biostudies-literatureMacPherson DSAcademy of FinlandNIBIB NIH HHSNCRR NIH HHSNIEHS NIH HHSNational Cancer InstituteNCI NIH HHSNational Institute of Biomedical Imaging and BioengineeringNIH HHS775-782https://www.ebi.ac.uk/biostudies/studies/S-EPMC10263003biostudies-literatureUnknown20(1)Site-specifically modified radioimmunoconjugates exhibit superior <i>in vitro</i> and <i>in vivo</i> behavior compared to analogues synthesized via traditional stochastic methods. However, the development of approaches to site-specific bioconjugation that combine high levels of selectivity, simple reaction conditions, and clinical translatability remains a challenge. Herein, we describe a novel solution to this problem: the use of dual-variable domain immunoglobulins (DVD-IgG). More specifically, we report the synthesis, <i>in vitro</i> evaluation, and <i>in vivo</i> validation of a ¹⁷⁷Lu-labeled radioimmunoconjugate based on ^HER2DVD, a DVD-IgG containing the HER2-targeting variable domains of trastuzumab and the catalytic variable domains of IgG h38C2. To this end, we first modified ^HER2DVD with a phenyloxadiazolyl methlysulfone-modified variant of the chelator CHX-A″-DTPA (PODS-CHX-A''-DTPA) and verified the site-specificity of the conjugation for the reactive lysines within the catalytic domains via chemical assay, MALDI-ToF mass spectrometry, and SDS-PAGE. The chelator-bearing immunoconjugate was subsequently labeled with [¹⁷⁷Lu]Lu³⁺ to produce the completed radioimmunoconjugate, [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD, in >80% radiochemical conversion and a specific activity of 29.5 ± 7.1 GBq/μmol. [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD did not form aggregates upon prolonged incubation in human serum, displayed 87% stability to demetalation over a 7 days of incubation in serum, and exhibited an immunoreactive fraction of 0.95 with HER2-coated beads. Finally, we compared the pharmacokinetic profile of [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD to that of a ¹⁷⁷Lu-labeled variant of trastuzumab in mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. The <i>in vivo</i> performance of [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD matched that of ¹⁷⁷Lu-labeled trastuzumab, with the former producing a tumoral activity concentration of 34.1 ± 12.1 %ID/g at 168 h and tumor-to-blood, tumor-to-liver, and tumor-to-kidney activity concentration ratios of 10.5, 9.6, and 21.8, respectively, at the same time point. Importantly, the DVD-IgG did not exhibit a substantially longer serum half-life than the traditional IgG despite its significantly larger size (202 kDa for the former vs 148 kDa for the latter). Taken together, these data suggest that DVD-IgGs represent a viable platform for the future development of highly effective site-specifically labeled radioimmunoconjugates for diagnostic imaging, theranostic imaging, and radioimmunotherapy.Molecular pharmaceuticsLeveraging a Dual Variable Domain Immunoglobulin to Create a Site-Specifically Modified Radioimmunoconjugate.PMC10263003S10 RR020892R01 CA244327331659K99 ES034053P30 CA008748R21 EB030275S10 OD016207U01CA221046U01 CA221046R01CA240963R01 CA174844R01 CA204167R01 CA204484R01CA204484R01 CA240963R21EB030275R01CA244327R01CA204167Rader CMacPherson DSHwang DKeinanen OSarrett SMRodriguez CZeglis BMfalseLeveraging a Dual Variable Domain Immunoglobulin to Create a Site-Specifically Modified Radioimmunoconjugate.Site-specifically modified radioimmunoconjugates exhibit superior <i>in vitro</i> and <i>in vivo</i> behavior compared to analogues synthesized via traditional stochastic methods. However, the development of approaches to site-specific bioconjugation that combine high levels of selectivity, simple reaction conditions, and clinical translatability remains a challenge. Herein, we describe a novel solution to this problem: the use of dual-variable domain immunoglobulins (DVD-IgG). More specifically, we report the synthesis, <i>in vitro</i> evaluation, and <i>in vivo</i> validation of a ¹⁷⁷Lu-labeled radioimmunoconjugate based on ^HER2DVD, a DVD-IgG containing the HER2-targeting variable domains of trastuzumab and the catalytic variable domains of IgG h38C2. To this end, we first modified ^HER2DVD with a phenyloxadiazolyl methlysulfone-modified variant of the chelator CHX-A″-DTPA (PODS-CHX-A''-DTPA) and verified the site-specificity of the conjugation for the reactive lysines within the catalytic domains via chemical assay, MALDI-ToF mass spectrometry, and SDS-PAGE. The chelator-bearing immunoconjugate was subsequently labeled with [¹⁷⁷Lu]Lu³⁺ to produce the completed radioimmunoconjugate, [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD, in >80% radiochemical conversion and a specific activity of 29.5 ± 7.1 GBq/μmol. [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD did not form aggregates upon prolonged incubation in human serum, displayed 87% stability to demetalation over a 7 days of incubation in serum, and exhibited an immunoreactive fraction of 0.95 with HER2-coated beads. Finally, we compared the pharmacokinetic profile of [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD to that of a ¹⁷⁷Lu-labeled variant of trastuzumab in mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. The <i>in vivo</i> performance of [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD matched that of ¹⁷⁷Lu-labeled trastuzumab, with the former producing a tumoral activity concentration of 34.1 ± 12.1 %ID/g at 168 h and tumor-to-blood, tumor-to-liver, and tumor-to-kidney activity concentration ratios of 10.5, 9.6, and 21.8, respectively, at the same time point. Importantly, the DVD-IgG did not exhibit a substantially longer serum half-life than the traditional IgG despite its significantly larger size (202 kDa for the former vs 148 kDa for the latter). Taken together, these data suggest that DVD-IgGs represent a viable platform for the future development of highly effective site-specifically labeled radioimmunoconjugates for diagnostic imaging, theranostic imaging, and radioimmunotherapy.2023-01-01T00:00:00Z2023 Jan2025-04-05T11:27:29.509Z2025-04-05T11:27:29.509ZS-EPMC102630033637769610.1021/acs.molpharmaceut.2c00700