Hilmar Ebersbach

Antigen generation and display in therapeutic antibody drug discovery – a neglected but critical player

Disease intervention by targeting a critical pathway molecule through a blocking antibody or interference by therapeutic proteins is currently en vogue. Generation of blocking antibodies or therapeutic proteins inevitably requires the production of recombinant proteins or cell‐based immunogens. Thus, one could call the antigen molecule the neglected player in antibody drug discovery. The variety of methods available for making recombinant proteins or recombinant cell lines that present the target on the cell surface is extensive. These need to be addressed in conjunction with biochemical and biophysical quality criteria and the experimental application intended. Fundamentally, successful production and isolation of monoclonal antibodies requires optimized antigen preparation and presentation to the immune host. This review summarizes the most important aspects of antigen generation and display, enabling logical decision making to give rise to potent high‐affinity antibodies.

Antigen presentation for the generation of binding molecules.

In the last few decades, several new methods have been established to isolate full antibodies and fragments thereof, some even using alternative scaffolds from in vivo and in vitro sources. These methods encompass robust techniques including immunization and hybridoma technology or phage display and also more laborious and novel approaches including ribosome display or B-cell immortalization. All methodologies are dependent upon proper antigen presentation for isolation, screening, and further characterization of the selected binding molecules. Here, antigens are classes of molecules including soluble or membrane proteins, part or domains thereof (extracellular domains of GPCRs), peptides, carbohydrates, and small-molecular-weight moieties. Presentation of the antigen in a functional state or perhaps even mimicking the intended application is crucial for successful isolation of useful binding molecules. Moreover, it is also necessary to consider the expression host and any posttranslational modifications of target proteins. The increasing demand to target more complex antigens, for instance, receptors and ion channels, is leading to the development of alternative procedures to present these proteins appropriately, for example by the use of virus-like particles and DNA immunization. This chapter describes in general approaches for the preparation of different forms of immunogens including synthetic peptides, proteins, cell-based antigens for immunization and in vitro display systems and in detail the preparation of a soluble protein as antigen.

Mechanistic Insights of an Immunological Adverse Event Induced by an Anti-KIT Antibody Drug Conjugate and Mitigation Strategies

Purpose: Hypersensitivity reactions (HSRs) were observed in three patients dosed in a phase I clinical trial treated with LOP628, a KIT targeted antibody drug conjugate. Mast cell degranulation was implicated as the root cause for the HSR. Underlying mechanism of this reported HSR was investigated with an aim to identifying potential mitigation strategies. Experimental Design: Biomarkers for mast cell degranulation were evaluated in patient samples and in human peripheral blood cell-derived mast cell (PBC-MC) cultures treated with LOP628. Mitigation strategies interrogated include pretreatment of mast cells with small molecule inhibitors that target KIT or signaling pathways downstream of FcϵR1, FcγR, and treatment with Fc silencing antibody formats. Results: Transient elevation of serum tryptase was observed in patients 1-hour posttreatment of LOP628. In agreement with the clinical observation, LOP628 and its parental antibody LMJ729 induced degranulation of human PBC-MCs. Unexpectedly, KIT small molecule inhibitors did not abrogate mast cell degranulation. By contrast, small molecule inhibitors that targeted pathways downstream of Fc receptors blunted degranulation. Furthermore, interference of the KIT antibody to engage Fc receptors by pre-incubation with IgG or using engineered Fc silencing mutations reduced or prevented degranulation. Characterization of Fcγ receptors revealed human PBC-MCs expressed both FcγRII and low levels of FcγRI. Interestingly, increasing the level of FcγRI upon addition of IFNγ, significantly enhanced LOP628-mediated mast cell degranulation. Conclusions: Our data suggest LOP628-mediated mast cell degranulation is the likely cause of HSR observed in the clinic due to co-engagement of the FcγR and KIT, resulting in mast cell activation. Clin Cancer Res; 24(14); 3465–74. ©2018 AACR.