Evaluation of targeting αVß3 in breast cancers using RGD peptide-based agents.

Patients with HER2-positive and triple negative breast cancer (TNBC) are associated with increased risk to develop metastatic disease including reoccurring disease that is resistant to standard and targeted therapies. The αVß3 has been implicated in BC including metastatic disease. The aims of this study were to investigate the potential of αVß3-targeted peptides to deliver radioactive payloads to BC tumors expressing αVß3 on the tumor cells or limited to the tumors' neovascular. Additionally, we aimed to assess the pharmacokinetic profile of the targeted α-particle therapy (TAT) agent [225Ac]Ac-DOTA-cRGDfK dimer peptide and the in vivo generated decay daughters. The expression of αVß3 in a HER2-positive and a TNBC cell line were evaluated using western blot analysis. The pharmacokinetics of [111In]In-DOTA-cRGDfK dimer, a surrogate for the TAT-agent, was evaluated in subcutaneous mouse tumor models. The pharmacokinetic of the TAT-agent [225Ac]Ac-DOTA-cRGDfK dimer and its decay daughters were evaluated in healthy mice. Selective uptake of [111In]In-DOTA-cRGDfK dimer was shown in subcutaneous tumor models using αVß3-positive tumor cells as well as αVß3-negative tumor cells where the expression is limited to the neovasculature. Pharmacokinetic studies demonstrated rapid accumulation in the tumors with clearance from non-target organs. Dosimetric analysis of [225Ac]Ac-DOTA-cRGDfK dimer showed the highest radiation absorbed dose to the kidneys, which included the contributions from the free in vivo generated decay daughters. This study shows the potential of delivering radioactive payloads to BC tumors that have αVß3 expression on the tumor cells as well as limited expression to the neovascular of the tumor. Furthermore, this work determines the radiation absorbed doses to normal organs/tissues and identified key organs that act as suppliers and receivers of the actinium-225 free in vivo generated α-particle-emitting decay daughters.

Assessment of Novel Mesothelin-Specific Human Antibody Domain VH-Fc Fusion Proteins-Based PET Agents.

Mesothelin (MSLN) is a tumor-associated antigen found in a variety of cancers and is a target for imaging and therapeutic applications in MSLN-expressing tumors. We have developed high affinity anti-MSLN human VH domain antibodies, providing alternative targeting vectors to conventional IgG antibodies that are associated with long-circulating half-lives and poor penetration of tumors, limiting antitumor activity in clinical trials. Based on two newly identified anti-MSLN VH binders (3C9, 2A10), we generated VH-Fc fusion proteins and modified them for zirconium-89 radiolabeling to create anti-MSLN VH-Fc PET tracers. The focus of this study was to assess the ability of PET-imaging to compare the in vivo performance of anti-MSLN VH-Fc fusion proteins (2A10, 3C9) targeting different epitopes of MSLN vs IgG1 (m912; a clinical benchmark antibody with an overlapped epitope as 2A10) for PET imaging in a mouse model of colorectal cancer (CRC). The anti-MSLN VH-Fc fusion proteins were successfully modified and radiolabeled with zirconium-89. The resulting MSLN-targeted PET-imaging agents demonstrated specific uptake in the MSLN-expressing HCT116 tumors. The in vivo performance of the MSLN-targeted PET-imaging agents utilizing VH-Fc showed more rapid and greater accumulation and deeper penetration within the tumor than the full-length IgG1 m912-based PET-imaging agent. Furthermore, PET imaging allowed us to compare the pharmacokinetics of epitope-specific VH domain-based PET tracers. Overall, these data are encouraging for the incorporation of PET imaging to assess modified VH domain structures to develop novel anti-MSLN VH domain-based therapeutics in MSLN-positive cancers as well as their companion PET imaging agents.

"Click-to-Clear": A Strategy to Minimize Radioactivity from the Blood Pool Utilizing Staudinger Ligation.

The availability of several bioorthogonal reactions that can proceed selectively and efficiently under physiologically relevant conditions has garnered the interest of biochemists and organic chemists alike. Bioorthogonal cleavage reactions represent the latest innovation in click chemistry. Here, we employed the Staudinger ligation reaction to release radioactivity from immunoconjugates, improving target-to-background ratios. In this proof-of-concept study, model systems, including the anti-HER2 antibody trastuzumab, radioisotope I-131, and a newly synthesized bifunctional phosphine, were used. Staudinger ligation occurred when biocompatible N-glycosyl azides reacted with this radiolabeled immunoconjugate, leading to cleavage of the radioactive label from the molecule. We demonstrated this click cleavage in vitro and in vivo. Biodistribution studies in tumor models showed that radioactivity was eliminated from the bloodstream, thereby improving tumor-to-blood ratios. SPECT imaging revealed that tumors could be visualized with enhanced clarity. Our simple approach represents a novel application of bioorthogonal click chemistry in the development of antibody-based theranostics.

Optimizing and determining the click chemistry mediated Cu-64 radiolabeling and physiochemical characteristics of trastuzumab conjugates.

Over the last decade, 64Cu-labeling of monoclonal antibody (mAb) via inverse electron demand Diels-Alder click chemistry (IEDDA) have received much attention. Despite the tetrazine-transcyclooctene (Tz-TCO) click chemistry's convenience and efficiency in mAb labeling, there is limited information about the ideal parameters in the development of click chemistry mediated (radio)immunoconjugates. This encourages us to conduct a systematic optimization while concurrently determining the physiochemical characteristics of the model mAb, trastuzumab, and TCO conjugates. To accomplish this, we investigated a few critical parameters, first, we determined the degree of conjugations with varying molar equivalents (eq.) of TCO (3, 5, 10, and 15 eq.). Through analytical techniques like size exclusion chromatography, dynamic light scattering, and zeta potential, qualitative analysis were performed to determine the purity, degree of aggregation and net charge of the conjugates. We found that as the degree of conjugation increased the purity of intact mAb fraction is compromised and net charge of conjugates became less positive. Next, all trastuzumab-PEG4-TCO conjugates with varying molar ratio and quantity (30, 50, 100, 200, 250 µg) were radiolabeled with 64Cu-NOTA-PEG4-Tz via IEDDA click chemistry and radiochemical yields were determined by radio-thin layer chromatography. The radiochemical yields of trastuzumab conjugates improved with increased amount and molar ratio. Next, we investigated the effect of the radioprotectant ascorbic acid (AA) of varied concentrations (0.25, 0.5, 0.75, 1 mM) on radiochemical yields and subsequent pharmacokinetics. A concentration of 0.25 mM of AA was found to be optimal for click reaction and in vivo biodistribution. Finally, we investigated the indirect influence of bioconjugation buffers on radiochemical yields and biodistribution in NIH3T6.7 tumor models that resulted approximately ∼11 %ID/g tumor uptake.

Preclinical Evaluation of hnRNPA2B1 Antibody in Human Triple-Negative Breast Cancer MDA-MB-231 Cells via PET Imaging.

Triple-negative breast cancer (TNBC) does not express estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Because TNBC lacks the expression of commonly targeted receptors, it is challenging to develop a new imaging agent for this cancer subtype. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA-protein complexes that have been linked to tumor development and progression. Considering the high expression of hnRNPA2B1, an hnRNP subtype, in TNBC MDA-MB-231 cells, this study aimed to develop a novel hnRNPA2B1 antibody-based nuclear imaging agent. The hnRNPA2B1-specific antibody was radiolabeled with 64Cu and evaluated in vitro and in vivo. The trans-cyclooctene (TCO) was functionalized on the antibody to obtain hnRNP-PEG4-TCO and reactive tetrazine (Tz) on the ultrastable bifunctional chelator PCB-TE2A-alkyne to yield PCB-TE2A-Tz for the inverse electron demand Diels-Alder reaction. The 64Cu-radiolabeled antibody was administered and imaged at 1-18 h time points for conventional imaging. Alternatively, the unlabeled antibody conjugate was administered, and 48 h later radiolabeled 64Cu-PCB-TE2A-Tz was administered to the same mice for the pretargeting strategy and imaged at the same time intervals for direct comparison. The tumor was successfully visualized in both strategies, and comparatively, pretargeting showed superior results. The 64Cu-PCB-TE2A-Tz was successfully clicked at the tumor site with hnRNP-PEG4-TCO and the non-clicked were concurrently eliminated. This led to increase the tumor uptake with extremely high tumor-to-background ratio manifested by positron emission tomography (PET) imaging and biodistribution studies.

In vivo detection of hydrogen sulfide in the brain of live mouse: application in neuroinflammation models.

PURPOSE: Hydrogen sulfide (H2S) plays important roles in brain pathophysiology. However, nuclear imaging probes for the in vivo detection of brain H2S in living animals have not been developed. Here, we report the first nuclear imaging probe that enables in vivo imaging of endogenous H2S in the brain of live mice. METHODS: Utilizing a bis(thiosemicarbazone) backbone, a fluorescent ATSM-FITC conjugate was synthesized. Its copper complex, Cu(ATSM-FITC) was thoroughly tested as a biosensor for H2S. The same ATSM-FITC ligand was quantitatively labeled with [64Cu]CuCl2 to obtain a radioactive [64Cu][Cu(ATSM-FITC)] imaging probe. Biodistribution and positron emission tomography (PET) imaging studies were performed in healthy mice and neuroinflammation models. RESULTS: The Cu(ATSM-FITC) complex reacts instantly with H2S to release CuS and becomes fluorescent. It showed excellent reactivity, sensitivity, and selectivity to H2S. Endogenous H2S levels in living cells were successfully detected by fluorescence microscopy. Exceptionally high brain uptake of [64Cu][Cu(ATSM-FITC)] (> 9% ID/g) was observed in biodistribution and PET imaging studies. Subtle changes in brain H2S concentrations in live mice were accurately detected by quantitative PET imaging. Due to its dual modality feature, increased H2S levels in neuroinflammation models were characterized at the subcellular level by fluorescence imaging and at the whole-body scale by PET imaging. CONCLUSION: Our biosensor can be readily utilized to study brain H2S function in live animal models and shows great potential as a novel imaging agent for diagnosing brain diseases.

Imaging Strategy that Achieves Ultrahigh Contrast by Utilizing Differential Esterase Activity in Organs: Application in Early Detection of Pancreatic Cancer.

Most nanoparticles show much higher uptake in mononuclear phagocyte system (MPS) organs than in tumors, which has been a long-lasting dilemma in nanomedicine. Here, we report an imaging strategy that selectively decreases MPS organ uptakes by utilizing the differential esterase activity in tumors and other organs. When an esterase-labile radiotracer loaded liposome was injected into the body, radioactivity was rapidly excreted from the liver and spleen after breakage of the ester bond by esterase. However, the lipophilic radiotracer delivered to the tumor remained in the tumor with minimal bond cleavage. The underlying mechanism was fully characterized in vitro and in vivo in colon tumor models. As a proof of concept, the liposomal radiotracer was further optimized for the early detection of pancreatic cancer. The folate-coated liposomal radiotracer showed highly selective tumor uptake. At 4 h postinjection, a pancreatic tumor a few millimeters in size was unambiguously visualized in orthotopic tumor models by PET imaging. At 24 h, an exceptionally high tumor-to-background ratio was achieved, enabling the visualization of tumors alone with minimal background noise. More than 9% of the total radioactivity was found in the tumor. Utilizing our imaging strategy, various tumor imaging agents can be developed for sensitive detection with ultrahigh contrast.

Correction: A short PEG linker alters the in vivo pharmacokinetics of trastuzumab to yield high-contrast immuno-PET images.

Correction for 'A short PEG linker alters the in vivo pharmacokinetics of trastuzumab to yield high-contrast immuno-PET images' by Woonghee Lee et al., J. Mater. Chem. B, 2021, 9, 2993-2997, DOI: 10.1039/D0TB02911D.

A short PEG linker alters the in vivo pharmacokinetics of trastuzumab to yield high-contrast immuno-PET images.

The prolonged blood circulation of the radiolabeled antibody conjugates is problematic when using immuno-PET imaging due to the increased radiation exposure and longer hospitalization required until sufficient contrast develops. In contrast to the prevailing belief that PEGylation prolongs blood retention time, we observed that a PEGylated antibody with a short PEG8 linker cleared much faster from the blood while maintaining tumor uptake compared to its non-PEGylated counterpart. Breast tumors were clearly visualized with a very high tumor-to-background ratio as early as 24 h after injection in immuno-positron emission tomography (PET) imaging.

Successful Application of CuAAC Click Reaction in Constructing 64Cu-Labeled Antibody Conjugates for Immuno-PET Imaging.

Immuno-positron emission tomography (immuno-PET) is a rapidly growing imaging technique in which antibodies are radiolabeled to monitor their in vivo behavior in real time. However, effecting the controlled conjugation of a chelate-bearing radioactive atom to a bulky antibody without affecting its immunoreactivity at a specific site is always challenging. The in vivo stability of the radiolabeled chelate is also a key issue for successful tumor imaging. To address these points, a facile ultra-stable radiolabeling platform is developed by using the propylene cross-bridged chelator (PCB-TE2A-alkyne), which can be instantly functionalized with various groups via the click reaction, thus enabling specific conjugation with antibodies as per choice. The PCB-TE2A-tetrazine derivative is selected to demonstrate the proposed strategy. The antibody trastuzumab is functionalized with the trans-cyclooctene (TCO) moiety in the presence or absence of the PEG linker. The complementary 64Cu-PCB-TE2A-tetrazine is synthesized via the click reaction and radiolabeled with 64Cu ions, which then reacts with the aforementioned TCO-modified antibody via a rapid biorthogonal ligation. The 64Cu-PCB-TE2A-trastuzumab conjugate is shown to exhibit excellent in vivo stability and to maintain a higher binding affinity toward HER2-positive cells. The tumor targeting feasibility of the radiolabeled antibody is evaluated in tumor models. Both 64Cu-PCB-TE2A-trastuzumab conjugates show high tumor uptakes in biodistribution studies and enable unambiguous tumor visualization with minimum background noise in PET imaging. Interestingly, the 64Cu-PCB-TE2A-PEG4-trastuzumab containing an additional PEG linker displays a much faster body clearance compared to its counterpart with less PEG linker, thus affording vivid tumor imaging with an unprecedentedly high tumor-to-background ratio.

Development of dansyl based copper(ii) complex to detect hydrogen sulfide in hypoxia.

Hydrogen sulfide (H2S) has been reported as a gaseous signaling molecule in cells. H2S modulation is dependent on the partial pressure of oxygen in cells, which means hypoxia can induce H2S production under various pathophysiological conditions. Hypoxia is a common condition in solid tumors and can lead to malignant tumors that may become aggressive and result in worse prognosis. We designed and synthesized probe Cu-CD for H2S detection under hypoxia conditions. It is selective and sensitive toward various biological thiols, reactive nitrogen species (RNS), and reactive oxygen species (ROS). The fluorescence intensity of Cu-CD in the cytoplasms of HeLa and EMT6 cells was enhanced in proportion to the concentration of exogenous/endogenous H2S. Moreover, Cu-CD can be able to detect endogenous H2S production accompanied by expression of HIF-1α. Therefore, Cu-CD can be a key tool to explore how H2S contributes to neovascularization and growth of solid tumor tissues in pathophysiological or hypoxic conditions.