Site-specific modification of native IgGs with flexible drug-load.

Homogeneous, site-specifically conjugated antibodies have shown to result in antibody-drug conjugates (ADCs) with improved efficacy and tolerability compared to stochastically conjugated ADCs. However, precisely controlling the drug-load as well as attaching multiple payload moieties on the antibody remains challenging. Here, we demonstrate the simple and direct modification of native IgG-antibodies at the residue glutamine 295 (Q295) without the need for any protein engineering at flexible drug-to-antibody ratios of one or multiple payloads. The conjugation is enabled through short, positively charged lysine containing peptides and native, commercially available microbial transglutaminase. In proof-of-concept studies, HER2-targeting ADCs based on trastuzumab were generated with drug-to-antibody ratios (DARs) of 2 and 4 of the same or different payloads using orthogonal conjugation chemistries. Quantitative biodistribution studies performed with 111In-radiolabeled conjugates showed high tumour uptake and low accumulation of radioactivity in non-targeted tissues. A single dose study of trastuzumab conjugated to the highly potent payload α-Amanitin demonstrated complete and long-lasting tumour remissions and was well-tolerated at all dose levels tested.

Unlocking Reactivity of TrpB: A General Biocatalytic Platform for Synthesis of Tryptophan Analogues.

Derivatives of the amino acid tryptophan (Trp) serve as precursors for the chemical and biological synthesis of complex molecules with a wide range of biological properties. Trp analogues are also valuable as building blocks for medicinal chemistry and as tools for chemical biology. While the enantioselective synthesis of Trp analogues is often lengthy and requires the use of protecting groups, enzymes have the potential to synthesize such products in fewer steps and with the pristine chemo- and stereoselectivity that is a hallmark of biocatalysis. The enzyme TrpB is especially attractive because it can form Trp analogues directly from serine (Ser) and the corresponding indole analogue. However, many potentially useful substrates, including bulky or electron-deficient indoles, are poorly accepted. We have applied directed evolution to TrpB from Pyrococcus furiosus and Thermotoga maritima to generate a suite of catalysts for the synthesis of previously intractable Trp analogues. For the most challenging substrates, such as nitroindoles, the key to improving activity lay in the mutation of a universally conserved and mechanistically important residue, E104. The new catalysts express at high levels (>200 mg/L of Escherichia coli culture) and can be purified by heat treatment; they can operate up to 75 °C (where solubility is enhanced) and can synthesize enantiopure Trp analogues substituted at the 4-, 5-, 6-, and 7-positions, using Ser and readily available indole analogues as starting materials. Spectroscopic analysis shows that many of the activating mutations suppress the decomposition of the active electrophilic intermediate, an amino-acrylate, which aids in unlocking the synthetic potential of TrpB.

Dual, Site-Specific Modification of Antibodies by Using Solid-Phase Immobilized Microbial Transglutaminase.

Microbial transglutaminase (MTG) was stably solid-phase immobilized on glass microbeads by using a second-generation dendronized polymer. Immobilized MTG enabled the efficient generation of site-specifically conjugated proteins, including antibody fragments, as well as whole antibodies through distinct glutamines and, unprecedentedly, also through lysines with various bifunctional substrates with defined stoichiometries. With this method, we generated dual, site-specifically modified antibodies comprising a fluorescent probe and a metal chelator for radiolabeling-a strategy anticipated to design antibodies for imaging and simultaneous therapy. Furthermore, we provide evidence that immobilized MTG features higher siteselectivity than soluble MTG.

Translational immunoPET imaging using a radiolabeled GD2-specific antibody in neuroblastoma.

Background: Antibodies targeting surface expressed disialoganglioside GD2 are increasingly used in neuroblastoma immunotherapy and might also have potential for use in radioimmunotherapy. As such targeted treatments might benefit from a dedicated theranostic approach, we studied the influence of radiolabeling on the binding characteristics of ch14.18 antibodies produced by Chinese hamster ovary (CHO) cells and evaluated the benefit of GD2-ImmunoPET as a potential tool for therapy planning. Methods:64Cu was used to reduce radiation burden, which is of high importance especially in a pediatric patient population. 64Cu-labeling was accomplished using the chelators NOTA- or DOTAGA-NCS. Radiolabeled antibodies were characterized in vitro. [64Cu]Cu-DOTAGA-ch14.18/CHO was studied in a neuroblastoma mouse model (subcutaneous CHP-134 xenografts). In vivo PET and MR images were acquired at 3 h, 24 h, and 48 h p.i. The specificity of binding was verified using GD2-negative tumors (HEK-293 xenografts), a control antibody and in vivo blocking. A first translational application was performed by PET/MRI in a patient with metastasized neuroblastoma. Results: Radiolabeling at an antibody-to-chelator ratio ≥1:10 yielded a product with a radiochemical purity of ≥90% and a specific activity of 0.2-1.0 MBq/µg. Radiochelation was stable over 48 h in PBS, mouse serum or EDTA, and 50.8 ± 3.5% and 50.8 ± 2.0% of the radiolabeled conjugates, prepared at antibody-to-chelator ratios of 1:10 or 1:15, were immunoreactive. In vivo, highly specific accumulation (31.6 ± 5.8% ID/g) in neuroblastoma was shown preclinically. Clinical PET/MR scans using [64Cu]Cu-NOTA-ch14.18/CHO (NOTA used for safety reasons) could visualize neuroblastoma metastases. Conclusions:In vivo,64Cu-labeled ch14.18/CHO is suitable for specific identification of neuroblastoma in PET. A first patient PET indicated the feasibility of the method for clinical translation and the potential utility in image-guided therapy.