Enzyme mediated incorporation of zirconium-89 or copper-64 into a fragment antibody for same day imaging of epidermal growth factor receptor

Rudd, Stacey E.; Zuylekom, Jessica Van; Raicevic, Anna; Pearce, Lesley A.; Cullinane, Carleen; Williams, Charlotte C.; Adams, Timothy E.; Hicks, Rodney J.; Donnelly, Paul S.

doi:10.1039/d1sc01422f

Cited by 12 publications

(12 citation statements)

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“…We note that different variations of DFO-glycine probes have recently been reported by the groups of Ploegh and Donnelly . Our triglycine chelate DFO-Gly 3 ( 1 ) is structurally different from these previously reported chelates and was synthesized and isolated in 29% overall yield by using standard chemical transformations.…”

Section: Resultsmentioning

confidence: 98%

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Sortase-Mediated Site-Specific Conjugation and ⁸⁹Zr-Radiolabeling of Designed Ankyrin Repeat Proteins for PET

et al. 2022

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Designed ankyrin repeat proteins (DARPins) are genetically engineered proteins that exhibit high specificity and affinity toward specific targets. Here, the G3-DARPin, which binds the HER2/neu receptor, was site-specifically modified with enzymatic methods and 89Zr-radiolabeled for applications in positron emission tomography (PET). Sortase A transpeptidation was used to install a desferrioxamine B (DFO) chelate bearing a reactive triglycine group to the C-terminal sortase tag of the G3-DARPin, and 89Zr-radiolabeling produced a novel 89ZrDFO-G3-DARPin radiotracer that can detect HER2/neu-positive tumors. The triglycine probe, DFO-Gly3 (1), was synthesized in 29% overall yield. After sortase A transpeptidation and purification from the nonfunctionalized protein component, the DFO-G3-DARPin product was radiolabeled to give 89ZrDFO-G3-DARPin. Binding specificity was assessed in HER2/neu-expressing BT-474 and SK-OV-3 cellular assays. The pharmacokinetics, tumor uptake, and specificity of 89ZrDFO-G3-DARPin were measured in vivo by PET imaging and confirmed by final time point (24 h) biodistribution experiments in female athymic nude mice bearing BT-474 xenografts. Sortase A transpeptidation afforded the site-specific and stoichiometrically precise functionalization of DFO-G3-DARPin with one chelate per protein. The modified DFO-G3-DARPin was purified from the nonfunctionalized DARPin by using Ni-NTA affinity chromatography. 89ZrDFO-G3-DARPin was obtained with a radiochemical purity of >95% measured by radio-size-exclusion chromatography. BT-474 tumor uptake at 24 h postadministration reached 4.41 ± 0.67 %ID/g (n = 3) with an approximate ∼70% reduction in tumor-associated activity in the blocking group (1.26 ± 0.29 %ID/g; 24 h postadministration, n = 5, P-value of <0.001). Overall, the site-specific, enzyme-mediated functionalization and characterization of 89ZrDFO-G3-DARPin in HER2/neu positive BT-474 xenografts demonstrate that DARPins are an attractive platform for generating a new class of protein-based radiotracers for PET. The specific uptake and retention of 89ZrDFO-G3-DARPin in tumors and clearance from most background tissues produced PET images with high tumor-to-background contrast.

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Section: Resultsmentioning

confidence: 98%

“…We note that different variations of DFO-glycine probes have recently been reported by the groups of Ploegh 38 and Donnelly. 39 Our triglycine chelate DFO-Gly 3…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Sortase-Mediated Site-Specific Conjugation and ⁸⁹Zr-Radiolabeling of Designed Ankyrin Repeat Proteins for PET

et al. 2022

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“…The conjugate was used for PET imaging of thrombosis and cancer, with excellent quality images. , The chelator has also been developed to allow incorporation into the scFv via a site-specific conjugation method and by click chemistry . The versatility of MeCOSar–NHS has been further demonstrated by the conjugation of the ligand to the human epidermal growth factor receptor binding antibody cetuximab and subsequent preclinical imaging (Figure b) which demonstrated excellent tumor uptake even at early time points as well as significant tumor retention (Figure c). The high spleen, liver, and bone uptake of the conjugate is consistent with the well-known phenomenon of Fc-mediated interactions in this immunocompromised mouse model .…”

Section: Theranostic Opportunities With “Matched” Pair Radionuclidesmentioning

confidence: 99%

Theranostic Nuclear Medicine with Gallium-68, Lutetium-177, Copper-64/67, Actinium-225, and Lead-212/203 Radionuclides

Morgan,

Rudd,

Noor

et al. 2023

Chem. Rev.

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Molecular changes in malignant tissue can lead to an increase in the expression levels of various proteins or receptors that can be used to target the disease. In oncology, diagnostic imaging and radiotherapy of tumors is possible by attaching an appropriate radionuclide to molecules that selectively bind to these target proteins. The term “theranostics” describes the use of a diagnostic tool to predict the efficacy of a therapeutic option. Molecules radiolabeled with γ-emitting or β+-emitting radionuclides can be used for diagnostic imaging using single photon emission computed tomography or positron emission tomography. Radionuclide therapy of disease sites is possible with either α-, β-, or Auger-emitting radionuclides that induce irreversible damage to DNA. This Focus Review centers on the chemistry of theranostic approaches using metal radionuclides for imaging and therapy. The use of tracers that contain β+-emitting gallium-68 and β-emitting lutetium-177 will be discussed in the context of agents in clinical use for the diagnostic imaging and therapy of neuroendocrine tumors and prostate cancer. A particular emphasis is then placed on the chemistry involved in the development of theranostic approaches that use copper-64 for imaging and copper-67 for therapy with functionalized sarcophagine cage amine ligands. Targeted therapy with radionuclides that emit α particles has potential to be of particular use in late-stage disease where there are limited options, and the role of actinium-225 and lead-212 in this area is also discussed. Finally, we highlight the challenges that impede further adoption of radiotheranostic concepts while highlighting exciting opportunities and prospects.

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“…In 2021, for example, Rudd et al used the transpeptidase sortase A to catalyze the attachment of glycine-bearing chelators to an epidermal growth factor receptor-targeting Fab bearing a C-terminal LPETG recognition sequence, ultimately creating both 64 Cu-and 89 Zr-labeled radioimmunoconjugates that displayed excellent in vivo performance in a murine model of epidermal growth factor receptor-positive epidermoid carcinoma (Figs. 3C and 3D) (22). Bridoux et al used a similar approach to append a variant of NOTA with a GGGYK tag to an hPD-L1-targeting sdAb bearing a C-terminal LPETG recognition element.…”

Section: Site-specific Bioconjugationmentioning

confidence: 99%

Antibody Engineering for Nuclear Imaging and Radioimmunotherapy

Rodriguez

Delaney²,

Sarrett³

et al. 2022

J Nucl Med

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Radiolabeled antibodies have become indispensable tools in nuclear medicine. However, the natural roles of antibodies within the immune system mean that they have several intrinsic limitations as a platform for radiopharmaceuticals. In recent years, the field has increasingly turned to antibody engineering to circumvent these issues while retaining the manifold benefits of the immunoglobulin framework. In this "Focus on Molecular Imaging" review, we cover recent advances in the application of antibody engineering to immunoPET, immunoSPECT, and radioimmunotherapy. Specifically, we address how antibody engineering has been used to improve radioimmunoconjugates on four fronts: optimizing pharmacokinetics, facilitating site-specific bioconjugation, modulating Fc interactions, and creating bispecific constructs.

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Enzyme mediated incorporation of zirconium-89 or copper-64 into a fragment antibody for same day imaging of epidermal growth factor receptor

Abstract: Identification of tumors which over-express Epidermal Growth Factor Receptor (EGFR) is important in selecting patients for anti-EGFR therapies. Enzymatic bioconjugation was used to introduce positron-emitting radionuclides (89Zr, 64Cu) into an...

Cited by 12 publications

References 59 publications

Sortase-Mediated Site-Specific Conjugation and ⁸⁹Zr-Radiolabeling of Designed Ankyrin Repeat Proteins for PET

Sortase-Mediated Site-Specific Conjugation and ⁸⁹Zr-Radiolabeling of Designed Ankyrin Repeat Proteins for PET

Theranostic Nuclear Medicine with Gallium-68, Lutetium-177, Copper-64/67, Actinium-225, and Lead-212/203 Radionuclides

Antibody Engineering for Nuclear Imaging and Radioimmunotherapy

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Enzyme mediated incorporation of zirconium-89 or copper-64 into a fragment antibody for same day imaging of epidermal growth factor receptor

Abstract: Identification of tumors which over-express Epidermal Growth Factor Receptor (EGFR) is important in selecting patients for anti-EGFR therapies. Enzymatic bioconjugation was used to introduce positron-emitting radionuclides (89Zr, 64Cu) into an...

Cited by 12 publications

References 59 publications

Sortase-Mediated Site-Specific Conjugation and 89Zr-Radiolabeling of Designed Ankyrin Repeat Proteins for PET

Sortase-Mediated Site-Specific Conjugation and 89Zr-Radiolabeling of Designed Ankyrin Repeat Proteins for PET

Theranostic Nuclear Medicine with Gallium-68, Lutetium-177, Copper-64/67, Actinium-225, and Lead-212/203 Radionuclides

Antibody Engineering for Nuclear Imaging and Radioimmunotherapy

Contact Info

Product

Resources

About

Sortase-Mediated Site-Specific Conjugation and ⁸⁹Zr-Radiolabeling of Designed Ankyrin Repeat Proteins for PET

Sortase-Mediated Site-Specific Conjugation and ⁸⁹Zr-Radiolabeling of Designed Ankyrin Repeat Proteins for PET