Spot on: Bacterial transglutaminase enables the site‐specific modification of Gln side chains of tumor‐targeting antibodies with various probes containing lysine or lysine surrogates. The method yields completely homogeneous immunoconjugates with a defined stoichiometry. In comparative in vivo studies with xenografted mice the pharmacological profiles for enzymatically conjugated antibodies were better than those of chemically modified analogues.

The L1‐cell adhesion molecule (L1‐CAM) is highly expressed in various cancer types including ovarian carcinoma but is absent from most normal tissue. A chimeric monoclonal antibody, chCE7, specifically binds to human L1‐CAM and exhibits anti‐proliferative effects on L1‐CAM‐expressing tumor cells. The goal of this study was to evaluate the efficacy of a novel 177Lu‐chCE7 radioimmunotherapeutic agent and to compare it to a treatment protocol with unlabeled, growth‐inhibiting chCE7 in a mouse xenograft model of disseminated ovarian cancer. chCE7agl, an aglycosylated IgG1 variant with improved pharmacokinetics, was conjugated with 1,4,7,10‐tetraazacyclododecane‐N‐N′‐N′‐N‴‐tetraacetic acid (DOTA) and labeled with the low‐energy β‐emitter 177Lu. Tumor growth and survival were assessed after a single i.v. dose of 8 MBq (60 μg) radioimmunoconjugate in nude mice bearing either subcutaneous or intraperitoneal SKOV3.ip1 human ovarian cancer tumors. Therapeutic efficacy was compared with three times weekly i.p. administration of 10 mg/kg unconjugated chCE7. In vivo analysis of 177Lu‐chCE7agl biodistribution demonstrated high and specific accumulation of radioactivity at the tumor site with maximal tumor uptake of up to 48.0 ± 8.1% ID/g at 168 h postinjection. A single treatment with 177Lu‐DOTA‐chCE7agl caused significant retardation of tumor growth and prolonged median survival from 33 to 71 days, while administration of a nontargeted 177Lu‐immunoconjugate had no beneficial effect. Three times weekly i.p. application of unlabeled chCE7 10 mg/kg similarly increased survival from 44 to 72 days. We conclude that a single dose of 177Lu‐DOTA‐chCE7agl is as effective as repeated administration of nonradioactive chCE7 for treatment of small intraperitoneal tumors expressing L1‐CAM.

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Site-specific enzymatic reactions with microbial transglutaminase (mTGase) lead to a homogenous species of immunoconjugates with a defined ligand/antibody ratio. In the present study, we have investigated the influence of different numbers of 1,4,7,10-tetraazacyclododecane-N-N′-N′′-N′′′-tetraacetic acid (DOTA) chelats coupled to a decalysine backbone on the in vivo behavior of the chimeric monoclonal anti-L1CAM antibody chCE7agl. The enzymatic conjugation of (DOTA)1-decalysine, (DOTA)3-decalysine or (DOTA)5-decalysine to the antibody heavy chain (via Gln295/297) gave rise to immunoconjugates containing two, six or ten DOTA moieties respectively. Radiolabeling of the immunoconjugates with 177Lu yielded specific activities of approximately 70 MBq/mg, 400 MBq/mg and 700 MBq/mg with increasing numbers of DOTA chelates. Biodistribution experiments in SKOV3ip human ovarian cancer cell xenografts demonstrated a high and specific accumulation of radioactivity at the tumor site for all antibody derivatives with a maximal tumor accumulation of 43.6±4.3% ID/g at 24 h for chCE7agl-[(DOTA)-decalysine]2, 30.6±12.0% ID/g at 24 h for chCE7agl-[(DOTA)3-decalysine]2 and 49.9±3.1% ID/g at 48 h for chCE7agl-[(DOTA)5-decalysine)]2. The rapid elimination from the blood of chCE7agl-[(DOTA)-decalysine]2 (1.0±0.1% ID/g at 24 h) is associated with a high liver accumulation (23.2±4.6% ID/g at 24 h). This behavior changed depending on the numbers of DOTA moieties coupled to the decalysine peptide with a slower blood clearance (5.1±1.0 (DOTA)3 versus 11.7±1.4% ID/g (DOTA)5, p<0.005 at 24 h) and lower radioactivity levels in the liver (21.4±3.4 (DOTA)3 versus 5.8±0.7 (DOTA)5, p<0.005 at 24 h). We conclude that the site-specific and stoichiometric uniform conjugation of the highly DOTA-substituted decalysine ((DOTA)5-decalysine) to an anti-tumor antibody leads to the formation of immunoconjugates with high specific activity and excellent in vivo behavior and is a valuable option for radioimmunotherapy and potentially antibody-drug conjugates (ADCs).

Wohldefiniert modifiziert: Die Anwendung der bakteriellen Transglutaminase ermöglicht die ortsspezifische Modifikation von Glutaminseitenketten tumorgerichteter Antikörper mit verschiedenen Lysin‐ähnlichen Sonden. Die Methode führt zu homogenen Immunkonjugaten mit definiertem stöchiometrischem Verhältnis. Vergleichenden In‐vivo‐Studien in xenotransplantierten Mäusen zufolge weisen die enzymatisch konjugierten Antikörper ein besseres pharmakologisches Profil auf als chemisch hergestellte Analoga.