A Pretargeted Imaging Strategy for EGFR Positive Colorectal Carcinoma via the Modulation of Tz-radioligand Pharmacokinetics

Objective: Previously, we successfully developed a pretargeted imaging strategy (Atezolizumab-TCO/99mTc-HYNIC-PEG11-Tz), which is a powerful tool for evaluating Programmed Cell Death Ligand-1 (PD-L1) expression in xenograft mice tumor models. However, the surplus unclicked 99mTc-HYNIC-PEG11-Tz is cleared somewhat sluggishly through the intestines. This is certainly not an ideal situation for imaging for colorectal cancer (CRC). In order to shift the excretion of the Tz-radioligand to the renal system, we have sought to develop a novel Tz-radioligand by adding a polypeptide linker between HYNIC and PEG11. Methods: Pretargeted molecular probes 99mTc-HYNIC-Polypeptide-PEG11-Tz and Cetuximab-TCO were synthesized. The stability of 99mTc-HYNIC-Polypeptide-PEG11-Tz was evaluated in vitro, and its blood pharmacokinetic test was performed in vivo. In vitro ligation reactivity of 99mTc-HYNIC-Polypeptide-PEG11-Tz towards Cetuximab-TCO was tested. The biodistribution and imaging of 99mTc-HYNIC-Polypeptide-PEG11-Tz was performed to observe the clear pathway of this novel Tz-radioligand. Pretargeted biodistribution of three different accumulation intervals was performed to determine the optimal pretargeted interval time. Comparison of pretargeted (Cetuximab-TCO 48 h/99mTc-HYNIC-PEG11-Tz 6 h) and (Cetuximab-TCO 48 h/99mTc-HYNIC-Polypeptide-PEG11-Tz 6 h) imagings was performed to show the effect of the two Tz-radioligands with different excretion pathway on tumor imaging. Results: 99mTc-HYNIC-Polypeptide-PEG11-Tz showed favorable in vitro stability and rapid blood clearance in mice. SEC-HPLC imaging strategy using Cetuximab-TCO/99mTc-HYNIC-Polypeptide-PEG11-Tz could be used for diagnosing CRC since the surplus unclicked 99mTc-HYNIC-Polypeptide-PEG11-Tz is cleared through urinary system and produces low abdominal uptake background. Conclusion: We developed a novel pretargeted imaging strategy (Cetuximab-TCO/99mTc-HYNIC-Polypeptide-PEG11-Tz) for imaging CRC since the surplus unclicked 99mTc-HYNIC-Polypeptide-PEG11-Tz produces low abdominal uptake background, which broadens the application scope of pretargeted imaging strategy. Atezolizumab-TCO Atezolizumab-TCO was binded specifically to PD-L1 overexpressed by tumor and concomitantly from slowly. (3) Then 99m Tc-HYNIC-PEG 11 -Tz was injected. (4) 99m Tc-HYNIC-PEG 11 binded pretargeted Atezolizumab-TCO in vivo click ligation Tz-radioligand. According to the results of pretargeted biodistribution study, we chose the relatively most suitable accumulation interval (48 h) to compare the pretargeted Cetuximab-TCO/ 99m Tc-HYNIC-PEG 11 -Tz and Cetuximab-TCO/ 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz biodistribution and SPECT imaging using subcutaneous HCT116 xenografts mice. The results demonstrated that the surplus unclicked 99m Tc-HYNIC-PEG 11 -Tz is cleared rapidly through the intestines into the faces and remained in it for a long


Introduction
Colorectal cancer (CRC) is a common malignant tumor of the gastrointestinal tract with its morbidity ranking 3th among all malignant tumors and its mortality ranking 4th in the world, which poses a serious threat to human health [1]. In recent years, with the changes of lifestyle, the incidence of CRC has been increased year by year. Early diagnosis and treatment will greatly improve the prognosis of CRC, most of which could be cured in the early stage with a 5-year survival rate of 90%, while only 12.5% for advanced CRC [2]. Therefore, it is greatly significant for the diagnosis of CRC, especially for the early diagnosis, to research and develop the specific targeted molecular probe for CRC. At present, the clinical diagnosis for CRC mainly relies on endoscopy and endoscopy-guided biopsy, imaging methods such as computed tomography (CT), magnetic resonance imaging (MRI), and 2deoxy-2-[ 18 F]fluoro-D-glucose ( 18 F-FDG) positron emission tomography/computed tomography (PET/CT). Invasive pathological biopsy guided by endoscopy is still the gold standard for clinical diagnosis of CRC. Common endoscopy mainly relies on morphological changes for the diagnosis of CRC, and it is easy to miss lesions with insignificant morphological changes, especially for early and micro lesions [3]. With the development of magnifying endoscopy, confocal laser endoscopy, and fluorescence endoscopy, endoscopic techniques could detect microscopic pathological changes and increase the diagnosis level of CRC. However, it is still difficult to detect surrounding tissue invasion and distant metastasis and achieve targeted imaging of CRC for these endoscopic methods.
Anatomical imaging methods such as endoscopic ultrasonography, CT, or MRI have obvious advantages in the evaluation of surrounding tissue infiltration, lymph node and distant metastasis. 4 But the imaging techniques are not specific for the diagnosis of CRC. As a functional imaging technology, 18 F-FDG PET/CT can reflect the tumor glucose metabolism. However, the most common positron radioactive tracer, 18 F-FDG, is still not a specific targeting molecular probe for CRC.
Currently, targeted molecular probes for CRC mainly include antibody [4][5][6], polypeptide [7,8], and nanoparticle [9,10] probes for epithelial growth factor receptor (EGFR) or vascular endothelial growth factor (VEGF). Antibody probes have exquisite affinity and selectivity for molecular targets such as EGFR or VEGF overexpressed by CRC. However, due to the slow pharmacokinetics, the use of antibodies as tracers requires labeling with isotopes with long half-lives (e.g., 111 In, 64 Cu, or 131 I), which significantly increases the radiation dose to non-target tissues. Polypeptide probes have several advantages over antibody probes including low molecular weight, reduced circulation time, easy access to target sites, and passivity to the immune system with little or no immunogenicity. However, the stability and affinity to target sites vary among different polypeptide tracers. Nanoparticle probes are equipped with different targeting units for different receptors, but this type of probe poses a potential threat of immunogenicity and renal toxicity.
EGFR has become a therapeutic target for CRC due to the close association of the EGFR expression with disease progression and metastasis [11]. The therapeutic antibody Cetuximab has been approved by FDA for treating CRC. As EGFR inhibitor, Cetuximab specifically targets the extracellular domain of the EGFR and block the intracellular tyrosine kinase activity. Moreover, molecular tracers using Cetuximab labeled with the long-lived radionuclides (such as 111 In, 89 Zr, 64 Cu, and 124 I) have been developed to detect the EGFR expression and evaluate the therapeutic response to EGFRblocking [12][13][14][15][16]. However, extended circulation time of radiolabeled antibodies creates prohibitively high radiation dose to healthy organs and low tumor/background imaging contrast. In this study, we will develop a novel pretargeted imaging strategy for evaluating EGFR expression of CRC.
Previously, we developed a pretargeted single photon emission computed tomography (SPECT) imaging strategy for evaluating immune checkpoint ligand PD-L1 expression in tumor based on bioorthogonal Diels-Alder click chemistry [17]. The molecular probe mainly includes two components: 5 TCO-modified Atezolizumab (Atezolizumab-TCO) that could target PD-L1 and 99m Tc labeled Tzradioligand ( 99m Tc-HYNIC-PEG 11 -Tz). Pretargeted (Atezolizumab-TCO/ 99m Tc-HYNIC-PEG 11 -Tz) imaging involve following steps: (1) Atezolizumab-TCO was first injected into the bloodstream. (2) Atezolizumab-TCO was binded specifically to PD-L1 overexpressed by tumor and concomitantly was cleared from the blood slowly. (3) Then 99m Tc-HYNIC-PEG 11 -Tz was injected. (4) 99m Tc-HYNIC-PEG 11 -Tz was binded to pretargeted Atezolizumab-TCO via in vivo click ligation of the two components, followed by the rapid clearance of the excess Tz-radioligand. The pretargeted imaging strategy clearly delineated PD-L1 expressing H1975 human lung cancer xenografts with high imaging contrast and significantly reduced background radiation dose to nontarget organs. However, the surplus unclicked 99m Tc-HYNIC-PEG 11 -Tz is cleared somewhat sluggishly through the intestines. This is, of course, not an ideal situation for imaging abdominal tumor, especially for CRC. Therefore, we will further try to develop a novel Tz-radioligand with more favorable pharmacokinetic profile.
We will add a polypeptide chain containing hydrophilic amino acids between the HYNIC and Tz to shift the excretion of the Tz-derivative to the renal system, thereby reducing abdominal uptake background and facilitating imaging abdominal tumors. Technetium ( 99m Tc) is the most widely used radionuclide for diagnosis in SPECT imaging, which is easily obtained from 99 Mo/ 99m Tc generator and presents a appropriate half-life (6.02 h) and electron emission energy (140 keV). Thus, 99m Tc was still chosen as the radionuclide in the study. Ultimately, we developed another pretargeted imaging strategy based on the combination of Cetuximab-TCO and 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz for EGFR expression of CRC due to the renal excretion of the novel Tz-radioligand, which further broaden the application scope of pretarged imaging strategy.

Reagents and materials
All reagents and solvents were obtained from commercial sources (Sigma-Aldrich, Peprotech, Merda, Thermo, BD Bioscience, Invitrogen, Millipore, Nest, Hyclone, and Corning) and used as received without further purification. Cetuximab solutions were obtained from Selleck Chemicals. Aminereactive TCO-NHS was purchased from Click Chemistry Tools. 3-(4-Benzylamino)-1,2,4,5-tetrazine (Tz) bearing an amine-reactive linker and O-(2-aminoethyl)-O′-[2-(boc-amino)ethyl]-decaethylene glycol (NH 2 -PEG 11 -NHBoc) were purchased from Xi'an KaiXin Biological. Na[ 99m Tc]TcO 4 was purchased from Shanghai Atom Kexing Pharmaceutical Co., Ltd. All solvents used for HPLC analysis within this project were HPLC grade. 3.2 µl N,N-Dimethylformamide (DMF) and 2.8 µl 8 mg/ml HYNIC-Polypeptide-PEG 11 -Tz solution in DMF were added to 24 µl of the product obtained in above step, resulting in a total volume of 30 µl and a Tz/Cetuximab ration of 20. As a control group, 3.2 µl DMF and 2.8 µl 8 mg/ml HYNIC-Polypeptide-PEG 11 -Tz solution in DMF were added to 24 µl PBS solution to make the total volume of the reaction solution to be 30 µl. The reaction solution was incubated with gentle shaking at 37 ℃ for 20 minutes in dark, and then the consumption of HYNIC-Polypeptide-PEG 11 -Tz was determined with reversedphase high performance liquid chromatography (RP-HPLC) to calculate the average number of TCO conjugated to each Cetuximab.

Synthesis of Tz derivatives, radiolabelling with 99m Tc and in vitro stability assay
We designed the chemical structures of HYNIC-PEG 11 -Tz and HYNIC-Polypeptide-PEG 11 -Tz, as indicated in Figure 1. Detailed synthesis route procedures are described in our previous work [17] and

Distribution coefficient assay
10 µl of radiolabelled 99m Tc-HYNIC-PEG 11 -Tz or 9m Tc-HYNIC-Polypeptide-PEG 11 -Tz was placed in an Eppendorf tube containing 500 μl of n-octanol and 500 μl of PBS (pH = 7.4) solutions. The tube was vortexed vigorously for 1 min and then centrifuged at 12000 rpm for 5 minutes. The n-octanol/PBS (pH = 7.4) distribution coefficient was calculated in triplicate for each product. For each replicate, 10 μl solution was collected from both phases. Finally, the radioactivity of each fraction was measured in a γ-counter. The distribution coefficient was calculated as logD (logD = log 10 n-octanol counts/aqueous phase counts).

Pretargeted cell immunoreactivity binding assay
HCT116 human CRC cells were obtained from Cell Bank of the Chinese Academy of Sciences. The cells were cultured in RPMI-1640 medium, which was supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were cultured at 37 °C in a humidified atmosphere with 5% CO 2 .
HCT116 cells were seeded in 24-well plate and grown to 80% confluence. Experimental group and control group cells were incubated for 2 h with Cetuximab-TCO (100 nM) and Cetuximab in 500 μl RPMI 1640 medium, respectively. Blocking group cells were pretreated by adding an excess of unmodified Cetuximab (100-fold) to the wells 2 h prior to incubation with Cetuximab-TCO. Then, the cells were washed with PBS three times and incubated for 30 min with 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz (0.1 μg, 4.63 MBq/μg) in 200 μl PBS. After the incubation period, the cells were washed with PBS three times and then lysed with aqueous NaOH (2 M). The membrane-bound activity of each group was determined by measuring the radioactivity of lysed cells using a γ-counter.

In vivo pharmacokinetics, biodistribution and imaging of 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz
The pharmacokinetics, biodistribution and SPECT imaging studies of 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz alone were performed to investigate its pharmacokinetic profile, organ biodistriution and excretion pathway. Animal care and experimental procedures were approved by the Animals Ethics Committee of Zhongshan Hospital, Fudan University. For pharmacokinetics study, tumor-bearing mice (n = 3) were injected intravenously with 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz and then blood from the tail vein was collected at serial time points (1, 3, 5, 7, 10, 15, 30 and 60 min). The blood samples were weighed and measured in a γ-counter along with standards to determine the percentage of the injected activity per gram of tissue mass (%IA/g).
For biodistribution and imaging studies, male athymic nude mice bearing subcutaneous HCT116 (right shoulder) xenografts were administered 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz (16.65-18.50 MBq in 100 μl 0.9% sterile saline) via intravenous tail vein. Micro-SPECT/CT scans were conducted on the Nano SPECT/CT scanner (BioScan) at 30 min, 2 h, and 6 h post Tz-radioligand injection. Anesthesia and SPECT scan methods refer to our previous work [17]. Biodistribution experiments were performed at the same time point post Tz-radioligand injection with SPECT imaging. The mice were anesthetized with isoflurane and euthanized by cervical dislocation. Blood was withdrawn by heart puncture, and each organ and tissue of interest was harvested, blotted dry, and weighed. The radioactivity of each sample was measured using a γ-counter along with standards to determine the %IA/g for each sample of interest.

Pretargeted biodistribution study
Male athymic nude mice bearing subcutaneous HCT116 xenografts were administered 100 μg Cetuximab-TCO in 100 μl PBS via intravenous tail vein injection (The injected dose of 100 µg mAb-TCO was according to the published pretargeted tumor imaging studies [17][18][19][20][21][22][23][24]). In order to validate the optimal pretargeted interval time, three different accumulation intervals of 24 h, 48 h, and 72 h were performed in the pretargeted biodistribution study (n = 3). After three pretargeted accumulation periods, the same mice were then administered 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz (16.65-18.50 MBq in 100 μl 0.9% sterile saline), also via tail vein injection. Six hours after the Tz-radioligand administration, the animals were anesthetized with isoflurane and blood was withdrawn by heart puncture (The time point of 6 h was chosen according to the results of our previous investigation [17]). Then the mice were euthanized by cervical dislocation, and tissue samples including tumor, heart, lung, liver, spleen, stomach, small intestine, large intestine, kidney, muscle, bone, skin, brain, thyroid, faces (in distal colon), and urine were harvested, blotted dry, and weighed. The radioactivity of all samples was measured in a γ-counter to determine the %IA/g.

Pretargeted biodistribution and imaging comparison
Nude mice bearing subcutaneous HCT116 xenografts were administered 100 μg Cetuximab-TCO via intravenous tail vein injection. After the appropriate accumulation interval (the highest tumor/blood ratio) of 48 h according to the results of pretargeted biodistribution study, the mice were then administered 99m Tc-HYNIC-PEG 11 -Tz or 99m Tc-HYNIC-Polypeptide-PEG 11  The in vitro stability of 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz was tested by incubation in NS, PBS, and FBS at 37 °C and subsequent HPLC analysis. As indicated in Figure 3, the Tz-radioligand remained relatively stable in the three media at early time point (generally above 85% radiochemical integrity in the three media after 2 h incubation). With time, however, decomposition of 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz becomes apparent, especially in FBS (only 72.93 ± 2.51% and 63.10 ± 1.77% of the Tz-radioligand remained intact after 4 h and 8 h incubation, respectively). The in vitro stability of 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz was similar to that of 99m Tc-HYNIC-PEG 11 -Tz [17]. The in vitro reactivity of 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz towards Cetuximab-TCO was tested in PBS at 37 ℃ for 30 min. Subsequently, a radio-SEC-HPLC analysis was performed. SEC-HPLC showed that the cycloaddition reaction between 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz and Cetuximab-TCO proceeded near completely, with the 8:1 Tz-to-mAb reaction stoichiometry providing an average yield of 87.83 ± 3.27% (Figure 4).

Pretargeted HCT116 cell immunoreactivity binding assay
Pretargeted cell immunoreactivity binding assay was performed using high EGFR-expressing HCT116 cell line. Pretargeted experimental group showed a high radioactivity retention in cells with an average counts per minute (CPM) value of 19,045 ± 557 ( Figure 5). The control and blocking group cells showed a significantly lower activity with an average CPM value of 323 ± 52 and 2,326 ± 341, respectively (P < 0.05). The pretargeted cell immunoreactivity binding assay confirmed the specific binding of Cetuximab-TCO to HCT116 cells and verified preliminarily the feasibility of the molecular probes used for in vivo pretargeted imaging.

Molecular probe in vivo experiments
The pharmacokinetics, biodistribution and SPECT imaging experiments of 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz alone were first performed to investigate its pharmacokinetic profile, tissue biodistribution and excretion pathway. The Tz-radioligand was cleared quickly from the circulation and distributed rapidly into various tissues and organs.  Table 2 and Figure 9). Biodistribution experiments demonstrated the accumulation in kidneys and urine was relatively high with a %IA/g of more than 3.0 at each accumulation interval. After filtering through the kidneys, the radiotracer concentrated in the bladder and finally excreted out of the body through urine. In addition to urinary system, high activity levels were observed in the tumor and blood (>1 %IA/g at each accumulation interval), suggesting a great incidence of in vivo click reactions in the blood in addition to ligations at the tumor. The tumor/blood ratio was 0.83 ± 0.13, 1.40 ± 0.31, and 1.15 ± 0.21, respectively after allowing 24 h, 48 h, and 72 h for accumulation of Cetuximab-TCO in HCT116 tumor. One-way analysis of variance revealed that the difference of tumor/blood ratio for 24 h, 48 h, and 72 h pretargeted interval was significant (F = 5.357, P < 0.05). Pair-wise comparisons of any two pretargeted intervals using LSD test revealed that, although tumor/blood ratio for 48 h and 72 h accumulation interval was not statistically different (P = 0.191), the difference of tumor/blood ratio for 24 h and 48 h accumulation interval was significant (P < 0.05). Thus, it becomes clear that 48 h represents a relatively more appropriate accumulation interval between the administration of mAb-TCO and the subsequent injection of Tz-radioligand. The accumulation and retention of the radiotracer in other organs including hypervascular lung, liver, and spleen remained generally very low, which suggested that the pretargeted strategy could efficiently improve the target/non-target ratio and significantly reduce background radiation dose to nontarget organs. Statistical analysis revealed that the radiotracer accumulation in HCT116 tumors was significantly higher than that in each organ/tissue (including heart, lung, liver, spleen, stomach, small intestine, large intestine, muscle, bone, skin, brain, and thyroid) for each pretargeted interval of 24 h, 48 h or 72 h (all P < 0.05).
According to the results of pretargeted biodistribution study, the most suitable accumulation interval (48 h) was chosen as the accumulation period of Cetuximab-TCO to compare the pretargeted Cetuximab-TCO/ 99m Tc-HYNIC-PEG 11 -Tz and Cetuximab-TCO/ 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz biodistribution and SPECT imaging (Supplementary Table 3 and Figure 10). As 99m Tc-HYNIC-PEG 11 -Tz was cleared rapidly via the hepatobiliary system, the radioactivity in the liver, small intestine, and large intestine was relatively low with a %IA/g of 0.19 ± 0.11, 0.08 ± 0.13, and 0.08 ± 0.16, respectively at 6 h post 99m Tc-HYNIC-PEG 11 -Tz injection. The amount of activity in faces was relatively high (28.55 ± 24.66 %IA/g), suggesting that the surplus unclicked 99m Tc-HYNIC-PEG 11 -Tz is cleared rapidly through the intestines into the faces and remained in it. Additionally, a small amount of 99m Tc-HYNIC-PEG 11 -Tz was cleared through urinary system and the accumulation of urine was 1.20 ± 1.98 %IA/g after injecting the Tz-radioligand for 6 h. Pretargeted Cetuximab-TCO/ 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz biodistribution demonstrated that the accumulation in kidneys and urine was relatively high with a %IA/g of 3.42 ± 0.61 and 21.98 ± 7.18, respectively. The uptake levels in liver, intestines, and faces were all less more 0.4 %IA/g, indicating that the surplus unclicked 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz is cleared through urinary system. The tumor/blood ratio was 1.61 ± 0.12 and 1.40 ± 0.31, respectively for pretargeted Cetuximab-TCO/ 99m Tc-HYNIC-PEG 11 -Tz and Cetuximab-TCO/ 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz biodistribution experiment. Both pretargeted Cetuximab-TCO/ 99m Tc-HYNIC-PEG 11 -Tz and Cetuximab-TCO/ 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz imagings delineated the HCT116 tumor clearly and the imaging results were consistent with the results of biodistribution, as indicated in Figure 11. Pretargeted Cetuximab-TCO/ 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz imaging avoided the retention of the surplus unclicked Tz-radioligand in faces, which favors to specific targeting imaging for CRC.

Discussion
The pretargeted strategy (Atezolizumab-TCO/ 99m Tc-HYNIC-PEG 11 -Tz) that we previously developed could evaluate immune checkpoint ligand PD-L1 expression in tumor and simultaneously reduce the radiation dose to non-target organs [17]. However, the surplus unclicked 99m Tc-HYNIC-PEG 11 -Tz was cleared through hepatobiliary system and remained in faces for a long time, which is not favorable for imaging abdominal tumor, especially for CRC. In order to further broaden the application scope of pretarged imaging strategy, we added a polypeptide chain between the HYNIC and Tz to shift the excretion of the Tz-radioligand to the renal system, thereby facilitating imaging CRC.
In order to improve the hydrophilicity of previous 99m Tc-HYNIC-PEG 11 -Tz to facilitate renal excretion, we chose hydrophilic lysine, glutamate, and arginine in designing polypeptide sequence. Ultimately, the polypeptide sequence was determined as Gly-Arg-Glu-Arg-Glu-Lys and was synthesized successfully through the Fmoc-method. In order not to increase the steric resistance of click reaction between Cetuximab-TCO and Tz-radioligand, we added the polypeptide chain between HYNIC and PEG 11 . Finally, the modified Tz-derivative structure was identified as HYNIC-Polypeptide-PEG 11 -Tz.
The urinary excretion of the polypeptide-modified 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz is probably related to the increased hydrophilicity of the Tz-derivative results from hydrophilic amino acids and the presence of positive charge and lysine residue in the polypeptide chain. It is known that the Tzscaffold part in the Tz-radioligands is hydrophobic. It is necessary to add hydrophilic structure to improve the hydrophilicity of the final Tz-derivative in the synthetic design of Tz-derivative. In the study conducted by Garcia et al. [18], 99m Tc-HYNIC-PEG 4 -Tz-Me was mainly cleared through the hepatobiliary system, and the relative absorption value of the liver and intestine was 9.91 ± 0.97 %IA/g and 23.35 ± 3.84 %IA/g, respectively at 1 h post the Tz-radioligand injection. The authors further added a polypeptide sequence between HYNIC and PEG 4 to increase the hydrophilicity of the 16 Tz-radioligand to a logD of −1.05 ± 0.02. The newly synthesized Tz-radioligand was excreted mainly through kidneys. The relative absorption value of urine was 81.92 ± 5.06 %IA at 1 h post injection. In addition, Nichols et al. [25] and Devaraj et al. [26] successfully synthesized 68 Ga and 18 F-labeled Tzcoated polymer by introducing a well-established hydrophilic aminodextran backbone into the Tzderivative, respectively. In our study, the n-octanol/PBS distribution coefficient of 99m Tc-HYNIC-PEG 11 -Tz and 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz was -3.75 ± 0.08 and -4.64 ± 0.21, respectively. The modified 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz showed a higher hydrophilic parameter compared to 99m Tc-HYNIC-PEG 11 -Tz.
It has been shown that the introduction of positive charge to radiopharmaceuticals can increase their renal clearance and retention [27][28][29]. Similarly, the introduction of positive charge in the Tzradioligands increases clearance through the kidneys, whereas Tz derivatives with no charge were mainly excreted via the hepatobiliary system. In the study performed by Zeglis et al., as 64 Cu-NOTA-Tz is cleared somewhat sluggishly through the gastrointestinal pathway [22], the authors further created two novel Tz radioligands ( 64 Cu-NOTA-PEG 7 -Tz and 64 Cu-SarAr-Tz) in order to improve their pharmacokinetic profiles [23]. For 64 Cu-SarAr-Tz, in addition to the change of the coordination environment from N 3 O 3 to N 6, more importantly, the overall charge of the Tz-radioligand was shifted from −1 to +2. Finally, the newly synthesized 64 Cu-SarAr-Tz eliminates quickly and cleanly through the urinary tract [23]. In a pharmacokinetic profile study including 25 different Tz derivatives radiolabeled with either Al[ 18 F] or 68 Ga, Meyer et al. [30] observed that 68 Ga-NODA-Tz was excreted through renal pathway and Al  3 -Tz (net charge: +2) were excreted mainly through the kidneys [30]. Besides, it has been reported that renal uptake and excretion may be related to the presence of lysine residues in the molecular structure [31,32].
After investigating three different Cetuximab-TCO accumulation internval of 24 h, 48 h, and 72 h, we found that the tumor/blood ratio was highest (48h: 1.40 ± 0.31 vs. 24 h: 0.83 ± 0.13 and 72 h: 1.15 ± 0.21) for allowing 48 h accumulation of Cetuximab-TCO, which represents a relatively more appropriate accumulation interval. After injecting Cetuximab-TCO for 24 h, the accumulation of the mAb-TCO at HCT116 tumor had not reached a saturation status due to its large molecular weight and poor tissue infiltration. Simultaneously, there existed a large of circulating Cetuximab-TCOs in the blood since most Cetuximab-TCOs had not been eliminated out of the body, which leads to a great incidence of in vivo click reactions in the blood in addition to ligations at the tumor after 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz injection. In fact, the biodistribution data for 24 h accumulation showed that the uptake in the blood (1.60 ± 0.48 %IA/g) was even higher than that at the tumor (1.32 ± 0.14 %IA/g).
The relatively lower activity level of blood and tumor in the accumulation interval of 72 h compared to 48 h was probably resulted from the slow isomerization conversion from TCO into less reactive ciscyclooctene (CCO) due to the prolonged in vivo period of Cetuximab-TCO. Rossin et al. [24] investigated the deactivation mechanism of mAb-TCO in vivo and found that the TCO-CCO isomerization conversion via copper-containing proteins such as transcuprein, mouse serum albumin, and ceruloplasmin was the sole deactivation pathway of mAb-TCO in serum for a long time. CCO is of 5 orders of magnitude less reactive toward tetrazines than TCO.
According to the results of pretargeted biodistribution study, we chose the relatively most suitable accumulation interval (48 h) to compare the pretargeted Cetuximab-TCO/ 99m Tc-HYNIC-PEG 11 -Tz and Cetuximab-TCO/ 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz biodistribution and SPECT imaging using subcutaneous HCT116 xenografts mice. The results demonstrated that the surplus unclicked 99m Tc-HYNIC-PEG 11 -Tz is cleared rapidly through the intestines into the faces and remained in it for a long 18 time. The surplus unclicked 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz is cleared completely through urinary system. The tumor/blood ratio was 1.61 ± 0.12 and 1.40 ± 0.31, respectively for pretargeted Cetuximab-TCO/ 99m Tc-HYNIC-PEG 11 -Tz and Cetuximab-TCO/ 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz biodistribution. Both pretargeted imaging strategies delineated the HCT116 tumor clearly. However, pretargeted Cetuximab-TCO/ 99m Tc-HYNIC-Polypeptide-PEG 11 -Tz imaging avoided the retention of the surplus unclicked Tz-radioligand in faces, which favors to specific targeting imaging for CRC. Shi  was excreted through hepatobiliary system and remained in faces for a long time, the authors were unable to resolve the problem that imaging agent remained in faces interfered with CRC imaging.

List Of Abbreviations
The animals were euthanized, and tissues of interest were harvested at 6 h after the Tzradioligand injection.