PHOTOCHEMICALLY INDUCED CONJUGATION OF RADIOMETALS TO SMALL MOLECULES, PEPTIDES AND NANOPARTICLES IN A SIMULTANEOUS ONE-POT REACTION
20220017433 · 2022-01-20
Assignee
Inventors
Cpc classification
C07B59/004
CHEMISTRY; METALLURGY
C07F7/00
CHEMISTRY; METALLURGY
C07C259/06
CHEMISTRY; METALLURGY
International classification
Abstract
The invention relates to a method for labeling a target compound with a radiometal by photochemically induced conjugation. Furthermore, a chelating compound for use in said method is provided. The chelating compound is characterized by an arylazide moiety which can be photo-conjugated to a target compound and a chelator moiety which can be radiolabelled. The photo-conjugation and radiolabelling are both performed at basic pH performed in a simultaneous one-pot reaction.
Claims
1. A method for preparing a photoradiolabelled compound comprising i. providing a reaction mixture comprising at least one chelating compound, and at least one target compound B comprising an amine and/or thiol and/or carboxylate moiety, particularly an amine and/or thiol moiety, and at least one radioactive ion of a radionuclide, ii. performing photoconjugation and radiolabelling in a photoradiolabelling step by adjusting the pH to pH>7, in particular pH>8, more particularly pH 8 to 11 irradiation of the reaction mixture with light at a wavelength selected from 200 nm to 420 nm, wherein the chelating compound is a compound of formula 1, ##STR00029## wherein A is a chelator suitable for coordinating an ion of a radionuclide at basic pH, L is a linker with z being 0 or 1, R.sup.1 is independently from each other selected from C.sub.1-6-alkyl, C.sub.2-6-alkenyl, C.sub.2-6-alkynyl, —NH.sub.2, —NHR.sup.2, —NR.sup.2R.sup.3, —OH, —OR.sup.4, —SR.sup.4, —CF.sub.3, —CH.sub.2F, —CHF.sub.2, —CH.sub.2—CF.sub.3, —CH.sub.2—CH.sub.2F, —CH.sub.2—CHF.sub.2, —SOCF.sub.3, —SO.sub.2CF.sub.3, —SC.sub.2—NR.sup.2R.sup.3, —CN, —NO.sub.2, —F, —Cl, —Br or —I, in particular —OH, —CN, —NO.sub.2, —F, —Cl, —Br, or —I, with R.sup.2 and R.sup.3 being independently selected from C.sub.1-6-alkyl, C.sub.2-6-alkenyl and C.sub.2-.sub.6-alkynyl, .sup.4 being selected from C.sub.1-6-alkyl, C.sub.2-6-alkenyl and C.sub.2-6-alkynyl which may optionally be substituted with —F, —Cl, —Br or —I n is 0, 1, 2 or 3, in particular 0 or 1, more particularly 0, and R.sup.1 and —N.sub.3 are positioned in such a way that at least one of the positions 2 to 6 of the phenyl moiety that are next to —N.sub.3 is unsubstituted.
2. The method according to any one of the preceding claims, wherein the radionuclide is selected from .sup.43Sc, .sup.44Sc, .sup.47Sc, .sup.45Ti, .sup.51Cr, .sup.51Mn, .sup.52Mn, .sup.52mMn, .sup.52Fe, .sup.55Co, .sup.57Ni, .sup.60Cu, 61.sub.Cu, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.65Zn, .sup.66Ga, .sup.67Ga, .sup.68Ga, .sup.69Ge, .sup.71As, .sup.72As, .sup.74As, .sup.76As, .sup.77As, .sup.82Rb, .sup.82mRb, .sup.82Sr, .sup.83.sub.Sr, .sup.89.sub.Sr, .sup.86Y, .sup.90Y, .sup.89.sub.Zr, .sup.97Zr, .sup.90Nb, .sup.94mTc, .sup.99mTc, .sup.97Ru, .sup.105Rh, .sup.111Ag, .sup.110mIn, .sup.111In, .sup.117mSn, .sup.153Sm, .sup.149Tb, .sup.152Tb, .sup.155Tb, .sup.161Tb, .sup.166Ho, .sup.165Er, .sup.177Lu, .sup.178Ta, .sup.186Re, .sup.188Re, .sup.192Ir, .sup.195mPt, .sup.198Au, .sup.197mHg, .sup.201Tl, .sup.212Pb, .sup.212Bi, .sup.213.sub.Bi, .sup.211At, .sup.223Ra, .sup.255Ac, in particular from .sup.153Sc, .sup.44Sc, .sup.47Sc, .sup.60Cu, .sup.61Cu, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.67Ga, .sup.68Ga, .sup.86Y, .sup.90Y, .sup.89Zr, .sup.99mTc, .sup.111In, .sup.153Sm, .sup.149Tb, .sup.152Tb, .sup.155Tb, .sup.161Tb, .sup.77Lu, .sup.186Re, .sup.188Re, .sup.212Pb, .sup.212Bi, .sup.213Bi, .sup.223Ra, .sup.255Ac.
3. The method according to any one of the preceding claims, wherein a co-ligand is added to the reaction mixture, in particular acetate, oxalate or chloride.
4. A chelating compound comprising formula 2, ##STR00030## wherein A is a chelator suitable for coordinating an ion of a radionuclide, particularly at basic pH, L is a linker with z being 0 or 1, R.sup.1 is independently from each other selected from C.sub.1-6-alkyl, C.sub.2-6-alkenyl, C.sub.2-6-alkynyl, —NH.sub.2, —NHR.sup.2, —NR.sup.2R.sup.3, —OH, —OR.sup.4, —SR.sup.4, —CF.sub.3, —CH.sub.2F, —CHF.sub.2, —CH.sub.2—CF.sub.3, —CH.sub.2—CH.sub.2F, —CH.sub.2—CHF.sub.2, —SOCF.sub.3, —SC.sub.2CF.sub.3, —SC.sub.2—NR.sup.2R.sup.3, —CN, —NO.sub.2, —F, —Cl, —Br or —I, in particular ——OH, —OR.sup.4, —CN, —NO.sub.2, —F, —Cl, —Br, or —I, with R.sup.2 and R.sup.3 being independently selected from C.sub.1-6-alkyl, C.sub.2-6-alkenyl and C.sub.2-.sub.6-alkynyl, R.sup.4 being selected from C.sub.1-6-alkyl, C.sub.2-6-alkenyl and C.sub.2-6-alkynyl which may optionally be substituted with —F, —Cl, —Br or —I, n is 0, 1, 2 or 3, wherein R.sup.1 and —N.sub.3 are positioned in such a way that at least one of the positions 2 to 6 of the phenyl moiety that are next to —N.sub.3 is unsubstituted, with the proviso that in case of z being 0, A is not EDTA, and with the proviso that in case of z being 1, A is not DTPA.
5. A radiolabelled intermediate compound comprising formula 3, ##STR00031## wherein A* is a chelator bound to a radionuclide by coordinate bonds, and L, z, R.sup.1 and n are defined as described above.
6. The compound according to any one of claim 1, 4 or 5, wherein —N.sub.3 is in meta or para position, particularly in para position.
7. A photoconjugated intermediate compound comprising formula 4a, 4b, 4c, 4d or 4e, ##STR00032## wherein A, L, z, R.sup.1, n and B are defined as described above.
8. A photoradiolabelled compound comprising formula 5a, 5b, 5c, 5d or 5e, ##STR00033## wherein A*, L, z, R.sup.1, n and B are defined as described above.
9. The compound according to any one of the preceding claims, wherein the chelator is selected from NODAGA, NOTA, DOTA, Desferrioxamine B (DFO), ATSM, DOTAGA, HBED-CC, SAAC, DTPA, DTPA-benzyl, DFO-Star, oxoDFO-Star, HOPO, p-SCN-Bn-HOPO ##STR00034## and derivatives thereof, in particular from NODAGA, NOTA, Desferrioxamine B (DFO), ATSM, DOTAGA, HBED-CC, SAAC, DFO-Star, oxoDFO-Star, p-SCN-Bn-HOPO, ##STR00035## and derivatives thereof.
10. The compound according to any one of the preceding claims, wherein L is a linker comprising one or more moieties, particularly 1 to 20 moieties, more particularly 1 to 15 moieties, selected from —C(═X)—, —NR.sup.6—, —C(═X)—NR.sup.6—, —NR.sup.6—C(═X)—, —NR.sup.6—C(═X)—NR.sup.6—, —O—C(═X)—NR.sup.6—, —NR.sup.6—C(═X)—O—, —O—, —C.sub.1-8-alkyl-, particularly selected from —C(═X)—, —NR.sup.6—, —C(═X)—NR.sup.6—, —NR.sup.6—C(═X)—, —NR.sup.6—C(═X)—NR.sup.6—, —O—, —C.sub.1-8-alkyl-, with R.sup.6 being H or C.sub.1-8-alkyl and X being O or S.
11. The compound according to any one of the preceding claims, wherein L is —C(═O)— or L comprises one or more moieties selected from —C(═X)—, —NR.sup.6—, —C(═X)—NR.sup.6—, —NR.sup.6—C(═X)—, —NR.sup.6—C(═X)—NR.sup.6—, —O—, —C.sub.1-8-alkyl- with R.sup.6 being H or C.sub.1-8-alkyl and X being O or S, wherein a moiety that comprises a heteroatom N, O or S alternates with an alkyl moiety, wherein in particular one or both ends of the linker are independently formed by a moiety that comprises a heteroatom N, O or S.
12. The compound according to any one of the preceding claims, wherein L is —C(═O)— or a moiety of formula 2, R.sup.a.sub.n—(C.sub.1-6-alkyl)-R.sup.b.sub.m—R.sup.c— (2), wherein R.sup.a is —C(═O)—, —NR.sup.6—C(═X)—NR.sup.6—, or —NR.sup.6—, particularly —C(═O)— or —NR.sup.6—, more particularly —NR.sup.6—, with R.sup.6 being H or C.sub.1-4-alkyl, or R.sup.a is a moiety —X.sup.1—C.sub.1-8-alkyl —X.sup.2— with X.sup.1 and X.sup.2 being a moiety independently selected from —C(═O)—, —NR.sup.6—, —C(═X)—NR.sup.6—, —NR.sup.6—C(═X)—, —NR.sup.6—C(═X)—NR.sup.6—, —O—C(═X)—NR.sup.6—, —NR.sup.6—C(═X)—O—, particularly —C(═O)—, —NR.sup.6—, —C(═O)—NR.sup.6—, —NR.sup.6—C(═O)—, n is 0 or 1, R.sup.b is a polyether moiety with p elements [—O—C.sub.u-alkyl], wherein u is independently selected for each element from an integer between 1 to 4 and p is an integer between 1 and 6, m is 0 or 1, R.sup.c is —NR.sup.5—C(═O)—, —NR.sup.5—C(═X)—NR.sup.5—, —O—C(═X)—NR.sup.5—, —NR.sup.5—C(═X)—O—, wherein R.sup.5 is independently from each other H or C.sub.1-4-alkyl X is O or S, particularly S.
13. The compound according to any one of claim 5 or 8, wherein the radionuclide is selected from .sup.43Sc, .sup.44Sc, .sup.47Sc, .sup.45Ti, .sup.51Cr, .sup.51Mn, .sup.52Mn, .sup.52mMn, .sup.52Fe, .sup.55Co, .sup.57Ni, .sup.60Cu, .sup.61Cu, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.65Zn, .sup.66Ga, .sup.67Ga, .sup.68Ga, .sup.69Ge, .sup.71As, .sup.72As, .sup.74As, .sup.76As, .sup.77As, .sup.82Rb, .sup.82mRb .sup.82Sr, .sup.83Sr, .sup.89Sr, .sup.86Y, .sup.90Y, .sup.89Zr, .sup.97Zr, .sup.90Nb, .sup.94mTc, .sup.99mTc, .sup.97Ru, .sup.105Rh, .sup.111Ag, .sup.110mIn, .sup.111In, .sup.117mSn, .sup.153Sm, .sup.149Tb, .sup.152Tb, .sup.155Tb, .sup.161Tb, .sup.166Ho, .sup.165Er, .sup.177Lu, .sup.178Ta, .sup.186Re, .sup.188Re, .sup.192Ir, .sup.195mPt, .sup.198Au, .sup.197mHg, .sup.201Tl, .sup.212Pb, .sup.212Bi, .sup.213Bi, .sup.211At, .sup.223Ra, .sup.255Ac, in particular .sup.43Sc, .sup.44Sc, .sup.47Sc, .sup.60Cu, .sup.61Cu, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.67Ga, .sup.68Ga, .sup.86Y, .sup.90Y, .sup.89Zr, .sup.99mTc, .sup.111In, .sup.153Sm, .sup.149Tb, .sup.152Tb, .sup.155Tb, .sup.161Tb, .sup.77Lu, .sup.186Re, .sup.188Re, .sup.212Pb, .sup.212Bi, .sup.213Bi, .sup.223Ra, .sup.255Ac. AC.
14. The compound according to any one of claim 1, 7 or 8, wherein the target compound B is selected from a small molecule, a peptide, a protein, an antibody, an antibody-like molecule, an antibody fragment or a nanoparticle.
15. The compound according to any one of claim 7, 8 or 14, wherein the target compound B is bound to the azepine moiety via said amine of the target compound B or a thioether moiety —S— derived from the thiol moiety —SH of the target compound B, in particular an amine —NH— derived from lysine.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0250]
[0251]
[0252]
[0253]
[0254]
[0255]
[0256] Radio-ITLC chromatograms showing the kinetics of formation of [.sup.89Zr] ZrDFO-azepin-antibody versus time using a pre-functionalised DFO-azepin-antibody sample prepared with an initial chelate-to-monoclonal antibody ratio of 26.4-to-1. (B) Plot of the percentage radiochemical conversion (RCC) versus time using samples of DFO-azepin-antibody pre-conjugated at different initial chelate-to-monoclonal antibodies ratios. (C) Radioactive SEC-UHPLC confirming that [.sup.89Zr] ZrDFO-azepin-antibody remains stable with respect to change in radiochemical purity during incubation in human serum at 37° C. for 92 h.
[0257]
[0258] Analytical PD-10-SEC elution profiles showing the [.sup.89Zr][ZrDTPA].sup.− control (green, equivalent to the no chelate control confirming no non-specific binding of .sup.89Zr.sup.4+ ions to the monoclonal antibody), a control reaction without monoclonal antibody (yellow), crude reaction mixtures after irradiation and DTPA quenching at 365 nm (black) and 395 nm (red), and the purified product (blue). (C) SEC-UHPLC chromatograms of the crude and purified product.
[0259]
[0260]
[0261]
[0262]
[0263]
[0264]
[0265]
[0266]
[0267]
[0268]
[0269]
[0270]
[0271]
[0272]
[0273]
EXAMPLES
[0274] The method according to the invention is directed towards simultaneous radiolabelling of a chelator moiety of a chelating compound and photoconjugation of an aryl-azide moiety of said chelating compound to a target compound. Irradiation of the aryl-azide releases N.sub.2 forming a singlet arylnitrene, which at room temperature undergoes extremely fast intramolecular rearrangement to give ketenimines (or benzazirine) intermediates. Ketenimines react relatively slowly with oxygen, protons and water, but undergo rapid nucleophilic addition with amines or thiols of the target compound B. The addition is facilitated if the amine or thiol moiety is deprotonated.
[0275] For instance, suitable target compounds are various peptides and proteins that comprise an amine or a thiol moiety e.g. in the side chain of amino acids such as lysine or cysteine. Suitable full-length antibodies may be selected from trastuzumab, cetuximab, bevacizumab, panitumumab, ibritumomab tiuxetan, J591, fresolimumab, rituximab, brentuximab, lumretuzumab, U36, R1507, ranibizumab, DN30, 7E11, particularly trastuzumab. A suitable antibody fragment is onartuzumab. Suitable proteins may be selected from albumin, transferrin, ceruloprotein, globulins (in general), fibrinogen and other proteins circulating in the blood pool, particularly serum albumin.
[0276] The invention is further demonstrated by the examples described herein showing photoradiolabelling using a full-length antibody, an antibody fragment and the protein albumin.
Example 1: Simultaneous Photoradiolabelling Using DFO-PEG.SUB.3.-ArN.SUB.3
[0277] The chelating compound DFO-PEG.sub.3-ArN.sub.3 (
Example 2: Simultaneous Photoradiolabelling of Antibodies
[0278] In proof-of-concept work, it was demonstrated that the photoradiochemical approach showed equivalent successful when radiolabelling either pre-purified fractions of monoclonal antibodies, or starting from fully formulated samples. Reactions were established in which [.sup.89Zr][Zr(C.sub.2O.sub.4].sup.4−, and a monoclonal antibody (at an initial chelate-to-monoclonal antibody ratio of ˜29-to-1) were mixed in water and the pH adjusted to ˜8-9. Control reactions were also performed in the absence of either the chelate or the monoclonal antibody. Reactions were then stirred and irradiated using the LED source (365 nm or 395 nm) at room temperature for 10 min.
[0279] After irradiation, the mixtures were quenched by the addition of DTPA. Aliquots of the crude mixtures were retained and a fraction was purified by SEC-methods. Crude and purified samples were then analysed by using radio-iTLC, analytical PD-10-SEC and SEC-UHPLC methods (
[0280] Comparison of the final RCYs measured between the two-step process and the simultaneous one-pot (one-step) process indicate that the photochemical conjugation efficiency increases from about 3.5% to >75%. This is a remarkable result that means that the chemical efficiency of simultaneous photoradiolabelling is comparable to some of the most efficient thermally mediated conjugation processes (typically ˜60-80%). Under the conditions employed, it is likely that the kinetics of metal ion complexation are similar to the photochemical conjugation step. If .sup.89Zr.sup.4+ ions are coordinated first by the DFO-ArN.sub.3 chelate, this limits the possibility of intramolecular reaction between the nucleophilic hydroxamate groups and the photo-generated intermediates. Such an elegant photoradiochemical process is also amenable to full automation which has potential to change the way in which radiolabelled monoclonal antibodies are produced in the clinic.
Example 3: Two-Step Photochemical Conjugation and .SUP.89.Zr-Radiolabelling of a Monoclonal Antibody
[0281] Prior to investigating a simultaneous one-pot photoradiochemical process, experiments were performed using the traditional two-step approach involving an initial photochemical conjugation between DFO-ArN.sub.3 and a monoclonal antibody, followed by .sup.89Zr-radiolabelling.
[0282] The photochemical conjugation between DFO-ArN.sub.3 and the monoclonal antibody was performed at room temperature for 35 min. using a Rayonet reactor. The DFO-azepin-antibody conjugate was purified by using a combination of size-exclusion chromatography (SEC) methods including spin-column centrifugation and preparative PD-10 gel filtration. Then aliquots of DFO-azepin-antibody were radiolabelled with .sup.89Zr using standard conditions..sup.[31, 33-35] Aliquots of the crude radiolabelling mixture were retained and the radiolabelled fraction of [.sup.89Zr]ZrDFO-azepin-antibody was purified and formulated in sterile PBS by standard SEC methods. Analytical measurements on the crude and purified samples of [.sup.89Zr]ZrDFO-azepin-antibody were performed using radioactive instant thin-layer chromatography (radio-iTLC), analytical PD-10-SEC and radioactive SEC-UHPLC.
[0283] Experiments confirmed that the DFO-azepin-antibody was sample radiolabelled efficiently with .sup.89Zr giving with a crude radiochemical conversion (RCC) of >98% after incubating the mixture at room temperature for 15 min. On scaling-up the radiolabelling reaction for use in subsequent cellular and animal experimentations, the final radiochemical yield (RCY) of the purified sample was >99% and the radiochemical purity (RCP) was measured at >99.5% (by analytical PD-10-SEC) and >98% (by SEC-UHPLC).
[0284] Additional .sup.89Zr-radiolabelling experiments were performed to measure the radiolabelling kinetics and overall RCC of DFO-azepin-antibody samples that were prepared using different initial chelate-to-monoclonal antibody ratios in the photochemical conjugation step (
[0285] The radiochemical stability of [.sup.89Zr]ZrDFO-azepin-antibody with respect to change in the RCP during incubation in human serum at 37° C. for up to 92 h was determined by SEC-UHPLC (
Example 4: One-Pot Pre-Radiolabelling Followed by Photoconjugation Using Two Different pH
[0286] The photoradiochemical reaction was tested further by using a one-pot approach. Compound 5 (NODAGA-PEG.sub.3-ArN.sub.3) was pre-radiolabelled with .sup.68Ga, re-buffered to pH 8.0, and then prepurified monoclonal antibody was added and the mixture irradiated (
[0287] Finally, with a view to expanding the utility of photoradiochemistry, the one-pot approach was tested using a preparation of an antibody. Clinical preparations of an antibody are typically stabilised by the addition of salts, amino acids, anti-oxidants and surfactants. The preparation used herein contains histidine, a,a-trehalose dehydrate and polysorbate 20.
[0288] Traditional coupling methods do not tolerate such additives which necessitates pre-purification of the mAb component (usually from a GMP source). Removing the stabilisers risks damaging the protein, and isolation/storage of an intermediate species raises other concerns regarding the long-term biological integrity of the radiolabelling precursor with respect to the parent compound. Methods that allow direct radiolabelling of the formulated
[0289] GMPgrade mAbs could potentially redefine the way in which radiopharmaceuticals are prepared for immuno-PET and RIT. Experiments showed that the photochemical approach using the antibody preparation produced [.sup.68Ga]GaNODAGA-azepin-antibody in a decay-corrected RCY of 23.3±3.4% (n=3). Interestingly, the presence of histidine only slightly reduced the RCY.
[0290] From a pharmacokinetic standpoint, the combination of .sup.68Ga with long-circulating, full-length mAbs is sub-optimal but this radionuclide is useful for radiolabelling lower molecular weight species like immunoglobulin fragments and peptides. Compound 5 can also be used for complexation of other radionuclides including .sup.64Cu. Based on these proof-of-concept studies, the approach was expanded by synthesising a range of compounds for radiolabelling with .sup.64Cu, .sup.89Zr, .sup.90Y, .sup.111In, .sup.177Lu, .sup.225Ac and others.
[0291] One-pot photochemical conjugation and radiolabelling of a monoclonal antibody One-pot photochemical conjugation and radiolabelling reactions were performed in accordance with the following general procedure. To a solution of NODAGA-PEG.sub.3-ArN.sub.3 (5) (160 μg, 2.21×10.sup.−7 mol, 2.21 mM) buffered with NaOAc (0.24 M, pH4.4) was added [.sup.68Ga][Ga(H.sub.2O).sub.6]Cl.sub.3(aq.) stock solution (31.8±2.0 MBq, generator 2, n=3) resulting in a total reaction volume of 100 μL. Note, one-pot reactions were not stirred because stirring was found to have no effect on the radiolabelling or the photochemical conjugation efficiency. Reactions were monitored by radio-iTLC. Formation of [.sup.68Ga]GaNODAGA-PEG.sub.3-ArN.sub.3, .sup.68Ga-5, was complete after <5 min. incubation at 23° C. with radiochemical conversion (RCC) >99% (n=3, R.sub.f=0.06−0.17 on iTLC,
[0292] Radio-iTLC analyses of the crude reactions after irradiation and quenching showed that ˜40% (n=3) of the radioactivity was bound to the antibody (R.sub.f=0.0). Note: integration of these radio-iTLC data is unreliable because the radiolabelled antibody fraction partially overlaps with the peak associated with .sup.68Ga-5 and the photodegraded .sup.68Ga-5 species (R.sub.f=0.06-0.17). Nevertheless, analytical PD-10-SEC measurements on the crude reaction mixtures confirmed this observation with an estimated RCP of 38.0±2.0% (n=3). Equivalent decay corrected SEC-UHPLC measurements indicated that the radiolabelled fraction of [.sup.68Ga]GaNODAGA-azepin-antibody in the crude mixture was 22.0±3.5% (n=3).
[0293] Crude reaction mixtures were then purified by preparative PD-10-SEC eluting with PBS (collecting only the high purity 0.0-1.6 mL fraction). Prior to analysis, samples were concentrated using an Amicon Ultra-4 mL centrifugal filter (Millipore, 30 kDa MWCO, 4000 RPM, ˜10 min.). The purified and formulated [.sup.68Ga]GaNODAGA-azepin-antibody products (pH7.4) were obtained in <15 min. with decay corrected radiochemical yields (RCY) of 33.9±0.7% (n=3). The estimated lower limit on the molar activity (A.sub.m/[MBq/nmol] of protein) of the formulated [.sup.68Ga]GaNODAGA-azepin-antibody samples was 1.02±0.07 MBq/nmol (n=3). Purified products were then reanalysed by radio-iTLC, analytical PD-10-SEC and SEC-UHPLC. The RCP of purified [.sup.68Ga]GaNODAGA-azepin-antibody was >99% (n=3) by radio-iTLC, 97.6±0.9% (n=3) by analytical PD-10-SEC, and 91.0 ±2.7% (n =3) by SEC-UHPLC.
[0294] Appropriate control reactions were also performed. In the absence of the NODAGA-PEG.sub.3-ArN.sub.3 chelate (5) no .sup.68Ga-radioactivity bound to the monoclonal antibody in the crude reaction mixtures after irradiation and quenching with EDTA (
[0295] One-Pot Photochemical Conjugation and Radiolabelling of a Preparation of a Monoclonal Antibody
[0296] From a mechanistic perspective, the presence of an amino acid (histidine) in the formulation limits the possibilities for conjugation of the antibody with a chelate in situ. The amine group of the amino acid competes with the ε-NH.sub.2 side-chain of accessible lysine residues on the protein in most standard conjugation chemistries. Therefore, radiolabelling antibodies typically requires a pre-purification step to isolate the antibody fraction from other components of the standard formulation.
[0297] For instance, a standard antibody preparation as used herein contains L-histidine hydrochloride (9.9 mg), L-histidine (6.4 mg), α,α-trehalose dihydrate (400 mg, α-D-glucopyranosyl-α-D-glucopyranoside), and polysorbate 20 (1.8 mg). After reconstitution with 20 mL of the supplied bacteriostatic water for injection (BWFI), containing 1.1% benzyl alcohol as a preservative, the injectate contains monoclonal antibody at 21 mg/mL, at pH ˜6.0. Thus, the formulation contains a total of 9.31×10.sup.−6 mol of histidine and 2.93×10.sup.−6 mol of antibody (assuming a molecule weight of about 150,000 Da). Therefore, the mole ratio of primary amine groups from histidine to total moles of mAb is approximately 31.7-to-1. The monoclonal antibody has approximately 90 lysine residues. Assuming that mAb_lysine groups are chemically accessible, the histidine-to-mAb_lysine ratio is approximately 0.156 (i.e. one histidine-NH.sub.2 group to 6.4 mAb_lysine groups). Hence, it should be possible to radiolabel the monoclonal antibody directly in the preparation without the need for a pre-purification step. The caveat is that the thermodynamics and kinetics of coupling to histidine-NH.sub.2 are potentially different to that of the antibody-lysine residues. Nevertheless, to test the hypothesis, one-pot photochemical conjugation and radiolabelling experiments were performed using non-purified preparation reconstituted in 18.2 MΩ.Math.cm water.
[0298] To a solution of NODAGA-PEG.sub.3-ArN.sub.3 (5) (160 μg, 2.21×10.sup.−7 mol, 2.21 mM) buffered with NaOAc (0.3 M, pH4.4) was added [.sup.68Ga][Ga(H.sub.2O).sub.6]Cl.sub.3(aq.) stock solution (35.1±0.5 MBq, generator 2, n=4) resulting in a total reaction volume of 100 μL. Reactions were monitored by radio-iTLC. Formation of [.sup.68Ga]GaNODAGA-PEG.sub.3-ArN.sub.3, .sup.68Ga-5, was complete after 5 min. incubation at 23° C. with radiochemical conversion (RCC) >99% (n=4, R.sub.f=0.06−0.17 on radio-iTLC). The pH of the reaction mixture was then adjusted to ˜8.0 by the addition of an aqueous solution of NaHCO.sub.3 (1.0 M, 40 μL added). After adjusting the pH, an aliquot of the preparation was added to the reaction (stock solution prepared by reconstituting 4.5 mg preparation in water (60 μL): reactions contained 28 μL of stock solution which was equivalent to 1.077 mg monoclonal antibody, 7.42×10.sup.−9 mol, final reaction concentration=6.41 mg/mL). The initial chelate-to-antibody ratio of was 29.8 at the start of the photochemical conjugation step (total reaction volume ˜168 μL). The reaction mixture was then irradiated using the LED (100% intensity, 365 nm) for 10 min. at room temperature without stirring. After irradiation, the reaction was quenched by the addition of EDTA (disodium form, 100 μL, 50 mM stock solution, pH 7.1, containing 5×10.sup.−6 mol EDTA, 22.6-fold excess with respect to the initial concentration of compound 5; final reaction volume ˜268 μL). Note, the pH of the reaction mixture did not change after addition of the EDTA solution. Aliquots of this crude, quenched reaction mixture were then analysed by using radio-iTLC and SEC-UHPLC analysis (Figure S40).
[0299] Radio-iTLC analyses of the crude reactions after irradiation and quenching showed that 37.3 ±2.4% (n=3) of the radioactivity was bound to the antibody (R.sub.f=0.0). Note: integration of these radio-iTLC data is unreliable because the radiolabelled antibody fraction partially overlaps with the peak associated with .sup.68Ga-5 and the photodegraded .sup.68Ga-5 species (R.sub.f=0.06−0.17). Equivalent decay corrected SEC-UHPLC measurements indicated that the radiolabelled fraction of [.sup.68Ga]GaNODAGA-azepin-antibody in the crude mixture was 10.8±1.9% (n=3).
[0300] Crude reaction mixtures were then purified by preparative PD-10-SEC eluting with PBS (collecting only the high purity 0.0-1.6 mL fraction). Prior to analysis, samples were concentrated using an Amicon Ultra-4 mL centrifugal filter (Millipore, 30 kDa MWCO, 4000 RPM, 10 min.). The purified and formulated [.sup.68Ga]GaNODAGA-azepin-antibody products (pH7.4) were obtained in <15 min. with decay corrected radiochemical yields (RCY) of 23.3±3.4% (n=3). The estimated lower limit on the molar activity (A.sub.m/[MBq/nmol] of protein) of the formulated [.sup.68Ga]GaNODAGA-azepin-antibody samples was 0.92±0.15 MBq/nmol (n=3). Purified products were then reanalysed by radio-iTLC and SEC-UHPLC (Figure S40). The RCP of purified [.sup.68Ga]GaNODAGA-azepin-antibody was >99% (n=3) by radio-iTLC, and 86.0±2.6% (n=3) by SEC-UHPLC. Lower RCP was observed in SEC-UHPLC analyses of these radiolabelling reactions starting from the antibody preparation because the capacity of the PD-10 columns for preparative purification was insufficient to allow complete purification of the radiolabelled mAb component from the large amount of radiolabelled histidine. In future optimisation work, this issue can be readily resolved by using higher capacity purification methods.
[0301] Appropriate control reactions were also performed. After radiosynthesis of [.sup.68Ga]GaNODAGA-PEG.sub.3-ArN.sub.3 (.sup.68Ga-5) and re-buffering, an aliquot of the antibody preparation was added to the reaction vessel and the mixture was incubated in the dark at room temperature for ˜10 min. After quenching, radio-iTLC and SEC-UH PLC analysis showed that no .sup.68Ga-radioactivity was bound to the monoclonal antibody. In the absence of light, all radioactivity in solution remained as unreacted .sup.68Ga-5.
Example 5: Photochemical Conjugation and .SUP.68.Ga-Radiolabelling of a Monoclonal Antibody Using NOTA-, DOTA- and DOTAGA-PEG.SUB.4.-ArN.SUB.3 .(Different pH for Conjugation and Radiolabelling)
[0302] Synthesis of the Photoactive Chelates
[0303] The photoactive chelates, NOTA-PEG.sub.3-ArN.sub.3 (1), DOTA-PEG.sub.4-ArN.sub.3 (3) and DOTAGA-PEG.sub.4-ArN.sub.3 (4) were synthesised via standard chemical transformations starting from 4-azidobenzoic acid and commercially available reagents (
[0304] DOTA-PEG.sub.4-ArN.sub.3 was synthesised in 89% yield via the reaction of DOTA-PEG.sub.4-NH.sub.2 with the activated NHS ester, 2,5-dioxopyrrolidin-1-yl-4-azidobenzoate (2). DOTAGA-PEG.sub.4-ArN.sub.3 was produced in 29% after direct coupling of DOTAGA-PEG.sub.4-NH.sub.2 with 4-azidobenzoic acid in the presence of HATU/DIPEA in DMF.
[0305] PEG linkers were introduced to increase the space between the chelate and the photoactivatable ArN.sub.3 group. PEG groups also have the additional benefit of increasing water solubility which is a limiting factor for some chelates. However, it is conceivable that shorter linkers or even direct coupling of ArN.sub.3 to one of the carboxylate arms of the chelates would also generate viable photoactive reagents.
[0306] Synthesis of Metal Complexes
[0307] In addition to the chelates, non-radioactive Ga complexes were produced and characterised by HR-ESI-MS and UHPLC (
[0308] Testing of the Photochemical Reactivity of the Metal Complexes
[0309] Following successful radiolabelling experiments on the chelates, the photochemical reactivity of the .sup.68Ga-complexes was tested. Samples of [.sup.68GaNOTA-PEG.sub.3-ArN.sub.3].sup.+, [.sup.68GaDOTA-PEG.sub.4-ArN.sub.3] and [.sup.68GaDOTAGA-PEG.sub.4-ArN.sub.3] were irradiated using an intense light-emitting diode (LED, 365 nm, 10-30 min, room temperature). Subsequent radio-iTLC and radio-UHPLC analysis confirmed that the radioactive complexes reacted rapidly under UV-irradiation to give essentially a single major new radioactive species (
[0310] Radiolabelling of Target Molecules
[0311] The two-step, one-pot photoradiochemical approach for radiolabelling a monoclonal antibody, and structures of the three products is shown in Scheme 2.
[0312] Standard .sup.68Ga.sup.3+ radiochemistry is not perfectly compatible with the photochemical conjugation step because the complexation reaction is performed under acidic conditions (pH˜4.4, NaOAc buffer). In contrast, the photochemical conjugation proceeds most efficiently under slightly basic conditions where the nucleophilicity of the lysine side-chain is increased via deprotonation of the primary ε-NH.sub.2 amine (pKa ˜10.5). For this reason, the chelates were pre-radiolabelled with [.sup.68Ga][Ga(H.sub.2O)6].sup.3+ before adjusting the pH in situ to >7.5 using NaHCO.sub.3 solution. Complex formation was monitored by radio-iTLC and radio-size-exclusion chromatography (SEC) UHPLC. After complete complexation, an aliquot of pre-purified monoclonal antibody was added with an initial chelate-to-monoclonal antibody ratio of ˜10-to-1. Reaction mixtures were then irradiated for 15 min at room temperature. Aliquots of the crude reaction mixtures were analysed by radio-iTLC, manual size-exclusion chromatography (PD-10-SEC) and radio-SEC-UHPLC. In addition, a fraction was purified by preparative PD-10 and spin-centrifugation methods to measure the absolute radiochemical yield (RCY), radiochemical purity (RCP) and molar activities of the purified .sup.68Ga-radiolabelled antibody (
[0313] One-pot photoradiochemistry using NOTA-PEG.sub.3-ArN.sub.3 (1)
[0314] To a solution of NOTA-PEG.sub.3-ArN.sub.3 (1) (50 μg, 7.68×10.sup.−8 mol, 1.02 mM) buffered with NaOAc (0.53 M, pH4.4) was added [.sup.68Ga][Ga(H.sub.2O).sub.6]Cl.sub.3(aq.) stock solution (30.8±4.3 MBq, n=3) resulting in a total reaction volume of 75 μL. Reactions were monitored by radio-iTLC. Formation of [68Ga][GaNOTA-PEG.sub.3-ArN.sub.3].sup.+ (.sup.68Ga-1.sup.+) was complete after 5 min. incubation at 23° C. with radiochemical conversion (RCC) >99% (n=3, R.sub.f=0.03−0.19 on iTLC). The pH of the reaction mixture was then adjusted to >7.5 by the addition of an aqueous solution of NaHCO.sub.3 (1.0 M, 50 μL added). After adjusting the pH, an aliquot of pre-purified monoclonal antibody (1.015 mg, 7.00×10.sup.−9 mol, reaction concentration=6.3 mg/mL) was added to give an initial chelate-to-antibody ratio of 11.0 at the start of the photochemical conjugation step (total reaction volume ˜160 μL). The reaction mixture was then irradiated using the LED (100% intensity, 365 nm) for 15 min. at room temperature without stirring. Aliquots of this crude reaction mixture were then analysed by using radio-iTLC, PD-10-SEC and SEC-UHPLC analysis.
[0315] Radio-iTLC analyses of the crude reactions after irradiation showed that ˜30% (n=3) of the radioactivity was bound to the antibody (R.sub.f=0.0). Note: integration of these radio-iTLC data is unreliable because the radiolabelled antibody fraction partially overlaps with the peak associated with .sup.68Ga-1.sup.+ and the photodegraded .sup.68Ga-1.sup.+ species (R.sub.f=0.03−0.19). Nevertheless, analytical PD-10-SEC measurements on the crude reaction mixtures confirmed this observation with an estimated RCP of 15.9±1.8% (n=3). Equivalent decay corrected SEC-UHPLC measurements indicated that the radiolabelled fraction of [.sup.68Ga]GaNOTA-azepin-antibody in the crude mixture was 15.5±1.5% (n=3).
[0316] Crude reaction mixtures were then purified by preparative PD-10-SEC eluting with PBS (collecting only the high purity 0.0-1.6 mL fraction). Prior to analysis, samples were concentrated using an Amicon Ultra-4 mL centrifugal filter (Millipore, 30 kDa MWCO, 4000 RPM, ˜10 min.). The purified and formulated [.sup.68Ga]GaNOTA-azepin-antibody products (pH7.4) were obtained in <25 min. with decay corrected radiochemical yields (RCY) of 10.1±0.7% (n=3). The estimated lower limit on the molar activity (A.sub.m/[MBq/nmol] of protein) of the formulated [.sup.68Ga]GaNOTA-azepin-antibody samples was 0.46±0.09 MBq/nmol (n=3, remeasured protein concentration). Purified products were then reanalysed by radio-iTLC, analytical PD-10-SEC and SEC-UHPLC. The RCP of purified [.sup.68Ga]GaNOTA-azepin-antibody was >99% (n=3) by radio-iTLC, 91.3±4.4% (n=3) by analytical PD-10-SEC, and 95.2±2.0% (n=3) by SEC-UHPLC.
[0317] One-Pot Photoradiochemistry Using DOTA-PEG.sub.4-ArN.sub.3 (3)
[0318] To a solution of DOTA-PEG.sub.4-ArN.sub.3 (3) (60 μg, 7.81×10.sup.−8 mol, 1.03 mM) buffered with NaOAc (0.53 M, pH4.4) was added [.sup.68Ga][Ga(H.sub.2O).sub.6]Cl.sub.3(aq.) stock solution (31.6±1.1 MBq, n=3) resulting in a total reaction volume of 76 μL. Reactions were monitored by radio-iTLC. Formation of [.sup.68Ga]GaDOTA-PEG.sub.4-ArN.sub.3 (.sup.68Ga-3) was complete after 10 min. incubation at 70° C. with radiochemical conversion (RCC)>99% (n=3, R.sub.f =0.06-0.21 on iTLC). The pH of the reaction mixture was then adjusted to >7.5 by the addition of an aqueous solution of NaHCO.sub.3 (1.0 M, 50 μL added). After adjusting the pH, an aliquot of pre-purified monoclonal antibody (1.015 mg, 7.00×10.sup.−9 mol, reaction concentration=6.3 mg/mL) was added to give an initial chelate-to-antibody ratio of 11.2 at the start of the photochemical conjugation step (total reaction volume ˜160 μL). The reaction mixture was then irradiated using the LED (100% intensity, 365 nm) for 15 min. at room temperature without stirring. Aliquots of this crude reaction mixture were then analysed by using radio-iTLC, PD-10-SEC and SEC-UHPLC analysis.
[0319] Radio-iTLC analyses of the crude reactions after irradiation showed that ˜30% (n=3) of the radioactivity was bound to the antibody (R.sub.f=0.0). Note: integration of these radio-iTLC data is unreliable because the radiolabelled antibody fraction partially overlaps with the peak associated with .sup.68Ga-3 and the photodegraded .sup.68Ga-3 species (R.sub.f=0.06−0.21). Nevertheless, analytical PD-10-SEC measurements on the crude reaction mixtures confirmed this observation with an estimated RCP of 16.2±0.3% (n=3). Equivalent decay corrected SEC-UHPLC measurements indicated that the radiolabelled fraction of [.sup.68Ga]GaDOTA-azepin-antibody in the crude mixture was 12.7±3.2% (n=3).
[0320] Crude reaction mixtures were then purified by preparative PD-10-SEC eluting with PBS (collecting only the high purity 0.0-1.6 mL fraction). Prior to analysis, samples were concentrated using an Amicon Ultra-4 mL centrifugal filter (Millipore, 30 kDa MWCO, 4000 RPM, ˜10 min.). The purified and formulated [.sup.68Ga]GaDOTA-azepin-antibody products (pH7.4) were obtained in <30 min. with decay corrected radiochemical yields (RCY) of 8.3±1.4% (n=3). The estimated lower limit on the molar activity (A.sub.m/[MBq/nmol] of protein) of the formulated [.sup.68Ga]GaDOTA-azepin-antibody samples was 0.37±0.08 MBq/nmol (n=3, remeasured protein concentration). Purified products were then reanalysed by radio-iTLC, analytical PD-10-SEC and SEC-UH PLC. The RCP of purified [.sup.68Ga]GaDOTA-azepin-antibody was >99% (n=3) by radio-iTLC, 90.7±1.1% (n=3) by analytical PD-10-SEC, and 93.0±3.0% (n=3) by SEC-UHPLC.
[0321] One-Pot Photoradiochemistry Using DOTAGA-PEG.sub.4-ArN.sub.3 (4)
[0322] To a solution of DOTAGA-PEG.sub.4-ArN.sub.3 (4) (60 μg, 7.14×10.sup.−8 mol, 0.94 mM) buffered with NaOAc (0.53 M, pH4.4) was added [.sup.68Ga][Ga(H.sub.2O).sub.6]Cl.sub.3(aq.) stock solution (31.6±3.0 MBq, n=3) resulting in a total reaction volume of 76 μL. Reactions were monitored by radio-iTLC. Formation of [.sup.68Ga][GaDOTAGA-PEG.sub.4-ArN.sub.3].sup.− (.sup.68Ga-4.sup.−) was complete after 10 min. incubation at 70° C. with radiochemical conversion (RCC) >99% (n=3, R.sub.f=0.06−0.22 on iTLC). The pH of the reaction mixture was then adjusted to >7.5 by the addition of an aqueous solution of NaHCO.sub.3 (1.0 M, 50 μL added). After adjusting the pH, an aliquot of pre-purified monoclonal antibody (1.015 mg, 7.00×10.sup.−9 mol, reaction concentration=6.3 mg/mL) was added to give an initial chelate-to-antibody ratio of 10.2 at the start of the photochemical conjugation step (total reaction volume ˜160 λL). The reaction mixture was then irradiated using the LED (100% intensity, 365 nm) for 15 min. at room temperature without stirring. Aliquots of this crude reaction mixture were then analysed by using radio-iTLC, PD-10-SEC and SEC-UHPLC analysis.
[0323] Radio-iTLC analyses of the crude reactions after irradiation showed that ˜30% (n=3) of the radioactivity was bound to the antibody (R.sub.f=0.0). Note: integration of these radio-iTLC data is unreliable because the radiolabelled antibody fraction partially overlaps with the peak associated with .sup.68Ga-4.sup.− and the photodegraded .sup.68Ga-4.sup.− species (R.sub.f=0.06−0.22). Nevertheless, analytical PD-10-SEC measurements on the crude reaction mixtures confirmed this observation with an estimated RCP of 18.3±0.7% (n=3). Equivalent decay corrected SEC-UHPLC measurements indicated that the radiolabelled fraction of [.sup.68Ga]GaDOTAGA-azepin-antibody in the crude mixture was 11.1±0.2% (n=3).
[0324] Crude reaction mixtures were then purified by preparative PD-10-SEC eluting with PBS (collecting only the high purity 0.0-1.6 mL fraction). Prior to analysis, samples were concentrated using an Amicon Ultra-4 mL centrifugal filter (Millipore, 30 kDa MWCO, 4000 RPM, —10 min.). The purified and formulated [.sup.68Ga]GaDOTAGA-azepin-antibody products (pH7.4) were obtained in <30 min. with decay corrected radiochemical yields (RCY) of 9.2±0.6% (n=3). The estimated lower limit on the molar activity (A.sub.m/[MBq/nmol] of protein) of the formulated [.sup.68Ga]GaDOTAGA-azepin-antibody samples was 0.37±0.07 MBq/nmol (n=3, remeasured protein concentration). Purified products were then reanalysed by radio-iTLC, analytical PD-10-SEC and SEC-UHPLC. The RCP of purified [.sup.68Ga]GaDOTAGA-azepin-antibody was >99% (n=3) by radio-iTLC, 92.2±1.0% (n=3) by analytical PD-10-SEC, and 92.2±1.8% (n=3) by SEC-UHPLC.
[0325] Experimental
[0326] The experiments described in this specification may be performed with any antibody or antibody fragment that comprises a free amine or thiol moiety such as cetuximab, bevacizumab, trastuzumab, panitumumab, ibritumomab tiuxetan, onartuzumab, J591, fresolimumab, rituximab, brentuximab, lumretuzumab, U36, R1507, ranibizumab, DN30, 7E11, particularly trastuzumab. As described above, the photoconjugation requires an amine or thiol moiety such as in the side chain of the amino acids lysine or cysteine. The experiments described herein using an antibody were performed with trastuzumab.
[0327] General Details
[0328] Unless otherwise stated, all chemicals were of reagent grade and purchased from SigmaAldrich (St. Louis, Mo.), Merck (Darmstadt, Germany), Tokyo Chemical Industry (Eschborn, Germany), abcr (Karlsruhe, Germany) or CheMatech (Dijon, France). Water (>18.2 MΩ.Math.cm at 25° C., Puranity TU 3 UV/UF, VWR International, Leuven, Belgium) was used without further purification. Solvents for reactions were of reagent grade, and where necessary, were dried over molecular sieves. Evaporation of the solvents was performed under reduced pressure by using a rotary evaporator (Rotavapor R-300, Buchi Labortechnik AG, Flawil, Switzerland) at the specified temperature and pressure. If the antibody is not specified otherwise, the experiments described herein were performed using the antibody trastuzumab.
[0329] .sup.1H and .sup.13C NMR spectra were measured in deuterated solvents on a Bruker AV-400 (.sup.1H: 400 MHz, .sup.13C: 100.6 MHz) or a Bruker AV-500 (.sup.1H: 500 MHz, .sup.13C: 125.8 MHz) spectrometer. Chemical shifts (6) are expressed in parts per million (ppm) relative to the resonance of the residual solvent peaks, for example, with DMSO 6.sub.H=2.50 ppm and δ.sub.C=39.5 ppm with respect to tetramethylsilane (TMS, δ.sub.H and δ.sub.C=0.00 ppm). Coupling constants (J) are reported in Hz. All resonances were assigned by using a combination of 1D and 2D NMR (HSQC, COSY) spectra. Peak multiplicities are abbreviated as follows: s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet), and br (broad).
[0330] High-resolution electrospray ionisation mass spectra (HR-ESI-MS) were measured by the mass spectrometry service at the Department of Chemistry, University of Zurich.
[0331] Column chromatography was performed by using Merck silica gel 60 (63-200 μm) with eluents indicated in the experimental section. Standard thin-layer chromatography (TLC) for synthesis employed Merck TLC plates silica gel 60 on an aluminium base with the indicated solvent system. The spots on TLC were visualised either by UV/visible light (254 nm) or by staining with KMnO.sub.4.
[0332] Semi-preparative high-performance liquid chromatography (HPLC) purifications were performed using a Rigol HPLC system (Contrec AG, Dietikon, Switzerland) equipped with a 018 reverse-phase column (VP 250/21 Nucleodur C18 HTec, 21 mm ID×250 mm, 5 μm) using a flow rate of 8 mL min.sup.−1 with a linear gradient of solvent A (distilled H.sub.2O containing 0.1% TFA) and B (MeOH): t=0-3 min., 60% A; t=25-30 min., 5% A; t=33-38 min., 60% A. Electronic absorption was measured at 254 nm.
[0333] Analytical ultra-high-performance liquid chromatography (UHPLC) experiments were performed using two separate Hitachi Chromaster Ultra Rs systems fitted with either a reverse phase VP 250/4 Nucleodur C18 HTec (4 mm ID×250 mm, 5 μm) column or a reverse phase Acquity UPLC column (BEH C18, 1.7 μm, 2.1 mm ID×50 mm). One of these systems was also connected to a radioactivity detector (FlowStar.sup.2 LB 514, Berthold Technologies, Zug, Switzerland) equipped with a 20 μL PET cell (MX-20-6, Berthold Technologies) for analysing radiochemical reactions. Proteins were analysed by using the same UHPLC system equipped with a size-exclusion column (Enrich SEC 70 column: 24 mL volume, 10±2 μm particle size, 10 mm ID×300 mm, Bio-Rad Laboratories, Basel, Switzerland). UHPLC using the Acquity column used a flow rate of 0.6 mL min.sup.−1 with a linear gradient of solvent A (distilled H.sub.2O containing 0.1% TFA) and B (acetonitrile): t=0-0.5 min., 30% A; t=9.5 min., 0% A; t=10 min., 0% A. Electronic absorption was measured at 254 nm.
[0334] Analytical high performance liquid chromatography (HPLC) experiments for photodegradation kinetics were performed using a Hitachi Chromaster system equipped with a reverse phase column (Reproshell 100 Dr. Maisch C18, 2.8 μm, 75×4.6 mm) using a flow rate of 1.5 mL min.sup.−1 with a linear gradient of solvent A (distilled H.sub.2O containing 0.1% HCOOH) and B (acetonitrile): t=0 min., 95% A; t=5.8 min., 0% A; t=6.8 min., 0% A; t=7.3 min., 90% A. Electronic absorption was measured at 260 nm.
[0335] Electronic absorption spectra were recorded using a Nanodrop™ One.sup.C Microvolume UV-Vis Spectrophotometer (ThermoFisher Scientific, supplied by Witec AG, Sursee, Switzerland). Protein concentration was determined in accordance with the manufacturers protocol.
[0336] Photochemistry
[0337] Photochemical conjugation experiments were performed in transparent glass vials at the indicated concentrations. Stock solutions were prepared in H.sub.2O (antibody and DFO-ArN.sub.3 [1]).
[0338] Photochemical reactions were stirred gently using a magnetic stir bar. Detail procedure and reaction times are indicated in the experimental section. Irradiations used three light sources. For pre-conjugation experiments, a high-powered Rayonet reactor.sup.[1] (350 nm, 16×8 W Sylvania BLB-lamps, 10 cm diameter) was used. For kinetic studies and for simultaneous one-pot photoradiochemical labelling reactions, portable, high-powered, light-emitting diodes (LEDs at either 365 nm or 395 nm) were used. The LED intensity was adjusted using a UV-LED controller (Opsytec Dr. Grobel GmbH, Ettlingen, Germany), where 100% corresponded to a power of approximately 263 mW and 355 mW for the 365 nm and 395 nm sources, respectively. LED intensity was measured using a S470C Thermal Power Sensor Head, Volume Absorber, 0.25-10.6 μm, 0.1mW-5W, ∅15 mm. Total irradiance power of the
[0339] Rayonet reactor was estimated to be approximately 92 mW (approximately 300 mW/cm.sup.3). Note that calculation of exact power incident to the reaction is non-trivial because it depends on the specific geometry of the experiment. The temperature of all photochemical conjugation reactions was typically 23±2° C. The Rayonet reactor had an experimentally measured λ.sub.max at 368 nm with full-width at half-maximum (FWHM) value of 16.0 nm. The LED (365 nm) had a maximum emission intensity at 364.5 nm (FWHM of 9.1 nm). The LED (395 nm) had a maximum emission intensity at 389.9 nm (FWHM of 9.1 nm).
[0340] Radioactivity and Radioactive Measurements
[0341] All instruments for measuring radioactivity were calibrated and maintained in accordance with previously reported routine quality control procedures..sup.[2] [.sup.89Zr][Zr(C.sub.2O.sub.4).sub.4].sup.4−(aq.) was obtained as a solution in ˜1.0 M oxalic acid from PerkinElmer (Boston, Ma., manufactured by the BV Cyclotron VU, Amsterdam, The Netherlands) and was used without further purification. Radioactive reactions were monitored by using instant thin-layer chromatography (radio-iTLC). Glass-fibre iTLC plates impregnated with silica-gel (iTLC-SG, Agilent Technologies) were developed in using aqueous mobile phases containing either EDTA (50 mM, pH7.1) or DTPA (50 mM, pH7.4) and were analysed on a radio-TLC detector (SCAN-RAM, LabLogic Systems Ltd, Sheffield, United Kingdom). Radiochemical conversion (RCC) was determined by integrating the data obtained by the radio-TLC plate reader and determining both the percentage of radiolabelled product (R.sub.f=0.0) and ‘free’ .sup.89Zr (R.sub.f=1.0; present in the analyses as either [.sup.89Zr]Zr(EDTA) or [.sup.89Zr]Zr(DTPA). Integration and data analysis were performed by using the software Laura version 5.0.4.29 (LabLogic). Appropriate background and decay corrections were applied as necessary. Radiochemical purities (RCPs) of labelled protein samples were determined by size-exclusion chromatography (SEC) using two different columns and techniques. The first technique used an automated size-exclusion column (Bio-Rad Laboratories, ENrich SEC 70, 10±2 μm, 10 mm ID×300 mm) connected to a UHPLC device (Hitachi ChromasterUltra Rs, VWR International, Leuven, Belgium) equipped with a UV/visible diode array detector (absorption measured at 220, 254 and/or 280 nm) as well as a radioactivity detector (FlowStar.sup.2 LB 514, Berthold Technologies, Zug, Switzerland). Isocratic elution with phosphate buffered saline (PBS, pH7.4) was used. The second method used a manual procedure involving size-exclusion column chromatography using a PD-10 desalting column (Sephadex G-25 resin, 85-260 μm, 14.5 mm ID×50 mm, >30 kDa, GE Healthcare). For analytical procedures, PD-10 columns were eluted with sterile saline or PBS. A total of 40×200 μL fractions were collected up to a final elution volume of 8 mL. Note that the loading/dead-volume of the PD-10 columns is precisely 2.50 mL which was discarded prior to aliquot collection. For quantification of radioactivity, each fraction was measured on a gamma counter (HIDEX Automatic Gamma Counter, Hidex AMG, Turku, Finland) using an energy window between 480-558 keV for .sup.89Zr (511 keV emission) and a counting time of 30 s. Appropriate background and decay corrections were applied throughout. PD-10 SEC columns were also used for preparative purification and reformulation of radiolabelled products by collecting a fraction of the eluate corresponding to the high molecular weight protein (>30 kDa fraction eluted in the range between 0.0 to 1.6 mL as indicated for each experiment).
[0342] Stability Studies
[0343] The stability of [.sup.89Zr]ZrDFO-azepin-antibody with respect to change in radiochemical purity due to loss of radioactivity from the protein fraction was investigated in vitro by incubation in human serum. Aliquots of [.sup.89Zr]ZrDFO-azepin-antibody (250 μL, 81.5 μg, 0.54 nmol, 6.59 MBq, A.sub.m ˜12.1 MBq/nmol) were added to human serum (400 μL) giving a total reaction volume of 650 μL. Solutions were incubated at 37° C. and SEC-UHPLC measurements recorded at the specified time points up to 45 h. The stability was monitored by quantifying the radioactivity associated with intact [.sup.89Zr]ZrDFO-azepin-antibody from integration of the decay corrected SEC-UHPLC radioactive chromatograms.
[0344] Synthesis and Chemical Characterisation
[0345] Chemical syntheses were performed in accordance with Scheme 2.
##STR00028##
[0346] All reactions involving photosensitive compounds were performed in the dark. The IgG.sub.1 antibody component was purified from an antibody preparation by spin column centrifugation (4000 RPM, 3×15 min., 1×20 min.) by using a membrane filter (Amicon Ultra-4 mL centrifugal filter, Millipore, 10 kDa MWCO). Briefly, aliquots of the antibody preparation (60 mg) were washed with H.sub.2O (4×4 mL) at room temperature and concentrated before use.
[0347] After concentration, protein samples were removed from the centrifugation filter by rinsing with water (500 μL) and the protein concentration was determined using a Nanodrop™ One.sup.C Microvolume UV-Vis Spectrophotometer. Typically, 25-30 mg of protein was obtained and samples were aliquoted into Eppendorf tubes and stored at −20° C. for future use.
[0348] Synthesis of Desferrioxamine-p-arylazide, DFO-ArN.sub.3 (1)
[0349] A solution of 4-azidobenzoic acid (206 mg, 1.26 mmol), HATU (506 mg, 1.33 mmol) and N,N-diisopropylethylamine (DIPEA, 130 μL) in dry DMF (8 mL) was stirred at room temperature for 40 min. Then desferrioxamine B mesylate (DFO, 407 mg, 0.725 mmol) was added to the mixture along with additional DIPEA (95 μL) and N-methylmorpholine (250 μL). After stirring at room temperature for 80 h, the mixture was transferred to a single-necked round bottom flask (100 mL) and the solvent was evaporated under reduced pressure (25 mbar). The orange-beige residue was washed by sonication with cold acetone (6×7 mL, −20 ° C.) and ice cold H.sub.2O (4×7 mL). Note that between each washing step, the solid residue was collected by centrifugation and cooled. Washing with acetone the orange colour and subsequent lyophilisation gave the crude product DFO-ArN.sub.3 (1, 40% yield, 228 mg, 0.291 mmol, estimated 68% purity measured by .sup.1H NMR) as a white amorphous powder. A portion of the crude product was purified by semi-preparative HPLC and after lyophilisation, purified compound 1 was obtained as a white amorphous powder. (Yield 4%, estimated purity >95% by UHPLC and by .sup.1H NMR).
[0350] Synthesis of [ZrDFO-ArN.sub.3].sup.+ (Zr-1.sup.+)
[0351] DFO-ArN.sub.3 (1, 0.68 mg, 0.964 μmol was dissolved in a mixture of H.sub.2O (50 μL) and NaOH(aq.) (0.1 M, 30 μL). After dissolution of compound 1, a clear, colourless solution was obtained. Then the pH of the mixture was reduced to ˜8-9 by the addition HCl(aq.) (0.1 M, 2×10 μL). Then an aliquot of ZrCl.sub.4(aq.) (112 μL, 6 M Zr.sup.4+ ions dissolved in 0.1 M HCl(aq.)) was added dropwise. The reaction was monitored by RP-UHPLC and after stirring at room temperature for 2 h, complete conversion was observed. Presence of desired product Zr-1.sup.+ was confirmed by a single peak in analytical HPLC that gave the expected mass of molecular ion as the base peak in high-resolution electrospray ionisation mass spectrometry (see
[0352] Radiochemistry and Photoradiochemistry
[0353] Molar Activity of the [.sup.89Zr][Zr(C.sub.2O.sub.4).sub.4].sup.4−(aq.) Stock Solution
[0354] The molar activity of the .sup.89Zr-oxalate stock solution was measured by isotopic dilution assays. Briefly, a stock solution of desferrioxamine B mesylate was prepared in water (3.77 mg, MW=656.79 g mol.sup.−1, 5.74 μmol, 1.0 mL, [DFO]=5.74 mM) and was diluted to give a secondary solution (2.87 μM). To microcentrifuge tubes (n=3) was added H.sub.2O (90 μL) and an aliquot of the secondary DFO stock solution (10 μL, 0.0287 nmol). Then an aliquot of a neutralised [.sup.89Zr][Zr(C.sub.2O.sub.4).sub.4].sup.4−(aq.) stock solution (see below for details on the neutralisation step) was added to each tube (1.637 MBq). Reactions were vortexed and incubated at room temperature for 90 min. to ensure complete reaction occurred. At the end of the reaction, aliquots were spotted onto iTLC plates and developed using aqueous mobile phase containing DTPA (50 mM, pH7.4) or EDTA (50 mM, pH7.1). Radio-iTLC analysis was used to measure the radiochemical conversion (RCC) with the product [.sup.89Zr]ZrDFO retained at the baseline (R.sub.f=0.0) and either [.sup.89Zr]Zr(EDTA) or [.sup.89Zr]Zr(DTPA) eluting at the solvent front (R.sub.f=1.0). The experimentally measured molar activity of the [.sup.89Zr][Zr(C.sub.2O.sub.4).sub.4].sup.4−(aq.) stock solution was A.sub.m=37.0±0.12 MBq/nmol.
[0355] Radiosynthesis and Characterisation of [.sup.89Zr][ZrDFO-ArN.sub.3].sup.+ (.sup.89Zr-1.sup.+)
[0356] A stock solution of DFO-ArN.sub.3 (1, 0.67 mg, 0.950 μmol) was dissolved in H.sub.2O (50 μL) and NaOH(aq.) (30 μL of a 0.1 M stock solution). The pH of the DFO-ArN.sub.3 solution was reduced to ˜8-9 by the addition of HCl(aq.) (2×10 μL of a 0.1 M stock solution). A stock solution of [.sup.89Zr][Zr(C.sub.2O.sub.4).sub.4].sup.4− was prepared by adding .sup.89Zr radioactivity from the source (68.7 MBq, 70 μL in ˜1.0 M aqueous oxalic acid) to a vial containing water (200 μL). The solution was neutralised and made slightly basic by the addition of aliquots of Na.sub.2CO.sub.3(aq.) (1.0 M stock solution, 55 μL added, final pH ˜8.3-8.5). Caution: Acid neutralisation with Na.sub.2CO.sub.3 releases CO.sub.2(g) and care should be taken to ensure that no radioactivity escapes the microcentrifuge tube. After CO.sub.2 evolution ceased, an aliquot of the neutralised [.sup.89Zr][Zr(C.sub.2O.sub.4).sub.4].sup.4− solution (20-40 μL, 4.66 MBq) was added to the reaction microcentrifuge vial containing an aliquot of the DFO-ArN.sub.3 stock solution (10 μL, 95 nmol, 9.5 mM) and water (50 μL) giving a clear, colourless solution (pH 7-8). The reaction was vortexed and incubated at room temperature. Reaction progress was monitored by radio-ITLC and complete radiochemical conversion to give of .sup.89Zr-1.sup.+ (R.sub.f=0.0) was observed in <10 min. Aliquots of the crude reaction mixture were analysed be radioactive HPLC (
[0357] Photochemical Conjugation
[0358] General procedure for photochemical conjugation: A stock solution of photoactive ligand was prepared by dissolving DFO-ArN.sub.3 (1, 0.85 mg, 1.21 μmol) in water (50 μL) and NaOH(aq.) (40 μL of a 0.1 M stock solution). Immediately before starting the photochemical conjugation reactions, the pH of the DFO-ArN.sub.3 solution was reduced to ˜9 by the addition of HCl(aq.) (2×10 μL of a 0.1 M stock solution). Note: DFO-ArN.sub.3 (1) is sparingly soluble at high pH but starts to precipitate slowly when the pH decreases below ˜9. For this reason, photochemical reactions should be initiated immediately after adding the HCl and the protein. After adjusting the pH, aliquots of the DFO-ArN.sub.3 stock solution were added to clear 2 mL glass vials equipped with small magnetic stirring bars and containing an aqueous solution of antibody (120 μL, 2.76 mg, 1.84×10.sup.−8 mol, stock protein concentration=23.0 mg/mL) and a variable amount of water (constant total reaction volume=200 μL). The chelate-to-mAb ratio was varied used 5.3-fold (9 μL), 10.7-fold (18 μL) or 26.4-fold (45 μL) excess of DFO-ArN.sub.3 stock solution. The final pH of the solutions was 8-8.5. The reaction mixture was then irradiated for 25 min. using the Rayonet reactor. The irradiated crude mixture was then purified by a three-step procedure. First, the mixture was taken in a 30 kDa MWCO membrane centrifugal filter (Amicon Ultra-4 mL centrifugal filter, Millipore,), concentrated and washed with PBS (2×4 mL) using centrifugation (4000 RPM, ˜15 min). Then the mixture was purified using a preparative PD-10-SEC column (eluted with PBS, collecting the 0.0-1.6 mL fraction immediately after discarding the 2.5 mL column dead volume). In the last step, the fraction from PD-10-SEC was taken in a new 30 kDa MWCO membrane centrifugal filter, washed and concentrated using PBS (2×4 mL) followed by water (2×4 mL) as described in first step. The purified protein was removed from the spin column filter in a final volume of ˜320 μL water. Protein concentration was measured using the Nanodrop. Stock solutions of DFO-azepin-antibody were aliquoted and stored at −20° C.
[0359] .sup.89Zr-Radiolabelling of DFO-azepin-antibody
[0360] For animal experiments, the radiochemical synthesis of [.sup.89Zr]ZrDFO-azepin-antibody was scaled up using a sample of DFO-azepin-antibody prepared from an initial chelate-to-antibody ratio of 26.4-to-1 in the photochemical conjugation reaction. To a microcentrifuge tube was added water (100 μL) and [.sup.89Zr][Zr(C.sub.2O.sub.4).sub.4].sup.4−(aq.) stock solution (70 μL, 88.66 MBq). The oxalic acid was neutralised and made slightly basic by the addition of aliquots of Na.sub.2CO.sub.3(aq.) (˜1.0 M, 55 μL, final pH8.1-8.3). Caution: Acid neutralisation with Na.sub.2CO.sub.3 releases CO.sub.2(g) and care should be taken to ensure that no radioactivity escapes the microcentrifuge tube. After CO.sub.2 evolution ceased, an aliquot of photochemically conjugated DFO-azepin-antibody (125 μL, 8.0 mg/mL, mass=1.0 mg of protein, 6.67 nmol) produced using an initial chelate-to-mAb ratio of 26.4-to-1 was added to the neutralised solution of [.sup.89Zr][Zr(C.sub.2O.sub.4].sup.4−(aq.). The pH decreased slightly to 6.6 and was readjusted to pH7.4-7.7 by the addition of Na.sub.2CO.sub.3(aq.) (˜1.0 M, 4 μL). The reaction was mixed gently and then incubated at room temperature for 1 h. The reaction was monitored by radio-iTLC. Control reactions performed in the absence of antibody showed complete formation of [.sup.89Zr]Zr(EDTA) under the conditions used to develop the iTLC plates with no activity retained at the baseline (R.sub.f=0.0). The reaction showed a RCC>95% after the 15 minutes but a slight improvement in RCC occurred by 40 min. (RCC>98%), which remained the same by 60 min. After 1 h, the reaction was quenched by the addition of a small aliquot of EDTA (5 μL, 50 mM, pH7.4) and incubating for a further 5 min. An aliquot of the crude mixture was retained for further analysis and then the major fraction (250 μL) was purified by preparative PD-10-SEC eluting with sterile PBS. All crude and purified mixtures were analysed by radio-iTLC, analytical PD-10-SEC and SEC-UHPLC.
[0361] The radiochemical purity (RCP) of the crude sample of [.sup.89Zr]ZrDFO-azepin-antibody was determined by analytical PD-10-SEC (>98%) as well as SEC-HPLC (>98%). Purification and formulation [.sup.89Zr]ZrDFO-azepin-antibody (pH7.4) was completed in <5 min. with a decay corrected radiochemical yield (RCY) of >99%, and a final activity concentration of 29.67 MBq/mL. After preparative PD-10-SEC (collecting the 0.0-1.8 mL fraction) the RCP was to >99.5% (measured by analytical PD-10-SEC) and >98% (measured by SEC-UHPLC).
[0362] Aliquots of the final [.sup.89Zr]ZrDFO-azepin-antibody product were then prepared for injection in the normal and blocking groups of animals (n=6 mice/group). Briefly, two aliquots of [.sup.89Zr]ZrDFO-azepin-antibody (350 μL, ˜10.4 MBq) were added to separate centrifuge tubes. For the normal group dose, the activity was diluted with sterile PBS (1.65 mL) giving a final volume of 2.0 mL. For the blocking group, the activity was diluted with sterile PBS (1.511 mL) and then an aliquot of non-radiolabelled antibody (stock protein concentration=57.7 mg/mL, 0.139 mL, 8.0 mg) was added and the solution mixed gently. A total of 7 syringes (250 μL/each) were drawn for both the normal and blocking formulations. The seventh syringe was used as a standard for accurate quantification of the biodistribution data (vide supra). In addition, aliquots of the normal and blocking formulations were retained and the protein concentration was re-measured using the Nanodrop. The measured molar activities (A.sub.m/[MBq/nmol] of protein) of the injectates (decay-corrected to the point of final formulation) were then calculated as 13.7 MBq/nmol for the normal doses and 0.14 MBq/nmol for the blocking doses. The blocking dose contained ˜98-fold higher concentration of mAb than the normal dose.
[0363] Chelate Number Estimation
[0364] The number of chemically accessible chelates per antibody produced after photochemically conjugating the monoclonal antibody with different initial chelate-to-antibody ratios were estimated by radiolabelling the DFO-azepin-antibody samples using an excess of [.sup.89Zr][Zr(C.sub.2O.sub.4).sub.4].sup.4−(aq.), ensuring that the RCC was <100%. Samples of the crude radiolabelling reactions forming [.sup.89Zr]ZrDFO-azepin-antibody were analysed by radio-ITLC eluting with EDTA. The fraction of .sup.89Zr radioactivity retained at the baseline (R.sub.f=0.0) and at the solvent front (R.sub.f =1.0, [.sup.89Zr]ZrEDTA) was determined by integration after appropriate background corrections. Radio-ITLC data for the reaction using the 26.4-fold initial chelate-to-mAb ratio is shown in
[0365] Simultaneous, One-Pot Photoradiochemical Synthesis of [.sup.89Zr]ZrDFO-azepin-antibody
[0366] Simultaneous, one-pot photochemical conjugation and radiolabelling reactions were performed in accordance with the following general procedure. A stock solution of DFO-ArN.sub.3 (1, 0.68 mg, 0.964 μmol) was dissolved in H.sub.2O (50 μL) and NaOH(aq.) (30 μμL of a 0.1 M stock solution). The pH of the DFO-ArN.sub.3 solution was reduced to ˜8-9 by the addition of HCl(aq.) (2×10 μL of a 0.1 M stock solution). Different reactions and control were performed at the same time using the same stock solutions. Details are given in Table S3 below. Details for reaction 1 are given here. To a transparent glass vial containing water (50 μL) was added an aliquot of pre-purified antibody stock solution (stock concentration=23.0 mg/mL, 50 μL added, 1.15 mg of protein, 7.69 nmol), an aliquot of the DFO-ArN.sub.3 stock solution (1, 23 μL, 0.222 μmol, ˜28.9-fold excess) and an aliquot of pre-neutralised [.sup.89Zr][Zr(C.sub.2O.sub.4).sub.4].sup.4− (aq.) stock solution (50 μL, 4.2 MBq). Note: see the radiochemistry sections above for details about neutralisation of oxalic acid in the .sup.89Zr stock solution. The total reaction volume was kept constant at 150 μL for all reactions. Reactions were stirred and irradiated at room temperature for 10 min. at the specified LED wavelength (100% power). Reactions were then quenched by the addition of DTPA (10 μL, 50 mM) and aliquots of the crude reaction mixtures were analysed by using radio-ITLC, analytical PD-10-SEC and SEC-UHPLC. Data are presented in
TABLE-US-00002 TABLE S3 Experimental data on the conditions used in the simultaneous photoradiolabelling reactions for the synthesis of [.sup.89Zr]ZrDFO-azepin-antibody. Reaction 2 Reaction 3 (no chelate (no antibody Solution Reaction 1 control) control) Reaction 4 Vol. water/μL 27 50 27 27 Vol. DFO-ArN.sub.3 23 0 23 23 stock/μL Vol. trastuzumab 50 50 0 50 stock/μL Vol. 50 50 50 50 [.sup.89Zr]Zr(C.sub.2O.sub.4).sub.4].sup.4− stock/μL Total volume/μL 150 150 150 150 Activity ~4.2 MBq ~4.2 MBq ~4.2 MBq ~4.2 MBq Irradiation λ/nm 365 365 365 395 Irradiation time/ 10 10 10 10 min.
REFERENCES
[0367] [1] N. Srinivas, P. Jetter, B. J. Ueberbacher, M. Werneburg, K. Zerbe, J. Steinmann, B. Van Der Meijden, F. Bernardini, A. Lederer, R. L. A. Dias, et al., Science (80-.). 2010, 327, 1010-1013.
[0368] [2] P. Zanzonico, J. Nucl. Med. 2008, 49, 1114-1131.
[0369] [3] T. Lindmo, E. Boven, F. Cuttitta, J. Fedorko, P. A. Bunn, J. Immunol. Methods 1984, 72, 77-89.
[0370] [4] Institute for Laboratory Animal Research, Guide for the Care and Use of Laboratory Animals: 8th Ed., 2011.
[0371] [5] R. Fridman, G. Benton, I. Aranoutova, H. K. Kleinman, R. D. Bonfil, Nat. Protoc. 2012, 7, 1138-1144.
[0372] [6] B. Bai, M. Dahlbom, R. Park, L. Hughes, G. Dagliyan, L. P. Yap, P. S. Conti, IEEE Nucl. Sci. Symp. Conf. Rec. 2012, bai, 3765-3768.
[0373] [31] M. J. W. D. Vosjan, L. R. Perk, G. W. M. Visser, M. Budde, P. Jurek, G. E. Kiefer, G. A. M. S. van Dongen, Nat. Protoc. 2010, 5, 739-743.
[0374] [33] J. P. Holland, V. Divilov, N. H. Bander, P. M. Smith-Jones, S. M. Larson, J. S. Lewis, J. Nucl. Med. 2010, 51, 1293-1300.
[0375] [34] J. P. Holland, E. Caldas-Lopes, V. Divilov, V. a Longo, T. Taldone, D. Zatorska, G. Chiosis, J. S. Lewis, PLoS One 2010, 5, e8859.
[0376] [35] J. P. Holland, M. J. Evans, S. L. Rice, J. Wongvipat, C. L. Sawyers, J. S. Lewis, Nat. Med. 2012, 18, 1586-1591.
[0377] [36] S. N. Rylova, L. Del Pozzo, C. Klingeberg, R. Tonnesmann, A. L. Illert, P. T. Meyer, H. R. Maecke, J. P. Holland, J Nucl Med 2016, 57, 96-102.