Impact of DOTA Conjugation on Pharmacokinetics and Immunoreactivity of [177Lu]Lu-1C1m-Fc, an Anti TEM-1 Fusion Protein Antibody in a TEM-1 Positive Tumor Mouse Model

Delage, Judith Anna; Faivre-Chauvet, Alain; Barbet, Jacques; Fierle, Julie K.; Schaefer, Niklaus; Coukos, George; Viertl, David; Dunn, Steven M.; Gnesin, Silvano; Prior, John O.

doi:10.3390/pharmaceutics13010096

Cited by 12 publications

(30 citation statements)

References 36 publications

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“…The biodistribution profile of [ 64 Cu]Cu-4DOTA-1C1m-Fc ( Figure 4 a) was very similar to the one of [ 177 Lu]Lu-1DOTA-1C1m-Fc ( Figure 4 b, data already published in [ 28 ]) or [ 177 Lu]Lu-3DOTA-1C1m-Fc ( Figure 4 c, data already published in [ 27 ]). The blood uptake at 4 h of [ 64 Cu]Cu-4DOTA-1C1m-Fc was closer to that of [ 177 Lu]Lu-1DOTA-1C1m-Fc.…”

Section: Resultssupporting

confidence: 59%

“…[ 177 Lu]Lu-1C1m-Fc-absorbed dose data were extrapolated from [ 64 Cu]Cu-1C1m-Fc biodistribution data and compared to the experimental absorbed doses previously published in [ 27 , 28 ] ( Table 4 ). Extrapolated absorbed doses for parenchymal organs (such as: liver, lung, spleen and kidneys) showed a 20% difference when compared with [ 177 Lu]Lu-1DOTA-1C1m-Fc.…”

Section: Resultsmentioning

confidence: 99%

“…In our previous works, we have validated the relevance of a bivalent Fc-fusion protein based on a novel single chain antibody, 1C1m-Fc, radiolabeled with 177 Lu for a therapeutic approach [ 27 , 28 ]. In the present study, we studied the detection of TEM-1 in tumors using 1C1m-Fc radiolabeled with 64 Cu for PET imaging.…”

Section: Discussionmentioning

confidence: 99%

“…The number of chelate per antibody was determined by mass spectrometry (MS) analysis using a Q Exactive HF Orbitrap (Thermo Fisher Scientific, Waltham, MA, USA), as previously described in Delage et al [ 27 , 28 ].…”

Section: Methodsmentioning

confidence: 99%

“…The impact of DOTA conjugation was evaluated. Using a pharmacokinetic model, the number of DOTA per fusion protein antibody was demonstrated to have an impact on the pharmacokinetics and immunoreactivity of [ 177 Lu]Lu-1C1m-Fc [ 28 ]. The biodistribution and imaging contrast was improved by decreasing the number of chelating agents per 1C1m-Fc molecule: one DOTA per 1C1m-Fc gave the best tumor-to-liver ratio, but the specific activity was only 200 MBq/mg.…”

Section: Introductionmentioning

confidence: 99%

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Copper-64-Labeled 1C1m-Fc, a New Tool for TEM-1 PET Imaging and Prediction of Lutetium-177-Labeled 1C1m-Fc Therapy Efficacy and Safety

Delage

Gnesin

Prior

et al. 2021

Cancers

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1C1m-Fc, a promising anti-TEM-1 DOTA conjugate, was labeled with 64Cu to target cancer cells for PET imaging and predicting the efficacy and safety of a previously studied [177Lu]Lu-1C1m-Fc companion therapy. DOTA-conjugated 1C1m-Fc was characterized by mass spectrometry, thin layer chromatography and immunoreactivity assessment. PET/CT and biodistribution studies were performed in human neuroblastoma xenografted mice. Absorbed doses were assessed from biodistribution results and extrapolated to 177Lu based on the [64Cu]Cu-1C1m-Fc data. The immunoreactivity was ≥ 70% after 48 h of incubation in serum, and the specificity of [64Cu]Cu-1C1m-Fc for the target was validated. High-resolution PET/CT images were obtained, with the best tumor-to-organ ratios reached at 24 or 48 h and correlated with results of the biodistribution study. Healthy organs receiving the highest doses were the liver, the kidneys and the uterus. [64Cu]Cu-1C1m-Fc could be of interest to give an indication of 177Lu dosimetry for parenchymal organs. In the uterus and the tumor, characterized by specific TEM-1 expression, the 177Lu-extrapolated absorbed doses are overestimated because of the lack of later measurement time points. Nevertheless, 1C1m-Fc radiolabeled with 64Cu for imaging would appear as an interesting radionuclide companion for therapeutic application with [177Lu]Lu-1C1m-Fc.

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Section: Resultssupporting

confidence: 59%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Copper-64-Labeled 1C1m-Fc, a New Tool for TEM-1 PET Imaging and Prediction of Lutetium-177-Labeled 1C1m-Fc Therapy Efficacy and Safety

Delage

Gnesin

Prior

et al. 2021

Cancers

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[²¹³Bi]Bi³⁺/[¹¹¹In]In³⁺-neunpa-cycMSH: Theranostic Radiopharmaceutical Targeting Melanoma─Structural, Radiochemical, and Biological Evaluation

Wharton

Zhang²,

Yang

et al. 2022

Bioconjugate Chem.

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With the emergence of [ 225 Ac]Ac 3+ as a therapeutic radionuclide for targeted α therapy (TAT), access to clinical quantities of the potent, short-lived α-emitter [ 213 Bi]Bi 3+ (t 1/2 = 45.6 min) will increase over the next decade. With this in mind, the nonadentate chelator, H 4 neunpa-NH 2 , has been investigated as a ligand for chelation of [ 213 Bi]Bi 3+ in combination with [ 111 In]In 3+ as a suitable radionuclidic pair for TAT and single photon emission computed tomography (SPECT) diagnostics. Nuclear magnetic resonance (NMR) spectroscopy was utilized to assess the coordination characteristics of H 4 neunpa-NH 2 on complexation of [ nat Bi]Bi 3+ , while the solid-state structure of [ nat Bi][Bi(neunpa-NH 3 )] was characterized via X-ray diffraction (XRD) studies, and density functional theory (DFT) calculations were performed to elucidate the conformational geometries of the metal complex in solution. H 4 neunpa-NH 2 exhibited fast complexation kinetics with [ 213 Bi]Bi 3+ at RT achieving quantitative radiolabeling within 5 min at 10 −8 M ligand concentration, which was accompanied by the formation of a kinetically inert complex. Two bioconjugates incorporating the melanocortin 1 receptor (MC1R) targeting peptide Nle-CycMSH hex were synthesized featuring two different covalent linkers for in vivo evaluation with [ 213 Bi]Bi 3+ and [ 111 In]In 3+ . High molar activities of 7.47 and 21.0 GBq/μmol were achieved for each of the bioconjugates with [ 213 Bi]Bi 3+ . SPECT/CT scans of the [ 111 In]In 3+ -labeled tracer showed accumulation in the tumor over time, which was accompanied by high liver uptake and clearance via the hepatic pathway due to the high lipophilicity of the covalent linker. In vivo biodistribution studies in C57Bl/6J mice bearing B16-F10 tumor xenografts showed good tumor uptake (5.91% ID/g) at 1 h post-administration with [ 213 Bi][Bi(neunpa-Ph-Pip-Nle-CycMSH hex )]. This study demonstrates H 4 neunpa-NH 2 to be an effective chelating ligand for [ 213 Bi]Bi 3+ and [ 111 In]In 3+ , with promising characteristics for further development toward theranostic applications.

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A Kinetically Controlled Bioconjugation Method for the Synthesis of Radioimmunoconjugates and the Development of a Domain Mapping MS-Workflow for Its Characterization

Pometti,

Di Natale,

Geremia

et al. 2024

Bioconjugate Chem.

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Immunoconjugates exploit the high affinity of monoclonal antibodies for a recognized antigen to selectively deliver a cytotoxic payload, such as drugs or radioactive nuclides, at the site of disease. Despite numerous techniques have been recently developed for site-selective bioconjugations of protein structures, reaction of ε-amine group of lysine residues with electrophilic reactants, such as activated esters (NHS), is the main method reported in the literature as it maintains proteins in their native conformation. Since antibodies hold a high number of lysine residues, a heterogeneous mixture of conjugates will be generated, which can result in decreased target affinity. Here, we report an intradomain regioselective bioconjugation between the monoclonal antibody Trastuzumab and the N-hydroxysuccinimide ester of the chelator 2,2′,2″,2‴-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) by a kinetically controlled reaction adding substoichiometric quantities of the activated ester to the mAb working at slightly basic pH. Liquid chromatography–mass spectrometry (LC–MS) analyses were carried out to assess the chelator-antibody ratio (CAR) and the number of chelating moieties linked to the mAb chains. Proteolysis experiments showed four lysine residues mainly involved in bioconjugation (K188 for the light chain and K30, K293, and K417 for the heavy chain), each of which was located in a different domain. Since the displayed intradomain regioselectivity, a domain mapping MS-workflow, based on a selective domain denaturation, was developed to quantify the percentage of chelator linked to each mAb domain. The resulting immunoconjugate mixture showed an average CAR of 0.9. About a third of the heavy chains were found as monoconjugated, whereas conjugation of the chelator in the light chain was negligible. Domain mapping showed the CH3 domain bearing 13% of conjugated DOTA, followed by CH2 and VH respectively bearing 12.5 and 11% of bonded chelator. Bioconjugation was not found in the CH1 domain, whereas for the light chain, only the CL domain was conjugated (6%). Data analysis based on LC–MS quantification of different analytical levels (intact, reduced chains, and domains) provided the immunoconjugate formulation. A mixture of immunoconjugates restricted to 15 species was obtained, and the percentage of each one within the mixture was calculated. In particular, species bearing 1 DOTA with a relative abundance ranging from 4 to 20-fold, in comparison to species bearing 2DOTA, were observed. Pairing of bioconjugation under kinetic control with the developed domain mapping MS-workflow could raise the standard of chemical quality for immunoconjugates obtained with commercially available reactants.

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Impact of DOTA Conjugation on Pharmacokinetics and Immunoreactivity of [177Lu]Lu-1C1m-Fc, an Anti TEM-1 Fusion Protein Antibody in a TEM-1 Positive Tumor Mouse Model

Cited by 12 publications

References 36 publications

Copper-64-Labeled 1C1m-Fc, a New Tool for TEM-1 PET Imaging and Prediction of Lutetium-177-Labeled 1C1m-Fc Therapy Efficacy and Safety

Copper-64-Labeled 1C1m-Fc, a New Tool for TEM-1 PET Imaging and Prediction of Lutetium-177-Labeled 1C1m-Fc Therapy Efficacy and Safety

[²¹³Bi]Bi³⁺/[¹¹¹In]In³⁺-neunpa-cycMSH: Theranostic Radiopharmaceutical Targeting Melanoma─Structural, Radiochemical, and Biological Evaluation

A Kinetically Controlled Bioconjugation Method for the Synthesis of Radioimmunoconjugates and the Development of a Domain Mapping MS-Workflow for Its Characterization

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Impact of DOTA Conjugation on Pharmacokinetics and Immunoreactivity of [177Lu]Lu-1C1m-Fc, an Anti TEM-1 Fusion Protein Antibody in a TEM-1 Positive Tumor Mouse Model

Cited by 12 publications

References 36 publications

Copper-64-Labeled 1C1m-Fc, a New Tool for TEM-1 PET Imaging and Prediction of Lutetium-177-Labeled 1C1m-Fc Therapy Efficacy and Safety

Copper-64-Labeled 1C1m-Fc, a New Tool for TEM-1 PET Imaging and Prediction of Lutetium-177-Labeled 1C1m-Fc Therapy Efficacy and Safety

[213Bi]Bi3+/[111In]In3+-neunpa-cycMSH: Theranostic Radiopharmaceutical Targeting Melanoma─Structural, Radiochemical, and Biological Evaluation

A Kinetically Controlled Bioconjugation Method for the Synthesis of Radioimmunoconjugates and the Development of a Domain Mapping MS-Workflow for Its Characterization

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[²¹³Bi]Bi³⁺/[¹¹¹In]In³⁺-neunpa-cycMSH: Theranostic Radiopharmaceutical Targeting Melanoma─Structural, Radiochemical, and Biological Evaluation