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Article 64

Site-specific Cu labelling of the serine protease, active site inhibited factor seven azide (FVIIai-N), using copper free click chemistry 3

Troels Elmer Jeppesen, Lotte Kellemann Kristensen, Carsten Haagen Nielsen, Lars Christian Petersen, Jesper Bøggild Kristensen, Carsten Behrens, Jacob Madsen, and Andreas Kjær Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.bioconjchem.7b00649 • Publication Date (Web): 05 Dec 2017 Downloaded from http://pubs.acs.org on December 11, 2017

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Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Bioconjugate Chemistry

Title: Site-specific 64Cu Labelling of the Serine Protease, Active Site Inhibited Factor Seven Azide (FVIIai-N3), Using Copper Free Click Chemistry

Authors: Troels E. Jeppesen1, Lotte K. Kristensen1,2, Carsten H. Nielsen1,2, Lars C. Petersen3, Jesper B. Kristensen3, Carsten Behrens3, Jacob Madsen1, Andreas Kjaer1

1Department

of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging,

Department of Biomedical Sciences, Rigshospitalet and University of Copenhagen; 2Minerva Imaging ApS; and 3Novo Nordisk A/S

Corresponding author: Andreas Kjaer 1Department

of Clinical Physiology,

Nuclear Medicine & PET and Cluster for Molecular Imaging, Department of Biomedical Sciences, Rigshospitalet and University of Copenhagen Blegdamsvej 9 DK-2100 Copenhagen Ø +45 27258614 [email protected]

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

Abstract

A method for site-specific radiolabelling of the serine protease active site inhibited factor seven (FVIIai) with 64Cu has been applied using a biorthogonal click reaction. FVIIai binds to tissue factor (TF), a trans-membrane protein involved in haemostasis, angiogenesis, proliferation, cell migration, and survival of cancer cells. First a single azide moiety was introduced in the active site of this 50 kDa protease. Then a NOTA moiety was introduced via a strain promoted azide-alkyne reaction and the corresponding conjugate was labelled with 64Cu. Binding to TF and the stability was evaluated in vitro. TF targeting capability of the radiolabelled conjugate was tested in vivo by positron emission tomography (PET) imaging in pancreatic human xenograft cancer mouse models with various TF expressions. The conjugate showed good stability (>91% at 16 hours), an immunoreactivity of 93.5% and a mean tumour uptake of 2.1 ± 0.2 %ID/g at 15 hours post injection. In conclusion, FVIIai was radiolabelled with 64Cu in single well defined position of the protein. This method can be utilized to prepare conjugates from serine proteases with the label at a specific position.

Introduction Positron emission tomography (PET) is a versatile imaging technique used for diagnostic and prognostic purposes.1 Copper-64 (64Cu; half-life 12.7 h, β+ 0.656 MeV, 17.4%) has received increasing interest as a PET radioisotope for labelling of biomolecules such as peptides, proteins and antibodies due to its relative long half-life, enabling imaging at late time points and central radiochemistry production for distribution. The traditional way of introducing radioactive metals into biomolecules is by chelation.2 Typically, the polydentate, cyclic bifunctional chelators DOTA or NOTA has been utilized due to the strong kinetic stability of the metal-chelator complex.3,4 The


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search for even more stable Cu-chelator complexes includes cross-bridged structures, e.g. CBTE2A, chelators with phosphine pendant arms, sarcophagine analogues and NODAGA. However, labelling of CB-TE2A requires high temperature (>90C)5, the kinetic inertness of 64Cu-chelators with phosphine pendant arms are not considerably higher than for NOTA6 and sarcophagine analogues are laborious to synthesize7. A NODAGA chelator could have been used, but the gain in stability, compared to NOTA, is not that notable8 and clinically DOTA and NOTA chelators have been preferred.2,9 The in vivo stability of 64Cu-NOTA appears to be superior to 64Cu-DOTA, and chelation to NOTA can be obtained at room temperature.10 This is important as complex biomolecules frequently display poor stability at elevated temperatures. Consequently, NOTA was chosen as chelator for this study.

Biomolecules often contain lysine residues which can react with NOTA functionalized with aminereactive groups, such as isothiocyanates or NHS-esters. This approach will provide an unpredictable distribution between the accessible reactions sites which can, potentially affect the binding affinity of the biomolecule, or it may partially un– or refold the protein, rendering the biomolecule inactive. In light of this, site specific radiolabelling of larger biomolecules, such as proteins, would be desirable. To this end, the glycan chains of antibodies have been investigated for specific radiolabelling with Zirconium-89 (89Zr; half-life 78.41 h, β+ 0.897 MeV, 22.7%).11 The glycan chains can be modified to expose four positions for such labelling. Showing comparable parameters to a random labelling (isothiocyanate), the authors suggested that less robust biomolecules might benefit more from site specific radiolabelling.11 For glycoproteins this method is not applicable due to the observation that removal of the terminal sialic acids of the glycan chains will result in fast hepatobiliary clearance from the bloodstream.12



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In recent years click chemistry has achieved greater attention by radiochemists, realizing the potential for labelling biomolecules by these efficient and water compatible reactions.13 Especially Cu catalysed Huisgen 1,3 dipolar cycloaddition and strain promoted azide-alkyne coupling (SPAAC) are attractive for labelling of biomolecules. Azides are fairly easy to incorporate, are stable towards degradation, and are inert with regard to biological reactions14–19. Cu-ions and ascorbate used for Huisgen 1,3 dipolar cycloaddition may, however, alter protein structures by virtue of its redox potential.20 Bertozzi and co-workers introduced the strain promoted azide-alkyne cycloaddition (SPAAC), to circumvent the use of copper, by introducing the strained cyclooctyne as reaction partner to the azide.21 The reaction kinetics were slow and therefore several modified cyclooctynes, including BCN and DIBO species, were developed to increase the ring strain and thereby the reaction rate.22–24 With an increasing number of cyclooctyne molecules commercially available, SPAAC is convenient to use as long as an azide can be introduced in the target molecule.

Tissue factor (TF) is a 47 kDa membrane-bound glycoprotein that initiates the coagulation cascade when it is exposed to the circulation and binds its natural ligand factor seven (FVIIa). TF is upregulated in several cancer forms and the TF:FVIIa complex has been shown to influence angiogenesis, proliferation, cell migration, and cell survival through protease activated receptors (PARs).25,26 FVIIa can be specifically blocked in the protease domain, producing active site inhibited factor seven (FVIIai). As a result FVIIai does not initiate the coagulation cascade, but still form complexes with TF with a fivefold higher affinity.27,28 Inhibition of FVIIa can be achieved by a chloromethylketone-tripeptide, e.g. H-D-Phe-Phe-Arg-chloromethylketone (fFRck), which can be modified in the N-terminus without abolishing its specific inhibitory effect.29


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Serine proteases constitute a large class of proteins, which accounts for almost one third of all proteases. All of these can be inhibited by chloromethyl ketones. Some serine proteases will, like FVIIai, still preserve a targeting ability when they are split in separate heavy and light chains upon activation.30 The heavy and light chains of all coagulation factor proteinases are linked via a disulphide bond after activation, and consequently retain binding to their receptors. They may therefore be applicable radioactive ligands for the receptors by a specific site labelling with radioactive nuclides attached to a single azide incorporated in the active site via chloromethylketone chemistry.

Previously, FVIIai has been labelled unspecifically with 18F and 64Cu via lysines in FVIIai by an NHS-ester and an isothiocyanate respectively.31–33 However, a specific labelling method could potentially improve the biodistribution, the immunoreactive fraction, the uptake of the tracer and regulatory approval.20 In this study, a single azide moiety was introduced in a 50 kDa serine protease, FVIIai and used as a single well defined specific site for 64Cu radiolabelling. In brief, the inhibitor fFRck was modified in the N-terminus with an azide (Figure 1), and used to form a new FVIIai analogue, namely FVIIai-N3 (4). This new analogue was coupled via SPAAC with ADIBOPEG4-NOTA (5), and chelated with 64Cu. The tracer was evaluated in vitro and in vivo.

Results and Discussion Synthesis 2-Azidoacetic acid (2) was produced following a literature procedure, Figure 1.34 The inhibitor N3fFRck (3) was synthesized by TSTU coupling of 2-azidoacetic acid (2) to the N-terminal of fFRck. In addition to the desired product (3) a side-product which was identified to be a cyclic adduct (supporting information) was formed. After preparative HPLC purification the resulting azide (3)



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was isolated and lyophilized. Excess N3-fFRck (3) was coupled to FVIIa using standard conditions for 19 hours in gly-gly buffer.28 Unreacted inhibitor was removed by dialysis, and the resulting FVIIai-N3 (4) was aliquoted, and stored at -80°C.

N3 O HO

O

O

a

Br

b

N3

HO

O

HN

O

H N

N H

Cl

O

(2) NH HN

NH 2

N 3-fFRck (3) N3 O

O

HN

N H

O

H N

Cl

c

FVIIai

N3 S

O

(4)

O H N

NH HN

O

R=

NH 2

O

O

N H

N H

N H O

O

N 3-fFRck (3)

O

O

HO N

HO

N N

O

d

N N

N

R

N

O

HO N

N

O

e

N N

FVIIai

O

N

N

R

N

O

N 64Cu

N

O

N

R

FVIIai

OH

OH

O (5)

HO

N

O

O (6)

O (7)

Figure 1. Synthesis of FVIIai-N3 (4), coupling to ADIBO-PEG4-NOTA (5) and labelling with 64Cu. a) NaN3 (2 eq, 14.5 mmol), H2O (8 mL), 24h, r.t. b) 1. TSTU (2.2 eq, 74.4 µmol), Et3N (2.2 eq, 10.3 µL), azidoacetic acid (2.5 eq, 82.2 µmol), DMF (150 µL), 30 min, 22°C. 2. fFRck (1 eq, 33.5 µmol), DMF(115 µL), Et3N (2.1 eq, 9.6 µL), 30 min 22°C. c) FVIIa (0.1 eq 10 mL, 1.39 mg/mL, 25 mM gly-gly, 50 mM NaCl, 25 mM CaCl2, pH 6.0), 19h r.t. d) FVIIai-N3 (4) (1 eq., 50 mM HEPES, pH 7.8, 150 mM NaCl, 10 mM CaCl2), 37°C, 20h. e) 64CuCl2, 0.1M NH4OAc (pH 5.5, 10 mM CaCl2, 150 mM NaCl), 25°C, 15 min.


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Efficiency of inhibition The efficiency of inhibition of FVIIa to obtain FVIIai-N3 (4) was determined by measuring the amount of FVIIa activity still present compared to the activity of the starting material. FVIIa kinetics was measured by a colorimetric assay in a spectrophotometer (supporting information). The degree of inhibition was 99.4%, designating full inhibition of FVIIa.

Degree of labelling The degree of labelling was determined by fluorescence coupling with DIBO-Alexa fluorescent markers, Alexa 488 and Alexa 647, at equivalents ranging from 0.5 to 5, Figure 2B. The degree of labelling was determined to be 0.95 ± 0.05 (n=4) by HPLC analysis, taking into account both fluorescent labels. As expected this correlate accurately to the degree of inhibition, by the additional notion that FVIIai-N3 (4) displays a single azide. A slightly lower observed degree of labelling than unity can be expected as the azide is in close vicinity to the protein, introducing a steric hindrance11, (details in supporting information). SDS-PAGE verified these results, displaying fluorescent labelling only at the heavy chain, which contains the proteolytic site inhibited by fFRck, Figure 2A. As expected the fluorescent band overlays closely the FVIIa band. Upon activation by hydrolysis of the peptide bond between arginine 152 and isoleucine 153 FVII is cleaved, and separated into two chains linked by a disulphide bond at cysteine 135 and cysteine 262. Cleavage of disulphide bonds under reducing conditions separate the proteins on the gel, producing heavy and light chains having apparent molecular weights of 30 and 28 kDa, respectively.35

SDS-PAGE was also used to verify that 64Cu was indeed attached to the heavy chain of the protein (radiolabelling as described below). Figure 2C shows the gel, and the radioactive band is coincident with the protein staining for FVIIai under both non-reducing and reducing conditions (lanes 3 and 4,



respectively). Only the heavy chain has been radiolabelled (lane 4), indicating attachment of NOTA at the proteolytic site. At random labelling conditions with 64Cu and 18F, two radioactive bands are present under reducing conditions (supporting information and reference31).

A

L 1 2 3 4 5 6 7

B 1.5 Degree of Labelling

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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Alexa 647 Alexa 488

1.0

0.5

0.0 0

C

kDa

2 4 Equivalents Alexa-Dye

L

1

2

3

6

4

62 49 38 28

Figure 2. Degree of labelling, Coomassie, fluorescence and radioactive labelling on SDS-PAGE. A) reducing conditions. Depicted for Click-IT Alexa Flour 488 DIBO Alkyne. L: Seeblue Plus 2 prestained standard. Lane 1: FVIIa + Alexa Flour 488 DIBO Alkyne. Lane 2: FVIIai-N3 (4). Lane 3-7 FVIIai-N3 (4) and 0.5, 1, 1.5, 2 and 5 eq. Alexa Flour 488 DIBO Alkyne. B) Degree of labelling ± SEM determined with Click-IT Alexa Fluor 488 DIBO Alkyne and Click-IT Alexa Fluor 647 DIBO Alkyne in increasing equivalents. The fluorescence signals levels off close to 1 equivalent (0.95 ± 0.05 (n=4), both fluorescent dyes included). The samples were analysed by a fluorescence detector on HPLC, Figure S4. C) Overlay of radiography on coomassie staining. L: Seeblue 2 prestained standard. 1: FVIIai, non-reducing conditions. 2: FVIIai, reducing conditions. 3: 64Cu-NOTA-PEG

4-FVIIai

(7), non-reducing conditions. 4: 64Cu-NOTA-PEG4-FVIIai (7), reducing conditions.


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Taken together, these results imply a complete reaction between N3-fFRck (3) and FVIIa to FVIIaiN3 (4), providing FVIIai conjugated with a single azide, which can be used in a number of reactions, including the stain promoted azide-alkyne click reaction.

Radiolabelling The bifunctional chelator ADIBO-PEG4-NOTA (5) was coupled to FVIIai-N3 (4) to provide a single chelation site for 64Cu. This was performed by overnight incubation of FVIIai-N3 (4) with ADIBO-PEG4-NOTA (5) at 37°C, followed by purification on a PD-10 desalting column. The resulting NOTA-PEG4-FVIIai (6) derivative was analysed by HPLC to assess concentration (Figure S9A), aliquoted and frozen at -80°C for later use. Physiological concentrations of Ca2+-ions are necessary for correct FVIIai-folding.36Thus 10 mM Ca2+ has to be present in the buffer used for formulation of NOTA-PEG4-FVIIai (6). Ca2+ ions in large excess could potentially interfere with 64Cu labelling of NOTA-PEG4-FVIIai (6) as Ca2+ has affinity for NOTA with a stability constant of 8.92, whereas Cu2+ has a stability constant of 21.63.37 The chelator ADIBO-PEG4-NOTA (5) was therefore labelled with 64Cu in a HEPES buffer with 10 mM Ca2+to rule out that a high concentration of Ca2+ compared to Cu2+ would affect the radiolabelling (supporting information, showing full incorporation). The minimum amount of protein required for labelling was investigated to obtain optimal molar activity of the resulting tracer. A series of test-reactions were set up with different protein amounts for a fixed radioactivity amount of 300-400 MBq 64CuCl2. The results are shown in Figure 3A. Full incorporation could be achieved with at least 2 nmol of protein.

For animal experiments the following reaction conditions were applied: An aliquot of 64CuCl2 (400500 MBq) was added to 2-3 nmol of NOTA-PEG4-FVIIai (6), and the reaction was shaken at 300



rpm, at room temperature for 15 minutes. The mixture was purified by a PD-10 desalting column, pre-eluted with BSA, using gly-gly as the formulation buffer. Radiochemical yield (RCY) 64.2 ± 3.5%, molar activity of 874 ± 198 GBq/µmol, a radiochemical purity (RCP) of 99.0 ± 0.5% (HPLC) and 95.6 ± 0.4% (protein precipitation) was achieved (n = 4). Representative HPLC chromatograms can be seen in Figure 3B

A

B

Cu-NOTA-PEG4-FVIIai UV (254 nm)

Radio (mV)

Radio 100

50

300 250

1000

UV (a.u.)

% incorporation

64

1500

150

200 500

150 100

0

0 0

2

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6

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5

nmol

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64

Cu + FVIIai-N3

1500

UV (254 nm)

10 Time(min)

UV (254 nm)

100

0

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500

UV (a.u.)

Radio (mV)

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Radio 200

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Cu + FVIIai

Radio 1000

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64

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UV (a.u.)

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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100

0 0

5

10 Time(min)

15

20

0

5

10 Time(min)

15

20

Figure 3. Incorporation and HPLC analysis. A) Incorporation at 0-5 nmoles NOTA-PEG 4-FVIIai (6), using 300-400 MBq of 64CuCl . 2

Full incorporation is achieved above 2 nmol. B) HPLC chromatogram of 64Cu-NOTA-PEG4-FVIIai (7). The peak at

9.5 min coincide in both UV and radio trace. C) 64CuCl2 and FVIIai-N3 (4). The UV peak at 9.5 min does not correspond to a radiopeak. The radiopeak at 4 min is 64Cu-EDTA. D) 64CuCl2 and FVIIai. The UV peak at 9.5 min does not correspond to a radiopeak. The radiopeak at 4 min is 64Cu-EDTA.


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Negative controls with both FVIIai and FVIIai-N3 (4) in combination with 64CuCl2 was conducted and analysed by HPLC, Figure 3C and D. In both cases, no protein-bound radioactivity could not be observed and no difference was seen upon EDTA addition.

Stability In vitro stability was tested with and without gentisic acid as radical scavenger, Figure 4A. Without gentisic acid the RCP was 87% after 5 hours which clearly indicate the need for a scavenger. With gentisic acid 64Cu-NOTA-PEG4-FVIIai (7) was intact up to 5 hours, >95% RCP retained, assuming no dilution or further formulation was performed. This is not surprising as for example thio-urea bonds are known to be unstable at higher radioactivity concentrations.38 In the final formulation, 0.1% BSA is added to avoid non-specific binding to plastic utensils. To reduce the radioactive concentation the formulation can be further diluted with gly-gly buffer prior to injection.



B 110

gly-gly buffer

100

Gentisic acid

90 80 70 60 50 0

C

5

10 Time (h)

15

20

% intact 64Cu-NOTA-FVIIai

% intact 64Cu-NOTA-FVIIai

A

[Immunoreactive fraction]-1

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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120

Plasma stability

100 80 60 40 0

10

20 Time (h)

30

40

2.0 1.5 1.0

X0 = 1.07 IRF = 93.5%

0.5 0.0 0.0

5.0×10-7 1.0×10-6 [Cell number] -1

1.5×10-6

Figure 4. Stability and immunoreactive fraction. A) Stability over time in gly-gly buffer with and without gentisic acid as scavenger (n=4). >95% stability is maintained for 5 hours. Analysed by HPLC. B) Stability in mouse-plasma, over time, assessed by SDS-PAGE and autoradiography (n=3). Normalised to 64Cu-NOTA-PEG4-FVIIai (7). C) Lindmo assay, showing the immunoreactive fraction (IRF) of 64Cu-NOTA-PEG 4-FVIIai (7). Performed with BxPC-3, high expressing TF cells.

Plasma stability was assessed by SDS-PAGE in mouse plasma, over 36 hours, Figure 4B and Figure S7. The tracer showed greater than 91% intact 64Cu-NOTA-PEG4-FVIIai (7) at 16 hours, implying low transchelation in the plasma. The immunoreactivity of 64Cu-NOTA-PEG4-FVIIai (7) was assessed in pancreatic adenocarcinoma BxPC-3 (TF high-expressing) cells by a method described by Lindmo, et al.39 An immunoreactive fraction of 93% (Figure 4C) implies that affinity to the biological receptor is retained.

Longitudinal PET imaging and ex vivo biodistribution


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The temporal in vivo distribution of 64Cu-NOTA-PEG4-FVIIai (7) was assessed by longitudinal PET/CT imaging of mice with subcutaneous BxPC-3 tumors. Coronal and axial images are depicted in Figure 5A for the same mouse at 1, 4, 15 and 36 hours post-injection of 64Cu-NOTA-PEG4FVIIai (7). A specific uptake and increased accumulation in the tumors was observed over time with mean and maximum uptakes peaking at 15 hours post-injection, Figure 5B. In addition, high accumulation of 64Cu-NOTA-PEG4-FVIIai (7) was seen in the liver and kidneys, indicating these organs as major routes of clearance of the tracer. Quantitative ROI analysis revealed a mean tumor uptake of 1.5 ± 0.2, 2.2 ± 0.2, 2.1 ± 0.2 and 1.5 ± 0.1 %ID/g at 1, 4, 15 and 36 hours post-injection, respectively (n=4 mice pr timepoint). Maximum uptake within the tumors was significantly higher (4.8 ± 0.4, 6.9 ± 0.6, 7.0 ± 0.6 and 5.6 ± 0.4 %ID/g for 1, 4, 15 and 36 hours), illustrating the heterogeneity of the tumors. The uptake pattern in the BxPC-3 model with 64Cu-NOTA-PEG4FVIIai (7) was comparable to the previous results with randomly labelled 64Cu-NOTA-FVIIai 32 and 18F-FVIIai33 (uptake evaluated up to 4 hours post injection). Conventional ex vivo biodistribution was performed at 4, 15 and 36 hours after injection of 64CuNOTA-PEG4-FVIIai (7) in BxPC-3 tumor-bearing mice (n=3 mice at 4 and 15 hours, n=4 mice at 36 hours). The biodistribution results, Figure 5C, were in agreement with the PET ROI quantification in tumors (Figure 5B) and confirmed high retention in the liver (6.4 ± 0.5 %ID/g) and kidneys (3.6 ± 0.3 %ID/g). The distribution of 64Cu-NOTA-PEG4-FVIIai (7) is similar to randomly labelled 64Cu-NOTA-FVIIai, which likewise reported high liver and kidney uptakes. 32 Preferably, future studies could include a dose optimization study to identify optimal protein dose for highcontrast images.



AA

A

1h

4h

15 h

36 h

7 %ID/g

0 %ID/g

C

B 8

30

Mean Max Uptake (%ID/g)

10

Uptake (%ID/g)

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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6 4 2

20

4h 15 h 36 h

10 6 4 2

0

0

1h

4h

15 h

36 h

d rt s s n er h e s le e in r oo ea ung rea lee Liv ac stin ney usc Bon Bra mo l B H L nc Sp om te id M Tu S t In K Pa

Figure 5. Longitudinal PET/CT imaging of 64Cu-NOTA-PEG4-FVIIai (7) in BxPC-3 tumor bearing mice. A) Axial (bottom panel) and coronal (top panel) PET/CT images of 64Cu-NOTA-PEG4-FVIIai (7) uptake in one mouse at 1, 4, 15 and 36 hours after injection of 4.3 MBq 64Cu-NOTA-PEG 4-FVIIai (7). Arrows designate the tumors. B) Quantification of in vivo uptake in BxPC-3 xenografts over 36 hours. C) Ex vivo biodistribution at 4, 15 and 36 hours post injection.


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Specificity The in vitro binding specificity of 64Cu-NOTA-PEG4-FVIIai (7) was evaluated using human cancer cell lines with varying TF surface expression. BxPC-3 (TF high-expressing), AsPC-1 (TF intermediate-expressing) and PANC-1 (TF low-expressing) cells were incubated with 64Cu-NOTAPEG4-FVIIai (7). The in vitro binding assay detected significantly higher uptake of 64Cu-NOTAPEG4-FVIIai (7) in BxPC-3 cells than in the AsPC-1 (P≤0.01) and PANC-1 (P≤0.001) cell lines, Figure 6B (in triplicates). Blocking with excess FVIIai significantly reduced the uptake of 64CuNOTA-PEG4-FVIIai (7) in all cell lines (P≤0.01), confirming the specificity of the uptake. Collectively, the in vitro binding assay shows that 64Cu-NOTA-PEG4-FVIIai (7) can be used to detect varying levels of TF expression in vitro and that the uptake is specific.

The ability of 64Cu-NOTA-PEG4-FVIIai (7) to image TF expression in vivo was tested in different pancreatic adenocarcinoma models established by subcutaneous injection of either PANC-1, AsPC1 or BxPC-3 cells (n=4 pr model). Based on the longitudinal PET imaging data, PET/CT images were obtained 15 hours post-injection of 64Cu-NOTA-PEG4-FVIIai (7), Figure 6A. ROI quantification analysis performed on PET/CT images revealed a mean tumor uptake of 1.0 ± 0.05, 1.5 ± 0.1 and 2.1 ± 0.2 %ID/g for PANC-1, AsPC-1 and BxPC-3 respectively, Figure 6C. Maximum tumor uptake was in line with these results, with values of 3.3 ± 0.2, 4.7 ± 0.3 and 7.0 ± 0.6 %ID/g for PANC-1, AsPC-1 and BxPC-3 respectively. The BxPC-3 model in vivo mean and maximum uptakes were significantly different from the other models (P≤0.01), whereas PANC-1 and AsPC-1 tumor uptakes were not found to be significantly different. The in vivo uptake pattern was in agreement with the in vitro obtained data, confirming 64Cu-NOTA-PEG4-FVIIai (7) as a ligand specific for TF expression.



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Figure 6. Uptake of 64Cu-NOTA-PEG4-FVIIai (7) in vitro and in vivo. A) Representative axial PET/CT images of 64CuNOTA-PEG 4-FVIIai (7) uptake in BxPC-3, AsPC-1 and PANC-1 mice 15 hours after injection of tracer. Arrows designate the tumors. B) In vitro uptake (CPM) of 64Cu-NOTA-PEG 4-FVIIai (7) in BxPC-3, AsPC-1 and PANC-1 cells with and without competition, normalized to protein mass in triplicates, after 2 hours of incubation. C) Quantitative ROI analysis of mean and maximum uptake (%ID/g) of 64Cu-NOTA-PEG4-FVIIai (7) within the tumors of the different models 15 hours post-injection (n=4 mice (8 tumors)/model).

Conclusion In conclusion, a single azide moiety was successfully incorporated into a large protein in one welldefined position, enabling specific labelling. This handle can be utilized for a number of purposes,


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including, but not limited to radioactive labelling. As a proof of principle, SPAAC was used to incorporate a NOTA chelator which could be labelled with 64Cu in good RCY (64.2 ± 3.5%). The labelled product was stable in plasma (>91%) for 16 hours. A high immunoreactive fraction (93.5%) was obtained. Specificity was tested in vitro with 3 cell lines with varying TF-expression, showing significantly higher uptake by high TF-expressing cells, and reduced uptake by blocking. This tendency is reflected by in vivo PET/CT with the same cell lines. The in vivo uptake and biodistribution of 64Cu-NOTA-PEG4-FVIIai (7) were in line with the result obtained by random labelling of lysines in FVIIai32. In vivo uptake giving a mean tumor uptake (2.1 ± 0.2 %ID/g, 15h). Biodistribution showing high liver uptake (16.0 ± 1.3 %ID/g, 15h), a kidney uptake (10.4 ± 0.3 %ID/g, 15h) and a tumor uptake comparable to the in vivo uptake (2.6 ± 0.8 %ID/g, 15h). In the present case, the specific labelling did not surpass random labelling, but a specific label due to the controlled chemistry is envisioned to facilitate approval for human application. Furthermore, other serine proteases can benefit from this labelling technique, as interference of important binding or structural sites can be avoided by specific labelling.

Experimental Procedures All chemicals were obtained from Sigma Aldrich, Copenhagen, Denmark, unless stated otherwise. FVIIa and sTF(1-219) was obtained from Novo Nordisk A/S, Måløv, Denmark, and was supplied as 1.39 mg/mL in 25 mM gly-gly, 50 mM NaCl, 25 mM CaCl2, pH 6.0. 64CuCl2 was obtained from Risø, Denmark. ADIBO-PEG4-NOTA (5) was obtained from Futurechem co., LTD, Seoul, Korea. fFRck was purchased from Bachem AG, Bubendorf, Switzerland. Chromogenic substrate S-2288 (H-D-Ile-Pro-Arg-p-nitroaniline) was obtained from Chromogenix, Mölndal, Sweden. PD-10 desalting column, Sephadex G-25, was purchased from GE Healthcare, Brøndby, Denmark. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the XCell



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SureLock system (Life Technologies, a Thermo Fisher Scientific Brand) and the power supply Power PAC 300 (Bio-Rad, Hercules). NuPAGE 4-12% Bis-Tris Gel, NuPage MES SDS running buffer, NuPAGE LDS sample buffer, NuPAGE sample reducing agent (10X), SimplyBlue SafeStain and SeeBlue Plus2 pre-staining standard were purchased from Thermo Fisher Scientific Inc, Life Technologies. Samples were denaturized (70°C for 10 min.) in NuPage LDS buffer with or without reducing agent before analysis. Electrophoresis was run for 5 min at 50 V and subsequently 35 min at 35 min. The gels were removed from the cassette and washed briefly with MilliQ water before staining. The radioactive gels were analysed with Optiquant™ software, Perkin Elmer, or ImageJ after imaging either on a Perkin Elmer Cyclone Phosphor System with MultiSensitive Phosphor Screens, Medium (for plasma stability), or a Packard InstantImager (for all other radioactive SDS-PAGE). Centrifugation was performed on a Megafuge 1.0R (Heraeus). Packard Cobra -Counter, with auto-gamma was used for measuring gamma counts. Analytical liquid chromatographic analysis was performed on a HPLC system (Gilson) equipped with a Dionex UVD170U photodiode array detector, or a Thermo Fischer Ultimate 3000 system and gamma detector (Scansys Laboratorieteknik) connected in series. Data collection and liquid chromatography control used the program Chromeleon 6.8. Analytical HPLC was performed using a gradient from 10-100% MeCN/H2O, 0.1% TFA, on a Grace Vydac C4 Protein column, 300 Å, 5 µm, 4.6 x 250 mm. Preparative HPLC was performed on a waters prep system, equipped with a UV detector and a waters Xbridge prep C18, 5 µm,50 x 250 mm, OBD column. Gradient: 0 to 5 min 10% B, 5 to 8 min 40% B, 8 to 48 min 65% B, where A: H2O, 0.1% TFA, B: MeCN, 0.1% TFA. HPLC analysis of fluorescence was performed on an Agilent HPLC 1200 SL system equipped with a fluorescence detector, 1260 FLD. A Zorbax 300-SB-C3, 4.6 x 50 mm, 3.8 µ column was used at 30C. Gradient: 0-4 min, 25% B, 4-14 min 25-46% B, 14-35 min 46-52% B, 35-40 min 52-90% B,


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40-41 min 90-25% B, with A and B as designated before. Protein precipitation was performed as described by Erlandsson et al.31 All data are represented as ± SEM where applicable.

Protein chemistry 2-azidoacetic acid (2) was produced as described by Haridas et al.34 1H and 13C NMR complied with the reference. 2-azidoacetic acid (2) (8.3 mg) was dissolved in DMF (150 µL). TSTU (22.4 mg) and then Et3N (10.3 µl) was added and the mixture turned yellow. The reaction was stirred for 30 min. at 22 °C. to produce the azide NHS-ester. fFRck (16.8 mg) was dissolved in DMF (115 µL) and Et3N (9.6 µL) and the Azide NHS-ester mixture (3.5 eq.) was added. The reaction was stirred for 30 min. at 22 °C. The reaction was neutralized with acetic acid (4 eq., 9.4 µL) and purified by preparative HPLC. The purified fractions were collected (20 mL) and lyophilized after dilution with H2O (15 mL) to produce N3-fFRck (3) in 21% yield. MS calculated 583.2, found, [M+H]+: 584.7.

N3-fFRck (3) (2mg, 3.4µmol, 12 eq.) was dissolved in 25 mM HEPES buffer, pH 7.2 (2mL). pH was 7.04 after addition. FVIIa (10 mL, 1.39 mg/mL, 25mM gly-gly, 50 mM NaCl, 25 mM CaCl2, pH 6.0) was added, pH was 6.97 and the mixture was gently rotated for 19 hours. Excess N3-fFRck (3) was removed by dialysis (Slide-A-Lyzer dialysis cassette, MWCO 10 kDa, 12-30 mL, Thermo Scientific) following the manufacturers protocol using 50 mM HEPES buffer (containing 100 mM NaCl and 10 mM CaCl2, pH 7.0), to produce FVIIai-N3 (4) in 91% yield (quantified by HPLC with FVIIa as reference, Figure S8). The protein was aliquoted and stored at -80 °C.

S-2288 (H-D-Ile-Pro-Arg-p-nitroaniline) assay was performed by preparing 200 nM stock solution of sTF (soluble tissue factor 1-219) in HEPES buffer (50 mM HEPES, 5 mM CaCl2, 100 mM NaCl,



Page 20 of 30

0.01% Tween80, pH 7.4) and 200 nM stock solutions of FVIIa reference and FVIIai-N3 (4). To an ELISA plate in triplicates, 135 µL HEPES buffer, 10 µL of 200 nM stock solution of the test entity and 50 µL 200 mM stock solution of sTF to the well. The plate was left for 5 minutes, and the reaction as initiated by adding 10 µL of 10 mM S-2288 stock (in H2O). The plate was analysed by kinetic setting in an ELISA reader (Absorbance 405 nm) for 15 minutes at room temperature, and the relative activities were calculated from the initial rates of FVIIa, FVIIai-N3 (4) and blank.

FVIIai-N3 (4) (830 µL, 20 nmol) was mixed with NOTA-PEG4-ADIBO (5) (20 nmol, 10 µL of 2 mM stock in HEPES, pH 7.8) and incubated at 37 °C for 14 h. The resulting NOTA-PEG4-FVIIai (6) was purified by PD-10 desalting column with 0.1M ammonium acetate buffer (containing 10 mM CaCl2, 150 mM NaCl, pH 5.5). The modified protein was aliquoted in 1 nmol portions (45.5 µL) and stored at -80°C. The total yield was 67% (13.46 nmol).

64CuCl (2000 2

MBq), dried from HCl, was dissolved in Traceselect H 2O, and left for 30 min. An

aliquot of 64Cu in H2O (400-500 MBq) was transferred to the aliquoted protein (2 nmol), and reacted for 15 min at 25 °C. 64Cu-NOTA-PEG4-FVIIai (7) was purified by PD-10, pre-eluted with BSA 0.1%, in 10 mM gly-gly buffer (containing 10 mM CaCl2, 150 mM NaCl, pH 7.5). The two fractions containing the most radioactivity was collected, and gentisic acid (20 mM, from a 735 mM stock in gly-gly buffer, pH 7.25) was added to the collected fractions. Prior to injection into mice, BSA (final concentration 0.1%) was added.

Stability was tested with and without gentisic acid (20 mM). 250 µL of the collected fractions was analysed at time points 0, 1, 2, 3, 4, 5, 6 and 15 hours, with and without gentisic acid, by HPLC. In vitro experiments were performed in mouse plasma and analysed by non-reducing SDS-PAGE,


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time points up to 36 hours. Here the radioactive trace at 50 kDa was compared to the rest of the lane, Figure S6.

Cell culture and animal models PANC-1 (ATCC CRL-1469, LGC Standards), AsPC-1 (ATCC CRL-1682, LGC Standards) and BxPC-3 (ATCC CRL-1687, LGC Standards) were cultured in RPMI-1640 (AsPC-1 and BxPC-3) or DMEM (PANC-1) media supplemented with 10% foetal bovine serum and 1% penicillinstreptomycin (Invitrogen) and maintained at 37˚C and 5% CO 2. Cells were harvested in their exponential growth phase and re-suspended at 1:1 in complete growth media and Matrigel TM (BD Biosciences) at a concentration of 5x107 cells/mL. 100 µL of cell suspension was injected subcutaneously with a 25G needle into each flank above the hind limbs of female NMRI nude mice (Taconic, Denmark). All animal experiments were performed under a protocol approved by the National Animal Experiments Inspectorate.

In vitro binding assay and estimation of immunoreactive fraction In vitro cell uptake of 64Cu-NOTA-PEG4-FVIIai (7) was performed by incubating 0.2x105 BxPC-3, AsPC-1 and PANC-1 cells with 6x10-2 MBq 64Cu-NOTA-PEG4-FVIIai (7) in a 24-well plate for 2 hours at 4°C. Blocking was performed with 5000-fold excess of unlabelled FVIIai. After ended incubation, cells were washed 2 times with PBS containing 1% BSA, dissolved in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) and counted in a gamma counter (Wizard2, PerkinElmer). Cell lysates were analysed for total protein content with the Pierce TM BCA Protein Assay Kit (ThermoFisher) and the radioactivity measured by the gamma counter was normalized to the total protein level in the sample. The assay was performed in triplicates.



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To determine the immunoreactive fraction the method described by Lindmo et al. 39 was used. Increasing concentrations of BxPC-3 cells in suspension (1.25x106 – 4x107 cells/mL) were incubated with 6 nM

64Cu-NOTA-PEG

4-FVIIai

(7) for 3 hours at 4°C. Cells were centrifuged at

500g for 5 minutes and the supernatants and pellets counted in a gamma counter (Wizard 2, PerkinElmer). Cell associated radioactivity was calculated as the ratio of cell bound radioactivity to the total amount of added radioactivity.

Small animal PET/CT imaging Small animal PET/CT imaging was performed with an Inveon Multimodality PET/CT scanner (Siemens). Static PET images were acquired in list mode 1, 4, 15, and 36 hours (acquisition time 300, 300, 600 and 900 seconds respectively) after intravenous injection of 4.3 ± 0.52 MBq

64

Cu-

NOTA-PEG4-FVIIai (7) in 200 µL. Images were reconstructed using a 3D maximum a posteriori algorithm with CT based attenuation correction. CT images were acquired at 180 projections at 65 kV and 500 µA with 400 ms exposure, and reconstructed with an isotropic voxel size of 104 µm. Mice were anesthetized with sevoflurane (Abbott Laboratories) during injection and PET/CT imaging. Image analysis was carried out using Inveon software (Siemens Medical Solutions) and 64Cu-NOTA-PEG

4-FVIIai

(7) uptake was expressed as percentage injected dose (%ID) per gram of

tissue.

Statistics One-way ANOVA corrected for multiple comparisons (Bonferroni) was applied to analyse the in vitro and in vivo uptake of

64Cu-NOTA-PEG

4-FVIIai

(7) across pancreatic cancer models. The

effect of competition was evaluated by Multiple t-tests corrected for multiple comparisons (Holm-


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Sidak). P values ≤ 0.05 were considered statistical significant. Statistical analyses were performed using GraphPad Prism 6.0d.

Biodistribution Conventional ex vivo biodistribution was performed in BxPC-3 tumor bearing mice injected with 5.1 ± 0.23 MBq

64Cu-NOTA-PEG

4-FVIIai

(7). Mice were euthanized at 4, 15 and 36 hours post-

injection (n=3 at 4 and 15 hours, n=4 at 36 hours), tumors and organs resected and weighted, and the radioactivity was counted in a gamma counter (Wizard 2, PerkinElmer).

Acknowledgements The authors wish to thank the staff at the PET and Cyclotron unit, and technician Sonja Bak at Novo Nordisk A/S for expert technical assistance. Financial support from the Innovations Fund Denmark, the Lundbeck Foundation, the Novo Nordisk Foundation, the Svend Andersen Foundation, the Arvid Nilsson Foundation, the John and Birthe Meyer Foundation, the Resarch Foundation of Rigshospitalet, the Research Foundation of the Capital Region, the Global Excellence Program, H2020 program and the ERC are gratefully acknowledged.

Supporting information Synthesis and analytical details, figures and illustrations. This information is available free of charge via the internet at http://pubs.acs.org.

Abbreviations NOTA

1,4,7-Triazacyclononane-1,4,7-triacetic acid

DOTA

1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid



CB-TE2A

2,2'-(1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diyl)diacetic acid

NODAGA

4-[4,7-Bis-(carboxymethyl)-[1,4,7]triazonan-1-yl]-4-carboxy-butyric acid

Conflicts of interest The authors have no conflicts of interest to disclose.

ORCID ID Troels Elmer Jeppesen orcid.org/0000-0002-5009-4522

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