Dendrimer Scaffold for the Amplification of In Vivo Pretargeting Ligations

The development of immunoconjugates requires a careful balance between preserving the functionality of the antibody and modifying the immunoglobulin with the desired cargo. Herein, we describe the synthesis, development, and in vivo evaluation of a novel bifunctional dendrimeric scaffold and its application in pretargeted PET imaging. The site-specific modification of the huA33 antibody with this dendrimeric scaffold yields an immunoconjugate^sshuA33-DEN-TCOdecorated with ∼8 <i>trans</i>-cyclooctene (TCO) moieties, a marked increase compared to the ∼2 TCO/mAb of a nondendrimeric control immunoconjugate (^sshuA33-PEG₁₂-TCO). Pretargeted PET imaging and biodistribution experiments were used to compare the in vivo performance of these two immunoconjugates in athymic nude mice bearing subcutaneous SW1222 human colorectal cancer xenografts. To this end, the mice were administered 100 μg of each immunoconjugate followed 120 h later by the injection of a tetrazine-bearing radioligand, [⁶⁴Cu]­Cu-SarAr-Tz. Pretargeting with ^sshuA33-DEN-TCO produced excellent tumoral uptake at 24 h (8.9 ± 1.9 %ID/g), more than double that created by ^sshuA33-PEG₁₂-TCO (4.1 ± 1.3 %ID/g). Criticallyand somewhat surprisinglythe attachment of the G_0.5 dendrimeric structures did not hamper the in vivo behavior of the immunoconjugate, suggesting that this versatile bifunctional scaffold may have applications beyond pretargeting

Click-Mediated Pretargeted Radioimmunotherapy of Colorectal Carcinoma

Pretargeted radioimmunotherapy (PRIT) based on the inverse electron demand Diels–Alder (IEDDA) reaction between tetrazine (Tz) and <i>trans</i>-cyclooctene (TCO) represents a promising strategy for leveraging the affinity and specificity of antibodies without their pharmacokinetic drawbacks. Herein, we present an investigation of the <i>in vivo</i> efficacy and dosimetry of a PRIT strategy for colorectal carcinoma based on the ligation between a ¹⁷⁷Lu-labeled Tz radioligand (¹⁷⁷Lu-DOTA-PEG₇-Tz) and a TCO-bearing immunoconjugate of the huA33 antibody (huA33-TCO). Biodistribution studies in tumor-bearing mice using intervals of 24, 48, and 72 h between the administration of huA33-TCO and ¹⁷⁷Lu-DOTA-PEG₇-Tz revealed that a 24 h lag time produced the most promising <i>in vivo</i> results: high activity concentrations in the tumor (21.2 %ID/g ± 2.9 at 24 h postinjection), low uptake in nontarget tissues, and favorable dosimetry (an effective dose of 0.054 mSv/MBq). A subsequent longitudinal therapy study revealed striking differences between both the survival and tumor growth of the treatment and control cohorts, clearly underscoring the promise of this approach for the radiotherapy of colorectal carcinoma

Site-Specifically Labeled Antibody–Drug Conjugate for Simultaneous Therapy and ImmunoPET

The conjugation of antibodies with cytotoxic drugs can alter their <i>in vivo</i> pharmacokinetics. As a result, the careful assessment of the <i>in vivo</i> behavior, and specifically the tumor-targeting properties, of antibody–drug conjugates represents a crucial step in their development. In order to facilitate this process, we have created a methodology that facilitates the dual labeling of an antibody with both a toxin and a radionuclide for positron emission tomography (PET). To minimize the impact of these modifications, this chemoenzymatic approach leverages strain-promoted azide–alkyne click chemistry to graft both cargoes to the heavy chain glycans of the immuoglobulin’s F_c domain. As a proof-of-concept, a HER2-targeting trastuzumab immunoconjugate was created bearing both a monomethyl auristatin E (MMAE) toxin as well as the long-lived positron-emitting radiometal ⁸⁹Zr (<i>t</i>_1/2 ≈ 3.3 days). Both the tumor targeting and therapeutic efficacy of the ⁸⁹Zr-trastuzumab-MMAE immunoconjugate were validated <i>in vivo</i> using a murine model of HER2-expressing breast cancer. The site-specifically dual-labeled construct enabled the clear visualization of tumor tissue via PET imaging, producing tumoral uptake of ∼70%ID/g. Furthermore, a longitudinal therapy study revealed that the immunoconjugate exerts significant antitumor activity, leading to a >90% reduction in tumor volume over the course of 20 days

Alternative Chelator for ⁸⁹Zr Radiopharmaceuticals: Radiolabeling and Evaluation of 3,4,3-(LI-1,2-HOPO)

Zirconium-89 is an effective radionuclide for antibody-based positron emission tomography (PET) imaging because its physical half-life (78.41 h) matches the biological half-life of IgG antibodies. Desferrioxamine (DFO) is currently the preferred chelator for ⁸⁹Zr⁴⁺; however, accumulation of ⁸⁹Zr in the bones of mice suggests that ⁸⁹Zr⁴⁺ is released from DFO in vivo. An improved chelator for ⁸⁹Zr⁴⁺ could eliminate the release of osteophilic ⁸⁹Zr⁴⁺ and lead to a safer PET tracer with reduced background radiation dose. Herein, we present an octadentate chelator 3,4,3-(LI-1,2-HOPO) (or HOPO) as a potentially superior alternative to DFO. The HOPO ligand formed a 1:1 Zr-HOPO complex that was evaluated experimentally and theoretically. The stability of ⁸⁹Zr-HOPO matched or surpassed that of ⁸⁹Zr-DFO in every experiment. In healthy mice, ⁸⁹Zr-HOPO cleared the body rapidly with no signs of demetalation. Ultimately, HOPO has the potential to replace DFO as the chelator of choice for ⁸⁹Zr-based PET imaging agents

Modular Strategy for the Construction of Radiometalated Antibodies for Positron Emission Tomography Based on Inverse Electron Demand Diels–Alder Click Chemistry

A modular system for the construction of radiometalated antibodies was developed based on the bioorthogonal cycloaddition reaction between 3-(4-benzylamino)-1,2,4,5-tetrazine and the strained dienophile norbornene. The well-characterized, HER2-specific antibody trastuzumab and the positron emitting radioisotopes ⁶⁴Cu and ⁸⁹Zr were employed as a model system. The antibody was first covalently coupled to norbornene, and this stock of norbornene-modified antibody was then reacted with tetrazines bearing the chelators 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA) or desferrioxamine (DFO) and subsequently radiometalated with ⁶⁴Cu and ⁸⁹Zr, respectively. The modification strategy is simple and robust, and the resultant radiometalated constructs were obtained in high specific activity (2.7–5.3 mCi/mg). For a given initial stoichiometric ratio of norbornene to antibody, the ⁶⁴Cu-DOTA- and ⁸⁹Zr-DFO-based probes were shown to be nearly identical in terms of stability, the number of chelates per antibody, and immunoreactivity (>93% in all cases). <i>In vivo</i> PET imaging and acute biodistribution experiments revealed significant, specific uptake of the ⁶⁴Cu- and ⁸⁹Zr-trastuzumab bioconjugates in HER2-positive BT-474 xenografts, with little background uptake in HER2-negative MDA-MB-468 xenografts or other tissues. This modular systemone in which the divergent point is a single covalently modified antibody stock that can be reacted selectively with various chelatorswill allow for both greater versatility and more facile cross-comparisons in the development of antibody-based radiopharmaceuticals

Chemoenzymatic Strategy for the Synthesis of Site-Specifically Labeled Immunoconjugates for Multimodal PET and Optical Imaging

The complementary nature of positron emission tomography (PET) and optical imaging (OI) has fueled increasing interest in the development of multimodal PET/OI probes that can be employed during the diagnosis, staging, and surgical treatment of cancer. Due to their high selectivity and affinity, antibodies have emerged as promising platforms for the development of hybrid PET/OI agents. However, the lack of specificity of many bioconjugation reactions can threaten immunoreactivity and lead to poorly defined constructs. To circumvent this issue, we have developed a chemoenzymatic strategy for the construction of multimodal PET/OI immunoconjugates that have been site-specifically labeled on the heavy chain glycans. The methodology consists of four steps: (1) the enzymatic removal of the terminal galactose residues on the heavy chain glycans; (2) the enzymatic incorporation of azide-bearing galactose (GalNAz) residues into the heavy chain glycans; (3) the strain-promoted click conjugation of chelator- and fluorophore-modified dibenzocyclooctynes to the azide-modified sugars; and (4) the radiolabeling of the immunoconjugate. For proof-of-concept, a model system was created using the colorectal cancer-targeting antibody huA33, the chelator desferrioxamine (DFO), the positron-emitting radiometal ⁸⁹Zr, and the near-infrared fluorescent dye Alexa Fluor 680. The bioconjugation strategy is robust and reproducible, reliably producing well-defined and immunoreactive conjugates labeled with ⁸⁹Zr, Alexa Fluor 680, or an easily and precisely tuned mixture of the two reporters. In <i>in vivo</i> PET and fluorescence imaging experiments, a hybrid ⁸⁹Zr- and Alexa Fluor 680-labeled huA33 conjugate displayed high levels of specific uptake (>45% ID/g) in athymic nude mice bearing A33 antigen-expressing SW1222 colorectal cancer xenografts

Exploring Structural Parameters for Pretargeting Radioligand Optimization

Pretargeting offers a way to enhance target specificity while reducing off-target radiation dose to healthy tissue during payload delivery. We recently reported the development of an ¹⁸F-based pretargeting strategy predicated on the inverse electron demand Diels–Alder reaction as well as the use of this approach to visualize pancreatic tumor tissue in vivo as early as 1 h postinjection. Herein, we report a comprehensive structure: pharmacokinetic relationship study of a library of 25 novel radioligands that aims to identify radiotracers with optimal pharmacokinetic and dosimetric properties. This investigation revealed key relationships between molecular structure and in vivo behavior and produced two lead candidates exhibiting rapid tumor targeting with high target-to-background activity concentration ratios at early time points. We believe this knowledge to be of high value for the design and clinical translation of next-generation pretargeting agents for the diagnosis and treatment of disease

Pretargeted PET Imaging Using a Site-Specifically Labeled Immunoconjugate

In recent years, both site-specific bioconjugation techniques and bioorthogonal pretargeting strategies have emerged as exciting technologies with the potential to improve the safety and efficacy of antibody-based nuclear imaging. In the work at hand, we have combined these two approaches to create a pretargeted PET imaging strategy based on the rapid and bioorthogonal inverse electron demand Diels–Alder reaction between a ⁶⁴Cu-labeled tetrazine radioligand (⁶⁴Cu-Tz-SarAr) and a site-specifically modified huA33-<i>trans</i>-cyclooctene immunoconjugate (^sshuA33-PEG₁₂-TCO). A bioconjugation strategy that harnesses enzymatic transformations and strain-promoted azide–alkyne click chemistry was used to site-specifically append PEGylated TCO moieties to the heavy chain glycans of the colorectal cancer-targeting huA33 antibody. Preclinical in vivo validation studies were performed in athymic nude mice bearing A33 antigen-expressing SW1222 human colorectal carcinoma xenografts. To this end, mice were administered ^sshuA33-PEG₁₂-TCO via tail vein injection andfollowing accumulation intervals of 24 or 48 h⁶⁴Cu-Tz-SarAr. PET imaging and biodistribution studies reveal that this strategy clearly delineates tumor tissue as early as 1 h post-injection (6.7 ± 1.7%ID/g at 1 h p.i.), producing images with excellent contrast and high tumor-to-background activity concentration ratios (tumor:muscle = 21.5 ± 5.6 at 24 h p.i.). Furthermore, dosimetric calculations illustrate that this pretargeting approach produces only a fraction of the overall effective dose (0.0214 mSv/MBq; 0.079 rem/mCi) of directly labeled radioimmunoconjugates. Ultimately, this method effectively facilitates the high contrast pretargeted PET imaging of colorectal carcinoma using a site-specifically modified immunoconjugate

Optimization of a Pretargeted Strategy for the PET Imaging of Colorectal Carcinoma via the Modulation of Radioligand Pharmacokinetics

Pretargeted PET imaging has emerged as an effective strategy for merging the exquisite selectivity of antibody-based targeting vectors with the rapid pharmacokinetics of radiolabeled small molecules. We previously reported the development of a strategy for the pretargeted PET imaging of colorectal cancer based on the bioorthogonal inverse electron demand Diels–Alder reaction between a tetrazine-bearing radioligand and a transcyclooctene-modified huA33 immunoconjugate. Although this method effectively delineated tumor tissue, its clinical potential was limited by the somewhat sluggish clearance of the radioligand through the gastrointestinal tract. Herein, we report the development and in vivo validation of a pretargeted strategy for the PET imaging of colorectal carcinoma with dramatically improved pharmacokinetics. Two novel tetrazine constructs, Tz-PEG₇-NOTA and Tz-SarAr, were synthesized, characterized, and radiolabeled with ⁶⁴Cu in high yield (>90%) and radiochemical purity (>99%). PET imaging and biodistribution experiments in healthy mice revealed that although ⁶⁴Cu-Tz-PEG₇-NOTA is cleared via both the gastrointestinal and urinary tracts, ⁶⁴Cu-Tz-SarAr is rapidly excreted by the renal system alone. On this basis, ⁶⁴Cu-Tz-SarAr was selected for further in vivo evaluation. To this end, mice bearing A33 antigen-expressing SW1222 human colorectal carcinoma xenografts were administered huA33-TCO, and the immunoconjugate was given 24 h to accumulate at the tumor and clear from the blood, after which ⁶⁴Cu-Tz-SarAr was administered via intravenous tail vein injection. PET imaging and biodistribution experiments revealed specific uptake of the radiotracer in the tumor at early time points (5.6 ± 0.7 %ID/g at 1 h p.i.), high tumor-to-background activity ratios, and rapid elimination of unclicked radioligand. Importantly, experiments with longer antibody accumulation intervals (48 and 120 h) yielded slight decreases in tumoral uptake but also concomitant increases in tumor-to-blood activity concentration ratios. This new strategy offers dosimetric benefits as well, yielding a total effective dose of 0.041 rem/mCi, far below the doses produced by directly labeled ⁶⁴Cu-NOTA-huA33 (0.133 rem/mCi) and ⁸⁹Zr-DFO-huA33 (1.54 rem/mCi). Ultimately, this pretargeted PET imaging strategy boasts a dramatically improved pharmacokinetic profile compared to our first generation system and is capable of clearly delineating tumor tissue with high image contrast at only a fraction of the radiation dose created by directly labeled radioimmunoconjugates

H₂azapa: a Versatile Acyclic Multifunctional Chelator for ⁶⁷Ga, ⁶⁴Cu, ¹¹¹In, and ¹⁷⁷Lu

Preliminary experiments with the novel acyclic triazole-containing bifunctional chelator H₂azapa and the radiometals ⁶⁴Cu, ⁶⁷Ga, ¹¹¹In, and ¹⁷⁷Lu have established its significant versatile potential as an alternative to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for metal-based radiopharmaceuticals. Unlike DOTA, H₂azapa radiolabels quantitatively with ⁶⁴Cu, ⁶⁷Ga, ¹¹¹In, and ¹⁷⁷Lu in 10 min at room temperature. In vitro competition experiments with human blood serum show that ⁶⁴Cu remained predominantly chelate-bound, with only 2% transchelated to serum proteins after 20 h. Biodistribution experiments with [⁶⁴Cu­(azapa)] in mice reveal uptake in various organs, particularly in the liver, lungs, heart, intestines, and kidneys. When compared to [⁶⁴Cu­(DOTA)]^2–, the lipophilic neutral [⁶⁴Cu­(azapa)] was cleared through the gastrointestinal tract and accumulated in the liver, which is common for lipophilic compounds or free ⁶⁴Cu. The chelator H₂azapa is a model complex for a click-based bifunctional chelating agent, and the lipophilic benzyl “place-holders” will be replaced by hydrophilic peptides to modulate the pharmacokinetics and direct activity away from the liver and gut. The solid-state molecular structure of [In­(azapa)­(H₂O)]­[ClO₄] reveals a very rare eight-coordinate distorted square antiprismatic geometry with one triazole arm bound, and the structure of [⁶⁴Cu­(azapa)] shows a distorted octahedral geometry. The present study demonstrates significant potential for bioconjugates of H₂azapa as alternatives to DOTA in copper-based radiopharmaceuticals, with the highly modular and “clickable” molecular scaffold of H₂azapa easily modified into a variety of bioconjugates. H₂azapa is a versatile addition to the “pa” family, joining the previously published H₂dedpa (^67/68Ga and ⁶⁴Cu), H₄octapa (¹¹¹In, ¹⁷⁷Lu, and ⁹⁰Y), and H₅decapa (²²⁵Ac) to cover a wide range of important nuclides