Synthesis and preclinical evaluation of [11C]EAI045 as a PET tracer for imaging tumors expressing mutated epidermal growth factor receptor.

BACKGROUND: Mutations in the epidermal growth factor receptor (EGFR) kinase domain are common in non-small cell lung cancer. Conventional tyrosine kinase inhibitors target the mutation site in the ATP binding pocket, thereby inhibiting the receptor's function. However, subsequent treatment resistance mutations in the ATP binding site are common. The EGFR allosteric inhibitor, EAI045, is proposed to have an alternative mechanism of action, disrupting receptor signaling independent of the ATP-binding site. The antibody cetuximab is hypothesized to increase the number of accessible allosteric pockets for EAI045, thus increasing the potency of the inhibitor. This work aimed to gain further knowledge on pharmacokinetics, the EGFR mutation-targeting potential, and the influence of cetuximab on the uptake by radiolabeling EAI045 with carbon-11 and tritium. RESULTS: 2-(5-fluoro-2-hydroxyphenyl)-2-((2-iodobenzyl)amino)-N-(thiazol-2-yl)acetamide and 2-(5-fluoro-2-hydroxyphenyl)-N-(5-iodothiazol-2-yl)-2-(1-oxoisoindolin-2-yl)acetamide were synthesized as precursors for the carbon-11 and tritium labeling of EAI045, respectively. [11C]EAI045 was synthesized using [11C]CO in a palladium-catalyzed ring closure in a 10 ± 1% radiochemical yield (decay corrected to end of [11C]CO2 production), > 97% radiochemical purity and 26 ± 1 GBq/µmol molar activity (determined at end of synthesis) in 51 min. [3H]EAI045 was synthesized by a tritium-halogen exchange in a 0.2% radiochemical yield, 98% radiochemical purity, and 763 kBq/nmol molar activity. The ability of [11C]EAI045 to differentiate between L858R/T790M mutated EGFR expressing H1975 xenografts and wild-type EGFR expressing A549 xenografts was evaluated in female nu/nu mice. The uptake was statistically significantly higher in H1975 xenografts compared to A549 xenografts (0.45 ± 0.07%ID/g vs. 0.31 ± 0.10%ID/g, P = 0.0166). The synergy in inhibition between EAI045 and cetuximab was evaluated in vivo and in vitro. While there was some indication that cetuximab influenced the uptake of [3H]EAI045 in vitro, this could not be confirmed in vivo when tumor-bearing mice were administered cetuximab (0.5 mg), 24 h prior to injection of [11C]EAI045. CONCLUSIONS: EAI045 was successfully labeled with tritium and carbon-11, and the in vivo results indicated [11C]EAI045 may be able to distinguish between mutated and non-mutated EGFR in non-small cell lung cancer mouse models. Cetuximab was hypothesized to increase EAI045 uptake; however, no significant effect was observed on the uptake of [11C]EAI045 in vivo or [3H]EAI045 in vitro in H1975 xenografts and cells.

Synthesis and Preclinical Evaluation of [Methylpiperazine-11C]brigatinib as a PET Tracer Targeting Both Mutated Epidermal Growth Factor Receptor and Anaplastic Lymphoma Kinase.

Brigatinib, a tyrosine kinase inhibitor (TKI) with specificity for gene rearranged anaplastic lymphoma kinase (ALK), such as the EML4-ALK, has shown a potential to inhibit mutated epidermal growth factor receptor (EGFR). In this study, N-desmethyl brigatinib was successfully synthesized as a precursor in five steps. Radiolabeling with [11C]methyl iodide produced [methylpiperazine-11C]brigatinib in a 10 ± 2% radiochemical yield, 91 ± 17 GBq/µmol molar activity, and ≥95% radiochemical purity in 49 ± 4 min. [Methylpiperazine-11C]brigatinib was evaluated in non-small cell lung cancer xenografted female nu/nu mice. An hour post-injection (p.i.), 87% of the total radioactivity in plasma originated from intact [methylpiperazine-11C]brigatinib. Significant differences in tumor uptake were observed between the endogenously EML4-ALK mutated H2228 and the control xenograft A549. The tumor-to-blood ratio in H2228 xenografts could be reduced by pretreatment with ALK inhibitor crizotinib. Tracer uptake in EGFR Del19 mutated HCC827 and EML4-ALK fusion A549 was not significantly different from uptake in A549 xenografts.

Synthesis and preclinical evaluation of two osimertinib isotopologues labeled with carbon-11 as PET tracers targeting the tyrosine kinase domain of the epidermal growth factor receptor.

INTRODUCTION: Osimertinib is a third-generation tyrosine kinase inhibitor (TKI) that is able to inhibit the EGFR treatment resistance mutation T790M and primary EGFR mutations Del19 and L858R. The aim of the study was to evaluate the potential of carbon-11 labeled osimertinib to be used as a tracer for the PET imaging of tumors bearing the T790M mutation. METHODS: Osimertinib was labeled with carbon-11 at two positions, and the effect of the labeling position on the metabolism and biodistribution was studied in female nu/nu mice. The mutation status specificity of osimertinib was confirmed in vitro in a cell growth inhibition experiment, and the tumor-targeting potential of the carbon-11 isotopologues was evaluated using female nu/nu mice xenografted with NSCLC cell lines; the wild-type EGFR expressing A549, the primary Del19 EGFR mutated HCC827 and the resistance T790M/L858R mutated H1975. One of the osimertinib tracers was selected based on the results acquired and evaluated for tracer specificity and selectivity by assessment of tumor uptake in a PET study where HCC827 tumor-bearing mice were pretreated with osimertinib or afatinib. RESULTS: [Methylindole-11C]- and [dimethylamine-11C]osimertinib were synthesized by 11C-methylation of precursors AZ5104 and AZ7550, respectively. Rapid metabolism of both analogs of [11C]osimertinib was observed. Although the tumor uptake and retention of [methylindole-11C]- and [dimethylamine-11C]osimertinib in tumors were similar, the tumor-to-muscle ratios appeared to be higher for [methylindole-11C]osimertinib. The highest uptake, tumor-to-blood, and tumor-to-muscle ratio were observed in the Del19 EGFR mutated HCC827 tumors. However, the specificity and selectivity of [methylindole-11C]osimertinib PET could not be demonstrated in HCC827 tumors. The uptake of [methylindole-11C]osimertinib was not significantly higher in T790M resistance mutated H1975 xenografts compared to the negative control cell line A549. CONCLUSIONS: Osimertinib was successfully labeled at two positions with carbon-11, yielding two EGFR PET tracers, [methylindole-11C]osimertinib and [dimethylamine-11C]osimertinib. The preclinical evaluation demonstrated uptake and retention in three NSCLC xenografts; A549, HCC827, and H1975. The highest uptake was observed in the primary Del19 EGFR mutated HCC827. The ability of [methylindole-11C]osimertinib to distinguish between the T790M resistance mutated H1975 xenografts and the wild-type EGFR expressing A549 could not be confirmed in the ex vivo study.

Imaging the TGFß type I receptor in pulmonary arterial hypertension.

Transforming growth factor ß (TGFß) activity is perturbed in remodelled pulmonary vasculature of patients with pulmonary arterial hypertension (PAH), cancer, vascular diseases and developmental disorders. Inhibition of TGFß, which signals via activin receptor-like kinase 5 (ALK5), prevents progression and development of experimental PAH. The purpose of this study was to assess two ALK5 targeting positron emission tomography (PET) tracers ([11C]LR111 and [18F]EW-7197) for imaging ALK5 in monocrotaline (MCT)- and Sugen/hypoxia (SuHx)-induced PAH. Both tracers were subjected to extensive in vitro and in vivo studies. [11C]LR111 showed the highest metabolic stability, as 46 ± 2% of intact tracer was still present in rat blood plasma after 60 min. In autoradiography experiments, [11C]LR111 showed high ALK5 binding in vitro compared with controls, 3.2 and 1.5 times higher in SuHx and MCT, respectively. In addition, its binding could be blocked by SB431542, an adenosine triphosphate competitive ALK5 kinase inhibitor. However, [18F]EW-7197 showed the best in vivo results. 15 min after injection, uptake was 2.5 and 1.4 times higher in the SuHx and MCT lungs, compared with controls. Therefore, [18F]EW-7197 is a promising PET tracer for ALK5 imaging in PAH.

Advancing 89Zr-immuno-PET in neuroscience with a bispecific anti-amyloid-beta monoclonal antibody - The choice of chelator is essential.

The accelerated approval of the monoclonal antibody (mAb) aducanumab as a treatment option for Alzheimer's Disease and the continued discussions about its efficacy have shown that a better understanding of immunotherapy for the treatment of neurodegenerative diseases is needed. 89Zr-immuno-PET could be a suitable tool to open new avenues for the diagnosis of CNS disorders, monitoring disease progression, and assessment of novel therapeutics. Herein, three different 89Zr-labeling strategies and direct radioiodination with 125I of a bispecific anti-amyloid-beta aducanumab derivate, consisting of aducanumab with a C-terminal fused anti-transferrin receptor binding single chain Fab fragment derived from 8D3 (Adu-8D3), were compared ex vivo and in vivo with regard to brain uptake and target engagement in an APP/PS1 Alzheimer's disease mouse model and wild type animals. Methods: Adu-8D3 and a negative control antibody, based on the HIV specific B12 antibody also carrying C-terminal fused 8D3 scFab (B12-8D3), were each conjugated with NCS-DFO, NCS-DFO*, or TFP-N-suc-DFO-Fe-ester, followed by radiolabeling with 89Zr. 125I was used as a substitute for 124I for labeling of both antibodies. 30 µg of radiolabeled mAb, corresponding to approximately 6 MBq 89Zr or 2.5 MBq 125I, were injected per mouse. PET imaging was performed 1, 3 and 7 days post injection (p.i.). All mice were sacrificed on day 7 p.i. and subjected to ex vivo biodistribution and brain autoradiography. Immunostaining on brain tissue was performed after autoradiography for further validation. Results: Ex vivo biodistribution revealed that the brain uptake of [89Zr]Zr-DFO*-NCS-Adu-8D3 (2.19 ±0.12 %ID/g) was as high as for its 125I-analog (2.21 ±0.15 %ID/g). [89Zr]Zr-DFO-NCS-Adu-8D3 and [89Zr]Zr-DFO-N-suc-Adu-8D3 showed significantly lower uptake (< 0.65 %ID/g), being in the same range as for the 89Zr-labeled controls (B12-8D3). Autoradiography of [89Zr]Zr-DFO*-NCS-Adu-8D3 and [125I]I-Adu-8D3 showed an amyloid-beta related granular uptake pattern of radioactivity. In contrast, the [89Zr]Zr-DFO-conjugates and the control antibody groups did not show any amyloid-beta related uptake pattern, indicating that DFO is inferior for 89Zr-immuno-PET imaging of the brain in comparison to DFO* for Adu-8D3. This was confirmed by day 7 PET images showing only amyloid-beta related brain uptake for [89Zr]Zr-DFO*-NCS-Adu-8D3. In wild type animals, such an uptake was not observed. Immunostaining showed a co-localization of all administered Adu-8D3 conjugates with amyloid-beta plaques. Conclusion: We successfully demonstrated that 89Zr-immuno-PET is suitable for imaging and quantifying amyloid-beta specific brain uptake using a bispecific aducanumab brain shuttling antibody, Adu-8D3, but only when using the novel chelator DFO*, and not DFO, for labeling with 89Zr.

Synthesis and preclinical evaluation of [11C]LR111 and [18F]EW-7197 as PET tracers of the activin-receptor like kinase-5.

The transforming growth factor ß (TGFß) pathway plays a complex role in cancer biology, being involved in both tumour suppression as well as promotion. Overactive TGFß signalling has been linked to multiple diseases, including cancer, pulmonary arterial hypertension, and fibrosis. One of the key meditators within this pathway is the TGFß type I receptor, also termed activin receptor-like kinase 5 (ALK5). ALK5 expression level is a key determinant of TGFß signalling intensity and duration, and perturbation has been linked to diseases. A validated ALK5 positron emission tomography (PET) tracer creates an opportunity, therefore, to study its role in human diseases. To develop ALK5 PET tracers, two small molecule ALK5 kinase inhibitors were selected as lead compounds, which were labelled with carbon-11 and fluorine-18, respectively. [11C]LR111 was synthesized with a yield of 17 ± 6%, a molar activity of 126 ± 79 GBq·µmol-1 and a purity of >95% (n = 44). [18F]EW-7197 was synthesized with a yield of 10 ± 5%, a molar activity of 183 ± 126 GBq·µmol-1 and a purity of >95% (n = 11). Metabolic stability was evaluated in vivo in mice, showing 39 ± 2% of intact [11C]LR111 and 21 ± 2% of intact [18F]EW-7197 in blood plasma at 45 min p.i. In vitro binding experiments were conducted in breast cancer MDA-MB-231 and lung cancer A431 cell lines. In addition, both tracers were used for PET imaging in MDA-MB-231 xenograft models. Selective uptake of [18F]EW-7197 and [11C]LR111 was observed in MDA-MB-231 cells, in the MDA-MB-231 tumour xenografts in vivo and in the autoradiograms. As [11C]LR111 and [18F]EW-7197 showed selectivity of binding to ALK5 in vivo and in vitro. Both tracers are thereby valuable tools for the detection of ALK5 activity.

Pretargeted PET Imaging with a TCO-Conjugated Anti-CD44v6 Chimeric mAb U36 and [89Zr]Zr-DFO-PEG5-Tz.

The recent advances in the production of engineered antibodies have facilitated the development and application of tailored, target-specific antibodies. Positron emission tomography (PET) of these antibody-based drug candidates can help to better understand their in vivo behavior. In this study, we report an in vivo proof-of-concept pretargeted immuno-PET study where we compare a pretargeting vs targeted approach using a new 89Zr-labeled tetrazine as a bio-orthogonal ligand in an inverse electron demand Diels-Alder (IEDDA) in vivo click reaction. A CD44v6-selective chimeric monoclonal U36 was selected as the targeting antibody because it has potential in immuno-PET imaging of head-and-neck squamous cell carcinoma (HNSCC). Zirconium-89 (t1/2 = 78.41 h) was selected as the radionuclide of choice to be able to make a head-to-head comparison of the pretargeted and targeted approaches. [89Zr]Zr-DFO-PEG5-Tz ([89Zr]Zr-3) was synthesized and used in pretargeted PET imaging of HNSCC xenografts (VU-SCC-OE) at 24 and 48 h after administration of a trans-cyclooctene (TCO)-functionalized U36. The pretargeted approach resulted in lower absolute tumor uptake than the targeted approach (1.5 ± 0.2 vs 17.1 ± 3.0% ID/g at 72 h p.i. U36) but with comparable tumor-to-non-target tissue ratios and significantly lower absorbed doses. In conclusion, anti-CD44v6 monoclonal antibody U36 was successfully used for 89Zr-immuno-PET imaging of HNSCC xenograft tumors using both a targeted and pretargeted approach. The results not only support the utility of the pretargeted approach in immuno-PET imaging but also demonstrate the challenges in achieving optimal in vivo IEDDA reaction efficiencies in relation to antibody pharmacokinetics.

Folate Receptor Beta for Macrophage Imaging in Rheumatoid Arthritis.

Non-invasive imaging modalities constitute an increasingly important tool in diagnostic and therapy response monitoring of patients with autoimmune diseases, including rheumatoid arthritis (RA). In particular, macrophage imaging with positron emission tomography (PET) using novel radiotracers based on differential expression of plasma membrane proteins and functioning of cellular processes may be suited for this. Over the past decade, selective expression of folate receptor ß (FRß), a glycosylphosphatidylinositol-anchored plasma membrane protein, on myeloid cells has emerged as an attractive target for macrophage imaging by exploiting the high binding affinity of folate-based PET tracers. This work discusses molecular, biochemical and functional properties of FRß, describes the preclinical development of a folate-PET tracer and the evaluation of this tracer in a translational model of arthritis for diagnostics and therapy-response monitoring, and finally the first clinical application of the folate-PET tracer in RA patients with active disease. Consequently, folate-based PET tracers hold great promise for macrophage imaging in a variety of (chronic) inflammatory (autoimmune) diseases beyond RA.

Novel Thienopyrimidine-Based PET Tracers for P2Y12 Receptor Imaging in the Brain.

The P2Y12 receptor (P2Y12R) is uniquely expressed on microglia in the brain, and its expression level directly depends on the microglial activation state. Therefore, P2Y12R provides a promising imaging marker for distinguishing the pro- and anti-inflammatory microglial phenotypes, both of which play crucial roles in neuroinflammatory diseases. In this study, three P2Y12R antagonists were selected from the literature, radiolabeled with carbon-11 or fluorine-18, and evaluated in healthy Wistar rats. Brain imaging was performed with and without blocking of efflux transporters P-glycoprotein and breast cancer resistance protein using tariquidar. Low brain uptake in healthy rats was observed for all tracers at baseline conditions, whereas blocking of efflux transporters resulted in a strong (6-7 fold) increase in brain uptake for both of them. Binding of the most promising tracer, [18F]3, was further evaluated by in vitro autoradiography on rat brain sections, ex vivo metabolite studies, and in vivo P2Y12R blocking studies. In vitro binding of [18F]3 on rat brain sections indicated high P2Y12R targeting with approximately 70% selective and specific binding. At 60 min post-injection, over 95% of radioactivity in the brain accounted for an intact tracer. In blood plasma, still 40% intact tracer was found, and formed metabolites did not enter the brain. A moderate P2Y12R blocking effect was observed in vivo by positron emission tomography (PET) imaging with [18F]3 (p = 0.04). To conclude, three potential P2Y12R PET tracers were obtained and analyzed for P2Y12R targeting in the brain. Unfortunately, the brain uptake appeared low. Future work will focus on the design of P2Y12R inhibitors with improved physicochemical characteristics to reduce efflux transport and increase brain penetration.

Retention of 64Cu-FLFLF, a Formyl Peptide Receptor 1-Specific PET Probe, Correlates with Macrophage and Neutrophil Abundance in Lung Granulomas from Cynomolgus Macaques.

Neutrophilic inflammation correlates with severe tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (Mtb). Granulomas are lesions that form in TB, and a PET probe for following neutrophil recruitment to granulomas could predict disease progression. We tested the formyl peptide receptor 1 (FPR1)-targeting peptide FLFLF in Mtb-infected macaques. Preliminary studies in mice demonstrated specificity for neutrophils. In macaques, 64Cu-FLFLF was retained in lung granulomas and analysis of lung granulomas identified positive correlations between 64Cu-FLFLF and neutrophil and macrophage numbers (R2 = 0.8681 and 0.7643, respectively), and weaker correlations for T cells and B cells (R2 = 0.5744 and 0.5908, respectively), suggesting that multiple cell types drive 64Cu-FLFLF avidity. By PET/CT imaging, we found that granulomas retained 64Cu-FLFLF but with less avidity than the glucose analog 18F-FDG. These studies suggest that neutrophil-specific probes have potential PET/CT applications in TB, but important issues need to be addressed before they can be used in nonhuman primates and humans.

Towards PET imaging of the dynamic phenotypes of microglia.

There is increasing evidence showing the heterogeneity of microglia activation in neuroinflammatory and neurodegenerative diseases. It has been hypothesized that pro-inflammatory microglia are detrimental and contribute to disease progression, while anti-inflammatory microglia play a role in damage repair and remission. The development of therapeutics targeting the deleterious glial activity and modulating it into a regenerative phenotype relies heavily upon a clearer understanding of the microglia dynamics during disease progression and the ability to monitor therapeutic outcome in vivo. To that end, molecular imaging techniques are required to assess microglia dynamics and study their role in disease progression as well as to evaluate the outcome of therapeutic interventions. Positron emission tomography (PET) is such a molecular imaging technique, and provides unique capabilities for non-invasive quantification of neuroinflammation and has the potential to discriminate between microglia phenotypes and define their role in the disease process. However, several obstacles limit the possibility for selective in vivo imaging of microglia phenotypes mainly related to the poor characterization of specific targets that distinguish the two ends of the microglia activation spectrum and lack of suitable tracers. PET tracers targeting translocator protein 18 kDa (TSPO) have been extensively explored, but despite the success in evaluating neuroinflammation they failed to discriminate between microglia activation statuses. In this review, we highlight the current knowledge on the microglia phenotypes in the major neuroinflammatory and neurodegenerative diseases. We also discuss the current and emerging PET imaging targets, the tracers and their potential in discriminating between the pro- and anti-inflammatory microglia activation states.

Performance of nanoScan PET/CT and PET/MR for quantitative imaging of 18F and 89Zr as compared with ex vivo biodistribution in tumor-bearing mice.

INTRODUCTION: The assessment of ex vivo biodistribution is the preferred method for quantification of radiotracers biodistribution in preclinical models, but is not in line with current ethics on animal research. PET imaging allows for noninvasive longitudinal evaluation of tracer distribution in the same animals, but systemic comparison with ex vivo biodistribution is lacking. Our aim was to evaluate the potential of preclinical PET imaging for accurate tracer quantification, especially in tumor models. METHODS: NEMA NU 4-2008 phantoms were filled with 11C, 68Ga, 18F, or 89Zr solutions and scanned in Mediso nanoPET/CT and PET/MR scanners until decay. N87 tumor-bearing mice were i.v. injected with either [18F]FDG (~ 14 MBq), kept 50 min under anesthesia followed by imaging for 20 min, or with [89Zr]Zr-DFO-NCS-trastuzumab (~ 5 MBq) and imaged 3 days post-injection for 45 min. After PET acquisition, animals were killed and organs of interest were collected and measured in a γ-counter to determine tracer uptake levels. PET data were reconstructed using TeraTomo reconstruction algorithm with attenuation and scatter correction and regions of interest were drawn using Vivoquant software. PET imaging and ex vivo biodistribution were compared using Bland-Altman plots. RESULTS: In phantoms, the highest recovery coefficient, thus the smallest partial volume effect, was obtained with 18F for both PET/CT and PET/MR. Recovery was slightly lower for 11C and 89Zr, while the lowest recovery was obtained with 68Ga in both scanners. In vivo, tumor uptake of the 18F- or 89Zr-labeled tracer proved to be similar irrespective whether quantified by either PET/CT and PET/MR or ex vivo biodistribution with average PET/ex vivo ratios of 0.8-0.9 and a deviation of 10% or less. Both methods appeared less congruent in the quantification of tracer uptake in healthy organs such as brain, kidney, and liver, and depended on the organ evaluated and the radionuclide used. CONCLUSIONS: Our study suggests that PET quantification of 18F- and 89Zr-labeled tracers is reliable for the evaluation of tumor uptake in preclinical models and a valuable alternative technique for ex vivo biodistribution. However, PET and ex vivo quantification require fully described experimental and analytical procedures for reliability and reproducibility.

Head-to-head comparison of DFO* and DFO chelators: selection of the best candidate for clinical 89Zr-immuno-PET.

PURPOSE: Almost all radiolabellings of antibodies with 89Zr currently employ the hexadentate chelator desferrioxamine (DFO). However, DFO can lead to unwanted uptake of 89Zr in bones due to instability of the resulting metal complex. DFO*-NCS and the squaramide ester of DFO, DFOSq, are novel analogues that gave more stable 89Zr complexes than DFO in pilot experiments. Here, we directly compare these linker-chelator systems to identify optimal immuno-PET reagents. METHODS: Cetuximab, trastuzumab and B12 (non-binding control antibody) were labelled with 89Zr via DFO*-NCS, DFOSq, DFO-NCS or DFO*Sq. Stability in vitro was compared at 37 °C in serum (7 days), in formulation solution (24 h ± chelator challenges) and in vivo with N87 and A431 tumour-bearing mice. Finally, to demonstrate the practical benefit of more stable complexation for the accurate detection of bone metastases, [89Zr]Zr-DFO*-NCS and [89Zr]Zr-DFO-NCS-labelled trastuzumab and B12 were evaluated in a bone metastasis mouse model where BT-474 breast cancer cells were injected intratibially. RESULTS: [89Zr]Zr-DFO*-NCS-trastuzumab and [89Zr]Zr-DFO*Sq-trastuzumab showed excellent stability in vitro, superior to their [89Zr]Zr-DFO counterparts under all conditions. While tumour uptake was similar for all conjugates, bone uptake was lower for DFO* conjugates. Lower bone uptake for DFO* conjugates was confirmed using a second xenograft model: A431 combined with cetuximab. Finally, in the intratibial BT-474 bone metastasis model, the DFO* conjugates provided superior detection of tumour-specific signal over the DFO conjugates. CONCLUSION: DFO*-mAb conjugates provide lower bone uptake than their DFO analogues; thus, DFO* is a superior candidate for preclinical and clinical 89Zr-immuno-PET.

Combined VLA-4-Targeted Radionuclide Therapy and Immunotherapy in a Mouse Model of Melanoma.

Very late antigen-4 (VLA-4; also known as integrin α4ß1) is expressed at high levels in aggressive and metastatic melanoma tumors and may provide an ideal target for imaging and targeted radionuclide therapy (TRT). 177Lu-DOTA-PEG4-LLP2A (177Lu-LLP2A) is a TRT that shows high affinity for VLA-4 and high uptake in B16F10 mouse melanoma tumors in vivo. Here, we report efficacy studies of 177Lu-LLP2A, alone and combined with immune checkpoint inhibitors (ICIs) (anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies), in B16F10 tumor-bearing mice. Methods: Tumor cells (1 × 106) were implanted subcutaneously in C57BL/6 mice. After 8-10 d, the mice were randomized into 8 groups. 177Lu-LLP2A was injected intravenously on day 8 or 9 (single dose), and ICI antibodies were administered intraperitoneally in 3 doses. Tumor growth was monitored over time via calipers. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining for apoptosis was performed on fixed tumors. In a separate study, Cy3-LLP2A or Cy3-scrambled LLP2A was injected in tumor-bearing mice, and tumors were collected 4 h after injection and then analyzed by flow cytometry and immunofluorescence microscopy using different immune cell markers. Results: TRT alone showed efficacy comparable to the dual-ICI anti-PD-1 + anti-CTLA-4 or anti-PD-L1 + anti-CTLA-4, whereas TRT + ICIs significantly enhanced survival. TUNEL staining showed that the highest levels of apoptosis were in the TRT + ICI groups. In addition to targeting tumor cells, TRT also bound immune cells in the tumor microenvironment. Flow cytometry data showed that the tumors consisted of about 77% tumor cells and fibroblasts (CD45-negative/CD49d-positive) and about 23% immune cells (CD45-positive/CD49d-positive) and that immune cells expressed higher levels of VLA-4. Cy3-LLP2A and CD49d colocalized with macrophages (CD68), T cells (CD8, CD4), and B cells (CD19). Immunohistochemical analysis identified a significant colocalization of Cy3-LLP2A and CD68. Conclusion: Combination treatment with TRT + ICIs targets both tumor cells and immune cells and has potential as a therapeutic agent in patients with metastatic melanoma.

In vivo evaluation of two tissue transglutaminase PET tracers in an orthotopic tumour xenograft model.

BACKGROUND: The protein cross-linking enzyme tissue transglutaminase (TG2; EC 2.3.2.13) is associated with the pathogenesis of various diseases, including cancer. Recently, the synthesis and initial evaluation of two high-potential radiolabelled irreversible TG2 inhibitors were reported by us. In the present study, these two compounds were evaluated further in a breast cancer (MDA-MB-231) tumour xenograft model for imaging active tissue transglutaminase in vivo. RESULTS: The metabolic stability of [11C]1 and [18F]2 in SCID mice was comparable to the previously reported stability in Wistar rats. Quantitative real-time polymerase chain reaction analysis on MDA-MB-231 cells and isolated tumours showed a high level of TG2 expression with very low expression of other transglutaminases. PET imaging showed low tumour uptake of [11C]1 (approx. 0.5 percentage of the injected dose per gram (%ID/g) at 40-60 min p.i.) and with relatively fast washout. Tumour uptake for [18F]2 was steadily increasing over time (approx. 1.7 %ID/g at 40-60 min p.i.). Pretreatment of the animals with the TG2 inhibitor ERW1041E resulted in lower tumour activity concentrations, and this inhibitory effect was enhanced using unlabelled 2. CONCLUSIONS: Whereas the TG2 targeting potential of [11C]1 in this model seems inadequate, targeting of TG2 using [18F]2 was achieved. As such, [18F]2 could be used in future studies to clarify the role of active tissue transglutaminase in disease.

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.

Positron Emission Tomography Imaging of Macaques with Tuberculosis Identifies Temporal Changes in Granuloma Glucose Metabolism and Integrin α4ß1-Expressing Immune Cells.

Positron emission tomography and computed tomography imaging (PET/CT) is an increasingly valuable tool for diagnosing tuberculosis (TB). The glucose analog [18F]fluoro-2-deoxy-2-d-glucose ([18F]-FDG) is commonly used in PET/CT that is retained by metabolically active inflammatory cells in granulomas, but lacks specificity for particular cell types. A PET probe that could identify recruitment and differentiation of different cell populations in granulomas would be a useful research tool and could improve TB diagnosis and treatment. We used the Mycobacterium-antigen murine inflammation model and macaques with TB to identify [64Cu]-labeled CB-TE1A1P-PEG4-LLP2A ([64Cu]-LLP2A), a high affinity peptidomimetic ligand for very late Ag-4 (VLA-4; also called integrin α4ß1) binding cells in granulomas, and compared [64Cu]-LLP2A with [18F]-FDG over the course of infection. We found that [64Cu]-LLP2A retention was driven by macrophages and T cells, with less contribution from neutrophils and B cells. In macaques, granulomas had higher [64Cu]-LLP2A uptake than uninfected tissues, and immunohistochemical analysis of granulomas with known [64Cu]-LLP2A uptake identified significant correlations between LLP2A signal and macrophage and T cell numbers. The same cells coexpressed integrin α4 and ß1, further supporting that macrophages and T cells drive [64Cu]-LLP2A avidity in granulomas. Over the course of infection, granulomas and thoracic lymph nodes experienced dynamic changes in affinity for both probes, suggesting metabolic changes and cell differentiation or recruitment occurs throughout granuloma development. These results indicate [64Cu]-LLP2A is a PET probe for VLA-4, which when used in conjunction with [18F]-FDG, may be a useful tool for understanding granuloma biology in TB.

Ex Vivo and In Vivo Evaluation of Overexpressed VLA-4 in Multiple Myeloma Using LLP2A Imaging Agents.

UNLABELLED: Very-late-antigen-4 (VLA-4, α4ß1 integrin, CD49d/CD29) is a transmembrane adhesion receptor that plays an important role in cancer and immune responses. Enhanced VLA-4 expression has been observed in multiple myeloma (MM) cells and surrounding stroma. VLA-4 conformational activation has been associated with MM pathogenesis. VLA-4 is a promising MM imaging and therapeutic biomarker. METHODS: Specificity of (64)Cu-LLP2A ((64)Cu-CB-TE1A1P-PEG4-LLP2A), a high-affinity VLA-4 peptidomimetic-based radiopharmaceutical, was evaluated in α4 knock-out mice and by competitive blocking in wild-type tumor-bearing mice. (64)Cu-LLP2A PET/CT (static and dynamic) imaging was conducted in C57BL6/KaLwRij mice bearing murine 5TGM1-GFP syngeneic tumors generated after intravenous injection via the tail. Blood samples were collected for serum protein electrophoresis. Bone marrow and splenic cells extracted from tumor-bearing and control mice (n= 3/group) were coincubated with the optical analog LLP2A-Cy5 and mouse B220, CD4, Gr1, and Mac1 antibodies and analyzed by fluorescence-activated cell sorting. Human radiation dose estimates for (64)Cu-LLP2A were extrapolated from mouse biodistribution data (6 time points, 0.78 MBq/animal, n= 4/group). Ten formalin-fixed paraffin-embedded bone marrow samples from deceased MM patients were stained with LLP2A-Cy5. RESULTS: (64)Cu-LLP2A and LLP2A-Cy5 demonstrated high specificity for VLA-4-positive mouse 5TGM1-GFP myeloma and nonmalignant inflammatory host cells such as T cells and myeloid/monocytic cells. Ex vivo flow cytometric analysis supported a direct effect of myeloma on increased VLA-4 expression in host hematopoietic microenvironmental elements. SUVs and the number of medullar lesions detected by (64)Cu-LLP2A PET corresponded with increased monoclonal (M) protein (g/dL) in tumor-bearing mice over time (3.29 ± 0.58 at week 0 and 9.97 ± 1.52 at week 3). Dynamic PET with (64)Cu-LLP2A and (18)F-FDG demonstrated comparable SUV in the prominent lesions in the femur. Human radiation dose estimates indicated urinary bladder wall as the dose-limiting organ (0.200 mGy/MBq), whereas the dose to the red marrow was 0.006 mGy/MBq. The effective dose was estimated to be 0.017 mSv/MBq. Seven of the ten human samples displayed a high proportion of cells intensely labeled with LLP2A-Cy5 probe. CONCLUSION: (64)Cu-LLP2A and LLP2A-Cy5 demonstrated binding specificity for VLA-4 in an immune-competent murine MM model. (64)Cu-LLP2A displayed favorable dosimetry for human studies and is a potential imaging candidate for overexpressed VLA-4.

In vivo inflammation imaging using a CB2R-targeted near infrared fluorescent probe.

Chronic inflammation is considered as a critical cause of a host of disorders, such as cancer, rheumatoid arthritis, atherosclerosis, and neurodegenerative diseases, although the exact mechanism is yet to be explored. Imaging tools that can specifically target inflammation are therefore important to help reveal the role of inflammation in disease progression, and allows for developing new therapeutic strategies to ultimately improve patient care. The purpose of this study was to develop a new in vivo inflammation imaging approach by targeting the cannabinoid receptor type 2 (CB2R), an emerging inflammation biomarker, using a unique near infrared (NIR) fluorescent probe. Herein, we report the first in vivo CB2R-targeted NIR inflammation imaging study using a synthetic fluorescent probe developed in our laboratory, NIR760-mbc94. In vitro binding assay and fluorescence microscopy study indicate NIR760-mbc94 specifically binds towards CB2R in mouse RAW264.7 macrophage cells. Furthermore, in vivo imaging was performed using a Complete Freund's Adjuvant (CFA)-induced inflammation mouse model. NIR760-mbc94 successfully identified inflamed tissues and the probe uptake was blocked by a CB2R ligand, SR144528. Additionally, immunofluorescence staining in cryosectioned tissues validated the NIR760-mbc94 uptake in inflamed tissues. In conclusion, this study reports the first in vivo CB2R-targeted inflammation imaging using an NIR fluorescent probe. Specific targeting of NIR760-mbc94 has been demonstrated in macrophage cells, as well as a CFA-induced inflammation mouse model. The combined evidence indicates that NIR760-mbc94 is a promising inflammation imaging probe. Moreover, in vivo CB2R-targeted fluorescence imaging may have potential in the study of inflammation-related diseases.

Theranostic nanoemulsions for macrophage COX-2 inhibition in a murine inflammation model.

Targeting macrophages for therapeutic and diagnostic purposes is an attractive approach applicable to multiple diseases. Here, we present a theranostic nanoemulsion platform for simultaneous delivery of an anti-inflammatory drug (celecoxib) to macrophages and monitoring of macrophage migration patterns by optical imaging, as measurement of changes in inflammation. The anti-inflammatory effect of the theranostic nanoemulsions was evaluated in a mouse inflammation model induced with complete Freund's adjuvant (CFA). Nanoemulsions showed greater accumulation in the inflamed vs. control paw, with histology confirming their specific localization in CD68 positive macrophages expressing cyclooxygenase-2 (COX-2) compared to neutrophils. With a single dose administration of the celecoxib-loaded theranostic, we observed a reduction in fluorescence in the paw with time, corresponding to a reduction in macrophage infiltration. Our data strongly suggest that delivery of select agents to infiltrating macrophages can potentially lead to new treatments of inflammatory diseases where macrophage behavior changes are monitored in vivo.