A Metal-Free DOTA-Conjugated 18F-Labeled Radiotracer: [18F]DOTA

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A Metal-Free DOTA-Conjugated 18F-Labeled Radiotracer: [18F]DOTAAMBF3-LLP2A for Imaging VLA-4 Over-Expression in Murine Melanoma with Improved Tumor Uptake and Greatly Enhanced Renal Clearance Aron Roxin, Chengcheng Zhang, Sungjoon Huh, Mathieu Louis Lepage, Zhengxing Zhang, Kuo-Shyan Lin, François Bénard, and David M. Perrin Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00146 • Publication Date (Web): 21 Mar 2019 Downloaded from http://pubs.acs.org on March 22, 2019

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Bioconjugate Chemistry

The first DOTA-appended 18F-labeled Peptidic Radiotracer 183x108mm (300 x 300 DPI)

ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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A Metal-Free DOTA-Conjugated 18F-Labeled Radiotracer: [18F]DOTA-AMBF3-LLP2A for Imaging VLA-4 Over-Expression in Murine Melanoma with Improved Tumor Uptake and Greatly Enhanced Renal Clearance Áron Roxin1, Chengcheng Zhang2, Sungjoon Huh1, Mathieu Lepage1, Zhengxing Zhang2, Kuo-Shyan Lin*2, François Bénard*2, David M. Perrin*,1 1Chemistry

Department, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia, V6T 1Z1, Canada; 2Molecular Oncology, British Columbia Cancer Agency Research Centre, 675 West 10th Avenue, Vancouver, BC, V5Z 1L3, Canada.

Addresses for correspondence: Francois Benard, M.D. BC Cancer Agency Research Centre 675 West 10th Avenue Vancouver, BC, V5Z 1L3, Canada Telephone: 604-675-8206 Email: [email protected] David Perrin, Ph.D. Chemistry Department 2036 Main Mall University of British Columbia Vancouver, BC, V6T 1Z1, Canada Telephone: 604-822-0567 Email: [email protected] Kuo-Shyan Lin, Ph.D. BC Cancer Agency Research Centre 675 West 10th Avenue Vancouver, BC, V5Z 1L3, Canada Telephone: 604-675-8203 Email: [email protected]


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ABSTRACT DOTA is commonly used for radiometal chelation in molecular imaging. Yet in the absence of a radiometal, DOTA is hypothesized to promote renal clearance of 18Flabeled peptide tracers. In light of an increasing interest in the use of F18 for PET, here the effect of DOTA is evaluated for the first time with an 18F-labeled tracer and is found to significantly improve the quality of images acquired through positron emission tomography (PET). We chose to image the peptide LLP2A that recognizes the transmembrane protein very-late antigen 4 (VLA-4) that is overexpressed by many cancers. Since it is known that [18F]RBF3-PEG2-LLP2A derivatives gave low tumor uptake values and significant GI tract accumulation, this ligand thus represents an ideal means of testing the additive effects of a DOTA group on clearance while permitting a facile, user-friendly, one-step 18F-labeling. A newly designed RBF3-LLP2A bioconjugate with an appended DOTA moiety increased tumor uptake nearly 3-fold and reduced GI accumulation by more than 10-fold. The DOTA-AMBF3-PEG2-LLP2A was radiolabeled by isotope exchange and was purified by semi-prep HPLC and C18 cartridge elution. Male C57BL/6J mice bearing B16-F10 melanoma tumors that overexpress the VLA-4 target were used to evaluate [18F]DOTA-AMBF3-PEG2-LLP2A using a combination of static and dynamic PET scans, biodistribution studies and blocking controls at 1h post injection (p.i.). The precursor peptide was synthesized and 18F-labeled to provide formulations with mean (±SD) radiochemical purities of 95.9 ± 1.8 %, in radiochemical yields of 4.8 ± 2.9 % having molar activities of 131.7 ± 50.3 GBq/μmol. In vivo static PET images of [18F]DOTA-AMBF3-PEG2-LLP2A provided clear tumor visualization, and biodistribution studies showed that tumor uptake was 9.46 ± 2.19 percent injected dose



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per gram of tissue (%ID/g) with high tumor:muscle and tumor:blood contrast ratios of ~8 and ~10, respectively. Blocking confirmed the specificity of [18F]DOTA-AMBF3-PEG2LLP2A to VLA-4 in the tumor and the bone marrow. Dynamic PET showed clearance of [18F]DOTA-AMBF3-PEG2-LLP2A mainly via the renal pathway, wherein accumulation in the intestines was reduced 10-fold compared to our previously investigated LLP2A’s, while spleen uptake was at levels similar to previously reported LLP2A-chelator radiotracers. [18F]DOTA-AMBF3-PEG2-LLP2A represents a promising VLA-4 radiotracer and provides key evidence as to how a DOTA appendage can significantly reduce GIuptake in favor of urinary excretion. Implications for the development of dual-isotope theranostics that exploit the use fluorine-18 for imaging and DOTA to chelate therapeutic metal cations for therapy are discussed.

KEY WORDS: 18F-trifluoroborates,

positron emission tomography, radiofluorination, F18,

pharmacokinetic linkers, PET imaging agents, theranostics


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INTRODUCTION

Molecular imaging is at the forefront of translational medicine with new developments in theranostic and multi-modal imaging applications on the horizon. In the past two decades, cancer subtypes are increasingly being distinguished by peptides and other high-affinity ligands that have emerged from advances in proteomics and combinatorial screens1-6, designed as target-based diagnostics and therapeutics7. Towards these ends, labeled peptides distinguish pathologically distinct cell types and can assess the presence of specific molecular targets, which is impossible to do with [18F]FDG8-11. Of the various imaging modalities, positron emission tomography (PET) is increasingly used for pre-clinical target validation and clinical cancer diagnosis due to its high sensitivity and dynamic spatio-temporal resolution 12-14. For PET, several β+emitting isotope are commonly used including fluorine-18, gallium-68, and copper-64. Whereas copper-64 and gallium-68 provide considerable convenience in terms of peptide labeling, there are several well-known limitations associated with these radiometals. For example, gallium-68 generators are limited to 1-3 doses per day. More significantly, gallium-68 provides lower resolution images than those obtained with fluorine-1815-17. Copper-64, which is now available on a cyclotron, still requires isolation from Zn or Ni targets by strong acid treatment, is often produced at moderate specific activity 18, may be contaminated with other radiometals 19, does not have a unique decay path (17.8% radiotoxic β-), is prone to transchelation once chelated20-22, and is not nearly as disseminated as fluorine-18. For these reasons, 18F-fluorine remains a



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mainstay isotope with several key advantages including: a very low β+-emission energy that enhances image resolution15, 16, negligible radiotoxicity due to a near-unique decay path (97% β+), and on-demand production at multiCurie-levels in hospital cyclotrons for scalable radiosyntheses23. Previously, the use of 18F-fluorine for peptide labeling was encumbered by multi-step reactions. Yet, several one-step labeling methods, recently reviewed24, now overcome the well-known challenges in 18F-labeling to make radiofluorination nearly as user-friendly as radiometallation25. To wit, 18F-labeled trifluoroborate radioprosthetic groups ([18F]RBF3s) have emerged as a viable means of achieving a simple, user-friendly, scalable, one-step, aqueous labeling of complex molecules e.g. peptides 26. Due to their polarity, [18F]RBF3s are salts that are distinctly hydrophilic compared to most other 18F-labeled radioprosthetic groups. In turn, [18F]RBF3s have demonstrably favored renal clearance over hepatobiliary clearance in the context of five classic peptide tracers: e.g. bombesin 27,

octreotate 28, bradykinin 29, melanocortin 30, and rhodamine-bis-RGD 31. In these

antecedent cases, the rapid clearance combined with good-to-high tumor uptake values is thought to contribute to high-contrast images, as well as characteristically low liver and intestinal uptake values (e.g. 0.05) affected.

Table 1. Biodistribution of [18F]6 at 1 h p.i. in B16-F10 tumor-bearing mice and with coinjection (200 μg) of the blocking agent, 1 (mean %ID/g with ±SD). Tissue [18F]6 (n = 6) [18F]6 with blocking (n = 5) Blood 0.94 ± 0.05 0.72 ± 0.09 Fat 0.31 ± 0.08 0.08 ± 0.01 Seminal 0.92 ± 0.24 0.27 ± 0.16 Testes 0.31 ± 0.02 0.22 ± 0.04 Intestine 4.55 ± 0.80 2.6 ± 0.55 Spleen 28.33 ± 4.28 3.49 ± 0.61 Pancreas 0.86 ± 0.14 0.22 ± 0.01 Stomach 1.24 ± 1.82 a 1.20 ± 0.20 Liver 1.61 ± 0.21 0.63 ± 0.06 Adrenal glands 1.51 ± 0.31 0.47 ± 0.17 Kidney 4.32 ± 0.50 3.70 ± 0.56 Heart 0.66 ± 0.09 0.26 ± 0.03 Lungs 6.86 ± 0.46 1.06 ± 0.03 Tumor 9.46 ± 2.19 2.37 ± 0.34 Bone & Marrow 8.23 ± 0.84 1.80 ± 0.17 Muscle 1.30 ± 0.33 0.27 ± 0.05 Brain 0.07 ± 0.02 0.04 ± 0.01 Tail 1.84 ± 0.41 1.10 ± 0.85 a. Includes one mouse with 4.45 %ID/g.



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Table 2. Tumor-to-tissue ratios of [18F]6 after 1 h p.i. in B16-F10 tumor-bearing mice and with co-injection (200 μg) of the blocking agent, 1 (mean ratio and ±SD). Ratios Tumor: Bone & Marrow Tumor: Muscle Tumor: Blood Tumor: Kidney

[18F]6 (n = 6) 1.98 ± 0.78 7.96 ± 3.37 10.08 ± 2.17 2.22 ± 0.60

[18F]6 with blocking (n = 5) 0.46 ± 0.07 0.60 ± 0.05 1.50 ± 0.18 2.55 ± 0.95

DISCUSSION Previously, we had provided the first-ever report of an 18F-labeled LLP2A radiotracer labeled by isotope exchange on an [18F]AMBF3-PEG2-LLP2A tracer. Given the high intestinal uptake previously observed for [18F]AMBF3-PEG2-LLP2A, this particular AMBF3-LLP2A conjugate also provided an ideal peptidic scaffold to test the hypothetical influence of DOTA to modulate tracer biodistribution. To minimize structural and chemical differences, we designed 6 such that the DOTA would be appended on an otherwise nearly-identical LLP2A-AMBF3 conjugate (see comparison of structures in Figure S1). The chemical synthesis of 6 proceeded in an efficient, step-wise manner with nearly quantitative yields for most chemical reactions. The synthesis of the FmocLys(AMBF3)-COOH represents a novel building block for introducing the trifluoroborate during peptide synthesis. The relatively low recovery yield of 6 in the final chemical step was attributed to sample loss as a result of stringent HPLC purification conditions. Nevertheless, 6 had high chemical purity and was obtained in sufficient quantities for all described 18F-radiolabelings and in vivo studies. Radiosynthesis of [18F]6 was achieved in a single aqueous step by 18F-IEX under aqueous conditions as previously demonstrated for other [18F]RBF3s evaluated by our lab 26. The radiosynthesis time, yields, purities and molar activities were suitable for in


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vivo studies and were similar to [18F]AMBF3-PEG2-LLP2A 32. While yields were low, sufficient activities of [18F]6, 0.37-1.85 GBq, were consistently obtained for each in vivo study (n>6). Notably, both the labeling and recovery methods are unoptimized and we are confident that these yields may be improved by modifying the radiolabeling and purification steps. To make this study consistent with prior published data, we imaged the wellestablished murine melanoma model, B16-F10, which expresses the target VLA-4. Besides high tumor uptake, [18F]6 displayed accumulation in the spleen and bone marrow at 1h p.i. as is characteristic of all tracers based on LLP2A. Gratifyingly, the uptake of [18F]6 in B16-F10 melanoma tumors and the corresponding tumor-to-muscle and tumor-to-blood ratios were similar to those observed for [64Cu]Cu-CB-TE1A1PPEG4-LLP2A, [64Cu]Cu-CB-TE2A-LLP2A, [64Cu]Cu-NODAGA-PEG4-LLP2A and [68Ga]Ga-NODAGA-PEG4-LLP2A with the same tumor model at similar time points (1h 2h p.i.) 55, 57-59. The high accumulation of [18F]6 in the spleen also recapitulates images obtained with 64Cu- and 68Ga-labeled LLP2A derivatives 55, 57-59. Our results also confirmed that the clearance route of [18F]6 was via the kidneys and bladder, as with antecedent studies with radiometallated LLP2A-based tracers 54, 56, 58, 62-64. While [18F]6, obtained at 94-98% radiochemical purity, showed small amounts of contaminating free [18F]fluoride ion (1-6%), which would likely have been taken up in bone, the significant and blockable bone uptake is most likely due to specific uptake of [18F]6 in bone marrow. Here, we suggest that the observed accumulation in bone is not due to defluorination for the following reasons: 1) relatively high bone marrow uptake is expected, as VLA-4 is expressed by haematopoietic stem cells found in bone marrow 35,



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15 42, 43, 65-68;

2) previously, 64Cu- and 68Ga-labeled LLP2A derivatives 55, 57-59 were found to

accumulate in bone with similar tumor:bone ratios: 2.3-2.6 56 compared to the tumor:bone ratio of 2.0 herein; 3) the co-injection of unlabeled LLP2A reduced uptake in bone and in tumor to a similar extent: 4.5 fold for bone and 4-fold for tumor, thus confirming the specificity of [18F]6 uptake in the bone marrow due to VLA-4 expression therein. Since we did not confirm metabolic stability, we note the possibility that [18F]6 could have been metabolized to an unstable metabolite that would subsequently release free [18F]fluoride ion. In this scenario, liberated [18F]fluoride ion would then accumulate in bone, whereas the coinjected unlabeled LLP2A (1) might have blocked metabolism to give the appearance of reduced specific uptake in bone. Yet, it would be unlikely that [18F]6 uptake in tumors is VLA-4 specific, while uptake in bone marrow were due to metabolic defluoridation that had been indirectly blocked at the level of metabolism. Finally, of note, several other classic peptides 27, 28, 30, 31, each of which was labeled with the same [18F]AMBF3 radioprosthetic group used herein, showed less than 1% bone uptake, an observation that further corroborates the in vivo stability of the radioprosthetic group in regards to metabolic/solvolytic liberation of [18F]fluoride ion. Taken together, the most parsimonious explanation for blocked uptake in bone marrow is direct competition by 1 for recognition of VLA-4, which is expressed in bone marrow. Whether uptake in bone marrow would pose a significant problem in a clinical setting is a complex question; in considering dosimetry of F-18 vs. Cu-64, the uptake value in bone marrow of both classes of LLP2A tracer is comparable (8-12% ID/g). Insofar as Cu-64 has a longer half-life than F-18 and produces toxic β--particles, it is


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expected that a radiocuprated tracer will result in higher collateral radiation doses to bone and marrow. Nevertheless, irrespective of which is isotope used, LLP2A may give higher radiation doses to bone marrow than other tracers that recognize targets that are not expressed in marrow. [18F]6 was labeled in a single aqueous step at higher molar activities than those reported previously for 64Cu-labeled LLP2A derivatives 56, 58, 59. Although high molar activity may have contributed to the high-contrast static PET images for [18F]6, similarly high molar activities were achieved for the previously reported [18F]AMBF3-PEG2-LLP2A tracer that exhibited 4.6-fold higher binding affinity. Interestingly, for [18F]AMBF3-PEG2LLP2A, tumor uptake values were 2-3 fold lower than those shown herein. These observations show an imperfect correlation between affinity and image quality and nicely illustrate how varying the peptide composition not only affects affinity but tumor uptake values, most notably (and counterintuitively) in distinctly opposite directions. The only appreciable difference between 6 and the antecedent AMBF3-PEG2LLP2A conjugate (Figure S1) is the DOTA moiety, which must be responsible for higher tumor uptake and reprogrammed clearance to the kidneys. By contrast, the previously reported [18F]AMBF3-PEG2-LLP2A exhibited ca. 4-fold lower spleen retention and ~11fold higher accumulation in the GI tract. In general, our results herein, along with others’ support the appendage of a hydrophilic group as a means of redirecting the route of clearance (i.e. renal vs. hepatobiliary) of LLP2A bioconjugates. It is noteworthy that while LLP2A has been extensively optimized over the past decade with regards to varying linker arms and new chelators to finally achieve excellent images with gallium68 and copper-64, the work herein represents only the second instance where LLP2A



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has been labeled with F18; without extensive optimization, the simple appendage of a metal-free DOTA now gives images that are comparable to those obtained with 64Cu. While the origins of the observed increased tumor uptake and decreased background have not been fully elucidated here, we speculate that the free carboxylates along with the cationic amines that impart an overall zwitterionic nature to the DOTA likely enhance the image quality. Yet, subtler effects, including increased water solubility and general polarity might also be contributing factors, all of which would be worthy of further investigation by testing discrete DOTA derivatives including ones where the carboxylates are replaced with carboxamides. Also, while we did not examine the affects of DOTA in the contexts of other 18Fradioprosthetic groups e.g. SiFA, SFB (as such would go far beyond the scope of this study), it is reasonable to suggest that DOTA may find use with many other 18F-labeled radioprosthetic groups, particularly less polar ones. Hence, this report should empower others with expertise in these alternative labeling methods to consider the use of DOTA for improved renal clearance. We also surmise that DOTA may prove similarly useful for other peptidic tracers in cases where intestinal uptake is high and we see no limitations in appending DOTA onto other peptide tracers e.g. TATE or PSMA. Several different chemical functionalities that are already known to favor renal clearance e.g. mono/di-saccharides 69, 70 aspartate residues 71, or Glu-His hexamers 72 might have been used. While we might have recapitulated their use, here we sought to use metal-free DOTA for the explicit reason that it had never been used to address the clearance of an 18F-labeled peptide. Unlike the aforementioned chemical functionalities that have been appended to favor renal clearance, DOTA affords the unique potential


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for incorporating non-radioactive metals (e.g. Zn2+) to study their effects on in vivo PK/PC via 18F-PET imaging. Herein we considered DOTA to be M2+-free. Yet, based on the MALDI-TOF data (Figure S4), we cannot fully exclude the possibility that the tracer production may have been contaminated with either Mg2+ or Ca2+ (although these metal cations were not used in labeling and would have likely been precipitated as their respective waterinsoluble difluoride salts). More interestingly, in vivo levels of Ca2+ and Mg2+ may affect the distribution of so-called DOTA-modified tracers. Hence, we cannot absolutely exclude the possibility that the clearance observed herein reflects adventitious interactions between DOTA and multivalent metal cations in vivo that may be chelated by DOTA. These results are portentous for developing new theranostics. A canonical theranostic is defined to “combine diagnostic and therapeutic capabilities into a single agent73” where the entire composition, including the radioisotope, must be chemically invariant. Apart from a few select isotope pairs that interchangeably permit PET and therapeutic applications (e.g. 86Y/90Y) 74, the use of a single element for diagnostic PET and targeted therapy has been impractical. Due to this difficulty, a pair of radiometals is often used. For example, DOTATATE, which is labeled with gallium-68 for diagnosis and lutetium-177 for therapy75, is commonly considered to be a theranostic. Yet whereas the peptide-chelator is invariant, the two isotopes differ in their coordination chemistry76. Consequently, the affinity of [68Ga]Ga-DOTA-TATE has been reported to be up to 20-fold higher than that of [177Lu]Lu-DOTATATE 77 (compare reports for GaDOTATATE: 0.2 nM 78 or 1.2 nM 79 with reports for Lu-DOTATATE: 2 nM 79 or 3.8 nM



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In human studies, these differences may account for noted discordance in lesion

detection whereby lesions are revealed by one tracer but not the other, and vice-versa 81.

Similar discrepancies are observed with PSMA-617: in vitro, the affinity of the Ga-

chelate is twice that of the Lu-chelate; in mice, tumor uptake values and T:NT ratios differ substantially between the two chelates82. Finally, for DOTA-PEG4-LLP2A, the Ki value the Ga-chelate is half that of the Lu-chelate 62. To meet the challenge of designing a canonical theranostic agent (not for semantic reasons but ultimately physiological ones), here we suggest an attractive solution: the production of “hot-cold/cold-hot” paired isotopologs (e.g. 18F/natLu and natF/177Lu)

that are absolutely identical in chemical composition. As envisaged, for

diagnostic use, the peptide is chelated to a nonradioactive metal cation and labeled with 18F,

whilst for therapeutic use, the peptide is chelated to the corresponding radiotoxic

metal cation in conjunction with an unlabeled trifluoroborate. Conceptually, this greatly expands the choice of radiotoxic metals for use in therapy whilst i) granting access to fluorine-18 as the choice isotope for small-molecule PET, ii) alleviating the supply problems associated with gallium-68 and iii) obviating the unfortunate dissimilarity in chelation chemistries that arise from using two different radiometals for PET and therapy. Furthermore, isotopologous labeling should be readily practicable with other fluorinated radioprosthetics e.g. SiFA, SFB provided that chemical compatibilities are addressed and convenient radiolabeling is ensured 83. In summary, this work represents the first investigation of the effects of metalfree DOTA on the clearance of an 18F-labeled peptidic radiotracer. Herein, we show that tumor uptake of an 18F-labeled RBF3-PEG2-LLP2A can be greatly enhanced by using a


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hydrophilic metal-free DOTA that can be introduced in a simple synthetic approach using Fmoc-Lys-AMBF3. 18F-labeling proceeds by isotope exchange at high molar activity. In vivo PET imaging and biodistribution studies demonstrate that the DOTA moiety diverts radiotracer accumulation from the GI tract to the bladder via the kidneys to lower intestinal uptake by more than 10-fold while significantly increasing tumor uptake values by more than 3-fold. Thus, [18F]DOTA-AMBF3-PEG2-LLP2A represents a promising VLA-4 radiotracer and provides evidence that a DOTA appendage can significantly reduce GI-uptake in favor of urinary excretion. A further extension of this approach would be to use the same molecule for PET imaging with F-18, and targeted radiotherapy with radiometals such as Lu-177.

MATERIALS AND METHODS

Synthesis of DOTA-AMBF3-PEG2-LLP2A (6) The LLP2A peptidomimetic was synthesized on the solid phase as described 33, while using a O-bis-(aminoethyl)ethyleneglycol trityl resin for anchoring a PEG2 spacer and an –NH2 conjugation handle, as previously reported 32. LLP2A-PEG2-NH2 (1) (4.2 mg, 4.46 µmol, 1 eq.) was dissolved in 200 µL of (1:19) DIPEA: DMF (v/v) and was subsequently used to dissolve Fmoc-Lys-(AMBF3)-NHS (2) (5.85 mg, 8.9 µmol, 2 eq.), the synthesis of which is given in Scheme 1 and detailed in the supplementary information. The conjugation proceeded at r.t. for 2 h. The mixture was concentrated by speed-vac (50-100 µL), precipitated with 1.0 mL Et2O and centrifuged. The supernatant was removed and the product was redissolved in 50 µL DMF. The described Et2O



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precipitation/centrifugation methods were repeated, and the final pellet was dried by speed-vac. These methods gave 6.5 mg (4.3 µmol) of LLP2A-PEG2-AMBF3-Fmoc (3) in near quantitative yield. ESI-MS(+): calculated for C76H98BF3N14O13, 1483.5 m/z; found, [M+Na]+ = 1506.8 m/z, [M+2Na-H]+ = 1528.8 m/z (Figure S2). TLC: [(1:19) NH4OH: EtOH, v/v], Rf = 0.46 (visible with 254 nm). The intermediate, 3 (1.5 mg, 1 µmol, 1 eq.), was dissolved with 200 µL of (1:4) piperidine: DMF (v/v), and Fmoc-removal was achieved within 2 h at rt. The mixture was concentrated and subjected to two rounds of the described Et2O precipitation/centrifugation methods. The final pellet was dried by speed-vac to provide ~1.3 mg (~1 µmol) of AMBF3-PEG2-LLP2A-NH2 (4) with a quantitative yield. ESI-MS(+): calculated for C61H88BF3N14O11, 1261.3 m/z; found, [M+H]+ = 1262.6 m/z, [M+Na]+ = 1284.6 m/z (Figure S3). TLC: [(1:19) NH4OH: EtOH, v/v], Rf = 0.18 (visible with 254 nm, stained with ninhydrin). The intermediate, 4 (1.8 mg, 1.4 µmol, 1 eq.), was dissolved in 100 µL of (1:19) DIPEA: DMF (v/v) and conjugated to DOTA-NHS (5) (1.6 mg, 2.1 µmol, 1.5 eq.) within 2 h at rt. The mixture was concentrated and subjected to two rounds of the described Et2O precipitation/centrifugation methods. The final pellet was dried by speed-vac to provide 2.4 mg (~1.4 µmol) of crude DOTA-AMBF3-PEG2-LLP2A (6). The aforementioned procedures were repeated to obtain 42.1 mg of crude 6 for HPLC purification. These combined samples were dissolved in 1.0 mL of (1:1) MeCN with 0.1% formic acid: H2O with 0.1% formic acid (v/v) and purified by HPLC method A. The product (6) was collected at tR = 8.9 min and diluted with an equivalent volume of H2O before freezing with dry ice and lyophilisation. These methods gave 3.1 mg (1.9 μmol) of the purified (>95% purity) radiotracer precursor, 6 (characterized by ESI-MS, Figure S4 and HPLC,


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Figure S5), for a 7.4% yield. ESI-MS(+): calculated for C77H114BF3N18O18, 1647.6 m/z; found, [M+H]+ = 1648.9 m/z, [M+Na]+ = 1670.9 m/z [M+K]+ = 1687.0 m/z. TLC: [(1:19) NH4OH: MeOH], Rf = 0.67 (visible with 254 nm, stained with light blue with bromocresol green). Mass spectra, and the HPLC method (A) and chromatogram can be found in the Supplementary Information.

Saturation binding assay In vitro binding saturation assays were performed on B16-F10 cells following published procedures (n = 3) 32. Cells were grown to near-confluence on 24 well poly-Dlysine plates. Growth media was removed, and reaction media (RPMI, 1% BSA, 100 U/mL penicillin/streptomycin) was added and allowed to incubate for at least 1 h at 37 °C. Increasing concentrations (5 pM to 20 nM) of [18F]6 were added to the cells and incubated for 1 h at 25 °C with mild agitation. Non-specific binding was determined by repeating the described incubations with [18F]6 while simultaneously adding 1 (10 μM). After incubation, cells were washed twice with ice-cold PBS, harvested following incubation with trypsin, and measured for radioactivity using a WIZARD 2480 gamma counter (PerkinElmer). Values from the non-specific binding assays were subtracted from the respective values of the specific binding assays. Dissociation constants (Kd’s) were determined using GraphPad PRISM 7 with a one-site specific binding model.

Radiolabeling of DOTA-AMBF3-PEG2-LLP2A (6) with [18F]fluoride anion Precursor 6 (80 nmol) was dissolved in 15 μL of 1 M pyridazine-HCl (pH = 2), 10 µL of DMF, 15 μL of MeOH and 4 μL of 5 mM KHF2 (aq) (50 mol% 19F- carrier) with a final



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pH of ~2.0 (Scheme 1). The 18F/ H218O (29 – 44 GBq) was passed through an activated 9 mg QMA cartridge (pre-conditioned with 1.7 mL saturated NaCl then rinsed with 2.5 mL water and dried with 3 mL air) to trap 18F (>95% efficiency), which was then eluted into a septum-sealed reaction vessel containing the precursor using 80-100 μL of 0.9% saline (>90% efficiency). The solution was heated at 82-84 °C for 5 min and then heated in vacuo for 15 min. After quenching with 2 mL of 40 mM NH4HCO2 (aq) (pH = 6.8), the solution was purified by semi-preparative HPLC with two successive isocratic solvent conditions (HPLC method B). The radiotracer [18F]6, was collected and directly diluted into 50 mL of H2O. By applying a small pressure of helium to the container, the resulting solution was passed through a Sep-Pak Light C18 cartridge (pre-washed successively with 9 mL EtOH, 9 mL H2O and 10 mL air). The trapped [18F]6 was eluted into a septum-sealed vial with 0.5 mL of (9:1) EtOH: 0.9% saline (v/v), and was finally formulated with 4 mL PBS to provide [18F]6 in (1:9) EtOH: PBS (v/v) at pH ~7 for animal injections. All purified formulations of [18F]6 were characterized by analytical HPLC (method C) to quantify the radiochemical purity, radiochemical yield, and molar activity based on a standard curve, (Figure S6) prior to animal injections. The HPLC methods (B and C) and the standard curve for 6 can be found in the Supplemental Materials.

PET imaging of [18F]DOTA-AMBF3-PEG2-LLP2A ([18F]6) in B16-F10 tumor-bearing mice B16-F10 cells (ATTC, CRL-6475) were cultured in DMEM with 10 % FBS (v/v), 100 U/ mL penicillin and 100 μg mL streptomycin at 37 °C under 5% CO2. All animal experiments were conducted following the guidelines of the Canadian Council on


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Animal Care and were approved by Animal Ethics Committee of the University of British Columbia. Male C57BL/6J mice were briefly sedated with 2% isoflurane, and injected with 1×106 B16-F10 cells subcutaneously at the right shoulder. Tumors were allowed to grow until reaching diameters of 7-9 mm. PET and CT imaging studies involved a microPET/CT scanner (Inveon, Siemens) as previously reported 27, 29, 32. For static PET scans, mice were first injected with 4-6 MBq of ([18F]6) via the tail vein while briefly sedated with 2% isoflurane. The mice were allowed to recover and roam free prior to being sedated for a baseline attenuation correction CT scan (approximately 10 min for 3 bed positions) followed by static PET imaging acquired over 10 minutes. For static PET images involving competitive VLA-4 blocking, 200 μg of 1 was co-injected with 4-6 MBq of [18F]6, and the same methods were used to obtain both baseline CT and PET images at 1h p.i. Dynamic PET scans were performed under 2% isoflurane anesthesia. A baseline CT scan was acquired as described above for attenuation correction. A 60minute dynamic acquisition was started and 4-6 MBq of [18F]6 injected intravenously immediately after starting the acquisition. PET images were rebinned to a total of 28 time intervals (12 x 10 sec, 8 x 1 min, 7 x 5 min and 1 x 15 min), ranging from 5 seconds for early time points to 10 minutes for later time points. Mice were finally euthanized using CO2 inhalation after each static PET imaging study and their organs were harvested for biodistribution measurements.



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Biodistribution of [18F]DOTA-AMBF3-PEG2-LLP2A ([18F]6) in B16-F10 tumorbearing mice Mice were briefly sedated using 2% isoflurane and injected with 2-3 MBq of [18F]6 via the tail vein. For competitive VLA-4 blocking studies, 200 μg of 1 was co-injected with 2-3 MBq of [18F]6. At 1h p.i., mice were euthanized by CO2 inhalation, blood was collected, and the organs were excised. After rinsing and drying the samples, organs were weighed, their radioactivity was recorded using a Wallac WIZARD2 gamma counter (PerkinElmer), and values were expressed as %ID/g for each organ. Two-tailed ANOVA Sidak’s multiple comparison tests (GraphPad PRISM) were used to evaluate statistical significance between the biodistribution of [18F]6 alone compared with competitive blocking of VLA-4 by co-injections of 1.

ACKNOWLEDGEMENTS: This work was supported by an innovation grant of the Canadian Cancer Society Research Institute (CCSRI# 704366). We thank cyclotron operators Wade English, Baljit Singh, and Milan Vuckovic, and PET imaging technologists, Nadine Colpo and Navjit Hundal-Jabal, for technical assistance.

CONFLICT OF INTEREST A provisional patent which entitles authors to eventual royalties has been filed on aspects herein. UBC holds issued patents on compositions of matter of trifluoroborates


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that would entitle DMP to royalties upon licensing. No other competing interests relevant to this article exist.

SUPPORTING INFORMATION STATEMENT NMR spectroscopic and MS data, IC50 analysis, and detailed synthetic procedures are found in a single word-document in the supporting information.

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production of diverse 18F-labeled PET tracers on the ELIXYS multireactor radiosynthesizer without hardware modification. Journal of nuclear medicine technology 42, 203-210.

GRAPHICAL ABSTRACT:

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ACS Paragon Environment DOTA-modified [18F]BFPlus 3-labeled Tracers: higher tumor uptake very low gut uptake

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A Metal-Free DOTA-Conjugated 18F-Labeled Radiotracer: [18F]DOTA

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