Preclinical evaluation of a brain penetrant PARP PET imaging probe in rat glioblastoma and nonhuman primates.

PURPOSE: Currently, there are multiple active clinical trials involving poly(ADP-ribose) polymerase (PARP) inhibitors in the treatment of glioblastoma. The noninvasive quantification of baseline PARP expression using positron emission tomography (PET) may provide prognostic information and lead to more precise treatment. Due to the lack of brain-penetrant PARP imaging agents, the reliable and accurate in vivo quantification of PARP in the brain remains elusive. Herein, we report the synthesis of a brain-penetrant PARP PET tracer, (R)-2-(2-methyl-1-(methyl-11C)pyrrolidin-2-yl)-1H-benzo[d]imidazole-4-carboxamide ([11C]PyBic), and its preclinical evaluations in a syngeneic RG2 rat glioblastoma model and healthy nonhuman primates. METHODS: We synthesized [11C]PyBic using veliparib as the labeling precursor, performed dynamic PET scans on RG2 tumor-bearing rats and calculated the distribution volume ratio (DVR) using simplified reference region method 2 (SRTM2) with the contralateral nontumor brain region as the reference region. We performed biodistribution studies, western blot, and immunostaining studies to validate the in vivo PET quantification results. We characterized the brain kinetics and binding specificity of [11C]PyBic in nonhuman primates on FOCUS220 scanner and calculated the volume of distribution (VT), nondisplaceable volume of distribution (VND), and nondisplaceable binding potential (BPND) in selected brain regions. RESULTS: [11C]PyBic was synthesized efficiently in one step, with greater than 97% radiochemical and chemical purity and molar activity of 148 ± 85 MBq/nmol (n = 6). [11C]PyBic demonstrated PARP-specific binding in RG2 tumors, with 74% of tracer binding in tumors blocked by preinjected veliparib (i.v., 5 mg/kg). The in vivo PET imaging results were corroborated by ex vivo biodistribution, PARP1 immunohistochemistry and immunoblotting data. Furthermore, brain penetration of [11C]PyBic was confirmed by quantitative monkey brain PET, which showed high specific uptake (BPND > 3) and low nonspecific uptake (VND < 3 mL/cm3) in the monkey brain. CONCLUSION: [11C]PyBic is the first brain-penetrant PARP PET tracer validated in a rat glioblastoma model and healthy nonhuman primates. The brain kinetics of [11C]PyBic are suitable for noninvasive quantification of available PARP binding in the brain, which posits [11C]PyBic to have broad applications in oncology and neuroimaging.

Poly (ADP-ribose) polymerases as PET imaging targets for central nervous system diseases.

Poly (ADP-ribose) polymerases (PARPs) constitute of 17 members that are associated with divergent cellular processes and play a crucial role in DNA repair, chromatin organization, genome integrity, apoptosis, and inflammation. Multiple lines of evidence have shown that activated PARP1 is associated with intense DNA damage and irritating inflammatory responses, which are in turn related to etiologies of various neurological disorders. PARP1/2 as plausible therapeutic targets have attracted considerable interests, and multitudes of PARP1/2 inhibitors have emerged for treating cancer, metabolic, inflammatory, and neurological disorders. Furthermore, PARP1/2 as imaging targets have been shown to detect, delineate, and predict therapeutic responses in many diseases by locating and quantifying the expression levels of PARP1/2. PARP1/2-directed noninvasive positron emission tomography (PET) has potential in diagnosing and prognosing neurological diseases. However, quantitative PARP PET imaging in the central nervous system (CNS) has evaded us due to the challenges of developing blood-brain barrier (BBB) penetrable PARP radioligands. Here, we review PARP1/2's relevance in CNS diseases, summarize the recent progress on PARP PET and discuss the possibilities of developing novel PARP radiotracers for CNS diseases.

Histone Methyltransferase SETDB1 Promotes Immune Evasion in Colorectal Cancer via FOSB-Mediated Downregulation of MicroRNA-22 through BATF3/PD-L1 Pathway.

Tumors may develop a variety of immune evasion mechanisms during the progression of colorectal cancer (CRC). Here, we intended to explore the mechanism of histone methyltransferase SETDB1 in immune evasion in CRC. The expression of SETDB1, microRNA-22 (miR-22), BATF3, PD-L1, and FOSB in CRC tissues and cells was determined with their interactions analyzed also. Gain-of-function and loss-of-function approaches were employed to evaluate the effects of the SETDB1/FOSB/miR-22/BATF3/PD-L1 axis on T cell function, immune cell infiltration, and tumorigenesis. Aberrant high SETDB1 expression in CRC was positively associated with PD-L1 expression. SETDB1 negatively regulated miR-22 expression by downregulating FOSB expression, while miR-22 downregulated PD-L1 expression via targeting BATF3. Furthermore, SETDB1 silencing promoted the T cell-mediated cytotoxicity to tumor cells via the FOSB/miR-22/BATF3/PD-L1 axis and hindered CRC tumor growth in mice while leading to decreased immune cell infiltration. Taken together, SETDB1 could activate the BATF3/PD-L1 axis by inhibiting FOSB-mediated miR-22 and promote immune evasion in CRC, which provides a better understanding of the mechanisms underlying immune evasion in CRC.

Feasibility of imaging synaptic density in the human spinal cord using [11C]UCB-J PET.

PURPOSE: Neuronal damage and synapse loss in the spinal cord (SC) have been implicated in spinal cord injury (SCI) and neurodegenerative disorders such as Amyotrophic Lateral Sclerosis (ALS). Current standards of diagnosis for SCI include CT or MRI imaging to evaluate injury severity. The current study explores the use of PET imaging with [11C]UCB-J, which targets the synaptic vesicle protein 2A (SV2A), in the human spinal cord, as a way to visualize synaptic density and integrity in vivo. RESULTS: First, simulations of baseline and blocking [11C]UCB-J HRRT scans were performed, based on SC dimensions and SV2A distribution to predict VT, VND, and VS values. Next, human baseline and blocking [11C]UCB-J HRRT images were used to estimate these values in the cervical SC (cSC). Simulation results had excellent agreement with observed values of VT, VND, and VS from the real human data, with baseline VT, VND, and VS of 3.07, 2.15, and 0.92 mL/cm3, respectively, with a BPND of 0.43. Lastly, we explored full SC imaging with whole-body images. Using automated SC regions of interest (ROIs) for the full SC, cSC, and thoracic SC (tSC), the distribution volume ratio (DVR) was estimated using the brain gray matter as a reference region to evaluate SC SV2A density relative to the brain. In full body imaging, DVR values of full SC, cSC, and tSC were 0.115, 0.145, and 0.112, respectively. Therefore, measured [11C]UCB-J uptake, and thus SV2A density, is much lower in the SC than in the brain. CONCLUSIONS: The results presented here provide evidence for the feasibility of SV2A PET imaging in the human SC, however, specific binding of [11C]UCB-J is low. Ongoing and future work include further classification of SV2A distribution in the SC as well as exploring higher-affinity PET radioligands for SC imaging.

Intravenous Delivery of RNA Encoding Anti-PD-1 Human Monoclonal Antibody for Treating Intestinal Cancer.

Recently, antibody-based therapeutic agents are becoming most leading biologics for treating many diseases, especially for cancer. However, large-scale application of antibody drugs is still hampered by high cost and complex technical process. Endogenous expression of proteins or antibodies can be achieved by applying in vitro transcription (IVT) technique to produce mRNA and then deliver into body, which supplies opportunity to avoid many disadvantages in antibody production as well as clinical applications. Here, we designed the IVT-mRNA encoding the Pembrolizumab, as a commercial anti-PD-1 monoclonal antibody (mAb). The in vitro functional properties and in vivo antitumor activities of the Pembrolizumab expressed from mRNA were both assessed. Maximized expression level of the Pembrolizumab from IVT-mRNA was achieved via optimizing the usage of signal peptide and molar ratio of heavy/light chain. Then the mRNA was further formulated by lipid nanoparticle (LNP), which enable efficient in vivo delivery and protect mRNA from degradation. Intravenously delivered the single dose of mRNA-LNPs in mice resulted in duration of serum Pembrolizumab level over 25 µg/mL more than 35 days. Pharmacokinetic study exhibited significantly enhanced drug exposure of mRNA-encoded mAbs compared with direct injection of Pembrolizumab at same dose. Chronic treatment of the tumor-bearing mice with LNP-encapsulated Pembrolizumab mRNA effectively downregulated the growth of intestinal tumors and improved the animal survival. In brief, our present research demonstrated that the application of LNP-encapsulated IVT-mRNA can change the human body into a protein drug manufacturing site to express full-size mAbs for treating cancer and hold potential to be a novel alternative to protein-based therapies.

Combination therapy with Nab-paclitaxel and the interleukin-15 fused with anti-human serum albumin nanobody as a synergistic treatment for colorectal cancer.

This study determines the effect of Nab-paclitaxel in combination with IL-15 fusion protein, containing IL-15 and an anti-HSA nanobody domain, on colorectal cancer bearing mice. In vitro binding test of IL15 fusion protein to HSA and Nab-paclitaxel, as well as CTLL-2 cell stimulation assay were performed. The tumor inhibitory effects of Nab-paclitaxel in combination with IL-15 fusion protein was evaluated in the HCT116 bearing murine model. Moreover, the population and function of cytotoxic T cells and M1 macrophages, as well as MDSCs and Treg cells, were also further examined. As a result, combination therapy of Nab-paclitaxel and IL-15 fusion protein effectively inhibits the tumor growth and produced a 78% reduction in tumor size for HCT116, as compared to vehicle group. In the TDLN for the combination group, there were 18% of CD8+ IFN-γ + T-cells and 0.47% CD4+CD25+FOXP3+ regulatory T-cells, as opposed to 5.0% and 5.1%, respectively, for the model control group. Combination therapy further exhibited enhanced suppressive effects on the accumulation of CD11b+GR-1+ MDSC in spleen and bone marrow. Furthermore, Nab-paclitaxel and IL-15 fusion protein showed a significant suppression of NF-κB-mediated immune suppressive markers and increased expression of CD8, Granzyme B, CD62L, CD49b, and CD86 without obvious organ toxicity. In conclusion, combination therapy of Nab-paclitaxel and IL-15 fusion protein can effectively stimulate the antitumor activity of immune effector cells, thereby inhibiting immunosuppressive cells within the TME of colorectal cancer, and the overall therapeutic effect has a significant advantage over monotherapy.AbbreviationsInterleukin 15, IL-15; Human serum albumin, HSA; Myeloid-derived suppressor cells, MDSC; Albumin binding domain, ABD; Tumor drainage lymph node, TDLN; Natural killer (NK); Tumor-draining lymph node (TDLN); Tumor infiltrating lymphocyte, TIL; Immunogenic cell death, ICD; Enhanced permeability retention, EPR; Liposomal doxorubicin, Doxil; 5-fluorouracil, 5-FU.

Engineered mRNA-expressed bispecific antibody prevent intestinal cancer via lipid nanoparticle delivery.

The potential of antibodies, especially for the bispecific antibodies, are limited by high cost and complex technical process of development and manufacturing. A cost-effective and rapid platform for the endogenous antibodies expression via using the in vitro transcription (IVT) technique to produce nucleoside-modified mRNA and then encapsulated into lipid nanoparticle (LNP) may turn the body to a manufactory. Coinhibitory pathway of programmed death ligand 1 (PD-L1) and programmed cell death protein 1 receptor (PD-1) could suppress the T-cell mediated immunity. We hypothesized that the coblocking of PD-L1 and PD-1 via bispecific antibodies may achieve more potential antitumor efficacies compare with the monospecific ones. Here, we described the application of mRNA to encode a bispecific antibody with ablated Fc immune effector functions that targets both human PD-L1 and PD-1, termed XA-1, which was further assessed the in vitro functional activities and in vivo antitumor efficacies. The in vitro mRNA-encoded XA-1 held comparable abilities to fully block the PD-1/PD-L1 pathway as well as to enhance functional T cell activation compared to XA-1 protein from CHO cell source. Pharmacokinetic tests showed enhanced area under curve (AUC) of mRNA-encoded XA-1 compared with XA-1 at same dose. Chronic treatment of LNP-encapsulated XA-1 mRNA in the mouse tumor models which were reconstituted with human immune cells effectively induced promising antitumor efficacies compared to XA-1 protein. Current results collectively demonstrated that LNP-encapsulated mRNA represents the viable delivery platform for treating cancer and hold potential to be applied in the treatment of many diseases.Abbreviations: IVT: in vitro transcription; LNP: lipid nanoparticle; hPD-1: human PD-1; hPD-L1: human PD-L1; ITS-G: Insulin-Transferrin-Selenium; Pen/Strep: penicillin-streptomycin; FBS: fetal bovine serum; TGI: tumor growth inhibition; IE1: cytomegalovirus immediate early 1; SP: signal peptide; hIgLC: human immunoglobulin kappa light chain; hIgHC: human IgG1 heavy chain; AUC: area under the curve; Cl: serum clearance; Vss: steady-state distributed volume; MLR: mixed lymphocyte reaction.

The Therapeutic Potential of Purinergic Receptors in Alzheimer's Disease and Promising Therapeutic Modulators.

Recent studies have proven that the purinergic signaling pathway plays a key role in neurotransmission and neuromodulation, and is involved in various neurodegenerative diseases and psychiatric disorders. With the characterization of the subtypes of receptors in purinergic signaling, i.e. the P1 (adenosine), P2X (ion channel) and P2Y (G protein-coupled), more attention has been paid to the pathophysiology and therapeutic potential of purinergic signaling in the central nervous system disorders. Alzheimer's disease (AD) is a progressive and deadly neurodegenerative disease that is characterized by memory loss, cognitive impairment and dementia. However, as drug development aimed to prevent or control AD has series of failures in recent years, more researchers have focused on the neuroprotection-related mechanisms such as purinergic signaling in AD patients to find a potential cure. This article reviews the recent discoveries of purinergic signaling in AD, and summarizes the potential agents as modulators for the receptors of purinergic signaling in AD-related research and treatments. Thus, our paper provides an insight into purinergic signaling in the development of anti- AD therapies.

Approaches to PET Imaging of Glioblastoma.

Glioblastoma multiforme (GBM) is the deadliest type of brain tumor, affecting approximately three in 100,000 adults annually. Positron emission tomography (PET) imaging provides an important non-invasive method of measuring biochemically specific targets at GBM lesions. These powerful data can characterize tumors, predict treatment effectiveness, and monitor treatment. This review will discuss the PET imaging agents that have already been evaluated in GBM patients so far, and new imaging targets with promise for future use. Previously used PET imaging agents include the tracers for markers of proliferation ([11C]methionine; [18F]fluoro-ethyl-L-tyrosine, [18F]Fluorodopa,[18F]fluoro-thymidine, and [18F]clofarabine), hypoxia sensing ([18F]FMISO, [18F]FET-NIM, [18F]EF5, [18F]HX4, and [64Cu]ATSM), and ligands for inflammation. As cancer therapeutics evolve toward personalized medicine and therapies centered on tumor biomarkers, the development of complimentary selective PET agents can dramatically enhance these efforts. Newer biomarkers for GBM PET imaging are discussed, with some already in use for PET imaging other cancers and neurological disorders. These targets include Sigma 1, Sigma 2, programmed death ligand 1, poly-ADP-ribose polymerase, and isocitrate dehydrogenase. For GBM, these imaging agents come with additional considerations such as blood-brain barrier penetration, quantitative modeling approaches, and nonspecific binding.

64Cu-Labeled Phosphonate Cross-Bridged Chelator Conjugates of c(RGDyK) for PET/CT Imaging of Osteolytic Bone Metastases.

OBJECTIVE: The goal of this research was to evaluate c(RGDyK) conjugated to phosphonate-based cross-bridged chelators using Cu-free click chemistry in the 4T1 mouse mammary tumor bone metastasis model in comparison with 64Cu-CB-TE2A-c(RGDyK), which previously showed selective binding to integrin αvß3 on osteoclasts. EXPERIMENTAL: Two phosphonate-based cross-bridged chelators (CB-TE1A1P and CB-TE1K1P) were conjugated to c(RGDyK) through bio-orthogonal strain-promoted alkyne-azide cycloaddition. In vitro and in vivo evaluation of the 64Cu-labeled TE1A1P-DBCO-c(RGDyK) (AP-c(RGDyK)), TE1K1P-PEG4-DBCO-c(RGDyK) (KP-c(RGDyK)), and CB-TE2A-c(RGDyK) were compared in the 4T1 mouse model of bone metastasis. The affinities of the unconjugated and chelator-c(RGDyK) analogs for αvß3 integrin were determined using a competitive-binding assay. For in vivo evaluation, BALB/c mice were injected with 1 × 105 4T1/Luc cells in the left ventricle. Formation of metastases was monitored by bioluminescence imaging (BLI) followed by small-animal PET/CT 2 h postinjection of radiotracers. RESULTS: The chelator-peptide conjugates showed similar affinity to integrin αvß3, in the low nM range. PET imaging demonstrated a higher uptake in bones having metastases for all 64Cu-labeled c(RGDyK) analogs compared with bones in nontumor-bearing mice. The correlation between uptake of 64Cu-AP-c(RGDyK) and 64Cu-KP-c(RGDyK) in bones with metastases based on PET/CT imaging, and osteoclast number based on histomorphometry, was improved over the previously investigated 64Cu-CB-TE2A-c(RGDyK). CONCLUSION: These data suggest that the phosphonate chelator conjugates of c(RDGyK) peptides are promising PET tracers suitable for imaging tumor-associated osteoclasts in bone metastases.

Bridging from Brain to Tumor Imaging: (S)-(-)- and (R)-(+)-[18F]Fluspidine for Investigation of Sigma-1 Receptors in Tumor-Bearing Mice.

Sigma-1 receptors (Sig1R) are highly expressed in various human cancer cells and hence imaging of this target with positron emission tomography (PET) can contribute to a better understanding of tumor pathophysiology and support the development of antineoplastic drugs. Two Sig1R-specific radiolabeled enantiomers (S)-(-)- and (R)-(+)-[18F]fluspidine were investigated in several tumor cell lines including melanoma, squamous cell/epidermoid carcinoma, prostate carcinoma, and glioblastoma. Dynamic PET scans were performed in mice to investigate the suitability of both radiotracers for tumor imaging. The Sig1R expression in the respective tumors was confirmed by Western blot. Rather low radiotracer uptake was found in heterotopically (subcutaneously) implanted tumors. Therefore, a brain tumor model (U87-MG) with orthotopic implantation was chosen to investigate the suitability of the two Sig1R radiotracers for brain tumor imaging. High tumor uptake as well as a favorable tumor-to-background ratio was found. These results suggest that Sig1R PET imaging of brain tumors with [18F]fluspidine could be possible. Further studies with this tumor model will be performed to confirm specific binding and the integrity of the blood-brain barrier (BBB).

miR-9 induces cell arrest and apoptosis of oral squamous cell carcinoma via CDK 4/6 pathway.

Oral cancer remains a major public concern with considerable socioeconomic impact in the world, especially in southeast Asia. Despite substantial advancements have been made in treating oral cancer, the five-year survival rate for OSCC remained undesirable, and 35-55% patients developed recurrence within two years even with multimodality therapeutic strategies. Hence, identification of novel biomarkers for diagnosis and evaluating clinical outcome is of vital importance. MicroRNAs are 22-24 nt non-coding RNAs that could bind to 3' UTR of target mRNAs, thereby inducing degradation of mRNA or inhibiting translation. Due to its implication in regulation of post-transcriptional processes, the role of miRNAs in malignancies has been extensively studied, among which the discovery of functional miR-9 in oral squamous cell carcinoma (OSCC) remained to be elucidated. We first demonstrated that miR-9 was down-regulated in 21 OSCC patients, and we further found that forced expression of miR-9 could result in deterred cell proliferation and decreased ability to migrate and form colonies. Flow cytometry displayed cell-cycle arrested at G0/G1 phase. We next used Targetscan to predict target genes of miR-9. CDK6, a protein highly implicated in cell cycle control, was chosen for verification. Down-regulation of CDK6 and Cyclin D1 in Tca8113 transfected with miR-9 mimics indicate that the complex formed by both proteins may be the effector of the antiproliferative function of miR-9 in OSCCs. Considering small molecules are developed to target CDK4/6, our finding may provide valuable scientific evidence for research and development of therapies and diagnostic methodology in OSCCs.

PET Imaging Evaluation of Four σ1 Radiotracers in Nonhuman Primates.

The σ1 receptors (S1Rs) are implicated in a variety of diseases including Alzheimer disease and cancer. Previous PET S1R radiotracers are characterized by slow kinetics or off-target binding that impedes their use in humans. Here, we report the first PET imaging evaluation in rhesus monkeys of 4 18F-labeled spirocyclic piperidine-based PET radiotracers (18F-1 to 18F-4). Methods: Baseline scans for the 4 radiotracers were obtained on an adult male rhesus monkey. Blocking scans were obtained with the S1R-selective agonist SA4503 to assess binding specificity of 18F-2 and 18F-4 Arterial input functions were measured, and binding parameters were determined with kinetic modeling analysis. Results: In the rhesus brain, all 4 radiotracers showed high and fast uptake. Tissue activity washout was rapid for 18F-2 and 18F-4, and much slower for 18F-1 and 18F-3, in line with their respective in vitro S1R-binding affinities. Both the 1-tissue-compartment and multilinear analysis-1 kinetic models provided good fits of time-activity curves and reliable estimates of distribution volume. Regional distribution volume values were highest in the cingulate cortex and lowest in the thalamus for all radiotracers. 18F-4 showed greater differential uptake across brain regions and 3-fold-higher binding potential than 18F-2 SA4503 at the dose of 0.5 mg/kg blocked approximately 85% (18F-2) and 95% (18F-4) of radiotracer binding. Conclusion: Tracers 18F-2 and 18F-4 displayed high brain uptake and fast tissue kinetics, with 18F-4 having higher specific binding signals than 18F-2 in the same monkey. Taken together, these data indicate that both 18F-2 and 18F-4 possess the requisite kinetic and imaging properties as viable PET tracers for imaging S1R in the human brain.

Synthesis and preliminary evaluation of an ¹8F-labeled oleic acid analog for PET imaging of fatty acid uptake and metabolism.

INTRODUCTION: Imaging fatty acid uptake and utilization has broad impact in investigating myocardial diseases, hepatic functions, tumor progression, and the metabolic state of adipose tissue. The SPECT tracer (123)I-15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (BMIPP) is a clinically used nuclear medicine tracer to image myocardial uptake of fatty acid. Although ((18)F-5) has been in clinical use for PET imaging of adipose tissue as well as the myocardium, here we developed a click oleate analog to compare to FTO, with the goal of improved stability to defluorination and suitability for imaging myocardial uptake and oxidation of fatty acids. METHODS: A rapid and convenient synthetic approach for a precursor to a (18)F-labeled oleate analog using click chemistry was developed and evaluated for PET imaging in fasted mice. RESULTS: The overall yield for the preparation of the labeling precursor of the clicked oleate analog was 12%. This precursor was efficiently radiolabeled with F-18 in 17% non-decay-corrected radiochemical yield. PET/CT imaging and biodistribution results show that this fatty acid analog had reasonable heart uptake (0.94±0.28 %ID/g at 0.5 h p.i.) and heart-to-muscle ratio (2.05±0.39 at 0.5h p.i.) and is a potential lead for developing new PET tracers to image fatty acid uptake and utilization using click chemistry methodologies. The synthetic route to FTO was optimized to three steps from known starting materials. CONCLUSION: While the uptake of the clicked oleic acid analog was sufficient for visualizing the myocardium in mice, the preliminary metabolism data suggest that only a fraction of the uptake was due to fatty acid beta-oxidation. Studies are under way to explore the uptake/oxidation mechanism and kinetics.

Cu(I)-assisted click chemistry strategy for conjugation of non-protected cross-bridged macrocyclic chelators to tumour-targeting peptides.

Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry has inherent challenges for copper-labeled radiopharmaceuticals. An azide-modified phosphonate-based cross-bridged macrocyclic chelator was synthesized for click chemistry conjugation with azide-modified Y3-TATE (a somatostatin analogue) on resin, without the need for protecting the chelator. The (64)Cu-labeled bioconjugate shows favourable in vitro and in vivo behaviour.

64Cu-labeled somatostatin analogues conjugated with cross-bridged phosphonate-based chelators via strain-promoted click chemistry for PET imaging: in silico through in vivo studies.

Somatostatin receptor subtype 2 (sstr2) is a G-protein-coupled receptor (GPCR) that is overexpressed in neuroendocrine tumors. The homology model of sstr2 was built and was used to aid the design of new somatostatin analogues modified with phosphonate-containing cross-bridged chelators for evaluation of using them as PET imaging radiopharmaceuticals. The new generation chelators were conjugated to Tyr3-octreotate (Y3-TATE) through bioorthogonal, strain-promoted alkyne azide cycloaddition (SPAAC) to form CB-TE1A1P-DBCO-Y3-TATE (AP) and CB-TE1K1P-PEG4-DBCO-Y3-TATE (KP) in improved yields compared to standard direct conjugation methods of amide bond formation. Consistent with docking studies, the clicked bioconjugates showed high binding affinities to sstr2, with Kd values ranging from 0.6 to 2.3 nM. Selected isomers of the clicked products were used in biodistribution and PET/CT imaging. Introduction of the bulky dibenzocyclooctyne group in AP decreased clearance rates from circulation. However, the additional carboxylate group and PEG linker from the KP conjugate significantly improved labeling conditions and in vivo stability of the copper complex and ameliorated the slower pharmacokinetics of the clicked somatostatin analogues.

Comparison of conjugation strategies of cross-bridged macrocyclic chelators with cetuximab for copper-64 radiolabeling and PET imaging of EGFR in colorectal tumor-bearing mice.

Epidermal growth-factor receptor (EGFR) is overexpressed in a wide variety of solid tumors and has served as a well-characterized target for cancer imaging and therapy. Cetuximab was the first mAb targeting EGFR approved by the FDA for the treatment of metastatic colorectal and head and neck cancers. Previous studies showed that (64)Cu (T1/2 = 12.7 h; ß(+) (17.4%)) labeled DOTA-cetuximab showed promise for PET imaging of EGFR-positive tumors; however the in vivo stability of this compound has been questioned. In this study, two recently developed cross-bridged macrocyclic chelators (CB-TE1A1P and CB-TE1K1P) were conjugated to cetuximab using standard NHS coupling procedures and/or strain-promoted azide-alkyne cycloaddition (SPAAC) methodologies. The radiolabeling and in vitro/vivo evaluation of the resulting cetuximab conjugates were compared. Improved Cu-64 labeling efficiency and high specific activity (684 kBq/µg, decay corrected to the end of bombardment) were obtained with the CB-TE1K1P-PEG4-click-cetuximab conjugate. Saturation binding assays indicated that the prepared cetuximab conjugates had comparable affinity (1.32-2.00 nM) in the HCT116 human colorectal tumor cell membranes. In the subsequent in vivo evaluation, (64)Cu-CB-TE1K1P-PEG4-click-cetuximab demonstrated more rapid renal clearance with a higher tumor/nontumor ratio than other (64)Cu-labeled cetuximab conjugates, and it shows the greatest promise for imaging and therapy of EGFR-positive tumors.