003cp\u003e\u0026sup1;⁷⁷Lu-NYM080 demonstrated a favorable safety profile. The ex vivo biodistribution of \u003csup\u003e177\u003c/sup\u003eLu-NYM080 showed minimal accumulation in kidneys and intestines (\u0026lt;1%ID/g at 48h; \u0026lt;0.1%ID/g at 168h), and there was no evidence of organ damage in normal tissues upon histopathological examination in both G2 and G3. This low toxicity profile supports its clinical viability, particularly for repeated dosing regimens.\u003c/p\u003e\u003cp\u003eRecently, PSMA PET/CT has been reported as a useful tool in ccRCC lesion detection, with pooled detection rate of 83% in primary or metastatic ccRCC[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Compared to conventional CT for detection of metastatic disease, Rhee et al. reported \u003csup\u003e68\u003c/sup\u003eGa-PSMA-HBED-CC PET/CT had significantly higher sensitivity (92% vs. 69%) and positive predictive value (97% vs. 80%) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. \u003csup\u003e18\u003c/sup\u003eF-DCFPyL PET/CT also demonstrated superior sensitivity to conventional CT or MRI (95 versus 79%)[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Besides, there is an ongoing phase I/II trial designed to assess the tolerability and effectiveness of \u003csup\u003e177\u003c/sup\u003eLu-PSMA-1 in individuals with PSMA-positive metastatic ccRCC[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. While prostate-specific membrane antigen (PSMA) has been explored as a theranostic target in ccRCC, its clinical utility is limited by its rapid washout due to expression on neovascular endothelial cells, leading to reduced tumor retention over time. In addition, PSMA is expressed on the neovasculature of many other solid malignancies, benign conditions and various RCC subtypes[\u003cspan additionalcitationids=\"CR47 CR48 CR49\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In contrast, CAIX demonstrates prolonged retention as it is stably over-expressed on the cell membrane of ccRCC. Combined with its high specificity for ccRCC, CAIX stands as a promising theranostic target.\u003c/p\u003e\u003cp\u003eCurrently, there are very few peptide-based CAIX-targeting small molecule \u003csup\u003e68\u003c/sup\u003eGa/\u003csup\u003e177\u003c/sup\u003eLu theranostic pair[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], and NYM080 offers a novel potential strategy for ccRCC imaging and therapy. The favorable tumor retention and dose-dependent antitumor efficacy demonstrated by NYM080 provide compelling evidence for the translational potential of the CAIX target. These findings strongly support the continued development of CAIX-targeted therapies in the future. Another novel CAIX-targeted theranostic molecule has recently been developed, and a corresponding Phase I clinical study is currently underway to evaluate its theranostic capabilities in metastatic ccRCC patients (ClinicalTrials.gov identifier: NCT06649682).\u003c/p\u003e\u003cp\u003eThis study has certain limitations. Firstly, due to the proof-of-concept nature of this study, human participant data and radiation dosimetry evaluations were not included, limiting the translation of these findings to clinical settings. Secondly, the pharmacokinetic properties and biodistribution observed in animal models may differ from those in humans, potentially affecting the predictability of therapeutic efficacy and toxicity profiles. Thirdly, while the remarkable tumor uptake and therapeutic efficacy highlight the value of NYM080 as a CAIX-targeted theranostic agent, its non-relatively high initial gastrointestinal and renal uptake necessitates further molecular optimization Furthermore, the short follow-up period did not allow for assessment of the long-term toxicity and side effects of the radiolabeled ligand, long-term toxicity safety profile needs to be assessed in future studies.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis is preclinical study demonstrated distinct tumor uptake of both \u003csup\u003e68\u003c/sup\u003eGa-NYM080 and \u003csup\u003e177\u003c/sup\u003eLu-NYM080. Furthermore, \u003csup\u003e177\u003c/sup\u003eLu-NYM080 showed marked anti-tumor effect in ccRCC xenograft models, highlighting its therapeutic potential for ccRCC patients.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCAIX: Carbonic anhydrase IX\u003c/p\u003e\n\u003cp\u003eccRCC: Clear cell renal cell carcinoma\u003c/p\u003e\n\u003cp\u003eVHL: von Hippel-Lindau\u003c/p\u003e\n\u003cp\u003eHIF-1\u0026alpha;: Hypoxia-inducible factor 1-alpha\u003c/p\u003e\n\u003cp\u003eTKI: Tyrosine kinase inhibitor\u003c/p\u003e\n\u003cp\u003eICI: Immune checkpoint inhibitor\u003c/p\u003e\n\u003cp\u003eSUV: Standardized uptake value\u003c/p\u003e\n\u003cp\u003e%ID/g: Percentage injected dose per gram\u003c/p\u003e\n\u003cp\u003eSD: Standard deviation\u003c/p\u003e\n\u003cp\u003eT/M: Tumor-to-muscle ratio\u003c/p\u003e\n\u003cp\u003eT/H: Tumor-to-heart ratio\u003c/p\u003e\n\u003cp\u003eDOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid\u003c/p\u003e\n\u003cp\u003eNOTA: 1,4,7-triazacyclononane-1,4,7-triacetic acid\u003c/p\u003e\n\u003cp\u003eANOVA: Analysis of variance\u003c/p\u003e\n\u003cp\u003ePDAC: Pancreatic ductal adenocarcinoma\u003c/p\u003e\n\u003cp\u003ePSMA: Prostate-specific membrane antigen\u003c/p\u003e\n\u003cp\u003eNCCN: National Comprehensive Cancer Network\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Peking Union Medical College Hospital. All procedures involving animals \u003cem\u003ewere performed following the National Research Council’s Guide for the Care and Use of Medical Laboratory Animals (Ministry of Health, China). All efforts were made to minimize animal suffering.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data will be shared on reasonable request to the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant No. U24A20758); Beijing Natural Science Foundation (Grant No. L242062); National Key R\u0026amp;D Program of China (Grant No. 2024YFC2419400); Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (Grant No. CIFMS-2024-I2M-ZD-001, CIFMS-2023-I2M-2-002, CIFMS-2021-I2M-1-002, CIFMS-2021-I2M-1-003, CIFMS-2021-I2M-1-025); National High Level Hospital Clinical Research Funding (Grant No. 2022-PUMCH-D-001, 2022-PUMCH-D-002).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXY: conceptualization; data curation and analysis; drafting original manuscript. WZ, ML and LH: writing guidance and revision. CR, YL, ZH, and HZ: technical support; data validation. GZ and XL: clinical consultation. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge\u0026nbsp;the National Natural Science Foundation of China (Grant No. U24A20758); Beijing Natural Science Foundation (Grant No. L242062); National Key R\u0026amp;D Program of China (Grant No. 2024YFC2419400); Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (Grant No. CIFMS-2024-I2M-ZD-001, CIFMS-2023-I2M-2-002, CIFMS-2021-I2M-1-002, CIFMS-2021-I2M-1-003, CIFMS-2021-I2M-1-025); National High Level Hospital Clinical Research Funding (Grant No. 2022-PUMCH-D-001, 2022-PUMCH-D-002) for supporting our work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eParkkila S, Parkkila A-K, Saarnio J, Kivel\u0026auml; J, Karttunen TJ, Kaunisto K, et al. 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Ann Nucl Med. 2015;29:877\u0026ndash;82. \u003c/li\u003e\n\u003cli\u003eKryza D, Vinceneux A, Bidaux A-S, Garin G, Tatu D, Cropet C, et al. A multicentric, single arm, open-label, phase I/II study evaluating PSMA targeted radionuclide therapy in adult patients with metastatic clear cell renal cancer (PRadR). BMC Cancer. 2024;24:163. \u003c/li\u003e\n\u003cli\u003ePyka T, Weirich G, Einspieler I, Maurer T, Theisen J, Hatzichristodoulou G, et al. \u003csup\u003e68\u003c/sup\u003eGa-PSMA-HBED-CC PET for Differential Diagnosis of Suggestive Lung Lesions in Patients with Prostate Cancer. J Nucl Med. 2016;57:367\u0026ndash;71. \u003c/li\u003e\n\u003cli\u003eArdies PJ, Gykiere P, Goethals L, De Mey J, De Geeter F, Everaert H. PSMA Uptake in Mediastinal Sarcoidosis. Clin Nucl Med. 2017;42:303\u0026ndash;5. \u003c/li\u003e\n\u003cli\u003eWang H, Wang S, Song W, Pan Y, Yu H, Si T, et al. Expression of Prostate-Specific Membrane Antigen in Lung Cancer Cells and Tumor Neovasculature Endothelial Cells and Its Clinical Significance. PLoS ONE. 2015;10:e0125924. \u003c/li\u003e\n\u003cli\u003eHangaard L, Jochumsen MR, Vendelbo MH, Bouchelouche K. Metastases From Colorectal Cancer Avid on 68Ga-PSMA PET/CT. Clin Nucl Med. 2017;42:532\u0026ndash;3. \u003c/li\u003e\n\u003cli\u003eSilver DA, Pellicer I, Fair WR, Heston WD, Cordon-Cardo C. Prostate-specific membrane antigen expression in normal and malignant human tissues. Clin Cancer Res. 1997;3:81\u0026ndash;5. \u003c/li\u003e\n\u003cli\u003eMassi\u0026egrave;re F, Wiedemann N, Borrego I, Hoehne A, Osterkamp F, Paschke M, et al. Preclinical Characterization of DPI-4452: A\u003csup\u003e68\u003c/sup\u003eGa/\u003csup\u003e177\u003c/sup\u003eLu Theranostic Ligand for Carbonic Anhydrase IX. J Nucl Med. 2024;65:761\u0026ndash;7. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejre","sideBox":"Learn more about [EJNMMI Research](http://ejnmmires.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ejre/default.aspx","title":"EJNMMI Research","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"carbonic anhydrase IX, clear cell renal cell carcinoma, theranostics, 68Ga/177Lu, radionuclide therapy","lastPublishedDoi":"10.21203/rs.3.rs-6954806/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6954806/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eCarbonic anhydrase IX (CAIX) is a hypoxia-regulated enzyme overexpressed in clear cell renal cell carcinoma (ccRCC) but minimally in normal tissues, making it a promising theranostic target. Current imaging modalities for ccRCC face limitations in accuracy and nephrotoxicity, while systemic therapies are constrained by resistance and variable efficacy. This study evaluates a novel CAIX-targeting theranostic pair, \u003csup\u003e68\u003c/sup\u003eGa/\u003csup\u003e177\u003c/sup\u003eLu-NYM080, for imaging and therapy in preclinical ccRCC models.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe in vivo and ex vivo biodistribution of \u003csup\u003e68\u003c/sup\u003eGa/\u003csup\u003e177\u003c/sup\u003eLu-NYM080 was evaluated in CAIX-positive OS-RC-2 xenograft bearing models, and the therapeutic efficacy of \u003csup\u003e177\u003c/sup\u003eLu -NYM080 (74 MBq single dose vs. 74 MBq\u0026times;2 doses) was also investigated. NYM080 exhibited high CAIX affinity (Kd\u0026thinsp;=\u0026thinsp;9.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 nM). \u003csup\u003e68\u003c/sup\u003eGa-NYM080 showed rapid tumor uptake (peak 10.05%ID/g at 30 min) and specificity (blocking reduced uptake by 55%). Tumor-to-muscle ratios reached 6.5 at 1h. \u003csup\u003e177\u003c/sup\u003eLu-NYM080 demonstrated prolonged tumor retention (8.79%ID/g at 168 h) and a trend toward enhanced therapeutic efficacy with increased dosing: single dose of 74 MBq delayed tumor doubling to 13.0 days (vs. control, P\u0026thinsp;=\u0026thinsp;0.064), while 74 MBq\u0026times;2 doses extended it to 19.5 days (P\u0026thinsp;=\u0026thinsp;0.0054 vs. control). Minimal off-target uptake and no acute toxicity were observed.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003e\u003csup\u003e68\u003c/sup\u003eGa/\u003csup\u003e177\u003c/sup\u003eLu-NYM080 is a promising theranostic pair for ccRCC, combining high tumor specificity, prolonged tumor retention, and favorable therapeutic efficacy.\u003c/p\u003e","manuscriptTitle":"Preclinical Evaluation of a Novel Carbonic Anhydrase IX-Targeting 68Ga/177Lu Theranostic Pair for Clear Cell Renal Cell Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-11 12:21:27","doi":"10.21203/rs.3.rs-6954806/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-09-06T02:09:18+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-04T20:49:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-04T08:48:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"EJNMMI Research","date":"2025-09-03T23:11:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejre","sideBox":"Learn more about [EJNMMI Research](http://ejnmmires.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ejre/default.aspx","title":"EJNMMI Research","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3e0e8975-153d-44db-a7c1-f2de48e045c7","owner":[],"postedDate":"September 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-09-11T12:21:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-11 12:21:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6954806","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6954806","identity":"rs-6954806","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}