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Pretargeted Immuno-PET based on Bioorthogonal Chemistry for Imaging EGFR Positive Colorectal Cancer Xudong Shi, Kai Gao, Hao Huang, and Ran Gao Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00023 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 18, 2018

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Bioconjugate Chemistry

Pretargeted Immuno-PET based on Bioorthogonal Chemistry for Imaging EGFR Positive Colorectal Cancer Xudong Shi, [a] Kai Gao, [a] Hao Huang,[a] and Ran Gao*[a] *Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) & Comparative Medicine Centre, Peking Union Medical Collage (PUMC), No. 5 Panjiayuan Nanli, Chaoyang District, Beijing, 10021, PR China Corresponding Author’s E-mail: [email protected]

Abstract: Pretargeted immuno-PET imaging based on the bioorthogonal chemistry between 18F-labeled Reppe anhydride derivatives and tetrazine conjugates of the EGFR-specific monoclonal antibodies cetuximab and panitumumab was performed. This pretargeting approach yielded high target-to-nontarget ratios. Furthermore, due to the fast clearance rate of the PET probe, the overall radiation burden to nontarget tissues was also substantially decreased.

Immuno-positron emission tomography (PET) technique uses positron emitter radiolabeled antibodies or engineered antibody fragments as imaging agents to visualize the functions of living systems and to reveal the biological state of cells. These antibodies and related molecules can recognize specific cell surface biomarkers such as growth factor receptors, adhesion molecules, enzymes and proteases. In recent years, immuno-PET using long-lived positron emitter radiolabeled monoclonal antibodies (mAbs) has promoted the diagnosis of disease and the development of mAbs-based immunotherapies.1-3 The long in vivo retention time of intact monoclonal antibodies means that an optimal target-to-nontarget (T/NT) ratio was achieved at 2-4 days post-injection. Long-lived isotopes such as 124I (half-life, 4.18 d), 89Zr (half-life, 3.27 d) with ideal physical characteristics were shown to be suitable for conjugation with the mAbs used in immuno-PET imaging.4, 5 However, the extended retention time in vivo resulted in an overall radiation burden on non-target tissues and organs. Since then, the pretargeted immuno-PET strategy based on bioorthogonal chemistry has been optimized.6-8 Bioorthogonal chemistry has fascinated researchers for decades, particularly for its applications in molecular imaging, cell detection and materials functionalization.9, 10 Moreover, immuno-PET imaging based on bioorthogonal chemistry has been used successfully to visualize and quantify monoclonal antibody activity in vivo.11 The latter deployed an inverse electron demand Diels-Alder reaction (IED-DAR) between diene (tetrazine) and strained dienophiles (trans-cyclooctene, cyclooctynes, norbornene). This tetrazine-strained dienophiles ligation has also been used for the pretargeted immuno-PET imaging of tumors. First, mAbs are modified with bioorthogonal functional molecules (tetrazine or strained dienophiles) and introduced to then accumulate specifically in the targeted region. Short-lived positron emitter (18F, 68Ga, 64Cu) radiolabeled dienophile reaction partners are subsequently introduced as the probe.12-14 As a result, a rapid and targeted [4+2] IED-DAR reaction between tetrazine and the strained dienophiles occurs at the tumor site, producing stable adducts between the two molecules without interfering with the innate biochemical processes. Research has shown that Reppe anhydride

derivatives were suitable to react with tetrazine through IED-DAR cycloaddition reactions. The strained four-membered ring system of a Reppe anhydride 15 can react with the tetrazine over a short period of time and under mild reaction conditions. Compared with other strained dienophiles, Reppe anhydride is also more stable and the tetrazine-Reppe anhydride ligation has a higher reaction rate. Overexpression of the epidermal growth factor receptor (EGFR) has previously been associated with colorectal cancer progression and metastasis. EGFR has since become a therapeutic target for the treatment of colorectal cancer.16 Moreover, the therapeutic antibodies cetuximab and panitumumab have been marketed for treating colorectal cancer. As EGFR inhibitors, they specifically target the extracellular domain of the EGFR and block the intracellular tyrosine kinase activity. Also, noninvasive molecular imaging using cetuximab and panitumumab has been developed for the diagnosis of cancer and for evaluating the therapeutic response to EGFR-blocking. Cetuximab and panitumumab are labeled with the long-lived radionuclides 89Zr and 124I respectively, in order to detect and quantify the EGFR expression using immuno-PET imaging.17-19 By comparison, an approach using short-lived radionuclides such as 18F or 68Ga would allow for a more targeted and rapid reaction with the IED-DAR cycloaddition partner in vivo. Compared to long-lived positron emitter labeled mAbs, the use of short-lived radionuclides is, therefore, believed to significantly decrease the radiation burden on non-target organs and tissues and, by extension, lead to a higher target to background ratio. Here, we developed a novel Reppe anhydride derivative-based PET probe for imaging EGFR expression in colorectal cancer tissues using a bioorthogonal immuno-PET strategy. The macrocyclic chelator NOTA was introduced to the Reppe anhydride derivative endo-tricyclo-[4.2.2.02.5]deca-3,9diene. The probe [18F]AlF-NOTA-Reppe anhydride was obtained using a facile and one-pot 18F-labeling method chelating NOTA and [18F]AlF. An IED-DAR cycloaddition reaction between the PET probes and tetrazine modified EGFR-specific monoclonal antibodies cetuximab and panitumumab pre-injected in mouse models of colorectal cancer followed. Immuno-PET imaging and biodistribution experiments demonstrated a rapid hepatobiliary and renal excretion and a low background accumulation of the probe, resulting in a clear and unobstructed image of EGFR expression in vivo. The study presented herein suggested that the bioorthogonal chemistry-based PET probe was a powerful imaging probe for the diagnosis of cancer and for monitoring the therapeutic response to EGFR-blocking in tumor models and also potentially humans. This immuno-PET imaging technique based on bioorthogonal chemistry will give physicians a robust tool for visualizing EGFR expression in cancer tissues.

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a 1) 20% CF3 COOH CH 3OH, rt, 4 h

O N

NHBoc 2) p-SCN-Bn-NOTA CH 3CN, rt, 1 h

O

O

O N O

2

N

AlCl3 ,18 F-fluoride

O

N

S

1

O

OH N

H NH N

O

O

HO

sodium acetate buffer 90 °C ,20 min

O OH

N O

H NH N S

TD-NOTA-Al-18F

55%

18

F

N Al N N

OH O

O O

30% RCY

b ε

Lys

NH2

+

NN NN

COOH

mAbs Figure 1. (a) Schematic of the synthesis of NOTA-Reppe anhydrides and conjugates where mAbs represents cetuximab or panitumumab.

The bioorthagnol chemistry between Reppe anhydride derivatives and tetrazine has successfully been used to synthesize peptide conjugates and other medical applications.20, 21 In this study, a Reppe anhydride derivative was chosen as the dienophile building block for an IED-DAR cycloaddition. In general, the isothiocyanate group was typically introduced as a reactive group and conjugated to biomolecules via thiourea covalent bonds. In this study, the hexadentate isothiocyanato-benzyl derivative of NOTA (p-SCN-Bn-NOTA) was conjugated to amino-Reppe anhydride derivatives. Conventionally, 18F-labeled amine reactive prosthetic group (N-succinimidyl 4-[18F] fluorobenzoate, 4-([18F]fluoromethyl) phenyl isothiocyanate, etc.) is used to label peptides or small amino-functionalized molecules. However, its procedures are laborious and time-consuming 22 and, recently, spurred the development of a more user-friendly method.23 This facile method is based on the chelation of [18F]aluminum fluoride by NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) and has been used to create stable radiolabeled peptides in a short period of time and with a high radiochemical yield and specific activity. For this study, 18F-radiolabeling of Reppe anhydride derivatives was achieved following the efficient binding between [18F]AlF and the bifunctional chelating agent NOTA in a two-step, one-pot reaction by mixing Al3+ and [18F]fluoride in sodium acetate buffer at 90℃ for 20 min (Figure 1a). [18F]AlF-NOTAReppe anhydride (Rf = 0.7~0.8) was obtained with a radiochemical yield of 30% under non-optimized conditions. Its radiochemical purity exceeded 95% after removing unchelated [18F]AlF (Rf=0.0~0.1), as determined by radio-TLC (Thin Layer Chromatography) (Figure S3). Such radiochemical purity was suitable for in vivo applications. Compared to the purification of other [18F]AlF radiolabeled peptides,24 the purification method

Figure 2. (a) Schematic of the IED-DAR cycloaddition reaction between 18 mAbs-tetrazine conjugates (50 µg in 100 µl PBS buffer) and [ F]-radiolabeled Reppe anhydride derivatives (7.4 MBq in 50 µl PBS buffer). (b) The reaction 18 between cetuximab-tetrazine conjugate and [ F]AlF-NOTA-Reppe anhydride in vitro was demonstrated by Radio-TLC.

NN

εH

EDC/NHS

N

CH3 CN/DMSO 4 °C ,5 h

Lys

NN O

mAbs-tetrazine 18

F-radiolabeled Reppe anhydride derivatives. (b) Synthesis of mAbs-tetrazine

employed in this study was simple and required no further HPLC purification. [18F]AlF-NOTA-Reppe anhydride had a specific activity of 1.25 GBq/µmol and was stable in human serum for up to 2 h. The octanol-water partition coefficient of the PET probe was determined by radio-TLC and shown to be appropriate lipophilic with a logP value of -2.1. There are 80-100 lysine residues on a typical IgG molecule and most of them are exposed on the surface of the antibody.25 These lysine side-chains (ε-amino group) can be used for a chemical coupling by acylation. EGFR-specific monoclonal antibodies (cetuximab or panitumumab) were, therefore, mixed with NHS-activated carboxyl-tetrazine using EDC/NHS conjugation chemistry (Figure 1b). According to UV/Vis analysis, 26 the molar ratio of conjugated tetrazine to cetuximab and panitumumab was estimated at 10.6:1 and 8.3:1, respectively. The mAbs-tetrazine conjugates were subsequently used to bind with the positron emitter labeled Reppe anhydride in vivo. In order to evaluate the conjugation of the tetrazine modified antibodies and the PET probe in vitro, a radio-TLC analysis was performed. As shown in Figure 2b, a peak was initially observed at the position corresponding to the [18F]AlF-NOTA-Reppe anhydride. After 10 min of incubation with the cetuximab-tetrazine conjugate (phosphate buffered saline, pH = 7.4 at room temperature), this peak had almost disappeared concomitant with the appearance of a new peak at a different position, indicating the formation of the “probe-antibody” complex. The IED-DAR cycloaddition reaction between the probe and antibody was swift in aqueous conditions without catalysts or hazardous side products. Finally, noninvasive imaging in vivo of the radiolabeled probe targeted at an antibody bound to the cell surface EGFR receptor resulted in a low background signal and a high T/NT ratio. Nuclear imaging visualized the biodistribution and metabolism of [18F]AlF-NOTA-Reppe anhydride in nude mice bearing HCT116 colorectal tumor xenografts. The localization of [18F]AlF-NOTA-Reppe anhydride in normal tissue and tumors is shown in Figure 3. These results showed non-specific EGFR receptor localization of [18F]AlF-NOTA-Reppe anhydride in the tumor. The tumor uptake of the PET tracer was 0.69 ± 0.36 percentage injected dose per gram (%ID/g) (the tumor to muscle ratio was 1.02 ± 0.55, 30 min post-injection [p.i.]). The PET probes are generally excreted via hepatobiliary and renal routes. The liver (9.04 ± 2.64 %ID/g) and the small intestine (5.30 ± 1.33 % ID/g) showed significant uptake of probes within 30 min p.i., while tracer accumulation in the kidney was comparatively low (2.45 ± 1.46 %ID/g). Due to the appropriate lipophilicity of [18F]AlF-NOTA-Reppe anhydride, a part of probe passed via the hepatobiliary system,27,28 causing high background activity in the


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abdominal region at 30 min p.i. Notably, the fast hepatobiliary clearance into the small intestine was also associated with the observed decrease in tracer accumulation in the liver and increased uptake in the gallbladder and small intestine. After 120 min, only low tracer levels were recorded in the abdomen (small intestine, 1.98 ± 0.65 %ID/g).

metabolic results of PET probe were similar with the PET imaging in vivo. The uptake of Figure 5b and 5d, show the remarkable enhancement in targeting efficacy as well as tumor-to-blood ratios (T/Bcetuximab-tetrazine = 13.02 ± 0.64, T/B panitumumab-tetrazine = 10.15 ± 1.56 at 120 min p.i.) and tumor-tomuscle ratios (T/Mcetuximab-tetrazine = 28.01 ± 0.41, T/Mpanitumumabtetrazine = 22.00 ± 2.63 at 120 min p.i.). The tumor-to-muscle ratios of PET probe in mice pre-treated with mAbs-tetrazine have a tendency to increase over time. The tumor-specific accumulation of PET probe demonstrated high affinity and selectivity of cetuximab and panitumumab for EGFR in EGFR-positive tumor model.

Figure 3. In vivo PET/CT imaging results of BALB/c nude mice bearing HCT116 and injected with [18F]AlF-NOTA-Reppe anhydride. Images were acquired at 15 min, 30 min and 120 min post injection. Abbreviations: L =liver; I =small intestine; G =gallbladder. The white dashed line encircles the tumor region (n =5/ group).

The study furthermore determined whether the PET probe was able to bind to the EGFR-specific antibodies in vivo. Tetrazine modified monoclonal cetuximab or panitumumab (200 µg in 200 µl of 0.9% saline) were injected intravenously into nude mice bearing an EGFR-positive HCT116 tumor. After 48 h, the [18F]AlF-NOTA-Reppe anhydride (14.9 ± 0.5 MBq) was injected. Mice in the control group were first injected with non-immunized control antibody IgG from mouse serum (mIgG) and then given the same dose of PET probe. Small animal PET/CT quantification analysis showed a significantly higher radioactivity in the tumor of nude mice treated with cetuximab (6.33 ± 0.71 %ID/g) and panitumumab (8.73 ± 1.04 %ID/g). The T/NT ratios (tumor-to-muscle) of [18F]AlF-NOTA-Reppe anhydride were much higher in the treated mice, 7.12 ± 1.23 (cetuximab) and 8.97 ± 1.82 (panitumumab), than in the controls (Figure 4). There was negligible accumulation of [18F]AlF-NOTAReppe anhydride in tumors (0.55 ± 0.23 % ID/g) of mice in the control group. Tetrazine modified cetuximab and panitumumab targeted the EGFRs specifically at the targeted region. Following an injection with [18F]AlF-NOTA-Reppe anhydride, the IED-DAR reaction between the PET probe and the pre-injected mAbs-tetrazine was shown to occur at the tumor site. The uptake of PET probes by the xenograft tumor denoted the level of anti-EGFR monoclonal antibodies binding to EGFR. Besides, any excess of PET probe was expelled rapidly with only the gallbladder (4.63 ± 1.27 %ID/g) and small intestine (3.65 ± 0.59 %ID/g) still showing traces of [18F]AlF-NOTA-Reppe anhydride at 120 min p.i. It was concluded that the pretargeting approach yielded high T/NT ratios. Furthermore, given the fast clearance rate of the PET probes and the short half-life of [18F], the overall radiation burden on non-target tissues and organs would also have been substantially decreased.29 Ex vivo biodistribution studies of HCT116 xenograft nude mice intravenously injected with [18F]AlF-NOTA-Reppe anhydride using the pretargeting approach were also performed. These results indicated a high retention in the tumor and a low uptake of mAbs-tetrazine by the other organs. The uptake of [18F]AlF-NOTA-Reppe anhydride in the tumor of nude mice treated with panitumumab-tetrazine (6.6 ± 0.90 %ID/g) showed higher than cetuximab-tetrazine (5.6 ± 0.31 %ID/g). The

Figure 4. A) The procedure followed for the pretargeted immuno-PET imaging study based on bioorthogonal chemistry. B) Transverse (upper) and coronal section (lower) PET-CT images of HCT116 tumor-bearing mouse at 120 min 18 after injection of F-labeled Reppe anhydride derivatives (18.5 MBq). Mice were pre-treated with (a) cetuximab-tetrazine conjugates (b) panitumumab-tetrazine conjugates (c) mIgG-tetrazine conjugates and recieved 18 another injection with F-labeled Reppe anhydride derivatives 48 h later. The white dashed line encircles the tumor region (n = 5/ group).

Figure 5. Biodistribution and tumor-to-blood (muscle) ratios of [18F]AlF-NOTA-Reppe anhydride in HCT116 xenograft nude mice pre-treated



with cetuximab-tetrazine conjugates (a, b) and panitumumab-tetrazine conjugates (c, d). Organs were collected at 15, 30 and 120 min post injection (n = 5/ group).

Conclusions Our study enabled the visualization EGFR activity using immuno-PET imaging based on bioorthogonal chemistry. The IED-DAR reaction between 18F-labeled Reppe anhydride derivatives and tetrazine conjugates of EGFR-specific monoclonal antibodies, cetuximab and panitumumab, was shown to be technically feasible and could significantly reduce the overall radiation burden to non-target tissues and organs. Immuno-PET imaging furthermore demonstrated rapid hepatobiliary excretion and a low background accumulation of probe, resulting in high-contrast images of EGFR expression in vivo. In brief, the work presented herein has shown that pretargeted immuno-PET imaging imaging based on bioorthogonal chemistry is a powerful tool for visualizing EGFR expression in xenograft mice tumor models. We also strongly believe that this technique could, in the future, also have applications in humans. Further studies will be performed to monitor therapeutic efficacy of specific monoclonal antibodies targeted against EGFR by bioorthogonal based immuno-PET.

■ ACKNOWLEDGMENTS AND FUNDING SOURCES This study was financially supported by the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (grant nos. 2016-12M-2-006-001). The authors also gratefully acknowledge Prof. Chunying Shu form Institute of Chemistry, Chinese Academy of Sciences.

■ ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Additional details on experimental methods, radiolabeling methods, and in vivo animal PET/CT imaging, biodistribution. (PDF) ■ AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Xudong Shi: 0000-0001-7496-3174 Notes The authors declare no competing financial interest. ■ REFERENCES (1) Van Dongen, G. A., Visser, G. W., Lub-de Hooge, M. N., De Vries, E. G., Perk, L. R. (2007). Immuno-PET: a navigator in monoclonal antibody development and applications. Oncologist, 12, 1379-1389. (2) Dijkers, E. C., Oude Munnink, T. H., Kosterink, J. G., Brouwers, A. H., Jager, P. L., Jong, J. D., Vries, E. G. (2010). Biodistribution of 89Zr ‐ trastuzumab and PET imaging of HER2-positive lesions in patients with metastatic breast cancer. Clin. Pharmacol. & Ther., 87, 586-592. (3) Paudyal, B., Paudyal, P., Oriuchi, N., Hanaoka, H., Tominaga, H., & Endo, K. (2011). Positron emission tomography imaging and biodistribution of vascular endothelial growth factor with 64Cu‐labeled bevacizumab in colorectal cancer xenografts. Cancer. Sci., 102, 117-121. (4) Lee, F. T., Hall, C., Rigopoulos, A., Zweit, J., Pathmaraj, K., O’Keefe, G. J. Scott, A. M. (2001). Immuno-PET of human colon xenograft–bearing BALB/c nude mice using 124I-CDR–grafted humanized A33 monoclonal antibody. J. Nucl. Med., 42, 764-769. (5) Verel, I., Visser, G. W., Boellaard, R., Stigter-van Walsum, M., Snow, G. B., van Dongen, G. A. (2003). 89Zr immuno-PET: comprehensive procedures for the production of 89Zr-labeled monoclonal antibodies. J. Nucl. Med.,44, 1271-1281.

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Pretargeted immuno-PET imaging based on bioorthogonal chemistry was proven to be an effective technique for detecting colorectal cancer in mice.


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Pretargeted immuno-PET imaging based on bioorthogonal chemistry was proven to be an effective technique for detecting colorectal cancer in mice. 226x68mm (300 x 300 DPI)


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