A genetically encoded protein tag for control and quantitative imaging of CAR T cell therapy.

Chimeric antigen receptor (CAR) T cell therapy has been successful for hematological malignancies. Still, a lack of efficacy and potential toxicities have slowed its application for other indications. Furthermore, CAR T cells undergo dynamic expansion and contraction in vivo that cannot be easily predicted or controlled. Therefore, the safety and utility of such therapies could be enhanced by engineered mechanisms that engender reversible control and quantitative monitoring. Here, we use a genetic tag based on the enzyme Escherichia coli dihydrofolate reductase (eDHFR), and derivatives of trimethoprim (TMP) to modulate and monitor CAR expression and T cell activity. We fused eDHFR to the CAR C terminus, allowing regulation with TMP-based proteolysis-targeting chimeric small molecules (PROTACs). Fusion of eDHFR to the CAR does not interfere with cell signaling or its cytotoxic function, and the addition of TMP-based PROTACs results in a reversible and dose-dependent inhibition of CAR activity via the proteosome. We show the regulation of CAR expression in vivo and demonstrate imaging of the cells with TMP radiotracers. In vitro immunogenicity assays using primary human immune cells and overlapping peptide fragments of eDHFR showed no memory immune repertoire for eDHFR. Overall, this translationally-orientied approach allows for temporal monitoring and image-guided control of cell-based therapies.

Regulation of eDHFR-tagged proteins with trimethoprim PROTACs.

Temporal control of protein levels in cells and living animals can be used to improve our understanding of protein function. In addition, control of engineered proteins could be used in therapeutic applications. PRoteolysis-TArgeting Chimeras (PROTACs) have emerged as a small-molecule-driven strategy to achieve rapid, post-translational regulation of protein abundance via recruitment of an E3 ligase to the target protein of interest. Here, we develop several PROTAC molecules by covalently linking the antibiotic trimethoprim (TMP) to pomalidomide, a ligand for the E3 ligase, Cereblon. These molecules induce degradation of proteins of interest (POIs) genetically fused to a small protein domain, E. coli dihydrofolate reductase (eDHFR), the molecular target of TMP. We show that various eDHFR-tagged proteins can be robustly degraded to 95% of maximum expression with PROTAC molecule 7c. Moreover, TMP-based PROTACs minimally affect the expression of immunomodulatory imide drug (IMiD)-sensitive neosubstrates using proteomic and biochemical assays. Finally, we show multiplexed regulation with another known degron-PROTAC pair, as well as reversible protein regulation in a rodent model of metastatic cancer, demonstrating the formidable strength of this system. Altogether, TMP PROTACs are a robust approach for selective and reversible degradation of eDHFR-tagged proteins in vitro and in vivo.

"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.

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 evaluation of the effects of combined boron and gadolinium neutron capture therapy in mouse models.

While boron neutron capture therapy (BNCT) depends primarily on the short flight range of the alpha particles emitted by the boron neutron capture reaction, gadolinium neutron capture therapy (GdNCT) mainly relies on gamma rays and Auger electrons released by the gadolinium neutron capture reaction. BNCT and GdNCT can be complementary in tumor therapy. Here, we studied the combined effects of BNCT and GdNCT when boron and gadolinium compounds were co-injected, followed by thermal neutron irradiation, and compared these effects with those of the single therapies. In cytotoxicity studies, some additive effects (32‒43%) were observed when CT26 cells were treated with both boron- and gadolinium-encapsulated PEGylated liposomes (B- and Gd-liposomes) compared to the single treatments. The tumor-suppressive effect was greater when BNCT was followed by GdNCT at an interval of 10 days rather than vice versa. However, tumor suppression with co-injection of B- and Gd-liposomes into tumor-bearing mice followed by neutron beam irradiation was comparable to that observed with Gd-liposome-only treatment but lower than B-liposome-only injection. No additive effect was observed with the combination of BNCT and GdNCT, which could be due to the shielding effect of gadolinium against thermal neutrons because of its overwhelmingly large thermal neutron cross section.

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.

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.

PEGylated liposome encapsulating nido-carborane showed significant tumor suppression in boron neutron capture therapy (BNCT).

Boron neutron capture therapy (BNCT) is a binary radiotherapy based on nuclear reactions that occur when boron-10 is irradiated with neutrons, which result in the ejection of high-energy alpha particles. Successful BNCT requires the efficient delivery of a boron-containing compound to effect high concentrations in tumor cells while minimizing uptake in normal tissues. In this study, PEGylated liposomes were employed as boron carriers to maximize delivery to tumors and minimize uptake in the reticuloendothelial system (RES). The water-soluble potassium salt of nido-7,8-carborane, nido-carborane, was chosen as the boron source due to its high boron content per molecule. Nido-carborane was encapsulated in the aqueous cores of PEGylated liposomes by hydrating thin lipid films. Repeated freezing and thawing increased nido-carborane loading by up to 47.5 ± 3.1%. The average hydrodynamic diameter of the prepared boronated liposomes was determined to be 114.5 ± 28 nm through dynamic light scattering (DLS) measurement. Globular liposomes approximately 100 nm in diameter were clearly visible in transmission electron microscope (TEM) images. The viability of tumor cells following BNCT with 70 µM nido-carborane was reduced to 17.1% compared to irradiated control cells, which did not contain boronated liposomes. Confocal microscopy revealed that fluorescently labeled liposomes injected into the tail veins of mice were deeply and evenly distributed in tumor tissues and localized in the cytoplasm of tumor cells. When mice were properly shielded with a 12 mm-thick polyethylene board during in-vivo irradiation at a thermal neutron flux of 1.94 × 104/cm2·sec, almost complete tumor suppression was achieved in tumor models injected with boronated liposomes (21.0 mg 10B/kg). Two BNCT cycles spaced 10 days apart further enhanced the therapeutic anti-tumor effect, even when the dose was lowered to 10.5 mg 10B/kg. No notable weight loss was observed in the tumor models during the BNCT study.

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.

High in Vivo Stability of 64Cu-Labeled Cross-Bridged Chelators Is a Crucial Factor in Improved Tumor Imaging of RGD Peptide Conjugates.

Although the importance of bifunctional chelators (BFCs) is well recognized, the chemophysical parameters of chelators that govern the biological behavior of the corresponding bioconjugates have not been clearly elucidated. Here, five BFCs closely related in structure were conjugated with a cyclic RGD peptide and radiolabeled with Cu-64 ions. Various biophysical and chemical properties of the Cu(II) complexes were analyzed with the aim of identifying correlations between individual factors and the biological behavior of the conjugates. Tumor uptake and body clearance of the 64Cu-labeled bioconjugates were directly compared by animal PET imaging in animal models, which was further supported by biodistribution studies. Conjugates containing propylene cross-bridged chelators showed higher tumor uptake, while a closely related ethylene cross-bridged analogue exhibited rapid body clearance. High in vivo stability of the copper-chelator complex was strongly correlated with high tumor uptake, while the overall lipophilicity of the bioconjugate affected both tumor uptake and body clearance.

Immobilization of the Gas Signaling Molecule H2 S by Radioisotopes: Detection, Quantification, and In Vivo Imaging.

Hydrogen sulfide (H2 S) has multifunctional roles as a gas signaling molecule in living systems. However, the efficient detection and imaging of H2 S in live animals is very challenging. Herein, we report the first radioisotope-based immobilization technique for the detection, quantification, and in vivo imaging of endogenous H2 S. Macrocyclic (64) Cu complexes that instantly reacted with gaseous H2 S to form insoluble (64) CuS in a highly sensitive and selective manner were prepared. The H2 S concentration in biological samples was measured by a thin-layer radiochromatography method. When (64) Cu-cyclen was injected into mice, an elevated H2 S concentration in the inflamed paw was clearly visualized and quantified by Cerenkov luminescence and positron emission tomography (PET) imaging. PET imaging was also able to pinpoint increased H2 S levels in a millimeter-sized infarcted lesion of the rat heart.