Side-by-side comparison of the two widely studied GRPR radiotracers, radiolabeled NeoB and RM2, in a preclinical setting

Introduction: NeoB and RM2 are the most investigated gastrin-releasing peptide receptor (GRPR)–targeting radiotracers in preclinical and clinical studies. Therefore, an extensive side-by-side comparison of the two radiotracers is valuable to demonstrate whether one has advantages over the other. Accordingly, this study aims to compare the in vitro and in vivo characteristics of radiolabeled NeoB and RM2 to guide future clinical studies. Method: The stability of the radiolabeled GRPR analogs was determined in phosphate buffered saline (PBS), and commercially available mouse and human serum. Target affinity was determined by incubating human prostate cancer PC-3 cells with [177Lu]Lu-NeoB or [177Lu]Lu-RM2, + / − increasing concentrations of unlabeled NeoB, RM2, or Tyr4-bombesin (BBN). To determine uptake and specificity cells were incubated with [177Lu]Lu-NeoB or [177Lu]Lu-RM2 + / − Tyr4-BBN. Moreover, in vivo studies were performed to determine biodistribution and pharmacokinetics. Finally, radiotracer binding to various GRPR-expressing human cancer tissues was investigated. Results: Both radiotracers demonstrated high stability in PBS and human serum, but stability in mouse serum decreased substantially over time. Moreover, both radiotracers demonstrated high GRPR affinity and specificity, but a higher uptake of [177Lu]Lu-NeoB was observed in in vitro studies. In vivo, no difference in tumor uptake was seen. The most prominent difference in uptake in physiological organs was observed in the GRPR-expressing pancreas; [177Lu]Lu-RM2 had less pancreatic uptake and a shorter pancreatic half-life than [177Lu]Lu-NeoB. Furthermore, [177Lu]Lu-RM2 presented with a lower tumor-to-kidney ratio, while the tumor-to-blood ratio was lower for [177Lu]Lu-NeoB. The autoradiography studies revealed higher binding of radiolabeled NeoB to all human tumor tissues. Conclusion: Based on these findings, we conclude that the in vivo tumor-targeting capability of radiolabeled NeoB and RM2 is similar. Additional studies are needed to determine whether the differences observed in physiological organ uptakes, i.e., the pancreas, kidneys, and blood, result in relevant differences in organ absorbed doses when the radiotracers are applied for therapeutic purposes.</p

Therapeutic applications of pretargeting

Targeted therapies, such as radioimmunotherapy (RIT), present a promising treatment option for the eradication of tumor lesions. RIT has shown promising results especially for hematologic malignancies, but the therapeutic efficacy is limited by unfavorable tumor-tobackground ratios resulting in high radiotoxicity. Pretargeting strategies can play an important role in addressing the high toxicity profile of RIT. Key to pretargeting is the concept of decoupling the targeting vehicle from the cytotoxic agent and administrating them separately. Studies have shown that this approach has the ability to enhance the therapeutic index as it can reduce side effects caused by off-target irradiation and thereby increase curative effects due to higher tolerated doses. Pretargeted RIT (PRIT) has been explored for imaging and treatment of different cancer types over the years. This review will give an overview of the various targeted therapies in which pretargeting has been applied, discussing PRIT with alpha-and beta-emitters and as part of combination therapy, plus its use in drug delivery systems

In Vivo Evaluation of Indium-111-Labeled 800CW as a Necrosis-Avid Contrast Agent.

Current clinical measurements for tumor treatment efficiency rely often on changes in tumor volume measured as shrinkage by CT or MRI, which become apparent after multiple lines of treatment and pose a physical and psychological burden on the patient. Detection of therapy-induced cell death in the tumor can be a fast measure for treatment efficiency. However, there are no reliable clinical tools for detection of tumor necrosis. Previously, we studied the necrosis avidity of cyanine-based fluorescent dyes, which suffered long circulation times before tumor necrosis could be imaged due to low hydrophilicity. We now present the application of radiolabeled 800CW, a commercially available cyanine with high hydrophilicity, to image tumor necrosis in a mouse model. We conjugated 800CW to DOTA via a PEG linker, for labeling with single-photon emission-computed tomography isotope indium-111, yielding [ &lt;sup&gt;111&lt;/sup&gt; In]In-DOTA-PEG &lt;sub&gt;4&lt;/sub&gt; -800CW. We then investigated specific [ &lt;sup&gt;111&lt;/sup&gt; In]In-DOTA-PEG &lt;sub&gt;4&lt;/sub&gt; -800CW uptake by dead cells in vitro, using both fluorescence and radioactivity as detection modalities. Finally, we investigated [ &lt;sup&gt;111&lt;/sup&gt; In]In-DOTA-PEG &lt;sub&gt;4&lt;/sub&gt; -800CW uptake into necrotic tumor regions of a 4T1 breast tumor model in mice. We successfully prepared a precursor and developed a reliable procedure for labeling 800CW with indium-111. We detected specific [ &lt;sup&gt;111&lt;/sup&gt; In]In-DOTA-PEG &lt;sub&gt;4&lt;/sub&gt; -800CW uptake by dead cells, using both fluorescence and radioactivity. Albeit with a tumor uptake of only 0.37%ID/g at 6 h post injection, we were able to image tumor necrosis with a tumor to background ratio of 7:4. Fluorescence and radioactivity in cryosections from the dissected tumors were colocalized with tumor necrosis, confirmed by TUNEL staining. [ &lt;sup&gt;111&lt;/sup&gt; In]In-DOTA-PEG &lt;sub&gt;4&lt;/sub&gt; -800CW can be used to image tumor necrosis in vitro and in vivo. Further research will elucidate the application of [ &lt;sup&gt;111&lt;/sup&gt; In]In-DOTA-PEG &lt;sub&gt;4&lt;/sub&gt; -800CW or other radiolabeled hydrophilic cyanines for the detection of necrosis caused by chemotherapy or other anti-cancer therapies. This can provide valuable prognostic information in treatment of solid tumors

Necrosis binding of Ac-Lys⁰(IRDye800CW)-Tyr³-octreotate: a consequence from cyanine-labeling of small molecules.

There is a growing body of nuclear contrast agents that are repurposed for fluorescence-guided surgery. New contrast agents are obtained by substituting the radioactive tag with, or adding a fluorescent cyanine to the molecular structure of antibodies or peptides. This enables intra-operative fluorescent detection of cancerous tissue, leading to more complete tumor resection. However, these fluorescent cyanines can have a remarkable influence on pharmacokinetics and tumor uptake, especially when labeled to smaller targeting vectors such as peptides. Here we demonstrate the effect of cyanine-mediated dead cell-binding of Ac-Lys &lt;sup&gt;0&lt;/sup&gt; (IRDye800CW)-Tyr &lt;sup&gt;3&lt;/sup&gt; -octreotate (800CW-TATE) and how this can be used as an advantage for fluorescence-guided surgery. Binding of 800CW-TATE could be blocked with DOTA &lt;sup&gt;0&lt;/sup&gt; -Tyr &lt;sup&gt;3&lt;/sup&gt; -octreotate (DOTA-TATE) on cultured SSTR &lt;sub&gt;2&lt;/sub&gt; -positive U2OS cells and was absent in SSTR &lt;sub&gt;2&lt;/sub&gt; negative U2OS cells. However, strong binding was observed to dead cells, which could not be blocked with DOTA-TATE and was also present in dead SSTR &lt;sub&gt;2&lt;/sub&gt; negative cells. No SSTR &lt;sub&gt;2&lt;/sub&gt; -mediated binding was observed in frozen tumor sections, possibly due to disruption of the cells in the process of sectioning the tissue before exposure to the contrast agent. DOTA-TATE blocking resulted in an incomplete reduction of 61.5 ± 5.8% fluorescence uptake by NCI-H69-tumors in mice. Near-infrared imaging and dead cell staining on paraffin sections from resected tumors revealed that fluorescence uptake persisted in necrotic regions upon blocking with DOTA-TATE. This study shows that labeling peptides with cyanines can result in dead cell binding. This does not hamper the ultimate purpose of fluorescence-guided surgery, as necrotic tissue appears in most solid tumors. Hence, the necrosis binding can increase the overall tumor uptake. Moreover, necrotic tissue should be removed as much as possible: it cannot be salvaged, causes inflammation, and is tumorigenic. However, when performing binding experiments to cells with disrupted membrane integrity, which is routinely done with nuclear probes, this dead cell-binding can resemble non-specific binding. This study will benefit the development of fluorescent contrast agents

A Flexible Synthesis of 68Ga-Labeled Carbonic Anhydrase IX (CAIX)-Targeted Molecules via CBT/1,2-Aminothiol Click Reaction

We herein describe a flexible synthesis of a small library of 68Ga-labeled CAIX-targeted molecules via an orthogonal 2-cyanobenzothiazole (CBT)/1,2-aminothiol click reaction. Three novel CBT-functionalized chelators (1⁻3) were successfully synthesized and labeled with the positron emitter gallium-68. Cross-ligation between the pre-labeled bifunctional chelators (BFCs) and the 1,2-aminothiol-acetazolamide derivatives (8 and 9) yielded six new 68Ga-labeled CAIX ligands with high radiochemical yields. The click reaction conditions were optimized to improve the reaction rate for applications with short half-life radionuclides. Overall, our methodology allows for a simple and efficient radiosynthetic route to produce a variety of 68Ga-labeled imaging agents for tumor hypoxia