Dataset Information

Site-Specific Dual-Labeling of a VHH with a Chelator and a Photosensitizer for Nuclear Imaging and Targeted Photodynamic Therapy of EGFR-Positive Tumors.

ABSTRACT: Variable domains of heavy chain only antibodies (VHHs) are valuable agents for application in tumor theranostics upon conjugation to both a diagnostic probe and a therapeutic compound. Here, we optimized site-specific conjugation of the chelator DTPA and the photosensitizer IRDye700DX to anti-epidermal growth factor receptor (EGFR) VHH 7D12, for applications in nuclear imaging and photodynamic therapy. 7D12 was site-specifically equipped with bimodal probe DTPA-tetrazine-IRDye700DX using the dichlorotetrazine conjugation platform. Binding, internalization and light-induced toxicity of DTPA-IRDye700DX-7D12 were determined using EGFR-overexpressing A431 cells. Finally, ex vivo biodistribution of DTPA-IRDye700DX-7D12 in A431 tumor-bearing mice was performed, and tumor homing was visualized with SPECT and fluorescence imaging. DTPA-IRDye700DX-7D12 was retrieved with a protein recovery of 43%, and a degree of labeling of 0.56. Spectral properties of the IRDye700DX were retained upon conjugation. ¹¹¹In-labeled DTPA-IRDye700DX-7D12 bound specifically to A431 cells, and they were effectively killed upon illumination. DTPA-IRDye700DX-7D12 homed to A431 xenografts in vivo, and this could be visualized with both SPECT and fluorescence imaging. In conclusion, the dichlorotetrazine platform offers a feasible method for site-specific dual-labeling of VHH 7D12, retaining binding affinity and therapeutic efficacy. The flexibility of the described approach makes it easy to vary the nature of the probes for other combinations of diagnostic and therapeutic compounds.

SUBMITTER: Renard E

PROVIDER: S-EPMC7865570 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Publications

Site-Specific Dual-Labeling of a VHH with a Chelator and a Photosensitizer for Nuclear Imaging and Targeted Photodynamic Therapy of EGFR-Positive Tumors.

Renard Emma E Collado Camps Estel E Canovas Coline C Kip Annemarie A Gotthardt Martin M Rijpkema Mark M Denat Franck F Goncalves Victor V van Lith Sanne A M SAM

Cancers 20210123 3

Variable domains of heavy chain only antibodies (VHHs) are valuable agents for application in tumor theranostics upon conjugation to both a diagnostic probe and a therapeutic compound. Here, we optimized site-specific conjugation of the chelator DTPA and the photosensitizer IRDye700DX to anti-epidermal growth factor receptor (EGFR) VHH 7D12, for applications in nuclear imaging and photodynamic therapy. 7D12 was site-specifically equipped with bimodal probe DTPA-tetrazine-IRDye700DX using the dic ...[more]

PMID: 33498707

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Project description:Site-specific protein labeling is an important technique in protein chemistry and is used for diverse applications ranging from creating protein conjugates to protein immobilization. Enzymatic reactions, including protein prenylation, have been widely exploited as methods to accomplish site-specific labeling. Enzymatic prenylation is catalyzed by prenyltransferases, including protein farnesyltransferase (PFTase) and geranylgeranyltransferase type I (GGTase-I), both of which recognize C-terminal CaaX motifs with different specificities and transfer prenyl groups from isoprenoid diphosphates to their respective target proteins. A number of isoprenoid analogues containing bioorthogonal functional groups have been used to label proteins of interest via PFTase-catalyzed reaction. In this study, we sought to expand the scope of prenyltransferase-mediated protein labeling by exploring the utility of rat GGTase-I (rGGTase-I). First, the isoprenoid specificity of rGGTase-I was evaluated by screening eight different analogues and it was found that those with bulky moieties and longer backbone length were recognized by rGGTase-I more efficiently. Taking advantage of the different substrate specificities of rat PFTase (rPFTase) and rGGTase-I, we then developed a simultaneous dual labeling method to selectively label two different proteins by using isoprenoid analogue and CaaX substrate pairs that were specific to only one of the prenyltransferases. Using two model proteins, green fluorescent protein with a C-terminal CVLL sequence (GFP-CVLL) and red fluorescent protein with a C-terminal CVIA sequence (RFP-CVIA), we demonstrated that when incubated together with both prenyltransferases and the selected isoprenoid analogues, GFP-CVLL was specifically modified with a ketone-functionalized analogue by rGGTase-I and RFP-CVIA was selectively labeled with an alkyne-containing analogue by rPFTase. By switching the ketone-containing analogue to an azide-containing analogue, it was possible to create protein tail-to-tail dimers in a one-pot procedure through the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. Overall, with the flexibility of using different isoprenoid analogues, this system greatly extends the utility of protein labeling using prenyltransferases.

Dataset Information

Site-Specific Dual-Labeling of a VHH with a Chelator and a Photosensitizer for Nuclear Imaging and Targeted Photodynamic Therapy of EGFR-Positive Tumors.

Publications

Site-Specific Dual-Labeling of a VHH with a Chelator and a Photosensitizer for Nuclear Imaging and Targeted Photodynamic Therapy of EGFR-Positive Tumors.

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