AAZTA5-squaramide ester: promising tool for 177Lu-labeling of monoclonal antibodies under mild conditions


 Background

Combining the advantages of both cyclic and acyclic chelator systems, AAZTA (1,4-bis(carboxymethyl)‐6‐[bis(carboxymethyl)]amino‐6‐methylperhydro‐1,4‐diazepine) is well suited for complexation of various diagnostic and therapeutic radiometals such as gallium-68, scandium-44 and lutetium-177 under mild conditions. Due to its specificity for primary amines and pH dependent binding properties, squaric acid (SA) represents an excellent tool for selective coupling of the appropriate chelator to different target vectors. Therefore, the aim of this study was to evaluate radiolabeling properties of the novel bifunctional AAZTA5-SA being coupled to a model antibody (bevacizumab) in comparison to DOTA-SA using the therapeutic nuclide lutetium-177.
Results

As proof-of-concept, bevacizumab was first functionalized with either AAZTA5-SA or DOTA-SA. After purification via fractionated size exclusion chromatography (SEC), the corresponding immunoconjugates were subsequently radiolabeled with lutetium-177 at pH 7 and room temperature (RT) as well as 37 °C. After 90 min, labeling of AAZTA5-SA-mAb resulted in almost quantitative radiochemical yields (RCY) of > 98% and > 99%, respectively. After purification via SEC, the radioconjugate [177Lu]Lu-AAZTA5-SA-mAb could be obtained with a purity of > 99% and an apparent specific activity of 4.5 GBq/µmol. In contrast, 177Lu-labeling of DOTA-SA-mAb showed negligible radiochemical yields of < 2% both at room temperature and 37 °C. In vitro complex stability measurements of [177Lu]Lu-AAZTA5-SA-mAb in human serum at 37 °C indicated > 99% protein bound activity within 15 days. In phosphate buffered saline (PBS), a slightly decreased stability of > 93% intact conjugate was observed over the same period.
Conclusion

Coupling of AAZTA5-SA to the monoclonal antibody bevacizumab allowed for 177Lu-labeling with almost quantitative radiochemical yields both at room temperature and 37 °C. Within 15 days, the resulting radioconjugate indicated very high in vitro complex stability both in human serum and PBS. Therefore, AAZTA5-SA is a promising tool for 177Lu-labeling of sensitive biomolecules such as antibodies for theranostic applications.


Background
The utilization of monoclonal antibodies (mAbs) or corresponding smaller protein fragments for immunotherapeutic strategies targeting cancer has gained increasing clinical importance and interest in recent years. Since the admission of the rst antibody Muromonab-CD3 (Orthoclone OKT3) in 1986, more than 90 therapeutic monoclonal antibodies or antibody-based drugs have been approved by the Food and Drug Administration (FDA) or the European Medicines Agency (EMA), about 55 of them in the last ve years and 32 of them aiming for treatment of cancer (Abramowicz et al. 1989 In addition to the native therapeutic anti-cancer effects of unmodi ed mAbs, their highly selective binding can also be used to transport either diagnostically or therapeutically relevant radionuclides to a speci c target. An essential factor in the selection of a suitable radionuclide is its half-life, which should match to the typically slow pharmacokinetics of intact antibodies. Although the utilization of such radioimmunoconjugates for diagnosis and treatment of cancer and other diseases represents a promising strategy, to date there is only a small number of actually clinically approved agents including 99m Tc-or 111 In-labeled antibodies like Scintimun®, LeukoScan®, CEA-scan®, ProstaScint®, Verluma® and OncoScint® for SPECT imaging as well as the 131 I-or 90 Y-labeled conjugates Bexxar® and Zevalin® for radioimmunotherapy (RIT) (Bohdiewicz 1998 There are several therapeutic radionuclides that are used in combination with antibodies or being subject of recent investigations aiming for radioimmunotherapeutic applications including the β − -emitters 67 Cu, 90 Y, 131 I and 177 Lu and α-emitters 213  Since it could be shown that smaller tumors and metastases are more accessible for radiolabeled antibodies than large ones and thus show a better response to this treatment, RIT is a promising tool especially for therapy of cancer even before tumors or lesions become detectable via imaging methods or in advanced stages of metastatic diseases (Barbet et al. 2012; Barbet et al. 2009). In this case, β − -emitting radionuclides with comparatively low β − -energy are the best choice to minimize the damage to surrounding healthy tissue while destroying malignant cells in the target. Due to the fact that it can be directly covalently bound to the protein by iodination of tyrosine residues and due to its dual emission (maximum β − -energy of 606 keV and primary γ-energy of 364 keV), iodine-131 is a candidate of great interest for both RIT and SPECT-imaging (Boros and Holland 2018; Kawashima 2014; Yeong et al. 2014). However, it could be shown, that the covalent bond between tyrosine residues and iodine provides insu cient stability leading to certain release of free 131 I or other proteolytic products even in the case of internalizing antigens (Repetto-Llamazares et al. 2014; Stein et al. 2003). Another frequently used radionuclide for RIT is 90 Y, which decays exclusively (100%) via β −emission. With a maximum electron energy of 2.288 MeV this radiometal provides a comparatively long effective range and therefore the opportunity to penetrate larger solid tumors (Boros and Holland 2018).
Compared to these isotopes, 177 Lu has several advantages regarding radioimmunotherapeutic applications for treatment of more accessible smaller tumors. In contrast to 90 Y, 177 Lu offers signi cantly lower beta-particle energies (E β,max = 498 keV) and compared to 131 I also the absence of high-energy gamma photons (iodine-131: E γ,max = 723 keV; lutetium-177: E γ,max = 208 keV). Furthermore, compared to 90 Y (t 1/2 = 64.1 h), the physical half-life of 177 Lu (t 1/2 = 6.7 d) matches better the slow pharmacokinetics of full-size antibodies and is shorter than that of 131 I (t 1/2 = 8.0 d) (Barbet et al. 2012;Dash et al. 2015).
Lutetium-177 therefore provides excellent properties both for RIT and immuno-SPECT-imaging of small solid tumors or metastatic lesions even at an early stage of the disease. A number of 177 Lu-labeled radiopharmaceuticals such as the somatostatin analogues 177 Lu-DOTATOC/DOTATATE have already demonstrated the great potential of 177 Lu- 177 Lu are typical candidates for mAb-based applications.
In recent years, several chelator scaffolds have been evaluated for the complexation of 177 Lu and 90 Y including linear systems like DTPA (Diethylenetriaminepentaacetic acid) and CHX-A"-DTPA (Cyclohexyldiethylenetriaminepentaacetic acid), as well as cyclic systems like DOTA (1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid), TETA (1,4,8,4,8,tetraacetic acid) and corresponding bifunctional derivatives (e.g. DOTAGA 1,4,7,10-Tetraazacyclododececane-1-glutaric acid-4,7,10-triacetic acid), respectively ( Stimmel et al. 1995). In most cases, DOTA complexes with trivalent radiometals are characterized by very high stability, even after long retention times in vivo. However, due to its macrocyclic structure, DOTA functionalized molecules typically require high temperatures up to 95 °C for fast and successful radiolabeling with high radiochemical yields. Milder conditions usually lead to signi cantly reduced yields or to the necessity of much longer reaction times (up to hours) ( 177 Lu up to 6-8 GBq per dose (Demirci et al. 2018;Forrer et al. 2005). To prevent or at least to reduce radiolytic degradation of important binding regions of an antibody at such high activity levels, the synthesis duration and the time between production and administration of the radiopharmaceutical needs to be minimized (Garrison 1987). Despite its advantages in kinetic and thermodynamic stability, DOTA therefore represents a rather inappropriate choice for radiolabeling of temperature-sensitive biomolecules such as antibodies. As a result, preclinical and clinical applications of 177 Lu- Wojdowska et al. 2015), there is a great demand for suitable bifunctional chelator molecules, which ensure both rapid and complete radiolabeling at low temperatures as well as high complex stability. In this study we wanted to evaluate the applicability of AAZTA 5 for radiolabeling of antibodies with 177 Lu regarding radioimmunotherapy compared to DOTA as the gold standard for complexation of this radiometal. For this purpose, we rst synthesized AAZTA 5 as recently reported by our group and extended the basic scaffold with an ethylenediamine unit (Sinnes et al. 2019). In the next step, we introduced a squaric acid diethyl ester (SA(OEt) 2 ) as linker entity via selective formation of a vinylogous monoamide ( Fig. 1). SA(OEt) 2 provides various excellent properties for coupling of an appropriate chelator to different target vectors. On the one hand, it reacts selectively with primary amines which often avoids the necessity of protective group strategies and therefore simpli es the synthesis of the tracer or corresponding precursor. On the other hand, it offers the possibility of a stepwise asymmetric and pH dependent amidation. In a rst step, squaric acid diethyl ester can be coupled to the amine of a chelator moiety at pH 7 via monoamide formation and subsequently be isolated and stored. In a second step the resulting precursor can then be coupled at pH 9 to the primary amine of appropriate target vectors such as antibodies. This unique property is based on a change of aromaticity during the rst reaction step (Tietze et al. 1991;Wurm and Klok 2013). Despite its advantages, the utilization of squaric acid as linker entity in radiopharmaceutical chemistry is still mostly uncommon and innovative. 2016, Rudd et al. introduced a deferoxamine squaramide ester for radiolabeling of mAbs with 89 Zr for Immuno-PET imaging. They were also able to show the effect of increasing 89 Zr-deferoxamine-complex stability due to the two additional oxygen atoms of the squaramide moiety leading to an octadentate coordination geometry (Rudd et al.

2016).
As proof of concept we used the commercially available monoclonal antibody bevacizumab (trade name Avastin®) which is applied for treatment of various types of cancer inhibiting the vascular endothelial growth factor A (VEGF-A). Yet it was not our intention to refer to the pharmacology of bevacizumab, in our experiments it simply served as biomolecule with mAb pro le. First, the protein was functionalized with AAZTA 5 -SA. Following optimized puri cation, the resulting conjugate was subsequently radiolabeled with 177 Lu under mild conditions. For comparison, we analogously synthesized, puri ed and radiolabeled DOTA-SA-bevacizumab. Prior to evaluation of the corresponding immunoconjugates, the unconjugated bifunctional chelators DOTA-SA and AAZTA 5 -SA were also radiolabeled and compared under the same conditions.

General
All chemicals were purchased from Sigma-Aldrich, Merck, VWR, TCI, Acros Organics, Fluka, AlfaAesar, Fisher Scienti c and Chematech and used without further puri cation unless otherwise declared. For radiolabeling reactions trace metal-free substances were used. Column chromatography was performed using silica gel 60 (0.063-0.200 mm, Acros Organics) as stationary phase and the respectively speci ed solvents as mobile phase. NMR measurements were performed using a Bruker Avance III HD 400 (400 MHz) or Avance III 600 (600 MHz). Mass spectrometry was measured via Agilent Technologies 1220 In nity LC system coupled to an Agilent Technologies 6130 Single Quadrupole LC/MS system. HPLC puri cation and analysis was performed using a Merck LaChrom system with Hitachi L7100 pump and L7400 UV-detector and the respectively mentioned column and conditions. Puri cation of the immunoconjugates was performed via fractionated SEC using PD-10 Desalting Columns (8.3 mL Sephadex™ G-25, GE Healthcare) and PBS as mobile phase. For radiolabeling experiments n.c.a. Radiochemical yields were determined via radio thin layer chromatography (radio-TLC, stationary phase: Merck Silica 60 F 254 TLC plates; mobile phase: 0.1 M citrate-buffer pH 4), image plate scanner (CR35-Bio, Elysia Raytest) and AIDA Image Analysis software (Elysia Raytest). Radiochemical purity was measured via radio-TLC and radio-SEC-HPLC monitoring (column: Phenomenex BioSep SEC-S 2000, mobile phase: 0.05 M sodium phosphate pH 7, ow: 1 mL/min).
To a mixture of 1 (1.05 g, 2.39 mmol) and Pd(OH) 2 /C (0.62 g, 10 wt%) in abs. ethanol (20 mL) acetic acid (411 µL, 7.19 mmol) was added and the resulting solution was stirred at room temperature overnight under an atmosphere of hydrogen. After completion of the reaction, the mixture was ltered over Celite® and the ltrate was evaporated under reduced pressure. The crude product 2 was used for the following reaction without further puri cation.
Determination of the chelator-to-antibody ratio (CAR) of AAZTA 5 -SA-bevacizumab . The resulting mixture (1 mL) was shaken for 90 min at room temperature and 550 rpm via thermomixer. After completion of the reaction the percentage of protein-bound activity was determined via radio-TLC. The chelator-to-antibody ratio was calculated via: Synthesis and puri cation of DOTA-SA-bevacizumab 120 µL of the bevacizumab solution (3.0 mg mAb, 20.1 nmol, 25 mg/mL, Avastin®, Roche) was diluted with 0.5 M Na 2 HPO 4 -buffer (pH = 9, 1 mL). A tenfold molar excess of DOTA-SA solution (115 µL, 201.3 nmol, 1 mg/mL) was added and the mixture was shaken overnight at room temperature via thermomixer. The resulting immunoconjugate was subsequently puri ed via fractionated SEC using a PD-10 Desalting Column (8.3 mL Sephadex™ G-25, GE Healthcare) and PBS as mobile phase. In detail, the column was rst preconditioned with 20 mL PBS. The reaction mixture was then completely applied to the column and the ow-through was collected in the rst fraction. Subsequently, a further 9 fractions with 0.5 mL PBS each were collected. In the rst approach, the protein containing fractions 6-8 were combined and homogenized before subsequent radiolabeling. In a further approach, fraction 6 was used separately for subsequent radiolabeling and fraction 7 was further puri ed via second SEC using the same procedure. The sixth and seventh fraction of the extended puri cation step were combined and homogenized before radiolabeling.

Determination of in vitro complex stability of [ 177 Lu]Lu-AAZTA 5 -SA-bevacizumab
The puri ed radioimmunoconjugate [ 177 Lu]Lu-AAZTA 5 -SA-bevacizumab (in 220 µL PBS) was added to either 500 µL human serum or 500 µL PBS and the resulting mixtures were shaken via thermomixer at 37 °C and 550 rpm for 15 d. The proportion of the intact conjugate versus released radionuclide was determined via radio-TLC at various times.

Organic Synthesis
The tert-butyl-protected derivative of AAZTA 5 (4, Fig. class="InternalRef">2) could be successfully synthesized with an overall yield of 32% within four steps according to the work recently published by our group (Sinnes et al. 2019). The initial step rst includes an in situ ring-opening reaction of 2nitrocyclohexane followed by formation of the diazepane scaffold via double Nitro-Mannich reaction of the resulting nucleophilic compound with N,N'-dibenzylethylenediamine and paraformaldehyde. In the next step the endocyclic amines were deprotected by hydrogenolysis and the nitro group was simultaneously reduced to an exocyclic amine. Without further puri cation, intermediate 2 was then directly processed with tert-butyl bromoacetate to yield the tetra alkylated product 3. In order to obtain a free linkable carboxylic acid group, the corresponding methyl ester was nally cleaved with lithium hydroxide leading to the desired AAZTA 5 ( t Bu) 4 (4). For later introduction of a squaramide as the actual linker structure, it was necessary to rst implement a terminal amine. For this purpose, compound 5 was synthesized via amide coupling of N-Boc-ethylenediamine to the carboxylic acid functionality of product 4.
Afterwards, all protective groups were deprotected with tri uoroacetic acid and nally squaric acid diethyl ester was coupled to the primary amine at pH 7 to yield the nal product AAZTA 5 -SA (6, Fig. 3). A severe pH control is required to prevent double amidation of the squaric acid, which occurs at more basic conditions. Furthermore, for comparison of the two chelator systems, squaric acid diethyl ester was coupled analogously to previously deprotected commercially available DOTA( t Bu) 3 -ethylenediamine. Both bifunctional chelator systems 6 and 7 were puri ed via HPLC.
Radiolabeling Of AAAZTA 5 -SA And DOTA-SA With 177 Lu Prior to their conjugation to an antibody, AAZTA 5 -SA and DOTA-SA were evaluated for their radiolabeling properties with lutetium-177 as stand-alone bifunctional chelators. For this purpose, 10 nmol of the corresponding compound were incubated with [ 177 Lu]LuCl 3 in HEPES-buffer at pH 7 and either room temperature or 37 °C. The respective radiochemical yield was analyzed at different times over a period of 30 min via radio-TLC. DOTA-SA showed a negligibly low radiolabeling e ciency at room temperature (0.8 ± 0.1% RCY) and even at 37 °C only a slightly increased radiochemical yield could be obtained after 30 minutes (6.9 ± 1.9% RCY). In contrast, a signi cantly higher radiochemical yield could be observed for AAZTA 5 -SA at room temperature as well as at 37 °C after just 1 min. At room temperature, a value of 68.1 ± 1.0% was achieved after 5 min, which did not change considerably in the further process of the reaction (69.6 ± 1.4% after 30 min). At 37 °C this maximum (69.6 ± 0.6%) could already be reached after 3 min. As shown in Fig. 4A, analysis of the radio-TLC indicated the presence of another not further investigated radiolabeled species with a R f value of 0.4 besides the desired [ 177 Lu]Lu-AAZTA 5 -SA at R f = 0.1. It may be assumed that this second species represents either a different protonation stage of the complex or a partially change in conformation or coordination geometry due to the in uence of the squaramide linker.
Via LC/MS analysis of the bifunctional compound, it was veri ed that no initial impurity could be responsible for the formation of a second radiolabeled product. Since the ratio of these two species did not change signi cantly during the reaction, this led to a maximum result of approximately 70% instead of quantitative radiochemical yields, despite the fact that no unbound radiometal could be observed.
For further analysis, radiolabeling of DOTA-SA was also performed using higher temperatures as well as slightly acidic pH (5.5). As shown in Fig. 5, at pH 7 (0.5 M HEPES) conditions up to 95 °C were necessary to produce acceptable radiochemical yields. Similar to the radiolabeling results of AAZTA 5 -SA, a second radiolabeled species could be observed with a proportion of approx. 20% after 60 min at 95 °C. Since both molecules have a squaramide ester as structural commonality, this might also be an indication for the in uence of the squaric acid moiety on the formation of a further species. Utilization of 1 M ammonium acetate buffer (pH 5.5) as reaction medium led to signi cantly faster complexation kinetics and higher radiochemical yields at 95 °C. Already after 30 min almost no unbound radiometal could be observed. However, these conditions evidently also facilitated the formation of the second radiolabeled species, resulting in a proportion of approx. 45% after 60 min (Fig. S1).

Antibody Coupling And Radiolabeling Of The Resulting Immunoconjugates
To evaluate its applicability as a bifunctional chelator system for radiolabeling of temperature and pH sensitive biomolecules such as antibodies with the therapeutic nuclide 177 Lu, AAZTA 5 -SA was coupled to bevacizumab as model antibody (Fig. 6). For this purpose, the pH dependent second amidation of the squaric acid linker was used to form a stable bond between the remaining ethyl ester and lysine side chains of the protein. A tenfold molar excess of AAZTA 5 -SA resulted in a ratio of 0.29 ± 0.04 bound chelator moieties per antibody. This value is su ciently low to assume the a nity of the antibody not to be affected by the modi cation. To remove excess unbound chelator molecules from the reaction mixture, the resulting immunoconjugate was puri ed by fractionated size exclusion chromatography using a gel ltration column and PBS as eluent. Fractions of 0.5 mL each were collected and then the protein-containing fractions 6 to 8 were combined and used for subsequent 177 Lu-labeling. Similarly, DOTA-SA was also coupled to bevacizumab and puri ed under the same conditions. Analogously to the unbound chelator systems, the resulting mAb-conjugates were subsequently incubated with [ 177 Lu]LuCl 3 in HEPES buffer at pH 7 both at room temperature and at 37 °C.
In the rst experiment, 177 Lu-labeling of the AAZTA 5 -functionalized antibody conjugate resulted in a radiochemical yield of 63.8 ± 4.3% at room temperature and 79.3 ± 3.6% at 37 °C, respectively. These values were already obtained at the rst measured time point after 15 min and only a small increase could be observed in the further progress of the reaction (64.4 ± 4.2% and 79.8 ± 3.3% after 60 min). In comparison, 177 Lu-labeling of the DOTA-functionalized analogue indicated a very low radiochemical yield of 2.6 ± 0.1% at room temperature after 60 min. At 37 °C, however, the yield increased to 19.5 ± 1.0% after the same duration. As expected, this value is signi cantly lower than that of [ 177 Lu]Lu-AAZTA 5 -SAbevacizumab, but nevertheless it exceeds the result of unbound DOTA-SA by a factor of almost three.
Analysis of the radio-TLC results of the AAZTA 5 -functionalized conjugate indicated the presence of a radiolabeled sideproduct at R f = 0.1 and at least one other impurity at R f = 0.4 besides the desired radioimmunoconjugate at R f = 0 (Fig. 7B). These species led to a decreased maximum radiochemical yield despite the absence of unbound 177 Lu but could be removed via subsequent SEC-puri cation providing a radiochemical purity of > 99% and an apparent speci c activity of 4.5 GBq/µmol.
Comparison of the obtained radio-TLC results of the AAZTA 5 -functionalized antibody with those of the unbound chelator led to the assumption that the previous SEC puri cation was insu cient (Fig. 7B). The For a more profound analysis, coupling of AAZTA 5 -SA to bevacizumab was repeated in a second approach. Instead of combining the protein-containing fractions 6 to 8 after SEC-puri cation, this time fractions 5 to 10 were used separately for subsequent 177 Lu-labeling. In this case, identical labeling conditions as previously described were chosen. As shown in Fig. 8, radiolabeling of fraction 5 resulted almost exclusively in free uncomplexed radiometal (R f = 0.8-1.0) indicating the absence of the AAZTA 5conjugated antibody as expected. Fraction 6, in contrast, showed a decreasing amount of free 177 Lu and the desired radiolabeled product (R f = 0) with a progressively increasing radiochemical yield. 177 Lulabeling of fraction 7 indicated the AAZTA 5 -functionalized protein being the main component with an additional impurity of non-separated AAZTA 5 -SA (approx. 28%). Furthermore, a small amount of the second species at R f = 0.4 was also detectable, con rming the presence of excess unbound chelator. Fraction 8 already consisted primarily of AAZTA 5 -SA and only a small amount represented the corresponding immunoconjugate. Finally, labeling of fractions 9 and 10 con rmed the exclusive presence of the unbound bifunctional chelator moiety with increasing elution volume.
Separate radiolabeling of individual fractions after SEC-puri cation of AAZTA 5 -SA-bevacizumab could therefore con rm the assumption that the excess unbound chelator was not completely separated in the rst approach. It could be shown that a certain breakthrough of AAZTA 5 -SA leads to contamination of the antibody-containing fractions even at lower elution volumes than expected.
In order to optimize the purity of the protein solution used for labeling, a further approach for coupling of the bifunctional chelator system to the model antibody bevacizumab and puri cation was performed.
Instead of combining the protein-containing fractions 6-8, this time only fraction 6, which provides the highest purity of modi ed antibody, was used for subsequent 177 Lu-labeling at room temperature as well as at 37 °C. Fraction 7, which consists mainly of the required immunoconjugate with a certain impurity of excess unbound chelator (Fig. 8), was further puri ed via second SEC before radiolabeling. For comparison, antibody functionalization and this extended puri cation was performed both with AAZTA 5 -SA and DOTA-SA. 177 Lu-labeling conditions were chosen analogous to the previously described experiments.
Radiolabeling of the 6th fraction after SEC-puri cation of AAZTA 5 -SA-mAb (175-457 µg protein) resulted after 90 min in almost quantitative radiochemical yields of 99.2 ± 0.3% at room temperature and 99.7 ± 0.1% at 37 °C, respectively. Already after 10 min at 37 °C a radiochemical yield of 92.0 ± 1.5% could be obtained. As expected, slightly slower reaction kinetics was observed at room temperature (86.7 ± 2.3% after 15 min). However, these results indicate again a very fast complexation of 177 Lu at mild temperatures and a neutral pH value by the AAZTA 5 -SA moiety. The twice-puri ed fraction 7 (199-419 µg protein after second SEC) showed also comparable outcomes of 97.30 ± 1.8% at room temperature and 97.90 ± 0.1% after 90 min at 37 °C, respectively using analogous labeling conditions.
In comparison, 177 Lu-labeling of both fraction 6 (278-382 µg protein) and the twice-puri ed fraction 7 (272-273 µg protein after second SEC) of the DOTA-functionalized immunoconjugate provided only negligible complexation rates at room temperature and 37 °C, con rming the unique advantages of AAZTA 5 -SA. Here, the obtained results (< 2% RCY at 37 °C) differ considerably from the rst radiolabeling approach of the DOTA-functionalized antibody (19.5 ± 1.0%). This can be explained by different concentrations of the modi ed protein in the individual experiments. While in the rst labeling approach 900 µg of the radioimmunoconjugate were used in each case, the optimized puri cation and separation of the fractions in the last approach led to a lower amount per labeling experiment (272382 µg) at constant volume.

In vitro complex stability of [ 177 Lu]Lu-AAZTA 5 -SA-bevacizumab
The protein-bound 177 Lu-AAZTA 5 -SA-complex indicated no measurable release of the radiometal in human serum (> 99% intact conjugate) over a period of at least 15 days. In PBS the complex remained stable within the rst 48 h (> 99%) and only a slightly decreased stability could be observed during the following 13 d resulting in 93.9 ± 0.9% intact conjugate (Fig. 10).

Conclusion
In the present study a novel AAZTA 5 squaramide ester was synthesized, evaluated and compared to the DOTA-functionalized analogue regarding its applicability as bifunctional chelator for radiolabeling of sensitive biomolecules such as antibodies with the theranostic radiometal lutetium-177 under mild conditions. Prior to evaluation of the corresponding immunoconjugates, the HPLC-puri ed bifunctional chelators were radiolabeled with 177 Lu as stand-alone systems at neutral pH and both at room temperature and 37 °C. While labeling of DOTA-SA resulted in negligibly low yields (6.9 ± 1.9% RCY after 30 min) even at slightly elevated temperatures (37 °C), [ 177 Lu]Lu-AAZTA 5 -SA achieved almost 70% RCY already after 3 min at room temperature.
Coupling of AAZTA 5 -SA to the model antibody bevacizumab resulted in a ratio of 0.29 ± 0.04 bound chelator moieties per protein. Puri cation and 177 Lu-labeling of the resulting immunoconjugate was evaluated and optimized within three approaches. In the rst approach, radiolabeling of the combined protein-containing volumes resulting from fractionated SEC already produced a radiochemical yield of 63.8 ± 4.3% at room temperature and 79.3 ± 3.6% at 37 °C after a short reaction time (15 min). The DOTA analogue, as expected, showed a signi cantly lower complexation rate resulting in 2.6 ± 0.1% RCY at RT and 19.5 ± 1.0% RCY at 37 °C after 60 min. Analysis of the radio-TLC results indicated the presence of a certain residue of unbound AAZTA 5 -SA leading to a radiolabeled sideproduct and therefore to reduced radiochemical yields of [ 177 Lu]Lu-AAZTA 5 -SA-mAb.
In a second approach the individual fractions after SEC-puri cation of AAZTA 5 -SA-mAb were radiolabeled with 177 Lu separately in order to determine the breakthrough of excess unbound AAZTA 5 -SA. It could be con rmed that with increasing elution volume a certain amount of excess unbound chelator is already present in the protein-containing fractions.
Thus, in a further approach, SEC-puri cation of the AAZTA 5 -modi ed protein was optimized via separate use of the fraction containing exclusively the corresponding immunoconjugate and via repeated SEC of the fraction containing a certain amount of excess unbound chelator. Subsequent 177 Lu-labeling successfully con rmed the separation of AAZTA 5 -SA via signi cantly improved radiochemical yields and purity of [ 177 Lu]Lu-AAZTA 5 -SA-mAb already after short reaction times of 10-15 min (> 90% at 37 °C and > 85% at room temperature, respectively). After approximately 30 min almost quantitative yields could be achieved for both temperatures. In contrast, the analogously puri ed DOTA-conjugate provided only negligibly low complexation rates both at room temperature and 37 °C underlining its disadvantages over the AAZTA 5 system.
While these labeling studies successfully veri ed the potency of the AAZTA 5 -SA moiety for fast and almost quantitative 177 Lu-coordination, the stability of the protein bound complex was investigated as second aspect. Indeed, [ 177 Lu]Lu-AAZTA 5 -SA-mAb appeared to be very stable both in human serum and PBS at 37 °C over a long period of more than two weeks (> 99% and > 93%, respectively).
The results of this study show the high potential of the novel AAZTA 5 squaramide ester as bifunctional chelator system for mild radiolabeling of sensitive target vectors such as antibodies with 177 Lu. It therefore represents a promising tool for formation of radioimmunoconjugates that can be used for diagnostic and therapeutic applications as well as for simultaneous imaging of the therapeutic progress.
Furthermore, we expect these advantages to be also transferable to applications with various other target vectors, e.g. those based on small molecules, peptides or polymeric structures.