New Bifunctional Chelators Incorporating Dibromomaleimide Groups for Radiolabeling of Antibodies with Positron Emission Tomography Imaging Radioisotopes

Positron Emission Tomography (PET) imaging with antibody-based contrast agents frequently uses the radioisotopes [64Cu]Cu2+ and [89Zr]Zr4+. The macrobicyclic chelator commonly known as sarcophagine (sar) is ideal for labeling receptor-targeted biomolecules with [64Cu]Cu2+. The siderophore chelator, desferrioxamine-B (dfo), has been widely used to incorporate [89Zr]Zr4+ into antibodies. Here, we describe new bifunctional chelators of sar and dfo: these chelators have been functionalized with dibromomaleimides (dbm), that enable site-specific and highly stable attachment of molecular cargoes to reduced, solvent-accessible, interstrand native disulfide groups. The new sar–dbm and dfo–dbm derivatives can be easily conjugated with the IgG antibody trastuzumab via reaction with reduced interstrand disulfide groups to give site-specifically modified dithiomaleamic acid (dtm) conjugates, sar–dtm–trastuzumab and dfo–dtm–trastuzumab, in which interstrand disulfides are rebridged covalently with a small molecule linker. Both sar– and dfo–dtm–trastuzumab conjugates have been radiolabeled with [64Cu]Cu2+ and [89Zr]Zr4+, respectively, in near quantitative radiochemical yield (>99%). Serum stability studies, in vivo PET imaging, and biodistribution analyses using these radiolabeled immunoconjugates demonstrate that both [64Cu]Cu-sar–dtm–trastuzumab and [89Zr]Zr-dfo–dtm–trastuzumab possess high stability in biological milieu. Dibromomaleimide technology can be easily applied to enable stable, site-specific attachment of radiolabeled chelators, such as sar and dfo, to native interstrand disulfide regions of antibodies, enabling tracking of antibodies with PET imaging.

M onoclonal IgG antibody therapies have been transformative in the treatment of many cancers and autoimmune diseases, and new antibody therapies are likely to have further clinical impact. The ability to quantitatively image antibody biodistribution at the whole-body level can help predict individual patient response, as well as aid in understanding treatment outcomes during clinical development. 1,2 Positron-emitting radiometallic isotopes attached to antibodies via chelators have been widely used to image antibody biodistribution using Positron Emission Tomography (PET).
Conventional labeling strategies have attached chelators to antibodies using electrophilic groups such as isothiocyanates, N-hydroxysuccinimides, and anhydrides that react with solvent accessible primary amines of lysine side chains. 3−5 These bioconjugation methods are simple, but as these reactions are not site-specific and lack stoichiometric control, the presence of multiple lysine side chains leads to heterogeneous product mixtures. The resulting chelator−antibody conjugates often demonstrate reduced affinity for target receptors, particularly at high chelator:antibody conjugation ratios. 6−8 Several recent and elegant site-specific strategies include enzyme-mediated conjugation of chelators to (i) glycan regions of IgG antibodies using a combination of β-1,4-galactosidase/β-1,4galactosyltransferase(Y289L), 9−11 (ii) hinge region glutamine residues of IgG antibodies using a combination of deglycosylation with N-glycosidase followed by chelator coupling with bacterial transglutaminase, 12 and (iii) modified C-termini of antibody fragment derivatives using the bacterial enzyme sortase A. 13 Alternatively, incorporation of a nonnatural amino acid containing an azide group in an IgG antibody has enabled orthogonal and site-selective conjugation of chelators using click chemistry. 14 These approaches have resulted in radiolabeled chelator−protein conjugates that display improved PET imaging capabilities relative to radiolabeled conjugates prepared using conventional, non-sitespecific methods. However, these enzyme-based or protein engineering-based conjugation methods are more difficult to implement than conventional, non-site-specific conjugation technologies. Furthermore, modification or removal of glycan regions can modify the pharmacokinetics of antibodies, as well as affect their Fc-receptor binding properties and antibodydependent cell-mediated toxicity. 15−17 Maleimide derivatives have been widely used to incorporate radiolabeled chelators into IgG antibodies and other proteins. 18−21 Maleimides react selectively with reduced disulfides at near-neutral pH, and in the case of IgG antibodies, reduction of the four solvent-accessible, interstrand disulfide bonds leads to the presence of eight available thiol attachment points. While maleimides enable site-selective attachment of chelators and other cargoes to IgG antibodies, these conjugates are unstable: the resulting thioether can undergo a retro-Michael reaction in biological milieu, converting back to the starting thiol and maleimide. 21−24 In vivo, the starting maleimide motif, still tethered to its payload, can then react with endogenous molecules containing bioavailable thiols, such as glutathione and albumin. In radionuclide imaging, this can potentially result in accumulation of radioactivity at off-target sites, decreasing image contrast, sensitivity, and the ability to quantify antibody distribution.
New conjugation platforms including bissulfone, 25 divinylpyrimidine, 26 divinyltriazine, 27 dibromoalkyl oxetane, 28 dibromopyridazinedione, 29,30 and disubstituted maleimide 31−36 derivatives enable site-specific attachment of cargo to dithiol groups of antibodies, including those at IgG hinge regions. Here, we focus on dibromomaleimide: this motif reacts specifically with two reduced thiol groups of antibodies, thus enabling concomitant attachment of cargo and rebridging of two cysteines. The resulting dithiomaleimide can be hydro-lyzed to a dithiomaleamic acid under mildly basic conditions to give homogeneous antibody conjugates. 32−35 Importantly, the dithiomaleamic acid conjugates are unreactive toward serum thiols and do not undergo retro-Michael reactions in biological media, unlike conventional maleimide derivatives. This dibromomaleimide platform has the potential to enable highly stable site-specific radiolabeling of antibodies at hinge region disulfides.
Zirconium-89 (t 1/2 = 78 h, β + E max = 897 keV, 23%) and copper-64 (t 1/2 = 12.7 h, β + E max = 656 keV, 18%) have both been used for imaging antibody distribution with PET: the half-lives of these isotopes match the time required for antibodies to clear circulation and accumulate in target tissue (1 day−1 week  2+ (1) as a precursor, 39 as following derivatization, Mg 2+ can be easily removed from the chelator by acidification. [Mg((NH 2 ) 2 sar)] 2+ was reacted with 3,4-dibromomaleimide-N-hexanoic acid (2) and coupling agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) in acetonitrile (Scheme 1). The resulting product mixture was treated with acetic acid, prior to purification using reverse-phase HPLC, to yield sar−dbm (3) in 8% yield. Similarly, dfo (4) was reacted with dibromomaleimide-Nglycine (5) and EEDQ in dimethyl sulfoxide, followed by purification using reverse-phase HPLC to give dfo−dbm (6, Scheme 2) in 22% yield. The inclusion of a C 6 aliphatic linker group in sar−dbm aided reverse-phase HPLC purification and isolation of sar− dbm: the sar chelator motif is very hydrophilic and is poorly retained on reverse-phase columns in the absence of hydrophobic appendages. In contrast, the dfo chelator had sufficient adsorption on reverse-phase stationary phases, and so a C 2 linker group, which has demonstrated improved conjugation properties relative to a C 6 linker (vide infra), 34 was utilized.
The yield of sar−dbm (8%) was significantly lower than the yield of dfo−dbm (22%). We attribute this to the lower reactivity of the apical primary amine groups of (NH 2 ) 2 sar and its complexes relative to other primary amines, possibly due to steric encumbrance of the macrobicyclic hexaamine rings. Previously reported syntheses, in which (NH 2 ) 2 sar complexes have been reacted with electrophilic motifs, have required significant heating. 37−40 Here, the reaction was not heated, as the dibromomaleimide group is sensitive to heat. Although the yield of sar−dbm was low under these reaction conditions, it could be obtained in a one-pot synthesis from [Mg- The compound sar−dbm 2 , in which both apical primary amines of (NH 2 ) 2 sar react with 2 equiv of 3,4dibromomaleimide-N-hexanoic acid to yield a sarcophagine chelator containing two dibromomaleimide groups, was also observed. Although we have detected and even isolated small amounts of sar−dbm 2 and its Mg 2+ complex, [Mg(sar− dbm 2 )] 2+ , analyses of crude reaction mixtures revealed that it was present in low yield (see the Supporting Information).  HER2-positive breast cancer. The four solvent-accessible interstrand disulfide bonds of trastuzumab were reduced with tris(2-carboxyethyl)phosphine) (TCEP), the reduced antibody was reacted with 8 mol equiv of either sar−dbm or dfo−dbm at pH 8.5, and the conjugation reactions were monitored over time by SDS-PAGE (vide infra). These reaction conditions were adapted from well-established methods for coupling drugdibromomaleimide or fluorophore-dibromomaleimides to IgG1 constructs. 31,34,41 Notably, increasing the molar equivalents of chelator−dbm relative to the trastuzumab antibody, for example, from 8 mol equiv of sar−dbm to either 16 or 24 mol equiv of sar−dbm, did not significantly affect the efficacy of the conjugation reaction ( Figure S1).
For reaction of either sar−dbm or dfo−dbm with reduced trastuzumab, thiol substitution to give dithiomaleimide conjugates proceeded rapidly (within 5 min). Subsequent hydrolysis of the dithiomaleimide to the dithiomaleamic acid (dtm) was achieved by further incubation of the reaction solutions at pH 8.5 for 2−48 h (Scheme 3). Prior studies on this class of compounds have shown that dithiomaleimide hydrolysis proceeds faster in the presence of electronwithdrawing imide substituents: significantly, the proximity of the amide bond to the maleimide motif increases the rate of hydrolysis. 34 For dfo−dtm−trastuzumab, with a C 2 glycinederived linker, hydrolysis (as monitored by SDS-PAGE) was complete within 2 h ( Figure S2). The sar−dtm−sarcophagine conjugate, with a longer C 6 aminohexanoate linker, was incubated for 48 h at 37°C to ensure complete hydrolysis ( Figure S1).
As expected, the SDS-PAGE of TCEP-reduced trastuzumab showed the presence of separate heavy and light chains under the denaturing analysis conditions (Figure 1, lane C). Following dithiol rebridging reactions with chelator−dbm (for example sar−dbm, Figure 1, lane D, and dfo−dbm, Figure  1, lane G) and subsequent hydrolysis, the two major products observed corresponded to the "full antibody", in which the maleamic acid groups bridge inter chain dithiols at the hinge region, and the "half antibody", in which the maleamic acid groups bridge intra chain dithiols at the hinge region. The "full antibody" rebridging pattern mirrors that of the native trastuzumab antibody. The "half antibody" corresponds to a covalently linked single heavy chain + single light chain and is typical of this type of reaction. 31,34 It arises as a result of "disulfide scrambling" and is only observed by harsh denaturing analysis. Less intense bands, corresponding to antibody heavy chains and light chains that remained "unbridged", were also discernible, and these were more prominent for sar−dtm−trastuzumab than dfo−dtm−trastuzumab.
The same conjugation products were also analyzed using reducing SDS-PAGE conditions: dithiothreitol (DTT) was incubated with the antibody conjugate prior to running the SDS-PAGE. The same two "full antibody" and "half antibody bands" were observed (Figure 1, lanes E and H), indicating that (i) the conjugation and subsequent hydrolysis reactions to yield maleamic acid-conjugated motifs were complete, and (ii) the rebridged products were stable in the presence of excess thiols, consistent with prior reports detailing the high stability of such conjugates. 31,34 The bioconjugates sar−dtm−trastuzumab and dfo−dtm− trastuzumab were also characterized by ESI-HRMS, which showed that both sar−dtm−trastuzumab and dfo−dtm− trastuzumab contain two−four copies of each chelator. The most intense signals in the deconvoluted ESI-HRMS of trastuzumab conjugates (Tables 1, S2, Figures S6, S7) corresponded to "half antibody". Signals corresponding to "full antibody" were weak, which is a common feature of such immunoconjugates as the smaller fragments are always detected with greater relative intensity. Additionally, all deconvoluted "half antibody" and "full antibody" signals were significantly broader than that observed for similarly prepared and characterized alkyne−trastuzumab immunoconjugates. 34,42 We attribute this peak broadening to sar−dtm−trastuzumab and dfo−dtm−trastuzumab readily complexing trace metal ions: we have previously observed trace metal binding to chelator conjugates in our mass spectrometric analyses. 43,44 The combination of SDS-PAGE and ESI-HRMS data indicates that the new immunoconjugates consist of a heterogeneous mixture, comprised of predominantly "full antibody" and "half antibody" constructs (with low amounts of conjugate in which some heavy and light chains remain "unbridged"), and contain two−four chelators per antibody. This heterogeneity, and in particular the presence of both "full antibody" and "half antibody" constructs, is unlikely to significantly modify the immunoconjugates' affinity for the target HER2 receptor or their biodistribution in vivo: we have previously shown that mixtures containing similar "disulfidescrambled" trastuzumab species exhibit comparable antigenbinding and Fc-binding properties to native trastuzumab. 42 The immunoconjugate sar−dtm−trastuzumab was radio-  (Figure 2b, bottom trace). In contrast, when a sample of unmodified trastuzumab was reacted with either [ 64 Cu]Cu 2+ or [ 89 Zr]Zr 4+ , <2% of radioactivity was associated with the antibody, with the majority of unreacted radiometal eluting at 11.6 min (Figure 2a,b).
[ 64 Cu]Cu-sar−dtm−trastuzumab and [ 89 Zr]Zr-dfo−dtm− trastuzumab were additionally analyzed by SDS-PAGE with bright field imaging and autoradiography: a radioactivity signal from radiolabeled immunoconjugates was coincident with  (Figure 2 a,b, S8, S9). In contrast, when [ 64 Cu]Cu 2+ was incubated in serum, SE-HPLC analysis showed that the majority of [ 64 Cu]Cu 2+ was complexed by a serum protein (retention time of 8.5 min) or low molecular weight species (retention time of 11.6 min). When [ 89 Zr]Zr 4+ was incubated in serum, the majority of radioactivity was associated with low molecular weight species (retention time of 11.8 min).
For preparations of radiopharmaceuticals based on chelator−protein bioconjugates, it is important that the amount of the precursor bioconjugate is relatively low. Typically, unlabeled immunoconjugate is not separated from the radiolabeled conjugate prior to in vivo administration, and high concentrations of unlabeled bioconjugate lead to in vivo receptor "blocking", compromising PET image contrast. Furthermore, the ability to radiolabel in near quantitative radiochemical yields obviates purification steps. To assess the ability of sar−dtm−trastuzumab or dfo−dtm−trastuzumab to complex [ 64 Cu]Cu 2+ or [ 89 Zr]Zr 4+ respectively, solutions containing radiometal were added to increasingly dilute solutions of immunoconjugate (Figure 2c Figure S10). We have previously assessed in vivo stability of radiometal− chelator complexes by quantifying the biodistribution of their IgG-based immunoconjugates in healthy mice over an extended period of time. 3,19 In healthy mice, antibodies such as trastuzumab have long blood half-lives in the absence of diseased tissues/tumors that are positive for the target receptor. Here, in vivo studies in healthy animals aimed to assess the biodistribution and stability of [ 64 Cu]Cu-sar−dtm− trastuzumab in vivo. [ 64 Cu]Cu-sar−dtm−trastuzumab (1.0− 2.5 MBq) was administered intravenously (via tail vein) to NOD scid gamma female mice. At 2 h, 1 day, and 2 days postinjection, PET/CT images were acquired, mice were culled, and organs and serum samples were collected for ex vivo analysis (Figures 3, S11).
PET/CT imaging and ex vivo biodistribution (Figures 3a,b, S11) showed that significant 64 Cu radioactivity remained in blood circulation over 1−2 days postinjection, although as typically observed, this activity decreased from 45.6 ± 4.3%ID (percentage injected dose) g −1 at 2 h postinjection to 18.7 ± 1.9%ID g −1 at 2 days postinjection. Importantly, 64 Cu radioactivity in the liver did not increase from 2 h to 2 days postinjection. Prior studies 3,45 have shown that loss of [ 64 Cu]Cu from a bioconjugate (likely via transchelation to endogenous proteins including albumin in blood 46 and superoxide dismutase 47 and ceruloplasmin 48 in the liver) results in increased accumulation of [ 64 Cu]Cu in the liver over time. Our biodistribution data is consistent with prior reports that detail exceptional stability of [ 64 Cu][Cu(sar)] 2+ conjugates (both peptide and protein conjugates) in vivo. 3,45 Radioactivity uptake and retention in healthy HER2expressing tissue also indicated that [ 64 Cu]Cu-sar−dtm− trastuzumab retains affinity for its target HER2 receptor (Figures 3a,b, S11). Notably, skin, uterine horn, and ovaries− healthy tissues that are known to express HER2 49,50 −showed either increased uptake or persistent retention of 64 Cu radioactivity over the course of the study. For example, radioactivity uptake in skin increased from 1.0 ± 0.2%ID g −1 at 2 h postinjection to 4.9 ± 0.7%ID g −1 at 1 day postinjection (p = 3.1 × 10 −5 ); at 2 days postinjection, skin samples showed retention of 64 Cu radioactivity (5.3 ± 0.4%ID g −1 ).
Serum samples were obtained from mice administered [ 64 Cu]Cu-sar−dtm−trastuzumab at 2 h, 1 day, and 2 days postinjection and analyzed by SE-HPLC and SDS-PAGE. In each SE-HPLC chromatogram, only a single signal was observed, with a retention time matching that of [ 64 Cu]Cusar−dtm−trastuzumab (Figure 3c). Serum samples were also subjected to SDS-PAGE separation, followed by autoradiography imaging, which revealed radioactive bands (Figure 3d  bioconjugates, resulting in transfer of maleimide groups and their cargo from bioconjugates to albumin, 21 Concluding Remarks: Usability of Chelators Containing Dibromomaleimide Groups for Site-Specific Radiolabeling of an IgG Antibody. The majority of chelator− antibody conjugates are prepared from chelators containing reactive electrophilic groups that react with solvent accessible primary amines of lysine side chains. While these conjugation protocols are very simple to implement (even for researchers inexperienced in protein chemistry), such methods mostly lead to heterogeneous mixtures of chelator−protein conjugates that can exhibit suboptimal pharmacokinetics and decreased affinity for target receptors. 6−8 Immunoconjugates containing conventional maleimide groups can enable site-specific attachment to reduced thiol groups of disulfide bonds of antibody derivatives, but the resulting conjugates undergo retro-Michael reactions in the biological milieu, resulting in loss of radioactive cargo. 21−24 Many new chemical innovations in site-specific modification of antibodies have been adapted to stably incorporate chelators into antibodies and their derivatives; 9−14 however, these methods involve enzymatic reactions, engineering of specific peptide sequences into the antibody and/or multistep procedures, and are therefore not simple to implement and tend to require extensive optimization for them to work reliably.
Dibromomaleimide chemistry is superbly suited to simple, stable, and site-specific incorporation of radionuclides into antibodies for receptor-targeted molecular imaging. We have prepared dibromomaleimide derivatives of (i) a dfo chelator, commonly used for [ 89 Zr]Zr 4+ radiolabeling of antibodies, and (ii) a sarcophagine chelator, which is considered state-of-theart for [ 64 Cu]Cu 2+ radiolabeling of proteins and peptides, and successfully incorporated both new bifunctional chelators into the HER2-targeted IgG antibody, trastuzumab. These are the first examples of radiometal-labeled chelator−antibody conjugates that have been prepared using dibromomaleimide technology, enabling site-specific attachment of two different chelators to the IgG trastuzumab antibody. Importantly, the methods we describe are simple to implement and produce chelator−antibody conjugates that can be readily radiolabeled with the PET radiometals  2+ to endogenous thiols, consistent with the reported stability of dithiomaleamic acid groups. Our future efforts will compare the biological properties of these sitespecifically radiolabeled immunoconjugates with radiolabeled immunoconjugates modified using either conventional maleimide conjugation chemistry or non-site-specific (stochastic) conjugation chemistry.