A rotaxane-based platform for tailoring the pharmacokinetics of cancer-targeted radiotracers

Radiolabelled monoclonal antibodies (mAbs) are a cornerstone of molecular diagnostic imaging and targeted radioimmunotherapy in nuclear medicine, but one of the major challenges in the field is to identify ways of reducing the radiation burden to patients. We reasoned that a rotaxane-based platform featuring a non-covalent mechanical bond between the radionuclide complex and the biologically active mAb could offer new ways of controlling the biophysical properties of cancer-specific radiotracers for positron emission tomography (PET). Herein, we present the photoradiosynthesis and characterisation of [⁸⁹Zr]ZrFe-[4]rotaxane-azepin-onartuzumab ([⁸⁹Zr]ZrFe-2), a unique rotaxane-antibody conjugate for PET imaging and quantification of the human hepatocyte growth factor receptor (c-MET). Multiple component self-assembly reactions were combined with simultaneous ⁸⁹Zr-radiolabelling and light-induced bioconjugation methods to give [⁸⁹Zr]ZrFe-2 in 15 ± 1% (n = 3) decay-corrected radiochemical yield, with >90% radiochemical purity, and molar activities suitable for PET imaging studies (>6.1 MBq mg⁻¹ of protein). Cellular assays confirmed the specificity of [⁸⁹Zr]ZrFe-2 binding to the c-MET receptor. Temporal PET imaging in athymic nude mice bearing subcutaneous MKN-45 gastric adenocarcinoma xenografts demonstrated specific binding of [⁸⁹Zr]ZrFe-2 toward c-MET in vivo, where tumour uptake reached 9.8 ± 1.3 %ID g⁻¹ (72 h, n = 5) in a normal group and was reduced by ∼56% in a control (blocking) group. Head-to-head comparison of the biodistribution and excretion profile of [⁸⁹Zr]ZrFe-2versus two control compounds, alongside characterisation of two potential metabolites, showed that the rotaxane-radiotracer has an improved clearance profile with higher tumour-to-tissue contrast ratios and reduced radiation exposure to critical (dose-limiting) organs including liver, spleen, and kidneys. Collectively, the experimental results suggested that non-covalent mechanical bonds between the radionuclide and mAb can be used to fine-tune the pharmacokinetic profile of supramolecular radiopharmaceuticals in ways that are simply not accessible when using traditional covalent design.

Mechanically interlocked rotaxanes are transformed into positron-emitting radiotracers for cancer biomarker imaging. Mechanical bonding offers new possibilities for controlling drug delivery, metabolism, and excretion profiles in vivo.     

Enzyme‐Mediated Site‐Specific Bioconjugation of Metal Complexes to Proteins: Sortase‐Mediated Coupling of Copper‐64 to a Single‐Chain Antibody

The enzyme‐mediated site‐specific bioconjugation of a radioactive metal complex to a single‐chain antibody using the transpeptidase sortase A is reported. Cage amine sarcophagine ligands that were designed to function as substrates for the sortase A mediated bioconjugation to antibodies were synthesized and enzymatically conjugated to a single‐chain variable fragment. The antibody fragment scFv_anti‐LIBS targets ligand‐induced binding sites (LIBS) on the glycoprotein receptor GPIIb/IIIa, which is present on activated platelets. The immunoconjugates were radiolabeled with the positron‐emitting isotope ⁶⁴Cu. The new radiolabeled conjugates were shown to bind selectively to activated platelets. The diagnostic potential of the most promising conjugate was demonstrated in an in vivo model of carotid artery thrombosis using positron emission tomography. This approach gives homogeneous products through site‐specific enzyme‐mediated conjugation and should be broadly applicable to other metal complexes and proteins.     

Tuning Tetrazole Photochemistry for Protein Ligation and Molecular Imaging

Photochemistry provides a wide range of alternative reagents that hold potential for use in bimolecular functionalisation of proteins. Here, we report the synthesis and characterisation of metal ion binding chelates derivatised with disubstituted tetrazoles for the photoradiochemical labelling of monoclonal antibodies (mAbs). The photophysical properties of tetrazoles featuring extended aromatic systems and auxochromic substituents to tune excitation toward longer wavelengths (365 and 395 nm) were studied. Two photoactivatable chelates based on desferrioxamine B (DFO) and the aza-macrocycle NODAGA were functionalised with a tetrazole and developed for protein labelling with ⁸⁹Zr, ⁶⁴Cu and ⁶⁸Ga radionuclides. DFO-tetrazole ( 1 ) was assessed by direct conjugation to formulated trastuzumab and subsequent radiolabelling with ⁸⁹Zr. Radiochemical studies and cellular-based binding assays demonstrated that the radiotracer remained stable in vitro retained high immunoreactivity. Positron emission tomography (PET) imaging and biodistribution studies were used to measure the tumour specific uptake and pharmacokinetic profile in mice bearing SK-OV-3 xenografts. Experiments demonstrate that tetrazole-based photochemistry is a viable approach for the light-induced synthesis of PET radiotracers.     

Dual reactivity disulfide bridging reagents; enabling new approaches to antibody fragment bioconjugation

Disulfide bridging, also known as disulfide stapling, is a powerful strategy for the construction of site-selective protein bioconjugates. Here we describe the first examples of a new class of such reagents, containing a ‘stable-labile’ design. These dual-reactive reagents are designed to form a stable bond to one cysteine and a labile bond to the second; resulting in a robust attachment to the protein with one end of the bridge, whilst the other end serves as a reactive handle for subsequent bioconjugation. By incorporating thioesters into these bridges, we demonstrate that they are primed for native chemical ligation (NCL) with N-terminal cysteines; offering an alternative to the requirement for C-terminal thioesters for use in such ligations. Alternatively, the use of hydrazine as the ligating nucleophile enables a separate cargo to be attached to each cysteine residue, which are exploited to insert variably cleavable linkers. These methodologies are demonstrated on an antibody fragment, and serve to expand the scope of disulfide bridging strategies whilst offering a convenient route to the construction of multifunctional antibody fragment conjugates.

Here we describe the first examples of a new class of disulfide bridging reagents, designed to insert a ‘stable-labile’ linkage; which can then be exploited to generate dual functional antibody fragment conjugates.     

In Vitro and In Vivo Characterization of 89Zirconium-Labeled Lintuzumab Molecule

Objective: Positron emission tomography (PET) imaging is a powerful non-invasive method to determine the in vivo behavior of biomolecules. Determining biodistribution and pharmacokinetic (PK) properties of targeted therapeutics can enable a better understanding of in vivo drug mechanisms such as tumor uptake, off target accumulation and clearance. Zirconium-89 (⁸⁹Zr) is a readily available tetravalent PET-enabling radiometal that has been used to evaluate the biodistribution and PK of monoclonal antibodies. In the current study, we performed in vitro and in vivo characterization of ⁸⁹Zr-lintuzumab, a radiolabeled anti-CD33 antibody, as a model to evaluate the in vivo binding properties in preclinical models of AML. Methods: Lintuzumab was conjugated to p-SCN-Bn-deferoxamine (DFO) and labeled with ⁸⁹Zr using a 5:1 µCi:µg specific activity at 37 °C for 1h. The biological activity of ⁸⁹Zr-lintuzumab was evaluated in a panel of CD33 positive cells using flow cytometry. Fox Chase SCID mice were injected with 2 × 10⁶ OCI-AML3 cells into the right flank. After 12 days, a cohort of mice (n = 4) were injected with ⁸⁹Zr-lintuzumab via tail vein. PET/CT scans of mice were acquired on days 1, 2, 3 and 7 post ⁸⁹Zr-lintuzumab injection. To demonstrate ⁸⁹Zr-lintuzumab specific binding to CD33 expressing tumors in vivo, a blocking study was performed. This cohort of mice (n = 4) was injected with native lintuzumab and 24 h later ⁸⁹Zr-lintuzumab was administered. This group was imaged 3 and 7 days after injection of ⁸⁹Zr-lintuzumab. A full ex vivo biodistribution study on both cohorts was performed on day 7. The results from the PET image and ex vivo biodistribution studies were compared. Results: Lintuzumab was successfully radiolabeled with ⁸⁹Zr resulting in a 99% radiochemical yield. The ⁸⁹Zr-lintuzumab radioconjugate specifically binds CD33 positive cells in a similar manner to native lintuzumab as observed by flow cytometry. PET imaging revealed high accumulation of ⁸⁹Zr-lintuzumab in OCI-AML3 tumors within 24h post-injection of the radioconjugate. The ⁸⁹Zr-lintuzumab high tumor uptake remains for up to 7 days. Tumor analysis of the PET data using volume of interest (VOI) showed significant blocking of ⁸⁹Zr-lintuzumab in the group pre-treated with native lintuzumab (pre-blocked group), thus indicating specific targeting of CD33 on OCI-AML3 cells in vivo. The tumor uptake findings from the PET imaging study are in agreement with those from the ex vivo biodistribution results. Conclusions: PET imaging of ⁸⁹Zr-lintuzumab shows high specific uptake in CD33 positive human OCI-AML3 tumors. The results from the image study agree with the observations from the ex vivo biodistribution study. Our findings collectively suggest that PET imaging using ⁸⁹Zr-lintuzumab could be a powerful drug development tool to evaluate binding properties of anti-CD33 monoclonal antibodies in preclinical cancer models.     

A homogeneous high-DAR antibody–drug conjugate platform combining THIOMAB antibodies and XTEN polypeptides

The antibody–drug conjugate (ADC) is a well-validated modality for the cell-specific delivery of small molecules with impact expanding rapidly beyond their originally-intended purpose of treating cancer. However, antibody-mediated delivery (AMD) remains inefficient, limiting its applicability to targeting highly potent payloads to cells with high antigen expression. Maximizing the number of payloads delivered per antibody is one key way in which delivery efficiency can be improved, although this has been challenging to carry out; with few exceptions, increasing the drug-to-antibody ratio (DAR) above ∼4 typically destroys the biophysical properties and in vivo efficacy for ADCs. Herein, we describe the development of a novel bioconjugation platform combining cysteine-engineered (THIOMAB) antibodies and recombinant XTEN polypeptides for the unprecedented generation of homogeneous, stable “TXCs” with DAR of up to 18. Across three different bioactive payloads, we demonstrated improved AMD to tumors and Staphylococcus aureus bacteria for high-DAR TXCs relative to conventional low-DAR ADCs.

Efficiency of targeted cell delivery of small molecules was enhanced in cells and animals via a novel well-defined bioconjugation platform combining site-specific antibody conjugation and XTEN polypeptides to enable high payload loading.     

A novel tyrosine hyperoxidation enables selective peptide cleavage

A novel tyrosine hyperoxidation enabling selective peptide cleavage is reported. The scission of the N-terminal amide bond of tyrosine was achieved with Dess–Martin periodinane under mild conditions, generating a C-terminal peptide fragment bearing the unprecedented hyperoxidized tyrosine motif, 4,5,6,7-tetraoxo-1H-indole-2-carboxamide, along with an intact N-terminal peptide fragment. This reaction proceeds with high site-selectivity for tyrosine and exhibits broad substrate scope for various peptides, including those containing post-translational modifications. More importantly, this oxidative cleavage was successfully applied to enable sequencing of three naturally occurring cyclic peptides, including one depsipeptide and one lipopeptide. The linearized peptides generated from the cleavage reaction significantly simplify cyclic peptide sequencing by MS/MS, thus providing a robust tool to facilitate rapid sequence determination of diverse cyclic peptides containing tyrosine. Furthermore, the highly electrophilic nature of the hyperoxidized tyrosine unit disclosed in this work renders it an important electrophilic target for the selective bioconjugation or synthetic manipulation of peptides containing this unit.

A Tyr-selective peptide cleavage was reported using Dess–Martin periodinane. The cleavage generates an unprecedented hyperoxidized tyrosine motif in the C-terminal fragment and showed excellent site-specificity and broad scope for various peptides.     

Tyrosine bioconjugation with hypervalent iodine

Hypervalent iodine reagents have recently emerged as powerful tools for late-stage peptide and protein functionalization. Herein we report a tyrosine bioconjugation methodology for the introduction of hypervalent iodine onto biomolecules under physiological conditions. Tyrosine residues were engaged in a selective addition onto the alkynyl bond of ethynylbenziodoxolones (EBX), resulting in stable vinylbenziodoxolones (VBX) bioconjugates. The methodology was successfully applied to peptides and proteins and tolerated all other nucleophilic residues, with the exception of cysteine. The generated VBX were further functionalized by palladium-catalyzed cross-coupling and azide–alkyne cycloaddition reactions. The method could be successfully used to modify bioactive natural products and native streptavidin to enable thiol-mediated cellular uptake.

A tyrosine bioconjugation for the introduction of hypervalent iodine onto biomolecules is described. The transformation was applied to peptides and proteins and was used to modify native streptavidin to enable thiol-mediated cellular uptake.     

[89Zr]-Pertuzumab PET Imaging Reveals Paclitaxel Treatment Efficacy Is Positively Correlated with HER2 Expression in Human Breast Cancer Xenograft Mouse Models

Paclitaxel (PTX) treatment efficacy varies in breast cancer, yet the underlying mechanism for variable response remains unclear. This study evaluates whether human epidermal growth factor receptor 2 (HER2) expression level utilizing advanced molecular positron emission tomography (PET) imaging is correlated with PTX treatment efficacy in preclinical mouse models of HER2+ breast cancer. HER2 positive (BT474, MDA-MB-361), or HER2 negative (MDA-MB-231) breast cancer cells were subcutaneously injected into athymic nude mice and PTX (15 mg/kg) was administrated. In vivo HER2 expression was quantified through [⁸⁹Zr]-pertuzumab PET/CT imaging. PTX treatment response was quantified by [¹⁸F]-fluorodeoxyglucose ([¹⁸F]-FDG) PET/CT imaging. Spearman’s correlation, Kendall’s tau, Kolmogorov–Smirnov test, and ANOVA were used for statistical analysis. [⁸⁹Zr]-pertuzumab mean standard uptake values (SUV_mean) of BT474 tumors were 4.9 ± 1.5, MDA-MB-361 tumors were 1.4 ± 0.2, and MDA-MB-231 (HER2−) tumors were 1.1 ± 0.4. [¹⁸F]-FDG SUV_mean changes were negatively correlated with [⁸⁹Zr]-pertuzumab SUV_mean (r = −0.5887, p = 0.0030). The baseline [¹⁸F]-FDG SUV_mean was negatively correlated with initial [⁸⁹Zr]-pertuzumab SUV_mean (r = −0.6852, p = 0.0002). This study shows PTX treatment efficacy is positively correlated with HER2 expression level in human breast cancer mouse models. Molecular imaging provides a non-invasive approach to quantify biological interactions, which will help in identifying chemotherapy responders and potentially enhance clinical decision-making.     

Chemotopological study of positron emitter radionuclides used in PET diagnostic imaging: physical, physico-chemical, dosimetric, quantum and nuclear properties

We carried out a chemotopological study of two groups of positron emitter radionuclides of current and potential use in positron emission tomography (PET) diagnostic imaging. The aim was to look for potential β⁺ radionuclides not yet in use for PET imaging, taking into account the similarities of these radionuclides with radionuclides already used in PET imaging. The similarity was studied through physical, physico-chemical, dosimetric, quantum and nuclear properties of the radionuclides. We applied cluster analysis (two similarity functions and three grouping methodologies) and generated six dendrograms. One dendrogram was selected in each group, which was used to build up a basis for a topology. From the calculation of topological properties of several subsets of interest, we propose β⁺ radionuclides ⁴⁷V, ⁴⁸V, ⁶³Zn, ⁷⁰As, ⁹⁰Nb, ¹⁰⁶Ag, ¹¹⁵Sb, ¹¹⁶Sb, ¹²⁰Sb, ¹³⁰Cs, ¹³⁴La and ¹⁴⁰Pr, as potential new radionuclides of use in PET imaging.     

中枢多巴胺系统正电子发射计算机断层扫描显像剂的研究进展

中枢多巴胺系统与多种神经行为障碍的病理生理学有关。一直以来,多巴胺系统正电子发射计算机断层扫描(PET)成像在研究活体大脑中多巴胺生物化学过程上有着重要价值。PET成像的基础是¹¹C、¹⁸F等发射正电子的放射性核素标记的显像剂,这些显像剂通过与多巴胺神经系统不同的靶点特异性结合从而反映多巴胺合成、囊泡储存、突触释放和受体结合以及再摄取过程,推动神经病学、精神病学、药物滥用和成瘾以及药物开发的研究进展。本文综述了以氨基酸脱羧酶、多巴胺转运体、多巴胺受体以及囊泡单胺转运体为靶点的¹¹C、¹⁸F标记的PET显像剂的研究进展。     

New Synthesis of 2-Deoxy-2-fluoro-D-hexoses by Fluorination in Water

Interest in fluorinated sugars labelled with {¹⁸F} positron-emitting radionuclides as tracers for the measurement of glucose utilization in man by positron emission tomography¹ (PET) and in animals by autoradiography² has resulted in the development of numerous syntheses^3-11 of 2-deoxy-2-fluoro-D-glucose (3a). Some of these syntheses are not practical because the carbohydrate substrates for the fluorination reactions are not readily available.     

Synthesis of 11C, 18F, 15O, and 13N radiolabels for positron emission tomography

Positron emission tomography (PET) is a powerful and rapidly developing area of molecular imaging that is used to study and visualize human physiology by the detection of positron-emitting radiopharmaceuticals. Information about metabolism, receptor/enzyme function, and biochemical mechanisms in living tissue can be obtained directly from PET experiments. Unlike magnetic resonance imaging (MRI) or computerized tomography (CT), which mainly provide detailed anatomical images, PET can measure chemical changes that occur before macroscopic anatomical signs of a disease are observed. PET is emerging as a revolutionary method for measuring body function and tailoring disease treatment in living subjects. The development of synthetic strategies for the synthesis of new positron-emitting molecules is, however, not trivial. This Review highlights key aspects of the synthesis of PET radiotracers with the short-lived positron-emitting radionuclides (11)C, (18)F, (15)O, and (13)N, with emphasis on the most recent strategies.     

中枢多巴胺系统正电子发射计算机断层扫描显像剂的研究进展

中枢多巴胺系统与多种神经行为障碍的病理生理学有关。一直以来,多巴胺系统正电子发射计算机断层扫描(PET)成像在研究活体大脑中多巴胺生物化学过程上有着重要价值。PET成像的基础是¹¹C、¹⁸F等发射正电子的放射性核素标记的显像剂,这些显像剂通过与多巴胺神经系统不同的靶点特异性结合从而反映多巴胺合成、囊泡储存、突触释放和受体结合以及再摄取过程,推动神经病学、精神病学、药物滥用和成瘾以及药物开发的研究进展。本文综述了以氨基酸脱羧酶、多巴胺转运体、多巴胺受体以及囊泡单胺转运体为靶点的¹¹C、¹⁸F标记的PET显像剂的研究进展。     

Chelate-free metal ion binding and heat-induced radiolabeling of iron oxide nanoparticles

A novel reaction for chelate-free, heat-induced metal ion binding and radiolabeling of ultra-small paramagnetic iron oxide nanoparticles (USPIOs) has been established. Radiochemical and non-radioactive labeling studies demonstrated that the reaction has a wide chemical scope and is applicable to p-, d- and f-block metal ions with varying ionic sizes and formal oxidation states from 2+ to 4+. Radiolabeling studies found that ⁸⁹Zr–Feraheme (⁸⁹Zr–FH or ⁸⁹Zr–ferumoxytol) can be isolated in 93 ± 3% radiochemical yield (RCY) and >98% radiochemical purity using size-exclusion chromatography. ⁸⁹Zr–FH was found to be thermodynamically and kinetically stable in vitro using a series of ligand challenge and plasma stability tests, and in vivo using PET/CT imaging and biodistribution studies in mice. Remarkably, ICP-MS and radiochemistry experiments showed that the same reaction conditions used to produce ⁸⁹Zr–FH can be employed with different radionuclides to yield ⁶⁴Cu–FH (66 ± 6% RCY) and ¹¹¹In–FH (91 ± 2% RCY). Electron magnetic resonance studies support a mechanism of binding involving metal ion association with the surface of the magnetite crystal core. Collectively, these data suggest that chelate-free labeling methods can be employed to facilitate clinical translation of a new class of multimodality PET/MRI radiotracers derived from metal-based nanoparticles. Further, this discovery is likely to have broader implications in drug delivery, metal separation science, ecotoxicology of nanoparticles and beyond.     

Low power threshold photochemical upconversion using a zirconium(iv) LMCT photosensitizer

The current investigation demonstrates highly efficient photochemical upconversion (UC) where a long-lived Zr(iv) ligand-to-metal charge transfer (LMCT) complex serves as a triplet photosensitizer in concert with well-established 9,10-diphenylanthracene (DPA) along with newly conceived DPA–carbazole based acceptors/annihilators in THF solutions. The initial dynamic triplet–triplet energy transfer (TTET) processes (ΔG ∼ −0.19 eV) featured very large Stern–Volmer quenching constants (K_SV) approaching or achieving 10⁵ M⁻¹ with bimolecular rate constants between 2 and 3 × 10⁸ M⁻¹ s⁻¹ as ascertained using static and transient spectroscopic techniques. Both the TTET and subsequent triplet–triplet annihilation (TTA) processes were verified and throughly investigated using transient absorption spectroscopy. The Stern–Volmer metrics support 95% quenching of the Zr(iv) photosensitizer using modest concentrations (0.25 mM) of the various acceptor/annihilators, where no aggregation took place between any of the chromophores in THF. Each of the upconverting formulations operated with continuous-wave linear incident power dependence (λ_ex = 514.5 nm) down to ultralow excitation power densities under optimized experimental conditions. Impressive record-setting η_UC values ranging from 31.7% to 42.7% were achieved under excitation conditions (13 mW cm⁻²) below that of solar flux integrated across the Zr(iv) photosensitizer''s absorption band (26.7 mW cm⁻²). This study illustrates the importance of supporting the continued development and discovery of molecular-based triplet photosensitizers based on earth-abundant metals.

The LMCT photosensitizer Zr(^MesPDP^Ph)₂ paired with DPA-based acceptors enabled low power threshold photochemical upconversion with record-setting quantum efficiencies.     

Biomedical radioisotope generator systems

Although the⁹⁹Mo^99mTc radionuclide generator system has evolved as the workhorse of nuclear medicine over the past three decades, recent developments in radionuclide generator technology have provided positron emitters for PET studies, radionuclides for therapy, and ultra short-lived radionuclides for repeated clinical applications. Attention is being paid to development ofin vivo generator systems and potential use of tandem generator systems. Separation of radionuclides in a generator system exploits differences in chemical properties of the parent-daughter pair; information is presented for these generator types based on the three chemical categories of chromatography, extraction, and volatilization and sublimation methods. This review focuses on chromatography-based systems.     

Heat‐Induced Radiolabeling of Nanoparticles for Monocyte Tracking by PET

Heat‐induced radiolabeling (HIR) yielded ⁸⁹Zr‐Feraheme (FH) nanoparticles (NPs) that were used to determine NP pharmacokinetics (PK) by positron emission tomography (PET). Standard uptake values indicated a fast hepatic uptake that corresponded to blood clearance, and a second, slow uptake process by lymph nodes and spleen. By cytometry, NPs were internalized by circulating monocytes and monocytes in vitro. Using an IV injection of HIR ⁸⁹Zr‐FH (rather than in vitro cell labeling), PET/PK provided a view of monocyte trafficking, a key component of the immune response.