EGFR binding Fc domain-drug conjugates: stable and highly potent cytotoxic molecules mediate selective cell killing

The exposition of cancer cells to cytotoxic doses of payload is fundamental for the therapeutic efficacy of antibody drug conjugates (ADCs) in solid cancers. To maximize payload exposure, tissue penetration can be increased by utilizing smaller-sized drug conjugates which distribute deeper into the tumor. Our group recently explored small human epidermal growth factor receptor 2 (HER2) targeting Fc antigen binding fragments (Fcabs) for ADC applications in a feasibility study. Here, we expand this concept using epidermal growth factor receptor (EGFR) targeting Fcabs for the generation of site-specific auristatin-based drug conjugates. In contrast to HER2-targeting Fcabs, we identified novel conjugation sites in the EGFR-targeting Fcab scaffold that allowed for higher DAR enzymatic conjugation. We demonstrate feasibility of resultant EGFR-targeting Fcab-drug conjugates that retain binding to half-life prolonging neonatal Fc receptor (FcRn) and EGFR and show high serum stability as well as target receptor mediated cell killing at sub-nanomolar concentrations. Our results emphasize the applicability of the Fcab format for the generation of drug conjugates designed for increased penetration of solid tumors and potential FcRn-driven antibody-like pharmacokinetics

From strain engineering to process development: monoclonal antibody production with an unnatural amino acid in Pichia pastoris

Background Expansion of the genetic code is a frequently employed approach for the modification of recombinant protein properties. It involves reassignment of a codon to another, e.g., unnatural, amino acid and requires the action of a pair of orthogonal tRNA and aminoacyl tRNA synthetase modified to recognize only the desired amino acid. This approach was applied for the production of trastuzumab IgG carrying p-azido-l-phenylalanine (pAzF) in the industrial yeast Pichia pastoris. Combining the knowledge of protein folding and secretion with bioreactor cultivations, the aim of the work was to make the production of monoclonal antibodies with an expanded genetic code cost-effective on a laboratory scale. Results Co-translational transport of proteins into the endoplasmic reticulum through secretion signal prepeptide change and overexpression of lumenal chaperones Kar2p and Lhs1p improved the production of trastuzumab IgG and its Fab fragment with incorporated pAzF. In the case of Fab, a knockout of vacuolar targeting for protein degradation further increased protein yield. Fed-batch bioreactor cultivations of engineered P. pastoris strains increased IgG and IgGpAzF productivity by around 50- and 20-fold compared to screenings, yielding up to 238 mg L−1 and 15 mg L−1 of fully assembled tetrameric protein, respectively. Successful site-specific incorporation of pAzF was confirmed by mass spectrometry. Conclusions Pichia pastoris was successfully employed for cost-effective laboratory-scale production of a monoclonal antibody with an unnatural amino acid. Applying the results of this work in glycoengineered strains, and taking further steps in process development opens great possibilities for utilizing P. pastoris in the development of antibodies for subsequent conjugations with, e.g., bioactive payloads.ISSN:1475-285

Self-Assembled Hybrid Aptamer-Fc Conjugates for Targeted Delivery: A Modular Chemoenzymatic Approach

Over the past decade, DNA and RNA aptamers have attracted keen research interest due to their ability to specifically bind targets of therapeutic relevance. However, their application is often hampered by a short serum half-life and missing effector functions. Conjugation of aptamers to antibody Fc fragments could improve pharmacokinetics, enable immune effector mechanisms, and provide an option for the introduction of desired payloads (e.g., toxins or fluorescent dyes). We developed a modular scaffold-supported system based on human IgG1 Fc fragments, which allows for its dual functionalization with moieties of interest. In our approach, two bioorthogonal, enzyme-mediated reactions were used in combination with oxime ligation and self-assembly based on PNA–DNA base pairing. Thus, an engineered synthetic peptide nucleic acid (PNA) oligomer was coupled to the <i>C</i>-termini of the Fc dimer upon sequence-specific sortase A-mediated transpeptidation. Hybridization of the resulting Fc-PNA conjugate with a tailored DNA aptamer that binds cancer-related hepatocyte growth factor receptor (c-MET) led to a hybrid construct which showed strong and specific binding to c-MET and was readily internalized by c-MET-overexpressing cells. To install an additional orthogonally addressable site, aldehyde tag technology was applied followed by oxime ligation with an aminooxy-bearing fluorescent dye as model cargo. Delivery of fluorescent probe specifically to c-MET-overexpressing cells was confirmed by flow cytometry. Our approach can provide access to engineered aptamer-Fc conjugates with desired target specificity and cytotoxic payloads

A Generic Approach for Miniaturized Unbiased High-Throughput Screens of Bispecific Antibodies and Biparatopic Antibody–Drug Conjugates

The toolbox of modern antibody engineering allows the design of versatile novel functionalities exceeding nature’s repertoire. Many bispecific antibodies comprise heterodimeric Fc portions recently validated through the approval of several bispecific biotherapeutics. While heterodimerization methodologies have been established for low-throughput large-scale production, few approaches exist to overcome the bottleneck of large combinatorial screening efforts that are essential for the identification of the best possible bispecific antibody. This report presents a novel, robust and miniaturized heterodimerization process based on controlled Fab-arm exchange (cFAE), which is applicable to a variety of heterodimeric formats and compatible with automated high-throughput screens. Proof of applicability was shown for two therapeutic molecule classes and two relevant functional screening read-outs. First, the miniaturized production of biparatopic anti-c-MET antibody–drug conjugates served as a proof of concept for their applicability in cytotoxic screenings on tumor cells with different target expression levels. Second, the automated workflow enabled a large unbiased combinatorial screening of biparatopic antibodies and the identification of hits mediating potent c-MET degradation. The presented workflow utilizes standard equipment and may serve as a facile, efficient and robust method for the discovery of innovative therapeutic agents in many laboratories worldwide