Simultaneous Photoradiochemical Labelling of Antibodies for Immuno-PET

Klingler, Simon; Eichenberger, Larissa S.; Holland, Jason P.

doi:10.2139/ssrn.3307531

Cited by 5 publications

(30 citation statements)

References 27 publications

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“…A combination of stability measurements, cellular assays, temporal PET imaging, and biodistribution studies confirmed that the 89 Zr-DFO-azepintrastuzumab obtained by photoradiochemistry was biochemically equivalent to 89 Zr-DFO-Nsucc-trastuzumab produced by a conventional thermochemical conjugation route (Fig. 3) (32,33). Recent work by Poot et al introduced fully automated radiolabeling and purification of 89 Zr-radiolabeled antibodies, but as with manual synthesis, the approach still relies on the use of preconjugated protein (34).…”

Section: Chemical Functionalization Methodsmentioning

confidence: 59%

“…As an alternative conjugation method, our group recently reported the development of a photochemically mediated conjugation and radiolabeling reaction based on chelates bearing aryl azide groups ( Fig. 2D) (30)(31)(32). The process is distinct from almost all other conjugation chemistries in that the active reagent is formed in situ by irradiating the samples with ultraviolet light (;365 nm).…”

Section: Chemical Functionalization Methodsmentioning

confidence: 99%

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The Impact of Emerging Bioconjugation Chemistries on Radiopharmaceuticals

Fay

Holland

2019

J Nucl Med

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The use of radiolabeled antibodies, immunoglobulin fragments, and other proteins are an increasingly important sector of research for diagnostic imaging and targeted radiotherapy in nuclear medicine. As with all radiopharmaceuticals, efficient radiochemistry is a prerequisite to clinical translation. For proteins, variations in the primary amino acid sequence, the secondary structures, and tertiary folds, as well as differences in the size, charge, polarity, lipophilicity, and the presence of posttranslational modifications, add complexity to the system. The choice of radionuclide or chelate, and its impact on the thermodynamic, kinetic, and metabolic stability of a radiotracer, has attracted much attention but the chemistry by which the radionuclide is conjugated to the protein scaffold is of equal importance. Recently, a wealth of creative advances in protein ligation methods based on chemical, photochemical, and enzyme-mediated processes has emerged. As radiochemists explore alternative bioconjugation strategies, this article considers their potential impact on radiotracer design.

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Section: Chemical Functionalization Methodsmentioning

confidence: 59%

Section: Chemical Functionalization Methodsmentioning

confidence: 99%

The Impact of Emerging Bioconjugation Chemistries on Radiopharmaceuticals

Fay

Holland

2019

J Nucl Med

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“…The synthesis, characterization, and reactivity of the photoactive chelate DFO-ArN3 (1) was reported elsewhere. (17) Briefly, a neutralized stock solution of [ 89 Zr(C2O4)4] 4-( 89 Zr-oxalate) was added to an open glass vial containing a mixture of compound 1 and formulated onartuzumab with an initial chelate-to-mAb ratio of 6.15-to-1, and a final pH ~8-9. The reaction was stirred gently at room temperature and irradiated directly for 10 min.…”

Section: Radiochemistrymentioning

confidence: 99%

“…Prior tests determined that this experimental geometry was sufficient to affect complete photochemical reaction of DFO-ArN3. (17) After quenching the reaction with excess DTPA to ensure that any free Page 9 89 Zr 4+ ions that may non-specifically bind to proteins were solubilized, crude aliquots were analyzed by radioactive instant thin-layer chromatography (radio-iTLC; Supplemental Figure 2), manual size-exclusion chromatography (SEC) using PD-10 gel filtration columns ( Figure 2A), and high-performance liquid chromatography coupled to a size exclusion gel column (SEC-HPLC; Figure 2B). Chromatographic methods on the crude reaction samples confirmed that 89 Zr-activity was bound to the protein as evidenced by a peak in the PD-10 chromatograms in the 0.0-1.6 mL fraction (radiochemical conversion, RCC ~25.0%, Figure 2A red trace) and by a radioactive peak in the SEC-HPLC that coincided with the retention time of onartuzumab at ~14.1 min.…”

Section: Radiochemistrymentioning

confidence: 99%

“…(8)(9)(10)(11) Our group has recently developed a one-pot photoradiosynthesis approach for the simultaneous conjugation and 89 Zr-radiolabeling of mAbs. (15)(16)(17)(18)(19) Here, we evaluated the photoradiosynthesis of 89 Zr-radiolabeled onartuzumab starting from fully formulated MetMAb without pre-purification of the protein or isolation of an intermediate (Figure 1).…”

Section: Introductionmentioning

confidence: 99%

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Light-Induced Radiosynthesis of ⁸⁹Zr-DFO-Azepin-Onartuzumab for Imaging the Hepatocyte Growth Factor Receptor

2020

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Methods that provide rapid access to radiolabeled antibodies are vital in the development of diagnostic and radiotherapeutic agents for positron emission tomography (PET) or radioimmunotherapy. The human hepatocyte growth factor receptor (c-MET) signaling pathway is dysregulated in a number of malignancies including gastric cancer, and is an important biomarker in drug discovery. Here, we used a photoradiochemical approach to produce 89Zr-radiolabeled onartuzumab (a monovalent, anti-human c-MET antibody), starting directly from the fully formulated drug (MetMAb). Methods: Simultaneous 89Zr-radiolabeling and protein conjugation was performed in one-pot reactions containing 89Zr-oxalate, the photoactive chelate DFO-aryl azide (DFO-ArN3) and MetMAb to give 89ZrDFOazepin-onartuzumab. As a control, 89ZrDFO-Bn-NCS-onartuzumab was prepared via a conventional two-step process using pre-purified onartuzumab and DFO-Bn-NCS. Radiotracers were purified by using size-exclusion methods and evaluated by radiochromatography. Radiochemical stability was studied in human serum and immunoreactivity was determined by cellular binding assays using MKN-45 gastric carcinoma cells. PET imaging at multiple time points (0-72 h) was performed in female athymic nude mice bearing subcutaneous MKN-45 xenografts. Biodistribution experiments were performed after the final image. Tumor specificity of 89ZrDFO-azepin-onartuzumab was assessed by competitive inhibition (blocking) studies. Results: Initial photoradiosynthesis experiments produced 89ZrDFO-azepin-onartuzumab in <15 min. with an isolated decay-corrected radiochemical yield (RCY) of 24.8%, a radiochemical purity (RCP) 90% and a molar activity (Am) of 1.5 MBq nmol-1. Reaction optimization improved the radiochemical conversion (RCC) of 89ZrDFO-azepin-onartuzumab to 56.9±4.1% (n = 3), with isolated RCYs of 41.2±10.6% (n = 3), and RCPs >90%. Conventional methods produced 89ZrDFO-Bn-NCSonartuzumab with isolated RCY >97%, RCP >97% and Am 14.0 MBq nmol-1. Both radiotracers were immunoreactive and stable in human serum. PET imaging and biodistribution studies showed high tumor uptake for both radiotracers. By 72 h, tumor and liver uptake reached 15.37±5.21 %ID g-1, 6.56±4.03 %ID g-1, respectively for 89ZrDFO-azepin-onartuzumab (n = 4), and 21.38±11.57 %ID g-1 and 18.84±6.03 %ID g-1 for 89ZrDFO-Bn-NCS-onartuzumab (n = 4). Blocking experiments gave a statistically significant reduction in tumor uptake (6.34±0.47 %ID g-1) of 89ZrDFO-azepin-onartuzumab (n = 4). Conclusion: Experiments demonstrate that photoradiosynthesis is a viable alternative approach for producing 89Zr-radiolabeled antibodies direct in protein formulation buffer which reduces protein aggregation and liver uptake.

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The Influence of a Polyethylene Glycol Linker on the Metabolism and Pharmacokinetics of a ⁸⁹Zr-Radiolabeled Antibody

et al. 2021

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Most experimental work in the space of bioconjugation chemistry focuses on using new methods to construct covalent bonds between a cargo molecule and a protein of interest such as a monoclonal antibody (mAb). Bond formation is important for generating new diagnostic tools, yet when these compounds advance to preclinical in vitro and in vivo studies, and later for translation to the clinic, understanding the fate of potential metabolites that arise from chemical or enzymatic degradation of the construct is important to obtain a full picture of the pharmacokinetic performance of a new compound. In the context of designing new bioconjugate methods for labeling antibodies with the positron-emitting radionuclide 89Zr, we previously developed a photochemical process for making 89Zr-mAbs. Experimental studies on [89Zr]ZrDFO-PEG3-azepin-mAb constructs revealed that incorporation of the tris-polyethylene glycol (PEG3) linker improved the aqueous phase solubility and radiochemical conversion. However, the use of a PEG3 linker also has an impact on the whole-body residence time of the construct, leading to a more rapid excretion of the 89Zr activity when compared with radiotracers that lack the PEG3 chain. In this work, we investigated the metabolic fate of eight possible metabolites that arise from the logical disconnection of [89Zr]ZrDFO-PEG3-azepin-mAb at bonds which are susceptible to chemical or enzymatic cleavage. Synthesis combined with 89Zr-radiolabeling, small-animal positron emission tomography imaging at multiple time points from 0 to 20 h, and measurements of the effective half-life for whole-body excretion are reported. The conclusions are that the use of a PEG3 linker is non-innocent in terms of its impact on enhancing the metabolism of [89Zr]ZrDFO-PEG3-azepin-mAbs. In most cases, degradation can produce metabolites that are rapidly eliminated from the body, thereby enhancing image contrast by reducing nonspecific accumulation and retention of 89Zr in background organs such as the liver, spleen, kidney, and bone.

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Simultaneous Photoradiochemical Labelling of Antibodies for Immuno-PET

Cited by 5 publications

References 27 publications

The Impact of Emerging Bioconjugation Chemistries on Radiopharmaceuticals

The Impact of Emerging Bioconjugation Chemistries on Radiopharmaceuticals

Light-Induced Radiosynthesis of ⁸⁹Zr-DFO-Azepin-Onartuzumab for Imaging the Hepatocyte Growth Factor Receptor

The Influence of a Polyethylene Glycol Linker on the Metabolism and Pharmacokinetics of a ⁸⁹Zr-Radiolabeled Antibody

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Simultaneous Photoradiochemical Labelling of Antibodies for Immuno-PET

Cited by 5 publications

References 27 publications

The Impact of Emerging Bioconjugation Chemistries on Radiopharmaceuticals

The Impact of Emerging Bioconjugation Chemistries on Radiopharmaceuticals

Light-Induced Radiosynthesis of 89Zr-DFO-Azepin-Onartuzumab for Imaging the Hepatocyte Growth Factor Receptor

The Influence of a Polyethylene Glycol Linker on the Metabolism and Pharmacokinetics of a 89Zr-Radiolabeled Antibody

Contact Info

Product

Resources

About

Light-Induced Radiosynthesis of ⁸⁹Zr-DFO-Azepin-Onartuzumab for Imaging the Hepatocyte Growth Factor Receptor

The Influence of a Polyethylene Glycol Linker on the Metabolism and Pharmacokinetics of a ⁸⁹Zr-Radiolabeled Antibody