In Vivo Chemistry for Pretargeted Tumor Imaging in Live Mice

Rossin, Raffaella; Verkerk, Pascal Renart; Bosch, Sandra M. van den; Vulders, Roland C. M.; Verel, Iris; Lub, Johan; Robillard, Marc S.

doi:10.1002/anie.200906294

Cited by 450 publications

(412 citation statements)

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“…11 These IEDDA reactions have second-order rate constants ranging from k 2 = 10 3 –10 6 M −1 s −1 , which are primarily driven by expulsion of N 2 and TCO strain relief following the cycloaddition 12,13 Furthermore, electron withdrawing substituents attached to the tetrazine confer increased reaction rates between the dienophile and diene. 14,15 Successful pretargeted in vivo nuclear imaging has been demonstrated in preclinical models using PET isotopes, including 11 C (t 1/2 20.3 minutes), 16,17 18 F (t 1/2 110 minutes) 18–20 and 64 Cu (t 1/2 12.7 hours), 4,21–24 as well as SPECT isotopes including 99m Tc (t 1/2 6 hours), 25–27 111 In (t 1/2 2.8 days) 28–30 and 177 Lu (t 1/2 6.7 days). 27,31–33 The recent success of IEDDA reactions for in vivo pretargeting and other chemical biology applications has been reviewed by several groups.…”

Section: Introductionmentioning

confidence: 99%

“…4,23,30 While these early imaging studies were successful, highly expressed non-internalizing targets only represent a fraction of clinically relevant oncology targets since many cell-surface cancer targets are internalizing. For instance, clinical imaging of human epidermal growth factor receptor 2 (HER2)-expressing tumors has been actively pursued via directly labeled antibodies, 40,41 antibody fragments 42 and affibodies 43 in an effort to improve patient selection and response monitoring for HER2-targeted cancer therapies.…”

Section: Introductionmentioning

confidence: 99%

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Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

Mandikian¹,

Rafidi²,

Adhikari³

et al. 2018

mAbs

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Antibody pretargeting is a promising strategy for improving molecular imaging, wherein the separation in time of antibody targeting and radiolabeling can lead to rapid attainment of high contrast, potentially increased sensitivity, and reduced patient radiation exposure. The inverse electron demand Diels-Alder ‘click’ reaction between trans-cyclooctene (TCO) conjugated antibodies and radiolabeled tetrazines presents an ideal platform for pretargeted imaging due to rapid reaction kinetics, bioorthogonality, and potential for optimization of both slow and fast clearing components. Herein, we evaluated a series of anti-human epidermal growth factor receptor 2 (HER2) pretargeting antibodies containing distinct molar ratios of site-specifically incorporated TCO. The effect of stoichiometry on tissue distribution was assessed for pretargeting TCO-modified antibodies (monitored by 125I) and subsequent accumulation of an 111In-labeled tetrazine in a therapeutically relevant HER2+tumor-bearing mouse model. Single photon emission computed tomography (SPECT) imaging was also employed to assess tumor imaging at various TCO-to-monoclonal antibody (mAb) ratios. Increasing TCO-to-mAb molar ratios correlated with increased in vivo click reaction efficiency evident by increased tumor distribution and systemic exposure of 111In-labeled tetrazines. The pharmacokinetics of TCO-modified antibodies did not vary with stoichiometry. Pretargeted SPECT imaging of HER2-expressing tumors using 111In-labeled tetrazine demonstrated robust click reaction with circulating antibody at ~2 hours and good tumor delineation for both the 2 and 6 TCO-to-mAb ratio variants at 24 hours, consistent with a limited cell-surface pool of pretargeted antibody and benefit from further distribution and internalization. To our knowledge, this represents the first reported systematic analysis of how pretargeted imaging is affected solely by variation in click reaction stoichiometry through site-specific conjugation chemistry.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

Mandikian¹,

Rafidi²,

Adhikari³

et al. 2018

mAbs

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“…This approach was inspired by the pretargeted method used frequently in the molecular imaging field. [18][19][20][21][22][23] The concept is schematically presented in Figure 1.A simplified model was proposed in which various surface receptors are expressed on cells A and B. Cell A may be selectively visualized by applying the fluorescently labeled probe C, which shows a high affinity toward a receptor overexpressed on the surface of A, i.e., at K D of nano molar level ( Figure 1A).…”

mentioning

confidence: 99%

“…This approach was inspired by the pretargeted method used frequently in the molecular imaging field. [18][19][20][21][22][23] The concept is schematically presented in Figure 1.…”

mentioning

confidence: 99%

In Situ Ligation of High‐ and Low‐Affinity Ligands to Cell Surface Receptors Enables Highly Selective Recognition

et al. 2017

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molecules that are highly expressed in tumor vasculature [11] are widely used to image breast, [15] brain, [16] and lung [17] cancers in small animal models; however, RGD peptides often provide poor imaging contrast because the integrins are also expressed on other endothelial cells. The RGD peptides act as "strongly" interacting integrin ligands that nonselectively bind to the various integrins and are rapidly captured by the cells via receptor-mediated endocytosis, which decreases the tumor/ background signal ratio.While most studies have focused on optimizing ligands with a high affinity and selectivity to the cell surface targets, we envisioned a new imaging approach that utilized one high-and one low-affinity ligand targeted to independent receptors on a target cell surface. This approach was inspired by the pretargeted method used frequently in the molecular imaging field. [18][19][20][21][22][23] The concept is schematically presented in Figure 1.A simplified model was proposed in which various surface receptors are expressed on cells A and B. Cell A may be selectively visualized by applying the fluorescently labeled probe C, which shows a high affinity toward a receptor overexpressed on the surface of A, i.e., at K D of nano molar level ( Figure 1A). The same receptor could also be expressed more This paper reports an entirely unexplored concept of simultaneously recognizing two receptors using high-and low-affinity ligands through ligating them in situ on the target cell surface. This de novo approach is inspired by the pretargeting strategy frequently applied in molecular imaging, and has now evolved as the basis of a new paradigm for visualizing target cells with a high imaging contrast. A distinct advantage of using a labeled low-affinity ligand such as glycan is that the excess labeled ligand can be washed away from the cells, whereas the ligand bound to the cell, even at the milli molar affinity level, can be anchored by a bioorthogonal reaction with a pretargeted high-affinity ligand on the surface. Consequently, nonspecific background is minimized, leading to improved imaging contrast. Importantly, despite previously unexplored for molecular imaging, a notoriously weak glycan/lectin interaction can now be utilized as a highly selective ligand to the targets. Cell ImagingMolecular imaging research has focused on noninvasively analyzing the molecular kinetics in small animals for use in diagnostic applications. [1][2][3][4][5][6][7] In vivo information about biologically active small molecules and biomolecules, such as the localization or expression levels of target receptors, may be readily imaged using fluorescence or radionuclide-based detection. Although many promising tracers can target specific organs or tumors, [8][9][10][11][12][13] the selectivity and specificity of these tracers toward target cells must be improved. For example, RGD peptides, highaffinity ligands of α V β 3 integrins, [9,14] which are cell adhesion

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“…28,29 Along these lines, it is important to note that the recent report from our laboratories was not the first to apply IEDDA chemistry to pretargeting: the first report of pretargeted imaging with IEDDA arose from the work of Rossin, et al and featured a SPECT methodology employing an 111 In-labeled tetrazine. 30 As we discussed above, the pretargeting methodology has four fairly simple steps (Figure 2). In the protocol at hand, a pretargeted strategy for the PET imaging of colorectal cancer that employs a 64 Cu-NOTA-labeled tetrazine radioligand and a TCO-modified conjugate of the huA33 antibody will be described.…”

mentioning

confidence: 99%

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

Reiner

Lewis

Zeglis

2015

JoVE

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Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for PET imaging. However, the necessity of labeling antibodies with radionuclides with long physical half-lives often results in high background radiation dose rates to non-target tissues. In order to circumvent this issue, we have employed a pretargeted PET imaging strategy based on the inverse electron demand Diels-Alder cycloaddition reaction. The methodology decouples the antibody from the radioactivity and thus exploits the positive characteristics of antibodies, while eschewing their pharmacokinetic drawbacks. The system is composed of four steps: (1) the injection of a mAb-trans-cyclooctene (TCO) conjugate; (2) a localization time period during which the antibody accumulates in the tumor and clears from the blood; (3) the injection of the radiolabeled tetrazine; and (4) the in vivo click ligation of the components followed by the clearance of excess radioligand. In the example presented in the work at hand, a 64 Cu-NOTA-labeled tetrazine radioligand and a trans-cyclooctene-conjugated humanized antibody (huA33) were successfully used to delineate SW1222 colorectal cancer tumors with high tumor-to-background contrast. Further, the pretargeting methodology produces high quality images at only a fraction of the radiation dose to non-target tissue created by radioimmunoconjugates directly labeled with 64 Cu or 89 Zr. Ultimately, the modularity of this protocol is one of its greatest assets, as the transcyclooctene moiety can be appended to any non-internalizing antibody, and the tetrazine can be attached to a wide variety of radioisotopes. Video LinkThe video component of this article can be found at

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In Vivo Chemistry for Pretargeted Tumor Imaging in Live Mice

Cited by 450 publications

References 10 publications

Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

In Situ Ligation of High‐ and Low‐Affinity Ligands to Cell Surface Receptors Enables Highly Selective Recognition

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

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