Design, synthesis and evaluation of novel bifunctional tetrahydroxamate chelators for PET imaging of 89Zr-labeled antibodies

Accepted Manuscript Design, synthesis and evaluation of novel bifunctional tetrahydroxamate chelators for PET imaging of 89Zr-labeled antibodies Julie Rousseau, Zhengxing Zhang, Gemma M. Dias, Chengcheng Zhang, Nadine Colpo, François Bénard, Kuo-Shyan Lin PII: DOI: Reference:

S0960-894X(17)30066-5 http://dx.doi.org/10.1016/j.bmcl.2017.01.052 BMCL 24628

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

3 December 2016 15 January 2017 16 January 2017

Please cite this article as: Rousseau, J., Zhang, Z., Dias, G.M., Zhang, C., Colpo, N., Bénard, F., Lin, K-S., Design, synthesis and evaluation of novel bifunctional tetrahydroxamate chelators for PET imaging of 89Zr-labeled antibodies, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.01.052

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Design, synthesis and evaluation of novel bifunctional tetrahydroxamate chelators for PET imaging of 89Zr-labeled antibodies Julie Rousseaua, Zhengxing Zhanga, Gemma M. Diasa, Chengcheng Zhanga, Nadine Colpoa, François Bénarda,b,c*, Kuo-Shyan Lina,b,c* a

Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC V5Z 1L3, Canada

b

Department of Functional Imaging, BC Cancer Agency, Vancouver, BC V5Z 4E6, Canada

c

Department of Radiology, University of British Columbia, Vancouver, BC V5Z 4E3, Canada

Keywords: Zirconium-89; bifunctional chelators; antibody; Trastuzumab; molecular imaging; positron emission tomography

*Corresponding authors:

François Bénard, M.D. Tel: 1-604-675-8206; Fax: 1-604-675-8218 E-mail: [email protected] Address: 675 West 10th Avenue, Rm 14-111, Vancouver, BC V5Z 1L3, Canada

Kuo-Shyan Lin, Ph.D. Tel: 1-604-675-8208; Fax: 1-604-675-8218 E-mail address: [email protected] Address: 675 West 10th Avenue, Rm 4-123, Vancouver, BC V5Z 1L3, Canada

Abstract Two compact and symmetrical bifunctional tetrahydroxamate chelators, 1 and 2, were synthesized and evaluated for labeling antibodies with 89Zr for imaging with positron emission tomography. Using 2,2’-iminodiacetamide as the backbone, four hydroxamate-containing moieties coupled to the diacetamide nitrogen were used for 89Zr labeling, while a pendant connected to the amino group provided an isothiocyanate group for coupling to the antibody. Both 1- and 2-conjugated Trastuzumab were labeled with 89Zr efficiently (> 90% radiolabeling yield), and their 89Zr-labeled products maintained comparable immunoreactivity to Trastuzumab. Compared to 89Zr-labeled deferoxamine-conjugated Trastuzumab, 89Zr-1- and 89 Zr-2-Trastuzumab showed faster demetalation in mouse plasma, and also displayed higher bone uptake in mice. Despite suboptimal stability of 89Zr complexes of 1 and 2, our design strategy led to tetrahydroxamate chelators for efficient 89Zr labeling, and could be potentially modified to provide novel chelators with improved stability.

Graphical Abstract

Positron Emission Tomography (PET) is a powerful non-invasive molecular imaging modality with high sensitivity, spatial resolution and quantification capability, and is routinely used in the clinic for cancer imaging.1 With high affinity and specificity for their targets, monoclonal antibodies (mAb) have been successfully used in the clinic as molecularly-targeted therapeutics.2 The development of innovative PET isotopes production and radiolabeling techniques further stimulates the application of immunoPET imaging to provide information on target expression and the pharmacokinetics of mAb.3 Zirconium-89 (89Zr) is an appealing radionuclide for immunoPET imaging with mAb.4,5 Its half-life (78.4 h) is well suited to the slow pharmacokinetics of mAb, and its low positron energy (Emax = 908 keV) results in high spatialresolution PET images. Moreover, efficient procedures for radiolabeling mAb with 89Zr have been established6 and several 89Zr-labeled mAb are currently under investigation in the clinic.710

Despite great potential of 89Zr for labeling mAb for PET imaging, the derivatives (maleimide and isothiocyanate) of the bacterial siderophore deferoxamine (DFO) are currently the only commercially available bifunctional chelators for 89Zr labeling. Significant bone uptake (up to 15.1 %ID/g, 5 days after injection) resulting from demetalation of 89Zr-labeled DFO-conjugated mAb has been constantly observed in mice in preclinical studies, especially in high bone remodeling regions.11 This has been suggested due to the fact that DFO can provide only three hydroxamate groups (hexadentate) for chelation but octadentate chelators are required to stably chelate Zr4+.12,13 The high bone uptake reduces the overall tumor uptake and the ability to detect smaller bone metastatic lesions. Therefore, development of novel bifunctional chelators that lead to higher in vivo stability of 89Zr-labeled mAb is needed to facilitate the application of 89Zr for immunoPET imaging. As shown in Fig.1, in the past 10 years, several bifunctional chelators have been synthesized and evaluated for labeling antibodies with 89Zr. Perk et al.14 reported that labeling MX-DTPAconjugated Zevalin with 89Zr resulted in < 0.1% labeling yield. Price et al.15 showed that labeling H6phospa-conjugated Trastuzumab with 89Zr resulted in up to 12% radiochemical yield, whereas no 89Zr chelation was observed using H4octapa-conjugated Trastuzumab. The tripodal tris(hydroxypyridinone) ligand YM-103 was reported by Ma et al.,16 and showed > 98% radiochemical yield for labeling Trastuzumab with 89Zr. However, massive demetalation was observed in mice as uptake in bone was 25.9 ± 0.58 %ID/g at seven days post-injection. Evaluation of a 3-hydroxypyridin-2-one based macrocyclic chelator BPDETLysH22-2,3-HOPO for labeling mAb with 89Zr was reported by Tinianow et al.17 Despite good radiolabeling yield (60 – 69%), 89Zr-labeled BPDETLysH22-2,3-HOPO-conjugated Trastuzumab showed higher bone uptake compared to that of 89Zr-DFO-Trastuzumab (15.1 ± 2.7 and 10.6 ± 1.0 %ID/g, respectively at Day 6. Deri et al.18 reported the evaluation of p-SCN-Bn-HOPO for labeling mAb with 89Zr. Compared to 89Zr-DFO-Trastuzumab, 89Zr-HOPO-Trastuzumab showed much less bone uptake in mice at Day 14 (17.0 ± 4.1 and 2.4 ± 0.3 %ID/g, respectively), demonstrating its superior in vivo stability. A cyclam-based trihydroxamate chelator L5 was reported by Boros et al.19 L5-conjugated Trastuzumab was radiolabeled with 89Zr in quantitative radiochemical yield, but 89Zr-L5-Trastuzumab suffered significant in vivo demetalation in mice (18.9 ± 1.1 % uptake in bone at Day 4). Recently, Vugts et al.20 reported adding an additional hydroxamate group to

the DFO chelator, so the resulting DFO* chelator could fulfill the octadentate requirement to stably complex Zr4+. Labeling DFO* with 89Zr was achieved in > 95% radiochemical yield. Furthermore, 89Zr-DFO*-Trastuzumab was demonstrated to be more stable than 89Zr-DFOTrastuzumab as their uptake values in knees in mice were 1.38 ± 0.23 and 8.20 ± 2.94 % ID/g, respectively at Day 7.

Figure 1: Reported bifunctional chelators for labeling antibodies with 89Zr.

Despite superior in vivo stability of 89Zr-HOPO and 89Zr-DFO* complexes, the synthesis of these two bifunctional chelators is not trivial.18,20 The reported synthesis methods involved multiple steps and HPLC purification, and only a few mg batch production of both chelators was demonstrated.18,20 To develop novel bifunctional chelators for labeling mAb with 89Zr, we designed and synthesized two compact and symmetrical bifunctional tetrahydroxamate chelators 1 and 2 (Fig. 1). 2,2’-Iminodiacetamide was used as the backbone for both chelators. A pendant arm stretching out from the central amino group was functionalized to provide an isothiocyanate group for coupling to antibodies. Four hydroxamate groups to fulfill the octadentate requirement were connected to the two amide nitrogen with different length of linker (Fig. 1, 1: X = CH2; 2: X = CH2CH2). The chelators were designed to be compact and symmetrical, so they could be facilely synthesized and characterized.

Figure 2: Synthesis of bifunctional tetrahydroxamate chelators 1 and 2 for labeling antibodies with 89Zr.

The synthetic scheme for the preparation of bifunctional chelators 1 and 2 is depicted in Fig. 2. To construct the hydroxamate-containing moieties, the N-Boc protected 2,2’-iminodiacetic acid 3 was acquired commercially, whereas the N-Boc protected 3,3’-iminodipropionic acid 4 was synthesized in 79% yield by treating 3,3'-iminodipropionic acid with di-tert-butyl dicarbonate. Compounds 3 and 4 were converted to their activated di-(2,3,5,6-tetrafluoro)phenyl diesters 5 and 6 in 57 and 78% yields, respectively. Di-O-benzyl protected dihydroxamates 7 and 8 were obtained in 78 and 53% yields, respectively, by coupling 5 and 6 with O-benzyl-Nmethylhydroxylamine. The free amines 9 and 10 were prepared in 80 and 98 % yields, respectively, after removing the N-Boc protecting group with trifluoroacetic acid. To construct the 2,2’-iminodiacetamide backbone with a pendant arm for mAb coupling, N-Boc1,4-butanediamine was coupled with 2 equivalents of benzyl 2-bromoacetate to form 11 in 96% yield. The O-benzyl protecting group was removed by Pd-catalyzed hydrogenation to give Nsubstituted 2,2’-iminodiacetic acid 12 quantitatively. Compound 12 was subsequently converted to the activated di-(2,3,5,6-tetrafluoro)phenyl diacetate 13 in 84% yield. Coupling 13 with amines 9 or 10 provided tetrahydroxamate-conjugated 2,2’-iminodiacetamides 14 and 15 in 76 and 58% yields, respectively. The N-Boc protecting group was removed by treating 14 and 15 with trifluoroacetic acid, and free amines 16 and 17 were obtained in 99 and 87% yields, respectively. The four O-benzyl protecting groups of 16 and 17 were removed by catalytic hydrogenation to afford 18 and 19 in 94 and 89% yields, respectively. Coupling 18 and 19 with 1,4-phenylene diisothiocyanate gave the desired bifunctional tetrahydroxamate chelators 1 and 2 in 66 and 72% yields, respectively. To assess the efficiency of bifunctional chelators 1 and 2 for labeling mAb with 89Zr, Trastuzumab was selected as the model mAb as it was used previously by others to test new 89 Zr chelators.15-20 Conjugation of Trastuzumab with 1, 2 and p-SCN-DFO, and radiolabeling of the resulting conjugates with 89Zr followed the protocol reported by Vosjan et al.6 On average, 0.55 ± 0.04, 0.91 ± 0.02 and 0.94 ± 0.04 of DFO, 1 and 2, respectively were conjugated to Trastuzumab. The radiolabeling yields of 89Zr-DFO-Trastuzumab, 89Zr-1-Trastuzumab and 89Zr-2Trastuzumab were very efficient (> 90%) as confirmed by iTLC-SG. After purification by PD-10 column, the radiochemical purities were > 99% for all three radioimmunoconjugates and their specific activities were in the range of 0.3 – 0.6 MBq/μg. Compared to unmodified Trastuzumab, no significant change in immunoreactivity of all three radioimmunoconjugates was observed (96, 104 and 101% immunoreactivity for 89Zr-DFO-Trastuzumab, 89Zr-1Trastuzumab, and 89Zr-2-Trastuzumab, respectively). This indicates that chelator conjugation and 89Zr labeling did not reduce the binding capability of Trastuzumab to the HER2 antigen. To assess the stability of 89Zr complexes of 1 and 2, in vitro stability assays were conducted for 89 Zr-1-Trastuzumab, 89Zr-2-Trastuzumab and 89Zr-DFO-Trastuzumab. These three radioimmunoconjugates were incubated in mouse plasma at 37 °C, and the extent of demetalation was monitored over time and quantified by iTLC. As shown in Table 1, progressive demetalation was observed for all three radioimmunoconjugates in mouse plasma. 89Zr-DFOTrastuzumab displayed the best plasma stability. While 77 % of 89Zr-DFO-Trastuzumab remained intact after one day, only 25% of 89Zr-1-Trastuzumab and 46% 89Zr-2-Trastuzumab

were still intact. On Day 3, there were 3, 16 and 55% of 89Zr-1-Trastuzumab, 89Zr-2-Trastuzumab and 89Zr-DFO-Trastuzumab, respectively, remained intact. Table 1: In vitro plasma stability of 89Zr-DFO-Trastuzumab, 89Zr-1-Trastuzumab and 89Zr-2Trastuzumab. Data are presented as the percentage of the total activity corresponding to the intact radioimmunoconjugates. 89

Day 0 Day 1 Day 2 Day 3

Zr-DFO-Trastuzumab > 99 77 69 55

89

Zr-1-Trastuzumab > 99 25 13 3

89

Zr-2-Trastuzumab > 99 46 18 16

Figure 3: Representative maximum intensity projection PET images of 89Zr-labeled Trastuzumab conjugates obtained at 1 and 3 days post-injection. Spleen is indicated by arrows.

In addition to in vitro stability assays, PET imaging was also conducted to assess the in vivo behaviour of these three radioimmunoconjugates. Mice were injected with 8.4 ± 2.0 μg of radioimmunoconjugates and PET/CT images were acquired at one and three days postinjection. As shown in Fig. 3, spleen was clearly visualized for all three radioimmunoconjugates on both images acquired on Day 1 and 3. Spleen is the organ that is typically observed in antibody imaging, and was used here as a surrogate target. Clear spleen visualization indicated that significant portions of radioimmunoconjugates remained intact and reached this target organ. Bone uptake (especially in the knees) was also observed and increased over time, suggesting there was progressive demetalation for all three radioimmunoconjugates. The bone uptake of 89Zr-1-Trastuzumab and 89Zr-2-Trastuzumab was higher than that of 89Zr-DFOTrastuzumab as observed from both images taken on Day 1 and 3. The observed inferior in vivo stability of complexes of 89Zr-1 and 89Zr-2 was consistent with the observations from the in vitro plasma stability assays.

Figure 4: Biodistribution of 89Zr-labeled Trastuzumab conjugates at three days post-injection. Data are expressed as mean ± SD (n = 6). For statistics analysis, * (p < 0.05) and *** (p < 0.001) indicate the difference is significant when compared to the uptake of 89Zr-DFO-Trastuzumab.

At Day 3, all mice were euthanized and tissues/organs of interest were harvested and counted to obtain comprehensive biodistribution data of all three radioimmunoconjugates. Compressive tissue uptake values were provided in Supplementary Table 1. As shown in Fig. 4, significantly higher spleen uptake (p < 0.001) was observed using 89Zr-DFO-Trastuzumab (48.9 ± 7.92 %ID/g) than 89Zr-1-Trastuzumab (24.6 ± 4.08 %ID/g) and 89Zr-2-Trastuzumab (27.64 ± 4.22 %ID/g). In addition, bone uptake was also significantly lower (p < 0.001) for 89Zr-DFO-Trastuzumab (7.58 ± 0.36 %ID/g) than 89Zr-1-Trastuzumab (19.5 ± 3.59%ID/g) and 89Zr-2-Trastuzumab (18.3 ± 2.91 %ID/g). These biodistribution data were consistent with observations from PET images, and confirmed that 89Zr-1-Trastuzumab and 89Zr-2-Trastuzumab had a higher extent of in vivo demetalation leading to higher bone uptake and lower spleen uptake. Both in vitro and in vivo studies of 89Zr-1-Trastuzumab and 89Zr-2-Trastuzumab suggested that chelators 1 and 2 did not form a stable complex with 89Zr. This could be due to for example (1) the Zr complexes of 1 and 2 were not the expected eight-coordinate complexes despite the presence of four hydroxamate groups per chelator; or (2) the eight-coordinate Zr complexes of 1 and 2 were not in an optimized coordination arrangement due to the steric constraints imposed by the 2,2’-iminodiacetamide backbone and the shorter arm (X = CH2 for 1 and X = CH2CH2 for 2, Fig. 1) of hydroxamate moieties. A similar finding was reported by Boros et al.19 on the comparison of stability between 89Zr complexes of two hydroxamate chelators L3 and L4 (Fig. 1) that shared a same cyclam backbone. L3 was an octadentate tetrahydroxamate chelator with an (N-methyl-N-hydroxy)carboxamidomethyl group connected to all four amino groups of the cyclam. For L4, it was a hexadentate trihydroxamate chelator with substitution of a hydroxamate group with a longer arm ((N-methyl-N-hydroxy)carboxamidoethylcarbonyl) at three of the four amino groups of the cyclam. Despite being a hexadentate chelator, the complex of L4 with 89Zr was more stable against EDTA (ethylenediaminetetracetic acid) challenge than the complex of the tetrahydroxamate chelator L3 with 89Zr. Computer calculations revealed that L3-Zr complex might not form the expected eight-coordinate complex, but a seven-coordinate species due to steric constraints. For L4 with longer hydroxamate arms, it could adopt a more ideal geometry with decreased ligand strain and form a more stable Zr complex. Therefore, based on these observations, it might be possible to improve the design of our tetrahydroxamate chelators by reducing steric constraints. This could be achieved for example by increasing the arm length of hydroxamate moieties to propylene (CH2CH2CH2) or longer (Fig. 1) and/or replacing the 2,2’-iminodiaetamide backbone with 3,3’iminodipropinamide. In conclusion, we designed a simple and modifiable template for the preparation of bifunctional tetrahydroxamate chelators for labeling mAb with 89Zr. Chelators 1 and 2 were synthesized via multistep reactions with a good to excellent (53 – 100%) yield for each reaction step. The octadentate chelators 1 and 2 were prepared in > 100 mg batch without the need of HPLC purification. Radiolabeling of 1- and 2-conjugated Trastuzumab was very efficient with > 90% radiochemical yields, and their radiolabeled immunoconjugates maintained full immunoreactivity of the mAb. In vitro study showed that compared to 89Zr-DFO-Trastuzumab,

89

Zr-labeled 1- and 2-conjugated Trastuzumab had faster demetalation in mouse plasma, and this was consistent with their higher bone uptake observed in imaging and biodistribution studies. The instability of 89Zr-1- and 89Zr-2 complexes could be due to steric constraints that prevent the formation of stable eight-coordinate complexes of these two chelators with Zr4+. Reducing steric constraints by extending the arm length between the hydroxamate and the amide nitrogen and/or replacing the 2,2’-iminodiacetamide backbone with 3,3’iminodipropinamide could potentially lead to novel tetrahydroxamate 89Zr chelators with improved in vivo stability.

Acknowledgements This work was supported by the Terry Fox Research Institute. The authors would like to thank Milan Vuckovic, Wade English and Baljit Singh for their technical assistance for 89Zr production. Supplementary data Supplementary data associated with this article can be found, in the online version, at XXXXX.

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