Template directed synthesis of antibody Fc conjugates with concomitant ligand release.

Antibodies are an attractive therapeutic modality for cancer treatment as they allow the increase of the treatment response rate and avoid the severe side effects of chemotherapy. Notwithstanding the strong benefit of antibodies, the efficacy of anti-cancer antibodies can dramatically vary among patients and ultimately result in no response to the treatment. Here, we have developed a novel means to regioselectively label the Fc domain of any therapeutic antibody with a radionuclide chelator in a single step chemistry, with the aim to study by SPECT/CT imaging if the radiolabeled antibody is capable of targeting cancer cells in vivo. A Fc-III peptide was used as bait to bring a carbonate electrophilic site linked to a metal chelator and to a carboxyphenyl leaving group in close proximity with an antibody Fc nucleophile amino acid (K317), thereby triggering the covalent linkage of the chelator to the antibody lysine, with the concomitant release of the carboxyphenyl Fc-III ligand. Using CHX-A''-DTPA, we radiolabeled trastuzumab with indium-111 and showed in biodistribution and imaging experiments that the antibody accumulated successfully in the SK-OV-3 xenograft tumour implanted in mice. We found that our methodology leads to homogeneous conjugation of CHX-A''-DTPA to the antibody, and confirmed that the Fc domain can be selectively labeled at K317, with a minor level of unspecific labeling on the Fab domain. The present method can be developed as a clinical diagnostic tool to predict the success of the therapy. Furthermore, our Fc-III one step chemistry concept paves the way to a broad array of other applications in antibody bioengineering.

Comparison of absorbed dose extrapolation methods for mouse-to-human translation of radiolabelled macromolecules.

BACKGROUND: Extrapolation of human absorbed doses (ADs) from biodistribution experiments on laboratory animals is used to predict the efficacy and toxicity profiles of new radiopharmaceuticals. Comparative studies between available animal-to-human dosimetry extrapolation methods are missing. We compared five computational methods for mice-to-human AD extrapolations, using two different radiopharmaceuticals, namely [111In]CHX-DTPA-scFv78-Fc and [68Ga]NODAGA-RGDyK. Human organ-specific time-integrated activity coefficients (TIACs) were derived from biodistribution studies previously conducted in our centre. The five computational methods adopted are based on simple direct application of mice TIACs to human organs (M1), relative mass scaling (M2), metabolic time scaling (M3), combined mass and time scaling (M4), and organ-specific allometric scaling (M5), respectively. For [68Ga]NODAGA-RGDyK, these methods for mice-to-human extrapolations were tested against the ADs obtained on patients, previously published by our group. Lastly, an average [68Ga]NODAGA-RGDyK-specific allometric parameter αnew was calculated from the organ-specific biological half-lives in mouse and humans and retrospectively applied to M3 and M4 to assess differences in human AD predictions with the α = 0.25 recommended by previous studies. RESULTS: For both radiopharmaceuticals, the five extrapolation methods showed significantly different AD results (p < 0.0001). In general, organ ADs obtained with M3 were higher than those obtained with the other methods. For [68Ga]NODAGA-RGDyK, no significant differences were found between ADs calculated with M3 and those obtained directly on human subjects (H) (p = 0.99; average M3/H AD ratio = 1.03). All other methods for dose extrapolations resulted in ADs significantly different from those calculated directly on humans (all p ≤ 0.0001). Organ-specific allometric parameters calculated using combined experimental [68Ga]NODAGA-RGDyK mice and human biodistribution data varied significantly. ADs calculated with M3 and M4 after the application of αnew = 0.17 were significantly different from those obtained by the application of α = 0.25 (both p < 0.001). CONCLUSIONS: Available methods for mouse-to-human dosimetry extrapolations provided significantly different results in two different experimental models. For [68Ga]NODAGA-RGDyK, the best approximation of human dosimetry was shown by M3, applying a metabolic scaling to the mouse organ TIACs. The accuracy of more refined extrapolation algorithms adopting model-specific metabolic scaling parameters should be further investigated.

Biological evaluation of new TEM1 targeting recombinant antibodies for radioimmunotherapy: In vitro, in vivo and in silico studies.

The tumour endothelial marker 1 (TEM1/endosialin/CD248) is a receptor overexpressed in several human solid tumours and silenced in normal adult tissues, representing a suitable and potentially safe target for radioimmunotherapy of sarcoma. To develop new tools with improved TEM1 targeting properties, a new panel of antibody fragments was for the first time evaluated preclinically following 125I radiolabelling. The antibody fragment 1C1m-Fc, with the highest human/murine TEM1 binding affinity, was extensively characterized in vitro and in vivo in a Ewing's sarcoma human xenograft mouse model. In silico studies were also performed to elucidate the influence of a single amino acid mutation in the complementarity-determining region (CDR3) of the heavy chain, upon affinity maturation of the parental clone 1C1-Fc. From this study, 1C1m-Fc emerged as a promising candidate for the development of TEM1-targeted radioimmunoconjugates, namely to be further explored for theranostic applications with other suitable medical radionuclides.

Correction to: Preclinical Evaluation and Dosimetry of [111In] CHX-DTPA-scFv78-Fc Targeting Endosialin/Tumor Endothelial Marker 1 (TEM1).

In the original article, some numerical values in the following paragraph were reported incorrectly.

Preclinical Evaluation and Dosimetry of [111In]CHX-DTPA-scFv78-Fc Targeting Endosialin/Tumor Endothelial Marker 1 (TEM1).

PURPOSE: Endosialin/tumor endothelial marker-1 (TEM1) is an attractive theranostic target expressed by the microenvironment of a wide range of tumors, as well as by sarcoma and neuroblastoma cells. We report on the radiolabeling and preclinical evaluation of the scFv78-Fc, a fully human TEM1-targeting antibody fragment cross-reactive with mouse TEM1. PROCEDURES: The scFv78-Fc was conjugated with the chelator p-SCN-Bn-CHX-A"-DTPA, followed by labeling with indium-111. The number of chelators per molecule was estimated by mass spectrometry. A conventional saturation assay, extrapolated to infinite antigen concentration, was used to determine the immunoreactive fraction of the radioimmunoconjugate. The radiopharmaceutical biodistribution was assessed in immunodeficient mice grafted with Ewing's sarcoma RD-ES and neuroblastoma SK-N-AS human TEM1-positive tumors. The full biodistribution studies were preceded by a dose-escalation experiment based on the simultaneous administration of the radiopharmaceutical with increasing amounts of unlabeled scFv78-Fc. Radiation dosimetry extrapolations to human adults were obtained from mouse biodistribution data according to established methodologies and additional assumptions concerning the impact of the tumor antigenic sink in the cross-species translation. RESULTS: [111In]CHX-DTPA-scFv78-Fc was obtained with a radiochemical purity > 98 % after 1 h incubation at 42 °C and ultrafiltration. It showed good stability in human serum and > 70 % immunoreactive fraction. Biodistribution data acquired in tumor-bearing mice confirmed fast blood clearance and specific tumor targeting in both xenograft models. The radiopharmaceutical off-target uptake was predominantly abdominal. After a theoretical injection of [111In]CHX-DTPA-scFv78-Fc to the reference person, the organs receiving the highest absorbed dose would be the spleen (0.876 mGy/MBq), the liver (0.570 mGy/MBq) and the kidneys (0.298 mGy/MBq). The total body dose and the effective dose would be 0.058 mGy/MBq and 0.116 mSv/MBq, respectively. CONCLUSIONS: [111In]CHX-DTPA-scFv78-Fc binds specifically to endosialin/TEM1 in vitro and in vivo. Dosimetry estimates are in the range of other monoclonal antibodies radiolabeled with indium-111. [111In]CHX-DTPA-scFv78-Fc could be potentially translated into clinic.

A Robust Method for Assaying the Immunoreactive Fraction in Nonequilibrium Systems.

The immunoreactive fraction r provides important information on the functional purity of radiolabeled proteins. It is traditionally determined by saturating the radioimmunoconjugate with an increasing excess of antigen, followed by linear extrapolation to infinite antigen excess in a double inverse "Lindmo plot". Although several reports have described shortcomings in the Lindmo plot, a systematic examination is lacking. Using an experimental and simulation-based approach, we compared-for accuracy, precision and robustness-the Lindmo plot with the "rectangular hyperbola" extrapolation method based on the Langmuir model. The differences between the theoretical and extrapolated r values demonstrate that nonequilibrium and antigen depletion are important sources of error. The mathematical distortions resulting from the linearization of the data in the Lindmo plot induce fragility towards stochastic errors and make it necessary to exclude low bound fractions. The rectangular hyperbola provides robust and precise r estimates from raw binding data, even for slow kinetics.

Internal radiation dosimetry of a 152Tb-labeled antibody in tumor-bearing mice.

BACKGROUND: Biodistribution studies based on organ harvesting represent the gold standard pre-clinical technique for dose extrapolations. However, sequential imaging is becoming increasingly popular as it allows the extraction of longitudinal data from single animals, and a direct correlation with deterministic radiation effects. We assessed the feasibility of mouse-specific, microPET-based dosimetry of an antibody fragment labeled with the positron emitter 152Tb [(T1/2 = 17.5 h, Eß+mean = 1140 keV (20.3%)]. Image-based absorbed dose estimates were compared with those obtained from the extrapolation to 152Tb of a classical biodistribution experiment using the same antibody fragment labeled with 111In. 152Tb was produced by proton-induced spallation in a tantalum target, followed by mass separation and cation exchange chromatography. The endosialin-targeting scFv78-Fc fusion protein was conjugated with the chelator p-SCN-Bn-CHX-A"-DTPA, followed by labeling with either 152Tb or 111In. Micro-PET images of four immunodeficient female mice bearing RD-ES tumor xenografts were acquired 4, 24, and 48 h after the i.v. injection of 152Tb-CHX-DTPA-scFv78-Fc. After count/activity camera calibration, time-integrated activity coefficients (TIACs) were obtained for the following compartments: heart, lungs, liver, kidneys, intestines, tumor, and whole body, manually segmented on CT. For comparison, radiation dose estimates of 152Tb-CHX-DTPA-scFv78-Fc were extrapolated from mice dissected 4, 24, 48, and 96 h after the injection of 111In-CHX-DTPA-scFv78-Fc (3-5 mice per group). Imaging-derived and biodistribution-derived organ TIACs were used as input in the 25 g mouse model of OLINDA/EXM® 2.0, after appropriate mass rescaling. Tumor absorbed doses were obtained using the OLINDA2 sphere model. Finally, the relative percent difference (RD%) between absorbed doses obtained from imaging and biodistribution were calculated. RESULTS: RD% between microPET-based dosimetry and biodistribution-based dose extrapolations were + 12, - 14, and + 17 for the liver, the kidneys, and the tumors, respectively. Compared to biodistribution, the imaging method significantly overestimates the absorbed doses to the heart and the lungs (+ 89 and + 117% dose difference, respectively). CONCLUSIONS: MicroPET-based dosimetry of 152Tb is feasible, and the comparison with organ harvesting resulted in acceptable dose discrepancies for body districts that can be segmented on CT. These encouraging results warrant additional validation using radiolabeled biomolecules with a different biodistribution pattern.

Progress in Lanthionine and Protected Lanthionine Synthesis.

Lanthionine (Lan), a non-proteinogenic natural amino acid, is an essential component of peptidoglycan found in the cell wall of Fusobacterium species. Lan and ß-methyllanthionine are also key constituents in lantibiotics, a prevalent class of peptide antibiotics. The development of those new antibacterial drugs with enhanced properties is the focus of recent research. Since multiple isomers of Lan are possible, a regio- and diastereoselective synthesis is challenging. This comprehensive review summarizes the known chemical syntheses of lanthionine from various precursors (e.g., ß-chloroalanine, cystine, dehydroalanine, ß-iodoalanine, aziridine, serine lactone, sulfamidate) since 1941. Methods for preparation of unprotected, protected, orthogonally protected, and mutually orthogonally protected lanthionine with relevant experimental details and perspectives on their usefulness are provided. The potential of these Lan derivatives is illustrated by one recent application.

Cardiac Radionuclide Imaging in Rodents: A Review of Methods, Results, and Factors at Play.

The interest around small-animal cardiac radionuclide imaging is growing as rodent models can be manipulated to allow the simulation of human diseases. In addition to new radiopharmaceuticals testing, often researchers apply well-established probes to animal models, to follow the evolution of the target disease. This reverse translation of standard radiopharmaceuticals to rodent models is complicated by technical shortcomings and by obvious differences between human and rodent cardiac physiology. In addition, radionuclide studies involving small animals are affected by several extrinsic variables, such as the choice of anesthetic. In this paper, we review the major cardiac features that can be studied with classical single-photon and positron-emitting radiopharmaceuticals, namely, cardiac function, perfusion and metabolism, as well as the results and pitfalls of small-animal radionuclide imaging techniques. In addition, we provide a concise guide to the understanding of the most frequently used anesthetics such as ketamine/xylazine, isoflurane, and pentobarbital. We address in particular their mechanisms of action and the potential effects on radionuclide imaging. Indeed, cardiac function, perfusion, and metabolism can all be significantly affected by varying anesthetics and animal handling conditions.

Enantioselective synthesis of α-benzylated lanthionines and related tripeptides for biological incorporation into E. coli peptidoglycan.

The synthesis of modified tripeptides (S)-Ala-γ-(R)-Glu-X, where X = (R,S) or (R,R) diastereomers of α-benzyl or α-(4-azidobenzyl)lanthionine, was carried out. The chemical strategy involved the enantioselective alkylation of a 4-MeO-phenyloxazoline. The reductive opening of the alkylated oxazolines, followed by cyclization and oxidation, led to four PMB-protected sulfamidates. Subsequent PMB removal, Boc protection and regioselective opening with cysteine methyl ester led to protected lanthionines. These compounds were further converted in a one pot process to the corresponding protected tripeptides. After ester and Boc deprotection, the four tripeptides were evaluated as potential analogues of the natural tripeptide (S)-Ala-γ-(R)-Glu-meso-A2pm. These compounds were evaluated for introduction, by means of the biosynthetic recycling pathway, into the peptidoglycan of Escherichia coli. A successful in vitro biosynthesis of UDP-MurNAc-tripeptides from the tripeptides containing α-benzyl lanthionine was achieved using purified murein peptide ligase (Mpl). Bioincorporation into E. coli W7 did not occur under different tested conditions probably due to the bulky benzyl group at the Cα carbon of the C-terminal amino acid.

Stereoselective synthesis of lanthionine derivatives in aqueous solution and their incorporation into the peptidoglycan of Escherichia coli.

The three diastereoisomers-(R,R), (S,S) and meso-of lanthionine were synthesized in aqueous solution with high diastereoselectivity (>99%). The (S) and (R) enantiomers of two differently protected sulfamidates were opened by nucleophilic attack of (R) or (S)-cysteine. Acidification and controlled heating liberated the free lanthionines. Using the same chemistry, an α-benzyl lanthionine was also prepared. The proposed method, which avoids the need of enrichment by recrystallization, opens the way to the labelling of these compounds with (35)S. Furthermore, in vivo bioincorporation into Escherichia coli W7 was studied. No incorporation of α-benzyl lanthionine was observed. In contrast, meso-lanthionine can effectively replace meso-diaminopimelic acid in vivo, while in the presence of (R,R)-lanthionine the initial increase of bacterial growth was followed by cell lysis. In the future, meso-[(35)S]lanthionine could be used to study the biosynthesis of peptidoglycan and its turnover in relation to cell growth and division.