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Optimization of the Synthesis of Ac-225-labelled DOTA-Radioimmunoconjugates for Targeted Alpha Therapy, based on Investigations on the Complexation of Trivalent Actinides by DOTA

Kannengießer, Stefanie

German Title: Optimierung der Synthese von Ac-225-markierten DOTA-Radioimmunkonjugaten für die zielgerichtete Alpha-Therapie, basierend auf Untersuchungen zur Komplexierung dreiwertiger Actiniden durch DOTA

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The successful application of alpha-emitters in targeted alpha therapy (TAT) goes together with developments in radionuclide production and labelling chemistry. Especially profound understanding of the coordination chemistry of the respective metal ion-ligand system is of major importance to develop protocols for the synthesis of radioimmunoconjugates and to predict the fate of radionuclides in vivo.

Radioconjugates of the therapeutic alpha-emitter Ac-225 with polyamino-polycarboxyl ligands as chelating agents are being actively studied [1, 2, 3, 4]. In particular the macrocyclic ligand 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) has shown promise due to the high kinetic- and thermodynamic stability (log K > 20) of its complexes with trivalent metal cations [5]. The scope of the presented work was the experimental characterisation and evaluation of DOTA as a suitable chelator for trivalent actinides (An(III)), particularly in terms of stable binding of the long-lived alpha-emitting radionuclide Ac-225 to biomolecules for safe application in radioimmunotherapy (RIT). The project included not only basic studies to contribute to a better understanding of the complexation mechanisms and kinetics, but, based on these findings, was aimed at the development of a robust labelling protocol for facile and effective synthesis of an Ac-225-DOTA-MabThera® conjugate. In the course of the investigations, focus was set on in vitro testing of the obtained radioimmunoconjugate, in particular the evaluation of the kinetic stability in presence of competing agents as well as in human blood serum. Assessment of the antigen binding affinity of the antibody conjugate completed the work.

The protocol for the design and synthesis of Ac-225 radiopharmaceuticals of McDevitt et al. provided the starting point for this study [6]. This research group developed a synthetic scheme to radiolabel DOTA-proteins with Ac-225. Later, the protocol was applied to antibodies in form of a two-step synthesis, with the first step being the Ac-225 DOTA-Bn-NCS complexation, followed by the coupling of the Ac-225-DOTA-Bn-NCS to the mAb. The idea behind this two-step approach was that, when the complexation of Ac(III) with DOTA is conducted at elevated temperatures and basic pH (first step), presumably a more stable complex is formed. However, since higher temperature / pH are known to have a negative effect on the antibody efficiency, the conjugation to the biomolecule can hence only be conducted at lower temperatures in a second step. This two-step synthesis though suffers from yields below 10 %, which is assumed to be due to the competing hydrolysis reaction of the isothiocyanate moiety occurring at the pH used during the complexation step. This makes the labelling protocol an interesting subject for further studies on how this synthesis can be improved.

From the findings of McDevitt et al it was apparent that a number of variables needed to be investigated in order to improve on the low efficiency of the protocol. These studies were conducted previously and are summarised in the Diploma Thesis, S. Kannengieÿer, 2009 [7]. A one-step synthesis protocol was tested and the labelling yields for Ac-225 were found to be dependent on temperature and especially on the pH of the reaction mixture. Eventually, an optimised protocol for radiolabelling of DOTA-peptides and MabThera® with Ac-225 activities up to 2 µCi (= 74 kBq) per 100 µg mAb was established, offering labelling yields > 95 %. The present work now aimed on translation of the developed synthesis protocol to higher specific activities of clinical relevance (>10 µCi (>370 kBq) per 100 µg mAb).

To gain profound knowledge about the complexation reaction mechanism and the thermodynamic and kinetic properties of the An(III)-DOTA system, the coordination chemistry was initially studied by means of time resolved laser fluorescence spectroscopy (TRLFS). Since Ac(III) has no suitable spectroscopic properties, the metal ion complexation by DOTA was investigated with Cm(III) as substitute for the trivalent actinides. A comparable study on [Eu(III)DOTA]- has been reported before [8, 9, 10]. Besides determination of the kinetic rate constants and thermodynamic parameters (log K, deltaG, deltaH, deltaS) at labelling-relevant temperatures up to 90 °C, attention was also paid to the detection of possible intermediate species which are frequently discussed in the literature [11].

TRLFS is a powerful speciation method which makes use of the excellent fluorescence properties of Eu(III) and Cm(III), both regarded as good representatives for trivalent lanthanides and actinides. With TRLFS it is possible to detect and characterise complex species in sub-micromolar concentrations without influencing the chemical equilibrium of the system. An experimental setup was chosen which allows for adjusting the concentration of the reactive DOTA species by variation of the pH of the reaction. Due to the slow kinetics of the complexation reaction at room temperature, experiments at 45 to 90 °C were conducted to identify the complex species and quantify their relative ratios by means of peak deconvolution. Based on the potentiometrically determined pKa,n values of HxDOTA(4-x)- for the respective conditions, from these ratios the conditional complex stability constants log K of [Cm(III)DOTA]- were calculated (I = 0.1, 45 to 90 °C). Application of the van't Hoff law allowed for extrapolation of the log K at 25 °C to be 22.0±0.4. The parameters deltaG, deltaH and deltaS obtained from the Gibbs Helmholtz relation indicate that the reaction is exergonic, endothermic and driven by the change of entropy.

Identification and further characterisation of the involved complex species was done by comparison of the fluorescence lifetimes, which give information about the first coordination sphere of the metal cation. Furthermore, additional investigations with NMR were executed to identify and understand the mechanism related to the complex formation with DOTA-Bn-NCS. Based on the results of the NMR study, the complexation kinetics of DOTA and DOTA-Bn-NCS were further investigated and compared to gain insight into the involved reaction mechanisms.

Proper understanding and interpretation of the thermodynamic behaviour of the Cm(III)-DOTA complex formation allowed for facile translation of the experimental settings to the Ac(III)-DOTA system. Since no spectroscopic methods are available for this system, Chelex cation exchange resin as well as Instant Thin Layer Chromatography (ITLC) were chosen as radiochemical speciation methods and were evaluated for their feasability. Determination and refinement of the stability constant log K of Ac(III)DOTA]- for the temperature range of 25 to 90 °C was done in analogy to the Cm(III)-DOTA system, resulting in a log K25°C = 19.5±0.4. To obtain reliable results, the protocol for the radiochemical separation of Ac-225 from Ra-225 required optimisation to ensure highest purity and quality of the radionuclide.

Based on these findings, the studies on the Ac-225-labelling of DOTA-Bn-NCSMabThera® for targeted alpha therapy of Non-Hodgkins-Lymphoma were subsequently continued. The previously established protocol was modified and further optimised in order to be applicable for facile clinical synthesis of radioimmunoconjugates with higher specific activities (SA) within 20 min. In this regard, the antibody labelling kinetics were reviewed respective reaction temperature and ideal pH of the high-yield radiolabelling, which permitted further improvement of the labelling effectiveness (pH 9, 37 - 42 °C, 5 - 15 min; ave. yields 94 - 96 %, > 98 % RCP after purification). The protocol was successfully evaluated for reliability with SA up to 50 µCi (= 1.85 MBq) per 100 µg mAb. The obtained radioconjugates were assessed for their kinetic stability in different buffers as well as under physiolocigal conditions in human blood serum and proved to be satisfyingly stable over up to 30 days ( > 85 % Ac-225 still bound).

Finally, a preliminary radiobiological study with cancer cells (K422 cell line, B-cell lymphoma) was conducted to determine the antigen binding afinity of the radiolabelled CD20-antibody (SA = 1 µCi (= 37 kBq)/100 µg mAb, Bmax= 8.88 nM, Kd = 52.55 nM). The results give rise to further preclinical in vitro studies. In summary, it was demonstrated that rapid, high-yield radiolabelling of DOTAchelated mAbs is possible under alkaline conditions at rather low temperatures. Under these conditions a thermodynamically and kinetically stable radioimmunoconjugates with specific activities suitable for application in clinical TAT studies is formed while the integrity of the antibody is preserved.

Item Type: Dissertation
Supervisor: Fanghänel, Prof. Dr. Thomas
Date of thesis defense: 28 June 2013
Date Deposited: 15 Jul 2013 12:33
Date: 2013
Faculties / Institutes: Fakultät für Chemie und Geowissenschaften > Institute of Physical Chemistry
Subjects: 500 Natural sciences and mathematics
540 Chemistry and allied sciences
600 Technology (Applied sciences)
Controlled Keywords: Actinium, Curium, DOTA, Radioimmunoconjugates, Targeted Alpha Therapy, Actinides, Complexation, Flourescence Spectroscopy
Uncontrolled Keywords: Ac-225, Cm-248, TRLFS
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